SYNTHESIS AND ANTIMICROBIAL EVALUATION OF NOVEL PYRAZOLE, IMIDAZOLE AND PYRIMIDINE DERIVATIVES POSSESSING IMIDAZO[4,5-B]INDOL MOIETY

In this study, new pyrazole, imidazole, pyrimidine derivatives having imidazo[4,5-b]indol moiety were successfully synthesized, elucidated by spectroscopic techniques, and evaluated as potential antimicrobial agents. The structure-activity relationship was investigated to obtain a better understanding of the relationship between the chemical structure of the synthesized compounds and their corresponding biological activity. Compounds 2b and 3b exhibited potent antibacterial activities against Bacillus subtilis bacteria comparable to that of Ampicillin standard. Structure-activity relationship studies revealed that the presence of withdrawing carbonyl group on 5-position of pyrazole moiety 2b, phenylpyrazole moiety 3b led to an enhancement in the antibacterial activity of pyrazole derivatives. Furthermore, the presence of carbonyl group on 2-position of the pyrimidine ring of compounds 4a, 5a and 6a has a significant effect on their antibacterial activity against Bacillus subtilis. The antifungal studies indicated that compounds 3b, 4b, 7 and 9 have comparable antifungal activity to that of standard Amphotericin B against Candida albicans and Aspergillus flavus fungi.

The existence of pyrimidine base in uracil, cytosine, and thymine -the main building blocks of nucleic acids DNA and RNA, is among the reasons for their extensive pharmacological applications [23]. The literature surveys depict that compounds encompassing pyrimidine moiety constitute an important class of natural and non-natural products, many of which display a wide range of biological activity [24,25]. Various pyrimidine derivatives have been found to exhibit remarkable antibacterial [26], antifungal [27], antihypertensive [28], antipyretic [29] and anticancer activity [30].
The recent drug design tendency is to reassemble two or three heterocyclic molecules possessing distinct sites of action to serve as new core structures of molecules towards the obtaining of new biologically active agents. In view of our global interest in the design of new potent antimicrobial agents, the main purpose of our study is to synthesize a new set of antimicrobial agents based on pyrazole, imidazole, pyrimidine derivatives bearing the imidazo [4,5-b]indol moiety.
In this study, the identification of the designed compounds was carried out by IR spectroscopy, NMR and mass spectrometry. The biological activity of these compounds was realized to evaluate their antibacterial and antifungal properties against various strains.

Generalities
Unless otherwise mentioned, reagents were provided from Sigma Aldrich (Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia) and used without further purification. Reaction progress was monitored using thin-layer chromatography (TLC) on silica gel pre-coated F254Merck plates (Darmstadt, Germany) and spots were visualized by ultraviolet irradiation.
The melting point values were determined by Gallenkamp electrothermal melting point device (Weiss-Gallenkamp, Loughborough, UK) and are uncorrected.
The 1 H and 13 C NMR spectra were measured on a Varian MercuryVXR-300 NMR spectrometer (Palo Alto, CA) at 400 and 125 MHz for using DMSO-d6 as solvents.
Mass spectra were recorded on a Hewlett Packard MS-5988 spectrometer (Palo Alto, CA) at 70 eV.
Elemental analysis was carried out at the Micro-analytical Center of Cairo University, Giza, Egypt.

General synthesis of compounds (1a-c)
A mixture of isatins (1 mmol), urea (1 mmol), and different active methylene reagents (malononitrile, ethylcyanoacetate, ethylacetoacetate) (1 mmol) dissolved in ethanol (10 mL) was stirred under reflux for 2 h until the reaction was completed. Thin-layer chromatography (TLC) on silica gel plates was carried out for reaction monitoring using ethanol : ethyl acetate (1:2). The reaction mixture was left to cool at room temperature and filtered off. The solution was concentrated under vacuum to afford the product 1a-c, which was purified by recrystallization in ethanol to afford the corresponding 1a-c in good yield. [4,5-b]

