Synthesis, preliminary structure-activity relationships, and in vitro biological evaluation of 6-aryl-3-amino-thieno[2,3-b]pyridine derivatives as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2013.02.059

In our previous study, a series of 6-aryl-3-amino-thieno[2,3-b]pyridine derivatives exhibited potent antiproliferative activities and an unique hepatocellular carcinoma (HCC)-specific anticancer activity was also observed. In further anti-inflammatory research, thienopyridine derivative 1a showed potent inhibition of nitric oxide (NO) production. So a series of thienopyridine analogues of 1a were synthesized and evaluated for anti-inflammatory activities. The structure-activity relationships (SARs) revealed that the most potent analogues 1f and 1o were identified as potent inhibitors of NO production with IC₅₀ values of 3.30 and 3.24 μM, respectively. These results suggest that these 6-aryl-3-amino-thieno[2,3-b]pyridine derivatives might potentially constitute a novel class of anti-inflammatory agents, which require further studies.

Full text of DOI:10.1016/j.bmcl.2013.02.059

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2349–2352
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, preliminary structure–activity relationships, and in vitro
biological evaluation of 6-aryl-3-amino-thieno[2,3-b]pyridine
derivatives as potential anti-inflammatory agents
Huan Liu a, , Yi Li a, , Xiang-Ying Wang a, Bo Wang a, Hai-Yun He a, Ji-Yan Liu b, Ming-Li Xiang a, Jun He a,
Xiao-Hua Wu a, , Li Yang a,
⇑
⇑
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medicinal School, Sichuan University, Chengdu 610041, Sichuan, China
b Department of Medical Oncology, Cancer Center, West China Hospital, West China Medicinal School, Sichuan University, Chengdu 610041, Sichuan, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In our previous study, a series of 6-aryl-3-amino-thieno[2,3-b]pyridine derivatives exhibited potent anti-
proliferative activities and an unique hepatocellular carcinoma (HCC)-specific anticancer activity was
also observed. In further anti-inflammatory research, thienopyridine derivative 1a showed potent inhibi-
tion of nitric oxide (NO) production. So a series of thienopyridine analogues of 1a were synthesized and
evaluated for anti-inflammatory activities. The structure–activity relationships (SARs) revealed that the
most potent analogues 1f and 1o were identified as potent inhibitors of NO production with IC50 values
of 3.30 and 3.24 lM, respectively. These results suggest that these 6-aryl-3-amino-thieno[2,3-b]pyridine
derivatives might potentially constitute a novel class of anti-inflammatory agents, which require further
studies.
Received 8 October 2012
Revised 31 January 2013
Accepted 13 February 2013
Available online 24 February 2013
Keywords:
6-Aryl-3-amino-thieno[2,3-b]pyridine
derivatives
Anti-inflammatory agent
Structure–activity relationships (SARs)
Nitric oxide (NO)
Ó 2013 Elsevier Ltd. All rights reserved.
Tumor necrosis factor-a (TNF-a)
Many studies have demonstrated that inflammation is involved
in various diseases such as cancer, atherosclerosis, rheumatoid
tory research, compound 1a was also identified as a potent inhib-
itor of NO production as shown in Table 1. Although there are some
anti-inflammatory reports that 3-amino-thieno[2.3-b]pyridine
arthritis and asthma.1 Cytokines such as TNF-
a, IL-8, nitric oxide
(NO) produced by macrophages and lymphocytes are local media-
tors and play a central role in the development of inflammation.2
So agents which can inhibit the production of cytokines may be a
beneficial therapeutic strategy.3 During inflammation reactions,
macrophages release various cytokines, among which NO is well
characterized.4 Various studies have demonstrated that overpro-
duction of NO has been found in macrophages of many inflamma-
tory diseases, including rheumatoid arthritis,5 atheroscleosis,6 and
hepatitis.7 Because of the important roles of NO and other proin-
flammatory mediators in the development of inflammation, drugs
which regulate these proinflammatory mediators production could
be used in controlling inflammatory diseases.8
Our research group has been interested in the design, screening,
synthesis and biological evaluation of anti-inflammatory
agents.9,19 In our previous study, a series of 6-aryl-3-amino-thie-
no[2,3-b]pyridine derivatives exhibited potent antiproliferative
activities and an unique hepatocellular carcinoma (HCC)-specific
anticancer activity was also observed.10 In further anti-inflamma-
derivatives, such as 1b, 1c and 1d are inhibitors of IjB kinase b
(IKK-b),11 as shown in Figure 1. To the best of our knowledge,
the anti-inflammatory activity of 6-aryl-3-amino-thieno[2,3-
b]pyridine derivative is being firstly reported by our group. So
herein we report the synthesis, the structure–activity relationship
(SAR) study, and preliminary biological evaluation of these novel
thienopyridine derivatives as anti-inflammatory agents.
