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Synthesis and biological evaluation of novel hybrid

Cite this: RSC Pharm., 2024, 1, 283
compounds bearing pyrazine and 1,2,4-triazole
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analogues as potent antitubercular agents†

Shivakumar Naik, a Dinesha Puttachari,a Vanishree A. L.,a Udayakumar D., *a
Varsha Prakash Shetty,b Chaitra Prabhub and Vijaya Kumar Deekshitb

In this study, we elucidate the conceptualization and synthesis of hybrid compounds (T1–T18) amalga-
mating pyrazine and 1,2,4-triazole scaffolds. A total of eighteen compounds were screened in vitro for
their efficacy against the Mycobacterium tuberculosis H37Rv strain via the MABA assay. The results
revealed that eight compounds (T4, T5, T6, T11, T14, T15, T16, and T18) manifested noteworthy activity
against Mtb, with minimum inhibitory concentration (MIC) values of ≤21.25 µM. Furthermore, we also
examined these compounds for their antibacterial and antifungal properties against various strains.
Compounds T4, T9, T10, T16, and T18 displayed significant antibacterial activity, while compounds T12
and T14 demonstrated significant antifungal activity. Subsequently, the most potent compounds were
evaluated for their potential cytotoxicity to the Vero cell line via the MTT assay, revealing IC50 values sur-
passing 375 µM, indicative of minimal cytotoxicity. Additionally, we conducted in silico studies on these
target molecules to better understand their action mechanisms. The in silico investigations suggest that
the target enzyme involved in the action of the compounds may be DprE1. However, further experimental
validation is necessary to ascertain the target responsible for the whole cell activity. All the target com-
pounds are docked within the active site of the DprE1 enzyme, demonstrating favorable binding inter-
Received 27th November 2023, actions. Furthermore, we predicted the ADME properties, physicochemical characteristics, and drug-like
Accepted 11th March 2024
qualities of the target compounds using in silico methods. We also performed DFT studies to examine
DOI: 10.1039/d3pm00054k their electronic properties. These findings collectively indicate that the active compounds hold substantial
rsc.li/RSCPharma promise as prospective contenders for the development of novel antitubercular agents.

Introduction with adverse side effects, underscoring the critical need for
innovative therapies.2 Over the preceding decades, notable
Tuberculosis (TB), an infectious disease caused by advancements have transpired in the exploration of novel anti-
Mycobacterium tuberculosis, persists as a formidable global microbial agents characterized by heightened efficacy and
health challenge. With millions of new cases and deaths diminished toxicity. In this context, hybrid compounds have
recorded annually, it endures as a predominant source of mor- emerged as a compelling strategy to address the growing threat
bidity and mortality worldwide.1 The advent of drug-resistant of drug-resistant TB.3,4 Hybrid compounds are designed by
variants, notably multidrug-resistant tuberculosis (MDR-TB) combining distinct pharmacophores in a single molecule,
and extensively drug-resistant tuberculosis (XDR-TB), has leveraging the unique properties of each component to
intensified the exigency for the formulation of groundbreaking enhance biological activity. Pyrazine and 1,2,4-triazole ana-
and more efficacious antitubercular agents.1 The current treat- logues, well-known for their diverse pharmacological pro-
ment regimens for TB are often lengthy, costly, and associated perties, have garnered substantial attention as potential build-
ing blocks for such hybrid compounds.5,6
Pyrazinamide serves as a primary pharmacotherapeutic
a
Organic and Medicinal Chemistry Laboratory, Department of Chemistry, National agent in the treatment of tuberculosis, commonly employed in
Institute of Technology Karnataka, Surathkal-575025, Mangalore, Karnataka, India. conjunction with other anti-TB medications. Its robust anti-TB
E-mail: [email protected], [email protected] efficacy plays a pivotal role in abbreviating the duration of
b
Division of Infectious Diseases, NITTE University Center for Science Education and tuberculosis therapy.7 Various modified versions of pyrazina-
Research, NITTE (Deemed to be University), Deralakatte-575018, Mangalore,
mide have been explored as effective antitubercular agents. For
Karnataka, India
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/ instance, Reddyrajula et al. investigated bioisosteric modifi-
10.1039/d3pm00054k cations of pyrazinamide derivatives, resulting in the develop-

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Paper RSC Pharmaceutics

ment of potent antitubercular compounds.8 Srinivasarao and 4.1.4). Furthermore, we acquired mass spectra of the syn-
team have focused on N-(6-(4-( pyrazine-2-carbonyl)piperazine/ thesized compounds employing a Waters Xevo QTOF MS
homopiperazine-1-yl)pyridin-3-yl)benzamide derivatives as system equipped with an electrospray ionization (ESI) source.
antitubercular agents.9 Additionally, Panda et al. reported pyra-
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zolopyridones as a novel class of noncovalent DprE1 inhibitors Chemistry

with strong anti-mycobacterial activity.10 Zhou and his team Synthesis and characterization
have reported pyrazine-2-carboxamide derivatives that possess Procedure for the synthesis of (Z)-pyrazine-2-carbohydrazona-
antitubercular properties.11 Kumar et al. reported a series of mide (2). In a clean and dry 100 mL round bottom flask, pyra-
pyrazine analogues as promising inhibitors of DprE1 for the zine-2-carbonitrile (1) (10 g, 95.14 mmol) was combined with
treatment of tuberculosis.12 In parallel, 1,2,4-triazoles, a sub- anhydrous methanol (50 mL). To this mixture, hydrazine
Open Access Article. Published on 13 March 2024. Downloaded on 7/18/2024 7:27:02 AM.

class of five-membered heterocyclic compounds, have a well- hydrate (8.33 mL, 166.50 mmol) was introduced. The reaction
established history of pharmacological importance. In recent mixture was then stirred at room temperature for 24 hours.
research, Karczmarzyk and team have explored derivatives of The progression of the reaction was scrutinized using thin-
1,2,4-triazoles combined with pyridine for their antitubercular layer chromatography. Following the reaction, the obtained
activity.13 Oh et al. reported a series of 1,2,4-triazole derivatives solid product was isolated by filtration, washed with cold
with antitubercular properties.14 Karabanovich et al. reported methanol, and subsequently dried. The purification process
a series of 3,5-dinitrophenyl-containing 1,2,4-triazoles and involved recrystallization in methanol, resulting in the for-
their trifluoromethyl analogues as potent inhibitors of DprE1 mation of yellow crystals.16 Yield: 12.83 g, 98%; m.p.:
for tuberculosis treatment.15 Combining these two structural 127–128 °C; 1H-NMR (CDCl3, 400 MHz, δ in ppm): 9.24 (s, 1H),
elements within a single hybrid molecule offers a unique 8.48 (d, J = 2.36 Hz, 1H), 8.40 (d, J = 1.28 Hz, 1H), 5.10 (s, 2H),
opportunity to leverage their combined strengths, thereby 4.70 (s, 2H); 13C-NMR (CDCl3, 100 MHz, δ in ppm): 146.60,
potentially culminating in the conception of more potent and 146.44, 144.11, 142.81, 142.40; ESI-MS (m/z) = 106.03 [M + H]+.
versatile antitubercular pharmaceutical agents. In light of the Procedure for the synthesis of 5-( pyrazin-2-yl)-4H-1,2,4-triazole-
information provided, this study aims to illustrate the syn- 3-thiol (3). Pyrazine-2-carbohydrazonide (2) (10 g, 72 mmol)
thesis of an array of novel hybrid molecules by linking the pyr- and KOH (4.5 g, 80 mmol) were taken in anhydrous methanol
azine ring with a biologically active 1,2,4-triazole moiety to (100 mL) in a clean 250 mL round bottom flask. Carbon disulfide
assess the effectiveness of these compounds in combating (4.40 g, 72 mmol) was introduced into the reaction mixture and
tuberculosis, evaluate their safety through cytotoxicity testing, refluxed for 24 hours at 65 °C. The progress of the reaction was
investigate their physicochemical and pharmacokinetic pro- scrutinized using TLC. The reaction mixture was then cooled to
perties, and explicate the plausible mechanism of action. room temperature and excess solvent was removed using a rotor-
vap. The obtained residue was poured into crushed ice and made
acidic (pH = 6) using 20% HCl, and the obtained solid was fil-
Materials and methods tered and dried. Recrystallization was performed using metha-
nol.17 Pale yellow solid, yield: 92%, m.p.: 176–177 °C, 1H-NMR
The required chemical reagents were procured from diverse (DMSO-d6, 400 MHz, δ in ppm): 14.1692 (s, 1H), 13.9546 (s, 1H),
commercial suppliers, including Sigma Aldrich, TCI, and Alfa- 9.1809 (s, 1H), 8.7571 (s, 2H); 13C-NMR (DMSO-d6, 100 MHz, δ in
Aesar. To monitor the progress of the chemical reaction, we ppm):167.9086, 148.3865, 145.8394, 144.3842, 142.3343,
employed TLC with alumina plates coated with silica gel 140.5931, 40.1253, 39.9179, 39.7089, 39.5005, 39.2919, 39.0836,
(Merck 60 F254) as the stationary phase and a mobile phase 38.8748; ESI-MS (m/z) = 180.03 [M + H]+.
comprising a 3 : 7 amalgamation of ethyl acetate and pet- General procedure for the synthesis of 1-phenyl-2-((5-( pyrazin-2-
roleum ether. Subsequently, we scrutinized the resultant spots yl)-4H-1,2,4-triazol-3-yl)thio)ethan-1-one derivatives (T1–T9). A
under a UV chamber. For the determination of the melting mixture of 5-( pyrazin-2-yl)-4H-1,2,4-triazole-3-thiol (3)
points of the synthesized compounds, we utilized a digital (1.0 mmol) and sodium hydroxide (1.1 mmol) was taken in
melting point apparatus without any adjustments. aqueous methanol (80%) (10 mL) in a clean 50 mL round
Furthermore, for in-depth structural analysis, we conducted bottom flask and the reaction mixture was stirred for
spectroscopic analysis on the synthesized compounds, encom- 10 minutes at room temperature. Then substituted phenacyl
passing proton nuclear magnetic resonance (1H-NMR) and bromide (1.0 mmol) was added and the reaction mixture was
carbon nuclear magnetic resonance (13C-NMR) spectroscopy. stirred at room temperature for 4 hours. The progression of
These spectroscopic assessments were carried out using a the reaction was scrutinized using thin-layer chromatography.
Bruker Avance Fourier transform-NMR (FT-NMR) spectrometer The reaction mixture was then poured into ice-cold water. The
operating at 400 MHz for 1H-NMR and 100 MHz for 13C-NMR. precipitated solid was filtered, washed with ice-cold water, and
CDCl3 or DMSO-d6 was employed as a solvent, with tetra- dried. Recrystallization was performed using methanol.18
methyl silane (TMS) as an internal standard. Chemical shifts 1-Phenyl-2-((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)thio)ethan-1-
are articulated in parts per million on the δ-scale, while coup- one (T1). White solid, yield: 86%; m.p.: 193–194 °C; 1H-NMR
ling constants are presented in Hertz (Hz). NMR spectral ana- (CDCl3, 400 MHz, δ in ppm): 12.7680 (s, 1H), 9.4716 (s, 1H),
lysis was conducted utilizing Bruker NMR software (TopSpin 8.6173 (s, 1H), 8.5612 (s, 1H), 8.0357 (s, 2H), 7.5983 (s, 1H),

