Discovery of Novel Aryl Carboxamide Derivatives as Hypoxia-Inducible Factor 1α Signaling Inhibitors with Potent Activities of Anticancer Metastasis

Article abstract of DOI:10.1021/acs.jmedchem.9b01313

In order to discover novel hypoxia-inducible factor 1 (HIF-1) inhibitors for the cancer metastasis treatment, 68 new aryl carboxamide compounds were synthesized and evaluated for their inhibitory effect by dual luciferase-reporter assay. Based on five rounds of investigation on structure-activity relationships step by step, compound 30m was discovered as the most active inhibitor (IC₅₀ = 0.32 μM) with no obvious cytotoxicity (CC₅₀ > 50 μM). It effectively attenuated hypoxia-induced HIF-1α protein accumulation and reduced transcription of vascular epidermal growth factor in a dose-dependent manner, which was further demonstrated by its inhibitory potency on capillary-like tube formation, angiogenesis of zebrafish as well as cellular migration and invasion. Importantly, compound 30m exhibited antimetastatic potency in breast cancer lung metastasis in the mice model, indicating its promising therapeutic potential for prevention and treatment of tumor metastasis. These results definitely merit attention for further rational design of more efficient HIF-1 inhibitors in the future.

Full text of DOI:10.1021/acs.jmedchem.9b01313

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Article
Discovery of Novel Aryl Carboxamide Derivatives as
Hypoxia-inducible Factor (HIF) 1# Signaling Inhibitors
with Potent Activities of Anticancer Metastasis
Mingming Liu, Yuru Liang, Zhongzhen Zhu, Jin Wang, Xingxing Cheng,
Jiayi Cheng, Binpeng Xu, Rong Li, XinHua Liu, and Yang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01313 • Publication Date (Web): 26 Sep 2019
Downloaded from pubs.acs.org on September 26, 2019
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Discovery of Novel Aryl Carboxamide Derivatives as
Hypoxia-inducible Factor (HIF) 1α Signaling Inhibitors with Potent
Activities of Anticancer Metastasis
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1,3,4,#
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Mingming Liu
, Yuru Liang , Zhongzhen Zhu , Jin Wang , Xingxing Cheng ,
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Jiayi Cheng , Binpeng Xu , Rong Li , Xinhua Liu * and Yang Wang *
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.
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School of Pharmacy, Anhui Medical University, Hefei 230032, China
School of Pharmacy, Fudan University, Shanghai 201203, China
Shanghai Key Laboratory of New Drug Design, East China University of Science
and Technology, Shanghai 200237, China
4.
Anhui Chem-Bright Bioengineering Co., Ltd., Huaibei 235025, China
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Abstract
In order to discover novel HIF-1 inhibitors for cancer metastasis treatment, 68 new
aryl carboxamide compounds were synthesized and evaluated for their inhibitory
effect by dual luciferase-reporter assay. Based on five rounds of investigation on
structure-activity relationships (SARs) step by step, compound 30m was discovered
as the most active inhibitor (IC =0.32 μM) with no obvious cytotoxicity (CC >50
0
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μM). It effectively attenuated hypoxia-induced HIF-1α protein accumulation and
reduced transcription of VEGF in a dose-dependent manner, which was further
demonstrated by its inhibitory potency on capillary-like tube formation, angiogenesis
of zebrafish as well as cellular migration and invasion. Importantly, compound 30m
exhibited anti-metastatic potency in breast cancer lung metastasis in mice model,
indicating its promising therapeutic potential for prevention and treatment of tumor
metastasis. These results definitely merit attention for further rational design of more
efficient HIF-1 inhibitors in the future.
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1
. Introduction
Cancer metastasis is a multiple process in which cancer cells disseminate from the
primary tumor to distant secondary organs through the circulatory system. The
migratory and invasive capabilities facilitate metastatic cells to invade through
basement membrane, intravasate into Lumina or blood vessels, and extravasate into
the parenchyma of distant tissues, thereby generating clinically detectable metastatic
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tumors . Metastasis remains the leading cause of therapeutic failure and responsible
for over 90% of cancer-associated patient mortality, mainly due to the reason that
most of the current chemotherapeutic agents control tumor growth rather than its
3
-4
metastasis. Recent advance in the understanding of the molecular signaling pathway
that regulates metastatic spread provides potential therapeutic targets for cancer
5-6
metastasis prevention and inhibition.
Hypoxia-inducible factor 1 (HIF-1), a transcription factor that responds to a
hypoxic environment, is a heterodimer composed of HIF-1α and HIF-1β subunits.
Under normoxia condition, the hydroxylated HIF-1α subunit is recognized by the von
Hippel–Lindau (VHL) E3-ubiquitin ligase complex, and subjected to proteasomal
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-8
degradation subsequently . Whereas, under hypoxic conditions, the absence of
oxygen and decreasing hydroxylation stabilize HIF-1α, resulting in its nuclear
translocation and dimerization with HIF-1β. The heterodimer binds to the hypoxia
responsive element (HRE) located in the region of target genes promotor, and
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therefore activates transcription of a number of downstream target genes.
These
genes encode proteins, including vascular epidermal growth factor (VEGF), matrix
metalloproteases (MMPs), snail1, twist and glucose transporter type 1 (GLUT1), et
11-15
al.,
which promote cancer development and metastasis through enhancing cellular
motility and invasiveness, inducing epithelial-mesenchymal transition (EMT) and
cancer stem cell properties, promoting angiogenesis, reprogramming glucose
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metabolism, and resisting apoptosis.
Recent studies have highlighted that dramatic
overexpression of HIF-1 was measured in many human cancers, such as breast cancer,
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ovarian cancer, hepatocellular carcinoma, cervical cancer and so on, leading to tumor
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metastasis and poor patient prognosis.
Therefore, HIF-l has been considered to be
a promising target for the development of novel therapeutics against cancer
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metastasis.
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In recent years, a number of small-molecule inhibitors have been identified with
the capacity to reduce the transcriptional activities of HIF-1 or to induce HIF-1α
degradation (Figure 1), including adamantyl-based inhibitors (1, LW6),
carborane-based inhibitors (2, GN26361,), manassantin A (3) and derivatives,
deguelin (4) and its ring-truncated derivatives (5, SH-1280), sulfonamide derivatives
2
6-33
(6, KCN), YC-1 (7) and derivatives etc.
Most of these inhibitors were discovered
through high throughput screening (HTS) or natural products screening with large
molecular weight, complicated chemical structures, relative hydrophobicity, poor
water solubility and low potency, which thereby limited their further development.
Unfortunately, there is no HIF-1 inhibitor has been approved by FDA for cancer
treatment up to date. Therefore, there is still an urgent need to discover novel HIF-1
inhibitors with sufficient potency, low toxicity, good druggability and novel chemical
diversity.
OH
H
O
O
N
H
B
HO
OH
OH
H
HO OH
O
N
O
O
O
=
=
BH
C
MeO
OMe
O
MeO
OMe
OMe
O
OMe
MeO
LW6 (1)
GN26361 2
Manassantin A(3)
(
)
OMe
OMe
MeO
MeO
O
O
O
O
OMe
N
N
H
H
O
S
N
OMe
OMe
O
O
O
O
O
OH
Deguelin(4)
SH-1280 5
(
)
( )
KCN 6
YC-1(7)
Figure 1. Structures of representative HIF-1α inhibitors.
