Expanding benzoxazole based inosine 5’-monophosphate dehydrogenase (IMPDH) inhibitor structure-activity as potential anti-tuberculosis agents

Shibin Chacko; Helena I M Boshoff; Vinayak Singh; Davide M Ferraris; Deviprasad R Gollapalli; Minjia Zhang; Ann P Lawson; Michael J Pepi; Andrzej Joachimiak; Menico Rizzi; Valerie Mizrahi; Gregory D Cuny; Lizbeth Hedstrom

doi:10.1021/acs.jmedchem.7b01839

. Author manuscript; available in PMC: 2019 Jun 14.

Published in final edited form as: J Med Chem. 2018 May 30;61(11):4739–4756. doi: 10.1021/acs.jmedchem.7b01839

Expanding benzoxazole based inosine 5’-monophosphate dehydrogenase (IMPDH) inhibitor structure-activity as potential anti-tuberculosis agents

Shibin Chacko ^§, Helena I M Boshoff ^#, Vinayak Singh ^†, Davide M Ferraris ^∬, Deviprasad R Gollapalli ^§, Minjia Zhang ^§, Ann P Lawson ^§, Michael J Pepi ^¶, Andrzej Joachimiak ^∫, Menico Rizzi ^∬, Valerie Mizrahi ^†, Gregory D Cuny ^‡,^*, Lizbeth Hedstrom ^§,^¶,^*

PMCID: PMC6166404 NIHMSID: NIHMS984968 PMID: 29746130

Abstract

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis (Mtb) inosine 5’-monophosphate dehydrogenase 2 (MtbIMPDH2) is a promising yet controversial potential target. Inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤ 1 μM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), derives from inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.

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INTRODUCTION

The worldwide burden of tuberculosis is staggering: more than 1 million deaths and 10 million new cases occur each year (https://www.cdc.gov/tb/statistics/default.htm, accessed 5/21/2017). Approximately one third of the world’s population is infected with the causative agent Mycobacterium tuberculosis (Mtb), many with latent infections that can recrudesce if the patient becomes immunosuppressed. Treatment requires administration with isoniazid, rifampicin pyrazinamide and ethambutol for two months followed by an additional four months with isoniazid and rifampicin. However, almost 0.5 million cases involve Mtb strains that are resistant to isoniazid and rifampicin, further complicating treatment. Extensively drug resistant strains have also emerged that are resistant to at least isoniazid and rifampicin, a fluoroquinolone, and any one of the second-line injectables, capreomycin, kanamycin and amikacin. Frustratingly, as illustrated by the newly approved tuberculosis drugs bedaquiline and delamanid ¹, resistance can emerge just a few years after introduction of a new therapy, underscoring the critical need for new drugs, drug targets, and drug regimens to improve tuberculosis treatment and combat the relentless evolution of resistance.

Inosine 5’-monophosphate dehydrogenase (IMPDH) is the enzyme that catalyzes the NAD⁺-dependent conversion of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). This reaction is the first and the rate limiting step in the de novo biosynthesis of guanine nucleotides and therefore controls the size of the guanine nucleotide pool.² IMPDH is an attractive target for the development of new antibiotics due to the essential role of guanine nucleotides in DNA and RNA synthesis, signal transduction, energy transfer, glycoprotein biosynthesis and many other processes involved in cell proliferation.³ Mtb has three genes, designated as guaB1, guaB2 and guaB3, which encode IMPDH homologs. Only guaB2 is essential, ^4–6 and only guaB2 encodes an active IMPDH (MtbIMPDH2).⁷ The proteins encoded by guaB1 and guaB3 are each missing key catalytic residues and neither display IMPDH activity ⁷. The functions of these proteins are currently unknown.

MtbIMPDH2 is a controversial target for tuberculosis. Several small molecule inhibitors of MtbIMPDH2 have been identified that display antibacterial activity in vitro and in macrophages ^7–13, but as yet no compounds have been reported with activity in animal models of tuberculosis. Recently, Park et al. suggested that high levels of guanine (200–2000 μM¹⁰) in the lungs of infected rabbits and humans can protect bacteria from MtbIMPDH2 inhibitors,¹⁰ calling into question the vulnerability of this target. However, the availability of free guanine for salvage is by no means certain since these concentrations exceed the solubility limit. More promisingly, Singh et al. constructed a conditional knockout of guaB2 in Mtb and found that depletion of MtbIMPDH2 is rapidly bactericidal in vitro.¹¹ They also showed that silencing of MtbIMPDH2 expression prevented infection in mice ¹¹, consistent with the lower levels of guanine found in mouse tissue (7–20 μM ¹⁰). On the basis of these findings, Singh et al. concluded that this enzyme is indeed vulnerable.¹¹

IMPDH also appears to have moonlighting functions of unknown physiological consequence, which further complicates the question of whether it is a vulnerable target. The protein contains a regulatory CBS subdomain in addition to the catalytic (β/α)₈ domain. CBS domains bind nucleotides and modulate the activity of other enzymes and channels ¹⁴. However, deletion of the CBS subdomain does not affect the activity of the catalytic domain in IMPDH ^15–18. The CBS subdomain binds ATP and/or GTP depending on the particular IMPDH ^19–22. These interactions modulate the oligomerization of tetramers to form octamers and higher order structures in vitro, with modest effects on enzymatic activity ^19–22. The physiological relevance of ATP/GTP binding and these higher order structures is uncertain. Most interestingly, deletion of CBS subdomain dysregulates the purine nucleotide pools in E. coli, leading to the toxic accumulation of ATP and other adenine nucleotides ^{23, 24}. IMPDH binds nucleic acid and can act as a transcription factor in eukaryotes, and this activity is mediated by the CBS subdomain ^25–27. IMPDH also interacts with numerous other proteins in bacteria, including the ribosome, RNAP, amino acyl tRNA synthetases, penicillin binding proteins, uridylate kinase, PRPP synthetase, adenylosuccinate synthetase and DNA helicase ^{28, 29}. Neither the sites nor the physiological consequences of these interactions have been elucidated. Therefore it is possible that the essentiality of guaB2 in Mtb derives from a moonlighting function rather than enzymatic activity. Moreover, the physiological consequences of enzyme inhibition may well be different from protein depletion, which downregulates both enzymatic and moonlighting activities ³⁰. More potent MtbIMPDH2 inhibitors are required to resolve this conundrum and determine the vulnerability of Mtb infections to IMPDH inhibition ³¹.

We have developed triazole (A-series), benzimidazole (C-series), phthalazinone (D-series), 4-oxo-[1]benzopyrano[4,3-c]pyrazole (N-series), urea (P-series) and benzoxazole (Q-series) inhibitors of prokaryotic IMPDHs, including MtbIMPDH2 ^{9, 32–40}. These inhibitors exploit the highly diverged cofactor binding site, which explains their selectivity for bacterial over eukaryotic orthologs of IMPDH ^{9, 17}. Individual members of the A, D, P and Q-series display antibacterial activity against Mtb as well as other pathogens ^{9, 41, 42}. The best Q compound, 3 (Figure 1A), was a potent inhibitor of MtbIMPDH2 (K_i,app = 14 nM) and displayed moderate antibacterial activity (MIC = 6.3 and 11 μM in minimal GAST/Fe and rich 7H9/ADC/Tween media, respectively). The values of MIC increased 3 to 7-fold in the presence of guanine, suggesting that antibacterial activity resulted from the on-target inhibition of MtbIMPDH2.

Figure 1. — **Inhibitors of *Mtb*IMPDH2. A**) Structures of 1, 2 and 3. B) Crystal structure of *Mtb*IMPDH2ΔCBS •IMP•3 (PDB 4ZQO)⁹. 3 is pink, IMP is blue, hydrogen bonds are shown in cyan, residues from the adjacent subunit are marked with ‘. C) Crystal structures of *M. thermoresistible* IMPDH2 in complex with IMP and cyclohexyl[4-(5-isoquinolinylsulfonyl)-1-piperazinyl]methanone (VCC234718, PDB 5J5R, protein and IMP are spring green, inhibitor is forest green) ¹¹, and N-1H-indazol-6-yl-3,5-dimethyl-1H-pyrazole-4-sulfonamide (6Q9, PDB 5K4X, protein and IMP are gray, inhibitor is blue) ¹⁰. The structure of *Mtb*IMPDH2ΔCBS •IMP•3 (PDB 4ZQO, protein and IMP are tan, 3 is pink)⁹ is also included. This figure was produced with UCSF Chimera ⁴⁴.

Herein we report an expansion of the structure-activity relationship (SAR) for the Q-series of MtbIMPDH2 inhibitors and improved antibacterial activity. Importantly, antibacterial activity is not reduced in the presence of guanine for the most potent compounds. Nonetheless, antibacterial activity appears to derive from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. These findings suggest that MtbIMPDH2 is indeed a vulnerable target.

RESULTS AND DISCUSSION

Design strategy.

IMPDHs are a square planar homotetramers, with each monomer composed of two domains, the catalytic domain and a smaller domain containing tandem cystathione β-synthetase (CBS) motifs.⁴³ The physiological role of the CBS domain is unknown. Deletion of this domain does not impact enzymatic properties or tetramer formation, but does improve solubility and crystallization. The crystal structure of the CBS deletion variant of MtbIMPDH2 (MtbIMPDH2ΔCBS)⁹ in complex with IMP and inhibitor 3 reveals that the aromatic ring of the 2,3-dichloroaniline is involved in a π- π interaction with the purine base of IMP, whereas the amide participates in an ionic-dipole interaction of the NH with the carboxylate of Glu458 and a hydrogen bond of the amide carbonyl with a water molecule (Figure 1B). The nitrogen atom of the 1,3-benzoxazole interacts with a water molecule that also engages the main chain nitrogen and oxygen atoms of His286. Moreover, the 4-pyridyl–1,3-benzoxazole moiety of 3 binds in a hydrophobic pocket formed by Val60, Pro61, and Tyr487 and the pyridyl nitrogen atom interacts with the backbone carbonyl of Ser57 via a water network.⁹ This pocket is not present in human IMPDHs, explaining the selectivity of the compounds for bacterial IMPDHs. Note that the two sulfonamide inhibitors in Figure 1C do not extend into this pocket, perhaps explaining the higher affinity and selectivity of the Q compounds. ^{10, 11}

Using this information, we explored several strategies to expand the SAR of the Q-series (Figure 2): (1) modifications to the pyridine or replacement of the 4-pyridyl with a phenyl containing various substituents that can interact (e.g. via hydrogen bonds and ionic-dipole interactions) with the side chain of Arg290; (2) modification of the 2,3-dichloroaniline with 2,3-difluorophenyl ethers to enhance cell permeability or phenyl ethers containing hydrophilic groups capable of forming interactions with IMP and Glu458; (3) modifications of the amide group via replacement with a thioamide or amine to further explore interactions with Glu458; (4) replacement of the benzoxazole with a imidazo[1,2-a]pyridine; (5) insertion of a methylene linker between the benzoxazole and phenyl that was used as a pyridine replacement. Chemistry. The synthesis used to generate ether linked Q-series derivatives is illustrated in Scheme 1. Arylbenzoxazoles 4 were directly synthesized by oxidative cyclization methods using 2-amino-4-nitrophenol and aldehydes in the presence of activated carbon (Darco KB) under an oxygen atmosphere. The nitro group was reduced using Pd/C under 1 atm hydrogen to give 5-amine-2-arylbenzoxazoles 5. Enantiomerically pure phenyl ethers 6 were synthesized from (+)-methyl D-lactate and the corresponding phenol using Mitsunobu reaction conditions. The esters 6 were hydrolyzed to the corresponding acids 7, and then coupled with various 5-amine-2-arylbenzoxazoles in the presence of EDC·HCl in DMF to yield 8.³⁹ Furthermore, 8 (Ar = 4-OMePh, R = 2,3-diCl) was treated with Lawesson’s reagent under standard conditions to generate thioamide 9 quantitatively. Ester 6a was also treated with 1M DIBAL-H in DCM at −78 ºC to generate aldehyde 10, which was subjected to reductive amination using 5 (Ar = 4-OMePh) and sodium triacetoxyborohydride in DCM to provide amine 11.

Scheme 1. — Synthesis of various aryl benzoxazoles 8, thioamide 9, amine 11, imidazo[1,2-a]pyridine 13, methylene linked derivative 15 and N-arylated benzoxazoles **17a**-b.^a

^aReagents and conditions: (a) ArCHO, DarcoKB, O₂ (1 atm), xylene 140 ºC, 6–8 h, 70–80%; (b) 10% Pd/C, H₂ (1 atm), EtOAc:MeOH, 3 h, 85–94%; (c) PPh₃, DEAD, (+)-methyl D-lactate, THF, 0 ºC to rt, 4 h, 85–90%, (d) LiOH, THF:MeOH, 4 h, 86–91%; (e) 5, EDC·HCl, DMF, 12 h, 62–79%. (f) Lawesson’s reagent, 1,4-dioxane, reflux, 2 h, 71%; (g) 1. DIBAL-H, DCM, −78 ºC, 1h, 89%, (h) 5 (Ar = 4-OMePh), NaBH(OAc)₃, DCE, 1.5 h, 81%; (i) 4-OMePhC(O)CH₂Br, NaHCO₃, EtOH, reflux, 12 h, 88%; (j) SnCl₂, EtOH:EtOAc, reflux, 2 h; (k) 7 (R = 2,3-diCl), EDC·HCl, DMF, rt, 12 h, 60%; (l) 2-amino-4-nitrophenol, DABCO, NMP, 100 ºC, 24 h, 60%; (m) 10% Pd/C, H₂, MeOH:EtOAc, 2–3 h; (n) 7 (R = 2,3-diCl), EDC·HCl, DMF, rt, 12 h, 67%; (o) L-alanine, CuI, Cs₂CO₃, DMF, 90 ºC, 24 h; (p) 5 (Ar = 4-CNPh), HATU, DMF, rt, 12 h, 45–54%.

The synthesis of an imidazo[1,2-a]pyridine analogue started by allowing 5-nitropyridin-2-amine to react with an α-bromoketone in the presence of NaHCO₃ in ethanol to give 12. Nitro group reduction using SnCl₂ in ethanol followed by coupling with carboxylic acid 7 (R = 2,3-diCl) generated imidazo[1,2-a]pyridine 13.

The synthesis of a derivative with a methylene linker between the benzoxazole and the benzene was undertaken. 2-Amino-4-nitrophenol was coupled with a 1,1-dibromoethylene derivative using DABCO in NMP at 100 ºC to generate 14. The nitro group was reduced using 10% Pd/C under a hydrogen atmosphere and the resulting amine was coupled with 7 (R = 2,3-diCl) using EDC·HCl in DMF to yield 15.

Enantiopure N-arylated benzoxazole derivatives were synthesized using copper catalyzed Ullman type reactions. L-alanine was treated with aryl iodides in the presence of cesium carbonate and CuI in DMF for 24 h to provide the N-arylated amino acids 16a-b.³⁹ Attempts to couple these materials with 5 (Ar = 4-CNPh) using EDC·HCl failed. However, using HATU as the coupling reagent in DMF resulted in formation of 17a-b.

