Design, synthesis and optimization of 7-substituted-pyrazolo[4,3-b]pyridine ALK5 (activin receptor-like kinase 5) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.03.026

A series of potent ALK5 inhibitors were designed using a SBDD approach and subsequently optimized to improve drug likeness. Starting with a 4-substituted quinoline screening hit, SAR was conducted using a ALK5 binding model to understand the binding site

Full text of DOI:10.1016/j.bmcl.2017.03.026

Accepted Manuscript
Design, Synthesis and Optimization of 7-substituted- pyrazolo[4,3-b]pyridine
ALK5 (activin receptor-like kinase 5) inhibitors
Mark Sabat, Haixia Wang, Nick Scorah, J. David Lawson, Joy Atienza, Ruhi
Kamran, Mark S. Hixon, Douglas R. Dougan
PII:
S0960-894X(17)30261-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.026
BMCL 24778
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 January 2017
8 March 2017
10 March 2017
Please cite this article as: Sabat, M., Wang, H., Scorah, N., Lawson, J.D., Atienza, J., Kamran, R., Hixon, M.S.,
Dougan, D.R., Design, Synthesis and Optimization of 7-substituted- pyrazolo[4,3-b]pyridine ALK5 (activin
receptor-like kinase 5) inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.03.026
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Design, Synthesis and Optimization of 7-substituted- pyrazolo[4,3-
b]pyridine ALK5 (activin receptor-like kinase 5) inhibitors
Mark Sabat *, Haixia Wang, Nick Scorah, J. David Lawson, Joy Atienza, Ruhi Kamran, Mark S. Hixon
Douglas R. Dougan
Takeda California; 10410 Science Center Drive, San Diego, CA, 92121
ABSTRACT: A series of potent ALK5 inhibitors were designed using a SBDD approach and subsequently optimized to improve drug likeness.
Starting with a 4-substituted quinoline screening hit SAR was conducted using a ALK5 binding model to understand the binding site and optimize
activity. The resulting inhibitors displayed excellent potency but were limited by high in vitro clearance in rat and human microsomes. Using a scaffold
morphing strategy, these analogs were transformed into a related pyrazolo[4,3-b]pyridine series with improved ADME properties.
ALK5 (activin-like kinase 5, also known as TGF-βRI / transforming
growth factor β receptor I) is a receptor serine-threonine kinase that has
a role in the regulation of a variety of physiological processes.1a TGF-β
ligands initially bind TGF-βRII which recruits and phosphorlyates
ALK5 (TGF-βRI). The activated ALK5 then phosphorylates
SMAD2/SMAD3 proteins which dissociate from the receptor and form a
heteromeric complex with SMAD4. This complex eventually translo-
cates to the nucleus and interacts with DNA cofactors to regulate gene
expression controlling cell growth, differentiation and development.
Thus, modulation of TGF-β signaling via small-molecule ALK5 inhibi-
tors has been considered a potential treatment of fibrotic disease1j,k and
cancer. The role of TGF-β in cancer biology is complex with both pro-
moter and suppressor functions depending on cancer stage. During late
stages of carcinogenesis tumor cells utilize the TG signaling system to
promote metastatic spread and overcome immune surveillance.1b,c This
later role of TGF-β makes it a particularly attractive target to combat the
most invasive and metastatic cancers particularly in brain and pancreas.
the C
−
H at position C2 of the quinoline ring and the carbonyl oxygen of
Asp281. The pyridyl ring of compound
2
is positioned to hydrogen bond
with the side chain amino group of Lys232 as well as with a conserved
water molecule that is coordinated by Asp351, Glu245 and Tyr249. Finally,
the phenyl ring (which is attached at the C2’ position of pyridine ring) is
oriented towards the back hydrophobic pocket.
Figure 2. Molecular model of early quinoline screening lead (2) docked
into the ALK5 nucleotide binding site.
Modulating ALK5 with small molecule inhibitors has received pharma-
ceutical industry attention, however to date only two small molecules
LY2157299 and EW-7197 have entered clinical trials.1b,d,2 In this paper,
we describe our efforts to identify potent and selective small molecule
inhibitors of ALK5 and their subsequent optimization to yield an orally
bio-available candidate which advanced into toxicological testing.
