Discovery of the disubstituted oxazole analogues as a novel class anti-tuberculotic agents against MDR- and XDR-MTB

Article abstract of DOI:10.1016/j.bmcl.2015.09.072

A high-throughput screening effort on 45,000 compounds resulted in the discovery of a disubstituted oxazole as a new structural class inhibitor of Mycobacterium tuberculosis (Mtb). In order to improve the activity and investigate the SAR of this scaffold, a series of disubstituted azole analogues have been designed and synthesized. The newly synthesized compounds 1a-y were evaluated for their in vitro anti-TB activity versus replicating, multi- and extensive drug resistant Mtb strains. All the compounds, except 1o, 1p and 1q, showed potent anti-TB activity with MIC of 1-64 mg/L. The test of broad spectrum panel revealed that this series are specific to Mtb. The cytotoxicity assessment indicated that the compounds were not cytotoxic against HEK 293 cells. The compounds could have a novel mechanism to anti-Mtb as they can inhibit drug sensitive and drug resistant Mtb.

Full text of DOI:10.1016/j.bmcl.2015.09.072

Accepted Manuscript
Discovery of the disubstituted oxazole analogues as a novel class anti-tubercu-
lotic agents against MDR- and XDR-MTB
Dongsheng Li, Nana Gao, Ningyu Zhu, Yuan Lin, Yan Li, Minghua Chen,
Xuefu You, Yu Lu, Kanglin Wan, Jian-Dong Jiang, Wei Jiang, Shuyi Si
PII:
S0960-894X(15)30106-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.09.072
BMCL 23153
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 May 2015
10 September 2015
30 September 2015
Please cite this article as: Li, D., Gao, N., Zhu, N., Lin, Y., Li, Y., Chen, M., You, X., Lu, Y., Wan, K., Jiang, J-D.,
Jiang, W., Si, S., Discovery of the disubstituted oxazole analogues as a novel class anti-tuberculotic agents against
MDR- and XDR-MTB, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2015.09.072
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Bioorganic & Medicinal Chemistry Letters
Discovery of the disubstituted oxazole analogues as a novel class anti-
tuberculotic agents against MDR- and XDR-MTB
Dongsheng Lia , Nana Gaoa , Ningyu Zhua , Yuan Linb , Yan Lia , Minghua, Chena , Xuefu You a , Yu Luc ,
Kanglin Wand , Jian-Dong Jiangb , Wei Jianga,  and Shuyi Sia, 
aInstitute of Medicinal Biotechnology, Peking Union Medical College & Chinese Academy of Medical Sciences, Tiantanxili No 1, Beijing 100050, P. R. China
bInstitute of Material Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Xiannongtan No 1, Beijing 100050, P. R. China
cBeijing Tuberculosis and Thoracic Tumor Research Institute, Beimachang No 97, Beijing 101149, P. R. China
dChinese Center for Disease Control and Prevention, Changbai Road No 155, Beijing 102206, P. R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A high-throughput screening effort on 45,000 compounds resulted in the discovery of a
disubstituted oxazole as a new structural class inhibitor of M. tuberculosis (Mtb). In order to
improve the activity and investigate the SAR of this scaffold, a series of disubstituted azole
analogues have been designed and synthesized. The newly synthesized compounds 1a-y were
evaluated for their in vitro anti-TB activity versus replicating, multi- and extensive drug resistant
Mtb strains. All the compounds, except 1o, 1p and 1q, showed potent anti-TB activity with MIC
of 1-64 mg/L. The test of broad spectrum panel revealed that this series are specific to Mtb. The
cytotoxicity assessment indicated that the compounds were not cytotoxic against HEK 293 cells.
The compounds could have a novel mechanism to anti-Mtb as they can inhibit drug sensitive and
drug resistant Mtb.
Keywords:
Disubstituted oxazole
Antituberculosis
MDR-MTB
.
XDR-MTB
Tuberculosis (TB) is a communicable disease caused by
mycobacterium tuberculosis (Mtb). TB remains a major health
risk globally, and ranks as the second cause of death from
infectious disease worldwide.1 According to the report of World
Health Organization (WHO), in 2013, an estimated 9.0 million
people developed TB and 1.5 million died from the disease
being made, new drugs are required to shorten and simplify
treatment, to improve the efficacy and tolerability of treatment for
TB and to improve the treatment of TB among people living with
HIV.3b
The slow growth and highly infectious nature of Mtb, along
with the complex laboratory infrastructure required, pose major
challenges for developing a comprehensive high-throughput
screening (HTS) method. Mycobacterium smegmatis is a fast-
growing, non-pathogenic model mycobacteria that shares many
features with the pathogenic Mtb.4 Mycobacterium smegmatis
can be used as a surrogate of Mtb to set up a whole-cell-based
HTS, thus avoiding the use of the highly pathogenic, slow-
growing Mtb in the early drug screening process. In fact, recent
success involving Bedaquiline have originated out of whole-cell
based approach using Mycobacterium smegmatis.5
In order to identify a new small molecular Mtb inhibitor, we
ran a whole-cell-based screen on 45,000 compounds library from
the National Center for Screening New Microbial Drug using
Mycobacterium smegmatis mc2-155. Compound 1a, (2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)(1-((2-(4-fluorophenyl)-oxazol-
4-yl)methyl)piperidin-4-yl)methanone, was identified as a potent
Mycobacterium smegmatis mc2-155 inhibitor with an in vitro
MIC of 0.3 mg/L. Compound 1a also shows good inhibitory
(including 360,000 deaths among HIV-positive people).1
A
combination therapy method (first-line drugs: rifampin, isoniazid,
pyrazinamide, and ethambutol) developed by the WHO is one of
the most efficient weapons against TB. However, the treatment
success rate struggles to reach the target of 85%.2 These
combination therapy drugs have been used for decades in clinical
and first-line treatment but can fail because of poor compliance,
leading to the emergence of multi-drug resistant (MDR-TB) and
extensively drug resistant (XDR-TB) strains of Mtb.3 In 2014, an
estimated 3.5% of new cases and 20.5% of previously treated
cases have MDR-TB, and, on average, an estimated 9.0% of
people with MDR-TB have XDR-TB.1
In the last three years, two drugs (bedaquiline and delamanid)
were approved by FDA for the MDR-TB treatment. Currently,
there are 10 new or repurposed molecules such as AZD5847,
Rifapentine, Gatifloxacin and Moxifloxasin are in phase II or
phase III clinical evaluation.1 Although there are many advances
———
 Corresponding author. e-mail: imbjiangv@163.com
 Corresponding author. Tel.: +86-010-63180604; e-mail: sisyimb@hotmail.com

