Use of 3-oxy-sulfamoyloxy-7β-methyl-D-en-6-oxaestra-1,3,5(10),8(9)-tetraen-17a-one for treating breast cancer

Abstract

The present invention relates to the pharmaceutical field and the chemical and pharmacological industries, and to a drug for treating breast cancer. In particular, the present invention relates to the use of 3-oxy-sulfamoyloxy-7β-methyl-D-enhanced-6-oxaestra-1,3,5(10),8(9)-tetraen-17a-one as an anticancer agent in monotherapy and adjuvant therapy for breast cancer (including triple-negative breast cancer).

Description

Technical Field

[0001] The present invention relates to the medical field and the chemical and pharmaceutical industries, and to a drug for treating breast cancer. Background Art

[0002] Breast cancer is a major neoplastic disease in women [Parkin DM, Bray F., Ferlay J., Pisani P., CA Cancer J. Clin., 2005, Vol. 55, pp. 74-108]. According to the World Health Organization, there are 2.09 million patients worldwide and 627,000 women die from breast cancer each year [http: / / www.who.int / news-room / fact-sheets / detail / cancer].

[0003] A large proportion of tumors that appear in this location develop under the action of estrogen [Yue W., Yager JD, Wang J.-P., Jupe ER, Santen RJ, Steroids, 2013, Vol. 78, pp. 161-170]. Immunohistochemical analysis revealed breast cancers that express receptors for estrogen (ER), progesterone (PR), and HER2NEU (which makes the cancer "Herceptin sensitive"). A large proportion of tumors have ER, PR, and / or HER2NEU 3+. If a tumor does not have ER and PR and is insensitive to Herceptin (ER0, PR0, HER2NEU 0-1), it is considered a triple-negative form (ER- / PR- / HER-2-). This is one of the most deadly forms of breast cancer because it has no target to inhibit its growth. Currently, there is no drug in the world to treat this form of cancer.

[0004] "Estrogen circulates in the blood and accumulates in tumors in the form of sulfates that are unable to bind to estrogen receptors, but after conversion to free hormones they activate tumor growth. Therefore, a promising line of therapeutic strategy consists in blocking the formation of this group of free hormones in tumors using inhibitors."

[0005] Tamoxifen is a drug from the group of selective estrogen receptor modulators that blocks the effects of estrogen on hormone-dependent tissues, including breast tissue. Tamoxifen is a standard hormone therapy for premenopausal and postmenopausal women. The most typical side effects that occur during the use of tamoxifen include: increased risk of venous thrombosis, worsening of the course of cardiovascular disease (including angina attacks), development of endometrial tumors (polyps and endometrial cancer), and uterine fibroids. Tamoxifen also has hepatotoxicity. According to the Biopharmaceutical Classification System (BCS) developed by Gordon Amidon et al. in 1995, tamoxifen is classified as a second class, i.e., a drug with low solubility and high permeability.

[0006] Aromatase inhibitors have been shown to be more effective than tamoxifen. Currently, aromatase inhibitors such as letrozole and anastrozole have been put into use in the field. According to the BCS system, they are classified into Class I, which represents drugs with high solubility and high permeability. Side effects of aromatase inhibitors include osteoporosis, hot flashes and headaches.

[0007] In addition to the above drugs, steroid sulfatase is also a topic of great concern because large amounts of circulating estrone sulfate can lead to local interstitial formation of estrogen. Steroid sulfatase catalyzes the hydrolysis of estrone sulfate to estrone and catalyzes the hydrolysis of DHEA sulfate to DHEA (Dibbelt I., Biol. Chem, Hoppe-Seyler, 1991, Vol. 372, pp. 173-185; Stein C., J. Biol. Chem., 1989, Vol. 264, pp. 13865-13872).

[0008] The most famous steroidal sulfatase inhibitor is EMATE-estrone sulfamate (Ahmed S., Curr. Med. Chem., 2002, Vol. 9, No. 2, pp. 263-273). However, it has a significant disadvantage. Estrone sulfatase inhibitors containing sulfamate groups lead to irreversible enzyme inactivation with the release of free ligands [Howarth NM, Purohit A., Reed MJ, J. Med. Chem., 1994, Vol. 37, pp. 219-221]. In particular, estrone sulfamate inhibits estrone sulfatase, but the release of free hormone leads to the emergence of strong uterotropic activity [Shields-Botella J. et al., J. Steroid Biochem. Mol. Biol., 2003, Vol. 84, pp. 327-335].

