Design, synthesis and anti-tubercular evaluation of new 2-acylated and 2-alkylated amino-5-(4-(benzyloxy)phenyl)thiophene-3-carboxylic acid derivatives. Part 1

Article abstract of DOI:10.1016/j.ejmech.2011.05.018

A series of 2-acylated and 2-alkylated amino-5-(4-(benzyloxy)phenyl) thiophene-3-carboxylic acid derivatives were synthesized and evaluated for anti-tubercular activity. Among these compounds, 10d, 15, 12h and 12k inhibited Mycobacterium tuberculosis (Mtb

Full text of DOI:10.1016/j.ejmech.2011.05.018

European Journal of Medicinal Chemistry 46 (2011) 3551e3563
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and anti-tubercular evaluation of new 2-acylated and
2-alkylated amino-5-(4-(benzyloxy)phenyl)thiophene-3-carboxylic acid
derivatives. Part 1
,
,
Xiaoyun Lu a b, Baojie Wan c, Scott G. Franzblau c, Qidong You a
*
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of 2-acylated and 2-alkylated amino-5-(4-(benzyloxy)phenyl)thiophene-3-carboxylic acid
Received 11 March 2011
Received in revised form
3 May 2011
Accepted 9 May 2011
Available online 19 May 2011
derivatives were synthesized and evaluated for anti-tubercular activity. Among these compounds, 10d,
15, 12h and 12k inhibited Mycobacterium tuberculosis (Mtb) growth with MIC values between 1.9 and
7.7 mM and low toxicity against VERO cells. The four compounds were also tested against multidrug-
resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) clinical strains,
which were found to show moderate activity. In addition, molecular docking simulation was performed
to position compounds 10d, 15, 12h and 12k into mtFabH active site to predict the probable binding
mode. These studies thus suggest that the designed 2-amino-5-phenylthiophene-3-carboxylic acid
scaffold may serve as new promising template for further elaboration as anti-TB drugs.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
2-acylated and 2-alkylated amino-5-(4-
(benzyloxy)phenyl)thiophene-3-carboxylic
acid derivatives
Anti-tubercular activity
MDR-TB
XDR-TB
1. Introduction
develop novel anti-tubercular drugs that can be equally effective
against Mtb and MDR-TB, and reduce the duration of therapy.
Tuberculosis (TB), a highly contagious and air-borne disease
caused by Mycobacterium tuberculosis (Mtb), emerged with multi-
drug-resistant strains (MDR-TB) [1] and acquired immune defi-
ciency syndrome (AIDS) in recent years [2]. According to World
Health Organization (WHO) data (2009) [3], one-third of the
world’s population are infected with Mtb and approximately 1.7
million deaths occured worldwide in 2007. In spite of the
increasing worldwide incidence of TB and its alarming threat
toward the public health, no new drugs have actually been brought
to the market over the past four decades. Short-course chemo-
therapy for TB-infected patients is based on the combined use of
multiple chemotherapeutics including isoniazid (INH), rifampin
(RIF), pyrazinamide (PZA), streptomycin (SM) and ethambutol
(EMB), over a period of 6e10 months in accordance with WHO’s TB
treatment guidelines [4]. Therefore, it is an imperative need to
Thiolactomycin (TLM, 1, Fig. 1) was the first example of a natu-
rally occurring thiolactone to exhibit antibiotic activity [5e7]. It is
a unique molecule that exhibits selective activity against the
dissociable type II fatty acid synthase (FAS-II) only. TLM inhibits
Mtb FAS-II by targeting
b-ketoacyl-ACP synthase (mtFabH and
mtFabB), in vitro and in vivo, leading to inhibition of cell wall
mycolic acid biosynthesis and subsequently to Mtb cell death
[8e10]. The characteristics of low toxicity in mice [11] and ideal
drug-likeness [12] of TLM make it an attractive lead molecule for
TB. Previous studies have shown that TLM analogues exhibit good
activity against mtFabH by chemical modifications of the C5 posi-
tion isoprenoid side of TLM [13e16] (1a and 1b, Fig. 1) and the C4
position [17], but limited improvements in activity against Mtb
compared to TLM. Further, the studies [15,18,19] have revealed that
the (R)-configuration at the C5 position and the free-rotational
isoprenoid 1,3-diene moiety of TLM are essential for activity.
However, the chiral quaternary carbon at C5 position makes the
chemical synthetic route to TLM analogues difficultly and costly
[20,21], which becomes one of the problems in this area [22]. In this
* Corresponding author. Tel./fax: þ86 25 83271351.
E-mail address: youqidong@gmail.com (Q. You).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.018

3552
X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
O
by systematic structural modifications on acylated (R1) (10aek) and
S
O
S
S
O
alkylated (R2) (12aej) substituents to explore the anti-tubercular
activity against Mtb. Besides, with the specific aim of study of the
structure activity relationship (SAR) of 2,6-dichlorobenzyloxy
group, we used 2-chloro and 3,4-dichlorobenzyloxy group (10l
and 10m) instead of 2,6-dichlorobenzyloxy group in the amide class
of compound (10d). In order to identify compounds with better
potency, and reasonable physicochemical parameter (LogP), incor-
poration of R1 and R2 substituents (15 and 12k, respectively) con-
taining an acidic group was made to increase aqueous solubility.
Biological evaluation indicated that compounds 10d,15,12h and 12k
displayed not only remarkable anti-tubercular activity against Mtb
H37Rv, but also favorable activity against MDR-TB and XDR-TB
clinical strains. In addition, molecular docking simulation was per-
formed to position compounds 10d, 15, 12h and 12k into mtFabH
active site to predict the probable binding mode, which would play
an important help in the design of new potent compounds.
OHC
HO
1b
HO
1a
OH
HOOC
1
Cl
O
O
OH
COOH
Cl
N
NH2
S
O
O
Cl
2
3
Fig. 1. The chemical structures of TLM (1), TLM analogues (1a and 1b), 2-amino-5-
phenylthiophene-3-carboxylic acid (2), and a FabH inhibitor (3).
regard, we have focused on the synthesis of alternative and easily
accessible five membered heterocyclic isosteres to investigate the
inhibitory activity against Mtb. Thiophene nucleus represents
a very important field in drug discovery because of its varied
biological activities [23], more recently found application in
development of antibacterial agents [24,25]. Herein, we have
identified 2-amino-5-phenylthiophene-3-carboxylic acid (2, Fig. 1)
as a promising scaffold for the development of new anti-tubercular
agents. According to the reported binding mode of the known FabH
inhibitor (3, Fig. 1) [26], the 2,6-dichlorobenzyloxy group was
selected to extend to the slender hydrophobic pocket directing the
Cys112-His244-Asn274 catalytic triad of mtFabH [27,28]. Moreover,
Botta et al. [29,30] suggested that the p-chlorobenzoyl moiety
might produce a good anti-tubercular activity.
2. Chemistry
2.1. Synthesis of amides derivatives (10aen)
Compounds 10aen possessing various amides were prepared
using the methods outlined in Scheme 1. The synthesis was started
with reaction of commercially available substituted benzyl chlo-
rides 4aec with 4-hydroxyphenethyl alcohol 5 to obtain the cor-
responding alcohols 6aec in good yield (91%). As for the next
oxidation step, alcohol 6a was originally oxidized by PCC in dry
CH2Cl2 at room temperature. However, we got only a trace amount
aldehyde 7a due to PCC-mediated carbonecarbon bond cleavage
reaction giving 4-(2,6-dichlorobenzyloxy)benzaldehyde [31].
Based on the above mentioned findings, we report in the present
work the design and synthesis of two series of 2-amino-5-(4-(2,6-
dichlorobenzyloxy)phenyl)thiophene-3-carboxylic acid derivatives
CH2CH2OH
b
CH2CH2OH
CH2Cl
a
+
R
O
R
HO
6a-c
4a: R = 2,6-Dichloro
4b: R = 2-Chloro
4c: R = 3,4-Dichloro
5
O
O
O
O
CH2CHO
O
d
c
NH2
R1
S
N
O
S
R
H
O
O
R
R
8a-c
9a-m
7a-c
8a: R = 2,6-Dichcloro
8b: R = 2-Chloro
8c: R = 3,4-Dichloro
e
f
O
O
O
OH
OH
N R2
O
g
O
N
R1
N R2
H
S
Cl
S
Cl
S
H
H
O
O
Cl
O
R
Cl
10a-m
12a-k
11a-k
9a, 10a: R = 2,6-Dichloro, R1 = Phenyl
11a, 12a: R2 = Benzyl
9b, 10b: R = 2,6-Dichloro, R1 = 2-Chlorophenyl
9c, 10c: R = 2,6-Dichloro, R1 = 3-Chlorophenyl
9d, 10d: R = 2,6-Dichloro, R1 = 4-Chlorophenyl
9e, 10e: R = 2,6-Dichloro, R1 = 2-Fluorophenyl
11b, 12b: R2 = 2-Chlorobenyl
11c, 12c: R2 = 4-Chlorobenyl
11d, 12d: R2 = n-Propyl
11e, 12e: R2 = Isobutyl
11f, 12f: R2 = Allyl
9f, 10f: R = 2,6-Dichloro, R1 = 2-Methoxyphenyl
9g, 10g: R = 2,6-Dichloro, R1 = 3-Methoxyphenyl
9h, 10h: R = 2,6-Dichloro, R1 = 4-Methoxyphenyl
9i, 10i: R = 2,6-Dichloro, R1 = 2-Pyridine
11g, 12g: R2 = 3-Methyl-2-butene
11h, 12h: R2 = n-Butyl
11i, 12i: R2 = n-Pentyl
9j, 10j: R = 2,6-Dichloro, R1 = 2-Furan
11j, 12j: R2 = n-Hexyl
9k, 10k: R = 2,6-Dichloro, R1 = 2-Thiophene
9l, 10l: R = 2-Chloro, R1 = 4-Chlorophenyl
9m, 10m: R =3,4-Chloro, R1 = 4-Chlorophenyl
11k, 12k: R2 = Carboxymethyl
Scheme 1. Reagents and conditions: (a) K2CO3, acetone, reflux, 24 h; 91% (b) TEMPO, NaBrO2, H2O-CH2Cl2, 0e5 ꢀC, 30 min; 92% (c) CNCH2COOEt, S, Et3N, absolute C2H5OH, 60 ꢀC,
overnight; 50% (d) R1COOH, SOCl2, reflux, 2 h, then R1COCl, Et3N, CH2Cl2, 0 ꢀC-rt, 12 h; 12e60% for two steps. (e) 1N NaOH, C2H5OH, reflux, 2 h; 27e74% (f) R2X, K2CO3, KI, DMF,
60 ꢀC, 24 h; 10e36% (g) 1N NaOH, C2H5OH, reflux, 2 h; 25e84%.

