Oxadiazole-isopropylamides as Potent and Non-covalent Proteasome Inhibitors

Abstract

Screening of the 50,000 ChemBridge compound library led to the identification of the oxadiazole-isopropylamide 1 (PI-1833) which inhibited CT-L activity (IC₅₀ 0.60 μM) with little effects on the other 2 major proteasome proteolytic activities, T-L and PGPH-L. LC/MS-MS and dialysis show that 1 is a non-covalent and rapidly reversible CT-L inhibitor. Focused library synthesis provided 11ad (PI-1840) with CT-L activity (IC₅₀ 27 nM). Detailed SAR studies indicate that the amide moiety and the 2 phenyl rings are sensitive toward modifications. Hydrophobic residues, such as propyl or butyl, in the para-position (not ortho or meta) of the A-ring and a meta-pyridyl group as B-ring significantly improve activity. Compound 11ad (IC₅₀ 0.37 μM) is more potent than 1 (IC₅₀ 3.5 μM) at inhibiting CT-L activity in intact MDA-MB-468 human breast cancer cells and inhibiting their survival. The activity of 11ad warrants further pre-clinical investigation of this class as non-covalent proteasome inhibitors.

INTRODUCTION

The ATP-dependent ubiquitin-proteasome pathway is responsible for the controlled degradation of proteins in eukaryotic cells.^1-6 The 26S proteasome is a multifunctional complex consisting of a 19S regulatory particle (RP) and a 20S core particle (CP).⁷ The three main catalytic activities of the proteasome; peptidylglutamyl peptide hydrolyzing-like (PGPH-L), trypsin-like (T-L), and chymotrypsin-like (CT-L) are mediated by three distinct catalytic β-1, β-2, and β-5 subunits, respectively.⁸ For each of the catalytic β-subunits, the N-terminal Thr-1 serves as the nucleophile that initiate cleavage of the peptide bond.^9,10,11 Development of inhibitors of CT-L activity has been the subject of considerable interest in the treatment of cancer due to its critical role in the degradation of apoptotic and tumor suppressor proteins.^8,10,12 The proteasome inhibitors advanced to clinic or clinical trials are derived from 3 structural classes (Figure 1A): 1. Boronic acid containing compounds such as bortezomib, a dipeptide boronic acid (clinically approved by FDA),^{13, 14} MLN9708^15-17 and CEP-18770;^18-21 (Figure 1A). 2. β-lactones such as salinosporamide A (NPI-0052)^22,23 which is a marine microbial natural product (Figure 1A) and 3. Epoxyketone containing tetrapeptide carfilzomib²⁴ (Figure 1A), clinically approved by FDA which is related to the natural product epoxomicin. Each inhibitor class reacts with the proteasome N-terminal threonine (Thr-1 at the active site) by a distinct mechanism. Peptide boronic acids form a covalent and slowly reversible tetrahedral adducts with the OH group of the catalytic Thr-1.^11,25 For the β-lactone salinosporamide A, attack of the lactone ring by catalytic Thr-1²⁶ forms an ester bond (that undergoes intramolecular rearrangement) which makes this compound an irreversible inhibitor. The epoxyketone¹⁴ moiety of carfilzomib reacts with the OH and the α-amino group of Thr-1 to form 2 covalent bonds, also making the inhibition irreversible.

Figure 1.

A. Structures of clinically advanced covalent proteasome inhibitors B. Structures of non-covalent small molecule proteasome inhibitors; hydroxyurea pharmacophore³⁸ and peptidic pharmacophore from Millennium.^11,45

Covalent proteasome inhibitors are classified as slow reversible or irreversible inhibitors according to their chemical structure and mechanism of inhibition. Covalent irreversible or covalent slow reversible inhibitors as described above possess a chemically reactive group that bind to the proteasome covalently. In contrast, non-covalent inhibitors inhibit the proteasome through a network of interactions (hydrophobic, hydrogen bonds, electrostatic and/or van der Waals). Although some proteasome inhibitors have been suggested to bind non-covalently^27-34 structural evidence to support this was provided for only three of these. The X-ray structures of TMC-95A,^35-37 hydroxyl urea³⁸ (Figure 1B) and the peptide¹¹ from Millenium (Figure 1B) bound to proteasome demonstrate that the binding mode of these compounds are non-covalent.

Since non-covalent inhibitors do not have a reactive electrophilic moiety or ‘warhead’, which is often associated with metabolic instability, poor specificity, and excessive reactivity, they have the advantage of exerting fewer side effects over the covalent ones. It has been shown that the proteasome activity recovers at the same rate with covalent irreversible inhibitors as with covalent slow reversible inhibitors, presumably via de novo proteasome synthesis.³⁹ The clinical advantages/benefits of non-covalent proteasome inhibitors in cancer treatment are not well understood. Figure 1B shows the structures of small molecules that have been identified as non-covalent proteasome inhibitors.^11,38 We have been actively engaged in the discovery of novel proteasome inhibitors.^40,41 We reported the discovery of the compound 1 as a proteasome inhibitor in a poster at the 2011 American Association for Cancer Research (AACR) meeting.⁴² Villoutreix et al also have reported oxadiazole-isopropylamide containing compounds as proteasome modulators.^43,44 Although Villoutreix et al and our group have independently identified similar scaffolds, each group focused on different modifications of the hits that led to important findings that are complementary but not overlapping. In our study, we have extensively explored SAR (Figure 2) on the oxadiazole-isopropylamide containing compounds as proteasome inhibitors by systematically synthesizing focused libraries around key features of the pharmacophore. We present compound 1 and its most potent analogs as non-peptidic, non-covalent and reversible proteasome inhibitors that have the potential to become clinical candidates.

Figure 2.

Modifications and library synthesis around 1 for design of new proteasome inhibitors and SAR studies.

CHEMISTRY

The screening hit 1 was identified as a CT-L proteasome inhibitor with an IC₅₀ value of 0.60 ± 0.18 μM (in-vitro). Validation of the synthetic route to 1 was undertaken to confirm both the structure and the in vitro CT-L inhibitory activity. Synthesis of 1 was achieved using the route shown in Scheme 1. The substituted acetyl chloride building block library 5 (Scheme 1) was synthesized from readily available phenol derivatives via the ester 3 and acid 4 using reported protocols.^46-50 The oxadiazole portion of the compound 1 was synthesized from readily available nitrile building blocks 6. The nitrile building blocks were reacted with hydroxylamine hydrochloride and sodium carbonate at 70 °C in water to yield the hydroxyamidines⁵¹ 7 (Scheme 1, condition g). The 5-substituted pyrimidinehydroxyamidine 7h (see supporting information) was synthesized starting from the commercially available methyl pyrimidine-5-carboxylate via amide 24 and nitrile 25.⁵² The intermediate hydroxyamidine library 7 was reacted with chloroacetyl chloride (Scheme 1, condition h) to provide the library 8,⁵³ which was cyclized in refluxing toluene to provide the oxadiazole portion of the pharmacophore 9. The library 9 was subsequently reacted with appropriate alkyl amines (isopropyl-, isobutyl-, methyl-. ethyl-, cyclopropyl- and tert-butyl amines) to obtain the amine building block library 10⁵⁴ (Scheme 1, condition j). For the synthesis of 10n (R³ = H), compound 9a (R² = para-tolyl, see supporting information compound 26) was first reacted with phthalimide in the presence of potassium carbonate in refluxing acetonitrile, followed by reaction with hydrazine to obtain the compound 10n in high yield.⁵⁵ We were able to generate library 10 with a variety of substituted aryl and hetero-aryl R² moieties and library 5 with substituted/unsubstituted aromatic R¹ moieties (Scheme 1). Modifications around 1 for library synthesis are shown in Figure 2, and initially we focused our synthetic efforts to modify R¹, R² and R³ moieties. The two key building block libraries 10 and 5 were then reacted in the presence of triethylamine to provide the compound 1, library 11 and 12 (Scheme 1, Tables 1 and 3) in good yields. The route described in Scheme 1 was efficient and convenient for rapid synthesis and optimization of the substituted phenyl and amide moieties. The final libraries 11, 12 and compound 1 were characterized using NMR, LC-MS, HRMS and the purity was > 95% as determined by HPLC. The final compound library 11 and 12 (including compound 1) showed formation of approximately 3:1 atropisomers (hindered rotation about the C-N amide bond) by ¹H NMR and ¹³C NMR spectroscopy (see the experimental section and supporting information). Analysis of ¹H NMR spectra of compound 1 at variable temperatures (20 °C to 50 °C) showed that the peaks from the minor rotamer coalesced with the major rotamer as the temperature increased.

Scheme 1.

Synthetic route to compound 1, libraries 11 and 12. Reagents and conditions: a. Ethyl bromoacetate, K₂CO₃, Acetone, reflux, 14 h. b. tert-Butyl bromoacetate, DMF, 80 °C, 14 h. c. Ethyl bromoacetate, K₂CO_3, DMF, r.t., 14 h. d. NaOH, THF, reflux, 2 h. (R= Ethyl). e. CF₃COOH, DCM, r.t., 2 h (R = ^tBu). f. SOCl₂, benzene, reflux, 3 h. g. NH₂OH.HCl, Na₂CO_3, water, 70 °C, 14 h. h. Chloroacetyl chloride, acetone, r.t., 30 min. i. toluene, reflux, 2 h. j. Alkylamine, K₂CO₃, CH₃CN, reflux, 30 min. k. Et₃N, THF, r.t., 15 min.

Table 1.

Synthetic analogs of Library 12 and SAR.

Entry	Compound ID	R3	IC50 (μM) CT-La
1	1	isopropyl	0.60 ± 0.18 (n=27)
2	12a	isobutyl	2.37 ± 0.40 (n=2)
3	12b	ethyl	6.04 ± 1.34 (n=3)
4	12c	methyl	29.90 ± 3.9 (n=2)
5	12d	H	No inhibition @ 10 μM
6	12e	tert-butyl	No inhibition @ 10 μM
7	12f	cyclopropyl	No inhibition @ 10 μM

^aIC₅₀ values are given as the average of 2 or more determinations; n = number of determinations.

Table 3.

Compound 1, synthetic analogs of library 11 and SAR.

Entry	Compound ID	R1	R2	IC50 (μM) CT-L a
13	1			0.60 ± 0.18 (n=27)
14	11a			1.08 ± 0.33 (n=4)
15	11b			0.43 ± 0.12 (n=4)
16	11c			2.53 ± 0.95 (n=5)
17	11d			0.94 ± 0.26 (n=8)
18	11e			1.12 ± 0.33 (n=5)
19	11f			1.41 ± 0.19 (n=2)
20	11g			1.07 ± 0.05 (n=4)
21	11h			0.51 ± 0.16 (n=6)
22	11i			0.45 ± 0.15 (n=4)
23	11j			6.20 ± 1.1 (n=2)
24	11k			8.50 ± 0.60 (n=2)
25	11l			11.20 ± 2.1 (n=3)
26	11m			0.31 ± 0.08 (n=4)
27	11n			0.97 ± 0.15 (n=2)
28	11o			No inhibition @ 10 μM
29	11p			No inhibition @ 10 μM
30	11q			No inhibition @ 10 μM
31	11r			No inhibition @ 10 μM
32	11s			10.09 ± 1.63(n=3)
33	11t			No inhibition @ 10 μM
34	11u			0.98 ± 0.50 (n=3)
35	11v			10.12 ± 4.5 (n=3)
36	11w			3.75 ± 1.47 (n=6)
37	11x			0.22 ± 0.084 (n=6)
38	11y			0.37 ± 0.05 (n=4)
39	11z			4.0 ± 0.90 (n=3)
40	11aa			0.26 ± 0.05 (n=6)
41	11ab			0.099 ± 0.032 (n=14)
42	11ac			2.71 ± 0.56 (n=5)
43	11ad			0.027 ± 0.014 (n=20)
44	11ae			0.039 ± 0.01 (n=7)
45	11af			0.120 ± 0.04 (n=3)
46	11ag			0.43 ± 0.04 (n=3)
47	11ah			1.36 ± 0.18 (n=3)
48	11ai			0.44 ± 0.07 (n=3)
49	11aj			46.49 ± 1.23(n=3)
50	11ak			0.140 ± 0.05 (n=3)
51	11al			0.032 ± 0.003 (n=3)
52	11am			0.105 ± 0.031 (n=3)
53	11an			0.107 ± 0.013 (n=3)
54	11ao			1.27 ± 0.22 (n=3)
55	11ap			0.273 ± 0.10 (n=3)
56	11aq			No inhibition @ 10 μM
57	11ar			1.93 ± 0.61 (n=4)
58	11as			0.14 ± 0.064 (n=4)

^aIC₅₀ values are given as the average of 2 or more determinations; n = number of determinations.

Next, we focused our efforts to modify the chemical space between the amide moiety, A and B rings (Figure 2) in compound 1. First, we introduced a urea moiety to assess the SAR. To install the urea moiety, the intermediate 10d was reacted with commercially available isocyanate 13 in the presence of triethylamine in refluxing benzene, and under these conditions urea 14 was obtained in good yield (Scheme 2, condition q). Further modifications included replacement of the ether moiety (H-bond acceptor) in 1 (Figure 2) with an NH (H-bond acceptor/donor) group. The amine 10d was first reacted with chloroacetyl chloride in the presence of triethylamine in THF at room temperature to obtain intermediate 15 (Scheme 2) followed by coupling 15 with para-methylaniline using sodium acetate in refluxing ethanol to obtain the final compound 16 (Scheme 2, conditions r and s respectively) also in good yield. The ether moiety in 1 (Figure 2) was also replaced by a methylene unit using 3-(4-(trifluoromethyl)phenyl)propanoic acid building block (17a). The acid starting material 17a (Scheme 2) was converted to the corresponding acid chloride 18a and coupled with 10d to provide the oxadiazole 19a (Scheme 2). The final compound 19b with bulky R-groups was synthesized following the route in Scheme 2 starting from benzofuran-2-carboxylic acid (17b) via the formation of acid chloride 18b and subsequent coupling with 10f. The intermediate 10d was chosen for synthesis of compounds 14, 16, and 19a since our early SAR indicated unsubstituted B ring is desirable to retain in vitro CT-L potency together with para-CH₃ group on the A ring and the isopropyl moiety in the amide group. Extension of the linker between the oxadiazole moiety and amide group in 1 (Figure 2) by one carbon atom was achieved by first reacting the intermediate 7d with 3-chloropropanoyl chloride to generate intermediate 20 which provided 21 upon refluxing in toluene. The intermediate 21 was next reacted with isopropylamine to obtain 22, which was further reacted with intermediate 5a (see supporting information) to obtain the final compound 23 in good yield. The intermediates and final compounds were characterized by ¹H NMR, ¹³C NMR, HRMS, LC-MS; the purity of final compounds was determined using HPLC.

Scheme 2.

Reagents and conditions: q. Et₃N, benzene, reflux, 14 h, 78%. r. Chloroacetyl chloride, Et₃N, THF, r.t., 15 min., 80%. s. para-Methylaniline, NaOAc, ethanol, reflux, 15 h, 78%. f. SOCl₂, benzene, reflux, 3 h, 94%. i. toluene, reflux, 2 h, 81%. j. Isopropylamine, K₂CO₃, CH₃CN, reflux, 30 min., 86%. k. Et₃N, THF, r.t., 15 min., 88% (19a), 82% (19b), 87%, (23). l. DCM, r.t., 14 h, 76%.

RESULTS AND DISCUSSIONS

Unlike lactacystin, 1 is a non-covalent and reversible CT-L inhibitor

To determine whether our hit compound 1 is a non-covalent CT-L inhibitor, we used two approaches, Liquid Chromatography Tandem Mass Spectrometry (LC/MS-MS) and dialysis. With both approaches we used lactacystin, a known covalent and irreversible CT-L inhibitor as a control.^56,57 For LC/MS-MS, 1 or lactacystin were incubated with the 20S proteasome, digested with trypsin and the resulting peptides purified by HPLC and analyzed by LC/MS-MS as described under Methods. Figure 3A shows that peptides purified from the vehicle-treated samples contained unmodified TTTLAFK peptide (observed as a protonated molecule at m/z 781.4401) corresponding to the N-terminal tryptic peptide of rabbit proteasome subunit β type-5. Figure 3B shows that peptides purified from the compound 1 treated samples also contained the unmodified Thr-1 containing TTTLAFK peptide (Figure 3D). Both intact mass spectrum and tandem mass spectrum indicate unmodified Thr-1 containing peptide. Figure 3C shows that lactacystin-treated samples contained a lactacystin-modified Threonine adduct corresponding to the doubly charged modified peptide at m/z 497.7795 (structure shown in Figure 3E). The searches matching experimental data to peptides, from the database of rabbit proteasome subunits produced only one modified peptide, which indicated that Thr-1 on β-5 was modified by lactacystin. No other modifications to β-5 subunit from samples treated with DMSO, compound 1 or lactacystin were observed. In addition, no modifications were detected for other proteasome subunits, such as β-1 and β-2 subunits included in the database (n =21). These results suggest that, unlike lactacystin, 1 does not bind covalently to the proteasome.

Figure 3.

A: LC/MS-MS analysis of tryptic digests from proteasome CT-L subunit after incubation with vehicle. LC/MS-MS analysis of Thr-1 containing peptide from the proteasome CT-L subunit β5 after tryptic digestion is shown. Singly charged unmodified peptide was observed at m/z 781.4401, which represents a mass error of 6.9 ppm. The tandem mass spectrum was matched to peptide TTTLAFK. The b ions (labeled in red), contain the N-terminus of the peptide; and y ions, (labeled in blue), contain the C-terminus of the peptide. The number associated with each ion indicates the number of amino acids in that fragment (for example, y4 contains LAFK from C-terminus of the peptide). B: LC/MS-MS analysis of tryptic digests from proteasome CT-L subunit after incubation with compound 1. The Thr-1 containing peptide didn’t show any modification. Both intact mass spectrum and tandem mass spectrum indicate unmodified Thr-1 containing peptide. C: LC/MS-MS analysis of tryptic digests from proteasome CT-L subunit after incubation with lactacystin shows clasto-lactacystin-modified Thr-1 containing peptide. Doubly charged lactacystin-Thr modified peptide was detected at m/z 497.7795, which represents a mass error 6.1 ppm. The tandem mass spectrum confirms the modification of the peptide by lactacystin. D: Structure of the unmodified TTTLAFK tryptic peptide. E: Structure of the clasto-lactacystin modified peptide.

To confirm these results, we incubated 1 and lactacystin with the 20S proteasome and determined the reversibility of binding by dialysis as described under Methods. The Figure 4 shows that more than 70% of the CT-L activity in the dialysis compartment from the sample that was treated with 1 was recovered within the first 20 min., and recovered fully by 2 hours of dialysis. In contrast, very little CT-L activity was recovered from the sample that was treated with lactacystin even after 18 hours of dialysis (Figure 4). Taken together these results confirm the well established fact that lactacystin is a covalent and irreversible CT-L inhibitor and indicate that 1 is a non-covalent and reversible CT-L inhibitor. This is consistent with the chemical structure of 1 that lacks any reactive groups, unlike clasto-lactacystin that contains the reactive beta-lactone moiety. The β-lactone moiety of the clasto-lactacystin covalently modifies the β-subunits of the proteasome and is responsible for inhibiting 20S proteasome in an irreversible mode of action⁵⁷.

Figure 4.

Recovery of CT-L activity upon dialysis of the 20S proteasome-compound complexes after pre-incubation with lead 1 and lactacystin.

Structure Activity Relationship studies and chemistry leading to compound 11ad

Our screening efforts of the 50,000 in-house ChemBridge compound library led to the discovery of the hit 1, an inhibitor of the CT-L activity of the proteasome. After confirming the structure and in vitro CT-L activity of the in-house synthesized 1 (Scheme 1), we embarked on synthetic modifications to develop structure and activity relationship (SAR) data to identify novel, potent and selective CT-L proteasome inhibitors that block the action of the proteasome in a non-covalent manner. Proteasome CT-L activity was measured using a fluorogenic assay as previously described.⁴¹ Focused library synthesis was undertaken by independently varying the R¹, R² and R³ groups in compound 1 (Figure 2). Initially, we replaced the isopropyl R³ group in 1 with H, isobutyl, ethyl, methyl, tert-butyl and cyclopropyl moieties (Table 1). The loss of CT-L activities of these analogs indicated that the isopropyl group is essential and optimal for proteasome inhibitory activity (see Table 1). The isobutyl amide 12a and ethyl amide 12b (Table 1, Entries 2 and 3) showed 4- to 10-fold loss of activity with IC₅₀ values of 2.37 and 6.02 μM respectively compared to 1. Methyl, H, tert-butyl and cyclopropyl as R³ groups were detrimental for CT-L activity (12c, 12d, 12e, 12f in Table 1 showed weak or no proteasome inhibition at 10 μM). The ¹H and ¹³C NMR spectra of the compound 1 with isopropyl amide group indicated the presence of a mixture of 3:1 atropisomers (isomers that exist due to the hindered rotation about the carbon-nitrogen bond). We observed a similar ratio of atropisomers with 12b (R³ = ethyl), and 12c (R³ = methyl) by analysis of ¹H and ¹³C NMR spectra. Compounds 12d with unsubstituted amide (Entry 5, R³ = H, Table 1), 12e with bulky tert-butyl group (Entry 6, R³ = ^tBu, Table 1) and 12f with cyclopropyl group (Entry 7, R³ = cyclopropyl) did not show any atropisomers by ¹H or ¹³C NMR spectra.

In the next generation of synthetic analogs, we retained the isopropyl R³ group (Figure 2) and modified the phenyl rings in 1 [i.e. library 11, (Scheme 1 and Table 3)]. Initial SAR studies demonstrated that substitutions on the para-position of the R¹ and R² with small hydrophobic groups are tolerable. For example, changing the para-methyl in R¹ and R² to trifluoromethyl or chlorine as in compounds 11a, 11c, 11d, 11e, 11f, 11g, 11i and 11n retained the in vitro CT-L inhibitory activities (Entries 14, 16-20, 22, 27, Table 3). Next we demonstrated that the R¹ methyl is required whereas the R² methyl is dispensable. Indeed, compounds 11b, 11h and 11m (Entries 15, 21 and 26, Table 3) with an unsubstituted phenyl ring as R² showed slightly improved IC₅₀ values around 0.3 to 0.5 μM indicating para-substitution of R² phenyl in 1 is not required for inhibitory activity. However, compound 11j with both unsustituted phenyl rings resulted in 10 fold loss of CT-L activity (Entry 23, Table 3, IC₅₀ = 6.22 μM). Removal of the R¹ para-methyl group as in compounds 11k and 11l (Entries 24 and 25, Table 3) also led to 14- and 18-fold loss of activity with IC₅₀ values of 8.5 and 11 μM, respectively. The loss of in vitro potency of compounds 11j, 11k and 11l suggest that para-substitution in R¹ phenyl ring is critical to maintain CT-L proteasome activity. Changing the R¹ para-methyl to meta- or ortho-positions as in compounds 11o and 11p (Entries 28 and 29, Table 3) was detrimental for in vitro potency further suggesting that R¹ para-substitution is important for CT-L proteasome activity. Overall our SAR indicated that the para-methyl group in R¹ but not in R² and the R³ isopropyl amide are key features that are responsible for retaining the CT-L proteasome activity of compound 1 and loss of activity was consistent with unsubstituted R¹ aromatic rings in this class of compounds.

We next modified the chemical space between the amide moiety and R¹/R² in the parent compound 1 (Figure 2) using the synthetic routes and protocols described in Scheme 2 (also see supporting information). First, replacement of the ether-oxygen by methylene showed significant loss of CT-L activity (19a, IC₅₀ 53.48 μM, Entry 8, Table 2). Furthermore, substituting the amide group by urea as in 14 (Entry 9, IC₅₀ > 10 μM, Table 2) also led to loss of activity. Replacement of the ether (H-bond acceptor) with NH (H-bond donor/acceptor) also decreased the in vitro CT-L activity (16, IC₅₀ 5.67 μM, Entry 10, Table 2). These modifications confirmed that the ether moiety, most likely, as H-bond acceptor, is critical for focused library synthesis and improving the CT-L inhibitory activity. Extending the spacer between the amide and the oxadiazole by one carbon as shown in 23 (Entry 11, IC₅₀ > 10 μM, Table 2) was detrimental for CT-L inhibitory activity, probably due to the increased flexibility of the molecule. Compound 19b (IC₅₀ 0.37 μM, Entry 12, Table 2), a bulky R¹ substituent with a rigid ether moiety and meta-pyridyl as R² showed improved inhibitory activity compared to 1 (pyridyl moiety was chosen for the synthesis of 19b, based on the most potent analogs described in Table 3). Overall, synthetic modifications of the linkers between R¹, R² and the amide moiety in 1 were not tolerated.

Table 2.

Modifications of the spacer between the amide, R¹ and R² groups and SAR.

Entry #	Compound Structure and ID	IC50 (μM) CT-La
8		53.48 ± 10.89 (n=3)
9		No inhibition @ 10 μM
10		5.67 ± 0.96 (n=4)
11		No inhibition @ 10 μM
12		0.379 ± 0.06 (n=3)

^aIC₅₀ values are given as the average of 2 or more determinations; n = number of determinations.

To gain more insight in the interactions between R¹ and the binding region, we modified the R¹ via several synthetic modifications. For example, synthesis of 11q and 11r that possessed large hydrophobic groups such as phenyl and Br-naphthyl as para-R¹ groups respectively further revealed that large hydrophobic groups are not tolerated in the binding region (Entries 30, 31, IC₅₀ > 10 μM, Table 3). Replacement of the R¹ methyl group in compound 1 by small hydrophobic fluorine as in 11s (Entry 32, Table 3) also displayed poor CT-L inhibitory activity. The 16-fold loss of CT-L inhibitory activity in compound 11s with para-fluorine compared to 11b with para-CF₃ (Entry 15, IC₅₀ = 0.43 μM) was not unexpected since fluorine is isosteric to hydrogen⁵⁸ and as described above we have already observed the detrimental effects of unsubstituted R¹ rings in compounds 11k, 11l and 11ac (Entries 24, 25 and 42, Table 3) toward the CT-L inhibitory activity. The hydrophilic COOH, COO^tBu and OH groups in the para-position of the R¹ as in compounds 11t, 11aq and 11v respectively also failed to maintain the CT-L inhibitory activity (Entries 33, 56 and 35, Table 3) indicating H-bond acceptor/donor moieties are not desirable. In contrast compound 11u (Entry 34, Table 3) with para-hydroxyphenyl as R¹ and meta-pyridyl as R² showed an IC₅₀ value of 1 μM, and comparison of in vitro CT-L activities of 11u and 11v (Entries 34 and 35, Table 3) with para-hydoxyphenyl as R¹ highlights the significance of the R² meta-pyridyl group toward CT-L activity in this class of compounds.

We next made 2 major observations that led us to our most potent compounds. First, we demonstrated that increasing the alkyl length from methyl in 1 to a propyl as in 11ap (IC₅₀ = 0.27 μM) increased potency (Entry 55, Table 3). Second, replacing the tolyl R² in 1 by a meta-pyridyl (as in 11x, Entry 37, Table 3) or para- pyridyl (as in 11y, Entry 38, Table 3) but not ortho-pyridyl (as in 11w, Entry 36, Table 3) improved potency with the meta-pyridyl (IC₅₀ 0.22 μM) being slightly better than the para-pyridyl (IC₅₀ 0.37 μ, Table 3). Substituting both, the R¹ methyl with a propyl and the R² tolyl with a meta-pyridyl as in 11ad (combined features of compounds 11ap and 11x) resulted in our most potent compound with an IC₅₀ value of 27 nM. Furthermore, the length of the alkyl group in para-position of the R¹-ring is critical with the propyl or butyl groups being the optimal size. Indeed, decreasing the size from propyl (or butyl) to ethyl, methyl and hydrogen as in 11ab, 11x and 11ac, resulted in progressive loss of inhibitory activity from 27 nM to 99 nM, 220 nM and 2710 nM, respectively. Increasing the size of the R¹ para-hydrophobic moiety also resulted in progressive loss of potency from propyl or butyl to pentyl, hexyl, isobutyl, isopropyl, cyclohexyl and tert-butyl as in 11af (IC₅₀ 120 nM), 11ag (IC₅₀ 430 nM), 11ak (IC₅₀ 140 nM), 11ai (IC₅₀ 440 nM), 11ah (IC₅₀ 1356 nM) and 11aj (IC₅₀ 46.49 μM), respectively. Furthermore, substituting the pyridyl in 11ad with 3,5-pyrazine as in 11al did not alter the potency (IC₅₀ 32 nM) which further supports the importance of a meta-positioned nitrogen atom in the ring. In contrast, substituting the pyridyl in 11ad by 2,5-pyrazine as in 11am (Entry 52, IC₅₀ 105 nM, Table 3) with one meta-positioned nitrogen or 2,6-pyrimidine as in 11ao (Entry 54, IC₅₀ 1269 nM) which possess two ortho-positioned nitrogen atoms, led to 3-fold and 40-fold loss of CT-L activity respectively (Table 3) further highlighting the importance of the nitrogen in the meta-position of the ring. Compounds 11am and 11an that show CT-L inhibitory activities with IC₅₀ values in the range of 100 nM also demonstrate propyl or butyl in the para-position of the R¹ are equally tolerated in the binding region. Interestingly, compound 11ar and 11as with hydrophobic, electron withdrawing fluorine and chlorine in the para-position of the R¹ and meta-pyridyl as R² (Entries 57 and 58, Table 3) showed 3-5-fold improvement in inhibitory activities respectively comparing to its related analogs 11s (Entry 32, Table 3) 11h and (Entry 21, Table 3). Overall, our detailed SAR studies signify the importance of meta-pyridyl, isopropylamide and para-propyl/-butyl phenyl groups in the oxadiazole pharmacophore shown in 1 for inhibition of CT-L activity of the proteasome.

The parent compound 1 and 11ad were further evaluated for T-L and PGPH-L activities of the proteasome as shown in the Table 4. The in vitro data (IC₅₀) indicated that this class of compounds shows excellent selectivity for CT-L inhibitory activity over both T-L and PGPH-L activities. This level of CT-L selectivity is impressive for a non-peptidic, small molecule as compared to the covalent proteasome inhibitors reported to date. Only a small number of compound classes have been reported with this level of activity. ^{11, 38}

Table 4.

IC₅₀ values of 1 and 11ad for CT-L, T-L and PGPH-L activities of the proteasome.

Compound	CT-L (μM)	T-L (μM)a	PGPH-L (μM)a
1	0.60 ± 0.18 ( n= 27)	>100	>100
11ad	0.027 ± 0.014 (n = 20)	>100	>100

^aThe values given are the means of 3 experiments; n = number of determinations.

Lead compound 11ad is more potent than the parent compound 1 at inhibiting CT-L activity and survival/proliferation of intact human breast cancer cells

Our in vitro SAR studies demonstrated that substituting the methyl of ring A with a propyl and replacing ring B tolyl with meta-pyridine in 1 (IC₅₀ 600 nM) led to 22 fold more potent 11ad (IC₅₀ 27 nM). To determine whether these CT-L proteasome inhibitors are cell permeable and inhibit CT-L activity in intact cells, human breast cancer MDA-MB-468 cells were treated with increasing concentrations of 1 or 11ad for 2 hours and the cells were processed for CT-L activity⁴¹ determination as described under Methods. Figure 5A shows that 1 inhibited CT-L activity in a dose dependent manner with an IC₅₀ value of 3.5 μM. Figure 5A also shows that consistent with the in vitro results, 11ad was more potent than the parent compound 1 and inhibited the CT-L activity with an IC₅₀ value of 0.37 μM. We next determined whether compounds 1 and 11ad are capable of inhibiting tumor cell survival/proliferation. To this end, MDA-MB-468 cells were treated with parent compound 1 and 11ad and processed for MTT assay as described under Methods. Figure 5B shows that treatment with both compounds inhibited survival/proliferation but that 11ad was more potent than the parent compound 1.

Figure 5.

A: Lead 11ad is more potent at inhibiting proteasomal CT-L activity in intact human MDA-MB-468 cancer cells compared to the parent compound 1. B: Lead 11ad is more potent at inhibiting proliferation/survival of human MDA-MB-468 cells compared to the parent hit 1.

CONCLUSIONS

In this study we have extensively explored the SAR of oxadiazole-isopropylamide containing analogs as non-covalent CT-L proteasome inhibitors. The structure of the hit, 1 (IC₅₀ = 0.60 ± 0.18 μM) served as a starting point for synthetic modifications and design and synthesis of novel, highly potent and non-covalent proteasome inhibitors. The LC/MS-MS and dialysis experiments confirmed that 1 binds to CT-L β-5 subunit of the proteasome in a non-covalent and rapidly reversible binding mode. Compound 11ad with an IC₅₀ value of 27 nM was identified as one of the most potent and cell permeable proteasome inhibitors. Our detailed SAR analysis indicated that extending the R¹-methyl in 1 to an optimal size with propyl or butyl in combination with meta-pyridyl as R² significantly improves the CT-L proteasome inhibitory activity (11ad and 11ae, IC₅₀ 27 and 39 nM respectively). The isopropyl amide and the ether moieties in 1 are critical for inhibitory activity and modification of these moieties substantially reduced the CT-L inhibitory activity. The SAR demonstrated the importance of the length and composition of the linking chain between the amide moiety, R¹ and R² groups. The potent lead 11ad demonstrated efficacy in cellular assays and showed anti-proliferative activity at low micromolar concentrations. Our in vitro data also highlighted that this class of compounds shows high level of selectivity for CT-L proteasome inhibition over T-L and PGPH-L activities. Moreover, activities observed for some of the other analogs presented herein (such as 11al [IC₅₀ 32 nM], 11am [IC₅₀ 105 nM] and 11an [IC₅₀ 107 nM]) could also be optimized in the development of novel non-covalent CT-L proteasome inhibitors.

