Site-Selectivity of the Reaction of 3-Amino-4-Cyano-5-Phenyl-1H-Pyrrole-2-Carboxylic Acid Amide with α-Halocarbonyl Compounds. Antimicrobial Activity and Docking Study for COVID-19 of the Products

Abstract In this context, we are interested to study the site-selectivity of the reaction of 3-amino-4-cyano-5-phenyl-1H-pyrrole-2-carboxylic acid amide 1 with different types of hydrazonoyl halides as well as the α-haloketones. The products of these reactions were confirmed from their spectral data and conformational studies to be pyrrolo[1,2-d][1,2,4]triazine and pyrrolo[1,2-a]pyrazine derivatives. We used docking studies to test the ability of the new synthesized pyrrolo[1,2-d][1,2,4]triazine and pyrrolo[1,2-a]pyrazine derivatives to dock and inhibit the microbes and COVID-19 proteins. The results of both docking studies revealed a strong fit of almost the tested derivatives into the active sites of peroxiredoxin 5 (Pdb: 2WFC; the overexpressed proteins in Candida albicans) and COVID-19 main protease (Pdb: 6LU7; severe acute respiratory syndrome coronavirus 2). Moreover, the antimicrobial activity of the products was investigated against two selected Fungi species and two G+ and two G− bacteria. The results of antimicrobial activity indicated that almost all derivatives showed no activity against fungi species, while all of them have moderate activity against bacteria strains (G+ and G− bacteria). Graphical Abstract


Introduction
The COVID-19 pandemic that swept the world at the end of 2019 is one of the largest health emergencies that humans have faced in the past 100 years. The number of deaths due to infection with the Coronavirus has reached nearly six million cases, in addition to the infections that exceeded four hundred million infections and resulted in serious side effects. Scientists are trying everyone in his researching field for a drug or a way to stop this epidemic by producing means to limit its spread, such as the muzzle or the transparent protective mask, or through theoretical researching way about the possibility of the effectiveness of some heterocyclic compounds against this epidemic. [1][2][3] Theoretical research that includes the docking study has shortened the time to predict the effectiveness of some heterocyclic compounds for many diseases. [4][5][6] On the other side, overuse as well as the misuse of antibiotics has led to the expansion of multidrug resistance between various types of microorganism's strains. 7 Accordingly, new antimicrobial agents must be developed that act on new targets to overcome the increasing incidence of microbial resistance to antibiotic therapy. Scientists are also working day and night to search for new compounds with antimicrobial activity. Pyrrole ring when fused with triazine or pyrazine afforded potent bioactive systems 8-11 ( Figure 1). As for instance, pyrrolo[1,2-a]pyrazine ring system is an essential part of several drugs having anticonvulsant, 12 nootropic, 13 neuroleptic, 14 antagonist, 15 antibiotic agent 16 and well known with its antidiabetic activity. 17 Combination of all the above findings with our research project concerning with the synthesis of bioactive heterocyclic compounds, [18][19][20][21][22][23][24][25] we focused our effort in this research project to study the site-selectivity of the reaction of 3-amino-4-cyano-5-phenyl-1H-pyrrole-2-carboxylic acid amide 1 with a-halocarbonyl compounds due to this reaction is expected to give different products as a result of the presence of several nucleophiles in compound 1; amino (NH 2 ), amide (CONH 2 ) and cyclic NH groups (Scheme 1). After confirmation of the structure of all new synthesized derivatives, they will be subjected to investigation their ability to fit in the active site of peroxiredoxin 5 (Pdb: 2WFC; the overexpressed proteins in Candida albicans) and COVID-19 main protease (Pdb: 6LU7; severe acute respiratory syndrome coronavirus 2), in addition, in vitro screening of all synthesized derivatives against bacteria (G þ & G À ) and Fungi species.

