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Synthesis: Small library of hybrid scaffolds of benzothiazole having hydrazone and evaluation of their β-glucuronidase activity
Bioorganic Chemistry 77 (2018) 47–55
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Synthesis: Small library of hybrid scaffolds of benzothiazole having hydrazone and evaluation of their b-glucuronidase activity Muhammad Taha a,⇑, Mastura Arbin b,c, Norizan Ahmat b,c, Syahrul Imran b,c, Fazal Rahim d a
Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam Campus, Malaysia c Faculty of Applied Science Universiti Teknologi MARA, 40450 ShahAlam, Selangor D.E, Malaysia d Department of Chemistry, Hazara University, Mansehra 21120, Pakistan b
a r t i c l e
i n f o
Article history: Received 7 August 2017 Revised 30 November 2017 Accepted 2 January 2018 Available online 4 January 2018 Keywords: Benzothiazole Synthesis b-Glucuronidase activity SAR
a b s t r a c t Due to the great biological importance of b-glucuronidase inhibitors, here in this study, we have synthesized a library of novel benzothiazole derivatives (1–30), characterized by different spectroscopic methods and evaluated for b-glucuronidase inhibitory potential. Among the series sixteen compounds i.e. 1–6, 8, 9, 11, 14, 15, 20–23 and 26 showed outstanding inhibitory potential with IC50 value ranging in between 16.50 ± 0.26 and 59.45 ± 1.12 when compared with standard D-Saccharic acid 1,4-lactone (48.4 ± 1.25 µM). Except compound 8 and 23 all active analogs showed better potential than the standard. Structure activity relationship has been established. Ó 2018 Elsevier Inc. All rights reserved.
1. Introduction The chemistry of fused ring heterocycles is recognized to be a key field of investigation in medicinal chemistry, subsequently, they have been establishing to show enhanced biological activity. Heterocyclic compounds display important role in medicinal chemistry and their structures in natural products. One of them is benzothiazole scaffold which have gain considerable attention in synthetic organic chemistry with valuable applications in the pharmaceutical industry. From the literature survey, benzothiazole analog have shown numerous biological activities [1–3] used in material science and in other wide range industrial applications [4,5]. Furthermore, these benzothiazole compounds have also shown enzymes inhibition, like protoporphyrinogen oxidase, which give free radicals that result in simultaneous toxicological effects on plants and animals [6,7]. The benzothiazole is an exceptional scaffold for further molecular study to synthesize innovative compounds. Benzothiazole analogs have been reported as antimicrobial agents [8,9], if having pyrazole or isatin moiety then exhibit significant antimicrobial potential [10–13]. b-Glucuronidase is an exoglycosidase enzyme catalyzes the cleavage of glucuronosyl-O-bonds [14]. This enzyme is broadly distributed in anaerobic bacteroides, also occur in human blood cells, ⇑ Corresponding author. E-mail address: [email protected] (M. Taha). https://doi.org/10.1016/j.bioorg.2018.01.002 0045-2068/Ó 2018 Elsevier Inc. All rights reserved.
gastric juice, body fluids, spleen, serum, urine and organs like liver, lung, bile kidney and muscle [15,16]. The deficit of b-glucuronidase in human body leads to Sly syndrome which is linked with the increase of glycosaminoglycans in cells [17,18]. Certain hepatic diseases and AIDS are reported due to the overreaction of the enzyme. Though, it over expressed in some pathological conditions like cancer, inflammation, epilepsy, in hepatic as well as renal diseases, in joints, bladder, and neoplasm of breast, testes, and larynx [19–21]. Literature survey revealed that the bacterial b-glucuronidase inhibitor lead to a reduction in carcinogen prompted colonic tumors [22]. Consequently, inhibitors of this enzyme are immediate need of the day to cure numerous pathological conditions. Recently our group has reported various classes of heterocyclic compounds as a potent inhibitor of b-glucuronidase [23]. In this connection here we are reporting the synthesis of new benzothiazole analogs and its b-glucuronidase inhibitory potential. 2. Experimental All nuclear magnetic resonance experiments had been carried out using an AvanceBruker 600 MHz. Elemental analysis was performed on Carlo Erba Strumentazion-Mod-1106, Italy. Electron impact mass spectra (EI-MS) were recorded on a Finnigan MAT-311A, Germany. Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany).
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M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
2.1. Synthetic procedure of methyl 4-(5-chlorobenzo[d]thiazol-2-yl) benzoate (I) The methyl 4-(5-chlorobenzo[d]thiazol-2-yl) benzoate (I) was prepared by the treatment of aldehyde (1 mmol) and 4-chloro-2aminothiophenol (1 mmol) in the presence of DMF for 5 h. After completion of reaction as determined by TLC analysis, the contents were cooled and dried. The impurities were removed washing with ethyl acetate and hexane. 2.2. Synthetic procedure of 4-(5-chlorobenzo[d]thiazol-2-yl)benzohydrazide (II) In second step, 4-(5-chlorobenzo[d]thiazol-2-yl) benzohydrazide (II) was synthesized by treating of the equimolar amount of methyl 4-(5-chlorobenzo[d]thiazol-2-yl) benzoate (I) (1 mmol) and hydrazine hydrate (1 mmol) in the presence of methanol for 6 h. The reaction completion was monitored by TLC. 2.3. Synthetic procedure of (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 (3-hydroxybenzylidene) benzohydrazide (1–30) In third step, 4-(5-chlorobenzo[d]thiazol-2-yl)benzohydrazide (II) (1 mmol) was further treated with different benzaldehydes (1 mmol) in the presence of acetic acid for 3 h. The reaction completion was monitored by TLC. Different spectroscopic techniques such as ESI-MS, 1H NMR and 13C NMR were used to determine the structure of all analogs. 2.3.1. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3hydroxybenzylidene)benzohydrazide (1) White solid, Yield: 81%. 1H NMR (500 MHz, DMSO d6):d 8.32 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.10 (d, J = 8.8 Hz, 2H, Ar), 7.94 (d, J = 8.6 Hz, 2H, Ar), 7.88 (d, J = 8.01 Hz, 1H, Ar), 7.54 (d, J = 7.8 Hz, 1H, Ar), 7.43 (s, 1H, Ar), 7.37 (d, J = 6.8 Hz, 1H, Ar), 7.23 (t, 1H, Ar), 7.01 (d, J = 6.6 Hz, 1H, Ar), 5.34 (s, 1H, AOH); 13C NMR (126 MHz, DMSO d6): d 170.2, 164.3, 159.4, 156.7, 147.3, 146.6, 139.3, 133.4, 132.5, 131.6, 131.4, 130.5, 130.3, 128.3, 128.1, 125.2, 123.5, 122.4, 121.6, 115.5, 118.4; HR-ESI-MS: m/z calcd for C21H14ClN3O2S, [M]+ 407.0752; Found 407.0751. 2.3.2. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2,3-dihydroxybenzylidene)benzohydrazide (2) White solid, Yield: 81%. 1H NMR (500 MHz, DMSO d6):d H 8.75 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.10 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.6 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.53 (d, J = 7.8 Hz, 1H, Ar), 7.20 (s, 1H, Ar), 6.83 (d, J = 7.2 Hz, 1H, Ar), 6.80 (t, J = 7.2 Hz, 1H, Ar), 5.33 (s, 2H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 157.7, 146.6, 156.2, 152.4, 147.4, 147.1, 146.2, 134.1, 133.2, 131.4, 130.6, 130.4, 128.2, 128.1, 125.5, 125.1, 124.2, 123.4, 122.1, 120.5, 120.2; HR-ESI-MS: m/z calcd for C21H14ClN3O3S, [M]+ 423.0503; Found 423.0501. 2.3.3. (E)-4-(5-chlorobenzo[d]thiazole-2-yl)-N0 -(4-hydroxybenzylidene) benzohydrazide (3) Yellow solid, Yield: 86%. 1H NMR (500 MHz, DMSO d6): dH 8.34 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.11 (d, J = 8.8 Hz, 2H, Ar), 7.96 (d, J = 8.6 Hz, 2H, Ar), 7.92 (d, J = 8.01 Hz, 1H, Ar), 7.75 (d, J = 8.1 Hz, 2H, Ar), 7.54 (d, J = 7.8 Hz, 1H, Ar), 6.83 (d, J = 7.3 Hz, 2H, Ar), 5.34 (s, 1H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 161.4, 156.2, 147.3, 147.2, 133.4, 132.0, 131.3, 131.3, 130.8, 130.6, 130.6, 128.4, 128.4, 127.2, 125.2, 124.2, 122.1, 117.1, 117.1; HRESI-MS: m/z calcd for C21H14ClN3O2S, [M]+ 407.0315; Found 408.0312.
