Cationic lipid, liposome containing cationic lipid, nucleic acid pharmaceutical composition containing liposome, preparation and application thereof
1. A cationic lipid having a structure represented by the general formula (1):
wherein N is a nitrogen branching center;
L1、L2is a bond or a divalent linking group each independently selected from-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CR) cRc)sO-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-and-NRcAny one of C (═ O) S-, wherein R iscEach occurrence independently is a hydrogen atom or C1-12Alkyl, s is 2, 3 or 4;
L3is a connecting bond, -L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5-or-L4-Z-L5-Z-; said L4、L5Are carbon chain linking groups, each independently is- (CR)aRb)t-(CRaRb)o-(CRaRb)p-, where t, o and p are each independently integers from 0 to 12 and are not 0 at the same time, RaAnd RbEach occurrence is independently a hydrogen atom or an alkyl group; each occurrence of Z is independently- (C ═ O) -, -O (C ═ O) -, - (C ═ O) O-, -S-, -C (═ O) S-, -SC (═ O) -, -NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC (═ O) S-andany one of (1), wherein RcEach occurrence independently of the others being a hydrogen atom orC1-12An alkyl group;
B1、B2each independently is a connecting bond or C1-30An alkylene group;
R1、R2each independently is C2-30An aliphatic hydrocarbon group;
R3is a hydrogen atom, -Rd、-ORd、-NRdRd、-SRd、-(C＝O)Rd、-(C＝O)ORd、-O(C＝O)Rd、-O(C＝O)ORdOrWherein R isdEach occurrence is independently C1-12Alkyl radical, G1A terminal branched group with a valence of k +1, j is 0 or 1, F contains a functional group R01When j is 0, G1In the absence of, when j is 1, G1K F is led out, and k is an integer of 2-8;
a is selected from- (CR)aRb)sO-、-O(CRaRb)s-、-(CRaRb)sS-、-S(CRaRb)s-、-(CRaRb)sO(CRaRb)sS-、-(CRaRb)sS(CRaRb)sO-、-(CRaRb)sNRc(CRaRb)sS-、-(CRaRb)sS(CRaRb)sNRc-、-(CRaRb)sNRc(CRaRb)sO-and- (CR)aRb)sO(CRaRb)sNRc-any of, wherein s is 2, 3 or 4, RaAnd RbEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
when A is- (CR)aRb)sO-or-O (CR) aRb)s-when n is an integer from 2 to 6; when A is not- (CR)aRb)sO-or-O (CR)aRb)s-when n is an integer from 1 to 6;
the alkyl, alkylene, alkoxy, aliphatic hydrocarbon groups are each independently substituted or unsubstituted.
2. The cationic lipid of claim 1, wherein B is1、B2Each independently is a connecting bond or C1-20An alkylene group; more preferably B1、B2One of them is a connecting bond and the other is C1-20An alkylene group; more preferably B1、B2Each independently is any of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene, and eicosylene.
3. The cationic lipid of claim 1, wherein L is L3Is- (CH)2)t-、-(CH2)tZ-、-Z(CH2)t-、-(CH2)tZ(CH2)t-and-Z (CH)2)tZ-, wherein t is an integer of 1 to 12; is preferably- (CH)2)t-、-(CH2)tO-、-(CH2)tC(＝O)-、-(CH2)tC(＝O)O-、-(CH2)tOC(＝O)-、-(CH2)tC(＝O)NH-、-(CH2)tNHC(＝O)-、-(CH2)tOC(＝O)O-、-(CH2)tNHC(＝O)O-、-(CH2)tOC(＝O)NH-、-(CH2)tNHC(＝O)NH-、-O(CH2)t-、-C(＝O)(CH2)t-、-C(＝O)O(CH2)t-、-OC(＝O)(CH2)t-、-C(＝O)NH(CH2)t-、-NHC(＝O)(CH2)t-、-OC(＝O)O(CH2)t-、-NHC(＝O)O(CH2)t-、-OC(＝O)NH(CH2)t-、-NHC(＝O)NH(CH2)t-、-(CH2)tO(CH2)t-、-(CH2)tC(＝O)(CH2)t-、-(CH2)tC(＝O)O(CH2)t-、-(CH2)tOC(＝O)(CH2)t-、-(CH2)tC(＝O)NH(CH2)t-、-(CH2)tNHC(＝O)(CH2)t-、-(CH2)tOC(＝O)O(CH2)t-、-(CH2)tNHC(＝O)O(CH2)t-、-(CH2)tOC(＝O)NH(CH2)t-、-(CH2)tNHC(＝O)NH(CH2)t-、-O(CH2)tO-、-C(＝O)(CH2)tC(＝O)-、-C(＝O)O(CH2)tC(＝O)O-、-OC(＝O)(CH2)tOC(＝O)-、-C(＝O)O(CH2)tOC(＝O)-、-OC(＝O)(CH2)tC(＝O)O-、-OC(＝O)O(CH2)tOC(＝O)O-、-C(＝O)NH(CH2)tC(＝O)NH-、-NHC(＝O)(CH2)tNHC(＝O)-、-NHC(＝O)(CH2)tC(＝O)NH-、-C(＝O)NH(CH2)tNHC(＝O)-、-NHC(＝O)O(CH2)tNHC(＝O)O-、-OC(＝O)NH(CH2)tOC(＝O)NH-、-NHC(＝O)O(CH2)tOC(＝O)NH-、-OC(＝O)NH(CH2)tNHC(＝O)O-、-NHC(＝O)NH(CH2)tNHC(＝O)NH-、-C(＝O)(CH2)tO-、-C(＝O)(CH2)tC(＝O)O-、-C(＝O)(CH2)tOC(＝O)-、-C(＝O)(CH2)tOC(＝O)O-、-C(＝O)(CH2)tNHC(＝O)O-、-C(＝O)(CH2)tOC (═ O) NH — and-C (═ O) (CH)2)tNHC (═ O) NH-.
4. The cationic lipid of claim 1, wherein F has the structure- (Z)2)q-(Z1)q1-R01Q, q1 are each independently 0 or 1; z 1、Z2Each independently is a divalent linking group, more preferably each independently is-L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5and-L4-Z-L5-Z-, and t is an integer from 1 to 12; r01Is a functional group capable of interacting with biologically relevant substances.
5. The cationic lipid of claim 4, wherein R is01Selected from: reactive groups, variations of reactive groups, functional groups with therapeutic targeting, fluorescent functional groups; wherein the variants include any of a precursor of a reactive group, an active form as a precursor thereof, a substituted active form, a protected form, a deprotected form; wherein the precursor of the reactive group refers to a structure which can be converted into the reactive group by at least one of oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexing, ionization, protonation and deprotonation; wherein, the change form of the reactive group refers to a form that one reactive group still has activity after undergoing at least one process of oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexing, ionization, protonation, deprotonation, substitution and deprotection, or an inactive form after being protected; the R is 01More preferably from the group consisting of any one of the following functional groups of groups A to H and their modifications or functional groups of groups I to J:
class A: activated ester groups, analogous structures of activated ester groups; wherein the active ester group comprises: a succinimide active ester group, a p-nitrophenyl active ester group, an o-nitrophenyl active ester group, a benzotriazole active ester group, a 1,3, 5-trichlorobenzene active ester group, a 1,3, 5-trifluorobenzene active ester group, a pentafluorobenzene active ester group and an imidazole active ester group; wherein, the analogous structure of the active ester group comprises: 2-thiothiazolidine-3-carboxylate, 2-thiopyrrolidine-N-carboxylate, 2-thiobenzothiazole-N-formate, 1-oxo-3-thioisoindoline-N-formate;
class B: sulfonate, sulfinate, sulfone, sulfoxide, 1, 3-disulfonyl-2-propylcarbonylphenyl, sulfone methacryl;
class C: hydroxylamino group, mercapto group, primary amino group, secondary amino group, halogen atom, haloacetamido group, tetramethylpiperidinoxy group, dioxopiperidinoxy group, ammonium salt, hydrazine group, dithio compound group, ester group, thioester group, thiocarbonate group, carbonate group, thiocarbonate group, dithiocarbonate group, trithiocarbonate group, xanthate group, peroxythiocarbonate group, tetrathiodiester group, O-carbonylhydroxylamino group, amide group, imide group, hydrazide group, sulfonylhydrazide group, hydrazone group, imine group, enamine group, alkynylamine group, carbamate group, monothiocarbamate group, dithiocarbamate group, protected amino group;
Class D: carboxyl, sulfonic, sulfenic, hydroxamic, thiohydroxamic, xanthic, acid halide, sulfonyl chloride, aldehyde, glyoxal, acetal, hemiacetal, hydrated aldehyde, keto, ketal, hemiketal, ketal, hydrated keto, orthoacid, orthoester, cyanate, thiocyanate, isocyanurate, isothiocyanate, ester, oxycarbonylhalide, oxazoline, isoxazoline, thioaldehyde, thioketone, thioacetal, thioketone hydrate, thiothiothiothiothioketal, thiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothioketal, thioester, thiohemiacetal, monothiohydrate, dithiohydrate, thiohydrate, thiocarbonylmonocarboxylic, thiothiocarboxylic, dithiocarboxylic, thiosemicarbazide, guanidino, and protonated forms thereof, Amidino group and protonated form thereof, acid anhydride group, squaric acid ester group, hemisquaric acid ester group, N-carbamoyl-3-imidazolyl group, N-carbamoyl-3-methylimidazolium iodide group, imido group, nitrone group, oximo group, pseudoureido group;
Class E: maleimide group, acrylate group, N-acrylamide group, methacrylate group, N-methacrylamide group, protected maleimide group, maleamido group, 1,2, 4-triazoline-3, 5-dione group, linear azo compound group, cyclic olefin group; wherein the cycloalkenylhydrocarbyl group includes cyclooctenylhydrocarbyl, norbornenyl, 7-oxa-bicyclo [2.2.1] hept-5-en-2-yl, bicycloheptedienyl, 7-oxabicycloheptedienyl;
class F: epoxy, vinyl, propenyl, alkenyl hydrocarbon, alkynyl hydrocarbon;
the class of the signal is a class G,
class Ga: cycloalkynylalkyl, cycloalkynheteroalkyl, linear conjugated dienyl, cyclic conjugated dienyl, hybrid cyclic conjugated dienyl, 1,2,4, 5-tetrazinyl;
class Gb: azido, nitrile oxide groups, cyano, isocyano, aldoximo, diazo, diazonium ions, azoxy, nitrilo imino, N-oxyaldoimino, tetrazolyl, 4-acetyl-2-methoxy-5-nitrophenoxy and diazotized forms thereof; other functional groups capable of undergoing a 1, 3-dipolar cycloaddition reaction;
class H: hydroxyl, protected hydroxyl, siloxy, protected bishydroxy, trihydroxysilyl, protected trihydroxysilyl; wherein the hydroxyl group comprises alcoholic hydroxyl group, phenolic hydroxyl group, enol hydroxyl group and hemiacetal hydroxyl group;
Class I: targeting groups and pharmaceutically acceptable salts thereof;
class J: the fluorescent group includes any one of fluorescein, rhodamine, anthracene, pyrene, coumarin, fluorescein 3G, carbazole, imidazole, indole and alizarin violet and the residue of any one of functional derivatives.
6. The cationic lipid of claim 5, wherein R is01Functional groups selected from any of the following classes A to J, and variants of classes A to HChemical forms, class I to class J functional derivatives; the variant is selected from any one of a precursor of a reactive group, an active form thereof as a precursor, a substituted active form, a protected form, a deprotected form:
class A:
or class B:
or class C:
or class D:
or class E:
or class F:
or class G:
class Ga:
or class Gb:
or class H:
or class I:
or class J:
wherein M is5Is a ring-forming atom selected from any one of carbon atom, nitrogen atom, phosphorus atom and silicon atom; m5The cyclic structure is a 3-50-membered ring, preferably a 3-32-membered ring, more preferably a 3-18-membered ring, and still more preferably a 5-18-membered ring; the cyclic structure is preferably any one of the following groups, a substituted form of any one, or a hybridized form of any one: cyclohexane, furanose ring, pyranose ring, benzene, tetrahydrofuran, pyrrolidine, thiazolidine, cyclohexene, tetrahydropyran, piperidine, 1, 4-dioxane, pyridine, pyridazine, pyrimidine, pyrazine, 1,3, 5-triazine, 1,4, 7-triazacyclononane, tripeptide, indene, indane, indole, isoindole, purine (iv) Perrin, naphthalene, Dihydroanthracene, xanthene, thioxanthene, Dihydrophenanthrene, 10, 11-dihydro-5H-Dibenzo [ a, d ]]Cycloheptane, dibenzocycloheptene, 5-dibenzocycloheptenone, quinoline, isoquinoline, fluorene, carbazole, iminodibenzyl, naphthylene ring, dibenzocyclooctyne, aza-dibenzocyclooctyne;
wherein, Y1Is a leaving group attached to sulfonyl, sulfinyl, oxysulfonyl or oxysulfinyl, selected from any one of methyl, ethyl, n-propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, vinyl, phenyl, benzyl, p-methylphenyl, 4- (trifluoromethoxy) phenyl, trifluoromethyl, 2,2, 2-trifluoroethyl;
wherein W is F, Cl, Br or I;
wherein, W2Is F, Cl, Br or I;
wherein, W3Is a leaving group selected from F, Cl, Br, I, PhS;
wherein the content of the first and second substances, each is a cyclic structure containing a nitrogen atom, a nitrogen onium ion, a double bond, an azo, a triple bond, a disulfide bond, an anhydride, an imide, a diene on the ring backbone, the cyclic structure being selected from a carbocycle, a heterocycle, a benzoheterocycle, a substituted carbocycle, a substituted heterocycle, or a substituted benzoheterocycle;
wherein M is a carbon atom, a nitrogen atom, a phosphorus atom or a silicon atom on the ring;
Wherein M is8Is a carbon atom, a nitrogen atom, a phosphorus atom or a silicon atom located on the ring; m8The number of ring-forming atoms of the ring is 4-50; preferably 4-32; more preferably 5 to 32;
wherein M is22Is a carbon atom, a nitrogen atom, a phosphorus atom or a silicon atom on an alicyclic or alicyclic ring; m22The number of ring atoms of the ring is 4, 5, 6, 7 or 8;
wherein R is22Is a terminal group or a divalent linking group to which an oxygen or sulfur atom is bonded, selected from a hydrogen atom, R21Or R33Any one atom or group;
wherein R is21Is a divalent linking group and participates in ring formation; r21Is selected from C1-20Alkylene, divalent C1-20Heterohydrocarbyl, substituted C1-20Alkylene, substituted divalent C1-20Any divalent linking group or any two or any three of divalent linking groups in the heterohydrocarbon group; r21Preferably methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, 1, 2-phenylene, benzylene, C1-20Oxaalkylene, C1-20Thiaalkylene group, C1-20Any one group of azaalkylene and azaaralkyl, a substituted form of any one group, any two or more of the same or different groups, or a combination of substituted forms thereof;
wherein R is 3Is a terminal group linked to an oxy or thio group, selected from C1-20Hydrocarbyl radical, C1-20Heterohydrocarbyl radical, C1-20Substituted hydrocarbyl radical, C1-20Any of substituted heterohydrocarbyl groups; preferably any one or substituted form of any one of methyl, ethyl, n-propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, benzyl, allyl;
wherein R is4Is- (R)4)C＝N+＝N—Or- (R)4)C--N+A hydrogen atom, a substituent atom or a substituent on C in the structure of [ identical to ] N; preferably any one atom or group of hydrogen atom, methyl group, ethyl group, n-propyl group, isopropyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, allyl group, propenyl group, vinyl group, phenyl group, methylphenyl group, butylphenyl group, benzyl group;
wherein R is8、R9、R10、R11、R12Each independently is a hydrogen atom, a substituent atom or a substituent on a double bond (-C-), and R is in the same molecule8、R9、R10、R11、R12May be the same as or different from each other; r8、R9、R10、R11、R12Each independently selected from: hydrogen atom, fluorine atom, methyl group; in class E3, R8Preferably methyl;
wherein R is24Is a terminal group linked to a disulfide bond selected from: c1-20Alkyl, aryl, hybrid phenyl;
wherein R is27Is a substituent attached to azo selected from: phenyl, substituted phenyl or hybrid phenyl;
Wherein R is30Is a hydrocarbyl group selected from: c1-20Alkyl, benzyl, phenyl ring hydrogen atoms by C1-20A hydrocarbyl-substituted benzyl group;
wherein M is19、M20、M21Each independently is an oxygen atom or a sulfur atom, and may be the same as or different from each other in the same molecule;
wherein, X6Is a terminal group attached to the oxygen atom of the ester group and is selected from a hydroxyl protecting group or the group LG4；LG4Is selected from C1-20Alkyl, aryl, aralkyl, C1-20Heteroalkyl, heteroaryl, heteroaralkyl, C1-20Alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, C1-20Heteroalkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, C1-20Alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, C1-20Alkylthio-carbonyl, arylthio-carbonyl, aralkylthiocarbonyl, C1-20Alkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl, C1-20Heteroalkyloxycarbonyl, heteroaryloxycarbonyl, heteroaralkyloxycarbonyl, C1-20Heteroalkylthio-carbonyl, heteroarylthio-carbonyl, heteroaralkylthio-carbonyl, C1-20Heteroalkylaminocarbonyl, heteroarylaminocarbonyl, heteroarylalkylaminocarbonyl, C1-20Alkylthio, arylthio, aralkylthiocarbonyl, C1-20Heteroalkylthiocarbonyl, heteroarylthiocarbonyl, heteroarylalkylthiocarbonyl, C 1-20Alkoxythiocarbonyl, aryloxylthiocarbonyl, aralkylAlkoxycarbonyl, C1-20Alkylthio thiocarbonyl, arylthio thiocarbonyl, aralkylthio thiocarbonyl, C1-20Alkylaminothiocarbonyl, arylaminothiocarbonyl, aralkylaminothiocarbonyl, C1-20Heteroalkyloxythiocarbonyl, heteroaryloxythiocarbonyl, heteroarylalkoxythiocarbonyl, C1-20Heteroalkylthio thiocarbonyl, heteroarylthio thiocarbonyl, heteroarylalkylthio thiocarbonyl, C1-20(ii) any one of a heteroalkylaminothiocarbonyl, heteroarylaminothiocarbonyl or a substituted version of any one of the groups; wherein, the substituent atom or the substituent group is fluorine atom, alkoxy or nitro;
wherein, X11Is a terminal group attached to a carbonyl or thiocarbonyl group, selected from C1-20An alkyl group;
wherein, X12Is a terminal group to which a carbonate or thiocarbonate group is attached, selected from C1-20A hydrocarbyl group;
wherein, X13Is a terminal group for attaching a sulfur group selected from: mercapto-protecting group, group LG2；
Wherein LG is2Selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, allyl, trityl, phenyl, benzyl, methylbenzyl, nitrobenzyl, t-butylthio, benzylthio, 2-pyridylthio, acetyl, benzoyl, methoxycarbonyl, ethoxycarbonyl, t-butyloxycarbonyl, phenoxycarbonyl, benzyloxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl, t-butylthiocarbonyl, phenylthiocarbonyl, benzylthiocarbonyl, 2-pyridylcarbonyl, methylaminocarbonyl, ethylaminocarbonyl, t-butylaminocarbonyl, benzylamino-carbonyl, ethylthiocarbonyl, phenylmethylthiocarbonyl, methylthiocarbonyl, Methoxythiocarbonyl, ethoxythiocarbonyl, tert-butyloxythiocarbonyl, phenoxythiocarbonyl, benzyloxythiocarbonyl, methylthiothiocarbonyl, ethyl Thiylthiocarbonyl, tert-butylthiocarbonyl, phenylthiocarbonyl, benzylthiocarbonyl, methylaminothiocarbonyl, ethylaminothiocarbonyl, tert-butylaminothiocarbonyl, benzylaminothiocarbonyl, C1-10Any one of a halogenated hydrocarbon group, a trifluoroacetyl group, a nitrophenyl group, or a substituted form of any one of the groups; wherein, the substituent atom or the substituent group is fluorine atom, alkoxy or nitro;
wherein Q is an atom or substituent contributing to the induction of unsaturated bond electrons, conjugation effect; when Q is on a ring, the number is one or more; when the number is multiple, the structure is the same, or the combination of two or more different structures; when a substituent group, Q has a linear structure, a branched structure containing a pendant group, or a cyclic structure;
wherein Q is3An H atom or a group contributing to the induction of the electrons of unsaturated bonds, a conjugation effect, selected from any atom or group of hydrogen atom, fluorine atom, chlorine atom, bromine atom, iodine atom, methyl group, ethyl group, n-propyl group, isopropyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, vinyl group, propenyl group, allyl group, propynyl group, propargyl group, cyclopropyl group, cyclopropenyl group, phenyl group, benzyl group, butylphenyl group, p-methylphenyl group, p-nitrophenyl group, o-nitrophenyl group, p-methoxyphenyl group, azaphenyl group, methoxy group, ethoxy group, phenoxy group, benzyloxy group, methylthio group, ethylthio group, phenylthio group, benzylthio group, trifluoromethyl group, 2,2, 2-trifluoroethyl group, or a substituted form of any group;
Wherein Q is5Is H atom, methyl, ethyl or propyl; when Q is5When located on a ring, the number is one or more; when more than 1, the structure is the same, or the combination of two or more different structures;
wherein Q is6Is a hydrogen atom or a methyl group; q7Is a hydrogen atom, a methyl group, a phenyl group or a substituted phenyl group; in the same molecule, Q6And Q7May be the same or different;
wherein Q is8Is a substituent atom or a substituent group on the imidazolyl group, and is selected from H atoms, methyl groups,Any one of ethyl, propyl, butyl, and phenyl; when Q is8Is one or more; when more than 1, the structure is the same, or the combination of two or more different structures;
wherein Q is11Is a substituent on the nitrogen atom of tetrazole, and is selected from any one of phenyl, substituted phenyl and aza-phenyl;
wherein PG2Is a thiol protecting group, the protected thiol group being denoted as SPG2Preferably any one of thioether, disulfide, silyl sulfide, thioester;
wherein PG3Is an alkynyl protecting group, preferably a silicon group;
wherein PG4Is a hydroxy protecting group, the protected hydroxy group being represented by OPG4Any one of ether, silyl ether, ester, carbonate and sulfonate is preferable;
wherein PG 5Is an amino protecting group, the protected amino group being represented by NPG5Preferably any one of carbamate, amide, imide, N-alkylamine, N-arylamine, imine, enamine, imidazole, pyrrole and indole;
wherein PG6Is a bishydroxy protecting group, and PG6An acetal structure which forms a five-membered ring or a six-membered ring with two oxygen atoms; PG (Picture experts group)6Is methylene or substituted methylene; wherein PG6The substituent(s) is a hydrocarbyl substituent or a heteroatom-containing substituent selected from: any one of methylene, 1-methylmethylene, 1-dimethylmethylene, 1-cyclopentylene, 1-cyclohexylene, 1-phenylmethylene, and 3, 4-dimethylphenylmethylene;
wherein PG8Protecting groups for orthocarbonic acid or orthosilicic acid.
7. The cationic lipid of claim 4, wherein L is L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2The structure of (a) is not particularly limited, and each independently is preferably a linear structure, a branched structure, or a cyclic structure; further, in the present invention,L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2the number of the non-hydrogen atoms of (b) is not particularly limited, and each is independently preferably 1 to 50 non-hydrogen atoms; more preferably 1 to 20 non-hydrogen atoms; more preferably 1 to 10 non-hydrogen atoms; wherein the non-hydrogen atom is C, O, S, N, P, Si or B; when the number of the non-hydrogen atoms is more than 1, the kind of the non-hydrogen atoms is 1, 2, or more than 2, and the non-hydrogen atoms are any combination of carbon atoms and carbon atoms, carbon atoms and heteroatoms, and heteroatoms.
8. The cationic lipid of claim 4, wherein the divalent linking group L is1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2Is not particularly limited, wherein any one of the divalent linking groups or any one of the divalent linking groups consisting of a group with an adjacent heteroatom is each independently a stably existing linking group STAG or a degradable linking group DEGG; the stable connecting group STAG can stably exist under any condition selected from light, heat, low temperature, enzyme, oxidation reduction, acidity, alkaline condition, physiological condition and in-vitro simulation environment, and preferably can stably exist under any condition selected from light, heat, enzyme, oxidation reduction, acidity and alkalinity; the DEGG degradable condition is degradable under any condition selected from light, heat, low temperature, enzyme, redox, acidity, alkalinity, physiological condition and in-vitro simulation environment, and preferably degradable under any condition selected from light, heat, enzyme, redox, acidity and alkalinity;
the linking group STAG which may be stably present is further preferably one containing an alkylene group, a divalent heteroalkyl group, a double bond, a triple bond, a divalent dienyl group, a divalent cycloalkyl group, a divalent cycloalkenyl group, a divalent cycloalkenylene group, an aromatic ring, an alicyclic ring, a hetero-fused heterocycle, a substituted alkylene group, a substituted heteroalkyl group, a substituted divalent heteroalkyl group, a substituted double bond, a substituted triple bond, a substituted diene, a substituted divalent cycloalkyl group, a substituted divalent cycloalkenyl group, a substituted divalent cycloalkenylene group, a substituted divalent cycloalkynylene group, a substituted aromatic ring, a substituted alicyclic ring, a substituted hetero-fused heterocycle, a substituted aromatic ring, a substituted hetero-fused heterocycle, an ether bond, a thioether bond, urea bond, thiourea bond, a carbamate group, a thiocarbamate group, -P (═ O) -, a divalent silyl group containing no active hydrogen, A boron atom-containing divalent connecting group, a secondary amino group, a tertiary amino group, a carbonyl group, a thiocarbonyl group, an amido group, a thioamido group, a sulfamide group, an enamine group, triazole, 4, 5-dihydroisoxazole, any divalent connecting group in the skeleton of amino acid and derivatives thereof, and a stable divalent connecting group consisting of any two or more than two groups;
The degradable linking group DEGG further preferably contains a disulfide bond, a vinyl ether bond, an ester group, a thioester group, a dithioester group, a carbonate group, a thiocarbonate group, a dithiocarbonate group, a trithiocarbonate group, a carbamate group, a thiocarbamate group, a dithiocarbamate group, an acetal, a cyclic acetal, a mercaptal, an azaacetal, an azacyclic acetal, a thioheterocyclic acetal, a dithioacetal, a hemiacetal, an azahemiacetal, a ketal, a thioketal, an azaketal, a thioheterocyclic ketal, an imine bond, a hydrazone bond, an acylhydrazone bond, an oxime bond, a sulfoximine ether group, a semicarbazone bond, a thiosemicarbazone bond, a hydrazine group, a hydrazide group, a thiocarbonyl group, an azohydrazide group, a thiocarbazohydrazide group, a carbazoyl group, a thiocarbazohydrazide group, a thiocarbamate group, a thiocarbazide, a thiocarbhydrazide group, a thiocarbamate group, a thiocarbazide group, a thiocarbhydrazide group, a hydrazine group, a thiocarbazide group, a, Azo groups, isothioureido groups, allophanate groups, thioallophanate groups, guanidino groups, amidino groups, aminoguanidino groups, aminoamidino groups, imino ester thioester groups, sulfonate groups, sulfinate groups, sulfonylhydrazide groups, sulfonylureido groups, maleimide groups, orthoester groups, phosphate groups, phosphite groups, phosphinate groups, phosphonate groups, phosphosilane groups, silane groups, carbonamide, thioamide groups, sulfonamide groups, polyamide, phosphoramide, pyrophosphamide, cyclophosphamide, ifosfamide, thiophosphoramide, aconityl groups, polypeptide fragments, a backbone of nucleotides and derivatives thereof, any divalent linking group in a backbone of deoxyribonucleotides and derivatives thereof, or a combination of any two or more divalent linking groups.
9. The cationic lipid of claim 1, wherein R is3Is a hydrogen atom, Rd、ORd、-(C＝O)Rd-、-(C＝O)ORd、-O(C＝O)Rd、-O(C＝O)ORdAndany one of (1), the R3More preferably contains any of a hydrogen atom, an alkyl group, an alkoxy group, an alcoholic hydroxyl group, a protected alcoholic hydroxyl group, a thiol hydroxyl group, a protected thiol hydroxyl group, a carboxyl group, a protected carboxyl group, an amino group, a protected amino group, an aldehyde group, a protected aldehyde group, an ester group, a carbonate group, a carbamate group, a succinimide group, a maleimide group, a protected maleimide group, a dimethylamino group, an alkenyl group, an enoate group, an azide group, an alkyne group, a folate group, a rhodamine group, and a biotin group; further preferably contains H, - (CH)2)tOH、-(CH2)tSH、-OCH3、-OCH2CH3、-(CH2)tNH2、-(CH2)tC(＝O)OH、-C(＝O)(CH2)tC(＝O)OH、-C(＝O)CH3、-(CH2)tN3、-C(＝O)CH2CH3、-C(＝O)OCH3、-OC(＝O)OCH3、-C(＝O)OCH2CH3、-OC(＝O)OCH2CH3、-(CH2)tN(CH3)2、-(CH2)tCHO、
10. The cationic lipid of claim 1, wherein R is3The number of atoms spaced from the nitrogen branching center N is greater than or equal to 6, preferably6 to 50, more preferably 6 to 35, more preferably 6 to 25, most preferably 6 to 15.
11. The cationic lipid of claim 1, wherein R is1、R2Each independently is a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a linear alkynyl group or a branched alkynyl group; preferably a linear alkyl group; more preferably each independently C 1-25A linear alkyl group; more preferably each independently C1-17A linear alkyl group; most preferably, each independently of the others, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, (Z) -tridec-8-enyl, (Z) -tetradec-9-enyl, (Z) -pentadec-8-enyl, (Z) -hexadec-9-enyl, (Z) -heptadec-5-enyl, (Z) -heptadec-8-enyl, (E) -heptadec-8-enyl, (Z) -heptadec-10-enyl, pentyl, (8Z,11Z) -heptadecane-8, 11-dienyl, (Z) -octadecan-6-enyl, (Z) -octadecan-9-enyl, (E) -octadecan-9-enyl, (Z) -octadecan-11-enyl, (9Z,12Z) -octadecan-9, 12-dienyl, (9Z,12Z,15Z) -octadecan-9, 12, 15-trienyl, (8Z,11Z,14Z) -octadecan-8, 11, 14-trienyl, (Z) -eicosan-11-enyl, (11Z,14Z) -eicosan-11, 14-dienyl, (Z) -nonadecane-10-enyl, (10Z,13Z) -nonadecane-10, 13-dienyl, 2,6, 10-trimethylundecane-1, 5, 9-trienyl, 3,7, 11-trimethyldodecane-2, 6, 10-trienyl or 3,7,11, 15-tetramethylhexadecan-2-enyltridecyl.
12. The cationic lipid of claim 11, wherein R is1、R2Each independently is a branched alkyl, branched alkenyl or branched alkynyl, each independently isWherein R ise、RfEach independently is C1-C15Alkyl radical, C2-C15Alkenyl and C2-C15Any one of alkynyl groups; more preferably, each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propenyl, allyl, butenyl, alkenylbutyl, pentenyl, alkenylpentyl, hexenyl, alkenylhexyl, heptenyl, alkenylheptyl, octenyl, nonenyl, decenyl, alkenyldecyl, ethynyl, propynyl, propargyl, butynyl, pentynyl, hexynyl, alkynhexyl, heptynyl, alkynheptyl, octynyl, nonynyl, decynyl, and alkyndecyl; more preferably, each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.
13. The cationic lipid of claim 12, wherein R is 1、R2Each independently selected from any one of the following structures:
wherein t is an integer of 0 to 12.
14. The cationic lipid of claim 1, wherein A is-CH2CH2O-、-OCH2CH2-、-CH(CH3)CH2O-、-OCH(CH3)CH2-、-CH2CH2S-、-SCH2CH2-、-CH2CH2NHCH2CH2O-、-CH2CH2OCH2CH2NH-、-CH2CH2SCH2CH2O-or-CH2CH2OCH2CH2S-, more preferably-CH2CH2O-、-OCH2CH2-、-CH2CH2SCH2CH2O-or-CH2CH2OCH2CH2S-, most preferably-CH2CH2O-or-OCH2CH2-。
15. The cationic lipid of claim 14, having the structure of any one of the following structural formulas:
16. the cationic lipid of claim 1, wherein L is L1、L2Each independently is a bond, -O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CH)2)sAny one of O-, -S-, -C (═ O) S-, -SC (═ O) -, -NHC (═ O) -, -C (═ O) NH-, -NHC (═ O) NH-, -OC (═ O) NH-, -NHC (═ O) O-, -SC (═ O) NH-, and-NHC (═ O) S-, is more preferable, and L-is more preferable1、L2One of which is a bond and the other is-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CH)2)sAny one of O-, -S-, -C (═ O) S-, -SC (═ O) -, -NHC (═ O) -, -C (═ O) NH-, -NHC (═ O) NH-, -OC (═ O) NH-, -NHC (═ O) O-, -SC (═ O) NH-, and-NHC (═ O) S-; more preferably L1And L2Each independently represents-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CH) 2)sO-、-S-、-C(＝O)S-、-SC(＝O)-、-NHC(＝O)-、-C(＝O)NH-、-NHC(＝O)NH-、-OC(＝Any one of O) NH-, -NHC (═ O) O-, -SC (═ O) NH-, and-NHC (═ O) S-; more preferably L1And L2Each independently is any one of-O (C ═ O) -, - (C ═ O) O-and-O (C ═ O) O-, more preferably L1And L2At the same time, -O (C ═ O) -or at the same time- (C ═ O) O-or at the same time-O (C ═ O) O-.
