阅读说明：本技术 3-o-氨基磺酸-16,16-二甲基-d-高马萘雌甾酮在治疗肿瘤疾病中的用途 (Use of 3-O-sulfamic acid-16, 16-dimethyl-D-homonaphthalenestrone for the treatment of tumor diseases ) 是由 沙米尔·肖诺维奇·伊利亚索夫 亚历山大·格里戈里耶维奇·沙夫瓦 斯韦特兰娜·尼古拉耶芙娜·莫罗兹 于 2019-09-19 设计创作，主要内容包括：本发明涉及医药领域以及化学和药理学行业,并且涉及一种用于治疗癌症的药剂。本发明提出了在诸如肝细胞癌、胃癌、肺癌、慢性髓细胞性白血病和乳腺癌(包括三阴性的乳腺癌)的肿瘤疾病的单一疗法和辅助疗法中将3-O-氨基磺酸16,16-二甲基-D-高马萘雌甾酮作为抗癌剂。(The present invention relates to the field of medicine and the chemical and pharmacological industries and to an agent for the treatment of cancer. The present invention proposes 3-O-sulfamic acid 16, 16-dimethyl-D-homoequilenin as an anticancer agent in monotherapy and adjuvant therapy of neoplastic diseases such as hepatocellular carcinoma, gastric cancer, lung cancer, chronic myelogenous leukemia, and breast cancer (including triple negative breast cancer).)

1. Use of 3-O-sulfamic acid-16, 16-dimethyl-D-homoequilenin of formula (3) as anticancer agent in monotherapy and adjuvant therapy of tumor diseases such as hepatocellular carcinoma, gastric cancer, lung cancer, chronic myelogenous leukemia, breast cancer, triple negative form of breast cancer

Technical Field

The present invention relates to the field of medicine and the chemical and pharmaceutical industries and relates to a medicament for the treatment of cancer, including breast cancer.

Background

Breast Cancer is a major neoplastic disease in women [ Parkin d.m., Bray f., Ferlay j., Pisani p., CA Cancer j.clin., 2005, volume 55, pages 74-108 ]. According to the world health organization data, 209 million patients are present worldwide, and 62.7 million women are dying each year in association with breast cancer [ http:// www.who.int/news-room/fact-sheets/detail/cancer ].

A large proportion of the tumors that appear at this location develop under the influence of estrogen [ Yue w., Yager j.d., Wang j.p., Jupe e.r., Santen r.j., Steroids, 2013, volume 78, page 161-170 ]. Immunohistochemical analysis indicated that breast cancer expressed Estrogen Receptor (ER), Progesterone Receptor (PR) and HER2NEU (rendering the cancer "herceptin sensitive"). A large fraction of tumors have ER, PR and/or HER2NEU 3 +. A tumor is considered to be a triple negative form (ER-/PR-/HER-2-) if it lacks both ER and PR and is insensitive to herceptin (ER0, PR0, HER2NEU 0-1). This is one of the most lethal forms of breast cancer, as it has no target to inhibit its growth. Currently, there is no drug in the world that treats this form of cancer.

Estrogens circulate in the blood and accumulate in tumors as sulfates, which do not bind to estrogen receptors, but after conversion to free hormones, they activate tumor growth. Thus, one promising strategy for treatment involves the use of inhibitors to block the formation of free hormones in this group in the tumor.

Tamoxifen is a drug from selective estrogen receptor modulators that blocks the effects of estrogen on hormone-dependent tissues, including breast tissue. Tamoxifen is the standard of hormone therapy for pre-and post-menopausal women. The most typical side effects that occur during tamoxifen use include: increased risk of venous thrombosis, exacerbation of the course of cardiovascular disease (including angina attacks), development of endometrial tumors (polyps and endometrial carcinomas), 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 into the second class, i.e., drugs with low solubility and high permeability.

Aromatase inhibitors have been shown to be more effective than tamoxifen. Currently, aromatase inhibitors such as letrozole and anastrozole are already in use in the art. According to the BCS system, they fall into the first category, i.e., highly soluble and highly permeable drugs. Side effects of aromatase inhibitors include osteoporosis, hot flashes and headaches.

In addition to the above drugs, steroid sulfatases are also a subject of great interest because the abundant circulating estrone sulfate pool (pool) forms estrogens in the local interstitium. Steroid sulfatase catalyzes the hydrolysis of estrone sulfate to estrone and the hydrolysis of DHEA sulfate to DHEA (Dibbelt I., biol. chem., Hopeller, 1991, vol 372, p 173-185; Stein C., J.biol. chem., 1989, vol 264, p 13865-13872).