2.3.4. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2,5-dihydroxybenzylidene)benzohydrazide (4) Yellowish solid, Yield: 83%. 1H NMR (500 MHz, DMSO d6):dH 8.73 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.08 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.5 Hz, 2HAr), 7.93 (d, J = 8.01 Hz, 1H, Ar), 7.55 (d, J = 7.8 H z, 1H, Ar), 7.27 (s, 1H, Ar), 6.83(d, J = 6.9 Hz, 2H, Ar), 5.33 (s, 2H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 154.4, 152.3, 147.2, 146.4, 133.4, 132.2, 131.4, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 122.1, 121.4, 120.2, 119.8, 117.3; HRESI-MS: m/z calcd for C21H14ClN3O3S, [M]+ 423.0404; Found 423.0401. 2.3.5. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3,4,dihydroxybenzylidene)benzohydrazide (5) White solid, Yield: 84%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.07 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.6 Hz, 2H, Ar), 7.92 (d, J = 8.01 Hz, 1H, Ar), 7.53 (d, J = 7.8 Hz, 1H, Ar), 7.32 (d, J = 8.1 Hz, 1H, Ar), 7.26 (s, 1H, Ar), 6.82 (d, J = 7.0 1 Hz, 1H, Ar), 5.32 (s, 2H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 150.5, 147.4, 147.1, 146.2, 133.4, 133.2, 132.0, 131.6, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 123.6, 118.4, 117.3; HR-ESI-MS: m/z calcd for C21H14ClN3O3S, [M]+ 423.0314; Found 424.0311. 2.3.6. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2,4-dihydroxybenzylidene)benzohydrazide (6) Yellowish solid, Yield: 82%. 1H NMR (500 MHz, DMSO d6):dH 8.73 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.06 (d, J = 8.8 Hz, 2H, Ar), 7.94 (d, J = 8.5 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.62 (d, J = 7.02 Hz, 1H, Ar), 7.52 (d, J = 7.8 Hz, 1H, Ar), 7.50 (s, 1H, Ar), 6.38 (d, J = 6.2 Hz, 1H, Ar), 5.34 (s, 2H, AOH); 13C NMR (126 MHz, DMSO d6): dC170.2, 164.3, 162.8, 162.7, 156.2, 147.1, 146.4, 134.1, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 121.4, 111.6, 109.3, 104.1; HR-ESI-MS: m/z calcd for C21H14ClN3O3S, [M]+ 423.0412; Found 424.0414. 2.3.7. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-methylbenzylidene) benzohydrazide (7) Yellowish solid, Yield: 76%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.23 (s, 1H, Ar), 8.06 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.6 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar) 7.53 (d, J = 7.8 H z, 1H, Ar), 7.67 (d, J = 8.1 Hz, 2H, Ar), 7.23 (d, J = 7.9 Hz, 2H, Ar), 2.31 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.4, 147.1, 141.1, 133.4, 132.0, 131.2, 130.8, 130.6, 130.6, 129.6, 129.6, 128.4, 128.4, 126.7, 126.7, 125.2, 124.2, 121.4, 21.7; HR-ESI-MS: m/z calcd for C22H16ClN3OS, [M]+ 405.0518; Found 406.0515. 2.3.8. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-nitrobenzylidene) benzohydrazide (8) Yellow solid, Yield: 82%. 1H NMR (500 MHz, DMSO d6):dH 8.32 (s, 1H, ACH), 8.29 (d, J = 8.3 Hz, 2H, Ar), 8.21 (s, 1H, Ar), 8.05 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H, Ar), 7.94 (d, J = 8.6 Hz, 2H, Ar), 7.92 (d, J = 8.02 Hz, 1H, Ar), 7.54 (d, J = 7.8 Hz, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 150.7, 147.4, 147.1, 140.2, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.6, 124.6, 124.5, 124.3, 124.2, 124.2; HR-ESI-MS: m/z calcd for C21H13ClN4O3S, [M]+ 436.0321; Found 437.0318. 2.3.9. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2-hydroxybenzylidene) benzohydrazide (9) Brown solid, Yield: 72%. 1H NMR (500 MHz, DMSO d6):dH 8.73 (s, 1H, ACH), 8.20 (s, 1H, Ar), 8.06 (d, J = 8.8 Hz, 2H, Ar), 7.93 (d, J = 8.6 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.64 (d, J = 7.8 Hz, 1H, Ar), 7.59 (d, J = 8.4 Hz, 1H, Ar), 7.04 (m, 1H, Ar), 7.48 (m, 1H), 7.01 (d, J = 7.2 Hz, 1H, Ar), 5.34 (s, 1H, AOH); 13C NMR (150 MHz,
M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
DMSO d6): dC 170.2, 164.3, 157.6, 156.2, 147.1, 146.6, 133.4, 132.8, 132.0, 130.8, 130.6, 130.6, 128.4, 128.1, 125.2, 124.2, 122.1, 121.6, 119.1, 118.2; HR-ESI-MS: m/z calcd for C21H14ClN3O2S, [M]+ 407.7312; Found 408.7311. 2.3.10. (E)-methyl4-((2-(4-(5-chlorobenzo[d]thiazol-2-yl)benzoyl) hydrazono)methyl)benzoate (10) Brown solid, Yield: 74%. 1H NMR (500 MHz, DMSO d6):dH 8.33 (s, 1H, Ar), 8.22 (s, 1H, Ar), 8.18 (d, J = 8.4 Hz, 2H, Ar), 8.04 (d, J = 8.7 Hz, 2H, Ar), 7.95 (d, J = 8.4 Hz, 2H, Ar), 7.92 (d, J = 8.01 Hz, 1H, Ar), 7.90 (d, J = 8.01 Hz, 2H, Ar), 7.51 (d, J = 7.4 Hz, 1H, Ar), 3.85 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC170.2, 166.2, 164.3, 156.2, 147.3, 147.0, 138.6, 133.4, 132.8, 132.0, 130.8, 130.6, 130.6, 130.4, 130.4, 129.6, 129.6, 128.4, 128.4, 125.2, 124.2, 121.6, 52.2; HR-ESI-MS: m/z calcd for C23H16ClN3O3S, [M]+ 449.0390; Found 449.0388. 2.3.11. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-chlorobenzylidene) benzohydrazide (11) White solid. Yield: 78%. 1H NMR (500 MHz, DMSO d6):d H 8.31 (s, 1H, ACH), 8.20 (s, 1H, Ar), 8.04 (d, J = 8.2 Hz, 2H, Ar), 7.94 (d, J = 8.1 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.74 (d, J = 7.9 Hz, 2H, Ar), 7.53 (d, J = 7.8 Hz, 1H, Ar), 7.49 (d, J = 8.7 Hz, 2H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.2, 147.0, 137.3, 133.4, 132.2, 132.0, 131.1, 131.1, 130.8, 130.6, 130.6, 129.4, 129.4, 128.4, 128.4, 125.2, 124.2, 121.6; HR-ESI-MS: m/z calcd for C21H13Cl2N3OS, [M]+ 425.0315; Found 425.0318. 2.3.12. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-hydroxy-2-iodo-4methoxybenzylidene)benzohydrazide (12) Yellowish solid. Yield: 73%. 1H NMR (500 MHz, DMSO d6):d H 8.32 (s, 1H, Ar), 8.22 (s, 1H, Ar), 8.03 (d, J = 8.3 Hz, 2H, Ar), 7.93 (d, J = 8.1 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.54 (d, J = 7.8 Hz, 1H, Ar), 7.01 (d, J = 7.4 Hz, 1H, Ar), 6.83 (d, J = 7.2 Hz, 1H, Ar), 5.33 (s, 1H, AOH), 3.80 (s, 3H, ACH3); 13C NMR (150 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 155.8, 152.4, 147.0, 143.8, 134.2, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 125.2, 124.2, 121.6, 111.8, 86.5, 56.6; HR-ESI-MS: m/z calcd for C22H15ClN3O3S, [M]+ 563.0327; Found 563.0324. 2.3.13. (E)-N0 -(3-bromo-4-hydroxybenzylidene)-4-(5-chlorobenzo[d] thiazol-2-yl)benzohydrazide (13) White solid. Yield: 78%. 1H NMR (500 MHz, DMSO d6):d H 8.32 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.05 (d, J = 8.3 Hz, 2H, Ar), 7.94 (d, J = 8.1 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.78 (s, 1H, Ar), 7.70 (d, J = 8.01 Hz, 1H, Ar), 7.52 (d, J = 7.6 Hz, 1H, Ar), 6.89 (d, J = 7.1 Hz, 1H, Ar), 5.32 (s, 1H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 159.4, 156.2, 147.2, 147.0, 133.4, 132.0, 130.8, 130.7, 130.6, 130.6, 130.1, 129.1, 128.4, 128.4, 125.2, 124.2, 121.6, 118.8, 114.3; HR-ESI-MS: m/z calcd for C21H13BrClN3O2S, [M]+ 485.0432; Found 486.0429. 2.3.14. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2-hydroxy-5methoxybenzylidene)benzohydrazide (14) White solid. Yield: 87%. 1H NMR (500 MHz, DMSO d6): dH 8.74 (s, 1H, ACH), 8.19 (s, 1H, Ar), 8.07 (d, J = 8.4 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.7 Hz, 1H, Ar), 7.30 (s, 1H), 6.86 (d, J = 7.1 Hz, 1H, Ar), 6.87 (d, J = 6.9 Hz, 1H, Ar), 5.32 (s, 1H, AOH), 3.78 (s, 3H, ACH3); 13C NMR (150 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 153.8, 153.6, 147.2, 146.4, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 121.6, 120.1, 118.6, 117.8, 113.9, 56.2; HR-ESI-MS: m/z calcd for C22H16ClN3O3S, [M]+ 437.0510; Found 437.0514.