activity against Mtb H37Rv with MIC of 8 mg/L in vitro. In this
report we present our efforts in exploring the structure-activity
relationship (SAR) of compound 1a, with the aim of developing
compounds with lower cytotoxicity and improved efficacy against
Mtb.
large hydrocarbon substituents, to examine the effect on anti-
tuberculosis activity. We also swapped the phenyl ring for
heteroaryls, such as pyridine, thiophene and furan to study the
influence of different aryl rings. Next, the oxazole segment was
modified, as unsaturated five-membered heterocyclic rings are
known to be important pharmacophores in many anti-Mtb
compounds.6 A 2-phenylthiazole ring and a 5-phenylisoxazole
ring were introduced to examine the effect of changing the five-
membered ring. Finally, we explored the influence on potency of
changing the 2,3-dihydrobenzo[b][1,4] dioxine to a mono- or bis-
substituted phenyl ring.
To facilitate the synthesis and subsequent SAR analysis,
compound 1a was divided into the four major segments shown
The synthetic tactics involved a convergent process in which
the two main fragments, disubstituted azole segment and
piperidine methanone segment, were synthesized separately and
then condensed. Compounds 1a–n were synthesized from
commercially available starting materials as described in Scheme
1. Stirring the appropriate aldehyde 2a–n with serine methyl ester
hydrochloride in the presence of K2CO3 in DMA for 12 h at
ambient temperature, followed by the addition of BrCCl3 and
DBU at 0 °C, afforded the desired oxazoles 5a–n in yields of 49–
71%.7 A lower yield of 20% was obtained for the synthesis of
methyl 2-(4-fluorophenyl)thiazole-4-carboxylate, so we altered
the method and instead reacted 4-chloro-thiobenzamide (or 4-
(tert-butyl)thiobenzamide) with 3-bromo-2-oxopropanoate to
afford 5o and 5p in yields of 74% and 69%, respectively.
Isoxazole 5q was synthesized from 4-fluorophenylacetylene and
ethyl 2-chloro-2-(hydroxyimino)acetate with yield of 68%. The
intermediate 6a–q were obtained by using lithium aluminum
hydride (LAH) as reducing agent and anhydrous THF as the
solvent in good yields. Then the alcohols 6a–q were reacted with
SOCl2 in the solvent of dichloromethane to yield the key
intermediate 7a–q without further purification. Piperidine-4-
carboxylic acid 8 was treated with Ac2O in the presence of
pyridine to afford 1-acetylpiperidine-4-carboxylic acid 9. The
carboxylic acid was converted to acyl chloride by treaing with
Figure 1. Systematic modifications and SAR of compound 1a
in Figure 1. Initial investigation into the SAR began with 2-
substitution of the oxazole core. A series of analogues were
synthesized with a range of substituted phenyl rings at the C-2
position, including electron-withdrawing, electron-donating and
Scheme 1. General synthesis of disubstituted azole analogues 1a-x. Reagents and conditions: (a) Ser-OMe·HCl, K2CO3, DMA,12 h, then BrCCl3, DBU, 0 – 25 °C, 12
h; (b) ethyl bromopyruvate, ethanol, reflux, 2 h; (c) ethyl 2-chloro-2-(hydroxyimino)acetate, THF, rt, 12 h; (d) LAH, THF, -5 °C, 1h; (e) SOCl2, DCM, reflux, 2 h; (f)
acetic anhydride, pyridine, 140°C, 2 h; (g) (1) SOCl2, 1,2-dichloroethane, 60°C, 4 h; (2) substituted benzene, AlCl3. 1,2-dichloroethane, 0 – 65°C, 16 h; (h) 3 mol/L
HCl(aq), reflux, 10h; (i) CH3CN, 1 mol/L NaOH(aq), reflux, 4 h.

