Synthesis of 3,4,5-trihydroxybenzohydrazone and evaluation of their urease inhibition potential

Muhammad Taha; Syed Adnan Ali Shah; Ajmal Khan; Fiza Arshad; Nor Hadiani Ismail; Muhammad Afifi; Syahrul Imran; Muhammad Iqbal Choudhary

doi:10.1016/j.arabjc.2015.06.036

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ORIGINAL ARTICLE

12 (

); 2973-2982

doi:

10.1016/j.arabjc.2015.06.036

Synthesis of 3,4,5-trihydroxybenzohydrazone and evaluation of their urease inhibition potential

Muhammad Taha^{^a,^b,⁎}, Syed Adnan Ali Shah^{^a,^c}, Ajmal Khan^{^e}, Fiza Arshad^{^d}, Nor Hadiani Ismail^{^a,^b}, Muhammad Afifi^{^a,^c}, Syahrul Imran^{^a,^b}, Muhammad Iqbal Choudhary^{^d}

a Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam Campus, Malaysia

b Faculty of Applied Science Universiti Teknologi MARA, 40450 Shah Alam, Selangor D. E., Malaysia

c Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, 42300 Selangor, Malaysia

d H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan

e Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan

⁎Corresponding author at: Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam Campus, Malaysia. Tel.: +60 193098141. taha_hej@yahoo.com (Muhammad Taha) muhamm9000@puncakalam.uitm.edu.my (Muhammad Taha)

Received: 2015-3-2, Accepted: 2015-6-27, Published: 2015-12-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

In the continuation of our work to synthesize enzyme inhibitors, we synthesized 3,4,5-trihydroxybenzohydrazones (1–19) from 3,4,5-trihydroxybenzohydrazide, which were obtained from methyl 3,4,5-trihydroxybenzoate by refluxing with hydrazine hydrate. All the synthesized compounds were characterized by different spectroscopic methods. The synthesized compounds were evaluated for urease inhibition and showed excellent results, close to the standards thiourea. The kinetic studies on the five most active compounds 6, 10, 14, 16 and 18 were carried out to determine their mode of inhibition and dissociation constant K_i. The compounds 6 and 16 were found to be competitive inhibitors with K_i values 19.1 and 10.53 μM, respectively, while the compounds 10, 14 and 18 were found to be mixed-type of inhibitors with K_i values in the range of 18.4–21.7 μM.

Keywords

3,4,5-Trihydroxybenzohydrazones

Urease inhibition

Kinetic study

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1 Introduction

Applications of benzohydrazones are reported in medicinal and analytical chemistry (Cimerman et al., 2000; Taha et al., 2014; Tarafder et al., 2002 ). Benzohydrazones having heterocyclic rings were reported to have antiglycation, anticonvulsant, Phosphodiesterase-1 inhibitors, antiproliferative, antifungal and anti-HIV activities (Kabak et al., 1999; Küçükgüzel et al., 2004; Khan et al., 2014a; Jamil et al., 2015; Pandeya et al., 1999 ). Several benzohydrazones reported interesting bioactivities, such as antifungal, anticonvulsant, anti-inflammatory, antibacterial, antimalarial, antiplatelets, analgesic, antituberculosis, antioxidant (Loncle et al., 2004; Küçükgüzel et al., 2003; Todeschini et al., 1998; Melnyk et al., 2006; Lima et al., 2000; Cunha et al., 2003; Kaymakçıoğlu et al., 2006; Aziz et al., 2014 ), antileishmanial, insecticidal, immunoconjugates, antimycobacterial, adriamycin proteinase inhibition and activity against protozoan parasite (Sawada et al., 2003; Taha et al., 2013; Küçükgüzel and Rollas, 2002; Greenfield et al., 1990; Caffery et al., 2002 ). Other benzohydrazone derivatives have reported β-glucuronidase (Jamil et al., 2014) and α-glucosidase inhibition activity (Taha et al., 2015a). On the other hand, substituted acylhydrazide Schiff bases have shown a broad range of bioactivities, including antiurease (Taha et al., 2015b), and antibacterial activities (Imran et al., 2014). Hydrazine derivatives also have several commercial applications (Ragnarsson, 2001).

Urease (E.C 3.5.1.5) plays an important role in the virulence of some bacterial pathogens as well as determinant in pathogenesis of many diseases in human. It is involved in the production of infectious stones; add to the pathogenesis of urolithiasis, pyelonephritis, and hepatic encephalopathy (Weatherburn, 1967). The urease results in pathologies by Helicobacter pylori (HP), by helping the bacteria to endure at low pH of the stomach during colonization. Thus, it plays a vital role in the pathogenesis of the gastric as well as peptic ulcers which may cause cancer (Devesa et al., 1998). Additionally, urease causes kidney stones formation but also engages in the growth of urolithiasis, pyelonephritis, and hepatic encephalopathy (Martelli et al., 1981). In agriculture, during urea fertilization, high urease activity results in significant environmental and economic losses by discharge of abnormally huge amounts of ammonia in atmosphere. This also leads to plant damage by depriving them from essential nutrients, secondary ammonia toxicity and increase in pH of the soil (Mobley and Hausinger, 1989). Urease inhibition, therefore, has been identified as first line of treatment of diseases caused by ureolytic bacteria (Saify et al., 2014). Recently reported urease inhibitors are hydroxamate complex (Cheng et al., 2014), homoserine lactone derivative (Czerwonka et al., 2014), thiophosphoric triamides (Ludden et al., 2000), oxadiazoles derivatives (Akhtar et al., 2014), thioureas (Khan et al., 2014b), ethyl 4-(3-benzothioureido) benzoates derivatives (Saeed et al., 2014), Oxindole derivatives (Taha et al., 2015c) and thiobarbituric acid derivatives (Khan et al., 2014c). However, currently available inhibitors are not efficient and the full potential of urease inhibition is yet to be discovered (Font et al., 2008).

2 Experimental

2.1

2.1 General experimental

Melting points were determined on a Büchi 434 melting point apparatus and are uncorrected. NMR was performed on Bruker AV 300, 400, and 500 MHz instruments, respectively. CHN analyses were determined on a Carlo Erba Strumentazion-Mod-1106, Italy instrument. Infrared (IR) spectra were recorded on a JASCO IR-A-302 spectrometer. Electron impact mass spectra (EI MS) were recorded on a Finnigan MAT-311A, Germany spectrometer. Thin layer chromatography (TLC) was performed on precoated silica gel glass plates (Kieselgel 60, 254, E. Merck, Germany).

2.2

2.2 Experimental protocol

2.2.1

2.2.1 General procedure for the synthesis of 3,4,5-trihydroxybenzohydrazide

The methyl 3,4,5-trihydroxybenzoate was refluxed with the mixture of hydrazine hydrated (5 mL) and methanol (15 mL) for 6 h. The excess hydrazine and methanol were evaporated to obtain crude product which was recrystallized by methanol and yielded 92% pure 3,4,5-trihydroxybenzohydrazide.

2.2.2

2.2.2 General procedure for the synthesis of 3,4,5-trihydroxybenzohydrazone derivatives

The 3,4,5-trihydroxybenzohydrazone derivatives were synthesized by refluxing in methanol a mixture of 2 mmol each of 3,4,5-trihydroxybenzohydrazide with different aldehydes and catalytic amount of acetic acid for 3 h. After the completion of the reaction, the solvent was evaporated by vacuum to afford crude products which were further recrystallized in methanol and got needle like pure product in most of the cases in good to excellent yields.

2.2.2.1

2.2.2.1 (E)-N′-(3-chlorobenzylidene)-3,4,5-trihydroxybenzohydrazide (1)

Yield: 82%. m.p. 172–173 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.64 (s, 1H, NH), 9.64 (br. s, 3H, OH), 8.34 (s, 1H, N⚌CH—Ar), 7.77 (s, 1H), 7.66 (d, 1H, J = 5.5 Hz), 7.06 (dd, 1H, J = 5.5, 2.0 Hz), (d, 1H, J = 2.0 Hz), 6.91(s, 2H); Anal. Calcd for Anal. Calcd for C₁₄H₁₁ClN₂O₄, C = 54.83, H = 3.62, N = 9.13, Found C = 54.84, H = 3.63, N = 9.15; EI MS m/z (% rel. abund.): 308 (M + 2, 6), 306 (M⁺, 20).

2.2.2.2

2.2.2.2 (E)-3,4,5-trihydroxy-N′-(4-methoxybenzylidene)benzohydrazide (2)

Yield: 78%. m.p. 174–175 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.70 (s, 1H, NH), 11.40 (s, 1H, OH) 9.88 (br. s, 3H, OH), 831 (s, 1H, N⚌CH—Ar), 7.92 (d, 2H, J = 8.5 Hz), 7.06 (d, 2H, J = 8.5 Hz), 6.90 (s, 2H), 3.81 (s, 3H, OCH₃); Anal. Calcd for Anal. Calcd for C₁₅H₁₄N₂O₅, C = 59.60, H = 4.67, N = 9.27, Found C = 59.61, H = 4.68, N = 9.26; EI MS m/z (% rel. abund.): 302.

2.2.2.3

2.2.2.3 (E)-3,4,5-trihydroxy-N′-(pyridin-2-ylmethylene)benzohydrazide (3)

Yield: 84%. m.p. 175–176 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.48 (s, 1H, NH), 10.54 (s, 1H, OH), 9.70 (br. s, 2H, OH), 9.06 (d, 1H, J = 2.0 Hz), 8.70 (dd, 1H, J = 7.0, 2.0 Hz), 8.41 (dd, 2H, J = 2.0, 6.5 Hz), 831 (s, 1H, N⚌CH—Ar), 7.82 (dd, 1H, J = 7.0, 6.5 Hz), 6.94 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₃H₁₁N₃O₄, C = 57.14, H = 4.06, N = 15.38, Found C = 57.15, H = 4.08, N = 15.37; EI MS m/z (% rel. abund.): 273.

2.2.2.4

2.2.2.4 (E)-3,4,5-trihydroxy-N′-(thiophen-2-ylmethylene)benzohydrazide (4)

Yield: 85%. m.p. 179–180 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.48 (s, 1H, NH), 9.60 (br. s, 3H, OH), 8.61 (s, 1H, N⚌CH—Ar), 7.49 (d, 1H, J = 5.0 Hz), 7.41 (d, 1H, J = 3.0 Hz), 7.22 (dd, 1H, J = 3.0, 5.0 Hz), 6.89 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₂H₁₀N₂O₄S, C = 51.79, H = 3.62, N = 10.07, Found C = 59.78, H = 3.63, N = 10.09; EI MS m/z (% rel. abund.): 278.