MATERIALS AND METHODS

CT-L, T-L, PGPH-L proteolytic activity assays

In the high-throughput screen, we used fluorogenic peptides as substrates to assay (at 10 μM) the 50,000 compounds library from ChemBridge for inhibitory activity against the CT-L proteolytic activity of the purified rabbit 20S proteasome, resulting in the identification of hit 1. To test for selectivity for CT-L over T-L and PGPH-L we used the corresponding fluorogenic peptides. Briefly, 1 nM of purified 20S rabbit proteasome was incubated with 20 μM Suc-Leu-Leu-Val-Tyr-AMC for the CT-L activity, Bz-Val-Gly-Arg-AMC for the T-L activity, and benzyloxycarbonyl Z-Leu-Leu-Glu-AMC for the PGPH-L activity for 1 h at 37 °C in 100 μl of assay buffer (50 mM Tris-HCl, pH 7.6) with or without compound. After incubation, production of hydrolyzed 7-amido-4-methyl-coumarin (AMC) groups was measured using a WALLAC Victor² 1420 Multilabel Counter with an excitation filter of 355 nm and an emission filter of 460 nm (Perkin Elmer Life Sciences, Turku, Finland). The amount of AMC released is within the linear range. Bortezomib was used as a positive control for IC₅₀ determinations (IC₅₀ of Bortezomib was typically around 10-30 nM). We used the following equation to calculate the in vitro IC₅₀ values, A=1/1+([I]/IC₅₀)ⁿ, where A is the fraction of activity remaining, [I] is the concentration of inhibitor, and n is the Hill Slope Coefficient. To determine proteasome activity in whole cell, extracts (5 μg) from cultured cell lysate was used instead of 20S rabbit proteasome, and followed the same assay mentioned above except using the following equation; Y = M + (L – M)/ (1+10^ ((X-logIC₅₀)) from GraphPad software, where Y = % inhibition, X = inhibitor concentration, M= maximum % inhibition, L = lowest % inhibition.

Cell culture and cell lysate preparation

Human MDA-MB-468 breast cancer cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) supplemented with 100 units/ml of penicillin and 100 μg/ml of streptomycin. Cells were maintained at 37 °C in a humidified incubator in an atmosphere of 5% CO₂.

Whole cell lysates were prepared as follows

Cells were harvested, washed with PBS twice, and homogenized in a lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) for 30 min at 4 °C. Cell lysates were centrifuged at 12,000 g for 15 min, and the supernatants were collected as whole cell lysates.

Dialysis using purified rabbit 20S proteasome

To measure the effect of dialysis on CT-L activity, compounds 1 (10 μM), lactacystin (2.5 μM) or vehicle (DMSO) were added to rabbit 20S proteasome at a final concentration of 1 nM in proteasome assay buffer (50 mM Tris-HCl, pH 7.6) and incubated at room temperature for 30 min. After 30 min of incubation, proteasome-compound mixtures were added to 3500 MWCO Thermo Scientific Slide-A-Lyzer Mini Dialysis Units (Rockford, IL) and dialyzed against proteasome assay buffer. Immediately (t = 0) and 0.25, 1, 2, 4, and 18 hour of dialysis at 4 °C, samples were removed from the dialysis cassette and the CT-L activity of 20S proteasome was determined as described above under “CT-L, T-L, and PGPH-L proteolytic activity assays” section. Proteasome activity was normalized against proteasome activity of DMSO control.

MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation/survival assay

Cells were plated in 96-well plates in 100 μl medium and allowed to attach overnight. Cells were then incubated for 120 h with varying concentrations of inhibitors. Media was aspirated and replaced with 100 μl complete media containing 1 mg/ml MTT and incubated for three hours at 37 °C in 5% CO₂ humidified incubator. Media was then aspirated and DMSO was added. Cells were incubated for 10 min at room temperature while shaking, and the absorbance was determined at 540 nm using a μQuant spectrophotometric plate reader (Bio-TEK, Winooski, VT). The IC₅₀ values were determined using equation under CT-L, T-L, PGPH-L proteolytic activity assays.

Protein Digestion and Peptide Purification

Rabbit 20S Proteasome (1 nM), inhibitors and buffer (50 mM Tris-Hcl, pH 7.6) were incubated (450 μl total reaction volume) for 30 min at room temperature. After incubation, 112.5 μl of acetonitrile was added and trypsin was added to quench the reaction and denature the protein. Trypsin was added with an enzyme-to-substrate ratio of 1:50. The digestion was carried out for 4 hours at 37 °C. The digest was concentrated by vacuum centrifugation (ISS110, Speedvac, Thermo), and the peptides were extracted with C18 reversed phase pipette tip columns (Ziptip, Millipore). An aliquot (25%) of the total digest was injected into mass spectrometer. To assess LC/MS-MS performance, tryptic peptides from horse apomyoglobin (25 fmol) were spiked in each LC/MS-MS analysis.

LC/MS-MS Analysis

Liquid chromatography-tandem mass spectrometry (LC/MS-MS) peptide sequencing experiments were performed using a nanoflow liquid chromatograph (U3000, Dionex, Sunnyvale, CA) interfaced with an electrospray ion trap mass spectrometer (LTQ-Orbitrap, Thermo, San Jose, CA) in order to detect and localize modified peptides from the proteasome. The sample was first loaded onto a trap column (5mm × 300 μm ID packed with C18 reversed-phase resin, 5μm particle size, 100Å pore size) and washed for 8 minutes with aqueous 2% acetonitrile and 0.04% trifluoroacetic acid. The trapped peptides were eluted onto the analytical column, (C18 Pepmap 100, 75 μm ID × 15 cm, Dionex, Sunnyvale, CA). The 120-minute gradient was programmed as: 95% solvent A (2% acetonitrile + 0.1% formic acid) for 8 minutes, solvent B (90% acetonitrile + 0.1% formic acid) from 5% to 50% in 90 minutes, then solvent B from 50% to 90% B in 7 minutes and held at 90% for 5 minutes, followed by solvent B from 90% to 5% in 1 minute and re-equilibration for 10 minutes. The flow rate on analytical column was 300 nl/min. Five tandem mass spectra were collected in a data-dependent manner following each survey scan. The MS survey scans were performed in Orbitrap to obtain accurate peptide mass measurement and the MS/MS scans were performed in linear ion trap using 60 second exclusion for previously sampled peptide peaks.

Database Searching and Data Analysis

Sequest⁵⁹ searches were performed against a database containing all rabbit proteasome protein sequences (n = 22) extracted from the UniProt (http://www.uniprot.org). Cleavage was set to fully tryptic, allowing up to two missed cleavages; the precursor mass tolerance was 1.08 Da and MS/MS mass tolerance was 0.8 Da. Dynamic modifications included oxidation (Met + 15.99492), and potential modifications on threonine (Thr +379.18957 for compound 1, Thr +394.20047 for compound 11ad and +213.10009 for β-lactone modification by lactacystin⁶⁰). The search results were summarized in Scaffold 3.0. (www.proteomesoftware.com). The integrated peak areas for peptide quantification were calculated from extracted ion chromatograms (EIC) using QuanBrowser from Xcalibur 2.0. These values were restricted by m/z (+/− 0.02) and retention time (120 seconds). The accuracy of the m/z values and the fragmentation patterns of the target peptides were manually inspected to insure proper sequence assignment.

EXPERIMENTAL

General

All reagents were purchased from commercial suppliers and used without further purification. Melting points were determined using a Barnstead international melting point apparatus or Optimelt automated melting point system (Stanford Research Systems) and remain uncorrected. Proton NMR spectra were recorded on an Agilent-Varian Mercury 400 MHz spectrometer with CDCl₃ or DMSO-d₆ as the solvent. Carbon (¹³C) NMR spectra are recorded at 100 MHz. All coupling constants are measured in Hertz (Hz) and the chemical shifts (δ_H and δ_C) are quoted in parts per million (ppm) relative to TMS (δ 0), which was used as the internal standard. Formula guided High resolution mass spectroscopy (HRMS) was carried out on an Agilent 6210 LC-MS (ESI-TOF). HPLC analysis was performed using a JASCO HPLC system equipped with a PU-2089 Plus quaternary gradient pump and a UV-2075 Plus UV-VIS detector, using an Alltech Kromasil C-18 column (150 × 4.6 mm, 5 μm) and Agilent Eclipse XDB-C18 (150 × 4.6 mm, 5 μm). Thin layer chromatography was performed using silica gel 60 F254 plates (Fisher), with observation under UV when necessary. Anhydrous solvents (acetonitrile, dimethylformamide, ethanol, isopropanol, methanol and tetrahydrofuran) were used as purchased from Aldrich. Burdick and Jackson HPLC grade solvents (methanol, acetonitrile and water) were purchased from VWR for HPLC and high resolution mass analysis. HPLC grade TFA was purchased from Fisher.

N-Isopropyl-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)-2-(p-tolyloxy)acetamide (1)

To a solution of 10a (187 mg, 0.81 mmol) and triethylamine (164 mg, 1.62 mmol) in THF (15 ml) at r.t., was added 5a (179 mg, 0.97 mmol) in THF (3 mL) dropwise (1-2 min). Upon addition of the acetyl chloride 5a, a precipitate was formed and the reaction was completed in 15 min (monitored by tlc, R_f = 0.75, TLC, EtOAc/Hexane [1:1]). The THF was evaporated and the residue was dissolved in EtOAc (20 ml), washed with HCl (4M, 2 ×15 ml) and water (15 ml). The organic phase was dried (MgSO₄), evaporated and the crude product obtained was purified by SiO₂ chromatography (EtOAc/Hexane gradient elution) to obtain 1 (270 mg, 88%) as a white solid. mp 142.1-143.4 °C. HPLC 100% (t_R = 11.8 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, J = 8.0 Hz, 2H, [7.93 minor isomer]), 7.30-7.25 (m, 2H), 7.10 (d, J = 8.3 Hz, 2H, [7.05 minor isomer]), 6.87 (d, J = 8.5 Hz, 2H, [6.82 minor isomer]), 4.78 (s, 2H, [4.84 minor isomer]), 4.70 (s, 2H, [4.83 minor isomer]), 4.45-4.39 (m, 1H), 2.40 (s, 3H, [2.42 minor isomer]), 2.28 (s, 3H, [2.25 minor isomer]), 1.29 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]); ¹H NMR (400 MHz, d₆-DMSO) δ 7.84 (d, J = 8.2 Hz, 2H, [7.87 minor isomer]), 7.36 (d, J = 7.9 Hz, 2H, [7.37 minor]), 7.01 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H, [6.75 minor isomer]), 4.88 (s, 2H, [4.98 minor isomer]), 4.71 (s, 2H, [4.82 minor isomer]), 4.31-4.21 (m, 1H, [4.62-4.52 minor isomer]), 2.37 (s, 3H), 2.18 (s, 3H), 1.26 (d, J = 6.6 Hz, 6H, [1.06 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.40 [176.54 minor isomer], 168.51, 168.56, 156.01 [155.81 minor isomer], 141.68, 131.23 [131.25 minor isomer shown], 130.29 [130.24 minor isomer], 129.68 [129.84 minor isomer shown], 127.65, 124.08, 114.68 [114.64 minor isomer], 67.99 [68.74 minor isomer], 48.96 [46.96 minor isomer], 37.20 [38.40 minor isomer], 21.49 [19.97 minor isomer], 20.81, 20.73; Anal. Calcd for C₂₂H₂₅N₃O₃: C, 69.64; H, 6.64; N, 11.07. Found: C, 69.51; H, 6.74; N, 11.13; LC-MS (ESI+) m/z 380.20 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₆N₃O₃ (M+H)⁺ 380.1969, found 380.1975.

N-Isopropyl-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)-2-(4-(trifluoromethyl)phenoxy)acetamide (11a)

This compound was prepared from 5b (149 mg, 0.62 mmol) and 10a (120 mg, 0.52 mmol) using triethylamine (105 mg, 1.04 mmol) in a similar manner as described for compound 1. The crude product was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11a (196 mg, 87%) as a white solid. mp 76.6-78.5 °C. HPLC 99.5% (t_R = 14.8 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, J = 8.1 Hz, 2H), 7.57-7.49 (m, 2H), 7.25 (d, J = 7.9 Hz, 2H, [7.29 minor isomer]),7.04 (d, J = 8.5 Hz, 2H, [6.99 minor isomer]), 4.88 (s, 2H, [4.94 minor isomer]), 4.71 (s, 2H, [4.76 minor isomer]), 4.38-4.32 (m, 1H), 2.41 (s, 3H, [2.43 minor isomer]), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100MHz, CDCl₃) δ 176.16 [176.23 minor isomer], 168.57 [167.84 minor isomer], 167.80 [167.87 minor isomer], 160.48, 141.86 [142.43 minor isomer], 129.73, 127.58, 127.29 (q, J = 3.67 Hz), 124.47 (q, J = 270 Hz), 124.11 (q, J = 32.5 Hz), 123.88 [123.86 minor isomer], 114.96 [114.82 minor isomer], 67.48 [67.90 minor isomer], 49.00 [47.05 minor isomer], 37.23 [38.35 minor isomer], 21.81, 21.45 [19.96 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.02 [−62.06 minor isomer]; LC-MS (ESI+) m/z 434.18 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₃F₃N₃O₃ (M+H)⁺ 434.1686, found 434.1711.

N-Isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-2-(4-(trifluoromethyl)phenoxy)acetamide (11b)

This compound was prepared from 5b (131 mg, 0.55 mmol) and 10d (100 mg, 0.46 mmol) using triethylamine (93 mg, 0.92 mmol) in a similar manner as described for compound 1. The crude product was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11b (172 mg, 89%) as a white solid. mp 97.9-99.3 °C. HPLC 100% (t_R = 10.4 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.02-7.98 (m, 2H), 7.55-7.43 (m, 5H), 7.04 (d, J = 8.8 Hz, 2H, [7.01 minor isomer]), 4.88 (s, 2H, [4.94 minor isomer]), 4.72 (s, 2H, [4.79 minor isomer]), 4.39-4.35 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100MHz, CDCl₃) δ 176.36 [176.47 minor isomer], 168.58 [168.54 minor isomer shown], 167.66 [166.71 minor isomer], 160.50 [160.47 minor isomer], 131.47 [131.92 minor isomer], 129.00 [129.20 minor isomer], 127.65, 127.29 (q, J = 3.74 Hz), 126.78 [126.17minor isomer], 124.46 (q, J = 270 Hz), 124.14 (q, J = 32.7 Hz), 114.97 [114.90 minor isomer], 67.56 [68.07 minor isomer], 48.96 [47.07 minor isomer], 37.21 [38.41 minor isomer], 21.48 [19.98 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.03 [−62.07 minor isomer]; LC-MS (ESI+) m/z 420.16 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₁F₃N₃O₃ (M+H)⁺ 420.1530, found 420.1547.

N-Isopropyl-2-(4-(trifluoromethyl)phenoxy)-N-((3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11c)

This compound was prepared from 5b (100 mg, 0.42 mmol) and 10b (100 mg, 0.35 mmol) using triethylamine (71 mg, 0.70 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11c (157 mg, 92%) as a white solid. mp 96.8-98.9 °C. HPLC 95% (t_R = 19.0 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H, [7.49 minor isomer]), 7.04 (d, J = 8.7 Hz, 2H, [6.97 minor isomer]), 4.88 (s, 2H, [4.91 minor isomer]), 4.72 (s, 2H, [4.84 minor isomer]), 4.42-4.38 (m, 1H), 1.34 (d, J = 6.6 Hz, 6H, [1.17 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.91, 167.87 [167.90 minor isomer], 167.60, 160.42 [160.02 minor isomer], 133.18 (q, J = 33 Hz), 130.16, 128.01 [128.12 minor isomer], 127.32 (q, J = 3.59 Hz), 126.03 (q, J = 3.81 Hz), 124.41 (q, J = 270 Hz), 124.23 (q, J = 32.59 Hz), 123.94 (q, J = 270 Hz), 114.94 [114.83 minor isomer], 67.51 [64.80 minor isomer], 49.08, 37.30, 29.94, 21.50 [19.97 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.07 [−62.02 minor isomer], −62.13 [−62.14 minor isomer], −62.45 [−62.52 minor isomer]; LC−MS (ESI+) m/z 488.14 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₀F₆N₃O₃ (M+H)⁺ 488.1403, found 488.1419.