N
Using the hypothesized binding insights, initial optimization efforts on
this series were targeted at improving enzymatic potency and obtaining a
better understanding of the importance of the various hydrophobic pock-
et groups (described in the ALK5 literature5) (Table 1). Additionally we
also attempted to optimally fill a small hydrophobic pocket (observed in
the docked model of lead molecule 2 in ALK5) located directly below
N
1
N
N
N
4
N
N
H
(2)
O
O
5
LY2157299 (1)
H2N
the C5 position of the pyridyl ring of compound
2 (see Fig 2).
Figure1. Structures of ALK5 inhibitor LY2157299 (1) and screening
hit based lead (2)
Synthetic methodology used to access the elaborated quinolines (
described in Scheme 1.6
2-11) is
Screening of our internal compound collection followed by deconstruc-
tion afforded the early lead molecule
potency in the ALK5 enzymatic assay (ALK5 pIC50 = 6.2)3. Compound
was docked into the ATP binding site of the ALK5 protein using coor-
dinates disclosed in the literature4 (see Fig 2).Compound
was predicted
2 which displayed reasonable
Scheme 1
2
2
to adopt a binding pose thatpositions the quinoline ring nitrogen to make
an H-bond with the backbone NH of His283 at the kinase’s hinge region.
In this pose, a potential pseudo hydrogen bond is also formed between

hERG14b inhibition. As an example analog 6 displayed an in vitro clear-
ance of 302(R) and 86(H) mL/min/kg. Additionally the series suffered
elevated hERG inhibition with compound 6 showing 94% inhibition at
10uM. Both liabilities were partially attributed to the high lipophilicity
of the series (compound 6 cLogD = 4.4).
Using a scaffold morphing strategy, the quinoline series was transformed
into the related 7-substituted-pyrazolo[4,3-b]pyridine series. The re-
placement of a phenyl ring with a pyrazole resulted in a reduction of
lipophilicity (contrast LLE values between Tables 1 & 2)
Scheme 1. Reagents and conditions: (a) Pd2(PPh3)4, K2CO3 aq, 1,4-
dioxane, 120 °C 0.5h MW; (b) Pd2(dba)3, NaOtBu, XANTPHOS, 1,4-
dioxane, 100 °C 1h MW
The chemistry methodology used to access the appropriate 7-substituted-
pyrazolo[4,3-b]pyridines (12-18) is described in Scheme 28,and their
biological properties are disclosed in Table 2.
Substitution at the meta position on the phenyl ring in 2 with a Cl atom
gave a modest increase in potency (3). Installation of a CH3 group at R1
further improved potency by filling a small lipophilic grove in the ALK5
model. Installation of a 5-chloro-2-fluorophenyl at R of the core further
improved potency (compound 5). Modeling suggested the incorporation
of a F atom (compound 5) helped to improve the orientation of the aro-
matic ring in the hydrophobic pocket. Next we looked at substituent size
at position R1 (compounds 6-8) holding the ring at position R (5-chloro-
2-fluorophenyl) constant. Unfortunately increased size at R1 resulted in
decreased enzyme potency (compare 6 vs. 7 & 8) and thus a methyl
group at R1 was judged optimal. Exchange of the 5-Cl for a methyl
group in analogs 9 and 10 afforded similarly potent analogs (compare 10
vs. 6). Finally introduction of a 5-fluoro-2-methylpyridine group at
position R afforded a potent analog with improved efficiency (compare 9
vs. 11; LLE7a 3.6 vs. 4.1).