17. The cationic lipid of claim 16, having the structure of any one of the following structural formulas:
wherein, in the formulae (2-1) to (2-10), B1And B2Are not a bond, B in formula (2-22)1Is a connecting bond; the A is preferably-CH2CH2O-、-OCH2CH2-、-CH2CH2S-、-SCH2CH2-、-CH2CH2NHCH2CH2O-、-CH2CH2OCH2CH2NH-、-CH2CH2SCH2CH2O-and-CH2CH2OCH2CH2Any of S-, more preferably-CH2CH2O-、-OCH2CH2-、-CH2CH2NHCH2CH2O-、-CH2CH2OCH2CH2NH-、-CH2CH2SCH2CH2O-and-CH2CH2OCH2CH2S-is any one of the above.
18. The cationic lipid according to claim 1, wherein when the terminal is a trivalent or higher-trivalent branched group, G is preferable1Is a trivalent terminal branching group or a tetravalent terminal branching group; preferably G1Is a trivalent terminal branching group selected from the group consisting of trivalent terminal branching groups of amino acid residues; preferably G1Is a trivalent residue of lysine or glycine.
19. The cationic lipid according to claim 1, wherein the structure thereof includes, but is not limited to, any of the following structures:
20. a cationic liposome comprising the cationic lipid of any one of claims 1-19.
21. The cationic liposome of claim 20, further comprising one or more of a neutral lipid, a steroid lipid, and a pegylated lipid; more preferably, the lipid composition contains neutral lipid, steroid lipid and pegylated lipid; wherein the neutral lipid is preferably a phospholipid.
22. The cationic liposome of claim 21, wherein the neutral lipid is selected from 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-didecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1, 2-dilinolacyl-sn-glycero-3-phosphocholine, 1, 2-didodecaoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didecylhexaenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), Dioleoylphosphatidylserine (DOPS), Dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), Distearoylphosphatidylethanolamine (DSPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-Stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and Lysophosphatidylethanolamine (LPE), and combinations thereof.
23. The cationic liposome of claim 21, wherein the steroid lipid is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.
24. The cationic liposome of claim 21, wherein the pegylated lipid is selected from the group consisting of polyethylene glycol-1, 2 dimyristoyl glyceride (PEG-DMG), polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically including polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-1, 2-oleoylphosphatidylethanolamine, polyethylene glycol 2000-1, 2-oleoylphosphatidylethanolamine, and polyethylene glycol 2000-2, any one of 3-dimyristoyl glycerol (PEG-DMG).
25. The cationic liposome of claim 21, wherein the pegylated lipid has the structure represented by formula (2):
Or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,
wherein L is7、L8Each independently is a bond or a divalent linking group selected from-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -S-, -C (═ O) S-, -SC (═ O) -, -NR (r) ScC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-and-NRcAny one of C (═ O) S-, wherein RcEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
L3is a connecting bond, -L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5-or-L4-Z-L5-Z-; said L4、L5Are carbon chain linking groups, each independently is- (CR)aRb)t-(CRaRb)o-(CRaRb)p-, where t, o and p are each independently integers from 0 to 12 and are not 0 at the same time, RaAnd RbEach occurrence is independently a hydrogen atom or an alkyl group; each occurrence of Z is independently- (C ═ O) -, -O (C ═ O) -, - (C ═ O) O-, -S-, -C (═ O) S-, -SC (═ O) -, -NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC (═ O) S-andany one of (1), wherein RcEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
B3、B4each independently is a connecting bond or C1-12An alkylene group;
R1、R2each independently is C1-30An aliphatic hydrocarbon group;
r is hydrogen, alkyl, alkoxy, - (C ═ O) Rd、-(C＝O)ORd、-O(C＝O)Rd、-O(C＝O)ORdOrWherein R isdIs C1-12Alkyl radical, G1A terminal branched group having a valence of k +1, j is 0 or 1, F contains a functional group, and when j is 0, G 1In the absence of, when j is 1, G1K F is led out, and k is an integer of 2-8;
a is- (CR)aRb)sO-or-O (CR)aRb)s-, where s is 2, 3 or 4, RaAnd RbEach independently is a hydrogen atom or C1-12An alkyl group;
n1is an integer from 20 to 250;
the alkyl, alkylene, alkoxy, aliphatic hydrocarbon groups are each independently substituted or unsubstituted.
26. The cationic liposome of claim 25, wherein the pegylated lipid has a structure selected from any one of the following structural formulas:
27. the cationic liposome of claims 21-26, comprising 20-80% cationic lipid, 5-15% neutral lipid, 25-55% steroid lipid, and 0.5-10% pegylated lipid of formula (2), said percentages being mole percent of each lipid based on total lipid in a solution comprising solvent.
28. The cationic liposome of claims 21-26, wherein the cationic lipid comprises a mole percentage of 30-65% of total lipid in the solution comprising the solvent; more preferably about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%.
29. The cationic liposome of claims 21-26, wherein the neutral lipid comprises 7.5-13% mole percent of total lipid in the solution comprising the solvent; more preferably about 8%, 9%, 10%, 11%, 12%.
30. A cationic liposome according to claims 21-26, characterized in that the molar percentage of steroid lipids to total lipids in the solvent-containing solution is 35-50%, more preferably about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.
31. The cationic liposome of claims 21-26, wherein the mole percentage of pegylated lipid based on total lipid in the solvent-containing solution is from 0.5% to 5%; preferably 1 to 3%; more preferably about 1.5%, 1.6%, 1.7%, 1.8%, 1.9%.
32. A cationic liposome nucleic acid pharmaceutical composition comprising the cationic liposome of any one of claims 21-26 and a nucleic acid drug.
33. The cationic liposome nucleic acid pharmaceutical composition of claim 32, wherein the nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, mRNA, interfering nucleic acid, aptamer, antagomir, miRNA, ribozyme, and siRNA; preferably any one of DNA, mRNA, miRNA and siRNA.
34. The cationic liposomal nucleic acid pharmaceutical composition of claim 33, wherein the pharmaceutical composition is used as a drug selected from any one of the following: a medicament for treating any one of cancer, malignancy, liver disease, hepatitis, diabetes, gout, rheumatism, rheumatoid, senile dementia, cardiovascular disease, an antiallergic drug, an anti-infective agent, an antibiotic agent, an antiviral agent, an antifungal agent, a vaccine, a central nervous system inhibitor, a central nervous system stimulant, a psychotropic drug, a respiratory tract drug, a peripheral nervous system drug, a drug that acts at a synaptic or neuroeffector junction, a smooth muscle active drug, a histaminergic agent, an antihistaminicergenic agent, a blood and hematopoietic drug, a gastrointestinal drug, a steroid agent, a cytostatic agent, an anthelmintic agent, an antimalarial agent, an antiprotozoal agent, an antimicrobial agent, an anti-inflammatory agent, an immunosuppressant agent, an Alzheimer's disease drug or compound, an imaging agent, an antidote, an antispasmodic, a muscle relaxant, an anti-inflammatory agent, an imaging agent, an anti-inflammatory agent, a cardiovascular agent, a pharmaceutical composition, a pharmaceutical, Appetite suppressants, migraine agents, muscle contractants, antimalarials, antiemetics/antiemetics, bronchodilators, antithrombotic agents, antihypertensive agents, antiarrhythmics, antioxidants, antiasthmatics, diuretics, lipid regulators, antiandrogens, antiparasitics, anticoagulants, neoplastic agents, hypoglycemic agents, nutritional agents, supplements, growth supplements, anti-enteritis agents, antibodies, diagnostic agents, contrast agents, hypnotics, sedatives, psychostimulants, tranquilizers, antiparkinson agents, analgesics, anxiolytics, muscle infectives, and auditory disease agents; the cationic liposome pharmaceutical composition is more preferably used for treating related diseases caused by abnormal gene expression, wherein the diseases are selected from any one of genetic diseases, malignant tumors, cardiovascular diseases, rheumatoid diseases and infectious diseases; more preferably for the treatment of any of the following diseases: hemophilia, cystic fibrosis, familial hypercholesterolemia, breast cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, esophageal cancer, gastric cancer, carcinoma of large intestine, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, AIDS and viral infection.
35. A cationic liposomal nucleic acid pharmaceutical composition formulation comprising the liposomal nucleic acid pharmaceutical composition of claims 34-36 and a pharmaceutically acceptable diluent or excipient, preferably any one of deionized water, ultrapure water, phosphate buffer, and physiological saline, more preferably phosphate buffer or physiological saline, and most preferably physiological saline.
Background
Liposomes refer to micro vesicles formed by encapsulating a drug within a lipid bilayer. The liposome nanoparticle comprises liposome and nucleic acid drugs, has a structure similar to a biological membrane, is a nontoxic nanometer material with good biocompatibility, can encapsulate water-soluble and fat-soluble drugs, and has the advantages of reducing drug dosage, realizing slow release and targeted drug release, protecting encapsulated nucleic acid from degradation and removal in serum, and the like. In addition, the nano liposome is an excellent antigen carrier, can wrap a series of antigens and immunologic adjuvants with different physicochemical properties, protects protein polypeptide antigens from being degraded, can promote phagocytosis and presentation of antigen presenting cells to the antigens, and improves the specific immunoreaction of organisms. Thus, liposomal nanoparticles are widely used in the field of drug delivery.
Based on the aforementioned advantages, liposome nanoparticles are receiving more and more attention as a novel vaccine carrier, and are being used for development of antiviral vaccines, antibacterial vaccines, antiparasitic vaccines, antitumor vaccines, and the like. Neutral liposomes and cationic liposomes carrying a positive charge on their surface are the most commonly used carriers for nano-vaccines. The cationic liposome is not only an excellent protein/polypeptide antigen carrier, but also a novel immunologic adjuvant, can directly activate antigen presenting cells and enhance immune response induced by the vaccine, and therefore, the cationic liposome is widely used for encapsulating and transporting nucleic acid drugs in the field of vaccines.
The process of encapsulating and transporting nucleic acid drugs by cationic liposome is that firstly, cationic liposome-nucleic acid drug complex is formed: the surface of the cationic liposome is positively charged, and the nucleic acid is negatively charged, so that the cationic liposome and the nucleic acid form a cationic liposome-nucleic acid drug complex through electrostatic interaction; secondly, the whole surface of the cationic liposome-nucleic acid drug compound has positive charges, is adsorbed to the surface of a negatively charged cell through electrostatic interaction, and enters the cell through endocytosis to form an endosome; furthermore, the cationic lipid in the cationic liposome and the negatively charged lipid in the endosome generate electrostatic interaction, the negatively charged lipid is transferred into the cavity from the outside of the endosome cavity to form a neutral ion pair with the positively charged lipid, and the nucleic acid drug enters cytoplasm after being separated from the cationic liposome; finally, nucleic acid drugs transfected into cells perform corresponding functions within the cell. Cationic liposomes play an important role throughout the delivery of nucleic acid drugs into cells, until nucleic acid molecules express in vivo peptides or protein (antigen) fragments that are characteristic of pathogens (e.g., bacteria and viruses) or tumor antigens, and finally, to elicit a specific immune response.
Cationic liposomes are typically complexed by combining a cationic lipid, which interacts electrostatically with the nucleic acid, and a co-lipid, which serves to prevent oxidation of the lipid or to attach a ligand to the surface of the liposome or to reduce aggregation of the lipid particles, such as 1, 2-oleoylphosphatidylethanolamine (DOPE), Distearoylphosphatidylethanolamine (DSPE) and/or cholesterol, more preferably DOPE, under appropriate conditions. Cationic lipids, typically amphiphilic molecules, have a lipid affinity region containing one or more hydrocarbon groups, and a hydrophilic region containing at least one positively charged polar head group. The cationic lipid and the giant molecules such as nucleic acid form a complex with positive charge based on the total charge, and the surface of the cell membrane is totally negatively charged, so that the giant molecules such as nucleic acid easily enter cytoplasm through the plasma membrane of the cell, and the transport rate of nucleic acid drugs is improved.
Despite recent advances in cationic lipids for drug delivery, there remains a need in the art for improved cationic lipids suitable for conventional therapeutic use.
Disclosure of Invention
The invention provides a novel cationic lipid, a cationic liposome containing the cationic lipid, a nucleic acid pharmaceutical composition containing the cationic liposome and a preparation thereof.
The above object of the present invention is achieved by the following technical means,
one embodiment of the present invention:
a cationic lipid has a structure represented by general formula (1):
wherein N is a nitrogen branching center;
L1、L2is a bond or a divalent linking group each independently selected from-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CR)cRc)sO-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、 -NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-and-NRcAny one of C (═ O) S-, wherein R iscEach occurrence independently is a hydrogen atom or C1-12Alkyl, s is 2, 3 or 4;
L3is a connecting bond, -L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5-or-L4-Z-L5-Z-; said L4、L5Are carbon chain linking groups, each independently is- (CR)aRb)t-(CRaRb)o-(CRaRb)p-, where t, o and p are each independently integers from 0 to 12 and are not 0 at the same time, RaAnd RbEach occurrence ofEach independently is a hydrogen atom or an alkyl group; each occurrence of Z is independently- (C ═ O) -, -O (C ═ O) -, - (C ═ O) O-, -S-, -C (═ O) S-, -SC (═ O) -, -NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、 -NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC (═ O) S-andany one of (1), wherein RcEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
B1、B2each independently is a connecting bond or C1-30An alkylene group;
R1、R2each independently is C2-30An aliphatic hydrocarbon group;
R3is a hydrogen atom, -Rd、-ORd、-NRdRd、-SRd、-(C＝O)Rd、-(C＝O)ORd、-O(C＝O)Rd、 -O(C＝O)ORdOrWherein R isdEach occurrence is independently C 1-12Alkyl radical, G1A terminal branched group with a valence of k +1, j is 0 or 1, F contains a functional group R01When j is 0, G1In the absence of, when j is 1, G1K F is led out, and k is an integer of 2-8;
a is selected from- (CR)aRb)sO-、-O(CRaRb)s-、-(CRaRb)sS-、-S(CRaRb)s-、-(CRaRb)sO(CRaRb)sS-、 -(CRaRb)sS(CRaRb)sO-、-(CRaRb)sNRc(CRaRb)sS-、-(CRaRb)sS(CRaRb)sNRc-、 -(CRaRb)sNRc(CRaRb)sO-and- (CR)aRb)sO(CRaRb)sNRc-any of, wherein s is 2, 3 or 4, RaAnd RbEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
when A is- (CR)aRb)sO-or-O (CR)aRb)s-when n is an integer from 2 to 6; when A is not- (CR)aRb)sO-or-O (CR)aRb)s-when n is an integer from 1 to 6;
the alkyl, alkylene, alkoxy, aliphatic hydrocarbon groups are each independently substituted or unsubstituted.
The invention also provides another implementation scheme:
a cationic liposome comprises cationic lipid with structure shown in formula (1).
The invention also provides another implementation scheme:
a liposome nucleic acid medicinal composition comprises cationic liposome and medicament, and the cationic liposome comprises cationic lipid and nucleic acid medicament with structure shown in formula (1).
The present invention also provides another embodiment:
a liposome nucleic acid pharmaceutical composition preparation comprises the liposome nucleic acid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.
Compared with the prior art, the invention has the following beneficial effects:
The novel cationic lipid is nitrogen-branched, the ammonia of a branched center is easy to protonate under the physiological pH condition to generate partial positive charges, and the cationic lipid can be combined with nucleic acid with negative charges, so that the loading rate of nucleic acid drugs is improved.
The cationic liposome nucleic acid medicine composition prepared from the novel cationic lipid has positive charges on the surface, is adsorbed to the surface of a negatively charged cell through electrostatic interaction, and enters the cell by virtue of endocytosis to form an endosome. The tail end of the cationic lipid contains hydroxyl, amino, carboxyl and other groups, the cationic lipid is easy to protonate under an acidic condition, an endosome is in an acidic state, partial positive charges are generated after protonation, the cationic lipid and negatively charged lipids in the endosome are subjected to electrostatic interaction, the negatively charged lipids are transferred into a cavity from the outside of the cavity of the endosome and form neutral ion pairs with the positively charged lipids, so that the drug is separated from the cationic liposome and enters cytoplasm after escaping from the endosome, and the transport rate of the nucleic acid drug is improved.
The cationic liposome nucleic acid medicinal composition prepared from the novel cationic lipid has higher serum stability, stronger gene recombination capability, higher biocompatibility and stronger gene transfection effect, and is beneficial to improving the gene treatment effect of medicaments, particularly the gene treatment effect of nucleic acid medicaments.
The terminal of the novel cationic lipid can also contain a fluorescent group or a targeting group, and the cationic liposome nucleic acid pharmaceutical composition of the cationic lipid can have the fluorescent or targeting function, so that the gene therapy and/or diagnosis effect of the nucleic acid pharmaceutical can be further improved.
Detailed description of the preferred embodiments
Description of the terms
In the present invention, unless otherwise specified, each term has the following meaning.
In the present invention, when the structure concerned has an isomer, any of the isomers may be used unless otherwise specified. For example, a cis-isomer or trans-isomer may be present in a structure; the structure of E/Z isomer exists, and can be an E structure or a Z structure; when the optical rotation property exists, the optical rotation property can be left-handed rotation or right-handed rotation.
In the present invention, the numerical value interval includes not only the numerical value interval marked by the short horizontal line (e.g. 1-6), but also the numerical value interval marked by the wavy line (e.g. 1-6). In the present invention, unless otherwise specified, the integer intervals marked as intervals may represent the group of all integers within the range of the interval, and the range includes both endpoints. If the integer range 1-6 represents the group consisting of 1, 2, 3, 4, 5, 6. The numerical ranges in the present invention, including but not limited to numerical ranges expressed as integers, non-integers, percentages, and fractions, include both endpoints unless otherwise specified.
In the present invention, the formulae (2-1) to (2-10) are meant to include the formulae (2-1), (2-2), (2-3), (2-4), (2-5), (2-6), (2-7), (2-8), (2-9) and (2-10).
The numerical values referred to herein as "about" and "about" generally refer to a range of values of + -10%, and in some cases may be scaled up to + -15%, but not more than + -20%. The preset value is used as a base number. For example, a molar percentage of steroid lipids to total lipids in a solution comprising a solvent is about 40%, which is generally considered to include cases where the molar percentage of steroid lipids is between 30% and 50%.
In the present invention, the terms "including", "comprising" and "containing" and similar referents shall be construed in an open and inclusive sense in this specification and claims, unless otherwise indicated.
Two or more objects in the present invention "are independently preferred" and, when there are multiple levels of preference, they are not required to be selected from the same group of preference, but may be one of wide-range preference, one of narrow-range preference, one of maximum range preference, the other of either preference, or the other of the same preference, and may be selected from the same group of preference, for example, "R" or 1、R2Each independently is preferably a linear alkyl group; more preferably C1-25A linear alkyl group; more preferably C1-17Linear alkyl "may be R1Is C1-25Linear alkyl radical, R2Is C1-17Linear alkyl, or R1Is C1-17Linear alkyl radical, R2Is C1-25Linear alkyl, or R1And R2At the same time is C1-25Linear alkyl, or R1And R2At the same time is C1-17A linear alkyl group.
In the present invention, the divalent linking group such as alkylene, arylene, amide bond and the like is not particularly limited, and any of the two linking ends may be selected when linking other groups, for example, C-CH2CH2-and-CH2When an amide bond is used as a divalent linking group between-D, it may be C-CH2CH2-C(＝O)NH-CH2-D or C-CH2CH2-NHC(＝O)-CH2-D。
In the structural formula of the invention, when the terminal group of the connecting group is easy to be confused with the substituent group contained in the connecting group, the method adoptsTo mark the position of the linking group to which other groups are attached, e.g. in formulaeIn (1), adoptTo mark two positions of the divalent linking group to which other groups are attached, the two aforementioned structural formulas each represent-CH (CH)2CH2CH3)2-、-CH2CH2CH(CH3)2-CH2CH2-。
In the present invention, the range of carbon atoms in a group is indicated in subscript form to C, indicating the number of carbon atoms the group has, e.g. C1-12Denotes "having 1 to 12 carbon atoms", C 1-30Means "having 1 to 30 carbon atoms". "substituted C1-12Alkyl "means C1-12A compound obtained by substituting a hydrogen atom of an alkyl group. "C1-12Substituted alkyl "refers to compounds having from 1 to 12 carbon atoms in the compound resulting from substitution of the hydrogen atom of the alkyl group. Also for example, when a group can be selected from C1-12When alkylene is used, it may be selected from alkylene having any number of carbon atoms within the range indicated by the subscript, i.e., it may be selected from C1、 C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12Any alkylene group of alkylene groups. In the present invention, unless otherwise specified, subscripts set forth as intervals each represent an integer selected from any one of the ranges, including both endpoints.
The heteroatom in the present invention is not particularly limited, but includes, but is not limited to, O, S, N, P, Si, F, Cl, Br, I, B and the like.
In the present invention, the heteroatom for substitution is referred to as "substituent atom", and any group for substitution is referred to as "substituent group".
In the present invention, "substituted" means any of the above groups (e.g., aliphatic hydrocarbyl, alkyl, or alkylene) wherein at least one hydrogen atom is substituted with a bond to a non-hydrogen atom such as, but not limited to: halogen atoms such as F, Cl, Br and I; oxo (═ O); hydroxyl (-OH); hydrocarbyloxy (-OR) dWherein R isdIs C1-12Alkyl groups); carboxyl (-COOH,); amine group (-NR)cRcTwo RcEach independently is H, C1-12Alkyl groups); c1-12Alkyl and cycloalkyl groups. In some embodiments, the substituent is C1-12An alkyl group. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.
As used herein, "atomic separation" or "atomic distance" refers to the number of atoms of the backbone chain spaced along the backbone chain, and is generally the shortest atomic distance, regardless of the side groups and side chains, and can be used to indicate the length of the linking group; for example, in A-CO-NH-B the atomic separation of A and B is 2, A-p-Ph-CH2The atomic spacing of A and B in-B is 5(p-Ph is p-phenylene), and as another example A-CH (CH)2CH2CH2CH3) The atomic spacing of B is 1. The "backbone atoms" participating in the formation of the atomic spacer can only be non-hydrogen atoms. Wherein, for a divalent linking group containing a cyclic structure, the atomic spacing thereof means the shortest atomic number calculated along the ring-forming atoms, for example, the atomic spacing of p-phenylene, i.e., 1, 4-phenylene, is 4, the atomic spacing of m-phenylene is 3, and the atomic spacing of o-phenylene is 2. And as-CH 2–、–CH(CH3)–、–C(CH3)2–、–CH(CH2Ph)2–、–C(CH2OX) -with an atomic spacing of 1.
As used herein, a "carbon chain linker" refers to a linker in which all of the main chain atoms are carbon atoms, while the side chain moieties allow for the substitution of a heteroatom or heteroatom-containing group for a hydrogen atom of a main chain carbon. When the "backbone atom" is a heteroatom, it is also referred to as a "backbone heteroatom", e.g., A-S-CH2-B、A-O-CH2-B、(the atomic separation is 4) is considered to contain a main chain heteroatom. The carbon chain linking group can be divided into alkylene and a carbon chain linking group of which the side group contains hetero atoms; heteroatom-containing carbon chain linking groups of the pendant groups include, but are not limited to, oxo (═ O), thio (═ S), amino (attached to the backbone carbon by a carbon-nitrogen double bond), oxahydrocarbyl groups in the form of ether linkages, thiahydrocarbyl groups in the form of thioether linkages, azahydrocarbyl groups in the form of tertiary amino groups, and the like. The "carbon chain linker" backbone is composed entirely of carbon atoms, and the pendant groups of the carbon chain are allowed to contain heteroatoms. I.e. linked by methylene or substituted methylene groups. The substituted methylene group may be substituted with one monovalent substituent, two monovalent substituents or one divalent substituent (e.g. divalent oxygen, e.g. together with the divalent methylene group to form a three-membered ring) And (4) substitution. The substituted methylene group may be one in which a hydrogen atom is substituted (e.g., -CH (CH) 3) -) or two hydrogen atoms may be independently substituted (e.g., - (CH)3)C(OCH3) -, it being possible for two hydrogen atoms to be substituted simultaneously (for example carbonyl, thiocarbonyl, -C (-))NH)-、 -C(＝N+H2) -) or may be cyclic side groups (e.g.The atomic separation is denoted as 1).
Secondary amino or hydrazine bonds in the context of the present invention mean that the "NH-" is capped at both ends by alkylene groups, e.g. -CH2-NH-CH2-; if-C (═ O) -NH-is referred to as an amide bond, it is not considered to contain a secondary amino bond.
In the context of the present invention, a compound, a group or an atom may be substituted and hybridized both at the same time by a hydrogen atom, for example by a nitrophenyl group, such as, in turn, -CH2-CH2-CH2-is replaced by-CH2-S-CH(CH3)-。
In the present invention, "linkage" means only linkage, and does not contain any atom, and when a group is defined as a linkage, that means that the group may not exist.
In the context of the present invention, "independently at each occurrence" means not only that the different groups may independently be any one of the items in the definition but also that the groups may independently be any one of the items in the definition when they occur at different positions in the same group, for example, -Z-L4-Z-wherein "Z is independently at each occurrence- (C ═ O) -, -O (C ═ O) -, - (C ═ O) O-, -S-, -C (═ O) S-, -SC (═ O) -, -NR-O cC(＝O)-、-C(＝O)NRc-、 -NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC (═ O) S-andany one of (1), wherein RcEach occurrence independently is a hydrogen atom or C1-12Alkyl radicals "at" -Z-L4In the radical-Z- "the two Z radicals may be identical or different, in the radical" -NRcC(＝O)NRc- "in two RcMay be the same or different and are each independently a hydrogen atom or C1-12An alkyl group.
A "group" as used herein, containing at least 1 atom, refers to a compound that has lost one or more atoms to form a free radical. With respect to compounds, the radicals formed after the loss of a partial group are also referred to as residues. The valence of the group is not particularly limited, and may be classified into monovalent group, divalent group, trivalent group, tetravalent group, … …, hundredth-valent group, and the like, as examples. Wherein, the groups with the valence of more than or equal to 2 are collectively called connecting groups. The linking group may also contain only one atom, such as oxy, thio.
In the present invention, "hydrocarbon" refers to a hydrocarbon compound composed of carbon atoms and hydrogen atoms.
In the present invention, hydrocarbons are classified into aliphatic hydrocarbons and aromatic hydrocarbons according to the hydrocarbon group type. The hydrocarbon having no structure of either benzene ring or hydrocarbon-substituted benzene ring is defined as an aliphatic hydrocarbon. Hydrocarbons containing at least one benzene ring or a hydrocarbyl-substituted benzene ring are defined as aromatic hydrocarbons. And the aromatic hydrocarbon can contain aliphatic hydrocarbon structures, such as toluene, diphenylmethane, 2, 3-indane and the like.
In the present invention, the hydrocarbons are classified into saturated hydrocarbons and unsaturated hydrocarbons according to the degree of saturation. All aromatic hydrocarbons are unsaturated hydrocarbons. Saturated aliphatic hydrocarbons are also known as alkanes. The degree of unsaturation of the unsaturated aliphatic hydrocarbon is not particularly limited. By way of example, but not limitation, alkenes (containing double bonds), alkynes (containing triple bonds), dienes (containing two conjugated double bonds), and the like. When the aliphatic hydrocarbon portion of the aromatic hydrocarbon is a saturated structure, it is also referred to as an aromatic hydrocarbon such as toluene.
In the present invention, the structure of the hydrocarbon is not particularly limited, and may be in the form of a linear structure containing no side group, a branched structure containing a side group, a cyclic structure, a tree structure, a comb structure, a hyperbranched structure, or the like. When not particularly defined, the linear structure containing no pendant group, the branched structure containing a pendant group, and the cyclic structure are preferably a linear hydrocarbon, a branched hydrocarbon, and a cyclic hydrocarbon, respectively. Wherein, the hydrocarbons without cyclic structure are collectively called open chain hydrocarbons, including but not limited to linear structure without side groups, branched structure with side groups. Open-chain hydrocarbons belong to the aliphatic hydrocarbons. Therefore, the linear hydrocarbon may be a linear aliphatic hydrocarbon. The branched hydrocarbon may be a branched aliphatic hydrocarbon.
In the present invention, a compound in which a carbon atom at any position of a hydrocarbon is substituted with a heteroatom is collectively referred to as a heterohydrocarbon.
In the present invention, the aliphatic heterohydrocarbon means a heterohydrocarbon derived from an aliphatic hydrocarbon, and includes aliphatic heterocyclic hydrocarbons, aliphatic heteroopen chain hydrocarbons and the like. The saturated aliphatic heterohydrocarbons are heteroalkanes.
In the present invention, "hydrocarbyl group" refers to a residue formed after a hydrocarbon has lost at least one hydrogen atom. According to the number of hydrogen atoms lost, the hydrocarbon group can be classified into a monovalent hydrocarbon group (one hydrogen atom is lost), a divalent hydrocarbon group (two hydrogen atoms are lost, also called as alkylene group), a trivalent hydrocarbon group (three hydrogen atoms are lost), and the like. The hydrocarbon group in the present invention means a monovalent hydrocarbon group unless otherwise specified.
The source of the hydrocarbon group in the present invention is not particularly limited, and may be derived from, for example, an aliphatic hydrocarbon or an aromatic hydrocarbon, a saturated hydrocarbon or an unsaturated hydrocarbon, a linear hydrocarbon, a branched hydrocarbon or a cyclic hydrocarbon, a hydrocarbon or a hetero hydrocarbon, or the like. From a saturation point of view, for example, they may be derived from alkanes, alkenes, alkynes, dienes, and the like; for cyclic hydrocarbons, for example, they may be derived from alicyclic or aromatic hydrocarbons, monocyclic or polycyclic hydrocarbons; for heterocyclic hydrocarbons, for example, they may be derived from aliphatic or aromatic heterocyclic hydrocarbons.
In the present invention, the "aliphatic hydrocarbon group" means a residue formed by losing at least one hydrogen atom from an aliphatic hydrocarbon. The aliphatic hydrocarbon group in the present invention means a monovalent aliphatic hydrocarbon group unless otherwise specified. The aliphatic hydrocarbon group includes a saturated aliphatic hydrocarbon group and an unsaturated aliphatic hydrocarbon group.
In the present invention, "alkyl" refers to a hydrocarbon group formed from an alkane, and unless otherwise specified, refers to a hydrocarbon group formed by losing a hydrogen atom at any position, and may be linear or branched, and may be substituted or unsubstituted. Specifically, for example, propyl means any of n-propyl and isopropyl, and propylene means any of 1, 3-propylene, 1, 2-propylene and isopropylene.
In the present invention, the "unsaturated hydrocarbon group" refers to a hydrocarbon group formed by losing a hydrogen atom from an unsaturated hydrocarbon. The hydrocarbon group formed by losing a hydrogen atom from an unsaturated carbon of an unsaturated hydrocarbon can be classified into an alkenyl group, an alkynyl group, a dienyl group and the like, such as an propenyl group and a propynyl group as examples. Unsaturated hydrocarbons the hydrocarbon group formed by losing a hydrogen atom on a saturated carbon is, depending on the unsaturated bond, for example, referred to as an alkenyl group, an alkynyl group, a dialkenyl group, etc., specifically, as an allyl group, a propargyl group.
In the present invention, "alkenyl" or "alkenyl group" means a substituted or unsubstituted straight or branched alkenyl group including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon double bond. The symbol "C 2-15Alkenyl "means a substituted or unsubstituted straight or branched alkenyl group comprising 2 to 15 carbon atoms and at least one carbon-carbon double bond, i.e. an alkenyl group may comprise one, two, three, four or more carbon-carbon double bonds. Unless otherwise specifically stated, alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups
As used herein, "alkynyl" or "alkynyl group" means an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon triple bond. The symbol "C2-15Alkynyl "means a substituted or unsubstituted straight or branched alkynyl group comprising 2 to 15 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups can include one, two, three, four, or more carbon-carbon triple bonds. Unless specifically stated otherwise, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.
In the present invention, "molecular weight" represents the mass size of one molecule of a compound, and "average molecular weight" represents the mass size of a component of a compound of the formula in a macroscopic substance, and "average molecular weight" generally means "number average molecular weight" when it is not particularly specified Quantum "Mn. The number average molecular weight may be the molecular weight of the polydisperse block or substance, or the molecular weight of the monodisperse block or substance. Unless otherwise specified, the unit of measure of "molecular weight" and "average molecular weight" is daltons, Da. The molecular weight of the polyethylene glycol chain can also be characterized by the degree of polymerization, specifically the number of repeating units (oxyethylene units, EO units) in one compound molecule. Accordingly, the average, number average, of the number of repeating units is characterized by an "average degree of polymerization", preferably a "number average degree of polymerization".