[0009] WO9933858 (A2) (published on July 8, 1999) describes a group of estrone sulfatase inhibitors, wherein the derivatives of the following general formula can be used as estrone sulfatase inhibitors for treating or preventing estrogen-dependent diseases, such as breast cancer.

[0010]

[0011] These compounds also possess uterotropic activity.

[0012] Therefore, when searching for new anticancer agents, it needs to be taken into account that the carrier of the sulfamate group of the estrone sulfatase inhibitor should not have hormonal activity.

[0013] These requirements have led to an expansion of the use of estrogen-based sulfamate esters, as they have a wide range of activities, in particular they can be used to treat recurrent breast cancer [Shah R. et al., "Sulfatase inhibitors for recidivist breast cancer treatment: A chemical review", Eur. J. Med. Chem., 2016, Vol. 114, pp. 170-190]. In particular, a 17α-substituted derivative of estradiol sulfamate is described.

[0014] The prior art also discloses a compound having the following chemical formula: 3,17αβ-diaminosulfonyloxy-7β-methyl-D-in-6-oxaestra-1,3,5(10),8,14-pentaene (RU 2619457, published on May 16, 2017) as a growth inhibitor of breast cancer cells MCF-7. This compound can be selected as the closest prior art of the present invention.

[0015] Summary of the invention

[0016] The technical problem of the present invention is to provide a compound and to develop an effective method for synthesizing the compound, which can be used as an anticancer agent in monotherapy and adjuvant therapy of breast cancer, including the most lethal forms of breast cancer, such as triple-negative breast cancer (ER- / PR- / HER-2-).

[0017] This problem is solved by providing the use of the compound of formula (2) 3-oxo-sulfamoyloxy-7β-methyl-oxo-6-oxaestra-1,3,5(10),8(9)-tetraene, which inhibits estrone sulfatase, as an anticancer agent in monotherapy and adjuvant therapy of breast cancer, including triple negative forms (ER- / PR- / HER-2-) of breast cancer.

[0018]

[0019] The following is a synthesis scheme for the target compound of formula (2).

[0020]

[0021] The compound of formula (3) is introduced into a reaction with vinylmagnesium bromide. The resulting condensation of vinylmethanol with 2-methylcyclohexane-1,3-dione leads to the formation of a ring-opened compound of formula (4). The cyclodehydration reaction of the compound of formula (4) is carried out in hydrochloric acid to obtain a compound of formula (5). The estratetraene of formula (6) is prepared by hydrogenating the compound of formula (5) on palladium / carbon in ethanol with a yield of 75%. The aminosulfonate of formula (2) is prepared with a yield of 86%.

[0022] The anticancer agent obtained belongs to the fifth category of dangerous substances (LD 50 >5000 mg / kg). The data obtained allow the doses used in the antitumor activity studies to be graded as safe. The therapeutically effective dose of the drug is preferably 0.1 to 20 mg / kg.

[0023] Mammary tumors (BT) of FBV / N female mice (HER-2 / neu transgenic) are adenocarcinomas characterized by low levels of estrogen receptor (ER) Figure 1 ) and lack progesterone receptor (PR). It can be differentiated into two adenocarcinoma subtypes, specifically ER+, PR- and ER-, PR-. Immunohistochemical analysis of ERα in breast tumors showed that only 10 of 45 samples had single positive staining in the nucleus ( Figure 1 ).

[0024] The model was sensitive to CAF (cyclophosphamide, doxorubicin, 5-fluorouracil) treatment, with observed tumor growth inhibition (TGI), resulting in stabilization of mean tumor volume ( Figure 2 ). The TGI on the 14th and 21st days of the experiment were 63% and 46%, respectively.

[0025] Since animals may have multiple tumors, the average total tumor volume of mice was analyzed at the beginning of the experiment ( Figure 3 ) and the relative changes in tumor volume ( Figure 4 ) is reasonable. The relative increase in tumor volume shows that this parameter changes linearly in the control group, which indicates that this parameter is suitable for evaluating the biological response of the tumor to treatment. For example, treatment with CAF therapy did not cause any changes in this parameter over a period of nearly 3 weeks, which indicates that the disease is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is illustrated by the following drawings:

[0027] Figure 1 Staining of breast tumors with ERα antibody is shown;

[0028] Visualization: Horseradish peroxidase + diaminobenzidine, magnification x100 (A). Specific staining of mammary ductal cell nuclei and individual tumor cell nuclei (B), magnification x400.