Scheme 2
Scheme 2. Reagents and conditions: (a) Lithium diisopropylamide, I2,
THF, -78 to 0°C; (b) NH2NH2, n-BuOH, 105°C; (c) NaNO2, HI, AcOH,
DME, 0 to 80°C; (d) NaH, 1-(chloromethyl)-4-methoxybenzene,
DMF;(e) NaOtBu, BINAP, Pd(OAc)2; (f) TFA, DCM
Table 1. Selected data for 4-substituted quinoline analogues
Table 2. Selected data for 7-substituted-pyrazolobpyridine
Cmpd
R
R1
LLE
pIC50
Cmpd
R
R1
LLE
pIC50 pEC50* pEC50†
Phenyl
H
2
2.6
6.2
5-chloro-2-fluorophenyl
H
5.3
8.2
7.8
8.1
7.8
7.3
-
-
12
3-chlorophenyl
3-chlorophenyl
H
2.4
3.2
6.5
7.5
3
4
5-methyl-2-fluorophenyl
2,5-difluorophenyl
H
H
5.1
5.8
-
13
14
15
CH3
7.4
7.5
6.7
-
5-chloro-2-
fluorophenyl
5-chloro-2-fluorophenyl
H
3.6
3.5
7.7
7.9
7.6
7.3
5
6
7
8
CH3 4.7
CH3 6.5
5-chloro-2-
fluorophenyl
2,5-difluorophenyl
CH3
9.0
8.1
7.5
16
5-chloro-2-
fluorophenyl
CH3CH2
6-methylpyridin-2-yl
CH3 6.5
CH3 6.5
8.3
8.6
7.5
-
6.7
7.5
17
18
5-chloro-2-
fluorophenyl
2.6
3-fluoro-6-
methylpyridin-2-yl
5-methyl-2-
fluorophenyl
9
LLE= p(IC50) - logD see ref. 7a
*Reporter Inh Cell Line 293T SBE-bla; †pSMAD2/3Calu-6
H
3.6
3.7
7.6
7.9
5-methyl-2-
fluorophenyl
CH3
10
11
The utilization of a pyrazolo[4,3-b]pyridine hinge binding element in
place of the quinoline afforded more efficient inhibitors of ALK5 as
evidenced by liphophilic ligand efficiencies values (compare analog
3-fluoro-6-
methylpyridin-2-yl
H
4.1
7.4
LLEs for 2-11 vs. 12-18) as well as improved enzyme and cell potencies.
LLE= p(IC50) - logD see ref. 7a
Installation of either a 5-chloro-2-fluorophenyl or 5-methyl-2-
fluorophenyl group (analogs 12 & 13) at position R of the pyrazolo[4,3-
b]pyridine core afforded improved potency with lower lipophilicity and
a marked improvement in LLE (compare 12 vs. 5 and 13 vs. 9). Addi-
Although the quinoline series of compounds showed good ALK5 en-
zyme and cellular potency (data not shown) most examples displayed
high in vitro clearance in rat and human microsomes and substantial
2

tionally, exchange of a F atom for Cl or CH3 (analogs 13 & 14) resulted
in comparable potency with a decrease in lipophilicity. Installation of a
CH3 group at R1 similar to analogs in the quinoline series at first did not
offer an increase in potency (compare 15 vs 12); however in case of the
R= 2,5-difluorophenyl analogs, a 8x increase in potency was observed
(compare 16 vs. 14). Keeping CH3 at position R1 constant and installing
2-methylpyridine (analog 17) or 5-fluoro-2-methylpyridine at position R
afforded slightly less potent but equally efficient ALK5 inhibitors.
Inhibitors 12-18 additionally showed cellular potency in both a function-
al assay (Reporter Inh Cell Line 293T SBE-bla) and in a MOA (mecha-
nism of action) cell assay (pSMAD). 9
F
22
23
24
25
CH3
CH3
H
9.0
8.5
8.5
7.8
7.9
5.6
7.6
8.1
7.6
7.5
7.7
-
7.7
7.7
7.2
-
<.23/.2
.64/.54
.57/.31
.51/.52
.54/.44
.91/.93
F
Cl
Cl
Cl
Cl
CH3
CH3
H
The most promising analogs from the 7-substituted- pyrazolo[4,3-
b]pyridine series (highest cellular potency) were further profiled to as-
sess their in vitro ADME properties. Compounds 15-16 & 18 displayed
high metabolic clearance and in vitro extraction ratios. Additionally
these analogs showed elevated Cyp profiles particularly isoforms 2C8
and 3A4 (Table 3) despite the reduction in lipophilicity.
