Synthesis, in vitro anti-HIV-1 reverse transcriptase evaluation, molecular modelling, DFT analysis and acute oral toxicity assessment of benzotriazole derivatives

Nigam Jyoti Maiti; Swastika Ganguly; Kiattawee Choowongkomon; Supaphorn Seetaha; Siriwan Saehlee; Thitinan Aiebchun; Rahul Ghosh; Mohammad Adnan Khan; Biplab Debnath; Rajarshi Nath; Sumel Ashique; Nisha Kumari Singh; Mohammad Yousuf Ansari; Mohd Tariq; Parwiz Niazi

doi:10.1038/s41598-026-44267-0

. 2026 Apr 13;16:17209. doi: 10.1038/s41598-026-44267-0

Synthesis, in vitro anti-HIV-1 reverse transcriptase evaluation, molecular modelling, DFT analysis and acute oral toxicity assessment of benzotriazole derivatives

Nigam Jyoti Maiti ^1,^2,^✉, Swastika Ganguly ², Kiattawee Choowongkomon ³, Supaphorn Seetaha ³, Siriwan Saehlee ³, Thitinan Aiebchun ³, Rahul Ghosh ², Mohammad Adnan Khan ⁴, Biplab Debnath ⁵, Rajarshi Nath ⁵, Sumel Ashique ⁵, Nisha Kumari Singh ⁶, Mohammad Yousuf Ansari ⁷, Mohd Tariq ^8,¹⁰, Parwiz Niazi ^9,^✉

PMCID: PMC13234155 PMID: 41974835

Abstract

In this study, we synthesized and assessed a series of benzotriazole derivatives (3–30) for their anti-human immunodeficiency virus (HIV)-1 reverse transcriptase (R.T.) activity and compared them to the standard drug efavirenz. Among these, compound 27 demonstrated noteworthy anti-HIV-1 R.T. activities. In vivo oral toxicity tests on the most potent compound, 27, indicating acceptable acute oral safety. We used molecular docking techniques to investigate the potential interactions between the synthesized compounds (ligands) and a target protein (PDB ID 1RT2) at the molecular level. Additionally, molecular dynamics studies were conducted on compound 27 within the active binding site of the co-crystallized HIV-1 RT structure (PDB ID 1RT2) to understand better the behaviour, interactions, and changes in biomolecular systems at the atomic or molecular scale over time. Finally, the DFT-derived HOMO and LUMO orbitals, along with an analysis of the molecular electrostatic potential map, provide insights into the reactivity characteristics of the molecule.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-44267-0.

Keywords: Anti-HIV, DFT, Molecular docking, Molecular dynamics, Oral toxicity

Subject terms: Biochemistry, Chemical biology, Chemistry, Computational biology and bioinformatics, Drug discovery

Introduction

Since the late 1980s, global efforts to combat human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) have shown promising results, with worldwide declines in new infections and deaths supporting the goal of ending AIDS by 2030. Despite therapeutic advancements preventing millions of deaths, a significant proportion of individuals with HIV remain untreated, and many treated individuals are not virally suppressed, particularly in sub-Saharan Africa, where AIDS remains a leading cause of death. Over the years, there has been a gradual decrease infection in children, indicating progress in HIV awareness efforts^1,2.

There are 39.0 million HIV-positive individuals worldwide, according to a World Health Organisation (WHO) report. Additionally, the number of new cases (1.3 million) and deaths from HIV-related causes (630 thousand) highlight the urgent need for effective therapies to control HIV infection and transmission³. One crucial target in AIDS treatment is reverse transcriptase (R.T.), a key enzyme in HIV-1 replication⁴. Nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) are classes of antiretroviral drugs that disrupt reverse transcriptase (R.T.) activity. NRTIs compete with natural substrates and act as chain terminators, whereas NNRTIs bind to a different site on the enzyme, adopting a ‘butterfly-like’ conformation as revealed by X-ray crystallography^5–7. Between 1996 and 2018, the U.S. Food and Drug Administration (FDA) approved six NNRTIs for treating HIV infections. These include the first-generation NNRTIs delavirdine (DLV), nevirapine (NVP), and efavirenz (EFV), as well as the second-generation NNRTIs etravirine (ETR), rilpivirine (RPV), and doravirine (DOR)⁸. While these inhibitors and other classes of antiretroviral medications are pivotal in suppressing viral replication and halting disease progression, they face challenges due to the emergence of drug resistance during their widespread clinical use^9,10. Benzotriazole derivatives, characterized by their unique triazole ring structure, have garnered attention for their diverse applications, including corrosion inhibition and pharmaceutical development^11–15. Moreover, their combined effects with current antiretroviral treatments demonstrate enhanced antiviral potency, suggesting promising advancements in treating drug-resistant strains^16,17. Benzotriazole derivatives have been reported to exhibit diverse biological activities, including antiviral effects; however, their application as HIV-1 reverse transcriptase inhibitors has remained structurally limited. Most previously reported analogues involve minimal substitution on the benzotriazole core, which may restrict optimal interaction within the NNRTI binding pocket. Our research focused on the synthesizing of compounds (3–30). We characterized the synthesized compounds by utilizing mass spectrometry, ¹H-NMR, and ¹³C-NMR. The anti-HIV-1 reverse transcriptase activity of benzotriazole derivatives was assessed through in vitro studies, followed by in vivo oral toxicity testing of the most active compounds, which validated their safety profiles. We performed molecular docking and dynamics studies to understand the interactions with target proteins. At the same time, we used density functional theory (DFT) to analyze the reactivity and structure of the most active compound, aiming to address the challenges in developing effective and safe anti-HIV drugs. In this study, perhalo-substitution of the benzotriazole nucleus using tetra-bromo and tetra-chloro derivatives was deliberately employed to enhance hydrophobicity, molecular rigidity, and non-covalent interactions critical for NNRTI binding. Halogen substitution was also expected to modulate electronic properties and improve binding stability. Based on this rationale, we hypothesized that perhalo-substituted benzotriazole derivatives would demonstrate improved HIV-1 RT inhibitory potential, guiding their synthesis and biological evaluation.Some of the established HIV-1 RT inhibitors are as follows Fig. 1.

Fig. 1 — Established HIV-1 RT inhibitors.

Results and discussion

Chemistry

In this study, we synthesized the target compounds (3–30) following the procedure outlined in Scheme 1. Initially, the starting materials 4,5,6,7-tetrabromo-1 H-benzo[d] [1,2,3] triazole (1a)¹⁸, 4,5,6,7-tetrachloro-1 H-benzo[d] [1,2,3] triazole (1b)¹⁹, and 2-chloro-N-(substituted phenyl)acetamide (2a-n)^25–28 were prepared using established procedures. Subsequently, the target compounds (3–30) were synthesized by reacting tetrabromobenzotriazole (1a) and tetrachlorobenzotriazole (1b) with substituted acetamides (2a-n) in N, N-dimethylformamide (DMF) using N, N-diisopropylethylamine (DIPEA) as a catalyst following the method described by Nigam et al.²⁰. We characterized the synthesized compounds using spectral data analysis.

HIV-1 RT inhibition study

In our previous studies, the synthesized compounds (17, 19–23, 25, 26, and 28–30) exhibited potent antimicrobial agent activity²⁰. Given benzotriazole derivatives’ broad spectrum of biological activities, we investigated the HIV-1 reverse transcriptase inhibitory potential of all 28 synthesized compounds (Scheme 1). The experimental approach was adapted from the methodology described by Silprasit et al., ensuring rigorous assessment of the antiviral efficacy of the compounds²¹. Efavirenz is the reference standard in this assay.

Among the 28 compounds evaluated, compounds 18, 24, and 27 exhibited exceptional reverse transcriptase (R.T.) inhibitory activity, with inhibition rates of 100%, 99.98%, and 93.02%, respectively, surpassing or closely approaching the efficacy of the reference drug efavirenz (Fig. 2a, b). Compared with the standard drug, the synthesized compounds such as (23, 20, 29, 19, 21, 26, 28, 22, 30, 12, and 5) exhibited moderate HIV-1 RT inhibitory activities, with inhibition rates of 88.51%, 86.11%, 86.00%, 84.50%, 84.45%, 84.45%, 79.34%, 78.71%, 78.32%, 73.31%, and 73.25% respectively (Table 1). The other test compounds displayed comparatively less anti-HIV-1 reverse transcriptase activity.

Fig. 2 — a, b R.T. inhibitory percentage of all synthesized compounds (**3–16**) & (**17–30**) compared to the standard drug efavirenz. c IC₅₀ value of potent synthesized compound 27.

Table 1.

The percentage of HIV-1 reverse transcriptase (R.T.) inhibitory activity of the test compounds against the wild-type (wt) strain.

Compound	% of R.T. inhibition	Compound	% of R.T. inhibition
3	48.22 ± 4.31	17	77.31 ± 5.88
4	69.14 ± 2.79	18	100 ± 1.13
5	73.25 ± 5.89	19	84.50 ± 0.97
6	47.84 ± 3.11	20	86.11 ± 0.45
7	59.49 ± 4.53	21	84.45 ± 0.58
8	63.40 ± 1.29	22	78.71 ± 18.83
9	71.52 ± 0.60	23	88.51 ± 6.57
10	65.02 ± 0.34	24	99.98 ± 0.43
11	19.60 ± 0.16	25	67.44 ± 2.14
12	68.57 ± 0.043	26	84.45 ± 0.78
13	73.31 ± 0.01	27	93.02 ± 1.33
14	64.76 ± 6.67	28	79.34 ± 2.61
15	68.70 ± 5.43	29	86.00 ± 0.22
16	68.69 ± 1.91	30	78.32 ± 1.65
EFV	92.78 ± 0.04	EFV	92.78 ± 0.04

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Based on the percentage of inhibition of the chosen compounds (18, 24, and 27), we calculated their IC₅₀ values. The IC₅₀ values were 20.26 ± 5.14 µM, 23.10 ± 0.21 µM, and 2.72 ± 0.07 µM for compounds 18, 24 and 27 respectively. Based on these IC₅₀ values, compound 27 was considered the most potent, with an IC₅₀ value of 2.72 ± 6.30 µM, (Fig. 2c) compared to the all synthesized compounds.

