CN111171102A - 2-methoxyestradiol-17 β -prolinamide analogue, synthetic method and application thereof - Google Patents

Abstract

the invention belongs to the field of pharmaceutical chemistry, and relates to a 2-methoxyestradiol-17 β -prolinamide analogue, a synthetic method and application thereof, wherein the analogue has the following structural general formula:

wherein m =0,1,2, n =0,1,2, r =0,1,2, X = H, OH, F, Cl, Br, OSO₂NH₂；Y=C,O,S,N；R₁=H,SO₂NH₂；R₂H, Me, Et, Ac; proved by in vitro antitumor cell proliferation activity test research, the derivatives maintain or enhance the activity of antitumor cells, have remarkably increased water solubility compared with 2-methoxyestradiol, and have better druggability, the in vivo metabolism half-life of the medicament reaches about 4 hours, and the in vivo bioavailability of the medicament is achievedAlso obviously improves the quality and can be used for developing anti-tumor medicinal preparations.

Description

2-methoxyestradiol-17 β -prolinamide analogue, synthetic method and application thereof

Technical Field

the invention relates to the field of pharmaceutical chemistry, and mainly relates to a novel 2-methoxyestradiol-17 β -prolinamide analogue taking estra as a mother nucleus, a preparation method and application thereof.

Background

According to the statistical data of Cancer in China in 2015 (Chen W, et al.Ca A Cancer Journal for Clinicians,2016,66(2):115.), 429 million new Cancer patients and 281 million new Cancer deaths were added in China 2015, wherein the main Cancer types are lung Cancer and gastric Cancer. The incidence of breast cancer in the female population exceeds lung cancer as found by gender differentiation, and the incidence of reproductive cancers in women is generally far greater than in men.

The female's physiological cycle and reproductive capacity are primarily regulated by estrogen, which was previously thought to be the only specific effects produced by estrogen, progesterone and other hormones like oxytocin, prolactin, etc. It is now recognized that hormones play only a part of the role in the human body, and that their metabolites play an important role in the regulation of reproductive physiological and pathological processes as well. These hormonal metabolic disorders can disrupt the balance of hormones in the body and alter the function of reproductive organs such as breast, ovary, uterus, etc., with consequent irregular menstruation, preeclampsia, endometriosis, infertility and cancer.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17 β -estradiol 2-Methoxyestradiol is present in urine and blood of normal persons, and 2-Methoxyestradiol is reported to be present in lower plasma levels (10-35 pg/ml for male and non-pregnant women, respectively)^-1And 18 to 138 pg.ml^-1) The content of the extract in the blood plasma of pregnant women is higher (up to 216-10690 pg/ml)^-1). 2-ME was once thought to be a non-functional metabolite, but it has now been found that 2-ME is important for the physiological regulation of reproductionThe ingredients have certain effects on cancer, rheumatoid arthritis, cardiovascular system, etc. (Dubey R K, oral&Metabolism,2009,20(8):374-379)。

2-methoxyestradiol is a promising candidate for anticancer and is currently under phase I and II clinical study. However, due to its poor water solubility, low bioavailability, short half-life in vivo and irregular absorption in vivo, clinical application of 2-ME still faces a number of difficulties. Therefore, the structural modification and the reconstruction of the 2-methoxyestradiol so as to improve the water solubility and the bioavailability of the 2-methoxyestradiol have practical significance and research value.

Disclosure of Invention

the invention aims to provide a 2-methoxyestradiol-17 β -prolinamide analogue with better anti-tumor activity and good water solubility, a preparation method thereof and application thereof in anti-tumor drugs.

In order to realize the purpose of the invention, the technical scheme is as follows:

the 2-methoxyestradiol-17 β -prolyl amine analogue is a 2-methoxyestradiol-17 β -prolyl amide analogue taking estra as a mother nucleus, and has the following general structure:

wherein m is 0,1,2; n is 0,1,2; r is 0,1,2; x ═ H, OH, F, Cl, Br, OSO₂NH₂；Y＝C,O,S,N；R₁＝H,SO₂NH₂；R₂＝H,Me,Et,Ac；

Preferably: m is 0,1,2; n is 0, 1; r is 0,1,2; x ═ H, OH, OSO₂NH₂；Y＝C,S；R₁＝H,SO₂NH₂；R₂＝H,Ac。

The substituent at the 17-position of the compound is in an L or D or DL configuration.

the present invention provides 2-methoxyestradiol-17 β -prolinamide analogues, obtainable by the following reaction scheme:

(1) dissolving the estra parent compound 1 in an organic solvent, adding Lewis base and a condensing agent to dissolve under stirring at normal temperature, then adding proline or proline analogue protected by amino N, and detecting a thin layer until the reaction is complete; then washing, extracting and separating, drying and concentrating the organic phase, and separating and purifying by recrystallization or column chromatography to obtain an amide condensed intermediate compound 2;

(2) dissolving the amide intermediate 2 in an organic solvent, removing a protective agent by adopting a catalytic hydrogenation, acidolysis, alkaline hydrolysis or trimethyl halogenated silane method, then carrying out suction filtration on a reaction solution, concentrating a filtrate, and carrying out recrystallization or column chromatography by using a solvent to obtain a compound 3 or a compound A;

(3) reacting the compound 3 with aminosulfonyl chloride in an organic solvent to generate a 3-site aminosulfonyl esterified compound 4, wherein the aminosulfonyl chloride can also be prepared by reacting chlorosulfonyl isocyanate with formic acid in an ice bath on site;

(4) dissolving amide compound 4 in organic solvent, removing protective agent by catalytic hydrogenation, acidolysis, alkaline hydrolysis or trimethyl halogenated silane method, suction filtering reaction solution, concentrating filtrate, and recrystallizing or column chromatography with solvent to obtain compound B.

In the step (1), the proline analogue can be proline, hydroxyproline, thioproline, high proline (2-pipecolic acid), 3-pipecolic acid and 4-pipecolic acid with L, D or DL configuration; the amino protecting group R' is selected from carbobenzoxy (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) or acetyl (Ac); wherein the molar ratio of the estrogen parent compound 1 to the proline analogue protected by amino is 1: 1.0-1: 3.0.

The Lewis base used is selected from triethylamine, N, N-diisopropylethylamine, ethylenediamine, N, N ' -dimethylethylenediamine, N, N, N ', N ' -tetramethylethylenediamine, pyridine, triethanolamine, triethylenediamine, N-methylmorpholine, 4-dimethylaminopyridine, pyrrolidinylpyridine, 1, 5-diazabicyclo, 1, 8-diazabicyclo [5.4.0] undec-7-ene, tetramethylguanidine, 1,5, 7-triazabicyclo [ 4.4.0 ] deca-5-ene, 7-methyl-1, 5, 7-triazabicyclo [ 4.4.0 ] deca-5-ene, or one of sodium carbonate, sodium acetate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, calcium hydride, sodium hydroxide, potassium acetate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, sodium hydride, sodium hydroxide, sodium methoxide, sodium ethoxide, sodium, One of potassium tert-butoxide, sodium tert-butoxide, n-butyllithium, tert-butyllithium, potassium hexamethyldisilazide, sodium hexamethyldisilazide, lithium diisopropylamide, and lithium 2,2,6, 6-tetramethylpiperidine (LiTMP); wherein the molar ratio of the female steroid parent compound 1 to the Lewis base is 1: 1.0-1: 5.0.

