Compositions and methods for preparing orally bioavailable guanidine prodrugs

Abstract

Due to their inherent polarity and basicity, guanidine-based drugs are frequently associated with poor oral bioavailability. While there are reported examples of orally bioavailable guanidine-containing drugs, improvements in adsorption are still needed. The present invention provides prodrugs of a potent, guanidine-based SphK2-selective inhibitor (Ki = 82 nM, 122-fold SphK2 selective) with validated in vivo activity. Modifications to the guanidine head of the inhibitor provided compounds that were effective in alleviating the observed poor oral bioavailability.

Description

COMPOSITIONS AND METHODS FOR PREPARING ORALLY BIOA VAILABLE GUANIDINE PRODRUGSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of the filing date of U.S. Provisional application no. 63 / 604,860 filed on November 30, 2023, which is hereby incorporated by reference herein in its entirety.STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under Grant No. R01 GM121075 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTIONField of the Invention

[0003] The present invention relates to chemical compounds, pharmaceutical compositions, and methods for inhibiting Sphingosine kinases. More specifically, the present invention provides pro-drugs for improvement of the bioavailability of sphingosine kinase inhibitors.Description of Related Art

[0004] In order for a drug to be efficacious in vivo, it is essential for the compound to reach the intended molecular target. Depending on the physiological location of the target, some routes of drug administration may be advantageous for reasons such as mechanism of action, metabolism, drug distribution, and other pharmacokinetic properties. The goal for most drug discovery campaigns is to develop a medication that can be administered to patients orally, i.e., per os (p.o.). Compared to other methods of drug delivery, oral administration is by far the most convenient route for individuals undergoing chronic treatment and ensures the greatest patient compliance for doctors prescribing these medications. Furthermore, orally bioavailable drugs provide the greatest cost-effectiveness in large scale manufacturing, fewest constraints on the sterility of the drug, and greatest flexibility in terms of the prescribed dose (Viswanathan, P. et al. Chapter 7 - Challenges in Oral Drug Delivery: A Nano-Based Strategy to Overcome. In Nanostructures for Oral Medicine, 1st ed.; Andronescu, E., Grumezescu, A. M., Eds.; 2017; pp 173-201; Tetteh, E.; Morris, S. Systematic Review of Drug Administration Costs and Implications for Biopharmaceutical Manufacturing. Appl. Health Econ. and Health Policy. 2013, 11, 445-456). Unfortunately, drugs administered p.o. suffer the greatest challenges associated with achieving the maximum delivered payload to the preferred target in vivo, and as such display the greatest variability in terms ofoverall bioavailability (Bardal, S. K. et al. Chapter 2 - Pharmacokinetics. In Applied Pharmacology, 1st ed.; Bardal, S. K., Waechter, J. E., Martin, D. S., Eds.; Saunders: Philadelphia, 2011; pp 17-34).

[0005] For a drug to be considered bioavailable, it must be present in the bloodstream (Bardal, 2011). As opposed to other routes of administration, such as intraperitoneal (IP) or intramuscular (IM) where access into systemic circulation is accomplished with relative ease, medications introduced p.o. are required to navigate and survive a myriad of barriers before entering into circulation. For a drug to be orally bioavailable, it must survive passage through the acidic stomach content and travel to the small intestine (SI). Next, the drug will need to diffuse across the lumen of the SI to reach the hepatic portal system leading to the liver before reaching systemic circulation. This process is often accompanied by a dramatic reduction in the concentration of active drug and is known as first pass metabolism (Zhu, L. et al. Chapter 11 - Oral Absorption Basics: Pathways and Physicochemical and Biological Factors Affecting Absorption. In Developing Solid Oral Dosage Forms Pharmaceutical Theory and Practice, 2nd ed.; Qiu, Y., Chen, Y., Zhang, G. G. Z., Yu, L., Mantri, R. V., Second E. Eds.; Academic Press: Boston, 2017; pp 297-329). Unfortunately, drug absorption in the gastrointestinal (GI) tract is greatly dependent on the intrinsic physical and chemical properties of the drug as well as the physiological aspects of the SI. Various factors that can affect adsorption and must be taken into consideration during a drug discovery campaign include local pH, inherent drug basicity or acidity, microbial colonization, and Lipinski’s rule of five (Viswanathan, 2017; Zhu, 2017; Lipinski, C. A. et al. Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings. Adv. Drug Deliv. Rev. 1997, 23, 3-25).

[0006] Guanidine-containing compounds have been extensively utilized in the rational drug design of various medications and are commonly observed in nature. As shown in FIG. 1, synthetic examples include compound 1 (zanamivir), an effective neuraminidase inhibitor approved by the FDA for the treatment of influenza type A and B, as well as FDA approved bi-guanidine medications 2 (metformin) and 3 (proguanil) for the treatment of Type 2 diabetes and malaria, respectively. The guanidine functionality is also found in many important naturally occurring compounds such as 4 (creatine), 5 (L-arginine), and 6 (streptomycin). Unfortunately, high basicity of the guanidine functional group (pKa = 13.6) causes protonation upon entering the acidic environment in the SI (pH = 6.0 - 7.4), leading to a condition known as ion trapping (Bardal,2011). Once in the GI tract, this positive charge prevents the drug from progressing toward the hepatic portal system leading to systemic circulation. This is due to the resulting high desolvation cost during passive diffusion and subsequent poor absorption across the lumen of the SI (Lam, P. Y. S. et al. Structure-Based Design of Novel Guanidine / Benzamidine Mimics: Potent and Orally Bioavailable Factor Xa Inhibitors as Novel Anticoagulants. J. Med. Chem. 2003, 46, 4405-4418). Taken together, guanidine-containing compounds are very frequently associated with poor oral bioavailability (Sun, J. et al. Mechanistic Enhancement of the Intestinal Absorption of Drugs Containing the Polar Guanidino Functionality. Expert Opin. Drug Metab. Toxicol. 2011, 7, 313-323).

[0007] Several strategies have been proposed to improve the oral bioavailability of guanidine containing drugs. Potential solutions range from chemical attachment of an acidic moiety such as a carboxylic acid to create an ion pair and an overall neutral drug (Miller, I. M. et al. Enabling the Intestinal Absorption of Highly Polar Antiviral Agents: Ion-Pair Facilitated Membrane Permeation of Zanamivir Heptyl Ester and Guanidino Oseltamivir. Mol. Pharmaceutics. 2010, 7, 1223-1234; Liu, K.-C. et al. Intramolecular Ion-Pair Prodrugs of Zanamivir and Guanidino-Oseltamivir. Bioorg. Med. Chem. 2011, 19, 4796-4802), and selective targeting of membrane transporters in the SI to facilitate transport across the epithelial cell wall (Sun, J. et al. Enhancing the Intestinal Absorption of Molecules Containing the Polar Guanidino Functionality: A Double-Targeted Prodrug Approach. J. Med. Chem. 2010, 53, 624-632; Sun, J. et al. Specificity of a Prodrug- Activating Enzyme hVACVase: The Leaving Group Effect. Mol. Pharmaceutics. 2010, 7, 2362- 2368). Perhaps the most extensively utilized approach is the chemical modification of the guanidine moiety with metabolically labile hydrophobic alkyl and aryl substituents to increase compound lipophilicity and serve as a platform for bioactivation. This strategy has shown moderate success in developing prodrugs that effectively hide the positively charged guanidine in order to promote passive diffusion across the SI lumen and subsequent metabolic activation once in the liver (Huttunen, K. M. et al. The First Bioreversible Prodrug of Metformin with Improved Lipophilicity and Enhanced Intestinal Absorption. J. Med. Chem. 2009, 52, 4142-4148; Hsu, P - H. et al. Acylguanidine Derivatives of Zanamivir and Oseltamivir: Potential Orally Available Prodrugs Against Influenza Viruses. Eur. J. Med. Chem. 2018, 154, 314-323; Saulnier, M. G. et al. An Efficient Method for the Synthesis of Guanidino Prodrugs. Bioorg. Med. Chem. Lett. 1994,4, 1985-1990; Arafa, R. K. et al. Novel Linear Triaryl Guanidines, N-Substituted Guanidines and Potential Prodrugs as Antiprotozoal Agents. Eur. J. Med. Chem. 2008, 43, 2901-2908).

[0008] For example, Huttunen and coworkers reported the benefit of increasing the lipophilicity of 2 to improve oral bioavailability. Impressively, they successfully developed prodrug 7 (2a) (FIG. 2). Through in vivo rat studies, 7 displayed quantitative bioconversion to active metabolite 2 when administered IV, as well as a 22% increase in bioavailability upon oral administration (Huttunen, 2009). In another report, Hsu et al. disclosed that the synthesis of the acylguanidine derivative of 9 (guanidino-oseltamivir carboxylic acid: GOC), a potent therapeutic against influenza type A and B, improved cell permeability and overall bioavailability. The production of acylguanidine derivatives represents an interesting prodrug strategy. It has been documented that acylguanidine moieties are susceptible to hydrolysis in vivo and could potentially serve as platforms for bioactivation (Humphreys, W. G. et al. Oxidative Activation of Acylguanidine Prodrugs: Intestinal Presystemic Activation in Rats Limits Absorption and Can Be Inhibited by Co-Administration of Ketoconazole. Xenobiotica 2003, 33, 93-106). Hsu et al. demonstrated that guanidino N-acylation of 9 with / / -octanoic acid to produce prodrug 8 (8a) resulted in a 28-fold increase in cellular uptake when administered to MDCK cells and roughly 10% of 8 being hydrolyzed to form active metabolite 9 in vitro (FIG. 2) (Hsu, P.-H., 2018). Another promising prodrug strategy is the synthesis of N-hydroxyguanidine derivatives (Schade, D. et al. Zanamivir Amidoxime- and N-Hydroxyguanidine-Based Prodrug Approaches to Tackle Poor Oral Bioavailability. J. Pharm. Sci. 2015, 104, 3208-3219; Schade, D. et al. Prodrug Design for the Potent Cardiovascular Agent Nco-Hydroxy-l- Arginine (NOHA): Synthetic Approaches and Physicochemical Characterization. Org. Biomol. Chem. 2011, 9, 5249-5259; Maryanoff, B. E. et al. Exploration of Potential Prodrugs of RWJ-445167, an Oxyguanidine-Based Dual Inhibitor of Thrombin and Factor Xa. Chem. Biol. Drug Des. 2006, 68, 29-36). Investigations have shown these moieties to aid in the smuggling of poorly adsorbed substrates into the liver while also being susceptible to metabolic reduction to form their corresponding guanidine derivatives (Schade, 2011; Kotthaus, J. et al. Reduction of Nco-Hydroxy-L- Arginine by the Mitochondrial Amidoxime Reducing Component (mARC). Biochem. J. 2010, 433, 383-391; Clement, B. et al. Microsomal Catalyzed N-Hydroxylation of Guanabenz and Reduction of the N-Hydroxylated Metabolite: Characterization of the Two Reactions and Genotoxic Potential of Guanoxabenzl. Chem. Res. Toxicol. 1996, 9, 682-688; Froehlich, A. K. et al. Metabolism of N-Hydroxyguanidines(N-Hydroxydebrisoquine) In Human and Porcine Hepatocytes: Reduction and Formation of Glucuronides. Drug Metab. Dispos. 2005, 33, 1532-1537). However, the mechanism by which this bio-reduction takes place is still debated. One proposed metabolic route is due to the action of liver homogenates and hepatocytes catalyzed by a microsomal NADH-dependent system (Clement, B. et al. Cytochrome P450 Dependent N-Hydroxylati on of a Guanidine (Debrisoquine), Microsomal Catalysed Reduction and Further Oxidation of the N-Hydroxy-Guanidine Metabolite to the Urea Derivative. Similarity with the Oxidation of Arginine to Citrulline and Nitric Oxide. Biochem. Pharmacol. 1993, 46, 2249-2267). Regardless, the production of A-hydroxyguanidine derivatives presents another possible method to enhance the oral bioavailability of guanidine containing drugs.

[0009] Recently, a small side cavity in the active site of sphingosine kinase 2 (SphK2) was discovered (Sibley, C. D. et al. Discovery of a Small Side Cavity in Sphingosine Kinase 2 That Enhances Inhibitor Potency and Selectivity. J. Med. Chem. 2020, 63, 1178-1198). In mammals and perhaps all vertebrates, sphingosine 1-phosphate (SIP) is produced exclusively from the ATP- dependent phosphorylation of sphingosine (Sph) by sphingosine kinases (SphKl and SphK2) (FIG. 3). It is well documented that SIP is implicated in various animal models of diseases such as renal fibrosis (Huwiler, A.; Pfeilschifter, J. Sphingolipid Signaling in Renal Fibrosis. Matrix Biol. 2018, 68-69, 230-247; Schwalm, S. et al. Sphingosine Kinase-2 Deficiency Ameliorates Kidney Fibrosis by Up-Regulating Smad7 in a Mouse Model of Unilateral Ureteral Obstruction. Am. J. Pathol. 2017, 187, 2413-2429), cancer (Pyne, N. J. et al. Sphingosine 1-Phosphate and Cancer. Adv. Biol. Regul. 2018, 68, 97-106; Yuza, K. et al. Different Roles of Sphingosine Kinase 1 and 2 in Pancreatic Cancer Progression. J. Surg. Res. 2018, 232, 186-194; LeBlanc, F. R. et al. Sphingosine Kinase-2 Is Overexpressed in Large Granular Lymphocyte Leukaemia and Promotes Survival through Mcl-1. Br. J. Haematol. [Online early access], DOI: 10.1111 / bjh.16530. Published Online: March 2, 2020), and multiple sclerosis (Farez, M. F.; Correale, J. Sphingosine 1-Phosphate Signaling in Astrocytes: Implications for Progressive Multiple Sclerosis. J. Neurol. Sci. 2016, 361, 60-65; Kappos, L. et al. Siponimod versus Placebo in Secondary Progressive Multiple Sclerosis (EXPAND): A Double-Blind, Randomised, Phase 3 Study. Lancet 2018, 391, 1263-1273). Furthermore, multiple studies have demonstrated that inhibition of SphKl or SphK2 has therapeutic potential in these disease models (Santos, W. L.; Lynch, K. R. Drugging Sphingosine Kinases. ACS Chem. Biol. 2015, 10, 225-233; Pyne, S. et al. Sphingosine1-Phosphate and Sphingosine Kinases in Health and Disease: Recent Advances. Prog. Lipid Res. 2016, 62, 93-106). Significant efforts by the present inventors have focused on the development of SphKl -selective, dual SphKl / 2, and SphK2-selective inhibitors. Recently, Sibley etal. reported the guanidine-based inhibitor 10 (SLM6071469) which is the most potent SphK2-selective inhibitor published in the literature to date (Table 1). Additional findings included molecular modeling studies that showed that the internal trifluoromethyl substituent likely interacts with a previously unrecognized side cavity resulting in enhanced inhibitor potency toward SphK2. Moreover, it was shown that the guanidine moiety in 10 forms a salt-bridge with the carboxylate of Asp211, which flanks the ATP active site when docked in the Sph binding pocket of SphK2. Further experiments were conducted assessing the in vivo properties of inhibitor 10. Measured blood SIP concentrations have proven to be an exceptional pharmacodynamic marker for SphK2 inhibition in mice. Curiously, SphK2 specific inhibition results in blood SIP concentrations to rise nearly 3-fold (Kharel, Y. et al. Sphingosine Kinase 2 Inhibition and Blood Sphingosine 1- Phosphate Levels. J. Pharmacol. Exp. Ther. 2015, 355, 23-31; Kharel, Y. et al. Sphingosine Kinase Type 2 Inhibition Elevates Circulating Sphingosine 1-Phosphate. Biochem. J. 2012, 447, 149— 157). Kharel et al. proposed that this phenomenon is the result of a cascade wherein Sph, formed at the hepatocyte surface via dephosphorylation of blood SIP, is captured and phosphorylated by SphK2 and in turn degraded by intracellular SIP lyase. Small molecule inhibition of SphK2 causes the recently formed Sph to then in turn be captured and phosphorylated by SphKl resulting in an overall increase in SIP concentrations (Kharel, Y. et al. Mechanism of Sphingosine 1-Phosphate Clearance from Blood. Biochem. J. 2020, 477, 925-935). Administration of a single 10 mg / kg dose of 10 in C57BL / 6 mice via IP injection displayed a 2-fold increase in blood SIP concentrations after 4 hours post injection and remained elevated for up to 6 hours. The observed rise in blood SIP levels is strong evidence that 10 is inhibiting SphK2 in an isoform selective manner when administered in vivo.

