Synthesis and biological evaluation of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase and as antiopportunistic infection and antitumor agents

A series of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) dihydrofolate reductase (DHFR) and as inhibitors of the growth of tumor cell lines in culture. Compounds 4-15 were designed as part of a continuing effort to examine the effects of substitutions at the 5-position, in the two-atom bridge, and in the side chain phenyl ring on structure-activity/selectivity relationships of 2,4-diaminopyrido[2,3-d]pyrimidines against a variety of DHFRs. Reductive amination of the common intermediate 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile 16 with the appropriate anilines afforded the target compounds 4-12. Nucleophilic substitution or reductive methylation afforded the N10-methyl target compounds 13-15. As predicted, compounds 4-15 were, in general, less potent against all three DHFRs compared to the corresponding 2,4-diamino-5-methyl analogues previously reported; however, the greater decrease in potency against rlDHFR compared to pcDHFR and tgDHFR resulted in appreciable selectivity toward pathogenic DHFRs from different pathogens. The 2',5'-dichloro analogue 8 showed selectivity ratios (IC(50) against rlDHFR/IC(50) against pcDHFR or tgDHFR) of 15.7 and 23 for pcDHFR and tgDHFR, respectively. Thus, the selectivity of 8 for pcDHFR is higher than the first line clinical agent trimethoprim (TMP). In a P. carinii cell culture study, analogue 8 exhibited 88% cell growth inhibition at a concentration of 10 muM and afforded marginal effects in an in vivo study in the T. gondii mouse model. Selected compounds were evaluated in the National Cancer Institute (NCI) in vitro preclinical antitumor screening program and inhibited the growth of tumor cells in culture at micromolar to submicromolar concentrations and were selected for evaluation in a NCI in vivo hollow fiber assay.     

Design, synthesis, and biological evaluation of 2,4-diamino-5-methyl-6-substituted-pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors.

2,4-diamino-5-methyl-6-(substituted-phenyl)thiopyrrolo[2,3-d]pyrimidines 4-11 were synthesized as dihydrofolate reductase (DHFR) inhibitors against opportunistic pathogens that afflict patients with AIDS. Synthesis was achieved from 2,4-diamino-5-methypyrrolo[2,3-d]pyrimidine and substituted phenylthiols under modified conditions reported by Gangjee et al. Some of these compounds were potent and selective against DHFR from both Toxoplasma gondii and Mycobacterium avium compared to mammalian DHFR. Compound 11 with a 1-naphthyl substituent is 16-fold more potent and equally selective against Toxoplasma gondii DHFR as the clinically used trimethoprim.     

CoMFA and CoMSIA analyses of Pneumocystis carinii dihydrofolate reductase, Toxoplasma gondii dihydrofolate reductase, and rat liver dihydrofolate reductase

In a continuing effort to develop potent and selective dihydrofolate reductase (DHFR) inhibitors against opportunistic pathogens, we developed three-dimensional quantitative structure-activity relationship (3D QSAR) models for the inhibitory activity against Pneumocystis carinii (pc) DHFR, Toxoplasma gondii (tg) DHFR, and rat liver DHFR, using a data set of 179 structurally diverse compounds. To ensure a balanced distribution of more potent and less potent drugs in the training set, three different 90-compound training sets taken from the main data set were used, one for each enzyme, while the remaining 89 compounds in the main data set in each case were used as the test set. Three methods, namely, conventional CoMFA, all orientation search (AOS) CoMFA, and CoMSIA were applied to the training sets. While the AOS CoMFA models gave the best internal predictions (cross-validated r(2) values from the training sets), which are satisfactory, CoMSIA models gave the best external predictions (predictive r(2) values from the test sets). Both AOS CoMFA and CoMSIA analyses were used to construct stdev*coefficient contour maps which can be used to design new compounds in an interactive fashion.     

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.     

Synthesis and biological evaluation of 2,4,6-functionalized derivatives of pyrido[2,3-d]pyrimidines as cytotoxic agents and apoptosis inducers

In the search for new derivatives with anticancer activity that are able to induce a selective pro-apoptotic mechanism in cancer cells, we have designed, synthesized, and evaluated a series of new 2-(alkylsulfanyl)-N-alkylpyrido[2,3-d]pyrimidine-4-amine derivatives as cytotoxic and apoptosis inducers. The potential antitumor activity of the compounds was evaluated in vitro by examining their cytotoxic effects against human breast, colon, and bladder cancer-cell lines. The IC(50)values of the compounds that showed cytotoxic activity were calculated. The cytotoxic compounds were then tested for their ability to induce caspase-3 activation and nuclear-chromatin degradation. Some compounds, such as 6c, 6d, 6e, 6j, 6o, and 6p, show significant in-vitro cytotoxicity in at least two of the three tested cell lines, induced apoptosis, and also produced a rapid dose-dependent increase in the caspase-3 level in some of the cell lines tested. In order to test the selectivity of the compounds, two non-tumoral human cell lines were used. Several compounds of the did not show cytotoxicity in these cell lines.     