The most well-known steroid sulfatase inhibitors are EMATE-estrone sulfamate (Ahmed s., curr. med. chem., 2002, vol. 9, No. 2, p. 263-273). However, it has a significant disadvantage. Estrone sulfatase inhibitors containing sulfamate groups lead to irreversible enzyme inactivation with the release of free ligand [ Howarth N.M., Purohit A., Reed M.J., J.Med.chem., 1994, Vol.37, p.219-221 ]. In particular, estrone sulfamate inhibits estrone sulfatase, but the release of free hormone leads to the emergence of strong uterotrophic activity (uterotropic) [ Shields-Botella j. et al, j. stemoid biochem. mol. biol., 2003, vol.84, p. 327-335 ].

Therefore, in the search for new anti-cancer drugs, it is considered that the vehicle for the sulfamate group of the estrone sulfatase inhibitor should not have hormonal (uterotrophic) activity.

It is known that D-equilenin, which has no carcinogenic properties, is used as a drug for preventing breast cancer. Preparations having a cholesterol-lowering effect but not having uterotropic and hypertriglyceridemic activity are prepared from equilenin derivatives [ Urusova E.A., Gluzdikov I.A., Selivanov S.I., Starova G.L., Nikolaev S.V., Shavva A.G., "Synthesis and study of equivalent derivatives and its modified analogues", Russ.J.org.chem., 2004, Vol.40, No. 4, p.506-512 ].

Malassestrone compounds as medicaments for hormone replacement therapyThe composition of (1). These properties are desirable because the use of steroidal estrogen metabolism inhibitors means their long-term use.

Disclosure of Invention

The technical problem underlying the present invention was to provide compounds which can be used as anticancer agents in monotherapy and in adjunctive therapy for tumor diseases such as hepatocellular carcinoma, gastric cancer, lung cancer, chronic myelogenous leukemia and breast cancer, including the most lethal forms thereof, e.g. triple negative breast cancer (ER-/PR-/HER-2-), and to develop a highly efficient method for the synthesis of these compounds.

This problem is solved by using the compound 3-O-sulfamic acid-16, 16-dimethyl-D-homoequilenin of formula (3) which inhibits estrone sulfatase.

The scheme for the synthesis of the target compound of formula (3) is shown below. A significant advantage of the proposed solution compared to the prior art is that it allows to scale up the production of the target compound (drug) to industrial scale.

The reaction of known isothiourea salts [ Al Safar, Zakharov A.V., Ananchenko S.N., Torgov I.V., Synthesis of sodium 16,16-dimethyl-D-homosteroids, Reports of Academy of Sciences of the USSR.Ser.chem., 1968, 10 th, p.2326-2332 ] with 2,4, 4-trimethylcyclopentane-1, 3-dione leads to the formation of ring-opened steroids whose cyclodehydration leads to the pentacene estrol nucleus of formula (1). Pentacene estrenol nucleus [ Gluzdikov i.a., Egorov m.s., Selivanov s.i., Starova g.l., Shavva a.g., New analoges of D-homoequilene with subsistents in the D ring, rus.j.org.chem., 2006, volume 42, phase 11, formula 1682 ] was treated with Ni/Ra under conditions previously proposed to synthesize structurally similar D-homoanalogs (homoanaloges) [ glumakov i.a., egov m.s., Selivanov s.i., Starova g g.l., shavva.g., New analoges of D-homoequilene with, russ.j.org.chem., 2006, volume 42, phase 11, formula 1682 ], and the methyl ester of 16.16-dimethyl-D-homonaphthalenestrone of (2) [ Gluzdikov i.a., besseh. chem. thesis, 2007, saint burg ]. Sulfamates of formula (3) [ Morozkina s.n., Gluzdikov i.a., Drozdov a.s., Selivanov s.i., kovav r.a., fillatov m.v., Shavva a.g., russ.j.org.chem., 2015, vol 51, are obtained according to well-known methods. Stage 3, page 425-430 ].

Results from steroid binding simulations indicate that the structure of this steroid is poorly compatible with the geometry of the ligand-bound receptor pocket. The distance between the oxygen in the steroid ketone group and the NH of His524 isThis hinders the formation of hydrogen bonds. The distance between the hydroxyl group at Czochralski and the oxygen atom in the carboxyl group Glu353 is calculated to be aboutFar greater than that in the complex containing the natural hormone estradiol

Incompatibility between the geometry of the steroid and the receptor domain responsible for ligand binding can result in low affinity of the target compound for the receptor, rendering the steroid devoid of uterotrophic activity.

The compound completely inhibits the proliferation of tumor MCF-7 cells.

This drug belongs to the fourth hazard class (4)^th hazard class)(LD₅₀>300-2000 mg/kg). The data obtained allow the determination of the dose used for the study of antitumor activity as a safe dose.

Studies of binding to estrogen receptors showed that sulfamates were devoid of hormonal activity at concentrations of-0.001 and 0.01, 0.1 and 1.0 μmol/ml.

The proposed compound (drug) is more effective than tamoxifen and the aromatase inhibitor letrozole used in clinical practice in animal models with triple negative breast cancer xenograft human tumors.

