Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
  • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
  • C07D241/04—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
  • A61P31/06—Antibacterial agents for tuberculosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals

Definitions

• the present invention relates to novel compounds based on substituted phenymydrazinyl- pyrazines, processes for their preparation, and their use as antituberculous drugs and pharmaceutical preparations containing them.
• Substituted pyrazine derivatives belong to compounds having significant biological activity, they are used as essential drugs (Miniyar P.B., Murumkar P.R., Patil P.S., et al.: Mini Rev. Med. Chem. 13, 1607-25, 2013; Ferreira S.B., Kaiser C.R. Exp. Opin. Ther. Patents 22, 1033- 51, 2012).
• Tuberculosis caused by Mycobacterium tuberculosis complex (MTB), has for many years belonged to the most widespread infectious diseases in the world. Its incidence and mortality has so far failed to reduce despite existing antituberculotic therapy (World Health Organization, Stop TB Partnership. Tuberculosis Global Facts 2014). The reason is the emergence and development of increasingly resistant forms of the disease agent and co- infection with HIV, the incidence in the Czech Republic is increasing (tJstav zdravotnickych informaci a statholic CR; Tuberkuloza a respiracni nemoci 2012, Prha UZIS, 2013, 111pp.).
• the most important first line anti-tuberculosis drugs include pyrazinamide (PZA).
• PZA pyrazinamide
• PZA does not act by a single mechanism, but we recognize still new ways of its action: a) PZA is a prodrug, which is converted with the enzyme nikotinamidase/pyrazinamidase (PncA) to the pyrazinoic acid (POA), as its active form. There is an accumulation of POA and intracellular acidification (Dolezal M., Kesetovic D., Zitko J. Curr. Pharm. Design, 2011, vol. 17, no. 32, p. 3506-3514). This disturbance leads to influencing the activity of enzymatic systems and disrupting the processes that are dependent on the pH gradient at the interface organelle / cytoplasmic compartment (Zhang H., et al.
• POA acts by inhibiting protein and RNA synthesis, serine uptake, disruption of membrane potential in the acidic pH (Wright H.T., Reynolds K.A. Curr. Opin. Microbiol. 10, 447-453, 2007);
• PZA influences RpsA - an enzyme system responsible for regulating biological processes, hcluding cell adhesion, migration and differentiation (Zimic M, et al. Microb. Drug Resist. 18, 372-375, 2012; Sayahi H. ⁇ et al. Chemistry & Biodiversity 9, 2582-2596, 2012).
• PZA inhibits tra «s-translation, which is a process to prevent the creation of non-functional proteins, and is associated with stress survival, virulence and recovery of nutrient starvation (Shi W., Zhang X., Jiang X., et al. Science 333, 1630-1632, 2011); c) estimated competition of PZA (and 5-chloro-pyrazine-2-carboxamide) with NADPH for binding to the complex FAS I (Fatty Acid Synthase). The synthesis of mycolic acids, which are part of the cell wall of mycobacteria, is inhibited. Also POA is binding to FAS I, but in different location of the enzyme (Zimic M, et al. Microb.
• FAS I is the primary goal of the pyrazinamide derivatives, including 5-chloropyrazinamide, which also inhibits FAS complex II.
• FAS I is involved in the synthesis of short chain-mycolic acids, FAS II mediates the synthesis of long chain-mycolic acids.
• FAD II is the enoyl- ACP reductase (Ngo S.C., et al. Antimicrob. Agents Chemother. 51, 2430-2435, 2007).
• ATs which would act against MDR-TB strains, or against latent forms of TB. They must be structurally novel compounds which act by novel mechanisms other than the currently used AT, or with an improved pharmacokinetic profile.
• New potential ATs which are now in preclinical and clinical stage of development, are often substances which contain a nitro moiety in their molecule. Relevant examples include the nitroimidazoles pretomanid, OPC-67683 and delamanid.
• Another promising group of AT are oxazolidinones, for example linezolid and Vozolid, containing, inter alia, the carboxamide functional moiety (Matthias Stehr M., Elamin A.A., Singh M. Curr. Top. Med. Chem. 14, 110-129, 2014).
• nitro or carboxamide moieties have proved essential for the antimycobacterial activity, but the mechanisms of action were different for each other (Matthias Stehr M., Elamin A.A., Singh M. Curr. Top. Med. Chem. 14, 110-129, 2014).
