WO2023155846A1 - 17-吡啶基-10α-甲基-甾体衍生物及中间体化合物、其制备方法、其用途、以及其药物组合物 - Google Patents

17-吡啶基-10α-甲基-甾体衍生物及中间体化合物、其制备方法、其用途、以及其药物组合物 Download PDF

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WO2023155846A1
WO2023155846A1 PCT/CN2023/076540 CN2023076540W WO2023155846A1 WO 2023155846 A1 WO2023155846 A1 WO 2023155846A1 CN 2023076540 W CN2023076540 W CN 2023076540W WO 2023155846 A1 WO2023155846 A1 WO 2023155846A1
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pyridyl
methyl
compound
steroid
reaction
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PCT/CN2023/076540
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English (en)
French (fr)
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刘喜荣
唐杰
蒋红平
何群
罗桂芳
张沐
李龙
吴四清
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上海醇健医药技术有限公司
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Priority to CN202380008382.XA priority Critical patent/CN116917306A/zh
Publication of WO2023155846A1 publication Critical patent/WO2023155846A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton

Definitions

  • the invention relates to the technical field of medicines, in particular to a 17-pyridyl-10 ⁇ -methyl-steroid derivative and an intermediate compound, a preparation method thereof, an application thereof, and a pharmaceutical composition thereof.
  • Abiraterone acetate the chemical name is 17-(3-pyridyl)-androst-5,16-dien-3 ⁇ -ol acetate (structural formula is as follows), is a CYP17 inhibitor, its In combination with prednisone in the clinical treatment of metastatic advanced prostate cancer that has been resistant to traditional hormone therapy, it can not only reduce the level of prostate specific antigen, but also help shrink tumors and prolong the life of advanced prostate cancer patients.
  • the invention proposes a 17-pyridyl-10 ⁇ -methyl-steroid derivative and an intermediate compound, its preparation method, its use, and its pharmaceutical composition.
  • the present invention has synthesized 17-pyridyl-10 ⁇ -methyl-steroid derivatives whose C-10-position methyl group is ⁇ -configuration, and the compound is a new class of compound, which is effective for prostate cancer, breast cancer, colon cancer, and lung cancer Both have inhibitory effect.
  • the present invention provides a 17-pyridyl-10 ⁇ -methyl-steroid derivative, the steroid derivative has the structure of the following formula I or a pharmaceutically acceptable salt thereof:
  • R2 is selected from C1 ⁇ C5 alkyl, C1 ⁇ C5 haloalkyl, phenyl or halophenyl;
  • R3 is selected from halogen, C1 ⁇ C5 alkyl, C1 ⁇ C5 haloalkyl, C1 ⁇ C5 alkoxy, hydroxyl or amino;
  • i, j, k, m and n are each independently selected from 0, 1, 2, 3, 4 or 5.
  • the steroid derivative has the following structural formula of formula II:
  • the -OH when the R 1 is selected from -OH, the -OH is ⁇ -OH or ⁇ -OH.
  • the R 3 is selected from C1-C5 alkyl or haloalkyl.
  • said k is 0.
  • said i, j, m and n are all 0, preferably said k is 0.
  • the halogen in R to R is selected from F, Cl, Br or I.
  • the steroid derivative is selected from the following structural formulas:
  • the steroid derivative is selected from the following structural formulas:
  • the present invention provides a method for preparing 17-pyridyl-10 ⁇ -methyl-steroid derivatives.
  • the method comprises using the intermediate represented by formula V as a raw material, and connecting substituted or unsubstituted at the C-17 position The pyridyl;
  • the substituted or unsubstituted pyridyl is correspondingly identical to the pyridyl structure on the 17-pyridyl-10 ⁇ -methyl-steroid derivative;
  • R 9 and R 4 correspond to the same or correspond to the same after the reaction
  • the R 10 and R 5 correspond to the same or correspond to the same after the reaction
  • the R 11 and R 6 correspond to the same or correspond to the same after the reaction
  • the R 12 and R 7 correspond to the same or correspond to the same after the reaction.
  • the present invention provides an intermediate compound for the preparation of 17-pyridyl-10 ⁇ -methyl-steroid derivatives, wherein the intermediate compound is selected from the following structural formulas:
  • R 8 is selected from -OH or a protected hydroxyl group
  • said R 8 is selected from -OH or OAc; and/or;
  • the intermediate compound is selected from the following structural formulas:
  • the invention provides a preparation method of an intermediate compound, the preparation method comprising the following steps: performing photochemical conversion on the compound of the following formula so that the methyl group at the C-10 position is reversed from the ⁇ configuration to the ⁇ configuration
  • the photochemical conversion is an ultraviolet photocatalytic reaction.
  • the ultraviolet photocatalytic reaction first makes the steroid ring open in the wavelength range of 260-290nm, and then at 295nm The steroid ring is closed within the wavelength range of -340nm, and the reaction temperature is -10-50°C;
  • the preparation method of the intermediate compound of 17-pyridyl-10 ⁇ -methyl-steroid derivative is as follows:
  • Compound 1 is protected by the 3-hydroxyl group (for example, using acetic anhydride and other reagents) and the 17-position carbonyl group (for example, using ethylene glycol reagent) to obtain compound 3.
  • 3-hydroxyl group for example, using acetic anhydride and other reagents
  • 17-position carbonyl group for example, using ethylene glycol reagent
  • Compound 3 is oxidized to carbonyl by allylic position (7-position) to obtain compound 4, such as using catalyst N-hydroxyphthalimide and initiator benzoyl peroxide for air oxidation.
  • the methyl group at the 10-position is reversed from the ⁇ -configuration to the ⁇ -configuration to obtain compound 7 (ie, the intermediate compound).
  • the photocatalytic reaction first makes the steroid ring open in the wavelength range of 260-290nm, and then closes the ring in the wavelength range of 295-340nm.
  • the reaction temperature is controlled at -10-50°C.
  • Compound 7 undergoes two-step catalytic hydrolysis with acid (such as p-toluenesulfonic acid) and alkali (such as sodium hydroxide, etc.) to remove the protecting groups at the 3-position and 17-position to obtain Compound 8.
  • acid such as p-toluenesulfonic acid
  • alkali such as sodium hydroxide, etc.
  • Compound 8 undergoes hydrazone formation, iodine, and coupling reactions at the 17th position to obtain TM1.
  • the coupling reaction reagents are diethyl-(3-pyridine)-borane, bis(triphenylphosphine) dichloro palladium as catalyst.
  • TM1 is acetylated at the 3-position to obtain TM2.
  • the acetylation reagent can be acetic anhydride, acetyl chloride, etc.
  • TM2 is hydrogenated under the catalysis of transition metals, and the 5, 7, 16 double bonds are reduced to obtain TM4.
  • Pd/CaCO 3 catalyst is used, the reaction temperature is 40-70 ° C, the reduction is under hydrogen atmosphere, selectivity and higher yields.
  • TM1 protected the 5,7 conjugated double bond, 3 Oxidation and deprotection of the hydroxy group to obtain TM8.
  • the oxidation system used for the hydroxy oxidation is Tempo (2,2,6,6-tetramethylpiperidine oxide)-NaClO-NaBr.
  • the deprotection is carried out at a temperature of 50-65° C., because the target compound cannot be obtained at normal temperature.
  • Compound 7 is hydrolyzed into compound IN1F under base catalysis.
  • the reaction solvent can be ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile, acetone, etc.
  • the base can be sodium hydroxide, potassium hydroxide, potassium carbonate, etc., and the reaction temperature is 0-60°C.
  • Compound IN1F is hydrogenated under transition metal catalysis to synthesize compound CK004-1A.
  • the reaction solvent is one or a mixture of ethyl acetate, ethanol, dioxane, tetrahydrofuran, and dichloromethane.
  • the catalyst is 5% palladium carbon, 10% palladium carbon, metal platinum, platinum dioxide, platinum acetate and the like.
  • the hydrogen pressure is 0.05-2.0MPa, and the reaction temperature is 40-70°C.
  • Compound CK004-1A is hydrolyzed into compound CK004-1B under acid catalysis.
  • the reaction solvent is ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile, acetone, etc.
  • the acid is p-toluenesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, acetic acid, etc., and the reaction temperature is 0-40°C.
  • Compound CK004-1B undergoes hydrazone formation, iodine, and coupling reactions at the 17th position to obtain TM5.
  • the coupling reaction reagent is diethyl-(3-pyridine)-borane, using bis(triphenylphosphine) ) palladium dichloride catalyst.
  • the synthesis scheme of synthesizing TM7 from compound TM5 is that compound TM5 undergoes an esterification reaction with acetic anhydride, acetyl chloride, etc. under the action of a catalyst and a base to obtain TM7.
  • the reaction solvent can be dichloromethane, chloroform , tetrahydrofuran, ethyl acetate, etc.
  • the catalyst is 4-dimethylaminopyridine
  • the base can be triethylamine, pyridine, etc.
  • the reaction temperature is 0-40°C.
  • the synthesis scheme of synthesizing TM6 from compound TM5 is that compound TM5 undergoes an oxidation reaction to synthesize compound TM6 under the action of an oxidizing agent.
  • the oxidizing agent can be Jones reagent, TEMPO/sodium hypochlorite, sodium hypochlorite, Euphennaol oxidation etc.
  • Compound IN1F is hydrogenated under transition metal catalysis to obtain compound CK004-1E.
  • the catalyst is 5% palladium carbon, 10% palladium carbon, metallic platinum, platinum dioxide, platinum acetate, etc., and the reaction temperature is 40-70°C .
  • Compound CK004-1E is hydrolyzed into compound CK004-1F under acid catalysis.
  • the acid is p-toluenesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, acetic acid, etc.
  • the reaction temperature is 0-40°C.
  • Compound TM23 is oxidized under the action of an oxidizing agent to synthesize compound TM22.
  • the oxidizing agent can be Jones reagent, TEMPO/sodium hypochlorite, sodium hypochlorite, Ovennaol oxidation, etc.
  • Compound TM23 undergoes an esterification reaction with acetic anhydride, acetyl chloride, etc. under the action of a catalyst and a base to obtain TM9.
  • the catalyst is 4-dimethylaminopyridine
  • the base can be triethylamine, pyridine, etc.
  • the invention provides a use of a 17-pyridyl-10 ⁇ -methyl-steroid derivative in the preparation of a medicine for treating cancer.
  • the cancer comprises prostate cancer, breast cancer, colon cancer or lung cancer.
  • the invention provides a pharmaceutical composition, comprising 17-pyridyl-10 ⁇ -methyl-steroid derivatives and pharmaceutically acceptable auxiliary materials.
  • the dosage form of the pharmaceutical composition can be common dosage forms such as oral preparations and injection preparations, and can be solid preparations, liquid preparations, etc., solid preparations such as tablets, capsules, granules, etc., liquid preparations such as solutions, suspensions, emulsions, etc. .
  • tablet excipients include fillers (diluents), binders, disintegrants, lubricants, glidants, and the like.
  • Fillers are selected from lactose, microcrystalline cellulose, mannitol, pregelatinized starch, and the like.