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2.3.15. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(pyridine-2-ylmethylene)benzohydrazide (15) White solid. Yield: 86%. 1H NMR (500 MHz, DMSO d6):d H 8.62 (d, J = 8.4 Hz, 1H, Ar), 8.19 (s, 1H, Ar), 8.04 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.1 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.8 Hz, 1H, Ar), 7.81 (d, J = 8.1 Hz, 1H, Ar), 7.76 (t, 1H), 7.61 (t, 1H), 7.10 (s, 1H, ACH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 154.3, 149.7, 147.2, 145.4, 136.7, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 126.8, 125.2, 124.2, 121.6, 120.6; HR-ESI-MS: m/z calcd for C20H13ClN4OS, [M]+ 392.0280; Found 392.0277. 2.3.16. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(pyridine-3-ylmethylene)benzohydrazide (16) White solid. Yield: 83%. 1H NMR (500 MHz, DMSO d6):dH 8.66 (d, J = 8.4 Hz, 1H, Ar), 8.30 (s, 1H, ACH), 8.26 (d, J = 8.2 Hz, 1H, Ar), 8.21 (s, 1H, Ar), 8.05 (d, J = 8.2 Hz, 2H, Ar), 7.94 (d, J = 8.3 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.54 (t, 1H, Ar), 7.52 (d, J = 7. 6 Hz, 1H, Ar), 9.04 (s, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 152.4, 149.7, 147.2, 144.8, 134.4, 133.4, 132.0, 130.8, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.4, 124.2, 121.6; HR-ESI-MS: m/z calcd for C20H13ClN4OS, [M]+ 392.0623; Found 392.0621. 2.3.17. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-methylbenzylidene) benzohydrazide (17) Yellowish solid. Yield: 85%. 1H NMR (500 MHz, DMSO d6):dH 8.31 (s, 1H, ACH), 8.20 (s, 1H, Ar), 8.04 (d, J = 8.2 Hz, 2H, Ar), 7.93 (d, J = 8.3 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.64 (s, 1H, Ar), 7.61 (d, J = 7.4 Hz, 1H, Ar), 7.52 (d, J = 7.7 Hz, 1H, Ar), 7.38 (t, 1H, Ar), 7.26 (d, J = 6.9 Hz, 1H, Ar), 2.32 (s, 3H, ACH3); 13C NMR (150 MHz, DMSO d6): dC170.2, 164.3, 156.2, 147.2, 147.1, 139.1, 134.2, 133.4, 132.0, 131.8, 130.8, 130.6, 130.6, 129.8, 129.1, 128.4, 128.4, 126.6, 125.2, 124.2, 121.6, 21.6; HR-ESI-MS: m/z calcd for C22H16ClN3OS, [M]+ 405.0312; Found 405.0314. 2.3.18. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2-methylbenzylidene) benzohydrazide (18) White solid. Yield: 81%. 1H NMR (500 MHz, DMSO d6):dH 8.32 (s, 1H, ACH), 8.19 (s, 1HAr), 8.06 (d, J = 8.1 Hz, 2H, Ar), 7.94 (d, J = 8.1 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.69 (d, J = 7.4 Hz, 1H, Ar), 7.50 (d, J = 7.8 Hz, 1H, Ar), 7.30 (t, 1H, Ar), 7.26 (d, J = 7.1 Hz, 1H, Ar), 7.20 (t, 1H, Ar), 2.46 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC170.2, 164.3, 156.2, 147.2, 143.1, 135.6, 133.4, 132.0, 131.4, 131.0, 130.8, 130.6, 130.6, 129.2, 128.4, 128.4, 126.3, 126.1, 125.2, 124.2, 121.6, 19.2; HR-ESI-MS: m/z calcd for C22H16ClN3OS, [M]+ 405.0323; Found 405.0321. 2.3.19. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(pyridin-4-ylmethylene) benzohydrazide (19) White solid. Yield: 83%. 1H NMR (500 MHz, DMSO d6):dH 8.64 (d, J = 8.7 Hz, 2H, Ar), 8.58 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.05 (d, J = 8.1 Hz, 2H, Ar), 8.96 (d, J = 8.8 Hz, 2H, Ar), 7.93 (d, J = 8.1 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.8 Hz, 1H, Ar); 13 C NMR (126 MHz, DMSO d6):dC170.2, 164.3, 156.2, 149.6, 149.6, 147.2, 147.0, 144.7, 133.4, 132.0, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.4, 121.6, 120.4, 120.4; HR-ESI-MS: m/z calcd for C20H13ClN4OS, [M]+ 392.0650; Found 392.0647. 2.3.20. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2-hydroxy-4-methoxybenzylidene)benzohydrazide (20) White solid. Yield: 87%. 1H NMR (500 MHz, DMSO d6):d H 8.74 (s, 1H, ACH), 8.20 (s, 1H, Ar), 8.04 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.69 (d, J = 7.4 Hz, 1H, Ar), 7.52 (d, J = 7.7 Hz, 1H, Ar), 6.58 (d, J = 6.8 Hz, 1H, Ar), 6.44 (s, 1H, Ar), 5.32 (s, 1H, AOH), 3.78 (s, 3H, ACH3); 13C NMR (126
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MHz, DMSO d6): dC 170.2, 164.4, 164.3, 162.3, 156.2, 147.0, 146.2, 133.4, 133.2, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 121.6, 111.2, 103.2, 107.4, 56.2; HR-ESI-MS: m/z calcd for C22H16ClN3O3S, [M]+ 437.0742; Found 437.0739. 2.3.21. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-hydroxy-4methoxybenzylidene)benzohydrazide (21) Brown solid. Yield: 85%. 1H NMR (500 MHz, DMSO d6):d H 8.34 (s, 1H, ACH), 8.19 (s, 1H, Ar), 8.05 (d, J = 8.6 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.7 Hz, 1H, Ar), 7.38 (d, J = 7.1 Hz, 1H, Ar), 7.32 (s, 1H, Ar), 6.86 (d, J = 6.6 Hz, 1H, Ar), 5.31 (s, 1H, AOH), 3.76 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 152.8, 147.2, 147.1, 147.0, 134.4, 132.0, 131.2, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 123.1, 121.6, 116.3, 112.1, 56.2; HR-ESI-MS: m/z calcd for C22H16ClN3O3S, [M]+ 437.0500; Found 437.0510. 2.3.22. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(thiophen-2ylmethylene)benzohydrazide (22) Brown solid. Yield: 85%. 1H NMR (500 MHz, DMSO d6):dH 8.32 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.04 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.50 (d, J = 7.7 Hz, 1H, Ar), 7.66 (d, J = 7.2 Hz, 1H, Ar), 7.60 (d, J = 7.1 Hz, 1H, Ar), 7.13 (t, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.0, 144.8, 133.4, 132.0, 130.8, 130.6, 130.6, 130.4, 128.8, 128.4, 128.4, 127.5, 125.3, 125.2, 124.2, 121.6; HR-ESI-MS: m/z calcd for C19H12ClN3OS2, [M]+ 397.0401; Found 397.0405. 2.3.23. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-hydroxy-3methoxybenzylidene)benzohydrazide (23) Yellowish solid. Yield: 84%. 1H NMR (500 MHz, DMSO d6):d H 8.33 (s, 1H, Ar), 8.20 (s, 1H, Ar), 8.05 (d, J = 8.6 Hz, 2H, Ar). 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.7 Hz, 1H, Ar), 7.49 (s, 1H, Ar), 7.30 (d, J = 6.9 Hz, 1H, Ar), 6.89 (d, J = 7.1, 1H, Ar), 5.34 (s, 1H, AOH), 3.76 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 150.9, 149.1, 147.1, 147.0, 133.2, 132.0, 131.2, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 123.3, 121.6, 117.4, 112.5, 56.4; HR-ESI-MS: m/z calcd for C22H16ClN3O3S, [M]+ 437.0322; Found 437.0320. 2.3.24. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-methoxybenzylidene) benzohydrazide (24) Yellowish solid. Yield: 86%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.23 (s, 1H, Ar), 8.06 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.88 (d, J = 8.01 Hz, 1H, Ar) 7.50 (d, J = 7.7 H z, 1H, Ar), 7.48 (s, 1H, Ar), 7.36 (d, J = 6.9 Hz, 1H, Ar), 7.29 (m, 1H, Ar), 7.02 (d, J = 6.7 Hz, 1H, Ar), 3.78 (s, 3H, ACH3); 13C NMR (150 MHz, DMSO d6): dC 170.2, 164.3, 161.1, 156.2, 147.1, 147.0, 138.5, 133.2, 132.0, 130.8, 130.6, 130.6, 130.1, 128.4, 128.4, 125.2, 124.2, 121.8, 121.6, 116.8, 112.5, 56.2; HR-ESI-MS: m/z calcd for C22H16ClN3O2S, [M]+ 421.0412; Found 421.0210. 2.3.25. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-methoxybenzylidene) benzohydrazide (25) Yellowish solid. Yield: 1H NMR (500 MHz, DMSO d6):dH 8.33 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.05 (d, J = 8.6 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.79 (d, J = 7.6 Hz, 2H, Ar), 7.51 (d, J = 7.6 Hz, 1H, Ar), 7.02 (d, J = 7.4 Hz, 2H, Ar), 3.80 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 163.2, 156.2, 147.1, 147.0, 133.4, 132.0, 130.8, 130.6, 130.6, 130.5, 130.5, 128.4, 128.4, 126.1, 125.2, 124.2, 121.6, 114.6, 114.6, 56.4; HR-ESI-MS: m/z calcd for C22H16ClN3O2S, [M]+ 421.0500; Found 421.0508.
2.3.26. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-flurobenzylidene) benzohydrazide (26) White solid. Yield: 87%. 1H NMR (500 MHz, DMSO d6):dH 8.32 (s, 1H, ACH), 8.23 (s, 1H, Ar), 8.07 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.88 (d, J = 8.01 Hz, 1H, Ar), 7.78 (d, J = 7.6 Hz, 2H, Ar), 7.52 (d, J = 7.6 Hz, 1H, Ar), 7.31 (d, J = 7.1, 2H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 165.4, 164.2, 156.2, 147.1, 147.0, 133.4, 132.0, 131.1, 131.1, 130.8, 130.6, 130.6, 129.5, 128.4, 128.4, 125.2, 124.2, 121.6, 116.1, 116.1; HR-ESI-MS: m/z calcd for C21H13ClFN3OS, [M]+ 409.0324; Found 409.0326. 2.3.27. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3,4-dimethoxybenzylidene)benzohydrazide (27) Yellowish solid. Yield: 86%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.06 (d, J = 8.6 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.54 (s, 1H, Ar), 7.50 (d, J = 7.6 Hz, 1H, Ar), 7.38 (d, J = 6.8, 1H, Ar), 6.90 (d, J = 6.5, 1H, Ar), 3.79 (s, 6H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 152.4, 150.1, 147.1, 147.0, 133.4, 132.0, 130.9, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.4, 122.8, 121.6, 112.1, 109.5, 56.4, 56.4; HR-ESI-MS: m/z calcd for C23H18ClN3O3S, [M]+ 451.0434; Found 451.0430. 2.3.28. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-chlorobenzylidene) benzohydrazide (28) Yellowish solid. Yield: 81%. 1H NMR (500 MHz, DMSO d6):d H 8.32 (s, 1H-CH), 8.21 (s, 1H, Ar), 8.04 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.89 (s, 1H, Ar), 7.88 (d, J = 8.01 Hz, 1H, Ar), 7.68 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H, Ar), 7.49 (d, J = 7.8 Hz, 1H, Ar), 7.41 (t, 1H, Ar); 13C NMR (150 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.1, 147.0, 135.6, 134.8, 133.4, 132.0, 131.5, 130.8, 130.6, 130.6, 130.4, 128.4, 128.4, 127.6, 127.5, 125.2, 124.2, 121.6; HR-ESI-MS: m/z calcd for C21H13Cl2N3OS, [M]+ 425.0324; Found 425.0329. 2.3.29. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(furan-2-ylmethylene) benzohydrazide (29) Yellowish solid. Yield: 84%. 1H NMR (500 MHz, DMSO d6):dH 8.41 (s, 1H, ACH), 8.23 (s, 1H, Ar), 8.06 (d, J = 8.6 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.52 (d, J = 7.6 Hz, 1H, Ar), 7.71 (d, J = 7.4 Hz, 1H, Ar), 6.90 (d, J = 6.4, 1H, Ar), 6.49 (t, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 149.5, 147.1, 144.8, 134.8, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 121.6, 119.3, 112.8; HR-ESI-MS: m/z calcd for C19H12ClN3O2S, [M]+ 382.0840; Found 382.0851. 2.3.30. (E)-N0 -benzylidene-4-(5-chlorobenzo[d]thiazol-2-yl) benzohydrazide (30) White solid. Yield: 88%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.07 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.80 (d, J = 7.9 Hz, 2H, Ar), 7.51 (d, J = 7.6 Hz, 1H, Ar), 7.49 (dd, J = 7.6, 1.4, Hz, 2H, Ar), 7.48 (dd, J = 7.4, 1.3, Hz, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.1, 147.0, 134.1, 133.4, 132.0, 131.4, 130.8, 130.6, 130.6, 129.5, 129.5, 129.1, 129.1, 128.4, 128.4, 125.2, 124.2, 121.6; HR-ESI-MS: m/z calcd for C21H14ClN3OS, [M]+ 391.0370; Found 391.0375. 2.4. Docking studies All compounds were prepared using Chem3D by CambridgeSoft. The geometry and energy of the structures were being optimized using Polak-Ribiere algorithm in HyperChem. AutoDock 4.2 [26] was used to identify the binding modes of active benzothiazole derivatives responsible for the activity. Genetic Algorithm (GA) with default settings was employed for the studies. Human
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b-D-glucuronidase crystal structure was first retrieved from the protein data bank (PDB code: 1BHG) [24,25]. The docking results had been visualized using Discovery Studio visualizer 4.0. 2.5. b-Glucuronidase assay b-Glucuronidase activity was determined in accordance to method used by Taha et al. [26] by measuring absorbance at 405 nm of p-nitrophenol formed substrate by spectrophotometric method. 250 µL was the volume of total reaction. Reaction mixture containing 5 µL of test compound solution, 185 µL of 0.1 M acetate buffer and 10 µL of enzyme solution were incubated for 30 min at 37 °C. At 405 nm the plates were recorded on multiplate reader (Specta Maxplus 384) after the addition of 50 µL of 0.4 mM pnitrophenyl-b-D-glucuronide. Experiments were performed for triplicate [27]. To avoid precipitation, compound concentration was decreased and the volume of reaction was increased (200 µL). Precipitation probability was less thus addition of detergents was not needed. 3. Result and discussion 3.1. Chemistry The synthesis of benzothiazole hybrid compounds (1–30) was established with the treatment aldehyde with 4-chloro-2aminothiophenol in DMF for 5 h to afford aryl ester (I) substituted benzothiazole. The benzothiazole having benzohydrazide ring (II) was afforded by refluxing compound (II) with hydrazine hydrate in methanol for 6 h (Scheme 1). The benzothiazole hybrid compounds (1–30) were synthesized by refluxing equal molar amount of compound (II) and different aryl aldehyde in the presence of acetic acid (see Fig. 1 and Table 1). 3.2. b-Glucuronidase activity Enzyme inhibition is an adynamic tool in governing the start and development of associated pathologies. The enzyme inhibition has been widely used in drugs discovery. Therefore, series of benzothiazole having hydrazone have been synthesized and screened for b-glucuronidase inhibitors potential. Out of 30, twelve compounds exhibit outstanding b-glucuronidase inhibitors potential better than the standard, one compound showed similar inhibitory potential to standard while three compounds showed less inhibitory potential than the standard. The remaining all compounds were found almost inactive. Structure activity relationship has been established mainly based on the substitution pattern on aromatic aldehyde ring.
Fig. 1. General structure of benzothiazole analog.
Compound (E)-N0 -(2,3-dihydroxybenzylidene)-4-(5-chlorobenzo [d]thiazol-2-yl)benzohydrazide (2) showed excellent inhibitory potential with IC50 value 16.50 ± 0.26 µM three folds more potent than the standard d-saccharic acid 1,4-lactone (IC50 = 48.4 ± 1.25 µM). Similarly, analog (E)-N0 -(2,3-dihydroxybenzylidene)-4-(5-chl orobenzo[d]thiazol-2-yl)benzohydrazide (5) showed outstanding inhibitory potential with IC50 value 18.10 ± 0.27 µM almost three fold better than the standard (see Fig. 2). The compounds 1, 3, 4, 6, 9, 11, 14, 15, 20, and 26 also showed potent inhibitory potential. The analogs 8, 22 and 23 also showed good inhibitory potential with IC50 value very close to standard. Here in this study, we observed that almost all hydroxyl substituted analogs showed inhibitory potential against this enzyme. The position and number of hydroxyl groups on aromatic ring slightly affect the inhibitory potential as shown in Table 2. The reason for the inhibitory activity of these compounds seems to due to hydrogen bonding. The fluoro and chloro substituted analogs also showed potential, but we observed the great effect of position difference as in analogs 11 and 28. However, in order to explain the SAR and interaction of molecules with the active site of enzyme, in silico studies was carried out. 3.3. Docking studies In order to obtain more insight into the binding mode of benzothiazole derivatives within the active site of b-D-glucuronidase and to obtain additional validations for experimental results, molecular docking studies were performed. In this study, the Xray crystal structure of human b-glucuronidase enzyme at 2.6 Å resolution (PDB ID: 1BHG) [25] was employed to further identify binding modes involved in the inhibition activity. The human bD-glucuronidase 3D structure was used for our structure-activity relationship (SAR) studies due to the absence of bovine b-Dglucuronidase structure. Prior to the docking of the benzothiazole derivatives, known substrate molecule p-nitrophenyl b-D-glucuronide was first docked into the active site of b-D-glucuronidase using docking program AutoDock 4.2 [26]. The modeled substrate-bound structure of human b-D-glucuronidase showed that the glycoside bond of p--
Scheme 1. Synthetic route benzothiazole hybrid compounds.
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M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
Table 1 Synthesis of various analogs of benzothiazole (1–30). S.No.
R
S.No.
R
S.No.
1
11
21
2
12
22
3
13
23
4
14
24
5
15
25
6
16
26
7
17
27
8
18
28
9
19
29
10
20
30
R
Fig. 2. Structure-activity relationship of compound 1 and 2.
Table 2 b-Glucuronidase inhibitory potential of benzothiazole analogs (1–30). S.No.
(IC50 ± SEM)
S.No.
(IC50 ± SEM)
S.No.
(IC50 ± SEM)
1 2 3 4 5 6 7 8 9 10
33.60 ± 0.84 16.50 ± 0.26 27.40 ± 0.37 22.90 ± 0.28 18.10 ± 0.27 36.20 ± 0.53 N.A. 59.45 ± 1.12 29.10 ± 0.41 N.A.
11 12 13 14 15 16 17 18 19 20
34.06 ± 0.65 N.A. N.A. 48.12 ± 1.01 43.60 ± 0.84 N.A. N.A. N.A. N.A. 38.10 ± 0.67 48.4 ± 1.25 µM
21 22 23 24 25 26 27 28 29 30
46.20 ± 0.93 51.40 ± 1.12 49.45 ± 1.10 N.A. N.A. 24.06 ± 0.45 N.A. N.A. N.A. N.A.
D-saccharic
acid 1,4-lactone
nitrophenyl b-D-glucuronide was properly oriented towards the catalytic residues Glu451, Glu540, and Tyr504 (Fig. 3). It has been proposed for human b-D-glucuronidase that during catalysis,
Glu451 acts as the acid/base catalyst while Glu540 serves as the nucleophilic residue [25]. Tyr504 was found to contribute significantly towards catalysis even though its function is still unclear.
M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
53
Fig. 5. Binding position for compound 2, 3, 4, and 26.
Fig. 3. b-D-glucuronide in the active site of b-glucuronidase.
It is also claimed that Asp207-Glu451 pair might form the nucleophile-acid-base catalyst pair in human b-D-glucuronidase, analogous to the Glu35-Asp52 pair in lysozyme. As shown in Fig. 3, the substrate neatly fits in the active site making various hydrogen bonding interactions with the active site residues including Asp207, His385, Glu451, Tyr504 and Glu540. Docking studies showed that active compounds occupied and interacted quite well with catalytic residues in the binding cavity of the enzyme. Analysis for putative binding conformation of the most active hydroxyl-substituted compound 2 (IC50 = 16.50 ± 0.2 6 µM) in Fig. 4 displayed that its extended benzohydrazone moiety positioned towards the interior of the active site’s core with benzothiazole moiety binding to the residues at the entrance. Visually inspecting the hydrogen bond interaction of best binding position for compound2displayed that the amine (NH) and carboxy (CO) of hydrazone linkage formed a strong hydrogen bond with the backbone (OD2) of Asp207 (2.60 Å) and hydroxy (OH) of Tyr504 (3.74 Å), respectively. Hydroxyl is at ortho position on its phenyl-end interacts with Tyr205, while the other hydroxyl group at meta position, through its oxygen atom, formed a hydrogen bond with Gly411 at a distance of 3.32 Å. Nitrogen on benzothiazole ring formed a hydrogen bonding with Tyr504 at a distance of 3.18 Å.
Further observation on Fig. 4 suggests that the center ring that connects between the benzothiazole and benzohydrazone moiety is supported by two hydrophobic interaction involving a p-p stacking with Tyr508 (4.73 Å) and a T-shape p-p interaction with Tyr504 (4.23 Å). Another hydrophobic interaction was observed between Ser503 and benzothiazole moiety, which formed ar-p interaction at a distance of 3.65 Å. All catalytically important amino acids formed electrostatic interactions with compound 2. The analysis suggests that Glu451 could interact with compound 2through three different electrostatic interaction involving anion-p and attractive charge interactions. Glu451 interacts with central and benzylidene ring via anion-pi interaction. The results also displayed that Asp207, Glu451, and Glu540 interacted with the nitrogen of imine (C@N) through the same type of interaction that is attractive charge interaction. Docking results of other active compounds (3, 4, and 26) showed that these compounds possess similar binding conformation as compound 2 (Fig. 5). With their benzothiazole moieties being aligned well in the binding cavity, the main difference in the binding conformations came from the benzohydrazone linkages, which possess higher rotatability features. Docking results showed that benzylidene moiety of compound 3 forms one hydrogen bonding with Tyr205 at a distance of 1.97 Å through its single hydroxyl substituent at para position (Fig. 6). As for compound 4, the two hydroxyl substituents on benzylidene moiety forms hydrogen bonding with Glu451 and His385 at the distance of 2.05 Å and 2.19 Å, respectively. On the other hand, thorough analysis on putative docking mode of fluoro
Fig. 4. Interaction of compound 2 with residues in the active site of b-glucuronidase.
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M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
Fig. 6. Binding position for (a) compounds 3, (b) compound 4, and (c) compound 26.
substituent at para position on benzylidene ring of compound 26 suggests that it interacts with Asp207 through a hydrophobic interaction.
Acknowledgement The authors would like to acknowledge Universiti Teknologi MARA for the financial support through research grant, UiTM 600-RMI/5/3 CIFI (142/2013). The first author would like to thank Ministry of Higher Education, Malaysia for the financial funding through MyBrain15 scholarship.
