RU2804771C1 - Complex antiangiogenic and hypoxia-targeted antitumor agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is related to a complex antiangiogenic and hypoxia-oriented antitumor agent containing the chemical compound 1-cyclohexanoyl-2-ethylisothioureaα-cyano-4-hydroxycinnamate as an active substance of general formula (I), which has NOS-inhibitory and MCT-inhibitory activity and has a combined antiangiogenic and hypoxia-oriented toxic effect on neoplasia.

EFFECT: complex antiangiogenic and hypoxia-oriented antitumor agent that provides a more pronounced, stable and long-lasting antitumor effect against neoplasia of various histogenesis, without increasing toxic effects.

2 cl, 3 tbl, 3 ex

Description

The invention relates to medicine, namely to new pharmacologically active compounds that have complex antiangiogenic and hypoxia-oriented effects, and can be used in oncology, as targeted therapy for solid malignant neoplasms.

Oncological diseases represent a serious problem for modern medicine. According to medical statistics, in 2020, in the Russian Federation alone, 3.2 million patients were registered with various oncological diseases (Kaprin A.D., Starinsky V.V., Shakhzadova A.O. State of oncological care for the population of Russia in 2020. M.: MNRIO named after P. A. Herzen - branch of the Federal State Budgetary Institution "National Medical Research Center of Radiology" of the Ministry of Health of Russia, 2021. 239 pp. ISBN 978-5-85502-262-9). Despite continuous improvements in radiotherapy, chemotherapy, and methods for early diagnosis of cancer, many common malignancies remain difficult to treat.

According to leading experts in the field of oncology, changing the current picture is possible only with the introduction into clinical practice of targeted antitumor drugs, the targets of which are biochemical or pathophysiological processes specific to malignant neoplasms or certain types of them (Willett C.G., Duda D.G., di Tomaso E. et al. Complete pathological response to bevacizumab and chemoradiation in advanced rectal cancer. Nat. Clin. Pract. Oncol. 2007. 4(5): 316-21; Lein S., Lowman H. B. Therapeutic anti-VEFG antibodies. Handb. Exp. Pharmacol 2008. 181: 131-50).

Thus, it is known that two broad groups of antiangiogenic drugs are currently approved for clinical use or are undergoing clinical trials. The first is drugs that narrowly selectively block the VGEF–VGEFR system (bevacizumab, aflibercept, ramucirumab), the second is multi-targeted tyrosine kinase inhibitors (sorafenib, sunitinib, axitinib, pazopanib, lenvantinib, vandetanib, cabozantinib, apatinib, regorafenib, imatinib, dasatinib, temsirolimus) , the action of which blocks several angiogenic receptors, signaling or hormonal pathways that stimulate angiogenesis. This diversity of drugs is due to the complex organization of tumor angiogenesis, which provides many molecular targets for therapeutic intervention.

However, the clinical results of modern antiangiogenic therapy remain quite limited due to the rapid adaptation of tumors to such influences. Moreover, malignant neoplasms acquire resistance not only to drugs that act on one signaling pathway (VEGF inhibitors), but also to multikinase inhibitors (Itatani Y.; Kawada K.; Yamamoto T. et al. Resistance to Anti-Angiogenic Therapy in Cancer- Alterations to Anti-VEGF Pathway. 2018. 19(4):1232). It has been shown that long-term use of antiangiogenic drugs causes the development of intratumoral hypoxia, which induces transcription factors that promote the adaptation of tumor cells to antiangiogenic effects (Mendez-Blanco C., Fondevila F., Garcia-Palomo A. et al. Sorafenib resistance in hepatocarcinoma: role of hypoxia-inducible factors. ExpMolMed. 2018. 50(10):1-9).

Along with synthetic antiangiogenic pharmacological drugs, agents of natural origin are also known that have similar activity. In particular, curcumin is known (Application for invention US No. 96116127, published on November 10, 1998), which is a polyphenolic compound extracted from the rhizomes of Curcuma longa, Curcuma zedoaria and Acorus calamus L., which has antiangiogenic, anti-inflammatory, antioxidant, and antiproliferative effects.