Thus, the compounds are useful in the field of breast cancer therapy, including triple negative forms of breast cancer.

In parallel comparison with the antineoplastic drug tamoxifen (control drug), preclinical studies of specific activity in breast cancer models were performed. In vivo drug comparison studies were performed on female Balb/c nude mice with subcutaneously (s.c.) transplanted tumor cells. The experiment lasted 29 days after the first dose (dosing started on day 5 after tumor strain implantation).

The drug and control drugs were administered intragastrically at doses of 30mg/kg and 10mg/kg daily for 21 days.

The placebo group was gavaged with a 1% starch solution on the same schedule and at equal doses. Tumor nodules were measured 2 times per week. Antitumor activity was assessed by a standard Tumor Growth Inhibition (TGI) index. After the treatment was completed, the last tumor measurement was performed, and the animals were weighed and euthanized.

All necessary operations on the animals (testing, weighing, measuring and animal necropsy) were performed from 9 to 12 am.

Animals were weighed on the first day of the experiment and then weighed 2 times per week throughout the experiment according to the study protocol.

Tumor nodules were measured 2 times per week after tumor implantation throughout the course of the experiment. Tumor nodule volume was determined as follows:

v ═ pi/6 × L × W × H, where L, W, H is the linear size of the tumor.

Determination of antitumor Activity

Tumor growth inhibition index (TGI) was used as a criterion for studying the efficacy of drugs and was calculated by the following formula:

TGI(％)＝|V_control–V_{Experiment of}|/V_Control*100

Wherein V_ControlAnd V_{Experiment of}Mean tumor volume (mm) in control and experimental groups, respectively³)。

After the treatment period, animals were euthanized by cervical dislocation.

Statistical data processing was performed using the SPSS 21 software package (license number 20130626-3). Descriptive statistics in the study included the following: arithmetic mean, Standard Deviation (SD), standard mean error (SE), median, quartile, interquartile range. To compare the quantitative characteristics of the groups, the Mann-Whitney-Wilcoxon test was used without correction for multiple comparisons. The results were considered statistically significant (p < 0.05).

Mammary tumors (BT) of HER-2/neu transgenic female FBV/N mice are adenocarcinoma characterized by low Estrogen Receptor (ER) levels (FIG. 1) and Progesterone Receptor (PR) deficiency. Two adenocarcinoma subtypes can be distinguished, in particular ER +, PR-and ER-, PR- [2 ]. Immunohistochemical analysis of era in breast tumors showed only 10 specimens with a single positive staining of the nuclei in 45 specimens (fig. 1).

This model was sensitive to cyclophosphamide, doxorubicin, 5-fluorouracil (CAF) treatment and tumor growth inhibition was observed, resulting in a stable mean tumor volume (fig. 2). Tumor Growth Inhibition (TGI) was 63% and 46% at day 14 and day 21 of the experiment, respectively.

Since animals may have multiple tumors, it is reasonable to analyze the average total tumor volume (fig. 3) of the mice and the relative change in their tumor volume (fig. 4) at the beginning of the experiment. The relative increase in tumor volume is shown by a linear change in this parameter in the control group, indicating that this parameter is suitable for assessing the biological response of the tumor to treatment. For example, treatment with CAF therapy did not cause any change in this parameter over a period of approximately 3 weeks, indicating that the disease was stable. By day 21, the frequency of individual tumor stabilization was approximately 60%.

This study was performed by the conventional class in HER-2/neu transgenic female FVB mice; this strain was obtained from the Charles River Laboratories (Italy) mouse library. At the beginning of the study, the animals were 21-41 weeks old and 25-30 grams in weight.

Tamoxifen Hexal (Hexal AG, 83607, batch HA1575, 1 month 2017, hall keng, germany) was used in the experiments, which represents a dosage form of a drug formulated as a round biconvex tablet with a white or yellowish coating, having a uniform smooth surface, containing 20mg of active compound.

The breeding conditions were selected according to The criteria specified in The Guide for Care and Use of Laboratory Animals (ILAR publication, 1996 edition, national academy of academic, 1996).

Animals were housed in separate groups (no more than 5 animals per group) in independent T2-type cages suitable for laboratory rodents in separate rooms. The cage size is 268X 215X 141mm³(bottom area of 370cm²) Each cage was fitted with a polycarbonate bucket, a stainless steel lid with a feed container, and a drinking bottle divider.

The straw mat in the cage is made of dust-free wood shavings.

These animals can obtain mixed whole pellet feed for laboratory rodents without limitation.

Drinking water was freely available from a standard 190ml drinking bottle made from TECNIPLAST, made of high temperature polysulfone with a silicone ring and a metal cap made of AISI 316 stainless steel.

The room temperature is maintained at a level of 20-26 deg.C and the relative humidity is 50-70%. The light cycle was set to 12:12 hours night/day with artificial illumination by fluorescent lamps.

