Indian Journal of Experimental Biology Vol. 48, March 2010, pp. 265-268

Synthesis, biological evaluation and molecular docking of aryl hydrazines and hydrazides for anticancer activity

Vikrantsinh M Gohila, Satyam K Agrawalc, Ajit K Saxenac, Divita Gargb, C Gopimohanb & Kamlesh K Bhutania*

aDepartment of Natural Products, bCentre for Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER) Sector-67, SAS Nagar, Punjab 160 062, India cDepartment of Pharmacology, Indian Institute of Integrative Medicine (CSIR),

Canal Road, Jammu 180 001, India

Received 4 November 2009; revised 14 December 2009

Aryl hydrazine and hydrazide analogues were synthesized based on p-tolyl hydrazine, isolated as a breakdown product of a secondary metabolite from the mushroom, Agaricus bisporus, and tested to be highly active molecule than 5-fluorouracil in in vitro anticancer studies. The synthesized analogues were tested for anticancer activity using NCI protocol. Anolgues 12 and 15 emerged as molecules with significant in vitro anticancer activity. Molecular docking study revealed the binding orientations of aryl hydrazines and hydrazides analogues in the active sites of thymidylate synthase.

Keywords: Anticancer activity, Aryl hydrazides, Aryl hydrazines, Molecular docking

Among the nitrogen-nitrogen bond containing chemicals, the hydrazines are well-known for their diverse biological actions. Even though both the beneficial and harmful effects of hydrazines had been recognized at the turn of this century, primarily their deleterious actions have been highlighted during the past four decades1. Although it was proposed in the early 1900s that hydrazine compounds are toxic to animals and to humans, they have been administered as antidepressant (e.g., iproniazid), antihypertensive (e.g., hydralazine), anti-parkinsonic (e.g., phenelzine) and anti-tuberculosis (e.g., isoniazid) drugs. The role of hydrazines in cancer has been controversial over the time. It is well-known that cancer induction and cancer inhibition sometimes occur simultaneously2. However, with an exception of procarbazine and hydrazine sulfate, none of the hydrazine compounds have shown promising contribution in anticancer drug development. Bioactive molecule from natural origin, p-tolyl hydrazine isolated as a degradation products from the mushroom Agaricus bisporus, was found to be highly active compared to 5-fluorouracil (5-FU) in in vitro anticancer studies3. The results prompted further investigation on the potential of aryl hydrazines and hydrazides as anticancer agents. In the

present study, several aryl hydrazines and hydrazides were synthesized and evaluated for anticancer and antioxidant activity and were also docked to study the important binding orientations. Materials and Methods

Synthesis of compounds—Aryl hydrazines and hydrazides were prepared by using previously reported methods4-6. The aryl hydrazides (Scheme1) were prepared by refluxing the aryl ester for 5 h with hydrazine hydrate to yield the desired hydrazides. For the preparation of aryl hydrazines analogues were synthesized by first carrying out diazotization of the aryl amine, followed by subsequent reduction with stannous chloride or sodium sulfite in presence of HCl (Scheme 2).

In vitro cytotoxicity—The NCI protocol of 60-line anticancer screen has been followed. The compounds have been tested on the one dose 5-cell line prescreen7. 5-fluoro uracil was used as a standard. The human tumor cell lines of the cancer-screening panel were grown in RPMI-1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated into 96-well microtitre plates in 100 µL at plating densities. After cell inoculation, the microtitre plates were incubated at 37°C, 5% CO2, 95% air and 100% RH for 24 h prior to addition of experimental compounds. After 24 h, two plates of each cell line

—————— *Correspondent author Telefax: + 91- 172- 214682 E-mail: kkbhutani@niper.ac.in

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were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of compound addition (Tz). Experimental compounds were solubilized in DMSO at 400 fold the desired final maximum test concentration and stored frozen prior to use. At the time of compound addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg/mL gentamicin. Additional 4, 10 fold or ½ log serial dilutions were made to provide a total of five compound concentrations plus control. Aliquots of 100 µL of these different compound dilutions were added to the appropriate microtitre wells already containing 100 µL of medium, resulting in the required final compound concentrations.

Following compound addition, the plates were incubated for an additional 48 h at 37°C, 5% CO2,

95% air, and 100% RH. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4°C. The supernatant was discarded, and the plates were washed five times with tap water and air-dried. Cells were plated out in 96-well plates at an appropriate cell density that has been determined for each cell line under study and relates to the doubling time of the cell line. The cells were left overnight to adhere and then dosed with either a fixed concentration or a range of concentrations of test compound. After a set incubation period (usually 72 h) the cells were stained using sulforhodamine B (SRB). Plates were read at an appropriate wavelength on a plate-reader and the reading for each well related to the number of cells present.