The condensing agent is selected from 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride, N-hydroxysuccinimide, N-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazol, N-hydroxyphthalimide, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate, O-benzotriazol-N, N, one or two of N ', N' -tetramethylurea hexafluorophosphate, O- (N-succinimide) -1,1,3, 3-tetramethylurea tetrafluoroborate and 2- (5-norbornene-2, 3-dicarboximido) -1,1,3, 3-tetramethylurea tetrafluoroborate quaternary ammonium salt; the molar ratio of the estra parent compound 1 to the condensing agent is 1: 1.0-1: 5.0.

The organic solvent used in the step (1) is one or two selected from N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetone, methyl ethyl ketone, toluene, chloroform, dichloromethane, ethyl acetate, methanol, ethanol, acetonitrile, isopropanol, isobutanol, diethyl ether, isopropyl ether, ethyl acetate, tetrahydrofuran, dioxane, nitromethane, pyridine, ethylene dichloride and the like;

the estra parent compound 1 has the following molecular structure general formula:

wherein R' is a hydrogen atom, acetyl, benzyl, methoxymethylene or tetrahydropyranyl; benzyl is preferred. The preparation method adopts Xiufang Shi, et al, Steroids,2017,128:6-14 literature method for synthesis.

In the step (2) and the step (4), the reaction temperature is preferably 50-90 ℃ in the deprotection process; selecting a palladium-carbon catalyst with the mass percentage of 10% by a catalytic hydrogenation method, wherein the using amount of the palladium-carbon catalyst is 5% -20% of the mass of the amide intermediate; the trimethylhalosilane is preferably trimethylbromosilane, trimethyliodosilane, or trimethylchlorosilane; the acidolysis solution is selected from Lewis acids such as hydrochloric acid, hydrobromic acid, trifluoroacetic acid, glacial acetic acid or acetic acid, formic acid, phosphoric acid, citric acid or tartaric acid, boron trifluoride, ferric trichloride, titanium tetrachloride, ceric amine nitrate and the like; the alkaline hydrolysis solution is selected from aqueous solution or sodium alkoxide of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, potassium bicarbonate, etc.; the organic solvent is selected from one or two of methanol, ethanol, acetonitrile, isopropanol, isobutanol, diethyl ether, isopropyl ether, ethyl acetate, dichloromethane, chloroform, acetone, methyl ethyl ketone, toluene, tetrahydrofuran, 1, 4-dioxane, nitromethane, pyridine and ethylene dichloride; the recrystallization or column chromatography solvent is preferably one or two of ethyl acetate, petroleum ether, methanol, ethanol, isopropanol, acetonitrile, acetone, dichloromethane, etc.

In the step (3), the organic solvent is preferably water, ethanol, acetonitrile, N-dimethylformamide, N-dimethylacetamide.

The structures of the series A compound and the series B compound prepared by the method are as follows:

the 2-methoxyestradiol-7 β -prolinamide analogue has the advantages that 2-methoxyestradiol is taken as a lead compound, a sulfamate group is introduced at the 3-position and prolinamide group is introduced at the 17 β -position to prepare the 2-methoxyestradiol derivative, the preparation method is simple, the conditions are mild, the yield is high and reaches more than 60%, and the 2-methoxyestradiol-17 β -prolinamide analogue can be conveniently prepared.

Drawings

FIG. 1 is a graph showing a comparison of half-lives of compounds A-1 and 2-ME of the present invention, wherein 1 is a compound A-1 and 2 is a 2-methoxyestradiol compound (2-ME).

Detailed Description

The following describes in detail embodiments of the present invention with reference to examples.

wherein the female parent 2-methoxy-3-benzyloxy-estra-1, 3,5(10) -trien-17 β -amine is synthesized according to XiufangShi, et al, Steroids,2017,128: 6-14.

example 1 preparation of the Compound 2-methoxy-3-hydroxy-17 β - (L-prolinamido) -estra-1, 3,5, (10) -triene (A-1)

2-methoxy-3-benzyloxy-estra-1, 3,5, (10) -triene-17 β -amine 400mg was accurately weighed and charged into a three-necked flask, 15ml of dichloromethane was added and dissolved with stirring at room temperature, then 329mg of N-t-butoxycarbonyl-L-proline, 422mg of dicyclohexylcarbodiimide, 235mg of N-hydroxysuccinimide and 0.55ml of N, N-diisopropylethylamine were successively added, and after stirring at room temperature for 6 hours, the reaction was completed, the reaction solution was filtered, and then extracted once with 10% hydrochloric acid, saturated sodium carbonate and saturated sodium chloride, respectively, the organic phase was dried over anhydrous sodium sulfate, purified by column chromatography (eluent: petroleum ether: ethyl acetate 4:1 by volume), to obtain 477mg of condensation product, yield 80%.

And (2) adding 426mg of the condensation product into a three-neck flask, dissolving the condensation product by using 12ml of acetonitrile, heating the mixture to 60 ℃ in an oil bath, dropwise adding 0.8ml of trimethyl bromosilane, reacting the mixture for 4 hours at 70 ℃, cooling, then carrying out suction filtration on the reaction solution, concentrating the filtrate, and recrystallizing the filtrate by using tetrahydrofuran to obtain 218mg of the product with the yield of 76%.¹H NMR(400MHz,Chloroform-d)δ7.57(d,J＝9.5Hz,1H),6.77(s,1H),6.64(s,1H),3.91(q,J＝9.1,8.7Hz,1H),3.85(s,3H),3.75(dd,J＝9.1,5.4Hz,1H),3.01(dt,J＝10.3,6.8Hz,1H),2.88(dt,J＝10.2,6.4Hz,1H),2.75(h,J＝7.4Hz,2H),2.27–2.18(m,2H),2.17–2.08(m,2H),1.97–1.89(m,1H),1.89–1.82(m,1H),1.82–1.74(m,2H),1.70(td,J＝7.0,5.2Hz,2H),1.44(t,J＝10.0Hz,2H),1.34(pd,J＝10.9,9.6,3.2Hz,5H),0.73(s,3H).¹³C NMR(101MHz,Chloroform-d)δ175.0,144.7,143.6,114.7,108.2,60.7,58.2,56.1,51.6,47.2,44.1,43.4,38.9,36.9,30.8,29.0,28.7,27.4,26.5,26.2,23.3,12.1.

example 2 preparation of the Compound 2-methoxy-3-sulfamate-17 β - (L-prolinamido) -estra-1, 3,5, (10) -triene (B-1)

200mg of the proline condensation product prepared in example 1 was dissolved in 20mL of ethanol in a 50mL Teflon pressure bottle, then 10% palladium on carbon catalyst was added in 30mg, the hydrogenation apparatus was put under hydrogen pressure of 0.3MPa, and the reaction was carried out in an oil bath at 60 ℃ for 3 hours until the hydrogenation reaction was complete. Then filtering, evaporating filtrate to dryness, and recrystallizing with appropriate amount of ethanol to obtain crude hydrogenated product 156mg with yield of 92%.