Considering the advantages of fragment-based approaches in discovering novel
leading compounds, an in-house fragments collection (M.W. < 300 Da) was screened
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using HRE luciferase reporter assay to identify hits of HIF-1 inhibitors for further
optimization. From the screening, a thiazole carboxamide fragment 8 (M.W. = 246 Da,
Figure 2) was identified as a preliminary hit containing moderate inhibitory effect
with the IC value of 55.7 μM. Biological evaluation showed that this hit inhibited
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the invasion and migration of breast cancer MDA-MB-231 cells without obvious
cytotoxicity. These results encouraged us to conduct further structural optimization
and structure-activity relationships (SARs) studies to improve the potency for
intervention of cancer metastasis. Therefore, based on the chemical structure of this
hit and rational drug design, a small-molecular library of 68 novel aryl carboxamide
derivatives was constructed and evaluated as HIF-1 inhibitors.
2
. Design
In order to carry out more extensive rational design, the modification and SAR
study of hit 8 were approached in a step-by-step manner. As shown in Figure 2, the
hit was divided into three regions, namely the methyl group (region A), the thiazole
ring (region B) and the phenyl group (region C) by the connection of N-ethyl amide
(Linker). Firstly, region C was modified with several substituted phenyl and
heterocyclic groups as well as the length of Linker was investigated, resulting in
2
-chlorophenyl and 4-pyridyl analogs with improved activities (step 1). For fragment
growing, phenyl or heterocycles were introduced to replace the methyl group (region
A). The results from SAR showed that introduction of phenyl into region A achieved
increasing activities (step 2). Next, the thiazole ring (region B) was replaced with a
variety of five- and six-membered aromatic rings, including pyridine-2-carbonamide
which showed relatively better activity (step 3). Then, the substituent on the phenyl
ring of region C was further optimized, and the replacement of chlorine with fluorine
dramatically increased activity (step 4). Finally, the phenyl group of region A was
replaced with a variety of heterocycles and substituted phenyl, and 2-methoxyl phenyl
substituent turned out to be the best alternative with activity of almost 170-fold
stronger than that of hit 8 (step 5).
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Figure 2. Structural optimization step by step.
3
. Chemistry
As depicted in Scheme 1, thiazole and other aryl carboxamides (8, 10a-h,12a-f)
were prepared by acylation of various substituted amines with corresponding aryl
carbonyl chlorides, which were generated by the reaction of commercial available
acids (9 and 11a-c) with oxalyl chloride.
Scheme 1. Synthesis of 8, 10a-h and 12a-f
S
S
H
i. COCl , CH Cl , cat. DMF, rt, 1 h
OH
(
)
2
2
2
N
R
H C
H C
3
3
N
N
ii. corresponding amines
Et N, CH Cl rt, 6-12 h
O
O
3
2
2,
9
8, 10a-h 59-93%
( )
8
1
1
: R = phenethyl
0a: R = benzyl
10c: R = 2-chlorophenethyl 10f: R = 2- pyridin-2-yl ethyl
( )
10d: R = 3-chlorophenethyl 10g: R = 2- pyridin-3-yl ethyl
( )
0b: R = 3-phenylpropyl 10e: R = 4-chlorophenethyl 10h: R = 2- pyridin-4-yl ethyl
(
)
O
i (
COCl , CH Cl , cat. DMF, rt, 1 h
.
O
)
2
2
2
H C
R
H C
3
3
ii di g
. corres on n am nes
Ar
N
Ar OH
p
i
H
Et N, CH Cl rt, 6-12 h
3
2
2,
1
1a-c
12a-f 61-80%
(
)
11a,12a-b: Ar =
11b, 12c-d: Ar =
12a, c, e: R = 2-chlorophenyl
12b, d, f: R = pyridin-4-yl
O
N
1
1c, 12e-f: Ar =
The synthesis of five-membered heterocycles carboxamides 15a-f and 18a-j was
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described in Scheme 2. As key intermediate, aryl thiazolyl carboxylic acid ethyl esters
(13a-c), phenyl oxazolyl carboxylic acid ethyl esters (16a-b), and phenyl oxadiazolyl
3
4-35
carboxylic acid ethyl esters (16c-e) were synthesis according to the literatures
,
respectively (see supporting information). Hydrolysis of the esters 13a-c and 16a-e
with LiOH in ethanol produced the corresponding acids 14a-c and 17a-e, respectively.
Finally, the targeted thiazole, oxazole and oxadiazole carboxamides 15a-f and 18a-j
were obtained by a similar procedure described in Scheme 1.
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Scheme 2. Synthesis of 15a-f and 18a-j
i. COCl , CH Cl
2
cat. DMF, rt, 1 h;
(
)
2
2
S
H
S
LiOH aq. , EtOH
S
R
N
(
)
Ar1
Ar1
Ar1
N
COOEt
rt, 1 h
COOH ii. corres ondin amines
N
N
p
g
O
Et N, CH Cl , rt, 6-12 h
3
2
2
13a-c
14a-c
1
5 -f 48-57% for two steps
a
(
)
1
1
1
1
3a, 15a-b: Ar = phenyl
15a, c, e: R = 2-chloro hen l
p
y
1
3b, 15c-d: Ar = pyridin-3-yl
15b, d, f: R = ridin-4- l
py
y
1
3c, 15e-f: Ar = pyridin-4-yl
i. COCl , CH Cl
cat. DMF, rt, 1 h;
(
)
2
2
2
H
Ar2
COOEt
Ar
2
Ar2
LiOH aq. ,
N
(
)
COOH
R
EtOH rt, 1 h
ii. corresponding amines
Et N, CH Cl , rt, 6-12 h
O
3
2
2
16a-e
17a-e
18a- 46-63% for two steps
j (
)
N O
O
18a, c, e, g, i: R = 2-chlorophenyl
6a, 18a-b: Ar2=
16d, 18g h A
r =
- :
2
1
N
N N
O
18b, d, f, h, : R = pyridin-4-yl
N
N
j
16b, 18c-d: Ar2=
16e, 18i- : Ar =
2
j
O
O N
N
6c, 18e-f: Ar2=
1
As outlined in Scheme 3, Suzuki coupling of phenylboronic acid (21) with
-bromopicolinic acid (22a) or 6-bromonicotinic acid (22b) in the presence of
Pd(PPh ) Cl afforded phenyl substituted pyridine carboxylic acids 23a-b.
5
3
2
2
pyrimidine-4-carboxamide 25a and pyrimidine-2-carboxamide 25b were synthesized
3
6
according to the literature (see supporting information) and were then hydrolyzed in
0% H SO (aq.) to give the corresponding pyrimidine carboxylic acids 26a-b,
2
2
4
respectively. Finally, the targeted amides 20a-b, 24a-d, and 27a-b were obtained by a
similar procedure described in Scheme 1.
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Scheme 3. Synthesis of 20a-b, 24a-d, and 27a-b.
i. COCl , CH Cl
(
)
2
2
2
O
cat. DMF, rt, 1 h;
20a: R = 2-chlorophenyl
R = ridin-4- l
COOH
20b
:
py
y
HN
ii. corresponding amines
Et N, CH Cl , rt, 6-12 h
R
3
2
2
1
9
20a-b 74%
( )
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0
Pd PPh 2Cl
i. COCl , CH Cl
( )
2 2 2
cat. DMF, rt, 1 h;
OH
B
(
3
)
2
Ar1
COOH
H
K CO
Ar1
1
2
3
N
A
r
OH
Br
Ph
R
COO
H
dioxane, H O
ii. corresponding amines
Et N, CH Cl , rt, 6-12 h
2
O
N protection
2
3
2 2
reflux, 12 h
21
22a-b
23a-b 61-69%
24a-d 72-77%
(
)
(
)
1
22a, 23a, 24a-b: Ar =
24a, c: R = 2-chloro hen l
p
y
N
2
4b, d: R = pyridin-4-yl
1
2
2b, 23b, 24c-d: Ar =
N
H
N
R
i. COCl , CH Cl
2
Ar2 NH2
O
r2
(
)
2
2
Ar2
A
OH
2
0% H SO
cat. DMF, rt, 1 h;
ii. corresponding amines
Et N, CH Cl , rt, 6-12 h
2
4
o
O
O
1
00 C
5
-10 h
3
2
2
2
5a-b
26a-b (34-39%)
27a-b (71-73%)
N
N
2
25a, 26a, 27a: Ar =
27a: R = 2-chlorophenyl
7b: R = pyridin-4-yl
2
N
2
25b, 26b, 27b: Ar =
N
As described in Scheme 4, the synthesis of series of 5-phenylpicolinic amides 28a-l
was similar to that of compounds 20a-b. Meanwhile, acylation of
2
-fluorophenethylamine by 5-bromopicolinic acid (22a), followed by Suzuki coupling
reaction with various aryl boronic acids, gave the target 5-aryl substituted picolinic
amides 30a-r.
