RU2506085C1 - Tetraethyl-2-(2,2,6,6-tetramethylpiperidin-4-ylamino)-ethylene-1,1-bisphosphonate possessing anticancer activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to an anticancer compound of formula

.

EFFECT: what is presented is the new anticancer compound possessing a high selectivity index in relation to cancer cell as compared to human and animal normal phenotype cells to be used in medicine and veterinary science for treating cancer and preventing metastases, including those including osseous lesion.

2 ex, 2 tbl

Description

The invention relates to new biologically active chemical compounds belonging to the class of bisphosphonates (polyphosphonates) with high antitumor activity, which can be used in medicine and veterinary medicine for the treatment of cancer and the prevention of tumor metastasis, including tumors with bone damage.

Oncological diseases are one of the most serious and dangerous human diseases, they are one of the main causes of human death in the modern world. To achieve the maximum effect of cancer treatment, a combination of different approaches is used. As a rule, a surgical method is used to remove the tumor, followed by treatment with chemotherapy and radiation therapy. However, the complexity of the treatment is due to the large number of various human tumors, metastasis and the resistance of many tumors to chemotherapy and radiation therapy. In recent years, new approaches such as photodynamic therapy, oncolytic viruses, antibody and immune cell immunotherapy, gene therapy, and cancer antigen vaccines have been tried out for cancer treatment. Experiments in animals have shown very encouraging results, but they also made it possible to draw the main conclusion - tumor treatment should be strictly individual, which necessitates the selection of a specific type (s) and treatment method (s).

Today, many different chemotherapeutic agents are known for the treatment of cancer. However, their number and the main quality do not completely cover the needs of the fight against cancer and require the creation of new antitumor drugs.

State of the art

One of the promising anticancer drugs is zoledronic acid, which belongs to polyphosphonates (V.G. Ivanov, V.F. Semiglazov, V.V. Semiglazov, PMPaltuev, P.V. Krivorotko, A.A. Malodusheva, S.S. Gurbanov, RV Donskikh. Bisphosphonates in clinical oncology: use in patients with solid tumors // Medical journal: Farmateka. - M .: Bionika, 2007. - No. 18. - P.23-28), widely used for the treatment of oncological bone diseases. The drug is available under the names "Rezorba", "Zometa", "Zomera", "Aclasta", "Reclast". Its anticancer activity is well understood. A medication is known based on bisphosphonates or aminogluconates in the form of salts for the prevention and treatment of osteoporosis, osteoarthritis, cancer, hypercalcinemia, multiple myeloma (Chinese patent No. CN 1475218, IPC A61K 31/7008, publ. 18.02.2004). The use of bisphosphonate in the preparation of a medicament for the treatment of osteoblastic metastases associated with prostate cancer is known. Bisphosphonate administered as part of the drug is used in addition to antitumor therapy.

Also known are some other bisphosphonates that have found application in medical practice. For example, compounds such as pamidronic, alendronic, risedronic, tiludronic, clodronic acids and some pharmaceutically acceptable salts thereof. Some bisphosphonates also include an imidazole, oxazole, isooxazole, oxadiazole, thiazole, thiadiazole, pyridine, 1,2,3-triazole, 1,2,4-triazole or benzimidazole groups, etc.

Bisphosphonates are also widely used to suppress osteoclast activity in various benign and malignant diseases that cause excessive or inappropriate bone resorption. More recently, they have been used to treat bone metastases and in multiple myeloma (see Fleisch H, 1997, Bisphosphonates clinical. In Bisphosphonates in Bone Disease. From the Laboratory to the Patient. Eds: The Parthenon Publishing Group, New York / London , pp. 68-163).

The closest analogue of the invention is a pharmaceutical product based on bisphosphonates, for example, in the form of a ready-to-use solution of zoledronic acid (formula I) or its pharmaceutically acceptable salt (RF patent No. 2358739, IPC A61K 31/663, publ. 06/20/2009) .

