EA042238B1 - APPLICATION OF 3-O-SULFAMOYLOXY-7β-METHYL-D-HOMO-6-OXAESTRA-1,3,5(10),8(9)-TETRAEN-17a-ONE FOR THE TREATMENT OF BREAST CANCER - Google Patents

Description

Technical field

The invention relates to the field of medicine and pharmaceutical industry and relates to agents for the treatment of breast cancer.

Prior Art

Breast cancer is the leading cancer in women [Parkin D.M., Bray F., Ferlay J., and Pisani P., CA Cancer J. Clin., 2005, vol. 55, p. 74-108]. According to WHO, there are 2.09 million patients in the world, and 627 thousand women die of breast cancer every year [http://www.who.int/news-room/fact-sheets/detail/cancer].

A significant part of the tumors of this localization progresses under the influence of estrogens [Yue W., Yager J.D., Wang J.-P., Jupe E.R., Santen R.J., Steroids, 2013, vol. 78, p. 161-170]. Immunohistochemical examination of breast cancer detects estrogen receptors (ER), progesterone (PR) and her2neu (herceptin sensitivity). A significant proportion of tumors contain estrogen, progesterone and/or HER2NEU 3+ receptors. If the tumor does not have receptors and is not sensitive to Herceptin (ERO, PRO, HER2NEU 0-1), it is considered as triple negative (ER-/PR-/HER-2-). It is one of the deadliest forms of breast cancer due to the lack of targets to inhibit its growth. To date, there are no drugs in the world to treat this form of cancer.

Estrogens circulate in the blood and accumulate in the tumor as sulfates that are unable to bind to estrogen receptors, but after being converted to free hormones, they activate tumor growth. Therefore, a promising line of treatment is based on the use of inhibitors that block the formation of this group of free hormones in the tumor.

Tamoxifen belongs to the group of drugs from the group of selective estrogen receptor modulators, which block the effects of estrogen on hormone-dependent tissues, including breast tissue. Tamoxifen is the standard hormone therapy for premenopausal and postmenopausal women. The most characteristic side effects that occur during the use of tamoxifen are: an increased risk of developing venous thrombosis, aggravation of the course of diseases of the cardiovascular system (including angina attacks), as well as the appearance of endometrial neoplasms (polyps and endometrial cancer), as well as uterine fibroids. In addition, tamoxifen is hepatotoxic. According to the biopharmaceutical classification system (BCS), developed by Gordon Amidon et al. in 1995, tamoxifen is assigned to the second class - the drug has low solubility and high permeability.

Aromatase inhibitors showed greater efficiency compared to tamoxifen, in particular letrozole and anastrozole, which belong to the first class, were used. They have high solubility and high permeability. Side effects of aromatase inhibitors are osteoporosis, hot flashes, headaches.

In addition, steroid sulfatase attracts attention in connection with the local interstitial formation of estrogens from the abundant pool of circulating estrone sulfate. Steroid sulfatase catalyzes the hydrolysis of estrone sulfate to estrone and DHEA sulfate to DHEA (Dibbelt l., Biol. Chem., Hoppe-Seyler, 1991, 372, 173-185; Stein C., J. Biol. Chem., 1989, 264, 13865-13872).

The best known steroid sulfatase inhibitor is EMATE - estrone sulfamate (Ahmed S., Curr. Med. Chem., 2002, vol 9, no. 2, p. 263-273). However, it has a significant drawback. Under the action of estrone sulfatase inhibitors, which have a sulfamate group in their composition, the enzyme is irreversibly deactivated with the release of a free ligand [Howarm N.M., Purohit A., Reed M.J. J. Med. Chem., 1994, vol. 37, p. 219-221]. In particular, estrone sulfamate inhibits estrone sulfatase, however, the release of free hormone leads to a strong uterotropic activity [Shields-Botella J. et al., J. Steroid Biochem. Mol. Biol, 2003, vol. 84, p. 327-335].

A number of estrone sulfatase inhibitors are protected in patent WO9933858 (A2) - 1999-07-08, where derivatives of the general formula can be used as estrone sulfatase inhibitors for the treatment or prevention of estrogen-dependent disorders such as breast cancer

These derivatives also have uterotropic activity.

Thus, when searching for new anticancer agents, it should be taken into account that the carrier of the sulfamate group of an estrone sulfatase inhibitor should not have hormonal activity.

The fulfillment of these conditions has led to an expansion of the scope of estrogen-based sulfamates due to their wide potential of action, in particular, in recurrent breast cancer [Shah R. Et al., Sulfatase inhibitors for recidivist breast cancer treatment: A chemical review, Eur. J. Med.

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Chem., 2016. vol. 114, p. 170-190]. In particular, 17a-substituted estradiol sulfamate derivatives are described.

The compound 3,17aβ-disulfamoyloxy-7β-methyl-D-homo-6-oxaestra-1,3,5(10),8,14-pentaene of the formula

as an inhibitor of the growth of breast cancer cells MCF-7 (RU 2619457, publ. 05/16/2017). This compound can be listed as the closest analogue.

Disclosure of invention

The objective of the present invention is to search for and develop an effective synthesis of compounds that can be used as anticancer agents in mono- and adjuvant therapy of breast cancer, including its most lethal form - triple-negative (ER-/PR-/HER- 2-).

