Mycobacterium tuberculosis growth inhibitors.A benzofuran series of inhibitors that targeted the Pks13 thioesterasedomain, failed to progress to preclinical development due to concernsover cardiotoxicity. Herein, we report the identification of a noveloxadiazole series of Pks13 inhibitors, derived from a high-throughputscreening hit and structure-guided optimization. This new series bindsin the Pks13 thioesterase domain, with a distinct binding mode comparedto the benzofuran series. Through iterative rounds of design, assistedby structural information, lead compounds were identified with improvedantitubercular potencies (MIC < 1 μM) and in vitro ADMET profiles.There is an urgentneed for new tuberculosis (TB) treatments, withnovel modes of action, to reduce the incidence/mortality of TB andto combat resistance to current treatments. Through both chemicaland genetic methodologies, polyketide synthase 13 (Pks13) has beenvalidated as essential for mycobacterial survival and as an attractivetarget for  Since the benzofuran series was terminatedbecause of a liability associated with its pharmacophore, a lipophilicbasic amine, it was decided to pursue an in vitro screening program to identify novel chemical starting points againstPks13, devoid of this cardiotoxicity flag. This report describes thescreening program and the optimization of the most promising hit,based on a novel oxadiazole core.Althoughoriginally identified as a phenotypic hit, lead optimizationof the benzofuran series had been pursued as a structure-based drugdiscovery program, as both an 1a was condensed with methyl 2-chloro-2-oxo-acetate to affordthe substituted oxadiazole methyl ester 2a. The amides 3–10 were formed directly from the methylester of 2a. The carboxylic acid 6 was convertedto amides 11–18. The m-phenol of 10 was alkylated to form the m-methoxyphenyl 19. Compound 20 wassynthesized from 3,4-dihydroxybenzonitrile 21—itwas converted to crude hydroxy amidine, which was condensed with methyl2-chloro-2-oxo-acetate to give the 1,2,4-oxadiazole 2b, and then the methyl ester of 2b was converted to theamide 20.To explore the structure–activityrelationship (SAR) of the phenethyl amide, the synthesis of severalanalogues was achieved using the route outlined in 22 and potassium (2-((tert-butoxycarbonyl)amino)ethyl)trifluoroborategave the Boc-protected amine 23. The Boc protecting groupwas removed to give the amine 24. The amine of 24 was then converted to the amide 25 by reactionwith ethyl 3--1,2,4-oxadiazole-5-carboxylate.A route used to introduce a substituted pyridyl isoutlined in 26 was converted to the primary amide 27. A palladium-catalyzed carbonylation reaction19 on the bromide of 27 gave the ethyl ester 28. Carboxylic acid 29 was formed from the hydrolysisof ester 28; the carboxylic acid 29 wasthen converted to the amide, and the Boc protecting group was removedfrom the amine to give 30. The amide 31 wasformed by reaction of the ester of 32 with the amine 30. The primary amide of 31 was converted tothe nitrile 33 by treatment with TFAA, triethylaminein THF. A similar route was used to make 34: the carboxylicacid 29 was converted to the amide, and after Boc deprotectionof the amine, the amide was yielded from the coupling with the lithiumsalt of the carboxylic acid 35, which was formed fromthe hydrolysis of ester 32, and finally the primary amidewas converted to the nitrile 34. A similar route wasalso used to make 38: the amide 37 was formedby reaction of the ester of 32 with the amine 36, and then the primary amide of 37 was converted tothe nitrile 38 by treatment with TFAA, triethylaminein THF.A routeused to introduce a nitrile on the linker chain is outlinedin 39 was converted to the amide 40. 2-(Benzhydrylideneamino)acetonitrilewas then alkylated with the chloride 40 to give the imine 41.20 Hydrolysis of the imine 41 yielded the aminonitrile 42. The sodiumsalt of the carboxylic acid 43 was formed from the hydrolysisof the ester 32. Amide formation21 was achieved by reaction of the sodium salt of the carboxylic acid 43 with aminonitrile 42, HATU, and DIPEAin DMF to afford 44. To synthesize 45, thehydroxy amidine 46 was condensed with ethyl 2-chloro-2-oxo-acetateto afford 47. 48 was formed by a Mitsunobu22 reaction on the phenol of 47. Theacid chloride 39 was converted to the amide 49. 2-(Benzhydrylideneamino)acetonitrile was thenalkylated with the chloride 49 to give the imine, andhydrolysis of the imine yielded the aminonitrile. The amide 45 was formed by reaction of the aminonitrile with theethyl ester 48. The route in 44 at a higher scale (410 mg) and high levels of purity  for in vivo studies.An alternativeroute to introduce a nitrile on the linker chainis outlined in 50 was alkylated with methyl iodide to givethe methyl amide 51. Alkylation of 2-phenylethanaminewith the chloride 52 gave the amine 53.Routes to amide modifications are outlined in 54–60 werecondensed with ethyl 2-chloro-2-oxo-acetate to afford substitutedoxadiazole ethyl esters, and the amides 61–67 were formed by reaction of the ethyl esters with amine 69. 69 was formed by converting the carboxylicacid of 68 to the amide, and then the Boc protectinggroup was removed from the amine group.A routeto explore dimethoxyphenyl ring modifications isoutlined in 70 was alkylated to form the ethers 71–73, and then the nitrile of 71–73 wasconverted to the hydroxyamidines 74–76. Condensation with ethyl 2-chloro-2-oxo-acetate and reactionwith the amine 83 gave the amides 77–79. Amine 83 was formed by converting the carboxylicacid of 68 to the amide, and then the Boc protectinggroup was removed from the amine group. To synthesize 80, the hydroxy amidine 81 was condensed with ethyl 2-chloro-2-oxo-acetateto afford substituted oxadiazole ethyl ester 82. Theamide was formed directly from the ethyl ester of 82,and 80 was formed by a Mitsunobu22 reaction.Routes to methoxy modificationsare outlined in 84 was protected to form 85. Formationof the hydroxy amidine and carbonyl deprotection and cyclization gavethe isoxazole 86. Reaction of the amine of 86 with 3-propanoic acid and thionylchloride gave the amide 87.A route to the isoxazole core with a reverse amide is outlinedin 89 was formed by reaction of the acid chloride 88 with ethyl 2-(hydroxyamino)-2-imino-acetate. The amide 90 was formed by reaction of the ester of 89 withphenethylamine.A routeto the alternative 1,2,4-oxadiazole isomer is outlinedin 23 on the bromide of 91 with (dimethoxyphenyl)boronicacid gave 92. Hydrolysis of the methyl ester of 92 formed the carboxylic acid 93, which was convertedto the amide 94.A route to the furan coreis outlined in 95 is converted to the amide 96.A Suzuki reaction23 on the bromide of 96 with (dimethoxyphenyl)boronicacid gave 97.A route to the phenyl core is outlined in 98 was reacted with ethyl 2-amino-2-thioxo-acetateto form intermediate 99, which was cyclized to the triazole 100. The ester of 100 was converted to the amide 101. The hydrazide 98 was reacted with ethyl2-chloro-2-oxoacetate to formthe intermediate 102, andcyclization gave the 1,3,4-oxadiazole 103. The esterof 103 was converted to the amide 104.Routes to thetriazole and 1,3,4-oxadiazole cores are outlinedin 24 These compoundswere tested at a single concentration  in an in vitro Pks13 TEenzyme assay.17 From the initial screen,there were ∼1500 compounds that warranted further follow up,an initial hit rate of ∼1%.A screening campaignwas run on ∼150,000 compounds thatcame from seven different chemical libraries. These libraries representeda diverse collection of potential start points including: known phenotypicactives; a small polar molecule collection; the standard Dundee DrugDiscovery Unit (DDU) compound collection; and the Gates Global HealthChemical Diversity Library.in vitro enzymeassay over a dose response curve; (2) assessment in a counter screento eliminate false positive compounds that interfered with fluorescenceof the TE reaction product, 4-methylumbelliferone (Ex. 350 nm/Em.450 nm); (3) assessment in a biolayer interferometry binding assayto confirm direct binding to the TE domain; and (4) evaluation ina Pks13 hypomorph strain . Only two compounds, 50 and 105, completed the assessment successfully,with the anticipated readout in the hypomorph strain in which overexpressionof Pks13 (+ATc) reduced potency of the compounds, while underexpressionof Pks13 (−ATc) increased compound potency . Ten closely related analogues of 105, with a chromone scaffold, were all active in the Pks13assay and have also been reported elsewhere as Pks13 inhibitors.25 Significantly, there was a clear structuralsimilarity between 105 and the original TE inhibitorTAM16; in particular, both contained the basic lipophilic piperidinegroup that was believed to be responsible for the off-target hERGchannel inhibition that had resulted in the termination of the benzofuranseries.18 It was determined experimentallythat 105 did indeed strongly inhibit the hERG channel(Q-patch IC50 = 0.8 μM), and, as a result, no furtherwork on this series was performed. On the other hand, 50 represented a very different, oxadiazole-based series with no obvioushERG liabilities (Q-patch IC50 > 20 μM) and, therefore,became the focus of further investigation.Hit confirmation involvedthe following: (1) assessment in the  potency 1. Both o50 withinthe Pks13 TE domain and to support the medicinal chemistry program,the X-ray co-crystal structure was determined. While conditions hadbeen published previously17 for obtainingcrystals of both the apo Pks13 TE domain (PDB 5V3W) and a back-soakingplatform to produce complexes with a benzofuran inhibitor series (PDB 5V3Y), modificationsto these conditions were required to allow suitable co-complexes tobe determined. The structure of Pks13 TE domain in complex with 50 was solved at 1.8 Å resolution . Upon 50 binding, the Arg1563side-chain orientation shifts outward, resulting in the opening ofthe tunnel. It appears that the Arg1563 side-chain orientation determinesthe length of the tunnel, and it is crucial for holding substrates.The dimethoxyphenyl (ring A) sits at the bottom of the tunnelin a hydrophobic groove formed by Trp1683, Ile1648, Ala1646, and thealiphatic side chain of Arg1563  was not tolerated in the Pks13 assay. Thiswas presumed to be due to the shorter linker not allowing the dimethoxyphenylring B to reach its solvent-exposed cavity, resulting in steric clasheswith the residues at the mouth of the catalytic active-site tunnel,mainly Asn1640, Tyr1674, and Ser1533. Compound 50 hasfour methoxy groups that make hydrogen-bond interactions with thewater network within the protein. To assess their contributions topotency, the methoxys were removed. From the crystal structure, itwas clear that the methoxy groups on ring B were in a