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. 2021 Nov 12;6(46):31348–31357. doi: 10.1021/acsomega.1c05222

(Benzylideneamino)triazole–Thione Derivatives of Flurbiprofen: An Efficient Microwave-Assisted Synthesis and In Vivo Analgesic Potential

Muhammad Zaheer †, Muhammad Zia-ur-Rehman †,*, Rubina Munir ‡,*, Nadia Jamil §, Saiqa Ishtiaq ∥, Rahman Shah Zaib Saleem ⊥, Mark R J Elsegood #
PMCID: PMC8613847  PMID: 34841178

Abstract

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Triazole is an imperative heterocycle renowned for its broad-spectrum biological significance. In this manuscript, facile microwave-assisted synthesis of a series of 4-(benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione 6(a–m) derivatives along with their in vivo analgesic activity is reported. 2-(2-Fluoro-[1,1′-biphenyl]-4-yl)propanoic acid (flurbiprofen) was converted to methyl 2-(2-fluoro-[1,1′-biphenyl]-4-yl)propanoate using microwave irradiation, followed by its hydrazinolysis with hydrazine monohydrate. 2-(2-Fluoro-[1,1′-biphenyl]-4-yl)propanehydrazide thus obtained was converted to 4-amino-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione, followed by its condensation with different aromatic aldehydes to get the title compounds. Structures of all the synthesized compounds were established using different methods (1H NMR and 13C NMR spectroscopies, mass spectrometry, and elemental analysis) and evaluated for their potential as analgesic agents by tail flick, hot plate, and writhing methods. The results of this in vivo study revealed several compounds as potent analgesic agents among which compound 6e showed significant analgesic effect for all the three assays employed.

1. Introduction

Synthesis of triazoles and their biological importance have received considerable attention in recent years, and this class of compounds have become one of the potential agents for drug discovery.1−5 Among these, 1,2,4-triazoles represent an important class of organic compounds with wide use in medicine, agriculture, and industry.6 A wide variety of therapeutically interesting drug candidates possessing analgesic, anti-inflammatory, anti-microbial, anti-cancer, anti-depressant, anti-fungal, anti-convulsant, and anti-viral activities are discussed in the literature possessing the 1,2,4-triazole moiety.7−16

Many strategies have been reported in the literature regarding the development of more effective NSAIDs (non-steroidal anti-inflammatory drugs) with reduced side effects.17−19 In certain cases, derivatization of the carboxylic functional group in NSAIDs led to increased analgesic and anti-inflammatory activities with reduced ulcerogenic potential.20,21

Flurbiprofen, 2-(2-fluoro-[1,1′-biphenyl]-4-yl)propanoic acid, is a non-selective cyclooxygenase (COX) inhibitor for the treatment of non-infectious inflammation, arthritis, and pain.22−24 However, like other drugs of this category, it is also associated with gastro-intestinal (GI) ulceration, nephrotoxicity, and bleeding.25,26 Keeping in view the analgesic activities of flurbiprofen and 1,2,4-triazoles, both the moieties are synergized in one hybrid unit as part of our ongoing program regarding the development of biologically active heterocycles. Flurbiprofen was esterified, followed by its hydrazinolysis, and subsequent reactions with carbon disulfide and hydrazine hydrate resulted in 4-amino-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (5). It was further reacted with different aldehydes to get a series of 4-(benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thiones 6(a–m) (Scheme 1). Besides chemical characterization, these compounds were checked for their analgesic activity on albino mice using tail flick, hot plate, and writhing methods.

Scheme 1. Synthesis of 4-(Benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thiones.

Scheme 1

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Reaction conditions: (i) MeOH/H+/microwaves (ii) hydrazine hydrate/MeOH/microwaves (iii) EtOH/KOH/CS2 (iv) EtOH/hydrazine hydrate/microwaves (v) RCHO/EtOH/H+/microwaves.

2. Results and Discussion

2.1. Chemistry

4-Amino-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (5), the main intermediate of the designed scheme, was synthesized using the multi-step synthesis strategy. Esterification of 2-(2-fluoro-[1,1′-biphenyl]-4-yl)propanoic acid (1) using microwaves followed by hydrazinolysis yielded 2-(2-fluoro-[1,1′-biphenyl]-4-yl)propanehydrazide (3).27 The use of microwaves in the esterification and hydrazinolysis steps plays a significant role as it assists in minimizing the reaction time and increases the yield of reactions.28,29 During this study, the reaction yield improved from 82 to 98.81% for the esterification step, while 74.94 to 95.12% for the hydrazinolysis reaction. 2-(2-Fluoro-[1,1′-biphenyl]-4-yl)propanehydrazide (3) was converted to potassium 2-(2-(2-fluoro-[1,1′-biphenyl]-4-yl)propanoyl)hydrazinecarbodithioate (4) with the reaction of carbon disulfide and potassium hydroxide, followed by its reaction with hydrazine hydrate to form 4-amino-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (5), followed by its condensation with different aldehydes under microwaves to get the title compounds. The use of microwaves in the condensation reactions was found more effective to attain better product yields and shorter reaction times (Table 1).

Table 1. Synthesis of 4-(Benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thiones (6a–6n) under Conventional and Microwave Conditions.

        conventional
 microwave
entry product R M.P (°C) reaction time (min) yield (%)a reaction time (min) yield (%)a reaction temperature (°C)
1 6a phenyl 213 270 77 11 93 287
2 6b 2-chlorophenyl 189 269 67 15 95 330
3 6c 3-chlorophenyl 190 236 65 13 90 305
4 6d 4-chlorophenyl 196 220 73 9 97 308
5 6e 2-hydroxyphenyl 195 290 66 18 89 303
6 6f 4-hydroxyphenyl 180 280 73 14 95 291
7 6g 2,4-dichlorophenyl 204 210 63 20 96 317
8 6h 2,3-dichlorophenyl 226 240 59 23 92 320
9 6i 4-nitrophenyl 208 200 79 17 94 300
10 6j 4-N,N-dimethylphenyl 210 260 74 12 90 328
11 6k 4-fluorophenyl 179 200 78 20 96 308
12 6l 3,4-dimethoxyphenyl 170 229 71 19 94 302
13 6m 2-furfural 197 190 75 10 97 280
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a

Isolated yields based on 4-amino-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione.