Scheme 2 depicts the functional group transformations to various substituents attached to the benzoxazole aryl group. Compounds 8i and 8j were subjected to alkylation reactions with ethyl 2-bromoacetate in the presence of K₂CO₃ and DMF to give 18a and 18b, respectively. These compounds were hydrolyzed using LiOH in THF:MeOH (1:3) to yield 19a and 19b, respectively. Carboxylic acid 19a was also converted to ester 20 using propargyl bromide and K₂CO₃ in dry DMF. Ester 8h was also converted to an array of derivatives. For example, it was treated with hydroxylamine hydrochloride to provide the corresponding hydroxamic acid 21. Base catalyzed ester hydrolysis of 8h in MeOH:THF:H₂O (3:1:1) yielded carboxylic acid 22. Finally, ester 8h was converted to hydrazide 23 using hydrazine hydrate in EtOH, followed by treatment with carbonyl diimidazole (CDI) in the presence of DIPEA in DMF yielding 1,3,4- oxadiazolone 24.

Scheme 3 illustrates the synthesis of an array of derivatives via functional group transformation of 8f. For example, 8f was treated with hydroxylamine hydrochloride in absolute ethanol in the presence of triethylamine to afford hydroxamidine 25, which upon treatment with CDI and DIPEA gave 1,2,4-oxadiazolone 26. The tetrazole derivative 27 was prepared by treatment of 8f with sodium azide in the presence of ammonium chloride in dry DMF. Primary amide 28 was generated by treating 8f with t-BuOK in t-BuOH.⁴⁵ Finally, 8f was treated with NiCl₂·6H₂O and NaBH₄ in the presence of Boc₂O in MeOH:THF to give the Boc protected primary amine, which was deprotected using trifluoroacetic acid in DCM to provide 29.⁴⁶

The synthesis of 2-pyridone derivative 30 was carried out by treating 8b with LiCl and p-toluenesulfonic acid in anhydrous methanol (Scheme 4). The pyridyl of 8a was oxidized using m-CPBA in DCM to give the corresponding pyridine N-oxide 31. The nitrile in 8u was reduced using NiCl₂·6H₂O and NaBH₄ then protected as the corresponding t-butyl carbamate. Removal of the protecting group using trifluoroacetic acid in DCM provided amine 32. The benzyl ether of 8v was removed by hydrogenolysis in presence of Pd/C under a hydrogen atmosphere to yield 33.

Finally, the synthesis of a benzoxaborole analogue is depicted in Scheme 5. A Mitsunobu reaction between 2-bromo-3-hydroxybenzaldehyde and (+)-methyl D-lactate was carried out in the presence of PPh₃ and DEAD in THF to give 34. This aldehyde was treated with NaBH₄ in ethanol to generate the primary alcohol, which was protected as the corresponding methoxymethyl ether 35 using methoxymethyl chloride (MOMCl) and DIPEA in DCM. Boronylation of the aryl bromide was carried out using bis(pinacolato)diboron in the presence of KOAc and a catalytic amount of Pd(Ph₃P)₂Cl₂ in 1,4-dioxane to give 36.⁴⁷ Ester hydrolysis followed by amine coupling with 5 (Ar = 4-CNPh) in the presence of EDC·HCl in DMF and then treatment with 6N HCl in THF gave benzoxaborole 37.

Evaluation of MtbIMPDH2 inhibition.

We previously described the MtbIMPDH2ΔCBS inhibition and antibacterial activity of benzoxazole (Q) based inhibitors.³⁹ The most promising compounds, 2 and 3 (Figure 1A), had K_i,app values of 76 nM and 14 nM, and MIC values of 16 μM and 12 μM, respectively. MtbIMPDH2ΔCBS was expressed and purified as previously described and the enzymatic activity was assayed by monitoring the production of NADH at sub-saturating NAD⁺ (3 × K_m) and saturating IMP concentrations and 20–50 nM enzyme.⁹ The K_i,appvalues reported herein were determined from the average of two independent experiments, unless otherwise noted. We also evaluated inhibition of the host enzymes hIMPDH2 and hGMPR2.

Initially, modifications of the pyridyl of 2 were evaluated (Table 1). Pyridine N-oxide 31 demonstrated comparable activity to the parent compound 2. Addition of a methoxy to the pyridyl 8b and 8c resulted in 5-fold more inhibition than 2. However, changing pyridyl to 2-pyridone (30) reduced inhibitory activity by 3-fold. Replacing pyridyl with phenyl having both electron donating groups (EDG) and electron withdrawing groups (EWG) was evaluated. Compounds 8d (4-OMe), 8e (4-OCF₃) and 8f (4-CN) resulted in 7-fold increase in inhibitory activity compared to 2. Fluorine (8g) and ester (8h) substituents were also tolerated. Replacing the pyridyl with a 4-hydroxy phenyl (8i) resulted in a 2-fold increase in inhibitory activity, whereas the 3-hydroxy (8j) and 2-hydroxy (8k) derivatives showed slightly decreased potency. Given the improved activityof 8d, the effect of other ethers was examined. For example, 18a and propargyl ester 20 shows a ~12-fold increase in activity compared to 2, while the corresponding carboxylic acid (19a) and tetrazole (27) were moderately less potent. Translocating the alkoxy from the para to meta position resulted in a 4-fold loss in activity (18b and 19b). Compounds containing hydrophilic substituents at the 4-position, such as hydroxamic acid 21, carboxylic acid 22 and aminomethyl 29, were found to be much less active. Replacing the carboxylate with hydrazide 23, 1,3,4-oxadiazolone 24, hydroxamidine 25, 1,2,3-oxadiazolone 26 and primary amide 28 showed 2 to 7-fold increase in activity.

Table 1.

MtbIMPDH2ΔCBS Inhibition of Benzoxazole Derivatives Containing Various Aryl Substituents.


Cmpd	Ar	K_i,app (nM)^a
Cmpd	Ar	MtbIMPDH2	hIMPDH2	hGMPR2
1 ^b	See Figure 1A	150 ± 50	>5000	>5000
2 (e.g. 8a) ^b	See Figure 1A	76 ± 27	>5000	>5000
3 ^b	See Figure 1A	14 ± 3	>5000	>5000
30	4-pyridin-2-one	220 ± 6	>5000	>5000
31	4-Py-N-oxide	70 ± 19	>5000	>5000
8b	4-(2-OMe)-Py	12 ± 0.1	>5000	>5000
8c	3-(6-OMe)-Py	20 ± 4	>5000	>5000
8d	4-OMe-Ph	6.9 ± 3.9	>5000	>5000
8e	4-OCF₃-Ph	4.3 ± 0.2	>5000	>5000
8f	4-CN-Ph	12 ± 3	2300	>5000
8g	4-F-Ph	35 ± 7	>5000	>5000
8h	4-PhCO₂Me	96 ± 43	>5000	>5000
8i	4-OH-Ph	50 ± 17	>5000	>5000
8j	3-OH-Ph	85 ± 30	>5000	>5000
8k	2-OH-Ph	96 ± 21	>5000	>5000
18a	4-Ph-OCH₂CO₂Et	6.6 ± 0.4	>5000	>5000
20	4-Ph-OCH₂CO₂CH₂CCH	12 ± 2	>5000	>5000
19a	4-Ph-OCH₂CO₂H	18 ± 3	>5000	>5000
18b	3-Ph-OCH₂CO₂Et	23 ± 1	>5000	>5000
19b	3-Ph-OCH₂CO₂H	55 ± 10	>5000	>5000
21	4-Ph-C(O)NHOH	630 ± 210	>5000	>5000
22	4-PhCO₂H	360 ± 140	>5000	>5000
23	4-Ph-C(O)NHNH₂	49 ± 6	>5000	>5000
24		2.3 ± 1.0	2900	>5000
25		39 ± 12	>5000	>5000
26		46 ± 12	>5000	>5000
27	4-Ph-tetrazole	51 ± 2	>5000	>5000
28	4-Ph-C(O)NH₂	64 ± 26	>5000	>5000
29	4-Ph-CH₂NH₂	240 ± 26	>5000	>5000

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^a.

Values are the average and range of at least two independent determinations.

Single determination.

^b.

Values from Makowska-Grzyska et al⁹.

Previous work on the benzoxazole series had established that substitution at the 2- and 3-positions of aryl ether were important for inhibition of both Cryptosporidium parvum IMPDH and MtbIMPDH2ΔCBS.^39,9 Several 2,3-dichlorophenyl compounds (2, 8c, 8d, 8f, 8g and 8i) were compared to the corresponding 2,3-difluorophenyl analogues (8l, 8o, 8m, 8p, 8p and 8n). Three of these compounds (8l, 8p and 8q) displayed similar activity whereas the others were 2 to 4-fold less active. Adding another substitution on the 4-position (8r) resulted in slightly reduced activity. Replacement of the 2-chlorophenyl with a 2-cyanophenyl (8s and 8t) was well tolerated. In addition, compounds with 2-cyano (8u), 2-aminomethyl (32), and 2-hydroxy (33), and 2- benzyloxy (8v) substituents on the phenyl ether demonstrated MtbIMPDH2ΔCBS K_i,app values < 45 nM. However, the benzoxaborole (37) was not as well tolerated.

Several other areas of the Q-series were examined (Table 3). For example, the amide was replaced with a thioamide (9), which demonstrated a modest loss of activity. This decrease in potency is likely due a loss of a hydrogen bond to water, while retaining the ionic-dipole interaction between the thioamide NH and the carboxylate of Glu458. Furthermore, replacement of the amide with an amine (11) was not tolerated. In addition, activity was significantly decreased when the benzoxazole was replaced with an imidazo[1,2-a]pyridine (13). Adding a methylene between the phenyl and benzoxazole (15) also reduced activity by a factor of 6 compared to 8d. However, replacing the aryl ether with aniline (17a and 17b) resulted in increased inhibitory activity.

Table 3.

MtbIMPDH2ΔCBS Inhibition of Other Benzoxazole Derivatives.

Cmpd	Structure	K_i,app^a(nM)
Cmpd	Structure	MtbIMPDH2	hIMPDH2	hGMPR2
9		27 ± 21	>5000	>5000
11		1290 ± 250^*	n.a.	>5000
13		160 ± 4	>5000	>5000
15		100 ± 40	>5000	>5000
17a		9 ± 4	3300	>5000
17b		18 ± 3	>5000	>5000

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^a.

Values are the average and range of at least two independent determinations.

Single determination.

Selectivity of inhibition.

The cofactor binding sites are widely diverged in bacterial and eukaryotic IMPDHs, and the MtbIMPDH2 inhibitors exploit this divergence ^{9, 17}. Only four compounds, 8f, 8p, 17a and 24, modestly inhibited human IMPDH2, and in these cases selectivity ranged from a factor of 200 to 1000. No inhibition of human IMPDH2 was observed for the other compounds (maximum concentration tested was 5 μM). GMP reductase (GMPR) is closely related to IMPDH, and catalyzes a similar reaction, the reduction of GMP by NADPH to produce IMP, NADP⁺ and ammonia ⁴⁸. The adenosine site of human GMPR2 contains the Ala-Tyr motif that characterizes the inhibitor binding site of MtbIMPDH2. However, none of the compounds inhibited human GMPR2 (maximum concentration tested was 5 μM). These experiments demonstrate that compounds selectively inhibit bacterial IMPDHs and do not affect related host enzymes.

Evaluation of antibacterial activity.

Antibacterial activity was determined for MtbIMPDH2ΔCBS inhibitors with K_i,app ≤ 40 nM and selected additional compounds by monitoring the growth of Mtb H37Rv (ATCC 27294) after one week (Table 4). Since in vitro antibiotic efficacy can vary unpredictably with growth conditions,⁴⁹ antibacterial activity was assessed in both GAST/Fe and 7H9/ADC/Tween media, both of which lack purines (e.g. –Gua). Two compounds, 18a and 20, displayed MICs less than 1 μM in both media. An additional 8 compounds, 8f, 8l, 8m, 8p, 8u, 17a, 17b and 18b, displayed MIC ≤ 5 μM in both media. Six of these compounds retained antibacterial activity over two weeks: 8l, 8u, 17a, 17b, 18a and 20. Two compounds (21 and 22) were active in GAST/Fe medium (MIC ≤ 5 μM) but considerably less effective in 7H9/ADC/Tween medium. These compounds also retained activity over two weeks.

Table 4.

Antitubercular activity of MtbIMPDH2 inhibitors against Mtb H37Rv. The number of independent determinations is shown in parentheses.

Compound	GAST/Fe MIC (μM) ^a		7H9/ADC/Tween MIC (μM) ^a
Compound	−Gua	+Gua	−Gua	+Gua
1 ^b	5.3 ± 0.9 (3)	24 ± 16 (3)	10 ± 2 (3)	21 ± 4 (3)
2 (e.g. 8a) ^b	9.4 ± 3.0 (2)	37 (1)	16 ± 6 (3)	28 ± 9 (2)
3 ^b	6.7 ± 3 (2)	50 (1)	12 ± 6 (3)	35 ± 15 (2)
8b	6.3 ± 0.0 (2)	6.3 ± 0.0 (2)	3.5 ± 1.2 (2)	14 ± 5 (2)
8c	7 ± 2 (3)	9 ± 7 (3)	6 ± 3 (3)	> 25 (3)
8d	>50 (2)	>50 (2)	>50 (2)	>50 (2)
8e	>50 (1)	>50 (1)	>50 (2)	>50 (2)
8f	4 ± 1 (3)	5 ± 1 (3)	3 ± 1 (4)	6 ± 1 (4)
8g	>50 (2)	>50 (2)	>50 (2)	>50 (2)
8l	4 ± 2 (2)	6.3 ± 0 (2)	1.9 ± 0.5 (2)	4 ± 3 (2)
8m	5 ± 2 (3)	8 ± 4 (3)	4 ± 4 (4)	14 ± 7 (4)
8n	6 (1)	19 (1)	7 ± 3 (2)	>30 (2)
8o	6 ± 5 (2)	8 ± 7 (2)	>50 (5)	>50 (5)
8p	3 ± 1 (3)	6.3 ± 0 (2)	1.5 ± 0.8 (6)	4 ± 3 (6)
8q	>50 (1)	>50 (2)	>50 (2)	>50 (2)
8r	>50 (2)	>50 (2)	>50 (5)	>50 (5)
8t	6 ± 4 (2)	11 ± 2 (2)	4 ± 1 (2)	8 ± 2 (2)
8u	3.1 ± 0.0 (2)	6.3 ± 0.0 (2)	1.2 ± 0.0 (2)	4.7 ± 0.0 (2)
9	37 (1)	>50 (1)	>50 (2)	>50 (2)
17a	4 ± 3 (2)	13 ± 9 (2)	3 ± 1 (2)	6 ± 1 (2)
17b	2 ± 1 (2)	6 ± 0 (2)	1.0 ± 0.3 (2)	1.4 ± 0.3 (2)
18a	0.4 ± 0.0 (2)	0.5 ± 0.1 (2)	0.2 ± 0.1 (2)	0.23 ± 0.16 (2)
18b	0.9 ± 0.2 (2)	1.4 ± 0.2 (2)	1.6 ± 0.0 (2)	1.6 ± 0.0 (2)
19a	2 ± 1 (4)	4 ± 2 (4)	9 ± 4 (5)	20 ± 10 (5)
20	0.4 ± 0.0 (2)	1.4 ± 0.2 (2)	0.2 ± 0 (2)	0.4 ± 0 (2)
21	3 ± 1 (4)	6 ± 1 (4)	30 ± 10 (5)	>50 (5)
22	4 ± 2 (2)	5 ± 0 (2)	>25 (2)	>50 (2)
24	>50 (2)	>50 (2)	>50 (2)	>50 (2)
25	25 (1)	25 (1)	>50 (2)	>50 (2)
29	11 ± 2 (2)	11 ± 2 (2)	25 ± 12 (2)	25 ± 12 (2)
32	9.4 ± 3.1 (2)	16 ± 3 (2)	5.5. ± 0.8 (2)	9.4 ± 0.0 (2)
33	22 ± 3 (2)	25 ± 0.0 (2)	9.4 ± 0.0 (2)	22 ± 3 (2)

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^a.

