Synthesis and biological evaluation of 1-(6-methylpyridin-2-yl)-5- (quinoxalin-6-yl)-1,2,3-triazoles as transforming growth factor-β type 1 receptor kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.12.008

A series of 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles has been synthesized and evaluated for their ALK5 inhibitory activity. The 1-(6-methylpyridin-2-yl)-1,2,3-triazoles were assembled by Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition. Following this, quinoxaline was introduced through Pd-catalyzed direct arylation. The synthesized 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles revealed significant selectivity differences with respect to p38α MAP kinase. In particular, 12k showed 80.8% ALK5 inhibitory activity at a concentration of 10 μM and IC₅₀ value of 4.69 μM, but did not show p38α MAP kinase inhibitory activity (-1.94% inhibition at a concentration of 10 μM).

Full text of DOI:10.1016/j.bmcl.2012.12.008

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1083–1086
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 1-(6-methylpyridin-2-yl)-
5-(quinoxalin-6-yl)-1,2,3-triazoles as transforming growth factor-b
type 1 receptor kinase inhibitors
Fei Li a, Yunjeong Park a, Jung-Mi Hah b, Jae-Sang Ryu a,
⇑
a Center for Cell Signaling & Drug Discovery Research, College of Pharmacy and Division of Life & Pharmaceutical Sciences, Ewha Womans University, 11-1 Daehyun-Dong,
Seodaemun-Gu, Seoul 120-750, Republic of Korea
b Department of Pharmacy, College of Pharmacy, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan, Kyeonggi-do 426-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles has been synthesized and evalu-
ated for their ALK5 inhibitory activity. The 1-(6-methylpyridin-2-yl)-1,2,3-triazoles were assembled by
Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition. Following this, quinoxaline was introduced
through Pd-catalyzed direct arylation. The synthesized 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-
Received 14 November 2012
Revised 30 November 2012
Accepted 7 December 2012
Available online 20 December 2012
1,2,3-triazoles revealed significant selectivity differences with respect to p38
12k showed 80.8% ALK5 inhibitory activity at a concentration of 10 M and IC50 value of 4.69
not show p38 l
a
MAP kinase. In particular,
M, but did
M).
l
l
Keywords:
ALK5
TGF-b type 1 receptor kinase
Anticancer
a
MAP kinase inhibitory activity (À1.94% inhibition at a concentration of 10
Ó 2012 Elsevier Ltd. All rights reserved.
Click chemistry
1,2,3-Triazole
In the last 30 years, a large family of secreted polypeptides,
known collectively as the transforming growth factor-b (TGF-b)
superfamily, has been revealed. This superfamily consists of three
transforming growth factor-b isoforms (TGF-b1, TGF-b2 and TGF-
b3),1 four activins, and over 20 bone morphogenetic proteins
(BMPs). Among them, TGF-b has been shown to participate in a
wide range of biological processes and to act as a multifunctional
regulator: a suppressor of cell growth, a promoter of apoptosis, a
stimulator of extracellular matrix production, and a controller of
proliferation and migration of endothelial and vascular smooth
muscle cells. In addition, TGF-b also regulates proliferation, differ-
entiation, and activation of immune cells. Therefore, deregulated
TGF-b signaling could be a provoking cause of various diseases2
such as cancer,3 tissue fibrosis,4 vascular disorders and autoim-
mune diseases.
phorylates Smad2/Smad3 proteins at the C-terminal SSXS-motif.
The phosphorylated Smad2/Smad3 proteins are dissociated from
the receptor and form a heteromeric complex with Smad4. This
Smad complex moves into the nucleus and regulates gene tran-
scription.6 Therefore, inhibition of ALK5 phosphorylation of
Smad2/Smad3 could reduce TGF-b-induced excessive accumula-
tion of extracellular matrix, which would finally lead to the treat-
ment of fibrotic diseases and cancer.
