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1.  Discovery of 5-substituted pyrrolo[2,3-d]pyrimidine antifolates as dual acting inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis: implications of inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase to AMPK activation and anti-tumor activity 
Journal of medicinal chemistry  2013;56(24):10016-10032.
We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5–10) with 1 to 6 bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analog and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Anti-proliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.
PMCID: PMC3917155  PMID: 24256410
2.  Tumor-Targeting with Novel Non-Benzoyl 6-Substituted Straight Chain Pyrrolo[2,3-d]pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and Inhibition of de novo Purine Nucleotide Biosynthesis 
Journal of medicinal chemistry  2013;56(21):10.1021/jm401139z.
A new series of 6-substituted straight side chain pyrrolo[2,3-d]pyrimidines 3a–d with varying chain lengths (n = 5–8) was designed and synthesized as part of our program to provide targeted antitumor agents with folate receptor (FR) cellular uptake specificity and glycinamide ribonucleotide formyltransferase (GARFTase) inhibition. Carboxylic acids 4a–d were converted to the acid chlorides and reacted with diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a–d. Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with 5a–d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a–d. Hydrolysis and subsequent coupling with diethyl L-glutamate and saponification afforded target compounds 3a–d. Compounds 3b–d showed selective cellular uptake via FRα and -β, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase, resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.
PMCID: PMC3880613  PMID: 24111942
3.  Structure activity-relationship and in vitro and in vivo evaluation of the potent cytotoxic anti-microtubule agent N-(4-methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminium chloride and its analogues as antitumor agents 
Journal of medicinal chemistry  2013;56(17):10.1021/jm400639z.
A series of 21 substituted cyclopenta[d]pyrimidines were synthesized as an extension of our discovery of the parent compound 1·HCl as an antimicrotubule agent. The structure-activity relationship indicates that the N-methyl and a 4′-methoxy groups appear important for potent activity. In addition, the 6-substituent in the parent analogue is not necessary for activity. The most potent compound 30·HCl was a 1–2 digit nanomolar inhibitor of most tumor cell proliferations and was up to 7-fold more potent than the parent compound 1·HCl. In addition, 30·HCl inhibited cancer cell proliferation regardless of Pgp or βIII-tubulin status, both of which are known to cause clinical resistance to several antitubulin agents. In vivo efficacy of 30·HCl was demonstrated against a triple negative breast cancer xenograft mouse model. Compound 30·HCl is water soluble, easily synthesized and serves as a lead compound for further preclinical evaluation as an antitumor agent.
PMCID: PMC3850778  PMID: 23895532
cyclopenta[d]pyrimidines; antimitotic; tubulin; colchicine site
4.  Design, synthesis and molecular modeling of novel pyrido[2,3-d]pyrimidine analogs as antifolates: Application of Buchwald-Hartwig aminations of heterocycles 
Journal of medicinal chemistry  2013;56(11):4422-4441.
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 analogs. 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.
PMCID: PMC3723128  PMID: 23627352
5.  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 
Journal of medicinal chemistry  2008;51(18):5789-5797.
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(3H)-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 (IC50 = 40 nM) and human DHFR (IC50 = 20 nM) known to date. The nonclassical analogues 5–13 were moderately potent against human TS with IC50 values ranging from 0.11 to 4.6 µM. 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.
PMCID: PMC3892769  PMID: 18800768
6.  N9-Substituted 2,4-Diaminoquinazolines: Synthesis and Biological Evaluation of Lipophilic Inhibitors of Pneumocystis carinii and Toxoplasma gondii Dihydrofolate Reductase 
Journal of medicinal chemistry  2008;51(19):10.1021/jm800694g.
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—CH3 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.
PMCID: PMC3885247  PMID: 18771252
7.  Design, Synthesis, and Biological Evaluation of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-6-methylpyrrolo[3,2-d]pyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors 
Journal of medicinal chemistry  2007;51(1):10.1021/jm701052u.
We designed and synthesized a classical antifolate N-{4-[(2-amino-6-methyl-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl]benzoyl}-l-glutamic acid 4 and 11 nonclassical analogues 5–15 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was N-(4-chloro-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)-2,2-dimethylpropanamide, 29, to which various 5-benzyl substituents were attached. For the classical analogue 4, the ester obtained from the N-benzylation reaction was deprotected and coupled with diethyl l-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC50 = 46 nM, about 206-fold more potent than pemetrexed) and DHFR (IC50 = 120 nM, about 55-fold more potent than pemetrexed). The nonclassical analogues were marginal inhibitors of human TS, but four analogues showed potent T. gondii DHFR inhibition along with >100-fold selectivity compared to human DHFR.
PMCID: PMC3885252  PMID: 18072727
8.  Discovery of Novel Antitumor Antimitotic Agents That Also Reverse Tumor Resistance1 
Journal of medicinal chemistry  2007;50(14):10.1021/jm070194u.
