Pyrido[2’,3’:4,5]pyrrolo[2,1-d ][1,2,3,5]tetrazine-4(3 H )-ones, a new class of temozolomide heteroanalogues

Twelve derivatives of new ring system pyrido[2’,3’:4,5]pyrrolo[2,1-d ][1,2,3,5]tetrazine were prepared in good yields by reaction of 2-diazo-3-ethoxycarbonyl-pyrrolo[3,2-b ]pyridine with alkyl-or aryl-isocyanates. Nine derivatives, screened by the National Cancer Institute (Bethesda, MD) for the in vitro one dose primary anticancer assay against a panel of about 60 human tumor cell lines, showed no significant activity

][11] We have previously reported on the synthesis and the antitumor activity of pyrrolo[2,1d] [1,2,3,5]tetrazine-4(3H)-ones (1) which exhibited a significant growth inhibition efficacy in many cancer cell lines, with GI 50 values in the low micromolar or sub-micromolar range and reaching, in some cases nanomolar concentrations. 12,13yrrolotetrazinones, did not show any selectivity with respect to the CNS cancer and melanoma sub-panels against which temozolomide showed curative properties, however they exhibited excellent responses in the breast cancer and leukaemia sub-panels. 13Nanostructured lipid carrier (NLC) loaded with pyrrolotetrazinones tested in vitro against hepatocellular carcinoma (HuH-6 and HuH-7) and human prostate cancer (PC-3) cell lines have shown an enhancement of cytotoxicity. 14SAR studies as well as the computerised analysis COMPARE 15 indicated that pyrrolotetrazinones have a mode of action different from that of temozolomide.Studies directed to elucidate the biochemical mechanism of action of this class of compounds indicated that they interfere with the microtubule network, block mitosis and that mitochondria and caspases play a central role in the activation of the executioner phase of pyrrolotetrazinonesinduced apoptosis. 16enzocondensation of the pyrrolotetrazinone ring led to [1,2,3,5]tetrazino [5,4-a]indole-4-one (2) derivatives which showed antiproliferative activity in the micromolar range.These latter, at variance with pyrrolotetrazinones, exhibited good selectivity with respect to the CNS sub-panel in which the lead compound, as already stated, showed curative properties. 17ncouraged by these results we thought it was interesting to verify whether the condensation of the pyrrolotetrazine system with a pyrido moiety increases the antineoplastic activity.
In this paper we focus our attention on the synthesis of the new ring system pyrido[2',
a For designation of R in 8a-l and 9a-l, see Table 1.

Scheme 2 a
Reaction between the 2-chloro-3-nitropyridine 3 and the potassium enolate of ethyl cyanoacetate 4 gave derivative 5 in high yield (92%).Reduction of this latter with iron and acetic acid at room temperature yielded the 2-amino-3-ethoxycarbonyl-pyrrolo[3,2-b]pyridine 6 (yield 80%).The 2-diazo-3-ethoxycarbonyl-pyrrolo[3,2-b]pyridine 7 was obtained, in excellent yield (96%), by diazotization of the corresponding amine 6.The reaction was carried out in 80% acetic acid with stoichiometric amount of sodium nitrite under nitrogen atmosphere in the dark followed by neutralization with sodium carbonate.The strict control of the temperature at 0°C both during diazotization and neutralization is crucial in obtaining a good yield.The structure of 2-diazo-pyrrolo[3,2-b]pyridine 7 was confirmed by analytical and spectral data (IR, 1 H and 13 C NMR).In particular the IR spectra showed a sharp and strong band at 2198 cm -1 .
The pyridopyrrolo-tetrazines 9a-l have been prepared in moderate to good yields (35-80%) by reaction of the diazo 7 with stoichiometric amounts of proper isocyanates 8a-l in DCM at room temperature for 12-48 h.The same reactions carried out under microwave irradiation, with a CEM discover apparatus, gave the pyridopyrrolo-tetrazines 9a-l with higher yield (62-95%) in a shorter time (3 min) (Table 1).The structure of all derivatives 9 was confirmed by spectroscopic data.Biological screenings were performed, on nine selected pyridopyrrolotetrazines (9a, 9b 9c, 9d, 9e, 9f, 9g, 9k, 9i), by the National Cancer Institute (Bethesda, MD), at one dose concentration (10 -5 M), for the in vitro disease-oriented antitumor screenings against a panel of about 60 human tumor cell lines that have grouped in disease sub-panel including leukaemia, non-small lung, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast tumors cell lines. 18The results obtained take into consideration the growth inhibitory power (GI 50 ).None of the tested compounds showed significant activity.

Experimental Section
General Procedures.All melting points were taken on a Büchi-Tottoli capillary apparatus and are uncorrected; IR spectra were determined in bromoform with a Jasco FT/IR 5300 spectrophotometer; 1 H and 13 C NMR spectra were measured at 200 and 50.3 MHz, respectively in DMSO-d6 or CDCl 3 solution, using a Bruker AC series 200 MHz spectrometer (TMS as internal reference).Column chromatography was performed with Merck silica gel 230-400 Mesh ASTM or with Büchi Sepacore chromatography module (prepacked cartridge system).Elemental analyses (C, H, N) were within ±0.4% of the theoretical values.Microwave experiments were carried out using a CEM Discover LabmateTM microwave apparatus.