N-((3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl)methyl)-N-isopropyl-2-(4-(trifluoromethyl)phenoxy) acetamide (11d)

This compound was prepared from 5b (115 mg, 0.48 mmol) and 10c (101 mg, 0.40 mmol) using triethylamine (81 mg, 0.80 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11d (154 mg, 85%) as a white solid. mp 79.0-80.5 °C. HPLC 100% (t_R = 16.5 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.93 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H, [7.50 minor isomer]), 7.43 (d, J = 8.6 Hz, 2H, [7.46 minor isomer]), 7.04 (d, J = 8.6 Hz, 2H, [6.99 minor isomer]), 4.88 (s, 2H, [4.92 minor isomer]), 4.70 (s, 2H, [4.80 minor isomer]), 4.41-4.35 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.60 [176.74 minor isomer], 167.81, 167.70, 160.46 [160.40 minor isomer], 137.61, 129.35 [129.54 minor isomer], 128.96, 127.31 (q, J = 3.7 Hz), 125.28, 124.45 (q, J = 270 Hz), 124.17(q, J = 32 Hz), 114.95 [114.87 minor isomer], 67.54 [68.25 minor isomer], 49.00 [ 47.16 minor isomer], 37.25 [38.50 minor isomer], 21.50 [19.98 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.03 [−62.08 minor isomer]; LC-MS (ESI+) m/z 454.12 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₀ClF₃N₃O₃ (M+H)⁺ 454.1140, found 454.1149.

2-(4-Chlorophenoxy)-N-isopropyl-N-((3-p-tolyl 1,2,4-oxadiazol-5-yl)methyl)acetamide (11e)

This compound was prepared from 5d (106 mg, 0.52 mmol) and 10a (100 mg, 0.43 mmol) using triethylamine (87 mg, 0.86 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11e (151 mg, 88%) as a white solid. mp 133.1-136.5 °C. HPLC 99.6% (t_R = 12.5 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, J = 8.3 Hz, 2H, [7.91 minor isomer]), 7.30-7.26 (m, 2H), 7.23 (d, J = 9.0 Hz, 2H, [7.20 minor isomer]), 6.86 (d, J = 9.0 Hz, 2H, [6.87 minor isomer]), 4.81 (s, 2H, [4.86 minor isomer]), 4.70 (s, 2H, [4.79 minor isomer]), 4.42-4.35 (m, 1H), 2.41 (s, 3H, [2.41 minor isomer]), 1.30 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.22 [176.34 minor isomer], 168.57, 168.01, 156.75, 141.75 [142.36 minor isomer], 129.90, 129.74 [129.72 minor isomer], 129.67, 127.61 [126.90 minor isomer], 123.98 [123.42 minor isomer], 116.24 [116.17 minor isomer], 67.95 [68.47 minor isomer], 48.93 [46.97 minor isomer], 37.17 [38.37 minor isomer], 21.82, 21.47 [19.98 minor isomer]; LC-MS (ESI+) m/z 400.15 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₃ClN₃O₃ (M+H)⁺ 400.1423, found 400.1448.

2-(4-Chlorophenoxy)-N-isopropyl-N-((3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)methyl) acetamide (11f)

This compound was prepared from 5d (86 mg, 0.42 mmol) and 10b (100 mg, 0.35 mmol) using triethylamine (71 mg, 0.70 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11f (129 mg, 81%) as a white solid. mp 106.4-108.9 °C. HPLC 99.8% (t_R = 17.1 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, J = 8.1 Hz, 2H, [8.13 minor isomer]), 7.73 (d, J = 8.2 Hz, 2H, [7.76 minor isomer]), 7.23 (d, J = 9.0 Hz, 2H, [7.18 minor isomer]), 6.91 (d, J = 9.0 Hz, 2H, [6.83 minor isomer]), 4.81 (s, 2H, [4.85 minor isomer]), 4.70 (s, 2H, [4.83 minor isomer]), 4.45-4.39 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.00 [177.20 minor isomer], 168.16, 167.64, 156.68, 133.11 (q, J = 32.6 Hz), 130.18, 129.76, 128.04, 126.96, 126.08 (q, J = 3.8 Hz), 123.96 (q, J = 270 Hz), 116.20 [116.08 minor isomer], 67.91 [68.88 minor isomer], 49.01 [47.09 minor isomer], 37.24 [38.52 minor isomer], 21.50 [19.88 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −63.40 [−63.46 minor isomer]; LC-MS (ESI+) m/z 454.11 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₀ClF₃N₃O₃ (M+H)⁺ 454.1140, found 454.1142.

2-(4-Chlorophenoxy)-N-((3-(4-chlorophenyl)-1,2,4-oxadiazol-5 yl)methyl)-N-isopropylacetamide (11g)

This compound was prepared from 5d (98 mg, 0.48 mmol) and 10c (101 mg, 0.40 mmol) using triethylamine (81 mg, 0.80 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11g (133 mg, 79%) as a white solid. mp 133.4-135.8 °C. HPLC 99.9% (t_R = 15.5 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 8.7 Hz, 2H, [7.96 minor isomer]), 7.44 (d, J = 8.6 Hz, 2H, [7.48 minor isomer]), 7.23 (d, J = 9.0 Hz, 2H, [7.19 minor isomer]), 6.91 (d, J = 9.1 Hz, 2H, [6.84 minor isomer]), 4.80 (s, 2H, [4.83 minor isomer]), 4.69 (s, 2H, [4.81 minor isomer]), 4.40-4.37 (m, 1H), 1.30 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.66 [176.83 minor isomer], 168.10, 167.78, 156.69, 137.56, 129.75, 129.36 [129.54 minor isomer], 128.99, 126.93, 125.29, 116.20 [116.11 minor isomer], 67.90 [68.68 minor isomer], 48.96, 37.20, 21.49 [19.98 minor isomer]; LC-MS (ESI+) m/z 420.08 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀₂₀Cl₂N₃O₃ (M+H)⁺ 420.0876, found 420.0891.

2-(4-Chlorophenoxy)-N-isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11h)

This compound was prepared from 5d (119 mg, 0.58 mmol) and 10d (104 mg, 0.48 mmol) using triethylamine (97 mg, 0.96 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11h (152 mg, 82%) as a white solid. mp 108.3-109.5 °C. HPLC 99.8% (t_R = 9.5 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.03−7.99 (m, 2H), 7.56−7.41 (m, 3H), 7.23 (d, J = 9.0 Hz, 2H, [7.19 minor isomer]), 6.91 (d, J = 9.0 Hz, 2H, [6.87 minor isomer]), 4.80 (s, 2H, [4.85 minor isomer]), 4.70 (s, 2H), 4.43−4.36 (m, 1H), 1.30 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.42, 168.56, 168.08, 156.73, 131.44 [131.87 minor isomer], 129.75 [129.68 minor isomer], 129.75 [129.02 minor isomer], 127.69, 126.91, 126.79, 116.23 [116.16 minor isomer], 67.92 [68.52 minor isomer], 48.97 [47.07 minor isomer], 37.19 [38.43 minor isomer], 21.47 [19.98 minor isomer]; LC-MS (ESI+) m/z 386.14 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₁ClN₃O₃ (M+H)⁺ 386.1266, found 386.1269.

N-((3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl)methyl)-N-isopropyl-2-(p-tolyloxy)acetamide (11i)

This compound was prepared from 5a (89 mg, 0.48 mmol) and 10c (101 mg, 0.40 mmol) using triethylamine (81 mg, 0.80 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11i (131 mg, 82%) as a white solid. mp 133.5-134.3 °C. HPLC 99.7% (t_R = 21.0 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.95 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H, [7.45 minor isomer]), 7.08 (d, J = 8.3 Hz, 2H, [7.03 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.79 minor isomer]), 4.78 (s, 2H, [4.86 minor isomer]), 4.69 (s, 2H, [4.81 minor isomer]), 4.46-4.40 (m, 1H), 2.28 (s, 3H, [2.25 minor isomer]), 1.30 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]); ¹³C NMR (101 MHz, CDCl₃) δ 176.85, [176.00 minor isomer], 168.56, 167.78, 156.26, 137.34, 131.26, 130.28 [130.24 minor], 129.30 [129.45 minor isomer], 129.04, 125.42, 114.67 [114.59 minor isomer], 67.98 [68.95 minor isomer], 48.96 [47.05 minor isomer], 37.22 [38.48 minor isomer], 21.50 [19.99 minor isomer], 20.74; LC-MS (ESI+) m/z 400.14 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₃ClN₃O₃ (M+H)⁺ 400.1423, found 400.1423.

N-Isopropyl-2-phenoxy-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11j)

This compound was prepared from 5c (47 mg, 0.28 mmol) and 10d checked (50 mg, 0.23 mmol) using triethylamine (47 mg, 0.46 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11j (61 mg, 76%). as a white solid. mp 77.9-79.0 °C. HPLC 94.4% (t_R = 11.6 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.02 (dd, J = 8.1, 1.6 Hz, 2H, partially overlapped, [8.05 minor isomer, overlapped]), 7.53-7.43 (m, 3H), 7.32-7.25 (m, 2H), 7.03-6.85 (m, 3H), 4.83 (s, 2H, [4.88 minor isomer]), 4.72 (s, 2H, [4.86 minor isomer]), 4.46-4.40 (m, 1H), 1.31 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.53 [176.65 minor isomer], 168.72 [168.80 minor isomer], 168.53, 158.01 [157.85 minor isomer], 156.40, 131.44 [131.81 minor isomer], 129.88 [129.82 minor isomer], 129.79, 129.00 [129.17 minor isomer], 127.73 [127.69 minor isomer], 127.04, 126.78, 122.00, 116.21, 114.86 [114.81 minor isomer shown], 67.66 [68.34 minor isomer shown], 49.09 [47.12 minor isomer], 37.28 [38.45 minor isomer], 21.43 [19.95 minor isomer]; LC-MS (ESI+) m/z 352.16 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₂N₃O₃ (M+H)⁺ 352.1656, found 352.1662.

N-Isopropyl-2-phenoxy-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11k)

This compound was prepared from 5c (80 mg, 0.47 mmol) and 10a (90 mg, 0.39 mmol) using triethylamine (78 mg, 0.77 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11k (111 mg, 78%) as a white solid. mp 97.8-99.5 °C. HPLC 99.8% (t_R = 8.5 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, J = 8.2 Hz, 2H, [7.93 minor isomer shown]), 7.34-7.22 (m, 4H), 7.02-6.94 (m, 3H), 4.82 (s, 2H [4.87 minor isomer]), 4.71 (s, 2H, [4.83 minor isomer]), 4.45-4.39 (m, 1H), 2.41 (s, 3H [2.42 minor isomer]), 1.30 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.49 [176.50 minor isomer], 168.34, 168.55, 158.08, 141.68 [142.23 minor isomer],129.86 [129.81 minor isomer],129.68, 127.65, 124.01, 121.95, 114.86 [114.83 minor isomer], 67.81 [68.49 minor isomer], 48.96 [46.89 minor isomer], 37.21 [38.37 minor isomer], 21.82, 21.48 [19.98 minor isomer]; LC-MS (ESI+) m/z 366.19 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₄N₃O₃ (M+H)⁺ 366.1812, found 366.1816.

N-Isopropyl-2-phenoxy-N-((3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11l)

This compound was prepared from 5c (57 mg, 0.34 mmol) and 10b (80 mg, 0.28 mmol) using triethylamine (57 mg, 0.56 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11l (95 mg, 81%) as a white solid. mp 122.8-123.8 °C. HPLC 100% (t_R = 11.6 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J = 8.1 Hz, 2H, [8.15 minor isomer]), 7.72 (d, J = 8.2 Hz, 2H, [7.75 minor isomer]), 7.35-7.26 (m, 2H), 7.03-6.94 (m, 3H, [6.90 minor isomer]), 4.83 (s, 2H, [4.89 minor isomer]), 4.72 (s, 2H, [4.86 minor isomer]), 4.48-4.43 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃ ) δ 177.15 [177.39 minor isomer], 168.45, 167.58, 158.04, 133.07 (q, J = 32.6 Hz), 130.29 [130.27 minor isomer shown], 129.87 [129.83 minor isomer],128.09, 125.97 (q, J = 3.7 Hz), 123.99 (q, J = 271 Hz), 121.99 [122.07 minor isomer], 114.83 [114.75 minor isomer], 67.75 [68.75 minor isomer], 49.01 [47.05 minor isomer], 37.27 [38.58 minor isomer], 21.50 [19.98 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −63.37, [−63.43 minor isomer]; LC-MS (ESI+) m/z 420.15 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₁F₃N₃O₃ (M+H)⁺ 420.1530, found 420.1530.

N-Isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-2-(p-tolyloxy)acetamide (11m)

This compound was prepared from 5a (102 mg, 0.55 mmol) and 10d (100 mg, 0.46 mmol) using triethylamine (93 mg, 0.92 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11m (153 mg, 91%). as a white solid. mp 134.7-136.5 °C. HPLC 96.3% (t_R = 8.5 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.02 (dd, J = 8.0, 1.6 Hz, 2H overlapped, [8.04 minor isomer overlapped]), 7.55-7.41 (m, 3H), 7.08 (d, J = 8.3 Hz, 2H, [7.04 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.81 minor isomer]), 4.78 (s, 2H, [4.85 minor isomer]), 4.71 (s, 2H, [4.83 minor isomer]), 4.45-4.40 (m, 1H), 2.28 (s, 3H, [2.25 minor isomer]), 1.30 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.62, 168.55, 168.53, 156.00 [155.80 minor isomer], 131.37 [131.74 minor isomer], 131.22 [131.25 minor isomer], 130.28 [130.24 minor isomer], 128.97 [129.13 minor isomer], 127.73, 126.89, 114.68 [114.62 minor isomer], 67.97 [68.75 minor isomer], 48.93 [46.94 minor isomer], 37.20 [38.44 minor isomer], 21.48, [19.98 minor isomer], 20.74; LC-MS (ESI+) m/z 366.18 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₄N₃O₃ (M+H)⁺ 366.1812, found 366.1828.

N-Isopropyl-2-(p-tolyloxy)-N-((3-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11n)

This compound was prepared from 5a (78 mg, 0.42 mmol) and 10b (100 mg, 0.35 mmol) using triethylamine (71 mg, 0.70 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11n (127 mg, 84%) as a white solid. mp 146.9-148.6 °C. HPLC 99% (t_R = 16.1 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H, overlapped, [7.75 minor isomer, overlapped]), 7.08 (d, J = 8.3 Hz, 2H, [7.02 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.77 minor isomer]), 4.79 (s, 2H, [4.89 minor isomer]), 4.70 (s, 2H, [4.81 minor isomer]), 4.48-4.42 (m, 1H), 2.28 (s, 3H, [2.23 minor isomer]), 1.31 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.20 [177.43 minor isomer], 168.63, 167.57, 155.98 [155.73 minor isomer], 133.05 (q, J = 32.5 Hz), 131.27 [131.37 minor isomer], 130.29 [130.24 minor isomer], 128.09, 125.97 (q, J = 3.8 Hz), 124.0 (q, J = 270.6 Hz), 114.65 [114.54 minor isomer], 67.94 [68.85 minor isomer], 48.97 [47.08 minor isomer], 37.26 [38.63 minor isomer], 21.50 [19.62 minor isomer], 20.74; ¹⁹F NMR (376 MHz, CDCl₃) δ −63.39 [−63.45 minor isomer]; LC-MS (ESI+) m/z 434.18 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₃F₃N₃O₃ (M+H)⁺ 434.1686, found 434.1693.

N-Isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-2-(m-tolyloxy)acetamide (11o)

This compound was prepared from 5h (66 mg, 0.36 mmol) and 10d (65 mg, 0.30 mmol) using triethylamine (61 mg, 0.60 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11o (83 mg, 76%) as a colorless viscous compound; HPLC 98.8% (t_R = 8.6 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.01 (dd, J = 8.0, 1.5 Hz, 2H, [8.08 minor isomer]), 7.52-7.42 (m, 4H), 7.17 (t, J = 7.6 Hz, 1H, overlapped, [7.15, minor isomer])), 6.82-6.69 (m, 2H), 4.79 (s, 2H, [4.85 minor isomer]), 4.71 (s, 2H, [4.84 minor isomer]), 4.46-4.40 (m, 1H), 2.31 (s, 3H, [2.25 minor isomer]), 1.31 (d, J = 6.6 Hz, 6H, [1.16 minor isomer shown]); ¹³C NMR (100 MHz, CDCl₃) δ 176.58, 168.56, 168.43, 158.09, 139.99, 131.36 [131.74 minor isomer], 129.58 [129.53 minor isomer], 128.96 [129.13 minor isomer], 127.72, 126.90, 122.80 [122.84 minor isomer], 115.64 [115.74 minor isomer], 111.70 [111.46 minor isomer], 67.79 [68.59 minor isomer], 48.97 [46.97 minor isomer shown], 37.22 [38.52 minor isomer], 21.76, 21.49 [19.98 minor isomer shown]; LC-MS (ESI+) m/z 366.19 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₄N₃O₃ (M+H)⁺ 366.1812, found 366.1817.

