Synthesis, structure, and antimicrobial activity of heterocyclic phenylsulfonyl- and 4-aminophenylsulfonyl-carboximidamides

Abstract A series of novel phenylsulfonyl- and 4-aminophenylsulfonyl-carboximidamides were synthesized by condensation of sulfonamides with heterocyclic methyl carbimidates obtained from heterocyclic carbonitriles and used ‘at its inception.’ The molecular structure of the obtained compounds is discussed. Compounds possessing heterocyclic systems with a nitrogen atom in the α position to the functional group showed a different single-crystal structure than expected. The synthesized derivatives were evaluated for antimicrobial activities: tuberculostatic, antibacterial, and antifungal. Graphical Abstract .


Introduction
At the end of the twentieth century, a number of new and ''reemerging'' pathogens were recognized [1]. These included S. pneumonia, L. pneumophila, M. avium, E. coli, H. pylori, S. aureus, C. albicans, and M. tuberculosis [2][3][4]. These microorganisms quickly develop a multidrug resistance (MDR) to used chemotherapeutics and antibiotics. A special case is M. tuberculosis, whose strains also develop extensive drug-resistance (XDR). Resistant strains of microorganisms are a major threat to immunocompromised individuals, and infections caused by them are the most common complication in HIV-positive patients [5]. At the same time, a lack of development of new antimicrobial drugs is observed, which can pose a serious threat to public health [6]. Thus, the interest of many research groups is focused on the search for new drugs active against resistant strains.
One of the research directions is to modify the structure of already used drugs. So the interest in chemical groups such as, for example, sulfonamides has not diminished. This group is characterized by multidirectional pharmacological activity. Sulfonamides act as anhydrase inhibitors [7], antifungal [8], antiviral [9], anticancer [10], antiinflammatory [11], and of course antibacterial agents.
There are few reports on the pharmacological activity of sulfonamidines. So far, only their in vitro ability to compete with triiodothyronine for binding to the thyroid hormone-a1 receptor (hTHR-a1) has been described [17]. These compounds can be obtained in several ways. They are formed as a result of the reaction of carbonitriles with primary sulfonamides [18] or in a reaction of amidines with sulfonyl chlorides [19]. The reports of reactions of sulfonamides with alkyl-or phenylcarbimidates could also be found in the chemical literature [20]. In the structure assigned to the products, two protons are connected to different nitrogen atoms of the amidine moiety [21]. That structure was adopted on the basis of 1 H NMR spectra in which two different signals for those protons were observed. The reaction of sulfonamides with heterocyclic carbimidates has not been described so far.
The above facts prompt us to synthesize sulfonyl-carboximidamides possessing in their structure phenylsulfonyl or 4-aminophenylsulfonyl moieties linked to heterocyclic rings of pyridine, pyrimidine, or pyrazine by the sulfonamidine group. Synthesized compounds have been evaluated for their antimicrobial activity in vitro: tuberculostatic, antibacterial, and antifungal.

