LV15518B - AZIRIDINE-2-CARBOXAMIDE DERIVATIVES AS THIOREDOXIN REDUCTASE INHIBITORS WITH ANTI-CANCER AND ANTIMETASTIC ACTIVITY AND THEIR SYNTHESIS - Google Patents

Abstract

The invention relates to new potentially therapeutic substances containing 1-acyl-aziridine-2-carboxamide derivatives and their analogues, which have anti-tumor and/or anti-proliferative or anti-metastatic properties. These compounds are thioredoxin reductase inhibitors.

Description

DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION

The invention relates to therapeutic substances, in particular to 1-acyl derivatives of aziridine-2-carboxamide and their analogues which have anti-cancer and / or antiproliferative and / or antimetastatic properties and / or which are inhibitors of thioredoxin reductase, and to the use of these compounds. use in the treatment and / or prevention of cancer.

Basis of the invention

The invention relates to the discovery of a new type of antitumor agent, the mechanism of action of which is based on, but not limited to, thioredoxin reductase inhibition, with further possibilities for their treatment in tumors such as connective tissue tumors, neuroblastomas and breast cancer, as well as bladder, bone, brain, central and peripheral nervous system, colon, endocrine gland, esophagus, endometrium, stem cells, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, kidney, tumors of tissues, testicles, stomach, skin, urethra, vagina, vulva, including congenital cancers derived from retinoblastoma and Wilms' tumor, leukemia, lymphoma, non-Hodgkin's disease, chronic and acute myelogenous leukemia, acute lymphoblastic leukemia, myeloma and T-cell lymphoma, myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndrome. Human connective tissue fibrosarcoma has been described since 1972 [1] and is thought to affect the human population between the ages of 30 and 40. Human neuroblastoma is a neuroendocrine tumor that develops in the elements of the sympathetic nervous system and affects children from the age of 2. [2] Human breast adenocarcinoma [3] is a type of cancer that develops mainly in breast tissue and mainly, but not always, affects women.

Thioredoxin reductases (TrxR, EC 1.8.1.9) are the only known enzymes that reduce thioredoxin (Trx) from its oxidized form. Trx, on the other hand, has one of the main / crucial roles in maintaining redox homeostasis in cells, including the capture of oxygen-active forms (ROS). Thus, TrxR-es is one of the elements that ensure cell survival and growth, in the case of cancer TrxR also provides oncogenesis. [4]

[004] Three TrxR isoforms are known - TrxR1, TrxR2 and TrxR3. TrxR1 is mainly found in the cytosol, [5] TrxR2 in mitochondria, [6] and TrxR3 in the testes [7].

[005] Since the survival of cancer cells also depends on the antioxidant system, so it is a specific vulnerability, an approach based on the interaction with the redox signaling system and major antioxidant systems such as the Trx system in the presence of ROS inducers is a new anticancer agent. tactics. [8]

Although TrxR1 inhibitors are defined as novel anticancer drugs, [9] no anticancer drug is used in the clinic whose mechanism of action is based on the target of TrxR as a drug. It should be noted that a small number of TrxR inhibitors have been reported in the literature, most of which have IC50 values in the millimolar or high micromolar range.

We found no written evidence of any aziridine-2-carboxamide derivatives as thioredoxin reductase inhibitors and / or their use in any type of tumor research or treatment based on TrxR enzyme inhibition.

Description of the invention

We have found that certain 1-acyl aziridine-2-carboxamide derivatives have the ability to inhibit TrxR and are highly cytotoxic, i.e., they are potent antiproliferative agents with potential anti-metastatic properties on various types of cancer cells for use in the treatment of cancer.

Object of the invention

At least one compound having TrxR inhibitory capacity selected from the group of compounds of Formula I

O

V7 ^X ^ ^N H2

N O ^ R

I,

Where

R is, but is not limited to, hydrogen, linear, branched and substituted alkyl groups, including alkene and alkyne groups, unsubstituted and substituted aryl and hetaryl groups;

Formula I also includes enantiomers, diastereomers, tautomers and / or pharmaceutically acceptable salts, hydrates and solvates.