2.2.2.5

2.2.2.5 (E)-N′-(4-fluorobenzylidene)-3,4,5-trihydroxybenzohydrazide (5)

Yield: 86%. m.p. 171–172 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.60 (s, 1H, NH), 9.80 (br. s, 3H, OH), 8.37 (s, 1H, N⚌CH—Ar), 7.78 (t, 2 H, J = 5.5 Hz), 7.33 (t, 1H, J = 6.0 Hz), 6.91 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₄H₁₁FN₂O₄, C = 57.93, H = 3.82, N = 9.65, Found C = 57.94, H = 3.83, N = 9.67; EI MS m/z (% rel. abund.): 290.

2.2.2.6

2.2.2.6 (E)-N’-(furan-2-ylmethylene)-3,4,5-trihydroxybenzohydrazide (6)

Yield: 81%. m.p. 178–179 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.42 (s, 1H, NH), 9.70 (br. s, 3H, OH), 8.42 (s, 1H, N⚌CH—Ar), 7.60 (d, 1H, J = 6.0 Hz), 7.35 (d, 1H, J = 4.0 Hz), 7.41 (d, 1H, J = 3.0 Hz), 7.10 (dd, 1H, J = 4.0, 6.0 Hz), 6.92 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₂H₁₀N₂O₅, C = 54.97, H = 3.84, N = 10.68, Found C = 54.96, H = 3.84, N = 10.69; EI MS m/z (% rel. abund.): 262.

2.2.2.7

2.2.2.7 (E)-3,4,5-trihydroxy-N′-(3-methylbenzylidene)benzohydrazide (7)

Yield: 83%. m.p. 175–176 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.51 (s, 1H, NH), 9.42 (br. s, 3H, OH), 8.42 (s, 1H, N⚌CH—Ar), 7.52 (s, 1H), 7.48 (d, 1H, J = 7.5 Hz), 7.35 (t, 1H, J = 7.5 Hz), 7.10 (d, 1H, J = 8.0 Hz), 6.93 (s, 2H), 2.36 (3H, CH₃); Anal. Calcd for Anal. Calcd for C₁₅H₁₄N₂O₄, C = 62.93, H = 4.93, N = 9.79, Found C = 62.94, H = 3.94, N = 9.81; EI MS m/z (% rel. abund.): 286.

2.2.2.8

2.2.2.8 (E)-3,4,5-trihydroxy-N′-(3-hydroxy-4-methoxybenzylidene)benzohydrazide (8)

Yield: 88%. m.p. 172–173 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.34 (s, 1H, NH), 9.51 (br. s, 3H, OH), 9.21 (s, 1H, OH), 8.25 (s, 1H, N⚌CH—Ar), 7.23 (s, 1H), 7.03 (d, 1H, J = 8.5 Hz), 6.97 (t, 1H, J = 8.5 Hz), 6.90 (s, 2H), 3.86 (3H, OCH₃); Anal. Calcd for Anal. Calcd for C₁₅H₁₄N₂O₆, C = 56.60, H = 4.43, N = 8.80, Found C = 56.62, H = 4.44, N = 8.82; EI MS m/z (% rel. abund.): 318.

2.2.2.9

2.2.2.9 (E)-3,4,5-trihydroxy-N′-(pyridin-4-ylmethylene)benzohydrazide (9)

Yield: 90%. m.p. 174–175 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.48 (s, 1H, NH), 10.54 (s, 1H, OH), 9.70 (br. s, 2H, OH), 8.79 (d, 2H, J = 6.0 Hz), 8.35 (s, 1H, N⚌CH—Ar), 7.66 (d, 2H, J = 6.0, Hz), 6.93 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₃H₁₁N₃O₄, C = 57.14, H = 4.06, N = 15.38, Found C = 57.16, H = 4.09, N = 15.36; EI MS m/z (% rel. abund.): 273.

2.2.2.10

2.2.2.10 (E)-3,4,5-trihydroxy-N′-(2-nitrobenzylidene)benzohydrazide (10)

Yield: 87%. m.p. 170–171 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.92 (s, 1H, NH), 9.84 (br. s, 3H, OH), 8.82 (s, 1H, N⚌CH—Ar), 8.13 (d, 1H, J = 7.5 Hz), 8.08 (dd, 1H, J = 2.0, 7.0 Hz), 7.89 (t, 1H, J = 7.5 Hz), 7.68 (ddd, 1H, J = 7.5, 2.0, 2.0 Hz), 6.92 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₄H₁₁N₃O₆, C = 53.00, H = 3.49, N = 13.24, Found C = 53.02, H = 3.50, N = 13.26; EI MS m/z (% rel. abund.): 317.

2.2.2.11

2.2.2.11 (E)-3,4,5-trihydroxy-N′-(3-nitrobenzylidene)benzohydrazide (11)

Yield: 92%. m.p. 171–172 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.79 (s, 1H, NH), 9.74 (br. s, 3H, OH), 8.52 (s, 1H), 8.46 (s, 1H, N⚌CH—Ar), 8.26 (dd, 1H, J = 6.5, 2.0 Hz), 8.13 (d, 1H, J = 7.5 Hz), 7.77 (t, 1H, J = 8.0 Hz), 6.95 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₄H₁₁N₃O₆, C = 53.00, H = 3.49, N = 13.24, Found C = 53.01, H = 3.51, N = 13.25; EI MS m/z (% rel. abund.): 317.

2.2.2.12

2.2.2.12 (E)-N′-(4-chlorobenzylidene)-3,4,5-trihydroxybenzohydrazide (12)

Yield: 90%. m.p. 180–181 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.60 (s, 1H, NH), 9.65 (br. s, 3H, OH), 8.35 (s, 1H, N⚌CH—Ar), 7.74 (d, 2H, J = 8.0 Hz), 7.50 (d, 2H, J = 8.0, Hz), 6.90 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₄H₁₁ClN₂O₄, C = 54.83, H = 3.62, N = 9.13, Found C = 54.84, H = 3.61, N = 9.14; EI MS m/z (% rel. abund.): 308 (M + 2, 20), 306 (M⁺, 60).

2.2.2.13

2.2.2.13 (E)-3,4,5-trihydroxy-N′-(4-methylbenzylidene)benzohydrazide (13)

Yield: 87%. m.p. 177–178 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.44 (s, 1H, NH), 9.80 (br. s, 3H, OH), 8.33 (s, 1H, N⚌CH—Ar), 7.60 (d, 2H, J = 8.0 Hz), 7.27 (d, 2H, J = 8.0, Hz), 6.90 (s, 2H), 2.33 (3H, CH₃); Anal. Calcd for Anal. Calcd for C₁₅H₁₄N₂O₄, C = 62.93, H = 4.93, N = 9.79, Found C = 62.94, H = 4.92, N = 9.80; EI MS m/z (% rel. abund.): 286.

2.2.2.14

2.2.2.14 (E)-N′-(3-fluorobenzylidene)-3,4,5-trihydroxybenzohydrazide (14)

Yield: 80%. m.p. 175–176 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.63 (s, 1H, NH), 9.70 (br. s, 3H, OH), 8.37 (s, 1H, N⚌CH—Ar), 7.54–7.46 (m, 3 H), 7.33 (td, 1H, J = 6.5, 2.0 Hz), 6.91 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₄H₁₁FN₂O₄, C = 57.93, H = 3.82, N = 9.65, Found C = 57.95, H = 3.81, N = 9.66; EI MS m/z (% rel. abund.): 290.

2.2.2.15

2.2.2.15 (E)-3,4,5-trihydroxy-N′-(2-methylbenzylidene)benzohydrazide (15)

Yield: 82%. m.p. 169–170 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.40 (s, 1H, NH), 9.60 (br. s, 3H, OH), 8.69 (s, 1H, N⚌CH—Ar), 7.85 (d, 1H, J = 7.5 Hz), 7.31–7.23 (m, 3H), 6.92 (s, 2H), 2.35 (3H, CH₃); Anal. Calcd for Anal. Calcd for C₁₅H₁₄N₂O₄, C = 62.93, H = 4.93, N = 9.79, Found C = 62.94, H = 4.94, N = 9.81; EI MS m/z (% rel. abund.): 286.

2.2.2.16

2.2.2.16 (E)-3,4,5-trihydroxy-N′-(3-hydroxybenzylidene)benzohydrazide (16)

Yield: 90%. m.p. 172–173 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.47 (s, 1H, NH), 10.21 (s, 1H, OH), 9.74 (br. s, 3H, OH), 8.32 (s, 1H, N⚌CH—Ar), 7.26 (t, 1H, J = 8.0 Hz), 7.16 (s, 1H), 7.07 (d, 1H, J = 8.0 Hz), 6.92 (s, 2H), 7.16 (dd, 1H, J = 8.0, 2.0 Hz); Anal. Calcd for Anal. Calcd for C₁₄H₁₂N₂O₅, C = 58.33, H = 4.20, N = 9.72, Found C = 58.34, H = 4.21, N = 9.71; EI MS m/z (% rel. abund.): 288.

2.2.2.17

2.2.2.17 (E)-3,4,5-trihydroxy-N′-(3-methoxybenzylidene)benzohydrazide (17)

Yield: 82%. m.p. 170–171 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.53 (s, 1H, NH), 9.74 (br. s, 3H, OH), 8.33 (s, 1H, N⚌CH—Ar), 7.26 (t, 1H, J = 7.5 Hz), 7.30 (s, 1H), 7.26 (d, 1H, J = 8.0 Hz), 6.99 (dd, 1H, J = 8.0, 2.0 Hz) 6.91 (s, 2H), (s, 3H, OCH₃); Anal. Calcd for Anal. Calcd for C₁₅H₁₄N₂O₅, C = 59.60, H = 4.67, N = 9.27, Found C = 59.61, H = 4.66, N = 9.28; EI MS m/z (% rel. abund.): 302.