As used herein, a percentage of "about" generally means. + -. 0.5%.
"Stable existence" and "degradable" of groups in the present invention are a pair of opposite concepts.
In the present invention, "degradable" (be degradable or can be degraded) refers to the breaking of the inventive chemical bond, and the breaking is at least two residues independently of each other. If the structure is altered by a chemical change, but the entire linker is still only one complete linker, the linker is still classified as "stably available". The degradable condition is not particularly limited, and may be an in vivo physiological condition, or an in vitro simulated physiological environment or other conditions, preferably an in vivo physiological condition and an in vitro simulated physiological condition. The physiological condition is not particularly limited, and includes, but is not limited to, serum, heart, liver, spleen, lung, kidney, bone, muscle, fat, brain, lymph node, small intestine, gonad, etc., and may refer to intracellular, extracellular matrix, normal physiological tissue, and pathological tissue (such as tumor, inflammation, etc.). The in vitro simulated environment is not particularly limited and includes, but is not limited to, physiological saline, buffer, culture medium, and the like. The degradation rate is not particularly limited, and may be, for example, rapid degradation by an enzyme, slow hydrolysis under physiological conditions, or the like. The physiological condition in vivo includes physiological condition during treatment, such as ultraviolet irradiation, thermotherapy, etc. Including but not limited to, degradable under conditions of light, heat, low temperature, enzymes, redox, acidic, basic, physiological conditions, in vitro simulated environments, and the like, preferably under conditions of light, heat, enzymes, redox, acidic, basic, and the like. Degradable refers to degradation under stimulation under any of the conditions described above. The light conditions include, but are not limited to, visible light, ultraviolet light, infrared light, near infrared light, mid-infrared light, and the like. The thermal conditions refer to temperature conditions above normal physiological temperature, typically above 37 ℃, and typically below 45 ℃, preferably below 42 ℃. The low temperature condition is lower than the physiological temperature of human body, preferably lower than 25 ℃, more preferably less than or equal to 10 ℃, and specific examples are refrigeration temperature, freezing temperature, liquid nitrogen treatment temperature, 2-10 ℃, 4-8 ℃, 4 ℃, 0 ℃, and-20 +/-5 ℃, and the like. The enzyme conditions are not particularly limited, and enzymes that can be produced under physiological conditions are included, and examples thereof include peptidases, proteases, lyases and the like. The redox conditions are not particularly limited, such as redox transition between thiol group and disulfide bond, and hydrogenation reduction transition. The acidic and alkaline conditions mainly refer to the pH conditions of the internal body parts such as normal tissues, pathological tissues, organs or tissues in the treatment period, for example, the stomach is acidic, and the tumor part is often acidic. Degradable herein refers to degradation by metabolic action in vivo (e.g., physiological action, such as enzymes, such as redox, etc.), degradation at specific sites in the body by micro-environmental stimuli (e.g., acidic, basic), or degradation under clinical therapeutic stimuli (e.g., light, such as heat, such as hypothermia), etc. It should be noted that some extreme conditions in organic chemistry relative to organisms, such as bond cleavage under strong acid, strong base, high temperature (e.g., above 100 ℃), etc., are not included in the scope of the degradable conditions of the present invention. For another example, although ether linkages can be cleaved under strong acid conditions such as hydrobromic acid, they are always classified as stably available linkers in the present invention.
In the present invention, "stably exist" means that a linker can remain as an intact linker (one linker is stably covalently linked to its neighboring group), and is defined as "stably exist" (be table or can remaining table) in which a chemical change capable of maintaining the integrity of the linker is allowed to occur. The chemical changes are not particularly limited and include, but are not limited to, isomerization, oxidation, reduction, ionization, protonation, deprotonation, substitution reactions, and the like. The conditions that can be stably present are not particularly limited, and include, but are not limited to, light, heat, low temperature, enzymes, redox, neutral, acidic, basic, physiological conditions, in vitro simulated environments, and the like, and preferably, light, heat, enzymes, redox, acidic, basic, and the like. The stable existence here means that stable linkage can be maintained in metabolic cycles in vivo without specific stimulation (e.g., pH condition at a specific site, light, heat, low temperature at the time of treatment, etc.) and molecular weight reduction due to chain cleavage (as long as integrity is maintained) does not occur.
In the present invention, the term "stably exist" with respect to the same linker is not an absolute concept, and for example, an amide bond is more stable under acidic or basic conditions than an ester bond, and the linker "stably exist" in the present invention includes an amide bond. However, an amide bond formed by dehydration condensation of an α -carboxyl group of a molecule of an amino acid and an α -amino group of a molecule of an amino acid, such as a peptide bond, can be cleaved when subjected to a particular enzyme, and is therefore also included in the "degradable" linker. Similarly, carbamate, thiocarbamate, and the like may be either a stably existing linker or a degradable linker. More generally, carbamate groups, thiocarbamate groups, and the like are more prone to slow degradation, while amide bonds other than peptide bonds may be stable during in vivo circulation. Also, for example, common ester bonds can be degraded under acid and alkali conditions, while ester bonds contained in a specific structure can be degraded under ultraviolet light conditions. For another example, even if some chemical bonds are degraded by a specific enzyme, if they are used clinically, the corresponding chemical bonds can be considered to be stably present if the circulating pathway does not pass through or substantially passes through the specific enzyme environment (such as in the case of site-specific administration).
In the present invention, in order to define the degradable nature of the compound structure more clearly, a reference criterion is provided, i.e. the chemical bond under investigation is kept at a certain percentage (e.g. 90%) for a limited time interval. In the case of 90%, the pharmacokinetic profile of the functionalized polyethylene glycol modified product is usually referenced to the percent of dose that meets clinical evaluation criteria. For example, for an intravenously administered pegylated drug, when the blood concentration (based on the active pharmaceutical ingredient, including the pegylated drug and the non-pegylated ingredient after degradation) is less than 15% of the initial concentration (or other ratio more consistent with clinical evaluation of the drug), based on the remaining 85%, it is a group that can exist stably in the present invention if the ratio of one linker group remaining chemically bonded exceeds 90%, and conversely, it is a degradable group if it is less than 90%. Hydrolytic stabilization, enzymatic degradation, and the like reported in the published literature are also incorporated into the present invention. Taking the hydrolysis stability as an example, the hydrolysis rate when the hydrolysis is stable as reported in the published literature is included, preferably the hydrolysis rate under physiological conditions is less than 1-2% (generally 2%), by mass or molar amount per day. The rate of hydrolysis of typical chemical bonds can be found in most standard chemical manuals.
In the present invention, the "hydroxyl-protecting group" includes all groups that can be used as protecting groups for general hydroxyl groups. A hydroxyl-protecting group is preferably an alkanoyl group (e.g., acetyl, tert-butyryl), an aralkanoyl group (e.g., benzoyl), benzyl, trityl, trimethylsilyl, tert-butyldimethylsilyl, allyl, acetal group or ketal group. The removal of acetyl groups is generally carried out under basic conditions, the most common being NH3Ammonolysis with MeOH and methanolysis with methanol anion catalysis; the benzyl can be easily removed by palladium catalytic hydrogenolysis in a neutral solution at room temperature, and can also be reduced and cracked by using metal sodium in ethanol or liquid ammonia; trityl groups are typically removed by catalytic hydrogenolysis; trimethylsilyl groups are typically removed using fluoride-containing reagents (e.g., tetrabutylammonium fluoride/anhydrous THF, etc.); the tert-butyl dimethyl silicon ether is stable and can bear ester hydrolysis conditions and mild reduction conditions (such as Zn/CH) of alcoholic potassium hydroxide3OH, etc.), fluoride ion (e.g., Bu) may be used4N+F-) It is removed in tetrahydrofuran solution or by using aqueous acetic acid at room temperature.
In the present invention, the "carboxyl protecting group" means a group capable of being protected by hydrolysisAnd converting the carboxyl protecting group into a carboxyl protecting group by deprotection reaction. The carboxyl-protecting group is preferably an alkyl group (e.g., methyl, ethyl, t-butyl) or an aralkyl group (e.g., benzyl), more preferably a t-butyl group (tBu), methyl group (Me) or ethyl group (Et). In the present invention, the "protected carboxyl group" refers to a group formed by protecting a carboxyl group with a suitable carboxyl protecting group, and is preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group. The carboxyl protecting group can be removed by hydrolysis under the catalysis of acid or base, and occasionally can be eliminated by pyrolysis, for example, tert-butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA and H 2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably TFA and H2A combination of O, LiOH and MeOH, or LiOH and EtOH. Deprotection of a protected carboxyl group to produce the corresponding free acid, said deprotection being carried out in the presence of a base which forms a pharmaceutically acceptable salt with the free acid formed by said deprotection.
In the present invention, the "amino-protecting group" includes all groups that can be used as protecting groups for general amino groups, for example, aryl C1-6Alkyl radical, C1-6Alkoxy radical C1-6Alkyl radical, C1-6Alkoxycarbonyl, aryloxycarbonyl, C1-6Alkylsulfonyl, arylsulfonyl, silyl, or the like. Preferred amino protecting groups are Boc t-butyloxycarbonyl, Moz p-methoxybenzyloxycarbonyl and Fmoc 9-fluorenylmethyloxycarbonyl. The reagent for removing the amino protecting group is selected from TFA and H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably TFA and H2A combination of O, LiOH and MeOH, or LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl/EA; TFA is preferred. The deprotection agent used in the Fmoc-protecting group removal reaction was a 20% piperidine in N, N-Dimethylformamide (DMF).
In the present invention, "carboxyl group activation" refers to activation treatment of carboxyl group with a carboxyl group activating agent, which promotes better condensation reaction after carboxyl group activation, such as: inhibiting the generation of racemization impurities in condensation reaction, catalyzing and accelerating the reaction speed, and the like. A "carboxy activating group" is the residue of a carboxy activating agent. The carboxyl activating agent is one or more of N-hydroxysuccinimide (NHS), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2, 3-dicarboximide (HONb) and N, N-Dicyclohexylcarbodiimide (DCC), preferably NHS/EDCI, NHS/DCC, HONb/DCC, most preferably NHS/EDCI.
In the present invention, "cationic" means that the corresponding structure is permanently, or non-permanently, capable of carrying a positive charge in response to certain conditions (e.g., pH). Thus, cations include both permanent cations and cationizable cations. By permanent cation is meant that the corresponding compound or group or atom is positively charged at any pH or hydrogen ion activity of its environment. Typically, a positive charge is generated by the presence of a quaternary nitrogen atom. When a compound carries multiple such positive charges, it may be referred to as a permanent cation. Cationizable means that a compound or group or atom is positively charged at a lower pH and uncharged at the higher pH of its environment. In addition, in non-aqueous environments where pH cannot be determined, cationizable compounds, groups or atoms are positively charged at high hydrogen ion concentrations and uncharged at low hydrogen ion concentrations or activities. It depends on the respective nature of the cationizable or polycationizable compound, in particular the pKa of the corresponding cationizable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In dilute aqueous environments, the fraction of cationizable compounds, groups or atoms carrying a positive charge can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. For example, in some embodiments, if a compound or moiety is cationizable, it is preferred that it is positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or even 6 to 8, more preferably at a pH equal to or lower than 9, equal to or lower than 8, equal to or lower than 7, most preferably at physiological pH (e.g., about 7.3 to 7.4), i.e., under physiological conditions, particularly under physiological salt conditions of cells in vivo. In other embodiments, it is preferred that the cationizable compound or moiety be predominantly neutral at physiological pH values (e.g., about 7.0-7.4), but become positively charged at lower pH values. In some embodiments, a preferred range of pKa of the cationizable compound or moiety is from about 5 to about 7.
In the present invention, "cationic lipid" refers to a lipid containing a positive charge or being ionizable as a whole. The cationic lipids include, but are not limited to, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), 3- (didodecylamino) -N1, N1, 4-tridodecyl-1-piperazineethylamine (KL10), N1- [2- (didodecylamino) ethyl ] -N1, n4, N4-tridodecyl-1, 4-piperazine diethylamine (KL22), 14, 25-ditridecyl-15, 18,21, 24-tetraaza-triacontane (KL25), 1, 2-dioleyloxy-N, any one of N-dimethylaminopropane (DLin-DMA), 2-dioleyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), thirty-seven carbon-6, 9,28, 31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC3-DMA) and 2, 2-dioleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC2-DMA) and mixtures thereof.
In the present invention, "pegylated lipids" refers to molecules comprising a lipid moiety and a polyethylene glycol moiety. The pegylated lipids include, but are not limited to, polyethylene glycol-1, 2 dimyristoyl glyceride (PEG-DMG), polyethylene glycol-distearoyl phosphatidyl ethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxy propyl (PEG-DAA), specifically, polyethylene glycol 500-dipalmitoyl phosphatidyl choline, polyethylene glycol 2000-dipalmitoyl phosphatidyl choline, polyethylene glycol 500-stearoyl phosphatidyl ethanolamine, polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine, polyethylene glycol 500-1, 2-oleoyl phosphatidyl ethanolamine, polyethylene glycol 2000-1, 2-oleoyl phosphatidyl ethanolamine, and polyethylene glycol 2000-2, in addition to those shown in the general structural formula (2) of the present invention, 3-dimyristoyl glycerol (PEG-DMG), and the like.
In the present invention, "neutral lipid" refers to any of a number of lipid substances, preferably phospholipids, that exist in the uncharged or neutral zwitterionic form at a selected pH. Such lipids include, but are not limited to, 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dicarballyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Ly PC), 1, 2-dilinolacyl-sn-glycero-3-phosphocholine, 1, 2-dineoyl-sn-glycero-3-phosphocholine, 1, 2-didodecanoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didecylhexaenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), Dioleoylphosphatidylserine (DOPS), Dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), Distearoylphosphatidylethanolamine (DSPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-Stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and Lysophosphatidylethanolamine (LPE), and combinations thereof. Neutral lipids may be of synthetic or natural origin.
In the present invention, the "steroid lipid" is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof
In the present invention, the "amino acid residue" includes an amino acid in which a hydrogen atom is removed from an amino group and/or a hydroxyl group is removed from a carboxyl group and/or a hydrogen atom is removed from a mercapto group and/or an amino group is protected and/or a carboxyl group is protected and/or a mercapto group is protected. Not to be precise, an amino acid residue may be referred to as an amino acid. The source of the amino acid in the present invention is not particularly limited unless otherwise specified, and may be a natural source, a non-natural source, or a mixture of both. The amino acid structure type in the present invention is not particularly limited unless otherwise specified, and may be either an L-type or a D-type, or a mixture of both.
The term "functional group source" as used herein refers to a source that is reactive or potentially reactive, photosensitive or potentially photosensitive, targeted, or potentially targeted. The term "latent" refers to a molecule that can be converted into a reactive group by a chemical process selected from the group consisting of, but not limited to, functional modification (e.g., grafting, substitution, etc.), deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation, change in leaving group, etc., and can emit light or generate targeting by external stimuli such as light, heat, enzymes, specific binding molecules, in vivo microenvironment, etc. The luminescence is not particularly limited, and includes, but is not limited to, visible light, fluorescence, phosphorescence, and the like.
The change form in the invention refers to a structure form which can be converted into a target reactive group through any chemical change process of oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexing, ionization, protonation, deprotonation, substitution, deprotection, change of a leaving group and the like.
"reactive group variant" as used herein refers to a form in which a reactive group remains active (remains a reactive group) after at least one chemical change of oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexing, ionization, protonation, deprotonation, substitution, deprotection, change of leaving group, or the like, or an inactive form after it has been protected.
The term "micro-modification" as used herein refers to a chemical modification process that can be accomplished by a simple chemical reaction process. The simple chemical reaction process mainly refers to chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, leaving group conversion and the like, and the 'micro change form' corresponds to 'micro modification' and refers to a structural form capable of forming a target reactive group after undergoing simple chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, leaving group conversion and the like. A transition of the leaving group, such as a transition from the ester form to the acid chloride form.
In the present invention, an "adjuvant or adjuvant component" is typically a (e.g. pharmacological or immunological) agent or composition that can alter (e.g. enhance) the efficacy of other agents (e.g. drugs or vaccines). Generally, the term refers in the context of the present invention to a compound or composition that acts as a carrier or auxiliary substance for an immunogen and/or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers to a wide range of substances capable of increasing the immunogenicity of antigens incorporated into or co-administered with the adjuvant in question. In the present invention, an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention. Typically, "adjuvant" or "adjuvant component" have the same meaning and may be used interchangeably. Adjuvants can be classified, for example, as immunopotentiators, antigen delivery systems, or even combinations thereof.
In the present invention, the "N/P ratio" refers to the molar ratio of nitrogen atoms in the cationic lipid to phosphoric acid in the nucleic acid.
In the present invention, "nucleic acid" refers to DNA or RNA or modified forms thereof, which contain purine or pyrimidine bases (adenine "a", cytosine "C", guanine "G", thymine "T") present in DNA or purine or pyrimidine bases (adenine "a", cytosine "C", guanine "G", uracil "U") present in RNA.
In the present invention, "RNA" refers to ribonucleic acid which may or may not occur naturally. For example, the RNA can include modified and/or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyadenylation sequence, and/or a polyadenylation signal. The RNA can have a nucleotide sequence encoding a polypeptide of interest. For example, the RNA may be messenger RNA (mrna). Translation of an mRNA encoding a particular polypeptide, for example, translation of an mRNA in vivo within a mammalian cell, can result in the encoded polypeptide. The RNA may be selected from the non-limiting group consisting of: small interfering rna (sirna), asymmetric interfering rna (airna), microrna (mirna), Dicer-substrate rna (dsrna), small hairpin rna (shrna), mRNA, single stranded guide rna (sgrna), cas9 mRNA, and mixtures thereof.
In the present invention, antisense oligonucleotides or small interfering RNAs (siRNAs) can inhibit the expression of target genes and target proteins in vitro or in vivo.
In the present invention, FLuc mRNA can express luciferase protein that emits bioluminescence in the presence of luciferin substrates, so FLuc is commonly used in mammalian cell culture to measure gene expression and cell activity.
In the present invention, "inhibiting expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit expression of a target gene. To test the extent of gene silencing, a test sample (e.g., a sample of cells in a medium expressing a target gene) is contacted with a nucleic acid that inhibits expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cells in medium expressing the target gene) that has not been contacted or administered the nucleic acid. The expression of the target gene in the control sample can be assigned a value of 100%. In particular embodiments, inhibition of expression of the target gene is achieved when the level of expression of the target gene in the test sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of expression of the target gene in a control sample or control mammal.
In the present invention, the method for determining the expression level of the target gene includes, but is not limited to, dot blotting, northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic assay.
In the present invention, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.
In the context of the present invention, an "antigen" typically refers to a substance that is recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example by forming antibodies and/or antigen-specific T cells as part of the adaptive immune response. Typically, the antigen may be or may comprise a peptide or protein that can be presented by MHC to a T cell. In the sense of the present invention, an antigen may be the translation product of a provided nucleic acid molecule, preferably an mRNA as defined herein. In this context, fragments, variants and derivatives of peptides and proteins comprising at least one epitope are also understood as antigens.
In the present invention, "delivery" refers to providing an entity to a target. For example, the drug and/or therapeutic and/or prophylactic agent is delivered to a subject, which is a tissue and/or cell of a human and/or other animal.
By "pharmaceutically acceptable carrier" in the context of the present invention is meant a diluent, adjuvant, excipient, or vehicle that is administered together with a therapeutic agent and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers that may be employed in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also optionally contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Specifically, for example, excipients include, but are not limited to, antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavorants, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. More specific excipients include, but are not limited to, Butylated Hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, croscarmellose sodium, crospovidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenyl paraben, retinol palmitate, shellac, silicon dioxide, carboxymethylcellulose sodium, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin a, vitamin E (alpha-tocopherol), Vitamin C, xylitol.
The pharmaceutical compositions of the present invention may act systemically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection (e.g. intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation. For these routes of administration, the pharmaceutical compositions of the present invention may be administered in suitable dosage forms. Such dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.
In the present invention, a vaccine is a prophylactic or therapeutic material that provides at least one antigen or antigenic function. The antigen or antigenic function may stimulate the adaptive immune system of the body to provide an adaptive immune response.
The treatment, as used herein, refers to the treatment and care of a patient for the purpose of combating a disease, disorder or condition, and is intended to include delaying the progression of the disease, disorder or condition, alleviating or alleviating symptoms and complications, and/or curing or eliminating the disease, disorder or condition. The patient to be treated is preferably a mammal, especially a human.
1.1. A cationic lipid having a structure represented by general formula (1):
wherein N is a nitrogen branching center;
L1、L2is a bond or a divalent linking group each independently selected from-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CR)cRc)sO-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-and-NRcAny one of C (═ O) S-, wherein R iscEach occurrence independently is a hydrogen atom or C1-12Alkyl, s is 2, 3 or 4;
L3is a connecting bond, -L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5-or-L4-Z-L5-Z-; said L4、L5Is carbonChain linking groups, each independently is- (CR)aRb)t-(CRaRb)o-(CRaRb)p-, where t, o and p are each independently integers from 0 to 12 and are not 0 at the same time, RaAnd RbEach occurrence is independently a hydrogen atom or an alkyl group; each occurrence of Z is independently- (C ═ O) -, -O (C ═ O) -, - (C ═ O) O-, -S-, -C (═ O) S-, -SC (═ O) -, -NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、 -NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC (═ O) S-andany one of (1), wherein RcEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
B1、B2each independently is a connecting bond or C1-30An alkylene group;
R1、R2each independently is C2-30An aliphatic hydrocarbon group;
R3is a hydrogen atom, -Rd、-ORd、-NRdRd、-SRd、-(C＝O)Rd、-(C＝O)ORd、-O(C＝O)Rd、 -O(C＝O)ORdOrWherein R isdEach occurrence is independently C1-12Alkyl radical, G1A terminal branched group with a valence of k +1, j is 0 or 1, F contains a functional group R 01When j is 0, G1In the absence of, when j is 1, G1K F is led out, and k is an integer of 2-8;
a is selected from- (CR)aRb)sO-、-O(CRaRb)s-、-(CRaRb)sS-、-S(CRaRb)s-、-(CRaRb)sO(CRaRb)sS-、 -(CRaRb)sS(CRaRb)sO-、-(CRaRb)sNRc(CRaRb)sS-、-(CRaRb)sS(CRaRb)sNRc-、 -(CRaRb)sNRc(CRaRb)sO-and- (CR)aRb)sO(CRaRb)sNRc-any of, wherein s is 2, 3 or 4, RaAnd RbEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
when A is- (CR)aRb)sO-or-O (CR)aRb)s-when n is an integer from 2 to 6; when A is not- (CR)aRb)sO-or-O (CR)aRb)s-when n is an integer from 1 to 6;
the alkyl, alkylene, alkoxy, aliphatic hydrocarbon groups are each independently substituted or unsubstituted.
1.1.1.L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2
In the present invention, L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2The structure of (a) is not particularly limited, and each independently includes, but is not limited to, a linear structure, a branched structure, or a cyclic-containing structure.
In the present invention, L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2The number of the non-hydrogen atoms of (b) is not particularly limited, and each is independently preferably 1 to 50 non-hydrogen atoms; more preferably 1 to 20 non-hydrogen atoms; more preferably 1 to 10 non-hydrogen atoms. The non-hydrogen atom is a carbon atom or a heteroatom. Such heteroatoms include, but are not limited to O, S, N, P, Si. B, and the like. When the number of non-hydrogen atoms is 1, the non-hydrogen atoms may be carbon atoms or hetero atoms. When the number of non-hydrogen atoms is more than 1, the kind of non-hydrogen atoms is not particularly limited; may be 1 species, or may be 2 or more than 2 species; when the number of non-hydrogen atoms is more than 1, any combination of carbon atoms and carbon atoms, carbon atoms and hetero atoms, and hetero atoms may be used.
In the present invention, two identical or different reactive groups can be reacted to form a divalent linking group. The reaction conditions, depending on the type of divalent linking group formed by the reaction, can be according to the prior art. For example: the amino is respectively reacted with active ester, formic acid active ester, sulfonate, aldehyde, alpha, beta-unsaturated bond, carboxylic acid group, epoxide, isocyanate and isothiocyanate to obtain bivalent connecting groups such as amido, urethane group, amino, imino (which can be further reduced into secondary amino), amino, amido, amino alcohol, urea bond, thiourea bond and the like; reacting a sulfhydryl group with a divalent linking group containing an active ester, a formic acid active ester, a sulfonic ester, a sulfhydryl group, maleimide, aldehyde, an alpha, beta-unsaturated bond, a carboxylic acid group, iodoacetamide and an anhydride to obtain a thioester group, a thiocarbonate, a thioether, a disulfide, a thioether, a hemithioacetal, a thioether, a thioester, thioether, imide and the like; unsaturated bonds react with sulfydryl to obtain thioether groups; carboxyl or acyl halide reacts with sulfhydryl and amino respectively to obtain thioester group, amide group and other groups; hydroxyl reacts with carboxyl, isocyanate, epoxide and chloroformyl to obtain divalent linking groups such as ester group, carbamate group, ether bond, carbonate group and the like; reacting carbonyl or aldehyde group with amino, hydrazine and hydrazide to obtain divalent connecting groups such as imine bond, hydrazone, acylhydrazone and the like; reactive groups such as azide, alkynyl, alkenyl, sulfydryl, azide, diene, maleimide, 1,2, 4-triazoline-3, 5-diketone, dithioester, hydroxylamine, hydrazide, acrylate, allyloxy, isocyanate, tetrazole and the like are subjected to click chemistry reaction to generate various divalent connecting groups with structures such as triazole, isoxazole, thioether bonds and the like.
L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2Is not particularly limited, and any one of the divalent linking groups or any one of the divalent linking groups consisting of a group with an adjacent heteroatom is independently a stably existing linking group STAG or a degradable linking group DEGG.
1.1.1.1.L1、L2
In the present invention, L1、L2Is a bond or a divalent linking group each independently selected from-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -S-, -C (═ O) S-, -SC (═ O) -, -NR-, -C (═ O) S-, -SC (═ O) -, -OcC(＝O)-、-C(＝O)NRc-、 -NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-and-NRcAny one of C (═ O) S-, and R iscEach occurrence independently is a hydrogen atom or C1-12An alkyl group.
In one embodiment of the present invention, L is more preferred1、L2Each independently is a bond, -O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CH)2)sAny one of O-, -S-, -C (═ O) S-, -SC (═ O) -, -NHC (═ O) -, -C (═ O) NH-, -NHC (═ O) NH-, -OC (═ O) NH-, -NHC (═ O) O-, -SC (═ O) NH-, and-NHC (═ O) S-.
In one embodiment of the present invention, L is more preferred1、L2One of which is a bond and the other is-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CH)2)sAny one of O-, -S-, -C (═ O) S-, -SC (═ O) -, -NHC (═ O) -, -C (═ O) NH-, -NHC (═ O) NH-, -OC (═ O) NH-, -NHC (═ O) O-, -SC (═ O) NH-, and-NHC (═ O) S-.
In one embodiment of the present invention, L is more preferred1And L2Each independently represents-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CH)2)sAny one of O-, -S-, -C (═ O) S-, -SC (═ O) -, -NHC (═ O) -, -C (═ O) NH-, -NHC (═ O) NH-, -OC (═ O) NH-, -NHC (═ O) O-, -SC (═ O) NH-, and-NHC (═ O) S-.
In one embodiment of the present invention, L is more preferred1And L2Each independently is any one of-O (C ═ O) -, - (C ═ O) O-, and-O (C ═ O) O-.
In one embodiment of the present invention, L is more preferred1And L2At the same time, -O (C ═ O) -or at the same time- (C ═ O) O-or at the same time-O (C ═ O) O-.
In one embodiment of the invention, RcPreferably a hydrogen atom; or RcPreferably C1-12Alkyl, more preferably C1-8The alkyl group is more preferably any of a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group.
1.1.1.2.L7、L8
In the present invention, L7、L8Each independently is a bond or a divalent linking group selected from-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -S-, -C (═ O) S-, -SC (═ O) -, -NR (r) ScC(＝O)-、-C(＝O)NRc-、 -NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-and-NRcAny one of C (═ O) S-, and R iscEach occurrence independently is a hydrogen atom or C1-12An alkyl group.
In one embodiment of the invention, RcPreferably a hydrogen atom; or RcPreferably C1-12Alkyl, more preferably C1-8The alkyl group is more preferably any of a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group.
1.1.1.3.L3
In the present invention, L3is-L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5-or-L4-Z-L5-Z-; said L4、L5Are carbon chain linking groups, each independently is- (CR)aRb)t-(CRaRb)o-(CRaRb)p-, where t, o, p are each independently an integer of 0 to 12, RaAnd RbEach occurrence is independently a hydrogen atom or an alkyl group; each occurrence of Z is independently- (C ═ O) -, -O (C ═ O) -, - (C ═ O) O-, -S-, -C (═ O) S-, -SC (═ O) -, -NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC (═ O) S-andany one of (1), wherein RcEach occurrence independently is a hydrogen atom or C1-12An alkyl group.
In one embodiment of the invention, RcPreferably a hydrogen atom.
In one embodiment of the present invention, L3Is- (CH)2)t-、-(CH2)tZ-、-Z(CH2)t-、-(CH2)tZ(CH2)t-and-Z (CH)2)tZ-wherein t is an integer of 1 to 12, and Z is- (C ═ O) -, -O (C ═ O) -, - (C ═ O) O-, -S-, -C (═ O) S-, -SC (═ O) -, -NR (r) —cC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、 -OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC (═ O) S-andany of the above. Preferably L3Is- (CH)2)t-、-(CH2)tO-、-(CH2)tC(＝O)-、-(CH2)tC(＝O)O-、-(CH2)tOC(＝O)-、 -(CH2)tC(＝O)NH-、-(CH2)tNHC(＝O)-、-(CH2)tOC(＝O)O-、-(CH2)tNHC(＝O)O-、 -(CH2)tOC(＝O)NH-、-(CH2)tNHC(＝O)NH-、-O(CH2)t-、-C(＝O)(CH2)t-、-C(＝O)O(CH2)t-、 -OC(＝O)(CH2)t-、-C(＝O)NH(CH2)t-、-NHC(＝O)(CH2)t-、-OC(＝O)O(CH2)t-、 -NHC(＝O)O(CH2)t-、-OC(＝O)NH(CH2)t-、-NHC(＝O)NH(CH2)t-、-(CH2)tO(CH2)t-、 -(CH2)tC(＝O)(CH2)t-、-(CH2)tC(＝O)O(CH2)t-、-(CH2)tOC(＝O)(CH2)t-、 -(CH2)tC(＝O)NH(CH2)t-、-(CH2)tNHC(＝O)(CH2)t-、-(CH2)tOC(＝O)O(CH2)t-、 -(CH2)tNHC(＝O)O(CH2)t-、-(CH2)tOC(＝O)NH(CH2)t-、-(CH2)tNHC(＝O)NH(CH2)t-、 -O(CH2)tO-、-C(＝O)(CH2)tC(＝O)-、-C(＝O)O(CH2)tC(＝O)O-、-OC(＝O)(CH2)tOC(＝O)-、 -C(＝O)O(CH2)tOC(＝O)-、-OC(＝O)(CH2)tC(＝O)O-、-OC(＝O)O(CH2)tOC(＝O)O-、 -C(＝O)NH(CH2)tC(＝O)NH-、-NHC(＝O)(CH2)tNHC(＝O)-、-NHC(＝O)(CH2)tC(＝O)NH-、 -C(＝O)NH(CH2)tNHC(＝O)-、-NHC(＝O)O(CH2)tNHC(＝O)O-、 -OC(＝O)NH(CH2)tOC(＝O)NH-、-NHC(＝O)O(CH2)tOC(＝O)NH-、 -OC(＝O)NH(CH2)tNHC(＝O)O-、-NHC(＝O)NH(CH2)tNHC(＝O)NH-、-C(＝O)(CH2)tO-、 -C(＝O)(CH2)tC(＝O)O-、-C(＝O)(CH2)tOC(＝O)-、-C(＝O)(CH2)tOC(＝O)O-、 -C(＝O)(CH2)tNHC(＝O)O-、-C(＝O)(CH2)tOC (═ O) NH — and-C (═ O) (CH)2)tNHC (═ O) NH-.
1.1.2. Description of the groups which can be stabilized and degraded
The linker STAG or the degradable linker DEGG which can be stably present in the present invention may be present in the above-mentioned L3、L4、L5、L7、L8、Z、Z1、Z2Any one of the divalent linking groups, or a divalent linking group present between any one of the divalent linking groups and an adjacent heteroatom group.
1.1.2.1. Stag, a bivalent connecting group stably existing in the present invention
The condition under which the stably existing bivalent linking group STAG can be stably existing is not particularly limited, and the stably existing bivalent linking group STAG can be stably existing under any condition including, but not limited to, light, heat, low temperature, enzyme, redox, acidic, basic condition, physiological condition, in vitro simulated environment, and the like, and preferably can be stably existing under any condition of light, heat, enzyme, redox, acidic, basic, and the like.