[0010] However, a need remains for an orally bioavailable SphK2-selective inhibitor. Unfortunately, as with other guanidine containing drugs, oral administration of 10 is ineffective. Therefore, the present inventors sought to design a guanidine prodrug that would improve the poor oral bioavailability of 10.SUMMARY OF THE INVENTION

[0011] The synthesis and evaluation of orally bioavailable prodrugs of guanidine-based SphK2-selective inhibitors are reported.

[0012] Aspect 1 of the invention is a compound or pharmaceutically acceptable salt thereof according to formula (I):(I)( '") * ” nU'"R2

[0013]

[0014] wherein:

[0015] X is phenyl, indolyl, or naphthyl;

[0016] R1is independently selected from the group consisting of H, OH, -(Ci-Cs)alkyl, halo, -(Ci-C6)haloalkyl, NH2, and CN;

[0017] or R1, if bound to adjacent carbon atoms, in combination with the existing carbon-carbon bond represent a double bond between the adjacent carbon atoms;

[0018] W is CH2, O, or NH;

[0019] V is selected from the group consisting of H, (Ci-Cio)alkyl, (C2-Ci2)alkenyl, -(Ci- Cio)alkyl-(C6-Cio)aryl, -(C2-Ci2)alkenyl-(C6-Cio)aryl, -(Ci-Cio)alkyl-(C6-Cio)aryl-(Ci-Cio)alkyl, - (Ci-Cio)alkyl-(C3-C8)cycloalkyl, -(Ci-Cio)alkyl-heterocyclyl containing from 1 to 3 ring heteroatoms selected from N, O, and S;

[0020] wherein any aryl is optionally fused to (Ce-Cio)aryl, (Cs-Csjcycloalkyl, or heterocyclyl containing from 1 to 3 ring heteroatoms selected from N, O, and S; and / or

[0021] wherein any alkyl, alkenyl, cycloalkyl, heterocyclyl, or aryl is optionally substituted by 1-4 substituents independently selected from the group consisting of F, Cl, Br, (Ci- C6)alkyl, -O-(Ci-C6)alkyl, (C2-C6)alkenyl, (Ci-C6)haloalkyl, (C6-Cio)aryl, and CN;

[0022] Y1, Y2, and Y3are independently selected from C, N, NH, O, and S;

[0023] m=0, l, or 2;

[0024] T is independently selected from the group consisting of C=O, SO2, CN, 0, or NH;

[0025] n=0, 1, 2, or 3;

[0026] U can be present or absent, but when present is independently selected from the group consisting of C, O, N, and S;

[0027] R2and R3can be present or absent, but when present are independently selected from the group consisting of H, C=O, -C=0(Ci-Cio)alkyl, (Ci-Cio)alkyl, (Ci-C8)cycloalkyl, (Ci- Ce)haloalkyl, (C6-Cio)aryl, (C2-Ci2)alkenyl, -(Ci-Cio)alkyl-(C6-Cio)aryl, -(C2-Ci2)alkenyl-(C6- Cio)aryl, -(Ci-Cio)alkyl-(C6-Cio)aryl-(Ci-Cio)alkyl, -(Ci-Cio)alkyl-(C3- C8)cycloalkyl, -(Ci- Cio)alkyl-heterocyclyl containing from 1 to 3 ring heteroatoms selected from N, O, and S;

[0028] wherein any aryl is optionally fused to (Ce-Cio)aryl, (C3-Cs)cycloalkyl, or heterocyclyl containing from 1 to 3 ring heteroatoms selected from N, O, and S; and / or

[0029] wherein any alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, R2or R3are optionally substituted by 1-4 substituents independently selected from the group consisting of F, Cl, Br, (Ci-C6)alkyl, -O-(Ci- C6)alkyl, -N-(Ci-C6)alkyl, (C2-C6)alkenyl, (Ci-C6)haloalkyl, (C6- Cio)aryl, and CN.

[0030] Aspect 2 is the compound or pharmaceutically acceptable salt of Aspect 1, wherein:

[0031] YHs N, Y2is N, and Y3is O.

[0032] Aspect 3 is the compound or pharmaceutically acceptable salt of Aspect 1, wherein:

[0033] Y1is N, Y2is O, and Y3is N.

[0034] Aspect 4 is the compound or pharmaceutically acceptable salt of Aspect 1, 2 or 3, wherein the compound is one of the following compounds:

[0035] Aspect 5 is a pharmaceutical composition comprising one or more compound or pharmaceutically acceptable salt thereof of any of Aspects 1-4 and one or more pharmaceutically acceptable excipient.

[0036] Aspect 6 is a method of treating a disease in a patient comprising:

[0037] administering to a subject a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof of any of Aspects 1-5.

[0038] Aspect 7 is the method of Aspect 6, wherein the disease is selected from one or more of autoimmune disease such as multiple sclerosis, type I diabetes, inflammatory bowel diseases including Crohn’s disease and ulcerative colitis, Grave’s disease, Addison’s disease, dermatomyositis, myasthenia gravis, systemic lupus erythematosus, scleroderma, psoriasis, atopic dermatitis, or a fibrotic disease (such as chronic kidney disease, acute kidney disease, or idiopathic pulmonary fibrosis).

[0039] Aspect 8 is a method of inhibiting a sphingosine kinase of a patient, comprising:

[0040] administering to the patient a compound, pharmaceutically acceptable salt thereof, or pharmaceutical composition of any of Aspects 1-5.

[0041] Aspect 9 is the method of Aspect 8, wherein the sphingosine kinase is selected from sphingosine kinase 1, sphingosine kinase 2, or a combination thereof.

[0042] Aspect 10 is the method of Aspect 8 or 9, wherein the sphingosine kinase is sphingosine kinase 2.

[0043] Aspect 11 is the method of any of Aspects 8-10, further comprising selectively inhibiting sphingosine kinase 2 relative to sphingosine kinase 1.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings illustrate certain aspects of implementations of the present disclosure and should not be construed as limiting. Together with the written description the drawings serve to explain certain principles of the disclosure.

[0045] FIG. 1 shows chemical structures of synthetic and natural guanidine-containing compounds.

[0046] FIG. 2 shows chemical structures of prodrugs and bioconversion into their active metabolites.

[0047] FIG. 3 shows the sphingosine / sphingosine 1 -phosphate equilibrium in vivo.

[0048] FIG. 4 is a scheme showing the synthesis of common intermediate 14 and analogue 19. Reagents and conditions: (a) 2-cyclohexylethanol, NaH, THF, 0 °C-rt, 18 h, 87%;(b) NH2OH«HCl, Et3N, EtOH, reflux, 3 h, 96%; (c) Boc-L-proline, DIEA, HCTU«PF6, DMF, 100 °C, 18 h, 84%; (d) TFA, CH2CI2, 3-12 h, 87%; (e) N,N’-t / z-Boc-lH-pyrazole-l- carboxamidine, DIEA, CH3CN, rt, 18 h, 86%; (f) HC1, Dioxane, rt, 18 h, 89-91%.

[0049] FIG. 5 is a scheme showing the synthesis of analogues 20a-al. Reagents and conditions: (a) carbamoyl chloride derivative, DIEA, CH2CI2, 10 min, 38-79%; (b) chloroformate derivative, DIEA, CH2CI2, 10 min, 66-94%; (c) BrCN, DIEA, CH2CI2, 40 °C, 2 h, 30%; (d) sulfonyl chloride derivative, DIEA, CH2CI2, 10 min, 30-66%; (e) benzoic acid, CDI, TEA, DMAP, CH2CI2, 18 h, 84%; (f) 2,2,2-trifluoroacetic anhydride, DIEA, CH2CI2, 10 min, 71%; (g) acyl chloride derivative, DIEA, CH2CI2, 10 min, 30-79%.

[0050] FIG. 6 is a scheme showing the synthesis of analogues 20am and 20an. Reagents and conditions: (a) TFA, CH2C12, 3-12 h, 87%; (b) BrCN, DIEA, CH3CN, 80 °C, 2 h, 87%;(C) NH2NH2«H2O, CS2CO3, THF, 65 °C, 18 h, 33%; (d) NH2OH«HC1, Cs2CO3, EtOH, 85 °C, 18 h, 40%.

[0051] FIG. 7 is a graph showing blood concentrations of active metabolite 14 in C57BL / 6 mice after 6 h post PO administration with 30 mg / kg test compound.DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

[0052] The detailed description provided below is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the examples and the sequence of steps for constructing and operating the examples. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0053] Previous work has been focused on the design and synthesis of highly potent and selective inhibitors of SphK2. Early experiments conducted examined whether there was a benefitof having a positively charged moiety on the inhibitor scaffold. It was rationalized that when docked in the active site of SphK2, a positively charged amine within the molecule could mimic the electrostatic interactions observed between Sph and catalytic amino acid Asp211 (Worrell, B. L. et al. In Silico Characterization of Structural Distinctions between Isoforms of Human and Mouse Sphingosine Kinases for Accelerating Drug Discovery. J. Chem. Inf. Model. 2019, 59, 2339-2351). A series of first-generation inhibitors which utilized various terminal amine functionalities lead to the development of compounds 11 (trans- la) and 12 (tran -12a) (Table 1) (Raje, M. R. et al. Design, Synthesis and Biological Activity of Sphingosine Kinase 2 Selective Inhibitors. Bioorg. Med. Chem. 2012, 20, 183-194). Investigation into the effect of a positively charged species with regards to SphK2 inhibition revealed that compared to neutral inhibitor 11, charged derivative 12 displayed increased potency K = 8.0 pM) and selectivity (8-fold) for SphK2 thus validating the theory that a positive charge is beneficial for kinase inhibition.

[0054] A library of second-generation inhibitors was synthesized, that replaced the quaternary ammonium salt moiety of amidine-based inhibitors or sphingosine kinases (Mathews, T. P. et al. Discovery, Biological Evaluation, and Structure-Activity Relationship of Amidine Based Sphingosine Kinase Inhibitors. J. Med. Chem. 2010, 53, 2766-2778; Kennedy, A. J. et al. Development of Amidine-Based Sphingosine Kinase 1 Nanomolar Inhibitors and Reduction of Sphingosine 1-Phosphate in Human Leukemia Cells. J. Med. Chem. 2011, 54, 3524-3548), with a guanidine functionality. This strategy led to the development of compound 13 (SLR080811) (Table 1) (Kharel, 2012; Patwardhan, N. N. et al. Structure-Activity Relationship Studies and In Vivo Activity of Guanidine-Based Sphingosine Kinase Inhibitors: Discovery of SphKl- and SphK2- Selective Inhibitors. J. Med. Chem. 2015, 58, 1879-1899). When tested for enzyme inhibition, 13 displayed improved potency (K = 1.3 pM) and selectivity (9-fold) for SphK2. Additional modifications lead to the discovery of 10 (Sibley, 2020). Inhibitor 10 contains a shorter, less flexible 4-trifluoromethyl benzyl ether tail in place of the / / -octyl moiety in 11-13, as well as the addition of a trifluoromethyl substituent on the internal aryl ring. Biologic evaluation of 10 revealed it to be the most potent (89 nM) SphK2-selective (73 -fold) inhibitor known at the time. Further structure-activity relationship (SAR) studies conducted with inhibitor 10 led to the synthesis of compound 14 (SLS1081832) (Table 1). It was observed that replacement of the 4-trifluoromethyl benzyl ether moiety for a 2-cyclohexylethoxy substituent imparted improvedpotency (82 nM) and selectivity (122-fold) for SphK2, thus making it the most potent SphK2-selective inhibitor currently known.

[0055] Table 1. K Values of Select SphK2 Inhibitorsa"Inhibitory constants for recombinant enzymes were obtained by kinetic analysis of SIP production using variable concentration of sphingosine and a fixed concentration of ATP in the presence or absence of compounds. Selectivity for each compound was determined by dividing the KmSphK2 by the Kmof SphKl.

[0056] Unfortunately, guanidine-based inhibitor 14 suffers the same deficiency of poor oral bioavailability. To provide compounds with improved bioavailability, guanidino A-substitutions on guanidine-based SphK2-selective inhibitors were developed. A wide range of substituents were attached to parent compound 14 and examined for their effect on oral bioavailability and subsequent metabolism, resulting in improved SphK2 inhibition.

[0057] To investigate the effect of A- substitution on the oral bioavailability of guanidine-based SphK2-selective inhibitors, a series of derivatives was synthesized. The synthesis started with preparation of the common intermediate 14 (FIG. 4). Compound 15 (4-fluoro-3- (trifluoromethyl)benzonitrile) is available for purchase from commercial sources. Completion of a nucleophilic aromatic substitution reaction of 15 with 2-cyclohexylethanol and sodium hydride was performed to generate benzonitrile intermediate 16. Next, intermediate 16 was treated with hydroxylamine hydrochloride and triethylamine (TEA) while refluxing in ethanol to produce amidoxime intermediate 17. Following amidoxime production, 17 was dissolved in DMF and heated to 100 °C in the presence of Boc-L-Proline, HCTU, and Hiinig’s base to afford 1,2,4-oxadiazole intermediate 18. Next, installation of the guanidine moiety was completed by removing the Boc protecting group with a mixture of trifluoroacetic acid (TFA), 18 and CH2CI2 followed by reaction with A,A’-A / -Boc- I A-pyrazole- l-carboxamidine and Hiinig’s base to afford the bis-Boc-protected intermediate. Subsequent removal of the Boc protecting groups was achieved with HC1 in dioxane (4.0 M) to yield the desired common intermediate 14 as an HC1 salt. Similarly, production of pyrrolidinium analogue 19 was carried out with a mixture of intermediate 18 and HC1 in dioxane (4.0 M) to yield the desired secondary amine HC1 salt.