[0029] Figure 2 The mean volume of tumors in mice treated with CAFs at the start of the experiment is shown;

[0030] Figure 3 The mean total volume of tumors in mice treated with CAFs at the start of the experiment is shown;

[0031] Figure 4 Relative changes in the mean total volume of tumors in mice treated with CAFs at the start of the experiment are shown;

[0032] Figure 5 The relative changes in the mean total volume of tumors in mice when the antitumor activity of the drug was evaluated using doses of 20 mg / kg and 100 mg / kg are shown;

[0033] Figure 6 Shown are the relative changes in the mean total volume of tumors in mice when the antitumor activity of the drug was evaluated using a dose of 2.0 mg / kg. DETAILED DESCRIPTION

[0034] The present invention will be further described below by way of the embodiments disclosed herein.

[0035] Example 1

[0036] Synthesis of 3-hydroxy-7β-methyl-D-eno-6-oxaestra-1,3,5(10),8(9),8,14-pentaen-17a-one (5).

[0037] The ring-opened compound of formula 4 was prepared from 6.0 g of chromanone (3).

[0038] To a solution of the secosteroid of formula 4 in 100 ml of methanol, 6 ml of hydrochloric acid was added, and the resulting mixture was boiled for 10 hours. The cooled reaction mixture was poured into cold water, washed with 5% sodium bicarbonate solution, then washed with water to neutral pH, and dried over sodium sulfate. The solvent was distilled on a rotary evaporator. The resulting product had a melting point of 237-240°C.

[0039] NMR spectroscopy 1H(CDCl3), ppm: 7.08; 6.45; 6.39; 5.84; 5.30; 5.00, using (1H, C3-OH); 2.79; 2.74-2.40; 2.14, 1.69; 1.28; 1.24, using (3H).

[0040] NMR spectroscopy 13 С(DMSO-d 6 ),ppm:214.6;158.6,152.6(С-3,С-5),137.4;C);123.9,125.4;124.1 ;115.7;118.3;108.7;103.8;69.9;45.0;35.4;28.6;24.1;21.1;18.1.

[0041] 3-Hydroxy-7β-methyl-D-eno-6-oxaestra-1,3,5(10),8(9)-tetraen-17a-one (6) was obtained from 100 mg of estrapentaene. The reaction product was crystallized from methanol and dried. 76 mg (75%) of the compound were obtained, T>254°C.

[0042] NMR spectroscopy 1 H(DMSO-d 6 ),ppm:9.44, using (С3-ΟH);6.96;6.33,6.22;4.84,2.80;2.51;2.46,2.18;2.12;2.06;1.91;1.83;1.62;1.52;1.16,1.08, using (ЗH).

[0043] NMR spectroscopy 13 С(DMSO-d 6 ), ppm:214.4;

[0044] Thin layer chromatography (TLC) was used on Silufol UV254 produced by Kavalier and Alugram SIL G / UV254 plates produced by Machereynagel in a solvent system of hexane / petroleum ether 40-70-ethyl acetate in different ratios in order to check the progress of the reaction and the identity of the compounds. max These compounds were detected by adsorption of iodine vapor at 400 nm (λ = 254 nm) or by reaction with a methanolic solution of sulfuric acid (3:1) upon heating.

[0045] The compounds were purified by column chromatography using silica gel 60 (0.035-0.070 mm; 0.060-0.200 mm) purchased from Acros Organics.

[0046] On the Bruker DPX-300, for 1 H and 13 С were recorded at 400.130 and 100.613 MHz at 298 K in CDCl3 (unless otherwise stated) 1 H and 13 C NMR spectra. A solution of 3-5 mg of compound in 0.6 ml CDCl3 was used to obtain 1 1H NMR spectra. Obtained in the same volume of solution containing 20-50 mg of compound 13 C NMR spectrum. The chemical shift relative to tetramethylsilane was measured, and the distribution value of the solvent signal (СDСl3 / СHСl3=99.9 / 0.1) was 7.26 ppm ( 1 H) and 77.16ppm( 13 С).

[0047] Example 2

[0048] Studies on pharmacological activity.

[0049] Cell culture and culture conditions.

[0050] The studies were performed on cultures of MCF-7 human cell cultures (breast adenocarcinoma) at passage 1. Early passage normal human dermal fibroblasts (HDF) were used as negative controls.