General procedure for the synthesis of pyrazole derivatives (2a-c)
A mixture of compounds 1a-c (mmol) and hydrazine hydrate (15 mL) in ethanol was refluxed until the reaction was completed. The reaction was checked by TLC using (ethyl acetate : hexane, 3:1) and the reaction mixture was cooled at room temperature to obtain a solid colorless product 2a-c. Subsequently, the precipitate was filtered and recrystallized with ethanol and dried. 4-(imidazo [4,5-

General procedure for the synthesis of compounds (3a-c)
A mixture of compounds 1a-c (1 mmol) and phenylhydrazine (15 mL) in ethanol/triethylamine was refluxed and the progress of the reaction was monitored by TLC using ethyl acetate: hexane (3:1). After completion of the reaction, the contents were cooled at room temperature, and the solid thus obtained was filtered and washed with ethanol and dried. [4,5-b] A mixture of compounds 1a-c (0.1 mmol), trimethylamine (few drops), and urea or thiourea (0.1 mmol) in ethanol (15 mL) was refluxed for 8 h. After the reaction was completed, the mixture was concentrated and the obtained solid was collected by filtration and recrystallized from ethanol.

General procedure for the synthesis of compounds 7, 8 and 9
A mixture of compounds 1a-c (10 mmol), guanidine hydrochloride (12.0 mmol), anhydrous K2CO3 (15.0 mmol), and absolute ethanol (20 mL) was heated and refluxed for 7 h. After cooling, the mixture was poured into ice water and neutralized with acetic acid. A solid product formed was filtered off and recrystallized from EtOH to afford the corresponding compounds 7, 8 and 9 with good yields. 5-(imidazo [4,5-b]

Antimicrobial activity assays
Antimicrobial activity (antibacterial and antifungal) of the tested samples was checked out according to a modified Kirby-Bauer disk diffusion method [31]. Briefly, 100 μL of each test bacteria/fungi were grown in 10 mL of fresh media until reaching a count of nearly 108 cells/mL for bacteria and 105 cells/mL for fungi. Subsequently, 100 µL of the microbial suspension was spread out onto Mueller-Hinton agar plates corresponding to the broth in which they were maintained. Plates impregnated with filamentous fungi like Aspergillus flavus at 25°C for 48 h; gram-positive bacteria as Staphylococcus aureus, Bacillus subtilis; gram-negative bacteria as Escherichia coli, Pseudomonas aeuroginosa were incubated at 35-37°C for 24-48 h and yeast as Candida albicans was incubated at 30°C for a period varying between 24 and 48 h. Subsequently, the diameters of the inhibition zone were measured in millimeters. The standard disk of ampicillin (antibacterial agent), amphotericin B (an antifungal agent), served as a positive control for antimicrobial activity and filter disks impregnated with 10 μL of solvent (distilled water, chloroform, and DMSO) were used as a negative control. All experiments were repeated and carried out in triplicate in the case of a significant difference in the results and mean values were reported. The mean inhibition zone diameters were measured in mm/mg sample.

Structure determination
The novel pyrazole, imidazole, and pyrimidine derivatives were synthesized according to the regioselective attack that occurred on the cyano group present in 2-(imidazo [4,5-b]indol-2(4H)-ylidene) derivatives 1a,b. Refluxing of 1a-c derivatives with hydrazine hydrate or phenyl hydrazine in the presence of ethanol gave pyrazole compounds derivatives 2a-c and 3a-c (Schemes 1 and 2). These reactions involve 1,2-dinucleophile cyclization on CN moiety in compounds 1a-c and concomitant aromatization in the presence of hydrazine and ethanol under reflux.
All reactions were prepared with satisfactory yields varying from 68% to 74% and the obtained products 2a-c were confirmed by spectral data including IR, 1 H and 13 C NMR. The IR spectra of 4-(imidazo [4,5-b]indol-2-yl)-1Hpyrazole-3,5-diamine 2a showed the characteristic NH2 amine absorption bands in the 3421-3257 cm -1 range and the absence of absorption bands corresponding to the cyano group. 1 H NMR spectrum of compound 2a displayed two different broad signals concentrated at 10.29, 13.25 ppm and attributed to NH2 and NH groups. Furthermore, the 13 C NMR spectrum of this compound showed signals at 146.25 ppm related to CH=N and further signals appeared in the regular regions were mentioned in the experimental data.