Results and discussion
The subject of this work was the synthesis of heterocyclic phenylsulfonyl-and 4-aminophenylsulfonyl-carboximidamides 1-13. The performed reactions are shown in Scheme 1.
The structures of all these new compounds were confirmed by IR and NMR spectra as well as elemental analyses. Two signals for the NH groups shifted from each other have been observed in the 1 H NMR spectra. These separated signals can be due to the taken amino-imine structure of compounds obtained (Fig. 1, structure a) as we suggest for 3-and 4-pyridine derivatives 3-6.  also be a result of the magnetic inequivalence of NH protons in the amine moiety upon formation of a hydrogen bond in the case of the heterocyclic compounds in which the amidine group is in the a position to the nitrogen atom of heterocyclic ring (structure b). X-ray diffraction analysis was performed for N 0 -(4-aminophenylsulfonyl)-4-chloropicolinimidamide to address that question. We have described the synthesis of this compound previously [24]. It was chosen because we were able to obtain its crystals of sufficient size. The results of the single-crystal diffraction study confirmed a tautomeric structure b (Fig. 1). If both hydrogen atoms are bonded to the same nitrogen atom in the solid state, their magnetic inequivalence in the solution is probably caused by formation of hydrogen bonds and reduction of symmetry.
The study also resolved the molecular structure of the products of the reaction between carbimidates and 4-aminobenzenesulfonamide, as the active group in that reaction could be both the amine group of the sulfonamide moiety, as it was in the case of the reaction of benzene sulfonamide, or the aromatic amine group in the para position to the sulfonamide moiety, since the reactions of aromatic and aliphatic amines with carbimidates have been described [25,26]. For that purpose, the reaction of methyl pyrazine-2-carbimidate with N-(4-sulfamoylphenyl)acetamide was carried out. The resulting product 9 was identical with the compound that was obtained by the acetic anhydride acylation of derivative 8, which was formed in the reaction of methyl pyrazine-2carbimidate with 4-aminobenzenesulfonamide. This showed that the sulfonamide group was the active group in the reactions carried out, and the resulting compounds had structure c (Fig. 2).
Crystal structure of N 0 -(4-aminophenylsulfonyl)-4chloropicolinimidamide The crystallographic data, data collection, and structure refinement of N 0 -(4-aminophenylsulfonyl)-4-chloropicolinimidamide are summarized in Table 1. The bond lengths and angles characterizing the geometry of the molecules are presented in Table 2. In the molecule of the title compound ( Fig. 2), the bond lengths and angles characterizing the geometry of the 4-aminophenylsulfonyl and pyridine fragments are typical for compounds containing them ( Table 2).
In the crystal structure of N 0 -(4-aminophenylsulfonyl)-4chloropicolinimidamide, the H atoms from the amino group bonded with the C7 atom participate in the intramolecular N16-H16AÁÁÁO18 and N16-H16BÁÁÁN1 hydrogen bond (Table 3; Fig. 2). In the packing, the molecules are linked into chains of rings along the c axis ( Fig. 3b). In these rings, four molecules of N 0 -(4-aminophenylsulfonyl)-4-chloropicolinimidamide are linked via N19-H19AÁÁÁO17 and N19-H19BÁÁÁN19 and form the R 4 4 (20) hydrogen bond ring motif (Fig. 3a). The parallel lying chains of rings are connected through the N16-H16AÁÁÁO17 hydrogen bond and form columns along the b axis (Table 3; Fig. 3a). In the crystal lattice, these columns form a zipper-type supramolecular motif.

Tuberculostatic activity
The synthesized phenylsulfonyl-and 4-aminophenylsulfonyl-carboximidamides 1-13 were examined in vitro for their tuberculostatic activity against M. tuberculosis H 37 Rv  (Table 4). Investigations were performed by a classical test-tube method of successive dilution in Youmans' modification of Proskauer and Beck's liquid medium containing 10 % of bovine serum [33,34]. Bacterial suspensions were prepared from 14-day-old cultures of slowly growing strains and from 48-h-old cultures of saprophytic strains [35,36]. Solutions of the compounds in ethylene glycol were tested. Stock solutions contained 10 mg of compounds in 1 cm 3 . Dilutions (in geometric progression) were prepared in Youmans' medium. The medium containing no investigated substances and containing isoniazid (INH) as a reference drug were used for comparison. Incubation was performed at a temperature of 37°C. The MIC values were determined as minimum concentration inhibiting the growth of tested tuberculous strains in relation to the probe with no tested compound. The influence of the compound on the growth of bacteria at a certain concentration, 3.1, 6.2, 12.5, 25, 50, and 100 lg/cm 3 , was evaluated.

