Synthesis and antiproliferative activity of novel 4-substituted-phenoxy-benzamide derivatives

Article abstract of DOI:10.1016/j.cclet.2015.06.017

A series of novel 4-substituted-phenoxy-benzamide derivatives bearing an aryl cycloaliphatic amine moiety were synthesized and evaluated for antiproliferative activity against SW620, HT29 and MGC803 cancer cell lines in vitro. The pharmacological data demonstrated that the majority of target compounds exhibited moderate efficacy in HT29 and MGC803 cell lines. Compound 10c showed promising inhibition of hedgehog (Hh) signaling pathway in an Hh-related assay. In addition, the superposition pattern of 10c showed a good fit for a pharmacophoric model generated by Hh inhibitors and provided a basis for further structural optimization.

Full text of DOI:10.1016/j.cclet.2015.06.017

Accepted Manuscript
Title: Synthesis and antiproliferative activity of novel
4-substituted-phenoxy-benzamide derivatives
Author: Chi-Yu Sun Yang-Sheng Li Ai-Long Shi Ya-Fei Li
Rui-Fang Cao Huai-Wei Ding Qing-Qing Yin Li-Juan Zhang
Hua-Chuan Zheng Hong-Rui Song
PII:
S1001-8417(15)00273-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2015.06.017
CCLET 3374
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
24-3-2015
13-4-2015
29-5-2015
Please cite this article as: C.-Y. Sun, Y.-S. Li, A.-L. Shi, Y.-F. Li, R.-F. Cao, H.-W.
Ding, Q.-Q. Yin, L.-J. Zhang, H.-C. Zheng, H.-R. Song, Synthesis and antiproliferative
activity of novel 4-substituted-phenoxy-benzamide derivatives, Chinese Chemical
Letters (2015), http://dx.doi.org/10.1016/j.cclet.2015.06.017
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Graphical Abstract
Synthesis and antiproliferative activity of novel 4-substituted-phenoxy-benzamide derivatives
Chi-Yu Suna, Yang-Sheng Lia, Ai-Long Shia, Ya-Fei Lia, Rui-Fang Caoa, Huai-Wei Dinga,*, Qing-Qing Yina, Li-Juan Zhanga,
Hua-Chuan Zhengb, Hong-Rui Songa,*
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China
b The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, China
H
O
N
N
N
O
N
N
N
F3CO
N
O
N
N
OH
connector
connector
O
O
O
Functional group reversion
F3CO
F3CO
N
HN
N
N
O
Aryl
N
Part A
X
Part A
Part B
Part B
I
II
A series of 4-substituted-phenoxy-benzamide derivatives bearing an aryl cycloaliphatic amine moiety were synthesized and evaluated for their
antiproliferative activity. Three compounds were further examined for their hedgehog pathway inhibition.
Page 1 of 6

Original article
Synthesis and antiproliferative activity of novel 4-substituted-phenoxy-benzamide
derivatives
Chi-Yu Suna, Yang-Sheng Lia, Ai-Long Shia, Ya-Fei Lia, Rui-Fang Caoa, Huai-Wei Ding a,*, Qing-Qing Yina,
Li-Juan Zhanga, Hua-Chuan Zhengb, Hong-Rui Song a,1
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
b The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, China
ARTICLE INFO
ABSTRACT
A series of novel 4-substituted-phenoxy-benzamide derivatives bearing an aryl cycloaliphatic
amine moiety were synthesized and evaluated for antiproliferative activity against SW620,
HT29 and MGC803 cancer cell lines in vitro. The pharmacological data demonstrated that the
majority of target compounds exhibited moderate efficacy in HT29 and MGC803 cell lines.
Compound 10c showed promising inhibition of hedgehog (Hh) signaling pathway in an Hh-
related assay. In addition, the superposition pattern of 10c showed a good fit for a
pharmacophoric model generated by Hh inhibitors and provided a basis for further structural
Article history:
Received 24 March 2015
Received in revised form 13 April 2015
Accepted 29 May 2015
Available online
Keywords:
optimization
.
aryl cycloaliphatic amine
antiproliferative activity
hedgehog signaling
1. Introduction
The Hedgehog (Hh) protein family, which includes Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehogs, regulated cell
growth and migration during embryonic development [1-3]. Activation of the Hh signaling pathway is initiated by Shh ligands
bound to its receptor Patched (Ptch), which relieves its inhibition of Smoothen (Smo) receptor. Smo activation triggers a series of
intracellular events ultimately leads to specific gene expression mediated by the Gli family transcription factors[4,5]. Hh signaling
pathway was normally silent in adult tissues, nevertheless aberrant activation of the Hh pathway was associated with certain
cancers. Thus, the blockade of Hh pathway had been investigated as a novel strategy in cancer chemotherapy [6,7].
