Protozoa aspartyl proteases and inhibitors for animal and human use

Abstract

The present disclosure relates to methods of treating infections caused by one or more protozoa such as Cryptosporidium, Eimeria, Babesia and Toxoplasma comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein.

Description

26098-2PROTOZOA ASPARTYL PROTEASES AND INHIBITORS FOR ANIMAL AND HUMAN USEFIELD

[0001] The present disclosure relates to use of an effective amount of an inhibitor of plasmepsin IX and / or X or pharmaceutically acceptable salts thereof, in the treatment of infections caused by one or more of Cryptosporidium, Eimeria, Babesia, and / or Toxoplasma protozoa and the like.BACKGROUND

[0002] The phylum Apicomplexa comprises a large group of protozoa (single-celled eukaryotes) many of which are obligate intracellular parasites. Apicomplexan species are classified by the presence of an 'apical complex' which is a collection of organelles and cytoskeletal structures found at the apical end of these polarized cells. These organelles and structures are involved in the invasion of their target host cell. The microneme organelles release their contents onto the parasite surface to bind to their host cells. The large clubbed-shaped rhoptry organelles secrete their contents upon strong contact with their host cell to facilitate invasion.

[0003] Secretory proteins that traffic to these apical organelles in apicomplexans are often proteolytically processed for correct function. Aspartyl proteases that reside in the secretory pathway have become of particular interest due to their important biological roles as well as their capacity to be inhibited by small drug-like molecules. For example, Toxoplasma gondii ASP3 (TgASP3) acts as a maturase of subsets of microneme and rhoptry proteins and is essential for growth and acute-stage disease (Dogga et al., Elife. 2017 Sep 12; 6:e27480. doi:10.7554 / eLife.27480. PMID: 28898199; PMCID: PMC5595437). The Golgi resident aspartyl protease, TgASP5 is important for virulence due to its role in maturation of dense granule proteins that are both secreted from the parasite and reside at the vacuole membrane and exported into the host cell (Hammoudi et al., PLoS Pathog. 2015 Oct 16; 1 l(10):el005211. doi:10.1371 / journal.ppat.1005211. PMID: 26473595; PMCID: PMC4608785). TgASP5 recognizes and cleaves proteins that possess Toxoplasma Export Element (TEXEL), a conserved motif consisting of ‘RRLxx’ (Coffey et al., mBio. 2018 Oct 30; 9(5):e01796-18. doi:10.1128 / mBio.01796-18. PMID: 30377279; PMCID: PMC6212819).

[0004] The Plasmodium falciparum genome encodes ten aspartyl proteases, termed ‘Plasmepsin I-X' (PfPMI-X). P. falciparum Plasmepsin I-III (PfPMI-III) are involved in26098-2hemoglobin degradation in the parasite’s food vacuole, whilst PMV is required for maturation and correct trafficking of proteins that are exported into the host red blood cell. Plasm epsin IX and X (PfPMIX and PfPMX) have become the topic of recent work as they fulfill important roles in maturation of rhoptry and micronemal proteins and can be inhibited by small drug-like molecules at several points across the complex life cycle (Nasamu et al., J. Biol Chem. 2020 June 19; 295(25):8425-8425; Pino et al., Science 2017, 358(6362): pg 522-8). A small inhibitor, 49c, was found to target PfPMX (Pino et al. Science 2017, 358, pg 522-8), but lacks features that would allow it to progress into the clinic. More recently, a new class of compounds (e.g., WM4 and WM382) that exhibits drug-like properties were identified. Intriguingly, WM382 potently inhibits both PfPMIX and PfPMX providing a harder path for parasites to gain resistance over these compounds (Favuzza et al., Cell Host Microbe. 2020 Apr 8; 27(4): 642-658. el 2; Hodder et al., Structure. 2022 Jul 7; 30(7):947-961). Additional aspartyl protease inhibitors found to be useful in inhibiting PfPMIX and / or PfPMX are disclosed in PCT Application PCT / US2024 / 033203, patent publications WO2021 / 155791, WO2023 / 107356, US2023 / 0013692, US2022 / 0331321 and US20210379020, and US Pat. No. 11,766,435, all herein incorporated by reference in the entirety. See also Favuzza et al., Cell Host Microbe. 2020 Apr 8; 27(4):642-658.el2; and de Lera Ruiz et al., ACS Med Chem Lett. 2022 Oct 12;13(11): 1745-1754.

[0005] Currently, aspartic acid proteases are prime targets for drug development: the HIV aspartic acid protease has been successfully targeted with a drug in clinical use; inhibitors that target human renin, BACE1 and gamma-secretase have been or are in clinical development. In the antimalarial drug space, P. falciparum aspartic acid proteases plasmepsin X and IX (PMX and PMIX) have been identified as potential targets since inhibitors block parasite egress and invasion of the host cell and prevent maturation of some rhoptry and micronemal proteins required for this process. Homologs of malaria parasite plasmepsin enzymes exist in other protozoa that cause infections in humans and animals including Cryptosporidium, Toxoplasma, Eimeria, and Babesia. See Shea et al., Pub Med, 2007 Aug; 8(8): 1018-34. doi: 10.1111 / j.1600-0854.2007.00589.x. Epub 2007 Jun 5; The importance of plasmepsins in many aspects of parasite biology makes them intriguing targets for antiparasitic therapy. Indeed, inhibitors of invasion and egress offer hope for a desperately needed new drug to combat these nefarious organisms. Inhibitors of these homologs such as those described herein could provide therapeutics for Cryptosporidium, Eimeria, Babesia, and / or Toxoplasma protozoan pathogens.

[0006] There are up to 1.7 billion cases of diarrhea diseases every year, which kill approximately 500,000 children under the age of five. Cryptosporidium is an enteric pathogen26098-2responsible for severe and life-threatening diarrhea and it is the second leading cause of diarrheal deaths in children globally after rotavirus. Cryptosporidium infection leads to cryptosporidiosis, which is characterized by watery diarrhea, nausea, vomiting, lethargy, and weight loss leading to the loss of eight million disability-adjusted life years (DALYs) and more than 100,000 deaths annually in young children. Cryptosporidiosis is largely self-limiting in healthy adults but can be life-threatening in people with immature and compromised immune systems such as young children and AIDS patients. Cryptosporidiosis is a particular problem in young children as the infection is prolonged and can have chronic effects including permanent growth deficits, vitamin A deficiency and increase vulnerability towards other pathogens.

[0007] Despite massive impact on human health, there is no highly effective preventative or curative treatment available for cryptosporidiosis. The only FDA-approved drug is Nitazoxanide, which has major limitations as it has limited efficacy in HIV-positive individuals and children, which are the most vulnerable groups (O’Connor et al., AIDS 25(5) 549-560, 2011) Other treatments have been tested including aminoglycoside antibiotics such as paromomycin and azithromycin, but these also have limited efficacy in immunocompromised individuals.Macrolide antibiotics, compounds from Medicines for Malaria venture (MMV) box, and lysyl-tRNA synthetase inhibitors, are all in development. Nevertheless, there has been no drug that yet clears all hurdles through the clinical trials and thus become an available treatment for cryptosporidiosis.

[0008] Close relative Plasmodium and Cryptosporidium species, Eimeria, can cause the disease coccidiosis in livestock (e.g., cattle, sheep, goats, horses, swine, and poultry (chickens and turkeys)) although most species of the genus are strictly host-specific. Coccidiosis is a significant economic burden to commercial cattle and sheep production, but the greatest losses occur within the poultry industry. Treatment of coccidiosis in poultry may vary depending on the nature and severity of the outbreak. For example, Amprolium, which blocks the parasite’s ability to uptake and multiply is a popular treatment for coccidiosis in poultry. Clopidol and folic acid antagonists (e.g., ethopabate) are other treatments for coccidiosis in poultry. However, the risk of resistance, amongst other things, are associated with these drugs.

[0009] Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, capable of infecting a broad range of vertebrates including humans. It causes toxoplasmosis, an infection that can be fatal in immunocompromised individuals and causes congenital diseases when acquired in utero. While there are drugs to treat acute stage Toxoplasma infection, there is no treatment that can clear latent stages, which can persist for the life of its host.26098-2

[0010] Babesiosis is a malaria-like parasitic infection of the red blood cells caused by apicomplexan parasites of the genus, Babesia, a close relative of Plasmodium species. The most common way to contract Babesia is a bite from an infected tick. Babesiosis is becoming an increasing health concern due to rising worldwide incidence and the risk of human-to-human transmission through blood transfusion. Treatment of human babesiosis is currently limited and consists of combinations of atovaquone and azithromycin or clindamycin and quinine. However, these compounds are associated with mild or severe adverse events and a rapid emergence of drug resistance, thereby underscoring the need for new therapeutic strategies. Renard, et al., Pathogens. 2021 Sep; 10(9): 1120.

[0011] Thus, there remains a need for new treatments and therapies for the treatment of parasitic infections caused by one or more protozoa such as Cryptosporidium, Eimeria, Babesia, and Toxoplasma.SUMMARY

[0012] The present disclosure relates to methods of treating infections caused by one or more protozoa such as Cryptosporidium, Eimeria, Babesia and Toxoplasma comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein. An embodiment is realized when the inhibitor is plasmepsin IX selected from compounds disclosed herein. Another embodiment is realized when the inhibitor is plasmepsin X selected from compounds disclosed herein. Another embodiment is realized when the inhibitor is a dual inhibitor of plasmepsin IX and X selected from compounds disclosed herein.

[0013] As shown in the Examples described herein, inhibitors of the Plasmodium aspartyl proteases plasmepsins IX and / or X (PMIX / X) are used to treat or prevent infections caused by one or more protozoa selected from Cryptosporidium, Eimeria, Babesia, and Toxoplasma. These compounds may serve as a causal prophylactic treatment by preventing one or more Cryptosporidium, Eimeria, Babesia, and Toxoplasma parasites. The compounds may also treat the disease-causing asexual blood stage parasites in either or both their asexual or sexual developmental stages.

[0014] The Aspartyl Proteases (ASPs) in related Apicomplexan parasites are druggable, however, little is known about these enzymes in Cryptosporidium parvum (C. parvum) or related protozoa. This disclosure relates to the use of compounds disclosed herein which are active against C. parvum, by binding to and inhibiting CpASP4. This disclosure further relates to use of compounds disclosed herein or a pharmaceutically acceptable salt, solvate and / or tautomer26098-2thereof, to block C. parvum growth in the treatment of cryptosporidiosis. This disclosure illustrates that CpASP4 is required for parasite survival and can proteolytically mature a rhoptry protein CpR0P4. Thus, this disclosure further relates to compounds disclosed herein, or a pharmaceutically acceptable salt, solvate and / or tautomer thereof, and their use to prevent CpASP4 activity which leads to parasite death. This disclosure further relates to use of the compounds disclosed herein or a pharmaceutically acceptable salt, solvate and / or tautomer thereof, to reduce parasite burden in immunocompromised patients.

[0015] An embodiment of this disclosure relates to methods of treating cryptosporidiosis, caused by the protozoan Cryptosporidium comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt, solvate and / or tautomer thereof. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0016] Another embodiment of this disclosure relates to methods of treating cocci diosis, caused by the protozoan Eimeria, comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt, solvate and / or tautomer thereof. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0017] Another embodiment of this disclosure relates to methods of treating toxoplasmosis, caused by the protozoan Toxoplasma, comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt, solvate and / or tautomer thereof. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0018] Another embodiment of this disclosure relates to methods of treating babesiosis, caused by the protozoan Babesia, comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt, solvate and / or tautomer thereof. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0019] The present disclosure is also directed to methods of treating parasitic infections caused by protozoans selected from Cryptosporidium, Eimeria, Babesia, and Toxoplasma comprising administering to a patient, an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, and an effective amount of one or more additional anti-antiparasitic agents. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.26098-2

[0020] The present disclosure is also directed to methods of treating cryptosporidiosis, comprising administering an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, to a patient wherein the patient does not have a parasitic infection containing homologs of Cryptosporidium plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0021] The present disclosure is also directed to methods of treating cryptosporidiosis, comprising administering a long-acting injectable formulation comprising an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, to a patient wherein the patient does not have a parasitic infection that contains homologs of Cryptosporidium plasmepsin, and wherein the patient is later exposed to a wild-type parasite containing homologs of Cryptosporidium plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0022] The present disclosure is also directed to methods of treating toxoplasmosis, comprising administering an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, to a patient wherein the patient does not have a parasitic infection containing homologs of Toxoplasma plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0023] The present disclosure is also directed to methods of treating toxoplasmosis, comprising administering a long-acting injectable formulation comprising an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, to a patient wherein the patient does not have a parasitic infection that contains homologs of Toxoplasma plasmepsin, and wherein the patient is later exposed to a wildtype parasite containing homologs of Toxoplasma plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0024] The present disclosure is also directed to methods of treating coccidiosis, comprising administering an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, to a patient wherein the patient does not have a parasitic infection containing homologs of Eimeria plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.26098-2

[0025] The present disclosure is also directed to methods of treating cocci diosis, comprising administering a long-acting injectable formulation comprising an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, to a patient wherein the patient does not have a parasitic infection that contains homologs Eimeria plasmepsin and wherein the patient is later exposed to a wild-type parasite containing homologs of Eimeria plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0026] The present disclosure is also directed to methods of treating babesiosis, comprising administering an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, to a patient wherein the patient does not have a parasitic infection containing homologs of Babesia plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0027] The present disclosure is also directed to methods of treating babesiosis, comprising administering a long-acting injectable formulation comprising an effective amount of an inhibitor of plasmepsin IX and / or X selected from compounds disclosed herein, or a pharmaceutically acceptable salt thereof, to a patient wherein the patient does not have a parasitic infection that contains homologs of Babesia plasmepsin, and wherein the patient is later exposed to a wild-type parasite containing homologs of Babesia plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0028] The present disclosure is also directed to the use of an inhibitor of plasmepsin IX and / or X selected from the compounds disclosed herein, or a pharmaceutically acceptable salt thereof for the treatment of parasitic infections selected from cryptosporidiosis, coccidiosis, babesiosis, and toxoplasmosis. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0029] The present disclosure is also directed to the use of an inhibitor of plasmepsin IX and / or X selected from the compounds disclosed herein, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cryptosporidiosis, coccidiosis, babesiosis, and / or toxoplasmosis wherein the patient does not have a parasitic infection containing homologs of Cryptosporidium, Eimeria, babesia, and / or Toxoplasma plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0030] The present disclosure is also directed the use of an inhibitor of plasmepsin IX and / or X selected from the compounds disclosed herein, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cryptosporidiosis, coccidiosis, babesiosis,26098-2and / or toxoplasmosis wherein the patient does not have a parasitic infection that contains homologs of Cryptosporidium, Eimeria, babesia, and / or Toxoplasma plasmepsin, and wherein the patient is later exposed to a wild-type parasite containing homologs of Cryptosporidium plasmepsin. An embodiment is realized when the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

[0031] An embodiment of this disclosure is directed to methods and uses described herein, wherein the inhibitor of plasmepsin IX and / or X is selected from compounds disclosed in PCT Application PCT / US2024 / 033203, patent publications WO2021 / 155791, WO2023 / 107356, US2023 / 0013692, US2022 / 0331321 and US20210379020, and US Pat. No. 11,766,435, all herein incorporated by reference in the entirety.

[0032] Also described herein are methods of inducing an immune response to a Plasmodium parasite infection linked to one or more of Cryptosporidium, Eimeria, Babesia, and Toxoplasma, comprising administering to a patient, an effective amount of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof. An embodiment is realized when the compounds disclosed herein functions via a dual inhibitor mechanism of action of plasmepsin IX and X.

[0033] Also described herein are compositions capable of inducing an immune response comprising a compound disclosed herein, or a pharmaceutically acceptable salt thereof; and an adjuvant.

[0034] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A depicts a graph of inhibition of C. parvum growth in HCT-8 cells in response to increasing concentrations WM382.

[0036] Figure IB depicts a graph of inhibition of C. parvum growth in HCT-8 cells in response to increasing concentrations WM4.

[0037] Figure 1C shows Solvent Induced Precipitation (SIP) and western blot analysis of C. parvum lysate upon treatment with WM4 and WM382 compounds probed by CpASP4 antibodies.

[0038] Figure ID are two plots of differential soluble protein abundance analysis of total C. parvum lysate as measured by mass spectrometry upon treatment with WM382 or WM4 compounds as compared to DMSO control. Non-significant (ns) proteins were plotted in gray circles, significantly stabilized proteins in hashed circles.26098-2

[0039] Figure 2 are structural Alphafold depictions of (A) CpASP4 structure, (B) CpASP4 structure (compared with PfPMIX and PfPMX, (C) a comparison of the orientation of aspartic acid residues of each enzyme as highlighted in box (D) structural and amino acid alignment of CpASP4 with PfPMX bound to compound WM4 and (E) when bound to WM382.

[0040] Figure 3 depicts bioactivity from the FRET-based protease cleavage assays of various (A) PfPMIX and PfPMX substrates, (B) PfRON3 mutant peptides.

[0041] Figure 4 depicts (A) Bioactivity results from the FRET-based protease cleavage assays showing CpASP4 activity towards itself (DABCYL-134DSESFLINKPKE145-ED ANS), CpROP4 (DABCYL-31LEVSFLNAASE42-EDANS) and CpROP6 (DABCYL-637KPKSFLEKVLE647-EDANS) peptides and (B)) Bioactivity results from the FRET-based peptide cleavage assay of CpROP4 with CpASP4 in the presence of compound WM382, WM4 and 49c.

[0042] Figure 5 depicts in vivo results of drug treated mice infected with C. parvum. Mice were infected with nanoluciferase (NLuc)-expressing C. parvum and treated by oral gavage once a day for four days with increasing concentrations of WM382 in milligrams per kilogram (mpk), as compared to Nitazoxanide or DMSO as a control in a 4xQD dosing regime; (A) Prophylactic treatment regime; whereby WT C57B6 mice were infected with C. parvum and treated with increasing concentrations of WM382 for the first 4 days; (B) Curative dosing regime where WT C57B6 mice were treated with increasing concentration WM382 at day 2 when NLuc signal is first observed in fecal material; (C) Prophylactic dosing regime in a immunocompromised IFNy" ’ model whereby mice were infected with C. parvum and treated with increasing concentrations of WM382 for the first 4 days (QD); (D) Kaplin-Meier survival curve of the mice infected in C with increasing concentrations of WM382, Nitazoxanide or DMSO; (E) Curative dosing regime of WM382 in an IFNy" ’ immunocompromised model whereby compounds were dosed with NLuc signal first observed at Day 5; and (F) Kaplin-Meier survival curve of the mice infected in E with increasing concentrations of WM382, Nitazoxanide or DMSO.

[0043] Figure 6 depicts inhibition of Growth of Toxoplasma gondi - dose curves show the (ECso) of WM382 (1.972pM) and WM4 (2.05pM) on Toxoplasma gondii (modified RH strain) tachyzoites and time of action curves of the WM382 and WM4 compounds.DETAILED DESCRIPTION

[0044] Described herein are methods of treating infections caused by one or more plasmepsin-like protozoa selected from Cryptosporidium, Eimeria, Babesia and Toxoplasma comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X of the compounds disclosed in PCT Application PCT / US2024 / 033203, patent26098-2publications WO2021 / 155791, WO2023 / 107356, US2023 / 0013692, US2022 / 0331321 and US20210379020, and US Pat. No. 11,766,435, all herein incorporated by reference in the entirety.

[0045] An embodiment is realized wherein methods of treating infections caused by one or more plasmepsin-like protozoa selected from Cryptosporidium, Eimeria, Babesia and Toxoplasma comprises administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X of Formula Id1:or a pharmaceutically acceptable salt, solvate and / or tautomer thereof, wherein:U is N or CH, wherein when U is N, X”, Y” and Z” are CH;X” is N or CH, wherein when X” is N, U, Y” and Z” are CH;Y” is N or CH, wherein when Y” is N, X”, U and Z” are CH;Z” is N or CH, wherein when Z” is N, X”, Y” and U are CH;R1is a heterocycloalkyl or Cs-Cncycloalkyl, wherein the heterocycloalkyl or Cs-Cncycloalkyl is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, oxo, COOCi-C6alkyl, phenyl, C3-C6cycloalkyl, Ci-C6alkyl, haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) and Ci-C6alkylN(R7”)(R8”);R2is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH;R3is halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, C3-C6cycloalkyl, Ci-C6alkyl, haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) or Ci-C6alkylN(R7)(R8);R4is hydrogen, halogen, CN, Ci-CealkylCN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-C6alkylOhaloCi-C6alkyl, Ci-CealkylOCi-CealkylOCi-Cealkyl, Ci-C6alkylCOOH, COOH, Ci-C6alkylSO2Ci-C6alkyl, Ci-Cealkylphenyl, phenyl, heterocycloalkyl, Ci-Cealkylheterocycloalkyl, heteroaryl, Ci-Cealkylheteroaryl, Cs-Cecycloalkyl, Ci-Cealkyl, Ci-Cealkenyl, Ci-Cealkynyl, haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”), Ci-C6alkyl(OCH2CH2)wN3, or Ci-CealkylN(R7)(R8), wherein the Ci-Cealkylphenyl, phenyl, heterocycloalkyl, Ci-26098-2Cealkylheterocycloalkyl, heteroaryl, Ci-Cealkylheteroaryl, Ci-CealkylOH, Ci-CealkylOCi-Cealkyl or Cs-Cecycloalkyl is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, oxo, COOCi-C6alkyl, Ci-C6alkyl, haloCi-C6alkyl, Ci-C6alkylOH, SO2Ci-C6alkyl, Ci-C6alkySO2Ci-C6alkyl, OSO2F, CON(R7”)(R8”), N(R7”)(R8”) and Ci-C6alkylN(R7)(R8);R5is hydrogen, halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, phenyl, Ci-CealkylCs-Cecycloalkyl, Cs-Cecycloalkyl, Ci-Csalkyl, haloCi-Cealkyl, Ci-CealkylOH, CON(R7)(R8”), N(R7)(R8”) or Ci-C6alkylN(R7)(R8”) or when taken with R6represents a C3-Cecycloalkyl or Cs-Ceheterocycloalkyl or when R5is Ci-Csalkyl and R1is a heterocycloalkyl or C3-Ci2cycloalkyl, R5optionally bonds to R1to form a macrocycle;R6is hydrogen, halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, phenyl, Ci-CealkylCs-Cecycloalkyl, Cs-Cecycloalkyl, Ci-Csalkyl, haloCi-Cealkyl, Ci-CealkylOH, CON(R7)(R8”), N(R7)(R8”) or Ci-C6alkylN(R7)(R8”) or when taken with R5represents a C3-Cecycloalkyl or Cs-Ceheterocycloalkyl;R7is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH;R8is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH;R9is hydrogen, halogen, or Ci-Cealkyl;w is 1, 2 or 3; andz is 0, 1, 2 or 3.

[0046] An embodiment is realized wherein methods of treating infections caused by one or more plasmepsin-like protozoa selected from Cryptosporidium, Eimeria, Babesia and Toxoplasma comprises administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X of Formula Idl:R2'R5'or a pharmaceutically acceptable salt, solvate and / or tautomer thereof, wherein:26098-2U is N or CH, wherein when U is N, X”, Y” and Z” are CH;X” is N or CH, wherein when X” is N, U, Y” and Z” are CH;Y” is N or CH, wherein when Y” is N, X”, U and Z” are CH;Z” is N or CH, wherein when Z” is N, X”, Y” and U are CH;R1is a Ci-6 alkyl, carbocycycle, heterocycloalkyl, Ci-6 alkylaryl, C3-6 cycloalkylaryl, -CH(C3-6 cycloalkyl)aryl, -CH(CO(O)Ci-6alkyl)aryl, heteroaryl, or Cs-Cncycloalkyl, wherein the alkyl, carbocycle, heterocycloalkyl, aryl, heteroaryl, or Cs-Cncycloalkyl is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, CN, OH, alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH, oxo, COOCi-C6alkyl, phenyl, C3-Cecycloalkyl, Ci-C6alkyl, haloCi-C6alkyl, -OhaloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) and Ci-C6alkylN(R7”)(R8”);R2is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH, or R1and R2combine with the nitrogen atom to which they are attached to form a heterocycloalkyl or heterocycle;R3is halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, C3-Cecycloalkyl, Ci-C6alkyl, haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) or Ci-C6alkylN(R7)(R8);R4is hydrogen, halogen, CN, Ci-CealkylCN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-C6alkylOhaloCi-C6alkyl, Ci-CealkylOCi-CealkylOCi-Cealkyl, Ci-C6alkylCOOH, Ci-C6alkylCOOCi-C6alkyl, COOH, Ci-C6alkylSO2Ci-C6alkyl, Ci-C6alkylphenyl, phenyl, heterocycloalkyl, Ci-Cealkylheterocycloalkyl, heteroaryl, Ci-Cealkylheteroaryl, Cs-Cecycloalkyl, Ci-Cealkyl, Ci-C6alkenyl, Ci-C6alkynyl, haloCi-Cealkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”), Ci-C6alkyl(OCH2CH2)wN3, or Ci-C6alkylN(R7”)(R8”), wherein the Ci-Cealkylphenyl, phenyl, heterocycloalkyl, Ci-Cealkylheterocycloalkyl, heteroaryl, Ci-Cealkylheteroaryl, Ci-CealkylOH, Ci-CealkylOCi-Cealkyl or Cs-Cecycloalkyl is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, CN, OH, alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH, oxo, COOCi-C6alkyl, Ci-Cealkyl, haloCi-Cealkyl, Ci-C6alkylOH, SO2Ci-C6alkyl, Ci-C6alkySO2Ci-C6alkyl, OSO2F, CON(R7”)(R8”), N(R7”)(R8”) and Ci-C6alkylN(R7”)(R8”);R5is hydrogen, halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, phenyl, CH2phenyl, Ci-CealkylCs-Cecycloalkyl, Cs-Cecycloalkyl, Ci-Csalkyl, haloCi-Cealkyl, Ci-Cealkyl haloCi-Cealkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) or Ci-C6alkylN(R7”)(R8”) or when taken with R6represents a Cs-Cecycloalkyl or Cs-Ceheterocycloalkyl or when R5is Ci-26098-2Cgalkyl and R1is a heterocycloalkyl or Cs-Cncycloalkyl, R5optionally bonds to R1to form a macrocycle, said phenyl optionally substituted with 1 to 3 groups of halogen and haloCi-Cealkyl; R6is hydrogen, halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, phenyl, CH2phenyl, Ci-CealkylCs-Cecycloalkyl, Cs-Cecycloalkyl, Ci-Csalkyl, haloCi-Cealkyl, Ci-C6alkyl haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) or Ci-C6alkylN(R7”)(R8”) or when taken with R5represents a Cs-Cecycloalkyl or Cs-Ceheterocycloalkyl;R7is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH;R8is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH;R9is hydrogen, halogen, or Ci-Cealkyl;w is 1, 2 or 3; andz is 0, 1, 2 or 3.

[0047] In certain embodiments of Formula Idor Id1, U is N or CH. In certain embodiments of Formula Id, U is N. In certain embodiments of Formula Idor Id1, U is CH. In certain embodiments of Formula Idor Id1, U is N, and X”, Y” and Z” are CH. In certain embodiments of Formula Idor Id1, X” is N or CH. In certain embodiments of Formula Idor Id1, X” is N. In certain embodiments of Formula Idor Id1, X” is CH. In certain embodiments of Formula Idor Id1, X” is N, and U, Y” and Z” are CH. In certain embodiments of Formula Idor Id1, Y” is N or CH. In certain embodiments of Formula Idor Id1, Y” is N. In certain embodiments of Formula Id, Y” is CH. In certain embodiments of Formula Idor Id1, Y” is N, and X”, U and Z” are CH. In certain embodiments of Formula Idor Id1, Z” is N or CH. In certain embodiments of Formula Idor Id1, Z” is N. In certain embodiments of Formula Idor Id1, Z” is CH. In certain embodiments of Formula Idor Id1, Z” is N, and X”, Y” and U are CH. In certain embodiments of Formula Idor Id1, each of U, Z”, Y” and Z” is CH.

[0048] In certain embodiments of Formula Idor Id1, R1is a heterocycloalkyl or C3-Cncycloalkyl, wherein the heterocycloalkyl or C3-Ci2cycloalkyl is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, CN, OH, alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH, oxo, COOCi-C6alkyl, phenyl, C3-Cecycloalkyl, spiroCs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl, Ci-CealkylOH, CON(R7)(R8”), N(R7”)(R8”) and Ci-C6alkylN(R7”)(R8”).

[0049] In certain embodiments of Formula Idor Id1, R1is26098-2wherein each occurrence of R* is independently selected from the group consisting of halogen, CN, OH, alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH, oxo, COOCi-C6alkyl, phenyl, C3-C6cycloalkyl, Ci-C6alkyl, haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) and Ci-CealkylN(R7)(R8”); and wherein q is 0, 1, 2, 3 or 4. In certain embodiments, each occurrence of R* is independently is selected independently from the group consisting of halogen, OH, Ci-Cealkyl, haloCi-Cealkyl, Cs-Cecycloalkyl, Ci-CealkylOCi-Cealkyl, Ci-CealkylOH, and phenyl. In certain embodiments, each occurrence of R* is independently selected independently from the group consisting of phenyl, bromine, fluorine, chlorine, methyl, OH, cyclopropyl, cyclobutyl, spirocyclobutyl, halogen, CN, hydroxymethyl, oxo, methoxymethyl, COOCH2CH3 and trifluoromethyl.

[0050] In certain embodiments of Formula Idor Id1, R2is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH.

[0051] In certain embodiments of Formula Idor Id1, R3is halogen, CN, OH, alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH, C3-C6cycloalkyl, Ci-C6alkyl, haloCi-C6alkyl, Ci-CealkylOH, CON(R7)(R8”), N(R7)(R8”) or Ci-C6alkylN(R7)(R8). In certain embodiments, R3is halogen. Suitable halogens include, but are not limited to, fluorine, chlorine, bromine, or iodine. In certain embodiments of Formula Idor Id1, R3is CN. In certain embodiments of Formula Idor Id1, R3is OH.

[0052] In certain embodiments of Formula Idor Id1, z is 0, meaning the ring containing U, X”, Y” and Z” is unsubstituted. In certain embodiments of Formula Idor Id1, z is 1, meaning a single substitutable hydrogen on the ring containing U, X”, Y” and Z” is replaced by R3. In certain embodiments of Formula Idor Id1, z is 2, meaning two substitutable hydrogens on the ring containing U, X”, Y” and Z” are replaced by R3. In certain embodiments of Formula Idor Id1, z is 3, meaning three substitutable hydrogens on the ring containing U, X”, Y” and Z” are replaced by R3.

[0053] In certain embodiments of Formula Idor Id1, R4is hydrogen, fluorine, methyl, ethyl, propyl, methoxymethyl, methoxypropyl, phenyl, pyridine, n-butyl, isobutyl, methoxyethyl, bicyclopentanyl, butenyl, butynyl, hydroxypropyl, cyanopropyl, hexynyl, methylbutadienyl, thiazole, pentynyl, CH2CH2OCH3, CH2CH2OCH2CH3, CH2OCH2CH2OCH3, CH2CH2CN, SO2CH3, CH2SO2CH3, CH2CH2SO2CH3, pyrimidine,26098-2

[0054] In certain embodiments of Formula Idor Id1, when R5is C?alkyl, and R1is a heterocycloalkyl or Cs-Cncycloalkyl, R5optionally bonds to R1to form a macrocycle, as shown in Formula IId

[0055] In certain embodiments of Formula Idor Id1, when R5is C?alkyl, and R1is a heterocycloalkyl or Cs-Cncycloalkyl, R5optionally bonds to R1to form a macrocycle, as shown in Formula IId2

[0056] In certain embodiments of Formula Id, Idl, IId, and IId2, R6is hydrogen, halogen, CN, OH, alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH, phenyl, Ci-C6alkylC3-Cecycloalkyl, Cs-Cecycloalkyl, Ci-CealkylCs-Cecycloalkyl, Ci-Csalkyl, haloCi-Cealkyl, Ci-CealkylOH, CON(R7)(R8”), N(R7)(R8”) or Ci-C6alkylN(R7)(R8”). In certain embodiments, R6and R5are independently selected from optionally substituted phenyl, CH2cycloalkyl,26098-2cycloalkyl, methyl, ethyl, propyl, isopropyl, butyl, and CH2CF3. Suitable cycloalkyls are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. In certain embodiments, R6is hydrogen. In certain embodiments, R6is halogen. Suitable halogens include, but are not limited to, fluorine, chlorine, bromine, or iodine. In certain embodiments, R6is CN. In certain embodiments, R6is OH.