The disadvantage of curcumin is its poor chemical stability and low oral bioavailability. The main reasons contributing to low levels of curcumin in plasma and tissues are due to poor absorption, rapid metabolism and rapid systemic excretion of curcumin from the body (Kharat M, Du Z, Zhang G, McClements DJ. Physical and chemical stability of curcumin in aqueous solutions and emulsions : impact of pH, temperature, and molecular environment. J Agric Food Chem. 2017; 65(8):1525–1532).

An antiangiogenic drug based on tannins is known (patent RU No. 2634253, published on October 24, 2017), which as an active substance contains dimeric macrocyclic tannin isolated from the shoots and inflorescences of Chamerion angustifolium, composition C68H48O44, which has an antiangiogenic effect.

A significant disadvantage of this drug is the lack of selectivity for vascular endothelial growth factor receptors and, as a consequence, the lack of significant antitumor effect.

In recent decades, the important role of endogenous nitric oxide (NO) in the processes of tumor angiogenesis has been established (Wang H., Wang L., Xie Z. et al. Nitric Oxide (NO) and NO Synthases (NOS)-Based Targeted Therapy for Colon Cancer. Cancers (Basel). 2020. 12(7):1881 ). There are a number of promising studies on the use of effective nitric oxide synthase inhibitors (NOS inhibitors) in the treatment of malignant tumors of certain locations. In particular, the authors of this invention have previously shown the promise of using some original isothioureas with NOS-inhibiting action (RU patents No. 2503450, published 01/10/2014; No. 2699558, published 09/06/2019; No. 2751776, published 07/16/2021) in as antiangiogenic agents (Filimonova M.V. et al. Comparative study of the effect of the NOS inhibitor T1023 and bevacizumab on the growth and morphology of Lewis lung carcinoma. Pat. Physiol. Exp. Ter. 2019. T. 63(2). P. 89-98 ; Filimonova M.V. et al. Experimental study of the antitumor activity of the new nitric oxide synthase inhibitor T1023. Mol. Med. 2015. No. 1. P. 61-64). At the same time, for NOS inhibitors there is also a drawback characteristic of pharmacopoeial antiangiogenic drugs - the rapid development of resistance in malignant tumors to such antiangiogenic effects.

The prototype of the proposed invention is the original isothiourea - 1-cyclohexanoyl-2-ethylisothiourea hydrobromide (compound T1059), with the structural formula:

,

which was previously obtained and studied by the authors of this invention (RU patent No. 2552529, published on June 10, 2013).

Compound T1059 is an effective competitive inhibitor of endothelial and inducible isoforms of NOS (eNOS and iNOS), which play a leading role in the regulation of vascular tone and the processes of tumor angiogenesis. According to experimental data, compound T1059, when administered chronically in doses of 50-80 mg/kg, causes the development of tumor vascularization disorders.

However, a significant drawback of the prototype is the short duration of its antiangiogenic effect due to the rapid development of resistance in experimental neoplasias.

To adapt to hypoxia, tumor cells use multiple mechanisms. Active expression of hypoxia-inducible factors (HIFs) activates metabolic pathways that contribute to increased hypoxic tolerance. In particular, this is facilitated by stimulation of glucose and lactate metabolism - high levels of HIF-1α induce the expression of the glucose transporter GLUT1. And the increased supply of glucose into hypoxic tumor cells ensures their energy metabolism along glycolytic pathways with the formation of lactate and H ⁺ . And the development of acidosis in these cells is also counteracted by HIF-1α-dependent expression of monocarboxylate lactate transporters (MCTs), which at the same time prevent acidification of the intracellular environment.

A number of studies have shown that overexpression of MCT1 and MCT4 isoforms plays an important role in the survival and proliferation of a pool of hypoxia-resistant tumor cells. Moreover, the use of MCTs inhibitors, by suppressing lactate excretion, can cause the death of hypoxic tumor cells (Guan X., Morris M. In Vitro and In Vivo Efficacy of AZD3965 and Alpha-Cyano-4-Hydroxycinnamic Acid in the Murine 4T1 Breast Tumor Model. 2020 22(4): 84; Wang Y., Qin L., Chen W. et al. Novel strategies to improve tumor therapy by targeting the proteins MCT1, MCT4 and LAT1. Eur J Med Chem. 2021. 226:113806).

