Discovery of a Small Side Cavity in Sphingosine Kinase 2 that Enhances Inhibitor Potency and Selectivity

Abstract

The sphingosine-1-phosphate (S1P) signaling pathway is an attractive drug target due to its involvement in immune cell chemotaxis and vascular integrity. The formation of S1P is catalyzed by sphingosine kinase 1 or 2 (SphK1 or SphK2) from sphingosine (Sph) and ATP. Inhibition of SphK1 and 2 to attenuate levels of S1P has been reported to be efficacious in animal models of diseases such as cancer, sickle cell disease and renal fibrosis. While inhibitors of both SphKs have been reported, improvements in potency and selectivity are still needed. Towards that end, we performed a structure-activity relationship profiling of 8 (SLM6031434) and discovered a heretofore unrecognized side cavity that increased inhibitor potency toward SphK2. Interrogating this region revealed that relatively small hydrophobic moieties are preferred with 10 being the most potent SphK2 selective inhibitor (K_i = 89 nM, 73-fold SphK2 selective) with validated in vivo activity.

Graphical Abstract

INTRODUCTION

Over the last two decades, sphingolipid signaling has emerged as an attractive candidate for drug development due to its involvement in a wide range of cellular processes. One sphingolipid in particular, sphingosine 1-phosphate (S1P), has garnered attention due to its implication in pathologic processes including cancer,^1–4 sickle cell disease,^5,6 renal fibrosis,^7–9 and autoimmunity.^10,11 S1P circulates at low micromolar concentrations in the blood of mammals and perhaps all vertebrates.¹² S1P is produced solely via ATP-dependent phosphorylation of sphingosine (Sph) by sphingosine kinases (SphK1 and SphK2) (Figure 1). The biologic roles of both enzymes are, to a certain extent, redundant in that mice lacking either isoform are viable, fertile and phenotypically unremarkable. However, elimination of both SphK1 and 2 is embryonically lethal at about day E13.5.^13,14 While both isoforms catalyze the conversion of Sph to S1P, the two enzymes differ in cellular localization and degree of substrate selectivity. SphK1 is predominantly located in the cytosol while SphK2 is found in the nucleus, mitochondria, and endoplasmic reticulum. The association of S1P with a wide array of cellular processes and diseases is, in part, due to its acting as both an intra- and extra-cellular signaling molecule. Extracellularly, S1P is a high affinity ligand for five G-protein coupled receptors (S1P_1–5) resulting in a downstream signaling cascade leading to cellular migration and survival.^3,15,16 In contrast, intracellular S1P is reported to interact with pathways influencing cellular functions ranging from cell arrest and apoptosis to proliferation and survival.¹⁷ In one such example, Laviad et al. reported findings of intracellular S1P acting as a noncompetitive inhibitor towards the pro-apoptotic protein ceramide synthase 2 (CerS2), thus conferring pro-survival effects.¹⁸ Conversely, studies reported by Chipuk et al. demonstrated that mitochondrial S1P can interact with Bcl-2 homologous antagonist killer (BAK) protein, resulting in an increase in mitochondrial membrane permeabilization. Subsequent elevated permeabilization leads to the release of the pro-apoptotic messenger cytochrome c into the cytosol inducing apoptosis.¹⁹ From a pharmaceutical viewpoint, S1P signaling and synthesis represent interesting targets to manipulate and evoke a broad range of cellular responses.

Figure 1.

The Sph/S1P equilibrium. Interconversion of Sph to S1P is catalyzed by SphK while the reverse is achieved by S1P phosphatase.

Recently, our laboratories as well as Pyne et al. disclosed findings on topographical differences between the two isoform ligand binding pockets.^20,21 The studies indicate that compared to SphK1, the Sph binding pocket of SphK2 extends deeper into the enzyme while the mid-section is narrower due to a isoleucine to valine substitution. Such differences afford the potential for ligand selectivity. A curious difference between the two isoforms is the change of blood S1P levels in response to isoform inhibition or genetic deficiency. Specifically, inhibition of SphK1 in mice or rats reduces blood S1P levels by >50%, while inhibition of SphK2 increases blood S1P concentrations by roughly 3-fold.^22–24 The rise in S1P in response to SphK2 deficiency has been ascribed to a cascade wherein Sph, which is formed at the hepatocyte surface by dephosphorylation of blood S1P, is captured by SphK2-mediated phosphorylation (Kharel et al., submitted for publication). Thus isoform-selective inhibition provides a method to either raise or lower blood S1P levels.

Since 2010, the S1P pathway has become a validated, druggable target with the FDA approval of compound 1 (fingolimod), a Sph analogue, for the treatment of relapsing remitting multiple sclerosis (rrMS) (Figure 2). Mechanistically, 1 is a prodrug that is phosphorylated by SphK2. Phosphorylated 1 ((S)-phospho-fingolimod) is then transported outside of cells via the S1P exporter, SPNS2²⁵, to act as a functional antagonist at the S1P1/3/4/5 receptors, triggering S1P receptor internalization and degradation. Specifically, the absence of the cell surface S1P1 receptor hinders the ability of naïve and central memory T lymphocytes to follow the lymph S1P concentration gradient out of secondary lymphoid tissues into the lymph and thence to the blood, ultimately resulting in therapeutic immunosuppression for patients with rrMS. More recently, the FDA granted marketing approval for 2 (siponimod) for the treatment of secondary progressive multiple sclerosis.²⁶

Figure 2.

Chemical structures of Fingolimod and Siponimod.

In addition to acting on the S1P receptors, targeting other portions of the S1P pathway such as SphK are also promising. To date, there are examples of SphK1 selective as well as SphK1/2 dual inhibitors. Compounds 3a (PF-543), 3b (Amgen 23), and 4 (SLP7111228) are three of the most potent SphK1 selective inhibitors reported and have served as valuable molecular tools to probe the S1P pathway for better understanding (Figure 3).^27–29 In an animal model for sickle cell disease (SCD), mice treated with 3a exhibited reduced blood and plasma S1P levels resulting in decreased erythrocyte sickling.⁵ In a separate study, rats dosed with 10 mg/kg of 4 displayed decreased blood S1P levels by about 80%. Further evaluation of 4 exhibited favorable in vivo stability, low nanomolar potency, and high (>200 fold) selectivity for SphK1. As is the case for SphK1 selective inhibitors, progress has been made to develop SphK1/2 dual inhibitors. A strategy employed by Pitson et al. utilizes both the substrate and ATP binding sites to afford compound 5 (MP-A08) with measured human SphK1 and SphK2 K_i values of 27 ± 3 μM and 6.9 ± 0.8 μM, respectively.³⁰ Another dual inhibitor is compound 6 (27a) developed by Schnute and coworkers at Pfizer (Figure 3).³¹ Inhibitor 6 exhibits an IC₅₀ of <1.7 nM for both kinase isoforms. Further computational docking revealed the phenyl ether moiety in 6 potentially allows for favorable rotation inside the enzyme binding pocket. In this manner, 6 avoids the deep hydrophobic pocket found in the Sph active site and instead participates in strong π−hydrophobic or π−π interactions between the phenyl substituent and F259.

Figure 3.

Select structures of SphK1, dual SphK1/2, and SphK2 inhibitors.

In contrast, highly potent (<100 nM) and selective (>100-fold) SphK2 inhibitors are scarce, thus prompting our interest in expanding the known chemical toolkit. Two early generation compounds designed by our group are 7 (SLR080811) and 8 (SLM6031434) (Figure 3).^23,22 Both of these guanidine-based inhibitors possess moderate selectivity, low micromolar SphK2 potency, and ability to lower cellular S1P levels in vitro with measured mouse SphK2 K_i values of 1.3 μM and 0.4 μM, respectively.²² Additionally, compound 8 treatment in mice was successful in recapitulating the inverse relationship observed with SphK2 inhibition or genetic deficiency and an increase in blood S1P concentration. Herein we report the development, synthesis, and biological evaluation of, to the best of our knowledge, the most potent SphK2 selective inhibitor with in vivo activity described to date.

RESULTS AND DISCUSSION

Inhibitor Design.

Previous work in our group was committed to the development of novel and potent SphK2 selective inhibitors, leading to the discovery of 7, a second generation SphK2 inhibitor. Evaluation of 7 revealed a 9-fold selectivity for the SphK2 over SphK1 with a SphK2 K_i value of 1.3 μM (12 μM for SphK1). Structurally, inhibitor 7 possesses an octylphenyl “tail” attached to a 1,2,4-oxadiazole linker, followed by a guanylated pyrrolidine head group. Molecular modeling studies conducted with similar SphK inhibitors possessing a guanidine head group predict that the guanidine moiety in our scaffold hydrogen bonds to catalytic amino acid residue Asp211 flanking the ATP active site when docked in the Sph binding pocket of SphK2.^33,34 Structure-activity relationship (SAR) studies conducted by Congdon et al. on the scaffold of 7 established that deletion of the aryl portion of the octylbenzene tail resulted in a significantly diminished inhibition towards SphK2, demonstrating that the aryl ring is essential for inhibitor efficacy.²¹ Further SAR experiments focused on the tail portion led to the development of inhibitors 8 and 9 (SLM6031422) (Table 1). Both analogues retain the 1,2,4-oxadiazole linker and guanylated pyrrolidine head group but with an additional ether linkage to the octylbenzene tail. In addition, 8 possesses a trifluoromethyl moiety at the 3-position of the internal aryl ring. Surprisingly, the trifluoromethyl addition in compound 8 imparted improved selectivity (50-fold) and potency (0.4 μM) for SphK2. In contrast, compound 9 exhibited reduced potency towards both SphK1 and SphK2 compared to 7. These results highlight the significance of the trifluoromethyl group in SphK2 selective binding.

Table 1.

Inhibitory activity of select compounds represented as % inhibition of SphK1 and SphK2^a

Compound	Structure	% hSphK1 inhibition (1.0 μM)	% hSphK2 inhibition (1.0 μM)	% hSphK2 inhibition (0.3 μM)
8		6 ± 5	80 ± 2	51 ± 4
9		11 ± 3	8 ± 2	3 ± 1
10		20 ± 1	77 ± 3	65 ± 3

^aSphK inhibition is presented as % control (no inhibitor added). Recombinant human SphK1 or SphK2 were isolated from cell lysates. Enzyme inhibition was measured with 5 μM (SphK1) or 10 μM (SphK2) sphingosine and 250 μM γ-[³²P] ATP. Compounds were assayed at 1.0 and 0.3 μM in triplicate.

The Sph binding site of SphKs can be divided up into three regions as defined by Worrell et al. They are as follows: (1) the head region adjacent to the ATP binding site, (2) the hydrophobic core region, and (3) the tail region.³⁴ Molecular docking of 8 and 9 demonstrate different ligand orientations in the Sph binding site of SphK2, giving insight into the role and influence the trifluoromethyl group has in regard to SphK2 selectivity (Figure 4). Compound 8 docks in the Sph binding pocket of SphK2 in a position that indicates more interactions in the tail region of the binding site, as influenced by the hydrophobic core of the pocket, in particular, the space for the trifluoromethyl group to sit between hydrophobic residues Phe548, Leu544, and Leu547 (Figure 4A, B). This positioning and space at helix α8 can be accommodated in SphK2, whereas in docked poses of 9 (Figure 4C, D) in SphK2, or docked poses of 8 in SphK1 (see supporting information), this is not observed. In comparison, compound 9 is positioned in the Sph binding site of SphK2 in the traditional “J-shape” conformation that has been observed in SphK1 structures crystallized with various inhibitors.^35,36 Additionally, interactions of 9 with residues in the head region near the ATP binding site are observed. These interactions are at the threshold for hydrogen bonding and strong electrostatic interactions, indicating a potentially weak and non-selective inhibitor based on previous docking studies.^33,34 The unique binding mode of 8 maximizes interactions in the hydrophobic core and tail regions within the Sph binding site of SphK2, a phenomenon not observed in SphK1 (see supporting information). There are small but significant differences between the Sph binding site residues of SphK1 versus SphK2 with variations Ile174 to Val304 in the hydrophobic core region and Phe288 to Cys533 in the tail region, respectively.³⁴ With SphK2 having the smaller amino acid, valine, in the hydrophobic core of the binding site, the trifluoromethyl group can favorably interact with the residues (Phe548, Leu544, and Leu547) that form a side cavity within the Sph binding pocket of SphK2. This interaction in the side cavity is not tolerated in SphK1 with the presence of the larger isoleucine residue. Additionally, the presence of the smaller cysteine residue in the tail region of SphK2 grants access for our inhibitors to migrate deeper towards the tail of the binding pocket which, in comparison, is blocked by Phe288 in SphK1, resulting in restricted inhibitor docking (see supporting information).

Figure 4.

Comparison of the docked poses of 8 (A, B), 9 (C, D), and 10 (E, F) in a homology model of hSphK2 (PDB ID: 3VZB used as a starting template). Key residues in the binding pocket are represented by grey sticks and are labeled, ATP is shown in orange and colored by element. The SphK2 protein structure is depicted in grey cartoon. Distances between interacting atoms are shown as dashed lines. Inhibitors are shown as stick and colored by element, with the carbon atom indicating inhibitor (8 – blue, 9 – teal, 10 – cyan). Panels B, D, and F show the inhibitor as Van der Waals radii in dots to represent volume occupancy of the inhibitors.

Further SAR experiments conducted in our group with the scaffold of 8 led to the development of molecule 10 (SLM6071469) (Table 1). This inhibitor replaces the octyloxy benzene moiety for a shorter, less flexible 4-trifluoromethyl benzyl tail that, when assessed in vitro, exhibited comparable activity to 8. Molecular docking of 10 in the Sph binding site of SphK2 demonstrates a similar pose as 8 (Figure 4E, F). These results highlight the superiority of the 4-trifluoromethylbenzyl tail with significantly reduced rotatable bonds, which allows favorable hydrophobic interactions at key residues in the tail region of the binding site. These presumably lead to the observed comparable inhibition towards SphK2. Further evaluation with SphK2 illustrated that 1 μM of 10 was successful in inhibiting SphK2 activity by roughly 77%, albeit with decreased SphK2 selectivity, and was more potent than 8 at lower (0.3 μM) concentrations (Table 1). In this study, we probed the importance of substituents spanning the internal phenyl ring and the potential presence of an unexplored side cavity within the Sph binding pocket of SphK2. A wide range of alkyl and aryl substituents were attached and examined for their effect on SphK2 inhibition.

Chemistry.

To investigate substitutions on the internal aryl ring of the 10 scaffold, analogues 15a-q, and 15s were synthesized containing various alkyl and aryl moieties at the 2- and 3-positions as shown in Scheme 1. Intermediates 11a-e were synthesized in a microwave reactor via two different Suzuki-Miyaura coupling conditions using 3-bromo-4-hydroxybenzonitrile (11g) and various boronic acids. Compounds 11f-o are available for purchase from commercial sources. Allylation of 4-hydroxybenzonitrile (11f) with allyl bromide and potassium carbonate was performed followed by microwave-assisted Claisen rearrangement to afford compound 11p. Subsequent hydrogenation of the allyl group with Pd/C in the presence of tetrahydroxydiboron afforded the propyl analog 11q.³⁷ Compound 11r is available for purchase from commercial sources. With the requisite materials in hand, derivatives 11a-r were reacted with 4-(trifluoromethyl)benzyl bromide and potassium carbonate at 80 °C in acetonitrile to afford intermediates 12a-r. Subsequently, a mixture of hydroxylamine hydrochloride, triethylamine (TEA) and 12a-r were refluxed in ethanol to generate amidoxime intermediates 13a-r. Afterwards, 13a-r were dissolved in DMF and heated to 100 °C in the presence of Boc-L-proline, HCTU and Hünig’s base to afford 1,2,4-oxadiazole intermediates 14a-r. Synthesis of 14s was performed via a Sonogashira coupling reaction with 14g and TMS-acetylene, followed by removal of the TMS group using TBAF and subsequent hydrogenation. Lastly, Boc deprotection of compounds 14a-s was completed using trifluoroacetic acid (TFA), which was followed by reaction with N,N’-di-Boc-1H-pyrazole-1-carboxamidine and Hünig’s base to install the guanidine moiety. Removal of the Boc protecting groups was performed with HCl in methanol to provide the desired analogues 15a-q, 15s, and 10 as HCl salts (see Table 2 for structures).

Scheme 1.