However, the above analogues have the following disadvantages: often they have a complex multi-stage production technology, which ultimately increases the cost of drugs. In addition, instructions for the use of zoledroic acid preparations contain a warning regarding their renal toxicity. The Food and Drug Administration (US Department of Health) has ordered zoledronic acid manufacturers to write in the annotations that this drug increases the risk of kidney failure. As a warning, zoledronic acid can also lead to severe dehydration of patients. Blood creatinine monitoring is recommended to evaluate the patient's kidneys.

Another important circumstance is that the experience of using various antitumor drugs shows: (a) the need to use a combination of several drugs to increase the effectiveness of tumor treatment; (b) the existence of tumors that are resistant to the action of one drug, which requires the use of several different chemotherapeutic agents, depending on the treatment tactics; (c) individual patient tolerance to a chemotherapy drug can also vary within very wide limits. This is one of the significant drawbacks of existing chemotherapy drugs. This drawback can be overcome only through the creation of new highly effective antitumor drugs for the complex chemotherapy of cancer.

The technical result of the invention is to obtain a new chemical compound having a production technology using available reagents, with a high selectivity index (the ability to destroy cancer cells without affecting cells with a normal phenotype) and a pronounced anticancer effect against various types of human and animal tumors.

The technical result is achieved by the creation of a new biologically active organic compound - tetraethyl-2- (2,2,6,6-tetramethylpiperidin-4-ylamino) ethylene-1,1-bisphosphonate, formula II:

exhibiting antitumor activity against various types of human and animal tumors, which may find application in medical practice as a drug for the treatment of cancer.

Compound II is prepared by reacting 4-amino-2,2,6,6-tetramethylpiperidine with tetraethyl-ethen-1,1-diylbis (phosphonate) taken in a 1: 1 ratio without solvent for 10 hours according to the following scheme:

Comparative analysis with analogues shows that the claimed compound is fundamentally different from the analogue [RF patent No. 2358739] in that it contains a tetramethylpiperidine molecule in the immediate vicinity of the bis (phosphonate) part.

Example 1. Obtaining tetraethyl-2- (2,2,6,6-tetramethylpiperidin-4-ylamino) ethylene-1,1-bisphosphonate (II). To 180 mg (0.6 mmol) of tetraethyl-ethen-1,1-diylbis (phosphonate), 94 mg (0.6 mmol) of 4-amino-2,2,6,6-tetramethylpiperidine is added. After 12 hours, there is a complete absence of the starting reagents (control by ¹ N NMR). Purification using column chromatography (SiO ₂ , CHCl ₃ : MeOH 18: 1, R _f = 0.8). Liquid. Yield: 85%. Found,%: C 49.75, H 9.36, N 6.10, P 13.38. C ₁₉ H ₄₂ N ₂ O ₆ P ₂ . Calculated,%: C 49.99, H 9.27, N 6.14, P 13.57.

IR (KBr, cm ^-1 ): 975.8, 1024.0, 1099.2, 1164.8, 1249.7, 1392.4, 1444.4, 1479.2, 1643.1, 2875.4, 2908.2, 2935.2, 2985.3

¹ H NMR (400 MHz, CDCl ₃ , δ ppm): 0.84 (ABd, 2H, J = 11.6 Hz, 12.7 Hz, 2 * CH ₂ ax); 1.10 (s, 6H, 2 * CH ₃ ); 1.18 (s, 6H, 2 * CH ₃ ); 1.34 (t, 12H, J = 7 Hz, 4 * CH ₃ ); 1.82 (AVd, 2H, J = 3.6 Hz, 12.7 Hz, 2 * CH ₂ , equiv); 2.58 (tt, 1H, J = 6 Hz, 23.5 Hz, P-CH-P); 2.90 (tt, 1H, J = 3.6 Hz, 11.6 Hz, CH-NH); 3.19 (dt, 2H, J = 6 Hz, 17 Hz, CH ₂ -NH); 4.12-4.22 (m, 8H, 4 * CH ₂ ). ¹³ C NMR (100 MHz, CDCl ₃ , δ ppm): 16.25 (dd, J = 2.4 Hz, 6.3 Hz, 4 * CH ₃ , Et); 28.47 (2 * CH ₃ ); 34.87 (2 * CH ₃ ); 37.71 (t, J = 132 Hz, P-CH-P); 42.33 (t, J = 4 Hz, NH-CH ₂ ); 45.63 (2 * CH ₂ ); 49.02 (CH-NH); 50.78 (2 * CMe ₂ ); 62.52 (dd, J = 6.7 Hz, 33.5 Hz, 4 * CH ₂ , Et).