Binding mode analysis of the potent compound as an HIV-1 RT inhibitor

The binding mode analysis of compound 27, a potent HIV-1 RT inhibitor, was carried out through docking experiments. The TNK 999 co-crystallized ligand was first docked into the Non-Nucleoside Inhibitor Binding Pocket (NNIBP) of 1RT2 for protocol validation, yielding an RMSD value of 0.46 Å, confirming the accuracy of the docking method. During docking, TNK 999 formed a hydrogen bond with the active binding pocket (NHpyrimidine—COLYS101 = 2.3 Å) (Fig. 3a), while the standard drug efavirenz exhibited a similar interaction (NHoxazinane—COLYS101 = 1.9 Å) (Fig. 3b) within the NNIBP.

Fig. 3 — a The ligand TNK 999 was redocked into the NNIBP of HIV-1 RT (1RT2) to analyze its binding mode. The ligand is shown as a stick model with interacting amino acids represented in a line format, whereas non-interacting protein regions were excluded from the visualization. b Illustrates the binding mode of efavirenz within the NNIBP of HIV-1 RT (1RT2). Efavirenz is shown as a stick model, whereas the interacting amino acid residues are depicted using a line model. We made the visualization more transparent by removing non-interacting regions of the protein. c The binding mode of the test compound (27) in the NNIBP of HIV-1 RT (PDB 1RT2) is shown with the ligand as a stick model and the interacting residues in a line model. Dotted spheres indicate hydrogen bonds between TYR 318 and LYS 103. For clarity, the remaining protein structures are omitted.

In the final docking experiment on the 1RT2 receptor, a comparison of compound 27 with efavirenz indicated that the carbonyl group (CO) of buta-2-one in compound 27 participated in hydrogen bond interactions with the NH of LYS 103 (COBUTA-2-ONE—NHLYS103 = 2.1 Å). Additionally, the nitrogen (N) of the triazole formed a hydrogen bond with the OH of TYR 318 (NTRIAZOLE—OHTYR318 = 2.9 Å) in the NNIBP of reverse transcriptase (Fig. 3c). Lysine 103 (Lys103) and tyrosine 318 (Tyr318) are well-established key residues of the non-nucleoside inhibitor binding pocket (NNIBP) of HIV-1 reverse transcriptase and play critical roles in NNRTI binding and resistance development^22,23.

Compound 27 achieved a docking score of – 10.3 kcal/mol, while the standard drug efavirenz showed a slightly higher docking score of -10.8 kcal/mol.

Molecular dynamics (M.D.)

Assessing protein flexibility is vital in docking studies, although it alone may not fully confirm binding accuracy. Desmond MD simulations were employed to ensure a robust understanding of the binding mechanism and stability of the interactions. These simulations were conducted on the 1RT2 protein to validate the ligand binding rigorously. By capturing the dynamic behavior of protein-ligand complexes, M.D. simulations significantly enhance the drug development process and contribute to advancing research on resistance mechanisms. The resulting data offer crucial insights into these compound’s binding stability and interaction dynamics.

Studies on the co-crystallized structure of the HIV-1 RT (1RT2)-27 complex

The study reported favourable RMSD values, reflecting the stability of the protein-ligand complex conformations. As shown in Fig. 4a, the protein exhibited stable behaviour between 50 and 300 ns, whereas ligand stability fluctuated between 100 and 300 ns. The overall RMSD values ranged from 0.3 to 0.9 Å for the protein and 1.5 to 4.8 Å for the ligand, which are within acceptable limits for stable ligand–protein interactions within acceptable RMSD limits. The RMSF analysis (Fig. 4b) revealed that most residues exhibited moderate fluctuations, indicating overall structural stability during the 300 ns simulation. Higher flexibility was observed in specific loop regions, while residues at the binding site showed comparatively lower fluctuations, suggesting stable ligand interactions. The increased mobility at the terminal regions is consistent with their inherent structural flexibility. During the simulation, the hydrogen bond interactions reached their peak, particularly between compound 27 and LYS-103, as illustrated in Fig. 4b. Figure 3c shows that this interaction was in agreement with protein-ligand docking studies (1RT2-27), which also found identical hydrogen bonding with LYS-103. The interactions shown in Fig. 4c have been simulated using molecular dynamics (M.D). The strength of the amino acid connections is indicated by the color intensity, with darker shades indicating more pronounced interactions.

Structural characterization using DFT and TD-DFT methods

Figure 5 shows the DFT-optimized crystal structure of 27, with the corresponding bond lengths and angles listed in Table 2. The structure was optimized using density functional theory (DFT) with the B3LYP/LanL2DZ basis set, yielding a stable configuration with an optimized energy of − 688,053.75 kcal/mol.

Fig. 5 — DFT/B3LYP/LanL2DZ optimized structure of 27.

Table 2.

Selected theoretical bond length and bond angles of 27.

Bond length (Å)	DFT	Bond length (Å)	DFT	Bond angles (˚)	DFT	Bond angles (˚)	DFT
O(26)-H(34)	0.942	C(8)-Cl(12)	1.420	H(33)-C(23)-C(22)	120.000	H(28)-C(14)-C(15)	108.800
C(23)-H(33)	1.100	C(8)-C(9)	1.719	H(33)-C(23)-C(18)	120.000	H(27)-C(14)-C(15)	108.800
C(22)-H(32)	1.100	C(7)-Cl(11)	1.420	H(32)-C(22)-C(23)	120.000	Cl(13)-C(9)-C(8)	118.800
C(22)-C(23)	1.420	C(7)-C(8)	1.719	H(32)-C(22)-C(21)	120.000	Cl(13)-C(9)-C(5)	118.800
C(21)-N(24)	1.456	C(6)-Cl(10)	1.420	N(24)-C(21)-C(22)	120.000	C(8)-C(9)-C(5)	120.000
C(21)-C(22)	1.420	C(6)-C(7)	1.420	N(24)-C(21)-C(20)	120.000	Cl(12)-C(8)-C(9)	118.800
C(20)-H(31)	1.100	C(5)-C(9)	1.420	C(22)-C(21)-C(20)	120.000	Cl(12)-C(8)-C(7)	118.800
C(20)-C(21)	1.420	C(4)-C(6)	1.420	H(31)-C(20)-C(21)	120.000	C(9)-C(8)-C(7)	120.000
C(19)-H(30)	1.100	C(4)-C(5)	1.470	H(31)-C(20)-C(19)	120.000	Cl(11)-C(7)-C(8)	118.800
C(19)-C(20)	1.420	N(3)-C(14)	1.364	H(30)-C(19)-C(20)	120.000	Cl(11)-C(7)-C(6)	118.800
C(18)-C(23)	1.420	N(3)-C(4)	1.328	H(30)-C(19)-C(18)	120.000	C(8)-C(7)-C(6)	120.000
C(18)-C(19)	1.420	N(2)-N(3)	1.358	C(23)-C(18)-C(19)	120.000	Cl(10)-C(6)-C(7)	118.800
N(17)-H(29)	1.012	N(1)-C(5)	1.333	C(23)-C(18)-N(17)	120.000	Cl(10)-C(6)-C(4)	118.800
N(17)-C(18)	1.345	N(1)-N(2)	1.420	C(19)-C(18)-N(17)	120.000	C(7)-C(6)-C(4)	120.000
C(15)-N(17)	1.369			H(29)-N(17)-C(18)	110.000	C(9)-C(5)-C(4)	120.000
C(15)-O(16)	1.208			H(29)-N(17)-C(15)	117.400	C(9)-C(5)-N(1)	120.000
C(14)-H(28)	1.113			N(17)-C(15)-O(16)	124.932	C(4)-C(5)-N(1)	120.000
C(14)-H(27)	1.113			N(17)-C(15)-C(14)	116.438	C(6)-C(4)-C(5)	120.000
C(14)-C(15)	1.509			O(16)-C(15)-C(14)	118.579	C(6)-C(4)-N(3)	120.000
C(9)-Cl(13)	1.719			H(28)-C(14)-H(27)	107.861	C(5)-C(4)-N(3)	120.000

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Frontier molecular orbital and MEP studies

The HOMO and LUMO orbitals were determined using the DFT method with the B3LYP/LanL2DZ basis set, as shown in Fig. 6. For compound 27, the HOMO wavefunctions are concentrated on the nitrobenzene group, with an energy of − 7.299 eV, while the LUMO wavefunctions are similarly localized, with an energy of − 3.845 eV. The energy gap (ΔE) between these orbitals is 3.454 eV, indicating the molecule’s reactivity. Suggesting moderate reactivity based on frontier orbital analysis (Table 3).

Table 3.

The global reactivity descriptors of compound 27 were calculated using Koopmans’ theorem equations²⁴.

E_HOMO	− 7.299 eV
E_LUMO	− 3.845 eV
Energy gap (∆E)	3.454 eV
Ionization Potential(I)	7.299 eV
Electron affinity (A)	3.845 eV
Hardness, (η = E_LUMO-E_HOMO/2)	1.727
Softness, (S = 1/ 2η)	0.289
Electrophilicity (ω = µ²/2η)	9.061
Chemical potential, [− µ = − (E_{LUMO +} E_HOMO)/2]	− 5.572
Electronegativity, [χ = − (E_{LUMO +} E_HOMO)/2]	5.572

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We generated a molecular electrostatic potential (MEP) map by evaluating the total surface density of 27, as illustrated in Fig. 7. The MEP map highlights the reactive regions of the molecule, with the most attractive potential area appearing as a reddish-orange color. Figure 7 shows that the oxygen atoms (O25 and O26) of the 4-nitrobenzene group are in the reddish regions, indicating their role in electrophilic reactions. In contrast, the blue regions, typically associated with carbon and hydrogen atoms, suggest areas likely to be involved in nucleophilic reactions. The electrostatic potential followed the trend red < orange < yellow < green < blue.

The molecular electrostatic potential map was generated to visualize the charge distribution of compound 27. Electron-rich regions localized around heteroatoms of the benzotriazole moiety correspond to potential interaction sites for hydrogen bonding and electrostatic interactions with NNIBP residues. This qualitative analysis complements the docking results by rationalizing the observed binding orientation.

In vivo acute oral toxicity

Our activity study indicated that the synthesized compound 27 had an inhibition percentage comparable to that of the standard drug. However, the standard drug had a significantly lower IC₅₀ value, implying that compound 27 required a higher concentration for equivalent inhibition. Compound 27 was tested for acute oral toxicity under OECD guideline 420 at doses up to 2000 mg/kg, with no observed fatalities, classifying it as non-toxic.

Experimental section

We sourced liquid and solid chemicals from reputable vendors, including Sigma-Aldrich, Merck, and Rankem. We measured melting/boiling points to ensure reagent quality and performed thin-layer chromatography (TLC). We continuously monitored TLC spots under U.V. light. Melting points were measured using an Opti-melting point device. For the 1 H NMR and 13 C NMR spectra (400 MHz), we utilized a JEOL spectrophotometer, referencing chemical shifts to tetramethyl silane and using deuterated DMSO as the solvent. An LC-MS system from Thermo Scientific was used to acquire mass spectra, while an Elemental Vario-EL instrument was employed for elemental analysis.

General procedure for the synthesis of tetrabromobenzotriazole (1)¹⁸

We synthesized tetrabromobenzotriazole by reacting 11.9 g of benzotriazole (0.10 mol) with 23 ml of bromine (0.45 mol) and 150 ml of nitric acid under reflux for 48 h. The precipitate was filtered and recrystallized with acetic acid to yield creamy crystals. [Yield: 75%, m.p: 264–266 °C]

General procedure for the synthesis of tetrachlorobenzotriazole (4)¹⁹

We synthesized tetrachlorobenzotriazole by reacting 4.76 g benzotriazole (0.040 mol) with 525 ml of hydrochloric acid and 175 ml of nitric acid under reflux for 4 h. The precipitate was then filtered and recrystallized from nitromethane to yield white crystals. [Yield: 80%, m.p: 259–261 °C]

Synthesis of acetamide derivatives 2a-n^25–28

We added a solution containing 0.033 moles of the respective aromatic amine in 15 ml of glacial acetic acid dropwise to 0.037 moles of 2-chloroacetyl chloride in an ice bath. After stirring the mixture in an ice bath for 30 min and then at room temperature for another 2 h, we poured it into a saturated sodium acetate solution. The resulting precipitate was filtered, washed with cold water, and dried. Recrystallization from dehydrated ethanol produced white crystals of 2-chloro-N-(substituted phenyl)acetamide. Derivatives of acetamides 2a-n with their respective yields and melting points, referenced to established data, are shown below.

2a) 2-chloro-N-phenylacetamide [Yield: 80%, m.p.: 138–140 °C], 2b) 2-chloro-N-(4-hydroxyphenyl) acetamide [Yield: 65%, m.p.: 124–126 °C], 2c) 2-chloro-N-(4-ethylphenyl) acetamide [Yield: 65%, m.p.: 90–92 °C], 2d) 2-chloro-N-(2,4-dimethylphenyl) acetamide [Yield: 74%, m.p.: 144–146 °C], 2e) 2-chloro-N-(3,4-dimethylphenyl) acetamide [Yield: 78%, m.p.: 147–149 °C], 2f) 2-chloro-N-(2-chlorophenyl)acetamide [Yield: 76%, m.p.: 98–100 °], 2 g) 2-chloro-N-(3-chlorophenyl)acetamide [Yield: 75%, m.p.: 101–103 °C], 2 h) 2-chloro-N-(4-chlorophenyl) acetamide [Yield: 78%, m.p.: 104–106 °C], 2i) 2-chloro-N-(4-isopropylphenyl)acetamide [Yield: 79%, m.p.: 174–176 °C], 2j) 2-chloro-N-(3-nitrophenyl)acetamide [Yield: 79%, m.p.: 174–176 °C], 2k) 2-chloro-N-(4-nitrophenyl) acetamide [Yield: 78%, m.p.: 180–182 °C], 2 L) 2-chloro-N-(3-chloro-2-methylphenyl) acetamide [Yield: 87%, m.p.: 156–158 °C], 2 m) 2-chloro-N-(3,4-dichlorophenyl) acetamide [Yield: 68%, m.p.: 92–93 °C], 2n) 2-chloro-N-(4-bromophenyl) acetamide [Yield: 71%, m.p:75–77 °C].

Synthesis of N-(aryl)-2-(perhalo-1H-benzo[d] [1,2,3] triazol-1-yl)-acetamides (3–30)

Initially tetrabromobenzotriazole (1a) (0.434 gm, 1 mmol) and tetrachlorobenzotriazole (0.256 gm, 1 mmol) were stirred with respective acetamide derivatives 2a-n (1 mmol) in DMF (20 ml), and N, N-diisopropylethylamine (0.26 gm, 1 mmol) was used as a catalyst. Reflux was carried out for 15 h, followed by standing at room temperature to yield a cream-colored compound. Recrystallization with ethanol produced a white powder.

N-(phenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (3)

White Solid; Reaction time (hour):15; Yield (%):75; m.p. (°C): 222–224; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.62 (s, 1H; NH-CO-CH₂), 7.55–7.04 (m, 5 H; Ar-H), 5.79 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 163.5, 143.3, 138.9, 138.7, 129.5, 126.5, 124.6, 124.4, 119.9, 60.2; For C₁₄H₈Br₄N₄O LC-MS: m/z = 563.74 [M-1H]⁺; Elemental Analysis, calc. C, 29.61; H, 1.42; Br, 56.28; N, 9.87; O, 2.82; Found: C, 29.55; H, 1.40; Br, 56.22; N, 9.83; O, 2.80.

N-(2-hydroxyphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (4)

White Solid; Reaction time (hour):15; Yield (%):75; m.p. (°C):221–223; ¹H-NMR (400 MHz, DMSO-D6) ppm 9.84 (d, 2 H; NH-CO-CH₂; OH) 7.97–6.69 (m, 4 H; Ar-H), 5.88 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 162.8, 148.5, 143.3, 126.5, 126.1, 125.9, 125.5, 122.6, 119.4, 115.8, 114.0, 60.1; For C₁₄H₈Br₄N₄O₂, LC-MS: m/z = 583.86 [M-1H]⁺; Elemental Analysis, calc. C, 28.80; H, 1.38; Br, 54.74; N, 9.60; O, 5.48; Found: C, 28.75; H, 1.32; Br, 54.70; N, 9.55; O, 5.43.

N-(4-ethylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (5)

White Solid; Reaction time (hour):15; Yield (%):80; m.p. (°C): 229–231; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.52 (s, 1H; NH-CO-CH₂), 7.28 (m, 4 H; Ar-H), 5.75 (s, 2 H; NH-CO-CH₂), 2.46 (dd, 2 H; CH₂-CH₃), 1.10 (t, 3 H; CH₂-CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm 163.2, 143.3, 140.1, 136.2, 129.2, 128.7, 126.5, 120.0, 113.9, 60.2, 28.1, 16.2; For C₁₆H₁₂Br₄N₄O, LC-MS: m/z = 595.92 [M-1H]⁺; Elemental Analysis, calc. C, 32.25; H, 2.03; Br, 53.63; N, 9.40; O, 2.68; Found: C, 32.28; H, 2.01; Br, 53.60; N, 9.44; O, 2.62.

N-(2,4-dimethylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (6)

White Solid; Reaction time (hour):15; Yield (%):78; m.p. (°C): 218–220; ¹H-NMR (400 MHz, DMSO-D6) ppm 9.82 (s, 1H; NH-CO-CH₂), 7.26–6.93 (m, 3 H; Ar-H), 5.81 (s, 2 H; NH-CO-CH₂), 2.20 (s, 3 H; CH₃), 2.18 (s, 3 H; CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm 163.6, 143.4, 135.4, 133.2, 132.2, 131.5, 127.1, 126.5, 125.6, 125.4, 114.3, 59.9, 21.0, 18.0; For C₁₆H₁₂Br₄N₄O, LC-MS: m/z = 595.92 [M-1H]⁺; Elemental Analysis, calc. C, 32.25; H, 2.03; Br, 53.63; N, 9.40; O, 2.68; Found: C, 32.29; H, 2.01; Br, 53.68; N, 9.35; O, 2.72.

N-(3,4-dimethylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (7)

White Solid; Reaction time (hour):15; Yield (%):75; m.p. (°C):223–225; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.45 (s, 1H; NH-CO-CH₂), 7.32–7.03 (m, 3 H; Ar-H), 5.75 (s, 2 H; NH-CO-CH₂), 2.14 (s, 3 H; CH₃), 2.13 (s, 3 H; CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm 163.1, 143.3, 137.1, 136.4, 132.4, 130.2, 126.6, 121.1, 117.4, 114.2, 60.2, 20.1; For C₁₆H₁₂Br₄N₄O, LC-MS: m/z = 595.92 [M-1H]⁺; Elemental Analysis, calc. C, 32.25; H, 2.03; Br, 53.63; N, 9.40; O, 2.68; Found: C, 32.28; H, 2.00; Br, 53.65; N, 9.42; O, 2.72.

N-(2-chlorophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (8)

White Solid; Reaction time (hour):15; Yield (%):75; m.p. (°C):221–223; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.35 (s, 1H; NH-CO-CH₂), 7.85–7.34 (m, 4 H; Ar-H), 6.05 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.9, 134.5, 134.3, 130.6, 129.3, 127.5, 127.1, 126.8, 126.2, 124.3, 114.2, 60.2; For C₁₄H₇Br₄ClN₄O, LC-MS: m/z = 602.30 [M-1H]⁺; Elemental Analysis, calc. C, 27.92; H, 1.17; Br, 53.07; Cl, 5.89; N, 9.30; O, 2.66; Found: C, 27.90; H, 1.21; Br, 53.05; Cl, 5.85; N, 9.33; O, 2.62.

N-(3-chlorophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (9)

White Solid; Reaction time (hour):15; Yield (%):82; m.p. (°C):232–234; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.83 (s, 1H; NH-CO-CH₂), 7.91–7.12 (m, 4 H; Ar-H), 5.81 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.5, 143.5, 140.2, 140.0, 133.4, 131.2, 126.6, 124.4, 124.1, 119.4, 118.3, 59.8; LC-MS: m/z = 602.30 [M-1H]⁺; Elemental Analysis, calc. C, 27.92; H, 1.17; Br, 53.07; Cl, 5.89; N, 9.30; O, 2.66; Found: C, 27.89; H, 1.19; Br, 53.09; Cl, 5.83; N, 9.25; O, 2.70.