  • the binder is selected from hydroxypropylmethylcellulose, polyvinylpyrrolidone, methylcellulose, povidone, starch and the like.
  • the disintegrating agent is selected from croscarmellose sodium, crospovidone, carboxymethyl starch sodium, corn starch and the like.
  • the lubricant is selected from magnesium stearate, stearic acid, sodium stearyl fumarate and the like.
  • the glidant is selected from talcum powder, micronized silica gel and the like. Tablets can be prepared by wet or dry granulation, or by direct powder compression or blank granulation.
  • the present invention provides the use of a steroid compound with the following structure in the preparation of medicines for treating colon cancer or lung cancer
  • the present invention at least obtains the following beneficial technical effects:
  • the 17-pyridyl-10 ⁇ -methyl-steroid derivative of the present invention is a brand-new compound, and its C-10 methyl group is in the ⁇ configuration, and it is effective for prostate cancer, breast cancer, colon cancer, and lung cancer cells. Strong inhibitory effect, it can not only act as a CYP17 inhibitor, have a strong inhibitory effect on prostate cancer and breast cancer, but also act on the Ano1 (Anoctamin 1) target, and have a strong inhibitory effect on colon cancer and lung cancer cells effect.
  • the compound of the present invention has an IC50 against human prostate cancer cells in the range of ⁇ 140 ⁇ M, preferably in the range of ⁇ 80 ⁇ M, more preferably in the range of ⁇ 40 ⁇ M, more preferably in the range of ⁇ 20 ⁇ M.
  • the compound of the present invention has an IC 50 against human colon cancer cells in the range of ⁇ 100 ⁇ M, preferably in the range of ⁇ 50 ⁇ M, more preferably in the range of ⁇ 30 ⁇ M, more preferably in the range of ⁇ 15 ⁇ M.
  • the compound of the present invention has an IC 50 against human non-small cell lung cancer cells in the range of ⁇ 100 ⁇ M, preferably in the range of ⁇ 50 ⁇ M, more preferably in the range of ⁇ 30 ⁇ M, more preferably in the range of ⁇ 15 ⁇ M.
  • the methyl group at the C-10 position of the 17-pyridyl-10 ⁇ -methyl-steroid derivatives of the present invention is in the ⁇ configuration.
  • the reactivity with enzymes is greatly reduced, the molecular stability is better, and it is not easy to It degrades under the action of enzymes and has good tolerance to enzymes in the body. It is expected that the metabolism in the body will be relatively slow, and the duration of the drug effect will be longer, which is conducive to the development of long-acting preparations.
  • the 17-pyridyl-10 ⁇ -methyl-steroid derivative of the present invention has high bioavailability and less side effects.
  • Fig. 1 is the single crystal structure figure of compound I
  • Fig. 2 is the TLC result graph of compound D4A in the tolerance experiment to enzyme
  • a is the TLC result of reaction monitoring 1 (0.5 hours);
  • b is the TLC result of reaction monitoring 2 (2 hours);
  • c is the TLC result of reaction monitoring 3 (5 hours);
  • Fig. 3 is the TLC result graph of compound TM22 in the tolerance experiment to enzyme
  • d is the TLC result of reaction monitoring 1 (0.5 hours);
  • e is the TLC result of reaction monitoring 2 (2 hours);
  • f is the TLC result of reaction monitoring 3 (5 hours);
  • the left point is the raw material
  • the middle point is the mixing point of the raw material and the enzyme reduction reaction solution
  • the right point is the enzyme reduction reaction solution.
  • Fig. 4 is the docking simulation result figure of abiraterone-3 ⁇ steroid dehydrogenase
  • Fig. 5 is the scoring data diagram of Abiraterone-3 ⁇ steroid dehydrogenase
  • Figure 6 is a diagram of the docking simulation results of TM23-3 ⁇ steroid dehydrogenase
  • Figure 7 is a scoring data graph of TM23-3 ⁇ steroid dehydrogenase
  • Figure 8 is a diagram of the docking simulation results of D4A-cholesterol oxidase
  • Fig. 9 is a scoring data diagram of D4A-cholesterol oxidase
  • Figure 10 is a diagram of the docking simulation results of TM22-cholesterol oxidase
  • Fig. 11 is a graph of scoring data of TM22-cholesterol oxidase.
  • mass yield refers to the mass ratio of the obtained product to the raw material. Taking the above example as an example, the mass yield of 100% means that the mass ratio of the obtained white solid (ie, compound 2) to the added raw material (ie, compound 1) is 100%.
  • the layers were separated, and the aqueous layer was extracted once with 40 mL of chlorobenzene, and the organic layers were combined and washed once with water.
  • the organic layer was dried with anhydrous sodium sulfate for 2h. After filtration, the filtrate was rotary evaporated to dryness under reduced pressure in a water bath at 55°C. After adding dichloromethane to fully dissolve, in a water bath at 35°C, rotate under reduced pressure to replace dichloromethane with methanol. After filtering, the filter cake was washed once with a small amount of methanol, and dried in a blast oven at 50°C for 12 hours to obtain 19 g of off-white solid 6 with a mass yield of 54.3%.
  • TM1 17-pyridyl-10 ⁇ -methyl-steroid derivatives
  • TLC monitors the completion of the raw material reaction, cools to room temperature, adds 20 mL of water, extracts with 20 mL of dichloromethane ⁇ 3, combines the organic layers, washes the organic layer with 20 mL of water ⁇ 3, and rotary evaporates to dryness under reduced pressure in a water bath at 50 ° C to obtain a brown oil Compound (TM1) 2.5g, mass yield 83.3%.
  • TM1 prepared in Example 2 is used for the synthesis of 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives, the compound synthesized here is denoted as TM2, and the synthetic steps of TM2 are as follows:
  • TM2 prepared in Example 3 is used for the synthesis of 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives, the compound synthesized here is denoted as TM4, and the synthetic steps of TM4 are as follows:
  • TM1 prepared in Example 2 is used to synthesize 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives, the compound synthesized here is denoted as TM8, and the synthetic steps of TM8 are as follows:
  • 13 C NMR 13C NMR (101MHz, CDCl3) ⁇ 152.10, 147.75, 133.49, 132.77, 66.40, 50.31, 47.40, 45.14, 37.88, 37.59, 35.87, 34.45, 34.01, 33.54, 32.50, 28.7 3,28.26,25.20,20.90, 17.14, 14.70.
  • TM5 prepared in Example 6 is used to synthesize 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives, the compound synthesized here is denoted as TM7, and the synthetic steps of TM7 are as follows:
  • 13 C NMR 13C NMR (101MHz, CDCl3) ⁇ 170.69, 152.10, 147.81, 133.47, 132.72, 128.49, 123.02, 70.05, 50.29, 47.41, 45.07, 38.86, 37.28, 35.11, 33.99, 3 3.52, 32.84, 32.49, 28.23, 25.80, 25.01, 21.58, 20.91, 17.14, 14.84.
  • TM5 prepared in Example 6 is used for the synthesis of 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives, the compound synthesized here is denoted as TM6, and the synthetic steps of TM6 are as follows:
  • TM23 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives
  • the compound synthesized here is denoted as TM23
  • the synthetic steps of TM23 are as follows:
  • 13C NMR is: 13C NMR (101MHz, CDCl3) ⁇ 167.71, 151.40, 147.61, 137.95, 133.06, 130.91, 128.85, 127.17, 123.16, 118.23, 66.32, 65.57, 51.44, 47.37, 45 .68, 35.83, 35.23, 34.59, 32.64, 31.61 ,30.64,29.18,23.67,19.10,13.73,10.24.
  • TM23 prepared in Example 9 is used to synthesize 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives, the compound synthesized here is denoted as TM9, and the synthetic steps of TM9 are as follows:
  • 13C NMR is: 13C NMR (101MHz, CDCl3) ⁇ 170.73, 151.36, 147.66, 138.00, 133.05, 131.74, 127.17, 123.17, 118.13, 69.81, 51.43, 47.29, 45.68, 36.12, 34. 32, 32.79, 32.58, 32.37, 30.54, 26.29 ,23.74,21.53,19.03,10.38.
  • TM23 prepared in Example 9 is used to synthesize 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives, the compound synthesized here is denoted as TM22, and the synthetic steps of TM22 are as follows:
  • the compound samples of each example were prepared into a 100 mM mother solution with the solvent dimethyl sulfoxide (DMSO), and diluted into a working solution with the complete medium corresponding to each cell culture, with concentrations of 100, 30, 10, 3, 1, and 0.3 ⁇ M. Set up a vehicle control group, a positive control group with different concentrations, and a sample treatment group with different concentrations.
  • DMSO solvent dimethyl sulfoxide
  • Human prostate cancer cell (DU 145) medium is MEM medium containing 10% FBS (fetal bovine serum); human colon cancer cell (HCT-116) medium is McCoy's 5A containing 10% FBS; human non-small cell lung cancer
  • the cells (A549) were Ham's F-12K medium containing 10% FBS, and the culture conditions were all 37°C and 5% CO 2 . When the growth status is good, subculture once every 2 days, and the subculture ratio is 1:3. Discard the medium in the purification workbench, wash it twice with 1 ⁇ PBS, and then add 600 ⁇ L of 0.25% trypsin to digest it. After about 1-3 minutes, after the cells fall off, add 3 mL of 10% FBS containing the corresponding culture of each cell.
  • step 1.1 Take the example compound sample working solution prepared in step 1.1, and add it to the corresponding well at a concentration of 100 ⁇ L/well, so that the final volume of each well is 200 ⁇ L (100 ⁇ L cell culture medium, 100 ⁇ L sample working solution), and the final concentration is 50, 15, 5, 1.5, 0.5, 0.15 ⁇ M, and set the vehicle control group at the same time, the positive control group with the concentration of 50, 15, 5, 1.5, 0.5, 0.15 ⁇ M respectively, and the number of multiple wells in each group is 3. Cultivate for 72 hours at 37°C and 5% CO 2 .
  • the OD value of the vehicle control group was set as 100% cell viability, and the ratio of the OD value of the other groups to the OD value of the vehicle control group was the relative cell viability.
  • the activity of the sample to DU145 or HCT-116 or A549 cells was evaluated by the cell proliferation rate. If there was a proliferation inhibition rate > 100%, it was judged as a systematic error and calculated as 100%.
  • inhibition rate (%) (1-OD sample /OD solvent ) ⁇ 100%
  • the half inhibitory rate (IC 50 ) was calculated using SPSS software.
  • DU145 is a human prostate cancer cell
  • HCT-116 is a human colon cancer cell
  • A549 is a human non-small cell lung cancer cell.
  • the 17-(pyridyl)-10 ⁇ -methyl-steroid derivative of the present invention is a brand-new compound, and its C-10 methyl group is in an ⁇ configuration, and it can inhibit prostate cancer, colon cancer, and lung cancer cells effect.
  • the IC 50 of the compounds of each embodiment of the present invention against human prostate cancer cells is in the range of ⁇ 140 ⁇ M, preferably in the range of ⁇ 80 ⁇ M, more preferably in the range of ⁇ 40 ⁇ M, and more preferably in the range of ⁇ 20 ⁇ M.