[0057] In certain embodiments of Formula Idor Id1, R7is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH; R8is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH; and R9is hydrogen, halogen, or Ci-Cealkyl.

[0058] In certain embodiments of Formula Id, Idl, IId, and IId2, z is 1. In certain embodiments of Formula Id, z is 2. In certain embodiments of Formula Id, Idl, IId, and IId2, z is 3.

[0059] In certain embodiments of Formula Id, Idl, IId, and IId2, the R1substituent is CH(CH3)3CO(O)Ci-6alkyl,wherein Rxis hydrogen, CO(O)Ci-6 alkyl, or Ci-6 alkyl, and the R1substituents are unsubstituted or substituted with 1 to 5 substituents selected from the group consisting of halogen, COOH, COOCH3, OH, alkoxy, Ci-Cealkyl, haloCi-Cealkyl, OhaloCi-Cealkyl, Cs-Cecycloalkyl, Ci-CealkylOCi-Cealkyl, Ci-CealkylOH, and phenyl.

[0060] In each of the various embodiments of the disclosure, the compounds described herein, including those in each of Formula Id, Idl, IId, and IId2, and the various embodiments thereof, may exit in different forms of the compounds such as, for example, any solvates, hydrates, stereoisomers, and tautomers of said compounds and of any pharmaceutically acceptable salts thereof.

[0061] In certain embodiments, methods for treating infections caused by one or more protozoa such as Cryptosporidium, Eimeria, and / or Toxoplasma comprises administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X selected from List A.List A:26098-2^ NH J I A xx< y~TI C ZZtXb X ' Vs YA p- ' -A- / oictX^ X? Crc ZCA ^-NH AXAVA X ''Mz / TIT 'BrXX YnKr~ Z l( JOAny O 4xyx Lxn XX Jxxx X; "ox A ^°Zo 0 x yNH NH HNXNZYXF. A xZoFA °¥ □¥¥ A^A O h < XNH O^ NH O’X'NH ANH NH NH NHxb?aFHNXN00 F HNAN^< VF0^ y AXOFO^ NH O^ NHO'^ N 'X ~ l ~^ <¥NH " AO ""■‘YQNH NH NH_A F_ HN^ N^V^V HN^ N AAfFrG x t J HN-^NAXX \_ / Ss / ^o A^ yp || O^NH CANH > A» ¥QZNAx< Q LAAXo26098-2NH NH HN NFNH o^f^ NH o\^HN^ N'^r^Y'FU 1 JJ 1HN^ N'^Sj'^ HN^N^'sjXA S^o V'''^A-AO AAQ-^-N'XUA O^N ^A O^ N ATN F - / / \\ \=JNH NH\ ozHN N^ ^A^AA AL~ / A O^ N ^\N= / N^A ACl=3A A^. H^IAY^ / ^O V A6fA■TA •TA-A’" A-HA^A AHHN^'AS9HN^N A,^, L / ^AA A yA AAA---,, / =A ° ACFJvAAF^HNAAX°’9AA A==AA V A--Y?\ A / A~NA PJ A W oNYv V ^J26098-2A<? Yx w -; t w-yax AW<1HCJ0 9yyX'NH^ ^ uNH NHuHN^N^Y |I / \Ac yx°V HN^N'Xr'XATXNH\J\ C^NH HN' x)YpXu uooxo / x^NH O xy NH o Y HN^NXX|X5X 11 1 _ HN^N^Yp^l / “AX / X-Q xy / " TAXA-Q ' O^NHXCY^NH oo Oo<1 '[AY. Y °FO O ° jT oo C JO26098-2NHY YkJ YYY Q Y °YHYYQ'b<3 HNYN '" JI'A Y Y Q V VYo M ^kuuHu O ° Y4 Y U YYO0»QH„Q nr b'' "''v^'"..H°z ^kAo U kbX^^XUQ kJ O^NH JI J. JI Y Y XS^Ao V " u i I IYYHL J YZx^^1} £ C JONH HNAN^X / ^ YY^Q ^Y j- CYNH sC<u u Y ^ Y^oVhuooNHHtYi'X'^yFHY Y Y(Y'NH U " u T A U U YA U U YYJ \m'^'rrb--«strAN»b-'L yk< UHU XA U u YAYT U / 70JU " V26098-2NH HN^N-yAHl \-pJU " u A U u yXy ■ u / —HkJo NHNH Y / I JC, / \ OH JLK 1X OH <1 HN'^N'^Y^l E HN^N^Xj^X] =yX y - u / _' 0^N* v\ OHXjNH NHJ-L, / \ OHHN^N^Xj^X ■ HN^NXX'Y u ^ ° x L' CANH*> AS kJY^Oyx VHuyXuHu yAIX" Uf^0Y^O A ^rf^A N*> As1 ^ ^ 1 1 1 XjxAo MHUU I I JhO A AVA U u yi\ i> " uNH |11 1 _ HN^N^^sp^ 0AA\ 0 YO^NH 9’ ■Tf'"'IT'^'l ’ '1'1 1 oX< V “ u XOu " u oo26098-2NH |0 YHN^N^X^i JH0^Y XAC V'^ > X55X^‘ N*^V A O^NH YAoy^A, Uhy HO^ / X^ rn^O*^X ^-O NH NH Y HN'''X'^rf''X"F1 1 / KX HNZxN / \p^ Y O^NH O^NHH0^X\^ xo \_FjXO'^S^NH |NHX _ _ F HNHO HN^N^Yf ^X -^N-Xf^, YJ^AQ O^NHX y u O^NH HOK A ^X^-O ooNH |NH YN x 1 H('ANX% H ^N^^X^lX xA^o U 7s* — ^oHO^NH V u ' CXNH HQ^XS^J. o ^XoxX v_TjNH HN Y X X / \HN^N^^X ^1jtca ’ / XA0U U O^NH ' CANHH0YQHOKX^--\V_TJJjX MHU xjXXX k)26098-2OHJjX V u YA X^0 / K Afi-k^A^NrL A^XA0VHU y\ M " u JAAQH\Y / TXV " y ”\X YY x^xx XXsY X^ XYU _ *QTA / pjjHU X ^-AAQ <jX ^AA^o AAm0X r«\ CAXH wr-'hl'— '<’^< '*1— ^=9, \ CX-NH A U “ uHOk^A— oo vXjNH11 F A?r »FtF^ HN-^N-^AY ^AXAOAJA NAA A. UHU \ - O^NH Y^ v uc^oHOX0NH HNAA / YFX, HN^N^YXNAA ■[^'''r^ 'J’’'l4^''.! O^NHX U U XX u u " Al HC’K / A-'Xv_Fj26098-2NH < 0, HN^'N'9<o%0LNrk^An? K!'F'^-'N;^-F|*'OOF', < Cv JHXJ YAO V u SXA U ANH HN'^N-'^Y'^ / ^Ao U / XAc U' O^NH Y ° X^O X ^ SxAo uj / fXOX'A / lhuJT XJ)NHli.. F S, HN-AYV AAVAV ’r^\" X)o ° = oTHO^Z\-A V_TJ0 0ff KIQAH’^O, HAAVI'. A')MA.. VHX)<yy-"? °" Vv f AW O XCko U “ X)U UF^LFSixAoUHU AxAo V " U VHu / XA, 17 " U26098-2 / >yCy - X) y ^ ^ XA, V U XX V “ u >^A yNH < NH Z 1 1 ^ u 1NHCHM“rxx^o y^OZ Z ~A\^A U U ' O^NH yx MHy ' CZNH ^0 UMONHHiZN^yy^AF1^A U YFT^° I Cl H N -^- N y AZZZZ)° n'yj0J Z 1VAU Uo o1 Z L ^ Z AA X U U ZOZZ)wJ 1 yzzy ““? °"°X\,yhyy \XA0V U oNHH \3 A A b TAJJ u A U UJ AV " U A U u A AA U UJ AMOA ljT A U u^pjCiHA A U "1 NH\1^^ 1 1 1 A N* YA1 'p.u “ W U^A V U MAOUHUNZZ~~1AA x A U UA0^ 0^NH NH X AHlXlXy HN-^'Nxyyyk, HNxY^AxX'^Nxkr^X1X AO~ —A I JHk AAJ U y^- — y AD I JHO AA x y y X y0^NH HN" XxX--''X x X a y NH x0r ^° y C^Y) AA U y x j, XA XJ - u;HHN^N-^XAXA^ / XAcV x X a y HNXAK w — — WAA, ' O^N y " u \ ^xxx iyx H ooHO-V x)NH HN^XtfX, N AX X L J Y V YJ, iA\AAyA HN^N^Sj^Y^0yA u u AA. U " U x^A aAAoIJAox $y A A U U 'o'kB'<O ^AAQ1X Z X HNX / V- Y / ■XY / y U YJ^X^nhA °HVk YA V X A )A Y'O'J'YOYh° r> HOo A A A A A X Xx A ' / ^A' '’Y=^'Jy, UhU ^ IA, UHUtII 1' AAAkkXA0U U yX MHuN^N | ~° NH y HN-YyY AA U U p Y U ^jkj ijYr^0H% V N^NA HNX^X— Y.IJ " U ^^yXXHO- EX JOX.4 YXX NH HN'^XX- A A X XX, HN^NY^ZO AH N N Y. X AW26098-2yX )SA U SLAO U y U UC J Y^lo A A^^ X ^ / _ A — — X / xX X*^ MAV ■ UQr ^o XC0 y xyF? S 4> U U \ < AX0 ^ X1X nrF? S X J< ° / - O^N-^f A ACC x w \=~z ^Al X X AX U u X1|X0X Y Y )26098-2X^ / HMV^OHNXN-AAAXXN< L -Xu I-IN^N^AA^ AUQ UHU Y / AAAOO / IA, U u pjj u ^ U UQfFXX QrFXX NASAA HUAIA-AA'0AA HNANAY^^AN< XAX1XAXTHU Y^AO U UJ J HN-^'N-AAA J1A, HN^N-AA / ^NAA '^^AAQ A'A HN^N^k^X^N^kX^ YX u uA >p>0 VHu uX^NH°z' 1 1 ^ J A X HN / XN / XZX k k ^^xko po HLANAAAO '>p XT U '^X^AAQA==ZOA'-AAHZJ uJ^ Y HN*^Y" A HN^N-^V^Ij ^NHY v p kk CX w X Y)kJ26098-2NHV °H°TX^xTO £XA^ yi J up r1X HN^N-^ JA^ X 1 X 1 L JI 1 1 ^ 1 L JJ '^Ayr""^X^O XX X XuJ X'Q J, HN^N-X><,V^N'TX> J X X^Q MA U x XAOUHU XCO x°XA^ U °X^I I NH J X x y X - o / =\ X X X U''-jXp X x x I U V(_Y Y j ° x IZNH NH^iccvHN-^-N-XfXA x YCX NH^X O^NH NHXX U UCl xNHKHI'-YN^VAJI JI NH X J1X VO^NHY^° T rx YY j oXXXHVa / 26098-226098-2or a pharmaceutically acceptable salt thereof, wherein * indicates the location of a chiral center.

[0062] In other certain embodiments, methods for treating infections caused by one or more protozoa such as Cryptosporidium, Eimeria, and / or Toxoplasma comprises administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X of selected from:or a pharmaceutically acceptable salt thereof.26098-2Definitions and Abbreviations:

[0063] The terms used herein have their ordinary meaning and the meaning of such terms is independent at each occurrence thereof. That notwithstanding and except where stated otherwise, the following definitions apply throughout the specification and claims. Chemical names, common names and chemical structures may be used interchangeably to describe that same structure. These definitions apply regardless of whether a term is used by itself or in combination with other terms, unless otherwise indicated. Hence the definition of “alkyl” applies to “alkyl” as well as the “alkyl” portion of “hydroxyalkyl”, “haloalkyl”, arylalkyl-, alkylaryl-, “alkoxy” etc.

[0064] It shall be understood that, in the various embodiments of the disclosure described herein, any variable not explicitly defined in the context of the embodiment is as defined in Formula (T).

[0065] In the various embodiments described herein, each variable is selected independently of the others unless otherwise indicated.

[0066] “Drug resistant” means, in connection with a Plasmodium parasite strain, a Plasmodium species which is no longer susceptible to at least one previously effective drug; which has developed the ability to withstand attack by at least one previously effective drug. A drug resistant strain may relay that ability to withstand to its progeny. Said resistance may be due to random genetic mutations in the bacterial cell that alters its sensitivity to a single drug or to different drugs.

[0067] " Patient" includes both human and non-human animals. Non-human animals include those research animals and companion animals such as mice, rats, primates, monkeys, chimpanzees, great apes, dogs, and house cats.

[0068] " Pharmaceutical composition" (or “pharmaceutically acceptable composition”) means a composition suitable for administration to a patient. Such compositions may contain the neat compound (or compounds) of the disclosure or mixtures thereof, or salts, solvates, prodrugs, isomers, or tautomers thereof, and one or more pharmaceutically acceptable carriers or diluents. The term “pharmaceutical composition” is also intended to encompass both the bulk composition and individual dosage units comprised of one or more (e.g., two) pharmaceutically active agents such as, for example, a compound of the present disclosure and an additional agent selected from the lists of the additional agents described herein, along with any pharmaceutically inactive excipients. The bulk composition and each individual dosage unit can contain fixed amounts of the afore-said "more than one pharmaceutically active agents". The bulk composition is material that has not yet been formed into individual dosage units. An illustrative dosage unit is an oral dosage unit such as tablets, pills and the like. Similarly, the herein-described method of treating a26098-2patient by administering a pharmaceutical composition of the present disclosure is also intended to encompass the administration of the afore-said bulk composition and individual dosage units.

[0069] “Halogen” and "halo" mean fluorine, chlorine, bromine, or iodine. Preferred are fluorine, chlorine and bromine.

[0070] " Alkyl" means an aliphatic hydrocarbon group which may be straight or branched and comprising about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups contain about 1 to about 12 carbon atoms in the chain. More preferred alkyl groups contain about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkyl chain. " Lower alkyl" means a group having about 1 to about 6 carbon atoms in the chain which may be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl and t-butyl.

[0071] “Haloalkyl” means an alkyl as defined above wherein one or more hydrogen atoms on the alkyl is replaced by a halo group defined above.

[0072] " Aryl" means an aromatic monocyclic or multicyclic ring system comprising about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. Non-limiting examples of suitable aryl groups include phenyl and naphthyl. " Monocyclic aryl" means phenyl.

[0073] " Cycloalkyl" means a non-aromatic mono- or multicyclic ring system comprising about 3 to about 12 carbon atoms, preferably about 3 to about 10 carbon atoms. Preferred cycloalkyl rings contain about 5 to about 10 ring atoms. The cycloalkyl can be optionally substituted with one or more substituents, which may be the same or different, as described herein. Monocyclic cycloalkyl refers to monocyclic versions of the cycloalkyl moieties described herein. Nonlimiting examples of suitable monocyclic cycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Multicyclic cycloalkyls refers to multicyclic, including bicyclic, rings that include a non-aromatic ring. Non-limiting examples of suitable multicyclic cycloalkyls include 1 -decalinyl, norbornyl, adamantyl and the like. In certain embodiments, a non-aromatic ring is fused to an aromatic ring. Further non-limiting examples of cycloalkyl include the following:“Heterocycloalkyl” (or "heterocyclyl") means a non-aromatic, saturated or partially saturated monocyclic or multicyclic ring system comprising about 3 to about 10 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination.26098-2There are no adjacent oxygen and / or sulfur atoms present in the ring system. Preferred heterocyclyls contain about 5 to about 6 ring atoms. The prefix aza, oxa or thia before the heterocyclyl root name means that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom. Any -NH in a heterocyclyl ring may exist protected such as, for example, as an -N(Boc), -N(CBz), -N(Tos) group and the like; such protections are also considered part of this disclosure. The heterocyclyl can be optionally substituted by one or more substituents, which may be the same or different, as described herein. The nitrogen or sulfur atom of the heterocyclyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S, S-di oxide. Thus, the term “oxide,” when it appears in a definition of a variable in a general structure described herein, refers to the corresponding N-oxide, S-oxide, or S, S-di oxide. “Heterocyclyl” also includes rings wherein =0 replaces two available hydrogens on the same carbon atom (i.e., heterocyclyl includes rings having a carbonyl group in the ring). Such =0 groups may be referred to herein as HN^^NI / n“oxo.” An example of such a moiety is pyrrolidinone (or pyrrolidone):. As used herein, the term “monocyclic heterocycloalkyl” refers monocyclic versions of the heterocycloalkyl moieties described herein and include a 4- to 7-membered monocyclic heterocycloalkyl groups comprising from 1 to 4 ring heteroatoms, said ring heteroatoms being independently selected from the group consisting of N, N-oxide, O, S, S-oxide, S(0), and S(0)2. The point of attachment to the parent moiety is to any available ring carbon or ring heteroatom. Non-limiting examples of monocyclic heterocycloalkyl groups include piperidyl, oxetanyl, pyrrolyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, beta lactam, gamma lactam, delta lactam, beta lactone, gamma lactone, delta lactone, and pyrrolidinone, and oxides thereof. A non-limiting example ofa monocyclic heterocycloalkyl group include the moiety:- °. Non-limiting examples of multicyclic heterocycloalkyl groups include, bicyclic heterocycloalkyl groups. Specific examples include, but are not limited to,

[0074] " Alkoxy" means an alkyl-O- group in which the alkyl group is as previously described. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, / / -propoxy, isopropoxy and / / -butoxy. The bond to the parent moiety is through the ether oxygen.26098-2

[0075] The term “substituted” means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom’s normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. By “stable compound’ or “stable structure” is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0076] The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties.

[0077] When a variable appears more than once in a group, e.g., R8in -N(R8)2, or a variable appears more than once in a structure presented herein, the variables can be the same or different.

[0078] A solid line, as a bond generally indicates a mixture of, or either of, the possible isomers, e.g., containing (R)- and (S)-stereochemistry. For example:

[0079] The wavy line, as used herein shown crossing a line representing a chemical bond, indicates a point of attachment to the rest of the compound.

[0080] Lines drawn into the ring systems, such as, for exampleindicates that the indicated line (bond) may be attached to any of the substitutable ring atoms.

[0081] “ Oxo” is defined as an oxygen atom that is double bonded to a ring carbon in a cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, or another ring described herein,

[0082] In this specification, where there are multiple oxygen and / or sulfur atoms in a ring system, there cannot be any adjacent oxygen and / or sulfur present in said ring system.

[0083] As well known in the art, a bond drawn from a particular atom wherein no moiety is depicted at the terminal end of the bond indicates a methyl group bound through that bond to the atom, unless stated otherwise. For example:26098-2H3CCH,represents

[0084] In another embodiment, the compounds useful in the methods of the disclosure, and / or compositions comprising them useful in said methods, are present in isolated and / or purified form. The term "purified", "in purified form" or “in isolated and purified form” for a compound refers to the physical state of said compound after being isolated from a synthetic process (e.g., from a reaction mixture), or natural source or combination thereof. Thus, the term "purified", "in purified form" or “in isolated and purified form” for a compound refers to thephysical state of said compound (or a tautomer or stereoisomer thereof, or pharmaceutically acceptable salt or solvate of said compound, said stereoisomer, or said tautomer) after being obtained from a purification process or processes described herein or well known to the skilled artisan (e.g., chromatography, recrystallization and the like), in sufficient purity to be suitable for in vivo or medicinal use and / or characterizable by standard analytical techniques described herein or well known to the skilled artisan.

[0085] It shall be understood that any carbon as well as heteroatom with unsatisfied valences in the text, schemes, examples and tables herein is assumed to have the sufficient number of hydrogen atom(s) to satisfy the valences.

[0086] When a functional group in a compound is termed “protected”, this means that the group is in modified form to preclude undesired side reactions at the protected site when the compound is subjected to a reaction. Suitable protecting groups will be recognized by those with ordinary skill in the art as well as by reference to standard textbooks such as, for example, T. W. Greene et al. Protective Groups in Organic Synthesis (1991), Wiley, New York.

[0087] Another embodiment provides prodrugs and / or solvates of the compounds of the disclosure. A discussion of prodrugs is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987) 14 of the A. C. S. Symposium Series, and in Bioreversible Carriers in Drug Design, (1987) Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press. The term “prodrug” means a compound (e.g., a drug precursor) that is transformed in vivo to yield a compound of the disclosure or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (e.g., by metabolic or chemical processes), such as, for example, through hydrolysis in blood. A discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A. C. S. Symposium Series, and in Bioreversible Carriers in26098-2Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergam on Press, 1987.

[0088] For example, if a compound useful in the methods of the disclosure or a pharmaceutically acceptable salt thereof, contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as, for example, (Ci-C8)alkyl, (C2-Ci2)alkanoyloxymethyl, l-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1 -methyl- l-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxy carbonyloxymethyl having from 3 to 6 carbon atoms, 1 -(alkoxy carbonyloxy)ethyl having from 4 to 7 carbon atoms, 1 -methyl- 1 -(alkoxy carbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3 -phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N, N-(Ci-C2)alkylamino(C2-C3)alkyl((such as P-dimethylaminoethyl), carbamoyl-(Ci-C2)alkyl, N, N-di (Ci-C2)alkylcarbamoyl-(C1-C2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-C3)alkyl, and the like.

[0089] Similarly, if a compound used in the methods of the disclosure contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as, for example, (Ci-C6)alkanoyloxymethyl, l-((Ci-C6)alkanoyloxy)ethyl, l-methyl-l-((Ci-C6)alkanoyloxy)ethyl, (Ci-C6)alkoxycarbonyloxymethyl, N-(Ci-C6)alkoxycarbonylaminomethyl, succinoyl, (Ci-C6)alkanoyl, -amino(Ci-C4)alkanyl, arylacyl and -aminoacyl, or -aminoacyl— aminoacyl, where each -aminoacyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH)2, -P(O)(O(Ci-Ce)alkyl)2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate), and the like.

[0090] If a compound used in the methods of the disclosure incorporates an amine functional group, a prodrug can be formed by the replacement of a hydrogen atom in the amine group with a group such as, for example, R-carbonyl, RO-carbonyl, NRR’ -carbonyl where R and R’ are each independently (Ci-Cio)alkyl, (C3-C7) cycloalkyl, benzyl, or R-carbonyl is a natural -aminoacyl or natural -aminoacyl, -C(OH)C(O)OY1wherein Y1is H, (Ci-Ce)alkyl or benzyl, -C(OY2)Y3wherein Y2is (C1-C4) alkyl and Y3is (Ci-Ce)alkyl, carboxy (Ci-Ce)alkyl, amino(Ci-C4)alkyl or mono-N- or di-N, N-(Ci-C6)alkylaminoalkyl, -C(Y4)Y5wherein Y4is H or methyl and Y5is mono-N- or di-N, N-(Ci-C6)alkylamino morpholino, piperidin-l-yl or pyrrolidin-l-yl, and the like.

[0091] One or more compounds used in the methods of the disclosure may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and26098-2the like, and it is intended that the disclosure embrace both solvated and unsolvated forms." Solvate" means a physical association of a compound of the disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. " Solvate" encompasses both solution-phase and isolatable solvates. Nonlimiting examples of suitable solvates include ethanolates, methanolates, and the like. " Hydrate" is a solvate wherein the solvent molecule is H2O.

[0092] One or more compounds used in the methods of the disclosure may optionally be converted to a solvate. Preparation of solvates is generally known. Thus, for example M. Caira et al. J. Pharmaceutical Sci., 1993, 3, 601-611, describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by E. C. van Tonder et al. AAPS PharmSciTech., 5(1), article 12 (2004); and A. L. Bingham et al. Chem. Commun., 603-604 (2001). Atypical, non-limiting, process involves dissolving the inventive compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example I. R. spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

[0093] " Effective amount" or "therapeutically effective amount" is meant to describe an amount of compound or a composition used in the methods of the present disclosure effective in inhibiting the above-noted diseases or enzyme activity and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect.

[0094] Another embodiment provides pharmaceutically acceptable salts of the compounds to be used in the methods of the disclosure. Thus, reference to a compound used in the methods of the disclosure herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic salts formed with inorganic and / or organic acids, as well as basic salts formed with inorganic and / or organic bases. In addition, when a compound of the disclosure contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein.

[0095] Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful. Salts of the compounds used in the methods of the disclosure may be formed, for example, by reacting a compound of the disclosure with an26098-2amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

[0096] Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates,) and the like.Additionally, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al. Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al. Journal of Pharmaceutical Sciences (1977)66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Anderson et al. The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D. C. on their website). These disclosures are incorporated herein by reference thereto.

[0097] Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quartemized with agents such as lower alkyl halides (e.g., methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g., decyl, lauryl, and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0098] All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the disclosure and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the disclosure.

[0099] Another embodiment provides pharmaceutically acceptable esters of the compounds used in the methods of the disclosure. Such esters include the following groups: (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, acetyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, Ci-4alkyl, or Ci-4alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkyl sulfonyl (for example, methanesulfonyl); (3) amino acid26098-2esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or triphosphate esters. The phosphate esters may be further esterified by, for example, a C1-20 alcohol or reactive derivative thereof, or by a 2,3 -di (Ce-24)acyl glycerol.

[0100] As mentioned herein, another embodiment provides tautomers of the compounds of the disclosure to be used in the methods herein, and salts, solvates, esters and prodrugs of said tautomers. It shall be understood that all tautomeric forms of such compounds are within the scope of the compounds used in the methods of the disclosure. For example, all keto-enol and imine-enamine forms of the compounds, when present, are included in the disclosure.

[0101] The compounds used in the methods of the disclosure may contain asymmetric or chiral centers, and, therefore, exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds used in the methods of the disclosure as well as mixtures thereof, including racemic mixtures, form part of the present disclosure. In addition, the present disclosure embraces use of all geometric and positional isomers. For example, if a compound used in the methods of the disclosure incorporates a double bond or a fused ring, both the cisand trans-forms, as well as mixtures, are embraced within the scope of the disclosure.

[0102] Another embodiment provides for diastereomeric mixtures and individual enantiomers of the compounds used in the methods of the disclosure. Diastereomeric mixtures can be separated into their individual diastereomers based on their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher’s acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. Also, some of the compounds used in the methods of the disclosure may be atropisomers (e.g., substituted biaryls) and are considered as part of this disclosure. Enantiomers can also be separated by use of chiral HPLC column.

[0103] All stereoisomers (for example, geometric isomers, optical isomers and the like) of the compounds used in the methods of the disclosure (including those of the salts, solvates, esters and prodrugs of the compounds as well as the salts, solvates and esters of the prodrugs), such as those which may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotameric forms, atropisomers, and diastereomeric forms, are contemplated as embodiments within the scope of this disclosure, as are positional isomers (such as, for example, 4-pyridyl and 3-pyridyl). (For example, if a compound of the disclosure incorporates a double bond or a fused ring, both the26098-2cis- and trans-forms, as well as mixtures, are embraced within the scope of the disclosure. Also, for example, all keto-enol and imine-enamine forms of the compounds are included in the methods of the disclosure).

[0104] Individual stereoisomers of the compounds of the disclosure may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The chiral centers of the present disclosure can have the S or R configuration as defined by the IUPAC 1974 Recommendations. The use of the terms "salt", "solvate", “ester”, "prodrug" and the like, is intended to equally apply to the salt, solvate, ester and prodrug of enantiomers, stereoisomers, rotamers, tautomers, positional isomers, racemates or prodrugs of the inventive compounds.

[0105] Another embodiment provides isotopically-labelled compounds to be used in the methods the disclosure. Such compounds are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,170,31P,32P,35S,18F, and36C1, respectively.

[0106] Certain isotopically-labelled compounds of the disclosure (e.g., those labeled with3H and14C) are useful in compound and / or substrate tissue distribution assays. Tritiated (i.e.,3H) and carbon-14 (i.e.,14C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e.,2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labelled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or in the Examples hereinbelow, by substituting an appropriate isotopically labelled reagent for a non-isotopically labelled reagent.

[0107] In the compounds used in the methods of the disclosure, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present disclosure is meant to include all suitable isotopic variations of the compounds of the disclosure. For example, different isotopic forms of hydrogen (H) include protium (1H) and deuterium (2H). The presence of deuterium in the compounds of the disclosure is indicated by " D". Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic26098-2advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds of the disclosure can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the schemes and examples herein using appropriate isotopically-enriched reagents and / or intermediates.

[0108] Polymorphic forms of the compounds used in the methods of the disclosure, and of the salts, solvates, esters and prodrugs of the compounds of the disclosure, are intended to be included in the present disclosure.

[0109] The methods of the present disclosure are useful for treating cr in that they inhibit the onset, growth, or progression of the condition, ameliorate the symptoms of the condition, cause regression of the condition, cure the condition, or otherwise improve the general well-being of a subject afflicted with, or at risk of, contracting the condition. Thus, in accordance with the presently disclosed subject matter, the terms “treat”, “treating”, and grammatical variations thereof, as well as the phrase “method of treating”, are meant to encompass any desired therapeutic intervention, including but not limited to a method for treating an existing infection in a subject of infection, such as in a subject that has been exposed to a parasite as disclosed herein.

[0110] Embodiments of the disclosure also include one or more of the compounds of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt thereof (i) for use in, (ii) for use as a medicament or composition for, or (iii) for use in the preparation of a medicament for: (a) therapy (e.g., of the human body); (b) medicine; (c) inhibition of parasite / Plasmodium growth, (d) treatment or prophylaxis of infection by Plasmodium species; (e) reduction of the progression, onset or severity of pathological symptoms associated with Plasmodium infection and / or reduction of the likelihood of severe Plasmodium infection or, (f) treatment, prophylaxis of, or delay in the onset, severity, or progression of Plasmodium -associated disease(s), including, but not limited to: malaria.

[0111] Accordingly, another embodiment provides methods for the treatment of parasitic infections selected from cryptosporidiosis, coccidiosis, babesiosis, and toxoplasmosis or for the treatment of infection derived from Cryptosporidum, Eimeria, Bebesia, and / or Toxoplasma, comprising administration of combinations comprising an amount of at least one compound of Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof, and an effective amount of one or more additional agents described below. In certain embodiments, described herein are methods for the treatment of treatment of parasitic infections selected from cryptosporidiosis, coccidiosis, babesiosis, and toxoplasmosis or for the treatment of infection26098-2derived from Cryptosporidum, Eimeria, Bebesia, and / or Toxoplasma, comprising administration of combinations comprising an amount of at least one compound of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof, and an effective amount of one or more additional anti-malarial agents. In certain embodiments, described herein are methods for the treatment of parasitic infections selected from cryptosporidiosis, coccidiosis, babesiosis, and toxoplasmosis by inhibition of plasmepsin X, IX and at least one other mechanism, comprising administration of combinations comprising an amount of at least one compound of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof, and an effective amount of one or more additional anti-malarial agents, wherein the additional anti-malarial agents act through a different mechanism than inhibiting plasmepsin IX or plasmepsin X. The pharmacological properties of the compounds of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt thereof may be confirmed by several pharmacological assays. Certain assays are exemplified herein.Dosage and Administration

[0112] Another embodiment provides suitable dosages and dosage forms of the compounds used in the methods of the disclosure. Suitable doses for administering compounds used in the methods of the disclosure to patients may readily be determined by those skilled in the art, e.g., by an attending physician, pharmacist, or other skilled worker, and may vary according to patient health, age, weight, frequency of administration, use with other active ingredients, and / or indication for which the compounds are administered. Doses may range from about 0.001 to 500 mg / kg of body weight / day of the compound of the disclosure. In one embodiment, the dosage is from about 0.01 to about 25 mg / kg of body weight / day of a compound of the disclosure, or a pharmaceutically acceptable salt or solvate of said compound. In another embodiment, the quantity of active compound in a unit dose of preparation may be varied or adjusted from about 1 mg to about 100 mg, in specific embodiments from about 1 mg to about 50 mg, in specific embodiments from about 1 mg to about 25 mg, according to the particular application. In another embodiment, a typical recommended daily dosage regimen for oral administration can range from about 1 mg / day to about 500 mg / day, in specific embodiments 1 mg / day to 200 mg / day, in two to four divided doses.