Scheme 2. Synthetic routes for the compounds 3a-c.
The appearance of bands between 3420-3256 (NH2), 3203 (NH), 1673 (C=O) and 1618 (C=N) cm -1 in the IR spectrum; a multiplet at δ value 7.20-7.36 ppm for phenyl ring support the formation of 2-(5-methyl-1-phenyl-1H-pyrazol-4yl)imidazo [4,5-b]indole 3c. Compounds 1a-c were subjected to condensation reactions with urea and thiourea under reflux to give pyrimidine  derivatives 4a,b, 5a,b and 6a,b as shown in (Scheme 3). The chemical structures of these compounds were confirmed by spectral data (see experimental section). The IR spectrum of all compounds displayed the characteristic absorption bands of C=C and C=N stretching frequencies at 1475 and 1620 cm -1 corresponding to pyrimidine moiety. The absorption of C=O at 1673 cm -1 was observed in the IR spectrum of compound 4a. The 1 H NMR spectrum of compounds 4a,b displayed a multiplet at δ value 7.20-7.36 ppm for C6H4, two singlets at 10.35 and 4.86 ppm for NH2 groups. Moreover, the 1 H NMR spectrum of compounds 5a,b exhibited two singlets for each NH of pyrimidine ring in the range of 11.25 and 9.26 ppm. The IR spectrum of compounds 8a,b revealed the presence of absorption bands at 1673 cm -1 due to (C=O). The absence of the CN group in compounds 6a,b (IR spectrum) and the singlet at 7.23 ppm related to NH2 ( 1 H NMR) ascertain their formation.
In this work, another procedure was adopted to design novel substituted pyrimidines having imidazo [4,5-b]indol moiety (Scheme 4). For this purpose, compounds 1a-c were treated with guanidine hydrochloride anhydrous K2CO3 in ethanol (20 mL) under reflux to give novel pyrimidine derivatives 7-9. All spectral data confirmed the formation of these compounds. 1 H NMR spectrum of compounds 7-9 displayed three singlets in different positions ranging from 4.83 to 5.62 for NH2 groups. Compound 9 showed a singlet peak at 2.36 ppm for CH3 group and a singlet peak at 4.25 ppm corresponding to NH2.