Synthesis of Analogues 15a–q, 15s, and 10^a

^aReagents and conditions: (a) boronic acid derivative, Cs2CO3, PdCl2(dppf), DMF, 100 °C, microwave, 60 min, 52–57%; (b) boronic acid derivative, K2PO4H, Pd(OAc)2, P(Cy)3, tol/H2O, 100 °C, 72 h, 50%; (c) allyl bromide, K2CO3, CH3CN, 80 °C, 4–6 h, 92%; (d) neat, 200 °C, microwave, 20 min, 76%; (e) Pd/C, B2(OH)4, H2O, CH2Cl2, rt, 18 h, 85%; (f) 4-(trifluoromethyl)benzyl bromide, K2CO3, CH3CN, 80 °C, 4–6 h, 48–99%; (g) NH2OH•HCl, Et3N, EtOH, reflux, 3 h, 47–96%; (h) Boc-L-proline, DIEA, HCTU•PF6, DMF, 100–110 °C, 18 h, 27–65%; (i) Et3N, CuI, TMS-acetylene, Pd(PPh)2Cl2, THF, reflux, 48 h, 74%; (j) TBAF, THF, rt, 3 h, 100%; (k) H2 (g), Pd/C, EtOH, rt, 18 h, 45%. (l) TFA, CH2Cl2, 3–12 h, 90–99%; (m) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN, 50 °C, microwave, 3 h, 47–96%; (n) HCl (g), MeOH, rt, 15 min, 90–99%.

Table 2.

Activity of SphK2 inhibitors represented as % inhibition of the enzyme.^a

Cmpd	R	% SphK2 inhibition (0.3 μM)	Cmpd	R	% SphK2 inhibition (0.3 μM)
10		65 ± 3	15n		33 ± 4
15f		2 ± 0.2	15k		15 ± 1
15a		28 ± 1	15h		23 ± 2
15b		31 ± 1.9	20		3 ± 0.4
15c		8 ± 0.8	15s		42 ± 4
15d		12 ± 1	15q		60 ± 4
15g		24 ± 3	15p		59 ± 4
15i		33 ± 2	27		67 ± 5
15j		23 ± 2	15e		52 ± 6
15l		40 ± 4	15o		66 ± 7
15m		35 ± 3

^aSphK inhibition is presented as % control (no inhibitor added). Recombinant human SphK2 was isolated from a cell lysate. Enzyme activity was measured with 10 μM (SphK2), sphingosine and 250 μM γ-[³²P] ATP. Compounds were assayed at 0.3 μM in triplicate.

Synthesis of methyl analogue 20 is outlined as shown in Scheme 2. Execution of a nucleophilic aromatic substitution reaction of 4-(trifluoromethyl)benzyl alcohol with commercially available 4-fluoro-3-methylbenzonitrile (16) and sodium hydride afforded benzonitrile intermediate 17. Next, a mixture of 17, hydroxylamine hydrochloride, and TEA in ethanol was heated to generate compound 18. Afterwards, HCTU coupling with Boc-L-proline, Boc deprotection, installation of the guanidine group and final deprotection yielded the desired analogue 20 as an HCl salt.

Scheme 2.

Synthesis of Analogue 20^a

^aReagents and conditions: (a) 4-(trifluoromethyl)benzyl alcohol, NaH, DMF, 0 °C-rt, 18 h, 54%; (b) NH2OH•HCl, Et3N, EtOH, reflux, 3 h, 89%; (c) Boc-L-proline, DIEA, HCTU•PF6, DMF, 100 °C, 18 h, 36%; (d) TFA, CH2Cl2, 12 h, 90%; (e) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN, 50 °C, microwave, 3 h, 86%; (f) HCl (g), MeOH, rt, 15 min, 90%.

Synthesis of isopropyl analogue 27 is shown in Scheme 3. Intermediate 22 was produced via the nucleophilic substitution of commercially available 3-acetyl-4-hydroxybenzonitrile (21) using 4-(trifluoromethyl)benzyl bromide and potassium carbonate. Next, reduction of the acetyl moiety was completed through a Wittig reaction using n-butyl lithium and methyltriphenylphosphonium bromide dissolved in THF and cooled to 0 °C for eighteen hours to generate compound 23. Next, refluxing 23 in ethanol for three hours in the presence of hydroxylamine hydrochloride and TEA generated amidoxime intermediate 24. Following the sequence of oxadiazole formation, hydrogenation, Boc deprotection, installation of guanidine and final removal of Boc groups produced derivative 27.

Scheme 3.

Synthesis of Analogue 27^a

^aReagents and conditions: (a) 4-(trifluoromethyl)benzyl bromide, K2CO3, CH3CN, 80 °C, 6 h, 97%; (b) n-BuLi, CH3P(Ph)3Br, THF, 0 °C-rt, 18 h, 59%; (c) NH2OH•HCl, Et3N, EtOH, reflux, 3 h, 90%; (d) Boc-L-proline, DIEA, HCTU•PF6, DMF, 100 °C, 18 h, 67%; (e) H2 (g), Pd/C, EtOH, rt, 18 h, 72%; (f) TFA, CH2Cl2, 12 h, 90%; (g) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN, 50 °C, microwave, 3 h, 62%; (h) HCl (g), MeOH, rt, 15 min, 90%.

Structure-Activity Relationship Studies and Biologic Evaluation of Derivatives.

The goal of this SAR was to improve SphK2 inhibition observed with 10 and probe the significance of substitutions on the internal phenyl ring with respect to SphK2 inhibition and selectivity. A library of compounds with various substituents spanning the 2-, 3- and 5-positions of the internal phenyl ring were synthesized and assayed according to a previously described protocol.^29,38 This assay, which utilizes recombinant SphKs and γ-[³²P]ATP, was used as a preliminary screen to identify inhibitors suitable for further evaluation. The compounds were assayed at 0.3 μM. To confirm the advantage of substituents attached to the 3-position of the internal phenyl ring, 15f was synthesized to represent the minimal scaffold with no substituents attached (Table 2). As expected, removal of the CF₃ group resulted in little to no inhibitory activity towards SphK2. Compounds 15a-d were designed to replace the relatively small CF₃ moiety for a much bulkier aryl substituent to probe the size of any potential docking space, as well as potential π-π stacking or hydrophobic interactions with nearby residues Phe-548 and His-556. Substitution with a phenyl ring (15a) resulted in poor SphK2 inhibition. Likewise, decoration of the phenyl substituent (15b-c) to exploit potential hydrophobic interactions, also led to poor inhibition of SphK2. Compound 15d was synthesized to mimic 10, but with an additional phenyl ring in between the scaffold backbone and CF₃. Our rationale was that the added phenyl linker could potentially extend the CF₃ moiety deeper into the previously unexplored side cavity leading to increased ligand affinity. However, this increase in steric bulk resulted in a significant loss of efficacy compared to 10. These results suggest that larger substituents sterically clashed inside that portion of the active site, directing us towards smaller moieties. Thus, halogen substituents at the 3-carbon were explored with compounds 15g and 15i-j. The identity of the halogen seemed to be important with the most successful being the chloro variant (15i), inhibiting normal SphK2 activity by about 33%. Compared to inhibitor 15i, it was observed that the fluoro (15j) and bromo (15g) derivatives did not have as large of an inhibitory impact towards SphK2. We next investigated the effect of migrating substituents over to the 2-position of the internal phenyl ring. However, compared to 10, both the 2-chloro (15l) and 2-CF₃ (15m) moieties had minimal impact on SphK2 inhibition. We also examined the effect of the electronics on the phenyl ring with electron withdrawing (15n) or donating (15k) groups. While the nitro derivative 15n was roughly twice as effective as methoxy variant 15k with inhibiting SphK2, neither was improved compared to 10. Next, the impact of 3,5-disubstitution was determined with compound 15h. Unfortunately, addition of a second moiety on our scaffold resulted in poor inhibition of SphK2. Taken together, our data indicate that monosubstitution at the 3-position of the aryl ring is conducive to further structure-activity relationship profiling. Thus, alkyl substituents were synthesized to probe the size of this potential side cavity within the binding site. Replacing the trifluoromethyl group with a smaller methyl (20) led to a significant decrease in potency while the slightly larger ethyl (15s) substituent had modest SphK2 inhibitory activity. To fine-tune the size of the side cavity, we synthesized compounds containing propyl (15q), allyl (15p), isopropyl (27), cyclopropyl (15e), and tert-butyl (15o) groups. Collectively, these moieties had similar inhibitory activity against SphK2 along with 10. Nonetheless, our studies indicate that there is a bona fide side binding cavity around the central phenyl ring that can accommodate a substituent larger than methyl group, but smaller than a phenyl ring.

Considering our observations summarized in Table 2, select inhibitors that displayed moderate (>42%) or greater SphK2 inhibitory activity were carried forward for further evaluation. Thus, inhibitors were subjected to an in vitro assay that utilizes the budding yeast Saccharomyces cerevisiae as a platform for assessing inhibitors of human SphK1 and 2 (hSphK1 and 2).³⁹ In short, this assay takes advantage of the observed toxicity of excessive levels of phosphorylated long chain bases, such as S1P, towards S. cerevisiae. Rescue of a modified yeast cell line (KYA1) designed to harbor plasmids expressing either hSphK1 or 2 in the presence of select inhibitors can be accomplished in a dose dependent fashion by measuring the growth of yeast culture. In this manner, dose-response curves with our lead compound 10 demonstrated hSphK2 inhibition with an EC₅₀ of 59 nM, while also displaying a 16-fold selectivity for hSphK2 over hSphK1 (Figure 5). As shown in Table 3, the n-propyl (15q) and isopropyl (27) derivatives were the most potent inhibitors with measured EC₅₀ values of 37 nM and 32 nM respectively. Interestingly, the trifluoromethyl (10), allyl (15p), cyclopropyl (15e) and tert-butyl (15o) analogs were slightly less potent with EC₅₀ values ranging from 50 nM to 84 nM, while the ethyl derivative (15s) was the least potent (EC₅₀ = 146 nM). From our experience, there are some nuances in using the yeast cells as a secondary screening tool. For example, inhibitor concentrations higher than 3 μM result in cell death; fortunately, our compounds are effective at much lower concentrations. Furthermore, guanidine containing compounds accumulate inside the yeast cells at varying degrees, thus, generating apparent improved efficacy. Nonetheless, this assay affords a rapid and simple process for validating SphK inhibition in vitro.

Figure 5.

Rescue of growth of yeast strain KYA1 expressing hSphK1 (A) or hSphK2 (B) in the presence of inhibitor over 24 hours.

Table 3.

EC₅₀ Values of Select SphK2 Inhibitors.^a

Compound	Structure	hSphK1 EC50 (nM)	hSphK2 EC50 (nM)
27		>550 ± 85	32 ± 6
15s		>1000 ± 160	146 ± 15
15e		>800 ± 100	50 ± 11
15q		>800 ± 105	37 ± 10
10		>950 ± 65	59 ± 12
15o		>500± 45	73 ± 9
15p		>1000 ± 135	84 ± 11

^aEC₅₀ values of various compounds were calculated from the growth curves of yeast strain KYA1 encoding either hSphK1 or hSphK2 in the presence of inhibitor over 24 hours. For detailed assay conditions, see the experimental section.

The inhibitory constant (K_i) was determined for the most potent derivatives in this library.²³ As shown in Table 4, evaluation of the propyl derivative (15q) displayed good SphK2 inhibition with a K_i of 201 nM whereas the tert-butyl (15o), isopropyl (27), and cyclopropyl (15e) substituents exhibited similar K_i’s ranging from 197–186 nM. Interestingly, the trifluoromethyl analogue (10) proved to be the most potent SphK2 inhibitor in the series with a K_i of 89 nM. This result is consistent with our molecular modeling studies that indicate a side cavity within the Sph binding pocket comprised of Phe548, Leu544, and Leu547. That is, the side cavity is relatively small, hydrophobic, and can best accommodate a trifluoromethyl group due to its optimal size and hydrophobic nature. Furthermore, the selectivity of the compounds between SphK2 vs. SphK1 follows the trend of increasing size leading to poor SphK2 selectivity attributing to the fact that SphK2 has an overall smaller binding pocket than SphK1. Specifically, SphK2 selectivity decreases from CF₃ > cyclopropyl ~ isopropyl > propyl > tert-butyl. One of the key residues that lines the SphK2 substrate binding site is Val304, which is Ile174 in SphK1. It is predicted that Ile174 of SphK1 causes a steric clash with the trifluoromethyl group forcing the molecule to shift upwards and away from the ATP binding site, and as a result, the inhibitor migrates away from the side cavity (see supporting information). However, the variation of Ile174 → Val304 in SphK2 does not cause this steric clash, thus allowing the molecule to slide deeper into the binding site permitting the trifluoromethyl group to lock into the side cavity leading to improved SphK2 selectivity. Likewise, when the phenyl substituent is a tert-butyl group, the steric clash with Ile174 in SphK1 becomes even greater, sending the inhibitor towards the head of the binding pocket. However, unlike 10, the increased steric clash orients the molecule towards the ATP binding site, leading to marginal SphK1 inhibition (see supporting information).

Table 4.

K_i Values of Select SphK2 Inhibitors.^a

Cmpd	Structure	hSphK1 Ki (nM)	hSphK2 Ki (nM)	hSphK2 selectivity
15q		9600 ± 2100	201 ± 11	48
15o		6100 ± 1700	197 ± 76	31
27		3500 ± 800	192 ± 39	18
15e		5300 ± 1700	186 ± 12	28
10		6500 ± 1500	89 ± 8	73

^aInhibitory constants for recombinant enzymes were obtained by kinetic analysis of S1P 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 K_m SphK2 by the K_m of SphK1.

To assess inhibition of SphK2 in vivo, we administered C57BL/6 male mice with a single 10 mg/kg intraperitoneal (i.p.) dose of 10 and measured blood S1P levels. S1P level has proven to be an excellent pharmacodynamic marker for SphK2 inhibition.^22–24 Thus, blood S1P levels were measured at the indicated time points up to 8 hours post administration of 10 via LC-MS/MS. As shown in Figure 6, the levels of 10 in blood peaked at ~8 μM, which increased blood S1P levels by nearly two-fold after 4 hours and remained elevated for up to 6 hours. The elevation of S1P levels indicates blockade of SphK2 activity in vivo and recapitulate the inverse relationship between blood S1P levels and SphK2 inhibition. Taken together, our investigations suggest that 10 not only inhibits SphK2 in vitro using recombinant protein and in yeast, but also in vivo using C57BL/6 mice.

Figure 6.

Blood concentrations of S1P and 10 in C57BL/6 male mice after IP administration with 10 mg/kg of 10. The level of significance is indicated for S1P concentrations (*P < 0.05 and **P < 0.01) using an unpaired t-test (compared to vehicle injected control).

CONCLUSIONS

Modulating S1P signaling at the level of S1P receptors represents a clinically validated approach for managing relapsing remitting multiple sclerosis. This fact encourages investigations as to whether other nodes in the S1P signaling cascades are also drug targets. Inhibition of the S1P generative enzymes SphK1 and 2 can facilitate the decrease or increase in blood S1P levels, respectively, which may have the potential for treating other diseases such as sickle cell disease, renal fibrosis and cancer. As a result of SphK1 and its inhibitors being a subject of intense investigation, potent and SphK1 selective compounds have been developed. However, potent and SphK2 selective inhibitors are lacking. In this report, we discovered a heretofore undisclosed side cavity within the Sph binding site. Interrogating this cavity with various functional groups revealed that relatively small hydrophobic moieties (i.e., trifluoromethyl) are best accommodated. Indeed, characterization of inhibitor 10 revealed the most potent SphK2 selective inhibitor to date (K_i 89 nM, 73-fold SphK2 selective). Molecular modeling studies suggest that the trifluoromethyl group fits securely into the side cavity generated by residues Phe548, Leu544, and Leu547 imparting increased potency towards SphK2, whereas the Val304 to Ile174 variation in SphK1 causes a steric clash that forces the CF₃ group out of the side cavity. Collectively, these phenomena increase SphK2 inhibitor potency and selectivity. Overall, our investigations provide a platform for developing SphK inhibitors that may lead to compounds useful for treating diseases where sphingosine mediated signaling events have gone awry.

EXPERIMENTAL

Sphingosine Kinase Biological Assays.

The percent inhibition of SphK2 from synthesized compounds was carried forward using a previously described protocol.^29,38 Recombinant human SphK2 (10 μM) was isolated from a cell lysate and incubated with (0.3 μM) or without compound, sphingosine, and 250 μM γ-[³²P] ATP via scintillation counting. Compounds were assayed in triplicate.

The growth of recombinant yeast (Saccharomyces cerevisiae) cells encoding either hSphK1 or hSphK2 was performed according to our previously described protocol.³⁹ Briefly, yeast strain KYA1 harboring a plasmid encoding either hSphK1 or hSphK2 was selected, maintained and grown in SC-URA media with 2% glucose overnight at 30 °C. Following overnight growth media was diluted 1:100 into SC-URA media supplemented with 2% galactose and various concentrations of test inhibitor. After another incubation period of 24 – 48 hours at 30 °C, cellular growth was quantified by measuring absorbance at 600 nm.

In vivo mouse studies were conducted as follows. Groups of 8 to 10 week old male C57BL/6 mice were injected intraperitoneally (i.p.) with either vehicle or 10 mg/kg SLM6071469 (2% solution of hydroxypropyl-β-cyclodextrin, Cargill Cavitron 82004). After 0.5, 1, 2, 4, 6, and 8 hours of injection, whole blood was collected from the retro-orbital sinus and 10 μL of blood was processed for LC/MS analysis as described previously.²² 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.