MICs determined after 1 week in culture.

^b.

Values from Makowska-Grzyska et al⁹. +Gua, 200 μM guanine.

Figure 3 shows the dependence of antibacterial activity on inhibition of MtbIMPDH2ΔCBS for the Q compounds described above as well as those reported previously ⁹. Uptake and metabolism also play important roles in antibacterial efficacy, so it is not surprising that some potent MtbIMPDH2ΔCBS inhibitors fail to display antibacterial activity. Nonetheless, more potent enzyme inhibition is generally associated with greater antibacterial activity, as expected if antibacterial activity derived from inhibition of MtbIMPDH2.

Many bacteria have the ability to salvage guanine or guanosine, and thus overcome inhibition of IMPDH. Mtb can salvage guanine, but not guanosine, and previously reported IMPDH inhibitors were much less effective in the presence of high guanine concentrations ^9–11. For example, the MIC values of indazole sulfonamide inhibitors of MtbIMPDH2 increased more than 25-fold in 100–125 μM guanine ^{10, 11}. Smaller increases were observed for previously reported Q compounds ⁹. However, the MIC of only one of the new Q compounds, 8n, increased substantially in the presence of guanine in both media. Intriguingly, this compound was one of the weaker inhibitors of MtbIMPDH2 (K_iapp = 121 nM). The MIC of one additional compound, 8c (K_i,app = 20 nM), increased in the presence of guanine in 7H9/ADC/Tween medium but not in GAST/Fe. The failure of guanine to decrease the antibacterial activity of most Q compounds would usually suggest that antibacterial activity derives from the engagement of another target. Alternatively, guanine salvage may not be sufficient to support growth in the presence of potent MtbIMPDH2 inhibition. It is also possible that the Q inhibitors stabilize/disrupt a protein complex, thereby perturbing a moonlighting activity of MtbIMPDH2 in addition to enzyme activity.

We performed two experiments to further address the question of whether antibacterial activity derived from on-target inhibition of MtbIMPDH2. First, we evaluated the effect of MtbIMPDH2 depletion on the antibacterial activity of four MtbIMPDH2 inhibitors, including one compound with guanine-dependent antibacterial activity (1)⁹, and three compounds with guanine-independent antibacterial activity (17b, 18a and 22; note that 22 is only active in GAST/Fe). The downregulation of MtbIMPDH2 was achieved using strain guaB2 cKD, in which guaB2 expression is suppressed by anhydrotetracycline (ATc).¹¹ The antibacterial activities of 1, 17b, 18a, and 22 against the wild-type strain were not affected by the addition of ATc (Table 5). In contrast, treatment with ATc hypersensitized the guaB2 cKD strain to all four compounds (Figure 4), suggesting that antibacterial activity derives primarily from the inhibition of MtbIMPDH2.

Table 5.

Antibacterial activity against SRMV2.6. H37Rv is the wild-type strain. b. SRMV2.6 contains MtbIMPDH2/Y487C, which confers resistance to the isoquinoline sulfonamide inhibitor VCC234718¹¹.

Compound	WT^a K_i,app (nM)	Y487C^b K_i,app (µM)	Ratio K_i,app (Y487C) /K_i,app (WT)	MIC (µM)^c
Compound	WT^a K_i,app (nM)	Y487C^b K_i,app (µM)	Ratio K_i,app (Y487C) /K_i,app (WT)	H37Rv	H37Rv (+ATc)	SRMV2.6
1	150 ± 50	>50^d	>300	12.5	12.5	>100
17b	18 ± 3	1.1 ± 0.1	60	3.1	3.1	>100
18a	6.6 ± 0.4	~13^f	~2000	3.1	3.1	3.1
22	360 ± 140	>15^e	>40	25	25	25

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^a.

Inhibition of purified wild-type MtbIMPDH2.

^b.

Inhibition of purified MtbIMPDH2/Y487C

^c.

Bacteria were cultured in 7H9/Glycerol/OADC/Tween and growth was measured with Alamar Blue as previously described ¹¹, +ATc, 100 ng/mL.

^d.

20% inhibition at 50 µM.

^e.

5-10% inhibition at 15 µM.

^f.

80% inhibition at 50 µM.

Figure 4. — Knockdown of *Mtb*IMPDH2 hypersensitizes *Mtb* to Q compounds. Regulated expression of *guaB2* is achieved in a TET-OFF system, as previously described ¹¹. Addition of anhydrotetracycline (ATc) represses expression of *guaB2*, decreasing the level of *Mtb*IMPDH2 within the bacteria. ATc concentrations (ng/mL) are 0 (dark orange), 0.08 (dark blue), 0.15 (green), 0.31 (sky blue), 0.62 (yellow), 1.25 (gray), 2.5 (orange) and 5 (royal blue).

The antibacterial activity of 1, 17b, 18a, and 22 was also assessed against Mtb strain SRMV2.6. This strain expresses the mutant MtbIMPDH2/Y487C, which is resistant to an isoquinoline sulfonamide inhibitor ¹¹. As noted above, Tyr487 interacts with the benzoxazole group, so the substitution of Cys is expected to disrupt the binding of all the Q inhibitors. SRMV2.6 was resistant to 1 and 17b (Table 5), further confirming that the antibacterial activity of these compounds resulted from on-target inhibition of MtbIMPDH2. Interestingly, however, SRMV2.6 remained sensitive to 18a and 22.

We measured the inhibition of recombinant MtbIMPDH2/Y487C to determine if the Y487C mutation decreased the affinity of all the Q compounds as expected (Table 5). The values of K_i,app for 1 and 17b were increased by more than 300-fold and 60-fold, respectively, which can account for the resistance of strain SRMV2.6. These observations further confirm that the antibacterial activity of 1 and 17b derive from inhibition of MtbIMPDH2. The values of K_i,app for 18a and 22 were similarly increased in MtbIMPDH2/Y487C, by at least 2000-fold and approximately 40-fold, respectively. These observations suggest that strain SRMV2.6 should also be resistant to 1 and 18a, yet it remained sensitive to these compounds. Perhaps 1 and 18a are concentrated or metabolized in the bacteria, or perhaps these compounds interfere with a moonlighting function. Alternatively, these compounds may engage an additional target(s). Irrespective of the ambiguous mechanism of action of 1 and 18a, 17b demonstrates that on-target inhibition of MtbIMPDH2 can be impervious to guanine rescue, suggesting that it is a vulnerable target.

Evaluation of Human Cytotoxicity Activity.

Compounds displaying potent antibacterial activity (8l, 8p, 8u, 17b, 18a and 20) were evaluated for cytotoxicity against HepG2 cells using a LDH release assay. Only compounds 8p, 8u and 17b displayed measurable cytotoxicity (9–13% lactate dehydrogenase release) at 25 μM (LD₅₀ > 25 μM, Figure 5). Since 18a and 20 are esters of 19a, we also examined the cytotoxicity of this compound, and again no cytotoxicity was observed. The cytotoxicity of 17b (25 μM) was also less than 10% in Hela, HEK293T and MCF7 cells. These experiments demonstrate that all of the compounds display a greater than 10-fold selectivity for antibacterial activity versus cytotoxicity (the recommended criteria for Mtb ⁵⁰), with the selectivity of some exceeding 100-fold.

Figure 5. — Cytotoxicity of select Q compounds in HepG2 cells after 24 h.

Preliminary pharmacokinetic evaluation of 17b.

The above observations prompted us to consider evaluating the pharmacokinetics of 17b and 18a as a prelude to possible testing in a mouse model of tuberculosis. We first evaluated the stability of 17b and 18a in mouse liver microsomes. 18a was rapidly metabolized (t_1/2 = 1.3 min) in an NADPH-independent process. Compound 19a, the ester of 18a was also metabolized in an NADPH-independent process, although with a much longer half-life (t_1/2 = 23 min). These observations suggest that both the ester and amide bonds of 18a may be liabilities. Compound 17b was metabolized in an NADPH-dependent process with t_1/2 = 26 min. No decomposition of 17b was observed when it was incubated in mouse plasma at 37 °C for 2 h. Based on these results 17b was selected for further analysis. This compound displayed promising pharmacokinetics in mice, with a single 20 mg/kg oral dose [formulated using Tween 80 (1%) and 0.5% (w/v) methylcellulose in water (99%)] producing a maximum plasma concentration level comparable to MIC (C_max = 3 μM) in 0.5 h (T_max) with a plasma elimination half-life of 5 h. However, 17b also displays high serum protein binding (>99%), which suggests that the free drug concentration is insufficient to achieve in vivo efficacy. Further optimization of 17b to increase antibacterial activity and decrease plasma protein binding is ongoing.

CONCLUSIONS

The SAR of the benzoxazole-based IMPDH inhibitors (Q-series) has been expanded by replacing the 4-pyridyl with phenyl or benzyl groups containing a variety of hydrogen bonding and ionic-dipole interacting substituents. Modifications to the central amide and benoxazole also reinforced the importance of interactions between the inhibitors and Glu458, and the spatial orientation provided by the central heterocycle, respectively. Two MtbIMPDH2 inhibitors displayed antibacterial activity with MIC values ≤ 1 μM, and another eight compounds displayed MICs ≤ 5 μM. The antibacterial activity of the best candidate, 17b, derives from inhibition of MtbIMPDH2, yet is not affected by the presence of guanine. This observation alleviates the concern that guanine salvage can rescue bacteria from MtbIMPDH2 inhibition. We note that previously reported MtbIMPDH2 inhibitors were not as potent as the current compounds, and suggest that the ability of guanine to protect bacteria derived from incomplete inhibition of MtbIMDPH2. The values of K_i,app for the best inhibitors are 25-fold more potent than the indazole sulfonamide inhibitor that called into question the vulnerability of MtbIMPDH2,¹⁰ suggesting that guanine salvage cannot overcome potent inhibition. 17b was also non-toxic at 25 μM in four human cell lines. Overall, this study provides further evidence for the vulnerability of MtbIMPDH2.

Experimental Section

Synthetic Chemistry.

Unless otherwise noted, all reagents and solvents were purchased from commercial sources and used without further purification. All reactions were performed under a nitrogen atmosphere in dried glassware unless otherwise noted. All NMR spectra were obtained using a 400 MHz spectrometer. For ¹H NMR, all chemical shifts are reported in δ units ppm and are referenced to tetramethylsilane (TMS). All chemical shift values are also reported with multiplicity, coupling constants, and proton count. Coupling constants (J) are reported in hertz. Column chromatography was carried out on SILICYCLE SiliaFlash silica gel F60 (40−63 μm, mesh 230−400). High-resolution mass spectra were obtained using a Q-tof UE521 mass spectrometer (University of Illinois, SCS, and Mass Spectrometry Laboratory). HPLC conditions: All final compounds have a chemical purity of >98% as determined by analysis using a Varian Prostar (380-LC) HPLC instrument equipped with a quaternary pump and a Varian Microsorb MV-100–5 C-8 column (250 mm × 4.6 mm). UV absorption was monitored at 332 nm. All samples were dissolved in THF (1–2 mg/mL) and the injection volume was 20 μL. HPLC gradient was 30% acetonitrile and 70% water (both solvents contain 0.05% formic acid) with a total run time of 20 min and a flow rate of 1.0 mL/min.

General Procedure for the Synthesis of 5-nitro-2-phenylbenzo[d]oxazoles (4):

To a stirred solution of 2-amino-4-nitrophenol (1 mmol) and aromatic aldehydes (1 mmol) in anhydrous xylene DarcoKB (300 mg) was added. The solution was stirred under O₂ atmosphere at 140 ºC for 6–8 h. After completion of the reaction as observed from TLC, reaction mixture was filtered with the aid of Celite, which was washed with hot ethyl acetate (3×20 mL). The filtrate was concentrated, and the products were either used directly or recrystallized using ethyl acetate and hexane.

General Procedure for the Synthesis of 5:

5-Nitro-2-arylbenzo[d]oxazole 4 (1 mmol) was dissolved in 10 mL EtOAc:MeOH (1:1) and 10% Pd/C (catalytic) was added and stirred well under a H₂ atmosphere for 3 h. After the successful completion, the reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The crude amines were used directly or recrystallized using ethyl acetate and hexane.

General Procedure for the Synthesis of Phenyl Ethers 6:

Substituted phenol (1 mmol) was added to the solution of methyl (+)-methyl D-lactate (1.38 mmol) in anhydrous THF (6 mL) under a nitrogen atmosphere. The solution was cooled to 0 °C, followed by PPh₃ (1.20 mmol) was added portion wise to the stirred solution. DEAD (1.50 mmol) was added dropwise to the above solution and stirred well at room temperature for 4 h. After completion, solvent was removed under reduced pressure and the crude residue was purified by column chromatography on silica gel using ethyl acetate/n-hexane (10:90) to yield corresponding phenyl ether as a colorless liquid (85–90% Yield).

General Procedure for Ester Hydrolysis for Preparation of 7:

LiOH (1.5 mmol) was added portion wise to the stirred solution of ester 6 (1 mmol) in 5 mL THF:MeOH (2:3) at 0 ºC. The reaction was brought to room temperature and stirred well for 4 h. The solvents were removed under reduced pressure, 1N HCl was added to a pH of 4 and then the mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried using anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude acids were used in the next step without purification.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8a):

Prepared and characterized previously.³⁹

General Procedure for Synthesis of 8b-8v:

EDC·HCl (1.5 mmol) was added to the stirred solution of acid 7 (1 mmol) and amine 5 (1 mmol) in dry DMF at 0 ºC under nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, excess water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSO₄. The solvent was removed under reduced pressure and the crude residue was purified through column chromatography using ethyl acetate/n-hexane.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(2-methoxypyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8b):

White solid (306 mg, 66%), ¹H NMR (DMSO-d₆) 10.34 (s, 1H, NH), 8.93 (d, J = 2.00 Hz, 1H, CH), 8.39–8.36 (m, 1H, CH), 8.06 (d, J = 2.00 Hz, 1H, CH), 7.69 (d, J = 8.80 Hz, 1H, CH), 7.52 (dd, J = 8.90 Hz, J = 2.00 Hz, 1H, CH), 7.30–7.26 (m, 1H, CH), 7.22 (t, J = 1.20 Hz, 1H, CH), 7.02–7.00 (m, 2H, 2×CH), 4.98 (q, J = 6.80 Hz, 1H, CH), 3.92 (s, 3H, -OCH₃), 1.59 (d, J = 6.80 Hz, 3H, CH₃). Purity 98% (t_R = 15.13). m.p. 165–166 C.