Specific compounds of Formula I within the scope of this invention include, but are not limited to:

1-acetylaziridine-2-carboxylic acid amide,

1- (2-methylpropanoyl) aziridine-2-carboxylic acid amide, 1-benzoylaziridine-2-carboxylic acid amide, 1- (2-iodobenzoyl) aziridine-2-carboxylic acid amide, 1- [2- (trifluoromethyl) benzoyl] aziridine- 2-carboxylic acid amide, 1- (thiophen-2-ylcarbonyl) aziridine-2-carboxylic acid amide,

1- (Furan-2-ylcarbonyl) aziridine-2-carboxylic acid amide.

Detailed description of the invention

In search of new anticancer agents with antiproliferative and / or antimetastatic properties, we have found that the 1-acyl-aziridine-2-carboxamide derivatives of Formula I are potent antiproliferative agents for various types of cancer cells for use in the treatment of cancer.

[014] We have also found that compounds of Formula I inhibit an enzyme important for the development of cancer cells, thioredoxin reductase.

The mode of synthesis of the compounds of Formula I is described in more detail in the Examples below and is illustrated in Scheme 1. The set of inventions should not be limited to the given examples, which are given for demonstration purposes. One skilled in the art can practice this invention based on the findings of the proposed patent application.

One skilled in the art can employ methods that can produce analogs and derivatives of the compounds of this invention that have improved therapeutic effects, namely, greater activity and / or selectivity for the target enzyme, better ability to cross the mammalian blood-brain barrier, lower side effects, and the like.

One skilled in the art will recognize that the compounds of this invention may contain chiral centers and the compounds may be prepared in optically active (enantiomers and / or diastereomers) or racemic form. Also, the compounds of this invention may be any racemate, optically active compound, tautomer, or stereoisomeric form of a compound of the invention having the properties described herein.

For therapeutic use, the compounds of Formula I may be in the form of pharmacologically acceptable salts or solvates. The term "pharmacologically acceptable" as used herein refers to therapeutically active, non-toxic salt forms which the compounds of Formula I are capable of forming. Such salt forms could be obtained by treating a base form of a compound of Formula I with acids such as inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc., or organic acids such as acetic acid, propanoic acid, hydroxyacetic acid, oxopropanoic acid, 2-hydroxypropane succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, methanesulfonic acid, phenylsulfonic acid, 4-methylphenylsulfonic acid, 2-hydroxybenzoic acid, etc. Also, the salts of the compounds of Formula I may be converted back to the free base form by treatment with a base or an alkali.

The synthesis of compounds of Formula I is detailed in Scheme 1.

Target compounds I-1 to I-7 were synthesized by treating aziridine-2-carboxylic acid amide 1 with the corresponding carboxylic acid chlorides in the presence of a base (Scheme 1).

Scheme 1.

NEt ₃ , MeCN i. t, 24 h

RCOCI

[021] Not all synthesized compounds of Formula I are known in the literature.

Examples

General method for the synthesis of 1-acyl-2-carboxylic acid amides I-1 to I-7

Aziridine-2-carboxylic acid amide (1) (2.32 mmol) is suspended in MeCN (5 mL), NEt 3 (2.32 mmol) is added, cooled to 0 ^° C, the corresponding acyl chloride (2.32 mmol) is added. Stir at room temperature for 24 h, filter. Evaporate the filtrate, add acetone, filter, evaporate. Recrystallize from acetonitrile.

1. Example

1-Acetylaziride-2-carboxylic acid amide (I-1)

Obtained from aziridine-2-carboxylic acid amide (1) (0.20 g, 2.32 mmol), NEt 3 (0.32 mL, 2.32 mmol) and acetyl chloride (0.17 mL, 2.32 mmol). ). 179 mg (60%) are obtained.

1 H-NMR (DMSO-d 6, 400 MHz), δ, m. d .: 2.18 (3H, s), 2.41 (1H, dd, J = 0.9 and 3.0 Hz), 2.56 (1H, dd, J = 0.9 and 6.4 Hz) ), 3.03 (1H, dd, J = 3.0 and 6.4 Hz), 5.95 (1H, pl s), 6.26 (1H, pl s).

AIMS (ESI, m / z): calcd. For C5H5N2O2Na [M + Na] + 151.0489, found 151.0583.

2. Example

1- (2-Methylpropanoyl) aziridine-2-carboxylic acid amide (I-2)

NH ₂

Obtained from aziridine-2-carboxylic acid amide (1) (0.20 g, 2.32 mmol), NEt 3 (0.32 mL, 2.32 mmol) and isobutyryl chloride (0.24 mL, 2.32 mmol). 117 mg (32%) are obtained.

1 H-NMR (DMSO-d 6, 400 MHz), δ, m. d .: 1.03 (3H, d, J = 6.9 Hz), 1.09 (3H, d, J = 6.9 Hz), 2.27 (1H, dd, J = 2.0 and 3 , 1 Hz), 2.37 (1H, dd, J = 2.0 and 3.1 Hz), 2.46 (1H, septet, J = 6.9 Hz), 3.07 (1H, dd, J = 3.1 and 5.5 Hz), 7.38 (1H, pl s), 7.87 (1H, pl s).

¹³ C-NMR (DMSO-d 6, 100 MHz), δ, md: 18.5; 19.6; 28.9; 35.3; 35.4; 168.7; 186.9.

AIMS (ESI, m / z): calcd. For C

3. Example

1-Benzoylaziridine-2-carboxylic acid amide (I-3)

Obtained from aziridine-2-carboxylic acid amide (1) (0.20 g, 2.32 mmol), NEt 3 (0.32 mL, 2.32 mmol) and benzoyl chloride (0.27 mL, 2.32 mmol). ). 57 mg (44%) are obtained.

1 H-NMR (DMSO-d 6, 400 MHz), δ, m. d .: 2.48-2.52 (1H, m), 2.59 (1H, dd, J = 1.6 and 5.5 Hz), 3.29 (1H, dd, J = 3.1 and 5.5 Hz), 7.34 (1H, pl. S), 7.48-7.54 (2H, m), 7.58-7.63 (1H, m), 7.887.92 (3H, m ).

¹³ C-NMR (DMSO-d 6, 100 MHz), δ, md: 29.5; 37.1; 128.2; 128.5; 132.7; 133.1; 168.2; 176.1. AIMS (ESI, m / z): calcd. For C10H11N2O2 [M + H] ⁺ 191.0821, found 191.0828.

4. Example

1- (2-Iodobenzoyl) aziridine-2-carboxylic acid amide (I-4)

Obtained from aziridine-2-carboxylic acid amide (1) (0.20 g, 2.32 mmol), NEt 3 (0.32 mL, 2.32 mmol) and 2-iodobenzoyl chloride (0.62 g, 2, 32 mmol). 258 mg (35%) are obtained.

1 H-NMR (DMSO-d 6, 400 MHz), δ, m. d .: 2.44 (1H, dd, J = 1.5 and 3.1 Hz), 2.57 (1H, dd, J = 1.5 and 5.4 Hz), 3.21 (1H, dd , J = 3.1 and 5.4 Hz), 7.22 (1H, dt, J = 1.6 and 7.8 Hz), 7.34 (1H, pl. S), 7.49 (1H, dt, J = 1.2 and 7.8 Hz), 7.72 (1H, dt, J = 1.6 and 7.8 Hz), 7.85 (1H, pl. s), 7.98 (1H , dd, J = 1.2 and 7.8 Hz).

