Pyrazolopyridine inhibitors of B-RafV600E. Part 2: Structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2011.06.097

Structure-activity relationships around a novel series of B-Raf ^V600E inhibitors are reported. The enzymatic and cellular potencies of inhibitors derived from two related hinge-binding groups were compared and3-methoxypyrazolopyridine proved to be superior. The 3-alkoxy group of lead B-Raf^V600E inhibitor 1 was extended and minimally affected potency. The propyl sulfonamide tail of compound 1, which occupies the small lipophilic pocket formed by an outward shift of the αC-helix, was expanded to a series of arylsulfonamides. X-ray crystallography revealed that this lipophilic pocket unexpectedly enlarges to accommodate the bulkier aryl group.

Full text of DOI:10.1016/j.bmcl.2011.06.097

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5533–5537
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazolopyridine inhibitors of B-RafV600E. Part 2: Structure–activity
relationships
Steve Wenglowsky a, , Kateri A. Ahrendt a, Alex J. Buckmelter a, Bainian Feng b, Susan L. Gloor a,
⇑
Stefan Gradl b, Jonas Grina a, Joshua D. Hansen a, Ellen R. Laird a, Paul Lunghofer a, Simon Mathieu b,
David Moreno a, Brad Newhouse a, Li Ren a, Tyler Risom a, Joachim Rudolph b, Jeongbeob Seo a,
Hillary L. Sturgis a, Walter C. Voegtli a, Zhaoyang Wen b
a ArrayBioPharma, 3200 Walnut Street, Boulder, CO 80301, United States
b Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, United States
a r t i c l e i n f o
a b s t r a c t
Structure–activity relationships around a novel series of B-RafV600E inhibitors are reported. The enzymatic
and cellular potencies of inhibitors derived from two related hinge-binding groups were compared and 3-
methoxypyrazolopyridine proved to be superior. The 3-alkoxy group of lead B-RafV600E inhibitor 1 was
extended and minimally affected potency. The propyl sulfonamide tail of compound 1, which occupies
Article history:
Received 14 June 2011
Accepted 21 June 2011
Available online 2 July 2011
the small lipophilic pocket formed by an outward shift of the aC-helix, was expanded to a series of aryl-
sulfonamides. X-ray crystallography revealed that this lipophilic pocket unexpectedly enlarges to accom-
modate the bulkier aryl group.
Keywords:
B-RafV600E
Pyrrolopyridine
Pyrazolopyridine
Structure–activity relationship
Ó 2011 Elsevier Ltd. All rights reserved.
The Ras/Raf/MEK/ERK (MAPK) signaling pathway transduces
signals from cell surface receptors to the nucleus leading to cellular
proliferation, differentiation and survival.1 Mutations in the BRAF
gene may lead to constitutive activation of B-Raf kinase which re-
sults in amplification of the MAPK pathway. Mutated B-Raf is pres-
ent in approximately 7% of all cancers and is most frequently
associated with melanoma.2 Over 90% of the detected mutations
in B-Raf are a glutamic acid for valine substitution at residue 600
(V600E).2 This mutation leads to constitutive kinase activity
500-fold greater than B-RafWT and correlates with increased
malignancy and decreased response to chemotherapy.3 Thus, the
development of small-molecule inhibitors of B-RafV600E is a prom-
ising strategy for cancer therapy, particularly in melanoma.4
Using structure-based design our laboratory discovered a series
of ATP-competitive B-RafV600E inhibitors which utilized 3-methoxy
pyrazolopyridine as a novel hinge-binding group.5 Initial struc-
ture–activity relationships demonstrated that bicyclic hinge-
binders such as pyrrolo- and pyrazolopyridine were more potent
than either pyridine or aminopyridine, and that a 3-methoxy
substituted pyrazolopyridine was more potent than 3-alkyl, 3-halo
and 3-amino. Optimization led to compounds 1 and 2, potent and
selective inhibitors of B-RafV600E which were highly active against a
broad panel of melanoma and colon cancer cell lines driven by this
activating mutation (Fig. 1). Compounds 1 and 2 possessed favor-
able ADME and pharmacokinetic profiles and displayed significant
antitumor activity in the Colo205 mouse xenograft model.5 On the
basis of their in vivo efficacy and preliminary safety profiles, 1 and
2 were selected for further preclinical evaluation.6
During the course of this work inhibitor 3 was also prepared.
Although compound 3 utilizes a pyrrolopyridine hinge-binder
common to inhibitors of several kinases4,7 it was notably less ac-
tive than compounds 1 and 2 against B-RafV600E in the enzymatic
and cellular assays.5 To further elucidate the nature of their bind-
ing to B-RafV600E, and as a broader comparison between these
two hinge-binders, thorough structure–activity relationships
around compounds 1 and 3 have been accomplished. Changes in-
cluded several central phenyl ring substitutions and replacement
of the propylsulfonamide tail with alkyl sulfonamides and sulfa-
mides. Additionally, 3-substituents of both hinge-binding cores
X
F
O
O
S
O O
H
N
H
N
S
N
N
N
N
H
H
O
F
O
F
HN
N
HN
1, X = F
2, X = Cl
3
OMe
⇑
Corresponding author.
E-mail address: wenglo1056@yahoo.com (S. Wenglowsky).
Figure 1. B-RafV600E inhibitors 1–3.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.097

