Synthesis and evaluation of anti-tubercular and antibacterial activities of new 4-(2,6-dichlorobenzyloxy)phenyl thiazole, oxazole and imidazole derivatives. Part 2

Article abstract of DOI:10.1016/j.ejmech.2012.01.007

A series of substituted 4-(2,6-dichlorobenzyloxy)phenyl thiazole, oxazole and imidazole derivatives were synthesized. The derivatives were screened for in vitro anti-tubercular activities against Mycobacterium tuberculosis H37Rv using the Microplate Alamar Blue Assay (MABA), and antibacterial activities with agar dilution method against clinical S. aureus, E. coli, S. pneumoniae and penicilin-resistant S. pneumoniae. Among 15 compounds, several thiazole derivatives exhibited good anti-tubercular activities with MIC values between 1 μM and 61.2 μM, and potent activities against S. pneumoniae with MIC values less than 0.134 μM. These studies suggest that the thiazole scaffold may serve as a new promising template for further elaboration as anti-tubercular and antibacterial drugs.

Full text of DOI:10.1016/j.ejmech.2012.01.007

European Journal of Medicinal Chemistry 49 (2012) 164e171
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of anti-tubercular and antibacterial activities of new
4-(2,6-dichlorobenzyloxy)phenyl thiazole, oxazole and imidazole derivatives.
Part 2
,
,
*
Xiaoyun Lu a b, Xiaobo Liu a, Baojie Wan c, Scott G. Franzblau c, Lili Chen d, Changlin Zhou d, Qidong You a
a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
b Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, No. 190, Kaiyuan Avenue,
Science Park, Guangzhou 510530, China
c Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, USA
d School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, 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 substituted 4-(2,6-dichlorobenzyloxy)phenyl thiazole, oxazole and imidazole derivatives were
synthesized. The derivatives were screened for in vitro anti-tubercular activities against Mycobacterium
tuberculosis H37Rv using the Microplate Alamar Blue Assay (MABA), and antibacterial activities with agar
dilution method against clinical S. aureus, E. coli, S. pneumoniae and penicilin-resistant S. pneumoniae.
Among 15 compounds, several thiazole derivatives exhibited good anti-tubercular activities with MIC
Received 24 October 2011
Received in revised form
4 January 2012
Accepted 5 January 2012
Available online 12 January 2012
values between 1
0.134 M. These studies suggest that the thiazole scaffold may serve as a new promising template for
further elaboration as anti-tubercular and antibacterial drugs.
Ó 2012 Elsevier Masson SAS. All rights reserved.
mM and 61.2 mM, and potent activities against S. pneumoniae with MIC values less than
m
Keywords:
4-(2,6-dichlorobenzyloxy)phenyl thiazole
derivatives
Anti-tubercular activity
Antibacterial activity
1. Introduction
are indispensable structural units for medicinal chemists and have
attracted continuing interest over the years as their varied biolog-
ical activities, such as antibacterial [5e8], anti-tubercular [9,10],
antifungal [11], anticancer [12,13] and anti-inflammatory [14], etc.
In continuation of our research efforts to develop new therapeutics
for treatment of TB, we envisioned the replacement of the thio-
phene nucleus by thiazole, oxazole and imidazole isostere to
provide the general structure (Fig. 2). The major suggestion derived
from previous study was that the compound containing p-chlor-
obenzamide group at thiophene ring was proved to be highly active
in the amide series. Accordingly, with the aim to further explore the
structureeactivity relationships (SARs) of diazoles, which are
presumed to be fundamental for anti-tubercular activity, a series of
4-(2,6-dichlorobenzyloxy)phenyl thiazole, oxazole and imidazole
derivatives (10aef, 11aee, 20, 21, 26 and 27) were synthesized and
evaluated for their anti-tubercular activity.
Tuberculosis (TB), a highly contagious and air-borne disease
caused by Mycobacterium tuberculosis, emerged with multi-drug
resistant strains (MDR-TB) and acquired immune deficiency
syndrome (AIDS) in recent years [1,2]. According to World Health
Organization (WHO) [3], one third of the world’s population is
infected with M. tuberculosis and approximately 1.7 million deaths
occured worldwide in 2009. In spite of the increasing worldwide
incidence of TB, no new drugs have been brought to the market
over the past four decades. Therefore, it is an imperative need to
develop novel anti-tubercular drugs that can be equally effective
against M. tuberculosis and MDR-TB, and also reduce the duration of
therapy.
Our research group have recently disclosed that 2-acylated
and 2-alkylated amino-5-(4-(benzyloxy)phenyl)thiophene-3-car-
boxylic acid, exemplified by compounds 1 and 2 (Fig. 1), were
potent anti-tubercular agents [4]. Thiazole, oxazole and imidazole
2. Chemistry
The synthetic strategies employed to obtain the target
compounds are depicted in Schemes 1e3. For thiazole series, the
key intermediate ethyl 2-amino-4-(4-(2,6-dichlorobenzyloxy)
* Corresponding author. Tel./fax: þ86 25 83271351.
E-mail address: youqidong@gmail.com (Q. You).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.007

X. Lu et al. / European Journal of Medicinal Chemistry 49 (2012) 164e171
165
3. Results and discussion
The title compounds along with the standard drugs (Isoniazid,
Moxifloxacin and Gentamycin) for comparison were firstly evalu-
ated for their activities against M. tuberculosis H37Rv (ATCC #
27294) by Microplate Alamar Blue Assay (MABA) [18,19] (Table 1). A
closer look into the biological results of these compounds revealed
that in thiazole series (10aef and 11aee), esters 10aef
Fig. 1. The chemical structures of thiophene-based lead compounds 1 and 2.
(MIC ¼ 1.0e61.2
11aee (MIC
compound 10b containing 4-chloro group was the most active
M), whereas other three compounds
mM) showed much better activities than acids
¼
45.4e128 M). Among benzamides (10aed),
m
compound (MIC ¼ 1.0
m
10a, 10c and 10d have were much less active. Compared to
10b, compound 10c showed a 25-fold reduction in potency
(MIC ¼ 25.4
mM), which suggested that the ortho-position of the
phenyl ring was unfavorable for anti-tubercular activity. However,
oxazole and imidazole derivatives (20, 21, 26 and 27) did not show
considerable activity (MIC > 128 mM). The results clearly revealed
that the thiazole scaffold had encouraging activity, while oxazole
and imidazole nucleus did not show encouraging activity.
M. tuberculosis are unique to surround by a thick and waxy
cell wall. Efficient anti-tubercular drugs should have a reasonable
lipophilicity to penetrate the cell wall. To investigate the obvious
activity difference between thiazole esters 10aef and acids 11aee,
the logp values for the above compounds were calculated
(Table 1). It may be explained that the esters posses preferable
lipophilicity than acids and show optimal membrane
permeability.
