Healthy individuals experience gastrointestinal distress that can last for two or more weeks. range of temperatures.[9] Infection occurs when an individual ingest oocyst in contaminated food or water. Healthy individuals experience gastrointestinal distress that can last for two or more weeks. However, infection rates are higher and more severe in children, the elderly, and immunocompromised individuals, where it leads to severe and life-threatening wasting disease.[8C10] Currently, nitazoxanide is the only FDA-approved drug for the treatment of cryptosporidiosis.[10] However, the efficacy of nitazoxanide is variable in immunocompetent patients, limited in children, and ineffective in immunocompromised patients, indicating a pressing need for improved therapies.[1, 9, 10] The essential enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), have long been chemotherapeutic targets for cancer and infectious diseases.[11, 12] In humans and most organisms, these two enzymes exist as separate polypeptide chains, whereas in both activities are encoded into a single bifunctional enzyme TS-DHFR.[13, 14] TS-DHFR has been extensively characterized and shown to be a promising target for the development of inhibitors.[12, 15, 16] TS catalyzes the synthesis of deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate (dUMP) and the cofactor 5,10-methylenetetrahydrofolate (CH2H4F).[11, 17] The dihydrofolate produced as the other product of the TS reaction is then reduced by DHFR utilizing the cofactor NADPH, to produce tetrahydrofolate and NADP+.[11, 18] The TS active site is highly conserved for both the and the human enzymes, however there are two distinct, variant amino acids in the region of the binding pocket that interact with the glutamate moiety of folate.[19] In TS ((29a) (hexanes to ethyl acetate). White powder, 0.54 g, 1.44 mmol, 37% yield. 1H NMR (400 MHz, CDCl3) 7.79 C 7.73 (m, 2H), 7.21 C 7.16 (m, 2H), 5.51 C 5.44 (m, 1H), 3.78 (s, 3H), 3.62 C 3.56 (m, 1H), 3.31 C 3.18 (m, 1H), 2.37 C 2.31 (m, 1H), 1.78 C 1.74 (m, 3H), 1.43 C 1.35 (m, 2H). LC-MS (ESI) m/z 374.1 [M+H]+. 4.2.2. (29b) (hexanes to hexanes/ dichloromethane 1:1). White powder, 0.54 g, 1.39 mmol, 32% yield. 1H NMR (400 MHz, CDCl3) 7.80 C 7.74 (m, 2H), 7.52 C 7.46 (m, 2H), 7.29 (s, 1H), 4.30 (qd, = 9.0, 7.9, 4.4 Hz, 1H), 3.72 (s, 3H), 2.91 (q, = 4.6 Hz, 1H), 2.24 C 2.13 (m, 1H), 1.83 C 1.51 (m, 5H), 1.51 C 1.40 (m, 1H), 1.33 C 1.20 (m, 1H). LC-MS (ESI) m/z 388.1 [M+H]+. 4.2.3. (29c) (hexanes/dichloromethane 8:2 to dichloromethane). White powder, 0.66 g, 1.73 mmol, 87% yield. 1H NMR (400 MHz, CDCl3) 12.07 (s, 1H), 8.90 (d, = 8.5 Hz, 1H), 8.12 CETP-IN-3 C 8.06 (m, 1H), 7.91 C 7.86 (m, 2H), 7.80 C 7.74 (m, 2H), 7.65 C 7.58 (m, 1H), 7.17 C 7.11 (m, 1H), 3.97 (s, 3H). LC-MS CETP-IN-3 (ESI) m/z 382.0 [M+H]+. 4.2.4. (29d) (hexanes to ethyl acetate). White powder, 0.97 g, 2.55 mmol, 68% yield. 1H NMR (400 MHz, CDCl3) 8.16 C 8.09 (m, 1H), 8.05 C 8.01 (m, 1H), 7.92 (s, 1H), 7.88 C 7.80 (m, 3H), 7.63 C 7.58 (m, 2H), 7.46 (t, = 7.9 Hz, 1H), 3.92 (s, 3H). LC-MS (ESI) m/z 382.1 [M+H]+. 4.2.5. (29e) (hexanes to ethyl acetate). White powder, 0.29 g, 0.76 mmol, 20% yield. 1H NMR (400 MHz, CDCl3) 8.09 C 8.05 (m, 2H), 7.88 C 7.85 (m, 2H), 7.75 C 7.70 (m, 2H), 7.63 C 7.59 (m, 2H), 3.92 (s, 3H). LC-MS (ESI) m/z 382.1 [M+H]+. 4.2.6. (29f) Vax2 (hexanes/dichloromethane 8:2 to dichloromethane). White powder, 1.15 g, 2.91 mmol, 78% CETP-IN-3 yield. 1H NMR (500 MHz, CDCl3) 9.78 (s, 1H), 8.02 (d, = 8.1 Hz, 1H), 7.90 C 7.84 (m, 2H), 7.80 C 7.74 (m, 2H), 7.39 C 7.35 (m, 1H), 7.27 C 7.22 (m, 1H), 7.18 C.