Under the protection of ice bath and nitrogen, 156mg of crude hydrogenation product is dissolved in 5mL of anhydrous N, N-dimethylformamide solution, then 0.1mL of aminosulfonyl chloride is slowly added dropwise, and the mixture is stirred for 2h under ice bath till the reaction is complete. Then, distilled water was slowly dropped in an amount of 8 times the volume of the reaction solution to precipitate a large amount of a white solid, which was then subjected to suction filtration to obtain 145mg of a sulfamoyl compound in a yield of 80.2%.

Putting 145mg of the sulfamide in a round-bottom flask, adding 5mL of ethanol, stirring for dissolving, then dropwise adding 0.5mL of trifluoroacetic acid at room temperature, reacting for 2h, evaporating the ethanol and the trifluoroacetic acid under reduced pressure, washing the residue three times with saturated sodium bicarbonate solution, and purifying by column chromatography, wherein the volume ratio of an eluent is dichloromethane: methanol: triethylamine 10: 1: 0.1 yield 95mg of product, 79.2%.¹H NMR(400MHz,Chloroform-d)δ7.27(s,1H),6.75(s,1H),6.45(d,J＝3.0Hz,1H),4.78(s,1H),4.45(s,1H),3.71(t,J＝2.5Hz,3H),3.24(dq,J＝22.1,7.5Hz,2H),2.75–2.58(m,2H),2.39–2.24(m,2H),2.14(t,J＝9.7Hz,2H),1.96(h,J＝7.5,6.6Hz,3H),1.78(d,J＝11.2Hz,2H),1.69(d,J＝9.7Hz,1H),1.66–1.58(m,1H),1.41(d,J＝11.9Hz,1H),1.37(s,2H),1.33(d,J＝10.5Hz,3H),1.23(s,1H),0.76(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ168.76,145.54,144.41,129.94,128.26,115.59,109.85,83.89,58.84,55.85,48.81,45.40,43.43,42.88,38.17,36.31,28.32,27.99,26.88,26.83,25.88,22.94,22.73,11.90.

EXAMPLE 3 preparation of the Compound 2-methoxy-3-hydroxy-17 β - (L-2-piperidincarboxamido) -estra-1, 3,5, (10) -triene (A-7)

accurately weighing 540mg of 2-methoxy-3-benzyloxy-estra-1, 3,5, (10) -triene-17 β -amine, adding into a three-neck flask, adding 30mL of toluene, stirring and dissolving at room temperature, adding 483mg of N-benzyloxycarbonyl-L-2 pipecolic acid, adding 847mg of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 0.7mL of triethylamine, detecting by TLC, reacting at room temperature for 4 hours, completely reacting, washing the filtered reaction liquid with 10% by mass of hydrochloric acid aqueous solution, saturated sodium carbonate solution and saturated salt water once, drying the organic phase with a proper amount of anhydrous sodium sulfate, concentrating, and purifying by column chromatography (the eluent is petroleum ether: ethyl acetate ═ 6: 1 in volume ratio) to obtain 648mg of condensation product with the yield of 73.8%.

Adding 648mg of the condensation product into a polytetrafluoroethylene pressure-resistant bottle, dissolving the condensation product by using a proper amount of ethanol, adding 48mg of 10% palladium-carbon catalyst, reacting for 4 hours at 60 ℃ under the hydrogenation pressure of 0.3Mpa, and detecting the reaction progress by TLC. Then, the reaction solution was filtered with thin layer silica gel, and recrystallized with ethyl acetate and petroleum ether to obtain 286mg of product with a yield of 68.3%.¹H NMR(400MHz,DMSO)δ8.59(br,1H,-OH),7.25(d,J＝9.0Hz,1H),6.75(s,1H),6.44(s,1H),3.85–3.74(m,1H),3.71(s,3H),3.07(dd,J＝10.0,1.9Hz,1H),2.91(d,J＝12.6Hz,1H),2.69–2.57(m,2H),2.23(d,J＝4.9Hz,1H),2.11(s,1H),1.95–1.83(m,1H),1.81–1.61(m,6H),1.46(d,J＝9.9Hz,2H),1.35–1.22(m,10H),0.66(s,3H).¹³C NMR(101MHz,DMSO)δ172.94,145.52,144.34,130.29,128.29,115.57,109.83,59.31,57.71,55.82,51.14,45.05,43.68,43.23,38.68,36.90,30.18,28.41,27.32,27.10,26.05,25.84,23.98,22.91,11.96。

example 4 preparation of the Compound 2-methoxy-3-sulfamate-17 β - (D-2-piperidinecarboxamido) -estra-1, 3,5, (10) -triene (B-8)

accurately weighing 617mg of 2-methoxy-3-benzyloxy-estra-1, 3,5, (10) -triene-17 β -amine, adding the 2-methoxy-3-benzyloxy-estra-1, 3,5 and (10) -triene-17 β -amine into a three-neck flask, adding 30mL of acetonitrile, stirring and dissolving at room temperature, then successively adding 620mg of N-tert-butoxycarbonyl-D-2-pipecolic acid, 1.0g of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and 1.0mL of N, N-diisopropylethylamine, reacting at room temperature for 4h, filtering reaction liquid, washing with 10 mass percent of hydrochloric acid aqueous solution, saturated sodium carbonate solution and saturated salt water, adding a proper amount of anhydrous sodium sulfate, drying, concentrating, and purifying by column chromatography, wherein an eluent is petroleum ether and ethyl acetate is 20: 3 in volume ratio, so as to obtain 626mg of a condensation product, and the yield is 65..

Adding the condensation product 626mg and 50mL of methanol into a 50mL polytetrafluoroethylene pressure bottle, dissolving, adding 10% palladium-carbon catalyst 70mg, introducing hydrogen into a hydrogenation device under the pressure of 0.3MPa, and reacting for 4 hours at 60 ℃ in an oil bath. Filtering, evaporating filtrate to dryness, and recrystallizing with appropriate amount of ethanol to obtain crude product of 536mg, with 100% yield.

Under the protection of ice bath and nitrogen, 0.176mL of anhydrous formic acid is added into 0.406mL of chlorosulfonyl isocyanate, the mixture is rapidly stirred, a large amount of white solid is generated in a bottle after 15min, 5mL of anhydrous acetonitrile is added, and then the mixture is stirred at room temperature for 7h to obtain an aminosulfonyl chloride solution. Then 8mL of anhydrous N, N-dimethylacetamide solution containing 536mg of crude hydride was slowly added dropwise to the above sulfamoyl chloride solution, and stirred for 2h under ice bath until the reaction was complete. Then, 8 times the volume of distilled water was slowly dropped to precipitate a large amount of white solid, which was then suction-filtered to obtain 546mg of the sulfamoyl compound with a yield of 88.3%.