An identification card is placed on the cage, and the number of the cage/card, the number of experimental groups, the number of animals in each group, the number and sex of the animals, the research number and the name of a person in charge of research are arranged on the identification card. The pinna of each animal were clipped with a metallic marker for small laboratory rodents made of Kent-Scientific nickel alloy in the united states (the marker having three digits imprinted by the manufacturer).

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

The LD of this drug in HER-2/neu transgenic FVB mice was determined using the OECD 423 assay (OECD chemical test guideline-acute oral toxicity-acute toxicity grading method)₅₀The value is obtained.

12 female mice were used in the experiment.

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

Since no animals died after administration at a dose of 300mg/kg, the same dose was given to 3 other animals. No deaths were found in this group. Thus, further administration was carried out in two steps at a dose of 2000mg/kg, and 3 animals were administered in each step.

Clinical observations were made and body weights were recorded according to the protocol shown in table 1. Animals were examined twice daily, with a separate clinical examination occurring within the first 60 minutes after dosing, with special attention given during the first 4 hours, with periodic examinations occurring during the first 24 hours and the second day, and then weekly examinations for up to 14 days. Each animal was thoroughly examined in a cage in the hands of an observer. The overall condition of the animals was recorded, such as their behavioral characteristics, the intensity and nature of the motor activity, the appearance of hair and skin.

The body weight of the animals was recorded using a validated high speed electronic laboratory balance, Ohaus Scout Pro (usa), which has a maximum load of 2000g and a measurement step of 0.1 g.

On day 15, animals were euthanized and necropsied and macroscopic changes were recorded. Data were collected in a dedicated individual necropsy table.

At the end of the study, all experimental animals were examined pathomorphologically. Animals euthanized were carefully examined for external pathological changes. The thorax and abdomen were examined and macroscopic analysis of the internal organs was performed. No microscopic analysis of organs and tissues of experimental animals was performed, as no animal death was recorded throughout the observation period. Furthermore, macroscopic analysis of the internal organs did not reflect any pathological changes.

Table 1.

Protocol for animal observation and weighing in assessing acute toxicity

The drug in question was administered intragastrically using metal non-invasive gavage (this procedure corresponds to the oral route of administration in a clinical setting). The drug was administered in a single daily dose (20mg/kg body weight) and 5 times the daily dose of 100mg/kg body weight. Extemporaneous solutions of medicaments for administration are prepared from olive oil (refined olive oil supplemented with unrefined extra virgin olive oil, sold under the trademark Global Village "claico", batch No. L:183351116, shelf life up to 26 days 4 and 2020, spanish BAIEO). The dose was 0.1ml per 10g mouse body weight (0.2 ml of ready-to-use formulation per 20g mouse). Dosing was started 24 hours after randomization. The administration course is 27-28 days.

The control drug tamoxifen was also administered using a metal atraumatic gavage (this procedure corresponds to oral administration in a clinical setting), at a daily dose of 4.0mg/kg, calculated from the clinical dose [5 ]. This dose corresponds to a daily human dose of 20 mg/kg. Tamoxifen tablets were pulverized in a triturator and a suspension in 40ml of olive oil was prepared using the resulting powder, administered in an amount of 0.08ml per 10g of mouse body weight, for the same duration as the experimental drug, for 27-28 days.

The control group was fed olive oil (placebo).

Evaluation criteria included clinical observations, animal body weight, time to death (if applicable), tumor growth over time, pathomorphic examination data (validation of tumors during necropsy, assessment of toxic effects by macroscopic manifestation of internal organ changes).

The body weight of the animals was recorded prior to the first dose using a validated high speed electronic laboratory balance Ohaus Scout Pro (usa) and then twice weekly, with a maximum load of 2000g on the balance and a measurement step size of 0.1 g.

Macroscopically detectable tumor nodules in the animal were measured once a week during weighing. Two linear dimensions were recorded for each nodule, including the largest dimension and the largest rectangular dimension relative to the former. The largest dimension is taken as the length of the tumor nodule (a) and the second dimension is taken as the width of the tumor nodule (b). Tumor volume was calculated as the following expression:

V＝(a×b)2/2，

the efficacy of treatment was assessed by recording the change in mean tumor volume, Tumor Growth Inhibition (TGI) and mean total tumor volume per mouse and their changes over time.

The percentage of tumor growth inhibition was calculated as follows:

TGI＝(V_control-V_{Experiment of})/V_Control×100(％)，

Wherein V_ControlIs the mean tumor volume, V, of the control group_{Experiment of}Is the mean tumor volume of the experimental group.

The raw data of each table was transferred to microsoft Excel 2007 table. Group arithmetic mean (M) and standard deviation (M) were calculated for all quantized data. Statistical analysis was performed using statistical software GraphPad prism 6.0. Differences between groups were assessed using ANOVA regression analysis and fisher's exact test.