Inhibition of Smo activity has shown some promise in the treatment of cancers driven by activating mutations of the Hh
pathway [8-10]. Furthermore, several Hh pathway antagonists have proceeded to clinical development, among which vismodegib
(1, Fig.1) has obtained marketing authorization in the United States in 2012 [11, 12]. Sonidegib (2, Fig.2), a clinical stage Hh
inhibitor developed by Novartis, is awaiting for the registration in the U.S. for the treatment of patients with advanced basal cell
carcinoma. Sonidegib (2) bearing a morpholinopyridine unit suppressed the growth of Hh pathway-dependent tumors by selective
inhibition of the positive regulator smoothened (Smo) [13, 14]. LEQ506 (3a), a second-generation Hh inhibitor, is currently being
investigated in a Phase I clinical trial for patients with solid tumors. SAR studies had demonstrated the replacement of the benzylic
methylene linker with an oxygen atom (3b) was well tolerated, whereas replacement with an NH group (3c) resulted in a 10-fold
decrease in inhibition of the Hh pathway [15].
Inspired by the structural characteristics of Sonidegib (2) and LEQ506 (3a), we envisioned that the merging of these two
bioactive components would afford a hybrid structure with the potential for antiproliferative activity. We therefore adopted the
biaryl ether (Part A) active fragment from LEQ506 analog (3b) and morpholino pyridine (Part B) unit from Sonidegib (2) in target
compounds, and a carbonyl group or an amide group was selected as the linker between the two parts. Additionally, the order of
heteroaryl or aryl cycloaliphatic amine units in the target compounds attaching to the linker was reversed based on the functional
group reversion principle. Eventually, a series of 4-substituted-phenoxy-benzamide derivatives (I, II Fig.2) were obtained. In this
paper, the synthesis of these benzamide derivatives was reported, and their biological activities were evaluated.
2. Experimental
The key intermediates 7a-d were synthesized according to the routes outlined in Scheme 1. Aryl iodides were synthesized from
aryl amines by a diazotization reaction. The generated diazonium salts were iodinated with KI to give compounds 5a-d [16]. The
———
1 Corresponding author.
E-mail address: dinghuaiwei627@163.com (H.-W. Ding); hongruisonghrs@126.com (H.-R. Song);
Page 2 of 6

substituted iodobenzenes 5a-d were coupled to methylparaben to afford compounds 6a-d, via an N,N-dimethyl glycine-promoted
Ullmann coupling. Compounds 6a-d were further hydrolyzed to compounds 7a-d in excellent yields under reflux [17].
Compounds 10a-f were synthesized according to the routes outlined in Scheme 2. 2-Chloro-5-nitropyridine was converted to
compounds 8a-b in the presence of morpholine or piperidine [18]. The resulting nitro compounds 8a-b were reduced to the amino
compounds 9a-b using stannous chloride dihydrate and hydrochloric acid in aqueous ethanol, and then alkalized with sodium
hydroxide solution [19]. 7a-d were treated with thionyl chloride to produce the corresponding acyl chloride. 9a-b reacted with acyl
chloride to give the target compounds 10a-f [20].
Compounds 14a-n, 15a-b, 16a-j were synthesized according to the routes outlined in Scheme 3. Compound 11 was synthesized
from 6-chloronicotinic acid via an esterification reaction and then converted to compound 12 using a substitution reaction [21].
Compound 12 was deprotected to give compound 13 under acid conditions [22]. Intermediate 13 was reacted with the
corresponding acyl chlorides 7a-d to give the target compounds 14a-d, which were further hydrolyzed to compounds 15a-b.
Similarly, compounds 16a-j were obtained by the reaction of the acyl chloride 7c and various aryl piperazine [23].
General procedure for preparation of target compounds was given in the Supporting Information.
3. Results and discussion
All the target compounds (10a-f, 14a-d, 15a-b and 16a-j) were evaluated for their antiproliferative activity against human
colorectal cancer cell lines (SW620 and HT29), and human gastric cancer cell line (MGC803) using the MTT assay with
vismodegib as a positive control. The results expressed as half maximal inhibitory concentration (IC50) values are summarized in
Table 1.