Fig. 2. The general structure designed by replacement of thiophene nucules by thia-
zole, oxazole and imidazole, respectively.
phenyl)thiazole-5-carboxylate 8 was prepared by cyclization of
a
-bromoketoesters 7 with thiourea [15]. Then, acylation of 8
The results from the anti-tubercular activity of thiazole series
(Table 1) prompted us to investigate their antibacterial activity
against clinical S. aureus, E. coli, S. pneumoniae, penicilin-resistant S.
pneumoniae (Table 2). The results revealed that compounds 10a,
10def, 11aeb and 11dee exhibited potent activities against the
Gram-positive clinical S. pneumoniae with MIC values better than
with different acyl chloride in the presence of DMAP led to
the desired esters 10aef. Hydrolysis of 10aed gave the corre-
sponding carboxylic acids 11aed. Compound 11e was obtained
by reacting intermediate 9 with butyric anhydride due to the
amide bond breakage under alkaline conditions. For oxazole series,
the intermediate 19 was prepared by cyclization of 4-(2,6-
dichlorobenzyloxy)benzoyl chloride 15 with ethyl amino-
cyanoacetate 4-toluenesulfonate 18 in NMP [16]. Acylation of 19
with p-chlorobenzoyl chloride afforded the ester 20, which
subsequently was hydrolyzed at reflux to give the corresponding
acid 21. For imidazole series, reaction of ethyl aminocyanoacetate,
triethylorthoformate and 4-(2,6-dichlorobenzyloxy)aniline 24 in
one-pot gave the intermediate 25 [17]. Acylation of 25 with p-
chlorobenzoyl chloride afforded ester 26, which subsequently was
hydrolyzed at reflux to give the corresponding acid 27.
the control drug ampicillin (MIC ¼ 0.168
activity against clinical S. aureus, E. coli, and penicilin-resistant S.
pneumoniae (MIC > 128 M). Compound 10b exhibited the activity
against clinical S. pneumoniae equal to the control drug gentamycin
(MIC ¼ 6.9 M), while did not show any considerable activity
against other three clinical strains (MIC > 128 M). On the contrary,
mM), but did not have
m
m
m
compounds comprising 2,4-dichloro substitution on phenyl ring
(10c and 11c) showed no inhibitory activity against all the tested
clinical strains (MIC > 128 mM).
Scheme 1. Reagents and conditions: (a) K2CO3, KI, DMF, reflux for 12 h; (b) CO(OEt)2, NaH, 1,4-dioxane, reflux for 4 h; (c) Br2, CH2Cl2, 10 ꢀCert, 1 h; (d) NH2CSNH2, C2H5OH; reflux for
2 h; rt for overnight; (e) RCOCl, DMAP, THF; rt; 10 h; (f) 1NNaOH, CH3OH; rt; 3 days; (g) (CH3CH2CH2CO)2O, DMAP, THF; rt; 10 h.

166
X. Lu et al. / European Journal of Medicinal Chemistry 49 (2012) 164e171
Scheme 2. Reagents and conditions: (a) K2CO3, acetone; reflux for 24 h; (b) KOH, C2H5OH; reflux for 2 h; (c) (COCl)2, CH2Cl2, DMF(Cat); reflux for 5 h; (d) NaNO2, H3PO4, HCl, H2O;
40e45 ꢀC; rt for overnight; (e) Na2S2O4, NaHCO3, NaCl, H2O; rt; 3 h; (f) TsOH, (Et)2O, THF; (g) NMP; rt; overnight; (h) 4-ClPhCOCl, Et3N, CH2Cl2; rt; 12 h; (i) 1NNaOH, C2H5OH; reflux
for 2 h.
4. Conclusions
expressed in parts per million (ppm, d units). Coupling constants
are in units of hertz (Hz). The spin multiplicities are indicated by
the symbols s (singlet), d (doublet), dd (doublet of doublets), t
(triplet), q (quartet), m (multiplet), and br (broad). MS spectra were
recorded on a Shimadzu GCeMS 2010 (EI) or a Mariner Mass
Spectrum (ESI). Elemental analyses (C, H, N) were determined on
on Carlo Erba 1106 elementary analysis apparatus and were within
ꢁ0.5% of the theoretical values. TLCs and preparative thin-layer
chromatography were performed on silica gel GF/UV 254, and the
chromatograms were performed on silica gel (100e200 mesh)
visualized under UV light at 254 nm and 365 nm. All solvents were
reagent grade and, when necessary, were purified and dried by
standards methods. Concentration of solutions after reactions and
extractions involved the use of a rotary evaporator operating at
a reduced pressure of ca. 20 Torr. Organic solutions were dried over
anhydrous sodium sulfate.
In conclusion, we have synthesized
a series of 4-(2,6-
dichlorobenzyloxy)phenyl thiazole, oxazole and imidazole deriva-
tives, and studied their anti-tubercular and antibacterial activities.
In particular, the thiazole 10b carrying p-chlorobenzoyl substituent
appeared to exhibit the highest anti-tubercular activity with MIC
value as low as 1 mM. Such SAR results were in agreement with
those reported from our previous studies [4]. The possible
improvements in the activity can be further achieved by modifi-
cations the substituents on phenyl ring. Furthermore, thiazoles 10a,
10def, 11aeb and 11dee appeared to exhibit potent antibacterial
activity against S. pneumoniae. This new class of compounds is now
being tested in more extensive in vitro studies to determine efficacy
against M. tuberculosis and S. pneumoniae.
5. Experimental
5.1.1. General procedure for the synthesis of compounds (10aef and
11aee)
5.1. Chemistry
5.1.1.1. 1-(4-(2,6-Dichlorobenzyloxy)phenyl)ethanone (5). To a solu-
tion of 1-(4-hydroxyphenyl)ethanone 4 (6.8 g, 0.05 mol) and K2CO3
(13.8 g, 0.1 mol) in acetone (100 mL), 2.6-dichlorobenzyl chloride 3
(10.78 g, 0.055 mol) was added. The mixture was stirred vigorously,
and heated to reflux for 24 h. On cooling the reaction mixture to
room temperature without down, the mixture was concentrated
Melting points were determined on a Mel-TEMP II melting point
apparatus and are uncorrected. Infrared (IR) spectra (KBr) were
recorded on a Nicolet Impact 410 instrument (KBr pellet). 1H-NMR
spectra were recorded with a Bruker Avance 300 MHz spectrometer
at 300 K, using TMS as an internal standard. Chemical shifts are
Scheme 3. Reagents and conditions: (a) K2CO3, acetone; reflux for 24 h; (b) KOH, C2H5OH; reflux for 16 h; (c) NCC(NH2)COOEt, (C2H5O)3CH, CH3CN; rt; 3 h; (d) 4-ClPhCOCl, Et3N,
CH2Cl2; rt; 12 h; (e) 1NNaOH, C2H5OH; reflux for 2 h.

X. Lu et al. / European Journal of Medicinal Chemistry 49 (2012) 164e171
167
99.3%) as a white solid. m.p.130e132 ꢀC. 1H-NMR (300 MHz,
CDCl3)：
Table 1
In vitro anti-tubercular activities of compounds 10aef, 11aee, 20, 21, 26 and 27
against M. Tuberculosis H37Rv and their calculated clogP.
d
2.51 (3H, s, CH3), 5.28 (2H, s, CH2), 6.99 (2H, d, J ¼ 8.7 Hz,
ArH), 7.22 (1H, m, ArH), 7.32 (2H, d, J ¼ 7.5 Hz, ArH), 7.91 (2H, d,
J ¼ 8.7 Hz, ArH). EI-MS (m/z): 294 (M)þ.
