Transforming Sphingosine Kinase 1 Inhibitors into Dual and Sphingosine Kinase 2 Selective Inhibitors: Design, Synthesis, and In Vivo Activity

Abstract

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that interacts with its five G-protein coupled receptors S1P_1-5 to regulate cell growth and survival and has been implicated in a variety of diseases including cancer and sickle cell disease. As the key mediators in the synthesis of S1P, sphingosine kinase (SphK) isoforms 1 and 2 have attracted attention as viable targets for pharmaceutical inhibition. In this report, we describe the design, synthesis, and biological evaluation of aminothiazole-based guanidine inhibitors of SphK. Surprisingly, combining features of reported SphK1 inhibitors generated SphK1/2 dual inhibitor 20l (SLC4011540) (hSphK1 K_i = 120 nM, hSphK2 K_i = 90 nM) and SphK2 inhibitor 20dd (SLC4101431) (K_i = 90 nM, 100-fold SphK2 selectivity). These compounds effectively decrease S1P levels in vitro. In vivo administration of 20dd validated that inhibition of SphK2 increases blood S1P levels.

Graphical abstract

INTRODUCTION

Sphingosine 1-phosphate (S1P) is a ubiquitous cellular signaling molecule that has been implicated in a variety of diseases including cancer,^1–4 fibrosis,^5–7 Alzheimer’s,^{8, 9} sickle cell disease,^{10, 11} and viral infections such as Chikungunya virus.¹² S1P interacts with proteins both within and outside of the cell. As an extracellular ligand, S1P promotes cell migration and survival by binding to five G-protein coupled receptors, S1P_1-5.^13–16 Although its function as an intracellular ligand is less well defined, S1P is reported to control epigenetic modifications through regulation of HDAC 1/2 activity via sphingosine kinase isotype 2 (SphK2) and alter erythrocyte glycolysis to increase O₂ release under hypoxic conditions.^17–19 S1P is synthesized by the phosphorylation of sphingosine (Sph) by SphK, which exists as two isotypes – SphK1 and SphK2. SphK1 and SphK2 share approximately 50% sequence identity²⁰ but differ with respect to their function, in part due to their differing localization within the cell. SphK1 is a cytosolic enzyme that promotes cell survival and proliferation,^{21, 22} whereas some SphK2 is found in the nucleus^{17, 18} but can relocate to the cytosol on phosphorylation.^{23, 24} Depending on this localization, SphK2 can promote either apoptosis^12,13 or cell proliferation.^{17, 18, 25, 26} Although isotype-specific SphK null mice are viable, fertile, and phenotypically unremarkable,²⁷ SphK1-null mice have about a two-fold reduction in blood S1P levels while SphK2-null mice have 2-4 fold increased blood S1P levels.^28–32 However, ablation of both SphK isotype genes in the mouse is embryonically lethal in mid gestation as a consequence of complications during vascular and neurological development.^{27, 32}

The therapeutic potential of drugging the S1P pathway was realized by the approval of Fingolimod by the Food and Drug Administration for the treatment of multiple sclerosis.^{33, 34} Fingolimod is a pro-drug that is phosphorylated by SphK2 to act as a functional antagonist of S1P receptors, S1P1/3. Other approaches have also been employed to manipulate S1P activity including targeting SphKs to control S1P synthesis.^{3, 35–37} There are currently multiple reports of potent SphK1-selective inhibitors and SphK1/SphK2-dual inhibitors (Figure 1). Inhibitors such as the SphK1-selective inhibitor 1 (PF-543)^{38, 39} and the SphK1/SphK2-dual inhibitor 5 (SKI-II)^40–42 have been co-crystallized with SphK1 and have been useful in developing next generation inhibitors.^{43, 44} Indeed, medicinal chemists at Amgen improved 5 to realize a selective SphK1 inhibitor, 2 (Amgen 23),⁴⁵ while preserving the aminothiazole region. While 5 is recently reported to have off-target activity against dihydroceramide desaturase,⁴⁶ 1 has shown promise for disease states such as sickle cell disease, where inflammation, cell sickling, and hemolysis were reduced in treated mouse models.¹¹

Figure 1.

Select SphK Inhibitors

In contrast with SphK1 inhibitors, highly potent SphK2-selective inhibitors are lacking. Early SphK2 inhibitors (8 (SLR080811),^{29, 47} 9 (ABC294640),⁴⁸ and 10 (K145)^{49, 50}), which have low micromolar potency and are modestly selective vs. SphK1, are active in cultured cells as measured by lowering of cellular S1P levels. Among these, 9 has been deployed in a variety of animal disease models, including ulcerative colitis,⁵¹ Crohns disease,⁵² ischaemia/reperfusion injury,⁵³ osteoarthritis,⁵⁴ and colon cancer.⁵⁵ However, 9 has recently been reported to have an off-target effect of acting as a tamoxifen-like molecule with the estrogen receptor.⁵⁶ The development of improved inhibitors that are SphK2 specific will help in understanding the physiological function of SphK2 in vivo. Recently, SphK2 was shown to play a role in endothelial cell barrier integrity as well as attenuation of kidney fibrosis through interferon gamma using the azetidine analogue of 8.^{57, 58} In addition, an oncogenic role for SphK2 is emerging where high-level overexpression is associated with reduced cell survival and proliferation.⁵⁹

The paucity of small molecule chemical biology tools for investigating SphK2 function prompted our interest in developing better selective SphK2 inhibitors. We previously reported two new guanidine-based inhibitors: the SphK1/SphK2 dual-inhibitor 7 (SLC5111312)^{28, 60} and the SphK2-selective inhibitor 11 (SLM6031434)²⁸ (>50-fold selective). Treatment of mice with these inhibitors slows the clearance of mass-labeled S1P from the bloodstream, which implicates SphK2 in the disposal of circulating S1P. Although the mechanism whereby SphK2 influences S1P clearance from blood is currently unclear, these results highlight the need for more potent and selective SphK2 and SphK1/SphK2 dual inhibitors to explore this phenomenon further. Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of guanidine-based aminothiazoles with in vivo activity as selective SphK2 inhibitors.

RESULTS AND DISCUSSION

Inhibitor Design and Development

Our group reported the synthesis of 3 (SLP7111228),⁴⁷ an SphK1-selective inhibitor with a K_i of 48 nM and >100-fold selectivity for SphK1 over SphK2 for both the rat and human enzymes. This compound was found to reduce S1P levels in both cultured human U937 leukemia cells and in the bloodstream of rats. The latter result recapitulates the phenotype of SphK1 null mice.^{32, 47} In developing this molecule, the addition of a methylene unit between the 1,2,4-oxadiazole and pyrrolidine rings was key in converting a 10-fold SphK2-selective inhibitor (8) into a >100-fold SphK1-selective inhibitor. In this work, we sought to improve 3’s potency and selectivity further by replacing the octyl chain with an aminothiazole group, as present in SphK1 inhibitors such as 2 (Figure 2). Docking of 2 into the binding pocket of a homology model of SphK2 and superimposition with 2’s “aminothiazole tail” linked to an oxadiazole phenyl ring suggested binding interactions mimicking 2 (Figure 3). We hypothesized that these altered ‘tail’ groups, represented by the 14-18 carbon aliphatic groups of sphingoid bases, would create inhibitors that can adopt the necessary “j-shape” for strong binding interactions with the sphingosine binding pocket, as established by published co-crystal structures of SphK1 with the aminothiazole-based inhibitors 2 and 5.^{44, 45} Gustin et al. recently developed potent aminothiazole-based SphK1/SphK2-dual inhibitors derived from 5, including 2.⁴⁵ Although most compounds reported were dual-inhibitors, some compounds in their series show a bias towards SphK1 inhibition with attractive pharmacokinetic properties. Vogt and co-workers subsequently reported the synthesis of aminothiazole-based derivatives of 5 that are SphK1/SphK2-dual inhibitors with several inhibitors showing a slight bias for SphK2 selectivity.⁶¹ However, these molecules show only micromolar potency, and extensive biological studies have yet to be reported. Aurelio et al. also recently reported the synthesis of derivatives of 5 that show limited success as SphK1/SphK2 dual inhibitors and SphK1- and SphK2-selective inhibitors.⁶² Due to the prominence of aminothiazole moieties in SphK inhibitor scaffolds, we incorporated aminothiazole moieties into our guanidine-based scaffold, specifically SphK1-selective inhibitors.

Figure 2.

Tail group modifications towards the development of new “j-shaped” SphK1 inhibitors.

Figure 3.

Superimposition of lowest energy docked poses of 2 (purple sticks, colored by atom) and aminothiazole analogue of 3 (teal stick, color by atom) in homology model of SphK2. Key residues in the binding cavity are shown as grey sticks, ATP is shown green colored by atom, and the overall protein structure is shown as grey cartoon for perspective.

Inhibitor Synthesis

In prior studies,^{47, 63} we established that the guanidine, 1,2,4-oxadiazole, and internal phenyl ring moieties were key features of the sphingosine kinase inhibitor scaffold. Therefore, we focused our attention on the ‘tail’ region by appending aminothiazoles decorated with diverse aryl structures. The synthesis of aryl-substituted aminothiazoles is shown in Scheme 1. 4-Trifluoromethylacetamide benzonitrile 13 was synthesized by the acetylation of 4-aminobenzonitrile 12 with trifluoroacetic anhydride. Benzonitrile 13 was then reacted with hydroxylamine hydrochloride and triethylamine in ethanol using a microwave reactor to yield amidoxime 14, which was reacted with homoproline using HCTU and Hunig’s base at 100 °C to afford the 1,2,4-oxadiazole 15. The trifluoroacetate group was removed using lithium hydroxide to afford amine 16. Treatment of 16 with thiocarbodiimidazole followed by ammonia provided the key intermediate thiourea 17. Aminothiazoles 18a-ee were produced by reacting the required α-bromoketone with thiourea 17 and Hunig’s base in ethanol in a microwave reactor. The Boc group was removed with trifluoroacetic acid and immediately reacted with N,N′-di-Boc-1H-pyrazole-1-carboxamidine in a microwave reactor to afford the bis-Boc-protected guanidines 19a-ee. Bubbling HCl gas subsequently provided the desired guanidine derivatives 20a-ee (see Tables 1 and 2 for structures).

Scheme 1.

Synthesis of aryl-substituted aminothiazole derivatives 20a-ee.^a

^a Reagents and conditions: (a) trifluoroacetic anhydride, DCM, 0 °C – rt, 19 h, 88%; (b) NH₂OH^.HCl, TEA, ACN, 150 °C microwave, 6 min, 57%; (c) Boc-L-homoproline, HCTU, DIEA, DMF, rt – 100 °C, 4 h, 67%; (d) 1:1 1M LiOH:MeOH, 100 °C, 3 h, 82%; (e) Thio-CDI, THF, rt, 4 h; (f) NH₃(g), 1 min, 86%; (g) α-bromoketone, DIEA, EtOH, 100 °C, microwave, 5 min, 30-90%; (h) 1:1 TFA:DCM (i) N, N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, ACN, 50 °C, microwave, 2 h, 26-87%; (j) HCl (g), MeOH, 90-100%.

Table 1.

SphKl and SphK2 Inhibitory Activity for Mono-Substituted Aminothiazole Derivatives.^a

Compound	R	X	hSphKl	hSphK2	Compound	R	X	hSphKl	hSphK2
20a		NH	100 ±6	83 ±4	20k		NH	57 ± 0.4	36 ± 1
20b		NH	100 ±5	94 ±4	201		NH	31 ± 12	28 ± 0.7
20c		NH	95 ± 10	94 ±7	20m		NH	69 ±4	72 ±6
20d		NH	100 ± 13	55 ±1	20n		NH	89 ± 0.7	44 ±12
20e		NH	70 ±3	51 ± 0	20o		NH	58 ±4	82 ±6
20f		NH	88 ±4	46 ±6	20p		NH	75 ± 0.2	80 ±7
20g		NH	60 ± 1	45 ±2	20q		NH	91± 2	100 ±2
20h		NH	64 ± 1	45 ±10	20r		NH	100 ±9	84 ±2
20i		NH	79 ± 12	44 ±8	20s		NH	90 ± 1	100 ±2
20j		NH	50 ±4	44 ±9

^aSphK activity is presented as % control (no inhibitor added). Recombinant human SphK1 or SphK2 was isolated from a cell lysate, and enzyme activity was measured with 5 μM (SphKl) or 10 μM (SphK2) sphingosine and 250 μM y-[³²P]ATP. Compounds were assayed at 0.3 μM in triplicate.

Table 2.

SphKl and SphK2 Inhibitory Activity for Di-Substituted and Bulky Aminothiazole Derivatives.^a

Compound	R	X	hSphKl	hSphK2	Compound	R	X	hSphKl	hSphK2
20t		NH	91 ±4	67 ±12	20cc		NH	65 ±2	48 ±1
20u		NH	100 ±7	100 ±7	20dd		NH	100 ±2	27 ± 12
20v		NH	100 ±0.2	71 ± 11	23a		NH	92 ±8	73 ±7
20w		NH	51 ± 5	51 ± 5	23b		NH	82 ±9	31 ±7
20x		NH	51 ±4	46 ± 11	23c		NH	100 ±2	96 ±1
20y		NH	58 ±5	33 ±5	23d		NH	99 ±6	85 ±3
20z		NH	68 ±7	43 ±8	20ee		NH	68 ±7	67 ±5
20aa		NH	51 ±4	30 ±15	31		O	62 ±4	55 ±2
20bb		NH	100 ±4	63 ±4	34		NCH3	100 ±5	100 ±5

^aSphK activity is presented as % control (no inhibitor added). Recombinant human SphK1 or SphK2 was isolated from a cell lysate, and enzyme activity was measured with 5 μM (SphKl) or 10 μM (SphK2) sphingosine and 250 μM y-[³²P]ATP. Compounds were assayed at 0.3 μM in triplicate.

Biphenyl derivatives were synthesized as outlined in Scheme 2. Using either compound 18f or 18i, Suzuki-Miyaura cross-coupling with various aryl boronic acids yielded the biaryl aminothiazoles 21a-d. The standard synthetic sequence of deprotection, guanylation, and deprotection was then followed to yield the desired biaryl aminothiazole derivatives 23 a-d.

Scheme 2.

Synthesis of biphenyl aminothiazole derivatives 23 a-d.^a

^aReagents and conditions: (a) aryl boronic acid, PdCl₂(dppf), Cs₂CO₃, DMF, 150 °C microwave, 90 min; (b) 1:1 TFA:DCM; (c) N, N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, ACN, 50 °C, microwave, 2 h; (d) HCl (g), MeOH.

To assess the significance of the hydrogen bond effect in this series of aminothiazole, a comparison of activity between a representative aminothiazole example of the series (20k, Table 1) and its analogous oxathiazole was done. The corresponding oxathiazole derivative was synthesized as presented in Scheme 3. 4-Tert-butoxy benzonitrile 25 was synthesized via nucleophilic aromatic substitution of 4-fluorobenzonitrile 24 with 1 M potassium tert-butoxide. The benzonitrile 25 was then reacted with hydroxylamine hydrochloride and triethylamine in ethanol using a microwave reactor to yield its amidoxime that was then reacted with homoproline using HCTU and Hunig’s base at 100 °C to afford the 1,2,4-oxadiazole 26. The phenol derivative 27 was achieved via treatment of 1,2,4-oxadiazole 26 with trifluoroacetic acid followed by reprotection with Boc-anhydride. Nucleophilic aromatic substitution with the phenol derivative 27 on 2,4-dibromothiazole afforded the oxathiazole 28. Suzuki-Miyaura cross-coupling with 4-trifluoromethylphenylboronic acid yielded the desired oxathiazole 29. The standard synthetic sequence of deprotection, guanylation, and deprotection was followed for compound 29 to yield the desired oxathiazole derivative 31.

Scheme 3.

Synthesis of the oxathiazole analogue of inhibitor 20k.^a

^aReagents and conditions: (a) 1M KO^tBu; THF, reflux, 15 h; (b) NH₂OH^.HCl, TEA, EtOH, 150 °C microwave, 6 min; (c) Boc-L-homoproline, HCTU, DIEA, DMF, rt – 100 °C, 4 h; (d) 1:1 TFA:DCM (e) di-tert-butyl dicarbonate, TEA, dioxane, 0 °C, 1 h; (f) 2, 5-dibromothiazole, K₂CO₃, DMF, 135 °C, 1 h; (g) 4-trifluoromethylphenyl boronic acid, PdCl₂(dppf), Cs₂CO₃, DMF, 150 °C, microwave, 90 min; (h) N, N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, ACN, 50 °C, microwave, 2 h; (i) HCl (g), MeOH.

To further probe the significance of the hydrogen bond effect of the amino moiety in 20dd, its methylated derivative was synthesized as illustrated in Scheme 4. The biaryl aminothiazole 18dd was methylated with methyl iodine in refluxing acetone to yield the 32. The standard synthetic sequence of deprotection, guanylation, and deprotection was then followed to yield the desired methylated aminothiazole derivative 34.

Scheme 4.

Synthesis of the methylated analogue of 20dd.^a

^aReagents and conditions: (a) MeI, K₂CO₃, acetone, reflux, 17 h; (b) 1:1 TFA:DCM; (c) N, N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, ACN, 50 °C, microwave, 2 h; (d) HCl (g), MeOH.

Structure-Activity Relationship Studies and Biological Evaluation of Derivatives

With the goal of defining the structure-activity profile of SphKs, a focused library of aminothiazoles bearing a guanidine group was synthesized. These analogues were assayed using a previously described protocol.^{47, 64} In particular, synthesized inhibitors were tested at 0.3 μM with human recombinant enzymes (Tables 1 & 2). All aminothiazoles vary with respect to the number and type of substituents on the appended aryl ring as well as the substitution pattern on this ring.

As shown in Table 1, substituting the thiazole ring with an unsubstituted phenyl ring (20a) resulted in poor inhibition of either SphK isotype in our assay. Replacement of the phenyl ring with either a 4’ (20b) or 3’ (20c) pyridyl ring also produced compounds that were inactive with both SphK isotypes. Halogen substituents at the para position of the phenyl ring inhibited both enzymes but with slight selectivity for SphK2. The selectivity and potency for SphK2 was unanticipated because these compounds were expected to inhibit SphK1, as previous work from our laboratories demonstrated that the homologated guanidine-pyrrolidine head group⁴⁷ generates an SphK1 inhibitor when the ‘tail’ is an unsubstituted octyl group. Furthermore, aminothiazoles in the Amgen series are also selective for SphK1,⁴⁵ although their scaffold differed in other aspects also. The identity of the halogen atom – fluorine, chlorine, or bromine – did not greatly affect the compounds’ (20d-f) SphK2 potency, reducing the enzyme activity to ~ 50%. Moving the halogen group to the meta or ortho position (20g-j) resulted in a retention of SphK2 potency, but with a loss in SphK2 selectivity, producing only moderately SphK2 selective inhibitors.

Because of the SphK2 selectivity observed with halogen moieties at the para position, a bulkier electron-withdrawing trifluoromethyl group at the para position (20k (SLC4071411)) was synthesized. 20k was more potent but less selective for SphK2. Interestingly, placement of the trifluoromethyl group at the meta position (20l) produced a more potent SphK inhibitor (28% inhibition). However, this molecule showed no selectivity for SphK1 vs. SphK2. The ortho trifluoromethyl analogue (20m) did not improve potency nor selectivity. These results suggest that the key binding interactions were lost with the ortho-substituted derivative likely due to a loss in CF₃ interactions with the residues Cys533, His556 and Tyr566 that are found at the end of the hydrophobic tunnel of the SphK2 binding pocket, which was shown⁶⁰ by our group to be important for strong inhibitor binding.

Replacement of the electron-withdrawing groups with electron-donating groups– methyl (20n), methyl ether (20o), and ethyl ether (20p) – at the para position markedly diminished SphK2 inhibition (<30%) (Table 1). Likewise, positioning the methyl ether to the meta position (20q) did not improve inhibitory activity at either enzyme isotype. A trifluoromethyl ether group was then tested at the para position for increased lipophilicity and to reestablish deactivating electronics^{65, 66} to the aryl ring. The resulting molecule (20r (SLC4081418)) decreased SphK2 activity to ~40%, which is likely due to a reestablishment of the fluorine bonding interactions mentioned (vide supra). Good selectivity was also observed with this molecule, as SphK1 activity was only diminished by ~10%. However, shifting the trifluoromethyl ether moiety from the para position to the meta position (20s) led to a switch in SphK potency and selectivity. Collectively, these results indicate that placement of electron-donating groups on the aryl ring is unfavorable for SphK2 inhibition.

The effects of di-substitution on the aryl ring of the aminothiazole were also explored (Table 2). Although poor inhibition activities were observed with mono-substituted aryl rings containing traditional electron-donating groups, we were curious about the effects of having di-substituted aryl rings. Compounds with either two methyl (20t) or two methyl ether moieties (20u) showed minimal activity. Placement of a methyl ether at the para position and the chlorine moiety at the meta position (20v) led to modest SphK2 activity (~70%) and no SphK1 inhibition. In contrast, di-substitution with electron-withdrawing groups reestablished SphK2 potency, particularly fluorines (20w, 20z), chlorine (20x), or a combination of fluorine with a trifluoromethyl group (20y (SLC4091423), 20aa (SLC4091424), 20cc). The substitution pattern for these molecules did not significantly affect their SphK2 potency; however, they did show SphK1/SphK2 dual inhibitor activity, affording good SphK2 inhibition (50-70%) and modest to good SphK1 inhibition (30-50%). Disubstitution of the aryl ring with two trifluoromethyl moieties (20bb) led to a loss in compound potency, but this loss in potency was met with a gain in selectivity, as it was inactive towards SphK1 at concentrations up to 1 μM. Together, these results reinforce the preference for electron deficient aryl ring.

Our previous studies^{63, 67} suggest that the SphK2 lipid-binding pocket is larger than that of SphK1. To explore the effects of bulky moieties on the aminothiazole ring, biphenyl derivatives were synthesized and tested (Table 2). We discovered that the para-substituted biphenyl derivative (20dd) was not only potent towards SphK2 but also selective for SphK2. The meta-substituted biphenyl derivative (23a) essentially lost activity. However, a fluorine on the 4-position (23b) maintained the potency and selectivity observed with 20dd. Attempts to substitute 20dd with a trifluoromethyl moiety at either the 4- (23c) or 3-position (23d) on the biphenyl ring led to compounds inactive in both SphKs, suggesting that the molecules may either be too large for the binding pocket or have poor solubility (clogP = 6.78). In an effort to introduce additional bulk onto the aminothiazole, a benzofuran moiety (20ee) was synthesized but was found to be ineffective as an inhibitor. Collectively, these results suggest that the biphenyl moiety has the optimal binding interaction as well as ‘bend’ to fit in the ‘J-shaped’ hydrophobic tunnel.

In addition to investigating the effects of various aryl groups on the aminothiazoles, we also explored the effects of modifying the exocyclic nitrogen of the aminothiazoles (Table 2). The co-crystal structure of SphK1 with 5 reported by Wang et al. notes a key hydrogen bonding interaction between the aminothiazole NH and SphK1 threonine-196.⁴⁴ Although our scaffold would most likely fit slightly deeper into the SphK1 binding pocket, we probed the significance of this hydrogen bonding capability by either replacing the NH with an ether linkage (31) or methylating (34) the amino group. The N-Me derivative 34 was found to be completely inactive in both SphKs up to 1 μM while the ether derivative 31 showed moderate activity towards both SphKs, decreasing SphK1 and SphK2 activity by ~ 45% at 0.3 μM. These results suggest that the amino moiety plays a key role in enzyme binding likely through hydrogen bonding (compare 34 with 20dd) although we cannot rule out the loss in SphK2 potency may also be due to steric effects created from the methylation. Comparison of 31 with the amino moiety 20k shows a loss in SphK2 potency with equipotent SphK1 inhibition, indicating that the molecules’ hydrogen bonding role (donor vs. acceptor) at this position is more significant in SphK2 than SphK1.

To quantify the aminothiazoles’ activity towards SphK1 and SphK2, we determined the inhibitory constant (K_i) of the most potent compounds in the library (Table 3). Consistent with the results of the initial screen (Tables 1 & 2), fluoro and trifluoromethyl positional isomers of disubstituted aryls 20y and 20aa had good activity with both enzymes and partial selectivity towards SphK2 (~5 fold) (entries 1-2). Fortunately, monosubstitution with a meta-trifluoromethyl group (20l) by removal of the fluorine atom from 20y or 20aa resulted in improved binding inhibition (entry 3). 20l is a dual inhibitor with K_i of 120 nM and 90 nM with SphK1 and SphK2, respectively. Moving the trifluoromethyl group to the para position had a negative inhibitory effect as expected (entry 4). However, switching to a trifluoromethylether on the para position (20r) improved the K_i towards SphK2 (250 nM) while decreasing inhibitory activity against SphK1 (K_i 5 μM), affording 20-fold selectivity towards SphK2 (entry 5). The introduction of a more lipophilic and larger substituent such as a phenyl ring on the para position (20dd) resulted in a potent and selective SphK2 inhibitor (K_i 90 nM). To the best of our knowledge, 20dd is the most potent SphK2 reported to date; this compound is 100-fold selective towards SphK2 vs SphK1. In comparing compounds 20k, 20r, and 20dd, it is interesting to note that as the steric bulk and lipophilicity of the molecules (cLogP 5.18, 5.42 and 6.18, respectively) increased, the inhibition constant and selectivity improved. These results are consistent with the observation that the hydrophobic binding pocket of SphK2 is larger than SphK1.

Table 3.

K_i and cLogP^a values of Selected Inhibitors of SphKl and SphK2.

Entry	Compound	Structure	hSphKl Ki(μM)	hSphK2 Ki(μM)	hSphK2 Selectivity	cLogP
1	20y		0.7 ±0.05	0.17 ±0.15	4	5.33
2	20aa		0.8 ±0.07	0.17 ±0.17	5	5.33
3	201		0.12 ±0.04	0.09 ±0.01	1	5.18
4	20k		0.82 ± 0.25	0.39 ± 0.09	2	5.18
5	20r		5 ±0.22	0.25 ± 0.10	20	5.42
6	20dd		9 ± 2	0.09 ± 0.02	100	6.18

^acLogP was calculated for the protonated inhibitor with Chemdraw Professional 13.0.

