The discovery of potent and selective 4-aminothienopyridines as B-Raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.11.020

A series of novel, potent 4-aminothienopyridine B-Raf kinase inhibitors was designed and synthesized using knowledge-based design. Compounds 5f, and 6k exhibited not only excellent potency in both enzyme assay (IC₅₀ = 5.1, 16.6 nM) and cellular

Full text of DOI:10.1016/j.bmcl.2012.11.020

Bioorganic & Medicinal Chemistry Letters 23 (2013) 66–70
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of potent and selective 4-aminothienopyridines as B-Raf
kinase inhibitors
⇑
Jun Tang , Karen E. Lackey, Scott H. Dickerson
GlaxoSmithKline Research & Development, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel, potent 4-aminothienopyridine B-Raf kinase inhibitors was designed and synthesized
Received 10 September 2012
Revised 31 October 2012
Accepted 7 November 2012
Available online 16 November 2012
using knowledge-based design. Compounds 5f, and 6k exhibited not only excellent potency in both
enzyme assay (IC50 = 5.1, 16.6 nM) and cellular assay (IC50 = 0.2, 0.2
selectivity profile against other kinases.
lM), but also had an outstanding
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf inhibitor
Knowledge-based design
The rationale for targeting the Raf-MEK-ERK signaling pathway
with small molecule inhibitors was well reviewed in recent pa-
pers.1 Sorafenib (Nexavar, BAY-43-9006) was approved in 2005
for use in renal cell carcinoma treatment and inhibits VEGFR2,
PDGFR, c-KIT, and Raf.2 However, multiple preclinical animal mod-
els and clinical trials showed mixed results using Sorafenib, which
raised some doubts about its therapeutic potential of targeting B-
Raf.3 Given the increasing interest in developing chemotherapeu-
tics to this protein kinase and the limited number of selective B-
Raf kinase inhibitors so far reported (Fig. 1), we aimed to discover
new B-Raf kinase inhibitors with improved kinase inhibition pro-
files. Herein we describe the successful identification of a 4-amino-
thienopyridine series.
With regard to designing an ATP competitive inhibitor, knowl-
edge-based design is emerging as the predominant design tool.7
The 2-D pharmacophore map shown in Figure 2 captured known
structural information in a simplified form. It depicts the seven
critical binding regions of the ATP-binding domain that can be ac-
cessed to construct potent, ATP-competitive kinase inhibitors. The
thienopyridine series was elaborated by designing functional
groups that interacted with key features of these regions. The scaf-
fold of an ATP-competitive kinase inhibitor typically includes a
hinge binder as the starting point for the design. From this pharma-
cophore model for ATP competitive kinase inhibitors, we can spec-
ulate that (1) the aminopyridine moiety of the thienopyridine
scaffold binds to the hinge region through hydrogen-bond donor-
acceptor interactions in a similar manner to N1 and N6 in the ade-
nine base of ATP; (2) the 3-aryl moiety would fill the inner hydro-
phobic pocket not generally occupied by ATP; (3) the 7-aryl
moiety would be exposed to the outer hydrophobic pocket. Based
upon the known B-Raf kinase inhibitors, the urea and amide are
reasonable linkers to connect the inner hydrophobic moiety to the
induced fit moiety.8
Synthetic methods to build the designed compounds are sum-
marized in Scheme 1. The key building block 4 used in this study
was prepared according to the literature from commercially avail-
able reagents.9 In this study, the 3-pyridyl group was carefully se-
lected and incorporated via the bronic ester 4a at the 7-position of
the 3-bromo-7-iodothieno[3,2-c]pyridin-4-amine 4. We chose 3-
pyridyl group as a moiety that occupies the outer hydrophobic pock-
et for the following reasons. The outer hydrophobic pocket appears
to prefer an aromatic ring or its bioisosteres in B-Raf ATP binding
site. We would expect pyridine to confer improved solubility com-
pared to a phenyl ring and the 3-pyridyl ring would most likely
have fewer liabilities with regard to p450 inhibitory activities in
comparison to 4-pyridyl ring. Boc protected 3-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)aniline 4c was subjected to
a
microwave mediated Suzuki coupling reaction with bromide 4b
in good yield. The protective Boc group in compound 4d was re-
moved by aqueous HCl in MeOH at an elevated temperature to pro-
vide key precursor 4e, which reacted with the corresponding acyl
chlorides and isocyanates to give the final compounds 5 and 6.
Thus, a novel series of thienopyridine analogues (4e, 5a–5g, 6a–
6k) were prepared and are highlighted in Tables 1 and 2. The com-
pounds were tested for B-Raf inhibition in a fluorescence anisot-
ropy binding assay.10 In this study, we decided to focus on the
meta amido and urea substituted compounds, since the para ana-
logues were found to be active inhibitors against C-FMS, PDGFR
and VEGFR.11 The parent amino compound 4e showed no inhibi-
tory activity (IC50 >10,000 nM). The amide functionality demon-
⇑
Corresponding author. Tel.: +1 919 483 4173.
E-mail address: jun.x.tang@gsk.com (J. Tang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.020

