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Dichlorobenzoprim and methylbenzoprim were the lead compounds to emerge from investigations on a series of lipophilic 2,4-diamino-5-aryl-6-ethylpyrimidines synthesized and evaluated for their inhibition of dihydrofolate reductase (DHFR). Here the results of further mechanism-of-action studies are summarized. As expected, growth inhibitory activity of these compounds in the National Cancer Institute 60-cell-line screen correlated positively with DHFR enzyme inhibitory activity. Interestingly, two other aspects of their activity have been revealed. First, as evidenced by reversal experiments using hypoxanthine and thymidine, the two compounds, dichlorobenzoprim and methylbenzoprim, have been shown to exert an additional non-folate mechanism. Secondly, by exploitation of the COMPARE algorithm, a positive correlation has been established between the activity of certain members of this series and the existence of a mutation in the Ki-ras gene of non-small-cell lung and colon cancer cell lines. These observations have suggested that modification of the lead structures may offer opportunities to generate novel molecules without DHFR-inhibitory activity, but which may interact with new molecular targets for anti-cancer drug design.

Methods have been developed to conjugate the antitumour imidazotetrazines mitozolomide and temozolomide to DNA minor and major groove-binding peptidic motifs by solid phase peptide synthesis. Side chain deprotection and resin cleavage steps were accomplished under acidic conditions to maintain the structural integrity of the imidazotetrazine nucleus. When mitozolomide was conjugated to the DNA minor groove-binding peptide (SPKK)2-NH2 (3) a strong preference for binding with [dA-dT]2 sequences was observed by circular dichroism studies, consistent with the construct making non-covalent interactions within the minor groove. This conjugate showed a > 100-fold DNA alkylating activity compared with the free imidazotetrazine as measured by a Taq polymerase assay. Unexpectedly, alkylation patterns of all conjugates were nearly identical to those elicited by the major groove interactive agents cisplatin and the unconjugated imidazotetrazines temozolomide and mitozolomide, indicating that covalent modification was restricted to guanine sites in the major groove of DNA irrespective of the targeting property of the peptidic ligand. The electrophilic reactive chloroethyldiazonium ion intermediate formed in the breakdown of the imidazotetrazine ring of mitozolomide (methyldiazonium ion from temozolomide) must be liberated from the DNA-bound conjugate prior to the alkylation event, and must diffuse to and react with more nucleophilic sites in the major groove.

The non-covalent interactions between a series of new tetracyclic acridine derivatives (5-11) and DNA have been studied by spectrophotometric analysis, fluorescences quenching, thermal denaturation, and circular and linear dichroism. In order to compare the extent of the DNA binding by compounds 5-11 in their neutral and cationic forms, all experiments were conducted at pH 7.4 (physiological pH) and 5.0. The results indicated that compounds 5-11 are strong DNA-binding ligands with DNA affinities comparable to that of m-AMSA (1) or even higher. They showed a stronger DNA binding activity at pH 5.0 as a result of the N-protonation of the pyridoacridine aromatic chromophore. Ethidium-DNA fluorescence assays showed an A-T base pair preference of the binding distinguishing these novel compounds from simple acridines which show a slight G-C base pair preference. Circular and linear dichroism studies indicated that the drugs bind to DNA by undergoing intercalation inside the duplex macromolecule at high DNA:drug ratios and revealed alternative binding modes at low DNA:drug ratios.

The noncovalent interactions between a series of new polycyclic acridine derivatives (1-5) and salmon testes DNA have been studied using several physicochemical techniques. These include spectrophotometric analysis, fluorescence quenching, thermal denaturation, and circular and linear dichroism. In order to compare the extent of the DNA binding by compounds 1-5 in their neutral and cationic forms, all experiments have been conducted at pH 7.4 and at pH 5.0. Other polynucleotides, including [Poly(dA-dT)]2 and [Poly(dG-dC)]2, were used in order to study the DNA base-pair binding specificity of these novel annelated acridine derivatives. The results indicate that the new polycyclic acridines display the following properties: (i) they are strong DNA-binding ligands with affinities 10- to 400-fold greater than that of acridine, 3- to 100-fold greater than that of m-AMSA (6) and 1- to 23-fold greater than that of proflavine at physiological pH (7.4); (ii) they have stronger DNA-binding activity at pH 5.0 as a result of the N-protonation of the aromatic chromophore; (iii) they bind more selectively to [Poly(dA-dT)]2 polynucleotide than to [Poly(dG-dC)]2 polynucleotide; (iv) within the series compound 3 binds to DNA less than compounds 1, 2, 4 and 5 at both pH values studied; and (v) the polycyclic acridines form a molecular complex with DNA undergoing intercalation inside the duplex macromolecule, as shown by linear and circular dichroism. Nevertheless, circular dichroism studies reveal alternative binding modes at low DNA: drug ratios.

The immunomodulatory agent bropirimine crystallizes from N-methylformamide (NMF) as a triply hydrogen-bonded duplex in the manner of a Watson-Crick guanine:cytosine base pair. The stoichiometry of the solvate is bropirimine:NMF (2:1) and all NMF molecules are in the Z-rotomeric form. The intermolecular hydrogen bonding is disrupted when the solvate is dissolved in deuteriated dimethyl sulphoxide at 37 degrees C. Bropirimine and other molecules which possess the 2-aminopyrimidin-4-one fragment are potentially capable of recognizing guanine or cytosine residues in single-stranded RNA or DNA by Watson-Crick bonding or double-stranded DNA by Hoogsteen bonds. The immunomodulatory properties of these compounds might be triggered by these encounters.