We selected the most potent, SphK2 selective (20dd) and SphK1/2 dual inhibitors (20l) towards their effect on S1P synthesis. U937 cells, a histiocytic lymphoma myeloid cell line that expresses both SphK isotypes, were incubated with either compound. Following incubation, the cells were lysed and sphingosine and S1P levels were determined by LC-MS-MS. Both inhibitors showed a concentration-dependent decrease in S1P levels (Figure 4A/C) while Sph levels remained constant (Figure 4B/D), indicating that the compounds are cell permeable and capable of inhibiting SphK activity in whole cells. Further, the selectivity of 20dd towards SphK2 in vitro is observed as its effect on S1P level is lower than 20l—S1P is still generated by functional SphK1. To assess the in vivo properties of 20dd, C57BL/6 mice were treated with a single 10 mg/kg intraperitoneal dose. S1P concentration in blood was monitored over a course of 24 hours via LC-MS-MS. As shown in Figure 5, blood S1P levels increased on treatment with the SphK2 selective inhibitor 20dd at two and a six-hour time points and returned to basal levels after 24 hours. Such an observation is in accordance with three SphK2 selective inhibitors^{28, 29, 47} and supports the notion that SphK2 selective inhibitors drive elevated S1P levels in whole blood.²⁸ To date, 20dd is the most potent and selective SphK2 inhibitor reported with in vivo activity.

Figure 4.

Effect of 20l and 20dd on sphingolipids in U937 cells. Following 2 h of incubations U937 cells were harvested by centrifugation, lysed, and levels of (A, C) S1P and (B, D) sphingosine were measured using LC-MS-MS. Amounts associated with cells are expressed as the number of pmoles per 10⁶ cells. The experiment was performed in duplicate. The level of significance is indicated for each experiment (**P<0.005, ***P<0.001) using an unpaired t test (compared to control).

Figure 5.

Detected S1P blood levels in mice following injection with 20dd. Wild-type mice were treated with a single 10 mg/kg ip dose of 20dd and blood samples were collected at the indicated time points. S1P levels from the blood samples were measured via LC-MS-MS. The standard deviations are values from a group of three to four mice. The level of significance is indicated for each experiment (**P < 0.01) using one-way analysis of variance with the Bonferroni multiple comparison test.

CONCLUSIONS

Herein, we disclose the discovery and development of the most potent and selective SphK2 inhibitor reported to date. The scaffold contains a guanidine head and aminothiazole ‘tail’ groups. A surprising result of our studies is that principles obtained from earlier SphK1 inhibitors – insertion of a methylene unit between the oxadiazole and pyrrolidine ring of 3 and aminothiazole from 2 – generated a potent and selective SphK2 inhibitor. Unfortunately, the X-ray crystal structure of SphK2 is not yet available. Thus, docking of inhibitors in the binding site using a validated homology model of SphK2 has been utilized to facilitate the development of inhibitors. Structure-activity relationship studies of these inhibitors indicate that potent inhibition of both SphK1 and SphK2 necessitates electron-deficient phenyl ring, but these substituents likely benefit from interacting with residues Cys533, His556 and Tyr566 at the end of the binding pocket. Our investigations also support the importance of hydrogen bonding with the exocyclic NH of the aminothiazole ring; removal of hydrogen bonding capacity resulted in significant loss in activity.

Biological analysis of the aminothiazole inhibitors 20l and 20dd effectively lower S1P levels in U937 cells. In vivo study of the SphK2 selective 20dd caused elevated blood S1P levels in wildtype mice, recapitulating our previous findings^{28, 29, 47, 60} with SphK2 selective inhibitors. Collectively, our work provides a novel chemical biology approach towards selective SphK2 and dual SphK inhibition. We expect that these studies will aid in elucidating the in vivo function of SphK2 as well as the development of improved SphK inhibitors.

EXPERIMENTAL SECTION

Sphingosine Kinase assays

The inhibitory activity of the synthesized compounds on human SphK1 and SphK2 was determined using a previously published method.^{28, 47} Recombinant human SphK1 or SphK2 isolated from a cell lysate was briefly incubated with (0.3 μM) or without compound, sphingosine, and γ-[³²P]ATP. The radiolabeled sphingosine 1-phosphate was isolated via extraction and thin-layer chromatography and then quantified via scintillation counting.

Sample Preparation and LC-MS-MS Analysis

Sample preparation protocols were from our previous publication²⁹ with minor modifications. Cell pellets (2–3 × 10⁶ cells), whole blood (10 μl) or plasma (10 μl) was mixed with 2 ml of a methanol:chloroform solution (3:1) and transferred to a capped glass vial. Suspensions were supplemented with 10 μl of internal standard solution containing 10 pmoles of deuterated (D7) S1P and deuterated (D7) sphingosine. The mixture was placed in a bath sonicator for 10 min and incubated at 48°C for 16 h. The mixture was then cooled to ambient temperature and mixed with 200 μl of 1M KOH in methanol. The samples were again sonicated and incubated a further 2 h at 37 °C. Samples were then neutralized by the addition of 20 μl of glacial acetic acid and transferred to 2 ml microcentrifuge tubes. Samples were then centrifuged at 12,000 × g for 12 min at 4 °C. The supernatant fluid was collected in a separate glass vial and evaporated under a stream of nitrogen gas. Immediately prior to LC-MS analysis, the dried material was dissolved in 0.3 ml of methanol and centrifuged at 12,000 × g for 12 min at 4 °C. Fifty μL of the resulting supernatant fluid were analyzed by Liquid Chromatography-ESI Mass Spectrometry (LC-MS) using a triple quadrupole mass spectrometer (AB-Sciex 4000 Q-Trap) coupled to a Shimadzu LC-20AD LC system. A binary solvent gradient with a flow rate of 1 ml/min was used to separate sphingolipids and drugs by reverse phase chromatography using a Supelco Discovery C18 column (50 mm × 2.1 mm, 5 μm bead size). Mobile phase A consisted of water : methanol : formic acid (79:20:1) while mobile phase B was methanol : formic acid (99:1). The run started with 100% A for 0.5 minutes. Solvent B was then increased linearly to 100% B in 5.1 minutes and held at 100% for 4.3 min. The column was finally re-equilibrated to 100% A for 1 min. Natural sphingolipids were detected using multiple reaction monitoring (MRM) as follows: S1P (380.4 → 264.4); deuterated (D7)C₁₈S1P (387.4 → 271.3); sphingosine (300.5 → 264.4); deuterated (D7) sphingosine (307.5 → 271.3).

Pharmacokinetic Analysis

Mouse studies were conducted using a previously reported method.⁴⁷ 20dd (10 mg/kg) and 20l (3 mg/kg) were administered intraperitoneally to groups of 3 to 4 mice (strain: C57BL6/j) or an equal volume of vehicle (2% solution of hydroxypropyl-β-cyclodextrin (Cargill Cavitron 82004)). Blood samples were then collected at the specified time points (ASAP time points were collected 1-2 min following drug addition). The blood samples were analyzed via LC-MS, as described (vide supra). Animal protocols were approved prior to experimentation by the University of Virginia’s School of Medicine Animal Care and Use Committee.

Molecular Docking

Molecular docking was performed using compounds (2, 20x) to assess potential difference in position in the 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.⁶⁰ Marvin was used for drawing, displaying and characterizing chemical structures, substructures and reactions for preparation in docking programs, Marvin 17.3.13, 2017, ChemAxon (http://www.chemaxon.com). AutoDock Tools⁶⁸ was used to prepare the protein and ligand files, while AutoDock Vina⁶⁹ was used to perform the docking for pose prediction. The grid box was set to 20 × 20 × 28 Angstrom, with a 1.000 Å grid spacing was used. The center of the box was placed at the approximate center of the ligand-binding cavity, with a part of the ATP binding cavity included as previously performed⁶⁰ and to ensure coverage and interaction with key Asp residues. Up to ten docked poses were predicted for each compound. The number of predicted poses is dependent on the fitness of the sampled compound orientations. The lowest energy pose for each docked ligand in SphK2 was then used for analysis of interactions with key residues in the SphK2 binding pocket. Free energy of binding scores were cataloged for each docked compound and used as one level of comparison between compounds.

General Material and Synthetic Procedures

All reactions conducted in a microwave were conducted in a Discover SP microwave synthesizer (CEM Corporation). All solvents were dried using the PureSolv solvent purification system prior to use. All chemical reagents were purchased from commercial sources and used without further purification. Thin layer chromatography (TLC) was performed on aluminum-backed silica gel, 200 μm, F254, and column chromatography was performed on flash grade silica gel, 40-63 μm, using a Combiflash Rf purification system. ¹H NMR spectra were recorded at 500 or 400 MHz; the corresponding ¹³C NMR resonant frequencies were 126 and 101 MHz, respectively; the corresponding ¹⁹F NMR resonant frequencies were 471 and 376 MHz, respectively. ¹H NMR chemical shifts are reported in ppm with the solvent resonance as an internal standard (CDCl₃: 7.26 ppm; CD₃OD: 4.87 ppm; acetone-d₆: 2.05 ppm). ¹³C NMR, chemical shifts are reported in ppm with the solvent resonance as the internal standard (CDCl₃: 77.16 ppm; CD₃OD: 49.00 ppm; acetone-d₆: 206.26 ppm). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m= multiplet), coupling constants (Hz), and integration. Rotamers are denoted by an asterisk (*). High resolution mass spectroscopy (HRMS) was performed on an LC/MS time-of-flight mass spectrometer using electrospray ionization (ESI). HPLC analyses were performed with a Thermo Electron TSQ triple quadrupole mass spectrometer equipped with an ESI source. All compounds tested in biological assays are >95% pure by ¹H NMR and HPLC analyses unless noted otherwise.

General Procedure A: Synthesis of amide-oxime derivatives

TEA (2.7 equiv) was added to a solution of the appropriate benzonitrile (1 equiv) with hydroxylamine hydrochloride (2.6 equiv) in ethanol (0.47 M solution). The mixture was reacted in a microwave for 6 min at 150 °C. The organic solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to yield the desired product.

General Procedure B: Coupling of amide-oxime derivatives with (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid

DIEA (1.80 equiv) was added to a solution of the appropriate amidoxime (1 equiv) and (S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid in DMF (0.2 M solution). HCTU (1.5 equiv) was then added to the resulting mixture at rt and stirred at 100 °C for 4 – 8 h. The reaction progress was followed by TLC. The solution was partitioned between EtOAc and LiBr aqueous solution. The aqueous solution was washed with EtOAc three times, and the combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated via vacuum. The resulting residue was purified by silica gel column chromatography.

General Procedure C: Coupling of 6 with alpha-bromoketones

DIEA (2 equiv) was added to a solution of tert-butyl (S)-2-((3-(4-thioureidophenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate 6 (1 equiv) and alpha-bromoketone (1 equiv) in ethanol (0.2 M solution). The resulting reaction mixture was then reacted in a microwave at 100°C for 5 min. The organic solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography.

General Procedure D: Suzuki Coupling of aryl bromides with phenyl boronic acids

Cs₂CO₃ (2 equiv) was added to a solution of of t-Boc protected aryl bromide intermediate (1 equiv) and the appropriate phenyl boronic acid derivative (3 equiv) in DMF (0.045 M solution). The resulting mixture was degassed for 10 min by bubbling N₂ through the solution. PdCl₂(dppf) (0.03 equiv) was then added to the mixture, and heated in a microwave reactor at 150 °C for 90 min. The solution was partitioned between EtOAc and LiBr aqueous solution. The aqueous solution was washed with EtOAc three times, and the combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated via vacuum. The resulting residue was purified by silica gel column chromatography.

General Procedure E: Deprotection of t-Boc protecting groups with TFA and guanylation of amines

To a solution of t-Boc protected intermediate in DCM, a 1N TFA solution in DCM was added. The reaction mixture was then stirred at rt for 4 h. At this time, TLC showed complete conversion of starting material. The organic solvent was removed under reduced pressure. The residue was then dissolved in ACN (0.02 M solution). Diisopropylethylamine (10 equiv) and (Z)-tert-butyl (((tert-butoxycarbonyl)imino)(1H-pyrazol-1-yl)methyl)carbamate (1.05 equiv) were added to the solution, and the resulting reaction mixture was reacted in a microwave reactor at 50° C for 2 h. The organic solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography.

General Procedure F: Deprotection of t-Boc protecting groups with HCl (g)

Hydrochloric acid gas was bubbled through a solution of the N-Boc protected compound in methanol for 2-5 minutes, until complete consumption of starting material was observed by TLC. The reaction mixture was concentrated under reduced pressure and triturated with diethyl ether to yield the corresponding free amine hydrochloride salt.

N-(4-cyanophenyl)-2,2,2-trifluoroacetamide (13)

4-aminobenzonitrile 12 (1.0 g, 8.46 mmol) was dissolved in DCM (8.5 mL) and cooled to 0°C. Triethylamine (1.3 mL, 9.31 mmol) was added dropwise and allowed to stir at 0°C for 10 min. Trifluoroacetic anhydride (1.8 mL, 9.31 mmol) was then added dropwise. The reaction mixture was allowed to warm up to rt and stirred for 19 h. The resulting reaction mixture was quenched with sat. aq. NH₄Cl, and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated via vacuum. The residue was purified by silica gel column chromatography (30% EtOAc/hexanes) to yield 13 (1.0 g, 88%) as white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.89 (d, J = 8.7 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H); ¹³C NMR (101 MHz, CD₃OD) δ 157.14 (q, ²J_CF = 38.4 Hz), 156.76 (q, ²J_CF = 38.4 Hz), 142.17, 122.19, 121.26 (q, ¹J_CF = 281.3 Hz), 119.35, 118.57 (q, ¹J_CF = 281.3 Hz), 115.70 (q, ¹J_CF = 281.3 Hz), 112.85 (q, ¹J_CF = 281.3 Hz), 109.84; ¹⁹F NMR (376 MHz, CD₃OD) δ −75.64 (s, 3F); HRMS (ESI−): Calcd for C₉H₄F₃N₂O [M-H]^-: 213.0275, Found: 213.0273.

(Z)-2,2,2-trifluoro-N-(4-(N′-hydroxycarbamimidoyl)phenyl)acetamide (14)

Synthesized by General Procedure A. 330 mg, 57% as white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.39 (d, J = 8.9 Hz, 2H), 6.70 (d, J = 8.9 Hz, 2H); ¹³C NMR (101 MHz, acetone-d₆) δ 156.22 (q, ²J_CF = 38.4 Hz), 155.85 (q, ²J_CF = 38.4 Hz), 155.48 (q, ²J_CF = 38.4 Hz), 155.11 (q, ²J_CF = 38.4 Hz), 152.17, 138.14, 131.54, 127.11, 121.23, 120.91 (q, ¹J_CF = 280.2 Hz), 118.29 (q, ¹J_CF = 280.2 Hz), 115.42 (q, ¹J_CF = 280.2 Hz), 112.56 (q, ¹J_CF = 280.2 Hz); ¹⁹F NMR (471 MHz, acetone-d₆) δ −76.14 (s, 3F); HRMS (ESI+): Calcd for C₉H₈F₃N₃O₂ [M+H]⁺: 248.0659, Found: 248.1822.

tert-butyl (S)-2-((3-(4-(2,2,2-trifluoroacetamido)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (15)

Synthesized by General Procedure B. 454 mg, 67%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 9.19 (s, 1H), 8.12 – 7.93 (m, 2H), 7.75 (dd, J = 14.9, 8.2 Hz, 2H), 4.35 – 4.19 (m, 1H), 3.46 – 3.21 (m, 3H), 3.07 (dt, J = 15.3, 7.3 Hz, 1H), 2.06 (q, J = 8.0, 6.9 Hz, 1H), 1.94 – 1.73 (m, 3H), 1.43 (s, 9H); ¹³C NMR (101 MHz, cdcl₃) δ 177.39, 167.67, 155.56, 155.19 (q, ²J_CF = 53.5 Hz), 154.79 (q, ²J_CF = 53.5 Hz), 154.47 (q, ²J_CF = 53.5 Hz), 153.94 (q, ²J_CF = 53.5 Hz) 138.54, 128.45, 124.30, 120.89, 120.11 (q, ¹J_CF = 289.9 Hz), 117.24 (q, ¹J_CF = 289.9 Hz), 114.37 (q, ¹J_CF = 289.9 Hz), 111.51 (q, ¹J_CF = 289.9 Hz), 80.54*, 80.00*, 55.32*, 55.18*, 46.82*, 46.37*, 31.68*, 31.05*, 30.27, 28.47, 23.53*, 22.81*; ¹⁹F NMR (470 MHz, CDCl₃) δ −74.47 (s, 3F); HRMS (ESI+): Calcd for C₂₀H₂₃F₃N₄NaO₄ [M+Na]⁺: 463.1569, Found: 463.1546.

tert-butyl (S)-2-((3-(4-aminophenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (16)

(S)-tert-butyl-2-(3-(4-iodophenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate 15 (54 mg, 0.123 mmol) was dissolved in MeOH (5 mL) and then 1 M LiOH (5 mL) was added. The reaction mixture was refluxed for 3 h. At this time, TLC showed complete conversion of starting material. The resulting product was extracted with EtOAc, and the combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated via vacuum to provide 16 (45 mg, 82%) as a clear solid without further purification. ¹H NMR (400 MHz, CDCl₃) δ 7.83 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.1 Hz, 2H), 4.34 – 4.17 (m, 1H), 4.04 (s, 2H), 3.49 – 3.19 (m, 3H), 3.13 – 2.90 (m, 1H), 2.02 (s, 1H), 1.93 – 1.69 (m, 3H), 1.45 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ ¹³C NMR (101 MHz, CDCl₃) δ 177.14, 177.01, 168.62, 154.81, 154.59, 149.68, 129.26, 116.93, 116.78, 115.03, 114.71, 80.34*, 79.92*, 55.55, 47.11*, 46.70*, 32.16*, 31.35*, 31.14*, 30.38*, 28.85, 23.91*, 23.13*; HRMS (ESI+): Calcd for C₁₈H₂₄N₄NaO₃ [M+Na]⁺: 367.1746, Found: 367.1743.

tert-butyl (S)-2-((3-(4-thioureidophenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (17)

tert-butyl (S)-2-((3-(4-aminophenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate 16 (100 mg, 0.290 mmol) was dissolved in THF (4 mL). Di(1H-imidazol-1-yl)methanethione (70 mg, 0.392 mmol) was added to the reaction mixture and allowed to stir at rt until TLC showed complete conversion of starting material. Ammonia gas was then passed through the solution for 1 min. The organic solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (35% -80% EtOAc/hexane) to yield 17 (101 mg, 86%) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.48 (d, J = 13.6 Hz, 1H), 8.12 (d, J = 8.1 Hz, 2H), 7.42 – 7.32 (m, 2H), 6.33 (s, 2H), 4.29 (d, J = 30.3 Hz, 1H), 3.52 – 3.25 (m, 3H), 3.08 (t, J = 7.6 Hz, 1H), 2.07 (s, 0H), 1.93 – 1.76 (m, 3H), 1.45 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 181.73, 177.91, 167.46, 154.36, 151.61, 139.13, 129.40, 125.85, 124.54, 80.28*, 79.89*, 55.28, 46.88*, 46.53*, 31.90*, 31.15*, 30.34, 28.62, 23.69*, 22.92.*; HRMS (ESI+): Calcd for C₁₉H₂₅N₅O₃S [M+H]⁺ : 404.1756, Found: 404.1751.

tert-butyl (S)-2-((3-(4-((4-phenylthiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18a)

Synthesized by General Procedure C. 42 mg, 56%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.10 – 8.04 (m, 2H), 7.91 – 7.84 (m, 2H), 7.71 – 7.61 (m, 1H), 7.59 – 7.51 (m, 2H), 7.42 (dd, J = 8.4, 6.9 Hz, 2H), 7.37 – 7.30 (m, 1H), 6.91 (s, 1H), 4.30 (m, 1H), 3.40 (m, 3H), 3.07 (m, 1H), 2.08 (m, 1H), 1.93 – 1.81 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.22, 168.00, 162.90, 154.37, 151.71, 142.84, 134.53, 128.96, 128.83, 128.19, 126.28, 120.75, 117.33, 102.84, 80.21, 55.28, 46.90*, 46.51*, 31.95*, 31.17*, 30.24*, 29.85*, 28.64, 23.72*, 22.93*; HRMS (ESI+): Calcd for C₂₇H₂₉N₅O₃S [M+H]⁺: 504.2069, Found: 504.2024.

tert-butyl (S)-2-((3-(4-((4-(pyridin-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18b)

Synthesized by General Procedure C. 25 mg, 39%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.67 (s, 2H), 8.09 (d, J = 8.3 Hz, 2H), 7.85 – 7.69 (m, 2H), 7.60 (t, J = 7.8 Hz, 2H), 7.15 (s, 1H), 4.41 – 4.21 (m, 1H), 3.51 – 3.27 (m, 3H), 3.15 – 2.99 (m, 1H), 2.15 – 2.01 (m, 1H), 1.96 – 1.78 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.33, 169.03, 167.92, 163.43, 150.29, 149.18, 142.56, 141.56, 131.98, 129.01, 121.16, 120.56, 117.59, 106.73, 80.22, 55.32, 46.90*, 46.53*, 31.94*, 31.18*, 30.28*, 29.85*, 28.65, 23.72*, 22.95*; HRMS (ESI+): Calcd for C₂₆H₂₉N₆O₃S [M+H]⁺: 505.2022, Found: 505.2016.

tert-butyl (S)-2-((3-(4-((4-(pyridin-3-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18c)

Synthesized by General Procedure C. 28 mg, 75%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 9.20 (d, J = 1.9 Hz, 1H), 8.57 (dd, J = 4.8, 1.7 Hz, 1H), 8.23 – 8.13 (m, 2H), 8.08 (d, J = 8.2 Hz, 2H), 7.65 – 7.54 (m, 2H), 7.36 (dd, J = 7.9, 4.7 Hz, 1H), 7.00 (s, 1H), 4.40 – 4.21 (m, 1H), 3.52 – 3.26 (m, 3H), 3.15 – 2.98 (m, 1H), 2.13 – 2.01 (m, 1H), 1.95 – 1.75 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.24, 167.95, 163.59, 154.40, 148.85, 148.62, 147.73, 142.84, 133.54, 130.48, 128.98, 123.73, 120.88, 117.47, 104.04, 80.23*, 79.81*, 55.29, 46.91*, 46.50*, 31.92*, 31.15*, 30.23*, 29.85*, 28.63, 23.71*, 22.93*; HRMS (ESI+): Calcd for C₂₆H₂₉N₆O₃S [M+H]⁺: 505.2022, Found: 505.2012.

tert-butyl (S)-2-((3-(4-((4-(4-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18d)

Synthesized by General Procedure C. 21 mg, 66%, light yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 8.05 (ap t, J = 10, 2H), 7.95 (br s, 1H), 7.86 – 7.81 (m, 2H), 7.58 – 7.50 (m, 2H), 7.09 (ap t, J = 10, 2H), 6.82 (s, 1H), 4.38 – 4.23 (m, 1H), 3.50 – 3.26 (m, 3H), 3.14 – 3.00 (m, 1H), 2.12 – 2.00 (m, 1H), 1.94 – 1.77 (m, 3H), 1.48 (br s, 9H); ³C NMR (101 MHz, CDCl₃) δ 177.23, 167.97, 164.00 (d,f ¹J_CF = 248.5 Hz), 163.13, 161.54 (d, ¹J_CF = 248.5 Hz), 158.61, 154.41, 151.31, 150.70, 142.80, 130.86 (d, ⁴J_CF = 3.0), 130.83 (d, ⁴J_CF = 3.0 Hz), 129.23, 128.95, 128.03 (d, ³J_CF = 8.1 Hz), 127.95 (d, ³J_CF = 8.1 Hz), 125.54, 120.95, 117.37, 115.83 (d, ²J_CF = 22.2), 115.6 (d, ²J_CF = 22.2 Hz), 102.34, 80.24*, 79.82*, 55.34, 46.90*, 46.52*, 31.94*, 31.17*, 31.01*, 30.26*, 28.64, 23.71*, 22.94*; ¹⁹F NMR (376 MHz, CDCl₃) δ −109.71 – −116.29 (m, 1F); HRMS (ESI+): Calcd for C₂₇H₂₈FN₅O₃S [M+H]⁺: 522.1975, Found: 522.1966.

tert-butyl (S)-2-((3-(4-((4-(4-chlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18e)

Synthesized by General Procedure C. 30 mg, 77%, yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (ap t, J = 10 Hz, 2H), 7.98 (br s, 1H), 7.79 (dt, J = 10, 2.5 Hz, 2H), 7.59 – 7.50 (m, 2H), 7.37 (dt, J = 10, 2.5 Hz, 2H), 6.87 (s, 1H), 4.40 – 4.23 (m, 1H), 3.50 – 3.26 (m, 3H), 3.14 – 3.00 (m, 1H), 2.12 – 2.03 (m, 1H), 1.94 – 1.77 (m, 3H), 1.48 (br s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.05, 167.80, 162.99, 154.50, 150.34, 142.63, 133.70, 132.87, 128.80, 128.77, 127.34, 120.62, 117.23, 102.96, 80.10*, 79.67*, 55.14, 46.74*, 46.36*, 31.75*, 31.00*, 30.10*, 29.68*, 28.48, 23.54*, 22.76*; HRMS (ESI+): Calcd for C₂₇H₂₈ClN₅NaO₃S [M+Na]⁺: 560.1499, Found 560.1502.

tert-butyl (S)-2-((3-(4-((4-(4-bromophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18f)