Structures of synthesized compounds 6(a–m) were confirmed through spectroscopic techniques and were found in agreement with the expected values. In 1H NMR spectra, the NH proton of the triazole ring appeared as a singlet around 14.00 ppm, whereas the imine N=CH proton emerged as a singlet near 9.20–10.78 ppm. Methyl (CH3) protons exhibited a doublet near 1.65 ppm and the methine (CH) proton gave a quartet around 4.40–4.65 ppm. Aromatic ring protons appeared between 6.76 and 8.34 ppm depending on their environment. In 13C NMR spectra, methyl carbon (CH3) and methine carbon (CH) appeared around 19.5 and 35.8 ppm individually. Thione carbon (C=S) was observed above 160.0 ppm and imine carbon (N=CH) near 153–154 ppm in all the synthesized derivatives 6(a–n). FT-IR data, elemental analysis results, and mass spectra too were in accordance with the structures of all the compounds.

2.2. Stereochemistry and X-ray Crystallography

A single crystal of 4-(benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6a) as a representative of the title series of compounds 6(a–m) was grown in 90% ethanol and examined by single-crystal X-ray diffraction. The data shows that the C=N bond exhibits the E configuration. It is evident from the crystal data that sulfur exists in the thione form instead of thiol (Figure 1). Molecules form H-bonded dimers via pairs of centrosymmetric N–H···S interactions (Figure 2). A planar conformation between the benzylidene-amino and triazole moieties is stabilized via a C–H···S interaction. F···F interactions at 2.871 Å and C–H···F interactions give rise to an overall 3D supramolecular network. Crystal data and structure refinement details are given in the Experimental Section.

Figure 1.

Figure 1

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ORTEP diagram of compound 6a with the numbering scheme. Displacement ellipsoids are drawn at the 50% probability level; H atoms are represented by circles of arbitrary radii.

Figure 2.

Figure 2

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Perspective view showing intra- and intermolecular hydrogen bonding in 6a.

2.3. Analgesic Activity

The newly synthesized 4-(benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thiones 6(a–m) were evaluated for their analgesic activities by three different methods, that is, tail flick, hot plate, and writhing methods. Albino mice for the current studies were housed and kept at the animal house situated at the University College of Pharmacy in the University of the Punjab Lahore, Pakistan, in groups of six and were acclimatized to experimental conditions for 2 days. Water was freely provided to the animals while access to feed was withdrawn 1 day before the experiments.

2.3.1. Tail Flick Method

Compounds 6(a–m) were tested for their analgesic activity using the tail flick method.30,31 This method is based on the phenomenon that certain drugs such as morphine can selectively prolong the duration of distinctive tail withdrawal reflex time when the distal end of mouse’s tail is immersed in water at 55 ± 1.0 °C. Test compounds were assessed at equimolar oral doses relative to 10 mg/kg of flurbiprofen. Using this method, the analgesia effect was calculated for 0.5 to 4.5 h period since it was found that most of the animals showed analgesia ranging from 1 to 3 h. Moderate to significant activities for all the synthesized derivatives were observed, which are shown in Table 2 along with the analgesic activity of reference standard, flurbiprofen. Among the 14 tested compounds, 6e, 6i, 6j, and 6m showed highly significant analgesic activity compared with the reference drug, while compounds 6b, 6d, and 6g showed significant analgesic activities. Compounds 6a, 6f, and 6l showed less to moderately significant analgesic activities, while 6c, 6h, and 6k showed the least activity compared with the reference drug (Figure 3).

Table 2. Evaluation of the Analgesic Activity of 6(a–m) by the Tail Flick Method.
  variation flicking time with ± SEM (time in sec at 55 ± 1 °C)
compound ID 0 h 0.5 h min 1 h 1.5 h 2 h 2.5 h 3 h 3.5 h 4 h 4.5 h
flurbiprofen 2.727 ± 0.197 2.862 ± 0.262 2.917 ± 0.280 3.203 ± 0.299 3.418 ± 0.344 3.562 ± 0.516 3.432 ± 0.450 3.172 ± 0.356 2.992 ± 0.324 2.873 ± 0.286
6a 2.787 ± 0.436 3.880 ± 0.554 3.562 ± 0.401 3.313 ± 0.357 3.645 ± 0.432 3.752 ± 0.688 3.448 ± 0.799 3.113 ± 0.356 2.975 ± 0.322 2.828 ± 0.296
6b 2.415 ± 0.214 3.713 ± 0.610 4.347 ± 0.585 4.140 ± 0.795 4.383 ± 0.697 4.432 ± 0.668 4.040 ± 0.442 3.457 ± 0.358 3.097 ± 0.220 2.683 ± 0.253
6c 2.670 ± 0.311 3.817 ± 0.354 3.935 ± 0.389 3.257 ± 0.267 3.568 ± 0.544 3.385 ± 0.375 3.085 ± 0.404 2.708 ± 0.398 3.043 ± 0.358 2.930 ± 0.340
6d 2.787 ± 0.398 3.880 ± 0.506 3.562 ± 0.366 3.313 ± 0.326 3.645 ± 0.395 3.752 ± 0.628 3.448 ± 0.730 3.113 ± 0.325 2.975 ± 0.294 2.828 ± 0.270
6e 2.787 ± 0.398 4.477 ± 0.306 4.050 ± 0.429 4.640 ± 0.540 4.620 ± 0.630 3.503 ± 0.234 4.138 ± 0.382 4.050 ± 0.349 2.817 ± 0.329 2.855 ± 0.294
6f 3.828 ± 0.462 4.062 ± 0.818 3.960 ± 0.257 3.500 ± 0.270 3.907 ± 0.410 3.375 ± 0.273 3.222 ± 0.287 3.050 ± 0.186 2.682 ± 0.236 2.668 ± 0.158
6g 2.717 ± 0.213 3.340 ± 0.290 3.185 ± 0.254 4.262 ± 0.673 4.183 ± 0.519 3.132 ± 0.387 3.387 ± 0.500 3.173 ± 0.362 3.013 ± 0.295 2.867 ± 0.272
6h 1.767 ± 0.092 2.963 ± 0.262 3.375 ± 0.240 2.802 ± 0.193 3.117 ± 0.256 3.688 ± 0.335 3.853 ± 0.194 3.572 ± 0.190 2.705 ± 0.124 2.393 ± 0.130
6i 3.318 ± 0.318 3.435 ± 0.391 4.062 ± 0.560 6.078 ± 2.520 4.407 ± 0.594 3.083 ± 0.273 2.997 ± 0.268 2.915 ± 0.173 2.812 ± 0.169 2.762 ± 0.155
6j 3.880 ± 0.161 6.135 ± 0.653 6.148 ± 0.719 6.390 ± 0.749 6.265 ± 0.947 3.752 ± 0.628 3.448 ± 0.730 3.113 ± 0.325 2.975 ± 0.294 2.828 ± 0.270
6k 3.345 ± 0.170 3.948 ± 0.487 3.980 ± 0.495 4.020 ± 0.544 3.665 ± 0.581 3.752 ± 0.628 3.448 ± 0.730 3.113 ± 0.325 2.967 ± 0.292 2.845 ± 0.270
6l 3.582 ± 0.372 4.062 ± 0.818 3.960 ± 0.257 3.500 ± 0.270 3.907 ± 0.410 3.375 ± 0.273 3.202 ± 0.278 2.993 ± 0.180 2.660 ± 0.235 2.648 ± 0.158
6m 1.992 ± 0.122 5.218 ± 1.191 4.535 ± 0.797 4.163 ± 0.679 4.210 ± 0.550 5.590 ± 1.068 4.388 ± 0.453 3.097 ± 0.192 2.763 ± 0.186 2.353 ± 0.123
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Figure 3.

