Synthesis of some quinoline-pyrazoline-based naphthalenyl thiazole derivatives and their evaluation as potential antimicrobial agents

Purpose: To prepare and evaluate some quinoline-pyrazoline-based naphthalenyl thiazole derivatives as antimicrobial agents. Methods: Some quinoline-pyrazoline-based naphthalenyl thiazoles (5a-5e and 6a-6e) were prepared by reacting 5-(2-chloroquinolin-3-yl)-3-substitutedphenyl-4,5-dihydro-1H-pyrazole-1-carbothiamides (4a4e) with 2-bromo-1-(1-naphthyl)ethanone and 2-bromo-1-(2-naphthyl)ethanone, respectively. Fourier transform infra-red (FTIR), C-Nuclear magnetic resonance (C-NMR), H-Nuclear magnetic resonance (H-NMR), elemental analysis, and mass spectrometry were used to elucidate and confirm the chemical structures of the target compounds. Serial plate dilution technique was used to evaluate the antimicrobial activity of the title compounds using ketoconazole and ofloxacin as standards, and their minimum inhibitory concentrations (MIC) were determined. Results: A total of ten compounds, (5a-5e) & (6a-6e) were prepared. Compound 6d (R = 4-F, naphthalen-2-yl derivative) exhibited antimicrobial activities that were higher than those of the standard drug (ofloxacin) against S. aureus (MIC = 25 μg/mL, p < 0.05), S. epidermidis (MIC = 25 μg/mL, p < 0.0001), K. pneumonia (MIC = 25 μg/mL, p < 0.0001), P. vulgaris (MIC = 25 μg/mL, p < 0.0001) and P. citrinum (MIC = 25 μg/mL, p < 0.0001). Compound 5d (R = 4-F, naphthalen-1-yl derivative) displayed higher antifungal activity than ketoconazole against C. albicans (MIC = 25 μg/mL, p <0.0001). Conclusion: The naphthalen-2-yl derivatives (6a-6e) are superior antimicrobial agents as compared to the naphthalen-1-yl derivatives (5a-5e) and the presence of 4-F substituent in 6d and 5d is essential for stronger antimicrobial activity. The compound 6d needs further investigations related to its safety and efficacy.


INTRODUCTION
Antimicrobial resistance (AMR), a current global concern for human health, is mostly related to the irrational use of antibiotics [1,2]. This has caused the emergence of multidrug-resistant (MDR) pathogens and has also made treatment of many bacterial infections difficult. It is reported that the infections caused by the antibiotic resistant bacteria kill about twenty thousand patients annually in the USA and also leads to economic loss [3]. The cases of AMR bacterial infections are also increasing in the Kingdom of Saudi Arabia due to irrational use of antibiotics as well as due to the socio-economic and demographic characteristics of Saudi, non-Saudi and pilgrim population [4][5][6][7]. Another factor contributing to the development of AMR is failure to discover new antimicrobial agents [8][9][10]. Accordingly, there is a need to take remedial actions with respect to the issues related to the antimicrobial resistance.

EXPERIMENTAL Chemistry
Open capillary tube method was used to determine the melting points of the synthesized compounds which are uncorrected. The FTIR spectra in KBr were generated using a 5PC FT-IR spectrophotometer (Nicolet, Browser Morner, USA). The 1 H-NMR and 13 C-NMR spectra were generated using a DRX-300 FT NMR (Germany, Bruker) spectrophotometer. Mass spectra were generated on a mass spectrometer (70 eV, Jeol-JMS-D-300, Japan). The C, H and N were analysed satisfactorily for the titled compounds within the range of ±0.4 % of theoretical value. Completion of reaction was monitored by checking on pre-coated commercial thin layer chromatography plates, by using ultra-violet cabinet for visualization purpose. The solvent system to run these plates consisted of a mixture of toluene, ethyl acetate and formic acid (5:4:1). Only analytical grade reagents were used for the present work. Ofloxacin was procured from Sun Pharmaceuticals, India, as a gift sample. Ketoconazole was procured from Cipla, India, as a gift sample. Figure 2 provides the route of synthesis of the desired compounds (5a-5e) & (6a-6e).  The compounds (4a-4e) were prepared according to the method provided in the literature [18]. The compounds 2-bromo-1-(1-naphthyl) ethanone and 2-bromo-1-(2-naphthyl)ethanone were prepared according to the method provided in our earlier report [22].

Antimicrobial activity
The compounds (5a-5e) & (6a-6e) were screened for their antimicrobial activity using the serial plate dilution technique [23][24] against five Gram-positive bacteria; five Gram-negative bacteria; and five fungi. The procedure provided in our earlier reports [19][20][21] was followed and the minimum inhibitory concentrations (MICs) values were also determined with respect to ketoconazole and ofloxacin.

Statistical analysis
The data presented in Table 1, Table 2 and Table 3 (n = 6) were analyzed using one-way analysis of variance along with Dunnett's comparison test in comparison to control group and standard group using GraphPad Prism version 5.00 for Windows (GraphPad Software (www.graphpad.com).
The results were considered significantly different at p < 0.05.
These compounds were characterized by their different melting points with respect to their respective starting materials, different R f values in a particular solvent system, spectral data (IR, 13 C-NMR and 1 H-NMR) and elemental analysis. The IR spectra of the compounds (5a-5e) & (6a-6e) showed IR peaks for C=N group ranging from 1575 cm -1 to 1580 cm -1 ; and for C=C group ranging from 1535 cm -1 to 1540 cm -1 . It also displayed characteristic IR peak of C-S group of thiazole moiety ranging from 1106 cm -1 to 1112 cm -1 . The 1 H-NMR spectra of the compounds (5a-5e) & (6a-6e) exhibited characteristic signals for the methylene protons (C 4 -H protons) of pyrazoline ring. One proton of the methylene group of pyrazoline ring appeared as doublet at (δ 3.59-3.65) and another proton also appeared as doublet (δ 3.87-3.91).
The C 5 -H proton of the pyrazoline ring appeared as doublet at δ 5.16 -5.21. The 1 H-NMR spectra also showed characteristic signals as multiplets at δ 7.10-8.15 for aromatic protons. The 13 C-NMR spectra also supported the assigned number of carbon atoms. It showed characteristic signal at δ 37.0 -37.5 due to the methylene carbon (C 4 ) of the pyrazoline ring and at δ 53.4 due to the methine carbon (C 5 ) of the pyrazoline ring. The signals at about δ 166.3 -166.6 arose due to the C 2 carbon of the thiazole ring. The mass spectra and the elemental analysis data for the compounds (5a-5e) & (6a-6e) were also in accordance with the assigned chemical structures.

Antimicrobial activity
The data of the antimicrobial activity of the compounds (5a-5e and 6a-6e) obtained by serial plate dilution technique are listed in Table 1,  Table 2 and Table 3

DISCUSSION
The spectral data and elemental analysis of the titled compounds (5a-5e and 6a-6e) were in accordance with the assigned chemical structures.
The disappearance of the characteristic IR peaks at about 3370 cm -1 to about 3442 cm -1 due to the N-H group, and the peaks at about 1330 cm -1 to about 1340 cm -1 due to the C=S groups present in the compounds (4a-4e) [23], supported the formation of the compounds (5a-5e and 6a-6e). The disappearance of signal of two amino protons of the compounds (4a-4e) at δ 8.44 to 8.68 [23] also supported the formation of the compounds (5a-5e and 6a-6e).
It is also expected that these compounds might be exhibiting their antimicrobial effect by the same mechanism as has been reported for similar type of compounds. It has also been observed that the naphthalen-2-yl derivatives (6a-6e) produced better antimicrobial activity than the naphthalen-1-yl derivatives (5a-5e). The compounds (5a-5e and 6a-6e) were also found to be better antibacterial agents against Gram negative bacteria. This effect may be because of the presence of more aromatic groups that impart lipophilic character to the compounds. The structure activity relationship of the compounds (5a-5e and 6a-6e) revealed that the presence of 4-F substituent in naphthalen-2-yl derivative (6d) and in naphthalen-1-yl derivative (5d) is required for superior antimicrobial activity against the gram positive bacteria, gram negative bacteria as well as fungi. The replacement of 4-F substituent with 4-NO 2 substituent (6e) or with 2-F substituent (6b) or with 2-Cl substituent (6a) provide compounds with lower antibacterial and antifungal potencies.

CONCLUSION
The findings for the synthesized compounds (5a-5e and 6a-6e) indicate that the naphthalen-2-yl derivative (6d) with 4-F substituent in the phenyl ring yields more potent antimicrobial activity against S. aureus, S. epidermidis, K. pneumonia, P. vulgaris and P. citrinum. Compound 6d is a potential lead compound for further development, and therefore, needs to be tested against other microbial strains to confirm its broad spectrum antimicrobial activity.