The method of obtaining the claimed substance is simple to implement (is implemented in one stage), the starting materials are available, the yield is 85% of theoretically calculated. The proposed compound is stable in air, readily soluble in chloroform, dimethylformamide and dimethyl sulfoxide, slightly soluble in water. It is well soluble in water with a slight acidification.

Example 2. The study of the antitumor activity of the claimed compounds.

We used A431 tumor cell cultures (human epidermoid carcinoma cells), SW480 (human colon adenocarcinoma cells), RD (human rhabdomyosarcoma), Mel-8 (human melanoma), and normal diploid culture MRC-5 (pulmonary epithelial cells of a human embryo). Cells were cultured in DMEM Needle medium (Gibco BRL) containing 5-10% fetal calf serum (Gibco BRL), 2 mM L-glutamine, 80 μg / ml gentamicin sulfate at 37 ° C in plastic culture bottles (Costar, USA). To remove cells from the substrate, a 0.25% trypsin solution and a 0.02-versene solution (FGUN SSC VB "Vector") were used in a 1: 1 ratio.

Determination of antitumor (cytotoxic) activity of compounds in vitro (MTT test). The MTT test was performed as described in [Niks M and Otto M. (1990) Towards an optimized MTT assay. J. Immunol. Meth. 130 (1), 149-151]. A431, SW480, MRC-5 cells at a concentration of 5 × 10 ⁴ in 100 μl of DMEM needle containing 10% fetal calf serum were placed in wells of 96-well culture microtiter plates and incubated for 24 hours at 37 ° C in an atmosphere containing 5% CO ₂ . Then, 50 μl of the test compounds dissolved in DMSO and their triplicate dilutions were added to the wells. Triple dilutions of DMSO in culture medium were added to control wells. Each experimental point was performed in triplicate (three wells for each dilution of the corresponding compound and control). After that, the culture microplates were incubated for 48 hours, 96 hours and 168 hours under the above conditions. Then, in the corresponding culture microplates, the medium containing the test compounds was removed, 200 μl of serum-free medium was added to the wells, 10 μl of MTT solution (initial concentration of 5 mg / ml in phosphate buffer) was added, and the microplates were further incubated for 6 hours. At the end of the incubation, the medium was removed and 150 μl of DMSO was added to dissolve the blue formazan crystals formed as a result of the reaction. The optical density of the formazan dissolved in DMSO was measured on a Multiscan microplate spectrophotometer at a wavelength of 492 nM. A 50% cytotoxic concentration (CC ₅₀ ) was calculated according to [Niks M and Otto M. (1990) Towards an optimized MTT assay. J. Immunol. Meth. 130 (1), 149-151].

Antitumor (cytotoxic) activity against tumor and normal human diploid cells are presented in table 1. The values of CC ₅₀ (μg / ml) are given taking into account the approximate solubility of the studied compounds in DMSO. The MRC-5 cell culture was used as control normal cells in order to determine the possible selectivity of the cytotoxic effects of the test compounds against tumor cells. The degree of selectivity of cytotoxicity was expressed through a selectivity index (IS), which is the ratio of the CC _{50 of the} test compound for cells of normal phenotype to TC ₅₀ (toxic concentration) for tumor cells. A sufficiently high antitumor activity is indicated for the claimed compound (II) and its analogue (I). The cytotoxicity of these compounds for tumor cell cultures varies in the range of CC ₅₀ values from 0.4 to 31 μg / ml and is comparable to the cytoxic activity of the commercially available antitumor preparation embihin (CC ₅₀ for A431-3 μg / ml cells.

