1. A compound represented by formula V, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof;

Y-L-M_WEE1 (V)

wherein the content of the first and second substances,

M_WEE1is a WEE1 binding moiety capable of binding to WEE1 protein kinase;

y is an E3 ubiquitin ligase ligand moiety; and

l is a linking group.

2. The compound of claim 1,

(i) y is a group shown as formula A;

wherein the content of the first and second substances,

z is selected from the group consisting of: c (═ O), C (R)^a)₂、N(R^b)；

V¹、V²、V³And V⁴One is C and the others are each independently selected from the group consisting of: c (R)^a) And N;

R³selected from the group consisting of: H. halogen, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl;

R^aeach independently selected from the group consisting of: H. halogen, hydroxy, cyano, C1-C6 alkyl, C1-C6 haloalkyl;

R^beach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl;

R^deach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl; and

subscript p1 is 0, 1, 2, or 3;

or Y is a group shown as formula B;

wherein the content of the first and second substances,

V¹、V²、V³and V⁴One is C and the others are each independently selected from the group consisting of: c (R)^a) And N;

V⁵and V⁷Each independently selected from the group consisting of: c (R)^a) And N;

V⁶selected from the group consisting of: c (R)⁶) And N;

R⁶selected from the group consisting of: H. cyano, C1-C6 alkyl, C1-C6 haloalkyl;

R^aeach independently selected from the group consisting of: H. halogen, hydroxy, cyano, C1-C6 alkyl, C1-C6 haloalkyl;

R^deach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl; and

subscript p1 ═ 0, 1, 2, or 3;

or Y is a group shown as formula C;

wherein the content of the first and second substances,

R⁴selected from the group consisting of: H. halogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl; and

R⁵selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl;

and/or the presence of a gas in the gas,

(ii) l is a linking group as shown in formula D

-(M_L)_s- (D)；

Wherein the content of the first and second substances,

M_Lare structural units each independently selected from the group consisting of: -C (R)^c)₂-、-C(R^c)₂-C(R^c)₂-O-、-C(R^c)₂-C(R^c)₂-S-、-O-、-S-、-N(Rⁿ)-、-C(＝O)-、-N(Rⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-、-N(Rⁿ)C(＝O)N(Rⁿ)-、-SO-、-SO₂-, -CH ═ CH-, -C ≡ C-, -C3-C8 cycloalkyl-, -4-to 7-membered heterocyclyl-, -C6-C10 aryl-, -5-or 6-membered heteroaryl-; wherein said cycloalkyl, heterocyclyl, aryl and heteroaryl are unsubstituted or optionally substituted with one or more (preferably, 1, 2, or 3) substituents selected from the group consisting of: halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl;

subscript s is an integer of 1-30; preferably, the subscript s is an integer of 3 to 25; more preferably, the subscript s is an integer of 3 to 20;

R^ceach independently selected from the group consisting of: H. hydroxy, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C3-C5 cycloalkyl; and

Rⁿeach independently selected from the group consisting of: H. C1-C4 alkyl, C1-C4 haloalkyl, C3-C5 cycloalkyl;

and/or the presence of a gas in the gas,

(iii)M_WEE1as shown in formula E:

wherein the content of the first and second substances,

Cy¹selected from the group consisting of: substituted or unsubstituted C6-C10 cycloalkyl, substituted or unsubstituted 5-to 10-membered heterocycloalkyl;

Cy²selected from the group consisting of: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 10-membered heteroaryl (preferably, 5-or 6-membered heteroaryl);

R^beach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl;

R^deach independently selected from the group consisting of: H. halogen, C1-C6 alkyl, C1-C6 haloalkyl; and

subscript p2 is 0, 1, 2, or 3;

R¹selected from the group consisting of: substituted or unsubstituted C1-C6 alkanesA group (preferably, C1-C4 alkyl), a substituted or unsubstituted C1-C6 alkenyl group (preferably, C1-C4 alkenyl group), a substituted or unsubstituted C1-C6 alkynyl group (preferably, C1-C4 alkynyl group), a substituted or unsubstituted benzyl group;

R²selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl (preferably, C1-C4 alkyl), substituted or unsubstituted C1-C6 alkoxy (preferably, C1-C4 alkoxy), substituted or unsubstituted C6-C10 aryl;

x is selected from the group consisting of: c (R)^a) And N;

R^aselected from the group consisting of: H. halogen, hydroxy, cyano, C1-C6 alkyl, C1-C6 haloalkyl;

unless otherwise specified, said substitution means that one or more (preferably, 1, 2, 3,4 or 5) hydrogens on the group are substituted with a substituent selected from the group consisting of: hydroxyl, halogen (preferably, F, Cl, Br), CN, C1-C6 alkyl, C1-C6 haloalkyl.

3. The compound of claim 1,

l is a linking group of formula D1:

-Q¹-(C(R^c)₂)_n-W-(C(R^c)₂)_n-W-(C(R^c)₂)_m- (D1)

wherein the content of the first and second substances,

Q¹selected from the group consisting of: none (absent), -O-, -S-, -CH ═ CH-, -C ≡ C-, -N (R ≡ C)ⁿ)-、-N(Rⁿ)C(＝O)-、-C(＝O)N(Rⁿ) -and-N (R)ⁿ)C(＝O)N(Rⁿ)-；

Each W is independently selected from the group consisting of: none (absent), -N (R)ⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-；

Each subscript n is independently 0, 1, 2, 3,4, 5, 6, 7, or 8;

subscript m is 0, 1, 2, 3,4, 5, or 6;

R^cand RⁿAs defined in formula D;

alternatively, L is a linking group as shown in formula D2:

-Q²-[C(R^c)₂-C(R^c)₂-O]_v-(C(R^c)₂)_o-W-(C(R^c)₂)_m- (D2)

wherein the content of the first and second substances,

Q²selected from the group consisting of: -N (R)ⁿ)-、-N(Rⁿ)C(＝O)-、-C(＝O)N(Rⁿ) -and-N (R)ⁿ)C(＝O)N(Rⁿ)-；

W is selected from the group consisting of: none (absent), -N (R)ⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-；

Subscript v is 0, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10;

subscript m is 0, 1, 2, 3,4, 5, or 6;

subscript o is 1, 2, or 3;

R^cand RⁿAs defined in formula D;

alternatively, L is a linking group as shown in formula D3:

-Q³-(C(R^c)₂)_r1-Ar¹-(C(R^c)₂)_r2-U-(C(R^c)₂)_n-W-(C(R^c)₂)_m- (D3)

wherein the content of the first and second substances,

Q³selected from the group consisting of: -N (R)ⁿ) -, -O-and-S-;

Ar¹selected from the group consisting of: -C3-C8 cycloalkyl-, 4-to 7-membered heterocyclyl-, -C6-C10 aryl-, -5-or 6-membered heteroaryl-;

w is selected from the group consisting of: none (absent), -N (R)ⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-；

U is selected from the group consisting of: -N (R)ⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-；

Subscript n is 0, 1, 2, 3,4, 5, 6, 7, or 8;

subscript m is 0, 1, 2, 3,4, 5, or 6;

r1 and r2 are each independently 0, 1 or 2;

R^cand RⁿAs defined in formula D.

4. The compound of claim 1,

(i) y is a group as shown in formula A1, A2, A3 or A4;

in the formulae (III), (III) Z, R³As defined above;

or Y is a group shown as formula B1;

or Y is a group shown as formula C1 or C2;

in the formulae, R⁴As defined above;

and/or the presence of a gas in the gas,

(ii) l is a linking group of formula D1 a:

-Q¹-(CH₂)_n-W-(CH₂)_n-W-(CH₂)_m- (D1a)

wherein Q is¹Subscripts m, W and subscript n are as defined in formula D1;

alternatively, L is a linking group as shown in formula D2 a:

-Q²-[CH₂-CH₂-O]_v-(CH₂)₂-W-(CH₂)_n- (D2a)

wherein Q is²Subscripts v, W and subscript n are as defined in formula D2;

alternatively, L is a linking group as shown in formula D3 a:

-Q³-(CH₂)-Ph-(CH₂)_r2-U-(CH₂)_n-W-(CH₂)_m- (D3a)

wherein Ph is phenyl and subscript r2 is 0 or 1; and Q³U, subscript m, W, subscript n and as defined in formula D3;

and/or the presence of a gas in the gas,

(iii)M_WEE1is a group represented by E1;

5. the compound of claim 1, wherein the compound is of formula I;

wherein R is¹、R²Y and L are as defined for formula V.

6. The compound of claim 1,

the compound is shown as a formula III;

wherein subscript n is 1, 2, 3,4, 5, 6, 7, or 8;

or, the compound is shown as formula IV;

wherein subscript n is 1, 2, 3,4, 5, 6, 7, or 8.

7. The compound of claim 1, wherein said compound is selected from the group consisting of the compounds of table 1.

8. A composition comprising (i) a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; and (ii) a pharmaceutically acceptable carrier.

9. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a composition of claim 8, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by the WEE1 protein.

10. A method for degrading WEE1 protein, comprising the steps of: contacting a subject with a compound of claim 1, thereby degrading WEE1 protein.

Background

Uncontrolled proliferation of tumor cells is a basic biological characteristic of malignant tumors, and relates to cell cycle regulation mechanism and cell conduction pathway change of DNA damage repair. The WEE1 protein is a cell cycle regulatory protein, is one of important members of a serine/threonine protein kinase family, and affects the combination of the WEE1 protein and CyclinB to block the transition from the G2 phase to the M phase by regulating the phosphorylation state of CDK1, thereby ensuring the DNA replication accuracy and the chromatin integrity. Thus, the WEE1 protein is a key protein kinase involved in the cell cycle G2/M checkpoint and DNA damage repair process. On the other hand, in normal cell cycle progression, the p53 protein can also monitor genome integrity by regulating the G1/S phase and DNA damage check, but most tumor cells have a deletion of the p53 gene, resulting in a defective G1/S checkpoint in their cell cycle. Therefore, the p 53-deleted tumor cells are more dependent on the G2/M checkpoint in the process of DNA replication and damage repair, so that WEE1 protein kinase is highly expressed. Theoretically, by inhibiting the activity of WEE1 protein kinase, the DNA damage of these p 53-deleted tumor cells cannot be repaired in time and enter M phase, causing genomic instability and chromosome deletion, triggering mitotic catastrophe, leading to tumor cell apoptosis. Although AZD1775 has made some clinical research progress as a WEE1 protein target inhibitor, there still exist some application limitations including significant clinical toxic side effects such as nausea, fatigue, thrombocytopenia, and insufficient targeting, for example, AZD1775 has been shown to have PLK1/WEE1 dual target inhibition.

In view of the above, there is an urgent need in the art to develop a new drug targeting the WEE1 protein with high selectivity.

Disclosure of Invention

The invention aims to provide a WEE1 protein degrading agent with high selectivity and excellent anti-tumor proliferation activity.

In a first aspect of the invention, there is provided a compound of formula V, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof;

Y-L-M_WEE1 (V)

wherein the content of the first and second substances,

M_WEE1is a WEE1 binding moiety capable of binding to WEE1 protein kinase;

y is an E3 ubiquitin ligase ligand moiety; and

l is a linking group.

In another preferred embodiment, the E3 ubiquitin ligase ligand moiety is capable of binding to CRBN or CRL4^CRBNAnd (4) combining.

In another preferred example, the E3 ubiquitin ligase ligand is CRBN ligand or CRL4^CRBNE3 ubiquitin ligase ligand.

In another preferred embodiment, Y is a group represented by formula A;

wherein the content of the first and second substances,

z is selected from the group consisting of: c (═ O), C (R)^a)₂、N(R^b)；

V¹、V²、V³And V⁴One is C and the others are each independently selected from the group consisting of: c (R)^a) And N;

R³selected from the group consisting of: H. halogen, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl;

R^aeach independently selected from the group consisting of: H. halogen, hydroxy, cyano, C1-C6 alkyl, C1-C6 haloalkyl;

R^beach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl;

R^deach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl; and

subscript p1 is 0, 1, 2, or 3.

In another preferred embodiment, R^aIs H.

In another preferred embodiment, R³Is H.

In another preferred embodiment, Z is C (═ O) or C (R)^a)₂(ii) a Preferably, Z is C (═ O) or CH₂。

In another preferred embodiment, V¹、V²、V³And V⁴One is C and the others are CH.

In another preferred embodiment, V¹Is C and V²、V³And V⁴Are all CH; or, V²Is C and V¹、V³And V⁴Are all CH; or, V³Is C and V¹、V²And V⁴Are all CH; or, V⁴Is C and V¹、V²And V³Are both CH.

In another preferred example, subscript p1 ═ 0.

In another preferred embodiment, Z, V¹、V²、V³、V⁴、R³、R^dAnd p1 are each independently the corresponding group in the specific compound in table 1.

In another preferred embodiment, Y is a group as shown in formula a1, a2, A3 or a 4;

in the formulae (III), (III) Z, R³As defined above.

In another preferred embodiment, Y is a group as shown in formula A1.

In another preferred embodiment, Y is a group as shown in formula A1, and wherein R is³H and/or Z is C (═ O). .

In another preferred embodiment, Y is a group represented by formula B;

wherein the content of the first and second substances,

V¹、V²、V³and V⁴One is C and the others are each independently selected from the group consisting of: c (R)^a) And N;

V⁵and V⁷Each independently selected from the group consisting of: c (R)^a) And N;

V⁶selected from the group consisting of: c (R)⁶) And N;

R⁶selected from the group consisting of: H. cyano, C1-C6 alkyl (preferably, C1-C4 alkyl), C1-C6 haloalkyl;

R^aeach independently selected from the group consisting of: H. halogen, hydroxy, cyano, C1-C6 alkyl, C1-C6 haloalkyl;

R^deach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl; and

subscript p1 ═ 0, 1, 2, or 3.

In another preferred embodiment, V¹、V²、V³And V⁴One is C and the others are CH.

In another preferred embodiment, V¹Is C and V²、V³And V⁴Are all CH; or, V²Is C and V¹、V³And V⁴Are all CH; or, V³Is C and V¹、V²And V⁴Are all CH; or, V⁴Is C and V¹、V²And V³Are both CH.

In another preferred embodiment, V⁵And V⁷Are all N.

In another preferred embodiment, V⁶Is C (R)⁶) And R is⁶Is C1-C6 alkyl (preferably, C1-C4 alkyl); preferably, V⁶Is C (CH)₃)。

In another preferred example, subscript p1 ═ 0.

In another preferred embodiment, V¹、V²、V³、V⁴、V⁵、V⁶、V⁷、R^dAnd subscript p1 are each independently the corresponding groups in the specific compounds in table 1.

