RU2699558C2 - Agent for targeted therapy of malignant growths - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: invention relates to 1-isobutanoyl-2-isopropyl isothiourea dichloroacetate (compound T1084) with the structural formula:as a target therapeutic agent for solid malignant growths, having a complex anti-non-regional action – NOS-inhibiting anti-angiogenic and PDK1-inhibiting hypoxia-oriented cytotoxic.EFFECT: providing effective and stable anti-tumor action and inhibiting (or inhibiting) development of tumor resistance to anti-angiogenic therapy, as well as broader capabilities for developing new pharmacological agents for complex targeted therapy of oncological diseases.5 cl, 1 dwg, 3 tbl, 5 ex

Description

The invention relates to new drugs that have complex antiangiogenic and hypoxia-oriented cytotoxic effects and can find application as targeted therapy for solid malignant neoplasms.

The problem of developing effective drugs for the treatment of cancer remains highly relevant for the entire world community. So, in the Russian Federation at present, there are about 3.3 million patients in need of effective treatment registered with various oncological diseases. But, despite the significant successes of oncology in recent decades, many common oncological diseases, as before, are difficult to treat. For example, breast cancer, which annually affects over 65 thousand women in Russia, causes the death of 35% of cases, and the mortality rate of lung cancer, which causes about 60 thousand Russians annually, reaches 85% (Kaprin A.D., Starinsky V.V. , Petrova V.G. Malignant neoplasms in Russia in 2015 (morbidity and mortality) - M.: P. Herzen MNIIOI - branch of FSBI NIIRTS MH RF, 2017).

According to many experts, changing the current picture in the treatment of oncological diseases is possible only with the introduction into clinical practice of effective targeted antitumor agents, in particular, antiangiogenic ones, the target of which are biochemical and pathophysiological processes specific for all malignant neoplasms or for their specific types (Willett CG, Duda DG, 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 HB Therapeutic anti-VEFG antibodies. Handb. Exp. Pharmacol. 2 008.181: 131-50).

So, regorafenib, a protein kinase inhibitor used in targeted therapy of colorectal cancer, is known (Grothey A., Van Cutsem E., Sobrero A. et al. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicenter, randomized, placebo -controlled, phase 3 trial. Lancet. 2013.381 (9863): 303-12).

The disadvantage of regorafenib, like other antiangiogenic agents, is the progressive weakening of the antitumor effect due to the development of tumor resistance to its effects. Thus, according to clinical studies, the increase in the survival period of cancer patients through the use of antiangiogenic agents does not exceed 1 year, and prolonged suppression of tumor growth during monotherapy with such drugs is extremely rare; Frandsen S., Kopp S., Wehland M. et al. Latest results for anti-angiogenic drugs in cancer treatment. Curr. Pharm. Des. 2016.22 (39): 5927-42).

Bevacizumab, a targeted drug containing recombinant monoclonal IgG1 antibodies, is known for the treatment of metastatic colorectal, ovarian and other types of cancer.

The disadvantages of bevacizumab are significant restrictions on the use associated with side effects (perforation of the gastrointestinal tract, difficulty in healing wounds, hemorrhage, arterial thromboembolism, hypertensive crisis, reversible leukoencephalopathic syndrome, neutropenia and infection, nephrotic syndrome, congestive heart failure), as well as its inability to increase congestive heart failure), as well as its inability to increase overall patient survival (Rossi L., Verrico M., Zaccarelli E., Papa A., Colonna M., Strudel M., Vici P., Bianco V., Tomao F. Bevacizumab in ovarian cancer: A critical review of phase III studies. Oncotarget. 2017 Feb 14; 8 (7): 12389-12405).

Known "Antitumor agent" according to patent RU 2503450 (01/10/2014). The agent is an effective NOS inhibitor, causing marked inhibition of the growth and metastasis of a number of transplanted animal tumors, due mainly to the antiangiogenic, hypoxic effect, against which proliferation decreases and tumor cell apoptosis is enhanced.

Disadvantages: the antitumor effect of this drug is realized mainly at the early stage of tumor development. Since the entire molecular apparatus involved in angiogenesis remains intact under the action of this agent, its “abolition” is accompanied by rapid revascularization and resumption of tumor growth (Filimonova M.V., Yuzhakov V.V., Shevchenko L.I. et al. Experimental study of the antitumor activity of a new nitric oxide synthase inhibitor T1023. Molec. honey. 2015. (1): 61-64).

The prototype of the proposed technical solution is "vasoconstrictor agent" according to the patent for the invention RU 2475479 (02.20.2013) with the general structural formula -

where R = (CH ₃ ) ₂ CH;

- 1-isobutanoyl-2-isopropylisothiourea hydrobromide (hereinafter, compound T1023). T1023 is an effective inhibitor of NOS, causes a pronounced vasoconstrictor effect, capable of creating hypoxia in biological tissues, including tumor tissues.