For the claimed compound (II), a selectivity of cytotoxicity was found immediately for two used tumor cell cultures A431 and SW-480 with a selectivity index of 5.9 and 9.1, respectively. The selectivity indices of currently used antitumor chemotherapeutic agents usually do not exceed 4-8 [Ring C.J. (2002) Cytolytic viruses as potential anti-cancer agents. J. of Gen. Virol., 83, 491-502]. It is noteworthy that compound (I) (analogue) showed its cytotoxic activity at a later incubation time (96 and 168 hours) with respect to only one type of A431 cells with a high selectivity index only after 168 hours incubation, whereas the claimed compound (II) was effective during the entire observation period, that is, it showed a pronounced antitumor activity after 48 hours of incubation.

Note: The tested compounds (III) and (IV) were synthesized by the applicants and were used as comparison compounds, not showing significant cytotoxicity in relation to the studied cell cultures (negative control).

The cytotoxicity of compound (II) was further investigated using human tumor cultures: MCF-7 (breast cancer), RD, Mel-8, and one human cell culture of normal phenotype — MCF-10A. Compound (II) also has selective cytotoxicity to tumor cells RD and Mel-8 with respect to both normal cell cultures (IP from 2.2 for MRC5 / RD and Mel-8 to 11.1 for MCF-10A / SW480).

Thus, the technical result of the invention is confirmed by the fact that the claimed compound, formula II, exhibits high antitumor activity against human cancer cells A431, SW-480, RD, Mel-8 with a selectivity index of 2.2 to 11.1.

Example 3. Evaluation of the antitumor effect of the claimed preparation (II) for C57BL / 6 mice with tumor inoculated with Lewis mouse pulmonary carcinoma tumor CRL-1642 LLC.

The study was carried out according to the methodology described in [Guide to the experimental (preclinical) study of new pharmacological substances. / Ed. By R.U. Khabriev, M .: 2005]. A Lewis murine pulmonary carcinoma cell culture CRL-1642 LLC was cultured as a monolayer in Eagle DME-F12 medium (Gibco BRL) containing 5-10% fetal calf serum (Gibco BRL), 2 mM L-glutamine, 80 μg / ml gentamicin sulfate at 37 ° C in culture plastic bottles (Costar, USA). Inoculation of tumor cells was carried out by subcutaneous injection of 10 ⁷ CRL-1642 LLC cells in 0.2 ml of DMEM-F12 medium to female C57BL / 6 mice weighing 16-18 g. C57BL / 6 mice were treated with test compound (II) twice by intraperitoneal injection of 0.2 ml a solution of compound (II) in physiological solution: the first injection one day after the introduction of tumor cells, the second after 13 days. Tetraethyl-2- (2,2,6,6-tetramethylpiperidin-4-ylamino) ethylene-1,1-bisphosphonate (II) was administered at a dose of 1.0 mg per mouse per injection. The choice of dose of compound (II) was determined by its toxicity to C57BL / 6 mice and was determined in preliminary experiments. Cyclophosphamide was used as a reference drug. Cyclophosphamide (analogue) is a widely used pharmacopoeial highly effective antitumor drug, also available under the trademarks Endoxan, Cytoxan, Neosar, Procytox, Revimmune, Cyclophosphamide (Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" in Pazdman R., Pazdur L. Cam. KA, Hoskins WJ. (Eds) Cancer Management: A Multidisciplinary Approach. 11 ed. 2008). The drug has long been widely used to treat people. Cyclophosphamide has a wide spectrum of antitumor activity against small cell lung cancer, ovarian cancer, breast cancer, cervical and uterine cancer, bladder cancer, prostate cancer, neuroblastoma, retinoblastoma, lymphogranulomatosis, lymphosarcoma, non-Hodgkin’s lymphoma, reticulosarcoma myoma, osteocarcoma myoma, , chronic lymphatic and myeloid leukemia, acute lymphatic, myeloid, and monoblastic leukemia, as well as a number of mycoses and autoimmune diseases.