In another preferred embodiment, Y is a group as shown in formula B1;

in another preferred embodiment, Y is a group represented by formula C;

wherein the content of the first and second substances,

R⁴selected from the group consisting of: H. halogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl; and

R⁵selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl.

In another preferred embodiment, R⁴Selected from the group consisting of: hydroxyl, C1-C6 alkoxy.

In another preferred embodiment, R⁵Selected from the group consisting of: H. C1-C6 alkyl (preferably, C1-C4 alkyl; more preferably, methyl).

In another preferred embodiment, R⁴And R⁵Each independently is a group corresponding to the particular compound in table 1.

In another preferred embodiment, Y is a group as shown in formula C1 or C2;

in the formulae, R⁴As defined above.

In another preferred embodiment, Y is a group as shown in formula C1.

In another preferred embodiment, Y is a group of formula C1 and R⁴Is a hydroxyl group.

In another preferred embodiment, L is a linking group as shown in formula D

-(M_L)_s- (D)；

Wherein the content of the first and second substances,

M_Lare structural units each independently selected from the group consisting of: -C (R)^c)₂-、-C(R^c)₂-C(R^c)₂-O-、-C(R^c)₂-C(R^c)₂-S-、-O-、-S-、-N(Rⁿ)-、-C(＝O)-、-N(Rⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-、-N(Rⁿ)C(＝O)N(Rⁿ)-、-SO-、-SO₂-, -CH ═ CH-, -C ≡ C-, -C3-C8 cycloalkyl-, -4-to 7-membered heterocyclyl-, -C6-C10 aryl-, -5-or 6-membered heteroaryl-; wherein said cycloalkyl, heterocyclyl, aryl and heteroaryl are unsubstituted or optionally substituted with one or more (preferably, 1, 2, or 3) substituents selected from the group consisting of: halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl;

subscript s is an integer of 1-30; preferably, the subscript s is an integer of 3 to 25; more preferably, the subscript s is an integer of 3 to 20;

R^ceach independently selected from the group consisting of: H. hydroxy, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C3-C5 cycloalkyl; and

Rⁿeach independently selected from the group consisting of: H. C1-C4 alkyl, C1-C4 haloalkyl, C3-C5 cycloalkyl.

In another preferred embodiment, subscript s is 3,4, 5, 6, 8, 9, 10, 11, 12, 13, 14, or 15.

In another preferred embodiment, R^cIs H.

In another preferred embodiment, RⁿIs H.

In another preferred embodiment, L is a linking group according to formula D1:

-Q¹-(C(R^c)₂)_n-W-(C(R^c)₂)_n-W-(C(R^c)₂)_m- (D1)

wherein the content of the first and second substances,

Q¹selected from the group consisting of: none (absent), -O-, -S-, -CH ═ CH-, -C ≡ C-, -N (R ≡ C)ⁿ)-、-N(Rⁿ)C(＝O)-、-C(＝O)N(Rⁿ) -and-N (R)ⁿ)C(＝O)N(Rⁿ)-；

Each W is independently selected from the group consisting of: none (absent), -N (R)ⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-；

Each subscript n is independently 0, 1, 2, 3,4, 5, 6, 7, or 8;

subscript m is 0, 1, 2, 3,4, 5, or 6;

R^cand RⁿAs defined in formula D.

In another preferred embodiment, subscript m and each n are not simultaneously 0.

In another preferred embodiment, Q¹Is linked to Y.

In another preferred embodiment, Q¹Selected from the group consisting of: none (absent), -O-, -CH-, -C.ident.C-, -NH-, -NHC (. ident.O) -and-NHC (. ident.O) NH-.

In another preferred embodiment, each W is independently selected from the group consisting of: none, -NHC (═ O) -, -C (═ O) NH-.

In another preferred embodiment, L is a linking group according to formula D1 a:

-Q¹-(CH₂)_n-W-(CH₂)_n-W-(CH₂)_m- (D1a)

wherein Q is¹Subscripts m, W, and subscript n are as defined in formula D1.

In another preferred embodiment, Q¹、Rⁿ、R^cW, subscript m, and subscript n are each independently the corresponding groups in the specific compounds in table 1.

In another preferred embodiment, L is a linking group according to formula D2:

-Q²-[C(R^c)₂-C(R^c)₂-O]_v-(C(R^c)₂)_o-W-(C(R^c)₂)_m- (D2)

wherein the content of the first and second substances,

Q²selected from the group consisting of: -N (R)ⁿ)-、-N(Rⁿ)C(＝O)-、-C(＝O)N(Rⁿ) -and-N (R)ⁿ)C(＝O)N(Rⁿ)-；

W is selected from the group consisting of: none (absent), -N (R)ⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-；

Subscript v is 0, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10;

subscript m is 0, 1, 2, 3,4, 5, or 6;

subscript o is 1, 2, or 3;

R^cand RⁿAs defined in formula D.

In another preferred embodiment, subscript v is 1, 2, 3,4, 5, 6, 7, 8, 9, or 10.

In another preferred embodiment, Q²Is linked to Y.

In another preferred embodiment, Q²Selected from the group consisting of: -NH-, -NHC (═ O) -and-NHC (═ O) NH-.

In another preferred embodiment, W is selected from the group consisting of: none, -NHC (═ O) -, -C (═ O) NH-.

In another preferred embodiment, L is a linking group according to formula D2 a:

-Q²-[CH₂-CH₂-O]_v-(CH₂)₂-W-(CH₂)_m- (D2a)

wherein Q is²Subscripts v, W and subscript m are as defined in formula D2.

In another preferred embodiment, Q²、Rⁿ、R^cW, subscript v, subscript o and subscript m are each independently the corresponding groups in the specific compounds of table 1.

In another preferred embodiment, L is a linking group according to formula D3:

-Q³-(C(R^c)₂)_r1-Ar¹-(C(R^c)₂)_r2-U-(C(R^c)₂)_n-W-(C(R^c)₂)_m- (D3)

wherein the content of the first and second substances,

Q³selected from the group consisting of: -N (R)ⁿ) -, -O-and-S-;

Ar¹selected from the group consisting of: -C3-C8 cycloalkyl-, 4-to 7-membered heterocyclyl-, -C6-C10 aryl-, -5-or 6-membered heteroaryl-;

w is selected from the group consisting of: none (absent), -N (R)ⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-；

U is selected from the group consisting of: -N (R)ⁿ)C(＝O)-、-C(＝O)N(Rⁿ)-；

Subscript n is 0, 1, 2, 3,4, 5, 6, 7, or 8;

subscript m is 0, 1, 2, 3,4, 5, or 6;

subscript r1 and subscript r2 are each independently 0, 1, or 2;

R^cand RⁿAs defined in formula D.

In another preferred embodiment, - (C (R)^c)₂)_r2-U-is-C (═ O) N (R)ⁿ) -or- (C (R)^c)₂)-N(Rⁿ)C(＝O)-。

In another preferred embodiment, - (C (R)^c)₂)_r2-U-is-C (═ O) NH or- (C (R)^c)₂)-NHC(＝O)-。

In another preferred embodiment, Q³Is linked to Y.

In another preferred embodiment, Q³Selected from the group consisting of: -NH-and-O-.

In another preferred embodiment, Ar¹Selected from the group consisting of: -C6-C10 aryl-, -5-or 6-membered heteroaryl-.

In another preferred embodiment, L is a linking group according to formula D3 a:

-Q³-(CH₂)-Ph-(CH₂)_r2-U-(CH₂)_n-W-(CH₂)_m- (D3a)

wherein Ph is phenyl, and r2 is 0 or 1; and Q³U, subscript m, W, and subscript n are as defined in formula D3.

In another preferred embodiment, Q³、W、U、Ar¹、Rⁿ、R^cSubscript r1, subscript r2, subscript m, and subscript n are each independently the corresponding groups of the particular compounds of table 1.

In another preferred embodiment, L is a linking group as shown in formula D1.

In another preferred embodiment, L is a linking group as shown in formula D1 a.

In another preferred embodiment, L is a linking group as shown in formula D1 or D1a, and Q¹Is NH.

In another preferred embodiment, L is a linking group as shown in formula D1 or D1a, and W is nothing.

In another preferred embodiment, L is a linking group as shown in formula D1 or D1a, and W are both absent, subscript m is 0, one of subscripts n is 0, and the other subscript n is 1, 2, 3,4, 5, 6, 7, or 8. In another preferred embodiment, L is-NH- (CH)₂)_n-。

In another preferred embodiment, M_WEE1Is a WEE1 binding moiety derived from a WEE1 kinase inhibitor.

In another preferred embodiment, M_WEE1Is a WEE1 binding moiety derived from a compound selected from the group consisting of: AZD 1775.

In another preferred embodiment, M_WEE1As shown in formula E:

wherein the content of the first and second substances,

Cy¹selected from the group consisting of: substituted or unsubstituted C6-C10 cycloalkyl, substituted or unsubstituted 5-to 10-membered heterocycloalkyl (preferably, 5-or 6-membered heterocycloalkyl);

Cy²selected from the group consisting of: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 10-membered heteroaryl (preferably, 5-or 6-membered heteroaryl);

R^beach independently selected from the group consisting of: H. C1-C6 alkyl, C1-C6 haloalkyl;

R^deach independently selected from the group consisting of: H. halogen, C1-C6 alkyl, C1-C6 haloalkyl; and

subscript p2 is 0, 1, 2, or 3;

R¹selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl (preferably, C1-C4 alkyl), substituted or unsubstituted C1-C6 alkenyl (preferably, C1-C4 alkenyl), substituted or unsubstituted C1-C6 alkynyl (preferably, C1-C4 alkynyl), substituted or unsubstituted benzyl;

R²selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl (preferably, C1-C4 alkyl), substituted or unsubstituted C1-C6 alkoxy (preferably,C1-C4 alkoxy), substituted or unsubstituted C6-C10 aryl;

x is selected from the group consisting of: c (R)^a) And N;

R^aselected from the group consisting of: H. halogen, hydroxy, cyano, C1-C6 alkyl, C1-C6 haloalkyl;

unless otherwise specified, said substitution means that one or more (preferably, 1, 2, 3,4 or 5) hydrogens on the group are substituted with a substituent selected from the group consisting of: hydroxyl, halogen (preferably, F, Cl, Br), CN, C1-C6 alkyl, C1-C6 haloalkyl.

In another preferred embodiment, X is N.

In another preferred embodiment, subscript p2 is 0.

In another preferred embodiment, Cy²Is a C6-C10 aryl group; preferably, it is phenyl.

In another preferred embodiment, Cy¹Is a 5 to 10 membered heterocycloalkyl group containing at least one N heteroatom.

In another preferred embodiment, Cy¹Is composed ofWherein, and Cy²And (4) connecting.

In another preferred embodiment, R¹Is substituted or unsubstituted C1-C6 alkenyl; preferably, it is a substituted or unsubstituted C1-C4 alkenyl group; more preferably, R¹Selected from: -CH₂CH＝CH₂、-CH₂CH₂CH＝CH₂、-CH(CH₃)CH＝CH₂、-CH₂CH＝CH(CH₃)、-CH₂CH＝C(CH₃)₂。

In another preferred embodiment, R²Selected from the group consisting of: H. halogen, C1-C4 alkyl (preferably methyl), C1-C4 alkoxy (preferably methoxy), C1-C4 haloalkyl (preferably trifluoromethyl), hydroxy-substituted C1-C4 alkyl (preferably-C (CH)₃)₂OH), substituted or unsubstituted phenyl.

In another preferred embodiment, Cy¹、Cy²、R¹、R²、R^b、R^dX, and subscript p2 are each independently the corresponding groups in the specific compounds in table 1.

In another preferred embodiment, M_WEE1Is a group represented by E1;

in a second aspect of the invention, there is provided a compound of formula I;

wherein R is¹、R²Y and L are as defined for formula V.

In another preferred embodiment, Y is a group represented by formula A; preferably, Y is a group as shown in formula A1.

In another preferred embodiment, Y is a group as shown in formula A1, and wherein R is³H and/or Z is C (═ O).

In another preferred embodiment, when Y is a group of formula A or formula A1, L is a linking group of formula D1; preferably, it is a linking group as shown in formula D1 a.

In another preferred embodiment, when Y is a group of formula A or formula A1, L is a linking group of formula D1a, and Q is in formula D1a¹is-NH-, further in formula D1a where W is all absent, further in formula D1a where m is 0, one of n is 0, and the other n is 1, 2, 3,4, 5, 6, 7 or 8.

In another preferred embodiment, when Y is a group of formula A or formula A1, L is-NH- (CH)₂)_n-。

In another preferred embodiment, the compound is represented by formula III;

wherein n is 1, 2, 3,4, 5, 6, 7 or 8.

In another preferred embodiment, Y is a group represented by formula C; preferably, Y is a group as shown in formula C1.

In another preferred embodiment, Y is a group of formula C1, and wherein R⁴Is a hydroxyl group.

In another preferred embodiment, when Y is a group of formula C or formula C1, L is a linking group of formula D1; preferably, it is a linking group as shown in formula D1 a.

In another preferred embodiment, when Y is a group of formula C or formula C1, L is a linking group of formula D1a, and Q is in formula D1a¹is-NHC (═ O) -, further in formula D1a W is absent, further in formula D1a m is 0.

In another preferred embodiment, when Y is a group of formula C or formula C1, L is-NHC (═ O) - (CH)₂)_n-。

In another preferred embodiment, the compound is represented by formula IV;

wherein n is 1, 2, 3,4, 5, 6, 7 or 8.

In another preferred embodiment, Y is a group of formula A (preferably, a group of formula A1) and L is a linking group of formula D1 (preferably, a linking group of formula D1 a).

In another preferred embodiment, the compound is selected from the compounds in table 1.

In another preferred embodiment, the compound is selected from the group consisting of compounds 1-22 and compounds 25-48 of Table 1; preferably, selected from compounds 1-18 and compounds 25-28 in Table 1; more preferably, it is selected from compounds 1-3 of Table 1.

In another preferred embodiment, the compound is compound 2 in table 1;

in a third aspect of the invention, there is provided a composition comprising (i) a compound as described in the first or second aspect, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; and (ii) a pharmaceutically acceptable carrier.

In a fourth aspect of the invention, there is provided the use of a compound according to the first or second aspects, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a composition according to the third aspect, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by the WEE1 protein.

In another preferred embodiment, the disease or disorder mediated by WEE1 protein is cancer.

In another preferred embodiment, the disease or condition mediated by WEE1 protein is selected from the group consisting of: lymphoma, osteosarcoma, neuroblastoma, glioblastoma, melanoma, ovarian cancer, head and neck cancer, colorectal cancer, pancreatic cancer, liver cancer, skin cancer, lung cancer, kidney cancer, breast cancer, leukemia.