5.1.1.2. Ethyl 3-(4-(2,6-dichlorobenzyloxy)phenyl)-3-oxopropanoate
(6). To a mixture of NaH/oil dispersion (0.528 g, 50% NaH by
weight, calculated by 0.264 g of NaH, 11 mmol), diethyl carbonate
(12 mL), and THF (15 mL) was added dropwise a solution of 5
(2.94 g, 10 mmol) in THF (15 mL). The mixture was heated at reflux
for 4 h. The reaction mixture was poured into water, the pH was
adjusted to 9, and mixture was extracted with methylene dichlor-
ide. The solvent was removed, and the residue was purified by flash
silica gel chromatography (petroleum ether: ethyl acetate ¼ 10 : 1)
as the eluent to yield 6 (2.49 g, 67.84%) as yellow solid. m.p.
Compd.
R
R1
MABA MIC (
mM)
clogPa
10a
11a
10b
11b
10c
11c
10d
11d
10e
11e
10f
Et
H
Et
H
Et
H
Et
H
Ph
Ph
61.2
>128
1.0
>128
25.4
45.5
7.6
>128
60.8
> 128
7.2
6.25
5.78
6.50
5.94
6.81
6.18
6.45
6.02
6.00
5.74
5.36
4-ClePh
4-ClePh
2,4-ClePh
2,4-ClePh
4eCH3OePh
4eCH3OePh
nePr
96e98 ꢀC. 1H-NMR (300 MHz, CDCl3)：
d
1.20 (3H, t, J ¼ 7.2 Hz, CH3),
3.89 (2H, s, COCH2), 4.15 (2H, q, J ¼ 7.2 Hz, COOCH2), 5.28 (2H, s,
CH2O), 7.00 (2H, d, J ¼ 8.7 Hz, ArH), 7.22 (1H, m, ArH), 7.32 (2H, d,
J ¼ 7.5 Hz, ArH), 7.90 (2H, d, J ¼ 8.7 Hz, ArH). EI-MS (m/z): 366 (M)þ.
Et
H
Et
nePr
Ethylene
5.1.1.3. Ethyl 2-bromo-3-(4-(2,6-dichlorobenzyloxy)phenyl)-3-oxop-
ropanoate (7). To a solution of 6 (1.83 g, 5 mmol) in CH2Cl2
(25 mL) was added a solution of Br2 (0.69 g, 4.3 mmol) in CH2Cl2
(5 mL) in a dropwise manner at 10 ꢀC. The reaction mixture was
stirred further for an additional 1 h and then treated with 10%
K2CO3 solution. The organic layer was separated, dried (MgSO4),
filtered, and concentrated in vacuo giving the crude bromide
7, which was used subsequently in the next step. EI-MS (m/z):
Compd.
R
MABA MIC (
mM)
clogPa
20
21
Et
H
> 128
> 128
6.52
6.06
444 (M) þ
.
5.1.1.4. Ethyl 2-amino-4-(4-(2,6-dichlorobenzyloxy)phenyl)thiazole-
5-carboxylate (8). To a solution of thiourea (0.38 g, 5 mmol) in
absolute EtOH (25 mL) compound 7 was added dropwise. After the
addition was completed, the solution was heated at reflux for 2 h
and stirred at room temperature overnight (Rf ¼ 0.4, PE: EA ¼ 4:1).
The mixture was then concentrated to dryness, and washed by
concentrated NH4OH. The solid was filtered and crystallized from
EtOH to yield 8 (1.6 g, 75.83% two steps) as yellow solid. m.p.
compd
R
MABA MIC (
mM)
clogPa
26
27
Et
H
e
e
e
> 128
> 128
0.81
0.50
0.44
5.74
5.28
e
171e173 ꢀC. 1H-NMR (300 MHz, CDCl3)：
d
1.20 (3H, t, J ¼ 7.2 Hz,
CH3), 4.15 (2H, q, J ¼ 7.2 Hz, COOCH2), 5.25 (2H, s, CH2O), 5.58 (2H, s,
NH2), 6.98 (2H, d, J ¼ 8.4 Hz, ArH), 7.20 (1H, t, J ¼ 4.8 Hz, ArH), 7.30
Isoniazid
Moxifloxacin
PA824
e
e
(2H, d, J ¼ 8.1 Hz, ArH), 7.65 (2H, d, J ¼ 8.7 Hz, ArH). EI-MS (m/z):
þ
a
The clogP values were calculated using Pallas software.
422 (M)
.
and diluted with water (100 mL) and then extracted with AcOEt
(2 ꢂ 50 mL). The organic layer was washed with 10% NaOH solution
(50 mL), dried with Na2SO4 and then concentrated in vacuo to
afford 1-(4-(2,6-dichlorobenzyloxy)phenyl)ethanone 5 (14.65 g,
5.1.1.5. 2-Amino-4-(4-(2,6-dichlorobenzyloxy)phenyl)thiazole-5-
carboxylic acid (9). To a solution of 8 (420 mg, 1 mmol) in EtOH
(40 mL) was added 1 N NaOH (5 mL, 5 mmol). The reaction mixture
was stirred at room temperature for 3 days and after poured into
water (30 mL) followed with 1 N AcOH to pH 4e5. The suspension
solution was extracted with AcOEt (2 ꢂ 50 mL). The combined
organic layers was dried over Na2SO4 and concentrated in vacuo to
afford crude prouct. Recrystallization from AcOEt gave 9 (0.09 g,
22.8%) as a white solid. m.p. 224e227 ꢀC (dec). ESI-MS (m/z): 393
Table 2
In vitro anti-microbial activities of compounds 10aef and 11aee.
Compd.
MIC (mM)
S. aureus
E. coli
S. pneumoniae
penicilin-resistant
S. pneumoniae
(M ꢃ H) þ
.
10a
11a
10b
11b
10c
11c
10d
11d
10e
11e
10f
Ampicillin
Gentamycin
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
2.7
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
10.8
ꢄ0.119
ꢄ0.125
7.12
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
10.8
5.1.2. General procedure for the synthesis of compounds 10aef
To a mixture of 8 (0.21 g, 0.5 mmol), DMAP (0.05 g, 0.4 mmol)
and dry THF (10 mL), the solution of various chloride (4 mmol) in
dry THF (10 mL) was added, while keeping the temperature at 0 ꢀC.
The whole mixture was stirred for 10 h at room temperature.
Ammonia was added to the mixture to pH 8e9 and extracted with
EtOAc (20 mL ꢂ 3). The combined organic layers were washed with
brine and dried with Na2SO4, then concentrated in vacuo. Purifi-
cation of the crude products by column chromatography eluting
with petroleum-ethyl acetate (1:1) gave 10aef.