J. Tang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 66–70
67
H
O
N
H
H
N
N
H
N
O
CF3
S
O
F
O
F
HN
N
N
O
N
N
N
N
N
H
O
HN
Cl
N
1
2
(SB-590885)
3
(PLX4720)
Figure 1. Representative selective B-Raf inhibitor 1 (SB-590885),4 compound 2,5 and 3 (PLX4720).6
Gate Keeper
induced fit
induced fit
O
A
NH
inner
hydrophobic
inner
hydrophobic
Lys
NH
H
NH2
O
Glu
HO
N
H
Scaffold
N H
Phosphate
Binding
O
O
OH
Asp
S
Sugar
Pocket
Sugar
Pocket
O
Outer
Hydrophobic
Outer
Hydrophobic
N
A = C, N
Solvent Front
Solvent Front
Figure 2. 2-D pharmacophore map of kinase ATP domain and the designs of the inhibitor.
NH2
Br
NH2
O
O
O
Br
B
O
N
O
a
N
N
b
B
N
+
S
+
S
HN
I
O
4
4a
4b
4c
H
N
H
H
N
H
N
N
NH2
R2
R1
O
NH2
NH2
NH2
NH2
O
c
O
d
N
N
N
N
or
S
S
S
S
N
N
N
N
4d
4e
5
6
Scheme 1. Reagents and conditions: (a) Pd(PPh3)4, aq Na2CO3, DME, MW heating, 120 °C, 15 min, 69%; (b) Pd(PPh3)4, aq Na2CO3, DME, MW heating, 120 °C, 1.5 h; (c) aq HCl,
MeOH, 80 °C, 2 h, 70% (two steps); (d) Acyl chlorides or isocyanates, pyridine, rt, 1–12 h.
strated greater potential than its sulphonamide analogue (compare
5a with 5d). As the pharmacophore map suggested, the terminal
moieties tethering to the amide, would be positioned to the induced
fit pocket, which prefers either substituted or unsubstituted hydro-

68
J. Tang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 66–70
Table 1
Table 2
B-Raf inhibitory activities of compounds 4e, 5a–5h
B-Raf inhibitory activities of compounds 6a–6k
H
N
H
H
N
N
R1
R2
NH2
NH2
O
N
N
N
S
S
N
Compound
R1
H
B-RAF(FP), nM
>10,000
B-RAF(ELISA),
N/A
lM
Compound
R2
B-RAF(FP), nM
7940
B-RAF(ELISA),
N/A
l
M
4e
CH3
CH3
O
6a
S
H3C
5a
>10,000
N/A
O
O
CH3
6b
692
>10
CH3
N
N
H3C
O
5b
5c
5d
4170
1550
93
N/A
>10
4.5
6c
6d
6e
30.9
20.9
13.8
4.1
1
O
O
O
1
F
F
CH3
O
6f
9.6
0.7
1.7
5e
5f
49
1.1
0.2
F
F
F
6g
19.5
F
F
5.1
O
O
6h
933
>10
5g
5h
19
45
1.5
2.5
O
F
F
6i
6j
25.1
21.4
0.3
0.3
S
O
F
F
F
F
F
phobic aromatic rings with regard to potency for B-Raf kinase. Con-
sistent with these considerations, the compounds possessing a
hydrophilic piece such as methylated pyrrole (5b), or a non-aro-
matic cyclohexyl ring (5c), were inactive. The phenyl ring deriva-
tive (5d) showed good enzyme inhibitory activity (IC50 = 93 nM),
F
F
F
6k
16.6
0.2
Cl
however, relatively weak cellular potency (IC50 = 4.5 l
M).10 Phenyl
rings bearing lipophilic substituents (5e, 5f) were more potent
than the unsubstituted phenyl derivative (5d). Direct connection
of an aromatic moiety to the amide linker was not crucial based
on the inhibitory activities observed for the homologated phenyl
analogue 5g (IC50 = 19 nM) and thiophenylmethyl analogue 5h
(IC50 = 45 nM). Among these compounds, compound 5f12 bearing
a meta-trifluoromethylphenyl ring demonstrated not only good en-
zyme inhibitory activity (IC50 = 5.1 nM), but also good cellular po-
Selectivity Profile of 5f
9
8
7
6
5
4
tency (IC50 = 0.2 lM). Furthermore, compound 5f showed more
than 40-fold selectivity against a variety of other kinases screened
(Fig. 3) except for Lyn kinase. Addition of the Lyn inhibitory activity
can be positive to this molecule, for the reason of that Lyn is
remarkably overexpressed at the protein level in leukemic cells.13
Urea derivatives were also studied and the results are shown in
Table 2. A survey of substitutions was performed to find the opti-
mal group for the induced fit pocket (6a–6k). The terminal alkyl
substituted urea compound (6a) was inactive. The hydrophilic
Figure 3. Kinase selectivity profile of compound 5f. Potency is represented by pIC50
.