Molecular Docking.

To visualize and rationalize differences in efficacy of 8, 9, 10, and 15o molecular docking was utilized to predict position and ligand-protein interactions of the inhibitors in the sphingosine binding pocket of SphK2. The SphK2 model, with ATP and Mg²⁺ bound, was generated with Molecular Operating Environment (MOE) and energy minimized as previously described.^33,34 In order to draw, display, and characterize chemical structures, substructures, and reactions for preparation in docking programs, Marvin 17.3.13, 2017 was used (ChemAxon; http://www.chemaxon.com) to create structure files that were cleaned in 3D. AutoDock Tools was used to prepare the protein and ligand files.⁴⁰ In order to perform the docking, AutoDock Vina was utilized to dock the inhibitors to SphK2, with 9 poses being created for each inhibitor.⁴¹ The grid box was set to 20 × 20 × 28 ų with grid spacing of 1.000 Å. To include all known key residues in the sphingosine binding cavity, the grid box was positioned at the approximate center of the ligand-binding cavity based on the position of sphingosine in the crystal structure (PDB ID: 3VZB).^33,35 The lowest energy docked pose for each molecule was then used to compare potential differences in ligand docking position inside the SphK2 active site.

General Material and Synthetic Procedures.

Where indicated, reactions utilizing a microwave reactor were performed with a Discover SP microwave synthesizer (CEM Corporation). 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 μm, F254 plates were utilized for all thin-layer chromatography (TLC) experiments. A Combiflash Rf purification system with flash grade silica gel, 40–63 μm was used for all column chromatography. All reported ¹H NMR spectra were measured at 400 MHz and the complementary ¹³C NMR frequency was 101 MHz. Reported ¹H NMR chemical shifts are in ppm with the solvent resonance as an internal standard (CDCl₃: 7.26 ppm; CD₃OD: 3.31 ppm; (CD₃)₂CO: 2.05 ppm). Reported ¹³C NMR chemical shifts are in ppm with the solvent resonance as the internal standard (CDCl₃: 77.16 ppm; CD₃OD: 49.00 ppm; (CD₃)₂CO: 206.26 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.

General Procedure A: Suzuki-Miyaura Coupling of Aryl Bromides with Phenyl Boronic Acids.

3-bromo-4-hydroxybenzonitrile (11g) (1.0 equiv) was dissolved in DMF (0.2 M solution) and put under a nitrogen gas atmosphere. The appropriate phenyl boronic acid (3.0 equiv) was then added followed by Cs₂CO₃ (2.0 equiv). The resulting mixture was degassed for 15 min by bubbling N₂ through solution. Next, Pd(dppf)Cl₂ (0.03 equiv) was added and the resulting mixture was heated in a microwave reactor at 100 °C for 60 min. Next, the solution was partitioned between EtOAc and LiBr aqueous solution. Using additional EtOAc, the aqueous LiBr solution was washed three times and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated via vacuum. The resulting concentrate was then purified by silica gel chromatography to yield a mixture of the major product detected by TLC as well as a minor impurity. Isolated fractions were then carried forward crude without further characterization.

General Procedure B: Williamson Ether Synthesis of Hydroxybenzonitrile Derivatives.

K₂CO₃ (4 equiv) was added to a solution of the CH₃CN (0.2 M solution) and the appropriate hydroxybenzonitrile derivative (1.0 equiv). 4-(trifluoromethyl)benzyl bromide (1.2 equiv) was then added followed by flushing the reaction container with nitrogen gas. The reaction mixture was refluxed at 80 °C for 4 – 6 hours. Afterward, the reaction was concentrated via vacuum and then purified by silica gel chromatography to yield the desired product.

General Procedure C: Synthesis of Amide-Oxime Analogs.

To a solution of ethanol (0.2 M) and the appropriate benzonitrile (1.0 equiv), hydroxylamine hydrochloride (2.0 equiv) was added followed by TEA (3.0 equiv). The reaction mixture was heated to 85 °C for 4 hours. The resulting mixture was then concentrated via vacuum and then purified by silica gel chromatography to yield the desired product.

General Procedure D: Coupling of Amide-Oxime Analogs with Boc-L-Proline.

The appropriate amide-oxime derivative (1.0 equiv) was dissolved in DMF (0.2 M solution) and put under an N₂ blanket. Boc-L-Proline (1.2 equiv) followed by Hünig’s base (1.8 equiv) and HCTU (1.2 equiv) were then added and stirred vigorously at 100 °C for 18 hours. The reaction progress was monitored by TLC. Subsequently, the resulting solution was partitioned between EtOAc and LiBr aqueous solution. Using additional EtOAc, the aqueous LiBr solution was washed three times and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated via vacuum. The resulting concentrate was purified by silica gel chromatography.

General Procedure E: Boc Deprotection of Pyrrolidine Intermediates With TFA, Guanylation of Deprotected Amines and Subsequent Boc Deprotection of Guanyl Groups with HCl (g).

To a solution of dichloromethane (0.2 M) and the appropriate Boc protected pyrrolidine (1.0 equiv), 1.0 M TFA (10 equiv) was added and allowed to stir at room temperature for 3 – 12 hours. The reaction progress was monitored by TLC. Next, the dichloromethane was removed via vacuum and replaced with diethyl ether to promote TFA salt precipitation. Once the desired product was precipitated, the diethyl ether was removed via vacuum and the resulting Boc deprotected product was carried forward crude. Thereafter, the resulting Boc deprotected product was dissolved in CH₃CN (0.2 M) and put under an N₂ atmosphere. To the reaction mixture, Hünig’s base (10 equiv) followed by N,N’-di-Boc-1H-pyrazole-1-carboxamidine (1.2 equiv) were added and the reaction mixture was heated to 50 °C inside a microwave reactor for 3 hours. The reaction mixture was then concentrated via vacuum, and the resulting concentrate was then purified by silica gel chromatography to afford bis-Boc-protected intermediates. Following silica gel chromatography, bis-Boc-protected intermediates were then dissolved in methanol (0.2 M) and bubbled with HCl gas for 2 – 5 minutes to yield the desired Boc deprotected moiety. Lastly, the organic mixture was concentrated via vacuum and triturated with diethyl ether to afford the corresponding analogue as an HCl salt.

CHARACTERIZATION

(S)-2-(3-(3-(trifluoromethyl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (10).

Synthesized by General Procedure E: 58 mg, 59%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.28 – 8.26 (m, 2H), 7.70 (q, J = 8.3 Hz, 4H), 7.44 (d, J = 9.2 Hz, 1H), 5.45 (dd, J = 8.0, 1.8 Hz, 1H), 5.42 (s, 2H), 3.78 (t, J = 9.4 Hz, 1H), 3.62 (q, J = 9.6 Hz, 1H), 2.61 – 2.44 (m, 2H), 2.28 – 2.19 (m, 1H), 2.13 – 2.02 (m, 1H); ¹³C NMR (101 MHz, CD₃OD) δ 179.28, 168.45, 159.92, 157.11, 141.91, 134.01, 131.26 (q, ²J_CF = 32.5 Hz), 128.54, 127.25 (q, ³J_CF = 5.0 Hz), 126.53 (q, ³J_CF = 3.6 Hz), 125.53 (q, ¹J_CF = 272.9 Hz), 124.63 (q, ¹J_CF = 271.9 Hz), 120.61 (q, ²J_CF = 30.4 Hz), 120.24, 115.48, 70.89, 56.48, 32.72, 24.32; ¹⁹F NMR (376 MHz, CD₃OD) δ −63.61 (s, 3F), −63.63 (s, 3F); HRMS (ESI+): calcd for C₂₂H₂₀F₆N₅O₂ [M]⁺ 500.1521; found, 500.1518.

6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-carbonitrile (12a).

Synthesized by General Procedure B: 165 mg, 91%, white solid. ¹H NMR (400 MHz, (CD₃)₂CO) δ 7.75–7.67 (m, 4H), 7.61 (d, J = 7.7 Hz, 4H), 7.45 (t, J = 7.3 Hz, 2H), 7.41 – 7.32 (m, 2H), 5.36 (s, 2H); ¹³C NMR (101 MHz, (CD₃)₂CO) 159.46, 141.93 (d, ⁴J _CF3 = 1.3 Hz), 137.23, 135.08, 134.09, 132.98, 130.34, 130.23 (q, ²J_CF3 = 32.3 Hz), 129.01, 128.66, 128.37, 126.15 (q, ³J_CF3 = 3.7 Hz), 125.16 (q, ¹J_CF3 = 271.1 Hz), 119.42, 114.47, 105.58, 70.28; HRMS (ESI+): calcd for C₂₁H₁₅F₃NO [M+H]⁺ 354.1100; found, 354.1104.

4’-fluoro-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-carbonitrile (12b).

Synthesized by General Procedure B: 151 mg, 87%, white solid. ¹H NMR (400 MHz, (CD₃)₂CO) δ 7.81–7.62 (m, 8H), 7.40 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 8.7 Hz, 2H), 5.43 (s, 2H); ¹³C NMR (101 MHz, (CD₃)₂CO) 163.37 (d, ¹J _CF = 245.8 Hz), 159.52, 142.03 (d, ⁴J _CF3 = 1.2 Hz), 135.12, 134.30, 133.51 (d, ⁴J _CF = 3.4 Hz), 132.45 (d, ³J _CF = 8.3 Hz), 131.97, 130.29 (q, ²J _CF3 = 32.0 Hz), 128.51, 126.27 (q, ³J _CF3 = 3.8 Hz), 125.17 (q, ¹J _CF3 = 271.0 Hz), 119.34, 115.84 (d, ²J _CF = 21.5 Hz), 114.61, 105.63, 70.41; HRMS (ESI+): calcd for C₂₁H₁₇F₄N₂O [M+NH₄]⁺ 389.1272; found, 389.1269.

3-(pyridin-4-yl)-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12c).

Synthesized by General Procedure B: 87 mg, 48%, yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.68 (s, 2H), 7.72–7.57 (m, 4H), 7.48–7.36 (m, 4H), 7.10 (d, J = 8.4 Hz, 1H), 5.24 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 158.43, 149.97, 143.95, 139.38 (d, ⁴J _CF3 = 1.2 Hz), 134.51, 134.47, 130.65 (q, ²J _CF3 = 32.6 Hz), 129.68, 127.02, 125.86 (q, ³J _CF3 = 3.7 Hz), 124.15, 123.97 (q, ¹J _CF3 = 271.1 Hz), 118.52, 113.36, 105.50, 69.98; HRMS (ESI+): calcd for C₂₀H₁₄F₃N₂O [M+H]⁺ 355.1053; found, 355.1083.

4’-(trifluoromethyl)-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-carbonitrile (12d).

Synthesized by General Procedure B: 361 mg, 78%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.74–7.59 (m, 8H), 7.41 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.6 Hz, 1H), 5.25 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 158.46, 139.84 (q, ⁴J _CF3 = 1.3 Hz), 139.62 (q, ⁴J _CF3 = 1.3 Hz), 134.72, 133.99, 131.11, 130.55 (q, ²J _CF3 = 32.7 Hz), 130.20 (q, ²J _CF3 = 32.6 Hz), 129.88, 127.00, 125.82 (q, ³J _CF3 = 3.7 Hz), 125.34 (q, ³J _CF3 = 3.8 Hz), 124.20 (q, ¹J _CF3 = 272.0 Hz), 124.13 (q, ¹J _CF3 = 272.0 Hz), 118.73, 113.36, 105.35, 69.93; HRMS (ESI−): calcd for C₂₂H₁₂F₆NO [M − H]⁻ 420.0829; found, 420.0807.

3-cyclopropyl-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12e).

Synthesized by General Procedure B: 83 mg, 49%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.63 (dd, J = 38.7, 8.0 Hz, 4H), 7.43 (d, J = 8.5 Hz, 1H), 7.15 (s, 1H), 6.90 (d, J = 8.5 Hz, 1H), 5.23 (s, 2H), 2.25–2.15 (m, 1H), 1.01 (q, J = 5.7 Hz, 2H), 0.69 (q, J = 5.4 Hz, 2H); ¹³C NMR (101 MHz CDCl₃) 160.45, 140.31 (d, ⁴J _CF3 = 1.3 Hz), 134.29, 131.30, 130.50 (d, ²J _CF3 = 33.0 Hz), 129.39, 127.27, 125.80 (q, ³J _CF3 = 3.8 Hz), 124.12 (q, ¹J _CF3 = 271.5 Hz), 119.45, 111.64, 104.56, 69.49, 9.69, 8.01; HRMS (ESI+): calcd for C₁₈H₁₅F₃NO [M+H]⁺ 318.1100; found, 318.1091.

4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12f).

Synthesized by General Procedure B: 270 mg, 97%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J = 8.0 Hz, 2H), 7.56 (dd, J = 8.8, 12.2 Hz, 4H), 7.02 (d, J = 8.8 Hz, 2H), 5.18 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 161.59, 139.87 (d, ⁴J _CF3 = 1.4 Hz), 134.10, 130.47 (q, ²J _CF3 = 32.8 Hz), 127.48, 125.70 (q, ³J _CF3 = 3.8 Hz), 124.01 (d, ¹J _CF3 = 272.3 Hz), 119.08, 115.58, 104.62, 69.32; HRMS (ESI+): calcd for C₁₅H₁₀F₃NO [M+H]⁺ 278.0787; found, 278.0769.

3-bromo-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12g).

Synthesized by General Procedure B: 372 mg, 98%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J = 2.0 Hz, 1H), 7.72–7.55 (m, 5H), 6.96 (d, J = 8.6 Hz, 1H), 5.27 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 158.25, 139.28 (d, ⁴J _CF3 = 1.4 Hz), 137.07, 133.21, 130.77 (q, ²J _CF3 = 32.6 Hz), 127.16, 125.93 (q, ³J _CF3 = 3.9 Hz), 124.08 (q, ¹J _CF3 = 271.2 Hz), 117.68, 113.32, 113.10, 106.09, 70.24; HRMS (ESI+): calcd for C₁₅H₁₃BrF₃N₂O [M+NH₄]⁺ 373.0158; found, 373.0162.

3,5-dimethyl-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12h).

Synthesized by General Procedure B: 209 mg, 92%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.63 (dd, J = 8.1, 39.0 Hz, 4H), 7.36 (s, 2H), 4.91 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 159.40, 140.84 (d, ⁴J _CF3 = 1.2 Hz), 133. 05, 132.81, 130.56 (q, ²J _CF3 = 32.6 Hz), 127.64, 125.73 (q, ³J _CF3 = 3.8 Hz), 124.15 (q, ¹J _CF3 = 272.3 Hz), 119.01, 108.05, 73.26, 16.45; HRMS (ESI+): calcd for C₁₇H₁₄F₃NNaO [M+Na]⁺ 328.0920; found, 328.0944.

3-chloro-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12i).

Synthesized by General Procedure B: 197 mg, 99%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.69–7.54 (m, 5H), 7.50 (dd, J = 2.0, 8.6 Hz, 1H), 7.01 (d, J = 8.7 Hz, 1H), 5.25 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 157.32, 139.28 (d, ⁴J _CF3 = 1.2 Hz), 133.76, 132.41, 130.50 (q, ²J _CF3 = 32.6 Hz), 127.14, 125.73 (q, ³J _CF3 = 3.8 Hz), 124.13, 124.00 (q, ¹J _CF3 = 272.8 Hz), 117.76, 113.56, 105.34, 70.01; HRMS (ESI−): calcd for C₁₅H₈ClF₃NO [M − H]⁻ 310.0252; found, 310.0232.

3-fluoro-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12j).

Synthesized by General Procedure B: 236 mg, 99%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (dd, J = 8.1, 38.4 Hz, 4H), 7.38 (t, J = 7.7 Hz, 2H), 7.05 (t, 8.70 Hz, 1H), 5.25 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 151.98 (d, ¹J _CF = 250.8 Hz), 150.54 (d, ²J _CF = 10.3 Hz), 139.29, 130.67 (q, ²J _CF3 = 32.6 Hz), 129.67 (d, ⁴J _CF = 4.0 Hz), 127.47, 125.79 (q, ³J _CF3 = 3.8 Hz), 123.91 (q, ¹J _CF3 = 272.5 Hz), 119.93 (d, ²J _CF = 21.2 Hz), 117.88 (d, ³J _CF = 2.5 Hz), 115.23 (d, ³J _CF = 2.2 Hz), 104.77 (d, ⁴J _CF = 8.2 Hz), 70.30; HRMS (ESI+): calcd for C₁₅H₁₀F₄NO [M + H]⁺ 296.0693; found, 296.0687.

3-methoxy-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12k).