N-Isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-2-(o-tolyloxy)acetamide (11p)

This compound was prepared from 5i (51 mg, 0.28 mmol) and 10d (50 mg, 0.23 mmol) using triethylamine (57 mg, 0.56 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11p (71 mg, 85%) as a white solid. mp 97.3-98.0 °C. HPLC 98.0% (t_R = 9.5 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.95 (broad dd, J = 7.9, 1.6 Hz, 2H), 7.51-7.43 (m, 3H), 7.17-7.09 (m, 2H), 6.92-6.87 (m, 2H), 4.82 (s, 2H, [4.81 minor isomer]), 4.71 (s, 2H), 4.48-4.38 (m, 1H), 2.28 (s, 3H, [2.20 minor isomer]), 1.29 (d, J = 6.6 Hz, 6H, [1.14 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.57 [176.68 minor isomer], 168.57, 168.50, 156.23, 131.38 [131.77 minor isomer], 131.18, 128.98 [129.15 minor isomer], 127.73, 127.25, 126.87, 126.84, 121.61 [121.69 minor isomer], 111.28 [111.63 minor isomer], 68.01 [68.86 minor isomer], 48.94 [48.90 minor isomer], 37.22 [38.26 minor isomer], 21.51 [20.07 minor isomer], 16.62; LC-MS (ESI+) m/z 366.18 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₄N₃O₃ (M+H)⁺ 366.1812, found 366.1821.

2-(Biphenyl-4-yloxy)-N-isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11q)

This compound was prepared from 5e (116 mg, 0.47 mmol) and 10d (85 mg, 0.39 mmol) using triethylamine (79 mg, 0.78 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11q (145 mg, 87%) as a white solid. mp 147.8-148.7 °C. HPLC 99.6% (t_R = 14.6 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.02 (dd, J = 8.2, 1.4 Hz, 2H), 7.55-7.36 (m, 9H), 7.31 (t, J = 7.4 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H, [7.00 minor isomer]), 4.87 (s, 2H, [4.91 minor isomer]), 4.73 (s, 2H), 4.48-4.42 (m, 1H), 1.33 (d, J = 6.6 Hz, 6H, [1.17 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.55 [176.69 minor isomer], 168.58, 168.30 [168.38 minor isomer], 157.63 [157.49 minor isomer], 140.78, 135.04 [135.07 minor isomer], 131.36 [131.77 minor isomer], 128.98 [129.16 minor isomer], 128.94, 128.53, 127.71, 127.04, 127.01, 126.88, 115.17 [115.10 minor isomer], 67.86 [68.62 minor isomer], 48.98 [47.02 minor isomer], 37.22 [38.50 minor isomer], 21.51 [20.00 minor isomer shown]; LC-MS (ESI+) m/z 428.19 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₆H₂₆N₃O₃ (M+H)⁺ 428.1969, found 428.1968.

2-(6-Bromonaphthalen-2-yloxy)-N-isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11r)

This compound was prepared from 5g (140 mg, 0.47 mmol) and 10d (85 mg, 0.39 mmol) using triethylamine (79 mg, 0.78 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11r (141 mg, 75%) as a white solid. mp 97.7-98.4 °C. HPLC 98.1% (t_R = 19.6 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.99-7.86 (m, 3H), 7.67 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.53-7.36 (m, 4H), 7.24 (dd, J = 9.0, 2.6 Hz, 1H), 7.19 (d, J = 2.3 Hz, 1H), 4.93 (s, 2H, [4.98 minor isomer]), 4.72 (s, 2H, [4.86 minor isomer]), 4.52-4.46 (m, 1H), 1.33 (d, J = 6.6 Hz, 6H, [1.18 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.47, 168.59, 168.14, 156.26, 133.02, 131.80, 131.36 [131.40 minor isomer], 130.63, 130.00 [129.94 minor isomer], 129.82, 129.03 [129.14 minor isomer], 128.94, 127.63 [127.68 minor isomer], 126.75, 119.65 [119.49 minor isomer], 117.90, 107.72, 67.89 [68.50 minor isomer], 49.03 [47.10 minor isomer], 37.23 [38.65 minor isomer], 21.53 [19.99 minor isomer]; LC-MS (ESI+) m/z 480.09 and 482.09 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₄H₂₃BrN₃O₃ (M+H)⁺ 480.0917, found 480.0914.

2-(4-Fluorophenoxy)-N-isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11s)

This compound was prepared from 5f (95 mg, 0.50 mmol) and 10d (91 mg, 0.42 mmol) using triethylamine (85 mg, 0.84 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11s (127 mg, 82%) as a white solid. mp 87.9-89.9 °C. HPLC 99.5 % (t_R = 16.00 min, 50% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.00 (dd, J = 8.1, 1.6 Hz, 2H, [8.04 minor isomer]), 7.54-7.44 (m, 3H), 7.02-6.88 (m, 4H), 4.80 (s, 2H, [4.85 minor isomer]), 4.72 (s, 2H, [4.83 minor isomer]), 4.44-4.38 (m, 1H), 1.31 (d, J = 6.7 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.49 [177.63 minor isomer], 168.82 [168.56 minor isomer], 168.24, 157.98 (d, J = 238 Hz), 154.24 (d, J = 2.07 Hz), 131.44 [131.86 minor], 129.00 [129.19 minor isomer], 127.69, 126.82, 116.34 (d, J = 23 Hz), 116.03 (d, J = 8.11 Hz), 68.30 [68.92 minor isomer], 48.91 [46.94 minor isomer], 37.18 [38.39 minor isomer], 21.46 [19.97 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −123.13; LC-MS (ESI+) m/z 370.15 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₁FN₃O₃ (M+H)⁺ 370.1562, found 370.1567.

N-Isopropyl-(3-phenyl-1,2,4-oxadiazol-5-yl)methylamino-2-oxoethoxy)benzoic acid (11t)

A solution of 11aq (50 mg, 0.11 mmol) in trifluoroacetic acid (3 ml) and dichloromethane (5 ml) were stirred at room temperature for 2 h. Acetone (5 ml) was added to the reaction mixture and the solvents were evaporated under vacuum to give the pure compound 11t (41 mg, 95%) as a white compound. mp 211.3-213.8 °C. HPLC 100 % (t_R = 14.8 min, 40% MeOH in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, DMSO) δ 12.58 (brs, 1H), 7.97-7.90 (m, 2H, [8.03 -7.98 minor isomer]), 7.82 (d, J = 8.8 Hz, 2H), 7.58-7.53 (m, 3H), 6.96 (d, J = 8.8 Hz, 2H, [6.99 minor isomer]), 5.09 (s, 2H, [5.02 minor isomer]), 4.75 (s, 2H, [5.00 minor isomer]), 4.32-4.20 (m, 1H, [4.66-4.60 minor isomer]), 1.28 (d, J = 6.5 Hz, 6H, [1.07 minor isomer]). ¹³C NMR (100 MHz, CDCl₃) δ 176.32, 171.41, 168.58, 167.80, 162.38, 132.70 [132.63 minor isomer], 131.48 [131.94 minor isomer], 129.22, 129.01 [129.22 minor isomer], 127.68 [126.71 minor isomer], 122.97 [122.90 minor isomer], 114.71 [114.68 minor isomer], 67.46 [67.96 minor isomer], 49.07 [47.24 minor isomer], 37.24 [38.43 minor isomer], 21.46 [19.96 minor isomer]; LC-MS (ESI+) m/z 396.15 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₂N₃O₅ (M+H)⁺ 396.1554, found 396.1566.

2-(4-Hydroxyphenoxy)-N-isopropyl-N-((pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11u)

To a solution of 10f (59 mg, 0.27 mmol) and triethylamine (55 mg, 0.54 mmol) in THF (10 ml) at r.t., was added 2-(4-hydroxyphenoxy)acetyl chloride (5s) (60 mg, 0.32 mmol) in THF (1 ml, dropwise 3-4 min). Upon addition of acetyl chloride, a precipitate was formed and the reaction was completed in 15 min. (monitored by tlc, R_f = 0.50, EtOAc/Hexane [2:1]). The THF was evaporated and the crude product obtained was purified by SiO₂ chromatography (EtOAc/Hexane gradient elution) to obtain pure 11u (83 mg, 83%) as a white solid. mp 140.0-142.4 °C. HPLC 95.5% (t_R = 5.47 min, 45% MeOH in 0.1% TFA water 20 min); The ¹H NMR showed 5:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.11 (s, 1H, [9.23 minor isomer]), 8.70 (dd, J = 4.8, 1.4 Hz, 1H, [8.74 minor isomer]), 8.30 (dt, J = 8.0, 1.9 Hz, 1H), 7.43 (dd, J = 7.9, 4.8 Hz, 1H), 6.88 (d, J = 9.1 Hz, 2H), 6.80 (d, J = 9.1 Hz, 2H), 4.78 (s, 2H, [4.88 minor isomer]), 4.69 (s, 2H), 4.60-4.52 (m, 1H), 1.29 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.39, 169.00, 166.13, 152.05, 151.05, 148.10, 135.85, 124.36, 117.00 [116.45 minor isomer] 115.97, 68.37, 48.88, 37.08, 21.49 [19.99 minor isomer]; LC-MS (ESI+) m/z 369.15 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₁₉H₂₁N₄O₄ (M+H)⁺ 369.1557, found 369.1571.

2-(4-Hydroxyphenoxy)-N-isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11v)

This compound was prepared from 5s (52 mg, 0.28 mmol) and 10d (50 mg, 0.23 mmol) using triethylamine (47 mg, 0.46 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11v (68 mg, 80%) as a white solid. mp 155.5-158.5 °C. HPLC 100% (t_R = 13.1 min, 40% CH₃CN in 0.1% TFA water 20 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.01 (dd, J = 7.8, 1.7 Hz, 2H, [8.04 minor isomer]), 7.51-7.43 (m, 3H), 6.85 (d, J = 9.0 Hz, 2H, [6.80 minor isomer]), 6.73 (d, J = 9.0 Hz, 2H, [6.70 minor isomer]), 4.75 (s, 2H, [4.85 minor isomer]), 4.71 (s, 2H, [4.80 minor isomer]), 4.48-4.41 (m, 1H), 1.30 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]); ¹³C NMR (100 MHz, DMSO) δ 178.76 [178.92 minor isomer], 168.57 [168.67 minor isomer], 168.12 [168.25 minor isomer], 152.16 [152.02 minor isomer], 151.45, 150.38, 132.27 [132.45 minor isomer], 129.97 [130.02 minor isomer], 127.61 [127.69 minor isomer], 126.78 [126.55 minor isomer], 116.21 [116.30minor isomer], 116.16 [116.13 minor isomer], 67.33 [67.42 minor isomer], 48.31, 37.77, 21.34 [19.94 minor isomer]. LC-MS (ESI+) m/z 368.16 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₂N₃O₄ (M+H)⁺ 368.1605, found 368.1615.

N-Isopropyl-N-((3-(pyridin-2-yl)-1,2,4-oxadizaol-5-yl)methyl)-2-(p-tolyloxy)acetamide (11w)

This compound was prepared from 5a (43 mg, 0.23 mmol) and 10e (42 mg, 0.19 mmol) using triethylamine (39 mg, 0.38 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11w (52 mg, 75%) as a white solid. mp 125.7-126.9 °C; HPLC 96.2% (t_R = 15.7 min, 40% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.79 (d, J = 4.7 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.82 (td, J = 7.8, 1.7 Hz, 1H), 7.42 (appdd, J = 6.7, 4.9 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H, [7.03 minor isomer]), 6.87 (d, J = 8.5 Hz, 2H, [6.80 minor isomer]), 4.79 (s, 2H, [4.93 minor isomer]), 4.77 (s, 2H, [4.81 minor isomer]), 4.46-4.38 (m, 1H), 2.29 (s, 3H, [2.24 minor isomer]), 1.29 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.38, 168.57, 168.37, 155.96, 150.60 [150.73 minor isomer], 146.46, 137.19, 131.25, 130.30, 125.72, 123.51, 114.66, 67.98 [68.99 minor isomer], 49.01, 37.24, 21.44 [19.95 minor isomer], 20.72; LC-MS (ESI+) m/z 367.17 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₃N₄O₃ (M+H)⁺ 367.1765, found 367.1774.

N-Isopropyl-N-((3-(pyridin-3-yl)-1,2,4-oxadizaol-5-yl)methyl)-2-(p-tolyloxy)acetamide 11x

This compound was prepared from 5a (51 mg, 0.28 mmol) and 10f (50 mg, 0.23 mmol) using triethylamine (47 mg, 0.46 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11x (69 mg, 82%) as a white solid. mp 126.5-128.3 °C. HPLC 98.3% (t_R = 10.6 min, 35% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.24 (s, 1H), 8.72 (appdd, J = 4.8, 1.2 Hz, 1H, [8.75 minor isomer]), 8.27 (dt, J = 7.9, 1.6 Hz, 1H), 7.39 (dd, J = 8.0, 4.9 Hz, 1H, [7.43 minor isomer]), 7.08 (d, J = 8.6 Hz, 2H, [7.02 minor isomer]), 6.86 (d, J = 8.5 Hz, 2H, [6.77 minor isomer]), 4.78 (s, 2H, [4.88 minor isomer]), 4.70 (s, 2H, [4.78 minor isomer]), 4.49-4.39 (m, 1H), 2.27 (s, 3H, [2.22 minor isomer]), 1.31 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.23, 168.60, 166.65, 155.94 [155.72 minor isomer], 152.24 [152.52 minor isomer], 148.92 [148.87 minor isomer], 134.97, 131.30, 130.28, 123.79 [123.86 minor isomer], 123.20, 114.64 [114.53 minor isomer], 67.91 [69.02 minor isomer], 48.99 [48.96 minor isomer], 37.27 [38.66 minor isomer], 21.52 [19.99 minor isomer], 20.72; LC-MS (ESI+) m/z 367.17 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₃N₄O₃ (M+H)⁺ 367.1765, found 367.1774.

N-Isopropyl-N-((3-(pyridin-4-yl)-1,2,4-oxadizaol-5-yl)methyl)-2-(p-tolyloxy)acetamide (11y)

This compound was prepared from 5a (21 mg, 0.12 mmol) and 10g (22 mg, 0.10 mmol) using triethylamine (19 mg, 0.19 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11y (29 mg, 78%) as a white solid. mp 150.6-151.7 °C. HPLC 96.6% (t_R = 14.7 min, 30% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.75 (d, J = 4.8 Hz, 2H), 7.87 (d, J = 4.6 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H, [7.02 minor isomer]), 6.87 (d, J = 8.5 Hz, 2H, [6.76 minor isomer]), 4.80 (s, 2H, [4.90 minor isomer]), 4.70 (s, 2H, [4.79 minor isomer]), 4.51-4.40 (m, 1H), 2.29 (s, 3H, [2.23 minor isomer]), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.57, 168.65, 167.05, 155.94, 150.79 [150.91 minor isomer], 134.35, 131.30, 130.29, 121.57, 114.64 [114.50 minor isomer], 67.91 [69.08 minor isomer], 49.01[48.97 minor isomer], 37.27 [38.61 minor isomer], 21.52 [19.98 minor isomer], 20.72; LC-MS (ESI+) m/z 367.18 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₃N₄O₃ (M+H)⁺ 367.1765, found 367.1780.

N-Isopropyl-2-(6-methylpyridin-3-yloxy)-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11z)

A solution of 6-methyl-3-hydroxypyridin (80 mg, 0.73 mmol), N-isopropyl-2-chloro-N-((3-phenyl-1,2,4-oxodiazol-5-yl)methyl)acetamide (15) (214 mg, 0.73 mmol) and potassium carbonate (510 mg, 3.67 mmol) in acetonitrile (25 ml) were refluxed for 14 h. The solvent was evaporated; and the residue was dissolved in ethyl acetate (20 ml), washed with water (2 × 20 ml). The organic phase was dried (MgSO₄), evaporated and the crude compound obtained was purified by SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford 11z as (193 mg, 72%) a white solid. mp 130.3-132.2 °C. HPLC 97.26% (t_R = 8.3 min, 30% CH₃CN in 0.1% TFA water 30 min); ¹H NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 2.9 Hz, 1H, [8.26 minor isomer]), 8.00 (dd, J = 7.9, 1.6 Hz, 1H, [8.04 minor isomer]), 7.52-7.44 (m, 4H), 7.20 (d, J = 8.5 Hz, 1H), 4.93 (s, 2H, [5.01 minor isomer]), 4.89-4.80 (m, 1H), 4.72 (s, 2H, [4.78 minor isomer]), 4.30-4.23 (m, 1H, [4.89-4.80 minor isomer]), 2.63 (s, 3H), 1.35 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.25, 168.58, 166.98, 153.61, 149.71, 131.50 [131.94 minor isomer], 129.26, 129.04, 127.69, 126.73, 125.54, 67.68, 48.90 [47.05 minor isomer], 37.21, 21.61, 21.51 [19.99 minor isomer]; LC-MS (ESI+) m/z 367.18 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₃N₄O₃(M+H)⁺ 367.1765, found 367.1759.