[0058] Synthesis of A-substituted guanidine analogues 20a-al was performed as shown in FIG.5. Analogues 20a-c were prepared through a nucleophilic substitution reaction of 14, Hü nig’s base and various carbamoyl chlorides dissolved in CH2CI2. Likewise, carbamate analogues 20d— i were synthesized via nucleophilic substitution reaction of intermediate 14 with various chloroformates and Hiinig’s base. Reaction of 14 with cyanogen bromide and Hiinig’s base in CH2CI2 resulted in the formation of cyanoguanidine analogue 20j . Sulfonamide analogues 20k-m were generated through nucleophilic substitution reaction of 14, Hiinig’s base, and various sulfonyl chlorides. Formation of benzamide derivatives 20n and 20p-r were completed with a mixture of 14, benzoic acid, carbonyldiimidazole (CDI), 4-dimethylaminopyridine (DMAP), and TEA while stirring in CH2CI2. Synthesis of trifluoroacetamide analogue 20s was performed via anucleophilic substitution reaction with 14, trifluoroacetic anhydride, and Htinig’s base. A mixture of 14, Htinig’s base and various acyl chlorides were stirred in CH2CI2 at room temperature to generate acylguanidine derivatives 20o, and 20t-al.

[0059] The synthesis of analogues 20am and 20an are shown in FIG. 6. Intermediate 18 was added to a mixture of TFA and CH2CI2 to promote the removal of the Boc protecting group resulting in the production of intermediate 21 as a TFA salt. Following Boc removal, cyanamide formation was accomplished by the reaction of 21 with Htinig’s base and cyanogen bromide while refluxing in acetonitrile to produce intermediate 22. Synthesis of amino guanidine analogue 20am was performed via a reaction with 22 in the presence of hydrazine hydrate and cesium carbonate while heating in THF. Similarly, production of hydroxy guanidine derivative 20an was accomplished via a reaction with 22 in the presence of hydroxylamine hydrochloride and cesium carbonate refluxed in ethanol.

[0060] Biologic Evaluation of Derivatives

[0061] A library of compounds with various substituents spanning the terminal guanidine head of the 14 scaffold was synthesized and assessed for improved oral bioavailability and SphK2 specific inhibitory activity, i.e. increased blood SIP levels. To assess improved oral bioavailability in vivo, the synthesized prodrugs were administered to C57BL / 6 mice with a single bolus (30 mg / kg p.o. dose) and blood SIP levels were measured. When considering in vivo investigations, an observed rise in blood SIP concentrations has been proven to be an exceptional pharmacodynamic marker for SphK2 specific inhibition (Kharel, 2015; Kharel, 2012; Kharel, 2020). To determine the amount of innate oral bioavailability, parent compound 14 was administered into mice (Table 2). Comparison of vehicle (no treatment) versus 14 displayed an 18% increase in blood SIP levels, indicating that inhibitor 14 is somewhat, albeit not significantly, orally bioavailable and is selective for SphK2 in vivo. Thus, any tested prodrug that is successful in inducing blood SIP concentrations to rise >18% would be considered significant.

[0062] Table 2. Activity of SphK2 Inhibitors Represented as % Increase of Blood S IP Levels versus Vehicle"Percent increase of blood SIP for various compounds was calculated from comparison of vehicle (no compound administered as control) versus administered test compound in C57BL / 6 mice after PO administration with 30 mg / kg test compound after 6 h. % values with * indicate test compound was administered at 20 mg / kg. % values with ** indicate test compound was administered at 30 mg / kg. For detailed assay conditions, see Experimental Section.

[0063] Next, to confirm the advantage of the guanidine head with respect to SphK2 inhibition, compound 19 was synthesized to represent the minimal scaffold without the guanidine functionality (Table 2). Removal of the guanidine head resulted in a small 10% increase of blood SIP, thus validating use of the guanidine moiety to achieve maximum SphK2 inhibition. Compounds 20a-c were designed to transform the guanidine into a urea-like derivative to probe any potential for improved adsorption and subsequent urea metabolism once in the liver. Conversion to a dimethyl urea moiety (20a) resulted in only a slight increase of blood SIP levels, indicating poor compound adsorption or metabolism. Likewise, Weinreb amide (20b) and acyl morpholine (20c) derivatives were unsuccessful with increasing blood SIP levels more than 14, thus indicating the use of urea moieties as a poor prodrug strategy. Accordingly, carbamate analogues were explored with compounds 20d-i. Transformation of the guanidine head to a carbamate moiety potentially allows for prodrug metabolism via hydrolysis and carbon dioxide release, thus exposing active metabolite 14. Utilization of aryl carbamates did not favor oral bioavailability as phenyl carbamate (20d) and benzyl carbamate (20g) derivatives resulted in a 10% and 11% rise in blood SIP levels, respectively. Interestingly, extension of the alkyl carbamate carbon chain had a positive impact on oral bioavailability, as indicated by ethyl carbamate variant 20f increasing blood SIP levels by nearly twice as much as methyl carbamate (20e). Encouraged from this observation, allyl (20h) and 2-methoxyethyl (20i) analogues were synthesized. However, compared to compound 20f, this resulted in a decrease in measured bloodSIP levels. Thereafter, the benefit of a cyanoguanidine (20j) moiety was explored for improved oral bioavailability. Excitingly, 20j was successful in increasing blood SIP concentrations by 30%; nearly 1.7-fold greater than 14.

[0064] Next, inspired from the potential leaving group ability of sulfonamide functional groups, mesyl (20k), triflyl (201), and tosyl (20m) sulfonamides were synthesized. Collectively, these derivatives had an overall poor effect on oral bioavailability as indicated by marginal increases in blood SIP. Next, the potential of benzamide functionalities as a method to improve oral bioavailability was investigated. Unfortunately, this resulted in poor SphK2 inhibition as indicated by minimal increases in blood SIP. All analogues (20n-p) failed to increase blood SIP levels following oral administration when compared to the direct oral administration of 14 in mice. Thereafter, the effect of trifluoroacetamide (20s), acetamide (20t), and propionamide (20u) variants was examined. Comparison of measured blood SIP levels revealed that 20t was nearly twice as effective as 20s and 20u in terms of SphK2 inhibition.

[0065] Inspired by these results, decoration of the guanidine head with a-alkoxy acetamides was explored with compounds 20v-ai. These derivatives proved to be the most effective in terms of improved oral bioavailability and SphK2 inhibition. Of the fourteen analogues synthesized, twelve derivatives displayed significant increases in blood SIP levels compared to 14. The 2- benzyloxyacetamide prodrug (20v, 62%) and 2-ethoxyacetamide (20x, 71%) demonstrated greater than a 60% increase in circulating SIP in mice following oral administration. Furthermore, comparison of 20w with parent compound 14 displayed a considerable 4-fold improvement with increasing blood SIP levels in vivo. These were joined in a category of a significant increase of over 50% increase in blood SIP levels by the 2-acetoxyacetamide (20w, 54%), 2- phenoxyacetamide (20z, 54%), and 2-methoxyacetamide (20aa, 52%) derivatives when administered orally at a 30 mg / kg dose of compound. While the aforementioned prodrugs impressed with significant increases in blood SIP levels, two prodrugs in this series failed to elicit a significant response. 20y, bearing a 2-hydroxyacetamide afforded a 23% increase while (S)-20ab bearing an (,S')-2-methoxypropanamide only increased circulating SIP by 16%, a stark decrease in activity with respect to the 2-methoxyacetamide analogue 20aa. To further probe this series, several 2-cycloalkoxy acetamides (20ac-af) and 2-heterocyclicoxy acetamides (20ag-ai) were synthesized and administered to mice. The 2-cycloalkoxy acetamides produced several effective prodrugs to the result of reducing the p.o. dosage from 30 mg / kg to 10 mg / kg.2-cyclopropoxyacetamide analogue 20ac increased blood SIP levels by 15%, while the larger aliphatic rings 20ad (2-cyclobutoxyacetimide, 35%) and 20ae (2-cyclopentoxyacetimide, 31%) proved to showcase the two highest increases of blood SIP concentrations at 10 mg / kg. The prodrug bearing the largest of the aliphatic rings 20af only managed to increase SIP levels to vehicle by 29% at a 30 mg / kg dose. In comparison, the 2-heterocyclicoxy acetamides yielded similar activity levels. (A)-20ag increased blood SIP levels by 22% while (R )-20ah increased blood SIP levels by 12% at a 10 mg / kg dose. The six-membered counterpart 20ai showed a 64% increase in circulating SIP in mice at a 30 mg / kg dose.

[0066] Following the success of the 2-alkoxy acetamides, 2-alkylthio acetamide analogues were synthesized to determine the importance of the oxygen atom at the 2-position of the acetamide prodrugs. 20aj-al can be compared directly to their 2-alkoxyacetamide counterparts 20v, 20x, and 20aa, respectively. The 2-benzylthioacetamide 20aj increased blood SIP concentration by 31%, a significant reduction in comparison to the 2-benzyloxyacetamide 20v (62%). Compared to one of the most effective prodrugs, the 2-ethylthioacetamide 20ak exhibited a 51% increase in blood SIP compared to a 71% increase from 20x. The lowest performing prodrug of this series, 20al bearing a 2-methylthioacetamide head group increased circulating SIP levels by 31% in mice in comparison to the 2-methoxyacetamide prodrug 20aa that increased blood SIP levels by 52%. Lastly, the effect of heteroatom substitution was explored with aminoguanidine (20am) and hydroxyguanidine (20an) derivatives. Measurement of blood SIP revealed that 20am was ineffective while 20an displayed significant increases in blood SIP by 35% compared to vehicle. It should be noted that 20am and 20an were assessed at a reduced concentration of 20 mg / kg.

[0067] Considering the observations summarized in Table 2, blood concentrations of active metabolite 14 were measured after treatment of test compound in vivo. Collectively, this would help determine whether the modified compounds were behaving as prodrugs and regenerating significant concentrations of the active metabolite. As shown in FIG.7, comparison of blood 14 levels for vehicle (no treatment control) versus 14 administered p.o. displayed a detected concentration of 0.028 pM, thus indicating marginal innate oral bioavailability. Next, to serve as a control, administration of pyrrolidine derivative 19 was assessed and as expected, did not register any detectable levels of 14. After, methyl (20e) and ethyl (20f) carbamates were examined, and surprisingly neither yielded significant concentrations of parent compound 14. Considering theobserved significant rise in blood SIP levels with 20f, these results suggest these compounds possibly possess inherent SphK2 inhibitory activity, although this remains to be further investigated. Examination of cyanoguanidine (20j) revealed detected parent compound levels that were similar to that of direct 14 p.o. administration. Given these results combined with 20j inducing only moderate increases in blood SIP, cyanoguanidines do not seem to be a viable prodrug strategy. Moving forward, compounds 20k and 20o were analyzed and registered no detected 14 levels in blood. These results are consistent with the observed poor SphK2 inhibitory activity of 20k and 20o. Administration of trifluoroacetamide (20s) analogue resulted in a 2.5-fold increase of detected 14, while the propionamide (20u) derivative displayed no prodrug activity. Excitingly, oral administration of three 2-alkoxy acetamides (20v-x) resulted in significant detected levels of active metabolite 14. When compared to 14 oral administration, 20w and 20x were successful in increasing active metabolite levels by 25-fold and 11-fold respectively. Furthermore, these results are consistent with the observed excellent SphK2 inhibitory activity, i.e. rise in blood SIP, observed for these compounds in vivo. Taken altogether, these results suggest that decoration of a guanidine functional group with 2-alkoxy acetamide moieties are a viable prodrug strategy in order to alleviate poor oral bioavailability.

[0068] When considering prescribed medications in humans and animals, it has been shown that in order to achieve maximum patient compliance and ensure the greatest ease of treatment, orally administered drugs are unsurpassed. Unfortunately, drugs that are administered orally face the greatest barriers in vivo on route to their intended biological target. Towards that end, it has been shown that certain functionalities that comprise the drug can also be problematic once administered orally into humans leading to poor oral bioavailability. Numerous investigations have revealed various strategies to alleviate this problem via chemical modification, thus effectively hiding the troublesome functionality. This in turn has been demonstrated to promote enhanced drug adsorption in vivo and subsequent metabolism to reveal the active parent compound. In this study, prodrugs of the highly potent, guanidine-based SphK2 inhibitor 14 (Ki= 82 nM, 122-fold SphK2 selective) were developed. SphK2 has proven to be implicated in a wide variety of disease states, and chemical inhibition has been demonstrated to be therapeutic. Structurally, the guanidine functionality on 14 has been shown to be crucial for the observed pharmacodynamic effect; unfortunately, it also diminished oral bioavailability. As such, guanidine containing drugs are frequently associated with poor oral bioavailability. Chemical modification of the guanidine headresulted in the synthesis of two highly effective prodrugs, 20w and 20x. Biological assessment demonstrated that these two compounds were successful in enhancing oral adsorption in mice and increased active metabolite 14 levels by up to 25-fold greater than previously observed. Additionally, measurement of potential SphK2 inhibitory activity revealed significant increases in blood SIP levels up to 71% compared to 14 treatment alone. Collectively, this data demonstrates that these modifications unquestionably enhance inhibitor oral bioavailability and magnify the delivered payload into systemic circulation in vivo. Overall, these investigations provide a strong foundation for the development of future guanidine containing prodrugs, and may lead to the clinical advancement of future medications where oral bioavailability is lacking.

[0069] EXAMPLES

[0070] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0071] Sphingosine Kinase Biological Assays. The growth of recombinant yeast (Saccharomyces cerevisiae) cells encoding either hSphKl or hSphK2 was performed according to our previously described protocol (Schade, 2015; Kharel, 2012). In short, yeast strain KYA1 harboring a plasmid encoding either hSphKl or hSphK2 was selected, maintained and grown in SC-URA media with 2% glucose overnight at 30 °C. Afterwards, growth media was diluted 1 : 100 into SC-URA media supplemented with 2% galactose and various concentrations of test compound. After another incubation period of 24 - 48 hours at 30 °C, cellular growth was quantified by measuring absorbance at 600 nm.

[0072] In vivo mouse studies were conducted as follows. Groups of 8 to 10 week old C57BL / 6 mice were orally gavaged (p.o.) with either vehicle or 30 mg / kg test compound (2% solution of hydroxypropyl-P-cyclodextrin, Cargill Cavitron 82004). After 6 hours of administration, whole blood was collected from the retro-orbital sinus and 10 L of blood was processed for LC / MS analysis as described previously. The use of mice for these studies was approved by the University of Virginia’s School of Medicine Animal Care and Use Committee prior to experimentation.

[0073] General Material and Synthetic Procedures. All solvents used were dried using a PureSolv solvent system and all chemical reagents were purchased from commercial sources without further purification. Aluminum-backed silica gel, 200 z / m, F254 plates were utilized for all thin-layer chromatography (TLC) experiments. A Combiflash Rf purification system with flash grade silica gel, 40-63 z / m was used for all column chromatography. All reported NMR spectrawere measured at 400 MHz and the complementary13C NMR frequency was 101 MHz. Reported1H NMR chemical shifts are in ppm with the solvent resonance as an internal standard (CDCh: 7.26 ppm; CD3OD: 3.31 ppm). Reported13C NMR chemical shifts are in ppm with the solvent resonance as the internal standard (CDCl3: 77.16 ppm; CD3OD: 49.00 ppm). High-resolution mass spectrometry (HRMS) data was obtained with an LC-MS time-of flight mass spectrometer by electrospray ionization (ESI). All compounds tested in biological assays possess >95% purity, as determined by HPLC analyses. HPLC was performed using a Thermo Electron TSQ triple quadrupole mass spectrometer fitted with an ESI source.

