Synthesis of indole analogs as potent β-glucuronidase inhibitors

Accepted Manuscript Synthesis of indole analogs as potent β-glucuronidase inhibitors Mohd Syukri Baharudin, Muhammad Taha, Syahrul Imran, Nor Hadiani Ismail, Fazal Rahim, Muhammad Tariq Javed, Khalid Mohammed Khan, Muhammad Ali PII: DOI: Reference:

S0045-2068(17)30093-7 http://dx.doi.org/10.1016/j.bioorg.2017.05.005 YBIOO 2065

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

14 February 2017 15 April 2017 1 May 2017

Please cite this article as: M.S. Baharudin, M. Taha, S. Imran, N. Hadiani Ismail, F. Rahim, M.T. Javed, K.M. Khan, M. Ali, Synthesis of indole analogs as potent β-glucuronidase inhibitors, Bioorganic Chemistry (2017), doi: http:// dx.doi.org/10.1016/j.bioorg.2017.05.005

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Synthesis of indole analogs as potent β-glucuronidase inhibitors Mohd Syukri Baharudina,b, Muhammad Tahaa,b , Syahrul Imrana,b, Nor Hadiani Ismaila,b, Fazal Rahim c, Muhammad Tariq Javed c, Khalid Mohammed Khand, Muhammad Ali a

Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), Universiti Teknologi MARA,

Puncak Alam Campus, 42300 Bandar PuncakAlam, Selangor D. E. Malaysia b c

Faculty of Applied Science, UiTM Shah Alam, 40450 Shah Alam, Selangor D.E. Malaysia

Depatment of Chemistry, Hazara University, Mansehra-21120, Pakistan

d

H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological

Sciences, University of Karachi, Karachi-75270, Pakistan Abstract Natural products are the main source of motivation to design and synthesize new molecules for drug development. Designing new molecules against β-glucuronidase inhibitory is utmost essential. In this study indole analogs (1–35) were synthesized, characterized using various spectroscopic techniques including 1H-NMR and EI-MS and evaluated for their β-glucuronidase inhibitory activity. Most compounds were identified as potent inhibitors for the enzyme with IC50 values ranging between 0.50 - 53.40 µM, with reference to standard D-saccharic acid 1,4-lactone (IC50= 48.4 ± 1.25 µM). Structure-activity relationship had been also established. The results obtained from docking studies for the most active compound 10 showed that hydrogen bond donor features as well as hydrogen bonding with (Oε1) of nucleophilic residue Glu540 is believed to be the most importance interaction in the inhibition activity. It was also observed that hydroxyl at fourth position of benzylidene ring acts as a hydrogen bond donor and interacts with hydroxyl (OH) on the side chain of catalysis residue Tyr508. The enzyme-ligand complexed were being stabilized through electrostatic π-anion interaction with acid-base catalyst Glu451 (3.96 Å) and thus preventing Glu451 from functioning as proton donor residue. Keywords: Synthesis, Indole, β-glucuronidase inhibition, Docking



Corresponding authors. E-mail addresses: [email protected] and [email protected] (Muhammad Taha)

1.

Introduction

β-Glucuronidase is a member of the lysosomal glycosidase family. It catalyzes the degradation of glycosaminoglycans of the cell membranes and extracellular matrix of normal and cancerous tissues, which is necessary to the metastasis, invasion and tumor cells proliferation [1,2]. Increase activities of β-glucuronidase have been previously reported in several cancers, such as colon carcinoma [3,4], liver cancer [5] and neoplasm of bladder [6]. Hence, β-glucuronidase has been used as a tumor marker [7,8], and the quantitative detection of β-glucuronidase has become an effective screening tool for the early-stage diagnosis, real-time monitoring, and evaluating clinical therapies. In addition, many evidences show that numerous physiological diseases including rheumatoid arthritis [9], urinary tract infection [10], renal diseases [11], epilepsy [12] are also associated with significantly elevated serum β-glucuronidase levels. Liver disease has become a major health issue globally [13]. Liver damage cause an increase in the level of βglucuronidase in blood [14] and liver cancer could be related to this enzyme [15]. Consequently, inhibition of β-glucuronidase enzyme is active in preventing numerous diseases. Indole moiety is frequently encountered in medicinal chemistry and is considered to be a privileged scaffold [16]. Several compounds possessing indole moiety showed antitumor activity [17] and can lead to inflammation and vessication of human skin [18]. More importantly, indolecontaining compounds have pronounced effects in many physiological processes, and indoles having 5-HT receptor activity as agonists [19] or antagonists [20] have aroused considerable synthetic interest during the last decade in many research groups. In this study, a series of indole derivatives had been synthesized and evaluated for βglucuronidase inhibitory potential. Previously our group has reported various β-glucuronidase inhibitors based on benzothiazole, bisindole and thiosemicarbazide hybrid analogs, disulfide and sulfone hybrid scaffolds [21]. 2.

Material and Methods

2.1

General methods

All nuclear magnetic resonance experiments had been carried out using on Avance Bruker 500 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). Chromatograms were visualized by UV at 254 and 365 nm.

2.2

Synthesis of 5-chloro-1H-indole carbohydrazone derivatives

Equimolar amount of 5-chloro-3H-indole-2-carbohydrazide (1 mmol) and aromatic aldehyde (1 mmol) were refluxed for 6 hours in the presence of catalytic amount of glacial acetic acid (1 ml) and methanol (25 ml) as a solvent. After 6 hours, TLC was done to observe reaction completion. When the reaction had completed, methanol was removed and residues collected were being washed thoroughly using diethyl ether to afford pure product. The residues were finally air-dried before being subjected through 1H NMR for structural elucidation and confirmation. 2.2.1 (E)-5-chloro-N'-(4-methylbenzylidene)-1H-indole-2-carbohydrazide White solid. Yield: 83 %. m.p.: 255-256 °C; 1H NMR (500 MHz, DMSO) δH 11.99 (s, 1H), 11.90 (s, 1H), 8.43 (s, 1H), 7.77 (s, 1H), 7.66 (d, J = 7.8 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.35 – 7.25 (m, 3H), 7.23 (dd, J = 8.7, 2.0 Hz, 1H), 2.35 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δC 157.8, 148.1, 140.4, 135.7, 132.1, 132.0, 129.9, 129.9, 128.5, 127.6, 127.6, 125.0, 124.4, 121.3, 114.5, 103.6, 21.5; HR-ESI-MS: m/z calcd for C17H14ClN3O, [M] + 311.7690; Found 311.7695; Anal. Calcd: C, 65.49; H, 4.53; N, 13.48; Found C, 65.51; H, 4.52; N, 13.46