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7.4901 (d, J = 5.68 Hz, 2H), 5.0396 (s, 2H); 13C-NMR (CDCl3, 197–198 °C; 1H-NMR (DMSO-d6, 400 MHz, δ in ppm): 12.9686
100 MHz, δ in ppm): 191.91, 166.40, 163.37, 146.63, 144.76, (s, 1H), 9.4573 (s, 1H), 8.6234 (t, J = 1.56 Hz, 2H), 8.0875 (d, J =
144.07, 139.41, 134.92, 134.49, 129.15, 128.69, 41.97; ESI-MS 5.56 Hz, 1H), 8.0679 (d, J = 5.56 Hz, 1H), 7.2400 (s, 1H), 7.1642
(m/z) = 298.11 [M + H]+. (t, J = 8.28 Hz, 1H), 5.0019 (s, 2H); 13C-NMR (DMSO-d6,
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2-((5-(Pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)thio)-1-( p-tolyl)ethan- 100 MHz, δ in ppm): 190.84, 166.21, 163.48, 146.70, 144.79,
1-one (T2). White solid, yield: 88%; m.p.: 190–191 °C; 1H-NMR 144.11, 141.14, 139.38, 130.11, 129.55, 41.64; ESI-MS (m/z) =
(CDCl3, 400 MHz, δ in ppm): 12.9680 (s, 1H), 9.4716 (s, 1H), 332.04 [M + H]+.
8.6260 (d, J = 2.16 Hz, 1H), 8.5711 (s, 1H), 7.9356 (d, J = 8.16 1-(4-Bromophenyl)-2-((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)
Hz, 2H), 7.2870 (d, J = 8.08 Hz, 2H), 5.0317 (s, 2H), 2.4134 (s, thio)ethan-1-one (T9). White solid, yield: 94%; m.p.:
3H); 13C-NMR (CDCl3, 100 MHz, δ in ppm): 191.53, 166.57, 203–204 °C; 1H-NMR (CDCl3, 400 MHz, δ in ppm): 12.8630 (s,
Open Access Article. Published on 13 March 2024. Downloaded on 7/18/2024 7:27:02 AM.