N-(4-chlorophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (10)

White Solid; Reaction time (hour):15; Yield (%):73; m.p. (°C):231–233; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.80 (s, 1H; NH-CO-CH₂), 7.57–7.32 (m, 4 H; Ar-H), 5.80 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 165.4, 137.8, 133.4, 129.4, 128.2, 126.6, 124.3, 121.8, 121.4, 60.2; For C₁₄H₇Br₄ClN₄O, LC-MS: m/z = 602.30 [M-1H]⁺; Elemental Analysis, calc. C, 27.92; H, 1.17; Br, 53.07; Cl, 5.89; N, 9.30; O, 2.66; Found: C, 27.96; H, 1.13; Br, 53.05; Cl, 5.90; N, 9.28; O, 2.68.

N-(4-isopropylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (11)

White Solid; Reaction time (hour):15; Yield (%):68; m.p. (°C):219–221; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.53 (s, 1H; NH-CO-CH₂), 7.46–7.14 (m, 4 H; Ar-H), 5.76 (s, 2 H; NH-CO-CH₂), 2.84–2.77 (m, 1H; CH₃⁻CH-CH₃), 1.13 (d, 6 H; CH₃⁻CH-CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm 163.1, 144.6, 143.3, 136.4, 133.6, 127.1, 126.2, 119.8, 114.2, 59.7, 31.0, 24.3; For C₁₇H₁₄Br₄N₄O, LC-MS: m/z = 609.94 [M-1H]⁺; Elemental Analysis, calc. C, 33.48; H, 2.31; Br, 52.40; N, 9.19; O, 2.62; Found: C, 33.45; H, 2.28; Br, 52.33; N, 9.15; O, 2.65.

N-(3-nitrophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (12)

White Solid; Reaction time (hour):15; Yield (%):72; m.p. (°C):222–224; ¹H-NMR (400 MHz, DMSO-D6) ppm 11.12 (s, H; NH-CO-CH₂), 8.52–7.58 (m, 4 H; Ar-H), 5.85 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.3, 148.5, 143.5, 140.0, 139.7, 131.0, 126.6, 125.9, 118.8, 114.2, 113.8, 60.1; For C₁₄H₇Br₄N₅O₃, LC-MS: m/z = 612.86 [M-1H]⁺; Elemental Analysis, calc. C, 27.44; H, 1.15; Br, 52.15; N, 11.43; O, 7.83; Found: C, 27.48; H, 1.14; Br, 52.13; N, 11.45; O, 7.81.

N-(4-nitrophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (13)

White Solid; Reaction time (hour):15; Yield (%):76; m.p. (°C):233–235,¹H-NMR (400 MHz, DMSO-D6) ppm 11.23 (s, 1H; NH-CO-CH₂), 8.25–7.77 (m, 4 H; Ar-H), 5.91 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.3, 145.2, 144.6, 143.4, 127.1, 125.6, 120.2, 119.8, 113.7, 59.9; For C₁₄H₇Br₄N₅O₃, LC-MS: m/z = 612.86 [M-1H]⁺; Elemental Analysis, calc. C, 27.44; H, 1.15; Br, 52.15; N, 11.43; O, 7.83; Found: C, 27.42; H, 1.13; Br, 52.17; N, 11.45; O, 7.82.

N-(3-chloro-2-methylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (14)

White Solid; Reaction time (hour):15; Yield (%):71; m.p. (°C):231–233; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.17 (s, 1H; NH-CO-CH₂), 7.35–7.18 (m, 3 H; Ar-H), 5.85 (s, 2 H; NH-CO-CH₂), 2.25 (s, 3 H; CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.3, 137.3, 134.4, 131.0, 127.5, 127.1, 126.7, 126.5, 125.0, 124.8, 114.2, 59.7, 15.5; For C₁₅H₉Br₄ClN₄O, LC-MS: m/z = 616.33 [M + 1H]⁺; Elemental Analysis, calc. C, 29.23; H, 1.47; Br, 51.86; Cl, 5.75; N, 9.09; O, 2.60; Found: C, 29.25; H, 1.42; Br, 51.89; Cl, 5.79; N, 9.05; O, 2.65.

N-(3,4-dichlorophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (15)

White Solid; Reaction time (hour):15; Yield (%):77; m.p. (°C):222–224,¹H-NMR (400 MHz, DMSO-D6) ppm 10.92 (s, 1H; NH-CO-CH₂), 7.91–7.43 (m, 3 H; Ar-H), 5.82 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.2, 143.3, 138.9, 138.7, 131.7, 131.4, 126.6, 126.1, 125.1, 121.1, 120.0, 60.1; For C₁₄H₆BrCl₄N₃O, LC-MS: m/z = 634.90 [M-1H]⁺; Elemental Analysis, calc. C, 26.41; H, 0.95; Br, 50.20; Cl, 11.13; N, 8.80; O, 2.51; Found: C, 26.41; H, 0.95; Br, 50.20; Cl, 11.13; N, 8.80; O, 2.51.

N-(4-bromophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (16)

White Solid; Reaction time (hour):15; Yield (%):68; m.p. (°C):229–231; ¹H- NMR (400 MHz, DMSO-D6) ppm 10.75 (s, 1H; NH-CO-CH₂), 7.50 (s, 4 H; Ar-H), 5.79 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 163.6, 143.5, 137.9, 135.8, 132.3, 131.9, 126.9, 122.4, 121.8, 60.2; For C₁₄H₇Br₅N₄O, LC-MS: m/z = 646.76 [M-1H]⁺; Elemental Analysis, calc. C, 26.00; H, 1.09; Br, 61.77; N, 8.66; O, 2.47; Found: C, 26.08; H, 1.04; Br, 61.77; N, 8.65; O, 2.49.

N-(phenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)-acetamide (17)

White Solid; Reaction time (hour):15; Yield (%):76; m.p.:202–204 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.59 (s, 1H; NH-CO-CH₂), 7.55–7.05 (m, 5 H; Ar-H), 5.83 (s, 2 H; NH-CO-CH₂);¹³C-NMR (101 MHz, DMSO-D6) ppm 165.0, 141.5, 138.7, 130.2, 129.2, 124.6, 124.3, 121.7, 120.2, 119.7, 60.3; For C₁₄H₈Cl₄N₄O LC-MS: m/z = 389.12 [M-1H]⁺; Elemental Analysis: C, 43.11; H, 2.07; Cl, 36.35; N, 14.36; O, 4.10; Found: C, 43.13; H, 2.09; Cl, 36.31; N, 14.38; O, 4.09.

N-(2-hydroxyphenyl)-2-(tetrachloro-1 H-benzo[d] [1,2,3] triazol-1-yl)acetamide(18) [202431017120

White Solid; Reaction time (hour):15; Yield (%):78; m.p.:208–210 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 9.95 (d, 2 H; NH-CO-CH₂; O.H.), 7.73–6.69 (m, 4 H; Ar-H), 5.93 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 165.2, 148.6, 141.5, 131.2, 125.9, 125.5, 124.2, 122.6, 121.5, 119.4, 118.3, 115.8, 60.2; For C₁₄H₈Cl₄N₄O LC-MS: m/z = 405.10 [M-1H]+; Elemental Analysis: C, 41.41; H, 1.99; Cl, 34.92; N, 13.80; O, 7.88; Found: C, 41.40; H, 1.98; Cl, 34.93; N, 13.79; O, 7.87.

N-(4-ethylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (19)

White Solid; Reaction time (hour):15; Yield (%):72; m.p.:201–203 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.55 (s, 1H; NH-CO-CH₂), 7.42–7.11 (m, 4 H; Ar-H), 5.84 (s, 2 H; NH-CO-CH₂), 2.52 (dd, 2 H; CH₂-CH₃), 1.11 (t, 3 H; CH₂-CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.5, 139.9, 136.5, 131.2, 129.8, 128.6, 127.8, 121.2, 120.2, 119.8, 53.0, 28.0, 15.9; For C₁₆H₁₂Cl₄N₄O LC-MS: m/z = 417.07 [M-1H]⁺; Elemental Analysis: C, 45.96; H, 2.89; Cl, 33.92; N, 13.40; O, 3.83; Found: C, 45.97; H, 2.88; Cl, 33.89; N, 13.43; O, 3.85.

N-(2,4-dimethylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (20)

White Solid; Reaction time (hour):15; Yield (%):75; m.p.:209–211 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 9.84 (s, 1H; NH-CO-CH₂), 7.25–6.93 (m, 3 H; Ar-H), 5.85 (s, 2 H; NH-CO-CH₂), 2.18 (s, 3 H; CH₃), 2.04 (s, 3 H; CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm 165.1, 141.5, 135.3, 133.2, 132.4, 132.1, 131.5, 129.9, 127.1, 125.5, 125.3, 121.5, 60.1, 20.6, 18.3; For C₁₆H₁₂Cl₄N₄O LC-MS: m/z = 417.15 [M-1H]⁺; Elemental Analysis: C, 45.96; H, 2.89; Cl, 33.92; N, 13.40; O, 3.83; Found: C, 45.98; H, 2.88; Cl, 33.90; N, 13.41; O, 3.84.

N-(3,4-dimethylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (21)

White Solid; Reaction time (hour):15; Yield (%): 70; m.p.:215–217 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.46 (s, 1H; NH-CO-CH₂), 7.32–7.03 (m, 3 H; Ar-H), 5.79 (s, 2 H; NH-CO-CH₂), 2.13 (s, 3 H; CH₃), 2.04 (s, 3 H; CH₃);¹³C-NMR (101 MHz, DMSO-D6) ppm 163.2, 141.4, 137.1, 136.5, 136.4, 132.4, 130.3, 129.9, 121.5, 121.0, 117.3, 60.6, 20.0; For C₁₆H₁₂Cl₄N₄O LC-MS: m/z = 417.12 [M-1H]⁺; Elemental Analysis, cald. C, 45.96; H, 2.89; Cl, 33.92; N, 13.40; O, 3.83; Found: C, 45.97; H, 2.87; Cl, 33.91; N, 13.42; O, 3.84.

N-(2-chlorophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (22)

White Solid; Reaction time (hour):15; Yield (%): 73; m.p.: 222–224 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.24 (s, 1H: NH-CO-CH₂), 7.71–7.18 (m, 4 H; Ar-H), 5.95 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.1, 141.8, 134.5, 130.3, 129.9, 128.1, 127.5, 127.0, 126.5, 121.5, 60.0; For C₁₄H₇Cl₅N₄O LC-MS: m/z = 423.06 [M-1H]⁺; Elemental Analysis, cald. C, 39.61; H, 1.66; Cl, 41.76; N, 13.20; O, 3.77; Found: C, 39.63; H, 1.65; Cl, 41.78; N, 13.22; O, 3.78.

