1. A steroid derivative having a chemical structure represented by the following formula (I):

2. use of a steroid derivative as claimed in claim 1, in the manufacture of a medicament for the treatment of inflammatory bowel disease.

3. Use according to claim 2, characterized in that said anti-inflammatory agent consists of the steroid derivative according to claim 1 and of a pharmaceutically acceptable adjuvant.

Background

Inflammatory enteritis (IBD) is a high autoimmune disease in humans, and is clinically manifested by repeated abdominal pain, diarrhea, abdominal mass, mucous bloody stool, intestinal obstruction, intestinal perforation, weight loss, and the like. With the rising incidence in western countries, inflammatory bowel disease has become an important disease affecting human health. According to the pathological characteristics and the different disease sites, the disease is classified into Ulcerative Colitis (UC) and Crohn's Disease (CD). When intestinal homeostasis is imbalanced, intestinal mucosal permeability is increased, leading to an impaired intestinal mucosal barrier function, mediating the development of IBD. Compared with normal mucosal lamina propria macrophages, the intestinal mucosal macrophages in lesion local colon tissues of patients with active IBD are remarkably increased in number and tend to be more activated, which indicates that the intestinal macrophages play an important role in the occurrence and development of IBD. Bone marrow-derived macrophages can be induced by different stimuli to polarize to form macrophages of different phenotypes and functions, mainly M1 type and M2 type. Among them, M1 type macrophages secrete a large amount of proinflammatory cytokines after being stimulated and activated, and decompose L-arginine to generate NO and Reactive Oxygen Species (ROS) through high expression Inducible Nitric Oxide Synthase (iNOS), participate in processes such as phagocytosis of bacteria, chemotaxis of inflammatory cells, promotion of Th1 and Th17 cell-mediated immune response, and exert host immune function, resulting in inflammatory injury of intestinal tissues of IBD patients.

The drugs currently used for treating IBD are various, and mainly comprise aminosalicylic acids (such as sulfasalazine or mesalamine (5-ASA)), adrenal glucocorticoids (such as prednisolone), immunosuppressants (such as thiopurine or methotrexate), biological agents (such as TNF inhibitors) and the like. The glucocorticoid can quickly relieve symptoms by combining with glucocorticoid receptor in nucleus, has anti-inflammatory effect, is suitable for acute attack of IBD, and has no effect on sufficient treatment of aminosalicylic acid. However, systemic side effects caused by gastrointestinal absorption, such as opportunistic infection, diabetes, osteoporosis and the like, limit the applicability of glucocorticoids. In recent years, oral formulations of Budesonide (Budesonide) and Beclomethasone (Beclomethasone) have received attention because of their high local anti-inflammatory activity and low systemic side effects. These drugs are delivered to the intestinal mucosa specifically after oral administration. The medicine can be quickly and effectively inactivated after being absorbed by intestinal tracts, thereby playing a local anti-inflammatory role.

Since glucocorticoids play an important role in the treatment of IBD, the development of new steroid skeleton drugs for the treatment of IBD is of great importance.

Disclosure of Invention

The technical problem to be solved by the invention is to provide the steroid derivative, and the anti-inflammatory effect of the steroid derivative is obvious.

The scheme for solving the technical problems is as follows:

a steroid derivative having a chemical structure represented by the following formula (I):

the preparation method of the steroid derivative comprises the following steps: firstly, carrying out a haloform reaction on pregnenolone acetate to generate a compound 2, and then carrying out a condensation reaction on the compound 2 to obtain the steroid derivative. The chemical reaction formula of the preparation method is as follows:

the steroid derivative has obvious inhibition effect on NO produced by macrophage stimulated by LPS, and can be used for preparing anti-inflammatory drugs, in particular to preparing drugs for treating inflammatory enteritis. Wherein, the medicine for treating inflammatory enteritis consists of the steroid derivative and medically acceptable auxiliary materials.

The medicine for treating inflammatory enteritis provided by the invention can obviously inhibit NO induced by LPS in RAW264.7 cells, and has an obvious effect of relieving colonic inflammation in a mouse body.

The present invention will be further described with reference to the following embodiments.

Drawings

Fig. 1 is a histogram of the effect of the steroid derivatives of the present invention in inhibiting LPS-induced NO in RAW264.7 cells, wherein p is <0.05 (n-3) compared to the positive control dexamethasone.

Fig. 2 is a histogram of experimental mouse DAI scores (n ═ 5), where a is the difference in mouse diarrhea scores and B is the difference in mouse bloody stool scores. In the figure, # # # denotes, # # # p <0.001, compared to the Control group; indicates p <0.001 compared to DSS group.