163.36, 146.63, 145.66, 144.77, 144.09, 139.45, 132.45, 129.84, 1H), 9.4631 (s, 1H), 8.6313 (s, 1H), 8.5737 (s, 1H), 7.9071 (d, J =
128.83, 42.07, 21.99; ESI-MS (m/z) = 312.09 [M + H]+. 7.00 Hz, 2H), 7.6406 (d, J = 7.08 Hz, 2H), 4.9843 (s, 2H);
1-(4-Methoxyphenyl)-2-((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl) 13
C-NMR (CDCl3, 100 MHz, δ in ppm): 191.07, 166.21, 163.50,
thio)ethan-1-one (T3). White solid, yield: 93%; m.p.: 146.72, 144.80, 144.13, 139.40, 133.73, 132.57, 130.18, 129.97,
198–199 °C; 1H-NMR (CDCl3, 400 MHz, δ in ppm): 12.7783 (s, 41.61; ESI-MS (m/z) = 375.99 [M + H]+.
1H), 9.4574 (s, 1H), 8.6143 (d, J = 2.44 Hz, 1H), 8.5622 (d, J = General procedure for the synthesis of 2-(5-(benzylthio)-
1.44 Hz, 1H), 8.0076 (d, J = 8.8 Hz, 2H), 6.9419 (d, J = 8.84, 2H), 4H-1,2,4-triazol-3-yl)pyrazine derivatives (T10–T18). A mixture of
5.0007 (s, 2H), 3.8567 (s, 3H); 13C-NMR (CDCl3, 100 MHz, δ in 5-( pyrazin-2-yl)-4H-1,2,4-triazole-3-thiol (3) (1.0 mmol) and pot-
ppm): 190.39, 166.70, 164.64, 163.35, 146.64, 144.79, 144.10, assium carbonate (1.0 mmol) was taken in acetone (10 mL) in
139.47, 131.15, 127.95, 114.35, 55.81, 41.94; ESI-MS (m/z) = a clean 50 mL round bottom flask and the reaction mixture
328.13 [M + H]+. was stirred for 10 minutes at room temperature. Then, substi-
1-(4-Hydroxyphenyl)-2-((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl) tuted benzyl bromide (1.0 mmol) was introduced and the reac-
thio)ethan-1-one (T4). White solid, yield: 87%; m.p.: tion mixture was stirred at room temperature for 2 hours. The
250–251 °C; 1H-NMR (DMSO-d6, 400 MHz, δ in ppm): 12.9889 progression of the reaction was scrutinized using thin-layer
(s, 1H), 10.5739 (s, 1H), 9.3073 (s, 1H), 8.8451 (t, J = 5.80 Hz, chromatography. The reaction mixture was then poured into
2H), 7.9481 (d, J = 8.52 Hz, 2H), 6.9016 (d, J = 8.52 Hz, 2H), ice-cold water. The precipitated solid was filtered, washed with
5.1359 (s, 2H); 13C-NMR (DMSO-d6, 100 MHz, δ in ppm): ice-cold water, and dried. Recrystallization was performed
190.28, 165.39, 162.90, 162.87, 146.95, 145.02, 143.43, 138.69, using methanol.18
131.21, 126.43, 115.48; ESI-MS (m/z) = 314.06 [M + H]+. 2-(5-(Benzylthio)-4H-1,2,4-triazol-3-yl)pyrazine (T10). Brown
1-(4-Nitrophenyl)-2-((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)thio) solid, yield: 96%; m.p.: 198–199 °C; 1H-NMR (CDCl3, 400 MHz,
ethan-1-one (T5). White solid, yield: 81%; m.p.: 229–230 °C; δ in ppm): 12.7482 (s, 1H), 9.5144 (d, J = 1.00 Hz, 1H), 8.6320
1
H-NMR (DMSO-d6, 400 MHz, δ in ppm): 12.6630 (s, 1H), (d, J = 2.44 Hz, 1H), 8.5723 (d, J = 1.44 Hz, 1H), 7.4541 (d, J =
9.3847 (d, J = 1.20 Hz, 1H), 8.8110 (d, J = 2.48 Hz, 1H), 8.7697 7.16 Hz, 2H), 7.3169 (m, J = 6.88 Hz, 3H), 4.6244 (s, 2H);
(d, J = 1.52 Hz, 1H), 8.2163 (d, J = 8.68 Hz, 2H), 7.7926 (d, J = 13
C-NMR (CDCl3, 100 MHz, δ in ppm): 168.19, 162.49, 160.02,
8.64 Hz, 2H), 4.8030 (s, 2H); 13C-NMR (DMSO-d6, 100 MHz, δ 145.89, 144.90, 144.38, 142.46, 131.67, 131.63, 130.20, 130.11,
in ppm): 191.83, 164.80, 162.96, 150.21, 146.90, 144.92, 143.35, 124.49, 31.46; ESI-MS (m/z) = 270.08 [M + H]+.
139.54, 138.53, 129.85, 123.88; ESI-MS (m/z) = 343.08 [M + H]+. 2-(5-((4-(Trifluoromethyl)benzyl)thio)-4H-1,2,4-triazol-3-yl)pyra-
4-(2-((5-(Pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)thio)acetyl)benzo- zine (T11). White solid, yield: 92%; m.p.: 154–155 °C; 1H-NMR
nitrile (T6). White solid, yield: 85%; m.p.: 215–216 °C; 1H-NMR (DMSO-d6, 400 MHz, δ in ppm): 12.9783 (s, 1H), 9.3917 (d, J =
(DMSO-d6, 400 MHz, δ in ppm): 12.7140 (s, 1H), 9.3132 (d, J = 1.36 Hz, 1H), 8.8125 (d, J = 2.56 Hz, 1H), 8.7720 (t, J = 1.52 Hz,
1.32 Hz, 1H), 8.8724 (d, J = 2.48 Hz, 1H), 8.8494 (t, J = 1.48 Hz, 1H), 7.7360 (s, 2H), 7.6738 (s, 2H), 4.7617 (s, 2H); 13C-NMR
1H), 8.4048 (d, J = 8.84 Hz, 2H), 8.3055 (d, J = 8.84 Hz, 2H), (DMSO-d6, 100 MHz, δ in ppm): 168.19, 162.49, 160.02,
5.2958 (s, 2H); 13C-NMR (DMSO-d6, 100 MHz, δ in ppm): 145.89, 144.90, 144.36, 142.46, 131.66, 131.63, 130.20, 130.11,
190.28, 165.39, 162.90, 162.87, 146.95, 145.02, 143.43, 138.69, 115.63; ESI-MS (m/z) = 338.11 [M + H]+.
131.21, 126.43, 121.44, 115.48; ESI-MS (m/z) = 323.10 [M + H]+. 2-(5-((4-Fluorobenzyl)thio)-4H-1,2,4-triazol-3-yl)pyrazine (T12).
1-(4-Fluorophenyl)-2-((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl) White solid, yield: 86%; m.p.: 150–151 °C; 1H-NMR (CDCl3,
thio)ethan-1-one (T7). White solid, yield: 92%; m.p.: 400 MHz, δ in ppm): 12.8980 (s, 1H), 9.4857 (d, J = 1.12 Hz,
202–203 °C; 1H-NMR (CDCl3, 400 MHz, δ in ppm): 12.9680 (s, 1H), 8.6161 (d, J = 2.44 Hz, 1H), 8.5516 (t, J = 1.52 Hz, 1H),
1H), 9.4573 (s, 1H), 8.6234 (t, J = 1.56 Hz, 2H), 8.0875 (d, J = 7.4241 (d, J = 5.40 Hz, 1H), 7.4030 (d, J = 5.36 Hz, 1H), 7.0115
5.56 Hz, 1H), 8.0679 (d, J = 5.56 Hz, 1H), 7.2400 (s, 1H), 7.1642 (s, 1H), 6.9791 (d, J = 8.60 Hz, 1H), 4.5731 (s, 2H); 13C-NMR
(t, J = 8.28 Hz, 1H), 5.0019 (s, 2H); 13C-NMR (CDCl3, 100 MHz, (CDCl3, 100 MHz, δ in ppm): 167.88, 163.78, 161.34, 145.90,
δ in ppm): 190.42, 167.87, 166.30, 165.32, 163.46, 146.70, 144.85, 144.37, 142.45, 131.74, 131.71, 131.17, 131.09, 115.78,
144.79, 144.11, 139.40, 131.57, 131.47, 116.56, 116.34, 41.69; 37.44; ESI-MS (m/z) = 288.08 [M + H]+.
ESI-MS (m/z) = 316.09 [M + H]+. 2-(5-((2-Fluorobenzyl)thio)-4H-1,2,4-triazol-3-yl)pyrazinepyra-
1-(4-Chlorophenyl)-2-((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl) zine (T13). Yellow solid, yield: 98%; m.p.: 130–131 °C; 1H-NMR
thio)ethan-1-one (T8). White solid, yield: 96%; m.p.: (CDCl3, 400 MHz, δ in ppm): 12.9637 (s, 1H), 9.5007 (d, 1H),

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8.6205 (d, J = 2.52 Hz, 1H), 8.5582 (t, J = 1.56 Hz, 1H), 7.5256 predict the pharmacokinetic properties of the target com-
(m, J = 1.56 Hz, 1H), 7.2660 (m, J = 2.04 Hz, 1H), 7.0876 (m, J = pounds, we have utilized the computational capabilities of
7.60 Hz, 2H), 4.6542 (s, 2H); 13C-NMR (CDCl3, 100 MHz, δ in Schrödinger’s qikprop program.20
ppm): 168.19, 162.49, 160.02, 145.89, 144.90, 144.38, 142.46, In silico molecular docking studies. In this investigation, we
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131.67, 131.63, 130.20, 130.11, 115.73, 31.46; ESI-MS (m/z) = utilized computational techniques to scrutinize how the ligand
288.10 [M + H]+. molecules interact with a target receptor. To do this, we
4-(((5-(Pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)thio)methyl)benzonitrile employed a software, AutoDock-Vina 1.1.2.21,22 First, we
(T14). White solid, yield: 89%; m.p.: 209–210 °C; 1H-NMR created the chemical structures of the ligand molecules we
(DMSO-d6, 400 MHz, δ in ppm): 12.9220 (s, 1H), 9.3871 (d, wanted to study using ChemDraw Professional 20.1.1 software.
1H), 8.8122 (d, J = 2.52 Hz, 1H), 8.7701 (t, J = 1.56 Hz, 1H), Then, we optimized their 3D structures using Chem3D 20.1.1
software.23 The receptor’s crystal structure was obtained from
Open Access Article. Published on 13 March 2024. Downloaded on 7/18/2024 7:27:02 AM.