[0074] General Procedure A: CDI Coupling

[0075] To a 6-dram vial containing was added the appropriate carboxylic acid (1.1 equiv) and DCM (0.2 M), followed by CDI (1.5 equiv). The reaction mixture was stirred for 10 minutes, followed by the addition of (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidine-l-carboximidamide hydrochloride (1.0 equiv), TEA (3.0 equiv), and DMAP (10 mol%). The reaction mixture was allowed to stir at room temperature for 18 hours or until complete consumption of starting material was observed on TLC. The resulting mixture was concentrated in vacuo to afford a yellow oil which was then subjected to flash chromatography with an appropriate ethyl acetate in hexanes solvent system to afford the pure product.

[0076] General Procedure B: Nucleophilic Substitution of Acyl Chlorides

[0077] (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5- yl)pyrrolidine-l-carboximidamide hydrochloride (1.0 equiv) was dissolved in DCM (0.2 M solution) and put under an argon gas atmosphere. Next DIEA (2.0 equiv) was added followed by dropwise addition of the appropriate acyl chloride (1.1 equiv). The reaction mixture was stirred at room temperature for 18 hours or until complete consumption of starting material was observed on TLC. Afterward, the reaction was concentrated via vacuum and then purified by silica gel chromatography with an appropriate ethyl acetate in hexanes solvent system to yield the desired product.

[0078] General Procedure C: Nucleophilic Substitution of Guanidine Derivatives with Sulfonyl Chlorides

[0079] (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5- yl)pyrrolidine-l-carboximidamide hydrochloride (1.0 equiv) was dissolved in CH2CI2 (0.2 M solution) and put under a argon gas atmosphere. Next A,A-diisopropylethylamine (2.0 equiv) wasdded followed by dropwise addition of the appropriate sulfonyl chloride (1.1 equiv). The reaction mixture was stirred at room temperature for 10 minutes. The reaction progress was monitored by TLC. Afterward, the reaction was concentrated via vacuum and then purified by silica gel chromatography to yield the desired product.

[0080] General Procedure D: 1,2,4-oxadiazole ring closure

[0081] 4-(2-cyclohexylethoxy)-A"-hydroxy-3-(trifluoromethyl)benzimidamide (1.0 equiv) was dissolved in DMF (0.2 M solution) and put under an argon blanket. Next, (S)-l-(tert- butoxycarbonyl)azetidine-2-carboxylic acid (1.2 equiv) was added to the reaction mixture followed by Hiinig’s base (1.8 equiv) and then HCTU (1.2 equiv) and stirred vigorously at 100 °C for 18 hours. The reaction progress was monitored by TLC. After completion of reaction, the resulting solution was partitioned between EtOAc and aqueous LiBr solution. Using additional EtOAc, the aqueous LiBr solution was washed three times and the combined organic layers were dried over NazSCU, filtered, and concentrated via vacuum. The resulting concentrate was purified by silica gel chromatography.

[0082] General Procedure E: Trifluoroacetic acid-assisted Boc Deprotection

[0083] To a solution of dichloromethane (0.2 M) and (S)-2-(3-(4-(2-cyclohexylethoxy)-3- (trifluoromethyl)phenyl)-l, 2, 4-oxadiazol-5-yl)azeti dine- 1 -carboxylate (1.0 equiv), 1.0 M TFA (10 equiv) was added and then allowed to stir at room temperature for 16 hours. The reaction progress was monitored by TLC. Next, the dichloromethane was removed via vacuum followed by addition of diethyl ether to promote TFA salt precipitation. Once the desired intermediate was precipitated as a tan solid, the diethyl ether was removed via vacuum and the resulting Boc deprotected intermediate was carried forward crude.

[0084] General Procedure F: Guanylation

[0085] (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5- yl)azetidin-l-ium 2,2,2-trifluoroacetate was dissolved in CH3CN (0.2 M) and put under an argon blanket. To the reaction mixture, Hünig ’s base (10 equiv) was added followed by N,N’- z-Boc- IH-pyrazole-l-carboxamidine (1.2 equiv) and the reaction mixture was allowed to stir at room temperature for 18 hours. The reaction progress was monitored by TLC. Next, the reaction mixture was then concentrated via vacuum, and the resulting concentrate was purified by silica gel chromatography to afford the bis-Boc-protected intermediate.

[0086] Example 1 : Synthesis of (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidine-l-carboximidamide hydrochloride (14)

[0087] Scheme 1

[0088] Reagents and conditions: (a) 2-cyclohexylethanol (1.2 equiv), NaH (1.5 equiv), THF, 0 °C-rt, 18 h, 87%; (b) NH2OH·HC1 (2.0 equiv), TEA (3.0 equiv), EtOH, reflux, 3 h, 96%;(c) Boc-L-proline (1.2 equiv), DIEA (2.0 equiv), HCTU«PF6(1.5 equiv), DMF, 100 °C, 18 h, 84%;(d) TFA (10 equiv), CH2CI2, 3-12 h, 87%; (e) N,N’-di-Boc-lH-pyrazole-l-carboxamidine (1.2 equiv), DIEA (3.0 equiv), CH3CN, rt, 18 h, 86%; (f) HC1 (10 equiv), Dioxane, rt, 18 h, 89-91%.

[0089] Example 2: Synthesis of (S)-N-(amino(2-(3-(4-(2-cyclohexylethoxy)-3- (trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-4-methoxybenzamide (20p)

[0090] Scheme 2

[0091] (a) 4-methoxybenzoic acid (1.1 equiv), CDI (1.5 equiv), TEA (3.0 equiv), DMAP (10 mol%), DCM, rt, 18 h.

[0092] Synthesized according to procedure A, white solid, 106 mg, 49% yield.1H NMR (400 MHz, CDCI3) 5 8.29 (d, J= 2.1 Hz, 1H), 8.16 (dd, J= 8.7, 2.2 Hz, 1H), 8.06 - 7.97 (m, 2H), 7.04(d, J= 8.7 Hz, 1H), 6.81 - 6.73 (m, 2H), 5.64 (br, 1H), 4.12 (t, J= 6.6 Hz, 2H), 3.77 (s, 3H), 3.75 - 3.70 (m, lH), 3.54 (br, 1H), 2.49 - 2.33 (m, 2H), 2.26 - 2.14 (m, 2H), 1.78 - 1.61 (m, 8H), 1.60 - 1.46 (m, 1H), 1.33 - 1.08 (m, 3H), 1.03 - 0.90 (m, 2H).13C NMR (101 MHz, CDC13) 6 177.0, 167.4, 162.2, 159.3, 158.8, 132.5, 131.1, 131.0, 126.9 (q, J = 5.3 Hz), 123.4 (q, J = 272.7 Hz), 119.5 (q, J = 31.1 Hz), 118.6, 113.1, 113.0, 77.5, 77.2, 76.8, 67.2, 55.3, 54.7, 45.5, 36.2, 34.4, 33.3, 31.2, 26.6, 26.3, 24.4. HRMS: (ESI) [M+H]+calc, for C30H35F3N5O4, 586.2636, observed, 586.2619.

[0093] Example 3: Synthesis of (S)-N-(amino(2-(3-(4-(2-cyclohexylethoxy)-3- (trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)acetamide (20t)

[0094] Scheme 3

[0095] (a) acetyl chloride (1.1 equiv), DIEA (2.0 equiv), DCM, rt, 18 h.

[0096] Synthesized by General Procedure B: 128 mg, 63%, yellow solid.1H NMR (400 MHz, CD3OD) δ 8.20-8.14 (m, 2H), 7.24 (d, J= 9.4 Hz, 1H), 5.50-5.43 (m, 1H), 4.16 (t, J= 6.4 Hz, 2H), 3.76-3.67 (m, 1H), 3.59-3.49 (m, 1H), 2.49-2.39 (m, 1H), 2.29-2.11 (m, 3H), 1.88-1.62 (m, 10H), 1.61-1.50 (m, 1H), 1.32-1.13 (m, 3H), 1.03-0.92 (m, 2H);13C NMR (101 MHz, CD3OD) δ 184.6, 182.3, 168.3, 160.6, 159.9, 133.8, 127.0 (q,J= 5.2 Hz), 124.7 (q, J= 271.6 Hz), 120.2 (q, J= 31.0 Hz), 119.7, 114.6, 68.2, 56.0, 46.8, 37.2, 35.6, 34.2, 32.1, 27.6, 26.9, 25.0; HRMS (ESI+): calc, for C24H31F3N5O3 [M+H]+, 494.2374; found, 494.2358.

[0097] Example 4: Synthesis of (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)-N'-(methylsulfonyl)pyrrolidine-l-carboximidamide (20k)