N-(3-chlorophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (23)

White Solid; Reaction time (hour):15; Yield (%):75; m.p.: 215–217 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 9.93 (s, 1H; NH-CO-CH₂), 7.72–6.89 (m, 4 H; Ar-H), 5.92 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 165.1, 140.8, 139.3, 133.7, 131.3, 130.1, 125.9, 124.3, 122.8, 119.3, 118.3, 115.8, 53.9; For C₁₄H₇Cl₅N₄O LC-MS: m/z = 423.10 [M-1H]⁺; Elemental Analysis, cald. C, 39.61; H, 1.66; Cl, 41.76; N, 13.20; O, 3.77; Found: C, 39.62; H, 1.64; Cl, 41.77; N, 13.21; O, 3.79.

N-(4-chlorophenyl)-2-(tetrachloro-1 H-benzo[d] [1,2,3] triazol-1-yl) acetamide (24) [202431017120

White Solid; Reaction time (hour):15; Yield (%):72; m.p.:219–221 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.79 (s, 1H; NH-CO-CH₂), 7.58–7.34 (m, 4 H; Ar-H), 5.84 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 163.6, 141.5, 137.7, 137.6, 130.1, 129.4, 128.2, 128.0, 127.9, 121.4, 60.3; For C₁₄H₇Cl₅N₄O LC-MS: m/z = 423.09 [M-1H]+; Elemental Analysis: C, 39.61; H, 1.66; Cl, 41.76; N, 13.20; O, 3.77; Found: C, 39.60; H, 1.65; Cl, 41.75; N, 13.21; O, 3.78.

N-(4-isopropylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (25)

White Solid; Reaction time (hour):15; Yield (%):73; m.p.:209–211 °C ¹H-NMR (400 MHz, DMSO-D6) ppm 10.55 (s, 1H; NH-CO-CH₂), 7.46–7.16 (m, 4 H; Ar-H), 5.80 (s, 2 H; NH-CO-CH₂), 2.85–2.78 (m, 1H; CH₃⁻CH-CH₃), 1.13 (d, 6 H; CH₃⁻CH-CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm: 163.3, 141.5, 136.8, 136.5, 131.7, 129.9, 128.2, 127.2, 121.4, 119.9, 60.2, 33.4, 24.4 ; For C₁₇H₁₄Cl₄N₄O LC-MS: m/z = 431.14 [M-1H]⁺; Elemental Analysis, cald. C, 47.25; H, 3.27; Cl, 32.81; N, 12.97; O, 3.70 Found: C, 47.27; H, 3.26; Cl, 32.80; N, 12.98; O, 3.73.

N-(3-nitrophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (26)

White Solid; Reaction time (hour):15; Yield (%):75; m.p.:214–216 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 11.15 (s, 1H; NH-CO-CH₂), 8.52–7.59 (m, 4 H; Ar-H), 5.94 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 165.7, 148.6, 148.3, 142.6, 139.9, 131.3, 130.9, 126.0, 121.5, 119.5, 119.0, 114.0, 52.8; For C₁₄H₇Cl₄N₅O₃ EI-MS: m/z = 434.06 [M-1H]⁺; Elemental Analysis, cald. C, 38.65; H, 1.62; Cl, 32.59; N, 16.10; O, 11.03; Found: C, 38.67; H, 1.61; Cl, 32.56; N, 16.12; O, 11.05.

N-(4-nitrophenyl)-2-(tetrachloro-1 H-benzo[d] [1,2,3] triazol-1-yl) acetamide (27) [202431017120

White Solid; Reaction time (hour):15; Yield (%):75; m.p.:222–224 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 11.24 (s, 1H; NH-CO-CH₂), 8.22–7.76 (m, 4 H; Ar-H), 5.96 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 165.7, 142.6, 139.9, 131.0, 127.9, 125.8, 121.5, 120.4, 119.5, 119.0, 53.2; For C₁₄H₇Cl₄N₅O₃, LC-MS: m/z = 434.11 [M-1H]+; Elemental Analysis: C, 38.65; H, 1.62; Cl, 32.59; N, 16.10; O, 11.03 ; Found: C, 38.67; H, 1.61; Cl, 32.57; N, 16.08; O, 11.05.

N-(3-chloro-2-methylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (28)

White Solid; Reaction time (hour):15; Yield (%):72; m.p.:219–221 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.18 (s, 1H; NH-CO-CH₂), 7.35–7.18 (m, 3 H; Ar-H), 5.90 (s, 2 H; 2 H; NH-CO-CH₂), 2.04 (s, 3 H; CH₃); ¹³C-NMR (101 MHz, DMSO-D6) ppm 164.3, 141.5, 137.4, 137.1, 134.4, 131.0, 129.9, 127.5, 127.1, 124.9, 121.5, 115.8, 59.7, 15.2; For C₁₅H₉Cl₅N₄O LC-MS: m/z = 437.07 [M-1H]⁺+; Elemental Analysis, cald. C C, 41.09; H, 2.07; Cl, 40.42; N, 12.78; O, 3.65; Found: C, 41.08; H, 2.05; Cl, 40.44; N, 12.77; O, 3.67.

N-(3,4-dichlorophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (29)

White Solid; Reaction time (hour):15; Yield (%):71; m.p.:220–222 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.95 (s, 1H; NH-CO-CH₂), 7.91–7.44 (m, 3 H; Ar-H), 5.86 (s, 2 H; NH-CO-CH₂);¹³C-NMR (101 MHz, DMSO-D6) ppm 163.8, 141.5, 138.8, 138.7, 131.7, 131.4, 130.0, 128.4, 126.1, 121.5, 121.1, 119.9, 60.2; For C₁₄H₆Cl₆N₄O LC-MS: m/z = 457.03 [M-1H]⁺; Elemental Analysis, cald. C, 36.64; H, 1.32; Cl, 46.35; N, 12.21; O, 3.49; Found: C, 36.66; H, 1.31; Cl, 46.33; N, 12.23; O, 3.47.

N-(4-bromophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (30)

White Solid; Reaction time (hour):15; Yield (%):73; m.p.:213–215 °C; ¹H-NMR (400 MHz, DMSO-D6) ppm 10.77 (s, 1H; NH-CO-CH₂), 7.51 (s, 4 H; Ar-H), 5.83 (s, 2 H; NH-CO-CH₂); ¹³C-NMR (101 MHz, DMSO-D6) ppm 163.9, 141.4, 138.2, 132.3, 130.0, 128.5, 126.8, 121.8, 121.3, 119.2, 116.2, 59.9; For C₁₄H₇BrCl₄N₄O LC-MS: m/z = 467.91 [M-1H]⁺; Elemental Analysis, cald. C C, 35.86; H, 1.50; Br, 17.04; Cl, 30.24; N, 11.95; O, 3.41; Found: C, 35.85; H, 1.51; Br, 17.03; Cl, 30.26; N, 11.94; O, 3.44.

Anti-HIV-1 RT activity

We performed the HIV-1 RT assay as per the protocol of Seetaha et al. (2016) with minor modifications²⁹. The p66 and p51 plasmids were transformed into E. coli BL21(DE3) for protein expression. After overnight culture in LB medium with ampicillin, 1% inoculum was added to 250 ml of LB with ampicillin and cultured at 37 °C until the OD600 reached 0.8. The cultures were then induced with 0.5 mM IPTG at 16 °C for 18 h. The cells were harvested by centrifugation, and protein expression was confirmed using 12% SDS-PAGE.

The expressed p66 and p51 cells were combined and suspended in 50 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, 0.5% Triton X-100, 1 mM PMSF). The cells were lysed by sonication, and debris was removed by centrifugation at 14,000 rpm for 45 min at 4 °C. The supernatant was loaded onto a nickel sulfate (Ni²⁺) column and washed with buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol), followed by buffer A with 10 mM imidazole. .T.R.T. proteins were eluted with buffer B (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole, 5% glycerol). The eluted fraction was dialyzed in a buffer (50 mM Tris-HCl pH 6.5, 50 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 5% glycerol, 5 mM MgCl2), then purified via ion-exchange on a Resource S column using gradient elution. The .T.R.T. protein was further purified by size-exclusion chromatography on a Superdex-200 column using an FPLC system. 12% SDS-PAGE verified the purity, and the protein was exchanged into storage buffer (50 mM Tris-HCl pH 7.6, 2 mM DTT, 20% glycerol) and stored at -80 °C until use.

After expressing and purifying HIV-1 RT, all the selected test compounds were dissolved in DMSO to prepare 20 mM stock solutions. These were then diluted with 10 mM Tris-HCl (pH 7.5) containing 50% DMSO to obtain 10 mM working solutions. For the inhibition assay, 2 µL of each inhibitor and 3 µL of 400 nM recombinant HIV-1 RT were gently mixed on ice. The reaction was initiated by adding 10 µL of the primer/template and incubating the mixture at room temperature for 30 min. Following incubation, we added 5 µL of 0.2 M EDTA to halt the polymerization reaction. We compared the relative inhibitory effect on HIV-1RT activity by calculating the percent inhibition using the following equation;

where RT_Control = fluorescence without a sample (normal R.T. reaction or positive control); RT_Blank = fluorescence without enzyme activity (negative control); RT_Sample = fluorescence with sample.

We used the EnzChek^® reverse transcriptase assay kit (Molecular Probes, USA) was used to determine the IC₅₀. For the assay, 2 µL of each test compound, serially diluted two-fold, was mixed with 2 µL of 30 ng/µL of purified HIV-1 RT. Template/primer polymerization buffer (4 µL) was added to each well, and the mixture was incubated at 37 °C for 10 min. The reactions were stopped by adding 5 µL 200 mM EDTA and immediately incubating on ice for 30 min. The activity was measured using the PicoGreen fluorometric method. The experiment was conducted in triplicate, and the results were analyzed using GraphPad Prism4 version 148 with a nonlinear regression model.

Molecular modeling

We utilized AutoDock 1.5.6^30–34 to prepare proteins and dock the test compounds into the NNIBP of 1RT2³⁵ to evaluate the potential of these molecular targets as anti-HIV agents. The crystal structure of HIV-1 reverse transcriptase complexed with TNK 999 (PDB ID: 1RT2) was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb). The high-resolution crystal structure of 1RT2 is ideal for molecular docking, as it shows HIV-1 reverse transcriptase (R.T.) bound to the non-nucleoside .T.R.T. inhibitor (NNRTI) nevirapine, providing a favorable pre-existing conformation of the RT-binding pocket. This structure not only accelerates drug discovery by enabling detailed analysis of the binding interactions of nevirapine with .T.R.T. but also aids in developing more potent NNRTIs. The insights gained from the NNRTI-RT interactions enhance our understanding of anti-HIV drug mechanisms and support the validation of docking studies. Therefore, selecting a protein structure such as PDB ID 1RT2 is essential for studying the interaction between the test compounds and the target enzyme in anti-HIV-1 RT research³⁶.

For the docking procedure, we retained chain A of 1RT2, removed the water molecules and chain B, and added hydrogen atoms to optimize the structure. The co-crystallized ligand TNK 999 was extracted for validation purposes. The re-docking procedure yielded an RMSD value of 0.46 Å between the docked and crystallographic ligand conformations, indicating good agreement and validating the accuracy of the docking protocol. Pymol³⁷ was used for visualization of docking poses and analysis of ligand–protein interactions. The synthesized compounds (3–30) were optimized and converted to the PDB format using the PRODRG³⁸ web server.

Molecular dynamics

Molecular dynamics simulations were carried out on the HIV-1 reverse transcriptase–compound 27 complex obtained from molecular docking. The protein structure was prepared by removing the co-crystallized ligand and solvent molecules prior to docking, and the best-scoring docked pose of compound 27 was used as the starting structure for MD simulations with molecule 27 using Desmond 3.8 from the Schrödinger package³⁹. The simulation was conducted for 300 ns by applying the OPLS-AA force field and following Maestro 2021-4 MD protocols. We recorded trajectories at 1000-frame intervals spaced 300 ps apart. The simulation used an NPT ensemble (isothermal–isobaric) with a temperature of 300 K and a pressure of 1.01325 bar. The model was relaxed before the simulation and allowed to proceed as planned^40–43.

DFT studies

The structure of synthesized compound 27 was refined using density functional theory at the B3LYP/LanL2DZ level using the Gaussian 09 program⁴⁴. Optimized structures. chk file was then used in Gauss View 6⁴⁵ to generate illustrations of the highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), and molecular electrostatic potential (MEP).

In vivo oral toxicity⁴⁶

Healthy, non-pregnant and non-lactating female Swiss albino mice were used for the study. All animals were obtained from the Animal House Facility of BIT Mesra, Ranchi, and housed under controlled laboratory conditions, including a temperature of 22 ± 3 °C, relative humidity below 30%, and a 12-hour light–dark cycle. Standard laboratory diet and water were supplied ad libitum. Each mouse was individually marked for identification and allowed to acclimatize for at least seven days before experimentation.

The acute oral toxicity evaluation was performed following the OECD Guideline 420 (Fixed Dose Procedure). Prior to dosing, the animals were fasted for approximately four hours. A single oral dose of 2000 mg/kg of Compound 27 was administered via gavage to a group of three mice. A brief fasting period was maintained for two hours after dosing in accordance with standard laboratory practice.

Animals were observed at 1, 2, 4, 8, 12, and 24 h post-administration for any immediate signs of toxicity, including alterations in skin and fur, behavioural changes, salivation, aggression, responsiveness to sound, pain reaction, and general morbidity. Thereafter, the animals were monitored daily for 14 days to detect any delayed toxic manifestations or mortality.

All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC), BIT Mesra, under approval number 1972/PH/BIT/27/23/IAEC, dated 21.01.2023, and were conducted in compliance with CPCSEA guidelines.

The primary outcome measures included

Clinical signs of acute toxicity, evaluated through behavioural and physiological observations.
Delayed toxicity, assessed through daily monitoring for 14 days.
Mortality, recorded throughout the study period.
General well-being, assessed by activity levels, grooming behaviour, posture, and appearance.

Throughout the 14-day observation period, no mortality or toxic clinical signs were detected. All mice remained active and exhibited normal behaviour, indicating that Compound 27 was well tolerated and showed no evidence of acute oral toxicity at a limit dose of 2000 mg/kg.

Conclusion

We synthesized, characterized, and evaluated N-(aryl)-2-(tetrahalo-1H-benzo[d] [1,2,3] triazol-1-yl)-acetamides (3–30) as potential HIV-1 reverse transcriptase (RT) inhibitors. Novel benzotriazoles were synthesized by reacting tetrahalobenzotriazoles with suitable acetamides. We assessed the HIV-1 RT inhibitory activity of the synthesized compounds. Compound 27 exhibited remarkable inhibitory activity, with a 93.02% inhibition rate and an IC₅₀ of 2.72 ± 0.07 µM, outperforming as most active derivative within the synthesized series. Molecular modeling and dynamic studies of compound 27 in the NNIBP of 1RT2 revealed its binding modes and interactions, which correlated well with its inhibitory activity. Oral toxicity supporting preliminary safety assessment of compound 27, highlighting its potential for further drug development. DFT studies have also demonstrated that compound 27, comparatively higher electron-donating tendency and moderate reactivity within the studied compound series. Overall, the benzotriazole scaffold shows promise for further drug development, with specific substitutions contributing favorably to reverse transcriptase inhibition.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1^{(11.8MB, docx)}

Acknowledgements

The authors thank CIF, BIT, Mesra, India for providing NMR, Mass spectral, and elemental analytical data. We thank the All-India Council for Technical Education, India under the Quality Improvement Program (AICTE-QIP) for providing fellowship to Nigam Jyoti Maiti for his doctoral research work.

Abbreviations

1RT2: Crystal structure of HIV-1 reverse transcriptase complexed with TNK-999
NMR: Proton nuclear magnetic resonance
¹³C NMR: Carbon nuclear magnetic resonance
DMSO: Dimethyl sulfoxide

Author contributions

NJM, SG, RG, MAK- Writing – original draft; Writing – review & editing; KC, SuS, SS TA - Conceptualization, Investigation, Validation; BD, RN, SA, NKS -Resources, Software, Validation; PN, MT, MYA -Project Administration, Resources, Supervision.

Funding

No funding received.

Data availability

All data generated or analysed during this study are included in this published article and in its supplementary files.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

ARRIVE statement

All methods are reported in accordance with ARRIVE guidelines.

Clinical trial

Not applicable.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Nigam Jyoti Maiti, Email: nigammaiti007@gmail.com.

Parwiz Niazi, Email: parwiz60@kdru.edu.af.