Putting 546mg of the sulfamide into a round-bottom flask, adding 10mL of dichloromethane, stirring for dissolving, then dropwise adding 0.840mL of trifluoroacetic acid at room temperature, reacting for 2h, evaporating dichloromethane and trifluoroacetic acid under reduced pressure, washing the residue three times with saturated sodium bicarbonate solution, and purifying by column chromatography, wherein the eluent has the volume ratio of dichloromethane: methanol: triethylamine 10: 1: 0.1 yield 345mg product, 76.1% yield.¹H NMR(400MHz,DMSO)δ8.14(d,J＝8.3Hz,1H),7.83(s,2H),6.99(s,1H),6.97(s,1H),3.82-3.79(m,1H),3.76(s,3H),3.74-3.71(m,1H),3.22(d,J＝11.8Hz,1H),3.15–3.06(m,1H),2.93(d,J＝11.6Hz,1H),2.77–2.67(m,2H),2.37–2.27(m,1H),2.26–2.17(m,1H),2.13–2.03(m,1H),2.00–1.88(m,1H),1.87–1.75(m,3H),1.74-1.65(m,2H),1.64–1.55(m,1H),1.53-1.43(m,3H),1.40–1.24(m,6H),0.71(s,3H).¹³C NMR(101MHz,DMSO)δ168.41,149.43,138.79,136.78,128.29,122.90,110.52,58.76,57.17,55.90,51.06,43.88,43.28,43.21,38.18,36.71,28.21,27.20,27.02,26.76,25.74,22.83,21.62,21.23,11.92。

Other compounds of the A series can be prepared by methods similar to those of examples 1 and 3.

The B series compounds can be prepared by analogy with examples 2 and 4.

The compounds A-1 to A-15 and B-1 to B-15 mentioned in the claims are not limited to these compounds, and the synthesis method thereof can be carried out by referring to the methods of examples 1 to 4. Among these, nuclear magnetic data for representative compounds are as follows:

the compound A-2 is a compound of formula (I),¹H NMR(400MHz,DMSO)δ8.02(d,J＝8.7Hz,1H),6.74(s,1H),6.45(s,1H),4.29(s,1H),4.04–3.97(m,1H),3.78(q,J＝9.2Hz,1H),3.70(s,3H),3.08(dd,J＝11.7,4.2Hz,1H),2.90(d,J＝11.8Hz,1H),2.68–2.58(m,2H),2.27–2.19(m,1H),2.12(dd,J＝12.8,7.6Hz,2H),1.97–1.90(m,1H),1.84–1.59(m,5H),1.53–1.42(m,1H),1.33–1.23(m,6H),0.67(s,3H).¹³C NMR(101MHz,DMSO)δ171.03,145.49,144.38,130.21,128.33,115.61,109.91,70.29,58.62,58.22,55.87,54.26,51.04,43.62,43.36,39.81,38.64,36.90,28.39,27.25,27.09,26.03,22.90,12.01。

the compound A-3 is a compound of formula (I),¹H NMR(400MHz,DMSO)δ8.13(d,J＝8.7Hz,1H),6.74(s,1H),6.45(s,1H),4.35–4.27(m,1H),4.06(dd,J＝9.6,5.6Hz,1H),3.83(q,J＝9.0Hz,1H),3.70(s,3H),3.16(dd,J＝11.4,4.8Hz,1H),2.87(dd,J＝14.5,7.3Hz,1H),2.67–2.57(m,2H),2.49–2.38(m,1H),2.23(d,J＝9.2Hz,1H),2.12(s,1H),1.98–1.92(m,1H),1.90–1.87(m,1H),1.86–1.74(m,2H),1.72–1.64(m,2H),1.63–1.50(m,1H),1.37–1.22(m,6H),0.68(s,3H).¹³C NMR(101MHz,DMSO)δ169.39,145.51,144.34,130.26,128.27,115.57,111.88,68.96,58.66,58.11,55.83,53.05,50.97,43.61,40.07,38.63,36.80,28.37,27.10,26.75,26.08,22.91,12.06。

the compound A-4 is a compound of formula (I),¹H NMR(400MHz,DMSO)δ8.58(s,1H),7.77(d,J＝9.5Hz,1H),6.75(s,1H),6.44(s,1H),4.65(s,1H),4.15–4.06(m,1H),3.75(q,J＝9.6Hz,1H),3.71(s,3H),3.48(dd,J＝9.4,5.4Hz,1H),2.94(dd,J＝10.8,5.3Hz,1H),2.68–2.60(m,3H),2.27–2.20(m,1H),2.17–2.08(m,2H),1.98–1.86(m,1H),1.82–1.74(m,1H),1.74–1.61(m,3H),1.49–1.38(m,1H),1.34–1.20(m,6H),0.69(s,3H).¹³C NMR(101MHz,DMSO)δ173.98,145.52,144.35,130.35,128.32,115.58,109.87,71.12,59.27,57.50,55.83,54.71,51.16,43.69,43.39,39.56,38.67,36.71,28.40,27.75,27.11,26.07,22.93,11.66。

the compound A-5 is a compound of formula (I),¹H NMR(400MHz,CDCl₃)δ6.77(s,1H),6.63(s,1H),4.58(d,J＝8.0Hz,1H),3.91–3.86(m,1H),3.86(s,4H),3.21(q,J＝7.1Hz,3H),2.73(dt,J＝13.9,9.7Hz,3H),2.47–2.34(m,2H),2.21(dd,J＝16.4,10.4Hz,3H),2.11(s,3H),1.95–1.84(m,6H),1.44(dd,J＝13.8,8.1Hz,4H),1.33–1.27(m,3H),0.75(s,3H).¹³C NMR(101MHz,CDCl₃)δ171.15,171.13,144.63,143.48,131.80,129.47,114.63,108.23,59.82,59.14,56.12,51.63,48.40,47.36,44.13,43.51,38.93,37.11,28.99,28.58,27.44,26.56,25.02,23.38,22.52,12.11。

the compound A-6 is a compound of,¹H NMR(400MHz,DMSO)δ8.58(s,1H),7.72(d,J＝8.8Hz,1H),6.75(s,1H),6.44(s,1H),4.22–4.15(m,1H),3.86–3.78(m,1H),3.78–3.72(d,J＝21.6Hz,2H),3.70(s,3H),3.24–3.13(m,1H),2.78(dd,J＝9.8,6.9Hz,1H),2.68–2.59(m,2H),2.30–2.20(m,1H),2.12(s,1H),1.98–1.84(m,2H),1.82–1.74(m,1H),1.71–1.62(m,2H),1.54–1.43(m,1H),1.35–1.21(m,6H),0.67(s,3H).¹³C NMR(101MHz,DMSO)δ170.00,145.52,144.34,130.27,128.31,115.57,109.81,65.89,58.25,55.81,54.02,51.06,43.64,43.34,38.64,36.94,36.38,28.39,27.20,27.09,26.06,22.90,12.05。