The problem is solved by using the compound 3-O-sulfamoyloxy-7β-methyl-D-homo-6-oxaestra1,3,5(10),8(9)-tetraene (2), which inhibits estrone sulfatase,

and can be used as an anti-cancer agent in mono- and adjuvant therapy of breast cancer, including triple-negative form (ER-/PR-/HER-2-) of breast cancer.

The scheme for the synthesis of the target compound (2) is presented below.

Compound (3) was reacted with vinylmagnesium bromide, condensation of the resulting vinylcarbinol with 2-methylcyclohexane-1,3-dione led to the formation of seco compound (4). The cyclodehydration reaction of compound 4 was carried out in hydrochloric acid. Estratetraene 6 was obtained by hydrogenation over Pd/C in ethanol, 75% yield. Sulfamate 2 was obtained in 86% yield.

The resulting anticancer agent belongs to the 5th hazard class (LD50>5000 mg/kg). The data obtained make it possible to assess the doses for the study of antitumor activity as safe. Therapeutically effective doses of the drug are preferably from 0.1 to 20 mg.

Despite significant progress in clinical oncology, the surgical method plays a leading role in the treatment of patients with malignant neoplasms of the breast. However, many patients subsequently die. This means that these patients, along with the primary tumor, already have micrometastases, which, unfortunately, cannot always be detected using the most modern research methods. For the purpose of long-term suppression of such micrometastases, as a rule, adjuvant therapy is used - an auxiliary chemotherapy treatment that supplements surgical or radiation methods. Adjuvant therapy is mainly carried out for 5-10 years, which can lead to various side effects on other tissues and organs. The proposed connection provides such therapy with greater efficiency.

Mammary tumors (MA) in female FBV/N mice transgenic for HER-2/neu are adenocarcinomas characterized by a low content of estrogen receptors (ER) (Fig. 1) and the absence of progesterone receptors (PR). Two subtypes of adenocarcinomas can be distinguished, namely ER+, PR-, and ER-, PR-. Immunohistochemical study of ERa in breast tumors revealed single positively stained nuclei only in 10 out of 45 samples (Fig. 1).

The model is sensitive to the CAF (cyclophosphamide, adriamycin, 5-fluorouracil) regimen, with inhibition of tumor growth resulting in stabilization of the mean tumor volume (Fig. 2). Inhibition of tumor growth was 63% on day 14 and 46% on day 21 of the experiment.

Since animals may develop multiple tumors, it seems appropriate to analyze the average total volume of tumors in mice (Fig. 3) and its relative change to the beginning of the experiment (Fig. 4). The graph of the relative increase in tumor volume shows that there is a linear change in this indicator in the control group, which indicates the expediency of using this indicator to assess the biological response of the tumor to treatment. So, when exposed to the CAF scheme, there is no change in this indicator for almost 3 weeks, indicating stabilization of the disease. The rate of stabilization of individual tumors is about 60% by 21 days.

Brief description of the figures of the drawings

The invention is illustrated by the following figures:

fig. 1 - staining with antibodies to ERa of a breast tumor; visualization horseradish peroxidase + diaminobenzidine, uv. x100 (A). Specific staining of the nuclei of the cells of the duct of the mammary gland and the nuclei of single tumor cells (B), sw. x400;

fig. 2 - dynamics of the average volume of tumors present at the beginning of the study in mice treated with the CAF regimen;

fig. 3 - dynamics of the average total volume of tumors present at the beginning of the study in mice treated with the CAF regimen;

fig. 4 - relative change in the average total volume of tumors present at the beginning of the study in mice treated with the CAF regimen;

fig. 5 - relative change in the average total volume of tumors in mice when assessing the antitumor activity of the drug at doses of 20 and 100 mg/kg;

fig. 6 - relative change in the average total volume of tumors in mice when assessing the antitumor activity of the drug at a dose of 2 mg/kg.

The best example of the invention

The invention can be illustrated by the following examples of the invention.

Example 1

Synthesis of 3-Hydroxy-7β-methyl-D-homo-6-oxestra-1,3,5(10),8(9),8,14-pentaen-17a-one (5).

Seco compound 4 was obtained from 6 g of chromanone (3).

To a solution of secosteroid 4 in 100 ml of methanol was added 6 ml of hydrochloric acid, boiled for 10 h. The cooled reaction mixture was poured into chilled water, successively washed with 5% NaHCO ₃ solution, water until neutral, dried over Na ₂ SO ₄ . The solvent was distilled off on a rotary evaporator. Received the product with so pl. 237-240°C.

1H NMR spectrum (CDCl ₃ ), ppm: 7.08; 6.45; 6.39; 5.84; 5.30; 5.00 s (1H, C3-OH); 2.79; 2.74-2.40; 2.14, 1.69; 1.28; 1.24 s (3H).

¹³ C NMR spectrum (DMSO-d ⁶ ), ppm: 214.6; 158.6, 152.6 (C-3, C-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.

3-Hydroxy-7β-methyl-D-homo-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 MeOH and dried. Received 76 mg (75%) of the compound, T>254°C.

1H NMR spectrum, (DMSO-d ⁶ ), ppm: 9.44 s (C ₃ -OH); 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 s (3H).

