Design, synthesis and evaluation of novel molecules with a diphenyl ether nucleus as potential antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2011.12.022

A series of compounds with a diphenyl ether nucleus were synthesized by incorporating various amines into the diphenyl ether scaffold with an amide bond. Their antitubercular activities were evaluated against Mycobacterium tuberculosis H₃₇Rv by a microdilution method, with MIC values ranging from 4 to 64 μg/mL. Through structure-activity relationship studies, the two chlorine atoms at 3 and 4 positions in the phenyl ring of R² group were found to play a significant role in the antitubercular activity. The most potent compound 6c showed an MIC value of 4 μg/mL and a good safety profile in HepG2 cell line by the MTT assay. Compound 6c was further found to be effective in a murine model of BCG infection, providing a good lead for subsequent optimization.

Full text of DOI:10.1016/j.bmcl.2011.12.022

Bioorganic & Medicinal Chemistry Letters 22 (2012) 954–957
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of novel molecules with a diphenyl ether
nucleus as potential antitubercular agents
Yinghong Yang a,b, , Zhenling Wang a, , Jianzhong Yang a, Tao Yang a, Weiyi Pi a, Wei Ang c, Yanni Lin a,
a,
a
Yuanyuan Liu a, Zicheng Li b, , Youfu Luo , Yuquan Wei
⇑
⇑
a State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China
b Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, PR China
c Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of compounds with a diphenyl ether nucleus were synthesized by incorporating various amines
into the diphenyl ether scaffold with an amide bond. Their antitubercular activities were evaluated
against Mycobacterium tuberculosis H37Rv by a microdilution method, with MIC values ranging from 4
Received 10 August 2011
Revised 29 November 2011
Accepted 3 December 2011
Available online 9 December 2011
to 64
in the phenyl ring of R2 group were found to play a significant role in the antitubercular activity. The most
potent compound 6c showed an MIC value of 4 g/mL and a good safety profile in HepG2 cell line by the
lg/mL. Through structure–activity relationship studies, the two chlorine atoms at 3 and 4 positions
l
Keywords:
Diphenyl ether nucleus
Antituberculosis
MTT assay. Compound 6c was further found to be effective in a murine model of BCG infection, providing
a good lead for subsequent optimization.
Ó 2011 Elsevier Ltd. All rights reserved.
H37Rv
BCG murine model
Tuberculosis (TB), one of the leading causes of death in the
world, poses a significant challenge to the public health. The World
Health Organization (WHO) has estimated that one-third of the
world’s population is currently infected with the TB bacillus.1
According to the annual report on global control of TB published
by the WHO in 2010, there were about 9.4 million incident cases
of TB globally in 2009, and approximately 1.7 million people died
of TB, among these deaths 23% were HIV-positive.2 People who
are HIV-positive are more likely to be infected with TB, so the
co-infection with HIV has been responsible for this serious situa-
tion. On the other hand, the emergence of multi drug resistant
tuberculosis (MDR-TB) and extensively drug resistant tuberculosis
(XDR-TB) also contributes to the increased morbidity and mortal-
ity. MDR-TB, describes strains of tuberculosis that are resistant to
at least the two main first-line TB drugs-isoniazid (INH) and rifam-
picin. XDR-TB is defined as resistance not only to rifampicin and
isoniazid but also fluoroquinolones and to at least one of the inject-
able second-line drugs. It is also said that there was an estimated
0.44 million cases of MDR-TB in 2008, and 58 countries and terri-
tories had reported at least one case of XDR-TB by July 2010.2 This
worsening situation leads to an urgent necessity for development
of new agents to fight against this disease.