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Contents lists available at ScienceDirect
Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg
Synthesis: Small library of hybrid scaffolds of benzothiazole having hydrazone and evaluation of their b-glucuronidase activity Muhammad Taha a,⇑, Mastura Arbin b,c, Norizan Ahmat b,c, Syahrul Imran b,c, Fazal Rahim d a
Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam Campus, Malaysia c Faculty of Applied Science Universiti Teknologi MARA, 40450 ShahAlam, Selangor D.E, Malaysia d Department of Chemistry, Hazara University, Mansehra 21120, Pakistan b
a r t i c l e
i n f o
Article history: Received 7 August 2017 Revised 30 November 2017 Accepted 2 January 2018 Available online 4 January 2018 Keywords: Benzothiazole Synthesis b-Glucuronidase activity SAR
a b s t r a c t Due to the great biological importance of b-glucuronidase inhibitors, here in this study, we have synthesized a library of novel benzothiazole derivatives (1–30), characterized by different spectroscopic methods and evaluated for b-glucuronidase inhibitory potential. Among the series sixteen compounds i.e. 1–6, 8, 9, 11, 14, 15, 20–23 and 26 showed outstanding inhibitory potential with IC50 value ranging in between 16.50 ± 0.26 and 59.45 ± 1.12 when compared with standard D-Saccharic acid 1,4-lactone (48.4 ± 1.25 µM). Except compound 8 and 23 all active analogs showed better potential than the standard. Structure activity relationship has been established. Ó 2018 Elsevier Inc. All rights reserved.
1. Introduction The chemistry of fused ring heterocycles is recognized to be a key field of investigation in medicinal chemistry, subsequently, they have been establishing to show enhanced biological activity. Heterocyclic compounds display important role in medicinal chemistry and their structures in natural products. One of them is benzothiazole scaffold which have gain considerable attention in synthetic organic chemistry with valuable applications in the pharmaceutical industry. From the literature survey, benzothiazole analog have shown numerous biological activities [1–3] used in material science and in other wide range industrial applications [4,5]. Furthermore, these benzothiazole compounds have also shown enzymes inhibition, like protoporphyrinogen oxidase, which give free radicals that result in simultaneous toxicological effects on plants and animals [6,7]. The benzothiazole is an exceptional scaffold for further molecular study to synthesize innovative compounds. Benzothiazole analogs have been reported as antimicrobial agents [8,9], if having pyrazole or isatin moiety then exhibit significant antimicrobial potential [10–13]. b-Glucuronidase is an exoglycosidase enzyme catalyzes the cleavage of glucuronosyl-O-bonds [14]. This enzyme is broadly distributed in anaerobic bacteroides, also occur in human blood cells, ⇑ Corresponding author. E-mail address: [email protected] (M. Taha). https://doi.org/10.1016/j.bioorg.2018.01.002 0045-2068/Ó 2018 Elsevier Inc. All rights reserved.
gastric juice, body fluids, spleen, serum, urine and organs like liver, lung, bile kidney and muscle [15,16]. The deficit of b-glucuronidase in human body leads to Sly syndrome which is linked with the increase of glycosaminoglycans in cells [17,18]. Certain hepatic diseases and AIDS are reported due to the overreaction of the enzyme. Though, it over expressed in some pathological conditions like cancer, inflammation, epilepsy, in hepatic as well as renal diseases, in joints, bladder, and neoplasm of breast, testes, and larynx [19–21]. Literature survey revealed that the bacterial b-glucuronidase inhibitor lead to a reduction in carcinogen prompted colonic tumors [22]. Consequently, inhibitors of this enzyme are immediate need of the day to cure numerous pathological conditions. Recently our group has reported various classes of heterocyclic compounds as a potent inhibitor of b-glucuronidase [23]. In this connection here we are reporting the synthesis of new benzothiazole analogs and its b-glucuronidase inhibitory potential. 2. Experimental All nuclear magnetic resonance experiments had been carried out using an AvanceBruker 600 MHz. Elemental analysis was performed on Carlo Erba Strumentazion-Mod-1106, Italy. Electron impact mass spectra (EI-MS) were recorded on a Finnigan MAT-311A, Germany. Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany).
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2.1. Synthetic procedure of methyl 4-(5-chlorobenzo[d]thiazol-2-yl) benzoate (I) The methyl 4-(5-chlorobenzo[d]thiazol-2-yl) benzoate (I) was prepared by the treatment of aldehyde (1 mmol) and 4-chloro-2aminothiophenol (1 mmol) in the presence of DMF for 5 h. After completion of reaction as determined by TLC analysis, the contents were cooled and dried. The impurities were removed washing with ethyl acetate and hexane. 2.2. Synthetic procedure of 4-(5-chlorobenzo[d]thiazol-2-yl)benzohydrazide (II) In second step, 4-(5-chlorobenzo[d]thiazol-2-yl) benzohydrazide (II) was synthesized by treating of the equimolar amount of methyl 4-(5-chlorobenzo[d]thiazol-2-yl) benzoate (I) (1 mmol) and hydrazine hydrate (1 mmol) in the presence of methanol for 6 h. The reaction completion was monitored by TLC. 2.3. Synthetic procedure of (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 (3-hydroxybenzylidene) benzohydrazide (1–30) In third step, 4-(5-chlorobenzo[d]thiazol-2-yl)benzohydrazide (II) (1 mmol) was further treated with different benzaldehydes (1 mmol) in the presence of acetic acid for 3 h. The reaction completion was monitored by TLC. Different spectroscopic techniques such as ESI-MS, 1H NMR and 13C NMR were used to determine the structure of all analogs. 2.3.1. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3hydroxybenzylidene)benzohydrazide (1) White solid, Yield: 81%. 1H NMR (500 MHz, DMSO d6):d 8.32 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.10 (d, J = 8.8 Hz, 2H, Ar), 7.94 (d, J = 8.6 Hz, 2H, Ar), 7.88 (d, J = 8.01 Hz, 1H, Ar), 7.54 (d, J = 7.8 Hz, 1H, Ar), 7.43 (s, 1H, Ar), 7.37 (d, J = 6.8 Hz, 1H, Ar), 7.23 (t, 1H, Ar), 7.01 (d, J = 6.6 Hz, 1H, Ar), 5.34 (s, 1H, AOH); 13C NMR (126 MHz, DMSO d6): d 170.2, 164.3, 159.4, 156.7, 147.3, 146.6, 139.3, 133.4, 132.5, 131.6, 131.4, 130.5, 130.3, 128.3, 128.1, 125.2, 123.5, 122.4, 121.6, 115.5, 118.4; HR-ESI-MS: m/z calcd for C21H14ClN3O2S, [M]+ 407.0752; Found 407.0751. 2.3.2. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2,3-dihydroxybenzylidene)benzohydrazide (2) White solid, Yield: 81%. 1H NMR (500 MHz, DMSO d6):d H 8.75 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.10 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.6 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.53 (d, J = 7.8 Hz, 1H, Ar), 7.20 (s, 1H, Ar), 6.83 (d, J = 7.2 Hz, 1H, Ar), 6.80 (t, J = 7.2 Hz, 1H, Ar), 5.33 (s, 2H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 157.7, 146.6, 156.2, 152.4, 147.4, 147.1, 146.2, 134.1, 133.2, 131.4, 130.6, 130.4, 128.2, 128.1, 125.5, 125.1, 124.2, 123.4, 122.1, 120.5, 120.2; HR-ESI-MS: m/z calcd for C21H14ClN3O3S, [M]+ 423.0503; Found 423.0501. 2.3.3. (E)-4-(5-chlorobenzo[d]thiazole-2-yl)-N0 -(4-hydroxybenzylidene) benzohydrazide (3) Yellow solid, Yield: 86%. 1H NMR (500 MHz, DMSO d6): dH 8.34 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.11 (d, J = 8.8 Hz, 2H, Ar), 7.96 (d, J = 8.6 Hz, 2H, Ar), 7.92 (d, J = 8.01 Hz, 1H, Ar), 7.75 (d, J = 8.1 Hz, 2H, Ar), 7.54 (d, J = 7.8 Hz, 1H, Ar), 6.83 (d, J = 7.3 Hz, 2H, Ar), 5.34 (s, 1H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 161.4, 156.2, 147.3, 147.2, 133.4, 132.0, 131.3, 131.3, 130.8, 130.6, 130.6, 128.4, 128.4, 127.2, 125.2, 124.2, 122.1, 117.1, 117.1; HRESI-MS: m/z calcd for C21H14ClN3O2S, [M]+ 407.0315; Found 408.0312.