Initially, target compounds were divided into two regions: diaryl ether (Part A), aryl cycloaliphatic amine moiety (Part B). In
general, most of them displayed high efficacy in HT29 and MGC803 cell lines. At the outset, our focus was on the modifications
of Part A, including the substitutions on the para position of phenyl. The para-trifluoromethoxy substituent 10c (IC50 = 1.15
µmol/L [HT29], IC50 = 0.56 µmol/L [MGC803]) showed decent activity, however, para-methoxy and para-chlorine led to a
significant loss of activity (10c vs 10a, 10d or 14a vs 14b, 14d) against MGC803 cells, confirming the beneficial impact of
trifluoromethoxy in the R1 position.
Further investigations focusing on Part B on the antiproliferative activity were performed. On comparing 10d with 10f, it was
found that morpholino pyridine surrogates were superior to piperidyl pyridine surrogates. Through functional group reversion,
pyridyl piperazidine analog 16c (IC50 = 4.33 µmol/L [HT29], IC50 = 5.35 µmol/L [MGC803]) was obtained and exhibited moderate
potencies. Addition of a polar methoxycarbonyl (14a, IC50 = 2.36 µmol/L [HT29]) group to pyridine increased the activity while
its hydrolysate 15a lost potency by nearly two-fold. When the pyridyl was changed to a pyrimidyl (16b, IC50 = 2.14 µmol/L
[HT29]), a two-fold increase of activity was observed. However, the trend did not hold in other analogs, as unsubstituted phenyl
piperazidine analogue 16j was less potent. Attempts to increase the antiproliferative activity were made by adding various groups
to the phenyl ring (Part B). It was worth noting that the para-fluorine (16h, IC50 =2.52 µmol/L [HT29]) and meta-methoxy (16a,
IC50 =1.95 µmol/L [HT29]) analogues were much more active than other substituted phenyl piperazidine analogues. Overall, the
structure-activity relationships (SAR) study revealed that the antiproliferative activity of 3 compounds (10a, 10c, 10d) was
comparable to that of the positive control vismodegib (1) in HT29 and MGC803 cells.
As shown in Table 2, the selected compound 10c, 10d, 16c were further tested for their Hh signaling inhibition using a
luciferase reporter assay [24, 25] in NIH3T3 cells carrying a stably transfected Gli-reporter construct (NIH3T3-Gli-luc reporter
cell line). This assay can effectively identify hedgehog signaling pathway inhibitors. It was suggested that compound 10c (IC50=
0.082 µmol/L) was less active as compared to the positive control vismodegib (IC50= 0.013 µmol/L). In addition, 10c and 10d
(IC50= 0.127 µmol/L) showed higher potency than 16c (IC50= 0.364 µmol/L) in parallel to their antiproliferative activity. Although
the hedgehog pathway inhibition of 10c was less potent than that of vismodegib, these results were encouraging and worthy of
further investigation owing to the large structural changes involved.
Then we examined if compound 10c fit the proposed pharmacophoric model for Hh antagonists. A conformational analysis was
performed using the Common feature program in the Discovery studio 3.0 software. This analysis was conducted on the three
compounds covering conformers with a range of 20 kcal/mol with respect to the global minimum that was used in building the
best pharmacophoric model. According to this, the low energy conformers of vismodegib (1), sonidegib (2) and LEQ506 (3a)
showed a very similar orientation and fulfilled all the pharmacophoric features of the model.
This model was built up by two hydrogen bond acceptor (HBA1-2) groups and two hydrophobic (HY1-2) regions. Fitting of
vismodegib (1), sonidegib (2) and LEQ506 (3a) to the pharmacophoric model was shown in Fig. 3A. Analysis of the superposition
pattern of 10c showed a good fit between the molecule and the pharmacophoric model (Fig. 3B). For instance, the carbonyl
oxygen in the amide moiety of compound 10c matched HBA1, and the oxygen atom in the morpholine moiety (Part B)
corresponded to the HBA2 feature of the model. Moreover, the two phenyl rings in the diaryl ether moiety (Part A) were
superimposed to the hydrophobic regions HY1 and HY2 perfectly.
4. Conclusion
In summary, a series of novel and distinctive 4-substituted-phenoxy-benzamide analogues were synthesized and characterized.
Furthermore, three human cancer cell lines were used to evaluate their antiproliferative activity; the majority of analogues
exhibited moderate activity and high selectivity in HT29 and MGC803 cell lines. In particular, the most promising compound 10c
Page 3 of 6

(Hh pathway inhibition IC50 = 0.082 µmol/L) displayed desirable antiproliferative activity with IC50 values of 1.15 µmol/L and
0.56 µmol/L against HT29 and MGC803 cell lines, respectively. The analysis of SAR indicated that compounds with a para-
trifluoromethoxyl group on Part A were more potent than those with other substituents. In addition, the morpholino pyridine
fragment in Part B exhibited higher efficacy as compared to the aryl piperazidine fragment. Further structural optimization studies
are presently in progress and will be reported in due course.