logical activities. A classic chemical with this nucleus is triclosan,
which is launched in the early 1970s. It is widely used in a variety
of cleaning and personal care products for its broad spectrum anti-
microbial activity against a variety of Gram-positive and Gram-
negative bacteria3 and continuous studies have been focused on
triclosan and its derivatives in recent years. 4 FAB-001 (Fig. 1), a FabI
inhibitor with the diphenyl ether nucleus developed by FAB Pharma
(Paris, France), is currently being evaluated in phase-I trials for the
treatment of drug-resistant staphylococci.5 Moreover, lots of novel
diphenyl ether analogues were designed to exhibit enhanced anti-
mycobacterial activity.6 Kini and co-workers synthesized a series
of heterocycle substituted diphenyl ether derivatives and all the
compounds showed good antitubercular activity against Mycobac-
terium tuberculosis strain H37Rv.7 Diaryl amide skeleton is also a
class of privileged structure with broad range of biological activities
such as antiviral,8 anti-inflammatory,9 analgesic,10 antibacterial,11
melanin synthesis inhibitor12 and S1P4 antagonist.13 Recently, in a
high throughput screening for inhibitors of M. tuberculosis H37Rv,
several diaryl amides14 (Fig. 1) were found to possess potent activ-
ity in combination with the relatively low toxicity. Encouraged by
the prominent activity of diphenyl ether derivatives and diaryl
amides, we proposed to synthesize some novel diphenyl ether ana-
logues containing the diaryl amide moiety by molecular hybridiza-
tion approach, aiming to search for potential candidates with better
antitubercular activities and lower toxicities.
Diphenyl ether nucleus is an important unit found in several
synthetic and natural agents possessing wide range of pharmaco-
In this Letter, we designed and synthesized 22 compounds with
a diphenyl ether nucleus, and their antitubercular activity against
M. tuberculosis H37Rv in vitro were evaluated by a microdilution
⇑
Corresponding authors. Tel./fax: +86 28 85503817 (Y.L.).
E-mail addresses: sculzc@scu.edu.cn (Z. Li), luo_youfu@scu.edu.cn (Y. Luo).
These authors contributed equally to this paper.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.022

Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 954–957
955
Scheme 2. Synthetic route of compound 6v. Reagents and conditions: (i) CuI, N,N-
dimethylglycine, Cs2CO3, 1,4-dioxane, 90 °C; (ii) NaOH, EtOH/H2O, reflux; (iii) SOCl2,
CHCl3, reflux; (vi) 3-chloroaniline (5a), CH2Cl2, Et3N, rt.
Figure 1. Chemical structures of triclosan, FAB-001 and diaryl amides.
atoms in 6c were replaced by two fluorine atoms in 6d (R2 = 3,4-
di F-Ph), the activity was decreased a lot, and so was 6e (R2 = 2-
Cl-4-Br-Ph) with a chlorine atom and a bromine atom. All these
data suggested that the two chlorine atoms at 3 and 4 positions
in the phenyl ring of R2 group are very important in the antituber-
cular activity. Compounds 6f (R2 = 2-CN-Ph), 6g (R2 = 4-CF3-Ph)
introduced with electron-withdrawing substituents and com-
pounds 6h (R2 = 4-OCF3-Ph), 6i (R2 = 2,4-di OCH3-Ph) with
electro-donating substituents all showed moderate activity
method. Compound 6c was found to be the most potent antituber-
cular agents with MIC of 4 lg/mL, and it was then assessed in vivo
using a BCG infection mice model.
The target compounds were synthesized utilizing the reaction
sequence as shown in Schemes 1 and 2. Iodobenzene or substituted
iodobenzene (1) was employed as starting material, it was conden-
sated with compound 2 to give compound 3, via the N,N-dimethyl
glycine-promoted Ullmann coupling reaction.15 Compound 3 was
further hydrolyzed to compound 4 in excellent yields under reflux
conditions in a solution of sodium hydroxide. Compound 4 was
then treated with thionyl chloride to obtain the corresponding acyl
chloride, and it was reacted with various amines 5 in presence of
triethylamine in anhydrous dichloromethane to give the target
compounds 6a–v. The structures of 6a–v were fully characterized
by 1H NMR, 13C NMR, and ESI-MS analysis and all the analytical
data were documented in the Supplementary data. The purity of
all compounds was above 97.0% determined by HPLC normaliza-
tion method.
