RU2426737C1 - 4-HETERO-16α, 17α-CYCLOHEXANOPREGNANES - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel 4-oxa- and 4-aza-16α, 17α-cyclohexanopregnanes (4-oxa- and 4-aza-pregna-D'-pentaranes), which can be used n medicine to treat malignant tumours, having general formula I

, where X=0 or NR, R=R₁=R₂=R₄=H where R₁+R₃ form a bond. Said compounds are obtained by oxidising ring A 16α, 17α-cyclohexano-progesterone with potassium permanganate and sodium periodate in the presence of sodium carbonate in 5-oxo-A-nor-3,5-secoacid followed by closure of ring A in 4-oxa-16α, 17α-cyclohexanopregn-5-en-20-one while treating with a sodium acetate dehydrating agent in Ac₂O and in 4-aza-16α, 17α-cyclohexanopregn-5-en-20-one using ammonium acetate in acetic acid.

EFFECT: compounds are effective inhibitors of oestrogen-stimulated cell proliferation HeLa.

2 cl, 2 ex, 1 tbl

Description

The invention relates to the field of chemistry of natural and physiologically active substances, in particular to the field of steroid hormones containing a heteroatom in the steroid nucleus, specifically to new 4-oxa and 4-aza-16α, 17α-cyclohexanopregnans (4-oxa and 4-aza -pregna-D′-pentaranam), which can be used in medicine for the treatment of malignant tumors.

It is known that steroids containing heteroatom (s) in the steroid skeleton can have different types of biological (hormonal and non-hormonal) activity depending on the structural features and type of steroid [M. Ibrahim-Quali. Recent advances in oxasteroid chemistry. Steroids, 2007, 72, 475; Recent advances in azasteroid chemistry. Steroids, 2008, 73, 375]. Modern therapy for hormone-dependent tumors needs drugs that stop the uncontrolled proliferation (growth) of cancer cells. Along with various cytostatic drugs, progestins and antiprogestins, progesterone receptor (RP) modulators, are widely used for this purpose. Among them, a promising group is pregna-D′-pentaranes - derivatives of progesterone with an additional carbocycle D ′ at the 16α, 17α-positions of the steroid skeleton, possessing progestogen and antiprogestagenic activity in vivo and suitable for oral use.

The closest in structure and properties is 16α, 17α-cyclohexanoprogesterone II, which is an active progestin, but its antiproliferative effect is small [A.V. Kamernitsky, I.S. Levin. Pregna-D′-pentarans. Progestins and antiprogestins. Bioorgan. Chemistry, 2005, v.31, c.115 and 227]. But among these described steroids, pentaranes containing a heteroatom in ring A are unknown.

The present invention is the creation of new steroids containing a heteroatom in position 4 of the ring A ring and a six-membered ring D ′ at the 16α, 17α positions of the steroid skeleton, having affinity for the progesterone receptor and exhibiting antiproliferative (inhibiting cancer cell growth) activity.

The problem is achieved by the new 4-oxa and 4-aza-16α, 17α-cyclohexanopregnans of the general formula I

where X = O or NR, R = H, lower alkyl, R ₁ = R ₂ = H or R ₁ + R ₂ form a bond, R ₁ = R ₃ = R ₄ = H or R ₁ + R ₃ form a bond, and wherein R ₄ = H.

The proposed compounds are obtained by oxidation of the ring A 16α, 17α-cyclohexanoprogesterone II with potassium permanganate and sodium periodate in the presence of sodium carbonate in 5-oxo-A-nor-3,5-secanoic acid III, followed by ring A closure in the latter to 4-oxasteroid Ia at treatment with sodium acetate in Ac ₂ O and 4-azasteroid Ib with a dehydrating agent by the action of ammonium acetate in acetic acid according to the following scheme:

The structure of the obtained compounds was confirmed by elemental analysis, mass and NMR ¹ H and ¹³ C spectra.

The effect of the substitution of the carbon atom at position 4 on the heteroatom in the pentaran molecule for binding was tested by the ability of the obtained compounds Ia and Ib to interact with the rat uterine cytosol RP. To assess the affinity, a competitive analysis was used (displacement of complexes of [ ³ H] proteins with progesterone II. Average relative competitive activity (OKA) using a number of 16α, 17α-cyclohexanoprogesterone II pentaranes as a standard, which can be considered as the basic structure for The claimed compounds are presented in the table.