the compound A-8 is a compound of,¹H NMR(400MHz,DMSO)δ8.85–8.34(br,1H,-OH),7.25(d,J＝9.0Hz,1H),6.75(s,1H),6.44(s,1H),3.79(q,J＝9.2Hz,1H),3.71(s,3H),3.08–3.03(m,1H),2.89(d,J＝12.7Hz,1H),2.67–2.55(m,3H),2.27–2.20(m,1H),2.16–2.08(m,1H),1.94–1.84(m,1H),1.80–1.62(m,5H),1.53–1.42(m,2H),1.38–1.23(m,10H),0.67(s,3H).¹³C NMR(101MHz,DMSO)δ172.86,145.51,144.34,130.29,128.30,115.57,109.84,59.21,57.75,55.82,51.14,44.90,43.67,43.29,38.67,36.97,29.69,28.41,27.36,27.10,26.03,25.72,23.72,22.91,12.01。

the compound A-9 is a compound of,¹H NMR(400MHz,DMSO)δ7.67–7.64(m,1H),6.74(s,1H),6.44(s,1H),3.77(q,J＝9.2Hz,1H),3.70(s,3H),2.93–2.79(m,2H),2.68–2.57(m,3H),2.36–2.27(m,1H),2.25–2.17(m,1H),2.10(s,1H),1.92–1.74(m,3H),1.69–1.51(m,5H),1.50–1.32(m,3H),1.31–1.20(m,6H),0.65(s,3H).¹³C NMR(101MHz,DMSO)δ173.75,145.53,144.40,130.31,128.31,115.60,109.87,59.71,57.87,55.84,51.14,47.94,45.43,43.67,43.29,42.18,38.69,37.07,28.40,27.11,26.07,24.51,22.89,20.71,14.05,12.08。

the compound A-10 is a compound of formula (I),¹H NMR(400MHz,CDCl₃)δ6.79(s,1H),6.65(s,1H),4.02–3.95(m,1H),3.87(s,3H),2.86–2.82(m,2H),3.54–3.47(m,1H),3.46–3.29(m,2H),2.81–2.74(m,2H),2.74–2.68(m,1H),2.43–2.36(m,1H),2.26–2.20(m,2H),2.15–2.13(m,1H),2.12(s,2H),1.90–1.83(m,2H),1.80–1.75(m,2H),1.73–1.63(m,2H),1.60–1.55(m,1H),1.49–1.44(m,2H),1.41–1.35(m,4H),0.69(s,3H)。

the compound A-11 is a compound of,¹H NMR(400MHz,DMSO)δ7.42(d,J＝8.6Hz,1H),6.74(s,1H),6.44(s,1H),3.77(q,J＝9.2Hz,1H),3.70(s,3H),2.97(d,J＝11.8Hz,2H),2.68–2.58(m,2H),2.54–2.42(m,2H),2.33–2.17(m,2H),2.10(s,1H),1.89–1.74(m,2H),1.68–1.58(m,3H),1.56–1.42(m,4H),1.32–1.21(m,7H),0.64(s,3H).13C NMR(101MHz,DMSO)δ174.42,145.52,144.38,130.31,128.29,115.59,109.84,57.93,55.82,51.16,45.18,44.97,43.67,43.34,42.12,38.69,37.10,29.70,28.47,28.40,27.11,26.96,26.07,22.88,12.08。

the compound A-12 is a compound of,¹H NMR(400MHz,CDCl₃)δ6.77(s,1H),6.63(s,1H),4.56(d,J＝13.6Hz,1H),3.97(dd,J＝16.8,8.2Hz,1H),3.85(s,3H),3.16–3.05(m,1H),2.78–2.64(m,4H),2.43–2.30(m,2H),2.27–2.15(m,4H),2.09(s,3H),1.90–1.80(m,3H),1.79–1.60(m,5H),1.47–1.34(m,5H),0.72(s,3H).¹³C NMR(101MHz,CDCl₃)δ174.22,169.14,144.73,143.56,131.64,129.38,114.72,108.30,58.72,56.13,51.61,47.19,44.09,43.50,41.05,38.95,37.12,28.97,28.53,27.40,26.53,23.29,21.43,12.12,8.78.¹³C NMR(101MHz,DMSO)δ173.75,171.22,144.40,130.31,128.31,115.60,109.87,59.71,57.87,55.84,51.14,47.94,45.43,43.67,43.29,42.18,38.69,37.07,28.40,27.11,26.07,24.51,22.89,20.71,14.05,12.60。

the compound B-2 is a mixture of,¹H NMR(400MHz,DMSO)δ7.97–7.70(m,3H),7.50(s,2H,),6.98(s,1H),6.97(s,1H),4.98–4.93(m,1H),3.83–3.77(m,1H),3.76(s,3H),3.75–3.70(m,1H),3.11(dd,J＝12.8,4.3Hz,1H),3.07–3.00(m,1H),2.76–2.67(m,2H),2.36–2.25(m,2H),2.24–2.18(m,1H),1.96–1.87(m,2H),1.83(d,J＝10.7Hz,1H),1.72–1.63(m,2H),1.62–1.40(m,2H),1.39–1.25(m,6H),0.68(s,3H).¹³C NMR(101MHz,DMSO)δ172.53,149.42,138.9,136.74,128.28,122.91,110.50,81.55,59.12,57.94,55.88,52.55,51.09,43.89,43.17,38.21,37.57,36.84,28.21,27.47,26.77,25.79,22.90,11.93。

the compound B-3 is a compound of formula,¹H NMR(400MHz,DMSO)δ8.17(d,J＝8.3Hz,1H),7.82(s,2H),7.65(s,2H),6.98(s,1H),6.96(s,1H),5.10–5.04(m,1H),4.18-410(m,1H),3.84(q,J＝9.2Hz,1H),3.76(s,3H),3.48–3.39(m,2H),2.78–2.66(m,3H),2.36–2.08(m,4H),2.02–1.90(m,1H),1.88–1.65(m,4H),1.60–1.46(m,1H),1.42–1.23(m,7H),0.69(s,3H).¹³C NMR(101MHz,DMSO)δ168.55,149.42,138.86,136.74,128.27,122.91,110.45,77.86,58.70,58.15,55.87,51.03,51.00,43.87,43.50,38.19,36.82,36.67,28.19,26.91,26.78,25.86,22.88,11.92。

the compound B-4 is a compound of formula,¹H NMR(400MHz,DMSO)δ7.89–7.76(m,3H),7.49(s,2H),6.98(s,1H),6.96(s,1H),4.97–4.90(m,1H),3.84–3.77(m,1H),3.76(s,3H),3.74–3.68(m,1H),3.24(dd,J＝12.3,5.1Hz,0H),2.77–2.67(m,2H),2.48–2.41(m,1H),2.36–2.28(m,1H),2.26–2.16(m,1H),2.08–2.00(m,1H),2.00–1.91(m,1H),1.88–1.58(m,4H),1.56–1.42(m,1H),1.41–1.23(m,7H),0.70(s,3H).¹³C NMR(101MHz,DMSO)δ149.42,139.24,136.85,128.43,123.04,110.47,79.10,58.04,55.81,52.08,51.25,43.91,43.20,38.21,36.72,36.62,28.32,27.47,26.83,25.77,22.90,11.67。