26
27
7.5
-
Table 3. Selected In Vitro ADME data for Compounds 15-18
Eh = Clh/Qh where Eh = Hepatic extraction ratio; Clh = Clearance hepatic (mL/min/kg); Qh =
Hepatic blood flow see ref. 7b-c
Eh
(H/R)
mL/min/kg
CYP2C8/
CYP3A4
Substitution at R1 with acetamide (19), N-methylcarboxamide (20) and
N-ethylcarboxamide (21) to the N1 position of the difluorophenyl ana-
logs indicated that N-methyl was optimal for enzyme potency. Incorpo-
ration of a CH3 group at R2 (compounds 22 and 23) further boosted
potency and reduced in vitro clearance for compound 22. A similar set
of design changes for the 5-chloro-2-fluorophenyl analogs 24-26 did not
have the same potency effect in regard to the CH3 group at R2 (data
consistent with trend seen in Table 2 ex. 15)
Cmpd
cLogD
2.9
.57/.75
.74/.84
.57/.74
6.4/5.5
15
2.5
2.2
6.5/5.8
5.6/5.2
16
18
A similar set of changes at position R2 of the pyridine ring (Table 5) was
also tolerated with compound 31 showing the highest cellular potency in
this series.
Table 5. Selected data for Compounds N2 substituted compounds
Eh = Clh/Qh where Eh = Hepatic extraction ratio; Clh = Clearance hepatic (mL/min/kg); Qh =
Hepatic blood flow see ref. 7b-c
Cytochromes P450 (CYPs) family (2C8 & 3A4) pIC50
To improve metabolic stability and reduce Cyp inhibition we decided to
further reduce lipophilicity through the installation of polar substituent
onto the lead examples (molecules 15-16 & 18, Table 2). Based on the
binding model developed for these compounds in ALK5 protein, both
the quinoline ring nitrogen (required for good hinge binding) and the
pyridine nitrogen (needed for interaction with the conserved water) did
not allow for nearby substitutions. Only the N1 and N2 positions of the
pyrazolo[4,3-b]pyridine in this series appeared to be open for modifica-
tion. We were gratified that many analogs having substituents at these
positions (Tables 4-7) showed enzyme and cell potency along with im-
proved metabolic profiles. The chemistry to access the N1 and N2 sub-
stituent involved simple alkylation, separation and characterization and
is described in our patent application.8
pEC50
pSMAD
2/3
pEC50
SBE-bla
Cmpd
X
R1
R2
pIC50
Eh(H/R)
.44/.42
F
28
H
7.6
6.6
6.9
7.0
7.9
6.0
5.8
6.8
-
.79/.68
.52/.54
.46/.56
.36/.16
Table 4. Selected data for Compounds N1 substituted compounds
F
29
30
CH3
H
7.7
7.9
7.8
7.0
Cl
Cl
Cl
31
32
CH3
H
7.5
-
pEC50
Cmp
d
pEC50
SBE-bla
pSM
AD2/
3
X
F
F
F
R1
R2
H
H
H
pIC50
7.3
Eh(H/R)
.53/.57
.55/.27
.53/.29
Eh = Clh/Qh where Eh = Hepatic extraction ratio; Clh = Clearance hepatic (mL/min/kg); Qh =
Hepatic blood flow see ref. 7b-c
19
20
21
6.7
7.7
7.5
5.9
6.9
6.8
The piperidine analogs 27 and 32 were made to increase solubility and
enhance the probability of obtaining a crystal structure of the chemotype
bound to ALK5. The basic nitrogen of the piperidine ring could also
form a salt bridge with Asp290 and the edge of the piperidine ring could
also makes a mainly hydrophobic interaction with the backbone of Gly322
and Thr323 in models.
8.6
8.3
3

pEC50
SBE-bla
pEC50
pSMAD2/3
The team was pleased that a structure was successfully determined with
2.6Å resolution with compound 32. Examination of the co-structure in
ALK510 confirmed the predicted binding mode of the series (Figure 3).