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Molecular docking analysis—All docking calculations were performed in FlexX module of Sybyl7.18 installed on SGI Tezro workstation running IRIX 6.4. The compounds were built in Sybyl7.1 and optimized using the semi empirical AM1 method9. The crystal structure of human thymidylate synthase complexed with raltitrexed, solved at a resolution of 1.9 Å, (PDB ID: 1HVY)10 was used as the starting structure for docking analysis. While creating the receptor description file (rdf), the active site had been defined as the area within 6.5Å around the co-crystallized ligand. Formal charges had been assigned to all the molecules followed by a FlexX run.

Results and Discussion Synthesis of compounds—A total of 13 compounds

were synthesized from their respective amines or esters as shown in Scheme 1 and Scheme 2. The compounds were characterized using NMR and mass spectrometry and comparison with their reported values. Compounds 8, 12, 13 were procured from Sigma-Aldrich.

In vitro cytotoxicity—The synthesized compounds were tested on 5 cancer cell lines namely A-549, SK-N-SH, PC-3, HEP-2 and MCF-7. The results of this study are shown in Table 1. Compounds 12 and 15 were found to be the most active particularly for MCF-7 and A-549 cell lines.

Molecular docking—The potential binding modes of the compounds to the active site of human thymidylate synthase were carried out and the results of the analysis are summarized in Table 2. The compounds 12 and 15, experimentally found to be most active were also the best docked ligands, having high scores, indicating highest binding propensity towards the enzyme. The docked compounds occupied the same binding site pocket as occupied by raltitrexed in the crystal structure. The nitro group at position 4 in 12 made hydrogen bonds with the imidazole moiety of His 256 and hydroxyl group of Tyr 258 (Fig. 1a). The nitro group at position 2 in 12 made hydrogen bonds with amino groups of Asn112 and Arg50 and the hydrazine moiety was anchoring to Asn112. Thus docking analysis shows that 12 interacts with the enzyme in a mode similar to

Table 1—In vitro cytotoxicity against human cancer cell lines

Cell line type Lung Liver CNS Prostate Breast Cell line code A-549 HEP-2 SK-N-SH PC-3 MCF-7 OD 1.91 1.30 1.87 1.62 2.94 *Compound Growth inhibition (%) p-toluene-sulfonyl-hydrazide (1) 0 12 21 7 5 2,4,6-tribromo-phenylhydrazine (2) 20 31 30 21 15 p-bromo-phenylhydrazine (3) 29 16 55 31 41 p-chloro-phenylhydrazine (4) 31 16 57 14 38 p-ethyl-phenylhydrazine (5) 35 9 47 24 40 p-nitro-phenyl-hydrazine (6) 18 0 29 8 12 p-methoxy-phenylhydrazine (7) 60 28 56 38 55 p-tolyl hydrazine (8) 39 6 44 14 39 2-chloro-4-methyl-phenylhydrazine (9) 17 21 14 13 5 p-trityl-phenylhydrazine (10) 14 19 28 6 33 2,4-dimethyl-phenylhydrazine (11) 73 33 55 71 67 2,4-dinitro phenylhydrazine (12) 80 42 74 52 94 p-isopropyl phenyl hydrazine (13) 36 16 64 20 39 p-iodo-phenylhydrazine (14) 42 25 60 28 46 Gallic acid hydrazide (15) 80 8 43 19 90 5-FU 68 32 58 24 69

*Concentration (M) 1 × 10-4

Table 2—Docking analysis on thymidylate synthase

Comp No.

Score Hydrogen Bonding Interactions

Residues Involved π -stacking Interaction

Residues involved

Hydrophobic Interactions

Residues involved

12 -22.695 6 Arg50, Asn112, His256, Tyr258 0 --- 2 Ile108, Met311

15 -18.288 5 Glu87, Ile108, Asn112, His196, Asn226

1 Trp109 4 Ile108, Leu192, Phe225, Met311

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ralitrexed in its crystal structure complex. 15 on the other hand interacted with a different set of residues (Fig. 1b). The hydroxyl group at position 4 of 15 made a double hydrogen bond with the hydroxyl moiety of Tyr135. The hydroxyls at positions 2 and 3 bonded to carboxylic group of Glu 87 and imidazole of His196 respectively. The hydrazine moiety anchored to Asn112 and to the backbone carboxyl of Ile108. π-stacking interactions with Trp109 and hydrophobic interactions with Ile108 and Met311 were observed for most of the present studied compounds.

Conclusion A total of 15 hydrazine and hydrazide derivatives

were screened on cancer cell lines. Compounds 12 and 15 were found to be the most active particularly for MCF-7 and A-549. These findings were corroborated with the molecular docking studies where both of these compounds exhibited the maximum docking score. Coupling of these compounds with other known antineoplastic natural products for the finding of more promising anticancer compounds are being investigated.

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Fig. 1—Docked compounds (a): 12; and (b): 15 into the active site of thymidylate synthase. [The possible hydrogen bonding interactions are rendered as yellow hashed lines.]