2.3.4. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2,5-dihydroxybenzylidene)benzohydrazide (4) Yellowish solid, Yield: 83%. 1H NMR (500 MHz, DMSO d6):dH 8.73 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.08 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.5 Hz, 2HAr), 7.93 (d, J = 8.01 Hz, 1H, Ar), 7.55 (d, J = 7.8 H z, 1H, Ar), 7.27 (s, 1H, Ar), 6.83(d, J = 6.9 Hz, 2H, Ar), 5.33 (s, 2H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 154.4, 152.3, 147.2, 146.4, 133.4, 132.2, 131.4, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 122.1, 121.4, 120.2, 119.8, 117.3; HRESI-MS: m/z calcd for C21H14ClN3O3S, [M]+ 423.0404; Found 423.0401. 2.3.5. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3,4,dihydroxybenzylidene)benzohydrazide (5) White solid, Yield: 84%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.07 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.6 Hz, 2H, Ar), 7.92 (d, J = 8.01 Hz, 1H, Ar), 7.53 (d, J = 7.8 Hz, 1H, Ar), 7.32 (d, J = 8.1 Hz, 1H, Ar), 7.26 (s, 1H, Ar), 6.82 (d, J = 7.0 1 Hz, 1H, Ar), 5.32 (s, 2H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 150.5, 147.4, 147.1, 146.2, 133.4, 133.2, 132.0, 131.6, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 123.6, 118.4, 117.3; HR-ESI-MS: m/z calcd for C21H14ClN3O3S, [M]+ 423.0314; Found 424.0311. 2.3.6. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2,4-dihydroxybenzylidene)benzohydrazide (6) Yellowish solid, Yield: 82%. 1H NMR (500 MHz, DMSO d6):dH 8.73 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.06 (d, J = 8.8 Hz, 2H, Ar), 7.94 (d, J = 8.5 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.62 (d, J = 7.02 Hz, 1H, Ar), 7.52 (d, J = 7.8 Hz, 1H, Ar), 7.50 (s, 1H, Ar), 6.38 (d, J = 6.2 Hz, 1H, Ar), 5.34 (s, 2H, AOH); 13C NMR (126 MHz, DMSO d6): dC170.2, 164.3, 162.8, 162.7, 156.2, 147.1, 146.4, 134.1, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 121.4, 111.6, 109.3, 104.1; HR-ESI-MS: m/z calcd for C21H14ClN3O3S, [M]+ 423.0412; Found 424.0414. 2.3.7. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-methylbenzylidene) benzohydrazide (7) Yellowish solid, Yield: 76%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.23 (s, 1H, Ar), 8.06 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.6 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar) 7.53 (d, J = 7.8 H z, 1H, Ar), 7.67 (d, J = 8.1 Hz, 2H, Ar), 7.23 (d, J = 7.9 Hz, 2H, Ar), 2.31 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.4, 147.1, 141.1, 133.4, 132.0, 131.2, 130.8, 130.6, 130.6, 129.6, 129.6, 128.4, 128.4, 126.7, 126.7, 125.2, 124.2, 121.4, 21.7; HR-ESI-MS: m/z calcd for C22H16ClN3OS, [M]+ 405.0518; Found 406.0515. 2.3.8. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-nitrobenzylidene) benzohydrazide (8) Yellow solid, Yield: 82%. 1H NMR (500 MHz, DMSO d6):dH 8.32 (s, 1H, ACH), 8.29 (d, J = 8.3 Hz, 2H, Ar), 8.21 (s, 1H, Ar), 8.05 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H, Ar), 7.94 (d, J = 8.6 Hz, 2H, Ar), 7.92 (d, J = 8.02 Hz, 1H, Ar), 7.54 (d, J = 7.8 Hz, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 150.7, 147.4, 147.1, 140.2, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.6, 124.6, 124.5, 124.3, 124.2, 124.2; HR-ESI-MS: m/z calcd for C21H13ClN4O3S, [M]+ 436.0321; Found 437.0318. 2.3.9. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2-hydroxybenzylidene) benzohydrazide (9) Brown solid, Yield: 72%. 1H NMR (500 MHz, DMSO d6):dH 8.73 (s, 1H, ACH), 8.20 (s, 1H, Ar), 8.06 (d, J = 8.8 Hz, 2H, Ar), 7.93 (d, J = 8.6 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.64 (d, J = 7.8 Hz, 1H, Ar), 7.59 (d, J = 8.4 Hz, 1H, Ar), 7.04 (m, 1H, Ar), 7.48 (m, 1H), 7.01 (d, J = 7.2 Hz, 1H, Ar), 5.34 (s, 1H, AOH); 13C NMR (150 MHz,
M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
DMSO d6): dC 170.2, 164.3, 157.6, 156.2, 147.1, 146.6, 133.4, 132.8, 132.0, 130.8, 130.6, 130.6, 128.4, 128.1, 125.2, 124.2, 122.1, 121.6, 119.1, 118.2; HR-ESI-MS: m/z calcd for C21H14ClN3O2S, [M]+ 407.7312; Found 408.7311. 2.3.10. (E)-methyl4-((2-(4-(5-chlorobenzo[d]thiazol-2-yl)benzoyl) hydrazono)methyl)benzoate (10) Brown solid, Yield: 74%. 1H NMR (500 MHz, DMSO d6):dH 8.33 (s, 1H, Ar), 8.22 (s, 1H, Ar), 8.18 (d, J = 8.4 Hz, 2H, Ar), 8.04 (d, J = 8.7 Hz, 2H, Ar), 7.95 (d, J = 8.4 Hz, 2H, Ar), 7.92 (d, J = 8.01 Hz, 1H, Ar), 7.90 (d, J = 8.01 Hz, 2H, Ar), 7.51 (d, J = 7.4 Hz, 1H, Ar), 3.85 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC170.2, 166.2, 164.3, 156.2, 147.3, 147.0, 138.6, 133.4, 132.8, 132.0, 130.8, 130.6, 130.6, 130.4, 130.4, 129.6, 129.6, 128.4, 128.4, 125.2, 124.2, 121.6, 52.2; HR-ESI-MS: m/z calcd for C23H16ClN3O3S, [M]+ 449.0390; Found 449.0388. 2.3.11. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-chlorobenzylidene) benzohydrazide (11) White solid. Yield: 78%. 1H NMR (500 MHz, DMSO d6):d H 8.31 (s, 1H, ACH), 8.20 (s, 1H, Ar), 8.04 (d, J = 8.2 Hz, 2H, Ar), 7.94 (d, J = 8.1 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.74 (d, J = 7.9 Hz, 2H, Ar), 7.53 (d, J = 7.8 Hz, 1H, Ar), 7.49 (d, J = 8.7 Hz, 2H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.2, 147.0, 137.3, 133.4, 132.2, 132.0, 131.1, 131.1, 130.8, 130.6, 130.6, 129.4, 129.4, 128.4, 128.4, 125.2, 124.2, 121.6; HR-ESI-MS: m/z calcd for C21H13Cl2N3OS, [M]+ 425.0315; Found 425.0318. 2.3.12. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-hydroxy-2-iodo-4methoxybenzylidene)benzohydrazide (12) Yellowish solid. Yield: 73%. 1H NMR (500 MHz, DMSO d6):d H 8.32 (s, 1H, Ar), 8.22 (s, 1H, Ar), 8.03 (d, J = 8.3 Hz, 2H, Ar), 7.93 (d, J = 8.1 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.54 (d, J = 7.8 Hz, 1H, Ar), 7.01 (d, J = 7.4 Hz, 1H, Ar), 6.83 (d, J = 7.2 Hz, 1H, Ar), 5.33 (s, 1H, AOH), 3.80 (s, 3H, ACH3); 13C NMR (150 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 155.8, 152.4, 147.0, 143.8, 134.2, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 125.2, 124.2, 121.6, 111.8, 86.5, 56.6; HR-ESI-MS: m/z calcd for C22H15ClN3O3S, [M]+ 563.0327; Found 563.0324. 2.3.13. (E)-N0 -(3-bromo-4-hydroxybenzylidene)-4-(5-chlorobenzo[d] thiazol-2-yl)benzohydrazide (13) White solid. Yield: 78%. 1H NMR (500 MHz, DMSO d6):d H 8.32 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.05 (d, J = 8.3 Hz, 2H, Ar), 7.94 (d, J = 8.1 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.78 (s, 1H, Ar), 7.70 (d, J = 8.01 Hz, 1H, Ar), 7.52 (d, J = 7.6 Hz, 1H, Ar), 6.89 (d, J = 7.1 Hz, 1H, Ar), 5.32 (s, 1H, AOH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 159.4, 156.2, 147.2, 147.0, 133.4, 132.0, 130.8, 130.7, 130.6, 130.6, 130.1, 129.1, 128.4, 128.4, 125.2, 124.2, 121.6, 118.8, 114.3; HR-ESI-MS: m/z calcd for C21H13BrClN3O2S, [M]+ 485.0432; Found 486.0429. 2.3.14. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2-hydroxy-5methoxybenzylidene)benzohydrazide (14) White solid. Yield: 87%. 1H NMR (500 MHz, DMSO d6): dH 8.74 (s, 1H, ACH), 8.19 (s, 1H, Ar), 8.07 (d, J = 8.4 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.7 Hz, 1H, Ar), 7.30 (s, 1H), 6.86 (d, J = 7.1 Hz, 1H, Ar), 6.87 (d, J = 6.9 Hz, 1H, Ar), 5.32 (s, 1H, AOH), 3.78 (s, 3H, ACH3); 13C NMR (150 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 153.8, 153.6, 147.2, 146.4, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 121.6, 120.1, 118.6, 117.8, 113.9, 56.2; HR-ESI-MS: m/z calcd for C22H16ClN3O3S, [M]+ 437.0510; Found 437.0514.
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2.3.15. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(pyridine-2-ylmethylene)benzohydrazide (15) White solid. Yield: 86%. 1H NMR (500 MHz, DMSO d6):d H 8.62 (d, J = 8.4 Hz, 1H, Ar), 8.19 (s, 1H, Ar), 8.04 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.1 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.8 Hz, 1H, Ar), 7.81 (d, J = 8.1 Hz, 1H, Ar), 7.76 (t, 1H), 7.61 (t, 1H), 7.10 (s, 1H, ACH); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 154.3, 149.7, 147.2, 145.4, 136.7, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 126.8, 125.2, 124.2, 121.6, 120.6; HR-ESI-MS: m/z calcd for C20H13ClN4OS, [M]+ 392.0280; Found 392.0277. 2.3.16. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(pyridine-3-ylmethylene)benzohydrazide (16) White solid. Yield: 83%. 1H NMR (500 MHz, DMSO d6):dH 8.66 (d, J = 8.4 Hz, 1H, Ar), 8.30 (s, 1H, ACH), 8.26 (d, J = 8.2 Hz, 1H, Ar), 8.21 (s, 1H, Ar), 8.05 (d, J = 8.2 Hz, 2H, Ar), 7.94 (d, J = 8.3 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.54 (t, 1H, Ar), 7.52 (d, J = 7. 6 Hz, 1H, Ar), 9.04 (s, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 152.4, 149.7, 147.2, 144.8, 134.4, 133.4, 132.0, 130.8, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.4, 124.2, 121.6; HR-ESI-MS: m/z calcd for C20H13ClN4OS, [M]+ 392.0623; Found 392.0621. 2.3.17. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-methylbenzylidene) benzohydrazide (17) Yellowish solid. Yield: 85%. 1H NMR (500 MHz, DMSO d6):dH 8.31 (s, 1H, ACH), 8.20 (s, 1H, Ar), 8.04 (d, J = 8.2 Hz, 2H, Ar), 7.93 (d, J = 8.3 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.64 (s, 1H, Ar), 7.61 (d, J = 7.4 Hz, 1H, Ar), 7.52 (d, J = 7.7 Hz, 1H, Ar), 7.38 (t, 1H, Ar), 7.26 (d, J = 6.9 Hz, 1H, Ar), 2.32 (s, 3H, ACH3); 13C NMR (150 MHz, DMSO d6): dC170.2, 164.3, 156.2, 147.2, 147.1, 139.1, 134.2, 133.4, 132.0, 131.8, 130.8, 130.6, 130.6, 129.8, 129.1, 128.4, 128.4, 126.6, 125.2, 124.2, 121.6, 21.6; HR-ESI-MS: m/z calcd for C22H16ClN3OS, [M]+ 405.0312; Found 405.0314. 2.3.18. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2-methylbenzylidene) benzohydrazide (18) White solid. Yield: 81%. 1H NMR (500 MHz, DMSO d6):dH 8.32 (s, 1H, ACH), 8.19 (s, 1HAr), 8.06 (d, J = 8.1 Hz, 2H, Ar), 7.94 (d, J = 8.1 Hz, 2H, Ar), 7.91 (d, J = 8.01 Hz, 1H, Ar), 7.69 (d, J = 7.4 Hz, 1H, Ar), 7.50 (d, J = 7.8 Hz, 1H, Ar), 7.30 (t, 1H, Ar), 7.26 (d, J = 7.1 Hz, 1H, Ar), 7.20 (t, 1H, Ar), 2.46 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC170.2, 164.3, 156.2, 147.2, 143.1, 135.6, 133.4, 132.0, 131.4, 131.0, 130.8, 130.6, 130.6, 129.2, 128.4, 128.4, 126.3, 126.1, 125.2, 124.2, 121.6, 19.2; HR-ESI-MS: m/z calcd for C22H16ClN3OS, [M]+ 405.0323; Found 405.0321. 2.3.19. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(pyridin-4-ylmethylene) benzohydrazide (19) White solid. Yield: 83%. 1H NMR (500 MHz, DMSO d6):dH 8.64 (d, J = 8.7 Hz, 2H, Ar), 8.58 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.05 (d, J = 8.1 Hz, 2H, Ar), 8.96 (d, J = 8.8 Hz, 2H, Ar), 7.93 (d, J = 8.1 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.8 Hz, 1H, Ar); 13 C NMR (126 MHz, DMSO d6):dC170.2, 164.3, 156.2, 149.6, 149.6, 147.2, 147.0, 144.7, 133.4, 132.0, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.4, 121.6, 120.4, 120.4; HR-ESI-MS: m/z calcd for C20H13ClN4OS, [M]+ 392.0650; Found 392.0647. 2.3.20. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(2-hydroxy-4-methoxybenzylidene)benzohydrazide (20) White solid. Yield: 87%. 1H NMR (500 MHz, DMSO d6):d H 8.74 (s, 1H, ACH), 8.20 (s, 1H, Ar), 8.04 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.69 (d, J = 7.4 Hz, 1H, Ar), 7.52 (d, J = 7.7 Hz, 1H, Ar), 6.58 (d, J = 6.8 Hz, 1H, Ar), 6.44 (s, 1H, Ar), 5.32 (s, 1H, AOH), 3.78 (s, 3H, ACH3); 13C NMR (126
