New Triazinoindole Bearing Thiazole/Oxazole Analogues Synthesis, Α-Amylase Inhibitory Potential and Molecular Docking Study

Bioorganic Chemistry 92 (2019) 103284

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Bioorganic Chemistry

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New triazinoindole bearing thiazole/oxazole analogues: Synthesis, α- T amylase inhibitory potential and molecular docking study ⁎ ⁎ Fazal Rahima, , Sundas Tariqa, Muhammad Tahab, , Hayat Ullaha, Khalid Zamana, Imad Uddina, Abdul Wadoodc, Aftab Ahmad Khana, Ashfaq Ur Rehmanc, Nizam Uddind, Salman Zafare, Syed Adnan Ali Shahf,g a Department of Chemistry, Hazara University, Mansehra 21300, Khyber Pakhtunkhwa, Pakistan b Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia c Department of Biochemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan d Department of Chemistry, University of Karachi, Karachi 75270, Pakistan e Institute of Chemical Science, University of Peshawar, Khyber Pakhtunkhwa, Pakistan f Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, 42300 Bandar Puncak Alam, Selangor D.E., Malaysia g Faculty of Pharmacy, Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, 42300 Bandar Puncak Alam, Selangor D.E., Malaysia

ARTICLE INFO ABSTRACT

Keywords: New triazinoindole bearing thiazole/oxazole analogues (1–21) were synthesized and characterized through Synthesis spectroscopic techniques such as HREI-MS, 1H and 13C NMR. The configuration of compound 2i and 2kwas Triazinoindole confirmed through NOESY. All analogues were evaluated against α-amylase inhibitory potential. Amongthe Thiazole synthesized analogues, compound 1h, 1i, 1j, 2a and 2f having IC50 values 1.80 ± 0.20, 1.90 ± 0.30, Oxazole 1.2 ± 0.30, 1.2 ± 0.01 and 1.30 ± 0.20 μM respectively, showed excellent α-amylase inhibitory potential α-amylase when compared with acarbose as standard (IC = 0.91 ± 0.20 µM). All other analogues showed good to Molecular docking 50 SAR moderate inhibitory potential. Structural activity relationship (SAR) has been established and binding interactions were confirmed through docking studies.

1. Introduction insulin demands, increasing the action of insulin at target tissue and inhibition of degradation of di- and oligosaccharides. Glucosidase in- Alpha-Amylase (E.C.3.2.1.1) is a metallo-enzyme containing Ca2+ hibitors such as biguanide, insulin, thiazolidinediones, aldose re- ions in its active site and speed up hydrolysis of starch into maltose and ductase, carbomoylmethyl benzoic acid, insulin like growth factor and glucose. This enzyme fascinated extensive attention due to its capability sulfonylureas are the clinically used drugs for controlling diabetes. of hydrolyzing α-1,4-glycosidic connection of starch and activities that Alpha-amylase and alpha-glucosidase are the enzymes that convert can be carried out due to hydrolysis. Some serious problems like obe- disaccharides and oligosaccharides into monosaccharide. Acarbose, sity, diabetes and oral illness are associated with the uptake of high miglitol and voglibose are the drug generally used to inhibit these en- quantity of carbohydrate [1–4]. Diabetes mellitus (DM) is a type zymes to prevent the absorption of glucose [5–8]. These drugs are chronic metabolic syndrome characterized by both fasting and post- usually used for type II diabetes and they have many adverse effects like prandial hyperglycemia and originate due to deficit in insulin action abdominal discomfort, diarrhoea, flatulence and meteorism. These (Type-II) or insulin secretion (Type-I) or both, thus encouraging dis- drugs are used along with other diabetic drugs for better efficacy. New order in metabolism of fats, proteins and carbohydrates. Other un- therapy with low risk of side effect for diabetes is the need to beex- controlled illnesses that are linked with long term problems of diabetes plored [9–12] (see Table 1). mellitus comprise microangiopathy, retinopathy, neuropathy and car- Compounds containing triazinoindole motif in their structure have diovascular disorders. Different therapeutic approaches used for cure of revealed to possess many biological applications like antiviral and an- diabetes are motivating endogenous secretion of insulin, reduction of timalarial. These compounds also showed antidepressant and

⁎ Corresponding authors. E-mail addresses: [email protected] (F. Rahim), [email protected] (M. Taha). https://doi.org/10.1016/j.bioorg.2019.103284 Received 10 April 2019; Received in revised form 12 September 2019; Accepted 14 September 2019 Available online 17 September 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved. F. Rahim, et al. Bioorganic Chemistry 92 (2019) 103284

Table 1 phenylethylidenehydrazine-1-carbothioamide (III) and (E)-2-(2-((3,5- Different analogues of triazinoindole bearing thiazole/oxazole. dihydro-2H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-phenylethylidene) hydrazine-1-carboxamide (IV) as third and fourth intermediate pro- S.No. R1 R2 S.No. R1 R2 ducts respectively. Different phenacyl bromides were then reacted with 1a 4-NO2 4-NO2 2a 4-NO2 4-NO2 intermediate (III) and (IV) separately in ethanol using trimethylamine 1b 4-OMe 4-OMe 2b 4-NO2 4-Br to obtain the final product having triazinoindole based thiazole (1a-j) 1c 4-OMe 3-NO2 2c 4-NO2 4-OMe and triazinoindole based oxazole derivatives (2a-k). TLC was used to 1d 4-OMe 4-Br 2d 4-NO2 3-NO2