2-(4-Ethylphenoxy)-N-isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11aa)

This compound was prepared from 5j (93 mg, 0.47 mmol) and 10d (85 mg, 0.39 mmol) using triethylamine (79 mg, 0.78 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11aa (123 mg, 83%) as a white solid. mp 106.7-109.2 °C. HPLC 95.5% (t_R = 11.5 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 2:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.03 (dd, J = 7.9, 1.4 Hz, 2H), 7.55-7.38 (m, 3H), 7.11 (d, J = 8.4 Hz, 2H, [7.07 minor isomer]), 6.90 (d, J = 8.5 Hz, 2H, [6.84 minor isomer]), 4.79 (s, 2H, [4.86 minor isomer]), 4.71 (s, 2H, [4.83 minor isomer]), 4.46-4.40 (m, 1H), 2.59 (q, J = 7.5 Hz, 2H, [2.55 minor isomer]), 1.31 (d, J = 6.6 Hz, 6H, [1.16 minor isomer partially overlapped]), 1.20 (t, J = 7.6 Hz, 3H, partially overlapped with the doublet at 1.16); ¹³C NMR (100 MHz, CDCl₃) δ 176.62 [176.77 minor isomer], 168.56 [168.66 minor isomer], 168.55 [168.65 minor isomer], 156.14 [155.94 minor isomer], 137.73, 131.38 [δ 131.75 minor isomer], 129.11, 128.98, 127.73, 126.90, 114.72 [114.65 minor isomer], 67.97 [68.74 minor isomer], 48.98, 37.23 [38.44 minor isomer], 28.21, 21.48 [19.99 minor isomer], 16.01: LC-MS (ESI+) m/z 380.21 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₆N₃O₃ (M+H)⁺ 380.1969, found 380.1964.

2-(4-Ethylphenoxy)-N-isopropyl-N-((3-pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methylacetamide (11ab)

This compound was prepared from 5j (50 mg, 0.25 mmol) and 10f (46 mg, 0.21 mmol) using triethylamine (43 mg, 0.42 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ab (65 mg, 81%) as a white solid. mp 104.4-106.3 °C. HPLC 95.7% (t_R = 5.3 min, 50% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.21 (s, 1H), 8.67 (brs, 1H), 8.23 (d, J = 7.7 Hz, 1H), 7.42-7.29 (broad m, 1H), 7.04 (d, J = 8.5 Hz, 2H, [6.98 minor isomer]), 6.82 (d, J = 8.5 Hz, 2H, [6.73 minor isomer]), 4.72 (s, 2H, [4.82 minor isomer]), 4.64 (s, 2H, [4.74 minor isomer]), 4.40-4.32 (m, 1H), 2.51 (q, J = 7.6 Hz, 2H partially overlapped, [2.47 minor isomer]), 1.25 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]), 1.11 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 177.26, [ 177.46 minor isomer], 168.59, [168.65 minor isomer], 166.65, [166.87 minor isomer], 156.09, 152.11, 148.82, 137.79, 135.02, 129.10, 114.68 [114.57 minor isomer], 67.97 [69.01 minor isomer], 49.00 [46.98 minor isomer], 37.29 [38.61 minor isomer], 28.19 [29.93 minor isomer], 21.49 [19.99 minor isomer], 16.00; LC-MS (ESI+) m/z 381.19 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₅N₄O₃(M+H)⁺ 381.1921, found 381.1912.

N-Isopropyl-2-phenoxy-N-((3-(pyridine-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide-(11ac)

This compound was prepared from 5c (55 mg, 0.32 mmol) and 10f (59 mg, 0.27 mmol) using triethylamine (55 mg, 0.54 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ac (81 mg, 85%) as a white solid. mp 83.0-85.5 °C. HPLC 97.5 % (t_R = 11.4 min, 30% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.29 (s, 1H), 8.79 (brs, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.45 (m, 1H), 7.33-7.22 (m, 2H), 7.02-6.87 (m, 3H), 4.82 (s, 2H, [4.89 minor isomer]), 4.71 (s, 2H, [4.84 minor isomer]), 4.51-4.37 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.21 [177.41 minor isomer]), 168.43, 166.62, [166.87 minor isomer], 158.01 [157.83 minor isomer], 152.20 [152.53 minor isomer], 148.85, 134.99, 129.85, 123.83, 123.19, 121.98, 114.81 [114.71 minor isomer], 67.65 [68.66 minor isomer], 48.97 [47.08 minor isomer], 37.28 [38.61 minor isomer], 21.49 [19.97 minor isomer]; LC-MS (ESI+) m/z 353.16 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₁₉H₂₁N₄O₃(M+H)⁺ 353.1608, found 353.1614.

N-Isopropyl-2-(4 propylphenoxy)-N-((3-pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11ad)

This compound was prepared from 5k (84 mg, 0.40 mmol) and 10f (72 mg, 0.33 mmol) using triethylamine (67 mg, 0.66 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ad (109 mg, 84%) as a white solid. mp 103.2-105.5 °C. HPLC 100% (t_R = 7.58 min, 50% CH₃CN in 0.1% TFA water 20 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.26 (s, 1H), 8.78-8.70 (m, 1H), 8.29 (dt, J = 8.0 Hz, J = 2.0 Hz, 1H), 7.42 (appdd, J = 8.0, 4.9 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H, [ 7.03 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.79 minor isomer]), 4.78 (s, 1H), 4.79 (s, 2H, [4.89 minor isomer]), 4.70 (s, 2H, [4.81 minor isomer shown]), 4.48-4.38 (m, 1H), 2.51 (t, J = 7.7 Hz, 2H, [2.47 minor isomer]), 1.63-1.52 (m, 2H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]), 0.91 (t, J = 7.3 Hz, 3H, [0.89 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.06 [177.24 minor isomer], 168.34 [168.40 minor isomer], 166.33 [166.57 minor isomer], 155.86 [155.62 minor isomer], 151.74 [152.16 minor isomer], 148.44 [148.50 minor isomer]), 135.98 [136.05 minor isomer], 134.93[134.83 minor isomer], 129.45 [129.41 minor isomer], 123.66 [123.09 minor isomer], 114.34 [114.24 minor isomer], 67.62 [68.73 minor isomer], 48.74 [46.81 minor isomer], 37.11 [38.41 minor isomer], 37.06, 24.69 [23.47 minor isomer], 21.26 [19.74 minor isomer], 13.78; LC-MS (ESI+) m/z 395.21 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₇N₄O₃ (M+H)⁺ 395.2078, found 395.2080.

2-(4-Butylphenoxy)-N-isopropyl-N-((3-(pyridine-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11ae)

This compound was prepared from 5l (76 mg, 0.34 mmol) and 10f (61 mg, 0.28 mmol) using triethylamine (57 mg, 0.56 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ae (94 mg, 82%) as a white solid. mp 94.5-95.2 °C. HPLC 98.13% (t_R = 10.2 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.29 (s, 1H, [9.26 minor isomer overlapped]), 8.80-8.73 (m, 1H), 8.46 (d, J = 7.7 Hz, 1H, [8.33 minor isomer]), 7.56-7.52 (m, 1H [7.50 minor isomer]), 7.09 (d, J = 8.6 Hz, 2H, [7.02 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.78 minor isomer]), 4.78 (s, 2H, [4.90 minor isomer]), 4.70 (s, 2H, [4.80 minor isomer]), 4.47-4.40 (m, 1H), 2.54 (t, J = 7.3 Hz, 2H, [2.48 minor isomer]), 1.59-1.46 (m, 2H), 1.36-1.29 [m, 8H and around 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]), 0.91 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 177.23, 168.58, 166.66, 156.07, 152.24 [152.54 minor isomer]), 148.93, 136.46, 134.97, 129.64, 123.80, 123.21, 114.59 [114.50 minor isomer]), 67.91, 48.97, 37.29, 34.95, 34.03, 22.53, 21.50 [19.99 minor isomer], 14.20; LC-MS (ESI+) m/z 409.23 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₃H₂₉N₄O₃(M+H)⁺ 409.2234, found 409.2238.

N-Isopropyl-2-(4-pentylphenoxy)-N-((3-pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11af)

This compound was prepared from 5m (78 mg, 0.32 mmol) and 10f (59 mg, 0.27 mmol) using triethylamine (55 mg, 0.54 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11af (95 mg, 83%) as a white solid. mp 93.0-95.9 °C. HPLC 96.5 % (t_R = 17.1 min, 70% MeOH in 0.1% TFA water 20 min); ¹H NMR (400 MHz, CDCl₃) δ 9.19 (s, 1H, [9.24 minor isomer]), 8.67 (appdd, J = 4.9, 1.6 Hz, 1H), 8.24-8.22 (m, 1H, [8.30 minor isomer]), 7.37-7.28 32 (m, 1H), 7.02 (d, J = 8.4 Hz, 2H, [6.95 minor isomer]), 6.81 (d, J = 8.4 Hz, 2H, [6.72 minor isomer]), 4.72 (s, 2H, [4.83 minor isomer]), 4.64 (s, 2H, [4.74 minor isomer]), 4.40-4.33 (m, 1H), 2.46 (t, J = 7.8 Hz, 2H, partially overlapped [2.41 minor isomer partially overlapped]), 1.52-1.41 (m, 2H), 1.36-1.13 [broad m, 10H, and around 1.25 (d, J = 6.8 Hz, 6H)] ), 0.81 (t, J = 6.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 177.29 [177.50 minor isomer], 168.62, [168.67 minor isomer], 166.56 [ 166.79 minor isomer], 156.07 [155.82 minor isomer], 152.19, 151.88 [152.19 minor isomer], 148.60, 136.51 [136.33 minor isomer], 135.25, 129.63 [129.59 minor isomer], 123.97, 114.60 [114.50 minor isomer], 67.90 [69.04 minor isomer], 49.00 [47.08 minor isomer], 37.30 [38.65 minor isomer], 35.24, 31.69, 31.57 [29.93 minor], 22.77, 21.51 [19.99 minor isomer], 14.29; LC-MS (ESI+) m/z 423.24 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₄H₃₁N₄O₃(M+H) 423.2391, found 423.2393.

2-(4-Hexylphenoxy)-N-isopropyl-N-((3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11ag)

This compound was prepared from 5n (98 mg, 0.38 mmol) and 10f (70 mg, 0.32 mmol) using triethylamine (65 mg, 0.64 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ag (122 mg, 87%) as a white solid. mp 94.4-96.1 °C. HPLC 93.8 % (t_R = 7.7 min, 80% MeOH in 0.1% TFA water 20 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.28 (s, 1H), 8.75 (brs, 1H), 8.33 (appd, J = 7.9 Hz, 1H, [8.29 minor isomer]), 7.50-7.43 (m, 1H), 7.09 (d, J = 8.6 Hz, 2H, [7.03 minor isomer]), 7.87 (d, J = 8.7 Hz, 2H, [6.79 minor isomer]), 4.79 (s, 2H, [4.90 minor isomer]), 4.71 (s, 2H, [4.81 minor isomer]), 4.49-4.36 (m, 1H), 2.53 (t, J = 7.8 Hz, 2H partially overlapped, [2.48 minor isomer partially overlapped]), 1.59-1.51 (m, 2H), 1.33-1.27 (m, 12H and around 1.32 [d, J = 6.6 Hz, 6H, [1.16 minor isomer]), 0.91-0.83 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 177.32 [177.51 minor isomer], 168.58, [168.64 minor isomer], 166.54, 156.07 [155.82 minor isomer], 151.91 [152.38 minor isomer], 148.59 [148.68 minor isomer], 136.48 [136.54 minor isomer], 135.25 [135.13 minor isomer], 129.62 [129.58 minor isomer], 123.99 [123.40 minor isomer], 114.59 [114.50 minor isomer], 67.86 [68.96 minor isomer], 48.97 [47.05 minor isomer], 37.30 [38.64 minor isomer], 35.27 [35.22 minor isomer], 31.96, 31.85, 29.17, 22.84, 21.49 [19.97 minor isomer], 14.35; LC-MS (ESI+) m/z 437.25 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₅H₃₃N₄O₃(M+H)⁺ 437.2547, found 437.2548.

N-Isopropyl-2-(4-cyclohexylphenoxy)-N-((3-pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11ah)

This compound was prepared from 5o (79 mg, 0.31 mmol) and 10f (57 mg, 0.26 mmol) using triethylamine (53 mg, 0.52 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ah (88 mg, 78%) as a sticky solid. HPLC 97.00 % (t_R = 17.0 min, 70% MeOH in 0.1% TFA water 30 min); ¹H NMR (400 MHz, CDCl₃) δ 9.27 (s, 1H), 8.78-8.72 (m, 1H), 8.30 (appdt, J = 8.0, 2.0 Hz, 1H), 7.42-7.39 (m, 1H), 7.12 (d, J = 8.7 Hz, 2H, [7.06 minor isomer]), 6.88 (d, J = 8.7 Hz, 2H, [6.80 minor isomer]), 4.78 (s, 2H, [4.89 minor isomer]), 4.71 (s, 2H, [4.81 minor isomer]), 4.46-4.39 (m, 1H), 2.48-2.38 (m, 2H), 1.83-1.71 (m, 4H), 1.38-1.24 (brm, 11H, and around 1.32 [d, J = 6.6 Hz, 6H, [1.17 (minor isomer]); ¹³C NMR (100 MHz, CDCl₃ ) δ 177.26 [177.47 minor isomer], 168.57, 166.66 [166.88 minor isomer ], 156.11 [155.86 minor isomer], 152.20 [152.49 minor isomer], 148.91 [148.86 minor isomer],141.82, 141.75, 134.96, 128.03, 123.86, 114.60 [114.50 minor isomer], 67.88 [69.03 minor isomer], 48.98 [47.07 minor isomer], 43.88, 37.30 [38.66 minor isomer], 34.85, 27.13 [29.93 minor isomer], 26.36, 21.51 [19.99 minor isomer]; LC-MS (ESI+) m/z 435.24 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₅H₃₁N₄O₃(M+H)⁺ 435.2391, found 435.2395.

N-Isopropyl-2-(4-isopropylphenoxy)-N-((3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11ai)

This compound was prepared from 5p (61mg, 0.29 mmol) and 10f (52 mg, 0.24 mmol) using triethylamine (49 mg, 0.48 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ai (77 mg, 81%) as a sticky white solid. HPLC 99.1% (t_R = 6.7 min, 50% CH₃CN in 0.1% TFA water 20 min); ¹H NMR (400 MHz, CDCl₃) δ 9.27 (s, 1H), 8.77-8.70 (m, 1H), 8.30 (dt, J = 8.0,1.8 Hz, 1H), 8.40 (appdd, J = 7.7, 5.0 Hz, 1H), 7.14 (d, J = 8.6 Hz, 2H, [7.08 minor isomer]), 6.89 (d, J = 8.6 Hz, 2H, [6.81 minor isomer]), 4.79 (s, 2H, [4.89 minor isomer]), 4.71 (s, 2H, [4.81 minor isomer]), 4.45-4.39 (m, 1H), 2.8-82.78 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.17 minor isomer]), 1.20 (d, J = 6.9 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 177.26 [177.47 minor isomer], 168.56, 166.65 [166.88 minor isomer], 156.11 [155.88 minor isomer], 152.22 [152.52 minor isomer], 148.91, 142.44, 134.96, 127.66, 123.81 [123.91 minor isomer], 123.25, 114.64 [114.53 minor isomer], 67.89 [69.01 minor isomer], 48.98 [47.06 minor isomer], 37.30 [38.65 minor isomer], 33.49, 24.37, 21.50 [19.99 minor isomer]; LC-MS (ESI+) m/z 395.20 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₇N₄O₃(M+H)⁺ 395.2078, found 395.2074.

N-Isopropyl-2-(4-tert-butylphenoxy)-N-((3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11aj)

This compound was prepared from 5r (63 mg, 0.28 mmol) and 10f (50 mg, 0.23 mmol) using triethylamine (47 mg, 0.46 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11aj (78 mg, 83%) as a sticky colorless solid. HPLC 97.96% (t_R = 9.1 min, 50% CH₃CN in 0.1% TFA water 20 min); ¹H NMR (400 MHz, CDCl₃) δ 9.28 (s, 1H), 8.73 (brs, 1H), 8.20 (appd, J = 8.0 Hz, 1H), 7.40 (appdd, J = 7.7, 4.9 Hz, 1H), 7.29 (d, J = 8.7 Hz, 2H, [7.24 minor isomer shown]), 6.89 (d, J = 8.7 Hz, 2H, [6.81 minor isomer]), 4.78 (s, 2H, [4.89 minor isomer]), 4.71 (s, 2H, [4.83 minor isomer]), 4.43-4.37 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]), 1.27 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 177.27 [177.48 minor isomer], 168.62, [168.53 minor isomer], 166.64 [166.86 minor isomer], 155.76 [155.53 minor isomer], 152.23 [152.52 minor isomer], 148.89, 144.69, 134.97, 126.64 [126.60 minor isomer], 123.87, 123.26, 114.26 [114.15 minor isomer], 67.75 [68.86 minor isomer], 48.98 [47.00 minor isomer], 37.32 [38.62 minor isomer], 34.35, 31.70 [31.67 minor isomer], 21.52 [20.01 minor isomer]; LC-MS (ESI+) m/z 409.22 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₃H₂₉N₄O₃(M+H)⁺ 409.2234, found 409.2233.