In assessing acute toxicity, animals were observed continuously for the first 60 minutes, and then examined hourly for 3 hours and again for 24 hours. On the following day, observations were made twice daily. And then observed once daily. After administration of the active compound, each animal was clinically examined every other day and then weekly. Each animal was thoroughly examined in a cage in the hands of an observer. The overall condition of the animals was recorded, such as their behavioral characteristics, the intensity and nature of the motor activity, the appearance of hair and skin.

During the experiment, no animal death was found when dosed at 300 mg/kg. No animals showed visual signs of intoxication throughout the observation period. The appearance and behavior of the animals were the same as usual. When taken up by hand, these animals showed a conventional weak response. No sound was found to be emitted by the animals at or shortly after the time of administration.

Daily monitoring of the overall condition and behavioral response of the animals indicated that a single oral trial drug did not affect the overall condition and activity of experimental animals exposed to poisoning.

Body weight data are shown in tables 2 and 3.

Table 2.

Changes in animal body weight following administration at a dose of 300mg/kg

According to the data in table 2, the average body weight of the animals receiving the 300mg/kg dose of the drug steadily increased during the observation period.

Since no animal died at the dose of 300mg/kg, the dose was set to 2000 mg/kg. The administration was carried out in two steps, 3 hours apart, at a dose of 0.1ml per 10g of body weight at each step, at a concentration of 100mg/ml, since the suspension in oil is thicker at higher concentrations and cannot be administered by gavage. After administration at a dose of 2000mg/kg, no clinical signs of intoxication were found. Regardless of the group, the width of the palpebral fissure of the animal was nearly unchanged throughout the observation period. No pathological secretion of the eye was detected. The nose is pink, moderately moist and free of pathological secretion. All mice had well-defined, shiny hair and no bald spots. The average body weight of the animals was observed to decrease by 1% from baseline on day 2 and 2% on day 7 (table 3). At the end of the observation period, the mean body weight exceeded the baseline value by 3%.

Table 3.

Changes in animal body weight following administration at a dose of 2000mg/kg

All animals scheduled to be euthanized at experimental day 15 were necropsied. Macroscopic examination did not reveal any abnormalities in visceral status.

Necropsy data were as follows:

the animal hair has clean and bright appearance and no bald spots. The animal has satisfactory nutritional status.

The natural orifice has no pathological secretion.

The submandibular lymph nodes are round, light pink and moderate in density. The salivary gland has normal shape, light yellow color and moderate density.

The peritoneum is smooth and glossy, and no free liquid exists in the cavity.

The spleen is not swollen, compact, and the envelope is smooth and glossy. The pancreas is light pink and has a leafy structure.

The liver has no change in size and shape, and the liver envelope has luster. Liver tissue is brown, moderately dense in texture.

The kidney is compact, the envelope is smooth and glossy, and the periphery of the kidney has moderate adipose tissue proliferation.

The ovary is not enlarged and the surface is glossy.

The stomach has a folded shiny mucosa with a small amount of mucus and food in the lumen.

The pleura is smooth and glossy, and free liquid is not in the pleural cavity. The thymus is triangular and white. The lung is light pink, and the ventilation is good.

Thus, according to the OECD 423 test, the drug may be classified into the fifth or unclassified class, LD₅₀Is equal to orGreater than 5000mg/kg, which corresponds to the low-hazard compound class.

The animals tolerated the administration of the drug satisfactorily and no clinical signs of toxicity were observed. In addition, the hair of the animals receiving the drug was shiny, while the hair of the animals of the control group was somewhat messy. Tumor nodules were also more pronounced in the control group.

No specific signs of toxic damage to internal organs were found after autopsy at the end of the experiment. The tumor lesions were significantly less voluminous when administered at a dose of 20mg/kg, especially at a dose of 100mg/kg, compared to the control animals.

The animals of all groups gained weight throughout the observation period. Weight gain may be associated with increased tumor nodule volume in mice (table 4).

Table 4.

Change in mean body weight of mice when evaluating antitumor Activity of drugs (g)

Analysis of mean tumor volume changes showed that administration of 20mg/kg and 100mg/kg doses produced statistically significant antitumor effects, so that on day 28 of the experiment, the TGI at the 20mg/kg dose was 27% and the TGI at the 100mg/kg dose was 42%; this effect is dose-dependent. It should be noted that the administration of 100mg/kg resulted in an increase of the mean tumor volume of only 60% from the baseline value, and 155% in the control group, during the 28-day observation period (fig. 5).

Table 5.

Change in mean volume of tumor (cm) at the beginning of experiment when evaluating antitumor Activity of drugs with doses of 20mg/kg and 100mg/kg³)

M is an average value; m is the average error; n is the number of tumors at the beginning of the treatment included in the analysis.

^#The control drug group was from the third series of experiments.

P <0.05 compared to control,

^ap in comparison with the control drug group<0.05。

Notably, administration of each dose of drug resulted in an increase in tumor growth inhibition at the end of the observation period, but the TGI effect reached a maximum on day 21 of the experiment after administration of the control drug. Therefore, prolonging the administration time is expected to improve the effect of the drug.

Analysis of the stable frequency showed that this parameter increased significantly from 8% to 27% of the control group by administration at a dose of 100 mg/kg. In addition, the mean volume of new tumors formed during the observation period and their number were small compared to the control group (table 6).

Table 6.

Frequency of tumor stabilization in the evaluation of the antitumor Activity of the drugs and the number and mean volume of new tumors that appeared at the end of the experiment

The percentage of disease stability is a parameter similar to RECIST criteria, which reflects the frequency of tumor development, with no more than a 20% increase in tumor volume at the end of observation relative to the time of treatment initiation.

P <0.05 compared to control.

^#The control drug group was from the third series of experiments.

The mean total tumor volume of mice dosed at doses of 20mg/kg and 100mg/kg was significantly lower than the control group values from day 14 to day 28. This parameter was statistically significantly reduced compared to the control drug for the 100mg/kg dose on day 28 of the experiment (table 7, fig. 6).

Table 7.

Average Total volume (cm) of tumors in mice when evaluating antitumor Activity of drugs³)

^#The control drug group was from the third series of experiments.

P <0.05 compared to control.

P <0.05 compared to control drug group.

Evaluation of the relative change in mean total tumor volume has demonstrated a dose-dependent effect of the drug and superior efficacy to tamoxifen (figure 7, table 8).

Table 8.

Relative change in mean total tumor volume in mice compared to day 1 (expressed as%) when evaluating the antitumor activity of the drug

To evaluate the hormonal effect of the drugs, the weight of the polled uterine bodies was measured on day 28 of the observation period (table 9). No significant difference in this parameter was observed.

Table 9.

Uterine body weight and body weight of mice having breast tumor in evaluation of antitumor activity of drug

Comprises 1 animal in anaphase estrus; e-estrus; d-intermittent period.

The drugs at the doses of 20mg/kg and 100mg/kg had significant antitumor effects, so at day 28 of the experiment, the TGIs were 27% and 42% at the 20mg/kg and 100mg/kg doses, which were dose-related (the difference was statistically significant). The frequency of tumor stabilization was significantly increased from 8% to 27% in the control group when administered at a dose of 100 mg/kg. The drug dose of 100mg/kg was statistically significantly more effective than the control drug tamoxifen (4.0mg/kg) by day 28 to the end of the observation period.

The study showed that the drug has no uterotrophic activity.

According to the OECD 423 test, the drug can be classified into the fifth or non-classified category, the LD of which₅₀Equal to or greater than 5000mg/kg, corresponding to the low-hazard compound classes defined by the approved national standards.

Drawings

The invention is illustrated by the following figures:

figure 1 shows staining of breast tumors with ER α antibodies.

Visualization of horseradish peroxidase + diaminobenzidine, 100-fold magnification (a). Specific staining of mammary ductal nuclei and single tumor nuclei, 400-fold magnification (B).

Figure 2 shows the mean volume of tumor in mice receiving CAF treatment at the start of the experiment.

Figure 3 shows the average total volume of tumors in mice receiving CAF treatment at the start of the experiment.

Figure 4 shows the relative change in the mean total volume of tumours formed in mice receiving CAF treatment at the start of the experiment.

Figure 5 shows the mean volume of tumors in mice when evaluating the antitumor activity of the drugs.

Figure 6 shows the average total volume of tumors in mice when evaluating the anti-tumor activity of the drug.

Figure 7 shows the relative change in mean total volume of tumors in mice when evaluating the anti-tumor activity of the drug.

Detailed Description

The invention will be further illustrated by the examples of the present disclosure.

Example 1

The study analysis was performed on passaged MCF-7 human cell cultures (breast cancer). Normal human skin fibroblasts (HDF) from early passages were used as negative controls. Cells were cultured in a Karl flask (Carrel virtual) at 37 ℃ in a 5% carbon dioxide atmosphere in DMEM/F12 medium (Biolot) supplemented with 1.0% antibiotic-free fetal bovine embryo serum (Biolot).

At 50x10 per bottle⁴The density of individual cells was seeded on a karel flask. To study the proliferative activity of cells under sulfatase-inhibiting conditions, the culture medium was replaced 24 hours after inoculation with a medium containing a sulfatase inhibitor at a final concentration of 50 μ g/ml, and these tumor cell lines were then incubated for various times (24 to 72 hours). The inhibitor is dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the medium did not exceed 0.5%. To rule out the cytotoxic effect of DMSO, control samples containing DMSO but no sulfatase inhibitor were prepared. To rule out the non-specific damaging effects of the compounds, normal human dermal fibroblasts were used. The cells were then detached using ethylenediaminetetraacetic acid-trypsin (versene-trypsin) solution (Biolot) and placed on a kara flask containing fresh complete medium. When the untreated control cells reached the maximum cell density per unit surface area of the flask (monolayer), the cells were counted and their number was set to 100%. The proliferative activity of all cell cultures studied exposed to a sulfatase inhibitor was determined in triplicate.

