Analysis of complexes of inhibitors with Cryptosporidium hominis DHFR leads to a new trimethoprim derivative.

Cryptosporidiosis, an opportunistic infection affecting immunocompromised patients, the elderly, and children, is still an untreatable disease since the causative agent, Cryptosporidium hominis, is essentially resistant to all clinically used antimicrobial agents. In order to accelerate the design of new potent and selective inhibitors targeting dihydrofolate reductase of C. hominis (ChDHFR), we determined the structural basis for the potency of existing DHFR inhibitors using superpositions of the structure of ChDHFR with other species and analysis of active site complexes of ChDHFR bound to ligands exhibiting a wide range of IC(50) values. This information was used to develop an accurate docking model capable of identifying potent inhibitors in silico. A series of C7-trimethoprim derivatives, designed to exploit a unique pocket in ChDHFR, was synthesized and evaluated; 7-ethyl TMP has four times higher activity than TMP against ChDHFR.

Design, synthesis, and antifolate activity of new analogues of piritrexim and other diaminopyrimidine dihydrofolate reductase inhibitors with omega-carboxyalkoxy or omega-carboxy-1-alkynyl substitution in the side chain.

As part of a search for dihydrofolate reductase (DHFR) inhibitors combining the high potency of piritrexim (PTX) with the high antiparasitic vs mammalian selectivity of trimethoprim (TMP), the heretofore undescribed 2,4-diamino-6-(2',5'-disubstituted benzyl)pyrido[2,3-d]pyrimidines 6-14 with O-(omega-carboxyalkyl) or omega-carboxy-1-alkynyl groups on the benzyl moiety were synthesized and tested against Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium DHFR vs rat DHFR. Three N-(2,4-diaminopteridin-6-yl)methyl)-2'-(omega-carboxy-1-alkynyl)dibenz[b,f]azepines (19-21) were also synthesized and tested. The pyridopyrimidine with the best combination of potency and selectivity was 2,4-diamino-5-methyl-6-[2'-(5-carboxy-1-butynyl)-5'-methoxy]benzyl]pyrimidine (13), with an IC(50) value of 0.65 nM against P. carinii DHFR, 0.57 nM against M. avium DHFR, and 55 nM against rat DHFR. The potency of 13 against P. carinii DHFR was 20-fold greater than that of PTX (IC(50) = 13 nM), and its selectivity index (SI) relative to rat DHFR was 85, whereas PTX was nonselective. The activity of 13 against P. carinii DHFR was 20 000 times greater than that of TMP, with an SI of 96, whereas that of TMP was only 14. However 13 was no more potent than PTX against M. avium DHFR, and its SI was no better than that of TMP. Molecular modeling dynamics studies using compounds 10 and 13 indicated a slight binding preference for the latter, in qualitative agreement with the IC(50) data. Among the pteridines, the most potent against P. carinii DHFR and M. avium DHFR was the 2'-(5-carboxy-1-butynyl)dibenz[b,f]azepinyl derivative 20 (IC(50) = 2.9 nM), whereas the most selective was the 2'-(5-carboxy-1-pentynyl) analogue 21, with SI values of >100 against both P. carinii and M. avium DHFR relative to rat DHFR. The final compound, 2,4-diamino-5-[3'-(4-carboxy-1-butynyl)-4'-bromo-5'-methoxybenzyl]pyrimidine (22), was both potent and selective against M. avium DHFR (IC(50) = 0.47 nM, SI = 1300) but was not potent or selective against either P. carinii or T. gondii DHFR.

Synthesis of the lipophilic antifolate piritrexim via a palladium(0)-catalyzed cross-coupling reaction.

[reaction: see text] A regiospecific and convergent route the lipophilic antifolate piritrexim (PTX) is described in which a key step is a Pd(0)-catalyzed cross-coupling reaction between 2-amino-3-cyano-4-methyl-5-bromopyridine and 2,5-dimethoxybenzylzinc chloride to form 2-amino-4-methyl-5-(2,5-dimethoxybenzyl)nicotinonitrile. To complete the synthesis, the amino group is replaced by a more reactive bromine atom via nonaqueous diazotization with tert-butyl nitrite, and the resultant bromo nitrile is cyclized with guanidine.

Synthesis of 2,4-diamino-6-[2'-O-(omega-carboxyalkyl)oxydibenz[b,f]azepin-5-yl]methylpteridines as potent and selective inhibitors of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductase.

Six previously undescribed N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepines with water-solubilizing O-carboxyalkyloxy or O-carboxybenzyloxy side chains at the 2'-position were synthesized and compared with trimethoprim (TMP) and piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic organisms known to cause significant morbidity and mortality in patients with AIDS and other disorders of the immune system. The ability of the new analogues to inhibit reduction of dihydrofolate to tetrahydrofolate by Pc, Tg, Ma, and rat DHFR was determined, and the selectivity index (SI) was calculated from the ratio IC(50)(rat DHFR)/IC(50)(Pc, Tg, or Ma DHFR). The IC(50) values of the 2'-O-carboxypropyl analogue (10), with SI values in parentheses, were 1.1 nM (1300) against Pc DHFR, 9.9 nM (120) against Tg DHFR, and 2.0 nM (600) against Ma DHFR. The corresponding values for the 2'-O-(4-carboxybenzyloxy) analogue (12) were 1.0 nM (560), 22 nM (21), and 0.75 nM (630). By comparison, the IC(50) and SI values for TMP were Pc, 13 000 nM (14); Tg, 2800 nM (65); and Ma, 300 nM (610). For the prototypical potent but nonselective inhibitors PTX and TMX, respectively, these values were Pc, 13 nM (0.26) and 47 nM (0.17); Tg, 4.3 nM (0.76) and 16 nM (0.50); Ma, 0.61 nM (5.4) and 1.5 nM (5.3). Thus 10 and 12 met the criterion for DHFR inhibitors that combine the high selectivity of TMP with the high potency of PTX and TMX.

Interferon-alpha/beta-receptor interactions: a complex story unfolding.

The Type I interferons (IFN-alpha/beta) exhibit pleiotropic biological activities. Notably, the different IFN subtypes activate the same cell surface receptor complex to mediate variable responses. Accumulating evidence suggests that distinct differences in critical amino acid residues among the different IFN-alphas and IFN-beta determine the nature of the ligand-receptor interaction and the subsequent responses. This review focuses on IFN-receptor interactions, the key residues involved in this interaction and the potential for targeted modifications of the ligand to enhance bioactivity.

Structural studies on bioactive compounds. Part 36: design, synthesis and biological evaluation of pyrimethamine-based antifolates against Pneumocystis carinii.

As part of a research effort to improve the quality of current chemotherapy of Pneumocystis carinii pneumonia, we report a structure-based design project to optimise activity, species selectivity and pharmaceutical properties of the triazenyl-pyrimethamine TAB (4) (IC(50)=0.17 microM; rat liver DHFR IC(50)/P. carinii DHFR IC(50)=114). This has led us to design, synthesise and evaluate four new series of pyrimethamine derivatives bearing triazole, triazolium, triazinium and amino moieties at the 3'-position of the p-chlorophenyl ring. Such stabilised 'triazene' derivatives address the potentially compromised pharmaceutical profile of TAB and the 3'-amine substituted agents afford conformationally flexible substitutes. The benzylamino-pyrimethamine derivative (24a) (IC(50)=0.12 microM, rat liver DHFR IC(50)/P. carinii DHFR IC(50): 5.26) was the most potent and the only P. carinii-selective antifolate of the new series.