Synthesized by General Procedure C. 73 mg, 84%, off-white amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 8.2 Hz, 2H), 7.77 – 7.70 (m, 2H), 7.53 (dd, J = 9.2, 2.7 Hz, 3H), 6.90 (s, 1H), 4.40 – 4.22 (m, 1H), 3.50-3.27 (m, 3H), 3.15 – 2.99 (m, 1H), 2.13 – 2.03 (m, 1H), 1.95-178 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.12, 168.01, 163.27, 154.44, 150.39, 142.98, 142.86, 133.45, 131.84, 128.82, 127.76, 121.95, 120.50, 117.35, 103.14, 80.33*, 79.87*, 55.34*, 55.22*, 46.86*, 46.51*, 31.84*, 31.13*, 31.05*, 30.99*, 30.25*, 29.80*, 28.61, 23.64*, 22.88*; HRMS (ESI+): Calcd for C₂₇H₂₈BrN₅NaO₃S [M+Na]⁺: 604.0993, Found: 604.0967.

tert-butyl (S)-2-((3-(4-((4-(3-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18g)

Synthesized by General Procedure C. 58 mg, 90%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 8.2 Hz, 2H), 7.74 (br. s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.60 – 7.51 (m, 3H), 7.37 (td, J = 8.0, 5.9 Hz, 1H), 7.01 (q, J = 8.5, 1H), 6.92 (s, 1H), 4.40 – 4.40 – 4.21 (m, 1H), 3.3.57 – 3.26 (m, 3H), 3.16 – 2.96 (m, 1H), 2.15 – 2.01 (m, 1H), 1.94 – 1.78 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.28, 167.98, 164.52(d, ¹J_CF = 246.4 Hz), 163.06, 162.08 (d, ¹J_CF = 246.4 Hz), 154.42, 150.44, 142.71, 136.72 (d, ³J_CF = 8.1 Hz), 136.64 (d, ³J_CF = 8.1 Hz), 130.35 (d, ³J_CF = 9.1 Hz), 130.26 (d, ³J_CF = 9.1 Hz), 128.97, 121.80 (d, ⁴J_CF = 2.0 Hz), 121.78 (d, ⁴J_CF = 2.0 Hz), 121.11, 120.88 117.44, 115.05 (d, ²J_CF = 21.2 Hz), 114.84 (d, ²J_CF = 21.2 Hz), 113.36 (d, ²J_CF = 23.2 Hz), 113.13 (d, ²J_CF = 21.2 Hz), 103.77, 80.26*, 79.84*, 55.32, 46.91*, 46.52*, 31.94*, 31.16*, 30.27*, 29.85*, 28.64, 23.72*, 22.93*; ¹⁹F NMR (376 MHz, CDCl₃) δ −112.92 – −113.24 (m, 1F); HRMS (ESI+): Calcd for C₂₇H₂₈FN₅NaO₃S [M+Na]⁺: 544.1795, Found: 544.1784.

tert-butyl (S)-2-((3-(4-((4-(3-chlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18h)

Synthesized by General Procedure C. 43 mg, 63%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.04 (d, J = 7.7 Hz, 3H), 7.90 – 7.79 (m, 1H), 7.76 – 7.68 (m, 1H), 7.56 (t, J = 9.6 Hz, 2H), 7.40 – 7.21 (m, 2H), 6.90 (s, 1H), 4.45 – 4.19 (m, 1H), 3.54 – 3.24 (m, 3H), 3.07 (ddd, J = 22.4, 14.7, 8.6 Hz, 1H), 2.16 – 1.99 (m, 1H), 1.87 (dd, J = 14.3, 7.1 Hz, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.19, 168.01, 163.18, 154.70, 154.44, 150.21, 142.87, 136.28, 134.76, 130.02, 128.90, 128.03, 126.36, 124.29, 120.69, 117.41, 103.78, 80.30*, 79.86*, 55.35*, 55.24*, 50.92*, 46.89*, 46.52*, 31.89*, 31.15*, 30.27*, 28.63*, 23.68*, 22.91*; HRMS (ESI+): Calcd for C₂₇H₂₈ClN₅O₃S [M+H]⁺: 538.1680, Found: 538.1679.

tert-butyl (S)-2-((3-(4-((4-(3-bromophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18i)

Synthesized by General Procedure C. 90 mg, 78%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.11 – 8.04 (m, 2H), 8.01 (d, J = 1.9 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.55 (t, J = 8.9 Hz, 1H), 7.47 – 7.41 (m, 1H), 7.27 (d, J = 5.5 Hz, 1H), 6.91 (s, 1H), 4.30 – 4.22 (m, 1H), 3.40 – 3.16 – 2.98 (m, 3H), 3.07 (m, 1H), 2.20 – 1.97 (m, 1H), 1.97 – 1.77 (m, 2H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.22, 167.99, 163.11, 154.39, 150.10, 142.70, 136.48, 131.01, 130.33, 129.28, 128.96, 128.63, 124.75, 122.99, 120.83, 117.40, 103.84, 80.25*, 79.80*, 55.34, 46.90*, 46.51*, 31.92*, 31.16*, 30.24*, 28.64*, 23.71*, 22.92*; HRMS (ESI+): Calcd for C₂₇H₂₈BrN₅NaO₃S [M+Na]⁺: 604.0993, Found: 604.0988.

tert-butyl (S)-2-((3-(4-((4-(2-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18j)

Synthesized by General Procedure C. 25 mg, 56%, off-white amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.16 (td, J = 7.7, 2.0 Hz, 1H), 8.06 (d, J = 7.2 Hz, 2H), 7.75 (s, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.32 – 7.18 (m, 3H), 7.13 (ddd, J = 12.0, 7.9, 1.5 Hz, 1H), 4.40 – 4.22 (m, 1H), 3.54 – 3.24 (m, 3H), 3.16 – 2.99 (m, 1H), 2.14 – 2.01 (m, 1H), 1.96 – 1.77 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 191.63, 177.19, 168.00, 161.95, 161.71 (d, ¹J_CF = 251.5 Hz), 159.22 (d, ¹J_CF = 251.5 Hz), 154.41, 145.30, 142.90, 130.07 (d, ⁴J_CF = 3.0 Hz), 130.04 (d, ⁴J_CF = 3.0 Hz), 129.17 (d, ³J_CF = 9.1 Hz), 129.08 (d, ³J_CF = 9.1 Hz), 128.92, 124.54, 124.50, 122.39 (d, ³J_CF = 11.1 Hz), 122.28 (d, ³J_CF = 11.1 Hz), 120.69, 117.34, 117.24, 116.15 (d, ²J_CF = 22.2 Hz), 115.93 (d, ²J_CF = 22.2 Hz), 108.05 (d, ³J_CF = 16.2 Hz), 107.89 (d, ³J_CF = 16.2 Hz), 80.25*, 79.80*, 55.35, 46.91*, 46.52*, 31.93*, 31.16*, 30.26, 28.65, 23.71*, 22.93*; ¹⁹F NMR (376 MHz, CDCl₃) δ −114.11 (s, 1F); HRMS (ESI+): Calcd for C₂₇H₂₈FN₅NaO₃S [M+Na]⁺: 544.1795, Found: 544.1782.

tert-butyl (S)-2-((3-(4-((4-(4-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18k)

Synthesized by General Procedure C. 83 mg, 70%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (br s, 1H), 8.10-8.00 (m, 2H), 7.96 (ap d, J = 8, 2H), 7.64 (ap d, J = 8, 2H), 7.62 – 7.53 (m, 2H), 6.98 (br s, 1H), 4.41 – 4.23 (m, 1H), 3.51 – 3.25 (m, 3H), 3.14 -3.01 (m, 1H), 2.14 – 2.02 (m, 1H), 1.95 – 1.78 (m, 3H), 1.48 (br s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.19, 167.99, 163.35, 154.44, 150.17, 142.83, 137.74, 130.26 (q, ²J_CF = 32.3 Hz), 129.94 (q, ²J_CF = 32.3 Hz), 129.62 (q, ²J_CF = 32.3 Hz), 129.30 (q, ²J_CF = 32.3 Hz), 128.89, 126.38, 125.82 (q, ³J_CF = 4.0 Hz), 125.78 (q, ³J_CF = 4.0 Hz), 125.74 (q, ³J_CF = 4.0 Hz), 125.70 (q, ³J_CF = 4.0 Hz), 122.97 (q, ¹J_CF = 224.2 Hz), 120.90, 120.75 (q, ¹J_CF = 224.2 Hz), 120.26, 117.44, 104.67, 104.65, 80.34*, 79.89*, 55.25, 46.89*, 46.53*, 31.86*, 31.13*, 30.27*, 29.84*, 28.62*, 23.67*, 22.90*; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.49 (S, 3F); HRMS (ESI+): Calcd for C₂₈H₂₉F₃N₅O₃S [M+H]⁺: 572.1943, Found: 572.1958.

tert-butyl (S)-2-((3-(4-((4-(3-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18l)

Synthesized by General Procedure C. 45 mg, 79%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.43 (d, J = 12.3 Hz, 1H), 8.10 (s, 1H), 8.03 (dt, J = 14.0, 7.3 Hz, 3H), 7.54 (ddt, J = 23.4, 15.4, 8.1 Hz, 4H), 6.95 (s, 1H), 4.42 – 4.23 (m, 1H), 3.54 – 3.25 (m, 3H), 3.13 – 3.02 (m, 1H), 2.13 – 2.02 (m, 1H), 1.96 – 1.77 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.12, 168.02, 163.39, 154.49, 150.03, 142.94, 135.24, 131.58 (q, ²J_CF = 32.3 Hz), 131.25 (q, ²J_CF = 32.3 Hz), 130.93 (q, ²J_CF = 32.3 Hz), 130.61 (q, ²J_CF = 32.3 Hz), 129.32, 129.22, 128.82, 125.58 (q, ¹J_CF = 165.2 Hz), 124.56 (q, ³J_CF = 4.0 Hz), 124.52(q, ³J_CF = 4.0 Hz), 124.49 (q, ³J_CF = 4.0 Hz; q, ¹J_CF = 165.2 Hz)), 124.45 (q, ³J_CF = 4.0 Hz), 122.95, 122.91 (q, ¹J_CF = 165.2 Hz), 122.87, 120.65 (q, ¹J_CF = 165.2 Hz), 120.50, 117.37, 103.95, 80.40*, 79.90*, 55.35*, 55.20*, 46.86*, 46.51⁸, 31.81*, 31.10*, 30.99*, 30.25*, 28.59, 23.62*, 22.86*; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.71 (s, 3F). HRMS (ESI+): Calcd for C₂₈H₂₈F₃N₅NaO₃S [M+Na]⁺: 594.1763, Found: 594.1767.

tert-butyl (S)-2-((3-(4-((4-(2-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18m)

Synthesized by General Procedure C. 45 mg, 79%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.30 (br s, 1H), 7.98 (d, J = 8.5 Hz, 2H), 7.78 – 7.73 (m, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.56 (td, J = 7.7, 1.5 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 6.76 (s, 1H), 4.38 – 4.22 (m, 1H), 3.49 – 3.27 (m, 3H), 3.16 – 2.97 (m, 1H), 2.13 – 2.00 (m, 1H), 1.95 – 1.77 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 191.63, 177.20, 167.97, 162.95, 154.38, 148.62, 142.83, 134.47, 132.09, 131.72, 129.01 (q, ²J_CF = 30.3 Hz), 128.86, 128.71 (q, ²J_CF = 30.3 Hz), 128.40, 128.38, 128.32 (q, ²J_CF = 30.3 Hz), 128.10 (q, ¹J_CF = 275.7 Hz), 126.66 (q, ³J_CF = 6.1 Hz), 126.60 (q, ³J_CF = 6.1 Hz), 126.55 (q, ³J_CF = 6.1 Hz), 126.49 (q, ³J_CF = 6.1 Hz), 125.59 (q, ¹J_CF = 275.7 Hz), 122.87 (q, ¹J_CF = 275.7 Hz), 120.93, 120.73, 120.14 (q, ¹J_CF = 275.7 Hz), 117.48, 106.95, 106.91, 80.22*, 79.77*, 55.34, 46.90*, 46.51*, 31.95*, 31.16*, 30.98*, 30.23*, 29.84, 28.63*, 28.52*, 23.70*, 22.92*; ¹⁹F NMR (376 MHz, CDCl₃) δ −57.78 (s, 3F); HRMS (ESI+): Calcd for C₂₈H₂₈F₃N₅NaO₃S [M+H]⁺: 572.1943, Found: 572.1942.

tert-butyl (S)-2-((3-(4-((4-(p-tolyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18n)

Synthesized by General Procedure C. 30 mg, 47%, off-white amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 7.8 Hz, 2H), 7.58 – 7.47 (m, 2H), 7.22 (d, J = 7.9 Hz, 2H), 6.84 (s, 1H), 4.41 – 4.21 (m, 1H), 3.42 (t, J = 20.8 Hz, 3H), 3.18 – 2.97 (m, 1H), 2.38 (s, 3H), 2.16 – 2.01 (m, 1H), 1.87 (s, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.30, 167.99, 162.98, 151.64, 142.84, 138.08, 131.75, 129.52, 128.96, 126.20, 120.69, 117.32, 102.00, 80.22*, 79.80*, 55.35, 46.91*, 46.51*, 31.97*, 31.17*, 30.24*, 29.85*, 28.64, 23.72*, 22.93*, 21.43; HRMS (ESI+): Calcd for C₂₈H₃₂N₅O₃S [M+H]⁺: 518.2226, Found: 518.2225.

tert-butyl (S)-2-((3-(4-((4-(4-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18o)

Synthesized by General Procedure C. 30 mg, 75%, clear amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.15 – 7.96 (m, 3H), 7.80 (d, 2H), 7.61 – 7.44 (m, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.75 (s, 1H), 4.40 – 4.20 (m, 1H), 3.83 (s, 3H), 3.50 – 3.24 (m, 3H), 3.21 – 2.97 (m, 1H), 2.13 – 1.99 (m, 1H), 1.96 – 1.76 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.14, 168.03, 162.96, 159.63, 154.64, 154.39, 151.36, 143.01, 128.85, 127.65, 127.55, 127.53, 120.60, 120.41, 117.23, 114.16, 100.95, 80.24*, 79.78*, 55.45*, 55.34, 55.21*, 46.88*, 46.50*, 31.89*, 31.13*, 30.96*, 30.21*, 28.62, 23.68*, 22.90*; HRMS (ESI+): Calcd for C₂₈H₃₂N₅O₄S [M+H]⁺: 534.2175, Found: 534.2160.

tert-butyl (S)-2-((3-(4-((4-(4-ethoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18p)

Synthesized by General Procedure C. 81 mg, 92%, light yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 8.3 Hz, 2H), 7.82 (s, 1H), 7.79 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 7.7 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.75 (s, 1H), 4.39 – 4.21 (m, 1H), 4.07 (q, J = 7.0 Hz, 2H), 3.51 – 3.26 (m, 3H), 3.07 (m, 1H), 2.13 – 2.02 (m, 1H), 1.95 – 1.78 (m, 4H), 1.48 (s, 9H), 1.43 (t, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 177.09, 167.84, 162.73, 158.91, 154.25, 151.25, 142.74, 128.75, 127.38, 127.16, 120.41, 117.11, 114.58, 100.72, 80.06*, 79.63*, 63.49, 55.14, 46.72*, 46.34*, 31.77*, 30.99*, 30.05, 28.47, 23.54*, 22.75*, 14.80; HRMS (ESI+): Calcd for C₂₉H₃₄N₅O₄S [M+H]⁺: 548.2332, Found: 548.2308.

tert-butyl (S)-2-((3-(4-((4-(3-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18q)

Synthesized by General Procedure C. 28 mg, 70%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.09 – 7.97 (m, 3H), 7.58 – 7.51 (m, 2H), 7.49 – 7.40 (m, 2H), 7.32 (t, J = 8.1 Hz, 1H), 6.96 – 6.81 (m, 2H), 4.43 – 4.18 (m, 1H), 3.86 (s, 3H), 3.54 – 3.24 (m, 4H), 3.19 – 2.97 (m, 1H), 2.17 – 2.00 (m, 1H), 1.97 – 1.74 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.18, 167.97, 162.95, 160.02, 154.64, 154.40, 151.45, 142.89, 135.90, 129.82, 129.16, 128.88, 120.55, 118.71, 117.30, 113.91, 111.80, 103.11, 80.24*, 79.79*, 55.44*, 55.35*, 55.22*, 46.89*, 46.50*, 31.91*, 31.14*, 30.99*, 30.22*, 28.62, 23.69*, 22.91*; HRMS (ESI+): Calcd for C₂₈H₃₂N₅O₄S [M+H]⁺: 534.2175, Found: 534.2182.

tert-butyl (S)-2-((3-(4-((4-(4-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18r)

Synthesized by General Procedure C. 27 mg, 30%, yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (ap. t, J = 7.5 Hz, 2H), 7.90 (s, 1H), 7.88 (d, J = 8.5 Hz, 2H), 7.59 – 7.51 (m, 2H), 7.28 – 7.23 (m, 2H), 6.88 (s, 1H), 4.39 – 4.22 (m, 1H), 3.54 – 3.27 (m, 2H), 3.14 – 2.99 (m, 1H), 2.13 – 1.99 (m, 1H), 1.95 – 1.77 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.04, 167.73, 163.03, 154.22, 150.12, 148.80, 142.54, 133.08, 128.76, 127.46, 121.72 (q, ¹J_CF = 258.6 Hz), 121.09, 119.16 (q, ¹J_CF = 258.6 Hz), 117.22, 103.12, 80.09*, 79.64*, 55.18*, 55.06*, 46.73*, 46.34*, 31.73*, 30.98*, 30.83*, 30.06*, 29.68, 28.46*, 23.53*, 22.74*; ¹⁹F NMR(376 MHZ, CDCl₃) δ −57.82 (s, 3F); HRMS (ESI+): Calcd for C₂₈H₂₈F₃N₅O₄S [M+H]⁺: 588.1892, Found: 588.1916.

tert-butyl (S)-2-((3-(4-((4-(4-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18s)

Synthesized by General Procedure C. 27 mg, 30%, yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J = 8.3 Hz, 2H), 7.78 (dt, J = 7.9, 1.3 Hz, 1H), 7.73 (dt, J = 2.9, 1.4 Hz, 1H), 7.64 (s, 1H), 7.57 (d, J = 7.7 Hz, 2H), 7.43 (t, J = 8.0 Hz, 1H), 7.17 (ddt, J = 8.1, 2.3, 1.1 Hz, 1H), 6.95 (s, 1H), 4.40 – 4.20 (m, 1H), 3.51 – 3.25(m, 3H), 3.16 – 2.98 (m, 1H), 2.16 – 2.00 (m, 1H), 1.96 – 1.76 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.24, 167.96, 163.09, 154.40, 150.19, 149.84, 142.65, 136.56, 130.14, 128.98, 124.42, 121.97 (q, ¹J_CF = 156.6 Hz), 120.96, 120.32 (q, ¹J_CF = 156.6 Hz), 119.41, 118.87 (q, ¹J_CF = 156.6 Hz), 117.44, 104.04, 80.23*, 79.82*, 55.33, 46.89*, 46.52*, 31.94,* 31.17*, 30.26, 28.64, 23.71*, 22.93*; ¹⁹F NMR(376 MHZ, CDCl₃) δ −57.82 (s, 3F); HRMS (ESI+): Calcd for C₂₈H₂₈F₃N₅NaO₄S [M+Na]⁺: 610.1712, Found: 610.1721.

tert-butyl (S)-2-((3-(4-((4-(3,4-dimethylphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18t)

Synthesized by General Procedure C. 30 mg, 75%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.30 – 8.08 (m, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.63 (s, 1H), 7.57 (d, J = 7.7, 2.1 Hz, 1H), 7.54 – 7.46 (m, 2H), 7.15 (d, J = 7.9 Hz, 1H), 6.82 (s, 1H), 4.30 (d, J = 29.1 Hz, 1H), 3.55 – 3.26 (m, 4H), 3.20 – 2.93 (m, 1H), 2.29 (s, 3H), 2.27 (s, 3H), 2.13 – 2.00 (m, 1H), 1.94 – 1.77 (m, 4H), 1.54 – 1.40 (m, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.15, 168.03, 163.03, 154.64, 154.39, 151.76, 143.01, 136.92, 136.69, 132.19, 130.04, 128.84, 128.29, 127.48, 123.71, 120.40, 117.27, 101.87, 80.25*, 79.80*, 55.34*, 55.21*, 46.88*, 46.49*, 31.89*, 31.12*, 30.96*, 30.20*, 28.61, 23.67*, 22.89*, 20.07, 19.99, 19.72; HRMS (ESI+): Calcd for C₂₉H₃₄N₅O₃S [M+H]⁺: 532.2382, Found: 532.2398.

tert-butyl (S)-2-((3-(4-((4-(3,4-dimethoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18u)

Synthesized by General Procedure C. 25 mg, 63%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.23 – 7.98 (m, 3H), 7.58 – 7.48 (m, 2H), 7.46 – 7.37 (m, 2H), 6.90 (d, J = 8.6 Hz, 1H), 6.78 (s, 1H), 4.30 (d, J = 30.3 Hz, 1H), 3.94 (s, 3H), 3.91 (s, 3H), 3.53 – 3.25 (m, 3H), 3.19 – 2.95 (m, 1H), 2.15 – 1.99 (m, 1H), 1.97 – 1.75 (m, 3H), 1.47 (s, 9H); ¹³C NMR (101 MHz, cdcl₃) δ 177.18, 167.99, 162.95, 154.37, 151.37, 149.16, 149.11, 142.96, 128.87, 128.32, 127.78, 120.50, 118.79, 117.22, 111.36, 109.63, 101.28, 80.23*, 79.78*, 56.18, 56.08*, 56.06*, 56.02*, 55.31*, 46.87*, 46.48*, 31.91*, 31.13*, 30.98*, 30.20*, 28.61, 23.68*, 22.89*; HRMS (ESI+): Calcd for C₂₉H₃₄N₅O₅S [M+H]⁺: 564.2281, Found: 564.2271.

tert-butyl (S)-2-((3-(4-((4-(3-chloro-4-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18v)

Synthesized by General Procedure C. 83 mg, 91%, light yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 2.2 Hz, 1H), 7.73 (dd, J = 8.6, 2.2 Hz, 1H), 7.59 – 7.50 (m, 2H), 6.95 (d, J = 8.6 Hz, 1H), 6.76 (s, 1H), 4.40 – 4.22 (m, 1H), 3.93 (s, 3H), 3.47 – 3.29 (m, 3H), 3.07 (ddd, J = 30.3, 14.3, 8.2 Hz, 1H), 2.14 – 2.02 (m, 1H), 1.94 – 1.78 (m, 4H), 1.47 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.24, 167.90, 163.26, 155.01, 149.42, 142.47, 128.97, 128.11, 127.80, 125.73, 122.84, 117.56, 112.20, 101.65, 80.23*, 79.79*, 56.38, 55.34*, 55.23*, 46.90*, 46.50*, 31.93*, 31.15*, 30.99*, 30.23*, 28.63, 23.72*, 22.92*; HRMS (ESI+): Calcd for C₂₈H₃₁ClN₅O₄S [M+H]⁺: 568.1785, Found: 568.1780.

tert-butyl (S)-2-((3-(4-((4-(3,4-difluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18w)

Synthesized by General Procedure C. 21 mg, 58%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.11 – 8.03 (m, 2H), 7.72 – 7.62 (m, 2H), 7.61 – 7.50 (m, 3H), 7.19 (dd, J = 10.1, 8.3 Hz, 1H), 6.84 (s, 1H), 4.29 (t, J = 18.7 Hz, 1H), 3.50 – 3.26 (m, 3H), 3.15 – 2.99 (m, 1H), 2.16 – 2.01 (m, 1H), 1.96 – 1.77 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.25, 167.94, 163.13, 154.42, 151.93 (dd, ²J_CF = 36.4 Hz, ³J_CF = 13.1 Hz), 151.80 (dd, ²J_CF = 36.4 Hz, ³J_CF = 13.1 Hz), 151.57 (dd, ²J_CF = 36.4 Hz, ³J_CF = 13.1 Hz), 151.45 (dd, ²J_CF = 36.4 Hz, ³J_CF = 13.1 Hz), 149.65, 149.47 (dd, ²J_CF = 37.4 Hz, ³J_CF = 13.1 Hz), 149.34 (dd, ²J_CF = 37.4 Hz, ³J_CF = 13.1 Hz), 149.10 (dd, ²J_CF = 37.4 Hz, ³J_CF = 13.1 Hz), 148.97 (dd, ²J_CF = 37.4 Hz, ³J_CF = 13.1 Hz), 142.64, 131.80 (d, ³J_CF = 4.0 Hz), 131.76 (d, ³J_CF = 4.0 Hz), 131.74 (d, ³J_CF = 4.0 Hz), 131.70 (d, ³J_CF = 4.0 Hz), 128.97, 122.21 (d, ³J_CF = 4.0 Hz), 122.17 (d, ³J_CF = 4.0 Hz), 122.15 (d, ³J_CF = 4.0 Hz), 122.11 (d, ³J_CF = 4.0 Hz), 120.97, 117.68 80 (dd, ¹J_CF = 227.8 Hz, ²J_CF = 18.2 Hz), 117.50 (dd, ¹J_CF = 227.8 Hz, ²J_CF = 18.2 Hz), 117.45, 115.43 (dd, ¹J_CF = 227.8 Hz, ²J_CF = 19.2 Hz), 115.24 (dd, ¹J_CF = 227.8 Hz, ²J_CF = 19.2 Hz), 103.26, 80.26*, 79.85*, 55.31, 46.91*, 46.53*, 31.93*, 31.17*, 30.28, 28.64, 23.71*, 22.94*; ¹⁹F NMR (376 MHz, CDCl₃) δ −137.57, −138.63; HRMS (ESI+): Calcd for C₂₇H₂₇F₂N₅NaO₃S [M+Na]⁺: 562.1695, Found: 562.1666.

tert-butyl (S)-2-((3-(4-((4-(3,4-dichlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18x)

Synthesized by General Procedure C. 42 mg, 59%, yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.09 – 7.98 (m, 2H), 7.91 (d, J = 8.5 Hz, 1H), 7.56 – 7.44 (m, 2H), 7.33 – 7.23 (m, 2H), 4.41 – 4.23 (m, 1H), 3.53 – 3.29 (m, 3H), 3.16 – 2.99 (m, 1H), 2.14 – 2.02 (m, 1H), 1.95 – 1.74 (m, 3H), 1.49 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.25, 167.95, 162.20, 154.67, 146.89, 142.77, 133.94, 132.53, 132.30, 131.74, 130.36, 128.90, 127.38, 117.45, 108.45, 80.27*, 79.84*, 55.34, 46.90*, 46.52*, 31.91*, 31.14*, 30.26, 28.64, 23.69*, 22.92*; HRMS (ESI+): Calcd for C₂₇H₂₈Cl₂N₅O₃S [M+H]⁺: 572.1290, Found: 572.1244.