Scheme 4. Synthesis of 28a-l and 30a-r
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i. COCl , CH Cl2
cat. DMF, rt, 1 h;
(
)
2
2
H
OH ii. corresponding amines
Et N, CH Cl , rt, 6-12 h
N
N
N
R
3
2
2
O
O
-
2
3a
2 a-l
8
63 78%
(
)
a: R = hen l
b: R = 3-chlorophenyl
c: R = 4-chloro hen l
e: R = 3-fluoro hen l
yp
i: R = 4-h drox hen l
p
y
p
y
y
y
f: R = 4-fluorophenyl
j: R = 4-aminophenyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
p
y
g: R = ridin-2- l
py
y
k: R = 4-meth l hen l
y p
y
d: R = 2-fluorophenyl
h: R = pyridin-3-yl
l: R = 4-nitrophenyl
Br
ArB OH
)
2
Ar
Br
i. COCl , CH Cl
(
)
2
2
2
(
cat. DMF, rt, 1 h;
H
Pd PPh3 2Cl2
H
(
)
N
N
OH
N
N
N
ii. corresponding amine
Et N, CH Cl , rt, 6-12 h
K CO , dioxane
H O, N protection
2
3
O
O
O
3
2
2
2
2
F
F
reflux, 12 h
30a-r 61-70%
2
2a
29 76
%)
(
)
(
a: Ar = furan-2- l
b: Ar = furan-3-yl
g: Ar = 2-fluoro hen l
m: Ar = 2-methox hen l
yp
n: Ar = 3-methoxyphenyl
o: Ar = 4-methox hen l
p: Ar = 2-cyanophenyl
y y
p
q: Ar = 3-c ano hen l
r: Ar = 4-cyanophenyl
y
p
y
y
h: Ar = 3-fluorophenyl
i: Ar = 4-fluoro hen l
c: Ar = thio hen-2- l
p
y
p
y
yp
y
d: Ar = thiophen-3-yl
e: Ar = ridin-4- l
f: Ar = 1H-pyrazol-4-yl
j: Ar = 2-hydroxyphenyl
py
y
k: Ar = 3-h drox hen l
y
yp
y
l: Ar = 4-hydroxyphenyl
3
3
. RESULT AND DISCUSSION
.1 Cell-based HRE Assay and SAR Studies.
To improve the potency of hit 8 and identify more effective inhibitors of HIF-1, 68
new compounds were designed and synthesized based on SAR studies step by step.
The inhibition rates (IR) of synthesized compounds were initially determined at 10
μM by HRE luciferase reporter assay in human hepatocellular carcinoma LM3 cells
under hypoxia condition. In the assay, LM3 cells were co-transfected with a HIF-1
reporter vector which contains five copies of HRE sequences located in the region of
firefly luciferase gene promotor along with pRL-SV40 plasmid encoding Renilla
luciferase. After transfection, cells were treated with tested compounds and incubated
under hypoxia. Under hypoxic condition, accumulated heterodimers of HIF-1α and
HIF-1β bound to the HRE sequences and prompted the transcription of firefly
luciferase. Inhibition of HIF-1 signaling by tested compounds reduced the expression
of firefly luciferase leading to diminish the firefly luminescence. To determine the
inhibitory effect of synthesized compounds, firefly luminescence signal was measured
by using a microplate reader and normalized to the luminescence of Renilla luciferase.
37
LW6 (1) was selected as positive control.
Firstly, we examined the effect of the phenethyl group (region C and Linker) of hit
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
on inhibitory activity. As listed in Table 1, replacement of phenethyl by benzyl (10a)
or 3-phenylpropyl (10b) decreases the potency, indicating that favorable length of two
C atoms between N and phenyl. Introduction of a chlorine atom at ortho- position of
the phenyl ring (10c) significantly increased the potency, whereas meta- or
para-substitution diminished it. Replacement of phenyl by pyridin-4-yl (10h) led to
increasing IR from 21.2% to 31.3%. Based on this findings, 2-chlorophenethyl and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
-(pyridin-4-yl)ethyl were fixed in the following structural optimization (Figure 2).
Table 1. HIF-1 Inhibition Activities of Hit 8 and Compounds 10a-h at 10 μM
S
H
N
R
H C
3
N
O
Compound
R
HRE Inhibition (%) ± SD
21.2 ± 3.1
3.1 ± 2.4
8
phenethyl
benzyl
1
0a
0b
0c
0d
0e
1
3-phenylpropyl
2-chlorophenethyl
3-chlorophenethyl
4-chlorophenethyl
6.5 ± 0.3
1
42.7 ± 6.6
1.2 ± 1.1
1
1
0.6 ± 1.1
10f
10g
2-(pyridin-2-yl)ethyl
2-(pyridin-3-yl)ethyl
2-(pyridin-4-yl)ethyl
5.1 ± 2.7
2.4 ± 1.9
10h
31.3 ± 5.2
73.5 ± 4.7
1 (LW6)
As listed in Table 2, keeping the 2-chlorophenethyl or 2-(pyridin-4-yl)ethyl
unchanged, thiazole ring (region B) was replaced by other aromatic rings, such as
phenyl (12a-b), furan (12c-d) and pyridine (12e-f), resulting in no obvious
improvement of activities. Replacing methyl group (region A) with phenyl ring led to
increasing potency as shown by compounds 15a-b, however, introduction of
pyridin-3-yl (15c-d) or pyridin-4-yl (15e-f) appeared to be unfavorable. Therefore, we
fixed phenyl ring as region A and focused on the modification of thiazole ring
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(
Figure 2).
Table 2. HIF-1 Inhibition Activities of Compounds 12a-f and 15a-f at 10 μM
O
R2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
Ar
N
H
HRE Inhibition
%) ± SD
1
2
Compound
2a
2b
2c
12d
2e
2f
5a
5b
15c
5d
5e
5f
R
Ar
R
(
1
Me
Me
2-chlorophenyl
pyridin-4-yl
45.3 ± 3.2
3.1 ± 5.1
1
1
Me
2-chlorophenyl
pyridin-4-yl
17.4 ± 14.3
12.8 ± 4.0
37.3 ± 9.2
36.4 ± 5.1
54.1 ± 6.6
48.7 ± 7.4
21.6 ± 3.4
33.9 ± 7.8
18.4 ± 2.1
19.9 ± 3.5
O
O
Me
1
Me
2-chlorophenyl
pyridin-4-yl
N
N
1
Me
S
N
1
phenyl
phenyl
pyridin-3-yl
pyridin-3-yl
pyridin-4-yl
pyridin-4-yl
2-chlorophenyl
pyridin-4-yl
S
N
1
S
N
2-chlorophenyl
pyridin-4-yl
S
N
1
S
N
1
2-chlorophenyl
pyridin-4-yl
S
N
1
To explore the influence of aromatic ring of region B against HIF-1 inhibitory
activity, a variety of five- and six-membered aromatic rings were screened. As listed
in Table 3, all of these phenyl substituted aromatic carboxamides showed moderate to
potent inhibitory activities with IR values ranging from 36.9-77.2%, indicating the
critical role of phenyl substitution of region A. In general, six-membered aromatic
ring derivatives (20a-b, 24a-d, and 27a-b) exhibited more effective inhibition than
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
five-membered heterocyclic ones (18a-j), possibly due to their appropriate ring size or
polarities for the binding site. More interestingly, N-2-chlorophenethyl substituted
biphenyl carboxamide (20a) showed the highest potency (IR = 77.2%), which may
imply that the aromatic ring in region C mainly exerts π-π stacking or hydrophobic
interaction instead of hydrogen-bond effects, whereas the corresponding
N-2-(pyridin-4-yl)ethyl substituent (20b) displayed reduced inhibitory activity (IR =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
2.0%). Considering that both picolinamides (24a-b) possessed acceptable potency,
with IR values of 68.4% and 60.7%, respectively, we next fixed 5-phenylpicolinamide
group to region B and further modified the substitution on phenyl group in region C
(Figure 2).