¹³ C NMR spectrum (DMSO-d ⁶ ), ppm: 214.4; 158.1, 152.4; 128.2; 122.3; 116.4; 123.8; 108.5; 103.7; 70.5; 46.8; 45.4; 37.0; 29.0; 25.9; 22.3; 21.8; 19.6; 16.4.

The completeness of the reactions and the identity of the compounds were checked using thin layer chromatography (TLC) on Silufol UV254 plates from Kavalier and Alugram SIL G/UV254 from Machereynagel in hexane/petroleum ether 40-70 - ethyl acetate solvent systems in various ratios. Detection of substances was carried out in ultraviolet light (λmax=254 nm), by adsorption of iodine vapor or by reaction with sulfuric acid in methanol (3:1) when heated.

Silica gel 60 (0.035–0.070 mm; 0.060–0.200 mm) from Acros Organics was used to purify the substances by column chromatography.

1H and ^13C NMR spectra were recorded in CDCl3 (unless otherwise stated) on a Bruker DPX-300 instrument, respectively, at frequencies of 400.130 and 100.613 MHz for 1H and ^13C nuclei, respectively, at 298 K. For 1H NMR spectra, solutions of 3–5 mg of the substance in 0.6 ml of CDCl3, and for ¹³ C NMR - 20-50 mg in the same volume. Chemical shifts were measured with respect to tetramethylsilane by assigning the solvent signal (CDCl ₃ /CHCl ₃ =99.9/0.1) values: 7.26 ppm. (1H) and 77.16 ppm ( ^13C ).

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Example 2

Study of pharmacological activity.

Cell cultures and cultivation conditions.

The work was carried out on a continuous human cell culture MCF-7 (breast adenocarcinoma). Normal human dermal fibroblasts (HFC) from early passages were used as a negative control. Cells were cultured in Carrel flasks in DMEM/F12 medium (Biolot) supplemented with 1% fetal bovine serum (Biolot), without antibiotics, in 5% CO ₂ atmosphere, at 37°C.

Assessment of proliferative activity of cells.

Cells were planted on Carrel flasks at 50 x 10 ⁴ cells per flask. To study the proliferative activity of cells under conditions of suppression of sulfatase, 24 h after inoculation, the culture medium was replaced with a medium containing sulfatase inhibitors to a final concentration of 50 μg/ml, after which these tumor cell lines were incubated for various periods of time (from 24 to 72 h) . The inhibitor is dissolved in DMSO. The final concentration of DMSO in the culture medium did not exceed 0.5%. To exclude the cytotoxic effect of DMSO, a control was placed with the addition of DMSO without a sulfatase inhibitor. Normal human skin fibroblasts were used to exclude the nonspecific destructive effect of the substances. Next, the cells were removed with a solution of versenatrypsin (Biolot), scattered on Carrel flasks containing a new complete culture medium. Cells were counted when the untreated control cells reached the maximum cell density per unit area of the culture flask surface (monolayer), and their number was defined as 100%. The proliferative activity of all studied cell cultures under the influence of sulfatase inhibitors was determined at least three times.

The study of the effect of the obtained sulfamate on the growth of transplanted human cell culture MCF-7 (breast adenocarcinoma) showed that steroid 2 completely blocks the growth of tumor cells at a concentration of 20 μg/ml, while it does not affect the growth of human skin fibroblasts that do not have estrogen receptors . The growth of tumor cells is inhibited to the same extent as under the action of tamoxifen, which has been used in medical practice for more than 30 years. This is very important, since sulfamates and tamoxifen have different mechanisms of action, so there are prospects for their combined use.

In experiments on FVB mice transgenic for HER-2/neu (ER-/PR-/HER2+) with mammary tumors, the agent inhibits tumor growth comparable to tamoxifen used in clinical practice, which suggests their joint use for the treatment of breast cancer. glands.

In the triple negative breast cancer cell line MDA-MB-231, the compound inhibits cell growth, IC ₅₀ =4.8 μM, which is comparable to the clinically used chemotherapy drug etoposide.

Example 3

The study was carried out on female FVB mice transgenic for HER-2/neu of the conventional category, the line was obtained from the nursery of Charles River Laboratories (Italy). The age of the animals at the beginning of the study was 21-41 weeks, weight - 25-30 g.

For the study, the dosage form of the drug Tamoxifen Geksal (Geksal AG, Germany, 83607 Holzkirchen, series HA1575 dated 01/17) was used, which is a white or slightly yellowish coated tablet, round, biconvex, with a uniform smooth surface, containing 20 mg active substance.

Housing conditions meet the standards specified in The Guide for Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press, 1996).

Animals were kept in a separate room in groups (no more than 5 individuals) in individual cages for laboratory rodents type T2. Dimensions 268 x 215 x 141 mm (base area 370 cm ² ). Polycarbonate bathtub, stainless steel lid with feed bin, drinker divider.

The bedding is dust-free wood shavings.

Animals had unrestricted access to combined complete granular food for laboratory rodents.

Drinking water was given ad libitum in standard 190 ml drinkers manufactured by TECNIPLAST from high-temperature polysulfone with a silicone ring and a metal cap made of AISI 316 stainless steel.

The room temperature was maintained at 20-26°C, relative humidity - 50-70%. The photoperiod is set to 12 hours night - 12 hours day under artificial lighting with fluorescent lamps.

