Aleem Gangjee

The human proton-coupled folate transporter: Biology and therapeutic applications to cancer

This review summarizes the biology of the proton-coupled folate transporter (PCFT). PCFT was identified in 2006 as the primary transporter for intestinal absorption of dietary folates, as mutations in PCFT are causal in hereditary folate malabsorption (HFM) syndrome. Since 2006, there have been major advances in understanding the mechanistic roles of critical amino acids and/or domains in the PCFT protein, many of which were identified as mutated in HFM patients, and in characterizing transcriptional control of the human PCFT gene. With the recognition that PCFT is abundantly expressed in human tumors and is active at pHs characterizing the tumor microenvironment, attention turned to exploiting PCFT for delivering novel cytotoxic antifolates for solid tumors. The finding that pemetrexed is an excellent PCFT substrate explains its demonstrated clinical efficacy for mesothelioma and non-small cell lung cancer, and prompted development of more PCFT-selective tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that derive their cytotoxic effects by targeting de novo purine nucleotide biosynthesis.

Synthesis and discovery of water-soluble microtubule targeting agents that bind to the colchicine site on tubulin and circumvent Pgp mediated resistance

Two classes of molecules were designed and synthesized based on a 6-CH(3) cyclopenta[d]pyrimidine scaffold and a pyrrolo[2,3-d]pyrimidine scaffold. The pyrrolo[2,3-d]pyrimidines were synthesized by reacting ethyl 2-cyano-4,4-diethoxybutanoate and acetamidine, which in turn was chlorinated and reacted with the appropriate anilines to afford 1 and 2. The cyclopenta[d]pyrimidines were obtained from 3-methyladapic acid, followed by reaction with acetamidine to afford the cyclopenta[d]pyrimidine scaffold. Chlorination and reaction with appropriate anilines afforded (±)-3·HCl-(±)-7·HCl. Compounds 1 and (±)-3·HCl had potent antiproliferative activities in the nanomolar range. Compound (±)-3·HCl is significantly more potent than 1. Mechanistic studies showed that 1 and (±)-3·HCl cause loss of cellular microtubules, inhibit the polymerization of purified tubulin, and inhibit colchicine binding. Modeling studies show interactions of these compounds within the colchicine site. The identification of these new inhibitors that can also overcome clinically relevant mechanisms of drug resistance provides new scaffolds for colchicine site agents.

Potent Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors: Classical and Nonclassical 2-Amino-4-oxo-5-arylthio-substituted-6-methylthieno[2,3-d]pyrimidine Antifolates

N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3- d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid (4) and nine nonclassical analogues 5-13 were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was 2-amino-6-methylthieno[2,3-d]pyrimidin-4(3 H)-one (16), which was converted to the 5-bromo-substituted compound 17 followed by an Ullmann reaction to afford 5-13. The classical analogue 4 was synthesized by coupling the benzoic acid derivative 19 with diethyl L-glutamate and saponification. Compound 4 is the most potent dual inhibitor of human TS (IC 50 = 40 nM) and human DHFR (IC 50 = 20 nM) known to date. The nonclassical analogues 5- 13 were moderately potent against human TS with IC 50 values ranging from 0.11 to 4.6 microM. The 4-nitrophenyl analogue 7 was the most potent compound in the nonclassical series, demonstrating potent dual inhibitory activities against human TS and DHFR. This study indicated that the 5-substituted 2-amino-4-oxo-6-methylthieno[2,3-d]pyrimidine scaffold is highly conducive to dual human TS-DHFR inhibitory activity.

Design, synthesis, and X-ray crystal structure of classical and nonclassical 2-amino-4-oxo-5-substituted-6-ethylthieno[2,3-d]pyrimidines as dual thymidylate synthase and dihydrofolate reductase inhibitors and as potential antitumor agents.

N-{4-[(2-Amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-L-glutamic acid 2 and 13 nonclassical analogues 2a-2m were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in the synthesis was 2-amino-6-ethyl-5-iodothieno[2,3-d]pyrimidin-4(3H)-one, 7, to which various arylthiols were attached at the 5-position. Coupling 8 with L-glutamic acid diethyl ester and saponification afforded 2. X-ray crystal structures of 2 and 1 (the 6-methyl analogue of 2), DHFR, and NADPH showed for the first time that the thieno[2,3-d]pyrimidine ring binds in a folate mode. Compound 2 was an excellent dual inhibitor of human TS (IC50 = 54 nM) and human DHFR (IC50 = 19 nM) and afforded nanomolar GI50 values against tumor cells in culture. The 6-ethyl substitution in 2 increases both the potency (by 2-3 orders of magnitude) as well as the spectrum of tumor inhibition in vitro compared to the 6-methyl analogue 1. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from Toxoplasma gondii.

2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6-16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6-16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6-16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.

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 <or= 10(-7) M. Nonclassical 17 (IC50 = 58 nM) was a potent inhibitor of Toxoplasma gondii (T. gondii) DHFR with >500-fold selectivity over human DHFR. Analogue 17 was 50-fold more potent than trimethoprim and about twice as selective against T. gondii DHFR.

Design, synthesis, and molecular modeling of novel pyrido[2,3-d]pyrimidine analogues as antifolates; application of Buchwald-Hartwig aminations of heterocycles.

Opportunistic infections caused by Pneumocystis jirovecii (P. jirovecii, pj), Toxoplasma gondii (T. gondii, tg), and Mycobacterium avium (M. avium, ma) are the principal causes of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). The absence of any animal models for human Pneumocystis jirovecii pneumonia and the lack of crystal structures of pjDHFR and tgDHFR make the design of inhibitors challenging. A novel series of pyrido[2,3-d]pyrimidines as selective and potent DHFR inhibitors against these opportunistic infections are presented. Buchwald-Hartwig coupling reaction of substituted anilines with pivaloyl protected 2,4-diamino-6-bromo-pyrido[2,3-d]pyrimidine was successfully explored to synthesize these analogues. Compound 26 was the most selective inhibitor with excellent potency against pjDHFR. Molecular modeling studies with a pjDHFR homology model explained the potency and selectivity of 26. Structural data are also reported for 26 with pcDHFR and 16 and 22 with variants of pcDHFR.

Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

ABSTRACT Pneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with Ki values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased Km values for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of kcat, as well as a Km for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.

Functional loss of the reduced folate carrier enhances the antitumor activities of novel antifolates with selective uptake by the proton-coupled folate transporter.

Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced folate carrier (RFC), the major facilitative folate transporter, RFC expression may alter drug efficacies by affecting cellular tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [3H]THF cofactors derived from [3H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors than toward WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antifolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.

Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors.

Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2-chlorophenyl with a sulfur bridge with a Ki of 8.83±0.67 nM is discussed in terms of several Van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors.