Defense Date


Graduation Date

Fall 12-21-2018


One-year Embargo

Submission Type


Degree Name



Medicinal Chemistry


School of Pharmacy

Committee Chair

Aleem Gangjee

Committee Member

Marc W. Harrold

Committee Member

Patrick T. Flaherty

Committee Member

Kevin Tidgewell

Committee Member

Lauren O’Donnell


Antifolates, Targeted cancer chemotherapy, Multiple enzyme inhibition, Pyrrolo[2, 3-d]pyrimidines, Heterocycles, Bioisosteric replacements, Regioisomeric replacements, Fluorine-substitution, Fluorine hydrogen bond


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 > 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.



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