7.8314 (d, J = 8.28 Hz, 2H), 7.7139 (d, J = 8.24 Hz, 2H), 4.7464
(s, 2H); 13C-NMR (DMSO-d6, 100 MHz, δ in ppm): 167.39, the Protein Data Bank (PDB ID: 4P8N). However, this structure
163.89, 159.34, 146.58, 144.95, 142.62, 141.39, 132.49, 130.13, had some missing components, including residues and atoms,
123.49, 120.13, 110.37; ESI-MS (m/z) = 295.07 [M + H]+. which we needed to rectify. We accomplished this by using the
2-(5-((4-Bromobenzyl)thio)-4H-1,2,4-triazol-3-yl)pyrazine (T15). Modeller tool to complete and refine the receptor’s structure.24
Brown solid, yield: 92%; m.p.: 155–156 °C; 1H-NMR (DMSO- Before we proceeded with the actual docking simulation, we
d6, 400 MHz, δ in ppm): 12.9880 (s, 1H), 8.8150 (d, J = 2.52 Hz, prepared the receptor by removing all non-protein elements,
1H), 8.7779 (d, J = 1.52 Hz, 1H), 7.5579 (d, J = 8.40 Hz, 2H), such as cofactors, ligands already attached to the receptor,
7.4731 (d, J = 8.44 Hz, 2H), 4.6444 (s, 2H); 13C-NMR (DMSO-d6, water molecules, and heteroatoms. Additionally, we added
100 MHz, δ in ppm): 168.09, 163.79, 161.34, 146.54, 144.93, polar hydrogens and applied Kollman charges to ensure an
143.72, 141.37, 135.99, 131.50, 131.35, 120.89; ESI-MS (m/z) = accurate representation of the receptor’s electrostatic pro-
347.98 [M + H]+. perties. To identify the exact location of the receptor’s active
2-(5-((2-Bromobenzyl)thio)-4H-1,2,4-triazol-3-yl)pyrazine (T16). site, we made use of the online COACH server.25
Brown solid, yield: 92%; m.p.: 156–157 °C; 1H-NMR (CDCl3, Understanding the active site is critical because it’s where the
400 MHz, δ in ppm): 12.9889 (s, 1H), 9.5008 (d, J = 1.28 Hz, ligands will bind and interact with the receptor. We then set
1H), 8.6200 (d, J = 2.48 Hz, 1H), 8.5581 (t, J = 1.56 Hz, 1H), up the docking simulation by defining a specific area around
7.6238 (m, J = 6.08 Hz, 1H), 7.5658 (m, J = 7.04 Hz, 1H), 7.2533 the protein where we wanted to explore binding interactions.
(m, J = 6.44 Hz, 1H), 7.1454 (m, J = 6.12 Hz, 1H), 4.7505 (s, This area was represented by a cubical grid with dimensions of
2H); 13C-NMR (CDCl3, 100 MHz, δ in ppm): 168.19, 162.49, 100 × 100 × 105, possessing intervals of −0.389 Å, −3.917 Å,
160.02, 145.89, 144.90, 144.38, 142.46, 131.67, 131.63, 130.20, and 4.861 Å along the x, y, and z axes, respectively. Finally, we
130.11, 115.94, 31.46; ESI-MS (m/z) = 347.97 [M + H]+. conducted docking using the Vina software, with an exhaus-
2-(5-((4-Chlorobenzyl)thio)-4H-1,2,4-triazol-3-yl)pyrazine (T17). tiveness parameter set to 16, which controls the thoroughness
White solid, yield: 94%; m.p.: 162–163 °C; 1H-NMR (CDCl3, of the search for optimal binding positions between the recep-
400 MHz, δ in ppm): 12.9881 (s, 1H), 9.4932 (d, J = 1.20 Hz, tor and the target molecules.
1H), 8.6239 (d, J = 2.48 Hz, 1H), 8.5600 (t, J = 1.60 Hz, 1H), DFT studies. The molecular structures of the title com-
7.3880 (d, J = 8.40 Hz, 2H), 7.2692 (t, J = 8.40 Hz, 2H), 4.5689 pounds were optimized employing density functional theory
(s, 2H); 13C-NMR (CDCl3, 100 MHz, δ in ppm): 168.09, 163.79, (DFT) coupled with a 6-31G++ (d, p) basis set, incorporating
161.34, 145.90, 144.85, 144.37, 142.45, 131.74, 131.71, 131.17, Beck’s three-parameter exchange function and the Lee–Yang–
131.09, 116.00, 37.44; ESI-MS (m/z) = 304.05 [M + H]+. Parr nonlocal correlation functional, denoted as B3LYP.26 The
2-(5-((4-Nitrobenzyl)thio)-4H-1,2,4-triazol-3-yl)pyrazine (T18). computational computations were executed utilizing the
White solid, yield: 97%; m.p.: 194–195 °C; 1H-NMR (DMSO-d6, Schrödinger materials science package.20 To enhance compre-
400 MHz, δ in ppm): 12.9680 (s, 1H), 9.3846 (d, J = 1.16 Hz, hension of the charge transfer phenomena and the disparities
1H), 8.8109 (d, J = 2.52 Hz, 1H), 8.7696 (d, J = 1.48 Hz, 1H), in energy among molecular orbitals, a meticulous analysis
8.2163 (d, J = 8.68 Hz, 2H), 7.7925 (d, J = 8.68 Hz, 2H), 4.8030 known as Frontier molecular orbital (FMO) evaluation was con-
(s, 2H); 13C-NMR (DMSO-d6, 100 MHz, δ in ppm): 167.83, ducted. This analytical methodology facilitated the determi-
167.34, 146.85, 146.57, 144.93, 144.78, 143.67, 141.37, 130.40, nation of the energy levels of the highest occupied molecular
123.67; ESI-MS (m/z) = 315.08 [M + H]+. orbital (HOMO) and the lowest unoccupied molecular orbital
(LUMO). After these energy differentials, we extracted indis-
Computational studies pensable chemical reactivity parameters providing insightful
Pharmacokinetic and physicochemical studies. The complex perspectives into the stability and reactivity attributes of the
processes governing how a pharmaceutical agent moves within synthesized compounds.
the body are summarized by the acronym ADME, representing
absorption, distribution, metabolism, and excretion. A deep Biological studies
understanding of these ADME intricacies is essential when In vitro antitubercular activity. The evaluation of the efficacy
developing pharmaceutical substances that achieve a balanced of the title compounds against M. tuberculosis entailed employ-
combination of safety and effectiveness.19 In our efforts to ing the microplate Alamar blue assay (MABA) technique. This

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methodology, characterized by its gentleness, leverages the broth (PDB). A 96-well microtiter plate was meticulously pre-
utilization of a robust, cell-permeable substance denominated pared, with each well receiving 95 µl of Mueller–Hinton Broth
resazurin.27 To obviate desiccation of the culture medium (MHB). Initially, 100 µl of a particular compound was added to
during the incubation phase, 200 µL of sterile deionized water the first well, thoroughly blending it using precise pipetting
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was instilled into all wells of sterile 96-well plates. The com- techniques. This process of transferring 100 µl from each well
pounds of interest, alongside standard pharmaceutical agents, to the subsequent one was rigorously executed until reaching
were meticulously prepared in two-fold dilutions (50.0, 25.0, the ninth well, after which the resultant suspension was care-
12.5, 6.25, 3.12, 1.56, and 0.78 µg mL−1) through dissolution fully discarded. At this stage, 5 µl of the previously prepared
in dimethyl sulfoxide (DMSO). After this, 100 µL of fungal spore suspension was uniformly introduced into all
Middlebrook 7H9 broth, augmented with 0.2% glycerol and wells, except for the tenth well, designated as the compound
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10% oleate-albumin dextrose-catalase (OADC), were introduced control, containing 95 µl of MHB and 100 µl of the compound.
into the wells. Concurrently, 100 µL of M. tuberculosis H37Rv The eleventh well served as a control for the culture, compris-
(ATCC27294) were inoculated into the 7H9 broth wells contain- ing 95 µl of MHB and 5 µl of fungal spores. Finally, the twelfth
ing ten-fold serial dilutions of the pharmaceutical agents per well functioned as the media control, solely containing 95 µl
milliliter. The plates were hermetically sealed with parafilm of MHB without any additional components.
and incubated at 37 °C for five days. Post this incubation
period, a freshly concocted amalgamation of Alamar blue Cytotoxicity studies
reagent and a 10% solution of Tween 80 in a 1 : 1 ratio was The Vero cell line, sourced from the National Center for Cell
added to the wells. A subsequent 24-hour incubation at 37 °C Sciences (NCCS) in Pune, India, comprises African green
ensued. Thereafter, a visual examination of the well contents monkey kidney cells designated by Catalog number 11965-092.
was carried out, with a pink hue signifying bacterial prolifer- These cellular entities were cultivated in 96-well flat-bottomed
ation and a blue tint indicating the suppression of bacterial microtiter plates utilizing DMEM supplemented with 10%
growth. The minimum inhibitory concentration was delineated heat-inactivated fetal calf serum (FBS) and 1% antibiotic–anti-
as the lowest concentration of a compound required to impede mycotic 100× solution.28 Subsequently, they were housed in an
bacterial growth. For comparative analysis, pyrazinamide incubator maintained at a temperature of 37 °C, 95% humid-
(PZA), ciprofloxacin (INN), and streptomycin (STM) were ity, and 5% CO2 concentration for 24 hours. The cellular enti-
employed as benchmark pharmaceuticals. ties were then exposed to these distinct drug concentrations
and subjected to an additional incubation period of 72 hours.
In vitro antibacterial and antifungal activity (broth
Following this incubation, a thorough washing of cells in each
microdilution method)
well was executed using a phosphate buffer solution. After this
Bacterial isolates. Overnight-cultured pure strains of two step, a meticulously prepared stock solution of MTT (20 µL,
Gram-positive bacteria (Staphylococcus aureus, Streptococcus 5 mg mL−1 in sterile phosphate-buffered saline) was instilled
mutans) and two Gram-negative bacteria (Escherichia coli, into each well, followed by an additional incubation for
Salmonella Typhi) were introduced into Mueller Hinton Broth 4 hours in an environment comprising 5% CO2. Upon removal
(MHB) and permitted to proliferate at 37 °C until reaching a of the supernatant, 100 µL of DMSO was introduced to dissolve
density akin to 0.5 McFarland unit. A 96-well microtiter plate the precipitated crystals. The absorbance levels of the wells
was employed, with 95 µl of MHB added uniformly across all housing the cells and the corresponding blanks were quanti-
wells. Initially, 100 µl of the compound was introduced into fied at 570 nm using a microplate reader. The determination
the initial well and thoroughly amalgamated through pipet- of the extent of growth inhibition was executed through the
ting. Sequentially, 100 µl from the initial well was transposed application of the following formula: % Growth Inhibition =
to the subsequent well, and this process continued up to the (mean optical density (OD) of the test compound/mean OD of
ninth well, while the suspension from the ninth well was dis- the negative control) × 100.
carded. Subsequently, 5 µl of the respective bacterial cultures
was added to all wells. The tenth well functioned as a com-
pound control, containing 95 µl of MHB and 100 µl of the Results and discussion
compound. The eleventh, well-acted as a culture control, har-
boring 95 µl of MHB and 5 µl of the bacterial culture. Finally, Chemistry
the twelfth well operated as a media control, containing solely Based on the favorable results obtained through in silico
95 µl of MHB. studies, we have progressed toward the synthesis of the envi-
Fungal isolates. The pure fungal culture of Aspergillus niger, sioned compounds by following the synthetic pathways out-
cultivated overnight, was strategically applied in small doses lined in Scheme 1. The process commenced with the reaction
onto potato dextrose agar (PDA) plates. These plates were sub- of readily available pyrazine-2-carbonitrile (1) with hydrazine
jected to an incubation period at 30 °C lasting 3 to 4 days, fos- hydrate in the presence of methanol, yielding (Z)-pyrazine-2-
tering the proliferation of an extensive layer of fungal spores. carbohydrazonamide (2). Subsequently, compound 2 under-
Subsequently, these spores were meticulously harvested using went cyclization when treated with KOH and CS2, resulting in
sterilized cotton swabs and then suspended in potato dextrose the formation of 5-( pyrazin-2-yl)-4H-1,2,4-triazole-3-thiol (3).