ꢄ0.117
>128
>128
ꢄ0.112
ꢄ0.118
ꢄ0.127
ꢄ0.134
ꢄ0.131
0.168
6.9
0.43
6.9
6.9

168
X. Lu et al. / European Journal of Medicinal Chemistry 49 (2012) 164e171
5.1.2.1. Ethyl 2-benzamido-4-(4-(2,6-dichlorobenzyloxy)phenyl)thia-
zole-5-carboxylate (10a). Yield: 87.5%. White solid. m.p.
207e212 ꢀC. IR (KBr): 3297, 1663, 1530, 1329, 1278, 1236, 1095,
5.1.3. General procedure for the synthesis of compounds 11aed
To a solution of 10aed (1 mmol) in EtOH (40 mL) was added 1 N
NaOH (15 mL, 15 mmol). The reaction mixture was stirred at room
temperature for 3 days. The reaction mixture was poured into
water (30 mL) followed with 1 N AcOH to pH 4e5. The suspension
solution was extracted with AcOEt (50 mL ꢂ 3). The combined
organic layers was dried with Na2SO4 and concentrated in vacuo to
afford crude product. Recrystallization from AcOEt gave pure
11aed.
1009 cmꢃ1.1H-NMR (300 MHz, CDCl3)：
d
1.34 (3H, t, J ¼ 7.2 Hz,
CH3), 4.31 (2H, q, J ¼ 7.2 Hz, COOCH2), 5.29 (2H, s, CH2O), 6.99 (2H,
d, J ¼ 8.7 Hz, ArH), 7.26 (1H, m, ArH), 7.38 (2H, d, J ¼ 8.7 Hz, ArH),
7.47 (2H, t, J ¼ 7.5 Hz, ArH), 7.59 (1H, t, J ¼ 7.5 Hz, ArH), 7.69 (2H, d,
J ¼ 8.7 Hz, ArH), 7.87 (2H, d, J ¼ 8.4 Hz, ArH),10.50 (1H, s, NH). EI-MS
(m/z): 526 (M)þ. Formula: C26H20Cl2N2O4S. Anal (C%, H%, N%) Calcd:
59.21, 3.82, 5.31. Found: 59.24, 3.59, 5.26.
5.1.3.1. 2-Benzamido-4-(4-(2,6-dichlorobenzyloxy)phenyl)thiazole-
5-carboxylic acid (11a). Yield: 46.1%. White solid. m.p. 280e282 ꢀC
(dec). IR (KBr): 3176, 3057, 1680, 1538, 1500, 1439, 1324, 1285, 1246,
5.1.2.2. Ethyl 2-(4-chlorobenzamido)-4-(4-(2,6-dichlorobenzyloxy)
phenyl)thiazole-5-carboxylate (10b). Yield: 64.3%. White solid. m.p.
259e262 ꢀC (dec). IR(KBr): 3296, 1664, 1529, 1436, 1331, 1240, 1093,
1116, 1013 cmꢃ1 1H-NMR (300 MHz, DMSO-d6):
d 5.30 (2H,s, CH2O),
1011 cmꢃ1 1H-NMR (300 MHz, DMSO-d6):
d
1.26 (3H, t, J ¼ 7.2 Hz,
7.13 (2H, d, J ¼ 8.7 Hz, ArH), 7.49 (1H, m, ArH), 7.57 (4H, m, ArH),
7.67 (1H, t, J ¼ 7.5 Hz, ArH), 7.80 (2H, d, J ¼ 8.7 Hz, ArH), 8.14 (2H, d,
J ¼ 7.2 Hz, ArH), 13.04 (2H,s, NH, COOH). EI-MS (m/z): 497 (M ꢃ H)þ.
Formula: C24H16Cl2N2O4S$2H2O. Anal (C%, H%, N%) Calcd: 53.84,
3.77, 5.23. Found: 53.90, 3.76,5.35.
CH3), 4.23 (2H, q, J ¼ 7.0 Hz, COOCH2), 5.30 (2H, s, CH2O), 7.14 (2H, d,
J ¼ 8.4 Hz, ArH), 7.49 (1H, m, ArH), 7.59 (2H, d, J ¼ 7.5 Hz, ArH), 7.65
(2H, d, J ¼ 8.4 Hz, ArH), 7.78 (2H, d, J ¼ 8.7 Hz, ArH), 8.15 (2H, d,
J ¼ 8.4 Hz, ArH), 13.24 (1H, s, NH). 13C NMR (300 MHz, DMSO-d6):
14.02, 60.75, 64.95, 128.79, 130.27, 131.36e131.66, 136.05, 137.92,
159.02, 161.47. EI-MS (m/z): 560 (M)þ. Formula: C26H19Cl3N2O4S.
Anal (C%, H%, N%) Calcd: 55.58, 3.41, 4.99. Found: 55.41, 3.26, 4.83.
5.1.3.2. 2-(4-Chlorobenzamido)-4-(4-(2,6-dichlorobenzyloxy)phenyl)
thiazole-5-carboxylic acid (11b). Yield: 60.0%. White solid. m.p.
259e261 ꢀC (dec). IR (KBr): 3175, 2958, 1681, 1603, 1536, 1495,
1436, 1324, 1286, 1248, 1148, 1094, 1010 cmꢃ1 1H-NMR (300 MHz,
5.1.2.3. Ethyl 2-(2,4-Dichlorobenzamido)-4-(4-(2,6-dichlorobenzy-
loxy)phenyl)thiazole-5-carboxylate (10c). Yield: 84.2%. White solid.
m.p. 177e183 ꢀC. IR(KBr): 1692, 1532, 1437, 1332, 1297, 1243, 1098,
DMSO-d6)：
d
5.30 (2H, s, CH2O), 7.13 (2H, d, J ¼ 8.7 Hz, ArH), 7.49
(1H, m, ArH), 7.62 (4H, m, ArH), 7.79 (2H, d, J ¼ 9.0 Hz, ArH), 8.15
(2H, d, J ¼ 8.7 Hz, ArH), 13.11(2H, s, NH, COOH). EI-MS (m/z): 533
(M ꢃ H)þ. Formula: C24H15Cl3N2O4S. Anal (C%, H%, N%) Calcd: 54.00,
2.83, 5.25. Found: 53.66, 2.64, 5.03.
1006 cmꢃ1 1H-NMR (300 MHz, DMSO-d6):
d
1.26 (3H, t, J ¼ 7.2 Hz,
CH3), 4.24 (2H, q, J ¼ 7.2 Hz, COOCH2), 5.30 (2H, s, CH2O), 7.13 (2H, d,
J ¼ 8.7 Hz, ArH), 7.49 (1H, m, ArH), 7.59 (3H, m, ArH), 7.75 (3H, m,
ArH), 7.82 (1H, s, ArH), 13.32 (1H, s, NH). EI-MS (m/z): 594 (M)þ.
Formula: C26H18Cl4N2O4S$0.5H2O. Anal (C%, H%, N%) Calcd: 51.59,
3.16, 4.63. Found: 51.27, 3.39, 4.52.