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
3553
Hence, we modified the method by the treatment of 6aec with
a catalytic amount of 2,2,6,6-tetramethylpiperidine- -oxyl (TEMPO)
keeping the 2,6-dichlorobenzyloxy group. For phenyl rings and
benzyl groups, introduction of halogen substituents (10aee and
12aec) was tolerated, 4-chloro yielding the best activity (4-Cl > 3-
Cl > H > 2-Cl). It has been found that amide derivatives (10aee)
have displayed superior anti-tubercular activity than amine deriv-
atives (12aec). In contrast, introduction of electron donating group
(OCH3) on the phenyl ring (10feh) had a detrimental effect on the
inhibitory activity compared to 10a. Compounds 10iek, incorpo-
rated a heterocycle, shown moderate activity against Mtb with
L
and 3 equiv of sodium bromite (NaBrO2) in a CH2Cl2-H2O (buffered
at PH 8.6e9.0) system [32] afforded aldehydes 7aec in excellent
yield (92%). The Gewald [33] thiophene synthesis constructed the
key intermediate amines 8aec by Knovenagel condensation of
aldehydes 7aec with ethyl cyanoacetate followed by cyclization
with elemental sulfur under basic condition. The derivatives 9aem
were synthesized from the freshly prepared acyl chloride in the
presence of Et3N in yields of 12e60%. After basic hydrolysis of
9aem in ethanol, the desired compounds 10aem were obtained in
moderate to good yields (27e74%).
MICs ranging from 27.4 to 59.2 mM. For alkyl groups, to investigate
the effect of length of alkyls on activity, seven compounds (12dej)
varying in length of attachment to nitrogen atom were designed as
displayed in Table 1. The data suggested that compounds with three
or four atom alkyls (12d, 12e and 12h) shown the best anti-
tubercular activity, while the potency decreased with the increase
of carbon chain (12i and 12j).
Next, with the above preliminary SAR in hand, the most potent
compound 10d was chosen as an interesting startingpoint forfurther
optimization. In order to study the SAR of 2,6-dichlorobenzyloxy
group, we used 2-chloro and 3,4-dichlorobenzyloxy group instead
of 2,6-dichlorobenzyloxy group to generate compound 10l and 10m.
Unfortunately, compound 10l was found to exhibit moderate activity
2.2. Synthesis of amide 15
Synthesis of compound 15 was started from the intermediate 8a
illustrated in Scheme 2. Amide bond formation was achieved with
ethyl chloroformylformate using basic acylating conditions to
afford 13 in good yield. Hydrolysis of 13 using the same method-
ology as that used in the preparation of 10aen from 9aen gave 14,
an amide bond fission product. Selective hydrolysis of one of the
ethyl esters in 13 was achieved with 1N NaOH in ethanol at room
temperature for 1 h to produce compound 15 in good yield.
(MIC ¼ 31.7
m
M) whilst compound 10m led to a complete loss of anti-
tubercular activity (MIC ¼ 110.2
mM). The results suggested that 2,6-
dichlorobenzyloxy group appears essential to retaining activity.
MTB are unique to surround by a thick and waxy cell wall.
Efficient antituberculosis drugs should have a reasonable lip-
ophilicity to penetrate the cell wall. The above newly synthesized
compounds (10aem and 12aej) are characterized by a calculated
logP within range from 5.26 to 7.28 (Table 1), which means that it is
unlikely to have good oral bioavailability by “Lipinski rule of five”
[36]. In order to increase aqueous solubility of compounds, an
acidic group in side chain was introduced. Thus, compounds 15 and
12k were prepared, which was the most potent anti-tubercular
2.3. Synthesis of amines derivatives (12aek)
Synthesis of the target compounds 12aek was also carried out
from the intermediate 8a as shown in Scheme 1. Treatment of 8a
with various aromatic and alkyl halides in N,N-dimethyl formamide
using K2CO3 as a base gave 11aek in moderate yields. The desired
compounds 12aek were synthesized from 11aek following the
same methodology as that used in the preparation of 10aem from
9aem in moderate to good yields (25e84%).
agent in the series, with excellent MICs of 1.9
mM and 3.7 mM,
3. Results and discussion
respectively. Compounds 15 and 12k possess clogP values of 5.08
and 4.23, respectively (see Table 1). Likely, these positive results are
a consequence of the good permeability of these compounds,
indicating that the good activities are in connection with an
acceptable lipophilic factor.
Moreover, the in vitro VERO cell toxicity of all compounds was
determined as previously described [35]. The cytotoxicity results
presented in Table 1 are expressed as the concentration inhibiting
50% of the cell growth (IC50). The studied compounds show
moderate cytotoxicity in comparison to the standard RIF with the
3.1. Antitubercular activity
All the final compounds along with the standard drugs for
comparison were firstly evaluated for their activity against the Mtb
strain H37Rv (ATCC #27294) and for the in vitro cytotoxicity against
VERO cells (Table 1) [34,35]. The compounds showing good anti-
tubercular activity and low toxicity were further evaluated
against MDR-TB and XDR-TB clinical strains (Table 2).
Several compounds were found to effectively inhibit the growth
of replicating Mtb by Microplate Alamar Blue Assay (MABA) with
low micromolar MICs (Table 1). In the first round of structural
modifications, the R1 and R2 substituents of two amide and amine
series with substituted phenyl ring (10aeh), heterocycle (10iek),
substituted benzyl group (12aec) and alkyl group (12dej) were
introduced to obtain the optimal substitutions pattern while
IC50 values in the range of 21.31e>128 mM. However, some potent
compounds (10c, 10d, 15) displayed the selectivity index (SI) value,
defined as a ratio of IC50 to MIC, higher than 10, which means that
they should be considered for further screening [37].
Finally, the four most active compounds in the series, 10d, 15,
12h and 12k, were evaluated against MDR-TB and XDR-TB clinical
O
OH
NH2
S
Cl
O
O
O
O
O
b
14
O
Cl
a
OEt
NH2
Cl
S
N
O
S
Cl
H
O
O
O
c
O
O
OH
8a
13
Cl
N
H
Cl
S
Cl
O
O
15
Cl
Scheme 2. Reagents and conditions: (a) ClCOCOOEt, Et3N, CH2Cl2, 0 ꢀC-rt; 73% (b) 1N NaOH, C2H5OH, reflux, 2 h; (c) 1N NaOH, C2H5OH, 0 ꢀC, 1 h; 68%.

3554
X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
Table 1
In vitro anti-tubercular activity and cytotoxicity of compounds 10aem, 15, and 12aek against Mtb H37Rv and their calculated clogP.
Compd
R
R1
MABA MIC (
m
M)
clogPa
IC50
(
mM)
SI
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
15
2,6-dichloro
2,6-dichloro
2,6-dichloro
2,6-dichloro
2,6-dichloro
2,6-dichloro
2,6-dichloro
2,6-dichloro
2,6-dichloro
2,6-dichloro
2,6-dichloro
2-chloro
Phenyl
10.6
96.0
6.6
5.88
6.07
6.30
6.17
6.22
6.03
6.07
6.13
5.78
5.66
5.26
5.93
6.35
5.08
85.74
88.56
89.78
53.81
28.64
50.66
62.13
>128
>128
91.73
8.1
0.9
13.6
14.5
0.6
<0.4
2.0
>2.0
>2.2
2.5
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2-fluorophenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2-pyridine
3.7
48.4
>128
31.2
>64
59.2
36.6
27.4
31.7
110.2
1.9
2-furan
2-thiophene
4-chlorophenyl
4-chlorophenyl
e
70.03
84.31
28.21
31.35
2.6
2.7
0.3
16.5
3,4-dichloro
e
Compd
R2
MABA MIC (
m
M)
clogPa
IC50
SIb
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
Isoniazid
Moxifloxacin
PA824
Rifampicin
Benzyl
2-chlorobenyl
4-chlorobenyl
n-propyl
Isobutyl
Allyl
3-methyl-2-butene
n-butyl
n-pentyl
n-hexyl
carboxymethyl
e
e
e
e
48.2
60.0
30.3
7.5
6.17
6.26
6.54
6.06
6.45
5.61
6.49
6.48
6.88
7.28
4.23
e
58.2
28.05
>128
21.31
34.5
27.53
>128
35.56
52.55
55.59
21.31
1.2
0.5
>4.2
2.8
4.4
1.9
>8.5
4.6
3.5
1.0
5.8
7.8
14.7
15.1
7.7
15.1
58.5
3.7
0.81
0.50
0.44
0.08
e
e
e
e
e
e
e
e
e
217.1
2713
a
The clogP values were calculated using Pallas software.
Selectivity Index (SI) ¼ IC50/MIC.
b
strains (Table 2). Compound 10d and 15 exhibited good activity
against XDR-TB strain with MICs of 12.0 M and 16.0 M, respec-
3.2. Molecular modeling
m
m
tively, suggesting this compound class holds promise as lead
compounds for treatment of drug-resistant TB.
From the abovein vitroresults, it may be concluded that 2-amino-
5-(4-(2,6-dichlorobenzyloxy)phenyl)thiophene-3-carboxylic acid
represents a reasonable starting scaffold in generating newchemical
entities possessing anti-tubercular properties. In an attempt to
understand the possible biological target for our compounds, we
investigated the binding pattern of 3 with the closely homology
Table 2
In vitro anti-tubercular activity of compounds 10d, 15, 12h and 12k against MDR-TB
and XDR-TB clinical strains.
enzymeecFabH fromEscherichiacoli [26]. Compound
3 inhibits
ecFabH by forming several key interactions: 2,6-dichlorophobic
group is positioned in complementary hydrophobic catalytic triad
defined by residues Cys112, His244, and Asn274, which are impor-
tantfor potency;the acidic group forms an ionic interactionwith one
of the arginine residues at the top of the active site tunnel; the 6-
chloropiperonyl group makes largely hydrophobic contacts with
residues at the mouth of the active-site tunnel; and the indole
moiety merely serve as a scaffold to arrange the binding elements.
Compd
MIC (mM)
MDR-TB
XDR-TB
10d
15
12h
12k
48.0
16.0
128.0
32.0
8.0
12.0
16.0
128.0
32.0
Isoniazid
64.0

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
3555
Based on this analysis, we postulated that our compounds share
a common pharmacophoric element 2,6-dichlorobenzyloxy group,
which could be expected to form equivalent interactions with
mtFabH in Mtb.
ing Cys112eHis244eAsn274 catalytic triad of mtFabH, while
common structure-thiolactone moiety locates in the surface of
active site [40].
On the basis of these previously studied results, we then utilized
the lauric acid-mtFabH complex structure (PDB code: 1HZP) [41],
for which the active site is known to be patotheinyl-binding site, to
investigate the fit of our compounds. An automated docking of
some of the active and less active compounds from our data set was
performed into mtFabH, using the advanced molecular docking
program GOLD 3.01. These molecules were found to fit reasonably
well into the active site, with one primary binding mode being
observed similar interactions to compound 3 with ecFabH. The
predicted binding modes for the highly active compounds 10d, 15,
12h and 12k are shown in Fig. 2.
MtFabH initiates the FAB cycle by catalyzing the first conden-
sation step between acyl-CoA and malonyl-ACP, which is closely
similar in topology and active-site structure to ecFabH, with 37.3%
identity and 46.8% similarity [27,38]. The crystal structure of
mtFabH active site is connected by two channels leading to catalytic
triad (Cys112, His244, and Asn274) with only a single solvent
entrance, one is the long-chain acyl group binding channel and the
other is the CoA binding channel (patotheinyl-binding site) [28,39].
To data, as no inhibitor-mtFabH co-crystal structures have been
solved, we initially applied computational studies to predict
the binding mode between TLM analogues and mtFabH. The
results show that unsaturated side chains at C5 position of
TLM analogues extend to slender hydrophobic pocket direct-
In general, all compounds used in docking studies bind effectively
in the binding site Cys112eHis244eAsn274, like the mtFabHelauric
acid binary complex, but some binding differences existing at the
Fig. 2. Docked structures of 10d (A), 15 (B), 12h (C) and 12k (D) with mtFabH as determined using GOLD software. The arrows represent the hydrogen bonds interactions and the
flower patterns represent the ionic interactions.

3556
X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
surfaces surrounding the mouth of the active site could be the reason
of their differing activity. The 2,6-dichlorobenzyloxy moiety of all
molecules was found to fit into the catalytic triad
Cys112eHis244eAsn274, with no direct interactionwith the catalytic
residues (Fig. 2). For compounds 10d,12h and 12k, the acidic group at
thiophene ring formed the ionic interactionwith Arg36 at the top-left
of the active site tunnel (Fig. 2A, C and D, respectively). For compound
15, containing the carboethoxyl group, was predicted to be close to
Arg36 and Arg249: one hydrogen bond formed between the carbonyl
oxygenandthe hydrogenatomof theaminogroupofArg249(Fig.2B).
Theacylated(R1)andalkylated(R2)sidechainsofallcompoundswere
predicted to be interacted with the Arg249, Phe213 and Arg244 at the
top-right of the active site tunnel: hydrogen bonds formed between
the carbonyl oxygen of side chains of compounds 10d,15 and 12k and
the hydrogen atom of the amino group of Arg249, and the butyl side
chain of compound 12h made largely hydrophobic contacts with
residues Phe213 and Arg244, which might account for the good
activity. In particular, the acidic side chain of compound 12k was
found to form the second ionic interactions with Arg249 and Arg214.
The intermediate phenylthiophenewas likely to serve as a scaffold on
which these three binding elements were linked. Apparently, our
compounds were predicted to share similar binding mode of ecFabH
with 3. While these modeling studies are very preliminary, further
evaluation is still in progress.
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
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.
5.1.1. General procedure for the synthesis of compounds 6aec
To a solution of 4-(2-hydroxyethyl)phenol 5 (6.9 g, 0.05 mol)
and K2CO3 (10.35 g, 0.075 mol) in acetone (50 mL), 4aec (0.05 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 (200 mL) and then extracted with EtOAc (2 ꢂ 100 mL). The
organic layers was washed with 10% NaOH solution, dried with
Na2SO4 and then concentrated in vacuo to afford 6aec.
4. Conclusion
5.1.1.1. 2-(4-(2,6-dichlorobenzyloxy)phenyl)ethanol (6a). Yield: 91%.
In this study, a new class of 2-acylated and 2-alkylated-amino-
5-(4-(benzyloxy)phenyl)thiophene-3-carboxylic acid derivatives
showing good anti-tubercular properties was identified, with
several compounds found to show low micromolar MICs against
replicating TB in the MABA, low toxicity against VERO cells (SI >10)
and moderate activity against MDR-TB and XDR-TB clinical strains.
Plausible SARs could be derived from R1 and R2 substituents of
amides and amines, suggesting that p-chlorobenzoyl group and
three or four atom alkyls are preferred. Notably, introducing an
acidic group in side chain, as such with an acceptable lipophilic
factor, provides compounds with good anti-tubercular activity. The
2,6-dichlorobenzyloxy group played a significant role in the
activity, indicating that these compounds are likely to have
a specific Mtb target. The potent compounds 10d, 15, 12h and 12k
were found to inhibit Mtb growth at micromolar concentrations,
Red oil. 1H NMR (300 MHz, CDCl3):
d 1.76 (s, 1H, OH), 2.68e2.73 (t,
J ¼ 6.6 Hz, 2H, CH2), 3.68e3.72 (t, J ¼ 6.6 Hz, 2H, CH2), 5.15 (s, 2H,
OCH2), 6.87e6.90 (d, 2H, J ¼ 8.4 Hz, AA0BB0, AreH), 7.05e7.08 (d, 2H,
J ¼ 8.4 Hz, AA0BB0, AreH), 7.11e7.14 (t, 1H, J ¼ 7.2 Hz, AreH),
7.23e7.26 (d, 2H, J ¼ 7.5 Hz, AreH); EI-MS (m/z): 296 (M)þ.
5.1.1.2. 2-(4-(2-chlorobenzyloxy)phenyl)ethanol (6b). Yield: 100%.
Yellow oil. EI-MS (m/z): 262 (M)þ.
5.1.1.3. 2-(4-(3,4-dichlorobenzyloxy)phenyl)ethanol (6c). Yield: 83%.
Colorless oil. EI-MS (m/z): 296 (M)þ.
5.1.2. General procedure for the synthesis of compounds 7aec
6aec (0.01 mol) and 2,2,6,6-tetramethylpiperidine-L-oxyl
(28 mg, 0.0001 mol) were dissolved in CH2Cl2 (50 mL) and
combined with aqueous 5% NaHCO3 (100 mL). On cooling to 0e5 ꢀC,
NaBrO2$3H2O (31 mL, 0.03 mol) was added to the above mixture
with moderate stirring (buffered at pH 8.6). Stirring was continued
for 0.5 h at room temperature. The organic layer was separated, and
the aqueous layer was extracted with CH2Cl2. The combined
organic layers were washed with brine, dried with Na2SO4. The
solvent was concentrated in vacuo to give 7aec as brown oil
without further purification for next step.
with MIC values between 1.9 and 7.7
10d and 15 also had good activity against XDR-TB with MICs of
12 M and 16 M, respectively. Preliminary molecular modeling
mM. Moreover, compounds
m
m
results suggested that the target of this 2-amino-5-(4-(benzyloxy)
phenyl)thiophene-3-carboxylic acid series might be associated
with mtFabH. In general, the overall studied results provided
a useful starting point in the quest for chemically distinctive classes
of anti-tubercular agents. In the light of our encouraging results,
and exploiting the information arising from the binding mode, we
are currently engaged in working at the design, synthesis, and
biological evaluation of a second generation of more efficient
thiophene core-based anti-tubercular agents.
5.1.2.1. 2-(4-(2,6-dichlorobenzyloxy)phenyl)acetaldehyde
(7a). Yield: 92%. 1H NMR (300 MHz, CDCl3):
d 2.73e2.78 (t,
J ¼ 7.5 Hz, 2H, CH2), 2.90e2.95 (t, J ¼ 7.5 Hz, 2H, CH2), 5.25 (s, 2H,
OCH2), 6.95e6.98 (d, 2H, J ¼ 8.4 Hz, AA0BB0, AreH), 7.13e7.15 (d, 2H,
J ¼ 8.4 Hz, AA0BB0, AreH), 7.241e7.26 (t, 1H, J ¼ 7.2 Hz, AreH),
7.35e7.37 (d, 2H, J ¼ 7.5 Hz, AreH), 9.82 (s, 1H, CHO); EI-MS (m/z):
294 (M)þ.
5. Experimental
5.1. Chemistry
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
5.1.2.2. 2-(4-(2-chlorobenzyloxy)phenyl)acetaldehyde (7b). Yield:
100%. EI-MS (m/z): 260 (M)þ.
5.1.2.3. 2-(4-(3,4-dichlorobenzyloxy)phenyl)acetaldehyde
(7c). Yield: 64%. EI-MS (m/z): 294 (M)þ.
expressed in parts per million (ppm,
d units). Coupling constants

