Expanding Benzoxazole-Based Inosine 5′-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure-Activity As Potential Antituberculosis Agents

Article abstract of DOI:10.1021/acs.jmedchem.7b01839

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

Full text of DOI:10.1021/acs.jmedchem.7b01839

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Article
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, and Lizbeth Hedstrom
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01839 • Publication Date (Web): 10 May 2018
Downloaded from http://pubs.acs.org on May 13, 2018
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Expanding benzoxazole based inosine 5’ꢀ
monophosphate dehydrogenase (IMPDH) inhibitor
structureꢀactivity as potential antiꢀtuberculosis
agents
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§
#
†
∬
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* and Lizbeth Hedstrom*
§
¶
Department of Biology and Department of Chemistry, Brandeis University, 415 South St.
Waltham, Massachusetts 02454, United States
#
Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, Bethesda,
Maryland 20892, United States
†
MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of
Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine
&
Department of Pathology, University of Cape Town, Anzio Road, Observatory 7925, South
Africa
∫
Center for Structural Genomics of Infectious Diseases, University of Chicago, Chicago, IL
6
0557 and Structural Biology Center, Biosciences, Argonne National Laboratory, 9700 S Cass
Ave. Argonne, IL 60439, USA
∬
Dipartimento di Scienze del Farmaco, University of Piemonte Orientale, Via Bovio 6, 28100
Novara, Italy
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‡
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University
of Houston, Health Building 2, 4849 Calhoun Rd., Houston, Texas 77204, United States
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*
corresponding authors: gdcuny@central.uh.edu, hedstrom@brandeis.edu
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ABSTRACT: New drugs and molecular targets are urgently needed to address the emergence
and spread of drugꢀresistant 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 structureꢀactivity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors
and demonstrate that minimum inhibitory concentrations (MIC) of ≤ 1 ꢀM 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 secondꢀline injectables, capreomycin, kanamycin and amikacin. Frustratingly, as
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illustrated by the newly approved tuberculosis drugs bedaquiline and delamanid , 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
2
nucleotides and therefore controls the size of the guanine nucleotide pool. 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
3
biosynthesis and many other processes involved in cell proliferation. Mtb has three genes,
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designated as guaB1, guaB2 and guaB3, which encode IMPDH homologs. Only guaB2 is
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essential,
and only guaB2 encodes an active IMPDH (MtbIMPDH2). The proteins encoded
by guaB1 and guaB3 are each missing key catalytic residues and neither display IMPDH activity
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.
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
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but as yet no compounds have been reported with activity in animal models of tuberculosis.
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Recently, Park et al. suggested that high levels of guanine (200ꢀ2000 ꢀM ) in the lungs of
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infected rabbits and humans can protect bacteria from MtbIMPDH2 inhibitors, 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
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MtbIMPDH2 is rapidly bactericidal in vitro. They also showed that silencing of MtbIMPDH2
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expression prevented infection in mice , consistent with the lower levels of guanine found in
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mouse tissue (7ꢀ20 ꢀM ). On the basis of these findings, Singh et al. concluded that this
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enzyme is indeed vulnerable.
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) domain. CBS domains bind
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nucleotides and modulate the activity of other enzymes and channels . However, deletion of
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the CBS subdomain does not affect the activity of the catalytic domain in IMPDH
. The CBS
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subdomain binds ATP and/or GTP depending on the particular IMPDH
. These interactions
modulate the oligomerization of tetramers to form octamers and higher order structures in vitro,
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with modest effects on enzymatic activity
. 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
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other adenine nucleotides
. IMPDH binds nucleic acid and can act as a transcription factor in
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eukaryotes, and this activity is mediated by the CBS subdomain
. 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
28, 29
synthetase and DNA helicase
. 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
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downregulates both enzymatic and moonlighting activities . More potent MtbIMPDH2
inhibitors are required to resolve this conundrum and determine the vulnerability of Mtb
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infections to IMPDH inhibition .
We have developed triazole (Aꢀseries), benzimidazole (Cꢀseries), phthalazinone (Dꢀseries), 4ꢀ
oxoꢀ[1]benzopyrano[4,3ꢀc]pyrazole (Nꢀseries), urea (Pꢀseries) and benzoxazole (Qꢀseries)
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, 32ꢀ40
inhibitors of prokaryotic IMPDHs, including MtbIMPDH2
. These inhibitors exploit the
highly diverged cofactor binding site, which explains their selectivity for bacterial over
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, 17
eukaryotic orthologs of IMPDH
antibacterial activity against Mtb as well as other pathogens
Figure 1A), was a potent inhibitor of MtbIMPDH2 (Ki,app = 14 nM) and displayed moderate
. Individual members of the A, D, P and Qꢀseries display
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, 41, 42
. The best Q compound, 3
(
antibacterial activity (MIC = 6.3 and 11 ꢀM in minimal GAST/Fe and rich 7H9/ADC/Tween
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media, respectively). The values of MIC increased 3 to 7ꢀfold in the presence of guanine,
suggesting that antibacterial activity resulted from the onꢀtarget inhibition of MtbIMPDH2.
Herein we report an expansion of the structureꢀactivity relationship (SAR) for the Qꢀseries 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.
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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 βꢀ
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synthetase (CBS) motifs. 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
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(
MtbIMPDH2ꢁCBS) in complex with IMP and inhibitor 3 reveals that the aromatic ring of the
2
,3ꢀdichloroaniline is involved in a πꢀ π interaction with the purine base of IMP, whereas the
amide participates in an ionicꢀdipole 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 1,3ꢀbenzoxazole interacts with a water molecule that also engages the main chain nitrogen
and oxygen atoms of His286. Moreover, the 4ꢀpyridyl–1,3ꢀbenzoxazole moiety of 3 binds in a
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hydrophobic pocket formed by Val60, Pro61, and Tyr487 and the pyridyl nitrogen atom interacts
with the
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Figure 1. Inhibitors of MtbIMPDH2. A) Structures of 1, 2 and 3. B) Crystal structure of
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MtbIMPDH2ꢁCBS •IMP•3 (PDB 4ZQO) . 3 is pink, IMP is blue, hydrogen bonds are shown in
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cyan, residues from the adjacent subunit are marked with ‘. C) Crystal structures of M.
thermoresistible IMPDH2 in complex with IMP and cyclohexyl[4ꢀ(5ꢀisoquinolinylsulfonyl)ꢀ1ꢀ
piperazinyl]methanone (VCC234718, PDB 5J5R, protein and IMP are spring green, inhibitor is
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forest green) , and Nꢀ1Hꢀindazolꢀ6ꢀylꢀ3,5ꢀdimethylꢀ1Hꢀpyrazoleꢀ4ꢀsulfonamide (6Q9, PDB
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K4X, protein and IMP are gray, inhibitor is blue) . The structure of MtbIMPDH2ꢁCBS
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•
IMP•3 (PDB 4ZQO, protein and IMP are tan, 3 is pink) is also included. This figure was
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produced with UCSF Chimera .
Figure 2. An overview of the SAR explored in this study.
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backbone carbonyl of Ser57 via a water network. This pocket is not present in human IMPDHs,
explaining the selectivity of the compounds for bacterial IMPDHs. Note that the two
sulfonamide
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inhibitors in Figure 1C do not extend into this pocket, perhaps explaining the higher affinity and
10, 11
selectivity of the Q compounds.
Using this information, we explored several strategies to expand the SAR of the Qꢀseries
(
Figure 2): (1) modifications to the pyridine or replacement of the 4ꢀpyridyl with a phenyl
containing various substituents that can interact (e.g. via hydrogen bonds and ionicꢀdipole
interactions) with the side chain of Arg290; (2) modification of the 2,3ꢀdichloroaniline with 2,3ꢀ
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[1,2ꢀa]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 Qꢀseries derivatives is illustrated in Scheme 1.