Figure 3

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Analgesic activity by the tail flick method.

2.3.2. Hot Plate Method

The title compounds 6(a–m) were also evaluated for analgesic activity by using the hot plate method.32,33 Compounds were tested at equimolar oral dose relative to 10 mg/kg flurbiprofen. The analgesia effect was checked from 0.5 to 4.5 h period, and it was found that most of the animals showed analgesia ranging from 0.5 to 3.5 h. Results showed moderate to significant analgesic activities for the test compounds 6(a–m) and are shown in Table 3 compared with flurbiprofen (reference standard). Compounds 6e, 6i, 6l, 6m, and 6g showed highly significant analgesic activities compared with the reference drug, while 6a, 6h, and 6j exhibited moderate activities. The analgesic effects by the hot plate method are depicted in Figure 4.

Table 3. Evaluation of the Analgesic Activity of the Synthesized Derivatives by the Hot Plate Method.
  variation of hot plate time with ± SEM (time in sec at 55 ± 1 °C)
compound ID 0 h 0.5 h min 1 h 1.5 h 2 h 2.5 h 3 h 3.5 h 4 h 4.5 h
flurbiprofen 6.982 ± 0.763 8.312 ± 0.904 14.588 ± 1.226 15.343 ± 2.198 15.058 ± 1.125 12.925 ± 1.001 15.402 ± 0.359 13.403 ± 1.517 10.497 ± 1.199 9.152 ± 0.695
6a 9.195 ± 0.913 15.228 ± 0.866 19.473 ± 1.508 18.547 ± 1.107 18.022 ± 1.600 22.628 ± 2.135 18.412 ± 1.551 14.828 ± 1.303 11.397 ± 0.702 9.218 ± 0.682
6b 7.655 ± 0.484 15.448 ± 0.985 17.745 ± 1.353 18.062 ± 1.240 19.267 ± 2.308 23.900 ± 1.340 23.332 ± 1.136 23.337 ± 2.010 16.233 ± 1.563 12.352 ± 0.904
6c 6.758 ± 0.454 14.023 ± 1.138 18.325 ± 1.000 19.703 ± 0.600 20.838 ± 1.367 23.928 ± 1.292 22.413 ± 1.327 18.330 ± 1.078 15.718 ± 1.457 13.255 ± 0.574
6d 7.462 ± 0.878 14.195 ± 1.109 14.645 ± 0.546 16.490 ± 0.706 18.463 ± 0.744 20.095 ± 1.048 21.192 ± 1.080 19.838 ± 1.360 15.588 ± 1.180 10.742 ± 1.972
6e 8.492 ± 0.708 11.450 ± 0.594 16.188 ± 1.108 18.902 ± 1.405 21.263 ± 1.144 23.280 ± 2.092 22.110 ± 1.667 17.265 ± 1.209 12.908 ± 0.863 11.797 ± 0.600
6f 7.472 ± 0.635 11.777 ± 0.635 16.052 ± 1.060 19.058 ± 1.313 21.273 ± 1.137 23.280 ± 2.092 22.358 ± 1.681 17.217 ± 1.197 12.908 ± 0.863 11.612 ± 0.573
6g 8.913 ± 0.731 11.507 ± 0.954 13.890 ± 1.372 22.338 ± 1.908 20.625 ± 2.336 21.125 ± 1.351 27.443 ± 2.887 24.308 ± 1.899 19.338 ± 1.336 14.372 ± 0.998
6h 8.125 ± 0.427 11.607 ± 0.829 15.588 ± 0.763 18.975 ± 0.400 22.832 ± 0.700 21.763 ± 0.737 24.245 ± 1.285 20.905 ± 1.516 15.713 ± 1.117 13.935 ± 1.016
6i 8.880 ± 0.983 15.382 ± 4.119 28.280 ± 2.292 30.052 ± 2.256 24.538 ± 3.015 23.367 ± 2.004 21.077 ± 2.704 19.075 ± 2.380 15.650 ± 2.295 11.935 ± 1.012
6j 7.217 ± 0.665 12.328 ± 0.778 18.018 ± 1.652 15.967 ± 0.892 16.570 ± 1.677 16.518 ± 1.821 15.208 ± 1.107 13.400 ± 1.014 11.303 ± 0.756 10.175 ± 0.546
6k 6.395 ± 0.612 8.527 ± 0.668 15.082 ± 1.877 18.757 ± 1.917 20.225 ± 2.697 21.762 ± 2.976 17.453 ± 1.851 14.413 ± 1.149 12.145 ± 0.787 11.123 ± 0.415
6l 9.180 ± 1.293 16.405 ± 1.836 18.095 ± 1.424 21.245 ± 0.604 21.778 ± 1.462 24.965 ± 1.462 26.450 ± 2.602 22.797 ± 3.007 18.278 ± 2.195 13.438 ± 1.065
6m 7.087 ± 0.669 12.118 ± 0.895 15.845 ± 1.103 17.353 ± 1.323 18.417 ± 0.725 20.087 ± 1.026 16.632 ± 0.832 15.397 ± 1.448 11.953 ± 0.910 10.780 ± 0.807
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Figure 4.

Figure 4

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Analgesic activity by the hot plate method.

2.3.3. Acetic Acid Induced Writhing Method

The analgesic activity by the acetic acid induced writhing method was studied on the title compounds.34,35 Albino mice were kept in the test cage for half an hour for acclimatization to the environment before acetic acid injection. Animals were dosed orally (10 mg per kg body mass) with the test compounds and flurbiprofen (reference drug), and the analgesia effect was checked for the first half hour due to the fact that these compounds were found active in this time range. The percentage inhibition in the writhing method was calculated in two phases: the first phase during the initial 15 min after dose administration and the second phase for the next 15 min (16th–30th minute). Compounds 6a, 6e, and 6l exhibited highly significant analgesic activities while 6b, 6i, 6j, 6m, and 6h were found moderately active compared with the reference standard drug (Table 4). Compounds 6c, 6d, 6f, 6g, and 6k were found to possess comparable analgesic activities to the reference drug, flurbiprofen, and this is depicted in Figure 5. All the title compounds exhibited higher activity than the standard drug flurbiprofen. It was further noted that the percentage inhibition of all the compounds increased in the second half of the trial time (from 16th minute to 30th minute) compared with the first half (1st to 15th minute).