Table 3. HIF-1 Inhibition Activities of Compounds 18a-j, 20a-b, 24a-d, and 27a-b
at 10 μM
O
R
Ar
N
H
Compound
8a
Ar
O
R
HRE Inhibition (%) ± SD
51.4 ± 6.4
1
2-chlorophenyl
pyridin-4-yl
N
O
N
N
O
18b
8c
18d
8e
8f
8g
8h
18i
8j
38.7 ± 3.8
52.3 ± 2.2
53.5 ± 1.7
40.5 ± 6.4
49.7 ± 4.8
44.1 ± 9.0
36.9 ± 7.2
41.8 ± 2.9
67.3 ± 1.2
1
2-chlorophenyl
pyridin-4-yl
N
O
O
O
N
N
1
2-chlorophenyl
pyridin-4-yl
N
N
1
N
O
O
N
1
2-chlorophenyl
pyridin-4-yl
N
N
1
N
N
2-chlorophenyl
pyridin-4-yl
O
N
N
1
O
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2
0a
0b
4a
24b
4c
4d
7a
27b
2-chlorophenyl
pyridin-4-yl
77.2 ± 4.8
42.0 ± 5.5
2
2
2-chlorophenyl
68.4 ± 5.6
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pyridin-4-yl
2-chlorophenyl
pyridin-4-yl
60.7 ± 6.4
45.1 ± 2.8
47.4 ± 2.6
2
N
N
2
N
N
2
2-chlorophenyl
pyridin-4-yl
56.7 ± 4.9
61.5 ± 3.4
N
N
Compared with 24a, unexpectedly, deletion of the chlorine atom of region C (28a)
significantly elevated the inhibitory activity (Table 4) with IR values increasing from
68.4% to 82.2%, suggesting no obvious correlation between the electronic effects of
substituent and activities. Subsequently, we examined the positional effect of the
chlorine and fluorine on the phenyl ring. It turned out that most of chlorine and
fluorine substitution (28b-c and 28e-f) showed weaker activities than that of
unsubstituted derivative (28a), except the ortho-fluorine substituted derivative (28d)
with IR value of 91.4%. In addition, pyridin-2-yl, pyridin-3-yl and para-hydroxyl,
amino, methyl, nitro substituted phenyl derivatives (28g-l) displayed unsatisfied
activities.
Table 4. HIF-1 Inhibition Activities of Compounds 28a-l at 10 μM
H
N
N
R
O
Compound
8a
8b
R
HRE Inhibition (%) ± SD
82.2 ± 4.1
2
phenyl
2
3-chlorophenyl
64.6 ± 5.7
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
8c
8d
8e
8f
8g
8h
4-chlorophenyl
58.2 ± 8.3
91.4 ± 4.4
58.7 ± 2.6
46.1 ± 11.2
43.7 ± 7.9
44.5 ± 0.9
36.9 ± 4.1
60.2 ± 3.7
31.8 ± 3.0
40.2 ± 2.8
2
2-fluorophenyl
3-fluorophenyl
4-fluorophenyl
pyridin-2-yl
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
pyridin-3-yl
2
8i
4-hydroxyphenyl
4-aminophenyl
4-methylphenyl
4-nitrophenyl
8j
2
8k
8l
2
On
N-(2-fluorophenethyl)picolinamide and optimized the phenyl ring of region A (Figure
). As listed in Table 5, eighteen compounds were designed, synthesized and
evaluated for HIF-1 inhibition. Among them, thiophen-3-yl substituted compound
30d) maintained HIF-1 inhibitory potency similar with that of 28d, while furanyl,
the
basis
of
SAR
studies
above,
we
maintained
2
(
pyridinyl and pyrazolyl substitutions (30a-b, and 30e-f) resulted in decreased
activities more or less. In regard to substituted phenyl derivatives (30g-r),
ortho-substitutions were more favorable than meta- and para-substitutions; fluorine
(
30g-i) and methoxy (30m-o) substitutions displayed acceptable activities.
Particularly, 2-methoxyphenyl derivative (30m) was the most active inhibitor with IR
value of 97.7%.
Table 5. HIF-1 Inhibition Activities of Compounds 30a-r at 10 μM
Ar
H
N
N
O
F
Compound
0a
0b
Ar
HRE Inhibition (%) ± SD
57.2 ± 7.7
3
furan-2-yl
furan-3-yl
3
73.4 ± 6.1
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3
0c
0d
0e
0f
0g
0h
thiophen-2-yl
80.0 ± 2.0
88.2 ± 3.5
57.5 ± 3.1
29.5 ± 4.1
83.8 ± 9.4
55.3 ± 14.2
41.2 ± 12.4
43.6 ± 5.0
0.3 ± 1.5
3
thiophen-3-yl
pyridin-4-yl
3
3
1H-pyrazol-4-yl
2-fluorophenyl
3-fluorophenyl
4-fluorophenyl
2-hydroxyphenyl
3-hydroxyphenyl
4-hydroxyphenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2-cyanophenyl
3-cyanophenyl
4-cyanophenyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
3
3
0i
0j
3
0k
0l
0m
0n
3
33.2 ± 4.1
97.7 ± 1.4
53.8 ± 3.8
45.6 ± 6.4
62.7 ± 2.7
44.5 ± 7.1
13.1 ± 6.9
3
3
3
0o
3
0p
30q
30r
Finally, fifteen compounds with IR more than 60% were selected for determination
of their IC values (Table 6). Among them, 6 compounds with IC values below 3
5
0
50
μM, showed better HIF-1 inhibitory activity than that of positive control 1, and no
obvious cytotoxicity was observed (CC > 50 μM). To summarize, of these 68 newly
5
0
synthesized compounds, 30m was the most active compound with an IC value of
50
37
0.32 μM, almost 10-fold more effective than that of 1. In comparison to the structure
of original hit 8, the 2-methoxyphenyl group (region A) is essential for the activity,
the replacement of thiazole by pyridine (region B) improves the potency, and the
introduction of fluorine atom to the phenyl ring (region C) is beneficial to the
inhibitory effect. Thus, through five rounds of optimization, 30m showed over
1
70-fold enhancement of inhibitory activity against HIF-1 compared with the hit 8
and therefore was selected for further bioactivity evaluation.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 6. IC Values of Selected Compounds against HIF-1
50
Compound
8j
20a
4a
HRE IC (μM)
Cytotoxicity CC (μM)
5
0
50
1
7.32
2.87
9.84
13.80
10.04
4.56
9.84
0.93
11.61
4.24
2.48
1.41
2.16
0.32
8.53
3.08
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
4b
7b
28a
2
2
8b
8d
28j
0b
0c
0d
30g
0m
3
3
3
3
3
0p
1 (LW6)
3
.2 Inhibition of HIF-1α Accumulation
To further confirm their inhibition of HIF-1 pathway, representative compounds 30f,
0b and 30m with low, moderate and high potency in HRE reporter assay,
3
respectively, hit 8, as well as 1 as positive control were evaluated for inhibition of
HIF-1α protein accumulation under hypoxia condition using western blot assay. As
shown in Figure 3A, the effects of the tested compounds on HIF-1α protein
accumulation were consistent with their potency in HRE luciferase reporter assay.
