Approaches towards the synthesis of a novel class of 2-amino-5-arylazonicotinate, pyridazinone and pyrido[2,3-d]pyrimidine derivatives as potent antimicrobial agents

Background Despite significant progresses in antimicrobial therapy, infectious diseases caused by bacteria and fungi remain a major worldwide health problem because of the rapid development of resistance to existing antimicrobial drugs. Therefore, there is a constant need for new antimicrobial agents. There are a large number of heterocyclic derivatives containing nitrogen atoms that possess a broad spectrum of biological activities including pyridine and pyridazine, which are two of the most important heterocycles in medicinal chemistry. Results The reaction of 3-oxo-2-arylhydrazonopropanals 2 with ethyl cyanoacetate and malononitrile 3a,b has led to the formation of 2-amino-5-arylazo-6-aryl substituted nicotinates 8a-k as sole isolable products when the aryl group in the arylazo moiety was substituted with an electron-withdrawing group like Cl, Br, NO2. The pyridazinones 10 were formed from the same reaction when the arylazo moiety was phenyl or phenyl substituted with an electron-donating group. The 2-aminoazonicotinates 8 were condensed with DMF-DMA to afford the amidines 13a,b, which then were cyclized to afford the targeted pyrido[2,3-d]pyrimidine derivatives 15a,b, respectively. The structures of all new substances prepared in this investigation were determined by using X-ray crystallographic analysis and spectroscopic methods. Most of the synthesized compounds were tested and evaluated as antimicrobial agents and the results indicated that many of the obtained compounds exhibited high antimicrobial activity comparable to ampicillin, which was used as the reference compound. Conclusion A general rule for the synthesis of 2-amino-5-arylazo-6-aryl substituted nicotinic acid and pyridazinone was established using 3-oxo-2-arylhydrazonopropanal as a precursor. Moreover, a novel route to pyrido[2,3-d]pyrimidine was achieved. Most of the synthesized compounds were found to exhibit strong inhibitory effects on the growth of Gram-positive bacteria especially Bacillus subtilis. Compounds 1a, 8a-h, 10a-c, 15b and 16 showed a broad spectrum of antimicrobial activity against B. subtilis.


Background
The emergence and spread of antimicrobial resistance has become one of the most serious public health concerns across the world. Antimicrobial resistance refers to microorganisms that have developed the ability to inactivate, exclude, or block the inhibitory or lethal effects of antimicrobial agents [1]. Despite significant progress in antimicrobial therapy, infectious diseases caused by bacteria and fungi remain a major worldwide health problem because of the rapid development of resistance to the existing antimicrobial drugs (antibacterial and antifungal). In other words, the increasing use and misuse of existing antimicrobial drugs have resulted in the development of resistant pathogens. In particular, the emergence of multidrug-resistant Gram-positive and -negative bacteria has caused life-threatening infectious diseases in many countries. The chemical and biological study of heterocyclic compounds has been of interest for many years for medicinal and agricultural reasons. There are a large number of heterocyclic derivatives containing nitrogen atoms such as pyridine and pyridazine that possess a broad spectrum of biological activities including antimicrobial [2][3][4][5][6], anti-inflammatory and analgesic [7][8][9], anti-HIV [10], antiplasmodial [11], anti-tubercular [3,12], antibacterial [3,13], anticonvulsant [14,15], inhibition of cyclo-oxygenase [16], antidiabetic [17], antihypertensive [18], anticancer [19][20][21][22], inhibition of blood platelet aggregation [23], antidepressant and anxiolytic [24,25], antioxidant [26] and antifungal [27]. Thus, the extensive biological activities of pyridine and pyridazine make them important in the design of druglike molecules. Encouraged by the afore-mentioned findings and in a continuation of an ongoing program aimed at finding new structural leads with potential potent antibacterial and antifungal agents [28,29], this study describes the synthesis of a new class of 2-amino-5-arylazo-6-aryl substituted nicotinic acid, pyridazinone, and pyrido [2,3-d] pyrimidine derivatives.

