Identification of novel 2-aminothiazole conjugated nitrofuran as antitubercular and antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2016.05.088

The emergence of antibiotic resistant pathogens is an ongoing main problem in the therapy of bacterial infections. In order to develop promising antitubercular and antibacterial lead compounds, we designed and synthesized a new series of derivatives of 2-aminothiazole conjugated nitrofuran with activities against both Mycobacterium tuberculosis and Staphylococcus aureus. Eight compounds 12e, 12k, 12l, 12m, 18a, 18d, 18e, and 18j emerged as promising antitubercular agents. Structure–activity relationships (SARs) were discussed and showed that the derivatives substituted at the position-3 of benzene of 5-nitro-N-(4-phenylthiazol-2-yl)furan-2-carboxamide exhibited superior potency. The most potent compound 18e, substituted with benzamide at this position, displayed minimum inhibitory concentrations (MICs) of 0.27 μg/mL against Mtb H37Ra and 1.36 μg/mL against S. aureus. Furthermore, compound 18e had no obvious cytotoxicity to normal Vero cells (IC₅₀= 50.2 μM). The results suggest that the novel scaffolds of aminothiazole conjugated nitrofuran would be a promising class of potent antitubercular and antimicrobial agents.

Full text of DOI:10.1016/j.bmcl.2016.05.088

Accepted Manuscript
Identification of novel 2-aminothiazole conjugated nitrofuran as antitubercular
and antibacterial agents
Kai Ran, Chao Gao, Hongxia Deng, Qian Lei, Xinyu You, Ningyu Wang, Yaojie
Shi, Zhihao Liu, Wei Wei, Cuiting Peng, Lu Xiong, Kunjie Xiao, Luoting Yu
PII:
S0960-894X(16)30598-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.088
BMCL 23947
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 January 2016
17 May 2016
30 May 2016
Please cite this article as: Ran, K., Gao, C., Deng, H., Lei, Q., You, X., Wang, N., Shi, Y., Liu, Z., Wei, W., Peng,
C., Xiong, L., Xiao, K., Yu, L., Identification of novel 2-aminothiazole conjugated nitrofuran as antitubercular and
antibacterial agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.05.088
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Identification of novel 2-aminothiazole conjugated nitrofuran as antitubercular
and antibacterial agents
Kai Ran a, †, Chao Gao a, †, Hongxia Deng a, Qian Leia, Xinyu You b, Ningyu Wang a,
Yaojie Shia, Zhihao Liu a, Wei Weia, Cuiting Peng b, Lu Xiong a, Kunjie Xiao a and
Luoting Yu a,b, *
a
State Key Laboratory of Biotherapy, West China Hospital, West China Medical
School, Sichuan University, Chengdu 610041, China
b Department of Pharmaceutical and Bioengineering, School of Chemical Engineering,
Sichuan University, Chengdu, Sichuan 610065, China
*Corresponding author. Tel: +86-28-85164063; fax: +86-28-85164060.
E-mail address: yuluot@scu.edu.cn (L.T.Yu).
† These authors contributed equally to this work.
Abstract: The emergence of antibiotic resistant pathogens is an ongoing main
problem in the therapy of bacterial infections. In order to develop promising
antitubercular and antibacterial lead compounds, we designed and synthesized a new
series of derivatives of 2-aminothiazole conjugated nitrofuran with activities against
both Mycobacterium tuberculosis and Staphylococcus aureus. Eight compounds 12e,
12k, 12l, 12m, 18a, 18d, 18e, and 18j emerged as promising antitubercular agents.
Structure-activity relationships (SARs) were discussed and showed that the
derivatives substituted at the position-3 of benzene of 5-nitro-N-(4-phenylthiazol-2-yl)

furan-2-carboxamide exhibited superior potency. The most potent compound 18e,
substituted with benzamide at this position, displayed minimum inhibitory
concentrations (MICs) of 0.27 μg/mL against Mtb H37Ra and 1.36 μg/mL against S.
aureus. Furthermore, compound 18e had no obvious cytotoxicity to normal Vero cells
(IC50 = 50.2 μM). The results suggest that the novel scaffolds of aminothiazole
conjugated nitrofuran would be a promising class of potent antitubercular and
antimicrobial agents.
Key words: Staphylococcus aureus, Mycobacterium tuberculosis, aminothiazole,
nitrofuran, structure-activity relationships (SARs).
Due to the widespread bacterial infection and the emergence of antibiotic
resistant pathogens, the infectious diseases with high rate morbidity and mortality
threaten public health seriously. 1 , 2 The major challenges of antibacterial drug
discovery in the therapy are the phenomena of drug resistance, and the critical
discussions about drug resistance are Mycobacterium tuberculosis (Mtb) and
Staphylococcus aureus (S. aureus) infections.3,4 In particular, tuberculosis (TB) that
infecting approximately a third of world population has become the most dangerous
5
disease among various deadly infections.
However, the standard clinical
antitubercular drugs, like isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and
pyrazinamide (PZA), have been used for decades. The consequent emergencies of
multidrug-resistant and extensively drug resistant tuberculosis (MDR and XDR-TB)