[0113] As discussed above, the amount and frequency of administration of the compounds of the disclosure and / or the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated.26098-2

[0114] Liquid form preparations include solutions, suspensions and emulsions. As an example, may be mentioned water or water-propylene glycol solutions for parenteral injection or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.

[0115] Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g., nitrogen.

[0116] Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

[0117] Another embodiment provides for use of compositions for the treatment of parasitic infections selected from cryptosporidiosis, coccidiosis, babesiosis, and toxoplasmosis comprising a compound of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt thereof formulated for transdermal delivery. The transdermal compositions can take the form of creams, lotions, aerosols and / or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

[0118] Another embodiment provides for use of compositions for the treatment of parasitic infections selected from cryptosporidiosis, coccidiosis, babesiosis, and toxoplasmosis comprising a compound of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt thereof formulated for subcutaneous delivery. Another embodiment provides for use of compositions suitable for oral delivery. In some embodiments, it may be advantageous for the pharmaceutical preparation comprising one or more compounds of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt thereof to be prepared in a unit dosage form. In such forms, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose. Each of the foregoing alternatives is considered as included in the various embodiments of the disclosure.

[0119] When used in combination with one or more additional therapeutic agents ("combination therapy"), the compounds used in the methods of this disclosure, i.e., the compounds of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt thereof, may be administered together or sequentially. When administered sequentially, compounds of the disclosure may be administered before or after the one or more additional therapeutic agents, as determined by those skilled in the art or patient preference.26098-2

[0120] If formulated as a fixed dose, such combination products employ the compounds of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt thereof within the dosage range described herein and the other pharmaceutically active agent or treatment within its dosage range.Combination Therapy

[0121] Another embodiment provides for methods of treatment using pharmaceutically acceptable compositions comprising a compound of the disclosure, either as the neat chemical or optionally further comprising additional ingredients. Such compositions are contemplated for preparation and use alone or in combination therapy. For preparing pharmaceutical compositions from the compounds of the disclosure, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art, e.g., magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington ’s Pharmaceutical Sciences, 18thEdition, (1990), Mack Publishing Co., Easton, Pennsylvania.

[0122] Non-limiting examples of additional drugs and active agents useful in combination therapies for the treatment of malaria, include the following: Coartem® (Novartis International AG, Basel, Switzerland; artemether + lumefantrine), Eurartesim® (Sigma-Tau Pharmaceuticals, Inc., Rome, Italy; dihydroartemisinin-piperaquine), Pyramax® (Shin Poong Pharmaceutical Co., Ltd., Seoul, Korea; pyronaridine-artesunate), ASAQ Winthrop® (Sanofi SA (Gentilly, France) / DNDi (Geneva, Switzerland); artesunate + amodiaquine), ASMQ (Cipla Limited (Mumbai, India) / DNDi, artesunate + mefloquine), SPAQ-CO™ (Guilin Pharmaceutical Co., Ltd. (Shanghai), amodiaquine + sulfadoxine, pyrimethamine), Artesun® (Guilin Pharmaceutical, artesunate), artemether, artesunate, dihydroartemisinin, lumefantrine, amodiaquine, mefloquine, piperaquine, quinine, chloroquine, atovaquone and proguanil and sulfadoxine-pyrimethamine, Tafenoquine (Glaxosmithkline), OZ439 / PQP (Sanofi), OZ439 / FQ (Sanofi), KAE609 (Novartis), KAF156 (Novartis), DSM265 (NIH / Takeda), and MK-4815 (Merck & Co., Inc., Powles et al., Antimicrobial Agents and Chemotherapy 56(5): 2414-2419(2012)). Selection of such additional active ingredients will be according to the diseases or disorders present for which treatment is desired, as determined by the attending physician or other health care provider.26098-2

[0123] Thus, the disclosure also provides methods of using the compounds of Formula Id, Idl, IId, and IId2, or a pharmaceutically acceptable salt thereof to inhibit plasmepsin X, plasmepsin IX or plasmepsin X and IX, to treat treat parasitic infections selected from cryptosporidiosis, coccidiosis, babesiosis, and toxoplasmosis wherein the method further comprises administering to a subject in need thereof, one or more additional anti-malarial agents. In some embodiments, the one or more additional anti-malarial agents are selected from the group consisting of: artemether, lumefantrine, dihydroartemisinin, piperaquine, pyronaridine, artesunate, amodiaquine, mefloquine, sulfadoxine, pyrimethamine, lumefantrine, quinine, chloroquine, atovaquone, and proguanil.EXAMPLES ACN =MeCN= acetonitrileAcOEt = ethyl acetateBU3P= Bis(tri-tert-butylphosphine)palladium(0)CAA=chloroacetic acidDAPI=2-(4-amidinophenyl)-1H-indole-6-carboxamidineDBU=1,8-diazabicyclo(5.4.0)undec-7-eneDCE= di chloroethaneDCM = dichloromethaneDIAD= Diisopropyl azodi carb oxy lateDIEA= N, N-Diisopropylethylamine, or Hunig's baseDMEM=Dulbecco’s Modified Eagle MediumDMF = N, N-DimethylformamideDMP= Dess-Martin periodinaneDMSO = dimethyl sulfoxideEDC =EDCI= 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideEtOAc = ethyl acetateFBS=fetal bovine serumh = hoursHFF=Human Foreskin Fibroblasts cellsHOBt= HydroxybenzotriazoleIPA-isopropyl alcoholLCMS=Liquid chromatography-mass spectrometryLDA=lithium diisopropylamide26098-2LHMDS = LiHMDS= lithium bis(trimethylsilyl)amideLiAlH4=lithium aluminum hydridemin = minutesMe = methylMeOH= CH3OH=methanolMn(TMHD)3=Tris(2,2,6,6-tetramethyl-3,5-heptanedionato)manganese(III)NaBH4 = sodium borohydrideNa2SO4= sodium sulfateNH4C1= Ammonium chloridePd-C=palladium on carbonPd(dppf)C12 = [l,l'-Bis(diphenylphosphino)ferrocene]palladium(II) dichloride Pet.=petroleumPBS=phosphate buffered salineSFC = Supercritical Fluid ChromatographyTB AF= tetra-n-butylammonium fluorideTBS=tris-buffered salineTEA= triethylamineTEAB=tetraethylammonium bromideTCEP=tris(2-carboxyethyl)phosphineTBAI=tetrabutylammonium iodideTFA = tri fluoroacetic acidTHF= tetrahydrofuranTMS = TrimethylsilylCDCh = heavy CDC13=deuterated chloroformCD3OD = heavy methanol1 Standard atmosphere [atm] = 101325 pascal [Pa] = 14.6959488 psi

[0124] The meanings of the abbreviations in the nuclear magnetic resonance spectra are shown below: s = singlet, d = doublet, dd = double doublet, dt = double triplet, ddd = double double doublet, sept = septet, t = triplet, m = multiplet, br = broad, brs = broad singlet, q = quartet, J = coupling constant and Hz = hertz.

[0125] The pathways for preparing compounds and analogous structures disclosed herein will be apparent to those skilled in the art and can be found in U. S. Patent Application 17 / 633,655 (publication# US2022 / 0331321) filed August 2, 2022, PCT Application PCT / US2024 / 033203, patent publications WO2021 / 155791, WO2023 / 107356, US2023 / 0013692, and US20210379020,26098-2and US Pat. No. 11,766,435, all herein incorporated by reference in the entirety. Processes for making compounds such as 1, 2, 3, 4, and the like are exemplified by the following illustrative processes which should not be construed to limit the scope of the disclosure. Starting materials and intermediates were purchased commercially from common catalog sources or were made using known procedures, or as otherwise illustrated. Frequently applied routes to some of the compounds of Formula Id, Idl, IId, and IId2, are described in the Schemes that follow. In some cases, the order of carrying out the reaction steps in the schemes may be varied to facilitate the reaction or to avoid unwanted reaction products. An asterisk (*) may be used in a chemical structure drawing that indicates the location of a chiral center.

[0126] Preparative HPLC purifications were usually performed using either a mass spectrometry directed system or a non-mass guided system. Usually they were performed on a Waters Chromatography Workstation configured with LC-MS System consisting of: Waters ZQ™ single quad MS system with Electrospray Ionization, Waters 2525 Gradient Pump, Waters 2767 Injecto / Collector, Waters 996 PDA Detector, the MS Conditions of: 150-750 amu, Positive Electrospray, Collection Triggered by MS, and a Waters SUNFIRE® C-18 5-micron, 30 mm (id) x 100 mm column. The mobile phases consisted of mixtures of acetonitrile (10-100%) in water containing 0.1% TFA. Flow rates were maintained at 50 mL / min, the injection volume was 1800 z / L, and the UV detection range was 210-400 nm. An alternate preparative HPLC system used was a Gilson Workstation consisting of: Gilson GX-281 Injector / Collector, Gilson UV / VIS-155 Detector, Gilson 333 and 334 Pumps, and either a Phenomenex Gemini-NX C-18 5-micron, 50 mm (id) x 250 mm column or a Waters XBridge™ C-18 5-micron OBD™, 30 mm (id) x 250 mm column. The mobile phases consisted of mixtures of acetonitrile (0-75%) in water containing 5mmol (NH4HCO3. Flow rates were maintained at 50 mL / min for the Waters Xbridge™ column and 90 mL / min for the Phenomenex Gemini column. The injection volume ranged from 1000-8000 z / L, and the UV detection range was 210-400 nm. Mobile phase gradients were optimized for the individual compounds. Reactions performed using microwave irradiation were normally carried out using an Emrys Optimizer manufactured by Personal Chemistry, or an Initiator manufactured by Biotage. Concentration of solutions was carried out on a rotary evaporator under reduced pressure. Flash chromatography was usually performed using either a Biotage® Flash Chromatography apparatus (Dyax Corp.), an ISCO CombiFlash® Rf apparatus, or an ISCO CombiFlash® Companion XL on silica gel (32-63 μm, 60 A pore size) in pre-packed cartridges of the size noted.1H NMR spectra were acquired at 500 MHz spectrometers in CDC13solutions unless otherwise noted. Chemical shifts were reported in parts per million (ppm).Tetramethylsilane (TMS) was used as internal reference in CDCh solutions, and residual CH3OH26098-2peak or TMS was used as internal reference in CD3OD solutions. Coupling constants (J) were reported in hertz (Hz). Chiral analytical chromatography was most commonly performed on one of CHIRALPAK® AS, CHIRALPAK® AD, CHIRALCEL® OD, CHIRALCEL®IA, or CHIRALCEL®OJ columns (250x4.6 mm) (Daicel Chemical Industries, Ltd.) with noted percentage of either ethanol in hexane (%Et / Hex) or isopropanol in heptane (%IPA / Hep) as isocratic solvent systems. Chiral preparative chromatography was conducted on one of CHIRALPAK AS, of CHIRALPAK AD, CHIRALCEL®OD, CHIRALCEL®IA, CHIRALCEL®OJ columns (20x250 mm) (Daicel Chemical Industries, Ltd.) with desired isocratic solvent systems identified on chiral analytical chromatography or by supercritical fluid (SFC) conditions.

[0127] It is understood that a chiral center in a compound may exist in the "5" or " A"stereo-configuration, or as a mixture of both. Within a molecule, each bond drawn as a straight line from a chiral center includes both the (R) and (5) stereoisomers as well as mixtures thereof. Intermediate 1 (INT 1)

[0128] (R)-tert-butyl (4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-2(lH)-NaH THF, r.t., 16 hINT1-8 Step 6 INTI-9

[0129] Step 1: To a solution 2-cyclopropylacetic acid (23 g, 230 mmol), N, O-dimethylhydroxylamine hydrochloride (33.6 g, 345 mmol), lH-benzo[d][l,2,3]triazol-l-ol (15.52 g, 115 mmol) and Nl-((ethylimino)methylene)-N3, N3-dimethylpropane-l,3-diamine hydrochloride (57.3 g, 299 mmol) in THF (200 mL) was added DIEA (120 mL, 689 mmol). The reaction was heated at 25 °C for 16h under N2 atmosphere. LC / MS showed major DP mass. The mixture was quenched with water (100 mL), and extracted with EtOAc (3 x 150 mL). The26098-2combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude was purified by flash column (SiCh, Pet.ether: EtOAc = 100:0 to 5:1) to afford 2-cyclopropyl-N-methoxy-N-methylacetamide.

[0130] MS (ESI) m / z 144.1 (M+H)+

[0131] 'H NMR (400 MHz, CDC13): 83.67 (s, 3H), 3.20 (s, 3H), 2.36 (d, J= 6.8 Hz, 2H), 1.07-1.12 (m, 1H), 0.53-0.58 (m. 2H), 0.15-0.19 (m, 2H).

[0132] Step 2: To a solution of 2-cyclopropyl-N-methoxy-N-methylacetamide (16 g, 112 mmol) in THF (150 mL) was added phenylmagnesium bromide (74.5 mL, 223 mmol) dropwise at 0 °C under N2 atmosphere. Then the mixture was stirred at 20 °C for 1 h. LC / MS showed major DP mass. The mixture was quenched with Sat. a.q. NH4CI (50 mL), and extracted with EA (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, concentrated in vacuo. The residue was purified by flash column (SiC>2, Pet.ether: EtOAc = 100:0 to 50:1) to afford 2-cyclopropyl-l -phenyl ethanone.

[0133] MS (ESI) m / z 161.1 (M+H)+

[0134] 1H NMR (400 MHz, CDC13): 67.95-7.97 (m, 2H), 7.54-7.58 (m, 1H), 7.45-7.50 (m, 2H), 2.90 (d, J = 6.8 Hz, 2H), 1.16-1.19 (m, 1H), 0.58-0.63 (m. 2H), 0.18-0.22 (m, 2H).

[0135] Step 3: To a solution of (S)-2-methylpropane-2-sulfinamide (7.26 g, 59.9 mmol) in anhydrous THF (100 mL) was added 2-cyclopropyl-l -phenylethanone (8 g, 49.9 mmol) followed by tetraethoxytitanium (20.52 mL, 100 mmol) and the reaction was stirred at 75 °C for 16 h. LC / MS showed DP mass and TLC showed the reaction was finished. The mixture was cooled to 0 °C, diluted with DCM (200 mL), stirred 15 min, then added ice cold- saturated aqueous sodium bicarbonate solution (32 mL), then filtered and concentrated in vacuo. The crude was purified by flash column (SiC>2, Pet.ether: EtOAc = 100:0 to 10:1) to afford (S, E)-N-(2-cyclopropyl-l-phenylethylidene)-2-methylpropane-2-sulfinamide.

[0136] MS (ESI) m / z 264.2 (M+H)+

[0137] 1HNMR (400 MHz, CDC13): 67.88-7.90 (m, 2H), 7.42-7.49 (m, 3H), 3.18-3.26 (m, 2H), 1.33 (s, 9H), 0.97-1.00 (m, 1H), 0.37-0.49 (m, 4H).

[0138] Step 5: To a solution of diisopropylamine (17.66 mL, 125 mmol) in anhydrous THF (70 mL) at -78 °C was added butyllithium (50.1 mL, 125 mmol) dropwise under N2 atmosphere. The reaction was stirred at -10 °C for 30 min then cooled to -78 °C and methyl acetate (8.41 mL, 104 mmol) was added followed Ih later by triisopropoxytitanium(IV) chloride (146 mL, 146 mmol).26098-2After 40 mins, a solution of (S, E)-N-(2-cyclopropyl-l-phenylethylidene)-2-methylpropane-2-sulfinamide (11 g, 41.8 mmol) in anhydrous THF (30 mL) was then added dropwise and the mixture was stirred at -78 °C for 3 h. LC / MS showed DP mass. The mixture was quenched with ice-cold half-saturated aqueous ammonium chloride solution (50 mL). The slurry was diluted with EtOAc (100 mL) then filtered, rinsing with EtOAc and water. The organic layer was separated, then washed with brine (50 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash column (SiCh, Pet.ether: EtOAc = 100:0 to 3:1) to afford (R)-methyl 4-cyclopropyl-3-((S)-l, l-dimethylethylsulfmamido)-3-phenylbutanoate.

[0139] MS (ESI) m / z 338.1 (M+H)+

[0140] 1H NMR (400 MHz, CDC13): 87.38-7.40 (m, 2H), 7.32-7.34 (m, 2H), 7.25-7.30 (m, 1H), 5.60 (s, 1H), 3.59 (s, 3H), 3.43 (d, J =17.2 Hz, 1H), 3.29 (d, J = 17.2 Hz, 1H), 2.28-2.33 (m, 1H), 1.65-1.71 (m, 1H), 1.33 (s, 9H), 0.30-0.38 (m, 1H), 0.26-0.28 (m, 2H), -0.18-0.08 (m, 2H).

[0141] Step 6: To a solution of BocNH? (7.24 g, 61.8 mmol) in THF (70 mL) was added sodium hydride (3.30 g, 82 mmol) at 0 °C and the mixture was stirred for 15 min. A solution of (isothiocyanatomethyl)benzene (6.15 g, 41.2 mmol) in THF (10 mL) was added dropwise at 0 °C. Then the mixture was stirred at 25 °C for 16 h. LC / MS showed major DP mass. The mixture was quenched with water (50 mL), extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude was purified by flash column (SiO2, Pet.ether: EtOAc = 20: 1 to 5: 1) to afford tertbutyl N-(benzylcarbamothioyl)carbamate.

[0142] MS (ESI) m / z 267.1 (M+H) +

[0143] 1HNMR (400 MHz, CDC13): 69.98 (s, 1H), 7.96 (s, 1H), 7.31-7.37 (m, 5H), 4.87 (d, J = 5.6 Hz, 2H), 1.48 (s, 9H).

[0144] Step 6a: To a solution (R)-methyl 4-cyclopropyl-3-((S)-l,l-dimethylethylsulfinamido)-3-phenylbutanoate (10.3 g, 30.5 mmol) in MeOH (100 mL) at 0 °C was added 4N HCl-dioxane (50 mL) dropwise. The reaction was stirred at 25 °C for 1.5 h. LC / MS showed no remaining starting material. The mixture was concentrated with toluene to afford (R)-methyl 3-amino-4-cy cl opropy 1 -3 -pheny Ibutanoate hydrochi ori de.

[0145] 'H NMR (400 MHz, CDCI3): 69.17 (br s, 3H), 7.59 (m,2H), 7.44 (m, 2H), 7.35 (m,lH), 3.67 (s, 3H), 3.46-3.56 (m, 2H), 2.28-2.32 (m,lH), 2.10-2.15 (m,lH), 0.41-0.45 (m,3H), 0.03-0.06 (m, 2H).26098-2

[0146] Step 7: To a solution of (R)-methyl 3-amino-4-cyclopropyl-3-phenylbutanoate hydrochloride (6.5 g, 24.09 mmol) in DMF (100 mL) was treated with DIEA (16.83 mL, 96 mmol) for 30 min then Nl-((ethylimino)methylene)-N3, N3-dimethylpropane-l,3-diamine hydrochloride (5.08 g, 26.5 mmol) was added followed by tert-butyl N-(benzylcarbamothioyl)carbamate (7.06 g, 26.5 mmol). The reaction was heated at 65 °C for 16 h. LC / MS showed major DP mass. The mixture was quenched with water (50 mL), and extracted with EtOAc (3 x 50 mL). The organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude was purified by flash column (SiCh, Pet.ether: EtOAc = 100:1 to 10:1) to afford (R, E)-tert-butyl (l-benzyl-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-2(lH)-ylidene)carbamate.

[0147] MS (ESI) m / z 434.2 (M+H)+.

[0148] 1H NMR (400 MHz, CDC13): 8 10.66 (s, 1H), 7.29-7.32 (m,4H), 7.27-7.29 (m, 1H), 7.09-7.10 (m, 3 H), 6.91 (d, J= 6.8 Hz, 2H), 5.17 (d, J=14.8 Hz, 1H), 4.98 (d, J= 14.4 Hz, 1H), 3.28 (d, J= 16.0 Hz, 1H), 3.02 (d, J= 16.0 Hz, 1H), 2.08-2.13 (m,lH), 1.53 (s, 9H), 1.49-1.51 (m,lH), 0.40-0.57 (m, 3H), 0.00-0.15 (m, 2H).

[0149] Step 8: To a solution of (R)-tert-butyl (l-benzyl-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-2(lH)-ylidene)carbamate (8.7 g, 20.07 mmol) in MeOH (100 mL) was added dihydroxypalladium (3 g, 10.68 mmol). The mixture was stirred at 60 °C for 3 days under H2 (50 psi) atmosphere. LC / MS showed no remaining starting material. Then the mixture was filtered through a pad of Celite. The filtrate was concentrated in vacuo. The crude was purified by flash column (SiC>2, Pet.ether: EtOAc = 100:0 to 3:1) to yield (R)-tert-butyl (4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-2(lH)-ylidene)carbamate.

[0150] MS (ESI) m / z 344.0 (M+H)+

[0151] 1H NMR (400 MHz, CDC13): 67.37-7.41 (m, 2H), 7.30-7.33 (m, 2H), 3.14 (d, J = 16.0 Hz, 1H), 2.98 (d, J= 16.4 Hz, 1H), 2.18-2.23 (m, 1H), 1.56-1.60 (m, 1H), 1.53 (s, 9H), 0.43-0.59 (m, 3H), 0.03-0.20 (m, 2H).Intermediate 2 (INT 2)

[0152] methyl 3-((3-(tert-butoxycarbonyl)thioureido)methyl)benzoate26098-2

[0153] Step 1: To a flask containing N, N'-Di-Boc-thiourea (999.9 mg, 868.0 pL, 1 Eq, 3.618 mmol) in anhydrous THF (50 mL) at 0 °C was added LiHMDS (1.211 g, 7.236 mL, 1 molar, 2 Eq, 7.236 mmol). Stirred at 0 °C for ~1 hr under an atmosphere of Nitrogen. Then Trifluoroacetic anhydride (2.029 g, 1.349 mL, 2.67 Eq, 9.661 mmol) was added slowly and stirred at 0 °C for another 1 hr. Then added a suspension of methyl 3-(aminomethyl)benzoate hydrochloride (875.5 mg, 1.2 Eq, 4.342 mmol) in THF (50 mL) at 0 °C, stirred for ~30 min at 0 °C, then warmed to room temperature. Followed by LC / MS. After an hour, the reaction mixture was diluted with EtOAc, and saturated NaHCO3 / water. The organic layer was separated, then washed with water, then brine. The organic layer was then dried over anhydrous sodium sulfate, then filtered & concentrated. The resulting residue was then purified by silica gel chromatography (0-60% EtOAc / Hexanes; 14 CV; 80 g ISCO silica column). The desired fractions were concentrated, then dissolved in DCM / MeOH & concentrated to yield methyl 3-((3-(tert-butoxycarbonyl)thioureido)methyl)benzoate.

[0154] MS (ESI) m / z 325.4 (M+H)Example 1

[0155] 3 -fluoro-5-[(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin-l-yl)methyl]-N-[(lS)-l-phenyl ethyl ]b enzami de26098-2NH3·MeOHNHj-MeOH? 28 °C, 3 h Step 2

[0156] Step 1: To a solution of 3 -cyano-5 -fluorobenzoic acid (1 g, 6.06 mmol) in THF (20 mL) was added DIEA (3.17 mL, 18.17 mmol) andHATU (4.61 g, 12.11 mmol) under N2 atmosphere, then followed by the addition of (5)-l-phenylethanamine (1.101 g, 9.08 mmol). The reaction was heated at 28 °C for 16 h. LC / MS showed major DP mass. The mixture was quenched with water(20 mL), and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give thecrude product which was purified by flash column (SiC>2, Pet.ether: EtOAc = 10: 1 to 3: 1) afford(5)-3-cyano-5-fluoro-A-(l-phenylethyl)benzamide.

[0157] MS (ESI) m / z 269.1 [M+H]+.

[0158] 'HNMR (400 MHz, CDCI3): 87.83 (t, J= 1.2 Hz, 1H), 7.78 - 7.74 (m, 1H), 7.50 -7.47(m, 1H), 7.39 (s, 2H), 7.38 (s, 2H), 7.35 -7.30 (m, 1H), 6.38 (d, J= 6.4 Hz, 1H), 5.31 (q, J = 7.06 Hz, 1H), 1.64 (d, J = 6.8 Hz, 3H).

[0159] Step 2: To a solution of (5)-3-cyano-5-fluoro-A-(l-phenylethyl)benzamide (1.62 g, 6.06 mmol, crude) in MeOH (100 mL), was added Raney Ni (200 mg, 6.71 mmol) and NHi-MeOH(16 mL) under N2 atmosphere. The mixture was degassed and backfilled with H2 (three times).The resulting mixture was stirred at 28 °C for 3 h under H2 (50 psi) atmosphere. LC / MS showed major DP mass. The mixture was filtered through a pad of Celite and the filtrate was26098-2concentrated in vacuo to yield the crude (5)-3-(aminomethyl)-5-fluoro-7V-(l-phenylethyl)benzamide. The crude product was used for the next step directly.

[0160] MS (ESI) m / z 273.1 [M+H]+.

[0161] 'H NMR (400 MHz, CDC13): 67.51 (s, 1H), 7.35-7.41 (m, 5H), 7.30-7.31 (m, 1H), 7.18 (d, J= 8.4 Hz, 1H), 6.40 (d, J= 1.6Hz, 1H), 5.31-5.35 (m, 1H), 3.93 (s, 2H), 1.62 (d, = 6.8 Hz, 3H).

[0162] Step 3: To a solution of tert-butyl 7V-({[(tert-butoxy)carbonyl]amino}methanethioyl)carbamate (1.1 g, 3.98 mmol) in THF (12 mL) was added sodium hydride (0.318 g, 7.96 mmol) at 0 °C under N2 atmosphere. After Ihr at this temperature, TFAA (0.664 mL, 4.78 mmol) was added dropwise. The mixture was stirred at 0 °C for Ih. Then the solution of (5)-3-(aminomethyl)-5-fluoro-A-(l-phenylethyl)benzamide (1.301 g, 4.78 mmol) in THF (8 mL) was added dropwise at 0 °C. The mixture was stirred at 25 °C for 3 h. LC / MS showed major DP mass. The mixture was quenched with water (20 mL), and extracted with EtOAc (3 x 30 mL). The organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude was purified by flash column (SiC>2, Pet.ether: EtOAc = 100:1 to 5:1) to afford tert-butyl A-{[(3-fluoro-5-{[(15)-l-phenylethyl]carbamoyl}phenyl)methyl]carbamothioyl}carbamate.

[0163] MS (ESI) m / z 432.2 [M+H]+.

[0164] 'H NMR (400 MHz, CDCI3): 6 10.08 (s, IH), 7.93 (s, IH), 7.53 (s, IH), 7.41 - 7.37 (m, 5H), 7.31 - 7.30 (m, IH), 7.23 - 7.20 (m, IH), 6.29 - 6.28 (m, IH), 5.35 - 5.30 (m, IH), 4.90 (d, J = 6 Hz, 2H), 1.62 (d, J= 7.2 Hz, 3H), 1.49 (s, 9H).

[0165] Step 4: To a solution of Benzophenone (8.12 g, 7.34 mL, 1 Eq, 44.6 mmol) in THF (90 mL) was added (5)-2-methylpropane-2-sulfinamide (6.48 g, 1.2 Eq, 53.5 mmol) followed by Titaniumethoxide (20.3 g, 18.5 mL, 2 Eq, 89.1 mmol) under N2 atmosphere, then the reaction was stirred at 75 °C for 16 h. TLC showed new spots and starting material was consumed. The final mixture was cooled to 0 °C, diluted with EtOAc (100 mL), stirred 15 min, then ice cold-saturated aqueous sodium bicarbonate solution (20 mL) was added, then filtered and concentrated in vacuum. The filtrate was concentrated to give a residue which was purified by flash silica gel chromatography (ISCO®; 80g Agela® Silica Flash Column, Eluent of ethyl acetate / hexane 0-20% gradient @ 100 mL / min) to yield (A)-A-(di phenyl methyl ene)-2-methylpropane-2-sulfmamide.

[0166] MS (ESI) m / z 286.1 [M+H]+.26098-2

[0167] 1H NMR (400 MHz, CDC13) 68.01 - 7.28 (m, 10H), 1.31 (s, 9H).

[0168] Step 5: To a solution of methyl acetate (1.04 g, 1.12 mL, 2.5 Eq, 14.0 mmol) in anhydrous THF (20 mL) was added LDA (1.80 g, 8.41 mL, 2 molar, 3 Eq, 16.8 mmol) (2 M in THF solution) dropwise under N2 atmosphere at -78 °C. The reaction was stirred at -78 °C for 30 min and then was added Chlorotitanium triisopropoxide (5.11 g, 19.6 mL, 1 molar, 3.5 Eq, 19.6 mmol) (1 M in hexane solution) dropwise at -78 °C. After stirring at -78 °C for 30 min, a solution of (A)-A-(diphenylmethylene)-2-methylpropane-2-sulfinamide (1.60 g, 1 Eq, 5.61 mmol) in anhydrous THF (10 mL) was added dropwise at -78 °C. The mixture was stirred at -78 °C for 3 h. LC / MS showed the starting material was consumed and desired product was formed. The mixture was quenched with sat. NH4CI (20 mL), extracted with EtOAc (3 x 30 mL). The organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 22 g SepaFlash® Silica Flash Column, eluent of 0-30% ethyl acetate / pet. ether gradient @ 45 mL / min) to yield methyl (R)-3-((tert-butylsulfmyl)amino)-3,3-diphenylpropanoate.

[0169] MS (ESI) m / z 360.1 [M+H]+.

[0170] 'H NMR (400 MHz, CDCI3) 87.36 - 7.28 (m, 10H), 6.14 (s, 1H), 3.96 (d, J= 16.0 Hz, 1H), 3.73 (d, J= 16.8 Hz, 1H), 3.51 (s, 3H), 1.29 (s, 9H).

[0171] Step 6: A solution of methyl (A)-3-((tert-butylsulfmyl)amino)-3,3-diphenylpropanoate (600 mg, 1 Eq, 1.67 mmol) in HCl-MeOH (10 mL, 2M) was stirred at 25 °C for 1 h. LC / MS showed the reaction was complete. The mixture was then concentrated in vacuo to yield methyl 3-amino-3,3-diphenylpropanoate hydrochloride.MS (ESI) m / z 256.2 [M+H]+.

[0172] Step 7: To a solution of methyl 3-amino-3,3-diphenylpropanoate hydrochloride (40.6 mg, 0.139 mmol) in DMF (6 mL) was treated with DIEA (0.081 mL, 0.463 mmol) for 30 min, and then EDCI (26.7 mg, 0.139 mmol) was added followed by tert-butyl A-{[(3-fluoro-5-{[(15)-l-phenylethyl]carbamoyl}phenyl)methyl]carbamothioyl}carbamate (50 mg, 0.116 mmol) under N2 atmosphere. The reaction was heated at 65 °C for 16 h until LC / MS showed the reaction was complete, The mixture was quenched with water (10 mL), and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude was purified by Prep-TLC (SiO2, Pet.ether: EtOAc = 2:1) to afford tert-butyl (5)-(l-(3-fluoro-5-((l-26098-2phenylethyl)carbamoyl)benzyl)-6-oxo-4,4-diphenyltetrahydropyrimidin-2(U7)-ylidene)carbamate.

[0173] MS (ESI) m / z 621.3 [M+H]+.

[0174] Step 8: A solution of tert-butyl (5)-(l-(3-fluoro-5-((l-phenylethyl)carbamoyl)benzyl)-6-oxo-4, 4-diphenyltetrahydropyrimidin-2(U7)-ylidene)carbamate (27 mg, 0.043 mmol) in DCM (4 mL) and TFA (1 mL) was stirred at 25 °C for 1 h until LC / MS showed the reaction was complete. The mixture was then concentrated in vacuo. The resulting residue was purified by reverse preparative HPLC (Column: Phenomenex Synergi C18 150*30mm*4pm; Condition: water (0.1%TFA)-ACN Begin B 33 End B 53 Gradient Time (min) 11 100% B Hold Time 2 Flow Rate (mL / min) 25 Injections 3) to afford (5)-3-fluoro-5-((2-imino-6-oxo-4,4-diphenyltetrahydropyrimidin-l(27 / )-yl)methyl)- / ' / -(l -phenylethyl jbenzamide.

[0175] MS (ESI) m / z 512.2 [M+H]+.

[0176] 'HNMR (400 MHz, METHANOL-d4) 87.42 - 7.26 (m, 17H), 6.37 (d, J= 8.8 Hz, 1H), 5.26 - 5.18 (m, 1H), 5.08 (s, 2H), 3.84 (s, 2H), 1.58 (d, J= 6.8 Hz, 1H).Example 2

[0177] 3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-N-[(lS)-l-phenylethyl]benzamide2-3 Example 2

[0178] Step 1: To a vial containing tert-butyl (R, E)-(4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-2(lH)-ylidene)carbamate (1.027 g, 1 Eq, 2.990 mmol) was added cesium carbonate (969 mg, 0.995 Eq, 2.97 mmol), followed by methyl 3 -bromomethylbenzoate (699.1 mg, 1.021 Eq, 3.052 mmol) and finally anhydrous DMF (10 mL). The reaction mixture was then capped & stirred at room temperature. Followed by LC / MS. After 1 night at room temperature, the reaction mixture was diluted with EtOAc, and saturated sodium bicarbonate / water. The organic layer was separated, then washed with water, then brine. The organic layer was then dried over anhydrous sodium sulfate, then filtered & concentrated. The resulting residue was then purified by silica gel chromatography (0-50% EtOAc / Hex; 14 CV; 80g ISCO silica column). The desired fractions were concentrated, then dissolved in DCM / MeOH & concentrated to yield methyl (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)benzoate (the desired regioisomer was the less polar isomer).

[0179] MS (ESI) m / z 492.5 (M+H)+

[0180] Step 2: To a vial containing To a vial containing methyl (R, E)-3 -(^-((tertbutoxy carbonyl)imino)-4-(cy cl opropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)benzoate (743.7 mg, 1 Eq, 1.513 mmol) was added Potassium trimethyl(oxido)silane (1.29 g, 6.65 Eq, 10.1 mmol), and finally anhydrous THF (20 mL). The reaction mixture was then capped & stirred at room temperature. Followed by LC / MS. After ~30 min at room temperature, the reaction mixture was cooled to 0°C (ice water bath), then quenched with IM HC1, then diluted with EtOAc. The organic layer was separated and then washed with IM HC1, then brine. The organic layer was then dried over anhydrous sodium sulfate, then filtered & concentrated to yield (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-l (2H)-yl)m ethyl )benzoic acid which was used without further modification for the next step.

[0181] MS (ESI) m / z 478.5 (M+H)+

[0182] Step 3: To a vial containing (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)benzoic acid (693.3 mg, 1 Eq, 1.452 mmol) & 7-Azabenzotriazol-l-Yloxytris(Dimethylamino)PhosphoniumHexafluorophosphate (999 mg, 1.55 Eq, 2.25 mmol)was added anhydrous DMF (8 mL), then DIPEA (594 mg, 800 pL, 3.16 Eq, 4.59 mmol), & finally (S)-l-phenylethan-l -amine (266 mg, 1.51 Eq, 2.20 mmol). The reaction mixture was then capped & stirred at room temperature. Followed by LC / MS. After 1 night at room temperature the reaction mixture was diluted with DMSO / MeOH then purified (without workup) by reverse phase chromatography (20-100% MeCN / H2O; 0.1% TFA modifier; 30 min gradient; Waters 50x250 mm Sunfire 5 micron C18 column; Flow = 118.1 mL / min). The desired fractions were then free based; suspended in EtOAc, washed with saturated sodium bicarbonate, then brine. The organic layer was then dried over anhydrous sodium sulfate, then filtered & concentrated to yield tert-butyl ((R, E)-4-(cyclopropylmethyl)-6-oxo-4-phenyl-l-(3-(((S)-l-phenylethyl)carbamoyl)benzyl)tetrahydropyrimidin-2(lH)-ylidene)carbamate.

[0183] MS (ESI) m / z 581.5 (M+H)+

[0184] Step 4: To a flask containing tert-butyl ((R, E)-4-(cyclopropylmethyl)-6-oxo-4-phenyl-l-(3-(((S)-l-phenylethyl)carbamoyl)benzyl)tetrahydropyrimidin-2(lH)-ylidene)carbamate (807.3 mg, 1 Eq, 1.390 mmol) was added DCM (5 mL) followed by Trifluoroacetic acid (1 g, 1 mL, 9 Eq, 0.01 mol). The reaction mixture was then capped & stirred at room temperature. Followed by LC / MS. After ~2.5 hrs at room temperature the reaction mixture was diluted with MeOH, then concentrated then suspended in MeOH / DMSO & purified (without workup) by reverse phase chromatography (10-100% MeCN / H2O; 0.1% TFA modifier; 30 min gradient; Waters 50x250 mm Sunfire 5 micron C18 column; Flow = 118.1 mL / min). The desired fractions were concentrated to yield 3-(((R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)-N-((S)-l-phenylethyl)benzamide.

[0185] MS (ESI) m / z 481.5 (M+H)+

[0186] 'H NMR (500 MHz, MeOD) 88.63 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.43 (s, 1H), 7.37 - 7.32 (m, 2H), 7.31 - 7.26 (m, 2H), 7.26 - 7.22 (m, 2H), 7.22 - 7.17 (m, 4H), 7.03 (t, J = 7.8 Hz, 1H), 6.48 (d, J = 7.7 Hz, 1H), 5.34 (d, J = 16.6 Hz, 1H), 5.20 - 5.11 (m, 1H), 4.59(d, J = 16.6 Hz, 1H), 3.64 (d, J = 16.4 Hz, 1H), 3.39 (d, J = 16.4 Hz, 1H), 2.00 - 1.92 (m, 1H),1.72 - 1.63 (m, 1H), 1.51 (d, J = 7.1 Hz, 3H), 0.63 - 0.52 (m, 1H), 0.46 - 0.38 (m, 2H), 0.05 - -0.05 (m, 2H).Example 3NH

[0187] 3-((( / ?)-4-(2-chlorophenyl)-4-(cyclopropylmethyl)-2-imino-6-oxotetrahydropyrimidin- 1 (2J7)-yl)methyl)-7V-((5')- 1 -phenylethyl)benzamideS EDCI, HOBt, A DIEA HCI-dioxane BocHN^NHBoc THF, 20 °C, 16 h 25 °C, 2 h TFAA, NaH, THF, 0-20 °C, 12 h Step 1 Step 2 Step 3A(1 eq), o (COCI)2. DMFBrZnxAo- GUGI, THF,' DCM.0-20 °C, 2 h. then 2, -15-25 °C, 3 h Ti(OEt)4. THF,75 °C, 16 h 25 °C, 16 h Step 4 3-4 Step 5 3-5 Step 6 3-6 Step 7 3-7HCI-dioxane MeOH, 25 °C, 1 h Step 8

[0188] Step 1: To a solution of 3-((( / c / 7-butoxycarbonyl)amino)methyl)benzoic acid (5.00 g, 1 Eq, 19.9 mmol), EDCI (5.72 g, 1.5 Eq, 29.8 mmol), HOBt (4.57 g, 1.5 Eq, 29.8 mmol) and DIEA (7.72 g, 10.4 mL, 3 Eq, 59.7 mmol) in THF (2 mL) was added (5)- 1 -phenyl ethan-1 -amine (2.89 g, 1.2 Eq, 23.9 mmol) under N2 atmosphere. The reaction was stirred at 20 °C for 16 h until26098-2LC / MS showed the reaction was complete. The mixture was quenched with water (50 mL), and extracted with EtOAc (3 x 50 mL). The organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, eluent of 0-20% ethyl acetate / Hexane gradient @ 45 mL / min) to yield / c / 7-butyl (5)-(3-((l -phenylethyl)carbamoyl)benzyl)carbamate.

[0189] MS (ESI) m / z 355.1 [M+H]+.

[0190] 'H NMR (400 MHz, METHANOL-d4) 87.78 - 7.64 (m, 2H), 7.50 - 7.37 (m, 4H), 7.32 (t, J= 7.6 Hz, 2H), 7.27 - 7.17 (m, 1H), 5.24 (q, J= 7.2 Hz, 1H), 4.27 (s, 2H), 1.56 (d, J= 7.2 Hz, 3H), 1.49 - 1.36 (m, 9H).

[0191] Step 2: A solution of tert-butyl (5)-(3-((l-phenylethyl)carbamoyl)benzyl)carbamate (6.5 g, 1 Eq, 18 mmol) in HCl-di oxane (50 mL, 2 M) was stirred at 25 °C for 2 h until LC / MS showed the reaction was complete. The mixture was then concentrated under vacuum to yield (5)-3-(aminomethyl)-A-(l-phenylethyl)benzamide hydrochloride.

[0192] MS (ESI) m / z 255.0 [M+H]+.

[0193] 'H NMR (400 MHz, METHANOL-d4) 67.96 - 7.85 (m, 2H), 7.67 - 7.60 (m, 1H), 7.59 - 7.50 (m, 1H), 7.45 - 7.38 (m, 2H), 7.36 - 7.29 (m, 2H), 7.27 - 7.20 (m, 1H), 5.25 (q, J= 7.2 Hz, 1H), 4.18 (s, 2H), 1.58 (d, J= 7.2 Hz, 3H).

[0194] Step 3: To a solution of tert-butyl 7V-({ [(tertbutoxy )carbonyl]amino}methanethioyl)carbamate (4.94 g, 1 Eq, 17.9 mmol) in THF (50 mL) was added NaH (1.43 g, 60% Wt, 2 Eq, 35.8 mmol) at 0 °C in portions under N2 atmosphere. After 1 h at this temperature, TFAA (3.76 g, 2.53 mL, 1 Eq, 17.9 mmol) in THF (20 mL) was added dropwise. The mixture was stirred at 0 °C for 1 h. Then a solution of (5)-3-(aminomethyl)-A-(l-phenylethyl)benzamide hydrochloride (5.20 g, 1 Eq, 17.9 mmol) in THF (30 mL) was added dropwise at 0 °C. The mixture was stirred at 20 °C for 12 h. LC / MS showed major DP mass. The mixture was quenched with sat. NH4CI (50 mL) and water (50 mL), and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0-30% EtOAc gradient @ 100 mL / min) to afford tert-butyl N-{[(3-{[(lS)-l-phenylethyl]carbamoyl}phenyl)methyl]carbamothioyl}carbamate.

[0195] MS (ESI) m / z 414.1[M+H]+.26098-2

[0196] 'H NMR (400 MHz, METHANOL-d4) 6 10.33 (br s, 1H), 7.81 (s, 1H), 7.74 (d, J= 7.6 Hz, 1H), 7.56 - 7.50 (m, 1H), 7.47 - 7.37 (m, 3H), 7.35 - 7.28 (m, 2H), 7.26 - 7.20 (m, 1H), 5.23 (q, J= 7.2 Hz, 1H), 4.92 (d, J= 5.6 Hz, 2H), 1.56 (d, J= 7.2 Hz, 3H), 1.50 (s, 9H).

[0197] Step 4: To a solution of 2-cyclopropylacetic acid (5.00 g, 1 Eq, 49.9 mmol) and DMF (73.0 mg, 77.3 pL, 0.02 Eq, 999 pmol) in DCM (50 mL) under N2 atmosphere, was added (COC1)2 (7.61 g, 5.25 mL, 1.2 Eq, 59.9 mmol) in DCM (5mL) dropwise at 0°C. The reaction was stirred at 20°C for 2 h. TLC showed the reaction was complete. The reaction was concentrated to afford 2-cyclopropylacetyl chloride, which was used in next step directly without any further purification.

[0198] Step 5: To a solution of l-bromo-2-chlorobenzene (6.00 g, 1 Eq, 31.3 mmol) in THF (50 mL) under N2 atmosphere, was added i-PrMgCl LiCl (4.55 g, 24.1 mL, 1.3 molar, 1 Eq, 31.3 mmol) dropwise at 0°C. The reaction was then stirred at -15 °C for 1 h. Then 2-cyclopropylacetyl chloride (4.09 g, 1.1 Eq, 34.5 mmol) in THF (30 mL) solution was added dropwise. Then warmed to room temperature, the reaction was stirred at 25 °C for 3 h. LC / MS showed desired target was formed. The mixture was quenched with sat. NH4CI (50 mL), and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure to give the crude product. The resulting residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, eluent of 0-10% ethyl acetate / hexane gradient @ 80 mL / min) to yield l-(2-chlorophenyl)-2-cyclopropylethan-l-one.

[0199] MS (ESI) m / z 195.0 [M+H]+.

[0200] 'H NMR (400 MHz, CDCI3) 87.47 - 7.28 (m, 4H), 2.85 (d, J= 6.8 Hz, 2H), 1.18 - 1.01 (m, 1H), 0.67 - 0.48 (m, 2H), 0.30 - 0.10 (m, 2H).

[0201] Step 6: To a solution of l-(2-chlorophenyl)-2-cyclopropylethan-l-one (1.70 g, 1 Eq, 8.73 mmol) in THF (30 mL) was added (5)-2-methylpropane-2-sulfinamide (1.27 g, 1.2 Eq, 10.5 mmol) followed by Titaniumethoxide (3.98 g, 3.64 mL, 2 Eq, 17.5 mmol), then the reaction was stirred at 75 °C. Followed by LC / MS. After 16 h at 75 °C, the mixture was quenched with NaHC 03(10 mL), then filtered and the solvent was evaporated under reduced pressure to give the crude product. The resulting residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, eluent of 15% ethyl acetate / pet. ether gradient @ 4026098-2mL / min) to give (5)-7V-(l-(2-chlorophenyl)-2-cyclopropylethylidene)-2-methylpropane-2-sulfinamide.

[0202] MS (ESI) m / z 298.0 [M+H]+.

[0203] 'H NMR (400 MHz, CDC13) 67.27 (s, 4H), 3.20 - 2.45 (m, 2H), 1.32 - 1.24 (m, 9H), 1.14 - 0.78 (m, 1H), 0.59 - 0.34 (m, 2H), 0.14 (br s, 2H).

[0204] Step 7: To a solution of (5)-7V-(l-(2-chlorophenyl)-2-cyclopropylethylidene)-2-methylpropane-2-sulfmamide (200 mg, 1 Eq, 672 pmol) in THF (4 mL) was added Cuprous chloride (66.5 mg, 17.5 pL, 1 Eq, 672 pmol) and (2-methoxy-2-oxoethyl)zinc(II) bromide (293 mg, 2.86 mL, 0.47 molar, 2 Eq, 1.34 mmol) at 0 °C under N2 atmosphere. The mixture was stirred at 25 °C for 16 h. LC / MS showed the desired product was formed. The mixture was quenched with sat. NH4CI (10 mL), and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure to give the crude product. The resulting residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, eluent of 0-20% ethyl acetate / pet. ether gradient @ 35 mL / min) to give methyl (J / )-3-(((S)-lert-butylsulfmyl)amino)-3-(2-chlorophenyl)-4-cyclopropylbutanoate.

[0205] MS (ESI) m / z 372.0 [M+H]+.

[0206] 'H NMR (400 MHz, CDCI3) 87.63 (d, J= 8.0 Hz, 1H), 7.33 (d, J= 7.6 Hz, 1H), 7.26 -7.18 (m, 2H), 3.94 (d, J= 17.6 Hz, 1H), 3.58 (s, 3H), 3.35 (d, J= 17.2 Hz, 1H), 2.70 (dd, J= 4.0, 14.8 Hz, 1H), 2.12 (dd, J= 8.4, 14.8 Hz, 1H), 1.36 (s, 9H), 0.53 - 0.39 (m, 1H), 0.35 - 0.21 (m, 1H), 0.16 - 0.05 (m, 1H), 0.05 - -0.06 (m, 1H), 0.12 - -0.24 (m, 1H).

[0207] Step 8: A solution of methyl (A)-3-(((5)-tert-butylsulfmyl)amino)-3-(2-chlorophenyl)-4-cyclopropylbutanoate (150 mg, 1 Eq, 403 pmol) in HCl-l,4-Dioxane (2 M) (2 mL) was stirred at 25 °C for 1 h. LC / MS showed the desired product was formed. The reaction was concentrated to afford methyl ( / ?)-3 -ami no-3 -(2-chlorophenyl)-4-cyclopropylbutanoate hydrochloride, which was used directly in the next step without any further purification.

[0208] MS (ESI) m / z 268.0 [M+H]+.

[0209] Step 9: To a solution of tert-butyl A-{[(3-{[(15)-l-phenylethyl]carbamoyl}phenyl)methyl]carbamothioyl}carbamate (179 mg, 1.1 Eq, 434 pmol), methyl (A)-3-amino-3-(2-chlorophenyl)-4-cyclopropylbutanoate hydrochloride (120 mg, 1 Eq, 394 pmol), 3-(((ethylimino)methylene)amino)-A, A-dimethylpropan-l-amine hydrochloride (18926098-2mg, 2.5 Eq, 986 pmol) in THF (6 mL) was added A-ethyl-A-isopropylpropan-2-amine (255 mg, 344 pL, 5 Eq, 1.97 mmol) under N2 atmosphere. The mixture was stirred at 25 °C for 12 h under N2 atmosphere. LC / MS showed the desired product was formed. The mixture was quenched with water (20 mL), and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure to give the crude product. The resulting residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, eluent of 0-60% ethyl acetate / hexane gradient @ 35 mL / min) and then SFC (Column: DAICEL CHIRALPAK IM (250*30mm,10pm); Condition: 0.1%NH3H2O / MeOH; Begin B 60%, End B 60%; Flow Rate (mL / min): 80) to yield / c / 7-butyl ((A)-4-(2-chlorophenyl)-4-(cyclopropylmethyl)-6-oxo- 1 -(3 -(((5)- 1 -phenyl ethyl)carbamoyl)benzyl)tetrahydropyrimidin-2(U7)-ylidene)carbamate (99.70% de).

[0210] MS (ESI) m / z 615.2 [M+H]+.

[0211] Step 10: A solution of Zc / V-butyl ((A)-4-(2-chlorophenyl)-4-(cyclopropylmethyl)-6-oxo-l-(3-(((5)-l-phenylethyl)carbamoyl)benzyl)tetrahydropyrimidin-2(U7)-ylidene)carbamate (110 mg, 1 Eq, 179 pmol) in DCM / TFA (v / v = 3:1, 2 mL) was stirred at 25 °C for 1 h. LC / MS showed the desired product was formed. The mixture was concentrated in vacuo and the resulting residue was purified by Prep-HPLC (Column Boston Prime C18 150*30mm*5pm Condition water (0.1%TFA)-ACN Begin B 35 End B 55 Gradient Time (min) 11 100%B Hold Time 2 Flow Rate (mL / min) 25 Injections 3) to yield 3-(((A)-4-(2-chlorophenyl)-4-(cyclopropylmethyl)-2-imino-6-oxotetrahydropyrimidin- 1 (27 / )-yl jmethyl )-M(CS')- 1 -phenylethyl)benzamide.

[0212] MS (ESI) m / z 515.2 [M+H]+.

[0213] 'HNMR (400 MHz, METHANOL-d4) 87.64 (d, J= 7.6 Hz, 1H), 7.47 - 7.38 (m, 3H), 7.38 - 7.15 (m, 6H), 7.14 - 7.04 (m, 2H), 6.61 (d, J= 8.0 Hz, 1H), 5.43 (d, J= 16.4 Hz, 1H), 5.29 - 5.13 (m, 1H), 4.62 (d, J= 16.4 Hz, 1H), 4.04 (d, J= 16.4 Hz, 1H), 3.43 (d, J= 16.4 Hz, 1H), 2.21 - 2.05 (m, 2H), 1.56 (d, J= 7.2 Hz, 3H), 0.66 - 0.55 (m, 1H), 0.52 - 0.44 (m, 1H), 0.44 -0.35 (m, 1H), 0.13 - 0.10 (m, 1H), 0.03 - -0.03 (m, 1H).26098-2Example 4

[0214] 3-[[(4R)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]-N-[(lS)-l-phenylethyl]benzamideLDA, Ti(0i-Pr)3cl> Ti(OEt)4 CH3COOMe HCI THF, -78C THF, -78C MeOH, RT Step 1 Step 2 Step 3EDC, DIPEA KOTMS THF, 50C THF, RT Step 4 Step S 4-3 INT 2AOP, DIPEA TFA DMF, RT DCM, RT Step 6 Step 7

[0215] Step 1: To a flask containing 2-cyclopropyl-l-(2-fluorophenyl)ethan-l-one (1.00 g, 1 Eq, 5.61 mmol) & (S)-2-methylpropane-2-sulfinamide (736.6 mg, 1.08 Eq, 6.078 mmol) was added anhydrous THF (12 mL) followed by Titaniumethoxide (2.56 g, 2.34 mL, 2 Eq, 11.2 mmol). The reaction mixture (clear red / brown solution) was then capped with a vent needle (not under Nitrogen) & stirred at 75°C with a tall air cooled condenser attached. Followed by LC / MS.After 1 night at 75°C the reaction mixture was cooled to 0°C (ice water bath) then quenched by addition of saturated sodium citrate, then diluted with EtOAc & warmed to room temperature.The mixture was stirred for 15 minutes. The organic layer was separated, then washed with saturated saturated sodium bicaronate, then brine. The organic layer was then dried over anhydrous sodium sulfate, then filtered & concentrated. The resulting residue was purified by silica gel chromatography (0-60% EtOAc / Hex; 14 CV; 120g ISCO silica column). The desired26098-2fractions were concentrated, then dissolved in DCM / MeOH & concentrated to yield (S, Z)-N-(2-cyclopropyl-l-(2-fluorophenyl)ethylidene)-2-methylpropane-2-sulfmamide.MS (ESI) m / z 282.7 (M+H)+

[0216] Step 2: To a solution of LDA in THF (944.3 mg, 8.815 mL, 1 molar, 3 Eq,8.815 mmol) under an atmosphere of Nitgroen was cooled to -78 °C, then methyl acetate (544.2 mg, 584 pL, 2.5 Eq, 7.346 mmol) was added. After 1 hour at -78 °C a solution of Chlorotitanium triisopropoxide in THF (2.680 g, 10.28 mL, 1 molar, 3.5 Eq, 10.28 mmol) was added at -78 °C. After an additonal 40 mintutes at -78 °C a solution of (S, Z)-N-(2-cyclopropyl-l-(2-fluorophenyl)ethylidene)-2-methylpropane-2-sulfmamide (826.8 mg, 1 Eq, 2.938 mmol) in andyrous THF was then added dropwise and the mixture was stirred at -78 °C (under an atmosphere of Nitrogen) for ~30 minutes, then allowed to warm to room tempeature and stirred overnight. After 1 night at room tempature, the reaction mixture was diluted with EtOAc and quenched with ice cold saturated aqueous NH4C1. The organic layer was then separated, then washed with water, then brine. The orgaic layer was then then dried over anhydrous sodium sulfate, then filtered & concentrated. The resulting residue was then purified by silica gel chromatography (0-60% EtOAc / Hex; 16 CV; 120g ISCO silica column). The desired fractions were concentrated, dried to yield methyl 3-(((S)-tert-butylsulfmyl)amino)-4-cyclopropyl-3-(2-fluorophenyl)butanoate.

[0217] MS (ESI) m / z 356.3 (M+H)+

[0218] Step 3: To a solution of methyl 3-(((S)-tert-butylsulfinyl)amino)-4-cyclopropyl-3-(2-fluorophenyl)butanoate (380.4 mg, 1 Eq, 1.070 mmol) in MeOH (3 mL) was added a saturated solution of HC1 in EtOAc (~4N) (195.1 mg, 1.338 mL, 4 molar, 5 Eq, 5.351 mmol). The reaction was stirred at room temperature for ~30 minutes then diluted with MeOH & concentrated to yield methyl (R)-3-amino-4-cyclopropyl-3-(2-fluorophenyl)butanoate (280.2 mg, 1.5 Eq, 1.115 mmol) was added methyl 3-((3-(tert-butoxycarbonyl)thioureido)methyl)benzoate which was used crude without further modification.MS (ESI) m / z 252.2 (M+H)+

[0219] Step 4: To a vial containing methyl (R)-3-amino-4-cyclopropyl-3-(2-fhiorophenyl)butanoate (280.2 mg, 1.5 Eq, 1.115 mmol) was added methyl 3-((3-(tert-butoxycarbonyl)thioureido)methyl)benzoate (241.1 mg, 1 Eq, 743.3 pmol),26098-2then EDC (855.0 mg, 6 Eq, 4.460 mmol), then anhydrous THF (15 mL), & finally DIPEA (576.5 mg, 777 pL, 6 Eq, 4.460 mmol).

[0220] The reaction mixture was then capped (not under Nitrogen) & stirred at 50°C. After 1 night at 50°C the reaction mixture was concentrated. The resulting residue was dissolved in MeOH / DMSO & purified (without workup) by reverse phase chromatography (40-100% MeCN / H2O; 5 mM Ammonium Bicarbonate modifier in AQ only; 30 min gradient; XB RIDGE 50x250 mm 5 micron C18 column; Flow = 118.1 mL / min). The desired fractions were concentrated, then dissolved in DCM / MeOH and concentrated to yield methyl (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-6-oxotetrahydropyrimidin-1 (2H)-yl)methyl)benzoate.

[0221] MS (ESI) m / z 510.6 (M+H)+

[0222] Step 5: To a vial containing methyl (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-6-oxotetrahydropyrimidin-l(2H)-yl)methyl)benzoate (143.3 mg, 1 Eq, 281.2 pmol) was added anhydrous THF, then KOTMS (252.5 mg, 7 Eq, 1.968 mmol). The reaction mixture was then capped (not under Nitrogen) & stirred at room temperature for 1 hour. The reaction mixture was then quenched with IM HC1, then diluted with EtOAc. The organic layer was separated and then washed with IM HC1, then brine. The organic layer was then dried over anhydrous sodium sulfate, then filtered & concentrated to yield (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-6-oxotetrahydropyrimidin-l(2H)-yl)methyl)benzoic acid which was used crude without further modification.

[0223] MS (ESI) m / z 496.6 (M+H)+

[0224] Step 6: To a vial containing (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-6-oxotetrahydropyrimidin- 1 (2H)-yl)methyl)benzoic acid (40.0 mg, 1 Eq, 80.7 pmol), (S)-(-)-l -Phenylethylamine (14.7 mg, 15.6 pL, 1.5 Eq, 121 pmol), & 7-Azabenzotriazol-l-Yloxytris(Dimethylamino)PhosphoniumHexafluorophosphate (53.7 mg, 1.5 Eq, 121 pmol) was added anhydrous DMF (1.5 mL), then DIPEA (31.3 mg, 42.2 pL, 3 Eq, 242 pmol). The reaction miture was then capped (not under Nitrogen) & stirred at room temperature. Followed by LC / MS. After 3 hours at room temperature the reaction was diluted with MeOH & concentrated. The resulting residue was dissolved in MeOH / DMSO then purified (without workup) by reverse phase chromatography (50-100% MeCN / H2O; 5 mM Ammonium Bicarbonate modifier in AQ only; 25 min gradient; XBRIDGE 50x250 mm 5 micron Cl 826098-2column; Flow = 118.1 mL / min). The desired fractions were concentrated, then dissolved in DCM / MeOH & concentrated to yield tert-butyl ((R, E)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-6-oxo-l-(3-(((S)-l-phenylethyl)carbamoyl)benzyl)tetrahydropyrimidin-2(lH)-ylidene)carbamate.

[0225] MS (ESI) m / z 599.7 (M+H)+

[0226] Step 7: To a vial containing tert-butyl ((R, E)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-6-oxo-l-(3-(((S)-l-phenylethyl)carbamoyl)benzyl)tetrahydropyrimidin-2(lH)-ylidene)carbamate (34.0 mg, 1 Eq, 56.8 pmol) was added DCM (3 mL) followed by 2,2,2-trifluoroacetic acid (0.4 g,.3 mL, 7e+l Eq, 4 mmol). After 1 hour at room temperature the reaction mixture was diluted with MeOH then concentrated. The resulting residue was then dissolved in MeOH / DMSO, then purified (without workup) by reverse phase chromatography (1 x 4.0 mL injection) (20-100% MeCN / H2O; 0.1% TFA modifier; 20 min gradient; Waters 50x250 mm Sunfire 5 micron C18 column; Flow = 118 mL / min). The desired fractions were concentrated, then dissolved in DCM / MeOH & concentrated, then dissolved in some MeCN / water, froze & lyophilized (~36 hrs) to yield 3-[[(4R)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]-N-[(lS)-l-phenylethyl]benzamide.

[0227] MS (ESI) m / z 499.3 (M+H)+

[0228] 'H NMR (500 MHz, MeOD) 87.58 (d, J = 7.8 Hz, 1H), 7.40 (s, 1H), 7.33 (d, J = 7.3 Hz, 2H), 7.27 (t, J = 7.7 Hz, 2H), 7.21 - 7.14 (m, 2H), 7.14 - 7.08 (m, 1H), 7.06 (t, J = 7.8 Hz, 1H), 6.98 - 6.86 (m, 2H), 6.65 (d, J = 7.8 Hz, 1H), 5.35 (d, J = 16.5 Hz, 1H), 5.14 (q, J = 7.0 Hz, 1H), 4.59 (d, J = 16.5 Hz, 1H), 3.68 (d, J = 16.4 Hz, 1H), 3.39 (d, J = 16.4 Hz, 1H), 2.04 - 1.97 (m, 1H), 1.87 - 1,79 (m, 1H), 1.48 (d, J = 7.1 Hz, 3H), 0.60 - 0.51 (m, 1H), 0.41 - 0.33 (m, 2H), 0.07 - -0.11 (m, 2H).Example 526098-2

[0229] 3-((4-cyclobutyl-2-imino-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)-N-((S)- 1 -phenylethyl)benzamideLDA, Ti(Oi-Pr)3Cl, CH3COOMe, EDCI, DIEA Ti(OEt)4. THF, THF, 25 °C, 16 h then SFC Step 15-1steP25-2 Step 4

[0230] Step 1: To a stirred solution of cyclobutyl(phenyl)methanone (6.00 g, 1 Eq, 37.4 mmol) and (5)-2-methylpropane-2-sulfinamide (5.45 g, 1.2 Eq, 44.9 mmol) in THF (60 mL) was added titaniumethoxide (17.1 g, 15.6 mL, 2 Eq, 74.9 mmol) at 25 °C under N2 atmosphere. The reaction mixture was stirred at 75 °C for 16 h. Followed by LC / MS. Then the reaction mixture was quenched with sat. NaHCCh (50 mL), filtered, the filter cake was washed with EtOAc (3 x 50 mL). The mixture was separated, then the aqueous layer was extracted with EtOAc (3 x 50 mL).The combined organic layers were dried over sodium sulfate, filtered, the filtrate was concentrated. The resulting residue was purified by flash silica gel chromatography (ISCO®; 80 g Agela Silica Flash Column, eluent of 0-10% ethyl acetate / hexane gradient @ 60 mL / min) to afford (5)-A-(cyclobutyl(phenyl)methylene)-2-methylpropane-2-sulfinamide.

[0231] MS (ESI) m / z 264.1 [M+H]+. 'H NMR (400 MHz, CDCI3) 87.42 - 7.30 (m, 5H), 3.98 -3.28 (m, 1H), 2.46 - 2.12 (m, 4H), 2.03 - 1.92 (m, 1H), 1.83 - 1.82 (m, 1H), 1.24 (s, 9H).

[0232] Step 2: To a solution of methyl acetate (2.04 g, 2.19 mL, 2.5 Eq, 27.5 mmol) in anhydrous THF (20 mL) was added LDA (3.54 g, 16.5 mL, 2 molar, 3 Eq, 33.0 mmol) (2 M in THF) dropwise under N2 atmosphere at -78 °C. The reaction mixture was stirred at -78 °C for 1 h and then was added chlorotitanium triisopropoxide (38.5 mL, 1 molar, 3.5 Eq, 38.5 mmol) dropwise at -78 °C. After stirred at -78 °C for 1 h, a solution of (S)-N-(cyclobutyl(phenyl)methylene)-2-methylpropane-2-sulfinamide (2.90 g, 1 Eq, 11.0 mmol) in anhydrous THF (30 mL) was added dropwise at -78 °C. The mixture was stirred at -78 °C for 2 h. Followed by LC / MS. After 2 h stirred at -78 °C the reaction mixture was quenched with ice-26098-2cold half-saturated aqueous ammonium chloride solution (20 mL), extracted with EtOAc (3 x 20 mL). The organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated. The resulting residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0~5% EtOAc / hexane gradient @ 30 mL / min) to afford methyl 3-((fS')- / c / 7-butylsulfinyl)amino)-3-cyclobutyl-3-phenylpropanoate.

[0233] MS (ESI) m / z 338.1 [M+H]+.

[0234] 'H NMR (400 MHz, CDC13) 87.44 - 7.40 (m, 1H), 7.30 - 7.28 (m, 2H), 7.21 - 7.20 (m, 2H), 3.55 - 3.50 (m, 3H), 3.24 (d, J= 16.5 Hz, 0.5H), 3.17 (d, J= 1.8 Hz, 1H), 3.12 - 2.89 (m, 1H), 2.73 (d, J= 16.5 Hz, 1H), 1.77 - 1.55 (m, 6H), 1.26 - 1.20 (m, 9H).

[0235] Step 3: A solution of methyl 3-(((5)-tert-butylsulfinyl)amino)-3-cyclobutyl-3-phenylpropanoate (1.00 g, 1 Eq, 2.96 mmol) in HCl-MeOH (2M, 10 mL) was stirred at 25 °C for 1 hour. Followed by LC / MS. Then the reaction mixture was concentrated to yield methyl 3-amino-3-cyclobutyl-3-phenylpropanoate hydrochloride. The crude product was used directly in the next step without further purification.

[0236] MS (ESI) m / z 234.1 [M+H]+.

[0237] 'H NMR (400 MHz, MeOD) 67.52 - 7.45 (m, 2H), 7.44 - 7.36 (m, 3H), 3.63 (s, 3H), 3.44 (d, J= 17.2 Hz, 1H), 3.06 (d, J= 17.2 Hz, 1H), 2.98 - 2.85 (m, 1H), 1.98 - 1.79 (m, 5H), 1.72 - 1.63 (m, 1H).

[0238] Step 4: To a solution of methyl 3-((3-( / c77-butoxycarbonyl)thioureido)methyl)benzoate (870 mg, 1 Eq, 2.68 mmol), methyl 3-amino-3-cyclobutyl-3-phenylpropanoate (690 mg, 1.10 Eq, 2.96 mmol) and EDCI (1.29 g, 2.5 Eq, 6.70 mmol) in THF (10 mL) was added DIEA (1.56 g, 2.10 mL, 4.5 Eq, 12.1 mmol). The reaction mixture was stirred at 25 °C for 16 h under N2 atmosphere. Followed by LC / MS. Then the reaction mixture was diluted with water (10 mL), extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated. The resulting residue was purified by flash column (ISCO®; 12 g Agela Silica Flash Column, eluent of 0-20% ethyl acetate / hexane gradient @ 30 mL / min) to afford methyl 3-((2-((terL butoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2Z / )-yl)methyl)benzoate.

[0239] MS (ESI) m / z 492.3 [M+H]+.26098-2

[0240] SFC: The racemic methyl 3-((2-((ter / -butoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)methyl)benzoate (900 mg, 1 Eq, 1.83 mmol) was separated by preparative SFC (Column REGIS (R, R)WHELK-Ol(250mm*25mm, 10pm); Condition: 0.1%NH3H2O / EtOH; Begin B 60%, End B 60%; FlowRate (mL / min): 80) to afford methyl (E)-3-((2-((ter / -butoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)benzoate. The second eluting isomer from SFC was the desired isomer.

[0241] MS (ESI) m / z 492.2 [M+H]+

[0242] Step 5: To a solution of methyl (E)-3-((2-((ter / -butoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)benzoate (100 mg, 1 Eq, 203 pmol) in THF (1 mL) was added potassium trimethyl(oxido)silane (157 mg, 0.17 mL, 6 Eq, 1.22 mmol). The reaction mixture was stirred at 25 °C for 1 hour. Followed by LC / MS. Then the reaction mixture was quenched with water (5 mL) and used HC1 (1 N) to neutralize the reaction mixture to pH = 7, extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated to afford 3-((2-((ter / -butoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)benzoic acid. The crude product was used directly in the next step without further purification.

[0243] MS (ESI) m / z: 478.3 [M+H]+

[0244] Step 6: To a solution of 3-((2-((ter / -butoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)methyl)benzoic acid (97.0 mg, 1 Eq, 203 pmol) in THF (3 mL) was added (5)-l-phenylethan-l-amine (32.0 mg, 1.3 Eq, 264 pmol), EDCI (156 mg, 4 Eq, 812 pmol), HOBt (93.3 mg, 3 Eq, 609 pmol) and DIEA (158 mg, 212 pL, 6 Eq, 1.22 mmol) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 16 hours. Followed by LC / MS. Then the reaction mixture was concentrated to afford tert-butyl (4-cyclobutyl-6-oxo-4-phenyl-l-(3-(((5 -l-phenylethyl)carbamoyl)benzyl)tetrahydropyrimidin-2(U7)-ylidene)carbamate. The crude product was used directly in the next step without further purification.

[0245] MS (ESI) m / z: 581.3 [M+H]+.

[0246] Step 7: A solution of tert-butyl (4-cyclobutyl-6-oxo-4-phenyl-l-(3-(((5)-l-phenylethyl)carbamoyl)benzyl)tetrahydropyrimidin-2( IT / )-ylidene)carbamate (110 mg, 1 Eq, 189 pmol) in TFA / DCM (v / v=2 / l, 1 mL) was stirred at 25 °C for 0.5 h. Followed by LC / MS. Then26098-2the reaction mixture was concentrated and the resulting residue was purified by reverse preparative HPLC (Column: YMC-Actus Triart C18 150*30mm*5pm; Condition: water(0.1%TFA)-ACN Begin B 23 End B 53 Gradient Time (min) 11 100% B Hold Time 1.1 Flow Rate (mL / min) 40) to afford 3-((4-cyclobutyl-2-imino-6-oxo-4-phenyltetrahydropyrimidin-1 (2 / / )-yl )methyl )- / f-(fS')- 1 -phenylethyl)benzamide.

[0247] MS (ESI) m / z 481.1 [M+H]+.:

[0248] 'H NMR (400 MHz, MeOD) 87.63 (d, J= 7.6 Hz, 1H), 7.46 (s, 1H), 7.43 - 7.38 (m, 2H), 7.37 - 7.31 (m, 2H), 7.28 - 7.20 (m, 6H), 7.06 (t, J= 7.6 Hz, 1H), 6.46 (d, J= 7.6 Hz, 1H), 5.40 (d, J= 16.4 Hz, 1H), 5.26 - 5.16 (m, 1H), 4.60 (d, J= 16.8 Hz, 1H), 3.55 (d, J= 16.4 Hz, 1H), 3.25 (d, J = 16.4 Hz, 1H), 2.89 - 2.84 (m, 1H), 2.01 - 1.68 (m, 6H), 1.56 (d, J = 7.2 Hz, 3H).Example 6

[0249] 3-((lA)-l-(4-cyclobutyl-2-imino-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)ethyl)-N- ((5)- 1 -phenylethyl)benzamide

[0250] Step 1: To a solution of Formic acid (3.87 g, 3.22 mL, 3 Eq, 84.2 mmol) and TEA (17.0 g, 23.5 mL, 6 Eq, 168 mmol) in DMF (50 mL) was stirred for 15 min under N2 atmosphere, then RuCl( / ?-cymene)[(5,5)-Ts-DPEN] (893 mg, 0.05 Eq, 1.40 mmol) and methyl 3 -acetylbenzoate26098-2(5.00 g, 1 Eq, 28.1 mmol) was added into above mixture. The mixture was stirred at 25 °C for 16 h. Followed by LC / MS. Then the mixture was diluted with water (30 mL), extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with water (20 mL x 5), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated. The resulting residue was purified by flash silica gel chromatography (ISCO®; Agela® Flash Column Silica-CS (40 g), Eluent of 0~5% Ethyl acetate / hexane gradient @40 mL / min) to afford methyl (S)-3-(l-hydroxyethyl)benzoate

[0251] MS (ESI) m / z: 163.1 [M-18+H]+.

[0252] 'H NMR (400 MHz, CDC13) 88.02 (s, 1H), 7.93 - 7.90 (m, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 4.96 - 4.91 (m, 1H), 3.90 (s, 3H), 2.24 (br s, 1H), 1.50 (d, J = 6.4 Hz, 3H).

[0253] Step 2: To a solution of methyl (5)-3-(l-hydroxyethyl)benzoate (4.70 g, 1 Eq, 26.1 mmol) in toluene (50 mL) was added diphenylphosphoryl azide (21.5 g, 16.9 mL, 3 Eq, 78.2 mmol) and DBU (11.9 g, 11.8 mL, 3 Eq, 78.2 mmol) at 25 °C. The mixture was stirred at 50 °C for 16 hours under N2 atmosphere. TLC showed new spots was formed and starting material was consumed completely. The mixture was then concentrate and the resulting residue was purified by flash silica gel chromatography (ISCO®; 80 g Agela Silica Flash Column, Eluent of 0-10% EtOAc / Hexane gradient @ 30 mL / min) to give methyl (A)-3-(l-azidoethyl)benzoate.

[0254] 'H NMR (400 MHz, CDCI3) 68.03 - 7.97 (m, 2H), 7.57 - 7.51 (m, 1H), 7.50 - 7.44 (m, 1H), 4.69 (q, J= 6.8 Hz, 1H), 3.94 (s, 3H), 1.56 (d, J= 6.4 Hz, 3H).

[0255] Step 3: To a solution of methyl (A)-3-(l-azidoethyl)benzoate (4.3 g, 1 Eq, 21 mmol) in THF (50 mL) was added Pd / C (2.2 g, 10% Wt, 0.1 Eq, 2.1 mmol) (10% Wt / Wt) at 25 °C under N2 atmosphere. The mixture was degassed and backfilled with H2 (3x). The mixture was stirred at 25 °C for 16 hours under H2 (15 psi) atmosphere. LC / MS and TLC showed new spots was formed and starting material was consumed completely. The mixture was filtered by Celite and the filter cake was washed with MeOH (3 x 10 mL). The filtrate was concentrated and the resulting residue was purified by flash silica gel chromatography (ISCO®; 40 g Agela Silica Flash Column, Eluent of 30%-100% EtOAc / Hexane gradient @ 30 mL / min) to give methyl (R)-3-(l-aminoethyl)benzoate.

[0256] MS (ESI) m / z 163.1 [M- 17+H]+.26098-2

[0257] 'H NMR (400 MHz, CDCh) 68.02 (s, 1H), 7.91 (d, J= 7.6 Hz, 1H), 7.55 (d, J= 7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 4.18 (q, J= 6.4 Hz, 1H), 3.91 (s, 3H), 1.64 (s, 2H), 1.40 (d, J = 6.4 Hz, 3H).

[0258] Step 4: To a solution of tert-butyl A-({[(tert-butoxy)carbonyl]amino}methanethioyl)carbamate (3.855 g, 1 Eq, 13.95 mmol) in THF (40 mL) was added sodium hydride (1.116 g, 60% Wt, 2 Eq, 27.90 mmol) (60% in mineral oil) at 0 °C in portions under N2 atmosphere. The mixture was stirred at 0 °C for 1 h. 2,2,2-trifluoroacetic anhydride (3.076 g, 2.036 mL, 1.05 Eq, 14.65 mmol) in THF (30 mL) was added dropwise at 0 °C. The mixture was stirred at 0 °C for another 1 h. Then methyl (A)-3-(l-aminoethyl)benzoate (2.500 g, 1 Eq, 13.95 mmol) in THF (30 mL) was added dropwise at 0 °C. The mixture was stirred at 25 °C for 16 h. LC / MS and TLC (EtOAc: Hex = 1:5) showed the starting material was consumed and desired product was formed. The mixture was quenched with NH4CI (50 mL), extracted with EtOAc (3 x 50 mL). The organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated. The resulting residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0~5% EtOAc / Hexane gradient @ 50 mL / min) to yield methyl (R)-3-(l-(3-(tert-butoxy carb ony 1 )thi ourei do)ethy l)b enzoate.

[0259] MS (ESI) m / z: 339.2 [M+H]+.

[0260] 'H NMR (400 MHz, CDCh) 8 10.06 (d, J = 6.8 Hz, 1H), 8.03 (s, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.88 (br s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 5.66 - 5.60 (m, 1H), 3.93 (s, 3H), 1.63 (d, J = 6.8 Hz, 3H), 1.50 (s, 9H).

[0261] Step 5: To a solution of methyl (A)-3-(l-(3-(tert-butoxycarbonyl)thioureido)ethyl)benzoate (700 mg, 1 Eq, 2.07 mmol), methyl 3-amino-3-cyclobutyl-3-phenylpropanoate (531 mg, 1.1 Eq, 2.28 mmol) and EDCI (991 mg, 2.5 Eq, 5.17 mmol) in THF (10 mL) was added DIEA (1.20 g, 1.62 mL, 4.5 Eq, 9.31 mmol). The reaction was stirred at 25 °C for 16 h under N2 atmosphere. Followed by LC / MS. Them the reaction mixture was diluted with water (10 mL), and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated in vacuum to give methyl 3-((A)-l-(2-(tert-butoxycarbonyl)-3-((5)-l-cyclobutyl-3-methoxy-3-oxo-l-phenylpropyl)guanidino)ethyl)benzoate. The crude product was used directly in the next step without further purification.

[0262] MS (ESI) m / z 538.8 [M+H]+.26098-2

[0263] Step 6: To a solution of methyl 3-((A)-l-(2-(ter / -butoxycarbonyl)-3-((5)-l -cyclobutyl-3-methoxy-3-oxo-l-phenylpropyl)guanidino)ethyl)benzoate (1.10 g, 1 Eq, 2.05 mmol) in THF (10 mL) was added DBU (934 mg, 925 pL, 3 Eq, 6.14 mmol). The reaction was stirred at 50 °C for 1 h under N2 atmosphere. Followed by LC / MS. Then the reaction mixture was diluted with water (10 mL), extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated. The resulting residue was purified by flash column (ISCO®; 12 g Agela Silica Flash Column, eluent of 5-15% ethyl acetate / hexane gradient @ 30 mL / min) to afford methyl 3-((A)-l-((5)-2-((tert-butoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2Z / )-yl)ethyl)benzoate.

[0264] MS (ESI) m / z 506.3 [M+H]+.

[0265] SFC: The racemic methyl 3-((lA)-l-(2-((terLbutoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)ethyl)benzoate (600 mg, 1 Eq, 1.19 mmol) was separated by preparative SFC (Column REGIS (R, R) WHELK-01(250 mm*25mm, 10 pm); Condition: 0.1%NH3H2O / EtOH; Begin B 45%, End B 45%; FlowRate (mL / min): 80) to afford methyl 3-((lA)-l-(2-((terLbutoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)ethyl)benzoate. The second eluting isomer from SFC was the desired isomer.

[0266] MS (ESI) m / z 506.4 [M+H]+.

[0267] Step 7: To a solution of methyl 3-((lA)-l-(2-((terLbutoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)ethyl)benzoate (100 mg, 1 Eq, 198 pmol) in THF (1 mL) was added potassium trimethyl(oxido)silane (152 mg, 0.17 mL, 6 Eq, 1.19 mmol) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 1 hour. Followed by LC / MS. Then the reaction mixture was quenched with water (5 mL) and used HC1 (1 N) to acid the reaction mixture to pH ~ 6-7, then extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated to afford 3-((lA)-l-(2-((terLbutoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)ethyl)benzoic acid. The crude product was used directly in the next step without further purification.

[0268] MS (ESI) m / z: 492.3 [M+H]+.26098-2

[0269] Step 8: To a solution of 3-((lA)-l-(2-((tert-butoxycarbonyl)imino)-4-cyclobutyl-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)ethyl)benzoic acid (67.0 mg, 1 Eq, 136 pmol) in THF (1 mL) was added (5)-l-phenylethan-l-amine (21.5 mg, 1.3 Eq, 177 pmol), EDCI (105 mg, 4 Eq, 545 pmol), HOBt (62.6 mg, 3 Eq, 409 pmol) and DIEA (106 mg, 142 pL, 6 Eq, 818 pmol) under N2 atmosphere. The reaction mixture was stirred at 25 °C for 16 hours under N2 atmosphere. Followed by LC / MS. Then the reaction mixture was concentrated to afford tert-butyl ((E)-4-cy clobutyl-6-oxo-4-phenyl- 1 -((R)- 1 -(3 -(((S)- 1 -phenylethyl)carbamoyl)phenyl)ethyl)tetrahydropyrimidin-2(lH)-ylidene)carbamate. The crude product was used directly in the next step without further purification.

[0270] MS (ESI) m / z: 595.3 [M+H]+.

[0271] Step 9: A solution of tert-butyl (4-cy cl obutyl-6-oxo-4-phenyl-l -(( / ?)- 1 -(3 -((($)- 1-phenylethyl)carbamoyl)phenyl)ethyl)tetrahydropyrimidin-2(U7)-ylidene)carbamate (157 mg, 1 Eq, 264 pmol) in TFA / DCM (v / v = 2 / 1, 1 mL) was stirred at 25 °C for 0.5 h. Followed by LC / MS. Then the reaction mixture was concentrated & the resulting residue was purified by reverse preparative HPLC (Column: YMC-Actus Triart C18 150*30mm*5pm; Condition: water (0.1%TFA)-ACN Begin B 27 End B 57 Gradient Time (min) 11 100% B Hold Time 1.1 Flow Rate (mL / min) 40) to afford 3-((lA)-l-(4-cyclobutyl-2-imino-6-oxo-4-phenyltetrahydropyrimidin-l(2J7)-yl)ethyl)-N-((5)-l-phenylethyl)benzamide.

[0272] MS (ESI) m / z 495.3 [M+H]+.

[0273] 'H NMR (400 MHz, MeOD) 87.71 (d, J= 7.6 Hz, 1H), 7.63 (s, 1H), 7.48 - 7.42 (m, 2H), 7.41 - 7.37 (m, 3H), 7.36 - 7.31 (m, 4H), 7.29 - 7.21 (m, 2H), 6.75 (d, J= 7.6 Hz, 1H), 5.69 - 5.63 (m, 1H), 5.28 - 5.18 (m, 1H), 3.48 (d, J = 16.4 Hz, 1H), 3.26 (d, J = 16.4 Hz, 1H), 2.89 -2.83 (m, 1H), 2.01 - 1.98 (m, 2H), 1.91 - 1.78 (m, 3H), 1.75 - 1.66 (m, 1H), 1.57 - 1.53 (m, 6H).Example 7HN26098-2

[0274] 3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-N-[(lS)-l-phenylethyl]-5-(trifluoromethyl)benzamideCS2CO3 KOTOS DMF, RT THF, RT Step 1 Step 2AOP, DIPEA TFA DMF, RT DCM, RT Step 3 Step 4

[0275] Step 1: To a vial containing tert-butyl (R, E)-(4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-2(lH)-ylidene)carbamate (1.241 g, 1 Eq, 3.614 mmol) was added cesium carbonate (1.491 g, 1.266 Eq, 4.576 mmol), followed by Methyl 3-(bromomethyl)-5-(trifluoromethyl)benzoate (1.24 g, 1.16 Eq, 4.17 mmol) and finally anhydrous DMF (10 mL).The reaction mixture was then capped (not under Nitrogen) & stirred at room temperature.Followed by LC / MS. After ~50 min the reaction mixture was diluted with EtOAc, and saturated sodium bicarbonate / water. The organic layer was separated, then washed with saturated sodium bicarbonate, then water, then brine. The organic layer was then dried over anhydrous sodium sulfate, then filtered & concentrated. The resulting residue was then dissolved in MeOH / DMSO & purified (60-100% MeCN / H2O; 5 mM Ammonium Bicarbonate modifier in AQ only; 30 min gradient; XBRIDGE 50x250 mm 5 micron C18 column; Flow = 118.1 mL / min). The desired fractions were concentrated then dissolved in DCM / MeOH & concentrated to yield methyl(R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)-5-(trifluoromethyl)benzoate.

[0276] MS (ESI) m / z 560.7 (M+H)+

[0277] Step 2: To a vial containing methyl (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)-5-(trifluoromethyl)benzoate (561.0 mg, 1 Eq, 1.003 mmol) was added anhydrous THF, then KOTMS (900.3 mg, 7 Eq, 7.018 mmol). The reaction mixture was then capped (not under26098-2Nitrogen) & stirred at room temperature. Followed by LC / MS. The reaction was stirred for 2 hours at room temperature then quenched with IM HC1, then diluted with EtOAc, then separated. The organic layer was then washed with IM HC1, then brine. The organic layer was then dried over anhydrous sodium sulfate, then filtered & concentrated to yield (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)-5-(trifluoromethyl)benzoic acid.

[0278] MS (ESI) m / z 546.6 (M+H)+

[0279] Step 3: To a vial containing (R, E)-3-((2-((tert-butoxycarbonyl)imino)-4-(cyclopropylmethyl)-6-oxo-4-phenyltetrahydropyrimidin-l(2H)-yl)methyl)-5-(trifluoromethyl)benzoic acid (280.4 mg, 1 Eq, 514.0 pmol), (S)-(-)-l -Phenylethylamine (93.42 mg, 99.3 pL, 1.5 Eq, 771.0 pmol), & 7-Azabenzotriazol-l-Yloxytris(Dimethylamino)PhosphoniumHexafluorophosphate (341.7 mg, 1.5 Eq, 771.0 pmol) was added anhydrous DMF (7 mL), then DIPEA (199.3 mg, 269 pL, 3 Eq, 1.542 mmol). The reaction mixture was then capped (not under Nitrogen) & stirred at room temperature. Followed by LC / MS. After 30 minutes at room temperature the reaction mixture was diltued with MeOH & concentrated. The resulting residue was then diluted with MeOH / DMSO then purified (without workup) by reverse phase chromatography (50-100% MeCN / H2O; 5 mM Ammonium Bicarbonate modifier in AQ only; 25 min gradient; XBRIDGE 50x250 mm 5 micron Cl 8 column; Flow = 118.1 mL / min). The desired fractions were concentrated then dissolved in DCM / MeOH & concentrated to yield tert-butyl ((R, E)-4-(cyclopropylmethyl)-6-oxo-4-phenyl-l-(3-(((S)-l-phenylethyl)carbamoyl)-5-(trifluoromethyl)benzyl)tetrahydropyrimidin-2(lH)-ylidene)carbamate.

[0280] MS (ESI) m / z 649.5 (M+H)+

[0281] Step 4: To a vial containing tert-butyl ((R, E)-4-(cyclopropylmethyl)-6-oxo-4-phenyl-l-(3-(((S)-l-phenylethyl)carbamoyl)-5-(trifluoromethyl)benzyl)tetrahydropyrimidin-2(lH)-ylidene)carbamate (250.0 mg, 1 Eq, 385.4 pmol) was added DCM (10 mL) followed by Trifluoroacetic acid (3.0 g, 2.0 mL, 68 Eq, 26 mmol). After 1 hour at room temperature the reaction mixture was concentrated. The resulting residue was then diluted with MeOH / DMSO then purified (without workup) by reverse phase chromatography (20-100% MeCN / H2O; 0.1% TFA modifier; 20 min gradient; Waters 50x250 mm Sunfire 5 micron C18 column; Flow = 118 mL / min). The desired fractions were concentrated then dissolved in DCM / MeOH & concentrated. Then dissolved in MeCN / water, froze & lyophilized (~36 hrs) to yield 3-[[(4R)-4-26098-2(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-N-[(lS)-l-phenyl ethyl ] - 5 -(tri fluor omethy l)b enzami de.

[0282] MS (ESI) m / z 549.7 (M+H)+

[0283] 'H NMR (500 MHz, MeOD) 68.93 (d, J = 7.4 Hz, 1H), 7.97 (s, 1H), 7.59 (s, 1H), 7.39(d, J = 7.4 Hz, 2H), 7.36 - 7.30 (m, 3H), 7.26 - 7.20 (m, 1H), 7.19 - 7.15 (m, 2H), 7.10 - 7.02 (m, 3H), 5.35 (d, J = 16.5 Hz, 1H), 5.23 - 5.17 (m, 1H), 4.72 (d, J = 16.5 Hz, 1H), 3.61 (d, J = 16.4Hz, 1H), 3.41 (d, J = 16.4 Hz, 1H), 1.99 - 1.92 (m, 1H), 1.71 - 1.63 (m, 1H), 1.55 (d, J = 7.0 Hz,3H), 0.62 - 0.51 (m, 1H), 0.46 - 0.37 (m, 2H), 0.06 - -0.05 (m, 2H).

[0284] Example 8

[0285] 3 -((A)- 1 -(2-imino-6-oxo-4,4-diphenyltetrahydropyrimidin- 1 (2J7)-yl)ethyl)-7V-((5)- 1 -phenyl ethyl)- 5 -(tri fluor omethy l)b enzami de

[0286] Step 1: To a solution of Formic acid (875 mg, 727 pL, 3 Eq, 19.0 mmol) and TEA (3.85 g, 5.30 mL, 6 Eq, 38.0 mmol) in DMF (5 mL) was stirred for 15 min, then RuCl(p-cymene) [(5, 5)-Ts-DPEN] (40.3 mg, 0.01 Eq, 63.4 pmol) and methyl 3-acetyl-5-(trifluoromethyl)benzoate (1.56 g, 1 Eq, 6.34 mmol) was added and the mixture was stirred for 25 h at 25 °C under N2 atmosphere. Followed by LC / MS and TLC. Then the reaction mixture was quenched with water (60 mL), extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine (40 mL), dried over anhydrous Na2SO4, filtered and concentrated. The resulting residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, eluent of 0-15% ethyl acetate / hexane gradient @ 45 mL / min) to afford methyl CS')-3-( l-hydroxyethyl)-5-(trifluoromethyl)benzoate.

[0287] 'H NMR (400 MHz, CDCI3) 88.22 (d, J= 7.2 Hz, 2H), 7.87 (s, 1H), 5.04 (q, J= 6.4 Hz, 1H), 3.97 (s, 3H), 1.55 (d, J= 6.4 Hz, 3H).

[0288] Step 2: To a solution of methyl (5)-3-(l-hydroxyethyl)-5-(trifluoromethyl)benzoate (1.40 g, 1 Eq, 5.64 mmol) in Toluene (30 mL) was added DPPA (4.66 g, 3.65 mL, 3 Eq, 16.9 mmol) and DBU (2.58 g, 2.55 mL, 3 Eq, 16.9 mmol) at 20 °C. Then the reaction mixture was heated to 50 °C for 12 h under N2 atmosphere. Followed by LC / MS and TLC. The reaction mixture was then concentrated and the resulting residue was purified by flash silica gel chromatography (ISCO®; 20 g Agela Silica Flash Column, Eluent of 0-10% ethyl acetate / hexane gradient @ 40 mL / min) to give methyl ( / )-3-(l -azidoethyl )-5-(trifluoromethyl)benzoate.

[0289] 'H NMR (400 MHz, CDCI3) 68.26 (s, 1H), 8.20 (s, 1H), 7.79 (s, 1H), 4.78 (q, J= 6.8 Hz, 1H), 3.98 (s, 3H), 4.78 (d, J= 6.8 Hz, 3H).

[0290] Step 3: To a solution of methyl (A)-3-(l-azidoethyl)-5-(trifluoromethyl)benzoate (1.050 g, 1 Eq, 3.843 mmol) in EtOH (20 mL) was added Pd / C (409.0 mg, 10% Wt, 0.1 Eq, 384.3 pmol) at 20 °C under N2 atmosphere. The mixture was degassed and backfilled with H2 (3x). The mixture was stirred at 25 °C for 2 h under H2 (15 psi). Followed by LC / MS and TLC. Then the reaction mixture was filtered through a pad of celite and the filtrate was concentrated. The resulting residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, eluent of 0-15% ethyl acetate / pet. ether gradient @ 40 mL / min) to give methyl (A)-3-(l -aminoethyl)-5-(trifluoromethyl)benzoate.

[0291] 'H NMR (400 MHz, CDCI3) 68.23 - 8.15 (m, 2H), 7.85 (s, 1H), 4.28 (q, J= 6.8 Hz, 1H), 3.97 - 3.94 (s, 3H), 1.43 (d, J= 6.8 Hz, 3H).

[0292] Step 4: To a solution of tert-butyl A-({[(tert-butoxy)carbonyl]amino}methanethioyl)carbamate (782 mg, 1 Eq, 2.83 mmol) in THF (20 mL) was added NaH (227 mg, 60% Wt, 2 Eq, 5.66 mmol) at 0 °C in portions under N2 atmosphere. After 1 h at this temperature, TFAA (624 mg, 420 pL, 1.05 Eq, 2.97 mmol) in THF (5 mL) was added dropwise. The mixture was stirred at 0 °C for 1 h. Then a solution of methyl (A)-3-(l-aminoethyl)-5-(trifluoromethyl)benzoate (700 mg, 1 Eq, 2.83 mmol) in THF (1 mL) was added dropwise at 0 °C. The mixture was stirred at 25 °C for 12 h. Followed by LC / MS. Then the reaction mixture was quenched with sat. NH4CI (20 mL) and water (30 mL), and extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 30% EtOAc / hexane gradient @ 40 mL / min) to afford methyl (A)-3-(l-(3-(tert-butoxycarbonyl)thioureido)ethyl)-5-(trifluoromethyl)benzoate.

[0293] MS (ESI) m / z 407.0 [M+H]+.

[0294] *H NMR (400 MHz, CDCI3)) 6 10.10 (br d, J= 6.8 Hz, 1H), 8.21 (br d, J= 1.6 Hz, 2H), 7.91 (s, 1H), 7.79 (s, 1H), 5.69 - 5.61 (m, 1H), 3.96 (s, 3H), 1.65 (d, J= 7.2 Hz, 3H), 1.51 (s, 9H).

