2-Phenyl and 2-heterocyclic-4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridines as inhibitors of TGF-β1 and activin A signalling

Article abstract of DOI:10.1016/j.bmcl.2010.12.120

Novel inhibitors of TGF-β1 and activin A signalling based on a 2-aryl-4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridine pharmacophore have been synthesised. Compounds containing phenyl or aromatic nitrogen heterocycle substituents inhibited both types of signalling with HEK-293T cells in culture, with a selectivity preference for TGF-β1. Synthetic compounds containing pyridin-3-yl, pyrazol-4-yl, pyrazol-1-yl or 1H-imidazoyl-1-yl substituents exhibited structural and functional attributes suitable for further investigation related to the development of more potent TGF-β inhibitors.

Full text of DOI:10.1016/j.bmcl.2010.12.120

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5642–5645
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Phenyl and 2-heterocyclic-4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridines
as inhibitors of TGF-b1 and activin A signalling
Rudy Ciayadi a, Mahesh Potdar a, Kelly L. Walton b, Craig A. Harrison b, Geoffrey F. Kelso a, Simon J. Harris a,
Milton T. W. Hearn a,
⇑
a Centre for Green Chemistry, Monash University, Clayton, Victoria 3800, Australia
b Prince Henry’s Institute, Monash Medical Centre, Clayton, Victoria 3168, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel inhibitors of TGF-b1 and activin A signalling based on a 2-aryl-4-(3-(pyridin-2-yl)-1H-pyrazol-4-
yl)pyridine pharmacophore have been synthesised. Compounds containing phenyl or aromatic nitrogen
heterocycle substituents inhibited both types of signalling with HEK-293T cells in culture, with a selec-
tivity preference for TGF-b1. Synthetic compounds containing pyridin-3-yl, pyrazol-4-yl, pyrazol-1-yl or
1H-imidazoyl-1-yl substituents exhibited structural and functional attributes suitable for further inves-
tigation related to the development of more potent TGF-b inhibitors.
Received 7 December 2010
Revised 22 December 2010
Accepted 23 December 2010
Available online 30 December 2010
Keywords:
Transforming growth factor-b
Activin A
Ó 2011 Published by Elsevier Ltd.
Low molecular weight inhibitors
Structure–activity relationships
ATP-competitive ALK4/ALK5 inhibitors
The transforming growth factor-b (TGF-b) superfamily of cyto-
kines includes a large number of signalling proteins, including
the archetypal members TGF-b1 and activin A. These proteins
modulate many different biological processes,1 including cell pro-
liferation,2 cell differentiation,3 extracellular matrix production,4
apoptosis5 and wound repair.6 Their biological effects are mani-
fested by signalling through transmembrane serine/threonine
receptor kinases. Both TGF-b1 and activin A are known to bind to
the type II serine/threonine receptors TGF-bRII and ActRII, respec-
tively, which sequentially leads to recruitment, phosphorylation
and activation of a type I serine/threonine receptor, activin-like ki-
nase (ALK), ALK5 and ALK4, respectively.7 Upon activation, the ALK
receptors phosphorylate a subset of cytoplasmic Smad proteins
(Smad 2 and Smad 3), which then bind to a common-partner, Smad
4, to form a complex that induces regulation of gene transcription.7
Increased TGF-b1 signalling has been implicated in pathological
fibrosis and associated with the growth of highly malignant can-
cers, whilst activin A has also been implicated in numerous malig-
nancies as well as in apoptosis, wound repair, cell differentiation
and proliferation, and endocrine functions.8 Consequently, ALK5
has been proposed as a therapeutic target for these diseases and
investigations with different competitive inhibitors of ATP-binding
to ALK5 have been previously undertaken based on five-membered
6-nitrogen heterocycles containing either a pyridin-2-yl or methyl-
pyridin-2-yl substituent and an ortho aromatic substituent (Fig. 1).
Examples of these compound classes include 2,4,5-substituted imi-
dazoles (1)9, 1,3,5-substituted 1,4,5-triazoles (2)10, 2,4,5-substi-
tuted 1,2,3-triazoles (3)11, 4,5-substituted 2-aminothiazoles (4)12
,
1,2,4-substituted imidazoles (5)13
, 1,3,5-substituted pyrazoles
(6)13, 2,3-substituted pyryolo-pyrazoles (7)12,14 and 4,5-substi-
tuted pyrazoles (8 and 9).15
Recent clinical and biological investigations have also shown in-
creased levels of activin A signalling are involved in pathological
fibrosis, the progression from pre-malignancy to advanced cancers
as well as pre-eclampsia.16 Therefore ALK4 inhibitors may be
potentially useful in treating these diseases. Given the high se-
quence homology between the ATP-binding sites of ALK4 and
ALK5 (>90%)17 it is feasible that ATP-competitive ALK5 inhibitors
may also inhibit activin A signalling. Indeed, recent work has
shown that specific members from the pharmacophore series 5
and 6 are capable of inhibiting both ALK4 and ALK5 kinase activity
with different selectivities.13
Compounds based on the 2-aryl-4-(3-(pyridin-2-yl)-1H-pyra-
zol-4-yl)pyridine pharmacophore 9 containing a para-substituted
phenyl ring as the aromatic substituent are potent inhibitors of
ALK5/TGF-b1 signalling, and can prevent kidney fibrosis in rats
and mice.15b,18 However, structure–activity relationships for inhi-
bition of activin A signalling have not been explored in detail for
this class of compound. Here we present work on the synthesis
⇑
Corresponding author. Tel.: +61 3 9905 4547; fax: +61 3 9905 8501.
E-mail address: milton.hearn@monash.edu (M.T.W. Hearn).
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.12.120