In a fifth aspect of the invention, there is provided a method of treating or preventing a disease or condition mediated by WEE1 protein, comprising the steps of: administering to a subject in need thereof a safe and effective amount of a compound according to the first or second aspect, or a composition according to the third aspect.

In another preferred embodiment, the subject is a mammal, preferably a human.

In a sixth aspect of the invention, a method of degrading WEE1 protein, comprising the steps of: contacting a subject with a compound according to the first or second aspect, thereby degrading WEE1 protein.

In another preferred embodiment, the method is non-therapeutic in vitro.

In another preferred embodiment, the subject is a cell line.

In another preferred embodiment, the subject is a MV-4-11 cell line.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows compounds 1-24 in IC₅₀Immunoblot experiments of concentration-induced WEE1 protein degradation on MV-4-11 tumor cell lines are shown.

FIG. 2 is a graphical representation of the immunoblot experiment of Compound 2 at different concentration gradients on MV-4-11 tumor cell lines inducing WEE1 protein degradation.

FIG. 3 is a graphical representation of the immunoblot experiment of Compound 2 acting on MV-4-11 tumor cell lines at different time gradients to induce WEE1 protein degradation.

Figure 4 is a graphical representation of an immunoblot experiment demonstrating that compound 2 induces WEE1 protein degradation by protein competition binding, relying on the formation of a "WEE 1 protein-compound 2-CRBN protein" ternary complex.

Figure 5 is a bar graph of compound 2 inducing degradation of WEE1 protein but not of WEE 1-expressing mRNA.

FIG. 6 is a bar graph of Compound 2 inducing MV-4-11 tumor cell line cycle arrest.

FIG. 7 is a bar graph of Compound 2 inducing apoptosis of MV-4-11 tumor cell lines.

Detailed Description

The inventors have conducted extensive and intensive studies. Surprisingly, the novel compound formed by connecting the WEE1 target drug with the E3 ligase ligand (especially with the E3 ligase ligand part shown in the formula A) through the connecting group (L) is found to overcome the defect of insufficient targeting selectivity of the existing WEE1 target drug (such as a WEE1 protein kinase inhibitor). In addition, the compound of the invention can effectively degrade WEE1 protein and has excellent anti-tumor proliferation activity. Based on this, the inventors have completed the present invention.

Term(s) for

Unless otherwise stated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. Further, terms such as "haloalkyl" are meant to include monohaloalkyl or polyhaloalkyl. For example, the term "C1-C6 haloalkyl" or "C1-C4 haloalkyl" includes, for example, trifluoromethyl, 2,2, 2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

As used herein, "C1-C6 alkyl" refers to a straight or branched chain alkyl group including 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, or the like. Similarly, C1-C4 alkyl refers to straight or branched chain alkyl groups including 1-4 carbon atoms.

As used herein, "C1-C6 alkoxy" includes straight or branched chain alkoxy groups of 1-6 carbon atoms. Such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, or the like.

As used herein, "cycloalkyl" refers to a saturated cyclic hydrocarbon group (including monocyclic, bicyclic, such as fused, etc.) containing the indicated number of carbon atoms, for example, "C3-C6 cycloalkyl" refers to a cycloalkyl group containing 3-6 carbon atoms, and "C6-C10 cycloalkyl" includes, examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. Similarly, as used herein, the term "heterocycloalkyl" refers to a group formed by replacement of one or more (e.g., 1, 2, 3, or 4) carbon atoms in a ring in a cycloalkyl group with a heteroatom (e.g., O, S or N).

The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of unsaturated alkyl groups include: ethenyl, 2-propenyl, ethynyl, 1-and 3-propynyl, 3-butynyl and higher homologs and isomers.

The term "alkylene" by itself or as part of another substituent refers to a divalent radical derived from an alkane, e.g., -CH₂CH₂CH₂CH₂-. Unless otherwise defined, alkyl (or alkylene) groups typically have 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein.

Unless otherwise defined, the term "aryl" denotes a polyunsaturated (usually aromatic) hydrocarbon group which may be a single ring or multiple rings (up to three rings, preferably bi-or mono-rings) fused together or covalently linked. The term "heteroaryl" refers to an aryl (or ring) containing 1 to 5 heteroatoms (e.g., selected from N, O, and S), wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl groups, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuranyl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridyl, thienopyrimidyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, and the like. Unless otherwise defined herein, "aryl" or "heteroaryl" can also be optionally substituted by a substituent selected from the group consisting of: halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl.

As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si); preferably O, N and S are included.

For the compounds provided herein, a bond from a substituent (typically an R group) to the center of an aromatic ring (e.g., benzene, pyridine, etc.) or cycloalkyl, etc., will be understood to refer to a bond that provides attachment at any available vertex of the aromatic or alkyl ring.

Targeted protein degradation chimera technology (PROTACs)

The target protein degradation chimeras technology (PROTACs) induces protein degradation in a targeted way through the ubiquitination-proteasome approach, initiates a new treatment mode, and becomes a hot field for new drug development in recent years. The PROTACs molecules are bifunctional small molecules formed by connecting a target protein ligand and an E3 ubiquitin ligase ligand through a Linker, and can be simultaneously combined with a target protein and E3 ubiquitin ligase to enable the target protein and the E3 ubiquitin ligase to be close to each other and marked by ubiquitination, so that the target protein and the E3526 ubiquitin ligase are recognized and degraded by proteasomes in cells.

WEE1 protein kinase degradation agent

The existing therapeutic drugs for WEE1 target are small molecule kinase inhibitors, which can inhibit the function of WEE1 protein by occupying the active site of WEE1 protein (or WEE1 protein kinase) to achieve the purpose of treating diseases, and different from the above, the compound of formula I provided by the invention is a WEE1 protein degradation agent, and develops a new therapeutic mode for WEE1 target diseases based on the degradation mechanism of ubiquitination-proteasome pathway. In addition, the compound of the formula I not only has excellent degradation capability of WEE1 protein, but also can selectively degrade WEE1 protein, and does not degrade other proteins such as PLK protein, so that the application limit of insufficient targeting of the existing WEE1 protein kinase inhibitor is overcome, and clinical toxic and side effects caused by the action of the WEE1 protein kinase inhibitor AZD1775 on PLK protein are overcome.

Typically, the invention provides a WEE1 protein degradation agent, as shown in formula V

Y-L-M_WEE1 (V)

Wherein M is_WEE1Y and L are as defined in the first aspect.

In one embodiment, the invention provides a class of WEE1 protein degrading agents, represented by formula I:

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein the content of the first and second substances,

R¹selected from: substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted benzyl;

R²selected from: hydrogen, halogen (F, Cl, Br, I), C1-C4 alkyl(s) (I)Such as methyl), C1-C4 alkoxy (such as methoxy), C1-C4 haloalkyl (such as trifluoromethyl), hydroxy-substituted C1-C4 alkyl (such as tert-butanol (-C (CH)₃)₂OH)), a substituted or unsubstituted phenyl group;

y is selected from:

wherein Z is selected from: -CH₂-and-C (═ O) -; r³Selected from: hydrogen, halogen, methyl and hydroxy; r⁴Selected from: hydrogen, halogen, methyl and hydroxy;

l is a linking group selected from the group consisting of;

-Q¹-(CH₂)_n-W-(CH₂)_n-W-(CH₂)_m-；

-Q²-[CH₂-CH₂-O]_v-(CH₂)₂-W-(CH₂)_m-；

-Q³-(CH₂)-Ph-(CH₂)_r2-U-(CH₂)_n-W-(CH₂)_m-；

wherein the content of the first and second substances,

Q¹selected from: -O-, C2 alkenyl (-CH ═ CH-), C2 alkynyl (-C ≡ C-), -NH-, -NHC (═ O) -, and-NHC (═ O) -NH-, or Q¹Is absent;

Q²selected from: -O-, -NH-, -NHC (═ O) -, and-NHC (═ O) NH-;

Q³selected from: -O-and-NH-;

w (each independently) is selected from: -NHC (═ O) -and-C (═ O) NH-, or W is absent;

u is selected from: -C (═ O) NH-and-NHC (═ O) -; and r2 is 0 or 1; n is 0, 1, 2, 3,4, 5, 6, 7 or 8; m is 0, 1, 2, 3,4, 5 or 6; v is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10.

In one embodiment, the compound is of formula II:

in one embodiment, Y is

Preferably, R³Is hydrogen. Preferably, Z is-C (═ O) -.

Preferably, L is-Q¹-(CH₂)_n-W-(CH₂)_n-W-(CH₂)_m-. Preferably, Q¹is-NH-. Preferably, W is absent. Preferably, one of n is 0 and m is 0.

Preferably, the compound is of formula III:

preferably, the compound of formula III has the following structural formula:

in another embodiment, Y is

Preferably, R⁴Is a hydroxyl group.

Preferably, L is-Q¹-(CH₂)_n-W-(CH₂)_n-W-(CH₂)_m-. Preferably, Q¹is-NH-C (═ O) -. Preferably, W is absent. Preferably, one of n is 0 and m is 0.

Preferably, the compound has formula IV:

preferably, the compound of formula IV has the following structural formula:

in another embodiment, examples of compounds of the present invention include, but are not limited to, any one or more of the compounds listed in table 1:

TABLE 1

Active ingredient

As used herein, the terms "compound of the invention" or "degradant of the invention" or "WEE 1 protein degradant" are used interchangeably and refer to a compound (or degradant) as shown in formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts or hydrates or solvates of the compounds of formula I.

Wherein the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or a base, which is suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc. Another preferred class of salts are those of the compounds of the invention with bases, for example alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

In addition, the compounds of the present invention also include prodrugs of the compounds of formula I. The term "prodrug" includes a class of compounds which are biologically active or inactive in nature and which, when administered by an appropriate method, undergo a metabolic or chemical reaction in the body to convert the compound to formula I, or a salt or solution of a compound of formula I. The prodrugs include, but are not limited to, carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amide, carbamate, azo, phosphoramide, glucoside, ether, acetal forms of the compounds.

Preparation method

The process for the preparation of the compounds of formula I or formula V according to the invention is described in more detail below, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains. In general, in the preparative schemes, each reaction is usually carried out in an inert solvent at a temperature ranging from room temperature to reflux temperature (e.g., from 0 ℃ to 200 ℃, preferably from 0 ℃ to 120 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 48 hours. In general, the process for the synthesis of the compounds represented by the first or second aspect of the invention comprises 2 main steps, namely: step (i) ligation of the Y fragment and the L fragment to give the Y-L fragment, or the L fragment and M_wee1Ligation of fragments to give L-M_weeFragment, or Y fragment and L1 fragment and L2 fragment and M_wee1Ligation of the fragments to give Y-L1 fragment and L2-M, respectively_wee1Fragments, and step (ii) Y-L fragments and M_wee1Ligation of fragments, or Y fragment and L-M_wee1Ligation of fragments, or Y-L1 fragment and L2-M_wee1Ligation of fragments; wherein Y fragment is L fragment or L-M_wee1The fragments are capable of forming an E3 ubiquitin junction as described in the first aspectA compound of an enzyme ligand moiety (Y-); l is a group which is linked to the Y fragment and M_wee1The fragments are reacted to form a compound having a linking group (-L-) as described in the first aspect, with fragment L1 and fragment L2 referring to fragments Y and M, respectively_wee1After reaction of the fragments and coupling of the fragments to each other, a compound, M, which together form a linking group (-L-) as described in the first aspect_wee1Fragment means that upon reaction with the L fragment or the Y-L fragment, a binding moiety (-M) capable of forming WEE1 as described in the first aspect_WEE1) The compound of (1). Each fragment may be modified or unmodified, and may be protected with a protecting group (e.g., Boc) or unprotected. For example,

the Y segment can beM_wee1The fragment can beL, L1 and L2 fragment may each independently beBocNH-(CH₂)₂-(OCH₂CH₂)_v-NH₂、BocNH-(CH₂)_n-COOH、Br-(CH₂)_n-COOH; wherein n and v are as defined above.

These fragments, after deprotection (e.g. deprotection by conventional methods or methods described in the specification) and/or modification or replacement of the active group, may be coupled to the remaining fragments according to methods described in the specification or other conventional methods to give compounds according to the first or second aspect.

Pharmaceutical compositions and methods of administration

Because the compound has excellent selective WEE1 protein degradation activity, the compound and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and a pharmaceutical composition containing the compound as a main active ingredient can be used for treating, preventing and relieving diseases mediated by WEE1 protein. According to the prior art, the compounds of the invention are useful for the treatment of the following diseases: cancer, and the like, particularly, for example: lymphoma, osteosarcoma, neuroblastoma, glioblastoma, melanoma, ovarian cancer, head and neck cancer, colorectal cancer, pancreatic cancer, liver cancer, skin cancer, lung cancer, kidney cancer, breast cancer, leukemia.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-500mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 20 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention include

(a) The selectivity is good, and the compound of the invention has no degradation effect on PLK protein.

(b) Has excellent antitumor proliferation effect. Preferred compounds of the invention even have an anti-tumor proliferative capacity superior to the positive control dual-target inhibition of AZD 1775.

(c) The compound of the present invention has excellent degradability of WEE1 protein.

(d) The compound of the invention can effectively induce tumor cell apoptosis.

In general, the WEE1 protein degradation agent (the compound shown in the formula I) provided by the invention can selectively degrade WEE1 protein without degrading PLK protein, has better target selectivity compared with the existing WEE1 kinase inhibitor such as AZD1775(PLK1/WEE1 double-target inhibition effect), and avoids clinical toxic and side effects caused by off-target effect. On the other hand, the WEE1 protein degradation agent based on the PROTAC technology has a catalytic effect, so that the protein degradation agent has the potential advantages of low dosage administration and long drug effect in clinical treatment.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, the starting materials or reagents used in the examples are commercially available.

Example 1: synthetic route of intermediate S15

Step 1: synthesis of Compound S3

Compound S1(10.0g,67.5mmol) and compound S2(9.40g,70.9mmol) were added to 110mL of a toluene solution, and the reaction mixtureThe reaction was carried out for 18 hours while the temperature was increased to reflux. After completion of the detection reaction, it was cooled to room temperature, filtered, washed with n-hexane, and the white solid product was collected and spin-dried to give compound S3(16.1g, 91% yield).¹H NMR(400MHz,DMSO)δ9.87(s,1H),7.95(d,J＝3.2Hz,4H),1.45(s,9H)。

Step 2: synthesis of Compound S5

Compound S3(16.1g,61.2mmol), potassium carbonate (16.1g,116mmol) and benzyltriethylammonium chloride (1.39g,6.12mmol) were added to 110mL of acetonitrile solvent and stirred for 5 minutes, and then compound S4(8mL,91.8mmol) was added to the reaction system and reacted at room temperature for 18 hours. After the detection reaction is finished, 100mL of water is added into the system, extraction is carried out for three times by ethyl acetate, organic phases are combined and washed by saturated saline solution, drying is carried out by anhydrous sodium sulfate, filtration is carried out, filtrate is collected and rotary drying is carried out to obtain a yellow oily crude product. Adding the crude product into n-hexane solution, cooling to 5 ℃, and precipitating a solid. Filtration and washing with n-hexane collected the product as a white solid which was spin dried to give compound S5(15.7g, 85% yield).

And step 3: synthesis of Compound S6

Methyl hydrazine (3.40mL,64.3mmol) was added to a 100mL solution of compound S5(15.6g,51.5mmol) in tetrahydrofuran under ice-bath, and after the addition, the ice-bath was removed and the reaction was allowed to proceed at room temperature for 18 hours. Filtering, collecting filtrate and spin-drying to obtain oily crude product. The crude product was added to n-hexane and a white solid precipitated, the filtrate was collected by filtration and spin-dried, and the same operation was repeated three times until no solid precipitated, and spin-dried to give product S6(8.47g, 96% yield) as a yellow oily liquid.¹H NMR(400MHz,DMSO)δ5.90-5.66(m,1H),5.16-

4.99(m,2H),4.46(s,2H),3.99-3.76(m,2H),1.40(s,9H)。

And 4, step 4: synthesis of Compound S8

DIPEA (20.8mL, 120mmol) and the compound S6(8.23g,47.8mmol) were added to a solution of the compound S7(11.1g,47.8mmol) in 100mL of tetrahydrofuran, and the reaction was heated to reflux for 72 hours. After the reaction was completed, the tetrahydrofuran solvent was removed by rotary evaporation under reduced pressure, and 50mL of diethyl ether was added to the system to precipitate a solid. The filtrate was collected by filtration, spin-dried, cooled to 0 deg.C, added with 40mL of TFA, warmed to room temperature for 1 hour, and then warmed to 70 deg.C for 1 hour. After the solvent of the reaction system was removed by spinning under reduced pressure, 50mL of ethanol was added, the mixture was cooled to 0 ℃ and 75mL of 6M sodium hydroxide solution was added at this temperature, and the mixture was allowed to warm to room temperature for 15 minutes. After completion of the reaction, 40mL of concentrated hydrochloric acid was added to the system, and a yellow solid was precipitated. Removing solvent in the system by rotary removal under reduced pressure, adding 100mL of chloroform and 100mL of aqueous solution, extracting and collecting organic phase, washing with saturated saline solution, drying with anhydrous magnesium sulfate, filtering and collecting filtrate, vacuum concentrating to obtain oily crude product, adding the crude product into n-hexane, and separating out yellow solid. Filtration and washing with ethanol and ether, respectively, collected the yellow solid product, which was spin dried to give compound S8(5.44g, 51% yield).¹H NMR(400MHz,DMSO)δ12.75(s,1H),8.68(s,1H),5.87(ddd,J＝15.6,10.1,5.0Hz,1H),5.29-4.91(m,2H),4.38(d,J＝4.3Hz,2H),2.53(s,3H)。

And 5: synthesis of Compound S10

Compound S8(900mg,4.05mmol), compound S9(1.14g,5.27mmol) and dimethylethylenediamine (1mL) were sequentially added to 10mL of a1, 4-dioxane solution, and after the system was sufficiently dissolved, potassium carbonate (1.12g,8.1mmol) and cuprous iodide (770mg,4.05mmol) were added to the reaction system, and the temperature was raised to 90 ℃ to react for 18 hours. After the reaction is finished, cooling to room temperature, adding 20mL of ammonia water solution, extracting with ethyl acetate for three times, and combiningThe organic phase was washed with brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was collected and concentrated to give a crude product. The crude product was isolated and purified by silica gel column chromatography to give S10 as a solid compound (1.04g, 72% yield).¹H NMR(400MHz,CDCl₃)δ8.96(s,1H),7.93(t,J＝7.9Hz,1H),7.78(dd,J＝8.1,0.7Hz,1H),7.42(dd,J＝7.7,0.8Hz,1H),5.71(ddt,J＝16.4,10.2,6.2Hz,1H),5.07(dd,J＝10.2,1.1Hz,1H),4.95(dq,J＝17.0,1.3Hz,1H),4.82(dt,J＝6.2,1.2Hz,2H),3.82(s,1H),2.60(s,3H),1.60(s,6H)。

Step 6: synthesis of Compound S12

Compound S10(2.23g,6.24mmol) was added to 60mL of a toluene solution, followed by m-CPBA (1.18g,6.86mmol) and stirred at room temperature for 1 hour, giving a yellow-green solution. DIPEA (5mL) and compound S11(1.9g,6.86mmol) were added to the reaction system and reacted at room temperature for 18 hours. After the reaction is finished, ethyl acetate is added for extraction, an organic phase is collected, saturated sodium bicarbonate and saturated saline are washed sequentially, anhydrous magnesium sulfate is dried, filtration is carried out, a filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S12 as a yellow solid (1.98g, 54% yield).¹H NMR(400MHz,DMSO)δ8.84(s,1H),8.10-7.99(m,1H),7.76(d,J＝7.9Hz,1H),7.65-7.56(m,3H),6.96(d,J＝8.8Hz,2H),5.67(dq,J＝10.6,5.9Hz,1H),5.35(s,1H),5.00(d,J＝10.2Hz,1H),4.83(d,J＝17.2Hz,1H),4.69(d,J＝4.9Hz,2H),3.47(s,4H),3.06(s,4H),2.88(s,1H),1.47(s,6H),1.43(s,9H)。

And 7: synthesis of Compound S13

Compound S12(1g,1.70mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S13, which was directly used in the next step without further purification.

And 8: synthesis of Compound S14

Compound S13(820mg,1.69mmol) and tert-butyl bromoacetate (300uL) were added to a 5mL DMF solution, and after the system was completely dissolved, potassium carbonate (466mg,3.88mmol) was added, and the temperature was raised to 60 ℃ for reaction for 6 hours. After the reaction was completed, 10mL of water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S14(648mg, 64% yield) as a yellow solid.¹H NMR(400MHz,DMSO)δ8.83(s,1H),8.06(t,J＝7.3Hz,1H),7.76(d,J＝7.8Hz,1H),7.67-7.50(m,3H),6.93(d,J＝8.9Hz,2H),5.67(ddt,J＝16.3,10.3,6.0Hz,1H),5.34(s,1H),5.00(dd,J＝10.2,0.8Hz,1H),4.83(dd,J＝17.1,1.0Hz,1H),4.69(d,J＝5.3Hz,2H),3.17(s,2H),3.11(s,4H),2.66(s,4H),1.47(s,6H),1.43(s,9H)。

And step 9: synthesis of Compound S15

Compound S14(600mg,1mmol) was added to a mixed solvent of 1mL TFA and 2mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S15, which was directly used in the next step without further purification.

Example 2: general synthetic route to Compounds 1-3

Synthetic route to compound 1:

step 1: synthesis of Compound S18

Compounds S13(250mg,0.51mmol) and S17(148mg,0.62mmol) were added to a 10mL DMF solution, and after the system was completely dissolved, potassium carbonate (141mg,1.02mmol) was added and the reaction was allowed to warm to 60 ℃ for 6 hours. After the reaction was completed, 100mL of water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S18 as a yellow solid (220mg, 67% yield).¹H NMR(400MHz,DMSO)δ10.17(s,1H),8.83(d,J＝1.0Hz,1H),8.05(s,1H),7.75(d,J＝7.3Hz,1H),7.65-7.46(m,3H),6.92(d,J＝7.6Hz,2H),6.83(s,1H),5.66(dd,J＝14.5,8.9Hz,1H),5.33(s,1H),4.99(d,J＝10.2Hz,1H),4.82(d,J＝17.1Hz,1H),4.72-4.63(m,2H),3.16-3.02(m,4H),2.94(t,J＝12.5Hz,2H),2.50(s,4H),2.37-2.23(m,2H),1.59(t,J＝12.0Hz,2H),1.46(s,6H),1.38(s,9H)。

Step 2: synthesis of Compound S19

Compound S18(150mg,0.23mmol) was added to a mixed solvent of 1mL TFA and 2mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S19, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 1

Compounds S16(56mg,0.20mmol) and S19(100mg,0.18mmol) were added to a 3mL DMF solution, DIPEA (70uL) was added under nitrogen, and the reaction was warmed to 90 ℃ for 12 hours. After the reaction is finished, 30mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography to give yellow solid Compound 1(98mg, 68% yield)Rate).¹H NMR (400MHz, DMSO) δ 11.09(s,1H),10.17(s,1H),8.83(s,1H),8.10-8.00(m,1H),7.75(d, J ═ 7.6Hz,1H),7.70-7.47(m,4H),7.16(d, J ═ 8.4Hz,1H),7.11-6.86(m,3H),6.79(t, J ═ 5.4Hz,1H),5.67(ddd, J ═ 17.1,10.8,5.9Hz,1H),5.34(s,1H),5.09-4.95(m,2H),4.82(d, J ═ 16.9Hz,1H),4.74-4.59(m,2H),3.53-3.34(m, 3.03H), 3.94 (m, 3.01-4H), 3.49 (m,1H), 1H, 3.49 (d, 1H), 3.49H, 1H, and 1H. HRMS (ESI) m/z calculated value C₄₂H₄₅N₁₁O₆[M+H]⁺: 800.3633, found: 800.3650.

synthesis of Compound 2: the concrete steps are as in the synthesis example of the compound 1.¹H NMR (400MHz, DMSO) δ 11.10(s,1H),10.15(s,1H),8.83(s,1H),8.05(s,1H),7.76(d, J ═ 7.3Hz,1H),7.70-7.49(m,4H),7.14(d, J ═ 8.3Hz,1H),7.03(d, J ═ 6.7Hz,1H),6.92(d, J ═ 8.0Hz,2H),6.59(s,1H),5.67(dd, J ═ 16.5,9.7Hz,1H),5.32(s,1H),5.06(dd, J ═ 12.6,2.9Hz,1H),5.00(d, J ═ 10.1Hz,1H),4.83(d, 17.0 (d, J ═ 3.65), 3.3.65 (d, J ═ 3.0, 3.3H), 3.3.3H, 3.3H, 3.3.3 (d, 3-2H), 3.3H, 3H, 3.65 (d, 3.3H), 3H, 3.3H, 3.3.3.3.0 (d, 3H), 1H) 1.70-1.51(m,4H),1.47(s, 6H). HRMS (ESI) m/z calculated value C₄₃H₄₇N₁₁O₆[M+H]⁺: 814.3789, found: 814.3760.

synthesis of Compound 3: the concrete steps are as in the synthesis example of the compound 1.¹H NMR (400MHz, DMSO) δ 11.10(s,1H),10.16(s,1H),8.83(s,1H),8.09-7.98(m,1H),7.76(d, J ═ 6.1Hz,1H),7.69-7.47(m,4H),7.11(d, J ═ 8.3Hz,1H),7.03(d, J ═ 6.6Hz,1H),6.91(d, J ═ 7.6Hz,2H),6.55(s,1H),5.67(dd, J ═ 14.7,7.9Hz,1H),5.33(s,1H),5.02(dd, J ═ 22.6,9.4Hz,2H),4.83(d, J ═ 17.0, 1H),4.69(s, 1H), 3.34H), 3.18 (m, 3.55H), 2.55H, 2H, 2.3.55 (m ═ 2H, 3.0, 3.55H, 2H, 1, 2H) 1.55-1.43(m,8H),1.40-1.32(m, 2H). HRMS (ESI) m/z calculated value C₄₄H₄₉N₁₁O₆[M+H]⁺: 828.3946, found: 828.3923.

example 3: general synthetic route to Compounds 4-7

Synthetic route for compound 4:

step 1: synthesis of Compound S21

Compounds S16(1g,3.62mmol) and S20(630mg,3.62mmol) were added to a 10mL DMF solution, DIPEA (1.5mL) was added under nitrogen, and the reaction was warmed to 90 ℃ for 12 hours. After the reaction is finished, 100mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S21 as a yellow solid (1.17g, 75% yield).¹H NMR(400MHz,DMSO)δ11.09(s,1H),7.57(t,J＝7.3Hz,1H),7.08(d,J＝8.4Hz,1H),7.02(d,J＝6.6Hz,1H),6.91(s,1H),6.66(s,1H),5.05(d,J＝9.1Hz,1H),3.29(t,J＝12.3Hz,2H),3.00(d,J＝4.4Hz,2H),2.89(t,J＝15.1Hz,1H),2.64-2.53(m,2H),2.09-1.96(m,1H),1.72-1.59(m,2H),1.38(s,9H)。

Step 2: synthesis of Compound S22

Compound S21(200mg,0.47mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S22, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 4

Compound S15(163mg,0.30mmol) and compound S22(100mg,0.30mmol) were added to 5mL of DMF and stirredAfter dissolution with stirring, HATU (230mg,0.60mmol) and DIPEA (200uL) were added and the mixture was reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 4 as a yellow solid (156mg, 61% yield).¹H NMR (400MHz, DMSO) δ 11.11(s,1H),10.18(s,1H),8.84(s,1H),8.18-7.99(m,2H),7.76(d, J ═ 7.4Hz,1H),7.67-7.48(m,4H),7.10(d, J ═ 8.6Hz,1H),7.03(d, J ═ 6.8Hz,1H),6.94(d, J ═ 8.0Hz,2H),6.75(s,1H),5.67(dd, J ═ 16.4,10.0Hz,1H),5.34(s,1H),5.07(d, J ═ 10.9Hz,1H),5.00(d, J ═ 10.0, 1H),4.83(d, 17.65, 1H),5.07(d, J ═ 2H), 3.73H, 3.0H, 3.73 (m, 3.0H), 3.73 (d, 2H, 3.0H), 3.73 (m, 3.0H, 3.0H), 3.0 (m-2H), 2H) 1.47(s,6H),1.31-1.18(m, 2H). HRMS (ESI) m/z calculated value C₄₄H₄₈N₁₂O₇[M+H]⁺: 857.3847, found: 857.3856.