A study of the effect of the sulfamate obtained on the proliferation of passaged human MCF-7 cell cultures (breast cancer) showed that the steroid of formula (3) at a concentration of 20. mu.g/ml completely prevented the proliferation of tumor cells, but it did not affect the growth of human dermal fibroblasts without estrogen receptors. The proliferation of tumor cells is inhibited to the same extent as that of tamoxifen which has been used clinically for more than 30 years. This is important because sulfamates and tamoxifen have different mechanisms of action, and it is possible to use them in combination.

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

In the triple negative breast cancer MDA-MB-231 cell line, the compound inhibits cell proliferation by 83%, which is similar to the chemotherapeutic drug etoposide used in clinical practice.

The medicine can be used for treating hepatocellular carcinoma SK-Hep-1 cell (IC)₅₀53.4 μ M), lung cancer a549 (IC)₅₀29.6 μ M), gastric adenocarcinoma SNU638 (IC)₅₀22.8 μ M) and chronic myelogenous leukemia cells (K562) were active.

Example 2

Control group: there were 10 mice, 10 tumors per group. At the beginning of the treatment, V_Average＝1.5±0.4mm³. Without specific treatment, the size of the tumor reached V at day 33 post-transplantation_Average＝233.2±86.5mm³. The number of dead mice in this group was 2 out of 10 deaths before the end of the observation period.

Drug group, 10 mg/kg: there were 7 mice, 7 tumors per group.

At the beginning of the treatment, V_Average＝1.0±0.4mm³And no significant difference from the control group. At day 7 after the start of treatment, an insignificant TGI of 74.3% (V) was recorded_Average＝5.1±2.3mm³And V of control group_Average＝20.0±6.0mm³，p<0.220). On day 10 after the start of treatment, TGI 68.0% (V)_Average＝24.8±12.2mm³And V of control group_Average＝77.3±32.0mm³，p<0.181). On day 14 after the start of treatment, 94.3% of the maximum significant TGI (V) was observed_Average＝6.9±2.2mm³And V of control group_Average＝120.6±44.4mm³，p<0.03), this value remained at approximately the same level by day 17 and day 21, at 90.6% (V)_Average＝17.6±0.7mm³And V of control group_Average＝186.4±65.0mm³，p<0.061) and 90.2% (V)_Average＝26.l±12.l mm³And V of control group_Average＝266.5±91.3mm³，p<0.061). The number of dead mice in this group was 5 out of 7 before the observation period ended.

Tamoxifen group, 30 mg/kg: there were 7 mice, 7 tumors per group. At the beginning of the treatment, V_Average＝0.9±0.2mm³And no significant difference from the control group. On day 7, the TGI was 12.6% (V)_Average＝18.8±7.4mm³And V of control group_Average＝20.0±6mm³，p<0.962) and these values are not exceeded by the end of the observation period. On days 17 and 21 after the start of treatment, negative TGI values were observed, reaching a maximum TGI of-44.2 on day 21 (V)_Average＝384.8±103.2mm³And V of control group_Average＝266.5±91.3mm³，p<0.106). The number of dead mice in this group was 6 out of 7 before the observation period ended.

Tamoxifen group, 10 mg/kg: there were 7 mice, 7 tumors per group. At the beginning of the treatment, V_Average＝0.7±0.1mm³And no significant difference from the control group. On day 7 after the start of treatment, an insignificant TGI of 63.9% (V) was recorded_Average＝7.2±3.9mm³And V of control group_Average＝20.2±6.0mm³，p<0.364). On day 10 after the start of treatment, TGI 56.9% (V) was recorded_Average＝33.4±16.l mm³And V of control group_Average＝77.3±32.0mm³，p<0.315). From day 14 to day 17, an increase in TGI of 60.6% (V), respectively, was observed_Average＝47.5±16.8mm³And V of control group_Average＝120.6±44.4mm³，p<0.193) and 68.9% (V)_Average＝58.0±18.2mm³V of control group_Average＝186.4±65.0mm³，p<0.070). At day 21 after the start of treatment and until the end of the observation period, TGI is below the biologically significant value(s) ((s))<50%). The number of dead mice in this group was 2 out of 7 before the observation period ended.

The final results of the measurements and the statistical significance of their differences between groups are shown in tables 10 and 11.

Table 10.

Mean tumor volume (M. + -.m) of mice in each group when observed by SEM

Table 11.

Effect of drug and tamoxifen on SKBR-3 growth inhibition, TGI%

Example 3

Control group: there were 12 mice, 12 tumors per group. At the beginning of the treatment, V_Average＝3.8±1.0mm³. Without specific treatment, the size of the tumor reached V at day 30 post-transplantation_Average＝299.l±100.5mm³. The number of dead mice in this group was 6 out of 12 before the end of the observation period.

