Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase

Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic <i>in vitro</i> toward FR- and PCFT-expressing tumor cells and <i>in vivo</i> with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in <i>de novo</i> synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess <i>in vitro</i> inhibition by eight novel thieno- and pyrrolo­[2,3-<i>d</i>]­pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with <i>K</i>_i values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition

Synthesis and Biological Activity of 6-Substituted Pyrrolo[2,3-<i>d</i>]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

We previously reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a three- (<b>3a</b>) and four-carbon (<b>3b</b>) bridge. Compound <b>3a</b> was more potent than <b>3b</b> against tumor cells. While <b>3b</b> was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over the reduced folate carrier (RFC), <b>3a</b> was not. To determine if decreasing the distance between the bicyclic scaffold and l-glutamate in <b>3b</b> would preserve transport selectivity and potency against human tumor cells, <b>3b</b> regioisomers with [1,3] (<b>7</b> and <b>8</b>) and [1,2] (<b>4</b>, <b>5</b>, and <b>6</b>) substitutions on the thienoyl ring and with acetylenic insertions in the four-atom bridge were synthesized and evaluated. Compounds <b>7</b> and <b>8</b> were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC

Novel 5‑Substituted Pyrrolo[2,3‑<i>d</i>]pyrimidines as Dual Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5‑Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase and as Potential Antitumor Agents

A new series of 5-substituted thiopheneyl pyrrolo­[2,3-<i>d</i>]­pyrimidines <b>6</b>–<b>11</b> with varying chain lengths (<i>n</i> = 1–6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo­[2,3-<i>d</i>]­pyrimidines <b>2c</b> and <b>2d</b> with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds <b>6</b>–<b>11</b> inhibited KB human tumor cells in the order <b>9</b> = <b>10</b> > <b>8</b> > <b>7</b> > <b>6</b> = <b>11</b>. Compounds <b>8</b>–<b>10</b> were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of <b>8</b> and <b>9</b> appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure–activity relationship for transport and dual target inhibition

Discovery of 5‑Substituted Pyrrolo[2,3‑<i>d</i>]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 Antitumor Activity

We synthesized 5-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidine antifolates (compounds <b>5</b>–<b>10</b>) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound <b>8</b> with a 4-carbon bridge was the most active analogue 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. Antiproliferative effects of compound <b>8</b> 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 <b>8</b> 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

Fluorine-Substituted Pyrrolo[2,3‑<i>d</i>]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of <i>de Novo</i> Purine Nucleotide Biosynthesis

Novel fluorinated 2-amino-4-oxo-6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidine analogues <b>7</b>–<b>12</b> were synthesized and tested for selective cellular uptake by folate receptors (FRs) α and β or the proton-coupled folate transporter (PCFT) and for antitumor efficacy. Compounds <b>8</b>, <b>9</b>, <b>11</b>, and <b>12</b> showed increased <i>in vitro</i> antiproliferative activities (∼11-fold) over the nonfluorinated analogues <b>2</b>, <b>3</b>, <b>5</b>, and <b>6</b> toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds <b>8</b>, <b>9</b>, <b>11</b>, and <b>12</b> also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, <b>9</b>, <b>11</b>, and <b>12</b> showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the <i>de novo</i> purine biosynthesis pathway. Molecular modeling studies validated <i>in vitro</i> cell-based results. NMR evidence supports the presence of an intramolecular fluorine–hydrogen bond. Potent <i>in vivo</i> efficacy of <b>11</b> was established with IGROV1 xenografts in severe compromised immunodeficient mice

Fluorine-Substituted Pyrrolo[2,3‑<i>d</i>]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of <i>de Novo</i> Purine Nucleotide Biosynthesis

Novel fluorinated 2-amino-4-oxo-6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidine analogues <b>7</b>–<b>12</b> were synthesized and tested for selective cellular uptake by folate receptors (FRs) α and β or the proton-coupled folate transporter (PCFT) and for antitumor efficacy. Compounds <b>8</b>, <b>9</b>, <b>11</b>, and <b>12</b> showed increased <i>in vitro</i> antiproliferative activities (∼11-fold) over the nonfluorinated analogues <b>2</b>, <b>3</b>, <b>5</b>, and <b>6</b> toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds <b>8</b>, <b>9</b>, <b>11</b>, and <b>12</b> also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, <b>9</b>, <b>11</b>, and <b>12</b> showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the <i>de novo</i> purine biosynthesis pathway. Molecular modeling studies validated <i>in vitro</i> cell-based results. NMR evidence supports the presence of an intramolecular fluorine–hydrogen bond. Potent <i>in vivo</i> efficacy of <b>11</b> was established with IGROV1 xenografts in severe compromised immunodeficient mice

Fluorine-Substituted Pyrrolo[2,3‑<i>d</i>]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of <i>de Novo</i> Purine Nucleotide Biosynthesis

Novel fluorinated 2-amino-4-oxo-6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidine analogues <b>7</b>–<b>12</b> were synthesized and tested for selective cellular uptake by folate receptors (FRs) α and β or the proton-coupled folate transporter (PCFT) and for antitumor efficacy. Compounds <b>8</b>, <b>9</b>, <b>11</b>, and <b>12</b> showed increased <i>in vitro</i> antiproliferative activities (∼11-fold) over the nonfluorinated analogues <b>2</b>, <b>3</b>, <b>5</b>, and <b>6</b> toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds <b>8</b>, <b>9</b>, <b>11</b>, and <b>12</b> also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, <b>9</b>, <b>11</b>, and <b>12</b> showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the <i>de novo</i> purine biosynthesis pathway. Molecular modeling studies validated <i>in vitro</i> cell-based results. NMR evidence supports the presence of an intramolecular fluorine–hydrogen bond. Potent <i>in vivo</i> efficacy of <b>11</b> was established with IGROV1 xenografts in severe compromised immunodeficient mice

