CN114702544B - Amino steroid compound, and preparation method and application thereof - Google Patents

Abstract

The application provides an amino steroid compound, a preparation method and application thereof, wherein the amino steroid compound comprises at least one of chemical formulas shown as a formula I or a formula II,wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Independently selected from H, halogen-containing groups, C ₁ ～C ₁₀ Alkyl, C of (2) ₂ ～C ₁₀ Alkenyl, C ₂ ～C ₁₀ Alkynyl, C ₃ ～C ₁₀ Cycloalkyl, C ₆ ～C ₁₀ Aryl or C ₅ ～C ₁₀ Heteroaryl; and R is ₁ 、R ₂ At least one of which is a halogen-containing group; r is R ₃ 、R ₄ 、R ₅ At least one of which is a halogen-containing group.

Description

Amino steroid compound, and preparation method and application thereof

Technical Field

The application relates to the field of chemistry, in particular to an amino steroid compound, a preparation method and application thereof.

Background

Kras (kirsten rat sarcoma viral oncogene) A murine sarcoma virus oncogene, ras gene family and human tumor related genes of three, H-ras, K-ras and N-ras, located on chromosome 11, 12 and 1, respectively. K-Ras is the greatest in relation to human cancers due to the Ras protein encoding 21kD, also known as p21 gene, among which is the molecular switch: the cell growth path can be controlled and regulated when normal; once abnormal, cells are caused to continue to grow and to prevent apoptosis. It is involved in intracellular signaling, and when the K-ras gene is mutated, it is permanently activated and does not produce normal ras protein, which results in disturbance of intracellular signaling, and thus uncontrolled proliferation of cells, resulting in cancerous changes.

K-ras gene mutations are quite common in cancer, with about 30% of cancer patients having K-ras mutations, including 90% of pancreatic cancer, 50% of colon cancer and 25% of lung cancer. In non-small cell lung cancer, K-ras gene mutations account for 20-30%, which are frequently found in lung adenocarcinoma, and rarely found in lung squamous cell carcinoma.

However, among K-ras mutant types, G12C mutations are most common, accounting for about 44% of all K-ras mutations, and the most common cancer species is non-small cell lung cancer. Wherein the KRAS G12C mutation occurs in about 13% of non-small cell lung cancers (NSCLC) and 3% of colorectal cancers.

Currently, the K-ras protein inhibitor Sotorasib (Sotorasib, code AMG 510) is approved by U.S. FDA breakthrough therapy for the treatment of locally advanced or metastatic Kras mutation-bearing non-small cell lung cancer (NSCLC) patients who have previously received at least one systemic chemotherapy.

Although K-ras G12C mutations are pharmaceutically acceptable, patients and doctors have to face another difficulty in actual treatment: cancer cells develop drug resistance. The most important drug-resistant mechanism is K-ras protein allosteric, and small molecule drugs cannot be combined in the pocket region of the protein, so that the inhibition effect on the K-ras protein is lost.

In this regard, there is a need to develop small molecule inhibitors against the K-ras gene that avoid cancer cell resistance to conventional protein drugs.

Disclosure of Invention

According to an aspect of the present application, there is provided an amino steroid compound comprising at least one of the formulae shown as formula I or formula II,

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Independently selected from H, halogen-containing groups, C ₁ ～C ₁₀ Alkyl, C of (2) ₂ ～C ₁₀ Alkenyl, C ₂ ～C ₁₀ Alkynyl, C ₃ ～C ₁₀ Cycloalkyl, C ₆ ～C ₁₀ Aryl, C ₅ ～C ₁₀ Heteroaryl or a group of formula III;

wherein the R is ₆ Selected from H, C ₁ ～C ₆ Alkyl, C of (2) ₂ ～C ₆ Alkenyl, C ₂ ～C ₆ Alkynyl, C ₃ ～C ₆ Cycloalkyl, C ₆ ～C ₁₀ Aryl or C ₅ ～C ₁₀ Heteroaryl;

and R is ₁ 、R ₂ At least one of which is a halogen-containing group; r is R ₃ 、R ₄ 、R ₅ At least one of which is a halogen-containing group.

Alternatively, the number of halogens in formula I and formula II is 1 to 10, specifically, the number of halogens may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In the present application, when the number of halogens in the compound of formula I is 2, the compound has a good anticancer effect; when the number of halogens in the compound of formula II is 3, the compound has good anticancer effect.

Furthermore, the applicant has unexpectedly found that, when halogen is introduced at a specific position of the compounds of the present application, i.e. at R of the compounds of the present application ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ When halogen groups are introduced at the positions, the binding sites and modes of the obtained compound and the G-tetrad target are changed obviously, so that the combination of the obtained compound and the G-tetrad target can be facilitated, the combination of the obtained compound and the G-tetrad target is tighter, the combined conformation is more stable, and the pharmacological effect is better.

Further, the applicant has unexpectedly found that R of the compounds of the present application ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ The halogen group introduced at the position has a good anticancer effect when the variety of the halogen group is more diversified. Specifically, in the compounds of formula I herein, R is ₁ And R is ₂ Are all halogen-containing groups and R ₁ Halogen and R of (2) ₂ The halogen of (2) is different, and has good anticancer effect; in the compounds of formula II of the present application, R is ₃ 、R ₄ And R is ₅ Are all halogen-containing groups and R ₃ 、R ₄ And R is ₅ The halogen of (C) is different, and has good anticancer effect.

Alternatively, the halogen-containing group is selected from halogenC substituted by halogen ₁ ～C ₁₀ C substituted by halogen ₂ ～C ₁₀ Alkenyl of (2), C substituted by halogen ₂ ～C ₁₀ Alkynyl, C substituted by halogen ₃ ～C ₁₀ Cycloalkyl, C substituted by halogen ₆ ～C ₁₀ Aryl, C substituted by halogen ₅ ～C ₁₀ Heteroaryl or R ₇ C (O), wherein R ₇ Selected from halogen, C substituted by halogen ₁ ～C ₆ C substituted by halogen ₂ ～C ₆ Alkenyl of (2), C substituted by halogen ₂ ～C ₆ Alkynyl, C substituted by halogen ₃ ～C ₆ Cycloalkyl, C substituted by halogen ₆ ～C ₁₀ Aryl or C substituted by halogen ₅ ～C ₁₀ Heteroaryl groups.

Alternatively, R ₂ 、R ₅ Independently selected from H, haloacetyl or halobenzoyl; r is R ₁ 、R ₃ 、R ₄ Selected from H or halogen.

Alternatively, the halogen is selected from F, cl, br or I.

Alternatively, when at least two halogen-containing groups are present in formula I or formula II, the halogen in the halogen-containing groups may be the same or different, preferably the halogen in the halogen-containing groups is different; for example, when two halogen-containing groups are present, the two halogens may each be selected from: f and Cl; f and Br; f and I; cl and Br; cl and I; or Br and I.

Alternatively, when at least three halogen-containing groups are present in formula I or formula II, preferably, the halogens in the halogen-containing groups are different; i.e. the halogens in the halogen-containing groups are not identical or completely different. For example, when three halogen-containing groups are present, the three halogens in the three halogen groups may be completely different and may be selected from: F. cl and Br; F. cl and I; F. br and I; cl, br and I; the halogens of the three halogen groups may also not be identical, that is, the halogens of the three halogen groups may be partially identical, for example, when three halogen-containing groups are present, the halogens may be selected from: F. f and Cl; F. f and Br; F. f and I; cl, cl and F; cl, cl and Br; cl, cl and I; br, br and F; br, and Cl; br, br and I; cl, cl and F; cl, cl and Br; cl, cl and I; I. i and F; I. i and Cl; I. i and Br, etc.