1e 4-OMe 4-NO2 2e 4-NO2 4-CH3 monitor reaction completion. The product was washed with hexane and 1f 3-NO2 3-NO2 2f 4-NO2 2-NO2 the final product was collected. 1g 4-NO2 4-Br 2g 4-Br 4-Br Different structures of triazinoindole bearing thiazole/oxazole 1h 4-NO 3-NO 2h 4-Br 4-NO 2 2 2 analogues are provided in supplementary file as Table-S1. antihypertensive activities [13,14]. Triazinoindole based derivatives 2.2. In vitro α-amylase inhibitory potential of triazinoindole bearing showed broad spectrum antibacterial, antifungal [15], anti-in- thiazole/oxazole analogues flammatory and antihypoxic activities [16,17], and thus it got con- siderable interest to be explored more. Common cold treatment in- All the synthesized triazinoindole based thiazole analogues (1a-j) volves some drugs contains triazinoindole analogues [18–21] (see were screen against α-amylase. All derivatives exhibited good in- Fig. 1). hibitory potential ranging from 1.2 ± 0.30 to 19.50 ± 1.10 µM by We as a research group working on the synthesis of heterocyclic comparing with standard acarbose having IC50 value 0.91 ± 0.20 µM compounds since many years for exploring the lead molecules and had (Table 2). found excellent results [22–27]. Due to this consideration medicinal Structural activity relationship was established for all compounds and synthetic chemist has been reported various heterocyclic motifs as which depends upon the different substituents on the phenyl ring. in vitro anti-diabetic agents [28–30]. On the basis of rational drug de- Compound 1j was found most potent (IC50 = 1.2 ± 0.30 µM) signing, it is totally clear that both α-glucosidase and α-amylase is the having nitro group on the 4th position of the phenyl ring ‘‘A’’ while fascinating target to treat diabetes mellitus (DM). In connection with methyl on 4th position of phenyl ring ‘‘B’’ [Fig.-S1]. this declaration we have previously reported triazinoindole analogues If we compare 1a (IC50 = 3.50 ± 0.20 µM) with two nitro groups, as potent α-glucosidase inhibitors [31]. Thus, we decided to design both the nitro groups are present at 4th position on phenyl ring ‘‘A’’ and such compound library more effective to treat diabetes mellitus (DM). ‘‘B’’ with analogue 1 h (IC50 = 1.80 ± 0.20 µM) with two nitro groups With the hopes, here we synthesized compound library of triazi- with one at 4-position on ring ‘‘A’’ and other nitro group at 3-position noindole analogues with more aromatic character by introducing on ring ‘‘B’’. The difference in the inhibitory potential of these two thiazole/oxazole moieties in our previous work [31] (see Scheme 1). analogues may be due to the different position of the nitro groups on Correspondingly, the goal of this present work is to explore the α- phenyl rings [Fig.-S2]. amylase inhibitory activity of new triazinoindole bearing thiazole/ox- If we compared the inhibitory potential of analogues 1a azole analogues in search of potent anti-diabetic agent. (3.50 ± 0.20 µM) and 2a (1.2 ± 0.01 µM), the inhibitory potentials of analogue 2a was found many fold better. Both analogues have the same

NO2 substituents on both phenyl ring ‘’A’’ and ‘’B’’, the only difference is 2. Results and discussion the five member heterocycle. Analogue 1a have thiazole moiety while analogue 2a have oxazole moiety, the analogue 2a displayed better 2.1. Chemistry potentials with argue that O and N atom of oxazole in analogue 2a interacted with active site of enzyme through hydrogen bonding.

Isatin was reacted with thiosemicarbazide in distilled H2O using Although in analogue 1a thiazole moiety have N and S atoms, the S K2CO3 to obtained intermediate (I). Different phenacyl bromide were atom can make hydrogen bond and similarly exhibited a preference for then reacted with intermediate (I) in ethanol using K2CO3 to obtained perpendicular directional approach toward donor. Due to this reason 2-(5H-[1,2,4]triazino[5,6-b]indole-3-ylthiol)-1-phenylethanone as in- hydrogen bond of S is weaker then O atom [32]. Thus analog 1a ex- termediate (II) [31]. Semicarbazide and thiosemicarbazide were hibited less potentials as compare to analogue 2a [Fig.-S3]. treated separately with intermediate (II) in ethanol using acetic acid to Comparing 1c (IC50 = 5.20 ± 0.40 µM) with methoxy at 4th posi- obtained (E)-2-(5H-[1,2,4]triazino[5, 6-b]indol-3-ylthio)-1- tion on ring ‘‘A’’ and nitro at 3rd position on ring ‘‘B’’ with analogue 1e

Fig. 1. Rationalization of the newly synthesized triazinoindole bearing thiazole/oxazole analogues with already reported triazinoindole analogues.

2 F. Rahim, et al. Bioorganic Chemistry 92 (2019) 103284

Scheme 1. Synthesis of triazinoindole bearing thiazole/oxazole analogues.

Table 2 than analogue 2e (IC50 = 4.60 ± 0.30 µM). Although, both analogue α-amylase activity of triazinoindole bearing thiazole (1a-j) and triazinoindole have the same basic skeleton, the only difference in their structures are bearing oxazole analogues (2a-k). the five member heterocycle (thiazole/oxazole) [Fig.-S5].

a a S.NO. IC50 ± SEM [µM] S.NO. IC50 ± SEM [µM] In the whole study, we have concluded that electron withdrawing or electron donating groups on the phenyl ring play an important role in 1a 3.50 ± 0.20 2a 1.2 ± 0.01 the potential. Position and number of the substituents also play an 1b 8.50 ± 0.5 2b 11.50 ± 0.6 important role in inhibition of the enzyme. Molecular docking study 1c 5.20 ± 0.40 2c 4.80 ± 0.40 1d 19.50 ± 1.10 2d 3.70 ± 0.30 was carried out to understand the binding interaction of the all com- 1e 5.50 ± 0.40 2e 4.60 ± 0.30 pounds. 1f 3.80 ± 0.30 2f 1.30 ± 0.20 1g 18.90 ± 1.20 2g N.A. 2.3. Docking studies 1h 1.80 ± 0.20 2h 16.80 ± 0.70 1i 1.90 ± 0.30 2i 17.40 ± 0.80 1j 1.2 ± 0.30 2j 15.80 ± 0.70 Molecular docking study was conducted in order to explore the Acarbose 0.91 ± 0.20 2k 21.50 ± 1.2 binding pattern of the synthesized triazinoindole bearing thiazole and oxazole derivatives in the active site of the Porcine alpha-amylase enzyme using the PDB code 1OSE. Generally, the docking results de- (IC50 = 5.50 ± 0.40 µM) having a methoxy group at 4-position on monstrate that all triazinoindole bearing thiazole and oxazole deriva- phenyl ring ‘‘A’’ and nitro groups at 4-position on phenyl ring ‘‘B’’. This tives had various substituted groups including electron withdrawing difference in activity in these two derivatives is due to the different groups (EWG) and electron donating groups (EDG) over benzene ring. position of nitro group on the phenyl ring ‘‘B’’ [Fig.-S4]. We have noticed that the variation of substituted groups with respect to Magnetic shielding around oxazole and thiazole moiety effect their their position ultimately alter the enzyme activity at different level. The aromaticity and bonding of analogues, by this consideration oxazole is docking results for triazinoindole bearing thiazole derivatives revealed leat aromatic while thiazole is most aromatic [33]. With this possible that all the compounds showed favourable interaction and best in- argue analogue 1j (IC50 = 1.2 ± 0.30 µM) exhibited better potentials hibitory activity against the corresponding enzyme. The most potent