An identification card was installed on the cage, which indicated the number of the card/cage, the number of the experimental group, the individual numbers of animals, their number and gender, the study number, and the name of the head of the study. Each animal was placed on the auricle of a metal tap for small laboratory rodents made of nickel alloy Kent-4 042238

Scientific, USA (the taps have three-digit numbers stamped by the manufacturer).

At the end of the experiment, the mice were euthanized with carbon dioxide. All animal corpses were subjected to autopsy with a macroscopic description.

The LD50 of the drug was determined in mice of the FVB line transgenic for HER-2 / neu according to the test

OECD 423 (OECD guideline for testing of chemicals. Acute Oral Toxicity - Acute Toxic Class Method).

12 female mice were used.

At the first step, 3 mice were injected with the drug at a dose of 300 mg/kg. The study drug was administered intragastrically using a metal atraumatic probe (according to the oral route of administration in the clinic). For intragastric administration, the drug substance was mixed with olive oil to obtain a suspension of the required concentration for administration at the rate of 0.1 ml of suspension per 10 g of animal body weight. The dosage form for administration was prepared ex tempore.

Due to the absence of death of animals when the drug was administered at a dose of 300 mg/kg, the same dose level of the drug was used to administer 3 more animals. There was also no death of animals, so the drug was then administered at a dose of 2000 mg/kg in 2 steps for 3 animals.

Conducted clinical observation and registration of body weight in accordance with the table. 1. Animals were examined daily 2 times a day, clinically examined individually after dosing for the first 60 minutes, with special attention during the first 4 hours, periodically during the first 24 hours, on day 2 and weekly up to 14 days. A detailed examination of the animal was carried out in the cage, in the hands. The general condition of the animals was recorded: the features of their behavior, the intensity and nature of motor activity, the condition of the hair and skin.

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

On the 15th day, euthanasia and autopsy were performed with registration of macroscopic changes. The data were entered into a special individual autopsy form.

All experimental animals were subject to pathomorphological examination at the end of the study. After euthanasia, the animals were carefully examined for external pathological signs. A study of the state of the chest and abdominal cavities and a macroscopic examination of the internal organs were carried out. Microscopic analysis of organs and tissues of laboratory animals was not carried out, since the death of animals was not recorded during the entire observation period, in addition, macroscopic analysis of internal organs did not reveal pathological changes.

The study drug was administered intragastrically using a metal atraumatic probe (in accordance with the intended oral route of administration in the clinic). The drug was administered in a single daily dose (20 mg/kg of body weight) and a 5-fold daily dose of 100 mg/kg of body weight. For ex tempore administration, a solution of preparations in olive oil was prepared (refined olive oil with the addition of unrefined Extra Virgen brand Global Village Clasico series L: 183351116, expiration date until 26.04.2020, BAIEO, Spain). The injection volume was 0.1 ml per 10 g of mouse body weight (0.2 ml of the formulation for a 20 g mouse). Start of drug administration 24 hours after randomization. The course of administration was 27-28 days.

The reference drug, tamoxifen, was also administered using a metal atraumatic probe (according to the oral route of administration in the clinic), the daily dose was 4 mg/kg and was determined when recalculating clinical doses. This dose corresponds to a daily human dose of 20 mg/kg. A tamoxifen tablet was crushed in a mortar, a suspension was prepared from the resulting powder in 40 ml of olive oil, the injection volume was 0.08 ml per 10 g of mouse body weight, the duration of administration corresponded to the duration of administration of the experimental preparations and amounted to 27-28 days.

The control groups received olive oil (placebo).

The evaluation criteria were clinical observation data, animal body weight, time of death (if any), tumor growth in dynamics, pathomorphological examination data (verification of the neoplasm during autopsy, assessment of the toxic effect according to the macroscopic picture of changes in internal organs).

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Measurement of body weight.

The body weight of the animals was recorded before the first injection of drugs and then twice a week using a verified high-speed electronic laboratory balance Ohaus Scout Pro (USA), with a maximum load of 2000 g, a measurement step of 0.1 g.

Measurement of tumor size.

Once a week during weighing, macroscopically detectable tumor nodes were measured in animals. For each node, two linear dimensions were measured: the largest and the largest perpendicular to it. The largest size was taken as the length (a) and the second size as the width (b) of the tumor nodes. Tumor volume was calculated using the formula:

V=(axb)2/2,

The effectiveness of therapy was assessed by the change in the average tumor volume, tumor growth inhibition (TRO), the dynamics of the average total tumor volume in each mouse and its relative change.

The percentage of inhibition of tumor growth was calculated by the formula:

where VK is the average tumor volume in the control group, and VO is the average tumor volume in the experimental group.

Primary data from individual forms were transferred to Microsoft Excel 2007 books. For all quantitative data, the group arithmetic mean (M) and standard deviation (m) were calculated. Statistical analysis was carried out using the statistical program GraphPad Prism 6.0. Differences between groups were assessed using ANOVA regression analysis and Fisher's exact test.

When assessing acute toxicity, the animals were under continuous observation for the first 60 minutes, then they were examined hourly for 3 hours, then after 24 hours. On the second day, the observation was carried out twice a day. Then observations were made once a day. Clinical examination of each animal was carried out after the introduction of the substance, in a day, then weekly. A detailed examination of the animal was carried out in the cage, in the hands. The general condition of the animals was noted: the features of their behavior, physical activity, the condition of the hair and skin.

With the introduction of the drug at a dose of 300 mg/kg during the experiment, the death of animals was not recorded. Visually, no signs of intoxication were observed in any animal during the entire observation period. The appearance and behavior of the animals were normal. When picked up, the reaction was standard low. Vocalization in animals was not observed, including immediately during and immediately after administration.

Daily monitoring of the general condition of the animals, behavioral reactions showed that a single oral administration of the test object had no effect on the general condition and activity of the experimental animals subjected to intoxication.

Data on body weight are given in table. 2 and 3.

table 2

Dynamics of body weight of animals with the introduction of the drug at a starting dose of 300 mg/kg

: doi 300 [ 1 mgkg________________G ......1..........g Experience Grids ........i U ____ Ί; _7_ ί 1 series ί 03.05 2018 1 04.05.2018 > 16 05 2018 ; mouse Ml____1 27.5 g _27.6_ J8.0___ ⁱ 28.4 Mouse #2_________: 26.4 ΐ JO 27.0 J27.0 : ₍ mouse Ml '' 29.6 “ί 29.2 - at - ] 29.8 j । Episode 2 ~ | 15 05 201b 16 95 2018 G 21 05 2018 .. yyshEN i 26.0'J 26 5 i 27.4* f 29.3 _ _ 270* '25.2 25.6_ .......j ..........' 26.0___ * mouse #6 280* i '2 28.2 . G ’ 27U™ ’ Average (% pemhshoshch ( 27.4 _{ 27J (-1) 27.6(1) 1 . . 28.1(2*1 Detail acet o ί _____ ' 0 6

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Table 3

Dynamics of body weight of animals with the introduction of the drug at a dose of 2000 mg / kg _______

d "la 2000 Days of experience K1 1 1 2 5 7 j 14 j _ 1 chain _ _ mouse no.>1 30 05 201"[*10*201" ^t 05 00 2018 । 12 06 2018 '26.4 1 27.0 Y 25.7 1' 27.0 1 . ¹ . _to mshiLa2________ _t mouse No. 3, 2 series 1709 18 , 1809 18 J 2*09 IS 300918 j ___28.4___1 27.9 _ J_ ___24.7 ___j_______28.3 1 25.0 ΐ 25.5 | _ 243__ . 1 . 24.9 _ _i *009 18 ^D 011O18 i '0610201"" ! '1*102018' _; mouse number 4 _ „ । mouse __________ _μ mouse №6 _t Mean (“initial? 26.6 j 26.5 j 25.1 1 26.4 i _ 24.0 ? 23.2 23.1 j 25.6 '26.7 ’ ’ I ' 26.2 25.2 i *25.4 1 26.2; 26.1(-1) 24.7(-6} J 263 (O) i

Lethal effect (sho total) [ 0 6

As follows from the data in Table. 2 recorded a transient decrease in average body weight on the 2nd day after injection by 1% in animals that received the drug at a dose of 300 mg/kg.

Since there was no death of animals at a dose of 300 mg/kg, we switched to the administration of the drug at a dose of 2000 mg/kg. The drug was administered in 2 doses with an interval of 3 hours, in a volume of 0.1 ml per 10 g of body weight per dose at a concentration of 100 mg / ml. the suspension in oil at a higher concentration is thick, which does not allow the drug to be administered through a probe. With the introduction of a dose of 2000 mg / kg, the clinical picture of intoxication was not observed. The width of the palpebral fissure of animals, regardless of the group, practically did not change during the entire observation period. Pathological discharge from the eyes was not observed. The nose is pink, moderately moist, there were no pathological discharges. The coat of all mice was neat, shiny, without foci of baldness. There was a decrease in the average body weight in animals by 1% on the 2nd day and by 6% on the 7th day from the initial one (Table 3). By the end of the observation period, the average body weight did not differ from the initial value.

All animals subjected to planned euthanasia on the 15th day of the experiment underwent autopsy. Macroscopic examination revealed no abnormalities in the state of internal organs.

The autopsy data were as follows:

animal hair had a neat appearance, was shiny, without foci of baldness;

the fatness of the animals was satisfactory;

there were no pathological discharges from natural orifices;

submandibular lymph nodes were rounded, pale pink in color and moderately dense;

salivary glands of normal shape, pale yellow, moderate density;

the peritoneum is smooth, shiny, there is no free fluid in the cavity;

the spleen is not enlarged, dense, the capsule is smooth and shiny;

pancreas pale pink, lobed structure;

the size and shape of the liver are not changed, the liver capsule is shiny;

liver tissue had a brownish color and a moderately dense texture;

the kidneys are dense, the capsule is smooth, shiny, there is a moderate growth of fatty tissue around the kidneys;

the ovaries are not enlarged with a shiny surface;

stomach with a folded shiny mucous membrane, a small amount of mucus and food content in the lumen;

the pleura is smooth, shiny, there is no free fluid in the chest cavity;

thymus triangular, whitish;

light pink, airy lungs.

In accordance with the OECD 423 test, the drug can be assigned to the 5th or not classified category with an LD50 equal to or more than 5000 mg/kg, which corresponds to the class of low-hazard substances according to GOST 12.1.007-76.

Animals satisfactorily tolerated the administration of the drug, no clinical signs of toxicity were observed. The body weight of the animals increased during the entire observation period in all groups; the increase in body weight may be associated with an increase in the volume of tumor nodes in mice (Tables 4, 5). Autopsy at the end of the experiment revealed no specific signs of toxic damage to internal organs.

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Table 4

Dynamics of the average body weight of mice (g) when assessing the antitumor activity of the drug at doses of 20 and 100 mg/kg

Group Index Date (day of experience) Data 07/10/18(1) 16.07.18(7) 07/23/18 (14) 07/30/18 (21) 06.08.18 (28) Kv1grvl-2 M 27.5 27.2 27.6 28.6 30.1 <N=10) "1 0.5 0.5 0.5 0.7 0.8 20 m 26.8 27.4 27.7 28.8 mg/kg (N=10) m 0.5 0.6 0.6 0.8 0.9 100 m 27.2 27 3 2 1 27.8 28.8 mg/kg (N=10) m 0.4 0.6 About 0.6 0.8 Tamoxifen-!. 06/27/2018 02.07.2018 07/09/2018 16.07.18 07/23/18 4 mg/kg (N= 10) date (1) (6) (S3) (20) (27) M 27.7 26.5 27 3 27.5 28.2 »...................................... ...............« 0.8 0.9 0.9 1.0

Table 5

Dynamics of the average body weight of mice (g) when assessing the antitumor activity of the drug at a dose of 2 mg/kg

Group Index D<pa(smkponshl) date 09/18/18(1) 24 09 18(7) 0! 10 18(14) 08 1018(21) 15.10.18(28) Kshproz-Z M 31.4 3Ϊ.8 35.7 35.8 36.7 (N=TO) m 0.9 And 1.2 1.4 1.5 AND" ..... 08/21/18(1) 27(17 18(7) (BtW 1.4(14) 10.09.18(21) 17.09.18(28) 2 m 30.5 30.8 32.7 32.9 35.3 mgLkg(№«) m 1.0 GO 1.0 1.0 1.2 Tamoxifen" m zgo 32.0 32.9 32.8 34.2 2.4 mg/kg (N=9) m 1.1 1.3 1.2 1.3 1.4

Data on the dynamics of the average volume of tumors available at the beginning of the experiment in groups Control-1, drug 20 mg/kg, drug 100 mg/kg and Tamoxifen-1 are shown in Fig. 1, in table. 6, and for groups Control-3, the drug 2 mg/kg and Tamoxifen-2 - in Fig. 2, in table. 7.

Table 6

Dynamics of the average volume of tumors (cm ³ ), available at the beginning of the experiment, when assessing the antitumor activity of the drug at doses of 20 and 100 mg/kg

Group Pokatagep. 07/09/18 (0) 07/16/18 (7) 07/23/18 C 4) 07/30/18 (21) 06.08.18 (28) %isheneshsh | w ref 1 Counter "u-2 (No. 23) M 0.08 0.13 0.28 0.43 0.61 663 1 sh 0.01 0.02 0.04 0.05 0.08 20 mrkg (№22) m 0.18 0.28 0.43 0.54 0.55 206 1 ...........1 sh 0.04 0.06 0.09 0.14 0.14 tgo.% -125 -115 -54 -26 10 too ww(#22) m 0.13 0.19 .......<U1..... 0.37 0.44 238 1 f | m 0.03 0.04 0.06 0.06 0.06 SRW, % -6.3 -46 4 14 28 Tamoxif- 1. 4 mg/kg 4 (#29) date 25.06.201 8(-1) 02.07.201 ..............8.(6)............ 09.07.201 8(13) 07/16/18 (20) 07/23/18 (27) - M 0.23 0.26 0.3 0.47 0.58 152 m 0.07 0.07 0.07 0.1 0.11 SRW, % •188 -100 -7 -9 5

M is the mean value, m is the error of the arithmetic mean, N is the number of tumors at the beginning of treatment and included in the analysis. # - the comparison group is from the first series of experiments.

Table 7

Dynamics of the average volume of tumors (cm ³ ), available at the beginning of the experiment, when assessing the antitumor activity of the drug at a dose of 2 mg/kg

Grisha Pokashgel 09/17/18 (ABOUT) 24.09 18 (7) 01.10.18 (14) 08.10.18 (21) 15 10.18 (28) % smssnnya from ref. Ko1pr "b-3 M 0.38 0.5 0.7 ............AND.............. 0.97 155 (#60) m 0.05 0.06 0.08 0.1 0.Ϊ I date 20.08.18 27.08 18 030918 10.09.18 09/17/18 - T (7) (14) (21) (28) 2 m 0.27 0.42 0.6 0.77 1.11 311 w kg sh (HH 0.06 0.07 0.09 0.13 (N 48) GRO.% 29 16 14 9 -14 Tamoxnfen-2. m 0.29 0.46 0.65 0.75 1.27 33S 4 mt kg m 0.04 0.07 0.08 0.09 0.16 (#39) IF) % 24 8 7 12 -31

In general, we can conclude that the drug in the studied dose range (2, 20 and

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100 mg/kg) and the reference drug Tamoxifen at a dose of 4 mg/kg (groups Tamoxifen-1, Tamoxifen-2) showed a pronounced antitumor effect on this model. For the drug at doses of 20 and 100 mg/kg, the TPO index by the end of observation on day 28 was 10 and 28%, respectively, in relation to the Control-2 group. As follows from the data presented (Fig. 1, Table 6, Fig. 2, Table 7), the average tumor volumes in mice of the drug groups 2 mg/kg and Tamoxifen-2 were 3 times higher than in the Control-2 group. 4 and 3.6 times, respectively, on day 0 of the experiment. Therefore, a comparison of the absolute values of the tumor volumes of these groups does not seem correct, since this could affect the subsequent growth dynamics, so it is more correct to assess the relative values of changes in these parameters (Table 11, Table 12). It should be noted that when assessing the TPO parameter in the Tamoxifen-2 group, inhibition was observed on days 7-21, and at the end of the experiment, the average volume of tumors present at the beginning of treatment exceeded this indicator in the control group by 31%, which may be due to the development of resistance. tumors to the reference drug as they grow.

When analyzing the frequency of stabilization of tumors and the development of new ones during the observation period (Table 8), it should be noted that there was a trend towards a smaller volume of new tumors, determined at the end of the observation period, so in the Control-2 group this indicator was 0.28 ± 0, 09 cm ³ , and in the groups 20 mg/kg drug and 100 mg/kg drug - 0.19±0.04 cm ³ and 0.18±0.04 cm ³ , which is 32 and 36% less than the control value, respectively . The drug at a dose of 2 mg/kg did not affect either the frequency of stabilization or the volume of developing new tumors.

Table 8 Frequency of tumor stabilizations, number and average volume of new tumors that developed by the end of the study, when assessing the antitumor activity of the drug

Group The number of tumors at the beginning of treatment and the frequency of their stabilization .Absolute number of new swollen The average volume of new οπνχο "by the end nlbdkgleipya, (eat') Koitrat-2 23 4% 22 M_____ m M___t m~~ m 0.28____ О.ОО” / \ “ 0.19 ................ 0.04~ 0.18 0.04 20 mi kg 22 9% 22 KYumVkg 22 9% 19 Tamoxifen-L 4 mg/kg 29 10% 21 m 0.16 sh 0.05 Control-3 60 8% 25 m 0.23 m 0.04 2 mg/kg 41 5% thirty 0.33 m 0.05 Tamokshfen-1 4 mg/kg 39 3% 32 m 0.20 m 0.05

% stabilization of the disease - an indicator similar to the RECIST criterion, reflecting the frequency of tumors, the volume of which has not increased by more than 20% by the end of observation in relation to the start of treatment.

When analyzing the dynamics of the average total volume of tumors (Fig. 3, Table 9, Fig. 4, Table 10), similar data were obtained; % and 30%, respectively.

Table 9

Mean total volume of tumors (cm ³ ) in mice when evaluating antitumor

drug activity at doses of 20 and 100 mg/kg Grint I Irnra wen 24 09 18 01 10 IS 08 10 IS B 10 IS » blue , YU) . ABOUT .R ⁴ » . CPU ,.(28) "tn ΚοΗΐραιι.-2 M 0 1*1 0 20 ΟΧή 0.991W 632 (#10) m 0 0* 0 0' 0 1* 021'(Η) ' 20 m 041 062 0 94 1.18s 1.22 108 mgkg sh 0.1 ¹ 0.15 0 2' 0.34! 0.34 (No. Yu) 1I) % -By ' ^li4 -45 -19 12 100 m 0 28 0.41 0 59 0 81 0.9'" 246 mg kg m 007 , Oh Mr. *021 028 .0 29 (#10) IPO. % 47 | -41 18 I 30 Tamoxifen-1. 2*06201 , 02 07 201 '09 (D 201 Ι607.Ϊ8 < 23 07 18 4 mg/kg * date . "LU , *4*3 . 8(13) (20) , (27) (#10) M 0.66 i 0.76 1 0.88 1 38 < 1.68 '155 m .0.17 j 0.18 j 0.28 0.37 1 0.41 ί GRO. % . -247 1-162 '45 -39 j -21 ί

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Table 10

The average total volume of tumors (cm ³ ) in mice when assessing the antitumor activity of the drug at a dose of 2 mg/kg

1 pvnita Noi.ne ιι> J 09/17/18 24 04 IX 01 10 IX 0X 10IX ' and io lx °<> (imputation I(0) (14) (21) , < ^2S . from fur M 12 26 , 4.02 4.G 4 (G 5.80 15' (N- IP m 0 tb 0 46 0 41 0 74 Dove" | 20OX IX 2-mg 1x (14 O*) | X KN" IX 1709IX G But" . OD j2h m [ 124 191 7| ; * 5 04 mg'kg m • 0 20 0 2' 0.52 O4< About 50 (#9) PC "o" ' 4' I7 d .35 31 IN Tamoxifen-?, m G 1 26 2 P1 ₍ 2X4 -3 AZ* 4 GC 4 mg kg* m . 0 22 0 V'" 0.49 0 55 1.05 (#9) 1141% . -m LA . 42 36 G h

When evaluating the relative change in the total volume of tumors in mice, it was found that this indicator in the control groups (Control-2, Control-3) changes linearly and constantly increases. In the experimental groups, when the drug was administered at doses of 20 and 100 mg/kg, this indicator was statistically significantly lower (Fig. 5, Table 11), which indicates the effect of the studied drugs on tumor growth, although this did not lead to a decrease in absolute values tumor volumes. With the introduction of the drug at a dose of 2 mg/kg, this indicator was higher than the control values, but it should be noted that in the Tamoxifen-2 group this indicator exceeded the value in the Control-3 group.

Table 11 Relative change in the average total volume of tumors to - 1 day (%) in mice when assessing the antitumor activity of the drug at doses of 20 and 100 mg/kg

Group Bye >aic 1b G 09 18(0) 2409 IXp) . 01 Hl lx (14) , ox 10 18(21) 15 10 IX (28) Kon ιρο 11,-2 M I 0 2Ί i 46? i 6X4 Shch 101 § in < 0 101 ; 130 2o j M § υ | 209" (65?AW MG CG 10) g m 10 = 4 12" ϋ 46 149 100 M ! ⁰ i 42 i i ♦ "* 1269*** ml kg (N-K0 i m I 0 1 20 1 29 i 45 1am<jsifen- i . 25O6 2OIX , 02 07 201X16) <»07 2018 113) 16 07 1X120) 2.4 07 18(27) 1 4 hg kg ~ 1 date μ η (x10) [M ⁰ '40 | SO** 1 161 *** _ ___ _ TO - i AND ^1u_ J and _ J_44 _ _ _ _ '4 _ _ __

*, **, *** - p<0.05, p<0.01, p<0.001, respectively, compared with the control group.

Table 12

Relative change in the average total volume of tumors to - 1 day (%) in mice when assessing the antitumor activity of the drug at a dose of 2 mg/kg.

Ddv.....,.............. pokamom 17 09 IS NO Eat wisp) 01 10 IS(14) OX 10IS (21) 1 Kwipo"-3 M Ιό 4SS ^! ! I. (N=io) m 1 about I 6 1 14 ίAA 24 Date of............. ^ΐIs <11 JO' iSj'L To 4 09 IX_£14) _t 1009 VSC) ' Ι10ΐ Μ24_ 2 m ! th 1 59 DP 210 t*p"*" MG CG 10 P h* 1 20 4X i 53 * There "zhspfen-2. m Ϊ in 141 rx 4μγκγ'1Ν 9) m 10 , IN I 21 i ⁴¹ £66

*** p<0.001, respectively, compared with the control group.

According to all analyzed indicators, the antitumor activity in the model of breast tumors in FBV mice transgenic for HER-2\neu of the drug is not inferior to the reference drug tamoxifen.

An analysis of the hormonal effects of the drug in relation to the uterus of the body was carried out, comparing the body weight of this organ without horns at the end of the observation period (Table 13).

There were no statistically significant differences in this indicator in relation to the control group in the drug in the dose range from 2 to 100 mg/kg.

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Table 13

Body weight of the uterus and body weight of mice with mammary tumors in the study of the antitumor activity of the drug

grupia Body weight of the uterus (mg) Estrous pict phase (number of mice) Body mass (G) Contrkhi -2 __ — ~D I , ~ 30.2*0.9,___ . .... . 53*6 All 30.1*0.8 Tamoxifen-2, 4 mgkg ™2"" mp'kg 20 mp kg 28*1 E-9 34.2*1.4 D-0 ....i 2fel 53*3'' uy__' _ __ ~37*6' 4>7v ’ ’ All ( 34.2 * 1.4 E 4 '' 35 / G2 * ~ D - 5_____ ~ ₎ 3 ^s 1Н5____ / All ' - E-6 ' 29 6^-1.5 D 4 '' 7 27.6t0.2 ' 7 50*4 All 28.8*0.9 | . 100 56*5 E-8 28.9*4.0i 34*13..........~...... ..... ........ ™ rt! st 28.5*0.4 1 All J 20*0.8 j

The effectiveness of the drug in the model of multiple breast tumors in FBV/N female mice transgenic for HER-2/neu in terms of TPO was 10 and 28% for doses of 20 and 100 mg/kg, respectively. This activity is higher than the effect of the reference drug tamoxifen (4 mg/kg), for which the TPO index at the end of the observation period was 5%.

It is shown that the drug has no uterotropic activity.

In accordance with the OECD 423 test, the drug can be assigned to the 5th or not classified category with an LD50 equal to or more than 5000 mg/kg, which corresponds to the class of low-hazard substances according to GOST 12.1.007-76.

Industrial Applicability

In animal models of human tumor-grafted triple-negative breast cancer, the drug showed higher activity than clinically used tamoxifen and the aromatase inhibitor letrozole.

As a result of the studies, the following recommendations for use have been established: mono- and adjuvant therapy for early hormone-positive breast cancer in postmenopausal women;

treatment of triple negative breast cancer;

mono- and adjuvant therapy of breast cancer in postmenopausal women after 2-3 years of tamoxifen use;

treatment of advanced advanced breast cancer.

Claims (1)

CLAIM Application of 3-O-sulfamoyloxy-7β-methyl-O-homo-6-oxaestra-1,3,5(10),8(9)-tetraen- 17a-one formula as an anti-cancer agent in mono- and adjuvant therapy of breast cancer, including triple negative form.