  • the IC 50 of each embodiment compound of the present invention against human colon cancer cells is in the range of ⁇ 100 ⁇ M, preferably in the range of ⁇ 50 ⁇ M, more preferably in the range of ⁇ 30 ⁇ M, and more preferably in the range of ⁇ 15 ⁇ M.
  • the IC 50 of the compounds of each embodiment of the present invention against human non-small cell lung cancer cells is in the range of ⁇ 100 ⁇ M, preferably in the range of ⁇ 50 ⁇ M, more preferably in the range of ⁇ 30 ⁇ M, and more preferably in the range of ⁇ 15 ⁇ M.
  • an IC 50 in the range of 50-140 ⁇ M indicates that the compound has a certain degree of inhibitory effect on cancer cells
  • an IC 50 ⁇ 50 ⁇ M indicates that the compound has a good inhibitory effect on cancer cells
  • abiraterone acetate (or abiraterone) is clinically used in combination with prednisone for the treatment of prostate cancer, yet, the applicant finds and confirms by experiment unexpectedly (as Shown in table 1 result), abiraterone, abiraterone acetate and abiraterone oxide not only have the effect of anti-prostate cancer, but also show unexpected significant inhibitory effect against colon cancer cells or lung cancer cells,
  • the IC 50 of abiraterone against human colon cancer cells is in the range of ⁇ 50 ⁇ M
  • the IC 50 of abiraterone acetate against human colon cancer cells is in the range of ⁇ 10 ⁇ M
  • the IC 50 of abiraterone oxide against human colon cancer cells is in the range of ⁇ In the range of 10 ⁇ M
  • the IC 50 of abiraterone acetate against human non-small cell lung cancer cells is in the range of ⁇ 50 ⁇ M
  • the IC 50 of the compound TM1 of the present invention against human prostate cancer cells shows that it has a good inhibitory effect on prostate cancer cells, and its inhibitory effect on prostate cancer cells is even better than that of Abbit
  • the IC 50 of the compound TM5 of the present invention against human prostate cancer cells shows that it has a certain inhibitory effect on prostate cancer cells.
  • the IC 50 against human colon cancer cells is 24.100, even superior Abiraterone
  • the IC 50 against human colon cancer cells is 41.910, indicating that the compound TM5 of the present invention has a good inhibitory effect on human colon cancer cells.
  • the compound TM23 of the present invention has an IC 50 against human prostate cancer cells in the range of ⁇ 80 ⁇ M, indicating that it has a certain inhibitory effect on prostate cancer cells.
  • the IC 50 against human colon cancer cells is 25.242, even Better than abiraterone for human colon cancer cell IC 50 of 41.910, compound TM23 of the present invention for human non-small cell lung cancer cell IC 50 is 41.485, even better than abiraterone for human non-small cell lung cancer cell IC 50 ; in other words, The compound TM23 of the present invention shows a good inhibitory effect on both human colon cancer cells and human non-small cell lung cancer cells, which was unexpected by those skilled in the art.
  • the compound TM4 of the present invention has an IC 50 against human prostate cancer cells in the range of ⁇ 40 ⁇ M, and at the same time, has an IC 50 against human colon cancer cells within the range of ⁇ 20 ⁇ M , the IC 50 for human non-small cell lung cancer cells is in the range of ⁇ 30 ⁇ M; it shows that the compound TM4 of the present invention has good inhibitory effects against human prostate cancer cells, human colon cancer cells and human non-small cell lung cancer cells.
  • the compound TM7 of the present invention has an IC50 of ⁇ 20 ⁇ M for human prostate cancer cells, and at the same time, has an IC50 of ⁇ 20 ⁇ M for human colon cancer cells, and has an IC50 of ⁇ 20 ⁇ M for human non-small cell lung cancer cells.
  • IC50 is in the range of ⁇ 80 ⁇ M.
  • the compound TM7 of the present invention shows a good inhibitory effect on human prostate cancer cells and human colon cancer cells, and has a certain inhibitory effect on human non-small cell lung cancer cells.
  • the compound TM9 of the present invention has an IC 50 of 11.389 against human prostate cancer cells, even better than abiraterone acetate against human prostate cancer cells with an IC 50 of 12.717, and an IC 50 of human colon cancer cells at ⁇ 10 ⁇ M Within the range, the IC50 for human non-small cell lung cancer cells is 24.850, even better than abiraterone acetate for 30.607 of IC50 for human non-small cell lung cancer cells; it shows that the compound TM9 of the present invention is aimed at human prostate cancer cells, human colon cancer cells and human non-small cell lung cancer cells showed good inhibitory effect.
  • the compound TM6 of the present invention has an IC 50 of ⁇ 70 ⁇ M against human prostate cancer cells and human non-small cell lung cancer cells, indicating that it is effective against prostate cancer cells and human non-small cell lung cancer cells.
  • the IC50 for human colon cancer cells is in the range of ⁇ 30 ⁇ M, indicating that the compound TM6 of the present invention has a good inhibitory effect against human colon cancer cells;
  • the IC50 for human prostate cancer cells is within the range of ⁇ 140 ⁇ M
  • the IC50 for human colon cancer cells is within the range of ⁇ 100 ⁇ M
  • the range for human non-small cell lung cancer cells is within the range of ⁇ 100 ⁇ M.
  • 3 ⁇ reductase is the abbreviation of 3 ⁇ hydroxysteroid oxidoreductase, purchased from Nanjing Dulai Biotechnology Co., Ltd., and the 3 ⁇ reductase belongs to Comamonas testosteroni.
  • Glucose dehydrogenase was purchased from Shanghai Macklin Biochemical Technology Co., Ltd.
  • Coenzyme 1 (NAD) nicotinamide adenine dinucleotide, purchased from Shanghai Yueteng Biotechnology Co., Ltd.