Considering that the antiangiogenic effect of NOS inhibitors increases intratumoral hypoxia, it can be assumed that their antitumor activity will be enhanced when combined with agents targeting the hypoxic pool of tumor cells - in particular, with MCTs inhibitors. To implement such a strategy, it seems appropriate to use α-cyano-4-hydroxycinnamic acid (CHC) with the structural formula:

,

which is a selective inhibitor of MCT1 and MCT4 (Puri S., Juvale K. Monocarboxylate transporter 1 and 4 inhibitors as potential therapeutics for treating solid tumors: A review with structure-activity relationship insights Eur J Med Chem. 2020. 199:112393).

Moreover, the chemical and physicochemical properties of the compound T1059 and CHC (T1059 is a weak base, and CHC is a strong organic acid) made it possible to realize the combination of these two compounds at the chemical level in the form of 1-cyclohexanoyl-2-ethylisothiourea salt α-cyano-4- hydroxycinnamate (hereinafter referred to as compound T1114), with the structural formula:

.

The molecular structure of compound T1114 contains a fragment that has a NOS-inhibiting, antiangiogenic effect (1-cyclohexanoyl-2-ethyl isothiourea), and a fragment that has an MCT-inhibitory effect that causes the death of a resistant hypoxic pool of tumor cells (α-cyano-4-hydroxycinnamate) .

The technical result of the proposed invention is to create a new complex antiangiogenic and hypoxia-oriented antitumor agent that provides a more pronounced, stable and long-lasting antitumor effect against neoplasia of various histogenesis, without increasing toxic effects.

The technical result is achieved by the fact that the complex antiangiogenic and hypoxia-oriented antitumor agent contains the chemical compound 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate as an active substance, with the general formula:

,

possessing NOS-inhibiting and MCT-inhibiting activity, and having a combined antiangiogenic and hypoxia-oriented toxic effect on neoplasia.

The chemical synthesis of compound T1114 and its antitumor activity, as well as the achievement of the stated technical result, are reflected in the examples.

The essence of the present invention is the production of the chemical compound 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate and the use of this compound as an antiangiogenic and hypoxia-oriented antitumor agent.

Example 1. Synthesis of the chemical compound 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate (compound T1114).

3 g (10 mmol) of 1-cyclohexanoyl-2-ethylisothiourea hydrobromide was dissolved in 10 ml of water. A concentrated aqueous solution of ammonia was added dropwise to pH 8 (5 ml). The separated oil was extracted with ether (2x 15 ml). The ethereal extract was washed with water and dried using Na ₂ SO ₄ . The solvent was evaporated, the oily residue was 2 g (9.3 mmol) of 1-cyclohexanoyl-2-ethylisothiourea base. Dissolve 2 g (9.3 mmol) of base in 2 ml of acetone. Then, 1.76 g (9.3 mmol) of α-cyano-4-hydroxycinnamic acid was dissolved under heating in 10 ml of acetone with the addition of 1 ml of dimethyl sulfoxide. The hot solution was poured into a solution of the base in acetone and left for 30 minutes at room temperature, and then put into the refrigerator. The resulting white precipitate was filtered off and recrystallized from ethyl acetate. Yield 1.6 g (39.7%). T _pl 105-108 ⁰ C (with decomposition). PMR spectrum (DMSO-d6, δ): 1.21-1.23 (m., 9 N); 1.60-1.67(m., 4H); 2.0 (s., 1H); 2.2 (t., 1H); 2.9 (t., 2H); 4.0 (t., 1H); 6.95 (d., 2H); 7.95(d.,2H); 8.2 (s., 1H); 10.4-11.0 (w., 2H). Calculated (%): C 57.0; H 6.46; N 9.97. Found (%): C 57.36; H 6.22; N 10.1. Chemical formula: C ₂₀ H ₂₇ N ₃ O ₅ S.

In the available scientific and patent literature, methods for preparing 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate and its properties are not described, which indicates that we have obtained this compound for the first time.

Example 2. Evaluation of the antitumor activity of the compound 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate (T1114) in comparison with the prototype compound (T1059) in a model of Ehrlich solid carcinoma (ESC) in mice.