3 F. Rahim, et al. Bioorganic Chemistry 92 (2019) 103284

Fig. 2. The ligand-protein interaction (LPI) profile for synthesized triazinoindole bearing thiazole derivatives against Porcine alpha amylase enzyme (PDBID 1OSE). (A) The binding mode of the most potent compound 1j in the series with active site resiudes, (B) for 1i, (C) for 1h. The binding mode of the most potent compound in the series of triazinoindole bearing oxazole derivatives (D) for compound 2a and (E) for compound 2f. (F) The docking conformation for least compound 1d triazinoindole bearing thiazole.

compound in the series (1j & 1i) showed not only best fit-well pattern of 3. Conclusion binding and activity, but also, we have observed favourable interactions with the active site residues. In case of compound 1j includes Arg195, Herein this work two compound library of new triazinoindole H299, and hydrophobic side chain Trp59 respectively as shown in bearing thiazole (1a-1j) and oxazole (2a-2k) analogues were synthe- Fig. 2A. While in case of 1i includes Arg195, Lys200 and Trp59 sized in search of potent anti-diabetic agent. In considerations all (Fig. 2B). Both the compounds showed share a common residues in- analogues were screened for their α-amylase inhibitory potential under teraction with active site residues (Arg195 and Trp59), which possess positive control of acarbose as standard drug (IC50 = 0.91 ± 0.20 µM). difference in one additional residue; H299 in caseof 1j while Lys200 in Among the synthesized analogues, analogue 1h, 1i, 1j, 2a and 2f case of 1i. It was well noticed that the high potency of these compounds having IC50 values 1.80 ± 0.20, 1.90 ± 0.30, 1.2 ± 0.30, might be due to the EDG’s and EWDG’s. The oxygen lone pair is well- 1.2 ± 0.01 and 1.30 ± 0.20 μM respectively showed excellent α- placed to delocalize and increase electron density within the benzene amylase inhibitory potential when compared with standard drug acar- conjugated system. This allows delocalization to better stabilize (+ve)- bose. SAR study was established for inhibitory potentials of analogues, charges over the benzene, and hence enhance the inhibitory activity of rationalized on the basis of various substituents on phenyl ring A and B the corresponding enzyme. Furthermore, in case of other potent com- and even through five membered heterocyle like thiazole and oxazole. pound 1h showed favourable interactions with active site residues Overall results show that analogues 1h, 1i, 1j, 2a and 2f having IC50 (Fig. 2C). The high potency might be due to mechanism of withdrawing values 1.80 ± 0.20, 1.90 ± 0.30, 1.2 ± 0.30, 1.2 ± 0.01 and electron from the benzene ring through inductive effect, and remain the 1.30 ± 0.20 μM respectively emerged with most potent α-amylase in- benzene ring partial positive, the consequences further trigger and hibitory potential and act as lead candidate for in vitro study in future. unable the benzene ring to adopts pi-stacking interaction with other groups to re-gain stabilization. 4. Material and methods Similarly, the docking results of triazinoindole bearing oxazole derivatives involved in various favourable interactions with residues in- 4.1. General procedure for the synthesis of triazinoindole based thiazole/ cluding Trp58-59, Tyr62, Leu162, Arg195, Asp197, Glu233, Asp300 oxazole analogues and Asp356 etc. Interactions and docking scores enlisted in Table-S2.

Compound 2a exhibited good interaction with active site residues in- Isatin was reacted with thiosemicarbazide in distilled H2O using cluding Tyr62, Thr163, Asp197 and Asp356 (Fig. 2D). The good in- K2CO3 to obtained intermediate (I). Different phenacyl bromide were hibitory potential of the compound 2a might be due to the position of then reacted with intermediate (I) in ethanol using K2CO3 to obtained the nitro moiety as compared to compound 2f (Fig. 2E). The feature on 2-(5H-[1,2,4]triazino[5,6-b]indole-3-ylthiol)-1-phenylethanone as in- these molecules is the presence of EWG. The docking conformation for termediate (II) [31]. Semicarbazide and thiosemicarbazide were least compound 1d triazinoindole bearing thiazole shown in Fig. 2F, treated separately with intermediate (II) in ethanol using acetic acid to which exhibited just a pi-pi interaction with active site residue W59, obtained (E)-2-(5H-[1,2,4]triazino[5, 6-b]indol-3-ylthio)-1-pheny- and it might give us clue about the least activity and potency of this lethylidenehydrazine-1-carbothioamide (III) and (E)-2-(2-((3,5-di- compound. hydro-2H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-phenylethylidene) Overall, the molecular docking results demonstrates that the com- hydrazine-1-carboxamide (IV) as third and fourth intermediate pro- pounds occupied by both EWG’s and EDG’s substituted groups showed ducts respectively. Different phenacyl bromides were then reacted with favourable pattern of interaction with active site residues. While those, intermediate (III) and (IV) separately in ethanol using trimethylamine occupied by either same EWG’s or EDG’s showed different activities to obtain the final product having triazinoindole based thiazole (1a-j) again the corresponding enzyme. Additionally, the molecular docking and triazinoindole based oxazole derivatives (2a-k). TLC was used to results of triazinoindole bearing thiazole and oxazole moiety best cor- monitor reaction completion. The product was washed with hexane and relate with biological activities of the compounds. the final product was collected.

4 F. Rahim, et al. Bioorganic Chemistry 92 (2019) 103284

+ 4.1.1. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- m/z Calcd for C27H20N8O3S2 [M] 568.1099; Found: 568.1087. nitro phenyl) ethylidene) hydrazinyl)-4-(4-nitrophenyl) thiazole (1a) 1 H NMR (500 MHz, DMSO‑d6): δ 12.50 (s, NH, 1H), 11.92 (s, NH, 4.1.6. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(3- 1H), 8.33 (dd, J = 7.3, 1.4 Hz, 2H, Ar), 8.22 (d, J = 7.4 Hz, 1H, Ar), nitrophenyl) ethylidene) hydrazinyl)-4-(3-nitrophenyl) thiazole (1f) 1 8.17 (d, J = 7.3 Hz, 2H, Ar), 8.11 (dd, J = 7.2, 1.3 Hz, 2H, Ar), 8.03 H NMR (500 MHz, DMSO‑d6): δ 12.39 (s, NH, 1H), 11.73 (s, NH, (dd, J = 7.5, 1.4 Hz, 2H, Ar), 7.60 (m, 1H, Ar), 7.46 (d, J = 7.3 Hz, 1H, 1H), 8.57 (s, 1H, Ar), 8.49 (s, 1H, Ar), 8.29 (d, J = 7.2 Hz, 1H, Ar), 8.26 13 Ar), 7.39 (m, 1H, Ar), 7.23 (s, 1H, CH), 4.82 (s, 2H, SCH2). C NMR (d, J = 7.1 Hz, 1H, Ar), 8.24 (d, J = 7.3 Hz, 1H, Ar), 8.18 (d, (125 MHz, DMSO‑d6): δ 172.1, 171.1, 156.1, 149.9, 149.9, 148.5, J = 7.5 Hz, 1H, Ar), 8.13 (d, J = 7.3 Hz, 1H, Ar), 7.79 (t, J = 7.1 Hz, 144.6, 144.0, 139.9, 135.1, 126.9, 126.9, 126.0, 126.0, 125.6, 125.6, 1H, Ar), 7.66 (t, J = 7.3 Hz, 1H, Ar), 7.61 (m, 1H, Ar), 7.47 (d, 123.9, 123.9, 121.9, 121.0, 120.1, 118.8, 110.5, 106.0, 101.5, 30.9. J = 7.2 Hz, 1H, Ar), 7.30 (m, 1H, Ar), 7.12 (s, CH, 1H, Ar), 4.76 (s, 2H, + 13 HREI-MS: m/z Calcd for C26H17N9O4S2 [M] 583.0844; Found: SCH2). CNMR (125 MHz, DMSO‑d6): δ 177.4, 175.6, 154.7, 151.4, 583.0831. 147.3, 145.2, 144.5, 137.6, 136.7, 135.5, 134.6, 133.9, 132.4, 127.5, 127.5, 126.5, 125.8, 124.9, 123.6, 121.6, 118.1, 116.9, 116.7, 109.5, + 4.1.2. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- 102.6, 32.2: HREI-MS: m/z Calcd for C26H17N9O4S2 [M] 583.0844; methoxyphenyl) ethylidene) hydrazinyl)-4-(4-methoxyphenyl) Found: 583.0831. thiazole (1b) 1 H NMR (500 MHz, DMSO‑d6): δ 12.48 (s, NH, 1H), 11.90 (s, NH, 4.1.7. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- 1H), 8.19 (d, J = 7.4 Hz, 1H, Ar), 7.88 (dd, J = 7.2, 1.2 Hz, 2H, Ar), nitrophenyl) ethylidene) hydrazinyl)-4-(4-bromophenyl) thiazole 7.50 (m, 1H, Ar), 7.39 (d, J = 7.1 Hz, 1H, Ar), 7.33 (dd, J = 7.3, 1.3 Hz, (1 g) 1 2H, Ar), 7.21 (m, 1H, Ar), 7.15 (s, CH, 1H), 7.01 (d, J = 7.4 Hz, 2H, Ar), HNMR (500 MHz, DMSO‑d6): δ 12.31 (s, NH, 1H), 11.55 (s, NH, 13 6.98 (d, J = 7.6 Hz, 2H, Ar), 4.81 (s, 2H, SCH2), 3.78 (s, OCH3, 6H). C 1H), 8.31 (dd, J = 7.3, 1.4 Hz, 2H, Ar), 8.23 (d, J = 7.2 Hz, 1H), 8.04 NMR (125 MHz, DMSO‑d6): δ 172.9, 171.7, 165.6, 161.4, 159.8, 155.1, (dd, J = 7.5, 1.3 Hz, 2H, Ar), 7.71 (dd, J = 7.1, 1.1 Hz, 2H, Ar), 7.56 150.0, 147.5, 135.3, 135.3, 133.9, 133.9, 130.6, 128.4, 127.0, 125.1, (m, 1H, Ar), 7.42 (d, J = 7.3 Hz, 1H, Ar), 7.40 (dd, J = 7.1, 1.2 Hz, 2H, 13 122.3, 122.0, 119.8, 119.8, 118.3, 118.3, 112.8, 108.1, 104.0, 50.9, Ar), 7.29 (m, 1H, Ar), 7.11 (s, CH, 1H, Ar), 4.81 (s, 2H, SCH2). CNMR + 50.9, 30.7: HREI-MS: m/z Calcd for C28H23N7O2S2 [M] 553.1354; (125 MHz, DMSO‑d6): δ 172.1, 170.3, 154.2, 152.3, 152.3, 147.4, Found: 553.1341. 146.9, 141.1, 136.0, 133.5, 133.5, 129.6, 127.5, 126.9, 125.8, 125.8, 124.9, 123.8, 121.6, 121.6, 119.5, 118.9, 113.7, 105.8, 103.6, 32.7: + 4.1.3. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- HREI-MS: m/z Calcd for C26H17BrN8O2S2 [M] 616.0099; Found: methoxyphenyl) ethylidene) hydrazinyl)-4-(3-nitrophenyl) thiazole 616.0083; [M + 2] 618.0072. (1c) 1 HNMR (500 MHz, DMSO‑d6): δ 12.51 (s, NH, 1H), 11.94 (s, NH, 4.1.8. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- 1H), 8.57 (s, 1H, Ar), 8.29 (d, J = 7.5 Hz, 1H, Ar), 8.26 (d, J = 7.4 Hz, nitrophenyl) ethylidene) hydrazinyl)-4-(3-nitrophenyl) thiazole (1 h) 1 1H, Ar), 8.20 (d, J = 7.2 Hz, 1H, Ar), 7.89 (dd, J = 7.3, 1.2 Hz, 2H, Ar), HNMR (500 MHz, DMSO‑d6): δ 12.37 (s, NH, 1H), 11.49 (s, NH, 7.81 (d, J = 7.1 Hz, 1H, Ar), 7.65 (m, 1H, Ar), 7.55 (d, J = 7.4 Hz, 1H, 1H), 8.58 (s, 1H, Ar), 8.31 (dd, J = 7.2, 1.5 Hz, 2H, Ar), 8.28 (d, Ar), 7.38 (m, 1H, Ar), 7.23 (s, 1H, CH, Ar), 7.03 (d, J = 7.2 Hz, 2H, Ar), J = 7.4 Hz, 1H, Ar), 8.23 (d, J = 7.5 Hz, 1H, Ar), 8.16 (d, J = 7.3 Hz, 13 4.78 (s, 2H, SCH2), 3.77 (s, OCH3, 3H). CNMR (125 MHz, DMSO‑d6): 1H, Ar), 8.02 (dd, J = 7.1, 1.3 Hz, 2H, Ar), 7.70 (t, J = 7.4 Hz, 1H, Ar), δ 178.1, 175.3, 170.2, 163.8, 162.9, 153.1, 150.1, 149.2, 140.0, 137.1, 7.59 (m, 1H, Ar), 7.37 (d, J = 7.1 Hz, 1H, Ar), 7.27 (m, 1H, Ar), 7.14 (s, 13 135.1, 131.9, 131.9, 130.1, 128.9, 127.0, 125.9, 123.7, 121.9, 121.9, CH, 1H, Ar), 4.74 (s, 2H, SCH2). CNMR (125 MHz, DMSO‑d6): δ 118.8, 118.8, 114.9, 109.4, 106.8, 60.9, 30.6: HREI-MS: m/z Calcd for 179.7, 172.6, 156.7, 154.9, 154.9, 147.9, 144.8, 143.7, 141.8, 139.6, + C27H20N8O3S2 [M] 568.1099; Found: 568.1087. 135.7, 132.6, 128.7, 128.7, 127.5, 126.9, 123.9, 123.9, 123.4, 121.3, 118.5, 118.5, 113.8, 109.5, 107.6, 30.6: HREI-MS: m/z Calcd for + 4.1.4. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- C26H17N9O4S2 [M] 583.0844; Found: 583.0831. methoxyphenyl)ethylidene)hydrazinyl)-4-(4-bromophenyl)thiazole (1d) 4.1.9. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- 1 H NMR (500 MHz, DMSO‑d6): δ 12.47 (s, NH, 1H), 11.83 (s, NH, nitrophenyl) ethylidene) hydrazinyl)-4-(4-methoxyphenyl) thiazole 1H), 8.28 (d, J = 7.3 Hz, 1H), 7.86 (d, J = 7.2 Hz, 2H), 7.72 (d, (1i) 1 J = 7.5 Hz, 2H), 7.63 (m, 1H), 7.50 (d, J = 7.1 Hz, 1H), 7.46 (dd, HNMR (500 MHz, DMSO‑d6): δ 12.29 (s, NH, 1H), 11.33 (s, NH, J = 7.6, 1.3 Hz, 2H), 7.38 (m, 1H), 7.18 (s, CH, 1H), 7.01 (dd, J = 7.4, 1H), 8.27 (dd, J = 7.1, 1.3 Hz, 2H, Ar), 8.21 (d, J = 7.3 Hz, 1H, Ar), 13 1.4 Hz, 2H), 4.78 (s, 2H, SCH2), 3.72 (s, OCH3, 3H). CNMR (125 MHz, 8.04 (dd, J = 7.5, 1.5 Hz, 2H, Ar), 7.51 (m, 1H, Ar), 7.37 (d, J = 7.3 Hz, DMSO‑d6): δ 178.1, 176.8, 175.6, 167.2, 160.1, 155.1, 150.5, 147.8, 1H, Ar), 7.32 (d, J = 7.4 Hz, 2H, Ar), 7.24 (m, 1H, Ar), 7.11 (s, 1H, CH), 147.8, 143.6, 140.4, 140.4, 138.5, 138.5, 135.1, 133.5, 132.6, 132.1, 7.01 (dd, J = 7.1, 1.2 Hz, 2H, Ar), 4.84 (s, 2H, SCH2). 3.75 (s, OCH3, 13 128.7, 127.9, 124.5, 124.5, 121.0, 119.6, 115.4, 66.8, 31.2: HREI-MS: 3H). CNMR (125 MHz, DMSO‑d6): δ 177.6, 175.9, 161.9, 154.6, + m/z Calcd for C27H20BrN7OS2 [M] 601.0354; Found: 601.0340. 601; 154.6, 153.9, 149.8, 143.6, 141.8, 127.9, 127.9, 127.6, 126.7, 125.6, [M+2] 603.0341. 125.6, 123.9, 123.5, 123.5, 118.7, 116.5, 115.8, 115.8, 115.6, 108.7, + 105.8, 58.7, 31.5: HREI-MS: m/z Calcd for C27H20N8O3S2 [M] 4.1.5. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- 568.1099; Found: 568.1088. methoxyphenyl)ethylidene)hydrazinyl)-4-(4-nitrophenyl) thiazole (1e) 4.1.10. (E)-2-(2-(2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)-1-(4- 1 HNMR (500 MHz, DMSO‑d6): δ 12.43 (s, NH, 1H), 11.73 (s, NH, nitrophenyl) ethylidene) hydrazinyl)-4-p-tolylthiazole (1j) 1 1H), 8.27 (d, J = 7.3 Hz, 1H), 8.22 (d, J = 7.2 Hz, 2H), 8.12 (d, HNMR (500 MHz, DMSO‑d6): δ 12.38 (s, NH, 1H), 11.57 (s, NH, J = 7.4 Hz, 2H), 7.85 (d, J = 7.5 Hz, 2H), 7.61 (m, 1H), 7.46 (d, 1H), 8.28 (dd, J = 7.1, 1.4 Hz, 2H, Ar), 8.23 (d, J = 7.5 Hz, 1H, Ar), J = 7.2 Hz, 1H), 7.33 (m, 1H), 7.17 (s, CH, 1H), 7.02 (d, J = 7.4 Hz, 8.01 (dd, J = 7.2, 1.2 Hz, 2H, Ar), 7.77 (dd, J = 7.3, 1.1 Hz, 2H, Ar), 13 2H), 4.76 (s, 2H, SCH2), 3.69 (s, OCH3, 3H). CNMR (125 MHz, 7.57 (m, 1H, Ar), 7.41 (d, J = 7.4 Hz, 1H, Ar), 7.25 (m, 1H, Ar), 7.17 (s, DMSO‑d6): δ 173.8, 172.9, 169.3, 166.1, 162.2, 159.6, 155.5, 151.6, CH, 1H), 7.13 (dd, J = 7.1, 1.1 Hz, 2H, Ar), 4.84 (s, 2H, SCH2). 2.22 (s, 13 148.5, 140.6, 140.6, 137.9, 136.5, 136.5, 133.7, 133.7, 128.6, 124.4, 3H, CH3). CNMR (125 MHz, DMSO‑d6): δ 178.6, 172.8, 155.9, 155.9, 122.9, 121.8, 118.4, 118.4, 115.7, 109.5, 105.7, 59.8, 30.8: HREI-MS: 154.6, 146.9, 145.6, 141.9, 134.9, 132.8, 127.6, 127.6, 126.5, 126.5,

5 F. Rahim, et al. Bioorganic Chemistry 92 (2019) 103284

126.2, 125.9, 125.9, 123.4, 123.2, 123.2, 119.7, 118.3, 115.9, 109.0, 4.1.16. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- + 103.2, 31.5, 21.4: HREI-MS: m/z Calcd for C27H20N8O2S2 [M] nitrophenyl) ethylidene) hydrazinyl)-4-(2-nitrophenyl) oxazole (2f) 1 552.1150; Found: 552.1138. HNMR (500 MHz, DMSO‑d6): δ 12.44 (s, NH, 1H), 11.34 (s, NH, 1H), 8.31 (d, J = 7.6 Hz, 2H, Ar), 8.21 (d, J = 7.2 Hz, 1H, Ar), 8.03 (d, 4.1.11. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- J = 7.3 Hz, 2H, Ar), 8.01 (d, J = 7.7 Hz, 1H, Ar), 8.00 (d, J = 7.4 Hz, nitrophenyl) ethylidene) hydrazinyl)-4-(4-nitrophenyl) oxazole (2a) 1H, Ar), 7.87 (m, 2H, Ar), 7.52 (m, 2H, Ar), 7.49 (d, J = 7.1 Hz, 1H, 1 13 HNMR (500 MHz, DMSO‑d6): δ 12.40 (s, NH, 1H), 11.57 (s, NH, Ar), 7.65 (s, 1H, Ar), 4.71 (s, 2H, SCH2). CNMR (125 MHz, DMSO‑d6): 1H), 8.34 (d, J = 6.2 Hz, 2H, Ar), 8.30 (d, J = 7.4 Hz, 1H, Ar), 8.27 (d, δ 170.9, 155.6, 150.6,150.2, 148.8, 144.2, 142.6, 140.1, 140.1, 139.9, J = 7.9 Hz, 2H, Ar), 8.07 (d, J = 6.6 Hz, 2H, Ar), 7.97 (d, J = 6.8 Hz, 135.3, 132.6, 129.6, 127.7, 127.7, 127.0, 127.0, 125.2, 124.4, 122.2, 2H, Ar), 7.70 (m, 2H, Ar), 7.68 (s, 1H, Ar), 7.57 (d, J = 8.1 Hz, 1H, Ar), 121.7, 119.9, 119.8, 111.1, 102.1, 37.8: HREI-MS: m/z Calcd for 13 + 4.78 (s, 2H, SCH2). CNMR (125 MHz, DMSO‑d6): δ 170.9, 155.6, C26H17N9O5S [M] 567.1073; Found: 567.1061. 150.6, 150.2, 147.9, 144.2, 142.6, 140.1, 140.1, 139.9, 136.8, 127.7, 127.7, 127.0 ,127.0, 126.2, 126.2, 124.4,124.4, 122.2 ,121.7, 119.9, 4.1.17. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- + 119.8, 111.1, 102.1, 30.8: HREI-MS: m/z Calcd for C26H17N9O5S [M] bromophenyl) ethylidene) hydrazinyl)-4-(4-bromophenyl) oxazole (2 g) 1 567.1073; Found: 567.1061. HNMR (500 MHz, DMSO‑d6): δ 12.40 (s, NH, 1H), 11.46 (s, NH, 1H), 8.23 (d, J = 7.5 Hz, 1H, Ar), 7.78 (d, J = 7.7 Hz, 2H, Ar), 7.70 (d, J = 7.4 Hz, 2H, Ar), 7.66 (s, 1H, Ar), 7.64 (d, J = 7.2 Hz, 2H, Ar), 7.62 4.1.12. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- (m, 2H, Ar), 7.50 (d, J = 7.7 Hz, 1H, Ar), 7.55 (d, J = 7.3 Hz, 2H, Ar), nitrophenyl) ethylidene) hydrazinyl)-4-(4-bromophenyl) oxazole (2b) 13 1 4.77 (s, 2H, SCH2), CNMR (125 MHz, DMSO‑d6): δ 170.9, 155.6, HNMR (500 MHz, DMSO‑d6): δ 12.33 (s, NH, 1H), 11.47 (s, NH, 150.6, 144.2, 142.6, 140.1, 139.9, 133.0, 132.1, 132.1, 131.7, 131.7, 1H), 8.32 (d, J = 8.3 Hz, 2H, Ar), 8.29 (d, J = 8.7 Hz, 1H, Ar), 7.91 (d, 129.7, 128.6, 128.6, 128.3, 128.3, 125.4, 123.1, 122.2, 121.7, 119.9, J = 8.9 Hz, 2H, Ar), 7.73 (d, J = 6.9 Hz, 2H, Ar), 7.69 (s, 1H, Ar), 7.62 119.8, 111.1, 102.1, 37.8: HREI-MS: m/z Calcd for C26H17Br2N7OS (m, 2H, Ar), 7.57 (d, J = 7.9 Hz, 1H, Ar), 7.54 (d, J = 6 Hz, 2H, Ar), + 13 [M] 632.9582; Found: 632.9572; [M + 2] 634.9568; [M + 4] 4.78 (s, 2H, SCH2). CNMR (125 MHz, DMSO‑d6): δ 170.9, 636.9547. 155.6,150.6, 150.2, 144.2, 142.6, 140.1, 140.1, 139.9, 132.1, 132.1, 129.7, 128.3, 128.3, 127.7,127.7, 127.0, 127.0, 123.1, 122.2, 121.7, 4.1.18. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- 119.9, 119.8, 111.1, 102.1, 30.8: HREI-MS: m/z Calcd for + bromophenyl) ethylidene) hydrazinyl)-4-(4-nitrophenyl) oxazole C26H17BrN8O3S [M] 600.0327; Found: 600.0316; [M + 2] 602.0316. (2 h) 1 HNMR (500 MHz, DMSO‑d6): δ 12.46 (s, NH, 1H), 11.55 (s, NH, 4.1.13. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- 1H), 8.27 (d, J = 7.6 Hz, 1H, Ar), 8.22 (d, J = 7.5 Hz, 2H, Ar), 7.97 (d, nitrophenyl) ethylidene) hydrazinyl)-4-(4-methoxyphenyl) oxazole J = 7.1 Hz, 2H, Ar), 7.69 (d, J = 7.7 Hz, 2H, Ar), 7.59 (s, 1H, Ar), 7.57 (2c) 1 (d, J = 7.4 Hz, 2H, Ar), 7.53 (d, J = 7.0 Hz, 1H, Ar), 7.50 (m, 2H, Ar), HNMR (500 MHz, DMSO‑d6): δ 12.45 (s, NH, 1H), 11.37 (s, NH, 13 4.88 (s, 2H, SCH2), CNMR (125 MHz, DMSO‑d6): δ 170.9, 155.6, 1H), 8.30 (d, J = 7.5 Hz, 2H, Ar), 8.26 (d, J = 7.6 Hz, 1H, Ar), 8.04 (d, 150.6, 147.9, 144.2, 142.6, 140.1, 139.9, 136.8, 132.1, 133.0, 131.7, J = 7.7 Hz, 2H, Ar), 7.66 (s, 1H, Ar), 7.63 (m, 2H, Ar), 7.51 (d, 131.7, 128.6, 128.6,126.2, 126.2,125.4, 124.4, 124.4, 122.2, 119.9, J = 7.3 Hz, 1H, Ar), 7.49 (d, J = 7.6 Hz, 2H, Ar), 7.03 (d, J = 7.2 Hz, 13 119.8, 111.1, 102.1, 37.8: HREI-MS: m/z Calcd for C26H17BrN8O3S 2H, Ar), 4.88 (s, 2H, SCH ), 3.80 (s, 3H, OCH ), CNMR (125 MHz, + 2 3 [M] 600.0328; Found: 600.0319; [M + 2] 602.0311. DMSO‑d6): δ 178.1, 175.3, 170.2, 163.8 162.9, 153.1, 150.1, 149.2, 140.0, 137.1, 135.1, 131.1, 130.1, 128.9, 128.9, 128.9, 127.0, 127.0, 4.1.19. (Z)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- 125.9, 123.7, 121.9, 118.8, 114.9, 109.4, 106.8, 56.5, 30.6: HREI-MS: bromophenyl) ethylidene) hydrazinyl)-4-(4-methoxyphenyl) oxazole m/z Calcd for C H N O S [M]+ 552.1328; Found: 552.1315. 27 20 8 4 (2i) 1 HNMR (500 MHz, DMSO‑d6): δ 12.46 (s, NH, 1H), 11.55 (s, NH, 4.1.14. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- 1H), 8.24 (d, J = 8.4 Hz, 1H, Ar), 7.67 (d, J = 7.9 Hz, 2H, Ar), 7.61 (s, nitrophenyl) ethylidene) hydrazinyl)-4-(3-nitrophenyl) oxazole (2d) 1H, Ar), 7.60 (d, J = 8.1 Hz, 2H, Ar), 7.48 (d, J = 8.8 Hz, 1H, Ar), 7.44 1 HNMR (500 MHz, DMSO‑d6): δ 12.49 (s, NH, 1H), 11.40 (s, NH, (d, J = 8.4 Hz, 2H, Ar), 7.41 (m, 2H, Ar), 7.02 (d, J = 6.9 Hz, 2H, Ar), 13 1H), 8.60 (s, 1H, Ar), 8.29 (d, J = 7.8 Hz, 2H, Ar), 8.25 (d, J = 7.7 Hz, 4.71 (s, 2H, SCH2), 3.87 (s, 3H, OCH3). CNMR (125 MHz, DMSO‑d6): 1H, Ar), 8.22 (d, J = 7.4 Hz, 1H, Ar), 8.20 (d, J = 7.5 Hz, 1H, Ar), 8.02 δ 170.9, 160.6, 156.8, 150.6, 144.3, 142.4, 140.1, 139.9, 133.0, 131.7, (d, J = 7.3 Hz, 2H, Ar), 7.80 (t, J = 7.9 Hz, 1H, Ar), 7.61 (m, 2H, Ar), 131.7, 128.7, 128.7, 128.6, 128.6, 125.4, 123.0, 122.2, 121.5, 119.9, 7.49 (d, J = 7.1 Hz, 1H, Ar), 7.63 (s, 1H, Ar), 4.77 (s, 2H, SCH2). 119.8, 114.8, 114.8, 111.1, 102.1, 55.7, 30.6: HREI-MS: m/z Calcd for 13 + CNMR (125 MHz, DMSO‑d6): δ 170.9, 155.6, 150.6, 150.2, 148.4, C27H20BrN7O2S [M] 585.0582; Found: 585.0571; [M + 2] 587.0562. 144.2, 142.6, 140.1,140.1, 139.9, 133.9, 133.6, 130.6, 127.7, 127.7, The NOESY spectrum of compound 2i shows a strong correlation 127.0, 127.0, 123.9, 122.7, 122.2 ,121.7, 119.9, 119.8, 111.1, 102.1, between the imine N-H proton (12.42 ppm) and the methylene protons + 30.6: HREI-MS: m/z Calcd for C26H17N9O5S [M] 567.1073; Found: (3.63 ppm), suggesting a z-configuration at the imine functional group. 567.1061. 4.1.20. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- 4.1.15. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- bromophenyl) ethylidene) hydrazinyl)-4-(3-nitrophenyl) oxazole (2j) 1 nitrophenyl) ethylidene) hydrazinyl)-4-(p-tolyl) oxazole (2e) HNMR (500 MHz, DMSO‑d6): δ 12.32 (s, NH, 1H), 11.44 (s, NH, 1 HNMR (500 MHz, DMSO‑d6): δ 12.43 (s, NH, 1H), 11.50 (s, NH, 1H), 8.53 (s, 1H, Ar), 8.30 (d, J = 6.4 Hz, 1H, Ar), 8.28 (d, J = 8.4 Hz, 1H), 8.32 (d, J = 7.8 Hz, 2H, Ar), 8.29 (d, J = 7.4 Hz, 1H, Ar), 8.06 (d, 1H, Ar), 7.92 (d, J = 6.4 Hz, 1H, Ar), 7.82 (t, J = 6.2 Hz, 1H, Ar), 7.69 J = 7.5 Hz, 2H, Ar), 7.63 (s, 1H, Ar), 7.58 (d, J = 7.4 Hz, 2H, Ar), 7.52 (d, J = 6.2 Hz, 2H, Ar), 7.67 (s, 1H, Ar), 7.44 (d, J = 8.3 Hz, 2H, Ar),