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MHz, DMSO d6): dC 170.2, 164.4, 164.3, 162.3, 156.2, 147.0, 146.2, 133.4, 133.2, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 121.6, 111.2, 103.2, 107.4, 56.2; HR-ESI-MS: m/z calcd for C22H16ClN3O3S, [M]+ 437.0742; Found 437.0739. 2.3.21. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-hydroxy-4methoxybenzylidene)benzohydrazide (21) Brown solid. Yield: 85%. 1H NMR (500 MHz, DMSO d6):d H 8.34 (s, 1H, ACH), 8.19 (s, 1H, Ar), 8.05 (d, J = 8.6 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.7 Hz, 1H, Ar), 7.38 (d, J = 7.1 Hz, 1H, Ar), 7.32 (s, 1H, Ar), 6.86 (d, J = 6.6 Hz, 1H, Ar), 5.31 (s, 1H, AOH), 3.76 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 152.8, 147.2, 147.1, 147.0, 134.4, 132.0, 131.2, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 123.1, 121.6, 116.3, 112.1, 56.2; HR-ESI-MS: m/z calcd for C22H16ClN3O3S, [M]+ 437.0500; Found 437.0510. 2.3.22. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(thiophen-2ylmethylene)benzohydrazide (22) Brown solid. Yield: 85%. 1H NMR (500 MHz, DMSO d6):dH 8.32 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.04 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.50 (d, J = 7.7 Hz, 1H, Ar), 7.66 (d, J = 7.2 Hz, 1H, Ar), 7.60 (d, J = 7.1 Hz, 1H, Ar), 7.13 (t, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.0, 144.8, 133.4, 132.0, 130.8, 130.6, 130.6, 130.4, 128.8, 128.4, 128.4, 127.5, 125.3, 125.2, 124.2, 121.6; HR-ESI-MS: m/z calcd for C19H12ClN3OS2, [M]+ 397.0401; Found 397.0405. 2.3.23. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-hydroxy-3methoxybenzylidene)benzohydrazide (23) Yellowish solid. Yield: 84%. 1H NMR (500 MHz, DMSO d6):d H 8.33 (s, 1H, Ar), 8.20 (s, 1H, Ar), 8.05 (d, J = 8.6 Hz, 2H, Ar). 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.51 (d, J = 7.7 Hz, 1H, Ar), 7.49 (s, 1H, Ar), 7.30 (d, J = 6.9 Hz, 1H, Ar), 6.89 (d, J = 7.1, 1H, Ar), 5.34 (s, 1H, AOH), 3.76 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 150.9, 149.1, 147.1, 147.0, 133.2, 132.0, 131.2, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 123.3, 121.6, 117.4, 112.5, 56.4; HR-ESI-MS: m/z calcd for C22H16ClN3O3S, [M]+ 437.0322; Found 437.0320. 2.3.24. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-methoxybenzylidene) benzohydrazide (24) Yellowish solid. Yield: 86%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.23 (s, 1H, Ar), 8.06 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.88 (d, J = 8.01 Hz, 1H, Ar) 7.50 (d, J = 7.7 H z, 1H, Ar), 7.48 (s, 1H, Ar), 7.36 (d, J = 6.9 Hz, 1H, Ar), 7.29 (m, 1H, Ar), 7.02 (d, J = 6.7 Hz, 1H, Ar), 3.78 (s, 3H, ACH3); 13C NMR (150 MHz, DMSO d6): dC 170.2, 164.3, 161.1, 156.2, 147.1, 147.0, 138.5, 133.2, 132.0, 130.8, 130.6, 130.6, 130.1, 128.4, 128.4, 125.2, 124.2, 121.8, 121.6, 116.8, 112.5, 56.2; HR-ESI-MS: m/z calcd for C22H16ClN3O2S, [M]+ 421.0412; Found 421.0210. 2.3.25. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-methoxybenzylidene) benzohydrazide (25) Yellowish solid. Yield: 1H NMR (500 MHz, DMSO d6):dH 8.33 (s, 1H, ACH), 8.21 (s, 1H, Ar), 8.05 (d, J = 8.6 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.79 (d, J = 7.6 Hz, 2H, Ar), 7.51 (d, J = 7.6 Hz, 1H, Ar), 7.02 (d, J = 7.4 Hz, 2H, Ar), 3.80 (s, 3H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 163.2, 156.2, 147.1, 147.0, 133.4, 132.0, 130.8, 130.6, 130.6, 130.5, 130.5, 128.4, 128.4, 126.1, 125.2, 124.2, 121.6, 114.6, 114.6, 56.4; HR-ESI-MS: m/z calcd for C22H16ClN3O2S, [M]+ 421.0500; Found 421.0508.
2.3.26. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(4-flurobenzylidene) benzohydrazide (26) White solid. Yield: 87%. 1H NMR (500 MHz, DMSO d6):dH 8.32 (s, 1H, ACH), 8.23 (s, 1H, Ar), 8.07 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.88 (d, J = 8.01 Hz, 1H, Ar), 7.78 (d, J = 7.6 Hz, 2H, Ar), 7.52 (d, J = 7.6 Hz, 1H, Ar), 7.31 (d, J = 7.1, 2H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 165.4, 164.2, 156.2, 147.1, 147.0, 133.4, 132.0, 131.1, 131.1, 130.8, 130.6, 130.6, 129.5, 128.4, 128.4, 125.2, 124.2, 121.6, 116.1, 116.1; HR-ESI-MS: m/z calcd for C21H13ClFN3OS, [M]+ 409.0324; Found 409.0326. 2.3.27. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3,4-dimethoxybenzylidene)benzohydrazide (27) Yellowish solid. Yield: 86%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.06 (d, J = 8.6 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.54 (s, 1H, Ar), 7.50 (d, J = 7.6 Hz, 1H, Ar), 7.38 (d, J = 6.8, 1H, Ar), 6.90 (d, J = 6.5, 1H, Ar), 3.79 (s, 6H, ACH3); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 152.4, 150.1, 147.1, 147.0, 133.4, 132.0, 130.9, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.4, 122.8, 121.6, 112.1, 109.5, 56.4, 56.4; HR-ESI-MS: m/z calcd for C23H18ClN3O3S, [M]+ 451.0434; Found 451.0430. 2.3.28. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(3-chlorobenzylidene) benzohydrazide (28) Yellowish solid. Yield: 81%. 1H NMR (500 MHz, DMSO d6):d H 8.32 (s, 1H-CH), 8.21 (s, 1H, Ar), 8.04 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.89 (s, 1H, Ar), 7.88 (d, J = 8.01 Hz, 1H, Ar), 7.68 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H, Ar), 7.49 (d, J = 7.8 Hz, 1H, Ar), 7.41 (t, 1H, Ar); 13C NMR (150 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.1, 147.0, 135.6, 134.8, 133.4, 132.0, 131.5, 130.8, 130.6, 130.6, 130.4, 128.4, 128.4, 127.6, 127.5, 125.2, 124.2, 121.6; HR-ESI-MS: m/z calcd for C21H13Cl2N3OS, [M]+ 425.0324; Found 425.0329. 2.3.29. (E)-4-(5-chlorobenzo[d]thiazol-2-yl)-N0 -(furan-2-ylmethylene) benzohydrazide (29) Yellowish solid. Yield: 84%. 1H NMR (500 MHz, DMSO d6):dH 8.41 (s, 1H, ACH), 8.23 (s, 1H, Ar), 8.06 (d, J = 8.6 Hz, 2H, Ar), 7.95 (d, J = 8.2 Hz, 2H, Ar), 7.90 (d, J = 8.01 Hz, 1H, Ar), 7.52 (d, J = 7.6 Hz, 1H, Ar), 7.71 (d, J = 7.4 Hz, 1H, Ar), 6.90 (d, J = 6.4, 1H, Ar), 6.49 (t, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 149.5, 147.1, 144.8, 134.8, 133.4, 132.0, 130.8, 130.6, 130.6, 128.4, 128.4, 125.2, 124.2, 121.6, 119.3, 112.8; HR-ESI-MS: m/z calcd for C19H12ClN3O2S, [M]+ 382.0840; Found 382.0851. 2.3.30. (E)-N0 -benzylidene-4-(5-chlorobenzo[d]thiazol-2-yl) benzohydrazide (30) White solid. Yield: 88%. 1H NMR (500 MHz, DMSO d6):dH 8.34 (s, 1H, ACH), 8.22 (s, 1H, Ar), 8.07 (d, J = 8.6 Hz, 2H, Ar), 7.94 (d, J = 8.2 Hz, 2H, Ar), 7.89 (d, J = 8.01 Hz, 1H, Ar), 7.80 (d, J = 7.9 Hz, 2H, Ar), 7.51 (d, J = 7.6 Hz, 1H, Ar), 7.49 (dd, J = 7.6, 1.4, Hz, 2H, Ar), 7.48 (dd, J = 7.4, 1.3, Hz, 1H, Ar); 13C NMR (126 MHz, DMSO d6): dC 170.2, 164.3, 156.2, 147.1, 147.0, 134.1, 133.4, 132.0, 131.4, 130.8, 130.6, 130.6, 129.5, 129.5, 129.1, 129.1, 128.4, 128.4, 125.2, 124.2, 121.6; HR-ESI-MS: m/z calcd for C21H14ClN3OS, [M]+ 391.0370; Found 391.0375. 2.4. Docking studies All compounds were prepared using Chem3D by CambridgeSoft. The geometry and energy of the structures were being optimized using Polak-Ribiere algorithm in HyperChem. AutoDock 4.2 [26] was used to identify the binding modes of active benzothiazole derivatives responsible for the activity. Genetic Algorithm (GA) with default settings was employed for the studies. Human
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b-D-glucuronidase crystal structure was first retrieved from the protein data bank (PDB code: 1BHG) [24,25]. The docking results had been visualized using Discovery Studio visualizer 4.0. 2.5. b-Glucuronidase assay b-Glucuronidase activity was determined in accordance to method used by Taha et al. [26] by measuring absorbance at 405 nm of p-nitrophenol formed substrate by spectrophotometric method. 250 µL was the volume of total reaction. Reaction mixture containing 5 µL of test compound solution, 185 µL of 0.1 M acetate buffer and 10 µL of enzyme solution were incubated for 30 min at 37 °C. At 405 nm the plates were recorded on multiplate reader (Specta Maxplus 384) after the addition of 50 µL of 0.4 mM pnitrophenyl-b-D-glucuronide. Experiments were performed for triplicate [27]. To avoid precipitation, compound concentration was decreased and the volume of reaction was increased (200 µL). Precipitation probability was less thus addition of detergents was not needed. 3. Result and discussion 3.1. Chemistry The synthesis of benzothiazole hybrid compounds (1–30) was established with the treatment aldehyde with 4-chloro-2aminothiophenol in DMF for 5 h to afford aryl ester (I) substituted benzothiazole. The benzothiazole having benzohydrazide ring (II) was afforded by refluxing compound (II) with hydrazine hydrate in methanol for 6 h (Scheme 1). The benzothiazole hybrid compounds (1–30) were synthesized by refluxing equal molar amount of compound (II) and different aryl aldehyde in the presence of acetic acid (see Fig. 1 and Table 1). 3.2. b-Glucuronidase activity Enzyme inhibition is an adynamic tool in governing the start and development of associated pathologies. The enzyme inhibition has been widely used in drugs discovery. Therefore, series of benzothiazole having hydrazone have been synthesized and screened for b-glucuronidase inhibitors potential. Out of 30, twelve compounds exhibit outstanding b-glucuronidase inhibitors potential better than the standard, one compound showed similar inhibitory potential to standard while three compounds showed less inhibitory potential than the standard. The remaining all compounds were found almost inactive. Structure activity relationship has been established mainly based on the substitution pattern on aromatic aldehyde ring.