Tumor Targeting with Novel Pyridyl 6‑Substituted Pyrrolo[2,3‑<i>d</i>]Pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of <i>De Novo</i> Purine Nucleotide Biosynthesis

Tumor-targeted specificities of 6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidine analogues of <b>1</b>, where the phenyl side-chain is replaced by 3′,6′ (<b>5</b>, <b>8</b>), 2′,5′ (<b>6</b>, <b>9</b>), and 2′,6′ (<b>7</b>, <b>10</b>) pyridyls, were analyzed. Proliferation inhibition of isogenic Chinese hamster ovary (CHO) cells expressing folate receptors (FRs) α and β were in rank order, <b>6</b> > <b>9</b> > <b>5</b> > <b>7</b> > <b>8</b>, with <b>10</b> showing no activity, and <b>6</b> > <b>9</b> > <b>5</b> > <b>8</b>, with <b>10</b> and <b>7</b> being inactive, respectively. Antiproliferative effects toward FRα- and FRβ-expressing cells were reflected in competitive binding with [³H]­folic acid. Only compound <b>6</b> was active against proton-coupled folate receptor (PCFT)-expressing CHO cells (∼4-fold more potent than <b>1</b>) and inhibited [³H]­methotrexate uptake by PCFT. In KB and IGROV1 tumor cells, <b>6</b> showed <1 nM IC₅₀, ∼2–3-fold more potent than <b>1</b>. Compound <b>6</b> inhibited glycinamide ribonucleotide formyltransferase in <i>de novo</i> purine biosynthesis and showed potent <i>in vivo</i> efficacy toward subcutaneous IGROV1 tumor xenografts in SCID mice

Structural Characterization of 5‑Substituted Pyrrolo[3,2‑<i>d</i>]pyrimidine Antifolate Inhibitors in Complex with Human Serine Hydroxymethyl Transferase 2

We previously discovered first-in-class multitargeted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates that inhibit serine hydroxymethyltransferase 2 (SHMT2), resulting in potent in vitro and in vivo antitumor efficacies. In this report, we present crystallographic structures for SHMT2 in complex with an expanded series of pyrrolo[3,2-d]pyrimidine compounds with variations in bridge length (3–5 carbons) and the side chain aromatic ring (phenyl, thiophene, fluorine-substituted phenyl, and thiophene). We evaluated structural features of the inhibitor-SHMT2 complexes and correlations to inhibitor potencies (i.e., Kis), highlighting conserved polar contacts and identifying 5-carbon bridge lengths as key determinants of inhibitor potency. Based on the analysis of SHMT2 structural data, we investigated the impact of mutation of Tyr105 in SHMT2 kinetic analysis and studies with HCT116 cells with inducible expression of wild-type and Y105F SHMT2. Increased enzyme inhibition potency by the pyrrolo[3,2-d]pyrimidine inhibitors with Phe105 SHMT2 accompanied an increased growth inhibition of Phe105-expressing HCT116 cells compared to wild-type SHMT2. Pyrrolo[3,2-d]pyrimidine inhibitors with polyglutamate modifications were evaluated for potencies against SHMT2. We determined the crystal structures of SHMT2 in complex with our lead antifolate AGF347 lacking L-glutamate, or as a diglutamate and triglutamate, for comparison with parent AGF347. These data provide the first insights into the influence of antifolate polyglutamylation on SHMT2:inhibitor interactions. Collectively, our results provide new insights into the critical structural determinants of SHMT2 binding by pyrrolo[3,2-d]pyrimidine inhibitors as novel antitumor agents, as well as the first structural characterization of human SHMT2 in complex with polyglutamates of an SHMT2-targeted antifolate

Multitargeted 6‑Substituted Thieno[2,3‑<i>d</i>]pyrimidines as Folate Receptor-Selective Anticancer Agents that Inhibit Cytosolic and Mitochondrial One-Carbon Metabolism

Multitargeted agents with tumor selectivity result in reduced drug resistance and dose-limiting toxicities. We report 6-substituted thieno[2,3-d]pyrimidine compounds (3–9) with pyridine (3, 4), fluorine-substituted pyridine (5), phenyl (6, 7), and thiophene side chains (8, 9), for comparison with unsubstituted phenyl (1, 2) and thiophene side chain (10, 11) containing thieno[2,3-d]pyrimidine compounds. Compounds 3–9 inhibited proliferation of Chinese hamster ovary cells (CHO) expressing folate receptors (FRs) α or β but not the reduced folate carrier (RFC); modest inhibition of CHO cells expressing the proton-coupled folate transporter (PCFT) by 4, 5, 6, and 9 was observed. Replacement of the side-chain 1′,4′-phenyl ring with 2′,5′-pyridyl, or 2′,5′-pyridyl with a fluorine insertion ortho to l-glutamate resulted in increased potency toward FR-expressing CHO cells. Toward KB tumor cells, 4–9 were highly active (IC50’s from 2.11 to 7.19 nM). By metabolite rescue in KB cells and in vitro enzyme assays, de novo purine biosynthesis was identified as a targeted pathway (at 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase)). Compound 9 was 17- to 882-fold more potent than previously reported compounds 2, 10, and 11 against GARFTase. By targeted metabolomics and metabolite rescue, 1, 2, and 6 also inhibited mitochondrial serine hydroxymethyl transferase 2 (SHMT2); enzyme assays confirmed inhibition of SHMT2. X-ray crystallographic structures were obtained for 4, 5, 9, and 10 with human GARFTase. This series affords an exciting new structural platform for potent multitargeted antitumor agents with FR transport selectivity