the compound B-5 is a compound of formula,¹H NMR(400MHz,CDCl₃)δ7.27(s,1H),7.17(d,J＝8.8Hz,1H),7.02(s,1H),6.90(s,1H),5.38(s,2H,-NH₂),4.58(d,J＝7.1Hz,1H),3.92–3.87(m,1H),3.86(s,3H),3.61–3.53(m,1H),3.47–3.37(m,1H),2.94(dd,J＝32.0,23.7Hz,1H),2.81–2.75(m,2H),2.48–2.40(m,1H),2.24–2.18(m,2H),2.11(s,3H),2.03–1.97(m,1H),1.90–1.82(m,3H),1.77–1.70(m,1H),1.51–1.40(m,3H),1.39–1.27(m,5H),0.74(s,3H).¹³C NMR(101MHz,CDCl₃)δ171.22,171.16,149.03,140.36,136.91,130.04,123.94,110.56,59.80,59.06,56.41,51.63,48.41,44.36,43.40,38.44,37.00,28.64,28.54,27.42,27.14,26.32,25.04,23.37,22.49,12.07。

the compound B-6 is a compound of the formula,¹H NMR(400MHz,DMSO)δ7.83(s,2H),6.75(s,1H),6.44(s,1H),4.22–4.15(m,1H),3.86–3.78(m,1H),3.78–3.72(d,J＝21.6Hz,2H),3.70(s,3H),3.24–3.13(m,1H),2.78(dd,J＝9.8,6.9Hz,1H),2.68–2.59(m,2H),2.30–2.20(m,1H),2.12(s,1H),1.98–1.84(m,2H),1.82–1.74(m,1H),1.71–1.62(m,2H),1.54–1.43(m,1H),1.35–1.21(m,6H),0.67(s,3H).¹³C NMR(101MHz,DMSO)δ170.00,145.52,144.34,130.27,128.31,115.57,109.81,65.89,58.25,55.81,54.02,51.06,43.64,43.34,38.64,36.94,36.38,28.39,27.20,27.09,26.06,22.90,12.05。

the compound B-7 is a compound of formula,¹H NMR(400MHz,Acetone)δ7.02(s,1H),7.01(s,1H),4.13(dd,J＝11.8,3.3Hz,2H),3.98(t,J＝9.6Hz,1H),3.83(s,3H),3.82(t,J＝12Hz,1H),3.53(d,J＝12.8Hz,1H),3.21–3.17(m,1H),2.81–2.76(m,2H),2.39–2.33(m,1H),2.32–2.23(m,3H),1.96-1.93(m,2H),1.92-1.87(m,3H),1.84–1.80(m,1H),1.70–1.66(m,1H),1.64–1.61(m,1H),1.48–1.44(m,2H),1.43–1.36(m,5H),0.8(s,3H).¹³C NMR(101MHz,DMSO)δ168.49,149.42,138.80,136.76,128.27,118.54,115.57,58.28,56.93,55.84,50.91,43.83,43.55,43.17,38.17,36.80,30.56,28.17,27.75,26.74,25.80,22.83,21.64,21.19,11.98。

the compound B-9 is a mixture of,¹H NMR(400MHz,CDCl₃)δ7.23(s,1H),6.74(s,1H),6.44(s,1H),3.77(q,J＝9.2Hz,1H),3.70(s,3H),2.93–2.79(m,2H),2.68–2.57(m,3H),2.36–2.27(m,1H),2.25–2.17(m,1H),2.10(s,1H),1.92–1.74(m,3H),1.69–1.51(m,5H),1.50–1.32(m,3H),1.31–1.20(m,6H),0.65(s,3H).¹³C NMR(101MHz,DMSO)δ173.75,145.53,144.40,130.31,128.31,115.60,109.87,59.71,57.87,55.84,51.14,47.94,45.43,43.67,43.29,42.18,38.69,37.07,28.40,27.11,26.07,24.51,22.89,20.71,14.05,12.08。

the compound B-10 is a compound of formula,¹H NMR(400MHz,CDCl₃)δ7.28(s,1H),7.03(s,1H),6.89(s,1H),5.67(s,2H),4.09–3.93(m,2H),3.86(s,3H),3.84-3.81(m,1H),3.61–3.47(m,1H),3.44–3.22(m,2H),2.81–2.74(m,2H),2.42–2.32(m,1H),2.24–2.15(m,2H),2.12–2.10(m,1H)2.09(s,2H),1.90–1.84(m,2H),1.80–1.64(m,4H),1.57–1.50(m,1H),1.49–1.41(m,2H),1.40–1.29(m,4H),0.71(s,3H)。

the compound B-11 is a compound of formula,¹H NMR(400MHz,CDCl₃)δ7.27(s,1H),6.74(s,1H),6.44(s,1H),3.77(q,J＝9.2Hz,1H),3.70(s,3H),2.97(d,J＝11.8Hz,2H),2.68–2.58(m,2H),2.54–2.42(m,2H),2.33–2.17(m,2H),2.10(s,1H),1.89–1.74(m,2H),1.68–1.58(m,3H),1.56–1.42(m,4H),1.32–1.21(m,7H),0.64(s,3H).13C NMR(101MHz,DMSO)δ174.42,145.52,144.38,130.31,128.29,115.59,109.84,57.93,55.82,51.16,45.18,44.97,43.67,43.34,42.12,38.69,37.10,29.70,28.47,28.40,27.11,26.96,26.07,22.88,12.08。

the compound B-12 is a compound of,¹H NMR(400MHz,CDCl₃)δ7.27(s,1H),7.03(s,1H),6.90(s,1H),5.47(d,J＝2.9Hz,2H),4.62–4.54(m,1H),4.04–3.96(m,1H),3.87(s,3H),3.15–3.06(m,1H),2.82–2.76(m,2H),2.74–2.60(m,2H),2.41–2.32(m,2H),2.27–2.19(m,2H),2.09(s,2H),1.93–1.84(m,3H),1.81-1.74(m,3H),1.50–1.44(m,2H),1.42–1.31(m,6H),0.70(s,3H).¹³CNMR(101MHz,CDCl₃)δ173.19,168.15,148.11,139.14,135.85,128.94,122.90,109.35,55.25,50.57,44.94,43.49,42.57,42.20,39.90,37.59,35.82,29.99,28.67,28.33,28.06,27.81,27.69,26.28,25.28,22.26,20.36,11.22。

EXAMPLE 5 evaluation of the antiproliferative Activity of 2-methoxyestradiol-17 β -prolinamide analogues

(1) Test sample and cancer cell line

the 2-methoxyestradiol-17 β -prolinamide analogue samples (30 types in total) and 2-ME are respectively prepared into 20mmol/L raw drug stock solution by dimethyl sulfoxide (DMSO), and the raw drug stock solution is stored in a refrigerator at 4 ℃ for standby application, wherein the raw drug stock solution is diluted into working solution by RPMI-1640 complete culture medium (or DMEM culture medium) with 10% of FBS by mass percentage before an experiment, and the working solution is filtered by a 0.22 mu m filter membrane for use.