[0051] Cells were cultured in Carrel flasks in DMEM / F12 medium (Biolot) supplemented with 1.0% antibiotic-free fetal calf serum (Biolot) at 37°C in a 5% carbon dioxide atmosphere.

[0052] Assessment of cell proliferation activity.

[0053] 50x10 per bottle 4 Cells were inoculated on Carrel flasks. To study the proliferation activity of cells under sulfatase inhibition conditions, 24 hours after inoculation, the culture medium was replaced with a culture medium containing a sulfatase inhibitor at a final concentration of 50 mg / ml, and then these tumor cell lines were incubated for different time periods (24 to 72 hours). The inhibitor was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the culture medium did not exceed 0.5%. In order to exclude the cytotoxic effect of DMSO, a control sample containing DMSO but not containing a sulfatase inhibitor was prepared.

[0054] In order to exclude the non-specific harmful effects of compound, normal human skin fibroblasts were used. Then Versene-trypsin solution (Biolot) was used to separate cells and placed on a Carrel bottle with fresh complete culture medium. When untreated control cells reached the maximum cell density (monolayer) per unit surface area of ​​culture bottle, cells were counted and their quantity was set to 100%. The proliferation activity of all studied cell cultures exposed to sulfatase inhibitors was measured in triplicate.

[0055] The study of the effect of the aminosulfonate on the proliferation of the human MCF-7 cell culture (breast adenocarcinoma) of passage shows that the steroid of formula 2 with a concentration of 20 mg / ml completely prevents the proliferation of tumor cells, but it does not affect the growth of human skin fibroblasts without estrogen receptors. The degree to which the proliferation of tumor cells is inhibited is the same as the effect of tamoxifen, which has been used clinically for more than 30 years. This is very important because aminosulfonate and tamoxifen have different mechanisms of action, which makes their combined use promising.

[0056] In experiments with HER-2 / neu (ER- / PR- / HER2+) transgenic FVB mice bearing breast tumors, the agent inhibited tumor proliferation more effectively than the clinically used tamoxifen, suggesting their potential as a combination treatment for breast cancer.

[0057] In the triple-negative breast cancer MDA-MB-231 cell line, the compound inhibited cell growth with an IC 50 =4.8μM, which is equivalent to the clinically used chemotherapy drug etoposide.

[0058] Example 3

[0059] This study was performed on conventionally stocked HER-2 / neu transgenic female FVB mice obtained from the mouse bank of Charles River Laboratories (Italy). At the start of the study, the animals were 21-41 weeks old and weighed 25-30 g.

[0060] Tamoxifen Hexal (Hexal AG, 83607 Holzkirchen, Germany, batch number HA1575, January 2017) was used in the experiments, which represents a pharmaceutical dosage form formulated as white or slightly yellowish coated round biconvex tablets with a uniform smooth surface containing 20 mg of active compound.

[0061] Housing conditions were selected according to the standards specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press, 1996).

[0062] Animals were housed in small groups (no more than 5 animals per group) in individual rooms in individual T2-type cages suitable for laboratory rodents. The cages had dimensions of 268 × 215 × 141 mm3 (bottom area of ​​370 cm2) and were equipped with a polycarbonate barrel, stainless steel lid with food container and water bottle divider.

[0063] The cage bedding is made of dust-free wood shavings.

[0064] The animals had unlimited access to a mixed complete pelleted diet for laboratory rodents.

[0065] Drinking water is available ad libitum from a standard 190 ml drinking bottle manufactured by TECNIPLAST, made of high temperature polysulfone with a silicone ring and a metal cap made of AISI 316 stainless steel.

[0066] The room temperature was maintained at 20-26°C and the relative humidity was 50-70%. The photoperiod was set at 12:12 hours night / day under artificial lighting with fluorescent lamps.

[0067] An identification card with the cage / card number, experimental group number, number of animals, their numbers and sexes, study number, and name of the responsible researcher was placed on the cage. The pinna of each animal was clamped with a small laboratory rodent metal tag made of nickel alloy from Kent-Scientific, USA (the tag had a three-digit number stamped by the manufacturer).

[0068] At the end of the experiment, mice were euthanized with carbon dioxide. All animal carcasses were dissected and then microscopically described.

[0069] The LD50 of the drug in HER-2 / neu transgenic FVB mice was determined using the OECD 423 test (OECD Guidelines for the Testing of Chemicals. Acute Oral Toxicity - Acute Toxicity Classification Method). 50 value.

[0070] Twelve female mice were used in the experiments.

[0071] In a first step, 3 mice were dosed with the drug at a dose of 300 mg / kg. The drug under study was administered intragastrically (ig) using a metal atraumatic gavage (this procedure corresponds to oral administration in a clinical setting). For intragastrically administering, the active compound of the drug was mixed with olive oil to prepare a suspension of the desired concentration for administration of 0.1 ml of suspension per 10 g of animal body weight. The dosage form for administration was prepared extemporaneously.

[0072] Since no animals died after administration at 300 mg / kg, the same dose was administered to another 3 animals. No mortality was found in either group. Therefore, further administration at 2000 mg / kg was performed in two steps, with 3 animals administered in each step.

[0073] Clinical observations were performed and body weights were recorded according to the protocol shown in Table 1. Animals were examined twice daily, with a separate clinical examination within the first 60 minutes after dosing, special attention within the first 4 hours, regular examinations within the first 24 hours, the next day, and then once a week for up to 14 days. The observer held the cage in his hand and performed a thorough examination of each animal. The general condition of the animals, such as their behavioral characteristics, the intensity and nature of motor activity, and the appearance of the coat and skin were recorded.

[0074] The body weight of the animals was recorded using a validated high-speed electronic laboratory balance, Ohaus Scout Pro (USA), with a maximum load of 2000 g and a measurement step size of 0.1 g.

[0075] On day 15, animals were euthanized and necropsy analysis was performed to record macroscopic changes. Data were collected on a dedicated individual necropsy form.

[0076] At the end of the study, all experimental animals were subjected to pathomorphological examination. The external pathological changes of the euthanized animals were carefully examined. The thoracic and abdominal cavities were examined, and macroscopic analysis of the internal organs was performed. No microscopic analysis of the organs and tissues of the experimental animals was performed, because no animal deaths were recorded during the entire observation period. In addition, macroscopic analysis of the internal organs did not reveal any pathological changes.

[0077] Table 1.

[0078] Protocol for observation and weighing of animals when assessing acute toxicity

[0079]

[0080]

[0081] The drug under study was administered by gavage using a metal non-invasive tube feeding method (this procedure corresponds to the oral route of administration in a clinical setting). The drug was administered at a single daily dose of 20 mg / kg body weight and 5 times the daily dose of 100 mg / kg body weight. The temporary solution of the medicament for administration was prepared with olive oil (refined olive oil with unrefined extra virgin olive oil added, trademarked as Global Village "Clasico", batch number L: 183351116, shelf life until April 26, 2020, BAIEO, Spain). The dosage was 0.1 ml per 10 grams of mouse body weight (0.2 ml of ready-to-use preparation per 20 grams of mouse). Administration began 24 hours after randomization. The course of administration was 27-28 days.

[0082] The reference drug, tamoxifen, was also administered via a metal non-invasive gavage (a procedure that corresponds to oral administration in a clinical setting) at a daily dose of 4.0 mg / kg, which was calculated based on clinical dosing [5]. This dose corresponds to a human daily dose of 20 mg / kg. The tamoxifen tablets were crushed in a pestle and the resulting powder was used to prepare a suspension in 40 ml of olive oil, administered in an amount of 0.08 ml per 10 g of mouse body weight, for the same duration as the experimental drug, for 27–28 days.

[0083] The control group was fed olive oil (placebo).

[0084] Evaluation criteria included clinical observation data, animal body weight, time of death (if applicable), tumor growth over time, and pathological morphological examination data (verification of tumors during autopsy, assessment of toxic effects by macroscopic manifestations of changes in internal organs).

[0085] Weight measurement

[0086] The body weight of the animals was recorded before the first dosing and then twice a week using a validated high-speed electronic laboratory balance Ohaus Scout Pro (USA) with a maximum load of 2000 g and a measurement step size of 0.1 g.

[0087] Tumor size measurement

[0088] During the weighing process, macroscopically detectable tumor nodules in the animals were measured once a week. Two linear dimensions were recorded for each node, including the largest dimension and the largest dimension at right angles to the former. The largest dimension was taken as the length of the tumor node (a), and the second dimension was taken as the width of the tumor node (b). Tumor volume was calculated as follows: V = (a × b)2 / 2.

[0089] The efficacy of treatment was evaluated by recording the change in mean tumor volume, tumor growth inhibition (TGI) and mean total tumor volume per mouse and their changes over time.

[0090] The percentage of tumor growth inhibition was calculated as follows: TGI = (V ctrl -V exp ) / V ctrl ×100(%), where V ctrl is the mean tumor volume of the control group, V exp is the mean tumor volume of the experimental groups.