[0098] Scheme 4

[0099] (a) methanesulfonyl chloride (1.1 equiv), DIEA (2.0 equiv), DCM, rt, 10 min.[000100] Synthesized by General Procedure C: 144 mg, 66%, white solid.1H NMR. (400 MHz, CD3OD) δ 8.24-8.14 (m, 2H), 7.28 (d, J = 8.5 Hz, 1H), 5.41-5.34 (m, 1H), 4.18 (t, J = 6.5 Hz,2H), 3.77-3.69 (m, 1H), 3.61-3.52 (m, 1H), 2.66 (s, 3H), 2.53-2.41 (m, 1H), 2.28-2.12 (m, 3H), 1.81-1.64 (m, 7H), 1.62-1.52 (m, 1H), 1.34-1.15 (m, 3H), 1.05-0.93 (m, 2H);13C NMR (101 MHz, CD3OD) δ 181.9, 168.4, 160.6, 156.4, 133.9, 127.0 (q, J= 5.4 Hz), 124.7 (q, J= 272.1 Hz), 120.2 (q, J= 31.1 Hz), 119.6, 114.8, 68.2, 56.2, 47.6, 41.1, 37.3, 35.6, 34.2, 32.1, 27.6, 27.3, 25.1; HRMS (ESI+): calc, for C23H31F3N5O4S [M+H]+, 530.2043; found, 530.2027.[000101] Example 5: Synthesis of (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidine-l-carbohydrazonamide (20am) and (S)-2-(3-(4-(2- cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)-N'-hydroxypyrrolidine-l- carboximidamide (20an)[000102] Scheme 5[000103] Reagents and conditions: (a) TFA (10 equiv), CH2CI2, 3-12 h, 87%; (b) BrCN (1.2 equiv), DIEA (2.0 equiv), CH3CN, 80 °C, 2 h, 87%; (c) NH2NH2«H2O (1.2 equiv), CS2CO3(2.0 equiv), THF, 65 °C, 18 h, 33%; (d) NH2OH*HCI (1.2 equiv), Cs2CO3(2.0 equiv), EtOH, 85 °C, 18 h, 40%.[000104] Example 6: Synthesis of (S)-2-(5-(4-octylphenyl)-l,2,4-oxadiazol-3-yl)pyrrolidine-l- carboximidamide hydrochloride (40)[000105] Scheme 6[000106] Reagents and conditions: (a) DIEA, HCTU·PF6, tert-butyl (S)-2-(N'- hydroxycarbamimidoyl)pyrrolidine-l -carboxylate, DMF, 80 °C, 18 h, 19%; (b) TFA, CH2Cl2, 89%; (c) N,N'-di-Boc- I H-pyrazole-l -carboxamidine, DIEA, CH3CN, rt, 3 days, 19%; (d) HCl(g), MeOH, 28%.[000107] (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5- yl)pyrrolidine-l-carbohydrazonamide (20am):[000109] Synthesized according to the following protocol: (S)-2-(3-(4-(2-cyclohexylethoxy)-3- (trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidine-l -carbonitrile (1.0 equiv) was dissolved in THF (0.2 M solution) and put under an argon gas atmosphere. Next, hydrazine hydrate (2.0 equiv) was added to the reaction mixture followed by addition of CS2CO3(3.0 equiv). After, the reaction mixture was heated to 65 °C and stirred for 18 hours. The reaction progress was monitored by TLC. Afterward, the reaction was concentrated via vacuum and then purified by silica gel chromatography to yield the desired product; 43 mg, 33%, white solid.1H NMR (400 MHz, CD3OD) δ 8.25-8.17 (m, 2H), 7.30 (d, J= 8.7 Hz, 1H), 5.38-5.32 (m, 1H), 4.20 (t, J= 6.4 Hz, 2H), 3.77-3.68 (m, 1H), 3.61-3.53 (m, 1H), 2.54-2.43 (m, 1H), 2.29-2.14 (m, 3H), 1.82-1.64 (m, 7H), 1.62-1.52 (m, 1H), 1.34-1.15 (m, 3H), 1.06-0.93 (m, 2H);13C NMR (101 MHz, CD3OD)5 181.3, 168.5, 160.7, 160.1, 133.9, 127.0 (q,3JCF3 = 5.3 Hz), 124.7 (q, J = 271.6 Hz), 120.2 (q, J = 31.4 Hz), 119.6, 114.8, 68.2, 55.7, 48.1, 37.4, 35.6, 34.2, 32.4, 27.6, 27.4, 25.2; HRMS (ESI+): calc, for C22H30F3N6O2 [M+H]+, 467.2377; found, 467.2365.[000110] (S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)- A-hydroxypyrrolidine-l-carboximidamide (20an):[000111][000112] Synthesized according to the following protocol: (S)-2-(3-(4-(2-cyclohexylethoxy)-3- (trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidine-l -carbonitrile (1.0 equiv) was dissolved in 200 proof ethanol (0.2 M solution) and put under an argon gas atmosphere. Next, hydroxylamine hydrochloride (2.0 equiv) was added to the reaction mixture followed by addition of CS2CO3(3.0 equiv). After, the reaction mixture was heated to 85 °C and stirred for 18 hours. The reaction progress was monitored by TLC. Afterward, the reaction was concentrated via vacuum and then purified by silica gel chromatography to yield the desired product; 95 mg, 40%, white solid.1H NMR (400 MHz, CD3OD) δ 8.28-8.13 (m, 2H), 7.33-7.22 (m, 1H), 5.28-5.21 (m, 1H), 4.19 (t, J= 6.3 Hz, 2H), 3.72-3.63 (m, 1H), 3.56-3.46 (m, 1H), 2.50-2.36 (m, 1H), 2.29- 2.05 (m, 3H), 1.82-1.65 (m, 7H), 1.62-1.53 (m, 1H), 1.33-1.18 (m, 3H), 1.05-0.94 (m, 2H);13C NMR (101 MHz, CD3OD) δ 182.5, 168.4, 160.6, 160.1, 133.8, 127.0 (q, J = 5.3 Hz), 124.7 (q, J= 272.5 Hz), 120.1 (q, J = 31.6 Hz), 119.8, 114.7, 68.2, 55.2, 47.5, 37.4, 35.6, 34.2, 32.4, 27.6, 27.4, 25.6; HRMS (ESI+): calc, for C22H29F3N5O3[M+H]+, 468.2217; found, 468.2221.[000113] Example 6: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-3-(dimethylamino)benzamide (20q):[000114][000115] Synthesized according to procedure A, white solid, 147 mg, 91% yield.1H NMR (400 MHz, CDCh) 8 8.25 (d, J= 2.2 Hz, 1H), 8.14 (dd, J= 8.7, 2.2 Hz, 1H), 7.52 (dd, J = 2.8, 1.5 Hz, 1H), 7.45 (d, .7= 7.6 Hz, 1H), 7.15 (t, J= 7.9 Hz, 1H), 7.03 (d, J= 8.7 Hz, 1H), 6.81 - 6.74 (m, 1H), 5.71 (br, 1H), 4.13 (t, J= 6.6 Hz, 2H), 3.81 - 3.72 (m, 1H), 3.61 - 3.51 (m, 1H), 2.93 (s, 6H), 2.51 - 2.33 (m, 2H), 2.30 - 2.15 (m, 2H), 1.81 - 1.61 (m, 5H), 1.59 - 1.47 (m, 1H), 1.34 - 1.07 (m, 6H), 1.04 - 0.92 (m, 2H).13C NMR (101 MHz, CDCh) 8 177.9, 167.4, 159.3, 159.0, 150.5, 138.9, 132.5, 128.5, 126.9 (q, J = 5.4 Hz), 123.4 (q, J = 272.6 Hz), 119.5 (q, J= 31.5 Hz), 118.6, 117.9, 115.7, 113.5, 113.0, 67.2, 54.7, 45.4, 40.9, 36.3, 34.4, 33.3, 31.3, 29.8, 26.6, 26.3, 24.3. HRMS: (ESI) [M+H]+calc, for C31H38F3N6O3, 599.2952, observed, 599.2931.[000116] Example 7: (S)-2-((amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)amino)-2-oxoethyl acetate (20w):[000117][000118] Synthesized according to procedure B, white solid, 73 mg, 32% yield.1H NMR (400 MHz, CDCh) 5 8.24 (d, J = 2.2 Hz, 1H), 8.15 (dd, J = 8.7, 2.2 Hz, 1H), 7.06 (d, J = 8.7 Hz, 1H), 5.48 (br, 1H), 4.61 - 4.41 (m, 1H), 4.38 - 4.27 (m, 1H), 4.13 (t, J = 6.6 Hz, 2H), 3.73 - 3.62 (m, 1H), 3.48 (br, 1H), 2.46 - 2.23 (m, 1H), 2.21 - 2.12 (m, 2H), 2.10 (s, 3H), 1.78 - 1.62 (m, 6H), 1.59 - 1.47 (m, 1H), 1.31 - 1.09 (m, 6H), 0.96 (qd, J = 12.1, 2.8 Hz, 2H).13C NMR (101 MHz, CDCh) 8 178.9, 171.0, 170.6, 167.4, 159.4, 158.5, 132.6, 126.9 (q, J= 5.3 Hz), 123.4 (q, J= 272.7 Hz), 119.5 (q, J= 31.4 Hz), 118.4, 113.1, 67.2, 65.3, 54.6, 45.4, 36.2, 34.4, 33.3, 31.2, 29.8, 26.6, 26.3, 24.1, 20.9. HRMS: (ESI) [M+H]+calc, for C26H33F3N5O5, 552.2428, observed, 552.2428.[000119] Example 8: (S)-N- (amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-(cyclohexyloxy)acetamide (20af):[000120][000121] Synthesized according to procedure A, white solid, 180 mg, 82% yield.1H NMR (500 MHz, CDCh) 8 8.25 (d, J = 2.1 Hz, 1H), 8.16 (dd, J= 8.7, 2.1 Hz, 1H), 7.06 (d, J= 8.8 Hz, 1H), 5.47 (br, 1H), 4.14 (t, J = 6.6 Hz, 2H), 3.98 - 3.91 (m, 1H), 3.85 - 3.78 (m, 1H), 3.73 - 3.65 (m, 1H), 3.54 - 3.43 (m, 1H), 3.27 - 3.18 (m, 1H), 2.43 - 2.24 (m, 2H), 2.24 - 2.12 (m, 2H), 1.90 - 1.82 (m, 2H), 1.79 - 1.71 (m, 5H), 1.69 - 1.61 (m, 4H), 1.59 - 1.50 (m, 1H), 1.50 - 1.43 (m, 1H), 1.31 - 1.05 (m, 8H), 0.97 (qd, J = 12.1, 3.2 Hz, 2H).13C NMR (126 MHz, CDCh) 8 183.3, 167.4, 159.4, 158.6, 132.5, 126.9 (q, J = 5.2 Hz), 123.4 (q, J = 272.8 Hz), 119.6 (q, . / = 31.3 Hz), 118.6, 113.1, 77.6, 70.1, 67.3, 54.5, 45.3, 36.2, 34.5, 33.3, 32.2, 32.1, 31.2, 26.6, 26.3, 25.9, 24.3, 24.2. HRMS: (ESI) [M+H]+calc, for C30H41F3N5O4, 592.3105, observed, 592.3077.[000122] Example 9: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-(cyclopentyloxy)acetamide (20ae)[000123][000124] Synthesized according to procedure A, white solid, 112 mg, 53% yield.1H NMR (500 MHz, CDCh) 8 8.25 (d, J = 2.1 Hz, 1H), 8.16 (dd, J= 8.7, 2.1 Hz, 1H), 7.06 (d, J= 8.8 Hz, 1H), 5.48 (br, 1H), 4.14 (t, J = 6.6 Hz, 2H), 3.93 - 3.85 (m, 2H), 3.80 - 3.65 (m, 2H), 3.55 - 3.45 (m, 1H), 2.44 - 2.25 (m, 2H), 2.24 - 2.12 (m, 2H), 1.79 - 1.69 (m, 6H), 1.68 - 1.60 (m, 6H), 1.60 - 1.48 (m, 1H), 1.46 - 1.39 (m, 2H), 1.32 - 1.10 (m, 5H), 0.97 (qd, J= 12.0, 3.2 Hz, 2H).13C NMR (126 MHz, CDCh) 6 183.1, 167.4, 159.4, 158.6, 132.5, 126.9 (q, J = 5.2 Hz), 123.4 (q, J = 272.6 Hz), 119.6 (q, J = 31.5 Hz), 118.6, 113.1, 81.5, 71.1, 67.3, 54.5, 45.4, 36.3,34.5, 33.3, 32.3, 32.1, 31.2, 29.8, 26.6, 26.3, 24.2, 23.6. HRMS: (ESI) [M+H]+calc, for C29H39F3N5O4, 578.2949, observed, 578.2952.[000125] Example 10: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-((tetrahydro-2H-pyran-4-yl)oxy)acetamide(20ai):[000127] Synthesized according to procedure A, white solid, 177 mg, 81% yield.1H NMR (500 MHz, CDCI3) 6 8.25 (d, J= 2.1 Hz, 1H), 8.15 (dd, J= 8.8, 2.1 Hz, 1H), 7.07 - 7.03 (m, 1H), 5.47 (br, 2H), 4.14 (t, J= 6.6 Hz, 2H), 3.99 - 3.94 (m, 1H), 3.90 - 3.80 (m, 3H), 3.72 - 3.67 (m, 1H), 3.54 - 3.44 (m, 2H), 3.36 - 3.29 (m, 2H), 2.44 - 2.25 (m, 2H), 2.24 - 2.12 (m, 2H), 1.87 - 1.79 (m, 2H), 1.78 - 1.60 (m, 6H), 1.60 - 1.47 (m, 3H), 1.31 - 1.09 (m, 4H), 0.97 (qd, J= 12.0, 3.1 Hz, 2H).13C NMR (126 MHz, CDCI3) 5 182.7, 167.4, 159.4, 158.6, 132.5, 126.8 (q, J= 5.2 Hz), 123.4 (1, J = 272.7 Hz), 119.6 (q, J = 31.4 Hz), 118.5, 113.1, 74.0, 69.8, 67.3, 65.9, 54.5, 45.4, 36.2, 34.5, 33.3, 32.4, 32.4, 31.2, 26.6, 26.3, 24.2. HRMS: (ESI) [M+H]+calc, for C29H39F3N5O5, 594.2898, observed, 594.2873.[000128] Example 11 : (S)-A-(amino((S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2- methoxypropanamide (20ab):[000129][000130] Synthesized according to procedure A, white solid, 151 mg, 75% yield.1H NMR(500 MHz, CDCI3) 5 8.25 (d, J = 2.3 Hz, 1H), 8.15 (dd, J = 8.7, 2.3 Hz, 1H), 7.06 (d, J = 8.7 Hz,1H), 5.47 (br, 1H), 4.14 (t, .7= 6.6 Hz, 2H), 3.74 - 3.65 (m, 1H), 3.59 - 3.45 (m, 2H), 3.21 (s, 3H), 2.46 - 2.28 (m, 2H), 2.25 - 2.13 (m, 2H), 1.81 - 1.60 (m, 8H), 1.59 - 1.48 (m, 1H), 1.31 - 1.10 (m, 7H), 0.97 (qd, J= 12.0, 3.1 Hz, 2H).13C NMR (126 MHz, CDC13) 5 185.6, 167.4, 159.4, 158.9, 132.5, 126.8 (q, .7= 5.3 Hz), 123.38 (q,J= 272.7 Hz), 119.61 (q, J= 31.7 Hz), 118.6, 113.1, 80.6, 77.4, 77.2, 76.9, 67.3, 57.2, 54.6, 45.5, 36.3, 34.5, 33.3, 31.2, 26.6, 26.3, 24.3, 19.0. HRMS: (ESI) [M+H]+calc, for C26H35F3N5O4, 538.2636, observed, 538.2622.[000131] Example 12: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-(ethylthio)acetamide (20ak):[000133] Synthesized according to procedure A, white solid, 151 mg, 73% yield.1H NMR (500 MHz, CDCI3) 8 8.25 (d, .7 = 2.1 Hz, 1H), 8.16 (dd, J= 8.7, 2.2 Hz, 1H), 7.06 (d, J= 8.8 Hz, 1H), 5.50 (br, 1H), 4.14 (t, J = 6.6 Hz, 2H), 3.74 - 3.67 (m, 1H), 3.56 - 3.43 (m, 1H), 3.07 (br, 2H), 2.53 - 2.43 (m, 2H), 2.43 - 2.28 (m, 2H), 2.23 - 2.15 (m, 2H), 1.78 - 1.63 (m, 7H), 1.59 - 1.49 (m, 1H), 1.30 - 1.14 (m, 4H), 1.10 (t, J= 7.3 Hz, 3H), 0.97 (qd, J= 11.9, 3.1 Hz, 2H).13C NMR (126 MHz, CDCI3) 8 182.9, 167.4, 159.4, 158.7, 132.5, 126.9 (q, J = 5.3 Hz), 123.4 (q, .7= 272.7 Hz), 119.6 (q, J = 31.2 Hz), 118.6, 113.1, 67.3, 54.6, 45.4, 40.0, 36.3, 34.5, 33.3, 31.2, 26.6, 26.5, 26.3, 24.2, 14.4.[000134] Example 13: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-(benzylthio)acetamide (20aj):[000136] Synthesized according to procedure A, white solid, 176 mg, 77% yield.1H NMR (400 MHz, CDCI3) 8 8.20 (d, .7 = 2.1 Hz, 1H), 8.09 (dd, J= 8.7, 2.2 Hz, 1H), 7.22 - 7.11 (m, 5H),7.00 (d, J= 8.7 Hz, 1H), 5.52 (br, 1H), 4.13 (t, J= 6.6 Hz, 2H), 3.78 - 3.59 (m, 3H), 3.57 - 3.44 (m, 1H), 3.01 (s, 2H), 2.46 - 2.11 (m, 4H), 1.83 - 1.62 (m, 7H), 1.59 - 1.47 (m, 1H), 1.34 - 1.10 (m, 4H), 1.05 - 0.90 (m, 2H).13C NMR (126 MHz, CDCh) 5 182.9, 167.7, 159.6, 159.1, 138.7,132.8, 129.4, 128.6, 127.1, 127.0 (q, 5.4 Hz), 123.6 (q, 272.7 Hz), 119.8 (q, J= 31.1 Hz),118.8, 113.3, 67.5, 54.9, 45.7, 39.9, 36.9, 36.5, 34.7, 33.5, 31.5, 26.9, 26.6, 24.4. HRMS: (ESI) [M+H]+calc, for C31H37F3N5O3S, 616.2564, observed, 616.2542.[000137] Example 14: (S)-7V-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-(methylthio)acetamide (20al):[000138][000139] Synthesized according to procedure A, white solid, 149 mg, 43% yield.1H NMR (500 MHz, CDCh) 5 8.25 (d, J= 2.1 Hz, 1H), 8.16 (dd, J= 8.7, 2.2 Hz, 1H), 7.06 (d, J= 8.7 Hz, 1H), 5.50 (br, 1H), 4.14 (t, J= 6.6 Hz, 2H), 3.74 - 3.66 (m, 1H), 3.55 - 3.43 (m, 1H), 3.04 (s, 2H), 2.45 - 2.26 (m, 2H), 2.24 - 2.14 (m, 2H), 2.01 (s, 3H), 1.84 - 1.59 (m, 7H), 1.60 - 1.48 (m, 1H), 1.34 - 1.07 (m, 4H), 0.97 (qd, J = 11.9, 3.2 Hz, 2H).13C NMR (126 MHz, CDCh) 5 182.6, 167.5, 159.4, 158.7, 132.5, 126.9 (q, J= 5.4 Hz), 123.4 (q, J= 272.7 Hz), 119.6 (q, J = 31.5 Hz), 118.6, 113.1, 67.3, 54.6, 45.5, 42.4, 36.3, 34.5, 33.3, 31.2, 26.6, 26.3, 24.2, 16.2. HRMS: (ESI) [M+H]+calc, for C25H33F3N5O3S, 540.2251, observed, 540.2243.[000140] Example 15: (S)-2-(3-(4-(2-cy cl ohexylethoxy)-3-(trifluoromethyl)phenyl)- 1,2,4- oxadiazol-5-yl)-N'-(dimethylcarbamoyl)pyrrolidine-l-carboximidamide (20a):[000141][000142] (Synthesized according to procedure B, white solid, 73 mg, 34% yield.1H NMR(500 MHz, CDCh) 5 8.25 (d, J = 2.1 Hz, 1H), 8.15 (dd, J= 8.7, 2.2 Hz, 1H), 7.05 (d, J= 8.7 Hz,1H), 5.42 - 5.36 (m, 1H), 4.14 (t, J = 6.6 Hz, 2H), 3.73 - 3.65 (m, 1H), 3.54 - 3.45 (m, 1H), 2.83 - 2.77 (m, 6H), 2.44 - 2.28 (m, 2H), 2.20 - 2.10 (m, 2H), 1.80 - 1.62 (m, 7H), 1.60 - 1.49 (m, 1H), 1.33 - 1.07 (m, 4H), 1.03 - 0.91 (m, 2H).13C NMR (126 MHz, CDCI3) 8 181.0, 167.3, 164.8, 159.3, 157.2, 132.5, 126.8 (q, J = 5.3 Hz), 123.4 (q, J = 272.6 Hz), 119.5 (q, J= 31.4 Hz), 118.7, 113.0, 67.2, 54.3, 45.5, 38.7, 36.9, 36.3, 35.3, 34.4, 33.3, 31.2, 26.6, 26.3, 24.4. HRMS: (ESI) [M+H]+calc, for C25H34F3N6O3, 523.2639, observed, 523.2635.[000143] Example 16: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-cyclobutoxyacetamide (20ad):[000144][000145] Synthesized according to procedure A, white solid, 149 mg, 43% yield.1H NMR (500 MHz, CDCI3) 8 8.26 (d, J = 2.1 Hz, 1H), 8.16 (dd, J= 8.7, 2.2 Hz, 1H), 7.06 (d, J= 8.7 Hz, 1H), 5,49 (br, 1H), 4.14 (t, J= 6.6 Hz, 2H), 4.00 - 3.88 (m, 1H), 3.88 - 3.77 (m, 1H), 3.76 - 3.64 (m, 2H), 3.51 (br, 1H), 2.44 - 2.26 (m, 2H), 2.23 - 2.15 (m, 2H), 2.14 - 2.05 (m, 2H), 1.90 (p, J= 11.3, 10.6 Hz, 2H), 1.79 - 1.49 (m, 7H), 1.42 - 1.32 (m, 1H), 1.30 - 1.10 (m, 6H), 1.04 - 0.91 (m, 2H).13C NMR (126 MHz, CDCI3) 8 182.5, 167.4, 159.4, 132.5, 126.8 (q, J= 5.3 Hz), 123.3 (q, J = 212.1 Hz), 119.5 (q, J = 31.2 Hz), 118.4, 113.0, 73.1, 69.7, 67.2, 54.5, 45.2, 36.2, 34.4, 33.2, 31.2, 30.2, 30.2, 29.8, 26.6, 26.3, 24.2, 12.4. HRMS: (ESI) [M+H]+calc, for C28H37F3N5O4, 564.2792, observed, 563.2776.[000146] Example 17: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-cyclopropoxyacetamide (20ac):[000147][000148] Synthesized according to procedure A, white solid, 163 mg, 72% yield.1H NMR (500 MHz, CDCh) 8 8.28 (d, J = 2.1 Hz, 1H), 8.19 (dd, J = 8.7, 2.3 Hz, 1H), 7.09 (d, .7= 8.7 Hz, 1H), 5.52 (br, 1H), 4.17 (t, .7= 6.6 Hz, 2H), 4.03 - 3.93 (m, 1H), 3.91 - 3.83 (m, 1H), 3.76 - 3.68 (m, 1H), 3.54 (br, 1H), 3.46 - 3.38 (m, 1H), 2.46 - 2.32 (m, 1H), 2.28 - 2.17 (m, 2H), 1.81 - 1.64 (m, 8H), 1.62 - 1.52 (m, 1H), 1.34 - 1.15 (m, 4H), 1.04 - 0.93 (m, 2H), 0.60 - 0.54 (m, 2H), 0.40 - 0.34 (m, 2H).13C NMR (126 MHz, CDCh) 8 182.5, 167.4, 159.4, 158.5, 132.5, 126.9 (q, J= 5.3 Hz), 123.4 (d, J = 272.5 Hz), 119.6 (d, J= 31.4 Hz), 118.5, 113.0, 72.8, 67.3, 54.5, 53.6, 45.2, 36.3, 34.4, 33.3, 31.2, 26.6, 26.3, 24.2, 5.8, 5.7. HRMS: (ESI) [M+H]+calc, for C27H35F3N5O4, 550.2636, observed, 550.2642.[000149] Example 18: A-(amino((S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-(((S)-tetrahydrofuran-3-yl)oxy)acetamide((S)-20ag):[000150][000151] Synthesized according to procedure A, white solid, 44 mg, 46% yield.1H NMR (500 MHz, CDCh) 8 8.25 (d, J = 2.1 Hz, 1H), 8.16 (dd, J= 8.7, 2.2 Hz, 1H), 7.07 (d, J= 8.7 Hz, 1H), 5.51 (br, 1H), 4.24 - 4.09 (m, 3H), 4.00 - 3.63 (m, 6H), 3.51 (br, 1H), 2.49 - 2.27 (m, 2H), 2.21 (q, J = 6.6 Hz, 2H), 2.02 - 1.85 (m, 2H), 1.79 - 1.63 (m, 7H), 1.59 - 1.49 (m, 1H), 1.31 - 1.13 (m, 5H), 0.97 (qd, J= 12.2, 3.1 Hz, 2H).13C NMR (126 MHz, CDCh) 8 167.4, 159.4, 132.5, 126.8 (q, J = 5.3 Hz), 123.3 (q, J = 272.8 Hz), 119.5 (q, J = 31.2 Hz), 113.0, 79.6, 72.9, 70.9, 67.2, 67.1, 54.6, 45.3, 36.2, 34.4, 33.3, 32.5, 31.2, 29.8, 26.6, 26.3, 24.1. HRMS: (ESI) [M+H]+calc, for C28H37F3N5O5, 580.2741, observed, 580.2726.[000152] Example 19: A-(amino((S)-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-(((A)-tetrahydrofuran-3-yl)oxy)acetamide((R )-20ah):[000153][000154] Synthesized according to procedure A, white solid, 21 mg, 22% yield.1H NMR (500 MHz, CDCI3) 8 8.25 (d, J= 2.2 Hz, 1H), 8.16 (dd, J= 8.7, 2.2 Hz, 1H), 7.07 (d, J= 8.7 Hz, 1H), 5.48 (br, 1H), 4.20 - 4.08 (m, 3H), 3.94 - 3.66 (m, 6H), 3.51 (br, 1H), 2.44 - 2.29 (m, 2H), 2.25 - 2.16 (m, 2H), 2.00 - 1.84 (m, 2H), 1.79 - 1.63 (m, 7H), 1.59 - 1.50 (m, 1H), 1.31 - 1.11 (m, 5H), 1.02 - 0.92 (m, 2H).13C NMR (126 MHz, CDCI3) 8 182.3, 167.5, 159.4, 158.6, 132.5, 126.9 (q, J = 5.3 Hz), 123.4 (q, J = 272.9 Hz), 119.6 (q, J = 31.5 Hz), 118.4, 113.1, 79.6, 72.8, 71.0, 67.3, 67.1, 54.6, 45.2, 36.2, 34.4, 33.3, 32.6, 31.2, 29.8, 26.6, 26.3, 24.2. HRMS: (ESI) [M+H]+calc, for C28H37F3N5O5, 580.2741, observed, 580.2726.[000155] Example 20: (S)-2-(3-(4-(2-cy cl ohexylethoxy)-3-(trifluoromethyl)phenyl)- 1,2,4- oxadiazol-5-yl)-N'-(methoxy(methyl)carbamoyl)pyrrolidine-l-carboximidamide (20b):[000156][000157] Synthesized by General Procedure B: 174 mg, 79%, white solid. 'H NMR (400 MHz, CD3OD) δ 8.19-8.12 (m, 2H), 7.22 (d, J= 9.5 Hz, 1H), 5.42-5.32 (m, 1H), 4.15 (t, J= 6.4 Hz, 2H), 3.76-3.68 (m, 1H), 3.57-3.45 (m, 4H), 2.96 (s, 3H), 2.48-2.37 (m, 1H), 2.33-2.22 (m, 1H), 2.20- 2.05 (m, 2H), 1.80-1.61 (m, 7H), 1.60-1.49 (m, 1H), 1.32-1.11 (m, 3H), 1.02-0.90 (m, 2H);13C NMR (101 MHz, CD3OD) δ 182.7, 168.3, 166.8, 160.6, 159.7, 133.7, 126.9 (q, J = 5.5 Hz), 124.7 (q, J= 272.0 Hz), 120.14 (q, J = 31.2 Hz), 119.7, 114.7, 68.2, 61.2, 55.9, 46.9, 37.3, 35.9, 35.6, 34.2, 32.1, 27.6, 27.3, 25.2; HRMS (ESI+): calc, for C25H33F3N6NaO4 [M+Na]+, 561.2408; found, 561.2391.[000158] Example 21 : (S)-7V-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-1,2,4 oxadiazol-5-yl)pyrrolidin-l-yl)methylene)morpholine-4-carboxamide (20c):[000160] Synthesized by General Procedure B: 145 mg, 63%, white solid.1H NMR (400 MHz, CDCI3) 6 8.30-8.05 (m, 2H), 7.04 (d, J= 9.1 Hz, 1H), 5,38-5.27 (m, 1H), 4.12 (t, J= 6.2 Hz, 2H), 3.70-3.22 (m, 10H), 2.43-2.25 (m, 2H), 2.20-2.03 (m, 2H), 1.84-1.57 (m, 7H), 1.57-1.45 (m, 1H), 1.30-1.07 (m, 3H), 1.03-0.80 (m, 2H);13C NMR (101 MHz, CD3OD) δ 182.9, 168.2, 164.4, 160.6, 159.4, 133.7, 126.9 (q, J= 5.3 Hz), 124.6 (q, J = 271.4 Hz), 120.2 (q, J= 32.0 Hz), 119.7, 114.7, 68.2, 67.8, 67.5, 55.8, 46.9, 37.3, 35.6, 34.2, 32.0, 27.6, 27.3, 25.3; HRMS (ESI+): calc, for C25H33F3N6NaO4[M+Na]+, 561.2408; found, 561.2391.[000161] Example 22: phenyl (5)-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)carbamate (20d)[000162][000163] Synthesized by General Procedure B: 70 mg, 66%, tan solid.1H NMR (400 MHz, CD3OD) δ 8.23-8.14 (m, 2H), 7.32-7.00 (m, 4H), 6.94-6.73 (m, 2H), 5.52-5.31 (m, 1H), 4.21-4.13 (m, 2H), 3.77-3.68 (m, 1H), 3.58-3.48 (m, 1H), 2.45-2.31 (m, 1H), 2.22-2.05 (m, 3H), 1.81-1.63 (m, 7H), 1.62-1.51 (m, 1H), 1.32-1.16 (m, 3H), 1.04-0.93 (m, 2H);13C NMR (101 MHz, CD3OD) 5 181.6, 168.3, 163.5, 160.6, 153.7, 133.9, 130.4, 130.0, 127.1 (q, J = 5.6 Hz), 125.6, 124.7 (q, J= 272.5 Hz), 122.8, 120.2 (q, J= 31.6 Hz), 119.8, 116.2, 114.7, 68.2, 47.2, 37.3, 35.6, 34.2, 32.2, 27.6, 27.3, 24.8; HRMS (ESI+): calc, for C29H33F3N5O4 [M+H]+, 572.2479; found, 572.2472.[000164] Example 23: methyl (5)-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)carbamate (20e):[000165][000166] Synthesized by General Procedure B: 197 mg, 94%, white solid.1H NMR (400 MHz, CD3OD) δ 8.19-8.09 (m, 2H), 7.20 (d, J= 9.5 Hz, 1H), 5.48 (d, J= 8.5 Hz, 1H), 4.13 (t, J= 6.1 Hz, 2H), 3.79-3.69 (m, 1H), 3.60-3.45 (m, 4H), 2.49-2.35 (m, 1H), 2.26-2.09 (m, 3H), 1.79-1.60 (m, 7H), 1.58-1.48 (m, 1H), 1.32-1.11 (m, 3H), 1.02-0.89 (m, 2H);13C NMR (101 MHz, CD3OD) δ 182.0, 168.3, 165.4, 160.9, 160.5, 133.8, 127.0 (q, J= 5.4 Hz), 124.6 (q,J= 272.6 Hz), 120.1 (q, J= 31.4 Hz), 119.7, 114.6, 68.2, 55.8, 53.2, 47.2, 37.3, 35.5, 34.2, 32.2, 27.6, 27.3, 24.8; HRMS (ESI+): calc, for C24H31F3N5O4 [M+H]+, 510.2323; found, 510.2304.[000167] Example 24: ethyl (S)-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)carbamate (20f):[000168][000169] Synthesized by General Procedure B: 188 mg, 88%, white solid.1H NMR (400 MHz, CD3OD) 6 8.19-8.11 (m, 2H), 7.21 (d, J = 8.9 Hz, 1H), 5.49 (d, J = 8.3 Hz, 1H), 4.14 (t, J= 6.7 Hz, 2H), 3.99-3.84 (m, 2H), 3.79-3.69 (m, 1H), 3.60-3.49 (m, 1H), 2.50-2.36 (m, 1H), 2.28-2.11 (m, 3H), 1.81-1.61 (m, 7H), 1.61-1.48 (m, 1H), 1.32-1.14 (m, 3H), 1.09 (t, J = 7.1 Hz, 3H), 1.03-0.90 (m, 2H);13C NMR (101 MHz, CD3OD) δ 182.0, 168.3, 164.9, 161.0, 160.6, 133.8, 127.0 (q, J= 5.4 Hz), 124.7 (q, J= 272.0 Hz), 120.1 (q, J= 30.9 Hz), 119.7, 114.6, 68.2, 62.3, 55.8, 47.2, 37.3, 35.6, 34.2, 32.2, 27.6, 27.3, 24.8, 15.0; HRMS (ESI+): calc, for C25H33F3N5O4 [M+H]+, 524.2479; found, 524.2466.[000170] Example 25: benzyl (5)-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)carbamate (20g):[000171][000172] Synthesized by General Procedure B: 225 mg, 94%, yellow solid.1H NMR (400 MHz, CD3OD) δ 8.14-8.03 (m, 2H), 7.22-7.05 (m, 6H), 5.44 (d, J = 7.3 Hz, 1H), 4.97-4.85 (m, 2H), 4.07 (t, J = 6.4 Hz, 2H), 3.74-3.63 (m, 1H), 3.54-3.44 (m, 1H), 2.41-2.29 (m, 1H), 2.20-2.04 (m, 3H), 1.77-1.59 (m, 7H), 1.56-1.46 (m, 1H), 1.29-1.09 (m, 3H), 0.99-0.86 (m, 2H);13C NMR (101 MHz, CD3OD) 5 181.9, 168.2, 164.4, 160.8, 160.5, 138.7, 133.8, 129.2, 128.5, 128.3, 127.0 (q, J = 5.2 Hz), 124.6 (q, J = 272.3 Hz), 120.0 (q, J = 31.7 Hz), 119.6, 114.5, 68.3, 68.1, 55.9, 47.2, 37.2, 35.5, 34.1, 32.1, 27.5, 27.3, 24.8; HRMS (ESI+): calc, for C30H35F3N5O4 [M+H]+, 586.2636; found, 586.2618.[000173] Example 26: allyl (S)-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)carbamate (20h):[000175] Synthesized by General Procedure B: 170 mg, 77%, white solid.1H NMR (400 MHz, CD3OD) δ 8.19-8.12 (m, 2H), 7.21 (d, J= 9.3 Hz, 1H), 5.87-5.72 (m, 1H), 5.48 (d, J = 7.8 Hz, 1H), 5.13 (d, J= 17.3 Hz, 1H), 5.00 (d, J= 10.6 Hz, 1H), 4.45-4.31 (m, 2H), 4.14 (t, J= 6.9 Hz, 2H), 3.77-3.69 (m, 1H), 3.60-3.51 (m, 1H), 2.50-2.37 (m, 1H), 2.26-2.11 (m, 3H), 1.79-1.61 (m, 7H), 1.59-1.48 (m, 1H), 1.32-1.12 (m, 3H), 1.02-0.90 (m, 2H);13C NMR (101 MHz, CD3OD) 6 182.0, 168.3, 164.5, 161.0, 160.5, 134.9, 133.8, 127.0 (q, J= 5.3 Hz), 124.6 (q, . / = 272.2 Hz),120.1 (q, J = 31.8 Hz), 119.7, 116.9, 114.6, 68.2, 67.3, 55.9, 47.2, 37.3, 35.5, 34.2, 32.2, 27.6, 27.3, 24.9; HRMS (ESI+): calc, for C26H33F3N5O4 [M+H]+, 536.2479; found, 536.2469.[000176] Example 27: 2-methoxyethyl (S)-(amino(2-(3-(4-(2-cyclohexylethoxy)-3- (trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)carbamate (20i):[000177][000178] Synthesized by General Procedure B: 197 mg, 87%, white solid.1H NMR (400 MHz, CD3OD) 5 8.19-8.12 (m, 2H), 7.23 (d, J = 9.4 Hz, 1H), 5.49 (d, J = 8.0 Hz, 1H), 4.15 (t, J= 6.2 Hz, 2H), 4.07-3.92 (m, 1H), 3.78-3.69 (m, 1H), 3.60-3.50 (m, 1H), 3.47-3.37 (m, 2H), 3.21 (s, 3H), 2.50-2.37 (m, 1H), 2.27-2.11 (m, 3H), 1.80-1.61 (m, 7H), 1.61-1.48 (m, 1H), 1.32-1.11 (m, 3H), 1.04-0.90 (m, 2H);13C NMR (101 MHz, CD3OD) δ 182.0, 168.3, 164.6, 160.9, 160.6, 133.8, 127.0 (q, J= 5.4 Hz), 124.6 (q, J = 271.8 Hz), 120.1 (q, J= 31.8 Hz), 119.7, 114.7, 72.0, 68.2, 65.8, 59.0, 55.8, 47.2, 37.3, 35.6, 34.2, 32.2, 27.6, 27.3, 24.8; HRMS (ESI+): calc, for C26H35F3N5O5 [M+H]+, 554.2585; found, 554.2562.[000179] Example 28: (S)-N'-cyano-2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidine-l-carboximidamide (20j)[000180][000181] Synthesized according to the following protocol: (S)-2-(3-(4-(2-cyclohexylethoxy)-3- (trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidine-l-carboximidamide hydrochloride (1.0 equiv) was dissolved in CH2Q2 (0.2 M solution) and put under an argon gas atmosphere. Next 7V,7V-diisopropylethylamine (2.0 equiv) was added followed by addition of cyanic bromide (1.1 equiv). The reaction mixture was then stirred at 40 °C for 2 hours. The reaction progress was monitored by TLC. Afterward, the reaction was concentrated via vacuum and then purified by silica gel chromatography to yield the desired product; 61 mg, 30%, yellow solid. NMR (400MHz, CD3OD) δ 8.26-8.14 (m, 2H), 7.28 (d, J= 8.5 Hz, 1H), 5.38-5.29 (m, 1H), 4.19 (t, J= 6.3 Hz, 2H), 3.79-3.68 (m, 1H), 3.61-3.50 (m, 1H), 2.51-2.38 (m, 1H), 2.27-2.10 (m, 3H), 1.84-1.63 (m, 7H), 1.62-1.52 (m, 1H), 1.35-1.15 (m, 3H), 1.05-0.93 (m, 2H);13C NMR (101 MHz, CD3OD) 8 181.6, 168.4, 161.0, 160.7, 133.9, 127.0 (q, J = 5.4 Hz), 124.8 (q, J = 273.0 Hz), 120.2 (q, J= 31.4 Hz), 119.6, 119.1, 114.7, 68.2, 56.3, 48.2, 37.4, 35.6, 34.2, 32.4, 27.6, 27.4, 25.1; HRMS (ESI+): calc, for C23H28F3N6O2 [M+H]+, 477.2220; found, 477.2215.[000182] Example 29: (S)-2-(3-(4-(2-cy cl ohexylethoxy)-3-(trifluoromethyl)phenyl)- 1,2,4- oxadiazol-5-yl)-N'-tosylpyrrolidine-l-carboximidamide (20m):[000183][000184] Synthesized by General Procedure C: 133 mg, 54%, white solid.1H NMR (400 MHz, CDCI3) 8 8.12-8.00 (m, 2H), 7.44 (d, J= 7.4 Hz, 2H), 7.06 (d, J= 8.6 Hz, 1H), 6.87 (d, J= 6.9 Hz, 2H), 6.59 (br s, 2H), 5.39 (d, J= 6.8 Hz, 1H), 4.15 (t, J= 6.6Hz, 2H), 3.69-3.60 (m, 1H), 3.57-3.47 (m, 1H), 2.43-2.31 (m, 1H), 2.23-2.00 (m, 6H), 1.80-1.62 (m, 7H), 1.60-1.49 (m, 1H), 1.31-1.10 (m, 3H), 1.05-0.90 (m, 2H);13C NMR (101 MHz, CDCI3) 8 179.8, 167.0, 159.2, 154.2, 141.7, 139.7, 132.1, 129.0, 126.6 (q, J= 5.4 Hz), 125.6, 123.3 (q, J = 273.4 Hz), 119.3 (q, J= 31.7 Hz), 118.5, 112.9, 67.2, 54.7, 46.3, 36.2, 34.4, 33.2, 31.1, 26.6, 26.3, 24.2, 21.1; HRMS (ESI+): calc, for C29H35F3N5O4S [M+H]+, 606.2356; found, 606.2338.[000185] Example 30: (S)-2-(3-(4-(2-cy cl ohexylethoxy)-3-(trifluoromethyl)phenyl)- 1,2,4- oxadiazol-5-yl)-N'-((trifluoromethyl)sulfonyl)pyrrolidine-l-carboximidamide (201):[000186][000187] Synthesized by General Procedure C: 71 mg, 30%, white solid.1H NMR (400 MHz, CD3OD) δ 8.23-8.13 (m, 2H), 7.30 (d, J= 8.4 Hz, 1H), 5.50-5.37 (m, 1H), 4.20 (t, J= 6.4 Hz, 2H),3.82-3.72 (m, 1H), 3.64-3.55 (m, 1H), 2.55-2.41 (m, 1H), 2.31-2.12 (m, 3H), 1.84-1.63 (m, 7H), 1.63-1.52 (m, 1H), 1.35-1.15 (m, 3H), 1.07-0.93 (m, 2H);13C NMR (101 MHz, CD3OD) δ 181.1, 168.4, 160.7, 157.2, 133.8, 127.1 (q, J= 5.4 Hz), 124.7 (q, J= 272.8 Hz), 120.2 (q, J= 31.1 Hz), 119.7, 114.7, 68.2, 56.7, 48.0, 37.4, 35.6, 34.2, 32.0, 27.6, 27.4, 25.0; HRMS (ESI+): calc, for C23H28F6N5O4S [M+H]+, 584.1761; found, 584.1753.[000188] Example 31 : (S)-7V-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)benzamide (20n):[000189][000190] Synthesized by General Procedure B: 190 mg, 84%, white solid.1H NMR. (400 MHz, CDC13) 5 8.36-8.01 (m, 4H), 7.39-7.25 (m, 3H), 7.04 (d, J = 8.7 Hz, 1H), 5.66 (br s, 1H), 4.12 (t, J= 6.6 Hz, 2H), 3.79-3.67 (m, 1H), 3.60-3.43 (m, 1H), 2.47-2,31 (m, 2H), 2,25-2, 12 (m, 2H), 1.80-1.63 (m, 7H), 1.59-1.48 (m, 1H), 1.32-1.11 (m, 3H), 1.02-0.88 (m, 2H);13C NMR (101 MHz, CDCI3) 5 180.4, 177.2, 167.4, 159.3, 159.0, 138.3, 132.5, 131.2, 129.2, 127.8, 126.8 (q, J= 5.3 Hz), 123.4 (q, J= 273.8 Hz), 119.5 (q, J= 31.3 Hz), 118.5, 113.0, 67.2, 54.8, 45.5, 36.2, 34.4, 33.3, 31.2, 26.6, 26.3, 24.3; HRMS (ESI+): calc, for C29H33F3N5O3 [M+H]+, 556.2530; found, 556.2513.[000191] Example 32: (S)-N-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)benzo[tZ][l,3]dioxole-5-carboxamide (20o):[000193] Synthesized by General Procedure B: 194 mg, 79%, tan solid.1H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 8.06 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.42 (s, 1H), 7.02 (d, J= 9.0 Hz, 1H), 6.60 (d, J= 8.0 Hz, 1H), 5.82 (d, J= 23.2 Hz, 2H), 5.60-5.46 (m, 1H), 3.99 (t, J = 6.4 Hz, 2H), 3.77-3.65 (m, 1H), 3.60-3.47 (m, 1H), 2.48-2.35 (m, 1H), 2.33-2.21 (m, 1H), 2.19-2.06 (m, 2H), 1.74-1.54 (m, 7H), 1.52-1.41 (m, 1H), 1.26-1.09 (m, 3H), 0.95-0.82 (m, 2H);13C NMR (101 MHz, CD3OD) δ 182.5, 177.2, 168.3, 160.5, 159.9, 151.6, 148.6, 134.0, 133.7, 127.2 (q, J= 5.3 Hz), 125.1, 124.6 (q, J = 272.7 Hz), 120.0 (q, J= 31.1 Hz), 119.5, 114.4, 110.1, 108.1, 102.6, 68.1, 56.3, 46.9, 37.3, 35.5, 34.2, 32.0, 27.6, 27.3, 25.3; HRMS (ESI+): calc, for C30H33F3N5O5 [M+H]+, 600.2428; found, 600.2410.