solvent-exposedarea of the pocket and therefore probably had a low contribution topotency due to a high desolvation penalty. As expected, the phenethylamide 106 retained potency against Pks13, although surprisinglyit did lose MIC potency. The methoxy groups in ring A sit in a hydrophobicpocket, not as solvent-exposed as ring B, and make direct interactionswith a water molecule. As such, it was anticipated that they wouldhave a lower desolvation penalty for the interactions in this pocketand therefore a higher contribution to potency. As anticipated, the3-phenyl-1,2,4-oxadiazole 107 had a modest impact onPks13 potency (∼5-fold reduction). Removal of all four methoxygroups (4) reduced the potency further. The phenol analogue 20 also had a detrimental effect on potency and had no MICactivity. From this initial set of modifications, and the knowledgefrom the crystal structure that the dimethoxyphenyl ring B wasin a solvent-exposed area, we anticipated that it was the most promisingvector to modulate physicochemical properties, while retainingpotency, by the introduction of polar groups or substituents thatreduce lipophilicity.Although the potency of ns to 50 1. Shorte50 were carried out to explore SAR, MIC potency, and in vitro metabolic stability, all of which needed to beimproved  and replacing the 3,4-dimethoxyphenyl with pyridin-3-yl (5) retained Pks13 potency, but none showed an improvementin overall properties. The morpholine 108 had good in vitro metabolic stability, likely due to the reductionin lipophilicity, but lost potency against the enzyme. The benzoicacid 6 also had good in vitro metabolicstability and retained potency in the Pks13 assay but had no MIC activity.A set of amides were prepared to explore SAR; all retained Pks13 potencyexcept for the dimethyl amide 13. The primary amide andmethyl amide 11 and 12, although potentagainst Pks13, with good microsomal stability, had only modest MICactivity and poor in vitro hepatocyte stability.Amides with azetidine rings  all retained good activity against the Pks13 TEdomain, and all had improved MIC potency. The crystal structure ofthe Pks13 TE domain bound with the azetidine amide 14 was solved and showed that the ligand binds in a similar orientationto 50. As predicted by computational modeling, 14 had gained an extra direct H-bond interaction between theside chain of His1664 and the carbonyl oxygen of the azetidine  was not tolerated in the Pks13 assay. Introducingmethyl groups on the linker next to the phenyl (7–9) was also detrimental to Pks13 potency. From the crystalstructure, 50 binds in the active site close to the catalyticSer1533; thus, there was the opportunity to target Ser1533 througha covalent interaction and thereby increase the potency of this series.The racemic nitrile analogue 38 was designed as a potentialcovalent inhibitor. However, while the nitrile was well toleratedand the Pks13 potency was maintained, it did not improve metabolicstability compared to 106.The crystal structure highlighted that theethyl linker occupies a hydrophobic region. Its length is importantto confer the correct geometry of ring B to avoid clashing with Asn1640,Tyr1674, and Ser1533, that line the tunnel and whose orientationsappear to box the ligand in 2E. Despi50 indicatedthat there were no interactions with the 1,2,4-oxadiazole and thatthe amide sits in the middle of the narrow tunnel, establishing aH-bond with Tyr1674. The methyl amide 51 was inactivein the Pks13 assay (111 had good Pks13potency but had no MIC activity and poor metabolic stability. Pks13potency was maintained when the amide was reversed with this isoxazolecore (87), but this compound still had no MIC activityand had poor microsomal stability. Removing the carbonyl (53) was detrimental to Pks13’s potency, suggesting basicitywas not well tolerated. The alternative 1,2,4-oxadiazole isomer 90 reduced Pks13 potency. Replacing the heterocycle with afuran, phenyl, and triazole  was detrimental to potency. As previously reported,26 the 1,3,4-oxadiazole 104 led toa reduction in lipophilicity and an improvement in mouse microsomaland hepatocyte stability but, disappointingly, displayed reduced potencycompared to 15. In brief, there were no improvementsin overall properties with these heterocycle modifications.The crystal structure of 13 assay 4. This p61, 62) modestly reduced Pks13 potency but eliminatedMIC activity, although replacing the 4-methoxy did appear to improvemetabolic stability. Removal of the 4-methoxy (67), althoughit had no impact on Pks13 inhibition, did not show the same improvementin stability, and it also reduced MIC activity. Replacing the 3,4-dimethoxyphenylwith benzimidazole (63) was detrimental to bothPks13 potency and MIC activity. The benzofuran, indole, and indazole(64–66) were all active in the Pks13assay, but only 66 had some MIC activity. Guided by structuralinformation, the 3-methoxy was replaced with other ethers to try andextend into a small lipophilic subpocket, to access additional molecularinteractions. The 3-cyclopropylmethoxy, 3-cyclobutoxy,and 3-cyclopropoxy (77–79)were all well tolerated, but none of them offered a significant improvementover 15. Introducing a hydroxy group on the cyclopropylring of 77 (80) reduced lipophilicity,resulting in an improvement in metabolic stability while displayinglow micromolar MIC activity. In summary, changes to the 3-ether weretolerated in the MIC assay, and 80 had the best profile,with good MIC activity and mouse metabolic stability.The phenylring attached to the 1,2,4-oxadiazole establishes hydrophobic stackinginteractions with Arg1563. The methoxy groups are positioned closeto a small hydrophobic groove, consisting of Ala1646, Ile1648, Val1537,and Trp1683, and they interact with a water molecule 2C. Howev38; the nitrile was of interestbecause it had the potential to form a covalent interaction with thecatalytic Ser1533. The azetidine amide 33 had very goodMIC activity but was not metabolically stable. A crystal structurewas obtained of 33, which adopted the same orientationin the elongated binding site as 50. However, analysisof the crystal structure showed that the cyano group of 33 was positioned away from the oxyanion hole and did not make theanticipated covalent interaction with the active-site Ser1533 . The compound was dosedorally at 200 mg/kg, as this was the dose planned to be used for initialefficacy studies. Unfortunately, even at this high dose, the in vivo exposure was poor, with the Cmax failing to get above that required to see antibacterialactivity (∼500 ng/mL). Not unexpectedly, the in vivo clearance was poor (59 mL/min/g), in agreement with the poor in vitro hepatocyte stability. The next compound profiledwas 80, which had significantly improved metabolic stabilityand retained an MIC potency of ∼1 μM. In this case, 80 was dosed at a more standard 10 mg/kg, while the low in vivo exposure was explored. As anticipated from the in vitro data, there was an improved in vivo clearance for 80 (36 mL/min/g), but again it had poor in vivo exposure, failing to get close to the MIC levels. 80 had poor solubility (33 μM) and PAMPA permeability(14 nm/s), both of which may have been contributing to the poor in vivo exposure. The final compound profiled invivo was 44. It was the most polar compoundand displayed good solubility (197 μM) but still suffered withpoor PAMPA permeability (6 nm/s). Despite the improvement in solubility,when dosed orally, 44 had very poor exposure. Therefore,none of the three compounds tested had suitable oral exposure forprogression into mouse models of acute TB infection. Potentially thiscould be related to the modest permeability for these three moleculesand a more general issue for this chemotype, because none of the compoundsfrom this series, tested in the PAMPA assay, demonstrated good permeability(>100 nm/s).As the SAR for the series progressed, overall,there was excellent correlation between extracellular MIC and intramacrophageICstudies 7.The th18 but failedto advance to clinical trials due to cardiotoxicity risks.18 To identify alternative drug discovery startingpoints against this target, 150,000 compounds were screened in an in vitro thioesterase assay; a novel oxadiazole series wasselected for further hit assessment. Testing against a Pks13 hypomorphstrain confirmed that the oxadiazole hit 50, killed bacteriaby targeting Pks13. As this was a structurally enabled drug discoveryprogram, the co-structure of 50 bound to Pks13 TE domainwas acquired and showed a different binding mode compared to thatof the previously published benzofuran series.17 Due to small conformational shifts on binding of the benzofuran,the two binding modes were incompatible and so did not allow the rationaldesign of hybrid inhibitors. The oxadiazoles lie deeper in the substrate-bindingpocket, close to the catalytic triad, and, as such, avoid the keyinteraction between benzofuran and protein, a H-bond between the protonatednitrogen of the piperidine with the carbonyl oxygen of the Asn1640side chain. This was encouraging because the piperidine within thebenzofuran series was responsible for the hERG inhibition and theresultant cardiovascular liability for that series.Pks13 is an attractive target for the identificationof new TBtreatments. Pks13 TE inhibitors have shown excellent activity in bothacute and chronic TB mouse models27 small changes in the chemicalstructure that had or would be expected to have minimal impact onenzyme inhibition, reduced or lost MIC potency. The reason for thisdisparity was not clear. The oxadiazole core was important for potency;alternative aromatic heterocycles were less potent or inactive. Changesto both phenyl rings were identified that improved MIC activity. Introductionof a nitrile into the ethyl linker, combined with changes in the phenylring, led to the greatest improvement in MIC potency  for the series. Despite thejump in potency, a co-crystal structure of 33 bound tothe Pks13 TE domain did not show evidence of a covalent interactionwith the catalytic serine in the active site. Iterative rounds ofdesign and synthesis improved the antitubercular potency by >200-foldand improved in vitro metabolic stability to be withinacceptable limits for evaluation in vivo (<5 mL/min/g).Three lead molecules were selected for progression to mouse pharmacokineticstudies but, unfortunately, did not show suitable exposure for progressionto in vivo efficacy studies. As oxadiazoles are componentsof known drugs, including antitubercular agents,29 there is potential for a successful further evaluation of this series,which is beyond the scope of this report. Moreover, while this optimizationof the oxadiazole series was not successful, this work does demonstratethat a diverse range of scaffolds can inhibit the Pks13 thioesterasedomain; therefore, screening for compounds with better drug-like propertiesshould be encouraged.Optimizationof the oxadiazole hit focused on improving potencyand metabolic stability. As seen for other Pks13 TE inhibitors,4 was used for visualization. Flash chromatographywas performed using Combiflash Companion Rf (Teledyne ISCO) and prepackedsilica gel columns purchased from Grace Davison Discovery Scienceor SiliCycle. Mass-directed preparative HPLC separations were performedusing a Waters HPLC  connected to a Waters 2998 photodiode arrayand a Waters 3100 mass detector. Preparative HPLC separations wereperformed with a Gilson HPLC  connected to a Gilson 155 UV/vis detector.On both instruments, HPLC chromatographic separations were conductedusing Waters XBridge C18 columns, 19 mm × 100 mm, 5 μmparticle size, using 0.1% ammonia in water (solvent A) and acetonitrile(solvent B) as mobile phase. 1H NMR spectra were recordedon a Bruker Advance II 500 or 400 spectrometer operating at 500 and400 MHz (unless otherwise stated) using CDCl3, DMSO-d6, or CD3OD solutions. Chemical shifts(δ) are expressed in ppm, recorded using the residual solventas the internal reference in all cases. Signal splitting patternsare described as singlet (s), doublet (d), triplet (t), multiplet(m), broadened (br), or a combination thereof. Coupling constants(J) are quoted to the nearest 0.1 Hz. Low-resolutionelectrospray (ES) mass spectra were recorded on a Bruker DaltonicsMicroTOF mass spectrometer run in positive mode. High-resolution massspectroscopy (HRMS) was performed using a Bruker Daltonics MicroTOfmass spectrometer. LC-MS analysis and chromatographic separation wereconducted with either a Bruker Daltonics MicroTOF mass spectrometerconnected to an Agilent diode array detector or a Thermo Dionex Ultimate3000 RSLC system with a diode array detector, where the column usedwas a Waters XBridge column  and the compounds were eluted with a gradient of 5–95%acetonitrile/water + 0.1% ammonia, or with an Agilent Technologies1200 series HPLC connected to an Agilent Technologies 6130 quadrupoleLC-MS, connected to an Agilent diode array detector, where the columnused was a Waters XBridge column  or a Waters X-select column  with a gradient of 5–90% acetonitrile/water+ 0.1% formic acid; or with an Advion Expression mass spectrometerconnected to a Thermo Dionex Ultimate 3000 HPLC with a diode arraydetector, where the column used was a Waters XBridge column  or a Waters X-select column with a gradientof 5–90% acetonitrile/water + 0.1% formic acid. All final compoundsshowed chemical purity of ≥95% as determined from the UV chromatogram(190–450 nm) obtained by LC-MS analysis. Microwave-assistedchemistry was performed using a CEM or a Biotage microwave synthesizer.Chemicals and solvents werepurchased from commercial vendors and were used as received, unlessotherwise stated. Dry solvents were purchased in Sure Seal bottlesstored over molecular sieves. Unless otherwise stated herein, reactionshave not been optimized. Analytical thin-layer chromatography (TLC)was performed on precoated TLC plates . Developedplates were air-dried and analyzed under a UV lamp (UV 254/365 nm),and/or KMnON′-hydroxy-3,4-dimethoxy-benzamidine and triethylamine  in DCM(10 mL) at 0 °C was added methyl 2-chloro-2-oxo-acetate . The reaction mixture was stirred at 0 °C for10 min and then heated to 40 °C for 16 h, concentrated in vacuo, dissolved in water (20 mL), extracted with EtOAc(3 × 20 mL), passed through a hydrophobic frit, and concentrated in vacuo. Purification by flash column chromatography affordedmethyl 3--1,2,4-oxadiazole-5-carboxylate as a white solid. 1H NMR : δ 7.65 , δ 7.51 , δ7.16 , δ 3.99 , δ3.86 , δ 3.84 . LC-MS: m/z 265 [M+H]+.To a solution of in vacuo, dissolved in EtOAc (10 mL), washed with water(3 × 10 mL), passed through a hydrophobic frit, and concentrated in vacuo.To a solution of methyl3--1,2,4-oxadiazole-5-carboxylate and amine(0.9 equiv) in methanol was added triethylamine (2–3equiv). The reaction mixture was stirred at 60 °C for 16 h, concentrated N-[methyl]-1,2,4-oxadiazole-5-carboxamidewas obtained from methyl 3--1,2,4-oxadiazole-5-carboxylate, 3,4-dimethoxyphenyl)methanamine, and triethylamine  in methanol (5 mL). Purification by trituration with 1:1 methanol:DMSOafforded 3--N-[methyl]-1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 9.89 , 7.65 , 7.54 , 7.17 ,6.99 , 6.92–6.87 ,4.42 , 3.85 , 3.75 , 3.73 . HRMS (ESI) calcd for [M+H]+ C20H22N3O6, 400.1509, found 400.1496.Following general procedure A, 3--N-(2-phenylethyl)-1,2,4-oxadiazole-5-carboxamidewas obtained from methyl 3-phenyl-1,2,4-oxadiazole-5-carboxylate , 2-phenylethanamine ,and triethylamine  in methanol (5 mL). Purificationby prep-HPLC afforded 3-phenyl-N-(2-phenylethyl)-1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 9.55 , 8.07–8.05 , 7.66–7.59 , 7.32–7.29 , 7.27–7.25, 7.23–7.20 , 3.56–3.53 , 2.89. LC-MS: m/z 294 [M+H]+.Following general procedure A, compound3-phenyl-N-[2-(3-pyridyl)ethyl]-1,2,4-oxadiazole-5-carboxamidewas obtained from methyl 3--1,2,4-oxadiazole-5-carboxylate, 2-(3-pyridyl)ethanamine hydrochloride, and triethylamine in methanol (5 mL). Purification by prep-HPLC afforded 3--N-[2-(3-pyridyl)ethyl]-1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 9.53 , 8.48 , 8.43 , 7.67 , 7.65 , 7.53, 7.33 , 7.17 , 3.85, 3.59–3.55 , 2.92 . LC-MS: m/z 355 [M+H]+.Following general procedure A, compound3--in vacuo, dissolved in EtOAc(10 mL), washed with 2 M HCl (3 × 10 mL), passed through a hydrophobicfrit, and concentrated in vacuo to afford 4-amino]ethyl]benzoicacid  as a white solid. 1H NMR : δ 12.76 , 9.58 , 7.83 ,7.6 , 7.53 , 7.27 , 7.16 , 3.85 , 3.57 , 2.92 . LC-MS: m/z 398 [M+H]+.To a solution of 4-(2-aminoethyl)benzoicacid hydrochloride  and methyl 3--1,2,4-oxadiazole-5-carboxylate in methanol (5 mL) was added triethylamine. The reaction mixture was stirred at 60 °Cfor 16 h, concentrated N-(2-methyl-2-phenyl-propyl)-1,2,4-oxadiazole-5-carboxamidewas obtained from methyl 3--1,2,4-oxadiazole-5-carboxylate, 2-methyl-2-phenyl-propan-1-amine hydrochloride, and triethylamine in methanol (5 mL). Purification by prep-HPLC afforded 3--N-(2-methyl-2-phenyl-propyl)-1,2,4-oxadiazole-5-carboxamide as a yellow oil. 1H NMR : δ 9.10 , 7.64 , 7.51 , 7.45–7.43 , 7.36–7.33 , 7.24–7.21, 7.16 , 3.85 ,3.51 , 1.33 . LC-MS: m/z 382 [M+H]+.Following general procedure A, compound3--N-[(2R)-2-phenylpropyl]-1,2,4-oxadiazole-5-carboxamide wasobtained from methyl 3--1,2,4-oxadiazole-5-carboxylate in MeOH (5 mL), (2R)-2-phenylpropan-1-amine, and Et3N . Purificationby mass-directed HPLC with 5–95% MeCN acidic afforded 3--N-[(2R)-2-phenylpropyl]-1,2,4-oxadiazole-5-carboxamide. 1H NMR : δ 9.44 , 7.65 , 7.53 , 7.35–7.27, 7.24–7.17 , 3.86 , 3.53–3.41, 3.16–3.10 , 1.25 . HRMS (ESI) calcd for [M+H]+ C20H22N3O4, 368.1605, found 368.1594.Following general procedure A, in a sealedmicrowavetube 3--N-(2-phenylpropyl)-1,2,4-oxadiazole-5-carboxamidewas obtained from methyl 3--1,2,4-oxadiazole-5-carboxylate, 2-phenylpropan-1-amine , andEt3N . Purification by mass-directedHPLC with 5–95% MeCN acidic afforded 3--N-(2-phenylpropyl)-1,2,4-oxadiazole-5-carboxamide. 1H NMR : δ 9.44 , 7.65 , 7.53–7.52 , 7.35–7.17 , 3.86, 3.53–3.41 , 3.16–3.10 , 1.25. HRMS (ESI) calcd for [M+H]+ C20H21N3O 368.1610, found368.1615.Following general procedure Ain a sealed microwave tube, compound 3--N-(3-hydroxyphenethyl)-1,2,4-oxadiazole-5-carboxamidewas obtained from methyl 3--1,2,4-oxadiazole-5-carboxylate, 3-(2-aminoethyl)phenol hydrochloride , and triethylamine in methanol (5 mL). Purification by prep-HPLC afforded 3--N-(3-hydroxyphenethyl)-1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 9.48 , 9.27 , 7.65 , 7.53 , 7.16 , 7.10–7.07 , 6.67–6.65 ,6.62–6.60 , 3.86 , 3.85 , 3.49 , 2.80 .LC-MS: m/z 370 [M+H]+.Following general procedure A, 3--in vacuo. Purification by prep-HPLC afforded N-[2-(4-carbamoylphenyl)ethyl]-3--1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 9.51 , 7.89 ,7.82 , 7.65 , 7.53 , 7.33, 7.26 , 7.17 , 3.85 , 3.58–3.55 ,2.94 . LC-MS: m/z 395 [M–H]−.To a solution of 4-amino]ethyl]benzoicacid  in THF (5 mL) were added ammonia HOBt , EDCI·HCl , and DIPEA . The reaction mixture was stirred at rt for 16 h, dilutedwith water (10 mL), extracted with EtOAc (3 × 10 mL), passedthrough a hydrophobic frit, and concentrated in vacuo, diluted with EtOAc (20 mL), washed with water(3 × 20 mL), passed through a hydrophobic frit, and concentrated in vacuo.To a solution of4-amino]ethyl]benzoicacid , T3P , and triethylamine in DMF (5 mL) was added amine (0.28 mmol). Thereaction mixture was stirred at rt for 16 h, concentrated N-[2-[4-(methylcarbamoyl)phenyl]ethyl]-1,2,4-oxadiazole-5-carboxamidewas obtained from 4-amino]ethyl]benzoicacid , T3P , triethylamine, and methylamine hydrochloride  in DMF (5 mL). Purification by prep-HPLC afforded 3--N-[2-[4-(methylcarbamoyl)phenyl]ethyl]-1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 9.52 , 8.35–8.33 , 7.77 , 7.65 , 7.52 , 7.34 ,7.17 , 3.84 , 3.58–3.54, 2.94 , 2.77 . LC-MS: m/z 411 [M+H]+.Following general procedure B, compound3--N-[2-[4-(dimethylcarbamoyl)phenyl]ethyl]-1,2,4-oxadiazole-5-carboxamidewas obtained from 4-amino]ethyl]benzoicacid , T3P , triethylamine, and dimethylamine hydrochloride  in DMF (5 mL). Purification by prep-HPLC afforded 3--N-[2-[4-(dimethylcarbamoyl)phenyl]ethyl]-1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 9.56 , 7.65 , 7.52 , 7.35–7.30 , 7.17 , 3.85 , 3.57–3.55 , 2.96–2.90 . LC-MS: m/z 425 [M+H]+.Following general procedure B, 3--N-[2-[4-(azetidine-1-carbonyl)phenyl]ethyl]-3--1,2,4-oxadiazole-5-carboxamidewas obtained from 4-amino]ethyl]benzoicacid , T3P , triethylamine, and azetidine  in DMF (5 mL).Purification by prep-HPLC afforded N-[2-[4-(azetidine-1-carbonyl)phenyl]ethyl]-3--1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 9.56 , 7.65 , 7.57 , 7.52 , 7.32 , 7.17 ,4.28 , 4.02 , 3.85 , 3.55 , 2.93 , 2.24 . HRMS (ESI): calcd for [M+H]+ C23H25N4O5, 437.1825,found 437.1840.Following general procedure B, 2SO4. After filtrationand concentration, the filtrate was concentrated in vacuo. The residue was dissolved in DMF (20 mL). The precipitate was collectedby filtration and washed with MeOH (20 mL). The filtrate cake wasdried in vacuo to afford 3--N-(4-(3-methoxyazetidine-1-carbonyl)phenethyl)-1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR: δ 9.56 , 7.65 , 7.58 , 7.52 , 7.34 ,7.17 , 4.41 , 4.21 , 4.11 , 3.85 , 3.56 , 3.21 , 2.94 . 