Use of 3-O-aminosulfonic acid-16,16-dimethyl-D-homoenanthrin in treating tumor diseases

Abstract

The present invention relates to the pharmaceutical field and the chemical and pharmacological industries, and to a medicament for treating cancer. The present invention proposes the use of 3-O-aminosulfonic acid 16,16-dimethyl-D-homoequinostranolone as an anticancer agent in monotherapy and adjuvant therapy for neoplastic diseases such as hepatocellular carcinoma, gastric cancer, lung cancer, chronic myeloid leukemia and 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 cancer (including 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 shows that breast cancer expresses estrogen receptors (ER), progesterone receptors (PR), and HER2NEU (making 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 cannot bind to estrogen receptors, but after conversion to free hormones they activate tumor growth. Therefore, a promising therapeutic strategy consists of using inhibitors to block the formation of free hormones in tumors from this group."

[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 of care for hormonal 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 cardiovascular disease (including angina attacks), development of endometrial tumors (polyps and endometrial cancer), and uterine fibroids. Tamoxifen is also hepatotoxic. According to the Biopharmaceutical Classification System (BCS) developed by Gordon Amidon et al. in 1995, tamoxifen is classified as Class II, i.e., drugs 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 as Class I, i.e., highly soluble and highly permeable drugs. 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 the abundant circulating estrone sulfate pool forms estrogens in the local stroma. Steroid sulfatase catalyzes the hydrolysis of estrone sulfate to estrone and catalyzes the hydrolysis of DHEA sulfate to DHEA (Dibbelt I., Biol. Chem., Hoppeseler, 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--estrogen 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 appearance of strong uterotropic activity [Shields-Botella J. et al., J. Steroid Biochem. Mol. Biol., 2003, Vol. 84, pp. 327-335].

[0009] Therefore, when searching for new anticancer drugs, it needs to be taken into account that the carrier of the aminosulfonate group of the estrone sulfatase inhibitor should not have hormonal (uterotropic) activity.

[0010] It is known that D-equilenin, which has no carcinogenic properties, is used as a drug for the prevention of breast cancer. Preparations with cholesterol-lowering effects but without uterotropic and hypertriglyceridemic activity have been prepared from equilenin derivatives [Urusova EA, Gluzdikov IA, Selivanov SI, Starova GL, Nikolaev SV, Shavva A.G., "Synthesis and study of equilenin derivatives and its modified analogs", Russ. J. Org. Chem., 2004, Vol. 40, No. 4, pp. 506-512].

[0011] Equine sterol compound is a drug used in hormone replacement therapy These properties are desirable because the use of steroidal estrogen metabolism inhibitors implies their long-term application. Summary of the invention

[0012] The technical problem underlying the present invention is to provide compounds and to develop an efficient process for the synthesis of these compounds, which can be used as anticancer agents in monotherapy and adjuvant therapy of tumor diseases such as hepatocellular carcinoma, gastric cancer, lung cancer, chronic myeloid leukemia and breast cancer, including its most lethal forms, such as triple-negative breast cancer (ER- / PR- / HER-2-).

[0013] This problem was solved by using the compound 3-O-aminosulfonic acid-16,16-dimethyl-D-homoequinolone of formula (3) which inhibits estrone sulfatase.

[0014]

[0015] The synthesis scheme of the target compound of formula (3) is shown below. A significant advantage of the proposed scheme compared to the prior art is that it allows the production of the target compound (drug) to be scaled up to an industrial scale.

[0016]

[0017] The reaction of known isothiouronium salts [Al Safar, Zakharychev AV, Ananchenko SN, Torgov IV, Synthesis of some 16,16-dimethyl-D-homosteroids, Reports of Academy of Sciences of the USSR. Ser. Chem., 1968, No. 10, pp. 2326-2332] with 2,4,4-trimethylcyclopentane-1,3-dione leads to the formation of ring-opened steroids, the cyclodehydration of which produces the pentaestradiol nucleus of formula (1). The methyl ester of 16.16-dimethyl-D-homoequinostranolone of formula (2) can be isolated by treating the pentaestradiol nucleus with Ni / Ra under conditions previously proposed for the synthesis of structurally similar D-homoanalogues [Gluzdikov IA, Egorov MS, Selivanov SI, Starova G.L., Shavva AG, New analogues of D-homoequilenene with substituents in the Dring, Rus. J. Org. Chem., 2006, Vol. 42, No. 11, pp. 1675-1682] [Gluzdikov IA, PhD thesis in chemistry, 2007, St. Petersburg]. The aminosulfonate of formula (3) was obtained according to a well-known method [Morozkina SN, Gluzdikov IA, Drozdov AS, Selivanov SI, Kovalev RA, Filatov MV, Shavva AG, Russ. J. Org. Chem., 2015, Vol. 51. No. 3, pp. 425-430].