(MIC = 32 lg/mL) without potency improvement. In particular,
introduction of a nitro unit in the phenyl ring of R2 group (com-
pound 6j, R2 = 3-NO2-Ph; 6k, R2 = 4-NO2-Ph) did not increase anti-
tubercular activity, although lots of compounds with the NO2
substituent, such as nitrofunans and nitroimidazoles, possessed
broad spectrum antimicrobial activities by DNA damage. Next,
compounds 6m–6q with more complex aromatic or heterocyclic
rings were synthesized, compound 6n (R2 = 6-methoxybenzo[d]-
thiazol-2-yl) exhibited relatively higher level of activity with MIC
of 16 lg/mL compared with 6m and 6o–6q. Further modifications
on the length of the aliphatic chain between the B ring and the
All the synthesized compounds (6a–v) were evaluated for their
antimycobacterial activity againstM. tuberculosis H37Rv by a micro-
dilution method in 96-wells plates.16 Briefly, M. tuberculosis was
grown in Middlebrook 7H9 broth containing oleic acid-albumin-
dextrose-catalase (OADC) enrichment, and was diluted with 7H9-
amide bond showed that all the derivatives (6s–6v, n = 0, 2, 3) pos-
sess similar potency (MIC = 32 lg/mL) with compound 6a (n = 1).
Compound 6s with amide bond at meta position of B ring and com-
pound 6v at para position demonstrated similar activities. Neither
molecule 6q with chlorine atom nor 6r with fluorine atom of R1
group in A ring exhibited increased antimycobacterial activity
compared with molecule 6a. Further studies with modifications
of the A and B rings are in progress in our lab. Thus, at this stage,
all the modifications indicated that the two chlorine atoms at 3
and 4 positions in the phenyl ring of R2 group play a significant role
in the antitubercular activity.
OADC medium to 104 CFU/mL. An amount of 100
lL of this bacte-
rial culture was then added to a microtiter well containing serial
dilutions of compounds 6a–v. The plates were then incubated at
37 °C in a moisturized incubator and visually examined for growth.
INH was used as a positive control. The MIC values are shown in
Table 1.
As shown in Table 1, all the compounds of this series exhibited
The safety profile of compounds 6a–v was tested on human
antitubercular activity with MIC ranging from 4 to 64
lg/mL, and
cancer cell lines HepG2 at the concentration of 20 lM using the
compound 6c was found to be the most potent with MIC of 4
MTT method. Briefly, cells (2500/well) were seeded in 96-well
plates and cultured for 24 h, followed by treatment with the target
compounds 6a–v for another 48 h. Twenty microliter of 5 mg/ml
MTT was added per well and incubated for another 2.5 h at
37 °C. Then the supernatant fluid was removed and MTT formazan
l
g/mL. Compounds 6a (R2 = 3-Cl-Ph) and 6b (R2 = 4-Br-Ph) exhibited
lower activity than compound 6c (R2 = 3,4-di Cl-Ph), and 3-Cl
substituted compound 6a was slightly more effective compared
with the 4-Br substituted compound 6b. When the two chlorine
Scheme 1. Synthetic route of compounds 6a–u. Reagents and conditions: (i) CuI, N,N-dimethylglycine, Cs2CO3, 1,4-dioxane, 90 °C; (ii) NaOH, EtOH/H2O, reflux; (iii) SOCl2,
CHCl3, reflux; (vi) NH2R2 (5a–u), CH2Cl2, Et3N, rt.