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Scheme 1 Synthesis protocol for the synthesis of target compounds T1–T18. Reagents and conditions: (a) N2H4·H2O, CH3OH, RT, 24 hours, (b)
CS2, KOH, CH3OH, reflux, 24 hours, (c) phenacyl bromide derivatives, 10% NaOH, CH3OH, RT, 6 hours and (d) benzyl bromide derivatives, K2CO3,
acetone, reflux, 2 hours.

Compounds T1–T9 (Table 1) were produced by reacting com- ponding to its molecular structure. The peaks at δ: 190.28 and
pound 3 with commercially accessible phenacyl bromides, 165.39 ppm were assigned to the carbonyl carbon and the
while compounds T10–T18 (Table 1) were synthesized by react- carbon attached to the hydroxy group respectively. The two
ing compound 3 with commercially available substituted carbons of the 1,2,4-triazole were denoted by peaks at δ 162.90
benzyl bromides. and 162.87 ppm. The four carbons of the pyrazine ring were
The validation of the intermediates and target compounds represented by peaks at δ: 146.95, 145.02, 143.43, and
(T1–T18) involved a combination of analytical techniques, 138.69 ppm. Five carbons from the phenyl ring were identified
including 1H NMR, 13C NMR, and mass spectrometry. The 1H by three peaks at δ: 131.21, 126.43, and 115.48 ppm.
NMR spectrum of compound T4 showed two singlet peaks at Furthermore, a peak at δ: 38.88 ppm confirmed the presence
δ: 12.98 and 10.57 ppm, corresponding to the –NH proton of of –CH2 carbon. The molecular mass of compound T4 was
1,2,4-triazole and the –OH proton, respectively. The pyrazine unambiguously confirmed through the mass spectrum, reveal-
ring’s three aromatic protons were observed with two singlet ing a molecular ion peak (M + H peak) observed at (m/z)
peaks at δ: 9.30 and 8.84 ppm and one doublet peak at δ: 314.06. Similarly, the 1H NMR spectrum of compound T11 pre-
8.85 ppm. Additionally, two doublet peaks at δ: 7.95 and sented a singlet peak at δ: 12.97 ppm, signifying the –NH
6.90 ppm indicated the presence of four aromatic protons proton of the 1,2,4-triazole. The three aromatic protons within
from the phenyl ring. Finally, a sharp singlet peak at δ: the pyrazine ring manifested as two doublet peaks at δ: 9.39
5.13 ppm was attributed to the –CH2 protons. The 13C NMR and 8.81 ppm, along with one triplet peak at δ: 8.77 ppm. In
spectrum of compound T4 showed characteristic peaks corres- the region of δ: 7.73–7.67 ppm four aromatic protons originat-
ing from the phenyl ring were detected. Additionally, a distinct
sharp singlet peak at δ: 4.76 ppm represents the presence of
–CH2 protons. In the 13C NMR spectrum of compound T11,
Table 1 Structural details of the target compounds (T1–T18)
characteristic peaks aligned with its molecular structure. Peaks
Compound code R Compound code R at δ: 168.19 and 162.49 ppm were assigned to two carbons of
1,2,4-triazole. The four carbons of the pyrazine ring were rep-
T1, T10 T7, T12 resented by peaks at δ: 160.02, 145.89, 144.90, and
T2 T8, T17
144.36 ppm. Furthermore, the phenyl ring contributed five
peaks in the range of δ: 142.46 to 130.11 ppm, reflecting the
T3 T9, T15 presence of six carbon atoms. Peaks at δ: 115.63 and
36.59 ppm were attributed to the –CF3 and –CH2 carbons,
T4 T11 respectively. The molecular mass of compound T11 was con-
clusively confirmed through mass spectrometry, revealing the
T5, T18 T13 presence of the molecular ion peak (M + H peak) at (m/z)
338.18, thus validating its molecular weight.
T6, T14 T16
Computational studies
Pharmacokinetic and physicochemical studies. The compre-
The symbol ‘*’ denotes the point of attachment. hensive summary of the pharmacokinetic and physico-

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chemical characteristics of the target molecules is outlined in demonstrate a human oral absorption rate exceeding 85%,
Table 2, outlining their potential as promising candidates for which is a strong indicator of their outstanding oral bio-
advancing drug development. Adhering closely to Lipinski’s availability. In summary, the comprehensive in silico ADME
rule of five, a well-established standard for assessing a com- predictions provide strong support for advancing these com-
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pound’s suitability for oral administration ensures that all pounds as promising candidates for further development in
target compounds possess favorable pharmaceutical attri- drug discovery.
butes. Compounds that violate more than one of these criteria In silico molecular docking studies. In silico molecular
often face challenges related to permeability or solubility and docking was performed to uncover the binding interactions
are considered less suitable for pharmaceutical advancement. between a group of target compounds (T1–T18) and the recep-
In the scope of this study, all the target compounds adhere to tor, decaprenylphosphoryl-D-ribose oxidase (DprE1) of
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Lipinski’s rule of five, thereby confirming their suitability for Mycobacterium tuberculosis (PDB ID: 4P8N). DprE1, a vital
oral delivery. The evaluation of a drug’s safety profile heavily enzyme in the synthesis of the M. tuberculosis cell wall.29 The
relies on the parameters associated with absorption, distri- computational tool Auto Dock-Vina 1.1.2 was utilized for this
bution, metabolism, and excretion (ADME). All of the target purpose. To validate the docking methodology, the ligands
compounds exhibit favorable ADME characteristics. When from the crystallographic structures of the protein–ligand com-
examining the polar surface area (PSA), a crucial factor in how plexes were reconstructed and then subjected to redocking.
well a drug is absorbed, we found that the values comfortably The crucial amino acid residues situated in the active site of
fall within the optimal range (ranging from 60.70 to 131.85 Å). DprE1, including Arg 58, Ser 59, Ala 64, Ala 72, Gly 76, Gly 125,
Moreover, the favorable values for Caco-2 cell permeability His 132, Gly 136, Thr 141, Ala 147, Cys 148, Ile 203, Cys387,
(QPPCaco) and the aqueous solubility parameter (QP log S) Tyr 434, and Tyr 415, play a significant role. Most of the target
(ranging from 54.31 to 1128.37 nm s−1 and −5.25 to −3.38, compounds demonstrated interactions with these amino acid
respectively) further enhance the potential for efficient absorp- residues and exhibited higher binding energy values compared
tion in the intestines. Significantly, the blood–brain partition to the reference drugs (PZA, INN, and STM). Compound T11
coefficient (QP log BB) values are within an acceptable range docked within the active site of DprE1 with a binding energy
(−2.02 to −0.24), indicating that these compounds can cross of −10.10 kcal mol−1 and formed five hydrogen bond inter-
the blood–brain barrier. Furthermore, the human serum actions with Arg 58, Ser 59, Gly 125, His 132, and Tyr 415
albumin binding coefficient (QP log Khsa) falls comfortably amino acid residues. It also showed a π–σ interaction with Ala
within the desired range (ranging from −0.50 to 0.20), under- 64 and Ile 131 and a π–alkyl interaction with Cys 129 and Ala
scoring the potential of these compounds to effectively bind to 417 (Fig. 1 and 2). Table 3 provides a comprehensive summary
human serum albumin, a critical factor in how drugs are dis- of the binding energies and the amino acid residues engaged
tributed in the body. Notably, a majority of these compounds by both the target compounds and reference compounds.