[000194] Example 33: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2,2,2-trifluoroacetamide (20s):[000195][000196] Synthesized according to the following protocol: (S)-2-(3-(4-(2-cyclohexylethoxy)-3- (trifluoromethyl)phenyl)-l,2,4-oxadiazol-5-yl)pyrrolidine-l-carboximidamide hydrochloride (1.0 equiv) was dissolved in CH2CI2 (0.2 M solution) and put under a argon gas atmosphere. Next, N,N -di isopropyl ethyl amine (2.0 equiv) was added followed by dropwise addition of 2,2,2-trifluoroacetic anhydride (1.1 equiv). The reaction mixture was stirred at room temperature for 10 minutes. The reaction progress was monitored by TLC. Afterward, the reaction was concentrated via vacuum and then purified by silica gel chromatography to yield the desired product; 80 mg, 71%, yellow solid.1H NMR (400 MHz, CD3OD) 5 8.22-8.14 (m, 2H), 7.26 (d, J= 8.6 Hz, 1H), 5.58-5.46 (m, 1H), 4.18 (t, J= 6.9 Hz, 2H), 4.01-3.55 (m, 2H), 2.62-2.43 (m, 1H), 2.33-2.11 (m, 3H), 1.82-1.62 (m, 7H), 1.61-1.51 (m, 1H), 1.33-1.14 (m, 3H), 1.04-0.92 (m, 2H);13C NMR (101 MHz, CD3OD) δ 181.8, 180.73, 168.4, 160.6, 133.9, 127.1 (q, J= 5.5 Hz), 124.7 (q, J = 271.9 Hz), 120.2 (q, . / = 32.4 Hz), 119.7, 114.6, 68.2, 57.0, 56.7, 47.2, 37.4, 35.6, 34.2, 32.4, 27.6, 27.3, 25.0; HRMS (ESI+): calc, for C24H28F6N5O3 [M+H]+, 548.2091; found, 548.2088.[000197] Example 34: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)propionamide (20u):[000198][000199] Synthesized by General Procedure B: 61 mg, 30%, yellow solid.1H NMR (400 MHz, CD3OD) δ 8.23-8.12 (m, 2H), 7.24 (d, J= 9.9 Hz, 1H), 5.47-5.37 (m, 1H), 4.16 (t, J= 6.5 Hz, 2H), 3.93-3.81 (m, 1H), 3.77-3.66 (m, 1H), 2.57-2.45 (m, 1H), 2.40-2.13 (m, 5H), 1.80-1.61 (m, 7H), 1.60-1.49 (m, 1H), 1.32-1.16 (m, 3H), 1.14-0.98 (m, 3H), 0.96-0.85 (m, 2H);13C NMR (101 MHz, CDCI3) 5 179.6, 167.4, 159.3, 153.0, 132.5, 126.8 (q, J = 5.3 Hz), 123.3 (q, J = 272.0 Hz), 119.5 (q, J = 30.7 Hz), 118.6, 113.0, 67.2, 56.3, 49.7, 36.2, 34.4, 33.2, 32.3, 31.1, 26.6, 26.3, 24.5, 9.5, 9.2; HRMS (ESI+): calc, for C25H33F3N5O3 [M+H]+, 508.2530; found, 508.2529.[000200] Example 35: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-3,5-dimethylbenzamide (20r):[000201][000202] Synthesized by General procedure A: 178 mg, 84%, white solid.1H NMR (400 MHz, CDCI3) 8 8.28 (d, J= 2.1 Hz, 1H), 8.16 (dd, J= 8.7, 2.2 Hz, 1H), 7.63 (s, 2H), 7.05 - 6.97 (m, 2H), 5.64 (s, 1H), 4.10 (t, 6.6 Hz, 2H), 3.76 - 3.66 (m, 1H), 3.54 - 3.43 (m, 1H), 2.47 - 2.31 (m, 2H),2.25 (s, 6H), 2.20 - 2.08 (m, 2H), 1.79 - 1.66 (m, 7H), 1.59 - 1.46 (m, 1H), 1.28 - 1.24 (m, 3H), 1.02 - 0.92 (m, 2H).13C NMR (101 MHz, CDCI3) 8 177.4, 167.3, 159.2, 159.2, 158.8, 138.1, 137.2, 132.8, 132.4, 126.8, 126.8 (q, J= 5.3 Hz), 123.3 (q, J= 272.7 Hz), 119.3 (q, J = 31.3 Hz), 118.5, 112.9, 67.1, 36.1, 34.3, 33.1, 31.1, 29.7, 26.5, 26.2, 24.2, 21.1. HRMS: (ESI) [M+H]+calc, for C31H37F3N5O3+584.2843, observed, 584.2835.[000203] Example 36: (,V)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-ethoxyacetamide (20x):[000204][000205] Synthesized by General Procedure B: 85 mg, 38%, white solid.1H NMR (400 MHz,CDC13) 5 8.28-8.22 (m, 1H), 8.18-8.12 (m, 1H), 7.06 (d, J = 8.2 Hz, 1H), 5.48 (br s, 1H),4.14 (t, J= 6.6 Hz, 2H), 3.94-3.75 (m, 2H) 3.74-3.64 (m, 1H), 3.57-3.39 (m, 3H), 2.45-2.08 (m, 4H), 1.80-1.46 (m, 8H), 1.29-1.12 (m, 6H), 1.03-0.90 (m, 2H);13C NMR (101 MHz CDC13) 5 182.8, 167.4, 159.4, 158.2, 132.5, 126.9 (q, J = 5.3 Hz), 123.4 (q, J = 272.5 Hz), 119.6 (q, J= 31.1 Hz), 118.5, 113.0, 72.8, 67.3, 66.6, 54.5, 45.7, 36.0, 34.4, 33.3, 31.2, 26.6, 26.3, 24.2, 15.3. HRMS: (ESI) [M+H]+calc, for C26H35F3N5O4+538.2636, observed, 538.2639.[000206] Example 37: (S)-7V-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-hydroxyacetamide (20y):[000208] Synthesized according to the following protocol: (S)-A-(amino(2-(3-(4-(2- cyclohexylethoxy)-3 -(trifluoromethyl)phenyl)- 1 ,2,4-oxadiazol-5-yl)pyrrolidin- 1 -yl)methylene)- 2-(benzyloxy)acetamide (1 equiv) was dissolved in DCM (0.2 M solution)and chilled to 0 °C. Tribromoborane (I M solution, 20 equiv) was then added. The reaction mixture was stirred at 0 °C for 5 h. Once complete, the reaction was quenched with saturated aq. NaHCO3. Ethyl acetate was added to the reaction and the organic layer was removed via separatory funnel and dried over sodium sulfate. After, the sodium sulfate was filtered off and the organic layer was concentrated via vacuum and purified by silica gel chromatography at 100% EtOAc to yield the desired product: 25 mg, 19%, white solid.1H NMR (400 MHz, CD3OD) δ 8.25 - 8.17 (m, 2H), 7.34 - 7.28 (m, 1H), 5.57 - 5.44 (m, 1H), 4.76 (d, J = 4.4 Hz, 1H), 4.67 (d, J = 4.4 Hz, 1H), 4.21 (t, J= 6.4 Hz,2H), 3.98 - 3.88 (m, 1H), 3.85 - 3.75 (m, 1H), 2.63 - 2.52 (m, 1H), 2.40 - 2.25 (m, 1H), 2.26 - 2.16 (m, 2H), 1.82 - 1.65 (m, 7H), 1.63 - 1.53 (m, 1H), 1.36 - 1.15 (m, 5H), 1.06 - 0.94 (m, 2H).13C NMR (101 MHz, CD3OD) 5 187.5, 187.2, 178.5, 167.0, 132.5, 125.7 (q, J = 5.6 Hz), 119.5 (q, ,7 = 32.2 Hz), 113.3, 72.1, 72.0, 66.8, 63.6, 56.2, 54.1, 49.1, 35.9, 34.2, 32.8, 31.4, 31.2, 26.2, 25.9, 23.4, 23.2. HRMS: (ESI) [M+H]+calc, for 510.2323, observed, 510.2327.[000209] Example 38: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-phenoxyacetamide (20z):[000210][000211] Synthesized by General procedure B: 199 mg, 83%, white solid.1H NMR (400 MHz, CD3OD) δ 8.21 - 8.17 (m, 2H), 7.28 (d, J = 8.9 Hz, 1H), 7.00 (t, J = 7.7 Hz, 2H), 6.73 (t, J = 7.3 Hz, 1H), 6.67 - 6.61 (m, 2H), 5.44 (dd, J = 8.3, 3.4 Hz, 1H), 4.32 (d, J = 15.6 Hz, 1H), 4.19 (t, J= 6.4 Hz, 3H), 3.73 - 3.67 (m, 1H), 3.59 - 3.49 (m, 1H), 2.49 - 2.37 (m, 1H), 2.25 - 2.08 (m, 2H), 1.81 - 1.64 (m, 7H), 1.62 - 1.51 (m, 1H), 1.32 - 1.12 (m, 3H), 1.05 - 0.92 (m, 2H).13C NMR (101 MHz, CD3OD) δ 177.9, 167.1, 159.4, 155.8, 132.5, 129.1, 127.1 (q, J= 5.3 Hz), 124.7 (q, J = 271 Hz), 121.1, 120.2, 117.9, 114.2, 114.1, 113.4, 66.8, 35.9, 34.2, 32.8, 31.2, 26.2, 25.9, 22.9. HRMS: (ESI) [M+H]+calc, for C3oH35F3N504+586.2636, observed, 586.2614.[000212] Example 39: (S)-A-(amino(2-(3-(4-(2-cyclohexylethoxy)-3-(trifluoromethyl)phenyl)- l,2,4-oxadiazol-5-yl)pyrrolidin-l-yl)methylene)-2-methoxyacetamide (20aa):[000213][000214] Synthesized by General procedure A: 138 mg, 65%, white solid.1H NMR (500 MHz,CDCl3) 5 8.30 - 8.26 (m, 1H), 8.18 (dd, . / = 8.7, 2.2 Hz, 1H), 7.09 (d, . / = 8.8 Hz, 1H), 5.50 (s,1H), 4.17 (t, J = 6.6 Hz, 2H), 3.93 - 3.70 (m, 3H), 3.63 - 3.45 (m, 1H), 3.33 (s, 3H), 2.46 - 2.31 (m, 2H), 2.29 - 2.16 (m, 2H), 1.81 - 1.68 (m, 7H), 1.61 - 1.51 (m, 1H), 1.34 - 1.15 (m, 4H), 1.04 - 0.95 (m, 2H).13C NMR (126 MHz, CDC13) 8 182.3, 167.3, 159.3, 158.4, 132.4, 126.7, 123.2 (q, 272.5 Hz), 118.4, 112.9, 74.4, 67.1, 58.8, 54.4, 45.3, 36.1, 34.3, 33.1, 31.1, 26.5,26.2, 24.0. HRMS: (ESI) [M+H]+calc, for C25H33F3N5O4+524.2479, observed, 524.2459.[000215] Example 40: (S)-amino(2-(5-(4-octylphenyl)-l,2,4-oxadiazol-3-yl)pyrrolidin-l- yl)methaniminium (40)[000217] Synthesized according to the following protocol: DIEA (1.8 equiv) was added to a solution of 4-octylbenzoic acid (1 equiv), and tert-butyl (5)-2-(A- hydroxycarbamimidoyl)pyrrolidine-l -carboxylate (1.2 equiv) in DMF (0.2 M solution). HCTU (1.5 equiv) was then added to the resulting mixture at rt and stirred at 80 °C for 18 h. The solution was partitioned between ethyl acetate and water. The organic layer was collected and washed twice with saturated LiBr. The aqueous solution was then back extracted with ethyl acetate. The organic layers were then combined and washed with saturated NaHCCh and brine, dried over Na2SO4, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography on silica gel to yield the desired product (19% yield). Then, to a solution of di chloromethane (0.2 M) and tert-butyl (S)-2-(5-(4-octylphenyl)-l,2,4-oxadiazol-3-yl)pyrrolidine-l-carboxylate (1.0 equiv), 1.0 M TFA (10 equiv) was added and then allowed to stir at room temperature for 16 hours. The reaction was concentrated in vacuo followed by addition of diethyl ether to promote TFA salt precipitation. Once the desired intermediate was precipitated as a white solid, the diethyl ether was removed via vacuum to afford the corresponding product as a TFA salt in good yield (89%). The product was then dissolved in acetonitrile (0.2 M solution) before the addition of DIEA (3 equiv) and (Z)-tert-butyl (((tert-butoxycarbonyl)imino)-(lH-pyrazol-l-yl)methyl)carbamate (1.05 equiv). The solution was stirred at room temperature for 72 hours. The solvent was then removed under reduced pressure, and the resulting colorless residue was purified by flash column chromatography over silica gel to yield (S)-(((tert-butoxycarbonyl)amino)(2-(5-(4-octylphenyl)- l,2,4-oxadiazol-3-yl)pyrrolidin-l-yl)methylene)carbamate (19% yield). Then, hydrochloric acid gas was bubbled through a solution of the Boc-protected guanidine in methanol for 5 minutes. Thereaction mixture was concentrated under reduced pressure and triturated with diethyl ether to yield the corresponding free amine hydrochloride salt, which was further purified by trituration with diethyl ether to afford (S)-5-(4-octylphenyl)-3-(pyrrolidin-2-yl)-l,2,4-oxadiazole. 28% yield, white solid.1HNMR (400 MHz, CD3OD) δ 8.05 (d, J = 6.9 Hz, 2H), 7.43 (d, J = 7.2 Hz, 2H), 5.29 (d, J = 7.3 Hz, 1H), 3.74-3.69 (m, 1H), 3.65-3.53 (m, 1H), 2.73 (t, J = 7.8 Hz, 2H), 2.52-2.39 (m, 2H), 2.19 (s, 2H), 1.70-1.63 (m, 2H), 1.32 (d, J = 20 Hz, 10H), 0.93-0.86 (m, 3H);13C NMR (126 MHz, CD3OD) δ 178.2, 171.1, 156.9, 150.6, 130.5, 129.2, 122.4, 56.3, 37.0, 33.0, 32.4, 32.3, 30.5, 30.4, 30.3, 24.1, 23.7, 14.4; HRMS: (ESI) [M+H]+calc, for C21H32N5O [M+H]+370.2606, observed, 370.2617.[000218] Embodiments of the invention include preparation of one or more pro-drug from (S)-amino(2-(5-(4-octylphenyl)-l,2,4-oxadiazol-3-yl)pyrrolidin-l-yl)methaniminium (40), such as a pro-drug comprising any R-group shown in Table 2, using any procedure disclosed herein.[000219] In any of the examples disclosed herein, it is to be understood that the N and O atoms in the 1,2,4-oxadiazole ring are not limited to N and O, and can be independently selected from any of C, N, NH, O and S (i. e., Y1, Y2, Y3for formula I), and are not limited to the specific positions shown, thus can be present at any of the 1, 2, and 4 positions.[000220] Definitions[000221] As used herein, “treating” or “treatment” means complete cure or incomplete cure, or it means that the symptoms of the underlying disease or associated conditions are at least reduced and / or delayed, and / or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced, delayed and / or eliminated. It is understood that reduced or delayed, as used in this context, means relative to the state of the untreated disease, including the molecular state of the untreated disease, not just the physiological state of the untreated disease.[000222] The term “effective amount” refers to an amount that is sufficient to affect treatment, as defined below, when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending upon the patient being treated, the weight and age of the patient, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.[000223] In embodiments, the pharmaceutical compositions may be administered in either single or multiple doses by oral administration. Administration may be by way of any one or more of capsule, tablet, gel, spray, drops, solution, suspensions, syrups, or the like.[000224] The term “about” used herein in the context of quantitative measurements means the indicated amount ±10%. For example, with a ±10% range, “about 5 mg” can mean 4.5-5.5 mg.[000225] As used herein, “pharmaceutical compositions” include one or more compound according to formula (I) and, optionally, one or more pharmaceutically acceptable excipients or carriers including but not limited to, inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers, disintegrants, lubricants, binders, glidants, adjuvants, and combinations thereof. The present invention has been described with reference to particular embodiments having various features. In light of the disclosure provided above, it will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that the disclosed features may be used singularly, in any combination, or omitted based on the requirements and specifications of a given application or design. When an embodiment refers to “comprising” certain features, it is to be understood that the embodiments can alternatively “consist of’ or “consist essentially of’ any one or more of the features. Any of the methods disclosed herein can be used with any of the compositions disclosed herein or with any other compositions. Likewise, any of the disclosed compositions can be used with any of the methods disclosed herein or with any other methods. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.[000226] It is noted in particular that where a range of values is provided in this specification, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range as well. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is intended that the specification and examples be considered as exemplary in nature and that variations that do not depart from the essence of the invention fall within the scope of the invention. Further, all of the references cited in this disclosure are each individually incorporated by reference herein in their entireties and as such are intended to providean efficient way of supplementing the enabling disclosure of this invention as well as provide background detailing the level of ordinary skill in the art.