Synthesized by General Procedure B: 229 mg, 99%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.58 (dd, J = 8.3, 36.7 Hz, 4H), 7.20 (dd, J = 1.9, 8.4 Hz, 1H), 7.10 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 5.22 (s, 2H), 3.89 (s, 3H); ¹³C NMR (101 MHz CDCl₃) 151.60, 149.74, 139.98 (d, ⁴J _CF3 = 1.4 Hz), 130.41 (q, ²J _CF3 = 32.1 Hz), 127.33, 126.22, 125.72 (q, ³J _CF3 = 3.8 Hz), 124.05 (q, ¹J _CF3 = 272.2 Hz), 119.10, 114.49, 113.32, 104.70, 70.00, 56.19; HRMS (ESI+): calcd for C₁₆H₁₃F₃NO₂ [M + H]⁺ 308.0893; found, 308.0897.

2-chloro-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12l).

Synthesized by General Procedure B: 221 mg, 99%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (dd, J = 7.9, 42.7 Hz, 5H), 7.09 (s, 1H), 6.95 (d, , J = 8.3 Hz, 1H), 5.18 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 162.10, 139.28 (d, ⁴J _CF3 = 1.3 Hz), 138.28, 135.15, 130.58 (q, ²J _CF3 = 32.6 Hz), 127.55, 125.73 (q, ³J _CF3 = 3.8 Hz), 124.03 (q, ¹J _CF3 = 272.3 Hz), 116.52, 116.28, 114.10, 105.55, 69.74; HRMS (ESI+): calcd for C₁₅H₁₀ClF₃NO [M + H]⁺ 312.0398; found, 312.0386.

2-(trifluoromethyl)-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12m).

Synthesized by General Procedure B: 270 mg, 98%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.75 (d, J = 8.7 Hz, 1H), 7.62 (dd, J = 8.1, 36.6 Hz, 4H), 7.39 (d, J = 2.4 Hz, 1H), 7.22 (dd, J = 2.4, 8.6 Hz, 1H), 5.26 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 161.51, 139.14 (q, ⁴J _CF3 = 1.4 Hz), 136.77, 134.67 (q, ²J _CF3 = 32.1 Hz), 130.72 (q, ²J _CF3 = 32.1 Hz), 127.67, 125.80 (q, ³J _CF3 = 3.5 Hz), 124.07 (q, ¹J _CF3 = 271.7 Hz), 122.19 (q, ¹J _CF3 = 272.9 Hz), 117.51, 115.82, 114.46 (q, ³J _CF3 = 4.9 Hz), 101.82 (q, ³J _CF3 = 2.0 Hz), 69.92; HRMS (ESI+): calcd for C₁₆H₁₀F₆NO [M + H]⁺ 346.0661; found, 346.0670.

3-nitro-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12n).

Synthesized by General Procedure B: 270 mg, 98%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.63 (dd, J = 7.8, 44.1 Hz, 4H), 7.21 (d, J = 8.8 Hz, 1H), 5.36 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 154.62, 138.24, 137.75, 131.95, 130.03, 127.19, 126.11 (q, ³J _CF3 = 3.6 Hz), 125.15 (q, ¹J _CF3 = 272.2 Hz), 116.72, 115.64, 110.15, 105.17, 70.85; HRMS (ESI+): calcd for C₁₅H₁₃F₃N₃O₃ [M + NH₄]⁺ 340.0904; found, 312.0916.

3-(tert-butyl)-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12o).

Synthesized by General Procedure B: 277 mg, 97%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.71 – 7.56 (m, 5H), 7.47 (dd, J = 2.1, 8.5 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 5.25 (s, 2H), 1.40 (s, 9H); ¹³C NMR (101 MHz CDCl₃) 160.47, 140.06 (q, ⁴J _CF3 = 1.2 Hz), 139.74, 131.95, 131.01, 130.40 (q, ²J _CF3 = 32.3 Hz), 127.55, 125.77 (q, ³J _CF3 = 3.8 Hz), 124.07 (q, ¹J _CF3 = 272.3 Hz), 119.68, 112.71, 104.22, 69.68, 35.15, 29.39; HRMS (ESI+): calcd for C₁₉H₁₉F₃NO [M + H]⁺ 334.1413; found, 334.1412.

3-allyl-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12p).

Synthesized by General Procedure B: 277 mg, 97%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (dd, J = 8.2, 35.4 Hz, 4H), 7.48 – 7.41 (m, 2H), 6.95 (d, J = 8.8 Hz, 1H), 6.03 – 5.91 (m, 1H), 5.20 (s, 2H), 5.16 – 5.06 (m, 2H), 3.45 (d, J = 6.3 Hz, 2H); ¹³C NMR (101 MHz CDCl₃) 159.21, 140.10 (q, ⁴J _CF3 = 1.2 Hz), 134.99, 133.35, 132.20, 130.38, 130.14 (q, ²J _CF3 = 32.4 Hz), 127.16, 125.52 (q, ³J _CF3 = 3.8 Hz), 124.02 (q, ¹J _CF3 = 272.0 Hz), 119.16, 116.83, 111.71, 104.20, 69.17, 33.82; HRMS (ESI+): calcd for C₁₈H₁₅F₃NO [M + H]⁺ 318.1100; found, 318.1121.

3-propyl-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12q).

Synthesized by General Procedure B: 525mg, 78%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (dd, J = 8.1, 43.9 Hz, 4H), 7.48 – 7.39 (m, 2H), 6.92 (d, J = 8.4 Hz, 1H), 5.20 (s, 2H), 2.67 (t, J = 7.4 Hz, 2H), 1.70 – 1.60 (m, 2H), 0.96 (t, J = 7.3 Hz, 2H); ¹³C NMR (101 MHz CDCl₃) 159.49, 140.30 (d, ⁴J _CF3 = 1.3 Hz), 133.48, 132.77, 131.84, 130.23 (q, ²J _CF3 = 32.5 Hz), 127. 08, 125.64 (q, ³J _CF3 = 3.7 Hz), 124.1 (q, ¹J _CF3 = 272.0 Hz), 119.40, 111.68, 104.10, 69.13, 31.92, 22.45, 13.84; HRMS (ESI+): calcd for C₁₈H₁₇F₃NO [M + H]⁺ 320.1257; found, 320.1259.

3-(trifluoromethyl)-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (12r).

Synthesized by General Procedure B: 200 mg, 98%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, J = 1.8 Hz, 1H), 7.79 (dd, J = 8.7, 2.1 Hz, 1H), 7.67 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.7 Hz, 2H), 7.12 (d, J=8.7 Hz, 1H), 5.32 (s, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 159.23, 138.96, 137.72, 131.70 (³ J_CF = 5.3 Hz), 130.80 (² J_CF = 30.2 Hz), 127.05, 125.95 (³ J_CF = 3.8 Hz), 123.90 (¹ J_CF = 274.7 Hz), 122.26 (¹ J_CF = 274.3 Hz), 120.66 (q,²J_CF = 33.4 Hz), 117.72, 113.91, 104.80, 70.07; HRMS (ESI+): calcd for C₁₆H₁₀F₆NO [M + H]⁺ 346.0661; found, 346.0666.

N’-hydroxy-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-carboximidamide (13a).

Synthesized by General Procedure C: 56 mg, 51%, white solid. ¹H NMR (400 MHz, (CD₃)₂CO) δ 7.75 – 7.59 (m, 8H), 7.43 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.2 Hz, 1H), 7.18 (d, J = 8.5, 1H), 5.55 (s, 2H), 5.29 (s, 2H); ¹³C NMR (101 MHz (CD₃)₂CO) 156.83, 151.90, 142.91 (d, ⁴J _CF3 = 1.3 Hz), 139.14, 131.56, 130.42, 129.98 (d, ²J _CF3 = 32.0 Hz), 128.98, 128.82, 128.33, 127.93, 127.74, 126.91, 126.07 (q, ³J _CF3 = 3.9 Hz), 125.25 (d, ¹J _CF3 = 271.7 Hz), 113.65, 70.11; HRMS (ESI+): calcd for C₂₁H₁₈F₃N₂O₂ [M+H]⁺ 387.1315; found, 387.1321.

4’-fluoro-N’-hydroxy-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-carboximidamide (13b).

Synthesized by General Procedure C: 70 mg, 64%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.91 – 7.80 (m, 1H), 7.66 – 7.41 (m, 7H), 7.18 – 7.03 (m, 3H), 5.15 (d, J = 18.6, 2H); ¹³C NMR (101 MHz CD₃OD) 171.72, 163.52 (d, ¹J _CF = 245.2 Hz), 159.33, 157.62, 155.13, 142.80 (d, ⁴J _CF3 = 1.3 Hz), 135.38 (d, ⁴J _CF = 3.4 Hz), 132.50 (d, ³J _CF = 8.1 Hz), 131.52, 129.93, 128.38, 127.88, 126.31 (q, ³J _CF3 = 3.9 Hz), 125.60 (q, ¹J _CF3 = 271.0 Hz), 115.78 (d, ²J _CF = 21.8 Hz), 114.03, 70.54; HRMS (ESI+): calcd for C₂₁H₁₇F₄N₂O₂ [M+H]⁺ 405.1221; found, 405.1206.

N’-hydroxy-3-(pyridin-4-yl)-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13c).

Synthesized by General Procedure C: 75 mg, 79%, white solid. ¹H NMR (400 MHz, (CD₃)₂CO) δ 8.95 (br s, 1H), 8.62 (d, J = 5.4 Hz, 2H), 7.82 – 7.60 (m, 8H), 7.25 (d, J = 8.3 Hz, 1H), 5.56 (s, 2H), 5.35 (s, 2H); ¹³C NMR (101 MHz (CD₃)₂CO) 156.86, 151.51, 150.44, 146.57, 142.63 (d, ⁴J _CF3 = 1.4 Hz), 130.15 (d, ²J _CF3 = 32.1 Hz), 128.67, 128.62, 128.49, 128.22, 128.03, 126.21 (q, ³J _CF3 = 3.8 Hz), 125.2 (d, ¹J _CF3 = 271.5 Hz), 125.10, 113.77, 70.26; HRMS (ESI+): calcd for C₂₀H₁₆F₃N₃O₂ [M]⁺ 387.1195; found, 387.1225.

N’-hydroxy-4’-(trifluoromethyl)-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-carboximidamide (13d).

Synthesized by General Procedure C: 290 mg, 74%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.95 – 7.59 (m, 8H), 7.50 (t, J = 7.3 Hz, 2H), 7.26 – 7.17 (m, 1H), 5.26 (d, J = 18.4 Hz, 2H); ¹³C NMR (101 MHz CD₃OD) 159.40, 157.69, 155.01, 143.41 (d, ⁴J _CF3 = 1.4 Hz), 142.75 (d, ⁴J _CF3 = 1.4 Hz), 131.30, 130.91 (q, ²J _CF3 = 32.2 Hz), 130.87, 130.22 (q, ²J _CF3 = 32.4 Hz), 129.93, 128.55, 127.53, 126.36 (q, ³J _CF3 = 3.8 Hz), 125.87 (q, ³J _CF3 = 3.8 Hz), 125.83 (d, ¹J _CF3 = 271.3 Hz), 125.63 (d, ¹J _CF3 = 271.1 Hz), 114.28, 70.77; HRMS (ESI+): calcd for C₂₂H₁₇F₆N₂O₂ [M+H]⁺ 455.1189; found, 455.1184.

3-cyclopropyl-N’-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13e).

Synthesized by General Procedure C: 73 mg, 80%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (dd, J = 8.0, 30.6 Hz, 4H), 7.37 (d, J = 8.4 Hz, 1H), 7.17 (s, 1H), 6.86 (d, J = 8.4 Hz, 1H), 5.20 (s, 2H), 4.83 (s, 2H), 2.26 – 2.19 (m, 1H), 0.96 (q, J = 5.3 Hz, 2H), 0.72 (q, J = 5.1 Hz, 2H); ¹³C NMR (101 MHz CDCl₃) 158.61, 141.23 (d, ⁴J _CF3 = 1.3 Hz), 133.06, 130.23 (d, ²J _CF3 = 32.5 Hz), 127.25, 125.70 (q, ³J _CF3 = 3.8 Hz), 125.57, 125.47, 124.17, 123.28, 111.50, 69.47, 29.85, 9.85, 7.94; HRMS (ESI+): calcd for C₁₈H₁₈F₃N₂O₂ [M + H]⁺ 351.1315; found, 351.1309.

N’-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13f).

Synthesized by General Procedure C: 73 mg, 80%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.83 – 7.53 (m, 6H), 6.99 (t, J = 11.3 Hz, 2H), 5.90 (br s, 1H), 5.15 (s, 2H) 4.84 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 168.89, 161.44, 159.85, 152.56, 140.79, 129.54, 127.50, 125.73, 115.02, 114.79, 69.29; HRMS (ESI+): calcd for C₁₅H₁₄F₃N₂O₂ [M + H]⁺ 311.1002; found, 311.1006.

3-bromo-N’-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13g).

Synthesized by General Procedure C: 250 mg, 62%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 – 7.86 (m, 1H), 7.77 – 7.51 (m, 5H), 6.97 – 6.89 (m, 1H), 5.85 (br s, 1H), 5.25 (d, J = 13.3 Hz, 2H), 4.81 (s, 2H); ¹³C NMR (101 MHz CDCl₃) 156.04, 151.52, 140.21, 139.89, 133.07, 131.24, 128.41, 127.15, 126.26, 125.82, 113.34, 112.86, 70.10; HRMS (ESI+): calcd for C₁₅H₁₃BrF₃N₂O₂ [M + H]⁺ 389.0107; found, 389.0112.

N’-hydroxy-3,5-dimethyl-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13h).

Synthesized by General Procedure C: 201 mg, 88%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.71 – 7.63 (m, 4H), 7.59 (s, 1H), 7.34 (s, 1H), 4.92 (d, J = 11.4 Hz, 2H), 2.28 (d, J = 9.0 Hz, 6H); ¹³C NMR (101 MHz CD₃OD) 172.09, 158.08, 143.41, 132.25, 131.03 (d, ²J _CF3 = 32.4 Hz), 129.67, 129.10, 127.95, 126.36, 125.63 (d, ¹J _CF3 = 272.6 Hz), 74.02, 16.62; HRMS (ESI+): calcd for C₁₇H₁₈F₃N₂O₂ [M + H]⁺ 339.1315; found, 339.1314.

3-chloro-N’-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13i).

Synthesized by General Procedure C: 193 mg, 89%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.98 – 7.62 (m, 5H), 7.52 (d, J = 8.3 Hz, 1H), 7.18 – 7.07 (m, 1H), 5.27 (d, J = 15.7 Hz, 2H); ¹³C NMR (101 MHz CD₃OD) 170.45, 157.90, 156.16, 154.02, 142.43, 130.90, 129.13, 128.54, 126.96, 126.42, 123.96, 114.66, 70.77; HRMS (ESI+): calcd for C₁₅H₁₃ClF₃N₂O₂ [M + H]⁺ 345.0612; found, 345.0610.

3-fluoro-N’-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13j).

Synthesized by General Procedure C: 243 mg, 93%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.65 (q, J = 7.6 Hz, 4H), 7.40 (q, J = 12.1 Hz, 2H), 7.22 – 7.10 (m, 1H), 5.24 (s, 1H); ¹³C NMR (101 MHz CD₃OD) 154.13 (d, ⁴J _CF = 2.3 Hz), 153.47 (d, ¹J _CF = 245.1 Hz), 148.83 (d, ²J _CF = 11.0 Hz), 142.47 (q, ⁴J _CF3 = 1.3 Hz), 131.10 (q, ²J _CF3 = 32.8 Hz), 128.80, 127.89 (d, ³J _CF = 6.9 Hz), 126.43 (q, ³J _CF3 = 3.8 Hz), 125.45 (q, ¹J _CF3 = 271.1 Hz), 123.43 (d, ⁴J _CF = 3.5 Hz), 116.18 (d, ³J _CF = 2.0 Hz), 115.14 (d, ²J _CF = 20.3 Hz), 71.10; HRMS (ESI+): calcd for C₁₅H₁₃F₄N₂O₂ [M + H]⁺ 329.0908; found, 329.0890.

N’-hydroxy-3-methoxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13k).

Synthesized by General Procedure C: 243 mg, 93%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.64 (q, 4.6 Hz, 4H), 7.27 (s, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 5.19 (s, 2H), 3.88 (s, 3H); ¹³C NMR (101 MHz CD₃OD) 155.36, 150.89, 150.50, 143.11, 130.92 (d, ²J _CF3 = 32.6 Hz), 128.85, 127.69, 126.33 (d, ³J _CF3 = 3.4 Hz), 122.17, 120.02, 114.97, 111.33, 71.03, 56.44; HRMS (ESI+): calcd for C₁₆H₁₆F₃N₂O₃ [M + H]⁺ 341.1108; found, 341.1109.

2-chloro-N’-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13l).