(S)-2-(2,3-dichlorophenoxy)-N-(2-(6-methoxypyridin-3-yl)benzo[d]oxazol-5-yl)propanamide (8c):

White solid (315 mg, 69%), ¹H NMR (DMSO-d₆) 10.34 (s, 1H, NH), 8.92 (m, 1H, CH), 8.39–8.37 (m, 1H, CH), 8.06–8.05 (m, 1H, CH), 7.69–7.67 (m, 1H, CH), 7.53– 7.51 (m, 1H, CH), 7.27–7.20 (m, 2H, 2×CH), 7.01–6.98 (m, 2H, 2×CH), 5.01 (q, J = 6.80 Hz, 1H, CH), 3.91 (s, 3H, -OCH₃), 1.58 (d, J = 6.80 Hz, 3H, CH₃). Purity 97% (t_R = 14.85). m.p. 156–157 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanamide (8d):

White solid (351 mg, 77%), ¹H NMR (DMSO-d₆) 10.32 (s, 1H, NH), 8.08 (d, J = 8.80 Hz, 2H, 2×CH), 8.03 (d, J = 2.00 Hz, 1H, CH), 7.65 (d, J = 8.80 Hz, 1H, CH), 7.50–7.48 (m, 1H, CH), 7.27 (t, J = 8.20 Hz, 1H, CH), 7.21–7.19 (m, 1H, CH), 7.10 (d, J = 8.80 Hz, 2H, 2×CH), 7.01–6.99 (m, 1H, CH), 4.98 (q, J = 6.80 Hz, 1H, CH), 3.82 (s, 3H, -OCH₃), 1.59 (d, J = 6.80 Hz, 3H, CH₃). Purity 99% (t_R = 15.30). m.p. 230 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-(trifluoromethoxy)phenyl)benzo[d]oxazol-5-yl)propanamide (8e):

White solid (357 mg, 70%), ¹H NMR (DMSO-d₆) 10.41 (s, 1H, NH), 8.31 (d, J = 8.80 Hz, 2H, 2×CH), 8.16 (m, 1H, CH), 7.77 (d, J = 9.00 Hz, 1H, CH), 7.61 (d, J = 8.00 Hz, 2H, 2×CH), 7.59 (m, 1H), 7.32 (t, J = 8.20 Hz, 1H, CH), 7.26–7.24 (m, 1H, CH), 7.05 (d, J = 6.40 Hz, 1H, CH), 5.03 (q, J = 6.20 Hz, 1H, CH), 1.64 (d, J = 6.40 Hz, 3H, CH₃). Purity 99% (t_R = 17.15). m.p. 140–141 C.

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenoxy)propanamide (8f):

Yellow solid (311 mg, 69%), ¹H NMR (DMSO-d₆) 10.38 (s, 1H, NH), 8.28 (dd, J = 8.20 Hz, J = 1.20, 2H, 2×CH), 8.14 (m, 1H, CH), 8.02 (dd, J = 8.20 Hz, J = 1.20 Hz, 2H, 2×CH), 7.75–7.73 (m, 1H, CH), 7.58–7.56 (m, 1H, CH), 7.26 (t, J = 1.60 Hz, 1H, CH), 7.20 (m, 1H, CH), 7.00 (d, J = 8.40 Hz m, 1H, CH), 5.01 (q, 1H, J = 6.80 Hz, CH), 1.58 (d, 3H, J = 6.80 Hz, CH₃). Purity 99% (t_R = 14.85). m.p. 183–184 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)propanamide (8g):

White solid (351 mg, 79%), ¹H NMR (DMSO-d₆) 10.42 (s, 1H, NH), 8.26–8.23 (m, 2H, 2×CH), 8.13 (m, 1H, CH), 7.76–7.74 (m, 1H, CH), 7.59–7.44 (m, 3H, 3×CH), 7.32–7.26 (m, 2H, 2×CH), 7.06–7.04 (m, 1H, CH), 5.04 (q, J = 6.00 Hz, 1H, CH), 1.64 (d, J = 6.00 Hz, 3H, CH₃). Purity 98% (t_R = 15.73). m.p. 213–214 C.

Methyl (S)-4-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)benzoate (8h):

White solid (329 mg, 68%), ¹H NMR (DMSO-d₆) 10.37 (s, 1H, NH), 8.29–8.26 (m, 1H, CH), 8.11 (d, J = 8.80 Hz, 2H, 2×CH), 7.86 (m, 1H, CH), 7.73 (dd, J = 8.80 Hz, J = 3.20 Hz, 1H, CH), 7.56 (d, J = 8.20 Hz, 1H, CH), 7.40 (m, 1H, CH), 7.27–7.20 (m, 2H, 2×CH), 7.00 (dd, J = 8.80 Hz, J = 2.00 Hz, 1H, CH), 5.02 ( q, 1H, J = 6.80 Hz,, 1H, CH), 3.85 (s, 3H, -COOCH₃), 1.59 (d, 3H, J = 6.00 Hz, CH₃). Purity 99% (t_R = 15.60). m.p. 197–198 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-hydroxyphenyl)benzo[d]oxazol-5-yl)propanamide (8i):

White solid (313 mg, 71%), ¹H NMR (DMSO-d₆) 10.30 (s, 1H, NH), 8.00–7.99 (m, 1H, CH), 7.97 (d, J = 8.40 Hz, 2H, 2×CH), 7.62 (d, J = 8.80 Hz, 1H, CH), 7.48–7.46 (m, 1H, CH), 7.27 (t, J = 8.00 Hz, 1H, CH), 7.00 (d, J = 8.40 Hz, 1H, CH), 6.91 (d, J = 8.80 Hz, 2H, 2×CH), 4.47 (q, J = 6.40 Hz, 1H, CH), 1.58 (d, J = 6.80 Hz, 3H, CH₃). Purity 99% (t_R = 13.11). m.p. 213.5–214.5 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(3-hydroxyphenyl)benzo[d]oxazol-5-yl)propanamide (8j):

White solid (300 mg, 68%), ¹H NMR (DMSO-d₆) 10.38 (s, 1H, NH), 9.75 (s, 1H, OH), 8.12 (s, 1H, CH), 7.74 (d, J = 9.20Hz, 1H, CH), 7.61–7.55 (m, 3H, 3×CH), 7.40 (t, J = 8.00 Hz, 1H, CH), 7.32 (t, J = 8.20 Hz, 1H, CH), 7.26 (m, 1H), 7.06–7.01 (m, 2H, 2×CH), 5.03 (q, J = 6.80 Hz, 1H, CH), 1.64 (d, J = 6.40 Hz, 3H, CH₃). Purity 98% (t_R = 13.36). m.p. 259 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(2-hydroxyphenyl)benzo[d]oxazol-5-yl)propanamide (8k):

White solid (287 mg, 65%), ¹H NMR (DMSO-d₆) 11.10 (s, 1H, OH), 10.41 (s, 1H, NH), 8.13 (d, J = 2.00 Hz, 1H, CH), 7.97 (dd, J = 7.60 Hz, J = 1.20 Hz, 1H, CH), 7.76 (d, J = 8.40 Hz, 1H, CH), 7.55 (dd, J = 8.40 Hz, J = 2.00 Hz, 1H, CH), 7.54–7.48 (m, 1H, CH), 7.26 (t, J = 8.00 Hz, 1H, CH), 7.19 (m, 1H, CH), 7.09–7.00 (m, 3H, 3×CH), 5.00 (q, J = 6.80 Hz, 1H, CH), 1.59 (d, J = 6.60 Hz, 3H, CH₃). Purity 99% (t_R = 16.99). m.p. 209–210 C.

(S)-2-(2,3-Difluorophenoxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8l):

White solid (268 mg, 68%), ¹H NMR (DMSO-d₆) 10.40 (s, 1H, NH), 8.79 (dd, J = 4.20 Hz, J = 1.20 Hz, 2H, CH), 8.16 (m, 1H, CH), 8.04 (t, J = 2.00 Hz, 2H, 2×CH), 7.77 (d, J = 8.80 Hz, 1H, CH), 7.62 (m, 1H, CH), 7.62–7.60 (m, 1H, CH), 4.96 (q, J = 6.80 Hz, 1H, CH), 1.58 (d, J = 6.80 Hz, 3H, CH₃). Purity 99% (t_R = 14.45). m.p. 189–190 C.

(S)-2-(2,3-Difluorophenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanamide (8m):

White solid (322 mg, 76%), ¹H NMR (DMSO-d₆) 10.32 (s, 1H, NH), 8.08 (d, J = 9.20 Hz, 2H, 2×CH), 8.02 (d, J = 1.60 Hz, 1H, CH), 7.64 (d, J = 8.40 Hz, 1H, CH), 7.50 (d, J = 6.80 Hz, 1H, CH), 7.10 (d, J = 8.80 Hz, 3H, 3×CH), 6.99 (m, 1H, CH), 6.89 (t, J = 2.00 Hz, 1H, CH), 4.95 (q, J = 6.80 Hz, 1H, CH), 1.57 (d, J = 6.40 Hz, 3H, CH₃). Purity 97% (t_R = 13.95). m.p. 179–180 C.

(S)-2-(2,3-Difluorophenoxy)-N-(2-(4-hydroxyphenyl)benzo[d]oxazol-5-yl)propanamide (8n):

White solid (291 mg, 71%), ¹H NMR (DMSO-d₆) 10.36 (s, 1H, OH), 10.33 (s, 1H, NH), 8.04 (d, J = 2.00 Hz, 2H, 2×CH), 8.01 (m, 1H, CH), 7.67 (d, J = 8.80 Hz, 1H, CH), 7.52 (d, J = 8.20 Hz,, 1H, CH), 7.15–6.97 (m, 3H, 3×CH), 6.96 (d, J = 8.40 Hz, 2H, 2×CH), 4.99 (q, 1H, J = 6.80 Hz, CH), 1.62 (d, 3H, J = 6.40 Hz, CH₃). Purity 98% (t_R = 11.36). m.p. 182–183 C.

(S)-2-(2,3-Difluorophenoxy)-N-(2-(6-methoxypyridin-3-yl)benzo[d]oxazol-5-yl)propanamide (8o):

White solid (311 mg, 69%), ¹H NMR (DMSO-d₆) 10.49 (s, 1H, NH), 8.97 (s,, 1H, CH), 8.40 (dd, J = 8.40 Hz, J = 2.00 Hz, 1H, CH), 8.11 (m, 1H, CH), 7.73 (d, J = 8.00 Hz, 1H, CH), 7.59–7.56 (m, 1H, CH), 7.19–6.98 (m, 3H, 3×CH), 6.94 (t, J = 2.40 Hz, 1H, CH), 5.00 (q, J = 6.40 Hz, 1H, CH), 3.17 (s, 3H, -OCH₃), 1.63 (d, J = 6.40 Hz, 3H, CH₃). Purity 99% (t_R = 13.25). m.p. 177–178 C.

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-difluorophenoxy)propanamide (8p):

White solid (289 mg, 69%), ¹H NMR (DMSO-d₆) 10.44 (s, 1H, NH), 8.34 (d, J = 8.40 Hz, 2H, 2×CH), 8.19 (d, J = 1.60 Hz, 1H, CH), 8.07 (d, J = 8.8 Hz, 2H, 2×CH), 7.79 (d, J = 8.80 Hz, 1H, CH), 7.65–7.62 (m, 1H, CH), 7.17–7.12 (m, 1H, CH), 7.06–7.02 (m, 1H, CH), 6.95 (t, J = 2.00 Hz, 1H, CH), 5.01 (q,, J = 6.40 Hz, 1H, CH), 1.63 (d, J = 6.8 Hz, 3H, CH₃). Purity 98% (t_R = 13.24). m.p. 176–177 C.

(S)-2-(2,3-Difluorophenoxy)-N-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)propanamide (8q):

White solid (317 mg, 77%), ¹H NMR (DMSO-d₆) 10.40 (s, 1H, NH), 8.24 (dd, J = 8.60 Hz, J = 5.40 Hz, 2H, 2×CH), 8.13 (d, J = 1.60 Hz,1H, CH), 7.74 (d, J = 9.20 Hz,, 1H, CH), 7.58 (dd, J = 8.80 Hz, J = 2.00 Hz, 1H, CH), 7.46 (td, J = 8.80, J = 1.00 Hz, 2H, 2×CH), 7.17–7.12 (m, 1H, CH), 7.06–7.02 (m, 1H, CH), 6.96–6.92 (m, 1H, CH), 5.00 (q, J = 6.40 Hz, 1H, CH), 1.63 (d, J = 6.80 Hz, 3H, CH₃). Purity 97% (t_R = 14.29). m.p. 194–195 C.

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3,4-trifluorophenoxy)propanamide (8r):

White solid (270 mg, 62%), ¹H NMR (DMSO-d₆) 10.42 (s, 1H, NH), 8.34 (d, J = 6.80 Hz, 2H, 2×CH), 8.19 (s, 1H, CH), 8.08 (d, J = 7.20 Hz, 2H, 2×CH), 7.80 (d, J = 8.00 Hz, 1H, CH), 7.63 (d, J = 8.40 Hz, 1H, CH), 7.27 (d, J = 10.00 Hz 1H, CH), 6.98 (m, 1H, CH), 4.98 (q, J = 6.40 Hz, 1H, CH), 1.61 (d, J = 6.80 Hz, 3H, CH₃). Purity 99% (t_R = 13.60). m.p. 184–185 C.

(S)-2-(2-Cyanophenoxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8s):

White solid (261 mg, 68%), ¹H NMR (DMSO-d₆) 10.49 (s, 1H, NH), 8.84 (dd, J = 4.00 Hz, J = 1.60 Hz, 2H, 2×CH), 8.21 (d, J = 2.00 Hz, 1H, CH), 8.09 (dd, J = 4.40 Hz, J = 1.60 Hz, 2H, 2×CH), 7.83–7.77 (m, 2H, 2×CH), 7.68–7.63 (m, 2H, 2×CH), 7.14–7.11 (m, 2H, 2×CH), 5.11 (q, J = 6.80 Hz, 1H, CH), 1.66 ( d, J = 6.80 Hz, 3H, CH₃). Purity 98% (t_R = 12.67). m.p. 229–230 C.

(S)-2-(2-Cyano-3-fluorophenoxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8t):

White solid (285 mg, 71%), ¹H NMR (DMSO-d₆) 10.51 (s, 1H, NH), 8.84 (dd, J = 6.00 Hz, J = 1.00 Hz, 2H, 2×CH), 8.20 (s, 1H, CH), 8.09 (dd, J = 6.00 Hz, J = 1.00 Hz, 2H, 2×CH), 7.84–7.81 (m, 1H, CH), 7.73–7.63 (m, 2H, 2×CH), 7.10 (t, J = 8.80 Hz, 1H, CH), 6.99–6.97 (d, J = 8.80 Hz, 1H, CH), 5.16 (q, J = 6.40 Hz, 1H, CH), 1.67 (d, J = 6.80 Hz, 3H, CH₃). Purity 98% (t_R = 13.44). m.p. 189-dec C.