Among the selected compounds, compound 30m showed the most activity against
HIF-1α accumulation, even stronger than the positive control 1. Accordingly, the
inhibitory effects of 30m on HIF-1α accumulation were also evaluated in other cancer
cell lines including breast cancer (MDA-MB-231), ovarian cancer (SKOV3) and
cervical cancer (HeLa). As expected, 30m attenuated HIF-1α accumulation in a
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dose-dependent manner in all of the tested cell lines (Figure 3B). Our recent results
from qRT-PCR of HIF-1α mRNA and HIF-1α protein degradation assay indicated that
3
0m probably prompted the degradation of HIF-1α protein (data not shown). Next,
we plan to synthesize the biotin-based photo-cross-linking probe of 30m and to
conduct affinity pull-down experiment in order to identify the direct target of 30m.
Taken together, our data preliminarily indicated that compound 30m could block the
HIF pathway by reduction of HIF-1α protein level and prompted us to further evaluate
the effects of this compound on angiogenesis and tumor cellular metastasis.
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Figure 3. Effects of selected compounds on hypoxia-induced accumulation of HIF-1α.
(A) The expression of HIF-1α was examined in LM3 cells treated with selected
compounds, including compounds 30b, 30f, 30m and 8 at the concentration of 10 μM
under hypoxia. 1 (LW6) was used as positive control. (B) The effects of compound
30m on hypoxia-induced HIF-1α accumulation were determined in various human
cancer cell lines, including MDA-MB-231, SKOV3, and HeLa at indicated
concentrations.
3
.3 Inhibition of Compound 50m on Angiogenesis
VEGF, the target gene directly regulated by HIF-1, promotes hypoxia-induced
11-12
angiogenesis, which is essential for cancer progression and metastasis.
Silencing
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HIF-1/VEGF pathway, which results in reduced ability of malignant cells to recruit
vasculature and colonize at distant sites, has emerged as promising strategy for cancer
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treatment. Accordingly, we firstly determined effects of compound 30m on
transcription of VEGF in human umbilical vein endothelial cells (HUVECs). Results
obtained from real-time assay showed that 30m effectively reduced the mRNA levels
of VEGF in a dose-dependent manner (Figure 4A). Next, capillary-like tube
formation assay was performed to further evaluate the anti-angiogenesis activity of
compound 30m in vitro. As shown in Figure 4B, hypoxia induced a complete
network structure formed by HUVECs, whereas treatment with 30m significantly
suppressed the tubule formation at non-antiproliferative concentrations (2 and 5 μM).
Particularly, at concentration of 5 μM, 30m completely disrupted the formation of
tube structures. Furthermore, the in vivo anti-angiogenic activity of 30m was further
determined by transgenic zebrafish, in which the vascular endothelial cells were
labeled with green fluorescent protein (GFP). Zebrafish embryos were selected,
dechlorinated, and incubated with 0.5 or 1 μM of 30m for 24 h, meanwhile 0.5 μM of
commercial VEGFR inhibitor apatinib served as a positive control. The affection of
zebrafish subintestinal vessels (SIVs) was observed under a fluorescence microscope.
Figure 4C illustrated the obvious destruction of zebrafish SIVs by exposure to
compound 30m, indicating that 30m effectively blocked the growth of normal
angiogenesis with similar activities of the positive control apatinib (Figure 4D).
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Figure 4. Effects of compound 30m on angiogenesis in vitro and in vivo. (A) Relative
expression of VEGF mRNA was determined by real-time PCR after treatment of
HUVECs with 0.5 or 1 μM of compound 30m. Relative expression of VEGF was
normalized against GAPDH. Results represent the means ± SD from three
##
independent experiments. p<0.01 vs normoxia group; *p<0.05, **p<0.01 vs
untreated control. (B) Inhibition of hypoxia-induced capillary-like tube formation.
HUVECs were seeded in the 96-well plate pre-coated with matrigel, and incubated
with 2 or 5 μM of compound 30m. Tubule branches were photographed. A
representative result from three independent experiments is shown. (C) Inhibitory
effect of compound 30m on the angiogenesis of transgenic zebrafish. Zebrafish was
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treated with 30m (0.5 and 1 μM) or DMSO (n = 10), and the subintestinal vessels
(SIVs) of zebrafish were observed using confocal microscopy. Apatinib was used as a
positive control. Representative images from three independent experiments were
shown. White arrows indicated the destruction of SIVs. (D) Histogram showed the
numbers of zebrafish SIVs per field under confocal microscopy. Results represented
the means ± SD. *p<0.05, **p<0.01 vs untreated control.
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.4 Inhibition of Compound 30m on Cellular Migration and Invasion
Increased evidence indicates that, through HIF-1 activation, hypoxia stimulates the
expression of certain genes promoting tumor cellular invasiveness and metastasis,
1
3-14, 18-19
such as MMPs and EMT regulators.
Therefore, we performed wound healing
and transwell assays to examine the inhibitory effects of compound 30m on migration
and invasion of breast cancer MDA-MB-231 cells under hypoxic condition. To avoid
induction of apoptosis or growth arrest, cells were treated with non-toxic
concentrations (2, 5 and 10 μM) of 30m for 24 h. Results from wound healing assay
showed that 30m dose-dependently impaired the ability of cells to close a wound,
indicating the inhibition of cellular migration in vitro (Figure 5A). In transwell assay,
MDA-MB-231 cells were seeded into upper chambers with or without pre-coated
matrigel stimulating base-membrane for determination of cellular invasion or
migration, respectively. As illustrated in Figure 5B and 5C, the numbers of both
migratory and invasive cells were significantly reduced by 30m in a dose-dependent
manner. Furthermore, western blot assay was used to investigate whether compound
3
0m could affect the expression of HIF-1 downstream genes related to cancer
metastasis. As shown in Figure 5D, treatment of 30m dose-dependently induced
E-cadherin expression but repressed vimentin expression in MDA-MB-231 cells,
indicating the reversal of hypoxia-mediated EMT process. In addition, western blot
analysis also showed that 30m dose-dependently down-regulated the expression of
MMP-2 and MMP-9, which mediate extracellular matrix (ECM) degradation and
thereby promotes cancer metastasis. Together, these results implied that compound
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0m inhibited migration and invasion of cancer cells with inhibition of EMT process
and down-regulation of MMPs.
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Figure 5. Effects of compound 30m on cellular migration and invasion. (A) Inhibition
of cellular migration by compound 30m in wound-healing assay. After a single wound
was scratched on cellular monolayer, MDA-MB-231 cells were treated with
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compound 30m (2, 5 and 10 μM) or DMSO under normoxia for 1 h and hypoxia for
another 24 h. Representative images from three independent experiments were shown.
(B) Inhibition of cellular migration and invasion by compound 30m in transwell
assays. MDA-MB-231 cells were seeded on chambers with or without pre-coated
matrigel for invasion or migration assay, respectively. After incubated with compound
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0m, cells that migrated through the chambers were stained with crystal violet.
Representative images from three independent experiments were shown. (C)
Histogram shows the numbers of migrated and invasive cells. The experiments were
repeated three times and results were indicated as means ± SD. *p<0.05, **p<0.01 vs
untreated control. (D) The expression of E-cadherin, Vimentin, MMP-2 and MMP-9
were determined by western blotting assay. GAPDH was selected as loading control.