Table 4. Analgesic Activity by the Acetic Acid Induced Writhing Method.
    mean no. of writhes ± SEM
 inhibition (%)
treatment dose mg/kg orally first phase second phase first phase second phase
control 0.5 mL saline 57 ± 1.86 29 ± 0.951    
flurbiprofen 10 mg/kg 20.83 ± 1.249 10.83 ± 0.601 20.45 62.64
6a 10 mg/kg 17.17 ± 0.872 5.33 ± 0.760 69.88 80.25
6b 10 mg/kg 20.17 ± 1.720 6.83 ± 1.351 64.62 74.69
6c 10 mg/kg 20 ± 1.483 5.67 ± 0.881 64.91 79.01
6d 10 mg/kg 18.67 ± 1.646 5.50 ± 0.992 67.25 79.63
6e 10 mg/kg 18.33 ± 2.108 5.67 ± 0.881 67.84 79.9
6f 10 mg/kg 18 ± 1.612 5.83 ± 1.612 68.42 78.4
6g 10 mg/kg 18.33 ± 1.429 6.17 ± 1.429 67.84 77.16
6h 10 mg/kg 19 ± 2.049 6.5 ± 1.176 66.67 75.93
6i 10 mg/kg 24 ± 2.082 7.5 ± 0.992 59.65 72.22
6j 10 mg/kg 21.83 ± 1.447 6.33 ± 0.666 61.7 76.54
6k 10 mg/kg 21 ± 1.825 8.33 ± 0.881 63.16 69.14
6l 10 mg/kg 21.17 ± 2.300 6 ± 0.966 62.87 77.78
6m 10 mg/kg 22.5 ± 1.746 6.33 ± 0.881 60.53 79.54
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Figure 5.

Figure 5

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Analgesic activity by the acetic acid induced writhing method.

An insight into the structure–activity relationship indicates that compounds bearing a substituent at the ortho or para positions of the phenyl ring (2-hydroxy, 4-nitro, 4-N,N-dimethylamino) attached to the triazole heterocycle are more active as analgesic agents. Chloro and methoxy analogues exhibited significant activities regardless of their position on the phenyl ring (3-chloro, 4-chloro, 2,4-dichloro and 3,4-dimethoxy substituted derivatives), while the fluoro substituent seems to have no role in analgesic activity as the analogue came out to be least active.

3. Conclusions

Urged by the well-established analgesic properties of triazoles, a distinguished analgesic drug flurbiprofen was derivatized to obtain a series of novel 4-(benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thiones 6(a–m) by a facile microwave-assisted method in excellent yields. Facilitation of microwave irradiation during the core steps of synthesis was found reasonably effective in achieving better product yields and purities. Reaction times for the condensation reactions were significantly reduced by microwave irradiations as compared with the conventional heating method. The successful synthesis of the title compounds was validated from spectroscopic techniques and elemental analyses. The analgesic potential of the synthesized (benzylideneamino)triazole-thione derivatives of flurbiprofen was investigated in vivo by employing three different protocols. All the derivatives exhibited analgesic activity to a different degree; however, 6e displayed significant analgesic potential as a result of all the three assays. These experiments revealed that the compounds under study possess prospective analgesic activities and may serve as an architype for forthcoming studies through further derivatization and/or structural alteration.

4. Experimental Section

4.1. Apparatus, Reagents, and Chemicals

Chemicals employed in the research work were purchased from Wako and E. Merck and were used as received. Solvents were purified through standard procedures before use. Fourier transform infrared (FT-IR) spectroscopy spectra were scanned on a Thermo Nicolet IR 200 spectrometer, while an LCQ Advantage Max Thermo Fisher instrument was used in the ESI mode for mass spectra. 1H NMR spectra were recorded on a Bruker AVANCE-III 400 MHz spectrometer using TMS as the internal standard and 13C NMR spectra were recorded at 101 MHz. Melting points were determined on a Gallenkamp instrument and are uncorrected. For microwave-assisted reactions, a customized microwave oven (Orient eNNe781JF) equipped with inverter technology (for realistic control of the microwaves) operating at multiples of 100 W up to 1000 W generating 2450 MHz frequency was used. The temperature of the microwave-assisted reactions was monitored by Redington 9975-IRT gun. Crystal data were collected on a Bruker APEX 2 CCDD diffractometer using graphite-monochromated Mo Kα radiation.

4.2. General Procedures for Synthesis

Compounds 2 and 3 were synthesized by using our reported methods.27

4.2.1. 4-Amino-3-(1-(2-Fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (5)

A mixture of 2-(2-fluoro-[1,1′-biphenyl]-4-yl)propanehydrazide (3) (0.500 g; 1.93 mmol), potassium hydroxide (0.108 g, 1.93 mmol), and carbon disulfide (0.147 g, 0.117 mL: 1.93 mmol) dissolved in ethanol (25 mL) was agitated for 16 h at room temperature. Ether was added to the mixture and the precipitated potassium 2-(2-(2-fluoro-[1,1′-biphenyl]-4-yl)propanoyl)hydrazinecarbodithioate (4) was collected by filtration. It was then washed with ether and dried under vacuum. The filtered precipitates thus obtained (0.500 g) were dissolved in hydrazine hydrate 20% (6 mL) and reacted under microwaves for 30 min until complete evolution of hydrogen sulfide. Dilute acetic acid (50 mL; 0.1%) was added to the resultant solution to get white precipitates, which were crystallized from ethanol; mp 196 °C; IR (KBr) cm–1: 3314, 3140, 1265; 1H NMR (DMSO-d6, 400 MHz): δ 1.58 (d, J = 7.2 Hz, 3H, CH3), 4.44 (q, J = 7.2 Hz, 1H, CH), 5.48 (s, 2H, NH2), 7.17–7.26 (m, 2H, ArH), 7.32–7.42 (m, 2H, ArH), 7.45–7.56 (m, 4H, ArH), 13.65 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.5, 34.9, 115.3 (d, J = 23.2 Hz), 123.9 (d, J = 3.0 Hz), 126.8 (d, J = 14.1 Hz), 127.9, 128.7, 128.8 (d, J = 3.0 Hz), 130.9 (d, J = 4.0 Hz), 135.0, 143.4 (d, J = 8.1 Hz), 154.2, 159.0 (d, J = 246.4 Hz), 166.4 ppm; Anal. Calcd for C16H15FN4S: C, 61.13; H, 4.81; N, 17.82. Found: C, 61.11; H, 4.77; N, 17.85; MS m/z: [M + H]+ 315.77.