5.1.3.3. 2-(2,4-Dichlorobenzamido)-4-(4-(2,6-dichlorobenzyloxy)
phenyl)thiazole-5-carboxylic acid (11c). Yield: 73.9%. white solid.
m.p. 212e215 ꢀC. IR (KBr): 3417, 3187, 1708, 1671, 1531，1494, 1436,
5.1.2.4. Ethyl
4-(4-(2,6-Dichlorobenzyloxy)phenyl)-2-(4-methoxy-
1249, 1147, 1093, 1003 cmꢃ1 1H-NMR (300 MHz, DMSO-d6):
d 5.29
benzamido)thiazole-5-carboxylate (10d). Yield: 71.9%. White solid.
(2H, s, CH2O), 7.11 (2H, d, J ¼ 8.7 Hz, ArH), 7.49 (1H, m, ArH), 7.59
(3H, m, ArH), 7.77 (4H, m, ArH), 13.16 (2H, s, NH, COOH). EI-MS (m/
z): 567 (M ꢃ H)þ. Formula: C24H14Cl4N2O4S$0.5H2O. Anal (C%, H%, N
%) Calcd: 49.94, 2.62, 4.85. Found: 49.72, 2.97, 4.88.
m.p. 239e242 ꢀC (dec). IR (KBr): 3323, 1660, 1437, 1326, 1247, 1186,
1083, 1003 cmꢃ1 1H-NMR (300 MHz, DMSO-d6):
d 1.26 (3H, t,
J ¼ 7.2 Hz, CH2CH3), 3.86 (3H, s, OCH3), 4.23 (2H, q, J ¼ 7.2 Hz,
COOCH2), 5.30 (2H, s, CH2O), 7.12 (4H, m, ArH), 7.49 (1H, m, ArH),
7.59 (2H, d, J ¼ 9.0 Hz, ArH), 7.78 (2H, d, J ¼ 8.4 Hz, ArH), 8.15 (2H, d,
J ¼ 8.7 Hz, ArH), 12.98 (1H, s, NH). EI-MS (m/z): 556 (M)þ. Formula:
C27H22Cl2N2O5S$CH3OH. Anal (C%, H%, N%) Calcd: 57.05, 4.45, 4.75.
Found: 57.42, 4.23, 4.31.
5.1.3.4. 4-(4-(2,6-Dichlorobenzyloxy)phenyl)-2-(4-methoxybenzam
ido)thiazole-5-carboxylic acid (11d). Yield: 64.3%. White solid. m.p.
247e250 ꢀC. IR (KBr): 3279, 2944,1670,1607,1512,1437,1336,1249,
1179, 1147, 1090, 1020 cmꢃ1 1H-NMR (300 MHz, DMSO-d6):
d 3.86
(3H, s, OCH3), 5.30 (2H, s, CH2O), 7.11 (4H, m, ArH), 7.49 (1H, m,
ArH), 7.59 (2H, d, J ¼ 7.5 Hz, ArH), 7.79 (2H, d, J ¼ 8.4 Hz, ArH), 8.15
(2H, d, J ¼ 8.7 Hz, ArH), 12.89 (2H, s, NH, COOH). EI-MS (m/z): 527
(M ꢃ H)þ. Formula: C25H18Cl2N2O5S$CH3OH. Anal (C%, H%, N%)
Calcd: 55.62, 3.95, 4.99. Found: 55.42, 3.54, 4.57.
5.1.2.5. Ethyl 2-butyramido-4-(4-(2,6-dichlorobenzyloxy)phenyl)thia-
zole-5-carboxylate (10e). Yield: 65.0%. White solid. m.p.178e182 ꢀC. IR
(KBr): 3262, 1693, 1670, 1530, 1488, 1437, 1372, 1327, 1240, 1152, 1094,
1007 cmꢃ1 1H-NMR (300 MHz, DMSO-d6)：
d
0.91 (3H, t, J ¼ 7.2 Hz,
CH2CH2CH3), 1.24 (3H, t, J ¼ 7.2 Hz, COOCH2CH3), 1.64 (2H, m,
J ¼ 7.2 Hz, CH2CH2CH3), 2.47 (2H, t, CH2CH2CH3), 4.21 (2H, q, J ¼ 7.2 Hz,
COOCH2), 5.29 (2H, s, CH2O), 7.12 (2H, d, J ¼ 8.7 Hz, ArH), 7.49 (1H, m,
ArH), 7.60 (2H, d, J ¼ 7.5 Hz, ArH), 7.74 (2H, d, J ¼ 8.7 Hz, ArH), 12.64
(1H, s, NH). EI-MS (m/z): 492 (M)þ. Formula: C23H22Cl2N2O4S. Anal (C
%, H%, N%) Calcd: 55.99, 4.49, 5.68. Found: 55.97, 4.39, 5.44.
5.1.3.5. 2-Butyramido-4-(4-(2,6-dichlorobenzyloxy)phenyl)thiazole-
5-carboxylic acid (11e). To a mixture of 9 (0.05 g, 0.13 mmol), DMAP
(0.05 g, 0.4 mmol) and dry THF (10 mL), the solution of butyric
anhydride (0.16 g, 1 mmol) in dry THF (10 mL) was added, while
keeping the temperature at 0 ꢀC. The whole mixture was stirred for
10 h at room temperature. Water was added to the mixture and
extracted with EtOAc (20 mL ꢂ 3). The combined organic layers
were washed with brine and dried with Na2SO4, then concentrated
in vacuo. Purification of the crude products by column chroma-
tography eluting with petroleum-ethyl acetate (2:1) gave 11e
a white solid (0.05 g, 84.7%). m.p. 255e258 ꢀC. IR (KBr): 3414, 3160,
2961, 1699, 1573, 1533, 1438, 1326, 1246, 1178, 1141, 1092, 1013 cmꢃ1
5.1.2.6. Ethyl
2-acrylamido-4-(4-(2,6-dichlorobenzyloxy)phenyl)
thiazole-5-carboxylate (10f). Yield: 12.2%. m.p. 200e203 ꢀC. IR
(KBr): 3237, 3190, 1683, 1657, 1609, 1533, 1436, 1329, 1243, 1175,
1088, 1009 cmꢃ1 1H-NMR (300 MHz, CDCl3):
d
1.34 (3H, t, J ¼ 7.2 Hz,
CH3), 4.31 (2H, q, J ¼ 7.2 Hz, COOCH2), 5.30 (2H,s, CH2O), 5.55 (2H,
m, CH]CH2), 6.36 (1H, m, CH]CH2), 7.07 (2H, d, J ¼ 8.7 Hz, ArH),
7.24 (1H, m, ArH), 7.39 (2H, d, J ¼ 7.2 Hz, ArH), 7.76 (2H, d, J ¼ 8.7 Hz,
ArH), 11.24 (1H, s, NH). EI-MS (m/z): 476 (M)þ. Formula:
C22H18Cl2N2O4S$0.25 C3H8O2. Anal (C%, H%, N%) Calcd: 55.32, 4.04,
5.61. Found: 55.78, 4.34, 5.51.
1H-NMR (300 MHz, DMSO-d6):
d
0.91(3H, t, J ¼ 7.2 Hz, CH2CH2CH3),
1.64 (2H, m, J ¼ 7.2 Hz, CH2CH2CH3), 2.46 (2H, t, CH2CH2CH3), 5.29
(2H, s, CH2O), 7.11 (2H, d, J ¼ 8.7 Hz, ArH), 7.49 (1H, m, ArH), 7.59
(2H, d, J ¼ 7.5 Hz, ArH), 7.74 (2H, d, J ¼ 8.7 Hz, ArH), 12.51 (1H, s,

X. Lu et al. / European Journal of Medicinal Chemistry 49 (2012) 164e171
169
COOH), 12.89 (1H, s, NH). EI-MS (m/z): 463 (M ꢃ H)þ. Formula:
C21H18Cl2N2O4S. Anal (C%, H%, N%) Calcd: 54.20, H 3.90, 6.02. Found:
54.09, 3.76, 5.93.
d
1.22e1.27 (t, J ¼ 7.2 Hz, 3H, CH3), 2.29 (s, 3H, CH3), 4.31e4.33 (q,
J ¼ 7.2 Hz, 2H, CH2), 7.25e7.28 (d, J ¼ 8.4 Hz, 2H), 7.57e7.60 (d,
J ¼ 8.4 Hz, 2H).
5.1.4. The procedure for synthesis of compounds 20 and 21
5.1.4.6. Ethyl 5-amino-2-(4-(2,6-dichlorobenzyloxy)phenyl)oxazole-
4-carboxylate (19). To a stirred solution of 18 (4 g, 0.016 mol) in 1-
methyl-2-pyrrolidinone (60 mL) were added a solution of 15 in 1-
methyl-2-pyrrolidinone (30 mL) at room temperature. The reac-
tion mixture was stirred at 23e25 ꢀC overnight (12 he18 h) and
then diluted with ethyl acetate (100 mL). This solution was washed
with water (2 ꢂ 100 mL) and then with 10% sodium hydrogen
carbonate. The organic layer was dried, the solvent was evaporated
in vacua. Purification of the crude products by column chroma-
tography eluting with petroleum-ethyl acetate (2:1) gave 19 (1.5 g,
23.43%) as a white solid. Mp 198e200 ꢀC. 1H-NMR (300 MHz,
CDCl3): 1.25e1.30 (t, J ¼ 7.2 Hz, 3H, CH3), 4.17e4.25 (q, J ¼ 7.2 Hz,
2H, CH2), 5.29 (s, 2H, CH2), 7.17e7.20 (d, J ¼ 8.7 Hz, 2H), 7.37(s, 2H,
NH2), 7.48e7.51 (t, J ¼ 8.4 Hz, 1H, Ar), 7.57e7.60 (d, J ¼ 7.2 Hz, 2H,
Ar), 7.74e7.76 (d, J ¼ 8.7 Hz, 2H); EI-MS (m/z): 406 (M)þ.
5.1.4.1. Ethyl 4-(2,6-dichlorobenzyloxy)benzoate (13). To a solution
of ethyl 4-hydroxybenzoate 12 (5.81 g, 0.035 mol) and K2CO3 (9.6 g,
0.07 mol) in acetone (80 mL), 3 (6.86 g, 0.035 mol) was added. The
suspension was stirred vigorously, and heated to reflux for 24 h. On
cooling the reaction mixture down to room temperature, the
suspension was concentrated and diluted with water (100 mL) and
then extracted with AcOEt (2 ꢂ 100 mL). The organic layers was
washed with 10% NaOH solution (100 mL), dried with Na2SO4 and
then concentrated in vacuo to afford 13 as a white solid (10.52 g,
92.20%). Mp 84e85 ꢀC. 1H-NMR (300 MHz, CDCl3):
d 1.36e1.40 (t,
J ¼ 7.2 Hz, 3H, CH3), 4.32e4.39 (q, J ¼ 7.2 Hz, 2H, CH2), 5.32 (s, 2H,
CH2), 7.02e7.05 (d, J ¼ 8.7 Hz, 2H, Ar), 7.23e7.28 (t, J ¼ 7.2 Hz, 1H,
Ar), 7.36e7.38 (d, J ¼ 7.2 Hz, 2H, Ar), 8.02e8.04 (d, J ¼ 8.7 Hz, 2H,
Ar); EI-MS (m/z): 324 (M)þ.
5.1.4.2. 4-(2,6-Dichlorobenzyloxy)benzoic acid (14). 13 (10.5 g,
0.032 mol) was dissolved in EtOH (80 mL) and water (10 mL).
Potassium hydroxide (6.4 g, 0.16 mol) was added and the resulting
mixture heated to reflux for 2 h. After cooling, the reaction mixture
was diluted with water (300 mL) and acidified by HCl to pH 4e5.
The solid was filtered, the filtrate washed with water and dried
overnight to yield 14 as a whitþe crystal (8.53 g, 89.78%). Mp
194e196 ꢀC. EI-MS (m/z): 296 (M) .
5.1.4.7. Ethyl 5-(4-Chlorobenzamido)-2-(4-(2,6-dichlorobenzyloxy)
phenyl)oxazole-4-carboxylate (20). To a mixture of 19 (0.4 g,
1 mmol), dry Et3N (3 mL) and dry CH2Cl2 (20 mL), 4-chlorobenzoyl
chloride (0.34 g, 0.002 mol) was added, while keeping the
temperature at 0 ꢀC. The whole mixture was stirred for 12 h at
room temperature. Water was added to the mixture and extracted
with dichloromethane (15 mL ꢂ 2). The combined organic layers
were washed with brine and dried with Na2SO4, then concentrated
in vacuo. Purification of the crude products by column chroma-
tography eluting with petroleum-ethyl acetate (8:1) gave 20
(0.21 g, 37.04%) as a yellow solid. m.p. 207e209 ꢀC. IR (KBr, cmꢃ1):
3125, 1705 (CO), 1620 (eCONH), 1493, 1317, 1262(eCeOeCe), 1101,
1004, 842, 779; 1H-NMR (300 MHz, CDCl3): 1.44e1.49 (t, J ¼ 7.2 Hz,
3H, CH3), 4.45e4.52 (q, J ¼ 7.2 Hz, 2H, CH2), 5.34 (s, 2H, CH2),
7.08e7.11 (d, J ¼ 9.0 Hz, 2H, Ar), 7.24e7.29 (t, J ¼ 8.4 Hz, 1H, Ar),
7.37e7.39 (d, J ¼ 8.4 Hz, 2H, Ar), 7.50e7.52 (d, J ¼ 8.4 Hz, 2H, Ar),
7.92e7.95 (d, J ¼ 8.4 Hz, 2H, Ar), 8.09e8.12 (d, J ¼ 9.0 Hz, 2H, Ar),
10.22 (s, 1H, NHCO); EI-MS (m/z): 544 (M)þ. Formula:
C26H19Cl3N2O5$2H2O; Anal (C%, H%, N%) 53.67, 3.98, 4.81; Calcd:
53.72, 3.84, 5.03.
5.1.4.3. 4-(2,6-Dichlorobenzyloxy)benzoyl chloride (15). 14 (4.87 g,
0.016 mol) was dissolved in dry dichloromethane (60 mL). Then
thionyl chloride (5.5 mL, 0.065 mol) and DMF (2 drops) were added
to the mixture. The reaction mixture heated to reflux for 5 h and
concentrated to give yellow solid 15 used for the next step without
further purification.
5.1.4.4. Cyano-hydroxyimino-acetic acid ethyl eater (17). A solution
of sodium nitrite (5.73 g, 0.083 mol) in water (71 mL) was treated
with ethyl cyanoacetate 16 (10 g, 0.083 mol). 85% orthiphosphoric
acid (3.66 mL, 0.055 mol) was added dropwise, while keeping the
temperature of the reaction mixture below 10 ꢀC with the aid of an
ice bath. At the end of the addition, the mixture was warmed to
40 ꢀC and stirred for 1 h. The reaction was quenched at 45 ꢀC with
fuming HCl (7.4 mL, 0.088 mol), and the mixture was then left to
cool to room temperature and at 0 ꢀC overnight to complete
precipitation. The solid was filtered, the filtrate washed with water
and dried under high vacuum overnight to yield 17 as white crystals
(7.73 g, 61.45%). Mp 126e127 ꢀC; 1H-NMR (300 MHz, CDCl3):
5.1.4.8. 5-(4-Chlorobenzamido)-2-(4-(2,6-dichlorobenzyloxy)phenyl)
oxazole-4-carboxylic acid (21). To a solution of 20 (103 mg,
0.18 mmol) in EtOH (20 mL) was added 1 N NaOH (2.2 mL,
2.3 mmol). The reaction mixture was heated to reflux for 2 h. After
cooling to room temperature, the reaction mixture was poured into
water (50 mL) followed with 1 N HCl to pH 4e5. The suspension
solution was extracted with AcOEt (2 ꢂ 25 mL). The combined
organic layers was dried with Na2SO4 and concentrated in vacuo
to afford crude product. Recrystallization from AcOEt gave
21 (0.05 g, 53.76%) as a yellow solid. m.p. 191e194 ꢀC. IR (KBr,
cmꢃ1): 3364e3520 (eOH), 1709 (CO), 1625 (eCONH), 1252,
1137(eCeOeCe), 1001, 840, 771, 624; 1H-NMR (300 MHz, DMSO):
5.33 (s, 2H, CH2), 7.24e7.27 (d, J ¼ 9.0 Hz, 2H, Ar), 7.50e7.52 (t,
J ¼ 8.4 Hz, 1H, Ar), 7.58e7.60 (d, J ¼ 8.4 Hz, 2H, Ar), 7.64e7.66 (d,
J ¼ 8.4 Hz, 2H, Ar), 7.94e7.96 (d, J ¼ 8.4 Hz, 2H, Ar), 8.00e8.03 (d,
J ¼ 9.0 Hz, 2H, Ar); EI-MS (m/z): 515 (M ꢃ 1)þ. Formula:
C24H15Cl3N2O5. Anal (C%, H%, N%) Calcd: 55.68, 2.92, 5.41. Found:
55.28, 2.97, 5.10.
d
1.39e1.44 (t, J ¼ 7.2 Hz, 3H, CH3), 4.41e4.49 (q, J ¼ 7.2 Hz, 2H, CH2),
10.49 (s, 1H, OH); EI-MS (m/z): 142 (M)þ.
5.1.4.5. 1-(Cyano-2-ethoxy)-2-oxoethanominium 4-toluenesulfonate
(18). 17 (4.3 g, 0.03 mol) was dissolved in water (43 mL) and
sat. aq NaHCO3 solution (35 mL). Na2S2O4 (15.6 g, 0.09 mol) was
added and the resulting mixture stirred at 35 ꢀC for 30 min and
then stirred at room temperature for 3 h. The mixture was satu-
rated with NaCl (20 g), extracted with CH2Cl2 (4 ꢂ 30 mL), the
combined extracts dried (Na2SO4) and concentrated to give ethyl
aminocyanoacetate as a red oil (1.0 g, 26.32%). EI-MS (m/z): 127
(M)þ.The crude product was dissolved in Et2O (20 mL) and the
solution treated with TsOH$H2O (1.45 g, 0.0079 mol). After the
addition of THF (10 mL) and cooling, the precipitate was removed
by filtration, washed with cold Et2O (20 mL), and dried under
reduced pressure to provide 18 as a yellow solid (1.86 g, 93.47%).
Mp 128e130 ꢀC (lit [16]: 128e130 ꢀC). 1H-NMR (300 MHz, D2O):
5.1.5. The procedure for synthesis of compounds 26 and 27
5.1.5.1. N-(4-(2,6-dichlorobenzyloxy)phenyl)acetamide (23). To a solu-
tion of 4-acetamino phenol 22 (4.9 g, 0.03 mol) and K2CO3 (8.28 g,
0.06 mol) in acetone (80 mL), 3 (6.46 g, 0.033 mol) was added. The
mixture was stirred vigorously, and heated to reflux for 24 h. On

170
X. Lu et al. / European Journal of Medicinal Chemistry 49 (2012) 164e171
cooling the reaction mixture down to room temperature, the mixture
was concentrated and diluted with water (100 mL) and then
extracted with EtOAc (2 ꢂ 100 mL). The organic layers was washed
with 10% NaOH solution (100 mL), dried with Na2SO4 and then
concentrated in vacuo to afford 23 (10.52 g, 92.20%) as a white solid.
m.p. 185e187 ꢀC. EI-MS (m/z): 309 (M)þ.
NMR (300 MHz, DMSO-d6): 6518, 115.20, 126.18, 127.78, 128.60,
128.76, 129.62, 131.25, 131.69, 131.79, 136.02, 136.98, 158.33, 163.33,
165.83, 200.39, 204.28. EI-MS (m/z): 514 (M ꢃ 1)þ. Formula:
C26H19Cl2NO5S. Anal (C%, H%, N%) Calcd: 55.78, 3.12, 8.13. Found:
55.85, 3.65, 7.71.
5.2. Biological assay
5.1.5.2. 4-(2,6-Dichlorobenzyloxy)aniline (24). To a solution of 23
(5.01 g, 0.016 mol) in 90% C2H5OH, potassium hydroxide (5.6 g,
0.1 mol) was added. The mixture heated to reflux for 16 h and
concentrated in vacuo. Purification of the crude products by column
chromatography eluting with petroleum-ethyl acetate (4:1) gave
24 (4 g, 93.46%) as red oil. EI-MS (m/z): 267 (M)þ.
5.2.1. Anti-tubercular activity against H37RV (MABA)
Briefly, the test compound MICs against TB were assessed by the
MABA using INH, Moxifloxacin and PA824 as positive controls.