[0018] The results of steroid binding simulations indicate that the structure of this steroid is poorly compatible with the geometry of the ligand binding receptor pocket. The distance between the oxygen in the steroid keto group and the NH of His524 is This prevents the formation of hydrogen bonds. The distance between the hydroxyl group at СЗ and the oxygen atom in the carboxyl group Glu353 is calculated to be approximately Much greater than the distance in complexes containing the natural hormone estradiol

[0019] The incompatibility between the geometry of the steroid and the receptor region responsible for ligand binding can result in a low affinity of the target compound for the receptor, rendering the steroid inactive for uterotropism.

[0020] The compound completely inhibited the proliferation of tumor MCF-7 cells.

[0021] This drug belongs to the fourth hazard category (4 th hazard class)(LD 50 >300-2000 mg / kg). The data obtained allowed the dose used in the antitumor activity studies to be determined as a safe dose.

[0022] Studies on binding to estrogen receptors showed that aminosulfonates had no hormonal activity at concentrations of -0.001 and 0.01, 0.1 and 1.0 μmol / ml.

[0023] In an animal model with triple-negative breast cancer xenograft human tumors, the proposed compound (drug) was more effective than tamoxifen and the aromatase inhibitor letrozole, which are used in clinical practice.

[0024] Therefore, the compound may be useful in the treatment of breast cancer, including triple-negative forms of breast cancer.

[0025] Preclinical studies of specific activity in breast cancer models were conducted in parallel with the anti-tumor drug tamoxifen (control drug). In vivo drug comparison studies were conducted in female Balb / c nude mice with subcutaneous (sc) transplanted tumor cells. The experiment lasted 29 days after the first dose (dosing started on the 5th day after tumor transplantation).

[0026] The drug and control drug were intragastrically administered multiple times at a dose of 30 mg / kg and 10 mg / kg per day for 21 days.

[0027] The placebo group was given 1% starch solution by gavage with the same regimen and equal dose. Tumor nodules were measured twice a week. Antitumor activity was evaluated by standard tumor growth inhibition (TGI) index. After treatment was completed, the last tumor measurement was performed, and the animals were weighed and euthanized.

[0028] All necessary manipulations of the animals (tests, weighings, measurements and necropsies) were performed between 9:00 a.m. and 12:00 p.m.

[0029] Animals were weighed on the first day of the experiment and then twice a week throughout the experiment according to the study protocol.

[0030] During the entire experiment, tumor nodules were measured twice a week after tumor implantation. The tumor nodule volume was determined as follows:

[0031] V = π / 6*L*W*H, where L, W, H are the linear dimensions of the tumor.

[0032] Determination of antitumor activity

[0033] The tumor growth inhibition index (TGI) was used as the standard for studying drug efficacy and was calculated by the following formula:

[0034] TGI (%) = |V 对照 –V 实验 | / V 对照 *100

[0035] Where V 对照 and V 实验 The average tumor volume (mm) in the control group and the experimental group 3 ).

[0036] At the end of the treatment session, animals were euthanized by cervical dislocation.

[0037] SPSS 21 software package (license number 20130626-3) was used for statistical data processing. Descriptive statistics in the study included the following: arithmetic mean, standard deviation (SD), standard error of the mean (SE), median, quartile, and 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).

[0038] Mammary tumors (BT) of HER-2 / neu transgenic female FBV / N mice are adenocarcinomas characterized by low levels of estrogen receptor (ER) Figure 1 ) and lack of progesterone receptor (PR). Two subtypes of adenocarcinoma can be distinguished, specifically ER+, PR- and ER-, PR-[2]. Immunohistochemical analysis of ERα in breast tumors showed single positive staining of the nuclei in only 10 of 45 samples ( Figure 1 ).

[0039] The model was sensitive to treatment with cyclophosphamide, doxorubicin, and 5-fluorouracil (CAF), and tumor growth inhibition was observed, resulting in stabilization of the mean tumor volume ( Figure 2 The tumor growth inhibition (TGI) on the 14th and 21st days of the experiment were 63% and 46%, respectively.

