Facile NMI-MsCl mediated synthesis of novel pyrazole derivatives bearing heteroaryl amides as potent antimicrobial agents

We herein report the convenient synthesis of a series of novel pyrazole derivatives linked to heteroaryl groups via amide functionality.NMI-MsCl mediated amide bond formation reaction has been successfully employed for the synthesis of novel pharmacologically relevant pyrazole derivatives. The antimicrobial potential of the newly synthesized compoundswere evaluated at the later stage


Introduction
The nitrogen, and oxygen containing heterocyclic compounds are of considerable interest nowadays owing to its various applications in drug-discovery, agrochemicals, and material science. [1][2][3] Among the various heterocyclic compounds discovered so far, pyrazole, and its derivatives have a dominant space in the area of drug discovery owing to their varied applications in pharmaceutical industries, and academics. [4][5][6] The molecules derived from pyrazoles are utilized as agrochemicals for the extensive protection of crops in the form of insecticides, fungicides, and herbicides. 7 Pyrazole based architectures are known to possess a significant role in the area of medicinal chemistry for designing, synthesizing, and developing novel biologically active molecules leading to drugs. 8,9 The presence of pyrazole nucleus in many FDA approved marketed drugs further underlines its significance (Fig 1). In addition to this, many pyrazole based molecules are under different phases of clinical trials, and could appear as possible drugs in near future. 10 Among the diverse pyrazole based architectures, 5-aminopyrazole is an important scaffold because of its synthetic versatility, and varied applications in drug-discovery. 11 5-Aminopyrazoles are included in many bioactive compounds that target Aurora kinases as well as polo-like (PLK), and cyclin-dependent (CDK) kinases. They also targets adenosine A1 receptor, neuropeptide Y receptor 5 (NPY5), alpha-7 nicotinic acetylcholine receptors (a7nAChR), and corticotrophin-releasing factor-1 (CRF-1) receptor. [12][13][14] In addition to this, 5-aminopyrazoles are useful building blocks for the synthesis of several fused nitrogen heterocycles of potential biological importance. 15 The role of amides, and thioamides as efficient linkers in the modern area of drug-discovery is well reported in literature. 16 The amides attached to various heterocyclic groups are hypothesized to be acting as

Results and Discussion
The synthetic methodology adopted by us has been depicted in Scheme 1. The reaction of 2-methoxyphenyl hydrazine 2 with 3-aminocrotononitrile 1 in presence of 12 N HCl, and water as solvent at reflux conditions procured the 1-(2-methoxyphenyl)-3-methyl-1H-pyrazol-5-amine precursor 3 at 90% yield. 17 This key amino precursor was then subjected to classical peptide coupling reaction conditions with various heteroaryl acids 4a-j for synthesizing an array of pharmacologically relevant pyrazole linked with amides 5a-j. Scheme 1. Synthesis of amino precursor, and its coupling with various heteroaryl acids.
The key amino precursor 3, and thiophene-2-carboxylic acid 4a was selected for our initial control experiments in view of identifying the optimum reaction reactions for obtaining the maximum yield of the desired products (Table 1). Our first screening experiment was carried out by employing EDC . HCl as the coupling reagent, and DIPEA as a base in DCM solvent for 3 hours at room temperature ( Table 1, entry 1). The reaction got completed within the specific time as monitored by TLC. However, the desired amide product 5a was not obtained as a major product. Instead, we obtained the anhydride 6 that was derived from the acid, as the major product. This obtained result enlightened us the need for a detailed optimization study to improve the yield of the desired product 5a by minimizing the formation of anhydride 6. In view of the above obtained result, we decided to screen different coupling reagents, bases, and solvents for our optimization studies (Table 1). However, the traditional coupling reagents like EDC . HCl, HATU, HBTU, HCTU, and T3P in different solvents, and bases afforded the anhydride 6 as the major product (Table 1, entries 1-9). In all these screening studies, a reasonable amount of the desired amide product 5a was obtained. To our delight, we got the required product 5a in 55% yield when methanesulfonyl chloride (MsCl) was employed as the coupling reagent (Table 1, entry 10). This observation prompted us to screen different bases by fixing MsCl as the coupling reagent (Table 1, entries 11,12). Accordingly, the desired product was obtained in 80% yield when Nmethylimidazole (NMI) was used as a base, and DCM as a solvent (Table 6.1, entry 11). We found that the usage of NMI as a base significantly minimized the generation of anhydride 6. Finally, the expected product 5a was obtained in 90% isolated yield when the reaction was carried out at 45 ℃ (Table 6.1, entry 12). The utilization of other sulfonyl halides like toslyl chloride (TsCl), and trifluoromethanesulfonyl chloride (TfCl) as a coupling reagent also decreased the formation of unwanted side-product, anhydride 6 ( Table 6.1, entries 13,14). However, the desired product was procured in lesser yield when compared to MsCl. Among the various solvents screened, DCM was found to be essential for the formation of the desired amide product 5a in high yield (Table 6.1).
After identifying the optimum reaction conditions for the synthesis of amide product 5a, our next attention was to evaluate the generality of this developed protocol. Keeping this in mind, a series of heteroaryl acids 4a-j were treated with the key amino precursor 3 in our optimized reaction conditions for synthesizing a variety of pyrazoles linked with amides 5a-j ( Table 2). To our delight, all the acids reacted well enough to generate the desired amides in good to excellent yields (80-94%). The different acids containing heterocyclic groups like furan, thiophene, imidazole, coumarin, isoxazole, and pyridine were tolerant in this reaction conditions to procure the corresponding amides in high yields (Table 2). However, the acid having imidazole heterocyclic group rendered the desired amide 5g in slightly lower yield (80%). It is noteworthy that thiophene-3-carboxylic acid yielded the desired amide 5c in 94% yield.
All the newly synthesized amides 5a-j were then subjected to evaluate their potential as antimicrobial agents. The in vitro antibacterial activity studies of the newly synthesized pyrazole based molecules 5a-j were carried out against three bacterial strains, namely, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus.The antifungal activity studies of 5a-j have been carried out against three different fungal strains, Candida albicans, Aspergillusflavus, and Rhizopus sp.These studies were carried out by serial plate dilution method, 18,19 and ciprofloxacin, and Amphotericin Bwas employed as the reference standard for antibacterial, and antifungal studies respectively. The minimum inhibitory concentration values (MIC) of all the newly synthesized pyrazole derivatives 5a-j were evaluated, and our results are summarized in Table 3.   From our studies, we found that the compounds 5e,5f, 5g, and 5jwere promising antibacterial agents when compared with the standard. The most active compoundsagainst all the bacterial strains tested in our investigation were found to be 5g, and 5j.The compounds5a,5c,5d,5h, and 5i exhibited promising antifungal acticvity with respect to the standard drug. However, the compound 5d was found to be the best antifungal agent when compared to the other synthesized molecules. All the other tested molecules exhibited either moderate or lower activity profile, and hence can be considered as inactive.