Optionally, the R ₆ Selected from C substituted by halogen ₁ ～C ₆ C substituted by halogen ₂ ～C ₆ Alkenyl of (2), C substituted by halogen ₂ ～C ₆ Alkynyl, C substituted by halogen ₃ ～C ₆ Cycloalkyl, C substituted by halogen ₆ ～C ₁₀ Aryl or C substituted by halogen ₅ ～C ₁₀ Heteroaryl groups.

Optionally, the R ₂ 、R ₅ Independently selected fromWherein X is selected from F, cl, br or I.

Optionally, the amino steroid comprises

According to another aspect of the present application, there is provided a process for the preparation of an amino steroid as defined in any one of the above.

Optionally, the preparation method comprises the following steps:

(A) Reacting a compound of the formula with a halogen-containing compound to give the compound of the formula I

Or (b)

(B) Reacting a compound of the formula with a halogen-containing compound to give the compound of the formula II

Optionally, the preparation method comprises the following steps: (1) Reacting 2 beta-piperazinyl-3 alpha-hydroxy-5 alpha-steroid-17-ketone with a halogenating reagent to obtain an intermediate; (2) Reacting the intermediate with 1- (quinoline-2-acid ester) -L-proline to obtain the amino steroid compound; the halogenating agent comprises a halogen-containing group, C, substituted with halogen ₁ ～C ₁₀ Alkyl, C of (2) ₂ ～C ₁₀ Alkenyl, C ₂ ～C ₁₀ Alkynyl, C ₃ ～C ₁₀ Cycloalkyl, C ₆ ～C ₁₀ Aryl or C ₅ ～C ₁₀ At least one of heteroaryl;

The structure of the intermediate is shown below,

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Independently selected from H, halogen-containing groups, C ₁ ～C ₁₀ Alkyl, C of (2) ₂ ～C ₁₀ Alkenyl, C ₂ ～C ₁₀ Alkynyl, C ₃ ～C ₁₀ Cycloalkyl, C ₆ ～C ₁₀ Aryl or C ₅ ～C ₁₀ Heteroaryl; and R is ₁ 、R ₂ At least one of which is a halogen-containing group; r is R ₃ 、R ₄ 、R ₅ At least one of which is a halogen-containing group.

Alternatively, step (1) and step (2) of the above preparation process may be exchanged, for example, a compound of formula IX or formula X having a halogen-containing group may be first produced to regenerate a compound of formula I or formula II; compounds of the following structure may also be produced first,

followed by a further halogenation to produce a compound of formula I or formula II.

Optionally, the molar ratio of the 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one to the halogenating agent is 1: (3-6).

Alternatively, the conditions of the reaction in step (1) are: the temperature is between-5 and 100 ℃ and the time is between 5 and 24 hours.

Optionally, the halogenating agent is selected from at least one of chloroacetyl chloride, cupric chloride, trimethylsilyl halogen acetylene, bromoacetyl chloride, and cupric bromide.

Alternatively, the trimethylsilyl halogen acetylene comprises trimethylsilyl fluoroacetylene, trimethylsilyl chloroacetylene, trimethylsilyl bromoacetylene, and trimethylsilyl iodoacetylene.

Alternatively, the molar ratio of the intermediate to 1- (quinolin-2-yl) -L-proline is 1: (1-3).

Alternatively, the conditions of the reaction in step (2) are: the temperature is between-5 and 30 ℃ and the time is between 8 and 18 hours.

Optionally, a coupling reagent may also be added in step (2); the coupling reagent comprises 6-chlorobenzotriazole-1, 3-tetramethylurea hexafluorophosphate.

According to another aspect of the present application, there is provided the use of an amino steroid compound as described in any one of the above for the preparation of a K-ras cancer gene promoter targeting inhibitor.

According to a further aspect of the present application there is provided a gene inhibitor which targets and specifically binds to a cancer gene promoter, the gene inhibitor comprising any of the above amino steroid compounds.

The halogenated compound is combined with a promoter of a K-ras gene, particularly with a G tetrad structure in a promoter structure, so that the promoter is prevented from being started, the synthesis of K-ras protein is inhibited, and the effect of inhibiting the growth of cancer cells is achieved.

According to a further aspect of the present application there is provided the use of an amino steroid as described in any of the above in the manufacture of a medicament for the prophylaxis and/or treatment of cancer.

According to a further aspect of the present application there is provided a formulation comprising an amino steroid as described above and a pharmaceutically acceptable adjuvant.

Optionally, the preparation can be prepared from pharmaceutically acceptable common auxiliary materials according to conventional dosage by adopting a conventional preparation method.

The auxiliary materials can be different according to different preparations, such as surfactants, diluents, preservatives, stabilizers, flavoring agents, thickening agents, glidants and the like which are commonly used in liquid preparation forms such as oral liquid; diluents, disintegrants, excipients, binders, lubricants, surfactants, fillers and the like which are commonly used in solid preparations such as tablets, capsules, granules and the like.

Optionally, the auxiliary material is selected from at least one of starch, lactose, sucrose, dextrin, maltodextrin, microcrystalline cellulose, mannitol, xylitol, polyethylene glycol, calcium carbonate, modified starch, sorbitol, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, sodium carboxymethyl starch, hydroxypropyl cellulose, povidone K30, pregelatinized starch, magnesium stearate, talcum powder, micro powder silica gel, stevioside, betaine, aspartame, citric acid, green tea sweetener, saccharin sodium, rock sugar, honey, citric acid, sodium bicarbonate, sodium carbonate, carrageenan, agar, gelatin, sodium alginate, xanthan gum, guar gum, tragacanth gum, acacia, locust bean gum, stearic acid, crosslinked sodium polyacrylate, polyvinyl alcohol, carbomer, sorbic acid, potassium sorbate, ethylparaben, benzyl alcohol, glycerin and propylene glycol.

Optionally, the preparation is in the form of decoction, granule, capsule, tablet, chewable tablet, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection.

Optionally, the cancer comprises one or more of lung cancer, pancreatic cancer, colon cancer, leukemia, lymphoma, breast cancer, brain cancer, esophageal cancer, gastric cancer, renal cancer, prostate cancer, uterine cancer, ovarian cancer.

Alternatively, the cancer is pancreatic cancer.

The amino steroid compound can be well combined with the G tetrad in the promoter of the K-ras gene through the introduction of halogen, has a larger combination pocket with the G tetrad, and is more tightly combined, so that the inhibition effect on the G tetrad structure of the K-ras gene promoter is enhanced, the expression of the K-ras protein of the oncogene is further inhibited, the death of cancer cells is promoted, and the aim of treating cancer is fulfilled. The invention aims at inhibiting effect of the K-ras gene promoter G tetrad structure, is not used for inhibiting K-ras protein in essence, but is used for preventing the promoter of the K-ras gene from being started so as to inhibit the synthesis of Kras protein, so that protein allosteric drug resistance cannot be generated, and the inhibition is superior to that of a K-ras protein inhibitor.