[0295] Step 5: To a solution of methyl (R)-3-(l-(3-(tert-butoxycarbonyl)thioureido)ethyl)-5-(trifluoromethyl)benzoate (500 mg, 1 Eq, 1.23 mmol) in THF (10 mL) was added methyl 3-amino-3,3-diphenylpropanoate hydrochloride (431 mg, 1.2 Eq, 1.48 mmol), 3-(((ethylimino)methylene)amino)-A, A-dimethylpropan-l -amine hydrochloride (590 mg, 2.5 Eq, 3.08 mmol) and A-ethyl-A-isopropylpropan-2-amine (795 mg, 1.07 mL, 5 Eq, 6.15 mmol), the mixture was stirred at 25 °C for 12 h then50 °C for 3 h under N2 atmosphere. Followed by LC / MS. Then the reaction mixture was quenched with water (20 mL), and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure to yield methyl ( / ) -3 -(1 -(2-(tert-butoxy carbonyl)-3 -(3 -m ethoxy-3 -oxo- 1,1-diphenylpropyl)guanidino)ethyl)-5-(trifluoromethyl)benzoate, which was used dirctly in the next step without any further purification.

[0296] MS (ESI) m / z 628.3 [M+H]+.

[0297] Step 6: To a solution of methyl (R)-3-(l-(2-(tert-butoxycarbonyl)-3-(3-methoxy-3-oxo-l,l-diphenylpropyl)guanidino)ethyl)-5-(trifluoromethyl)benzoate (500 mg, 1 Eq, 797 pmol) inTHF (10 mL) was added 2,3,4,6,7,8,9,10-octahydropyrimido[l,2-a]azepine (364 mg, 3 Eq, 2.39 mmol), and the mixture was stirred at 50 °C for 1 h under N2 atmosphere. Followed by LC / MS. Then the reaction mixture was concentrated and the resulting residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, eluent of 0-20% ethyl acetate / hexane gradient @ 35 mL / min) to yield methyl ( / ?)-3-(l-(2-(( / c77-butoxycarbonyl)imino)-6-oxo-4,4-diphenyltetrahydropyrimidin-l(27 / )-yl)ethyl)-5-(trifluoromethyl)benzoate.

[0298] MS (ESI) m / z 596.3 [M+H]+.

[0299] Step 7: To a solution of methyl ( / ?)-3-(l-(2-(( / c77-butoxycarbonyl)imino)-6-oxo-4,4-diphenyltetrahydropyrimidin-l(2J7)-yl)ethyl)-5-(trifluoromethyl)benzoate (50 mg, 1 Eq, 84 pmol) in THF (1 mL) was added Potassium trimethyl(oxido)silane (65 mg, 71 pL, 6 Eq, 0.50 mmol), the mixture was stirred at 25 °C for 30 min under N2 atmosphere. Followed by LC / MS. Then added HC1 (1 N) to acidify the reaction mixture to pH = 6-7, then extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure to yield (7?)-3 -( 1 -(2-(( / c77-butoxycarbonyl)imino)-6-oxo-4,4-diphenyltetrahydropyrimidin-l(2T / )-yl)ethyl)-5-(trifluoromethyl)benzoic acid, which was used directly in the next step without any further purification.

[0300] MS (ESI) m / z 582.2 [M+H]+.

[0301] Step 8: To a solution of (A)-3-(l-(2-((terLbutoxycarbonyl)imino)-6-oxo-4,4-diphenyltetrahydropyrimidin-l(2J7)-yl)ethyl)-5-(trifluoromethyl)benzoic acid (65 mg, 1 Eq, 0.11 mmol) in THF (3 mL) was added lH-benzo[d][l,2,3]triazol-l-ol hydrate (51 mg, 3 Eq, 0.34 mmol), 3-(((ethylimino)methylene)amino)-A, A-dimethylpropan-l-amine hydrochloride (86 mg, 4 Eq, 0.45 mmol), A-ethyl-A-isopropylpropan-2-amine (87 mg, 0.12 mL, 6 Eq, 0.67 mmol) and (5)-l-phenylethan-l-amine (18 mg, 1.3 Eq, 0.15 mmol), and the mixture was stirred at 25 °C for 12 h under N2 atmosphere. Followed by LC / MS. Then the reaction mixture was quenched with water (10 mL), and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure to yield / c / V-butyl (6-oxo-4,4-diphenyl-l-((A)-l-(3-(((5)-l-phenylethyl)carbamoyl)-5-(trifluoromethyl)phenyl)ethyl)tetrahydropyrimidin-2(U7)-ylidene)carbamate.

[0302] MS (ESI) m / z 685.3 [M+H]+.26098-2

[0303] Step 9: A solution of tert-butyl (6-oxo-4,4-diphenyl-l-((A)-l-(3-(((5)-l-phenylethyl)carbamoyl)-5-(trifluoromethyl)phenyl)ethyl)tetrahydropyrimidin-2(l / / )-ylidene)carbamate (60 mg, 1 Eq, 88 pmol) in DCM / TFA (v / v = 3:1, 2 mL) was stirred at 25 °C for 1 h. Followed by LC / MS. Then the reaction mixture was concentrated in vacuo and the residue was purified by SFC (Column: DAICEL CHIRALPAK IG (250 mm*30mm,10pm); Condition: 0.1%NH3H2O / EtOH; Begin B 45%, End B 45%; Flow Rate (mL / min): 80) and then re-purified by Prep-HPLC (Column Boston Prime C18 150*30mm*5pm Condition water(0.1%TFA)-ACN Begin B 40 End B 60 Gradient Time (min) 11 100% B Hold Time 2 Flow Rate(mL / min) 25 Injections 1) to yield 3-((R)-l-(2-imino-6-oxo-4,4-diphenyltetrahydropyrimidin-l(2H)-yl)ethyl)-N-((S)-l-phenylethyl)-5-(trifluoromethyl)benzamide.

[0304] MS (ESI) m / z 585.3 [M+H]+.

[0305] 'H NMR (400 MHz, METHANOL-d4) 88.02 (s, 1H), 7.70 (s, 1H), 7.56 (s, 1H), 7.47 -7.21 (m, 15H), 5.39 (q, J= 6.4 Hz, 1H), 5.32 - 5.23 (m, 1H), 3.79 - 3.61 (m, 2H), 1.73 (d, J= 7.2 Hz, 3H), 1.61 (d, J= 7.2 Hz, 3H).Biological Assays

[0306] Unless specified the reagents (e.g., Tween-20, sodium acetate, formic acid, ROP4 peptide substrate ChinaPeptides (Catalog# 04010069108)) disclosed herein are publicly available.

[0307] Recombinant CpASP4 (MW = 73455.59Da) was made as described above.Mouse models of infection

[0308] IFNy" "mice were bred according to well known husbandry.

[0309] Animal infection and purification of C. parvum oocysts

[0310] For parasite amplification, 6- to 8-weeks old C57B6WT(wild type) or IFNy" ’ mice were infected with the oocysts by oral gavage. Faecal collection was started from 3 days post-infection until the end of experiment. For generation of transgenic parasites, 4- to 6-weeks old IFNy- ’ mice were treated with antibiotic cocktail in the drinking water (1 mg / ml ampicillin, 1 mg / ml streptomycin and 0.5 mg / ml vancomycin) a week prior to infection. On the day of infection, mice were treated with 8% sodium bicarbonate 5 minutes before the infection with transfected sporozoites by oral gavage. One day post-infection, mice were treated with paromomycin (16 g / L) for up to 3 weeks in the drinking water. Weighing and visual check was performed daily, and fecal collection started from 7 days post-infection for 3 to 4 weeks.26098-2

[0311] Purification of oocysts was done as previously described (Pawlowic et al., 2017).Briefly, fecal material was homogenized and filtered. The filtrate was overlaid with sucrose flotation solution and spun down. The supernatant was washed with 0.85% NaCl in PBS and spun down. The pellet was resuspended in saline and used to overlay caesium chloride solution. The pellet containing the oocysts were collected and resuspended in lx Pen / Strep solution. The purified oocysts were stored at 4°C prior to use.Preparation of C. parvum parasite lysate

[0312] Oocysts were excysted in 0.8% sodium taurocholate for 10 minutes, followed by incubation in PBS at 37°C for 1 hour. Parasites were then spun down and incubated in RIP A lysis buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Triton X-100, supplemented with lx EDTA-free protease inhibitor cocktail (Roche) and lx Benzonase (Merck) for 30 minutes on ice, vortexed every 5 minutes. Protein yield was measured by Pierce™ BCA Protein Assay Kit (Thermo Scientific™).For in vitro culture samples, 24-well plates containing coverslips were seeded with IxlO5HCT-8 cells (ATTC, CCL-244) per well and grown to become 65% confluent overnight in RPMI supplemented with Pen / Strep + 10% BCS + 1% GlutaMAX. On the next day, oocysts were primed in 0.8% sodium taurocholate for 10 minutes at 37 degrees, washed with PBS and inoculated into the host cells. Parasites were let to grow for 30-45 hours at 37°C, 10% CO2 in DMEM supplemented with Pen / Strep + 2% FBS +1% GlutaMAX. Samples were washed with 20 mg / ml D-galactose prior to fixation.High content imaging and EC50 determination of C. parvum growth

[0313] HCT-8 cells (ATCC, CCL-244) were seeded on 96-well PhenoPlates (PerkinElmer) (25,000 cells per well) and grown to 65% confluency overnight. On the next day, oocysts (50,000 per well) were bleached and primed prior to inoculation into the host cells in DMEM supplemented with Pen / Strep + 2% FBS + 1% GlutaMAX. For the in vitro drug assay, parasites were treated with compound Example 1 A (WM382) or WM43 hours post-infection and incubated at 37°C, 10% CO2 for 45 more hours. To prepare samples for high content imaging, the plate was washed with 20 mg / ml D-galactose, fixed, permeabilized and blocked. The intracellular parasites were stained with Fluorescein isothiocyanate (FITC)-conjugated Vicia villosa lectin (VVL) (Vector laboratories, FL1231) (0.3 pg / ml per well) for 1 hour at room temperature. The fluorescent dye, DAPI (Stemcell Technologies, Cat. No. 75004) was used to26098-2stain both parasite and host nuclei. The samples were washed and stored in PBS at 4°C prior to imaging. The samples were imaged with Opera Phenix and the analysis performed on Columbus (PerkinElmer) software. Parasites were selected based on parasite area, roundness and mean FITC intensity. Non-linear regression analysis was performed on GraphPad Prism ver 9.5.1 (www.graphpad.com) to determine the ECsos of compound Example 1 A (WM382) and WM4. Western blot analysis

[0314] C. parvum lysate was added 2x SDS-PAGE loading buffer (with or without P-mercaptoethanol) and run in 4-12% Bis-Tris polyacrylamide gels (Thermo Scientific™) at 100 V for 1 hour and at 150 V for 30 minutes. Proteins were transferred to Amersham™ Protran® Western blotting nitrocellulose membrane (Cytiva™) at 100 V for 1 hour and membranes were blocked in 5% skim milk in PBS overnight at 4°C. Membranes were probed by primary antibodies for 1 hour at room temperature and washed with 0.05% Tween-20 in PBS for 4 times. The membranes were probed with primary rabbit CpASPl antibody which was then detected by horse radish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotech, 1030-05) for 1 hour at room temperature, washed and imaged on Chemidoc™ (Bio-Rad) with HRP chemiluminescence reagents (Thermo Scientific™). Images were analyzed by Image Lab (BioRad).In vivo analysis of C. parvum growth

[0315] Drug assays were performed in wild type C57BL / 6 and IFNg" ’ mice (Jackson Laboratories, #002287). Mice were infected with 5,000 mouse-adapted, NLuc-expressing C. parvum parasites (Gullicksrud et al., Mucosal Immunol. 2022 Feb; 15(2):362-72). Mice were dosed 3 hours post-infection with compound Example 1 A once a day for 4 consecutive days (10, 20, 40, 80 mg per kg (mpk) in vehicle containing 20% DMSO and 60% polyethylene glycol). Mouse health was monitored by recording the body weights and visual check daily. Fecal sample was collected 3 days post-infection for 3 weeks. Parasite burden in fecal material was monitored by performing NLuc assay, as herein previously described.Expression of recombinant CpASP4

[0316] For plasmid construction, CpASP4 (CryptoDB accession number: cgd6_660, Puiiu et al. Nucleic Acids Res. 2004 Jan 1; 32(Database issue): D329-31; doi: 10.1093 / nar / gkh050. PMID: 14681426; PMCID: PMC308784) was codon optimized for expression in mammalian cells and cloned into linearized pCDNA3.4 vector by Gibson Assembly® Cloning Kit (New England26098-2Biolabs) to genetically fuse the coding sequence of CpASP4 with IL-2 signal sequence at the N-terminus and the Tobacco Etch Virus (TEV) protease site and 8xHis tag at the C-terminal end.

[0317] For protein expression in human embryonic kidney (HEK) Expi293F™ cells (Gibco-CatNo A14635), transfection was done using Expi293™ Expression System Kit and according to manufacturer’s protocol. The transfected cells were grown in culture at 37°C, 5% CO2 and harvested 3-7 days post-transfection. Protein purification was done by HisPur™ Ni-NTA Resin (Thermo Scientific™CatNo88221) followed by size exclusion chromatography (SEC) with Superdex® 200 Increase 10 / 300 GL (Cytiva, Cat#: 17-1069-01) or HiLoad® 16 / 600 Superdex® 200 pg (Cytiva, Cat#: 28-9893-35). Fractions containing the recombinant protein were pooled, concentrated and buffer exchanged into TBS pH 8.0. Protein concentration was measured using Nanodrop. Protein was stored at 4°C prior to usage.

[0318] Modelling of CpASP4 by AlphaFoldSequence of full-length and mature CpASP4 were used for structure prediction by AlphaFold ver.2.1.2 (DeepMind) (deepmind. google / technologies / alphafold / , and referenced in Jumper, J. et al. Nature 596, 583-589 (2021)

[0319] Biochemical assay for CpASP4 cleavage of peptide substratesFluorogenic substrates and recombinant enzyme, CpASP4, at concentration starting from 0.0625 pM to 1 pM was mixed in 20 mM sodium acetate pH 7.5 / 0.005% Tween-20 on 384-well plate (Coming #4514) and incubated at 37°C for 4 hours, protected from light. The sample was excited at 340 nm and fluorescence intensity of the sample was measured by Clariostar (BMG Labtech) at emission wavelength of 492 nm. For in vitro assay with compound WM382 or WM4, Kmconcentration of CpROP4 peptide was used and the drug was added at the start of the reaction and fluorescence was measured after 2 hours of incubation at 37°C, protected from light. GraphPad Prism ver. 9.5.1 was used to record the fluorescence intensity and the values were normalized to no enzyme control.

[0320] Solvent Proteome Integral Solubility Alteration (PISA) assay C. parvum cells were frozen liquid N2, followed by lOx mechanical sheering with a 29 g needle syringe, and soluble protein isolation through ultracentrifugation (100.000g for 20 minutes). Protein lysate was exposed to the drug (10 pM or 100 pM of WM4 or compound WM382) or the vehicle control (DMSO) for 3 minutes and subsequently incubated with varying concentration of the solvent mixture ‘AEF’ (50% Ethanol, 50% Acetone, 0.1% Formate) to a final concentration of 7-15% (v / v) with 2% intervals, for 20 minutes at 37°C at 800 rpm on a Thermomixer (Eppendorf). The experiment was carried out in triplicate. Denatured protein was pelleted through centrifugation (4°C, 18.000g, for 20 minutes), the soluble phase was recovered, analyzed via western blot (for26098-2SIP experiments) or pulled together in equal volume ratios and analyzed by Mass Spectrometry (for DIA Solvent experiment)

[0321] Mass spectrometry sample preparation and analysisSample preparation for proteomic analysis was carried out as follows. Protein was reduced (20 mM TCEP, lOOmM TEAB) for 20 minutes at 55°C and alkylated with 55 mM CAA for 30min, followed by precipitation on glass beads in 80% ACN with a 6-minute centrifugation at 21.000 g and 3x wash with 80% Ethanol. Speed-vac-dried beads were resuspended in lOOmM TEAB and subjected to sequential digestion with LysC (for 3 hours, 1:50 protein: enzyme ratio) and trypsin (overnight 1:50 protein: enzyme ratio) and the resulting digest was acidified with TFA to 1% final concentration and desalted on T3 Cl 8 stage tips (Affinisep) according to manufacturer’s instructions. Dried peptide samples were solubilised in 0.1% Formate, 2% ACN and loaded on to aC18 fused silica column (inner diameter 75 pM, OD360x 15cmlength, 1.6pM Cl 8 beads) packed into emitter tip (lonOptics). Samples were separated on a 45 min analytical gradient on a Neo Vanquish liquid chromatography system (Thermo Scientific) interfaced with MS (Orbitrap Eclipse Tribrid Mass Spectrometer, Thermo Scientific) and analysed in a DIA mode. Peptide identification was carried out in DIA-NN 1.8.1 using is silico spectral library generated from Uniprot C. parvum reference proteome.Table 1. List of amino acid residues that interact with compound WM382 or WM4.Residues that interact with compound WM382Present in CpASP4 / WM4D210 NoS211 No1274 No1281 Yes1318 YesS425 YesY395 YesT428 YesV494 YesP495 YesQ212 No1229 YesD231 YesG233 YesS234 YesF276 YesS278 NoF319 Yes1322 Yes26098-2F324 No1327 NoD421 YesG423 YesT424 YesS425 YesM490 Yes1492 YesL503 Yes

[0322] The disclosure will be more readily understood by reference to Examples 3-16, which are included merely for purposes of illustration of certain embodiments and embodiments of the present invention and are not intended to limit the invention.Example 7Compounds WM382 and WM4 are effective at killing C. parvum in vitro and interact with CpASP4 in parasites

[0323] P. falciparum PfPMIX and PfPMIX compound WM4 and compound WM382 were tested for their capacity to kill C. parvum using a in vitro growth assay. Both compounds were found to inhibit C. parvum growth with an ECso of compound WM382 and WM4 to be 0.27 and 0.85 pM, respectively (Fig. 1A, B).Example 8Compound WM382and WM4 can bind to CpASP4 in C. parvum lysate.

[0324] Solvent-induced Protein Precipitation (SIP) is an assay that assesses whether small molecules, such as these compounds, bind to the target proteins, can protect the solubility and therefore suggest an interaction between protein and compound. Herein the assay was performed according to Wang et al., Anal. Chem., 92, pg 1363-1371, 2020, to determine which aspartyl protease was targeted by compound WM382 and WM4 in C. parvum parasites. To do this, parasite lysate was subjected to increasing concentrations of a solvent mixture (50% acetone, 50% ethanol, 0.1% formic acid), in the presence and absence of both these compounds at concentrations of lOpM and lOOpM. Western blot in combination with antibodies against CpASP4 was then used to determine whether WM4 and WM382 could stabilize the amount of this aspartyl protease within the soluble fraction (Fig. 1C). At solvent concentrations above 11%, the solubility differences were observed between the DMSO control and both drug-treated26098-2samples at 100 pM but less so at 10 pM, consistent with both of these compounds binding to CpASP4 (Fig. 1C), suggesting that these compounds can bind to CpASP4 in C. parvum lysate.

[0325] Building upon the principle of this SIP assay, the inventors combined the findings with Data Independent Acquisition Mass Spectrometry (DIA-MS) and Proteome Integral Solubility Alteration((PISA) assay format to interrogate WM4 and compound WM382 induced proteinstabilizations in an unbiased manner across the wider proteome. Here soluble protein extract derived from whole excysted sporozoites was exposed to lOOpM of both WM4 and WM382 compounds or the DMSO vehicle, followed by organic solvent induced protein denaturation across 7-15% (v / v) gradient (Fig. ID). In both cases the relative abundance of CpASP4 (cgd6_660) in the soluble fraction increased in the presence of the compounds further suggesting an interaction. (Fig ID).Example 9CpASP4 is structurally similar to PfPMIX and PfPMX

[0326] The degree of similarity between CpASP4, PfPMIX and PfPMX was assessed to determine if either WM4 or WM382 is likely to engage with the C. parvum enzyme in a similar manner. In the absence of being able to generate crystals of CpASP4 with or without compound a structural prediction based on AlphaFold was generated (Fig. 2A). Overall, CpASP4 adopted a similar fold to other aspartyl proteases and contained the two canonical aspartic acid residues within the catalytic cleft. CpASP4, however, has an apparent large unstructured loop, which appears to be an elaboration on a flap that sits over the catalytic pocket of some family members as described in Dogga SK et al., Elife. 2017 Sep 12; 6. pii: e27480(Fig 2 A, B). To compare structural features of CpASP4, the modelled structure was aligned with an AlphaFold model of PfPMIX and the solved structure of PfPMX as described in Hodder AN et al. Structure. 2022 Jul 7; 30(7):947-961 and the results are shown in Fig. 2C. The residues within the catalytic pocket were observed to be highly conserved across all three aspartyl proteases (Fig. 2C). Interestingly, CpASP4 seemed to have characteristics of both PfPMIX and PfPMX. For example, like PfPMIX, CpASP4 has an extended loop region, but lacks a long a-helix which is more characteristic of PfPMX (as compared to PfPMIX).

[0327] The conservation and positioning of residues that interact with WM382 and WM4 between CpASP4, PfPMIX and PfPMX were also assessed. Sequence alignment analysis of these three aspartyl proteases showed that most, but not all residues that interact with WM382 and WM4 are conserved in CpASP4 (Table 1)26098-2

[0328] The predicted structure of CpASP4 to PfPMX structure when bound to WM382 or to Plasmodium vivax PMX (PvPMX) structure when bound to WM4 (Fig. 2D, E) was also aligned. When PfPMX and PvPMIX are in complex with WM382 (and WM4), most interactions reside at the front and back of the substrate binding cleft. In PvPMX- WM4 and PfPMX- WM382 complexes, hydrogen bonds occur between the amine and imine moieties of the compound and the active sites of PvPMX and between the carbonyl amide moiety of the compound and PvPMX main chain carbonyl group. A similar pattern in CpASP4 model was observed, where the hydrogen bonds form between WM4 and aspartic acid residues DI 89 and D438, and the main chain of G440, which also is a similar arrangement for PfPMX active site engagement with WM382 (D265, D456 and G458).Example 10CpASP4 can cleave a motif containing a serine-phenylalanine-leucine residues

[0329] To understand the biology of CpASP4, the enzyme was expressed in Expi293F™ cells. To determine whether a potential substrate motif of CpASP4 has activity towards PfPMIX, PfPMX and PfPMV different substrates were tested. Previously described peptides were explored using a FRET assay based on a quencher DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid) at the N-terminus and a fluorophore EDANS (5-((2-Aminoethyl)amino)naphthalene-l-sulfonic acid) at the C-terminus (Favuzza et al. Cell Host and Microbe, 2020 April 8; 27(4):642-658). When DABCYL and EDANS are in proximity (i.e., on the same peptide), DABCYL quenched EDANS and fluorescence is limited. Upon protease cleavage, DABCYL and EDANS are separated allowing fluorescence emission to occur in a dose dependent fashion. Fluorescence intensity, as a measure of cleavage activity, was recorded as a measure of CpASP4 activity after 4 hour-incubation with the substrate peptides at 37°C. The protease cleavage assay was performed with a range of PfPMIX / PfPMX substrate peptides. Out of all peptides tested, CpASP4 showed the highest activity against PfRON3 peptide (DABCYL-1838KEISFLERRE1848-EDANS) at 15- to 20- fold activity above background and some activity towards P. falciparum Rhoptry-associated, Leucine zipper-like Protein 1 (RALP1) peptide (DABCYL-53EKKSNMESVN63-EDANS) and Erythrocyte Binding Antigen 181 (EBA181) peptide (DABCYL-116NRNSFVQRSY125-EDANS) (Fig. 3A). As compared to other peptide sequences that CpASP4 does not cleave, this suggests that this enzyme can cleave ‘[S][FN][LM][E]’ but does not tolerate ‘[S][F][L][Q]’ based motifs.

[0330] The substrate specificity of CpASP4 was also explored. To do this, a series of alanine mutants of PfRON3 peptide were applied, in which every amino acid of the peptide was replaced26098-2with alanine (Fig. 3B). CpASP4 activity dramatically decreased when residue SI 840, Fl 841 and LI 842 were mutated, but not residues more distal to the cleavage site.Example 11Identification of CpROP4 as the potential substrate of CpASP4

[0331] Using the information gleaned from Example 10, a search for potential native substrates of CpASP4 using CryptoDB.org was conducted. This was done by searching for proteins that have both a signal peptide, for entry into the secretory pathway, as well as an ‘ [S][F][L]’ motif. Two hundred twenty nine (229) candidates were obtained in which three were selected for further characterization: 1) CpASP4 itself, which was predicted to be an autocatalytic site similar to PfPMX, 2) The rhoptry protein CpROP4 (CryptoDB.org accession number: cgd3_1730) and 3) the rhoptry protein, CpROP6 (CryptoDB.org accession number: cgd6_3630), both of which were recently characterized Guerin et al. Cell Host and Microbe 2021 Sep 8; 29(9): 1407-1420.Fluorogenic peptides were designed consisting of 10 amino acids, with ‘ [S][F] [L] ’ motif being approximately in the middle and performed the cleavage assay. At least 8-fold activity of CpASP4 towards CpROP4 (DABCYL-31LEVSFLNAASE42-EDANS) compared to the background control was observed, while negligible activity towards CpASP4 (DABCYL-134DSESFLINKPKE145-EDANS), and CpROP6 (DABCYL-637KPKSFLEKVLE647-EDANS) was found (Fig. 4A).Example 12WM4 and WM382 inhibits CpASP4 activity

[0332] The inhibition of CpASP4’s activity by WM382 and WM4 was tested. Using the same FRET -based cleavage assay, each compound was dosed, percentage of cleavage measured and the ICsos of WM382 and WM4 determined to be 17 nM and 86 nM, respectively (Fig. 4B). The effect of the compounds were compared with 49c and the IC50 was found to be 2.87 pM, -30-fold higher than WM382 and WM4 (Fig4B). This suggests that both WM382 and WM4 are more potent, than compound 49c, likely through more efficient or stronger binding to CpASP4.Overall, this showed that CpASP4 can be effectively inhibited by WM382 and WM4 likely through a similar mechanism to Plasmodium PMIX and X.Example 13WM382 is effective in treating cryptosporidiosis in mice -

[0333] To test whether inhibition of CpASP4 could treat a C. parvum infection in a mouse model, a mouse adapted strain of C. parvum that expressed NLuc and could efficiently infect26098-2adult wild type mice was used as described in Gullicksrud JA et al. Mucosal Immunology 2022 Feb(2):362-372. The capacity of compound WM382 to protect mice from a subsequent challenge of C. parvum (prophylactic treatment) was first tested. Here, mice were orally gavaged with 5,000 oocysts at the same time as administering the first treatment of compound WM382 (Fig 5 A). Dosing was given at 10, 20 or 80 mg per kg (mpk) once per day (quaque die, QD) for 4 consecutive days. NLuc activity was then monitored in the faecal material as a measure of oocyst shedding. In vehicle control group, the NLuc activity peaked 4 days post-infection and is cleared by 2 weeks (Fig. 6A). In contrast, in both 10-, and 20 mpk-treated groups, the peak of infection was delayed and the intensity was lowered as compared to the vehicle control. Interestingly, in 80 mpk-treated mice, the NLuc activity from the faecal material was almost undetectable, suggesting oocysts shed in these animals is minimal (Fig 5A).

[0334] The capacity of WM382 to reduce infection in an already established infection (i.e., curative) (Fig, 6B) was also tested. In this case, each group of mice was orally-gavaged with 5,000 oocysts and infection allowed to establish for two days, at which time oocysts shedding had started. Mice were then treated with 10, 20 and 80 mpk of WM382 (and oocysts shedding was monitored over the next 14 days. In each case, it was found that the treatment of mice reduced NLuc values signifying reduced oocyst shedding (Fig 5B).

[0335] The efficacy of WM382 on eliminating parasites in an immunodeficient mouse model was tested. This is particularly important because, as discussed earlier, cryptosporidiosis is predominantly problematic in people with compromised immune systems. In this study, TFNy’7’ mice were infected with 5000 mouse-adapted C. parvum and an additional cohort of mice treated at 40 mpk was added. Mice were then treated on the same day of infection (prophylactically) at the same doses as in wild type mice (Fig. 5C). Nitazoxanide was also included, which has been reported to have very limited efficacy in immunodeficient individuals as a comparator. NLuc in fecal material was monitored in both as well as time to death. NLuc activity in the vehicle control peaked in the second week of infection and all mice in this group had to be euthanized due to significant weight loss (Fig. 5D). Nitazoxanide-treated mice also presented with relatively high NLuc activity and some mice needed to be discarded due to ethical weight loss limits. In 10-mpk treated mice, the NLuc activity is comparable to the vehicle control, but in 20-, 40-mpk 80-mpk treated groups, the NLuc activity was at least 2-fold less than the vehicle and 10-mpk groups.

[0336] Also tested was the curative capacity of WM382 in IFNy" ’ mice (Fig. 5E). This experiment was done as described for wild type mice, by starting treatment when NLuc activity was detected and mice started showing symptoms. As expected, both vehicle and Nitazoxanide-treated groups showed significantly high NLuc activity and all mice needed to be euthanized due26098-2to severe weight loss (Fig. 5F). In 10-mpk and 20-mpk groups, mice showed slightly delayed peak of infection and some, but not all, recovered from infection. In contrast, 40-mpk and 80-mpk treated mice showed delayed, less severe infection and eventually cleared the parasites. Overall, the data suggests that WM382 can limit C. parvum infection in vivo.Example 14Potency Assay PrococolsCrypto Biochemical Assay Protocol #1 (“a”)

[0337] Compounds were assayed for CpASP4 inhibitor activity using a FRET-based assay that involves the cleavage of ROP4 peptide (Dabcyl)-LEVSFLNAASE(Edans). When uncleaved, the DABCYL quenches the fluorescent signal from the EDANS tag and upon cleavage by CpASP4, fluorescent signal from EDANS is measured at excitation 340nm and emission 480nm.Assay plates (Coming #C3728, 1536 well, NB) were prepared in 10-point dilution series of compounds using an Echo acoustic dispenser (Labcyte). Appropriate volumes of 10 mM compound stocks were transferred into the assay plates and all wells were backfilled with DMSO such that this remained constant at 1% DMSO. The 0% inhibition control was 1% DMSO and the 100% inhibition control was 30 pM WM382. Assay plates were stored at -20°C until use.The assay plates were thawed at room temperature for at least 60 minutes before assaying.Recombinant CpASP4 and ROP4 peptide were prepared in assay buffer containing 25 mM sodium acetate, pH 7.0 + 0.005% Tween-20. Firstly, 2 pL of 2x CpASP4 working solution (20 nM final) was added into columns 1-48 of the 1536w assay plate using a Multidrop dispenser (small cassette, high speed). The plate was incubated at room temperature for 20 minutes. Then 2 pL of 2x ROP4 working solution (25 pM final) was added into columns 1-48 of the assay plate using a Multidrop dispenser (small cassette, high speed). The plate was covered with a plate cover and centrifuged at 1000 rpm for 1 min. The plate was then incubated at 37°C, for 60 minutes in a humidified incubator at 37°C. To stop the reaction, 2 pL of 3x formic acid (67 mM final) was dispensed into columns 1-48 of the assay plate. The FRET signal was measured at excitation 340nm, emission 480nm in the PHERAstar FSX Reader (BMG Labtech Co.) IC50 values (Table 4) were calculated by Dotmatics 5.3 and Spotfire 7.11.1 software using a nonlinear regression four-parameter fit analysis. The equation used is sigmoidal dose response (variable slope), Y = bottom + (top - bottom) / (l+10((logEC50 - X)*Hill Slope)).26098-2Crypto Biochemical Assay Protocol #2 (“b”)

[0338] Compounds were assayed for CpASP4 inhibitor activity using a FRET-based assay that involves the cleavage of ROP4 peptide (Dabcyl)-LEVSFLNAASE(Edans) as per above.Assay plates (Coming #CLS3820, 384 well, NB) were prepared in 10-point dilution series of compounds using an Echo acoustic dispenser (Labcyte). Appropriate volumes of 10 mM compound stocks were transferred into the assay plates and all wells were backfilled with DMSO such that this remained constant at 1% DMSO. The 0% inhibition control was 1% DMSO and the 100% inhibition control was 30 pM WM382. Assay plates were stored at -20°C until use.The assay plates were thawed at room temperature for at least 60 minutes before assaying.Recombinant CpASP4 and ROP4 peptide were prepared in assay buffer containing 50 mM Tris, pH 7.0 + 0.005% Tween-20 + 1.25 mM DTT. Firstly, 7.5 pl of 2x CpASP4 working solution (1.25 nM final) was added into columns 1-24 of the 384w assay plate using a Multidrop dispenser (small cassette,high speed). The plate was incubated at room temperature for 15 minutes. Then 7.5 pl pL of 2x ROP4 working solution (2.5 pM final) was added into columns 1-24 of the assay plate using a Multidrop dispenser (small cassette, high speed). The plate was covered with a plate cover and centrifuged at 1000 rpm for 1 min. The plate was then incubated at 37°C, for 4 hours in a humidified incubator at 37°C. To stop the reaction, 5 pL of 4x formic acid (50 mM final) was dispensed into columns 1-24 of the assay plate. The FRET signal was measured at excitation 340nm, emission 480nm in the PHERAstar FSX Reader (BMG Labtech Co.) IC50 values (Table 4) were calculated by Dotmatics 5.3 and Spotfire 7.11.1 software using a nonlinear regression four-parameter fit analysis. The equation used is sigmoidal dose response (variable slope), Y = bottom + (top - bottom) / (l+10((logEC50 - X)*Hill Slope)).Crypto Cellular Assay Protocol

[0339] The Cryptosporidium growth inhibition assay was conducted using Wild-type Cryptosporidium parvum oocysts that were excysted and transfected to generate a reporter strain expressing Nanoluciferase. The proliferation of the reporter strain over a 42h infection of human epithelia HCT8 cells was measured by Nanoluciferase activity providing a measure of cryptosporidium proliferation in host cells.