  • Coenzyme II (NADP), nicotinamide adenine dinucleotide phosphate was purchased from Hangzhou Weitai Biological Pharmaceutical Co., Ltd.
  • the compounds of the remaining examples of the present invention only convert a small part or even do not convert in 0.5 to 5 hours, which also shows that the reactivity of the compounds of the present invention with reductases is greatly reduced or has no reactivity, and the molecular stability Better, it is not easy to degrade under the action of enzymes, and it has better tolerance to metabolic enzymes in the body. It can be expected that the metabolism in the body will be relatively slow, the excretion time will be prolonged, the action time will also be prolonged, and the drug effect will last longer, which is beneficial to Develop long-acting formulations.
  • the docking software MOE (Molecular Operating Environment, a comprehensive software system for pharmaceuticals and life sciences developed by the Canadian Chemical Computing Group ULC) was used to score the docking of the following compounds, and the specific results are shown in Table 2.
  • the scoring of the MOE software is calculated based on parameters such as electrostatic parameters between molecules and enzymes, hydrogen bonding, molecular attraction, and molecular orbitals. The more negative the score, the lower the free energy, the more stable the conformation and the better the binding.
  • the 17-(3-pyridyl)-10 ⁇ -methyl-steroid derivatives of the present invention have better tolerance to enzymes in vivo, and it is expected
  • the metabolism in the body is relatively slow, the excretion time will be prolonged, the action time will also be prolonged, and the duration of the drug effect will be longer, which is conducive to the development of long-acting preparations.
  • UV ultraviolet detector (205nm)

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Abstract

本发明涉及一种17-吡啶基-10α-甲基-甾体衍生物及中间体化合物、其制备方法、其用途、以及其药物组合物。本发明的甾体衍生物的C-10位甲基为翻转的α构型，是一类全新的化合物，对前列腺癌、乳腺癌、结肠癌、肺癌细胞均有较强的抑制作用。为了制备上述甾体衍生物，本发明还合成了一系列甾体衍生物对应的中间体。本发明的甾体衍生物可以作为药物有效治疗癌症，尤其是包括前列腺癌、乳腺癌、结肠癌或肺癌等方面的癌症。本发明的甾体衍生物C-10位甲基为α构型，相比β构型，与酶的反应活性大大降低，对体内酶的耐受性较好，预期在体内代谢比较慢，药效持续时间更长，有利于开发长效制剂。

Description

17-吡啶基-10α-甲基-甾体衍生物及中间体化合物、其制备方法、其用途、以及其药物组合物 技术领域
本发明涉及药物技术领域，具体涉及一种17-吡啶基-10α-甲基-甾体衍生物及中间体化合物、其制备方法、其用途、以及其药物组合物。
背景技术
醋酸阿比特龙(Abiraterone acetate)，化学名为17-(3-吡啶基)-雄甾-5,16-二烯-3β-醇乙酸酯(结构式如下)，是一种CYP17抑制剂，其在临床上与泼尼松联用治疗已发生对传统激素治疗耐药的转移性晚期前列腺癌，不但可以降低其前列腺特异性抗原水平，而且有助于缩小肿瘤，可延长晚期前列腺患者的生命。
在结构上，其衍生自如下的甾烷骨架结构，该骨架结构(以下称甾环)具有如下所示的四个环，各环上碳的标号(1-17)如下。雄甾指的是在C-10和C-13位各连接一个甲基。对于醋酸阿比特龙，C-10和C-13位连接的甲基均为β构型，可记为10β-甲基、13β-甲基。
发明内容
本发明提出一种17-吡啶基-10α-甲基-甾体衍生物及中间体化合物、其制备方法、其用途、以及其药物组合物。本发明合成了C-10位甲基为α构型的17-吡啶基-10α-甲基-甾体衍生物，该化合物是一类新的化合物，对前列腺癌、乳腺癌、结肠癌、肺癌均有抑制作用。
本发明提供一种17-吡啶基-10α-甲基-甾体衍生物，所述甾体衍生物具有如下式I的结构 或其药学可接受的盐：
R1选自＝O、-OH、卤素或-OC(O)R2；
R2选自C1～C5烷基、C1～C5卤代烷基、苯基或卤代苯基；
R3选自卤素、C1～C5烷基、C1～C5卤代烷基、C1～C5烷氧基、羟基或氨基；
R4、R5、R6、R7相同或不同，各自独立地选自-OH、＝O、卤素、氨基、C1～C5烷基、C1～C5卤代烷基、C1～C5烷氧基、C1～C5烯基或C1～C5酯基；
表示单键或双键；
当某一为双键时，与其相邻的为单键；
i、j、k、m和n各自独立地选自0、1、2、3、4或5。
在本发明的实施方案中，所述甾体衍生物具有如下式Ⅱ的结构式：
在本发明的实施方案中，所述R1选自＝O、-OH或OAc。
在本发明的实施方案中，所述R1选自-OH时，所述-OH为β-OH或α-OH。
在本发明的实施方案中，所述R3选自C1～C5烷基或卤代烷基。
在本发明的实施方案中，所述k为0。
在本发明的实施方案中，所述i、j、m和n均为0，优选所述k为0。
在本发明的实施方案中，所述R1至R7中的卤素选自F、Cl、Br或I。
在本发明的实施方案中，所述甾体衍生物选自如下结构式：
在本发明的实施方案中，所述甾体衍生物选自如下结构式：
本发明提供一种17-吡啶基-10α-甲基-甾体衍生物的制备方法，所述方法包括以式V所示的中间体为原料，在C-17位上连接取代或未被取代的吡啶基；
所述取代或未被取代的吡啶基与所述17-吡啶基-10α-甲基-甾体衍生物上的吡啶基结构对应相同；
所述R9与R4对应相同或者经过反应后对应相同；
所述R10与R5对应相同或者经过反应后对应相同；
所述R11与R6对应相同或者经过反应后对应相同；
所述R12与R7对应相同或者经过反应后对应相同。
本发明提供一种用于制备17-吡啶基-10α-甲基-甾体衍生物的中间体化合物，所述中间体化合物选自下述结构式：
R8选自-OH或被保护的羟基；
R13选自＝O或被保护的羰基。
在本发明的实施方案中，所述R8选自-OH或OAc；和/或；
所述R13选自＝O或
在本发明的实施方案中，所述中间体化合物选自下述结构式：

本发明提供一种中间体化合物的制备方法，所述制备方法包括以下步骤：将下式化合物进行光化学转化使C-10位的甲基由β构型翻转为α构型
在本发明的实施方案中，可选地，所述光化学转化为紫外光光催化反应，可选地，紫外光光催化反应先在260-290nm的波长范围内使甾环开环，然后在295-340nm的波长范围内使甾环闭环，反应温度为-10-50℃；
在本发明的实施方案中，17-吡啶基-10α-甲基-甾体衍生物的中间体化合物的制备方法如下：
具体地，在本发明的实施方案中，
化合物1经3位羟基保护(例如采用乙酸酐等试剂)和17位羰基保护(例如采用乙二醇试剂)，得到化合物3。
化合物3经烯丙位(7位)氧化为羰基，得到化合物4，如采用催化剂N-羟基邻苯二甲酰亚胺、引发剂过氧化苯甲酰，进行空气氧化。
化合物4经7位羰基腙化、脱腙形成5,7双键，得到化合物6。
化合物6经过紫外光光催化反应，10位甲基由β构型翻转位α构型，得到化合物7(即中间体化合物)。光催化反应先在260-290nm的波长范围内使甾环开环，然后在295-340nm的波长范围内闭环。反应温度控制在-10-50℃。
化合物7先后经酸(例如对甲苯磺酸)、碱(例如氢氧化钠等)两步催化水解，脱除3位和17位的保护基团，得到化合物8。
在本发明中，17-吡啶基-10α-甲基-甾体衍生物中部分化合物通过以下方法制备：
化合物8经17位成腙、碘代、偶联反应，得到TM1，可选地，偶联反应反应试剂为二乙基-(3-吡啶)-硼烷，二(三苯基膦)二氯化钯作为催化剂。
TM1在催化剂和碱的作用下，经3位乙酰化得到TM2，可选地，乙酰化试剂可以是乙酸酐、乙酰氯等。
TM2在过渡金属催化下加氢反应，5,7,16双键被还原，得到TM4，可选地，采用Pd/CaCO3催化剂，反应温度为40-70℃，在氢气气氛下还原，选择性和收率较高。
TM1通过采用PTAD(4-苯基-1,2,4-三唑啉-3,5-二酮)发生类似D-A反应(狄尔斯-阿尔德反应)保护5,7共轭双键、3位羟基氧化、脱保护，得到TM8，可选地，羟基氧化采用氧化体系为Tempo(2,2,6,6-四甲基哌啶氧化物)-NaClO-NaBr。在一个具体实施例中，脱保护在50～65℃的温度下进行，这是因为在常温下无法得到目标化合物。
在本发明中，17-吡啶基-10α-甲基-甾体衍生物中部分化合物通过以下方法制备：
化合物7在碱催化下水解成化合物IN1F，可选地，反应溶剂可以为乙酸乙酯、四氢呋喃、二氯甲烷、乙腈、丙酮等。碱可以为氢氧化钠、氢氧化钾、碳酸钾等，反应温度为0～60℃。
化合物IN1F在过渡金属催化下加氢合成化合物CK004-1A，可选地，反应溶剂为乙酸乙酯、乙醇、二氧六环、四氢呋喃、二氯甲烷中的一种或几种的混合物。催化剂为5％钯碳、10％钯碳、金属铂、二氧化铂、醋酸铂等。氢气压力为0.05-2.0MPa，反应温度为40-70℃。
化合物CK004-1A在酸催化下水解成化合物CK004-1B，可选地，反应溶剂为乙酸乙酯、四氢呋喃、二氯甲烷、乙腈、丙酮等。酸为对甲苯磺酸、甲磺酸、盐酸、硫酸、醋酸等，反应温度为0-40℃。
化合物CK004-1B经17位成腙、碘代、偶联反应，得到TM5，可选地，偶联反应反应试剂为二乙基-(3-吡啶)-硼烷，采用二(三苯基膦)二氯化钯催化。
从化合物TM5合成TM7的合成方案是，化合物TM5在催化剂和碱的作用下，和乙酸酐、乙酰氯等发生酯化反应，得到TM7，可选地，反应溶剂可以是二氯甲烷、三氯甲烷、四氢呋喃、乙酸乙酯等。催化剂为4-二甲氨基吡啶，碱可以是三乙胺、吡啶等，反应温度为0-40℃。
从化合物TM5合成TM6的合成方案是，化合物TM5在氧化剂作用下发生氧化反应合成化合物TM6，可选地，氧化剂可以是琼斯试剂、TEMPO/次氯酸钠、次氯酸钠、欧芬脑尔 氧化等。
在本发明中，17-吡啶基-10α-甲基-甾体衍生物中部分化合物通过以下方法制备：
化合物IN1F在过渡金属催化下加氢制得化合物CK004-1E，可选地，催化剂为5％钯碳、10％钯碳、金属铂、二氧化铂、醋酸铂等，反应温度为40-70℃。
化合物CK004-1E在酸催化下水解成化合物CK004-1F，可选地，酸为对甲苯磺酸、甲磺酸、盐酸、硫酸、醋酸等，反应温度为0-40℃。
化合物CK004-1F经17位成腙、碘代、偶联反应，得到TM23。
化合物TM23在氧化剂作用下发生氧化反应合成化合物TM22，可选地，氧化剂可以是琼斯试剂、TEMPO/次氯酸钠、次氯酸钠、欧芬脑尔氧化等。
化合物TM23在催化剂和碱作用下，和乙酸酐、乙酰氯等发生酯化反应，得到TM9，可选地，催化剂为4-二甲氨基吡啶，碱可以是三乙胺，吡啶等。
本发明提供一种17-吡啶基-10α-甲基-甾体衍生物在制备治疗癌症的药物中的用途。
在本发明的实施方案中，所述癌症包括前列腺癌、乳腺癌、结肠癌或肺癌。