rendered the scanty drug supply to be tighter.6 It was also clear from the WHO data
that TB conjugating with AIDS lowered immunity and caused higher mortality rate,
with 360000 deaths amongst HIV-sufferers in 2013.5 Unfortunately, there was a drop
in the number of authorization of new antimicrobial agents by the regulatory agencies
and there was low probability to discover a novel lead compound in the pre-clinical
study.7 Hence, there is a great need for increased drug discovery efforts in this
important area.
Our endeavors to develop a novel series of antibacterial agents led to the study of
privileged structure-based libraries. 2-amino-1, 3-thiazole derivatives were known to
exhibit a broad range of biological activities, some thiazole-2-amine agents had been
used in clinical antibacterial applications successfully (e.g., Sulfatizole, Cetraxone,
8
Aztreonam, Riluzole).
Of interest were compounds possessing the
4-phenylthiazol-2-amine scaffold with a benzoyl substituent on the amino group that
had the inhibitory activity against S. aureus and Escherichia coli by targeting
β-Ketoacyl-acyl carrier protein (ACP) synthase III (FabH). One of the compounds,
3-bromo-N-(4-(4-bromophenyl) thiazol-2-yl) benzamide (1) (Figure 1), exhibited a
MIC of 6.25 μg/mL against S. aureus and FabH inhibitory activity IC50 of 5.8 μM.9

2
4
MIC(H37Rv): 3 ng/mL
MIC(H37Rv): 6 ng/mL
3
5
MIC(H37Rv): 24 ng/mL
MIC(H37Rv): 0.1 μg/mL (R)
0.2 μg/mL (NR)
1
MIC (S. aureus): 6.25 μg/mL
IC50 (E. coli FabH): 5.8 μM
6 anti-TB
7
12b
Inh%(H37Ra, 20 μM): 99.5%
Inh%( S. aureus, 20 μM): 99.5%
Figure 1 Aminothiazole and nitrofuranyl containing antimicrobial and antitubercular
molecules.
On another hand, nitrofuran derivatives were also precursors with a
broad-spectrum activity against both Gram-negative and Gram-positive bacteria.10,11
A recent literature survey revealed that nitrofuran containing compounds owed
anti-TB potential.12-16 Compound 2 (Lee-562)13 and compound 3 (Lee-1106)14 that

generated from the nitrofuranylamides displayed nanomolar potency against H37Rv.
On the basis of Lee’s study, Yempalla and co-workers had rediscovered nitrofuranyl
methyl piperazie 4 with optimal pharmacokinetic property and comparatively better
aqueous solubility as an eff ective compound against sensitive and resistant strains of
MTB.15 Likewise, the nitrofuran introduction prompted calanolides 5 to eradicate both
replicating (R) and nonreplicating (NR) Mtb.16
Motivated by these findings, we adopted the strategy of combinatorial chemistry
and proposed the combination of 4-phenylthiazol-2-amine and 5-nitrofuran to
generate novel structure 7 (Figure 1), in anticipation of having the capacity of
fighting both antimicrobial and anti-tubercular activity. The initial
N-(4-(3-methoxyphenyl) thiazol-2-yl)-5-nitrofuran-2-carboxamide 12b was found to
exhibit the same inhibitory rate of 99.5% against Mtb and S. aureus at the
concentration of 20 μM, which proved the practicability of our design stategy. In
order
to
develop
more
potent
agents,
a
series
of
5-nitro-N-(4-phenylthiazol-2-yl)furan-2-carboxamide
derivatives,
compounds
11a-11b, 12a-12m, 13, 16, 17 and 18a-18j, were designed, synthesized and evaluated
for their antibacterial activities.
For synthesizing these designed compounds, we initially prepared a series of
4-phenylthiazol-2-amine analogues as shown in Scheme 1. Brominating of the
commercially available acetophenone 8a-8l with tetrabutylammonium afforded the
α-brominated ketone 9a-9l and Friedel-Crafts acylation of 8m-8n generated 9m-9n.
Subsequently the classical Hantzsch synthesis of 9a-9n with thiourea provided the

desired anminothiazole 10a-10n. Then condensation of 10a-10n with the
corresponding carboxylic acid afforded the products 11a-11b and 12a-12n. The
product 13 was synthesized by nitro-reduction of 12b.
Scheme 1 Reagents and conditions: (i) tetrabutylammonium tribromide, acetonitrile,
12 h; (ii) bromoacetyl bromide, AlCl3 , DCM, 0 ℃, 5 min; (iii) thiourea, EtOH,
reflux, 3 h, 50%-96%; (iv) the corresponding carboxylic acid, EDCI, DMAP, DCM,
r.t., 1 d; (vi) Fe, NH4Cl, EtOH, H2O, reflux, 6 h.
Another series of compounds 18a-18j were started from the above 10l (Scheme
2), which was hydrolyzed firstly via hydrochloric acid to produce the de-acetylated 14.
After the process of Boc-protection, 15 condensed with 5-nitro-2-furoic acid to
generate 16. The de-protection of 16 afforded compound 17. Finally, the uncovered
phenylamine 17 was reacted with various alkyl, aromatic acyl chloride or sulfuryl
chloride to generate the products 18a-18j.

Scheme 2 Agents and conditions: (vi) HCl, H2O, reflux, 12 h; (vii) (Boc)2O, TEA,
MeOH, r.t., 12 h; (viii) 5-nitro-2-furoic acid, EDCI, DMAP, DCM, r.t., 1 d; (ix) TFA,
0-5 ℃, 5 h; (x) the corresponding chloride or sulfonyl chloride, TEA, DCM, r.t.,
overnight.
The structure-activity relationships of all compounds were studied for their
inhibitory rate (%Inh) against M. tuberculosis H37Ra at the concentration of 10 μM
and 1μM in vitro, while ciprofloxacin was used as positive control. The cytotoxicity
was tested on Vero cells by a MTT assay (Table 1). The role of nitrofuran was
investigated firstly by synthesizing analogues 11a-11b, 13, in which the nitrofuran
group was replaced by furan, 4-nitrobenzene and 2-aminofuran, respectively.
Although the three compounds showed less toxicity than 12b, they lost anti-TB
activity. When the methoxyl of 12b was replaced by a hydrogen atom (12a), a sharp
loss of activity was observed at 1μM concentration, suggesting that the substituent on
benzene ring was very important. Then moving the methoxyl from the meta-position
to the para- and the ortho-position, the obviously declined tendency of 12c and 12d
(%Inh = -8.3% and %Inh = -3.0% respectively at 1 μM concentration) indicated that
the meta-position substitute was beneficial. Next, we introduced different substituents

in this position to investigate the polar and electronic effects on the potency. Among
the electron-withdrawing halogenic substituted 12e-12g, trifluoromethyl substituted
12h and acetamide 12l, the smallest 3-fluoro substituted 12e (%Inh = 95.7% at 1 μM
concentration) had better activity than 12b and 12h remained the equal grade, while
others were less potent in general. Among the electron-donating alkyoxy substituted
analogs, ethoxyl, isopropoxyl and cyclopentyloxyl substituted 12i, 12j, 12k (%Inh =
95.7%, 97.8% and 97.9% respectively at 1 μM concentration) resulted in the
enormous improvement of activity and lower cytotoxicity (IC50 > 90 μM, = 69.5 and
81.1 μM respectively) in comparison to 12b. Besides, the 2, 4, 6-trimethyl and
trifluoro substituted 12m and 12n showed no activity at all at 10 μM concentration.
Table 1 Inhibition rate (%Inh) against TB and Vero cell toxicity of all synthesized
compounds in vitro.
12a-12n, 16, 17, 18a-18j
Comps
R1
TBa（%Inh）
10 μM 1 μM
-0.9 1.9
Vero
IC50 (μM)
>90
11a
-1.5
-0.4
>90
11b
H
99.5
37.2
68.8
41.7
58.7
12a
12b
3
CH3O
100.2