To date, a number of attempts have been made to block TGF-b
effects. In particular, a study targeting ALK5 has led to the
development of potent imidazole and pyrazole inhibitors such as
1 (SB-505124),7 2 (SB-525334),8 3 (GW6604),9 4 (LY2157299),10
5 (IN-1166),11,12 6 (IN-1130) and 7 (IN-1233),13 which have shown
anti-fibrosis or anticancer activities (Fig. 1). Compounds 2 (SB-
525334) and 3 (GW6604) were reported to slow down the progress
of fibrosis in the kidney and liver, respectively. Compound 4
(LY2157299) exhibited strong anticancer activity in animal
models. Recently, Kim and co-workers have reported a 2-pyridyl-
substituted novel class of ALK5 inhibitors, 5 (IN-1166), 6 (IN-
1130) and 7 (IN-1233). As a preclinical candidate, 6 (IN-1130)
effectively suppressed the fibrogenic process of unilateral ureteral
obstruction in rats,14 ameliorated murine autoimmune encephalo-
myelitis,15 induced significant regression of fibrotic plaque in rats
with Peyronie’s disease,16 and inhibited tumor progress and en-
hanced immune response in mice with prostate cancer.17 Another
preclinical candidate 7 (IN-1233) prevented pulmonary arterial
TGF-b signals5 are transduced through a complex of two struc-
turally and functionally distinct serine/threonine kinase receptors,
called type I (TbR-I) and type II (TbR-II). Initially, TGF-b ligand
binds to TbR-II. This is recognized by TbR-I (also named activin
receptor-like kinase 5 or ALK5), which is then recruited into the
complex. Subsequently, TbR-II hyperphosphorylates serine/threo-
nine residues in the GS region of ALK5. After this, the activated
ALK5 creates a binding site for Smad2/Smad3 proteins and phos-
⇑
Corresponding author. Tel.: +82 2 3277 3008; fax: +82 2 3277 2851.
E-mail address: ryuj@ewha.ac.kr (J.-S. Ryu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.008

1084
F. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1083–1086
O
N
N
O
O
NH2
N
N
N
N
N
NH
N
H
N
H
N
N
N
N
N
N
1 (SB-505124)
2 (SB-525334)
3 (GW6604)
4 (LY2157299)
N
CN
N
N
O
N
O
N
HN
N
NH2
N
NH2
N
N
H
N
H
N
H
N
N
N
5 (IN-1166)
6 (IN-1130)
7 (IN-1233)
Figure 1. Small molecule ALK5 inhibitors.
hypertension in monocrotaline rats,18 and stopped granulation tis-
sue formation after bare metallic stent placement in a rat urethral
model.19
N
X
n
4
N
5
N
3
N
In 5, 6, and 7, the insertion of a methylene amino or a methy-
lene linkage between an imidazole ring and a phenyl ring signifi-
cantly improved ALK5 inhibitory activity and selectivity. It was
also found that the methylene phenyl moiety or methylene amino
phenyl at the 2-position of the imidazole ring served as an ex-
tended pharmacophore to the ATP-binding site of ALK5.11
With regard to the design of new ALK5 inhibitors, we carefully
looked at the binding pose of 5 built by a docking model and found
that the additional phenyl ring was oriented toward the quinoxa-
line side rather than the pyridine side.11 A similar conformation
was also observed for 7 (Fig. 2).13 On the basis of these findings,
we have designed and synthesized 1-(6-methylpyridin-2-yl)-
5-(quinoxalin-6-yl)-1,2,3-triazoles as novel ALK5 inhibitors
(Fig. 3). It was of our particular interest whether incorporation of
a substituent at the 4-position of the 1,2,3-triazole ring might help
this series of compounds to adopt the active conformation.
The importance of 1,2,3-triazole compounds in medicinal
chemistry is indubitable. 1,2,3-Triazole is metabolically stable
and capable of hydrogen bonding, thereby improving water solu-
bility and in turn favorable toward binding of drugs and its tar-
gets.20 Although 1,2,3-triazole does not occur in nature, the
synthetic molecules containing 1,2,3-triazole moieties show bio-
logical properties such as anticancer, antifungal, antibacterial,
anti-HIV, and antituberculosis activities.21 As part of our ongoing
efforts to develop 1,2,3-triazole anticancer agents,22 we have car-
N 2
1
N
n = 0~1
Figure 3. Design of novel 1,2,3-triazole ALK5 inhibitors.
ried out research which has produced potent 1,2,3-triazole kinase
inhibitors. In general, using a 1,2,3-triazole scaffold in the develop-
ment of kinase inhibitors offers several advantages: (i) a 1,2,3-
triazole can be a mimic of the purine of ATP that targets the kinase
ATP-binding site; (ii) a 1,2,3-triazole can function as the bioisostere
of flat heteroaromatic rings such as imidazole, pyrazole and purine,
which are frequently found in kinase inhibitors; (iii) a 1,2,3-
triazole can be easily assembled by ‘click chemistry’; and (iv) a
variety of substituents can be easily attached by using commercial
alkynes or azides. Therefore, in order to develop novel ALK5
inhibitors of 1,2,3-triazoles containing pyridine and quinoxaline,
we envisioned a short and efficient synthetic route through
Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC).23
Diverse substituents could be introduced by CuAAC at the
4-position of 1,2,3-triazole. After this, quinoxaline was coupled
through Pd-catalyzed direct arylation. All required functionalities
could be assembled in three steps as shown in Scheme 1. First,
2-bromo-6-methylpyridine (8) was treated with NaN3 in DMSO
at 120 °C, which gave 2-azido-6-methylpyridine (9) in 90% yield.