N-Isopropyl-2-(4-isobutylphenoxy)-N-((3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11ak)

This compound was prepared from 5q (74 mg, 0.32 mmol) and 10f (59 mg, 0.27 mmol mmol) using triethylamine (55 mg, 0.54 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ak (97 mg, 88%) as a white solid. mp 121.5-123.7 °C. HPLC 98.3% (t_R = 10.8 min, 50% CH₃CN in 0.1% TFA water 20 min); ¹H NMR (400 MHz, CDCl₃) δ 9.27 (s, 1H), 8.74 (brs, 1H), 8.29 (d, J = 7.8 Hz, 1H), 7.41(brm, 1H), 7.05 (d, J = 8.6 Hz, 2H, [7.00 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.79 minor isomer]), 4.79 (s, 2H, [4.90 minor isomer]), 4.71 (s, 2H, [4.81 minor isomer]), 4.47-4.40 (m, 1H), 2.40 (d, J = 7.2 Hz, 2H, partially overlapped, [2.36 minor isomer partially overlapped ]), 1.85-1.73 (m, 1H), 1.31 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]), 0.87 (d, J = 6.6 Hz, 6H, [0.85 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.24, 168.59, 166.72, 156.15 [155.92 minor isomer], 152.14, 148.89, 135.26 [135.34 minor isomer], 134.93, 130.35, 114.46 [114.37 minor isomer], 67.93 [69.06 minor isomer], 48.98 [47.06 minor isomer], 44.73, 37.30 [38.65 minor isomer], 30.53 [30.49 minor isomer], 22.52, 21.51 [19.98 minor isomer]; LC-MS (ESI+) m/z 409.23 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₃H₂₉N₄O₃(M+H)⁺ 409.2234, found 409.2231.

N-Isopropyl-2-(4-propylphenoxy)-N-((3-(pyrimidin-2-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11al)

This compound was prepared from 5k (46 mg, 0.22 mmol) and 10h (40 mg, 0.18 mmol) using triethylamine (36 mg, 0.36 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11al (58 mg, 81%) as a white solid. mp 92.7-94.0 °C. HPLC 97.2 % (t_R = 12.1 min, 50% CH₃CN in 0.1% TFA water 20 min); ¹H NMR (400 MHz, CDCl₃) δ 9.39-9.28 (m, 3H), 7.09 (d, J = 8.5 Hz, 2H, [7.01 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.75 minor isomer]), 4.78 (s, 2H, [4.92 minor isomer]), 4.70 (s, 2H), 4.49-4.41 (m, 1H), 2.52 (t, J = 7.6 Hz, 2H, [2.45 minor isomer]), 1.64-1.47 (m, 2H), 1.32 (d, J = 6.8 Hz, 6H, [1.17 minor isomer]), 0.91 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 177.87, 168.67, 164.52, 160.62 [160.82 minor isomer], 155.72 [156.04 minor isomer], 136.33, 129.71, 121.78, 114.54 [114.40 minor isomer], 67.83, 49.03 [49.00 minor isomer], 37.34 [38.87 minor isomer], 24.92, 21.53 [20.05 minor isomer],14.02; LC-MS (ESI+) m/z 396.20 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₆N₅O₃(M+H)⁺ 396.2030, found 396.2031.

N-Isopropyl-2-(4-propylphenoxy)-N-((3-(pyrazin-2-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11am)

This compound was prepared from 5k (77 mg, 0.36 mmol) and 10j (66 mg, 0.30 mmol) using triethylamine (61 mg, 0.60 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11am (101 mg, 85%) as a colorless sticky solid. HPLC 97.3 % (t_R = 11.1 min, 50% MeOH in 0.1% TFA water 30 min); ¹H NMR (400 MHz, CDCl₃) δ 9.29 (appd, J = 1.4 Hz, 1H, [9.26 minor isomer]), 8.73-8.70 (m, 2H), 7.08 (d, J = 8.6 Hz, 2H, [7.01 minor isomer]), 6.86 (d, J = 8.6 Hz, 2H, [6.77 minor isomer]), 4.78 (s, 2H, [4.94 minor isomer]), 4.76 (s, 2H, [4.79 minor isomer]), 4.46-4.36 (m, 1H), 2.50 (t, J = 7.8 Hz, 2H, [2.45 minor isomer]), 1.6381.51 (m, 2H), 1.30 (d, J = 6.6 Hz, 6H, [1.15 minor isomer]), 0.90 (t, J = 7.5 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 178.00, 168.64, 166.65 [166.86 minor isomer], 156.08 [155.82 minor isomer], 146.96, [146.67 minor isomer], 145.02 [145.09 minor isomer], 144.62, 142.47, 136.29, 129.72 [129.67 minor isomer], 114.58 [114.50 minor isomer], 67.92 [ 69.16 minor isomer], 49.02 [47.08 minor isomer], 37.35 [38.85 minor isomer], 24.92, 21.49 [19.96 minor isomer], 14.02; LC-MS (ESI+) m/z 396.19 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₆N₅O₃(M+H)⁺ 396.2030, found 396.2028.

2(-4-Butylphenoxy)-N-isopropyl-N-((3-(pyrazin-2-yl)-1,2,4-oxadiazol-5-yl)methylecetamide (11an)

This compound was prepared from 5l (54 mg, 0.24 mmol) and 10j (44 mg, 0.20 mmol) using triethylamine (41 mg, 0.40 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11an (64 mg, 78%) as a yellow/green sticky solid. HPLC 97.97 % (t_R = 9.7 min, 70% MeOH in 0.1% TFA water 30 min); ¹H NMR (400 MHz, CDCl₃) δ 9.30 (d, J = 1.4 Hz, 1H), 9.27 (brs, 1H), 8.74-8.71 (m, 1H), 7.09 (d, J = 8.6 Hz, 2H, [7.02 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.78 minor isomer]), 4.80 (s, 2H, [4.95 minor isomer]), 4.76 (s, 2H, [4.80 minor isomer]), 4.48-4.39 (m, 1H), 2.53 (t, J = 7.7 Hz, 2H, [2.47 minor isomer])), 1.63-1.48 (m, 3H), 1.37-1.14 [m, 10H and around 1.30 (d, J = 6.7 Hz, 6H)] 1.16 minor isomer]), 0.90 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 178.00, 168.64, 166.64, 156.05 [155.78 minor isomer], 146.65 [146.94 minor isomer], 145.00 [145.08 minor isomer], 144.61, 142.47 [142.08 minor isomer], 136.49 [136.57 minor isomer], 129.65 [129.80 minor isomer], 114.60 [114.53 minor isomer], 67.93 [69.17 minor isomer], 49.01 [47.13 minor isomer], 37.34 [38.85 minor isomer], 34.94, 34.00, 22.52 [21.11 minor isomer], 21.47 [19.95 minor isomer shown], 14.17; LC-MS (ESI+) m/z 410.222 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₈N₅O₃(M+H)⁺ 410.2187, found 410.2185.

N-Isopropyl-2-(4-propylphenoxy)-N-((3-(pyrimidin-2-yl)-1,2,4-oxadiazol-5-yl)methyl)acetamide (11ao)

This compound was prepared from 5k (46 mg, 0.22 mmol) and 10i (40 mg, 0.18 mmol) using triethylamine (36 mg, 0.36 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ao (63 mg, 88%) as a colorless sticky solid; HPLC 97.9 % (t_R = 7.1 min, 50% CH₃CN in 0.1% TFA water 20 min); ¹H NMR (400 MHz, CDCl₃) δ 8.96 (d, J = 4.9 Hz, 2H), 7.45 (t, J = 4.9 Hz, 1H, [7.48 minor isomer]), 7.10 (d, J = 8.6 Hz, 2H, [7.04 minor isomer]), 6.88 (d, J = 8.6 Hz, 2H, [6.81 minor isomer]), 4.83 (s, 2H, [4.99 minor isomer]), 4.80 (s, 2H, [4.81 minor isomer]), 4.44-4.34 (m, 1H), 2.52 (t, J = 7.7 Hz, 2H, partially overlapped [2.48 minor isomer, partially overlapped]), 1.66-1.51 (m, 2H, [1.46-1.38 minor isomer]), 1.28 (d, J = 6.6 Hz, 2H, [1.14 minor isomer]), 0.92 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 178.04, 168.62 [167.47 minor isomer], 167.85, 158.19 [158.27 minor isomer], 156.27, 156.09, 136.24, 129.73, 122.37, 114.59, 67.97 [69.02 minor isomer], 49.02 [47.00 minor isomer], 37.36 [38.85 minor isomer], 37.34, 24.93, 21.42 [19.90 minor isomer], 14.02; LC-MS (ESI+) m/z 396.19 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₆N₅O₃(M+H)⁺ 396.2030, found 396.2025.

N-Isopropyl-2-(4-propylphenoxy)-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)acetamide (11ap)

This compound was prepared from 5k (140 mg, 0.66 mmol) and 10a (127 mg, 0.55 mmol) using triethylamine (111 mg, 1.10 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ap (195 mg, 87%) as a white solid. mp 121.7-122.3 °C. HPLC 99.3 % (t_R = 11.1 min, 45% MeOH in 0.1% TFA water 30 min); ¹H NMR (400 MHz, CDCl₃) δ 7.90 (d, J = 8.2 Hz, 2H), 7.29-7.20 (m, 2H), 7.07 (d, J = 8.6 Hz, 2H, [7.03 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.82 minor isomer]), 4.77 (s, 2H, [4.83 minor isomer]), 4.69 (s, 2H, [4.82 minor isomer]), 4.45-4.34 (m, 1H), 2.54-2.46 (m, 2H), 2.39 (s, 3H, [2.40 minor isomer]), 1.60-1.50 (m, 2H), 1.29 (d, J = 6.6 Hz, 6H, [1.13 minor isomer]), 0.91 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 176.43 [δ 176.57 minor isomer]), 168.56 [168.80 minor isomer]), 168.50, 156.18 [155.99 minor isomer]), 141.66 [142.14 minor isomer]), 136.16, 129.69 [129.83 minor isomer]), 127.65, 124.09 [123.58 minor isomer]), 114.64 [114.59 minor isomer]), 67.99 [68.74 minor isomer]), 48.94 [46.89 minor isomer]), 37.37 [38.41 minor isomer]), 37.22, 24.92, 21.82, 21.47 [19.98 minor isomer]), 14.03; LC-MS (ESI+) m/z 408.22 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₄H₃₀N₃O₃(M+H)⁺ 408.2282, found 408.2279.

tert-Butyl-4-(2-(isopropyl((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)amino)-2-oxoethoxy)benzoate (11aq)

A solution of tert-Butyl-4-hydroxybenzoate (27, see supporting information for 27) (27 mg, 0.14 mmol), 15 (41 mg, 0.14 mmol) and potassium carbonate (97 mg, 0.70 mmol) in acetonitrile (20 ml) was heated under reflux overnight. Acetonitile was evaporated and the residue was dissolved in ethyl acetate (20 ml) and washed with water (20 ml × 2). The organic phase was dried (MgSO₄) and the product obtained was purified by SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11aq (51 mg, 80% as a white solid). mp 143.0-144.4 °C. HPLC 99.7% (t_R = 14.1 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.97 (d, J = 8.8 Hz, 2H, [8.01 minor isomer]), 7.94 (d, J = 8.8 Hz, 2H, [7.88 minor isomer]), 7.50-7.38 (m, 3H), 6.96 (d, J = 8.9 Hz, 2H, overlapped, [6.93 minor isomer, overlapped]), 4.86 (s, 2H, [4.91 minor isomer]), 4.69 (s, 2H, [4.78 minor isomer]), 4.41-4.31 (m, 1H), 1.55 (s, 9H), 1.29 (d, J = 6.6 Hz, 6H, [1.14 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) 176.14 [176.24 minor isomer], 168.32, 167.60 [167.68 minor isomer], 165.40 [165.65 minor isomer],161.11 [161.06 minor isomer], 159.73, 131.60 [131.64 minor isomer], 131.51 [131.45 minor isomer],128.77 [128.96 minor isomer], 127.45 [126.50 minor isomer], 125.51 [125.46 minor isomer], 124.25, 114.96, 114.11 [114.06 minor isomer], 80.74 [80.51 minor isomer], 67.30 [67.84 minor isomer], 48.78 (46.83 minor isomer), 36.98 [38.22 minor isomer], 28.24 [29.70 minor isomer], 21.24 [19.75 minor isomer]; LC-MS (ESI+) m/z 469.26 (M+NH )⁺ 4 ; HRMS (ESI+ve) m/z calculated for C₂₅H₃₀N₃O₅ (M+H)⁺ 452.2180, found 452.2191.

2-(4-Fluorophenoxy)-N-isopropyl-N-((3-(pyridin-3-yl)1,2,4-oxadiazol-5-yl)methyl)acetamide (11ar)

This compound was prepared from 5f (93 mg, 0.49 mmol) and 10f (90 mg, 0.41 mmol) using triethylamine (83 mg, 0.82 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11ar (132 mg, 87%) as a white solid. mp 83.8-84.6 °C. HPLC 100% (t_R = 10.8 min, 30% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4.5:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.25 (s, 1H), 8.73 (d, J = 4.1 Hz, 1H, [8.76 minor isomer]), 8.25 (dt, J = 8.0, 1.9 Hz, 1H), 7.40 (dd, J = 7.9, 4.9 Hz, 1H, partially overlapped, [7.43 minor isomer]), 7.05-6.83 (m, 4H, [6.88-6.78 minor isomer]), 4.79 (s, 2H, [4.81 minor isomer]), 4.71 (s, 2H, [4.86 minor isomer]), 4.47-4.35 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) 176.97 [177.10 minor isomer], 168.06, 166.25 [166.62 minor isomer], 157. 72 (d, J = 239 Hz), 153.93 (d, J = 2.06 Hz), 151.39 [152.19 minor isomer], 148.07 [148.41 minor isomer], 135.18 [134.86 minor isomer], 123.80, 123.16, 116.03 (d, J = 23 Hz), 115.75 (d, J = 8 Hz), 67.91 [68.67 minor isomer], 48.72 [46.89 minor isomer], 37.03 [38.34 minor isomer], 21.26 [19.75 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −122.60 [−122.53 minor isomer]; LC-MS (ESI+) m/z 371.15 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₁₉H₂₀FN₄O₃ (M+H)⁺ 371.1514, found 371.1517.

2-(4-Chlorophenoxy)-N-isopropyl-N-((3-(pyridin-3-yl)1,2,4-oxadiazol-5-yl)methyl)acetamide (11as)

This compound was prepared from 5d (66 mg, 0.32 mmol) and 10f (59 mg, 0.27 mmol) using triethylamine (55 mg, 0.54 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 11as (91 mg, 87%) as a white solid. mp 118.1-119.9 °C. HPLC 99.0% (t_R = 10.4 min, 35% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 9.25 (s, 1H), 8.73 (dd, J = 4.8, 1.4 Hz, 1H, overlapped [8.76 minor isomer, overlapped]), 8.24 (dt, J = 7.9, 1.9 Hz, 1H), 7.41 (dd, J = 8.0, 4.9 Hz, 1H), 7.22 (d, J = 9.0 Hz, 2H, [7.18 minor isomer]), 6.90 (d, J = 9.0 Hz, 2H, [6.83 minor isomer]), 4.80 (s, 2H, [4.85minor isomer]), 4.70 (s, 2H, [4.83 minor isomer]), 4.48-4.31 (m, 1H), 1.32 (d, J = 6.6 Hz, 6H, [1.17 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) 176.74, 167.82, 166.35, 156.39, 152.39 [152.03 minor isomer], 148.59 [148.55 minor isomer], 134.59, 129.51 [129.47 minor isomer], 126.68, 123.63 [122.89 minor isomer], 115.89 [115.83 minor isomer], 67.59 [68.57 minor isomer], 48.75 [46.93 minor isomer], 37.03 [38.38 minor isomer], 21.24 [18.67 minor isomer]; LC-MS (ESI+) m/z 387.03 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₁₉H₂₀ClN₄O₃ (M+H)⁺ 387.1218, found 387.1212.

