Structure–Activity Relationship and in Vitro and in Vivo Evaluation of the Potent Cytotoxic Anti-microtubule Agent <i>N</i>‑(4-Methoxyphenyl)‑<i>N</i>,2,6-trimethyl-6,7-dihydro‑5<i>H</i>‑cyclopenta[<i>d</i>]pyrimidin-4-aminium Chloride and Its Analogues As Antitumor Agents

A series of 21 substituted cyclopenta­[<i>d</i>]­pyrimidines were synthesized as an extension of our discovery of the parent compound (±)-<b>1</b>·HCl as an anti-microtubule agent. The structure–activity relationship indicates that the <i>N</i>-methyl and a 4<i>N</i>-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 <b>30</b>·HCl was a one to two digit nanomolar inhibitor of most tumor cell proliferations and was up to 7-fold more potent than the parent compound (±)-<b>1</b>·HCl. In addition, <b>30</b>·HCl inhibited cancer cell proliferation regardless of Pgp or βIII-tubulin status, both of which are known to cause clinical resistance to several anti-tubulin agents. In vivo efficacy of <b>30</b>·HCl was demonstrated against a triple negative breast cancer xenograft mouse model. Compound <b>30</b>·HCl is water-soluble and easily synthesized and serves as a lead compound for further preclinical evaluation as an antitumor agent

ENOblock Does Not Inhibit the Activity of the Glycolytic Enzyme Enolase

<div><p>Inhibition of glycolysis is of great potential for the treatment of cancer. However, inhibitors of glycolytic enzymes with favorable pharmacological profiles have not been forthcoming. Due to the nature of their active sites, most high-affinity transition-state analogue inhibitors of glycolysis enzymes are highly polar with poor cell permeability. A recent publication reported a novel, non-active site inhibitor of the glycolytic enzyme Enolase, termed ENOblock (N-[2-[2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]amino]1,3,5-triazin-2-yl]amino]benzeneacetamide). This would present a major advance, as this is heterocyclic and fully cell permeable molecule. Here, we present evidence that ENOblock does not inhibit Enolase enzymatic activity <i>in vitro</i> as measured by three different assays, including a novel ³¹P NMR based method which avoids complications associated with optical interferences in the UV range. Indeed, we note that due to strong UV absorbance, ENOblock interferes with the direct spectrophotometric detection of the product of Enolase, phosphoenolpyruvate. Unlike established Enolase inhibitors, ENOblock does not show selective toxicity to <i>ENO1</i>-deleted glioma cells in culture. While our data do not dispute the biological effects previously attributed to ENOblock, they indicate that such effects must be caused by mechanisms other than direct inhibition of Enolase enzymatic activity.</p></div

Non-selective toxicity of ENOBlock to ENO1-deleted glioma cells.

<p>A representative plate of cancer cells treated with ENOblock is shown in panel <b>a</b>, with quantification shown in panel <b>b</b> A plate treated with SF2312 is shown in panel <b>c</b>, with quantification shown in panel <b>d</b>. Cell were treated for 7 days. <b>(b, d)</b> D423 <i>ENO1</i>-deleted (red diamonds), D423 <i>ENO1</i>-rescued (blue squares) and LN319 <i>ENO1</i> WT (grey circles) were treated with the indicated doses of ENOblock in panel <b>b</b> (N = 4 ± S.D) or SF2312 in panel <b>d</b> (N = 4 ± S.D). Cell density was quantified by crystal violet and expressed relative to vehicle control as a function of inhibitor concentration. At high concentrations, SF2312 selectively killed D423 <i>ENO1</i>-deleted cells as compared to D423 <i>ENO1</i>-rescued cells (p<0.05, Repeated Measures one-way ANOVA with Bonferroni correction). ENOblock failed to show such selectivity regardless of dose.</p

Design, Synthesis, and Molecular Modeling of Novel Pyrido[2,3‑<i>d</i>]pyrimidine Analogues As Antifolates; Application of Buchwald–Hartwig Aminations of Heterocycles

Opportunistic infections caused by Pneumocystis jirovecii (P. jirovecii, <i>pj</i>), Toxoplasma gondii (T. gondii, <i>tg</i>), and Mycobacterium avium (M. avium, <i>ma</i>) 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 <i>pj</i>DHFR and <i>tg</i>DHFR make the design of inhibitors challenging. A novel series of pyrido­[2,3-<i>d</i>]­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-<i>d</i>]­pyrimidine was successfully explored to synthesize these analogues. Compound <b>26</b> was the most selective inhibitor with excellent potency against <i>pj</i>DHFR. Molecular modeling studies with a <i>pj</i>DHFR homology model explained the potency and selectivity of <b>26</b>. Structural data are also reported for <b>26</b> with <i>pc</i>DHFR and <b>16</b> and <b>22</b> with variants of <i>pc</i>DHFR

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

6‑Substituted Pyrrolo[2,3‑<i>d</i>]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors

2-Amino-4-oxo-6-substituted-pyrrolo­[2,3-<i>d</i>]­pyrimidine antifolate thiophene regioisomers of AGF94 (<b>4</b>) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (<b>5</b>) and AGF154 (<b>7</b>)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC₅₀s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC₅₀ < 7 nM). Compounds <b>5</b> and <b>7</b> inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (<b>6</b>) and AGF163 (<b>8</b>), 2,4-diamino-5-substituted-furo­[2,3-<i>d</i>]­pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with <b>5</b> and <b>7</b> were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, <b>7</b> was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting

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

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in <b>1</b> by N (<b>4</b>), O (<b>8</b>), or S (<b>9</b>), or with N-substituted formyl (<b>5</b>), acetyl (<b>6</b>), or trifluoroacetyl (<b>7</b>) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by <b>4</b>–<b>9</b> over the CH₂ analogue <b>1</b>. Compounds <b>4</b>–<b>9</b> inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for <b>4</b> with FRα and GARFTase showed that the bound conformations of <b>4</b> required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, <b>4</b> was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidines related to <b>1</b> provide targeted antifolates that warrant further evaluation as anticancer agents

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

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in <b>1</b> by N (<b>4</b>), O (<b>8</b>), or S (<b>9</b>), or with N-substituted formyl (<b>5</b>), acetyl (<b>6</b>), or trifluoroacetyl (<b>7</b>) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by <b>4</b>–<b>9</b> over the CH₂ analogue <b>1</b>. Compounds <b>4</b>–<b>9</b> inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for <b>4</b> with FRα and GARFTase showed that the bound conformations of <b>4</b> required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, <b>4</b> was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo­[2,3-<i>d</i>]­pyrimidines related to <b>1</b> provide targeted antifolates that warrant further evaluation as anticancer agents