¹³ C-NMR (DMSO-d 6, 100 MHz), δ, md: 30.2; 36.9; 128.1; 130.0; 132.3; 138.4; 140.8; 167.8; 176.5.

AIMS (ESI, m / z): calcd. For C10H10N2O2I [M + H] + 316.9787, found 316.9794.

5. Example

1- [2- (Trifluoromethyl) benzoyl] aziridine-2-carboxylic acid amide (I-5)

Obtained from aziridine-2-carboxylic acid amide (1) (0.20 g, 2.32 mmol), NEt 3 (0.32 mL, 2.32 mmol) and 2- (trifluoromethyl) benzoyl carbon (0.28 mL). , 2.32 mmol). 53 mg (11%) are obtained.

1 H-NMR (DMSO-d 6, 400 MHz), δ, m. d .: 2.45 (1H, dd, J = 1.6 and 3.1 Hz), 2.55 (1H, dd, J = 1.6 and 5.6 Hz), 3.17 (1H, dd , J = 3.1 and 5.6 Hz), 7.37 (1H, pl. S), 7.70-7.75 (1H, m), 7.78 (1H, dt, J = 1.3 and 7.4 Hz), 7.82-7.87 (3H, m).

AIMS (ESI, m / z): calcd. For C11H10N2O2F3 [M + H] ⁺ 259.0694, found 259.0688.

6. Example

1- (Thiophen-2-ylcarbonyl) aziridine-2-carboxylic acid amide (I-6)

Obtained from aziridine-2-carboxylic acid amide (Leakadina) (0.20 g, 2.32 mmol), NEt 3 (0.32 mL, 2.32 mmol) and 2-thiophenecarbonylchloride (0.25 mL, 2, 32 mmol). 41 mg (46%) are obtained.

1 H-NMR (DMSO-d 6, 400 MHz), δ, m. d .: 2.49-2.51 (1H, m), 2.61 (1H, dd, J = 1.7 and 5.7 Hz), 3.29 (1H, dd, J = 3.3 and 5.7 Hz), 7.21 (1H, dd, J = 3.7 and 4.8 Hz), 7.40 (1H, pl. S), 7.72 (1H, dd, J = 1.2 and 3.7 Hz), 7.90 (1H, dd, J = 1.2 and 4.8 Hz), 7.94 (1H, pl. s).

¹³ C-NMR (DMSO-d 6, 100 MHz), δ, md: 29.8; 37.1; 128.2; 131.6; 133.1; 137.9; 168.1; 170.7. AIMS (ESI, m / z): calcd. For C8H9N2O2S [M + H] + 197.0385, found 197.0394.

7. Example

1- (Furan-2-ylcarbonyl) aziridine-2-carboxylic acid amide (I-7)

Obtained from aziridine-2-carboxylic acid amide (1) (0.20 g, 2.32 mmol), NEt 3 (0.32 mL, 2.32 mmol) and 2-furoyl chloride (0.23 mL, 2, 32 mmol). 60 mg (14%) are obtained.

1 H-NMR (DMSO-d 6, 400 MHz), δ, m. d .: 2.46 (1H, dd, J = 1.6 and 3.3 Hz), 2.57 (1H, dd, J = 1.6 and 5.4 Hz), 3.28 (1H, dd , J = 3.3 and 5.4 Hz), 6.67 (1H, dd, J = 1.6 and 3.5 Hz), 7.19 (1H, dd, J = 0.8 and 3.5 Hz), Hz), 7.38 (1H, pl. S), 7.90 (1H, dd, J = 0.8 and 1.6 Hz), 7.93 (1H, pl. S).

¹³ C-NMR (DMSO-d 6, 100 MHz), δ, md: 29.2; 36.7; 112.1; 116.5; 146.7; 147.7; 166.6; 168.2.

AIMS (ESI, m / z): calcd. For C8H9N2O3 [M + H] ⁺ 181.0613, found 181.0615.