Subsequently, various commercial alkynes 10a–k were coupled
with azide 9 in the presence of copper(I) catalyst under standard
‘click’ conditions (CuI/DIPEA in DMF, or CuSO4/NaHCO3/sodium
ascorbate in H2O/DMF) to afford 1,4-disubstituted 1,2,3-triazoles
11a–k. The click reactions with the aliphatic alkynes 10a–c or
10k were efficient to afford the corresponding 1,2,3-triazoles in
98%, 64%, 75% and 84% yields, respectively. However, the click reac-
tions with the aromatic alkynes 10d–j were relatively sluggish and
provided the corresponding 1,2,3-triazoles 11d–j in 8–42% yields
due to the low solubility of substrates and/or products in the reac-
tion solvents. Next, we directly incorporated a quinoxaline moiety
at the C-5 position of 1,2,3-triazoles. We considered Pd-catalyzed
direct arylation reaction of 1,2,3-triazole which was recently devel-
oped by Gevorgyan and co-workers.24 This methodology allows for
Asp290
H
N
H
CN
N
His283
His283
O
N
N
Asn338
N
H
N
N
N
H
N
H
Asp351
N
N
Ser280
7
Ser280
5
Hydrophobic pocket
Figure 2. Active conformation of ALK5 inhibitors 5 and 7.
Hydrophobic pocket

F. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1083–1086
1085
Table 1
b
a
Inhibitory activity of 1,2,3-triazoles 12a–k on ALK5 and P38a
/MAPKa
N
N3
R
N
Br
10a-k
8
9 (90%)
N
N
R
10a: R = n-Butyl
10b: R = Isopentyl
10c: R = 4-Hydroxybutyl
10d: R = 3,5-Dimethoxyphenyl
10e: R = Phenyl
N
N
N
N
10f: R = 4-Fluorophenyl
10g: R = 2-Methoxyphenyl
10h: R = 4-Methoxyphenyl
10i: R = 3,5-Difluorophenyl
10j: R = p-Tolyl
12a-k
Compound
R
%Inhibitionb
ALK5 P38
IC50 (lM)
10k: R = Benzyl
a/
ALK5
N
MAPK
À2.52
À5.91
R
R
12a
12b
70.0
73.1
7.38
8.60
N
c
N
N
N
N
N
N
N
N
Br
N
OH
12c
20.4
À11.2
50.8
N
OCH3
11a-k
11a: R = n-Butyl (98%)
11b: R = Isopentyl (64%)
11c: R = 4-Hydroxybutyl (75%)
12a-k
12d
23.4
À6.87
ND
12a: R = n-Butyl (19%)
OCH3
F
12b: R = Isopentyl (56%)
12c: R = 4-Hydroxybutyl (22%)
12e
12f
À4.94
31.2
30.9
ND
ND
11d: R = 3,5-Dimethoxyphenyl (8%) 12d: R = 3,5-Dimethoxyphenyl (12%)
11e: R = Phenyl (42%)
12e: R = Phenyl (14%)
11f: R = 4-Fluorophenyl (26%)
11g: R = 2-Methoxyphenyl (26%)
11h: R = 4-Methoxyphenyl (16%)
11i: R = 3,5-Difluorophenyl (33%)
11j: R = p-Tolyl (27%)
12f: R = 4-Fluorophenyl (53%)
12g: R = 2-Methoxyphenyl (14%)
12h: R = 4-Methoxyphenyl (34%)
12i: R = 3,5-Difluorophenyl (19%)
12j: R = p-Tolyl (35%)
8.19
H3CO
12g
12h
34.9
0.26
18.4
34.5
ND
11k: R = Benzyl (84%)
12k: R = Benzyl (22%)
OCH3
Scheme 1. Synthesis of 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-tria-
zoles 12a–k. Reagents and conditions: (a) NaN3, DMSO, 120 °C, 20 h; (b) CuI,
DIPEA, DMF, 100 °C (11a–b, 11d, 11f); CuSO4, sodium ascorbate, NaHCO3, H2O/DMF,
100 °C (11c, 11e, 11g–k); (c) Pd(OAc)2, Bu4NOAc, NMP, 100 °C.