The 2-amino-6-phenylpyrimidin-4(3H)-ones bropirimine (1) and the debrominated analogue (4) undergo electrophilic nitration and sulphonation in the meta-position of the 6-phenyl group in strongly acidic conditions. Subsequent electrophilic substitutions take place in the 5-position of the pyrimidine ring, if vacant. Rearrangement of the 2-nitramino-6-phenyl-pyrimidines (22) and (23) furnishes the 2-amino-6-(3-nitrophenyl)pyrimidines (9) and (18), respectively. Nucleophilic substitutions, in contrast, occur readily in the pyrimidine 2-, 4- and 6-positions. Debromination of bropirimine by hydrazine hydrate can be rationalized by invoking an initial attack by the nucleophile at the electron-deficient pyrimidine 6-position. The reactive 4-chloro group in 5-bromo-4-chloro-2-formylamino-6-phenylpyrimidine (25) can be displaced with a range of nucleophiles (azide ion, ethanol, hydrazine, and primary and secondary aliphatic amines), without loss of the bromo substituent.

Methylbenzoprim and ethylbenzoprim undergo debenzylation in hydrochloric acid. Two reaction mechanisms are proposed to explain this reaction, both of which involve a neighbouring group participation by the nitro group ortho to the tertiary amine fragment. The physical stability of dihydrofolate reductase inhibitors in the benzoprim class may influence the choice of a clinical candidate.

Molecular mechanics modelling was carried out on the antimicrobial aromatic diamidine, pentamidine, bound to the minor groove of several AT-rich DNA octamers. The pentamidine molecule was found to span four base pairs, with its highly charged amidinium groups forming hydrogen bonds to the O2 of thymine or the N3 of adenine, but not to the backbone phosphate groups. The mean binding energy of the pentamidine-DNA complexes was -52 kcal/mol, of the same order as that of berenil-DNA complexes. There was no significant energy difference between those models which contained GC base pairs and those which did not. The presence of bound pentamidine resulted in some distortion of helix geometry involving helix opening towards the minor groove in most cases and a decrease in the number of residues per turn.

The new lipophilic dihydrofolate reductase inhibitor methylbenzoprim is a flexible substituted 2,4-diamino-5-arylpyrimidine bearing an ortho-disposed aromatic nitro group and an N-methylbenzylamine residue. Molecular modelling studies indicate that modifications which incorporate these structural features into a planar benzazole or benzazole-N-oxide arrangement could mimic the overall geometry of methylbenzoprim. Target compounds were synthesized by exploiting known reaction pathways involving ortho-interactions between diazonium-, azido- or nitro-substituents and the adjacent substituted amino group: these syntheses proceeded without detrimental effect on the diaminopyrimidine residue. None of the heterocyclic analogues were more potent than the lead molecule methylbenzoprim.

The structural and electronic properties of the major groove-binding anti-tumour imidazotetrazinones, mitozolomide and temozolomide were studied using molecular orbital techniques. Structure-activity relationships of mitozolomide derivatives emphasized the importance of a hydrogen bond donor on the C8-substituent and showed that good activity would be expected for derivatives carrying a small C8-substituent with restricted negative potential. The coplanarity of the carboxamide substituent in mitozolomide is unlikely to be disrupted by hydrogen bonding in the major groove. Semi-empirical M.O. calculations gave a very high partial positive charge on C4, indicating an enhanced susceptibility to nucleophilic attack leading to ring opening at this position and the formation of a triazene alkylating agent.

Two properties of the aromatic azido group have been exploited in the design of a novel dihydrofolate reductase inhibitor--lipophilicity and biotransformability. 2,4-Diamino-5-(3-azido-4-chlorophenyl)-6-ethylpyrimidine (MZP, 4), which can be prepared in three high-yielding synthetic steps from the antimalarial drug pyrimethamine (1), has a pKa of 7.19, a log P of 2.81 and inhibitory activity against the P388, L1210, B16 and M5076 tumours in vivo. The ethanesulphonic acid salt of MZP (MZPES, 6) is a potent inhibitor of rat liver dihydrofolate reductase (Ki 1.60 nM) and L1210 cells in vitro. Injections of MZPES in water (10 mg ml-1) at pH 4.1 can be sterilised by filtration and are stable provided they are protected from light.

C8H7N7.HCl has a formula weight Mr237.7, and crystallizes in space group P21/n with a = 6.498(1), b = 10.333(8), c = 15.406(5) A, beta = 91.72(2) degrees. The final R factor was 0.049 for 1010 unique observed reflections. The azido substituent is almost linear, coplanar with the heterocycle, and parallel to the C(7)-C(6) ring bond. The heterocycle is protonated at N(1) and extensively hydrogen bonded. The 6-azido compound is at least a 100-fold better inhibitor of Escherichia coli dihydrofolate reductase than the analogue lacking the azido group, according to I50 values. Superimposing the quinazoline moiety upon the pteridine ring of the methotrexate-dihydrofolate reductase (E. coli) complex reveals two orientations of the N3 group in which it can fit the enzyme, making van der Waals contacts. Ab initio molecular orbital calculations suggest that one of these conformations, with torsion angle phi = C(5)-C(6)-N(61)-N(62) = -163.8 degrees, is low in energy.