The type of divalent linking group STAG that can be stably present is not particularly limited, and includes, but is not limited to, alkylene, divalent heteroalkyl, double bond, triple bond, divalent dienyl, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloalkenylene, divalent cycloalkynylene, aromatic ring, alicyclic ring, heterocyclic ring, fused heterocyclic ring, substituted alkylene, substituted heteroalkyl, substituted divalent heteroalkyl, substituted double bond, substituted triple bond, substituted diene, substituted divalent cycloalkyl, substituted divalent cycloalkenyl, substituted divalent cycloalkenylene, substituted divalent cycloalkynylene, substituted aromatic ring, substituted heterocyclic aromatic ring, substituted heterocyclic ring, substituted fused heterocyclic ring, ether bond, thiourea bond, carbamate group, thiocarbamate group, -P (═ O) -, divalent silicon group containing no active hydrogen, divalent silicon group, A divalent linking group containing boron atoms, a secondary amino group, a tertiary amino group, a carbonyl group, a thiocarbonyl group, an amido group, a thioamido group, a sulfamide group, an enamine group, triazole, 4, 5-dihydroisoxazole, any divalent linking group in the skeleton of amino acid and derivatives thereof, and a stable divalent linking group consisting of any two or more groups.
In particular, the bivalent linking group STAG that can be stably present includes, but is not limited to, the structures described and exemplified in documents CN104530413A, CN104530415A, CN 104530417A. For example, CN104530417A corresponds to the segments [0627] to [0704 ]. The manner in which two or more species of divalent linking groups that can be stably present are combined to form STAG is not particularly limited. Including but not limited to segment [704] of CN 104530417A.
1.1.2.2. Degradable divalent linking group DEGG in the invention
The conditions under which the degradable divalent linking group DEGG is degradable are not particularly limited, and may be degradable under any conditions including, but not limited to, light, heat, low temperature, enzyme, redox, acidic, basic, physiological conditions, in vitro simulated environment, and the like, and preferably may be degradable under any conditions of light, heat, enzyme, redox, acidic, basic, and the like.
The divalent linking group formed by combining any degradable divalent linking group DEGG and any stably existing divalent linking group STAG is still a degradable linking group. For the degradable divalent linking group containing the aromatic ring, the aromatic ring and the degradable divalent linking group can be combined.
The type of degradable divalent linking group DEGG is not particularly limited and includes, but is not limited to, those containing disulfide bonds, vinyl ether bonds, ester groups, thioester groups, dithioester groups, carbonate groups, thiocarbonate groups, dithiocarbonate groups, trithiocarbonate groups, carbamate groups, thiocarbamate groups, dithiocarbamate groups, acetals, cyclic acetals, mercaptals, azaacetals, azaketals, dithioacetals, hemiacetals, thiohemiacetals, azahemiacetals, ketals, thioketals, azaketals, thioketals, imine bonds, hydrazone bonds, acylhydrazone bonds, oxime bonds, thiooxime groups, semicarbazone bonds, thiosemicarbazone bonds, hydrazine groups, hydrazide groups, thiocarbohydrazide groups, hydrazinoformate groups, hydrazinothiocarbamate groups, thioformate groups, thiosemicarbazide groups, semicarbazide groups, thiosemicarbazide groups, thiosemicarbazides groups, thiosemicarbazides groups, thiosemicarbazides groups, thiosemicarbazides groups, thiosemicarbazides, and the like, thiosemicarbazides, and the like, Carbohydrazide, thiocarbohydrazide, azo group, isoureido group, isothioureido group, allophanate group, thioallophanate group, guanidino group, amidino group, aminoguanidino group, amidino group, imido group, thioesterite group, sulfonate group, sulfinate group, sulfonylhydrazide group, sulfonylurea group, maleimide group, orthoester group, phosphate group, phosphite group, hypophosphite group, phosphonate group, phosphosilicate group, silane group, carbonamide, thioamide, sulfonamide group, polyamide, phosphoramide, phosphoramidite, pyrophosphamide, cyclophosphamide, ifosfamide, thiophosphoramide, aconityl group, a polypeptide fragment, a nucleotide and its derivative skeleton, any one divalent linking group in deoxynucleotide and its derivative skeleton, a combination of any two or more divalent linking groups.
The carbamate group, the thiocarbamate group, the carbamide group, the phosphoramide group and the like may be used as a linker which can exist stably or as a degradable linker. Depending on the environmental characteristics of its use.
In particular, the degradable divalent linking group DEGG includes, but is not limited to, the structures described and exemplified in documents CN104530413A, CN104530415A, CN 104530417A. For example, CN104530417A corresponds to segments [705] to [0725 ].
1.1.3.B1、B2、B3、B4
In the present invention, B1、B2Each independently is a connecting bond or C1-30An alkylene group.
In one embodiment of the present invention, B is preferred1、B2Each independently is a connecting bond or C1-20An alkylene group; more preferably B1、B2One of them is a connecting bond and the other is C1-20An alkylene group; more preferably B1、B2Each independently of the others, is methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecyleneAnd eicosylene.
In the present invention, B3、B4Each independently is a connecting bond or C1-12An alkylene group.
1.1.4.R1、R2
In the present invention, R 1、R2Each independently is C1-30An aliphatic hydrocarbon group.
In one embodiment of the invention, R is preferred1、R2Each independently is C5-30Aliphatic hydrocarbon group, more preferably C10-30Aliphatic hydrocarbon radical, most preferably C10-20An aliphatic hydrocarbon group.
In one embodiment of the present invention, R is preferred1、R2Each independently is a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a linear alkynyl group or a branched alkynyl group; preferably a linear alkyl group; more preferably each independently C1-25A linear alkyl group; more preferably each independently C1-17A linear alkyl group; most preferably, each independently of the others, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, (Z) -tridec-8-enyl, (Z) -tetradec-9-enyl, (Z) -pentadec-8-enyl, (Z) -hexadec-9-enyl, (Z) -heptadec-5-enyl, (Z) -heptadec-8-enyl, (E) -heptadec-8-enyl, (Z) -heptadec-10-enyl, pentyl, (8Z,11Z) -heptadecane-8, 11-dienyl, (Z) -octadecan-6-enyl, (Z) -octadecan-9-enyl, (E) -octadecan-9-enyl, (Z) -octadecan-11-enyl, (9Z,12Z) -octadecan-9, 12-dienyl, (9Z,12Z,15Z) -octadecan-9, 12, 15-trienyl, (8Z,11Z,14Z) -octadecan-8, 11, 14-trienyl, (Z) -eicosan-11-enyl, (11Z,14Z) -eicosan-11, 14-dienyl, (Z) -nonadecane-10-enyl, (10Z,13Z) -nonadecane-10, 13-dienyl, 2,6, 10-trimethylundecane-1, 5, 9-trienyl, 3,7, 11-trimethyldodecane-2, 6, 10-trienyl or 3,7,11, 15-tetramethyl Any of hexadecan-2-enyltridecyl.
In one embodiment of the invention, R is preferred1、R2Each independently is a branched alkyl, branched alkenyl or branched alkynyl, each independently isWherein R ise、RfEach independently is C1-C15Alkyl radical, C2-C15Alkenyl and C2-C15Any one of alkynyl groups; more preferably, each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propenyl, allyl, butenyl, alkenylbutyl, pentenyl, alkenylpentyl, hexenyl, alkenylhexyl, heptenyl, alkenylheptyl, octenyl, nonenyl, decenyl, alkenyldecyl, ethynyl, propynyl, propargyl, butynyl, pentynyl, hexynyl, alkynhexyl, heptynyl, alkynheptyl, octynyl, nonynyl, decynyl, and alkyndecyl; more preferably, each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Further preferred is R1、R2Each independently selected from any one of the following structures:
Wherein t is an integer of 0 to 12, preferably t is 0 or an integer of 1 to 12.
1.1.5.R3、R
In the present invention, R3Is a hydrogen atom, -Rd、-ORd、-NRdRd、-SRd、-(C＝O)Rd、-(C＝O)ORd、-O(C＝O)Rd、 -O(C＝O)ORdOrWherein R isdEach occurrence is independently C1-12Alkyl radical, G1A terminal branched group with a valence of k +1, j is 0 or 1, F contains a functional group R01When j is 0, G1In the absence of, when j is 1, G1K are introduced, and k is an integer of 2-8.
In the present invention, R is a hydrogen atom, an alkyl group, an alkoxy group, or- (C ═ O) Rd、-(C＝O)ORd、-O(C＝O)Rd、 -O(C＝O)ORdOrWherein R isdIs C1-12Alkyl radical, G1A terminal branched group having a valence of k +1, j is 0 or 1, F contains a functional group, and when j is 0, G1In the absence of, when j is 1, G1K are introduced, and k is an integer of 2-8.
1.1.5.1.Rd
In the present invention, RdEach occurrence is independently C1-12An alkyl group.
In one embodiment of the invention, RdPreferably C1-8Alkyl, in particular RdPreferably methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl.
1.1.5.2.G1
In the present invention, j is 0 or 1, and G is 01In the absence of, when j is 1, G1There are terminal branching groups with a valence of k +1, leading k functional group-containing F's, where k is an integer from 2 to 8, preferably k is 2, 3 or 4.
In one embodiment of the present invention, G is preferred1In the case of a trivalent or more-trivalent branched group, G is preferred 1Is a trivalent branching group or a tetravalent branching group; preferably G1Is a trivalent branching group; more preferably G1Is a trivalent branching group of glycerol and amino acid residue.
In the present invention, when the terminal is difunctionalized, G1Preferably derived from an alcohol, thiol, primary amine, secondary amine, sulfonate or halide containing two naked or two protected hydroxyl groups (again, for example, triethanolamine p-toluenesulfonate, glycerol monothioglycolate, 3, 4-dihydroxy-2' -chloroacetophenone, and hydroxyl protected forms), an alcohol, thiol, primary amine, secondary amine, sulfonate or halide containing two or two protected thiol groups (again, for example, dimercaprol and its thiol protected forms), an alcohol, thiol, primary amine, secondary amine, sulfonate or halide containing two primary amino groups, two secondary amino groups, two protected primary amino groups, or two protected secondary amino groups, and the like. Examples of alcohols containing two primary amines include 1, 3-diamino-2-propanol, aldehydes containing 1 epoxy group, alcohols containing 1 epoxy group (e.g., alcohol containing 1 epoxy group)) Sulfonate containing 1 epoxy group, halide containing 1 epoxy group, compound containing one epoxy group and 1 other reactive group. Also included are combinations of Michael addition reactions of primary amines with 2 molecules of acrylates. Or the lipoic acid is adopted to carry out end capping, and then the disulfide bond is reduced to open the ring, so that two sulfydryl groups at the tail end are obtained.
In the present invention, when the terminal is trifunctional, G1Preferably derived from tetrafunctional small molecule htetraSM containing three hydroxyl groups and another reactive group, including but not limited to: N-Trimethylol-2-aminoethanesulfonic acid, Trimethylol methylaminopropanesulfonic acid, methyl-6-O-p-toluenesulfonyl-alpha-D-glucoside, 2- (bromomethyl) -2- (hydroxymethyl) -1, 3-propanediol, Trimethylolaminomethane, 2-amino-1, 3, 4-octadecanetriol, 3-aminopropylsilanetriol, 4- (2-amino-1-hydroxyethyl) -1, 2-benzenediol, 4- [ (2-isopropylamino-1-hydroxy) ethyl]-1, 2-benzenediol, 3, 4-dihydroxy-alpha- ((methylamino) methyl) benzyl alcohol, 2, 5-anhydro-1-azido-1-deoxy-D-glucitol, 2,3, 4-trihydroxybutyraldehyde (L-erythrose, D-erythrose, L- (+) -threose, D- (+) -threose), 2,3, 4-trihydroxybenzaldehyde, 3,4, 5-trihydroxybenzaldehyde, tris (hydroxymethyl) methylglycine, 2,3, 4-trihydroxybutyric acid (including but not limited to erythronic acid, threonic acid), 2,4, 6-trihydroxybenzoic acid, shikimic acid, 3,4, 5-trihydroxybenzoic acid, 2,3, 4-trihydroxybenzoic acid, arjunolic acid, 1,4, 7-tri-tert-butoxycarbonyl-1, 4,7, 10-tetraazacyclododecane, tri-tert-butoxycarbonyl spermine, 1,4, 7-tri-tert-butoxycarbonyl-1, 4,7, 10-tetraazacyclododecane and the like and any of the above hydroxyl protected forms. The surfactant may be selected from the group consisting of citric acid, agaricic acid, N-hydroxyethylethylenediaminetriacetic acid, pentaerythritol triacrylate, aminomethane tripropionic acid, and tri-tert-butyl aminomethane tripropionate. Also included are terminal branching reactions based on alkenyl, trichlorosilane and allylmagnesium chloride, see Macromolecules, Vol.33, No.12,2000, to form tetravalent silicon-based branching centers. Also included are terminal branching reactions based on alkenyl, trichlorosilane, and allyl alcohol to form tetravalent siloxane branching centers. Also included are tri-functionalized small molecules such as 1,4, 7-tris (t-butoxycarbonylmethyl) -1,4,7, 10-azacyclotetradecane (NOTA), which require an excess of such tri-functionalized small molecules.
1.1.5.3. Containing functional groups R01F of (A)
In one embodiment of the present invention, it is preferred that F has the structure- (Z)2)q-(Z1)q1-R01Wherein q, q1 are each independently 0 or 1; z1, Z2 are each independently a divalent linking group; r01Is a functional group capable of interacting with biologically relevant substances.
In a more specific embodiment of the invention, R01Preferably selected from: reactive groups, variations of reactive groups, functional groups with therapeutic targeting, fluorescent functional groups; wherein the variants include any of a precursor of a reactive group, an active form as a precursor thereof, a substituted active form, a protected form, a deprotected form; wherein the precursor of the reactive group is referred to as a channelAt least one of peroxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexing, ionization, protonation, deprotonation, convertible to the structure of the reactive group; wherein, the change form of the reactive group refers to a form that one reactive group still has activity after undergoing at least one process of oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexing, ionization, protonation, deprotonation, substitution and deprotection, or an inactive form after being protected; the R is 01More preferably from the group consisting of any one of the following functional groups of groups A to H and their modifications or functional groups of groups I to J:
class A: activated ester groups, analogous structures of activated ester groups; wherein the active ester group comprises: a succinimide active ester group, a p-nitrophenyl active ester group, an o-nitrophenyl active ester group, a benzotriazole active ester group, a 1,3, 5-trichlorobenzene active ester group, a 1,3, 5-trifluorobenzene active ester group, a pentafluorobenzene active ester group and an imidazole active ester group; wherein, the analogous structure of the active ester group comprises: 2-thione-3-thiazolidineformate, 2-thiothioxothiazolidine-3-carboxylate, 2-thiothioxlidine-N-formate, 2-thiothioxothiazole-N-formate, 1-oxo-3-thiothioxoisoindoline-N-formate;
class B: sulfonate, sulfinate, sulfone, sulfoxide, 1, 3-disulfonyl-2-propylcarbonylphenyl, sulfone methacryl;
class C: hydroxylamino group, mercapto group, primary amino group, secondary amino group, halogen atom, haloacetamido group, tetramethylpiperidinoxy group, dioxopiperidinoxy group, ammonium salt, hydrazine group, dithio compound group, ester group, thioester group, thiocarbonate group, carbonate group, thiocarbonate group, dithiocarbonate group, trithiocarbonate group, xanthate group, peroxythiocarbonate group, tetrathiodiester group, O-carbonylhydroxylamino group, amide group, imide group, hydrazide group, sulfonylhydrazide group, hydrazone group, imine group, enamine group, alkynylamine group, carbamate group, monothiocarbamate group, dithiocarbamate group, protected amino group;
Class D: carboxyl, sulfonic, sulfenic, hydroxamic, thiohydroxamic, xanthic, acid halide, sulfonyl chloride, aldehyde, glyoxal, acetal, hemiacetal, hydrated aldehyde, keto, ketal, hemiketal, ketal, hydrated keto, orthoacid, orthoester, cyanate, thiocyanate, isocyanurate, isothiocyanate, ester, oxycarbonylhalide, oxazoline, isoxazoline, thioaldehyde, thioketone, thioacetal, thioketone hydrate, thiothiothiothiothioketal, thiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothiothioketal, thioester, thiohemiacetal, monothiohydrate, dithiohydrate, thiohydrate, thiocarbonylmonocarboxylic, thiothiocarboxylic, dithiocarboxylic, thiosemicarbazide, guanidino, and protonated forms thereof, Amidino group and protonated form thereof, acid anhydride group, squaric acid ester group, hemisquaric acid ester group, N-carbamoyl-3-imidazolyl group, N-carbamoyl-3-methylimidazolium iodide group, imido group, nitrone group, oximo group, pseudoureido group;
Class E: maleimide group, acrylate group, N-acrylamide group, methacrylate group, N-methacrylamide group, protected maleimide group, maleamido group, 1,2, 4-triazoline-3, 5-dione group, linear azo compound group, cyclic olefin group; wherein the cycloalkenylhydrocarbyl group includes cyclooctenylhydrocarbyl, norbornenyl, 7-oxa-bicyclo [2.2.1] hept-5-en-2-yl, bicycloheptedienyl, 7-oxabicycloheptedienyl;
class F: epoxy, vinyl, propenyl, alkenyl hydrocarbon, alkynyl hydrocarbon;
the class of the signal is a class G,
class Ga: cycloalkynylalkyl, cycloalkynheteroalkyl, linear conjugated dienyl, cyclic conjugated dienyl, hybrid cyclic conjugated dienyl, 1,2,4, 5-tetrazinyl;
class Gb: azido, nitrile oxide groups, cyano, isocyano, aldoximo, diazo, diazonium ions, azoxy, nitrilo imino, N-oxyaldoimino, tetrazolyl, 4-acetyl-2-methoxy-5-nitrophenoxy and diazotized forms thereof; other functional groups capable of undergoing a 1, 3-dipolar cycloaddition reaction;
class H: hydroxyl, protected hydroxyl, siloxy, protected bishydroxy, trihydroxysilyl, protected trihydroxysilyl; wherein the hydroxyl group comprises alcoholic hydroxyl group, phenolic hydroxyl group, enol hydroxyl group and hemiacetal hydroxyl group;
Class I: targeting groups and pharmaceutically acceptable salts thereof;
class J: the fluorescent group includes any one of fluorescein, rhodamine, anthracene, pyrene, coumarin, fluorescein 3G, carbazole, imidazole, indole and alizarin violet and the residue of any one of functional derivatives.
Further, R01Preferably a functional group selected from any one of the following classes A to J, variants of classes A to H, functional derivatives of classes I to J; the variant is selected from any one of a precursor of a reactive group, an active form thereof as a precursor, a substituted active form, a protected form, a deprotected form:
class A:
or class B:
or class C:
or class D:
or class E:
or class F:
or class G: class Ga:
or class Gb:
or class H:
or class I:
or class J:
wherein M is5Is a ring-forming atom selected from any one of carbon atom, nitrogen atom, phosphorus atom and silicon atom; m5The cyclic structure is a 3-50-membered ring, preferably a 3-32-membered ring, more preferably a 3-18-membered ring, and still more preferably a 5-18-membered ring; the cyclic structure is preferably any one of the following groups, a substituted form of any one, or a hybridized form of any one: cyclohexane, furanose ring, pyranose ring, benzene, tetrahydrofuran, pyrrolidine, thiazolidine, cyclohexene, tetrahydropyran, piperidine, 1, 4-dioxane, pyridine, pyridazine, pyrimidine, pyrazine, 1,3, 5-triazine, 1,4, 7-triazacyclononane, tripeptide, indene, indane, indole, isoindole, purine, naphthalene, dihydroanthracene, xanthene, thioxanthene, dihydrophenanthrene, 10, 11-dihydro-5H-dibenzo [ a, d ] s ]Cycloheptane, dibenzocycloheptene, 5-dibenzocycloheptenone, quinoline, isoquinoline, fluorene, carbazole, iminodibenzyl, naphthylene ring, dibenzocyclooctyne, aza-dibenzocyclooctyne;
wherein, Y1Is a leaving group attached to sulfonyl, sulfinyl, oxysulfonyl or oxysulfinyl, selected from any one of methyl, ethyl, n-propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, vinyl, phenyl, benzyl, p-methylphenyl, 4- (trifluoromethoxy) phenyl, trifluoromethyl, 2,2, 2-trifluoroethyl;
wherein W is F, Cl, Br or I;
wherein, W2Is F, Cl, Br or I;
wherein, W3Is a leaving group selected from F, Cl, Br, I, PhS;
wherein the content of the first and second substances, each of which is a cyclic structure having a ring skeleton containing a nitrogen atom, a nitrogen onium ion, a double bond, an azo group, a triple bond, a disulfide bond, an acid anhydride, an imide, or a diene, the cyclic structure being selected from a carbocyclic ring and a hetero ringA ring, benzo-heterocycle, substituted carbocycle, substituted heterocycle, or substituted benzo-heterocycle;
wherein M is a carbon atom, a nitrogen atom, a phosphorus atom or a silicon atom on the ring;
wherein M is8Is a carbon atom, a nitrogen atom, a phosphorus atom or a silicon atom located on the ring; m 8The number of ring-forming atoms of the ring is 4-50; preferably 4-32; more preferably 5 to 32;
wherein M is22Is a carbon atom, a nitrogen atom, a phosphorus atom or a silicon atom on an alicyclic or alicyclic ring; m22The number of ring atoms of the ring is 4, 5, 6, 7 or 8;
wherein R is22Is a terminal group or a divalent linking group to which an oxygen or sulfur atom is bonded, selected from a hydrogen atom, R21Or R33Any one atom or group;
wherein R is21Is a divalent linking group and participates in ring formation; r21Is selected from C1-20Alkylene, divalent C1-20Heterohydrocarbyl, substituted C1-20Alkylene, substituted divalent C1-20Any divalent linking group or any two or any three of divalent linking groups in the heterohydrocarbon group; r21Preferably methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, 1, 2-phenylene, benzylene, C1-20Oxaalkylene, C1-20Thiaalkylene group, C1-20Any one group of azaalkylene and azaaralkyl, a substituted form of any one group, any two or more of the same or different groups, or a combination of substituted forms thereof;
wherein R is3Is a terminal group linked to an oxy or thio group, selected from C1-20Hydrocarbyl radical, C1-20Heterohydrocarbyl radical, C1-20Substituted hydrocarbyl radical, C 1-20Any of substituted heterohydrocarbyl groups; preferably any one or substituted form of any one of methyl, ethyl, n-propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, benzyl, allyl;
wherein R is4Is- (R)4)C＝N+＝N—Or- (R)4)C--N+A hydrogen atom, a substituent atom or a substituent on C in the structure of [ identical to ] N; preferably any one atom or group of hydrogen atom, methyl group, ethyl group, n-propyl group, isopropyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, allyl group, propenyl group, vinyl group, phenyl group, methylphenyl group, butylphenyl group, benzyl group;
wherein R is8、R9、R10、R11、R12Each independently is a hydrogen atom, a substituent atom or a substituent on a double bond (-C-), and R is in the same molecule8、R9、R10、R11、R12May be the same as or different from each other; r8、 R9、R10、R11、R12Each independently selected from: hydrogen atom, fluorine atom, methyl group; in class E3, R8Preferably methyl;
wherein R is24Is a terminal group linked to a disulfide bond selected from: c1-20Alkyl, aryl, hybrid phenyl;
wherein R is27Is a substituent attached to azo selected from: phenyl, substituted phenyl or hybrid phenyl;
wherein R is30Is a hydrocarbyl group selected from: c1-20Alkyl, benzyl, phenyl ring hydrogen atoms by C 1-20A hydrocarbyl-substituted benzyl group;
wherein M is19、M20、M21Each independently is an oxygen atom or a sulfur atom, and may be the same as or different from each other in the same molecule;
wherein, X6Is a terminal group attached to the oxygen atom of the ester group and is selected from a hydroxyl protecting group or the group LG4；LG4Is selected from C1-20Alkyl, aryl, aralkyl, C1-20Heteroalkyl, heteroaryl, heteroaralkyl, C1-20Alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, C1-20Heteroalkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, C1-20Alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, C1-20Alkylthio-carbonyl, arylthio-carbonyl, aralkylthiocarbonyl, C1-20Alkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl, C1-20Heteroalkyloxycarbonyl, heteroaryloxycarbonyl, heteroaralkyloxycarbonyl, C1-20Heteroalkylthio-carbonyl, heteroarylthio-carbonyl, heteroaralkylthio-carbonyl, C1-20Heteroalkylaminocarbonyl, heteroarylaminocarbonyl, heteroarylalkylaminocarbonyl, C1-20Alkylthio, arylthio, aralkylthiocarbonyl, C1-20Heteroalkylthiocarbonyl, heteroarylthiocarbonyl, heteroarylalkylthiocarbonyl, C1-20Alkoxythiocarbonyl, aryloxylthiocarbonyl, aralkyloxythiocarbonyl, C 1-20Alkylthio thiocarbonyl, arylthio thiocarbonyl, aralkylthio thiocarbonyl, C1-20Alkylaminothiocarbonyl, arylaminothiocarbonyl, aralkylaminothiocarbonyl, C1-20Heteroalkyloxythiocarbonyl, heteroaryloxythiocarbonyl, heteroarylalkoxythiocarbonyl, C1-20Heteroalkylthio thiocarbonyl, heteroarylthio thiocarbonyl, heteroarylalkylthio thiocarbonyl, C1-20(ii) any one of a heteroalkylaminothiocarbonyl, heteroarylaminothiocarbonyl or a substituted version of any one of the groups; wherein, the substituent atom or the substituent group is fluorine atom, alkoxy or nitro;
wherein, X11Is a terminal group attached to a carbonyl or thiocarbonyl group, selected from C1-20An alkyl group;
wherein, X12Is a terminal group to which a carbonate or thiocarbonate group is attached, selected from C1-20A hydrocarbyl group;
wherein, X13Is a terminal group for attaching a sulfur group selected from: mercapto-protecting group, group LG2；
Wherein LG is2Selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, and mixtures thereof, Nonadecyl, eicosyl, allyl, trityl, phenyl, benzyl, methylbenzyl, nitrobenzyl, tert-butylthio, benzylthio, 2-pyridylthio, acetyl, benzoyl, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, phenoxycarbonyl, benzyloxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl, tert-butylthiocarbonyl, phenylthiocarbonyl, benzylthiocarbonyl, 2-pyridylcarbonyl, methylaminocarbonyl, ethylaminocarbonyl, tert-butylaminocarbonyl, benzylaminocarbonyl, ethylthiocarbonyl, phenylmethylthiocarbonyl, methoxythiocarbonyl, ethoxythiocarbonyl, tert-butyloxythiocarbonyl, phenoxythiocarbonyl, benzyloxythiocarbonyl, methylthiothiocarbonyl, ethylthiocarbonyl, tert-butylthiocarbonyl, phenylthiocarbonyl, methylbenzylthio, benzylthio, ethylthiocarbonyl, benzylthio, ethylthio, tert-butylthio, phenylthio, benzylthio, ethylthio, phenylthio, tert-thiocarbonyl, benzylthio, phenylthio, thiothio, or, Benzylthiothiocarbonyl, methylaminothiocarbonyl, ethylaminothiocarbonyl, tert-butylaminothiocarbonyl, benzylaminothiocarbonyl, C1-10Any one of a halogenated hydrocarbon group, a trifluoroacetyl group, a nitrophenyl group, or a substituted form of any one of the groups; wherein, the substituent atom or the substituent group is fluorine atom, alkoxy or nitro;
Wherein Q is an atom or substituent contributing to the induction of unsaturated bond electrons, conjugation effect; when Q is on a ring, the number is one or more; when the number is multiple, the structure is the same, or the combination of two or more different structures; when a substituent group, Q has a linear structure, a branched structure containing a pendant group, or a cyclic structure;
wherein Q is3Is H atom or a group contributing to the induction of the electrons of unsaturated bonds, conjugation effect, selected from the group consisting of hydrogen atom, fluorine atom, chlorine atom, bromine atom, iodine atom, methyl group, ethyl group, n-propyl group, isopropyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, vinyl group, propenyl group, allyl group, propynyl group, propargyl group, cyclopropyl group, cyclopropenyl group, phenyl group, benzyl group, butylphenyl group, p-methylphenyl group, p-nitrophenyl group, o-nitrophenyl group, p-methoxyphenyl group, azaphenyl group, methoxy group, ethoxy group, phenoxy group, benzyloxy group, methylthio group, ethylthio group, phenylthio group, benzylthio group, trifluoro phenyl groupAny one atom or group of methyl, 2,2, 2-trifluoroethyl, or a substituted form of any one group;
wherein Q is5Is H atom, methyl, ethyl or propyl; when Q is 5When located on a ring, the number is one or more; when more than 1, the structure is the same, or the combination of two or more different structures;
wherein Q is6Is a hydrogen atom or a methyl group; q7Is a hydrogen atom, a methyl group, a phenyl group or a substituted phenyl group; in the same molecule, Q6And Q7May be the same or different;
wherein Q is8Is a substituent atom or a substituent group on the imidazolyl group, and is selected from any one of H atoms, methyl groups, ethyl groups, propyl groups, butyl groups and phenyl groups; when Q is8Is one or more; when more than 1, the structure is the same, or the combination of two or more different structures;
wherein Q is11Is a substituent on the nitrogen atom of tetrazole, and is selected from any one of phenyl, substituted phenyl and aza-phenyl;
wherein PG2Is a thiol protecting group, the protected thiol group being denoted as SPG2Preferably any one of thioether, disulfide, silyl sulfide, thioester;
wherein PG3Is an alkynyl protecting group, preferably a silicon group;
wherein PG4Is a hydroxy protecting group, the protected hydroxy group being represented by OPG4Any one of ether, silyl ether, ester, carbonate and sulfonate is preferable;
wherein PG5Is an amino protecting group, the protected amino group being represented by NPG 5Preferably any one of carbamate, amide, imide, N-alkylamine, N-arylamine, imine, enamine, imidazole, pyrrole and indole;
wherein PG6Is a bishydroxy protecting group, and PG6An acetal structure which forms a five-membered ring or a six-membered ring with two oxygen atoms; PG (Picture experts group)6Is methylene or substituted methylene; wherein PG6The substituent being a hydrocarbyl substituent or a hetero atomSubstituents of the subgroups selected from: any one of methylene, 1-methylmethylene, 1-dimethylmethylene, 1-cyclopentylene, 1-cyclohexylene, 1-phenylmethylene, and 3, 4-dimethylphenylmethylene;
wherein PG8Protecting groups for orthocarbonic acid or orthosilicic acid.
1.1.5.4.R3Specific examples of (1)
In one embodiment of the present invention, R preferably contains any one of a hydrogen atom, an alkyl group, an alkoxy group, an alcoholic hydroxyl group, a protected alcoholic hydroxyl group, a thiol hydroxyl group, a protected thiol hydroxyl group, a carboxyl group, a protected carboxyl group, an amino group, a protected amino group, an aldehyde group, a protected aldehyde group, an ester group, a carbonate group, a carbamate group, a succinimide group, a maleimide group, a protected maleimide group, a dimethylamino group, an alkenyl group, an enoate group, an azide group, an alkynyl group, a folate group, a rhodamine group, and a biotin group; more preferably contains H, - (CH) 2)tOH、-(CH2)tSH、-OCH3、-OCH2CH3、 -(CH2)tNH2、-(CH2)tC(＝O)OH、-C(＝O)(CH2)tC(＝O)OH、-C(＝O)CH3、-(CH2)tN3、 -C(＝O)CH2CH3、-C(＝O)OCH3、-OC(＝O)OCH3、-C(＝O)OCH2CH3、-OC(＝O)OCH2CH3、 -(CH2)tN(CH3)2、-(CH2)tCHO、
1.1.5.5.R3Atomic separation to nitrogen branching centres
The length of a carbon chain in the compound is short, and the atomic separation of two groups has great influence on the performance of the compound.
One of the inventionIn a particular embodiment, R3The number of atomic separations to the nitrogen branching center N is greater than or equal to 6, preferably from 6 to 50, more preferably from 6 to 35, more preferably from 6 to 25, and most preferably from 6 to 15.
Specific examples are R in example 13The atomic distance from (hydrogen atom) to the nitrogen branching center N being-CH2CH2OCH2CH2O-, then R3The number of atomic separations to the nitrogen branching center N is 6; in example 2, example 4, R3The atomic distance from (-OH) to the nitrogen branching centre N being-CH2CH2OCH2CH2OCH2CH2-, then R3The number of atomic separations to the nitrogen branching center N is 8; in example 8, R3The atomic distance from (hydrogen atom) to the nitrogen branching center N being-CH2CH2NHC(＝O)CH2CH2OCH2CH2O-, then R3The number of atom separations to the nitrogen branching center N is 10.
1.1.6.A
In the present invention, A is selected from- (CR)aRb)sO-、-O(CRaRb)s-、-(CRaRb)sS-、-S(CRaRb)s-、 -(CRaRb)sO(CRaRb)sS-、-(CRaRb)sS(CRaRb)sO-、-(CRaRb)sNRc(CRaRb)sS-、 -(CRaRb)sS(CRaRb)sNRc-、-(CRaRb)sNRc(CRaRb)sO-and- (CR)aRb)sO(CRaRb)sNRc-any of, wherein s is 2, 3 or 4, Ra、RbAnd RcEach independently is a hydrogen atom or C1-12An alkyl group.
In one embodiment of the present invention, A is preferably-CH2CH2O-、-OCH2CH2-、-CH(CH3)CH2O-、 -OCH(CH3)CH2-、-CH2CH2S-、-SCH2CH2-、-CH2CH2NHCH2CH2O-、-CH2CH2OCH2CH2NH-、 -CH2CH2SCH2CH2O-or-CH2CH2OCH2CH2S-, more preferably-CH2CH2O-、-OCH2CH2-、 -CH2CH2SCH2CH2O-or-CH2CH2OCH2CH2S-, most preferably-CH2CH2O-or-OCH2CH2-。
1.1.7. Specific structural formula is exemplified
In a specific embodiment of the present invention, when A in the general structural formula (1) is-CH 2CH2O-、-OCH2CH2-、 -CH(CH3)CH2O-、-OCH(CH3)CH2-、-CH2CH2S-、-SCH2CH2-、-CH2CH2NHCH2CH2O-、 -CH2CH2OCH2CH2NH-、-CH2CH2SCH2CH2O-or-CH2CH2OCH2CH2S-, the structure of the cationic lipid of the present invention preferably satisfies any one of the following structural formulae:
wherein L is1、L2、L3、B1、B2、R1、R2、R3N is as defined in formula (1).
In one particular embodiment of the present invention,when L in the general structural formula (1)1、L2Each independently is a bond, -O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -O (CH)2)sWhen any one of O-, -S-, -C (═ O) S-, -SC (═ O) -, -NHC (═ O) -, -C (═ O) NH-, -NHC (═ O) NH-, -OC (═ O) NH-, -NHC (═ O) O-, -SC (═ O) NH-, and-NHC (═ O) S-, is selected, the structure of the cationic lipid of the present invention preferably satisfies any one of the following structural formulae:
wherein L is3、B1、B2、R1、R2、R3A, n are as defined in the general formula (1), in the formulae (2-1) to (2-10), B1And B2Are not a bond, B in formula (2-22)1Is a connecting bond; the A is preferably-CH2CH2O-、-OCH2CH2-、-CH2CH2S-、-SCH2CH2-、-CH2CH2NHCH2CH2O-、-CH2CH2OCH2CH2NH-、 -CH2CH2SCH2CH2O-and-CH2CH2OCH2CH2Any of S-, more preferably-CH2CH2O-、-OCH2CH2-、 -CH2CH2NHCH2CH2O-、-CH2CH2OCH2CH2NH-、-CH2CH2SCH2CH2O-and-CH2CH2OCH2CH2Any one of S-。
1.1.8. Specific examples of the construction
Some embodiments of the present invention, ultimately result in a cationic lipid having the structure shown below, including but not limited to any of the following structures:
2. preparation of cationic lipids
In the present invention, any one of the aforementioned cationic lipids can be prepared by a method including, but not limited to:
2.1. The method comprises the following steps:
step one, using carboxyl activating agent to react the carboxyl of acid A-1 or A-1' containing naked carboxylActivating the terminal to obtain carboxyl terminal activated ester A-2 or A-2', wherein B1’、B2' each independently is a connecting bond or a ratio B1、B2Alkylene with at least one methylene group; r1’、R2' each independently is R1、R2Or ratio R1、R2Aliphatic hydrocarbon groups having at least one methylene group; r7Is a carboxyl activating group, and when B is1’、L1When any one of them is not a connecting bond, R1' is R1When B is present1’、L1When both are connecting bonds, R1Is a ratio of R1Aliphatic hydrocarbon group having less than 1 methylene group, when B2’、L2When any one of them is not a connecting bond, R2' is R2When B is present2’、L2When both are connecting bonds, R2Is a ratio of R2An aliphatic hydrocarbon group which is 1 methylene group less;
step two: carrying out condensation reaction on ester A-2 or A-2 ' with activated carboxyl end and primary amine derivative A-3 or A-3 ' with nitrogen source end group to obtain amide intermediate A-4 or A-4 ';
step three: reducing the amide intermediate A-4 or A-4 'to a secondary amine intermediate A-5 or A-5' with a reducing agent;
step four: performing coupling reaction on the secondary amine intermediate and the double-end functionalized micromolecule A-6 to obtain a cationic lipid derivative A-7 or A-7'; wherein, the functional groups at the two ends of A-6 can be the same or different; wherein R of A-63' end containing a reactive group R 01Or contain R01A micro-variation of (a); the micro-variation form can be converted into R through any one of chemical processes of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving group01A group of (a); wherein, F of A-61Contains active functional group and can react with amino of secondary amine intermediate A-5 or A-5' to generate branched central nitrogen atom and divalent linking group L3；
When R is3Is equal to R3When the structure A-7 or A-7' is obtained, the structure corresponds to the structure shown in the general formula (1);
when R is3' not equal to R3Then, the A-7 or A-7 'is subjected to terminal micro-modification to obtain a structure of A-8 or A-8' corresponding to the general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, change of leaving group.
Step one
Step two
Step three
Step four
Wherein L is3、L1、L2、B1、B2、R3、R1、R2And n is the same as that described in the general formula (1), and will not be described herein.
In one embodiment of the present invention, A-1 in the aforementioned method 1 is preferably R1' -COOH or R2' -COOH, said A-3 being preferably R1-NH2Or R2-NH2The carboxyl activating agent is preferably any one of N-hydroxysuccinimide (NHS), 1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride (EDCI), N-hydroxy-5-norbornene-2, 3-dicarboximide (HONb) and N, N-Dicyclohexylcarbodiimide (DCC), and the cationic lipid is preferably prepared by the following route:
2.2. The method 2 comprises the following steps:
step one, performing coupling reaction on a double-end functionalized small molecule B-1 and a primary amino derivative B-2 or B-2 'containing a nitrogen source end group to obtain a secondary amine derivative B-3 or B-3'; wherein the functional groups at both ends of B-1 may be the same or different, R of B-13' end containing a reactive group R01Or contain R01A micro-variation of (a); the micro-variation form can be converted into R through any one of chemical processes of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving group01A group of (a); wherein, F1Containing reactive functional groups capable of reacting with the amino groups of primary amines B-2 or B-2' to form a compound containing a divalent linking group L3The secondary amine derivative of (A) B-3 or B-3';
step two, secondary amine derivative B-3 or B-3' with a compound containing a reactive group FNThe compound B-4 or B-4 'is subjected to alkyl reaction to obtain a cationic lipid derivative B-5 or B-5'; said FNReactive groups capable of reacting with amino or secondary amino groups, preferably-OMs, -OTs, -CHO, -F, -Cl, -Br;
when R is3Is equal to R3When the structure B-5 or B-5' is obtained, the structure corresponds to the structure shown in the general formula (1);
when R is3' not equal to R3Then, carrying out terminal micro modification on B-5 or B-5 'to obtain a structure of B-6 or B-6' corresponding to the general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, change of leaving group.
Step one
Step two
Wherein L is3、L1、L2、B1、B2、R3、R1、R2And n is the same as that described in the general formula (1), and will not be described herein.
In a specific embodiment of the present invention, B-3 or B-3' in the aforementioned Process 2 is R1-NH2Or R2-NH2Wherein B-4 or B-4' is R2-L2-B2-Br or R1-L1-B1-Br, said F1containing-OMSAnd (4) a base.
2.3. The method 3 comprises the following steps:
step one, reacting small molecule C-1 with small molecule C-2 to generate bivalent connecting group L1One end of which is a reactive group FNOne end of which is an aliphatic hydrocarbon group R1The small molecule intermediate C-3; wherein, F3And F4Each independently a reactive group capable of reacting to form a divalent linking group L1(ii) a Wherein C-2 is F containing heterofunctional group pair3And FNSaid FNReactive groups capable of reacting with amino or secondary amino groups, preferably-OMs, -OTs, -CHO, -F, -Cl, -Br;
step two, carrying out alkylation reaction on the two-molecule small-molecule intermediate C-3 and a primary amino derivative C-4 containing a nitrogen source end group to obtain a cationic lipid C-5, wherein R3' end containing a reactive group R01Or contain R01A micro-variation of (a); the micro-variation form can be converted into R through any one of chemical processes of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving group 01A group of (a);
when R is3Is equal to R3When the structure C-5 is obtained, the structure corresponds to the structure shown in the general formula (1);
when R is3' not equal to R3Then, carrying out terminal micro-modification on C-5 to obtain a structure shown by a corresponding general formula (1) of C-6; the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexing, ionization, protonation,Deprotonation, change of leaving group; wherein R is1And R2Same as B1And B2Same, L1And L2The same is true.
Wherein L is3、L1、L2、B1、B2、R3、R1、R2And n is the same as that described in the general formula (1), and will not be described herein.
Step one
Step two
2.4. The method 4 comprises the following steps:
step one, reacting small molecule D-1 with small molecule D-2 to generate bivalent connecting group L1One end is hydroxyl and the other end is aliphatic hydrocarbon radical R1The small molecule intermediate D-3; wherein, F3And F4Each independently a reactive group capable of reacting to form a divalent linking group L1(ii) a Wherein D-2 contains a heterofunctional group pair F3And a hydroxyl group;
step two, oxidizing hydroxyl of the small molecular intermediate D-3 into aldehyde group to obtain a small molecular intermediate D-4 containing aldehyde group, wherein B1Is a ratio of B1Alkylene with at least one methylene group;
step three, carrying out addition reaction on two molecules of the micromolecule intermediate D-4 containing aldehyde group and the primary amino derivative D-5 containing nitrogen source terminal group to obtain the cationic lipid D-6, wherein R 3' end containing a reactive group R01Or contain R01A micro-variation of (a); the micro-variation form can be converted into R through any one of chemical processes of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving group01A group of (a);
when R is3Is equal to R3When the structure D-6 is obtained, the structure corresponds to the structure shown in the general formula (1);
when R is3' not equal to R3Then, carrying out terminal micro-modification on D-6 to obtain a structure of which D-7 corresponds to the general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, change of leaving group, wherein R1And R2Same as B1And B2Same, L1And L2The same is true.
Step one
Step two
Step three
Wherein L is3、L1、L2、B1、B2、R3、R1、R2And n is the same as that described in the general formula (1), and will not be described herein.
2.5. Description of related materials and/or Steps in the preparation Process
2.5.1. Carboxyl group activator, condensing agent, oxidizing agent, and reducing agent
In the present invention, "carboxyl group activation" refers to activation treatment of carboxyl group with a carboxyl group activating agent, which promotes better condensation reaction after carboxyl group activation, such as: inhibiting the generation of racemization impurities in condensation reaction, catalyzing and accelerating the reaction speed, and the like. A "carboxy activating group" is the residue of a carboxy activating agent. The carboxyl activating agent is one or more of N-hydroxysuccinimide (NHS), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2, 3-dicarboximide (HONb) and N, N-Dicyclohexylcarbodiimide (DCC), preferably NHS/EDCI, NHS/DCC, HONb/DCC, most preferably NHS/EDCI.
In the present invention, the condensing agent used in the reaction is not limited, but is preferably N, N ' -Dicyclohexylcarbonyldiimine (DCC), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCl), 2- (7-azobenzotriazol) -N, N ' -tetramethyluronium Hexafluorophosphate (HATU), benzotriazol-N, N ' -tetramethyluronium Hexafluorophosphate (HBTU), and most preferably DCC. While the amount of the condensing agent is generally 1 to 20 times, preferably 5 to 10 times, the molar equivalent of the carboxylic acid, and a suitable catalyst (e.g., 4-dimethylaminopyridine) may be added for this reaction.
In the present invention, the oxidizing agent used in the reaction is not particularly limited as long as it is a compound or a combination of compounds capable of increasing the valence of the substrate, and is preferably phenyliodobis (trifluoroacetate), 1, 4-benzoquinone, benzyltrimethylammonium tribromide, pyridinium dichromate, potassium dichromate, ozone, oxygen, hypofluoric acid, sodium hypochlorite, cobaltous acetate, cobalt acetate, manganese acetate, palladium acetate, copper acetate, monoperoxyphthalic acid, iodine, N-iodosuccinimide, iodobenzene, 2-iodobenzoic acid, dimethyldioxirane, dimethylsulfoxide-oxalyl chloride, dimethylsulfoxide-acetic anhydride, DDQ, dichlorotris (triphenylphosphine) ruthenium, manganese dioxide, diacetoxyiodobenzene, periodic acid, sodium periodate-osmium tetraoxide, potassium permanganate, sodium perborate, perbenzoic acid, sodium perbenzoate, sodium periodate, sodium triphenoxide, or a combination of a plurality of compounds, Dibenzoyl peroxide, nickel peroxide, hydrogen peroxide, cumene hydroperoxide, t-butyl peroxy alcohol, peracetic acid, m-chloroperoxybenzoic acid, N-chlorosuccinimide, pyridinium chlorochromate, copper chloride palladium-chloride, urea hydrogen peroxide complex, triphenylmethyl tetrafluoroborate, tributyl tin oxide, cobalt trifluoride, vanadium trifluoride oxide, chromium trioxide, manganese triacetate, TEMPO, cerium ammonium nitrate, bromine, N-pyridine oxide, silver oxide, O-ethyl peroxycarbonate, manganese acetylacetonate, vanadyl acetylacetonate, aluminum isopropoxide, potassium hydrogen persulfate, dichloroiodobenzene, etc., more preferably one or a combination of oxygen, sodium hypochlorite, hydrogen peroxide, dichloroiodobenzene, potassium hydrogen persulfate, etc., in an amount of 1 to 50 times, preferably 1 to 20 times, the molar equivalent of the hydroxyl groups in the intermediate compound, more preferably 5 to 10 times.
In the present invention, the reducing agent used in the reaction is not particularly limited as long as it can reduce the Schiff base formed from ammonia and aldehyde or ketone to an amino group; preferably one or a combination of sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, borane, diborane, diisobutylaluminum hydride, diisopinocampheylborane, lithium borohydride, zinc borohydride, borane-pyridine, borane-methyl sulfide, borane-tetrahydrofuran, and the like; more preferably sodium cyanoborohydride, and the equivalent of the reducing agent is 1 to 50 times, preferably 1 to 20 times, more preferably 5 to 10 times the molar equivalent of the amino group to be modified.
In the present invention, the reaction temperature is 0 to 200 ℃, preferably 0 to 100 ℃, more preferably 0 to 25 ℃, and the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours. The obtained product can be purified by extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis or supercritical extraction.
In the present invention, the solvent for the reaction may be an aprotic or aprotic solvent, and the aprotic solvent includes toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, methyl tert-butyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, preferably tetrahydrofuran, dichloromethane, dimethyl sulfoxide, dimethylformamide.
In the present invention, the base used in the reaction is generally an organic base (e.g., triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine), and triethylamine or pyridine is preferred. The amount of the base used is 1 to 50 times, preferably 1 to 10 times, and more preferably 2 to 3 times the molar equivalent of the carboxylic acid.
2.5.2. Involving "protection" and "deprotection" of the relevant groups during the reaction "
In the invention, the reaction process also relates to the processes of 'protection' and 'deprotection' of related groups. In order to prevent the functional group from affecting the reaction, the functional group is usually protected. When the number of functional groups is 2 or more, only the target functional group is selectively reacted, and thus the other functional groups are protected. The protecting group is required to be easily removed as needed in addition to stably protecting a functional group to be protected. It is therefore important in organic synthesis to deprotect only the protecting group bonded to the specified functional group under appropriate conditions.
In the present invention, the "carboxyl-protecting group" refers to a protecting group which can be converted into a carboxyl group by hydrolysis or a deprotection reaction of the carboxyl-protecting group. The carboxyl-protecting group is preferably an alkyl group (e.g., methyl, ethyl, t-butyl) or an aralkyl group (e.g., benzyl), more preferably a t-butyl group (tBu), methyl group (Me) or ethyl group (Et). In the present invention, the "protected carboxyl group" refers to a group formed by protecting a carboxyl group with a suitable carboxyl protecting group, and is preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group. The carboxyl protecting group can be removed by hydrolysis under the catalysis of acid or base, and occasionally can be eliminated by pyrolysis, for example, tert-butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA and H 2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably TFA and H2A combination of O, LiOH and MeOH, or LiOH and EtOH. Deprotection of a protected carboxyl group to produce the corresponding free acid, said deprotection being carried out in the presence of a base which forms a pharmaceutically acceptable salt with the free acid formed by said deprotection.
In the present invention, the "amino-protecting group" includes all groups that can be used as protecting groups for general amino groups, for example, aryl C1-6Alkyl radical, C1-6Alkoxy radical C1-6Alkyl radical, C1-6Alkoxycarbonyl, aryloxycarbonyl, C1-6Alkylsulfonyl, arylsulfonyl, silyl, or the like. Preferred amino protecting groups are Boc t-butyloxycarbonyl, Moz p-methoxybenzyloxycarbonyl and Fmoc 9-fluorenylmethyloxycarbonyl. The reagent for removing the amino protecting group is selected from TFA and H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably TFA and H2A combination of O, LiOH and MeOH, or LiOH and EtOH. Threshing deviceThe reagent for removing the Boc protecting group is TFA or HCl/EA; TFA is preferred. The deprotection agent used in the Fmoc-protecting group removal reaction was a 20% piperidine in N, N-Dimethylformamide (DMF).
In the present invention, the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and may be, for example, an alcoholic hydroxyl group, a phenolic hydroxyl group or the like. The amino group of the amino-protecting group is not particularly limited, and may be derived from, for example, a primary amine, a secondary amine, a diamine, an amide, or the like. The amino group in the present invention is not particularly limited, and includes, but is not limited to, primary amino group, secondary amino group, tertiary amino group, quaternary ammonium ion.
In the present invention, deprotection of a protected hydroxyl group is related to the type of hydroxyl protecting group. The type of the hydroxyl protecting group is not particularly limited, and for example, benzyl, silyl ether, acetal, and tert-butyl are used to protect the terminal hydroxyl group, and the corresponding deprotection methods are as follows:
a: deprotection of benzyl groups
The benzyl deprotection can be realized by utilizing hydrogenation of a hydrogenation reducing agent and a hydrogen donor, the water content in the reaction system is less than 1%, and the reaction can be smoothly carried out.
The hydrogenation reduction catalyst is not limited, and is preferably palladium and nickel, but is not limited to a carrier, but is preferably alumina or carbon, and more preferably carbon. The amount of palladium is 1 to 100% by weight of the protected hydroxy compound, preferably 1 to 20% by weight of the protected hydroxy compound.
The reaction solvent is not particularly limited as long as both the starting material and the product can be dissolved, but methanol, ethanol, ethyl acetate, tetrahydrofuran, acetic acid; methanol is more preferred. The hydrogen donor is not particularly limited, but hydrogen gas, cyclohexene, 2-propanol, ammonium formate and the like are preferable. The reaction temperature is preferably 25 to 40 ℃. The reaction time is not particularly limited, and the reaction time is inversely related to the amount of the catalyst, and is preferably 1 to 5 hours.
B: deprotection of acetals and ketals
The acetal or ketal compounds used for such hydroxyl protection are preferably ethyl vinyl ether, tetrahydropyran, acetone, 2-dimethoxypropane, benzaldehyde, and the like. And the deprotection of the acetal and ketal is realized under acidic conditions, and the pH of the solution is preferably 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid are preferable, and hydrochloric acid is more preferable. The reaction solvent is not particularly limited as long as it can dissolve the reactants and the product, and water is preferred. The reaction temperature is preferably 0 to 30 ℃.
C: deprotection of the silyl Ether
Compounds useful for such hydroxy protection include trimethylsilyl ether, triethylsilyl ether, dimethyl t-butylsilyl ether, t-butyldiphenylsilyl ether, and the like. The deprotection of the silicon ether is carried out by a compound containing fluoride ions, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid and potassium fluoride, and more preferably tetrabutylammonium fluoride and potassium fluoride. The amount of fluorine-containing agent used is 5 to 20 times, preferably 8 to 15 times, the molar equivalent of the protected hydroxyl group, and if the amount of fluorine is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection may result; when the amount of the deprotecting reagent is more than 20 times the molar equivalent of the protected hydroxyl group, an excessive amount of the reagent or the compound causes troubles in purification and may be incorporated into the subsequent step, thereby causing side reactions. The reaction solvent is not particularly limited as long as it can dissolve the reactants and the product, and is preferably an aprotic solvent, and more preferably tetrahydrofuran or dichloromethane. The reaction temperature is preferably 0 to 30 ℃ and when the temperature is lower than 0 ℃, the reaction speed is slow and the protecting group cannot be completely removed.
D: deprotection of the tert-butyl group
Deprotection of the t-butyl group is carried out under acidic conditions, and the solution pH is preferably 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid are preferable, and hydrochloric acid is more preferable. The reaction solvent is not particularly limited as long as it can dissolve the reactants and the product, and water is preferred. The reaction temperature is preferably 0 to 30 ℃.
In the terminal functionalization method, q is preferably 0, q1＝1，Z1Is 1, 2-methylene. When q is not 0, A and R01With a linking group such as an amino acid, succinyl group, etc., Z can be formed by the art2Or Z1(including but not limited to alkylation, condensation, click reactions, and the like), andthe preparation was carried out with reference to the linear functionalization scheme described below.
2.5.3. Alkylation reaction
The alkylation reaction according to the invention is preferably based on the alkylation of hydroxyl, mercapto or amino groups, which in turn corresponds to the formation of ether bonds, thioether bonds, secondary or tertiary amino groups. Examples are as follows:
2.5.3.1. alkylation of substrate alcohol with sulfonate and halide
In the presence of alkali, nucleophilic substitution of substrate alcohol, sulfonic acid ester derivative and halide to obtain amine intermediate. Wherein, the molar equivalent of the sulfonate and the halide is 1 to 50 times, preferably 1 to 5 times of that of the substrate alcohol. When the molar equivalent of the sulfonate or the halide is less than 1 time the molar equivalent of the substrate alcohol, the reaction substitution is incomplete and purification is difficult. When the molar equivalent of the sulfonate or the halide is more than 50 times of that of the substrate alcohol, an excessive amount of the reagent causes troubles in purification, and may be mixed in subsequent steps, thereby causing an increase in side reactions in the next step and increasing the difficulty in purification.
The resulting product is a mixture of the ether intermediate and excess sulfonate, halide, which can be purified by anion exchange resin, osmosis, ultrafiltration, and the like. The anion exchange resin is not particularly limited as long as the target product can be ion-exchanged and adsorbed on the resin, and is preferably a tertiary amine or quaternary ammonium salt ion exchange resin having a skeleton of dextran, agarose, polyacrylate, polystyrene, polystyrol, or the like. The solvent for the permeation and ultrafiltration is not limited, and may be water or an organic solvent, wherein the organic solvent is not particularly limited as long as the product can be dissolved therein, and dichloromethane, chloroform and the like are preferred.
The reaction solvent is not limited, but is preferably an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, and more preferably dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran.
The base includes an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or an inorganic base (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate or potassium hydroxide), preferably an organic base, more preferably triethylamine, pyridine. The molar amount of base is 1 to 50 times, preferably 1 to 10 times, more preferably 3 to 5 times the molar equivalent of sulfonate or halide.
2.5.3.2. Alkylation of substrate amine with sulfonate and halide
A. Alkylation of substrate amine with sulfonate and halide
In the presence of alkali, the nucleophilic substitution of substrate amine, sulfonic acid ester derivative and halide can obtain amine intermediate. Wherein, the molar equivalent of the sulfonate and the halide is 1 to 50 times, preferably 1 to 5 times of that of the substrate amine. When the molar equivalent of the sulfonate or halide is less than 1 time the molar equivalent of the substrate amine, the reaction substitution is incomplete and purification is difficult. When the molar equivalent of the sulfonate or the halide is more than 50 times of that of the substrate amine, the excessive reagent brings trouble to purification and may be mixed in subsequent steps, thereby causing increased side reactions in the next step and increasing the purification difficulty.
The resulting product is a mixture of the amine intermediate and excess sulfonate, halide, which can be purified by anion exchange resin, osmosis, ultrafiltration, and the like. The anion exchange resin is not particularly limited as long as the target product can be ion-exchanged and adsorbed on the resin, and is preferably a tertiary amine or quaternary ammonium salt ion exchange resin having a skeleton of dextran, agarose, polyacrylate, polystyrene, polystyrol, or the like. The solvent for the permeation and ultrafiltration is not limited, and may be water or an organic solvent, wherein the organic solvent is not particularly limited as long as the product can be dissolved therein, and dichloromethane, chloroform and the like are preferred.
The reaction solvent is not limited, but is preferably an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, and more preferably dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran.
The base includes an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or an inorganic base (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate or potassium hydroxide), preferably an organic base, more preferably triethylamine, pyridine. The molar amount of base is 1 to 50 times, preferably 1 to 10 times, more preferably 3 to 5 times the molar equivalent of sulfonate or halide.
2.5.3.3. Alkylation reaction of substrate amine and aldehyde derivative
After an imine intermediate is obtained by reacting substrate amine with an aldehyde derivative, the intermediate is obtained under the action of a reducing agent. Wherein the molar equivalent of the aldehyde derivative is 1 to 20 times, preferably 1 to 2 times, more preferably 1 to 1.5 times that of the substrate amine. When the molar equivalent of the aldehyde derivative is more than 20 times that of the substrate amine, an excessive amount of the reagent causes troubles in purification, may be incorporated into the subsequent step, and increases the difficulty of purification. When the molar equivalent of the aldehyde derivative is less than 1 time that of the substrate amine, the reaction is incomplete, increasing the difficulty of purification. Wherein, the product after the reaction can be purified by means of cation exchange resin, permeation, ultrafiltration and the like to obtain an intermediate. The cation exchange resin is not particularly limited as long as it can exchange with the quaternary ammonium cation to achieve a separation effect. The solvent for the permeation and ultrafiltration is not limited, and may be water or an organic solvent, wherein the organic solvent is not particularly limited as long as the product can be dissolved therein, and dichloromethane, chloroform and the like are preferred.
The reaction solvent is not limited, and is preferably an organic solvent such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide; more preferably water and methanol.
The reducing agent is not particularly limited as long as the imine can be reduced to an amine, and sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, Zn/AcOH and the like are preferred, and sodium cyanoborohydride is more preferred. The reducing agent is generally used in an amount of 0.5 to 50 times, more preferably 1 to 10 times, the amount of the aldehyde derivative substance.
2.5.4. Linear functionalization of the termini
The method of terminal linear functionalization is not particularly limited, in relation to the type of the final functional group or protected form thereof. Mainly including functionalization of the terminal hydroxyl group and conversion to the target functional group or protected form thereof based on reactive groups.
A method for preparing a functional group of class A to class J or a protected form thereof by functionalizing a functional group of class A to class J starting from a terminal hydroxyl group of A is described in paragraphs [0960] to [1205] of CN 104530417A. The reaction formula is as follows:
Wherein q and Z2、q1、Z1、R01The definitions of (a) and (b) are consistent with those described above.
Based on the conversion of the reactive group to the target functional group or protected form thereof, this can be achieved by any of the following means:
the first method is as follows: direct modification, based on direct modification of a reactive group, results in a functional group of interest or a protected form thereof. By way of example, such as the conversion of a carboxyl group to an acid halide, hydrazide, ester, thioester, dithioester, such as the conversion of a hydroxyl, thiol, alkynyl, amino, carboxyl, etc., to the corresponding protected structure, and the like. For example, the acid anhydride modifies a hydroxyl group, an amino group, or the like.
The second method comprises the following steps: the coupling reaction between two reactive groups uses a heterofunctionalizing agent containing 1 reactive group and a target functional group or a protected form thereof as a raw material, and introduces the target functional group or the protected form thereof through the reaction between one of the reactive groups and the reactive group at the A terminal. The reaction method and method between two reactive groups are not particularly limited, and the reaction conditions are dependent on the type of divalent linking group formed by the reaction, and the prior art can be applied. Such as alkylation, alkenyl addition reaction, alkynyl addition reaction, Schiff base reaction combined reduction reaction, condensation reaction and the like. Among these, the alkylation reaction is preferably a reaction based on the alkylation of a mercapto group or an amino group, which in turn corresponds to the formation of a thioether bond, a secondary amino group or a tertiary amino group. Wherein the condensation reaction includes, but is not limited to, a condensation reaction to form an ester group, a thioester group, an amide group, an imine linkage, a hydrazone linkage, a carbamate group, and the like. For example, the target functional group or the protected form thereof is introduced by click reaction using a group containing azide, alkynyl, alkenyl, trithiocarbonate, mercapto, dienyl, furyl, 12,4, 5-tetrazinyl, cyanate, etc. and a heterofunctionalizing agent of the target functional group or the protected form thereof as a raw material. The reaction between the two reactive groups is accompanied by the formation of a new bond, and typical examples of the newly formed divalent linking group are an amide bond, a urethane bond, an ester group, a secondary amine bond, a thioether bond, a triazole group, and the like.
The third method comprises the following steps: the target functional group or protected form thereof is obtained by a combination of direct modification and coupling reactions.
In the present invention, the raw materials used in the respective production methods may be obtained commercially or obtained by self-synthesis.
The intermediates and final products prepared in the present invention can be purified by purification methods including, but not limited to, extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, or supercritical extraction. The final product structure and molecular weight can be confirmed by characterization methods including but not limited to nuclear magnetism, electrophoresis, ultraviolet-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism method, etc.
3.1. Cationic liposome
In the present invention, a cationic liposome comprises any of the cationic lipids described above and having a structure represented by general formula (1).
In one embodiment of the present invention, the cationic liposome comprises one or more of neutral lipid, steroid lipid and pegylated lipid in addition to the cationic lipid having the structure represented by the general formula (1); more preferably, the lipid composition contains neutral lipid, steroid lipid and pegylated lipid. The neutral lipid is preferably a phospholipid
In a specific embodiment of the invention, the neutral lipids in the cationic liposomes preferably include, but are not limited to, 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-didecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1, 2-dilinolacyl-sn-glycero-3-phosphocholine, 1, 2-didodecaoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didecylhexaenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), Dioleoylphosphatidylserine (DOPS), Dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), Distearoylphosphatidylethanolamine (DSPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-Stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and Lysophosphatidylethanolamine (LPE), and combinations thereof.
In a specific embodiment of the present invention, the steroid lipid in the cationic liposome is preferably any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.
In a specific embodiment of the present invention, the pegylated lipid in the cationic liposome is preferably polyethylene glycol-1, 2 dimyristoyl glyceride (PEG-DMG), polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically including polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-1, 2-oleoylphosphatidylethanolamine, polyethylene glycol 2000-1, 2-oleoylphosphatidylethanolamine and polyethylene glycol 2000-2, any one of 3-dimyristoyl glycerol (PEG-DMG).
In one embodiment of the present invention, the structure of the pegylated lipid in the cationic liposome is preferably represented by the general formula (2):
Or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,
wherein L is7、L8Each independently is a bond or a divalent linking group selected from-O (C ═ O) -, - (C ═ O) O-, -O (C ═ O) O-, -C (═ O) -, -O-, -S-, -C (═ O) S-, -SC (═ O) -, -NR (r) ScC(＝O)-、-C(＝O)NRc-、 -NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-and-NRcAny one of C (═ O) S-, wherein RcEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
L3is a connecting bond, -L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5-or-L4-Z-L5-Z-; said L4、L5Are carbon chain linking groups, each independently is- (CR)aRb)t-(CRaRb)o-(CRaRb)p-, where t, o and p are each independently integers from 0 to 12 and are not 0 at the same time, RaAnd RbEach occurrence is independently a hydrogen atom or an alkyl group; each occurrence of Z is independently- (C ═ O) -, -O (C ═ O) -, - (C ═ O) O-, -S-, -C (═ O) S-, -SC (═ O) -, -NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、 -NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC (═ O) S-andany one of (1), wherein RcEach occurrence independently is a hydrogen atom or C1-12An alkyl group;
B3、B4each independently is a connecting bond or C1-12An alkylene group;
R1、R2each independently is C1-30An aliphatic hydrocarbon group;
r is hydrogen, alkyl, alkoxy, - (C ═ O) Rd、-(C＝O)ORd、-O(C＝O)Rd、-O(C＝O)ORdOrWherein R isdIs C1-12Alkyl radical, G1A terminal branched group having a valence of k +1, j is 0 or 1, F contains a functional group, and when j is 0, G 1In the absence of, when j is 1, G1K F is led out, and k is an integer of 2-8;
a is- (CR)aRb)sO-or-O (CR)aRb)s-, where s is 2, 3 or 4, RaAnd RbEach independently is a hydrogen atom or C1-12An alkyl group;
n1is an integer from 20 to 250;
the alkyl, alkylene, alkoxy, aliphatic hydrocarbon groups are each independently substituted or unsubstituted.
In a specific embodiment of the present invention, the structure of the pegylated lipid in the cationic liposome is selected from any one of the following structural formulas:
in a specific embodiment of the present invention, it is preferred that any one of the aforementioned cationic liposomes comprises 20 to 80% of the cationic lipid represented by formula (1), 5 to 15% of the neutral lipid, 25 to 55% of the steroid lipid, and 0.5 to 10% of the pegylated lipid, said percentages being mole percentages of the respective lipids based on total lipid in the solution comprising the solvent.
In a particular embodiment of the invention, preferably in any one of the aforementioned cationic liposomes, the cationic lipid comprises 30-65% by mole of the total lipid in the solution comprising the solvent; more preferably about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%.
In a particular embodiment of the invention, preferably in any one of the aforementioned cationic liposomes, the molar percentage of neutral lipids to total lipids in the solvent containing solution is 7.5-13%; more preferably about 8%, 9%, 10%, 11%, 12%.
In a particular embodiment of the invention, preferably in any one of the aforementioned cationic liposomes, the molar percentage of steroid lipids to total lipids in the solvent containing solution is between 35 and 50%, more preferably about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.
In a particular embodiment of the invention, preferably in any one of the aforementioned cationic liposomes, the mole percentage of pegylated lipid based on total lipid in the solvent containing solution is between 0.5 and 5%; preferably 1 to 3%; more preferably about 1.5%, 1.6%, 1.7%, 1.8%, 1.9%.
3.2. Preparation of cationic liposomes
In the present invention, the cationic liposome can be prepared by a method including, but not limited to, a thin film dispersion method, an ultrasonic dispersion method, a reverse phase evaporation method, a freeze-drying method, a freeze-thawing method, a multiple emulsion method and/or an injection method, preferably a thin film dispersion method, an ultrasonic dispersion method and/or a reverse phase evaporation method.
In the method for preparing the cationic liposome of the present invention, the thin film dispersion method may include the steps of:
(1) weighing cationic lipid, steroid lipid, neutral lipid and polyethylene glycol lipid, dissolving in organic solvent, shaking, removing organic solvent by rotary evaporation under reduced pressure to form oil film, and vacuum pumping to remove organic solvent;
(2) Adding phosphate buffer solution dissolved with cryoprotectant, and performing ultrasonic treatment in water bath to form semitransparent emulsion;
(3) adding the emulsion into a high-pressure homogenizer for overpressure, then adding the emulsion subjected to overpressure into a liposome extruder for membrane passing to form cationic liposome; and
(4) optionally, drying the cationic liposomes in a lyophilizer to form a cationic liposome powder;
wherein, preferably, the organic solvent is dichloromethane, trichloromethane and/or methanol, more preferably trichloromethane and methanol; preferably, the rotation speed of the reduced pressure rotary evaporation is 30-300 rpm, more preferably 50-200 rpm, and most preferably 100-170 rpm; preferably, the temperature of the reduced pressure rotary evaporation is 10-200 ℃, more preferably 20-100 ℃, and most preferably 40-80 ℃;
preferably, the vacuum pump is used for pumping for 1-72 hours, more preferably 5-48 hours, and most preferably 15-36 hours;
preferably, the mass concentration of the cryoprotectant dissolved in the phosphate buffer solution is 0.1-80%, preferably 1-50%, and more preferably 5-20%;
preferably, the frequency of the water bath ultrasound is 10-300, more preferably 30-200, and most preferably 60-150;
preferably, the time of the water bath ultrasound is 0.1-5 h, more preferably 0.2-2 h, and most preferably 0.25-1 h;
Preferably, the pressure of the high-pressure homogenizer is 50-240 MPa, more preferably 80-200 MPa, and most preferably 100-150 MPa;
preferably, the overpressure frequency of the high-pressure homogenizer is any integer between 1 and 50, more preferably between 3 and 20, and most preferably between 5 and 10;
preferably, the pressure of the liposome extruder is 50-300 MPa, more preferably 80-250 MPa, and most preferably 120-200 MPa;
preferably, the number of membrane passing times of the liposome extruder is any integer between 1 and 50, more preferably 3 to 30, and most preferably 5 to 20;
preferably, the drying time of the freeze dryer is 1-120 h, more preferably 5-72 h, and most preferably 10-36 h.
In the method for preparing the cationic liposome of the present invention, the cationic liposome: the phosphate buffer with cryoprotectant dissolved may be 1 mg: (0.1-100) mL, preferably 1 mg: (0.3-50) mL, more preferably 1 mg: (0.5-5) mL.
4.1. Cationic liposome nucleic acid pharmaceutical composition
In one embodiment of the present invention, a cationic liposome nucleic acid pharmaceutical composition comprises any one of the cationic liposomes described above and a nucleic acid drug, wherein the cationic liposome comprises any one of the cationic lipids described above and having a structure represented by general formula (1).
In a specific embodiment of the present invention, in the cationic liposome nucleic acid pharmaceutical composition, the nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, mRNA (messenger RNA), interfering nucleic acid, aptamer, m iRNA inhibitor (antagomir), micro RNA (mirna), ribozyme, and small interfering RNA (sirna); preferably any one of RNA, miRNA, and siRNA.
In a specific embodiment of the present invention, the cationic liposome nucleic acid pharmaceutical composition is preferably used as a medicament selected from any one of the following: a medicament for treating any one of cancer, malignancy, liver disease, hepatitis, diabetes, gout, rheumatism, rheumatoid, senile dementia, cardiovascular disease, an antiallergic drug, an anti-infective agent, an antibiotic agent, an antiviral agent, an antifungal agent, a vaccine, a central nervous system inhibitor, a central nervous system stimulant, a psychotropic drug, a respiratory tract drug, a peripheral nervous system drug, a drug that acts at a synaptic or neuroeffector junction, a smooth muscle active drug, a histaminergic agent, an antihistaminicergenic agent, a blood and hematopoietic drug, a gastrointestinal drug, a steroid agent, a cytostatic agent, an anthelmintic agent, an antimalarial agent, an antiprotozoal agent, an antimicrobial agent, an anti-inflammatory agent, an immunosuppressant agent, an Alzheimer's disease drug or compound, an imaging agent, an antidote, an antispasmodic, a muscle relaxant, an anti-inflammatory agent, an imaging agent, an anti-inflammatory agent, a cardiovascular agent, a pharmaceutical composition, a pharmaceutical, Appetite suppressants, migraine agents, muscle contractants, antimalarials, antiemetics/antiemetics, bronchodilators, antithrombotic agents, antihypertensive agents, antiarrhythmics, antioxidants, antiasthmatics, diuretics, lipid regulators, antiandrogens, antiparasitics, anticoagulants, neoplastic agents, hypoglycemic agents, nutritional agents, supplements, growth supplements, anti-enteritis agents, vaccines, antibodies, diagnostic agents, contrast agents, hypnotics, sedatives, psychostimulants, tranquilizers, anti-parkinson's disease agents, analgesics, anxiolytics, muscle infectives, and auditory disease agents, more preferably for the treatment of any of the following conditions: hemophilia, cystic fibrosis, familial hypercholesterolemia, breast cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, esophageal cancer, gastric cancer, carcinoma of large intestine, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, bone cancer, AIDS and viral infection.
In the present invention, it is further preferred that the drug includes, but is not limited to, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, mechlorethamine, thiotepa, chlorambucil, lacrimycin, melphalan, carmustine, lomustine, busulfan, dibromomannitol, mitomycin C, cisplatin (II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine, cocaine, chlorpromazine, propranolol, morroni, labetalol, clonidine, hydralazine, imipramine, amitriptyline, doxepin, phenytoin, diphenhydramine, chlorpheniramine, promethazine, daptomycin, ciprofloxacin, cefoxitin, vintin, vinblastine, cytosine arabinoside, and other drugs, Miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, netitifen, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives and imaging agents, taxol, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, dihydroxyanthracenedione, mithramycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycin, maytansinoids.
In one embodiment of the present invention, it is preferable that the N/P ratio of the cationic liposome to the nucleic acid is (0.1 to 100): 1, more preferably (0.2 to 30): 1, most preferably (0.5-20): 1;
in a specific embodiment of the present invention, preferably, the working solution of the nucleic acid pharmaceutical composition preparation is deionized water, ultrapure water, phosphate buffer or normal saline, more preferably phosphate buffer or normal saline, and most preferably normal saline; preferred cationic liposomes: (0.05-20) g of working solution: 100mL, more preferably (0.1 to 10) g: 100mL, most preferably (0.2-5) g: 100 mL.
5. Cationic liposome nucleic acid pharmaceutical composition preparation
In the present invention, a cationic liposome nucleic acid pharmaceutical composition preparation comprises the aforementioned cationic liposome nucleic acid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any one of deionized water, ultrapure water, a phosphate buffer solution and physiological saline, more preferably a phosphate buffer solution or physiological saline, and most preferably physiological saline.
In the invention, the preparation of the cationic liposome nucleic acid pharmaceutical composition preparation comprises the following steps:
(1) Equilibrating the cationic liposomes in the diluent or excipient;
(2) adding the nucleic acid medicine into the mixture of the balanced cationic liposome and the diluent or excipient for compounding;
wherein, the balancing time is preferably 0.1-12 h, preferably 0.2-6 h, and more preferably 0.5-3 h; preferably, the compounding time is 0.1-12 h, preferably 0.2-5 h, and more preferably 0.5-2 h.
The following examples are further described in order to illustrate the present invention in detail, and not to limit the scope of the present invention. In the examples where cationic lipids were prepared, the final product was characterized by nuclear magnetism for structure, or confirmed by MALDI-TOF for molecular weight.
Example 1: cationic lipid (E1-1)
Corresponding to general formula (1), E1-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. Total molecular weightAbout 782 Da.
The preparation process is as follows:
Step a: dissolving the compound 2-hexyldecanoic acid (S1-1, 100.00g, 390.6mmol) in anhydrous DCM (1L), placing in a nitrogen protected flask, after the temperature of the mixture is reduced to 0-10 ℃, carefully adding 1, 6-hexanediol (S1-2, 92.19g, 781.3mmol) and 4-dimethylaminopyridine (DMAP, 57.18 g, 468.7mmol) into the solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 82.56g, 430.0mmol) in batches, and returning the reaction solution to room temperature for further reaction. After 16h of reaction, TLC showed complete consumption of S1-1, the reaction solution was washed twice with 500mL of a 0.4N HCl/10% NaCl mixture and once with saturated brine, and the combined organic phases were MgSO 84Drying, filtering and concentrating to obtain a crude product. And separating and purifying the crude product by using a silica gel column, collecting target eluent, and concentrating to obtain a small molecular intermediate alcohol derivative S1-3(79.02 g).
Step b: the condensation product (S1-3, 50.02g, 140.5mmol) is dissolved in 500mL of a solution of LPCM, after the temperature of the solution is reduced to 0 ℃,2,6, 6-tetramethylpiperidine nitroxide (Tempo, 11.00mg) and a KBr solution (20.06g, 168.6mmol) are added to the solution, the solution is dissolved in 50mL of purified water, NaClO solution (182.6mmol) is slowly added dropwise, and after the dropwise addition is finished, TLC is used for tracking the raw materials until the consumption is finished. Then sodium sulfite solution is added to quench the reaction, the reaction solution is returned to room temperature and extracted twice by 500mL DCM, and the organic phases are combined and anhydrous MgSO 4Drying, filtering and concentrating to obtain a crude product. The crude product was separated and purified by a silica gel column, and the target eluate was collected and concentrated to give oxidized product S1-4(23.12 g).
Step c: dissolving the above oxidation product (S1-4, 20.00g, 56.5mmol) in a mixed solution of 200mL THF and 20mL methanol, cooling the above solution to 0 deg.C, adding diglycolamine (S1-5, 2.82g, 26.9mmol) and glacial acetic acid (1.61g, 26.9mmol) to the above solution, slowly adding sodium triacetoxyborohydride (NaBH (OAc))317.49g, 82.5mmol), after the addition is finished, the reaction is continued for 2h, and TLC tracking is carried out until the consumption of the raw materials is finished. After the reaction is finished, adding saturated sodium bicarbonate solution for quenchingThe reaction was allowed to come to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 200mL DCM, and the combined organic phases were over anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E1-1(12.09 g). The nuclear magnetic hydrogen spectrum of E1-1 mainly comprises the following data:1H NMR(400MHz,CDCl3) Delta.4.07 (t,4H),3.70(t,2H),3.63(t,4H),2.65(t,2H),2.49(m,4H),2.32(m,2H),1.71-1.22 (m,64H),0.87(t, 12H). The molecular weight of E1-1 was determined to be 781.76Da by MALDI-TOF testing.
Example 2: cationic lipid (E2-1)
Corresponding to general formula (1), E2-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3is-CH3. The total molecular weight was about 796 Da.
The preparation process is as follows:
step a: the oxidized product (S1-4, 10.00g, 28.2mmol) of example 1 was dissolved in a mixed solution of THF 100mL and 10mL of methanol, and after the temperature of the above solution was lowered to 0 deg.C, 1- (2-aminoethoxy) -2-methoxyethane (S2-1, 1.61g, 13.5mmol) and glacial acetic acid (0.81g, 13.5mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(9.86g, 47.1mmol), after the addition was complete, the reaction was continued for 2h with TLC follow-up until the starting material was consumed. After the reaction is completed, addingThe reaction was quenched with saturated sodium bicarbonate solution, the reaction was returned to room temperature, the THF and methanol were concentrated and removed, the concentrate was extracted twice with 100mL DCM, and the combined organic phases were over anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E2-1(5.93 g). The nuclear magnetic hydrogen spectrum of E2-1 mainly comprises the following data: 1H NMR(400MHz, CDCl3) Delta.4.07 (t,4H),3.71(t,2H),3.63(t,4H),3.30(s,3H),2.65(t,2H),2.49(m,4H),2.32 (m,2H),1.70-1.22(m,64H),0.89(t, 12H). The molecular weight of E2-1 was determined to be 795.71Da by MALDI-TOF testing.
Example 3: cationic lipid (E3-1)
Corresponding to general formula (1), E3-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sS-，RaAnd RbAre each a hydrogen atom, s is 2, n is 1, R3is-CH2CH2And (5) OH. The total molecular weight was about 782 Da.
The preparation process is as follows:
step a: the oxidized product (S1-4, 5.00g, 14.1mmol) of example 1 was dissolved in a mixed solution of 50mL of THF and 5mL of methanol, 2- (2-aminoethylmercapto) ethanol (S3-1, 0.81g, 6.7mmol) and glacial acetic acid (0.40g, 6.7mmol) were added to the above solution after the temperature of the above solution was lowered to 0 deg.C, and NaBH (OAc) was slowly added in portions3(5.00g, 23.6mmol), after the addition was complete, the reaction was continued for 2h, followed by TLC untilAnd (5) finishing the consumption of the raw materials. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 50mL DCM, the combined organic phases were extracted with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E3-1(2.64 g). The nuclear magnetic hydrogen spectrum of E3-1 mainly comprises the following data: 1H NMR(400MHz,CDCl3) Delta.4.06 (t, 4H),3.70(t,2H),2.75-2.45(m,10H),2.32(m,2H),1.70-1.22(m,64H),0.87(t, 12H). The molecular weight of E3-1 was determined to be 782.37Da by MALDI-TOF testing.
Example 4: cationic lipid (E4-1)
Corresponding to general formula (1), E4-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3is-CH2CH2-，B1、B2Are each hexamethylene, R1、R2Are all made ofA is-O (CR)aRb)s-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3is-OH. The total molecular weight was about 826 Da.
The preparation process is as follows:
step a: the oxidized product of example 1 (S1-4, 5.00g, 14.1mmol) was dissolved in a mixed solution of 50mL THF and 5mL methanol, after the temperature of the above solution was lowered to 0 deg.C, triethylene glycol amine (S4-1, 1.00g, 6.7mmol) and glacial acetic acid (0.40g, 6.7mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(5.00g, 23.6mmol), after the addition was complete, the reaction was continued for 2h, followed by TLC until the starting material was recoveredAnd (5) finishing the consumption. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 50mL DCM, the combined organic phases were extracted with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E4-1(3.15 g). The nuclear magnetic hydrogen spectrum of E4-1 mainly comprises the following data: 1H NMR(400MHz,CDCl3) Delta.4.08 (t,4H),3.81-3.58(m,10H), 2.65(t,2H),2.49(m,4H),2.32(m,2H),1.70-1.23(m,64H),0.88(t, 12H). The molecular weight of E4-1 was determined to be 825.73Da by MALDI-TOF testing.
Example 5: cationic lipid (E5-1)
Corresponding to general formula (1), E5-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3is-CH2CH2O-，B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3is-CH2CH2And (5) OH. The total molecular weight was about 869 Da.
The preparation process is as follows:
step a: the oxidized product of example 1 (S1-4, 5.00g, 14.1mmol) was dissolved in a mixed solution of 50mL THF and 5mL methanol, after the temperature of the above solution was lowered to 0 deg.C, tetraethylene glycol amine (S5-1, 1.29g, 6.7mmol) and glacial acetic acid (0.40g, 6.7mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(5.00g, 23.6mmol) and after the addition was complete, continuingThe reaction is carried out for 2h, and TLC tracking is carried out until the consumption of the raw materials is finished. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 50mL DCM, the combined organic phases were extracted with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E5-1(3.54 g). The nuclear magnetic hydrogen spectrum of E5-1 mainly comprises the following data: 1H NMR(400MHz,CDCl3) Delta.4.07 (t,4H),3.85-3.55(m,14H), 2.64(t,2H),2.49(m,4H),2.32(m,2H),1.70-1.22(m,64H),0.87(t, 12H). The molecular weight of E5-1 was determined to be 869.37Da by MALDI-TOF testing.
Example 6: cationic lipid (E6-1)
Corresponding to general formula (1), E6-1, wherein L1、L2Are identical and are all-O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 750 Da.
The preparation process is as follows:
step a: dissolving a compound 2-hexyldecanol (S6-1, 24.20g, 100.0mmol) in 250mL of THF solution, placing the solution in a nitrogen-protected flask, cooling the temperature of the mixture to 0-10 ℃, carefully adding sodium hydride (6.00g, 150.0mmol, 60%) into the solution, reacting for 0.5h, slowly dropwise adding (6-bromohexyloxy) -tert-butyldimethylsilane (S6-2, 26.46g, 90.0mmol), and returning the reaction solution to the reaction kettleThe reaction was continued at room temperature for 12 h. TLC showed almost complete consumption of S6-1, the reaction was quenched with 100mL of water, THF was concentrated and the concentrate was extracted twice with 100mL of DCM, and the combined organic phases were dried over anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give product S6-3(25.25 g).
Step b: the above substituted product (S6-3, 25.00g, 54.8mmol) was dissolved in 250mL THF solution, placed in a nitrogen blanketed flask, and tetrabutylammonium fluoride solution (TBAF, 110mL, 1N in THF) was added. The reaction was carried out for 1h, TLC showed complete consumption of starting material, the reaction was quenched with 100mL of water, THF was concentrated and the concentrate was extracted twice with 100mL of DCM, the combined organic phases were over anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was separated and purified by silica gel column, and the target eluate was collected and concentrated to give product S6-4(16.33 g).
Step c: the above condensation product (S6-4, 16.28g, 47.6mmol) was dissolved in 200mL of DCM, after the temperature of the above solution was lowered to 0 deg.C, Tempo (4.00mg) and KBr solution (0.29g, 2.4mmol) were added to the above solution, dissolved in 20mL of purified water, and NaClO solution (57.2mmol) was slowly added dropwise, after the addition was completed, TLC tracing was performed until the consumption of the starting material was completed. Then sodium sulfite solution is added to quench the reaction, the reaction solution is returned to room temperature and extracted twice with 200mL DCM, the organic phases are combined and anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was separated and purified by a silica gel column, and the objective eluent was collected and concentrated to obtain oxidized product S6-5(12.94g, 80%).
Step d: the above oxidized product (S6-5, 12.92g, 38.0mmol) was dissolved in a mixed solution of 150mL THF and 15mL methanol, and after the temperature of the above solution was lowered to 0 deg.C, S1-5(1.90g, 18.1mmol) in step a of example 1 and glacial acetic acid (1.09g, 18.1mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(13.44g, 63.4mmol), after the addition was complete, the reaction was continued for 2h and TLC followed until the starting material was consumed. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, the THF and methanol were concentrated to remove, and the concentrate was extracted with 200mL of DCMTaking twice, combining organic phases and using anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E6-1 (6.37 g). The nuclear magnetic hydrogen spectrum of E6-1 mainly comprises the following data:1H NMR(400MHz,CDCl3) Delta.3.84-3.55 (m, 14H),2.65(t,2H),2.48(m,4H),1.82-1.22(m,66H),0.86(t, 12H). The molecular weight of E6-1 was determined to be 753.96Da by MALDI-TOF testing.
Example 7: cationic lipid (E7-2)
Corresponding to general formula (1), E7-2, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a link key, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd R bAre each a hydrogen atom, s is 2, n is 2, R3Is composed ofThe total molecular weight was about 854 Da.
The preparation process is as follows:
step a: dissolving the compound 2-hexyloctanoic acid (S7-1, 100.00g, 438.6mmol) in anhydrous DCM (1L), placing in a nitrogen protected flask, cooling the temperature of the mixture to 0-10 ℃, carefully adding S1-2(92.19g, 781.3mmol) and DMAP (57.18g, 468.7mmol) in step a of example 1 into the solution, adding EDCI (82.56g, 430.0mmol) in portions, and returning the reaction solution to room temperature to continue the reaction. After 16h of reaction, TLC showed complete consumption of S7-1, and the reaction solution was diluted with 500mL of 0.4N HCl/10% NaClWashing the mixed solution twice, washing the mixed solution once with saturated brine, and washing the organic phase with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give product S7-2(84.90 g).
Step b: the condensation product (S7-2, 50.00g, 152.4mmol) is dissolved in 500mL of a solution of LPCM, after the temperature of the solution is reduced to 0 ℃, Tempo (11.00mg) and KBr solution (20.06 g, 168.6mmol) are added into the solution, the solution is dissolved in 50mL of purified water, NaClO solution (182.6mmol) is slowly added dropwise, and after the dropwise addition is finished, TLC tracking is carried out until the consumption of the raw materials is finished. Then sodium sulfite solution is added to quench the reaction, the reaction solution is returned to room temperature and extracted twice with 500mL DCM, the organic phases are combined and anhydrous MgSO 4Drying, filtering and concentrating to obtain a crude product. The crude product was separated and purified by a silica gel column, and the target eluate was collected and concentrated to give oxidized product S7-3(23.05 g).
Step c: the above oxidized product (S7-3, 20.00g, 61.3mmol) was dissolved in a mixed solution of 200mL THF and 20mL methanol, after the temperature of the above solution was lowered to 0 deg.C, the compound S7-4(21.73 g, 32.1mmol, S7-4 is the product obtained by coupling bis Fmoc-protected lysine with S1-5 in step c of example 1) and glacial acetic acid (1.61g, 26.9mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(17.49g, 82.5mmol), after the addition was complete, the reaction was continued for 2h and TLC followed until the starting material was consumed. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 200mL DCM, the combined organic phases were extracted with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E7-1 (24.00g) containing two Fmoc-protected amino groups. The nuclear magnetic hydrogen spectrum of E7-1 mainly comprises the following data:1H NMR(400MHz,CDCl3)δ:7.20-7.80(m, 16H)，4.37-4.45(d,4H),4.42(m,1H),4.25(t,2H),4.20(m,2H),4.07(t,4H),3.63(t,4H), 2.69(m,2H),2.66(t,2H),2.49(m,4H),2.32(m,2H),1.76-2.00(m,2H),1.72-1.22(m,56H), 1.24-1.66(m,4H),0.86(t,12H)。
step d: and removing the Fmoc protecting group. The concentrated product E7-1(10.00g, 7.7mmol) was treated with 20% piperidine/DMF solution, solvent removed by rotary evaporation, dissolved in dichloromethane, precipitated with dry ether, filtered and recrystallized from isopropanol to give cationic lipid E7-2(6.45g, 98.1%) containing two naked amino groups. The nuclear magnetic hydrogen spectrum of E7-2 mainly comprises the following data: 4.36(t, 1H). In the nuclear magnetic hydrogen spectrum, the characteristic peak of Fmoc disappears. The molecular weight of E7-2 was determined to be 853.85Da by MALDI-TOF testing.
Example 8: cationic lipid (E8-1)
Corresponding to general formula (1), E8-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3is-CH2CH2-，B1、B2Are each hexamethylene, R1、R2Are all made ofA is-S (CR)aRb)s-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3is-OH. The total molecular weight was about 718 Da.
The preparation process is as follows:
step a: dissolving compound 2-hexylnonanoic acid (S8-1, 100.00g, 537.6mmol) in anhydrous DCM (1L), placing in a nitrogen protected flask, cooling the mixture to 0-10 deg.C, adding S1-2(92.19g, 781.3mmol) and DMAP (57.18g, 468.7mmol) carefully in step a of example 1 into the solution, adding EDCI (82.56g, 430.0mmol) in portions, and returning the reaction solution to room temperature to continue the reaction. After 16h of reaction, TLC showed complete consumption of S8-1, the reaction solution was washed twice with 500mL of a 0.4N HCl/10% NaCl mixture and once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was separated and purified by silica gel column, and the target eluate was collected and concentrated to give product S8-2(87.35 g).
Step b: the condensation product (S8-2, 50.00g, 174.8mmol) is dissolved in 500mL of LDCM solution, after the temperature of the solution is reduced to 0 ℃, Tempo (11.00mg) and KBr solution (20.06 g, 168.6mmol) are added into the solution, the solution is dissolved in 50mL of purified water, NaClO solution (182.6mmol) is slowly added dropwise, and after the dropwise addition is finished, TLC tracking is carried out until the consumption of the raw materials is finished. Then sodium sulfite solution is added to quench the reaction, the reaction solution is returned to room temperature and extracted twice with 500mL DCM, the organic phases are combined and anhydrous MgSO 4Drying, filtering and concentrating to obtain a crude product. The crude product was separated and purified by a silica gel column, and the target eluate was collected and concentrated to give oxidized product S8-3(24.78 g).
Step c: the above oxidized product (S8-3, 20.00g, 70.4mmol) was dissolved in a mixed solution of 200mL of THF and 20mL of methanol, and after the temperature of the above solution was lowered to 0 deg.C, the above solution was added(S8-4, 6.41g, 35.4mmol) and glacial acetic acid (1.61g, 26.9mmol) were slowly added in portions NaBH (OAc)3(17.49g, 82.5mmol), after the addition was complete, the reaction was continued for 2h and TLC followed until the starting material was consumed. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 200mL DCM, the combined organic phases were extracted with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E8-1(11.79 g). The nuclear magnetic hydrogen spectrum of E8-1 mainly comprises the following data:1H NMR(400MHz, CDCl3) Delta.4.07 (t,4H),3.70(t,2H),2.81(m,4H),2.75-2.45(m,10H),2.32(m,2H),1.72-1.22 (m,44H),0.88(t, 12H). The molecular weight of E8-1 was determined to be 717.50Da by MALDI-TOF testing.
Example 9: cationic lipid (E9-2)
Corresponding to general formula (1), E9-2, wherein L1、L2Are identical and are each-O (C ═ O) O-, L3is-CH2CH2NHC(＝O)-， B1、B2Are all-CH2CH2-，R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 744 Da.
The preparation process is as follows:
step a: amino Fmoc protected N, N-diethyl-1, 3-propanediamine succinimide carbonate in a dry, clean 1000mL round-bottomed flask(S9-2, 96.54g, 233.2mmol) was dissolved in dichloromethane, DMAP (62.59g, 513.0mmol) was added thereto, and the mixture was stirred, 2-hexyloctanol S9-1(100.0 g, 467.3mmol) was added to the reaction mixture, and the reaction was stirred at room temperature for 16 hours. After completion of the reaction, the reaction mixture was spin-dried, recrystallized from isopropanol, and purified by ion exchange resin to obtain amino Fmoc-protected ethylamine derivative S9-3(127.86g, 93.9%).
Step b: deprotecting Fmoc-protected amino-containing S9-3, treating S9-3(50.00g, 85.6mmol) with 20% piperidine/DMF solution, removing solvent by rotary evaporation, dissolving with dichloromethane, cooling the obtained crude reaction solution to 0-10 ℃, and adding a compound into the reaction system under the protection of nitrogen(S9-4, 13.00 g, 52.4mmol) and TEA (11.49g, 77.1mmol), and the reaction solution was returned toThe reaction was carried out at room temperature overnight. TLC showed complete consumption of starting material, the reaction solution was quenched with 100mL of water, extracted twice with 100mL of DCM, and the combined organic phases were over anhydrous MgSO 4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E9-1(27.54 g). The nuclear magnetic hydrogen spectrum of E9-1 mainly comprises the following data:1H NMR(400MHz, CDCl3)δ:4.22(m,8H),3.75(t,2H),3.65(m,2H),3.59(t,2H),3.14(m,2H),2.65(t,2H), 2.60(t,2H),2.51(t,4H),2.32(m,2H),1.82-1.22(m,40H),0.98(m,9H),0.88(t,12H),0.21 (m,6H)。
step c: the above substituted product (E9-1, 20.00g, 23.2mmol) was dissolved in 250mL THF solution, placed in a nitrogen blanketed flask, and tetrabutylammonium fluoride solution (TBAF, 110mL, 1N in THF) was added. The reaction was carried out for 1h, TLC showed complete consumption of starting material, the reaction was quenched with 100mL of water, THF was concentrated and the concentrate was extracted twice with 100mL of DCM, the combined organic phases were over anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E9-2(15.14g, 87.7%).
In the nuclear magnetic hydrogen spectrum, the characteristic peak of TBS disappeared. The molecular weight of E9-2 was determined to be 744.52Da by MALDI-TOF testing.
Example 10: cationic lipid (E10-1)
Corresponding to general formula (1), E10-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a connecting bond, B1、B2Are all heptylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is composed ofThe total molecular weight was about 772 Da.
The preparation process is as follows:
Step a: the compound dipentylacetic acid (S10-1, 100.00g, 499.5mmol) is dissolved in anhydrous DCM (1L) solution, the solution is placed in a flask protected by nitrogen, after the temperature of the mixture is reduced to 0-10 ℃, 1, 7-heptanediol (S10-2, 132.00g, 999.1mmol) and DMAP (73.12g, 599.4mmol) are carefully added into the solution, EDCI (105.48g, 549.4mmol) is added in portions, and the reaction solution returns to room temperature for continuous reaction. After 16h of reaction, TLC showed complete consumption of S10-1, the reaction solution was washed twice with 500mL of a 0.4N HCl/10% NaCl mixture and once with saturated brine, and the combined organic phases were MgSO 84Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give product S10-3(89.20 g).
Step b: the condensation product (S10-3, 50.00g, 159.1mmol) is dissolved in 500mL of a solution of LPCM, after the temperature of the solution is reduced to 0 ℃, Tempo (12.40mg) and KBr solution (22.72 g, 190.9mmol) are added to the solution, the solution is dissolved in 50mL of purified water, NaClO solution (206.8mmol) is slowly added dropwise, and after the dropwise addition is finished, TLC tracking is carried out until the consumption of the raw materials is finished. Then sodium sulfite solution is added to quench the reaction, the reaction solution is returned to room temperature and extracted twice with 500mL DCM, the organic phases are combined and anhydrous MgSO 4Drying, filtering and concentrating to obtain a crude product. The crude product was separated and purified by silica gel column, and the target eluate was collected and concentrated to give oxidized product S10-4(25.90 g).
Step c: dissolving the above oxidation product (S10-4, 20.00g, 64.0mmol) in a mixed solution of 200mL THF and 20mL methanol, cooling the above solution to 0 deg.C, adding S10-5(5.50g, 30.7mmol) and glacial acetic acid (1.84g, 30.7mmol) into the above solution, and mixing S10-5 with S1-5And (3) reacting to obtain the compound. Slowly add NaBH (OAc) in portions3(19.80g, 93.4mmol), after the addition was complete, the reaction was continued for 2h and TLC followed until the starting material was consumed. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 200mL DCM, the combined organic phases were extracted with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E10-1(13.30 g). The nuclear magnetic hydrogen spectrum of E10-1 mainly comprises the following data:1H NMR(400 MHz,CDCl3) Delta.4.13 (m,4H),3.65-3.77(m,6H),3.48(d,4H),3.30(m,1H),3.01(m,4H), 2.51(t,2H),2.13(m,2H),1.60-1.26(m,52H),0.87(t, 12H). The molecular weight of E10-1 was determined to be 771.27Da by MALDI-TOF testing.
Example 11: cationic lipid (E11-2)
Corresponding to general formula (1), E11-2, wherein L1、L2Are identical and are each- (C ═ O) O-, L3is-CH2CH2O-，B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is composed ofThe total molecular weight was about 869 Da.
The preparation process is as follows:
step a: oxygen in example 1The product (S1-4, 10.00g, 28.2mmol) was dissolved in a mixed solution of 100mL THF and 10mL methanol, and after the temperature of the above solution was lowered to 0 deg.C, S11-1(3.11 g, 13.5mmol) and glacial acetic acid (0.81g, 13.5mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(10.00g, 47.1mmol), after the addition was complete, the reaction was continued for 2h, followed by TLC until the starting material was consumed. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 100mL DCM, the combined organic phases were extracted with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give amino Boc-protected cationic lipid E11-1 (6.31 g).
Step b: and removing the Boc protecting group. A solution of trifluoroacetic acid in dichloromethane (1:2, v/v) was prepared in a dry, clean 500mL round-bottomed flask, and a solution of E11-1(5.00g, 5.1mmol) in dichloromethane was slowly added dropwise under ice-bath conditions, followed by reaction at room temperature for 2 hours. The reaction solution was concentrated, purified water was added, extraction was performed with methylene chloride, the extract was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated, and recrystallization was performed to obtain cationic lipid E11-2(4.00g, 90.3%) having a naked amino group. The nuclear magnetic hydrogen spectrum of E11-2 mainly comprises the following data: 1H NMR(400MHz,CDCl3) Delta.4.05 (m,4H),3.71-3.57(m, 10H),3.49(t,2H),3.01(m,4H),2.85(t,2H),2.51(t,2H),2.13(m,2H),1.60-1.25(m,64H), 0.88(t, 12H). The molecular weight of E11-2 was determined to be 868.70Da by MALDI-TOF testing.
Example 12: cationic lipid (E12-1)
Corresponding to general formula (1), E12-1, R1，R2Are each tetradecyl, B1，B2Is a connecting bond, L1，L2Is a connecting bond, L3Is a connecting bond, A is-CH2CH2OCH2CH2NH-, n is 1, R3Is composed ofThe overall molecular weight was about 568D a.
The preparation process is as follows:
step a: compound S12-1(8.80g, 50.0mmol) and toluene (200mL) were azeotropically removed at 140 ℃ and 60mL of the solvent was distilled off, and then the reaction temperature was lowered to room temperature. Triethanolamine (TEA, 14.90g, 100.0mmol) and methylsulfonyl chloride (MsCl, 10.31g, 90.0mmol) were added and the reaction stirred at room temperature overnight. After the reaction was complete, the reaction was poured into water (200mL), extracted twice with EtOAc (100mL × 2), the aqueous phase was retained, the aqueous phase was extracted twice with dichloromethane (100mL × 2), the organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 ℃, recrystallized in ice bath, and filtered to give compound S12-2(11.68g, 92%).
Step b: the above-mentioned compound S12-2(10.00g, 39.4mmol) was added to 100mL of water, and dissolved with stirring at room temperature. Potassium carbonate (54.37g, 394.0mmol), the compound S12-3(41.96g, 197.0mmol) and tetra-n-butylammonium bromide (1.26g, 3.9mmol) were added to the solution, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, extraction was performed twice with dichloromethane (100mL × 2), the organic phases were combined, back-washed once with saturated aqueous sodium chloride solution (100mL), the organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S12-4. Purification by column chromatography, concentration and oil pump drainage gave the title compound S12-4(9.59 g).
Step c: compound S12-4(9.00g, 24.3mmol) was dissolved in dry THF (120mL), NaH (60%, 9.72g, 243.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. After completion of the reaction, compound S12-5(8.05g, 29.2mmol) was added thereto, and the mixture was stirred in an ice bath for 1 hour, after which the reaction mixture was slowly returned to room temperature and reacted overnight. After completion of the reaction, the reaction was placed in an ice bath, and 2mL of methanol was slowly added to quench the reaction, followed by stirring for 30 minutes, followed by addition of water (300mL) and mixing with stirring. Extracted twice with EtOAc (150 mL. times.2)The aqueous phase was retained, extracted twice with dichloromethane (100mL x 2), the organic phases were collected and combined, back washed once with saturated sodium chloride (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E12-1. Purification by column chromatography (DCM: MeOH ═ 15:1), concentration, and oil pump-drying afforded cationic lipid E12-1(10.24g, 75%).1H NMR(500MHz,CDCl3) Delta. 3.83-3.45(m,4H),2.64(m,4H),2.52(t,4H),2.42(t,4H),2.23 (s,6H),1.56-1.46(m,4H),1.26-1.19(m,44H),0.87(t, 6H). The molecular weight of E12-1 was determined to be 567.72Da by MALDI-TOF testing.
Example 13: cationic lipid (E13-1)
Corresponding to general formula (1), E13-1, R1Is tridecyl, R 2Is tetradecyl, B1，B2Is a connecting bond, L1Is a carbonyl group, L2Is a connecting bond, L3Is a connecting bond, A is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3is-CH2CHO. The total molecular weight was about 554 Da.
The preparation process is as follows:
step a: compound S13-1(7.40g, 50.0mmol) and toluene (200mL) were azeotropically removed at 140 ℃ and 60mL of the solvent was distilled off, and then the reaction temperature was lowered to room temperature. TEA (14.9g, 100.0mmol) and MsCl (10.31g, 90.0mmol) were added and the reaction stirred at room temperature overnight. After the reaction was completed, the reaction solution was poured into water (200mL), extracted twice with EtOAc (100mL × 2), the aqueous phase was retained, the aqueous phase was extracted twice with dichloromethane (100mL × 2), the organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 ℃, recrystallized in ice bath, and filtered to obtain compound S13-2(10.17g, 90%).
Step b: the above-mentioned compound S13-2(10.17g, 45.0mmol) was added to 250mL of water, and dissolved with stirring at room temperature. Potassium carbonate (62.10g, 450.0mmol), compound S12-3(47.93g, 225.0mmol) and tetra-n-butylammonium bromide (1.45g, 4.5mmol) were added to the solution, and the reaction solution was stirred at room temperature for 72 hours. After the reaction was completed, extraction was performed twice with dichloromethane (250mL × 2), the organic phases were combined, back-washed once with a saturated aqueous sodium chloride solution (250mL), the organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude compound S13-3. Purification by column chromatography, concentration and oil pump drainage gave the title compound S13-3(10.08 g).
Step c: the above-mentioned compound S13-3(10.00g, 29.2mmol), compound S13-4(11.39g, 35.0mmol) and TEA (6.53g, 43.8mmol) were dissolved in methylene chloride (100mL) and the reaction was stirred at room temperature overnight. After concentration, the reaction was dissolved in 100mL of water, extracted twice with EtOAc (100mL x 2), the aqueous phase was retained, sodium chloride was added, extracted twice with dichloromethane (100mL x 2), the organic phases were combined and back-washed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered and the filtrate was concentrated to give a crude product. Purification by column chromatography, concentration, and oil pump-drying gave cationic lipid E13-1(13.47g, 83.4%).1H NMR(500MHz,CDCl3) Delta 9.87(s,1H), 4.17(s,2H),3.83-3.45(m,6H),3.35(t,2H),3.16(t,2H),2.23(t,2H),1.56-1.46(m,4H), 1.26-1.19(m,42H),0.86(t, 6H). The molecular weight of E13-1 was determined to be 553.54Da by MALDI-TOF testing.
Example 14: cationic lipid (E14-1)
Corresponding to general formula (1), E14-1, R1Is tridecyl, R2Is tetradecyl, B1，B2Is a connecting bond, L1Is a carbonyl group, L2Is a connecting bond, L3Is a connecting bond, A is- (CR)aRb)sO-, in which RaAnd RbIs a hydrogen atom, s is 2, n is 2, R3Is composed ofThe total molecular weight was about 620 Da.
The preparation process is as follows:
step a: compound S14-1(10.70g, 50.0mmol) and toluene (200mL) were azeotropically removed at 140 ℃ and 60mL of the solvent was distilled off, and then the reaction temperature was lowered to room temperature. TEA (14.90g, 100.0mmol) and MsCl (10.31g, 90.0mmol) were added and the reaction stirred at room temperature overnight. After the reaction was completed, the reaction solution was poured into water (200mL), extracted twice with EtOAc (100mL × 2), the aqueous phase was retained, the aqueous phase was extracted twice with dichloromethane (100mL × 2), the organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 ℃, recrystallized in ice bath, and filtered to obtain compound S14-2(12.96g, 88.8%).
Step b: the above-mentioned compound S14-2(9.34g, 32.0mmol) was added to 100mL of water and dissolved with stirring at room temperature. Potassium carbonate (44.16g, 320.0mmol), compound S12-3(34.08g, 160.0mmol) and tetra-n-butylammonium bromide (1.03g, 3.2mmol) were added to the solution, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, extraction was performed twice with dichloromethane (100mL × 2), the organic phases were combined, back-washed once with saturated aqueous sodium chloride solution (100mL), the organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S14-3. Purification by column chromatography, concentration and oil pump drying gave the title compound S14-3(8.39g, 64.1%).
Step c: the above-mentioned compound S14-3(7.65g, 18.7mmol), compound S13-4(7.29g, 22.4mmol) and TEA (4.18g, 28.1mmol) were dissolved in methylene chloride (100mL) and the reaction was stirred at room temperature overnight. After concentration, the reaction was dissolved in 100mL of water, extracted twice with EtOAc (100mL x 2), the aqueous phase was retained, sodium chloride was added, extracted twice with dichloromethane (100mL x 2), the organic phases were combined and back-washed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered and the filtrate was concentrated to give a crude product. Purifying by column chromatography, concentrating, Oil pump-dried to give cationic lipid E14-1(9.56g, 82.6%).1H NMR(500MHz,CDCl3) Delta.3.83-3.45 (m,8H), 3.35(t,2H),3.16(t,2H),2.62-2.42(m,4H),2.34(t,2H),2.23(t,2H),2.00(s,1H),1.56-1.46 (m,4H),1.26-1.19(m,42H),0.87(t, 6H). The molecular weight of E14-1 was determined to be 619.15Da by MALDI-TOF testing.
Example 15: cationic lipid (E15-2)
Corresponding to general formula (1), E15-2, R1Is tetradecyl, R2Is tridecyl, B1，B2Is a connecting bond, L1Is a connecting bond, L2Is a carbonyl group, L3Is a connecting bond, A is- (CR)aRb)sO-, in which RaAnd RbIs a hydrogen atom, s is 2, n is 2, R3is-C (═ O) CH2CH2COOH. The overall molecular weight was about 612 Da.
The preparation process is as follows:
step a: compound S15-1(15.30g, 50.0mmol) and toluene (250mL) were azeotropically removed at 140 ℃ and 80mL of the solvent was distilled off, and the reaction temperature was lowered to room temperature. TEA (14.90g, 100.0mmol) and MsCl (10.31g, 90.0mmol) were added and the reaction stirred at room temperature overnight. After the reaction was complete, the reaction was poured into water (200 mL), extracted twice with EtOAc (100mL × 2), the aqueous phase was retained, the aqueous phase was extracted twice with dichloromethane (150mL × 2), the organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 ℃, recrystallized in ice bath, and filtered to give compound S15-2(16.88g, 87.9%).
Step b: the above-mentioned compound S15-2(10.00g, 26.0mmol) was added to 100mL of water, and dissolved with stirring at room temperature. Potassium carbonate (35.88g, 260.0mmol), compound S12-3(27.69g, 130.0mmol) and tetra-n-butylammonium bromide (0.84g, 2.6mmol) were added to the solution, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, extraction was performed twice with dichloromethane (200mL × 2), the organic phases were combined, back-washed once with a saturated aqueous sodium chloride solution (200mL), the organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude compound S15-3. Purification by column chromatography, concentration and oil pump drainage gave the title compound S15-3(8.64 g).
Step c: the above-mentioned compound S15-3(8.00g, 16.0mmol), compound S13-4(6.24g, 19.2mmol) and TEA (3.58g, 24.0mmol) were dissolved in methylene chloride (100mL) and the reaction was stirred at room temperature overnight. After concentration, the reaction was dissolved in 100mL of water, extracted twice with EtOAc (100mL x 2), the aqueous phase was retained, sodium chloride was added, extracted twice with dichloromethane (100mL x 2), the organic phases were combined and back-washed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered and the filtrate was concentrated to give a crude product. Purification by column chromatography, concentration and oil pump drying gave the title compound E15-1(9.52g, 83.6%).
Step d: and removing the Boc protecting group. A solution of trifluoroacetic acid in dichloromethane (1:2, v/v) was prepared in a dry, clean 500mL round-bottomed flask, and a solution of E15-1(9.52g, 13.4mmol) in dichloromethane was slowly added dropwise under ice-bath conditions, followed by reaction at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated, purified water was added thereto, and the mixture was extracted with methylene chloride, and the extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and recrystallized to obtain cationic lipid E15-2(7.53g, 92.1%).1H NMR(500MHz,CDCl3) Delta.4.17 (t,2H),3.71-3.60(m,4H),3.35(t,2H),3.16(t,2H), 2.70-2.47(m,4H),2.23(t,2H),1.56-1.46(m,4H),1.26-1.19(m,42H),0.87(t, 6H). The molecular weight of E15-2 was determined to be 611.51Da by MALDI-TOF testing.
Example 16: cationic lipid (E16-2)
Corresponding to general formula (1), E16-2, R1Is tridecyl, R2Is composed ofB1Is a connecting bond, B2Is hexamethylene, L1Is a carbonyl group, L2Is- (C ═ O) O-, L3is-CH2CH2-, A is-O (CR)aRb)s-, in which RaAnd RbIs a hydrogen atom, s is 2, n is 2, R3Is a hydroxyl group. The total molecular weight was about 712 Da.
The preparation process is as follows:
step a: dissolving the compound 2-heptyldecanoic acid (S16-1, 10.00g, 37.0mmol) in anhydrous DCM (100mL), placing in a nitrogen protected flask, after the temperature of the mixture is reduced to 0-10 ℃, carefully adding 1, 6-hexanediol (S1-2, 8.73g, 74.0mmol) and DMAP (5.42g, 44.4mmol) into the solution, adding EDCI (78.14g, 407.0mmol) in batches, and returning the reaction solution to room temperature to continue the reaction. After 16h of reaction, TLC showed complete consumption of S16-1, the reaction solution was washed twice with 500mL of a 0.4N HCl/10% NaCl mixture and once with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated to give a crude product. The crude product was separated and purified by silica gel column, and the target eluate was collected and concentrated to give product S16-2(8.10 g).
Step b: the condensation product (S16-2, 8.00g, 21.6mmol) was dissolved in 500mL of a solution of LDCM, after the temperature of the solution was lowered to 0 ℃, Tempo (1.20mg) and KBr solution (3.08g, 25.9mmol) were added to the solution, dissolved in 50mL of purified water, and NaClO solution (17.5mmol) was slowly added dropwise, after the addition was completed, and TLC tracing was performed until the consumption of the starting material was completed. Then sodium sulfite solution is added to quench the reaction, the reaction solution is returned to room temperature and extracted twice with 50mL DCM, the combined organic phases are dried with anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give oxidized product S16-3(3.98 g).
Step c: the oxidation product S16-3 (3.98)g, 10.8mmol) was dissolved in a mixed solution of 50mL of THF and 5mL of methanol, and after the temperature of the above solution was lowered to 0 deg.C, an amine derivative (S16-4, 2.58 g, 9.8mmol) and glacial acetic acid (0.58g, 9.8mmol) were added to the above solution, and NaHB (OAc) was slowly added in portions3(6.23g, 29.4 mmol), after the addition was complete, the reaction was continued for 2h, followed by TLC until the starting material was consumed. After the reaction was completed, a saturated sodium bicarbonate solution was added to quench the reaction, the reaction solution was returned to room temperature, THF and methanol were concentrated to remove, then the concentrate was extracted twice with 200mL of DCM, the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give final product S16-5(3.45 g).
Step d: the above-mentioned compound S16-5(3.45g, 5.6mmol), compound S16-6(1.66g, 6.7mmol) and TEA (1.25g, 8.4mmol) were dissolved in methylene chloride (100mL) and the reaction was stirred at room temperature overnight. After concentration, the reaction was dissolved in 100mL of water, extracted twice with EtOAc (100mL x 2), the aqueous phase was retained, sodium chloride was added, extracted twice with dichloromethane (100mL x 2), the organic phases were combined and back-washed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered and the filtrate was concentrated to give a crude product. Purification by column chromatography, concentration and oil pump drying gave the hydroxy TBS protected cationic lipid E16-1(3.91g, 84.5%).
Step e: the above product E16-1(3.91g, 4.7mmol) was dissolved in THF (50mL), placed in a nitrogen-blanketed flask, and tetrabutylammonium fluoride solution (TBAF, 2mL, 1N in THF) was added. After 1h of reaction, TLC shows complete consumption of the raw material, the reaction solution is quenched with 10mL of water, THF is concentrated and removed, then the concentrated solution is extracted twice with 10mL of DCM, the combined organic phases are dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give the product cationic lipid E16-2(2.92g, 87.5%). 1H NMR (500MHz,CDCl3) Delta.4.08 (t,2H),3.75-3.43(m,10H),3.35(t,2H),3.15(t,2H),2.26-2.25 (m,3H),1.60-1.46(m,10H),1.32-1.25(m,46H),0.88(t, 9H). The molecular weight of E16-2 was determined to be 711.64Da by MALDI-TOF testing.
Example 17: cationic lipid (E17-2)
Corresponding to general formula (1), E17-2, R1Is tetradecyl, R2Is composed ofB1Is a connecting bond, B2Is hexamethylene, L1Is a linking bond, L2Is- (C ═ O) O-, L3is-CH2CH2-, A is-O (CR)aRb)s-, in which RaAnd RbIs a hydrogen atom, s is 2, n is 2, R3Is a hydroxyl group. The total molecular weight was about 698 Da.
The preparation process is as follows:
step a: compound S16-5(3.45g, 5.6mmol) was dissolved in dry THF (120mL), NaH (60%, 2.24g, 56.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. After completion of the reaction, compound S12-5(1.85g, 6.7mmol) was added thereto, and the mixture was stirred in an ice bath for 1 hour, after which the reaction mixture was slowly returned to room temperature and reacted overnight. After completion of the reaction, the reaction was placed in an ice bath, and 2mL of methanol was slowly added to quench the reaction, followed by stirring for 30 minutes, followed by addition of water (300mL) and mixing with stirring. Extraction was performed twice with EtOAc (150mL x 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (100mL x 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E17-1. Purification by column chromatography, concentration and oil pump drying gave the title compound E17-1(3.44g, 75.8%).
Step b: the above substitution product E17-1(3.44g, 4.2mmol) was dissolved in THF (50mL), placed in a nitrogen-blanketed flask, and tetrabutylammonium fluoride solution (TBAF, 2mL, 1N in THF) was added. Reaction for 1hTLC showed complete consumption of starting material, the reaction was quenched with 10mL of water, THF was concentrated and the concentrate was extracted twice with 10mL of DCM, the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to give the crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E17-2(2.57g, 87.0%).1H NMR(500MHz,CDCl3) Delta.4.09 (t,2H),3.73-3.48(m,10H),2.51(t,4H),2.26-2.25(m, 3H),1.56-1.46(m,10H),1.32-1.25(m,48H),0.87(t, 9H). The molecular weight of E17-2 was determined to be 697.51Da by MALDI-TOF testing.
Example 18: cationic lipid (E18-1)
Corresponding to general formula (1), E18-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is composed ofThe total molecular weight was about 956 Da.
The preparation process is as follows:
the oxidized product of example 1 (S1-4, 8.85g, 25.0mmol) was dissolved in a mixed solution of 100mL THF and 10mL methanol, and after the temperature of the above solution was lowered to 0 deg.C, S18-1(3.35g, 12.0mmol) and glacial acetic acid (0.72g, 12.0mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions 3(8.90g, 42.0mmol), and after the addition was complete, the reaction was continued for 2hAnd tracking by TLC until the raw materials are completely consumed. After completion of the reaction, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with DCM (100mL × 2), the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to give a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E18-1(6.02 g).1H NMR(500 MHz,CDCl3) δ:4.26(s,2H),4.21(t,2H),4.07(t,4H),3.67(t,2H),3.52(t,2H),3.48(t,4H), 3.30(d,2H),2.63(t,2H),2.53(t,4H),2.24(m,2H),1.66-1.50(m,13H),1.43-1.17(m,56H), 0.87(t, 12H). The molecular weight of E18-1 was determined to be 955.61Da by MALDI-TOF testing.
Example 19: cationic lipid (E19-2)
Corresponding to general formula (1), E19-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is composed ofThe total molecular weight was about 882 Da.
The preparation process is as follows:
step a: the oxidized product (S1-4, 8.85g, 25.0mmol) of example 1 was dissolved in a mixed solution of 100mL of THF and 10mL of methanol, and after the temperature of the above solution was lowered to 0 deg.C, S19-1(2.46 g, 12.0 mmol) was added to the above solution ) And glacial acetic acid (0.72g, 12.0mmol), add NaBH (OAc) slowly in portions3(8.90g, 42.0mmol), after the addition was complete, the reaction was continued for 2h, followed by TLC until the starting material was consumed. After the reaction was completed, a saturated sodium bicarbonate solution was added to quench the reaction, the reaction solution was returned to room temperature, THF and methanol were concentrated to remove, then the concentrate was extracted twice with 100mL of DCM, the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give compound E19-1(5.48 g).
Step b: and removing the Boc protecting group. A solution of trifluoroacetic acid in dichloromethane (1:2, v/v) was prepared in a dry, clean 500mL round-bottomed flask, and a solution of E19-1(5.00g, 5.1mmol) in dichloromethane was slowly added dropwise under ice-bath conditions, followed by reaction at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated, purified water was added thereto, and the mixture was extracted with methylene chloride, and the extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and recrystallized to obtain cationic lipid E19-2(4.13g, 91.8%).1H NMR(500MHz,CDCl3) δ:4.21(t,2H),4.07(t,4H),3.64(t,2H),3.52(t,2H),3.20(t, 2H),3.06(t,2H),2.63(t,2H),2.52(t,4H),2.24(m,2H),1.87(m,2H),1.66-1.50(m,12H), 1.45-1.17(m,52H),0.88(t, 12H). The molecular weight of E19-2 was determined to be 881.78Da by MALDI-TOF testing.
Example 20: cationic lipid (E20-1)
Corresponding to general formula (1), E20-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is composed ofThe total molecular weight was about 923 Da.
The preparation process is as follows:
the oxidized product of example 1 (S1-4, 8.85g, 25.0mmol) was dissolved in a mixed solution of 100mL THF and 10mL methanol, and after the temperature of the above solution was lowered to 0 deg.C, S20-1(2.94g, 12.0mmol) and glacial acetic acid (0.72g, 12.0mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(8.90g, 42.0mmol), after the addition was complete, the reaction was continued for 2h, followed by TLC until the starting material was consumed. After the reaction was completed, a saturated sodium bicarbonate solution was added to quench the reaction, the reaction solution was returned to room temperature, THF and methanol were concentrated to remove, then the concentrate was extracted twice with 100mL of DCM, the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E20-1(6.00 g).1H NMR(500MHz, CDCl3) Delta.4.30 (t,2H),4.07(t,4H),3.67(t,2H),3.52(t,2H),2.86(t,4H),2.63(t,2H),2.53(t, 4H),2.25(m,2H),1.66-1.50(m,12H),1.45-1.17(m,52H),0.88(t, 12H). The molecular weight of E20-1 was determined to be 922.79Da by MALDI-TOF testing.
Example 21: cationic lipid (E21-1)
Corresponding to general formula (1), E21-1, wherein L1、L2Are identical and are each- (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is composed ofThe total molecular weight was about 933 Da.
The preparation process is as follows:
the oxidized product of example 1 (S1-4, 8.85g, 25.0mmol) was dissolved in a mixed solution of 100mL THF and 10mL methanol, and after the temperature of the above solution was lowered to 0 deg.C, S21-1(3.06g, 12.0mmol) and glacial acetic acid (0.72g, 12.0mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(8.90g, 42.0mmol), after the addition was complete, the reaction was continued for 2h, followed by TLC until the starting material was consumed. After the reaction was completed, a saturated sodium bicarbonate solution was added to quench the reaction, the reaction solution was returned to room temperature, THF and methanol were concentrated to remove, then the concentrate was extracted twice with 100mL of DCM, the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E21-1(5.65 g).1H NMR(500MHz, CDCl3) δ 6.49(s,2H),4.25-4.10(m,6H),3.62(t,2H),3.52(t,2H),3.15(t,1H),2.63(t, 2H),2.50(t,4H),2.32(t,2H),2.24(m,2H),1,89(m,2H),1.66-1.50(m,12H),1.45-1.17(m, 52H),0.88(t, 12H). The molecular weight of E21-1 was determined to be 932.79Da by MALDI-TOF testing.
Example 22: cationic lipid (E22-1)
Corresponding to general formula (1), E22-1, wherein L1、L2Are identical and are each-O (C ═ O) -, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 781 Da.
The preparation process is as follows:
step a: the compound 7-hydroxyheptanoic acid (S22-1, 14.60g, 100.0mmol) is dissolved in anhydrous DCM (200 mL) and placed in a nitrogen protected flask, after the temperature of the mixture is reduced to 0-10 ℃, 7-pentadecanol (S22-2, 45.60g, 200.0mmol) and DMAP (14.64g, 120.0mmol) are carefully added into the solution, EDCI (21.12g, 110.0mmol) is added in portions, and the reaction solution returns to room temperature for further reaction. After 16h of reaction, TLC showed complete consumption of S22-1, the reaction solution was washed twice with 100mL of a 0.4N HCl/10% NaCl mixture and once with saturated brine, and the combined organic phases were MgSO 84Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give small molecule intermediate alcohol derivative S22-3(20.79 g).
Step b: the above condensation product (S22-3, 20.00g, 56.2mmol) was dissolved in 200mL DCM solution, after the temperature of the above solution was lowered to 0 deg.C, Tempo (3.00mg) and KBr solution (8.02g, 67.4mmol) were added to the above solution, dissolved in 50mL purified water, NaClO solution (73.1mmol) was slowly added dropwise, and after the addition was completed, the starting material was followed by TLC until the end of consumption. Then sodium sulfite solution is added to quench the reaction, the reaction solution is returned to room temperature and extracted twice by 200mL DCM, and the organic phases are combined and anhydrous MgSO 4Drying, filtering and concentrating to obtain a crude product. The crude product was separated and purified by a silica gel column, and the target eluate was collected and concentrated to give oxidized product S22-4(9.59 g).
Step c: dissolving the above oxidation product (S22-4, 8.00g, 22.6mmol) in a mixed solution of 100mL THF and 10mL methanol, cooling the above solution to 0 deg.C, adding S1-5(1.19g, 11.3mmol) and glacial acetic acid (0.68g, 11.3mmol) to the above solution, and addingSlow addition in portions of NaBH (OAc)3(7.19g, 33.9mmol), after the addition was complete, the reaction was continued for 2h and TLC followed until the starting material was consumed. After the reaction was complete, the reaction was quenched by addition of saturated sodium bicarbonate solution, the reaction was returned to room temperature, THF and methanol were concentrated and removed, the concentrate was extracted twice with 100mL DCM, the combined organic phases were extracted with anhydrous MgSO4Drying, filtering and concentrating to obtain a crude product. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E22-1(4.76 g). The nuclear magnetic hydrogen spectrum of E22-1 mainly comprises the following data:1H NMR(400MHz,CDCl3) Delta.4.87 (m,2H),3.70(t,2H),3.63(t,4H), 2.65(t,2H),2.49(m,4H),2.30(t,4H),1.71-1.22(m,64H),0.87(t, 12H). The molecular weight of E22-1 was determined to be 781.42Da by MALDI-TOF testing.
Example 23: cationic lipid (E23-1)
Corresponding to general formula (1), E23-1, wherein L1、L2Are identical and are each-O (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 814 Da.
The preparation process is as follows:
step a: 6-bromohexyl-4-nitrophenyl carbonate (S23-1, 4.48g, 13.0mmol, where S23-1 was prepared from the reaction of p-nitrophenyl chloroformate and 6-bromohexanol) was dissolved in DCM (15mL) under nitrogen, S22-2(11.86g, 52.0mmol) was added dropwise with stirring at room temperature, followed by slow addition of pyridine (1.28mL, 16.25 mmol) over 10min, followed by the addition of DMAP (0.32g, 2.6mmol) in one portion. The reaction was stirred at room temperature for 16h, after completion of the reaction, extracted twice with DCM, the organic phases were combined and washed with brine, then dried over anhydrous magnesium sulphate, filtered and concentrated to give the crude product. The crude product was separated and purified by silica gel column, and the target eluate was collected and concentrated to give product S23-2 (5.86 g).
Step b: a solution of S23-2(5.56g, 12.8mmol), S1-5(0.60g, 5.8mmol) and N, N-dimethylethylenediamine (0.68g, 7.7mmol) in DMF (10mL) was reacted at 77 ℃ for 18 h. After the reaction was completed, the reaction mixture was cooled and extracted with hexane (3 × 10 mL). The hexane extracts were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give cationic lipid E23-1(3.67 g). The nuclear magnetic hydrogen spectrum of E23-1 mainly comprises the following data: 1H NMR(400MHz,CDCl3) Delta.4.21 (t,4H),4.12(m,2H),3.70(t, 2H),3.63(t,4H),2.65(t,2H),2.51(m,4H),1.72-1.25(m,64H),0.90(t, 12H). The molecular weight of E22-1 was determined to be 813.84Da by MALDI-TOF testing.
Example 24: cationic lipid (E24-1)
Corresponding to general formula (1), E24-1, wherein L1、L2Are identical and are each-O (C ═ O) O-, L3Is a connecting bond, B1、B2Are all made ofR1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre all hydrogenAtom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 814 Da.
The preparation process is as follows:
step a: 5-Bromopentyl-4-nitrophenyl carbonate (S24-1, 4.30g, 13.0mmol, where S24-1 was prepared from the reaction of p-nitrophenyl chloroformate and 5-bromo-n-pentanol) was dissolved in DCM (15mL) under nitrogen, 2-hexyldecanol (S24-2, 12.58g, 52.0mmol) was added dropwise with stirring at room temperature, followed by slow addition of pyridine (1.28 mL, 16.25mmol) over 10min, followed by the addition of DMAP (0.32g, 2.6mmol) in one portion. The reaction was stirred at room temperature for 16h, after completion of the reaction, extracted twice with DCM, the organic phases were combined and washed with brine, then dried over anhydrous magnesium sulphate, filtered and concentrated to give the crude product. The crude product was separated and purified by silica gel column, and the target eluate was collected and concentrated to give compound S24-3(6.14 g).
Step b: a solution of S24-3(5.56g, 12.8mmol), S1-5(0.60g, 5.8mmol) and N, N-dimethylethylenediamine (0.68g, 7.7mmol) in DMF (10mL) was reacted at 77 ℃ for 18 h. After the reaction was complete, the reaction mixture was cooled and extracted with hexane (3 × 10 mL). The hexane extracts were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give cationic lipid E24-1(3.58 g). The nuclear magnetic hydrogen spectrum of E24-1 mainly comprises the following data:1H NMR(400MHz,CDCl3)δ:4.26(d,4H),4.20(t,4H),3.71(t, 2H),3.64(t,4H),2.65(t,2H),2.50(m,4H),2.08(m,2H),1.72-1.18(m,60H),0.89(t,12H)。
the molecular weight of E24-1 was determined to be 813.67Da by MALDI-TOF testing.
Example 25: cationic lipid (E25-1)
Corresponding to general formula (1), E25-1, wherein L1、L2Are identical and are each-O (C ═ O) O-, L3Is a connecting bond, B1Is pentylene, B2Is heptylene, R1Is undecyl, R2Is composed ofA is- (CR)aRb)sO-， RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight is about 786Da
The preparation process is as follows:
step a: s24-2(10.59g, 32.0mmol) was dissolved in DCM (30mL) under nitrogen, 1-undecanol (S25-1, 22.02g, 128.0mmol) was added dropwise with stirring at room temperature, followed by slow addition of pyridine (3.2mL, 40.0mmol) over 25min, followed by DMAP (0.78g, 6.4mmol) in one portion. The reaction was stirred at room temperature for 16h, after completion of the reaction, extracted twice with DCM, the organic phases were combined and washed with brine, then dried over anhydrous magnesium sulphate, filtered and concentrated to give the crude product. The crude product was separated and purified by a silica gel column, and the target eluate was collected and concentrated to give 5-bromopentylundecyl carbonate (S25-2, 3.03 g).
Step b: the above product S25-2(2.91g, 8.0mmol), S1-5(1.01g, 9.6mmol) and N, N-dimethylethylenediamine (0.36g, 4.0mmol) were reacted in DMF (50mL) at 77 ℃ for 18 h. After the reaction was complete, the reaction mixture was cooled and extracted with hexane (3 × 50 mL). The hexane extracts were combined, dried over sodium sulfate, filtered and concentrated. It was combined with the 2 nd reaction. The crude product was purified by column chromatography on silica gel to give the product S25-3(2.33 g).
Step c: the above compound S25-4(2.33g, 6.0mmol) was dissolved in dry THF (120mL), NaH (60%, 2.40g, 60.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. Compound S25-4 (3.43g, 7.2mmol, S25-4 was obtained by reacting 7-bromoheptyl-4-nitrophenylcarbonate and 9-heptadecanol, as in referential example 22) was added, and after stirring in an ice bath for 1 hour, the reaction was slowly returned to room temperature overnight. After the reaction is finished, the reaction is placed in an ice bath, 2mL of methanol is slowly added to quench the reaction,after stirring for 30 minutes, water (100mL) was added and mixed with stirring. Extraction was performed twice with EtOAc (150mL x 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (100mL x 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E25-1. Purification by column chromatography, concentration and oil pump drying gave product E25-1(3.56 g). The nuclear magnetic hydrogen spectrum of E25-1 mainly comprises the following data: 1H NMR(400MHz, CDCl3) δ:4.22(t,2H),4.16(t,4H),4.13(m,1H),3.70(t,2H),3.62(t,4H),2.65(t,2H),2.48 (m,4H), 1.72-1.25(m,62H),0.90(t, 9H). The molecular weight of E25-1 was determined to be 785.66Da by MALDI-TOF testing.
Example 26: cationic lipid (E26-1)
Corresponding to general formula (1), E26-1, wherein L1、L2Are identical and are each-O (C ═ O) O-, L3Is a connecting bond, B1Is pentylene, B2Is hexamethylene, R1Is undecyl, R2Is composed ofA is- (CR)aRb)sO-， RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 786 Da.
The preparation process is as follows:
step a: compound S23-1 from example 23 (11.04g, 32.0mmol) was dissolved in DCM (30mL) under nitrogen, S26-1(34.56g, 128.0mmol) was added dropwise with stirring at room temperature, followed by slow dropwise addition of pyridine (3.2mL, 16.3mmol) over 25min, followed by the addition of DMAP (0.78g, 6.4mmol) in one portion. The reaction was stirred at room temperature for 16h, after completion of the reaction, extracted twice with DCM, the organic phases were combined and washed with brine, then dried over anhydrous magnesium sulphate, filtered and concentrated to give the crude product. Separating and purifying the crude product with silica gel column, collecting target eluate, and concentrating to obtain product S26-2(3.66g)
Step b: compound S25-3(2.33g, 6.0mmol) from example 25 was dissolved in dry THF (120 mL), NaH (60%, 2.40g, 60.0mmol) was added slowly under ice bath, and the reaction was carried out for 1 hour under ice bath. Compound S26-2(3.43g, 7.2mmol) was added thereto, and the mixture was stirred in an ice bath for 1 hour, and then the reaction was slowly returned to room temperature overnight. After completion of the reaction, the reaction was placed in an ice bath, quenched by slowly adding 2mL of methanol, stirred for 30 minutes, and then added with water (100mL) and mixed with stirring. Extraction was performed twice with EtOAc (150mL x 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (100mL x 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E26-1. Purification by column chromatography, concentration and oil pump drying gave product E26-1(3.57 g). The nuclear magnetic hydrogen spectrum of E26-1 mainly comprises the following data: 1H NMR(400 MHz,CDCl3) Delta.4.26 (d,2H),4.21(t,2H),4.16(t,4H),3.70(t,2H),3.63(t,4H),2.65(t,2H), 2.49(m,4H),2.07(m,1H),1.72-1.18(m,60H),0.90(t, 9H). The molecular weight of E26-1 was determined to be 785.67Da by MALDI-TOF testing.
Example 27: cationic lipid (E27-1)
Corresponding to general formula (1), E27-1, wherein L1is-O (C ═ O) -, L2is-O (C ═ O) O-, L3Is a connecting bond, B1Is pentylene, B2Is heptylene, R1Is undecyl, R2Is composed ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 770 Da.
The preparation process is as follows:
step a: a solution of compound S25-4(10.00g, 21.0mmol) from example 25, compound S1-5(1.84g, 17.5mmol) and N, N-dimethylethylenediamine (0.92g, 10.5mmol) in DMF (20mL) was reacted at 77 ℃ for 18 h. After the reaction was complete, the reaction mixture was cooled and extracted with hexane (3 × 20 mL). The hexane extracts were combined, dried over sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography on silica gel to give S27-1(7.54 g).
Step b: compound S27-1(5.68g, 12.0mmol) was dissolved in dry THF (60mL), NaH (60%, 4.80g, 120.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. Compound S27-2(5.01g, 14.4mmol, where S27-2 is prepared by reacting 8-bromooctanoic acid with 1-nonanol, as in example 16) was added, and the reaction was stirred in ice bath for 1 hour, and then slowly returned to room temperature for overnight reaction. After completion of the reaction, the reaction was placed in an ice bath, and 2mL of methanol was slowly added to quench the reaction, followed by stirring for 30 minutes, followed by addition of water (200mL) and mixing with stirring. Extraction was performed twice with EtOAc (200mL × 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (200mL × 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (200mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E27-1. Purification by column chromatography, concentration and oil pump drying gave cationic lipid E27-1(6.93 g). The nuclear magnetic hydrogen spectrum of E27-1 mainly comprises the following data: 1H NMR(400MHz,CDCl3) δ:4.21(t,2H),4.12(m,1H),4.07(t,2H),3.70(t,2H),3.63(t,4H),2.65(t,2H),2.51(m,4H), 2.37(t,2H),1.72-1.25(m,62H),0.90(t, 9H). The molecular weight of E27-1 was determined to be 769.69Da by MALDI-TOF testing.
Example 28: cationic lipid (E28-1)
Corresponding to general formula (1), E28-1, wherein L1Is- (C ═ O) O-, L2is-O (C ═ O) O-, L3Is a connecting bond, B1Is pentylene, B2Is heptylene, R1Is undecyl, R2Is composed ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 770 Da.
The preparation process is as follows:
compound S27-1(5.68g, 12.0mmol) was dissolved in dry THF (60mL), NaH (60%, 4.80g, 120.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. Compound S28-1(5.01 g, 14.4mmol, where S28-1 is prepared by reacting lauric acid with 5-bromo-1-pentanol as in example 16) was added, and after stirring the reaction for 1 hour in ice bath, the reaction was slowly returned to room temperature overnight. After completion of the reaction, the reaction was placed in an ice bath, and 2mL of methanol was slowly added to quench the reaction, followed by stirring for 30 minutes, followed by addition of water (200mL) and mixing with stirring. Extraction was performed twice with EtOAc (200mL × 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (200mL × 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (200mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E28-1. Purification by column chromatography, concentration and oil pump drying gave cationic lipid E28-1(6.87 g). The nuclear magnetic hydrogen spectrum of E28-1 mainly comprises the following data: 1H NMR(400MHz,CDCl3) δ:4.20 (t,2H),4.12(m,1H),4.08(t,2H),3.70(t,2H),3.63(t,4H),2.64(t,2H),2.50(m,4H),2.37(t, 2H),1.72-1.25(m,62H),0.88(t, 9H). The molecular weight of E28-1 was determined to be 769.58 Da by MALDI-TOF testing.
Example 29: cationic lipid (E29-1)
Corresponding to general formula (1), E29-1, wherein L1is-O (C ═ O) O-, L2is-O (C ═ O) -, L3Is a connecting bond, B1Is pentylene, B2Is heptylene, R1Is undecyl, R2Is composed ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 770 Da.
The preparation process is as follows:
compound S25-3(4.67g, 12.0mmol) was dissolved in dry THF (60mL), NaH (60%, 4.80g, 120.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. Compound S29-1(6.62 g, 14.4mmol, where S29-1 is prepared by reacting 8-bromooctanoic acid with 9-heptadecanol, as in example 16) was added, and the reaction was stirred in ice bath for 1 hour and then slowly returned to room temperature for overnight reaction. After completion of the reaction, the reaction was placed in an ice bath, and 2mL of methanol was slowly added to quench the reaction, followed by stirring for 30 minutes, followed by addition of water (200mL) and mixing with stirring. Extraction was performed twice with EtOAc (200mL × 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (200mL × 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (200mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E29-1. Purification by column chromatography, concentration and oil pump drying gave cationic lipid E29-1(6.52 g). The nuclear magnetic hydrogen spectrum of E29-1 mainly comprises the following data: 1H NMR(400MHz,CDCl3) δ:4.86 (m,1H),4.16(t,4H),3.70(t,2H),3.62(t,4H),2.65(t,2H),2.48(m,4H),2.30(t,2H), 1.72-1.25(m,62H),0.88(t, 9H). Determination of E29 by MALDI-TOF testMolecular weight of-1 is 769.47 Da.
Example 30: cationic lipid (E30-1)
Corresponding to general formula (1), E30-1, wherein L1is-O (C ═ O) O-, L2Is- (C ═ O) O-, L3Is a connecting bond, B1Is pentylene, B2Is heptylene, R1Is undecyl, R2Is composed ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 770 Da.
The preparation process is as follows:
compound S25-3(4.67g, 12.0mmol) was dissolved in dry THF (60mL), NaH (60%, 4.80g, 120.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. Compound S30-1(6.62 g, 14.4mmol, where S30-1 is prepared by reacting 2-octyldecanoic acid with 7-bromo-1-heptanol as described in example 16) was added, and the reaction was stirred in ice bath for 1 hour and then allowed to slowly return to room temperature overnight. After completion of the reaction, the reaction was placed in an ice bath, and 2mL of methanol was slowly added to quench the reaction, followed by stirring for 30 minutes, followed by addition of water (200mL) and mixing with stirring. Extraction was performed twice with EtOAc (200mL × 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (200mL × 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (200mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E30-1. Purification by column chromatography, concentration and oil pump drying gave cationic lipid E30-1(6.79 g). The nuclear magnetic hydrogen spectrum of E30-1 mainly comprises the following data: 1H NMR(400MHz,CDCl3) δ:4.16(t,4H),4.08(t,2H),3.70(t,2H),3.62(t,4H),2.65(t,2H),2.48(m,4H),2.25(m,1H), 1.72-1.25(m,62H),0.88(t, 9H). The molecular weight of E30-1 was determined to be 769.38Da by MALDI-TOF testing.
Example 31: cationic lipid (E31-1)
Corresponding to general formula (1), E31-1, wherein L1Is- (C ═ O) O-, L2is-O (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 798 Da.
The preparation process is as follows:
step a: a solution of compound S23-2 from example 23 (3.47g, 8.0mmol), S1-5(0.70g, 6.7mmol) and N, N-dimethylethylenediamine (0.36g, 4.0mmol) in DMF (50mL) was reacted at 77 ℃ for 18 h. After the reaction was complete, the reaction mixture was cooled and extracted with hexane (3 × 50 mL). The hexane extracts were combined, dried over sodium sulfate, filtered and concentrated. It was combined with the 2 nd reaction. The crude product was purified by column chromatography on silica gel to give the product S31-1(2.70 g).
Step b: compound S31-1(2.75g, 6.0mmol) was dissolved in dry THF (120mL), NaH (60%, 2.40g, 60.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. Adding compound S31-2 (3.01g, 7.2mmol, S31-2 is obtained by reacting 6-bromo-n-hexanol and 2-hexyldecanoic acid, the reaction process can refer to example 16), stirring under ice bath for 1 hour, slowly returning to room temperature, and reacting Should be allowed to stand overnight. After completion of the reaction, the reaction was placed in an ice bath, quenched by slowly adding 2mL of methanol, stirred for 30 minutes, and then added with water (100mL) and mixed with stirring. Extraction was performed twice with EtOAc (150mL x 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (100mL x 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E31-1. Purification by column chromatography, concentration and oil pump drying gave cationic lipid E31-1(3.52 g). The nuclear magnetic hydrogen spectrum of E31-1 mainly comprises the following data:1H NMR(400MHz,CDCl3) δ:4.21 (t,2H),4.12(m,1H),4.08(t,2H),3.70(t,2H),3.63(t,4H),2.65(t,2H),2.51(m,4H),2.25 (m,1H),1.72-1.25(m,64H),0.90(t, 12H). The molecular weight of E31-1 was determined to be 797.72Da by MALDI-TOF testing.
Example 32: cationic lipid (E32-1)
Corresponding to general formula (1), E32-1, wherein L1is-O (C ═ O) -, L2is-O (C ═ O) O-, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO-，RaAnd RbAre each a hydrogen atom, s is 2, n is 2, R3Is a hydrogen atom. The total molecular weight was about 798 Da.
The preparation process is as follows:
compound S31-1(2.75g, 6.0mmol) from example 31 was dissolved in dry THF (120mL), NaH (60%, 2.40g, 60.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. Adding compound S32-1(3.01g, 7.2mmol, S32-1 is composed of 7-bromoheptanoic acid and 7-pentadecanoic acid) Alkanol reaction, the reaction process can refer to example 16), stirring reaction under ice bath for 1 hour, then slowly returning to room temperature for reaction overnight. After completion of the reaction, the reaction was placed in an ice bath, quenched by slowly adding 2mL of methanol, stirred for 30 minutes, and then added with water (100mL) and mixed with stirring. Extraction was performed twice with EtOAc (150mL x 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (100mL x 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E32-1. Purification by column chromatography, concentration and oil pump drying gave cationic lipid E32-1(3.63 g). The nuclear magnetic hydrogen spectrum of E32-1 mainly comprises the following data:1H NMR(400MHz,CDCl3) δ:4.87(m,1H),4.21(t,2H),4.13(m,1H),3.70(t,2H),3.63(t,4H),2.65(t,2H),2.51(m, 4H),2.30(t,2H),1.72-1.25(m,64H),0.88(t, 12H). The molecular weight of E32-1 was determined to be 797.72Da by MALDI-TOF testing.
Example 33: cationic lipid (E33-1)
Corresponding to general formula (1), E33-1, wherein L1、L2Are identical and are all ester bonds, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO(CRaRb)sNRc，Ra、RbAnd RcAre each a hydrogen atom, s is 2, n is 1, R3Is composed ofThe total molecular weight was about 1204 Da.
The preparation process is as follows:
step a: the oxidized product of example 1 (S1-4, 8.85g, 25.0mmol) was dissolved in a mixed solution of 100mL THF and 10mL methanol, and after the temperature of the above solution was lowered to 0 deg.C, S1-5(1.25g, 12.0mmol) and glacial acetic acid (0.72g, 12.0mmol) were added to the above solution, and NaBH (OAc) was slowly added in portions3(8.90g, 42.0mmol), after the addition was complete, the reaction was continued for 2h, followed by TLC until the starting material was consumed. After the reaction was completed, a saturated sodium bicarbonate solution was added to quench the reaction, the reaction solution was returned to room temperature, THF and methanol were concentrated to remove, then the concentrate was extracted twice with 100mL of DCM, the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column, and the target eluate was collected and concentrated to give product S33-1(4.89 g).
Step b: in a dry clean 1000mL round bottom flask, folic acid (S33-2, 3.75 g, 8.5mmol) dissolved in DMSO, S33-1(3.90g, 5.0mmol), pyridine (40mL) and DCC (4.64g, 22.5mmol) were added and the reaction stirred at room temperature for 4 h. After the reaction is finished, the pyridine is removed by vacuum concentration through a rotary evaporator, then the concentrated solution is extracted twice by 100mL DCM, the combined organic phase is dried by anhydrous magnesium sulfate, and the crude product is obtained by filtration and concentration. The crude product was purified by silica gel column separation, and the target eluate was collected and concentrated to give cationic lipid E33-1(5.59g, 92.8%). 1H NMR(500MHz,CDCl3) Δ:8.68(s,1H),7.68(d,1H),6.67(d,1H),4.51(d,2H),4.36(m, 1H),4.06(m,4H),3.83-3.45(m,6H),2.63(t,2H),2.53(t,4H),2.34(t,2H),2.18(m,2H), 2.08-1.94(m,2H),1.56-1.46(m,8H),1.36-1.19(m,56H),0.88(t, 12H). The molecular weight of E33-1 was determined to be 1203.87Da by MALDI-TOF testing.
Example 34: cationic lipid (E34-1)
Corresponding to general formula (1), E34-1, wherein L1、L2Are identical and are all ester bonds, L3Is a connecting bond, B1、B2Are each hexamethylene, R1、R2Are all made ofA is- (CR)aRb)sO(CRaRb)sNRc，Ra、RbAnd RcAre each a hydrogen atom, s is 2, n is 1, R3Is composed ofThe overall molecular weight was about 1006 Da.
The preparation process is as follows:
in a dry clean 1000mL round bottom flask, biotin succinimidyl ester (S34-1, 1.88g, 5.5mmol) in DMF and S33-1(3.90g, 5.0mmol) in DCM were added, mixed well before triethylamine (2.3mL, 16.5mmol) was added and the reaction was monitored by TLC at room temperature until complete. After the reaction, filtration, concentration, column purification, collection of combined eluates, concentration and lyophilization gave cationic lipid E34-1(3.65g, 72.5%).1H NMR(CDCl3) δ:4.18(s,2H),4.06(m,4H),3.84-3.55(m,6H),3.27(m,1H),2.83(d,2H), 2.63(t,2H),2.52(t,4H),2.22-2.18(m,4H),1.70-1.40(m,12H),1.36-1.19(m,58H),0.87(t, 12H). The molecular weight of E34-1 was determined to be 1006.62Da by MALDI-TOF testing.
Example 35: preparation of cationic liposomes
In this example, 5 groups of cationic liposomes were prepared for comparison, two of which were positive control groups, three of which were experimental groups, and corresponding blank control groups were also provided as necessary.
The composition of the 5 groups of cationic liposomes contained neutral lipids as DSPC and sterol lipids as cholesterol, which are different from cationic lipids and pegylated lipids, wherein the ratio of positive control group 1: contains polyethylene glycol lipid PEG-DMG and no cationic lipid; positive control group 2: the cationic lipid was DLin-MC3-DMA and contained the pegylated lipid PEG-DMG, panel 1: the cationic lipid is P-9, and the pegylated lipid is PEG-DMG; experimental group 2: the cationic lipid is P-10, the pegylated lipid is PEG-DMG, and the ratio of experimental group 3: the cationic lipid is P-9 of the present invention and contains the pegylated lipid E35-1 of the present invention.
Example 35.1 preparation of cationic Liposome 1 (Positive control 1)
Step a: distearoylphosphatidylcholine (DSPC, 3.0. mu. mol), cholesterol (12.0. mu. mol) and PEG-conjugated 1, 2-dimyristoyl-sn-glycerol (PEG2k-DMG,0.45 μmol) into a 100mL round-bottom flask, adding 30mL of chloroform to fully dissolve the solid, and shaking up;
Step b: performing reduced pressure rotary evaporation to remove chloroform solvent by using a rotary evaporator under the conditions that the rotating speed is 140rpm and the temperature is 55 ℃ to form a thin-layer oil film, and performing vacuum pumping for 12 hours by using a vacuum pump to ensure that all chloroform is removed;
step c: adding 30mL of phosphate buffer solution (PBS, pH 7.4) containing 10% lactose into the flask, and performing ultrasonic treatment for 30min at 90% frequency by using an ultrasonic cleaning machine to form semitransparent emulsion;
step d: adding the emulsion into a high-pressure homogenizer, and carrying out overpressure for 5 times under the condition that the pressure is 100 MPa; and then adding the emulsion into a liposome extruder, carrying out overpressure for 10 times under the condition of 150MPa of pressure to prepare the cationic liposome 1, and carrying out freeze drying for 24 hours to obtain cationic liposome 1 powder.
Example 35.2 preparation of cationic Liposome 2 (Positive control 2)
Step a: weighing cationic lipid DLin-MC3-DMA (15.0. mu. mol), distearoyl phosphatidylcholine (DSPC, 3.0. mu. mol), cholesterol (12.0. mu. mol), and PEG2k-DMG (0.45. mu. mol) in a 100mL round-bottomed flask, 30mL of chloroform was added to dissolve the solid thoroughly, and the mixture was shaken well;
step b: performing reduced pressure rotary evaporation to remove chloroform solvent by using a rotary evaporator under the conditions that the rotating speed is 140rpm and the temperature is 55 ℃ to form a thin-layer oil film, and performing vacuum pumping for 12 hours by using a vacuum pump to ensure that all chloroform is removed;
Step c: adding 30mL of phosphate buffer solution (PBS, pH 7.4) containing 10% lactose into the flask, and performing ultrasonic treatment for 30min at 90% frequency by using an ultrasonic cleaning machine to form semitransparent emulsion;
step d: adding the emulsion into a high-pressure homogenizer, and carrying out overpressure for 5 times under the condition that the pressure is 100 MPa; and then adding the emulsion into a liposome extruder, carrying out overpressure for 10 times under the condition of 150MPa of pressure to prepare the cationic liposome 2, and carrying out freeze drying for 24 hours to obtain cationic liposome 2 powder.
Example 35.3 preparation of cationic Liposome 3 (Experimental group 1)
Step a: weighing the cationic lipid P-9(15.0 mu mol), distearoyl phosphatidylcholine (DSPC, 3.0 mu mol), cholesterol (12.0 mu mol) and PEG2k-DMG (0.45 mu mol) of the invention into a 100mL round-bottom flask, adding 30mL of chloroform to fully dissolve the solid, and shaking up;
step b: performing reduced pressure rotary evaporation to remove chloroform solvent by using a rotary evaporator under the conditions that the rotating speed is 140rpm and the temperature is 55 ℃ to form a thin-layer oil film, and performing vacuum pumping for 12 hours by using a vacuum pump to ensure that all chloroform is removed;
step c: adding 30mL of phosphate buffer solution (PBS, pH 7.4) containing 10% lactose into the flask, and performing ultrasonic treatment for 30min at 90% frequency by using an ultrasonic cleaning machine to form semitransparent emulsion;
Step d: adding the emulsion into a high-pressure homogenizer, and carrying out overpressure for 5 times under the condition that the pressure is 100 MPa; and then adding the emulsion into a liposome extruder, carrying out overpressure for 10 times under the condition of 150MPa of pressure to prepare cationic liposome 3, and carrying out freeze drying for 24 hours to obtain cationic liposome 3 powder.
Example 35.4 preparation of cationic Liposome 4 (Experimental group 2)
Step a: weighing cationic lipid P-10 (15.0. mu. mol), distearoyl phosphatidylcholine (DSPC, 3.0. mu. mol), cholesterol (12.0. mu. mol) and PEG2k-DMG (0.45. mu. mol) into a 100mL round-bottomed flask, adding 30mL of chloroform to dissolve the solid sufficiently, and shaking up;
step b: performing reduced pressure rotary evaporation to remove chloroform solvent by using a rotary evaporator under the conditions that the rotating speed is 140rpm and the temperature is 55 ℃ to form a thin-layer oil film, and performing vacuum pumping for 12 hours by using a vacuum pump to ensure that all chloroform is removed;
step c: adding 30mL of phosphate buffer solution (PBS, pH 7.4) containing 10% lactose into the flask, and performing ultrasonic treatment for 30min at 90% frequency by using an ultrasonic cleaning machine to form semitransparent emulsion;
step d: adding the emulsion into a high-pressure homogenizer, and carrying out overpressure for 5 times under the condition that the pressure is 100 MPa; and then adding the emulsion into a liposome extruder, carrying out overpressure for 10 times under the condition of 150MPa of pressure to prepare cationic liposome 4, and carrying out freeze drying for 24 hours to obtain cationic liposome 4 powder.
Example 35.5 preparation of cationic Liposome 5 (Experimental group 3)
Example 35.5.1 preparation of Pegylated lipid E35-1
Corresponding to the general formula (2), E35-1, R1、R2Is tetradecyl, B3、B4Is a connecting bond, L7、L8Is a connecting bond, L3is-CH2CH2O-or-CH2CH2-, A is-OCH2CH2-or-CH2CH2O-, wherein RaAnd RbIs a hydrogen atom, s is 2, n1About 45, R is methyl or methoxyl, and the total molecular weight is about 2450 Da.
The preparation process is as follows:
step a: compound S35-1(20.00g, 10.0mmol, mPEG-OH, Mw about 2000, n1Approximatively 45, PDI 1.03), toluene (200mL) was azeotropically removed at 140 ℃ and 60mL of solvent was distilled off, and the reaction was cooled to room temperature. TEA (2.98g, 20.0mmol) and MsCl (2.05g, 18.0mmol) were added and the reaction stirred at room temperature overnight. After the reaction was complete, the reaction was poured into water (200mL), extracted twice with EtOAc (100mL × 2), the aqueous phase was retained, the aqueous phase was extracted twice with dichloromethane (100mL × 2), the organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 ℃, recrystallized in ice bath, and filtered to give compound S35-2(18.00g, 90%).
Step b: 80mL of water was added to the above-mentioned compound S35-2(18.00g, 9.0mmol), and the mixture was dissolved with stirring at room temperature. Potassium carbonate (12.42g, 90.0mmol), compound S35-3(9.58g, 45.0mmol) and tetra-n-butylammonium bromide (0.29g, 0.9mmol) were added to the reaction solution, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, extraction was performed twice with dichloromethane (100mL × 2), the organic phases were combined, back-washed once with saturated aqueous sodium chloride solution (100mL), the organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S35-4. Purification by column chromatography, concentration and oil pump drainage gave the title compound S35-4(12.00 g).
Step c: the above compound S35-4(12.00g, 6.0mmol) was dissolved in dry THF (120mL), NaH (60%, 2.40g, 60.0mmol) was slowly added under ice bath, and the reaction was carried out for 1 hour under ice bath. Compound S35-5(8.28g, 30.0mmol) was added thereto, and the mixture was stirred in an ice bath for 1 hour, and then the reaction was allowed to return to room temperature overnight. After completion of the reaction, the reaction was placed in an ice bath, and 2mL of methanol was slowly added to quench the reaction, followed by stirring for 30 minutes, followed by addition of water (300mL) and mixing with stirring. Extraction was performed twice with EtOAc (150mL x 2), the aqueous phase was retained, extraction was performed twice with dichloromethane (100mL x 2), the organic phases were collected and combined, back-washed once with saturated sodium chloride (100mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude compound E35-1. Purification by column chromatography, concentration, and oil pump-out afforded pegylated lipid E35-1(9.00g, 75%).1H NMR(500MHz,CDCl3) δ:3.85-3.45(m,182H),3.37(s,3H),3.15(t,2H),2.94(t,2H),2.62(t,2H),1.58-1.48(m,4H), 1.36-1.19(m,44H),0.86(t, 6H). The molecular weight of E35-1 was determined to be 2447Da and PDI 1.03 by MALDI-TOF testing.
EXAMPLE 33.5.2 preparation of cationic Liposome 5 (Experimental group 3)
Step a: cationic lipid P-9 (15. mu. mol), distearoyl phosphatidylcholine (DSPC, 3. mu. mol), cholesterol (12. mu. mol), and pegylated lipid E35-1 (0.45. mu. mol) prepared in inventive example 35.5.1 were weighed into a 100mL round-bottomed flask, 30mL of chloroform was added to dissolve the solids thoroughly, and shaken well;
Step b: performing reduced pressure rotary evaporation to remove chloroform solvent by using a rotary evaporator under the conditions that the rotating speed is 140rpm and the temperature is 55 ℃ to form a thin-layer oil film, and performing vacuum pumping for 12 hours by using a vacuum pump to ensure that all chloroform is removed;
step c: adding 30mL of phosphate buffer solution (PBS, pH 7.4) containing 10% lactose into the flask, and performing ultrasonic treatment for 30min at 90% frequency by using an ultrasonic cleaning machine to form semitransparent emulsion;
step d: adding the emulsion into a high-pressure homogenizer, and carrying out overpressure for 5 times under the condition that the pressure is 100 MPa; and then adding the emulsion into a liposome extruder, carrying out overpressure for 10 times under the condition of 150MPa of pressure to prepare the cationic liposome 5, and carrying out freeze drying for 24 hours to obtain cationic liposome 5 powder.
Example 36: preparation of cationic liposome nucleic acid pharmaceutical composition preparation
Step a: taking 0.03mL of physiological saline as working solution of the nucleic acid pharmaceutical composition preparation;
step b: 0.1mg of cationic liposome was weighed, dissolved in physiological saline, and equilibrated for 30 min. Weighing 1.00 mu g of siRNA dissolved in 10 mu L of normal saline according to the N/P ratio of 10/1, and compounding the siRNA with normal saline in which cationic liposome is dissolved for 30min to prepare the cationic liposome nucleic acid pharmaceutical composition preparation of the invention, wherein the cationic liposome comprises 5 groups of cationic liposomes (control group 1: cationic liposome does not contain polyethylene glycol lipid PEG-DMG, control group 2: cationic liposome DLin-MC3-DMA and polyethylene glycol lipid PEG-DMG, experiment group 1: cationic liposome P-9 and polyethylene glycol lipid PEG-DMG, experiment group 2: cationic liposome P-10 and polyethylene glycol lipid PEG-DMG, experiment group 3: cationic liposome P-9 and polyethylene glycol lipid E35-1 of the invention in example 35, and the neutral lipid in the 5 groups of cationic liposomes is DSPC, and the sterol lipid is cholesterol).
Example 37: biological activity testing of cationic liposome nucleic acid pharmaceutical composition formulations
(1) Study of Gene recombination Capacity
In this example, the gene recombination ability of the cationic liposome nucleic acid pharmaceutical composition preparation of the present invention was examined by gel permeation electrophoresis experiments. 0.8g of agarose is weighed and dissolved in 40mL of TAE solution, the agarose particles are completely dissolved by heating in a microwave oven, the solution is cooled, 5 microliter of nucleic acid dye GelGreen is added into the cooled agarose gel, and the gel is added into a gel tank and naturally dried. The mixture of different cationic liposome/siRNA nucleic acid pharmaceutical composition preparations (described in example 36) and 2. mu.L of LoadingBuffer was added into agarose gel wells, and electrophoresis was performed at 90V for 10min at room temperature. The results show that the experimental group has no free siRNA basically, and the cationic liposome nucleic acid pharmaceutical composition preparation prepared by the cationic lipid has stronger gene (mRNA) complexing capability.
(2) Investigation of cytotoxicity (biocompatibility)
The cytotoxicity test of the cationic liposome nucleic acid pharmaceutical composition preparation is carried out by adopting an MTT staining method, and the inoculation density is 1 multiplied by 10 by taking liver cancer cell HepG2 as a cell model 4Cells per well, 100. mu.L/well of cell suspension was seeded into 96-well plates. After inoculation, 4% CO at 37 ℃2The cell culture box is used for incubation and culture for 24 hours. Dissolving the cationic liposome nucleic acid pharmaceutical composition preparation in culture medium to obtain desired concentration, and optionally adding appropriate amount of cosolventReagent, old medium was discarded, 100. mu.L of medium containing cationic liposome nucleic acid pharmaceutical composition preparation (10/1N/P) at three concentrations of 1nM, 10nM and 100nM was added to each well, and 100. mu.L of fresh medium was added to the blank control, each at 6 duplicate wells. After the cationic liposome nucleic acid pharmaceutical composition preparation was incubated with HepG2 cancer cells for 24 hours, 5mg/mL of MTT in PBS buffer 20 μ L of MTT was added to each well, and the cancer cells were incubated for 4 hours, the mixture of the medium and MTT buffer was aspirated, DMSO 150 μ L/well was added to dissolve formazan, a purple crystal of living cells, and after shaking sufficiently, absorbance at 490nm was measured with a microplate reader. The results of calculation and drawing according to the measured light absorption values show that compared with a blank control group, the cell survival rate of the cationic liposome nucleic acid pharmaceutical composition preparation prepared by the invention is greater than 93% under different N/P conditions, which indicates that the cationic liposome nucleic acid pharmaceutical composition preparation modified by the pegylated lipid has good biocompatibility.
(3) Study of nucleic acid transport efficiency
In order to examine the nucleic acid transport rate of each group of cationic liposome nucleic acid pharmaceutical composition preparations prepared from the cationic lipid of the present invention, the experiment was conducted by using Luc-Hela cells stably expressing luciferase as a model and examining the nucleic acid transport rate of each group of cationic liposome nucleic acid pharmaceutical composition preparations (described in example 36) having N/P of 10/1. Culturing cells in 96-well plate until cell density reaches 80%, adding the cationic liposome nucleic acid pharmaceutical composition preparation into culture medium, and culturing at 37 deg.C and 4% CO2The cell culture box is used for incubation and culture for 24 hours, then the old culture medium is removed by suction, and the fresh culture medium is replaced for continuous culture for 24 hours. The cells were treated with lysis buffer, the fluorescence intensity was measured on a substrate luminometer using a luciferase assay kit, and a blank control was added to the cultured cells with the same amount of naked siRNA. Since the siRNA transcribed into the cells can inhibit the expression of luciferase gene Fluc mRNA, the untreated cells are used as a negative control group, and the expression (namely, fluorescence intensity) of the target gene in the negative control group is designated as 100%, the result shows that the fluorescence intensity of the blank control group is 78%, and the fluorescence intensity of the positive control group 1 is The intensity was 65%, the fluorescence intensity of the positive control group 2 was 43%, the fluorescence intensity of the experimental group 1 was 33%, the fluorescence intensity of the experimental group 2 was 35%, and the fluorescence intensity of the experimental group 3 was 25%. Compared with a blank control group, the positive control group 1, the positive control group 2, the experiment group 1, the experiment group 2 and the experiment group 3 have better luciferase gene inhibition effect, which indicates that the liposome can improve the transport rate of nucleic acid drugs; compared with the positive control group 1, the positive control group 2, the experimental group 1, the experimental group 2 and the experimental group 3 have better luciferase gene inhibition effect, which shows that the cationic liposome containing the cationic lipid can improve the transport rate of nucleic acid drugs; compared with a positive control group 2, the experiment group 1 and the experiment group 2 have better luciferase gene inhibition effect, which shows that compared with the existing cationic lipid, the cationic lipid can improve the nucleic acid transport rate of the liposome; compared with the experimental group 1, the experimental group 3 has a better luciferase gene inhibition effect, namely a good gene silencing effect, and the pegylated lipid and the cationic liposome prepared from the cationic lipid can further improve the transfection rate of genes.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be understood that it is capable of further modifications. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
