Synthesis and Aminomethylation of 2-Amino-4-(2-chlorophenyl)-6-(dicyanomethyl)-1,4-dihydropyridine-3,5-dicarbonitrile N-Methylmorpholinium Salt

Sequential reaction of 2-chlorobenzaldehyde, cyanothioacetamide, and malononitrile dimer in the presence of an excess of N-methylmorpholine resulted in the formation of N-methylmorphlinium salt of 2-amino-4-(2-chlorophenyl)-6-(dicyanomethyl)-1,4-dihydropyridine-3,5-dicarbonitrile. The resulting salt reacts under Mannich conditions with primary amines and an excess of formaldehyde to form substituted 2-alkylamino-4-(dicyanomethylene)-3,7-diazabicyclo[3.3.1]non-2-ene-1,5-dicarbonitriles. Structure of the key compound was confirmed by single crystal X-ray diffraction analysis.

Despite the rich synthetic potential, there are scarce data on the preparation of such pyridine derivatives [2,3]. The potential for the practical use of such compounds has not been fully realized. Thus, anticancer [18], molluscicidal [8], and antibacterial [19,20] effects of a number of 2-(dicyanomethyl)pyridines have been reported. A series of 3-cyano-2-(dicyanomethylene)pyridines with pronounced fluorescent and photoluminescent properties has been described in [21][22][23].
The reaction tolerates both aliphatic and aromatic amines. Presumably, in each case, the process begins with aminomethylation at the most active nucleophilic positions 3 and 5 of compound 11 to form the 3,7-diazabicyclo[3.3.1]- non-2-ene system. The choice of the reaction direction in favor of the formation of products 17 or 18 is apparently determined by the presence or absence of an excess of amine in the reaction medium. Closure of the 1,3,5-triazine ring was not observed in any of the cases. In our opinion, this is due to the low nucleophilicity of the endocyclic nitrogen atom owing to the strong electronwithdrawing effect of the dicyanomethylene fragment. The nature of the amines used and the chosen solvents do not have a fundamental effect on regioselectivity of the aminomethylation process. At the same time, the reaction of the salt 11 with p-toluidine in ethanol resulted in the isolation of the well-known [36] product of its reaction with formaldehyde, 1,3,5-tri(4-methylphenyl)-1,3,5-perhydrotriazine 19. However, it should be noted that compound 19 is also an aminomethylating agent and can successfully act as this agent in the absence of any catalysts (see, for example, recent examples in the review [37]). The absence of reaction with compound 11 can be explained by the relatively low solubility of 1,3,5-perhydrotriazine 19 in aqueous alcohol and removal from the reaction medium.
Structure of compounds 17-19 was determined using mass spectroscopy, 1 Н and 13 С NMR spectroscopy (DEPTQ), as well as IR spectroscopy method. In addition, structure of compound 17c was studied using singlecrystal X-ray diffraction analysis (Fig. 1).
3,7-Diazabicyclo[3.3.1]nonanes (bispidins) and related compounds are widely used as biologically active compounds [38,39], complexing agents for metals [40], radionuclides [41], as initial reagents for the synthesis of natural compounds [42], materials for imaging in positron emission tomography [43], platforms for chiral catalysts [44,45]. Recently, 3,7-diazabicyclononane derivatives have been successfully used as initial templates for constructing complex supramolecular assemblies [46][47][48][49]. Recently, submicromolar inhibitors of the main SARS-CoV-2 protease have been found among bispidins [50]. Based on the foregoing, it seemed appropriate to study the profile of the possible biological activity of new compounds by means of molecular docking. Possible protein targets for the obtained compounds were predicted using the GalaxySagittarius protein ligand docking protocol [51] based on the GalaxyWeb web server [52,53]. The 3D structures of the studied compounds were preliminarily optimized by means of molecular mechanics in the MM2 force field to optimize the geometry and minimize the energy. Docking using the GalaxySagittarius protocol was carried out in the Binding compatability prediction and Re-ranking using docking modes. Table S1 (see Supplementary Materials) shows docking results for each of compounds 17a-17g, 18a-18c for 20 target-ligand complexes with the lowest binding free energy ΔG bind and the best estimate of the protein-ligand interaction. Predicted protein targets are identified by ID in the Protein Data Bank (PDB) and in the UniProt database. As you can see from Table S1, common receptors for compounds 17a-17g, 18a-18c are blood coagulation factor XI (PDB ID 5e2p) and ephrin receptor (ephrin type-A receptor 2, PDB ID 5ia5), which plays an important role in the pathogenesis of some cancer forms (melanoma, Kaposi's sarcoma). Threedimensional visualization of the docking results was implemented using the UCSF Chimera software package [54,55] and is shown in Fig. 2. In general, compounds 17a-17g, 18a-18c can be considered as promising objects for further screening in order to find new agents for the treatment and therapy of oncological diseases and circulatory system diseases.
EXPERIMENTAL NMR spectra were recorded on a Bruker DPX-400 NMR spectrometer [400.40 ( 1 Н), 100.63 ( 13 С), 40.55 MHz ( 15 N)] in DMSO-d 6 relative to internal TMS or residual solvent signals. IR spectra were recorded on a Bruker Vertex 70 IR Fourier spectrometer with an ATR attachment on a diamond crystal. Mass spectra were obtained on a MX1321 instrument using a direct sample injection system at an ionization chamber temperature of 200°C and an ionizing electron energy of 70 eV. Elemental analysis was carried out on a C,H,N analyzer Carlo Erba 1106, measurement error ±0.4%. Purity of the obtained compounds was monitored by TLC on Silufol UV254 plates, eluting with acetone-hexane (1 : 1) and developing with iodine vapor or UV detector. Melting Scheme 5. points were determined on a Kofler instrument and were not corrected.
Commercially available reagents were used; cyanothioacetamide [56] and malononitrile dimer 1 [1] were obtained by known methods.      Single crystal X-ray diffraction analysis. Experimental material for a crystal of compound 17c (C 32 H 29 ClN 8 O 3 ) was obtained on a Bruker APEX-II CCD diffractometer at 296.15 K. The structure was solved by a direct method using the Olex2 [57] and ShelXD [58] software package and refined using the SHELXL package [59]. The structure was refined by full-matrix least squares in the anisotropic approximation for non-hydrogen atoms by F 2 . The main characteristics and unit cell parameters of 17c: crystal size 0.32 × 0.3 × 0.29 mm, triclinic crystal system, space group P-1, M 609.08; unit cell parameters: а 11.6884(7), b 12.1570(7), с 12.2224 (7)