2.2.2

(E)-5-chloro-N'-(2-methylbenzylidene)-1H-indole-2-carbohydrazide

White solid. Yield: 85 %. m.p.: 247-248 °C; 1H NMR (500 MHz, DMSO) δH 12.02 (s, 1H), 11.95 (s, 1H), 8.75 (s, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.78 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.30 (dt, J = 22.3, 7.3 Hz, 4H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H), 2.47 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δC 157.7, 146.6, 137.4, 135.7, 132.7, 132.1, 131.3, 130.3, 128.5, 126.7, 126.3, 125.0, 124.4, 121.3, 114.5, 103.5, 19.4; HR-ESI-MS: m/z calcd for C17H14ClN3O, [M]

+

311.7690; Found 311.7683; Anal. Calcd: C, 65.49; H, 4.53; N, 13.48; Found C, 65.51; H, 4.51; N, 13.49

2.2.3 (E)-5-chloro-N'-(4-nitrobenzylidene)-1H-indole-2-carbohydrazide Yellow solid. Yield: 86 %. m.p.: 293-294 °C; 1H NMR (500 MHz, DMSO) δH 12.24 (s, 1H), 12.05 (s, 1H), 8.55 (s, 1H), 8.32 (d, J = 8.7 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H), 7.80 (s, 1H), 7.49

(d, J = 8.7 Hz, 1H), 7.35 (s, 1H), 7.25 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 157.8, 148.3, 141.0, 141.0, 135.8, 132.1, 128.5, 127.8, 127.8, 125.0, 124.7, 124.7, 124.5, 121.4, 114.5, 104.2; HR-ESI-MS: m/z calcd for C16H11ClN4O3, [M]

+

342.7390; Found

342.7385; Anal. Calcd: C, 56.07; H, 3.24; N, 16.35; Found C, 56.08; H, 3.26; N, 16.33

2.2.4 (E)-5-chloro-N’-(3-nitrobenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 89 %. m.p.: 281-282 °C; 1H NMR (500 MHz, DMSO) δH 12.22 (s, 1H), 12.05 (s, 1H), 8.57 (s, 2H), 8.27 (dd, J = 8.1, 1.5 Hz, 1H), 8.18 (d, J = 6.7 Hz, 1H), 7.77 (dd, J = 11.4, 4.8 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.34 (s, 1H), 7.25 (dd, J = 8.7, 1.9 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 157.6, 148.7, 145.5, 136.6, 135.8, 133.8, 132.2, 130.9, 128.4, 125.0, 124.7, 124.6, 121.3, 114.5, 104.0, 49.0; HR-ESI-MS: m/z calcd for C16H11ClN4O3, [M]

+

342.7390; Found 342.7397; Anal. Calcd: C, 56.07; H, 3.24; N, 16.35; Found C, 56.09; H, 3.22; N, 16.33

2.2.5 (E)-5-chloro-N’-(3-hydroxy-4-methoxybenzylidene)-1H-indole-2-carbohydrazide White solid. Yield: 87 %. m.p.: 274-276 °C; 1H NMR (500 MHz, DMSO) δH 11.98 (s, 1H), 11.81 (s, 1H), 9.35 (s, 1H), 8.31 (s, 1H), 7.76 (s, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.35 – 7.25 (m, 2H), 7.23 (d, J = 8.6 Hz, 1H), 7.10 (dd, J = 8.2, 1.2 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 3.82 (s, 3H).

13

C NMR (126 MHz, DMSO-d6): δC 157.62, 150.33, 148.23, 147.36, 135.63, 132.22,

128.53, 127.57, 124.94, 124.32, 121.21, 120.84, 114.45, 112.89, 112.41, 103.36, 56.08; HR-ESIMS: m/z calcd for C17H14ClN3O3, [M]

+

343.7670; Found 343.7685; Anal. Calcd: C, 59.40; H,

4.11; N, 12.22; Found C, 59.38; H, 4.13; N, 12.23

2.2.6 (E)-5-chloro-N’-(2-hydroxy-5-methoxybenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 84 %. m.p.: 271-272 °C; 1H NMR (500 MHz, DMSO) δH 12.16 (s, 1H), 11.99 (s, 1H), 10.57 (s, 1H), 8.65 (s, 1H), 7.78 (d, J = 1.7 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.32 (s, 1H), 7.24 (dd, J = 8.7, 2.1 Hz, 1H), 7.18 (s, 1H), 6.93 (dd, J = 8.9, 3.0 Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 3.75 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δC 157.60, 152.72, 151.87 147.54, 135.77, 131.71, 128.50, 125.03, 124.54, 121.34, 119.63, 118.75, 117.77, 114.50, 112.58, 103.86, 56.04; HR-ESI-MS: m/z calcd for C17H14ClN3O3, [M] + 343.7670; Found 343.7657; Anal. Calcd: C, 59.40; H, 4.11; N, 12.22; Found C, 59.38; H, 4.13; N, 12.23

2.2.7 (E)-5-chloro-N’-(2-chlorobenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 78 %. m.p.: 232-234 °C; 1H NMR (500 MHz, DMSO) δH 12.19 (s, 1H), 12.04 (s, 1H), 8.87 (s, 1H), 8.06 (d, J = 4.7 Hz, 1H), 7.78 (d, J = 11.3 Hz, 1H), 7.58 – 7.40 (m, 5H), 7.34 (s, 1H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 157.8, 143.8, 135.7, 133.7, 132.8, 132.0, 131.9, 130.4, 128.5, 128.1, 127.5, 125.0, 124.6, 121.4, 114.5, 103.9; HR-ESI-MS: m/z calcd for C16H11Cl2N3O, [M] + 332.1840; Found 332.1848; Anal. Calcd: C, 57.85; H, 3.34; N, 12.65; Found C, 57.83; H, 3.36; N, 12.67

2.2.8 (E)-5-chloro-N’-(2-nitrobenzylidene)-1H-indole-2-carbohydrazide Yellow solid. Yield: 82 %. m.p.: 279-281 °C; 1H NMR (500 MHz, DMSO) δH 12.30 (s, 1H), 12.04 (s, 1H), 8.88 (s, 1H), 8.19 – 8.12 (m, 1H), 8.09 (dd, J = 8.2, 0.9 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.79 (d, J = 1.9 Hz, 1H), 7.73 – 7.63 (m, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.36 (s, 1H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 158.0, 148.7, 143.1, 135.8, 134.2, 132.1, 131.7, 131.1, 129.1, 128.5, 125.1, 125.0, 124.7, 121.4, 114.5, 104.2; HR-ESI-MS: m/z calcd for C16H11ClN4O3, [M]

+

342.7390; Found 342.7381; Anal. Calcd: C, 56.07; H, 3.24;

N, 16.35; Found C, 56.08; H, 3.22; N, 16.34

2.2.9 (E)-5-chloro-N’-(2,4,6-trihydroxybenzylidene)-1H-indole-2-carbohydrazide Brown solid. Yield: 78 %. m.p.: Above 320 °C; 1H NMR (500 MHz, DMSO) δH 12.02 (s, 1H), 11.96 (s, 1H), 11.09 (s, 2H), 9.89 (s, 1H), 8.80 (s, 1H), 7.77 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.26 (s, 1H), 7.23 (dd, J = 8.7, 2.0 Hz, 1H), 5.86 (s, 2H). 13C NMR (150 MHz, DMSO-d6): δC 162.1, 160.1, 157.0, 147.0, 147.0, 135.7, 131.8, 128.5, 128.5, 125.0, 124.4, 121.3, 114.5, 103.4, 99.6, 94.9; HR-ESI-MS: m/z calcd for C16H12ClN3O4, [M]

+

345.7390; Found 345.7396; Anal.