№№ Compound OKA,% P Progesterone 93 ± 8 (8) * II 16α, 17α-Cyclohexanoprogesterone one hundred Iba 16α, 17α-Cyclohexano-4-oxapregn-5-en-3,20-dione 0.72 ± 0.21 (8) Ib 16α, 17α-Cyclohexano-4-azapregn-5-en-3,20-dione 0.84 ± 0.26 (8) * Mean ± standard deviation. In parentheses is the number of measurements.

The effect of pentaranes Ia and Ib on the viability of tumor cells was studied on the cervical cancer cell line HeLa. For the experiment, a cell suspension was placed in a 96-well plate. The test substances were dissolved in dimethyl sulfoxide to a concentration of 10 ^-2 M, then on incubation medium to final concentrations in the culture of 10 ^-6 and 10 ^-5 M. Estradiol was also added to all wells (except for the “pure” control) to a final concentration of 10 ^-8 M and incubated for 6 days. Cell viability was assessed by the color reaction of living cells with MTT tetrazolium dye. The optical density of the control samples was taken as 100% cell survival. The value of estrogen-stimulated proliferation was calculated as the difference between the viability of control cells with estrogen (10 ^-8 M estradiol) and in its absence. Both compounds Ia and Ib in the studied doses proved to be effective inhibitors of estrogen-stimulated proliferation of HeLa cells: they inhibit cell viability by 90-100%.

Example 1

To a solution of 1.105 g (3 mmol) of 16α, 17α-cyclohexanopregn-4-ene-3,20-dione II in a mixture of 40 ml of tertbutanol and 3 ml of 40% aqueous Na ₂ CO _{3, a} solution of 5.6 g (26.18 mmol) is added at 60 ° C. ) NaJO ₄ and 0.18 g (1.13 mmol) of KMnO ₄ in 25 ml of water and kept at this temperature for 3 hours. Then the reaction mixture was cooled to 30 ° C and after 15 minutes the solid precipitate was filtered off, washed on the filter with water and the combined filtrates were evaporated. The aqueous residue was acidified (pH 2) with 6 M HCl, extracted with CH ₂ Cl ₂ , the organic extract was washed with water and dried with Na ₂ SO ₄ . After removal of the solvent, 1.04 g (90%) of the acid is obtained as a colorless foam, which is used in the next step without further purification. Part of the acid is purified by column chromatography. Elution with a mixture of pet. Ether-acetone-CH ₂ Cl ₂ (85: 10: 5) afforded crystalline sec. III acid with a melting point of 179-182 ° C (from ether-hexane). IR spectrum (CHCl ₃ ), √ cm ^-1 : 3600-3000, 1692. ¹ H NMR spectrum (δ, ppm, J / Hz): 0.75 (s, 3H, Me (18)); 1.15 (s, 3H, Me (19)); 2.15 (s, 3H, Me (21)); 3.0 (s, 1H, 16H).

A mixture of 0.39 g (1 mmol) of keto acid III and 0.82 g (10 mmol) of AcONa dried at 180 ° C in 10 ml of Ac ₂ O is boiled for 4 hours. EtOAc and H ₂ O are added to the residue after removal of Ac ₂ O, the organic layer is washed with saturated NaCl solution, dried Na ₂ SO ₄ and the solvent removed in vacuo. The residue was purified by chromatography. Elution with a mixture of pet. Ether-acetone (93: 7) gives 0.198 g (53%) of crystalline 4-oxasteroid Ia with a melting point of 158-160 ° C (from a mixture of acetone-hexane). Found (%): C, 77.94; H, 8.99. C ₂₄ H ₃₄ O ₃ . Calculated (%): C, 77.80; H, 9.25. ¹ H NMR spectrum (δ, ppm, J / Hz): 0.72 (s, 3H, Me (18)); 1.12 (s, 3H, Me (19)); 2.14 (s, 3H, Me (21)); 2.97 (s, 1H, 16H); 5.25 (d, 1H, H (6)).

Example 2

The keto acid III (0.34 g, 0.87 mmol) and 0.40 g (5.2 mmol) of AcONH ₄ in 10 ml of ice obtained analogously to Example 1 are boiled for 6 hours. Water is added to the residue after removal of AcOH, the precipitated brown powder is filtered off, washed with water and dried on in the air. After chromatographic purification on a column (elution with a mixture of pet. Ether-acetone 90: 10 → 88: 12), 0.21 g (65%) of crystalline 4-azasteroid Ib with so pl. 305-308 ° C (from a mixture of acetone-hexane). ¹ H NMR spectrum (δ, ppm, J / Hz): 0.72 (s, 3H, Me (18)); 1.10 (s, 3H, Me (19)); 2.14 (s, 3H, Me (21)); 2.97 (s, 1H, 16H); 4.85 (m, 1H, H (6)); 7.85 (br s, 1H, NH).