tert-butyl (S)-2-((3-(4-((4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18y)

Synthesized by General Procedure C. 21 mg, 24%, clear yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.15 – 7.98 (m, 4H), 7.82 (s, 1H), 7.60 – 7.51 (m, 2H), 7.31 – 7.17 (m, 1H), 6.90 (s, 1H), 4.40 – 4.23 (m, 1H), 3.52 – 3.27 (m, 3H), 3.15 – 3.01 (m, 1H), 2.14 – 2.03 (m, 1H), 1.94 – 1.81 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.27, 167.92, 163.40, 160.64 (d, ¹J_CF = 257.6 Hz), 158.09 (d, ¹J_CF = 257.6 Hz), 154.42, 149.25, 142.64, 131.46 (d, ³J_CF = 8.1 Hz), 131.38 (d, ³J_CF = 8.1 Hz), 131.14 (d, ⁴J_CF = 4.0 Hz), 131.10 (d, ⁴J_CF = 4.0 Hz), 128.96, 126.76 (q, ¹J_CF = 272.7 Hz), 125.04 (qd, ³J_CF = 5.1 Hz, ⁴J_CF = 1.0 Hz), 125.03 (qd, ³J_CF = 5.1 Hz, ⁴J_CF = 1.0 Hz), 125.00 (qd, ³J_CF = 5.1 Hz, ⁴J_CF = 1.0 Hz), 124.98 (qd, ³J_CF = 5.1 Hz, ⁴J_CF = 1.0 Hz), 124.95, 124.94, 124.05 (q, ¹J_CF = 272.7 Hz), 121.35 (q, ¹J_CF = 272.7 Hz), 120.99, 118.96 (d, ³J_CF = 13.1 Hz), 118.83 (d, ³J_CF = 13.1 Hz), 118.63 (q, ¹J_CF = 272.7 Hz), 118.50, 117.48, 117.43, 117.38, 117.22, 103.56, 80.30*, 79.88*, 55.33, 46.90*, 46.54*, 31.91*, 31.17*, 30.29, 28.63, 23.69*, 22.93*; ¹⁹F NMR (376 MHz, CDCl₃) δ −61.47 (d, J = 12.6 Hz 3F), −115.74 (br. s, 1F); HRMS (ESI+): Calcd for C₂₈H₂₇F₄N₅NaO₃S [M+Na]⁺: 612.1668, Found: 612.1663.

tert-butyl (S)-2-((3-(4-((4-(3,5-difluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18z)

Synthesized by General Procedure C. 37 mg, 76%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (t, J = 10.4 Hz, 2H), 7.94 (s, 1H), 7.62 – 7.52 (m, 2H), 7.40 – 7.32 (m, 1H), 6.92 (s, 1H), 6.75 (tt, J = 8.9, 2.5 Hz, 1H), 4.44 – 4.21 (m, 1H), 3.53 – 3.24 (m, 3H), 3.15 – 3.00 (m, 1H), 2.15 – 2.01 (m, 1H), 1.95 – 1.75 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.21, 168.01, 164.70 (dd, ¹J_CF = 248.5 Hz, ³J_CF = 13.1 Hz), 164.57 (dd, ¹J_CF = 248.5 Hz, ³J_CF = 13.1 Hz), 163.17, 162.24 (dd, ¹J_CF = 248.5 Hz, ³J_CF = 13.1 Hz), 162.11 (dd, ¹J_CF = 248.5 Hz, ³J_CF = 13.1 Hz), 154.47, 149.46, 149.42, 142.70, 137.75 (d, ²J_CF = 20.2 Hz), 137.65, 137.55 (d, ²J_CF = 20.2 Hz), 128.93, 120.87, 117.48, 109.16 (d, ²J_CF = 27.3 Hz), 109.08 (d, ³J_CF = 11.1 Hz), 108.97 (d, ³J_CF = 11.1 Hz), 108.89 (d, ²J_CF = 27.3 Hz), 104.66, 103.48 (d, ²J_CF = 51.5 Hz), 103.23, 102.97 (d, ²J_CF = 51.5 Hz), 80.34*, 79.89*, 55.34, 46.90*, 46.54*, 31.88*, 31.16*, 30.30, 28.74*, 28.63*, 23.68*, 22.92*; ¹⁹F NMR (376 MHz, CDCl₃) δ −109.78 (br. S, 2F); HRMS (ESI+): Calcd for C₂₇H₂₈F₂N₅O₃S [M+H]⁺: 540.1881, Found: 540.1845.

tert-butyl (S)-2-((3-(4-((4-(3-fluoro-5-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18aa)

Synthesized by General Procedure C. 39 mg, 44%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.12 – 8.02 (m, 2H), 7.91 (d, J = 12.9 Hz, 2H), 7.75 (d, J = 9.6 Hz, 0H), 7.64 – 7.51 (m, 2H), 7.28 – 7.23 (m, 1H), 7.00 (s, 1H), 4.41 – 4.23 (m, 1H), 3.52 – 3.27 (m, 3H), 3.14 – 3.01 (m, 1H), 2.14 – 2.02 (m, 1H), 1.97 – 1.75 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.24, 167.98, 164.16(d, ¹J_CF = 248.5 Hz), 163.39, 161.70 (d, ¹J_CF = 248.5 Hz), 154.47, 149.04, 142.61, 137.75 (d, ³J_CF = 8.1 Hz), 137.67 (d, ³J_CF = 8.1 Hz), 133.44 (qd, ²J_CF = 33.3 Hz, ³J_CF = 8.1 Hz), 133.36 (qd, ²J_CF = 33.3 Hz, ³J_CF = 8.1 Hz), 133.11(qd, ²J_CF = 33.3 Hz, ³J_CF = 8.1 Hz), 133.03, 132.78 (qd, ²J_CF = 33.3 Hz, ³J_CF = 8.1 Hz), 132.70 (qd, ²J_CF = 33.3 Hz, ³J_CF = 8.1 Hz), 132.45 (qd, ²J_CF = 33.3 Hz, ³J_CF = 8.1 Hz), 132.37 (qd, ²J_CF = 33.3 Hz, ³J_CF = 8.1 Hz), 128.96, 127.50 (q, ¹J_CF = 272.7 Hz), 124.81 (q, ¹J_CF = 272.7 Hz), 122.10 (q, ¹J_CF = 272.7 Hz), 121.00, 119.36 (q, ¹J_CF = 272.7 Hz),118.70, 118.66, 118.63, 117.53, 116.59 (d, ²J_CF = 23.2 Hz), 116.36 (d, ²J_CF = 23.2 Hz),, 112.11 (dq, ²J_CF = 23.2 Hz, ³J_CF = 4.0 Hz), 112.08 (dq, ²J_CF = 23.2 Hz, ³J_CF = 4.0 Hz), 111.87 (dq, ²J_CF = 23.2 Hz, ³J_CF = 4.0 Hz), 111.83 (dq, ²J_CF = 23.2 Hz, ³J_CF = 4.0 Hz), 105.06, 80.35*, 79.91*, 55.33, 46.90*, 46.54*, 31.89*, 31.16*, 30.31, 28.63, 23.69*, 22.93*; ¹⁹F NMR (376 MHz, cdcl₃) δ ¹⁹F NMR (376 MHz, CDCl₃) δ −62.87 (s, 3F), −110.68 (q, J = 8.2, 7.6 Hz, 1F); HRMS (ESI+): Calcd for C₂₈H₂₈F₄N₅O₃S [M+H]⁺: 590.1849, Found: 590.1843.

tert-butyl (S)-2-((3-(4-((4-(3,5-bis(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18bb)

Synthesized by General Procedure C. 33 mg, 49%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 2H), 8.08 (dd, J = 13.4, 8.2 Hz, 2H), 7.89 (s, 1H), 7.80 (s, 1H), 7.57 (t, J = 9.0 Hz, 2H), 7.08 (s, 1H), 4.41 – 4.23 (m, 1H), 3.52 – 3.26 (m, 3H), 3.16 – 3.00 (m, 1H), 2.15 – 2.03 (m, 1H), 1.97 – 1.80 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.28, 167.93, 163.64, 154.46, 148.68, 142.53, 136.44, 132.65 (d, ²J_CF = 33.3 Hz), 132.32 (q, ²J_CF = 33.3 Hz), 131.99 (q, ²J_CF = 33.3 Hz), 131.66 (d, ²J_CF = 33.3 Hz), 129.00, 127.56 (q, ¹J_CF = 273.7 Hz), 126.12, 124.85 (q, ¹J_CF = 273.7 Hz), 122.14 (q, ¹J_CF = 273.7 Hz), 121.35, 119.42 (q, ¹J_CF = 273.7 Hz), 117.59, 105.45, 80.36*, 79.91*, 55.31, 46.90*, 46.55*, 31.91*, 31.18*, 30.32, 28.64, 23.69*, 22.94*; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.96 (s, 6F); HRMS (ESI+): Calcd for C₂₉H₂₇F₆N₅NaO₃S [M+Na]⁺: 662.1636, Found: 662.1627.

tert-butyl (S)-2-((3-(4-((4-(2-fluoro-5-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18cc)

Synthesized by General Procedure C. 57 mg, 78%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.47 (dd, J = 7.1, 2.5 Hz, 1H), 8.07 (ap. t, J = 9.6 Hz, 2H), 7.91 (s, 1H), 7.63 – 7.48 (m, 3H), 7.32 – 7.15 (m, 2H), 4.42 – 4.21 (m, 1H), 3.52 – 3.27 (m, 3H), 3.16 – 3.02 (m, 1H), 2.15 – 2.02 (m, 1H), 1.97 – 1.77 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.24, 167.94, 163.19 (d, ¹J_CF = 256.5 Hz), 162.29, 160.65 (d, ¹J_CF = 256.6 Hz), 154.46, 143.80, 142.71, 128.95, 128.00 (q, ¹J_CF = 272.7 Hz), 127.72, 127.67, 127.61, 127.38 (q, ²J_CF = 33.3 Hz), 127.05 (q, ²J_CF = 33.3 Hz), 126.72 (q, ²J_CF = 33.3 Hz), 126.00, 125.30 (q, ¹J_CF = 272.7 Hz), 123.11 (d, ³J_CF = 12.1 Hz), 122.99 (d, ³J_CF = 12.1 Hz), 122.59 (q, ¹J_CF = 272.7 Hz), 120.89, 119.89 (q, ¹J_CF = 272.7 Hz), 117.43, 117.08, 116.81 (d, ²J_CF = 24.2 Hz), 116.57 (d, ²J_CF = 24.2 Hz), 109.27 (d, ²J_CF = 16.2 Hz), 109.11 (d, ²J_CF = 16.2 Hz), 80.34*, 79.88*, 55.31, 46.89*, 46.54*, 31.91*, 31.17*, 31.04*, 30.29*, 28.63, 23.69*, 22.92*; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.08 (s, 3F), −109.18 (br. s, 1F); HRMS (ESI+): Calcd for C₂₈H₂₇F₄N₅NaO₃S [M+Na]⁺: 612.1668, Found: 612.1621.

tert-butyl (S)-2-((3-(4-((4-([1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18dd)

Synthesized by General Procedure C. 13 mg, 19%, light yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J = 8.3 Hz, 2H), 7.95 (d, J = 8.0 Hz, 2H), 7.74 – 7.53 (m, 3H), 7.46 (t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.4 Hz, 1H), 6.95 (s, 1H), 4.40 – 4.22 (m, 1H), 3.52 – 3.27 (m, 3H), 3.17 – 2.98 (m, 1H), 2.14 – 2.02 (m, 1H), 1.95 – 1.78 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.22, 167.98, 162.92, 154.64, 151.40, 142.85, 140.86, 140.81, 133.55, 129.00, 128.97, 128.95, 127.51, 127.31, 127.13, 126.67, 117.35, 102.90, 80.21*, 79.79*, 55.32, 46.52, 31.95*, 31.17*, 30.23, 28.65, 23.71*, 22.93*; HRMS (ESI+): Calcd for C₃₃H₃₄N₅O₃S [M+H]⁺: 580.2382, Found: 580.2377.

tert-butyl (S)-2-((3-(4-((4-(benzofuran-3-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (18ee)

Synthesized by General Procedure C. 24 mg, 71%, tan amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.12 (s, 1H), 8.07 (d, J = 8.3 Hz, 2H), 7.99 – 7.93 (m, 1H), 7.77 (s, 1H), 7.61 – 7.51 (m, 3H), 7.40 – 7.31 (m, 2H), 6.94 (s, 1H), 4.40 – 4.22 (m, 1H), 3.53 – 3.27 (m, 3H), 3.17 – 2.98 (m, 1H), 2.15 – 2.01 (m, 1H), 1.97 – 1.77 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.25, 167.98, 163.02, 155.87, 154.39, 143.94, 143.76, 142.75, 128.96, 125.51, 124.82, 123.32, 120.99, 120.90, 117.43, 117.29, 117.13, 111.95, 103.23, 80.25_*, 79.81*, 55.33, 46.90, 46.52, 31.94*, 31.18*, 31.02*, 30.26*, 28.65, 23.71*, 22.94*; HRMS (ESI+): Calcd for C₂₉H₂₉N₅NaO₄S [M+Na]⁺: 566.1838, Found: 566.1813.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-phenylthiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19a)

Synthesized by General Procedure E. 21 mg, 82%, off-white oily solid. ¹H NMR (500 MHz, CDCl₃) δ 8.07 (d, J = 8.3 Hz, 2H), 7.87 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 8.3 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.34 (dd, J = 7.4 Hz, 1H), 6.90 (s, 1H), 4.79 (dd, J = 8.3, 4.9 Hz, 1H), 3.72 – 3.61 (m, 2H), 3.50 (s, 1H), 3.14 (dd, J = 15.2, 8.5 Hz, 1H), 2.32 – 2.25 (m, 1H), 1.93 (dt, J = 10.2, 5.1 Hz, 1H), 1.82 (dd, J = 14.0, 7.5 Hz, 2H), 1.48 (s, 18H); ¹³C NMR (126 MHz, CDCl₃) δ 177.01, 167.91, 163.01, 154.20, 151.38, 142.61, 134.29, 129.06, 128.87, 128.28, 126.27, 117.38, 102.66, 77.73, 56.66, 50.25, 30.81, 30.45, 28.31; HRMS (ESI+): Calcd for C₃₃H₄₀N₇O₅S [M+H]⁺: 646.2812, Found: 646.2805.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(pyridin-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19b)

Synthesized by General Procedure E. 3 mg, 11%, off-white oily solid. ¹H NMR (500 MHz, CDCl₃) δ 10.41 (s, 1H), 8.65 (ap. s, 2H), 8.09 (s, 1H), 8.01 (d, J = 7.8 Hz, 2H), 7.74 (ap. s, 2H), 7.60 (d, J = 7.9 Hz, 2H), 7.13 (s, 1H), 4.81 – 4.73 (s, 1H), 3.81 – 3.62 (m, 2H), 3.59 – 3.40 (m, 1H), 3.08 (dd, J = 15.2, 8.8 Hz, 1H), 2.36 – 2.26 (m, 1H), 1.98 – 1.89 (m, 1H), 1.84 – 1.75 (m, 2H), 1.48 (s, 18H); ¹³C NMR (126 MHz, CDCl₃) δ 176.85, 167.83, 163.35, 162.48, 159.25, 156.79, 154.30, 150.41, 150.01, 149.24, 142.56, 141.49, 128.97, 127.21, 122.29, 121.09, 120.50, 117.42, 106.53, 82.01, 79.68, 56.60, 50.32, 30.94, 30.46, 28.32, 24.52; HRMS (ESI+): Calcd for C₃₂H₃₉N₈O₅S [M+H]⁺: 647.2764, Found: 647.2772.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(pyridin-3-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19c)

Synthesized by General Procedure E. 18 mg, 78%, yellow oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.42 (s, 1H), 9.18 (s, 1H), 8.90 (s, 1H), 8.56 (d, J = 4.8 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.35 (dd, J = 8.0, 4.8 Hz, 1H), 6.96 (s, 1H), 4.81 – 4.71 (m, 1H), 3.85 – 3.62 (m, 2H), 3.59 – 3.42 (m, 1H), 3.04 (dd, J = 15.7, 9.0 Hz, 1H), 2.37 – 2.26 (m, 1H), 1.96 – 1.87 (m, 1H), 1.82 – 1.71 (m, 2H), 1.57 – 1.36 (m, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.57, 167.78, 163.53, 162.51, 154.41, 148.70, 148.51, 147.62, 142.92, 133.59, 130.59, 128.80, 123.71, 120.40, 117.17, 103.81, 82.25, 79.85, 56.54, 50.41, 31.02, 30.43, 28.29, 24.64; HRMS (ESI+): Calcd for C₃₂H₃₈N₈NaO₅S [M+Na]⁺: 669.2584, Found: 669.2589.

tert-butyl (S,E)-(((tert-butoxycarbonyl)amino)(2-((3-(4-((4-(3-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methylene)carbamate (19d)

Synthesized by General Procedure E. 10 mg, 43%, light yellow oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.40 (s, 1H), 8.01 (d, J = 8.7 Hz, 2H), 7.88 – 7.81 (m, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.14 – 7.06 (m, 2H), 6.81 (s, 1H), 4.82 – 4.73 (m, 1H), 3.79 – 3.62 (m, 2H), 3.57 – 3.46 (m, 1H), 3.09 (dd, J = 15.5, 8.8 Hz, 1H), 2.35 – 2.25 (m, 1H), 1.96 – 1.88 (m, 1H), 1.85 – 1.74 (m, 2H), 1.48 (s, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.79, 167.82, 163.97 (d, ¹J_CF = 247.5 Hz), 163.03, 161.52 (d, ¹J_CF = 247.5 Hz), 154.28, 150.72, 150.53, 142.67, 130.91, 130.76, 128.93, 128.00 (d, ³J_CF = 8.1 Hz), 127.92 (d, ³J_CF = 8.1 Hz), 120.72, 117.21, 115.83 (d, ²J_CF = 21.2 Hz), 115.62 (d, ²J_CF = 21.2 Hz), 102.16, 81.89, 79.74, 56.61, 50.34, 30.94, 30.44, 28.30; ¹⁹F NMR (376 MHz, CDCl₃) δ −113.86 (p, J = 6.7 Hz, 1F); HRMS (ESI+): Calcd for C₃₃H₃₉FN₇O₅S [M+H]⁺: 664.2717, Found: 664.2710.

tert-butyl (S,E)-(((tert-butoxycarbonyl)amino)(2-((3-(4-((4-(4-chlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methylene)carbamate (19e)

Synthesized by General Procedure E. 16 mg, 57%, light yellow oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.36 (s, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.85 (s, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 6.88 (s, 1H), 4.77 (qd, J = 7.2, 3.9 Hz, 1H), 3.79 – 3.62 (m, 2H), 3.57 – 3.45 (m, 1H), 3.09 (dd, J = 15.5, 8.8 Hz, 1H), 2.31 (dt, J = 12.3, 6.5 Hz, 1H), 1.96 – 1.88 (m, 1H), 1.86 – 1.74 (m, 1H), 1.72 – 1.59 (m, 1H), 1.48 (s, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.84, 167.84, 163.03, 154.28, 153.95, 151.48, 150.56, 142.69, 133.84, 133.34, 133.10, 128.96, 127.52, 120.83, 117.25, 103.04, 102.80, 82.17, 81.67, 56.61, 50.28, 30.93, 30.46, 28.32; HRMS (ESI+): Calcd for C₃₃H₃₈ClN₇O₅S [M+H]⁺: 680.2422, Found: 680.2425.

tert-butyl (S,Z)-((2-((3-(4-((4-(4-bromophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)((tert-butoxycarbonyl)imino)methyl)carbamate (19f)

Synthesized by General Procedure E. 10 mg, 83%, off-white oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.56 – 10.37 (m, 1H), 8.65 – 8.42 (m, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.2 Hz, 2H), 6.86 (s, 1H), 4.75 (dq, J = 11.1, 6.7, 6.0 Hz, 1H), 3.90 – 3.72 (m, 1H), 3.72 – 3.61 (m, 1H), 3.57 – 3.43 (m, 1H), 3.00 (dd, J = 15.9, 9.2 Hz, 1H), 2.39 – 2.27 (m, 1H), 1.97 – 1.85 (m, 1H), 1.82 – 1.70 (m, 1H), 1.48 (d, J = 5.7 Hz, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.42, 167.73, 162.96, 154.49, 150.48, 142.82, 133.57, 131.86, 128.73, 127.78, 121.90, 120.23, 116.99, 102.96, 82.26, 79.99, 56.52, 50.47, 31.13, 30.44, 28.63, 28.29, 28.10, 24.60; HRMS (ESI+): Calcd for C₃₃H₃₉BrN₇O₅S [M+H]⁺: 724.1917, Found: 724.1912.

tert-butyl (S,E)-(((tert-butoxycarbonyl)amino)(2-((3-(4-((4-(3-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methylene)carbamate (19g)

Synthesized by General Procedure E. 10 mg, 57%, yellow oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.43 (s, 1H), 8.21 – 8.04 (m, 0H), 7.98 (d, J = 8.3 Hz, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 8.6 Hz, 3H), 7.37 (ap q, J = 8.0, 5.8 Hz, 1H), 7.01 (td, J = 8.5, 2.7 Hz, 1H), 6.91 (s, 1H), 4.81 – 4.72 (m, 1H), 3.82 – 3.63 (m, 2H), 3.57 – 3.44 (m, 1H), 3.06 (dd, J = 15.6, 8.9 Hz, 1H), 2.38 – 2.27 (m, 1H), 1.96 – 1.87 (m, 1H), 1.84 – 1.72 (m, 2H), 1.49 (s, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.61, 167.80, 164.50 (d, ¹J_CF = 245.4 Hz), 162.93, 162.55, 162.07 (d, ¹J_CF = 245.4 Hz), 154.39, 150.44, 142.75, 136.84 (d, ³J_CF = 8.1 Hz), 136.76 (d, ³J_CF = 8.1Hz), 130.30 (d, ³J_CF = 8.1 Hz), 130.22 (d, ³J_CF = 8.1 Hz), 128.85, 121.76, 120.52, 117.11, 114.93 (d, ²J_CF = 21.2 Hz), 114.72 (d, ²J_CF = 21.2 Hz), 113.31 (d, ²J_CF = 23.2 Hz), 113.08 (d, ²J_CF = 23.2 Hz), 105.33, 103.58, 82.25, 79.80, 56.57, 50.45, 31.04, 30.43, 28.35, 28.25, 28.13; ¹⁹F NMR (376 MHz, CDCl₃) δ −113.07 – −113.17 (m, 1F); HRMS (ESI+): Calcd for C₃₃H₃₉FN₇O₅S [M+H]⁺: 664.2717, Found: 664.2727.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3-chlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19h)

Synthesized by General Procedure E. 37 mg, 77%, light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.43 (s, 1H), 8.35 (s, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.86 (s, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.37 – 7.24 (m, 2H), 6.89 (s, 1H), 4.80 – 4.71 (m, 1H), 3.86 – 3.73 (m, 1H), 3.72 – 3.62 (m, 1H), 3.57 – 3.45 (m, 1H), 3.02 (dd, J = 15.8, 9.1 Hz, 1H), 2.38 – 2.30 (m, 1H), 1.96 – 1.87 (m, 1H), 1.84 – 1.71 (m, 2H), 1.48 (ap d. rot, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.51, 167.78, 162.95, 162.52, 154.47, 150.43, 150.27, 142.77, 136.40, 134.73, 130.02, 128.80, 127.97, 126.32, 124.34, 120.42, 117.09, 103.61, 82.28, 79.89, 56.55, 50.43, 31.10, 30.46, 28.36, 28.25; HRMS (ESI+): Calcd for C₃₃H₃₈ClN₇O₅S [M+H]+: 680.2422, Found: 680.2435.

tert-butyl (S,Z)-((2-((3-(4-((4-(3-bromophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)((tert-butoxycarbonyl)imino)methyl)carbamate (19i)

Synthesized by General Procedure E. 21 mg, 57%, off-white oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.39 (s, 1H), 8.03 – 8.00 (m, 2H), 7.98 (s, 1H), 7.79 (dt, J = 7.8, 1.3 Hz, 1H), 7.66 (s, 1H), 7.58 – 7.54 (m, 1H), 7.46 – 7.42 (m, 1H), 7.29 (d, J = 7.9 Hz, 1H), 6.90 (s, 1H), 4.82 – 4.73 (m, 1H), 3.79 – 3.62 (m, 2H), 3.58 – 3.46 (m, 1H), 3.07 (dd, J = 15.6, 8.8 Hz, 1H), 2.37 – 2.26 (m, 1H), 1.97 – 1.87 (m, 1H), 1.86 – 1.73 (m, 2H), 1.48 (s, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.60, 167.81, 163.23, 154.59, 154.32, 150.03, 142.82, 136.65, 130.91, 130.32, 129.23, 128.84, 124.80, 122.96, 120.53, 117.15, 103.57, 83.05, 56.54, 50.33, 31.02, 30.45, 28.30, 24.45; HRMS (ESI+): Calcd for C₃₃H₃₉BrN₇O₅S [M+H]⁺: 724.1917, Found: 724.1909.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(2-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19j)