Calcd: C, 55.58; H, 3.50; N, 12.15; Found C, 55.56; H, 3.48; N, 12.14

2.2.10 (E)-5-chloro-N’-(2,4,5-trihydroxybenzylidene)-1H-indole-2-carbohydrazide Brown solid. Yield: 79 %. m.p.: 278-280 °C; 1H NMR (500 MHz, DMSO) δH 11.96 (s, 1H), 11.91 (s, 1H), 10.48 (s, 1H), 9.58 (s, 1H), 8.60 (s, 1H), 8.47 (s, 1H), 7.77 (d, J = 1.7 Hz, 1H),

7.47 (d, J = 8.7 Hz, 1H), 7.26 (s, 1H), 7.23 (dd, J = 8.7, 1.9 Hz, 1H), 6.94 (s, 1H), 6.36 (s, 1H). 13

C NMR (126 MHz, DMSO-d6): δC 157.2, 152.3, 149.8, 148.5, 139.1, 135.6, 132.0, 128.5,

124.9, 124.3, 121.2, 114.9, 114.4, 110.1, 104.0, 103.3; HR-ESI-MS: m/z calcd for C16H12ClN3O4, [M]

+

345.7390; Found 345.7390; Anal. Calcd: C, 55.58; H, 3.50; N, 12.15;

Found C, 55.56; H, 3.51; N, 12.16

2.2.11 (E)-5-chloro-N’-(4-hydroxybenzylidene)-1H-indole-2-carbohydrazide White solid. Yield: 76 %. m.p.: 290-291 °C; 1H NMR (500 MHz, DMSO) δH 11.96 (s, 1H), 11.77 (s, 1H), 9.96 (s, 1H), 8.37 (s, 1H), 7.76 (s, 1H), 7.60 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.28 (s, 1H), 7.22 (dd, J = 8.7, 2.0 Hz, 1H), 6.87 (d, J = 8.1 Hz, 2H). 13C NMR (126 MHz, DMSO-d6): δC 159.9, 157.6, 148.4, 135.6, 132.3, 129.4, 129.4, 128.6, 125.7, 125.7, 124.9, 124.3, 121.2, 116.2, 114.4, 103.3; HR-ESI-MS: m/z calcd for C16H12ClN3O2, [M]

+

313.7410;

Found 313.7418; Anal. Calcd: C, 61.25; H, 3.86; N, 13.39; Found C, 61.24; H, 3.85; N, 13.38

2.2.12 (E)-5-chloro-N’-(3-hydroxybenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 78 %. m.p.: 287-289 °C; 1H NMR (500 MHz, DMSO) δH 11.98 (s, 1H), 11.90 (s, 1H), 9.66 (s, 1H), 8.37 (s, 1H), 7.77 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.35 – 7.20 (m, 4H), 7.14 (d, J = 7.4 Hz, 1H), 6.86 (dd, J = 8.0, 1.8 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δC 158.2, 148.2, 136.0, 135.7, 132.1, 130.4, 128.6, 127.4, 125.0, 124.4, 121.3, 119.2, 118.0, 114.5, 113.3, 103.6; HR-ESI-MS: m/z calcd for C16H12ClN3O2, [M]

+

313.7410; Found

313.7398; Anal. Calcd: C, 61.25; H, 3.86; N, 13.39; Found C, 61.26; H, 3.87; N, 13.40

2.2.13 (E)-5-chloro-N’-(4-methoxybenzylidene)-1H-indole-2-carbohydrazide White solid. Yield: 81 %. m.p.: 271-273 °C; 1H NMR (500 MHz, DMSO) δH 11.99 (s, 1H), 11.85 (s, 1H), 8.40 (s, 1H), 7.77 (s, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.6 Hz, 1H), 7.29 (s, 1H), 7.23 (dd, J = 8.7, 1.9 Hz, 1H), 7.04 (d, J = 8.4 Hz, 2H), 3.82 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δC 161.4, 157.6, 147.9, 135.6, 132.2, 129.2, 129.2, 128.5, 127.3, 124.9, 124.3, 121.2, 114.9, 114.9, 114.4, 103.4, 55.8; HR-ESI-MS: m/z calcd for C17H14ClN3O2, [M]

+

327.7680; Found 327.7672; Anal. Calcd: C, 62.30; H, 4.31; N, 12.82; Found C, 62.32; H, 4.30; N, 12.80

2.2.14 (E)-4-((2-(5-chloro-1H-indole-2-carbonyl)hydrazono)methyl)benzoic acid White solid. Yield: 87 %. m.p.: 301-303 °C; 1H NMR (500 MHz, DMSO) δH 12.12 (s, 1H), 12.04 (s, 1H), 8.51 (s, 1H), 8.04 (d, J = 8.1 Hz, 2H), 7.88 (d, J = 8.1 Hz, 2H), 7.79 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.33 (s, 1H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δC 168.0, 157.5, 148.8, 136.5, 133.2, 131.7, 131.6, 129.4, 129.4, 129.1, 129.1, 128.5, 122.5, 121.4, 121.3, 111.9, 109.8; HR-ESI-MS: m/z calcd for C17H12ClN3O3, [M]

+

341.7510; Found

341.7514; Anal. Calcd: C, 59.75; H, 3.54; N, 12.30; Found C, 59.77; H, 3.56; N, 12.29

2.2.15 Methyl (E)-4-((2-(5-chloro-1H-indole-2-carbonyl)hydrazono)methyl)benzoate White solid. Yield: 90 %. m.p.: 281-282 °C; 1H NMR (500 MHz, DMSO) δH 12.11 (s, 1H), 12.02 (s, 1H), 8.51 (s, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.90 (d, J = 8.2 Hz, 2H), 7.79 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.33 (s, 1H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H), 3.89 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δC 166.7, 157.5, 148.8, 136.5, 134.7, 131.6, 131.6, 129.1, 129.1, 128.9, 128.9, 128.5, 122.5, 121.4, 121.3, 111.9, 109.8, 52.2; HR-ESI-MS: m/z calcd for C18H14ClN3O3, [M]

+

355.7780; Found 355.7788; Anal. Calcd: C, 60.77; H, 3.97; N, 11.81; Found C, 60.78; H, 3.96; N, 11.80 2.2.16 (E)-5-chloro-N’-(2-hydroxy-4-methoxybenzylidene)-1H-indole-2-carbohydrazide White solid. Yield: 89 %. m.p.: 275-277 °C; 1H NMR (500 MHz, DMSO) δH 12.09 (s, 1H), 12.00 (s, 1H), 11.46 (s, 1H), 8.57 (s, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.29 (s, 1H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H), 6.55 (dd, J = 8.6, 2.3 Hz, 1H), 6.51 (d, J = 2.3 Hz, 1H), 3.79 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δC 166.3, 157.8, 146.6, 139.2, 135.7, 132.1, 131.0, 130.1, 128.5, 127.7, 127.7, 125.0, 124.6, 121.4, 114.5, 103.9, 52.7; HR-ESI-MS: m/z calcd for C17H14ClN3O3, [M]