4.2.2. General Procedure for the Synthesis of 4-(Benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thiones 6(a–m)

A mixture of 4-amino-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (5) (200 mg; 0.774 mmol), aromatic aldehyde (0.774 mmol), ethanol (25 mL), and glacial acetic acid (1–2 drops) was irradiated to reflux under microwaves till completion of the reaction. After removal of excess ethanol under vacuum, the contents were neutralized with ice-cooled aqueous sodium bicarbonate solution (4% w/w). The products were filtered and recrystallized from alcohol.

4.2.2.1. (E)-4-(Benzylideneamino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6a)

Off-white crystalline powder; mp 213 °C. IR (KBr) cm–1: 3083, 1579, 1219; 1H NMR (DMSO-d6, 400 MHz): δ 1.65 (d, J = 6.8 Hz, 3H, CH3), 4.55 (q, J = 6.8 Hz, 1H, CH), 7.20 (dd, J = 8.0 Hz, 1.2 Hz, 1H, ArH), 7.26 (dd, J = 11.6 Hz, 1.2 Hz, 1H, ArH), 7.39 (t, J = 6.4 Hz, 1H, ArH), 7.44–7.62 (m, 8H, ArH), 7.83 (d, J = 7.2 Hz, 2H, ArH), 9.90 (s, 1H, N=CH), 14.02 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.6, 35.8, 115.7 (d, J = 23.2 Hz), 124.3 (d, J = 3.3 Hz), 127.2 (d, J = 13.1 Hz), 128.3, 128.9, 129.0, 129.1 (d, J = 3.0 Hz), 129.6, 131.3 (d, J = 4.0 Hz), 133.1, 132.6, 135.2, 143.7 (d, J = 8.1 Hz), 153.4, 159.3 (d, J = 247.5 Hz), 162.1, 163.6 ppm; Anal. Calcd for C23H19FN4S: C, 68.63; H, 4.76; N, 13.92. Found: C, 68.23; H, 4.75; N, 13.89; MS m/z: [M + H]+ 403.20.

4.2.2.2. 4-((2-Chlorobenzylidene)amino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6b)

Yellow crystalline powder; mp 189 °C. IR (KBr) cm–1: 3058, 1582, 1222; 1H NMR (DMSO-d6, 400 MHz): δ 1.64 (d, J = 7.2 Hz, 3H, CH3), 4.60 (q, J = 7.2 Hz, 1H, CH), 7.21 (dd, J = 8.0, 1.6 Hz, 1H, ArH), 7.28 (dd, J = 11.9, 1.7 Hz, 1H, ArH), 7.32–7.52 (m, 7H, ArH) 7.55–7.64 (m, 2H, ArH), 8.02 (d, J = 7.6 Hz, 1H, ArH), 10.72 (s, 1H, N=CH), 14.08 (s, 1H, NH); 13C NMR (DMSO-d6, 101 MHz): δ 19.3, 35.4, 115.3 (d, J = 23.2 Hz), 123.9, 126.9 (d, J = 14.1 Hz), 127.6, 127.9, 128.0, 128.7, 128.8 (d, J = 3.0 Hz), 130.0, 130.4, 131.0 (d, J = 3.0 Hz), 134.1, 134.8, 135.2, 143.5 (d, J = 8.1 Hz), 153.4, 156.4, 159.0 (d, J = 247.5 Hz), 161.7 ppm; Anal. Calcd for C23H18ClFN4S: C, 63.22; H, 4.15; N, 12.82. Found: C, 63.23; H, 4.16; N, 12.79; MS m/z: [M + H]+ 437.58.

4.2.2.3. 4-((3-Chlorobenzylidene)amino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6c)

White powder; mp 190 °C. IR (KBr) cm–1: 3152, 1562, 1239; 1H NMR (DMSO-d6, 400 MHz): δ 1.64 (d, J = 6.8 Hz, 3H, CH3), 4.57 (q, J = 7.1 Hz, 1H, CH), 7.18 (d, J = 8.0 Hz, 1H, ArH), 7.26 (d, J = 11.6 Hz, 1H, ArH), 7.36–7.46 (m, 6H, ArH), 7.56 (t, J = 7.6 Hz, 1H, ArH), 7.66 (d, J = 7.6 Hz, 1H, ArH), 7.77 (d, J = 7.6 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 10.01 (s, 1H, N=CH), 14.04 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.6, 35.8, 115.7 (d, J = 23.2 Hz), 124.2 (d, J = 3.0 Hz), 127.2 (d, J = 13.1 Hz), 127.9, 128.3, 129.0, 129.1 (d, J = 3.0 Hz), 131.4 (d, J = 3.0 Hz), 131.5, 132.7, 134.4, 134.8, 135.1, 135.2, 143.8 (d, J = 8.1 Hz), 153.5, 159.4 (d, J = 247.5 Hz), 161.2, 162.2 ppm. Anal. calculated for C23H18ClFN4S: C, 63.22 H, 4.15 N, 12.82; Found: C, 63.21; H, 4.17; N, 12.83; MS m/z: [M + H]+ 437.93.

4.2.2.4. 4-((4-Chlorobenzylidene)amino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6d)

Light brown powder; mp 196 °C. IR (KBr) cm–1: 3085, 1554, 1220; 1H NMR (DMSO-d6, 400 MHz): δ 1.64 (d, J = 6.8 Hz, 3H, CH3), 4.58 (q, J = 7.1 Hz, 1H, CH), 7.21 (dd, J = 8.0, 1.6 Hz, 1H, ArH), 7.28 (dd, J = 11.9, 1.7 Hz, 1H, ArH), 7.39–7.48 (m, 6H, ArH), 7.84 (d, J = 7.6 Hz, 2H, ArH), 8.00 (d, J = 7.6 Hz, 2H, ArH), 10.43 (s, 1H, N=CH), 14.01 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.6, 35.8, 115.7 (d, J = 23.2 Hz), 124.0 (d, J = 3.0 Hz), 124.3, 126.9 (d, J = 13.1 Hz), 127.9, 128.7, 128.8 (d, J = 3.0 Hz), 129.7, 131.1 (d, J = 3.0 Hz), 134.8, 138.1, 143.3 (d, J = 8.1 Hz), 149.5, 153.3, 159.0 (d, J = 247.5 Hz), 159.2, 163.0 ppm; Anal. Calcd for C23H18ClFN4S: C, 63.22; H, 4.15; N, 12.82. Found: C, 63.21; H, 4.18; N, 12.83; MS m/z: [M + H]+ 437.07.