FIG. 3 is a photomicrograph (200X) of HE stained sections of mouse colon tissue, wherein Panel A is a HE stained section of mouse colon tissue of group A, Panel B is a HE stained section of mouse colon tissue of group B, Panel C is a HE stained section of mouse colon tissue of group C, Panel D is a HE stained section of mouse colon tissue of group D, Panel E is a HE stained section of mouse colon tissue of group E, and Panel F is a HE stained section of mouse colon tissue of group F.

FIG. 4 is a photomicrograph (400X) of a CD86 immunohistochemical analysis of mouse colon tissue, wherein panel A is a CD86 immunohistochemical analysis of group A mouse colon tissue, panel B is a CD86 immunohistochemical analysis of group B mouse colon tissue, panel C is a CD86 immunohistochemical analysis of group C mouse colon tissue, panel D is a CD86 immunohistochemical analysis of group D mouse colon tissue, panel E is a CD86 immunohistochemical analysis of group E mouse colon tissue, and panel F is a CD86 immunohistochemical analysis of group F mouse colon tissue.

Detailed Description

Example 1 preparation of a Compound

The steroid derivative in this example consists of the following steps:

the method comprises the following steps:

dissolving 2.1g of sodium hydroxide in 18mL of water, dropwise adding 2.88g of bromine in an ice bath, and adding the mixture into 12mL of dioxane to obtain a mixed solution when the bromine is completely dissolved; the mixture was added to a solution containing 1.44g pregnenolone acetate (1) (a mixed solution of 56mL dioxane and 16mL water), stirred overnight, sodium sulfite was added, the mixture was heated under reflux until the solid was completely dissolved, acidified with hydrochloric acid to precipitate a white solid, and the solid was washed with water until the solution was colorless.

The chemical reaction formula of the first step is as follows:

and identifying the obtained white solid by adopting a nuclear magnetic resonance spectrum, wherein the identification result is as follows: 1H NMR (400MHz, DMSO) δ 11.88(s,1H),5.26(d, J ═ 4.7Hz,1H),4.61(s,1H),3.25(ddd, J ═ 15.2,10.4,4.4Hz,1H),2.26(t, J ═ 9.2Hz,1H),2.18 to 2.02(m,2H),1.95(dd, J ═ 19.3,11.9Hz,3H),1.76(dd, J ═ 13.3,3.2Hz,1H),1.73 to 1.64(m,2H),1.63 to 1.47(m,3H),1.45 to 1.30(m,3H),1.28 to 1.14(m,2H),1.12 to 1.04(m,1H),1.00 (m,3H), 3.6 (ddh), 0.86 (ddh), 3H), and the obtained compound was identified as a white solid.

Step two:

318mg (1mmol) of the compound 2 obtained in the first step is dissolved in anhydrous DMF, HOBt 135mg is added, triethylamine 4 drops is added, EDCI 192mg is added, finally imidazole 68mg is added and stirring is carried out overnight. After the reaction, water was added to precipitate a white solid.

The chemical reaction formula of the second step is as follows:

the obtained compound is identified by nuclear magnetic resonance spectrum, and the identification result is¹H NMR (400MHz, DMSO) δ 8.44(s,1H),7.73(s,1H),7.06(s,1H),5.28(d, J ═ 3.1Hz,1H),4.59(d, J ═ 4.4Hz,1H),3.41(t, J ═ 8.8Hz,1H),3.29 to 3.20(m,1H),2.14(dt, J ═ 35.9,11.9Hz,3H),1.93(dd, J ═ 33.6,11.3Hz,2H),1.71(dd, J ═ 29.3,10.3Hz,3H),1.55(dd, J ═ 27.2,11.6Hz,4H),1.40 to 1.21(m,5H),1.08 to 0.88(m,5H),0.65(s, 3H). From the above identification results, the obtained product was compound 3, i.e., a steroid derivative represented by formula (I).

Example 2 (cell experiment study)

First, experimental purpose and principle

Purpose of the experiment: and detecting the NO inhibition effect of the synthesized series of steroid compounds on macrophages stimulated by LPS by adopting an NO inhibition test.

The experimental principle is as follows: NO is very easily oxidized in vivo or in aqueous solution to generate NO₂ ^-Under acidic conditions, NO₂ ^-Diazo reaction with diazo sulphamide and generation of diazo compounds, which are further coupled with naphthyl ethylene diamine, the concentration of the products of this reaction being determined by the NO coupling reaction₂ ^-The concentration has a linear relation, and the maximum absorption peak exists between 540nm and 560 nm.