Synthesis of N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid as dual inhibitors of dihydrofolate reductase and thymidylate synthase and as potential antitumor agents

Two novel classical antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR-TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the growth of several human tumor cell lines in culture with GI50 < 10(-8) M. This study shows that the pyrrolo[2,3-d]pyrimidine scaffold is conducive to dual DHFR-TS and tumor inhibitory activity, and the potency is determined by the 4-position substituent.     

Novel antitumor and radioprotective sulfonamides containing pyrrolo [2,3-d]pyrimidines

Several sulfonamides having pyrrole (5a-c, 8, 11b-19, 23, 24), pyrrolo[2,3-d] pyrimidine (6, 7, 10, 20, 21, 25) and pyrrolo[2,3-b]pyridine (22) were synthesized and evaluated for their antitumor and radioprotective activities. The structure of the synthesized compounds was elucidated by elemental analyses and spectral data. Compounds 5a, 16, 17, 19, and 23 displayed more potent antitumor activities than the reference drug, doxorubicin. On the other hand compounds 19, 23 and 25 exhibited radioprotective activities.     

Synthesis of certain new thiieno [2,3-d] pyrimidines as potential antitumor and radioprotective agents

During research on anticancer and radioprotective heterocyclic compounds containing thiophene ring 5-10, 15, 19, thieno[2,3-d]pyrimidines 11-14 and biscompound having thieno[2,3-d]pyrimidine 18 were synthesized. The synthesized compounds were characterized by elemental ananlysis, IR, 1H-NMR and mass spectral data. Some of the obtained compounds showed interesting antitumor and radioprotective activities.     

Synthesis and biological activity of pyridol[2,3-d]pyrimidines

Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 25, No. 12, pp. 31–33, December, 1991.     

Synthesis and antitumor activity of new pyrido[2,3-d]pyrimidine derivatives

New series of pyrido[2,3-d]pyrimidines such as; 5-(4-aryl-5-sulfanyl-4H-[1,2,4]triazol-3-yl) 1H,3H,8H-pyrido[2,3-d]pyrimidine-2,4,7-triones 6, 7; S-[3-(2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidin-5-yl)-4-(4-substituted phenyl)-4H-[1,2,4]triazol-5-yl]-2-(4-phenylpiperazin-1-yl)ethanethioates 10, 11; 2,4,7-trioxo-N′-[(4-substituted piperazin-1-yl)acetyl]-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazides 13–16 and N′-arylidene-2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazides 17–19 was synthesized through the reaction of the key intermediate 2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazide 3 with different reagents. The structures of the newly synthesized compounds were elucidated through microanalysis, IR, ¹H NMR, ¹³C NMR, and mass spectroscopy. These compounds have been subjected to in vitro antitumor evaluation by bleomycin-dependant DNA damage assay. The most active antitumor compound 6 was selected for further in vivo evaluation of antineoplastic activity against Ehrlich ascites carcinoma in mice. It was observed that our target compound has a potent antitumor activity.     

Design and synthesis of classical and nonclassical 6-arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidines as antifolates

The classical antifolate N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-l-glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (20) served as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid derivative 18 with diethyl l-glutamate followed by saponification. The classical compound 2 was an excellent inhibitor of human DHFR (IC50 = 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium avium) that cause opportunistic infections in patients with compromised immune systems.     

Synthesis and antiviral activity of certain 4-substituted and 2,4-disubstituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidines

Treatment of the sodium salt of 4-chloro-2-(methylthio)pyrrolo[2,3-d]pyrimidine (2) with (2-acetoxyethoxy)methyl bromide (3) has provided 4-chloro-2-(methylthio)-7[(2-acetoxyethoxy)methyl]pyrrolo[2,3- d]pyrimidine (4). Ammonolysis of 4 at room temperature gave 4-chloro-2-(methylthio)-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3- d]pyrimidine (5). However, ammonolysis of 5 at 130 degrees C furnished 4-amino-2-(methylthio)-7-[(2-hydroxyethoxy)methyl]-pyrrolo[2,3- d]pyrimidine (6), which on desulfurization with Raney Ni yielded 4-amino-7-[(2-hydroxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidine (7) (acyclic analogue of tubercidin). The oxidation of 6 with m-chloroperbenzoic acid provided the sulfone derivative 8. A nucleophilic displacement of the 2-methylsulfonyl group from 8 with methoxide anion provided 4-amino-2-methoxy-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (9). Demethylation of 9 with iodotrimethylsilane gave 4-amino-2-hydroxy-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (10). Treatment of 2,4-dichloropyrrolo[2,3-d]pyrimidine (11) with 3 gave the protected acyclic compound 12, which on deacetylation and ammonolysis under controlled reaction conditions gave 2,4-dichloro-7-[(2-hydroxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidine (13) and 4-amino-2-chloro-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3- d]pyrimidine (14), respectively. The condensation of 2-acetamido-4-chloropyrrolo[2,3-d]pyrimidine (15) with 3 gave the protected acyclic compound 16, which on concomitant deacetylation and ammonolysis with methanolic ammonia at an elevated temperature yielded 2,4-diamino-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (17) in moderate yield. In tests involving human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1), only slight activity and cytotoxicity were observed. The most active compounds (12 and 13) were slightly more active against HCMV than acyclovir, but both compounds were inactive against HSV-1. The activity against HCMV, however, was not well separated from cytotoxicity leading to the conclusion that these compounds did not merit further study.     