4.2.2.5. 3-(1-(2-Fluoro-[1,1′-biphenyl]-4-yl)ethyl)-4-((2-hydroxybenzylidene)amino)-1H-1,2,4-triazole-5(4H)-thione (6e)

Off-white powder; mp 195 °C. IR (KBr) cm–1: 3109, 1584, 1211; 1H NMR (DMSO-d6, 400 MHz): δ 1.62 (d, J = 7.2 Hz, 3H, CH3), 4.51 (q, J = 7.2 Hz, 1H, CH), 6.90–6.97 (m, 2H, ArH), 7.16 (dd, J = 8.0, 1.6 Hz, 1H, ArH), 7.23 (dd, J = 11.6, 1.6 Hz, 1H, ArH), 7.37–7.49 (m, 7H, ArH), 7.77 (dd, J = 7.6, 1.6 Hz, 1H, ArH), 9.01 (s, 1H, OH), 10.03 (s, 1H, N=CH), 13.94 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.3, 35.5, 115.3 (d, J = 23.2 Hz), 116.7, 118.4, 119.7, 123.8, 126.9 (d, J = 13.1 Hz), 127.2, 127.9, 128.7, 128.8 (d, J = 3.0 Hz), 131.0 (d, J = 3.0 Hz), 134.3, 134.9, 143.4 (d, J = 8.1 Hz), 152.9, 157.8, 158.5, 160.2, 161.6 ppm; Anal. Calcd for C23H19FN4OS: C, 66.01; H, 4.58; N, 13.39. Found: C, 66.03; H, 4.59; N, 13.41; MS m/z: [M + H]+ 419.23.

4.2.2.6. 3-(1-(2-Fluoro-[1,1′-biphenyl]-4-yl)ethyl)-4-((4-hydroxybenzylidene)amino)-1H-1,2,4-triazole-5(4H)-thione (6f)

White powder; mp 180 °C. IR (KBr) cm–1: 3142, 1575, 1210; 1H NMR (DMSO-d6, 400 MHz): δ 1.62 (d, J = 6.8 Hz, 3H, CH3), 4.58 (q, J = 7.1 Hz, 1H, CH), 6.92 (d, J = 7.8 Hz, 2H, ArH), 7.18 (dd, J = 8.0, 1.6 Hz, 1H, ArH), 7.25 (dd, J = 11.6, 1.6 Hz, 1H, ArH), 7.36–7.48 (m, 6H, ArH), 7.58 (d, J = 7.5 Hz, 2H, ArH), 9.98 (s, 1H, OH), 10.38 (s, 1H, N=CH), 14.00 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.3, 35.5, 111.2, 115.3 (d, J = 23.2 Hz), 118.6, 123.7, 126.5 (d, J = 13.1 Hz), 127.8, 128.7, 128.8 (d, J = 3.0 Hz), 130.3, 131.0 (d, J = 3.0 Hz), 134.9, 143.3 (d, J = 7.1 Hz), 152.5, 153.7, 159.0 (d, J = 246.5 Hz), 161.7, 163.0; Anal. Calcd for C23H19FN4OS: C, 66.01; H, 4.58; N, 13.39. Found: C, 65.99; H, 4.56; N, 13.43; MS m/z: [M + H]+ 419.48.

4.2.2.7. 4-((2,4-Dichlorobenzylidene)amino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6g)

Yellow powder; mp 204 °C. IR (KBr) cm–1: 3094, 1536, 1212; 1H NMR (DMSO-d6, 400 MHz): δ 1.65 (d, J = 7.2 Hz, 3H, CH3), 4.62 (q, J = 7.2 Hz, 1H, CH), 7.23 (dd, J = 8.0 Hz, 1.2 Hz, 1H, ArH), 7.28 (dd, J = 12.0 Hz, 0.8 Hz, 1H, ArH), 7.37–7.50 (m, 6H, ArH), 7.61 (dd, J = 8.4, 1.6 Hz, 1H, ArH), 7.83 (d, J = 2.0 Hz, 1H, ArH), 8.05 (d, J = 8.4 Hz, 1H, ArH), 10.78 (s, 1H, N=CH), 14.09 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.7, 35.7, 115.6 (d, J = 24.2 Hz), 124.3 (d, J = 3.0 Hz), 127.3 (d, J = 14.1 Hz), 128.3, 128.8, 129.0, 129.1, 129.2 (d, J = 3.0 Hz), 129.5, 130.3, 131.4 (d, J = 4.0 Hz), 135.2, 136.3, 138.2, 143.8 (d, J = 8.1 Hz), 153.8, 155.3, 159.4 (d, J = 247.5 Hz), 162.0 ppm; Anal. Calcd for C23H17Cl2FN4S: C, 58.60; H, 3.64; N, 11.89. Found: C, 58.60; H, 3.63; N, 11.92; MS m/z: [M + H]+ 472.18.

4.2.2.8. 4-((2,3-Dichlorobenzylidene)amino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6h)

Off-white powder; mp 226 °C. IR (KBr) cm–1: 3100, 1558, 1209; 1H NMR (DMSO-d6, 400 MHz): δ 1.65 (d, J = 7.2 Hz, 3H, CH3), 4.62 (q, J = 7.2 Hz, 1H, CH), 7.23 (dd, J = 8.0 Hz, 1.6 Hz, 1H, ArH), 7.30 (dd, J = 11.6 Hz, 1.6 Hz, 1H, ArH), 7.37–7.50 (m, 6H, ArH), 7.54 (t, J = 8.0 Hz, 1H, ArH), 7.87 (dd, J = 8.0 Hz, 1.6 Hz, 1H, ArH), 8.00 (dd, J = 8.0 Hz, 1.2 Hz, 1H, ArH), 10.15 (s, 1H, N=CH), 10.90 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.8, 35.7, 115.8 (d, J = 24.2 Hz), 124.3, 126.6, 127.2 (d, J = 17.1 Hz), 128.3, 129.0, 129.1 (d, J = 3.0 Hz), 129.2, 131.4, 133.0, 133.2 (d, J = 4.0 Hz), 134.3, 135.2, 143.9 (d, J = 8.1 Hz), 153.8, 155.7, 155.8, 159.3 (d, J = 247.5 Hz), 162.0 ppm; Anal. Calcd for C23H17Cl2FN4S: C, 58.60; H, 3.64; N, 11.89. Found: C, 58.63; H, 3.65; N, 11.92; MS m/z: [M + H]+ 471.00.

4.2.2.9. 3-(1-(2-Fluoro-[1,1′-biphenyl]-4-yl)ethyl)-4-((4-nitrobenzylidene)amino)-1H-1,2,4-triazole-5(4H)-thione (6i)

Dark yellow powder; mp 208 °C. IR (KBr) cm–1: 3098, 1579, 1278; 1H NMR (DMSO-d6, 400 MHz): δ 1.65 (d, J = 7.2 Hz, 3H, CH3), 4.59 (q, J = 7.2 Hz, 1H, CH), 7.23 (dd, J = 8.0 Hz, 1.6 Hz, 1H, ArH), 7.27 (dd, J = 11.6 Hz, 1.6 Hz, 1H, ArH), 7.35–7.48 (m, 6H, ArH), 8.09 (d, J = 8.8 Hz, 2H, ArH), 8.34 (d, J = 8.8 Hz, 2H, ArH), 10.32 (s, 1H, N=CH), 14.10 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.3, 35.4, 115.2 (d, J = 23.2 Hz), 124.0 (d, J = 3.0 Hz), 124.3, 126.9 (d, J = 13.1 Hz), 127.9, 128.7, 128.8 (d, J = 3.0 Hz), 129.7, 131.1 (d, J = 3.0 Hz), 134.8, 138.3, 143.3 (d, J = 8.1 Hz), 149.5, 153.3, 159.0 (d, J = 247.5 Hz), 159.2, 161.8 ppm; Anal. Calcd for C23H18 FN5O2S: C, 61.46; H, 4.48; N, 15.58. Found: C, 61.49; H, 4.49; N, 15.61; MS m/z: [M + H]+ 448.11.

4.2.2.10. 4-((4-(Dimethylamino)benzylidene)amino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6j)

Yellow-colored powder; mp 210 °C. IR (KBr) cm–1: 3133, 1554, 1199; 1H NMR (DMSO-d6, 400 MHz): δ 1.61 (d, J = 7.2 Hz, 3H, CH3), 3.02 (s, 6H, N(CH3)2), 4.44 (q, J = 7.2 Hz, 1H, CH), 6.77 (d, J = 8.8 Hz, 2H, ArH), 7.15 (dd, J = 8.0 Hz, 1.6 Hz, 1H, ArH), 7.20 (dd, J = 12.0 Hz, 1.2 Hz, 1H, ArH), 7.36–7.48 (m, 6H, ArH), 7.61 (d, J = 9.2 Hz, 2H, ArH), 9.27 (s, 1H, N=CH), 13.84 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.1, 35.5, 39.7, 111.6, 115.3 (d, J = 23.2 Hz), 118.7, 123.9, 126.8 (d, J = 13.1 Hz), 127.9, 128.7, 128.8 (d, J = 3.0 Hz), 130.4, 130.9 (d, J = 3.0 Hz), 134.9, 143.4 (d, J = 7.1 Hz), 152.7, 153.2, 159.0 (d, J = 246.5 Hz), 161.7, 165.0 ppm; Anal. Calcd for C25H24FN5S: C, 67.39; H, 5.43; N, 15.72. Found: C, 67.42; H, 5.45; N, 15.73; MS m/z: [M + H]+ 446.13.

4.2.2.11. 3-(1-(2-Fluoro-[1,1′-biphenyl]-4-yl)ethyl)-4-((4-fluorobenzylidene)amino)-1H-1,2,4-triazole-5(4H)-thione (6k)

White powder; mp 179 °C. IR (KBr) cm–1: 3176, 1511, 1236; 1H NMR (DMSO-d6, 400 MHz): δ 1.63 (d, J = 7.2 Hz, 3H, CH3), 4.53 (q, J = 7.2 Hz, 1H, CH), 7.19 (dd, J = 8.0 Hz, 1.6 Hz, 1H, ArH), 7.24 (dd, J = 12.0 Hz, 1.6 Hz, 1H, ArH), 7.36–7.48 (m, 8H, ArH), 7.90 (dd, J = 8.8 Hz, 5.6 Hz, 2H, ArH), 9.85 (s, 1H, N=CH), 14.01 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.2, 35.4, 115.3 (d, J = 24.2 Hz), 116.5 (d, J = 22.2 Hz), 124.0 (d, J = 3.0 Hz), 126.9 (d, J = 13.1 Hz), 127.9, 128.7, 128.8 (d, J = 3.0 Hz), 128.9 (d, J = 3.0 Hz), 131.0 (d, J = 4.0 Hz), 131.2 (d, J = 9.1 Hz), 134.8, 143.3 (d, J = 7.1 Hz), 153.0, 159.0 (d, J = 247.5 Hz), 161.7, 162.3, 164.8 (d, J = 252.5 Hz) ppm; Anal. Calcd for C23H18F2N4S: C, 65.70; H, 4.31; N, 13.32. Found: C, 65.71; H, 4.33; N, 13.35; MS m/z: [M + H]+ 421.47.

4.2.2.12. 4-((3,4-Dimethoxybenzylidene)amino)-3-(1-(2-fluoro-[1,1′-biphenyl]-4-yl)ethyl)-1H-1,2,4-triazole-5(4H)-thione (6l)

Brown powder; mp 170 °C. IR (KBr) cm–1: 3139, 1574, 1214; 1H NMR (DMSO-d6, 400 MHz): δ 1.62 (d, J = 7.2 Hz, 3H, CH3), 3.82–3.83 (2 s (merged), 6H, OCH3), 4.51 (q, J = 7.2 Hz, 1H, CH), 7.07 (d, J = 8.4 Hz, 1H, ArH), 7.16 (dd, J = 8.0 Hz, 1.6 Hz, 1H, ArH), 7.27 (dd, J = 11.6 Hz, 1.6 Hz, 1H, ArH), 7.32 (dd, J = 8.4 Hz, 2.0 Hz, 1H, ArH), 7.37–7.45 (m, 7H, ArH), 9.68 (s, 1H, N=CH), 13.94 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.2, 35.6, 55.5, 55.8, 108.6, 111.5, 115.4 (d, J = 23.2 Hz), 123.8 (d, J = 3.0 Hz), 124.7, 126.8 (d, J = 13.1 Hz), 127.9, 128.7, 128.8 (d, J = 3.0 Hz), 131.0 (d, J = 3.0 Hz), 134.8, 143.6 (d, J = 8.1 Hz), 149.2, 152.8, 152.9, 153.3, 159.0 (d, J = 247.5 Hz), 161.7, 163.0 ppm; Anal. Calcd for C25H23FN4O2S: C, 64.92; H, 5.01; N, 12.11; Found: C, 64.95; H, 5.02; N, 12.13; MS m/z: [M + H]+ 463.18.