Table 1
In Vitro Evaluation of Compound 1a-x, and Four Controls against Replicating Mtb H37Rv (MIC in mg/L) and HEK-
293Celluar Toxicity (IC50 in mg/L)
Strains Range of anti-MTB MIC (mg/L)
IC50 (mg/L)
HEK-293
MDRa
XDRb
Cmpd
ClogP11
H37Rv
FJ05120
FJ05195
HRc
8
HRSCOc
3.53
4.1
8
8
> 150
> 150
> 150
> 150
> 150
> 150
> 150
> 150
> 150
> 150
> 150
> 150
> 150
> 150
ND
1a
1b
1c
2
8
8
4
4.25
3.37
4.28
3.61
3.53
3.53
3.39
3.87
5.19
1.98
2.75
2.54
4.75
5.86
3.84
3.68
5.05
4.43
3.98
3.48
3.38
3.70
6.60
8
8
4
8
8
1d
1e
32
8
64
8
64
8
1f
8
16
16
8
8
1g
4
8
1h
1i
8
4
64
1
64
8
64
2
1j
1k
1l
32
16
32
> 128
> 128
> 128
1
32
32
32
> 128
> 128
> 128
4
32
32
64
> 128
> 128
> 128
4
1m
1n
1o
ND
1p
1q
1r
ND
> 150
> 150
> 150
> 150
> 150
> 150
> 150
> 150
2
8
8
1s
2
4
4
1t
8
8
8
1u
1v
4
16
16
8
8
8
16
8
1w
1x
4
8
4
4
1y
INH
RIF
SM
EMB
<0.5
<0.5
<0.5
<0.5
1
＞256
<0.5
1
4
>256
64
8
a MDR-Mtb = multidrug resistant Mtb;
b XDR-Mtb = extensively drug resistant Mtb;
c drugs abbreviated as H = isoniazid, R = rifampin, S = streptomycin, C = capreomycin, O = ofloxacin
SOCl2. The another key intermediate 11a, 11r–x were
synthesized using a Friedel–Crafts reaction by treating acyl
chloride with corresponding aromatic rings respectively and
followed by removing acetyl in 3 mol/L HCl(aq). The final desired
products 1a–y were obtained from the reaction of compounds 7a–
q with compounds 11a, 11r–x in yields of 40-76%. All the
Assay (MABA).10 The clinical drugs INH, RIF, SM and EMB
were used as the reference(Table 1). SAR analysis based on the
whole-cell assay readout indicates that the presence of a phenyl
ring at the C-2 position of the oxazole is essential for cellular
potency. Heterocyclic rings such as 3-pyridyl (1l), 2-furanyl (1m)
and 3-furanyl (1n) had diminished activity (MICs of 16-64 mg/L)
compared with the phenyl ring analog (1d, MICs of 4-8 mg/L).
Left phenyl rings (R1) with electron-withdrawing substituents
(1a–c, 1f-h) show potent anti-TB activity (MICs of 2-8 mg/L),
except for the 4-trifluoromethyl analog (1e, MICs > 32 mg/L).
Phenyl rings (R1) with electron-donating substituents (1i-k) also
show potent anti-TB activity (MICs of 1-64 mg/L). Specially,
compound 1k, with the 4-tert-butyl substituent, was more potent
1
disubstituted azole analogues 1a-y were identified by their H
NMR, 13C NMR and mass (ESI-MS, HR-MS) spectral data. The
key compounds 1b, 1k and 1r also identified by their IR.8
All the 25 newly synthesized disubstituted azole analogues
1a-y were assessed for their in vitro activities against M.
tuberculosis H37Rv, MDR strain (FJ05120) and XDR strain
(FJ05195) in 7H9 media,9 using the Microplate Alamar Blue