Compound stock solutions were prepared in DMSO at a concen-
tration of 12.8 mM, and the final test concentrations ranged from
128 to 0.5
Middlebrook 7H12 medium (7H9 broth containing 0.1% w/v casi-
tone, 5.6 g/mL palmitic acid, 5 mg/mL bovine serum albumin,
4 mg/mL catalase, filter-sterilized) in a volume of 100 L in 96-well
microplates (black viewplates). M. tuberculosis H37RV (100
inoculum of 2 ꢂ 105 cfu/mL) was added, yielding a final testing
volume of 200
L. The plates were incubated at 37 ꢀC. On the 7th
day of incubation 12.5 L of 20% Tween 80 and 20 L of Alamar Blue
(Trek Diagnostic, Westlake, OH) were added to the test plate. After
incubation at 37 ꢀC for 16e24 h, fluorescence of the wells was
measured (ex 530, em 590 nm). The MICs ware defined as the
lowest concentration effecting a reduction in fluorescence of ꢅ90%
relative to the mean of replicate bacteria-only controls.
mM. Two-fold dilutions of compounds were prepared in
5.1.5.3. Ethyl 5-amino-1-(4-(2,6-dichlorobenzyloxy)phenyl)-1H-imid-
azole-4-carboxylate (25). To
a solution of ethyl 2-amino-2-
m
cyanoacetate (1.27 g, 0.01 mol) in MeCN was added triethylortho-
formate (1.48 g, 0.01 mol) and the resulting solution heated to 90 ꢀC.
After 45 min the reaction solution was cooled to room temperature
and a solution of 4-(2,6-dichlorobenzyloxy)aniline (2.68 g, 0.01 mol)
in MeCN was added. The reaction was stirred at room temperature
for 3 h. The solid was filtered, the filtrate recrystallized from EtOH
and dried under high vacuum overnight to yield 25 as white crystals
(2.32 g, 57.14%). m.p. 218e220 ꢀC. 1H-NMR (300 MHz, CDCl3):
1.39e1.44 (t, 3H, J ¼ 7.2 Hz, CH3), 4.34e4.41 (q, 2H, J ¼ 7.2 Hz, CH2),
5.33 (s, 2H, CH2), 7.07e7.10 (d, 2H, J ¼ 9.0 Hz, Ar), 7.24e7.27 (t,
J ¼ 7.8 Hz, 1H, Ar), 7.27e7.31 (d, J ¼ 9.0 Hz, 2H, Ar), 7.37e7.40 (d,
J ¼ 7.8 Hz, 2H, Ar), 7.55 (s,1H, H2-imidazole); EI-MS (m/z): 405 (M)þ.
m
mL
m
m
m
5.2.2. Antibacterial activity against clinical strains
Agar dilution method was used to test the MIC of the test
compounds against four S. aureus, E. coli, S. pneumoniae and pen-
icilin-resistant S. pneumoniae, which was performed as described
previously [20]. Briefly, a series of two-fold dilutions of the test
compounds, ampicillin and gentamicin were prepared in molten
Mueller-Hinton Agar (MHA) media, and kept at 37 ꢀC to obtain the
desired final concentrations. Media with concentrations ranging
5.1.5.4. Ethyl 5-(4-chlorobenzamido)-1-(4-(2,6-dichlorobenzyloxy)
phenyl)-1H-imidazole-4-carboxylate (26). To a mixture of 25 (0.4 g,
1 mmol), dry Et3N (3 mL) and dry CH2Cl2 (20 mL), 4-chlorobenzoyl
chloride (0.34 g, 2 mmol) was added, while keeping the tempera-
ture at 0 ꢀC. The whole mixture was stirred for 12 h at room
temperature. Water was added to the mixture and extracted with
CH2Cl2 (15 mL ꢂ 2). The combined organic layers were washed with
brine and dried with Na2SO4, then concentrated in vacuo. Purifi-
cation of the crude products by column chromatography eluting
with petroleum-ethyl acetate (4:1) gave 26 (0.23 g, 42.28%) as
a white solid. m.p. 215e216 ꢀC. IR (KBr, cmꢃ1): 3549, 3415, 1709
(COOEt), 1591, 1513, 1393, 1244 (eCeOeCe), 1099, 1006, 849; 1H-
NMR (300 MHz, CDCl3): 1.13e1.18 (t, 3H, J ¼ 7.2 Hz, CH3), 4.17e4.22
(q, 2H, J ¼ 7.2 Hz, CH2), 5.24 (s, 2H, CH2), 7.17e7.20 (d, 2H, J ¼ 9 Hz),
7.40e7.43 (d, 2H, J ¼ 9 Hz), 7.46e7.49 (t, 1H, J ¼ 6.9 Hz, Ar),
7.54e7.57 (d, 2H, J ¼ 6.9 Hz, Ar), 7.57e7.60 (d, 2H, J ¼ 8.4 Hz),
7.86e7.89 (d, 2H, J ¼ 8.4 Hz), 8.01 (s, 1H, H2-imidazole); EI-MS (m/
z): 543 (M)þ. Formula: C26H20Cl3N3O4$0.5H2O; Anal (C%, H%, N%)
Calcd: 56.39, 3.82, 7.59. Found: 56.36, 3.77, 7.47.
from 1280 to 0.125 mg/mL was obtained, and media without any
drugs was made as control. Plates were inoculated with 1e2 mL
culture containing 104 CFU/mL of the appropriate microorganism,
and the plates were incubated at 37 ꢀC. The MICs were the lowest
concentration in solid media at which no growth was observed
after 16e20 h.
Acknowledgements
The authors gratefully acknowledged financial support from the
“ Significant new drugs development” Science and Technology
Major Projects of China (No 2009ZX09501-003) and the Innovation
Program for the Postgraduates in China Pharmaceutical University,
P.R.China. We also thank CompuDrug International Inc. for allowing
us to use the Pallas software.
5.1.5.5. 5-(4-Chlorobenzamido)-1-(4-(2,6-dichlorobenzyloxy)phenyl)
-1H-imidazole-4-carboxylic acid (27). To a solution of 26 (157 mg,
0.29 mmol) in EtOH (20 mL) was added 1 N NaOH (3.6 mL,
3.7 mmol). The reaction mixture was heated to reflux for 2 h. After
cooling to room temperature, the reaction mixture was poured into
water (50 mL) followed with 1 N HCl to pH 4e5. The suspension
solution was extracted with AcOEt (2 x 25 mL). The combined
organic layers was dried with Na2SO4 and concentrated in vacuo
to afford crude product. Recrystallization from CH2Cl2 gave 27
(0.08 g, 53.33%) as a white solid. m.p. 165e167 ꢀC. IR (KBr, cmꢃ1):
3499e3445 (eOH), 3109, 1689 (CO), 1594 (eCONH), 1513, 1244
(eCeOeCe), 1095, 1009, 841, 767; 1H-NMR (300 MHz, DMSO-d6):
5.24 (s, 2H, CH2), 7.17e7.20 (d, 2H, J ¼ 9 Hz), 7.40e7.43 (d, 2H,
J ¼ 9 Hz), 7.46e7.49 (t, 1H, J ¼ 6.9 Hz, Ar), 7.54e7.57 (d, 2H,
J ¼ 6.9 Hz, Ar), 7.57e7.60 (d, 2H, J ¼ 8.4 Hz), 7.86e7.89 (d, 2H,
J ¼ 8.4 Hz), 8.01 (s, 1H, H2-imidazole), 10.34 (br, 1H, COOH); 13C
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