As presented in Scheme 1, 6-aryl-3-amino-thieno[2,3-b]pyri-
dine derivatives 1a and 1e–1w were readily synthesized as previ-
ously reported.10 Firstly, commercially available acetophenones
(2a–2g) reacted with N,N-dimethylformamide dimethyl acetal
(DMF–DMA) to give enaminones (3a–3g). Subsequent condensa-
tion with cyanothioaceamide, catalyzed by DABCO, yielded the
key intermediate 6-aryl-3-cyanopyridine-2-(1H)-thiones (4a–4g).
Using the same synthetic strategy shown in Scheme 1, 1a and
1e–1w were readily prepared through the condensation of 4a–4g
with 5a–5e, via the Thorpe–Ziegler cyclization.
The initial research showed the poor solubility of these 6-aryl-
3-amino-thieno[2,3-b]pyridine derivatives made it difficult to per-
form in vivo studies. To solve this problem, a lead optimization was
conducted through introducing N-methyl-piperazine group at the
2-position to obtain analogues 1t and 1w The preparation of key
⇑
Corresponding authors. Tel.: +86 28 85164063; fax: +86 28 85164060.
E-mail addresses: wuxh@scu.edu.cn (X.-H. Wu), yangli@scu.edu.cn (L. Yang).
These authors equally contributed to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.059

2350
H. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2349–2352
Table 1
The inhibition data for NO production of 1a and 1e–1w
R3
R2
R1
R4
N
S
a
Compound
R1
R2
R3
R4
NO IC50 (lM)
1a
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
1t
1u
1v
1w
1b
N-(4-Cl-Bn)NHCO–
N-(4-Cl-Bn)NHCO–
–CONH2
–CN
–COOEt
–COOH
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
NH2
NH2
NH2
NH2
NH2
NH2
NHAc
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
3-MeO-Ph
3,4-di Cl-Ph–
3-MeO-Ph
3-MeO-Ph
3-MeO-Ph
3-MeO-Ph
3-MeO-Ph
–Ph
2-Thiazolyl
3-Pyridyl
2-Furanyl
3-F-Ph–
3-NHCOCH3-Ph–
3-N(CH3)2-Ph–
4-OH-Ph–
4-MeO-Ph–
4-COOH-Ph
Naphthyl
3-F-Ph
17.7
13.5
3.30
8.13
>40.0
6.32
>40.0
10.5
>40.0
11.3
>40.0
3.24
15.2
13.9
5.95
6.10
>40.0
>40.0
7.46
5.80
7.01
–CONH2
–CONH2
N-(4-(4-Methylpiperazin-1-yl)phenyl)NHCO–
N-(4-(4-Methylpiperazin-1-yl)phenyl)NHCO–
–CONH2
3-MeO-Ph
Me
a
Inhibition of NO production by various compounds on murine RAW264.7 macrophage cell line stimulated by LPS were determined by the Griess reaction, and the results
were expressed as the mean IC50 calculated from three independent experiments.