Example 3. A method for determining the binding of compounds Ia and Ib with RP cytosol of rat uterus

We use sexually mature virgin female rats of a mixed population weighing 200-250 g. Animals are injected intramuscularly with estradiol of 10 μg in 200 μl of propylene glycol daily for 4 days and they are killed by decapitation on the 5th day. The uterus is crushed and homogenized in a glass homogenizer at 0-4 ° C for 5 min in a buffer solution containing 10 mm Tris-HCl (pH 7.5), 10 mm KCl, 1 mm EDTA, 0.5 mm phenylmethyl sulfonyl fluoride (PMSF), 1 mm dithiothreitis , 10% glycerol, with a tissue / buffer weight ratio of 1: 6. The homogenate is centrifuged at 4 ° C for 1 h at 50,000 g. The supernatant fraction (cytosol) with a protein concentration of 4-6 mg / ml is used immediately. Use [1,2,6,7- ³ H] progesterone to a specific radioactivity of 86 Ci / mmol (St. Petersburg).

The measurement of the relative competitive activity (OKA) of RP steroids is performed using [ ³ H] progesterone in the presence of 3 μM hydrocortisone. Uterine cytosol (100 μl) is incubated at 0-4 ° C for 20 h with 100 μl of a mixture of steroids in a buffer including (60-80) · 10 ³ cpm (final concentration 3-6 nM) [ ³ H] ligand and unlabeled competitor (final concentrations from 0 to 10 μM). The protein bound and free ligand is separated by incubation with 100 μl of a 2% suspension of Norit A activated carbon (Serva, Germany), coated with dextran-70 (Fluka, Switzerland) for 5 min at 0-4 ° C. After centrifugation for 5 min at 3000 g, aliquots (250 μl) of the supernatant fraction were collected to measure radioactivity. Incubation is carried out in quartz tubes coated with BSA. Radioactivity is measured using a dioxane scintillator with a counting efficiency of 20%. Protein content is determined using Coomassie blue dye. All measurements are carried out in two parallel samples. Each experiment is reproduced 3-4 times. The dimensionless OKA values are calculated by the ratio of K _d for progesterone and the compared ligand obtained in separate experiments, and the calculated OKA values are averaged.

Example 4. Evaluation of the effect of the claimed compounds on the viability of the cell line cancer of the cervical cancer HeLa

Cultivation of HeLa cells is carried out under sterile conditions using Laminar Box LB-B (Russia). Cells are incubated at 37 ° C. under 5% CO ₂ . When culturing cells using standard DMEM medium with the addition of 10% fetal calf serum, L-glutamine at a concentration of 100 μg / ml and the antibiotic gentamicin sulfate at a concentration of 40 μg / ml. Cells are grown at 37 ° C in the presence of 10 ^-8 M estradiol in DMEM medium containing 10% fetal calf serum. For the experiment, a cell suspension is placed in a 96-well flat-bottom plate. The test substances are dissolved in dimethyl sulfoxide to a concentration of 10 ^-2 M, then in the incubation medium to final concentrations in the culture of 10 ^-6 -10 ^-5 M. The concentration of dimethyl sulfoxide in the cell culture is 10 ^-3 -10 ^-6 M. All wells are also added estradiol to a final concentration of 10 ^-8 M, except for pure control. Incubated for 6 days, after which the results are taken into account by the MTT method. The optical density of the samples recorded at a wavelength of 530 nm on the analyzer "UNIPLAN" AIFR-01 (Russia). The optical density of the control samples in the presence of estradiol alone is taken as 100% cell survival.

Thus, as can be seen from the above examples, we obtained new 4-oxa and 4-aza-16α, 17α-cyclohexanopregnans Ia and Ib, which bind to the rat uterus progesterone receptor and are highly effective inhibitors of cancer cell growth, i.e. exhibit high antiproliferative activity. The claimed compounds may be of interest in medicine as antitumor agents, for example, in the treatment of hormone-dependent cancers.

Claims (2)

1. 4-Hetero-16α, 17α-cyclohexane-pregnanes of the general formula I

where X-O or NR, R = R ₁ = R ₂ = R ₄ = H, with R ₁ + R ₃ forming a bond. 2. The compounds according to claim 1, 16α, 17α-cyclohexano-4-oxapregn-5-ene-3,20-dione and 16α, 17α-cyclohexano-4-azapregn-5-en-3,20-dione having affinity to the progesterone receptor and antiproliferative activity.