(S)-2-(2-Cyanophenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanamide (8u):

White solid (318 mg, 77%), ¹H NMR (DMSO-d₆) 10.41 (s, 1H, NH), 8.13 (d, J = 8.80 Hz, 2H, 2×CH), 8.07 (m, 1H, CH), 7.78 (d, J = 8.80 Hz, 1H, CH), 7.72 (m, 2H, 3×CH), 7.54 (d, J = 8.00 Hz, 1H, CH), 7.21–7.04 (m, 4H, 4×CH), 5.09 (q, J = 6.00 Hz, 1H, CH), 3.82 (s, 3H, OCH₃), 1.65 (d, J = 6.40 Hz, 3H, CH₃). Purity 99% (t_R = 12.99). m.p. 162–163 C.

(S)-2-(2-(Benzyloxy)phenoxy)-N-(2-(4-cyanophenyl)benzo[d]oxazol-5-yl)propanamide (8v):

White solid (337 mg, 69%), ¹H NMR (DMSO-d₆) 10.23 (s, 1H, NH), 8.34 (d, J = 8.00 Hz, 2H, 2×CH), 8.21 (m, 1H, CH), 8.08 (d, J = 8.00 Hz, 2H, 2×CH), 7.76 (d, J = 8.80 Hz, 1H, CH), 7.61–7.58 (m, 1H, CH), 7.50 (d, J = 7.60 Hz, 2H, 2×CH), 7.38–7.29 (m, 3H, 3×CH), 7.08 (m, 2H, 2×CH), 6.93(m, 2H, 2×CH), 5.17 (s, 2H, -OCH₂-), 4.82 (q, J = 6.40 Hz, 1H, CH), 1.57 (d, J = 6.80 Hz, 3H, CH₃). Purity 99% (t_R = 15.31). m.p. 144–145 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanethioamide (9):

A mixture of amide 8 (Ar = 4-OMePh, R = 2,3-diCl, 1 mmol) and Lawesson’s reagent (1 mmol) was dissolved in dry dioxane (5 mL) and refluxed well for 2h. After cooling to room temperature, dioxane was removed under reduced pressure and the crude product was purified through column chromatography. White solid (335 mg, 71%), ¹H NMR (DMSO-d₆) 11.72 (s, 1H, NH), 8.10–8.08 (m, 3H, 3×CH), 7.76–7.70 (m, 1H, CH), 7.51–7.50 (m, 1H, CH), 7.32–7.29 (m, 1H, CH), 7.22–7.20 (m, 1H, CH), 7.14–7.02 (m, 2H, 2×CH), 7.02–7.00 (m, 1H, CH), 5.20 (q, J = 6.80 Hz, 1H, CH), 1.70 (d, J = 6.80 Hz, 3H, CH₃). Purity 98% (t_R = 17.09). m.p. 132–139 C.

(S)-2-(2,3-Dichlorophenoxy)propanal (10):

DIBAL-H (1.0 M, in cyclohexane, 2 mmol) was added dropwise to the stirred solution of ester 6a (1 mmol) in dry CH₂Cl₂ (4 mL) under a nitrogen atmosphere at −78 ºC. After completion of the addition, stirring was continued at the same temperature for 1 h. Aq. HCl (1 N, 3 mL) was added carefully. The bath was removed and the mixture was extracted with Et₂O (3 ×30 mL). The combined organic layers were washed with water (10 mL) and brine (10 mL) and dried over anhydrous MgSO₄. The solvent was removed under reduced pressure and the crude reaction mixture was purified through column chromatography. Colorless oil (193 mg, 89%), ¹H NMR (CDCl₃) 9.72 (s, 1H, CH), 7.10 (m, 2H, 2×CH), 6.74–6.71 (m, 1H, CH), 4.62–4.60 (m, 1H, CH), 1.51 (d, J = 7.20 Hz, 3H, CH₃).

(S)-N-(2-(2,3-Dichlorophenoxy)propyl)-2-(4-methoxyphenyl)benzo[d]oxazol-5-amine (11):

Aldehyde 10 (1 mmol) was dissolved in DCE (25 mL) at 0 °C. Benzoxazole amine 5 (Ar = 4-OMePh, 1 mmol) and sodium triacetoxyborohydride (2 mmol) were added to the above solution and stirred well. The reaction mixture was stirred for 1.5 h and quenched with satd. NaHCO₃ solution (20 mL), and the product was extracted with CH₂Cl₂ (2 × 20 mL). The organic layer was washed with brine and dried using anhydrous MgSO₄. The solvent was removed under reduced pressure and crude mixture was purified through column chromatography. Colorless oil (351 mg, 81%), ¹H NMR (CDCl₃) 8.11 (dd, J = 9.20 Hz, J = 2.00 Hz, 2H, 2×CH), 7.29 (dd, J = 8.80 Hz, J = 2.40 Hz, 1H, CH), 7.05–7.02 (m, 2H, 2×CH), 6.96 (dd, J = 9.00 Hz, J = 2.60 Hz, 2H, 2×CH), 6.92–6.91 (m, 1H, CH), 6.81–6.78 (m, 1H, CH), 6.59 (dd, J = 8.60 Hz, J = 2.20 Hz, 1H, CH), 4.64 (m, 1H, CH), 4.21 (bs, 1H, NH), 3.84 ( s, 3H, -OCH₃), 3.47–3.39 (m, 2H, -CH₂-), 1.39 (d, J = 6.20 Hz, 3H, CH₃). Purity 98% (t_R = 17.11).

2-(4-Methoxyphenyl)-6-nitroimidazo[1,2-a]pyridine (12):

5-Nitropyridin-2-amine (1 mmol) and NaHCO₃ (2 mmol) was added to a stirred solution of 2-bromo-4′-methoxyacetophenone (1 mmol) in ethanol at room temperature and the reaction mixture was refluxed for overnight. After completion, the solvent was removed under reduced pressure and the crude material was dissolved in water and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSO₄, filtered and concentrated under reduced pressure. The product was recrystallized using ethyl acetate and hexane. Brown solid (237 mg, 88%), ¹H NMR (DMSO-d₆) 9.76 (m, 1H, CH), 8.47 (s, 1H, CH), 7.90–7.88 (m, 3H, 3×CH), 7.67–7.65 (m, 1H, CH), 7.61 (d, 2H, J = 8.80 Hz, 2×CH, 3.78 (s, 3H, -OCH₃).

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-6-yl)propanamide (13):

To the stirred solution of 2-(4-methoxyphenyl)-6-nitroimidazo[1,2-a]pyridine 12 (1 mmol) in ethanol and ethyl acetate, SnCl₂ (2 mmol) was added and refluxed for 2h. Solvent was removed under reduced pressure and the reaction was quenched by adding a saturated solution of NaHCO₃ solution and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO₄, filtered and concentrated under reduced pressure. The crude amine was used in the next step without any purification. EDC·HCl (1.5 mmol) was added to the stirred solution of acid 7 (R = 2,3-diCl, 1 mmol) and amine (1 mmol) in dry DMF at 0 ºC under a nitrogen atmosphere. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSO₄. The solvent was removed under reduced pressure. The crude residue was purified through column chromatography to give the desired product. Brown solid (273 mg, 60%), ¹H NMR (DMSO-d₆) 10.35 (s, 1H, NH), 9.13 (s, 1H, CH), 8.33 (s, 1H, CH), 7.84 (d, J = 8.40 Hz, 2H, 2×CH), 7.53 (d, J = 8.00 Hz, 1H, CH), 7.24–6.92 (m, 6H, 6×CH), 5.02 (q, J = 6.40 Hz, 1H, CH), 3.79 (s, 3H, -OCH₃), 1.62 (d, J = 6.40 Hz, 3H, CH₃). Purity 97% (t_R = 12.00). m.p. 146–147 C.

2-(4-Methoxybenzyl)-5-nitrobenzo[d]oxazole (14):

DABCO (2 mmol) was added to the stirred solution of 1-(2,2-dibromovinyl)-4-methoxybenzene (1 mmol) and 2-amino-4-nitrophenol (1 mmol) in NMP (5 mL) under nitrogen atmosphere. The solution was stirred well for 24 h at 100 ºC. After completion, reaction was quenched by the addition of water and extracted with ethyl acetate. The organic layer was washed with brine and dried using MgSO₄, filtered and concentrated under reduced pressure. The crude product was purified using column chromatography. Yellow solid (171 mg, 60%), ¹H NMR (CDCl₃) 8.57–8.56 (m, 1H, CH), 8.28–8.25 (m, 1H, CH), 7.57–7.55 (m, 1H, CH), 7.31–7.29 (m, 2H, 2×CH), 6.91–6.89 (m, 2H, 2×CH), 4.66 (s, 2H, -CH₂-), 3.80 (s, 3H, -OCH₃).

(S)-2-(2,3-dichlorophenoxy)-N-(2-(4-methoxybenzyl)benzo[d]oxazol-5-yl)prop-anamide (15):

5-Nitro-2-arylbenzo[d]oxazoles (1 mmol) was dissolved in 10 mL EtOAc:MeOH (1:1) and 10% Pd/C (catalytic) was added and stirred well under a H₂ atmosphere for 2–3 h. After the successful completion, reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The crude amine was recrystallized using ethyl acetate and hexane. The amine was proceeded to next step without any chromatographic purification. EDC·HCl (1.5 mmol) was added to the stirred solution of acid 7 (R = 2,3-diCl, 1 mmol), amine (1 mmol) in dry DMF at 0 ºC under nitrogen atmosphere. Reaction mixture was stirred well for overnight at room temperature. After completion of the reaction, excess water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using MgSO₄. The solvent was removed under reduced pressure and crude residue was purified through column chromatography using ethyl acetate/n-hexane. White solid (315 mg, 67%), ¹H NMR (CDCl₃) 8.71 (s, 1H, NH), 7.97 (bs, 1H, CH), 7.46 (d, J = 8.00 Hz, 1H, CH), 7.39 (d, J = 8.20 Hz, 1H, CH), 7.28 (d, J = 8.00 Hz, 2H, 2×CH), 7.18–7.16 (m, 2H, 2×CH), 6.91–6.86 (m, 3H, 3×CH), 4.86 (q, J = 6.80 Hz, 1H, CH), 4.19 (s, 2H, -CH₂-), 3.78 (s, 3H, -OCH₃), 1.73 (d, J = 7.20 Hz, 3H, CH₃). Purity 97% (t_R = 14.78). m.p. 143–147 C.

Synthesis of 17:

A mixture of L-alanine (163 mg, 1.82 mmol), Cs₂CO₃ (1.19 g, 3.65 mmol), and CuI (69.7 mg, 0.36 mmol) was added to the stirred solution of 1,2-dichloro-3-iodobenzene or 1,2-difluoro-3-iodobenzene (1.83 mmol) in dry DMF (3 mL) under a nitrogen atmosphere. The reaction mixture was heated at 90 °C for 24 h. After completion, the reaction mixture was allowed to cool and water was added. The pH was adjusted to 3−5 by the addition of 2N HCl and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO₄, filtered and concentrated under reduced pressure. The crude acid 16 was used in the next step without further purification. Acid 16 (1 mmol) was added to the stirred solution of amine 5 (Ar = 4-CNPh, 1 mmol) in dry DMF at 0 ºC followed by HATU (1.2 mmol) and stirred well for overnight at room temperature. After completion, excess water was added to the reaction and extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous MgSO₄, filtered, and concentrated. The residue was purified by column chromatography using ethyl acetate/n-hexane.

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-((2,3-dichlorophenyl)amino)propanamide (17a):

Light brown powder (202 mg, 45%), ¹H NMR (DMSO-d₆) 10.39 (s, 1H, NH), 8.34 (d, J = 8.00 Hz, 2H, 2×CH), 8.20 (s, 1H, CH), 8.07 (d, J = 8.40 Hz, 2H, 2×CH), 7.81 (d, J = 8.40 Hz, 1H, CH), 7.60–7.58 (m, 1H, CH), 7.18 (t, J = 8.20 Hz, 1H, CH), 6.88 (d, J = 7.60 Hz, 1H, CH), 6.65 (d, J = 8.40 Hz, 1H, CH), 5.61 (d,J = 8.00 Hz, 1H, NH), 4.26 (m, 1H, CH), 1.54 (d, J = 6.8Hz, 3H, CH₃). Purity 99% (t_R = 14.95). m.p. 201–202 C.

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-((2,3-difluorophenyl)amino)propanamide (17b):

Yellow powder (225 mg, 54%) ¹H NMR (DMSO-d₆) 10.29 ( s, 1H, NH), 8.33 (d, J = 8.40 Hz, 2H, 2×CH), 8.20 (m, 1H, CH), 8.07 (d, J = 8.40 Hz, 1H, CH), 7.77 (d, J = 8.80 Hz, 1H, CH), 7.60 (d, J = 8.80 Hz, 1H, CH), 6.96 (q, J = 7.60 Hz, 1H, CH), 6.59 (m, 1H, CH), 6.51 (t, J = 7.60 Hz, 1H, CH), 5.92 (d,J = 7.60 Hz, 1H, NH), 4.18 (q, J = 6.80 Hz, 1H, CH), 1.51 (d, J = 6.40 Hz, 3H, CH₃). Purity 99% (t_R = 12.95). m.p. 197–198 C.

Synthesis of 18:

Arylbenzoxazole 8i or 8j (1 mmol) was dissolved in dry DMF (5 mL) in an oven dried round bottom flask and cooled to 0 ºC. K₂CO₃ (2 mmol) was added to the cold solution and stirred well for ten minutes, then ethyl bromoacetate (1.5 mmol) was added and stirred well at room temperature for 6 h. After completion, the reaction was quenched with water and extracted with ethyl acetate (20 mL) and the organic layer was washed with brine. The organic layer was dried using anhydrous MgSO₄, filtered and concentrated under reduced pressure. The crude residue was purified through column chromatography using hexane: ethyl acetate.

Ethyl (S)-2-(4-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)phenoxy)acetate (18a):

White powder (438 mg, 83%), ¹H NMR (DMSO-d₆) 10.31 (s, 1H, NH), 8.08–8.03 (m, 3H, 3×CH), 7.66–7.64 (m, 1H, CH), 7.50–7.48 (m, 1H, CH), 7.29–6.98 (m, 5H, 5×CH), 4.97 (q, J = 6.40 Hz, 1H, CH), 4.87 (s, 2H, -OCH₂-), 4.13 (q, J = 7.20 Hz, 2H, -OCH₂-), 1.58 (d, J = 6.80 Hz, 3H, CH₃), 1.17 (t, J = 7.20 Hz, 3H, CH₃). Purity 99% (t_R = 15.02). m.p. 144–145 C.