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
3557
5.1.3. General procedure for the synthesis of compounds 8aec
7aec (0.02 mol), powdered sulphur (0.64 g, 0.02 mol) and ethyl
cyanoacetate (2.2 g, 0.02 mol) were stirred at room temperature in
absolute EtOH (80 mL). Et3N (2.2 g, 0.02 mol) was added dropwise
and after the addition was completed the mixture was heated
overnight at 60 ꢀC. The mixture was cooled and the precipitated
solid was filtered off and recrystallised from EtOH. The solid was
filtered off, washed with ice cold EtOH (2 ꢂ 20 mL) and dried to
afford 8aec, then recrystallization from EtOH gave the pure product.
1293, 1246, 1207, 1179, 1037, 1020, 824, 783, 742, 677, 514; 1H NMR
(CDCl3, ppm):
d
1.40 (t, J ¼ 7.2 Hz, 3H, CH3), 4.37 (q, J ¼ 7.2 Hz, 2H,
CH2), 5.31 (s, 2H, OCH2), 7.09 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.29 (s,
1H, H-4), 7.37e7.43 (m, 4H, AreH), 7.46e7.51 (m, 2H, AreH), 7.58 (d,
J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.86 (d, J ¼ 1.2 Hz, 1H, AreH), 11.76 (s,
1H, CONH)ppm; EI-MS (m/z): 559 (M)þ.
5.1.4.3. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(3-chlorobenza-
mido)thiophene-3-carboxylate (9c). Yield: 52.38%. Yellow solid, mp:
189e191 ꢀC; IR (cmꢃ1): 3414, 3259, 1660, 1606, 1561, 1525, 1436,
1383, 1258, 1242, 1221, 1182, 1016, 825, 779, 764, 695; 1H NMR
5.1.3.1. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-aminothiophene-
3-carboxylate (8a). Yield: 50%. Yellow solid, mp: 179e181 ꢀC.
IR(KBr): 3451, 3305, 1670, 1588, 1240, 1090, 1012, 777 cmꢃ1; 1H NMR
(DMSO-d6, ppm):
d
1.35e1.40 (t, J ¼ 7.2 Hz, 3H, CH3), 4.38e4.40 (q,
J ¼ 7.2 Hz, 2H, CH2), 5.28 (s, 2H, OCH2), 7.12e7.15 (d, J ¼ 8.1 Hz, 2H,
AreH, AA0BB0), 7.51 (s, 1H, H-4), 7.57e7.60 (d, J ¼ 8.1 Hz, 2H, AreH,
AA0BB0), 7.57e7.60 (1H, overlapped), 7.65e7.67 (d, J ¼ 7.8 Hz, 2H,
AreH), 7.72e7.75 (d, J ¼ 8.7 Hz, 2H, AreH), 7.92e7.99 (m, 2H, AreH),
11.80 (s, 1H, CONH); EI-MS (m/z): 559 (M)þ.
(300 MHz, CDCl3):
d
1.30e1.34 (t, 3H, J ¼ 6.9 Hz, CH3), 4.27e4.33(q,
2H, J ¼ 6.9 Hz, CH2), 5.29 (s, 2H, OCH2), 7.03e7.05 (d, 2H, J ¼ 8.1 Hz,
AA0BB0, AreH), 7.18e7.23 (t, 1H, J ¼ 7.2 Hz, AreH), 7.30e7.33 (d, 2H,
J ¼ 7.2 Hz, AreH), 7.95e7.97 (d, 2H, J ¼ 8.1 Hz, AA0BB0, AreH), 8.12
(1H, s, H-4) ppm. MS (EI): 421 (M)þ.
5.1.4.4. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(4-chlorobenza-
mido)thiophene-3-carboxylate (9d). Yield: 26%. Yellow solid, mp:
225e227 ꢀC (dec); IR (cmꢃ1): 3437, 3274, 1790, 1664, 1565, 1530,
1439, 1381, 1264, 1247, 1222, 1175, 1092, 1012, 827, 777, 524；1H
5.1.3.2. Ethyl 2-amino-5-(4-(2-chlorobenzyloxy)phenyl)thiophene-3-
carboxylate (8b). Yield: 22%. Yellow solid, mp: 138e140 ꢀC. 1H
NMR (300 MHz, CDCl3):
d
1.34e1.39 (t, 3H, J ¼ 7.2 Hz, CH3),
4.26e4.33 (q, 2H, J ¼ 7.2 Hz, CH2), 5.18 (s, 2H, OCH2), 6.94e6.97 (2H,
d, J ¼ 8.7 Hz, AA0BB0, AreH), 7.10 (1H, s, H-4), 7.28e7.32 (m, 2H,
AreH), 7.36e7.39 (2H, d, J ¼ 8.7 Hz, AA0BB0, AreH), 7.41e7.42 (1H,
m, AreH), 7.53e7.56 (1H, m, AreH) ppm. EI-MS (m/z): 387 (M)þ.
NMR (300 MHz, CDCl3):
d
1.36e1.41 (t, J ¼ 6.9 Hz, 3H, CH3),
4.35e4.42 (q, J ¼ 6.9 Hz, 2H, CH2), 5.27 (s, 2H, OCH2), 7.12e7.15
(d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.48e7.49 (t, 1H, AreH), 7.51 (s,
1H, H-4), 7.57e7.60 (d, J ¼ 8.4 Hz, 2H, AreH), 7.64e7.67 (d,
J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.69e7.71 (m, 1H, overlapped),
7.78e7.81 (d, J ¼ 8.4 Hz, 2H, AreH), 7.90e7.92 (d, J ¼ 7.8 Hz, 2H,
AreH), 7.96 (s, 1H, AreH), 11.74 (s, 1H, CONH); EI-MS (m/z):
559 (M)þ.
5.1.3.3. Ethyl 2-amino-5-(4-(3,4-dichlorobenzyloxy)phenyl)thiophene-
3-carboxylate (8c). Yield: 51%. Yellow solid, mp: 126e128 ꢀC. 1H
NMR (300 MHz, CDCl3):
d
1.34e1.38 (t, 3H, J ¼ 7.2 Hz, CH3),
4.26e4.33(q, 2H, J ¼ 7.2 Hz, CH2), 5.15(s, 2H, OCH2), 6.89e6.92 (d, 2H,
J ¼ 8.7 Hz, AA0BB0, AreH), 7.11 (br, 2H, NH2), 7.21(d, 1H, J ¼ 8.1 Hz,
AreH), 7.29 (1H, s, H-4), 7.44e7.47 (d, 1H, J ¼ 8.1 Hz, AreH), 7.54 (s,
1H, AreH), 7.90e7.93 (d, 2H, J ¼ 8.7 Hz, AA0BB0, AreH). EI-MS (m/z):
421 (M)þ.
5.1.4.5. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(2-fluorobenz-
amido)thiophene-3-carboxylate (9e). Yield: 83%. Yellow solid, mp:
225e227 ꢀC; 1H NMR (300 MHz, CDCl3):
d
1.42e1.45 (t, J ¼ 6.9 Hz,
3H, CH3), 4.39e4.47 (q, J ¼ 6.9 Hz, 2H, CH2), 5.31 (s, 2H, OCH2),
7.05e7.07 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.21e7.29 (m, 4H, AreH,
H-4, overlapped), 7.37e7.40 (d, J ¼ 8.4 Hz, 2H, AreH), 7.55e7.58 (m,
1H, AreH), 7.58e7.61 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 8.21e8.27
(m, 1H, AreH), 12.22e12.26 (d, J ¼ 9.9 Hz, 1H, CONH); EI-MS (m/z):
543 (M)þ.
5.1.4. General procedure for the synthesis of compounds 9aem
A mixture of substituted benzoic acid (0.008 mol) in thionyl
chloride (5 mL) was stirred under refluxed for 2 h. After cooling to
room temperature, the mixture was concentrated in vacuo to give
crude product substituted benzoyl chloride as brown oils without
further purification. To a mixture of 8aec (0.002 mol), dry trie-
thylamine (3 mL) and dry dichloromethane (10 mL), the solution of
substituted benzoyl chloride in dry dichloromethane (10 mL)
previous prepared was added. The whole mixture was stirred for
overnight at room temperature. Water was added to the mixture
and extracted with dichloromethane (25 ꢂ 2 mL). The combined
organic layers were washed with brine and dried with Na2SO4, then
concentrated in vacuo. Purification of the crude products by column
chromatography eluting with petroleumeethyl acetate (10:1) gave
9aem.
5.1.4.6. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(2-methoxybe-
nzamido)thiophene-3-carboxylate (9f). Yield: 83%. Yellow solid,
mp: 203e205 ꢀC; IR (KBr): 3437, 3215, 1680, 1652, 1552, 1521,
1294, 1246, 1206, 1179, 783, 754, 688 cmꢃ1 1H NMR (300 MHz,
.
CDCl3):
d
1.40e1.44 (t, J ¼ 7.2 Hz, 3H, CH3), 4.18 (s, 3H, OCH3),
4.36e4.43 (q, J ¼ 7.2 Hz, 2H, CH2), 5.30 (s, 2H, OCH2), 7.03e7.06
(d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.05e7.08 (1H, AreH, over-
lapped), 7.11e7.16 (t, J ¼ 7.5 Hz, 1H, AreH), 7.23e7.28 (m, 1H,
overlapped), 7.36 (s, 1H, H-4), 7.39 (br, 2H, AreH), 7.52e7.54 (d,
J ¼ 7.2 Hz, AreH), 7.58e7.61 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
8.31e8.34 (d, J ¼ 6.6 Hz, AreH), 13.02 (s, 1H, CONH). EI-MS (m/z):
555 (M)þ.
5.1.4.1. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-benzamidothioph-
ene-3-carboxylate (9a). Yield: 48%. White solid, mp: 244e246 ꢀC. IR
(cmꢃ1): 3259, 1666, 1561, 1529, 1435, 1384, 129, 1240, 1215, 1179, 1027,
5.1.4.7. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(3-methoxybenz-
amido)thiophene-3-carboxylate (9g). Yield: 55%. Yellow solid, mp:
142e144 ꢀC; IR (KBr): 3435, 3326, 1646, 1563, 1508, 1437, 1236, 1175,
872, 822, 790, 777, 691, 677; 1H NMR (CDCl3, ppm):
d 1.44 (t,
J ¼ 7.2 Hz, 3H, CH3), 4.42 (q, J ¼ 7.2 Hz, 2H, CH2), 5.31 (2H, s, OCH2),
7.03 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.28 (s, 1H, H-4), 7.73 (s, 2H,
AreH), 7.39 (s, 1H, AreH), 7.56 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.52e7.63 (m, 3H, AreH, overlapped), 8.05 (d, J ¼ 6.9 Hz, 2H, AreH),
12.03 (s, 1H, CONH); EI-MS (m/z): 525 (M)þ.
1018, 776 cmꢃ1. 1H NMR (300 MHz, CDCl3):
d
1.41e1.46 (t, J ¼ 7.2 Hz,
3H, CH3), 3.90 (s, 3H, OCH3), 4.38e4.45 (q, J ¼ 7.2 Hz, 2H, CH2), 5.31
(s, 2H, OCH2), 7.04e7.07 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.12e7.15
(dd, J ¼ 2.4 Hz, J ¼ 8.4 Hz, 1H, AreH), 7.22e7.28 (m, 2H, overlapped,
AreH, H-4), 7.36e7.39 (d, J ¼ 7.5 Hz, 2H, AreH), 7.41e7.46 (t,
J ¼ 7.8 Hz, J ¼ 8.1 Hz, 1H, AreH), 7.56e7.59 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.55e7.62 (m, 2H, AreH), 11.99 (s, 1H, CONH). EI-MS (m/z):
555 (M)þ.
5.1.4.2. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(2-chlorobenza-
mido)thiophene-3-carboxylate (9b). Yield: 38%. Yellow solid, Mp
182e184 ꢀC; IR (cmꢃ1): 3417, 3256, 1681, 1646, 1553, 1522, 1439, 1380,