[0040] 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, indicating that this parameter is suitable for evaluating the biological response of tumors to treatment. For example, treatment with CAF therapy did not cause any changes in this parameter over a period of nearly 3 weeks, indicating stable disease. By day 21, the frequency of individual tumor stability was approximately 60%.

[0041] This study was performed in HER-2 / neu transgenic female FVB mice by conventional assortment; this strain was 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.

[0042] Tamoxifen Hexal (Hexal AG, Holzkirchen, Germany, 83607, batch number HA1575, January 2017) was used in the experiments, which represents a dosage form of a drug formulated as white or slightly yellow coated round biconvex tablets with a uniform smooth surface containing 20 mg of active compound.

[0043] The housing conditions were selected according to the standards specified in The Guide for the Care and Use of Laboratory Animals (ILAR Publication, 1996 Edition, National Academy Press, 1996).

[0044] Animals were housed in separate rooms in groups (no more than 5 animals per group) in individual T2 cages suitable for laboratory rodents. The dimensions of the cages were 268 × 215 × 141 mm. 3 (Bottom area is 370cm 2 ), each cage was equipped with a polycarbonate bucket, a stainless steel lid with a feed container, and a water bottle divider.

[0045] The cage bedding is made of dust-free wood shavings.

[0046] The animals had unlimited access to a mixed complete pelleted diet for laboratory rodents.

[0047] Drinking water was 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.

[0048] The room temperature was maintained at 20-26°C and the relative humidity was 50-70%. The light cycle was set to artificial lighting with fluorescent lamps for 12:12 hours night / day.

[0049] An identification card was placed on the cage with the cage / card number, experimental group number, number of animals in each group, their number and sex, study number, name of the responsible researcher. The pinna of each animal was clamped with a metal marker made of nickel alloy from Kent-Scientific, USA, for small laboratory rodents (the marker had a three-digit number stamped by the manufacturer).

[0050] At the end of the experiment, mice were euthanized with CO. All animal carcasses were necropsyed and then microscopically described.

[0051] The LD50 of the drug in HER-2 / neu transgenic FVB mice was determined using the OECD 423 test (OECD Guidelines for Testing of Chemicals - Acute Oral Toxicity - Acute Toxicity Classification Method). 50 value.

[0052] Twelve female mice were used in the experiments.

[0053] In the first step, 3 mice were given a dose of 300 mg / kg of the drug. The drug under study was administered intragastrically (ig) using a metal atraumatic gavage (this procedure corresponds to oral administration in a clinical setting). For intragastric administration, 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.

[0054] Since no animals died after administration at a dose of 300 mg / kg, the same dose was administered to another 3 animals. No deaths were found in this group either. Therefore, further administration at a dose of 2000 mg / kg was performed in two steps, with 3 animals administered in each step.

[0055] Clinical observations were performed and body weights were recorded according to the protocol shown in Table 1. Animals were examined twice daily, with individual clinical examinations within the first 60 minutes after dosing, special attention within the first 4 hours, regular examinations within the first 24 hours and the next day, and then weekly for up to 14 days. Each animal was thoroughly examined in its cage in the hands of the observer. The overall condition of the animals, such as their behavioral characteristics, the intensity and nature of motor activity, and the appearance of the coat and skin, was recorded.

[0056] 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 of 0.1 g.

[0057] On day 15, animals were euthanized and necropsy analysis was performed to record macroscopic changes. Data were collected on a dedicated individual necropsy form.

[0058] At the end of the study, all experimental animals were subjected to pathological morphological 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 reflect any pathological changes.

[0059] Table 1.

[0060] Protocol for observation and weighing of animals when assessing acute toxicity

[0061]

[0062] The drug under study was intragastrically administered using a 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 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, sold under the trademark of Global Village "Clasico", batch number L: 183351116, shelf life until April 26, 2020, BAIEO, Spain). The dosage was 0.1 ml per 10 g mouse body weight (0.2 ml ready-to-use preparation per 20 g mouse). Administration began 24 hours after randomization. The course of administration was 27-28 days.

[0063] The control drug, tamoxifen, was also administered by noninvasive gavage with a metal tube (a procedure that corresponds to oral administration in the clinical setting) at a daily dose of 4.0 mg / kg, which was calculated based on the clinical dose [5]. This dose corresponds to the 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, which was administered in an amount of 0.08 ml per 10 g of mouse body weight for the same duration as the experimental drug, which lasted for 27-28 days.

[0064] The control group was fed olive oil (placebo).

[0065] 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).

[0066] 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.

