Antitumor imidazotetrazines. 38. New 8-substituted derivatives of the imidazo[5,1-d ]-1,2,3,5-tetrazines temozolomide and mitozolomide

The imidazo[5,1-d ]-1,2,3,5-tetrazine ring-system is hypersensitive to attack by nucleophiles. Therefore, acidic conditions were applied successfully to synthesise new 8-substituted derivatives of the antitumor agents temozolomide (1a) and mitozolomide (1b). The 8-cyanoimidazotetrazines (7a, b) were prepared by dehydration of temozolomide and mitozolomide with thionyl chloride and 7b was converted to the corresponding 8-thioamide (9) with thioacetamide in DMF-HCl. Generation of tertiary carbocations from substituted alkenes by concentrated H 2 SO 4 in the presence of 8-cyano-imidazotetrazines afforded secondary amide congeners (13, 14) of imidazotetrazine-8-carboxamides (Ritter reaction). Peptidic-type coupling of imidazotetrazine-8-carboxylic acids (15a, b) with the protected hydrazine reagent, t -butylcarbazate (BOC-NHNH 2 ), followed by acidic deprotection, gave salts of the carbohydrazides (17, 18) which could be derivatised with aromatic aldehydes. Attempts to synthesise the unknown 8-amino-imidazotetrazine (24) from appropriate 8-substituted substrates by modifications of Hofmann, Curtius and Lossen rearrangements under acidic conditions were all unsuccessful.


Introduction
The antitumor imidazotetrazine temozolomide (1a) has been launched on the market under the brand name Temodal in Europe and Temodar in the USA for the treatment of certain brain tumors; desirable features of the drug include its oral bioavailability and a favourable side-effect profile. 1 The drug undergoes ring-opening at physiological pH to generate the corresponding open chain monomethyltriazene (MTIC; 2) which then undergoes proteolytic decomposition to 5-aminoimidazole-4-carboxamide (AIC; 3) and the electrophilic methanediazonium reactive species (4).The cytotoxic event is the methylation of guanine residues of DNA (5) at the O( 6) position (Scheme 1) leading to the incorporation of the mispairing base O 6 -methylguanine (6). 2 The 3-(2-chloroethyl)-derivative (mitozolomide; 1b), a DNA cross-linker and the most potent of the imidazotetrazines in preclinical evaluations, induced life-threatening bone-marrow suppression in patients and was withdrawn from clinical trial. 3The 3-benzyl-(1c) and 3-ethyl-analogues (1d) were devoid of antitumour activity in experimental systems. 4

Scheme 1
One approach to extend the clinical spectrum of action of temozolomide has concentrated on modifying the DNA-binding signature of the imidazotetrazines by adaptation of the 8-carboxamide group.Many factors might play a role on the activity of C-8 modified imidazotetrazines.The C-8 moiety could be involvedin: (i) modifying the transport properties of the bicyclic systems; (ii) perturbing the initial non-covalent encounter between the intact (prodrug) imidazotetrazine and guanine sequences in DNA; (iii) exerting kinetic control of the activating ring-opening reaction; and (iv) stabilizing the ring-opened triazene intermediates.Accordingly, structureactivity relationships for C(8) derivatives of the 3-(2-chloroethyl) series are complex to interpret.The 8-N-methyl-carboxamide was equiactive with the parent carboxamide (1b), but activity decreased as the bulk of N-alkyl substitution increased.Replacement of the 8-carboxamide residue with sulphonamide, N-alkylsulphonamide, sulphoxide and sulphone residues led to highly active compounds: conversely, activity was lost in the case of the 8phenyl, 8-cyano and 8-nitro derivatives and the 8-carboxylic acid, 8-esters and 8-thioesters wereonly active at toxic doses. 5Temozolomide and mitozolomide have been conjugated through the carboxylic function to heterocyclic Hbonding motifs and spermidine 6 and linked to peptides and lexitropsins by solid state automated chemistry. 7Peptidic motifs which recognise AT sequences in the minor groove of DNA (eg.SPPK-SPPK-NH 2 , the tandem repeat of the histone DNA-binding motif, or PRGRP, a fragment of the nonhistone chromosomal protein HMG-I/Y) or the major groove (eg. the guaninebinding tryptophan-containing tetrapeptide WDGW-NH 2 , or the helix fragment of the trp repressor IATITGSN-NH 2 ) have been linked to temozolomide and mitozolomide. 8Unexpectedly, DNA alkylation patterns of all conjugates were nearly identical to those elicited by the unconjugated imidazotetrazines, indicating that covalent modification of DNA was restricted to guanine sites in the major groove, irrespective of the putative targeting property of the peptidic ligand.In the present work we have returned to business left unfinished in our original work on imidazotetrazines in the 1980s.We now report chemistries exploiting the acid stability of the tetrazine ring which have allowed us to access further novel C(8) modifications of imidazotetrazines (1).

Results and Discussion
8-Cyano-imidazotetrazines. The cyano analogues (7a) and (7b) of temozolomide and mitozolomide have been prepared previously from the unstable 4-cyano-5-diazoimidazole and methyl 9 and 2-chloroethyl isocyanates, 5 respectively.Attempted dehydration of the carboxamide residue of 1a with P 2 O 5 alone, or in methanesulphonic acid at temperatures > 85 o C, led to decomposition of the tetrazine ring; similarly decomposition ensued under Swern oxidation conditions and following interaction of 1a,b with benzenesulphonyl chloride in pyridine.
More successful was the formation of the nitrile (7a) (59%) from 1a in POCl 3 at 90 o C. Optimum conversion of carboxamides (1a-d) to nitriles (7a-d) was achieved in thionyl chloride-DMF at 60 o C, the cyano analogues of temozolomide (7a) and mitozolomide (7b) being obtained in 85 and 49% yields, respectively.Similarly, the bis(imidazotetrazine)-dicarboxamide (8a) 10 afforded the corresponding dinitrile (8b) (55%) in thionyl chloride-DMF.Interestingly, 7a was also formed (35%) from temozolomide and formic acidacetonitrile in the presence of paraformaldehyde.This transamidation reaction is mechanistically intriguing: it has been reported as a general method for dehydrating alkyl-and arylamides 11 requiring formal transfer of water from the amide (ie 1a) to solvent (acetonitrile) with the aldehyde serving as a relay for the water transfer.8-Thiotemozolomide (9).Temozolomide was recovered following attempted direct thionation with P 2 S 5 in THF, toluene or xylene, or from Lawesson's reagent in HMPA at temperature < 80 o C. With the more soluble Belleau's reagent in DCM at room temperature a small yield (10%) of the yellow 8thiotemozolomide (9) was isolated.
Conversion of nitriles to thioamides with hydrogen sulphide is a standard process but typically requires the presence of bases, conditions inimical to tetrazine ring integrity.Cyanotemozolomide (7a) was recovered from attempted thionation using hydrogen sulphide in DMF at 45-65 o C; above 65 o C decomposition of substrate was observed.More success attended thionations conducted in strongly acidic conditions: conversion of 7a to thiotemozolomide (9) employing thiolacetic acid in 10M-hydrochloric acid (27% yield) was improved (to 49%) using a thioacetamide-DMF reaction medium saturated with HCl gas.
The 1 H NMR spectrum of 8-thiotemozolomide was comparable to that of temozolomide in the chemical shifts of the imidazole and methyl protons: however two exchangeable protons at d9.47 and 9.94 suggest that 9 exists in the imino-thiol tautomeric form (cf. the two NH absorptions in temozolomide at d7.68 and 7.81 which are characteristic of primary carboxamides).Secondary amide derivatives of imidazotetrazines ( 13) and (14).Nsubstituted derivatives of the 8-carboxamide group of imidazotetrazines have been prepared from the corresponding 8-acid chlorides and amines. 12However this route suffers the disadvantage that stoichiometric precision in the incorporation of the amine component is required to avoid base-induced degradation of the bicyclic nucleus.In the present work we have adapted the Ritter reaction (the electrophilic addition of olefins to nitriles under acidic conditions) to yield secondary amides after hydrolysis.A mixture of concentrated H 2 SO 4 in acetic acid was used both as a reactant, to generate carbocations, and as a solvent in which the imidazotetrazines were stable and soluble.