+

343.7670; Found 343.7679; Anal. Calcd: C, 59.40; H, 4.11; N,

12.22; Found C, 59.42; H, 4.13; N, 12.20

2.2.17 (E)-5-chloro-N’-(pyridin-2-ylmethylene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 85 %. m.p.: 268-269 °C; 1H NMR (500 MHz, DMSO) δH 12.16 (s, 1H), 12.06 (s, 1H), 8.64 (d, J = 4.6 Hz, 1H), 8.49 (s, 1H), 8.02 (d, J = 6.8 Hz, 1H), 7.91 (t, J = 7.3 Hz, 1H), 7.80 (s, 1H), 7.54 – 7.39 (m, 2H), 7.34 (s, 1H), 7.25 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δC 162.6, 157.3, 148.7, 135.7, 131.8, 131.4, 128.5, 125.0, 124.4, 121.3,

114.5, 112.4, 107.0, 103.6, 101.7; HR-ESI-MS: m/z calcd for C15H11ClN4O, [M]

+

298.7300;

Found 298.7289; Anal. Calcd: C, 60.31; H, 3.71; N, 18.76; Found C, 60.32; H, 3.73; N, 18.7

2.2.18 (E)-5-chloro-N’-(pyridin-3-ylmethylene)-1H-indole-2-carbohydrazide White solid. Yield: 82 %. m.p.: 259-261 °C; 1H NMR (500 MHz, DMSO) δH 12.13 (s, 1H), 12.03 (s, 1H), 8.90 (s, 1H), 8.63 (d, J = 4.6 Hz, 1H), 8.51 (s, 1H), 8.18 (d, J = 7.3 Hz, 1H), 7.79 (s, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.33 (s, 1H), 7.24 (dd, J = 8.7, 1.7 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 157.5, 148.8, 148.7, 141.6, 136.5, 133.2, 131.6, 131.3, 128.5, 123.7, 122.5, 121.4, 121.3, 111.9, 109.8; HR-ESI-MS: m/z calcd for C15H11ClN4O, [M]

+

298.7300; Found

298.7311; Anal. Calcd: C, 60.31; H, 3.71; N, 18.76; Found C, 60.30; H, 3.69; N, 18.74

2.2.19 (E)-5-chloro-N’-(2,3-dihydroxybenzylidene)-1H-indole-2-carbohydrazide Brown solid. Yield: 87 %. m.p.: 263-265 °C; 1H NMR (500 MHz, DMSO) δH 12.21 (s, 1H), 12.04 (s, 1H), 10.97 (s, 1H), 9.25 (s, 1H), 8.61 (s, 1H), 7.79 (d, J = 1.9 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.31 (s, 1H), 7.24 (dd, J = 8.7, 1.9 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 6.88 (dd, J = 7.8, 1.2 Hz, 1H), 6.76 (t, J = 7.8 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δC 157.6, 153.6, 150.0, 148.1, 137.4, 135.7, 132.1, 128.5, 127.4, 127.1, 124.9, 124.7, 121.4, 120.5, 114.5, 104.0; HRESI-MS: m/z calcd for C16H12ClN3O3, [M] + 329.7400; Found 329.7405; Anal. Calcd: C, 58.28; H, 3.67; N, 12.74; Found C, 58.30; H, 3.69; N, 12.72

2.2.20 (E)-5-chloro-N’-(2,4-dihydroxybenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 87 %. m.p.: 295-296 °C; 1H NMR (500 MHz, DMSO) δH 11.97 (s, 2H), 11.33 (s, 1H), 9.98 (s, 1H), 8.52 (s, 1H), 7.77 (d, J = 1.6 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.23 (dd, J = 8.7, 2.0 Hz, 1H), 6.39 (dd, J = 8.4, 2.1 Hz, 1H), 6.35 (s, 1H). 13C NMR (126 MHz, DMSO-d6): δC 161.2, 159.8, 157.3, 149.2, 135.7, 131.8, 131.6, 128.5, 125.0, 124.3, 121.2, 114.4, 111.1, 108.2, 103.5, 103.1; HR-ESI-MS: m/z calcd for C16H12ClN3O3, [M]

+

329.7400; Found 329.7408; Anal. Calcd: C, 58.28; H, 3.67; N, 12.74;

Found C, 58.29; H, 3.66; N, 12.73

2.2.21 (E)-5-chloro-N’-(3,5-dimethoxybenzylidene)-1H-indole-2-carbohydrazide White solid. Yield: 81 %. m.p.: 246-247 °C; 1H NMR (500 MHz, DMSO) δH 11.99 (s, 2H), 8.39 (s, 1H), 7.77 (d, J = 1.6 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.32 (s, 1H), 7.24 (dd, J = 8.7, 1.6 Hz, 1H), 6.92 (d, J = 1.9 Hz, 2H), 6.58 (t, J = 1.8 Hz, 1H), 3.81 (s, 6H). 13C NMR (126 MHz, DMSO-d6): δC 157.6, 149.0, 146.5, 146.1, 146.1, 135.8, 131.6, 128.5, 125.1, 124.5, 124.5, 121.3, 120.4, 119.7, 119.4, 117.9, 114.5, 103.9; HR-ESI-MS: m/z calcd for C18H16ClN3O3, [M] + 357.7940; Found 357.7931; Anal. Calcd: C, 60.43; H, 4.51; N, 11.74; Found C, 60.44; H, 4.49; N, 11.72

2.2.22 (E)-5-chloro-N’-(3,4-dimethoxybenzylidene)-1H-indole-2-carbohydrazide White solid. Yield: 87 %. m.p.: 272-273 °C; 1H NMR (500 MHz, DMSO) δH 11.96 (s, 1H), 11.85 (s, 1H), 8.39 (s, 1H), 7.77 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.38 (s, 1H), 7.32 – 7.19 (m, 3H), 7.04 (d, J = 8.3 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δC 161.2, 159.9, 157.3, 149.3, 135.7, 131.9, 131.6, 128.5, 125.0, 124.4, 121.3, 114.5, 111.1, 108.3, 103.5, 103.2, 56.1, 56.0; HR-ESI-MS: m/z calcd for C18H16ClN3O3, [M]

+

357.7940; Found

357.7947; Anal. Calcd: C, 60.43; H, 4.51; N, 11.74; Found C, 60.41; H, 4.52; N, 11.75