References

1.Clercq, D. Erik. Anti-HIV drugs: 25 compounds approved within 25 years after the discovery of HIV. Int. J. Antimicrob. Agents. 33, 307–320 (2009). [DOI] [PubMed] [Google Scholar]
2.Zhan, P., Liu, X., Li, Z., Pannecouque, C. & Clercq, E. D. Design strategies of novel NNRTIs to overcome drug resistance. Curr. Med. Chem.16, 3903–3917 (2009). [DOI] [PubMed] [Google Scholar]
3.HIV data and statistics. https://www.who.int/teams/global-hiv-hepatitis-and-stisprogrammes/hiv/strategic-information/hiv-dataand-statistics
4.Jonckheere, H., Anne, J. & Clercq, E. D. The HIV-1 reverse transcription (R.T.) process as target for R.T. inhibitors. Med. Res. Rev.20, 129–154 (2000). [DOI] [PubMed] [Google Scholar]
5.Clercq, E. D. Novel compounds in preclinical early clinical development for the treatment of HIV infections. Rev. Med. Virol.10, 255–277 (2000). [DOI] [PubMed] [Google Scholar]
6.Hajos, G., Riedi, S. & Molnar, J. Szabo, non-nucleoside reverse transcriptase inhibitors. Drugs Future. 25, 47–62. 10.1128/JVI.80.9.4440-4446.2006 (2000). [Google Scholar]
7.Ren, J. et al. Structural basis for the resilience of Efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse transcriptase. Strucure8, 1089–1094. 10.1016/S0969-2126(00)00513-X (2000). [DOI] [PubMed] [Google Scholar]
8.Namasivayam, V. et al. The journey of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) from lab to clinic. J. Med. Chem.62, 4851–4883 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Du, S., Hu, X., Menéndez-Arias, L., Zhan, P. & Liu, X. Target-based drug design strategies to overcome resistance to antiviral agents: opportunities and challenges. Drug Resist. Update73, 101053 (2024). [DOI] [PubMed] [Google Scholar]
10.Du, J., Guo, J., Kang, D., Li, Z. & Wang, G. New techniques and strategies in drug discovery. Chin. Chem. Lett.31, 1695–1708 (2020). [Google Scholar]
11.Borowski, P. et al. Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses. Eur. J. Biochem.270, 1645–1645. 10.1046/j.1432-1033.2003.03540.x (2003). [DOI] [PubMed] [Google Scholar]
12.El-Kardocy, A. et al. CK2 inhibition, lipophilicity and anticancer activity of new N 1 versus N 2-substituted tetrabromobenzotriazole regioisomers. New J. Chem.44, 13007–13017 (2020). [Google Scholar]
13.Prichard, R. K., Hall, C. A., Kelly, J. D., Martin, I. C. A. & Donald, A. D. The problem of anthelmintic resistance in nematodes. Aust. Vet. J.56, 239–250 (1980). [DOI] [PubMed] [Google Scholar]
14.Waller, P. J. The development of anthelmintic resistance in ruminant livestock. Acta Trop.56, 233–243 (1994). [DOI] [PubMed] [Google Scholar]
15.Kaplan, R. M. Anthelmintic resistance in nematodes of horses. Vet. Res.33, 491–507 (2002). [DOI] [PubMed] [Google Scholar]
16.Fobofou, S. A. T. et al. Isolation and anticancer, anthelminthic, and antiviral (HIV) activity of acylphloroglucinols, and regioselective synthesis of empetrifranzinans from Hypericum roeperianum. Bioorg. Med. Chem.23, 6327–6334 (2015). [DOI] [PubMed] [Google Scholar]
17.Mayer, A. M., Rodríguez, A. D., Berlinck, R. G. & Hamann, M. T. Marine pharmacology in 2005–6: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action. Biochim. et Biophys. Acta (BBA)-General Subj.1790, 283–308 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Wiley Richard, H., Hussung Karl, F. & Benzotriazoles, H. J. Am. Chem. Soc.79 4395–4400. (1957). [Google Scholar]
19.Wiley, R. H., Hussung, K. F. & Moffat, J. Preparation, structure, and properties of 4,4,6,7-Tetrachlorobenzotriazoleand its 1-and 2- substitution products. J. Am. Chem. Soc.77, 5105–5109 (1955). [Google Scholar]
20.Maiti, N. J. & Ganguly, S. Some new benzotriazole derivatives: Synthesis, antimycobacterial evaluation, antimicrobial efficacy, ADME studies, and molecular docking studies. Indian J. Heterocycl. Chem.33, 385–392 (2023). 10.59467/IJHC.2023.33.385
21.Silprasit, K., Thammaporn, R., Tecchasakul, S., Hannongbua, S. & Choowongkomon, K. Simple and rapid determination of the enzyme kinetics of HIV-1 reverse transcriptase and anti-HIV-1 agents by a fluorescence-based method. J. Virol. Methods. 171, 381–387 (2011). [DOI] [PubMed] [Google Scholar]
22.Kohlstaedt, E. W., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. Crystal structure at 3.5 Å resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science256, 1783–1790 (1992). [DOI] [PubMed] [Google Scholar]
23.Ren, J., Esnouf, R. L., Garman, E., Stuart, D. I. & Stammers, J. K. High-resolution structures of HIV-1 reverse transcriptase reveal binding interactions of non-nucleoside inhibitors. Structure3, 915–926 (1995). [DOI] [PubMed] [Google Scholar]
24.Tsuneda, T., Song, J. W., Suzuki, S. & Hirao, K. On ‘’’Koopmans’ theorem in density functional theory. J. Chem. Phys.133, 174101 (2013). [DOI] [PubMed] [Google Scholar]
25.Tarikogullari, A. H., Fatma, S. K., Erol, K. & Pabuccuoglu, V. Arzneimittelforschung60 593–598. (2010). [DOI] [PubMed] [Google Scholar]
26.Yadav, G., Ganguly, S., Murugesan, S. & Dev, A. Anti Infect. Agents13 65–77. (2015). [Google Scholar]
27.Soyer, Z., Kılıç, F. S., Erol, K. & Pabuçcuoğlu, V. Il Farmaco59 595–600. (2004). [DOI] [PubMed]
28.Kataev, V. A. et al. Russ. J. Gen. Chem.91 785–791. (2021). [Google Scholar]
29.Seetaha, S. et al. Application of site-specific spin labeling for NMR detecting inhibitor-induced conformational change of HIV-1 reverse transcriptase. ChemMedChem11, 363–366 (2016). [DOI] [PubMed] [Google Scholar]
30.Lindstrom, W., Morris, G. M. & Weber, C. R. Huey,2008, Using AutoDock for Virtual Screening. La. Jolla 1 (2008).
31.Goodsell, D. S., Morris, G. M. & Olson, A. J. Automated Docking of Flexible Ligands: Applications AutoDock. J. Mol. Recognit.9, 1–5 (1996). [DOI] [PubMed] [Google Scholar]
32. Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K. & Olson, A. J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem.19, 1639–1662 (1998).
33.Sousa, S. F., Fernandes, P. A. & Ramos, M. J. Protein–ligand docking: Current status and future challenges, proteins: Structure. Funct. Bioinf.65, 15–26 (2006). [DOI] [PubMed] [Google Scholar]
34.Huey, R., Morris, G. M., Olson, A. J. & Goodsell, D. S. A. Semiempirical free energy force field with charge-based desolvation. J. Comput. Chem.28, 1145–1152 (2007). [DOI] [PubMed] [Google Scholar]
35.Hopkins, A. L. et al. Complexes of HIV-1 reverse transcriptase with inhibitors of the HEPT series reveal conformational changes relevant to the design of potent non-nucleoside inhibitors. J. Med. Chem.39, 1589–1600 (1996). [DOI] [PubMed] [Google Scholar]
36.Silvestri, R. et al. 1-[2-(Diphenylmethoxy) ethyl]-2-methyl-5-nitroimidazole: a potent lead for the design of novel NNRTIs. Bioorg. Med. Chem. Lett.10, 253–25630 (2000). [DOI] [PubMed] [Google Scholar]
37.Yuan, S., Chan, H. C. S. & Hu, Z. Using PyMOL as a platform for computational drug design. Wiley Interdiscip Rev. Comput. Mol. Sci.7 (2017), 1298 (2017). [Google Scholar]
38.Schüttelkopf, A. W. & Van Aalten, D. M. F. A tool for high-throughput crystallography of protein–ligand complexes. Acta Crystallogr. D Biol. Crystallogr.60, 1355–1363 (2004). [DOI] [PubMed] [Google Scholar]
39.Carmena-Bargueño, M., Martínez-Cortés, C. & Banegas-Luna, A. J. H. Pérez-Sánchez,2023, TOLEDO: Accelerated Maestro GUI molecular dynamics simulations. (2023). [DOI] [PubMed]
40.Mark, P. & Nilsson, L. Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K. J. Phys. Chem. A. 105, 9954–9960 (2001). [Google Scholar]
41.Jorgensen, W. L., Maxwell, D. S. & Tirado-Rives, J. development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J. Am. Chem. Soc.118 (1996), 11225–11236 (1996). [Google Scholar]
42.Kumar, B. K. et al. Pharmacophore based virtual screening, molecular docking, molecular dynamics and MM-GBSA approach for identification of prospective SARS-CoV-2 inhibitor from natural product databases. J. Biomol. Struct. Dyn.. 40 (2022), 1363–1386 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Acobson, M. P. et al. hierarchical approach to all-atom protein loop prediction. Proteins Struct. Funct. Bioinform.55, 351–367 (2004). [DOI] [PubMed] [Google Scholar]
44.Frisch, M. J. et al. Gaussian 09, Revision D.01 (Gaussian, Inc., 2009).
45.Dennington, R., Keith, T. A. & Millam, J. M. GaussView (version 6) (Shawnee Mission (Semichem Inc., 2016).
46.Shanker, K., Naradala, J., Mohan, G. K., Kumar, G. S. & Pravallika, P. L. A sub-acute oral toxicity analysis and comparative in vivo anti-diabetic activity of zinc oxide, cerium oxide, silver nanoparticles, and Momordica charantia in streptozotocin-induced diabetic Wistar rats. RSC Adv.7, 37158–37167 (2017). [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(11.8MB, docx)}

Data Availability Statement

All data generated or analysed during this study are included in this published article and in its supplementary files.

[CR1] 1.Clercq, D. Erik. Anti-HIV drugs: 25 compounds approved within 25 years after the discovery of HIV. Int. J. Antimicrob. Agents. 33, 307–320 (2009). [DOI] [PubMed] [Google Scholar]

[CR2] 2.Zhan, P., Liu, X., Li, Z., Pannecouque, C. & Clercq, E. D. Design strategies of novel NNRTIs to overcome drug resistance. Curr. Med. Chem.16, 3903–3917 (2009). [DOI] [PubMed] [Google Scholar]

[CR3] 3.HIV data and statistics. https://www.who.int/teams/global-hiv-hepatitis-and-stisprogrammes/hiv/strategic-information/hiv-dataand-statistics

[CR4] 4.Jonckheere, H., Anne, J. & Clercq, E. D. The HIV-1 reverse transcription (R.T.) process as target for R.T. inhibitors. Med. Res. Rev.20, 129–154 (2000). [DOI] [PubMed] [Google Scholar]

[CR5] 5.Clercq, E. D. Novel compounds in preclinical early clinical development for the treatment of HIV infections. Rev. Med. Virol.10, 255–277 (2000). [DOI] [PubMed] [Google Scholar]

[CR6] 6.Hajos, G., Riedi, S. & Molnar, J. Szabo, non-nucleoside reverse transcriptase inhibitors. Drugs Future. 25, 47–62. 10.1128/JVI.80.9.4440-4446.2006 (2000). [Google Scholar]

[CR7] 7.Ren, J. et al. Structural basis for the resilience of Efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse transcriptase. Strucure8, 1089–1094. 10.1016/S0969-2126(00)00513-X (2000). [DOI] [PubMed] [Google Scholar]

[CR8] 8.Namasivayam, V. et al. The journey of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) from lab to clinic. J. Med. Chem.62, 4851–4883 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR9] 9.Du, S., Hu, X., Menéndez-Arias, L., Zhan, P. & Liu, X. Target-based drug design strategies to overcome resistance to antiviral agents: opportunities and challenges. Drug Resist. Update73, 101053 (2024). [DOI] [PubMed] [Google Scholar]

[CR10] 10.Du, J., Guo, J., Kang, D., Li, Z. & Wang, G. New techniques and strategies in drug discovery. Chin. Chem. Lett.31, 1695–1708 (2020). [Google Scholar]

[CR11] 11.Borowski, P. et al. Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/helicase activities of hepatitis C and related viruses. Eur. J. Biochem.270, 1645–1645. 10.1046/j.1432-1033.2003.03540.x (2003). [DOI] [PubMed] [Google Scholar]

[CR12] 12.El-Kardocy, A. et al. CK2 inhibition, lipophilicity and anticancer activity of new N 1 versus N 2-substituted tetrabromobenzotriazole regioisomers. New J. Chem.44, 13007–13017 (2020). [Google Scholar]

[CR13] 13.Prichard, R. K., Hall, C. A., Kelly, J. D., Martin, I. C. A. & Donald, A. D. The problem of anthelmintic resistance in nematodes. Aust. Vet. J.56, 239–250 (1980). [DOI] [PubMed] [Google Scholar]

[CR14] 14.Waller, P. J. The development of anthelmintic resistance in ruminant livestock. Acta Trop.56, 233–243 (1994). [DOI] [PubMed] [Google Scholar]

[CR15] 15.Kaplan, R. M. Anthelmintic resistance in nematodes of horses. Vet. Res.33, 491–507 (2002). [DOI] [PubMed] [Google Scholar]

[CR16] 16.Fobofou, S. A. T. et al. Isolation and anticancer, anthelminthic, and antiviral (HIV) activity of acylphloroglucinols, and regioselective synthesis of empetrifranzinans from Hypericum roeperianum. Bioorg. Med. Chem.23, 6327–6334 (2015). [DOI] [PubMed] [Google Scholar]

[CR17] 17.Mayer, A. M., Rodríguez, A. D., Berlinck, R. G. & Hamann, M. T. Marine pharmacology in 2005–6: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action. Biochim. et Biophys. Acta (BBA)-General Subj.1790, 283–308 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR18] 18.Wiley Richard, H., Hussung Karl, F. & Benzotriazoles, H. J. Am. Chem. Soc.79 4395–4400. (1957). [Google Scholar]