Synthesis and anticancer activities of 4-oxobenzopyrano[2,3-d]pyrimidines

Several 2-aryl-4-oxoxbenzopyrano[2,3-d]pyrimidines have previously been shown to exhibit in vivo antitumor activity in mice with P388 lymphocytic leukemia. In the present study, a series of novel substituted benzopyrano[2,3-d]pyrimidines have been prepared and tested for cytotoxic activity against a panel of cancer cell lines including the P388 lymphocytic leukemia cell line. The unsubstituted parent compound, some methoxylated derivatives and a cyclohexyl derivative all exhibited potent cytotoxic activity (IC50 values 0.3-0.64 microM). A number of derivatives, including the unsubstituted parent pyrimidine, were shown to cause a significant perturbation in cell cycle kinetics with an observed 2- to 3-fold increase in cells in the G2/M phase of the cell cycle. Furthermore, a polymethoxylated derivative, 2-(3,4,5-trimethoxyphenyl)-9-methoxy-4-oxo-2,3-dihydrobenzopyrano[ 2,3-d]pyrimidine 13, was shown to be selectively active against a number of human ovarian cell lines.     

N9-substituted 2,4-diaminoquinazolines: synthesis and biological evaluation of lipophilic inhibitors of pneumocystis carinii and toxoplasma gondii dihydrofolate reductase

N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available 2,4-diamino-6-nitroquinazoline 14 with Raney nickel afforded 2,4,6-triaminoquinazoline 15. Reductive amination of 15 with the appropriate benzaldehydes or naphthaldehydes, followed by N9-alkylation, afforded the target compounds 5- 13. In the 2,5-dimethoxybenzylamino substituted quinazoline analogues, replacement of the N9-CH 3 group of 4 with the N9-C2H5 group of 8 resulted in a 9- and 8-fold increase in potency against pcDHFR and tgDHFR, respectively. The N9-C2H5 substituted compound 8 was highly potent, with IC50 values of 9.9 and 3.7 nM against pcDHFR and tgDHFR, respectively. N9-propyl and N9-cyclopropyl methyl substitutions did not afford further increases in potency. This study indicates that the N9-ethyl substitution is optimum for inhibitory activity against pcDHFR and tgDHFR for the 2,4-diaminoquinazolines. Selectivity was unaffected by N9 substitution.     

Synthesis and biological evaluation of some pyrrolo[2,3-d]pyrimidines

Pyrrolo[2,3-d]pyrimidine and tetrazolopyrimidine derivatives 2a, b-5a, b were prepared. Also, acyclic and cyclic C-nucleosides 7a, b-12a, b were prepared by treating compound 6 with some aldoses. All prepared products were tested for antiviral activity against hepatitis-A virus (HAV, MBB-cell culture adapted strain) and herpes simplex virus type-1 (HSV-1). Plaque reduction infectivity assay was used to determine virus count reduction as a result of treatment with tested compounds. Compound 2a showed the highest effect on HAV, while compound 11b showed the highest effect on the HSV-1 virus.     

Novel synthesis of pyrrolo [2,3-d] pyrimidines bearing sulfonamide moieties as potential antitumor and radioprotective agents

A novel series of pyrrolo[2,3-d] pyrimidines bearing sulfonamide moieties have been synthesized and tested for their antitumor activity. Among them, compounds 4, 8b, Bd, 8g and 14 showed promising antitumor activity. Moreover, compound 8d exhibited radioprotective activity. The structures of the newly synthesized compounds were established by their elemental analyses and spectral data.     