[0091] The raw data of each table were transferred to a Microsoft Excel 2007 workbook. Group arithmetic means (M) and standard deviations (m) were calculated for all quantitative data. Statistical analysis was performed using the statistical software GraphPad Prism 6.0. Differences between groups were assessed using ANOVA regression analysis and Fisher's exact test.

[0092] When evaluating acute toxicity, the animals were observed continuously for the first 60 minutes, then checked every hour for 3 hours and again for 24 hours. They were observed twice on the second day. They were then observed once a day. After the administration of the active compound, each animal was clinically examined every other day and then once a week. The observer held the cage in his hand and examined the animal thoroughly. The general condition of the animals, such as their behavioral characteristics, the intensity and nature of motor activity, and the appearance of the fur and skin, was recorded.

[0093] During the experiment, no animal mortality was observed at the dose of 300 mg / kg. None of the animals showed visible signs of intoxication during the entire observation period. The animals looked and behaved as usual. When picked up by hand, the animals showed conventional weak reactions. No animals were observed to make any sounds during or shortly after dosing.

[0094] Daily monitoring of the animals' general condition and behavioral responses indicated that a single oral dose of the test drug did not affect the general condition and activity of the intoxicated animals.

[0095] Body weights are shown in Tables 2 and 3.

[0096] Table 2. Changes in animal body weight after administration of the initial dose of 300 mg / kg

[0097]

[0098] Table 3. Changes in animal body weight after administration of 2000 mg / kg

[0099]

[0100]

[0101] Starting from the date indicated in Table 2, the animals dosed at 300 mg / kg had a mean body weight decrease of 1% on day 2.

[0102] Since no animals died at the dose of 300 mg / kg, the dose was set to 2000 mg / kg. The dose was administered in two steps, 3 hours apart, with 0.1 ml per 10 g of body weight in each step, at a concentration of 100 mg / ml, because the suspension in oil is thicker at higher concentrations and cannot be administered by gavage. No clinical signs of intoxication were found after administration of 2000 mg / kg. The width of the palpebral fissures of the animals showed little change throughout the observation period, regardless of the group. No pathological discharge from the eyes was detected. The nose was pink, moderately moist, and without pathological discharge. The fur of all mice was neat and shiny, with no bald spots. The weight of the animals decreased by 1% from the initial body weight on day 2 and by 6% on day 7 (Table 3). By the end of the observation period, the average body weight did not differ from the initial body weight.

[0103] On day 15, the animals were euthanized and subsequently subjected to necropsy analysis. No changes in the internal organs were found upon macroscopic analysis.

[0104] The autopsy data are as follows:

[0105] The coat of the animal is neat and shiny, with no bald spots. The nutritional status of the animal is satisfactory.

[0106] There is no pathological secretion from the natural orifices.

[0107] The submandibular lymph nodes were round, light pink, and moderately dense. The salivary glands were normal in shape, light yellow, and moderately dense.

[0108] The peritoneum was smooth and shiny, with no free fluid in the cavity.

[0109] The spleen was not enlarged, dense, and had a smooth and shiny capsule. The pancreas was light pink and had a lobed structure.

[0110] The size and shape of the liver remain unchanged, the liver capsule is shiny, the liver tissue is brown and of medium density.

[0111] The kidneys are dense, with a smooth, shiny capsule and moderate adipose tissue outgrowth surrounding the kidneys.

[0112] The ovaries were not enlarged and had a shiny surface.

[0113] The stomach has a folded, shiny mucosa with a small amount of mucus and food in the cavity.

[0114] The pleura is smooth and shiny, and there is no free fluid in the pleural cavity. The thymus is triangular and white. The lungs are light pink and airy.

[0115] Therefore, according to the OECD 423 test, this drug can be classified in the fifth or unclassified category, and the LD 50 Equal to or greater than 5000 mg / kg, which corresponds to the category of low-hazard compounds defined in GOST 12.1.007-76.

[0116] Animals tolerated the administration of the drug satisfactorily, and no clinical signs of toxicity were observed. During the entire observation period, the weight of animals in each group was increasing, and the increase in weight may be attributed to the increase in the volume of tumor nodules in mice (Tables 4 and 5). The autopsy analysis at the end of the experiment did not reveal specific signs of toxic damage to internal organs.