The obtained compounds of Formula I (I-1 to I-7) showed both growth inhibition (cytotoxicity) and inhibition of TrxR1 in cancer cell cultures.

Cell culture and cell viability

[031] In vitro cytotoxicity assays were performed on monolayer tumor cell lines: HT1080 (Human connective tissue fibrosarcoma), SH-SY5Y (Human neuroblastoma), MCF-7 (Human breast adenocarcinoma). The results are summarized in Table 1.

Compared to the anti-cancer drug Leakadine 1 known in the literature, almost all compounds of Formula I showed a better cytotoxic effect on all three cancer cell lines (Table 1). Almost all compounds of Formula I showed moderate to high cytotoxic (antiproliferative) effects on all three cancer cell cultures. One compound of Formula I (I-5) showed very good cytotoxic effect on HT-1080 cells with IC50 values less than 200 μΜ (Table 1).

A compound of Formula I (I-5) showed very good cytotoxic effect on SH-SY5Y cells with IC50 values less than 200 μM (Table 1). In contrast, the cytotoxic effect on MCF7 cells is lower, where the best IC50 values are for compounds of Formula I (I-4, I-5 and I-6) (Table 1).

[034] In vitro cytotoxicity assay: HT-1080 or SH-SY5Y, or MCF-7 cells are seeded in a 96-well plate in DMEM (Dulbecco's modified Eagle's) containing 10% fetal serum, 4 mM L-glutamine, and cultured in 72 hours with the test compound at various concentrations. Cell viability is determined using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT). After incubation with the compound, the medium above the cells is changed to a new one with 0.2 mg / ml MTT in each plot of the plate. After incubation for 3 h at 37 ° C, 5% CO 2, the medium is removed with MTT solution and the bound dye is extracted with 200 μl DMSO. The optical density of the samples at 540 nm is determined using a multichannel spectrophotometer (Tecan multiplate reader InfinitelOO). The IC50 is calculated with Graph Pad Prism® 3.0.

1. Table. In vitro cytotoxicity of compounds of Formula I on monolayer tumor cell lines HT-1080 (Human connective tissue fibrosarcoma), SH-SY5Y (Human neuroblastoma) and MCF-7 (Human breast adenocarcinoma).

No. Formulas Cytotoxicity, IC50 (μM) HT-1080 SH-SY5Y MCF-7 1 0 N H 2400 ± 60 3100 ± 105 > 11600 I-1 О N 3200 ± 285 3100 ± 147 no I-2 0 VT '^ Nhķ N 5600 ± 311 2600 ± 95 no

I-3 О V7 ^ NH ₂ N ° ^ o 1400 ± 122 1200 ± 178 1400 ± 258 I-4 0 V7 ^ NH ₂ N ° όο 230 ± 18 1300 ± 32 530 ± 62 I-5 τι О ю 170 ± 72 170 ± 11 770 ± 52 I-6 О Ν 230 ± 28 780 ± 25 710 ± 23 I-7 о Vy ^ NH ₂ Ν ^оЛ й 1200 ± 174 1300 ± 123 1100 ± 251

no - no effect.

Determination of thioredoxin reductase activity in vitro

All compounds of Formula I showed the ability to inhibit TrxR (moderate to very good) at a concentration of 200 μM inhibitor (Table 2). Using a concentration of 50 μM inhibitors, two compounds of Formula I showed the ability to inhibit TrxR (Table 2).

It should be noted that the anticancer drug Leakadine 1, known in the literature, practically does not inhibit TrxR (Table 2).

Thioredoxin reductase inhibition assay: Human neuroblastoma SHSY5Y (ATCC collection) cells are grown in standard cell culture medium DMEM (Sigma) containing 10% fetal serum, 4 mM glutamine, in a thermostat at 37 ° C and 5% СО2. Cells (1x10 ⁸ ) are collected and washed from the culture medium. Lysate in 50 mM K-phosphate buffer containing 1 mM EDTA, pH 7.4.

The lysate is centrifuged at 10,000 rpm for 15 min and the supernatant is used as a source of TrxR. The effect of the compounds on TrxR was determined in the Thioredoxin Reductase Assay kit (Cat. No. 10007892, Cayman Chemical) in the test system. Incubate the 96-well TrxR plate with the compound (final concentration 10-50-200 μM) for 30 min. The TrxR detector dye is then added and the OD increase is read on a spectrophotometer at 405 nm for 10 min. The inhibitory effect of the compounds is calculated in% relative to the control.

2. Table. TrxR inhibition data for compounds of Formula I.

No. TrxR inhibition (%) IC50 (μΜ) inhibitor concentration 200 μΜ inhibitor concentration 50 μΜ 1 12 ± 5 2 ± 0.2 no I-1 89 ± 10 no 97 I-2 49 ± 7 e.g. 200 I-3 61 ± 7 no 163 I-4 100 ± 0 1 ± 6 62 I-5 58 ± 7 e.g. 200 I-6 86 ± 11 14 ± 5 100 I-7 28 ± 5 e.g. no

no - no effect; nt - not specified.

[038] All of the compounds of Formula I described herein correlate between their ability to inhibit TrxR and their antiproliferative properties, but the compound of Formula I (I-6), which has very good cytotoxic properties on all cancer cell lines described herein, is particularly noteworthy ( HT-1080, SH-SY5Y and MCF-7) and good TrxR inhibition.

[039] Several compounds of Formula I were found to show anti-metastatic activity in in vivo studies in mice (Balb / c mice with implanted breast cancer cells 4T1), reducing metastases by 80% or more. Good results were observed for compounds I-1 to I-7, with the best activity for compound I-6.

LITERATURE USED

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Claims (5)

1. Compounds of Formula I O Vy ^ NH N O ^ R I, where R is, but is not limited to, hydrogen, linear, branched and substituted alkyl groups, including alkene and alkyne groups, unsubstituted and substituted aryl and hetaryl groups; as well as their enantiomers, diastereomers, tautomers and / or pharmaceutically acceptable salts, hydrates and solvates. A compound according to claim 1, wherein the compound is selected from the group consisting of: 1-acetylaziridine-2-carboxylic acid amide, 1- (2-methylpropanoyl) aziridine-2-carboxylic acid amide, 1-benzoylaziridine-2-carboxylic acid amide , 1- (2-iodobenzoyl) aziridine-2-carboxylic acid amide, 1- [2- (trifluoromethyl) benzoyl] aziridine-2-carboxylic acid amide, 1- (thiophen-2-ylcarbonyl) aziridine-2-carboxylic acid amide, 1 - (furan-2-ylcarbonyl) aziridine-2-carboxylic acid amide. A compound according to claim 1 or 2 for use as an inhibitor of thioredoxin reductase (TrxR) in connective tissue tumors, neuroblastoma and breast cancer, as well as in the bladder, bone, brain, central and peripheral nervous system, colon, endocrine gland, esophagus, endometrium , stem cells, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testicles, stomach, skin, urethra, vagina, vulvar tumor also congenital cancer derived from retinoblastoma and Williams tumor, leukemia, lymphoma, non-Hodgkin's disease, chronic and acute myelogenous leukemia, acute lymphoblastic leukemia, Hodgkin's disease, multiple myeloma and T-cell lymphoma, plasmlasmic syndrome, myelodysplastic syndrome for the treatment or prevention of paraneoplastic syndromes, including reduction or prevention of metastasis. The compound of claim 1 or 2, wherein the compound is used in combination with one or more other chemotherapeutic agents, surgery, chemotherapy, radiotherapy, immunotherapy, or a combination thereof. A pharmaceutical composition for parenteral or oral administration comprising a therapeutically effective amount of a compound according to claim 1 or 2 and a pharmaceutically acceptable excipient or excipient.