The structure shows the N4 of the pyrazolo[4,3-b]pyridine accepting an
H-bond from the backbone NH of His283 at the kinase’s hinge region
while the C5 C-H makes a pseudo hydrogen bond10g to the carbonyl
oxygen of Asp281. The pyridyl ring of compound 32 is positioned to
hydrogen bond with the conserved water molecule that is coordinated by
Asp351, Glu245 and Tyr249. The phenyl ring, attached at the C2’ position of
pyridine ring, is oriented towards the back hydrophobic pocket. As com-
Cmpd
R
pIC50
Eh(H/R)
.56/.61
-CH3
33
34
35
8.4
8.7
7.9
8.2
8.1
7.7
7.4
7.3
-
.69/.69
-
-CH2CH3
8.1
8.1
7.0
7.2
6.3
6.4
.3/.3
.4/.3
36
37
pared to our model of compound 2, Lys232 now shifts away from the
ligand, no longer H-bonding to the pyridyl N. This is likely due to subtle
shift in the H-bonding network induced by an additional H-bond accep-
tor on the ligand of the parent crystal structure (ref 4) used for modeling
compound 2. The parent structure’s ligand has a 2-pyridyl ring in the
place of compound 32's phenyl ring.
<.23/.21
.28/<.15
38
39
8.5
7.8
-
6.3
5.8
Figure 3. Co crystal structure of compound 32 in ALK5 protein
6.9
Eh = Clh/Qh where Eh = Hepatic extraction ratio; Clh = Clearance hepatic (mL/min/kg); Qh =
Hepatic blood flow see ref. 7b-c
Table 7. Selected data for Compounds N2 substituted compounds
N
F
N
N
N
H
N
N
R
pEC50
SBE-bla
pEC50
pSMAD2/3
Eh
(H/R)
Cmpd
R
pIC50
.66/.46
-CH3
Several representative analogs from the 7-substituted- pyrazolo[4,3-
b]pyridine series with substituents at the N1 and N2 positions (Tables
40
41
8.4
8.0
7.1
7.8
6.5
6.8
6
&
7
) which displayed the highest cellular potency were further profiled
-
-CH2CH3
to assess their in vitro properties. Analog 22 which initially appeared
promising due to cell potency unfortunately had high Cyp 3A4 inhibition
(pIC50 = 6.1) and was no longer considered useful.
.38/.43
8.1
8.1
6.6
6.8
-
42
43
44
Compounds 26, 30-31, did not display elevated Cyps (3A4 values of 4.9,
4.9, and <4.3 respectively). Compounds 26 displayed moderate in vivo
rat clearance (42 mL/kg/min) with good oral bioavailability (F% =71).
Unfortunately compounds 26, 30-31 all displayed elevated hERG signal
6.3
.29/.3
(59% & 41%@ 10uM respectively).
To overcome the hERG inhibition observed in these analogs, a further
reduction in lipopholicity was achieved by introducing a 5-fluoro-2-
methylpyridine group in place of the 2,5-difluorophenyl or 5-chloro-2-
fluorophenyl group (see analog 18 in Table 3 and derivatives thereof in
Tables 4 & 5).
8.1
8.0
7.1
-
6.5
6.6
.4/.3
.25/.16
45
46
Substituent groups R were better tolerated at position N1 (Table 6) than
N2 (Table 7) in this series. Simple alkyl groups at position R (33
-34 &
40 41) were similarly potent as the parent 18 with no improvement in in
-
7.5
6.9
6.3
.37/.24
vitro clearance. More polar substitution particularly those in 36-39 and
43-46 appeared to mitigate clearance, and many were advanced to in
vivo PK experiments. The most promising analogs (36 F%= 38; 37
F%= 21 and 43 F%= 40 were tested for hERG inhibition and unfortu-
nately showed elevated hERG signal (42%, 51% and 56%@ 10uM
respectively).
Eh = Clh/Qh where Eh = Hepatic extraction ratio; Clh = Clearance hepatic (mL/min/kg); Qh =
Hepatic blood flow see ref. 7b-c
At this point in the program we learned of a potential cardiac toxicity
associated with modulating ALK5.11 The chemistry team was advancing
additional ALK5 inhibitors described in a previous article in this jour-
nal12a and had only begun to fully examine compound 36 (limited selec-
tivity13 and in vitro safety data was obtained) when a decision was made
to do in vitro safety testing thus necessitating a choice of lead molecule.