Results and discussion
3-Amino-4-cyano-5-phenyl-1H-pyrrole-2-carboxylic acid amide 1 was subjected to react with hydrazonoyl chloride 2 in dioxane/Et 3 N under reflux (Scheme 1). Such reaction was expected to afford one of the three products 6, 7, or 8 via the nucleophilic substitution reaction followed by the cyclization reaction with the elimination of ammonia or water molecule as illustrated in Scheme 1. The TLC test indicated the isolation of only one product and IR data of the isolated product revealed the presence of amidic carbonyl absorption band (C¼O) at ¼ 1642 cm À1 , these data excluded the triazole product 8. Both IR and 1 H NMR data of the isolated product proved structure 6 rather than pyrrolotriazepine derivative 7 due to the appearance of a broad signal with integration of two protons for NH 2 group rather than two singlet signals for two NH groups ( Figure 2).
In continuation of this investigation, the reaction of pyrrole derivative 1 with another type of hydrazonoyl chlorides 9 under the same reaction condition can afford four different types of heterocyclic derivatives, namely: pyrrolopyrazines 13, pyrrolodiazipenes (14 & 15) or pyrrolyltriazoles 16 (Scheme 2). After examining all the spectroscopic analyzes of the separated compounds, we found that the structure of these products is consistent with the structure 13 rather than 14-16.
For instance, the 1 H NMR spectra of the products 13a-c revealed two NH singlet signals at d ¼ 9.21-9.80 ppm and 10.21-11.23 ppm in addition to another singlet signal for one NH 2 at d ¼ 8.67-8.80 ppm while, the other regioisomers 14 and 15 need to confirm four singlet signals for 4NH protons. Furthermore, the mass spectra of derivatives 13a-c indicated the expected molecular ion peaks and the expected fragmentations as illustrated for derivative 13a in Figure 3. Another way to confirm the formation of derivatives 13a-c, the reaction of pyrrole derivative 1 with analidehydrazonoyl chloride 17 afforded identical product to 13a in all respects via elimination of HCl molecule to form intermediate 18 followed by elimination of aniline molecule to cyclized with the formation of 13a (Scheme 3). Another alternative synthesis of derivative 13a through the reaction 1 with a-chloro-ethylacetoacetate 19 in dioxane/Et 3 N to give acetyl-pyrrolopyrazine derivative 21. The structure of 21 was assured based on its spectral analyses as for example its 1 H NMR revealed all expected signals ( Figure 4). Coupling of the latter pyrrolopyrazine derivative 21 with diazotized aniline in pyridine in ice bath afforded an identical product to 13a (Scheme 3).
Two pathways for the formation of another series of pyrrolo[1,2-a]pyrazine derivatives 24 were illustrated in Scheme 4. The first pathway started with the reaction of 1 with acetyl-hydrazonoyl chlorides 22a-d in dioxane/Et 3 N to form an intermediate 23,  Finally, a new series of pyrrolopyrazines 30a-d were synthesized by the same way from the reaction of pyrrole derivative 1 with phenacyl bromide derivative 28a-d (Scheme 5).   Biological activity

Docking study
Docking study for COVID-19 proliferation The molecular docking simulation was performed using the MOE 2014 program 26  and the results were tabulated in Table 1. All investigated derivatives revealed docking score ranging from À6.6925 to À5.4203 kcal/mol. It is noted that cyano-group CN was involved in the interaction of derivatives 27, 24a, 24b, 30a, and 30b with the amino acid residues GLU166, SER144, and CYS145 ( Figure 5). Also, carbonyl group C¼O in derivatives 13a, 13b, 24c, and 27 involved in the interaction with HIS163, SER144, and GLY143. All pyrrolo[1,2-a]pyrazine derivatives except 13c and 24d are strongly fitted in the active site of 6lu7 with two types of hydrogen bond (H-acceptor and pi-H) ( Table 1 and Figure 5). The promising data for docking simulation using the major protease of COVID-19 (Pdb: 6LU7) encourage us to recommend these pyrrolo[1,2-a] pyrazine derivatives for in vitro investigation as COVD-19 inhibitors.
Docking study for microbial activity  (Figure 6). All tested derivatives showed the interaction of the cyano group CN with amino acids ASP422, GLY408, ALA315, THR413, and LEU404 (Table 2). Also, the second common interacted group is carbonyl C¼O group in derivatives 13a-c, 24a-c, and 30d interacted    21, and 27 on a number of fungi and bacteria species to verify the effectiveness of these derivatives as antifungals or anti-bacteria. The inhibition zone diameter (IZD) was used to estimate the antimicrobial activity for the tested derivatives, using ketoconazole and gentamicin as reference drugs for antifungal and antibacterial, respectively. The antimicrobial results are listed in Tables 3-5.
All derivatives didn't show any activity against the two fungi strains used except pyrrolo[1,2-a] pyrazine derivative 24a showed activity against Candida albicans with IZD ¼ 14 mm (about 70% activity compared with Ketoconazole (IZD ¼ 20 mm). In the case of the examination as anti-bacterial, the situation was different, as most of the derivatives showed remarkable activity through measuring the IZD as listed in Tables 3-5. Concerning G þ Bacteria, it was found that Staphylococcus aureus (S A) and Bacillis subtilis (B S) were sensitive to almost tested derivatives, with IZD ranging from 10 to 17 mm for SA (IZD for Gentamicin ¼ 24) and IZD ¼ 10-20 mm for BS ((IZD for Gentamicin ¼ 26). Derivative 24d is the most reactive derivative against G þ bacteria.    For G À bacteria, the most reactive derivative in the investigated series is compound 24c against E. coli, while in the case of Proteus vulgaris (P V), it was found three derivatives 21, 27, and 30 are sensitive with IZD ¼ 19-20 mm (Table 5).

Conclusion
Finally, we synthesized a series of pyrrolo[1,2-a]pyrazine derivatives and one derivative of pyrrolo [1,2-d] [1,2,4]triazine through the reaction of 3-amino-4-cyano-5-phenyl-1H-pyrrole-2-carboxylic acid amide 1 with different types of hydrazonoyl halides as well as the a-haloketones. The site-selectivity and the structure of isolated product were studied and confirmed. The docking study of all synthesized pyrrolo [1,2-d] [1,2,4]triazine and pyrrolo[1,2-a]pyrazine derivatives were performed to inhibit the microbes and COVID-19 proteins. The results of both docking studies revealed a strong fit of almost the tested derivatives into the active sites of peroxiredoxin 5 (Pdb: 2WFC; the overexpressed proteins in Candida albicans) and COVID-19 main protease (Pdb: 6LU7; severe acute respiratory syndrome coronavirus 2). Moreover, the antimicrobial activity of the products was investigated against two selected Fungi species and two G þ and two G À bacteria. The results of antimicrobial activity indicated that almost all derivatives showed no activity against fungi species, while all of them have moderate activity against bacteria strains (G þ and G À bacteria).

Chemistry
All pictures as well as the characterization of each device that were utilized for recording the spectral data were illustrated in the supplementary file.
Reaction of 3-amino-4-cyano-5-phenyl-1H-pyrrole-2-carboxylic acid amide 1 with hydrazonoyl chlorides 2, 9a-c, 17 and 22a-d and the a-haloketones 19, 25 and 28a-d 3-Amino-4-cyano-5-phenyl-1H-pyrrole-2-carboxylic acid amide 1 (2.5 mmol) was added in a suitable round flask and dissolved in 20 mL dioxane and 0.35 mL of Et 3 N, then, the respective 2.5 mmol of hydrazonoyl chlorides 2, 9a-c, 17, and 22a-d or the a-haloketones 19, 25, and 28a-d was added to the reaction mixture. The round flask with the reaction mixture was refluxed for 15 h. After the reaction was completed, the whole solution was poured into acidified cold water and the colored solid was collected by usual way and crystalized from the dioaxane/ethanol mixture. Alternative synthesis of derivatives 13a and 24a through coupling reactions of 21 and 27 The coupling reaction proceeded as usual way through the dissolving 0.002 mole of compounds 21 or 27 in 15 mL of pyridine with stirring in an ice bath. The diazotized aniline (prepared as reported in literature 27 ) was added to the pyridine solution portion wise with stirring in a period of time about 10 min. After the addition was completed, the solution was left to stir about 2 h then the formed colored precipitate was collected and crystalized from the dioaxane/ethanol mixture to give derivatives 13a and 24a, respectively. 4-Acetyl-8-amino-3-methyl-1-oxo-6-phenyl-1,4-dihydropyrrolo[1,2-a]pyrazine-7carbonitrile (27)