Synthetic chemistry
The reaction of the 3-oxo-2-arylhydrazonopropanals 2 with the active methylene reagents has been investigated in the past [30]. Recently, it was shown that this reaction affords either arylazo-2-oxonicotinates 6 or pyridazinones 10 [31]. However, the factors that control the nature of the end product could not be defined. In the present article, we report the synthesis of several derivatives of 2 with electron-donating and -withdrawing substituents on the arylazo moiety and identified the exact structure of the products of their reaction with the active methylene reagents 3a,b. It could be concluded that the reaction of 3 with 2 having an electron-donating substituent on the arylazo moiety afforded only the pyridazinones 10 while reacting 3 with 2 having an electron-withdrawing substituent on the arylazo moiety either in the p, m, or o position or a mix of them affords only the 2-amino-5-arylazo-6-aryl substituted nicotinic acid derivatives 8. Thus compounds 2a-k were prepared via coupling of 1 with aromatic diazonium salts [30] (cf. Scheme 1 and Figure 1). Reacting 2a-g with ethyl cyanoacetate 3a or with malononitrile 3b affords the 2-amino-5-arylazo-6-aryl substituted nicotinates 8a-k as confirmed from accurate mass determination and elemental analyses. Moreover, the structures were also confirmed from the X-ray single crystal structure determination for 8a, 8b, 8c, and 8h (cf. Figures 2, 3, 4, and 5, Tables 1, 2, and Scheme 1). It is believed that initially the acyclic condensation products 4 were formed and then these cyclize to the pyranimine 5 that reacts with ammonia from the reaction medium to yield the acyclic intermediate 7 that further cyclizes into the final isolable 2-aminonicotinic acid derivatives 8. Under these conditions, no traces of the arylazo-2-oxonicotinates 6 or 2hydroxy-5-arylazonicotinates were isolated as reported by Al-Mousawi et al. [31,32]. On the other hand, the reaction of 2h-k having a phenyl or a phenyl substituted with an electron-donating group on the arylhydrazone moiety with 3a afforded the pyridazinones 10a-d. It is believed that also in this case, the acyclic intermediate 4 was formed and then cyclized via attack of the arylhydrazone moiety at CN to afford the pyridazine imine intermediate 9 that was hydrolyzed under the reaction conditions to yield the final isolable pyridazinone 10. The structure of 10 was also supported by both the classical analytical analyses and through the X-ray crystal structure determination for 10a (cf. Figure 6, Table 3, and Scheme 1). It is believed that the basicity of the hydrazone moiety of 2 controls the nature of the final product as it facilitates the reversible cyclization of the intermediate 4 and at the same time helps to stabilize the cyclized 9, thus allowing the hydrolysis step to proceed to form the pyridazinone 10. In contrast, cyclization of 4 is highly reversible and a competing cyclization reaction takes place resulting in formation of the pyranimine 5, which in the presence of ammonium ion led to the formation of the stable aromatic 2-aminonicotinic acid derivatives 8.
The obtained arylazoaminonicotinates are interesting precursors for the synthesis of a variety of a novel arylazoheterocycles that may possess interesting biological activities. Reaction of the 2-amino-5-arylazonicotinates 8 with acetic anhydride afforded the mono-and the diacetylated products 11 and 12, respectively, depending upon the reaction time. The structures of the products 11a and 12 were confirmed by X-ray single crystal determination (cf. Scheme 2, Figures 7, 8).
Moreover, the 2-amino-5-arylazonicotinates 8 reacted with dimethylformamide dimethylacetal (DMF-DMA) to yield the corresponding amidines 13. The amidines 13a,b reacted with ammonia in refluxing acetic acid to yield the corresponding pyrido[2,3-d]pyrimidine derivatives 15a,b. The structures of these products were also confirmed by different spectroscopic analyses as illustrated in the experimental section. Furthermore, fusion of the azonicotinates 8f with thiourea afforded the corresponding pyrido[2,3-d]pyrimidine derivatives 16 (cf. Scheme 3).