Representative results from three independent experiments were shown.
4
.5 Inhibition of Compound 30m on Tumor Metastasis In Vivo
With great interests to discover anti-metastatic agents, we evaluated the inhibitory
potency of compound 30m using breast cancer lung metastasis mice model, which
was established by intravenous injection of MDA-MB-231 cells stable expressing
luciferase. Mice were treated with 30m or vehicle intraperitoneally (i.p.) every 2 days
during 3 weeks. After the final administration, bioluminescent imaging techniques
were used to assess the ability of malignant cells to generate lung metastases. After
the mice were sacrificed, the lung metastatic nodules were examined by histology.
Results obtained from quantitative analysis of bioluminescent signal (Figure 6A and
6B) and hematoxylin and eosin (H&E) staining (Figure 6D) revealed that treatment
with compound 30m significantly reduced lung colonization of tumor cells in
dose-dependent manner without obvious body-weight loss (Figure 6C). These results
indicated the therapeutic effects and low toxicity of compound 30m in metastatic
breast cancer mice model.
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Figure 6. Compound 30m inhibited the formation of metastasis of human breast
cancer cells in mice model. (A) MDA-MB-231 cells stably transfected with luciferase
were injected directly into the tail vein of BALB/c nude mice. Mice then received
vehicle or compound 30m (15 and 30 mg/kg) starting 24 h after injection of cells.
Animals received therapy every 2 days during 20 days. Bioluminescent images were
acquired 24 h after the final dose of inhibitor. (B) Quantification of bioluminescence.
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Data are expressed as the mean ± SD (n = 4 per group) *p<0.05. (C) The body
weights of the mice recorded at the end of the experiment. (D) Photomicrographs of
mice lung tissue sections stained with H&E illustrated the formation of breast cancer
metastasis. Black arrows indicated the metastatic nodules of breast cancer.
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. CONCLUSION
In the present study, in order to discover novel therapeutics for cancer metastasis,
series of aryl carboxamide derivatives were synthesized and evaluated for inhibition
of HIF-1 signaling pathway by dual luciferase-reporter assay. Starting from the
fragment hit 2-methyl-N-phenethylthiazole-4-carboxamide (8), systematic lead
optimization was carried out, in an attempt to improve potency. In particular,
replacement of methyl with 2-methoxyl substituted phenyl, conversion of the thiazole
moiety to pyrimidine moiety and introduction of F atom to the phenyl ring of
N-phenethyl led to increasing the HIF-1 inhibitory activities. After five rounds of
in-depth study on SAR step by step, compound 30m was discovered as the most
active inhibitor (IC50 = 0.32 μM) without obvious cytotoxicity (CC50 > 50 μM).
Further biological evaluation showed that it could dose-dependently block
hypoxia-induced HIF-1α protein accumulation in several human cancer cell lines.
Besides, it down-regulated transcription of VEGF, a downstream gene of HIF-1,
which was consistent with its inhibitory potency on capillary-like tube formation in
vitro and angiogenesis of zebrafish in vivo. Moreover, compound 30m inhibited
migration and invasion of tumor cells in vitro and blocked breast cancer lung
metastasis in mice model, indicating the promising therapeutic potential of compound
3
0m as an effective HIF-1 inhibitor to prevent tumor metastasis and progression.
Therefore, the above results are of great importance in the development of more
efficient HIF-1 inhibitors for cancer therapy.
5
. EXPERIMENTAL SECTION
5.1 Chemistry
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All commercial reagents and solvents were purchased from commercial suppliers
and used without further purification. Reactions were monitored by thin-layer
chromatography (TLC) and visualized under UV light at 254 and 365 nm. The
chromatograms were conducted on silica gel (300-400 mesh) using combiflash
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chromatography systems (Teledyne ISCO Rf200). H and C NMR spectral data
were recorded with a Bruker 600 MHz spectrometer in CDCl or DMSO-d using
3
6
tetramethylsilane (TMS) as the internal standard. High-resolution mass spectrometry
HRMS) was recorded on an Agilent Technologies LC-TOF instrument. Targeted
(
compounds were analyzed by a Shimadzu LC20A HPLC instrument with a
UV/visible detector (5 μm, 4.6 mm×150 mm C column). The purities of all targeted
1
8
compounds were determined by monitoring at 254 nm and were confirmed to be more
than 95% (Table S1).
General procedure for the synthesis of compounds 8, 10a-h, 12a-f, 15a-f,
1
8a-j,
20a-b,
24a-d,
27a-b,
28a-l,
and
(8).
29
exemplified
solution
by
2-methyl-N-phenethylthiazole-4-carboxamide
To
a
of
2-methylthiazole-4-carboxylic acid (143 mg, 1 mmol) in dry CH Cl (5 mL), oxalyl
2
2
chloride (0.17 mL) and DMF (1 drop) were added at 0 °C. Then, the resulting mixture
was stirred at room temperature for 1 h and concentrated in vacuo to give the
2
2
-methylthiazole-4-carbonyl chloride as crude product. To a solution of
-phenylethanamine (121 mg, 1 mmol) in CH Cl (5 mL) was added Et N (0.16 mL)
2
2
3
and the 2-methylthiazole-4-carbonyl chloride redissolved in CH Cl (1 mL). The
2
2
reaction mixture was stirred at room temperature for additional 6-12 h. The solution
was concentrated in vacuo. The residue was purified by flash chromatography to yield
1
compound 8 as a white solid (216 mg, 93%). H NMR (600 MHz, CDCl ) δ 7.93 (s,
3
1
2
1
H), 7.40 (brs, 1H), 7.33-7.30 (m, 2H), 7.25-7.22 (m, 3H), 3.69 (q, J = 6.0 Hz, 2H),
13
.93 (t, J = 6.0 Hz, 2H), 2.68 (s, 3H); C NMR (151 MHz, CDCl ) δ 165.26, 160.52,
3
49.12, 138.31, 128.16, 127.98, 125.84, 122.17, 40.02, 35.35, 18.46; ESI-HRMS (m/z)
+
calcd C H N OS (M + H ) 247.0900, found 247.0899.
1
3
14
2
1
N-Benzyl-2-methylthiazole-4-carboxamide (10a). White solid, yield 73%. H
NMR (600 MHz, CDCl ) δ 7.97 (s, 1H), 7.62 (brs, 1H), 7.36-7.32 (m, 4H), 7.29-7.26
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(
m, 1H), 4.63 (d, J = 6.0 Hz, 2H), 2.67 (s, 3H); C NMR (151 MHz, CDCl ) δ 165.36,
3
1
60.42, 148.95, 137.57, 128.07, 127.28, 126.86, 122.48, 42.75, 18.43; ESI-HRMS
+
(
m/z) calcd C H N OS (M + H ) 233.0743, found 233.0741.
1
2
12
2
2
-Methyl-N-(3-phenylpropyl)thiazole-4-carboxamide (10b). Colorless oil,
0
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2
3
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6
7
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9
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3
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9
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yield 64%. H NMR (600 MHz, CDCl ) δ 7.93 (s, 1H), 7.36 (brs, 1H), 7.29-7.26 (m,
3
2
H), 7.20-7.17 (m, 3H), 3.48-3.45 (m, 2H), 2.72-2.71 (m, 2H), 2.69 (s, 3H), 1.97-1.92
13
(
m, 2H); C NMR (151 MHz, CDCl ) δ 165.82, 161.12, 149.82, 141.40, 128.39,
3
1
28.35, 125.91, 122.65, 38.89, 33.24, 31.25, 19.05; ESI-HRMS (m/z) calcd
+
C H N OS (M + H ) 261.1056, found 261.1059.