Synthesized by General Procedure C: 220 mg, 90%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.64 (d, J = 19.8 Hz, 4H), 7.44 (dd, J = 8.2, 61.5 Hz, 1H), 7.11 (s, 1H), 6.99 (s, 1H), 5.19 (s, 2H); ¹³C NMR (101 MHz CD₃OD) 171.81, 161.03, 154.08, 142.40, 135.14, 133.05, 131.66, 131.04 (d, ²J _CF3 = 32.5 Hz), 126.40 (d, ³J _CF3 = 3.8 Hz), 125.50 (q, ¹J _CF3 = 271.4 Hz), 117.30, 114.46, 70.26; HRMS (ESI+): calcd for C₁₅H₁₃ClF₃N₂O₂ [M + H]⁺ 345.0612; found, 341.0600.

N’-hydroxy-2-(trifluoromethyl)-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13m).

Synthesized by General Procedure C: 139 mg, 47%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.66 (q, J = 7.9 Hz, 4H), 7.51 (dd, J = 8.5, 22.5 Hz, 1H), 7.36 (s, 1H), 7.28 (d, J = 8.8 Hz, 1H), 5.27 (s, 2H); ¹³C NMR (101 MHz CD₃OD) 173.08, 160.42, 154.00, 142.33 (q, ⁴J _CF3 = 1.2 Hz), 134.20, 131.64 (q, ²J _CF3 = 31.6 Hz), 131.45, 128.90, 126.47 (q, ³J _CF3 = 3.8 Hz), 125.54 (q, ¹J _CF3 = 271.0 Hz), 124.93 (q, ¹J _CF3 = 272.9 Hz), 118.61 (q, ⁴J _CF3 = 1.2 Hz), 114.63 (q, ³J _CF3 = 5.3 Hz), 70.42; HRMS (ESI+): calcd for C₁₆H₁₃F₆N₂O₂ [M + H]⁺ 379.0876; found, 379.0866.

N’-hydroxy-3-nitro-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13n).

Synthesized by General Procedure C: 220 mg, 85%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.13 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.64 (s, 4H), 7.29 (d, J = 8.8 Hz, 1H), 5.32 (s, 2H); ¹³C NMR (101 MHz CD₃OD) 169.28, 153.32, 141.66, 140.95, 132.65, 131.10 (d, ²J _CF3 = 32.3 Hz), 128.45, 127.24, 125.34 (q, ¹J _CF3 = 271.80 Hz), 126.41 (q, ³J _CF3 = 3.8 Hz), 124.16, 116.13, 71.20; HRMS (ESI+): calcd for C₁₅H₁₃F₃N₃O₄ [M + H]⁺ 356.0853; found, 356.0864.

3-(tert-butyl)-N’-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13o).

Synthesized by General Procedure C: 268 mg, 88%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.85 – 7.64 (m, 3H), 7.79 – 7.40 (m, 3H), 6.90 (t, J = 7.60 Hz, 1H), 6.12 (s, 1H), 5.21 (d, J = 11.6 Hz, 2H), 4.87 (br s, 2H), 1.42 (s, 9H); ¹³C NMR (101 MHz CDCl₃) 169.82, 160.22, 158.63, 152.92, 141.05, 138.76, 127.45, 126.86, 125.78, 124.96, 112.37, 111.98, 69.56, 35.18, 29.77; HRMS (ESI+): calcd for C₁₉H₂₂F₃N₂O₂ [M + H]⁺ 367.1628; found, 367.1647.

3-allyl-N’-hydroxy-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13p).

Synthesized by General Procedure C: 504 mg, 96%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.63 (q, J = 8.4 Hz, 4H), 7.49 – 7.44 (m, 2H), 6.97 (d, J = 9.2 Hz, 1H), 6.05 – 5.94 (m, 1H), 5.17 (s, 2H), 5.06 – 5.00 (m, 2H), 3.44 (d, J = 6.5 Hz, 2H); ¹³C NMR (101 MHz CD₃OD) 172.05, 158.60, 155.44, 143.13 (d, ⁴J _CF3 = 1.2 Hz), 137.73, 130.89 (q, ²J _CF3 = 32.3 Hz), 130.06, 129.12, 128.56, 126.98, 126.70, 126.36 (q, ³J _CF3 = 3.9 Hz), 125.62 (q, ¹J _CF3 = 271.7 Hz), 112.58, 70.04, 35.51; HRMS (ESI+): calcd for C₁₈H₁₈F₃N₂O₂ [M + H]⁺ 351.1315; found, 351.1340.

N’-hydroxy-3-propyl-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13q).

Synthesized by General Procedure C: 504 mg, 96%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.75 – 7.58 (m, 5H), 7.47 – 7.44 (m, 1H), 7.02 – 6.94 (m, 1H), 5.18 (s, 2H), 2.67 (t, J = 7.3 Hz, 2H), 1.64 (q, J = 7.5 Hz, 2H), 0.94 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz CD₃OD) 172.20, 158.82, 155.58, 143.30 (d, ⁴J _CF3 = 1.3 Hz), 132.39, 130.86 (q, ²J _CF3 = 32.3 Hz), 129.14, 128.50, 126.40 (q, ³J _CF3 = 3.9 Hz), 126.26, 125.59 (q, ¹J _CF3 = 271.5 Hz), 112.51, 69.99, 33.52, 24.15, 14.38; HRMS (ESI+): calcd for C₁₈H₂₀F₃N₂O₂ [M + H]⁺ 353.1471; found, 353.1479.

N’-hydroxy-3-(trifluoromethyl)-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (13r).

Synthesized by General Procedure C: 304 mg, 96%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.93 (d, J = 2.1 Hz, 1H), 7.83 (dd, J = 8.7, 2.2 Hz, 1H), 7.69 (d, J = 8.3 Hz, 2H), 7.65 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 8.7 Hz, 1H), 5.34 (s, 2H); ¹³C NMR (101 MHz, CD₃OD) δ 158.37, 154.01, 142.26, 132.55, 131.13 (q, ²J_CF = 32.3 Hz), 128.44, 126.97, 126.46 (q, ³J_CF = 3.8 Hz), 126.17 (q, ³J_CF = 5.4 Hz), 125.49 (q, ¹J_CF = 264 Hz), 125.02 (q, ¹J_CF = 277 Hz), 119.92 (q,²J_CF = 30.9 Hz), 114.61, 70.61; HRMS (ESI+): calcd for C₁₆H₁₃F₆N₂O₂ [M + H]⁺ 379.0876; found 379.0879.

tert-butyl (S)-2-(3-(6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-yl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14a).

Synthesized by General Procedure D: 33 mg, 40%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.63 – 7.56 (m, 4H), 7.48 – 7.34 (m, 5H), 7.11 – 7.05 (m, 1H), 5.21 (s, 2H), 5.09 – 5.02 (m, 1H), 3.76 – 3.63 (m, 1H), 3.61 – 3.45 (m, 1H), 2.46 – 2.30 (m, 1H), 2.21 – 2.09 (m, 2H), 2.04 – 1.95 (m, 1H), 1.46 (s, 1H), 1.31 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.63, 168.05, 157.58, 153.80, 140.74, 137.46, 132.08, 130.41, 129.72, 128.21, 127.65, 126.91, 125.61 (q, ³J _CF3 = 3.7 Hz), 124.44 (q, ¹J _CF3 = 272.6 Hz), 120.21, 113.15, 80.71, 69.70, 53.95, 46.50, 32.52, 31.61, 28.52, 28.30, 23.82; HRMS (ESI+): calcd for C₃₁H₃₁F₃N₃O₄ [M + H]⁺ 566.2261; found, 566.2291.

tert-butyl (S)-2-(3-(4’-fluoro-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-yl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14b).

Synthesized by General Procedure D: 43 mg, 43%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.08 – 7.99 (m, 2H), 7.65 – 7.51 (m, 4H), 7.42 (d, J = 8.1 Hz, 2H), 7.17 – 7.04 (m, 3H), 5.21 (s, 2H), 5.10 – 5.02 (m, 1H), 3.76 – 3.63 (m, 1H), 3.61 – 3.45 (m, 1H), 2.47 – 2.29 (m, 1H), 2.21 – 2.09 (m, 2H), 2.06 – 1.95 (m, 1H), 1.46 (s, 3H), 1.30 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.73, 167.97, 162.38 (d, ¹J _CF = 245.4 Hz), 157.50, 153.69, 140.59 (d, ⁴J _CF3 = 1.5 Hz), 133.39 (d, ⁴J _CF = 2.9 Hz), 131.37 (d, ³J _CF = 8.0 Hz), 130.26, 128.34, 126.94, 125.69 (q, ³J _CF3 = 3.8 Hz), 124.20 (d, ¹J _CF3 = 271.8 Hz), 120.30, 115.14 (d, ²J _CF = 20.9 Hz), 113.14, 80.60, 69.77, 53.96, 46.50, 32.56, 31.66, 29.85, 28.32, 23.86; HRMS (ESI+): calcd for C₃₁H₃₀F₄N₃O₄ [M + H]⁺ 584.2167; found, 584.2175.

tert-butyl (S)-2-(3-(3-(pyridin-4-yl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14c).

Synthesized by General Procedure D: 65 mg, 59%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.66 (s, 2H), 8.10 (s, 2H), 7.61 (d, J = 8.1 Hz, 2H), 7.56 – 7.49 (m, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.16 – 7.08 (m, 1H), 5.23 (s, 2H), 5.09 – 5.02 (m, 1H), 3.76 – 3.62 (m, 1H), 3.59 – 3.44 (m, 1H), 2.45 – 2.33 (m, 1H), 2.20 – 2.09 (m, 2H), 2.05 – 1.96 (m, 1H), 1.45 (s, 3H), 1.30 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.89, 167.68, 157.51, 153.64, 149.70, 145.38, 140.15, 130.00, 129.80,125.76 (d, ³J _CF3 = 3.2 Hz) 124.12 (d, ¹J _CF3 = 272.0 Hz), 120.51, 113.19, 80.58, 69.85, 53.92, 46.47, 32.53, 31.63, 29.82, 28.50, 28.28, 24.52, 23.84; HRMS (ESI+): calcd for C₃₀H₃₀F₃N₄O₄ [M + H]⁺ 567.2214; found, 567.2244.

tert-butyl (S)-2-(3-(4’-(trifluoromethyl)-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-yl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14d).

Synthesized by General Procedure D: 206 mg, 51%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.12 – 8.01 (m, 2H), 7.73 – 7.67 (m, 4H), 7.60 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 7.7 Hz, 2H), 7.15 – 7.06 (m, 1H), 5.23 (s, 2H), 5.12 – 5.02 (m, 1H), 3.77 – 3.63 (m, 1H), 3.62 – 3.46 (m, 1H), 2.47 – 2.32 (m, 1H), 2.20 – 2.10 (m, 2H), 2.04 – 1.95 (m, 1H), 1.47 (s, 3H), 1.31 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.81, 167.82, 157.48, 153.74, 141.17, 140.36, 130.51, 130.31, 130.03, 129.00, 125.70 (q, ³J _CF3 = 3.8 Hz), 125.12 (q, ³J _CF3 = 3.6 Hz), 124.40 (d, ¹J _CF3 = 272.2 Hz), 124.19 (q, ¹J _CF3 = 272.4 Hz), 120.41, 113.23, 80.66, 69.83, 53.94, 46.49, 32.50, 31.60, 28.49, 28.27, 24.49, 23.81; HRMS (ESI+): calcd for C₃₂H₃₀F₆N₃O₄ [M + H]⁺ 634.2135; found, 634.2105.

tert-butyl (S)-2-(3-(3-cyclopropyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14e).

Synthesized by General Procedure D: 15 mg, 27%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (d, J = 7.5 Hz, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 6.4 Hz, 2H), 7.27 (q, J = 8.0 Hz, 2H), 6.99 – 6.90 (m, 1H), 5.29 – 4.99 (m, 3H), 3.78 – 3.63 (m, 1H), 3.61 – 3.40 (m, 1H), 2.44 – 2.30 (m, 1H), 2.29 – 2.22 (m, 1H), 2.21 – 2.07 (m, 2H), 2.05 – 1.96 (m, 1H), 1.46 (s, 3H), 1.30 (s, 6H), 1.04 – 0.95 (m, 2H), 0.82 – 0.72 (m, 2H); ¹³C NMR (101 MHz CDCl₃) 180.46, 168.26, 159.53, 153.73, 141.06, 134.06, 133.60, 132.44, 130.30 (d, ²J _CF3 = 32.7 Hz), 129.62, 127.28, 126.25, 125.72 (q, ³J _CF3 = 3.7 Hz), 124.68, 124.19 (q, ¹J _CF3 = 272.1 Hz), 119.71, 111.58, 80.59, 69.43, 53.96, 46.49, 28.30, 7.99; HRMS (ESI+): calcd for C₂₈H₃₁F₃N₃O₄ [M + H]⁺ 530.2261; found, 530.2242.

tert-butyl (S)-2-(3-(4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14f).

Synthesized by General Procedure D: 148 mg, 63%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.05 – 7.97 (m, 2H), 7.58 (dd, J = 7.8, 36.4 Hz, 4H), 7.07 – 6.97 (m, 2H), 5.15 (s, 2H), 5.07 – 5.01 (1H), 3.74 – 3.60 (m, 1H), 3.58 – 3.43 (m, 1H), 2.43 – 2.26 (m, 1H), 2.17 – 2.06 (m, 2H), 2.03 – 1.91 (m, 1H), 1.44 (s, 3H), 1.28 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.52, 167.94, 160.71, 153.57, 140.59, 130.23 (q, ²J _CF3 = 32.7 Hz), 129.18, 127.43, 125.59 (q, ³J _CF3 = 3.2 Hz), 124.07 (d, ¹J _CF3 = 272.0 Hz), 119.87, 115.13, 80.40, 69.13, 46.37, 32.38, 28.40, 28.16, 23.71; HRMS (ESI+): calcd for C₂₅H₂₆F₃N₃NaO₄ [M+Na]⁺ 512.1768; found, 512.1773.

tert-butyl (S)-2-(3-(3-bromo-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14g).

Synthesized by General Procedure D: 172 mg, 47%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 8.00 – 7.91 (m, 1H), 7.62 (dd, J = 7.8, 21.5 Hz, 4H), 7.01 – 6.92 (m, 1H), 5.24 (s, 2H), 5.07 – 5.00 (m, 1H), 3.74 – 3.62 (m, 1H), 3.57 – 3.44 (m, 1H), 2.43 – 2.30 (m, 1H), 2.17 – 2.07 (m, 2H), 2.03 – 1.95 (m, 1H), 1.44 (s, 3H), 1.28 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.88, 166.98, 156.87, 153.54, 140.03, 132.62, 130.30 (q, ²J _CF3 = 31.4 Hz), 127.98, 127.07, 125.66 (d, ³J _CF3 = 3.1 Hz),, 124.12 (q, ¹J _CF3 = 271.8 Hz), 121.14, 113.30, 80.49, 69.94, 53.83, 46.41, 32.43, 29.75, 28.19, 23.75; HRMS (ESI+): calcd for C₂₅H₂₅BrF₃N₃NaO₄ [M+Na]⁺ 590.0873; found, 590.0877.

tert-butyl (S)-2-(3-(3,5-dimethyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14h).

Synthesized by General Procedure D: 190 mg, 62%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.76 (s, 2H), 7.61 (dd, J = 8.2, 26.3 Hz, 4H), 5.08 – 5.00 (m, 1H), 4.89 (s, 2H), 3.72 – 3.65 (m, 1H), 3.57 – 3.47 (m, 1H), 2.32 (s, 6H), 2.17 – 2.08 (m, 2H), 2.04 – 1.87 (m, 2H), 1.44 (s, 3H), 1.28 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.62, 168.07, 158.10, 153.56, 141.36, 131.84, 130.16 (q, ²J _CF3 = 32.3 Hz), 128.23, 127.56, 125.51 (d, ³J _CF3 = 3.6 Hz), 122.79, 122.53, 80.42, 73.00, 53.86, 46.40, 32.46, 28.17, 23.74, 16.42; HRMS (ESI+): calcd for C₂₇H₃₁F₃N₃O₄ [M+H]⁺ 518.2261; found, 518.2267.

tert-butyl (S)-2-(3-(3-chloro-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14i).

Synthesized by General Procedure D: 190 mg, 62%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.17 – 8.05 (m, 1H), 7.95 – 7.83 (m, 1H), 7.60 (dd, J = 8.1, 25.0 Hz, 4H), 7.05 – 6.94 (m, 1H), 5.23 (s, 2H), 5.07 – 5.01 (m,1H), 3.76 – 3.62 (m, 1H), 3.60 – 3.44 (m, 1H), 2.46 – 2.28 (m, 1H), 2.18 – 2.07 (m, 2H), 2.04 – 1.93 (m, 1H), 1.45 (s, 3H), 1.28 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.84, 167.12, 156.04, 153.65, 140.03, 130.33 (q, ²J _CF3 = 32.5 Hz), 129.55, 127.13, 125.67 (q, ³J _CF3 = 3.8 Hz), 124.13 (q, ¹J _CF3 = 272.5 Hz), 123.86, 120.69, 113.57, 80.61, 69.90, 53.85, 46.43, 32.41, 28.42, 28.19, 23.74; HRMS (ESI+): calcd for C₂₅H₂₅ClF₃N₃NaO₄ [M+Na]⁺ 546.1378; found, 546.1384.

tert-butyl (S)-2-(3-(3-fluoro-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14j).

Synthesized by General Procedure D: 217 mg, 58%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.83 – 7.70 (m, 2H), 7.57 (dd, J = 7.9, 21.3 Hz, 4H), 7.08 – 6.97 (m, 1H), 5.26 – 4.99 (m, 3H), 3.74 – 3.60 (m, 1H), 3.58 – 3.42 (m, 1H), 2.43 – 2.25 (m, 1H), 2.17 – 2.04 (m, 2H), 1.99 – 1.91 (m, 1H), 1.43 (s, 3H), 1.27 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.81, 167.23 (d, ⁴J _CF = 2.2 Hz), 153.61, 152.63 (d, ¹J _CF = 247.4 Hz), 148.84 (d, ²J _CF = 10.9 Hz), 140.05, 130.38 (q, ²J _CF3 = 32.2 Hz), 127.41, 125.63 (q, ³J _CF3 = 3.8 Hz), 124.00 (q, ¹J _CF3 = 272.3 Hz), 123.97 (d, ⁴J _CF = 3.7 Hz), 120.43 (d, ³J _CF = 7.2 Hz), 115.56 (d, ²J _CF = 20.7 Hz), 115.20, 80.55, 70.24, 53.81, 46.40, 32.35, 28.14, 23.69; HRMS (ESI+): calcd for C₂₅H₂₅F₄N₃NaO₄ [M+Na]⁺ 530.1673; found, 530.1679.

tert-butyl (S)-2-(3-(3-methoxy-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14k).

Synthesized by General Procedure D: 175 mg, 65%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.80 – 7.51 (m, 6H), 6.95 – 6.82 (m, 1H), 5.25 – 5.02 (m, 3H), 3.94 (s, 2H), 3.89 (s, 1H), 3.77 – 3.63 (m, 1H), 3.59 – 3.45 (m, 1H), 2.43 – 2.30 (m, 1H), 2.18 – 2.06 (m, 2H), 2.02 – 1.93 (m, 1H), 1.45 (s, 3H), 1.29 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.43, 168.04, 153.81, 150.26, 149.77, 140.62, 130.15 (q, ²J _CF3 = 33.0 Hz), 127.33, 125.55 (d, ³J _CF3 = 3.8 Hz), 124.14 (q, ¹J _CF3 = 272.0 Hz), 120.77, 120.11, 113.48, 110.41, 80.72, 70.02, 56.11, 53.87, 46.45, 32.37, 28.17, 23.71; HRMS (ESI+): calcd for C₂₆H₂₉F₃N₃O₅ [M+H]⁺ 520.2054; found, 520.2032.

tert-butyl (S)-2-(3-(2-chloro-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14l).

Synthesized by General Procedure D: 110 mg, 33%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.91 – 7.83 (m, 1H), 7.58 (dd, J = 7.5, 35.9 Hz, 4H), 7.14 – 7.07 (m, 1H), 6.98 – 6.89 (m, 1H), 5.22 – 5.00 (m, 3H), 3.73 – 3.60 (m, 1H), 3.57 – 3.42 (m, 1H), 2.43 – 2.29 (m, 1H), 2.18 – 2.07 (m, 2H), 2.02 – 1.93 (1H), 1.44 (s, 3H), 1.29 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.08, 166.85, 160.44, 153.53, 139.97, 134.56, 132.83, 130.45 (q, ²J _CF3 = 31.6 Hz), 127.48, 125.70 (d, ³J _CF3 = 3.6 Hz), 124.08 (q, ¹J _CF3 = 272.5 Hz), 119.03, 117.22, 113.73, 80.49, 69.46, 53.79, 46.40, 32.47, 28.20, 23.73; HRMS (ESI+): calcd for C₂₅H₂₆ClF₃N₃O₄ [M+H]⁺ 524.1558; found, 524.1574.

tert-butyl (S)-2-(3-(2-(trifluoromethyl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14m).

Synthesized by General Procedure D: 68 mg, 33%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J = 8.5 Hz, 1H), 7.58 (dd, J = 7.9, 29.1 Hz, 4H), 7.42 – 7.36 (m, 1H), 7.19 – 7.11 (m, 1H), 5.22 – 5.02 (m, 3H), 3.70 – 3.58 (m, 1H), 3.56 – 3.40 (m, 1H), 2.43 – 2.27 (m, 1H), 2.16 – 2.05 (m, 2H), 2.00 – 1.92 (m, 1H), 1.42 (s, 3H), 1.30 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.63, 167.20, 159.90, 153.46, 139.81, 133.58, 130.82 (q, ²J _CF3 = 32.3 Hz), 130.47 (q, ²J _CF3 = 32.4 Hz), 127.50, 125.67 (q, ³J _CF3 = 3.7 Hz), 124.05 (q, ¹J _CF3 = 272.3 Hz), 123.07 (q, ¹J _CF3 = 273.3 Hz), 117.24, 114.20 (q, ³J _CF3 = 5.6 Hz), 80.47, 69.49, 53.72, 46.35, 32.47, 28.31, 28.06, 23.60; HRMS (ESI+): calcd for C₂₆H₂₅F₆N₃NaO₄ [M + Na]⁺ 580.1641; found, 580.1637.

tert-butyl (S)-2-(3-(3-nitro-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14n).

Synthesized by General Procedure D: 149 mg, 39%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.58 – 8.49 (m, 1H), 8.23 – 8.13 (m, 1H), 7.59 (q, J = 7.1 Hz, 4H), 7.24 – 7.14 (m, 1H), 5.32 (s, 2H), 5.20 – 5.00 (m, 1H), 3.72 – 3.61 (m, 1H), 3.57 – 3.45 (m, 1H), 2.44 – 2.30 (m, 1H), 2.18 – 2.06 (m, 2H), 2.04 – 1.94 (m, 1H), 1.43 (s, 3H), 1.26 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 181.36, 166.39, 153.33, 139.10, 132.86, 130.45 (q, ²J _CF3 = 32.8 Hz), 127.05, 125.72 (d, ³J _CF3 = 3.7 Hz), 124.97, 123.99 (q, ¹J _CF3 = 271.3 Hz), 120.05, 115.16, 80.50, 70.36, 53.78, 46.39, 32.39, 31.47, 28.36, 28.15, 23.72; HRMS (ESI+): calcd for C₂₅H₂₅F₃N₄NaO₆ [M + Na]⁺ 557.1618; found, 524.1588.

tert-butyl (S)-2-(3-(3-(tert-butyl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14o).

Synthesized by General Procedure D: 210 mg, 53%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.07 – 8.01 (m, 1H), 7.92 – 7.84 (m, 1H), 7.61 (dd, J = 8.8, 31.7 Hz, 4H), 6.99 – 6.91 (m, 1H), 5.23 – 5.01 (m, 3H), 3.73 – 3.62 (m, 1H), 3.58 – 3.45 (m, 1H), 2.41 – 2.30 (m, 1H), 2.16 – 2.06 (m, 2H), 2.00 – 1.93 (m, 1H), 1.43 (s, 12H), 1.30 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.32, 168.35, 159.51, 153.58, 140.79, 138.98, 130.09 (q, ²J _CF3 = 33.2 Hz), 127.44, 126.89, 126.27, 125.62 (d, ³J _CF3 = 3.7 Hz), 124.12 (q, ¹J _CF3 = 271.7 Hz), 119.30, 112.48, 80.33, 69.44, 53.82, 46.33, 35.06, 32.36, 29.68, 28.13, 23.68; HRMS (ESI+): calcd for C₂₉H₃₅F₃N₃O₄ [M + H]⁺ 546.2574; found, 546.2576.

tert-butyl (S)-2-(3-(3-allyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14p).

Synthesized by General Procedure D: 444 mg, 58%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.89 (s, 2H), 7.56 (dd, J = 7.6, 35.7 Hz, 4H), 6.94 – 6.86 (m, 1H), 6.05 – 5.92 (m, 1H), 5.19 – 4.99 (m, 5H), 3.70 – 3.60 (m, 1H), 3.54 – 3.41 (m, 3H), 2.39 – 2.27 (m, 1H), 2.14 – 2.03 (m, 2H), 1.98 – 1.90 (m, 1H), 1.42 (s, 3H), 1.27 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.38, 167.96, 158.28, 153.48, 140.77, 135.94, 129.98 (q, ²J _CF3 = 33.3 Hz), 129.60, 129.13, 127.09, 125.45 (d, ³J _CF3 = 3.3 Hz), 124.22 (q, ¹J _CF3 = 272.0 Hz), 119.50, 116.11, 111.50, 80.25, 68.99, 53.75, 46.28, 34.37, 32.28, 28.26, 28.03, 23.61; HRMS (ESI+): calcd for C₂₈H₃₁F₃N₃O₄ [M + H]⁺ 530.2261; found, 530.2274.

tert-butyl (S)-2-(3-(3-propyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14q).

Synthesized by General Procedure D: 480 mg, 58%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.91 – 7.82 (m, 2H), 7.58 (dd, J = 7.4, 38.8 Hz, 4H), 6.94 – 6.86 (m, 1H), 5.20 – 5.01 (m, 3H), 3.74 – 3.62 (m, 1H), 3.57 – 3.42 (m, 1H), 2.73 – 2.64 (m, 2H), 2.41 – 2.28 (m, 1H), 2.16 – 2.05 (m, 2H), 1.99 – 1.90 (m, 1H), 1.72 – 1.61 (m, 2H), 1.43 (s, 3H), 1.28 (s, 6H), 1.00 – 0.88 (m, 3H); ¹³C NMR (101 MHz CDCl₃) 180.37, 168.12, 158.55, 153.55, 141.00, 132.05, 130.00 (q, ²J _CF3 = 32.2 Hz), 129.24, 127.00, 126.65, 125.52 (d, ³J _CF3 = 3.8 Hz), 124.14 (q, ¹J _CF3 = 271.1 Hz), 119.31, 111.46, 80.33, 68.94, 53.81, 46.33, 32.34, 28.32, 28.09, 23.66, 22.88, 14.00; HRMS (ESI+): calcd for C₂₈H₃₃F₃N₃O₄ [M + H]⁺ 532.2418; found, 532.2397.

tert-butyl (S)-2-(3-(3-(trifluoromethyl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14r).

Synthesized by General Procedure D: 146 mg, 64%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 8.20 (d, J = 8.6 Hz, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.11 (t, J = 8.7 Hz, 1H), 5.31 (s, 2H), 5.20 – 5.05 (m, 1H), 3.75 – 3.63 (m, 1H), 3.62 – 3.42 (m, 1H), 2.49 – 2.31 (m, 1H), 2.22 – 1.96 (m, 3H), 1.46 (s, 3H), 1.30 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 181.13, 167.22, 158.21, 153.63, 139.76, 132.66, 130.48 (q, ²J_CF = 30.3 Hz), 127.00, 125.82 (t, ³J_CF = 3.8 Hz), 123.99 (q, ¹J_CF = 272.8 Hz), 123.17 (q, ¹J_CF = 273.1 Hz), 119.69, 113.45, 80.64, 69.73, 53.92, 46.49, 32.53, 31.60, 28.50, 28.28, 23.85; HRMS (ESI+): calcd for C₂₆H₂₆F₆N₃O₄Na [M + Na]⁺ 580.1641; found, 580.1652.

tert-butyl (S)-2-(3-(3-ethyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (14s).

Synthesis was carried forward via a three step process according to the following protocol: To a solution of THF (0.2 M) under an atmosphere of argon gas, 370 mg (1 equiv) of tert-butyl (S)-2-(3-(3-bromo-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (compound 14g), 197 mg (3 equiv) of triethylamine, and 77 mg (1.2 equiv) of trimethylsilylacetylene were sequentially added to reaction vessel. Reaction solution was then degassed for 30 minutes by bubbling argon gas through solution while stirring. Next, 46 mg (0.1 equiv) of bis(triphenylphosphine) palladium(II) dichloride was added followed by 6 mg (0.05 equiv) copper(I) iodide. Reaction mixture was refluxed at 60 °C for 48 hours. The reaction progress was monitored by TLC. Subsequently, the resulting solution was partitioned between EtOAc and brine solution. Using additional EtOAc, the brine solution was washed three times and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated via vacuum. The resulting concentrate was purified by silica gel chromatography to yield 280 mg (74% yield) of intermediate product. Next, purified intermediate was then dissolved in a solution of THF (0.2 M) containing 250 mg (2 equiv) of tetra-n-butylammonium fluoride (TBAF) and stirred at room temperature for 3 hours. The reaction progress was monitored by TLC and concentrated via vacuum. The resulting concentrate was purified by silica gel chromatography to yield 245 mg (100% yield) of intermediate product. Finally, the resulting intermediate was hydrogenated by dissolving in ethanol (190 proof, 0.2 M) followed by addition of 10% palladium on carbon (0.1 equiv). Reaction mixture was then put under light vacuum followed by purge of hydrogen gas. Vacuum followed by addition of hydrogen gas was repeated three times. The resulting mixture was stirred at room temperature for 18 hours followed by concentration via vacuum and purification by silica gel chromatography to yield final product: 114 mg, 45%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.05 – 7.86 (m, 2H), 7.61(dd, J = 7.8, 39.3 Hz, 4H), 7.06 – 6.91 (m, 1H), 5.22 – 5.01 (m, 3H), 3.76 – 3.64 (m, 1H), 3.59 – 3.46 (m, 1H), 2.83 – 2.70 (m, 2H), 2.45 – 2.32 (m, 1H), 2.19 – 2.10 (m, 2H), 2.04 – 1.95 (m, 1H), 1.46 (s, 3H), 1.30 (s, 9H); ¹³C NMR (101 MHz CDCl₃) 180.46, 168.25, 158.55, 153.70, 141.01, 133.71, 130.23 (q, ²J _CF3 = 32.2 Hz), 128.47, 127.19, 126.75, 125.71 (d, ³J _CF3 = 3.3 Hz), 124.21 (q, ¹J _CF3 = 272.6 Hz), 119.55, 115.21, 111.44, 80.54, 69.12, 53.94, 46.46, 32.52, 28.26, 23.53, 14.13; HRMS (ESI+): calcd for C₂₇H₃₁F₃N₃O₄ [M + H]⁺ 518.2261; found, 518.2259.

(S)-2-(3-(6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-yl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15a).

Synthesized by General Procedure E: 15 mg, 90%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.06 – 7.97 (m, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.54 (dd, J = 7.5, 12.4 Hz, 4H), 7.47 – 7.35 (m, 3H), 7.30 (d, J = 8.6 Hz, 1H), 5.44 (d, J = 7.8 Hz, 1H), 5.29 (s, 2H), 3.81 – 3.74 (m, 1H), 3.61 (q, J = 9.31 Hz, 1H), 2.62 – 2.52 (m, 1H), 2.52 – 2.43 (m, 1H), 2.28 – 2.18 (m, 1H), 2.16 – 2.02 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.87, 169.28, 159.27, 157.07, 142.73, 138.86, 133.38, 130.90, 130.88 (q, ²J _CF3 = 31.9 Hz), 130.58, 129.27, 129.17, 128.60, 128.44, 126.34 (q, ³J _CF3 = 3.7 Hz), 125.56 (q, ¹J _CF3 = 271.8 Hz), 120.60, 114.58, 70.65, 56.47, 32.72, 24.34; HRMS (ESI+): calcd for C₂₇H₂₅F₃N₅O₂ [M + H]⁺ 508.1955; found, 508.1963.

(S)-2-(3-(4’-fluoro-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-yl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15b).

Synthesized by General Procedure E: 112 mg, 63%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.99 (d, J = 8.3 Hz, 1H), 7.94 (s, 1H), 6.60 – 7.45 (m, 6H), 7.24 (d, J = 8.3 Hz, 1H), 7.12 (t, J = 8.7 Hz, 2H), 5.49 (d, J = 7.1 Hz, 1H), 5.22 (s, 2H), 3.79 (t, J = 8.4 Hz, 1H), 3.63 (q, J = 9.3 Hz, 1H), 2.61 – 2.50 (m, 1H), 2.48 – 2.42 (m, 1H), 2.27 – 2.18 (m, 1H), 2.13 – 2.03 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.86, 169.13, 163.60 (d, ¹J _CF = 245.4 Hz), 159.05, 157.03, 142.48 (d, ²J _CF3 = 1.2 Hz), 134.83 (d, ⁴J _CF = 3.3 Hz), 132.45 (d, ³J _CF = 8.2 Hz), 131.97, 130.83 (q, ²J _CF3 = 32.5 Hz), 130.72, 129.45, 128.42, 126.32 (q, ³J _CF3 = 3.8 Hz), 125.49 (q, ¹J _CF3 = 271.2 Hz), 120.57, 115.87 (d, ²J _CF = 21.5 Hz), 114.48, 70.63, 56.43, 32.69, 24.34; HRMS (ESI+): calcd for C₂₇H₂₄F₄N₅O₂ [M + H]⁺ 526.1861; found, 526.1863.