[0067] During the weighing process, the macroscopically detectable tumor nodules in the animals were measured once a week. Two linear dimensions were recorded for each nodule, including the largest dimension and the largest rectangular dimension relative to the former. The largest dimension was taken as the length of the tumor nodule (a), and the second dimension was taken as the width of the tumor nodule (b). Tumor volume was calculated as follows:

[0068] V = (a × b)2 / 2,

[0069] 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.

[0070] The percentage of tumor growth inhibition was calculated as follows:

[0071] TGI=(V 对照 -V 实验 ) / V 对照 ×100(%),

[0072] Where V 对照 is the mean tumor volume of the control group, V 实验 is the mean tumor volume of the experimental groups.

[0073] The raw data of each table were transferred to Microsoft Excel 2007. The arithmetic mean (M) and standard deviation (m) of the group were calculated for all quantitative data. Statistical analysis was performed using the statistical software GraphPad Prism 6.0. ANOVA regression analysis and Fisher's exact test were used to evaluate the differences between the groups.

[0074] 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. On the second day, they were observed twice a day. Then they were observed once a day. After the administration of the active compound, each animal was clinically examined every other day and then once a week. Each animal was thoroughly examined in the cage in the hands of the observer. The general condition of the animals, such as their behavioral characteristics, the intensity and nature of motor activity, the appearance of the fur and skin, was recorded.

[0075] During the experiment, no animal deaths were found at the dose of 300 mg / kg. During the entire observation period, no animal showed visual signs of intoxication. The appearance and behavior of the animals were normal. When picked up by hand, the animals showed conventional weak reactions. No animal was found to make any sounds during or shortly after the administration of the drug.

[0076] 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.

[0077] Body weight data are shown in Tables 2 and 3.

[0078] Table 2.

[0079] Changes in animal body weight after administration of 300 mg / kg

[0080]

[0081] According to the data in Table 2, the average body weight of the animals receiving the drug at a dose of 300 mg / kg increased steadily during the observation period.

[0082] Since no animals died at a dose of 300 mg / kg, the dose was set to 2000 mg / kg. The drug was administered in two steps, 3 hours apart, with a dose of 0.1 ml per 10 g of body weight in each step and 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 poisoning were found after administration of 2000 mg / kg. Regardless of the group, the width of the palpebral fissure of the animals remained almost unchanged throughout the observation period. No pathological secretions from the eyes were detected. The nose was pink, moderately moist, and there was no pathological secretion. The fur of all mice was neat and shiny, with no bald spots. The average body weight of the animals was observed to have decreased by 1% from baseline on the second day and by 2% on the seventh day (Table 3). At the end of the observation period, the average body weight exceeded the baseline value by 3%.

[0083] Table 3.

[0084] Changes in animal body weight after administration of 2000 mg / kg

[0085]

[0086] Necropsy was performed on all animals scheduled for euthanasia on experimental day 15. Macroscopic examination did not reveal any abnormalities in the condition of the internal organs.

[0087] The autopsy data are as follows:

[0088] The animal's coat is neat and shiny, with no bald spots. The animal's nutritional status is satisfactory.

[0089] There is no pathological secretion from the natural orifices.

[0090] The submandibular lymph nodes were round, light pink, and of moderate density. The salivary glands were normal in shape, light yellow, and of moderate density.

[0091] The peritoneum was smooth and shiny, with no free fluid in the cavity.

[0092] The spleen was not enlarged, dense, and had a smooth and shiny capsule. The pancreas was light pink and had a lobed structure.

[0093] The size and shape of the liver remain unchanged, the liver capsule is shiny, the liver tissue is brown and of medium density.

[0094] The kidneys are dense, with a smooth, shiny capsule and moderate proliferation of adipose tissue around the kidneys.

[0095] The ovaries were not enlarged and had a shiny surface.

[0096] The stomach has a folded, shiny mucosa with a small amount of mucus and food in the lumen.

[0097] The pleura is smooth and shiny, with no free fluid in the pleural cavity. The thymus is triangular and white. The lungs are light pink and well ventilated.

[0098] 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.

[0099] The animals tolerated the administration of the drug satisfactorily, and no clinical signs of toxicity were observed. In addition, the fur of the animals receiving the drug was shiny, while the fur of the control animals was somewhat unkempt. Tumor nodules were also more obvious in the control group.

[0100] After necropsy analysis at the end of the experiment, no specific signs of toxic damage to internal organs were found. The volume of tumor lesions was significantly smaller when compared with control animals at the dose of 20 mg / kg and especially at the dose of 100 mg / kg.