Table 2 Pharmacokinetic and physicochemical parameters of the target compounds (T1–T18)

QPPCaco
(<25 nm s−1 is
MW HBD HBA QP log P QP log S nRB PSA low; >500 nm s−1 QP log Khsa QP log BB %OA (>80% is high:
Comp. (≤500 Da) (≤5) (≤10) (o/w) (≤5) (≤0.5) (0–15) (≤140 Å) is high) (−1.5 to 1.5) (−3.0 to 1.2) <25% is low)

T1 297.33 1 6.5 1.84 −3.49 4 87.14 454.69 −0.32 −0.95 85.31

T2 311.36 1 6.5 2.10 −4.03 4 87.16 454.22 −0.17 −0.99 86.83
T3 327.36 1 7.25 1.95 −3.73 5 95.37 453.58 −0.32 −1.05 85.90
T4 313.33 2 7.25 1.21 −3.38 5 109.73 137.55 −0.40 −1.57 72.31
T5 342.33 1 7.5 1.13 −3.60 5 131.86 54.32 −0.39 −2.03 64.61
T6 322.34 1 8 1.08 −4.41 5 112.94 96.13 −0.51 −1.78 68.78
T7 315.32 1 6.5 2.02 −3.78 4 87.15 454.64 −0.28 −0.85 86.31
T8 331.78 1 6.5 2.35 −4.23 4 87.14 454.89 −0.22 −0.81 88.27
T9 376.23 1 6.5 2.42 −4.33 4 87.14 454.87 −0.20 −0.80 88.70
T10 269.32 1 4.5 2.61 −3.85 3 60.78 1109.17 −0.05 −0.48 96.73
T11 337.32 1 4.5 3.58 −5.26 3 60.78 1109.04 0.20 −0.24 100
T12 287.31 1 4.5 2.83 −4.19 3 60.78 1109.26 −0.01 −0.38 100
T13 287.31 1 4.5 2.78 −4.08 3 60.77 1109.81 −0.02 −0.40 100
T14 294.33 1 6 1.83 −4.78 4 86.58 234.64 −0.22 −1.29 80.10
T15 348.22 1 4.5 3.17 −4.68 3 60.78 1109.31 0.09 −0.32 100
T16 348.22 1 4.5 3.08 −4.48 3 60.70 1128.37 0.06 −0.33 100
T17 303.77 1 4.5 3.09 −4.57 3 60.78 1109.50 0.06 −0.33 100
T18 314.32 1 5.5 1.88 −3.99 4 105.46 132.99 −0.10 −1.54 75.99

Comp.: compound, MW: molecular weight, HBD: number of hydrogen bond donors, HBA: number of hydrogen bond acceptors, QP log P (o/w):
logarithm of the partition coefficient between n-octanol and water, QP log S: aqua solubility parameter, nRB: number of rotatable bonds, PSA:
polar surface area, QPPCaco: caco-2 cell permeability, QP log Khsa: human serum albumin binding co-efficient, QP log BB: blood/brain partition
co-efficient, and %OA: percentage of oral absorption.

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Fig. 1 The 2D docking poses of compound T11 with receptor 4P8N.

Fig. 2 The 3D docking poses of compound T11 with receptor 4P8N.

DFT studies. The target molecular entities (T1–T18) were orbital, collectively denoted as the frontier molecular orbitals
subjected to geometric optimization employing density func- (FMOs). The HOMO signifies an electron-abundant orbital,
tional theory (DFT) with the B3LYP functional and the 6-31G++ whereas the LUMO denotes an electron-deficient one. These
(d, p) basis set. It is imperative to scrutinize the highest occu- FMOs assume a pivotal role in dictating a molecule’s inter-
pied molecular orbital and the lowest unoccupied molecular actions with its target counterparts. Generally, a molecule’s

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Table 3 Binding energy and interactions of target compounds (T1–T18) with receptor 4P8N

Comp. Binding energy (kcal mol−1) Inhibition constant (Ki) (nM) No. of H-bonds Interacting amino acid residues

T1 −8.3 393.27 03 GLY57, ARG58, SER59, CYS129

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T2 −9.4 125.14 04 GLY55, GLY57, ARG58, SER59, CYS129

T3 −9.1 156.21 02 ARG58, SER59, TYR60, ALA64
T4 −9.4 125.38 03 ALA53, ARG58, SER59, GLY125
T5 −8.8 277.76 04 SER59, GLY117, THR118, LYS134, GLN336
T6 −9.7 79.26 04 ARG58, SER59, CYS129, ASN63
T7 −9.3 149.61 03 ARG58, SER59, CYS129, ASN63
T8 −8.8 277.93 02 ALA53, SER59, GLY124, GLY125
T9 −8.9 281.47 02 ALA53, SER59, GLY124, GLY125, VAL121
−9.0
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T10 243.35 02 SER59, ALA64, VAL121, GLY125, CYS129

T11 −10.1 39.47 05 ARG58, SER59, GLY125, HIS132, TYR415
T12 −9.2 151.87 02 SER59, ALA64, GLY125, CYS129
T13 −9.3 149.82 02 SER59, ALA64, GLY125, CYS129
T14 −9.7 74.65 04 SER59, ALA64, GLY117, GLY125, CYS129
T15 −9.3 149.47 05 SER59, GLY117, THR118, LYS134, TYR415
T16 −9.1 156.07 02 SER59, ALA64, GLY125, CYS129, LYS418
T17 −9.1 156.03 02 SER59, ALA64, GLY125, CYS129
T18 −9.8 69.08 04 ARG58, SER59, ALA64, HIS132, TYR415
PZA −5.3 21.74 µM 04 GLY57, ARG58, SER59, GLY125, CYS129
INN −8.2 17.93 µM 04 ALA53, ARG54, THR122, GLY125, ILE184
STM −9.1 201.09 06 GLY117, HIS132, SER228, GLN336, TYR415

Comp.: compound, PZA: pyrazinamide, INN: ciprofloxacin, STM: streptomycin.

propensity to engage in binding interactions with other mole- tron cloud across the pyrazine ring. The dimension of the
cules tends to diminish as its HOMO energy decreases and its energy differential between the HOMO and the LUMO
LUMO energy escalates. Table 4 presents the HOMO and emerges as a pivotal metric for gauging the compound’s reac-
LUMO energy values for the target molecules (T1–T18), tivity, hardness, and softness. A diminutive energy gap con-
gleaned from meticulous DFT calculations. Fig. 3a elucidates notes heightened chemical reactivity and an augmented pro-
the visual depictions of the FMOs for the potent molecule T11. clivity for intermolecular interactions, thereby categorizing the
The studies conducted via DFT discern that within compound molecule as intrinsically soft. Conversely, molecules featuring
T11, the electron cloud of the HOMO predominantly localizes a substantial energy gap exhibit heightened thermal stability
itself across the 1,2,4-triazole ring and the concomitant sulfur and a diminished propensity for intermolecular interactions,
atom. Conversely, the LUMO expansively disseminates its elec- thereby classifying them as intrinsically rigid entities.

