Synthesis and cytotoxicity against human cancer cells of novel diazenecarboxamides

The synthesis as well as the cytotoxic activity of a series of diazenecarboxamides 1a - 1j , which are stable representatives of the diazene family, in a human cancer cell line panel are described. Compounds 1j and especially 1g showed a high cytotoxic activity against some leukemia cell lines, and can be considered as new leads for further development


Introduction
Diazenecarboxylic acid esters have already been evaluated decades ago in several biological systems by Kosower. 1 Unfortunately, most of them were found to be unstable.Their degradation involved free radicals, causing fatal damage throughout the cell.Having those results in mind, as well as the fact that amides are more stable towards hydrolysis than the corresponding esters, unsymmetrical diazenecarboxamides were supposed to be more appropriate for such studies than diazenecarboxylic acid esters.We described recently an application of diazenecarboxamides as convenient and selective oxidants of glutathione (GSH), cysteamine, dithiothreitol, and other thiols to disulfides. 2 Some of diazenecarboxamides were then demonstrated to act as potential modulators of drug resistance to cisplatin for certain types of tumours.Their effectiveness appears to be related to the ability to lower the intracellular level of GSH. 3 A particular diazene, i.e. a diazenecarboxamide similar to 1f−1i with the 2-pyridyl group attached directly to the diazene functionality, was found to exhibit a cytotoxic activity on 10 human cancer cell lines, on parental cells as well as on their drug-resistant sublines. 4As a continuation of this work we describe herein the synthesis and the evaluation of the cytotoxic activity in an in vitro human disease-oriented tumour cell line screening panel of a series of 10 selected diazenes (1a−1j; Figure 1).

Results and Discussion
Our recent reports on mono-and disubstituted hydrazines demonstrated their synthesis and a variety of applications in organic chemistry. 5Diazenes are easily available from monosubstituted ones in a two-step procedure.Thus, the addition of the monosubstituted hydrazine derivative 3 to the isocyanate 2 resulted in the formation of the corresponding 1,4-disubstituted semicarbazide 4.
The oxidation of the latter type of compounds was performed with N-bromosuccinimide/pyridine (NBS/Py; the preparation of 1a−1d), ceric(IV) ammonium nitrate (CAN; 1f, 1g, 1i and 1j) or with Br 2 /pyridine (Br 2 /Py; 1h; Scheme 1).The most convenient route to unsymmetrical diazenedicarboxamide 1e was the substitution of the ethoxy group in the diazene 5, 6 employing 2-picolylamine (6) as a nucleophile.The synthesis and spectroscopic data of 1a, 1c, 1d, and 1i have already been published. 2,7Other diazenes were obtained following the reaction conditions, depicted in Scheme 1, and were characterised as described in experimental section.The cytotoxic activity against human cancer cell lines was evaluated first in a 3-cell line, one dose primary anticancer assay.Results for each test compound are reported as the percentage of growth of the treated cells when compared to the untreated control cells, and are listed for the diazenes 1a-1j in Table 1.The complete set of data obtained in the screening panel demonstrated that especially these leukemia cell lines were relatively more sensitive to diazenes 1f-1j than were other cell lines.

Issue in
Diazene 1g turned out to be the most active compound.Against the leukemia cell lines CCRF-CEM and HL-60 (TB) its GI 50 values were in the nanomolar range (68 nM and 23 nM, respectively).The TGI values of 1g against both cell lines were 1.5 µM and 55 nM, and the LC 50 values 9.1 µM and 13.8 µM, respectively.Also diazene 1j was an order of magnitude more active against these two cell lines (GI 50 values 1.7 µM and 1.0 µM, respectively), than any other diazene tested.The TGI values of 1j were 26.3 µM and 5.2 µM, respectively, whereas the LC 50 values were >100 µM.In conclusion, the diazenecarboxamides 1j and especially 1g can be considered as new leads for the development of antitumoural agents, active against leukemia, which deserve further exploration.

Experimental Section
General Procedures.Melting points were determined on a hot stage and were uncorrected.IR spectra were measured as KBr pellets.NMR spectra were recorded at 300.13 and 75.

Cytotoxicity.
The cytotoxic activity of the diazenes 1a−1j was evaluated at the National Cancer Institute (NCI, Bethesda, Maryland, USA).The 3-cell line panel, consisting of MCF7 (breast Issue in Honor of Prof. cancer), NCI-H460 (non-small cell lung cancer), and SF-268 (central nervous system cancer, CNS) was used as a pre-screen for the large 60 cell line panel.Briefly, each cell line was inoculated and preincubated on a microtiter plate.Test compounds were then added at a single dose (100 µM) and the culture was incubated for 48 h.End-point determinations were made with sulforhodamine B, a protein-binding dye.The diazenes 1b and 1e−1j were then tested at a minimum of five concentrations at 10fold dilutions, 100 µM being the highest test concentration, in the NCI's screening panel consisting of 60 human tumour cell lines.In this assay results are evaluated in terms of specificity and potency.The cytotoxic effects of each compound can be expressed as the molar drug concentrations required for 50% growth inhibition (GI 50 ), total growth inhibition (TGI), and 50% cell kill (LC 50 ).8

Honor of Prof. Miha Tišler ARKIVOC 2001 (v) 42-50 ISSN 1424-6376 Page 45 © ARKAT USA, Inc Table 1. Growth percentages of diazenes 1a-1j in
Compounds which reduced the growth of any of the cell lines to less than 32% (negative numbers indicate cell kill), were passed on for evaluation in the full panel of 60 cell lines over a 5-log dose range.Since this condition was fulfilled for diazenes 1b, 1e, 1f, 1g, 1h, 1i, and 1j, they were tested in the NCI's screening panel consisting of 60 human tumour cell lines, which is largely derived from solid tumours, plus some leukemia cell lines.Nine subpanels represent diverse histologies, i.e. non-small cell lung, colon, central nervous system, renal, ovarian, prostate, and breast cancers, melanoma, and leukemia.The average GI 50 values calculated from all cell lines tested, as well as the GI 50 values for two leukemia cell lines, i.e.CCRF-CEM and HL-60 (TB), are displayed in Table2.Issue in Honor of Prof. Miha Tišler ARKIVOC 2001 (v) 42-50 ISSN 1424-6376 Page 46 © ARKAT USA, Inc

Table 2 .
GI 50 values (µM) a of diazenes 1b and 1e-1j in the 60-cell line panel, and against leukemia cell lines CCRF-CEM and HL-60 (TB) a GI 50 is the concentration of the diazene required for 50% growth inhibition.