Drawings

FIG. 1 is a schematic representation of the mechanism of the halogenated compounds of the present application to inhibit the K-ras gene.

FIG. 2 is a graph showing the effect of 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) and K-ras protein inhibitor Sotorasib (Sotorradib, AMG 510) on A549 cell proliferation activity and K-ras gene expression;

A. b, C: effect results of different concentrations of 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) and the K-ras protein inhibitor sotorastib (sotoracicb, AMG 510) co-cultured with a549 cells for 24h, 48h or 96 h; the abscissa is the control group, AMG510, and CK1002; the ordinate is cellular activity in units;

d: inhibition profile of 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) for a549 cells; the abscissa is the concentration logarithm of CK1002 without units; the ordinate is OD value, no unit;

e: results of co-culturing 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) and K-ras protein inhibitor sotorastib (AMG 510) with a549 cells for 24h, and graph of the effect on K-ras gene levels; the abscissa is the control, AMG510 (50. Mu.M) and CK1002 (100. Mu.M); the ordinate is the expression level of K-ras gene, and the reference group is used as a standard;

f: results of co-culturing 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) and K-ras protein inhibitor sotorastib (AMG 510) with a549 cells for 48h, and graph of the effect on K-ras gene levels; the abscissa is the control, AMG510 (50. Mu.M) and CK1002 (50. Mu.M); the ordinate is the expression level of K-ras gene, and the reference group is used as a standard;

G: results of co-culturing 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) and K-ras protein inhibitor sotorastib (AMG 510) with a549 cells for 96 h; the abscissa is the control, AMG510 (50. Mu.M) and CK1002 (50. Mu.M); the ordinate indicates the K-ras gene expression level, and the control group is used as a standard.

FIG. 3 is a graph showing the effect of the 2 beta-piperazinyl-3 alpha-hydroxy-5 alpha-steroid-17-one and the K-ras protein inhibitor Sotorasib on PANC-1 cell proliferation activity and K-ras gene expression;

A. b, C: effect results plots after co-culturing 2 beta-piperazinyl-3 alpha-hydroxy-5 alpha-steroid-17-ketone (CK 1002) and K-ras protein inhibitor sotorastib (AMG 510) with PANC-1 cells for 24h, 48h or 96h at different concentrations; the abscissa is the control group, AMG510, and CK1002; the ordinate is cellular activity in units;

d: inhibition profile of 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) for PANC-1 cells; the abscissa is the concentration logarithm of CK1002 without units; the ordinate is OD value, no unit;

e: results of co-culturing 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) and K-ras protein inhibitor sotorassib (AMG 510) with PANC-1 cells for 24h, and graph of the effect on K-ras gene levels; the abscissa is the control, AMG510 (50. Mu.M) and CK1002 (100. Mu.M); the ordinate is the expression level of K-ras gene, and the reference group is used as a standard;

F: results of co-culturing 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) and K-ras protein inhibitor sotorassib (AMG 510) with PANC-1 cells for 48h, and graph of the effect on K-ras gene levels; the abscissa is the control, AMG510 (50. Mu.M) and CK1002 (50. Mu.M); the ordinate is the expression level of K-ras gene, and the reference group is used as a standard;

g: results of co-culturing 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (CK 1002) and K-ras protein inhibitor sotorastib (AMG 510) with PANC-1 cells for 96 h; the abscissa is the control, AMG510 (50. Mu.M) and CK1002 (50. Mu.M); the ordinate indicates the K-ras gene expression level, and the control group is used as a standard.

FIG. 4 is a graph showing the effect of the 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one and the K-ras protein inhibitor Sotorasib on tumor volume in A549 lung cancer tumor-bearing mice in the present application; the abscissa is the number of days, the units are days; the ordinate is tumor volume in mm ³ 。

FIG. 5 is a graph showing the results of the docking of the compound of comparative example 2 of the present application with the G tetrad of the K-ras gene; a: a full view; b: a partial enlarged view of the white circle in fig. a.

FIG. 6 is a graph showing the results of the docking of the compound of example 5 of the present application with the G tetrad of the K-ras gene; a: a full view; b: a partial enlarged view of the white circle in fig. a.

Detailed Description

Embodiments will now be described in detail with reference to the accompanying drawings. Although the embodiments have been described in connection with the accompanying drawings and the related description, it is not intended to limit the scope to the embodiments disclosed herein. On the contrary, the intent is to cover all alternatives, modifications and equivalents as included within the scope of the invention as defined by the appended claims.

As shown in FIG. 1, in the present application, by using the compound of the present invention as a small molecule inhibitor, it is possible to bind to G tetrads which are easily formed in the DNA structure of the promoter sequence of the Kras gene (the promoter structure of the K-ras gene is a G-base-rich sequence, and a G tetrad (G-quatrupplex) structure is easily formed at the time of starting), thereby preventing the start of the promoter, inhibiting the synthesis of K-ras protein, and thus achieving the effect of inhibiting the growth of cancer cells.

Unlike conventional K-ras protein allosteric causes that small molecule drugs cannot bind to pocket regions of the protein, and thus the inhibition effect on the K-ras protein is lost, resulting in the generation of drug resistance, the present application relates to small molecule inhibitors of the K-ras gene, which can inhibit the gene as such, regardless of the allosteric of the K-ras protein, if inhibition occurs at the gene DNA level. In other words, the gene inhibitor of the present application is the gene inhibitor at the root of the target gene, and theoretically, does not cause the problem of drug resistance of the protein inhibitor.

Further description of the present application is provided below in connection with examples.

Example 1

The compound of example 1 of the present application having structure IV below can be prepared by the following two process routes.

The first process route is as follows:

(1.1) quinolinic acid (10 g,57.7 mmol) was dissolved in 200ml anhydrous DMF (N, N-dimethylformamide), pyBOP (benzotriazo-1-yl-oxy-tris-pyrrosidin-o-phospho-fluorous-phosphate, benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, 36g,69.2 mmol) and HOBt (N-hydroxybenzotriazo-le, 1-hydroxybenzotriazole, 9.43g,69.2 mmol) was added, and after stirring for 5min proline terbutyrate was added followed by DIPEA (N, N-diisopropylethylamine, 40mL,0.22 mol). The above solution was stirred at room temperature for reaction overnight for 12 hours, then the reaction solution was poured into 1L of water, extracted three times with ethyl acetate, dried over sodium sulfate, and evaporated to give 13.1g of N- (quinolinic acid-2-formate) -L-proline terbutyrate (Compound 9).

(1.2) to a solution of 150ml TFA/DCM (trifluoroacetic acid/dichloromethane, 95:5) was added N- (quinolinic acid-2-formate) -L-proline terbutyric acid ester (compound 9, 13.0g,35.8 mmol), and the mixture was stirred at room temperature for 3h, and evaporated to dryness to give 13.2g of 1- (quinolin-2-yl) -L-proline (compound 10).

(1.3) epiandrosterone (4 g) (14 mmol) was dissolved in anhydrous pyridine (20 ml), and p-toluenesulfonyl chloride (4.6 g) (24 mmol) was added thereto, followed by stirring and reaction for 3 hours, and then reflux was conducted for 4 hours, whereby pyridine was recovered to obtain a semisolid. Adding water solution to precipitate solid, filtering, and recrystallizing to obtain white crystal, namely 5α -steroid-2-alkene-17-ketone (compound 1), with yield of about 60%.