Synthesized by General Procedure E. 25 mg, 86%, light yellow oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.42 (s, 1H), 8.18 (td, J = 7.8, 2.0 Hz, 1H), 8.09 (s, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.63 – 7.56 (m, 2H), 7.25 – 7.18 (m, 2H), 7.17 – 7.09 (m, 1.5 Hz, 1H), 4.82 – 4.72 (m, 1H), 3.86 – 3.62 (m, 2H), 3.57 – 3.47 (d, J = 11.3 Hz, 1H), 3.04 (dd, J = 15.7, 9.0 Hz, 1H), 2.38 – 2.29 (m, 1H), 1.96 – 1.87 (m, 1H), 1.84 – 1.74 (m, 2H), 1.48 (ap. d., rot., 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.57, 167.83, 162.58, 161.80, 161.73 (d, ¹J_CF = 250.5 Hz), 159.25 (d, ¹J_CF = 250.5 Hz), 154.46, 150.45, 145.33 (d, ⁴J_CF = 3.0 Hz), 145.30 (d, ⁴J_CF = 3.0 Hz), 142.87, 130.15 (d, ⁴J_CF = 4.0 Hz), 130.11 (d, ⁴J_CF = 4.0 Hz), 129.05 (d, ³J_CF = 8.1 Hz), 128.97 (d, ³J_CF = 8.1 Hz), 128.83, 124.55, 124.52, 122.48 (d, ³J_CF = 11.1 Hz), 122.37 (d, ³J_CF = 11.1Hz), 120.46, 117.06, 116.11 (d, ²J_CF = 23.2 Hz), 115.88 (d, ²J_CF = 23.2 Hz), 107.92(d, ³J_CF = 16.2 Hz), 107.76 (d, ³J_CF = 16.2 Hz), 82.24, 79.79, 77.36, 56.56, 50.42, 31.07, 30.46, 28.38, 28.25, 28.13; ¹⁹F NMR (376 MHz, CDCl₃) δ − 114.04 – −114.17 (m, 1F); HRMS (ESI+): Calcd for C₃₃H₃₈FN₇NaO₅S [M+Na]⁺: 686.2537, Found: 686.2550.

tert-butyl (S,E)-(((tert-butoxycarbonyl)amino)(2-((3-(4-((4-(4-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methylene)carbamate (19k)

Synthesized by General Procedure E. 52 mg, 45%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.45 (br s, 1H), 8.54 (br s, 1H), 7.98 (d, J= 8 Hz, 2H), 7.93 (d, J = 8 Hz, 2H), 7.65 (d, J = 8 Hz, 2H), 7.61 (dt. J = 8, 4 Hz, 2H), 6.98 (br s. 1H), 4.81 – 4.72 (m, 1H), 3.85 – 3.62 (m, 2H), 3.58 – 3.43 (m, 1H). 3.02 (dd, J = 16, 8 Hz, 1H), 2.39 – 2.27 (m, 2H), 1.97 – 1.87 (m, 1H), 1.82 – 1.70 (m, 2H), 1.52-1.44 (m, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.49, 167.75, 163.18, 154.41, 150.11, 149.03, 142.73, 137.81, 130.19 (q, ²J_CF = 32.8 Hz), 129.86 (q, ²J_CF = 32.8 Hz), 129.54 (q, ²J_CF = 32.8 Hz), 129.22 (q, ²J_CF = 32.8 Hz), 128.77, 128.39, 126.38, 125.80 (q, ⁴J_CF = 4.0 Hz), 125.75 (q, ⁴J_CF = 4.0 Hz), 125.71 (q, ⁴J_CF = 4.0 Hz), 125.68 (q, ⁴J_CF = 4.0 Hz), 122.98, 120.45, 117.13, 104.45, 83.52, 66.00, 56.54, 50.45, 31.08, 30.42, 28.28, 28.10; ¹⁹F NMR(376 MHZ, CDCl₃) δ −62.53 (s, 3F); HRMS (ESI+): Calcd for C₃₄H₃₉F₃N₇O₅S [M+H]⁺: 714.2685, Found: 714.2703.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19l)

Synthesized by General Procedure E. 5 mg, 26%, off-white oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.46 (s, 1H), 8.60 (s, 1H), 8.11 (s, 1H), 8.07 (d, J = 7.5 Hz, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 7.57 – 7.48 (m, 2H), 6.95 (s, 1H), 4.81 – 4.71 (m, 1H), 3.87 – 3.74 (m, 1H), 3.73 – 3.62 (m, 1H), 3.57 – 3.46 (m, 1H), 2.99 (dd, J = 16.0, 9.3 Hz, 1H), 2.41 – 2.30 (m, 1H), 1.96 – 1.86 (m, 1H), 1.82 – 1.71 (m, 2H), 1.48 (d, rot., J = 22.4 Hz, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.38, 167.74, 163.12, 162.49, 154.55, 150.41, 150.18, 142.79, 135.41, 131.61 (q, ²J_CF = 32.3 Hz), 131.29 (q, ²J_CF = 32.3 Hz), 130.97 (q, ²J_CF = 32.3 Hz), 130.65 (q, ²J_CF = 32.3 Hz), 129.45, 129.24, 128.88, 128.73, 125.66, 124.53 (q, ⁴J_CF = 3.0 Hz), 124.48 (q, ⁴J_CF = 3.0 Hz), 124.45 (q, ⁴J_CF = 3.0 Hz), 124.41 (q, ⁴J_CF = 3.0 Hz), 122.96 (q, ⁴J_CF = 3.0 Hz), 122.92 (q, ⁴J_CF = 3.0 Hz), 122.89 (q, ⁴J_CF = 3.0 Hz), 122.85 (q, ⁴J_CF = 3.0 Hz), 120.34, 117.04, 103.79, 82.32, 79.98, 56.50, 50.48, 31.17, 30.47, 28.35, 28.22, 28.09; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.66; HRMS (ESI+): Calcd for C₃₄H₃₉F₃N₇O₅S [M+H]⁺: 714.2685, Found: 714.2682.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(2-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19m)

Synthesized by General Procedure E. 10 mg, 37%, yellow oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.33 (s, 1H), 8.01 (d, J = 8.2 Hz, 2H), 7.84 – 7.74 (m, 2H), 7.70 (d, J = 7.7 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.52 – 7.44 (m, 3H), 6.77 (s, 1H), 4.78 (qd, J = 7.3, 4.1 Hz, 1H), 3.74 – 3.61 (m, 2H), 3.56 – 3.44 (m, 1H), 3.12 (dd, J = 15.4, 8.5 Hz, 1H), 2.33 – 2.23 (m, 1H), 1.92 (dq, J = 10.4, 4.8 Hz, 1H), 1.87 – 1.75 (m, 2H), 1.48 (d, J = 9.0 Hz, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 191.61, 176.91, 167.90, 162.66 48 (q, ³J_CF = 19.2 Hz), 162.47 48 (q, ²J_CF = 319.2 Hz), 154.20, 150.48, 148.79, 142.69, 134.55 48 (q, ¹J_CF = 285.8 Hz), 132.10, 131.72 (q, ¹J_CF = 285.8 Hz), 128.97 48 (q, ¹J_CF = 285.8 Hz), 128.65, 128.32, 126.58 48 (q, ¹J_CF = 285.8 Hz), 120.91, 117.31, 107.12, 82.14, 79.54, 56.61, 50.27, 30.83, 30.46, 28.39*, 28.24*, 24.56; ¹⁹F NMR (376 MHz, CDCl₃) δ −57.78; HRMS (ESI+): Calcd for C₃₄H₃₈F₃N₇NaO₅S [M+Na]⁺: 736.2505, Found: 714.2500.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(p-tolyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19n).Synthesized by General Procedure E

21 mg, 82%, off-white oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.39 (s, 1H), 8.00 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 7.9 Hz, 2H), 6.83 (s, 1H), 4.81 – 4.73 (m, 1H), 3.80 – 3.60 (m, 2H), 3.56 – 3.45 (m, 1H), 3.08 (dd, J = 15.4, 8.8 Hz, 1H), 2.38 (s, 3H), 2.35 – 2.25 (m, 1H), 1.98 – 1.87 (m, 1H), 1.85 – 1.74 (m, 2H), 1.48 (s, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.75, 167.87, 162.80, 154.28, 151.70, 142.87, 137.94, 131.88, 129.49, 129.22, 128.90, 126.16, 120.47, 117.09, 101.88, 81.88, 79.77, 56.61, 50.32, 30.93, 30.44, 29.81, 28.30, 24.53, 21.44; HRMS (ESI+): Calcd for C₃₄H₄₂N₇O₅S [M+H]+: 660.2968, Found: 660.2991.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(4-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19o)

Synthesized by General Procedure E. 23 mg, 61%, clear oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.40 (s, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.84 – 7.75 (m, 2H), 7.62 (d, J = 2.1 Hz, 1H), 7.59 – 7.55 (m, 1H), 6.96 – 6.91 (m, 2H), 6.72 (s, 1H), 4.82 – 4.69 (m, 1H), 3.83 (s, 3H), 3.74 – 3.58 (m, 2H), 3.55 – 3.42 (m, 1H), 3.03 (dd, J = 15.7, 8.9 Hz, 1H), 2.36 – 2.23 (m, 1H), 1.96 – 1.83 (m, 1H), 1.81 – 1.71 (m, 1H), 1.58 – 1.35 (m, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.75, 167.87, 162.80, 154.28, 151.70, 142.87, 137.94, 131.88, 129.49, 129.22, 128.90, 126.16, 120.47, 117.09, 101.88, 81.88, 79.77, 56.61, 50.32, 30.93, 30.44, 29.81, 28.30, 24.53, 21.44; HRMS (ESI+): Calcd for C₃₄H₄₂N₇O₆S [M+H]⁺: 676.2917, Found: 676.2903.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(4-ethoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19p)

Synthesized by General Procedure E. 67 mg, 68%, light yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 6.74 (s, 1H), 4.82 – 4.73 (m, 1H), 4.08 (q, J = 7.0 Hz, 2H), 3.77 – 3.62 (m, 2H), 3.56 – 3.45 (m, 1H), 3.11 (dd, J = 15.4, 8.6 Hz, 1H), 2.34 – 2.24 (m, 1H), 1.97 – 1.88 (m, 2H), 1.86 – 1.75 (m, 2H), 1.52 – 1.40 (m, 21H); ¹³C NMR (126 MHz, CDCl₃) δ 176.96, 167.94, 162.78, 159.07, 153.98, 152.88, 151.53, 142.85, 134.03, 129.00, 127.54, 127.43, 117.19, 114.75, 105.35, 100.89, 83.18, 77.73, 63.67, 56.64, 50.25, 30.79, 30.45, 29.85, 28.31, 28.19, 15.00; HRMS (ESI+): Calcd for C₃₅H₄₄N₇O₆S [M+H]⁺: 690.3074, Found: 690.3084.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19q)

Synthesized by General Procedure E. 18 mg, 51%, clear oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.41 (s, 1H), 7.96 (d, J = 8.2 Hz, 2H), 7.63 (s, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.48 – 7.41 (m, 2H), 7.32 (t, J = 8.0 Hz, 1H), 6.92 – 6.83 (m, 2H), 6.36 (s, 1H), 4.82 – 4.71 (m, 1H), 3.86 (s, 3H), 3.80 – 3.61 (m, 2H), 3.58 – 3.44 (m, 1H), 3.05 (dd, J = 15.6, 8.9 Hz, 1H), 2.39 – 2.22 (m, 0H), 1.97 – 1.87 (m, 1H), 1.84 – 1.70 (m, 2H), 1.55 – 1.39 (m, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.65, 167.84, 163.00, 162.58, 160.02, 154.31, 153.02, 151.37, 150.40, 142.95, 136.02, 133.88, 129.80, 128.83, 120.35, 118.71, 117.07, 113.79, 111.79, 105.30, 102.87, 82.19, 79.75, 56.56, 55.45, 50.33, 30.96, 30.43, 29.83, 28.29, 28.10, 24.50; HRMS (ESI+): Calcd for C₃₄H₄₂N₇O₆S [M+H]⁺: 676.2917, Found: 676.2900.

tert-butyl (S,E)-(((tert-butoxycarbonyl)amino)(2-((3-(4-((4-(4-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methylene)carbamate (19r)

Synthesized by General Procedure E. 27 mg, 70%) as a yellow oily solid. ¹H NMR (500 MHz, CDCl₃) δ 10.41 (s, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.85 (s, 1H), 7.57 (d, J = 8.1 Hz, 2H), 6.88 (s, 1H), 4.81 – 4.71 (m, 1H), 3.79 – 3.61 (m, 2H), 3.56 – 3.44 (m, 1H), 3.14 – 3.04 (dd, J = 15.5, 8.6 Hz, 1H), 2.34 – 2.25 (m, 1H), 1.95 – 1.88 (m, 1H), 1.85 – 1.74 (m, 2H), 1.48 (s, 18H); ¹³C NMR (126 MHz, CDCl₃) δ 176.70, 168.89, 167.68, 162.92, 154.13, 150.83, 150.19, 148.84, 142.49, 133.15, 128.82, 127.48, 121.10, 120.75, 119.45, 117.11, 103.06, 81.60, 79.46, 56.46, 50.14, 30.75, 30.29, 29.67, 28.16, 24.33; ¹⁹F NMR (470 MHz, CDCl₃) δ −57.82 (s, 3F); HRMS (ESI+): Calcd for C₃₄H₃₈F₃N₇O₆S [M+H]⁺: 730.2634, Found: 730.2672.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19s)

Synthesized by General Procedure E. 20 mg, 83%, yellow oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.38 (s, 1H), 8.04 – 8.00 (m, 2H), 7.80 (ddd, J = 7.8, 1.6, 1.0 Hz, 1H), 7.73 (dt, J = 2.5, 1.3 Hz, 1H), 7.60 – 7.54 (m, 2H), 7.43 (t, J = 8.0 Hz, 1H), 7.19 – 7.15 (m, 1H), 6.94 (s, 1H), 4.82 – 4.73 (m, 1H), 3.82 – 3.67 (m, 2H), 3.57 – 3.45 (m, 1H), 3.09 (dd, J = 15.5, 8.8 Hz, 1H), 2.32 (d, J = 11.6 Hz, 1H), 1.97 – 1.88 (m, 1H), 1.86 – 1.75 (m, 1H), 1.67 – 1.56 (m, 1H), 1.50 (s rot., 9H), 1.46 (s rot., 9H); ¹³C NMR (101 MHz, CDCl₃) δ 176.81, 163.00, 150.47 (q, ³J_CF = 32.8 Hz), 150.20 (q, ³J_CF = 32.8 Hz), 149.82 (q, ³J_CF = 32.8 Hz), 142.60, 136.61, 130.13, 128.96, 124.46, 120.90, 120.28, 118.85, 117.26, 103.93, 82.20, 79.66, 56.59, 50.34, 30.95, 30.47, 28.38*, 28.24*, 28.14; ¹⁹F NMR (376 MHz, CDCl₃) δ −57.66; HRMS (ESI+): Calcd for C₃₄H₃₈F₃N₇O₆S [M+H]⁺: 730.2634, Found: 730.2635.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3,4-dimethylphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19t)

Synthesized by General Procedure E. 35 mg, 92%, clear oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.38 (s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.67 – 7.51 (m, 6H), 7.17 (d, J = 7.8 Hz, 1H), 6.81 (s, 1H), 6.36 (d, J = 2.4 Hz, 1H), 4.82 – 4.72 (m, 1H), 3.81 – 3.60 (m, 2H), 3.50 (s, 1H), 3.07 (dd, J = 15.5, 8.7 Hz, 1H), 2.30 (d, J = 11.3 Hz, 6H), 1.98 – 1.85 (m, 1H), 1.85 – 1.72 (m, 2H), 1.47 (s, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.70, 167.88, 163.10, 162.57, 154.72, 154.24, 153.05, 151.67, 150.44, 143.05, 136.92, 136.63, 133.77, 132.34, 130.07, 128.85, 127.44, 123.69, 120.31, 117.05, 105.24, 101.66, 82.97, 82.15, 79.69, 56.57, 50.32, 30.90, 30.42, 28.29, 28.11, 24.55, 20.04, 19.74; HRMS (ESI+): Calcd for C₃₅H₄₄N₇O₅S [M+H]⁺: 674.3125, Found: 674.3134.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3,4-dimethoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19u)

Synthesized by General Procedure E. 25 mg, 78%, clear oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.41 (s, 1H), 8.56 – 8.24 (m, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.47 – 7.39 (m, 2H), 7.22 (s, 1H), 6.76 (s, 1H), 4.76 (dq, J = 11.7, 7.1, 5.1 Hz, 1H), 3.93 (d, J = 18.3 Hz, 6H), 3.83 – 3.60 (m, 3H), 3.56 – 3.43 (m, 1H), 3.04 (dd, J = 15.6, 9.0 Hz, 1H), 2.38 – 2.23 (m, 1H), 1.97 – 1.84 (m, 1H), 1.82 – 1.70 (m, 2H), 1.54 – 1.38 (m, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.63, 167.81, 162.82, 162.56, 154.36, 151.37, 150.41, 149.11, 142.93, 128.79, 127.90, 120.32, 118.80, 117.01, 111.35, 109.61, 101.06, 83.08, 82.20, 79.74, 56.54, 56.08, 56.06, 50.36, 30.98, 30.46, 28.26, 28.09; HRMS (ESI+): Calcd for C₃₅H₄₄N₇O₇S [M+H]⁺: 706.3023, Found: 706.3028.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3-chloro-4-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19v)

Synthesized by General Procedure E. 17 mg, 34%, light yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 10.38 (s, 1H), 8.03 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 2.2 Hz, 1H), 7.75 (dd, J = 8.5, 2.2 Hz, 1H), 7.59 – 7.51 (m, 2H), 6.97 (d, J = 8.6 Hz, 1H), 6.78 (s, 1H), 4.82 – 4.72 (m, 1H), 3.94 (s, 3H), 3.84 – 3.62 (m, 2H), 3.58 – 3.42 (m, 1H), 3.10 (dd, J = 15.4, 8.7 Hz, 1H), 2.36 – 2.23 (m, 1H), 1.97 – 1.87 (m, 1H), 1.88 – 1.74 (m, 1H), 1.74 – 1.56 (m, 1H), 1.54 – 1.40 (m, 18H); ¹³C NMR (126 MHz, CDCl₃) δ 176.89, 167.87, 162.97, 154.91, 150.17, 142.71, 128.98, 128.46, 128.11, 125.70, 122.84, 117.23, 112.21, 101.84, 100.14, 83.18, 77.73, 56.62, 56.40, 30.88, 30.46, 29.85, 28.31, 28.18; HRMS (ESI+): Calcd for C₃₄H₄₁ClN₇O₆S [M+H]⁺: 710.2528, Found: 710.2542.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3,4-difluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19w)

Synthesized by General Procedure E. 10 mg, 43%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.42 (s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.96 – 7.87 (m, 1H), 7.74 – 7.65 (m, 1H), 7.62 – 7.52 (m, 3H), 7.23 – 7.15 (m, 1H), 6.84 (s, 1H), 4.81 – 4.72 (m, 1H), 3.86 – 3.61 (m, 2H), 3.57 – 3.43 (m, 1H), 3.07 (dd, J = 15.6, 8.8 Hz, 1H), 2.38 – 2.26 (m, 1H), 1.97 – 1.88 (m, 1H), 1.84 – 1.72 (m, 2H), 1.48 (d, J = 17.1 Hz, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.74, 167.79, 162.99, 162.59, 150.44, 149.63, 142.58, 128.91, 122.12, 120.79, (dd, ¹J_CF = 228.3 Hz, ³J_CF = 17.2 Hz), 117.49 (dd, ¹J_CF = 228.3 Hz, ³J_CF = 17.2 Hz), 117.19, 115.40 (dd, ¹J_CF = 228.3 Hz, ³J_CF = 17.2 Hz), 115.22 (dd, ¹J_CF = 228.3 Hz, ³J_CF = 17.2 Hz), 103.12, 82.23, 79.74, 56.57, 50.43, 31.01, 30.44, 28.36, 28.24; ¹⁹F NMR (376 MHz, CDCl₃) δ −137.54 – −137.74 (m, 1F), −138.64 – −138.84 (m, 1F); HRMS (ESI+): Calcd for C₃₃H₃₈F₂N₇O₅S [M+H]⁺: 682.2623, Found: 682.2590.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3,5-dichlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19x)

Synthesized by General Procedure E. 20 mg, 45, clear yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.36 (s, 1H), 7.94 (d, J = 8.5 Hz, 3H), 7.55 – 7.49 (m, 2H), 7.45 (d, J = 2.1 Hz, 1H), 7.29 (dd, J = 8.5, 2.2 Hz, 1H), 7.24 (s, 1H), 4.79 – 4.70 (m, 1H), 3.82 – 3.60 (m, 2H), 3.55 – 3.41 (m, 1H), 3.04 (dd, J = 15.6, 8.9 Hz, 1H), 2.33 – 2.23 (m, 1H), 1.95 – 1.85 (m, 1H), 1.82 – 1.68 (m, 2H), 1.48 (s, 12H), 1.44 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 176.68, 167.81, 162.57, 161.89, 150.45, 146.89, 142.75, 133.80, 132.44, 132.39, 131.76, 130.38, 128.87, 127.40, 120.65, 117.18, 108.44, 82.23, 79.76, 56.58, 50.34, 30.99, 30.45, 28.38, 28.24, 28.13; HRMS (ESI+): Calcd for C₃₃H₃₈Cl₂N₇O₅S [M+H]⁺: 714.2032, Found: 714.2042.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19y)

Synthesized by General Procedure E. 12 mg, 48%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.38 (s, 1H), 8.13 – 7.91 (m, 5H), 7.56 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 9.4 Hz, 1H), 6.90 (s, 1H), 4.84 – 4.71 (m, 1H), 3.83 – 3.63 (m, 2H), 3.57 – 3.46 (m, 1H), 3.07 (dd, J = 15.6, 8.9 Hz, 1H), 2.37 – 2.27 (m, 1H), 1.98 – 1.88 (m, 1H), 1.85 – 1.72 (m, 2H), 1.55 – 1.42 (m, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.79, 167.79, 163.28, 162.59, 160.66, 158.10, 154.35, 150.46, 149.30, 142.55, 140.47, 137.78, 131.52 (d, ³J_CF = 9.1 Hz), 131.43 (d, ³J_CF = 9.1 Hz), 131.19, 128.94, 126.81 (q, ¹J_CF = 273.7 Hz), 124.94, 124.09 (q, ¹J_CF = 273.7 Hz), 121.38 (q, ¹J_CF = 273.7 Hz), 120.93, 119.32, 118.64 (q, ¹J_CF = 273.7 Hz), 118.51, 117.43, 117.29, 117.22, 103.45, 82.24, 79.76, 56.58, 50.34, 30.99, 30.49, 28.36*, 28.26*; ¹⁹F NMR (376 MHz, CDCl₃) δ −61.44 (d, J = 12.7 Hz, 3F), −115.54 – −116.19 (m, 1F); HRMS (ESI+): Calcd for C₃₄H₃₈F₄N₇O₅S [M+H]⁺: 732.2591, Found: 732.2542.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3,5-difluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19z)

Synthesized by General Procedure E. 26 mg, 79%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 10.83 (s, 1H), 8.92 (s, 1H), 8.26 (d, J = 8.3 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.62 (s, 1H), 7.15 – 7.07 (m, 1H), 5.16 – 5.05 (m, 1H), 4.24 – 4.09 (m, 1H), 4.09 – 3.98 (m, 1H), 3.93 – 3.80 (m, 1H), 3.36 (dd, J = 15.9, 9.2 Hz, 1H), 2.78 – 2.65 (m, 1H), 2.34 – 2.23 (m, 1H), 2.18 – 2.05 (m, 2H), 1.92 – 1.74 (m, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.41, 167.72, 164.69 (dd, ¹J_CF = 248.5 Hz, ³J_CF = 13.1 Hz), 164.56 (dd, ¹J_CF = 248.5 Hz, ³J_CF = 13.1 Hz), 162.95, 162.49, 162.23 (dd, ¹J_CF = 248.5 Hz, ³J_CF = 13.1 Hz), 162.10 (dd, ¹J_CF = 248.5 Hz, ³J_CF = 13.1 Hz), 154.55, 150.38, 149.46, 142.68, 137.86 (dd, ³J_CF = 10.1 Hz), 137.76 (dd, ³J_CF = 10.1 Hz), 137.66 (dd, ³J_CF = 10.1 Hz), 128.77, 120.40, 117.07, 109.14 (dd, ³J_CF = 19.2 Hz, ⁴J_CF = 7.1 Hz), 109.07 (dd, ³J_CF = 19.2 Hz, ⁴J_CF = 7.1 Hz), 108.95 (dd, ³J_CF = 19.2 Hz, ⁴J_CF = 7.1 Hz), 108.88 (dd, ³J_CF = 19.2 Hz, ⁴J_CF = 7.1 Hz), 104.44, 103.40 (dd, ²J_CF = 25.8 Hz), 103.14 (dd, ²J_CF = 25.8 Hz), 102.89 (dd, ²J_CF = 25.8 Hz), 82.33, 79.97, 56.53, 50.56, 31.17, 30.44, 28.34*, 28.24*, 24.68; ¹⁹F NMR (376 MHz, CDCl₃) δ −109.87 (t, J = 8.2 Hz, 2F); HRMS (ESI+): Calcd for C₃₃H₃₈F₂N₇O₅S [M+H]⁺: 682.2623, Found: 682.2615.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3-fluoro-5-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19aa)