13C NMR : δ 169.5, 169.4, 168.4, 153.4,152.3, 149.6, 142.6, 131.5, 129.1, 128.3, 121.2, 118.2, 112.5, 110.2,69.4, 60.2, 56.2, 56.1, 55.8, 40.8, 34.7. HRMS (ESI): calcd for [M+H]+ C24H27N4O6, 467.1931,found 467.1925.To a solution of 4-amino]ethyl]benzoicacid  and 3-methoxyazetidine hydrochloride in dry DMF (20 mL) were added HATU and DIPEA  in turns below 0 °C. The resulting mixture was stirredat 20 °C for 1 h. The reaction mixture was poured into water(20 mL) and extracted with ethyl acetate (30 mL × 5). The combinedorganic layers were washed with water (20 mL × 2) and brine (20mL) and dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by prep-HPLC.To a mixture of 4--1,2,4-oxadiazole-5-carboxamido)ethyl)benzoicacid  and the corresponding amine(1.2 equiv) in DMF (1 mL) were added HATU  and DIPEA in turns at 25 °C. The mixture was stirred at 25 °C for1 to 12 h. The residue was poured into water (10 mL). The aqueousphase was extracted with ethyl acetate (5 mL × 3). The combinedorganic phase was washed with brine (5 mL), dried with anhydrous NaN-(4-(3-fluoroazetidine-1-carbonyl)phenethyl)-1,2,4-oxadiazole-5-carboxamidewas obtained from 4--1,2,4-oxadiazole-5-carboxamido)ethyl)benzoicacid  and 3-fluoroazetidine after 1 h under stirring.Purification by prep-HPLC  afforded 3--N-(4-(3-fluoroazetidine-1-carbonyl)phenethyl)-1,2,4-oxadiazole-5-carboxamideas a white solid . 1H NMR : δ 9.54 ,7.75–7.46 , 7.35 , 7.16 ,5.68–5.26, 4.70–4.27 , 4.04 , 3.85 ,3.56 , 2.94 . HRMS (ESI): calcd for [M+H]+ C23H24N4O5F,455.1731, found 455.1744.Following general procedure C, 3--N-phenethyl)-3--1,2,4-oxadiazole-5-carboxamidewas obtained from 4--1,2,4-oxadiazole-5-carboxamido)ethyl)benzoicacid  and 3,3-difluoroazetidine after 12 h under stirring. Afterpurification by prep-HPLC , N-phenethyl)-3--1,2,4-oxadiazole-5-carboxamidewas obtained as a white solid . 1H NMR : δ 9.55, 7.69–7.61 , 7.53, 7.38 ,7.18 , 4.88–4.33, 3.86 , 3.61–3.54 , 2.96 . HRMS (ESI): calcd for [M+H]+ C23H22N4O5F2, 473.1637, found 473.1647.Following general procedure C, N-(4-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)phenethyl)-3--1,2,4-oxadiazole-5-carboxamidewas obtained from 4--1,2,4-oxadiazole-5-carboxamido)ethyl)benzoicacid  and 2-oxa-6-azaspiro[3.3]heptane after 12 h under stirring. Afterpurification by prep-HPLC , N-(4-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)phenethyl)-3--1,2,4-oxadiazole-5-carboxamidewas obtained as a white solid . 1H NMR : δ 9.55, 7.66 , 7.60–7.51 , 7.34 , 7.18 , 4.67 , 4.46 , 4.20 , 3.86 , 3.61–3.52 , 2.95 . LC-MS: m/z 479[M+H]+.Following general procedure C, N-(3-hydroxyphenethyl)-1,2,4-oxadiazole-5-carboxamide in DMF (5 mL) were added potassium carbonate  and methyl iodide . The reactionmixture was stirred at rt for 16 h, concentrated in vacuo, dissolved in EtOAc (10 mL), washed with water (3 × 10 mL),passed through a hydrophobic frit, and concentrated in vacuo. Purification by prep-HPLC afforded 3--N-[2-(3-methoxyphenyl)ethyl]-1,2,4-oxadiazole-5-carboxamide as a colorless oil. 1H NMR : δ 9.52 , 7.65 , 7.52 , 7.24–7.16 , 6.83–6.77 , 3.85, 3.73 , 3.53 ,2.86 . HRMS (ESI): calcd for [M+H]+ C20H22N3O5, 384.1570,found 384.1557.To a solution of 3--in vacuo, and then dissolved in DCM (5 mL). Triethylamine was added, and the reaction mixture was cooled to0 °C. Methyl 2-chloro-2-oxo-acetate  was added,and the reaction mixture was heated at 40 °C for 16 h, concentrated in vacuo, dissolved in water (10 mL), extracted with EtOAc(3 × 10 mL), passed through a hydrophobic frit, and concentrated in vacuo. Purification by flash column chromatography affordedmethyl 3--1,2,4-oxadiazole-5-carboxylate as a yellow solid. 1H NMR : δ 9.72 , 9.50 , 7.43, 7.40 , 6.90 , 3.98.To a solution of 3,4-dihydroxybenzonitrile and hydroxylamine hydrochloride  in ethanol (5 mL) was added DIPEA . Thereaction mixture was heated at 80 °C for 16 h, concentrated N-[2-ethyl]-1,2,4-oxadiazole-5-carboxamidewas obtained from methyl 3--1,2,4-oxadiazole-5-carboxylate, 4-(2-aminoethyl)benzene-1,2-diol hydrochloride, and triethylamine in methanol (5 mL). Purification by prep-HPLC afforded 3--N-[2-ethyl]-1,2,4-oxadiazole-5-carboxamide as an off-white solid. 1H NMR : δ 9.70 , 9.47 , 9.40, 8.76 , 8.66 ,7.43 , 7.37 , 6.90 , 6.65–6.61, 6.47 , 3.44–3.40, 2.68 . HRMS (ESI): calcdfor [M+H]+ C17H16N3O6, 358.1034, found 358.1028.Following general procedure A, compound3--tert-butoxycarbonyl)amino)ethyl)trifluoroborate in a solution of toluene (3 mL) andH2O (1 mL) were added Pd(dppf)Cl2  and Cs2CO3  in turns at 25 °C, and the resulting mixturewas stirred at 100 °C for 12 h under N2. The residuewas poured into water (20 mL). The aqueous phase was extracted withethyl acetate (10 mL × 3). The combined organic phase was washedwith brine (5 mL), dried with anhydrous Na2SO4, filtered, and concentrated in vacuo. The crudeproduct was purified by reversed-phase HPLC (0.1% FA condition). tert-Butyl (2-(6-(azetidine-1-carbonyl)pyridin-3-yl)ethyl)carbamate was obtained as a white solid. LC-MS: m/z 306 [M+H]+.To a mixture of azetidin-1-yl-(5-bromopyridin-2-yl)methanone and potassium (2-((tert-butyl(2-(6-(azetidine-1-carbonyl)pyridin-3-yl)ethyl)carbamate in DCM (2 mL) was added TFA  in one portion at 0 °C.The mixture was stirred at 25 °C for 1 h and concentrated toget the residue. The residue was used in the next step without purification.(5-(2-Aminoethyl)pyridin-2-yl)(azetidin-1-yl)methanone  was obtained as a white oil. LC-MS: m/z 206 [M+H]+.To a mixture of 2SO4, filtered, and concentrated in vacuo. The residuewas purified by prep-HPLC . N-(2-(6-(azetidine-1-carbonyl)pyridin-3-yl)ethyl)-3--1,2,4-oxadiazole-5-carboxamide was obtained as a white solid. LC-MS: m/z 438.2 [M+H]+. 1H NMR: (400 MHz DMSO-d6): δ 8.68, 7.63 , 7.05–6.93 , 6.78 , 6.65 , 6.30, 3.68 , 3.19 , 2.98 ,2.73 , 2.10 , 1.39 .To a mixture of (5-(2-aminoethyl)pyridin-2-yl)(azetidin-1-yl)methanone and ethyl 3--1,2,4-oxadiazole-5-carboxylate in MeOH (1 mL) was added triethylamine 25 °C, and theresulting mixture was stirred at 60 °C for 12 h. The residuewas poured into water (20 mL). The aqueous phase was extracted withethyl acetate (10 mL × 3). The combined organic phase was washedwith brine (5 mL), dried with anhydrous Natert-butoxycarbonyl)amino)propanoic acid , HATU , and DIPEA in DCM (600 mL) was addedNH4Cl . The mixture wasstirred at 25 °C for 16 h, poured into H2O (500 mL),and extracted with EtOAc (700 mL × 3). The combined organic layerwas washed with brine (500 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by columnchromatography . tert-butyl (1-amino-3-(4-bromophenyl)-1-oxopropan-2-yl)carbamate  was obtained as a white solid. 1H NMR : δ 7.46 , 7.38 , 7.22 , 7.02 , 6.83 , 4.13–4.02 , 2.93 , 2.75–2.69 , 1.29 .To a solution of 3-(4-bromophenyl)-2-((tert-butyl(1-amino-3-(4-bromophenyl)-1-oxopropan-2-yl)carbamate  in EtOH (250 mL) were added triethylamine  and Pd(dppf)Cl2 . The mixture was stirred at 80 °C underCO  for 20 h, filtered, and concentrated. Theresidue was purified by column chromatography . Ethyl 4-(3-amino-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)benzoate was obtained as a brown solid. 1H NMR : δ 7.86, 7.40 , 7.03 , 6.87 , 4.29 , 4.12 , 3.03 , 2.80 ,1.30–1.26 , 1.25–1.09 .To a solution of tert-butoxycarbonyl)amino)-3-oxopropyl)benzoate  in EtOH (120 mL) was added a solution of LiOH in water (30 mL) at 25 °C. Themixture was stirred at 25 °C for 16 h, poured into water (150mL),and diluted with EtOAc (200 mL), and the aqueous phase was acidifiedto pH 2 with HCl (1 N) and diluted with EtOAc (100 mL × 3).The combined organic layer was washed with brine (100 mL), dried overNa2SO4, filtered, and concentrated. 4-(3-Amino-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)benzoic acid was obtained as a yellow solid. 1H NMR : δ 12.86–12.42, 7.84 , 7.37 , 7.03 , 6.86 , 4.12 , 3.02, 2.79 , 1.35–1.23 .To a solution of ethyl 4-(3-amino-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)benzoicacid  and azetidine  in DCM (60 mL) were added HATU  and DIPEA in turns at 25 °C. The mixture was stirred at 25 °C for16 h, poured into water (100 mL), and extracted with EtOAc (100 mL× 3). The combined organic layer was washed with brine (100 mL),dried over Na2SO4, filtered, and concentrated.The residue was purified by column chromatography . tert-Butyl (1-amino-3-(4-(azetidine-1-carbonyl)phenyl)-1-oxopropan-2-yl)carbamate was obtained as a yellow solid. To a solutionof tert-butyl (1-amino-3-(4-(azetidine-1-carbonyl)phenyl)-1-oxopropan-2-yl)carbamate in DCM (32 mL) was added TFA . The mixture was stirred at 25 °Cfor 1 h. The mixture was poured into NaHCO3 (40 mL) andextracted with EtOAc (40 mL × 3). The combined organic layerwas washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by reversed-phaseHPLC (0.1% NH3·H2O). 2-Amino-3-(4-(azetidine-1-carbonyl)phenyl)propanamide was obtained as a yellow solid. 1H NMR : δ 7.53, 7.36 , 7.29 , 6.98 , 4.29 , 4.03, 3.40–3.37 ,2.95 , 2.66 , 2.25 .To a solution of 4-. The mixture was stirredat 60 °C for 2 h, poured into H2O (15 mL), and extractedwith EtOAc (20 mL × 3). The combined organic layer was washedwith brine (20 mL), dried over Na2SO4, filtered,and concentrated. The residue was purified by prep-HPLC  and lyophilized. N-(1-Amino-3-(4-(azetidine-1-carbonyl)phenyl)-1-oxopropan-2-yl)-3--1,2,4-oxadiazole-5-carboxamide was obtained as a yellow solid. 1H NMR : δ 9.47, 7.71 , 7.65 , 7.52 , 7.37 , 7.30 ,7.17 , 4.77–4.58 ,4.25 , 4.00 , 3.85 ,3.25–3.21 , 3.15–3.06 , 2.22 .To a solution of 2-amino-3-(4-(azetidine-1-carbonyl)phenyl)propanamide and ethyl 3--1,2,4-oxadiazole-5-carboxylate in MeOH (10 mL) was added EtN-(1-amino-3-(4-(azetidine-1-carbonyl)phenyl)-1-oxopropan-2-yl)-3--1,2,4-oxadiazole-5-carboxamide in THF (1 mL) were added triethylamine and TFAA  in turns below 0 °C. The mixturewas stirred at 25 °C under N2 for 0.5 h, poured intoNaHCO3 (5 mL), and extracted with EtOAc (10 mL × 3).The combined organic layer was washed with brine (10 mL), dried overNa2SO4, filtered, and concentrated. The residuewas purified by prep-TLC  to give N-(2-(4-(azetidine-1-carbonyl)phenyl)-1-cyanoethyl)-3--1,2,4-oxadiazole-5-carboxamide as a pink solid. 1H NMR : δ 10.35–10.32 , 7.68–7.65 , 7.60–7.53 , 7.45- 7.42 , 7.20–7.17, 5.30–5.25 , 4.29–4.23 , 4.05–3.99, 3.88–3.83 , 3.37–3.27 , 2.29–2.20. HRMS (ESI): calcd for [M+H]+ C24H24N5O5, 462.1772, found 462.1790.To a solution of 2O  in water(3.00 mL) at 0 °C. The mixture was stirred at 50 °C for2 h and concentrated in vacuo. Lithium 3--1,2,4-oxadiazole-5-carboxylicacid  was obtained as a white solid.1H NMR: δ 7.60 , 7.50 , 7.12 , 3.83 .To a solution of ethyl 3--1,2,4-oxadiazole-5-carboxylate in MeOH (10.0 mL) was added a mixtureof LiOH·Htert-butoxycarbonyl) amino)-3-oxopropyl) benzoic acid , 3-methoxyazetidine , and HATU  in DMF(6 mL) was added DIPEA .The mixture was stirred at 20 °C for 5 h, poured into water (8mL), and extracted with EtOAc (10 mL × 3). The combined organiclayer was washed with brine (15 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified bycolumn chromatography . Impure tert-butyl(1-amino-3-(4-(3-methoxyazetidine-1-carbonyl)phenyl)-1-oxopropan-2-yl)carbamate was obtained as a white solid. To asolution of tert-butyl (1-amino-3-(4-(3-methoxyazetidine-1-carbonyl)phenyl)-1-oxopropan-2-yl)carbamate in DCM (2 mL) was added TFA . The mixture was stirred at 20°C for 1 h and concentrated in vacuo. The crudeproduct was used for next step directly. 2-Amino-3-(4-(3-methoxyazetidine-1-carbonyl)phenyl)propanamide was obtained as yellow oil. To a solutionof 2-amino-3-(4-(3-methoxyazetidine-1-carbonyl)phenyl)propanamide in DMF (1 mL) were addedHATU  and lithium 3--1,2,4-oxadiazole-5-carboxylicacid  at 0 °C. Then DIPEA was added to themixture at 0 °C and stirred at 20 °C for 12 h. The mixturewas poured into water (10 mL) and extracted with EtOAc (10 mL ×3). The combined organic layer was washed with brine (20 mL), driedover Na2SO4, filtered, and concentrated. Theresidue was purified by prep-HPLC –ACN];B%: 20–50%, 10 min) and lyophilized. N-(1-Amino-3-(4-(3-methoxyazetidine-1-carbonyl)phenyl)-1-oxopropan-2-yl)-3--1,2,4-oxadiazole-5-carboxamideimpure  was obtained as a white solid.To a solution of N-(1-amino-3-(4-(3-methoxyazetidine-1-carbonyl)phenyl)-1-oxopropan-2-yl)-3--1,2,4-oxadiazole-5-carboxamide in THF (0.20 mL) were added triethylamine and TFAA  at 0 °C under N2. The mixture was stirred at 20 °C for 0.5 h, pouredinto water (3 mL), and extracted with EtOAc (5 mL × 3). The combinedorganic layer was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. The residue was purifiedby prep-HPLC –ACN];B%: 20–50%, 10 min). N-(1-Cyano-2-(4-(3-methoxyazetidine-1-carbonyl)phenyl)ethyl)-3--1,2,4-oxadiazole-5-carboxamide was obtained as a yellow solid. 1H NMR : δ 10.00 , 7.69–7.66, 7.62–7.53 , 7.46–7.43 , 7.21–7.18, 5.29 , 4.44–4.39, 4.26–4.17 , 4.13–4.07 , 3.90–3.78, 3.37–3.31 , 3.21 . HRMS (ESI): calcdfor [M+H]+ C25H26N5O6, 492.1878, found 492.1882.To a solution of 4- and filtered to give N-(1-amino-1-oxo-3-phenylpropan-2-yl)-3--1,2,4-oxadiazole-5-carboxamide as a gray solid. 1H NMR : δ 9.42 , 7.61–7.73 , 7.52 , 7.24–7.31 , 7.15–7.21, 4.65 , 3.85 ,3.16–3.25 , 3.06 .To a solution of ethyl 3--1,2,4-oxadiazole-5-carboxylate in MeOH (25 mL) were added 2-amino-3-phenylpropanamide and triethylamine . The mixture was stirred at 60 °C for16 h, poured into water (40 mL), and extracted with EtOAc (50 mL ×3). The combined organic layers were washed with brine (50 mL), driedover NaN-(1-amino-1-oxo-3-phenylpropan-2-yl)-3--1,2,4-oxadiazole-5-carboxamide in THF (2 mL) were added triethylamine and TFAA  at 0 °C. The mixture was stirredat 25 °C for 0.5 h, poured into saturated NaHCO3 aqueoussolution (5 mL), and extracted with EtOAc (10 mL × 3). The combinedorganic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The residue was purifiedby prep-TLC  to give N-(1-cyano-2-phenylethyl)-3--1,2,4-oxadiazole-5-carboxamide as a white solid. 1H NMR : δ 10.35 ,7.67 , 7.53 , 7.30–7.40 , 7.23–7.29, 7.18 , 5.22 , 3.86 , 3.27 . HRMS (ESI): calcd for [M+H]+ C20H19N4O4, 379.1401, found 379.1391.To a solution of 2 and cooled in an ice bath. Triethylamine was added dropwise, followed by 2-oxa-6-azaspiro[3.3]heptane, and the reaction mixture was stirred with coolingfor 1 h, then a further hour at room temperature. The mixture wastreated with water (30 mL) and extracted with DCM (3 × 30 mL).The combined organics were dried (MgSO4), filtered, evaporated,and purified on silica, eluting with 100:0 to 99:1 DCM/MeOH. (4-(Chloromethyl)phenyl)(2-oxa-6-azaspiro[3.3]heptan-6-yl)methanonewas obtained as a white solid . 1H NMR : δ 7.66–7.63 , 7.48–7.45, 4.88–4.79 , 4.63 , 4.45–4.39. LC-MS: m/z 252 [M+H]+.4-(Chloromethyl)benzoyl chloride and DMAP  were dissolved in DCM(30 mL) under N4), filtered,evaporated, and purified on silica, eluting with 100:0 to 0:100 heptane/EtOActo give 3-(4-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)-2-((diphenylmethylene)amino)propanenitrile as a yellow foam. 1H NMR : δ 7.64–7.53 , 7.50–7.45 , 7.38 , 7.21–7.18 , 6.91 , 4.82 , 4.46–4.33, 3.33–3.23 .2-(Benzhydrylideneamino)acetonitrile and [4-(chloromethyl)phenyl]-(2-oxa-6-azaspiro[3.3]heptan-6-yl)methanone were dissolved in DCM (30 mL). Sodium hydroxide was added dropwise, followed by benzyltriethylammoniumchloride . The reaction mixture was stirred vigorouslyfor 1 h, diluted with water (30 mL), and extracted with DCM (3 ×50 mL). The combined organics were dried  and extracted with DCM (3 × 50mL). The combined organics were dried (MgSO4), filtered,and evaporated. The residue was triturated with heptane, and the resultingwhite solid was filtered and dried in vacuo overthe weekend to give impure 3-(4-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)-2-aminopropanenitrile. 1H NMR : δ7.66–7.63 , 7.40–7.37 , 4.84 ,4.47–4.45 , 4.39 , 4.02–3.92 ,3.11–3.07 . LC-MS: m/z 272 [M+H]+.3-(4-(2-Oxa-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)-2-((diphenylmethylene)amino)propanenitrile was dissolved in THF (52 mL), and hydrogen chloride was added dropwise. After stirring for 1h, the THF was removed in vacuo at 50 °C to give sodium 3--1,2,4-oxadiazole-5-carboxylate. 1H NMR : δ 7.60 , 7.51 , 7.14–7.11 , 3.86–3.84 .Ethyl 3--1,2,4-oxadiazole-5-carboxylate was suspended in ethanol (220 mL), and sodiumhydroxide  was added dropwise (exotherm to 28°C). The reaction mixture was stirred at room temperature for105 min and then evaporated, and the residual white solid was azeotropedwith toluene (×3) and then dried overnight 2, and DIPEA was added dropwise. The mixture was stirred at room temperature for75 min. The bulk of the DMF was removed in vacuo,and the residue was treated with water (75 mL) and extracted withEtOAc (3 × 200 mL). The combined organics were washed (brine),dried (Na2SO4), filtered, and evaporated. Theresidue was stirred with warm EtOAc and loaded onto a pad of silica.The pad was washed with EtOAc (3 column volumes) then 9:1 EtOAc/acetoneto elute the product. The filtrate was evaporated, and the residuewas triturated with EtOAc, filtered, and purified on silica, elutingwith 100:0 to 0:100 heptane/EtOAc. The product-containing cleanestfractions were combined and evaporated to a give a white foam anddried over the weekend at 50 °C. The foam was dissolved in DCM,evaporated to a white foam, and then dried at 50 °C under highvacuum for 2 h. NMR showed a trace of EtOAc and some DCM. The foamwas redissolved in ACN and evaporated then dried under high vacuumat 50 °C to give N-(2-(4-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)-1-cyanoethyl)-3--1,2,4-oxadiazole-5-carboxamide. 1H NMR : δ 10.35 , 7.69–7.67 , 7.60–7.53, 7.46–7.43 , 7.21–7.18 , 5.29, 4.66 , 4.47–4.40 , 4.23–4.17 , 3.87–3.86, 3.37–3.30 . 13C NMR :δ 168.9, 168.5, 168.4, 153.3, 152.4, 149.6, 138.7, 132.5, 129.8,128.3, 121.3, 118.4, 118.0, 112.5, 110.2, 80.0, 62.3, 58.2, 56.2,56.1, 42.2, 38.2, 37.2. HRMS (ESI): calcd for [M+H]+ C26H26N5O6, 504.1883, found504.1865.Sodium 3--1,2,4-oxadiazole-5-carboxylate, 3-(4-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)phenyl)-2-aminopropanenitrile, and HATU  were partially dissolvedin DMF (30 mL) under NZ)-N′,3-dihydroxy-4-methoxybenzimidamide and DIPEA  in THF (30.0 mL) was added ethyl 2-chloro-2-oxoacetate at 0 °C. The mixturewas stirred at 80 °C for 2 h and then concentrated invacuo. The crude product was purified by column chromatography. The eluant was concentrated in vacuo to give 2.38 g of yellow solid. Impure ethyl 3-(3-hydroxy-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate was obtained as a yellowsolid. 1H NMR : δ 7.75–7.64, 6.99–6.91 , 5.77 , 4.57 , 3.97 , 1.49 . LC-MS: m/z 265[M+H]+.To a solution of  and DEAD  at 0 °C under N2. The mixturewas stirred at 25 °C for 2 h and then concentrated. The crudeproduct was purified by prep-HPLC –ACN];B%:38–68%, 9 min). The eluent was concentrated and lyophilized.Impure ethyl 3-[3-[(1-hydroxycyclopropyl)methoxy]-4-methoxy-phenyl]-1,2,4-oxadiazole-5-carboxylatewas obtained . LC-MS: m/z 335 [M+H]+.To a solution of ethyl 3-(3-hydroxy-4-methoxy-phenyl)-1,2,4-oxadiazole-5-carboxylate and (1-tetrahydropyran-2-yloxycyclopropyl)methanol in THF (12 mL) were added PPh3N  at 0 °C. The mixturewas stirred at 0 °C for 2 h, diluted with DCM (100 mL), washedwith sat. NH4Cl (30 mL × 2) and brine (20 mL), driedover Na2SO4, filtered, and concentrated in vacuo. [4-(Chloromethyl)phenyl]-(3- methoxyazetidin-1-yl)methanone was obtained as a colorless oil. 1H NMR : δ 7.66–7.58 ,7.44 , 4.60 , 4.42–4.34, 4.29–4.22 , 4.17 , 4.08 , 3.32 .To a solution of 4-(chloromethyl)benzoylchloride  and 3-methoxyazetidine  in DCM (40.0 mL) was added Etin vacuo to give a residue. The residue was purified by silica gel chromatography(PE:EtOAc = 5:1–0:1). Impure 2-(benzhydrylideneamino)-3-[4-(3-methoxyazetidine-1-carbonyl)phenyl]propanenitrile was obtained as yellow oil. To a solutionof 2-(benzhydrylideneamino)-3-[4-(3-methoxyazetidine-1-carbonyl)phenyl]propanenitrile in dioxane (30.0 mL) was added HCl (1 M in water) at 20 °C. The mixturewas stirred at 20 °C for 3 h, poured into water (100 mL), andthen extracted with EtOAc (100 mL × 2). The aqueous phase wasbasified with sat. NaHCO3 adjusted to pH 12 and then extractedwith DCM:MeOH = 10:1 (100 mL × 4). The combined organic layerswere dried over Na2SO4, filtered, and concentrated in vacuo. Impure 2-amino-3-[4-(3-methoxyazetidine-1-carbonyl)phenyl]propanenitrile  was obtained as a yellowoil. To a solution of 2-amino-3-[4-(3-methoxyazetidine-1-carbonyl)phenyl]propanenitrile and ethyl 3-[3-[(1-hydroxycyclopropyl)methoxy]-4-methoxy-phenyl]-1,2,4-oxadiazole-5-carboxylate in MeOH (10.0 mL) was added Et3N  at 20 °C.The mixture was stirred at 40 °C for 16 h and then concentrated in vacuo to give a residue. The residue was purified byPrep-HPLC (water (0.225%FA)–ACN; B%: 30–60%, 10 min). N-[1-Cyano-2-[4-(3-methoxyazetidine-1-carbonyl)phenyl]ethyl]-3-[3-[(1-hydroxycyclopropyl)methoxy]-4-methoxy-phenyl]-1,2,4-oxadiazole-5-carboxamide was obtained as a white solid. 1H NMR : δ 10.38–10.32 ,7.67 , 7.61–7.57, 7.45–7.42 , 7.21–7.18 , 5.55, 5.28 , 4.43–4.36, 4.26–4.18 , 4.13–4.02 , 3.90–3.78, 3.37–3.31 , 3.25 , 0.76–0.68. LC-MS: m/z 548 [M+H]+.To a solution of 2-(benzhydrylideneamino)acetonitrile in THF (40.0 mL) was added NaOH  at 20 °C. The mixture was stirredat 20 °C for 30 min. A solution of [4-(chloromethyl)phenyl]-(3-methoxyazetidin-1-yl)methanone in THF (10.0 mL) was added to themixture at 0 °C. The mixture was stirred at 20 °C for 2h and then filtered. The filter cake was washed with EtOAc (10.0 mL× 3). The combined filtrate was concentrated N-[2-ethyl]-1,2,4-oxadiazole-5-carboxamide in DMF (5 mL) was added 60% sodium hydride , and the reaction mixture was stirred at rt for 10min. Methyl iodide  was added, and the reactionmixture was stirred at rt for 16 h, concentrated in vacuo, dissolved in EtOAc (10 mL), washed with water (5 × 10 mL),passed through a hydrophobic frit, and concentrated in vacuo. Purification by prep-HPLC afforded 3--N-[2-ethyl]-N-methyl-1,2,4-oxadiazole-5-carboxamide  as a colorlessoil. 