Scheme 3
Cleavage of the protected hydrazides (16a, b) with HCl or TFA led to the formation of the hydrochloride or trifluoroacetate salts of the hydrazides of temozolomide (17) and mitozolomide (18), respectively (Scheme 3).Surprisingly, catalytic hydrogenation of the N-nitroamide analogue of mitozolomide (1b; X = NHNO 2 ) (see Scheme 4), formed by nitration of mitozolomide in a nitric-sulphuric acid mixture, 12 did not produce the free base of hydrazide (18): instead, hydrogenolysis of the N-N double bond furnished only the carboxamide (1b: X = NH 2 ) (Scheme 4).Hydrazide (18) reacted with substituted benzaldehydes to yield the hydrazones (19a-c) but efforts to generate and trapdipolarreactive intermediates (20) with lead (IV) acetate (LTA) in acetic acid led only to recovery of unchanged carboxylic acid (15b).

Scheme 4
Unsuccessful Routes to 8-Aminoimidazotetrazines.Furtherefforts to synthesize the potentially useful isocyanate (23), which might have provided a source of the elusive 8-amino-imidazotetrazine (24), were unsuccessful.Attempted Hofmann rearrangement of mitozolomide (1b) by the hypervalent iodine oxidants iodosobenzene in formic acid or iodobenzene bis(trifluoroacetate) (PIFA) in aqueous acetonitrile, or LTA in acetic acid, have all led to the recovery of unchanged amide.A modified Curtius reaction involving interaction of the carboxylic acid (15b) with diphenylphosphoryl azide (DPPA) 13 in t-butanol failed to lead to the corresponding t-butylurethane of isocyanate (23): only degradation of the bicyclic nucleus was observed.
We have previously shown 12 that thermal degradation of the tetrazine ring preceeds Curtius rearrangement of the acylazide derivative (21) even in the low boiling anhydrous solvents acetone, chloroform, benzene or toluene, and no 23 was isolated.Attempted Lossen rearrangement of the hydroxamic acid (22) 12 in refluxing thionyl chloride was also unsuccessful.
As a last resort the 8-nitroimidazotetrazine (25) 5 was hydrogenated in 1,4dioxane over 5% Pd-charcoal catalyst at atmospheric pressure to yield an unstable brown product.However, its 1 H NMR spectrum was not consistent with that expected for the target amine (24): although the methylene triplets were shifted upfield compared to other imidazotetrazines, there was no upfield shift of the imidazole proton at C(6) in comparison to imidazotetrazines with an EWG at C(8); also, the presence of a triplet at δ 9.5 indicated that a ringopened product was formed.Clearly, unlike those stable imidazotetrazines with EWGs at C(8) (eg.temozolomide and mitozolomide), the presence of a +M electron-donating group in 8-aminoimidazotetrazine (24) renders the bicyclic system too unstable to be isolated.8-Cyano-3-methylimidazo[5,1-d]-1,2,3,5-tetrazine-4(3H)-one (7a).Thionyl chloride (0.2 mL) was added to a stirred suspension of temozolomide (1a) (0.5 g) in DMF (5 mL) and the mixture was maintained at 60 o C for 5 h.The mixture was then concentrated in vacuo and quenched with water.The resulting precipitate was collected and recrystallised from aqueous acetone to yield the cyano-imidazotetrazine (85%) as a white solid, mp 183-185 o C (decomp) (lit. 9185-187 o C) with IR, 1 H NMR and 13 C NMR spectra identical to material prepared (52%) from 4-cyano-5-diazoimidazole and methyl isocyanate in dry DMSO. 9Compound 7a was also formed (59%) from temozolomide and phosphorous oxychloride at 85-90 o C. To a stirred solution of temozolomide (0.2 g) in acetonitrile (2.5 mL) were added formic acid (0.4 mL) and paraformaldehyde (0.155 g).The mixture was heated at 65 o C for 12 h and the resulting yellow solution was concentrated under reduced pressure to give an oil which was triturated with ethyl acetate.

General method for the Ritter Synthesis of N-substituted imidazo[ 5
To a solution of the appropriate 8-cyanoimidazotetrazine (7) (0.3 g) in acetic acid (5 mL) containing concentrated H 2 SO 4 (0.1 mL) was added the alkene (10) (1.2 mol equiv) and the mixture was stirred at 25 o C for 12 h.The mixture was diluted with water, stirred at 25 o C for 2 h, and products were collected.Yields and physical properties of N-substituted imidazotetrazinecarboxamides are listed in Table.