(S)-2-(3-(3-(pyridin-4-yl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15c).

Synthesized by General Procedure E: 20 mg, 93%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.89 (s, 2H), 8.33 (d, J = 36.0 Hz, 4H), 7.66 (dd, J = 7.8, 28.2 Hz, 4H), 7.54 – 7.43 (m, 1H), 5.54 – 5.38 (m, 3H), 3.82 – 3.73 (m, 1H), 3.68 – 3.60 (m, 1H), 2.63 – 2.54 (m, 1H), 2.53 – 2.45 (m, 1H), 2.29 – 2.20 (m, 1H), 2.15 – 2.04 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 179.29, 168.66, 159.51, 157.07, 156.93, 142.70, 141.73, 133.22, 131.35 (q, ²J _CF3 = 32.1 Hz), 131.06, 129.25, 128.82, 126.88, 126.64 (q, ³J _CF3 = 3.7 Hz), 124.19, 121.51, 115.34, 71.44, 56.50, 32.79, 24.35; HRMS (ESI+): calcd for C₂₆H₂₄F₃N₆O₂ [M + H]⁺ 509.1907; found, 509.1919.

(S)-2-(3-(4’-(trifluoromethyl)-6-((4-(trifluoromethyl)benzyl)oxy)-[1,1’-biphenyl]-3-yl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15d).

Synthesized by General Procedure E: 102 mg, 86%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.11 – 8.02 (m, 2H), 7.77 – 7.71 (m, 4H), 7.58 (dd, J = 7.7, 44.6 Hz, 4H), 7.34 (d, J = 9.2 Hz, 1H), 5.45 (d, J = 7.2 Hz, 1H), 5.32 (s, 2H), 3.81 – 3.74 (m, 1H), 3.67 – 3.58 (m, 1H), 2.63 – 2.43 (m, 2H), 2.28 – 2.19 (m, 1H), 2.15 – 2.01 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.97, 169.10, 159.19, 157.07, 142.86 (q, ⁴J _CF3 = 1.4 Hz), 142.44 (q, ⁴J _CF3 = 1.4 Hz), 131.67, 131.26, 131.06 (d, ²J _CF3 = 32.3 Hz), 130.84, 130.56 (d, ²J _CF3 = 32.3 Hz), 130.15, 128.60, 126.42 (q, ³J _CF3 = 3.8 Hz), 126.04 (q, ³J _CF3 = 3.9 Hz), 125.73 (d, ¹J _CF3 = 271.5 Hz), 125.55 (d, ¹J _CF3 = 271.1 Hz), 120.81, 114.76, 70.84, 56.48, 32.73, 24.34; HRMS (ESI+): calcd for C₂₈H₂₄F₆N₅O₂ [M]⁺ 576.1829; found, 576.1838.

(S)-2-(3-(3-cyclopropyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15e).

Synthesized by General Procedure E: 102 mg, 86%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.84 (dd, J = 2.1, 8.5 Hz, 1H), 7.71 (s, 4H), 7.58 (d, J = 2.1 Hz, 1H), 5.42 (d, J = 7.9 Hz, 1H), 5.32 (s, 2H), 3.82 – 3.74 (m, 1H), 3.66 – 3.57 (m, 1H), 2.61 – 2.44 (m, 2H), 2.31 – 2.19 (m, 2H), 2.14 – 2.03 (m, 1H), 1.01 (q, J = 5.7 Hz, 2H), 0.70 (q, J = 5.2 Hz, 2H); ¹³C NMR (101 MHz CD₃OD) 178.70, 169.51, 161.25, 157.08, 143.06, 134.59, 131.02 (d, ²J _CF3 = 32.2 Hz), 128.69, 127.34, 126.48 (q, ³J _CF3 = 3.8 Hz), 125.37, 122.60, 120.09, 113.01, 70.38, 56.47, 32.73, 28.12, 24.35, 10.66, 8.11; HRMS (ESI+): calcd for C₂₄H₂₅F₃N₅O₂ [M + H]⁺ 472.1955; found, 472.1980.

(S)-2-(3-(4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15f).

Synthesized by General Procedure E: 68 mg, 38%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.01 (d, J = 8.9 Hz, 2H), 7.69 (q, J = 8.2 Hz, 4H), 7.17 (d, J = 8.2 Hz, 2H), 5.43 (d, J = 8.2 Hz, 1H), 5.28 (s, 2H), 3.77 (t, J = 9.3 Hz, 1H), 3.62 (q, J = 9.1 Hz, 1H), 2.62 – 2.44 (m, 2H), 2.29 – 2.19 (m, 1H), 2.16 – 2.03 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.74, 169.34, 162.61, 157.07, 142.79 (d, ⁴J _CF3 = 1.3 Hz), 130.17, 128.85, 126.47 (q, ³J _CF3 = 3.7 Hz), 124.30, 120.37, 116.43, 70.17, 56.46, 32.70, 24.32; HRMS (ESI+): calcd for C₂₁H₂₁F₃N₅O₂ [M + H]⁺ 432.1642; found, 432.1643.

(S)-2-(3-(3-bromo-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15g).

Synthesized by General Procedure E: 42 mg, 80%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.23 (d, J = 2.0 Hz, 1H), 8.00 (dd, J = 2.1, 8.6 Hz, 1H), 7.74 – 7.67 (m, 4H), 7.26 (d, J = 8.5 Hz, 1H), 5.46 (d, J = 8.0 Hz, 1H), 5.35 (s, 2H), 3.78 (t, J = 9.5 Hz, 1H), 3.63 (q, J = 9.1 Hz, 1H), 2.62 – 2.43 (m, 2H), 2.28 – 2.18 (m, 1H), 2.15 – 2.02 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 179.09, 168.27, 158.64, 157.05, 142.20 (d, ⁴J _CF3 = 1.2 Hz), 133.25, 131.13 (q, ²J _CF3 = 32.4 Hz), 129.21, 128.60, 126.48 (q, ³J _CF3 = 3.7 Hz), 125.58 (q, ¹J _CF3 = 271.5 Hz), 121.67, 114.99, 113.49, 71.02, 56.45, 32.69, 24.31; HRMS (ESI+): calcd for C₂₁H₂₀BrF₃N₅O₂ [M + H]⁺ 510.0747; found, 510.0750.

(S)-2-(3-(3,5-dimethyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15h).

Synthesized by General Procedure E: 67 mg, 91%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.75 (s, 2H), 7.70 (t, J = 9.6 Hz, 4H), 5.46 (d, J = 7.9 Hz, 1H), 4.98 (s, 2H), 3.78 (t, J = 9.6 Hz, 1H), 3.63 (q, J = 8.9 Hz, 1H), 2.62 – 2.51 (m, 1H), 2.51 – 2.43 (m, 1H), 2.33 (s, 6H), 2.28 – 2.19 (m, 1H), 2.15 – 2.02 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.83, 169.32, 159.69, 157.06, 143.26 (d, ⁴J _CF3 = 1.2 Hz), 133.32, 131.12 (q, ²J _CF3 = 32.4 Hz), 129.18, 129.15, 126.41 (q, ³J _CF3 = 3.7 Hz), 125.55 (q, ¹J _CF3 = 272.1 Hz), 123.30, 74.12, 56.45, 32.71, 24.32, 16.62; HRMS (ESI+): calcd for C₂₃H₂₅F₃N₅O₂ [M + H]⁺ 460.1955; found, 460.1969.

(S)-2-(3-(3-chloro-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15i).

Synthesized by General Procedure E: 62 mg, 88%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.03 (d, J = 2.1 Hz, 1H), 7.94 (dd, J = 2.1, 8.5 Hz, 1H), 7.69 (s, 4H), 7.27 (d, J = 8.7 Hz, 1H), 5.47 (d, J = 8.0 Hz, 1H), 5.33 (s, 2H), 3.79 (t, J = 9.4 Hz, 1H), 3.63 (q, J = 9.1 Hz, 1H), 2.63 – 2.51 (m, 1H), 2.50 – 2.42 (m, 1H), 2.29 – 2.18 (m, 1H), 2.16 – 2.02 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 179.06, 168.37, 157.68, 157.04, 142.16 (d, ⁴J _CF3 = 1.3 Hz), 131.10 (q, ²J _CF3 = 32.5 Hz), 130.95, 128.61, 128.50, 126.46 (q, ³J _CF3 = 3.8 Hz), 125.53 (q, ¹J _CF3 = 271.4 Hz), 124.57, 121.20, 115.20, 70.93, 56.42, 32.67, 24.31; HRMS (ESI+): calcd for C₂₁H₂₀ClF₃N₅O₂ [M]⁺ 466.1252; found, 466.1235.

(S)-2-(3-(3-fluoro-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15j).

Synthesized by General Procedure E: 132 mg, 96%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.82 – 7.72 (m, 2H), 7.67 (dd, J = 8.8, 12.8 Hz, 4H), 7.29 (t, J = 8.5 Hz, 1H), 5.48 (d, J = 7.8 Hz, 1H), 5.31 (s, 2H), 3.78 (t, J = 9.4 Hz, 1H), 3.63 (q, J = 8.2 Hz, 1H), 2.62 – 2.51 (m, 1H), 2.48 – 2.42 (m, 1H), 2.27 – 2.19 (m, 1H), 2.14 – 2.02 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 179.06, 168.55 (d, ⁴J _CF = 2.6 Hz), 157.04, 153.72 (d, ¹J _CF = 246.4 Hz), 150.52 (d, ²J _CF = 10.8 Hz), 142.16 (q, ⁴J _CF3 = 1.2 Hz), 131.17 (q, ²J _CF3 = 32.0 Hz), 128.87, 126.47 (q, ³J _CF3 = 3.8 Hz), 125.47 (q, ¹J _CF3 = 271.0 Hz), 125.28 (d, ⁴J _CF = 3.6 Hz), 120.84 (d, ³J _CF = 7.1 Hz), 116.57 (d, ³J _CF = 1.6 Hz), 115.98 (d, ²J _CF = 20.8 Hz), 71.10, 56.41, 32.65, 24.30; HRMS (ESI+): calcd for C₂₁H₂₀F₄N₅O₂ [M + H]⁺ 450.1548; found, 450.1555.

(S)-2-(3-(3-methoxy-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15k).

Synthesized by General Procedure E: 132 mg, 96%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.72 – 7.60 (m, 6H), 7.14 (d, J = 8.1 Hz, 1H), 5.44 (d, J = 7.7 Hz, 1H), 5.27 (s, 2H), 3.93 (s, 3H), 3.77 (t, J = 9.4 Hz, 1H), 3.62 (q, J = 8.0 Hz, 1H), 2.62 – 2.43 (m, 2H), 2.28 – 2.18 (m, 1H), 2.16 – 2.02 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.79, 169.43, 157.07, 152.23, 151.39, 142.83 (d, ⁴J _CF3 = 1.3 Hz), 131.06 (d, ²J _CF3 = 32.9 Hz), 128.88, 126.41 (q, ³J _CF3 = 3.8 Hz), 125.61 (d, ¹J _CF3 = 271.9 Hz), 122.05, 120.75, 115.06, 111.66, 70.96, 56.60, 56.47, 32.74, 24.34; HRMS (ESI+): calcd for C₂₂H₂₃F₃N₅O₃ [M + H]⁺ 462.1748; found, 462.1757.

(S)-2-(3-(2-chloro-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15l).

Synthesized by General Procedure E: 28 mg, 59%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.89 (d, J = 8.4 Hz, 1H), 7.68 (q, J = 8.7 Hz, 4H), 7.27 (s, 1H), 7.13 (d, J = 8.1 Hz, 1H), 5.46 (d, J = 7.0 Hz, 1H), 5.28 (s, 2H), 3.76 (t, J = 8.7 Hz, 1H), 3.62 (q, J = 8.4 Hz, 1H), 2.63 – 2.43 (m, 2H), 2.28 – 2.19 (m, 1H), 2.14 – 2.02 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.23, 168.14, 162.38, 157.04, 142.22 135.51, 134.01, 131.18 (q, ²J _CF3 = 32.2 Hz), 128.95, 126.50 (q, ³J _CF3 = 3.7 Hz), 125.55 (q, ¹J _CF3 = 271.4 Hz), 119.37, 118.37, 115.04, 70.57, 56.43, 32.71, 24.32; HRMS (ESI+): calcd for C₂₁H₁₉ClF₃N₅O₂ [M + H]⁺ 466.1252; found, 466.1241.

(S)-2-(3-(2-(trifluoromethyl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15m).

Synthesized by General Procedure E: 100 mg, 63%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.82 (d, J = 8.6 Hz, 1H), 7.69 (dd, J = 8.5, 13.8 Hz, 4H), 7.50 (d, J = 2.4 Hz, 1H), 7.40 (d, J = 9.0 Hz, 1H), 5.51 (d, J = 7.9 Hz, 1H), 5.34 (s, 2H), 3.75 (t, J = 9.4 Hz, 1H), 3.62 (q, J = 8.9 Hz, 1H), 2.63 – 2.52 (m, 1H), 2.48 – 2.41 (m, 1H), 2.28 – 2.20 (m, 1H), 2.12 – 1.99 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.73, 168.57, 161.81, 157.04, 142.09 (q, ⁴J _CF3 = 1.3 Hz), 134.93, 131.70 (q, ²J _CF3 = 32.1 Hz), 131.22 (q, ²J _CF3 = 32.1 Hz), 128.97, 126.52 (q, ³J _CF3 = 3.9 Hz), 125.58 (q, ¹J _CF3 = 271.4 Hz), 124.53 (q, ¹J _CF3 = 272.3 Hz), 118.70, 118.34 (q, ³J _CF3 = 2.0 Hz), 115.51 (q, ³J _CF3 = 5.6 Hz), 70.63, 56.30, 32.65, 24.18; HRMS (ESI+): calcd for C₂₂H₂₀F₆N₅O₂ [M + H]⁺ 500.1516; found, 500.1516.

(S)-2-(3-(3-nitro-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15n).

Synthesized by General Procedure E: 100 mg, 63%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.41 (s, 1H), 8.21 (br s, 1H), 7.67 (s, 4H), 7.54 – 7.45 (m, 1H), 5.52 (s, 1H), 5.42 (s, 2H), 3.85 – 3.77 (m, 1H), 3.65 (br s, 1H), 2.52 (d, J = 43.2 Hz, 2H), 2.18 (d, J = 51.4 Hz, 2H); ¹³C NMR (101 MHz CD₃OD) 179.42, 167.73, 156.98, 154.78, 141.38 (d, ⁴J _CF3 = 1.3 Hz), 141.13, 134.00, 131.13 (q, ²J _CF3 = 31.8 Hz), 128.58, 126.45 (q, ³J _CF3 = 3.7 Hz), 125.42, 125.39 (q, ¹J _CF3 = 271.5 Hz), 120.35, 117.17, 71.61, 56.45, 32.72, 24.36; HRMS (ESI+): calcd for C₂₁H₂₀F₃N₆O₄ [M + H]⁺ 477.1493; found, 477.1493.

(S)-2-(3-(3-(tert-butyl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15o).

Synthesized by General Procedure E: 25 mg, 58%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.02 (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 2.2, 8.6 Hz, 1H), 7.72 (dd, J = 9.0, 13.0 Hz, 4H), 7.17 (d, J = 9.0 Hz, 1H), 5.44 (d, J = 8.1 Hz, 1H), 5.32 (s, 2H), 3.78 (t, J = 9.5 Hz, 1H), 3.62 (q, J = 8.9 Hz, 1H), 2.62 – 2.51 (m, 1H), 2.51 – 2.44 (m, 1H), 2.28 – 2.20 (m, 1H), 2.16 – 2.04 (m, 1H), 1.44 (s, 9H); ¹³C NMR (101 MHz CD₃OD) 178.67, 169.75, 161.33, 157.13, 142.77 (d, ⁴J _CF3 = 1.3 Hz), 140.19, 131.20 (q, ²J _CF3 = 32.3 Hz), 129.20, 128.12, 127.03, 126.57 (q, ³J _CF3 = 3.8 Hz), 125.67 (q, ¹J _CF3 = 272.7 Hz), 119.84, 114.11, 70.70, 56.47, 35.95, 32.71, 30.12, 24.36; HRMS (ESI+): calcd for C₂₅H₂₉F₃N₅O₂ [M + H]⁺ 488.2268; found, 488.2293.

(S)-2-(3-(3-allyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15p).