J. Tang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 66–70
69
fold projected into the outer hydrophobic pocket without any spe-
cific contact of its nitrogen with the protein.
Selectivity Profile of 6k
9
8
7
6
5
4
In conclusion, we discovered a series of novel, potent B-Raf
inhibitors using knowledge-based design. As exemplified by com-
pounds 5f and 6k, we have achieved not only excellent potency
in both enzyme and cellular assays, but also an outstanding selec-
tivity profile against other kinases. The addition of the Lyn inhibi-
tory activity to these molecules is considered a desired kinase
inhibitory profile, which might work in Raf mediated tumors as
well as B-cell chronic lymphocytic leukemia due to the highly
overexpressed Lyn in B-CLL.
3
1
1
a
0
1
F
2
b
R
I
2
R
N
K
A
K
A
B
a
B
8
7
T
K5
r
K
T
F
a
r
F1
P
CK
BB
KK NK LY
J
RA
B
PK
RO
SY
o
or
-
P3
AL
AK
SK3
r
SG
EG
IG
ZA
ER
G
Au Au
Acknowledgments
Figure 4. Kinase selectivity profile of compound 6k. Potency is represented by
pIC50
.
We are grateful to the enzymologists for the contributions to
the screening work; Hiroshi Sootome (GlaxoSmithKline, Japan)
for the evaluation of cellular potencies.
3,5-dimethylisoxazole group in 6b resulted in a significant loss of
potency due to its hydrophilicity. Substitution of the urea with a
terminal cyclohexyl ring (6c) was relatively well tolerated and
demonstrated moderate enzyme activity (IC50 = 30.9 nM) com-
pared to the amide version 5c (IC50 = 1550 nM). Tethering a benzyl
group to the urea (6d) was also well tolerated and moderate cellu-
References and notes
1. (a) McCubrey, A. J.; Milella, M.; Tafuri, A.; Martelli, M. A.; Lunghi, P.; Bonati, A.;
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4. King, A. J.; Patrick, D. R.; Batorsky, R. S.; Ho, M. L.; Do, H. T.; Zhang, S. Y.; Kumar,
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Lougheed, J. C.; Lee, J.; Chau, D.; Stout, T. J.; May, E. W.; Rominger, C. M.;
Schaber, M. D.; Luo, L.; Lakdawala, A. S.; Adams, J. L.; Contractor, R. G.; Smalley,
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lar potency was observed (IC50 = 1.0 lM). Compounds with a phe-
nyl ring (6e), mono-substituted phenyl ring (6f, 6i), and di-
substituted phenyl ring (6g, 6j, 6k) were generally very potent
with IC50 values ranging from 9.6 to 25.1 nM, regardless of the
positions of the substituents. However, incorporation of an over-
sized substituent, for example, 4-benzyloxyphenyl (6h), resulted
in decrease in activity. Evaluation of these compounds in a cellular
assay via measuring the phosphorylation of MEK1 demonstrated
that compound 6k14 containing a 30-trifluoromethyl-40-chloro ben-
zyl group provided one of the best cellular potencies
5. Tang, J.; Hamajima, T.; Nakano, M.; Sato, H.; Dickerson, S. H.; Lackey, K. E.
Bioorg. Med. Chem. Lett. 2008, 18, 4610.
6. Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.; Bremer, R.; Gillette, S.;
Kong, J.; Haass, N. K.; Sproesser, K.; Li, L.; Smalley, K. S. M.; Fong, D.; Zhu, Y.-L.;
Marimuthu, A.; Nguyen, H.; Lam, B.; Liu, J.; Cheung, I.; Rice, J.; Suzuki, Y.; Luu,
C.; Settachatgul, C.; Shellooe, R.; Cantwell, J.; Kim, S.-H.; Schlessinger, J.; Zhang,
K. Y. J.; West, B. L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P. N.; Hirth, P.;
Artis, D. R.; Herlyn, M.; Bollag, G. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3041. In
this Letter, Cys531 was numbered as Cys532.
(IC50 = 0.2
showed greater than 100-fold selectivity against a variety of other
kinases screened except for p38 and Lyn kinase (Fig. 4).
lM) against B-Raf. Furthermore, compound 6k also
a
Illustrated in Figure 5 is a docking model of 5f complexed with
an inactive conformation of B-Raf.15 From this docking analysis, it
is noted that aminopyridine binds to the kinase at the hinge region
with its nitrogen and hydrogen atoms forming H-bond interactions
with the backbone CO of Gln529 and NH of Cys531.6 In this bound
conformation, the amide linker directed its terminal moiety into
the induced fit pocket and with its NH and CO interacting with
Glu500 and Asp593 of B-Raf kinase. The terminal trifluoromethyl-
phenyl group rested in a hydrophobic region comprised of the res-
idues of Leu504, Val503, Leu566, and His573. Based on this docking
analysis, the pyridyl ring at the 7-position of thienopyridine scaf-
7. (a) McGregor, M. J. J. Chem. Inf. Model. 2007, 47, 2374; (b) Liao, J. J.-L. J. Med.
Chem. 2007, 50, 409; (c) Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2, 358; (c)
Southall, N. T.; Ajay J. Med. Chem. 2006, 49, 2103.
8. (a) Perez, L. B.; Sheng, T. PCT Int. Appl. WO 2006102079.; (b) Riedl, B.; Dumas,
J.; Khire, U.; Lowinger, T. B.; Scott, W. J.; Smith, R. A.; Wood, J. E.; Monahan, M.-
K.; Natero, R.; Renick, J.; Sibley, R. N. PCT Int. Appl. WO 2000042012.
9. Miyazaki, Y. PCT Int. Appl. WO 2004100947.
10. Enzyme and cellular assay description can be found: Tang, J. PCT Int. Appl. WO
2007056625. Cellular assays: B-Raf mediated phosphorylation of MEK1 was
measured in the cellular assay. Expression constructs for B-Raf and FLAG-
tagged MEK1 were co-transfected in 3T3 cells and gene expression was
induced using the GeneSwitch (TM) system for inducible mammalian
expression (Invitrogen). Four hours following the induction of expression of
B-Raf and MEK1, cells were exposed to the test compounds for 2 h. The cells
Figure 5. Docking model of compound 5f bound to inactive form of B-Raf. Inhibitor atoms colored as follows: C, gray; N, blue; O, red; S, yellow.