Table 4 Global reactivity parameters calculated using DFT

Comp. EHOMO (eV) ELUMO (eV) ΔE (eV) IP (eV) EA (eV) η (eV) σ (eV) µ (eV) ω (eV) χ (eV)

T1 −6.0954 −1.8858 4.2096 6.0954 1.8858 2.1048 0.2376 −2.1048 1.0524 3.9906
T2 −6.0437 −1.8531 4.1906 6.0437 1.8531 2.0953 0.2386 −2.0953 1.0476 3.9484
T3 −6.0518 −1.7905 4.2613 6.0518 1.7905 2.1307 0.2347 −2.1307 1.0653 3.9212
T4 −6.0300 −1.8449 4.1851 6.0300 1.8449 2.0926 0.2389 −2.0926 1.0463 3.9375
T5 −6.3212 −3.1266 3.1946 6.3212 3.1266 1.5973 0.3130 −1.5973 0.7987 4.7239
T6 −6.3049 −2.6694 3.6354 6.3049 2.6694 1.8177 0.2751 −1.8177 0.9089 4.4872
T7 −6.1362 −1.9184 4.2178 6.1362 1.9184 2.1089 0.2371 −2.1089 1.0544 4.0273
T8 −6.1743 −2.0871 4.0872 6.1743 2.0871 2.0436 0.2447 −2.0436 1.0218 4.1307
T9 −6.1824 −2.1034 4.0790 6.1824 2.1034 2.0395 0.2452 −2.0395 1.0197 4.1429
T10 −6.0437 −1.8585 4.1851 6.0437 1.8585 2.0926 0.2389 −2.0926 1.0463 3.9511
T11 −6.2178 −1.9619 4.2559 6.2178 1.9619 2.1279 0.2350 −2.1279 1.0640 4.0899
T12 −6.1144 −1.8994 4.2150 6.1144 1.8994 2.1075 0.2372 −2.1075 1.0538 4.0069
T13 −6.0573 −1.8558 4.2014 6.0573 1.8558 2.1007 0.2380 −2.1007 1.0504 3.9565
T14 −6.3130 −2.0218 4.2912 6.3130 2.0218 2.1456 0.2330 −2.1456 1.0728 4.1674
T15 −6.1688 −1.9347 4.2341 6.1688 1.9347 2.1170 0.2362 −2.1170 1.0585 4.0518
T16 −6.0709 −1.8613 4.2096 6.0709 1.8613 2.1048 0.2376 −2.1048 1.0524 3.9661
T17 −6.1661 −1.9293 4.2368 6.1661 1.9293 2.1184 0.2360 −2.1184 1.0592 4.0477
T18 −6.3457 −2.6096 3.7361 6.3457 2.6096 1.8681 0.2677 −1.8681 0.9340 4.4776
PZA −6.7838 −1.9347 4.8491 6.7838 1.9347 2.4246 0.2062 −2.4246 1.2123 4.3592
INN −5.7007 −1.2027 4.4980 5.7007 1.2027 2.2490 0.2223 −2.2490 1.1245 3.4517

Comp.: compound, bandgap (ΔE) = EHOMO − ELUMO, ionization potential (IP) = −EHOMO, electron affinity (EA) = −ELUMO, chemical hardness (η) =
(IA − EA)/2, chemical softness (σ) = 1/2η, chemical potential (µ) = −η, electrophilicity index (ω) = η/2, and electronegativity (χ) = (IP + EA)/2.

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Fig. 3 (a) Frontier molecular orbitals, (b) molecular electrostatic potentials, and (c) the electron density surface of compound T11.

Comprehending overarching chemical reactivity descriptors ities of these target compounds based on their physico-
on a global scale is imperative for establishing intricate corre- chemical attributes and comprehending their predisposition
lations among the structural attributes, stability, and reactivity toward electrophilic and nucleophilic reactions.32 Through the
of a molecule.30 These descriptors are ascertained through the employment of DFT, we ascertained the MEP profiles of the
meticulous evaluation of the HOMO and LUMO energy levels specified compounds. In Fig. 3b, the MEPs of compound T11
of the molecules, with precise numerical values delineated in are delineated in detail, with the MEP surface meticulously
Table 4.31 An elevated chemical softness parameter signifies color-coded for optimal elucidation. The blue domains in the
an augmented proclivity for binding with the receptor, figure delineate regions characterized by a positive electrostatic
whereas an increased chemical hardness value implies a potential, while the red domains signify areas distinguished by
diminished inclination for binding. Significantly, all scruti- a negative electrostatic potential. The white regions denote a
nized compounds manifest heightened chemical softness and neutral electrostatic potential. Across the entirety of these
diminished chemical hardness in contrast to the reference molecules, regions characterized by a negative electrostatic
pharmaceutical agents. With the augmentation of the chemi- potential are chiefly congregated in the proximity of nitrogen,
cal potential, the discernibility of interaction between the sulfur, and fluorine atoms. Conversely, zones exhibiting a posi-
ligand and the receptor intensifies, and notably, all target tive electrostatic potential are predominantly situated in the
compounds manifest elevated chemical potential values in vicinity of hydrogen atoms and an intricately structured phenyl
comparison to the benchmark pharmaceuticals. The electro- ring.
philicity index and electronegativity serve as metrics gauging a
molecule’s capacity to attract electrons from its ambient Biological studies
milieu. Molecules endowed with a heightened electrophilicity Antitubercular activity. The MABA technique was employed
index and electronegativity values evince an augmented predis- to assess the efficacy of target compounds (T1–T18) against
position for forging interactions with receptors. It is of signifi- Mycobacterium tuberculosis H37Rv (ATCC27294). The results
cance that all target compounds exhibit electronegativity and presented in Table 5 indicate that the minimum inhibitory
electrophilicity index values akin to those inherent in the refer- concentration (MIC) values range from 9.25 to 46.45 µM (3.12
ence pharmaceuticals. Fig. 3c elucidates the electron density to 12.5 µg mL−1). Notably, compound T11 exhibited the
surface of compound T11. The ionization potential (IP) and highest potency among the tested compounds, displaying a
electron affinity (EA) serve as quantitative measures for MIC of 9.25 µM (3.12 µg mL−1), which is comparable to the
appraising a molecule’s proclivity to either gain or relinquish effectiveness of INN and STM, and surpasses PZA by a factor of
electrons within its proximate milieu. In the general context, two. Furthermore, compounds T4, T5, T6, T14, T15, T16, and
diminished values of IP and EA are correlated with an T18 demonstrated promising antitubercular activity with MIC
increased proclivity toward toxicity. It merits emphasis that all values ranging from 18.01 to 21.25 µM (6.25 µg mL−1). While
investigated compounds align within the stipulated IP and EA not as potent as STM and INN, they exhibited effectiveness
value range characteristic of the FDA-approved pharmaceuti- comparable to that of PZA. The remaining compounds exhibi-
cals, with EA values spanning the range from −3 to 7 eV and IP ted moderate antitubercular activity, with MIC values ranging
values encompassing the interval of 4–15 eV. from 33.33 to 46.45 µM (12.5 µg mL−1). These experimental
To evaluate the reactivity of these compounds, we under- findings closely align with predictions from computer-based
took molecular electrostatic potential (MEP) computations, a simulations. Docking analyses suggest that the most potent
pivotal methodology for prognosticating the interaction modal- compounds possess the strongest binding energies.

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Table 5 Antitubercular activity of compounds T1–T18 with Table 6 Antibacterial activity (MIC in mg mL−1) of the target com-
M. tuberculosis H37Rv pounds (T1–T18)

M. tuberculosis M. tuberculosis MIC (mg mL−1)

H37Rv H37Rv
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Comp. S. aureus S. mutans E. coli S. typhi

MIC MIC MIC MIC
Comp. (µg mL−1) (µM) Comp. (µg mL−1) (µM) T1 2.5 >5.0 5.0 >5.0
T2 2.5 >5.0 5.0 >5.0
T1 12.5 42.07 T12 12.5 43.54 T3 5.0 5.0 5.0 >5.0
T2 12.5 40.18 T13 12.5 43.54 T4 2.5 1.25 0.625 >5.0
T3 12.5 38.21 T14 6.25 21.25 T5 1.25 >5.0 >5.0 >5.0
T4 6.25 19.96 T15 6.25 18.01 T6 5.0 >5.0 >5.0 >5.0
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T5 6.25 18.27 T16 6.25 18.01 T7 2.5 >5.0 5.0 >5.0