Second, basic information of reagent

Name of reagent	Brand
		NaNO2	Macklin
N-1-naphthyl ethylenediamine hydrochloride	Macklin
		Sulfanilic acid	Macklin
RPMI-1640 basic culture medium	Gibco
		Fetal bovine serum	Gibco
Penicillin-streptomycin solution	Gibco
		Dimethyl sulfoxide (DMSO)	Macklin

Third, reagent preparation

1. RPMI-1640 complete medium

50mL of serum is suspended and poured into 450mL of RPMI-1640 culture medium, and then 5mL of penicillin-streptomycin solution is added according to the proportion of 1:100 to form complete culture medium, so that the culture medium can be used for cell culture.

2. Preparation of Griess reagent

2.110 mM NaNO2 Standard stock solution preparation: accurately weighing NaNO₂6.9mg, adding deionized water to make the volume to 10mL, namely 10mM NaNO2 storage solution.

2.2Griess reagent preparation

Reagent A: 6mL of concentrated phosphoric acid (85%); 70mL of deionized water; anhydrous sulfanilic acid 1.0 g. The above reagents were dissolved sufficiently and then the volume was adjusted to 100 mL.

And (3) reagent B: 0.1g of N-1-naphthyl ethylenediamine hydrochloride is dissolved in deionized water, and the volume is fixed to 100 mL.

3. Compound preparation

1mg of the steroid derivative (compound I) prepared in example 1 was weighed into an autoclaved EP tube, and then 136. mu.l of DMSO was added to the EP tube to prepare a 20mM stock solution, which was diluted to 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, and 2.5. mu.M with the complete medium before use.

Fourth, the experimental process

(1) Taking RAW264.7 cells in logarithmic growth phase, adjusting the concentration of the number of the cells to be 5 multiplied by 10 after digestion⁴Perml, 100. mu.L/well into 96-well plates. At 37 ℃ 5% CO₂Culturing in a cell culture box overnight until the cells adhere to the wall.

(2) The original medium was aspirated, 100. mu.L of LPS (100ng/mL) containing medium was added to each well, 100. mu.L of medium without LPS stimulation was added to the blank, and 100. mu.L of the formulated compounds at different concentrations, i.e., the actual treatment concentrations of the compounds were 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M and 1.25. mu.M, were added to each well after 2 hours. 0.1% DMSO was used as a negative control, and dexamethasone (Dex) was used as a positive control. After further incubation in the cell incubator for 24h, 50. mu.L of medium was taken per well for use.

(3) NO standard solution is diluted as follows

(4) Sample adding: 50 μ L of each diluted standard application solution or sample to be tested was added to the 96-well plate. Then adding 50 mu L of reagent A into each well, and reacting for 10min in an incubator at 37 ℃; then, 50. mu.L of reagent B was added to each well, and the mixture was reacted in an incubator at 37 ℃ for 10 min.

(5) And (3) lightly oscillating the 96-well plate for a plurality of times, and detecting the OD value of each well at the wavelength of 540nm by using an enzyme-linked immunosorbent assay (ELISA) detector after reaction liquid of each well is completely mixed. And fitting an NO reaction standard curve according to the obtained OD value, and calculating the NO content of each sample to be detected according to the standard curve.

(6) The NO inhibition was calculated according to the following formula:

the inhibition rate was [ (Al-Ab) - (As-Ab) ]/(Al-Ab) × 100%

Al: absorbance of control wells (stimulated with LPS, no compound treatment); as: absorbance of assay wells (stimulated with LPS, compound treatment); ab: absorbance of blank well (cell-free)

The inhibition rate of the compound at different concentrations was calculated by GraphPad Prism 8.2.1 software based on the inhibition rate of the drug on cell proliferation at different doses. The results of measuring the inhibition of LPS-induced NO by the compounds are shown in table 1:

inhibitory Effect of the Compounds of Table 1 on NO

Fifth, experimental results

The results of the NO inhibition experiments are shown in figure 1, when the concentration is 5-20 mu M, the NO induced by the compound I on LPS shows the activity equivalent to or higher than that of dexamethasone, wherein, the inhibitory activity of the compound I and the dexamethasone on the NO shows a significant difference when the concentration is 10 mu M. The results show that compound i has significant NO production inhibitory activity at concentrations greater than 10 μ M.

Example 3 (animal experiment study)

Animal model

In the development of new drugs for inflammatory enteritis, Dextran Sulfate Sodium Salt (DSS) induced colitis (UC) model is most widely used. By freely drinking DSS aqueous solutions with different concentrations for mice, two acute and chronic colitis models can be prepared according to the medication time and the medication period. The symptom of the model is very similar to human UC, and mainly shows diarrhea, mucus-like stool, fecal occult blood, naked eye bloody stool, weight reduction, activity reduction, hair color deterioration and the like. The chronic colitis model mainly shows obvious colon shortening, mucosa thickening, lymph node swelling, goblet cell deletion, crypt deletion, adenomatous polypus and tumor-like change of a small part of animals. Acute phase colitis model mainly shows colonic congestion, edema, shortening, embrittlement, weight-length ratio increase, colonic ulcer of different degrees, mucosal edema, goblet cell loss, crypt swelling damage, inflammatory cell infiltration of different degrees of mucosa and submucosa, and epithelial cell injury.

Second, experimental animals and reagents

Animals: c57 mouse, Male, 5 weeks old

Reagent: reagent information is shown in the following table

Name of reagent	Brand
		Dextran Sulfate Sodium (DSS)	MeilunBio
Castor oil	aladdin
		Polyethylene glycol	Macklin
Ethanol	Macklin
		Dimethyl sulfoxide (DMSO)	Macklin

Third, reagent preparation

DSS solution: DSS is dissolved in disinfected drinking water to prepare DSS solution with the weight percentage concentration of 3.5%.

2. Compound preparation: the steroid derivative (compound I) prepared in the example 1 is dissolved in saline, and castor oil, ethanol and DMSO (70: 10: 15: 5) are added to prepare solutions with different concentrations of 20mg/mL,10mg/mL,5mg/mL and the like for standby; dexamethasone was prepared as a 5mg/mL solution in the same manner and was used.

Fourth, the experimental procedure

1. Purchased 5-week-old C57 mice were housed in SPF-grade animal houses and acclimatized with normal water for one week.

2. When the mice grew to about 20g, the mice were randomly divided into 6 groups, and group a was given normal drinking water; group B was given 3.5% DSS solution for ad libitum consumption; group C was given 3.5% DSS solution for ad libitum drinking, and injected intraperitoneally with 20mg/mL of Compound I solution at 100. mu.L/body (100 mg/kg); group D was given 3.5% DSS solution for ad libitum drinking, and administered with intraperitoneal injection of 10mg/mL compound I solution 100. mu.L/body (50 mg/kg); group E was given 3.5% DSS solution for ad libitum drinking, and injected intraperitoneally with 5mg/mL of Compound I solution at 100. mu.L/body (25 mg/kg); group F was given 3.5% DSS solution for ad libitum consumption, and was injected intraperitoneally with 100. mu.L/body (25mg/kg) of 5mg/mL dexamethasone solution.

3. The mice were recorded for diarrhea, hematochezia, etc. Grading standard: (1) stool consistency: 0, good particles are formed; 2, pasty and unformed excrement which does not stick to the anus; 4, liquid defecates and adheres to the anus. (2) Bleeding of stool: 0, no blood is present in the blood; 2, bleeding; 4, heavy bleeding.

4. HE staining of colon tissue was performed. One week later, the mice were sacrificed and their colons dissected out, samples fixed in 10% formalin, paraffin embedded, sliced, stained with Hematoxylin and Eosin (HE), and observed under a microscope at a magnification of 200X.

5. Immunohistochemical analysis was performed on colon tissue. The sections were dewaxed and the antigen recovered in citrate buffer, then the samples were incubated with primary antibody (CD86, 1: 200 dilution) and then IgG-HRP antibody (1: 200 dilution). After incubation, the samples were developed under a diaminobiphenyl microscope, re-stained with hematoxylin, dehydrated, and sealed into microtablets. Colon structures were observed using an optical microscope at 400X magnification.

Fifth, experimental results

1. As shown in figure 2, stool viscosity and bleeding fractions were significantly increased in DSS-treated mice compared to non-DSS-treated mice, showing significant inflammatory symptoms of loose stool blood. After the treatment group mice are treated by different doses of the compound I, the stool viscosity and the stool blood fraction are both obviously reduced, wherein the disease activity index of the mice in the treatment group of 50mg/kg of the compound I is the lowest. The results show that the compound I can effectively relieve the colonic inflammation of mice.

2. As shown in fig. 3, DSS-treated mice exhibited pathological features such as crypt abscesses, goblet cell loss, mucus layer loss, neutrophil infiltration into the lamina propria (fig. 3B), reduction of inflammatory cells in the colon D (50mg/kg compound i), and an intact structure (fig. 3D), as compared to non-DSS-treated mice. The results show that the compound I can effectively relieve the inflammation of the colon of the mouse.

3. As shown in fig. 4, CD86(+) cells aggregated and formed several significant positive colonies in colon tissue of DSS-treated mice (fig. 4B), compared to non-DSS-treated mice, whereas CD86(+) cells were in a dispersed state in the colon of mice of the groups of DSS-untreated, DSS-treated and compound i (fig. 4A, C, D, E, F). The results show that the compound I can effectively relieve the DSS-induced colon inflammation of mice. Based on the compound I, the compound I is expected to be applied to inflammatory enteritis, namely IBD.