Design, synthesis and antimycobacterial activity of novel nitrobenzamide derivatives

Article abstract of DOI:10.1016/j.cclet.2018.08.005

We report herein the design and synthesis of a series of novel nitrobenzamide derivatives. Results reveal that many of them display considerable in vitro antitubercular activity. Four N-benzyl or N-(pyridine-2-yl)methyl 3,5-dinitrobenzamides A6, A11, C1 and C4 have not only the same excellent MIC values of <0.016 μg/mL against both drug-sensitive MTB strain H37Rv and two drug-resistant clinical isolates as PBTZ169 and the lead 1, but also acceptable safety indices (SI > 1500), opening a new direction for further development.

Full text of DOI:10.1016/j.cclet.2018.08.005

Accepted Manuscript
Title: Design, synthesis and antimycobacterial activity of
novel nitrobenzamide derivatives
Authors: Hongjian Wang, Kai Lv, Xiaoning Li, Bo Wang,
Apeng Wang, Zeyu Tao, Yunhe Geng, Bin Wang, Menghao
Huang, Mingliang Liu, Huiyuan Guo, Yu Lu
PII:
DOI:
Reference:
S1001-8417(18)30331-0
https://doi.org/10.1016/j.cclet.2018.08.005
CCLET 4626
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Chinese Chemical Letters
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Accepted date:
9-7-2018
9-8-2018
10-8-2018
Please cite this article as: Wang H, Lv K, Li X, Wang B, Wang A, Tao Z, Geng Y,
Wang B, Huang M, Liu M, Guo H, Lu Y, Design, synthesis and antimycobacterial
activity of novel nitrobenzamide derivatives, Chinese Chemical Letters (2018),
https://doi.org/10.1016/j.cclet.2018.08.005
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Communication
Design, synthesis and antimycobacterial activity of novel nitrobenzamide derivatives
Hongjian Wanga,1, Kai Lva,1, Xiaoning Lia, Bo Wanga, Apeng Wanga, Zeyu Taoa, Yunhe Genga, Bin
Wangb, Menghao Huangc, Mingliang Liua,*, Huiyuan Guoa, Yu Lub,*
aInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100050, China
bBeijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research
Institute, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
cDivision of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, United States
Graphical abstract
We report herein the design and synthesis of a series of novel nitrobenzamide derivatives. Results reveal that A6, A11, C1 and C4 have not only the same excellent
MIC values of <0.016 μg/mL against drug-resistant clinical isolates as lead 1, but also acceptable safety indices (SI>1500), opening a new direction for further
development.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We report herein the design and synthesis of a series of novel nitrobenzamide
derivatives. Results reveal that many of them display considerable in vitro
antitubercular activity. Four N-benzyl or N-(pyridine-2-yl)methyl 3,5-
dinitrobenzamides A6, A11, C1 and C4 have not only the same excellent MIC values
of <0.016 μg/mL against both drug-sensitive MTB strain H37Rv and two drug-
resistant clinical isolates as PBTZ169 and the lead 1, but also acceptable safety
indices (SI>1500), opening a new direction for further development.
Available online
Keywords: nitrobenzamides
synthesis
antimycobacterial activity
tuberculosis
Tuberculosis (TB) has existed for millennia and remains a major global health problem [1]. It is a widespread
infectious disease predominantly caused by Mycobacterium tuberculosis (MTB), which can be transmitted through
the air as droplets and affects the lungs [2]. The World Health Organization (WHO) estimated that approximately
10.4 million people were infected and 1.3 million died from TB worldwide in 2016 [1]. The spread of multidrug-
resistant TB (MDR-TB) and the emergence of extensively drug-resistant TB (XDR-TB) have reinvigorated drug
discovery efforts in search of novel agents [3-6]. Despite the introduction of Bedaquiline [7] and Delamanid [8] to
the repertoire of anti-TB therapies for MDR-TB, some adverse events have been noted [9]. Therefore, it is urgently
needed to develop antimycobacterial molecules with new mechanisms of action and that are active against MDR-
and XDR-TB [10].
Decaprenyl phosphoryl-β-D-ribose 2′-epimerase (DprE1) was identified as a potential target for developing
potent and safer anti-TB agents [11-13]. Some new chemical entities (NCEs) were found to have potent activity
against MDR/XDR-MTB as covalent or noncovalent inhibitors of the DprE1 enzyme [14-22], such as nitroaromatic
compounds DNB1, MTX and PBTZ 169 (Fig. S1 in Supporting information). As the most advanced scaffold among
* Coerresponding authors.
E-mail addresss: lmllyx@126.com (M. Liu), luyu4876@hotmail.com (Y. Lu).
1 These authors contributed equally to this work.

these NCEs, nitrobenzothiazinones (BTZs) have garnered great interest recently, and many series of BTZ derivatives
were reported [23-26]. Above all, candidate PBTZ169 entered in Phase II clinical trials in 2017 [1].
In our previous studies, many BTZs containing various cyclic ketoximes, spiro-heterocycles and piperidines
moieties were found to have considerable antitubercular activity [27-29]. Recently, N-(4-(4-
trifluoromethyl)piperidin-1-yl)benzyl nitrobenzamides 1 and 2 (Fig. 1) were identified as new anti-TB agents by the
thiazinone ring opening of PBTZ169 in our lab [30]. Both of them with simpler structures than PBTZ169, show
potent activity against MTB H37Rv strain (MIC ≤ 0.016 μg/mL). Moreover, compound 1 also displays acceptable
safety and better PK properties than PBTZ169.
Inspired by the above research results, compounds 1 and 2 were employed as lead compounds, and the three
moieties (A, B and C ring) were all explored in this study. We started with the modification of A ring and B ring.
Replacement of X group on ring A with various substituents (Y) leaded to 3-nitrobenzamides bearing N-benzyl (A1-
4); introduction of pyridine as A ring while reserving the nitro group gave 5-nitronicotinamides A5. Subsequently,
the B ring was changed to pyrin-3-yl or pyrin-2-yl leading to compounds B1-5 or C1-3 (Fig. 1). After identifying the
optimal A and B rings, C ring was then further investigated. Our primary objective was to find optimized
benzamides with potent antimycobacterial activity. A preliminary structure-activity relationship (SAR) study was
also explored to facilitate the further development of these compounds.
Detailed synthetic pathways to side chains 68, leads 1, 2 and targets AC are shown in Schemes S1 and S2
(Supporting information), respectively. Commercially unavailable benzylamines and pyridinylmethylamines 68
were first prepared according to Scheme S1. 4-Fluorobenzonitrile 3, 6-fluoronicotinonitrile 4 and 5-
fluoropicolinonitrile 5 were treated with various nitrogen heterocyclic amines ZH in DMSO in the presence of K2CO3
at 80 oC, and the resulting condensates were subsequently reduced with LiAlH4 in THF to produce the desired
compounds 6, 7 and 8, respectively.
Leads 1, 2 and targets A1-11, B1-21, C1-4 were easily obtained by coupling 3-nitrobenzoic acids 913 and 5-
nitronicotinic acid 14 with the above side chain compounds 68 or commercially available benzylamines 15ad in
the presence of triethylamine and condensation agent bis(2-oxo-3-oxazolidinyl) phosphonic chloride (BOP-Cl)
(Scheme S2).
Table 1
Structures and activity of compounds AC against MTB H37Rv.
Compd.
1
Y
MIC (μg/mL)
<0.016
0.016
1.357
0.459
>16
Compd.
B3
Y
MIC (μg/mL)
>16
5-NO2
5-CF3
5-F
4,6-di-Cl
H
2
B4
15.354
15.176
<0.016
31.088
15.732
<0.016
0.0781
0.0781
A1
A2
A3
A4
A5
B1
B5
5-Br
4,6-di-Cl
H
C1
5-NO2
H
C2
>16
C3
14.735
0.059
0.944
PBTZ169
INH
RFP
5-NO2
5-Br
B2
INH: isoniazid; RFP: rifampicin.
The target compounds A15, B15 and C13 bearing different kinds of substituents to ensure A and B rings
flexibility and structure diversity, were first synthesized. They were preliminarily screened for in vitro activity
against MTB H37Rv ATCC27294 strain, using the Microplate Alamar Blue Assay (MABA) [31,32]. The minimum
inhibitory concentration (MIC) is defined as the lowest concentration effecting a reduction in fluorescence of >90%

relative to the mean of replicate bacterium-only controls. The MIC values of the compounds along with the leads 1
and 2, PBTZ169, isoniazid (INH), and rifampicin (RFP) for comparison were obtained from three independent
experiments and presented in μg/mL in Table 1.
Effect of the substituents on A ring was first investigated. The nature and position of the substituents greatly
influence activity. Replacement of one nitro group of 1 or the trifluoromethyl of 2 with halogen in compounds A1
（F）and A2 (Br) leads to decreased activity (MIC: 1.357 and 0.459 μg/mL, respectively). Introduction of 4,6-
dichloro (A3) or reservation of one nitro (A4) destroys activity. Moreover, N-benzyl nicotinamide analogue (A5)
displays very poor potency. Overall, these results reveal that the presence of a strong electron-withdrawing group
(CF3, NO2) at C-5 position of nitrobenzamide core is essential for excellent activity (Table 1).
Table 2
Structures and activity of 3,5-dinitrobenzamides AC against MTB H37Rv.
Compd.
Z
MIC (μg/mL)
Compd.
Z
MIC (μg/mL)
A6
<0.016
B12
0.452
0.235
A7
A8
0.060
0.120
B13
B14
F
0.480
A9
CF3
0.059
0.033
<0.016
0.094
0.030
B15
B16
B17
B18
B19
1.255
0.210
0.178
0.233
0.491
A10
A11
B6
OCF3
OCH3
B7
B8
B9
0.030
0.108
B20
B21
0.973
0.143
B10
B11
0.059
0.119
C4
<0.016
<0.016
PBTZ169
In further modifications, the benzene ring (B ring) was replaced by a pyridine ring. As shown in Table 1, in
accordance with SAR of N-benzyl analogues (A15), N-(pyridin-3-yl)methyl and N-(pyridin-2-yl)methyl 3,5-
dinitrobenzamides (B1, C1) demonstrate potent MIC values of 0.059 and <0.016 μg/mL against this strain,
respectively, indicating that N-pyridinylmethyl on the amide is also acceptable.
Based on the above SAR, and better activity of lead compound 1 than 2, N-benzyl and N-pyridinylmethyl 3,5-
dinitrobenzamides with various groups at para-position of B ring were further designed and synthesized. As shown
in Table 2, all of them show good to excellent activity against MTB H37Rv strain (MIC: <0.016–0.973 μg/mL), with