14
16
2
N-(2-Chlorophenethyl)-2-methylthiazole-4-carboxamide (10c). Colorless oil,
1
yield 84%. H NMR (600 MHz, CDCl ) δ 7.94 (s, 1H), 7.42 (brs, 1H), 7.36 (dd, J =
3
7
.4, 1.4 Hz, 1H), 7.28-7.26 (m, 1H), 7.20-7.17 (m, 2H), 3.70 (q, J = 7.4 Hz, 2H), 3.06
13
(
t, J = 7.4 Hz, 2H), 2.68 (s, 3H); C NMR (151 MHz, CDCl ) δ 165.85, 161.17,
3
149.70, 136.52, 134.15, 130.92, 129.57, 127.97, 126.92, 122.77, 38.97, 33.66, 19.04;
+
ESI-HRMS (m/z) calcd C H ClN OS (M + H ) 281.0510, found 281.0512.
13
13
2
N-(3-Chlorophenethyl)-2-methylthiazole-4-carboxamide (10d). White solid,
1
yield 59%. H NMR (600 MHz, CDCl ) δ 7.94 (s, 1H), 7.37 (brs, 1H), 7.24-7.21 (m,
3
3H), 7.13-7.12 (m, 1H), 3.67 (q, J = 7.3 Hz, 2H), 2.90 (t, J = 7.3 Hz, 2H), 2.69 (s, 3H);
1
3
C NMR (151 MHz, CDCl ) δ 165.39, 160.56, 148.92, 140.32, 133.71, 129.25,
3
1
28.34, 126.37, 126.10, 122.31, 39.74, 35.03, 18.48; ESI-HRMS (m/z) calcd
+
C H ClN OS (M + H ) 281.0510, found 281.0513.
13 13
2
N-(4-Chlorophenethyl)-2-methylthiazole-4-carboxamide (10e). White solid,
1
yield 70%. H NMR (600 MHz, CDCl ) δ 7.93 (s, 1H), 7.37 (brs, 1H), 7.28-7.27 (m,
3
2
H), 7.18-7.16 (m, 2H), 3.66 (q, J = 7.2 Hz, 2H), 2.90 (t, J = 7.2 Hz, 2H), 2.68 (s, 3H);
13
C NMR (151 MHz, CDCl ) δ 165.38, 160.54, 148.94, 136.75, 131.66, 129.52,
3
+
1
28.09, 122.30, 39.85, 34.69, 18.50; ESI-HRMS (m/z) calcd C H ClN OS (M + H )
1
3
13
2
2
81.0510, found 281.0510.
2
-Methyl-N-(2-(pyridin-2-yl)ethyl)thiazole-4-carboxamide (10f). Colorless oil,
1
yield 72%. H NMR (600 MHz, CDCl ) δ 8.57 (d, J = 4.7 Hz, 1H), 7.92 (s, 1H), 7.86
3
(
brs, 1H), 7.62-7.59 (m, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.16-7.14 (m, 1H), 3.85 (q, J =
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.7 Hz, 2H), 3.10 (t, J = 6.7 Hz, 2H), 2.68 (s, 3H); C NMR (151 MHz, CDCl ) δ
3
65.77, 161.13, 159.29, 149.88, 149.35, 136.45, 123.31, 122.62, 121.47, 38.65, 37.50,
+
9.08; ESI-HRMS (m/z) calcd C H N OS (M + H ) 248.0852, found 248.0849.
1
2
13
3
2
-Methyl-N-(2-(pyridin-3-yl)ethyl)thiazole-4-carboxamide (10g). White solid,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
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4
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4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
yield 85%. H NMR (600 MHz, CDCl ) δ 8.49 (d, J = 1.6 Hz, 1H), 8.48 (dd, J = 4.8,
3
1
7
.6 Hz, 1H), 7.92 (s, 1H), 7.57 (dt, J = 7.8, 1.6Hz, 1H), 7.41 (brs, 1H), 7.24 (dd, J =
13
.8, 4.8 Hz, 1H), 3.69 (q, J = 7.0 Hz, 2H), 2.94 (t, J = 7.0 Hz, 2H), 2.67 (s, 3H); C
NMR (151 MHz, CDCl ) δ 165.41, 160.60, 149.56, 148.84, 147.43, 135.61, 133.70,
3
+
1
2
22.88, 122.36, 39.62, 32.52, 18.45; ESI-HRMS (m/z) calcd C H N OS (M + H )
1
2
13
3
48.0852, found 248.0856.
2
-Methyl-N-(2-(pyridin-4-yl)ethyl)thiazole-4-carboxamide (10h). White solid,
1
yield 77%. H NMR (600 MHz, CDCl ) δ 8.52 (d, J = 5.8 Hz, 2H), 7.93 (s, 1H), 7.38
3
(
brs, 1H), 7.17 (d, J = 5.8 Hz, 2H), 3.71 (q, J = 7.2 Hz, 2H), 2.93 (t, J = 7.2 Hz, 2H),
1
3
2.67 (s, 3H); C NMR (151 MHz, CDCl ) δ 165.45, 160.59, 149.33, 148.78, 147.27,
3
+
123.51, 122.41, 38.89, 34.63, 18.45; ESI-HRMS (m/z) calcd C H N OS (M + H )
1
2
13
3
248.0852, found 248.0855.
1
N-(2-Chlorophenethyl)-4-methylbenzamide (12a). White solid, yield 80%. H
NMR (600 MHz, CDCl ) δ 7.62-7.60 (m, 2H), 7.38-7.37 (m, 1H), 7.27-7.26 (m, 1H),
3
7
2
1
.22-7.17 (m, 4H), 6.17 (brs, 1H), 3.73 (q, J = 6.8 Hz, 2H), 3.09 (t, J = 6.8 Hz, 2H),
1
3
.38 (s, 3H); C NMR (151 MHz, CDCl ) δ 166.87, 141.20, 136.08, 133.51, 131.10,
3
30.50, 129.05, 128.59, 127.48, 126.42, 126.19, 39.09, 32.73, 20.79; ESI-HRMS (m/z)
+
calcd C H ClNO (M + H ) 274.0993, found 274.0993.
1
6
16
4
-Methyl-N-(2-(pyridin-4-yl)ethyl)benzamide (12b). White solid, yield 80%.
1
H NMR (600 MHz,CDCl ) δ 8.50 (d, J = 5.7 Hz, 2H), 7.60 (d, J = 7.8 Hz, 2H), 7.20
3
(
d, J = 7.8 Hz, 2H), 7.15 (d, J = 5.7 Hz, 2H), 6.28 (brs, 1H), 3.71 (q, J = 7.0 Hz, 2H),
13
2
.93 (t, J = 7.0 Hz, 2H), 2.38 (s, 3H); C NMR (151 MHz, CDCl ) δ 167.00, 149.26,
3
1
47.58, 141.43, 130.83, 128.63, 126.24, 123.62, 39.65, 34.51, 20.82; ESI-HRMS (m/z)
+
calcd C H N O (M + H ) 241.1335, found 241.1337.
1
5
16
2
N-(2-Chlorophenethyl)-5-methylfuran-2-carboxamide (12c). Colorless oil,
1
yield 67%. H NMR (600 MHz, CDCl ) δ 7.37 (dd, J = 7.5, 1.5 Hz, 1H), 7.28-7.26 (m,
3
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H), 7.22-7.17 (m, 2H), 6.99 (d, J = 3.3 Hz, 1H), 6.37 (brs, 1H), 6.08 (d, J = 3.3 Hz,
13
1
H), 3.68 (td, J = 7.0 Hz, 2H), 3.05 (t, J = 7.0 Hz, 2H), 2.32 (s, 3H); C NMR (151
MHz, CDCl ) δ 158.59, 154.32, 146.38, 136.51, 134.13, 131.00, 129.59, 128.02,
3
1
26.96, 115.28, 108.43, 38.77, 33.62, 13.74; ESI-HRMS (m/z) calcd C H ClNO (M
14 14 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
H ) 264.0786, found 264.0789.