R. Ciayadi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5642–5645
5643
Figure 1. Structures of ATP-competitive inhibitors of ALK-5.
of novel analogues containing different nitrogen heterocycle sub-
stituents on the 4-pyridine ring and document their activities,
along with other synthetic compounds containing phenyl substitu-
ents, as inhibitors of both TGF-b1 and activin A signalling using hu-
man embryonic kidney (HEK)-293T cells in culture.
To synthesise analogues of the pharmacophore series 9 contain-
ing nitrogen heterocycle substituents at position 2 of the 4-pyridine
ring, the precursor 2-bromo-4-(3-(pyridin-2-yl)-1-trityl-1H-pyra-
zol-4-yl)pyridine (13) was first synthesised from 2-bromo-4-
methyl pyridine (10) and ethyl picolinate with some improved
modifications (Scheme 1).19 Firstly, treatment of 10 with KHDMS
and then ethyl picolinate gave 2-(2-bromopyridin-4-yl)-1-(pyri-
din-2-yl)ethanone (11). We found that on the multigram scale, mild
heating following addition of ethyl picolinate gave an improved
yield of 11 and removed the need for purification by column chro-
matography as previously reported.15b Treatment of 11 with
dimethylformamide dimethyl acetal followed by hydrazine gave
crude 12 which was purified on a multigram scale by simply titur-
ation with hexane/dichloromethane to give 12 an excellent yield.
Protection of 12 with the trityl group in acetone containing K2CO3
gave poor yields of 13 and significant impurities in our hands.
Hence, we protected 12 in dichloromethane using triethylamine
as the base which gave an improved yield of 13 and removed the
need for subsequent column chromatography. The trityl intermedi-
ate 13 was reacted with different aromatic heterocyclic boronic
acids in a Suzuki–Miyaura coupling followed by trityl deprotection
to yield the corresponding arylated derivatives 9a–g. Alternatively,
trityl intermediate 13 was reacted with different aromatic or
aliphatic heterocyclic secondary amines at high temperature in a
focused microwave reactor followed by trityl deprotection to give
the corresponding aminated derivatives 9h–l. Moreover, the pre-
cursor 12 was readily hydrodebrominated to give the unsubstituted
analogue 9m.
Scheme 1. Reagents and conditions: (a) KHMDS, À50 °C; (b) ethyl picolinate,
À50 °C to rt 18 h, 45 °C 1 h; (c) DMF–DMA, AcOH; (d) NH2NH2ÁH2O, 50 °C 2 h to rt
22 h; (e) trityl chloride, Et3N; (f) ArB(OH)2, Pd(PPh3)4, Na2CO3 or heterocyclic
secondary amine, K2CO3, microwave heating; (g) methanolic 1 M HCl; (h) H2, Pd/C.
Table 1
Inhibition of TGF-b1 and activin A signalling by compounds 9a–m
Compound
TGF-b1a activity (% of
Activin Aa activity (% of
control)b
control)b
1
l
M
100 nM
1
l
M
100 nM
9a
9b
9c
9d
9e
9f
9g
9h
9i
14 (2–26)
1 (0–6)
7 (3–11)
29 (22–36)
46 (32–60)
58 (55–61)
1 (0–2)
na
59 (41–77)
58 (42–74)
62 (53–70)
na
na
na
1 (0–5)
37 (32–42)
62 (51–73)
62 (61–63)
na
na
na
na
na
na
na
63 (55–71)
72 (49–95)
69 (55–83)
na
na
3 (0–7)
42 (37–47)
50 (45–55)
70 (61–79)
na
83 (80–86)
1 (0–9)
na
na
na
na
na
na
11 (9–13)
34 (30–38)
80 (66–94)
90 (89–91)
50 (44–56)
9j
9k
9l
na
na
na
9m
87 (86–88)
a
100 pM.
b
The compounds were assessed as inhibitors of TGF-b signalling
in HEK-293T cell culture by measuring their inhibition of TGF-b1
and activin A dependent luciferase transcription (Table 1).20 This
cell line has previously been shown to endogenously express the
ALK-4, ALK-5 and associated type II co-receptors required for
TGF-b1 and activin A signalling.21a,b The cells were transfected
with a luciferase expression vector (A3-LUX) containing the activin
Values are means of duplicate experiments, 95% confidence intervals are given
in parentheses (na = not active).
responsive binding elements (ARE) in the promoter region, and a
fork head activin signal transducer-2 (FAST-2) expression vec-
tor.21a,22 The smad 2/3-smad 4 complex, arising from TGF-b signal-
ling through ALK receptors, targets the ARE elements which in turn