4.2.2.13. 3-(1-(2-Fluoro-[1,1′-biphenyl]-4-yl)ethyl)-4-((furan-2-ylmethylene)amino)-1H-1,2,4-triazole-5(4H)-thione (6m)

Dark brown powder; mp 197 °C. IR (KBr) cm–1: 3141, 1569, 1221; 1H NMR (DMSO-d6, 400 MHz): δ 1.62 (d, J = 7.2 Hz, 3H, CH3), 4.46 (q, J = 7.2 Hz, 1H, CH), 6.76 (dd, J = 3.6 Hz, 2.0 Hz, 1H, ArH), 7.18 (dd, J = 8.0 Hz, 2.0 Hz, 1H, ArH), 7.24 (dd, J = 12.0 Hz, 1.6 Hz, 1H, ArH), 7.29 (dd, J = 3.6 Hz, 0.4 Hz, 1H, ArH), 7.35–7.40 (m, 1H, ArH), 7.42–7.50 (m, 5H, ArH), 8.06 (d, J = 2.0 Hz, 1H, ArH), 9.77 (s, 1H, N=CH), 13.98 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 101 MHz): δ 19.2, 35.3, 113.1, 115.5 (d, J = 24.2 Hz), 120.0, 124.0 (d, J = 3.0 Hz), 126.9 (d, J = 13.1 Hz), 127.9, 128.7, 128.8 (d, J = 3.0 Hz), 130.9 (d, J = 4.0 Hz), 134.9, 143.1 (d, J = 8.1 Hz), 147.4, 148.2, 151.5, 153.0, 159.0 (d, J = 247.5 Hz), 161.6 ppm; Anal. Calcd for C21H17FN4OS: C, 64.27; H, 4.37; N, 14.28. Found: C, 64.30; H, 4.38; N, 14.33; MS m/z: [M + H]+ 393.16.

4.3. X-ray Data Collection and Structure Determination

Crystal data for 6a: C23H19FN4S, M = 402.48, triclinic, a = 7.4736(4), b = 10.2302(5), c = 13.6434(7) Å, α = 100.3807(7), β = 100.3547(8), γ = 91.5795(8) °, U = 1007.40(9) Å3, T = 150(2) K, and space group P̅1. Data were collected on a Bruker APEX 2 CCDD diffractometer using graphite-monochromated Mo Kα radiation, λ = 0.71073 Å.36Z = 2, Dc = 1.327 g cm–3, F(000) = 420, yellow, dimensions 0.47 × 0.27 × 0.11 mm3, μ = 0.19 mm–1, 3.20 < 2θ < 61.2°, 16217 reflections measured, 6117 unique, and Rint = 0.018. The structure was solved by direct methods and refined by full-matrix least squares on F2.37,38wR2 = 0.116 (all data, 277 parameters); R1 = 0.040 [5213 data with F2 > 2σ(F2)]. The F atom was modeled as two-fold disordered at atoms C(14) and C(16) with major occupancy {75.5(2)%}at C(14). CCDC 1511104 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

4.4. Analgesic Activity Methods

4.4.1. Tail Flick Method

The analgesic activity was determined by the tail immersion method.30,31 The distal 5 cm portion of tails of swiss albino mice of either sex weighing between 20 and 40 g were marked and immersed into the water bath maintained at exactly 55 ± 1.0 °C. The reaction time for withdrawal of tails as a reflex action from the water was recorded with and without oral administration of the test compounds 6(a–m) along with standard flurbiprofen (10 mg/kg). The readings for each test compound were recorded for six albino mice at the time intervals of 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 h, which are tabulated in Table 2. All the results are presented as mean ± SEM, and one-way ANOVA considering P < 0.05 as significant is used to calculate the statistical significance.

4.4.2. Hot Plate Method

The central analgesic effect was measured by the hot plate method originally developed by MacDonald and co-workers.39 Animals (20–40 g) were divided into groups of six, and each Swiss albino mouse was placed individually on the surface of the hot plate maintained at 52 ± 1 °C until it licked its forepaw or jumped, and the time was noted cautiously. The cut off time in the absence of response was set to 15 s to prevent tissue damage. The first group served as the positive control with no oral administration; the second group of animals were given a dose of the standard flurbiprofen (10 mg/kg) while other groups were administered with the test compounds 6(a–m). Readings were recorded after each 30 min interval till 4.5 h and are given in Table 3. The analgesic activity for each mouse was calculated as a percentage of the maximum possible effect (MPE %), where MPE % = [{(test latency – control latency)/(cut-off point – control latency)} × 100].

Statistical Analysis: The results are presented as mean ± SEM, one-way ANOVA considering P < 0.05 as significant is used to calculate the statistical significance.

4.4.3. Acetic Acid Induced Writhing Method

Swiss albino mice divided into groups of six, weighing between 20 and 40 g, were used for the evaluation of analgesic activity by acetic acid induced writhing method.34,35 The animals of group I, serving as control, were treated with 10 mL/kg of normal saline administered orally, group II was treated with 10 mg/kg of flurbiprofen as the standard drug, while the rest of the groups were treated with 10 mg/kg of the test compounds. After 30 min, each mouse was administered with acetic acid (0.6% in normal saline) and was immediately shifted into a visible glass chamber for counting of induced writhes during the next 30 min. Percent analgesic activity of each group was determined as

4.4.3.

where X = average count of writhes for the control group and Y = average count of writhes for the group administered with test compound.

The results of acetic acid induced writhing are presented in Table 4.

Acknowledgments

The authors extended their gratitude to PCSIR Laboratories Complex, Lahore, and the University College of Pharmacy, University of Punjab, Lahore, for the provision of research facilities. No external funding was obtained for this research.

Glossary

Abbreviations

NMR

nuclear magnetic resonance

NSAIDs

non-steroidal anti-inflammatory drugs

GI

gastrointestinal

COX

cyclooxygenase

DMSO

dimethyl sulfoxide

mp

melting point

FT-IR

Fourier transform infrared

TLC

thin-layer chromatography

MPE

maximum possible effect

CCDC

Cambridge Crystallographic Data Centre

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c05222.

  • Procedures for the synthesis of compound 2 and 3, 1H NMR, 13C NMR, and mass spectra of the synthesized compounds (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao1c05222_si_001.pdf (1,004.4KB, pdf)

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