1H NMR : δ 7.65–7.61 , 7.51–7.49 , 7.16, 6.89–6.62 , 3.86–3.81, 3.78 , 3.74–3.67 , 3.58 , 3.15–3.10, 2.90–2.85 . HRMS (ESI): calcd for [M+H]+ C22H26N3O6, 428.1822,found 428.1812.To a solution of 3--in vacuo. Purificationby prep-HPLC afforded N-methyl]-2-phenyl-ethanamine as a yellow oil. 1H NMR : δ 7.59 , 7.48 , 7.29–7.26, 7.23–7.21 , 7.19–7.16 , 7.13, 4.07 , 3.83 , 2.86–2.82 , 2.75–2.72. LC-MS: m/z 340 [M+H]+.To a solution of 5-(chloromethyl)-3--1,2,4-oxadiazole and 2-phenylethanamine  in DCM (5 mL) was added triethylamine . The reaction mixture was stirred at 40 °C for 16 h, dilutedwith DCM (10 mL), washed with water (3 × 10 mL), passed througha hydrophobic frit, and concentrated Z)-3-fluoro-N′-hydroxy-4-methoxybenzimidamide  and DIPEA  in THF(3 mL) was added ethyl 2-chloro-2-oxo-acetate  at 0 °C. The mixture was stirred at 25°C for 0.5 h and then at 80 °C for 2 h, poured into water(5 mL), and extracted with EtOAc (10 mL × 3). The combined organiclayer was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. Ethyl 3-(3-fluoro-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate was obtained as brown solid. To a solution ofethyl 3-(3-fluoro-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate in MeOH (2 mL) were added [4-(2-aminoethyl)phenyl]-(azetidin-1-yl)methanone and triethylamine . The mixture was stirred at 60 °Cfor 16 h, poured into water (5 mL), and extracted with EtOAc (10 mL× 3). The combined organic layer was washed with brine (10 mL),dried over Na2SO4, filtered, and concentrated.The crude product was washed with MeOH (10 mL) and filtered to give N-(4-(azetidine-1-carbonyl)phenethyl)-3-(3-fluoro-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxamide as a gray solid. 1H NMR : δ 9.54 , 7.91–7.76 , 7.56, 7.41 , 7.33 , 4.28, 4.02 , 3.94 , 3.55 , 2.93 , 2.27–2.20. LC-MS: m/z 425 [M+H]+.To a solution of (Z)-4-fluoro-N′-hydroxy-3-methoxybenzimidamide  and DIPEA  in THF(3 mL) was added ethyl 2-chloro-2-oxo-acetate  at 0 °C. The mixture was stirred at 25°C for 0.5 h and then stirred at 80 °C for 2 h, poured intowater (5 mL), and extracted with EtOAc (10 mL × 3). The combinedorganic layer was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. Ethyl 3-(4-fluoro-3-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate was obtained as a brown oil. To a solution ofethyl 3-(4-fluoro-3-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate and [4-(2-aminoethyl)phenyl]-(azetidin-1-yl)methanone in MeOH (2 mL) was added triethylamine. The mixture was stirredat 60 °C for 16 h, poured into water (5 mL), and extracted withEtOAc (10 mL × 3). The combined organic layer was washed withbrine (10 mL), dried over Na2SO4, filtered,and concentrated. The residue was purified by prep-TLC . N-(4-(azetidine-1-carbonyl)phenethyl)-3-(4-fluoro-3-methoxyphenyl)-1,2,4-oxadiazole-5-carboxamide was obtained as a white solid. 1H NMR : δ 9.58 , 7.73 , 7.66 , 7.56 , 7.46 , 7.33, 4.27 , 4.02 , 3.95, 3.56 , 2.94 , 2.24 . LC-MS: m/z 425 [M+H]+.To a solution of (Z)-N′-hydroxy-1H-benzo[d]imidazole-6-carboximidamide  and DIPEA in THF (3 mL) was dropwiseadded ethyl 2-chloro-2-oxo-acetate  at 0 °C, then the solution was stirred at 25 °Cfor 0.5 h, then at 80 °C for 2 h. The mixture was concentratedto get the residue. The residue was poured into water (30 mL). Theaqueous phase was extracted with ethyl acetate (10 mL × 3). Thecombined organic phase was washed with brine (5 mL), dried with anhydrousNa2SO4, filtered, and concentrated invacuo. The crude product was used to the next step withoutpurification . To a solution of the residue and [4-(2-aminoethyl)phenyl](azetidin-1-yl) in MeOH (1 mL) was added triethylamine, and the reaction was stirredat 60 °C for 12 h. The mixture was cooled to rt, and the precipitatewas collected, washed with MeOH (5 mL), and concentrated to afford N-(4-(azetidine-1-carbonyl)phenethyl)-3-(1H-benzo[d]imidazol-6-yl)-1,2,4-oxadiazole-5-carboxamide was obtained as a purple solid. 1H NMR : δ 12.87 , 9.59 , 8.40 , 8.30 , 7.92, 7.79 , 7.57 , 7.35 , 4.29 , 4.03 , 3.58 , 2.96 , 2.25 . LC-MS: m/z 417 [M+H]+.To a mixture of  and DIPEA  in THF (3 mL) was dropwise added ethyl 2-chloro-2-oxo-acetate at 0 °C. The solutionwas stirred at 25 °C for 0.5 h and then at 80 °C for 2 h.The mixture was concentrated to get the residue, which was pouredinto water (30 mL). The aqueous phase was extracted with ethyl acetate(10 mL × 3). The combined organic phase was washed with brine(5 mL), dried with anhydrous Na2SO4, filtered,and concentrated in vacuo. The residue was purifiedby prep-TLC . Ethyl 3-(benzofuran-6-yl)-1,2,4-oxadiazole-5-carboxylate was obtained as yellow solid. To a solution of[4-(2-aminoethyl)phenyl]-(azetidin-1-yl)methanone  and ethyl 3-(benzofuran-6-yl)-1,2,4-oxadiazole-5-carboxylate in MeOH (1 mL) was added triethylamine, and the resulting mixturewas stirred at 60 °C for 12 h. The mixture was concentrated toget the residue. The residue was purified by prep-HPLC –ACN]; B%: 32–62%, 10 min). N-(4-(Azetidine-1-carbonyl)phenethyl)-3-(benzofuran-6-yl)-1,2,4-oxadiazole-5-carboxamide was obtained as a white solid. 1H NMR : δ 8.26 , 8.02 , 7.93 , 7.79, 7.59 , 7.40 , 6.96 , 4.37 , 4.19 , 3.70 , 3.03 ,2.36 . HRMS (ESI): calcd for[M+H]+ C23H21N4O4, 417.1563, found 417.1578.To a mixture of Z)-N′-hydroxy-1H-indole-6-carboximidamide and DIPEA  in THF (3 mL) was added ethyl 2-chloro-2-oxo-acetate  at 0 °C. The mixture wasstirred at 25 °C for 0.5 h and then at 80 °C for 2 h, pouredinto water (5 mL), and extracted with EtOAc (10 mL × 3). Thecombined organic layer was washed with brine (15 mL), dried over Na2SO4, filtered, and concentrated. Ethyl 3-(1H-indol-6-yl)-1,2,4-oxadiazole-5-carboxylate  was obtained as a white solid. To a solution of ethyl 3-(1H-indol-6-yl)-1,2,4-oxadiazole-5-carboxylate  and [4-(2-aminoethyl)phenyl]-(azetidin-1-yl)methanone in MeOH (3 mL) was added triethylamine. The mixture was stirredat 60 °C for 16 h. The mixture was poured into water (5 mL) andextracted with EtOAc (10 mL × 3). The combined organic layerwas washed with brine (15 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by prep-HPLC–ACN]; B%: 25–55%,10 min) and lyophilized to give N-(4-(azetidine-1-carbonyl)phenethyl)-3-(1H-indol-6-yl)-1,2,4-oxadiazole-5-carboxamide  as yellow solid. 1H NMR : δ = 11.52 , 9.55 , 8.13 , 7.65–7.80 ,7.46–7.63 , 7.34 ,6.55 , 4.21–4.38, 4.02 , 3.51–3.63, 2.95 , 2.20–2.27. HRMS (ESI): calcd for [M+H]+ C23H22N5O3, 416.1717, found 416.1728.To a solution of (Z)-N′-hydroxy-1H-indazole-6-carboximidamide and DIPEA  in THF (3 mL) was added ethyl 2-chloro-2-oxo-acetate  at 0 °C. The mixture wasstirred at 25 °C for 0.5 h and then at 80 °C for 2 h, pouredinto water (8 mL), and extracted with EtOAc (15 mL × 3). Thecombined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. Ethyl 3-(1H-indazol-6-yl)-1,2,4-oxadiazole-5-carboxylate  was obtained as a brown solid. To a solution of ethyl3-(1H-indazol-6-yl)-1,2,4-oxadiazole-5-carboxylate and [4-(2-aminoethyl)phenyl]-(azetidin-1-yl)methanone in MeOH (3 mL) was added triethylamine. The mixture was stirredat 60 °C for 16 h. The mixture was poured into water (5 mL) andextracted with EtOAc (10 mL × 3). The combined organic layerwas washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by prep-HPLC–ACN];B%: 20–50%,10 min) and lyophilized to give N-(4-(azetidine-1-carbonyl)phenethyl)-3-(1H-indazol-6-yl)-1,2,4-oxadiazole-5-carboxamide  as a white solid. 1H NMR : δ 13.48 , 9.47–9.72, 8.23 , 7.98 , 7.78 ,7.56 , 7.34 , 4.28 , 3.98–4.08 , 3.54–3.65, 2.95 , 2.21–2.27. HRMS (ESI): calcd for [M+H]+ C22H21N6O3, 417.1670, found 417.1670.To a solution of (Z)-N′-hydroxy-3-methoxybenzimidamide  and DIPEA  in THF(3 mL) was added ethyl 2-chloro-2-oxo-acetate  at 0 °C. The mixture was stirred at 25°C for 0.5 h and then at 80 °C for 2 h, poured into water(5 mL), and extracted with EtOAc (10 × 3). The combined organiclayer was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. Ethyl 3-(3-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate was obtained as a brown oil.To a solution of ethyl 3-(3-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate and [4-(2-aminoethyl)phenyl]-(azetidin-1-yl)methanone in MeOH (2 mL) was added triethylamine. The mixture was stirredat 60 °C for 16 h. The mixture was poured into water (5 mL) andextracted with EtOAc (10 mL × 3). The combined organic layerwas washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by prep-HPLC–ACN]; B%: 30–60%,10 min) and lyophilized to give N-(4-(azetidine-1-carbonyl)phenethyl)-3-(3-methoxyphenyl)-1,2,4-oxadiazole-5-carboxamide was obtained as a yellow solid.1H NMR : δ 9.45–9.70, 7.64 , 7.47–7.60 , 7.33 , 7.21 , 4.28 , 4.03 ,3.85 , 3.52–3.64 , 2.94 , 2.22–2.27 . HRMS (ESI): calcd for [M+H]+ C22H23N4O4, 407.1730,found 407.1719.To a solution of (tert-butoxycarbonylamino)ethyl]benzoicacid  and azetidine  in DCM (30 mL) were added DIPEA  and HATU . The mixture was stirred at25 °C for 4 h, poured into water (20 mL), and extracted withEtOAc (30 mL × 3). The combined organic layer was washed withbrine (40 mL), dried over Na2SO4, filtered,and concentrated. The residue was purified by column chromatography. Compound tert-butyl N-[2-[4-(azetidine-1-carbonyl) phenyl]ethyl]carbamate was obtained as yellow oil. To a solutionof tert-butyl N-[2-[4-(azetidine-1-carbonyl)phenyl]ethyl]carbamate in DCM (20 mL) was added TFA . The mixture was stirred at 25 °C for 0.5 h, poured intoNaHCO3 (70 mL), and extracted with EtOAc (100 mL ×3). The combined organic layer was washed with brine (10 mL), driedover Na2SO4, filtered, and concentrated. Thecrude product was purified by reversed-phase HPLC (0.1% NH3·H2O). (4-(2-Aminoethyl)phenyl)(azetidin-1-yl)methanone was obtained as a brown oil. LC-MS: m/z 205 [M+H]+.To a solution of 4-+.To a solution of 3-hydroxy-4-methoxy-benzonitrile and bromomethylcyclopropane  in DMF (20 mL) was added K2O (20 mL), and extracted with EtOAc(20 mL × 3). The combined organic layer was washed with brine(20 mL), dried over Na2SO4, filtered, and concentrated.