5
-Methyl-N-(2-(pyridin-4-yl)ethyl)furan-2-carboxamide (12d). White solid,
1
yield 70%. H NMR (600 MHz, CDCl ) δ 8.51 (d, J = 5.8, 2H), 7.15 (d, J = 5.8 Hz,
3
2
2
1
H), 6.99 (d, J = 3.3 Hz, 1H), 6.43 (brs, 1H), 6.07 (d, J = 3.3, 1H), 3.68 (q, J = 7.0 Hz,
13
H), 2.91 (t, J = 7.0 Hz, 2H), 2.29 (s, 3H); C NMR (151 MHz, CDCl ) δ 158.00,
3
53.94, 149.31, 147.32, 145.50, 123.56, 114.99, 107.95, 38.68, 34.67, 13.18;
+
ESI-HRMS (m/z) calcd C H N O (M + H ) 231.1128, found 231.1128.
13
14
2
2
N-(2-Chlorophenethyl)-6-methylpicolinamide (12e). Colorless oil, yield 61%.
1
H NMR (600 MHz, CDCl ) δ 8.23 (brs, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.71 (t, J = 7.7
3
Hz, 1H), 7.37 (dd, J = 7.5, 1.5 Hz, 1H), 7.29 (d, J = 7.5 Hz, 1H), 7.27-7.25 (m, 1H),
13
7.22-7.16 (m, 2H), 3.74 (q, J = 7.1 Hz, 2H), 3.09 (t, J = 7.1 Hz, 2H), 2.54 (s, 3H); C
NMR (151 MHz, CDCl ) δ 164.56, 157.05, 149.21, 137.38, 136.66, 134.18, 130.99,
3
129.57, 127.95, 126.89, 125.74, 119.14, 39.05, 33.63, 24.17; ESI-HRMS (m/z) calcd
+
C H ClN O (M + H ) 275.0946, found 275.0949.
15
15
2
6
-Methyl-N-(2-(pyridin-4-yl)ethyl)picolinamide (12f). Colorless oil, yield 73%.
1
H NMR (600 MHz, CDCl ) δ 8.53 (d, J = 5.9 Hz, 2H), 8.25-8.15 (m, 1H), 7.98 (d, J
3
=
7.7 Hz, 1H), 7.72 (t, J = 7.7 Hz, 1H), 7.27 (d, J = 7.7 Hz, 1H), 7.19 (d, J = 5.9 Hz,
1
3
2
H), 3.74 (q, J = 7.0 Hz, 2H), 2.96 (t, J = 7.0 Hz, 2H), 2.53 (s, 3H); C NMR (151
MHz, CDCl ) δ 164.59, 157.13, 149.93, 148.97, 147.96, 137.46, 125.91, 124.13,
3
+
1
19.19, 39.58, 35.30, 24.18; ESI-HRMS (m/z) calcd C H N O (M + H ) 242.1288,
1
4
15
3
found 242.1289.
General procedure for the synthesis of compounds 14a-c and 17a-e
exemplified by 2-phenylthiazole-4-carboxylic acid (14a). 2N LiOH aqueous
solution was added to a solution of carboxylic ester 13a (1 eq) in ethanol at ambient
temperature. The reaction mixture was stirred for 1 h, and ethanol was then removed
by evaporation in vacuo. The resulting mixture was adjusted to pH = 5-6 with 1N HCl.
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The precipitated white solid was collected by filtration and dried to give the
carboxylic acid 14a as a white solid (68% yield), which was used in next step without
further purification.
N-(2-Chlorophenethyl)-2-phenylthiazole-4-carboxamide (15a). White solid,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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0
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8
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0
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2
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8
9
0
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2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
yield 48% from 9a. H NMR (600 MHz, CDCl ) δ 8.09 (s, 1H), 7.94-7.92 (m, 2H),
3
7
.59 (brs, 1H), 7.47-7.46 (m, 3H), 7.39 (dd, J = 7.4, 1.7 Hz, 1H), 7.31 (dd, J = 7.4, 1.7
13
Hz, 1H), 7.24-7.19 (m, 2H), 3.76 (q, J = 7.0 Hz, 2H), 3.12 (t, J = 7.0 Hz, 2H); C
NMR (151 MHz, CDCl ) δ 167.46, 160.57, 150.19, 135.94, 133.59, 132.25, 130.45,
3
1
29.99, 129.04, 128.44, 127.46, 126.37, 125.99, 122.14, 38.47, 32.99; ESI-HRMS
+
(
m/z) calcd C H ClN OS (M + H ) 349.0714, found 349.0702.
1
8
15
2
2
-Phenyl-N-(2-(pyridin-4-yl)ethyl)thiazole-4-carboxamide (15b). White solid,
1
yield 49% from 9a. H NMR (600 MHz, CDCl ) δ 8.56 (d, J = 5.8 Hz, 2H), 8.09 (s,
3
1
H), 7.92-7.90 (m, 2H), 7.56 (brs, 1H), 7.47-7.46 (m, 3H), 7.23 (d, J = 5.8 Hz, 2H),
1
3
3.77 (q, J = 7.2 Hz, 2H), 2.99 (t, J = 7.2 Hz, 2H); C NMR (151 MHz, CDCl ) δ
3
167.65, 160.57, 149.91, 149.39, 147.25, 132.13, 130.09, 128.47, 125.96, 123.56,
+
122.38, 38.99, 34.72; ESI-HRMS (m/z) calcd C H N OS (M + H ) 332.0828, found
1
7
15
3
332.0831.
N-(2-Chlorophenethyl)-2-(pyridin-3-yl)thiazole-4-carboxamide (15c). White
1
solid, yield 53% from 9b. H NMR (600 MHz, CDCl ) δ 9.16 (s, 1H), 8.70 (d, J = 5.4
3
Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.15 (s, 1H), 7.42-7.38 (m, 2H), 7.30 (d, J = 7.8 Hz,
1
3
1
H), 7.24-7.19 (m, 2H), 3.77 (t, J = 7.0 Hz, 2H), 3.12 (t, J = 7.0 Hz, 2H); C NMR
(
151 MHz, CDCl ) δ 164.08, 160.22, 150.72, 150.65, 147.08, 135.84, 133.59, 133.11,
3
1
30.43, 129.08, 128.26, 127.55, 126.39, 123.16, 122.88, 38.54, 32.90; ESI-HRMS
+
(
m/z) calcd C H ClN OS (M + H ) 344.0619, found 344.0618.
1
7
14
3
2
-(Pyridin-3-yl)-N-(2-(pyridin-4-yl)ethyl)thiazole-4-carboxamide
(15d).
1
White solid, yield 51% from 9b. H NMR (600 MHz, CDCl ) δ 9.13 (d, J = 1.5 Hz,
3
1
H), 8.68 (dd, J = 4.8, 1.5 Hz, 1H), 8.54 (d, J = 6.0 Hz, 2H), 8.20-8.18 (m, 1H), 8.19
(s, 1H), 7.53 (brs, 1H), 7.40 (dd, J = 8.0, 4.8 Hz, 1H), 7.19 (d, J = 6.0 Hz, 2H), 3.76 (q,
13
J = 7.2 Hz, 2H), 2.98 (t, J = 7.2 Hz, 2H); C NMR (151 MHz, CDCl ) δ 164.27,
3
1
60.24, 150.81, 150.39, 149.40, 147.15, 147.06, 133.08, 128.16, 123.53, 123.19,
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