Although the on target cardiac valvulopathy that was hypothesized from
ALK5 inhibition was unrelated to hERG signal14a, the teams nevertheless
choose an alternative molecule to compound 36 for the toxicological
studies to remove any potential to confound the results.12b
Table 6. Selected data for Compounds N1 substituted compounds
N
F
N
N
N
H
N
N
R
In summary, a novel series of ALK5 inhibitors was identified and devel-
oped. Starting from screening hit 2, by using SBDD and medicinal
4

chemistry knowledge, the team optimized potency, ascertained binding
pose (compound 32) and finally improved metabolic stability and bioa-
vailability of this series, culminating in the identification of lead com-
pound 36. However, inhibition of ALK5 was shown to produce signifi-
cant cardiac toxicity12 and lead to the program’s termination.
5.
a) Li, H. Y.; McMillen, W. T.; Heap, C. R.; McCann, D. J.; Yan,
L.; Campbell, R. M.; Mundla, S. R.; King, C. H.; Dierks, E. A.;
Anderson, B. D.; Britt, K. S.; Huss, K. L.; Voss, M. D.; Wang, Y.;
Clawson, D. K.; Yingling, J. M.; Sawyer, J. S. J. Med. Chem.
2008, 51, 2302. b) ref 4 c) Kim, D.; Kim, J.; Park, H. Bioorg &
Med. Chem. 2004, 12, 2013 d) Singh, J.; Chuaqui, C. E.; Boriack-
Sjodin, P. A.; Lee, W.; Pontz, T.; Corbley, M. J.; Cheung, H. K.;
Arduini, R. M.; Mead, J. N.; Newman, M. N.; Papadatos, J. L.;
Bioorg. Med. Chem. Lett. 2003, 13, 4355.
Acknowledgments
6.
Representative experimental for analogs in Table 1. Compound 3
step a: 2-chloropyridin-4-amine (200mg, 1.56 mmol), 3-
chlorophenylboronic acid (243 mg, 1.56 mmol) and Pd(PPh3)4 (450
mg, 0.39 mmol) were mixed in dioxane (4mL). K2CO3 saturated
solution (2 mL) was added and the mixture was heated at 120 °C
for 30 minutes in a microwave reactor. The solvent was removed
and the crude residue was purified on a silica column eluted with a
0-5% MeOH in DCM gradient. Concentration of product contain-
ing fractions gave 2-(3-chlorophenyl)pyridin-4-amine (150mg,
47.1 % yield) as a light yellow oil.
We are grateful to Matthew J. Cukierski, Mary E Carsillo, Ron
Eydelloth and Vic Kadambi for the toxicity studies and the staff of the
Berkeley Center for Structural Biology for support of beam line 5.0.3 at
the Advanced Light Source. The Advanced Light Source is supported by
the Director, Office of Science, Office of Basic Energy Sciences, of the
U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
References and Notes
Compound 3 step b: 4-bromo-5-methoxyquinoline (174 mg, 0.733
mmol), 2-(3-chlorophenyl)pyridin-4-amine (150 mg, 0.733 mmol),
Pd2(dba)3 (168 mg, 0.183 mmol), XANTPHOS (106 mg, 0.183
mmol) and sodium 2-methylpropan-2-olate (211 mg, 2.199 mmol)
were mixed in 1,4-Dioxane (4mL) and heated at 140 °C for 1h in a
microwave reactor. The reaction was concentrated; the crude resi-
due was taken up in MeOH, filtered and subjected to preparative
HPLC. N-(2-(3-chlorophenyl)pyridin-4-yl)-5-methoxyquinolin-4-
amine (compound 3) was obtained as a light yellow oil (55mg,
0.152 mmol, 20.74 % yield). 1H NMR (400 MHz, METHANOL-
d4) δ ppm 4.09 (s, 3 H) 7.26 (d, J=8.08 Hz, 1 H) 7.42 - 7.53 (m, 4
H) 7.71 - 7.81 (m, 2 H) 7.84 - 7.93 (m, 2 H) 8.08 (d, J=2.27 Hz, 1
H) 8.49 (d, J=6.82 Hz, 1 H) 8.63 (d, J=6.32 Hz, 1 H)
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M.; Lala, D. S.; Pocalyko, D. Plast. Reconstr. Surg. 2011, 128, (5),
e451. k) Boys, M. L.; Biana, F.; Kramera, J. B.; Chioa, C. L.; Re-
na, X.-D.; Chena, H.; Barretta, S. D.; Sextona, K. E.; Iulaa, D. M.;
Filzena, G. F.; Nguyena, M. N.; Angella, P.; Downsa, V. L.;
Wanga, Z. W.; Rahejaa, N.; Ellswortha E. L.; Fakhourya, S.;
Brattona, L. D.; Kellera, P. R.; Gowana, R.; Drummonda E. M.;
Maitib, S. N.; Henab M. A.; Lub, L.; McConnella, P.; Knafelsa, J.