synthesis of Compound 5: the concrete steps are as in the synthesis example of the compound 4.¹H NMR (400MHz, DMSO) δ 11.10(s,1H),10.17(s,1H),8.84(s,1H),8.05(s,1H),7.90(s,1H),7.76(d, J ═ 7.0Hz,1H),7.68-7.52(m,4H),7.12(d, J ═ 8.4Hz,1H),7.02(d, J ═ 6.7Hz,1H),6.92(d, J ═ 8.0Hz,2H),6.58(s,1H),5.67(dd, J ═ 15.9,9.5Hz,1H),5.34(s,1H),5.03(dd, J ═ 21.8,10.1, 2H),4.83(d, 17.0, 1H),4.76 (s,1H), 3.6.6.6, 6, 13H, 3.06H, 3.6.6, 6.6, 6, 2H, 6.6, 2H, 4.6.6, 4(dd, 15, 2H), 4.6.6, 3, 3.6.6, 6, 6.6.6, 6, 6.6, 6, 3, 6, 3, 6.6, 2H, 3, 2H, 6, 3, etc. HRMS (ESI) m/z calculated value C₄₅H₅₀N₁₂O₇[M+Na]⁺: 893.3823, found: 893.3845.

synthesis of Compound 6: the concrete steps are as in the synthesis example of the compound 4.¹H NMR(400MHz,DMSO)δ11.10(s,1H),10.15(s,1H),8.83(s,1H),8.05(t,J＝6.8Hz,1H),7.85-7.72(m,2H),7.67-7.51(m,4H),7.10(d,J＝8.4Hz,1H),7.02(d,J＝6.8Hz,1H),6.92(d,J＝8.1Hz,2H),6.60-6.48(m,1H),5.67(ddt,J＝16.0,10.7,5.2Hz,1H),5.33(s,1H),5.11-4.93(m,2H),4.84(d,J＝17.2Hz,1H),4.75-4.61(m,2H),3.35-3.27(m,4H),3.22-3.09(m,6H),3.01-2.79(m,3H),2.67-2.55(m,5H),2.07-1.97(m,1H),1.65-155(m,2H),1.47(s,6H),1.40-1.28(m, 2H). HRMS (ESI) m/z calculated value C₄₆H₅₂N₁₂O₇[M+Na]⁺: 907.3980, found: 907.3984.

synthesis of compound 7: the concrete steps are as in the synthesis example of the compound 4.¹H NMR (400MHz, DMSO) δ 11.10(s,1H),10.16(s,1H),8.84(s,1H),8.05(s,1H),7.86-7.70(m,2H),7.67-7.52(m,4H),7.09(d, J ═ 8.5Hz,1H),7.02(d, J ═ 6.9Hz,1H),6.93(d, J ═ 8.0Hz,2H),6.54(s,1H),5.67(td, J ═ 15.8,5.8Hz,1H),5.33(s,1H),5.10-4.96(m,2H),4.83(d, J ═ 17.1Hz,1H),4.74-4.61(m,2H),3.29(d, 6.5, 2H),3.7 (m,2H), 3.15, 2H), 3.0H, 2H), 3.7.7-7.7 (m,1H), 3.7H), 5.7 (d, 2H), 3.7H, 3.8H), 5.7 (m ═ 2H), 3.7H, 2H), 1.67-1.52(m,2H),1.48-1.24(m, 12H). HRMS (ESI) m/z calculated value C₄₇H₅₄N₁₂O₇[M+H]⁺: 899.4317, found: 899.4307.

example 4: synthetic route to Compound 8

Step 1: synthesis of Compound S24

Compounds S16(1g,3.62mmol) and S23(700mg,4.34mmol) were added to a 10mL DMF solution, DIPEA (1.9mL) was added under nitrogen, and the reaction was warmed to 90 ℃ for 12 hours. After the reaction is finished, 100mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S24 as a yellow solid (950mg, 63% yield).¹H NMR(400MHz,DMSO)δ11.09(s,1H),7.58(t,J＝7.8Hz,1H),7.14(d,J＝8.6Hz,1H),7.03(d,J＝7.1Hz,2H),6.71(s,1H),5.05(dd,J＝12.9,5.2Hz,1H),3.40-3.34(m,2H),3.12(dd,J＝11.4,5.8Hz,2H),2.96-2.83(m,1H),2.59(d,J＝17.4Hz,1H),2.52-2.49(m,1H),2.07-1.96(m,1H),1.36(s,9H)。

Step 2: synthesis of Compound S25

Compound S24(190mg,0.46mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S25, which was directly used in the next step without further purification.

And step 3: synthesis of Compound S27

Compound S25(150mg,0.48mmol) and compound S26(114mg,0.52mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (360mg,0.95mmol) and DIPEA (340uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S27 as a yellow solid (139mg, 57% yield).¹H NMR(400MHz,DMSO)δ11.11(s,1H),8.03(s,1H),7.59(t,J＝7.7Hz,1H),7.18(d,J＝8.5Hz,1H),7.03(d,J＝6.9Hz,1H),6.84-6.69(m,2H),5.06(dd,J＝12.6,4.8Hz,1H),3.37(d,J＝6.0Hz,2H),3.23(d,J＝5.3Hz,2H),2.97-2.79(m,3H),2.65-2.52(m,2H),2.04(t,J＝6.8Hz,3H),1.51-1.42(m,2H),1.36(s,11H)。

And 4, step 4: synthesis of Compound S28

Compound S27(140mg,0.27mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S28, which was directly used in the next step without further purification.

And 5: synthesis of Compound 8

Compound S15(50mg,0.09mmol) and compound S28(42mg,0.10mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (70mg,0.18mmol) and DIPEA (200uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 8 as a yellow solid (61mg, 71% yield).¹H NMR (400MHz, DMSO) δ 11.10(s,1H),10.17(s,1H),8.83(s,1H),8.04(t, J ═ 5.1Hz,2H),7.75(d, J ═ 7.8Hz,2H),7.66-7.52(m,4H),7.17(d, J ═ 8.6Hz,1H),7.02(d, J ═ 7.0Hz,1H),6.93(d, J ═ 8.6Hz,2H),6.74(t, J ═ 6.0Hz,1H),5.73-5.61(m,1H),5.33(s,1H),5.06(dd, J ═ 12.9,5.3, 1H),5.00(d, J ═ 10.2, 1H), 4.33 (s,1H),5.06(dd, 3, 1H),5.00(d, J ═ 10.9, 3.3, 1H),5.00(d, 3, 3.3, 3, 2H),3.3, 3H), 5.3, 3.3H, 3H, 3.3.3, 3, 3.3, 3H, 3H, 3H, etc., 5H) 2.14-1.96(m,3H),1.56-1.43(m,8H),1.43-1.35(m, 2H). HRMS (ESI) m/z calculated value C₄₈H₅₅N₁₃O₈[M+H]⁺: 942.4375, found: 942.4369.

example 5: synthetic route to compound 9

Step 1: synthesis of Compound S31

Compounds S29(100mg,0.37mmol) and S30(68mg,0.31mmol) were added to a 3mL DMF solution, and after the system was completely dissolved, potassium carbonate (84mg,0.61mmol) was added and the reaction was allowed to warm to 60 ℃ for 6 hours. After the reaction was completed, 30mL of water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S31 as a yellow solid (92mg, 72% yield).¹H NMR(400MHz,DMSO)δ11.11(s,1H),7.81(t,J＝7.5Hz,1H),7.54(d,J＝8.3Hz,1H),7.46(d,J＝6.9Hz,1H),6.98(s,1H),5.09(dd,J＝12.0,4.4Hz,1H),4.23(s,2H),3.34(s,2H),2.89(t,J＝13.3Hz,1H),2.68-2.53(m,2H),2.13-1.95(m,1H),1.37(s,9H)。

Step 2: synthesis of Compound S32

Compound S31(224mg,0.54mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S32, which was directly used in the next step without further purification.

And step 3: synthesis of Compound S33

Compound S32(150mg,0.47mmol) and compound S26(114mg,0.52mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (360mg,0.95mmol) and DIPEA (340uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S33(132mg, 55% yield) as a yellow solid.¹H NMR(400MHz,CDCl₃)δ9.06(s,1H),7.71(t,J＝7.8Hz,1H),7.52(d,J＝7.3Hz,1H),7.27(s,2H),6.76(s,1H),4.98(d,J＝5.9Hz,1H),4.39-4.13(m,2H),3.81-3.63(m,2H),3.23-3.03(m,3H),2.93-2.80(m,2H),2.24(t,J＝7.6Hz,2H),2.19-2.11(m,1H),1.70-1.56(m,2H),1.43(s,11H)。

And 4, step 4: synthesis of Compound S34

Compound S33(85mg,0.16mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S34, which was directly used in the next step without further purification.

And 5: synthesis of Compound 9

Compound S15(50mg,0.09mmol) and compound S34(42mg,0.10mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (70mg,0.18mmol) and DIPEA (200uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography to give compound 9 as a yellow solid (45mg, 53% yield).¹H NMR (400MHz, DMSO) δ 11.11(s,1H),10.16(s,1H),8.83(s,1H),8.05(t, J ═ 5.2Hz,2H),7.90-7.70(m,3H),7.68-7.51(m,4H),7.46(d, J ═ 7.2Hz,1H),6.93(d, J ═ 8.7Hz,2H),5.67(dq, J ═ 10.4,6.0Hz,1H),5.33(s,1H),5.09(dd, J ═ 12.8,5.3Hz,1H),5.00(d, J ═ 10.1Hz,1H),4.83(d, J ═ 17.1Hz,1H),4.69(d, J ═ 1H, 5.24, 24H), t, 2.5.5.5.5 (d, J ═ 10.5.1 Hz, 5H), 5.83 (d, J ═ 2H, 5.06 Hz, 5.06H, 5.5.5.5, 5.5.5.5.5, 5, 5.5.5 (d, 3H, 2H, 5, 5.06, 2H, 5.06H, 5, 2H, 5, 3H, 5H, 5, 2H, 5, 2H, 3H, 2H, 5, 2H, 5, 3H, 5, 2H, 5, 2H, 3H, 2H, 5, 2H, 5, 2H, 3H, 2H, 3H, 2H, 3H, 2H, 3H, 2H, 3H, 2H, 3H, 2H, 3H, 2H, 3H, 2H, etc., 1H) 1.57-1.34(m, 10H). HRMS (ESI) m/z calculated value C₄₈H₅₄N₁₂O₉[M+Na]⁺: 965.4034, found: 965.4001.

example 6: synthetic route to Compound 10

Step 1: synthesis of Compound S36

Compounds S16(200mg,0.73mmol) and S35(180mg,0.76mmol) were added to a 5mL solution of DMF and DIPEA (390uL) was added under nitrogen, and the reaction was warmed to 90 ℃ for 12 hours. After the reaction was completed, 50mL of water was added to the system, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentratedAnd (5) condensing to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S36 as a yellow solid (235mg, 66% yield).¹H NMR(400MHz,DMSO)δ11.11(s,1H),7.50(t,J＝6.9Hz,1H),7.40-7.27(m,3H),7.20(s,3H),7.01(d,J＝6.6Hz,1H),6.94(d,J＝8.2Hz,1H),5.07(dd,J＝11.5,3.6Hz,1H),4.53(d,J＝3.4Hz,2H),4.08(s,2H),2.89(t,J＝13.4Hz,1H),2.66-2.54(m,2H),2.11-1.98(m,1H),1.38(s,9H)。

Step 2: synthesis of Compound S37

Compound S36(160mg,0.33mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S37, which was directly used in the next step without further purification.

And step 3: synthesis of Compound S38

Compound S37(250mg,0.64mmol) and compound S26(153mg,0.70mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (485mg,1.28mmol) and DIPEA (500uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S38(207mg, 54% yield) as a yellow solid.¹H NMR(400MHz,DMSO)δ11.12(s,1H),8.25(s,1H),7.50(t,J＝7.6Hz,1H),7.31(d,J＝7.6Hz,2H),7.19(d,J＝7.7Hz,3H),7.01(d,J＝7.0Hz,1H),6.93(d,J＝8.4Hz,1H),6.79(s,1H),5.07(dd,J＝12.8,4.9Hz,1H),4.53(d,J＝5.3Hz,2H),4.22(d,J＝5.1Hz,2H),2.89(d,J＝4.9Hz,3H),2.67-2.53(m,2H),2.10(t,J＝7.1Hz,2H),2.06-1.97(m,1H),1.47(d,J＝6.9Hz,2H),1.36(s,11H)。

And 4, step 4: synthesis of Compound S39

Compound S38(100mg,0.17mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S39, which was directly used in the next step without further purification.

And 5: synthesis of Compound 10

Compound S15(50mg,0.09mmol) and compound S39(50mg,0.10mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (70mg,0.18mmol) and DIPEA (200uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 10(49mg, 54% yield) as a yellow solid.¹H NMR (400MHz, DMSO) δ 11.12(s,1H),10.18(s,1H),8.83(s,1H),8.28(t, J ═ 5.8Hz,1H),8.05(t, J ═ 7.4Hz,1H),7.77(t, J ═ 12.3Hz,2H),7.59(t, J ═ 14.1Hz,3H),7.50-7.45(m,1H),7.30(d, J ═ 8.1Hz,2H),7.23-7.18(m,3H),7.01(t, J ═ 5.3Hz,1H),6.92(t, J ═ 9.3Hz,3H),5.67(dq, J ═ 11.1,5.9, 1H),5.34(s, 1.5, 5H), 6.92(t, J ═ 9.3Hz,3H),5.67 (ddq, 11.1,5.9, 1H), 3H, 5, 4H, 4, 3H, 5, 4H, 3H, 4H, 3H, 4H, 3H, 4H, 3H, 4H, 3H, 4H, 3H, 3H, 3H, 3H, 4H, 3H, 4H, 6, 4H, 3H, 4H, 3H, 3H, 3H, 4H, 3H, and so on (d, 6.6.6.6.6.6, 6.6, 6, 6.6.6, 6, 6.6, 6H, 6H, 6, 6H) 2.95-2.83(m,3H),2.70-2.53(m,5H),2.14(t, J ═ 7.2Hz,2H),2.07-2.01(m,1H),1.56-1.38(m, 10H). HRMS (ESI) m/z calculated value C₅₄H₅₉N₁₃O₈[M+H]⁺: 1018.2663, found: 1018.2642.

example 7: synthetic route to compound 11

Step 1: synthesis of Compound S41

Compounds S29(100mg,0.37mmol) and S40(82mg,0.37mmol) were added to a 3mL DMF solution, and after the system was completely dissolved, potassium carbonate (100mg,0.74mmol) was added and the reaction was allowed to warm to 60 ℃ for 6 hours. After the reaction was completed, 30mL of water was added, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S41 as a yellow solid (65mg, 43% yield).¹H NMR(400MHz,DMSO)δ11.12(s,1H),7.83(t,J＝7.7Hz,1H),7.59(d,J＝8.5Hz,1H),7.55(d,J＝7.5Hz,2H),7.48(d,J＝7.1Hz,1H),7.43(d,J＝7.5Hz,2H),5.39(s,2H),5.11(dd,J＝12.6,4.8Hz,1H),4.47(s,2H),2.97-2.81(m,1H),2.66-2.53(m,2H),2.11-1.97(m,1H)。

Step 2: synthesis of Compound S42

Compound S41(60mg,0.14mmol) was added to a mixed solution of 1M HCl (2mL) and ether (2mL), triphenylphosphine (38mg, 0.14mmol) was added under ice bath, and after completion of addition, the mixture was warmed to room temperature to react for 12 hours. After the reaction, 4M sodium carbonate solution was added to adjust PH to 10, 5mL water was added, ethyl acetate was extracted, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S42(34mg, 61% yield) as a yellow solid.

And step 3: synthesis of Compound S43

Compound S42(350mg,0.92mmol) and compound S26(200mg,0.92mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (700mg,1.85mmol) and DIPEA (500uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S43 as a yellow solid (377mg, 69% yield).

And 4, step 4: synthesis of Compound S44

Compound S43(350mg,0.59mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S44, which was directly used in the next step without further purification.

And 5: synthesis of Compound 11

Compound S15(50mg,0.09mmol) and compound S44(50mg,0.10mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (70mg,0.18mmol) and DIPEA (200uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 11 as a yellow solid (62mg, 68% yield).¹H NMR (400MHz, DMSO) δ 11.08(s,1H),10.17(s,1H),8.83(s,1H),8.33(s,1H),8.05(s,1H),7.84-7.72(m,3H),7.69-7.52(m,4H),7.44(d, J-7.2 Hz,3H),7.28(d, J-7.3 Hz,2H),6.92(d, J-7.5 Hz,2H),5.66(dt, J-15.9, 8.0Hz,1H),5.42-5.23(m, dd3H), 5.09(d, J-12.3, 4.6Hz,1H),5.00(d, J-10.0, 1H),4.83(d, 17.17H), 4.63 (d, 2H), 4.27H, 2H, 3, 2H), 3.9-2H, 3H, 2H, 3H, 2H, 3H, 5.00(d, 2H, 3H, 2H, 3H, 2H, 3H, 2H, 3H, etc., 1H) 1.57-1.39(m, 10H). HRMS (ESI) m/z calculated value C₅₄H₅₈N₁₂O₉[M+H]⁺: 1019.4528, found: 1019.4504.

example 8: synthetic route to compound 12

Step 1: synthesis of Compound S46

Compounds S16(500mg,1.81mmol) and S45(450mg,1.81mmol) were added to a 10mL DMF solution, DIPEA (900uL) was added under nitrogen, the reaction was warmed to 90 ℃ and reacted for 12 hours. After the reaction is finished, 150mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S46(572mg, 63% yield) as a yellow solid.¹H NMR(400MHz,DMSO)δ11.09(s,1H),7.59(t,J＝7.8Hz,1H),7.15(d,J＝8.6Hz,1H),7.04(d,J＝7.0Hz,1H),6.72(s,1H),6.61(t,J＝5.5Hz,1H),5.06(dd,J＝12.9,5.3Hz,1H),3.62(t,J＝5.3Hz,2H),3.58-3.54(m,2H),3.53-3.50(m,2H),3.47(d,J＝5.4Hz,2H),3.38(t,J＝6.0Hz,2H),3.06(dd,J＝11.5,5.6Hz,2H),2.94-2.81(m,1H),2.66-2.53(m,2H),2.08-1.97(m,1H),1.36(s,9H)。

Step 2: synthesis of Compound S47

Compound S46(520mg,1.03mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane, and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S47, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 12

Compound S15(50mg,0.09mmol) and compound S47(42mg,0.10mmol) were added to 3mL of DMF, and after dissolving with stirring, HATU (70mg,0.18mmol) and DIPEA (200uL) were added and reacted at room temperature for two hours. After the reaction is completed, the reaction solution is fed into the systemAdding 30mL of water, extracting with ethyl acetate, combining organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering, collecting filtrate and concentrating to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 12 as a yellow solid (44mg, 53% yield).¹H NMR (400MHz, DMSO) δ 11.02(s,1H),10.08(s,1H),8.74(s,1H),7.96(t, J ═ 7.2Hz,1H),7.75-7.60(m,2H),7.59-7.35(m,4H),6.99(d, J ═ 8.6Hz,1H),6.92(d, J ═ 7.0Hz,1H),6.82(d, J ═ 8.6Hz,2H),6.51(t, J ═ 5.5Hz,1H),5.64-5.51(m,1H),5.25(s,1H),4.96(dd, J ═ 13.0,5.4Hz,1H),4.91(d, J ═ 10.2, 1H),4.73(d, 1H),4.96(dd, 3.7H), 3.7H), 3.7 (d, 3.7H), 3.7 (d, 3.6, 3.7H, 3, 3.7 (d, 3.7H), 1H) 2.56-2.44(m,5H),2.00-1.86(m,1H),1.37(s, 6H). HRMS (ESI) m/z calculated value C₄₇H₅₄N₁₂O₉[M+H]⁺: 931.4215, found: 931.4231.

example 9: synthetic route to compound 13

Step 1: synthesis of Compound S49

Compounds S16(236mg,0.86mmol) and S48(250mg,0.86mmol) were added to a 5mL solution of DMF and DIPEA (600uL) was added under nitrogen, and the reaction was warmed to 90 ℃ for 12 hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S49 as a yellow solid (248mg, 46% yield).¹H NMR(400MHz,DMSO)δ11.10(s,1H),7.58(t,J＝7.8Hz,1H),7.15(d,J＝8.6Hz,1H),7.04(d,J＝7.0Hz,1H),6.74(s,1H),6.61(t,J＝5.3Hz,1H),5.06(dd,J＝12.8,5.2Hz,1H),3.62(t,J＝5.1Hz,2H),3.59-3.42(m,10H),3.36(d,J＝6.0Hz,2H),3.05(dd,J＝11.3,5.5Hz,2H),2.96-2.80(m,1H),2.65-2.52(m,2H),2.08-1.96(m,1H),1.36(s,9H)。

Step 2: synthesis of Compound S50

Compound S49(220mg,0.40mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S50, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 13

Compound S15(120mg,0.22mmol) and compound S50(100mg,0.22mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (170mg,0.45mmol) and DIPEA (100uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 13 as a yellow solid (115mg, 53% yield).¹H NMR (400MHz, DMSO) δ 11.11(s,1H),10.16(s,1H),8.83(s,1H),8.07(t, J ═ 16.1Hz,1H),7.75(d, J ═ 5.4Hz,2H),7.64-7.51(m,4H),7.09(d, J ═ 8.6Hz,1H),7.02(d, J ═ 6.9Hz,1H),6.93(d, J ═ 8.2Hz,2H),6.61-6.51(m,1H),5.67(dq, J ═ 10.5,5.7Hz,1H),5.34(s,1H),5.10-4.96(m,2H),4.83(d, J ═ 17.2Hz,1H),4.67 (J ═ 4.67, 3.7H), 3.7H), 3.3.7H, 3.7H, 3H, 3.7H, 3H, 2.07-2.01(m,1H),1.47(s, 6H). HRMS (ESI) m/z calculated value C₄₉H₅₈N₁₂O₁₀[M+H]⁺: 975.4477, found: 975.4524.

example 10: synthetic route to compound 14

Step 1: synthesis of Compound S52

Compounds S16(500mg,1.81mmol) and S51(600mg,1.81mmol) were added to a 10mL DMF solution, DIPEA (600uL) was added under nitrogen, the reaction was warmed to 90 ℃ and reacted for 12 hours. After the reaction is finished, 150mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S52 as a yellow solid (728mg, 68% yield).¹H NMR(400MHz,DMSO)δ11.10(s,1H),7.58(t,J＝7.6Hz,1H),7.15(d,J＝8.5Hz,1H),7.04(d,J＝6.9Hz,1H),6.74(s,1H),6.61(s,1H),5.06(dd,J＝12.5,4.1Hz,1H),3.71-3.61(m,2H),3.59-3.44(m,14H),3.36(s,2H),3.05(d,J＝5.1Hz,2H),2.89(t,J＝13.4Hz,1H),2.65-2.52(m,2H),2.09-1.94(m,1H),1.37(s,9H)。

Step 2: synthesis of Compound S53

Compound S52(200mg,0.34mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S53, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 14

Compound S15(55mg,0.10mmol) and compound S53(50mg,0.10mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (76mg,0.20mmol) and DIPEA (100uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 14 as a yellow solid (63mg, 65% yield).¹H NMR(400MHz,DMSO)δ11.10(s,1H),10.18(s,1H),8.83(s,1H),8.09-8.01(m,1H),7.75(d, J ═ 7.4Hz,1H),7.68-7.51(m,4H),7.12(d, J ═ 8.4Hz,1H),7.03(d, J ═ 6.9Hz,1H),6.95(d, J ═ 8.0Hz,2H),6.59(s,1H),5.67(td, J ═ 15.7,6.8Hz,1H),5.34(s,1H),5.10-4.95(m,2H),4.83(d, J ═ 17.1Hz,1H),4.75-4.62(m,2H),3.62-3.42(m,18H),3.33-3.09(m,8H),2.94-2.53(m,6H), 2.07-1H (m,1H), 1.07-1H), 1.46 (m, 4H). HRMS (ESI) m/z calculated value C₅₁H₆₂N₁₂O₁₁[M+H]⁺: 1019.4739, found: 1019.4739.

example 11: synthetic route to compound 15

Step 1: synthesis of Compound S55

Compounds S16(240mg,0.86mmol) and S54(350mg,0.86mmol) were added to a 5mL DMF solution, DIPEA (300uL) was added under nitrogen, and the reaction was warmed to 90 ℃ for 12 hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S55 as a yellow solid (428mg, 79% yield).¹H NMR(400MHz,DMSO)δ11.12(s,1H),7.59(t,J＝7.8Hz,1H),7.15(d,J＝8.6Hz,1H),7.04(d,J＝7.0Hz,1H),6.77(s,1H),6.62(t,J＝5.4Hz,1H),5.06(dd,J＝12.9,5.3Hz,1H),3.62(t,J＝5.1Hz,2H),3.57-3.46(m,18H),3.37(d,J＝6.0Hz,2H),3.05(dd,J＝11.5,5.6Hz,2H),2.95-2.81(m,1H),2.66-2.53(m,2H),2.08-1.97(m,1H),1.37(s,9H)。

Step 2: synthesis of Compound S56

Compound S55(200mg,0.31mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S56, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 15

Compound S15(50mg,0.09mmol) and compound S56(54mg,0.10mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (70mg,0.18mmol) and DIPEA (200uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 15(49mg, 51% yield) as a yellow solid.¹H NMR (400MHz, DMSO) δ 11.12(s,1H),10.18(s,1H),8.84(s,1H),8.06(s,1H),7.77(d, J ═ 4.8Hz,2H),7.70-7.49(m,4H),7.12(d, J ═ 8.6Hz,1H),7.04(d, J ═ 7.0Hz,1H),6.94(d, J ═ 8.3Hz,2H),6.60(t, J ═ 5.1Hz,1H),5.67(dq, J ═ 10.9,5.9Hz,1H),5.35(s,1H),5.06(dd, J ═ 12.9,5.1, 1H),5.00(d, J ═ 10.1, 1H), 4.81H, 4(s,1H), 3.3H, 2H), 3.3.3H, 3H, 3.3H, 3H, 3.3H, 3H, etc., 1H) 2.69-2.55(m,5H),2.09-2.01(m,1H),1.47(s, 6H). HRMS (ESI) m/z calculated value C₅₃H₆₆N₁₂O₁₂[M+H]⁺: 1063.5001, found: 1063.4985.

example 12: synthetic route to compound 16

Step 1: synthesis of Compound S58

Compounds S16(1g,3.62mmol) and S57(1.54g,3.62mmol) were added to a 10mL DMF solution, DIPEA (1.3mL) was added under nitrogen, and the reaction was warmed to 90 ℃ for 12 hours. After the reaction is finished, 150mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. Coarse product warp-knitted fabricSeparation and purification by gel column chromatography gave compound S58 as a yellow solid (1.82g, 74% yield).¹H NMR(400MHz,DMSO)δ11.10(s,1H),7.59(t,J＝7.7Hz,1H),7.15(d,J＝8.5Hz,1H),7.04(d,J＝6.9Hz,1H),6.75(s,1H),6.61(s,1H),5.06(dd,J＝12.5,4.3Hz,1H),3.72-3.62(m,2H),3.58-3.46(m,22H),3.37(s,2H),3.06(d,J＝5.3Hz,2H),2.89(t,J＝13.6Hz,1H),2.66-2.52(m,2H),2.09-1.95(m,1H),1.37(s,9H)。

Step 2: synthesis of Compound S59

Compound S58(1g,1.47mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S59, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 16

Compound S15(45mg,0.08mmol) and compound S59(48mg,0.08mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (63mg,0.17mmol) and DIPEA (100uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 16(53mg, 58% yield) as a yellow solid.¹H NMR (400MHz, DMSO) δ 11.10(s,1H),10.17(s,1H),8.83(s,1H),8.12-8.00(m,1H),7.76(d, J ═ 5.1Hz,2H),7.63-7.54(m,4H),7.13(d, J ═ 8.6Hz,1H),7.04(d, J ═ 6.8Hz,1H),6.93(d, J ═ 8.0Hz,2H),6.67-6.58(m,1H),5.67(dd, J ═ 16.0,9.2Hz,1H),5.33(s,1H),5.09-4.97(m,2H),4.83(d, J ═ 17.0, 1H), 4.75-4.61H, 3.75 (m, 3.60H), 3.50-4.52H (m, 3.50H), 3.52H, 3.70-4.70H, 3.52 (m, 3.50H, 3.52H, 3.50H, 3.70H, 3.52H, 3.70H, 3H, 1H) 1.47(s, 6H). HRMS (ESI) m/z calculated value C₅₅H₇₀N₁₂O₁₃[M+Na]⁺: 1129.5083, found: 1129.5112.

example 13: synthetic route to compound 17

Step 1: synthesis of Compound S61

Compounds S16(700mg,2.56mmol) and S60(1.2g,2.56mmol) were added to a 10mL DMF solution, DIPEA (800uL) was added under nitrogen, and the reaction was warmed to 90 ℃ for 12 hours. After the reaction is finished, 150mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S61 as a yellow solid (1.44g, 78% yield).¹H NMR(400MHz,DMSO)δ11.11(s,1H),7.59(t,J＝7.8Hz,1H),7.15(d,J＝8.6Hz,1H),7.04(d,J＝7.0Hz,1H),6.76(t,J＝5.2Hz,1H),6.61(t,J＝5.6Hz,1H),5.06(dd,J＝12.9,5.3Hz,1H),3.62(t,J＝5.2Hz,2H),3.57-3.46(m,26H),3.39-3.35(m,2H),3.05(dd,J＝11.8,5.9Hz,2H),2.95-2.82(m,1H),2.64-2.53(m,2H),2.08-1.97(m,1H),1.37(s,9H)。

Step 2: synthesis of Compound S62

Compound S61(350mg,0.49mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S62, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 17

Compound S15(87mg,0.16mmol) and compoundS62(100mg,0.16mmol) was added to 5mL of DMF, and after dissolving with stirring, HATU (125mg,0.32mmol) and DIPEA (100uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 17(94mg, 51% yield) as a yellow solid.¹H NMR (400MHz, DMSO) δ 11.10(s,1H),10.18(s,1H),8.84(s,1H),8.30-8.18(m,1H),8.06(t, J ═ 11.6Hz,1H),7.76(d, J ═ 7.1Hz,1H),7.68-7.51(m,4H),7.14(d, J ═ 7.8Hz,1H),7.04(d, J ═ 6.4Hz,1H),6.95(d, J ═ 7.5Hz,2H),6.60(s,1H),5.67(dd, J ═ 16.2,8.7Hz,1H),5.33(s,1H),5.06(d, J ═ 10.9, 1H),5.00(d, J ═ 10.9, 1H), 10.00 (d, J ═ 10.8.7H), 3.69 (d, 3.18H), 3.18(d, 3.3.3H), 3.3.3.3H, 3.3.3 (m,3, 3.3, 3, 1H) 1.47(s, 6H). HRMS (ESI) m/z calculated value C₅₇H₇₄N₁₂O₁₄[M+H]⁺: 1151.5526, found: 1151.5562.

example 14: synthetic route to compound 18

Step 1: synthesis of Compound S64

Compounds S16(360mg,1.29mmol) and S63(660mg,1.29mmol) were added to a 5mL DMF solution, DIPEA (500uL) was added under nitrogen, the reaction was warmed to 90 ℃ and reacted for 12 hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S64 as a yellow solid (674mg, 68% yield).¹H NMR(400MHz,DMSO)δ11.09(s,1H),7.59(t,J＝7.6Hz,1H),7.15(d,J＝8.5Hz,1H),7.04(d,J＝6.8Hz,1H),6.73(s,1H),6.61(s,1H),5.06(dd,J＝12.5,3.9Hz,1H),3.67-3.60(m,2H),3.57-3.44(m,30H),3.37(s,2H),3.06(d,J＝5.0Hz,2H),2.89(t,J＝13.4Hz,1H),2.67-2.52(m,2H),2.09-1.96(m,1H),1.37(s,9H)。

Step 2: synthesis of Compound S65

Compound S64(250mg,0.33mmol) was added to a mixed solvent of 2mL TFA and 4mL dichloromethane and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain compound S65, which was directly used in the next step without further purification.

And step 3: synthesis of Compound 18

Compound S15(80mg,0.15mmol) and compound S65(98mg,0.15mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (112mg,0.30mmol) and DIPEA (80uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 18(112mg, 64% yield) as a yellow solid.¹H NMR (400MHz, DMSO) δ 11.10(s,1H),10.16(s,1H),8.83(s,1H),8.07(d, J ═ 19.8Hz,1H),7.84-7.73(m,2H),7.66-7.53(m,4H),7.14(d, J ═ 8.5Hz,1H),7.04(d, J ═ 6.9Hz,1H),6.94(d, J ═ 8.1Hz,2H),6.68-6.54(m,1H),5.67(ddd, J ═ 14.3,8.7,4.2Hz,1H),5.33(s,1H),5.11-4.96(m,2H),4.83(d, J ═ 17.1, 1H), 4.63-4.73 (m, 3.3H), 3.85 (m,3H), 3.3H, 3H), 3.6.6.6.06 (m,3H), 3H), 3.6.7 (d, 3H), 3H), 2.72-2.53(m,6H),2.06-1.98(m,1H),1.47(s, 6H). HRMS (ESI) m/z calculated value C₅₉H₇₈N₁₂O₁₅[M+H]⁺: 1195.5788, found: 1195.5774.

example 15: synthetic route to compound 19

Step 1: synthesis of Compound S68

Compound S66(166mg,0.45mmol), compound S67(38mg,0.45mmol), PdCl under nitrogen₂(PPh₃)₂(16mg,0.02mmol) and cuprous iodide (4mg,0.02mmol) were sequentially added to 10mL of DME, stirred to dissolve, triethylamine (182mg,1.8mmol) was added, and the reaction was refluxed for 12 hours. After the reaction is finished, 10mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S68 as a white solid (60mg, 41% yield). LC-Mass (ESI) m/z C₁₈H₁₈N₂O₄[M+H]⁺：327.24。

Step 2: synthesis of Compound S69

Compound S68(50mg,0.15mmol) was added to 10mL of dichloromethane under nitrogen, and phosphorus tribromide (182mg,1.8mmol) was added under ice-bath for reaction for 15 minutes. After the reaction is finished, 10mL of water is added into the system, dichloromethane is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, filtrate is collected and concentrated to obtain a product S69, and the product S69 is quickly put into the next step without purification.

And step 3: synthesis of Compound 19

Compound S69(58mg,0.15mmol) and compound S13(73mg,0.15mmol) were added to 5mL of DMF under nitrogen, potassium carbonate (62mg,0.45mmol) was added at room temperature, the mixture was warmed to 60 ℃ and reacted for 6 hours. After completion of the reaction, 50mL of water was added to the system, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to give compound 19 as a white solid (62mg, 53% yield))。1H NMR(400MHz,DMSO)δ11.04(s,1H),10.19(s,1H),8.68–8.05(m,2H),7.74(d,J＝7.3Hz,2H),7.62–7.48(m,2H),7.01(d,J＝7.9Hz,2H),5.75–5.54(m,1H),5.32(s,1H),5.22(d,J＝13.1Hz,1H),5.03(d,J＝11.2Hz,1H),4.81(t,J＝19.5Hz,1H),4.70(s,2H),4.38(dd,J＝57.2,16.8Hz,2H),3.95–3.66(m,6H),3.45–3.34(m,2H),3.18–3.03(m,4H),2.93(d,J＝13.2Hz,1H),2.70(d,J＝32.4Hz,4H),2.61(d,J＝16.2Hz,1H),2.49–2.37(m,1H),2.04(s,1H),1.47(s,6H)。

Example 16: synthetic route to compound 20

Step 1: synthesis of Compound S70

Compound S68(50mg,0.15mmol) and 10% palladium on carbon catalyst (5mg) were added to 10mL of methanol under nitrogen atmosphere, and the hydrogen gas was replaced, followed by reaction for 2 hours. After the reaction was completed, the palladium on carbon was removed by filtration, and the filtrate was collected and concentrated to give S70(48mg, 98% yield) as a solid. LC-Mass (ESI) m/z C₁₈H₂₄N₂O₄[M+H]⁺：331.46。

Step 2: synthesis of Compound S71

Compound S70(50mg,0.15mmol) was added to 10mL of dichloromethane under nitrogen, and phosphorus tribromide (182mg,1.8mmol) was added under ice-bath for reaction for 15 minutes. After the reaction is finished, 10mL of water is added into the system, dichloromethane is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, filtrate is collected and concentrated to obtain a product 71, and the product is quickly put into the next step without purification.

And step 3: synthesis of Compound 20

Compound S71(58mg,0.15mmol) and compound S13(73mg,0.15mmol) were added to 5mL of DMF under nitrogen, potassium carbonate (62mg,0.45mmol) was added at room temperature, the mixture was warmed to 60 ℃ and reacted for 6 hours. After completion of the reaction, 50mL of water was added to the system, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to give compound 20 as a white solid (56mg, 47.7% yield). 1H NMR (400MHz, DMSO) δ 11.05(s,1H),10.18(s,1H), 8.68-8.13 (m,3H),7.92(d, J ═ 7.3Hz,1H), 7.52-7.36 (m,2H),6.95(d, J ═ 7.2Hz,2H), 5.73-5.54 (m,1H),5.32(s,1H),5.20(d, J ═ 12.8Hz,1H),5.02(d, J ═ 11.2Hz,1H),4.81(d, J ═ 12.5Hz,1H),4.70(s,2H),4.38(dd, J ═ 56.8,16.7Hz,2H), 3.91-3.60 (m,8H), 3.51-3.41 (m,

4H),3.16–2.99(m,4H),2.92(d,J＝13.4Hz,1H),2.71(d,J＝32.4Hz,4H),2.61(d,J＝16.6Hz,1H),2.48–2.35(m,1H),2.04(s,1H),1.46(s,6H)。

example 17: synthetic route to compound 21

Step 1: synthesis of Compound S73

Compound S66(166mg,0.45mmol), compound S72(44mg,0.45mmol), PdCl under nitrogen₂(PPh₃)₂(16mg,0.02mmol) and cuprous iodide (4mg,0.02mmol) were sequentially added to 10mL of DME, stirred to dissolve, triethylamine (182mg,1.8mmol) was added, and the reaction was refluxed for 12 hours. After the reaction is finished, 10mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S73 as a white solid (68mg, 45% yield). LC-Mass (ESI) m/z C₁₈H₁₆N₂O₅[M+H]⁺：341.64。

Step 2: synthesis of Compound 21

Compound S73(51mg,0.15mmol) and compound S13(73mg,0.15mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (112mg,0.30mmol) and DIPEA (80uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 21 as a yellow solid (82mg, 69% yield).

1H NMR(400MHz,DMSO)δ11.04(s,1H),10.19(s,1H),8.84(s,1H),8.05(s,1H),7.74(dd,J＝19.8,7.3Hz,2H),7.61(s,3H),7.52(t,J＝7.4Hz,1H),6.95(d,J＝7.9Hz,2H),5.78–5.58(m,1H),5.34(s,1H),5.17(d,J＝12.7Hz,1H),5.00(d,J＝10.0Hz,1H),4.81(t,J＝19.5Hz,1H),4.70(s,2H),4.39(dd,J＝58.5,17.8Hz,2H),3.64(s,4H),3.45–3.31(m,2H),3.18–2.99(m,4H),2.93(t,J＝13.4Hz,1H),2.70(d,J＝36.9Hz,4H),2.60(d,J＝16.6Hz,1H),2.48–2.37(m,1H),2.04(s,1H),1.47(s,6H)。

Example 18: synthetic route to compound 22

Step 1: synthesis of Compound S74

Compound S73(70mg,0.20mmol) and 10% palladium on carbon catalyst (7mg) were added to 10mL of methanol under nitrogen atmosphere, and the hydrogen gas was replaced, followed by reaction for 2 hours. After the reaction was completed, the palladium on carbon was removed by filtration, and the filtrate was collected and concentrated to give S74(66mg, 95% yield) as a solid. LC-Mass (ESI) m/z C₁₈H₂₀N₂O₅[M+H]⁺：344.93。

Step 2: synthesis of Compound 22

Compound S74(51mg,0.15mmol) and compound S13(73mg,0.15mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (112mg,0.30mmol) and DIPEA (80uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound 22 as a yellow solid (68mg, 57% yield). 1H NMR (400MHz, DMSO) δ 11.04(s,1H),10.18(s,1H),8.83(s,1H),8.02(s,1H),7.83(d, J ═ 7.6Hz,2H),7.61(s,3H),7.52(t, J ═ 7.5Hz,1H), 7.01-6.58 (m,2H), 5.80-5.59 (m,1H),5.34(s,1H),5.17(d, J ═ 12.7Hz,1H),5.00(d, J ═ 10.0Hz,1H),4.81(t, J ═ 19.7Hz,1H),4.70(s,2H),4.39 (J ═ 58.5,17.8, 2H),3.64(s, 3.64, 3.51H), 3.7 Hz,1H, 4.70(s,2H),4.39(d, J ═ 58.5,17.8, 2H),3.64 (d, 3.51, 3.7H), 3.7H, 1H), 4.51, 3.50H, 3.31H, 3.51 (d, 3.50H), 3.6H, 1H), 4.50H, 1H, and so as to 3H.

Example 19: synthetic route to compound 23

Step 1: synthesis of Compound S77

Compound S75(64mg,0.15mmol) and compound S76(25mg,0.15mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (112mg,0.30mmol) and DIPEA (80uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S77(42mg, 49% yield) as a yellow solid. LC-Mass (ESI) m/z C₂₆H₃₅BrN₄O₄S[M+H]⁺：579.17。

Step 2: synthesis of Compound 23

Under the protection of nitrogen, the compoundS77(87mg,0.15mmol) and compound S13(73mg,0.15mmol) were added to 5mL of DMF, potassium carbonate (62mg,0.45mmol) was added at room temperature, the temperature was raised to 60 ℃ and the reaction was allowed to proceed for 6 hours. After completion of the reaction, 50mL of water was added to the system, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to give compound 23(61mg, 42.3% yield) as a white solid. HRMS (ESI) m/z calculated value C₅₂H₆₄N₁₂O₆S[M+H]⁺: 985.5191, found: 985.5174.

example 20: synthetic route to compound 24

Step 1: synthesis of Compound S79

Compound S75(64mg,0.15mmol) and compound S78(29mg,0.15mmol) were added to 5mL of DMF, and after dissolving with stirring, HATU (112mg,0.30mmol) and DIPEA (80uL) were added and reacted at room temperature for two hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and filtrate is collected and concentrated to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography to give compound S79 as a yellow solid (41mg, 47% yield). LC-Mass (ESI) m/z C₂₈H₃₉BrN₄O₄S[M+H]⁺：607.01。

Step 2: synthesis of Compound 24

Compound S79(91mg,0.15mmol) and compound S13(73mg,0.15mmol) were added to 5mL of DMF under nitrogen, potassium carbonate (62mg,0.45mmol) was added at room temperature, the mixture was warmed to 60 ℃ and reacted for 6 hours. After the reaction is finished, 50mL of water is added into the system, ethyl acetate is used for extraction, organic phases are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, filtrate is collected and concentrated to obtain white solid compoundSubstance 24(63mg, 41% yield). HRMS (ESI) m/z calculated value C₅₄H₆₈N₁₂O₆S[M+H]⁺: 1013.5184, found: 1013.5160.

test example 1: kinase binding assays.