The disadvantage of the prototype is a decrease in its antitumor effectiveness in the long term after its exposure, which is manifested by the development of hypoxic resistance of experimental neoplasias: a progressive decrease in the activity of apoptosis of tumor cells, weakening of the inhibition of tumor growth and enhancing their recurrent growth.

The technical result of the invention is to create a new antitumor agent with a longer duration and severity of antitumor action, as well as capable of suppressing (or inhibiting) the development of neoplasia resistance.

Our studies confirm the opinion of several authors that one of the promising ways to overcome the hypoxic resistance of neoplasia is the use of dichloroacetate (DCA), which is an inhibitor of PDK1 and has the properties of a hypoxia-oriented cytotoxin. A number of experimental studies have shown the ability of DCA to suppress the development of hypoxic resistance of tumors during therapy with some pharmacopoeial antiangiogenic agents (Koblyakov V.A. Hypoxia and glycolysis as possible objects of antitumor effects. Successes of molecular oncol. 2014, 2: 44-49; Duan Y. , Zhao X., Ren W. et al Antitumor activity of dichloroacetate on C6 glioma cell: in vitro and in vivo evaluation. Onco. Targets Ther. 2013.6: 189-98; Kumar A., Kant S., Singh S. Antitumor and chemosensitizing action of dichloracetate implicates modulation of tumor microenvironment: A role of reorganized glucose metabolism, cell survival regulation and macrophage differentiation. Toxicol. Appl. Pharmacol. 2013.273 (1): 196-20 8; Kumar K, Wigfield S, Gee HE, et al. Dichloroacetate reverses the hypoxic adaptation to bevacizumab and enhances its antitumor effects in mouse xenografts. J. Mol. Med. (Berl). 2013.91 (6): 749-58 )

The authors of the present invention shows the feasibility and prospects of combining DHA and compounds T1023 in the form of a new tool for the treatment of cancer.

The essence of the technical solution, including 1-isobutanoyl-2-isopropylisothiourea hydrobromide and the use of dichloroacetate, is the synthesis of 1-isobutanoyl-2-isopropylisothiourea dichloroacetate (hereinafter referred to as compound T1084) and the use of this compound as an active substance of antitumor agents.

List of figures

FIG. 1. The structural formula of 1-isobutanoyl-2-isopropylisothiourea dichloroacetate (compound T1084).

The chemical synthesis of compound T1084, its toxicological properties and antitumor activity, as well as the achievement of the claimed technical result are given in the examples below.

Synthesis of Compound T1084

Example 1. Obtaining compounds T1023

A mixture of 6 g (40 mmol) of isobutanoylthiourea, 9.8 g (80 mmol) of isopropyl bromide and 15 ml of dry acetonitrile in a sealed ampoule is heated in a boiling water bath for 20 hours. The solvent was evaporated, the residue was filtered off and recrystallized from 4-methyl-2-pentanone. Yield 3.6 g (33%). _Mp 129-131 ° C. PMR spectrum (DMSO-d ₆ ) δ 1.1 (d, 6H); 1.36 (d, 6H); 2.73 (m, 1H); 4.1 (m, 1H); 10.9 (broad t, 2H).

Calculated,%: C 35.69; H 6.37; N 10.41 C ₈ H ₁₆ N ₂ OS⋅HBr

Found,%: C 35.76; H 6.51; N 10.45

As a result, 1-isobutanoyl-2-isopropylisothiourea hydrobromide was obtained.

Example 2. Obtaining compounds T1084

1.3 g of 1-isobutanoyl-2-isopropylisothiourea hydrobromide are dissolved in 10 ml of water, concentrated aqueous ammonia solution is added dropwise to pH 8 and extracted twice with diethyl ether (2 × 10 ml). Dry with anhydrous Na ₂ SO ₄ and then evaporate. The yield of the base is 0.7 g.

Dissolve the base in 5 ml of diethyl ether and add a solution of 0.5 g of dichloroacetic acid in 3 ml of diethyl ether to it. Cool. The precipitate formed is filtered off and recrystallized from hexane. Yield 0.8 g (68%).

_Mp 67-69 ° C.

Calculated,%: C 37.86; H 5.72; N 8.83 C ₈ H ₁₆ N ₂ OS⋅C ₂ H ₂ Cl ₂ O ₂

Found,%: C 37.91; H 6.09; N, 9.04

The result is 1-isobutanoyl-2-isopropylisothiourea dichloroacetate (compound T1084).

