Synthesis, X-ray crystal structure and antimycobacterial activity of enantiomerically pure 1-ethyl-2,3-dicyano-5-(het)aryl-6-hetaryl-1,6-dihydropyrazines

The Petasis reaction of 6-alkoxy adducts of 1-alkyl-2,3-dicyano-5-arylpyrazinium salts with aromatic boronic acids, such as 2-thienylboronic, 2-furanylboronic and 3-thienylboronic acids, or their benzo analogs in dichloromethane proceeds smoothly at room temperature with the formation of the corresponding 5-aryl-6-hetaryl substituted 1,6-dihydropyrazine derivatives. All dihydropyrazines were separated as pure enantiomers by chiral HPLC, and their absolute configurations for each pair of enantiomers have been determined by X-ray analysis. Individual enantiomers were screened in vitro for their antimycobacterial activities against Mycobacterium tuberculosis H 37 Rv, avium, terrae and extensively drug-resistant and multi-drug-resistant strains isolated from tuberculosis patients in Ural region (Russia) . It has been shown that several compounds exhibit a good level of antituberculosis activity compared to the reference drugs.


Introduction
Tuberculosis is an illness that results from infection with Mycobacterium tuberculosis (MTB).2][3] Almost one-third to one-half of the world's population is infected with M. tuberculosis and approximately 10% of infected individuals will experience active disease at some time in their life. 46][7] Failure of patients to complete a full treatment protocol and the outbreak of acquired immune deficiency syndrome (AIDS) has resulted in the emergence of multi-drug resistance (MDR).Recent estimates show that 10% of all new tuberculosis infections are resistant to at least one antituberculosis drug.Subsequently, the World Health Organization (WHO) has declared tuberculosis as global public health emergency. 8For multi-drug resistance (MDR) and extensively drug resistance (XDR) combinations of the first line drugs and the second line drugs, such as aminoglycosides, fluoroquinolones, thioamides, cycloserine and pamino salicylic acid are used. 9Since the discovery of rifampicin in 1960, there is no more drugs developed to treat tuberculosis.The third line drugs include rifabutin, clarithromicin, linezolid, thiacetone, arginine and vitamin D, which are still being developed, have less or unproven efficacy and are very expensive. 10MTB have the cell with a high lipid content, thus resulting in a high degree of lipophilicity and resistance to alcohol, acids, alkali and some disinfectants. 11t is worth noting that practically all drugs used to treat tuberculosis are capable of causing toxic side effects. 11Therefore, it is obvious that new original drugs that are more active and effective than the existing ones are supposed to be discovered.In addition, pyrazinecarbonitrile analogs (pyrazinecarboxamide and pyrazine-2-carboxylic acid derivatives) are important and famous as the first-line antituberculosis drugs. 113][14][15][16][17] In order to cause these transformations azaaromatic compounds are usually activated by quaternization to obtain N-alkyl-1,4-diazinium salts. 17,188][19][20][21][22][23][24][25][26] On the other hand, the Petasis reaction is one of the multicomponent condensation reactions, which provides a convenient method for the preparation of α-amino acids by reacting an amine 1 with a carbonyl compound 2 and a boronic acid 3. [27][28][29][30][31][32][33][34] The reaction proceeds via the formation of aminal intermediate 4 (Scheme 1).
2][43] The reaction is a very efficient with alkenyl and electron-rich aromatic boronic acids.In addition to primary amines, the secondary amines, sterically hindered primary amines, and tertiary aromatic amines can also participate in these transformations.A mild, practical, and novel method for the C-C bond formation in the series of pyrazine (quinoxaline) derivatives by using the Petasis reaction has been developed through intermediacy of 6-alkoxy-1-ethyl-1,6-dihydropyrazines.All obtained dihydropyrazines were screened in vitro for their antimycobacterial activities against Mycobacterium tuberculosis H 37 Rv, avium, terrae and extensively drug-resistant and multi-drug-resistant strains isolated from tuberculosis patients in Ural region (Russia).It has been found that some of these compounds exhibit a high tuberculostatic activity comparable with that of pyrazinamide.