Folate antagonists. 21. Synthesis and antimalarial properties of 2,4-diamino-6-(benzylamino)pyrido[3,2-d]pyrimidines

The synthesis and antimalarial activity of a series of 2,4,6-triaminopyrido[3,2-d]pyrimidines (4) is described. Several 6-substituted benzylmethylamino analogues were more active against trophozoite induced Plasmodium berghei in mice than the corresponding quinazoline analogues. These agents, however, are cross-resistant to other antifolate compounds and are thus of limited potential as human agents.     

Dual inhibitors of thymidylate synthase and dihydrofolate reductase as antitumor agents: design, synthesis, and biological evaluation of classical and nonclassical pyrrolo[2,3-d]pyrimidine antifolates(1)

We designed and synthesized a classical analogue N-[4-[(2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogues 5-17 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in their synthesis was 2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine, 22, to which various aryl thiols were conveniently attached at the 5-position via an oxidative addition reaction using iodine. For the classical analogue 4, the ester obtained from the reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC(50) = 90 nM) and human DHFR (IC(50) = 420 nM). Compound 4 was not a substrate for human FPGS. Metabolite protection studies established TS as its principal target. Most of the nonclassical analogues were only inhibitors of human TS with IC(50) values of 0.23-26 microM.     

Inhibition of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductases by 2,4-diamino-5-[2-methoxy-5-(omega-carboxyalkyloxy)benzyl]pyrimidines: marked improvement in potency relative to trimethoprim and species selectivity relative to piritrexim.

A series of previously undescribed 2,4-diamino-5-[2-methoxy-5-alkoxybenzyl]pyrimidines (3a-e) and 2,4-diamino-5-[2-methoxy-5-(omega-carboxyalkyloxy)benzyl]pyrimidines (3f-k) with up to eight CH2 groups in the alkoxy or omega-carboxyalkyloxy side chain were synthesized and tested for the ability to inhibit partially purified dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat liver in comparison with two standard inhibitors, trimethoprim (1) and piritrexim (2). The latter drug is known to be extremely potent but shows a marked preference for binding to mammalian DHFR, whereas the former is very selective for the parasite enzymes but is a much weaker inhibitor. The underlying strategy for the synthesis of compounds 3a-k was that a hybrid structure embodying some features of both 1 and 2 might possess a more favorable combination of potency and selectivity than either parent drug. The choice of analogues 3f-k was based on the idea that the acidic omega-carboxyl group might interact preferentially with a basic center in the active site of DHFR from any of the parasite species relative to the active site of mammalian DHFR. In addition, the omega-carboxyl group was expected to improve water solubility relative to 1 or 2. In standardized spectrophotometric assays with dihydrofolate as the substrate and NADPH as the cofactor, 2,4-diamino-5-[(2-methoxy-4-carboxybutyloxy)benzyl]pyrimidine (3g) inhibited Pc DHFR with an IC(50) of 0.049 microM and rat DHFR with IC(50) of 3.9 microM. Its potency against Pc DHFR was 140-fold greater than that of 1 and close to that of 2, and its selectivity index, defined as the ratio IC(50)(rat liver)/IC(50)(P. carinii), was 8-fold higher than that of 1 and >10(4)-fold higher than that of 2. Although it was less potent and less selective against Tg than Pc DHFR, it was very potent as well as highly selective against Ma DHFR, with an IC(50) of 0.0058 microM and an IC(50)(rat liver)/IC(50)(M. avium) ratio of >600. Because of this favorable combination of potency and selectivity relative to 1 and 2, compound 3g may be viewed as a promising lead in the search for new antifolates with potential clinical activity against P. carinii and other opportunistic pathogens in patients with AIDS.     

The effect of 5-alkyl modification on the biological activity of pyrrolo[2,3-d]pyrimidine containing classical and nonclassical antifolates as inhibitors of dihydrofolate reductase and as antitumor and/or antiopportunistic infection agents

Novel classical antifolates (3 and 4) and 17 nonclassical antifolates (11-27) were synthesized as antitumor and/or antiopportunistic infection agents. Intermediates for the synthesis of 3, 4, and 11-27 were 2,4-diamino-5-alkylsubstituted-7H-pyrrolo[2,3-d]pyrimidines, 31 and 38, prepared by a ring transformation/ring annulation sequence of 2-amino-3-cyano-4-alkyl furans to which various aryl thiols were attached at the 6-position via an oxidative addition reaction using I2. The condensation of alpha-hydroxy ketones with malonodinitrile afforded the furans. For the classical analogues 3 and 4, the ester precursors were deprotected, coupled with diethyl-L-glutamate, and saponified. Compounds 3 (IC50 = 60 nM) and 4 (IC50 = 90 nM) were potent inhibitors of human DHFR. Compound 3 inhibited tumor cells in culture with GI50 500-fold selectivity over human DHFR. Analogue 17 was 50-fold more potent than trimethoprim and about twice as selective against T. gondii DHFR.