key: cord-328176-fck2ktxi authors: Mahapatra, Manojkumar; Kulandaivelu, Umasankar; Saiko, Philipp; Graser, Geraldine; Szekeres, Thomas; Andrei, Graciela; Snoeck, Robert; Balzarini, Jan; Jayaprakash, Venkatesan title: Methyl-2-arylidene hydrazinecarbodithioates: synthesis and biological activity date: 2013-02-19 journal: Chem Zvesti DOI: 10.2478/s11696-013-0346-4 sha: doc_id: 328176 cord_uid: fck2ktxi Methyl-2-arylidene hydrazine-carbodithioate has not been of particular interest to researchers even though its metal complexes are extensively reported on due to their biological activity. This study examined the cytostatic and antiviral activity of twelve methyl-2-arylidene hydrazinecarbodithioates reported by many researchers as intermediates for the synthesis of thiosemicarbazides and the preparation of their metal complexes. Compounds IIc, IIi, and IIl with tridentate ligand features were found to have the lowest IC(50) value (6.5 μM, ≈ 1 μM, and 0.8 μM, respectively) against HL60 human promyelocytic leukemia cells. They were also most inhibitory to human embryonic lung (HEL) fibroblast proliferation (5.3 μM, 17 μM, and 2.6 μM). Compound IIc and IIl show antiviral activity against wild-type herpes simplex virus (HSV), varicella zoster virus (VZV), and acyclovirresistant HSV; however, these activities were observed at concentrations at which the compounds also markedly inhibit HL60 and HEL cell proliferation. Many N-substituted thiosemicarbazones were prepared by the condensation of Schiff's bases of methyl-2-arylidene hydrazinecarbodithioate (MAHCD) with amines (Casero et al., 1980; Collins et al., 1982; Klayman et al., 1979 Klayman et al., , 1991 Scovill et al., 1982; Shipman et al., 1981) . While thiosemicarbazones and their metal complexes were studied for their biological activity (Beraldo & Gambinob, 2004; Ettari et al., 2010; Jiang et al., 2006; Katz, 1987; Liberta & West, 1992; Matesanz & Souza, 2009; Pandeya & Dimmock, 1993; Wnuk & Robins, 2006; Yu et al., 2009) , the intermediate MAHCD was rarely studied for its metal complexation behaviour and the complexes for their biological activity (Ali et al., 2002; Kanwar et al., *Corresponding author, e-mail: venkatesanj@bitmesra.ac.in 2008; Neelam et al., 2000; Saxena & Tandon, 1983; Singh et al., 1997) . Very few investigators have reported biological activity of compounds with a dithiocarbamate side chain (Cao et al., 2005; Huang et al., 2009; Kumar et al., 2010) . It is interesting to note that MAHCD shares structural similarity with Schiff's bases of N-hydroxy semicarbazides (SNHS) and Nhydroxy aminoguanidines (SNHAG) (Fig. 1) . Both SNHS and SNHAG were rigorously documented for their anticancer activity and antiviral properties. It was proposed that these compounds act by inhibiting ribonucleotide reductase (RR) through metal chelation (Das et al., 1999; Ren et al., 2002; T'ang et al., 1985) . With this background, twelve MAHCD derivatives were synthesised and evaluated for anticancer activity against HL60 human promyelocytic leukemia cells and antiviral activity against a panel of viral cell lines. Synthesis of methyl hydrazinecarbodithioate (I) and substituted benzaldehyde hydrazones of methyl hydrazine carbodithioate (II) To an ice cooled solution (< 10 • C) of 19.8 g (0.300 mol) of potassium hydroxide in 24 mL of water and 20 mL of propan-2-ol, 17.1 mL of 80 % pure hydrazine hydrate were added and constantly stirred. The amount of 18.2 mL (0.300 mol) of ice cooled carbon disulfide was added drop wise to the stirred solution, maintained at < 10 • C for over about 1-1.5 h. The bright yellow mixture formed was stirred for an additional 1 h after which ice cooled iodomethane (18.7 mL, 0.300 mol) was added drop wise over a 2 h period. Stirring was continued for an additional 1 h, and the white precipitate obtained was filtered and washed with ice-cold water. The crude product was recrystallised from dichloromethane: yield of 43 %; Audrieth et al. (1954) ; 81-83 • C, Klayman et al. (1979) ). Methyl hydrazinecarbodithioate (20.0 mmol) was dissolved in 20 mL of methanol and then, equimolar amount of aromatic/heteroaromatic aldehyde was added. The mixture was refluxed for 6-12 