Results and Discussion
5][46][47][48][49][50] It has been shown that they are capable of reacting with various types of nucleophiles under mild conditions to form products of nucleophilic addition to the unsubstituted positions of the pyrazine ring.The aim of the present study is to examine the further modifications of 6-alkoxy adducts 6-8 derived from the reaction of 5-(het)aryl-2,3-dicyano-1ethylpyrazinium tetrafluoroborates 5a-c with O-nucleophiles (water and alcohols) (Scheme 2). 23
The reactions of alkoxy adducts 6a-c,7a,8a with -rich heteroaromatic boronic acids proved to proceed in good yields (entries 1-17), however phenylboronic acid did not enter the reaction at all due to its low reactivity. 51It should be noted that the Petasis type reaction for 1,4-diazines is observed for the first time, although similar transformations have been described earlier for derivatives of quinoline and isoquinoline. 51ccording to the X-ray data, all racemic compounds 11a,c and 12a are crystallized in centrosymmetric P2 1 /n space group of monoclinic crystal system, and are characterized by the similarity of magnitudes in unit cell parameters (Tables 2, 4).Low variations in unit cell parameters for these structures are rationalised by the very similar packing requirements of the thiophene and benzene ring substituents in compounds 11a/12a and 11c.The dihydropyrazine ring in compounds is non-planar, the sp 3 -hybridized atom is deviated from the least-squared plane of the ring on 0.4-0.5 Å, and dihedral angles between the planes C6N1C2C3 and C3N4C5C6 are in limits 20-30 ° (numeration of atoms is given according to the figure for Table 2).However, atoms C3, N4, C5 are placed approximately in the plane of (het)aryl substituent at C5 and the double C=N bond of the azadienic fragment is obviously conjugated with the π-electronic system of the substituent.Measured sterical parameters of the dihydropyrazinic system, in particular, deviation of the sp 3 -atom from a plane of the pyrazine ring d(С6-N1C2C3C4C5), dihedral angles between planes C6N1C2C3/C3N4C5C6 and N1C2C3C4C5/C5C6N1, are depending not from the type of substituents at the sp3-hybridised Catom (Ar'), but from effects of the dihydropyrazine azadiene system conjugation with Ar substituent at С5.For pair 11а/12а a good structural correspondence is observed, however at the same time the structural parameters for the pair 11а/11c show significant differences (see Table 2).The next structural differences of dihydropyrazines are connected with their conformations and molecular packing.Dihedral angle between the plane of substitient Ar' at the sp3-hybridized C-atom and the plane C5C6C(Ar') for the pair 11а/12а is ~50°, the same angle for compound 11c is ~20° (exact definition of magnitudes is not possible due to disordering of substituent Ar').Despite on proximity of unit cell parameters, molecular packing for the pair 11а/12а and compound 11c show some variations.In particular, molecular packing for the pair 11а/12а is characterized by the presence of π-contacts between C≡N-groups and the azadiene system of the dihydropirazinis ring with distance ~3.3Å.This type of interaction is absent in packing of compound 11c, and their molecules form layers parallel 0b axe by means of axial N…S type contacts with distances ~3.25 Å between C≡N-groups and S-atoms of thienyl substituent at C5.All dihydropyrazines (±)-11, 12, 15, 16 and 18 were separated as pure enantiomers [enantiomeric excess approximately 95-99.9%] by semi-preparative chiral HPLC using Kromasil 5-CelluCoat (150 × 4.6 mm, 5 μm) column.Chiral HPLC and polarimetric analysis indicated that for all racemates, the first eluted enantiomers rotated polarized light in the negative direction, accordingly to a similar nature of substituents at the stereogenic center C (6)

. Crystals of enantiomeric pairs R-(-)-11b/S-(+)-11b and R-(-)-11c /S-(+)-11c suitable for X-ray experiments were grown by crystallization from acetonitrile solution. Enantiomeres R-(-)-11b/S-(+)-11b and R-(-)-11c/S-(+)-11c
were crystallized in very typical chiral P2 1 2 1 2 1 space group of orthorhombic crystal system with the nearest magnitudes in unit cell parameters (Tables 5, Figs. 4 and 5).All deviations in magnitudes in unit cell parameters for the pair of enantiomeres can to be explained by variation in temperatures of experiments and experimental errors.The presence of sulfur atoms in their structures permits to establish an absolute configuration of compounds.The absolute configuration of compounds was established on the basis of anomalous scattering effect (by Flack parameters), however, only for the R-(-)-11b and S-(+)-11c isomers their parameters are significant (Table 5).For the isomers R-(+)-11b and S-(-)-11c their parameters are less than the measurement mistakes, but are in accordance with measurements for enantiopodes.General stereochemistry of the enantiomeres is in good agreement with the stereochemistry of racemates.Some variations in dihedral angles due to rotation of a substituent around the single C-C bond's axis can to be explained by changing in the molecular packing environment.Any shortened intermolecular contacts in the molecular packing are absent.