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R. Ciayadi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5642–5645
are mediated by direct interaction with FAST-2.21 Hence, the assay
system used in these investigations enables TGF-b and activin A
signalling via the ALK-smad pathway to be selectively measured
in cell culture by quantifying the level of luciferase expression.
The results of these assays showed that compounds containing
pyridinyl (9b and 9c) or the smaller 1-methyl-1H-pyrazol-4-yl
(9g), 1H-pyrazol-1-yl (9h) or 1H-imidazoly-1-yl (9i) substituents
were more potent inhibitors of TGF-b1 signalling compared to
the phenyl derivative 9a, and had comparable potency to 9a as
inhibitors of activin A signalling, except for 9g which had signifi-
cantly greater potency. The compounds containing quinolinyl sub-
stituents (9d and 9e), the pyrmidin-5-yl substituent (9f) or no
substituent (9m) were comparable to 9a as inhibitors of TGF-b1
signalling but were weaker inhibitors of activin A signalling. In
addition, the benzimidazol-1-yl derivative 9j showed comparable
activity against both types of signalling. The compounds contain-
ing aliphatic piperidine or piperazine substituents (9k and 9l)
had much reduced or no activity compared to 9a. Overall, most
of the compounds that inhibited TGF-b1 signalling showed more
selective inhibition of TGF-b1 relative to activin A in these initial
screening assays.
Compounds in the pharmacophore class 9 containing para-
2-(dimethylamino)ethoxyphenyl (9n) or para-N-(tetrahydro-2H-
pyran-4-yl)benzamide (9o) substituents have been shown to be
considerably more potent inhibitors of ALK5 kinase activity and
TGF-b1 signalling compared to 9a whereas analogues containing
ortho or meta substituents are known to be relatively poor inhibi-
tors.15b However, the effect of different substituents or substitution
pattern on inhibition of activin A signalling has not been explored
in detail for this class. Hence we have synthesised compounds con-
taining ortho, meta and para substituents on the phenyl ring along
with novel pyridin-3-yl analogues containing 3-(dimethyl-
amino)propan-1-oxy (9r) and 4-methyl-piperazine (9s) substitu-
ents at position 6 of the pyridin-3-yl ring and assessed their
To obtain a more accurate assessment of structure–activity rela-
tionships, the IC50 values for signalling inhibition were determined
from dose–response curves of the more potent phenyl and pyridin-
3-yl derivatives, and the potent inhibitors containing the smaller
five-membered nitrogen heterocycle substituents identified in
the initial screening assays (Table 3).20 The compounds containing
para-substituted phenyl or 6-substituted pyridin-3-yl substituents
were of similar potency and had a slight selectivity toward TGF-b1,
except for 9r which had a comparable IC50 value for inhibition of
both types of signalling. Compounds containing the relatively
smaller five-membered nitrogen heterocycle substituents were
also potent inhibitors of TGF-b1 signalling and showed greater
selectivity toward TGF-b1 over activin A compared to the com-
pounds with the larger aromatic substituents.
In conclusion, the 2-aryl-(4-(pyridin-4-yl)-1H-pyrazol-3-yl)pyr-
idine pharmacophore 9 incorporating different aromatic nitrogen
heterocycles as aryl substituents to give compounds that inhibit
both TGF-b1 and activin A signalling, with selectivity for the for-
mer. In terms of drug development, these different structural fea-
tures and selectivities are expected to be important determinants
of pharmacokinetics, metabolism and side-effects. We have also
shown that compounds containing five-membered aromatic nitro-
gen heterocycle substituents are comparatively potent to those
containing larger aromatic substituents and are more selective
for TGF-b1. Given that specific substitutions of the larger phenyl
and pyridin-3-yl substituents lead to improved potency, it can be
concluded that similar substitutions of the smaller five-membered
nitrogen heterocycles may also provide compounds with improved
activity. The synthesis of a library of these compounds is the focus
of our current research and will be reported in due course.
activity as inhibitors of TGF-b1 and activin
A signalling
(Table 2).19,20
Compound 9q containing the ortho-methoxyphenyl substituent
inhibited both types of signalling with potency comparable to 9a
but did not show any selectivity. The meta-methoxyphenyl deriva-
tive 9p was not active at all. Compounds 9n and 9o containing the
para-substituted phenyl substituents were significantly more po-
tent as inhibitors of TGF-b1 and activin A signalling compared to
9a. We also found that the novel substituted pyrdin-3-yl analogues
9r and 9s inhibited both types of signalling with potency compara-
ble to 9n. As observed with the active compounds in the initial
screening assays, most of these more potent compounds also
showed selectivity toward TGF-b1 (Fig. 2).
Figure 2. Structures of synthetic compounds 9n–s.
Table 2
Inhibition of TGF-b1 and activin A signalling by compounds containing different
phenyl and pyrdin-3-yl substituents
Compound TGF-b1c activity (% of
Activin Ac activity (% of
control)d
Table 3
control)d
IC50 values for inhibition of TGF-b1 and activin A signalling
b
b
Compound
TGF-b1a activity IC50 (nM)
Activin Aa activity IC50 (nM)
9na
9o
1 (0–2)
52 (48–56)a
na
54 (46–62)
1 (0–3)
1 (0–5)
4 (0–30)
42 (31–53)b
na
9g
9h
9i
9n
9o
9r
9s
68 (49–95)
77 (58–103)
88 (33–234)
19 (16–24)
446 (288–689)
11 (6–19)
224 (155–322)
882 (357–3021)
956 (368–2495)
46 (30–70)
>1000
18 (11–30)
9pb
9qb
9ra
9sa
71 (53–89)
38 (28–48)
31 (27–45)
a
b
c
100 nM.
M.
100 pM.
32 (23–43)
108 (46–256)
1
l
a
100 pM.
d
Values are means of duplicate experiments, 95% confidence intervals are given
b
Calculated from non-linear regression analysis of dose–response curves, 95%
confidence intervals are given in parentheses.
in parentheses (na = not active).

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