2.2.23 (E)-5-chloro-N’-(2,5-dihydroxybenzylidene)-1H-indole-2-carbohydrazide Brown solid. Yield: 89 %. m.p.: 291-292 °C; 1H NMR (500 MHz, DMSO) δH 12.09 (s, 1H), 12.01 (s, 1H), 10.28 (s, 1H), 9.01 (s, 1H), 8.60 (s, 1H), 7.78 (d, J = 1.4 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.31 (s, 1H), 7.24 (dd, J = 8.7, 1.7 Hz, 1H), 7.04 (s, 1H), 6.82 – 6.70 (m, 2H). 13C NMR (126 MHz, DMSO-d6): δC 157.53, 150.62, 150.40, 147.53, 135.74, 131.78, 128.52, 125.02, 124.50, 121.33, 119.68, 119.47, 117.58, 114.48, 114.04, 103.73; HR-ESI-MS: m/z calcd for C16H12ClN3O3, [M]

+

329.7400; Found 329.7410; Anal. Calcd: C, 58.28; H, 3.67; N, 12.74;

Found C, 58.29; H, 3.65; N, 12.73

2.2.24 (E)-5-chloro-N’-(3,4-dihydroxybenzylidene)-1H-indole-2-carbohydrazide Brown solid. Yield: 85 %. m.p.: 268-269 °C; 1H NMR (500 MHz, DMSO) δH 11.96 (s, 1H), 11.73 (s, 1H), 9.36 (s, 2H), 8.27 (s, 1H), 7.76 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.32 – 7.19 (m, 3H), 6.98 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 157.52, 148.59, 148.49, 146.21, 135.60, 132.32, 128.56, 126.19, 124.92, 124.26, 121.19, 121.08,

116.10, 114.43, 113.32, 103.24; HR-ESI-MS: m/z calcd for C16H12ClN3O3, [M]

+

329.7400;

Found 329.7404; Anal. Calcd: C, 58.28; H, 3.67; N, 12.74; Found C, 58.27; H, 3.69; N, 12.72

2.2.25 (E)-5-chloro-N’-(3-methoxybenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 86 %. m.p.: 205-206 °C; 1H NMR (500 MHz, DMSO) δH 11.99 (s, 2H), 8.44 (s, 1H), 7.78 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.32 (s, 3H), 7.24 (dd, J = 8.7, 1.9 Hz, 1H), 7.08 – 6.99 (m, 1H), 3.83 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δC 157.5, 150.6, 150.4, 147.5, 135.7, 131.8, 128.5, 125.0, 124.5, 121.3, 119.7, 119.5, 117.6, 114.5, 114.0, 103.7, 39.7; HR-ESI-MS: m/z calcd for C17H14ClN3O2, [M] + 327.7680; Found 327.7686; Anal. Calcd: C, 62.30; H, 4.31; N, 12.82; Found C, 62.28; H, 4.33; N, 12.80

2.2.26 (E)-5-chloro-N’-(2-hydroxybenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 92 %. m.p.: 263-265 °C; 1H NMR (500 MHz, DMSO) δH 12.20 (s, 1H), 12.03 (s, 1H), 11.15 (s, 1H), 8.66 (s, 1H), 7.79 (d, J = 1.6 Hz, 1H), 7.60 (d, J = 7.3 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.36 – 7.27 (m, 2H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H), 6.99 – 6.90 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δC 157.80, 157.56, 148.13, 135.78, 131.89, 131.64, 129.68, 128.49, 125.03, 124.55, 121.36, 119.89, 119.34, 116.87, 114.50, 103.84; HR-ESI-MS: m/z calcd for C16H12ClN3O2, [M]

+

313.7410; Found 313.7402; Anal. Calcd: C, 61.25; H, 3.86; N, 13.39;

Found C, 61.23; H, 3.84; N, 13.40

2.2.27 (E)-5-chloro-N’-(pyridin-4-ylmethylene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 83 %. m.p.: 279-280 °C; 1H NMR (500 MHz, DMSO) δH 12.24 (s, 1H), 12.06 (s, 1H), 8.67 (d, J = 5.1 Hz, 2H), 8.44 (s, 1H), 7.80 (s, 1H), 7.70 (d, J = 4.8 Hz, 2H), 7.50 (d, J = 8.7 Hz, 1H), 7.35 (s, 1H), 7.25 (dd, J = 8.7, 1.7 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 160.1, 136.2, 135.7, 132.0, 130.5, 128.5, 125.0, 124.5, 121.3, 120.5, 116.7, 114.5, 112.0, 103.7, 55.7; HR-ESI-MS: m/z calcd for C15H11ClN4O, [M]

+

298.7300; Found 298.7308; Anal.

Calcd: C, 60.31; H, 3.71; N, 18.76; Found C, 60.33; H, 3.69; N, 18.75

2.2.28 (E)-5-chloro-N’-(4-fluorobenzylidene)-1H-indole-2-carbohydrazide White solid. Yield: 87 %. m.p.: 266-267 °C; 1H NMR (500 MHz, DMSO) δH 12.01 (s, 1H), 11.99 (s, 1H), 8.46 (s, 1H), 7.91 – 7.79 (m, 2H), 7.78 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.39 –

7.26 (m, 3H), 7.23 (dd, J = 8.7, 1.4 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 157.8, 157.6, 148.1, 135.8, 131.9, 131.6, 129.7, 128.5, 125.0, 124.6, 121.4, 119.9, 119.3, 116.9, 114.5, 103.8; HR-ESI-MS: m/z calcd for C16H11ClFN3O, [M]

+

315.7324; Found 315.7335; Anal. Calcd: C,

60.87; H, 3.51; N, 13.31; Found C, 60.85; H, 3.49; N, 13.29

2.2.29 (E)-5-chloro-N’-(2-fluorobenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 88 %. m.p.: 242-243 °C; 1H NMR (500 MHz, DMSO) δH 12.11 (s, 1H), 12.05 (s, 1H), 8.71 (s, 1H), 7.99 (t, J = 7.0 Hz, 1H), 7.79 (s, 1H), 7.56 – 7.44 (m, 2H), 7.37 – 7.27 (m, 3H), 7.24 (dd, J = 8.7, 1.8 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 158.1, 150.9, 150.7, 145.5, 141.9, 135.8, 132.1, 131.6, 128.5, 127.5, 125.1, 124.7, 123.3, 121.5, 114.5, 104.2; HR-ESI-MS: m/z calcd for C16H11ClFN3O, [M]

+

315.7324; Found 315.7313; Anal. Calcd: C,

60.87; H, 3.51; N, 13.31; Found C, 60.89; H, 3.53; N, 13.30

2.2.30 (E)-5-chloro-N’-(3-fluorobenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 81 %. m.p.: 261-262 °C; 1H NMR (500 MHz, DMSO) δH 12.08 (s, 1H), 12.03 (s, 1H), 8.47 (s, 1H), 7.78 (s, 1H), 7.66 – 7.43 (m, 4H), 7.33 (s, 1H), 7.28 (td, J = 8.5, 2.2 Hz, 1H), 7.24 (dd, J = 8.7, 2.1 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δC 157.8, 146.9, 135.7, 132.0, 131.3, 129.8, 129.7, 128.5, 126.7, 125.0, 124.5, 121.3, 116.5, 116.3, 114.5, 103.7; HR-ESI-MS: m/z calcd for C16H11ClFN3O, [M]