Claims

CLAIMS1. A compound or pharmaceutically acceptable salt thereof according to formula (I):wherein:X is phenyl, indolyl, or naphthyl;R1is independently selected from the group consisting of H, OH, -(Ci-Csjalkyl, halo, -(Ci-C6)haloalkyl, NH2, and CN; or R1, if bound to adjacent carbon atoms, in combination with the existing carbon-carbon bond represent a double bond between the adjacent carbon atoms;W is CH2, 0, or NH;V is selected from the group consisting of H, (Ci-Cio)alkyl, (C2-Ci2)alkenyl, -(Ci- Cio)alkyl-(C6-Cio)aryl, -(C2-Ci2)alkenyl-(C6-Cio)aryl, -(Ci-Cio)alkyl-(C6-Cio)aryl-(Ci- Ciojalkyl, -(Ci-Cio)alkyl-(C3-Cs)cycloalkyl, -(Ci-Cio)alkyl-heterocyclyl containing from 1 to 3 ring heteroatoms selected from N, 0, and S; wherein any aryl is optionally fused to (C6-Cio)aryl, (Cs-Csjcycloalkyl, or heterocyclyl containing from 1 to 3 ring heteroatoms selected from N, 0, and S; and / or wherein any alkyl, alkenyl, cycloalkyl, heterocyclyl, or aryl is optionally substituted by 1-4 substituents independently selected from the group consisting of F, Cl, Br, (Ci-Ce)alkyl, -O-(Ci-Ce)alkyl, (C2-C6)alkenyl, (Ci-Cejhaloalkyl, (Cg-Cio)aryl, and CN;Y1, Y2, and Y3are independently selected from C, N, NH, 0, and S; m=0, 1, or 2;T is independently selected from the group consisting of C=0, S02, CN,0, or NH;n=0, 1, 2, or 3;U can be present or absent, but when present is independently selected from the group consisting of C, 0, N, and S;R2and R3can be present or absent, but when present are independently selected from the group consisting of H, C=0, -C=0(Ci-Cio)alkyl, (Ci-Cio)alkyl, (Ci- Cs)cycloalkyl, (Ci-Cg)haloalkyl, (Cg-Cio)aryl, (C2-Ci2)alkenyl, -(Ci-Cio)alkyl-(Cg- Cio)aryl, -(C2-Ci2)alkenyl-(C6-Cio)aryl, -(Ci-Cio)alkyl-(Cg-Cio)aryl-(Ci-Cio)alkyl, -(Ci- Cio)alkyl-(C3- Cs)cycloalkyl, -(Ci-Cio)alkyl-heterocyclyl containing from 1 to 3 ring heteroatoms selected from N, 0, and S; wherein any aryl is optionally fused to (Cg-Cio)aryl, (C3-Cs)cycloalkyl, or heterocyclyl containing from 1 to 3 ring heteroatoms selected from N, 0, and S; and / or wherein any alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, R2or R3are optionally substituted by 1-4 substituents independently selected from the group consisting of F, Cl, Br, (Ci-C6)alkyl, -O-(Ci- Cg)alkyl, -N-(Ci-Cg)alkyl, (C2-Cg)alkenyl, (Ci-Cg)haloalkyl, (Cg-Cio)aryl, and CN.

2. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein: Y1is N, Y2is N, and Y3is O.

3. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein:Y1is N, Y2is O, and Y3is N.

4. A compound or pharmaceutically acceptable salt thereof, wherein the compound is one of5. A pharmaceutical composition comprising one or more compound or pharmaceutically acceptable salt thereof of any of claims 1-4 and one or more pharmaceutically acceptable excipient.

6. A method of treating a disease in a patient comprising: administering to a subject a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof of claim 5.

7. The method of claim 6, wherein the disease is selected from one or more of autoimmune disease such as multiple sclerosis, type I diabetes, inflammatory bowel diseases including Crohn’s disease and ulcerative colitis, Grave’s disease, Addison’s disease, dermatomyositis, myasthenia gravis, systemic lupus erythematosus, scleroderma, psoriasis, atopic dermatitis, or a fibrotic disease (such as chronic kidney disease, acute kidney disease, or idiopathic pulmonary fibrosis).

8. A method of inhibiting a sphingosine kinase of a patient, comprising: administering to the patient a compound, pharmaceutically acceptable salt thereof, or pharmaceutical composition of claim 5.

9. The method of claim 8, wherein the sphingosine kinase is selected from sphingosine kinase 1, sphingosine kinase 2, or a combination thereof.

10. The method of claim 8, wherein the sphingosine kinase is sphingosine kinase 2.

11. The method of claim 10, further comprising selectively inhibiting sphingosine kinase 2 relative to sphingosine kinase 1.

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