S-(+)-11b.
On the basis of combined chromatographic and X-ray studies, the absolute configuration assignment was unambiguously completed, and configurations were assigned to the fifteen enantiomeric pairs: R-(-)-11a-c, 12a-c, 15a-c, 16a-c, 18a-c/S-(+)-11a-c, 12a-c, 15a-c, 16a-c,  18a-c.Due to comparable chiral centers in this group of molecules, the results clearly indicate at the (S)-absolute configuration for all dextrorotatory enantiomers of the series and at the (R)absolute configuration for the corresponding levorotatory isomers.
The results obtained show that antimycobacterial activity of several derivatives of the series of 1-alkyl-2,3-dicyano-5-aryl-6-hetaryl-1,6-dihydropyrazines is similar to the standard drugs.In addition, the compounds of this series are active against extensively drug-resistant and multidrug-resistant strains of Mycobacterium tuberculosis.

Conclusions
It has been shown that the Petasis reaction provides a mild, practical, and novel method for noncatalytic synthesis of dihydropyrazine derivatives.Novel 1-alkyl-2,3-dicyano-5-(het)aryl-6hetaryl-1,6-dihydropyrazines have been obtained and characterized.The structures of these compounds have been established by X-ray crystallography analysis.Several compounds of this series proved to possess a better antimycobacterial activity, than those from the family of classic antituberculosis drugs, for example Pyrazinamide.Indeed, 6-thienyl and 6-furyl substituted 1alkyl-2,3-dicyano-5-(het)aryl-6-hetaryl-1,6-dihydropyrazines can be regarded as promising candidates for the further development of antitubercular agents.