NH2
NH2
3
O
O
MeO
S
HN
N
6
S
NH2
N
1b
1a
Cl
NH2
NH2
O
O
S
NH2
N
N
S
NH2
N
N
S
N
HN
1c
H
1d
OH
Figure 1. The structure of thienopyridine derivatives 1a–1d.
NH2
CN
O
O
R
c
5
a
b
S
Ar
N
Ar
N
Ar
N
H
S
Ar
2a-2g
1a, 1e-1w
3a-3g
4a-4g
5a
:2-chloro-N-(4-chlorobenzyl)acetamide
5b
5c
5d
:chloroacetonitrile
:ethyl chloroacetate
:chloroacetamide
5e:2-chloro-N-(4-(4-methylpiperazin-1-yl)phenyl)acetamide
Scheme 1. Synthesis of analogues 1a and 1e–1w.12 Reagents and conditions: (a) DMF–DMA (2.0 equiv), reflux, 4–24 h, 75–95%; (b) DABCO (0.5 equiv), cyanothioaceamide
(1.5 equiv), EtOH, reflux, 2–4 h, 40–97%; (c) compound 5, 10% aq KOH/DMF, rt 10–15 min and then 85 °C, 6 h, 50–80%.
intermediate 5e is outlined in Scheme 2, para-fluoronitrobenzene
reacted with N-methylpiperazine to give 6, subsequent reduction
of nitro group and then was treated with chloroacetyl chloride to
yield 5e.
As shown in Table 1, the SARs of the 2-, 3- and 6-position on the
thieno[2,3-b]pyridine scaffold were discussed by evaluating the
anti-inflammatory inhibitory activity in murine RAW264.7 macro-
phage cell line stimulated by LPS,13 determined by the Griess

H. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2349–2352
2351
a
b
N
N
O
NO2
F
NO2
N
N
NH2
6
7
H
c
N
N
N
Cl
5e
Scheme 2. Synthesis of 5e. Reagents and conditions: (a) N-methylpiperazine (1.1 equiv), rt 0.5–1 h, 95%; (b) Fe (4.0 equiv), NH4Cl (2.0 equiv), EtOH/H2O, reflux, 0.5–1 h, 93%;
(c) chloroacetyl chloride (3 equiv), CH2Cl2, reflux, 1 h, 100%.
reaction assay.14 With compounds 1a, 1e–1i and 1v–1w in hand,
we started our SAR studies at the 2-position (R1). Among them,
the most potent analogue 1f significantly inhibited NO production
at low concentrations (IC50 = 3.30 lM). It was about fivefold great-
er than that of the hit compound 1a, Furthermore, a remarkable in-
creased potency was observed when the substituted formamide
group was replaced by cyano group (1g), carboxylic group(1i),
whereas formate ester (1h) led to total loss of activity. These re-
sults demonstrate the importance of unsubstituted formamide
group at the 2-position (R1). To our delight, the N-methyl-pipera-
zine substituted analogue 1w showed improved potency in vitro
with better water solubility compared with 1a.15
After identifying the most potent 2-position substituent, we
next turned to examining the SAR at the 3-position (R2). The acyl-
ation of amino group at 3-position (1j) resulted in completely loss
of activity, which revealed the indispensablity of the unsubstituted
amino group.
Figure 2. Effects of the compound 1f on TNF-a secretion in the supernate of murine
RAW264.7 macrophage cell line stimulated by LPS. Data are means SD; and ⁄p
<0.05 versus LPS; ⁄⁄p <0.01 versus LPS.
Having identified the most potent substitutional group at 2- and
3-positions, respectively, then we focused our attention on 6-posi-
tion for further study. As shown in Table 1, the unsubstituted phe-
nyl ring in R4 (1k) showed remarkable reduced activity with that of
1f. Replacement of the phenyl ring in R4 with heteroaryl rings, such
as thiazole (1l), pyridine (1m), and furan (1n) groups also yielded
compounds with remarkable decreased potency compared with
1f. The introduction of a meta-fluoro substituent on the phenyl ring
(1o) in the R4 group showed comparable activity with that of 1f.
