Rapid generation of a high quality lead for transforming growth factor-β (TGF-β) type I receptor (ALK5)

Article abstract of DOI:10.1021/jm900807w

A novel class of 4-pyridinoxy-2-anilinopyridine-based TGF-β type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors is reported. The binding mode of this scaffold was successfully predicted by analyzing possible docked binding modes of li

Full text of DOI:10.1021/jm900807w

J. Med. Chem. 2009, 52, 7901–7905 7901
DOI: 10.1021/jm900807w
Rapid Generation of a High Quality Lead for Transforming Growth Factor-β (TGF-β)
Type I Receptor (ALK5)†,‡
ꢀ
Frederick W. Goldberg,* Richard A. Ward,* Steven J. Powell, Judit E. Debreczeni, Richard A. Norman,
Nicola J. Roberts, Allan P. Dishington, Helen J. Gingell, Kate F. Wickson, and Andrew L. Roberts
AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, U.K.
Received June 4, 2009
A novel class of 4-pyridinoxy-2-anilinopyridine-based TGF-β type I receptor (also known as activin-like
kinase 5 or ALK5) inhibitors is reported. The binding mode of this scaffold was successfully predicted by
analyzing possible docked binding modes of literature inhibitors and novel synthetic ideas. Compounds
such as 19 are potent ALK5 inhibitors with good physicochemical and pharmacokinetic properties and
thus represent high quality leads for further optimization.
Introduction
ALK5 inhibitors from the literature4-14 were modeled using
protein-ligand docking software along with manual ligand
overlaying. A published ALK5 structure that had a pyrrolopyr-
azole-based ALK5 inhibitor bound (1RW8) was used for the
docking studies.12 The inhibitor binding modes showed some
conserved interactions that were consistent across a number of
chemical series, in particular a hydrogen bond acceptor to the
kinase hinge region (His-283 in ALK5) that is characteristic of
the majority of ATP-competitive kinase inhibitors. Interest-
ingly, there was also a hydrogen bond acceptor, usually from a
pyridyl (or bipyridyl) group in the inhibitor, which can make a
water-mediated hydrogen bond to the protein. This water
molecule was observed in the “selectivity pocket”15 of the
1RW8 crystal structure and maintained in the docking calcula-
tions due to its likely essential involvement in the observed
binding mode. Including this water molecule in the docking
studies increased the consistency of the docked poses in a
number of inhibitors, suggesting this interaction might be
important. We therefore designed and synthesized a number
of compounds that would exploit these two key interaction
points but which offered scope for further optimization. On the
basis of these modeled binding modes, the published inhibitors
were manually fragmented into their respective hinge, selectivity
pocket, and solvent channel binding regions. Additional hinge
region fragments were added from in-house kinase inhibitor
structures. The fragments were then hybridized together in
different combinations, with all generated compounds contain-
ing a hinge region and selectivity pocket group and in some cases
a solvent channel group. All generated ideas were docked and
suitable structures were then prioritized by synthetic tractability
and novelty. This process ultimately resulted in the identifi-
cation of novel compound 1 (Figure 1), which possesses a
bipyridyl group that can access the selectivity pocket and make
the aforementioned water-mediated hydrogen bond to the
protein. This compound was also predicted to form a key
interaction to the hinge with the 2-amino-4-oxo pyridyl (ring
3) and place the trimethoxyaniline fragment (ring 4) into the
solvent channel of the kinase binding pocket.
Thetransforminggrowth factor family of ligands, including
transforming growth factor-β TGFβ,a activins, inhibins, no-
dal, and bone morphogenic proteins play a key role in
controlling cellular functions such as differentiation, migra-
tion, proliferation, adhesion, and development.1 This family
of ligands signals through heteromeric complexes of type I
(e.g., ALK5) and type II serine/threonine kinase (e.g.,
TGFβRII) receptors. Seven type I receptors have been identi-
fied (ALK1 through ALK7) in mammals; signaling specificity
is determined by the ligand/receptor complex and the cellular
context. Inhibitors of TGFβ signaling may have therapeutic
potential in targeting both fibrosis and cancer. While TGFβ
can act as a tumor suppressor as well as a tumor promoter, it
has been established that levels of TGFβ ligand and its
signaling axis are elevated in a wide range of tumor types
including breast, colon, gastric, lung, hepatocellular, and
pancreatic.2 There is mounting evidence that TGFβ levels
within the tumor and local environment correlate to a more
aggressive metastatic phenotype, increased angiogenesis, and
tumor progression manifested by effects on tumor cells,
endothelial cells, and infiltrating cells.3
Hit Identification
The ALK5 literature was reviewed to generate ideas for a
novel chemicalstart point. Thelikely bindingmodes of known
†We acknowledge the 100th anniversary of the Division of Medicinal
Chemistry of the American Chemical Society and are very pleased to be
able to contribute to the Centennial Issue of the Journal of Medicinal
Chemistry.
‡Crystal structures of ALK5 in complex with compounds 1 and 19
have been deposited in the Protein Data Bank (PDB accession codes
2WOT and 2WOU, respectively).
*To whom correspondence should be addressed. For F.W.G.: phone,
þ44(0)1625 519064; fax, ; E-mail Frederick.Goldberg@AstraZeneca.
com. For R.A.W.: phone, þ44(0)1625 519045; fax, ; E-mail Richard.A.
Ward@AstraZeneca.com.
a Abbreviations: ALK, activin-like kinase; TGFβ(R), transforming
growth factor-β (receptor); DMA, dimethylacetamide; DMF, N,N-
dimethylformamide; DMSO, dimethylsulfoxide; SAR, structure-activ-
ity relationship.
Compound 1 had excellent enzyme and cell potency
(Table 1) but poor oral bioavailability (rat F = 4%). The
r
2009 American Chemical Society
Published on Web 09/08/2009
pubs.acs.org/jmc

7902 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Goldberg et al.
ALK5 enzyme assay used a His-tagged fragment (residues
162-503) of the human enzyme, and the cellular assay was a
nuclear translocation assay of a GFP/Smad2 fusion repor-
ted in MDA-MB-468 cells (see Supporting Information for
further details). This compound had acceptable aqueous
solubility (52 μM in pH 7.4 phosphate buffer) and in vivo
clearance (20 mL/min/kg), so our hypothesis was that the
observed low bioavailability was due to poor absorption and/
or high efflux. Although the lipophilicity of 1 as an initial hit
was acceptable (logD7.4 = 3.2), it was felt that identifying a
smaller analogue while retaining potency (i.e., improving
ligand efficiency)16 would improve our chances of ultimately
developing a compound that would have improved absorp-
tion andtherefore efficacy invivo.17 Identifying which partsof
the scaffold were important for potency therefore became a
priority, and again X-ray crystallography was used to help
prioritize our efforts. A ligand bound ALK5 crystal structure
was generated in-house for 1 (Figure 2A). The observed
binding mode corresponded to that predicted by modeling
studies and gave us confidence as to how other parts of the
active site could be explored from this template.
The importance of the nitrogens on the bipyridyl motif was
assessed by systematically removing them. Although “ring 2”
(Figure 1) appeared to be very important to the binding of the
molecule (through the conserved water molecule), it was less
clear whether or not the interaction of the “ring 1” pyridine
with Lys-232 was required for potency. Further ideas were
also generated to probe other regions of the active site,
particularly the solvent channel and ribose pocket, as the
aniline was predicted to be able to interact with both regions.
Chemistry
The initial hit 1 was synthesized from phenol 313 by base-
mediated addition to 2,4-dichloropyridine and subsequent
Buchwald-Hartwig amination18 with 3,4,5-trimethoxyani-
line (Scheme 1). For compounds where the required phenol
was difficult to synthesize, an alternative route was also
developed via iodo intermediate 4. Negishi coupling19-21 of
4
with 2-pyridylzinc bromide and subsequent Buch-
wald-Hartwig amination as before afforded the desired
compound 5. The Negishi coupling is our preferred method
to synthesize bipyridyls due to the known instability of
2-pyridyl boronic acids22 and the toxicity of the equivalent
stannanes.
In some cases, the base-mediated addition of the phenol
group with 2,4-dichloropyridine did not yield the desired
product, so to expand the range of phenols that could be
used, Ullmann coupling23 with 2-chloro-4-iodo-chloropyri-
dine or base-mediated additions to 2-chloro-4-fluoropyridine
were also utilized (Scheme 2). This latter approach has the
advantage that the order of the steps can be easily reversed to
allow flexible assembly of the target molecules and therefore
rapid optimization.
Figure 1. Predicted binding mode of 1 overlaid with the modeled
binding mode of 2, an example from a Kirin patent13 (A). Schematic
view of 1 with key areas of the active site highlighted in (B) and the
structure of the Kirin compound 2 (C).
Figure 2. ALK5 ligand bound crystal structures of (A) the initial hit 1 and (B) the lead compound 19. PDB Accession Codes are 2WOT and
2WOU, respectively.

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7903
Table 1
c
compd
R1
R2
R3
R4
enzyme IC50, nMa
LEb
cell IC50, nMa
logD7.4
rat, F% (AUC)d
4 (0.15)
1
2-pyridyl
2-pyridyl
Me
Me
Me
Me
H
Me
H
3,4,5-trimethoxy
3,4,5-trimethoxy
3,4,5-trimethoxy
3,4,5-trimethoxy
3,4,5-trimethoxy
3,4,5-trimethoxy
2-OMe
44 (27-73)
13 (11-14)
35 (19-62)
227 (107-482)
108 (40-294)
107 (61-187)
7 (2-21)
739 (615-887)
4 (4-4)
6 (5-7)
30 (22-40)
109 (79-150)
21 (15-30)
12 (12-13)
129 (88-188)
72 (63-82)
0.22
0.24
0.27
0.26
0.26
0.26
0.27
0.18
0.28
0.24
0.25
0.2
55 (39-77)
20 (17-25)
21 (17-26)
>5800
3.2
3.4
3
5
6
H
21 (0.34)
7
H
H
2.7
3.2
3.2
3.4
3.1
nd
8
H
Me
Me
H
H
282 (221-361)
628 (291-1355)
114 (86-150)
>3000
9
H
10
11
12
13
14
15
16
17
18
19
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
2-morpholino
3-OMe
32 (17-60)
62 (56-69)
35 (22-54)
146 (85-251)
27 (23-31)
31 (27-36)
63 (33-120)
22 (19-25)
3-morpholino
4-OMe
3.3
3.5
2.6
2.3
3.4
3.2
2.5
4-morpholino
4-SO2NH2
0.24
0.24
0.24
0.27
20 (0.47)
75 (4.95)
4-CONMe2
4-F
4-SO2NH2
a IC50 data are the geometric mean of at least 2 independent values (66% confidence limits are given in parentheses, as calculated from the standard
error of the mean). b Enzyme pIC50/heavy atom count. To convert to ΔG-based ligand efficiencies as used by, e.g., Astex, multiply by 1.37. c The partition
coefficient between pH7.4 sodium phosphate buffer and octanol. d Oral bioavailability (with oral AUC in μM.h in parentheses) observed when dosing
compound as a 5 μmol/kg solution to male Han Wistar rats. nd = not determined.
Scheme 1a
Scheme 2a
a Reagents and conditions: (i) ROH, Cs2CO3, CuI, DMA, 25-77%;
(ii) ArNH2, Pd(OAc)2, Xantphos, Cs2CO3, 1,4-dioxane or DMA,
38-75%; (iii) ROH, K2CO3, DMF, 50-82%.
methyl at R3 improved the cellular potency (cf. 1 and 5). Even
more importantly, the pyridyl at R1 could be replaced with a
methyl group (cf. 5 and 6) while maintaining potency and thus
improving ligand efficiency. Both methyls of 6 were important
for potency, as evidenced by the fact that 7, 8, and 9 are all less
potent than 6. The 2,6-dimethylpyridyl group was therefore
identifiedasthe mostattractiveselectivitypocket groupdue to
its potency and ligand efficiency (and therefore favorable
predicted physicochemical properties). Simultaneously, we
examined the SAR of the aniline group (Table 1). Thus,
ortho substitution by a methoxy group as in 10 was well
tolerated, although the larger morpholine 11 did slightly
reduce potency. Both methoxy and morpholine groups
gave excellent levels of potency in the meta and para
positions (12-15) along with small and large electron
withdrawing groups (e.g., 16-18 shown for para sub-
stitution). As a wide range of substitution patterns was
tolerated on the aniline group, varying this group was
predicted to be a successful approach to optimize DMPK
and physical properties.
a Reagents and conditions: (i) NaH, DMF, 2,4-dichloropyridine,
51%; (ii) Pd(OAc)2, Xantphos, Cs2CO3, 1,4-dioxane, 3,4,5-trimethox-
yaniline, 38%; (iii) NaH, DMF, 2,4-dichloropyridine, 100%; (iv) 2-
pyridylzinc bromide, Pd(PPh3)4, THF/DMA, 56%; (v) Pd(OAc)2,
Xantphos, Cs2CO3, 1,4-dioxane, 3,4,5-trimethoxyaniline, 14%.
Results and Discussion
Guided by the X-ray crystallography results, we examined
the SAR of the 4-pyridinoxy-2-anilinopyridine scaffold with
the goal of improving the PK properties and cellular potency
of the original hit compound 1. To assess PK properties, we
used in vivo rat PK as we did not have a convincing correla-
tion between in vitro hepatocyte data and in vivo clearance.
Variation of the phenolgroup (Table1) focused on optimizing
R2 and R3 while aiming to replace the 2-pyridyl at R1 with a
smaller equipotent group to optimize ligand efficiency
(defined in this article as pIC50/heavy atom count) and
predicted physicochemical properties. Thus removal of the
With the preliminary SAR established, we were able to
rapidly identify lead compounds with improved pharmacoki-
netic profiles over the initial hit 1. Thus replacement of
the bipyridyl phenol with the smaller, more ligand efficient

7904 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Goldberg et al.
2,6-dimethylpyridyl group gave a modest improvement in rat
bioavailability and oral exposure (from F = 4%, AUC =
0.29 μM h for 1 to F = 21%, AUC = 0.67 μM h for 6) as did
replacement of the 3,4,5-trimethoxyaniline with groups
such as 4-aminobenzenesulfonamide (F = 20%, AUC =
gave 3-(2-chloropyridin-4-yl)oxy-2,6-dimethyl-pyridine (0.212 g,
53%) as a yellow solid. 1H NMR (DMSO): 2.28 (3H, s), 2.48 (3H,
s), 6.90 (1H, dd), 6.99 (1H, d), 7.22 (1H, d), 7.52 (1H, d), 8.29 (1H,
d); m/z: MHþ 235.7. A mixture of 3-(2-chloropyridin-4-yl)oxy-
2,6-dimethyl-pyridine (0.2 g, 0.85 mmol), sulfanilamide (0.191 g),
Cs2CO3 (0.417 g), Pd(OAc)2 (0.013 g), Xantphos (49 mg), and
DMA (2 mL) was heated at 150 °C for 10 min in a microwave.
After cooling, the crude product was semipurified by ion ex-
change chromatography, eluting with 7 M NH3/methanol.
Further purification by preparative HPLC using decreasingly
polar mixtures of water (containing 0.1% TFA) and acetonitrile
as eluent gave 19 (0.17 g, 54%) as a solid. 1H NMR (DMSO): 2.30
(3H, s), 2.49 (3H, s), 6.13 (1H, d), 6.52-6.56 (1H, m), 7.10 (2H, s),
7.23 (1H, d), 7.50 (1H, d), 7.68 (2H, d), 7.79 (2H, d), 8.14 (1H, d),
9.38 (1H, s); m/z: MHþ 371.0.
3
3
0.47 μM h for 16), although neither of these changes reduced
3
rat IV clearance. Combining these changes to give 19, how-
ever, gave a compound with high bioavailability and oral
exposure (F = 75%, AUC = 4.95 μM h) with low clearance
3
(12 mL/min/kg). This compound also had excellent cell
potency (22 nM), low molecular weight (370), and suitable
lipophilicity (logD7.4 = 2.5) to make it a novel, high quality
lead compound for further optimization. The crystal structure
of this compound was solved to assess further ways in which
this series couldbe modified (Figure 2B). Thebinding mode of
19 was consistent with 1 but with an additional interaction
between the NH2 of the sulfonamide group and the carboxyl
group of Asp-290. The general kinase selectivity of 19 was
also assessed by submission to a panel of 80 representative
kinases24 at Dundee University; only five kinases showed
>50% inhibition at 1 μM, of which only two showed inhibi-
tion of >80%.
Acknowledgment. We thank Jason Kettle, Ray Finlay,
Brian Law, Nabil Asaad, and Philip Jewsbury for their advice
and support. Our heartfelt thanks are also given to Robert
Cheung, who performed the insect cell culture and who is
sorely missed by all.
Supporting Information Available: General experimental
procedure, procedures for preparation of all final compounds
and their precursors and NMR data and purity statements,
enzymatic and cell-based assay procedures, procedures for
protein expression and X-ray crystallography, computational
chemistry techniques. This material is available free of charge
via the Internet at http://pubs.acs.org.
In conclusion, we have developed a novel series of ALK5
inhibitors based upon the 4-pyridinoxy-2-anilinopyridine scaf-
fold. The binding mode of the initial hit compound was suc-
cessfully predicted by docking studies prior to synthesis and
then later confirmed by X-ray crystallography. Optimization of
this scaffold focused on improving ligand efficiency and con-
trolling lipophilicity in order to improve pharmacokinetic
properties. Combining the optimal structural changes to each
position on the central core gave 19, which has excellent cell
potency and suitable physicochemical and pharmacokinetic
properties for further optimization. Compound 19 also demon-
strates an excellent selectivity profile across a panel of 80
kinases.
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