h and the reaction was monitored by TLC. The hot solution was poured onto crushed ice and the precipitate obtained was filtered (Klayman et al., 1979; Scovill et al., 1982) : yield of 60-80 %. The HL-60 human promyelocytic leukemia cell line was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were grown in a RPMI 1640 medium supplemented with a 10 % heat inactivated fetal calf serum (FCS), 1 % L-glutamine and 1 % penicillin-streptomycin in a humidified atmosphere containing 5 % of CO 2 . All media and supplements were obtained from Life Technologies (Paisley, Scotland, UK). Cell counts were determined using a micro cell counter CC-108 (SYSMEX, Kobe, Japan). Cells growing in the logarithmic phase of growth were used in all experiments described below. HL-60 cells (10 5 per mL) were seeded in 25 cm 2 Nunc tissue culture flasks and incubated with increasing concentrations of drugs (IIa-IIl) at 37 • C under cell culture conditions. Cell counts and IC 50 values were determined after 24 h, 48 h, and 72 h using the micro cell counter CC-108. Viability of the cells was determined by trypan blue exclusion. Results were calculated as the number of viable cells. Antiviral assays (except anti-human immunodeficiency virus (HIV) assays) were based on the inhibition of virus-induced cytopathic effect in HEL For the HCMV assays, confluent HEL fibroblasts were grown in 96-well microtiter plates and infected with the human cytomegalovirus strains Davis and AD-169 at 100 PFU per well. After a 2 h incubation period, the residual virus was removed and the infected cells were further incubated with a medium containing different concentrations of the test compounds (in duplicate). After incubation for 7-days at 37 • C, the virus-induced cytopathic effect was monitored microscopically after ethanol fixation and staining with Giemsa dye. Antiviral activity was expressed as the EC 50 or compound concentration required for the reduction of virus-induced cytopathogenicity by 50 %. The varicella zoster virus (VZV) drug susceptibility tests were performed on confluent HEL cells in 96well microtiter plates by the plaque reduction assay. Monolayers were infected with 20 plaque forming units (PFU) of the cell-associated virus per well. For each assay, virus controls (infected-untreated cells) were included. After a 2 h incubation period, the virus inoculum was removed and the media were replaced by different dilutions (in duplicate) of the tested molecules. Serial dilutions of test compounds were incubated with the infected monolayers for five days. After a five day incubation period, the cells were fixed and stained with Giemsa, and the level of the virus-induced cytopathic effect was determined by counting the number of plaques for each dilution. Activity was expressed as EC 50 (effective compound concentration required to reduce virus plaque formation by 50 %) compared to the untreated control. The anti-HIV activity of the compounds was evaluated against the wild type HIV-1 strain IIIB in MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) method. Briefly, virus stocks were titrated in MT-4 cells and expressed as the 50 % cell culture infective dose (CCID50). MT-4 cells were suspended in culture medium at 10 5 cells per mL and infected with HIV at the multiplicity of infection of 0.02. Immediately after viral infection, 100 µL of the cell suspension were placed in each well of a flat-bottomed microtiter tray containing various concentrations of the test compounds. The test compounds were dissolved in DMSO at 50 mM or more as stock solutions. Then, serial dilutions of the test compounds were made. The highest concentration tested was 100 µM of the test compound containing 0.2 % of DMSO, which is by itself not toxic to cell proliferation. After four days of incubation at 37 • C, the number of viable cells was determined using the MTT method. Compounds IIa-IIl were synthesised following the reaction sequence outlined in Fig. 2 . Methyl hydrazinecarbodithioate (I ) was prepared by the reaction of hydrazine hydrate (85 mass %) with carbon disulfide in the presence of potassium hydroxide at a temperature below 10 • C; followed by the addition of methyl iodide (Audrieth et al., 1954) . Condensation of I with aromatic aldehydes/ketones in methanol provided compounds IIa-IIl (Klayman et al., 1991; Scovill etal., 1982) . All compounds were characterised by their 1 H-NMR and FAB-MS data. Physicochemical characterisation data are presented Table 1 and spectral data of compounds IIa-IIl are presented in Table 2 . All compounds were tested for their anticancer activity against the HL-60 human promyelocytic leukemia cell line and the results are presented in Table 3 . The compounds were also tested for their antiviral activity against herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), herpes simplex virus-1 TK − (KOS ACV r ), varicella-zoster virus (OKA and 07/1), cytomegalovirus (Davis and AD169), vaccinia virus, vesicular stomatitis virus, feline corona virus, feline herpes virus, human immunodeficiency virus (HIV) type 1 (III B ) and type 2 (ROD), coxsackie virus B4, respiratory syncytial virus, parainfluenza 3 virus, re- Table 4 . Compound IIl, IIi, and IIc showed IC 50 values against HL60 cells at the concentration of 0.8 µM, ≈ 1 µM, and 6.5 µM, respectively, after 72 h. It is interesting to note that preferably compounds with tridentate ligand characteristics (IIl, IIi, and IIc) showed potent anti-proliferative activity against the HL60 cells (Table 3 ). The activity of compound IIi (2 -hydroxy acetophenone derivative) was higher than that of IIc (2-hydroxy benzaldehyde derivative) and almost equally potent to IIl (pyridine 2carboxaldehyde derivative). All the other compounds (IIa, IIb, IId-IIh, and IIk) were inhibitory at concentrations between 55 µM and 63 µM after 72 h, except for IIj, irrespective of the nature and position of the substitution. Compound IIj did not exert any inhibitory activity on the HL60 cells at the maximum concentration studied (100 µM, after 72 h). All the compounds exhibited a cytotoxic concentration (CC 50 ) of ≥ 100 µM in the HeLa cell cultures except for IIl (36 µM), (Table 3) . Compounds IIi and IIl showed the best selectivity towards the HL60 cells and therefore, further investigations are required. Compounds IIc and IIi resemble the iron chelator 311 (N -[(2-hydroxynaphthalen-1-yl)-methylidene]pyridine-4-carbohydrazide) and IIl resembles triapine (3-aminopyridine-2-carbaldehyde thiosemicarbazone). Compound IIi having a ketonic methyl group showed activity comparable to that of iron chelator 311 at 48 h. Compound IIl also exhibited an anti-proliferative activity comparable to that of Triapine. The analogue of IIl with a ketonic methyl group has to be considered for further improvement of its activity. It is interesting to observe that only compounds with tridentate ligand characteristics (IIc, IIi, and IIl) have shown antiviral activity against a few viruses and are presented in Table 4 . Compound IIl displayed pronounced antiviral activity against HSV-1 (8-42 µM), HSV-2 (9-11 µM ), VZV (6.5-42 µM), and vaccinia virus (15-45 µM). It should be noted that IIl inhibits the wild-type as well as the acyclovir-resistant HSV at comparable compound concentrations (8-20 µM, Table 4). Compound IIc with a 2-hydroxy substitution and compound IIi, an acetophenone analogue of IIc, show antiviral activity at somewhat higher concentrations than IIl. Compound IId that differs from IIc by a hydroxy functional group at the para position did not show any appreciable antiviral activity. Additional analogues of IIc and IIl are currently under further investigation. It should, however, be noted that IIc, IIi, and IIl are poorly cytotoxic but they show pronounced cytostatic activity in proliferating HEL (and HL60) cell cultures. Therefore, the observed antiviral activity might not be due to direct antiviral activity but rather due to indirect inhibitory action caused by the underlying cytostatic potential of these compounds. The present investigation revealed that: i) compounds with a metal chelating functional group (IIc, IIi, and IIl ) at their aryl end were found to be active and ii) the methyl dithioate (-CSSCH 3 ) group is not an bioisosteric equivalent of hydroxamate (-CONHOH) or amidoxime (-CNHNHOH) (Fig 1.) . 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