(1.4) A chloroform solution of 5α -steroid-2-en-17-one (Compound 1) (1.7 g in 13ml of chloroform) was added dropwise to 17ml of 8% peracetic acid, 14ml of water and 1.7g of sodium acetate. Stirring and reacting for 17h, separating an organic layer, washing to be neutral, concentrating the solid, and recrystallizing to obtain white crystals, namely 2 alpha, 3 alpha-epoxy-5 alpha-steroid-17-ketone (compound 2), wherein the yield is about 70%.

(1.5) 1.0g (3.5 mmol) of 2α,3α -epoxy-5α -steroid-17-ketone (compound 2) was refluxed in 5ml of piperazine and 2ml of water for 50 hours, the piperazine was recovered, the aqueous solution was added to precipitate a solid, the solid was collected by filtration, and after recrystallization, 2β -piperazinyl-3α -hydroxy-5α -steroid-17-ketone (compound 3) was obtained in a yield of about 40%.

(1.6) to 200ml of anhydrous diethyl ether, adding 6ml (42.3 mmol) of trimethylsilylacetylene, dropping MeLi (1.6M in THF) at 0 degree, stirring the mixture at room temperature for 1 hour, adding 2 beta-piperazinyl-3 alpha-hydroxy-5 alpha-steroid-17-one (compound 3) (4g,10.6mmol in THF 200ml) at 0 degree, stirring the mixture at room temperature for 7 hours, adding ice water to terminate the reaction, extracting with ethyl acetate, drying with sodium sulfate, and evaporating to obtain 10.3g (2 beta, 3 alpha, 5 alpha, 17 alpha) -2- (piperazin-1-yl) androstane-20-acetylene-3, 17-diol (compound 6) with the following structure;

(1.7) 1- (quinolin-2-yl) -L-proline (compound 10) (11.4 g,30.7 mmol) and HCTU (2- (6-chloro-1H-benzotriazol-1-yl) -1, 3-tetra methylaminium hexa-fluoroforsphate, 2- (6-chloro-1H-benzotriazol-1-yl) -1, 3-tetramethylammonium hexafluorophosphate, 12.7g,30.7 mmol) were added to 430ml of anhydrous DMF, the solution was stirred for 5min, then (2β,3α,5α,17α) -2- (piperazin-1-yl) androstane-20-acetylene-3, 17-diol (compound 6) (10.3 g,25.6 mmol) and DIPEA (26.7 ml,153.4 mmol) were added, the mixture was stirred overnight at room temperature, poured into 2L of water, extracted three times with ethyl acetate, and the organic phase was evaporated to give { 5.75g, 5 α,5α,17α) -2- (piperazin-1-yl) androstane-3, 17-diol (compound 6) (26.7 mmol), and { 2- [ (35.5 α,5 α,17 α) -2-hydroxy ] 2-prolinate.

(1.8) into a reaction flask, 196mg of Compound 11, 10ml of dichloromethane and 0.38ml of triethylamine were charged, and dissolved with stirring. Then 190mg of chloroacetyl chloride was slowly added. The reaction was stirred at room temperature overnight. After the completion of the reaction, the mixture was concentrated to dryness, and the residue was diluted with dichloromethane, washed successively with water, a saturated aqueous ammonium chloride solution, a potassium carbonate solution and a saturated brine, and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (DCM/meoh=150/1) to give compound IV of example 1 as a yellow powder in 41% yield.

Through the detection, the detection results show that, ¹ H NMR(400MHz,CDCl ₃ )δ8.22(t,J＝9.0Hz,1H),8.15–7.93(m,2H),7.84(d,J＝8.1Hz,1H),7.77–7.68(m,1H),7.62–7.52(m,1H),5.75and5.33(m,1H),5.18–5.08(m,1H),4.33–4.18(m,1H),4.06(d,J＝17.9Hz,2H),4.02–3.85(m,1H),3.77–3.68(m,1H),3.62(s,1H),3.22(d,J＝70.9Hz,2H),2.81(d,J＝41.8Hz,1H),2.58(d,J＝3.1Hz,1H),2.54–2.12(m,5H),1.99(ddd,J＝20.9,15.4,6.5Hz,8H),1.76–1.41(m,13H),1.03(s,1H),0.92(s,2H),0.89–0.86(m,3H),0.85–0.83(m,3H)。

¹³ C NMR(101MHz,CDCl ₃ )δ170.45,170.28,166.92,166.78,154.50,149.34,149.30,146.09,145.60,139.09,139.07,136.97,136.87,130.04,129.92,129.74,129.63,129.44,128.46,128.43,128.07,127.78,127.75,127.65,127.59,124.51,123.62,121.92,119.18,119.00,87.78,62.48,59.24,54.55,50.60,47.01,41.39,41.31,39.07,36.09,35.98,35.73,35.12,34.58,32.83,32.07,31.65,31.58,30.34,30.29,29.84,29.50,29.15,23.20,22.83,22.63,20.93,14.26,13.36,12.97,1.29,1.16.

mass spectrometry (MS, EI), C ₄₂ H ₅₃ ClN ₄ O ₅ ，728.8。

A second process route:

compound IV of example 1 was prepared in the same manner as in process route one, except that the order of the process steps was adjusted as follows: starting from compound (3) (2β -piperazinyl-3α -hydroxy-5α -steroid-17-one), in process route one, acetylene is first introduced to obtain compound (6) ((2β,3α,5α,17α) -2- (piperazin-1-yl) androsta-20-acetylene-3, 17-diol), followed by a quinoline and proline containing moiety to obtain compound 11 ({ 4- [ (2β,3α,5α,17α) -3, 17-dihydroxyandrosta-20-yn-2-yl ] piperazin-1-yl } [ (2S) -1- (quinoline-2-carboxylate) proline-2-yl ] formate) and finally halogenated to obtain compound IV of example 1; in scheme two, a halogen-containing group is first introduced in compound (3) to obtain compound (12) having the structure below, followed by acetylene to obtain compound (13), and finally a quinoline and proline containing moiety is introduced to obtain compound IV of example 1:

example 2

The compound of example 2 of the present application having structure V below can be prepared by the following two process routes.

The first process route is as follows:

(2.1)

starting from compound (3) (2β -piperazinyl-3α -hydroxy-5α -steroid-17-one), this compound (3) is reacted with 1- (quinolin-2-yl) -L-proline (compound 10) in which a 1- (quinolin-2-yl-carbonyl) -L-proline moiety is introduced to give compound D having the following structural formula

(2.2) into the reaction flask, 313mg of Compound D, 447mg of copper bromide and 20ml of methanol were charged, and the temperature was raised to 80℃to reflux the mixture for 15 hours. The reaction solution was cooled to room temperature, water and methylene chloride were added thereto, and the mixture was stirred for ten minutes, followed by separation. The organic phase was washed with saturated brineOnce, concentrate to dryness under reduced pressure. The residue was purified by column chromatography (DCM/MeOH/NH ₄ Oh=100/1/0.1), 157mg of the compound of example 2 was obtained as a yellow powder in 44% yield.