70
J. Tang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 66–70
were then lysed, and then an immunoassay was performed using anti-
phospho-MEK1/2 (Cell Signaling Technlogoies) to detect the percent inhibition
of MEK1 phosphorylation.
7.61 (s, 1H), 7.54–7.58 (m, 2H), 7.27 (dt, J = 1.2, 7.8 Hz, 1H), 5.68 (s, 2H). LC/MS:
m/z 491 (M+H)+.
13. Contri, A.; Brunati, A. M.; Trentin, L.; Cabrelle, A.; Miorin, M.; Cesaro, L.; Pinna,
L. A.; Zambello, R.; Semenzato, G.; Donella-Deana, A. J. Clin. Invest. 2005, 2, 369.
14. Analytic data of potent inhibitor 6k: 1H NMR (400 MHz, DMSO-d6) ppm 9.28 (s,
1H), 9.11 (s, 1H), 8.88 (dd, J = 0.8, 2.3 Hz, 1H), 8.62 (dd, J = 1.5, 4.8 Hz, 1H),
8.09–8.12 (m, 2H), 7.98 (s, 1H), 7.54–7.68 (m, 6H), 7.47 (t, J = 7.6 Hz,1H), 7.13
(dt, J = 1.5, 7.6 Hz, 1H), 5.68 (s, 2H). LC/MS: m/z 540 [M+H]+.
15. Model built from X-ray crystal structure PDB ID 1UWH with MacroModel,
Schrödinger LLC.
11. (a) Unpublished data; (b) Heyman, H. R.; Frey, R. R.; Bousquet, P. F.; Cunha, G.
A.; Moskey, M. D.; Ahmed, A. A.; Soni, N. B.; Marcotte, P. A.; Pease, L. J.; Glaser,
K. B.; Yates, M.; Bouska, J. J.; Albert, D. H.; Black-Schaefer, C. L.; Dandliker, P. J.;
Stewart, K. D.; Rafferty, P.; Davidsen, S. K.; Michaelides, M. R.; Curtin, M. L.
Bioorg. Med. Chem. Lett. 2007, 17, 1246.
12. Analytic data of potent inhibitor 5f: 1H NMR (400 MHz, DMSO-d6) ppm 10.67 (s,
1H), 8.89 (dd, J = 0.8, 2.3 Hz, 1H), 8.62 (dd, J = 1.8, 4.8 Hz, 1H), 8.31 (s, 1H), 8.28
(d, J = 7.8 Hz,1H), 8.10–8.13 (m, 1H), 7.92–8.00 (m, 4H), 7.81 (t, J = 7.8 Hz, 1H),