4
2
3
3
3
3
3
3
3
CH3O
CH3O
F
99.3
99.5
99.6
99.2
98.9
98.6
99.4
99.2
99.3
-8.3
-3.0
95.7
40.5
70.4
67.0
95.7
97.8
97.9
63.0
42.0
51.6
43.7
71.0
23.9
>90
12c
12d
12e
12f
12g
12h
12i
Cl
Br
CF3
CH3CH2O
(CH3)2CHO
69.5
81.1
12j
12k
3
99.4
40.6
57.6
12l
2, 4, 6
2, 4, 6
CH3
F
-4.3
-10.1
NTb
0.9
-4.1
5.2
30.4
19.9
NT
12m
12n
13
3
NT
40.0
NT
16
3
3
NH2
NT
-1.1
NT
17
99.6
99.3
35. 7
18a
3
3
3
3
NT
NT
1.9
0.2
NT
NT
18b
18c
18d
18e
99.1
99.5
98.9
98.9
21.9
50.2

3
NT
39.8
NT
18f
3
3
NT
NT
13.7
-2.7
NT
NT
18g
18h
3
3
82.1
NT
-4.1
<2.8125
59.3
18i
18j
93.9
Ciprofloxacin
99.7
99.6
NT
a Inhibitory rate (%Inh) test against M. tuberculosis H37Ra (ATCC25177).
b Not tested
Now that the prolonged O-alkylation, increasing compounds’ lipophilicity but
leading to poor aqueous solubility, contributed to anti-TB activity, we had a try of the
increasing lipophilic derivation with alkyl/aryl amide or sulfamide on the basis of
compound 12l as well. The first compound in this series we got was the
Boc-protection compound 16 (%Inh = 40.0%), which did not have any improved
activity. The barely amino-substituted compound 17 (%Inh = -1.1%) exhibited no
inhibition against Mtb. Compounds 18a and 18d among the C3-C6 cycloalkyl
substituted analogues emerged comparatively better inhibition of 99.3% and 98.9%,
respectively. In the further aryl amide modifications, compound 18e, with the
benzamide group on the benzene ring, inhibited the 98.9% strain growth at 1 μM

concentration. However, compounds 18f-18h, with some usually substituted groups
on benzamide, showed decreased activity in different degree, in which 18f and 18g
indicated lower inhibition (%Inh = 39.8% and 13.7% respectively) and 18h had no
activity. Therefore, we did not continue to explore the effect of other substituted
benzamide. In addition, two sulfamide compounds 18i and 18j, with methane
sulfamide and benzenemethane sulfamide respectively, were -4.1% and 93.9%
inhibition rate against H37Ra strain, which also clearly verified that the hydrophobic
substituent at the meta-position of benzene of structure 7 was beneficial for the
activity improvement.
Based on the initially measured inhibitory levels against Mtb, the eight most
active compounds, 12e, 12k, 12l, 12m, 12a, 18d, 18e, and 18j, whose inhibitory rate
higher than 90% against H37Ra, were further tested for MIC against Mtb and S.
aureus and aqueous solubility was also studied (Table 2). All the tested compounds
showed the MICs of 0.27-1.00 μg/mL against Mtb and 1.36-4.99 μg/mL against S.
aureus. Among them, compound 18e exhibited the lowest anti-TB MIC of 0.27 μg/mL
and the lowest anti-S. aureus MIC of 1.36 μg/mL. Compound 12e had the highest
aqueous solubility (14.83 μg/mL). The 3-alkoxyl substituted compounds 12k, 12l,
12m and sulfamide compound 18j remained the weaker inhibition, and the poor
aqueous solubility (<1 μg/mL) confirmed our previous prediction. Compound 18d
almost showed the equivalent activity against Mtb and 2-fold lower against S. aureus
than 18e. By the way, 18e was safer by comparing the cytotoxity of compound 18d
and 18e (IC50 = 21.9 and 50.2 μM respectively). These results declared that the

nitrofuranyl introduction of structure 7 satisfied our original design of inhibiting both
Mtb and S. aureus. Moreover, the meta-amide substitutions on the benzene ring of the
key structure 7 exhibited good activity. The most potent compound 18e exhibited
about 5-fold improvement than lead 1 against S. aureus, as well as possessed the
submicromolar anti-tubercular activity. Overall, these scaffolds could be potential
single antitubercular agents or broadly antibacterial agents.
Table 2 The solubility, MIC value against Mtb and S. aureus of compounds 12e, 12i,
12j, 12k, 12a, 18d, 18e and 18j.
Comps
Structure
Solubility MIC(μg/mL) MIC(μg/mL)
(μg/mL)a
Mtbb
S. aureusc
14.83
0.83
4.17
12e
0.34
0.27
0.90
0.47
1.00
0.50
4.49
4.67
4.99
2.49
12i
12j
12k
18a
0.027
0.47

3.46
1.27
0.48
0.28
0.27
0.61
1.04
2.75
1.36
1.52
0.26
18d
18e
18j
Ciprofloxacin
a Aqueous solubility at room temperature.
b MIC test against M. tuberculosis H37Ra (ATCC 25177).
c Inhibitory rate (%Inh) against S. aureus (ATCC 6538).
In conclusion, we had designed and synthesized novel scaffolds of
2-aminothiazole conjugating nitrofuran to effectively inhibit both the strains of Mtb
and S. aureus. The SARs indicated that substitutions at the meta-position of benzene
ring of 5-nitro-N-(4-phenylthiazol-2-yl) furan-2-carboxamide exhibited superior
activity. Eight compounds had the inhibitory rate > 90% against Mtb at 1 μM
concentration, and compound 18e showed the lowest antitubercular MIC of 0.27
μg/mL against H37Ra and the lowest antibacterial MIC of 1.36 μg/mL against S.
aureus. According to the above data, the novel scaffolds conjugating aminothiazole
with nitrofuran could be potential antitubercular and antibacterial agents and the more
potent molecules were currently underway.
Acknowledgments

This work was supported by The National Science and Technology Major Project
of China (2012ZX09103101-021). We thank Shanghai Chempartner for in vitro
antitubercular and antimicrobial screening. We also thank Lihua Zhou of State Key
Laboratory of Biotherapy (Sichuan University) for NMR measurements.
References and notes
1 Wright, G D. ACS Chem. Biol. 2012, 19: 3-10.
2 Heymann, D. L. Cell 2006, 124, 671.
3 Perri, G.D.; Bonora, S. J. Antimicrob. Chemother. 2004, 54, 593–602.
4 Nickerson, E. K.; West, T. E.; Day, N. P.; Peacock, S. J. Lancet Infect. Dis. 2009, 9, 130-135.
5 Global Tuberculosis Report 2014. World Health Organization, 2014.
6 Poce, G.; Cocozza, M.; Consalvi, S.; Biava, M. Eur. J. Med. Chem. 2014, 86, 335.
7 Demain, A. L. ACS Chem. Biol. 2011, 18, 939.
8 Kanehisa, M.; Goto, S. Nucleic Acids Res. 2000, 28, 27-30.
9 Li, J. R.; Li, D. D.; Wang, R. R.; Sun, J.; Dong, J. J.; Du, Q. R.; Fang, F.; Zhang, W. M.; Zhu, H.
L. Eur. J. Med. Chem. 2014, 75, 438.
10 Tavares, L. C.; Chisté, J. J.; Santos, M. G.; Penna, T. C. Boll. Chim. Farm. 1999, 138, 432-436.
11 Jorge, S. D.; Masunari, A.; Rangel-Yagui, C. O.; Pasqualoto, K. F. M.; Tavares, L. C. Bioorg.
Med. Chem. 2009, 17, 3028.
12 Tangallapally, R. P.; Lee, R. E.; Yendapally, R.; Hevener, K.; Jones, V. C.; Lenaerts, A. J. M.;
McNeil, M. R.; Wang, Y.; Franzblau, S.; Lee, R. E. J. Med. Chem. 2004, 47, 5276.
13 Tangallapally, R. P.; Yendapally, R.; Lee, R. E.; Lenaerts, A. J.M.; Lee, R. E. J. Med. Chem.
2005, 48, 8261.
14 Rakesh, B. D.; Bruhn, D. F.; Scherman, M. S.; Woolhiser, L. K.; Madhura, D. B.; Maddox, M.;
Singh, A. P.; Lee, R. B.; Hurdle, J. G.; McNeil, M. R.; Lenaerts, A. J.; Meibohm, B.; Lee, R. E.
PLoS One 2014, 9, e87909.
15 Yempalla, K. R.; Munagala, G.; Singh, S.; Magotra, A.; Kumar, S.; Rajput, V. S.; Bharate, S. S.;

Tikoo, M.; Singh, G. D.; Khan, I. A.; Vishwakarma, R. A.; Singh, P. P. ACS Med. Chem. Lett.
2015, 6, 1041.
16 Zheng, P.; Somersan-Karakaya, S.; Lu, S.; Roberts, J.; Pingle, M.; Warrier, T.; Little, D.; Guo,
X.; Brickner, S. J.; Nathan, C. F.; Gold, B.; Liu, G. J. Med. Chem. 2014, 57, 3755.