Example 3. The toxic characteristics of the compounds T1084

The toxicity of compound T1084 was studied in male outbred mice using the acute toxicity test at the age of 2-2.5 months with a body weight of 22-25 g with a single intraperitoneal administration (Guidelines for preclinical studies of drugs. Part One / Ed. A .N. Mironova - M.: Grif and K, 2012). T1084 working solutions were prepared on the basis of a 0.9% aseptic sodium chloride solution (Dalkhimpharm, RF). Observation of the animals was carried out for 14 days. The calculation of indicators of "acute" toxicity was carried out by probit analysis according to the Litchfield-Wilcoxon method.

The results of these studies showed that, like T1023, T1084 according to GOST 12.1.007-76 belongs to the third class of toxicity and danger (moderately toxic). The indicator of its average lethal dose (LD ₅₀ ) was 447 ± 9 mg / kg for mice (Table 2).

A comparison of the indicators of the “acute” toxicity of the compound T1084 with the corresponding indicators for T1023, which we obtained earlier on the same animals and with the same method of administration, shows that their difference in molar expression does not exceed 10%. This indicates that a change in the salt-forming acid from hydrobromide to dichloroacetate does not significantly change the toxicity of 1-isobutanoyl-2-isopropylisothiourea salts, and the differences observed in the acute toxicity T1023 and T1084 in terms of mg / kg are almost completely determined by the differences in molecular weight these compounds.

Example 4. Antitumor activity of compound T1084 on a model of solid Ehrlich carcinoma (CE).

The aim of the experiment was a comparative study of the effects of T1023, Na-DHA (DHA in the form of sodium salt) and T1084 in their chronic use on the growth of solid CE in mice.

The study was performed on 81 female F ₁ hybrid mice (CBA × C ₅₇ Bl / 6j) aged 2-2.5 months with a body weight of 19-22 g, randomized into 4 experimental groups: control and three experimental, 20 -21 individuals in each. CE was transplanted to all animals according to the method described in the Guidelines for conducting preclinical studies of drugs (2012). Control animals subsequently did not receive any experimental effects. Animals of the experimental groups from the 6th to the 20th day of CE growth were given daily iv administration, respectively, of T1023, Na-DCA and T1084 in equimolar doses: T1023 - 60 mg / kg; Na-DHA - 33.6 mg / kg; T1084 - 70.7 mg / kg. T1023 and T1084 solutions were prepared on the basis of a 0.9% aseptic sodium chloride solution (Dalchimpharm, RF), Na-DCA solutions were prepared on the basis of water for injection (Dalchimpharm, RF). Antitumor effects were studied according to the method described in the Guidelines for conducting preclinical studies of drugs (2012).

The results of this experiment showed that, with the administration schemes and doses of T1023 and Na-DCA, they had a similar effect on the development of CE (Table 3). Almost throughout the observation period, the action of these compounds was accompanied by a statistically significant antitumor effect. The maximum antitumor effect developed by the end of the first week of application of T1023 and Na-DCA, and was manifested by inhibition of CE growth by approximately 40%. But subsequently, the antitumor efficacy of T1023 and Na-DHA progressively decreased and in the later stages the TPO index was 21-24% (about 50% of the maximum efficiency).

The initial stages of development of the antitumor effect of the compound T1084 were similar to the action of T1023. But the increase in the antitumor effect of T1084 was more prolonged and reached a maximum by the 10th day from the start of its use. And during these periods, the antitumor effect of T1084 at the level of the statistical trend (p = 0.12) was 20-25% higher than the effects of T1023 and Na-DCA. And in the future, the dynamics of the antitumor effect of the T1084 compound qualitatively differed from the action of T1023 and Na-DCA. If in the late stages the inhibition of CE growth under the influence of T1023 and Na-DCA progressively weakened, the action of T1084 suppressed the adaptation of the tumor to this effect and maintained stable inhibition of tumor growth at the level of 45-50% (90-100% of maximum efficiency) until the end of the observation. . Moreover, in the later stages, the antitumor effect of T1084 was statistically significantly superior to the effects of T1023 and Na-DCA.

This reflects the ability of compound T1084 to realize both types of pharmacological activity - NOS-inhibiting, anti-angiogenic and hypoxia-oriented cytotoxic effects.

Example 5. Antitumor activity of compound T1084 in a model of cervical cancer.

The aim of the experiment was a comparative study of the effects of T1023, Na-DCA, and T1084 in their chronic use on the growth of cervical cancer (cervical cancer) in mice.

The study was performed on 74 female F ₁ hybrid mice (CBA × C ₅₇ Bl / 6j) aged 2-2.5 months with a body weight of 19-22 g, randomized into 4 experimental groups: control and three experimental, 18 each -20 individuals in each. All animals were transplanted with cervical cancer-5 in the area of the lateral surface of the right thigh by subcutaneous injection of 2 × 10 ⁶ tumor cells in 0.1 ml of a suspension based on medium 199 (Pan-Eco, RF). Control animals subsequently did not receive any experimental effects. As in Example 4, the animals of the experimental groups from the 7th to the 21st day of the growth of cervical cancer-5 were given a daily iv administration of T1023, Na-DCA and T1084 in equimolar doses of 60, 33.6 and 70.7 mg / kg, respectively. Antitumor effects were studied according to the method described in the Guidelines for conducting preclinical studies of drugs (2012).