本发明提供一种药物组合物，包含17-吡啶基-10α-甲基-甾体衍生物和药学上可接受的辅料。
药物组合物的剂型可以是口服制剂、注射制剂等常见剂型，可以是固体制剂、液体制剂等，固体制剂例如片剂、胶囊剂、颗粒剂等，液体制剂例如溶液剂、混悬剂、乳剂等。
可以选用药学可接受的常用辅料，按常规用量，采用常规的制剂方法制备。例如，片剂辅料包括填充剂(稀释剂)、粘合剂、崩解剂、润滑剂、助流剂等。填充剂选自乳糖、微晶纤维素、甘露醇、预胶化淀粉等。粘合剂选自羟丙基甲基纤维素、聚乙烯吡咯烷酮、甲基纤维素、聚维酮、淀粉等。崩解剂选自交联羧甲纤维素钠、交联聚维酮、羧甲淀粉钠、玉米淀粉等。润滑剂选自硬脂酸镁、硬脂酸、硬脂富马酸钠等。助流剂选自滑石粉、微粉硅胶等。片剂的制备可以采用湿法或干法制粒压片法，或采用直接粉末压片法、空白颗粒压片法。
本发明提供具有如下结构的甾体化合物在制备治疗结肠癌或肺癌的药物中的用途
与现有技术相对比，本发明至少获得例如以下的有益技术效果：
本发明的17-吡啶基-10α-甲基-甾体衍生物是一类全新的化合物，其C-10位甲基为α构型，对前列腺癌、乳腺癌、结肠癌、肺癌细胞均有较强的抑制作用，其不仅可以作为CYP17抑制剂，对前列腺癌、乳腺癌有较强的抑制作用，而且可以作用于Ano1(Anoctamin 1)靶点，对结肠癌、肺癌细胞也有较强的抑制作用。
本发明的化合物针对人前列腺癌细胞IC50在＜140μM范围内，优选在＜80μM范围内，更优选在＜40μM范围内，更优选在＜20μM范围内。本发明的化合物针对人结肠癌细胞IC50在＜100μM范围内，优选在＜50μM范围内，更优选在＜30μM范围内，更优选在＜15μM范围内。本发明的化合物针对人非小细胞肺癌细胞IC50在＜100μM范围内，优选在＜50μM范围内，更优选在＜30μM范围内，更优选在＜15μM范围内。
本发明的17-吡啶基-10α-甲基-甾体衍生物的C-10位甲基为α构型，相比β构型，与酶的反应活性大大降低，分子稳定性更好，不易在酶作用下降解，对体内酶的耐受性较好，预期在体内代谢比较慢，药效持续时间更长，有利于开发长效制剂。
此外，本发明的17-吡啶基-10α-甲基-甾体衍生物生物利用度高，其他副作用较少。
附图说明
图1为化合物I的单晶结构图；
图2为对酶的耐受性实验中化合物D4A的TLC结果图；
其中，a为反应监控1(0.5小时)的TLC结果；b为反应监控2(2小时)的TLC结果；c为反应监控3(5小时)的TLC结果；
图3为对酶的耐受性实验中化合物TM22的TLC结果图；
其中，d为反应监控1(0.5小时)的TLC结果；e为反应监控2(2小时)的TLC结果；f为反应监控3(5小时)的TLC结果；
TLC中左边点为原料，中间点为原料和酶还原反应液的混合点，右边点为酶还原反应液。
图4为阿比特龙-3α类固醇脱氢酶的对接模拟结果图；
图5为阿比特龙-3α类固醇脱氢酶的打分数据图；
图6为TM23-3α类固醇脱氢酶的对接模拟结果图；
图7为TM23-3α类固醇脱氢酶的打分数据图；
图8为D4A-胆固醇氧化酶的对接模拟结果图；
图9为D4A-胆固醇氧化酶的打分数据图；
图10为TM22-胆固醇氧化酶的对接模拟结果图；
图11为TM22-胆固醇氧化酶的打分数据图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明了，下面对本发明进行进一步详细说明。但是应该理解，此处所描述仅仅用以解释本发明，并不用于限制本发明的范围。
除非另有定义，本文所使用的所有的技术术语和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同，本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在限制本发明。本文中所使用的试剂和仪器均商购可得，所涉及的表征手段均可参阅现有技术中的相关描述，本文中不再赘述。
为了进一步了解本发明，下面结合实施例对本发明作进一步的详细说明。
本发明中涉及的10位为α甲基的具体化合物(实施例中化合物，包括中间体)默认构型如下
如果某化合物上与甾环连接的某基团或H与上述构型不同，则单独标明，例如14β。
对于通式化合物(包括至少两个化合物)而言，除了已标示出的基团的构型(例如两个角甲基)，与甾环连接的其它基团或H可以为α或β构型。
实施例1
在2000mL三口烧瓶中，加入DHEA(3β-羟基-5-雄烯-17-酮)(即，化合物1)200g，DCM(二氯甲烷)800mL，三乙胺140g，氮气置换3次，加入DMAP(4-二甲氨基吡啶)4g，氮气置换3次。在室温下进行搅拌使其充分溶解后，滴加乙酸酐140g，2h滴完。滴加完毕，继续搅拌15min。TLC监控原料反应完，加甲醇40mL，继续搅拌30min。用5％盐酸200mL洗涤1次，5％碳酸氢钠溶液200mL洗涤1次。有机相在水浴35℃下，减压旋蒸，用甲醇置换DCM。过滤，滤饼用少量甲醇洗涤1次，并于50℃鼓风干燥箱中干燥12h，得白色固体200g(即，化合物2)，质量收率100％。在本文中，“质量收率”指代所得产物与原料的质量之比。以上述实施例为例，其质量收率为100％指的是：所得白色固体(即，化合物2)与加入的原料(即，化合物1)的质量的比值为100％。
在2000mL三口烧瓶中，加入DHEA醋酸酯(即，前述获得的化合物2)200g，乙二醇1200mL，PTS(对甲苯磺酸)4g，原甲酸三乙酯260g。在50℃下进行搅拌，TLC监控原料反应完。冷却至室温，加三乙胺8mL，继续搅拌30min。倒入1600mL水中，搅拌30min。过滤，滤饼用少量水洗涤2次。滤饼加二氯甲烷800mL溶解，加三乙胺4mL，搅拌15min。 分去水层，有机相在水浴35℃下，减压旋蒸，用甲醇置换二氯甲烷。过滤，滤饼用少量甲醇洗涤1次，并于50℃鼓风干燥箱，干燥12h，得210g白色固体3，质量收率105％。
在2000mL三口烧瓶中，加入100g化合物3，环己酮800mL，通入干燥的空气，在50℃下进行搅拌使其充分溶解后，加入NOP(N-羟基邻苯二甲酰亚胺)32g，过氧化苯甲酰0.50g。TLC监控原料反应完。冷却至室温，水浴55℃下，减压旋蒸至干。加二氯甲烷200mL，石油醚500mL，室温搅拌30min。过滤，滤饼用少量石油醚洗涤1次。滤液加三乙胺60g，冰水浴滴加醋酸酐60g，继续搅拌30min。静置12h。水浴55℃下，减压旋蒸至干。加二氯甲烷充分溶解后，在水浴35℃下，减压旋蒸，用甲醇置换二氯甲烷。过滤，滤饼用少量甲醇洗涤1次，并于50℃鼓风干燥箱中干燥12h，得75g白色固体4，质量收率75％。
在2000mL三口烧瓶中，加入100g化合物4，TSH(对甲苯磺酰肼)66g，甲苯400mL，正己烷600mL，搅拌下加热至回流分水。TLC监控原料反应完。冷却至室温，水浴55℃下，减压旋蒸至干。加甲醇660mL，正己烷130mL，室温搅拌30min。过滤，滤饼用少量甲醇洗涤1次，并于50℃鼓风干燥箱中干燥12h，得126g白色固体5，质量收率126％。
在1000mL三口烧瓶中，加入氨基锂5.6g，氯苯175mL，减压除氨气。氨气除完后，加入化合物5的氯苯溶液(35g化合物5，氯苯350mL)，将烧瓶移入120℃油浴内，搅拌保温反应1h。TLC监控原料反应完。冷却至室温，冰水浴搅拌下，用5％磷酸调pH6～8。分液，水层用氯苯40mL萃取1次，合并有机层，并用水洗涤1次。有机层用无水硫酸钠干燥2h。 过滤，滤液在水浴55℃下，减压旋蒸至干。加二氯甲烷充分溶解后，在水浴35℃下，减压旋蒸，用甲醇置换二氯甲烷。过滤，滤饼用少量甲醇洗涤1次，并于50℃鼓风干燥箱中干燥12h，得19g类白色固体6，质量收率54.3％。
称取50g化合物6，0.5gBHT(抗氧化剂)，加入1.5L乙酸乙酯溶解，倒入光化反应器中，开启内冷却系统，用260-270nm的LED紫外灯(100W)光照3h，然后用310-330nm的LED紫外灯(100W)光照3h，取样HPLC监控反应，反应完成后，浓缩反应液至油状，加入150ml甲醇，搅拌析出化合物6，抽滤，回收化合物6(20g)，母液浓缩至干后，硅胶拌样，过层析柱，得到10.5g化合物7。
经检测，化合物7的1H NMR为：1H NMR(400MHz,CDCl3)δ5.57(dd,J＝5.5,2.1Hz,1H),5.41(dt,J＝5.3,2.5Hz,1H),4.71(tt,J＝11.4,4.5Hz,1H),4.03–3.80(m,4H),2.51(ddd,J＝14.2,4.8,2.2Hz,1H),2.42–2.25(m,2H),2.08–1.98(m,5H),1.97–1.86(m,3H),1.87–1.76(m,1H),1.76–1.63(m,3H),1.61–1.47(m,4H),1.44–1.31(m,1H),0.96(s,3H),0.79(s,3H).
HRMS质谱(EI)m/z：理论计算值373.5：测试值：372.9。
在250mL三口烧瓶中，加入4.2g化合物7，丙酮42g，PTS 0.84g，水21g。室温搅拌12h。TLC监控原料反应完，加氢氧化钠1.26g，在回流下，搅拌1h，TLC监控原料反应完。在水浴40℃下，减压旋蒸除丙酮。用二氯甲烷20mL×3萃取，合并有机层，并用水洗涤1次，在水浴45℃下，减压旋蒸至干，得化合物8，为淡黄色油状物2.9g，质量收率69.0％。
实施例2
将实施例1中制备的化合物8用于合成17-吡啶基-10α-甲基-甾体衍生物，此处合成的化合物记为TM1，TM1的合成步骤如下：
在250mL三口烧瓶中，加入3.6g化合物8，乙醇14.4g，水合肼2.60g，硫酸肼0.015g。在35℃下搅拌12h。TLC监控原料反应完，加冰水115.2g，搅拌30min，过滤，滤饼用少量水洗涤1次，并于50℃鼓风干燥箱，干燥12h，得类白色固体(即化合物9)2.5g，质量收率70％。
在250mL三口烧瓶中，加入THF(四氢呋喃)30g，冰水浴搅拌下，加碘5.16g，保温搅拌15min。滴加四甲基胍5.88g，10min滴完，保温搅拌15min。滴加化合物9的THF溶液(化合物9 3g，THF 36g)，120min滴完。冰水浴继续保温搅拌60min。TLC监控原料反应完，加硫代硫酸钠4.20g，在50℃下搅拌30min。过滤，滤饼用少量THF洗涤2次，滤液，在水浴50℃下，减压旋蒸至干。加THF 30g，充分溶解后，缓慢加入5％盐酸20mL，在50℃下搅拌30min。分液，水层用THF 20mL×2萃取，合并有机层，加饱和碳酸氢钠溶液20mL，室温搅拌30min。分液，有机层用饱和氯化钠溶液20mL洗涤1次。有机层加无水硫酸钠干燥2h。过滤，滤饼用少量THF洗涤2次。滤液，在水浴50℃下，减压旋蒸至干，得棕色油状物(即化合物10)3.6g，质量收率120％。
在100mL三口烧瓶中，加入3g化合物10，二乙基-3-吡啶基硼烷1.3g，二三苯基膦二氯化钯0.015g，DMF(N,N-二甲基甲酰胺)36g，碳酸钾1.8g，水4.5g，氮气置换3次，升温至80℃，保温搅拌12h。TLC监控原料反应完，冷却至室温，加水20mL，用二氯甲烷20mL×3萃取，合并有机层，用水20mL×3洗涤有机层，在水浴50℃下，减压旋蒸至干，得棕色油状 物(TM1)2.5g，质量收率83.3％。
经检测，化合物TM1 1H NMR为：1H NMR(400MHz,CDCl3)δ8.69(d,J＝1.8Hz,1H),8.44(dd,J＝4.7,1.3Hz,1H),7.70(s,1H),7.23(dd,J＝8.0,4.2Hz,1H),6.14(d,J＝3.0Hz,1H),5.71(dd,J＝5.2,2.4Hz,1H),5.65–5.59(m,1H),4.14–4.11(m,1H),3.06–2.94(m,1H),2.56–2.44(m,2H),2.40–2.26(m,3H),2.15–2.06(m,1H),2.04(s,1H),1.92–1.76(m,5H),1.75–1.63(m,6H),1.27(dt,J＝12.5,5.0Hz,9H),1.06(s,3H),0.78(s,3H).
HRMS质谱(EI)m/z：理论计算值：348.5，测试值：348.1。
实施例3
将实施例2中制备的TM1用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，此处合成的化合物记为TM2，TM2的合成步骤如下：
在50mL三口烧瓶中，加入化合物(TM1)500mg，二氯甲烷10mL，DMAP 0.015g，三乙胺0.36g，氮气置换3次。在室温下搅拌充分溶解后，滴加乙酸酐0.36g，30min滴完。滴完后继续搅拌30min。TLC监控原料反应完，水浴50℃下，减压旋蒸至干，加乙醇1g溶解，室温搅拌下，滴加水30g，滴完继续搅拌30min。过滤，滤饼用少量水洗涤1次，并于50℃鼓风干燥箱，干燥12h，得类白色固体(TM2)400mg，质量收率80％。
经检测，化合物TM2的1H NMR为：1H NMR(400MHz,CDCl3)δ8.70(s,1H),8.44(s,1H),7.71(d,J＝7.9Hz,1H),7.23(dd,J＝7.7,4.7Hz,1H),6.14(s,1H),5.63(s,2H),5.08(s,1H),3.01(s,1H),2.45(s,6H),2.07(s,5H),1.96–1.47(m,13H),1.27(d,J＝10.9Hz,7H),1.09(s,3H),0.79(s,3H).