[CR19] 19.Wiley, R. H., Hussung, K. F. & Moffat, J. Preparation, structure, and properties of 4,4,6,7-Tetrachlorobenzotriazoleand its 1-and 2- substitution products. J. Am. Chem. Soc.77, 5105–5109 (1955). [Google Scholar]

[CR20] 20.Maiti, N. J. & Ganguly, S. Some new benzotriazole derivatives: Synthesis, antimycobacterial evaluation, antimicrobial efficacy, ADME studies, and molecular docking studies. Indian J. Heterocycl. Chem.33, 385–392 (2023). 10.59467/IJHC.2023.33.385

[CR21] 21.Silprasit, K., Thammaporn, R., Tecchasakul, S., Hannongbua, S. & Choowongkomon, K. Simple and rapid determination of the enzyme kinetics of HIV-1 reverse transcriptase and anti-HIV-1 agents by a fluorescence-based method. J. Virol. Methods. 171, 381–387 (2011). [DOI] [PubMed] [Google Scholar]

[CR22] 22.Kohlstaedt, E. W., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. Crystal structure at 3.5 Å resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science256, 1783–1790 (1992). [DOI] [PubMed] [Google Scholar]

[CR23] 23.Ren, J., Esnouf, R. L., Garman, E., Stuart, D. I. & Stammers, J. K. High-resolution structures of HIV-1 reverse transcriptase reveal binding interactions of non-nucleoside inhibitors. Structure3, 915–926 (1995). [DOI] [PubMed] [Google Scholar]

[CR24] 24.Tsuneda, T., Song, J. W., Suzuki, S. & Hirao, K. On ‘’’Koopmans’ theorem in density functional theory. J. Chem. Phys.133, 174101 (2013). [DOI] [PubMed] [Google Scholar]

[CR25] 25.Tarikogullari, A. H., Fatma, S. K., Erol, K. & Pabuccuoglu, V. Arzneimittelforschung60 593–598. (2010). [DOI] [PubMed] [Google Scholar]

[CR26] 26.Yadav, G., Ganguly, S., Murugesan, S. & Dev, A. Anti Infect. Agents13 65–77. (2015). [Google Scholar]

[CR27] 27.Soyer, Z., Kılıç, F. S., Erol, K. & Pabuçcuoğlu, V. Il Farmaco59 595–600. (2004). [DOI] [PubMed]

[CR28] 28.Kataev, V. A. et al. Russ. J. Gen. Chem.91 785–791. (2021). [Google Scholar]

[CR29] 29.Seetaha, S. et al. Application of site-specific spin labeling for NMR detecting inhibitor-induced conformational change of HIV-1 reverse transcriptase. ChemMedChem11, 363–366 (2016). [DOI] [PubMed] [Google Scholar]

[CR30] 30.Lindstrom, W., Morris, G. M. & Weber, C. R. Huey,2008, Using AutoDock for Virtual Screening. La. Jolla 1 (2008).

[CR31] 31.Goodsell, D. S., Morris, G. M. & Olson, A. J. Automated Docking of Flexible Ligands: Applications AutoDock. J. Mol. Recognit.9, 1–5 (1996). [DOI] [PubMed] [Google Scholar]

[CR32] 32. Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K. & Olson, A. J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem.19, 1639–1662 (1998).

[CR33] 33.Sousa, S. F., Fernandes, P. A. & Ramos, M. J. Protein–ligand docking: Current status and future challenges, proteins: Structure. Funct. Bioinf.65, 15–26 (2006). [DOI] [PubMed] [Google Scholar]

[CR34] 34.Huey, R., Morris, G. M., Olson, A. J. & Goodsell, D. S. A. Semiempirical free energy force field with charge-based desolvation. J. Comput. Chem.28, 1145–1152 (2007). [DOI] [PubMed] [Google Scholar]

[CR35] 35.Hopkins, A. L. et al. Complexes of HIV-1 reverse transcriptase with inhibitors of the HEPT series reveal conformational changes relevant to the design of potent non-nucleoside inhibitors. J. Med. Chem.39, 1589–1600 (1996). [DOI] [PubMed] [Google Scholar]

[CR36] 36.Silvestri, R. et al. 1-[2-(Diphenylmethoxy) ethyl]-2-methyl-5-nitroimidazole: a potent lead for the design of novel NNRTIs. Bioorg. Med. Chem. Lett.10, 253–25630 (2000). [DOI] [PubMed] [Google Scholar]

[CR37] 37.Yuan, S., Chan, H. C. S. & Hu, Z. Using PyMOL as a platform for computational drug design. Wiley Interdiscip Rev. Comput. Mol. Sci.7 (2017), 1298 (2017). [Google Scholar]

[CR38] 38.Schüttelkopf, A. W. & Van Aalten, D. M. F. A tool for high-throughput crystallography of protein–ligand complexes. Acta Crystallogr. D Biol. Crystallogr.60, 1355–1363 (2004). [DOI] [PubMed] [Google Scholar]

[CR39] 39.Carmena-Bargueño, M., Martínez-Cortés, C. & Banegas-Luna, A. J. H. Pérez-Sánchez,2023, TOLEDO: Accelerated Maestro GUI molecular dynamics simulations. (2023). [DOI] [PubMed]

[CR40] 40.Mark, P. & Nilsson, L. Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K. J. Phys. Chem. A. 105, 9954–9960 (2001). [Google Scholar]

[CR41] 41.Jorgensen, W. L., Maxwell, D. S. & Tirado-Rives, J. development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J. Am. Chem. Soc.118 (1996), 11225–11236 (1996). [Google Scholar]

[CR42] 42.Kumar, B. K. et al. Pharmacophore based virtual screening, molecular docking, molecular dynamics and MM-GBSA approach for identification of prospective SARS-CoV-2 inhibitor from natural product databases. J. Biomol. Struct. Dyn.. 40 (2022), 1363–1386 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Acobson, M. P. et al. hierarchical approach to all-atom protein loop prediction. Proteins Struct. Funct. Bioinform.55, 351–367 (2004). [DOI] [PubMed] [Google Scholar]

[CR44] 44.Frisch, M. J. et al. Gaussian 09, Revision D.01 (Gaussian, Inc., 2009).

[CR45] 45.Dennington, R., Keith, T. A. & Millam, J. M. GaussView (version 6) (Shawnee Mission (Semichem Inc., 2016).

[CR46] 46.Shanker, K., Naradala, J., Mohan, G. K., Kumar, G. S. & Pravallika, P. L. A sub-acute oral toxicity analysis and comparative in vivo anti-diabetic activity of zinc oxide, cerium oxide, silver nanoparticles, and Momordica charantia in streptozotocin-induced diabetic Wistar rats. RSC Adv.7, 37158–37167 (2017). [Google Scholar]

PERMALINK

Synthesis, in vitro anti-HIV-1 reverse transcriptase evaluation, molecular modelling, DFT analysis and acute oral toxicity assessment of benzotriazole derivatives

Nigam Jyoti Maiti

Swastika Ganguly

Kiattawee Choowongkomon

Supaphorn Seetaha

Siriwan Saehlee

Thitinan Aiebchun

Rahul Ghosh

Mohammad Adnan Khan

Biplab Debnath

Rajarshi Nath

Sumel Ashique

Nisha Kumari Singh

Mohammad Yousuf Ansari

Mohd Tariq

Parwiz Niazi

Abstract

Supplementary Information

Introduction

Fig. 1.

Results and discussion

Chemistry

Scheme 1.

HIV-1 RT inhibition study

Fig. 2.

Table 1.

Binding mode analysis of the potent compound as an HIV-1 RT inhibitor

Fig. 3.

Molecular dynamics (M.D.)

Studies on the co-crystallized structure of the HIV-1 RT (1RT2)-27 complex

Fig. 4.

Structural characterization using DFT and TD-DFT methods

Fig. 5.

Table 2.

Frontier molecular orbital and MEP studies

Fig. 6.

Table 3.

Fig. 7.

In vivo acute oral toxicity

Experimental section

General procedure for the synthesis of tetrabromobenzotriazole (1)18

General procedure for the synthesis of tetrachlorobenzotriazole (4)19

Synthesis of acetamide derivatives 2a-n25–28

Synthesis of N-(aryl)-2-(perhalo-1H-benzo[d] [1,2,3] triazol-1-yl)-acetamides (3–30)

N-(phenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (3)

N-(2-hydroxyphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (4)

N-(4-ethylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (5)

N-(2,4-dimethylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (6)

N-(3,4-dimethylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (7)

N-(2-chlorophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (8)

N-(3-chlorophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (9)

N-(4-chlorophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (10)

N-(4-isopropylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (11)

N-(3-nitrophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (12)

N-(4-nitrophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (13)

N-(3-chloro-2-methylphenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (14)

N-(3,4-dichlorophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (15)

N-(4-bromophenyl)-2-(tetrabromo-1H-benzo[d] [1,2,3] triazol-1-yl) acetamide (16)

N-(phenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)-acetamide (17)

N-(2-hydroxyphenyl)-2-(tetrachloro-1 H-benzo[d] [1,2,3] triazol-1-yl)acetamide(18) [202431017120

N-(4-ethylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (19)

N-(2,4-dimethylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (20)

N-(3,4-dimethylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (21)

N-(2-chlorophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (22)

N-(3-chlorophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (23)

N-(4-chlorophenyl)-2-(tetrachloro-1 H-benzo[d] [1,2,3] triazol-1-yl) acetamide (24) [202431017120

N-(4-isopropylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (25)

N-(3-nitrophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (26)

N-(4-nitrophenyl)-2-(tetrachloro-1 H-benzo[d] [1,2,3] triazol-1-yl) acetamide (27) [202431017120

N-(3-chloro-2-methylphenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (28)

N-(3,4-dichlorophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (29)

N-(4-bromophenyl)-2-(tetrachloro-1H-benzo[d] [1,2,3] triazol-1-yl)acetamide (30)

Anti-HIV-1 RT activity

Molecular modeling

Molecular dynamics

DFT studies

In vivo oral toxicity46

The primary outcome measures included

Conclusion

General procedure for the synthesis of tetrabromobenzotriazole (1)¹⁸

General procedure for the synthesis of tetrachlorobenzotriazole (4)¹⁹

Synthesis of acetamide derivatives 2a-n^25–28

In vivo oral toxicity⁴⁶