The results of this experiment showed that the used experimental neoplasia was weakly sensitive to the action of T1023 and Na-DCA (Table 4). A short-term statistically significant antitumor effect developed by the 3rd day after the start of the use of compound T1023 and Na-DCA, and was manifested by inhibition of cervical cancer growth in mice of these groups by 30-35%. But subsequently, neoplasia rapidly acquired almost complete resistance to the action of these compounds. After 5-7 days from the start of T1023 and Na-DCA administration, the inhibition of cervical cancer growth sharply weakened, and until the end of the observation, their antitumor effects were completely absent even at the level of the statistical trend.

At the same time, the experimental tumor used was quite sensitive to T1084, and the antitumor effects of this compound were significantly more pronounced and stable compared to the effects of T1023 and Na-DCA. By the 3-5th day from the start of T1084, inhibition of cervical cancer growth reached 45%. And subsequently, until the end of the T1084 injection course (on the 21st day), there was no significant adaptation of the tumor to the action of T1084 - the inhibition of cervical cancer growth stably remained at the level of 35-40% (80-90% of maximum efficiency). Moreover, the antitumor effect of the compound T1084 on cervical cancer-5 was not only stable, but also long-term - as shown in Table. 4, a statistically significant effect persisted for the next 5 days after the end of the application of T1084.

Proof of achieving a technical result

The claimed technical result of the invention is to provide a more pronounced and stable antitumor effect of the compound T1084 in comparison with the prototypes 1-isobutanoyl-2-isopropylisothiourea hydrobromide (compound T1023) and sodium dichloroacetate (Na-DCA), as well as inhibition (or inhibition) development of neoplasia resistance.

In the given experiment (see example 4), it was found that the combined use of the NOS T1023 inhibitor and Na-DCA is accompanied by a more pronounced and stable antitumor effect on solid CE of mice and inhibits the development of carcinoma resistance to the action of T1023 and Na-DCA. The obtained result indicates that two unidirectional (antitumor) types of pharmacological activity - the NOS-inhibiting antiangiogenic effect of T1023 and the PDKl-inhibiting hypoxia-oriented cytotoxic effect of DCA, are capable of pharmacodynamic synergistic interaction. And in this regard, the combination of these types of pharmacological activity, implemented by various mechanisms, is a promising approach for the development of effective integrated tools and methods for targeted therapy of cancer. The combination of the above data indicates the feasibility of the synthesis of the compound T1084 - dichloroacetate 1-isobutanoyl-2-isopropylisothiourea (see examples 1,2).

In toxicological studies it was established (see example 3) that the replacement of salt-forming acid from hydrobromide (T1023) to dichloroacetate (T1084) does not change the “acute” toxicity of 1-isobutanoyl-2-isopriliziourea salts, and, formally, does not increase the risks of negative effects in if T1084 is used as a medicine. Moreover, in studies on two tumor models, Ehrlich solid carcinoma (EC; see Example 4) and cervical cancer (cervical cancer-5; see Example 5) in mice, the compound T1084 showed a significantly more pronounced and stable antitumor activity compared to T1023 and Na-DHA. In both tumor models, the chronic effects of T1023 and Na-DHA were accompanied by the development of tumor resistance to their action.

In general, on both tumor models used, the effect of T1084 qualitatively reproduced the effects observed with the combined use of T1023 and Na-DCA. Consequently, T1084 - 1-isobutanoyl-2-isopropylisothiourea dichloroacetate can be considered as a combined means of targeted therapy of malignant neoplasms, which allows creating new opportunities for the development of new, more effective pharmacological antitumor agents.

Claims (6)

1. An agent for targeted therapy of malignant neoplasms, containing 1-isobutanoyl-2-isopropylisothiourea dichloroacetate as the active substance with the structural formula:

2. The tool according to p. 1, characterized in that it has NOS-inhibiting and PDK1-inhibiting activity and has anti-angiogenic and hypoxia-oriented cytotoxic effects at doses of the active substance of 10-100 mg / kg 3. The tool according to paragraphs. 1 and 2, characterized in that it contains 0.1-10% of the active substance in the composition of solutions for injection or infusion. 4. The tool according to paragraphs. 1 and 2, characterized in that it contains 1-50 mg of the active substance in the composition of solid dosage forms. 5. The tool according to paragraphs. 1-4, characterized in that it is used at doses of the active substance 10-100 mg / kg as monotherapy, or as part of a pharmaceutical composition for chemotherapy, or in combination with radiation therapy.

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