(Z)-3-(Cyclopropylmethoxy)-N′-hydroxy-4-methoxybenzimidamide was obtained as a white solid. The crudeproduct was used to the next step directly. LC-MS: m/z 237 [M+H]+.To a solution of 3-(cyclopropylmethoxy)-4-methoxybenzonitrile and hydroxylamine  in EtOH (15 mL) was added DIPEA . The mixture was stirred at 80 °Cfor 12 h, poured into HZ)-3-(cyclopropylmethoxy)-N′-hydroxy-4-methoxybenzimidamide  and DIPEA  inTHF (6 mL) was added ethyl 2-chloro-2-oxo-acetate  at 0 °C. The mixture was stirred at20 °C for 0.5 h and then at 60 °C for 2 h, poured into water(10 mL), and extracted with EtOAc (10 mL × 3). The combined organiclayer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. Ethyl 3-[3-(cyclopropylmethoxy)-4-methoxy-phenyl]-1,2,4-oxadiazole-5-carboxylate was obtained as colorless oil. To a solutionof ethyl 3-[3-(cyclopropylmethoxy)-4-methoxy-phenyl]-1,2,4-oxadiazole-5-carboxylate and [4-(2-aminoethyl)phenyl]-(3-methoxyazetidin-1-yl)methanone in MeOH (4 mL) was added triethylamine . The mixture was stirred at 60 °C for 2h, poured into H2O (10 mL), and extracted with EtOAc (30mL × 3). The combined organic layer was washed with brine (20mL), dried over Na2SO4, filtered, and concentrated.The residue was purified by prep-HPLC –ACN]; B%: 30–60%, 11.5min) and lyophilized. 3-[3-(cyclopropylmethoxy)-4-methoxy-phenyl]-N-[2-[4-(3-methoxyazetidine-1-carbonyl)phenyl]ethyl]-1,2,4-oxadiazole-5-carboxamide was obtained as a white solid. 1H NMR : δ 7.67 , 7.64–7.58 , 7.56 , 7.31 , 7.18 , 6.97 ,4.39 , 4.25 , 4.21–4.02, 3.95 , 3.95 , 3.93 , 3.78 , 3.32 , 3.03 , 1.45–1.31 , 0.75–0.61 ,0.46–0.32 . HRMS (ESI): calcd for [M+H]+ C27H31N4O6, 507.2238, found507.2238.To a solution of  at 20 °C. The mixture was stirred at40 °C for 16 h and then at 60 °C for 3 h, poured into water(100 mL), and then extracted with EtOAc (50 mL × 3). The combinedorganic layers were dried over Na2SO4, filtered,and concentrated in vacuo to give a residue. Theresidue was purified by silica gel chromatography (PE:EtOAc = 100:1–10:1).3-Cyclobutoxy-4-methoxybenzonitrile  was obtainedas a white solid. 1H NMR : δ7.27 , 6.95–6.86 , 4.71–4.58 ,3.92 , 2.56–2.42 , 2.34–2.18 ,1.96–1.83 , 1.80–1.66 . LC-MS: m/z 204 [M+H]+.To a solution of 3-cyclobutoxy-4-methoxybenzonitrile and bromocyclobutane  in DMF (16 mL) was added K2O (10 mL), and extracted with EtOAc (30 mL × 3).The combined organic layer was washed with brine (20 mL), dried overNa2SO4, filtered, and concentrated. The crudeproduct was washed with EtOH (5 mL) and filtered. (Z)-3-Cyclobutoxy-N′-hydroxy-4-methoxybenzimidamide was obtained as a white solid. 1H NMR : δ 9.46, 7.20 , 7.10 , 6.93 ,5.70 , 4.70–4.55 , 3.76 , 2.44–2.30, 2.15–1.94 , 1.84–1.55 . LC-MS: m/z 237 [M+H]+.To a solutionof 3-(cyclobutoxy)-4-methoxybenzonitrile  and hydroxylamine in EtOH (20 mL) was added DIPEA . The mixture was stirred at 80 °C for 12 h, poured intoHZ)-3-cyclobutoxy-N′-hydroxy-4-methoxybenzimidamide  and DIPEA in THF (6 mL) was added ethyl 2-chloro-2-oxo-acetate  at 0 °C. The mixture was stirredat 20 °C for 0.5 h and then at 60 °C for 2 h, poured intoH2O (10 mL), and extracted with EtOAc (10 mL × 3).The combined organic layer was washed with brine (20 mL), dried overNa2SO4, filtered, and concentrated. Ethyl 3-(3-cyclobutoxy-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate was obtained as yellow oil. To a solution ofethyl 3-[3-(cyclobutoxy)-4-methoxyphenyl]-1,2,4-oxadiazole-5-carboxylate and [4-(2-aminoethyl)phenyl]-(3-methoxyazetidin-1-yl)methanone in MeOH (4 mL) was added triethylamine. The mixture was stirredat 60 °C for 2 h, poured into H2O (6 mL), and extractedwith EtOAc (10 mL × 3). The combined organic layer was washedwith brine (20 mL), dried over Na2SO4, filtered,and concentrated. The residue was purified by prep-HPLC –ACN]; B%: 30–60%,11.5 min) and lyophilized. 3-[3-(cyclobutoxy)-4-methoxy-phenyl]-N-[2-[4-(3-methoxyazetidine-1-carbonyl)phenyl]ethyl]-1,2,4-oxadiazole-5-carboxamide was obtained as a white solid. 1H NMR : δ 7.72–7.57, 7.42 , 7.31 , 7.17 ,6.96 , 4.77 , 4.39 , 4.30–4.22, 4.21–4.03 , 3.95 , 3.79 , 3.32 , 3.03 , 2.61–2.47 , 2.40–2.19 ,1.90–1.70 , 1.81–1.66 . HRMS (ESI): calcdfor [M+H]+ C27H31N4O6, 507.2238, found 507.2246.To a solution of  and KI .The mixture was stirred at 140 °C for 12 h, poured into H2O (30 mL), and extracted with EtOAc (20 mL × 3). Thecombined organic layer was washed with brine (40 mL), dried over Na2SO4, filtered, and concentrated. The residue waspurified by column chromatography . 3-Cyclopropoxy-4-methoxybenzonitrile  was obtained as a white solid. 1H NMR : δ 7.48 ,7.30 , 6.89 , 3.91 , 3.80–3.71 ,0.92–0.81 . LC-MS: m/z 190 [M+H]+.To a solution of 3-hydroxy-4-methoxybenzonitrile and bromocyclopropane  in dry DMF (30 mL) were added Cs2O (10 mL), and extracted with EtOAc(10 mL × 3). The combined organic layer was washed with brine(20 mL), dried over Na2SO4, filtered, and concentrated.(Z)-3-cyclopropoxy-N′-hydroxy-4-methoxybenzimidamide  was obtained as a whitesolid. LC-MS: m/z 223 [M+H]+.To a solution of 3-cyclopropoxy-4-methoxybenzonitrile and hydroxylamine  in EtOH (6 mL) was added DIPEA . The mixture was stirred at 70 °Cfor 12 h, poured into HZ)-3-cyclopropoxy-N′-hydroxy-4-methoxybenzimidamide  and DIPEA  inTHF (8 mL) was added ethyl 2-chloro-2-oxo-acetate  at 0 °C. The mixture was stirred at60 °C for 2 h, poured into H2O (10 mL), and extractedwith EtOAc (10 mL × 3). The combined organic layer was washedwith brine (20 mL), dried over Na2SO4, filtered,and concentrated. The residue was purified by column chromatography to afford ethyl 3-(3-cyclopropoxy-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate. To a solution of ethyl 3-(3-cyclopropoxy-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate and [4-(2-aminoethyl)phenyl]-(3-methoxyazetidin-1-yl)methanone in MeOH (4 mL) was addedtriethylamine .The mixture was stirred at 70 °C for 2 h, poured into H2O (10 mL), and extracted with EtOAc (10 mL × 3). The combinedorganic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The residue was purifiedby prep-HPLC –ACN]; B%: 45–75%,9 min) and lyophilized. 3-(3-Cyclopropoxy-4-methoxyphenyl)-N-(4-(3-methoxyazetidine-1-carbonyl) phenethyl)-1,2,4-oxadiazole-5-carboxamide was obtained as a white solid. 1H NMR : δ 9.53 , 7.84 , 7.67 , 7.58 , 7.34 , 7.17 , 4.41 ,4.29–4.17 , 4.16–4.05 , 3.93 , 3.83 , 3.63–3.51 , 3.21 , 2.94 , 0.88–0.79, 0.76–0.66 . LC-MS: m/z 493 [M+H]+.To a solution of  and DEAD  at 0 °C under N2. The mixturewas stirred at 25 °C for 2 h and then concentrated. The crudeproduct was purified by prep-HPLC –ACN];B%: 38–68%, 9 min). The eluent was concentrated and lyophilized.Impure ethyl 3-[3-[(1-hydroxycyclopropyl)methoxy]-4-methoxy-phenyl]-1,2,4-oxadiazole-5-carboxylatewas obtained .To a solutionof (4-(2-aminoethyl)phenyl)(3-methoxyazetidin-1-yl)methanone and triethylamine  in MeOH (10.0 mL) was added ethyl3-(3-((1-hydroxycyclopropyl)methoxy)-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate, and the reaction was stirred at 60°C for 2 h. The mixture was concentrated to 5.00 mL and purifiedby prep-HPLC –ACN]; B%: 25–55%, 11.5 min), and the eluent was concentratedand lyophilized. 3-(3-((1-Hydroxycyclopropyl)methoxy)-4-methoxyphenyl)-N-(4-(3-methoxyazetidine-1-carbonyl)phenethyl)-1,2,4-oxadiazole-5-carboxamide was obtained as a white solid. 1H NMR : δ 9.52 , 7.68–7.65 , 7.60–7.57 ,7.36–7.33 , 7.20–7.18 , 5.55 ,4.46–4.49 , 4.26–4.21 , 4.12–4.10, 4.05–4.04 , 3.90–3.53 , 3.61–3.53, 3.23–3.22 , 2.95 , 0.73–0.64 . LC-MS: m/z 454 [M+H]+.To a solution of ethyl 3-(3-hydroxy-4-methoxy-phenyl)-1,2,4-oxadiazole-5-carboxylate and (1-tetrahydropyran-2-yloxycyclopropyl)methanol in THF (12 mL) were added PPhZ)-N′,3-dihydroxy-4-methoxybenzimidamide  and DIPEA in THF (30.0 mL) was added ethyl 2-chloro-2-oxoacetate  at 0 °C. The mixture was stirred at80 °C for 2 h and then concentrated in vacuo. The crude product was purified by column chromatography , and the eluant was concentrated in vacuo. Impure ethyl 3-(3-hydroxy-4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate was obtained as a yellowsolid. 1H NMR : δ 7.75–7.64, 6.99–6.91 , 5.77 , 4.57 , 3.97 , 1.49 . LC-MS: m/z 265[M+H]+.To a solution of (tert-butoxycarbonylamino)ethyl]benzoicacid , 3-methoxyazetidine , and HATU  inDCM (30 mL) was added DIPEA .The mixture was stirred at 25 °C for 16 h and then concentrated in vacuo to remove DCM. The residue was diluted with H2O (50 mL) and extracted with EtOAc (50 mL × 3). The organiclayers were combined, washed with brine (50 mL), dried over Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography , the eluent was concentrated in vacuo. tert-Butyl N-[2-[4-(3-methoxyazetidine-1-carbonyl)phenyl]ethyl]carbamate  was obtained as a colorless oil. 1HNMR : δ 7.55 , 7.26 ,6.91 , 4.41 , 4.27–4.16 , 4.11 , 3.82 , 3.21 , 3.19–3.10 , 2.73 , 1.35 . To a solution of tert-butyl N-[2-[4-(3-methoxyazetidine-1-carbonyl)phenyl]ethyl]carbamate in dioxane (5 mL) was added HCl/dioxane at 0 °C. The mixture was stirred at25 °C for 1 h and then concentrated in vacuo. (4-(2-Aminoethyl)phenyl)(3-methoxyazetidin-1-yl)methanone was obtained as a yellow oil. LC-MS: m/z 235 [M+H]+.To a solution of 4-acetonitrile as a yellow solid. 1H NMR : δ 7.00 , 6.94 , 4.10, 3.83 , 3.76 , 3.75 , 3.27 .To a solution of 3--3-oxo-propanenitrile in toluene (10 mL) were added ethylene glycol and 4-methylbenzenesulfonic acid hydrate . The reaction mixture was heated at 110 °C for 16h, concentrated 3/methanol , and the reaction mixture was stirred at rt for 30min. Quinolin-8-ol  was added, followed by a solutionof -1,3-dioxolan-2-yl]acetonitrile  in methanol (1 mL). The reaction mixture was heatedat 70 °C for 16 h, concentrated in vacuo, azeotropedwith toluene (3 × 10 mL), dissolved in ethanol (5 mL), acidifiedto pH 1 with conc. HCl, and heated at 120 °C overnight. The reactionmixture was cooled and concentrated in vacuo thendissolved in DCM (10 mL) and washed with sat. NaHCO3 (10mL). The aqueous layer was extracted with DCM (2 × 10 mL), andthe combined organics were passed through a hydrophobic frit and concentrated in vacuo. Purification by flash column chromatography afforded5-isoxazol-3-amine  as a yellowsolid. 1H NMR : δ 7.31 , 7.3 , 7.04 ,6.21 , 5.57 , 3.82 , 3.80 . LC-MS: m/z 221 [M+H]+.To a solution of hydroxylamine hydrochloride  in methanol (1 mL) was added 7 M NHinvacuo, and azeotroped with DCM (3 × 10 mL) then dissolvedin DMF (5 mL). 5-isoxazol-3-amine  was added followed by triethylamine , and the reaction mixture was stirred at 50 °C for16 h. The reaction mixture was concentrated in vacuo then diluted with EtOAc (10 mL), washed with water (3 × 10mL), passed through a hydrophobic frit, and concentrated invacuo. Purification by prep-HPLC afforded 3--N-[5-isoxazol-3-yl]propenamide as a white solid. 1H NMR : δ 10.96 , 7.43 , 7.39 , 7.28 , 7.08 ,6.86–6.84 , 6.74 , 3.85 , 3.82 , 3.73 , 3.71 , 2.85, 2.69 . LC-MS: m/z 413 [M+H]+.To a solution of 3-propanoicacid  in DCM (5 mL) was added thionyl chloride , and the reaction mixture was heated at 40 °Covernight. The reaction mixture was cooled, concentrated in vacuo, DMF (3 mL) was added, and the reaction mixtureheated at 150 °C overnight. The cooled reaction mixture was partitionedbetween water (20 mL) and EtOAc (20 mL). The EtOAc extract was washedwith water (2 × 20 mL) and evaporated in vacuo. The residue was purified by silica (12 g) eluting with 0–100%EtOAc/heptane to afford ethyl 5--1,2,4-oxadiazole-3-carboxylate. 