2.2.2.18

2.2.2.18 (E)-N′-(2-fluorobenzylidene)-3,4,5-trihydroxybenzohydrazide (18)

Yield: 90%. m.p. 171–172 °C; ¹H-NMR (500 MHz, DMSO-d₆): δ 11.68 (s, 1H, NH), 9.50 (br. s, 3H, OH), 8.66 (s, 1H, N⚌CH—Ar), 7.92 (t, 3 H, J = 6.5 Hz), 7.48–7.46 (m, 1H), 7.31–7.26 (m, 2H), 6.94 (s, 2H); Anal. Calcd for Anal. Calcd for C₁₄H₁₁FN₂O₄, C = 57.93, H = 3.82, N = 9.65, Found C = 57.94, H = 3.83, N = 9.64; EI MS m/z (% rel. abund.): 290.

2.2.2.19

2.2.2.19 (E)-Methyl 4-((2-(3,4,5-trihydroxybenzo)hydrazono)methyl)benzoate (19)

Yield: 92%. m.p. 180–181 °C; ¹H NMR (500 MHz, DMSO-d₆): δ 11.60 (s, 1H, NH), 9.40 (br. s, 3H, OH), 8.36 (s, 1H, N⚌CH—Ar), 8.20 (d, 2H, J = 8.0 Hz), 7.84 (d, 2H, J = 8.0, Hz), 6.91 (s, 2H), 3.80 (3H, OCH₃); Anal. Calcd for Anal. Calcd for C₁₆H₁₄N₂O₆, C = 58.18, H = 4.27, N = 8.48, Found C = 58.19, H = 4.28, N = 8.49; EI MS m/z (% rel. abund.): 314.

2.2.3

2.2.3 Urease assay and inhibition

The reaction mixtures, comprising 25 μL of enzyme (jack bean urease) solution and 55 μL of buffers containing 100 mM urea, were incubated with 5 μL of the test compounds (0.5 mM concentration) at 30 °C for 15 min in 96-well plates. For the kinetics assessment the urea concentrations were changed from 2 to 24 mM. Urease activity was determined by measuring ammonia production using the indophenol method as described by Weatherburn (1967). Briefly, 45 μL of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and, 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 μL. The results (change in absorbance per min) were processed by using SoftMaxPro software (molecular Device, USA). The entire assays were performed at pH 6.8. Percentage inhibition was calculated from the formula 100-(OD_{test well}/OD_control) × 100. Thiourea was used as the standard inhibitor for urease (Khan et al., 2014d).

3 Results and discussion

3.1

3.1 Chemistry

In the continuation of our research on enzyme inhibition (Rahim et al., 2015a,b; Taha et al., 2015d; Abdullah et al., 2015; Khan et al., 2014e ) 3,4,5-trihydroxybenzohydrazones (1–19) were synthesized from 3,4,5-trihydroxybenzohydrazide which were obtained from methyl 3,4,5-trihydroxybenzoate by refluxing with hydrazine hydrate for 4 h. The 3,4,5-trihydroxybenzohydrazide obtained was recrystallized from methanol. 3,4,5-Trihydroxybenzohydrazones were prepared by refluxing 3,4,5-trihydroxybenzohydrazide with differently substituted aldehydes 1–19 in methanol for 3–4 h (Scheme 1). The crude products obtained were recrystallized in methanol and mostly needle like crystals were obtained in 74–87% yield. The structures of 3,4,5-trihydroxybenzohydrazones were deduced by using various spectroscopic techniques and CHN analyses.

Synthesis of 3,4,5-Trihydroxybenzohydrazones (1–19).

Table 1 Derivatives of 3,4,5-Trihydroxybenzohydrazone 1–19.

3.2

3.2 Urease inhibition

In the continuation of our work, on enzyme inhibition we synthesized 1–19 3,4,5-trihydroxybenzohydrzones. They were evaluated for urease inhibition. The compounds 16, 6, 18, 14 and 10 showed good activities. The compounds 1, 4, 7, 5, 11 and 9, 3, 19 showed moderate activities while, compounds 8, 12, 15, 13 and 17 showed weak activity (Table 1).

The compound 16 (IC₅₀ = 27.20 ± 1.2 μM) was found to be the most active among the series of nineteen compounds. It was found that this activity is due to the hydroxyl group present at the 3’ position and this was confirmed by compound 17 (IC₅₀ = 83.20 ± 2.2 μM) in which hydroxyl group is replaced by methoxy substituent which resulted in the decrease of activity by almost 3-fold. Compound 8 (IC₅₀ = 50.80 ± 1.9 μM) having 3′-hydroxy-4′-methoxy showed less activity than compound 16. This might be due to the bulky methoxy group at 4’-position, which causes compound 8 to become structurally non-compatible toward urease enzyme. The compound 2 (IC₅₀ = 57.40 ± 2.2 μM) having 4′-methoxy showed only weak activity further proved the significance of hydroxy at position 3’.

The compound 6 (IC₅₀ = 28.90 ± 1.2 μM) was found to be the second most active compound of the series and its activity is due to the furan ring which would be having interaction with enzyme.

Compound 4 (IC₅₀ = 38.40 ± 1.4 μM), which is a thiophene derivative, showed less activity as compared to the compound 6 (consists of furan ring) and this may be due to large size and low electronegative nature of sulfur as compared to oxygen. Higher electronegativity effect of oxygen allows furan-containing derivative 6 to bind better through hydrogen bonding with urease enzyme as compared to thiophene moiety of compound 4.