Cyclophosphamide was administered at the dose of 1.0 mg / mouse as described above. A group of control mice with inoculated tumors were injected with 0.2 ml PBS per injection. Each group consisted of 10 animals. Tumor progression was evaluated visually and by measurement on days 21 and 32 after grafting. The volume of the tumors was determined as follows: the maximum length of the tumor, multiplied by its width squared, divided by two. For each group of animals, the average life expectancy (LSS) was calculated.

The toxicity level of the claimed compound (II) and the effective chemotherapeutic anticancer comparison drug (cyclophosphamide) for mice was determined with a single intraperitoneal administration. Toxicity was expressed through LD ₅₀ (50% lethal dose). LD ₅₀ for the studied compounds amounted to:

> 5.0 mg / mouse for tetraethyl-2- (2,2,6,6-tetramethylpiperidin-4-ylamino) ethylene-1,1-bisphosphonate (claimed compound);

> 5.0 mg / mouse for cyclophosphamide.

The results of evaluating the antitumor activity of compound (II) are presented in Table 2. 100% efficiency of grafting of Lewis sarcoma was noted, accompanied by varying degrees of progression of grafted tumors in experimental and control groups of animals. At the time of the first measurement of tumor volumes (21 days after inoculation of tumors, 20 days after the first injection and 7 days after the second injection of the test compounds), a pronounced inhibitory effect was observed in groups of animals treated with cyclophosphamide and tetraethyl-2- (2,2,6,6 -tetramethyl-piperidin-4-ylamino) ethylene-1,1-bisphosphonate (II). Moreover, the maximum effect for this period was observed in the group of animals after treatment with cyclophosphamide. So, the average volume of tumors in the group of cyclophosphamide was 0.26 ml, and in the group of tetraethyl-2- (2,2,6,6-tetramethylpiperidin-4-ylamino) ethylene-1,1-bisphosphonate (II) - 0.92 ml and 2.47 ml in the control group. At the time of the second measurement of tumor volumes (32 days after the inoculation of tumors, 31 days after the first injection and 18 days after the second administration of the test compounds), significant inhibition of tumor progression was noted in groups of animals treated with bisphosphonate (II) and cyclophosphamide. In animals, after administration of the claimed compound (II), the effect is somewhat more pronounced for this period than in those receiving cyclophosphamide: the average tumor volume is 2.70 ml and 3.22 ml, respectively, against 6.05 ml in the control group of mice. The results of determining the average life expectancy in groups of animals are in good agreement with these data: an increase in life expectancy in relation to the control group of animals by approximately 25 percent (8 days) in the group of the claimed compound (II) and 15% (5 days) in the group of animals treated cyclophosphamide.

It is possible that changing the treatment regimen taking into account the pharmacodynamics and pharmacokinetics of the claimed compound will allow for more pronounced inhibition of tumor growth, which will be accompanied by a significant increase in average life expectancy.

The mouse model shows that tetraethyl-2- (2,2,6,6-tetramethylpiperidin-4-ylamino) ethylene-1,1-bisphosphonate (II) (the claimed compound) has a pronounced antitumor activity, which is characterized by a decrease in tumor size , a significant increase in the average life expectancy of animals. According to its antitumor activity, the claimed compound is superior to the activity of the highly active pharmacopeia anticancer drug cyclophosphamide.

Thus, the technical result of the invention is confirmed by the fact that the claimed preparation has a pronounced antitumor activity in relation to various tumor cell cultures of a person, such as A431, SW-480, RD, Mel-8, with a selectivity index of 2.2 to 11, one. The inventive preparation is capable of exerting a significant antitumor effect in the treatment of mice inoculated with a tumor inoculated with Lewis mouse lung carcinoma CRL-1642 LLC. In animal experiments, it provides a reduction in tumor size and a significant increase in animal life span.

Claims (1)

Tetraethyl-2- (2,2,6,6-tetramethylpiperidin-4-ylamino) ethylene-1,1-bisphosphonate of the following formula:

possessing antitumor activity in relation to human and animal tumors in vitro and in vivo.