N-Isobutyl-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)-2-(p-tolyloxy)acetamide (12a)

This compound was prepared from 5a (95 mg, 0.52 mmol) and 10m (106 mg, 0.43 mmol) using triethylamine (87 mg, 0.86 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 12a (147 mg, 87%) as a white solid. mp 78.4-79.8 °C. HPLC 99.1 % (t_R = 11.8 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed approximately 2:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 8.2 Hz, 2H), 7.30-7.25 (m, 2H), 7.08 (d, J = 8.3 Hz, 2H, [7.04 minor isomer]), 6.86 (d, J = 8.6 Hz, 2H, [6.81 minor isomer]), 4.85 (s, 2H, [4.93 minor isomer]), 4.79 (s, 2H, [4.86 minor isomer]), 3.36-3.34 (m, 2H), 2.41 (s, 3H, [2.42 minor isomer]), 2.27 (s, 3H, [2.24 minor isomer]), 2.06-1.94 (m, 1H), 1.01 (d, J = 6.6 Hz, 6H, [0.86 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 175.43 [175.48 minor isomer], 169.22 [169.08 minor isomer], 168.61 [168.79 minor isomer], 156.04 [155.63 minor isomer], 141.82 [142.15 minor isomer], 131.21 [131.33 minor isomer], 130.24, 129.74 [129.84 minor isomer], 127.66 [127.67 minor isomer], 123.92 [123.53 minor isomer], 114.82 [114.54 minor isomer], 67.23 [68.55 minor isomer], 55.50 [54.51 minor isomer], 42.10 [43.59 minor isomer], 27.73 [26.86 minor isomer], 21.85, 20.75 [20.70 minor isomer], 20.28 [20.15 minor isomer]; LC-MS (ESI+) m/z 394.20 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₃H₂₈N₃O₃ (M+H)⁺ 394.2125, found 394.2127.

N-Ethyl-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)-2-(p-tolyloxy)acetamide (12b)

This compound was prepared from 5a (51 mg, 0.28 mmol) and 10l (50 mg, 0.23 mmol) using triethylamine (47 mg, 0.46 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 12b (69 mg, 82%) as a white solid. mp 97.4-100.1 °C. HPLC 99.6% (t_R = 9.0 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 2:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 8.1 Hz, 2H, partially overlapped, [7.89 minor isomer]), 7.31-7.27 (m, 2H), 7.09 (d, J = 8.2 Hz, 2H, [7.03 minor isomer]), 6.87 (d, J = 8.6 Hz, 2H, [6.79 minor isomer]), 4.84 (s, 2H, [4.91 minor isomer]), 4.78 (s, 2H, [4.82 minor isomer]), 3.63 (q, J = 7.1 Hz, 2H, partially overlapped, [3.59 minor isomer overlapped]), 2.41 (s, 3H, [2.42 minor isomer]), 2.29 (s, 3H, [2.24 minor isomer]), 1.29 (t, J = 7.1 Hz, 3H, [1.16 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 175.56 [175.49 minor isomer], 168.86 [168.80 minor isomer], 168.66 [168.48 minor isomer], 156.00 [155.65 minor isomer], 141.85 [142.12 minor isomer], 131.27 [131.32 minor isomer], 130.30 [130.27 minor isomer], 129.75 [129.82 minor isomer], 127.66, 123.89 [123.56 minor isomer], 114.72 [114.52 minor isomer], 67.61 [68.52 minor isomer], 43.31 [42.97 minor isomer], 41.30 [42.72 minor isomer], 21.83, 20.74 [20.68 minor isomer], 14.14 [12.56 minor isomer]; LC-MS (ESI+) m/z 366.19 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₄N₃O₃ (M+H)⁺ 366.1812, found 366.1810.

N-Methyl-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)-2-(p-tolyloxy)acetamide (12c)

This compound was prepared from 5a (67 mg, 0.36 mmol) and 10k (61 mg, 0.30 mmol) using triethylamine (61 mg, 0.60 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 12c (100 mg, 95%) as a white solid. mp 99.1-100.9 °C. HPLC 96.7% (t_R = 7.4 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 2:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 8.3 Hz, 2H, overlapped, [7.90 minor isomer, overlapped]), 7.30-7.26 (m, 2H), 7.08 (d, J = 8.1 Hz, 2H, [7.03 minor isomer]), 6.87 (d, J = 8.8 Hz, 2H, [6.78 minor isomer]), 4.87 (s, 2H [4.94 minor isomer]), 4.78 (s, 2H, [4.82 minor isomer]), 3.28 (s, 3H, [3.12 minor isomer]), 2.41 (s, 3H, [2.42 minor isomer)], 2.28 (s, 3H, [2.23 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 175.13 [175.03 minor isomer], 169.08 [168.92 minor isomer shown], 168.80 [168.68 minor isomer shown], 155.58 [155.89 minor isomer], 141.91 [142.12 minor isomer], 131.32 [131.38 minor isomer], 130.29, 129.77 [129.82 minor isomer], 127.65, 123.82 [123.54 minor isomer], 114.76, [114.44 minor isomer], 67.53 [67.49 minor isomer], 43.98 [45.45 minor isomer], 35.77 [35.22 minor isomer], 21.85 [21.83 minor isomer], 20.75 [20.74 minor isomer]; LC-MS (ESI+) m/z 352.17 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₂₂N₃O₃ (M+H)⁺ 352.1657, found 352.1658.

N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)-2-(p-tolyloxy)acetamide (12d)

This compound was prepared from 5a (58 mg, 0.31 mmol) and 10n (49 mg, 0.26 mmol) using triethylamine (54 mg, 0.53 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 12d (69 mg, 79%) as a white solid. mp 113.7-115.5 °C. HPLC 99.9% (t_R = 6.5 min, 60% CH₃CN in 0.1% TFA water 30 min); ¹H NMR (400 MHz, CDCl₃) δ 7.94 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 4.84 (d, J = 5.9 Hz, 2H), 4.59 (s, 2H), 2.42 (s, 3H), 2.31 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 175.46, 169.14, 168.66, 155.20, 142.02, 131.98, 130.49, 129.82, 127.64, 123.67, 114.82, 67.70, 35.46, 21.85, 20.77; LC-MS (ESI+) m/z 360.14 (M+Na)⁺; HRMS (ESI+ve) m/z calculated for C₁₉H₂₀N₃O₃ (M+H)⁺ 338.1499, found 338.1505.

N-tert-Butyl-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl-2-(p-tolyloxy)acetamide (12e)

This compound was prepared from 5a (40 mg, 0.22 mmol) and 10o (44 mg, 0.18 mmol) using triethylamine (36 mg, 0.36 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 12e (53 mg, 75%) as a white solid. mp 129.1-130.0 °C. HPLC 96.2% (t_R = 18.3 min, 60% CH₃CN in 0.1% TFA water 30 min); ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, J = 8.2 Hz, 2H), 7.27 (m, 2H), 7.02 (m, 2H), 6.78 (d, J = 8.6 Hz, 2H), 4.95 (s, 2H), 4.76 (s, 2H), 2.42 (s, 3H), 2.24 (s, 3H), 1.47 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 176.97, 169.45, 168.78, 155.88, 142.11, 131.07, 130.17, 129.81, 127.66, 123.62, 114.55, 69.81, 59.15, 40.71, 28.47, 21.86, 20.72; LC-MS (ESI+) m/z 394.22 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₃H₂₈N₃O₃ (M+H)⁺ 394.2125, found 394.2114.

N-Cyclopropyl-N-((3-p-tolyl-1,2,4-oxadiazol-5-yl)methyl)-2-(p-tolyloxy)acetamide (12f)

This compound was prepared from 5a (74 mg, 0.40 mmol) and 10p (76 mg, 0.33 mmol) using triethylamine (67 mg, 0.66 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 12f (98 mg, 79%) as a white solid. mp 113.8-115.2 °C. HPLC 94.8% (t_R = 6.2 min, 70% MeOH in 0.1% TFA water 20 min); ¹H NMR (400 MHz, CDCl₃) δ 7.93 (d, J = 8.0 Hz, 2H), 7.31-7.23 (m, 2H), 7.06 (d, J = 8.2 Hz, 2H), 6.86 (d, J = 8.3 Hz, 2H), 4.97 (s, 2H), 4.88 (s, 2H), 3.10-3.02 (m, 1H), 2.42 (s, 3H), 2.26 (s, 3H), 1.05-0.91 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 175.92, 171.51, 168.61, 156.24, 141.86, 131.02, 130.19, 129.75, 127.65, 123.91, 114.81, 67.14, 43.44, 29.96, 21.82, 20.72, 9.29; LC-MS (ESI+) m/z 378.18 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₄N₄O₃(M+H)⁺ 378.1812, found 378.1811.

1-Isopropyl-3-(4-methylbenzyl)-1-((3 phenyl-1,2,4-oxadiazol-5-yl)methyl)urea (14)

A solution of 10d (70 mg, 0.32 mmol), 4-methylbenzyl isocyanate 13 (47 mg, 0.32 mmol) and triethylamine (40 mg, 0.39 mmol) were heated under reflux in benzene overnight (14-15h). The organics were evaporated and the residue was dissolved in ethyl acetate (20 ml) and washed with HCl (4M, 3×10 mL) and water (2×20 mL). The organic phase was dried (MgSO₄), evaporated, and the crude product obtained was purified by SiO₂ chromatography (EtOAc/Hexane gradient elution) to obtain pure 14 (91 mg, 78%) as a white solid. mp 87.6-89.1 °C. HPLC 96.26% (t_R = 14.98 min, 50% CH₃CN in 0.1% TFA water 30 min); ¹H NMR (400 MHz, CDCl₃) δ 7.98-7.95 (m, 2H), 7.44-7.49 (m, 1H), 7.49-7.44 (m, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 7.8 Hz, 2H), 5.52 (apparent t, 1H), 4.63 (s, 2H), 4.43 (d, J = 4.7 Hz, 2H), 4.40-4.34 (m, 1H), 2.33 (s, 3H), 1.22 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 177.63, 168.47, 158.01, 137.13, 136.32, 131.58, 129.52, 129.03, 128.03, 127.71, 126.53, 47.12, 45.27, 37.62, 21.36, 20.96; LC-MS (ESI+) m/z 365.20 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₅N₄O₂ (M+H)⁺ 365.1972, found 365.1988.

N-Isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-2-(p-tolylamino)acetamide (16)

A solution of p-toluidine (19 mg, 0.18 mmol), 15 (65 mg, 0.22 mmol) and sodium acetate (18 mg, 0.22 mmol) in ethanol (20 ml) were refluxed for 15 h. Ethanol was evaporated and the product was purified by SiO₂ chromatography (EtOAc/Hexane gradient elution) to obtain 16 as a yellow-brown sticky solid (51 mg, 78%); HPLC 96.59% (t_R = 12.2 min, 45% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 4:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.07-8.04 (m, 2H), 7.54-7.44 (m, 3H), 7.01 (d, J = 8.1 Hz, 2H), 6.59 (d, J = 8.3 Hz, 2H), 4.76 (s, 2H, [4.71 minor isomer]), 4.29-4.16 (m, 1H, [4.98-4.90 minor isomer]), 4.05 (s, 2H), 2.25 (s, 3H, [2.23 minor isomer]), 1.34 (d, J = 6.6 Hz, 6H, [1.18 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 176.66 [176.22 minor isomer], 169.91 [169.69 minor isomer], 168.65 [168.92 minor isomer], 145.14, 131.43 [131.83 minor isomer], 130.01 [130.14 minor isomer], 129.00 [129.18 minor isomer], 127.74 [127.70 minor isomer], 127.33, 126.82, 113.51 [113.65 minor isomer], 47.98 [49.59 minor isomer], 46.18 [46.74 minor isomer], 37.25 [37.44 minor isomer], 21.32 [19.96 minor isomer], 20.65 [20.14 minor isomer]; LC-MS (ESI+) m/z 365.19 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₁H₂₅N₄O₂(M+H)⁺ 365.1972, found 365.1978.

N-Isopropyl-N-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-3-(4-(trifluoromethyl)phenyl)propanamide (19a)

This compound was prepared from 18a (131 mg, 0.55 mmol) and 10d (100 mg, 0.46 mmol) using triethylamine (93 mg, 0.92 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 19a (169 mg, 88%) as a white solid. mp 96.1-97.7 °C. HPLC 98.5% (t_R = 5.27 min, 60% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.06 (dd, J = 7.9, 1.7 Hz, 2H, overlapped [8.03 minor isomer overlapped]), 7.56-7.42 (m, 5H), 7.37 (d, J = 8.1 Hz, 2H, [7.33 minor isomer]), 4.70 (s, 2H, [4.61 minor isomer]), 4.25-4.18 (m, 1H, [4.97 minor isomer]), 3.08 (t, J = 7.6 Hz, 2H, [3.02 minor isomer]), 2.79 (t, J = 7.6 Hz, 2H, [2.71minor isomer]), 1.24 (d, J = 6.7 Hz, 6H, [1.12 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.07 [176.66 minor isomer], 171.92 [172.02 minor isomer], 168.58, 145.48, 131.40 [131.84 minor isomer], 129.19, 129.07, 129.00 [128.88 minor isomer], 127.70 [127.67 minor isomer], 126.94, 125.64 (q, J = 3.76 Hz), 124.50 (q, J = 271 Hz), 48.78 [45.82 minor isomer], 37.16 [38.72 minor isomer], 34.81 [35.30 minor isomer], 31.09 [31.17 minor isomer], 21.33 [20.20 minor isomer]; ¹⁹F NMR (376 MHz, CDCl₃) δ −62.75,[−62.78 minor isomer]; LC-MS (ESI+) m/z 418.18 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₃F₃N₃O₂ (M+H)⁺ 418.1737, found 418.1745.

Benzofuran-2-carboxylic acid isopropyl-(3-pyridin-3-yl-[1,2,4]oxadiazol-5-yl)methyl) amide (19b)

This compound was prepared from 18b (63 mg, 0.35 mmol) and 10f (63 mg, 0.29 mmol using triethylamine (59 mg, 0.58 mmol) in a similar manner as described for compound 11u. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 19b (86 mg, 82%) as a sticky colorless solid. HPLC 96.1% (t_R = 4.1 min, 70% MeOH in 0.1% TFA water 20 min); ¹H NMR (400 MHz, CDCl₃) δ 9.27 (s, 1H), 8.71 (appd, J = 4.0 Hz, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.46-7.38 (m, 3H, [7.57-7.46 minor isomer]), 7.31-7.25 (m, 2H), 5.03-4.93 (m, 1H), 4.89 (brs, 2H), 1.37 (d, J = 3.1 Hz, 6H, [7.50 minor isomer]). ¹³C NMR (100 MHz, CDCl₃) δ 166.69, 161.31, 154.93, 152.05, 148.69, 135.19, 127.05 [126.98 minor isomer], 123.98 [123.95 minor isomer], 122.63 [123.26 minor isomer] 113.13, 112.13, 49.89, 38.21, 22.78, 21.66: LC-MS (ESI+) m/z 363.16 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₀H₁₉N₄O₃(M+H)⁺ 363.1452, found 363.1455.

N-Isopropyl-N-(2-(3-phenyl-1,2,4-oxadiazol-5-yl)ethyl)-2-(p-tolyloxy)acetamide (23)

This compound was prepared from 5a (56 mg, 0.55 mmol) and 22 (106 mg, 0.46 mmol, see supporting information for the synthesis of 22) using triethylamine (93 mg, 0.92 mmol) in a similar manner as described for compound 1. The crude product obtained was purified using SiO₂ chromatography (EtOAc/Hexane gradient elution) to afford pure 23 (152 mg, 87%) as a sticky colorless solid. HPLC 97.51% (t_R = 22.7 min, 50% CH₃CN in 0.1% TFA water 30 min); The ¹H NMR showed 3:1 ratio of atropisomers: ¹H NMR (400 MHz, CDCl₃) δ 8.07-8.02 (m, 2H), 7.52-7.44 (m, 3H), 7.09 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 4.69 (s, 2H), 4.29-4.20 (m, 2H, [4.55-4.44 minor isomer]), 3.72 (t, J = 7.4 Hz, 2H, [3.89 minor isomer]), 3.28 (t, J = 7.4 Hz, 2H), 2.28, (s, 3H, [2.26 minor isomer]), 1.28 (d, J = 6.7 Hz, 6H, [1.20 minor isomer]); ¹³C NMR (100 MHz, CDCl₃) δ 177.76, 168.50, 168.40, 155.99, 131.37 [131.18 minor isomer], 130.29, 129.05, 127.66 [126.99 minor isomer], 114.59 [114.67 minor isomer], 68.31 [68.99 minor isomer], 48.80 [47.87 minor isomer shown], 38.56 [41.31 minor isomer], 26.18 [28.64 minor isomer], 21.36 [20.45 minor isomer]; 20.72. LC-MS (ESI+) m/z 380.20 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₂₂H₂₆N₃O₃(M+H)⁺ 380.1969, found 380.1965.

ABBREVIATIONS USED

CT-L

chymotrypsin like

T-L

trypsin like

SAR

structure activity relationship

PGPH-L

postglutamylpeptidase hydrolysis-like

DCM

dichloromethane

THF

tetrahydrofuran

DMF

dimethylformamide

DMSO

dimethylsulfoxide

TFA

trifluoroacetic acid

LC/MS-MS

Liquid chromatography-tandem mass spectrometry

LC-MS

Liquid chromatography mass spectrometry

HRMS

High resolution mass spectroscopy

ESI

Electrospray ionization

HPLC

High performance liquid chromatography

AMC

7-amino-4-methyl-coumarin