than the 4-methoxyl and 4-methyl analog (1i and 1j, MICs of 8-
64 mg/L). Obviously, compound 1k showed an excellent activity
of anti MDR-MTB and XDR-MTB (MIC = 2 and 1 mg/L,
Table 2
compounds do not have MICs up to 128 mg/L, indicating
specificity to Mtb (Table 2).
In conclusion, we have designed and synthesized 25
compounds of disubstituted azole analogues. All the compounds
1a-y were tested for their anti-Mtb activities, including
replicating H37Rv, MDR-Mtb and XDR-Mtb strains, and also
assessed their cytotoxicity. The result reflected that 22
compounds (1a-n, 1r-y) of the disubstituted azole analogues have
potent activities and low cytotoxicity. The broad spectrum panel
of compounds 1b, 1k and 1r indicated these compounds have a
good selectivity for anti-Mtb. In addition, compared with the
current clinical drugs, this series appears to work through a novel
mechanism of action. Therefore, for future study and research,
this class can be further optimized through analog synthesis and
systematic medicinal chemistry evaluation.
Studies on Broad Spectrum Panel
MIC (mg/L)
species
1b
1k
1r
> 128*
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
S. aureus
E. coli
K. pneumoniae
P. aeruginosa
P. vulgaris
P. mirabilis
S. marcescens
A. calcoacetious
Acknowledgments
This work was supported by State Mega Programs
(2012ZX09301002-003/006), and grants from the National
Natural Science Foundation of China (81302816, 81001461,
81072672, 81321004).
* >, end points not determined
respectively). It is 4 and 8-fold than the lead compound 1a (MICs
= 8 mg/L). It seemed to be a good preference for higher ClogP
(ClogP = 5.19 for 1k vs 3.39 and 3.87 for 1i and 1j) and larger
area place in potency. Comparing thiazoles (1o, 1p, MICs >128
mg/L) and isoxazole (1q, MICs >128 mg/L) with the
corresponding oxazole analogs (1a, 1b, and 1k) indicates that the
oxazole ring is crucial for cellular potency. Investigating
substitution on the right phenyl ring (R2 and R3) (1r–x) gave
MICs ranging from 1 to 16 mg/L. A 2 to 8-fold improvement in
potency could be achieved by replacing the 1,4-Benzodioxan of
compound 1a with 1,2,3,4-tetrahydronaphthalene (1r).As the
compound 1k and 1r both perform a excellent anti-TB activity,
the hybrid compound 1y was synthesized and also showed a
potent anti-TB activity (MICs of 4-8 mg/L). However, the
compound 1y did not show a higher anti-TB activity than the
compound 1k or 1r. This may be caused by its high ClogP
(ClogP = 6.60).
More importantly, all the compounds, except 1o, 1p and 1q,
showed excellent potency against the MDR strain (FJ05120, a
strain that is resistant to isoniazid and rifmpin) and XDR strain
(FJ05195, a strain that is resistant to isoniazid, rifmpin,
streptomycin, capreomycin, and ofloxacin) with MICs of 1-64
mg/L (16 compounds have MICs of 1-16 mg/L and 5 compounds
of 32-64 mg/L)), which were much lower than the reference drug
rifmpin (MICs > 256 mg/L). Compound 1k (MICs of 1-2 mg/L)
was comparable to or slightly lower than the reference drugs,
isoniazid (MICs of 1-4 mg/L) and ethambutol (MICs of 1-8
mg/L). The exciting anti-Mtb result of the compounds encourage
us to believe that this class of molecules is likely to be effective
against a wide range of clinical isolates and is likely to act
through a novel mechanism.
Supplementary Date
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/
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Discovery of the disubstituted oxazole
analogues as a novel class anti-tuberculotic
agents against MDR- and XDR-MTB
Dongsheng Lia , Nana Gaoa , Ningyu Zhua , Yuan Linb , Yan Lia , Minghua, Chena , Xuefu You a , Yu Luc ,
Kanglin Wand , Jian-Dong Jiangb , Wei Jianga,  and Shuyi Sia, 