Ethyl(S)-2-(3-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)phenoxy)acetate (18b):

White powder (422 mg, 80%), ¹H NMR (DMSO-d₆) 10.35 (s, 1H, NH), 8.09 (m, 1H, CH), 7.76 (d, J = 8.00 Hz, 1H, CH), 7.70 (d, J = 8.00 Hz, 1H, CH), 7.62–7.61 (m, 1H, CH), 7.55–7.47 (m, 2H, 2×CH), 7.27 (t, J = 6.20 Hz, 1H, CH), 7.21–7.18 (m, 2H, 2×CH), 7.02–7.00 (m, 1H, CH), 4.98 (q, J = 6.80 Hz, 1H, CH), 4.89 (s, 2H, -OCH₂-), 4.15 (q, J = 7.20 Hz, 2H, -OCH₂-), 1.59 (d, J = 6.40 Hz, 3H, CH₃), 1.18 (t, J = 6.80 Hz, 3H, CH₃). Purity 99% (t_R = 13.28). m.p. 175–176 C.

Synthesis of 19:

The ester 18 was subjected to ester hydrolysis using the general procedure described for 7.

(S)-2-(4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)phenoxy)acetic acid (19a):

White powder (440 mg, 88%), ¹H NMR (DMSO-d₆) 10.36 (s, 1H, NH), 8.12 (d, J = 8.80 Hz, 2H, 2×CH), 8.08 (m, 1H, CH), 7.70 (d, J = 8.80 Hz, 1H, CH), 7.50 (m, 1H, CH), 7.32 (t, J = 8.40 Hz, 1H, CH), 7.25 (m, 1H), 7.13 (d, J = 8.80 Hz, 2H, 2×CH), 7.05 (d, J = 8.00 Hz, 1H, CH), 5.02 (q, J = 6.40 Hz, 1H, CH), 4.82 (s, 2H, -OCH₂-), 1.63 (d, J = 6.40 Hz, 3H, CH₃). Purity 98% (t_R = 12.64). m.p. 238–239 C.

(S)-2-(3-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)phenoxy)acetic acid (19b):

White powder (430 mg, 86%), ¹H NMR (DMSO-d₆) 13.07 (bs, 1H, COOH), 10.35 (s, 1H, NH), 8.09 (m, 1H, CH), 7.76–7.70 (m, 2H, 2×CH), 7.59–7.46 (m, 3H, 3×CH), 7.26 (t, J = 8.00 Hz, 1H, CH), 7.21–7.14 (m, 2H, 2×CH), 7.01 (d, J = 8.00 Hz, 1H, CH), 4.98 (q, J = 6.40 Hz, 1H, CH), 4.79 (s, 2H, -OCH₂-), 1.59 (d, J = 6.40 Hz, 3H, CH₃). Purity 97% (t_R = 12.77). m.p. 191-dec C.

Prop-2-yn-1-yl (S)-2-(4-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo[d]oxa-zol-2-yl)phenoxy)acetate (20):

Arylbenzoxazole 19a (1 mmol) was dissolved in dry DMF (5 mL) in an oven dried round bottom flask and cooled to 0 ºC. K₂CO₃ (2 mmol) was added to the cold solution and stirred well for ten minutes, followed by propargyl bromide 80% solution in toluene (1.5 mmol) was added and stirred well at room temperature for 6 h. After completion, reaction was quenched with water and extracted with ethyl acetate (20 mL) and organic layer was washed with brine. The organic layer was dried using MgSO₄, filtered and concentrated under reduced pressure. The crude product was purified through column chromatography. White solid (435 mg, 81%), ¹H NMR (DMSO-d₆) 10.37 (s, 1H, NH), 8.12 (d, J = 8.80 Hz, CH, 2×CH), 8.08 (m, 1H, CH), 7.72–7.70 (d, J = 8.80 Hz, 1H, CH), 7.55 (d, J = 8.00 Hz, 1H, CH), 7.34–7.26 (m, 2H, 2×CH), 7.17 (d, J = 8.40 Hz, 2H, 2×CH), 7.05 (d, J = 8.00 Hz, 1H, CH), 5.03–5.00 (m and s, 3H, CH, -OCH₂-), 4.83 (s, 2H, -OCH₂-), 3.64 (bs, 1H, CH), 1.63 (d, J = 6.00 Hz, 3H, CH₃). Purity 98% (t_R = 14.69). m.p. 132–133 C.

(S)-4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)-N-hydroxybenzamide (21):

The ester 8h (1 mmol) was dissolved in methanol and hydroxylamine hydrochloride (2 mmol) was added at 0 ºC and stirred well. KOH (3 mmol) was added to the cold solution and stirred overnight at room temperature. After completion, the solvent was removed under reduced pressure and the residue was dissolved in water and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSO₄. Solvent was removed under reduced pressure and the product was purified using column chromatography. White powder (310 mg, 65%), ¹H NMR (DMSO-d₆) 13.29 (bs, 1H, -OH), 10.37 (s, 1H, NH), 8.26 (d, J = 8.00 Hz, 2H, 2×CH), 8.11 (t, J = 8.40 Hz, 3H, 3×CH), 7.74(d, J = 9.20 Hz, 1H, CH), 7.56 (d, J = 8.80 Hz, 1H, CH), 7.26 (t, J = 8.00 Hz, 1H, CH), 7.20 (m, 1H, CH), 7.00 (d, J = 8.00 Hz, 1H, CH), 4.99 (q, J = 6.80 Hz, 1H, CH), 1.59 (d, J = 6.80 Hz, 3H, CH₃). Purity 98% (t_R = 13.37). m.p. 253–254 C.

(S)-4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)benzoic acid (22):

Ester 8h (1 mmol) was dissolved in THF:MeOH:H₂O (3:1:1) at room temperature and NaOH (1 mmol) was added and stirred well for 2 h. After completion, the solvents were removed under reduced pressure. The crude residue was dissolved in water and pH was adjusted to 3−5 by the addition of 2N HCl and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSO₄. The crude acid was recrystallized using ethyl acetate and hexane. White powder (423 mg, 90%), ¹H NMR (DMSO-d₆) 10.43 (s, 1H, NH), 8.30 (d, J = 8.40 Hz, 2H, 2×CH), 8.15 (t, J = 9.00, 3H, 3×CH), 7.79 (d, J = 8.80 Hz, 1H, CH), 7.61 (d, J = 8.80 Hz, 1H, CH), 7.32 (t, J = 8.20 Hz, 1H, 1×CH), 7.26–7.24 (m, 1H, CH), 7.05 (d, J = 8.00 Hz, 1H, CH), 5.04 (q, J = 6.80 Hz, 1H, CH), 1.64 (d, J = 6.40 Hz, 3H, CH₃). Purity 98% (t_R = 13.33). m.p. 244 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-(hydrazinecarbonyl)phenyl)benzo[d]oxazol-5-yl)propanamide (23):

Hydrazine hydrate (3 mmol) was added to the stirred solution of 8h (1 mmol) in ethanol (10 mL) at room temperature and refluxed for 5 h. After completion, the solvent was removed under reduced pressure, water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSO_4. The solvent was removed under reduced pressure and the crude mixture was purified through column chromatography. White powder (324 mg, 67%), ¹H NMR (DMSO-d₆) 10.41 (s, 1H, NH), 9.97 (s, 1H, NH), 8.25 (d, J = 6.80 Hz, 2H, 2×CH ), 8.16 (m, 1H, CH), 8.03 (d, J = 6.80 Hz, 2H, 2×CH), 7.77 (d, J = 9.2 Hz, 1H, CH), 7.60 (d, J = 8.40 Hz, 1H, CH), 7.32 (t, J = 7.60, 1H, CH), 7.25 (m, 1H), 7.05 (d, J = 8.40 Hz, 1H, CH), 5.03 (q, J = 6.80 Hz, 1H, CH), 4.60 (bs, 2H, NH₂), 1.58 (d, J = 6.80 Hz, 3H, CH₃). Purity 97% (t_R = 10.37). m.p. 247–248 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl)benzo[d]oxazol-5-yl)propanamide (24):

To the stirred solution of 23 (1 mmol) in anhydrous DMF (2 mL), carbonyldiimidazole (1.2 mmol) and diisopropylethylamine (1.2 mmol) was added. The reaction mixture was stirred for 2 h at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined extract was washed with brine, dried using anhydrous MgSO₄, filtered and concentrated. The residue was purified through colum chromatography (2−8% MeOH gradient in CH₂Cl₂) to give the title compound. White solid (336 mg, 66%), ¹H NMR (DMSO-d₆) 12.78 (s, 1H, NH), 10.44 (s, 1H, NH), 8.33 (d, J = 7.60 Hz, 2H, 2×CH), 8.17(m, 1H, CH), 8.01 (d, J = 8.00 Hz, 2H, 2×CH), 7.78 (d, J = 5.20 Hz, 1H, CH), 7.61 (d, J = 8.80 Hz, 1H, CH), 7.32 (t, J = 8.40 Hz, 1H, CH), 7.25 (m, 1H, CH), 7.06 (d, J = 8.00 Hz, 1H, CH),, 5.04 (q, J = 6.80 Hz, 1H, CH), 1.64 (d, J = 6.00 Hz, 3H, CH₃). Purity 99% (t_R = 13.05). m.p. 288-dec C.

(S,Z)-2-(2,3-Dichlorophenoxy)-N-(2-(4-(N’-hydroxycarbamimidoyl)phenyl)benzo[d]oxazol-5-yl)propanamide (25):

(S)-N-(2-(4-cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenox-y)propanamide 8f (1 mmol) was dissolved in anhydrous ethanol (5 mL) followed by hydroxylamine hydrochloride (3 mmol) and triethylamine (6 mmol) was added. The reaction mixture was refluxed for 4 h and the solvent was removed under reduced pressure. The residue was suspended in water and extracted with DCM (20 mL). Organic layer was washed with brine and dried using anhydrous MgSO₄. The solvent was removed under reduced pressure and the crude product was purified through column chromatography (2−8% MeOH gradient in CH₂Cl₂) to give the title compound. White solid (396 mg, 82%), ¹H NMR (DMSO-d₆) 10.34 (s, 1H, NH), 9.85 (s, 1H, OH), 8.13–8.08 (m, 2H, 2×CH), 7.86–7.84 (m, 2H, 2×CH), 7.71–7.68 (m, 1H, CH), 7.53–7.51 (m, 1H, CH), 7.28–7.18 (m, 3H, 3×CH), 7.00–6.98 (m, 1H, CH), 5.90 (s, 2H, NH₂), 4.97 (m, 1H, CH), 1.57 (m, 3H, CH₃). Purity 97% (t_R = 14.97). m.p. 196–197 C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl)benzo-[d]oxazol-5-yl)propanamide (26):

To the stirred solution of 25 (1 mmol) in anhydrous DMF (2 mL), carbonyldiimidazole (1.2 mmol) and diisopropylethylamine (1.2 mmol) was added. The reaction mixture was stirred for 2 h at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined extract was washed with brine, dried using anhydrous MgSO₄, filtered and concentrated. The residue was purified through colum chromatography (2−8% MeOH gradient in CH₂Cl₂) to give the title compound. White solid (321 mg, 61%), ¹H NMR (DMSO-d₆) 10.49 (s, 1H, NH), 9.60 (bs, 1H, NH), 8.32 (d, J = 8.40 Hz, 2H, 2×CH), 8.15 (m, 1H, CH), 7.99 (d, J = 8.40 Hz, 2H, 2×CH), 7.72 (m, 1H, CH), 7.58 (m, 1H, CH), 7.27 (t, J = 7.60 Hz, 1H, CH), 7.21 (m, 1H, CH), 7.02 (d, J = 8.40 Hz, 1H, CH), 5.03 (q, J = 6.80 Hz, 1H, CH), 1.59 (d, J = 6.80 Hz, 3H, CH₃). Purity 98% (t_R = 13.04). m.p. 260 C.

(S)-N-(2-(4-(1H-Tetrazol-5-yl)phenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenoxy)propanamide (27):

A mixture of (S)-N-(2-(4-cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenoxy)propanamide 8f (1 mmol), NaN₃ (2 mmol), and NH₄Cl (2 mmol) in DMF (1.5 mL) was heated at 100 °C for 6 h. Solvent was removed under reduced pressure, water was added and extracted with ethyl acetate. The organic layer was washed with brine, dried using anhydrous MgSO₄, filtered and concentrated under reduced pressure. The crude residue was purified through column chromatography. White solid (306 mg, 62%), ¹H NMR (DMSO-d₆) 11.11 (s, 1H, NH), 10.38 (s, 1H, NH), 8.36–8.34 (m, 1H, CH), 8.22 (d, J = 5.80 Hz, 2H, 2×CH), 8.13 (m, 1H, CH), 7.90 (m, 1H, CH), 7.76–7.70 (m, 1H, CH), 7.59–7.50 (m, 1H, CH), 7.30–7.21 (m, 2H, 2×CH), 7.00 (m, 1H, CH), 5.01 (m, 1H, CH), 1.59 (m, 3H, CH₃). Purity 99% (t_R = 12.25). m.p. 257 C.

(S)-4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)benzamide (28):

To the stirred solution of (S)-N-(2-(4-cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenoxy)prop-anamide 8f (1 mmol) in dry tert-butyl alcohol (4 mL/mmol), KOtBu (3 mmol), was added. The reaction mixture was stirred at room temperature for 12 h under a nitrogen atmosphere, and progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine, dried using anhydrous MgSO₄ and solvent was removed under reduced pressure. The crude mixture was purified through column chromatography. White solid (253 mg, 54%), ¹H NMR (DMSO-d₆) 10.36 (s, 1H, NH), 8.21 (d, J = 8.00 Hz, 2H, 2×CH), 8.12 (s, 2H, NH₂), 8.03 (d, J = 8.00 Hz, 2H, 2×CH), 7.73 (d, J = 8.40 Hz, 1H, CH), 7.55 (d, J = 8.40 Hz, 1H, CH), 7.51 (m, 1H, CH), 7.26 (t, J = 8.00 Hz, 1H, CH), 7.20 (m, 1H), 7.00 (d, J = 8.40 Hz, 1H, CH) 4.98 (q, J = 6.80 Hz, 1H, CH), 1.59 (d, J = 6.80 Hz, 3H, CH₃). Purity 98% (t_R = 11.56). m.p. 232 C.

(S)-N-(2-(4-(Aminomethyl)phenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenoxy)propanamide (29):

To a stirred solution of (S)-N-(2-(4-cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichloroph-enoxy)propanamide 8f ( 2.0 mmol) in THF: MeOH (15 mL), Boc₂O (873 mg, 4.0 mmol) and NiCl₂·6H₂O (48 mg, 0.2 mmol) were added at 0 ºC and stirred for five minutes. After five minutes, NaBH₄ (530 mg, 14.0 mmol) was added in small portions over 30 min followed by stirring at room temperature for 2h. The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The crude residue was poured into 1N HCl and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSO₄. Solvent was removed under reduced pressure and crude reaction mixture was purified through column chromatography. Trifluoroacetic acid (1 mL) was added to the stirred solution of Boc protected amine (1 mmol) in dry DCM (5 mL) at 0 ºC. After 4 h, the reaction was quenched with a saturated NaHCO₃ solution and extracted with DCM. The organic layer was washed with brine and dried using anhydrous MgSO₄. The solvent was removed under reduced pressure and the product was recrystallized using ethyl acetate and hexane. White solid (305 mg, 67%), ¹H NMR (DMSO-d₆) 10.40 (s, 1H, NH), 8.14–8.10 (m, 3H, 3×CH), 7.73 (d, J = 9.20 Hz, 1H, CH), 7.58–7.56 (m, 3H, 3×CH), 7.32 (t, J = 8.20 Hz, 1H, CH), 7.26–7.24 (m, 1H, CH), 7.05 (d, J = 8.00 Hz, 1H, CH), 5.03 (q, J = 6.80 Hz, 1H, CH), 3.82 (s, 3H, -OCH₃), 1.64–1.63 (d, J = 6.00 Hz, 3H, CH₃). Purity 97% (t_R = 14.63). m.p. 189-dec C.