Synthesized by General Procedure E. 22 mg, 51%, clear yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.43 (s, 1H), 8.34 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.89 (s, 1H), 7.78 (dt, J = 9.6, 1.9 Hz, 1H), 7.61 – 7.55 (m, 2H), 7.25 (d, J = 6.2 Hz, 2H), 6.99 (s, 1H), 4.81 – 4.72 (m, 1H), 3.86 – 3.62 (m, 2H), 3.59 – 3.44 (m, 1H), 3.02 (dd, J = 15.7, 9.0 Hz, 1H), 2.41 – 2.29 (m, 1H), 1.98 – 1.87 (m, 1H), 1.83 – 1.71 (m, 2H), 1.51 (s, 8H), 1.45 (s, 10H); ¹³C NMR (101 MHz, CDCl₃) δ 176.54, 167.74, 164.19 (d, ¹J_CF = 248.5 Hz), 163.20, 162.52, 161.73 (d, ¹J_CF = 248.5 Hz), 154.49, 150.43, 149.10, 142.57, 137.89 (q, ³J_CF = 9.1 Hz), 137.80 (d, ³J_CF = 9.1 Hz), 133.40 (qd, ²J_CF = 34.3 Hz, ³J_CF = 9.1 Hz), 133.32 (qd, ²J_CF = 34.3 Hz, ³J_CF = 9.1 Hz), 133.08 (qd, ²J_CF = 34.3 Hz, ³J_CF = 9.1 Hz), 132.99 (qd, ²J_CF = 34.3 Hz, ³J_CF = 9.1 Hz), 132.74 (qd, ²J_CF = 34.3 Hz, ³J_CF = 9.1 Hz), 132.66 (qd, ²J_CF = 34.3 Hz, ³J_CF = 9.1 Hz), 132.41 (qd, ²J_CF = 34.3 Hz, ³J_CF = 9.1 Hz), 132.33 (qd, ²J_CF = 34.3 Hz, ³J_CF = 9.1 Hz), 128.84, 127.56 (q, ¹J_CF = 274.7 Hz), 124.85 (q, ¹J_CF = 274.7 Hz), 122.13 (q, ¹J_CF = 274.7 Hz), 120.72, 119.41 (q, ¹J_CF = 274.7 Hz), 118.61, 117.20, 116.68 (d, ²J_CF = 23.2 Hz), 116.45 (d, ²J_CF = 23.2 Hz), 112.02 (d, ²J_CF = 25.3 Hz), 111.77 (d, ²J_CF = 25.3 Hz), 104.90, 82.32, 79.92, 56.54, 50.46, 31.13, 30.49, 28.37*, 28.24*, 24.62; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.84 (s, 3F), −110.73 (t, J = 9.0 Hz, 1F); HRMS (ESI+): Calcd for C₃₄H₃₈F₄N₇O₅S [M+H]⁺: 732.2591, Found: 732.2602.

tert-butyl (S,Z)-((2-((3-(4-((4-(3,5-bis(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)((tert-butoxycarbonyl)imino)methyl)carbamate (19bb)

Synthesized by General Procedure E. 31 mg, 84%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.40 (s, 1H), 8.30 (s, 2H), 8.11 (d, J = 19.0 Hz, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.80 (s, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.07 (s, 1H), 4.82 – 4.73 (m, 1H), 3.84 – 3.63 (m, 2H), 3.58 – 3.47 (m, 1H), 3.05 (dd, J = 15.6, 9.0 Hz, 1H), 2.39 – 2.28 (m, 1H), 1.99 – 1.88 (m, 1H), 1.85 – 1.74 (m, 2H), 1.48 (d, J = 18.2 Hz, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.75, 167.74, 163.51, 162.55, 150.45, 148.74, 142.42, 136.53, 132.64 (q, ²J_CF = 33.3 Hz), 132.31 (q, ²J_CF = 33.3 Hz), 131.98 (q, ²J_CF = 33.3 Hz), 131.66 (q, ²J_CF = 33.3 Hz), 128.94, 127.60(q, ¹J_CF = 274.7 Hz), 126.15, 124.88 (q, ¹J_CF = 274.7 Hz), 122.16 (q, ¹J_CF = 274.7 Hz), 121.35, 121.05, 119.45 (q, ¹J_CF = 274.7 Hz), 117.35, 105.34, 82.27, 79.83, 56.56, 50.40, 31.04, 30.53, 28.37*, 28.24*; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.94 (s, 6F); HRMS (ESI+): Calcd for C₃₅H₃₈F₆N₇O₅S [M+H]⁺: 782.2559, Found: 782.2541.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(2-fluoro-5-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (19cc)

Synthesized by General Procedure E. 27 mg, 51%, clear yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.34 (s, 1H), 8.48 (dd, J = 7.1, 2.5 Hz, 1H), 8.04 (d, J = 8.3 Hz, 2H), 7.83 (s, 1H), 7.55 (d, J = 8.4 Hz, 3H), 7.25 – 7.18 (m, 1H), 4.83 – 4.73 (m, 1H), 3.80 – 3.62 (m, 2H), 3.58 – 3.45 (m, 1H), 3.09 (dd, J = 16.9, 8.4 Hz, 1H), 2.36 – 2.24 (m, 1H), 1.99 – 1.89 (m, 1H), 1.86 – 1.72 (m, 2H), 1.49 (d, J = 9.5 Hz, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.91, 167.83, 163.22 (d, ¹J_CF = 256.5 Hz), 162.25, 160.68 (d, ¹J_CF = 256.5 Hz), 158.33, 154.26, 148.80, 143.85, 142.54, 129.01, 127.72, 126.04, 123.36, 123.26 (q, ³J_CF = 12.1 Hz), 123.15 (q, ³J_CF = 12.1 Hz), 123.03 (q, ³J_CF = 12.1 Hz), 122.90 (q, ³J_CF = 12.1 Hz), 122.61, 121.09, 117.33, 116.82, 116.58, 109.22 (d, ³J_CF = 16.2 Hz), 109.06 (d, ³J_CF = 16.2 Hz), 83.56, 56.61, 50.26, 30.92, 30.50, 29.85, 28.31, 28.14; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.05 (s,3F), −109.15 – −109.28 (m, 1F); HRMS (ESI+): Calcd for C₃₄H₃₈F₄N₇O₅S [M+H]⁺: 732.2591, Found: 732.2605.

tert-butyl (S,Z)-((2-((3-(4-((4-([1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)((tert-butoxycarbonyl)imino)methyl)carbamate (19dd)

Synthesized by General Procedure E. 7 mg, 83%, light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 10.43 – 10.31 (m, 1H), 8.05 (d, J = 8.3 Hz, 2H), 7.99 – 7.92 (m, 2H), 7.79 – 7.69 (m, 1H), 7.65 (dd, J = 8.5, 6.9 Hz, 4H), 7.61 – 7.56 (m, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 6.94 (s, 1H), 4.84 – 4.74 (m, 1H), 3.78 – 3.62 (m, 2H), 3.59 – 3.44 (m, 1H), 3.11 (dd, J = 15.5, 8.7 Hz, 1H), 2.36 – 2.25 (m, 1H), 1.97 – 1.88 (m, 1H), 1.87 – 1.75 (m, 2H), 1.53 – 1.44 (m, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.82, 167.87, 162.83, 162.58, 154.31, 151.36, 150.33, 142.77, 140.80, 133.58, 128.95, 128.67, 127.49, 127.27, 127.12, 126.93, 126.65, 120.68, 117.18, 113.25, 102.79, 82.17, 79.67, 56.60, 50.33, 30.91, 30.45, 29.86, 28.31, 24.60; HRMS (ESI+): Calcd for C₃₉H₄₄N₇O₅S [M+H]⁺: 722.3125, Found: 722.3107.

tert-butyl (S,Z)-((2-((3-(4-((4-(benzofuran-2-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)((tert-butoxycarbonyl)imino)methyl)carbamate (19ee)

Synthesized by General Procedure E. 18 mg, 69%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.39 (s, 1H), 8.13 (br. s, 2H), 8.05 – 7.92 (m, 3H), 7.62 – 7.50 (m, 3H), 7.40 – 7.31 (m, 2H), 6.92 (s, 1H), 4.83 – 4.73 (m, 1H), 3.83 – 3.62 (m, 1H), 3.57 – 3.45 (m, 1H), 3.08 (dd, J = 15.3, 8.6 Hz, 1H), 2.36 – 2.27 (m, 1H), 1.97 – 1.87 (m, 1H), 1.85 – 1.71 (m, 2H), 1.49* (ap. d, J = 5.9 Hz, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 176.75, 167.84, 163.01, 155.88, 154.34, 148.93, 143.88, 143.82, 142.76, 128.91, 125.53, 124.78, 123.31, 120.92, 120.74, 117.26, 117.16, 111.95, 103.02, 83.55, 82.15, 79.75, 56.58, 50.33, 36.83, 30.97, 30.48, 29.85, 28.32, 28.13, 24.84, 24.61; HRMS (ESI+): Calcd for C₃₅H₄₀N₇O₆S [M+H]⁺: 686.2761, Found: 686.2760.

(S)-amino(2-((3-(4-((4-phenylthiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20a)

Synthesized by General Procedure E. 8 mg, 85%, yellow solid. ¹H NMR (500 MHz, CD₃OD) δ 8.10 (d, J = 8.3 Hz, 2H), 7.85 (dd, J = 16.1, 8.0 Hz, 4H), 7.46 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.4 Hz, 1H), 7.19 (s, 1H), 4.56 – 4.50 (m, 1H), 3.56 (ddd, J = 12.1, 10.3, 5.8 Hz, 1H), 3.51 – 3.42 (m, 1H), 3.33 (s, 2H), 2.33 – 2.24 (m, 1H), 2.17 – 1.97 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.97, 169.20, 166.40, 156.47, 144.36, 129.88, 129.72, 129.63, 127.27, 122.36, 119.87, 57.22, 31.61, 30.08, 23.59; HRMS (ESI+): Calcd for C₂₃H₂₄N₇OS [M+H]⁺: 446.1763, Found: 446.1755.

(S)-amino(2-((3-(4-((4-(pyridin-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium trifluoroacetate (20b)

Synthesized by General Procedure E. 3 mg, 100%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.75 (d, J = 6.0 Hz, 2H), 8.47 – 8.41 (m, 2H), 8.10 – 8.02 (m, 3H), 7.95 – 7.89 (m, 2H), 4.58 – 4.49 (m, 1H), 3.59 – 3.52 (m, 1H), 3.51 – 3.42 (m, 1H), 3.34 – 3.31 (m, 2H), 2.36 – 2.23 (m, 1H), 2.19 – 1.96 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 176.41, 167.86, 163.98, 155.01, 148.91, 146.25, 143.48, 142.84, 128.02, 122.18, 119.62, 117.04, 114.31, 55.77, 30.14, 28.63, 22.15; HRMS (ESI+): Calcd for C₂₂H₂₃N₈OS [M+H]⁺: 447.1716, Found: 447.1704. HPLC purity: 87%.

(S)-amino(2-((3-(4-((4-(pyridin-3-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20c)

Synthesized by General Procedure E. 8 mg, 85%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 9.31 (s, 1H), 8.96 (dt, J = 8.4, 1.7 Hz, 1H), 8.69 (d, J = 5.6 Hz, 1H), 8.05 – 7.97 (m, 3H), 7.91 – 7.82 (m, 2H), 7.66 (s, 1H), 4.55 – 4.48 (m, 1H), 3.57 – 3.50 (m, 1H), 3.48 – 3.40 (m, 1H), 3.31 – 3.29 (m, 1H), 2.33 – 2.21 (m, 1H), 2.17 – 1.95 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.82, 169.29, 165.60, 156.45, 145.97, 145.00, 142.24, 142.04, 141.36, 135.43, 129.44, 128.13, 120.86, 118.37, 109.98, 57.18, 31.57, 30.06, 23.58; HRMS (ESI+): Calcd for C₂₂H₂₃N₈OS [M+H]⁺: 447.1716, Found: 447.1698.

(S)-amino(2-((3-(4-((4-(4-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20d)

Synthesized by General Procedure E. 5 mg, 100%, yellow solid. ¹H NMR (500 MHz, CD₃OD) δ 8.09 (d, J = 8.3 Hz, 2H), 7.89 (dd, J = 8.4, 5.3 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.19 (t, J = 8.6 Hz, 2H), 7.15 (s, 1H), 4.57 – 4.51 (m, 1H), 3.60 – 3.51 (m, 1H), 3.51 – 3.41 (m, 1H), 3.36 – 3.33 (m, 2H), 2.29 (ddd, J = 18.0, 10.1, 5.4 Hz, 1H), 3.39 – 3.36 (m, 0H), 2.20 – 1.96 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 176.54, 167.77, 165.04, 163.86 (d, ¹J_CF = 199.0 Hz), 161.89 (d, ¹J_CF = 199.0 Hz), 155.04, 146.83, 142.85, 129.13, 128.29, 127.95 (d, ⁴J_CF = 6.1 Hz), 127.89 (d, ⁴J_CF = 6.1 Hz), 120.97, 118.47, 115.30 (d, ³J_CF = 18.2 Hz), 115.12 (d, ³J_CF = 18.2 Hz), 55.80, 36.84, 30.19, 28.67, 22.18; ¹⁹F NMR (376 MHz, CD₃OD) δ −116.45 – −116.59 (m, 1F); HRMS (ESI+): Calcd for C₂₃H₂₃FN₇OS [M+H]⁺: 464.1669, Found: 464.1681.

(S)-amino(2-((3-(4-((4-(4-chlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20e)

Synthesized by General Procedure E. 5 mg, 100%, off-white solid. ¹H NMR (500 MHz, CD₃OD) δ 8.12 – 8.05 (m, 2H), 7.92 – 7.80 (m, 4H), 7.50 – 7.42 (m, 2H), 7.22 (d, J = 4.4 Hz, 1H), 4.55 (q, J = 6.6 Hz, 1H), 3.60 – 3.52 (m, 1H), 3.52 – 3.43 (m, 1H), 2.35 – 2.24 (m, 1H), 2.19 – 1.98 (m, 3H); ¹³C NMR (151 MHz, CD₃OD) δ 177.90, 169.24, 156.48, 148.86, 144.56, 135.11, 133.30, 129.92, 129.63, 128.72, 121.91, 119.47, 64.45, 57.24, 31.62, 30.10, 23.60; HRMS (ESI+): Calcd for C₂₃H₂₃ClN₇OS [M+H]⁺: 481.1452, Found: 481.1456.

(S)-amino(2-((3-(4-((4-(4-bromophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20f)

Synthesized by General Procedure E. 5 mg, 100% light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.06 (d, J = 8.7 Hz, 2H), 7.84 (dd, J = 10.2, 8.6 Hz, 4H), 7.59 (d, J = 8.5 Hz, 2H), 7.22 (s, 1H), 4.59 – 4.50 (m, 1H), 3.59 – 3.51 (m, 1H), 3.51 – 3.43 (m, 1H), 3.30 – 3.28 (m, 2H), 2.35 – 2.23 (m, 1H), 2.19 – 1.98 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.71, 169.35, 164.59, 164.52, 156.43, 151.28, 151.16, 145.44, 135.19, 134.57, 132.73, 129.38, 128.78, 123.98, 122.39, 120.31, 118.08, 104.75, 57.20, 31.60, 30.06, 23.58; HRMS (ESI+): Calcd for C₂₃H₂₃BrN₇OS [M+H]⁺: 524.0868, Found: 524.0873.

(S)-amino(2-((3-(4-((4-(3-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (9g)

Synthesized by General Procedure E. 5 mg, 100%, yellow solid. ¹H NMR (500 MHz, CD₃OD) δ 8.08 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 10.1 Hz, 1H), 7.49 – 7.41 (m, 1H), 7.26 (s, 1H), 7.09 (td, J = 8.4, 2.1 Hz, 1H), 4.56 – 4.48 (m, 1H), 3.56 – 3.50 (m, 1H), 3.48 – 3.41 (m, 1H), 3.32 – 3.30 (m, 2H), 2.33 – 2.22 (m, 1H), 2.17 – 1.97 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.96, 169.19, 166.35, 165.52 (d, ¹J_CF = 195.9 Hz), 163.58 (d, ¹J_CF = 195.9 Hz), 156.46, 148.11, 144.28, 136.47 (d, ⁴J_CF = 6.1 Hz), 136.41 (d, ⁴J_CF = 6.1 Hz), 131.74 (d, ⁴J_CF = 7.1 Hz), 131.67 (d, ⁴J_CF = 7.1 Hz), 129.71, 123.04, 123.02, 122.35, 119.82, 116.20 (d, ³J_CF = 17.2 Hz), 116.03 (d, ³J_CF = 17.2 Hz), 114.06 (d, ³J_CF = 18.2 Hz), 113.88 (d, ³J_CF = 18.2 Hz), 105.55, 57.22, 31.62, 30.08, 23.60; ¹⁹F NMR (376 MHz, CD₃OD) δ −77.20 (s, 1F); HRMS (ESI+): Calcd for C₂₃H₂₃FN₇OS [M+H]⁺: 465.1747, Found: 465.1724.

(S)-amino(2-((3-(4-((4-(3-chlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20h)

Synthesized by General Procedure E. 26 mg, 100%, light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.13 (d, J = 8.2 Hz, 2H), 7.89 (s, 1H), 7.80 (t, J = 8.2 Hz, 3H), 7.51 – 7.39 (m, 2H), 7.31 (s, 1H), 4.61 – 4.52 (m, 1H), 3.63 – 3.53 (m, 1H), 3.53 – 3.44 (m, 1H), 2.37 – 2.25 (m, 1H), 2.20 – 2.00 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 178.03, 169.05, 167.18, 156.42, 143.65, 135.88, 135.09, 131.50, 129.81, 129.73, 127.24, 125.65, 123.22, 120.58, 57.19, 31.60, 30.09, 23.59; HRMS (ESI+): Calcd for C₂₃H₂₃ClN₇OS [M+H]⁺: 480.1373, Found: 480.1370.

(S)-amino(2-((3-(4-((4-(3-bromophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20i)

Synthesized by General Procedure E. 3 mg, 100%, off-white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.09 – 8.05 (m, 1H), 8.04 – 7.98 (m, 2H), 7.91 – 7.82 (m, 3H), 7.47 – 7.42 (m, 1H), 7.31 (t, J = 7.9 Hz, 1H), 7.21 (s, 1H), 4.56 – 4.48 (m, 1H), 3.58 – 3.50 (m, 1H), 3.48 – 3.40 (m, 1H), 3.29 – 3.27 (m, 2H), 2.33 – 2.21 (m, 1H), 2.16 – 1.97 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 180.23, 171.87, 167.12, 158.95, 153.37, 147.95, 140.81, 134.06, 133.94, 132.42, 131.90, 128.20, 126.20, 122.87, 120.59, 107.96, 59.72, 34.12, 32.58, 26.10; HRMS (ESI+): Calcd for C₂₃H₂₃BrN₇OS [M+H]⁺: 524.0868, Found: 524.0869.

(S)-amino(2-((3-(4-((4-(2-fluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20j)

Synthesized by General Procedure E. 14 mg, 100%, light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.17 – 8.06 (m, 3H), 7.87 (d, J = 8.3 Hz, 2H), 7.47 – 7.39 (m, 1H), 7.38 – 7.21 (m, 3H), 4.62 – 4.54 (m, 1H), 3.66 – 3.56 (m, 1H), 3.55 – 3.44 (m, 1H), 3.39 – 3.35 (m, 2H), 2.40 – 2.27 (m, 1H), 2.22 – 2.02 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.92, 169.20, 165.30, 162.76 (d, ¹J_CF = 250.5 Hz), 160.28 (d, ¹J_CF = 250.5 Hz), 156.45, 156.01, 152.88, 148.68, 144.41, 143.41, 131.07, 130.97, 130.93, 129.66, 125.73 (d, ⁴J_CF = 3.0 Hz), 125.70 (d, ⁴J_CF = 3.0 Hz), 122.10, 121.98, 119.61, 117.13 (d, ²J_CF = 23.2 Hz), 116.90 (d, ²J_CF = 23.2 Hz), 57.22, 31.62, 30.09, 23.60; ¹⁹F NMR (471 MHz, CD₃OD) δ −116.05 – −116.22 (m, 1F); HRMS (ESI+): Calcd for C₂₃H₂₃FN₇OS [M+H]⁺: 464.1669, Found: 464.1658.

(S)-amino(2-((3-(4-((4-(4-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20k)

Synthesized by General Procedure E. 24 mg, 100%, yellow solid. ¹H NMR (500 MHz, CD₃OD) δ 8.10 (d, J = 8.1 Hz, 2H), 8.06 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.0 Hz, 2H), 7.37 (s, 1H), 4.57 – 4.50 (m, 1H), 3.60 – 3.51 (m, 1H), 3.51 – 3.42 (m, 1H), 3.32 (s, 1H), 2.34 – 2.24 (m, 1H), 2.19 – 1.98 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.81, 169.32, 165.27, 156.46, 149.94, 145.07, 139.07, 130.70 (q, ²J_CF = 25.3 Hz), 130.45 (q, ²J_CF = 25.3 Hz), 129.51, 127.53, 126.84, 126.70 (q, ⁴J_CF = 3.0 Hz), 126.67 (q, ⁴J_CF = 3.0 Hz), 126.64 (q, ⁴J_CF = 3.0 Hz), 126.60 (q, ⁴J_CF = 3.0 Hz), 124.69, 121.06, 118.72, 106.64, 57.24, 31.63, 30.09, 23.60; ¹⁹F NMR (471 MHz, CD₃OD) δ −64.02 (s, 3F); HRMS (ESI+): Calcd for C₂₄H₂₃F₃N₇OS [M+H]⁺: 515.1715, Found: 515.1718.

(S)-amino(2-((3-(4-((4-(3-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20l)

Synthesized by General Procedure E. 3 mg, 100%, light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.21 (s, 1H), 8.19 – 8.15 (m, 1H), 8.06 – 7.98 (m, 2H), 7.89 – 7.84 (m, 2H), 7.63 – 7.56 (m, 2H), 7.32 (s, 1H), 4.56 – 4.49 (m, 1H), 3.63 – 3.59 (m, 1H), 3.53 – 3.45 (m, 1H), 2.34 – 2.22 (m, 1H), 2.18 – 1.96 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.72, 169.34, 164.73, 156.46, 150.89, 145.42, 137.08, 134.58, 132.49 (q, ²J_CF = 31.3 Hz), 132.17 (q, ²J_CF = 31.3 Hz), 131.86 (q, ²J_CF = 31.3 Hz), 131.54 (q, ²J_CF = 31.3 Hz), 130.49, 130.43, 129.95, 129.80 (q, ¹J_CF = 272.7 Hz), 129.37, 127.10, 126.52 (q, ¹J_CF = 272.7 Hz), 125.14 (q, ⁴J_CF = 4.0 Hz), 125.11 (q, ⁴J_CF = 4.0 Hz), 125.06 (q, ⁴J_CF = 4.0 Hz), 125.03 (q, ⁴J_CF = 4.0 Hz), 124.40 (q, ¹J_CF = 272.7 Hz), 123.98, 123.59 (q, ⁴J_CF = 4.0 Hz), 123.56 (q, ⁴J_CF = 4.0 Hz), 123.51 (q, ⁴J_CF = 4.0 Hz), 123.48 (q, ⁴J_CF = 4.0 Hz), 121.70 (q, ¹J_CF = 272.7 Hz), 120.38, 118.08, 105.77, 57.22; ¹⁹F NMR (376 MHz, CD₃OD) δ −64.27 (s, 3F); HRMS (ESI+): Calcd for C₂₄H₂₃F₃N₇OS [M+H]⁺: 514.1637, Found: 514.1625.

(S)-amino(2-((3-(4-((4-(2-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20m)

5 mg, 93%, off-white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.15 – 8.09 (m, 2H), 7.89 (d, J = 7.8 Hz, 1H), 7.83 – 7.65 (m, 5H), 7.04 (s, 1H), 4.61 – 4.53 (m, 1H), 3.62 – 3.55 (m, 1H), 3.54 – 3.46 (m, 1H), 3.40 – 3.35 (m, 2H), 2.37 – 2.26 (m, 1H), 2.21 – 2.00 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 178.06, 169.06, 166.70, 156.45, 148.60, 144.53, 143.47, 133.49, 133.34, 132.47, 130.78, 129.82, 129.34 (q, ²J_CF = 29.3 Hz), 129.05 (q, ²J_CF = 29.3 Hz), 127.68 (q, ³J_CF = 5.1 Hz), 127.63 (q, ⁴J_CF = 5.1 Hz), 127.57 (q, ⁴J_CF = 5.1 Hz), 127.52 (q, ⁴J_CF = 5.1 Hz), 123.47, 120.86, 108.89, 57.20, 31.60, 30.08, 23.59; ¹⁹F NMR (376 MHz, CD₃OD) δ −59.48 (s, 3F); HRMS (ESI+): Calcd for C₂₄H₂₃F₃N₇OS [M+H]⁺: 515.1715, Found: 515.1663.

(S)-amino(2-((3-(4-((4-(p-tolyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20n)

Synthesized by General Procedure E. 6 mg, 85%, light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.02 (d, J = 8.7 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 7.8 Hz, 2H), 7.23 (d, J = 7.7 Hz, 2H), 7.06 (s, 1H), 4.57 – 4.49 (m, 1H), 3.58 – 3.55 (m, 1H), 3.50 – 3.46 (m, 1H), 3.30 – 3.24 (m, 2H), 2.37 (s, 3H), 2.34 – 2.22 (m, 1H), 2.19 – 1.98 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.71, 169.39, 164.38, 156.44, 152.69, 145.64, 138.70, 133.46, 130.22, 129.38, 126.99, 120.16, 117.99, 103.13, 57.22, 31.61, 30.06, 23.59, 21.28; HRMS (ESI+): Calcd for C₂₄H₂₆N₇OS [M+H]⁺: 460.1920, Found: 460.1920.

(S)-amino(2-((3-(4-((4-(4-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20o)

Synthesized by General Procedure E. 15 mg, 100%, off-white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.05 – 7.97 (m, 2H), 7.90 – 7.79 (m, 4H), 6.99 – 6.93 (m, 3H), 4.57 – 4.46 (m, 1H), 3.83 (s, 3H), 3.58 – 3.50 (m, 1H), 3.50 – 3.40 (m, 1H), 3.29 (d, J = 3.7 Hz, 1H), 2.34 – 2.22 (m, 1H), 2.17 – 1.95 (m, 4H); ¹³C NMR (101 MHz, CD₃OD) δ 178.23, 168.80, 168.62, 162.30, 156.38, 144.37, 142.34, 135.53, 131.81, 130.58, 130.11, 129.43, 128.98, 125.05, 123.38, 122.10, 118.64, 115.55, 114.71, 57.17, 56.02, 31.65, 30.14, 23.66; HRMS (ESI+): Calcd for C₂₄H₂₆N₇O₂S⁺ [M+H]⁺: 476.1869, Found: 476.1858. HPLC purity: 85%.