Antimicrobial activity
The novel chemical compounds synthesized in this study showed promising antimicrobial activities. In general, most of the tested compounds revealed better activity against Gram-positive rather than the Gram-negative bacteria and yeast. The results as depicted in Table 4 show strong activities against Gram-positive bacteria because all of the tested chemicals showed highly positive antimicrobial activities against B. subtilis with inhibition zones >10 mm. Only the tested chemical 1a displayed strong inhibitory effects on the growth of Escherichia coli (Gram-negative bacteria), Bacillus subtilis, and Staphylococcus aureus (Gram-positive bacteria), which showed inhibition zones exceeding 10 mm. It also strongly inhibited the growth of Candida albicans (yeast) while the cycloheximide did not inhibit growth of this yeast. None of the chemicals except 1a inhibited the growth of Gramnegative bacteria or yeast. Moreover compounds 2a, 2c, 2d, and 2g had high inhibitory activities against the Grampositive bacteria S. aureus. The tested chemicals 8a-h and 10a-c displayed very strong inhibitory effects toward the growth of the Gram-positive bacteria B. subtilis with inhibition zones exceeding the reference chemotherapeutic ampicillin (cf. Table 4). Compounds 8a and 10c were also nearly as active as ampicillin against B. subtilis (MIC = 12.5 μg/mL). It was found that transformation of the enaminones 1 into the corresponding arylhydrazonals 2 generally decreased the inhibitory effects while transformation of the latter into the corresponding 2amino-5-arylazo-6-aryl substituted nicotinates 8 or the pyridazinone 10 resulted in inhibition of the growth of only B. subtilis (Gram-positive bacteria) as revealed by the diameters of their inhibition zones. Conversely, conversion of the 2-aminoazonicotinates derivatives into the corresponding acetyl, diacetyl, or amidine derivatives exemplified by compounds 11, 12, and 13 unfortunately resulted in a decrease in the inhibitory effects but still had inhibition zones >10 mm. Fusing the pyridine ring into the bicyclic pyrido[2,3-d]pyrimidine derivatives 15a,b and 16 enhanced the antimicrobial activity because the majority of these compounds were active against only the Grampositive bacteria B. subtilis and S. aureus.
Structure activity relationship By comparing the experimental biological activity of the compounds reported in this study with their structures, the following structural activity relationship assumptions are postulated. ➢ The pyridine or pyridazine moieties are necessary to observe the higher antibacterial activities towards the Gram-positive bacteria B. subtilis. ➢ It is interesting to point out that for the azonicotinates 8 having an electron-withdrawing group in the arylazo moiety in the para-, metaand orthopositions like compounds 8a-e or having two electron-withdrawing groups in the arylazo moiety as in 8f and 8h results in higher antibacterial activity as evidenced by the inhibition zones that were similar (Table 4), and from the minimum inhibitory concentration (MIC) values presented in Table 5. This indicates that high antimicrobial activity may be correlated with the low electron density of the ring systems and the role of an electron-withdrawing group in increasing the  antimicrobial potency is similar to the results of Sharma et al. [33]. ➢ It is worth mentioning that changing the COOEt group to a CN group as in 8f and 8h has no significant effect on the biological activity.

