The activities of 28 6-substituted 2 4 2 4 6 7 8 and 2 4 against were tested. Institute Boston Mass. by methods described elsewhere (5) and are outlined by name in Table ?Table1.1. Pyrimethamine and CID 755673 DDS were CID 755673 purchased from Sigma Aldrich Co. St. Louis Mo. Chlorcycloguanil was a gift from AstraZeneca Cheshire United Kingdom. TABLE 1. In vitro activities of 2 4 2 4 6 7 8 2 4 3 of the benzyl group. Elongation of the bridge by one carbon as with compound 5 led to 157-fold less potency relative to that of compound 1. Three of the tetrahydroquinazoline analogues (compounds 8 9 and 10) were reasonably active with IC50s <50 nM. However the thienopyrimidine and pteridine analogues proved to be very fragile inhibitors with IC50s >1 0 nM in CID 755673 the majority of cases and thus they were clearly of less interest than the quinazolines. Even though compounds in Table ?Table11 were not tested for his or her effects on mammalian cells as part of this study it may be noted that compounds 1 and 2 had IC50s of 85 ± 8.0 and 22 ± 4.0 nM respectively when they were tested in vitro in the Dana-Farber Malignancy Institute against CCRF-CEM human being leukemic lymphoblasts grown for 72 h in standard RPMI 1640 medium supplemented with 10% fetal bovine serum (unpublished results). Therefore while CID 755673 compound 1 was found to be more potent than compound 2 against in the present work the opposite appears to be the case with regard to human being cells presumably reflecting delicate species-specific variations in the three-dimensional structure of the active site of DHFR in versus that in humans. However bearing in mind the antimalarial assays were based on [3H]purine (from hypoxanthine) incorporation into nucleic acids whereas the assays of activities against human being cells were based on cell growth this conclusion would have to become verified by directly comparing the activities of these compounds against purified enzymes. It has been known for more than 50 years the combination of a DHPS inhibitor and a DHFR inhibitor can synergistically block de novo folate synthesis in and the additional microorganisms in which this pathway is essential for growth (2 3 Pyrimethamine-sulfadoxine and chlorproguanil-DDS are examples of drug mixtures that take advantage of this effect. Because a quantity of the quinazolines tested Rabbit Polyclonal to TIE2. in this study experienced previously been found to inhibit the DHFR gene indicated in candida (5) we postulated that these dicyclic compounds too would similarly take action synergistically with DHPS inhibitors in retarding the growth of intact organisms in culture. Accordingly the most potent compound in Table ?Table1 1 compound 1 was tested in tradition in the presence of numerous concentrations of DDS. The results are offered in Table ?Table2.2. The IC50s of compound 1 and DDS only were 9 and 184 300 nM respectively. In the presence of 9 200 6 100 CID 755673 and 4 600 nM DDS compound 1 IC50s were reduced to 0.08 0.09 and 0.12 nM respectively; sFICs were between 0.037 and 0.061 a definite indication that DDS acts in synergy with compound 1. In comparison we have included data on the activity of chlorcycloguanil a well-established DHFR inhibitor in combination with DDS (Table ?(Table2).2). DDS improved the activity of chlorcycloguanil; however the range of chlorcycloguanil-DDS sFICs was higher (0.26 to 0.38) than that for compound 1-DDS an indication the latter combination is more synergistic. All this info helps our hypothesis that compound 1 and presumably the additional active compounds in Table ?Table11 are inhibitors of DHFR. TABLE 2. In vitro activities of the mixtures of compound 1 and-DDS and chlorcyloguanil-DDS against V1S In summary this paper reports that several 6-substituted 2 4 and 2 4 6 7 8 are potent inhibitors of the growth of the highly pyrimethamine-resistant strain V1S with IC50s of <50 nM. One compound compound 1 (IC50 9 nM) was more potent against this strain than pyrimethamine by 2 orders of magnitude and was nearly as potent as WR99210. These results point to compound 1 like a encouraging lead for further structure-activity optimization with the goal of.