(S)-amino(2-((3-(4-((4-(4-ethoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20p)

Synthesized by General Procedure E. 26 mg, 76%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.15 (d, J = 8.2 Hz, 2H), 7.81 – 7.65 (m, 4H), 7.04 – 6.96 (m, 2H), 4.58 – 4.49 (m, 1H), 4.09 (q, J = 7.0 Hz, 2H), 3.62 – 3.51 (m, 1H), 3.49 – 3.42 (m, 1H), 3.35 – 3.30 (m, 2H), 2.35 – 2.22 (m, 1H), 2.18 – 1.97 (m, 3H), 1.41 (t, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 178.18, 174.32, 168.95, 161.43, 156.42, 130.58, 130.00, 129.43, 128.88, 121.55, 118.52, 115.95, 115.22, 81.41, 64.74, 57.18, 31.59, 30.06, 23.59, 15.09; HRMS (ESI+): Calcd for C₂₅H₂₈N₇O₂S [M+H]⁺: 490.2025, Found: 490.2024.

(S)-amino(2-((3-(4-((4-(3-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20q)

Synthesized by General Procedure E. 12 mg, 86%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.29 (s, 1H), 8.12 (d, J = 6.5 Hz, 2H), 7.78 – 7.66 (m, 2H), 7.40 – 7.27 (m, 3H), 6.97 (d, J = 7.1 Hz, 1H), 6.82 (s, 1H), 4.58 – 4.45 (m, 1H), 3.84 (s, 3H), 3.59 – 3.49 (m, 1H), 3.50 – 3.37 (m, 1H), 3.34 – 3.28 (m, 2H), 2.35 – 2.20 (m, 1H), 2.15 – 1.93 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 178.16, 168.87, 168.24, 161.59, 156.38, 145.44, 142.77, 132.82, 131.27, 130.05, 124.52, 121.75, 119.71, 116.02, 113.06, 57.21, 56.09, 31.69, 30.19, 23.69; HRMS (ESI+): Calcd for C₂₄H₂₆N₇O₂S⁺ [M+H]⁺: 476.1869, Found: 476.1847.

(S)-amino(2-((3-(4-((4-(4-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20r)

Synthesized by General Procedure E. 9 mg, 90%, off-white solid. ¹H NMR (500 MHz, CD₃OD) δ 8.06 (d, J = 8.3 Hz, 4H), 7.92 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.24 (s, 1H), 4.61 – 4.50 (m, 1H), 3.63 – 3.55 (m, 1H), 3.54 – 3.45 (m, 2H), 3.33 – 3.21 (m, 1H), 2.20 – 2.14 (m, 1H), 2.14 – 2.02 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.73, 169.41, 164.67, 156.47, 151.16, 149.87, 145.53, 135.35, 129.40, 128.63, 122.19, 120.33, 118.09, 104.99, 57.25, 31.62, 30.08, 23.59; ¹⁹F NMR (470 MHz, CD₃OD) δ −59.47 (s, 3F); HRMS (ESI+): Calcd for C₂₄H₂₃F₃N₇O₂S [M+H]⁺: 531.1664, Found: 531.1672. HPLC purity: 81%.

(S)-amino(2-((3-(4-((4-(3-(trifluoromethoxy)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20s)

Synthesized by General Procedure E. 9 mg, 100%, light yellow solid. ¹H NMR (400 MHz, v) δ 8.04 (d, J = 8.5 Hz, 2H), 7.96 – 7.83 (m, 4H), 7.51 (t, J = 8.0 Hz, 1H), 7.29 (s, 1H), 7.25 – 7.19 (m, 1H), 4.58 – 4.50 (m, 1H), 3.60 – 3.52 (m, 1H), 3.51 – 3.43 (m, 1H), 2.34 – 2.23 (m, 1H), 2.19 – 1.99 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.75, 169.36, 164.61, 156.44, 150.98, 150.87, 145.47, 138.41, 131.34, 129.38, 125.52, 120.85, 120.37, 119.46, 118.07, 105.73, 57.23, 31.60, 30.07, 23.58; ¹⁹F NMR (376 MHz, CD₃OD) δ −59.32 (s, 3F); HRMS (ESI+): Calcd for C₂₄H₂₃F₃N₇O₂S [M+H]⁺: 531.1664, Found: 531.1712.

(S)-amino(2-((3-(4-((4-(3,4-dimethylphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20t)

Synthesized by General Procedure E. 25 mg, 89%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.28 (s, 1H), 8.14 (d, J = 7.9 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 6.82 (s, 1H), 4.60 – 4.48 (m, 1H), 3.61 – 3.40 (m, 2H), 3.37 – 3.30 (m, 1H), 2.33 (s, 3H), 2.30 (s, 3H), 2.19 – 1.94 (m, 3H), 1.40 – 1.12 (m, 1H); ¹³C NMR (101 MHz, CD₃OD) δ 178.21, 168.91, 168.29, 156.42, 145.71, 142.81, 139.57, 138.58, 135.47, 131.26, 130.04, 129.15, 128.37, 124.92, 124.60, 121.76, 57.18, 31.62, 30.10, 23.62, 19.89, 19.69; HRMS (ESI+): Calcd for C₂₅H₂₈N₇O₃S⁺ [M+H]⁺: 474.2076, Found: 474.2077.

(S)-amino(2-((3-(4-((4-(3,4-dimethoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20u)

Synthesized by General Procedure E. 19 mg, 100%, orange solid. ¹H NMR (400 MHz, CD₃OD) δ 8.17 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.6 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.60 – 7.54 (m, 1H), 7.33 (d, J = 6.9 Hz, 2H), 7.06 (d, J = 8.5 Hz, 1H), 4.58 – 4.49 (m, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.60 – 3.51 (m, 1H), 3.49 – 3.42 (m, 1H), 3.33 (d, J = 6.0 Hz, 2H), 2.34 – 2.23 (m, 1H), 2.17 – 2.01 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 178.31, 168.99, 168.80, 156.42, 152.07, 150.90, 143.92, 142.18, 130.17, 129.46, 125.53, 123.36, 122.47, 120.66, 118.81, 113.03, 112.57, 111.17, 57.17, 56.76, 56.53, 31.60, 30.09, 23.60; HRMS (ESI+): Calcd for C₂₅H₂₈N₇OS⁺ [M+H]⁺: 506.1969, Found: 506.1948.

(S)-amino(2-((3-(4-((4-(3-chloro-4-methoxyphenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20v)

Synthesized by General Procedure E. 11 mg, 100%, off-white solid. ¹H NMR (500 MHz, CD₃OD) δ 8.07 (d, J = 8.5 Hz, 2H), 7.90 (s, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 4.57 – 4.52 (m, 1H), 3.94 (s, 3H), 3.60 – 3.52 (m, 1H), 3.50 – 3.42 (m, 2H), 2.33 – 2.25 (m, 1H), 2.19 – 1.99 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.87, 169.26, 165.70, 156.49, 144.75, 129.59, 128.86, 128.54, 126.89, 123.76, 121.61, 119.20, 113.56, 57.24, 56.77, 31.63, 30.10, 23.60; HRMS (ESI+): Calcd for C₂₄H₂₅ClN₇O₂S [M+H]⁺: 510.1479, Found: 510.1495.

(S)-amino(2-((3-(4-((4-(3,4-difluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20w)

Synthesized by General Procedure E. 6 mg, 100%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.09 (d, J = 8.2 Hz, 2H), 7.91 – 7.79 (m, 3H), 7.77 – 7.70 (m, 1H), 7.35 (q, J = 8.9 Hz, 1H), 7.24 (s, 1H), 4.63 – 4.53 (m, 1H), 3.64 – 3.56 (m, 1H), 3.56 – 3.46 (m, 1H), 2.40 – 2.24 (m, 1H), 2.23 – 2.03 (m, 3H¹³C NMR (101 MHz, CD₃OD) δ 177.84, 169.26, 165.51, 156.44, 148.70, 144.82, 132.67, 129.56, 123.68 (dd, ⁴J_CF = 4.0 Hz), 123.64 (dd, ⁴J_CF = 4.0 Hz), 123.62 (dd, ⁴J_CF = 4.0 Hz), 123.58 (dd, ⁴J_CF = 4.0 Hz), 121.39, 118.99, 118.70 (d, ²J_CF = 18.2 Hz), 118.52 (d, ²J_CF = 18.2 Hz), 116.18 (d, ²J_CF = 19.2 Hz), 115.99 (d, ²J_CF = 19.2 Hz), 57.25, 31.66, 30.13, 23.63; ¹⁹F NMR (376 MHz, CD₃OD) δ −140.17 – −140.55 (m, 1F), −141.28 – −141.63 (m, 1F); HRMS (ESI+): Calcd for C₂₃H₂₂F₂N₇OS [M+H]⁺: 482.1575, Found: 482.1571.

(S)-amino(2-((3-(4-((4-(3,4-dichlorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20x)

Synthesized by General Procedure E. 15 mg, 100%, white solid. ¹H NMR (500 MHz, CD₃OD) δ 8.07 (d, J = 8.5 Hz, 2H), 7.90 (s, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 4.57 – 4.52 (m, 1H), 3.56 – 3.49 (m, 1H), 3.48 – 3.39 (m, 1H), 2.33 – 2.25 (m, 1H), 2.19 – 1.99 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.85, 169.26, 156.45, 146.18, 144.76, 135.36, 133.91, 133.59, 132.58, 131.14, 129.55, 128.49, 121.49, 119.08, 57.23, 54.79, 31.62, 30.08, 23.59; HRMS (ESI+): Calcd for C₂₃H₂₂Cl₂N₇OS [M+H]⁺: 514.0984, Found: 514.0973.

(S)-amino(2-((3-(4-((4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20y)

Synthesized by General Procedure E. 9 mg, 100%, off-white solid. ¹H NMR (500 MHz, CD₃OD) δ 8.23 – 8.10 (m, 2H), 8.04 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.39 (t, J = 9.4 Hz, 1H), 7.29 (s, 1H), 4.55 – 4.47 (m, 1H), 3.57 – 3.48 (m, 1H), 3.48 – 3.38 (m, 1H), 2.31 – 2.21 (m, 1H), 2.14 – 1.95 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.94, 169.17, 166.32, 161.64 (d, ¹J_CF = 205.0 Hz), 159.61 (d, ¹J_CF = 205.0 Hz), 156.44, 147.17, 144.34, 133.33 (d, ³J_CF = 7.1 Hz), 133.26 (d, ³J_CF = 7.1 Hz), 131.44 (d, ⁴J_CF = 2.0 Hz), 131.42 (d, ⁴J_CF = 2.0 Hz), 129.64, 127.31 (q, ¹J_CF = 218.2 Hz), 125.97 (q, ⁴J_CF = 4.0 Hz), 125.93 (q, ⁴J_CF = 4.0 Hz), 125.90 (q, ⁴J_CF = 4.0 Hz), 125.86 (q, ⁴J_CF = 4.0 Hz), 125.14 (q, ¹J_CF = 218.2 Hz), 122.98 (q, ¹J_CF = 218.2 Hz), 122.16, 120.82 (q, ¹J_CF = 218.2 Hz), 119.63, 119.36 (q, ³J_CF = 11.1 Hz), 119.25 (q, ³J_CF = 11.1 Hz), 118.67 (d, ³J_CF = 17.2 Hz), 118.50 (d, ³J_CF = 17.2 Hz), 57.22, 31.61, 30.10, 23.60; ¹⁹F NMR (376 MHz, CD₃OD) δ −62.93 (d, J = 12.8 Hz, 3F), −117.55 – −117.83 (m, 1F); HRMS (ESI+): Calcd for C₂₄H₂₂F₄N₇OS [M+H]⁺: 532.1543, Found: 532.1538.

(S)-amino(2-((3-(4-((4-(3,5-difluorophenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20z)

Synthesized by General Procedure E. 8 mg, 100%, off-white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.03 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.56 – 7.48 (m, 2H), 7.31 (s, 1H), 6.91 – 6.83 (m, 1H), 4.59 – 4.48 (m, 1H), 3.71 (d, J = 2.8 Hz, 1H), 3.60 – 3.51 (m, 1H), 3.49 – 3.41 (m, 1H), 3.16 – 3.10 (m, 1H), 2.34 – 2.22 (m, 1H), 2.18 – 1.97 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.73, 169.35, 166.08 (dd, ¹J_CF = 246.5 Hz), 165.95 (dd, ¹J_CF = 246.5 Hz), 164.57, 163.63 (dd, ¹J_CF = 246.5 Hz), 163.50 (dd, ¹J_CF = 246.5 Hz), 156.44, 150.23 (d, ⁴J_CF = 4.0 Hz), 150.19 (d, ⁴J_CF = 4.0 Hz), 145.35, 139.76 (dd, ³J_CF = 9.1 Hz), 139.66(dd, ³J_CF = 9.1 Hz), 139.56 (dd, ³J_CF = 9.1 Hz), 129.40, 120.43, 118.09, 109.79 (d, ²J_CF = 27.3 Hz), 109.71 (d, ³J_CF = 11.1 Hz), 109.59 (d, ³J_CF = 11.1 Hz), 109.52 (d, ²J_CF = 27.3 Hz), 106.65, 103.70 (dd, ²J_CF = 26.3 Hz), 103.44 (dd, ³J_CF = 26.3 Hz), 103.18 (dd, ²J_CF = 26.3 Hz), 71.34*, 67.91*, 57.20, 40.56, 31.61, 30.05, 23.59; ¹⁹F NMR (376 MHz, CD₃OD) δ −111.61 – −111.72 (m, 2F); HRMS (ESI+): Calcd for C₂₃H₂₂F₂N₇OS [M+H]⁺: 482.1575, Found: 482.1534.

(S)-amino(2-((3-(4-((4-(3-fluoro-5-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20aa)

Synthesized by General Procedure E. 6 mg, 100%, yellow solid. ¹H NMR (500 MHz, CD₃OD) δ 8.23 – 8.10 (m, 2H), 8.04 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.39 (t, J = 9.4 Hz, 1H), 7.29 (s, 1H), 4.55 – 4.47 (m, 1H), 3.57 – 3.48 (m, 1H), 3.48 – 3.38 (m, 1H), 2.31 – 2.21 (m, 1H), 2.14 – 1.95 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.77, 169.32, 165.57, 164.91, 163.12, 156.44, 149.32, 145.21, 139.62, (d, ³J_CF = 9.1 Hz) 139.53 (d, ³J_CF = 9.1 Hz), 134.07 (d, ³J_CF = 8.1 Hz), 133.99 (d, ³J_CF = 8.1 Hz), 133.74 (d, ³J_CF = 9.1 Hz), 133.65 (d, ³J_CF = 9.1 Hz), 129.42, 126.24, 123.51, 120.70, 119.55 (q, ⁴J_CF = 3.0 Hz), 119.52 (q, ⁴J_CF = 3.0 Hz), 119.51 (q, ⁴J_CF = 3.0 Hz), 119.48 (q, ⁴J_CF = 3.0 Hz), 118.30, 117.37 (d, ³J_CF = 17.2 Hz), 117.13 (d, ³J_CF = 17.2 Hz), 112.44, 112.17, 107.28, 57.25, 31.64, 30.12, 23.61; ¹⁹F NMR (376 MHz, CD₃OD) δ −64.34 (s, 1F), −112.36 (t, J = 9.1 Hz, 1F); HRMS (ESI+): Calcd for C₂₄H₂₂F₄N₇OS [M+H]⁺: 532.1543, Found: 532.1520.

(S)-amino(2-((3-(4-((4-(3,5-bis(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20bb)

Synthesized by General Procedure E. 22 mg, 100%, white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.51 (s, 2H), 8.07 (d, J = 8.4 Hz, 2H), 7.93 – 7.86 (m, 3H), 7.60 (s, 1H), 4.62 – 4.54 (m, 1H), 3.64 – 3.55 (m, 1H), 3.55 – 3.46 (m, 1H), 2.39 – 2.27 (m, 1H), 2.21 – 2.03 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 178.12, 168.96, 167.94, 156.43, 145.08, 143.15, 133.46, 133.42, 132.82 (q, ²J_CF = 32.3 Hz), 132.50 (q, ²J_CF = 32.3 Hz), 132.18 (q, ²J_CF = 32.3 Hz), 131.85 (q, ²J_CF = 32.3 Hz), 131.02, 130.99, 129.91, 126.79, 126.64 (q, ⁴J_CF = 3.0 Hz), 126.61 (q, ⁴J_CF = 3.0 Hz), 126.57 (q, ⁴J_CF = 3.0 Hz), 126.53 (q, ⁴J_CF = 3.0 Hz), 124.09, 124.06, 124.01, 123.98, 121.24, 57.18, 31.60, 30.09, 23.59; ¹⁹F NMR (376 MHz, CD₃OD) δ −64.49; HRMS (ESI+): Calcd for C₂₅H₂₂F₆N₇OS [M+H]⁺: 582.1511, Found: 582.1516.

(S)-amino(2-((3-(4-((4-(2-fluoro-5-(trifluoromethyl)phenyl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20cc)

Synthesized by General Procedure E. 6 mg, 100%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.57 – 8.52 (m, 1H), 8.11 – 8.03 (m, 2H), 7.90 (d, J = 8.6 Hz, 2H), 7.73 – 7.66 (m, 1H), 7.50 – 7.40 (m, 2H), 4.62 – 4.54 (m, 1H), 3.65 – 3.56 (m, 1H), 3.56 – 3.46 (m, 1H), 2.40 – 2.27 (m, 1H), 2.22 – 2.04 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 183.96, 177.78, 175.04, 169.33, 164.57 (d, ¹J_CF = 256.5 Hz), 164.00, 162.03 (d, ¹J_CF = 256.5 Hz), 156.45, 145.34, 144.51, 129.37, 128.20 (q, ¹J_CF = 112.1 Hz), 127.09 (q, ¹J_CF = 112.1 Hz), 124.59 (d, ³J_CF = 12.1 Hz), 124.47 (d, ³J_CF = 12.1 Hz), 124.32, 123.78, 120.62, 118.54, 118.20, 117.98, 110.58, 106.88, 103.88, 57.26, 31.61, 30.11, 23.58; ¹⁹F NMR (376 MHz, CD₃OD) δ −63.69 (s, 3F), −110.28 – −110.99 (m, 1F); HRMS (ESI+): Calcd for C₂₄H₂₂F₄N₇OS [M+H]⁺: 532.1543, Found: 532.1520. HPLC purity: 85% pure.

(S)-(2-((3-(4-((4-([1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)(amino)methaniminium chloride (20dd)

Synthesized by General Procedure E. 5 mg, 100%, light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.11 (d, J = 8.5 Hz, 2H), 8.03 (d, J = 8.0 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.73 (dd, J = 14.9, 7.9 Hz, 4H), 7.50 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.3 Hz, 1H), 7.26 (s, 1H), 4.65 – 4.51 (m, 1H), 3.65 – 3.56 (m, 1H), 3.55 – 3.47 (m, 1H), 2.41 – 2.27 (m, 1H), 2.24 – 2.02 (m, 4H); ¹³C NMR (101 MHz, CD₃OD) δ 176.60, 167.69, 165.59, 155.03, 146.50, 142.48, 141.43, 140.13, 130.85, 128.55, 128.38, 127.30, 126.99, 126.46, 126.36, 121.61, 119.03, 55.78, 30.18, 28.66, 22.17; HRMS (ESI+): Calcd for C₂₉H₂₈N₇OS [M+H]⁺: 522.2076, Found: 522.2046.

(S)-amino(2-((3-(4-((4-(benzofuran-2-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (20ee)

Synthesized by General Procedure E. 13 mg, 100%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.20 (d, J = 6.5 Hz, 1H), 7.98 (t, J = 7.7 Hz, 3H), 7.79 (d, J = 7.9 Hz, 2H), 7.49 (d, J = 7.5 Hz, 2H), 7.37 – 7.25 (m, 2H), 4.52 – 4.42 (m, 1H), 3.52 – 3.44 (m, 1H), 3.43 – 3.35 (m, 2H), 2.30 – 2.13 (m, 1H), 2.13 – 1.90 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.93, 168.95, 166.98, 156.94, 156.73, 156.32, 145.31, 143.60, 139.64, 129.74, 126.42, 126.20, 126.02, 124.57, 123.13, 121.58, 120.62, 115.61, 112.61, 57.14, 31.56, 30.08, 23.56; HRMS (ESI+): Calcd for C₂₅H₂₄N₇O₂S [M+H]⁺: 486.1712, Found: 486.1683.

tert-butyl (S)-2-((3-(4-((4-([1,1′-biphenyl]-3-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (21a)

Synthesized by General Procedure D. 12 mg, 30%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.12 – 8.02 (m, 3H), 7.85 (dt, J = 7.7, 1.5 Hz, 1H), 7.77 (s, 1H), 7.69 – 7.62 (m, 2H), 7.59 – 7.43 (m, 6H), 7.40 – 7.33 (m, 1H), 6.97 (s, 1H), 4.40 – 4.22 (m, 1H), 3.52 – 3.27 (m, 3H), 3.17 – 2.98 (m, 1H), 2.14 – 2.02 (m, 1H), 1.95 – 1.78 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.20, 167.90, 162.96, 154.37, 151.61, 142.82, 141.84, 141.22, 135.00, 129.26, 128.96, 128.92, 127.54, 127.37, 127.04, 125.21, 125.15, 120.66, 117.40, 117.29, 103.13, 80.23, 79.78*, 55.35, 55.22*, 46.90*, 46.50*, 31.93, 31.15*, 30.98*, 30.21*, 29.85*, 28.63, 23.71*, 22.91*; HRMS (ESI+): Calcd for C₃₃H₃₄N₅O₃S [M+H]⁺: 580.2382, Found: 580.2384.

tert-butyl (S)-2-((3-(4-((4-(4′-fluoro-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (21b)

Synthesized by General Procedure D. 14 mg, 34%, off-white amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 8.3 Hz, 2H), 7.93 (d, J = 8.0 Hz, 2H), 7.70 – 7.65 (br s, 1H), 7.63 – 7.50 (m, 6H), 7.14 (t, J = 8.7 Hz, 2H), 6.95 (s, 1H), 4.39 – 4.22 (m, 1H), 3.50 – 3.27 (m, 3H), 3.15 – 2.97 (m, 1H), 2.13 – 2.01 (m, 1H), 1.95 – 1.79 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.22, 168.00, 163.88 (d, ¹J_CF = 247.5 Hz), 162.96, 161.43 (d, ¹J_CF = 247.5 Hz), 154.37, 151.25, 150.57, 142.75, 139.84, 136.93 (d, ⁴J_CF = 3.0 Hz), 136.90 (d, ⁴J_CF = 3.0 Hz), 133.53, 133.45, 131.92, 128.95, 128.84, 128.72 (d, ³J_CF = 8.1 Hz), 128.64 (d, ³J_CF = 8.1 Hz), 127.80, 127.35, 126.71, 122.07, 120.73, 117.39, 117.35, 115.92 (d, ²J_CF = 21.2 Hz), 115.71 (d, ²J_CF = 21.2 Hz), 103.27, 102.96, 80.23, 55.34, 46.90*, 46.51*, 31.93*, 31.15*, 31.00*, 30.23*, 29.85*, 28.64*, 23.71*, 22.92*; ¹⁹F NMR (376 MHz, CDCl₃) δ −114.71 – −116.15 (m, 1F); HRMS (ESI+): Calcd for C₃₃H₃₃FN₅O₃S [M+H]⁺: 598.2288, Found: 598.2234.

tert-butyl (S)-2-((3-(4-((4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (21c)

Synthesized by General Procedure D. 11 mg, 37%, yellow amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J = 8.2 Hz, 2H), 7.97 (d, J = 8.0 Hz, 1H), 7.78 – 7.54 (m, 8H), 6.98 (s, 1H), 4.40 – 4.21 (m, 1H), 3.52 – 3.27 (m, 3H), 3.17 – 2.97 (m, 1H), 2.13 – 2.00 (m, 1H), 1.96 – 1.78 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.41, 167.99, 163.01, 154.38, 151.06, 144.29, 142.73, 139.28, 134.41, 129.69 (q, ²J_CF = 32.3 Hz), 129.37 (q, ²J_CF = 32.3 Hz), 128.98, 128.83 (q, ²J_CF = 32.3 Hz), 127.67, 127.54, 127.37, 127.23, 126.84, 125.91 (q, ⁴J_CF = 4.0 Hz), 125.87 (q, ⁴J_CF = 4.0 Hz), 125.79 (q, ⁴J_CF = 4.0 Hz), 117.39, 103.40, 80.23*, 79.81*, 55.30, 46.90*, 46.50*, 31.93*, 31.15*, 30.23*, 29.83*, 28.63, 23.71*, 22.92*; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.43 (s, 3F); HRMS (ESI+): Calcd for C₃₄H₃₂F₃N₅NaO₃S [M+H]⁺: 670.2075, Found: 670.2068.

tert-butyl (S)-2-((3-(4-((4-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (21d)

Synthesized by General Procedure D. 12 mg, 35%, tan amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.12 – 8.03 (m, 2H), 7.98 (d, J = 7.9 Hz, 2H), 7.88 (s, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.70 – 7.51 (m, 6H), 6.98 (s, 1H), 4.39 – 4.23 (m, 1H), 3.52 – 3.27 (m, 3H), 3.15 – 2.96 (m, 1H), 2.13 – 2.01 (m, 1H), 1.95 – 1.79 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.08, 167.78, 162.84, 154.24, 150.90, 142.60, 141.42, 139.12, 134.10, 131.77, 131.65, 131.34 (q, ²J_CF = 30.3 Hz), 131.02 (q, ²J_CF = 30.3 Hz), 130.22, 129.27, 128.81, 128.68, 127.64 (q, ¹J_CF = 155.5 Hz), 127.41, 126.69, 126.10 (q, ¹J_CF = 155.5 Hz), 125.51, 123.99 (q, ²J_CF = 30.3 Hz), 123.69 (q, ²J_CF = 30.3 Hz), 122.81, 120.63, 117.20, 103.18, 80.10, 55.15, 46.74*, 46.35*, 31.75*, 30.98*, 30.07*, 28.47, 23.54*, 22.76*; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.60 (s, 3F); HRMS (ESI+): Calcd for C₃₄H₃₃F₃N₅O₃S [M+H]⁺: 648.2256, Found: 648.2209.

tert-butyl (S,Z)-((2-((3-(4-((4-([1,1′-biphenyl]-3-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)((tert-butoxycarbonyl)imino)methyl)carbamate (22a)