• the pyrazinecarboxamide motif can be found in a wide range of modern drugs such as the antineoplastic agent bortezomib (Palle Raghavendracharyulu Venkata, Kadaboina Rajasekhar, Murki Veerendeer et al., Bortezomib and proces for producing same.
• a PZA fragment is also contained in a large number of drugs which are in the clinical trial phase, for example pyrazine derivatives patented as kinase inhibitors (Charrier J.D., Durrant S.J., Kay D., et al. Compounds useful as inhibitors of ATR kinase; US2014113005 (2014); Song Y., Xu Q., Jia Z., et al. Preparation of pyrazine derivatives for use as Syk kinase activity inhibitors. US20130131040 (2013); Boyle R.G., Boyce R.J.
• microwave synthesis methods Over the past decades, methods of microwave synthesis methods and their applications suitable for the development of new drugs have got the forefront of interest of pharmaceutical chemists. These reactions usually provide a higher yield, require significantly shorter reaction time, and reduce the consumption of solvents compared to conventional methods of organic synthesis. In some cases, the desired products can be obtained only by using a microwave synthesis. Advantages of these reactions can be explained by the interaction of microwaves and molecules; the possibility of reaching a temperature greater than the boiling point of the solvent at atmospheric pressure (in a sealed system) is also very advantageous (Hayes B.L. Microwave Synthesis: Chemistry at the Speed of Light; CEM Publishing: Matthews, NC, USA, 2002; De la Hoz A., Diaz-Ortiz A., Moreno A.
• microwaves in organic synthesis Thermal and non-thermal microwave effects. Chem. Soc. Rev. 34, 164-178, 2005).
• microwaves act directly on the molecules present in the reaction mixture and heating is hence not dependent on the thermal conductivity of the container, unlike in classical heating.
• the transmission of energy from the microwave heating to the substance proceeds by two basic mechanisms, either by dipole rotation, or by ion conductivity. Dipole rotation is an interaction of the spin at which polar molecules attempt to align with rapidly changing electric field of the microwaves. This rotational motion of molecules results in energy transfer. The second way is to transmit energy through the ion conduction.
• the important characteristics of used solvents include their polarity, the rule for which is that the more polar solvent, the higher the ability to interact with microwaves will be. Even solvents having a low boiling point can be used in this synthesis because the microwave energy reaches and exceeds the boiling point of most of solvents in a few seconds. Using of pressurized reaction vessels further ensures recovery of solvents with a low boiling point. Another important factor in selecting the solvent is how efficiently the solvent molecules interact with microwaves and convert the microwave energy into thermal energy. The microwave synthesis can be applied even in systems without using any solvent. There are three main types of reactions without solvent: reaction mixtures adsorbed on mineral oxides, reactions of a phase transfer catalyst and a clean reactions (Hayes B.L. Microwave Synthesis: Chemistry at the Speed of Light; CEM Publishing: Matthews, NC, USA, 2002).
• the compound 5-chloro-6-methyl-pyrazine-2,3-dicarbonitrile (II) was used in the past for the preparation of a series of substituted 5-benzylamino-6-memylpyrazme-2,3-dicarborutriles; the obtained products did not show significant biological properties in the tests (Jandourek, O.; Dolezal, M.; Paterova, P.; Kubicek, V.; Pesko, M.; unes, J.; Coffey, A.; Guo, J.; ralova, . Molecules 19, 651-671,2014).
• the compound 3-cWoropyrazme-2-carboxarrude (III) was used in the past for the preparation of a series of substituted 3-(alkylamino)pyrazine-2- carboxamides; the products obtained did not show significant biological properties in the assays (Jandourek, O.; Dolezal, M.; Kunes, J.; Kubicek, V.; Paterova, P.; Pesko, M.; Buchta, V.; Kralova, K.; Zitko, J. Molecules 19, 9318-9338, 2014).
• the invention relates to novel substituted 2-(2-phenymydrazinyl)pyrazine of the general formula I
• each R 1 , R 2 is independently H or CN; R 3 is CH 3 or CONH 2 ; each R 4 , R s , R 6 , R 7 , R 8 is independently H, CI, or N0 2 .
• Another object of the invention is a method for preparing of a substituted 2-(2- phenylhydrazinyl)pyrazine of general formula I, consisting in that the substituted chloropyrazine of general formula
• each R 4 , R 5 , R 6 , R 7 , R 8 is independently H, CI, or N0 2 , in a polar solvent, under the conditions of microwave synthesis to form a substituted phenymydrazinylpyrazine of general formula I.
