Pyrrolo[2,3-d]pyrimidine Classical Antifolates for Targeted Cancer Chemotherapy- Applications of Bioisosteric and Regioisomeric Substitutions for Improved Tumor-Selectivity and Potency

In 2018, it is estimated that 1,735,350 new cases of cancer and 609,640 deaths from the disease will be diagnosed in the United States alone. Conventional chemotherapy is by far the most successful category of clinical oncology, having cured (complete remission (CR), without return) or provided clinical benefit to millions of people. However, since its earliest discovery, the major causes of failure of conventional cancer chemotherapy have been dose-limiting toxicities and development of resistance. There is a desperate ongoing search for new cancer therapies as tumor-targeted agents (without harming normal cells or tissues) with low propensity for the development of resistance. Clinically used antifolates methotrexate (MTX; DHFR inhibitor), pemetrexed (PMX; TS and GARFTase inhibitor), pralatrexate (PTX; DHFR inhibitor), and raltitrexed (RTX; TS inhibitor), have two major disadvantages: (1) dose-limiting toxicities due to ubiquitous transport via the reduced folate carrier (RFC) and (2) drug-resistance; mutation and/or overexpression of RFC and target enzymes resulting in inadequate transport, reduced cellular retention, and decreased potency. However, two other folate transporters that are narrowly expressed in healthy tissue while overexpressed in many different types of cancer are the folate receptors (FRs) (epithelial ovarian cancer (EOC), NSCLC, renal, endometrial, colorectal, breast cancers, hematologic malignancies, etc.) and the proton-coupled folate transporter (PCFT) (ovarian and NSCLC). Our aim is to design classical antifolates for selective uptake by FRs and PCFT over RFC and inhibition of multiple folate metabolizing enzymes in their monoglutamate forms. Successful identification of such small molecule single agents will potentially (a) have tumor-targeting action and (b) combat resistance development. This dissertation discusses the bioisosteric and regioisomeric optimization of 5-and/or 6-substituted pyrrolo[2,3-d]pyrimidine antifolates for improved tumor-targeted activity. Molecular modeling with the help of known X-ray crystal structures of the transporters and the intracellular enzymes was used to rationalize the design of the analogs. NMR studies that were performed to provide structural evidence for the presence of a conformationally restricting intramolecular fluorine hydrogen bond have been described. The dissertation also discusses the synthetic efforts for obtaining the pyrrolo[2,3-d]pyrimidine analogs with regioisomeric substitutions, modified linkers and bioisoteric replacements (general structure I) for selective uptake, and inhibition of one or more enzymes in the de novo purine and/or pyrimidine biosynthetic pathway. The present work identified that, depending on the regioisomeric position, fluorine substitution on the side-chain (het)aryl ring improves both potency as well as selectivity, most significantly towards PCFT-expressing cell lines. NMR-based structural evidence led to one of our hypotheses that entropic benefit due to conformational restriction caused by an intramolecular fluorine hydrogen bond may partly be responsible for improved biological activity. The current work led to the identification of a fluorinated pyrrolo[2,3-d]pyrimidine analog 184, the most potent inhibitor of PCFT-expressing Chinese hamster ovarian (CHO) cells (PCFT4 IC50 = 1.5 (0.4) nM). Another important contribution of the current work is the discovery of multi-enzyme inhibitor 201, a C6-methylated version of the clinically used anticancer agent PMX, which showed selectivity over RFC (PMX PC43-10 IC50 = 26.2 nM; 201 PC43-10 IC50 \u3e 1000 nM). Such selectivity can potentially help overcome the dose-limiting toxicities of PMX. The fluorinated and methylated analogs described herein are testimonials for the profound effects that minor structural changes can have on polypharmacology

Evaluation of physiological and operative severity score for enumeration of mortality and morbidity and Portsmouth modification of possum scores in patients with hollow viscus perforation

Background: The physiological and operative severity score for the enumeration of mortality and morbidity (POSSUM) is widely used to predict the morbidity and mortality in a variety of surgical settings and provides a tool for risk adjustment and comparison. The aim of this study was to assess the predicting morbidity and mortality in hollow viscus perforation by applying POSSUM and Portsmouth modification of POSSUM scores. Methods: This study was a prospective study conducted on 33 patients admitted in the Department of General Surgery, Chalmeda Anand Rao Institute of Medical Sciences, Karimnagar, from September 2016 to August 2017, and present detailed records of the parameters under evaluation of patients admitted to surgical wards with the clinical diagnosis of hollow viscus perforation (gastric/duodenal/ileal perforation). Results: During the present study period, a total of 136 patients were operated on small bowel, and of these 33 cases fulfilled all the criteria and were selected for this study. The overall mortality in this study was 6 (18.18% of the study), while the morbidity was noted in 17 cases (51% of study). Mortality rates differed with POSSUM scores over predicting the mortality. On applying POSSUM, using exponential analysis, we found that the expected number of deaths for our study group is 10 (O:E = 1.66). Conclusion: On applying POSSUM using the exponential analysis, we found that the expected number of deaths for our study group is 10 (O:E = 1.66). We found the difference between expected and observed mortality rates using the exponential analysis

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

Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment

Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRβ is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRβ-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy

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