Cheryl A. Johnson

Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution.

The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.

In Vitro Activities of Several Diaminomethylpyridopyrimidines against Mycobacterium avium Complex

ABSTRACT Three recently synthesized dihydrofolate reductase (DHFR) inhibitors designated SoRI 8890, 8895, and 8897 were evaluated for their in vitro activities against 25 isolates of Mycobacterium avium complex. The MICs at which 50 and 90% of isolates were inhibited were 1 and 2, 4 and 8, and 4 and 8 μg/ml for SoRI 8890, 8895, and 8897, respectively. Although the addition of dapsone at 0.5 μg/ml did not significantly enhance the in vitro activities of these compounds, their activities alone were comparable to, if not better than, results seen with other DHFR inhibitors, such as pyrimethamine or WR99210.

Lipophilic Antifolates as Candidates against Opportunistic Infections

The problems in treating immunocompromised patients infected by Pneumocystis carinii and Toxoplasma gondii have prompted intense efforts to develop improved therapy against these pathogens.1,2 Lipophilic antifolates trimethoprim and pyrimethamine (structures shown under Table 1) are used in combination with sulfa drugs in current treatment regimens which inhibit the ability of the microorganism to synthesize reduced folates. In such treatment, the antifolate inhibits dihydrofolate reductase (DHFR), and the sulfa drug inhibits utilization by the microorganism of 4-aminobenzoic acid in its vital biosynthesis of dihydropteroic acid.2–4 Adverse reactions that frequently occur with these regimens often necessitate discontinuation of the therapy. New agents or combinations of agents of greater therapeutic effectiveness in terms of lower toxicity and shorter treatment periods are needed.

Synthesis and antifolate evaluation of the aminopterin analogue with a bicyclo[2.2.2]octane ring in place of the benzene ring

N-[4-[[2,4-diamino-6-pteridinyl)methyl]amino]bicyclo[2.2.2]octane-1-carbonyl]-L-glutamic acid (1) was synthesized and tested for antifolate activity. N-(4-Aminobicyclo[2.2.2]octane-1-carbonyl-L-glutamic acid dimethyl ester (6), the side chain precursor to subject compound 1, was synthesized readily via reported bicyclo[2.2.2]octane-1,4-dicarboxylic acid monoethyl ester (2). The side chain precursor 6 was alkylated by 6-(bromomethyl)-2,4-pteridinediamine (7). Subsequent ester hydrolysis then afforded 1. Antifolate and antitumor evaluation of 1 verses L1210 dihydrofolate reductase (DHFR) and three tumor cell lines (L1210, S180, and HL60) showed it to be ineffective. Although compound 1 was very similar to aminopterin structurally, the bicyclo[2.2.2]octane ring system in place of the phenyl ring in the p-aminobenzoate moiety effectively negates the stoichiometric binding displayed by many classical DHFR inhibitors bearing appropriate aromatic ring systems in the side chain.

Analogues of Classical Antifolates Bearing Naphthoyl in Place of Benzoyl

Molecular graphics approximating the binding of methotrexate (MTX) to human dihydrofolate reductase (DHFR) reveal a large open space in the protein structure which could accommodate potential inhibitors bearing in their side chains groups of greater bulk than the benzoyl of the normal folate or a classical antifolate. Candidate structures having larger groups may be readily designed to retain conformation needed for binding by key groups, such as the salt-bridge forming α-carboxyl group of the glutamic acid part. These observations prompted us to synthesize 4-amino-l-naphthoyl analogues of known antifolates in order to test the indications that inclusion of such larger or different groups would not lower the tight binding of MTX-related structural types to DHFR.