The study was carried out on 60 female F ₁ hybrid mice (CBA x C57Bl/6j) aged 2-2.5 months with a body weight of 19-22 g, randomized into 3 experimental groups of 20 animals each. All animals were transplanted with a suspension of Ehrlich carcinoma (ECS), containing 2.5 * 10 ⁶ million cells in 0.1 ml of medium 199 (PanEko, Russia), into the previously depilated lateral surface of the right thigh . Control animals did not subsequently receive any experimental treatments. Animals of the experimental group, from the 7th to the 20th day of SCE growth, received daily intraperitoneal administration of compound T1114 and compound T1059 in equimolar doses of 96 and 70 mg/kg, respectively (0.1 ml per 10 g of animal body weight). Compound T1114 was used in the form of a suspension, which was prepared on the basis of water for injection (Dalkhimfarm, Russia) with 0.5% 2-hydroxypropyl cellulose ether (hyprolose, Nipponsoda, Japan), solution T1059 was prepared on the basis of water for injection. Antitumor effects were studied according to the method described in the Guidelines for conducting preclinical studies of drugs (Mironov A.N. Guidelines for conducting preclinical studies of drugs. Part one. - M.: Grif i K, 2012. - 944 p.).

In a comparative study of antitumor activity (Table 1), the prototype compound NOS inhibitor T1059, at the dose and administration regimen used, independently had an extremely weak antitumor effect - a statistically significant effect was observed only at the beginning of use. Analysis of the data on inhibition of the growth of tumor nodes showed that the weak effect of the prototype compound T1059 had an extremely weak effect on the inhibition of tumor growth - only by 15-20%.

At the same time, compound T1114, when administered daily i.p. at a dose of 96 mg/kg, had a more stable effect on the growth of SCE in comparison with the prototype compound T1059. The antitumor activity of compound T1114 was realized by the 3-4th day from the start of use, and at the same time it reached its maximum values. The activity of compound T1114 during the entire observation period was moderately pronounced and was accompanied by significant statistical differences in the volumes of tumor nodes in comparison with tumor-bearing animals of the control group. At the same time, compound T1114 stably inhibited tumor growth by 20-35%.

Table 1 - Effect of compounds T1114 and T1059 in equimolar doses on the growth and growth inhibition of SCE in female F1 mice (CBA x C ₅₇ Bl/6j), n=16-18

Observation period, days Average tumor volume (V), relative units (M±σ); SRW index, % Control T1059, 70 mg/kg T1114, 96 mg/kg V V TRO V TRO 6 1.0 1.0 1.0 9 3.1 ± 0.7 2.3 ± 0.5* 25.2 ± 15.7 2.0 ± 0.5* 34.7 ± 17.3 12 4.6 ± 1.2 3.9 ± 1.1 20.3 ± 16.5 3.5 ± 0.7* 24.9 ± 14.4 14 7.4 ± 1.9 6.4 ± 1.7 18.0 ± 16.2 5.8 ± 1.0* 21.6 ± 14.0 16 10.6 ± 3.1 9.1 ± 2.1 17.0 ± 15.9 8.4 ± 1.3* 21.0 ± 12.0 19 16.3 ± 4.0 14.4 ± 4.0 15.9 ± 18.0 12.9 ± 1.3* 20.9 ± 8.3 21 21.3 ± 4.2 18.6 ± 5.0 17.5 ± 17.6 17.7 ± 2.2* 17.1 ± 10.5 23 26.0 ± 4.7 21.2 ± 5.2* 19.9 ± 18.2 21.7 ± 2.6* 18.6 ± 10.0

Symbol * – statistically significant differences with the control group according to the Mann-Whitney U test with the Holm-Bonferroni correction

Analysis of weight dynamics (Table 2) of experimental animals did not reveal statistically significant differences. The relative body weights of animals in the groups receiving treatment corresponded to those of control tumor carriers. At the same time, the development of lethal effects from the use of compound T1114 was not observed, which indicates the safety of the doses used and, in general, the safety of the use of such treatment.

Table 2 - Dynamics of relative body weight in the groups receiving compounds T1114 and T1059.

Observation period, days Body weight, % (M ± SD); Control T1059, 70 mg/kg T1114, 96 mg/kg 6 100.0 100.0 100.0 9 102.9 ± 3.5 103.5 ± 2.5 104.1 ± 4.0 12 103.2 ± 4.0 102.8 ± 2.8 102.9 ± 3.1 14 107.3 ± 4.5 108.1 ± 3.3 107.0 ± 5.1 16 111.8 ± 5.5 111.6 ± 3.9 110.8 ± 4.9 19 117.6 ± 4.8 115.9 ± 4.6 117.4 ± 6.2 21 119.3 ± 4.9 120.2 ± 4.9 121.3 ± 7.4 23 121.3 ± 5.6 122.9 ± 5.0 124.2 ± 6.7

Example 3. Evaluation of the antitumor activity of the compound 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate in comparison with the prototype compound (T1059) in a model of cervical cancer (CC-5) in mice.