We have discovered a novel series of 7-benzyl-4-methyl-5-[(2-substituted phenyl)ethyl]-7H-pyrrolo[2,3-d]-pyrimidin-2-amines, which possess antimitotic and antitumor activities against antimitotic-sensitive as well as resistant tumor cells. These agents bind to a site on tubulin that is distinct from the colchicine, vinca alkaloid, and paclitaxel binding sites and some, in addition to their antitumor activity, remarkably also reverse tumor resistance to antimitotic agents mediated via the P-glycoprotein efflux pump. The compounds were synthesized from N-(7-benzyl-5-ethynyl-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-2,2-dimethylpro-panamide 11 or the corresponding 5-iodo analog 14 via Sonogashira couplings with appropriate iodobenzenes or phenylacetylene followed by reduction and deprotection to afford the target analogs. Sodium and liquid NH3 afforded the debenzylated analogs. The most potent analog 1 was one to three digit nanomolar against the growth of both sensitive and resistant tumor cells in culture. Compounds of this series are promising novel antimitotic agents that have the potential for treating both sensitive and resistant tumors.
PMCID: PMC3858178  PMID: 17567121
9.  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 Agents1a-e 
Journal of medicinal chemistry  2008;51(15):10.1021/jm800244v.
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 α-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 ≤ 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.
PMCID: PMC3858179  PMID: 18605720
10.  Design and Synthesis of Classical and Nonclassical 6-Arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidines as Antifolates 
Journal of medicinal chemistry  2007;50(13):10.1021/jm070165j.
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.
PMCID: PMC3850752  PMID: 17552508
11.  Synthesis and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers as inhibitors of de novo purine biosynthesis with selectivity for cellular uptake by high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier 
Journal of Medicinal Chemistry  2012;55(4):1758-1770.
We reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a 3- (3a) and 4-carbon (3b) bridge. Compound 3a was more potent than 3b against tumor cells; While 3b was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and L-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3] (7 and 8) and [1,2] (4, 5 and 6) substitutions on the thienoyl ring, and with acetylenic insertions in the 4-atom bridge, were synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC.
PMCID: PMC3288238  PMID: 22243528
12.  Synthesis, biological and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase 
Journal of medicinal chemistry  2011;54(20):7150-7164.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1–3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with L-glutamate diethyl ester, followed by saponification, afforded 1–3. Compound 3 selectively inhibited proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including human tumor cells KB and IGROV1 much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1, 2 and 4-atom bridge lengths for the activity of this series.
PMCID: PMC3209708  PMID: 21879757
13.  Synthesis and Biological Activities of (R)- and (S)-N-(4-methoxy-phenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminium Chloride as Potent Cytotoxic Antitubulin Agents 
Journal of medicinal chemistry  2011;54(17):6151-6155.
(R,S)-1 is a potent antimitotic compound. (R)-1•HCl and (S)-1•HCl were synthesized from (R)- and (S)-3-methyladipic acid. Both enantiomers were potent inhibitors of cell proliferation, and caused cellular microtubule loss and mitotic arrest. They inhibited purified tubulin assembly and the binding of [3H]colchicine to tubulin, with (S)-1, being about twice as potent. Cytotoxicity against a panel of 60 tumor cell lines, however, indicated that the (S)-isomer was 10- to 88-fold more potent than the (R)-isomer.
PMCID: PMC3184787  PMID: 21786793
Antimitotic agents; Cytotoxicity; Enantiomers
14.  Synthesis and Discovery of Water Soluble Microtubule Targeting Agents that Bind to the Colchicine Site on Tubulin and Circumvent Pgp Mediated Resistance 
Journal of medicinal chemistry  2010;53(22):8116-8128.
Two classes of molecules were designed and synthesized based on a 6-CH3 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-diethoxybutanate and acetamidine, which in turn was chlorinated and reacted with the appropriate anilines to afford 1 and 2. The cyclopenta[d]pyrimidineswere 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 (add chemical compound designation) and (±)-3•HCl (add compound chemical designation) 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 microtubles, 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 resistence provides new scaffolds for colchicine site agents.
PMCID: PMC2988971  PMID: 20973488
15.  Single Agents with Designed Combination Chemotherapy Potential: Synthesis and Evaluation of Substituted Pyrimido[4,5-b]indoles as Receptor Tyrosine Kinase and Thymidylate Synthase Inhibitors and as Antitumor Agents 
Journal of medicinal chemistry  2010;53(4):1563-1578.
Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise and several such clinical trials are currently underway. We have designed, synthesized and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet derived growth factor receptor-beta (PDGFR-β) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-β and hTS is better than or close to standards. In a COLO-205 xenograft mouse model one of the analogs significantly decreased tumor growth (TGI = 76% at 35 mg/kg), liver metastases and tumor blood vessels compared to a standard drug and to control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents.
PMCID: PMC2825747  PMID: 20092323
16.  Synthesis and biological activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier for cellular entry† 
Journal of medicinal chemistry  2010;53(3):1306-1318.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and 4-6 carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FRα, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR- and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.
PMCID: PMC2836843  PMID: 20085328
17.  Design, Synthesis, and X-ray Crystal Structure of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-6-ethyl-thieno[2,3-d]pyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors and as Potential Antitumor Agents 
Journal of medicinal chemistry  2009;52(15):4892-4902.
N-{4-[(2-amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-L-glutamic acid 2 and thirteen 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 aryl thiols were attached at the 5-position. Coupling 8 with L-glutamic acid diethyl ester and saponification afforded 2. X-ray crystal structure 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 two- to three-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.
PMCID: PMC2739130  PMID: 19719239
18.  Synthesis and Discovery of High Affinity Folate Receptor-Specific Glycinamide Ribonucleotide Formyltransferase Inhibitors With Antitumor Activity 
Journal of medicinal chemistry  2008;51(16):5052-5063.
A series of 6-substituted classical pyrrolo[2,3-d]pyrimidine antifolates with a 3- to 6-carbon bridge between the heterocycle and the benzoyl-L-glutamate (compounds 2, 3, 4 and 5, respectively) was synthesized starting from methyl 4-formylbenzoate and a Wittig reaction with the appropriate triphenylphosphonium bromide, followed by reduction and conversion to the α-bromomethylketones. Cyclocondensation of 2,4-diamino-4-oxopyrimidine with the α-bromoketones, coupling with diethyl-L-glutamate and saponification afforded 2–5. Compounds 2–5 had negligible substrate activity for RFC but showed variably potent (nanomolar) and selective inhibitory activities toward Chinese hamster ovary cells that expressed FRα or FRβ, and toward FRα-expressing KB and IGROV1 human tumor cells. Inhibition of KB cell colony formation was also observed. Glycinamide ribonucleotide formyl transferase (GARFTase) was identified as the primary intracellular target of the pyrrolo[2,3-d]pyrimidines. The combined properties of selective FR targeting, lack of RFC transport, and GARFTase inhibition resulting in potent antitumor activity are unprecedented and warrant development of these analogs as antitumor agents.
PMCID: PMC2748117  PMID: 18680275
19.  Synthesis and biological activity of a novel series of 6-substituted thieno[2,3-d]pyrimidine antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors over the reduced folate carrier and proton-coupled folate transporter for cellular entry 
Journal of medicinal chemistry  2009;52(9):2940-2951.
A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines, with bridge length variations (from 2-8 carbon atoms) were synthesized as selective folate receptor (FR) α and β substrates and as antitumor agents. The syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes which were converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chlorformamidine afforded the thieno[2,3-d]pyrimidines 30-36 which were hydrolyzed and coupled with diethyl-L-glutamate, followed by saponification to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC50 4.7 to 334 nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory activity of the active thieno[2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory.
PMCID: PMC2730022  PMID: 19371039
20.  Dual Inhibitors of Thymidylate Synthase and Dihydrofolate Reductase as Antitumour Agents: Design, Synthesis and Biological Evaluation of Classical and Nonclassical Pyrrolo[2,3-d]pyrimidine Antifolates1 
Journal of medicinal chemistry  2006;49(3):1055-1065.
We designed and synthesized a classical analog 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 analogs 5-17 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumour 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 analog 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 (IC50 = 90 nM) and human DHFR (IC50 = 420 nM). Compound 4 was not a substrate for human FPGS. Metabolite protection studies established TS as its principal target. Most of the nonclassical analogs were only inhibitors of human TS with IC50 values of 0.23-26 μM.
PMCID: PMC2547132  PMID: 16451071
21.  Synthesis of classical, four-carbon bridged 5-substituted furo[2,3-d]pyrimidine and 6-substituted pyrrolo[2,3-d]pyrimidine analogues as antifolates1 
Journal of medicinal chemistry  2005;48(16):5329-5336.
We report, for the first time, the biological activities of four carbon atom bridged classical antifolates on dihydrofolate reductase (DHFR), thymidylate synthase (TS) and folylpolyglutamate synthetase (FPGS) as well as on antitumor activity. Extension of the bridge homologation studies of classical two-carbon bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine (2) afforded two, four-carbon bridged antifolates, analogues 5 and 6, with enhanced FPGS substrate activity and inhibitory activity against tumor cells in culture (EC50 values of ≤ 10−7 M) compared with the two-carbon bridged analogues. These results support our original hypothesis that the distance and orientation of the side chain para-aminobenzoyl-L-glutamate moiety with respect to the pyrimidine ring is a crucial determinant of biological activity. In addition, this study demonstrates that, for classical antifolates that are substrates for FPGS, poor inhibitory activity against isolated target enzymes is not necessarily a predictor of a lack of antitumor activity.
PMCID: PMC2538949  PMID: 16078850

Results 1-21 (21)