The cancer cell lines selected in the experiment are human cervical cancer HeLa cells, human ovarian cancer cells SKOV3, human breast cancer cells MCF-7, human prostate cancer cells PC-3 and human esophageal cancer cells EC-109. Wherein human cervical cancer HeLa cells are cultured in DMEM medium, human ovarian cancer cells SKOV3, human breast cancer cells MCF-7, human prostate cancer cells PC-3 and human esophageal cancer cells EC109 cells are cultured in RPMI 1640 medium.

(2) Experimental methods

The MTT staining method is used for detecting the inhibition effect of 30 2-methoxyestradiol analogues on the proliferation of different cancer cells, and compounds with good inhibition effect are screened out.

Is taken at logarithmDigesting the cells in the growth phase with pancreatin, preparing cell suspension with prepared RPMI-1640 medium, counting, and adjusting and diluting the cell density to 5 × 10⁴At one/ml, cell suspensions were seeded zigzag-wise into 96-well plates at 100. mu.L/well and marginal wells were filled with 200. mu.L sterile PBS to eliminate marginal effects. Standing for 3min after adding all the components, and placing the culture plate at 37 deg.C for 5 vol% CO₂And culturing for 24h in a saturated humidity incubator. Diluting the drug to be tested to a certain concentration with RPMI-1640 culture medium, setting zero adjustment group (no cells in blank culture medium), blank group (no drug added in blank cell suspension) and solvent group (adding blank solvent DMSO) as required, sucking out the culture medium of each well, adding 200 μ L of drug-containing culture medium (seven drug concentrations are set for each compound, 1.25 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L, 40 μmol/L and 80 μmol/L, three duplicate wells are set for each drug concentration), transferring the culture plate to constant temperature CO after adding drugs, and transferring the culture plate to a constant temperature₂In an incubator, at 37 ℃ and 5% by volume of CO₂And culturing for 72h under saturated humidity conditions. After 72h dosing, 20 μ of LMTT was added to each well. After continuing culturing for 4h, sucking out the culture medium, adding 150 mu L of DMSO, uniformly oscillating, detecting the absorbance value at 490nm by using a microplate reader, and calculating the inhibition rate by the following formula: the inhibition rate (%) × (1-absorbance value of administration group/absorbance value of blank group) × 100%. Then, statistical software SPSS is used for calculating the half Inhibitory Concentration (IC) of the 2-ME derivative on different tumor cell strains₅₀) Each experiment was repeated three times, and the values are expressed as inhibition (x. + -. s).

(3) Results of the experiment

The results are shown in tables 1 and 2.

Table 1 IC of Compounds on 5 cells₅₀Values (μmol/L, 72h)

As can be seen from the preliminary pharmacological activity data in Table 1, the 2-methoxyestradiol-17 β -prolinamide analog provided by the patent shows better anti-tumor cell proliferation effect, for example, five cells of the compounds A-1 and B-1 show better proliferation inhibition effect on Hela cells, SKOV3 cells, PC-3 cells, MCF-7 cells and EC-109 cells, the compound A-7 with activity better than or equal to 2-ME. has better selective inhibition effect on three cells of PC-3 cells, MCF-7 cells and EC-109 cells, the compounds B-7 and A-8 have better selective inhibition effect on two cells of MCF-7 cells and EC-109 cells, and A-9 has selective inhibition activity on EC-109 cells.

example 6 solubility of 2-methoxyestradiol-17 β -prolinamide (A-1)

(1) Chromatographic conditions

A chromatographic column: an Agilent Eclipse XDB-C18 column (4.6 mm. times.250 mm, 5 μm);

mobile phase: methanol-water (65:35, v/v);

flow rate: 0.8 mL/min;

column temperature: 30 ℃;

detection wavelength: lambda [ alpha ]_Excitation＝286nm,λ_Launching＝317nm；

Sample introduction amount: 20 mu L of the solution;

a detector: a fluorescence detector.

(2) Determination of drug solubility

0.5mg, 1.0mg and 2.0mg of compound A-1 are respectively weighed and put into a penicillin bottle, 5mL of ultrapure water is added, a preservative film is sealed, and the mixture is shaken for 72 hours at the constant temperature of 37 ℃ at 120 r/min. Taking 4mL of saturated solution, centrifuging at 4000rpm for 10min, taking a proper amount of supernatant, filtering through a 0.45-micron filter membrane, collecting in a liquid-phase sample injection bottle, and injecting samples according to the HPLC determination condition (1).

(3) Determination of the lipid-water partition coefficient

Precisely weighing the compound A-110.0 mg, placing the compound A-110.0 mg in a 10mL volumetric flask, adding 10mL of n-octanol solution saturated with water to a constant volume to a scale, dissolving the n-octanol solution by ultrasonic treatment, and filtering the dissolved n-octanol solution by using an organic microporous filter membrane of 0.45 mu m to remove insoluble substances to prepare an A-1 n-octanol solution saturated with water. Measuring 5mL of A-1 water-saturated n-octanol solution, and adding 5mL of n-octanolAnd deionized water. Shaking at 37 deg.C for 72h at 120rpm to balance the distribution, centrifuging, and measuring the water phase drug concentration and the organic phase drug concentration after balance distribution according to HPLC measurement conditions. According to the formula P ═ C_o/C_wTo the lipid water partition coefficient.

TABLE 22 solubility comparison of ME and its derivative A-1 (37 deg.C)

As can be seen from the experimental results in Table 2, the water solubility of 2-methoxyestradiol-17 β -prolinamide (A-1) is obviously better than that of 2-ME, and the derivatives can effectively improve the defect of poor water solubility of 2-ME.

EXAMPLE 7 pharmacokinetic Studies of the Compound 2-methoxyestradiol-17 β -prolinamide (A-1)

(1) Materials and reagents

Testing compound a-1 of the invention; control 2-methoxyestradiol (2-ME);

SPF rats (clean grade) weighing 200. + -.20 g in males and females, provided by the university of Zhengzhou laboratory animal center.

(2) Experimental methods

SPF-grade rats 12 were randomly divided into 2 groups (groups A-1 and 2-ME), 6 rats in each group were fasted for 12h before administration, allowed to drink water freely and were administered by intravenous injection at the trailing edge, A-1 and 2-ME were administered with hydrogenated castor oil: ethanol: normal saline l: 2: 7, administered at a dose of 10mg/kg body weight, about 0.5mL of blank plasma was taken before administration, about 0.5mL of blood was taken from orbital venous plexus of rats at several time points of 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 480, 720min after administration, placed in heparinized centrifuge tubes, centrifuged at 4000rmp for 10min, 200 μ L of upper plasma was aspirated for sample treatment, and the plasma concentration was calculated according to the established chromatographic conditions and the standard curve.