(d, J = 7.3 Hz, 1H, Ar), 7.45 (m, 2H, Ar), 7.11 (d, J = 7.3 Hz, 2H, Ar), 7.22 (d, J = 6.4 Hz, 1H, Ar), 7.39 (m, 2H, Ar), 4.78 (s, 2H, SCH2). 13 13 4.81 (s, 2H, SCH2), 2.29 (s, 3H, CH3). CNMR (125 MHz, DMSO‑d6): δ CNMR (125 MHz, DMSO‑d6): δ 170.9, 155.6, 150.6, 148.4, 144.2, 170.9, 155.6, 150.6, 150.2, 144.2, 142.6, 140.1, 140.1, 142.6, 140.1, 139.9, 133.9, 133.6, 133.0, 131.7, 131.7, 130.6, 128.6 139.9,131.7,129.5, 129.5, 127.7, 127.7, 127.7, 127.0, 127.0, 125.7, ,128.6, 125.4, 123.9, 122.7, 122.2, 121.7, 119.9, 119.8, 111.1, 102.1, + 125.7, 122.2, 121.7, 119.9, 119.8, 111.1, 102.1, 37.8, 21.3: HREI-MS: 30.6: HREI-MS: m/z Calcd for C26H17BrN8O3S [M] 600.0327; Found: + m/z Calcd for C27H20N8O3S [M] 536.1379; Found: 536.1367. 600.0315; [M + 2] 602.0327

6 F. Rahim, et al. Bioorganic Chemistry 92 (2019) 103284

4.1.21. (E)-2-(2-(2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(4- References bromophenyl) ethylidene) hydrazinyl)-4-(p-tolyl) oxazole (2 k) 1 HNMR (500 MHz, DMSO‑d6): δ 12.36 (s, NH, 1H), 11.31 (s, NH, 1H), [1] A. Sundarram, T.P.K. Murthy, J. Appl. Environ. Microbiol. 2 (2014) 166–175. 8.25 (d, J = 7.3 Hz, 1H, Ar), 7.66 (d, J = 7.5 Hz, 2H, Ar), 7.63 (s, 1H, Ar), [2] P.M. Sales, P.M. Souza, L.A. Simeoni, P.O. Magalhaes, D. Silveira, J. Pharm. Pharmaceut. Sci. 15 (2012) 141–183. 7.59 (d, J = 7.1 Hz, 2H, Ar), 7.51 (d, J = 7.2 Hz, 2H, Ar), 7.46 (d, [3] M. Taha, M.T. Javid, S. Imran, M. Selvaraj, S. Chigurupati, H. Ullah, F. Rahim, J = 7.3 Hz, 1H, Ar), 7.42 (m, 2H, Ar), 7.14 (d, J = 7.4 Hz, 2H, Ar), 4.71 (s, J.I. Mohammad, K.M. Khan, Bioorg. Chem. 74 (2017) 179–186. 13 [4] S.S. Nair, V. Kavrekar, A. Mishra, Eur. J. Exp. Biol. 3 (2013) 128–132. 2H, SCH2), 3.19 (s, 3H, CH3). CNMR (125 MHz, DMSO‑d6): δ 170.9, [5] I.G. Tamil, B. Dineshkumar, M. Nandhakumar, M. Senthilkumar, A. Mitra, Ind. J. 155.8, 150.6, 144.2, 142.6, 140.1, 139.9, 133.0, 131.7, 131.7,131.7, 129.5, Pharm. 42 (2010) 280–282. 129.5, 128.6, 128.6, 127.7,125.7,125.7,125.4,122.2, 121.7, 119.8, 119.8, [6] S. Shahidpour, F. Panahi, R. Yousefi, M. Nourisefat, M. Nabipoor, A. KhalafiNezhad, + Med. Chem. Res. 24 (2015) 3086–3096. 111.1, 102.1, 30.6, 21.3: HREI-MS: m/z Calcd for C27H20BrN7OS [M] [7] M.N. Wickramaratne, J.C. Punchihewa, D.B.M. Wickramaratne, BMC Compl. 569.0633; Found: 569.0622; [M + 2] 571.0610. No such cross peaks could Alternat. Med. 16 (2016) 466–470. be seen in compound 2k between the imine N-H and methylene protons, [8] K. Alagesan, P.K. Raghupathi, S. Sankarnarayanan, IJPLS 3 (2012) 1407–1412. [9] F. Ali, K.M. Khan, U. Salar, M. Taha, N.H. Ismail, A. Wadood, M. Riaz, S. Perveen, which suggests an E-configuration at the same site in 2k. Eur. J. Med. Chem. 138 (2017) 255–272. [10] F. Rahim, H. Ullah, M.T. Javid, A. Wadood, M. Taha, M. Ashraf, A. Shaukat, M. Junaid, S. Hussain, W. Rehman, R. Mehmood, M, Sajid, M.N. Khan, K.M. Khan, 4.2. Molecular docking Bioorg. Chem. 62 (2015) 15–21. [11] F. Rahim, F. Malik, H. Ullah, A. Wadood, F. Khan, M.T. Javid, M. Taha, W. Rehman, Molecular docking study was conducted by using Molecular A.U. Rehman, K.M. Khan, Bioorg. Chem. 60 (2015) 42–48. [12] M. Taha, S. Imran, N.H. Ismail, M. Selvaraj, F. Rahim, S. Chigurupati, H. Ullah, Operating Environment (MOE) software package [34,35], in order to F. Khan, U. Salar, M.T. Javid, S. Vijayabalan, K. Zaman, K.M. Khan, Bioorg. Chem. explore the binding mode of the synthesized compounds in the active 74 (2017) 1–9. [13] S. Shelke, S. Bhosale, Bioorg. Med. Chem. Lett. 20 (2010) 4661. site of the amylase enzyme. The structural coordinates of Porcine alpha [14] A. Monge, J.A. Palop, C. Ramierz, M. Font, A.E. Fernandez, Eur. J. Chem. 26 (1991) amylase (PDB code 1OSE) was retrieved from Protein Data Bank 179. (www.rcsb.org). Solvent were removed and 3D protonation and energy [15] J.L. Kgokong, P.P. 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