T6 6.25 19.40 T17 12.5 41.25 T8 5.0 5.0 5.0 >5.0
T7 12.5 39.67 T18 6.25 19.90 T9 0.01953 1.25 >5.0 1.25
T8 12.5 37.76 PZA 3.12 25.34 T10 0.625 2.5 5.0 >5.0
T9 12.5 33.33 STM 6.25 10.74 T11 5.0 5.0 1.26 5.0
T10 12.5 46.45 INN 3.12 9.41 T12 2.5 5.0 5.0 5.0
T11 3.12 9.25 T13 2.5 5.0 5.0 5.0
T14 2.5 >5.0 >5.0 >5.0
Comp.: compound, PZA: pyrazinamide, STM: streptomycin, INN: T15 2.5 5.0 2.5 >5.0
ciprofloxacin. T16 0.01953 0.01953 0.625 2.5
T17 2.5 >5.0 >5.0 5.0
T18 0.625 >5.0 1.25 5.0
STM 0.012 0.010 0.010 0.011
Additionally, density functional theory analyses indicate that CHL 0.010 0.010 0.012 0.010
Control 00 00 00 00
the most effective compounds exhibit higher values for pro-
perties such as chemical softness, chemical potential, electro- Comp.: compound, STM: streptomycin, CHL: chloramphenicol,
control: DMSO.
philicity, and electronegativity.
Antibacterial activity. The in vitro antibacterial activity of the
target compounds (T1–T18) was tested using the broth micro-
dilution method and the MIC values were measured in mg Table 7 Antifungal activity (MIC in mg mL−1) of the target compounds
mL−1. All the synthesized compounds were screened against (T1–T18)
two Gram-positive bacterial strains (S. aureus and S. mutans),
and two Gram-negative bacterial strains (E. coli and S. typhi) MIC (mg mL−1) MIC (mg mL−1)
Comp. A. niger Comp. A. niger
using streptomycin (STM) and chloramphenicol (CHL) as the
standard drugs. The MIC values of the target compounds T1 2.5 T11 1.25
T2 5.0 T12 0.625
along with those of standard drugs for comparison are pre-
T3 5.0 T13 5.0
sented in Table 6. Among the screened compounds, T9, T10, T4 5.0 T14 0.625
T16, and T18 demonstrated significant inhibition activity T5 5.0 T15 1.25
T6 5.0 T16 2.5
against S. aureus, whereas compound T16 was active against
T7 5.0 T17 5.0
S. mutans. Compounds T4, and T16 were potent against both T8 2.5 T18 5.0
E. coli. None of the compounds showed notable inhibition T9 5.0 AmB 0.004
T10 5.0 Control 00
activity against S. typhi.
Antifungal activity. The in vitro antifungal activity of the title Comp.: compound, AmB: amphotericin B, control: DMSO.
compounds (T1–T18) was screened using the broth microdilu-
tion method and the MIC values were measured in mg mL−1.
All the synthesized compounds were screened against the 092) at concentrations spanning 25, 50, 75, and 100 µM.
fungal strain A. niger using amphotericin B (AmB) as a stan- Notably, it was observed that all the tested potent compounds
dard drug. The MIC values of the target compounds along did not exhibit significant toxicity, as indicated by IC50 values
with the standard drugs for comparison are presented in exceeding 375 µM, as shown in Table 9. Additionally, it is
Table 7. Among the screened compounds T12 and T14 showed important to highlight that none of these compounds had
significant inhibition activity against A. niger. Compounds T11 adverse effects on normal cells, indicating the absence of
and T15 demonstrated moderate inhibition activity against the general cellular toxicity.
tested strain. Structure–activity relationship. The effectiveness of the
Cytotoxicity studies. The MTT assay served as the modality target compounds (T1–T18) in terms of their anti-tubercular
for the assessment of cytotoxicity of compounds exhibiting a and antimicrobial properties reveals specific structure–activity
MIC of 21.25 µM or lower in the context of antituberculosis relationships (SAR). The introduction of substituents that
screening. Table 8 shows the inhibitory impact these effica- enhance the aromaticity and electron density within the pyra-
cious compounds exerted on the proliferation of Vero cells zine or 1,2,4-triazole ring resulted in improved antitubercular
(African green monkey kidney cell line, Catalog number 11965- activity. Both 1-phenyl-2-((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)

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Table 8 Cell growth inhibition of active compounds against Vero cells went in vitro testing to assess their potential as antimicrobial
agents against diverse bacteria, fungi, and the tuberculosis-
% of inhibition inducing M. tuberculosis H37Rv strain. Among the tested com-
Compound 25 µM 50 µM 75 µM 100 µM pounds, eight exhibited notable activity against tuberculosis,
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with a MIC ≤ 6.25 µg mL−1. Particularly noteworthy was com-

T4 3.21 ± 0.34 6.46 ± 0.21 9.61 ± 0.18 12.92 ± 0.26
T5 3.05 ± 0.23 6.38 ± 0.17 9.53 ± 0.15 12.87 ± 0.19 pound T11, showcasing the most potent activity with a MIC of
T6 3.14 ± 0.27 6.35 ± 0.32 9.89 ± 0.23 13.05 ± 0.23 3.12 µg mL−1. To ascertain their suitability for pharmaceutical
T11 2.97 ± 0.15 6.03 ± 0.22 9.08 ± 0.27 12.10 ± 0.14 development, cytotoxicity evaluations were conducted, reveal-
T14 3.02 ± 0.18 6.24 ± 0.31 9.63 ± 0.20 13.09 ± 0.17
T15 2.99 ± 0.31 6.12 ± 0.19 9.74 ± 0.38 12.64 ± 0.27 ing that these active compounds did not induce harm to
T16 3.04 ± 0.25 6.16 ± 0.27 9.37 ± 0.16 12.35 ± 0.22 normal cells. Additionally, computational analysis was
Open Access Article. Published on 13 March 2024. Downloaded on 7/18/2024 7:27:02 AM.

T18 3.17 ± 0.23 6.38 ± 0.22 9.71 ± 0.21 12.59 ± 0.25 employed to scrutinize the binding interactions between these
The values represent the mean ± standard error of the mean obtained compounds and the active site of the DprE1 enzyme through
from three separate determinations after 72 hours of exposure. in silico molecular docking. Although these studies indicated
strong binding interactions, further experimental validation is
necessary to confirm the mechanism of inhibition action. DFT
Table 9 IC50 values of active compounds obtained through an MTT studies provided insights into the electronic properties of
assay against Vero cells these compounds, indicating a narrower HOMO–LUMO energy
gap, augmented chemical softness, diminished chemical hard-
Compound IC50 (µM) Compound IC50 (µM)
ness, lower electron affinity, and higher chemical potential in
T4 387.53 ± 10.15 T14 379.86 ± 11.47 comparison with reference compounds. These findings collec-
T5 387.29 ± 12.03 T15 389.47 ± 10.73 tively imply heightened reactivity and more robust interactions
T6 379.88 ± 10.49 T16 402.72 ± 13.48
T11 412.32 ± 11.63 T18 393.80 ± 12.44 with the target receptor. Beyond their antitubercular attributes,
compounds T9, T10, and T18 manifested significant antibac-
The IC50 values represent the concentration of a substance required to
terial activity against S. aureus, whereas compound T16 exhibi-
inhibit 50% of cell viability calculated after a 72-hour exposure.
ted antibacterial activity against S. aureus, S. mutans, and
E. coli. Compounds T12 and T14 evinced substantial antifun-
thio)ethan-1-one (T1–T9) and 2-(5-(benzylthio)-4H-1,2,4-triazol- gal activity against A. niger. This exhaustive study emphasizes
3-yl)pyrazine derivatives (T10–T18) exhibited superior activity the importance of further delving into the pharmacological
when equipped with electron-withdrawing groups. For characteristics of these compounds, inclusive of in vivo investi-
instance, T5 and T18 with a nitro group as well as T6 and T14 gations, to comprehensively fathom their potential in combat-
with a cyano group are equipotent (MIC of 6.25 µg mL−1). The ing infectious diseases.
introduction of a –CF3 (trifluoromethyl) group (T11) into the
active pharmacophore further improved the antimycobacterial
activity (MIC of 3.12 µg mL−1). This improvement is attributed Author contributions
to the small size of fluorine, which closely resembles hydrogen
Shivakumar: conceptualization, methodology, computational
and can meet the steric requirements for interaction with the
studies, data curation, visualization, biological studies, and
receptor enzyme. Additionally, it enhances lipid solubility,
writing – original draft. P Dinesha: data curation and visualiza-
thereby improving the absorption rate. Fluorinated analogues
tion. Vanishree A. L.: data curation and visualization.
also offer increased thermal and oxidative stability to the com-
Udayakumar D.: supervision, validation, and writing – review
pounds, as the carbon–fluorine (C–F) bond exhibits greater
and editing. Varsha Prakash Shetty: bacterial and fungal
bond strength relative to the carbon–hydrogen (C–H) bond.
activity. Vijaya Kumar Deekshit: bacterial and fungal activity.
Furthermore, the fluorine substituent can form hydrogen
bonds with target enzymes. The substitution of the –CF3 group
is more favorable than the –F group, as it is more lipophilic.
Hence it can be concluded that compound T11 exhibited the
Conflicts of interest
highest potency among the tested compounds primarily due to The authors declare that they have no known competing finan-
the presence of the –CF3 substituent. cial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Conclusions
Acknowledgements
This study entailed the synthesis of a new series of 1-phenyl-2-
((5-( pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)thio)ethan-1-one (T1–T9) The authors thank the National Institute of Technology
and 2-(5-(benzylthio)-4H-1,2,4-triazol-3-yl)pyrazine (T10–T18) Karnataka (NITK), Surathkal, India for providing all the necess-
derivatives. The synthesized compounds were characterized ary infrastructure and resources to carry out this research work
using various analytical techniques. The compounds under- and CRF, NITK for the instrumentation facilities.

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