Arylbenzoxazoles 4 were directly synthesized by oxidative cyclization methods using 2ꢀaminoꢀ
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 5ꢀamineꢀ2ꢀ
arylbenzoxazoles 5. Enantiomerically pure phenyl ethers 6 were synthesized from (+)ꢀmethyl Dꢀ
lactate and the corresponding phenol using Mitsunobu reaction conditions. The esters 6 were
hydrolyzed to the corresponding acids 7, and then coupled with various 5ꢀamineꢀ2ꢀ
3
9
arylbenzoxazoles in the presence of EDC·HCl in DMF to yield 8. Furthermore, 8 (Ar = 4ꢀ
OMePh, R = 2,3ꢀdiCl) was treated with Lawesson's reagent under standard conditions to
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2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
generate thioamide 9 quantitatively. Ester 6a was also treated with 1M DIBALꢀH in DCM at ꢀ78
ºC to generate aldehyde 10, which was subjected to reductive
0
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8
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Scheme 1. Synthesis of various aryl benzoxazoles 8, thioamide 9, amine 11, imidazo[1,2ꢀ
a
a]pyridine 13, methylene linked derivative 15 and Nꢀarylated benzoxazoles 17aꢀb.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
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3
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0
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2
2
2
2
2
2
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3
4
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4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
a
Reagents and conditions: (a) ArCHO, DarcoKB, O (1 atm), xylene 140 ºC, 6ꢀ8 h, 70ꢀ80%;
2
(
b) 10% Pd/C, H (1 atm), EtOAc:MeOH, 3 h, 85ꢀ94%; (c) PPh , DEAD, (+)ꢀmethyl Dꢀlactate,
2
3
THF, 0 ºC to rt, 4 h, 85ꢀ90%, (d) LiOH, THF:MeOH, 4 h, 86ꢀ91%; (e) 5, EDC·HCl, DMF, 12 h,
0
1
2
3
4
5
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7
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3
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2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
1
2ꢀ79%. (f) Lawesson's reagent, 1,4ꢀdioxane, reflux, 2 h, 71%; (g) 1. DIBALꢀH, DCM, ꢀ78 ºC,
h, 89%, (h) 5 (Ar = 4ꢀOMePh), NaBH(OAc) , DCE, 1.5 h, 81%; (i) 4ꢀOMePhC(O)CH Br,
3
2
NaHCO , EtOH, reflux, 12 h, 88%; (j) SnCl , EtOH:EtOAc, reflux, 2 h; (k) 7 (R = 2,3ꢀdiCl),
3
2
EDC·HCl, DMF, rt, 12 h, 60%; (l) 2ꢀaminoꢀ4ꢀnitrophenol, DABCO, NMP, 100 ºC, 24 h, 60%;
(
m) 10% Pd/C, H , MeOH:EtOAc, 2ꢀ3 h; (n) 7 (R = 2,3ꢀdiCl), EDC·HCl, DMF, rt, 12 h, 67%;
2
(
o) Lꢀalanine, CuI, Cs CO , DMF, 90 ºC, 24 h; (p) 5 (Ar = 4ꢀCNPh), HATU, DMF, rt, 12 h, 45ꢀ
2
3
54%.
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amination using 5 (Ar = 4ꢀOMePh) and sodium triacetoxyborohydride in DCM to provide amine
1
1.
The synthesis of an imidazo[1,2ꢀa]pyridine analogue started by allowing 5ꢀnitropyridinꢀ2ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
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8
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2
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8
9
0
amine to react with an αꢀbromoketone in the presence of NaHCO in ethanol to give 12. Nitro
3
group reduction using SnCl in ethanol followed by coupling with carboxylic acid 7 (R = 2,3ꢀ
2
diCl) generated imidazo[1,2ꢀa]pyridine 13.
The synthesis of a derivative with a methylene linker between the benzoxazole and the
benzene was undertaken. 2ꢀAminoꢀ4ꢀnitrophenol was coupled with a 1,1ꢀdibromoethylene
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 = 2,3ꢀ
diCl) using EDC·HCl in DMF to yield 15.
Enantiopure Nꢀarylated benzoxazole derivatives were synthesized using copper catalyzed
Ullman type reactions. Lꢀalanine was treated with aryl iodides in the presence of cesium
3
9
carbonate and CuI in DMF for 24 h to provide the Nꢀarylated amino acids 16a-b. Attempts to
couple these materials with 5 (Ar = 4ꢀCNPh) 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 K CO and DMF to give 18a and 18b, respectively. These
2 3
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
K CO in dry DMF. Ester 8h was also converted to an array of derivatives. For example, it was
2
3
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2
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6
treated with hydroxylamine hydrochloride to provide the corresponding hydroxamic acid 21.
Base catalyzed
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a
Scheme 2. Synthesis of derivatives 18aꢀb, 19aꢀb and 20ꢀ24.
A
O
N
O
N
O
O
O
O
a
O
CO R
OH
N
2
N
H
H
Cl
Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
Cl
b
8i para
18a para, R = Et
18b meta, R = Et
19a para, R = H
19b meta, R = H
8
j meta
g
2
0 para, R = H C
H
2
B
O
N
O
O
O
O
O
O
O
OR
N
H
N
OH
N
d
H
Cl
Cl
22
8h R = OMe
21 R = NHOH
c
Cl
e
Cl
O
N
O
O
N
N
O
O
O
NH
O
O
N
NHNH2
N
H
f
O
H
Cl
Cl
24
23
Cl
Cl
a
Reagents and conditions: (a) BrCH COOEt, K CO , DMF, rt, 6 h, 80ꢀ83%; (b) LiOH,
2
2
3
THF:MeOH, rt, 86ꢀ88%; (c) NH OH·HCl, KOH, MeOH, rt, 12 h, 65%; (d) NaOH,
2
THF:MeOH:H O (3:1:1), 2 h, rt, 90%; (e) NH NH ·H O, EtOH, reflux, 5 h, 67%; (f) CDI,
2
2
2
2
DIPEA, DMF, rt, 2 h, 66%; (g) CH≡CCH Br, K CO , DMF, rt, 6 h, 81%.
2
2
3
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6
ester hydrolysis of 8h in MeOH:THF:H O (3:1:1) yielded carboxylic acid 22. Finally, ester 8h
2
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 1,3,4ꢀ 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 1,2,4ꢀoxadiazolone 26. The tetrazole derivative 27 was prepared by
treatment of 8f with sodium azide in the presence of ammonium chloride in dry DMF. Primary
0
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3
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7
8
9
0
4
5
amide 28 was generated by treating 8f with tꢀBuOK in tꢀBuOH. Finally, 8f was treated with
NiCl ·6H O and NaBH in the presence of Boc O in MeOH:THF to give the Boc protected
2
2
4
2
46
primary amine, which was deprotected using trifluoroacetic acid in DCM to provide 29.
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Scheme 3. Synthesis of derivatives 25ꢀ29 from 8f.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
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5
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5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) NH OH·HCl, Et N, EtOH, reflux, 4 h, 82%; (b) CDI, DIPEA,
2
3
DMF, rt, 2 h, 61%; (c) NaN , NH Cl, DMF, 100 ºC, 6 h, 62%; (d) tꢀBuOK, tꢀBuOH, 12 h, 54%;
3
4
(
e) 1. NiCl ·6H O, NaBH , Boc O, MeOH:THF, rt, 2 h, 2. TFA, DCM, rt, 4 h, 67%.
2 2 4 2
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2
2
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4
4
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4
4
4
4
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5
5
5
5
5
5
6
The synthesis of 2ꢀpyridone derivative 30 was carried out by treating 8b with LiCl and pꢀ
toluenesulfonic acid in anhydrous methanol (Scheme 4). The pyridyl of 8a was oxidized using
mꢀCPBA in DCM to give the corresponding pyridine Nꢀoxide 31. The nitrile in 8u was reduced
using NiCl ·6H O and NaBH then protected as the corresponding tꢀbutyl carbamate. Removal
0
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9
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9
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1
2
3
4
5
6
7
8
9
0
2
2
4
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
3
3.
Finally, the synthesis of a benzoxaborole analogue is depicted in Scheme 5. A Mitsunobu
reaction between 2ꢀbromoꢀ3ꢀhydroxybenzaldehyde and (+)ꢀmethyl Dꢀlactate was carried out in
the presence of PPh and DEAD in THF to give 34. This aldehyde was treated with NaBH in
3
4
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
47
KOAc and a catalytic amount of Pd(Ph P) Cl in 1,4ꢀdioxane to give 36. Ester hydrolysis
3
2
2
followed by amine coupling with 5 (Ar = 4ꢀCNPh) in the presence of EDC·HCl in DMF and
then treatment with 6N HCl in THF gave benzoxaborole 37.
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Scheme 4. Synthesis of derivatives 30ꢀ33.
O
O
N
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH
O
N
H
a
b
R
N
Cl
3
0
8
b
O
N
O
Cl
Cl
O
N
O
N
H
N O
8
a
O
N
H
Cl
8b R = OMe
Cl
Cl
8a R = H
31
c, d
32 R = CH NH , R = 4-OMePh
1
2
2
2
O
N
R1
O
8
u
R2
O
N
R1
O
O
R2
N
O
H
N
H
8
v
e
3
3 R = OH, R = 4-CNPh
1 2
8
u R = CN, R = 4-OMePh
1 2
8v R = OBn, R = 4-CNPh
1
2
a
Reagents and conditions: (a) LiCl, pꢀTSA, DMF, 120 ºC, 2 h, 77%; (b) mꢀCPBA, DCM, rt, 12
h, 74%; (c) NiCl ·H O, NaBH , and then Boc O, THF, MeOH, rt, 4 h; (d) TFA, DCM, 68%; (e)
2
2
4
2
10% Pd/C, H , MeOH:EtOAc, rt, 3 h, 90%.
2
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 5. Synthesis of benzoxaborole 37.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) (+)ꢀMethyl Dꢀlactate, DEAD, PPh , THF, 0 ºC, 2 h, 83%; (b)
3
NaBH , EtOH, rt, 1 h; (c) MOMCl, DIPEA, DCM, rt, 12 h, 72%; (d) Pin B , Pd(Ph P) Cl ,
4
2
2
3
2
2
KOAc, 1,4ꢀdioxane, 95 ºC, 12 h, 64%; (e) LiOH, MeOH:THF, 4 h, 0 ºC; (f) 5 (Ar = 4ꢀCNPh),
EDC·HCl, DMF, rt, 12 h; (g) 4N HCl, THF, 4 h, 28%.
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Evaluation of MtbIMPDH2 inhibition. We previously described the MtbIMPDH2ꢁCBS
3
9
inhibition and antibacterial activity of benzoxazole (Q) based inhibitors. The most promising
compounds, 2 and 3 (Figure 1A), had Ki,app values of 76 nM and 14 nM, and MIC values of 16
ꢂM and 12 ꢂM, respectively. MtbIMPDH2ꢁCBS was expressed and purified as previously
described and the enzymatic activity was assayed by monitoring the production of NADH at subꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
9
saturating NAD (3 x K ) and saturating IMP concentrations and 20ꢀ50 nM enzyme. The
m
Ki,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 Nꢀoxide 31
demonstrated comparable activity to the parent compound 2. Addition of a methoxy to the
pyridyl 8b and 8c resulted in 5ꢀfold more inhibition than 2. However, changing pyridyl to 2ꢀ
pyridone (30) reduced inhibitory activity by 3ꢀfold. Replacing pyridyl with phenyl having both
electron donating groups (EDG) and electron withdrawing groups (EWG) was evaluated.
Compounds 8d (4ꢀOMe), 8e (4ꢀOCF ) and 8f (4ꢀCN) resulted in 7ꢀfold increase in inhibitory
3
activity compared to 2. Fluorine (8g) and ester (8h) substituents were also tolerated. Replacing
the pyridyl with a 4ꢀhydroxy phenyl (8i) resulted in a 2ꢀfold increase in inhibitory activity,
whereas the 3ꢀhydroxy (8j) and 2ꢀhydroxy (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 ~12ꢀfold 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 4ꢀfold loss in activity (18b and 19b). Compounds
containing hydrophilic substituents at the 4ꢀposition, such as hydroxamic acid 21, carboxylic
23
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2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acid 22 and aminomethyl 29, were found to be much less active. Replacing the carboxylate with
hydrazide 23, 1,3,4ꢀoxadiazolone 24,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Page 25 of 96
Journal of Medicinal Chemistry
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8
9
Table 1. MtbIMPDH2ꢁCBS Inhibition of Benzoxazole Derivatives Containing Various Aryl
Substituents. a. Values are the average and range of at least two independent determinations. *
9
Single determination. b. Values from MakowskaꢀGrzyska et al .