one exception B15. Among them, nine compounds A6, 7, 911, B7, 8, 10 and C4 (MIC: <0.0160.060 μg/mL) are
more active than INH/RFP (MIC: 0.0781 μg/mL), and roughly comparable to PBTZ169.
b
c
aMDR-TB 16833 and MDR-TB 16995 were isolated from patients in Beijing Chest Hospital; the 50% cytotoxic concentration; SI: selectivity index for MTB
H37Rv, CC50 / MIC
For N-benzyl 3,5-dinitrobenzamides, the presence of a halogen atom instead of trifluoromethyl at para-position
of the piperidine ring (C ring) was found to be also favorable. For example, compound A6 shows the same MIC
value of <0.016 μg/mL as the lead 1. Introduction of an additional aromatic moiety on C ring, such as 4-
fluorophenyl (A7, MIC: 0.059 μg/mL), is also acceptable. More interestingly, removal of C ring and direct
attachment of a simple group to B ring remain considerable activity (A811), and an electron-donating group
(OCH3) is preferred over an electron-withdrawing one (CF3, OCF3) or a halogen atom (F).
For N-(pyridin-3-yl)methyl 3,5-dinitrobenzamides, the presence of a halogen atom (Cl, Br) instead of
trifluoromethyl on C ring is more beneficial to activity (B1 vs. B7 and B8), and replacement of C ring in B1 with
thiomorpholine in compound B10 maintains the same potent activity (MIC: 0.059 μg/mL). However, introduction of
4-substituented phenyls on C ring, or replacement of the piperidine with piperazines bearing a substituted phenyl
moiety leads decreased activity (B1 vs B1121). Conversely, N-(pyridin-2-yl)methyl compound C4 with a 4-
(fluorophenyl)piperazine as C ring, displays the same potent MIC value of <0.016 μg/mL as C1, much more active
than the corresponding N-(pyridin-3-yl)methyl analogue B16 (MIC: 0.210 μg/mL) (Table 2).
Encouraged by their strong potency against the drug sensitive MTB H37Rv strain (MIC: <0.0160.060 μg/mL),
eleven 3,5-dinitrobenzamide derivatives A6, 7, 911, B1, 7, 8, 10 and C1, 4 were further evaluated against two
clinical isolated MTB-MDR (16833 and 16995) strains resistant to both INH and RFP. The cytotoxic potential of
these compounds was also investigated in a mammalian Vero cell line by MTS assay. As shown in Table 3, all of
them exhibit potent MIC values of <0.0160.071 μg/mL, similar to that against MTB H37Rv. Among of them,
compounds A6, A11, C1 and C4 have the same excellent activity (MIC: <0.016 μg/mL) as PBTZ169 and the lead 1.
With a few exceptions, these compounds (CC50: 22.63–34.57 μg/mL) are less cytotoxic than the lead 1, although
generally more cytotoxic than PBTZ169.
Lipinski’s rules are important guidelines for determining drug-likeness compounds [33]. The related values of
most potent compounds A6, A11, C1 and C4 were calculated using the online chemo-informatics software
molinspiration (http://www.molinspiration.com). As shown in Table S1 (Supporting information), none violation of
Lipinski’s rule-of-five was found among compounds A6, A11, and C1. The hydrogen bond acceptors of compound
C4 (HBA = 11) are more than the recommended number (HBA <10). However, compound C4 is still incorporate
with the Lipinski’s rule-of-five (violations ≤1). Thus, these compounds display good drug like properties, are all
deserved further development.
In conclusion, a series of nitrobenzamide derivatives containing N-benzyl or N-pyridinylmethyl moieties, based
on lead compounds 1 and 2 discovered in our lab, were designed and synthesized as new anti-TB agents. Many of
them exhibit potent in vitro antitubercular activity. Especially, N-benzyl 3,5-dinitrobenzamides A6 and A11, and N-
(pyridine-2-yl)methyl analogues C1 and C4 have not only the same excellent activity (MIC: <0.016 μg/mL) against
both drug-sensitive MTB strain H37Rv and two drug-resistant clinical isolates as PBTZ169 and the lead 1, but also
have acceptable safety indices (SI: >1500). In addition, compounds A6, A11, C1 and C4 display good drug like
properties, suggesting these compounds may serve as new and promising candidates for further antitubercular
drug discovery. By the way, the further expansion of the 3,5-dinitrobenzamides is underway to find potent anti-TB
agents.
Acknowledgments
This work is supported by the National Mega-project for Innovative Drugs (Nos. 2015ZX09102007-008,
2015ZX09102007-015, 2015ZX09304006-016, 2018ZX09721001-004-007, 2018ZX09711001-007-002), CAMS
Initiative for Innovative Medicine (No. 2016-I2M-1-010), CAMS Innovation Fund for Medical Science (Nos. CAMS-
2016-I2M-1-010, CAMS-2017-I2M-1-011), PUMC Youth Fund (No. 2017350011).
References
[1] Global Tuberculosis Report 2017, World Health Organization, www.who.Int/tb/publications/lpa-mdr-diagnostics/en/.