The study was carried out on female CBA mice aged 2-2.5 months with a body weight of 19-22 g, randomized into 3 experimental groups: control and two experimental, 20 animals each. All animals were transplanted with a RSM-5 suspension containing 2.5*10 ⁶ million cells in 0.3 ml of medium 199 (PanEco, Russia) into the previously depilated lateral surface of the right thigh. The compounds were administered in the regimens and doses described in example 2.

The results of the study of the antitumor activity of compounds T1059 and T1114 on the RSM-5 model (Table 3), in general, repeat the results obtained on the SCE tumor model in example 2. Independent use of the prototype compound T1059 (70 mg/kg, i.p. daily) had a rather weak effect on the growth of CC-5 in mice, which was accompanied by inhibition of tumor growth by 12-18%. The statistically significant antitumor effect of compound T1059 developed only in the later stages of use.

The independent effect of compound T1114 at an equimolar dose (96 mg/kg, i.p., daily) on the growth of CC-5 was significantly more pronounced. A significant antitumor effect of compound T1114 developed by the end of the first week of use and then remained at a stable level - TPO 22-25%.

Thus, the presence of a fragment of α-cyano-4-hydroxycinnamic acid in the composition of compound T1114 significantly enhances its antitumor activity in comparison with the prototype compound.

Table 3 - Effect of compounds T1114 and T1059 in equimolar doses on growth and growth inhibition of CC-5 in female mice CBA x C ₅₇ Bl/6j, n=16-18

Observation period, days Average tumor volume (V), relative units (M±σ); SRW index, % Control T1059, 70 mg/kg T1114, 96 mg/kg V V TRO V TRO 8 1.0 1.0 1.0 12 3.77 ± 0.56 3.36 ± 0.71 14.6 ± 12.8 3.43 ± 0.68 11.9 ± 13.6 15 7.04 ± 1.23 6.81 ± 1.60 9.9 ± 10.5 5.68 ± 1.57 * 22.0 ± 18.2 18 11.83 ± 2.98 10.69 ± 2.77 14.2 ± 13.8 9.08±1.38* 23.2 ± 11.7 20 16.44 ± 2.99 13.63 ± 2.64 17.6 ± 15.3 12.34 ± 1.87 * 24.9 ± 11.4 22 22.53 ± 3.05 18.47 ± 2.50 * 18.0 ± 11.1 17.27 ± 1.60 * 23.4 ± 7.1

Symbol * – statistically significant difference with the control group according to the Mann-Whitney U test with the Holm-Bonferroni correction

Thus, the claimed invention achieves a technical result consisting in a more pronounced, stable and long-lasting antitumor effect, compared with the prototype, against neoplasia of various origins, without increasing toxic effects.

Thus, in experimental studies on transplantable animal tumors of various histogenesis (examples 2-3), it was established that 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate (T1114) with chronic parenteral administration causes a significantly more pronounced and stable antitumor effect, than the prototype compound 1-cyclohexanoyl-2-ethylisothiourea hydrobromide (T1059), applied in a similar manner in equimolar doses.

The safety of the proposed compound is supported by observations in experiments with tumor-bearing animals, where T1114 did not cause lethal or toxic effects in animals when administered chronically.

The totality of the declared essential features indicates the achievement of the declared technical result. Compound T1114, 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate, can create new opportunities in targeted therapy of human malignancies.

Claims (4)

1. Complex antiangiogenic and hypoxia-oriented antitumor agent containing the chemical compound 1-cyclohexanoyl-2-ethylisothiourea α-cyano-4-hydroxycinnamate as an active substance of the general formula , possessing NOS-inhibiting and MCT-inhibiting activity and exerting a combined antiangiogenic and hypoxia-oriented toxic effect on neoplasia. 2. The agent according to claim 1, characterized in that the chemical compound exhibits NOS-inhibitory and MCT-inhibitory activity at a dose of 96 mg/kg of the active substance.