[0101] The body weight of animals in all groups increased throughout the observation period. The increase in body weight may be related to the increase in the volume of tumor nodules in the mice (Table 4).

[0102] Table 4.

[0103] Average body weight change of mice when evaluating the anti-tumor activity of drugs (g)

[0104]

[0105] Analysis of changes in mean tumor volume showed that administration of 20 mg / kg and 100 mg / kg doses produced a statistically significant antitumor effect, such that on day 28 of the experiment, the TGI was 27% for the 20 mg / kg dose and 42% for the 100 mg / kg dose; this effect was dose-related. It should be noted that during the 28-day observation period, administration of 100 mg / kg resulted in an increase in mean tumor volume of only 60% from baseline values, while an increase of 155% was observed in the control group ( Figure 5 ).

[0106] Table 5.

[0107] The changes in the mean tumor volume (cm2) at the beginning of the experiment when the antitumor activity of the drug was evaluated using the doses of 20 mg / kg and 100 mg / kg 3 )

[0108]

[0109] M is the mean; m is the mean error; N is the number of tumors at the start of treatment included in the analysis.

[0110] # The control drug group was from the third series of experiments.

[0111] *Compared with the control group, p<0.05,

[0112] a Compared with the control drug group, p<0.05.

[0113] It is noteworthy that administration of each dose of the drug resulted in increased tumor growth inhibition by the end of the observation period, but after administration of the control drug, the TGI effect reached a maximum on day 21 of the experiment. Therefore, extending the administration time is expected to improve the effect of the drug.

[0114] Analysis of the stable frequency showed that this parameter significantly increased from 8% in the control group to 27% by administration at a dose of 100 mg / kg. In addition, the average volume of new tumors formed during the observation period and their number were smaller compared to the control group (Table 6).

[0115] Table 6.

[0116] The frequency of tumor stabilization and the number and average volume of new tumors that appeared at the end of the experiment were used to evaluate the antitumor activity of the drug.

[0117]

[0118] The percentage of disease stabilization is a parameter similar to the RECIST criteria, which reflects the frequency of tumors that, at the end of observation, did not increase by more than 20% in volume relative to the start of treatment.

[0119] *P<0.05 compared with the control group.

[0120] # The control drug group was from the third series of experiments.

[0121] From day 14 to day 28, the mean total tumor volume of mice dosed with 20 mg / kg and 100 mg / kg was significantly lower than that of the control group. For the 100 mg / kg dose on day 28 of the experiment, this parameter was statistically significantly reduced compared with the control drug (Table 7, Figure 6 ).

[0122] Table 7.

[0123] The average total tumor volume (cm) in mice when evaluating the antitumor activity of the drug 3 )

[0124]

[0125]

[0126] # The control drug group was from the third series of experiments.

[0127] *P<0.05 compared with the control group.

[0128] *P<0.05 compared with the control drug group.

[0129] Evaluation of relative changes in mean total tumor volume has demonstrated a dose-dependent effect and superior efficacy to tamoxifen ( Figure 7 , Table 8).

[0130] Table 8.

[0131] Relative change in the average total tumor volume in mice compared with day 1 when evaluating the antitumor activity of the drug (expressed in %)

[0132]

[0133] To assess the hormonal effects of the drug, the weight of the uterine body without horns was measured on day 28 of the observation period (Table 9). No significant differences were observed in this parameter.

[0134] Table 9.

[0135] Uterine body weight and body weight of mice bearing breast tumors when evaluating the antitumor activity of drugs

[0136]

[0137] *Including 1 animal in metestrus; E - estrus; D - diestrus.

[0138] The drug at doses of 20 mg / kg and 100 mg / kg had a significant antitumor effect, so that on day 28 of the experiment, the TGI with the 20 mg / kg dose and the 100 mg / kg dose was 27% and 42%, respectively, and this effect was dose-related (the difference was statistically significant). Administration at a dose of 100 mg / kg significantly increased the frequency of tumor stabilization from 8% in the control group to 27%. From day 28 to the end of the observation period, the drug at a dose of 100 mg / kg was statistically significantly more effective than the control drug tamoxifen (4.0 mg / kg).

[0139] Studies have shown that this drug has no uterotropic activity.

[0140] 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 by the approved national standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] The present invention is illustrated by the following drawings:

[0142] Figure 1 Shown is staining of breast tumors with ERα antibody.