• Another object of the invention relates to the use of the above mentioned substituted 2-(2- fenylhydrazinyl)pyrazine of the formula I according to the invention for use as an antituberculotic against Mycobacterium tuberculosis and against its atypical strains (mycobacteriosis pathogens), including pathogenic strains isolated from the sick patients.
• All compounds of the general formula I contain, in their molecule, the hydrazino group, but their toxicity in the tests carried out is very low. These substances are exceptional with by their low toxicity since the hydrazine group in the molecule of a drug usually bears higher toxicity or irritation. Based on current knowledge of pharmaceutical chemistry such substitution in the field of synthesis of new promising drugs previously has always been considered undesirable.
• the essence of the invention consists is a combination of the six-membered heterocycle pyrazine and an aromatic part, linked each other with a linking hydrazine bridge.
• the heteroaromatic ring is substituted with a carboxamide or carbonitrile moieties, while the aromatic ring is unsubstituted, or substituted with nitro groups or chlorine atoms in various positions.
• the starting compounds II and III are accessible by conventional methods of organic synthesis (Takematsu T. f Segawa H., Miura, T. et al. A. 2,3-Dicyanopyrazines. USP4259489, 1981; Dlabal K., Palat K., Lycka A., Odlerova Z. Synthesis and 1H- and 13 C-NMR spectra of sulfur derivatives of pyrazine derived from amidation product of 2-chloropyrazine and 6- chloro-2-pyrazinecarbonitrile. Tuberculostatic activity. Collect. Czechoslov. Chem. Commun. 55, 2493-2500, 1990).
• Microwave Synthesis Chemistry at the Speed of Light; CEM Publishing: Matthews, NC, USA, 2002; De la Hoz A., Diaz-Ortiz A., Moreno A. Microwaves in organic synthesis. Thermal and non- thermal microwave effects. Chem. Soc. Rev. 34, 164-178, 2005).
• the apparatus used is equipped with a so-called focused field, which is advantageous since the microwaves are aimed directly to the reaction mixture placed in a waveguide.
• Another positive aspect of the method used is also time, energy and solvents saving.
• the prepared compounds of general formula I have been evaluated in vitro against the strains of M. kansasii 235/80, M. avium 80/72, M. avium 152/73 and M. tuberculosis H37Rv using a fluid Sula's semisynthetic medium (Trios, Prague, Czech Republic) by the microdilution method in comparison with pyrazinamide (PZA) at pH 5.6.
• the tested compounds were dissolved in dimethylsulfoxide (DMSO) and diluted with the medium to final concentrations of 100, 50, 25, 12,5, 6,25, 3,125 and 1,563 ⁇ g mL. The results were read after two or three weeks. Pyrazinamide (PZA) was used as the standard. The results are shown in Table 1.
• Results indicate high antimycobacterial activity, for all tested compounds of formula I it was 2-16x higher than that of the used standard PZA. High activity was discovered against M. ⁇ s sii for compounds 1-5 of general formula I and against M. avium in the case of compounds 2-4 of general formula I.
• pH / buffer 7.0 / MOPS (0,165 M)
• the prepared derivatives corresponding to the general formula I were also tested for antiviral activity at Rega Institute for Medical Research, KathoUeke Universiteit, Leuven, Belgium (Prof. Dr. Lieve Naesens and co-workers).
• the viral infections against which efficacy was evaluated included: Influenza virus A ( ⁇ ; H3N2) and B, Herpes simplex virus- 1 and -2, Vesicular stomatitis virus, Vaccinia virus, Parainfluenza-3 virus, Reovirus-1, Sindbisvirus, Coxsackie virus B4, Punta Toro virus, Respiratory syncytial virus, Feline corona virus, Feline herpes virus.
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are as defined above.
• TLC Thin Layer Chromatography
• Elemental analyses were measured with an EA 1110 CHNS Analyzer (Fisons Instruments S. p. A., Carlo Erba, Milano, Italy). All measured values are given in percent.
• Log k calculated from the capacity factor k, is used as the Upophilicity index converted to log P scale. Values of log P and Clog P were calculated with the PC programme CS ChemBioDraw Ultra 13.0 (CambridgeSoft, Cambridge, MA, USA).
• Compound 1 is prepared by reaction of phenylhydrazine (3 mmol) with 5-chloro-6- memylpyrazine-2,3-dicarbomtrile (II, 1 mmol) in 3 mL of methanol and pyridine (1 mmol). The reaction is performed in a microwave reactor at the temperature 140°C, pressure 15 kPa and an output of 120 W during 30 min. After completing the reaction, product 1 was isolated and purified by column chromatography on silica gel (mobile phase: hexane / ethyl acetate 1:1), yield 29%. Analytical data for compound 1: Brown-red crystalline solid; Mp.
• Compound 2 is prepared by reaction of 3-chlorophenylhydrazine (3 mmol) with 5-chloro-6- methylpyrazine-2,3-dicarbonitrile (II, 1 mmol) in 3 mL of methanol and pyridine (1 mmol). The reaction is performed in a microwave reactor at the temperature 140°C, pressure 15 kPa and an output of 120 W during 30 min. After completing the reaction, product 2 was isolated and purified by column chromatography on silica gel (mobile phase: hexane / ethyl acetate 1:1), yield 65%. Analytical data for compound 2: Green-brown crystalline solid; Mp.
• Compound 3 is prepared by reaction of 2-chlorophenylhydrazine (3 mmol) with 5-chloro-6- methylpyrazine-2, 3 -dicarbonitrile (II, 1 mmol) in 3 mL of methanol and pyridine (1 mmol). The reaction is performed in a microwave reactor at the temperature 140°C, pressure 15 kPa and an output of 120 W during 30 min. After completing the reaction, product 3 was isolated and purified by column chromatography on silica gel (mobile phase: hexane / ethyl acetate 1:1), yield 67%. Analytical data for compound 3: Green-brown crystalline solid; Mp.
• Compound 4 is prepared by reaction of 4-chlorophenylhydrazine (3 mmol) with 5-chloro-6- memylpyrazine-2,3-dicarbonitrile (II, 1 mmol) in 3 mL of methanol and pyridine (1 mmol). The reaction is performed in a microwave reactor at the temperature 140°C, pressure 15 kPa and an output of 120 W during 30 min. After completing the reaction, product 4 was isolated and purified by column chromatography on silica gel (mobile phase: hexane / ethyl acetate 1: 1), yield 21%. Analytical data for compound 4: Light-brown crystalline solid; Mp.
• Compound 5 is prepared by reaction of 2-nitrophenylhydrazine (3 mmol) with 5-chloro-6- memylpyrazine-2,3-dicarbonitrile (II, 1 mmol) in 3 mL of methanol and pyridine (1 mmol). The reaction is performed in a microwave reactor at the temperature 140°C, pressure 15 kPa and an output of 120 W during 30 min. After completing the reaction, product 5 was isolated and purified by column chromatography on silica gel (mobile phase; hexane / ethyl acetate 1:1), yield 38%. Analytical data for compound 5: Dark-brown crystalline solid; Mp.
• Compound 10 is prepared by reaction of 2-nitrophen .hydrazine (3 mmol) with 3- chloropyrazine-2-carboxamide (III, 1 mmol) in 3 mL methanol and pyridine (1 mmol). The reaction is performed in a microwave reactor at the temperature 140 °C, pressure 15 kPa and an output of 120 W during 30 min. After completing the reaction, product 10 was ' isolated and purified by column chromatography on silica gel (mobile phase: hexane / ethyl acetate 1:1), yield 17%. Analytical data for compound 10: Red-braun crystalline solid; Mp.
• Example 11 (content of the active ingredient 100 mg):
• Example 12 (content of the active ingredient 200 mg):
• Example 13 (content of the active ingredient 300 mg):
• Example 14 (content of the active ingredient 400 mg):
• Example 15 (content of the active ingredient 500 mg):
• the active ingredient is mixed with the individual ingredients and the tableting blend is compressed in a tablet machine in the usual manner.
• Example 16 (content of the active ingredient 100 mg):
• Example 17 (content of the active ingredient 200 mg :
• Example 18 (content of the active ingredient 300 mg):
• Example 19 (content of the active ingredient 400 mg):
• Example 16 (content of the active ingredient 500 mg):
• the active ingredient is gradually mixed with lactose, potato starch, the mixture is granulated in an aqueous solution of povidone, the dried granulate is mixed with sodium carboxymethyl starch, magnesium stearate and talc and the resulting blend is compressed in a tablet machine in the usual way.

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