HRMS质谱(EI)m/z：理论计算值：390.5测试值：390.1。
实施例4
将实施例3中制备的TM2用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，此处合成的化合物记为TM4，TM4的合成步骤如下：
在50mL三口烧瓶中，加入化合物(TM2)300mg，乙醇10mL，钯碳酸钙0.1g，氢气置换3次。在氢气球加压下，室温搅拌24h。TLC监控原料反应完，过滤，滤饼用少量乙醇洗涤1次。滤液，在水浴50℃下，减压旋蒸至干，加乙醇1g溶解，室温搅拌下，滴加水30g，滴完继续搅拌30min。过滤，滤饼用少量水洗涤1次，并于50℃鼓风干燥箱，干燥12h，得类白色固体(TM4)200mg，质量收率67％。
实施例5
将实施例2中制备的TM1用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，此处合成的化合物记为TM8，TM8的合成步骤如下：
在50mL三口烧瓶中，加入化合物(TM1)500mg，PTAD(4-苯基-1,2,4-三唑啉-3,5-二酮)170mg，三氯甲烷10mL。室温搅拌1h。TLC监控原料反应完。在水浴30℃下，减压旋蒸，用石油醚置换三氯甲烷。过滤，滤饼用少量石油醚洗涤1次，并于50℃鼓风干燥箱中干燥12h，得486mg类白色固体12，质量收率97.2％。

(注：其中“protect”指保护基团)
在50mL三口烧瓶中，加入500mg的化合物12，Tempo(2,2,6,6-四甲基哌啶氧化物)50mg，溴化钠50mg，三氯甲烷10mL，饱和碳酸氢钠溶液0.5mL。在室温搅拌充分溶解后，滴加次氯酸钠1.1g。滴完继续搅拌30min。TLC监控原料反应完。在水浴30℃下，减压旋蒸，用石油醚置换三氯甲烷。过滤，滤饼用少量石油醚洗涤1次，并于50℃鼓风干燥箱，干燥 12h，得类淡黄色固体500mg，质量产率100％。
在50mL三口烧瓶中，加入500mg化合物13，甲醇10mL，氯化氢乙醇溶液2mL。在60℃搅拌1h。TLC监控原料反应完。在水浴55℃下，减压旋蒸至干。加入甲醇10mL溶解，加水1mL，氢氧化钠0.2g，在室温下搅拌1h。在水浴55℃下，减压旋蒸至干。加入水50mL，二氯甲烷30mL，溶解后分液，水层用二氯甲烷20mL萃取1次。合并有机层，在水浴55℃下，减压旋蒸，用石油醚置换二氯甲烷。过滤，滤饼用少量石油醚洗涤1次，并于50℃鼓风干燥箱，干燥12h，得淡黄色固体(TM8)100g，质量产率20％。
实施例6
将实施例1中制备的化合物7用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，合成的化合物记为TM5，TM5的合成步骤如下：
在250mL三口烧瓶中，加入5g化合物7，丙酮30g，加氢氧化钠1.26g，室温搅拌1h，TLC监控原料反应完。减压旋蒸除丙酮，加入二氯甲烷萃取，浓干，得淡黄色油状物IN1F4.3g，质量产率86.0％。
于250ml加氢釜，加入10g IN1F，150ml无水乙醇，2g5％钯碳，氮气置换三次，氢气置换三次，升温至55-60℃，氢气压力0.15-0.20MPa反应4小时，中控反应完成，过滤除去 钯碳，浓缩，柱层析(洗脱剂石油醚：乙酸乙酯＝10:1～石油醚：乙酸乙酯5:1)得到固体6.3g，收率63.0％。
于50ml反应瓶加入1.5g CK004-1A，丙酮22ml，水7.5ml，对甲苯磺酸0.3g，反应完毕，浓缩无馏分，加入10ml二氯甲烷，分层，5ml的5％碳酸氢钠洗涤一次，分层，有机层浓干得到CK004-1B1.2g，收率80％。
于反应瓶加入1.2gCK004-1B，0.86g水合肼(85％)，5g无水乙醇，0.006g硫酸肼，升温至30-35℃反应过夜，反应完成将反应液缓慢倒入50ml水中，搅拌1小时，过滤，水淋洗，50℃烘干得白色固体1.15g，质量收率95.8％。
于反应瓶加入1.05gCK004-1C，再加入12ml四氢呋喃，搅拌溶清。于另一反应瓶加入10ml四氢呋喃降温至-5～5℃，加入1.72g碘，搅拌半小时。于-5～5℃滴加四甲基胍，滴加完毕搅拌半小时。于-5～5℃滴加CK004-1C的四氢呋喃溶液，滴完，于-5～5℃反应1小时，反应完毕，加入1.2g硫代硫酸钠，升温至40℃搅拌半小时，过滤，滤液浓缩至无馏分，加入10ml四氢呋喃，加入5ml 1M盐酸洗涤，再用5ml 5％碳酸氢钠溶液洗涤，再用5ml饱和硫代硫酸钠溶液洗涤分层，有机层浓干得1.21g，质量收率115.2％。
于反应瓶加入800mg CK004-1D，340mg二乙基-(3-吡啶)-硼烷，4mg二(三苯基膦)二氯化钯，10.0ml N,N-二甲基甲酰胺，480mg无水碳酸钾，1.2g水，升温至85-90℃反应。反应完毕，过滤，滤液加入10ml二氯甲烷，用饱和氯化钠洗涤3次，浓干，柱层析(洗脱剂：石油醚：乙酸乙酯＝13:1～石油醚：乙酸乙酯＝6:1)得到白色固体(TM5)590g，质量收率73.8％。
经检测，化合物TM5 1H NMR为：1H NMR(400MHz,CDCl3)δ8.64(d,J＝1.7Hz,1H),8.44(d,J＝4.7Hz,1H),7.66(d,J＝7.9Hz,1H),7.20(dd,J＝7.9,4.8Hz,1H),6.02(d,J＝2.9Hz,1H),4.04(s,1H),2.35–2.15(m,2H),2.15–2.01(m,1H),1.94(dd,J＝13.9,11.0Hz,1H),1.89–1.53(m,13H),1.50(dd,J＝12.8,2.7Hz,1H),1.47–1.35(m,2H),1.36–1.11(m,4H),1.06(s,3H),0.94(s,3H).
13C NMR为：13C NMR(101MHz,CDCl3)δ152.10,147.75,133.49,132.77,66.40,50.31,47.40,45.14,37.88,37.59,35.87,34.45,34.01,33.54,32.50,28.73,28.26,25.20,20.90,17.14,14.70.
HRMS质谱(EI)m/z：理论计算值：352.2测试值：352.0。
申请人以下利用CK004-1B合成了化合物I，并通过对化合物I的单晶结构进行确定以对CK004-1B的构型进行确定。
氮气保护下，于反应瓶加入540mg CK004-1B，12ml吡啶，加入760mg4-氯苯甲酰氯，室温搅拌反应2小时，检测原料反应完全，加入100ml二氯甲烷，用5％稀盐酸调pH至5-6，分液，有机层用5％碳酸氢钠洗涤，水洗，分层浓缩干，柱层析(石油醚：乙酸乙酯＝20:1～10:1)，得510mg化合物I。
化合物I 1H NMR为：1H NMR(400MHz,CDCl3)δ7.98(d,J＝8.7Hz,2H),7.42(d,J＝8.7Hz,2H),5.26(s,1H),2.45(dd,J＝19.2,9.0Hz,1H),2.16–1.91(m,4H),1.88–1.73(m,5H),1.67–1.56(m,8H),1.42(s,1H),1.34–1.23(m,3H),1.15(d,J＝13.4Hz,1H),0.97(s,3H),0.91(s,3H).
化合物I的单晶结构图如图1所示，证明化合物I的构型符合上式。
实施例7
将实施例6中制备的TM5用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，此处合成的化合物记为TM7，TM7的合成步骤如下：
于反应瓶加入130mgTM5,5ml二氯甲烷，75.4mg三乙胺，0.6mg 4-二甲氨基吡啶，于10-20℃加入75.4mg乙酸酐，反应完毕，加入0.5ml甲醇，浓缩干，柱层析(石油醚：乙酸乙酯＝5:1)得到白色固体(TM7)131mg，质量收率100.7％。
经检测，化合物TM7 1H NMR为：1H NMR(400MHz,CDCl3)δ8.64(d,J＝1.8Hz,1H),8.45(dd,J＝4.8,1.5Hz,1H),7.67(dt,J＝7.9,1.9Hz,1H),7.24–7.18(m,1H),6.11–5.93(m,1H),5.01(d,J＝2.4Hz,1H),2.35–2.17(m,2H),2.14–2.04(m,4H),2.00–1.89(m,1H),1.86–1.61(m,11H),1.60–1.45(m,5H),1.34–1.13(m,4H),1.07(s,3H),0.96(d,J＝4.7Hz,3H).
13C NMR为：13C NMR(101MHz,CDCl3)δ170.69,152.10,147.81,133.47,132.72,128.49,123.02,70.05,50.29,47.41,45.07,38.86,37.28,35.11,33.99,33.52,32.84,32.49,28.23,25.80,25.01,21.58,20.91,17.14,14.84.
HRMS质谱(EI)m/z：理论计算值：394.5测试值：394.2。
实施例8
将实施例6中制备的TM5用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，此处合成的化合物记为TM6，TM6的合成步骤如下：
于反应瓶加入250mg TM5，1.5ml二氯甲烷，0.25ml 5％碳酸氢钠溶液，0.01g溴化钠，控温0-5℃滴加10％次氯酸钠，反应完毕，分层，水层用二氯甲烷反萃，合并有机相，加入饱和硫代硫酸钠溶液洗涤，再用水洗，有机相浓缩至干，柱层析(石油醚：乙酸乙酯＝5:1)得到120mg白色固体TM6，质量收率48.0％。
实施例9
将实施例1中制备的化合物7用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，此处合成的化合物记为TM23，TM23的合成步骤如下：
于反应瓶加入3.0g IN1F，0.6g 5％的Pd/C，15mL二氧六环，水浴60℃，氮气置换，氢气置换，氢气球加压，反应完毕，过滤，浓缩至干，柱层析(石油醚：乙酸乙酯＝15:1)得到1.2g白色固体CK004-1E，质量收率40.0％。
于反应瓶加入2g CK004-1E，15ml丙酮，0.8g对甲苯磺酸，5ml水，室温反应，反应完毕后，浓缩除去丙酮，加入10ml二氯甲烷，5ml的5％碳酸氢钠溶液洗涤一次，5ml水洗一次，分层，有机层浓缩干得1.7g的CK004-1F，质量收率85.0％。
于反应瓶加入850mg CK004-1F，无水乙醇3.5ml，612mg水合肼，4mg硫酸肼，30-35℃反应。反应完毕，将反应液缓慢倒入50ml冰水中，搅拌1小时，过滤，水洗，50℃烘干得820mg白色固体CK004-1G，质量收率96.47％。
于反应瓶加入1.55g的CK004-1G，加入15ml四氢呋喃，40℃溶解。于另一反应瓶加入15ml四氢呋喃，降温至-5-5℃，加入2.66g碘，再滴加3g四甲基胍。控温-5-5℃滴加CK004-1G的四氢呋喃溶液，滴加完毕，于-5-5℃反应1小时，反应完毕，加入1.86g硫代硫酸钠，升温至40℃搅拌半小时，过滤，滤液浓缩至无馏分，加入15ml四氢呋喃，加入8ml的1M盐酸洗涤，再用8ml的5％碳酸氢钠溶液洗涤，再用8ml饱和硫代硫酸钠溶液洗涤分层，有机层浓干得1.8g的CK004-1H，质量收率116.1％。
于反应瓶加入1.0mg的CK004-1H，425mg二乙基-(3-吡啶)-硼烷，5mg二(三苯基膦)二氯化钯，12ml的N,N-二甲基甲酰胺，600mg无水碳酸钾，1.52g水，升温至85-90℃反应。反应完毕，过滤，滤液加入15ml二氯甲烷，用饱和氯化钠洗涤3次，浓干，柱层析(洗脱剂：石油醚：乙酸乙酯＝13:1～石油醚：乙酸乙酯＝6:1)得到化合物TM23，为410mg的白色固体，质量收率41.0％。
经检测，化合物TM23 1H NMR为：1H NMR(400MHz,CDCl3)δ8.69(s,1H),8.43(d,J＝3.8Hz,1H),7.78–7.67(m,1H),7.26–7.18(m,1H),6.12(s,1H),5.32(s,1H),4.31(s,1H),4.13 –4.07(m,1H),2.76(d,J＝2.4Hz,1H),2.38(dd,J＝6.6,3.3Hz,1H),2.35–2.13(m,3H),2.12–1.90(m,4H),1.90–1.59(m,9H),1.60–1.35(m,7H),1.26(s,2H),1.17(s,3H),0.96(t,J＝7.4Hz,1H),0.69(s,3H).