956
Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 954–957
Table 1
Antimycobacterial activity and toxicity of compounds 6a–v
H
O
n
O
N
R2
H
N
A
B
A
B
Cl
O
R1
O
6a-u
R2
6v
Compound
R1
n
H37Rv (
l
g/ml)
HepG2 (%)
MICb
IRc@20
lM
a
MIC90
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
2
3
—
3-Cl-Ph–
4-Br-Ph–
3,4-Di Cl-Ph–
3,4-Di F-Ph–
2-Cl-4-Br-Ph-
2-CN-Ph–
4-CF3-Ph–
4-OCF3-Ph–
2,4-Di OCH3-Ph–
3-NO2-Ph–
4-NO2-Ph–
Naphthalen-1-yl–
Diphenylmethyl–
6-Methoxybenzo[d]thiazol-2-yl–
5-Methylpyridin-2-yl–
Thiazol-2-yl–
3-Cl-Ph–
3-Cl-Ph–
3-Cl-Ph–
3-Cl-Ph–
3-Cl-Ph–
16
16
NTd
8
16
NT
8
16
NT
16
8
32
8
32
64
4
7.95
15.70
7.00
26.73
12.81
19.08
10.31
4.71
4.11
0.95
36.41
3.31
4.84
32
32
32
32
32
32
32
32
64
32
16
32
32
32
32
32
32
32
32
8
8
4.29
12.21
39.89
27.06
23.52
7.07
25.05
NT
29.20
3.51
16
16
16
16
16
16
16
6s
6t
6u
6v
INH
H
H
H
—
—
0.0625
0.0625
a
b
c
MIC90 is defined as the minimal drug concentration of the well in which the growth of the bacterial is similar to that with 100 CFU of bacteria inoculated merely.
MIC is defined as the minimal drug concentration of the well in which the growth of the bacterial is similar to that with 10 CFU of bacteria inoculated merely.
IR is the mean inhibitory rate calculated from three independent experiments measured at 48 h after treatment with the test compound at the concentration of 20
NT means not tested.
lM.
d
precipitate was dissolved in 150 lL of DMSO, shaken mechanically
for 15–20 min. The optical density of each well was measured at
Table 2
Multiplication of M. bovis BCG
570 nm, using a SpectraMAX M5 microplate spectrophotometer.
The inhibitory rates at 20 lM are given in Table 1, all the active
compounds showed low toxicity in this assay and the most potent
compound 6c exhibited good safety profile with inhibitory rates as
low as 7%.
Group
Na
Log10 CFU (mean SD)
Spleen
Lung
BCG + vehicle
BCG + 6c
BCG + INH
6
8
8
4.64 0.17
4.79 0.25
2
2
0.89⁄
0.45⁄
2
3
0.74⁄
0.24⁄
The most potent derivative 6c was further tested for in vivo
antitubercular activity using a murine model of BCG infection17
as described in detail in the Supplementary data. Female BALB/c
mice were infected intravenously with bacterial culture containing
approximately 1 Â 105 CFU of M. bovis BCG, and compound 6c was
orally administered at a dose of 25 mg/kg once daily. After six
weeks of treatment, mice were sacrificed. Lung and spleen tissues
were homogenized and plated on Modified Lowenstein-Jensen
Medium Base. BCG colonies were counted after six weeks of incu-
bation at 37 °C. Data were expressed as mean log10 CFU per organ
Significantly different: ⁄P <0.01 versus vehicle-treated group.
a
N means the number of treated mice in each group.
and results of one several representative experiments were shown
in Table 2. Compound 6c-treated mice had a significant reduction
in bacterial numbers in the lungs, as well as in spleens, compared
to vehicle controls. And there is no remarkable difference in
bacterial numbers between compound 6c group and INH group,
both in lungs and spleens.
Figure 2. Histopathological appearance of lungs of infected mice treated with vehicle (A), infected mice treated with compound 6c (B), and infected mice treated with INH
(C). Magnification, Â100.

Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 954–957
957
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This work was supported by the National Major Program of Chi-
na during the 12th Five-Year Plan Period(2012ZX09103-101-068).
The authors are grateful to Shanghai Pulmonary Hospital for
in vitro antitubercular screening, and thank Dr. Ruiliang Jin for
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Supplementary data
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Supplementary data associated with this article can be found, in
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References and notes
1. http://www.who.int/mediacentre/factsheets/fs104/en/.
2. Global Tuberculosis Control, 2010. http://www.who.int/tb/publications/
global_report/2010/en/index.html.