However, the introduction of a nitrogen-containing substituent
on the phenyl ring in the R4 group(1p and 1q) had a detrimental
effect on the potency, that is, a 4- to 5-fold decrease in potency,
respectively, compared with that of 1f. The moderate reduced po-
tency of para-hydroxy and para-methoxy substituted analogue (1r
and 1s) suggested that the para-hydroxy and para-methoxy substi-
tutes at the phenyl ring are tolerated. In addition, the introduction
of carboxylic group (1t) and a bulky naphthyl ring (1u) led to al-
most total loss of activity.
To summarize, a series of 6-aryl-3-amino-thieno[2,3-b]pyridine
derivatives have been synthesized and evaluated for its anti-
inflammatory activity. To the best of our knowledge, the anti-
inflammatory activity of 6-aryl-3-amino-thieno[2,3-b]pyridine
derivative is being firstly reported by our group. The structure–
activity relationships (SARs) revealed that the most potent ana-
logues 1f and 1o were identified as potent inhibitors of NO produc-
tion with IC50 values of 3.30 and 3.24 lM, respectively. To our
delight, the N-methyl-piperazine substituted analogues 1v and
1w showed comparable potency in vitro with better water solubil-
ity compared with 1f and 1o, which are candidates for the in vivo
anti-inflammatory research. Further in vivo research and mecha-
nism studies are ongoing in our laboratory.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (NSFC-81000982). We thank the Analytical and
Testing Center of Sichuan University for NMR spectral
measurements.
It was noteworthy that the most potent analogue 1f
(IC50 = 3.30
improvement in inhibitory activity against NO production over
that of 1b11b as a positive control (IC50 = 7.01
M). To our delight,
lM) and 1o (IC50 = 3.24 lM) exhibited a nearly twofold
l
Supplementary data
the N-methyl-piperazine substituted analogues 1v and 1w showed
comparable potency in vitro with better water solubility compared
with 1f and 1o, which are promising candidates for the in vivo
anti-inflammatory research.15
To further study the anti-inflammatory profile, the most potent
analogue 1f was selected for further evaluation of inhibitory activ-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.
059.
References and notes
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The results in Figure 2 demonstrate the favourable dependence
between concentration and inhibition, which are consistant with
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l
g/mL) for 24 h. The nitrite accumulation in the supernatant was
l of
assessed by the Griess reaction (Nath and Powledge, 1997). Each 50
l
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incubated at room temperature for 10 min. The absorbance at 540 nm was
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12. Typical procedure for the synthesis of 1a and 1e–1w. Compound 1f was
prepared as follows: to a suspension of 4f (1.0 g, 4.2 mmol) in DMF (6 mL) was
added aq KOH solution (1.8 M, 3.0 mL) followed by the addition of
chloroacetamide (0.5 g, 5 mmol) at room temperature. The resulting mixture
was stirred for 10–15 min at room temperature and then heated to 85 °C for
6 h after the addition of additional aqueous KOH solution (1.8 M, 3.0 ml). After
the reaction mixture was allowed to cool to room temperature, the precipitate
was collected by filtration, and washed with cold ethanol, then recrystallized
from ethanol to give a yellow solid (0.95 g, 75% yield), mp: 253–255 °C; 1H
NMR (400 MHz, DMSO-d6): d 8.54 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H),
8.08 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 10.4 Hz, 1H), 7.58 (q, J = 7.3 Hz, 1H), 7.33 (dt,
J = 8.4, 2.2 Hz, 1H), 7.23 (br, 2H), 7.22 (br, 2H); 13C NMR (100 MHz, DMSO-d6): d
166.86, 163.47, 161.86, 158.66, 154.76, 145.39, 140.43, 140.38, 131.77, 130.84,
130.79, 125.65, 122.91, 116.36, 116.20, 113.48, 113.33, 97.76; ESI-MS: m/z
286.24 [MÀH]+.
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