Among the pyridine derivatives, compound 9 (IC₅₀ = 42.80 ± 1.5 μM), which is 4′-pyridinyl derivative, showed good activity as compared to its positional isomer 2′-pyridene analogue 3 (IC₅₀ = 47.40 ± 1.8 μM).

The compound 18 (IC₅₀ = 30.10 ± 1.2 μM) having fluorine atom at 2′-postion and compound 14 (IC₅₀ = 30.20 ± 1.3 μM) having fluorine atom at 3′-postion showed similar activity while interestingly its other positional isomer compound 5 (IC₅₀ = 39.70 ± 1.4 μM), having fluorine atom at 4′-postion, exhibited slightly lower reactivity. Therefore, the order of reactivity of fluorinated derivative was found to be 2′-F > 3′-F > 4′-F.

Other halogen substituted, compounds 1 (IC₅₀ = 37.30 ± 1.4 μM) and 12 (IC₅₀ = 53.90 ± 1.8 μM) exhibited the same activity trend, such as, 3′-Cl > 4′-Cl. The compound 10 (IC₅₀ = 30.80 ± 1.2 μM) having 2′-nitro substituent showed good activity but its other isomer having 3′-nitro showed weak activity.

The compound 7 having 3′-methyl showed good activity as compared to its other analogues having 2′-methyl, compound 15 (IC₅₀ = 54.00 ± 1.8 μM) and 4′-methyl (compound 13 IC₅₀ = 55.40 ± 1.9 μM). Their order of reactivity was found to be 3′-methyl > 2′-methyl > 4′-methyl, while the compound 19 (IC₅₀ = 48.80 ± 1.6 μM) having ester group showed moderate activity.

To investigate the inhibition mechanism of this series, the kinetic studies on five most active compounds 6, 10, 14, 16 and 18 were performed, with different concentrations of test compounds and substrates. Enzyme reaction is the first order reaction and the enzyme kinetics were used for only the determination of type of inhibition and Ki value. From kinetic studies, it was inferred that compounds 6 and 16 are competitive inhibitors with K_i values 19.1 ± 0.007 and 10.53 ± 0.02 μM, respectively (Figs. 1 and 2). The type of inhibition was determined by Lineweaver–Burk plots. The reciprocal of the rate of the reaction was plotted against the reciprocal of substrate concentration to monitor the effect of inhibitor on both K_m and V_max. Figs. 1 and 2 showed that in the presence of compounds 6 and 16, the V_max of jack bean urease enzyme was not affected, while the K_m of enzyme increased, which indicates the competitive inhibition (Table 2).

The inhibition of urease by compound 6. (A) Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (urea) in the absence (△), and in presence of 20 μM (■), 25 μM (□), 30 μM (●), and 35 μM (○) of compound 6. (B) Secondary replot of Lineweaver–Burk plot between the slopes of each line on Lineweaver–Burk plot vs different concentration of compound 6.

The inhibition of urease by compound 10. (A) Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (urea) in the absence (△), and in presence of 20 μM (■), 30 μM (□), 40 μM (●), and 50 μM (○) of compound 10. (B) Secondary replot of Lineweaver–Burk plot between the slopes of each line on Lineweaver–Burk plot vs different concentration of compound 10.

Table 2 The mode of inhibition of compounds 6, 10, 14, 16, and 18.

Compounds	K_i (μM)	Type of inhibition
6	19.10 ± 0.007	Competitive inhibition
10	19.90 ± 0.003	Mixed-type inhibition
14	21.71 ± 0.007	Mixed-type inhibition
16	10.53 ± 0.02	Competitive inhibition
18	18.41 ± 0.003	Mixed-type inhibition
Standard (thiourea)	20.01 ± 0.02	Competitive inhibition

The secondary replots of Lineweaver–Burk plots (Lodhi et al., 2007; Muhammad Et al., 2014) were plotted to determine the K_i value (Fig. 2). The K_i values were calculated by plotting the slope of each line in the Lineweaver–Burk plots against different concentrations of compounds 6 and 16. The K_i value was confirmed from Dixon plot, by plotting the reciprocal of the rate of reaction against the different concentrations of compounds 6 and 16.

The kinetic studies of compounds 10, 14 and 18 indicated that these are mixed-type of inhibitors with K_i values between 18.41 and 21.71 μM (Figs. 3–5 ). The Lineweaver–Burk plots of compounds 10, 14 and 18 showed that in the presence of compounds 10, 14 and 18, both the V_max and K_m of jack bean urease were affected. In the presence of compounds 10, 14 and 18 the V_max of jack bean urease was decreased, while the K_m was increased, which indicated the mixed-type of inhibition. Again for K_i determination of compounds 10, 14 and 18 the secondary replots of Lineweaver–Burk plots and Dixon plot were used.

The inhibition of urease by compound 14. (A) Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (urea) in the absence (■), and in presence of 20 μM (□), 30 μM (●), and 40 μM (○) of compound 14. (B) Secondary replot of Lineweaver–Burk plot between the slopes of each line on Lineweaver–Burk plot vs different concentration of compound 14.