Through the detection, the detection results show that, ¹ H NMR(400MHz,CDCl ₃ )δ8.22(dd,J＝12.6,8.5Hz,1H),8.14–7.91(m,2H),7.87–7.80(m,1H),7.77–7.67(m,1H),7.63–7.51(m,1H),5.85 and 5.34(m,1H),5.10 and 4.52(m,1H),4.30–3.29(m,7H),3.08(s,1H),2.80–1.39(m,24H),1.08(d,J＝13.3Hz,2H),0.92(s,1H),0.88(dd,J＝5.1,2.5Hz,3H),0.86–0.81(m,3H)。

mass spectrometry (MS, EI), C ₃₈ H ₄₉ BrN ₄ O ₄ ，704.7。

A second process route:

compound V of example 2 was prepared in the same manner as in process route one, except that the order of the process steps was adjusted as follows: starting from compound (3) (2β -piperazinyl-3α -hydroxy-5α -steroid-17-one), in process scheme one, a quinoline and proline containing moiety is first introduced to obtain compound D, followed by halogenation to obtain compound V of example 2; in scheme two, a halogen-containing group is first introduced in compound (3) to obtain compound (14) having the structure, followed by a quinoline and proline containing moiety to obtain compound V of example 2:

example 3

The compound of example 3 of the present application having structure VI below can be prepared by the following procedure.

The compound of structure VI above was prepared in the same manner as in scheme one of example 1, except that: in step (1.6), trimethylsilyl chloroacetylene is substituted for the trimethylsilyl acetylene therein, and since halogen has been attached to acetylene, the halogenation process in step (1.8) is omitted here. The compound VI thus obtained is characterized as follows.

Through the detection, the detection results show that, ¹ H NMR(400MHz,CDCl ₃ )δ8.21(dd,J＝12.3,8.6Hz,1H),8.13–7.92(m,2H),7.85–7.79(m,1H),7.72(m,1H),7.62–7.52(m,1H),5.86 and 5.11(m,1H),4.23–3.11(m,8H),2.77–2.58(m,2H),2.51–2.04(m,5H),2.01–0.98(m,22H),0.86(2s,3H),0.82(2s,3H),0.76–0.64(m,1H)。

mass spectrometry (MS, EI), C ₄₀ H ₅₁ ClN ₄ O ₄ ，687.1。

Example 4

The compound of example 4 of the present application having structure VII below can be prepared by the following two process routes.

The first process route is as follows:

(4.1) in the same manner as in (1.6), starting from compound 3 (2β -piperazinyl-3α -hydroxy-5α -steroid-17-one), acetylene was introduced therein to give (2β,3α,5α,17α) -2- (piperazin-1-yl) androstane-20-acetylene-3, 17-diol (compound 6) of the following structure

(4.2) in the same manner as in (1.7), a portion containing quinoline and proline was introduced into the compound 6 to obtain a compound (15)

(4.3) in the same manner as in (2.2), a halogen bromine atom was introduced into the compound 15 to obtain a compound of the formula VII of example 4.

Through the detection, the detection results show that, ¹ H NMR(400MHz,CDCl ₃ )δ8.21(dd,J＝12.3,8.6Hz,1H),8.14–7.91(m,2H),7.86–7.78(m,1H),7.73(m,1H),7.61–7.51(m,1H),5.84 and 5.08(m,1H),4.22–3.10(m,9H),2.76–2.57(m,2H),2.56(2s,1H),2.51–2.04(m,5H),2.01–0.98(m,20H),0.87(2s,3H),0.81(2s,3H),0.77–0.63(m,1H)。

mass spectrometry (MS, EI), C ₄₀ H ₅₁ BrN ₄ O ₄ ，731.0。

Process route two

Compound VII of example 4 was prepared in the same manner as in process route one, except that the order of the process steps was adjusted as follows: starting from compound (3) (2β -piperazinyl-3α -hydroxy-5α -steroid-17-one), in process route one, acetylene is first introduced to obtain compound (6) ((2β,3α,5α,17α) -2- (piperazin-1-yl) androsta-20-acetylene-3, 17-diol), followed by a quinoline and proline containing moiety to obtain compound 15, and finally halogenation to obtain compound VII of example 4; in scheme two, a halogen-containing group is first introduced in compound (3) to obtain compound (16) having the structure below, followed by acetylene to obtain compound (17), and finally a quinoline and proline containing moiety is introduced to obtain compound VII of example 4:

Example 5

The compound of example 5 of the present application having structure VIII below can be prepared by the following two process routes.

The first process route is as follows:

(5.1) A compound having the following structure (17) was produced in the same manner as in (1.6), except that: replacement of trimethylsilyl acetylene with trimethylsilyl fluoroacetylene

(5.2) introducing a quinoline-and-proline-containing moiety into the compound (17) in the same manner as in (1.7) to obtain a compound having the following structure (18)

(5.3) introducing a halogen-containing moiety, i.e., a chlorine-containing moiety, into the compound (18) in the same manner as in (1.8) to obtain a compound having the following structure (19)

(5.4) in the same manner as in (2.2), a halogen bromine atom was introduced into the compound (19) to obtain a compound of the formula VIII of example 5.

Through the detection, the detection results show that, ¹ H NMR(400MHz,CDCl ₃ )δ8.22(t,J＝9.0Hz,1H),8.16–7.93(m,2H),7.84(d,J＝8.1Hz,1H),7.76–7.68(m,1H),7.62–7.51(m,1H),5.74 and 5.33(m,1H),5.17–5.08(m,1H),4.33–4.20(m,1H),4.06(d,J＝17.9Hz,2H),4.02–3.89(m,1H),3.75–3.69(m,1H),3.22(d,J＝70.9Hz,2H),2.81(d,J＝41.8Hz,1H),2.59(d,J＝3.1Hz,1H),2.53–2.12(m,4H),1.99(ddd,J＝20.9,15.4,6.5Hz,8H),1.77–1.41(m,13H),1.02(s,1H),0.93(s,2H),0.89–0.86(m,3H),0.85–0.82(m,3H)。

mass spectrometry (MS, EI), C ₄₂ H ₅₁ BrClFN ₄ O ₅ ，824.9。

Other process routes:

after the process step (5.1) is carried out to obtain the compound of structure (17), the order of the above process steps (5.2) to (5.4) may be adjusted, i.e. the halogenation may be carried out first to introduce halogen, followed by the introduction of the quinoline and proline containing moiety to obtain the compound of structure VIII of example 5.

EXAMPLE 6 preparation of oral tablets of the Compound of the invention

Each tablet contains 40mg of the compound of example 1, and the tablet composition is as follows:

mixing the above materials (except magnesium stearate), sieving with 120 mesh sieve, adding warm distilled water, stirring, granulating, drying, adding magnesium stearate, mixing, and tabletting.

Comparative example 1

The compound of comparative example 1 isNamely, compound 3.

The procedure (1.3) to (1.5) in scheme one of example 1 were employed to prepare the above compound 3.

Comparative example 2

The compound of comparative example 2 is

Namely, compound 11.

The procedure (1.3) to (1.7) of procedure one of example 1 were employed to prepare the above-mentioned compound 11.

Comparative example 3

The compound of comparative example 3 is

Namely, compound D.

The above compound D was obtained by the procedure (2.1) in the first process route of example 2.

Comparative example 4

The compound of comparative example 4 is the Kras protein inhibitor Sotorasib (Sotorasib) (AMG-510).