[0340] HCT8 cells were cultured in RPMI1640 medium supplemented with 10% fetal calf serum (FCS), 1% GlutaMAX Supplement + 1% Penicillin-Streptomycinnin a humidified incubator at 37°C and 10% CO2. One day in advance of seeding into assay plates, Cryptosporidium oocytsts were prepared by treating with ice cold bleach (4% final) for 526098-2minutes. Bleach solution was removed through centrifugation at 1600 rpm for 3 minutes. Oocysts were resuspended in PBS and stored at 4 °C until use.

[0341] Assay plates (Greiner #781098, 384 well, white, tissue culture treated) were prepared in 10-point dilution series of the compounds using an Echo acoustic dispenser (Labcyte).Appropriate volumes of 10 mM compound stocks were transferred into the assay plates and all wells were backfilled with DMSO such that this remained constant at 0.25% DMSO. The 0% growth inhibition control was 0.25% DMSO and the 100% growth inhibition control was 1.25 pM L797. Assay plates were stored at -20°C until use.The assay plates containing compounds were thawed at room temperature for at least 60 minutes before adding cells. HCT8 cells were seeded with 6000 cells in 15 pl DMEM (no phenol red) with 2%FBS, 1% GlutaMAX Supplement and 1% Penicillin-Streptomycin, into each well of the assay plates using a Multidrop Combi dispenser (Thermo Scientific). Plates were incubated at 37°C and 10% CO2 for 24 hours. Cryptosporidium oocysts were added into each well of the assay plates (with HCT8 cells) with 3000 oocysts in 15 pl DMEM (no phenol red) with 2%FBS, 1% GlutaMAX Supplement and 1% Penicillin-Streptomycin using a Multidrop Combi dispenser (Thermo Scientific). Plates were incubated at 37°C and 10% CO2 for 42 hours. Nanoluciferase was measured using Nano-Gio (Promega) and calculated as a percentage using DMSO as the positive growth control and 1.25 pM L797 as a negative growth control. EC50 values (Table 4) were calculated by Dotmatics 5.3 and Spotfire 7.11.1 software using a nonlinear regression four- parameter fit analysis. The equation used is sigmoidal dose response (variable slope), Y = bottom + (top - bottom) / (l+10((logEC50 - X)xHill Slope)).TABLE 4Crypt CryptoToxoo Cell BiocheCellExam MS Poten mical Name Structure Potencypie M+l cy PotencECsoECso y ICso(nM)(nM) (nM)F 3-fluoro-5-[(2-imino-6-oxo-4,4- A diphenyl-hexahydropyrimidin-1- 1 521.2 334 15a970yl)methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de° AO3-[[(4R)-4-(cyclopropylmethyl)-HII^NLA(A 2-imino-6-oxo-4-phenyl- 2 481.5 288 3aND hexahydropyrimidin-1- Oyl]methyl]-N-[(lS)-l- Apheny 1 ethyl ]b enzami deNH3-[[(4R)-4-(2-chlorophenyl)-4- <1 HN^N^j^,'"7' - (cyclopropylmethyl)-2-imino-6- 515.2 97 1bND oxo-hexahydropyrimidin-1- O-ciyl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deNH3-[[(4R)-4-(cyclopropylmethyl)- HN'^N'^Y^^ 4-(2-fluorophenyl)-2-imino-6-FHN^O 499.3 288 3bND oxo-hexahydropyrimidin-1- QA yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deNH3-[(4-cyclobutyl-2-imino-6-oxo- 4-phenyl-hexahydropyrimidin-l- OHJ^° 481.3 240 1bNDyl)methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deN QHzJNJA N 3-[(lR)-l-(4-cyclobutyl-2-imino- 6-oxo-4-phenyl- 495.3 495.3 11bNDO hexahydropyrimidin-l-yl)ethyl]- N-[(lS)-l-phenylethyl]benzamide Qj3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- hexahydropyrimidin-1- 549.5 28 1bNDyl]methyl]-N-[(lS)-l- C^ phenylethyl]-5- (trifluoromethyl)benzamide 3-[(lR)-l-(2-imino-6-oxo-4,4- diphenyl-hexahydropyrimidin-1- O x ° 585.3 541 92bNDyl)ethyl]-N-[(lS)-l-phenylethyl]- C^ 5-(trifluoromethyl)benzamide (6S)-2-imino-3-[[4-(isoindoline- 2-carbonyl)phenyl]methyl]-6- 405.1 372 ND NDisopropyl-6-methyl- hexahydropyrimidin-4-one 3-[[(4S)-4-ethyl-2-imino-4- isopropyl-6-oxo- V 439.3 570 27aND hexahydropyrimidin-1- yl]methyl]-5-fluoro-N-[(lS)-l- pheny 1 ethyl ]b enzami de3-[(4,4-diethyl-2-imino-5-methyl- 6-oxo-hexahydropyrimidin- 1 - N^y 439.1 908 ND NDH, u yl)methyl]-5-fluoro-N-[(lS)-l- pheny 1 ethyl ]b enzami de3-[(4,4-diethyl-2-imino-5-methyl- ■yyy 6-oxo-hexahydropyrimidin- 1 - 439.2 921 ND NDyl)methyl]-5-fluoro-N-[(lS)-l-, AO pheny 1 ethyl ]b enzami de3-[[(4R)-4-ethyl-2-imino-4- isopropyl-6-oxo- 439.3 1160 ND ND hexahydropyrimidin-1- yl]methyl]-5-fluoro-N-[(lS)-l- pheny 1 ethyl ]b enzami de5-[ 1 -(4,4-diethyl-2-imino-6-oxo- hexahydropyrimidin-l-yl)butyl]- Y': OT " o 450.4 1420 ND NDN-[(lS)-l-phenylethyl]pyridine- 3 -carboxamide3-[[(4S)-4-ethyl-2-imino-4- H, ZN-YYFisobutyl-6-oxo- 453.3 544 11aND hexahydropyrimidin-1- | CANZ\Z%u yl]methyl]-5-fluoro-N-[(lS)-l- pheny 1 ethyl ]b enzami de yF3-fluoro-5-[[(4R)-2-imino-4- methyl-6-oxo-4-phenyl- O'^X 459.2 1080 ND ND hexahydropyrimidin-1- ■^o yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de XV 3-[(5,5-diethyl-3-imino-l,l- dioxo-l,2,4-thiadiazinan-2- 461.2 891 ND NDyl)methyl]-5-fluoro-N-[(lS)-l- 0 pheny 1 ethyl ]b enzami deN-[(4S)-chroman-4-yl]-3-[[(4S)- 4-ethyl-2-imino-4-isopropyl-6- V_l L 1 JHI J 467.3 1010 ND ND\ oxo-hexahydropyrimidin-1- yl]methyl]-5-fluoro-benzamide (6R)-6-ethyl-3-[[3-fluoro-5- A A (isoindoline-2- vxv 471.2 1080 ND ND carbonyl)phenyl]methyl]-2-o imino-6-phenyl- hexahydropyrimidin-4-one JI ^F3-[[(4R)-4-ethyl-2-imino-6-oxo- 4-phenyl-hexahydropyrimidin-l- 473.2 454 12aNDo yl]methyl]-5-fluoro-N-[(lS)-l- pheny 1 ethyl ]b enzami deN-[(4S)-chroman-4-yl]-5-[l-(4,4- diethyl-2-imino-6-oxo-b n m 478.4 1920 ND NDhexahydropyrimidin-1- yl)butyl]pyridine-3 -carboxamide (6R)-6-(cyclopropylmethyl)-2- imino-3-[[3-(isoindoline-2- 479.3 769 ND ND carbonyl)phenyl]methyl]-6- phenyl-hexahydropyrimidin-4- oneN-[(4S)-chroman-4-yl]-3-[[(4S)- 4-ethyl-2-imino-4-isobutyl-6-oxo- y 481.3 835 28aNDhexahydropyrimidin-1- do yl]methyl]-5-fluoro-benzamidex A NH 4-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- > A M hexahydropyrimidin-1- Ohi^° 482.4 782 18bNDyl]methyl]-N-[(lS)-l- 0^ phenylethyl]pyridine-2- carb oxami deNH 2-[[(4R)-4-(cyclopropylmethyl)- < HNAN-Y% 2-imino-6-oxo-4-phenyl- hexahydropyrimidin-1- 482.6 1054 50bNDO 0 ° yl]methyl]-N-[(lS)-l- phenylethyl]pyridine-4- carb oxami deNH 6-[[(4R)-4-(cyclopropylmethyl)- <1 HN'^N'^Y^l 2-imino-6-oxo-4-phenyl- SkAo v hexahydropyrimidin-1- 482.5 1795 229bNDyl]methyl]-N-[(lS)-l- QHjY°phenylethyl]pyridine-2- QA carb oxami deN-[(4S)-chroman-4-yl]-3-[[(4R)- 4-ethyl-2-imino-6-oxo-4- ^■UoUp 493.3 1690 ND ND (trifluoromethyl)hexahydropyrimiHu din-l-yl]methyl]-5-fluoro- benzamideNH3-[[(4R)-4-(cyclopropylmethyl)- <^JHNAN^Y^2-imino-6-oxo-4-phenyl- hexahydropyrimidin-1- 0 HA 493.6 1175 12bNDyl]methyl]-N-(l- (A pheny 1 cy cl opropy l)b enzami de NH / i JL<1 HN-^N YjA 3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- 493.5 1213 13bND hexahydropyrimidin-1- OH7yl]methyl]-N-[(lR,2S)-2- pheny 1 cy cl opropy 1 ]b enzami de NH>. JI 3-[[(4R)-4-(cyclopropylmethyl)- <1 HN^A / A2-imino-6-oxo-4-phenyl- 495.6 277 2bND hexahydropyrimidin-1- O Y° yl]methyl]-N-[(lS)-l-(p- JCA toly l)ethy 1 ]b enzami deNHx Xi| HI N A 3-[[(4R)-4-(cyclopropylmethyl)- N2-imino-6-oxo-4-phenyl- V495.6 323 2bND hexahydropyrimidin-1- OH0 yl]methyl]-5-methyl-N-[(lS)-l-(Jj pheny 1 ethyl ]b enzami de26098-2NH3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- ^2^ HN"^O 495.6 379 2bND hexahydropyrimidin-1- yl]methyl]-N-[(lS)-l-(m- toly l)ethy 1 ]b enzami de3-[l-[(4R)-4- (cyclopropylmethyl)-2-imino-6- MA U 495.3 1090 ND ND oxo-4-phenyl- O Nhexahy dropy rimi din- 1 -y 1 ] ethyl ] - N-[(l S)- 1 -phenylethyl]benzamide 3-[(lR)-l-(4,4-diethyl-2-imino-6- oxo-hexahydropyrimidin-1- 495.2 1370 ND NDv L U yl)butyl]-N-(8-fluorochroman-4- yl)benzamideNH>4 ji _ _ F (6R)-6-(cyclopropylmethyl)-3- [[3-fluoro-5-(isoindoline-2- ^AAQ AA 497.5 423 5bND carbonyl)phenyl]methyl]-2- ^£jpNimino-6-phenyl- hexahydropyrimidin-4-one 3-[(lR)-l-[(4R)-4-ethyl-2-imino- 6-oxo-4-phenyl- yx U 497.4 1330 ND NDhexahydropyrimidin-l-yl]butyl]- N-[(l S)- 1 -phenylethyl]benzamide 3-[[(4R)-4-(cyclopropylmethyl)- F2-imino-6-oxo-4-phenyl- 499.4 134 2bND hexahydropyrimidin-1- 6 °X yl]methyl]-5-fluoro-N-[(lS)-l- " u pheny 1 ethyl ]b enzami deNH3-[[(4R)-4-(cyclopropylmethyl)- AAO AA 2-imino-6-oxo-4-phenyl- HN^O 499.5 201 2bND hexahydropyrimidin-1- yl]methyl]-N-[(lS)-l-(4- JAJ fluoropheny 1 ) ethyl ]b enzami de NH HN'^'N'^X^ 3-[[(4R)-4-(cyclopropylmethyl)- A> — Ao2-imino-6-oxo-4-phenyl- OHA> 499.5 326 3bND hexahydropyrimidin-1- yl]methyl]-N-[(lS)-l-(3- fluoropheny 1 ) ethyl ]b enzami de 3-[[(4S)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- u 499.3 942 ND ND hexahydropyrimidin-1- yl]methyl]-5-fluoro-N-[(lS)-l-pheny 1 ethyl ]b enzami de26098-23 -[ 1 -bicyclof 1.1.1 ]pentanyl-(4,4- diethyl-2-imino-6-oxo- 501.3 1290 ND 290 hexahydropyrimidin-1- Dp %xyl)methyl]-N-[(4S)-chroman-4- ^ ^^ yl]benzamideN-[(4S)-chroman-4-yl]-3-[l-(4,4- diethyl-2-imino-6-oxo- 501.6 1810 ND 610hexahydropyrimidin-l-yl)hex-5- ynyl]benzamide3-[(2-imino-6-oxo-4,4-diphenyl- X^ / TKX^O hexahydropyrimidin-1- 503.6 428 3bNDQ Tf° yl)methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de u3 -[ 1 -(4,4-diethyl-2-imino-6-oxo- ^hexahydropyrimidin-l-yl)butyl]- c xY 505.3 1520 ND NDN-(2,2-dimethylchroman-4- yl)benzamide3 -[ 1 -(4,4-diethyl-2-imino-6-oxo- HN^N'NJ'^Ihexahydropyrimidin-l-yl)butyl]-AY 505.3 1840 ND NDN-(2,2-dimethylchroman-4- yl)benzamideJYO3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- 473.7 340 7bND hexahydropyrimidin-1- O r yl]methyl]-N, N-dimethyl-5- (trifluoromethyl)benzamide 3-[[(4R)-4-(cyclopropylmethyl)-. HN^ XN — J< 2-imino-6-oxo-4-phenyl- 487.6 129 2bND hexahydropyrimidin-1- o yl]methyl]-N-isopropyl-5- (trifluoromethyl)benzamide 3-[[(4R)-4-(cyclobutylmethyl)-2- imino-6-oxo-4-phenyl- 495.7 131 1bND hexahydropyrimidin-1- o JY yl]methyl]-N-[(lS)-l- CJ pheny 1 ethyl ]b enzami de3-[[(4R)-4-(cyclopropylmethyl)- HN^N^Y^I4-(4-fluorophenyl)-2-imino-6- 499.6 194 1bND oxo-hexahydropyrimidin-1- QA0yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de- JL J< (6R)-6-(cyclopropylmethyl)-2- '-4- — AoA-A 499.7 310 3bND imino-6-phenyl-3 -[[3 - O 0^° (pyrrolidine- 1 -carbonyl)-5-26098-2(trifluoromethyl)phenyl]methyl]h exahydropyrimidin-4-oneNH3-[[(4R)-4-(cyclopropylmethyl)- 4-(3 -fluorophenyl)-2-imino-6- 499.4 213 2bND oxo-hexahydropyrimidin-1- Q yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de3-cyano-5-[[(4R)-4- \I H (cyclopropylmethyl)-2-imino-6- A oxo-4-phenyl- 506.6 1275 11bNDhexahydropyrimidin-1- 0^ yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deNH 3-[[(4R)-4-(cyclopropylmethyl)- \| HN^N^Y^I 2-imino-6-oxo-4-phenyl- hexahydropyrimidin-1- 507.6 212 2bNDHN^O yl]methyl]-N-[(S)- cy cl opropy 1 (pheny l)m ethyl ]b enza Q%mideNH3-[[2-imino-6-oxo-4-phenyl-4- Fto^(2,2,2- OHv / ° 509.2 1633 107bND trifluoroethyl)hexahydropyrimidi n-l-yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de3-[[2-imino-6-oxo-4-phenyl-4- A (2,2,2- HNA) 509.2 199 6bND trifluoroethyl)hexahydropyrimidi n-l-yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deN-[(4S)-chroman-4-yl]-3-[[(4R)- <1 AN'^\J^'^rk^A04-(cyclopropylmethyl)-2-imino-6- QANH509.3 539 12aND oxo-4-phenyl- r^o hexahydropyrimidin-1- yl]methyl]benzamideN-chroman-4-yl-3-[[(4R)-4- ^" XACA^C^ (cyclopropylmethyl)-2-imino-6- 509.3 988 ND ND oxo-4-phenyl- hexahydropyrimidin-1-0to yl]methyl]benzamidel_[3-[[(4R)-4- (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- 510.5 1322 5bND hexahydropyrimidin-1- Q A yl]methyl]-5- (trifluoromethyl)benzoyl]azetidine-3 -carbonitrile26098-2NHX < O1 3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- 511.6 289 2bND hexahydropyrimidin-1- O op yl]methyl]-N-[(lS)-l-(2- CiXW" <m m methoxyphenyl)ethyl]benzamide NH<1 3-[[(4R)-4-(cyclopropylmethyl)- '"'NX XX 2-imino-6-oxo-4-phenyl- QHjCo 511.6 503 3bND hexahydropyrimidin-1- yl]methyl]-N-[(lS)-l-(3- methoxyphenyl)ethyl]benzamideU\NH / I ji _ O 3-[[(4R)-4-(cyclopropylmethyl)- <1 HN^N^X^''2-imino-6-oxo-4-phenyl- HNXO511.6 953 10bND hexahydropyrimidin-1- yl]methyl]-5-methoxy-N-[(lS)-l- 0^ pheny 1 ethyl ]b enzami deF / , F 3-[(lR)-l-(4,4-diethyl-2-imino-6- NH / J 0 X -> X O oxo-hexahydropyrimidin-1- H N ^ N NCC 513.3 414 ND 680\_J 1 J H^--"''^P '''^ yl)butyl]-N-(6,8- difluorochroman-4-yl)benzamide NHii _ _ 3-[[(4R)-4-(cyclopropylmethyl)- <1 HN^N'^X^XX F2-imino-6-oxo-4-phenyl- ^C^x^o513.4 855 ND ND hexahydropyrimidin-1- O °^r yl]methyl]-5-fluoro-N-methyl-N- x. f( 1 R) - 1 -phenyl ethyl ]b enzami de NHzi ii 3-[[(4R)-4-(cyclopropylmethyl)- <1 HN^N^X^XX FX'y'xCo2-imino-6-oxo-4-phenyl- 513.5 961 ND ND hexahydropyrimidin-1- oyl]methyl]-5-fluoro-N-methyl-N- Ox f( 1 S)- 1 -phenylethyl]benzamide (6R)-6-(cyclopropylmethyl)-2- imino-3 - [[3 -(morpholine-4- carbonyl)-5- 515.7 350 4bND(trifluoromethyl)phenyl]methyl]- 6-phenyl-hexahydropyrimidin-4- oneNHN-[(lS)-l-(3-chlorophenyl)ethyl]- 3-[[(4R)-4-(cyclopropylmethyl)- Q 515.5 249 1bND 2-imino-6-oxo-4-phenyl- Ox hexahydropyrimidin-1- yl]methyl]benzamideClNH<3 HN^N^^^. N-[(lS)-l-(4-chlorophenyl)ethyl]-; X^o ^X 3-[[(4R)-4-(cyclopropylmethyl)- O 515.5 357 1bND 2-imino-6-oxo-4-phenyl-HJC°hexahydropyrimidin-1- JLJ yl]methyl]benzamideCIX'^5^26098-2NH 3 -chi oro-5 - [ [(4R)-4- < / I1 HN j^LN^X^ACl(cyclopropylmethyl)-2-imino-6- Ax oxo-4-phenyl- 515.4 130 1bNDQHA° hexahydropyrimidin-1- QA yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deNH3-[[(4S)-4-(2-chlorophenyl)-4- (cyclopropylmethyl)-2-imino-6- HN^CD 515.2 1154 64bND oxo-hexahydropyrimidin-1- yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deNH<1 HI'AN''^^ 3-[[(4R)-4-(4-chlorophenyl)-4- 'A — No(cyclopropylmethyl)-2-imino-6- 515.2 133 1bND oxo-hexahydropyrimidin-1- O T^° yl]methyl]-N-[(lS)_l_ClN QH x phenyl ethyl ]b enzami de3-[[(4S)-4-(4-chlorophenyl)-4- <1 HN-^N^^j^sAyAAo A1(cyclopropylmethyl)-2-imino-6- 515.2 902 26bND oxo-hexahydropyrimidin-1- OHJA°yl]methyl]-N-[(lS)_l_Cl(J phenyl ethyl ]b enzami de NH<.1 HNzJAIzAZ% 3 - [ [(4R)-4-(3 -chlorophenyl)-4- y (cyclopropylmethyl)-2-imino-6-ci—C2^ 515.2 103 1bND oxo-hexahydropyrimidin-1- HNADyl]methyl]-N-[(lS)_l_phenyl ethyl ]b enzami deNH3 -[ [(4 S)-4-(3 -chlorophenyl)-4- < / I1 HN^ JL N^X^’yAx^o Ax (cyclopropylmethyl)-2-imino-6- Cl— Z HN^O 515.2 848 14bND oxo-hexahydropyrimidin-1- yl]methyl]-N-[(lS)_l_phenyl ethyl ]b enzami deNH=3-[(lR)-l-(2-imino-6-oxo-4,4- OXA?0 diphenyl-hexahydropyrimidin-1- / HN^O 517.3 1949 228bNDyl)ethyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de / =\NHV_ / HN^N^X^ 3-[[(4R)-4-benzyl-2-imino-6-oxo- ^KAo Ax 4-phenyl-hexahydropyrimidin-l- HN^O 517.3 360 2bNDyl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de(6R)-6-(cyclopropylmethyl)-3- NH F [[3 -(3,3 -difluoroazetidine- 1 - <.1 HN^ AN^XAp AFcarbonyl)-5- 521.6 71 2bND(trifluoromethyl)phenyl]methyl]- OF?GNXO2-imino-6-phenyl-hexahydropyrimidin-4-one26098-23-cyclopropyl-5-[[(4R)-4- (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- 521.4 307 2bNDhexahydropyrimidin-1- yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deNH N-[(S)- cyclobutyl(phenyl)methyl]-3- K [[(4R)-4-(cyclopropylmethyl)-2- 521.6 773 8bNDCD*HK° imino-6-oxo-4-phenyl- CT v3 hexahydropyrimidin-1- yl]methyl]benzamide3-[(lR)-l-[2-imino-6-oxo-4- phenyl-4-(2,2,2- 523.2 725 99bND trifluoroethyl)hexahydropyrimidi OHj / ° n-l-yl]ethyl]-N-[(l S)-l- Qj pheny 1 ethyl ]b enzami de3-[(lR)-l-[(4R)-4- (cyclopropylmethyl)-2-imino-6- MAUHU 523.3 746 ND ND oxo-4-phenyl- 0 hexahydropyrimidin-l-yl]butyl]- N-[(l S)- 1 -phenylethyl]benzamide N-[(4S)-chroman-4-yl]-3-[l- [(4R)-4-(cyclopropylmethyl)-2- 523.3 1800 ND ND imino-6-oxo-4-phenyl- co hexahydropyrimidin-1- y 1 ] ethyl ]b enzami de(6S)-3-[[4-bromo-3-(morpholine- 4-carbonyl)phenyl]methyl]-6- b uo> 527.4 822 ND ND (cyclopropylmethyl)-2-imino-6- phenyl-hexahydropyrimidin-4- one3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- hexahydropyrimidin-1- 527.7 83 2bNDO yl]methyl]-N-(2,2,2- trifluoroethyl)-5- (trifluoromethyl)benzamide N-[(4S)-chroman-4-yl]-3-[[(4R)- 4-(cyclopropylmethyl)-2-imino-6- 527.3 149 6aND oxo-4-phenyl- co hexahydropyrimidin-1- yl]methyl]-5-fluoro-benzamide N-chroman-4-yl-3-[[(4R)-4- V (cyclopropylmethyl)-2-imino-6- Q K 527.3 323 9aND oxo-4-phenyl-Co hexahydropyrimidin-1- yl]methyl]-5-fluoro-benzamide26098-2N-[(4R)-chroman-4-yl]-3-[[(4R)- 4-(cyclopropylmethyl)-2-imino-6- M' Q 527.3 736 ND ND oxo-4-phenyl- O ™hexahydropyrimidin-1- yl]methyl]-5-fluoro-benzamide \ d / = methyl (2S)-2-[[3-[(2-imino-6- oxo-4, 4-diphenyl- 527.6 1954 77bND hexahy dropyrimidin- 1- G yl)methyl]benzoyl]amino]-3,3- dimethyl-butanoate3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- 528.6 203 3bND hexahydropyrimidin-1- O ^ yl]methyl]-N-thiazol-5-yl-5- 6 (trifluoromethyl)benzamide N-[(4S)-chroman-4-yl]-3-[l-(4,4- diethyl-2-imino-6-oxo- 529.3 1270 ND ND hexahy dropyrimidin- 1 -y 1 ) -3 - ^AoVhU (difluoromethoxy)propyl]benzami de___NH- 3-[(lR)-l-[(4R)-4-benzyl-2- imino-6-oxo-4-phenyl- 531.3 692 31bNDO ^ hexahy dropy rimi din- 1 -y 1 ] ethyl ] - N-[(lS)-l-phenylethyl]benzamide (6R)-6-(cyclopropylmethyl)-3- [[3 -(3, 3 -difluoropyrrolidine- 1- carbonyl)-5- 535.6 163 2bNDdpr^ (trifluoromethyl)phenyl]methyl]- 2-imino-6-phenyl- Bohexahydropyrimidin-4-one NH 3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- V Vp hexahy dropyrimidin- 1 - HN^O 535.6 204 2bNDyl]methyl]-N-[(lR)-2,2,2- QrY trifluoro- 1 -phenylethyl ]benzami de3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- hexahy dropyrimidin- 1- 535.6 447 5bNDyl]methyl]-N-[(l S)-2,2,2- trifluoro- 1 -phenylethyl ]benzami deN-benzyl-3-[[(4R)-4- (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- 535.5 92 1bNDhexahy dropyrimidin- 1 - yl]methyl]-5-(trifluoromethyl)benzamide26098-2NHmethyl 2-[(lS)-l-[[3-[[(4R)-4- (cyclopropylmethyl)-2-imino-6- HN^O oxo-4-phenyl- 539.5 318 2bNDhexahydropyrimidin-1- yl]methyl]benzoyl]amino]ethyl]bu\ enzoateNHmethyl 4-[(lS)-l-[[3-[[(4R)-4- <1(cyclopropylmethyl)-2-imino-6- — 'Nooxo-4-phenyl- Q A 539.5 861 5bNDhexahydropyrimidin-1- yl]methyl]benzoyl]amino]ethyl]b V3enzoateNHmethyl 3-[(lS)-l-[[3-[[(4R)-4- (cyclopropylmethyl)-2-imino-6- O A oxo-4-phenyl- 539.5 924 4bNDhexahydropyrimidin-1- QA yl]methyl]benzoyl]amino]ethyl]b 0^0 enzoate1(6R)-6-(cyclopropylmethyl)-2-. JL imino-3-[[3-(4- ^TNAO A^ methoxypiperidine- 1 -carbonyl)-5- 543.6 378 4bNDOO / \ C / J (trifluoromethyl)phenyl]methyl]- 1 6-phenyl-hexahydropyrimidin-4- oneNHN-benzhydryl-3-[[(4R)-4- <] HN^N^y^X^AA AA (cyclopropylmethyl)-2-imino-6- 543.6 702 6bND oxo-4-phenyl- O A°hexahydropyrimidin-1- 0 *0 yl]methyl]benzamideN-benzyl-2-bromo-5-[[(4S)-4- (cyclopropylmethyl)-2-imino-6- 547.2 1370 ND ND oxo-4-phenyl- hexahydropyrimidin-1- yl]methyl]benzamideNH. A 3-[[(4R)-4-(cyclopropylmethyl)- <1 HN^N'^'Y^l^-JAA^O 2-imino-6-oxo-4-phenyl- AAhexahydropyrimidin-1- OHJ^° 549.5 287 2bNDyl]methyl]-N-[(lS)-l-[4- vCA (trifluoromethyl)phenyl]ethyl]benFA^ zamideNH 3-[[(4R)-4-(cyclopropylmethyl)- < / i1 HN^ JLN'^'Y^.AAA^O2-imino-6-oxo-4-phenyl- AAhexahydropyrimidin-1- Oh^° 549.6 345 3bNDyl]methyl]-N-[(lS)-l-[2- cC (trifluoromethyl)phenyl]ethyl]benl^Fzamide26098-23-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- hexahydropyrimidin-1- OHA° 549.5 487 3bNDyl]methyl]-N-[(lS)-l-[3- QA (trifluoromethyl)phenyl]ethyl]ben zamide3-[[(4S)-4-(cyclopropylmethyl)- yy2-imino-6-oxo-4-[4- 549.3 1208 94bND (trifluoromethyl)phenyl]hexahydr opyrimidin- 1 -yl]methyl]-N-[( 1 S)- A Q 1 -phenylethyl]benzamide3-[[(4R)-4-(cyclopropylmethyl)- y2-imino-6-oxo-4-[4- 549.2 177 2bND (trifluoromethyl)phenyl]hexahydr opyrimidin- 1 -yl]methyl]-N-[( 1 S)- 1 -phenylethyl]benzamide3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-[3 -FAOH549.3 142 1bND (trifluoromethyl)phenyl]hexahydr?^°opyrimidin- 1 -yl]methyl]-N-[( 1 S)- 1 -phenylethyl]benzamide3-[[(4S)-4-(cyclopropylmethyl)- ^ 2-imino-6-oxo-4-[3 - V549.2 798 33bND (trifluoromethyl)phenyl]hexahydr opyrimidin- 1 -yl]methyl]-N-[( 1 S)- C^ 1 -phenylethyl]benzamide (6R)-6-(cyclopropylmethyl)-3- [[3 -(4,4-difluoropiperidine- 1 - carbonyl)-5- 549.7 162 3bNDO y (trifluoromethyl)phenyl]methyl]- 2-imino-6-phenyl- hexahydropyrimidin-4-one 3-[(lR)-l-[(4R)-4- (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- 551.3 1380 ND NDhexahydropyrimidin-l-yl]butyl]- N-[(lR,2R)-2-hydroxyindan-l- yl]benzamideN-[(4S)-chroman-4-yl]-3-[(lR)-l- [(4R)-4-(cyclopropylmethyl)-2- AJATAA) 551.3 1780 ND ND imino-6-oxo-4-phenyl- 0 hexahydropyrimidin-1- yl]butyl]benzamidemethyl 4-[(lS)-l-[[3-[[(4R)-4- < Y1 yyA / A0Ay (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- A 557.5 334 ND NDhexahydropyrimidin-1- yl]methyl]-5-fluoro-b enzoy 1 ] amino] ethyl ]b enzoate26098-2(6S)-3-[[4-bromo-3-(5,7-4 0 dihydropyrrolo[3,4-b]pyrazine-6- carbonyl)phenyl]methyl]-6- 561.2 1140 33aND(cyclopropylmethyl)-2-imino-6- phenyl-hexahydropyrimidin-4- m m oneNHmethyl 4-[(lS)-l-[[3-[(2-imino-6-X X / O / NNAAXQ O oxo-4, 4-diphenyl- OHJP° 561.7 1103 9bND hexahydropyrimidin-1-xyl)methyl]benzoyl]amino]ethyl]b o ij enzoateNH 3-[[(4R)-4-(cyclopropylmethyl)- <1 ^ 2-imino-6-oxo-4-phenyl- hexahydropyrimidin-1- HN^O 565.5 570 3bNDyl]methyl]-N-[(lS)-l-[4- (trifluoromethoxy)phenyl]ethyl]b 4O'OAenzamideNH<1 HN^N'^y^A. 3-[[(4R)-4-(cyclopropylmethyl)-ksy^ 2-imino-6-oxo-4-phenyl- QHJP° hexahydropyrimidin-1- 565.5 664 4bNDyl]methyl]-N-[(lS)-l-[3- (tri fluoromethoxy )phenyl]ethyl]b 0 F^5F enzamide°yA methyl (4R)-4-[(4R)-4- NH A (cyclopropylmethyl)-2-imino-6- <3 HN-^N — Y4 oxo-4-phenyl- A 567.3 1265 46bND hexahydropyrimidin-l-yl]-4-[3- OHJ^° [[(1S)-1- pheny 1 ethyl ] carb amoy 1 ] pheny 1 ]bu ex tanoateNH 3-[[(4R)-4-(cyclopropylmethyl)- <1 2-imino-6-oxo-4-phenyl- OX hexahydropyrimidin-1- Q - °O 571.4 680 21bND>< vOH yl]methyl]-5-fluoro-N-[(3 S,4R)- OCX 3-hydroxy-2,2-dimethyl- chroman-4-yl]benzamide methyl (2S)-2-[[3-[[(4R)-4- (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- 573.6 177 2bND hexahydropyrimidin-1- yl]methyl]-5- (trifluoromethyl)benzoyl]amino]- 3,3 -dimethyl-butanoate3 -[ [4-bromo-3 -(5,7- dihydropyrrolo[3,4-b]pyrazine-6- CTAO'A 583.1 1900 ND ND carbonyl)phenyl]methyl]-2- o imino-6,6-diphenyl-hexahydropyrimidin-4-one26098-23-[(lS)-l-(2-imino-6-oxo-4,4- diphenyl-hexahydropyrimidin-1- 585.3 791 332bNDO yl)ethyl]-N-[(lS)-l-phenylethyl]- 5-(trifluoromethyl)benzamide NH<7 1 methyl (2S)-2-[[3-[[(4R)-4- N HN^N^Y^IX II (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- Q 593.7 1981 21bNDhexahydropyrimidin-1- yl]methyl]benzoyl]amino]-2-[4- vOX(trifluoromethyl)phenyl]acetate 4-[(lS)-l-[[3-[[(4R)-4- (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- 593.7 1178 2bND hexahydropyrimidin-1- yl]methyl]-5- (trifluoromethyl)benzoyl]amino]e thyl]benzoic acidmethyl (2R)-2-[[3-[[(4R)-4- J< (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- ^x^ci593.6 88 1bND hexahydropyrimidin-1- o, ° yl]methyl]-5- Y e (trifluoromethyl)benzoyl]amino]- 2-phenyl-acetatemethyl (2S)-2-[[3-[[(4R)-4- (cyclopropylmethyl)-2-imino-6- oxo-4-phenyl- 593.6 435 1bND hexahydropyrimidin-1- yl]methyl]-5- 1 15 (trifluoromethyl)benzoyl]amino]- 2-phenyl-acetate(6S)-6-(cyclopropylmethyl)-3 -[ 1 - [3-(5,7-dihydropyrrolo[3,4- b]pyrazine-6-carbonyl)-4- XO XX 605.3 1670 ND ND(trifluoromethyl)phenyl]-3- methyl-butyl]-2-imino-6-phenyl- hexahydropyrimidin-4-one E HN^N'^Y^Y F 3-fluoro-5-[(2-imino-6-oxo-4,4- AAdiphenyl-hexahydropyrimidin-1- 607.7 70 1bNDyl)methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami de3-[[(4R)-4-(cyclopropylmethyl)- 2-imino-6-oxo-4-phenyl- OHr^° 643.3 896 140bND hexahydropyrimidin-1- yl]methyl]-N-[(lS)-l-pheny 1 ethyl ]b enzami de- Ill -26098-2NHiF- ji I >F3-[[(4R)-4-(2-chlorophenyl)-4- (cyclopropylmethyl)-2-imino-6- O A 643.3 748 372bND oxo-hexahydropyrimidin-1- yl]methyl]-N-[(lS)-l- pheny 1 ethyl ]b enzami deNHF c<7 A > 3-[[(4R)-4-(cyclopropylmethyl)- 4-(2-fluorophenyl)-2-imino-6- Z fl 661.7 159 4bND oxo-hexahydropyrimidin-1- yl]methyl]-N-[(lS)-l- LOY'pheny 1 ethyl ]b enzami dea=Crypto Biochemical Assay Protocol #1;b=Crypto Biochemical Assay Protocol #2 ND=not determined; *=chiral center resolvedExample 15Schizont maturation assay (E. tenella)