3558
X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
5.1.4.8. Ethyl
5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(4-methoxy-
5.1.5. General procedure for the synthesis of compounds 10aem
To a solution of 9aem (1 equiv) in EtOH (20 mL) was added 1N
NaOH (12.5 equiv). 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 1N HCl to pH 1. The suspension
solution was extracted with EtOAc (2 ꢂ 25 mL). The combined
organic layers was dried with NaSO4 and concentrated in vacuo to
afford 10aem. Recrystallization from EtOAc gave pure 10aem.
benzamido)thiophene-3-carboxylate (9h). Yield: 16%. Yellow solid,
mp: 207e209 ꢀC; IR (KBr): 3416, 3252, 1657, 1606, 1561, 1530, 1515,
1436, 1382, 1257, 1242, 1222, 1174, 1017, 825, 780, 763, 534,
511 cmꢃ1. 1H NMR (300 MHz, CDCl3):
d
1.41e1.46 (t, J ¼ 7.2 Hz, 3H,
CH3), 3.89 (s, 3H, OCH3), 4.38e4.45 (q, J ¼ 7.2 Hz, 2H, CH2), 5.31 (s,
2H, OCH2), 7.01e7.07 (m, 4H, AreH, overlapped), 7.23e7.28 (m, 1H,
AreH, overlapped), 7.36 (s, 1H, H-4), 7.37e7.39 (d, J ¼ 7.5 Hz, 2H,
AreH), 7.56e7.59 (d, J ¼ 8.7 Hz, 2H, AreH), 7.99e8.02 (d, J ¼ 8.7 Hz,
2H, AreH), 11.94 (s, 1H, CONH). EI-MS (m/z): 555 (M)þ.
5.1.5.1. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-benzamidothiophene-
3-carboxylic acid (10a). Compound 9a (0.35 g, 0.67 mmol), 1N
NaOH (8.3 mL) in EtOH (20 mL) gave compound 10a (0.18 g, 55%)
according to general procedure (5.1.5.) as a yellow solid, mp:
178e180 ꢀC (dec); IR (cmꢃ1): 3457, 3062, 1639, 1555, 1528, 1437,
1240, 1178, 1027, 824, 699; 1H NMR (DMSO-d6, ppm): 5.27 (s, 2H,
OCH2), 7.08 (s, 1H, H-4), 7.11e7.14 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.47e7.51 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.55e7.72 (m, 7H, AreH,
overlapped), 7.95e7.97 (d, J ¼ 6.9 Hz, 2H, AreH), 12.56 (s, 1H, OH);
ESI-MS (m/z): 497 (M ꢃ 1)þ. Formula: C26H19Cl2NO4S$1.5H2O; Anal
(C%, H%, N%) Calcd: 57.15, 3.84, 2.67. Found: 56.68, 4.03, 2.97.
5.1.4.9. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(picolinamido)
thiophene-3-carboxylate (9i). Yield: 32%. Yellow solid, mp:
245e247 ꢀC; IR (KBr): 3235, 2978, 1686, 1669, 1555, 1522, 1424,
1242, 1217, 1181, 1016, 826, 690 cmꢃ1. 1H NMR (300 MHz, CDCl3):
d
1.46e1.49 (t, J ¼ 6.9 Hz, 3H, CH3), 4.42e4.49 (q, J ¼ 6.9 Hz, 2H,
CH2), 5.31 (s, 2H, OCH2), 7.05e7.08 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.23e7.29 (m, 1H, AreH, overlapped), 7.37 (s, 1H, H-4), 7.37e7.40 (d,
J ¼ 6.9 Hz, 2H, AreH), 7.51e7.57 (m, 1H, AreH), 7.57e7.60 (d,
J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.90e7.95 (m, 1H, AreH), 8.29e8.31
(d, J ¼ 7.8 Hz, 1H, AreH), 8.77e8.75 (d, J ¼ 4.2 Hz, 1H, AreH), 12.90
(s, 1H, CONH). EI-MS (m/z): 526 (M)þ.
5.1.5.2. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(2-chlorobenzamido)
thiophene-3-carboxylic acid (10b). Yield: 43%. Yellow solid, mp:
260e262 ꢀC (dec). IR (KBr): 3033, 2953, 1662, 1553, 1526, 1463,
5.1.4.10. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(furan-2-carboxa-
mido)thiophene-3-carboxylate (9j). Yield: 12%. Yellow solid, mp
245e247 ꢀC; IR (KBr): 3414, 1677, 1660, 1583, 1560, 1526, 1485, 1436,
1383, 1277, 1243, 1219, 1183, 1014, 824, 779, 748 cmꢃ1; 1H NMR (CDCl3,
1437, 1246, 1223, 1014, 820, 777, 745, 726, 565 cmꢃ1 1H NMR
;
(300 MHz, DMSO-d6): 5.27 (s, 2H, OCH2), 7.12e7.15 (d, J ¼ 8.7 Hz,
2H, AreH, AA0BB0), 7.48 (s, 1H, H-4, overlapped), 7.46e7.51 (m, 1H,
AreH, overlapped), 7.53e7.55 (m, 1H, AreH), 7.57e7.60 (d,
J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.63e7.66 (m, 4H, AreH, overlapped),
7.83e7.86 (d, J ¼ 6.9 Hz, 1H, AreH), 11.85 (s, 1H, OH). ESI-MS (m/z):
531.8 (M ꢃ H)þ. Formula: C25H16Cl3NO4S$H2O, Anal (C%, H%, N%)
Calcd: 54.51, 3.29, 2.54 Found: 55.03, 3.23, 2.40.
ppm):
d
1.44 (t, J ¼ 7.2 Hz, 3H, CH3), 4.42 (q, J ¼ 7.2 Hz, 2H, CH2), 5.31 (s,
2H, OCH2), 6.60 (s, 1H, AreH), 7.03 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.28 (s, 1H, H-4), 7.30e7.34 (d, J ¼ 2.4 Hz, 1H, AreH), 7.36e7.37 (d,
J ¼ 2.4 Hz, 2H, AreH), 7.39 (s, 1H, AreH), 7.56e7.59 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.62 (s, 1H, AreH), 11.85 (s, 1H, CONH); EI-MS (m/z):
515 (M)þ.
5.1.5.3. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(3-chlorobenzamido)
thiophene-3-carboxylic acid (10c). Yield: 45%. Yellow solid, mp:
195-193 ꢀC (dec). IR (KBr): 3162, 3063, 1681, 1562, 1528, 1244, 1191,
5.1.4.11. Ethyl5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(thiophene-2-
carboxamido)thiophene-3-carboxylate (9k). Yield: 9%. Yellow solid,
mp 243e245 ꢀC (dec); IR (KBr): 3415, 3245, 1668, 1647, 1561, 1528,
1436,1384,1268,1242,1221,1183,1090, 825, 777, 717 cmꢃ1. 1H NMR
1005, 822, 703, 668 cmꢃ1 1H NMR (300 MHz, DMSO-d6): 5.25 (s,
;
2H, OCH2), 7.07e7.10 (d, J ¼ 8.4 Hz, 2H, AreH, AA0BB0), 7.30e7.34 (d,
J ¼ 8.4 Hz, 2H, AreH, AA0BB0), 7.48e7.59 (m, 5H, AreH, overlapped),
7.79e7.92 (m, 3H, AreH, overlapped). ESI-MS (m/z): 531.8 (M ꢃ H)þ.
Formula: C25H16Cl3NO4S, Anal (C%, H%, N%) Calcd: 56.35, 3.03, 2.63.
Found: 56.07, 3.21, 2.40.
(300 MHz, CDCl3):
d
1.41e1.46(t, J ¼ 6.9 Hz, 3H, CH3), 4.38e4.45(q,
J ¼ 6.9 Hz, 2H, CH2), 5.31 (s, 2H, OCH2), 7.04e7.07 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.16e7.19 (q, J ¼ 3.6 Hz, J ¼ 1.2 Hz, 1H, AreH),
7.23e7.28 (m, 1H, AreH, overlapped), 7.35 (s, 1H, H-4), 7.37e7.39 (t,
J ¼ 7.2 Hz, J ¼ 1.2 Hz, 2H, AreH), 7.55e7.58 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.62e7.64(d, J ¼ 4.8 Hz, 1H, AreH), 7.78e7.80(d, J ¼ 3.9 Hz,
1H, AreH), 11.86(s, 1H, CONH). EI-MS (m/z): 531 (M)þ.
5.1.5.4. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(4-chlorobenzamido)
thiophene-3-carboxylic acid (10d). Yield: 54%. Yellow solid, mp:
200e202 ꢀC (dec). IR (KBr): 3024, 2626,1639, 1550,1504,1487, 1437,
5.1.4.12. Ethyl 2-(4-chlorobenzamido)-5-(4-(2-chlorobenzyloxy)phen-
1262, 1175, 994, 828, 772, 723, 642, 528 cmꢃ1 1H NMR (300 MHz,
;
yl)thiophene-3-carboxylate (9l). Yield: 12%. Yellow solid, mp:
DMSO-d6): 5.25 (s, 2H, OCH2), 7.02e7.05 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.08 (s,1H, H-4), 7.37e7.40 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.47e7.50 (m,1H, AreH), 7.55e7.58(m, 3H, AreH), 7.59 (d,1H, AreH),
7.74 (d, 1H, AreH), 7.79 (d, 1H, AreH), 12.00 (s, 1H, CONH), 12.20 (s,
1H, OH). ESI-MS (m/z): 531.8 (M ꢃ H)þ. Formula: C25H16Cl3NO4S. Anal
(C%, H%, N%) Calcd: 56.35, 3.03, 2.63; Found: 56.48, 3.02, 2.57.
164e167 ꢀC. 1H NMR (300 MHz, CDCl3):
d 1.42e1.46 (t, 3H,
J ¼ 7.2 Hz, CH3), 4.38e4.45 (q, 2H, J ¼ 6.9 Hz, CH2), 5.20 (s, 2H,
OCH2), 7.00e7.03 (d, 2H, J ¼ 8.7 Hz, AreH, AA0BB0), 7.29e7.33 (m,
2H, AreH), 7.38 (s, 1H, H-4), 7.40e7.43 (m, 1H, AreH), 7.50e7.53
(d, 2H, J ¼ 8.4 Hz, AreH, AA0BB0), 7.53e7.56 (m, 1H, AreH,
overlapped), 7.55e7.57 (d, 2H,
J
¼
8.4 Hz, AreH, AA0BB0),
7.96e7.99 (d, 2H, J ¼ 8.7 Hz, AreH, AA0BB0), 12.04(s, 1H, eNHCO).
5.1.5.5. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(2-fluorobenzamido)
thiophene-3-carboxylic acid (10e). Yield: 49%. Yellow solid, mp:
252e256 ꢀC (dec); IR (KBr): 3279, 2946, 2599, 1657, 1551, 1523,
1454, 1288, 1246, 1015, 779, 665 cmꢃ1; 1H NMR (300 MHz, DMSO-
d6): 5.27 (s, 2H, OCH2), 7.12e7.15 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.44e7.51 (m, 4H, AreH, H-4, overlapped), 7.57e7.60 8.06e8.09 (t,
J ¼ 7.5 Hz, 2H, AreH), 7.63e7.66 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.74e7.76 (m, 1H, AreH), 8.08e8.13 (m, 1H, AreH), 12.21e12.25 (d,
J ¼ 10.8 Hz, 1H, CONH), 13.47 (br, 1H, COOH). ESI-MS (m/z): 513.9
(M ꢃ H)þ. Formula: C25H16Cl2FNO4S$H2O. Anal (C%, H%, N%) Calcd:
56.19, 3.40, 2.62. Found: 56.13, 3.22, 2.24.
EI-MS (m/z): 525 (M)þ.
5.1.4.13. Ethyl 2-(4-chlorobenzamido)-5-(4-(3,4-dichlorobenzyloxy)
phenyl)thiophene-3-carboxylate (9m). Yield: 59%. Yellow solid, mp:
167e169 ꢀC. 1H NMR (300 MHz, CDCl3):
d 1.41e1.46 (t, 3H,
J ¼ 7.2 Hz, CH3), 4.38e4.45(q, 2H, J ¼ 7.2 Hz, CH2), 5.04(s, 2H,
OCH2), 6.95e6.98 (d, 2H, J ¼ 8.7 Hz, AA0BB0, AreH), 7.29(d, 1H,
J ¼ 8.1 Hz, AreH), 7.35 (1H, s, H-4), 7.45e7.56 (m, 6H, AreH),
7.96e7.99 (d, 2H, J ¼ 8.7 Hz, AA0BB0, AreH), 12.03 (s, 1H, eCONH).
EI-MS (m/z): 561 (M)þ.