+

315.7324; Found 315.7329; Anal. Calcd: C,

60.87; H, 3.51; N, 13.31; Found C, 60.86; H, 3.49; N, 13.30

2.2.31 (E)-5-chloro-N’-(3-chlorobenzylidene)-1H-indole-2-carbohydrazide Yellowish solid. Yield: 82 %. m.p.: 241-242 °C; 1H NMR (500 MHz, DMSO) δH 12.10 (s, 1H), 12.03 (s, 1H), 8.44 (s, 1H), 7.79 (d, J = 11.7 Hz, 2H), 7.72 (s, 1H), 7.52 – 7.46 (m, 3H), 7.33 (s, 1H), 7.24 (dd, J = 8.7, 1.6 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 160.3, 157.8, 140.7, 135.8, 132.4, 131.8, 128.5, 126.9, 125.4, 125.0, 124.6, 122.2, 121.4, 116.4, 114.5, 103.8; HRESI-MS: m/z calcd for C16H11Cl2N3O, [M] + 332.1840; Found 332.1851; Anal. Calcd: C, 57.85; H, 3.34; N, 12.65; Found C, 57.86; H, 3.33; N, 12.63

2.2.32 (E)-N’-(3-bromo-4-hydroxybenzylidene)-5-chloro-1H-indole-2-carbohydrazide

White solid. Yield: 88 %. m.p.: 251-252 °C; 1H NMR (500 MHz, DMSO) δH 11.99 (s, 1H), 11.90 (s, 1H), 10.84 (s, 1H), 8.32 (s, 1H), 7.90 (s, 1H), 7.77 (s, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.29 (s, 1H), 7.23 (dd, J = 8.7, 1.6 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 157.9, 146.6, 137.3, 135.8, 131.9, 131.3, 128.5, 125.0, 124.6, 124.0, 123.9, 121.3, 117.2, 114.5, 113.4, 103.9; HR-ESI-MS: m/z calcd for C16H11BrClN3O2, [M] + 392.6370; Found 392.6375; Anal. Calcd: C, 48.94; H, 2.82; N, 10.70; Found C, 48.95; H, 2.80; N, 10.69

2.2.33 (E)-N’-(3-bromo-4-fluorobenzylidene)-5-chloro-1H-indole-2-carbohydrazide White solid. Yield: 83 %. m.p.: 257-258 °C; 1H NMR (500 MHz, DMSO) δH 12.11 (s, 1H), 12.02 (s, 1H), 8.41 (s, 1H), 8.08 (d, J = 6.1 Hz, 1H), 7.82 (s, 1H), 7.78 (d, J = 1.4 Hz, 1H), 7.52 – 7.44 (m, 2H), 7.32 (s, 1H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δC 157.9, 146.3, 137.0, 135.8, 134.2, 131.8, 130.2, 128.5, 126.8, 126.3, 125.0, 124.6, 121.4, 114.5, 103.9, 39.7; HR-ESI-MS: m/z calcd for C16H10BrClFN3O, [M]

+

394.6284; Found 394.6288;

Anal. Calcd: C, 48.70; H, 2.55; N, 10.65; Found C, 48.68; H, 2.52; N, 10.68

2.2.34 (E)-N’-(4-chlorobenzylidene)-5-chloro-1H-indole-2-carbohydrazide White solid. Yield: 80 %. m.p.: 271-272 °C; 1H NMR (500 MHz, DMSO) δH 12.06 (s, 2H), 8.45 (s, 1H), 7.85 – 7.75 (m, 3H), 7.55 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.7 Hz, 1H), 7.32 (s, 1H), 7.24 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δC 157.7, 156.3, 156.3, 146.7, 135.7, 131.8, 128.5, 127.5, 124.9, 124.4, 121.2, 117.0, 114.5, 114.5, 110.4, 103.5; HR-ESI-MS: m/z calcd for C16H11Cl2N3O, [M]

+

332.1840; Found 332.1832; Anal. Calcd: C, 57.85; H, 3.34; N,

12.65; Found C, 57.84; H, 3.32; N, 12.67

2.2.35 (E)-N’-(2,4-dichlorobenzylidene)-5-chloro-1H-indole-2-carbohydrazide White solid. Yield: 83 %. m.p.: 251-252 °C; 1H NMR (500 MHz, DMSO) δH 12.23 (s, 1H), 12.05 (s, 1H), 8.80 (s, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.72 (d, J = 1.8 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.33 (s, 1H), 7.24 (dd, J = 8.7, 1.8 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): δC 160.5, 158.6, 157.9, 145.3, 135.8, 133.1, 132.1, 129.0, 128.5, 125.0, 124.6, 121.3, 117.7, 114.5, 109.1, 103.9; HR-ESI-MS: m/z calcd for

C16H10Cl3N3O, [M]

+

366.6260; Found 366.6269; Anal. Calcd: C, 52.42; H, 2.75; N, 11.46;

Found C, 52.44; H, 2.77; N, 11.45

2.3

Docking

Docking studies had been performed in accordance to Taha et al. (2016) [22] using human β-Dglucuronidase crystal structure retrieved from the protein data bank (PDB code: 1BHG) [23]. The structure of all compounds were prepared using Chem3D by CambridgeSoft. The geometry and energy of the structures were being optimized using Steepest-Descent and Polak-Ribiere algorithm in HyperChem. AutoDock 4.2 [24] was used to identify the binding modes of active derivatives responsible for the activity. Genetic Algorithm (GA) with default settings was employed for the studies. The program was set to collect a maximum of 150 conformations. The conformations were then clustered in groups with RMSD less than 1.0 A. The clusters were ranked by the lowest energy representative of each cluster. For the binding energy, we considered the entropic effect as well. We selected the lowest energy structure in the best cluster as a final model. For all other parameters, the default values were used with AutoDock Tools. The docking results had been visualized using Discovery Studio visualizer 3.5 [25] and PyMol [26].

2.4

β-Glucuronidase assay

β-glucuronidase activity was determined in accordance to method used by Taha et al. (2016) [27] 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 0.1M acetate buffer and 10 µL of enzyme solution were incubated for 30 minute at 37 °C. At 405 nm the plates were recorded on multiplate reader (SpectaMax plus 384) after the addition of 50 µL of 0.4mM p-nitrophenyl-β-D-glucuronide. Experiments were performed for triplicate [28]. 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

Most of natural products consist of a heterocyclic core. Amongst nitrogen containing heterocycles, indole is a ubiquitous structural unit of a number of natural products. Indole moiety has been employed in the designing of new heterocyclic compounds with diverse biological and pharmacological properties [29]. In the family of heterocyclic compounds, nitrogen containing heterocycles are of greater significance as they have contributed enormously in understanding various life processes from the biological and industrial point of view [30]. However, indole derivatives bearing carbohydrazide moiety have been found to exhibit potent β-glucuronidase. 3.1 Chemistry The title compounds (1-35) were synthesized through Scheme-1. Indole hydrazones 1-35 (Table-1) were synthesized through a single step reaction of 5-chloro-1H-indole-2carbohydrazide II with benzaldehydes, in the presence of catalytic amount of glacial acetic acid. Compound II, which is the key intermediate for the synthesis of target compounds 1-35 was prepared through a single step reaction of indole ester I with hydrazine hydrate [31-33].

Scheme-1: Mechanism of formation 5-chloro-3H-indole-2-carbohydrazide. Reagents and condition: (a) NH2-NH2, Methanol, reflux, 6H; (b) Benzaldehydes, Methanol, acetic acid, reflux, 6H

Table-1. Synthesis of indole derivatives (1-35)

(1-35) No.

1

R

No.

13

R

No.

25

R

2

14

26

3

15

27

4

16

28

5

17

29

6

18

30

7

19

31

8

20

32

9

21

33

10

22

34

11

23

35

12

24

3.2 β-Glucuronidase activity In the continuation of our research work on enzyme inhibition [34-43]. Indole analogs (1-35) have been synthesized and evaluated for β-glucuronidase inhibitory potential. The results obtained from β-glucuronidase inhibition activity showed that 31out of 35 analogs that were synthesized in this study displayed varying degree of β-glucuronidase inhibitory potential ranging between 0.50 - 53.40 µM, when compared to standard D-saccharic acid 1,4-lactone (IC50= 48.4 ± 1.25 µM). Analogs 10 (IC50 = 0.50 ± 0.010 µM), 9 (IC50 = 2.40 ± 0.10 µM), 29 (IC50 = 2.50 ± 0.10 µM), 14 (IC50 = 2.70 ± 0.10 µM), 28 (IC50 = 6.5 ± 0.10 µM), 23 (IC50 = 7.50 ± 0.10 µM), 20 (IC50 = 8.70 ± 0.10µM), 11 (IC50 = 12.50 ± 0.30µM), 19 (IC50 = 13.40 ± 0.20µM), 7 (IC50 = 13.60 ± 0.15 µM), 30 (IC50 = 14.40 ± 0.10µM), 24 (IC50 = 14.60 ± 0.20µM), 16 (IC50 = 21.5 ± 0.30µM), 5 (IC50 = 22.50 ± 0.30 µM), 15 (IC50 = 22.60 ± 0.30 µM), 17 (IC50 = 22.80 ± 0.30µM), 6 (IC50 = 24.30 ± 0.30µM), 35 (IC50 = 24.60 ± 0.30µM), 26 (IC50 = 25.20 ± 0.20µM), 8 (IC50 = 32.50 ± 0.40µM), 12 (IC50 = 34.30 ± 0.45µM), 34 (IC50 = 37.20 ± 0.50µM), 2 (IC50 = 39.40 ± 0.50µM), 3 (IC50 = 41.40 ± 0.70µM), 27 (IC50 = 42.40 ± 0.80µM), 1 (IC50 = 43.50 ± 0.70µM), and 18 (IC50 = 44.50 ± 0.90 µM) showed outstanding β-glucuronidase activities, superior than the standard D-saccharic acid 1,4-lactone (IC50= 48.4 ± 1.25µM). Analogs 31 (IC50 = 53.40 ± 1.30µM), 13 (IC50 = 53.60 ± 1.30µM), and 4 (IC50 = 56.5 ± 1.20µM) exhibited moderate β-glucuronidase potential. Furthermore, compounds 21, 22, 25 and 32 were found inactive (Table-2). Structure-activity relationship had been also established. The SAR was based on the substitution pattern on aromatic ring. Analog 10, a 2,4,5-trihydroxy was found to be the most potent molecule among the series. The greater potential of the compound seems to be mainly due to these hydroxyl groups on phenyl ring, which might be involved in hydrogen bonding. Analog 9, a 2,4,6-trihydroxy analog was found to be the second most active compound among the series. The small difference in activity between these two analogs is due to position difference of substituents on aromatic ring. It was observed in this study that dihydroxy analogs like 19, 20,

24, 26 and monohydroxy analogs like 11, 12, are also supreme active analogs of the series with many fold better than the standard. Analogs 5, 6, 16 having hydroxyl and methoxy groups also showed good inhibitory but less than the dihydroxy or monohydroxy analogs. The less potential shown by these analogs might be due to steric hindrance of methoxy group. Analogs having fluoro substituent (28, 29 and 30), carboxylic acid (14), acetate (15), methoxy (23), chloro (7, 34 and 35) substituents on phenyl residue also showed good inhibitory potential. Compounds with methyl (1 and 2) and pyridine (17, 18 and 27) substituents showed moderate to good βglucuronidase inhibitory potential. Analogs 4 having nitro group at para position and 13 having methoxy group at para position and 31 having chloro group at meta position displayed moderate activity. Analog 33 having bromo and fluoro group at ortho, para position showed outstanding inhibition, while analogs 8 and 4 having nitro group at ortho and meta position displayed good and moderate β-glucuronidase activity, respectively. It was concluded from this study that the nature, number and position of substituents greatly influence the inhibitory potential of compounds. Table-2: β-Glucuronidase inhibitory potential of indole carbohydrazide derivatives No.

IC50 (µM) ± SEMa

No.

IC50 (µM) ± SEMa

1

43.50 ± 0.70

19

13.40 ± 0.20

2

39.40 ± 0.50

20

8.70 ± 0.10

3

41.40 ± 0.70

21

N.A.

4

56.5 ± 1.20

22

N.A.

5

22.50 ± 0.30

23

7.50 ± 0.10

6

24.30 ± 0.30

24

14.60 ± 0.20

7

13.60 ± 0.15

25

N.A.

8

32.50 ± 0.40

26

25.20 ± 0.20

9

2.40 ± 0.10

27

42.40 ± 0.80

10

0.50 ± 0.010

28

6.5 ± 0.10

11

12.50 ± 0.30

29

2.50 ± 0.10

12

34.30 ± 0.45

30

14.40 ± 0.10

13

53.60 ± 1.30

31

53.40 ± 1.30

14

2.70 ± 0.10

32

N.A.

15

22.60 ± 0.30

33

14.40 ± 0.10

16

21.5 ± 0.30

34

37.20 ± 0.50

17

22.80 ± 0.30

35

24.60 ± 0.30

44.50 ± 0.90

D-saccharic acid 1,4lactone

48.4 ± 1.25

18 a

3.3

Standard error mean

Molecular Docking studies

The ability of indole-2-carbohydrazone (1-35) derivatives to inhibit β-glucuronidase activity induced by had been further investigated using molecular docking studies. In this study, the aim of the docking was to predict the binding mode of active compounds. The results obtained from docking studies showed that active compounds are aligned in such a way that benzylidene moieties are oriented towards the inner core of the active site and interacts with polar residues in the active site (Figure-1).