À27.7
F
12i
12j
17.4
40.4
ND
F
efficient introduction of a quinoxaline group without the need of
an additional functionalization such as halogenation or borylation
at the C-5 position of the 1,2,3 triazole ring. Indeed, the treatment
of 1,4-disubstituted 1,2,3-triazoles 11a–k with Pd(OAc)2, tetrabu-
tylammonium acetate, and 6-bromoquinoxaline in NMP at 100 °C
produced C-5 arylated 1,2,3-triazoles 12a–k in 12–56% yields. A
variety of 1,4-disubstituted 1,2,3-triazoles, containing electron-
withdrawing or electron-donating aryl groups as well as aliphatic
groups at the C-4 position was shown to undergo C-5 arylation
successfully. Our synthetic strategy, a click reaction coupled with
direct C-5 arylation, allowed facile and flexible access to various
1,4,5-trisubstituted 1,2,3-triazoles which can be potential ALK5
inhibitors.
20.7
80.8
À5.07
À1.94
ND
12k
4.69
Staurosporine IC50
SB202190 IC50 (nM)
(l
M)
—
—
—
4.55
6.87
—
a
Enzymatic assay was conducted by Reaction Biology Corporation (http://
www.reactionbiology.com).26
b
Compounds were tested at 10 lM in the presence of 1 lM ATP. Data shown are
the average of duplicated assays. %Inhibition is calculated by subtracting % enzyme
activity from 100.
The kinase domain of p38
a MAP kinase is known as one of the
To evaluate the kinase inhibitory activity of synthesized 1,4,5-
trisubstituted 1,2,3-triazoles 12a–k, an in vitro kinase assay was
most homologous to that of ALK5.25 Therefore, we selected p38
a
MAP kinase to confirm the selectivity profile of this series of
performed at a single dose concentration of 10
lM over ALK5
compounds. Most of the synthesized 1,4,5-trisubstituted 1,2,3-
and p38 MAP kinase. Table 1 shows %inhibition against kinase
a
triazoles, 12a–k, were devoid of p38
activity at the tested concentration. In particular, compound 12k
was found to be very selective in this series of compounds (80.8%
inhibition for ALK5 vs À1.94% inhibition for p38
a MAP kinase inhibitory
activity as an average of duplicate assays and IC50 values. Alkyl
substituted 1,2,3-triazoles showed relatively high ALK5 inhibitory
activities. Among alkyl substituted 1,2,3-triazoles 12a–c, isopentyl
substituted 12b exhibited significant inhibitory activity (73.1%
a MAP kinase).
In conclusion, a series of novel 1-(6-methylpyridin-2-yl)-5-
(quinoxalin-6-yl)-1,2,3-triazole derivatives has been designed
based on a molecular docking study and the fully substituted
1,2,3-triazoles were efficiently synthesized from 2-bromo-6-
methylpyridine (8) in three steps. The synthetic strategy was
concise and straightforward. 2-Azido-6-methylpyridine (9) and
various alkynes were assembled by Cu(I)-catalyzed azide–alkyne
1,3-dipolar cycloaddition. The resulting 1-(6-methylpyridin-2-yl)-
1,2,3-triazoles were coupled with 6-bromoquinoxaline through
Pd-catalyzed direct arylation. The synthesized 1-(6-methylpyri-
din-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles exhibited moderate
inhibition at 10 lM, IC50 = 8.60 lM). However, aryl substituted
1,2,3-triazoles 12d–j exhibited rather low inhibitory activity
against ALK5 in a range of À4.94 to 34.9%. The aryl moiety directly
attached to the 1,2,3-triazole did not seem to be favorably accom-
modated into the ATP binding pocket of ALK5. This was also con-
firmed by IC50 value of 12g (34.5
than those of 12a and b (7.38 and 8.60
result was observed for benzyl 1,2,3-triazole 12k (80.8% inhibition
at 10 M, IC50 = 4.69 M). The length of alkyl chain between C-4 of
l
M), which was much higher
lM). Interestingly, the best
l
l
the central triazole and a phenyl ring also influenced ALK5
inhibition.

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F. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1083–1086
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selectivity differences against p38 MAP kinase. In particular,
12k showed 80.8% ALK5 inhibitory activity at a concentration of
10 M and IC50 value of 4.69 M, but did not show p38 MAP
kinase inhibitory activity (À1.94%). We believe that this compound
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Acknowledgments
This work was supported by a grant from Basic Science Re-
search Program (2009-0075425) through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology.
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Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.008.
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