Conclusions
The NMI-MsCl mediated convenient synthesis of novel pyrazole derivatives containing amides 5a-j have been efficiently carried out in good to excellent yields. The developed methodology paved the way for the synthesis of pyrazoles linked to diverse heteroaryl groups via amide functionality. The newly synthesized molecules were evealuated for their antimicrobial potential employing ciprofloxacin, and amphotericin B as the reference standard for antibacterial, and antifungal activity respectively.The compounds 5g, and 5jwere found to be promising antibacterial agents whereas the compound 5d was identified as the best antifungal agent when compared to respective standards. The detailed studies on the mode of action of these compounds, and the synthesis of more specialized derivatives are currently in process in our laboratory.

Experimental Section
General. All solvents, and reagents were purchased from commercial suppliers, and used without any further purification. Analytical TLC was performed on pre-coated aluminum sheets of silica (60 F254 nm), and visualized by short-wave UV light at λ 254. Melting points were noted on an EZ-Melt automated apparatus. 1 H, and 13 C NMR spectra were recorded at 400 or 600 MHz, and 100 or 150 MHz respectively. Chemical shifts were reported in parts per million (ppm), and coupling constants in Hertz (Hz). Tetramethylsilane (TMS) (δ0.00 ppm) or residual solvent peakin DMSO-d6 (δ2.50 ppm), and CDCl3 (δ 7.26 ppm) served as internal standard for recording. 20 Molecular weights of new compounds were measured by GCMS-QP2010 Ultra gas chromatograph operating at an ionization potential of 70 eV (EI). Microanalyses were performed on PerkinElmer Series II CHNS/O 2400 elemental analyzer. Melting points were determined using a Stuart SMP 3 apparatus.
Procedure for the synthesis of amino precursor 3. 3-Aminocrotononitrile(1, 10 mmol)was added to a suspension of 2-methoxyphenylhydrazine 2 (10 mmol) in 12N HCl-H2O (12 mL, 1:3), and the resulting mixture was refluxed for 12 hours. After the specified time, the reaction mixture was cooled, and neutralized with 2.5M sodium hydroxide solution. The suspension was extracted with CH2Cl2 (3x30 mL). The combined organic phases were washed with brine (20 mL), dried over sodium sulphate, and the solvent was distilled off under reduced pressure. The obtained residue oil was triturated with hexane to obtain a solid which was recrystallized in ethanol to obtain the entitled 1-(2-methoxyphenyl)-3-methyl-1H-pyrazol-5-amine precursor 3 as colourless oil in 90% yield. 171  General procedure for the synthesis of final compounds 5a-j. To a solution of amino precursor 3 (1 mmol, 1 equiv.) in DCM (10 vol) taken in a round bottomed flask, NMI (2 mmol, 2 equiv.), and MsCl (1 mmol, 1 equiv.) was added at 0 ℃. The reaction mixture was then warmed to room temperature, and stirred for 20 minutes. After 20 minutes, acids 4a-j (1.2 mmol, 1.2 equiv.) was added at 0 ℃, and the reaction mixture was then brought to room temperature. After that, the reaction mixture was heated at 45 ℃ for about 3 hours. The completion of the reaction was monitored by TLC. After reaction completion, the reaction mixture was diluted with ice water, and the aqueous layer was extracted thrice with DCM. The combined organic layers were washed with brine, and dried over anhydrous sodium sulfate, and distilled under reduced pressure to get crude residue. The crude mixture was purified by column chromatography using hexane, and ethyl acetate as eluent to obtain the titled pyrazole derivatives 5a-j in varying yields.   13