[0143] Visualization with horseradish peroxidase + diaminobenzidine, 100x magnification (A). Specific staining of mammary ductal cell nuclei and individual tumor cell nuclei, 400x magnification (B).

[0144] Figure 2 Shown are the mean volumes of tumors in mice treated with CAFs at the start of the experiment.

[0145] Figure 3 Shown are the mean total volumes of tumors in mice treated with CAFs at the start of the experiment.

[0146] Figure 4 Shown are the relative changes in the mean total volume of tumors formed in mice treated with CAFs at the start of the experiment.

[0147] Figure 5 Shown are the mean volumes of tumors in mice when evaluating the anti-tumor activity of the drugs.

[0148] Figure 6Shown are the mean total volumes of tumors in mice when evaluating the anti-tumor activity of the drugs.

[0149] Figure 7 Shown are the relative changes in the mean total volume of tumors in mice when evaluating the anti-tumor activity of the drugs. DETAILED DESCRIPTION

[0150] The present invention will be further described below by way of the embodiments disclosed herein.

[0151] Example 1

[0152] The analysis was performed on a culture of human MCF-7 cells (breast cancer) at passage. Normal human skin fibroblasts (HDF) at an early passage were used as negative controls. The cells were cultured in Carrel vials at 37°C in a 5% carbon dioxide atmosphere in DMEM / F12 medium (Biolot) supplemented with 1.0% fetal calf serum (Biolot) without antibiotics.

[0153] 50x10 per bottle 4 Cells were inoculated on Carrel bottles at a density of 10 cells. In order 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 sulfatase inhibitors at a final concentration of 50 μg / ml, and then these tumor cell lines were incubated for different times (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 sulfatase inhibitors was prepared. In order to exclude the nonspecific damaging effect of the compound, normal human skin fibroblasts were used. Then ethylenediaminetetraacetic acid-trypsin (versene-trypsin) solution (Biolot) was used to separate the cells and put them on a Carrel bottle containing fresh complete culture medium. When the untreated control cells reached the maximum cell density (monolayer) per unit surface area of ​​the culture bottle, the cells were counted and their number was set to 100%. The proliferation activity of all studied cell cultures exposed to sulfatase inhibitors was determined in triplicate.

[0154] Studies on the effects of aminosulfonates on the proliferation of cultured human MCF-7 cells (breast cancer) have shown that a steroid of formula (3) at a concentration of 20 μg / ml completely prevents the proliferation of tumor cells, but it does not affect the growth of human skin fibroblasts without estrogen receptors. The extent to which the proliferation of tumor cells is inhibited is the same as the effect of tamoxifen, which has been in clinical use for more than 30 years. This is very important because aminosulfonates and tamoxifen have different mechanisms of action and it is possible to use them in combination.

[0155] 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 use as a combination treatment for breast cancer.

[0156] In the triple-negative breast cancer MDA-MB-231 cell line, the compound inhibited cell proliferation by 83%, which is similar to the chemotherapy drug etoposide used in clinical practice.

[0157] The drug has an inhibitory effect on hepatocellular carcinoma SK-Hep-1 cells (IC 50 =53.4μM), lung cancer A549 (IC 50 =29.6μM), gastric adenocarcinoma SNU638 (IC 50 =22.8 μM) and chronic myeloid leukemia cells (K562).

[0158] Example 2

[0159] Control group: There were 10 mice, each with 10 tumors. At the beginning of treatment, V 平均 =1.5±0.4mm 3 Without specific treatment, the tumor size reached V 平均 =233.2±86.5mm 3 Before the end of the observation period, the number of dead mice in this group was 2 out of 10.

[0160] Drug group, 10 mg / kg: There were 7 mice and 7 tumors in each group.

[0161] At the start of treatment, V 平均 =1.0±0.4mm 3 On the 7th day after the start of treatment, an insignificant TGI of 74.3% (V 平均 =5.1±2.3mm 3 , while the control group's V 平均 =20.0±6.0mm 3, p<0.220). On the 10th day after the start of treatment, TGI=68.0% (V 平均 =24.8±12.2mm 3 , while the control group's V 平均 =77.3±32.0mm 3 , p<0.181). On day 14 after the start of treatment, the maximum significant TGI of 94.3% was observed (V 平均 =6.9±2.2mm 3 , while the control group's V 平均 =120.6±44.4mm 3 , p<0.03), and by days 17 and 21, the value remained at approximately the same level of 90.6% (V 平均 =17.6±0.7mm 3 , while the control group's V 平均 =186.4±65.0mm 3 , p<0.061) and 90.2% (V 平均 =26.1±12.1 mm 3 , while the control group's V 平均 =266.5±91.3mm 3 , p<0.061). Before the end of the observation period, the number of dead mice in this group was 5 out of 7.