Fig. 1. General structure of benzothiazole analog.
Compound (E)-N0 -(2,3-dihydroxybenzylidene)-4-(5-chlorobenzo [d]thiazol-2-yl)benzohydrazide (2) showed excellent inhibitory potential with IC50 value 16.50 ± 0.26 µM three folds more potent than the standard d-saccharic acid 1,4-lactone (IC50 = 48.4 ± 1.25 µM). Similarly, analog (E)-N0 -(2,3-dihydroxybenzylidene)-4-(5-chl orobenzo[d]thiazol-2-yl)benzohydrazide (5) showed outstanding inhibitory potential with IC50 value 18.10 ± 0.27 µM almost three fold better than the standard (see Fig. 2). The compounds 1, 3, 4, 6, 9, 11, 14, 15, 20, and 26 also showed potent inhibitory potential. The analogs 8, 22 and 23 also showed good inhibitory potential with IC50 value very close to standard. Here in this study, we observed that almost all hydroxyl substituted analogs showed inhibitory potential against this enzyme. The position and number of hydroxyl groups on aromatic ring slightly affect the inhibitory potential as shown in Table 2. The reason for the inhibitory activity of these compounds seems to due to hydrogen bonding. The fluoro and chloro substituted analogs also showed potential, but we observed the great effect of position difference as in analogs 11 and 28. However, in order to explain the SAR and interaction of molecules with the active site of enzyme, in silico studies was carried out. 3.3. Docking studies In order to obtain more insight into the binding mode of benzothiazole derivatives within the active site of b-D-glucuronidase and to obtain additional validations for experimental results, molecular docking studies were performed. In this study, the Xray crystal structure of human b-glucuronidase enzyme at 2.6 Å resolution (PDB ID: 1BHG) [25] was employed to further identify binding modes involved in the inhibition activity. The human bD-glucuronidase 3D structure was used for our structure-activity relationship (SAR) studies due to the absence of bovine b-Dglucuronidase structure. Prior to the docking of the benzothiazole derivatives, known substrate molecule p-nitrophenyl b-D-glucuronide was first docked into the active site of b-D-glucuronidase using docking program AutoDock 4.2 [26]. The modeled substrate-bound structure of human b-D-glucuronidase showed that the glycoside bond of p--
Scheme 1. Synthetic route benzothiazole hybrid compounds.
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M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
Table 1 Synthesis of various analogs of benzothiazole (1–30). S.No.
R
S.No.
R
S.No.
1
11
21
2
12
22
3
13
23
4
14
24
5
15
25
6
16
26
7
17
27
8
18
28
9
19
29
10
20
30
R
Fig. 2. Structure-activity relationship of compound 1 and 2.
Table 2 b-Glucuronidase inhibitory potential of benzothiazole analogs (1–30). S.No.
(IC50 ± SEM)
S.No.
(IC50 ± SEM)
S.No.
(IC50 ± SEM)
1 2 3 4 5 6 7 8 9 10
33.60 ± 0.84 16.50 ± 0.26 27.40 ± 0.37 22.90 ± 0.28 18.10 ± 0.27 36.20 ± 0.53 N.A. 59.45 ± 1.12 29.10 ± 0.41 N.A.
11 12 13 14 15 16 17 18 19 20
34.06 ± 0.65 N.A. N.A. 48.12 ± 1.01 43.60 ± 0.84 N.A. N.A. N.A. N.A. 38.10 ± 0.67 48.4 ± 1.25 µM
21 22 23 24 25 26 27 28 29 30
46.20 ± 0.93 51.40 ± 1.12 49.45 ± 1.10 N.A. N.A. 24.06 ± 0.45 N.A. N.A. N.A. N.A.
D-saccharic
acid 1,4-lactone
nitrophenyl b-D-glucuronide was properly oriented towards the catalytic residues Glu451, Glu540, and Tyr504 (Fig. 3). It has been proposed for human b-D-glucuronidase that during catalysis,
Glu451 acts as the acid/base catalyst while Glu540 serves as the nucleophilic residue [25]. Tyr504 was found to contribute significantly towards catalysis even though its function is still unclear.
M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
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Fig. 5. Binding position for compound 2, 3, 4, and 26.
Fig. 3. b-D-glucuronide in the active site of b-glucuronidase.
It is also claimed that Asp207-Glu451 pair might form the nucleophile-acid-base catalyst pair in human b-D-glucuronidase, analogous to the Glu35-Asp52 pair in lysozyme. As shown in Fig. 3, the substrate neatly fits in the active site making various hydrogen bonding interactions with the active site residues including Asp207, His385, Glu451, Tyr504 and Glu540. Docking studies showed that active compounds occupied and interacted quite well with catalytic residues in the binding cavity of the enzyme. Analysis for putative binding conformation of the most active hydroxyl-substituted compound 2 (IC50 = 16.50 ± 0.2 6 µM) in Fig. 4 displayed that its extended benzohydrazone moiety positioned towards the interior of the active site’s core with benzothiazole moiety binding to the residues at the entrance. Visually inspecting the hydrogen bond interaction of best binding position for compound2displayed that the amine (NH) and carboxy (CO) of hydrazone linkage formed a strong hydrogen bond with the backbone (OD2) of Asp207 (2.60 Å) and hydroxy (OH) of Tyr504 (3.74 Å), respectively. Hydroxyl is at ortho position on its phenyl-end interacts with Tyr205, while the other hydroxyl group at meta position, through its oxygen atom, formed a hydrogen bond with Gly411 at a distance of 3.32 Å. Nitrogen on benzothiazole ring formed a hydrogen bonding with Tyr504 at a distance of 3.18 Å.
Further observation on Fig. 4 suggests that the center ring that connects between the benzothiazole and benzohydrazone moiety is supported by two hydrophobic interaction involving a p-p stacking with Tyr508 (4.73 Å) and a T-shape p-p interaction with Tyr504 (4.23 Å). Another hydrophobic interaction was observed between Ser503 and benzothiazole moiety, which formed ar-p interaction at a distance of 3.65 Å. All catalytically important amino acids formed electrostatic interactions with compound 2. The analysis suggests that Glu451 could interact with compound 2through three different electrostatic interaction involving anion-p and attractive charge interactions. Glu451 interacts with central and benzylidene ring via anion-pi interaction. The results also displayed that Asp207, Glu451, and Glu540 interacted with the nitrogen of imine (C@N) through the same type of interaction that is attractive charge interaction. Docking results of other active compounds (3, 4, and 26) showed that these compounds possess similar binding conformation as compound 2 (Fig. 5). With their benzothiazole moieties being aligned well in the binding cavity, the main difference in the binding conformations came from the benzohydrazone linkages, which possess higher rotatability features. Docking results showed that benzylidene moiety of compound 3 forms one hydrogen bonding with Tyr205 at a distance of 1.97 Å through its single hydroxyl substituent at para position (Fig. 6). As for compound 4, the two hydroxyl substituents on benzylidene moiety forms hydrogen bonding with Glu451 and His385 at the distance of 2.05 Å and 2.19 Å, respectively. On the other hand, thorough analysis on putative docking mode of fluoro
Fig. 4. Interaction of compound 2 with residues in the active site of b-glucuronidase.
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M. Taha et al. / Bioorganic Chemistry 77 (2018) 47–55
Fig. 6. Binding position for (a) compounds 3, (b) compound 4, and (c) compound 26.
substituent at para position on benzylidene ring of compound 26 suggests that it interacts with Asp207 through a hydrophobic interaction.
Acknowledgement The authors would like to acknowledge Universiti Teknologi MARA for the financial support through research grant, UiTM 600-RMI/5/3 CIFI (142/2013). The first author would like to thank Ministry of Higher Education, Malaysia for the financial funding through MyBrain15 scholarship.
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