Experimental
General Melting points were recorded on a Griffin melting point apparatus and are reported uncorrected. IR spectra were recorded using KBr disks using a Perkin-Elmer System 2000 FT-IR spectrophotometer. 1 H-NMR (400 MHz) or (600 MHz) and 13 C-NMR (100 MHz) or (150 MHz) spectra were recorded at 25°C in CDCl 3 or DMSO-d 6 as solvent with TMS as internal standard on a Bruker DPX 400 or 600 super-conducting NMR spectrometer. Chemical shifts are reported in ppm. Mass spectra were measured using a high resolution GC-MS (DFS) thermo spectrometers with EI (70 EV). Microanalyses were performed on a LECO CHNS-932 Elemental Analyzer. Follow up of the reactions and checking homogeneity of the prepared compounds was made by thin layer chromatography (TLC). All single crystal data collections were made either on Rigaku R-AXIS RAPID diffractometer using Mo-Kα radiation (for samples 8a, 8c, 8h, 11a and 12) or on Bruker X8 Prospector using Cu-Kα radiation (for compounds 2a, 2h, 8b, and 10a). The data were  collected at room temperature. The structure was solved by direct methods and was expanded using Fourier techniques. The non-hydrogen atoms were refined anisotropically. In the case of compounds 8a, 8c, 8h, 11a and 12, all calculations were performed using the Crystal Structure [34] crystallographic software package except for refinement, which was performed using SHELXL-97 [35]. In the case of 2a, 2h, 8b, and 10a the structure was solved and refined using the Bruker SHELXTL Software Package (Structure solution program-SHELXS-97 and Refinement program-SHELXL-97) [35] (cf. Additional files 1, 2, 3, 4, 5, 6, 7, 8, 9 and Table 6). Data were corrected for the absorption effects using the multi-scan method (SADABS). The enaminones 1a,b and the arylhydrazonals 2a-k were prepared according to the literature procedure [30,31].
General procedure for the preparation 2-amino-5-arylazo-6aryl substituted nicotinates 8a-k Independent mixtures of 2a-g (10 mmol), active methylenenitrile derivatives 3a,b (10 mmol), and ammonium acetate (2 g) in acetic acid (20 mL) were stirred at reflux for 1-2 h. (the progress of the reactions was monitored by using TLC using 1:1 ethyl acetatepetroleum ether as eluent). The mixtures were cooled and then poured into ice-water. The solids that so formed were collected by filtration and crystallized from the proper solvents to give 8a-k as pure products.
2-Amino-6-(4-chlorophenyl)-5-(4-chlorophenylazo) nicotinic acid ethyl ester (8a) Recrystallized from an EtOH/dioxane         General procedure for the preparation pyridazine derivatives 10a-d Independent mixtures of 2h-k (10 mmol), ethyl cyanoacetate 3a (1.15 g, 10 mmol), and ammonium acetate (2 g) in acetic acid (20 mL) were stirred at reflux for 1-2 h. (the progress of the reactions was monitored by using TLC using 1:1 ethyl acetate-petroleum ether as eluent). The mixtures were cooled and then poured into iced water. The solids that so formed were collected by filtration and recrystallized from the proper solvents to give 10a-d as pure products.      General procedure for the preparation compounds 11a-c and 12 Independent solutions of the azonicotinates 8a,c,f (10 mmol) in acetic anhydride (10 mL) were stirred at reflux for 4 h. in case of compounds 11a-c and for 12 h. in case of compound 12. Then, the reaction mixture was allowed to cool to room temperature, the formed crude product was collected by filtration washed with ethanol and recrystallized from the proper solvent.
2-Acetylamino-5-(2-chloro-5-nitrophenylazo)-6-(4chlorophenyl)nicotinic acid ethyl ester (11a) Recrystallized from EtOH/dioxane 2-Acetylamino-6-(4-chlorophenyl)-5-(4-chlorophenylazo) nicotinic acid ethyl ester (11b) and cycloheximide (Sigma, St. Louis, MO, USA) both used as references in the experiment where ampicillin was used as an antibacterial drug, which is known to inhibit prokaryotes organisms while cycloheximide was used as an antifungal drug, which is known to inhibit eukaryotic organisms. The MIC measurement was determined for compounds with inhibition zones >12 mm using a two-fold serial dilution technique [40]. The inhibition zone diameters values cited in Table 4 are attributed to the tested original concentration (1 mg/mL) as a preliminary test and the MIC (μg/mL) values are recorded in Table 5.