The inhibition of urease by compound 16. (A) Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (urea) in the absence (▴), and in presence of 10 μM (△), 15 μM (■), 20 μM (□), 25 μM (●), and 30 μM (○) of compound 16. (B) Secondary replot of Lineweaver–Burk plot between the slopes of each line on Lineweaver–Burk plot vs different concentration of compound 16.

The inhibition of urease by compound 18. (A) Lineweaver–Burk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (urea) in the absence (■), and in presence of 20 μM (□), 30 μM (●), and 40 μM (○) of compound 18. (B) Secondary replot of Lineweaver–Burk plot between the slopes of each line on Lineweaver–Burk plot vs different concentration of compound 18.

4 Difference in IR and NMR data responsible for inhibition activity

The first ring of compounds (1–19) is common. The variation in inhibition potential is mainly due to the functional group present on second ring. We can easily distinguish by IR and NMR by observing the pick of functional group. In case of NMR inactive functional group we can see the variation in Chemical shift of adjacent Proton Table 3.

Table 3 Variation in the IR and H NMR of all functional groups present in compound (1–19).

Compound	R	IR (cm⁻¹)	¹H NMR
1	3-Cl	715	Due to chlorine electronegativity adjacent proton shifted to downfield
2	4-OCH3	1210	The presence of OCH₃ at δ 3.81
3	2-C⚌N—C	1640	Due to nitrogen electronegativity adjacent proton shifted to downfield
4	Thiophene	540	Due to small ring its proton coupling constants are 4–5 Hz only
4	C—S—C	540
5	4-F	1340	Its adjacent proton gave multiple splitting due to fluorine
6	Furan	1230	Due to small ring its proton coupling constants are 4–5 Hz only
6	C—O—C	1230
7	3-C—CH₃	2950	The presence of CH₃ at δ 2.36
7	3-C—CH₃	1440	The presence of CH₃ at δ 2.36
8	3-OH	3320	The presence of OH δ 9.20 and at 3.86 for OCH₃
8	4-OCH₃	1190	The presence of OH δ 9.20 and at 3.86 for OCH₃
9	4-C⚌N—C	1635	Due to nitrogen electronegativity adjacent proton shift to downfield
10	2-NO₂	1460	Due to nitrogen electronegativity adjacent proton shift to downfield
11	3-NO₂	1430	Due to nitrogen electronegativity adjacent proton shift to downfield
12	4-Cl	740	Due to chlorine electronegativity adjacent proton shift to downfield
13	4-CH₃	2930	The presence of CH₃ at δ 2.33
13	4-CH₃	1460	The presence of CH₃ at δ 2.33
14	3-F	1342	Its adjacent proton gave multiple splitting due to fluorine
15	2-CH₃	2937	The presence of CH₃ at δ 2.35
15	2-CH₃	1452	The presence of CH₃ at δ 2.35
16	3-OH	3340	The presence of OH δ 10.21
17	4-OCH₃	1190	The presence of OCH₃ at δ 3.80
18	2-F	1310	Its adjacent proton gave multiple splitting due to fluorine
19	4-COOMe	1710	The presence of OCH₃ at δ 3.84
19	4-COOMe	1220	The presence of OCH₃ at δ 3.84

5 Conclusion

In this study, we synthesized 3,4,5-trihydroxybenzohydrazones (1–19) and evaluated them for their urease inhibition activity. The results for urease inhibition showed excellent activity, close to the standard thiourea. We found new class of urease inhibitors. The kinetic studies on the five most active compounds 6, 10, 14, 16 and 18 were carried out. The compounds 6 and 16 were found to be competitive inhibitors and the compounds 10, 14 and 18 were found to be mixed-type of inhibitors.

Acknowledgments

Authors want to thank Universiti Teknologi Mara (UITM) Puncak Alam Campus for providing excellent laboratory facilities for the research and all technical and nontechnical staff of Atta-ur-Rahman Institute for Natural Product Discovery (RiND) for a lot of support for this work.

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Introduction

Experimental

General experimental
Experimental protocol

Results and discussion

Chemistry
Urease inhibition

Difference in IR and NMR data responsible for inhibition activity

Conclusion

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[1] Abdullah N.K.N.Z., Taha M., Ahmat N., Wadood A., Ismail N.H., Rahim F., Ali M., Abdullah N., Khan K.M., . Novel 2,5-disubstituted-1,3,4-oxadiazoles with benzimidazole backbone: a new class of β-glucuronidase inhibitors and in silico studies. Bioorg. Med. Chem.. 2015;23:3119-3125.
[Google Scholar]

[2] Akhtar T., Khan M.A., Iqbal J., Jones P.G., Hameed S., . A facile one pot synthesis of 2-arylamino-5-aryloxylalkyl-1,3,4-oxadiazoles and their urease inhibition studies. Chem. Biol. Drug. 2014;84:92-98.
[Google Scholar]

[3] Aziz A.N., Taha M., Ismail N.H., Anouar E.H., Yousuf S., Jamil W., Awang K., Ahmat N., Khan K.M., Kashif S.M., . Synthesis, crystal structure, DFT studies and evaluation of the antioxidant activity of 3,4-dimethoxybenzenamine Schiff bases. Molecules. 2014;19:8414-8433.
[Google Scholar]

[4] Caffery C.R., Schanz M., Nkemgu-Njinkeng J., Brush M., Hansell E., Cohen F.E., Flaherty T.M., Mckerrow J.H., Steverding D., . Screening of acyl hydrazide proteinase inhibitors for antiparasitic activity against Trypanosoma brucei. Int. J. Anitimicrob. Agents. 2002;19:227-231.
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[5] Cheng X.-S., Zhang J.-C., You Z.-L., Wang X., Li H.-H., . Synthesis, structures, and Helicobacter pylori urease inhibition of hydroxamate-coordinated oxovanadium complexes with benzohydrazone ligands. Trans. Metal Chem.. 2014;3:291-297.
[Google Scholar]

[6] Cimerman Z., Miljanić S., Galic N., . Schiff bases derived from aminopyridines as spectrofluorimetric analytical reagents. Croat. Chem. Acta. 2000;73:81-95.
[Google Scholar]

[7] Cunha A.C., Figueiredo J.M., Tributino J.L.M., Miranda A.L.P., Castro H.C., Zingali R.B., Fraga C.A.M., de Souza M.C.B.V., Ferreira V.F., Barreiro E.J., . Antiplatelet properties of novel N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives. Bioorg. Med. Chem.. 2003;11:2051-2059.
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[8] Czerwonka G., Arabski M., Wasik S., Jablonska-Wawrzycka A., Rogala P., Kaca W., . Morphological changes in Proteus mirabilis O18 biofilm under the influence of a urease inhibitor and a homoserine lactone derivative. Arch. Micro.. 2014;196:169-177.
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[9] Devesa S.S., Blot W.J., Fraumeni J.F., . Changing patterns in the incidence of esophageal and gastriccarcinoma in the United States. J. Cancer. 1998;83:2049-2053.
[Google Scholar]

[10] Font M., Domínguez M.J., Sanmartín C., Palop J.A., San-Francisco S., Urrutia O., Houdusse F., García-Mina J.M., . Structural characteristics of phosphoramide derivatives as urease inhibitors. Requirements for activity. J. Agric. Food Chem.. 2008;56:8451-8460.
[Google Scholar]

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Synthesis of 3,4,5-trihydroxybenzohydrazone and evaluation of their urease inhibition potential

Abstract

Keywords

3,4,5-Trihydroxybenzohydrazones

Urease inhibition

Kinetic study

1 Introduction

2 Experimental

2.1 General experimental

2.2 Experimental protocol

2.2.1 General procedure for the synthesis of 3,4,5-trihydroxybenzohydrazide

2.2.2 General procedure for the synthesis of 3,4,5-trihydroxybenzohydrazone derivatives

2.2.2.1 (E)-N′-(3-chlorobenzylidene)-3,4,5-trihydroxybenzohydrazide (1)

2.2.2.2 (E)-3,4,5-trihydroxy-N′-(4-methoxybenzylidene)benzohydrazide (2)

2.2.2.3 (E)-3,4,5-trihydroxy-N′-(pyridin-2-ylmethylene)benzohydrazide (3)

2.2.2.4 (E)-3,4,5-trihydroxy-N′-(thiophen-2-ylmethylene)benzohydrazide (4)

2.2.2.5 (E)-N′-(4-fluorobenzylidene)-3,4,5-trihydroxybenzohydrazide (5)

2.2.2.6 (E)-N’-(furan-2-ylmethylene)-3,4,5-trihydroxybenzohydrazide (6)

2.2.2.7 (E)-3,4,5-trihydroxy-N′-(3-methylbenzylidene)benzohydrazide (7)

2.2.2.8 (E)-3,4,5-trihydroxy-N′-(3-hydroxy-4-methoxybenzylidene)benzohydrazide (8)

2.2.2.9 (E)-3,4,5-trihydroxy-N′-(pyridin-4-ylmethylene)benzohydrazide (9)

2.2.2.10 (E)-3,4,5-trihydroxy-N′-(2-nitrobenzylidene)benzohydrazide (10)

2.2.2.11 (E)-3,4,5-trihydroxy-N′-(3-nitrobenzylidene)benzohydrazide (11)

2.2.2.12 (E)-N′-(4-chlorobenzylidene)-3,4,5-trihydroxybenzohydrazide (12)

2.2.2.13 (E)-3,4,5-trihydroxy-N′-(4-methylbenzylidene)benzohydrazide (13)

2.2.2.14 (E)-N′-(3-fluorobenzylidene)-3,4,5-trihydroxybenzohydrazide (14)

2.2.2.15 (E)-3,4,5-trihydroxy-N′-(2-methylbenzylidene)benzohydrazide (15)

2.2.2.16 (E)-3,4,5-trihydroxy-N′-(3-hydroxybenzylidene)benzohydrazide (16)

2.2.2.17 (E)-3,4,5-trihydroxy-N′-(3-methoxybenzylidene)benzohydrazide (17)

2.2.2.18 (E)-N′-(2-fluorobenzylidene)-3,4,5-trihydroxybenzohydrazide (18)

2.2.2.19 (E)-Methyl 4-((2-(3,4,5-trihydroxybenzo)hydrazono)methyl)benzoate (19)

2.2.3 Urease assay and inhibition

3 Results and discussion

3.1 Chemistry

3.2 Urease inhibition

4 Difference in IR and NMR data responsible for inhibition activity

5 Conclusion

Acknowledgments

References

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