[0162] Tamoxifen group, 30 mg / kg: There were 7 mice, 7 tumors in each group. At the beginning of treatment, V 平均 =0.9±0.2mm 3 On the 7th day, the TGI was 12.6% (V 平均 =18.8±7.4mm 3 , while the control group's V 平均 =20.0±6mm 3 , p<0.962), and did not exceed these values ​​at the end of the observation period. Negative TGI values ​​were observed on days 17 and 21 after the start of treatment, reaching a maximum value of TGI = -44.2 on day 21 (V 平均 =384.8±103.2mm 3 , while the control group's V 平均 =266.5±91.3mm 3 , p<0.106). Before the end of the observation period, the number of dead mice in this group was 6 out of 7.

[0163] Tamoxifen group, 10 mg / kg: There were 7 mice, 7 tumors in each group. At the beginning of treatment, V 平均 =0.7±0.1mm 3On the 7th day after the start of treatment, an insignificant TGI of 63.9% (V 平均 =7.2±3.9mm 3 , while the control group's V 平均 =20.2±6.0mm 3 , p<0.364). On the 10th day after the start of treatment, TGI=56.9% (V 平均 =33.4±16.1 mm 3 , while the control group's V 平均 =77.3±32.0mm 3 , p<0.315). From day 14 to day 17, TGI was observed to increase to 60.6% (V 平均 =47.5±16.8mm 3 , while the control group's V 平均 =120.6±44.4mm 3 , p<0.193) and 68.9% (V 平均 =58.0±18.2mm 3 , V of the control group 平均 =186.4±65.0mm 3 , p<0.070). On the 21st day after the start of treatment and until the end of the observation period, TGI was below the biologically significant value (<50%). Before the end of the observation period, the number of dead mice in this group was 2 out of 7.

[0164] The final results of the measurements and the statistical significance of the differences between the groups are shown in Tables 10 and 11.

[0165] Table 10.

[0166] The average tumor volume of mice in each group observed by SEM (M±m)

[0167]

[0168] Table 11.

[0169] Effects of drugs and tamoxifen on SKBR-3 growth inhibition during the entire observation period, TGI%

[0170]

[0171] Example 3

[0172] Control group: There were 12 mice, each with 12 tumors. At the beginning of treatment, V 平均 =3.8±1.0mm 3 Without specific treatment, the tumor size reached V平均 =299.1±100.5mm 3 Before the end of the observation period, the number of dead rats in this group was 6 out of 12.

[0173] Drug group, 10 mg / kg: There were 10 mice, 10 tumors in each group. At the beginning of treatment, V 平均 =1.6±0.4mm 3 On the 7th day after the start of treatment, a significant TGI of 66.1% (V 平均 =27.0±5.1 mm 3 , while the control group's V 平均 =79.6±14.2mm 3 , p<0.002). On the 10th day after the start of treatment, TGI=78.7% (V 平均 =21.6±4.9mm 3 , while the control group's V 平均 =101.2±22.5mm 3 , p<0.007). On the 14th day after the start of treatment, the maximum non-significant TGI was recorded = 83.1% (V 平均 =27.6±2.6mm 3 , while the control group's V 平均 =163.3±31.0mm 3 , p<0.06). On the 17th day after the start of treatment, TGI decreased to 76.1% (V 平均 =46.3±5.3mm 3 , while the control group's V 平均 =193.6±39.6mm 3 , p<0.04). Before the end of the observation period, the number of dead mice in this group was 10 out of 10.

[0174] Tamoxifen group, 30 mg / kg: There were 10 mice, 10 tumors in each group. At the beginning of treatment, V 平均 =4.1±1.2mm 3 On day 7 and until the end of the observation period, TGI was below the biologically significant threshold and did not exceed 44.5% (V 平均 =44.1±8.8mm 3 , while the control group's V 平均 =79.6±14.2mm 3 , p<0.10). Before the end of the observation period, the number of dead mice in this group was 4 out of 10.

[0175] Tamoxifen group, 10 mg / kg: There were 10 mice, 10 tumors in each group. At the beginning of treatment, V 平均 =2.3±0.8mm 3 , which was not significantly different from the control group. In this group, no tumor growth inhibition was detected during the entire observation period. Before the end of the observation period, the number of deaths in this group was 6 out of 9 mice.