Experimental Section
General.All solvents and reagents were purified and prepared according to the literature data. 52ompounds 6-8 were synthesized according to the procedures described in the literature. 23 1H and 13 C NMR spectra were recorded on a Bruker AVANCE-500 instrument using Me 4 Si as an internal standard.All signals in the 1 H and 13 C NMR spectra were assigned on the basis of 2D 1 H-1 H COSY, 1 H- 13 C HSQC and HMBC experiments.Elemental analysis was carried on a Eurovector EA 3000 automated analyzer.Melting points were determined on Boetius combined heating stages and were not corrected.The GC-MS analysis of all samples was carried out using an Agilent GC 7890A MS 5975C Inert XL EI/CI GC-MS spectrometer with a quadrupole massspectrometric detector with electron ionization (70 eV) and scan over the total ionic current in the range m/z 20-1000 and a quartz capillary column HP-5MS (30 m × 0.25 mm, film thickness 0.25 mm).Helium was used as a carrier gas, the split ratio of the flow was 1 : 50, and the consumption through the column was 1.0 mL min -1 ; the initial temperature of the column was 40 °C (storage 3 min), programming rate was 10 °C min -1 to 290 °C (storage 20 min), the temperature of the evaporator was 250 °C, the temperature of the source was 230 °C, the temperature of the quadrupole was 150 °C, and the temperature of the transition chamber was 280 °C.Solutions of the samples with a concentration of 3-4 mg mL -1 were prepared in acetonitrile.Samples of 1 mL of the obtained solutions were analyzed.Column chromatography was carried out using Lancaster silica gel 0.040-0.063mm (230-400 mesh), eluting with ethyl acetate-hexane, 1 : 2. The progress of reactions and the purity of compounds were checked by TLC on Sorbfil plates (Russia), in which the spots were visualized with UV light (λ 254 or 365 nm).Microwave experiments were carried out in a Discover unimodal microwave system (CEM, USA) with a working frequency of 2.45 GHz and the power of microwave radiation ranged from 0 to 300 W. The reactions were carried out in a 10 mL reaction tube with the hermetic Teflon cork.The temperature of the reaction was monitored using an inserted IR sensor by the external surface of the reaction vessel.Semi-preparative HPLC enantioseparations were performed on an Agilent 1100 instrument using stainless-steel Kromasil 5-CelluCoat (150 × 4.6 mm, 5 μm) column.The analytical HPLC to determine the enantiomeric excess of the optically pure compounds was performed on the same instrument using chiral Kromasil CelluCoat (150 × 4.6 mm, 5 μm) column and mobile phase of CH 3 CN-H 2 O at the different ratio.The values of retention time (t R ) were given in minutes.Enantiomeric excess values were calculated from peak areas using the formula (A1-A2)/(A1+A2), where A1 is the major enantiomer.Specific rotations of enantiomers were measured at 589 nm with a Perkin-Elmer polarimeter model 341 equipped with a Na lamp.The volume of the cell was 1 mL, and the optical path was 10 cm.A standard solution was prepared by dissolving 5 mg of compound into 2 mL of CH 3 CN (c = 0.25).X-ray diffraction analysis of single crystals of 11a,c, 12a and R-(-)-11c was performed on an automated X-ray diffractometer "Xcalibur 3" equipped with CCD detector.Compounds R-(-)-11b, S-(+)-11b, S-(+)-11c were analyzed on an automated X-ray diffractometer "Xcalibur E" on a standard procedure (λMoK α , graphite monochomator, ω/2θ scanning with step 1 o ).Empirical absorption correction was performed.Structures of compounds 11a,c, 12a and R-(-)-11c were solved and refined by full-matrix least-squared method on F 2 with using SHELXTL program package. 53 Compounds R-(-)-11b, S-(+)-11b, S-(+)-11c were solved and refined by using Olex2 program. 54Non hydrogen atoms were refined in anisotropic approximation; H-atoms were refined in isotropic approximation in riding model.General X-Ray data are summarized in the Table 4.The X-ray crystallography data for structures 11a-c and 12a reported in this paper have been deposited with Cambridge Crystallography Data Centre as supplementary publications no CCDC 991536 for (±)-11a, CCDC 991537 for (±)-11c, CCDC 991538 for (±)-12a, CCDC 991532 for (+)-11b, CCDC 991533 for (-)-11b, CCDC 991534 for (+)-11c and CCDC 991535 for (-)-11c.These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Table 5. General X-ray structural data for enantiomeric compounds R-(-)-11b /S-(+)-11b and R-(-)-11c /S-(+)-11c
Compound   1, entry 15), yellow-orange powder, mp 137-140 °C.Racemate was separated by chiral HPLC with a Kromasil 5-CelluCoat (150 × 4.6 mm, 5 μm) column.The Antimycobacterial assay.To evaluate the inhibitory efficiency of molecules on Mycobacterium tuberculosis, M. tuberculosis H 37 Rv, which is susceptible to all classical antituberculosis drugs, was used.The minimal inhibitory concentration (MIC) for M. tuberculosis H 37 Rv for each compound was determined by a micro broth dilution method.All molecules tested were dissolved in dimethylsulfoxide and their 1/2 dilutions were prepared in 5 mL tubes using Löwenstein-Jensen medium.A few colonies from freshly grown M. tuberculosis H 37 Rv were suspended in Löwenstein-Jensen medium to obtain 1.0 McFarland turbidity and diluted ten times using the same medium and the tubes were incubated at 37 °C medium with a different concentration of the tested molecule and to a positive control tube containing only clear growth medium.After 24 hours the tubes were placed in a vertical position and the free edge of the buried 0.3 mL of the substance in the test compounds concentrations: 12.5, 6.2, 3.1, 1.5, 0.75 μg/mL.The tubes were then placed in an thermostat at a temperature of 37 °C and incubated for 10 days.Growth estimate for the MTB were determined by standard methods, where the appearance of zones of growth retardation MTB (over 10 mm) indicated the presence of tuberculostatic properties in concentration of the compounds under study.Penetration size stunting MTB (in mm) is proportional to the degree of tuberculostatic activity.Growth delay of 100 mm or more is considered as a complete growth inhibition MTB.The extensively drugresistant (XDR) and multi-drug-resistant (MDR) tuberculosis strains have been isolated from tuberculosis patients in Ural Research Institute for Phthisiopulmonology (Russia).The minimal inhibitory concentrations against Mycobacterium tuberculosis avium, Mycobacterium tuberculosis terrae, XDR and MDR tuberculosis strains were evaluated similarly.

Table 1 .
Reaction conditions and yields of compounds

11, 12, 15, 16 and 18
n.d.-not determined Table2.Some stereometric characteristics of dihydropyrazines according to the X-Ray data.A numeration of ring's atoms is given according to the figure below
n.d.-not determined; MDR (multi-drug-resistant tuberculosis strain) -Rifampin and isoniazid resistant Mycobacterium tuberculosis strain having Beijing genotype with a combination of mutations

Table 4 .
General X-ray structural data for racemic compounds 11a,c and 12a