Antimicrobial activity assays Antibacterial activity
After successful synthesis and elucidation of the chemical structures of the newly synthesized compounds, the in vitro screening for their antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa bacteria was performed. The obtained results are presented in Table 1. Structure-activity relationship (SAR) studies allowed a better understanding of the relationship between the chemical structure of the synthesized compounds and their corresponding biological activity against the tested bacteria.
Most of the synthesized compounds displayed moderate to good activities and the SARs of these compounds were discussed. Among these compounds, compounds 2b and 3b showed comparable efficacy as ampicillin against Bacillus subtilis strains (d= 25 mm/mg sample). Firstly, the introduction of carbonyl group on the 5-position of pyrazole moiety of compound 2b and phenylpyrazole moiety of compound 3b afforded more potent antibacterial activities against the tested strains than compounds 2a,c and 3a,c containing amino and methyl groups on the 5-position (Table 1, Figure 1). Meanwhile, the relationship between various substituents on the 5-position of pyrazole ring were investigated, the presence of electro-donating groups such as amino 2a and methyl 2c showed lower activities against the bacterial strains compared to compound 2b having a carbonyl withdrawing group in 5-position of the pyrazole ring.
Secondly, compounds 4a and 5a with C=O group on the 2-position of pyrimidine moiety exhibited higher activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa bacteria strains compared to compounds 4b and 5b containing C=S group on the 2-position. The substitution of amino groups on 4-position in compounds 4a,b, and 5a,b with a carbonyl group in 6a,b improved the antibacterial activity against Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa bacteria. It is also noteworthy to indicate that compound 6a bearing a carbonyl group on 2-position of pyrimidine ring exhibited higher antibacterial properties against Bacillus subtilis, Staphylococcus aureus and Escherichia coli strains than compound 6b possessing a thiol (C=S) group on the same position.
These finding ascertain that the presence of more withdrawing groups on the 5-position of pyrazole moiety 2b, 3b and on 2-position of pyrimidine ring 4a, 5a and 6a of the synthesized compounds enhanced their antibacterial activities against the tested bacterial strains. Compound 9 exhibited better antibacterial activity against Escherichia coli, this finding was assigned to the absence of electro-donating amino group on 6-position and to the presence of methyl group in 6-position of the pyrimidine ring. Furthermore, compound 7 exhibited higher antibacterial activity against Bacillus subtilis compared to 8 and 9.

Antifungal activity
Antifungal properties of the synthesized compounds were tested against Aspergillus flavus and Candida albicans fungi using the standard Amphotericin B antifungal agent. The obtained results are summarized in Table 2. Antifungal data of targeted compounds have obviously shown that distinct electronic varieties are responsible for wide spectrum activity. Compounds 3b, 4b, 7 and 9 displayed similar antifungal activities as that of standard Amphotericin B against Candida albicans and Aspergillus flavus fungi. Compound 3b afforded more important antifungal activity against the tested fungi compared to compounds 3a and 3c, this finding was attributed to the presence of the carbonyl withdrawing group on the 5-position of the phenylpyrazole moiety of compound 3b. On the other hand, the antifungal activity of compound 4b against Candida albicans was associated with the presence of the C=S group on the 2-position of the pyrimidine ring. SARs studies also indicated that the introduction of the electron releasing groups such as amino (2,4,6-position) in compound 7 and methyl (4position) in compound 9 enhanced their antifungal activity against Candida albicans fungi.

Conclusions
In this study, novel pyrazole, imidazole, pyrimidine derivatives bearing imidazo [4,5b]indol moiety were successfully synthesized and their chemical structures were identified and confirmed by different spectral techniques. All the synthesized compounds were assessed for their antibacterial activities against four bacterial strains (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) and antifungal activities against two fungi (Aspergillus flavus and Candida albicans).
Structure-activity relationship studies revealed that the introduction of the electronwithdrawing group contributes substantially to the antibacterial activity of the synthesized compounds. It was interesting to note that the compounds bearing a carbonyl group on 5-position of pyrazole moiety 2b, phenylpyrazole moiety 3b displayed outstanding antibacterial activity against Bacillus subtilis bacteria and are almost similar to the Ampicillin standard. Furthermore, the presence of carbonyl group in 2-position of the pyrimidine ring of compounds 4a, 5a and 6a has significantly improved their antibacterial activity against Bacillus subtilis bacteria and the other tested bacterial strains.
The antifungal studies indicated that compounds 3b, 4b, 7 and 9 displayed comparable antifungal activity to that of standard Amphotericin B against Candida albicans and Aspergillus flavus fungi. Structure-activity relationship studies indicated that the presence of electrodonating groups such as amino (2,4,6-position) in compound 7 and methyl (4-position) in compound 9 enhanced their antifungal activity against Candida albicans fungi. These synthesized compounds could find fruitful applications as antibacterial and antifungal agents in pharmaceutical chemistry and additional studies are currently conducted by our group to evaluate their anti-inflammatory and anti-cancer activity.