Performance testing

1. The compounds of the present application and the compounds of the comparative examples have anti-A549 (human non-small cell lung cancer cells) and PANC-1 (human pancreatic cancer cells) proliferative activity in vitro and their effect on K-ras gene expression

The main reagent comprises: DMEM high sugar medium (pramoxine, WHO1122012XP 01), RPMI1640 medium (pramoxine, WHO1122007 XP), fetal bovine serum (bie xplorer, 2041296), 0.25% trypsin solution (pramoxine, EHO1122012SP 01), penicillin-streptomycin solution (pramoxine, WHO1122009 XP), phosphate buffer (pramoxine, WHO1132101SP 01), cell Counting Kit-8 reagent (Biosharp, PB 180327), total RNA flash extraction kit (Shanghai strapdown biotechnology limited, 220010), cDNA Synthesis SuperMix (Novoprotein, 0516971), SYBR qPCR SuperMix Plus (Novoprotein, 0519371), AMG510 (MCE, 2296729-00-3).

The main instrument is as follows: microscope, CO ₂ Incubator, ultra-clean bench, multifunctional enzyme-labeled instrument, nanoDrop concentration measuring instrument, bio-Rad fluorescent quantitative PCR instrument, high-speed low-temperature centrifuge, high-temperature pressure sterilizing pot, enzyme-free Tip head, pipettor, etc.

Cell culture: a549 cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS), PANC-1 cells were cultured in RPMI1640 medium containing 10% FBS; placing at 37deg.C and 5% CO ₂ Culturing in an incubator, and when the cells in the culture flask grow to 80% -90%, adopting pancreatin to digest the cells for passage, and taking the cells in the logarithmic growth phase for experiment.

CCK8 assay for cellular activity: a549 cells (about 5000 cells/well) or PANC-1 cells (about 6000 cells/well) were inoculated into 96-well plates, after the next day of adherence, 10. Mu.L of CKI002 (i.e., compound 3) or AMG510 (i.e., kras protein inhibitor Sotorasib) at different concentrations (0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M, and DMSO) were added according to the experimental design, the control group (without drug addition, replaced with corresponding volumes of DMSO) was simultaneously set, 5 duplicate wells were set up, after 24 hours, 48 hours, or 96 hours of incubation was performed, 10. Mu.L of CCK8 solution was added to each well, incubated at 37℃for 1 to 4 hours in the dark, absorbance values were measured at a wavelength of 450nm, and half inhibition concentration values (IC were calculated ₅₀ Values).

RT-PCR method for detecting K-ras gene expression

Total cell RNA extraction: after 24h, 48h, 96h (at 10) of cells were cultured and dosed using 6-well plates ^-6 Adding medicine in mol/L dosage, co-culturing with cells), extracting total RNA according to instruction using total RNA extraction kit, measuring total RNA concentration using Nanodrop concentration measuring instrument, adjusting concentration to 500-1000 ng/. Mu.L, and storing in refrigerator at-80deg.C.

RNA reverse transcription (using 20. Mu.L system): taking 1. Mu.g of total RNA and adding DEPC water (ultra-pure water treated with diethyl pyrocarbonate and sterilized at high temperature) to adjust to 12. Mu.l, and denaturing at 65℃for 5min; adding 4 mu L of 4-factor (namely, 4 times X DNA degrading enzyme reagent) into the mixed solution of the previous step, and incubating at 37 ℃ for 5min to remove gDNA; adding 4 mu L of 5-point reagent into the mixed solution in the previous step, and carrying out reverse transcription at 37 ℃ for 15min, 50 ℃ for 5min and 98 ℃ for 5min; the reverse transcription product PCR was taken and stored at-20 ℃.

qPCR (using 20 μl system): taking cDNA after reverse transcription, and diluting 5 times for standby; KRAS gene primer working solution concentration is 10 mu M; preparing qPCR mix+primer (wherein qPCR Mix refers to cDNA Synthesis SuperMix (Novoprotein, 0516971) in the reagent, and Primer refers to front and rear Primer sequences), uniformly configuring 10 mu L of Mix and 1 mu L of Primer F/R for each well; reasonably distributing plates, operating on ice, and adding 12 mu L of the mixed solution into each hole; 2 mu L of template cDNA of different treatment groups are respectively added, and 20 mu L of DEPC water is complemented; setting amplification parameters: preheating at 95 ℃ for 3min, amplifying (circulation is 40) at 95 ℃ for 15s and at 60 ℃ for 30s; the relative expression levels of the genes were calculated. The results are shown in FIGS. 2 and 3.

As shown in fig. 2, 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (compound 3, also referred to as CKI 002) has a better effect of inhibiting cell proliferation and is able to down regulate K-ras gene expression at the same concentration as AMG-510, with more significant differences from the control group and AMG-510. As shown in fig. 3, 2 beta-piperazinyl-3 alpha-hydroxy-5 alpha-steroid-17-one (compound 3, also referred to as CKI 002) has a good inhibitory effect against PANC-1 cell proliferation, but has no good advantage in inhibiting K-ras gene expression. Mutations in the K-ras gene typically occur in PANC-1 (pancreatic cancer) cells, and therefore, it is speculated that 2β -piperazinyl-3α -hydroxy-5α -steroid-17-one (compound 3, also referred to herein as CKI 002) inhibits the K-ras protein, and not the K-ras gene, similar to the therapeutic mechanism of AMG-510 for pancreatic cancer.

2. Effect of the Compounds of the present application and the Compounds of the comparative examples on A549 lung cancer tumor-bearing mice

SPF-class Balb/c-nu nude mice 70, male and female halves, body weight: 12.0-15.0 g, 10 mice are selected as blank groups, and the rest mice are used for preparing lung cancer tumor-bearing mice. Inoculating 1×107/ml0.2mL of logarithmic growth phase A549 lung cancer cells into right armpit of 60 Balb/c-nu nude mice, and treating tumor volume reaching 100mm ³ Above, 50 mice were selected and randomly divided into 9 groups according to tumor volume, respectively: the model control group, AMG510 control group (30 mg/kg) and the low, medium and high dose groups (5 mg/kg, 10mg/kg, 20 mg/kg) of compound 3 of comparative example 1 were given by intraperitoneal injection of the respective liquid medicine, each group of 10 mice, with a dose of 20mL/kg, 1 time a day for 14 days continuously, and the normal control group and the model control group were given by intraperitoneal injection of the same volume of vehicle. Weighing 1 time per week, and counting the weekly food intake and water intake of the mice; detecting the length and width of the tumor by using a vernier caliper every 3 days after administration, and calculating the tumor volume and the relative tumor proliferation rate; after the last administration, mice were sacrificed, spleen and tumor tissues were taken, and organ coefficients were calculated. The results are shown in fig. 4, and table 1 below also shows the effect of compound 3 on tumor volume in a549 lung cancer tumor-bearing mice.

Table 1: effect of Compound 3 on tumor volume of A549 lung cancer tumor-bearing mice

The results show that:

(1) Animal quality of life and time of survival evaluation: the mortality of the mice in the low, medium and high dose groups of the compound 3 is 10%, 20% and 20% respectively.

(2) Effect of compound 3 on body weight, food intake and water intake of a549 lung cancer tumor-bearing mice: the low, medium and high doses of compound 3 had no significant effect on the body weight, food intake and water intake of the mice.