Synthesized by General Procedure E: 24 mg, 79%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.91 (d, J = 8.6 Hz, 1H), 7.86 (s, 1H), 7.69 (dd, J = 8.2, 15.5 Hz, 4H), 7.15 (d, J = 8.6 Hz, 1H), 6.09 – 5.96 (m, 1H), 5.44 (d, J = 7.5 Hz, 1H), 5.29 (s, 2H), 5.11 – 5.03 (m, 2H), 3.78 (t, J = 9.2 Hz, 1H), 3.62 (q, J = 10.1 Hz, 1H), 3.50 (d, J = 6.4 Hz, 2H), 2.61 – 2.51 (m, 1H), 2.50 – 2.42 (m, 1H), 2.27 – 2.18 (m, 1H), 2.14 – 2.02 (m, 1H); ¹³C NMR (101 MHz CD₃OD) 178.71, 169.45, 160.17, 157.13, 142.91, 137.49, 131.09 (q, ²J _CF3 = 32.4 Hz), 131.01, 130.12, 128.71, 128.38, 126.46 (q, ³J _CF3 = 3.7 Hz), 125.58 (q, ¹J _CF3 = 271.3 Hz), 120.16, 116.35, 113.23, 70.32, 56.44, 35.34, 32.69, 24.34; HRMS (ESI+): calcd for C₂₄H₂₅F₃N₅O₂ [M + H]⁺ 472.1955; found, 472.1947.

(S)-2-(3-(3-propyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15q).

Synthesized by General Procedure E: 218 mg, 77%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.84 (d, J = 8.3 Hz, 1H), 7.79 (s, 1H), 7.62 (q, J = 8.3 Hz, 4H), 7.05 (d, J = 8.0 Hz, 1H), 5.53 (d, J = 7.5 Hz, 1H), 5.18 (s, 2H), 3.80 (t, J = 9.1 Hz, 1H), 3.64 (q, J = 8.5 Hz, 1H), 2.64 (t, J = 7.5 Hz, 2H), 2.59 – 2.50 (m, 1H), 2.46 – 2.39 (m, 1H), 2.25 – 2.17 (m, 1H), 2.11 – 2.00 (m, 1H), 1.61 (sx, J = 7.1 Hz, 2H), 0.91 (t, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz CD₃OD) 178.58, 169.33, 160.10, 156.96, 142.81 (d, ⁴J _CF3 = 1.3 Hz), 132.99, 130.77 (q, ²J _CF3 = 32.4 Hz), 129.96, 128.44, 127.94, 126.36 (q, ³J _CF3 = 3.8 Hz), 125.56 (q, ²J _CF3 = 271.5 Hz), 119.80, 112.93, 70.02, 56.35, 33.33, 32.61, 24.31, 23.90, 14.33; HRMS (ESI+): calcd for C₂₄H₂₇F₃N₅O₂ [M + H]⁺ 474.2111; found, 474.2112.

(S)-2-(3-(3-ethyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (15s).

Synthesized by General Procedure E: 32 mg, 77%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.89 – 7.84 (m, 2H), 7.68 (q, J = 6.9 Hz, 4H), 7.11 (q, J = 8.3 Hz, 1H), 5.45 (d, J = 7.0 Hz, 1H), 5.27 (s, 2H), 3.78 (t, J = 8.7 Hz, 1H), 3.62 (q, J = 8.7 Hz, 1H), 2.76 (q, J = 7.7 Hz, 2H), 2.61 – 2.51 (m, 1H), 2.49 – 2.43 (m, 1H), 2.27 – 2.19 (m, 1H), 2.14 – 2.04 (m, 1H), 1.24 (m, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz CD₃OD) 178.66, 169.45, 160.16, 157.03, 142.97 (d, ⁴J _CF3 = 1.4 Hz), 134.76, 130.98 (q, ²J _CF3 = 32.0 Hz), 129.17, 128.64, 127.92, 126.47 (q, ³J _CF3 = 3.8 Hz), 125.63 (q, ¹J _CF3 = 271.1 Hz), 120.03, 112.95, 70.15, 56.45, 32.71, 24.48, 24.34, 14.55; HRMS (ESI+): calcd for C₂₃H₂₅F₃N₅O₂ [M]⁺ 460.1955; found, 460.1955.

3-methyl-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (17).

Synthesized according to the following protocol: To a solution of THF (0.2 M), 4-(trifluoromethyl)benzyl alcohol (1.2 equiv) was added followed by blanking with nitrogen gas and chilled to 0 °C. Next, sodium hydride (1.1 equiv) was added and stirred for 5 minutes. 4-fluoro-3-methylbenzonitrile was then added and the reaction was allowed to warm to room temperature and stirred for 16 hours. Subsequently, the resulting solution was partitioned between EtOAc and brine solution. Using additional EtOAc, the brine solution was washed three times and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated via vacuum. The resulting concentrate was purified by silica gel chromatography to yield the final product; 234 mg, 54%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (dd, J = 8.1, 49.1 Hz, 4H), 7.50 – 7.43 (m, 2H), 6.89 (d, J = 8.6 Hz, 1H), 5.19 (s, 2H), 2.30 (s, 3H); ¹³C NMR (101 MHz CD₃OD) 159.87, 140.23 (d, ⁴J _CF3 = 1.4 Hz), 134.34, 132.03, 130.54 (q, ²J _CF3 = 32.4 Hz), 128.67, 127.25, 125.82 (q, ³J _CF3 = 3.8 Hz), 124.12 (q, ¹J _CF3 = 271.7 Hz), 119.38, 111.47, 104.30, 69.31, 16.33; HRMS (ESI+): calcd for C₁₆H₁₃F₃NO [M + H]⁺ 292.0944; found, 292.0924.

N’-hydroxy-3-methyl-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (18).

Synthesized by General Procedure C: 183 mg, 70%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.65 (q, J = 9.3 Hz, 4H), 7.47 – 7.41 (m, 2H), 6.95 (d, J = 8.5 Hz, 1H), 5.20 (s, 2H), 2.29 (s, 3H); ¹³C NMR (101 MHz CD₃OD) 159.10, 155.59, 143.29 (d, ⁴J _CF3 = 1.3 Hz), 130.88 (q, ²J _CF3 = 32.3 Hz), 129.70, 128.55, 127.96, 126.47, 126.39 (q, ³J _CF3 = 3.9 Hz), 126.20, 125.58 (d, ¹J _CF3 = 272.5 Hz), 112.25, 69.99, 16.53; HRMS (ESI+): calcd for C₁₆H₁₆F₃N₂O₂ [M + H]⁺ 325.1158; found, 325.1125.

tert-butyl (S)-2-(3-(3-methyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (19).

Synthesized by General Procedure D: 47 mg, 32%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.94 – 7.83 (m, 2H), 7.60 (dd, J = 7.9, 37.2 Hz, 4H), 6.95 – 6.87 (m, 1H), 5.22 – 5.02 (m, 3H), 3.76 – 3.62 (m, 1H), 3.60 – 3.46 (m, 1H), 2.46 – 2.29 (m, 4H), 2.19 – 2.08 (m, 2H), 2.04 – 1.95 (m, 1H), 1.46 (s, 3H), 1.29 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.49, 168.18, 158.92, 153.68, 140.98, 130.00, 127.84, 127.21, 126.74, 125.67 (d, ³J _CF3 = 3.7 Hz), 124.16 (d, ¹J _CF3 = 272.4 Hz), 119.38, 111.29, 80.52, 69.12, 53.93, 46.46, 32.51, 28.49, 28.25, 23.82, 16.44; HRMS (ESI+): calcd for C₂₆H₂₈F₃N₃NaO₄ [M + Na]⁺ 526.1924; found, 526.1935.

(S)-2-(3-(3-methyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (20).

Synthesized by General Procedure E: 75 mg, 65%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.88 – 7.84 (m, 2H), 7.68 (dd, J = 8.7, 14.6 Hz, 4H), 7.09 (d, J = 9.3 Hz, 1H), 5.45 (d, J = 7.8 Hz, 1H), 5.27 (s, 2H), 3.78 (t, J = 9.6 Hz, 1H), 3.62 (q, J = 8.6 Hz, 1H), 2.61 – 2.51 (m, 1H), 2.49 – 2.42 (m, 1H), 2.33 (s, 3H), 2.27 – 2.19 (m, 1H), 2.14 – 2.03 (m, 1H); ¹³C NMR (101 MHz CDCl₃) 178.65, 169.42, 160.57, 157.05, 142.99 (d, ⁴J _CF3 = 1.3 Hz), 130.98 (q, ²J _CF3 = 32.5 Hz), 130.63, 128.84, 128.63, 127.88, 126.46 (q, ³J _CF3 = 3.9 Hz), 125.59 (q, ¹J _CF3 =271.2 Hz), 119.87, 112.71, 70.12, 56.43, 32.68, 24.32, 16.50; HRMS (ESI+): calcd for C₂₂H₂₃F₃N₅O₂ [M + H]⁺ 446.1798; found, 446.1800.

3-acetyl-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (22).

Synthesized by General Procedure B: 219 mg, 92%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.69 – 7.50 (m, 5H), 7.11 (d, J = 8.3 Hz, 1H), 5.29 (s, 2H), 2.55 (s, 3H); ¹³C NMR (101 MHz CDCl₃) 197.12, 160.21, 138.93 (d, ⁴J _CF3 = 1.2 Hz), 136.97, 134.67, 130.54 (q, ²J _CF3 = 32.4 Hz), 129.12, 127.77, 125.72 (q, ³J _CF3 = 3.7 Hz), 123.82 (q, ¹J _CF3 = 272.7 Hz), 118.05, 113.67, 104.71, 70.25, 31.69; HRMS (ESI+): calcd for C₁₇H₁₃F₃NO₂ [M + H]⁺ 320.0893; found, 320.0896.

3-(prop-1-en-2-yl)-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (23).

Synthesized according to the following protocol: To a solution of THF (0.2 M) methyltriphenylphosphonium bromide (2 equiv) was added followed by cooling reaction to 0 °C and put under an N₂ atmosphere. Next, n-BuLi (2.0 equiv) was added to reaction mixture dropwise and allowed to stir for 10 minutes. 1 equivalent of 3-acetyl-4-((4-(trifluoromethyl)benzyl)oxy)benzonitrile (compound 22) was added portion wise, and the resulting reaction mixture was allowed to warm to room temperature and stir for 18 hours. Subsequently, the resulting solution was partitioned between EtOAc and brine solution. Using additional EtOAc, the brine solution was washed three times and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated via vacuum. The resulting concentrate was purified by silica gel chromatography to yield the final product; 60 mg, 61%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.68 – 7.48 (m, 6H), 6.94 (d, J = 8.4 Hz, 1H), 5.25 – 5.20 (m, 3H), 5.11 (s, 1H), 2.12 (s, 3H); ¹³C NMR (101 MHz CDCl₃) 158.70, 142.04, 139.98 (d, ⁴J _CF3 = 1.3 Hz), 134.52, 133.42, 133.01, 130.47 (q, ²J _CF3 = 32.5 Hz), 127.27, 125.80 (q, ³J _CF3 = 3.8 Hz), 124.08 (q, ¹J _CF3 = 272.4 Hz), 119.13, 117.19, 112.47, 104.62, 69.63, 22.95; HRMS (ESI+): calcd for C₁₈H₁₅F₃NO [M + H]⁺ 318.1100; found, 318.1091.

N’-hydroxy-3-(prop-1-en-2-yl)-4-((4-(trifluoromethyl)benzyl)oxy)benzimidamide (24).

Synthesized by General Procedure C: 556 mg, 90%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.68 – 7.47 (m, 6H), 6.98 (d, J = 9.0 Hz, 1H), 5.19 – 5.07 (m, 4H), 2.10 (s, 3H); ¹³C NMR (101 MHz CDCl₃) 157.86, 155.21, 145.03, 142.90 (d, ⁴J _CF3 = 1.4 Hz), 134.31, 130.83 (q, ²J _CF3 = 32.0 Hz), 128.63, 128.51, 127.51, 126.75, 126.33 (q, ³J _CF3 = 3.8 Hz), 125.54 (d, ¹J _CF3 = 271.4 Hz), 116.19, 113.28, 70.33, 23.51; HRMS (ESI+): calcd for C₁₈H₁₈F₃N₂O₂ [M + H]⁺ 351.1315; found, 351.1295.

tert-butyl(S)-2-(3-(3-(prop-1-en-2-yl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (25).

Synthesized by General Procedure D: 420 mg, 50%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.99 – 7.87 (m, 2H), 7.56 (dd, J = 8.1, 37.8 Hz, 4H), 6.99 – 6.90 (m, 1H), 5.22 – 5.00 (m, 5H), 3.73 – 3.61 (m, 1H), 3.56 – 3.43 (m, 1H), 2.41 – 2.28 (m, 1H), 2.16 – 2.04 (m, 5H), 2.00 – 1.91 (m, 1H), 1.43 (s, 3H), 1.28 (s, 6H); ¹³C NMR (101 MHz CDCl₃) 180.46, 167.91, 157.63, 153.53, 142.94, 140.68, 133.80, 130.02 (q, ²J _CF3 = 32.4 Hz), 128.76, 127.85, 127.16, 125.52 (d, ³J _CF3 = 3.7 Hz), 124.09 (q, ¹J _CF3 = 271.9 Hz), 119.61, 116.22, 112.24, 80.35. 69.37, 53.79, 46.33, 32.35, 28.10, 23.67, 23.11; HRMS (ESI+): calcd for C₂₈H₃₁F₃N₃O₄ [M + H]⁺ 530.2261; found, 530.2231.

tert-butyl (S)-2-(3-(3-isopropyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (26).

Synthesized according to the following protocol: To a solution of ethanol (190 proof, 0.2 M) addition of tert-butyl(S)-2-(3-(3-(prop-1-en-2-yl)-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (1 equiv, compound 25) followed by 10% palladium on carbon (0.1 equiv) was performed and stirred. Reaction mixture was then put under light vacuum followed by purge of hydrogen gas. Vacuum followed by addition of hydrogen gas was repeated three times. The resulting mixture was stirred at room temperature for 18 hours followed by concentration via vacuum and purification by silica gel chromatography to yield final product; 178 mg, 72%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (s, 1H), 7.90 – 7.84 (m, 1H), 7.59 (dd, J = 7.9, 36.9 Hz, 4H), 6.98 – 6.88 (m, 1H), 5.21 – 5.01 (m, 3H), 3.74 – 3.63 (m, 1H), 3.57 – 3.38 (m, 2H), 2.41 – 2.29 (m, 1H), 2.17 – 2.05 (m, 2H), 1.99 – 1.91 (m, 1H), 1.44 (s, 3H), 1.35 – 1.22 (m, 12H); ¹³C NMR (101 MHz CDCl₃) 180.34, 168.26, 157.88, 153.58, 140.96, 137.93, 130.06 (q, ²J _CF3 = 32.1 Hz), 127.14, 126.49, 125.56 (q, ³J _CF3 = 3.6 Hz), 124.18 (q, ¹J _CF3 = 272.2 Hz), 119.62, 111.52, 80.35, 69.11, 53.83, 46.35, 32.37, 28.35, 28.13, 27.08, 23.69, 22.49; HRMS (ESI+): calcd for C₂₈H₃₃F₃N₃O₄ [M + H]⁺ 532.2418; found, 532.2408.

(S)-2-(3-(3-isopropyl-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboximidamide (27).

Synthesized by General Procedure E: 24 mg, 55%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.70 (q, J = 7.2 Hz, 4H), 7.14 (d, J = 8.5 Hz, 1H), 5.44 (d, J = 7.6 Hz, 1H), 5.29 (s, 2H), 3.79 (t, J = 9.4 Hz, 1H), 3.62 (q, J = 9.0 Hz, 1H), 3.45 (p, J = 6.6 Hz, 1H), 2.62 – 2.53 (m, 1H), 2.51 – 2.43 (m, 1H), 2.28 – 2.20 (m, 1H), 2.14 – 2.05 (m, 1H), 1.28 (d, J = 7.1 Hz, 6H); ¹³C NMR (101 MHz CDCl₃) 178.70, 169.60, 159.65, 157.07, 142.98 (d, ⁴J _CF3 = 1.4 Hz), 139.09, 131.04 (q, ²J _CF3 = 32.2 Hz), 128.73, 127.73, 126.51 (q, ³J _CF3 = 3.8 Hz), 126.34, 125.61 (q, ¹J _CF3 = 271.6 Hz), 120.16, 113.18, 70.32, 56.47, 32.74, 28.28, 24.36, 22.88; HRMS (ESI+): calcd for C₂₄H₂₇F₃N₅O₂ [M]⁺ 474.2111; found, 474.2115.

ABBREVIATIONS USED

S1P

Sphingosine-1-phosphate

Sph

sphingosine

SphK

sphingosine kinase

HCTU

O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

TBAF

tetra-n-butylammonium fluoride