D.; Thanabala, V.; Suna, F.; Alessia, D.; McCarthya, A.; Zhanga,
E.; Finzela, B. C.; Patela, S.; Ciottia, S. M.; Eismaa, R.; Paynea,
N.A.; Gilbertsena, R. B.; Kostlana, C. R.; Pocalykoa, D. J.; Lalaa
D. S. Bioorg. Med. Chem. Lett.2012, 22, (10), 3392.
a) Clinical Trials Identifiers: NCT01246986; NCT01582269;
NCT01722825; NCT01220271 and NCT01373164 for LY2157299
and NCT 02160106 for EW-7197 b) Herbertz, S.; Sawyer, J. S.;
Stauber, A. J.; Gueorguieva, I.; Driscoll, K. E.; Estrem, S. T.;
Cleverly, A. L.; Desaiah, D.; Guba, S. C.; Benhadji, K. A.; Slapak,
C. A.; Lahn, M. M.; Drug. Des. Devel. Ther. 2015, 9, 4479.
Test compounds were screened for TGFBR1 ALK5 (PV5837
Invitrogen) inhibition by use of a LanthaScreen™ activity assay
(Invitrogen Corp. Madison, WI). Solutions of test compounds in
DMSO were diluted two-fold serially. Assays were conducted at
15 µL volumes consisting of 50 mM HEPES pH 7.4, 10 mM NaCl,
10 mM MgCl2, 0.01% Brij 35, 1 mM DTT, 2 µM ATP, 1.2 µM of
Fluorescein-SMAD3 Peptide FAM-NH2-
7.
a) LLE= p(IC50) – logD see Leeson and Springthorpe Nat. Rev.
Drug Discovery 2007, 6(11) 881; LE ≈ 1.37× p(IC50)/HA where
HA = Heavy Atoms see Hopkins et al., Drug Discov. Today 2004,
9, 430. b) Obach Curr. Top. Med Chem 2011, 11, 334. c) Davies
and Morris Pharm. Res. 1993, 10, (7), 1093.
8.
9.
For a more detailed experimental see WO/2011/146287
Test compounds were screened for cellular TGFBR1 ALK5 inhibi-
tion by use of a CellSensor SBE-bla HEK 293T (cat # K1550,
Invitrogen Corp. Madison, WI) cell line. The CellSensor® SBE-bla
HEK 293T cell line contains a beta-lactamase reporter gene under
control of the SBE response element stably integrated into HEK
293T cells. The SBE response element is a DNA region that binds
activated SMADs which results from the binding of TGFβ to, and
subsequent activation of ALK5.
10. a) PDB ID: 5USQ b) The ALK5 catalytic domain (residues 192-
500) was cloned into the pFastBacHTb vector and recombinant
baculovirus was generated and expressed in fusion with an N-
terminal 6x poly-histidine tag and TEV cleavage site. Large scale
production of recombinant protein was carried out in Spodoptera
frugiperda Sf9 cells. The cell pellet was suspended into lysis buff-
er consisting of 25 mM HEPES (pH 8.0), 1 M NaCl, 20 mM imid-
azole, 3 Roche Complete tablets. The lysate was centrifuged, and
clarified supernatant was batch bound with 10 ml of Probond Ni
resin (Invitrogen). The protein was then eluted with buffer con-
taining 25 mM HEPES (pH 7.6), 150 mM NaCl, and 250 mM im-
idazole. After tag cleavage, the protein was further purified by
size-exclusion chromatography utilizing a Superdex 200 column
equilibrated in 25 mM HEPES (pH 7.6), 150 mM NaCl, 5% Glyc-
erol. Fractions containing the protein of interest were pooled, con-
centrated to ~2 mg/ml and flash-frozen in liquid nitrogen for stor-
age at -80oC. Crystals suitable for data collection were obtained by
vapor diffusion in sitting drops at 20 °C. Reservoirs contained 14 -
18% PEG 8K and 100 mM CHES pH 9.0 -9.4. Crystals that took 7
-10 days to grow were immersed in mother liquor solution contain-
ing 22% ethylene glycol for cryoprotection and flash frozen in liq-
uid nitrogen. Crystals of ALK5 complex grew in the orthorhombic
space group P212121 and contain one molecule in the asymmetric
unit. Diffraction data to 2.55 Å were collected from single cryo-
genically protected crystals at beam line 5.0.3 of the Advanced
Light Source at Lawrence Berkeley National Laboratory. Data was
reduced using the HKL2000 software package.1The structure was
determined by molecular replacement with either MOLREP2 or
PHASER3 of the CCP4 program suite and refined with the pro-
gram REFMAC.4 several cycles of model building with
2.
3.
KVLTQMGSPSIRCSS[PO4]VS (M4337 Invitrogen), 15nM
ALK5 and test compounds at 1% final DMSO concentration. Re-
action was initiated by the addition of ALK5. Assays were incu-
bated for 90 min at room temperature and then quenched by the
addition of 60 mM EDTA and 8 nM Terbium labeled anti-
pSMAD3 Antibody (M4337 from Invitrogen) giving a final con-
centration of 15 mM EDTA and 2 nM Terbium labeled anti-
pSMAD3 Antibody. Phosphorylated peptide product was quanti-
fied by measuring an increase in TR-FRET on a BMG LABTECH
PHERAstar plus. For further details see WO/2011/146287
PDB ID: 3HMM (Research Collaboration for Structural Bioinfor-
matics) Gellibert, F.; Fouchet, M. H.; Nguyen, V. L.; Wang, R.;
Krysa, G.; de Gouville, A. C.; Huet, S.; Dodic, N. Bioorg. Med.
Chem. Lett. 2009, 19, 2277.
4.
5

XtalView5 and refinement were performed for improving the qual-
ity of the model. The coordinates and structure factors have been
deposited in Protein Data Bank with accession code 5USQ. c)
Otwinowski, Z.; Minor, W. 1997, Methods Enzymol. 276, 307. d)
Vagin, A.; Teplyakov, A. 1997, MOLREP: an automated program
for molecular replacement. J. Appl. Cryst. 30, 1022. e) McCoy, A.
J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni,
L. C.; Read, R. J.; 2007, Phaser Crystallographic Software. J. Appl.
Cryst. 40, 658. f) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J.
1997, Acta Crystallogr. Sect. D 53, 240–255. g) McRee, D. E.;
1999 J. Struct. Biol. 125, 156. h) In accord with Definition of the
hydrogen bond (IUPAC Recommendations 2011) Pure Appl.
Chem., 83, 8, 1637.
11. Anderton, M. J.; Mellor, H. R.; Bell, A.; Sadler, C.; Pass, M.;
Powell, S.; Steele, S. J.; Roberts, R. R. A.; Heier, A. Toxicol.
Pathol. 2011 39; 916.
12. a) Wang, H.; Lawson, J. D.; Scorah, N.; Kamran, R.; Hixon, M. S.;
Atienza, J.; Dougan, D. R.; Sabat, M. Bioorg. Med. Chem. Lett.,
2016, 26, 4334.
13. Compound 36 ALK5 family isozyme profiling selectivity: ALK4
is 39-fold, ALK2 and ALK3 (BMPR1α) are each >200-fold, based
on 2pt screening done at 10 & 1 uM, and compared to the in-house
determined of ALK5 IC50 value.
14. a) We do not believe hERG activity has any relationship to cardio
valvulopathy however we did not want potential AE to be possibly
clouded by or attributed to hERG risk. b) Automated whole cell
patch-clamp (Qpatch 16) or conventional whole cell patch-clamp
technique was used to record outward potassium currents from a
single cell. Conducted at: www.cerep.com
6

*To whom correspondence should be addressed. Phone (1) 858 731 3637. E-mail mark.sabat@takedasd.com
7