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(2-oxo-1,2-dihydropyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (30):

To the stirred solution of 8b (1 mmol) in DMF, LiCl (5 mmol) and p-TSA (5 mmol) was added and stirred well for 2 h at 120 ºC. After completion, reaction was quenched with saturated NaHCO₃ solution and extracted with ethyl acetate. The organic layer was washed with brine and dried using MgSO₄. Solvent was removed under reduced pressure and crude residue was purified through column chromatography. White solid (341 mg, 77%), ¹H NMR (DMSO-d₆) 12.20 (bs, 1H, NH), 10.30 (s, 1H, NH), 8.19 (s, 1H, CH), 8.02 (dd, J = 9.80 Hz, J = 2.20 Hz, 1H, CH), 7.98 (m, 1H, CH), 7.61 (d, J = 9.20 Hz, 1H, CH), 7.46 (m, 1H, CH), 7.27 (t, J = 8.00 Hz, 1H, CH), 7.19 (m, 1H), 6.99 (d, J = 8.80 Hz, 1H, CH), 6.47 (d, J = 9.60 Hz, 1H, CH), 4.97 (q, J = 6.40 Hz, 1H, CH), 1.58 (d, J = 6.80 Hz, 3H, CH₃). Purity 97% (t_R = 10.25). m.p. 240–241 C.

(S)-4-(5-(2-(2,3-Dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)pyridine1-oxide (31):

m-Chloroperoxybenzoic acid (2 mmol) was added to the stirred solution of 8a (1 mmol) in dry DCM at rt and stirred well for overnight. After completion, reaction was quenched with saturated NaHCO₃ solution and extracted with DCM. The organic layer was washed with brine, dried using MgSO₄, filtered and concentrated under reduced pressure. The crude product was purified through column chromatography. White solid (327 mg, 74%), ¹H NMR (DMSO-d₆) 10.43 (s, 1H, NH), 8.38 (d, J = 6.80 Hz, 2H, 2×CH), 8.17 (m, 1H, CH), 8.09 (d, J = 6.80 Hz, 2H, 2×CH), 7.78–7.76 (m, 1H, CH), 7.63–7.60 (m, 1H, CH), 7.16–7.12 (m, 1H, CH), 7.06–7.02 (m, 1H, CH), 6.95–6.92 (m, 1H, CH), 5.00 (q, J = 6.80 Hz, 1H, CH), 1.62 (d, J = 6.80 Hz, 3H, CH₃). Purity 99% (t_R = 10.80). m.p. 125-dec C.

(S)-2-(2-(Aminomethyl)phenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanamide (32):

The reaction was proceeded with 8u using the procedure, which used for the synthesis of 29. White solid (283 mg, 68%), ¹H NMR (DMSO-d₆) 11.10 (bs, 1H, NH), 8.13–8.11 (d, J = 9.2 Hz, 2H, 2×CH), 8.02–8.01 (m, 1H, CH), 7.67 (d, J = 8.2 Hz, 1H, CH), 7.54–7.51 (m, 1H, CH), 7.29–7.14 (m, 3H, 3×CH), 7.04–7.02 (m, 1H, CH), 6.93–6.91 (m, 1H, CH), 5.06 (q, J = 6.80 Hz, 1H, CH), 3.71 (s, 3H, -OCH₃), 1.62 (d, J = 6.80 Hz, 3H, CH₃). Purity 97% (t_R = 14.31). m.p. 160–161 C.

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2-hydroxyphenoxy)propanamide (33):

Compound 8v (1 mmol) was dissolved in 10 mL EtOAc: MeOH (1:1) and 10% Pd/C (catalytic) was added and stirred well under a H₂ atmosphere for 3 h. After the successful completion, reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The crude product was purified through column chromatography. White solid (359 mg, 90%), ¹H NMR (DMSO-d₆) 10.26 (s, 1H, NH), 9.29 (s, 1H, OH), 8.34 (d, J = 8.4 Hz, 2H, 2×CH), 8.22–8.21 (m, 1H, CH), 8.08 (d, J = 8.80 Hz, 2H, 2×CH), 7.81 (d, J = 8.80 Hz, 1H, CH), 7.68–7.66 (m, 1H, CH), 7.05–7.03 (d, J = 7.6 Hz, 1H, CH), 6.88–6.83 (m, 2H, 2×CH), 6.71–6.73 (m, 1H, CH), 4.87 (q, J = 6.80 Hz, 1H, CH), 1.57 (d, J = 6.80 Hz, 3H, CH₃). Purity 99% (t_R = 11.10). m.p. 185–186 C.

Methyl (S)-2-(2-bromo-3-formylphenoxy)propanoate (34):

2-Bromo-3-hydroxybenzaldehyde (1 mmol) was added to the solution of (+)-methyl-D-lactate (1.38 mmol) in anhydrous THF (6 mL) under a nitrogen atmosphere and the solution was cooled to 0 °C. PPh₃ (1.20 mmol) was added portion wise and stirred for 10 min followed by the dropwise addition of DEAD (1.50 mmol) over 20 min. The reaction mixture was stirred for 2 h at room temperature. After completion, the solvent was removed under reduced pressure and the crude mixture was purified through column chromatography using ethyl acetate/n-hexane (10:90) to yield methyl ((S)-2-(2-bromo-3-formylphenoxy)propanoate (236 mg, 83%) as a white solid.

Methyl (S)-2-(2-bromo-3-((methoxymethoxy)methyl)phenoxy)propanoate (35):

Under a nitrogen atmosphere, NaBH₄ (1.5 mmol) was added to the stirred solution of ((S)-2-(2-bromo-3-formylphenoxy)propanoate 34 (1 mmol) in ethanol at 0 ºC. After 1 h at room temperature, the reaction was quenched with a saturated NaHCO₃ solution and extracted with ethyl acetate. The organic layer was washed with brine and dried using anhydrous MgSO₄, filtered and concentrated under reduced pressure. The crude product was dissolved in dry DCM and cooled to 0 ºC. DIPEA (5 mmol) was added, followed by dropwise addition of MOMCl (3 mmol) and stirred for overnight at room temperature. After completion, the reaction was quenched with saturated NH₄Cl and extracted with DCM. The organic layer was dried using anhydrous MgSO₄ and the solvent was removed under reduced pressure. The crude residue was purified through column chromatography. White solid (239 mg, 72%), ¹H NMR (DMSO-d₆) 7.32–7.28 (m, 1H, CH), 7.12–7.09 (m, 1H, CH), 6.90–6.88 (m, 1H, CH), 5.06 (m, 1H, CH), 4.70 (bs, 3H, -OCH₃), 4.61–4.51 (m, 4H, 2×-OCH₂-), 3.68 (s, 3H, -COOCH₃), 1.55 (m, 3H, CH₃).

Methyl (S)-2-(3-((methoxymethoxy)methyl)-2-(4,4,5,5-tetramethyl-1,3,2-dioxa-borolan-2-yl)phenoxy)propanoate (36):

To a solution of methyl (S)-2-(2-bromo-3-((methoxymethoxy)methyl)phenoxy)propanoate 35 (400 mg, 1.145 mmol) in 1,4-dioxane (6 mL) was added KOAc (483 mg, 4.923 mmol), Pin₂B₂ (349 mg, 1.37 mmol) and Pd(Ph₃P)₂Cl₂ (80 mg, 0.114 mmol) under an argon atmosphere. The reaction flask was placed under vacuum and then backfilled with argon (two times). Then the reaction was stirred at 95 ºC for overnight. The solvent was removed under reduced pressure and the residue was suspended in water and extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous MgSO₄, filtered and concentrated. The crude product was purified through column chromatography using hexane/ethyl acetate. White solid (243 mg, 64%), ¹H NMR (DMSO-d₆) 7.28–7.24 (m, 1H, CH), 6.93–6.88 (m, 1H, CH), 6.68–6.66 (m, 1H, CH), 4.90–4.86 (m, 1H, CH), 4.56 (bs, 4H, 2×-OCH₂-), 4.47 (bs, 3H, -OCH₃), 3.67 (s, 3H, -COOCH₃), 1.45 (m, 3H, CH₃), 1.29 (bs, 12H, 4×CH₃).

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-((1-hydroxy-1,3-dihydrobenzo-[c][1,2]oxaborol-7-yl)oxy)propanamide (37):

Ester 36 (1 mmol) was dissolved in 5 mL THF: MeOH (2:3) and stirred at 0 ºC. LiOH (1.5 mmol) was added portion wise to the solution and stirred well at room temperature for 4 h. The solvents were removed under reduced pressure, crude residue was dissolved in ethyl acetate and 1N HCl was added until a pH of 4 was reached. The product was extracted with ethyl acetate and the organic extracts were combined, washed with brine, dried over anhydrous MgSO₄. The solvent was removed under reduced pressure and crude acid was used in the next step without chromatographic purification. EDC·HCl (1.5 mmol) was added to the stirred solution of acid (1 mmol), amine 5 (Ar = 4-CNPh, 1 mmol) in dry DMF at 0 ºC under nitrogen atmosphere. Reaction mixture was stirred well for overnight at room temperature. After completion of the reaction, excess water was added and extracted with ethyl acetate. The organic layer was washed with brine and dried using MgSO₄. The solvent was removed under reduced pressure. The crude residue was purified through column chromatography give desired coupled product. To a solution of this MOM protected material (140 mg, 0.363 mmol) in THF (0.9 mL) 4 N HCl (0.43 mL, 18.1 mmol) was added. The reaction was stirred at room temperature for 4 h, upon completion ethyl acetate was added and extracted. The organic phase was washed with brine, dried over anhydrous MgSO₄, filtered and concentrated under reduced pressure. The crude product was purified through column chromatography using hexane/ethyl acetate. Brown solid (122 mg, 28%).¹H NMR (DMSO-d₆) 10.13 (s, 1H, NH), 9.20 (s, 1H, OH), 8.34 (d, J = 8.4 Hz, 2H, 2×CH), 8.22 (m, 1H, CH), 8.08 (d, J = 8.40 Hz, 2H, 2×CH), 7.80 (d, J = 9.2 Hz, 1H, CH), 7.70 (m, 1H, CH), 7.45 (m, 1H, CH), 7.04 (d, J = 7.60 Hz, 1H, CH), 6.93 (d, J = 7.60 Hz, 1H, CH), 5.03 (q, J = 6.80 Hz, 1H, CH), 4.98 (s, 2H, -OCH₂-), 1.62 (d, J = 6.8 Hz, 3H, CH₃). Purity 97% (t_R = 11.27). m.p. hygroscopic.

Inhibition of MtbIMPDH.

The K_i,app values were determined by measuring the initial velocities at varying concentrations of the inhibitors (1–10,000 nM) with fixed concentrations of IMP (0.5 mM) and NAD⁺ (1.5 mM) and MtbIMPDH2 (20–50 nM). Inhibition of human IMPDH2 was assayed IMP using (0.25 mM) and NAD⁺ (0.060 mM) and hIMPDH2 (250 nM). Inhibition of human GMPR2 was assayed using GMP (0.050 mM), NADPH (0.045 mM) and enzyme (100 nM). The assay buffer contained 50 mM TrisCl, pH 8.0, 100 mM KCl and 1 mM dithiothreitol.

The values of K_i,app were obtained using the equations (1) and (2)

v_{i} = v_{0} / (1 + [I] / {IC}_{50})

(1)

K_{i,app} = {IC}_{50} - [E] / 2

(2)

where v_i is the initial velocity in the presence of inhibitor and v₀ is the initial velocity in the absence of the inhibitor. If the IC₅₀ value is comparable to the enzyme concentration, the Morrison tight binding equation was used to determine K_i,app (3)

v_{i} / v_{0} = 1 - (([E] + [I] + K_{i,app}) - {({([E] + [I] + K_{i,app})}^{2} - 4 [E] [I])}^{0.5}) / (2 [E])

(3)

where [E] is the concentration of the enzyme. All the initial velocity measurements were performed in triplicates. The K_i,app values reported are the average of at least two independent experiments unless otherwise noted.

MIC determinations.

MICs were determined as previously described.⁹ MIC values were determined in at least triplicate according to the broth microdilution methods using compounds from DMSO stock solutions. Isoniazid was used as a positive control and DMSO was utilized as a negative control. Isolated Mtb cells (ATCC 27294) were cultured to an OD 0.2–0.3 in the required medium, then diluted to deliver approximately 1 × 10⁴ bacteria per well of a 96 well clear round-bottom plate. Plates were read after 1 week with an inverted enlarging mirror plate reader and graded as either growth or no growth. GAST/Fe medium (per liter) consisted of 0.3 g of Bacto Casitone (Difco), 4.0 g of dibasic potassium phosphate, 2.0 g of citric acid, 1.0 g of L-alanine, 1.2 g of magnesium chloride hexahydrate, 0.6 g of potassium sulfate, 2.0 g of ammonium chloride, 1.80 ml of 10 N sodium hydroxide, and 10.0 ml of glycerol, 0.05% Tween 80 and 0.05 g of ferric ammonium citrate adjusted to pH 6.6. 7H9/glycerol/glucose/BSA/Tween medium consisted of Middlebrook 7H9 broth base supplemented per liter with 0.2% glucose, 0.2% glycerol, 0.5% BSA fraction V, 0.08% NaCl and 0.05% Tween 80. Cultures were supplemented with 200 μM guanine as noted.

MtbIMPDH2 downregulation and susceptibility of SRMV2.6.

Mtb strains guaB2 cKD (guaB2-B3 Tet-OFF attB::guaB3) and SRMV2.6, which carries guaB2^Y487C, were cultured in Middlebrook 7H9 media (Difco) supplemented with 0.2% glycerol, Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment (Difco) and 0.05% Tween 80 (7H9/Glycerol/OADC/Tween). Hygromycin (Hyg), kanamycin (Km) and gentamycin (Gm) were used in guaB2 cKD culture at final concentrations of 50, 25 and 2.5 μg/mL, respectively. ATc (Sigma) was used at concentrations up to 100 ng/mL. For pairwise combination (checkerboard) assays, a two-dimensional array of serial dilutions of test compound and ATc was prepared in 96-well plates, as previously described.¹¹ MIC testing was carried out by broth microdilution using the AlamarBlue (AB, Invitrogen) assay ^{51, 52}.

Mammalian Cell Culture.

Hep G2 cells (ATCC, purchased February 2017) were cultivated in EMEM supplemented with 10% heat inactivated FBS and 1X penicillin/streptomycin under standard conditions (37 °C in a 5% CO₂ humidified atmosphere). HEK293T, MCF7 and HELA cells were cultured in DMEM with 10% heat inactivated FBS and 1X penicillin/streptomycin. Active cell cultures routinely tested for presence of Mycoplasma (MycoAlert™ detection kit, Lonza) and confirmed to be Mycoplasma free.