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
3559
5.1.5.6. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(2-methoxybenzam-
ido)thiophene-3-carboxylic acid (10f). Yield: 27%. Yellow solid, mp:
220e222 ꢀC (dec). IR (KBr): 3481, 3168,1704, 1632,1552,1519,1465,
(t, J ¼ 4.2 Hz, J ¼ 4.5 Hz, 1H, AreH), 7.45 (s, 1H, H-4), 7.47e7.50 (t,
J ¼ 2.4 Hz, J ¼ 6.6 Hz, 1H, AreH), 7.56e7.59 (m, 2H, AreH, over-
lapped), 7.59e7.62 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.76e7.77 (d,
J ¼ 3.6 Hz, 1H, AreH), 7.98e8.00 (d, 1H, J ¼ 4.8 Hz, AreH), 12.46 (s,
1H, OH). ESI-MS (m/z): 502 (M ꢃ H)þ. Formula: C23H15Cl2NO4S2;
Anal (C%, H%, N%) Calcd: 54.77, 3.00, 2.78; Found: 54.55, 3.14, 2.71.
1437, 1244, 1179, 1013, 822, 782, 750 cmꢃ1 1H NMR (300 MHz,
;
DMSO-d6): 4.01 (s, 3H, OCH3), 5.25 (s, 2H, OCH2), 7.11e7.14 (d,
J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.17e7.22 (t, J ¼ 7.5 Hz, J ¼ 4.5 Hz, 1H,
AreH), 7.31e7.33 (d, J ¼ 7.8 Hz, 1H, AreH), 7.7e7.51 (m, 2H, AreH,
H-4), 7.57e7.59 (d, J ¼ 7.5 Hz, 2H, AreH), 7.61e7.64 (d, J ¼ 8.7 Hz,
2H, AreH, AA0BB0), 7.67e7.69 (m, 1H, AreH), 8.14e8.16 (d, 1H,
J ¼ 6.6 Hz, AreH), 12.98(s, 1H, CONH). EI-MS (m/z): 527 (M)þ.
Formula: C26H19Cl2NO5S; Anal (C%, H%, N%) Calcd: 59.10, 3.62, 2.65.
Found: 59.26, 3.85, 2.50.
5.1.5.12. 2-(4-chlorobenzamido)-5-(4-(2-chlorobenzyloxy)phenyl)thi-
ophene-3-carboxylic acid (10l). Yield: 54%. Yellow solid, mp:
206e209 ꢀC (dec); IR (KBr): 3453, 3430,1650,1554,1528,1462,1383,
1253, 1095, 832, 749 cmꢃ1. 1H NMR (300 MHz, DMSO-d6):
d5.20(s,
2H, OCH2), 7.07e7.10 (d, 2H, J ¼ 8.7 Hz, AreH, AA0BB0), 7.34e7.41(m,
2H, AreH), 7.47 (s, 1H, H-4), 7.51e7.54 (m, 1H, AreH), 7.71e7.73 (d,
2H, J ¼ 8.4 Hz, AreH, AA0BB0), 7.71e7.73 (m, 1H, AreH, overlapped),
7.79e7.80 (d, 2H, J ¼ 8.4 Hz, AreH, AA0BB0), 7.94e7.96 (d, 2H,
J ¼ 8.7 Hz, AreH, AA0BB0), 12.17(s, 1H, eNHCO). EI-MS (m/z): 497
(M ꢃ 1)þ. Formula: C25H17Cl2NO4S$H2O; Anal (C%, H%, N%) Calcd:
58.15, 3.71, 2.71; Found: 58.03, 3.60, 2.71.
5.1.5.7. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(3-methoxybenzam-
ido)thiophene-3-carboxylic acid (10g). Yield: 40%. Yellow solid, mp:
257e259 ꢀC (dec); IR (KBr): 3430, 3229, 1646, 1522, 1508, 1437,
1295, 1248, 1019, 782, 755, 695 cmꢃ1 1H NMR (300 MHz, DMSO-
;
d6): 3.86 (s, 3H, OCH3), 5.27 (s, 2H, OCH2), 7.12e7.15 (d, J ¼ 8.7 Hz,
2H, AreH, AA0BB0), 7.27e7.29 (d, J ¼ 7.8 Hz, 2H, AreH), 7.48e7.54
(m, 4H, AreH, H-4, overlapped), 7.57e7.60 (d, J ¼ 8.4 Hz, 2H, AreH),
7.62e7.65 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 12.17 (s, 1H, CONH). ESI-
MS (m/z): 525.9 (M ꢃ H)þ. Formula: C26H19Cl2NO5S$H2O. Anal (C%,
H%, N%) Calcd: 57.15, 3.87, 2.56. Found: 56.90, 3.60, 2.53.
5.1.5.13. 2-(4-chlorobenzamido)-5-(4-(3,4-dichlorobenzyloxy)phe-
nyl)thiophene-3-carboxylic acid (10m). Yield: 69%. Yellow solid,
mp: 217e220 ꢀC (dec); IR (KBr): 3313, 3086, 1678, 1649, 1549,
1524, 1464, 1247, 1178, 1091, 1031, 812, 723, 510 cmꢃ1 1H NMR
.
(300 MHz, DMSO-d6):
d5.20(s, 2H, OCH2), 7.05e7.08 (d, 2H,
5.1.5.8. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(4-methoxybenzam-
ido)thiophene-3-carboxylic acid (10h). Yield: 70%. Yellow solid, mp:
261e263 ꢀC (dec); IR (KBr): 3441, 2895, 1669, 1606, 1561, 1531, 1515,
J ¼ 8.7 Hz, AreH, AA0BB0), 7.45e7.47 (d, 1H, J ¼ 8.1 Hz, AreH), 7.47
(1H, s, H-4), 7.60e7.73 (m, 6H, AreH), 7.93e7.96 (d, 2H, J ¼ 8.7 Hz,
AA0BB0, AreH), 12.09 (s, 1H, eCONH), 12.40 (br, 1H, COOH). ESI-MS
(m/z): 532 (M ꢃ 1)þ. Formula: C25H16Cl3NO5S$H2O. Anal (C%, H%,
N%) Calcd: 54.51, 3.29, 2.54; Found: 54.37, 3.09, 2.51.
1462, 1437, 1252, 1183, 1024, 828, 777 cmꢃ1 1H NMR (300 MHz,
;
DMSO-d6): 3.87 (s, 3H, OCH3), 5.27 (s, 2H, OCH2), 7.11e7.14 (d,
J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.17e7.19 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.46 7.48e7.51 (m, 2H, AreH, H-4, overlapped), 7.57e7.60
(d, J ¼ 8.4 Hz, 2H, AreH), 7.61e7.64 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.90e7.93 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 12.05 (s, 1H, CONH). ESI-
MS (m/z): 525.9 (M ꢃ H)þ. Formula: C26H19Cl2NO5S$1.5H2O. Anal (C
%, H%, N%) Calcd: 56.22, 3.99, 2.52. Found: 55.90, 3.77, 2.45.
5.1.6. General procedure for the synthesis of compounds 11aek
Halides (1.5e2.0 equiv) were added to a mixture of 8a (2 mmol),
K2CO3 (1.1 g, 8 mmol), and KI (100 mg) in DMF (10 mL). The
resulting mixture was stirred at room temperature for 30 min and
at 60 ꢀC for a further 24 h. After being cooled, the mixture was
poured into ice water and the resulting mixture was extracted with
EtOAc (25 ꢂ 2 mL). The combined organic layer was washed with
brine and dried (Na2SO4). After evaporation of the solvent in vacuo,
the residue was purified by column chromatography eluting with
petroleum-ethyl acetate to give 11aek.
5.1.5.9. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(picolinamido)thio-
phene-3-carboxylic acid (10i). Yield: 42%. Yellow solid, mp:
276e278 ꢀC (dec); IR (KBr): 3444, 3238, 2590, 1658, 1554, 1554,
1523, 1437, 1240, 1179, 1017, 819, 690 cmꢃ1 1H NMR (300 MHz,
;
DMSO-d6): 5.26 (s, 2H, OCH2), 7.08e7.11 (d, J ¼ 8.4 Hz, 2H, AreH,
AA0BB0), 7.30 (s, 1H, H-4), 7.45e7.67 (m, 5H, AreH, overlapped),
7.89e7.91 (m, 1H, AreH), 8.06e8.09 (t, J ¼ 7.5 Hz, 2H, AreH),
8.14e8.17 (d, J ¼ 7.5 Hz, 1H, AreH), 8.74 (d, J ¼ 7.5 Hz, 1H, AreH),
14.76 (s, 1H, CONH). ESI-MS (m/z): 496.9 (M ꢃ H)þ. Formula:
C24H20Cl2N2O6S$2H2O; Anal (C%, H%, N%) Calcd: 53.84, 3.77, 5.23.
Found: 54.06, 3.70, 5.25.
5.1.6.1. Ethyl 2-(benzylamino)-5-(4-(2,6-dichlorobenzyloxy)phenyl)
thiophene-3-carboxylate (11a). Compound 8a (0.84 g, 2 mmol),
Benzyl bromide (0.51 g, 3 mmol), K2CO3 (1.1 g, 8 mmol), and KI
(100 mg) in DMF (10 mL) gave 11a (0.10 g, 14%) according to general
procedure (5.1.6.) as a yellow solid. m.p. 147e149 ꢀC. IR (KBr): 3415,
3325, 2929, 1661, 1556, 1504, 1463, 1233, 1201, 830, 773 cmꢃ1 1H
;
NMR (300 MHz, CDCl3): 1.33e1.38 (t, J ¼ 6.9 Hz, 3H, CH3), 4.24e4.31
(q, J ¼ 6.9 Hz, 2H, CH2), 4.48 (br, 2H, NCH2), 5.27 (s, 2H, OCH2),
6.97e7.00 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.18 (s, 1H, H-4),
7.20e7.27 (m, 3H, AreH, overlapped), 7.30e7.41 (m, 8H, AreH,
overlapped), 7.81 (br, 1H, NH). EI-MS (m/z): 510 (M)þ.
5.1.5.10. -(4-(2,6-dichlorobenzyloxy)phenyl)-2-(furan-2-carboxam-
ido)thiophene-3-carboxylic acid (10j). Yield: 48%. Yellow solid,
mp: 213e215 ꢀC (dec); IR (cmꢃ1): 3396, 2919, 1723, 1384, 1243,
1154, 1092, 1023, 747, 600, 559; 1H NMR (DMSO-d6, ppm): 5.27 (s,
2H, OCH2), 6.80 (s, 1H, H-4), 7.11e7.14 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.40e7.41 (d, J ¼ 3.6 Hz, 1H, AreH), 7.46e7.51 (m, 2H,
AreH), 7.57e7.59 (d, J ¼ 7.5 Hz, 2H, AreH), 7.61e7.64 (d, J ¼ 8.7 Hz,
2H, AreH, AA0BB0), 8.08 (s, 1H, AreH), 12.00 (s, 1H, CONH), 13.48 (s,
5.1.6.2. Ethyl 2-(2-chlorobenzylamino)-5-(4-(2,6-dichlorobenzyloxy)
phenyl)thiophene-3-carboxylate (11b). Yield: 26%. Yellow solid, mp:
126e128 ꢀC. IR (KBr): 355, 3479, 1660, 1550, 1531, 1437, 1241, 1216,
1035, 1018, 828, 772 cmꢃ1; 1H NMR (300 MHz, CDCl3): 1.34e1.39 (t,
J ¼ 6.9 Hz, 3H, CH3), 4.26e4.33 (q, J ¼ 6.9 Hz, 2H, CH2), 4.59 (br, 2H,
NCH2) 5.27 (s, 2H, OCH2), 6.97e7.00 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.18 (s, 1H, H-4), 7.20e7.27 (m, 3H, AreH, overlapped),
7.34e7.47 (m, 5H, AreH), 7.55e7.58 (m,1H, AreH), 7.95 (br,1H, NH).
EI-MS (m/z): 545 (M)þ.
1H, COOH); ESI-MS (m/z): 487 (M
C23H15Cl2NO5S$0.5H2O; Anal (C%, H%, N%) Calcd: 55.54, 3.24, 2.82.
ꢃ
1)þ. Formula:
Found: 55.45, 3.48, 3.05.
5.1.5.11. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(thiophene-2-carbo-
xamido)thiophene-3-carboxylic acid (10k). Yield: 74%. Yellow solid,
mp: 178e180 ꢀC (dec). IR (KBr): 3416, 3088, 1644, 1557, 1529, 1437,
1244, 1016, 824, 778, 725 cmꢃ1; 1H NMR (300 MHz, DMSO-d6): 5.31
(s, 2H, OCH2), 7.11e7.13 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.29e7.32
5.1.6.3. Ethyl 2-(4-chlorobenzylamino)-5-(4-(2,6-dichlorobenzyloxy)
phenyl)thiophene-3-carboxylate (11c). Yield: 10%. Yellow solid, mp:

3560
X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
185e187 ꢀC. IR (KBr): 3409, 3323, 2967, 1646, 1555,1543, 1426,1385,
1240, 1217, 1178,1011, 819, 776 cmꢃ1; 1H NMR (300 MHz, DMSO-d6):
1.33e1.38 (t, J ¼ 6.9 Hz, 3H, CH3), 4.25e4.32 (q, J ¼ 6.9 Hz, 2H, CH2),
4.38 (br, 2H, NCH2), 5.27 (s, 2H, OCH2), 6.97e7.00 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.18 (s, 1H, H-4), 7.24e7.39 (m, 9H, AreH, AA0BB0,
overlapped), 7.87(br, 1H, NH). EI-MS (m/z): 545 (M)þ.
5.28 (s, 2H, OCH2), 6.98e7.01 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.17
(s,1H, H-4), 7.22e7.29 (m, 1H, AreH, overlapped), 7.35e7.42 (m, 4H,
AreH, AA0BB0, overlapped), 7.46 (br, 1H, NH). EI-MS (m/z): 491 (M)þ.
5.1.6.10. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(hexylamino)
thiophene-3-carboxylate (11j). Yield: 36%. White solid, mp:
96e98 ꢀC. IR (KBr): 3330,1659, 1554, 1541, 1504, 1412, 1239, 1232,
5.1.6.4. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(propylamino)
thiophene-3-carboxylate (11d). Yield: 12%. White solid, mp:
116e118 ꢀC. IR (KBr): 3469, 2967, 1658, 1551, 1437, 1382, 1242, 1214,
1203, 1019, 831, 774, 727 cmꢃ1 1H NMR (300 MHz, CDCl3):
;
0.88e0.92 (t, J ¼ 6.9 Hz, 3H, CH3), 1.32e1.43 (m, 9H, 3(CH2) and
CH3), 1.66e1.73 (2H, m, CH2), 3.26 (br, 2H, NCH2), 4.24e4.31 (q,
J ¼ 6.9 Hz, 2H, CH2), 5.28 (s, 2H, OCH2), 6.98e7.01 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.17 (s, 1H, H-4), 7.22e7.29 (m, 1H, AreH, over-
lapped), 7.35e7.43 (m, 4H, AreH, AA0BB0, overlapped), 7.47 (br, 1H,
NH). EI-MS (m/z): 491 (M)þ.
1023, 832, 773 cmꢃ1 1H NMR (300 MHz, CDCl3): 1.03e1.05 (t,
;
J ¼ 7.2 Hz, 3H, CH3), 1.33e1.38 (t, J ¼ 7.2 Hz, 3H, CH3), 1.68e1.80 (m,
2H, CH2CH2CH3), 3.43 (br, 2H, NCH2), 4.29e4.37 (q, J ¼ 6.9 Hz, CH2,
CH2), 5.29 (s, 2H, OCH2), 6.98e7.01 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.22e7.29 (m, 4H, AreH, H-4, overlapped), 7.35e7.38 (d, J ¼ 7.2 Hz,
2H, AreH), 8.13e8.17 (br, 1H, NH). EI-MS (m/z): 463 (M)þ.
5.1.6.11. Ethyl5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(2-ethoxy-2-
oxoethylamino)thiophene-3-carboxylate (11k). Yield: 26%. Yellow
solid, mp: 136e138 ꢀC. IR (KBr): 3454, 3310, 2979, 1744, 1671, 1556,
5.1.6.5. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(isobutylamino)
thiophene-3-carboxylate (11e). Yield: 8%. White solid, mp:
120e122 ꢀC. IR (KBr): 3327, 2954, 1660, 1554 1503, 1465, 1243, 1216,
1138, 1013, 824, 778 cmꢃ1; 1H NMR (300 MHz, CDCl3): 1.01e1.03 (d,
J ¼ 6.9 Hz, 6H, (CH3)2), 1.33e1.38 (t, J ¼ 6.9 Hz, 3H, CH3), 1.95e2.06
(m, 1H, CH), 3.08 (br, 2H, NCH2), 4.24e4.31 (q, J ¼ 6.9 Hz, 2H, CH2),
5.28 (s, 2H, OCH2), 6.98e7.01 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.16
(s, 1H, H-4), 7.24e7.27 (m, 3H, AreH, overlapped), 7.35e7.38 (d,
J ¼ 7.8 Hz, 2H, AreH), 7.60 (br, 1H, NH). EI-MS (m/z): 476 (M)þ.
1541, 1506, 1437, 1240, 1215, 1017, 824, 776 cmꢃ1
;
1H NMR
(300 MHz, CDCl3): 1.26e1.31 (t, J ¼ 7.2 Hz, 3H, CH3), 1.34e1.39 (t,
J ¼ 7.2 Hz, 3H, CH3), 4.03e4.05 (d, J ¼ 5.4 Hz, 2H, NCH2), 4.24e4.34
(m, 4H, 2(CH2)), 5.28 (s, 2H, OCH2), 6.98e7.01 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.20 (s, 1H, H-4), 7.24e7.27 (m, 1H, AreH, over-
lapped), 7.35e7.38 (d, J ¼ 7.5 Hz, 2H, AreH), 7.39e7.42 (d, J ¼ 8.7 Hz,
2H, AreH, AA0BB0), 7.78 (t, J ¼ 6 Hz, 1H, NH). EI-MS (m/z): 507 (M)þ.
5.1.7. General procedure for the synthesis of compounds 12aek
To a solution of 11aek (1 equiv) in EtOH (15 mL) was added 1N
NaOH (12.5 equiv). The reaction mixture was heated to reflux for 2 h.
After cooling to room temperature, the reaction mixture poured into
water (50 mL) and extracted by diethyl ether. The resulting water
player was treated with 1N HCl to pH 4e5. The suspension solution
was extracted with EtOAc (2 ꢂ 25 mL). The combined organic layers
was dried with NaSO4 and concentrated in vacuo to afford 12aek.
Recrystallization from EtOAc gave pure product.
5.1.6.6. Ethyl 2-(allylamino)-5-(4-(2,6-dichlorobenzyloxy)phenyl)thiop-
hene-3-carboxylate (11f). Yield: 16%. Yellow solid, mp: 104e106 ꢀC. IR
(KBr): 3416, 3338,1657,1545,1438,1240,1210,1020, 831, 775 cmꢃ1; 1H
NMR (300 MHz, CDCl3): 1.34e1.38 (t, J ¼ 7.2 Hz, 3H, CH3), 3.92 (br, 2H,
NCH2), 4.25e4.31 (q, J ¼ 7.2 Hz, 2H, CH2), 5.28 (s, 2H, OCH2), 5.22 (dd,
J ¼ 1.2 Hz, J ¼ 10.2 Hz, 1H, C]CH), 5.32e5.39 (dd, J ¼ 1.2 Hz, J ¼ 10.2 Hz,
1H, C]CH), 5.87e6.00 (m, 1H, CH]C), 6.98e7.01 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.18 (s, 1H, H-4), 7.25e7.27 (t, 1H, J ¼ 7.2 Hz, AreH),
7.35e7.38 (d, J ¼ 7.2 Hz, 2H, AreH), 7.44e7.47 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.60 (br, 1H, NH). EI-MS (m/z): 461 (M)þ.
5.1.7.1. 2-(benzylamino)-5-(4-(2,6-dichlorobenzyloxy)phenyl)thioph-
ene-3-carboxylic acid (12a). Yield: 60%. Mp: 205e207 ꢀC. IR (KBr):
5.1.6.7. Ethyl5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(3-methylbut-2-
enylamino)thiophene-3-carboxylate (11g). Yield: 27%. Yellow solid,
mp: 118e120 ꢀC. IR (KBr): 3438, 3337, 1658, 1533, 1242, 1213, 1074,
828, 773 cmꢃ1; 1H NMR (300 MHz, CDCl3): 1.33e1.37 (t, J ¼ 7.2 Hz,
3H, CH3), 1.74e1.76 (d, J ¼ 6.3 Hz, 6H, (CH3)2), 3.92 (d, J ¼ 6.9 Hz, 2H,
NCH2), 4.23e4.30 (q, J ¼ 7.2 Hz, 2H, CH2), 5.28 (s, 2H, OCH2),
5.31e5.42 (t, J ¼ 6.0 Hz, 1H, CH]C), 6.98e7.01 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.18 (s, 1H, H-4), 7.25e7.27 (t, 1H, J ¼ 7.2 Hz, AreH),
7.35e7.38 (d, J ¼ 7.2 Hz, 2H, AreH), 7.40e7.44 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.60 (br, 1H, NH). EI-MS (m/z): 489 (M)þ.
3339, 3031, 1645, 1545, 1435, 1238, 1011, 819, 723, 574 cmꢃ1 1H
;
NMR (300 MHz, DMSO-d6): 4.48e4.50 (br, 2H, NCH2), 5.21 (s, 2H,
OCH2), 7.01e7.03 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.18 (s, 1H, H-4),
7.28e7.41 (m, 7H, AreH, overlapped), 7.44e7.49 (t, J ¼ 6.9 Hz, 1H,
AreH), 7.55e7.58 (d, J ¼ 7.2 Hz, 2H, AreH), 8.18e8.22 (t, J ¼ 6.3 Hz,
1H, NH). 12.15 (s, 1H, COOH). ESI-MS (m/z): 481.9 (M ꢃ 1)þ.
Formula: C25H19Cl2NO3S$H2O; Anal (C%, H%, N%) Calcd: 59.77, 4.21,
2.79; Found: 59.43, 4.11, 2.80.
5.1.7.2. 2-(2-chlorobenzylamino)-5-(4-(2,6-dichlorobenzyloxy)phe-
nyl)thiophene-3-carboxylic acid (12b). Yield: 25%. Yellow solid,
mp: 190e192 ꢀC (dec). IR (KBr): 3471, 3035, 2931, 1644, 1549, 1457,
5.1.6.8. Ethyl 2-(butylamino)-5-(4-(2,6-dichlorobenzyloxy)phenyl)thiop-
hene-3-carboxylate (11h). Yield: 20%. Yellow solid, mp: 113e115 ꢀC. IR
(KBr): 3346, 2967, 1664, 1538, 1503, 1238, 1212, 1027, 831, 774 cmꢃ1; 1H
NMR (300 MHz, CDCl3): 0.94e0.99 (t, J ¼ 7.2 Hz, 3H, CH3), 1.33e1.38 (t,
J ¼ 7.2 Hz, 3H, CH3), 1.40e1.52 (2H, CH2CH2CH3), 1.65e1.75 (m, 2H,
CH2CH2CH2CH3), 3.27 (br, 2H, NCH2), 4.24e4.31 (q, J ¼ 6.9 Hz, 2H, CH2),
5.28 (s, 2H, OCH2), 6.98e7.01 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.17 (s,
1H, H-4), 7.22e7.29 (m, 1H, AreH, overlapped), 7.35e7.42 (m, 4H,
AreH, AA0BB0, overlapped), 7.46 (br, 1H, NH). EI-MS (m/z): 477 (M)þ.
1438, 1245, 1014, 824, 776, 764 cmꢃ1 1H NMR (300 MHz, DMSO-
;
d6): 4.57e4.59 (d, J ¼ 6.3 Hz, 2H, NCH2), 5.21 (s, 2H, OCH2),
7.00e7.03 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.21 (s, 1H, H-4),
7.33e7.51 (m, 7H, AreH, AA0BB0, overlapped), 7.55e7.58 (d,
J ¼ 7.2 Hz, 2H, AreH), 8.20e8.25 (t, J ¼ 6.3 Hz, 1H, NH), 12.23 (s, 1H,
OH). ESI-MS (m/z): 515.9 (M ꢃ 1)þ. Formula: C25H18Cl3NO3S$H2O;
Anal (C%, H%, N%) Calcd: 55.93, 3.75, 2.61; Found: 55.56, 3.32, 2.61.
5.1.7.3. 2-(4-chlorobenzylamino)-5-(4-(2,6-dichlorobenzyloxy)phe-
nyl)thiophene-3-carboxylic acid (12c). Yield: 60%. Yellow solid,
mp: 154e156 ꢀC (dec). IR (KBr): 3437, 3338, 1636, 1551, 1539, 1437,
5.1.6.9. Ethyl 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(pentylamino)
thiophene-3-carboxylate (11i). Yield: 18%. White solid, mp:
117e119 ꢀC. IR (KBr): 3323, 2955, 1661, 1556, 1504, 1437, 1232, 1207,
1247, 1015, 823, 727, 574 cmꢃ1 1H NMR (300 MHz, DMSO-d6):
;
1021, 831, 774 cmꢃ1
;
1H NMR (300 MHz, CDCl3): 0.90e0.95 (t,
4.48e4.50 (d, J ¼ 6.3 Hz, 2H, NCH2), 5.21 (s, 2H, OCH2), 7.00e7.03
(d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.19 (s, 1H, H-4), 7.36e7.39 (d,
J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.42 (br, 4H, AreH, overlapped),
J ¼ 7.2 Hz, 3H, CH3), 1.33e1.43 (m, 7H, 2(CH2) and CH3), 1.67e1.74
(2H, m, CH2), 3.26 (br, 2H, NCH2), 4.24e4.31 (q, J ¼ 6.9 Hz, 2H, CH2),

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
3561
7.44e7.49 (t, 1H, J ¼ 6.9 Hz, AreH), 7.55e7.58 (t, J ¼ 7.2 Hz, 2H,
AreH), 8.23e8.27 (t, J ¼ 6.3 Hz, 1H, NH), 12.18 (s, 1H, COOH). ESI-
MS (m/z): 515.9 (M ꢃ 1)þ. Formula: C25H18Cl3NO3S$0.5H2O; Anal
(C%, H%, N%) Calcd: 56.89, 3.63, 2.65; Found: 56.56, 3.71, 2.61.
(M ꢃ 1)þ. Formula: C22H21Cl2NO3S; Anal (C%, H%, N%) Calcd: 58.67,
4.70, 3.11; Found: 58.43, 4.61, 2.78.
5.1.7.9. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(pentylamino)thio-
phene-3-carboxylic acid (12i). Yield: 48%. Yellow solid, mp:
143e145 ꢀC (dec). IR (KBr): 3480, 3360, 2953, 1647, 1548, 1504,
5.1.7.4. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(propylamino)thio-
phene-3-carboxylic acid (12d). Yield: 70%. Yellow solid, mp:
179e181 ꢀC (dec). IR (KBr): 3443, 3340, 2963,1644,1548,1453,1239,
1014, 821, 774, 574 cmꢃ1; 1H NMR (300 MHz, DMSO-d6): 0.91e0.96 (t,
J ¼ 7.2 Hz, 3H, CH3), 1.62e1.69 (m, 2H, CH2), 3.18e3.25 (q, J ¼ 6.6 Hz,
J ¼ 6.9 Hz, 2H, NCH2), 5.23 (s, 2H, OCH2), 7.03e7.06 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.19 (s, 1H, H-4), 7.41e7.44 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.47e7.50 (t, J ¼ 6.9 Hz, 1H, AreH), 7.55e7.58 (d, J ¼ 7.2 Hz,
2H, AreH), 7.69 (t, J ¼ 6.0 Hz,1H, NH),12.06 (s,1H, COOH). ESI-MS (m/
z): 433.9 (M ꢃ 1)þ. Formula: C21H19Cl2NO3S; Anal (C%, H%, N%) Calcd:
57.80, 4.39, 3.21; Found: 57.56, 4.51, 2.92.
1448, 1236, 1178, 1014, 827, 793, 571 cmꢃ1 1H NMR (300 MHz,
;
DMSO-d6): 0.87e0.91 (t, J ¼ 6.9 Hz, 3H, CH3), 1.32e1.35 (m, 2H,
2(CH2)),1.58e1.65 (m, 2H, CH2), 3.23e3.25 (q, J ¼ 6.6 Hz, 2H, NCH2),
5.23 (s, 2H, OCH2), 7.03e7.06 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.18
(s,1H, H-4), 7.41e7.44 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.46e7.50 (t,
J ¼ 6.9 Hz, 1H, AreH), 7.55e7.58 (d, J ¼ 7.2 Hz, 2H, AreH), 7.64e7.68
(t, J ¼ 6.6 Hz, 1H, NH), 12.05 (s, 1H, COOH). ESI-MS (m/z): 462
(M ꢃ 1)þ. Formula: C23H23Cl2NO3S; Anal (C%, H%, N%) Calcd: 59.48,
4.99, 3.02; Found: 59.11, 5.42, 2.88.
5.1.7.10. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(hexylamino)thio-
phene-3-carboxylic acid (12j). Yield: 50%. Yellow solid, mp:
150e152 ꢀC (dec). IR (KBr): 3467, 3411, 2941, 1649, 1548, 1503, 1453,
1383, 1236, 1013, 827, 793, 773, 725, 571 cmꢃ1; 1H NMR (300 MHz,
DMSO-d6): 0.83e0.89 (t, J ¼ 6.3 Hz, 3H, CH3), 1.17e1.37(m, 6H,
3(CH2)), 1.59e1.66 (m, 2H, CH2), 3.21e3.27 (q, J ¼ 6.6 Hz, 2H, NCH2),
5.23 (s, 2H, OCH2), 7.03e7.06 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.18
(s,1H, H-4), 7.41e7.44 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.46e7.49 (t,
J ¼ 6.9 Hz, 1H, AreH), 7.55e7.58 (d, J ¼ 7.2 Hz, 2H, AreH), 7.73e7.78
(t, J ¼ 5.7 Hz, 1H, NH), 12.03 (s, 1H, COOH). ESI-MS (m/z): 476
(M ꢃ 1)þ. Formula: C24H25Cl2NO3S; Anal (C%, H%, N%) Calcd: 60.25,
5.27, 2.93; Found: 60.42, 5.49, 3.01.
5.1.7.5. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(isobutylamino)thio-
phene-3-carboxylic acid (12e). Yield: 80%. Yellow solid, mp: 164 ꢀC
(dec). IR (KBr): 3470, 3351, 2961, 1642, 1547, 1451, 1383, 1244, 1017,
820, 571 cmꢃ1 1H NMR (300 MHz, DMSO-d6): 0.93e0.95 (d,
;
J ¼ 6.6 Hz, 6H, (CH3)2), 1.94e2.00 (m, 1H, CH), 3.06e3.11 (t,
J ¼ 6.6 Hz, 2H, NCH2), 5.23 (s, 2H, OCH2), 7.03e7.06 (d, J ¼ 8.7 Hz,
2H, AreH, AA0BB0), 7.18 (s, 1H, H-4), 7.41e7.44 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.46e7.49 (t, J ¼ 6.9 Hz, 1H, AreH), 7.55e7.58 (d,
J ¼ 7.2 Hz, 2H, AreH), 7.73e7.78 (t, J ¼ 6.3 Hz, 1H, NH), 12.07 (s, 1H,
COOH). ESI-MS (m/z): 448 (M ꢃ 1)þ. Formula: C22H21Cl2NO3S; Anal
Calcd: 58.35, 5.11, 2.98; Found: 58.40, 4.75, 3.18.
5.1.7.11. 2-(carboxymethylamino)-5-(4-(2,6-dichlorobenzyloxy)phe-
nyl)thiophene-3-carboxylic acid (12k). Yield: 67%. Yellow solid, mp:
158e160 ꢀC (dec). IR (KBr): 3438, 3068, 1724, 1652, 1551, 1437,
5.1.7.6. 2-(allylamino)-5-(4-(2,6-dichlorobenzyloxy)phenyl)thioph-
ene-3-carboxylic acid (12f). Yield: 84%. Yellowsolid, mp: 173e175 ꢀC
(dec). IR (KBr): 3343, 2956, 2571, 1639, 1550, 1538, 1505, 1454, 1438,
1242, 1016, 822, 776, 570 cmꢃ1; 1H NMR (300 MHz, DMSO-d6): 3.92
(d, J ¼ 4.2 Hz, 2H, NCH2), 5.28 (s, 2H, OCH2), 5.22 (dd, J ¼ 1.2 Hz,
J ¼ 10.2 Hz,1H, C]CH), 5.32e5.39 (dd, J ¼ 1.2 Hz, J ¼ 10.2 Hz, 1H, C]
CH), 5.85e5.96 (m, 1H, CH]C), 7.03e7.06 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.19 (s, 1H, H-4), 7.40e7.43 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0),
7.46e7.49 (t, 1H, J ¼ 7.8 Hz, AreH), 7.55e7.58 (d, J ¼ 7.8 Hz, 2H, AreH),
7.86 (br, 1H, NH), 12.15 (br, 1H, COOH). ESI-MS (m/z): 432 (M ꢃ 1)þ.
Formula: C21H17Cl2NO3S$0.5H2O; Anal (C%, H%, N%) Calcd: 56.89,
4.09, 3.16; Found: 56.76, 3.93, 3.20.
1248, 1018, 823, 776 cmꢃ1
;
1H NMR (300 MHz, DMSO-d6):
4.02e4.04 (d, J ¼ 6.0 Hz, 2H, NCH2), 5.23 (s, 2H, OCH2), 7.02e7.08
(d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.22 (s, 1H, H-4), 7.42e7.45 (d,
J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.46e7.49 (t, J ¼ 6.9 Hz, 1H, AreH),
7.55e7.58 (d, J ¼ 7.2 Hz, 2H, AreH), 7.85e7.87 (t, J ¼ 6.0 Hz, 1H,
NH),12.54 (br, 2H, 2(COOH)). ESI-MS (m/z): 450 (M ꢃ 1)þ. Formula:
C20H15Cl2NO5S; Anal (C%, H%, N%) Calcd: 53.11, 3.34, 3.10; Found:
53.02, 3.34, 3.14.
5.1.8. Ethyl5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(2-ethoxy-2-ox-
oacetamido)thiophene-3-carboxylate (13)
5.1.7.7. 5-(4-(2,6-dichlorobenzyloxy)phenyl)-2-(3-methylbut-2-eny-
lamino)thiophene-3-carboxylic acid (12g). Yield: 79%. Yellow solid,
mp: 203e205 ꢀC (dec). IR (KBr): 3480, 3330, 2918, 1644, 1547, 1455,
To a mixture of 8a (0.63 g, 0.0015 mol), dry triethylamine (3 mL)
and dry dichloromethane (10 mL), the solution of ethyl 2-chloro-2-
oxoacetate (0.68 mL, 0.006 mol) was added. The whole mixture was
stirred for overnight at room temperature. Water was added to the
mixture and extracted with dichloromethane (15 ꢂ 2 mL). The
combined organic layers were washed with brine and dried with
Na2SO4, then concentrated in vacuo. Recrystallization from EtOAc
gave pure 13 (0.57 g, 73%) as a yellow solid. mp: 168e170 ꢀC; 1H
1236, 1014, 822 cmꢃ1 1H NMR (300 MHz, DMSO-d6): 1.72 (s, 6H,
;
(CH3)2), 3.83e3.87 (t, J ¼ 6.0 Hz, 2H, CH2), 5.23 (s, 2H, OCH2),
5.27e5.29 (t, J ¼ 6.0 Hz, 1H, CH]C), 7.03e7.06 (d, J ¼ 8.7 Hz, 2H,
AreH, AA0BB0), 7.19 (s, 1H, H-4), 7.41e7.44 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 7.46e7.49 (t, J ¼ 6.9 Hz, 1H, AreH), 7.55e7.58 (d, J ¼ 7.2 Hz,
2H, AreH), 7.73e7.78 (t, J ¼ 6.0 Hz, 1H, NH), 12.03 (s, 1H, COOH).
ESI-MS (m/z): 460 (M ꢃ 1)þ. Formula: C23H21Cl2NO3S$H2O; Anal (C
%, H%, N%) Calcd: 57.50, 4.83, 2.92; Found: 57.48, 4.86, 2.87.
NMR (CDCl3, ppm):
d 1.39e1.45 (m, 6H, 2(CH3)), 3.05e3.14 (q,
J ¼ 6.9 Hz, 2H, CH2), 4.38e4.50 (q, J ¼ 6.9 Hz, 2H, CH2), 5.31 (s, 2H,
OCH2), 7.04e7.07 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.23e7.28 (t, 1H,
AreH, overlapped), 7.36e7.39 (d, J ¼ 7.8 Hz, 2H, AreH, overlapped),
7.38 (s, 1H, H-4, overlapped), 7.54e7.57 (d, J ¼ 8.7 Hz, 2H, AreH,
AA0BB0), 12.23 (s, 1H, CONH); EI-MS (m/z): 521 (M)þ.
5.1.7.8. 2-(butylamino)-5-(4-(2,6-dichlorobenzyloxy)phenyl)thioph-
ene-3-carboxylic acid (12h). Yield: 66%. Yellow solid, mp:
155e157 ꢀC (dec). IR (KBr): 3470, 3340, 2954,1631,1538,1453,1239,
1014, 825, 776, 574 cmꢃ1; 1H NMR (300 MHz, DMSO-d6): 0.91e0.96
(t, J ¼ 7.2 Hz, 3H, CH3), 1.28e1.41 (m, 2H, CH2), 1.58e1.65 (m, 2H,
CH2), 3.22e3.28 (q, J ¼ 6.9 Hz, 2H, NCH2), 5.23 (s, 2H, OCH2),
7.03e7.06 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.18 (s, 1H, H-4),
7.41e7.44 (d, J ¼ 8.7 Hz, 2H, AreH, AA0BB0), 7.47e7.50 (t, J ¼ 6.9 Hz,
1H, AreH), 7.55e7.58 (d, J ¼ 7.2 Hz, 2H, AreH), 7.64e7.68 (t,
J ¼ 6.0 Hz, 1H, NH), 12.05 (s, 1H, COOH). ESI-MS (m/z): 447.9
5.1.9. 2-(5-(4-(2,6-dichlorobenzyloxy)phenyl)-3-(ethoxycarbonyl)
thiophen-2-ylamino)-2-oxoacetic acid (15)
To a solution of 13 (0.08 g, 0.15 mmol) in THF (20 mL) was
added 1N NaOH (3.5 mL, 3.8 mmol). The reaction mixture was
stirred for 1 h at rt. Then, the reaction mixture was poured into
water (50 mL) followed extracted with diethyl ether (30 ml). The
water layer was adjusted with 1N HCl to pH 3e4. The suspension

3562
X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
solution was extracted with EtOAc (2 ꢂ 25 mL). The combined
organic layers was dried with NaSO4 and concentrated in vacuo to
afford 16p. Recrystallization from EtOAc gave pure product (0.05 g,
68%) as a yellow solid. mp: 193e195 ꢀC; IR (KBr): 3439, 3274, 2985,
5.3. Molecular docking
All the 3D structures of compounds were built in molecular
modeling software package Sybyl6.9. Partial atomic charges were
calculated by the GasteigereHuckel method, and energy minimi-
zations were performed by using the Tripos force field. The original
ligand-lauric acid as well as water molecules in mtFabH were
removed from the coordinated set. Hydrogen atoms were added to
the protein and kollman-all-atom charge was assigned.
The computer simulated automated docking studies were per-
formed using the widely distributed molecular docking software
(GOLD) based on the genetic algorithm (GA) method. The genetic
operators were 100 for the population size, 1.1 for the selection, 5
for the number of subpopulations, 100,000 for the maximum
number of genetic applications, and 2 for the size of the niche used
to increase population diversity. The weights were chosen so that
crossover mutations were applied with equal probability (95/95 for
the values) and migration was applied 5% of the time. ChemScore
function encoded in GOLD was applied to predict the binding mode
of mtFabH and compounds. The center of the bound lauric acid was
defined the binding site. Ten docking runs were performed per
structure. All poses were output into a single *.sdf file.
1770, 1711, 1565, 1510, 1383, 1251, 1175, 1018, 777, 717, 520 cmꢃ1
;
1H NMR (300 MHz, DMSO-d6):
d 1.33e1.38 (t, 3H, CH3), 4.31e4.38
(q, 2H, CH2), 5.27 (s, 2H, OCH2), 7.11e7.13 (d, J ¼ 8.4 Hz, 2H, AreH,
AA0BB0), 7.46e7.51 (m, 2H, AreH, H-4, overlapped), 7.56e7.59 (d,
J ¼ 8.4 Hz, 2H, AreH), 7.60e7.63 (d, J ¼ 8.4 Hz, 2H, AreH, AA0BB0),
12.05 (s, 1H, CONH). 13C NMR (300 MHz, DMSO-d6):
d
14.09, 60.7,
65.06, 114.63, 115.35, 118.70, 126.14, 126.63, 128.75, 131.50, 131.58,
133.22, 136.02, 145.15, 158.19, 163.84. ESI-MS (m/z): 492 (M ꢃ H)þ.
Formula: C22H17Cl2NO6S$H2O. Anal (C%, H%, N%) Calcd: 51.57, 3.74,
2.73. Found: 51.83, 3.63, 2.70.
5.2. Biological analysis
5.2.1. Antitubercular 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 concentration of 12.8 mM, and the final test concentrations
ranged from 128 to 0.5
prepared in Middlebrook 7H12 medium (7H9 broth containing
0.1% w/v casitone, 5.6 g/mL palmitic acid, 5 mg/mL bovine
serum albumin, 4 mg/mL catalase, filter-sterilized) in a volume of
mM. 2-fold dilutions of compounds were
Acknowledgement
m
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 allow-
ing us to use Pallas software.
100
(100
m
m
L in 96-well microplates (black viewplates). Mtb H37RV
L inoculum of 2 ꢂ 105 cfu/mL) was added, yielding a final
L. The plates were incubated at 37 ꢀC. On
L of 20% Tween 80 and 20 L of
testing volume of 200
the 7th day of incubation 12.5
m
m
m
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 ꢄ90% relative to the mean of replicate bacteria-
only controls.
References
[1] R. Johnson, E.M. Streicher, G.E. Louw, R.M. Warren, P.D. van Helden, T.C. Victor,
Curr. Issues Mol. Biol. 8 (2006) 97e112.
[2] J.T. Brooks, J.E. Kaplan, K.K. Holmes, C. Benson, A. Pau, H. Masur, Clin. Infect.
Dis. 48 (2009) 609e611.
[3] Global Tuberculosis Control, Epidemiology, Strategy, Financing WHO Report
2009 (2009). http://www.who.int/tb/publications/global_report/2009/en/
index.html.
[4] J.J. Ellner, M.J. Goldberger, D.M. Parenti, J. Infect. Dis. 163 (1991) 1326e1335.
[5] H. Oishi, T. Noto, H. Sasaki, K. Suzuki, T. Hayashi, H. Okazaki, K. Ando,
M. Sawada, J. Antibiot. (Tokyo) 35 (1982) 391e395.
5.2.2. Cytotoxicity assay
Vero cells (ATCC CRL-1586) were cultured in 10% fetal bovine
serum (FBS) in minimum essential medium Eagle. J774A.1 cells were
cultured in 10% FBS in Dulbecco’s modified Eagle’s medium (DMEM).
The cells were incubated at 37 ꢀC under 5% CO2 until confluent and
then diluted with phosphate-buffered saline to 106 cells/mL. In
a transparent 96-well plate (Falcon Microtest 96), threefold serial
dilutions of the macrolide stock solutions resulted in final concen-
[6] T. Noto, S. Miyakawa, H. Oishi, H. Endo, H. Okazaki, J. Antibiot. (Tokyo) 35
(1982) 401e410.
[7] R.A. Slayden, R.E. Lee, J.W. Armour, A.M. Cooper, I.M. Orme, P.J. Brennan,
G.S. Besra, Antimicrob. Agents Chemother. 40 (1996) 2813e2819.
[8] L. Kremer, J.D. Douglas, A.R. Baulard, C. Morehouse, M.R. Guy, D. Alland,
L.G. Dover, J.H. Lakey, W.R. Jacobs Jr., P.J. Brennan, D.E. Minnikin, G.S. Besra,
J. Biol. Chem. 275 (2000) 16857e16864.
[9] M.L. Schaeffer, G. Agnihotri, C. Volker, H. Kallender, P.J. Brennan, J.T. Lonsdale,
J. Biol. Chem. 276 (2001) 47029e47037.
[10] L. Kremer, L.G. Dover, S. Carrere, K.M. Nampoothiri, S. Lesjean, A.K. Brown,
P.J. Brennan, D.E. Minnikin, C. Locht, G.S. Besra, Biochem. J. 364 (2002)
423e430.
[11] S. Miyakawa, K. Suzuki, T. Noto, Y. Harada, H. Okazaki, J. Antibiot. (Tokyo) 35
(1982) 411e419.
[12] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 46
(2001) 3e26.
[13] S.J. Senior, P.A. Illarionov, S.S. Gurcha, I.B. Campbell, M.L. Schaeffer,
D.E. Minnikin, G.S. Besra, Bioorg. Med. Chem. Lett. 14 (2004) 373e376.
[14] S.J. Senior, P.A. Illarionov, S.S. Gurcha, I.B. Campbell, M.L. Schaeffer,
D.E. Minnikin, G.S. Besra, Bioorg. Med. Chem. Lett. 13 (2003) 3685e3688.
[15] P. Kim, Y.M. Zhang, G. Shenoy, Q.A. Nguyen, H.I. Boshoff, U.H. Manjunatha,
M.B. Goodwin, J. Lonsdale, A.C. Price, D.J. Miller, K. Duncan, S.W. White,
C.O. Rock, C.E. Barry 3rd, C.S. Dowd, J. Med. Chem. 49 (2006) 159e171.
[16] V. Bhowruth, A.K. Brown, S.J. Senior, J.S. Snaith, G.S. Besra, Bioorg. Med. Chem.
Lett. 17 (2007) 5643e5646.
trations of 102.4 to 0.42
bation at 37 ꢀC for 72 h, medium was removed and monolayers
were washed twice with 100 L of warm Hanks’ balanced salt
solution (HBSS). One hundred microliters of warm medium and
20 l of freshly made MTS-PMS[3-(4,5-dimethylthiazol-2-yl)-5-
mM in a final volume of 200 mL. After incu-
m
m
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-tetrazolium and
phenylmethasulfazone] (100:20) (Promega) were added to each
well, plates were incubated for 3 h, and absorbance was determined
at 490 nm.
5.2.3. Antitubercular activity against MDR-TB and XDR-TB clinical
strains
The MICs were determined using MDR-TB and XDR-TB clinical
strains according to published method by Wu et al. [42] with INH as
a positive control. Compound stock solutions were prepared in
DMSO at a concentration of 6.4 mM. The final test concentrations of
[17] A. Kamal, A.A. Shaik, R. Sinha, J.S. Yadav, S.K. Arora, Bioorg. Med. Chem. Lett.
15 (2005) 1927e1929.
[18] K. Ohata, S. Terashima, Bioorg. Med. Chem. Lett. 17 (2007) 4070e4074.
[19] K. Ohata, S. Terashima, Bioorg. Med. Chem. Lett. 18 (2008) 5598e5600.
[20] J.M. McFadden, G.L. Frehywot, C.A. Townsend, Org. Lett. 4 (2002) 3859e3862.
compound 10d ranged from 384 to 0.75
final test concentrations of compounds 15,12h,12k and INH ranged
from 64 to 0.5 M with 10 grads.
mM with 10 grads, and the
m

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 3551e3563
[31] R.A. Fernandes, P. Kumar, Tetrahedron Lett. 44 (2003) 1275e1278.
3563
[21] K.L. Dormann, R. Bruckner, Angew. Chem. Int. Ed. Engl. 46 (2007) 1160e1163.
[22] Y.M. Zhang, S.W. White, C.O. Rock, J. Biol. Chem. 281 (2006) 17541e17544.
[23] A. Kleemann, J. Engel, B. Kutscher, D. Reichert, Pharmaceutical Substances;
Syntheses, Patents, Applications, fourth ed., vols. 1e2, Thieme, 2001.
[24] M.C. Silva Lourenco, F. Rodrigues Vicente, M.G. de Oliveira Henriques,
A.L. Peixoto Candea, R.S. Borges Goncalves, T.C. Nogueira, M. de Lima Ferreira,
M.V. Nora de Souza, Bioorg. Med. Chem. Lett. 17 (2007) 6895e6898.
[25] A.M. Khalil, M.A. Berghot, M.A. Gouda, Eur. J. Med. Chem. 44 (2009)
4434e4440.
[32] T. Inokuchi, S. Matsumoto, T. Nishiyama, S. Torii, J. Org. Chem. 55 (1990)
462e466.
[33] F.D. Therkelsen, M. Rottlander, N. Thorup, E.B. Pedersen, Org. Lett. 6 (2004)
1991e1994.
[34] L. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997)
1004e1009.
[35] K. Falzari, Z. Zhu, D. Pan, H. Liu, P. Hongmanee, S.G. Franzblau, Antimicrob.
Agents Chemother. 49 (2005) 1447e1454.
[26] R.A. Daines, I. Pendrak, K. Sham, G.S. Van Aller, A.K. Konstantinidis,
J.T. Lonsdale, C.A. Janson, X. Qiu, M. Brandt, S.S. Khandekar, C. Silverman,
M.S. Head, J. Med. Chem. 46 (2003) 5e8.
[36] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Fernet, Adv. Drug Deliv. Rev. 46
(2001) 3e26.
[37] http://www.taacf.org/Process-text.htm.
[27] K.H. Choi, L. Kremer, G.S. Besra, C.O. Rock, J. Biol. Chem. 275 (2000)
28201e28207.
[38] X. Qiu, C.A. Janson, W.W. Smith, M. Head, J. Lonsdale, A.K. Konstantinidis,
J. Mol. Biol. 307 (2001) 341e356.
[28] J.N. Scarsdale, G. Kazanina, X. He, K.A. Reynolds, H.T. Wright, J. Biol. Chem. 276
(2001) 20516e20522.
[39] F. Musayev, S. Sachdeva, J.N. Scarsdale, K.A. Reynolds, H.T. Wright, J. Mol. Biol.
346 (2005) 1313e1321.
[29] F. Manetti, M. Magnani, D. Castagnolo, L. Passalacqua, M. Botta, F. Corelli,
M. Saddi, D. Deidda, A. De Logu, Chem. Med. Chem. 1 (2006) 973e989.
[30] D. Castagnolo, A. De Logu, M. Radi, B. Bechi, F. Manetti, M. Magnani, S. Supino,
R. Meleddu, L. Chisu, M. Botta, Bioorg. Med. Chem. 16 (2008) 8587e8591.
[40] X.Y. Lu, Y.D. Chen, Q.D. You, J. Enzyme Inhib. Med. Chem. 25 (2010) 240e249.
[41] http://www.rcsb.org/pdb/explore/explore.do%3FstructureId%3D1HZP.
[42] X.Y. Wu, Z.H. Liu, X.D. Shi, W.H. Zhang, G.M. Zhou, Chin. J. Clin. Lab. Sci. 25
(2007) 430e431.