[0117] Table 4. Changes in mean body weight of mice when evaluating the antitumor activity of the drug at doses of 20 mg / kg and 100 mg / kg (g)

[0118]

[0119]

[0120] Table 5. Changes in mean body weight of mice when evaluating the antitumor activity of the drug at a dose of 2.0 mg / kg (g)

[0121]

[0122] exist Figure 1 Table 6 shows the data of the average tumor volume changes over time in the control-1 group, the 20 mg / kg administration group, the 100 mg / kg administration group and the tamoxifen-1 group at the beginning of the experiment. Figure 2 Table 7 shows the data on the change in the average tumor volume over time in the control-3 group, the 2.0 mg / kg administration group, and the tamoxifen-2 group.

[0123] Table 6. Changes in mean tumor volume (cubic centimeters) at the start of the experiment when evaluating the antitumor activity of the drug at doses of 20.0 mg / kg and 100.0 mg / kg

[0124]

[0125] M is the mean, m is the mean error, and N is the number of tumors at the start of treatment included in the analysis. # is the control group of the first trial.

[0126] Table 7. Changes in mean tumor volume (cubic centimeters) at the start of the experiment when evaluating the antitumor activity of the drug at a dose of 2.0 mg / kg

[0127]

[0128]

[0129] Overall, it can be concluded that, similar to the reference drug tamoxifen at a dose of 4.0 mg / kg (Tamoxifen-1, Tamoxifen-2 groups), this drug had no significant antitumor effect in this model within the examined dose range (2.0, 20.0 and 100.0 mg / kg). For the drug at doses of 20 mg / kg and 100 mg / kg, the TGI was 10% and 28%, respectively, compared to control group 2 at the end of the observation period on day 28. Based on the data obtained ( Figure 1 , Table 6, Figure 2 , Table 7), on day 0 of the experiment, the average tumor volume in the mice of the 2.0 mg / kg dosing group and the tamoxifen-2 group was 3.4 times and 3.6 times larger than that of the control-2 group, respectively. Therefore, it seems inaccurate to compare the absolute values ​​of tumor volume across these groups, as this may affect the subsequent growth rate over time. Therefore, it is more accurate to evaluate the relative changes in these parameters (Table 11, Table 12). It should be noted that when evaluating the TGI of the tamoxifen-2 group, inhibition was observed from day 7 to day 21, and at the end of the experiment, the average volume of the tumor at the beginning of treatment exceeded this parameter in the control group by 31%, which may be related to the development of resistance of the tumor to the reference drug during its growth.

[0130] When analyzing the frequency of tumor stabilization and the development of new tumors throughout the observation period (Table 8), it should be noted that the trend determined at the end of the observation period was a decrease in the volume of new tumors. Thus, in the control-2 group, this parameter was 0.28 ± 0.09 cubic centimeters, in the 20 mg / kg administration group and the 100 mg / kg administration group, this parameter was 0.19 ± 0.04 cubic centimeters and 0.18 ± 0.04 cubic centimeters, respectively, which are 32% and 36% lower than the control (reference) values. The drug at a dose of 2.0 mg / kg had no effect on the frequency of stabilization or the volume of newly developed tumors.

[0131] Table 8. The frequency of stable tumors during the evaluation of the antitumor activity of the drugs and the number and average volume of new tumors that appeared at the end of the experiment

[0132]

[0133] The percentage (%) of disease stability is a parameter similar to the RECIST criteria, which reflects the frequency of tumors whose volume at the end of the observation period has not increased by more than 20% relative to the volume at the start of treatment.

[0134] When analyzing the changes in the mean total tumor volume ( Figure 3, Table 9, Figure 4 , Table 10) obtained similar data. In terms of TGI, the drug at doses of 20 and 100 mg / kg had antitumor effects of 12% and 30% at day 28, respectively.

[0135] Table 9. Average total tumor volume (cubic centimeters) in mice when evaluating the antitumor activity of the drug at doses of 20.0 mg / kg and 100.0 mg / kg

[0136]

[0137] Table 10. Average total tumor volume (cubic centimeters) in mice when evaluating the antitumor activity of the drug at a dose of 2.0 mg / kg

[0138]

[0139] When evaluating the relative changes in the total tumor volume of the mice, it was determined that this parameter was linear and increasing in the control groups (Control-2, Control-3). At doses of 20 and 100 mg / kg, this parameter was statistically significantly lower for all experimental groups ( Figure 5 , Table 11), which confirms the effect of the drug under study in regulating tumor growth, although it did not lead to a decrease in the total number of tumors. At a dose of 2.0 mg / kg, this parameter was greater than the control value, but it should be noted that this parameter in the tamoxifen-2 group also exceeded the value of the control-3 group.

[0140] Table 11. Relative changes (in %) in the mean total volume of tumors of mice when evaluating the antitumor activity of the drug at doses of 20 mg / kg and 100 mg / kg relative to day 1

[0141]

[0142] *, **, ***, respectively, p<0.05, p<0.01, p<0.001 compared with the control group.

[0143] Table 12. Relative changes (in %) in the mean total volume of tumors of mice when evaluating the antitumor activity of the drug at a dose of 2.0 mg / kg relative to day 1

[0144]

[0145]

[0146] ***p<0.001 compared with the control group.

[0147] For all analyzed data, the antitumor activity of this drug in the breast tumor model of HER-2\neu transgenic FBV mice was not inferior to that of the reference drug tamoxifen.

[0148] At the end of the observation period, the hormonal effects of the drugs on the uterine body were analyzed by comparing the weight of the uterus (excluding the uterine horns) (Table 13). No statistically significant differences were observed in this parameter for the drugs administered in the dose range of 2.0 to 100 mg / kg compared to the control group.

[0149] Table 13. Uterine body weight and body weight of mice bearing breast tumors during evaluation of antitumor activity of drugs

[0150]

[0151]

[0152] The drug's TGI, tested in a model of multiple mammary tumors in HER-2 / neu transgenic FBV / N female mice, was 10% and 28% for doses of 20 mg / kg and 100 mg / kg, respectively. This activity is similar to that of the reference drug tamoxifen (4.0 mg / kg), for which the TGI value was 5% at the end of the observation period.

[0153] The results showed that the drug had no uterotropic activity.

[0154] According to the OECD 423 test, this drug can be classified as Class V or non-classified, and its LD 50 Equal to or greater than 5000 mg / kg, which corresponds to the category of low-hazard compounds defined in GOST 12.1.007-76.

[0155] Industrial Applicability

[0156] In an animal model inoculated with triple-negative breast cancer human tumors, the compound showed better efficacy than the clinically used tamoxifen and aromatase inhibitor letrozole.

[0157] As a result of the experiments conducted, the following recommendations were formed for the use of this compound: monotherapy and adjuvant therapy of early hormone-positive breast cancer in postmenopausal women; treatment of triple-negative breast cancer; monotherapy and adjuvant therapy of breast cancer in postmenopausal women who have been treated with tamoxifen for 2-3 years; treatment of advanced breast cancer.

Claims

1. 3-O-sulfamoyloxy-7β-methyl-D-en-6-oxaestra-1,3,5(10),8(9)-tetraen-17a-one of formula (2), 2. Use of 3-oxo-sulfamoyloxy-7β-methyl-D-increased-6-oxaestra-1,3,5(10),8(9)-tetraen-17a-one of formula (2) in the preparation of an anticancer agent for the treatment of patients with breast cancer, 3. The use according to claim 2, wherein the patient is a female patient suffering from advanced breast cancer.

4. The use according to claim 2, wherein the breast cancer is a triple negative form of breast cancer.

5. The use according to claim 2, wherein the patient is a postmenopausal woman.

6. The use according to claim 5, wherein the patient has been treated with tamoxifen.

7. Use according to claim 6, wherein the treatment is monotherapy or adjunctive therapy.

8. The use according to claim 7, wherein the patient suffers from early stage breast cancer.

9. The use according to any one of claims 2 to 8, wherein the 3-oxo-sulfamoyloxy-7β-methyl-D-en-6-oxaestra-1,3,5(10),8(9)-tetraen-17a-one of formula (2) is for oral administration.

10. A method for preparing 3-oxygen-sulfamoyloxy-7β-methyl-D-en-6-oxaestra-1,3,5(10),8(9)-tetraene-17a-one of formula (2), the method comprising the following reaction:

11. The method according to claim 10, wherein the compound of formula (4) is obtained by reacting the chromanone of formula (3) with vinylmagnesium bromide and condensing the obtained vinylcarbinol with 2-methylcyclohexane-1,3-dione.

12. The method according to claim 10, wherein the cyclodehydration reaction of the compound of formula (4) is carried out in a methanol solution of hydrochloric acid to obtain the compound of formula (5).

13. The process of claim 10, wherein the estraene of formula (6) is prepared by hydrogenating the compound of formula (5) over palladium on carbon in ethanol.

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