(3) Data processing and results

According to the PKSolver software, the plasma concentration data of the compound are fitted, and it can be seen that the plasma concentration model of A-1 and 2-ME is a two-chamber model, and the half-life period of A-1 is 246.8min, which is obviously longer than that of 2-ME (26.9 min).

2-methoxyestradiol-17 β -prolinamide (A-1) and analogues thereof need to be subjected to deeper medicinal activity evaluation, and an antitumor medicament with better clinical curative effect is hopefully obtained and is used for developing antitumor medicinal preparations such as breast cancer resistance, prostatic cancer resistance, ovarian cancer resistance, cervical cancer resistance or esophageal cancer resistance.

Claims (7)

1.2-methoxyestradiol-17 β -prolinamide analogues characterized in that the compounds have the following general structural formula:

wherein m is 0,1,2; n is 0,1,2; r is 0,1,2; x ═ H, OH, F, Cl, Br, OSO₂NH₂；Y＝C,O,S,N；R₁＝H,SO₂NH₂；R₂＝H,Me,Et,Ac。

2. the 2-methoxyestradiol-17 β -prolinamide analog of claim 1,

m＝0,1,2；n＝0,1；r＝0,1,2；X＝H,OH,OSO₂NH₂；Y＝C,S；R₁＝H,SO₂NH₂；R₂＝H,Ac。

3. 2-methoxyestradiol-17 β -prolinamide analogue according to claim 1 or 2, characterized in that the substituent at position 17 is in L or D or DL configuration.

4. 2-methoxyestradiol-17 β -prolinamide analogue according to claim 1, characterized in that it is selected from the following compounds:

5. a process for the preparation of 2-methoxyestradiol-17 β -prolinamide analogues according to claim 1, characterized by the following reaction steps:

wherein R' is a hydrogen atom, acetyl, benzyl, methoxymethylene or tetrahydropyranyl;

(1) dissolving the estra parent compound 1 in an organic solvent, adding Lewis base and a condensing agent to dissolve under stirring at normal temperature, then adding proline or proline analogue protected by amino N, and detecting a thin layer until the reaction is complete; then washing, extracting and separating, drying and concentrating the organic phase, and separating and purifying by recrystallization or column chromatography to obtain an amide condensed intermediate 2;

(2) dissolving the amide intermediate 2 in an organic solvent, removing a protective agent by adopting a catalytic hydrogenation, acidolysis, alkaline hydrolysis or trimethyl halogenated silane method, then carrying out suction filtration on a reaction solution, concentrating a filtrate, and carrying out recrystallization or column chromatography by using a solvent to obtain a compound 3 or a compound A;

(3) reacting the compound 3 with aminosulfonyl chloride in an organic solvent to generate a 3-sulfamate esterified compound 4;

(4) dissolving amide compound 4 in an organic solvent, removing a protective agent by adopting a catalytic hydrogenation, acidolysis, alkaline hydrolysis or trimethyl halogenated silane method, then carrying out suction filtration on a reaction solution, concentrating a filtrate, and obtaining a compound B by using a solvent recrystallization or column chromatography method;

in the step (1), the proline analogue is proline, hydroxyproline, thioproline, high proline (2-pipecolic acid), 3-pipecolic acid and 4-pipecolic acid with L or D or DL configuration; the amino protecting group R' is selected from carbobenzoxy (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) or acetyl (Ac);

in the step (1), the Lewis base is selected from triethylamine, N, N-diisopropylethylamine, ethylenediamine, N, N ' -dimethylethylenediamine, N, N, N ', N ' -tetramethylethylenediamine, pyridine, triethanolamine, triethylenediamine, N-methylmorpholine, 4-dimethylaminopyridine, pyrrolidinylpyridine, 1, 5-diazabicyclo, 1, 8-diazabicyclo [5.4.0] undec-7-ene, tetramethylguanidine, 1,5, 7-triazabicyclo [ 4.4.0 ] deca-5-ene, 7-methyl-1, 5, 7-triazabicyclo [ 4.4.0 ] deca-5-ene, sodium carbonate, sodium acetate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, sodium acetate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, sodium hydroxide, sodium ethoxide, or a mixture thereof, One of calcium hydride, sodium hydride, potassium tert-butoxide, sodium tert-butoxide, n-butyllithium, tert-butyllithium, potassium hexamethyldisilazide, sodium hexamethyldisilazide, lithium diisopropylamide, and lithium 2,2,6, 6-tetramethylpiperidine (LiTMP);

in the step (1), the condensing agent is selected from 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride, N-hydroxysuccinimide, N-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazol, N-hydroxyphthalimide, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate and O-benzotriazol-N, one or two of N, N ', N' -tetramethylurea hexafluorophosphate, O- (N-succinimide) -1,1,3, 3-tetramethylurea tetrafluoroborate and 2- (5-norbornene-2, 3-dicarboximidyl) -1,1,3, 3-tetramethylurea tetrafluoroborate quaternary ammonium salt;

in the step (1), the organic solvent is one or two of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetone, methyl ethyl ketone, toluene, chloroform, dichloromethane, ethyl acetate, methanol, ethanol, acetonitrile, isopropanol, isobutanol, diethyl ether, isopropyl ether, tetrahydrofuran, dioxane, nitromethane, pyridine and ethylene dichloride;

in the step (2), catalytic hydrogenation is carried out on a palladium-carbon catalyst with the mass percentage of 10%;

in the step (2) and the step (4), the catalyst is selected from palladium carbon catalyst with the mass percentage of 10%; the trimethyl halogenated silane is selected from trimethyl bromine silane, trimethyl iodine silane or trimethyl chlorosilane; the acidolysis solution is selected from hydrochloric acid, hydrobromic acid, trifluoroacetic acid, glacial acetic acid or acetic acid, formic acid, phosphoric acid, citric acid or tartaric acid, boron trifluoride, ferric trichloride, titanium tetrachloride and ceric amine nitrate; the alkaline hydrolysis solution is selected from aqueous solution or sodium alkoxide of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate and potassium bicarbonate; the organic solvent is selected from one or two of methanol, ethanol, acetonitrile, isopropanol, isobutanol, diethyl ether, isopropyl ether, ethyl acetate, dichloromethane, chloroform, acetone, methyl ethyl ketone, toluene, tetrahydrofuran, 1, 4-dioxane, nitromethane, pyridine and ethylene dichloride;

in the step (3), the organic solvent is selected from water, ethanol, acetonitrile, N-dimethylformamide and N, N-dimethylacetamide.

6. the use of 2-methoxyestradiol-17 β -prolinamide analogues according to any of claims 1 to 4 in the preparation of medicaments, characterized in that the compounds are used as active ingredients or combined with other medicaments, mixed with pharmaceutically acceptable auxiliary and/or additional ingredients, and then prepared into anti-tumor pharmaceutical preparations according to conventional pharmaceutical methods and processes.

7. the use of a 2-methoxyestradiol-17 β -prolinamide analogue in the manufacture of a medicament according to claim 6, wherein the anti-tumor agent is an anti-breast, anti-prostate, anti-ovarian, anti-cervical or anti-esophageal cancer agent.

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