Synthesis of 2,4-diamino-6-(thioarylmethyl)pyrido[2,3-d]pyrimidines as dihydrofolate reductase inhibitors.

Six 2,4-diaminopyrido[2,3-d]pyrimidines with a 6-methylthio bridge to an aryl group were synthesized and biologically evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). The syntheses of analogues 3-8 were achieved by nucleophilic displacement of 2,4-diamino-6-bromomethylpyrido[2,3-d]pyrimidine 14 with various arylthiols. The alpha-naphthyl analogue 4 showed the highest selectivity ratios of 3.6 and 8.7 against pcDHFR and tgDHFR, respectively, versus rat liver (rl) DHFR. The beta-naphthyl analogue 5 exhibited the highest potency within the series with an IC(50) value against pcDHFR and tgDHFR of 0.17 and 0.09 microM, respectively. Analogue 4 was evaluated for in vitro antimycobacterium activity and was shown to inhibit the growth of Mycobacterium tuberculosis H(37)Rv cells by 58% at a concentration of 6.25 microg/mL.

Isolation of rat dihydrofolate reductase gene and characterization of recombinant enzyme.

While assays of many antifolate inhibitors for dihydrofolate reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we report the isolation of the rat DHFR gene through screening of a rat liver cDNA library. The rat liver DHFR gene has an open reading frame of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat enzyme with those from other species indicate a high level of conservation at the primary sequence level and more so for the amino acid residues comprising the active site of the enzyme. Expression of the rat DHFR gene in bacteria produced a recombinant protein with high enzymatic activity. The recombinant protein also paralleled the human enzyme with respect to the inhibition by most of the antifolates tested with PT652 and PT653 showing a reversal in their patterns. Our results indicated that rat DHFR can be used as a model to study antifolate compounds as potential drug candidates. However, variations between rat and human DHFR enzymes, coupled with unique features in the inhibitors, could lead to the observed differences in enzyme sensitivity and selectivity.

Pneumocystis carinii dihydrofolate reductase used to screen potential antipneumocystis drugs.

Pneumocystis carinii was obtained in high yield from the lungs of immunosuppressed rats by rupturing mammalian host cells, washing away the soluble mammalian dihydrofolate reductase, and harvesting intact organisms in association with the mammalian plasma membranes. P. carinii dihydrofolate reductase, measured in the 100,000 x g supernatant from sonicated organisms, was obtained in yields ranging up to 62 IU per rat. The enzyme prepared in the presence of protease inhibitors was stable when frozen in liquid nitrogen. P. carinii dihydrofolate reductase differed from the mammalian enzyme in that the former was slightly inhibited by 150 mM KCl, whereas the latter was stimulated over twofold by 150 mM KCl. The standard assay for P. carinii dihydrofolate reductase contained 0.12 mM NADPH and 92 microM dihydrofolic acid. Under these conditions, the 50% inhibitory concentrations of the known inhibitors trimethoprim, trimetrexate, and pyrimethamine were 12 microM, 42 nM, and 3.8 microM, respectively. These standard compounds were also tested against dihydrofolate reductase from rat liver to allow an assessment of the selectivity of the drugs. Although it was the least potent, trimethoprim was the most selective. Pyrimethamine was more potent but was nonselective. Trimetrexate was extremely potent but was selective for mammalian dihydrofolate reductase. A series of experimental compounds was obtained from the National Cancer Institute and other sources through the Developmental Therapeutics Branch of the Division of AIDS at the National Institute of Allergy and Infectious Diseases. Among the first 87 compounds tested, 11 had 50% inhibitory concentrations below that of trimetrexate and 3 were more selective than trimethoprim. The most promising compounds in this original group were chemically related to methotrexate.

In vitro activities of acridone alkaloids against Pneumocystis carinii.

Acridone alkaloids isolated from plants of the family Rutaceae have antiplasmodial activity in rodent models of malaria. Because a variety of antimalarial agents have also been shown to have activity against Pneumocystis carinii, we tested six of these alkaloids in an established culture model for P. carinii. Atalaphillinine and glycobismine A inhibited growth of cultured P. carinii at concentrations of 2.7 and 1.7 microM, respectively. This potency of effect is similar to that of chloroquine (3 microM) but somewhat less than that of primaquine (0.4 microM), which was previously evaluated in the same system.

Acquired resistance to parathyroid hormone.

Studies are presented in a patient with pseudohypoparathyroidism who showed a partial response to parathyroid extract. Resistance to the extract was observed after its short-term administration for the gourth time. Serum from the patient contained antibodies of the gamma G globulin class which bound 125I-labelled bovine parathyroid hormone. Prior incubation of parathyroid hormone with the serum prevented the activation in vitro of adenylate cyclase from pork renal cortex. The antibodies were directed primarily toward the C-terminal portion of the molecule. Thus, clinical resistance to parathyroid hormone is attributed to specific antibodies.