13C NMR为：13C NMR(101MHz,CDCl3)δ167.71,151.40,147.61,137.95,133.06,130.91,128.85,127.17,123.16,118.23,66.32,65.57,51.44,47.37,45.68,35.83,35.23,34.59,32.64,31.61,30.64,29.18,23.67,19.10,13.73,10.24.
HRMS质谱(EI)m/z：理论计算值：350.3，测试值：349.9。
实施例10
将实施例9中制备的TM23用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，此处合成的化合物记为TM9，TM9的合成步骤如下：
于反应瓶加入160mgTM23,5ml二氯甲烷，92.8mg三乙胺，0.8mg4-二甲氨基吡啶，于10-20℃加入92.8mg乙酸酐，反应完毕，加入0.8ml甲醇，浓缩干，柱层析(石油醚：乙酸乙酯＝5:1)得到白色固体(TM9)150mg，质量收率93.8％。
经检测，化合物TM9的1H NMR为：1H NMR(400MHz,CDCl3)δ8.70(d,J＝1.5Hz,1H),8.44(d,J＝3.7Hz,1H),7.81–7.67(m,1H),7.22(dd,J＝7.9,4.8Hz,1H),6.13(dd,J＝3.0,2.0Hz,1H),5.45–5.24(m,1H),5.16–4.88(m,1H),2.87–2.71(m,1H),2.40(d,J＝3.3Hz,1H),2.24(dd,J＝10.7,1.6Hz,2H),2.07(s,3H),2.02(d,J＝4.5Hz,2H),1.91(s,2H),1.85–1.74(m,2H),1.73–1.52(m,6H),1.42(ddd,J＝14.4,8.3,3.3Hz,3H),1.19(s,3H),0.71(s,3H).
13C NMR为：13C NMR(101MHz,CDCl3)δ170.73,151.36,147.66,138.00,133.05,131.74,127.17,123.17,118.13,69.81,51.43,47.29,45.68,36.12,34.32,32.79,32.58,32.37,30.54,26.29,23.74,21.53,19.03,10.38.
HRMS质谱(EI)m/z：理论计算值：392.5测试值：391.9。
实施例11
将实施例9中制备的TM23用于合成17-(3-吡啶基)-10α-甲基-甾体衍生物，此处合成的化合物记为TM22，TM22的合成步骤如下：
于反应瓶加入140mg TM23，5ml二氯甲烷，0.5ml 5％碳酸氢钠溶液，5mg溴化钠，控温0-5℃滴加1.5ml10％次氯酸钠，反应完毕，分层，水层用二氯甲烷反萃，合并有机相，加入饱和硫代硫酸钠溶液洗涤，再用水洗，有机相浓缩至干，柱层析(石油醚：乙酸乙酯＝5:1)得到白色固体(TM22)48mg，质量收率34.28％。
经检测，化合物TM22 1H NMR为：1H NMR(400MHz,CDCl3)δ8.70(s,1H),8.45(s,1H),7.74–7.66(m,1H),7.23(dd,J＝7.8,4.8Hz,1H),6.13(dd,J＝3.2,2.0Hz,1H),5.44–5.30(m,1H),2.79(dd,J＝6.3,2.3Hz,1H),2.54–2.37(m,2H),2.37–2.28(m,1H),2.29–2.18(m,4H),2.16(d,J＝1.7Hz,1H),2.15–1.96(m,4H),1.91–1.38(m,9H),1.28(dd,J＝13.6,10.4Hz,2H),1.16(s,3H),0.94(d,J＝3.8Hz,3H).
HRMS质谱(EI)m/z：理论计算值：348.3测试值：347.9。
除上述实施例制备的化合物外，通过与前述实施例相同/相似的制备工艺，本发明还制备得到其它一些实施例化合物，现将所有实施例化合物列举如下：
实施例12
一、药理实验：对癌细胞的抑制作用
1.试验方法
1.1实验分组及样品配制
各实施例化合物样品均采用溶媒二甲亚砜(DMSO)配制成100mM的母液，用各细胞培养对应的完全培养基稀释成工作液，浓度为100、30、10、3、1、0.3μM。设置溶媒对照组、不同浓度的阳性对照组、不同浓度的样品处理组。
1.2细胞培养
人前列腺癌细胞(DU 145)培养基为含10％FBS(胎牛血清)的MEM培养基；人结肠癌细胞(HCT-116)培养基为含10％FBS的McCoy’s 5A；人非小细胞肺癌细胞(A549)为含10％FBS的Ham's F-12K培养基，培养条件均为37℃、5％CO2。生长状态良好时，每2天传代一次，传代比例为1：3。在净化工作台中弃去培养基，用1×PBS清洗2次，然后加入600μL的0.25％胰蛋白酶消化，约1～3min后，待细胞脱落，加入3mL的含10％FBS的各细胞对应的培养基以终止胰酶的消化作用，吹打成单细胞悬液，转入EP管中，以1000rpm离心5min。弃去培养基，加入新鲜的培养基重悬，按一定比例(细胞密度约为105/mL)接种到新的培养瓶中，放置于37℃、5％CO2培养箱中培养。
1.3细胞接种
取生长状态良好的细胞，常规消化收集细胞，调节DU 145细胞密度至2×104个/mL，调节HCT-116细胞至2×104个/mL，调节A549细胞至3×104个/mL，各细胞悬液均以100μL/孔的密度分别接种至96孔培养板中，十字交叉振摇各10次，使细胞均匀铺于孔底，将培养板放置于CO2培养箱中培养24h。
1.4细胞处理
取步骤1.1中配制的实施例化合物样品工作液，分别浓度以100μL/孔加入至对应孔内，使各孔终体积为200μL(100μL细胞培养基，100μL样品工作液)，终浓度分别为50、15、5、1.5、0.5、0.15μM，同时设置溶媒对照组，浓度分别为50、15、5、1.5、0.5、0.15μM的阳性对照组，各组复孔数均为3个。在37℃、5％CO2条件下培养72h。
1.5细胞增殖OD值检测
细胞处理72h后，在每孔加入噻唑兰(MTT)20μL，在37℃、5％CO2条件下继续培养4h，小心吸弃各孔内液体，在孔内加入150μL/孔的DMSO，震荡10min。
在酶标仪上设定A1-H1孔(8孔)的OD值均值为调零值，在492nm处检测各孔OD值。
1.6结果计算
以溶媒对照组OD值设定为100％细胞活力，其余各组OD值与溶媒对照组OD值的比值为相对细胞活力。以细胞增殖率评价样品对DU 145或HCT-116或A549细胞的活性，若出现有增殖抑制率＞100％时，判定为系统误差，按100％计。
抑制率计算公式为：抑制率(％)＝(1-OD样品/OD溶媒)×100％
使用SPSS软件计算半数抑制率(IC50)。
2.实验结果
表1部分实施例和对比例的化合物对各个癌细胞的IC50值


注：DU145为人前列腺癌细胞，HCT-116为人结肠癌细胞，A549为人非小细胞肺癌细
胞。
本发明的17-(吡啶基)-10α-甲基-甾体衍生物是一类全新的化合物，其C-10位甲基为α构型，对前列腺癌、结肠癌、肺癌细胞均有抑制作用。本发明各实施例化合物针对人前列腺癌细胞IC50在＜140μM范围内，优选在＜80μM范围内，更优选在＜40μM范围内，更优选在＜20μM范围内。本发明各实施例化合物针对人结肠癌细胞IC50在＜100μM范围内，优选在＜50μM范围内，更优选在＜30μM范围内，更优选在＜15μM范围内。本发明各实施例化合物针对人非小细胞肺癌细胞IC50在＜100μM范围内，优选在＜50μM范围内，更优选在＜30μM范围内，更优选在＜15μM范围内。
在本发明中认为，IC50在50～140μM范围内，表明化合物对癌细胞有一定程度的抑制作用，IC50＜50μM表明化合物对癌细胞有良好的抑制作用。
此外，申请人指出，在现有技术中，醋酸阿比特龙(或阿比特龙)在临床上与泼尼松联用以治疗前列腺癌，然而，本申请出人意料地发现并通过试验证实了(如表1结果所示)，阿比特龙、醋酸阿比特龙和阿比特龙氧化物不仅具有抗前列腺癌的作用，而且针对结肠癌细胞或肺癌细胞也显示出了出乎意料的显著的抑制作用，特别是，阿比特龙针对人结肠癌细胞IC50在＜50μM范围内，醋酸阿比特龙针对人结肠癌细胞IC50在＜10μM范围内，阿比特龙氧化物针对人结肠癌细胞IC50在＜10μM范围内；醋酸阿比特龙针对人非小细胞肺癌细胞IC50在＜50μM范围内，阿比特龙氧化物针对人非小细胞肺癌细胞IC50在＜20μM范围内。由于不同癌症致病机理不同，药物作用机制也不同，在本申请之前，本领域技术人员无法预期阿比特龙、醋酸阿比特龙和阿比特龙氧化物对特定的癌种即结肠癌或人非小细胞肺癌能具有如此良好的抑制作用。
进一步地，从表1的结果可以看出，本发明的化合物TM1针对人前列腺癌细胞的IC50表明对前列腺癌细胞显示出良好的抑制作用，其对前列腺癌细胞的抑制作用甚至优于阿比特龙；此外，与阿比特龙相比，本发明的化合物TM5针对人前列腺癌细胞的IC50表明对前列腺癌细胞有一定抑制作用，与此同时，针对人结肠癌细胞IC50为24.100，甚至优于阿比特龙 针对人结肠癌细胞IC50的41.910，表明本发明的化合物TM5针对人结肠癌细胞显示出了良好的抑制作用。与阿比特龙相比，本发明的化合物TM23针对人前列腺癌细胞IC50在＜80μM范围内，表明对前列腺癌细胞有一定抑制作用，与此同时，针对人结肠癌细胞IC50为25.242，甚至优于阿比特龙针对人结肠癌细胞IC50的41.910，本发明的化合物TM23针对人非小细胞肺癌细胞IC50为41.485，甚至优于阿比特龙针对人非小细胞肺癌细胞IC50；换言之，本发明的化合物TM23针对人结肠癌细胞和人非小细胞肺癌细胞均显示出了良好的抑制作用，这也是本领域技术人员之前所无法预期的。
更进一步地，申请人指出，与醋酸阿比特龙相比，本发明的化合物TM4针对人前列腺癌细胞IC50在＜40μM范围内，与此同时，针对人结肠癌细胞IC50在＜20μM范围内，针对人非小细胞肺癌细胞IC50在＜30μM范围内；表明本发明的化合物TM4针对人前列腺癌细胞、人结肠癌细胞和人非小细胞肺癌细胞均显示出了良好的抑制作用。与醋酸阿比特龙相比，本发明的化合物TM7针对人前列腺癌细胞IC50在＜20μM范围内，与此同时，针对人结肠癌细胞IC50在＜20μM范围内，针对人非小细胞肺癌细胞IC50在＜80μM范围内。换言之，本发明的化合物TM7针对人前列腺癌细胞、人结肠癌细胞均显示出了良好的抑制作用，针对人非小细胞肺癌细胞有一定抑制作用。与醋酸阿比特龙相比，本发明的化合物TM9针对人前列腺癌细胞IC50为11.389，甚至优于醋酸阿比特龙针对人前列腺癌细胞IC50的12.717，针对人结肠癌细胞IC50在＜10μM范围内，针对人非小细胞肺癌细胞IC50为24.850，甚至优于醋酸阿比特龙针对人非小细胞肺癌细胞IC50的30.607；表明本发明的化合物TM9针对人前列腺癌细胞、人结肠癌细胞和人非小细胞肺癌细胞均显示出了良好的抑制作用。