Figure-1. Active compounds in the active site of β-glucuronidase

Cluster analysis was performed for all compounds using an rmsd tolerance of 1.0 Å. The cluster analysis results for compounds 9 and 10 were displayed in figure-3. Cluster analysis for

compound 9 showed that the conformations obtained from 150 docking runs for compound 9 can be divided into 12 clusters (Figure-2a). The cluster analysis for compound 9 displayed that first cluster for compound 9 contained the highest number of analysed conformations (35 out of 150 conformations) and it was found to be the lowest on the energy scale. Hence, it was the most energetically favourable cluster, possessing an estimated docking energy of about -8.0 kcal mol−1 (Figure-2b). Meanwhile for compound 10, a total of 7 conformational clusters were obtained from 150 docking runs (Figure-2b). Cluster analysis of the docked poses showed that major cluster for compound 10 displayed the lowest binding energy and it contains nearly half of the analysed conformations (64 out of 150 conformations). Predicted binding models with the lowest docking energy were therefore being used for further binding orientation analysis (Figure-3). a)

b)

Figure-2. Cluster analysis of the AutoDock docking runs of compound 9 (a) and compound 10 (b) in the binding site of β-glucuronidase enzyme. Based on figure-3(a), hydroxyl at fifth position on the benzylidene ring of the most active compound 10 played the most important role by acting as a hydrogen bond donor and interacts with the backbone (Oε1) of nucleophilic residue Glu540 at a distance of 1.91 Å. The hydroxyl was also able to interact with the backbone (O15) of Glu451 within the distance of 2.09 Å. On the other hand, hydroxyl (OH) on the side chain of catalysis residue Tyr508 acts as a hydrogen bond acceptor to form hydrogen bonding (2.14 Å) with hydroxyl at fourth position of benzylidene ring. It was observed that hydrogen of NH on indole ring and amine linkage interacts with the backbone (O) of Asn502 at the distance of 1.98 Å and 2.33 Å, respectively. The main interaction which played the most significant role in stabilizing the enzyme-inhibitor complex is the interaction of indole withTrp528 by forming a hydrophobic π-alkyl interaction with methyl at a distance of 4.18 Å.

Docking studies that for the second most active compound 9 showed that hydroxyl at sixth position on the benzylidene ring is able to interact with both catalytically important catalytic residue Glu451 and nucleophilic residue Glu540. Despite having similar binding pose, comparing with compound 10 showed that the hydroxyl at sixth position interacts with the backbone (Oε1) on Glu540 at a longer distance that is 2.86 Å. However, despite not being able to form hydrogen bonding with Glu540, the hydroxyl at sixth position is able to interact closely with the backbone (Oε2) of Glu451 at a distance of 2.12 Å. For compound 9, the hydroxyl (OH) on the side chain of Tyr508 forms hydrogen bonding (1.68 Å) with hydroxyl at fourth position on benzylidene ring. It was observed that hydrogen of NH on indole ring and amine linkage interacts with the backbone (O) of Asn502 at the distance of 1.84 Å and 2.47 Å, respectively. Enzyme-ligand complex was stabilized through electrostatic π-anion interaction of benzylidene ring for compound 9 with Glu451 (3.99 Å). On the other hand, methyl on indole moiety enhances stability of the complex by forming a hydrophobic π -alkyl interaction with Trp528 at a distance of 4.19 Å. (a)

(b)

Figure-2. Binding position of active compound 10 (a) and 9 (b) in the active site of βglucuronidase. It was observed from the docking studies that for the compound 31 (Figure-4), the most significant interaction is the ability of fluoro substituent at ortho position on benzylidene ring to form hydrogen bonding with Glu451. This can be clearly observed in Figure-4, which showed that fluoro substituent is in the position to interact with Glu451 and Glu540. Distance monitor suggest that the fluoro substituent is nearer to Glu451 (2.95 Å) as compared to Glu540 (3.18 Å).

It was observed that hydrogen of NH on indole ring and amine linkage interacts with the backbone (O) of Asn502 at the distance of 1.78 Å and 2.31 Å, respectively. As for compound 30, similar interaction as for compound 31 was observed in which the fluoro substituent at para for compound 30 forms an electrostatic interaction with OH of Tyr508 (2.74 Å). Unlike results observed for fluoro substituted compounds 30 and 31, chloro substituted compounds like 7 showed lower inhibition activity. The results from docking studies suggest that compounds having chloro substituent on the benzylidene ring are not able to accommodate the binding cavity and thus results in the chloro substituent not being able to form hydrogen bonding with important amino acids. It was also observed that the distances monitored between chloro and important amino acids are around 4.60 to 5.10 Å.

(a)

(b)

Figure-4. Binding positions of (a) compound 31 and (b) compound 7 in binding cavity of βglucuronidase.

Conclusion Indole analogs (1-35) have been synthesized and evaluated for β-glucuronidase inhibitory potential, 31out of 35 analogs displayed varying degree of β-glucuronidase inhibitory potential ranging between 0.50 - 53.40 µM. Molecular docking studies revealed the binding mode of the indole derivatives and the key moieties that are responsible for the inhibition activity taking

place. Analysis on docked binding mode suggests that the binding poses are governed by the hydrophilic functional groups present and how these hydrophilic functional groups affect the inhibition activity. It was observed that the most active compound played the most important role by acting as a hydrogen bond donor and interacts with Glu540, Tyr508, Asn502, and Glu451. The enzyme-inhibitor complex was also being stabilized by formation of a hydrophobic π-alkyl interaction between indole and Trp528.

Acknowledgement The authors would like to acknowledge Universiti Teknologi MARA for the financial support under the Research Intensive Faculty grant scheme with reference number UiTM 600RMI/DANA 5/3/LESTARI (54/2015).

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Synthesis of indole analogs as potent β-glucuronidase inhibitors Mohd Syukri Baharudina,b, Muhammad Tahaa,b , Syahrul Imrana,b, Nor Hadiani Ismaila,b, Fazal Rahim c, Muhammad Tariq Javed c, Khalid Mohammed Khand a

Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), Universiti Teknologi MARA,

Puncak Alam Campus, 42300 Bandar PuncakAlam, Selangor D. E. Malaysia b c

Faculty of Applied Science, UiTM Shah Alam, 40450 Shah Alam, Selangor D.E. Malaysia

Depatment of Chemistry, Hazara University, Mansehra-21120, Pakistan

d

H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological

Sciences, University of Karachi, Karachi-75270, Pakistan

Compound 10 (IC50 = 0.50 ± 0.010μM) Potent Inhibitor of β-Glucuronidase Standard Inhibitor D-saccharic acid 1,4-lactone (IC50 = 48.4 ± 1.25μM)



Corresponding authors. E-mail addresses: [email protected] and [email protected] (Muhammad Taha)

Highlights:  Synthesis of Indole Derivatives  In vitro β-glucuronidase inhibitory activity  Identification of a novel class of β-glucuronidase inhibitors  Molecular docking