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
O
Ar
O
N
H
Cl
Cl
a
Cmpd
Ar
Ki,app (nM)
MtbIMPDH2 hIMPDH2 hGMPR2
b
1
See Figure 1A
See Figure 1A
See Figure 1A
4ꢀpyridinꢀ2ꢀone
4ꢀPyꢀNꢀoxide
4ꢀ(2ꢀOMe)ꢀPy
3ꢀ(6ꢀOMe)ꢀPy
4ꢀOMeꢀPh
150 ± 50
76 ± 27
14 ± 3
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
2300
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
b
2 (e.g. 8a)
b
3
3
0
220 ± 6
70 ± 19
12 ± 0.1
20 ± 4
31
8
8
b
c
8d
6.9 ± 3.9
4.3 ± 0.2
12 ± 3
8
8
e
f
4ꢀOCF ꢀPh
3
4ꢀCNꢀPh
4ꢀFꢀPh
8g
35 ± 7
>5000
>5000
>5000
>5000
8
8
h
i
4ꢀPhCO
2
Me
96 ± 43
50 ± 17
85 ± 30
4ꢀOHꢀPh
3ꢀOHꢀPh
8j
25
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
1
k
2ꢀOHꢀPh
96 ± 21
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
2900
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
8a
4ꢀPhꢀOCH
4ꢀPhꢀOCH
4ꢀPhꢀOCH
3ꢀPhꢀOCH
3ꢀPhꢀOCH
2
2
2
2
2
CO
CO
CO
CO
CO
2
2
2
2
2
Et
CH
H
6.6 ± 0.4
12 ± 2
20
CCH
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
9a
8b
18 ± 3
Et
H
23 ± 1
19b
55 ± 10
630 ± 210
360 ± 140
49 ± 6
2
2
2
2
1
2
3
4
4ꢀPhꢀC(O)NHOH
4ꢀPhCO
4ꢀPhꢀC(O)NHNH
H
2
2
2.3 ± 1.0
2
2
5
6
39 ± 12
46 ± 12
>5000
>5000
>5000
>5000
2
2
7
8
4ꢀPhꢀtetrazole
4ꢀPhꢀC(O)NH
4ꢀPhꢀCH NH
2
51 ± 2
>5000
>5000
>5000
>5000
>5000
>5000
64 ± 26
240 ± 26
2
29
2
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Page 27 of 96
Journal of Medicinal Chemistry
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9
Table 2. MtbIMPDH2ꢁCBS Inhibition of Benzoxazole Derivatives Containing Various Phenyl
Ether Substituents. a. Values are the average and range of at least two independent
determinations. * Single determination, error of the fit is listed.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
O
Ar
O
R1
N
H
R3
R2
a
Cmpd R1
R2 R3 Ar
Ki,app (nM)
MtbIMPDH2 hIMPDH2
hGMPR2
>5000
8
8
l
F
F
F
F
H
H
4ꢀPy
4ꢀ
55 ± 9
19 ± 2
>5000
>5000
>5000
m
OMeꢀ
Ph
4ꢀOHꢀ 121 ± 21
Ph
>5000
>5000
>5000
>5000
8
n
F
F
F
F
H
H
3
ꢀ(6ꢀ
42 ± 17
8o
OMe)ꢀ
Py
4
ꢀCNꢀ 7.2 ± 2.5
2000
>5000
8
8
8
p
q
r
F
F
F
F
F
F
H
H
F
Ph
4ꢀFꢀPh 40 ± 3
>5000
>5000
>5000
>5000
4
ꢀCNꢀ 22 ± 11
Ph
27
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
s
t
CN
CN
H
F
H
H
4ꢀPy
4ꢀPy
4ꢀ
76 ± 35
40 ± 2
33 ± 5
>5000
>5000
>5000
>5000
>5000
>5000
8
8
u
CN
H
H
H
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OMePh
4
ꢀCNꢀ 43 ± 12
>5000
>5000
>5000
>5000
8v
OBn
Ph
4
ꢀ
23 ± 9
3
3
2
3
CH
2
NH
2
H
H
H
OMeꢀ
Ph
4
ꢀCNꢀ 36 ± 12
>5000
>5000
>5000
>5000
OH
H
H
Ph
4ꢀCNꢀ 370 ± 70 *
Ph
37
B(OH)OCH
2
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Page 29 of 96
Journal of Medicinal Chemistry
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Table 3. MtbIMPDH2ꢁCBS Inhibition of Other Benzoxazole Derivatives. a. Values are the
average and range of at least two independent determinations. * Single determination.
a
Ki,app (nM)
Cmpd
Structure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MtbIMPDH2
hIMPDH2 hGMPR2
2
1
1
7 ± 21
>5000
>5000
>5000
>5000
>5000
9
290 ± 250*
60 ± 4
n.a.
1
1
>5000
>5000
13
100 ± 40
1
5
9 ± 4
3300
>5000
>5000
1
1
7a
7b
18 ± 3
>5000
29
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