Acknowledgment
The work was supported by Program for Innovative Research Team of the Ministry of Education of China and Program for
Liaoning Innovative Research Team in University. Authors wish to thank to the Innovation and entrepreneurship training program
for college students in Liaoning Province (No. 201410163009).
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smoothened antagonists, Mol. Pharmacol. 78 (2010) 658-664.
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Ann. Pharmacother. 48 (2014) 99-106.
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volunteers, Cancer Chemother. Pharmacol. 74 (2014) 63-75.
[15] S. Peukert, F. He, M. Dai, et al., Discovery of NVP-LEQ506, a second-generation inhibitor of smoothened, Chem. Med. Chem. 8 (2013) 1261-1265.
[16] A.R. Hajipour, M. Seddighi, Application of [Hcpy] HSO4 brönsted acidic ionic liquid for the synthesis of aryl iodides from aromatic amines, Org.
Prep. Procd. Inter. 43 (2011) 292-296.
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derivatives, Molecules 17 (2012) 4703-4716.
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14071-14076.
[25] M.H. Xin, L.D. Zhang, F. Tang, et al., Design, synthesis, and evaluation of pyrrolo[2,1-f][1,2,4]triazine derivatives as novel hedgehog signaling
pathway inhibitors, Bioorg. Med. Chem. 22 (2014) 1429-1440.
Cl
O
Cl
N
H
N
S
O
O
1
Fig. 1 The structure of vismodegib (1)
H
N
N
connector
O
connector
O
O
Part A
I
F3CO
N
O
O
F3CO
2
F3CO
N
HN
N
N
Functional group reversion
O
X
Aryl
N
N
X
N
N
Part A
N
Part B
N
Part B
N
3a
X=CH2
II
OH
3b X=O
3c
X=NH
Fig. 2 Sonidegib (2), LEQ506 analogues (3a-c) and general structure of the target compounds (I, II).
O
O
NH2
I
a
b
c
4a, 5a, 6a, 7a
4b, 5b, 6b, 7b
4c, 5c, 6c, 7c: R1= -OCF3
4d, 5d, 6d, 7d: R1= -Cl
: R1= -OCH3
: R1= -CH3
O
O
R1
R1
R1
R1
O
OH
6a-d
7a-d
5a-d
4a-d
Scheme 1 Reagents and conditions: (a) (1) NaNO2, H2SO4, 0 °C, 2h; (2) KI, dichloromethane, 0 oC, 6h; (b) 4-methyl-1-iodobenzene, CuI, N,N-
dimethylglycine, Cs2CO3, 1,4-dioxane, reflux under nitrogen atmosphere, 24h; (c) NaOH, ethanol solution, reflux, 3h
O
H2N
O2N
O
O2N
a
b
c
R1
10a: R1= -OCH3, X= O
10c: R1= -OCF3, X= O
10e: R1= -CH3, X= CH2 10f: R1= -Cl, X= CH2
10b: R1= -CH3, X= O
10d: R1= -Cl, X= O
HN
N
N
N
N
N
N
Cl
X
X
9a: X= O
9b: X= CH2
8a: X= O
8b: X= CH2
N
X
9a-b
10a-f
8a-b
Scheme 2 Reagents and conditions: (a) morpholine or piperidine, K2CO3, THF, reflux, 4h; (b) SnCl2·2H2O, ethanol, HCl, reflux, 8h; (c) acyl chloride of
7a-d, Et3N, dichloromethane, 0 °C, 12h.