Synthesized by General Procedure E. 13mg, 68%, off-white oily solid. ¹H NMR (500 MHz, CDCl₃) δ 10.39 (s, 1H), 8.09 (s, 1H), 8.02 (d, J = 8.5 Hz, 2H), 7.86 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 7.4 Hz, 2H), 7.59 – 7.53 (m, 4H), 7.52 – 7.44 (m, 3H), 7.37 (t, J = 7.3 Hz, 1H), 6.96 (s, 1H), 4.82 – 4.72 (m, 1H), 3.81 – 3.62 (m, 3H), 3.57 – 3.43 (m, 1H), 3.09 (dd, 1H), 2.36 – 2.26 (m, 1H), 1.95 – 1.87 (m, 1H), 1.85 – 1.73 (m, 2H), 1.48 (d, J = 14.1 Hz, 18H); ¹³C NMR (126 MHz, CDCl₃) δ 176.87, 167.90, 162.91, 162.64, 154.28, 151.66, 150.48, 142.80, 141.84, 141.27, 135.10, 129.26, 128.95, 127.54, 127.38, 127.00, 125.26, 125.12, 120.74, 117.20, 103.03, 82.17, 79.61, 56.62, 50.29, 30.88, 30.47, 28.38, 28.25, 24.51; HRMS (ESI+): Calcd for C₃₉H₄₄N₇O₅S [M+H]⁺: 722.3125, Found: 722.3107.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(4′-fluoro-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (22b)

Synthesized by General Procedure E. 25 mg, 50%, off-white oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.39 (s, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H), 7.89 – 7.78 (m, 1H), 7.65 – 7.55 (m, 5H), 7.13 (t, J = 8.5 Hz, 2H), 6.94 (s, 1H), 4.84 – 4.71 (m, 1H), 3.81 – 3.61 (m, 2H), 3.58 – 3.43 (m, 1H), 3.09 (dd, J = 15.4, 8.7 Hz, 1H), 2.37 – 2.24 (m, 1H), 1.96 – 1.87 (m, 1H), 1.86 – 1.72 (m, 2H), 1.54 – 1.41 (m, 18H); ¹³C NMR (126 MHz, CDCl₃) δ 176.85, 167.88, 163.65 (d, ¹J_CF = 100 Hz), 162.89, 162.65 (d, ¹J_CF = 100 Hz), 154.31, 151.29, 150.51, 142.78, 139.81, 136.97, 133.61, 131.92, 128.97, 128.71 (d, ⁴J_CF = 7.1 Hz), 128.64 (d, ⁴J_CF = 7.1 Hz), 127.82, 127.35 (d, ²J_CF = 63.6 Hz), 126.72 (d, ²J_CF = 63.6 Hz), 120.77, 117.23, 115.91 (d, ³J_CF = 17.2 Hz), 115.74 (d, ³J_CF = 17.2 Hz), 103.17, 102.86, 82.17, 79.64, 56.61, 50.31, 30.92, 30.46, 29.85, 28.32, 28.17, 24.56; ¹⁹F NMR (376 MHz, CDCl₃) δ −115.56 – −115.66 (m, 1F); HRMS (ESI+): Calcd for C₃₉H₄₃FN₇O₅S [M+H]⁺: 740.3030, Found: 740.2979.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (22c)

Synthesized by General Procedure E. 10 mg, 75%, off-white oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.32 (s, 1H), 8.09 (d, J = 8.3 Hz, 2H), 7.98 (d, J = 7.9 Hz, 2H), 7.81 – 7.63 (m, 6H), 7.58 (d, J = 8.3 Hz, 2H), 7.46 (s, 1H), 6.98 (s, 1H), 4.79 (dt, J = 11.9, 5.8 Hz, 1H), 3.79 – 3.62 (m, 2H), 3.57 – 3.44 (m, 1H), 3.15 (dd, J = 15.4, 8.3 Hz, 1H), 2.33 – 2.22 (m, 1H), 1.98 – 1.91 (m, 1H), 1.89 – 1.74 (m, 2H), 1.53 – 1.44 (m, 18H); ¹³C NMR (126 MHz, CDCl₃) δ 176.80, 167.74, 162.82, 162.54, 154.05, 150.95, 148.99, 144.17, 142.55, 139.09, 134.34, 129.52, 128.86, 127.50, 127.21, 126.69, 125.78 (q, ⁴J_CF = 2.0 Hz), 125.75 (q, ⁴J_CF = 2.0 Hz), 125.73 (q, ⁴J_CF = 2.0 Hz), 125.70 (q, ⁴J_CF = 2.0 Hz), 121.05 (q, ⁴J_CF = 2.0 Hz), 120.79, 117.15, 103.17, 101.06, 83.41, 81.99, 56.47, 50.10, 30.69, 30.30, 29.69, 28.15, 27.98; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.44 (s, 3F); HRMS (ESI+): Calcd for C₄₀H₄₃F₃N₇O₅S [M+H]⁺: 790.2998, Found: 790.2989.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (22d)

Synthesized by General Procedure E. 7 mg, 37%, off-white oily solid. ¹H NMR (400 MHz, CDCl₃) δ 10.39 (s, 1H), 8.06 – 7.94 (m, 4H), 7.87 (s, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.68 – 7.52 (m, 6H), 6.96 (s, 1H), 4.82 – 4.73 (m, 1H), 3.82 – 3.62 (m, 2H), 3.57 – 3.45 (m, 1H), 3.09 (dd, J = 15.5, 8.8 Hz, 1H), 2.31 (dt, J = 11.5, 6.7 Hz, 1H), 1.97 – 1.88 (m, 1H), 1.84 – 1.75 (m, 1H), 1.71 – 1.62 (m, 2H), 1.55 – 1.42 (m, 18H); ¹³C NMR (126 MHz, CDCl₃) δ 176.83, 167.87, 163.01, 151.07, 150.47, 142.78, 141.61, 139.21, 134.37, 131.91, 131.75 (q, ²J_CF = 25.3 Hz), 131.49 (q, ²J_CF = 25.3 Hz), 131.24 (q, ²J_CF = 25.3 Hz), 130.98 (q, ²J_CF = 25.3 Hz), 130.36, 129.42, 128.96, 128.81 (q, ¹J_CF = 100.0 Hz), 127.81 (q, ¹J_CF = 100.0 Hz), 127.55, 126.86, 126.26, 125.41, 124.17 (q, ⁴J_CF = 3.0 Hz), 124.14 (q, ⁴J_CF = 3.0 Hz), 124.11 (q, ⁴J_CF = 3.0 Hz), 124.09 (q, ⁴J_CF = 3.0 Hz), 123.90 (q, ⁴J_CF = 3.0 Hz), 123.87 (q, ⁴J_CF = 3.0 Hz), 123.84 (q, ⁴J_CF = 3.0 Hz), 123.81 (q, ⁴J_CF = 3.0 Hz), 123.25, 120.75, 117.24, 117.16, 105.35, 103.20, 82.19, 79.67, 56.61, 50.32, 30.92, 30.45, 28.36, 28.28, 28.16; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.63; HRMS (ESI+): Calcd for C₄₀H₄₃F₃N₇O₅S [M+H]⁺: 790.2998, Found: 790.2997.

(S)-(2-((3-(4-((4-([1,1′-biphenyl]-3-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)(amino)methaniminium chloride (23a)

Synthesized by General Procedure E. 5 mg, 93%, light yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.19 – 8.14 (m, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.95 – 7.86 (m, 3H), 7.73 – 7.66 (m, 2H), 7.60 – 7.55 (m, 1H), 7.53 – 7.44 (m, 3H), 7.40 – 7.33 (m, 1H), 7.25 (s, 1H), 7.22 (s, 1H), 4.57 – 4.50 (m, 1H), 3.55 (ddd, J = 9.8, 8.0, 4.2 Hz, 1H), 3.51 – 3.41 (m, 1H), 2.36 – 2.19 (m, 1H), 2.19 – 1.97 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 177.73, 169.37, 164.53, 156.44, 152.44, 145.59, 142.93, 142.47, 136.66, 130.19, 129.92, 129.40, 128.48, 128.08, 127.47, 126.06, 125.64, 120.26, 118.04, 104.46, 57.23, 31.60, 30.07, 23.58; HRMS (ESI+): Calcd for C₂₉H₂₈N₇OS [M+H]⁺: 522.2076, Found: 522.2072. HPLC purity: 88%.

(S)-amino(2-((3-(4-((4-(4′-fluoro-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (23b)

Synthesized by General Procedure E. 8 mg, 90%, off-white solid. ¹H NMR (500 MHz, CD₃OD) δ 8.09 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 7.8 Hz, 2H), 7.84 (d, J = 8.5 Hz, 3H), 7.72 – 7.63 (m, 3H), 7.23 (s, 1H), 7.22 – 7.15 (m, 2H), 4.57 – 4.49 (m, 1H), 3.58 – 3.50 (m, 1H), 3.49 – 3.42 (m, 1H), 3.33 – 3.31 (m, 2H), 2.35 – 2.22 (m, 1H), 2.18 – 1.97 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.96, 169.20, 167.04, 166.33 (d, ¹J_CF = 128.3 Hz), 165.06 (d, ¹J_CF = 128.3 Hz), 156.46, 149.01, 144.38, 141.46, 138.03, 132.86, 129.76, 129.70, 129.54, 128.90, 128.26 (d, ²J_CF = 49.5 Hz), 127.77 (d, ²J_CF = 49.5 Hz), 119.81, 118.95, 116.73 (d, ³J_CF = 17.3 Hz), 116.56 (d, ³J_CF = 17.3 Hz), 57.22, 31.62, 30.08, 28.13, 23.59; ¹⁹F NMR (376 MHz, CD₃OD) δ −117.18 – −117.54 (m, 1F); HRMS (ESI+): Calcd for C₂₉H₂₇FN₇OS [M+H]⁺: 540.1982, Found: 540.1978.

(S)-amino(2-((3-(4-((4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (23c)

Synthesized by General Procedure E. 2 mg, 100%, off-white solid. ¹H NMR (500 MHz, CD₃OD) δ 8.11 – 7.99 (m, 3H), 7.96 – 7.83 (m, 4H), 7.80 – 7.73 (m, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.28 – 7.18 (m, 2H), 4.58 – 4.51 (m, 1H), 3.59 – 3.51 (m, 1H), 3.51 – 3.45 (m, 1H), 2.36 – 2.25 (m, 1H), 2.19 – 1.99 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 177.72, 169.41, 156.48, 152.04, 151.44, 145.62, 145.54, 139.94, 136.20, 135.32, 132.74, 131.64, 129.40, 128.80, 128.45, 128.38, 127.72, 126.84 (q, ⁴J_CF = 3.0 Hz), 126.81 (q, ⁴J_CF = 3.0 Hz), 126.78 (q, ⁴J_CF = 3.0 Hz), 126.75 (q, ⁴J_CF = 3.0 Hz), 122.39, 120.29, 118.08, 104.78, 57.26, 31.63, 30.09, 23.60; ¹⁹F NMR (376 MHz, CD₃OD) δ −63.98 (s, 3F): Calcd for C₃₀H₂₇F₃N₇OS [M+H]⁺: 590.1950, Found: 590.1954.

(S)-amino(2-((3-(4-((4-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (23d)

Synthesized by General Procedure E. 5 mg, 100%, off-white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.16 – 8.03 (m, 2H), 8.02 – 7.97 (m, 1H), 7.94 (d, J = 4.1 Hz, 2H), 7.88 – 7.82 (m, 2H), 7.79 – 7.75 (m, 2H), 7.68 – 7.65 (m, 2H), 4.57 – 4.50 (m, 1H), 3.60 – 3.50 (m, 1H), 3.50 – 3.41 (m, 1H), 3.34 – 3.31 (m, 1H), 2.34 – 2.22 (m, 1H), 2.17 – 2.00 (m, 4H); ¹³C NMR (101 MHz, CD₃OD) δ 177.96, 169.17, 166.36, 156.43, 148.71, 144.30, 142.73, 140.75, 133.87, 132.47, 131.64, 130.89, 129.70, 128.53, 127.96, 127.34, 127.06, 125.26 (q, ⁴J_CF = 4.0 Hz), 125.22 (q, J_CF = 4.0 Hz), 125.18 (q, J_CF = 4.0 Hz), 125.14 (q, J_CF = 4.0 Hz), 124.41, 124.37, 122.32, 119.83, 57.20, 31.60, 30.07, 23.59; ¹⁹F NMR (376 MHz, CD₃OD) δ −64.14 (s, 3F); HRMS (ESI+): Calcd for C₃₀H₂₇F₃N₇OS [M+H]⁺: 590.1950, Found: 590.1949.

N-(4-cyanophenyl)-2,2,2-trifluoroacetamide (25)

4-Fluorobenzonitrile (0.5 g, 4.13 mmol) was dissolved in THF (20.6 mL) and 1 M potassium tert-butoxide in THF (10.3 mL, 10.32 mmol) was then added. The reaction mixture was refluxed for 4 h, after which the organic solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography (5% EtOAc/hexanes) to yield 2 (207 mg, 29%) as a clear liquid. ¹H NMR (400 MHz, CDCl₃) δ 7.52 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 1.38 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 159.90, 133.36, 122.94, 119.11, 105.58, 80.17, 28.80; HRMS (ESI+): Calcd for C₁₁H₁₄NO [M+H]⁺: 176.1075, Found: 176.1083.

(Z)-4-(tert-butoxy)-N′-hydroxybenzimidamide

Synthesized by General Procedure A. 215 mg, 87% as white solid. ¹H NMR (400 MHz, acetone-d₆) δ 9.11 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.6 Hz, 1H), 5.48 (s, 2H), 1.34 (s, 9H); ¹³C NMR (101 MHz, acetone-d₆) δ 157.36, 151.95, 128.89, 126.94, 123.92, 78.87, 28.95; HRMS (ESI+): Calcd for C₁₁H₁₇N₂O₂ [M+H]⁺: 209.1290, Found: 209.1290.

tert-butyl (S)-2-((3-(4-(tert-butoxy)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (26)

Synthesized by General Procedure B. 214 mg, 52%, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.98 – 7.93 (m, 2H), 7.05 (d, J = 8.3 Hz, 2H), 4.35 – 4.18 (m, 1H), 3.46 – 3.23 (m, 3H), 3.04 (ddd, J = 37.3, 14.5, 8.7 Hz, 1H), 2.04 (q, J = 6.4, 4.4 Hz, 1H), 1.91 – 1.74 (m, 3H), 1.44 (s, 9H), 1.37 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.30, 168.09, 158.39, 154.24, 128.43, 123.86, 121.52, 80.05, 79.37, 55.26, 46.82, 46.41, 31.83, 31.03, 30.86, 30.10, 28.97, 28.54, 23.63, 22.84; HRMS (ESI+): Calcd for C₂₂H₃₂N₃O₄ [M+H]⁺: 402.2393, Found: 402.2407.

tert-butyl (S)-2-((3-(4-hydroxyphenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (27)

26 (212 mg, 0.529 mmol) was dissolved in DCM (2 mL) and then 1 N TFA (2 mL) was added. The reaction mixture was stirred for 4 h. At this time, TLC showed complete conversion of starting material. Organic solvent was removed under reduced pressure. The resulting product was then dissolved in dioxane (1 mL), and a mixture of di-tert-butyl dicarbonate (0.146 mL, 0.635 mmol) and TEA (0.192 mL, 1.38 mmol) was added dropwise to the solution. The reaction mixture was stirred for 1 h, after which the organic solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (30% EtOAc/hexane) to provide 27(73 mg, 40%) as a white solid. ¹H NMR (400 MHz, acetone-d₆) δ 7.92 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 4.29 – 4.20 (m, 1H), 3.41 – 3.25 (m, 2H), 3.13 (dd, J = 14.6, 8.2 Hz, 1H), 2.95 (s, 1H), 2.15 – 2.06 (m, 1H), 1.95 – 1.80 (m, 3H), 1.47 – 1.39 (m, 9H); ¹³C NMR (101 MHz, CD₃OD) δ 178.74, 169.35, 161.71, 156.09, 130.01, 118.99, 116.75, 81.52, 81.01, 56.89*, 56.53*, 47.81*, 47.31*, 32.38*, 32.10*, 31.53*, 31.16*, 28.73*, 28.59*, 24.33*, 23.51*; HRMS (ESI+): Calcd for C₁₈H₂₂N₃O₄ [M-H]^-: 344.1610, Found: 344.1620.

tert-butyl (S)-2-((3-(4-((4-bromothiazol-2-yl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (28)

2, 4-dibromothiazole (0.025 g, 0.101 mmol) was added to a mixture of 27 (0.035 g, 0.101 mmol) and K₂CO₃ (0.017 g, 0.122 mmol) in DMF (1 mL). The reaction mixture was refluxed for 17 h, after which the solution was partitioned between EtOAc and LiBr aqueous solution. The aqueous solution was washed with EtOAc three times, and the combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated via vacuum. The resulting residue was purified by silica gel column chromatography (30% EtOAc/hexane) to yield 28 (20 mg, 39%) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.17 – 8.10 (m, 2H), 7.40 (d, J = 8.4 Hz, 2H), 6.78 (s, 1H), 4.38 – 4.21 (m, 1H), 3.49 – 3.28 (m, 3H), 3.27 – 2.98 (m, 1H), 2.13 – 2.01 (m, 1H), 1.93 – 1.77 (m, 3H), 1.47 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.64, 172.10, 167.55, 156.88, 154.30, 129.48, 129.19, 124.79, 120.39, 120.10, 119.76, 110.97, 80.18*, 79.77*, 55.27*, 55.20*, 46.88*, 46.49*, 31.95*, 31.16*, 31.01*, 30.24*, 28.62, 23.71*, 22.92*; HRMS (ESI+): Calcd for C₂₁H₂₃BrN₄NaO₄S [M+Na]⁺: 529.0466, Found: 529.0493.

tert-butyl (S)-2-((3-(4-((4-(4-(trifluoromethyl)phenyl)thiazol-2-yl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (29)

Synthesized by General Procedure D. 30 mg, 45%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.16 (d, J = 8.6 Hz, 2H), 7.91 (d, J = 8.1 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 7.17 (s, 1H), 4.39 – 4.21 (m, 1H), 3.51 – 3.30 (m, 3H), 3.17 – 3.00 (m, 1H), 2.14 – 2.03 (m, 1H), 1.94 – 1.80 (m, 3H), 1.48 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.78, 172.08, 167.71, 157.42, 148.58, 137.36, 129.50, 129.41, 126.27, 125.83, 124.50, 120.36, 110.97, 108.87, 80.20*, 79.80*, 55.29, 46.89*, 46.52*, 31.96*, 31.17*, 30.27*, 28.64, 23.73*, 22.94*; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.61 (s, 3F); HRMS (ESI+): Calcd for C₂₁H₂₃BrN₄NaO₄S [M+Na]⁺: 529.0466, Found: 529.0493.

tert-butyl (S,Z)-(((tert-butoxycarbonyl)imino)(2-((3-(4-((4-(4-(trifluoromethyl)phenyl)thiazol-2-yl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methyl)carbamate (30)

Synthesized by General Procedure E. 10 mg, 83%, white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.33 (s, 1H), 8.21 – 8.13 (m, 2H), 7.91 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.50 – 7.45 (m, 2H), 7.42 – 7.37 (m, 1H), 7.17 (s, 1H), 4.85 – 4.75 (m, 1H), 3.74 – 3.61 (m, 2H), 3.56 – 3.43 (m, 1H), 3.20 (dd, J = 15.2, 8.2 Hz, 1H), 2.32 – 2.23 (m, 1H), 1.97 – 1.89 (m, 1H), 1.88 – 1.76 (m, 2H), 1.47 (d, J = 7.6 Hz, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 177.49, 172.13, 167.61, 162.67, 157.36, 156.85, 154.23, 150.45, 148.55, 137.35, 130.41, 130.24, 129.92, 129.58, 129.49, 126.26, 125.89 (q, ⁴J_CF = 3.5 Hz), 125.85 (q, ⁴J_CF = 3.5 Hz), 125.81 (q, ⁴J_CF = 3.5 Hz), 125.77 (q, ⁴J_CF = 3.5 Hz), 124.69, 121.91, 120.41, 120.35, 119.78, 110.93, 108.84, 82.12, 79.45, 56.61, 50.28, 30.76, 30.45, 28.36, 28.23, 24.54; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.62 (s, 3F); HRMS (ESI+): Calcd for C₃₄H₃₈F₃N₆O₆S [M+H]⁺: 715.2526, Found: 715.2499.

(S)-amino(2-((3-(4-((4-(4-(trifluoromethyl)phenyl)thiazol-2-yl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)methaniminium chloride (31)

Synthesized by General Procedure E. 6 mg, 100%, yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.24 – 8.15 (m, 2H), 8.02 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.63 (s, 1H), 7.61 – 7.53 (m, 2H), 4.59 – 4.51 (m, 1H), 3.61 – 3.52 (m, 1H), 3.53 – 3.43 (m, 1H), 3.38 – 3.34 (m, 2H), 2.38 – 2.25 (m, 1H), 2.21 – 2.01 (m, 3H); ¹³C NMR (101 MHz, cd₃od) δ 178.31, 173.59, 168.87, 159.02, 156.45, 149.34, 138.97, 130.41, 130.32, 127.36, 126.75 (q, ⁴J_CF = 4.0 Hz), 126.71 (q, ⁴J_CF = 4.0 Hz), 126.67 (q, ⁴J_CF = 4.0 Hz), 126.64 (q, ⁴J_CF = 4.0 Hz), 125.48, 121.77, 121.60, 113.12, 111.34, 57.17, 31.60, 30.05, 23.58; ¹⁹F NMR (376 MHz, CD₃OD) δ −64.16 (s, 3F); HRMS (ESI+): Calcd for C₂₄H₂₂F₃N₆O₂S [M+H]⁺: 515.1477, Found: 515.1445.

tert-butyl (S)-2-((3-(4-((4-([1,1′-biphenyl]-4-yl)thiazol-2-yl)(methyl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-carboxylate (32)

To a solution of tert-butyl (S)-2-((3-(4-18dd (0.025 g, 0.043 mmol) in acetone (1 mL) was added K₂CO₃ (0.024 g, 0.172 mmol) and methyl iodide (0.061 g, 0.431 mmol). The reaction mixture was refluxed for 17 h. The organic solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (30% EtOAc/hexane) to yield 32 (15 mg, 73%) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J = 8.3 Hz, 2H), 7.94 (d, J = 7.9 Hz, 2H), 7.63 (t, J = 7.2 Hz, 5H), 7.45 (t, J = 7.5 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 6.85 (s, 1H), 4.40 – 4.22 (m, 1H), 3.68 (s, 3H), 3.52 – 3.28 (, 3H), 3.20 – 2.99 (m, 1H), 2.16 – 2.02 (m, 1H), 1.97 – 1.78 (m, 3H), 1.49 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 177.62, 168.41, 154.64, 151.48, 148.51, 140.96, 140.54, 134.00, 128.93, 128.79, 127.43, 127.40, 127.33, 127.12, 126.59, 123.52, 102.76, 80.17, 55.29, 46.92*, 46.53*, 40.39, 31.98*, 31.13*, 30.25*, 29.85*, 28.65, 23.74*, 22.94*; HRMS (ESI+): Calcd for C₃₄H₃₆N₅O₃S [M+H]⁺: 594.2538, Found: 594.2553.

tert-butyl (S,Z)-((2-((3-(4-((4-([1,1′-biphenyl]-4-yl)thiazol-2-yl)(methyl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)((tert-butoxycarbonyl)imino)methyl)carbamate (33)

Synthesized by General Procedure E. 15 mg, 73%, off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.33 (s, 1H), 8.15 (d, J = 8.2 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H), 7.62 (dd, J = 10.9, 8.2 Hz, 6H), 7.45 (t, J = 7.6 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 6.84 (s, 1H), 4.84 – 4.76 (m, 1H), 3.73 – 3.61 (m, 5H), 3.55 – 3.48 (m, 1H), 3.19 (dd, J = 15.2, 8.2 Hz, 1H), 2.32 – 2.24 (m, 1H), 1.98 – 1.90 (m, 1H), 1.89 – 1.76 (m, 2H), 1.48 (s, 18H); ¹³C NMR (101 MHz, CDCl₃) δ 177.30, 168.44, 167.82, 158.56, 155.56, 154.18, 151.41, 148.46, 140.94, 140.50, 133.98, 128.91, 128.85, 128.71, 127.39, 127.11, 126.57, 123.65, 123.55, 102.70, 81.99, 79.53, 56.63, 50.23, 40.37, 30.76, 30.48, 28.30, 28.12, 24.54; HRMS (ESI+): Calcd for C₄₀H₄₆N₇O₅S [M+H]⁺: 736.3281, Found: 736.3274.

(S)-(2-((3-(4-((4-([1,1′-biphenyl]-4-yl)thiazol-2-yl)amino)phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)(amino)methaniminium chloride (34)

Synthesized by General Procedure F. 5mg, 100% yield, off-white solid. ¹H NMR (500 MHz, CD₃OD) δ 8.13 (d, J = 8.6 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 8.6 Hz, 2H), 7.66 – 7.59 (m, 5H), 7.43 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 7.10 (s, 1H), 4.58 – 4.50 (m, 1H), 3.66 (s, 3H), 3.55 (td, J = 9.2, 8.5, 3.9 Hz, 1H), 3.49 – 3.42 (m, 1H), 3.33 – 3.31 (m, 2H), 2.34 – 2.22 (m, 1H), 2.17 – 2.12 (m, 1H), 2.11 – 1.97 (m, 2H); ¹³C NMR (126 MHz, CD₃OD) δ 178.12, 169.97, 169.08, 156.47, 152.27, 150.03, 141.96, 141.78, 135.08, 129.89, 129.60, 128.41, 128.07, 127.81, 127.58, 124.81, 124.41, 57.20, 40.80, 31.61, 30.09, 23.59; HRMS (ESI+): Calcd for C₃₀H₃₀N₇OS [M*]⁺: 536.2233, Found: 536.2230.

ABBREVIATIONS

Sph

sphingosine

S1P

sphingosine-1-phosphate

SphK

sphingosine kinase

HCTU

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