[2] N. Nayak, J. Ramprasad, U. Dalimba, et al., Chin. Chem. Lett. 27 (2016) 365-369.
[3] A. Wang, Y. Yang, J. Yang, et al., Bioorg. Med. Chem. 26 (2018) 2073-2084.
[4] Z. Xu, S. Zhang, C. Gao, et al., Chin. Chem. Lett. 28 (2017) 159-167.
[5] K. Lv, L. Li, B. Wang, et al., Eur. J. Med. Chem. 137 (2017) 117-125.
[6] R.S. Wallis, M. Maeurer, P. Mwaba, et al., Lancet Infect Dis.16 (2016) 34-46.
[7] A.K. Kakkar, N. Dahiya, Tuberculosis (Edinb) 94 (2014) 357-362.
[8] M.T. Gler, V. Skripconoka, E. Sanchez-Garavito, et al., N. Engl. J. Med. 366 (2012) 2151-2160.
[9] D.T. Hoagland, J. Liu, R.B. Lee, R.E. Lee, Adv. Drug Deliv. Rev. 102 (2016) 55-72.
[10] Beena, D.S. Rawat, Med. Res. Rev. 33 (2013) 693-764.
[11] G. Manina, M.R. Pasca, S. Buroni, E. de Rossi, G. Riccardi, Curr. Med. Chem. 17 (2010) 3099-3108.
[12] G. Riccardi, M.R. Pasca, L.R. Chiarelli, et al., Appl. Microbiol. Biotechnol. 97 (2013) 8841-8848.
[13] M. Brecik, I. Centarova, R. Mukherjee, et al., ACS Chem. Biol. 10 (2015) 1631-1636.
[14] P.K. Crellin, R. Brammananth, R.L. Coppel, PloS One 6 (2011) e16869.
[15] S.A. Stanley, S.S. Grant, T. Kawate, et al., ACS Chem. Biol. 7 (2012) 1377-1384.
[16] F. Wang, D. Sambandan, R. Halder, et al., Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 2510-2517.
[17] M. Naik, V. Humnabadkar, S.J. Tantry, et al., J. Med. Chem. 57 (2014) 5419-5434.
[18] M. Panda, S. Ramachandran, V. Ramachandran, et al., J. Med. Chem. 57 (2014) 4761-4771.
[19] P.S. Shirude, R. Shandil, C. Sadler, et al., J. Med. Chem. 56 (2013) 9701-9708.
[20] T. Christophe, M. Jackson, H.K. Jeon, et al., PLoS Pathog. 5 (2009) e100645.
[21] K.J. Schaper, M. Pickert, A.W. Frahm, Archiv der Pharmazie 332 (1999) 91-102.
[22] C. Trefzer, M. Rengifo-Gonzalez, M.J. Hinner, et al., J. Am. Chem. Soc. 132 (2010) 13663-13665.
[23] V. Makarov, G. Manina, K. Mikusova, et al., Science 324 (2009) 801-804.
[24] V. Makarov, B. Lechartier, M. Zhang, et al., EMBO Mol. Med. 6 (2014) 372-383.
[25] A.L.D.L. Ribeiro, G. Degiacomi, F. Ewann, et al., PloS One 6 (2011) e26675.
[26] T. Karoli, B. Becker, J. Zuegg, et al., J. Med. Chem. 55 (2012) 7940-7944.
[27] R. Zhang, K. Lv, B. Wang, et al., RSC Adv. 7 (2017) 1480-1483.
[28] K. Lv, X. You, B. Wang, et al., ACS Med. Chem. Lett. 8 (2017) 636-641
[29] K. Lv, Z. Tao, Q. Liu, et al., Eur. J. Med. Chem. 151 (2018) 1-8.
[30] L. Li, K. Lv, Y. Yang, et al., ACS Med. Chem. Lett. 9 (2018) 741-745.
[31] L. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997) 1004-1009.
[32] Y. Lu, M. Zheng, B. Wang, et al., Antimicrob. Agents Chemother. 55 (2011) 5185-5193.
[33] C. A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney. Adv. Drug Deliv. Rev. 46 (2001) 3-26.
Fig. 1. Design of the new molecules.
Table 3
Activity against MDR-MTB, cytotoxicity and selectivity index (SI) values for selected compounds.

b
Compd.
CC50
SIc
MIC (g/mL)
(μg/mL)
MDR-MTB
16833a
MDR-MTB
16995a
1
<0.016
<0.016
0.071
0.070
0.029
<0.016
0.043
0.030
0.030
0.063
<0.016
<0.016
<0.016
>40
<0.016
<0.016
0.056
0.042
0.056
<0.016
0.028
0.056
0.029
0.060
<0.016
<0.016
<0.016
>40
20.15
24.74
10.51
33.62
31.21
28.02
16.80
23.17
22.63
17.60
26.61
34.57
36.68
NT
>1259
>1546
175
A6
A7
A9
569
A10
A11
B1
945
>1751
284
B7
772
B8
754
B10
C1
298
>1663
>2160
>2292
C4
PBTZ169
INH
RFP
>40
>40
NT