(3) Effect of compound 3 on tumor volume of a549 lung cancer tumor-bearing mice: the low dose of the compound 3 can obviously reduce the tumor volume of mice when D7, D13 and D14 are dosed, and the medium dose and the high dose of the compound 3 can obviously reduce the tumor volume of mice when D4-14 are dosed; the relative tumor proliferation rates of compound 3 at the 14 th day of low, medium and high dose administration are 61.57%, 58.58% and 50.75%, which indicates that the low, medium and high dose groups of compound 3 also show an ascending trend after 13 days of administration, indicating that drug resistance is also generated in mice; in contrast, AMG510 had a greatly reduced tumor inhibition effect and a dramatic increase in tumor volume after 10 days of administration.

(4) Effect of compound 3 on tumor weight and spleen organ coefficient of a549 lung cancer tumor-bearing mice: the high dose of the compound 3 can obviously reduce the tumor weight and the tumor body ratio of mice, and the low and medium doses of the compound 3 have a decreasing trend on the tumor weight and the tumor body ratio of mice, but have no statistical difference. The low, medium and high doses of the compound 3 have no obvious effect on the spleen organ coefficients of mice.

The above results indicate that: the compound 3 has obvious tumor inhibiting effect on A549 lung cancer tumor-bearing mice, reduces the death rate of the mice, but has the problem of drug resistance in the later period.

In view of the above problems, the present application was modified based on 2 beta-piperazinyl-3 alpha-hydroxy-5 alpha-steroid-17-ketone, namely, compound 3, and as described above, the compounds of examples 1 to 5 of the present application were prepared, and the properties of the compounds of examples and the compounds of comparative examples were examined.

3. Molecular modeling

MOE (Molecular Operating Environment ) is a comprehensive software system developed by canadian chemical computing group Chemical Computing Group ulc for pharmaceutical and life sciences. The system is a comprehensive application environment and technology development platform, and integrates visualization, simulation and application development. The system calculates the fraction of the binding between the molecule and the target according to the parameters of static electricity, hydrogen bonding, molecular attraction, molecular orbit and the like between the molecule and the target, and the more negative the fraction is, the lower the free energy after the binding is, the more stable the conformation is, the better the binding is, and the system can also show the specific binding conformation between the molecule and the target.

The compounds of examples 1 to 5 and comparative examples 1 to 3 were subjected to a docking simulation with G-tetrad by MOE and the docking results were scored. Wherein, the PDB file of the K-ras gene promoter DNA G-tetrad is downloaded from the RCSB website, and the PDB structure file of the amino steroid halogen is generated by chemBioDraw software. The docking results are shown in table 2, and figures 5 and 6 further show the conformation of the compound after halogen substitution in combination with G-tetrad.

Table 2: docking scoring of each compound with G tetrad

As can be seen from table 2, the score of the compound of each of examples 1 to 5 for butt joint with G tetrad is smaller than that of the comparative example, and as described above, the score is calculated based on parameters such as electrostatic parameters, hydrogen bonding, molecular attraction, molecular orbitals, and the like between the molecules and the target; the more negative the score, the lower the free energy after binding, the more stable the conformation, the better the binding, and therefore the compounds of the present application bind better to the G tetrad than the comparative compounds.

Further, as shown in fig. 5 and 6, the compound in example 5 introduces halogen, so that the binding site and mode between the molecule and the G-tetrad target are different from those of comparative example 2, and the binding site and mode between the molecule and the target are more favorable for tight binding and lower free energy. The compound contains strong electron withdrawing groups such as halogen and the like, and can form strong electrostatic action when encountering G-tetrad which is rich in electrons. As can be seen from FIG. 6, the halogen in the compound of example 5 has better binding to the G-tetrad, and the pocket binding to the target is larger and the binding site is increased, thus further enhancing the inhibition of the G-tetrad, relative to the docking of FIG. 5.

4. EXAMPLE 1 Down-Regulation of K-ras protein in A549 cells

The protein (Kras) enzyme-linked immunoassay (ELISA) kit of human and mouse sarcoma virus gene is adopted for determination. The principle is that the Kras protein level in the specimen is measured by a double antibody sandwich method. Coating a micro-pore plate with a purified Kras protein antibody to prepare a solid-phase antibody, adding a sample protein containing the Kras protein into the micro-pore, combining with an HRP-marked detection antibody to form an antibody-antigen-enzyme-labeled antibody complex, developing color by using TMB, detecting an OD value at a wavelength of 450nm by using an enzyme-labeled instrument, positively correlating the color with the content, and calculating the content of the Kras protein by using a standard curve.

After treating the cells with 10 μl of the compound of example 1 for 5d, a control group (adding only the same volume of DMSO) was set at the same time; collecting cells, adding cell lysate to break cells, extracting total cell protein, correcting protein content of each hole during measurement, making a standard curve, calculating K-ras protein content, and comparing difference of each group in ng/mg.

Table 3: down-regulating action of the Compounds of example 1 against K-ras protein in A549 cells

	Experimental conditions	K-ras protein content
			Blank control	0	4.3787ng/mg
Example 1 Compounds	Treatment with 0.6. Mu.M for 5 days	2.6797ng/mg

As shown in Table 3, the compound of example 1 was able to significantly inhibit K-ras protein content, from 4.3787ng/mg to 2.6797ng/mg, by 38.8%; in addition, although the K-ras gene in the A549 cells is easier to mutate, the compound in the embodiment 1 of the application can still generate a larger reduction effect at a lower concentration, which proves that the compound in the application can break through the drug resistance of the conventional drugs and has better development prospect.

5. Inhibition of pancreatic cancer (PANC-1) cells by the compounds of the present application and the compounds of the comparative examples

PANC-1 cells were cultured in RPMI1640 medium containing 10% FBS, and the medium was placed at 37℃in 5% CO ₂ Culturing in an incubator; when the cells in the culture flask grow to 80% -90%, the cells are digested by pancreatin for passage, and the cells in logarithmic growth phase are taken for experiment.

The above PANC-1 cells (about 6000 cells/well) in logarithmic growth phase were inoculated into 96-well plates, 10. Mu.L of the compound of example 1, the compound of comparative example 1 and the compound of comparative example 2 were added according to the experimental design for treatment after the next day of adherence, a control group (only the same volume of DMSO was added), 5 to 6 complex wells were set per group, and after culturing for 72 hours, 10. Mu.L of CCK8 solution was added per well, incubated at 37℃for 1 to 4 hours in the absence of light, absorbance values were measured at a wavelength of 450nm, and half maximal inhibitory concentration values (IC were calculated ₅₀ Values).

The preparation method of the compounds with different concentrations comprises the following steps: the compounds of each example and comparative example were formulated with DMSO, the mother liquor concentration was 100mM, and the dissolution was assisted by shaking with a vortex shaker for 20 min. The preparation concentration is respectively as follows: 100. Mu.M, 30. Mu.M, 10. Mu.M, 3. Mu.M, 1. Mu.M, 0.3. Mu.M, 0. Mu.M.

By the method, the following steps can be obtained: for PANC-1, the IC50 value of the compound of example 1 of this application was 1.532. Mu.M, the IC50 value of the compound of example 2 was 7.628. Mu.M, and the IC of the compound of comparative example 1 ₅₀ IC of the compound of comparative example 2 with a value of 58.06. Mu.M ₅₀ A value of 2.575. Mu.M; from the above comparison, the compound of example 1 has much higher inhibitory effect on PANC-1 than the compound of comparative example 1 and is highly compatible with the compound of comparative example 2, and as described above, since the compound of the present application is directly combined with the G tetrad in the K-ras gene, it has not only good effect of inhibiting pancreatic cancer cells, but also overcomes the problem of developing drug resistance of the compound of comparative example 1.

6. Inhibition of other tumor cells by the Compounds of the examples herein

Culturing cancer cells (DU 145, A549 and HCT-116) in DMEM culture solution containing 10% Fetal Bovine Serum (FBS), placing the culture solution at 37deg.C and 5% CO ₂ Culturing in an incubator; when the cells in the culture flask grow to 80% -90%, the cells are digested by pancreatin for passage, and the cells in logarithmic growth phase are taken for experiment.

Cancer cells (about 5000 cells/well) were inoculated into 96-well plates, 10. Mu.L of the compound of examples at different concentrations (100. Mu.M, 30. Mu.M, 10. Mu.M, 3. Mu.M, 1. Mu.M, 0.3. Mu.M) were added according to the experimental design after the next day of adherence, a control group (only DMSO of the same volume was added), 5-6 multiplex wells were provided per group, and after 72 hours of culture, 10. Mu.L of CCK8 solution was added per well, incubated at 37℃for 1-4 hours in the absence of light, absorbance values were measured at a wavelength of 450nm, and half inhibition concentration values (IC were calculated ₅₀ Values). The calculation results are shown in Table 4.

Table 4: IC of Compounds of examples on individual cancer cells ₅₀ Value of

DU145 is a prostate cancer cell, A549 is a lung cancer cell, and HCT-116 is a colon cancer cell.

As shown in Table 4, the amino steroid compound of the application has good inhibition effect on prostate cancer cells, lung cancer cells and colon cancer cells, and part of IC ₅₀ Even reaching below 10 mu M, and has good inhibiting effect on various cancers, and good therapeutic and prophylactic effects on cancersAnd (3) prospect.

7. Inhibition of KRAS gene expression by the compounds of the present application

The inhibition of KRAS gene expression by the compounds of example 1 of the present application was tested as follows.

7.1. Cell inoculation:

taking conventional cultured human non-small cell lung cancer cell A549 in logarithmic phase, digesting with 0.25% pancreatin, collecting cells, and adjusting cell density to 2×10 ⁴ Seed at 2.5 mL/well to 6 well plate;

7.2. cell treatment:

after the cells are attached, preparing the compound of the example 1 into a working solution of 3 mu M by using a complete culture medium corresponding to each cell, discarding the cell original culture solution, replacing the prepared working solution of the compound of the example 1, 2.5 mL/hole, and setting a solvent control hole;

7.3. Cell harvesting:

cells were digested with pancreatin and collected 24h, 48h, 96h after the treatment of cells with the compound working solution of example 1, respectively;

7.4. total cell RNA extraction:

extracting total RNA of cells by using an animal tissue total RNA extraction kit, and measuring the concentration of RNA;

KRAS Gene amplification:

7.5.1 preparation of reagents: specific primers were synthesized and used with ddH ₂ O was formulated at 100. Mu.M. Completely thawing the specific primer, the template RNA and corresponding reagents in the one-step RT-PCR kit, and placing the kit on an ice bath;

primer information is shown in table 5 below.

Table 5: primer information

7.5.2 the reaction system was formulated as shown in table 6 below.

Table 6: reaction system

Composition of components	Reaction system
		2X FastKing One Step RT-PCR Master mix (one-step fluorescence quantitative RT-PCR)	12.5μL
25 xRT-PCR Enzyme Mix (Enzyme mixture)	2μL
		Upstream specific primer (10. Mu.M)	0.625μL
Downstream specific primer (10. Mu.M)	0.625μL
		RNA template	100ng
RNase Free ddH 2 O (RNase-free ribonuclease double distilled water)	Is added to 50 mu L
		Total system	25μL

7.5.3 The PCR reaction conditions are shown in Table 7 below.

Table 7: PCR reaction conditions

7.5.4 electrophoretic analysis: preparing 1.5% agarose solution, adding Gel-Red (Gel Red) according to the proportion of 0.01%, and cooling to about 60 ℃ to prepare agarose Gel. And adding the product after the PCR reaction into a comb hole at a concentration of 10 mu L/hole, carrying out electrophoresis in TBE buffer solution, setting the electrophoresis voltage to 90V, finishing electrophoresis when the blue strip is electrified to a position which is about 1cm away from the bottom of the gel, and detecting the KRAS gene and GAPDH gene expression under a gel imaging system.

7.5.5 PCR band analysis: and analyzing the gel strips by adopting Image J software, measuring integrated optical density values, and calculating the relative gray value of KRAS.

7.6. The test results are shown in table 8 below.

Table 8: test results

From the above, it can be seen that the compound IV of example 1 of the present application acts on the human non-small cell lung cancer cell a549 for 24,48,96 hours, respectively, and can significantly down-regulate the Kras oncogene, which is related to the action mechanism of the compound for inhibiting the Kras gene promoter G-tetrad. Furthermore, as previously mentioned, the applicant has unexpectedly found that the inhibition of tumor cells by the halogenated amino steroid compounds of the present application occurs at the level of the gene DNA, which is essentially a small molecule inhibitor of the K-ras gene, in which case the K-ras protein can be inhibited regardless of its allosteric structure, and therefore does not create problems of resistance to protein inhibitors.

Claims (8)

1. An amino steroid compound is characterized in that the structural formula of the amino steroid compound is shown as a formula I or a formula II,

i is a kind of

Or (b)

II (II)

Wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Independently selected from H or a halogen-containing group;

and R is ₁ 、R ₂ At least one of which is a halogen-containing group; r is R ₃ 、R ₄ 、R ₅ At least one of which is a halogen-containing group;

And wherein R is ₁ 、R ₃ 、R ₄ Independently selected from H or halogen; r is R ₂ 、R ₅ Independently selected from H, haloacetyl or halobenzoyl.

2. The amino steroid according to claim 1, characterized in that said halogen is selected from F, cl, br or I.

3. The amino steroid according to claim 1, characterized in that it is selected from the group consisting of

(formula IV),

(V),

(formula VI),

(formula VII) or

(formula VIII).

4. A process for preparing an amino steroid according to any one of claims 1 to 3, characterized in that it comprises:

(A) Reacting a compound of the formula with a halogen-containing compound to give the compound of the formula I

The method comprises the steps of carrying out a first treatment on the surface of the Or (b)

(B) Reacting a compound of the formula with a halogen-containing compound to give the compound of the formula II

。

5. The use of an amino steroid compound according to any one of claims 1 to 3 for the preparation of a K-ras cancer gene promoter targeting inhibitor.

6. Use of an amino steroid compound according to any one of claims 1 to 3 for the preparation of a medicament for the prophylaxis and/or treatment of cancer, wherein the cancer is selected from one or more of pancreatic cancer, lung cancer and colon cancer.

7. A K-ras cancer gene promoter-targeted gene inhibitor, characterized in that the gene inhibitor comprises an amino steroid compound according to any one of claims 1-3.

8. A formulation comprising an amino steroid compound according to any one of claims 1 to 3 and a pharmaceutically acceptable adjuvant.