Screening model WEE1 kinase protein;

the screening method comprises the following steps of preparing a Protocol name WEE 1;

instrument, Envision (Perkinelmer, USA);

materials WEE1 protein was purchased from eurofins; kinase Tracer178 from Thermo Fisher Scientific; Anti-GST was purchased from Cisbio;

the binding reaction is carried out for 1h by adding Eu labeled GST antibody, WEE1 and Tracer 178. The binding of Tracer178 and the antibody to the kinase results in a high degree of FRET, whereas the use of a kinase inhibitor instead of a Tracer results in a loss of FRET.

WEE1 Activity assay Using a LanthaScreen^TMKinase binding assays. The GST label carried by the N end of WEE1 protein molecule interacts with Eu-labeled anti-GST antibody, and Eu luminescent group can be specifically combined with Tracer178 carrying fluorescent group, when Eu and Tracer178 are close to each other, fluorescence resonance energy transfer can occur, and fluorescence signal is generated. In Envisin^TMThe fluorescence values are read at the emission wavelengths of 620nm and 665nm by using 320nm laser excitation, and the change of the read fluorescence values reflects the binding capacity of WEE1 protein and the probe. The specific experimental procedures and considerations are as follows: 1, diluting a compound to be detected according to a certain proportion, and adding the diluted compound to a 384-well plate; 2 enzyme buffer, antibody and Tracer178 were added to the respective assay wells, while DMSO solvent control empty and AZD1775 positive compound control empty were set. The total reaction volume was 10. mu.L, and the reaction system contained: containing the band assay compounds dissolved in DMSO at a final concentration of 2%, 0.4 ng/. mu.L WEE1, 1nM Eu-Anti-GST, 70nM Tracer178, 10mM MgCl2,1mM DTT,1 Xkinase buffer, in 3 replicates per experimental well; 3, incubating at room temperature for 1h, detecting fluorescence values of 620nm and 665nm by using Envisin TM, and calculating a ratio of 665nm to 620 nm; 4 data were analyzed using GraphPad, curves were fitted, and IC50 was calculated.

As shown in table 3 below, with WEE1 inhibitorAZD1775 as a positive control, IC of Compounds 1-24 on WEE1 protein₅₀All values are greater than AZD1775 (IC)₅₀17.28 ± 2.58nM), indicating that compound 1-24 binds less strongly to WEE1 protein than the positive control AZD 1775.

TABLE 3

Test example 2: and (4) performing a cell toxicity test.

Screening models:MV-4-11；SUM149PT；A498；

the screening method comprises the following steps:protocol name suspension cells (3 days);

instrument SpectraMAX 340;

96plate (spring) material; cells; medium; DMSO; MTS;

the process is that the cell survival rate is detected by using an MTS method: 1. sucking the culture medium of the cells growing in the logarithmic phase, slightly blowing and beating, and counting; 2. seeding 90ul in 96-well plates at the corresponding cell density; 3. adding 10ul of compound, each compound having a concentration gradient, each concentration having three or more wells, each concentration being added to a corresponding well; the final concentration of DMSO was 0.2%; 4.5% CO₂Culturing at 37 deg.C for 3days, and adding 20ul MTS; after 3 hours incubation at 5.37 ℃ 490nm (L1) light absorption was measured using SpectraMAX 340, reference wavelength 690nm (L2), values (L1-L2) were plotted against different inhibitor concentrations, percent activity (%) -Compound OD-BLANK OD/DMSO OD-BLANK OD 100%, IC was fit using GraphPad to the formula₅₀。

Data processing and results description:

the activity of the sample is tested under a single concentration condition, e.g., 20. mu.g/ml, for primary screening. For samples that exhibit activity under certain conditions, e.g., an Inhibition% Inhibition of greater than 50, the testActive dose dependence, i.e. IC₅₀/EC₅₀Values, obtained by nonlinear fitting of sample concentrations through sample activity, were calculated as Graphpad Prism 4, the model used for fitting was sigmoidal dose-response (variable slope), and for most inhibitor screening models, the bottom and top of the fitted curve were set at 0 and 100. In general, each sample was tested with multiple wells (n.gtoreq.2) and the results were expressed as Standard Deviation (SD) or Standard Error (SE).

As shown in table 4 below, compounds 1-24 acted on three tumor cell lines with the WEE1 inhibitor AZD1775 as a positive control, including: MV-4-11 (leukemia cell line), SUM149PT (breast cancer cell line) and A498 (renal cancer cell line). Among them, compound 2, in particular, showed extremely superior anti-tumor proliferation activity in vitro compared to other compounds in table 4, including the positive control AZD 1775: MV-4-11(IC50 ═ 46.00 ± 6.87nM), SUM149PT (IC50 ═ 217.67 ± 38.57nM), and a498 (IC)₅₀＝163.86±16.20nM)。

TABLE 4

Test example 3: western blotting (Western blotting) experiment

The method comprises the following steps: 1) inoculating cells in a 6-well plate according to an experimental scheme, inoculating 100 ten thousand cells in each well, stabilizing for several hours, and then treating the cells in each well with corresponding compounds; 2) after compound treatment was complete, cells were harvested by centrifugation, washed twice with PBS and aspirated off; 3) and (3) denaturing and cracking: mu.l of 1 × loading (62.5mM Tris-HCI, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 1% DTT) was added to each well and the cells were blown up, avoiding blowing up air bubbles as much as possible, and stored in a 100 ℃ metal water bath for 15min at-20 ℃. 4) SDS-PAGE electrophoresis: preparing protein electrophoresis gel according to a Tricine gel formula, adding an inner tank electrophoresis buffer solution and an outer tank electrophoresis buffer solution, loading 10 mu l of each lane, after loading is finished, carrying out constant current electrophoresis, and carrying out 20mA treatment on each gel; 5) protein membrane conversion: after electrophoresis is finished, transferring the protein to a nitrocellulose membrane by using a BioRad wet-conversion electrophoresis instrument system, and calculating membrane conversion time frequently according to 1min of every 1KD protein under the condition of constant voltage of 100 volts; 6) sealing the strip: after the membrane transfer is finished, cutting off the target strip, placing the target strip in 5% skimmed milk sealing solution prepared by TBST in advance, sealing the target strip for 1 hour on a rocking bed at room temperature, and sealing phosphorylated proteins for 1 hour by using 5% BSA sealing solution at room temperature; 7) primary antibody incubation: after the blocking is finished, abandoning the supernatant blocking solution, simply washing the strip with TBST, removing the floating color, diluting the primary antibody to be detected with TBST according to the antibody using instruction, and incubating overnight at 4 ℃; after incubation, the strips were washed 3 times with 5mL TBST per strip, 5min each time; 8) and (3) secondary antibody incubation: anti-rabbit and anti-mouse fluorescent secondary antibodies were expressed as 1: 10000 dilution, incubation for 1h at room temperature, and washing the membrane on a shaking table for 3 times, 5min each time according to 5mL TBST of each strip after the incubation is finished; 9) exposure: and (3) sweeping the membrane by an Odyssey instrument to detect the change condition of the target protein.

Test example 3.1: the compounds of the invention induce WEE1 degradation in the MV-4-11 cell line.

The compounds of the invention were tested for cytotoxic IC using the WEE1 inhibitor AZD1775 as a positive control₅₀The cells were treated in MV-4-11 cell line at a concentration for 6 hours, and cell lysates were collected and subjected to Western blotting analysis. As shown in figure 1, the levels of WEE1 protein after the effect of compounds 1-24 of the present invention were significantly reduced compared to compound AZD1775, indicating that WEE1 protein was degraded. As a proteomic control, PLK1 protein was not degraded.

Test example 3.2: compound 2 induced WEE1 degradation in MV-4-11 cell line at different concentrations.

Compound 2 was treated in MV-4-11 cell line for 6 hours at concentrations ranging from 0.2nM to 100nM using the WEE1 inhibitor AZD1775 as a positive control, and cell lysates were collected and subjected to Western blotting analysis. As shown in figure 2, compound 2 started to decrease in the level of WEE1 protein at the concentration of 0.39nM, with a significant decrease in the level of WEE1 protein at the concentration of 50 nM. As a proteomic control, PLK1 protein was not degraded.

Test example 3.3: compound 2 induced WEE1 degradation in MV-4-11 cell line at different times.

Two groups were set, one group was the compound 2 at 50nM concentration on MV-4-11 cell line and set for 0.5 h-6 h for 7 gradients, and one group was the compound 2 at 3nM concentration on MV-4-11 cell line and set for 1 h-5 h for 5 gradients, and cell lysates were collected and subjected to Western blotting analysis. As shown in fig. 3, compound 2 significantly reduced the protein level of WEE1 after 0.5 hour at 50nM and WEE1 after 1 hour at 3 nM. As a proteomic control, PLK1 protein was not degraded.

It can be seen that the compounds of the present invention have excellent ability to selectively degrade WEE1 protein.

Test example 3.4: protein target binding competition assay

The MV-4-11 cell line was pretreated with CRBN inhibitors Iberdomide (CC-220) and Lenalidomide (Lenalidomide) 10uM, proteasome inhibitors Crafizomib (PR171) and Bortezomib (PS341)1uM for 2 hours in the control group, and then treated with compound 2 at a final concentration of 50nM for 3 hours, and total protein samples were collected for Western blotting analysis. As shown in fig. 4, compound 2, in the experimental group not pretreated with protein inhibitor, at a concentration of 50nM on MV-4-11 cell line, induced significant degradation of WEE1 protein and resulted in a corresponding down-regulation of downstream phosphorylated PCDC 2; after pretreatment with CRBN inhibitor, compound 2 was unable to induce WEE1 protein degradation at a concentration of 50nM on the MV-4-11 cell line; after proteasome inhibitor pretreatment, compound 2 was unable to induce WEE1 protein degradation at 50nM concentration on the MV-4-11 cell line. As a proteomic control, PLK1 protein was not degraded. Protein target competitive binding experiments prove that the compound 2 is formed by relying on a ternary complex of WEE1 protein-compound 2-CRBN protein and passing through CRL4^CRBNE3 ubiquitin ligase recruits WEE1 protein and then degrades WEE1 protein by a proteasome degradation system.

Test example 4: compound 2 induced degradation of WEE1 protein but not of WEE1 expressed mRNA.

As shown in fig. 5, compound 2 did not exhibit down-regulation of WEE 1-expressed mRNA transcriptome levels after 1,3 and 6 hours of action at 50nM, and it can be seen that the compound of the present invention did not affect the expression level of WEE1 transcriptome mRNA levels, and in combination with the results of the decreased expression level of WEE1 protein levels observed in the previous immunoblot experiments, it can be confirmed that: the compounds of the invention down-regulate WEE1 protein expression rather than mRNA transcriptome levels by degrading post-translational protein levels.

Test example 5: MV-4-11 tumor cell line G1 cycle arrest was induced.

Cell cycle arrest experimental principle and experimental method: the PI method is a classical periodic detection method. PI is a nucleic acid dye that specifically binds to DNA. After PI staining, the fluorescence intensity of G2/M phase cells is generally 2 times that of G0/G1 phase cells, and 1-2 times that of S phase cells undergoing DNA replication. By flow method, the proportion of cells with different DNA fluorescence intensity in the whole cell population can be examined, thereby determining the distribution of different cell cycles. The specific experimental steps are as follows:

1) cell culture: planting cells in a six-well plate, wherein each well is 1 × 106, the cells are in a growth logarithmic phase, and after the cells are stabilized for 3-4h and treated by different compounds for corresponding time, collecting the cells for detection; 2) cell treatment: collecting cells in 2mL centrifuge tube, 1000rpm centrifugation for 5min, 1mL precooling PBS heavy suspension washing, carefully suction supernatant; 3) cell fixation: fully resuspending 300 μ L PBS, adding 700 μ L precooled absolute ethyl alcohol while shaking to a final concentration of 75%, slightly shaking to make the cells become single cells, standing at 4 ℃ and fixing overnight; 4) the overnight fixed cells were washed twice with pre-chilled PBS, centrifuged at 1000rpm for 10min and 1mL PBS was added. Moving the cells into a 1.5mL EP tube, and slightly blowing up the dispersed cells to avoid cell clustering; 5) adding 2 μ L RNase (working concentration 20 μ g/mL), digesting RNA, resuspending cells, and digesting in water bath at 37 deg.C for 15 min; 6) adding PI dye 5 μ L to final concentration of 10 μ g/mL, dyeing at 4 deg.C in dark for 30min, and performing up-flow detection within 1 h; 7) fully mixing the cells, filtering by a 200-mesh filter screen, and performing cell cycle by using a flow cytometer; 8) collecting and analyzing data; red fluorescence was detected by flow cytometry. Cellular DNA content analysis was performed using FlowJo software.

As shown in FIG. 6, as the drug concentration of Compound 2 increased, the concentration was set to 5 gradients of 12.5nM, 25nM, 50nM, 100nM and 200nM on MV-4-11 tumor cell line for 24 hours, which was seen to have a block at stage G1.

Test example 6: compound 2 induces apoptosis in the MV-4-11 tumor cell line.

The experimental steps are as follows: 1) inoculating cells in a 6-hole plate according to an experimental scheme, inoculating 50W cells in each hole, stabilizing for several hours, treating the cells with compounds with different concentrations after the cells return to a normal culture state, and collecting the cells after 48 hours; 2) when the end point of the experiment is reached, all the following steps need to be gentle and slow, so that false positive of the experiment is avoided. Cells were first harvested by centrifugation (1000rpm, 5min) and washed twice with PBS; 3) sucking 500 mu L of Binding Buffer soft and heavy suspension cell sample of the apoptosis detection kit; 4) then adding 5 mul of Annexin V-FITC reagent provided by the kit into each sample, gently mixing the sample and the cells uniformly, and dyeing for 15min in a dark place at room temperature; 5) then, adding 5 mul of PI (propidium iodide) dye solution provided by the kit into each sample, gently mixing the PI (propidium iodide) dye solution with the cells, and dyeing for 5min in a dark place at room temperature; 6) then, the cells were mixed well, and the cells were filtered with a 200-mesh filter, and the cells were examined on the machine, and the examination by flow cytometry 7) and the examination by flow cytometry were carried out within 1 hour (C6 flow cytometer, BD corporation): firstly, a living cell group is circled, then a single cell group is circled after adhesion is removed, then 30000 cells are collected from each sample, Annexin V-FITC fluorescence is detected by using an FL1 channel, and PI fluorescence is detected by using an FL3 channel; 8) after data collection, data analysis and processing was performed using Flowjo10 software.

As shown in FIG. 7, as the concentration of compound 2 was increased, the concentration was set to 5 gradients of 12.5nM, 25nM, 50nM, 100nM and 200nM, and the apoptosis was significantly increased with increasing concentration in MV-4-11 tumor cell lines for 48 hours, with approximately 20% of the cells undergoing apoptosis.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.