再进一步地，申请人指出，与阿比特龙氧化物相比，本发明的化合物TM6针对人前列腺癌细胞和人非小细胞肺癌细胞IC50在＜70μM范围内，表明对前列腺癌细胞和人非小细胞肺癌细胞有一定抑制作用，与此同时，针对人结肠癌细胞IC50在＜30μM范围内，表明本发明的化合物TM6针对人结肠癌细胞显示出了良好的抑制作用；与阿比特龙氧化物相比，本发明的化合物TM23针对人前列腺癌细胞IC50在＜140μM范围内、针对人结肠癌细胞IC50在＜100μM范围内、针对人非小细胞肺癌细胞在＜100μM范围内，均显示出了一定的抑制作用。
二、对酶的耐受性实验
在洁净的反应瓶中加入9ml水，搅拌下加入0.5g化合物D4A(纯度：99.1％)和1.5ml无水乙醇，待化合物D4A全部溶清后，用1N氯化氢溶液调节pH至7～8，并调节反应温度30～35℃，加入12g葡萄糖、0.1g 3α还原酶、0.22g葡萄糖脱氢酶、0.015g辅酶一、0.015g辅酶二。搅拌均匀后，用1N氢氧化钠溶液调节pH＝7～8反应，反应0.5～3小时，TLC监控反应(展开剂为石油醚：二氯甲烷：乙酸乙酯＝5:2:1，显色剂为磷钼酸)，具体的结果如图2所示，TLC结果显示化合物D4A 0.5小时全部反应完全。
3α还原酶是3α羟基甾族化合物氧化还原酶的简称，购自南京都莱生物技术有限公司，该3α还原酶属于睾丸酮丛毛单胞菌来源。葡萄糖脱氢酶，购自上海麦克林生化科技有限公司。辅酶一(NAD)：烟酰胺腺嘌呤二核苷酸，购自上海跃腾生物技术有限公司。辅酶二(NADP)，烟酰胺腺嘌呤二核苷酸磷酸盐，购自杭州唯泰生物药业有限公司。
化合物TM22的酶还原反应，同上操作，具体的结果如图3所示，TLC结果显示化合物TM22在0.5～5小时均转化少部分。
以上结果表明，相对于D4A，TM22与还原酶的反应活性大大降低，分子稳定性更好，不易在酶作用下降解，对体内代谢酶的耐受性较好，可以预期在体内代谢比较慢，排出体外时间会延长，作用时间也会延长，药效持续时间更长，有利于开发长效制剂。
与TM22相类似的，本发明的其余实施例化合物在0.5～5小时也仅仅转化少部分甚至不转化，同样表明：本发明的化合物与还原酶的反应活性大大降低或无反应活性，分子稳定性更好，不易在酶作用下降解，对体内代谢酶的耐受性较好，可以预期在体内代谢比较慢，排出体外时间会延长，作用时间也会延长，药效持续时间更长，有利于开发长效制剂。
三、与酶分子(3α类固醇脱氢酶/胆固醇氧化酶)对接
采用对接软件MOE(Molecular Operating Environment，由加拿大化学计算集团公司Chemical Computing Group ULC开发的针对制药和生命科学的综合软件系统)对以下化合物进行对接打分，具体结果如表2所示。
其中，阿比特龙-3α类固醇脱氢酶的对接模拟结果如图4所示，打分数据如图5所示；
TM23-3α类固醇脱氢酶的对接模拟结果如图6所示，打分数据如图7所示；
D4A-胆固醇氧化酶的对接模拟结果如图8所示，打分数据如图9所示；
TM22-胆固醇氧化酶的对接模拟结果如图10所示，打分数据如图11所示。
表2
MOE软件的打分是根据分子与酶之间的静电参数、氢键结合、分子吸引、分子轨道等等参数计算的结果。分数越负，自由能越低，构象越稳定，结合越好。
以上结果显示：本发明制备的17-(3-吡啶基)-10α-甲基-甾体衍生物中，在C10位甲基翻转后，与酶的对接没有未翻转化合物好，说明翻转后的化合物对以上2种酶不敏感，或酶对翻转化合物无活性，可知本发明的17-(3-吡啶基)-10α-甲基-甾体衍生物对体内酶的耐受性较好，预期在体内代谢比较慢，排出体外时间会延长，作用时间也会延长，药效持续时间更长，有利于开发长效制剂。
与TM22和TM23相类似的，本发明的其余实施例化合物，在C10位甲基翻转后，与酶的对接没有未翻转化合物好，说明翻转后的化合物对以上2种酶不敏感，或酶对翻转化合物无活性，可知本发明的17-(3-吡啶基)-10α-甲基-甾体衍生物对体内酶的耐受性较好，预期在体内代谢比较慢，排出体外时间会延长，作用时间也会延长，药效持续时间更长，有利于开发长效制剂。
四、溶解度测试
称取各测试样品(TM1、阿比特龙、醋酸阿比特龙)约20mg，置于不同的10ml量瓶中，加不同pH的缓冲介质(pH为1.0的盐酸溶液、pH为4.5的乙酸-乙酸钠缓冲溶液、pH为6.8的磷酸二氢钾-氢氧化钠缓冲溶液)稀释至刻度，摇匀，振荡24小时，保持有固体未溶解状态，用0.45μm的滤膜过滤，取上清液各1.0ml与对应的不同pH缓冲介质0.5ml混匀，即得相应的不同pH的缓冲介质溶液，采用HPLC进行溶解度(每mL溶液中所含TM1、阿比特龙、醋酸阿比特龙的质量/μg)的定量分析。HPLC条件如下：
色谱柱：Waters(沃特世)的Waters SunFire C18(150mm*4.6mm；3.5μm)
流动相：乙腈-水(75/25；V/V)
流速：1.0ml/分钟
检测器：UV紫外检测器(205nm)
进样量：20μl
结果如表3所示：
表3
由此可知，TM1在酸性条件下在水中的溶解度远高于阿比特龙，有利于药物在胃肠道的溶出，预期具有更高的生物利用度。
以上所述仅为本发明的实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本发明的保护范围之内。

Claims (19)

  1. 一种17-吡啶基-10α-甲基-甾体衍生物，其特征在于，所述甾体衍生物具有如下式I的结构或其药学可接受的盐：
    R1选自＝O、-OH、卤素或-OC(O)R2；
    R2选自C1～C5烷基、C1～C5卤代烷基、苯基或卤代苯基；
    R3选自卤素、C1～C5烷基、C1～C5卤代烷基、C1～C5烷氧基、羟基或氨基；
    R4、R5、R6、R7相同或不同，各自独立地选自-OH、＝O、卤素、氨基、C1～C5烷基、C1～C5卤代烷基、C1～C5烷氧基、C1～C5烯基或C1～C5酯基；
    表示单键或双键；
    当某一为双键时，与其相邻的为单键；
    i、j、k、m和n各自独立地选自0、1、2、3、4或5。
  2. 根据权利要求1所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，所述甾体衍生物具有如下式Ⅱ的结构式：
  3. 根据权利要求2所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，
    所述R1选自＝O、-OH或OAc。
  4. 根据权利要求3所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，
    所述R1选自-OH时，所述-OH为β-OH或α-OH。
  5. 根据权利要求2所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，
    所述R3选自C1～C5烷基或卤代烷基。
  6. 根据权利要求5所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，
    所述k为0。
  7. 根据权利要求2所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，
    所述i、j、m和n均为0，优选所述k为0。
  8. 根据权利要求1-7中任一项所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，
    所述R1至R7中的卤素选自F、Cl、Br或I。
  9. 根据权利要求1-7中任一项所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，所述甾体衍生物选自如下结构式：
  10. 根据权利要求9所述的17-吡啶基-10α-甲基-甾体衍生物，其特征在于，所述甾体衍生物选自如下结构式：
  11. 一种权利要求1-10中任一项所述的17-吡啶基-10α-甲基-甾体衍生物的制备方法，其特征在于，所述方法包括以式V所示的中间体为原料，在C-17位上连接取代或未被取代的 吡啶基；
    所述取代或未被取代的吡啶基与所述17-吡啶基-10α-甲基-甾体衍生物上的吡啶基结构对应相同；
    所述R9与R4对应相同或者经过反应后对应相同；
    所述R10与R5对应相同或者经过反应后对应相同；
    所述R11与R6对应相同或者经过反应后对应相同；
    所述R12与R7对应相同或者经过反应后对应相同。
  12. 一种用于制备17-吡啶基-10α-甲基-甾体衍生物的中间体化合物，其特征在于，所述中间体化合物选自下述结构式：
    R8选自-OH或被保护的羟基；
    R13选自＝O或被保护的羰基。
  13. 根据权利要求12所述的中间体化合物，其特征在于，
    所述R8选自-OH或OAc；和/或；
    所述R13选自＝O或
  14. 根据权利要求12所述的中间体化合物，其特征在于，所述中间体化合物选自下述结构式：
  15. 一种权利要求12-14中任一项所述的中间体化合物的制备方法，其特征在于，所述制备方法包括以下步骤：将下式化合物进行光化学转化使C-10位的甲基由β构型翻转为α构型
  16. 一种根据权利要求1-10中任一项所述的17-吡啶基-10α-甲基-甾体衍生物在制备治疗癌症的药物中的用途。
  17. 根据权利要求16所述的用途，其特征在于，所述癌症包括前列腺癌、乳腺癌、结肠癌或肺癌。
  18. 一种药物组合物，包含权利要求1-10中任一项所述的17-吡啶基-10α-甲基-甾体衍生物和药学上可接受的辅料。
  19. 一种具有如下结构的甾体化合物在制备治疗结肠癌或肺癌的药物中的用途
PCT/CN2023/076540 2022-02-18 2023-02-16 17-吡啶基-10α-甲基-甾体衍生物及中间体化合物、其制备方法、其用途、以及其药物组合物 WO2023155846A1 (zh)

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