[0176] The measured results and the statistical significance of the differences between the groups are shown in Tables 12 and 13.

[0177] Table 12.

[0178] The average tumor volume of mice in each group observed by SEM (M±m)

[0179]

[0180] Table 13.

[0181] Effect of drugs and tamoxifen on TNBC_Kad growth inhibition during the entire observation period, TGI%

[0182]

[0183] The antitumor activity of the drug was compared in two phenotypically different subcutaneous xenograft models of human SKBR-3 and TNBC-Kad breast cancer in athymic, immunodeficient Balb / c nude mice after multiple administrations.

[0184] Preliminary experiments on the antitumor activity of the drug in the SKBR3 model compared with tamoxifen (dosage form) showed that treatment with the drug at a dose of 10 mg / kg was the most effective among all the treatment regimens studied: on the 10th day after the start of treatment, a significant antitumor effect was manifested, i.e., the maximum TGI = 91.2% (V 平均 =2.2±2.0mm 3 , while the control group's V 平均 =24.9±12.5mm 3 , p<0.018), and the drug was well tolerated. Tamoxifen at a dose of 10 mg / kg had no effect: no biologically significant TGI values ​​were reached during the entire observation period. On the contrary, tumor growth stimulation was recorded from day 10 to day 17 after the start of treatment.

[0185] Repeated experiments on the antitumor activity of the drug in the same SKBR3 model compared to tamoxifen (control drug) showed that the drug at a dose of 10 mg / kg had a high antitumor effect, reaching the maximum significant TGI on day 14 (94.3%, p<0.03) and day 24 (86.3%, p<0.04), which reproduced the results obtained in the first experiment.

[0186] Compared with tamoxifen (control drug), experiments on the antitumor activity of the drug in the TNBC_Kad model showed that on the 7th and 10th days after the start of treatment, the drug at a dose of 10 mg / kg inhibited tumor growth by 66.1% and 78.7% (significant differences were observed between the control group and the 30 mg / kg tamoxifen group). This effect lasted for 10 days and was roughly at the same level. Tamoxifen at a dose of 30 mg / kg produced a weaker and less significant tumor growth inhibition effect on the 7th day, with an inhibition rate of 44.5%, which decreased at the end of the observation. Tamoxifen at a dose of 10 mg / kg had no effect in this model.

[0187] Industrial Applicability

[0188] The experiments carried out show that the claimed use of the compounds is characterized by:

[0189] - No uterotropic activity,

[0190] - Broad anticancer activity, including activity against triple-negative breast cancer,

[0191] - Non-toxic,

[0192] - has cholesterol-lowering activity and has no effect on serum triglyceride levels,

[0193] -No adverse effects on the endometrium.

[0194] The present invention can be used for:

[0195] -Monotherapy and adjuvant therapy for early-stage hormone-positive breast cancer in postmenopausal women;

[0196] - Early-stage monotherapy and adjuvant therapy for hormone-positive breast cancer in postmenopausal women who have been treated with tamoxifen for 2-3 years;

[0197] - Treatment of advanced breast cancer;

[0198] -Treatment of triple-negative breast cancer;

[0199] - Monotherapy and adjuvant therapy for hepatocellular carcinoma;

[0200] - Monotherapy and adjuvant therapy for gastric cancer;

[0201] - Monotherapy and adjuvant therapy for lung cancer;

[0202] -Monotherapy and adjuvant therapy for chronic myeloid leukemia.

Claims

1. Use of 3-O-aminosulfonic acid-16,16-dimethyl-D-homoenanthrin of formula (3) in the preparation of anticancer agents, The anticancer agent is used for the treatment of a patient having a disease selected from the group consisting of: Hepatocellular carcinoma, Gastric cancer, Lung cancer, Chronic myeloid leukemia, and Triple-negative form of breast cancer.

2. The method according to claim 1, wherein the patient is a female patient suffering from early-stage triple-negative form of breast cancer.

3. The use according to claim 1, wherein the patient is a female patient suffering from advanced triple-negative form of breast cancer.

4. The use according to any one of claims 1 to 3, wherein the treatment is monotherapy or adjunctive therapy.

5. The use according to any one of claims 1 to 4, wherein the patient has been treated with tamoxifen.

6. The use according to any one of claims 1 to 5, wherein the patient is a postmenopausal woman.

7. The use according to any one of claims 1 to 6, wherein 3-O-aminosulfonic acid-16,16-dimethyl-D-homoequinostranolone is for oral administration.

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