[0342] Mammalian MDBK cells (Madin Darby Bovine Kidney cells) infected with Eimeria tenella sporozoites were incubated in 384- well microtiter plates with the test compounds JI, J2 and J3 at declining concentrations of 50 pM - 0.85 nM (11 concentrations in 1:3-dilution steps). After 48 hours, the development of mature E. tenella schizonts was assessed using an indirect immunofluorescence assay as readout. The four-parameter equation 205 from ActivityBase (IDBS, London, UK) was used to calculate ECso values. In addition, toxicity against MDBK cells, which would influence the assay read-out resulting in false positives, was determined using a Cellblue assay (CellTiter-Blue®Reagent; Promega, Madison, WI, USA).Proliferation assay Toxoplasma (T. gondii)

[0343] Vero-cells (originally isolated from kidney epithelial cells extracted from African green monkey) infected with Toxoplasma tachyzoites were incubated in 384- well microtiter plates with the test compounds JI, J2 and J3 at declining concentrations of 50 pM - 0.85 nM (11 concentrations in 1: 3 -dilution steps). After 42 hour incubation, the proliferation of the tachyzoites was assessed using an indirect immunofluorescence assay as readout. The four- parameter equation 205 from ActivityBase (IDBS, London, UK) was used to calculate ECso values. In addition, toxicity against Vero-cells, which would influence the assay read-out resulting in false positives, was determined using a Cellblue assay (CellTiter-Blue®Reagent; Promega, Madison, WI, USA).Cytotoxicity assay26098-2

[0344] Cytotoxicity was assessed by the AlamarBlue® assay in MDBK and Vero cells (384- well format). Cells were incubated with different test compounds (JI, J2 and J3) concentrations 50 pM - 0.85 nM (11 concentrations in 1:3-dilution steps) for 24 h. Then, metabolic formation of the fluorescent resorufin was measured (550 / 595 nm) with the fluorescence signal being proportional to metabolically active and viable cells. Finally, CTD50 values were calculated reflecting the test compound concentration that reduced cell viability by 50%.

[0345] Eimeria and Toxoplasma homologs (ASP3 and Etl982, respectively) of plasmepsin X were identified by BLASTP (version 2.6 (see Altschul, Stephen, et al., Nucleic Acids Research, 1997, Vol. 25, No. 173389-3402)) search of the taxa using protein sequence from the Plasmodium falciparum 3D7 strain (see Gardner, Malcolm et si., Nature, 2002 October 3; 419 (6906):. doi: 10.1038 / nature01097. Prior studies noted high sequence similarity of plasmepsins and Eimeria tenella and Toxoplasma gondii (SheaM et al., Traffic. 2007 Aug; 8(8): 1018-34; and KamyingkirdK et al., J. Protozool. Res. 201424:18-25) homologs and antibodies against Toxoplasma ASP3 afford protective immunity (Zhao G et al., Acta Trop. 2017 Jul; 171:17-23). Availability of aspartyl protease three dimensional structures aided identification of ASP3 inhibitors (Dogga SK et al., Elife. 2017 Sep 12; 6. pii: e27480) and homology modeling of their enzyme interactions (Mukherjee B et al., EMBO J. 2018 Apr 3; 37(7). pii: e98047). JI, J2 and J3 were tested using In vitro cellular assays run in triplicate for prevention of Eimeria schizont maturation and merozoite egress, and prevention of Toxoplasma tachyzoite development.Cytotoxicity was measured in two cell lines. Compound JI, J2, and J3 identified with in vitro activity against Eimeria tenella having an EC50 of - 5pM and had similar Toxoplasma gondii activity (Table 1). J3 was docked to a homology model of Eimeria Etl982 built from the Plasmodium vivax V crystal structure(RCSB 4ZL4)[ Hodder AN et al., Nat Struct Mol Biol. 2015 Aug; 22(8):590-6] (RCSB 4ZL4) using the MOE program.

[0346] Plasmepsin X inhibitors compounds JI, J2 and J3 are represented in structural Formula JA:wherein with Jl=Rj is CF3 and X is N; J2=R> is H and X is N; and J3=R> is H and X is C.26098-2Table 5- Inhibitor ActivityCompound Inhibition of Inhibition of Celltox MBDK Celltox Vero maturation of maturation of cells EC50 in cells EC 50 in Eimeria tenella Toxoplasma [pM] [pM] Mean EC50 in gondi Mean[pM] EC50 in [pM]JI 2.746 5.359 36.660 40.979J2 4.904 13.316 >50.000 45.175J3 6.248 10.009 17.686 30.125Example 16 - Inhibition of Maturation of Toxoplasma gondi

[0347] Additional Toxoplasmosis experiments were conducted with WM4 and WM382 and also found to work with EC50's at low pM as shown in Figure 6. The dose curves (two top graphs in Fig. 6) show the (ECso) of WM382 (1.972pM) and WM4 (2.05pM) on Toxoplasma gondii (modified RH strain) tachyzoites. Each repeat (dots; N=4) is the average of 3-4 technical replicates. The best-fit curve (solid line) is used to determine ECso.

[0348] The ECso / 9o / 99values of the dose curves were used to determine time of action of WM382 and WM4 compounds (two bottom graphs in Fig. 6). In both WM382 and WM4 parasite death started to occur between 18-24 hours, correlating with the first cycle of egress from the host cell and re-invasion of new cells. Each dot represents the average of 3 technical replicates, error bars are in standard deviation, solid line represents the best-fit decay curve.Methodology:

[0349] Toxoplasma RH Nano-Luc expressing tachyzoites (1000 / well) were add to a confluent layer of HFFs in a 96 well plate in DMEM + 1% FBS media. Compounds WM382 and WM4 were diluted from lOmM DMSO stocks to a final DMSO concentration of 1% across all dilutions. No compound control was 1% DMSO only. GraphPad (www.graphpad.com / quickcalcs / ECanythingl / ) was used to determine the EC90 and EC99. WM382 and WM4 and parasites were added at the same time to a final well volume of lOOpl. Each repeat (N) was conducted with 3-4 technical replicates. To readout, 50pl Nano-Gio substrate in Assay Buffer (1:50 dilution; Nano-Gio® Luciferase Assay System, Promega, N1110) was added on top of the lOOpl and incubated for lOmins at room temperature. 130pl from each well was moved to a white 96 well plate and readout using CLARIOStar. Final ratio was determined by this calculation, %survival = (technical repeat / DMSO average)* 100.Technical repeats were then averaged across N. For dose curve, final read out was at 72 hours. For time to death assays, read out times were as indicated. Non-linear curve ([Inhibitor] vs.26098-2response — Variable slope (four parameters) in Prism (GraphPad) was used to fit curves to data points and determine EC50.

[0350] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

26098-2WHAT IS CLAIMED IS:

1. A method of treating infections caused by one or more plasmepsin-like protozoa selected from Cryptosporidium, Eimeria, Babesia and Toxoplasma comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X of Formula Idl:or a pharmaceutically acceptable salt, solvate and / or tautomer thereof, wherein:U is N or CH, wherein when U is N, X”, Y” and Z” are CH;X” is N or CH, wherein when X” is N, U, Y” and Z” are CH;Y” is N or CH, wherein when Y” is N, X”, U and Z” are CH;Z” is N or CH, wherein when Z” is N, X”, Y” and U are CH;R1is a Ci-6 alkyl, carbocycycle, heterocycloalkyl, Ci-6 alkylaryl, C3-6 cycloalkylaryl, -CH(C3-6 cycloalkyljaryl, -CH(CO(O)Ci-6alkyl)aryl, heteroaryl, or C3-Ci2cycloalkyl, wherein the alkyl, carbocycle, heterocycloalkyl, aryl, heteroaryl, or C3-Ci2cycloalkyl is unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of halogen, CN, OH, alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH, oxo, COOCi-C6alkyl, phenyl, C3-C6cycloalkyl, Ci-C6alkyl, haloCi-C6alkyl, -OhaloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) and Ci-C6alkylN(R7”)(R8”);R2is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH, or R1and R2combine with the nitrogen atom to which they are attached to form a heterocycloalkyl or heterocycle;R3is halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, C3-C6cycloalkyl, Ci-C6alkyl, haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) or Ci-C6alkylN(R7)(R8);R4is hydrogen, halogen, CN, Ci-CealkylCN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-C6alkylOhaloCi-C6alkyl, Ci-CealkylOCi-CealkylOCi-Cealkyl, Ci-C6alkylCOOH, Ci-C6alkylCOOCi-C6alkyl, COOH, Ci-C6alkylSO2Ci-C6alkyl, Ci-C6alkylphenyl, phenyl, heterocycloalkyl, Ci-Cealkylheterocycloalkyl, heteroaryl, Ci-Cealkylheteroaryl, Cs-Cecycloalkyl, Ci-C6alkyl, Ci-C6alkenyl, Ci-C6alkynyl, haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”),26098-2N(R7”)(R8”), Ci-C6alkyl(OCH2CH2)wN3, or Ci-C6alkylN(R7”)(R8”), wherein the Ci-Cealkylphenyl, phenyl, heterocycloalkyl, Ci-Cealkylheterocycloalkyl, heteroaryl, Ci-Cealkylheteroaryl, Ci-CealkylOH, Ci-CealkylOCi-Cealkyl or Cs-Cecycloalkyl is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, CN, OH, alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH, oxo, COOCi-C6alkyl, Ci-C6alkyl, haloCi-C6alkyl, Ci-C6alkylOH, SO2Ci-C6alkyl, Ci-C6alkySO2Ci-C6alkyl, OSO2F, CON(R7”)(R8”), N(R7”)(R8”) and Ci-C6alkylN(R7”)(R8”);R5is hydrogen, halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, phenyl, CH2phenyl, Ci-CealkylCs-Cecycloalkyl, Cs-Cecycloalkyl, Ci-Csalkyl, haloCi-Cealkyl, Ci-C6alkyl haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) or Ci-C6alkylN(R7”)(R8”) or when taken with R6represents a Cs-Cecycloalkyl or Cs-Ceheterocycloalkyl or when R5is Ci-Csalkyl and R1is a heterocycloalkyl or Cs-Cncycloalkyl, R5optionally bonds to R1to form a macrocycle, said phenyl optionally substituted with 1 to 3 groups of halogen and haloCi-Cealkyl; R6is hydrogen, halogen, CN, OH, alkoxy, Ci-CealkylOCi-Cealkyl, Ci-CealkylCOOH, COOH, phenyl, CH2phenyl, Ci-CealkylCs-Cecycloalkyl, Cs-Cecycloalkyl, Ci-Csalkyl, haloCi-Cealkyl, Ci-C6alkyl haloCi-C6alkyl, Ci-C6alkylOH, CON(R7”)(R8”), N(R7”)(R8”) or Ci-C6alkylN(R7”)(R8”) or when taken with R5represents a Cs-Cecycloalkyl or Cs-Ceheterocycloalkyl;R7is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH;R8is hydrogen, Ci-CealkylCOOH, COOH, Cs-Cecycloalkyl, Ci-Cealkyl, haloCi-Cealkyl or Ci-CealkylOH;R9is hydrogen, halogen, or Ci-Cealkyl;w is 1, 2 or 3; andz is 0, 1, 2 or 3.

2. The method of claim according to claim 1 wherein the inhibitor of plasmepsin IX and / or X is a compound selected from Formula Id, Formula Idl, Formula IId, and Formula IId2, or a pharmaceutically acceptable salt thereof,.

3. The method according to any one of claims 1 and 2 wherein the inhibitor of plasmepsin IX and / or X is a compound selected from Table 4, or a pharmaceutically acceptable salt thereof.26098-24. The method according to any one of claims 1 through 3 wherein, in Formula Idl, R1is selected from the following groups:wherein Rxis hydrogen, CO(O)Ci-6 alkyl, or Ci-6 alkyl, said groups unsubstituted or substituted with 1 to 5 substituents selected from the group consisting of halogen, CN, OH, Ci-C6alkoxy, Ci-C6alkylOCi-C6alkyl, Ci-C6alkylCOOH, COOH,oxo, COOCi-C6alkyl, C3-C6cycloalkyl, C C6alkyl, haloCrC6alkyl, Ci-C6alkylOH, CON(R7)(R8), N(R7)(R8) and Ci-C6alkylN(R7)(R8), wherein R7is hydrogen, Ci-C6alkylCOOH, COOH, C3-C6cycloalkyl, Ci-C6alkyl, halo Ci-C6alkyl or Ci-C6alkylOH; and R8is hydrogen, Ci-C6alkylCOOH, COOH, C3-C6cycloalkyl, Ci-C6alkyl, halo Ci-C6alkyl or Ci-C6alkylOH,or a pharmaceutically acceptable salt thereof.

5. The method according to any one of claims 1 through 4 wherein thecompound of Formula Idl, or a pharmaceutically acceptable salt thereof, isrepresented by structural Formula IId:26098-26. The method according to any one of claims 1 through 5 wherein the compound of Formula Idl, or a pharmaceutically acceptable salt thereof, is represented by structural Formula IId2:

7. The method according to claim 6 wherein in the compound of Formula Idl, or a pharmaceutically acceptable salt thereof, R6and R5are independently selected from optionally substituted phenyl, CFhcycloalkyl, cycloalkyl, methyl, ethyl, propyl, isopropyl, butyl, and CH2CF3.

8. The method according to any one of claims 1 through 3, wherein in the compound of Formula Id, Formula Idl, Formula IId, and Formula IId2, or a pharmaceutically acceptable salt thereof, R1and R2combine with the nitrogen atom to which they are attached to represent a heterocycloalkyl or heterocycle.

9. A method of treating infections caused by one or more plasmepsin-like protozoa selected from Cryptosporidium, Eimeria, Babesia and Toxoplasma comprising administering to a subject in need thereof an effective amount of an inhibitor of plasmepsin IX and / or X compound selected from List A and Table 4, or a pharmaceutically acceptable salt thereof.

10. The method according to claim 9 wherein the inhibitor of plasmepsin IX and / or X of selected from:26098-2or a pharmaceutically acceptable salt thereof.

11. The method according to any one of claims 1 through 10 for treating cryptosporidiosis, derived from protozoa Cryptosporidium.

12. The method according to any one of claims 1 through 10 for treating coccidiosis, derived from protozoa Eimeria.

13. The method according to any one of claims 1 through 10 for treating toxoplasmosis, derived from protozoa Toxoplasma.

14. The method according to any one of claims 1 through 10 for treating babesiosis, derived from protozoa Babesia.

15. The method according to any one of claims 1 through 10 for treating parasitic infections caused by protozoans selected from Cryptosporidium, Eimeria, Babesia, and Toxoplasma wherein an effective amount of one or more additional anti-antiparasitic agents is used.26098-216. The method according to any one of claims 1 through 15 for treating cryptosporidiosis, coccidiosis, taxoplamosis, and / or babesiosis wherein the patient does not have a parasitic infection containing homologs of Cryptosporidium, Eimeria, Babesia, and / or Toxoplasma plasmepsin.

17. The method according to any one of claims 1 through 15 for treating cryptosporidiosis, coccidiosis, taxoplamosis, and / or babesiosis wherein the patient does not have a parasitic infection containing homologs of Cryptosporidium, Eimeria, Babesia, and / or Toxoplasma plasmepsin, and wherein the patient is later exposed to a wild-type parasite containing homologs of Cryptosporidium, Eimeria, Babesia, and / or Toxoplasma plasmepsin.

18. A method of inducing an immune response to a Plasmodium parasite infection linked to one or more of Cryptosporidium, Eimeria, Babesia, and Toxoplasma, comprising administering to a patient, an effective amount of the compounds of Formula Id, Formula Idl, Formula IId, Formula IId2according to claim 1, or a pharmaceutically acceptable salt thereof.

19. The method according to any one of claims 1 through 18, wherein the inhibitor of plasmepsin IX and / or X is a dual inhibitor of plasmepsin IX and X.

20. A compound which is:3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - phenylethyl] -5 -(trifluoromethyl)benzamide3 -[( 1 R)- 1 -[2-imino-6-oxo-4-phenyl-4-(2,2,2-trifluoroethyl)hexahydropyrimidin- 1 -yl] ethyl] -N- [ ( 1 S) - 1 - phenylethyl] benzamidemethyl (4R)-4-[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]-4-[3- [ [( 1 S)- 1 -phenylethyl]carbamoyl]phenyl]butanoate3 -[ [2-imino-6-oxo-4-phenyl-4-(2,2,2-trifluoroethyl)hexahydropyrimidin- 1 -yl]methyl] -N- [ ( 1 S) - 1 - phenylethyl] benzamide3 -[ [2-imino-6-oxo-4-phenyl-4-(2,2,2-trifluoroethyl)hexahydropyrimidin- 1 -yl]methyl] -N- [ ( 1 S) - 1 - phenylethyl] benzamide3 -[ [(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N- isopropyl-5-(trifluoromethyl)benzamide3-[[(4R)-4-(cyclopropylmethyl)-4-(3-fluorophenyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide26098-2methyl 4-[( 1 S)- 1 -[[3-[(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin- 1 - yl)methyl]benzoyl]amino]ethyl]benzoate3 -[ [(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N, N- dimethyl-5-(trifluoromethyl)benzamide(6R)-6-(cyclopropylmethyl)-2-imino-6-phenyl-3-[[3-(pyrrolidine-l-carbonyl)-5- (trifluoromethyl)phenyl]methyl]hexahydropyrimidin-4-onel-[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-5- (trifluoromethyl)benzoyl]azetidine-3-carbonitrile(6R)-6-(cyclopropylmethyl)-2-imino-3-[[3-(morpholine-4-carbonyl)-5- (trifluoromethyl)phenyl]methyl]-6-phenyl-hexahydropyrimidin-4-one(6R)-6-(cyclopropylmethyl)-3 -[ [3 -(3,3 -difluoroazetidine- 1 -carbonyl)-5 - (trifluoromethyl)phenyl]methyl]-2-imino-6-phenyl-hexahydropyrimidin-4-one3 -[ [(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-(2,2,2- trifluoroethyl)-5-(trifluoromethyl)benzamide3 -[ [(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-thiazol- 5 -yl -5 -(trifluoromethyl)benzamide(6R)-6-(cyclopropylmethyl)-3 -[ [3 -(3,3 -difluoropyrrolidine- 1 -carbonyl)-5 - (trifluoromethyl)phenyl]methyl]-2-imino-6-phenyl-hexahydropyrimidin-4-oneN-benzyl-3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]- 5 -(trifluoromethyl)benzamide(6R)-6-(cyclopropylmethyl)-2-imino-3-[[3-(4-methoxypiperidine-l-carbonyl)-5- (trifluoromethyl)phenyl]methyl]-6-phenyl-hexahydropyrimidin-4-one(6R)-6-(cyclopropylmethyl)-3-[[3-(4,4-difluoropiperidine-l-carbonyl)-5- (trifluoromethyl)phenyl]methyl]-2-imino-6-phenyl-hexahydropyrimidin-4-onemethyl (2S)-2-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl] -5 -(trifluoromethyl)benzoyl] amino] -3,3 -dimethyl -butanoatemethyl (2R)-2-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl] -5 -(trifluoromethyl)benzoyl] amino] -2 -phenyl -acetatemethyl (2S)-2-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl] -5 -(trifluoromethyl)benzoyl] amino] -2 -phenyl -acetate3 -[( 1 R)- 1 -(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin- 1 -yl)ethyl] -N-[(1S)-1- phenylethyl] benzamide3 -[( 1 R)- 1 -[(4R)-4-benzyl-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl] ethyl] -N-[(1S)-1- phenylethyl] benzamidemethyl 4-[( 1 S)- 1 -[ [3 -[( 1 S)- 1 -(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin- 1 -yl)ethyl] -5 - (trifluoromethyl)benzoyl]amino]ethyl]benzoate26098-2methyl 4-[( 1 S)- 1 -[ [3 -[( 1 R)- 1 -(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin- 1 -yl)ethyl] -5 - (trifluoromethyl)benzoyl]amino]ethyl]benzoate3-[[(4R)-4-(cyclopropylmethyl)-4-(2-fluorophenyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3 -[( 1 R)- 1 -(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin- 1 -yl)ethyl] -N- [ ( 1 S ) - 1 -phenylethyl] -5 - (trifluoromethyl)benzamide3 -[( 1 S)- 1 -(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin- 1 -yl)ethyl] -N- [ ( 1 S ) - 1 -phenylethyl] -5 - (trifluoromethyl)benzamidemethyl (2S)-2-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]benzoyl]amino]-2-[4-(trifluoromethyl)phenyl]acetate4-[(lS)-l-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]-5-(trifluoromethyl)benzoyl]amino]ethyl]benzoic acidmethyl 4-[(lS)-l-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]-5-(trifluoromethyl)benzoyl]amino]ethyl]benzoate3 -[[(4R)-4-benzyl-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl]-N-[( 1 S)- 1 - phenylethyl] benzamide3 -[( 1 R)- 1 -(4-cyclobutyl-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl)ethyl] -N-[(1S)-1- phenylethyl] benzamide3 -[ [(4R)-4-(cyclobutylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N- [ ( 1 S) - 1 - phenylethyl] benzamide3 -[ [(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N- [(lR,2S)-2-phenylcyclopropyl]benzamide3-[[(4R)-4-(cyclopropylmethyl)-4-(4-fluorophenyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3 -[(4-cyclobutyl-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl)methyl] -N- [ ( 1 S) - 1 - phenylethyl] benzamide3-cyano-5-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-N- [( 1 S)- 1 -phenylethyl]benzamide3 -[(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin- l-yl)methyl]-N-[( 1 S)- 1 -phenylethyl] benzamide methyl (2S)-2-[[3-[(2-imino-6-oxo-4,4-diphenyl-hexahydropyrimidin-l-yl)methyl]benzoyl]amino]-3,3- dimethyl-butanoate3 -[ [(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 R)- 2,2,2-trifluoro- 1 -phenyl -ethyl] benzamide3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-N-(l- phenylcyclopropyl)benzamide26098-2N-[(lS)-l-(3-chlorophenyl)ethyl]-3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl- hexahydropyrimidin- 1 -yl]methyl] benzamideN-[(lS)-l-(4-chlorophenyl)ethyl]-3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl- hexahydropyrimidin- 1 -yl]methyl] benzamide3-[[(4R)-4-(3-chlorophenyl)-4-(cyclopropylmethyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3-[[(4S)-4-(3-chlorophenyl)-4-(cyclopropylmethyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 2,2,2-trifluoro- 1 -phenyl -ethyl] benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - [4-(trifluoromethoxy)phenyl]ethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - [3-(trifluoromethoxy)phenyl]ethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - (m-tolyl)ethyl]benzamide3-cyclopropyl-5-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]-N-[( 1 S)- 1 -phenylethyl]benzamideN-benzhydryl-3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - [2-(trifluoromethyl)phenyl]ethyl]benzamide2-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - phenylethyl] pyridine -4-carboxamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[(S)- cyclopropyl(phenyl)methyl]benzamide3-[[(4R)-4-(4-chlorophenyl)-4-(cyclopropylmethyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3-[[(4S)-4-(4-chlorophenyl)-4-(cyclopropylmethyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3-[[(4R)-4-(2-chlorophenyl)-4-(cyclopropylmethyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3-[[(4S)-4-(2-chlorophenyl)-4-(cyclopropylmethyl)-2-imino-6-oxo-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3-[[(4S)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-[4-(trifluoromethyl)phenyl]hexahydropyrimidin-l- yl]methyl]-N-[( 1 S)- 1 -phenylethyl]benzamide26098-23-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-[4-(trifluoromethyl)phenyl]hexahydropyrimidin-l- yl]methyl]-N-[( 1 S)- 1 -phenylethyl]benzamide3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-[3-(trifluoromethyl)phenyl]hexahydropyrimidin-l- yl]methyl]-N-[( 1 S)- 1 -phenylethyl]benzamide3-[[(4S)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-[3-(trifluoromethyl)phenyl]hexahydropyrimidin-l- yl]methyl]-N-[( 1 S)- 1 -phenylethyl]benzamideN-[(S)-cyclobutyl(phenyl)methyl]-3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl- hexahydropyrimidin- 1 -yl]methyl] benzamide3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-5-methyl- N-[(1S)-1 -phenylethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - (3 -fluorophenyl)ethyl]benzamidemethyl 2-[(lS)-l-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]benzoyl]amino]ethyl]benzoate3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - [3-(trifluoromethyl)phenyl]ethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - (p-tolyl)ethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - (4-fluorophenyl)ethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - (2-methoxyphenyl)ethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - (3 -methoxyphenyl)ethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - [4-(trifluoromethyl)phenyl]ethyl]benzamide4-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - phenylethyl] pyridine -2 -carboxamide(6R)-6-(cyclopropylmethyl)-3-[[3-fluoro-5-(isoindoline-2-carbonyl)phenyl]methyl]-2-imino-6-phenyl- hexahydropyrimidin-4-onemethyl 3-[(lS)-l-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]benzoyl]amino]ethyl]benzoate3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-5- methoxy-N-[( 1 S)- 1 -phenylethyl]benzamidemethyl 4-[(lS)-l-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]benzoyl]amino]ethyl]benzoate26098-26-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - phenylethyl] pyridine -2 -carboxamide3-chloro-5-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]- N-[(1S)-1 -phenylethyl]benzamide3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-5-fluoro- N-[(3S,4R)-3-hydroxy-2,2-dimethyl-chroman-4-yl]benzamidemethyl 4-[(lS)-l-[[3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l- yl]methyl]-5-fluoro-benzoyl]amino]ethyl]benzoate3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-5-fluoro- N-methyl-N-[( 1R)- 1 -phenylethyl]benzamide3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-5-fluoro- N-methyl-N-[( 1 S)- 1 -phenylethyl]benzamide3 -[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin- 1 -yl]methyl] -N-[( 1 S)- 1 - phenylethyl] benzamide3-[[(4R)-4-(cyclopropylmethyl)-2-imino-6-oxo-4-phenyl-hexahydropyrimidin-l-yl]methyl]-5-fluoro- N-[(1S)-1 -phenylethyl]benzamideor a pharmaceutically acceptable salt thereof.

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