Antibacterial and antifungal activities
Antibacterial and antifungal activities of newly synthesized compounds were also examined. In the study of antibacterial activity three recommended reference strains S. aureus ATCC 25923, E. coli ATCC 25922, and P. aeruginosae ATCC 27853 were used [37]. Antifungal activity was determined with use of two strains: C. albicans ATCC 90028 and C. parapsilosis ATCC 22019 [38]. The susceptibility of the microorganisms to the agents was determined by the broth microdilution assay according to the procedures outlined by the National Committee for Clinical Laboratory Standards [37,38]. The stock solutions of the agents were prepared by dissolving the chemicals in DMSO. The final concentration of the agents in 200 mm 3 of Mueller-Hinton broth (for bacterial strains) or in RPMI 1,640 (for fungi) ranged over 0.125-256 lg/cm 3 .
In order to prepare the bacterial suspension, overnight culture of bacteria in 3 % Triptic soy broth was diluted in sterile saline to the final concentration of approximately 10 7 CFU/cm 3 . Aliquots (10 mm 3 ) of bacterial suspension were added to each agent solution. The MIC was defined as the lowest concentration of the agent that completely inhibited growth of the bacteria after 18 h incubation at 35°C.
Inocula of candida strains were prepared by suspension of five colonies picked from 24 h old cultures on Saburaud agar in sterile saline to the concentration of 10 6 cells per cm 3 . The final concentration of the working suspension was approximately 10 4 cells per cm 3 . Aliquots (10 mm 3 ) of the suspension were added to each agar solution. The MIC was defined as the lowest concentration of the agent that completely inhibited growth of the fungi after 48 h incubation at 35°C. The final results were average values from two independent experiments.
The study showed no antibacterial and antifungal activity of the tested compounds. All of the synthesized sulfonylcarboximidamides 1-13 exhibited activity with MIC [ 256 lg/cm 3 , which meant that those values did not fit standard test concentrations.

Conclusion
In conclusion, a series of novel sulfonyl-carboximidamides with different six-membered nitrogen heterocyclic systems were synthesized successfully in a reaction of heterocyclic methyl carbimidates with benzene sulfonamide and 4-aminobenzenesulfonamide. All these new compounds were confirmed by IR and NMR spectra as well as elemental analysis. The molecular structure of the obtained compounds was discussed. Compounds possessing heterocyclic systems with a nitrogen atom in the a position to the functional group showed a single-crystal structure different from expected and described for that chemical group in the literature. Antimicrobial activity of the synthesized compounds was evaluated against M. tuberculosis, S. aureus, E. coli, P. aeruginosae, C. albicans, and C. parapsilosis. Unfortunately, all of the studied compounds were practically inactive towards microbial strains tested. Infrared spectra were determined as KBr pellets of the solids on a Satellite FT-IR spectrophotometer (Mattson Instruments, Madison, WI). Melting points were determined with a Boethius apparatus (Franz Küstner Nachf. KG, Dresden, Germany). Methyl pyrazine-2-carbimidate and methyl 6-methoxypyrazine-2-carbimidate required for further syntheses were obtained according to the method described earlier by Foks and co-workers [22,23].
General method for the synthesis of sulfonylcarboximidamides 1-8 The respective carbonitrile (1 mmol) and 0.4 cm 3 (2 mmol) of DBU were refluxed in 10 cm 3 of methanol for 0.5 h. Then 0.8 mmol of benzene sulfonamide or 4-aminobenzenesulfonamide was added. The mixture was refluxed for another 3 h. Then methanol was evaporated in vacuo, and 30 cm 3 of water was added to the residue. The precipitate of the product was filtered off, dried, and purified by recrystallization from a suitable solvent.      13   Method B: sulfonylcarboximidamide 8 (0.28 g, 1 mmol) was refluxed for 0.5 h in a solution of 0.5 cm 3 (5 mmol) of acetic anhydride in 5 cm 3 of pyridine. Then pyridine was evaporated in vacuo, and 20 g of ice was added to the residue. The precipitate was filtered off, dried, and SHELXS-97 package solving the structures by direct methods and carrying out refinements by full-matrix leastsquares on F 2 using the SHELXL-97 program [28]. All H atoms bound with aromatic C atoms were placed geometrically and refined using a riding model with C-H = 0.93 Å and U iso (H) = 1.2 U eq (C). All H atoms bound with N atoms were placed geometrically and refined using a riding model with N-H = 0.86 Å and U iso (H) = 1.5 U eq (N). The -NH 2 group containing the N19 atom was assumed to be planar-trigonal and coplanar with the mean plane of the benzene ring. The -NH 2 group containing the N16 atom was assumed to be planar-trigonal and coplanar with the mean plane delineated by C2, C7, and N8 atoms. All interactions demonstrated were found by the PLATON program [29]. The programs used to prepare molecular graphics were: ORTEPII [30], PLUTO-78 [31], and Mercury [32]. Full crystallographic details, excluding structural features, have been deposited (deposition no. 849210) with the Cambridge Crystallographic Data Center. These data may be obtained, on request, from the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (Tel.: ?44-1223-336408; Fax: ?44-1223-336033; e-mail:depo-sit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
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