1H NMR : δ7.88 , 7.70 , 7.04–7.01 , 4.57 , 4.01 ,1.50 . LC-MS: m/z 279 [M+H]+.A solution of ethyl 2-(hydroxyamino)-2-iminoacetate in DCM (5 mL) was cooled to 0 °C, and triethylamine was added, followed by 3,4-dimethoxybenzoyl chloride portionwise. The reaction mixture was stirredat room temperature overnight. The reaction mixture was evaporated 3N . The reactionmixture was stirred at 60 °C overnight, concentrated in vacuo, dissolved in EtOAc (10 mL), and washed with water(10 mL), and the aqueous phase was extracted further with EtOAc (2× 10 mL). The EtOAc extracts were evaporated in vacuo. The residue was purified by mass-directed HPLC 5–95% MeCNbasic to afford 5--N-phenethyl-1,2,4-oxadiazole-3-carboxamide. 1H NMR : δ 9.09–9.04, 7.78 , 7.62 , 7.34–7.22 , 3.90–3.89, 3.56–3.50 , 2.91–2.87 . HRMS(ESI): calcd for [M+H]+ C19H20N3O4, 354.1454, found 354.1444.To a solution of ethyl 5--1,2,4-oxadiazole-3-carboxylate and phenethylamine  inMeOH (5 mL) was added Et3PO4 , and the reaction mixturewas degassed with nitrogen for 10 min. Pd(dtbpf)Cl2  was added, and the reaction mixture was degassed withnitrogen for 10 min then heated at 80 °C for 16 h. The reactionmixture was cooled, filtered through Celite, and washed with EtOAc(3 × 5 mL). The combined organics were washed with water (3 ×10 mL), passed through a hydrophobic frit, and concentrated in vacuo to afford methyl 4-furan-2-carboxylateas an orange solid. 1H NMR : δ 8.41 ,7.84 , 7.28 , 7.23 ,6.97 , 3.84 , 3.82 , 3.77 . LC-MS: m/z 263[M+H]+.To a mixture of methyl 4-bromofuran-2-carboxylate and boronic acid  in 1,4-dioxane (4 mL) and water (1 mL) was added Kin vacuo to afford 4-furan-2-carboxylicacid as a brown solid. 1H NMR : δ 13.07 , 8.34 , 7.71 ,7.26 , 7.21 , 6.97 , 3.82 , 3.77 . LC-MS: m/z 248 [M+H]+.To a solution of methyl 4-furan-2-carboxylate in water (5 mL) and ethanol (5 mL) was added sodiumhydroxide . The reaction mixture was heated at80 °C for 1 h, cooled, neutralized with 2 M HCl, extracted withEtOAc (3 × 10 mL), passed through a hydrophobic frit, and concentrated in vacuo, diluted with EtOAc(20 mL), washed with water (5 × 20 mL), passed through a hydrophobicfrit, and concentrated in vacuo. Purification byprep-HPLC afforded 4--N-(2-phenylethyl)furan-2-carboxamide  as anoff-white solid. 1H NMR : δ 8.44 ,8.25 , 7.52 , 7.31–7.28 , 7.25–7.16 ,6.97 , 3.82 , 3.77 , 3.49–3.45 , 2.84 . HRMS (ESI): calcd for [M+H]+ C21H22NO4, 352.1557, found 352.1549.To a solution of 4-furan-2-carboxylic, HATU , and triethylamine in DMF (5 mL) was added 2-phenylethanamine. The reaction mixture was stirred at rt overnightfor 16 h, concentrated in vacuo, diluted with EtOAc (10 mL), washed with water(5 × 10 mL), passed through a hydrophobic frit, and concentrated in vacuo. Purification by flash column chromatography afforded3-bromo-N-(2-phenylethyl)benzamide  as a white solid. 1H NMR : δ 8.69 ,7.98 , 7.83–7.80 ,7.74–7.71 , 7.43 ,7.32–7.18 , 3.50–3.45 , 2.84 . LC-MS: m/z 304/306 [M+H]+.To a solutionof 3-bromobenzoic acid ,HATU , and triethylamine  in DMF (5 mL) was added 2-phenylethanamine . The reaction mixture was stirred at rt for 16 h, concentrated N-(2-phenylethyl)benzamide  and boronicacid  in water (1 mL) and 1,4-dioxane (4 mL) wasadded K3PO4 , and the reactionmixture was degassed with nitrogen for 10 min. Pd(dtbpf)Cl2  was added, and the reaction mixture was degassedwith nitrogen for 10 min then heated at 80 °C for 16 h. The reactionmixture was cooled, filtered through Celite, and washed with EtOAc(3 × 5 mL). The combined organics were washed with water (3 ×10 mL), passed through a hydrophobic frit, and concentrated in vacuo. Purification by prep-HPLC afforded 3--N-(2-phenylethyl)benzamide  as an off-whitesolid. 1H NMR : δ 8.03 , 7.79 , 7.79 , 7.75 , 7.51, 7.32–7.24 , 7.22–7.20, 7.08–7.06 , 3.86 , 3.81 , 3.54–3.50, 2.87 . HRMS (ESI): calcdfor [M+H]+ C23H24NO3,362.1751, found 362.1767.To a mixture of 3-bromo-Z)-2-amino-2-hydrazono)acetate was obtained as a white solid. 1HNMR : δ 9.94 , 7.50 , 7.43–7.35, 7.04 , 6.76 ,4.26 , 3.82 6H), 1.29 .A mixture of 3,4-dimethoxybenzohydrazide and ethyl 2-amino-2-thioxo-acetate  was stirred at 180 °C for 1 h. To themixture was added DMSO (10 mL), and the precipitate was washed withEtOH (5 mL) and concentrated to get the residue. The residue was usedto the next step without purification. Ethyl (Z)-2-amino-2-hydrazono)acetate in AcOH (3 mL) was stirred at 100 °Cfor 1 h. The pH of solution was adjusted to 8 by aq. NaHCO3, the aqueous phase was extracted with ethyl acetate (10 mL ×3). The combined organic phase was washed with brine (5 mL), driedwith anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by prep-HPLC . Ethyl 3--1H-1,2,4-triazole-5-carboxylate was obtained as a white solid. LC-MS: m/z 278 [M+H]+.To a solution of  and(4-(2-aminoethyl)phenyl)(azetidin-1-yl)methanone  in MeOH (1 mL) was added triethylamine. The mixture was stirredat 60 °C for 12 h and then concentrated to get the residue. Theresidue was purified by prep-HPLC . N-(4-(Azetidine-1-carbonyl)phenethyl)-3--1H-1,2,4-triazole-5-carboxamide  was obtained as a yellow solid. 1H NMR :δ 7.65–7.55 , 7.38 , 7.05 , 4.35 , 4.17 , 3.90 , 3.66 , 2.99 ,2.34 . HRMS (ESI): calcd for[M+H]+ C23H26N5O4, 436.1979, found 436.1976.To a solution of ethyl 3--1in vacuo, and purified by column chromatography , and the eluant was concentrated.Ethyl 2-hydrazinyl)-2-oxoacetate was obtainedas a white solid.  1H NMR : δ 9.84 , 9.05 ,7.49–7.33 , 6.90 ,4.43 , 3.94 , 1.43 .To a solution of 3,4-dimethoxybenzohydrazide and triethylamine  in DCM (6.00 mL) was added ethyl 2-chloro-2-oxoacetate dropwise at 0 °C.The mixture was stirred at 25 °C for 16 h, concentrated 2, PE:EtOAc = 1:0 to 2:1), and the eluant was concentrated.Ethyl 5--1,3,4-oxadiazole-2-carboxylatewas obtained as a white solid . 1H NMR : δ 7.77 , 7.66 , 7.00 , 4.56 , 3.99 ,1.50 . LC-MS: m/z 279 [M+H]+.To a solution of ethyl 2-hydrazinyl)-2-oxoacetate and triethylamine  in DCM (6.00 mL) was added a solutionof pTsOH  in DCM (1.00 mL) at 0 °C.The mixture was stirred at 25 °C for 16 h and then concentratedto 2 mL. The crude product was purified by column chromatography (SiON-(4-(3-methoxyazetidine-1-carbonyl)phenethyl)-1,3,4-oxadiazole-2-carboxamidewas obtained as a brown solid. . 1H NMR : δ 9.43 , 7.67 ,7.60–7.53 , 7.36–7.32 , 7.22–7.19, 4.42 , 4.23–4.19 , 4.13–4.05, 3.89–3.80 , 3.56 , 3.21 , 2.97–2.91 . HRMS (ESI) calcdfor [M+H]+ C24H27N4O6, 467.1931, found 467.1935.A solution of ethyl 5--1,3,4-oxadiazole-2-carboxylate, (4-(2-aminoethyl)phenyl)(3-methoxyazetidin-1-yl)methanone, and triethylamine in MeOH (4.00 mL) wasstirred at 60 °C for 16 h. The crude product was purified byprep-HPLC (water 0.05% ammonia hydroxide v/v)–ACN;B%: 25–45%,10min), and the desired eluant was concentrated to remove ACN and lyophilized.5--50, 106–111 were all commercial compounds.Compounds 17The plasmid construct design, protein expression, and purificationwere carried out as described previously.Mtb Pks13 TE domaincrystallization conditions under vapor diffusion setting have previouslybeen determined and optimized using 0.1 M Tris-HCl pH 8.5, 1.8–2.0M ammonium sulfate, and 2–5% (v/v) of polypropylene glycolP-400 as an additive17 to allow back soakingusing the benzofuran ligands. However, a direct co-crystallizationplatform was developed due to the crystals being very fragile anddifficult to manipulate between drops. Ligands were first solubilizedin 100% DMSO to a final concentration of 100 mM as stock solution.For Mtb Pks13 TE domain-ligand co-crystallizationexperiment, ligand was added to the protein solution (15–20mg/mL) to a final concentration of 0.5–2 mM keeping the finalDMSO concentration at <5%, mixed with a gentle stirring using apipet tip, and incubated on ice for approximately 2 h. One μLof protein–ligand solution was mixed with 1 μL of reservoirsolution , and hanging dropswere set at 20 °C. The co-crystals grew within a week, reached maximum size within 2 weeks, and did notrequire back soaking with ligands as previously determined.17The Mtb Pks13 TE ligand co-crystalswere cryo-protected using Fomblin (Sigma) and flash frozen in liquidnitrogen. X-ray diffraction data were collected at the Diamond LightSource (DLS), Macromolecular Crystallography (MX) beamlines I04-1and I24 at 100 K. The Mtb Pks13 TE-50 and Mtb Pks13 TE-14 data was integrated with XDS30 and scaled using Aimless31 as part ofthe Xia2 DIALS auto processing pipeline at DLS, while the Mtb Pks13TE-33 data was integrated and scaled using autoPROC.32 Data processing statistics are given in For X-ray diffractiondata collection, the Mtb Pks13 TE domain in complex with the ligandswere solved by molecular replacement using the crystal structure ofthe apo form of Mtb Pks13 TE domain . PDB ID codes: Structurefactors and atomic coordinates have been deposited with the RCSB ProteinData Bank with the codes 8Q0T, 8Q0U, and 8Q17.Files may be retrieved online at http://www.rcsb.org/pdb/home/home.doThe co-crystal structuresof (PDB 5V3W) as a se17 In short, IC50 determinations were tested in10-point concentration curves using a 384 well assay format with compoundspredispensed using an Echo 550 (Labcyte). Final assay conditions were50 mM Tris pH 7.0, 100 mM NaCl, 0.1 mM TCEP, 0.5 μM Pks13 TEdomain. The reaction was initiated by the addition of 4-methylumbelliferylheptanoate substrate  to a final assayconcentration of 25 μM. Fluorescence was measured after 180 min incubation at rt using a PheraStar2 platereader, and data was analyzed using ActivityBase XE.these were performed as described previously.37All methodsused for both extra- and intracellular MIC determinationshave been described previously.18MIC analyses for H37Rv and pks13-TetONstrains were performed as described previously.In vitro ADME experiments were performed exactly as reported.4042Test compoundswere prepared as 0.5 mM solutions in 50:50 acetonitrile:water andanalyzed by reversed-phase HPLC-UV (wavelength 254 nm) using a PhenomenexLuna C18 100 Å 150 × 4.6 mm 5 μm column with a gradientof aqueous phase  and mobile phase(acetonitrile) as described.40 Allregulated procedures, at the University of Dundee, on living animalswas carried out under the authority of a project license issued bythe Home Office under the Animals (Scientific Procedures) Act 1986,as amended in 2012 (and in compliance with EU Directive EU/2010/63).License applications have been approved by the University’sEthical Review Committee (ERC) before submission to the Home Office.The ERC has a general remit to develop and oversee policy on all aspectsof the use of animals on university premises and is a subcommitteeof the University Court, its highest governing body.Murine pharmacokinetic studieswere performed exactly as reported.