LDH Cytotoxicity Assay.

All compounds were dissolved in DMSO and further diluted with culture medium before use in tissue culture assays (final DMSO concentrations were ≤ 0.1%). To determine cytotoxicity, LDH release was measured with the LDH Cytotoxicity Assay Kit (Pierce) according to manufacturer’s protocol. Briefly, 96 well plates were seeded with 13,000 Hep G2 cells (all other cell lines seeded at 6,000 to 8,000 cells per well) and the cells were cultured for 24 h prior to drug treatment. The cells were incubated in 110 μL of EMEM containing compound or DMSO (vehicle only, control) for 24 h at 37°C. At least four wells from each plate were used as either ‘spontaneous’ LDH controls or as ‘maximum’ LDH controls per manufacturer’s instructions. Cytotoxicity was determined by measuring absorbance on a microplate reader. Data represent two independent experiments each performed in quadruplicate (n =8).

ADMET studies were performed by GVK Biosciences, (Hyderbad India).

Supplementary Material

Spectra

NIHMS984968-supplement-Spectra.pdf^{(3.3MB, pdf)}

datafile

NIHMS984968-supplement-datafile.csv^{(6.8KB, csv)}

Table 2.

MtbIMPDH2ΔCBS Inhibition of Benzoxazole Derivatives Containing Various Phenyl Ether Substituents.


Cmpd	R₁	R₂	R₃	Ar	K_i,app^a(nM)
Cmpd	R₁	R₂	R₃	Ar	MtbIMPDH2	hIMPDH2	hGMPR2
8l	F	F	H	4-Py	55 ± 9	>5000	>5000
8m	F	F	H	4-OMe-Ph	19 ± 2	>5000	>5000
8n	F	F	H	4-OH-Ph	121 ± 21	>5000	>5000
8o	F	F	H	3-(6-OMe)-Py	42 ± 17	>5000	>5000
8p	F	F	H	4-CN-Ph	7.2 ± 2.5	2000	>5000
8q	F	F	H	4-F-Ph	40 ± 3	>5000	>5000
8r	F	F	F	4-CN-Ph	22 ± 11	>5000	>5000
8s	CN	H	H	4-Py	76 ± 35	>5000	>5000
8t	CN	F	H	4-Py	40 ± 2	>5000	>5000
8u	CN	H	H	4-OMePh	33 ± 5	>5000	>5000
8v	OBn	H	H	4-CN-Ph	43 ± 12	>5000	>5000
32	CH₂NH₂	H	H	4-OMe-Ph	23 ± 9	>5000	>5000
33	OH	H	H	4-CN-Ph	36 ± 12	>5000	>5000
37	B(OH)OCH₂		H	4-CN-Ph	370 ± 70 ^*	>5000	>5000

Open in a new tab

^a.

Values are the average and range of at least two independent determinations.

Single determination, error of the fit is listed.

Acknowledgment:

This work was funded with funds from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Department of Health and Human Services (AI093459 to LH, AI125362 to GDC and contracts HHSN272200700058C and HHSN272201200026C to the Center of Structural Genomics of Infectious Diseases); the Intramural Research Program of NIAID (HB); and the South African Medical Research Council and National Research Foundation (to VM). We thank Xingyou Wang for assistance with the melting points.

Abbreviations Used:

BSA: bovine serum albumin
CBS: cystathione β-synthetase
CDI: carbonyl diimidazole
DCM: dichloromethane
DEAD: diethylazodicarboxylate
DIPEA: diisopropylethylamine
EDC•HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
EDG: electron donating group
EWG: electron withdrawing group
Gua: guanine
HATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate)
IMP: inosine 5’-monophosphate
IMPDH2: inosine 5’-monophosphate dehydrogenase 2
LDH: lactate dehydrogenase
m-CPBA: meta-chloroperoxybenzoic acid
MIC: minimum inhibitory concentration
Mtb: Mycobacterium tuberculosis
NAD: nicotinamide adenine dinucleotide
NMP: N-methyl-2-pyrrolidone
p-TSA: para-toluene sulfonic acid
SAR: structure-activity relationship
TEA: triethylamine
TFA: trifluoroacetic acid
XMP: xanthosine 5’-monophosphate

Footnotes

Supporting Information: CSV file with molecular formula strings and the associated biochemical and biological data.

REFERENCES

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2.Hedstrom L IMP Dehydrogenase: structure, mechanism and inhibition. Chem. Rev 2009, 109, 2903 – 2928. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Hedstrom L; Liechti G; Goldberg JB; Gollapalli DR The antibiotic potential of prokaryotic IMP dehydrogenase inhibitors. Curr. Med Chem 2011, 18, 1909–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Spectra

NIHMS984968-supplement-Spectra.pdf^{(3.3MB, pdf)}

datafile

NIHMS984968-supplement-datafile.csv^{(6.8KB, csv)}

[R1] 1.Bloemberg GV; Keller PM; Stucki D; Trauner A; Borrell S; Latshang T; Coscolla M; Rothe T; Homke R; Ritter C; Feldmann J; Schulthess B; Gagneux S; Bottger EC Acquired resistance to bedaquiline and delamanid in therapy for tuberculosis. N. Engl. J. Med 2015, 373, 1986–1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] 2.Hedstrom L IMP Dehydrogenase: structure, mechanism and inhibition. Chem. Rev 2009, 109, 2903 – 2928. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Hedstrom L; Liechti G; Goldberg JB; Gollapalli DR The antibiotic potential of prokaryotic IMP dehydrogenase inhibitors. Curr. Med Chem 2011, 18, 1909–18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Cole ST; Brosch R; Parkhill J; Garnier T; Churcher C; Harris D; Gordon SV; Eiglmeier K; Gas S; Barry CE 3rd; Tekaia F; Badcock K; Basham D; Brown D; Chillingworth T; Connor R; Davies R; Devlin K; Feltwell T; Gentles S; Hamlin N; Holroyd S; Hornsby T; Jagels K; Krogh A; McLean J; Moule S; Murphy L; Oliver K; Osborne J; Quail MA; Rajandream MA; Rogers J; Rutter S; Seeger K; Skelton J; Squares R; Squares S; Sulston JE; Taylor K; Whitehead S; Barrell BG Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393, 537–544. [DOI] [PubMed] [Google Scholar]

[R5] 5.Sassetti CM; Boyd DH; Rubin EJ Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol 2003, 48, 77–84. [DOI] [PubMed] [Google Scholar]

[R6] 6.DeJesus MA; Gerrick ER; Xu W; Park SW; Long JE; Boutte CC; Rubin EJ; Schnappinger D; Ehrt S; Fortune SM; Sassetti CM; Ioerger TR Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis. MBio 2017, 8, e02133–16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] 7.Usha V; Gurcha SS; Lovering AL; Lloyd AJ; Papaemmanouil A; Reynolds RC; Besra GS Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis. Microbiology 2011, 157, 290–299. [DOI] [PubMed] [Google Scholar]

[R8] 8.Chen L; Wilson DJ; Xu Y; Aldrich CC; Felczak K; Sham YY; Pankiewicz KW Triazole-linked inhibitors of inosine monophosphate dehydrogenase from human and Mycobacterium tuberculosis. J. Med. Chem 2010, 53, 4768–4778. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Makowska-Grzyska M; Kim Y; Gorla SK; Wei Y; Mandapati K; Zhang M; Maltseva N; Modi G; Boshoff HI; Gu M; Aldrich C; Cuny GD; Hedstrom L; Joachimiak A Mycobacterium tuberculosis IMPDH in complexes with substrates, products and antitubercular compounds. PLoS One 2015, 10, e0138976. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Park Y; Pacitto A; Bayliss T; Cleghorn LA; Wang Z; Hartman T; Arora K; Ioerger TR; Sacchettini J; Rizzi M; Donini S; Blundell TL; Ascher DB; Rhee K; Breda A; Zhou N; Dartois V; Jonnala SR; Via LE; Mizrahi V; Epemolu O; Stojanovski L; Simeons F; Osuna-Cabello M; Ellis L; MacKenzie CJ; Smith AR; Davis SH; Murugesan D; Buchanan KI; Turner PA; Huggett M; Zuccotto F; Rebollo-Lopez MJ; Lafuente-Monasterio MJ; Sanz O; Diaz GS; Lelievre J; Ballell L; Selenski C; Axtman M; Ghidelli-Disse S; Pflaumer H; Bosche M; Drewes G; Freiberg GM; Kurnick MD; Srikumaran M; Kempf DJ; Green SR; Ray PC; Read K; Wyatt P; Barry CE 3rd; Boshoff HI Essential but not vulnerable: indazole sulfonamides targeting inosine monophosphate dehydrogenase as potential leads against Mycobacterium tuberculosis. ACS Infect. Dis 2017, 3, 18–33. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Singh V; Donini S; Pacitto A; Sala C; Hartkoorn RC; Dhar N; Keri G; Ascher DB; Mondesert G; Vocat A; Lupien A; Sommer R; Vermet H; Lagrange S; Buechler J; Warner DF; McKinney JD; Pato J; Cole ST; Blundell TL; Rizzi M; Mizrahi V The inosine monophosphate dehydrogenase, GuaB2, is a vulnerable new bactericidal drug target for tuberculosis. ACS Infect. Dis 2017, 3, 5–17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Usha V; Hobrath JV; Gurcha SS; Reynolds RC; Besra GS Identification of novel Mt-Guab2 inhibitor series active against M. tuberculosis. PLoS One 2012, 7, e33886. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Cox JA; Mugumbate G; Del Peral LV; Jankute M; Abrahams KA; Jervis P; Jackenkroll S; Perez A; Alemparte C; Esquivias J; Lelievre J; Ramon F; Barros D; Ballell L; Besra GS Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen. Sci. Rep 2016, 6, 38986. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] 14.Ignoul S; Eggermont J CBS domains: structure, function, and pathology in human proteins. Am. J. Physiol. Cell Physiol 2005, 289, C1369– C1378. [DOI] [PubMed] [Google Scholar]

[R15] 15.Nimmesgern E; Black J; Futer O; Fulghum JR; Chambers SP; Brummel CL; Raybuck SA; Sintchak MD Biochemical analysis of the modular enzyme inosine monophosphate dehydrogenase. Protein Expr. Purif 1999, 17, 282–289. [DOI] [PubMed] [Google Scholar]

[R16] 16.Gan L; Petsko GA; Hedstrom L Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5’-monophosphate dehydrogenase: NAD⁺ orients the active site loop for catalysis. Biochemistry 2002, 41, 13309–13317. [DOI] [PubMed] [Google Scholar]

[R17] 17.Makowska-Grzyska M; Kim Y; Maltseva N; Osipiuk J; Gu M; Zhang M; Mandapati K; Gollapalli DR; Gorla SK; Hedstrom L; Joachimiak A A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity. J. Biol. Chem 2015, 290, 5893–5911. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Expanding benzoxazole based inosine 5’-monophosphate dehydrogenase (IMPDH) inhibitor structure-activity as potential anti-tuberculosis agents

Shibin Chacko

Helena I M Boshoff

Vinayak Singh

Davide M Ferraris

Deviprasad R Gollapalli

Minjia Zhang

Ann P Lawson

Michael J Pepi

Andrzej Joachimiak

Menico Rizzi

Valerie Mizrahi

Gregory D Cuny

Lizbeth Hedstrom

Abstract

Table of Content graphic

INTRODUCTION

Figure 1.

RESULTS AND DISCUSSION

Design strategy.

Figure 2.

Scheme 1.

Scheme 2.

Scheme 3.

Scheme 4.

Scheme 5.

Evaluation of MtbIMPDH2 inhibition.

Table 1.

Table 3.

Selectivity of inhibition.

Evaluation of antibacterial activity.

Table 4.

Figure 3.

Table 5.

Figure 4.

Evaluation of Human Cytotoxicity Activity.

Figure 5.

Preliminary pharmacokinetic evaluation of 17b.

CONCLUSIONS

Experimental Section

Synthetic Chemistry.

General Procedure for the Synthesis of 5-nitro-2-phenylbenzo[d]oxazoles (4):

General Procedure for the Synthesis of 5:

General Procedure for the Synthesis of Phenyl Ethers 6:

General Procedure for Ester Hydrolysis for Preparation of 7:

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8a):

General Procedure for Synthesis of 8b-8v:

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(2-methoxypyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8b):

(S)-2-(2,3-dichlorophenoxy)-N-(2-(6-methoxypyridin-3-yl)benzo[d]oxazol-5-yl)propanamide (8c):

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanamide (8d):

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-(trifluoromethoxy)phenyl)benzo[d]oxazol-5-yl)propanamide (8e):

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-dichlorophenoxy)propanamide (8f):

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)propanamide (8g):

Methyl (S)-4-(5-(2-(2,3-dichlorophenoxy)propanamido)benzo[d]oxazol-2-yl)benzoate (8h):

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-hydroxyphenyl)benzo[d]oxazol-5-yl)propanamide (8i):

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(3-hydroxyphenyl)benzo[d]oxazol-5-yl)propanamide (8j):

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(2-hydroxyphenyl)benzo[d]oxazol-5-yl)propanamide (8k):

(S)-2-(2,3-Difluorophenoxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8l):

(S)-2-(2,3-Difluorophenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanamide (8m):

(S)-2-(2,3-Difluorophenoxy)-N-(2-(4-hydroxyphenyl)benzo[d]oxazol-5-yl)propanamide (8n):

(S)-2-(2,3-Difluorophenoxy)-N-(2-(6-methoxypyridin-3-yl)benzo[d]oxazol-5-yl)propanamide (8o):

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3-difluorophenoxy)propanamide (8p):

(S)-2-(2,3-Difluorophenoxy)-N-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)propanamide (8q):

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-(2,3,4-trifluorophenoxy)propanamide (8r):

(S)-2-(2-Cyanophenoxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8s):

(S)-2-(2-Cyano-3-fluorophenoxy)-N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)propanamide (8t):

(S)-2-(2-Cyanophenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanamide (8u):

(S)-2-(2-(Benzyloxy)phenoxy)-N-(2-(4-cyanophenyl)benzo[d]oxazol-5-yl)propanamide (8v):

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-methoxyphenyl)benzo[d]oxazol-5-yl)propanethioamide (9):

(S)-2-(2,3-Dichlorophenoxy)propanal (10):

(S)-N-(2-(2,3-Dichlorophenoxy)propyl)-2-(4-methoxyphenyl)benzo[d]oxazol-5-amine (11):

2-(4-Methoxyphenyl)-6-nitroimidazo[1,2-a]pyridine (12):

(S)-2-(2,3-Dichlorophenoxy)-N-(2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-6-yl)propanamide (13):

2-(4-Methoxybenzyl)-5-nitrobenzo[d]oxazole (14):

(S)-2-(2,3-dichlorophenoxy)-N-(2-(4-methoxybenzyl)benzo[d]oxazol-5-yl)prop-anamide (15):

Synthesis of 17:

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-((2,3-dichlorophenyl)amino)propanamide (17a):

(S)-N-(2-(4-Cyanophenyl)benzo[d]oxazol-5-yl)-2-((2,3-difluorophenyl)amino)propanamide (17b):

Synthesis of 18: