RU2462466C1 - Substituted azolo[1,2,4,5]tetrazines - inhibitors of antibacterial serine-threonine protein kinases - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: described are novel azolo[1,2,4,5]tetrazine derivatives

of general formula I, where Het=3,5-dimethylpyrazol-1-yl, X=N, R=cyclopropylmethylthio or pentylthio; or Het=3,5-dimethylpyrazol-1-yl, X=CH, R=isopropylthil; or Het=imidazol-1-yl, X=CH, R=H, and use of said compounds and additionally compounds of general formula (I), where Het=3,5-dimethylpyrazol-1-yl, X=N, K=cyclopentylthio; or Het=3,5-dimethylpyrazol-1-yl, X=CH, R=H or phenylthio based on disclosed activity as inhibitors of protein kinase PknA, PknB Mycobacterium tuberculosis when treating tuberculosis patients.

EFFECT: high efficiency of treatment.

2 cl, 2 tbl, 10 ex

Description

FIELD OF THE INVENTION

The invention relates to pharmaceutical chemistry and relates to derivatives of triazolo [4,3-b] - and imidazo [1,2-b] [1,2,4,5] tetrazines containing a 3,5-dimethylpyrazolyl or imidazolyl substituent in the tetrazine ring and an alkylthio group in the azole moiety. These compounds can be used as inhibitors of Mycobacterium tuberculosis protein kinases primarily for the treatment of tuberculosis patients.

State of the art

Existing anti-TB drugs were created 30-40 years ago (Gagneux, S., and Small P.M. Lancet Infect Dis: 2007; 7 (5): 328-337). One of the reasons for the difficulty in treating tuberculosis is the massive occurrence of multiple drug resistance (MDR or MDR - Multiple Drug Resistance) in the causative agent of tuberculosis both to traditional anti-TB drugs, the so-called first-line drugs (especially rifampicin and isoniazid), and second-line drugs (fluoroquinolones and aminoglycosides) (Edlin et al. N Engl J Med: 1992; 326 (23): 1514-1521; Fischl et al., Ann Intern Med: 1992; 117 (3): 177-183). Among the MDR strains, strains with the so-called extensive drug resistance (XDR or XDR - Extensively Drug-Resistant) are especially dangerous; they are resistant to at least 4 (sometimes up to 9) drugs. Therefore, overcoming the problems facing phthisiology in connection with the emergence of MDR is a task of paramount importance.

To date, at least 25 drugs of various chemical groups of the 1st and 2nd row that are used in the treatment of tuberculosis are known (Shi et al., Mini Rev Med Chem: 2007; 7 (11): 1177-1185). The targets for them are the most diverse aspects of bacterial cell metabolism: transcription (for rifampicin (Sensi, Rev Infect Dis: 1983; 5 Suppl 3S402-406)); translation and protein synthesis (for streptomycin (Bloom and Murray, Science: 1992; 257 (5073): 1055-1064), amikacin, capreomycin, kanamycin); DNA replication (for fluoroquinolones (O'brien, A J Respir Crit Care Med: 2003; 168 (11): 1266-1268)); synthesis of cell wall components (for isoniazid (Heifets, Semin Respir Infect: 1994; 9 (2): 84-103), ethambutol (Forbes et al. J Bacteriol: 1965; 891299-1305), ethionamide); processes associated with a change in the membrane potential of the cell (for pyrazinamide). Obviously, new approaches and the search for new targets for next-generation drugs are needed.

Problem to be solved: the search for anti-TB drugs with a new mechanism of action (serine-threonine protein kinase inhibitors) active against multidrug-resistant strains.

The compounds disclosed in the present invention are new, not previously described, derivatives of azolo [1,2,4,5] tetrazines. Methods for preparing the present compounds are described [ZhORKh, 1999, 35 (9), 1379-1383; Izv. AN Ser. Chem., 2011, 5]. The activity of azolo [1,2,4,5] tetrazines against PknA and PknB protein kinases of Mycobacterium tuberculosis is not known and is not described.

Analogues of the above substances in structure are temozolomide of formula A, used for the treatment of cancer [Patent US 0286088 from 11/10/2010], and imidazo [1,2-b] [1,2,4] triazines of formula B, which are gamma receptor ligands -aminobutyric acid, which can be used to treat various disorders of the psyche and central nervous system, including Parkinson's and Alzheimer's diseases [Patent US 6936608 from 08.30.2005].

An analogue in effectiveness, kanamycin of formula C, is an anti-tuberculosis drug that acts on the ribosomal complex, however, its disadvantage is the high percentage of strains resistant to this antibiotic.

Disclosure of invention

The objective of the invention is the use as inhibitors of protein kinases of Mycobacterium tuberculosis - enzymes important for cell division - in the treatment of patients with tuberculosis for the first time synthesized derivatives of [1,2,4] triazolo [4,3-b] - and imidazo [1,2-b] [1,2,4,5] tetrazines of the general formula I, where

1) Het = 3,5-dimethylpyrazol-1-yl, X = N, R = cyclopropylmethylthio (Ia) or pentylthio (Ib);

2) Het = 3,5-dimethylpyrazol-1-yl, X = CH, R = isopropylthio (Ic);

3) Het = imidazol-1-yl, X = CH, R = H (Id);

and previously known derivatives of [1,2,4] triazolo [4,3-b] - and imidazo [1,2-b] [1,2,4,5] tetrazines of the general formula I, where

1) Het = 3,5-dimethylpyrazol-1-yl, X = N, R = cyclopentylthio (Ie) [Eur. J. Org. Chem., 2011, 2309-2318];

2) Het = 3,5-dimethylpyrazol-1-yl, X = CH, R = H (If) or phenylthio (Ig) [Izv. AN Ser. Chem., 2011, 5].

The substances presented in the present invention can be obtained using known methods for the synthesis of derivatives of 3- (alkylthio) - [1,2,4] triazolo [4,3-b] [1,2,4,5] tetrazines [ZhORh, 1999, 35 (9), 1379-1383] and 6- (alkylthio) imidazo [1,2-b] [1,2,4,5] tetrazines [Izv. AN Ser. Chem., 2011, 5]. The structure of the new compounds presented in the invention is confirmed by ¹ H NMR spectroscopy (Avance DRX-400 spectrometer (Bruker) with an operating frequency of 400 MHz) and elemental analysis (automatic analyzer CHN PE 2400 ser. II (Perkin Elmer)).

Examples of specific performance.

Example 1

6- (3,5-Dimethylpyrazol-1-yl) -3- (cyclopropylmethylthio) - [1,2,4] triazolo [4,3-b] [1,2,4,5] tetrazine (Ia)

To a suspension of 412 mg (2.0 mmol) of 3-hydrazino-6- (3,5-dimethylpyrazol-1-yl) -s-tetrazine in 10 ml of DMF, 0.120 ml (2.0 mmol) of carbon disulfide was added with stirring. After 5 minutes, 168 mg (2.0 mmol) of NaHCO _{3 was} added to the resulting solution. 0.21 ml (2.2 mmol) of bromomethylcyclopropane was added to the reaction mixture and stirred until the starting tetrazine disappeared on TLC, filtered. The filtrate was poured into 100 ml of cold water, the product was extracted with chloroform, and purified by column chromatography (eluent CHCl ₃ -MeON 9: 1, Rf = 0.8). Yield 411 mg (68%). Mp 112-113 ° C. Found, (%): C 47.55; H 4.78; N, 36.67. C ₁₂ H ₁₄ N ₈ S. Calculated, (%): C 47.67; H 4.67; N, 36.67. ¹ H NMR spectrum (CDCl ₃ , δ, ppm): 0.66-0.71, 0.39-0.43 (both m, 2H, 2CH ₂ in cyclopropyl); 1.29-1.35 (m, 1H, CH in cyclopropyl); 2.40, 2.71 (both c, 3H, 2Me each in pyrazolyl); 3.50 (d, 2H, 2SCH ₂ , J = 7.4 Hz); 6.22 (s, 1H, H (4) in pyrazolyl).

Example 2

6- (3,5-Dimethylpyrazol-1-yl) -3- (pentylthio) - [1,2,4] triazolo [4,3-b] [1,2,4,5] tetrazine (Ib)

Obtained analogously to example 1 from 3-hydrazino-6- (3,5-dimethylpyrazol-1-yl) -s-tetrazine and 1-bromopentane. Yield 41%. Mp 109 ° C. Found, (%): C 49.47; H 5.22; N 35.32. C ₁₃ H ₁₈ N ₈ S. Calculated, (%): C 49.04; H 5.70; N 35.19. ¹ H NMR Spectrum (CDCl ₃ , δ, ppm): 0.92 (t, 3H, (CH ₂ ) ₄ CH ₃ , J = 7.3 Hz); 1.38, 1.48, 1.88 (all m, 2H, 3CH ₂ each); 2.40, 2.71 (both c, 3H, 2Me each in pyrazolyl); 3.54 (t, 2H, SCH ₂ , J = 7.4 Hz); 6.22 (s, 1H, H (4) in pyrazolyl).

Example 3

6- (Isopropylthio) -3- (3,5-dimethylpyrazol-1-yl) imidazo [1,2-b] [1,2,4,5] tetrazine (Ic)

To a solution of 215 mg (1.0 mmol) of 3- (3,5-dimethylpyrazol-1-yl) imidazo [1,2-b] [1,2,4,5] tetrazine in 7 ml of acetonitrile was added 91.2 mg (1.2 mmol) isopropyl mercaptan and 101 mg (1 mmol) of triethylamine. Stirred for 5 hours at room temperature. The solvent was evaporated, the residue was washed with hexane, and recrystallized from diethyl ether. Yield 95%. Mp 108-109 ° C. Found, (%): C 49.78; H 5.30; N 33.58. C ₁₂ H ₁₅ N ₇ S. Calculated, (%): C 49.81; H 5.23; N 33.88. ¹ H NMR spectrum (DMSO-d ₆ , δ, ppm): 1.30 (d, 6H, 2Me in Pr ⁱ , J = 6.7 Hz); 2.27, 2.58 (both c, 3H, 2Me each in pyrazolyl); 3.74 (m, 1H, CH in Pr ⁱ ); 6.30 (s, 1H, H (4) in pyrazolyl); 8.76 (s, 1H, CH).

Example 4

3- (Imidazol-1-yl) imidazo [1,2-b] [1,2,4,5] tetrazine (Id)

251 mg (1 mmol) of 3- (imidazol-1-yl) -6- (2,2-dimethoxyethylamino) -1,2,4,5-tetrazine was boiled for 4 hours in 7 ml of CH ₃ COOH. The solvent was removed under reduced pressure. The residue was washed with ethanol. Yield 86%. Mp 226-227 ° C. Found, (%): C 44.82; H 2.46; N 52.19. C ₇ H ₅ N ₇ . Calculated, (%): C 44.92; H 2.69; N, 52.39. ¹ H NMR Spectrum (DMSO-d ₆ , δ, ppm): 7.29, 8.05, 8.76 (all m, 1H, 3CH in imidazolyl); 8.68, 8.83 (both d, 1H, 2CH, J = 0.9 Hz).

Example 5

6- (3,5-Dimethylpyrazol-1-yl) -3- (pentylthio) - [1,2,4] triazolo [4,3-b] [1,2,4,5] tetrazine (Ie)

Obtained analogously to example 1 from 3-hydrazino-6- (3,5-dimethylpyrazol-1-yl) -s-tetrazine and bromocyclopentane. Yield 72%. Mp 119 ° C. Found, (%): C 49.47; H 5.22; N 35.32. C ₁₃ H ₁₆ N ₈ S. Calculated, (%): C 49.35; H 5.10; N, 35.42. ¹ H NMR Spectrum (CDCl ₃ , δ, ppm): 1.63-1.77 (m, 6H, 3CH ₂ ); 2.17 (m, 2H, CH ₂ ); 2.29, 2.59 (s, 3H, CH ₃ in pyrazolyl); 4.17 (m, 1H, SCH); 6.38 (s, 1H, H (4) in pyrazolyl).

Example 6

3- (3,5-Dimethylpyrazol-1-yl) imidazo [1,2-b] [1,2,4,5] tetrazine (If)

Obtained analogously to example 4 by boiling 3- (3,5-dimethylpyrazol-1-yl) -6- (2,2-dimethoxyethylamino) -1,2,4,5-tetrazine in acetic acid. Yield 86%. Mp 194-195 ° C. Found, (%): C 50.29; H 4.21; N 45.36. C ₉ H ₉ N ₇ . Calculated, (%): C 50.23; H 4.22; N 45.56. ¹ H NMR Spectrum (DMSO-d ₆ , δ, ppm): 2.39, 2.77 (both s, 3H, 2Me each in Pyr); 6.18 (s, 1H, H (4) in pyrazolyl); 8.19, 8.44 (both d, 1H, 2CH, J = 0.8 Hz).

Example 7

3- (3,5-Dimethylpyrazol-1-yl) -6- (phenylthio) imidazo [1,2-b] [1,2,4,5] tetrazine (Ig).

Obtained analogously to example 3 of 3- (3,5-dimethylpyrazol-1-yl) imidazo [1,2-b] [1,2,4,5] tetrazine and thiophenol. Yield 61 mg (38%). Mp 137-140 ° C. Found, (%): C 55.61; H 3.76; N 30.31. C ₁₅ H ₁₃ N ₇ S. Calculated, (%): C 55.71; H 4.05; N 30.32. ¹ H NMR spectrum (DMSO-d ₆ , δ, ppm): 2.25, 2.36 (both s, 3H, 2Me each in pyrazolyl); 6.26 (s, 1H, H (4) in pyrazolyl); 7.26-7.34 (m, 5H, Ph); 8.79 (s, 1H, CH).

Example 8

Test for activity against serine-threonine protein kinases.

In the Streptomyces lividans APHVIII + test system [Danilenko V.N., Simonov A.Y., Lakatosh S.A., Kubbutat M.H., Totzke F., Schächtele C., Elizarov S.M., Bekker O.B., Printsevskaya S.S., Luzikov Y.N., Reznova M.I.I. // J Med Chem., 2008, 51, 24, 7731-7736], 7 substances of the azolotetrazine class were tested for their ability to inhibit serine-threonine protein kinases. All substances showed activity to varying degrees as protein kinase inhibitors in in vitro experiments. Selected Ic and If. Docking was conducted with a model of the PknB structure of Mycobacterium tuberculosis. Docking was performed using the FRED program (Fast Rigid Exhaustive Docking, OpenEye Scientific Software Inc.). According to the results of docking, Ic and If showed the ability to interact with the adenine-binding pocket of PknB Mycobacterium tuberculosis. Thus, the biological target of the action of substances Ic and If was established (see Fig. 1).

Example 9

Determination of minimum inhibitory concentrations against Mycobacterium smegmatis.

The determination of minimum inhibitory concentrations was carried out by the disk method. The minimum inhibitory concentration for Ic is 1.45 μg / disk, for If it is 0.65 μg / disk.

Table 1 No. of substance Like weight Active concentration Zone of growth inhibition, mm nmole / disk mcg / disk Ic 1,0 0.29 0,0 289.36 5,0 1.45 8.5 7.0 2.03 12.0 If 0.5 0.32 0,0 323.38 2.0 0.65 8.5 5,0 1,60 11.0 Kanamycin 484.5 - 0.5 8.5 The diameter of the disc is 7 mm.

Example 10

Determination of the biological activity of Ic against bacterial microflora.

table 2 No. of substance Concentration Zone of growth inhibition, mm nmol / disk mcg / disk Bacillus subtilis 1A2 BGSC Lactobacillus plantarum NK-1 Escherichia coli BL21 DE3 Saccharom yces cerevisiae S288C Ic 5,0 1.45 0 0 0 0 10.0 2.89 8.0 0 0 0 20,0 5.78 12.0 0 0 0 50,0 14.50 15.0 0 0 8.0 100.0 28.93 17.0 8.0 0 9.0 200,0 57.86 19.0 9.0 7.5 9.5 500,0 145.0 20,0 10.0 8.5 10.0 The diameter of the disc is 7 mm.

Ic in the concentrations indicated in the table does not affect the growth of the human microbiota component Bifidobacterium longum, affects Lactobacillus plantarum at a concentration 20 times higher than MIC on Mycobacterium smegmatis, affects the growth of Escherichia coli at a concentration 100 times higher than MIC on Mycobacterium smegmatis, affects on Bacillus subtilis in a concentration exceeding 2 times the MIC on Mycobacterium smegmatis.

Therefore, Ic inhibits the growth of the bacterial flora shown in the table at concentrations higher than the inhibitory concentrations for Mycobacterium smegmatis.

Claims (2)

1.3- (Alkylthio) - [1,2,4] triazolo [4,3-b] [1,2,4,5] tetrazines and imidazo [1,2-b] [1,2,4,5 ] tetrazines of the general formula I

where Het is 3,5-dimethylpyrazol-1-yl, X is N,
R is cyclopropylmethylthio or pentylthio; or
Het - 3,5-dimethylpyrazol-1-yl, X - CH, R - isopropylthio; or Het is imidazol-1-yl, X is CH, R is H. 2. Inhibitors of protein kinases PknA, PknB Mycobacterium tuberculosis, which are compounds of formula I, where Het is 3,5-dimethylpyrazol-1-yl, X is N, R is cyclopropylmethylthio, pentylthio or cyclopentylthio; or Het — 3,5-dimethylpyrazol-1-yl, X — CH, R — isopropylthio, H or phenylthio; or Het is imidazol-1-yl, X is CH, R is H.

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