Page 5 of 6

O
Cl
N
Cl
N
a
b
c
NH
N
O
HCl
N
N
N
N
O
COOH
O
O
O
O
O
11
12
13
N
O
O
14a
: R1= -OCF3,
R2=
R2=
R2=
R2=
COOCH3
R1
N
N
R1
N
N
N
d
15a: R1= -OCF3,
R2=
R2=
e
14b: R1= -Cl,
N
COOH
COOH
COOCH3
COOCH3
COOCH3
N
R2
O
R2
O
N
N
15b
: R1= -Cl,
14c: R1= -CH3,
14d: R1= -OCH3,
14a-d
15a-b
O
Cl
Cl
Cl
O
N
F3CO
f
N
16i: R2=
16j: R2=
16g
: R2=
16a: R2=
16c: R2=
16e: R2=
16f: R2=
7c
N
O
F
O
R2
N
N
16h: R2=
F
16b
: R2=
16d
: R2=
16a-j
CF3
Scheme3 Reagents and conditions: (a) Methanol, concentrated sulfuric acid, 65°C; (b) 1-boc-piperazine, K2CO3, DMAP, DMF, 110 oC, 24h; (c)
concentrated hydrochloric acid in dioxane, dichloromethane 30 oC, 2h; (d) chloride of 7a-d, Et3N, dichloromethane, 0 oC, 4h; (e) NaOH, ethanol solution,
reflux, 3h; (f) chloride of 7a-d, aryl piperazidine or heteroaryl piperazidine, Et3N, dichloromethane, 0 oC, 4h.
Table 1 In vitro cell growth inhibition by the target compounds and vismodegib (1).
IC50 (µmol/L) ± SDaa
IC50 (µmol/L) ± SDaa
Compd.
Compd.
SW620
HT29
MGC803
2.36 ± 0.35
3.08 ± 0.63
0.56 ± 0.11
1.77 ± 0.48
3.82 ± 1.02
2.80 ± 0.91
3.16 ± 0.87
5.53 ± 1.66
SW620
HT29
MGC803
10a
10b
10c
10d
10e
10f
14a
14b
14c
26.76 ± 3.56
79.49 ± 10.28
7.24 ± 0.80
23.19 ± 3.03
>100
48.49 ± 6.17
14.85 ± 3.12
15.96 ± 4.25
>100
1.87 ± 0.22
2.82 ± 0.73
1.15 ± 0.24
1.53 ± 0.50
4.41 ± 1.35
3.45 ± 0.28
2.36 ± 0.96
3.88 ± 1.05
15b
16a
16b
16c
16d
16e
16f
16g
16h
16i
26.07 ± 3.64 16.20 ± 4.60 13.29 ± 2.43
12.56 ± 2.87
10.71 ± 1.98
19.08 ± 3.62
46.58 ± 7.41 14.58 ± 3.81 10.84 ± 2.59
20.34 ± 3.53 8.26 ± 2.27 6.27 ± 0.82
72.35 ± 9.60 34.93 ± 6.54 33.21 ± 5.88
65.62 ± 9.75 26.31 ± 4.82 38.54 ± 5.76
14.27 ± 2.82
51.83 ± 8.36 13.27 ± 2.37 14.18 ± 2.52
32.95 ± 4.51 26.08 ± 4.12 25.36 ± 3.35
1.95 ± 0.62
2.14 ± 0.49
4.33 ± 1.65
2.91 ± 0.57
2.63 ± 0.61
5.35 ± 1.40
16.20 ± 4.65 20.65 ± 2.90
2.52 ± 0.71
3.43 ± 0.94
14d
5.19 ± 1.21
57.31 ± 8.65
10.92 ± 2.76
11.74 ± 3.03
5.00 ± 1.07
1.08 ± 0.16
9.98 ± 1.78
9.19 ± 1.32
2.39 ± 0.17
15a
16j
Vismodegibb
Bold values show the IC50 values of target compounds lower than the values of the positive control.
a IC50: concentration of the compound (µmol/L) producing 50% cell growth inhibition after 48 h of drug exposure, as determined by the MTT assay. Each experiment was
carried out in triplicate.
b Used as a positive control.
Table 2 The hedgehog pathway inhibition of compound 10c, 10d, 16c and vismodegib (1).
Compd.
10c
10d
NIH3T3-Gli-luc IC50 (µmol/L)a
0.082
0.127
0.364
0.013
16c
vismodegibb
a The values are an average of triplicate separate determinations.
b Used as a positive control.
Fig. 3 A. Pharmacophore models for vismodegib, sonidegib and LEQ506. Graphical representation of vismodegib (pink), sonidegib (blue), LEQ506 (yellow)
fitted to the proposed pharmacophoric model for hedgehog antagonists. Pharmacophoric features are color coded: green for hydrogen bond acceptor groups
(HBA1-2) and cyan for hydrophobic regions (HY1-2). HBA features are constituted by a smaller sphere accommodating the hydrogen bond acceptor group, by a
directionality vector represented by an arrow, and by a larger sphere intended to allocate the hydrogen bond donor group of the target macromolecule. B.
Pharmacophore model for 10c. The atoms are color coded: Orange, carbon; white, hydrogen; red, oxygen; blue, nitrogen
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