Drug group, 10 mg/kg: there were 10 mice, 10 tumors per group. At the beginning of the treatment, V_Average＝1.6±0.4mm³And no significant difference from the control group. At day 7 after the start of treatment, a significant TGI of 66.1% (V) was recorded_Average＝27.0±5.l mm³And V of control group_Average＝79.6±14.2mm³，p<0.002). On day 10 after the start of treatment TGI 78.7% (V) was recorded_Average＝21.6±4.9mm³And V of control group_Average＝101.2±22.5mm³，p<0.007). On day 14 after the start of treatment, a maximum insignificant TGI of 83.1% (V) was recorded_Average＝27.6±2.6mm³And V of control group_Average＝163.3±31.0mm³，p<0.06). On day 17 after the start of treatment, TGI decreased to 76.1% (V)_Average＝46.3±5.3mm³And V of control group_Average＝193.6±39.6mm³，p<0.04). The number of dead mice in this group was 10 deaths out of 10 before the end of the observation periodOnly.

Tamoxifen group, 30 mg/kg: there were 10 mice, 10 tumors per group. At the beginning of the treatment, V_Average＝4.1±1.2mm³And no significant difference from the control group. At day 7 and until the end of the observation period, TGI was below the biologically significant threshold and did not exceed 44.5% (V)_Average＝44.1±8.8mm³And V of control group_Average＝79.6±14.2mm³，p<0.10). The number of dead mice in this group was 4 out of 10 deaths before the end of the observation period.

Tamoxifen group, 10 mg/kg: there were 10 mice, 10 tumors per group. At the beginning of the treatment, V_Average＝2.3±0.8mm³And no significant difference from the control group. In this group, no tumor growth inhibition was detected throughout the observation period. The number of dead mice in this group was 6 out of 9 before the observation period ended.

The results of the measurements and the statistical significance of their differences between groups are shown in tables 12 and 13.

Table 12.

Mean tumor volume (M. + -.m) of mice in each group when observed by SEM

Table 13.

Effect of drug and tamoxifen on growth inhibition of TNBC Kad, TGI%

A comparative study of the antitumor activity of this drug was conducted after multiple administrations to athymic immunodeficient Balb/c nude mice in two subcutaneous graft models of human SKBR-3 and TNBC-Kad breast cancers of different phenotypes.

Preliminary experiments on the antitumor activity of the drug in the SKBR3 model compared to tamoxifen (dosage form) showed that in all the treatment regimens studied, a dose of 10mg/kg was usedThe treatment with the drug is most effective: on day 10 after the start of treatment, a significant antitumor effect was exhibited, i.e., maximum TGI 91.2% (V)_Average＝2.2±2.0mm³And V of control group_Average＝24.9±12.5mm³，p<0.018), and the drug tolerance is better. Tamoxifen at a dose of 10mg/kg had no effect: no biologically significant TGI values were reached throughout the observation period. In contrast, tumor growth stimulation was recorded on days 10 to 17 after the start of treatment.

Repeated experiments on the antitumor activity of the drug in the same SKBR3 model compared to tamoxifen (control drug) showed that the 10mg/kg dose of drug had a very high antitumor effect, reaching the maximum significant TGI at day 14 (94.3%, p <0.03) and day 24 (86.3%, p <0.04), which reproduces the results obtained in the first experiment.

Experiments on the antitumor activity of the drug in the TNBC Kad model compared to tamoxifen (control drug) showed that the 10mg/kg dose of drug inhibited tumor growth by 66.1% and 78.7% on days 7 and 10 after the start of treatment (significant differences were observed between the control and 30mg/kg tamoxifen groups). The effect lasted for 10 days, approximately at the same level. Tamoxifen at a dose of 30mg/kg produced a weak and insignificant inhibition of tumor growth at day 7, with an inhibition rate of 44.5%, which was reduced towards the end of the observation. Tamoxifen at a dose of 10mg/kg had no effect in this model.

Industrial applicability

The experiments carried out show that the claimed use of the compounds is characterized in that:

-the absence of uterotrophic activity,

a broad range of anti-cancer activities, including activity against triple negative breast cancer,

-the absence of toxicity,

has hypocholesterolemic activity and has no effect on the triglyceride content of the serum,

no adverse effect on the endometrium.

The invention can be used for:

-monotherapy and adjuvant therapy of early hormone-positive breast cancer in postmenopausal women;

early monotherapy and adjuvant therapy of hormone-positive breast cancer in postmenopausal women who have been treated for 2-3 years with tamoxifen;

-treatment of advanced breast cancer;

-treatment of triple negative breast cancer;

monotherapy and adjuvant therapy of hepatocellular carcinoma;

mono-and adjuvant therapy of gastric cancer;

monotherapy and adjuvant therapy of lung cancer;

monotherapy and adjuvant therapy of chronic myeloid leukemia.