5534
S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5533–5537
Table 1
B-RafV600E activity of central phenyl ring analogs 1–21
5
2
5
2
R
6
R
6
O
O
O O
H
N
H
N
S
S
N
N
N
N
H
H
O
O
HN
N
HN
1, 2, 4-21
3, 13-21
OMe
a
a
a
a
R
Compd
B-Raf IC50 (nM)
pERK IC50 (nM)
Compd
B-Raf IC50 (nM)
pERK IC50 (nM)
2,6-diF
2-F.6-CI
2,6-diCI
2-F
6-CI
6-F
2,5-diF
2,5,6-triF
2-OMe,6-F
2-F,6-Me
2-Me,6-F
2-F,6-OMe
2-CF3,6-F
1
2
4
5
6
7
8
9
10
—
—
11
12
4.8
1.7
1.4
13
19
20
40
38
250
350
80
100
180
—
3
38
17
8.3
87
88
210
76
19
—
94
13
14
15
16
17
18
19
—
190
206
290
2100
—
400
410
—
61
150
7.5
2.3
110
—
—
2600
530
20
21
—
170
680
—
1400
—
—
—
—
—
—
—
—
a
Values are means of at least two experiments.
Table 2
B-RafV600E activity of sulfonamides and sulfamides 22–43
F
F
O
O
O O
H
H
N
N
S
S
N
N
R
N
N
H
R
H
O
F
O
F
HN
N
HN
1, 22-33
3, 34-43
OMe
a
a
a
a
R
Compd
B-Raf IC50 (nM)
pERK IC50 (nM)
Compd
B-Raf IC50 (nM)
pERK IC50 (nM)
n-Pr
Et
n-Bu
CH2CH2CH2F
n-Bu
CH2-cyclopropyl
CH2CH2CF3
CH2CF3
Cyclopropyl
Bn
NHCH2CH3
NHCH(CH3)2
NHCH2CHF2
N(CH3)2
N(CH3)CH2CH3
N-Pyrrolidyl
1
22
—
23
24
25
—
4.8
25
—
1.3
4.5
17
—
—
63
190
18
110
13
18
29
8.6
19
62
—
13
11
31
—
—
170
830
180
630
500
63
3
—
38
—
94
—
120
75
200
230
270
2600
710
—
—
—
—
260
340
170
34
35
36
37
38
39
40
—
—
—
—
41
42
43
40
19
140
29
33
38
99
—
—
—
—
200
400
120
—
26
27
28
29
30
31
32
33
28
18
a
Values are means of at least two experiments.
were compared, and aryl sulfonamides in the pyrazolopyridine ser-
ies were examined. Details of this evaluation are provided herein.
The dihalo aryl ring of compounds 1–3 resides in the hydropho-
bic pocket adjacent to the gatekeeper residue Thr529. Molecular
modeling indicated that there was scope for varying the substitu-
tions at the 2- and 6-positionsof this aryl ring, as well as space
for a substituent at the 5-position. Halogen, alkyl and alkoxy
groups were examined at these positions, and these changes were
compared between the pyrrolo- and 3-methoxypyrazolopyridine
hinge-binding groups. Table 1 provides the enzymatic and cellular
activities of these inhibitors.8
difluoro (5 vs 1 and 15 vs 3), while a mono-halo substitution at
the 6-position resulted in a significant loss of potency (6, 7, 16
and 17), which indicates that a 2-substituent is critical for binding.
Addition of a fluorine at the 5-position of the central phenyl ring (8,
9, 18 and 19) resulted in a 2–5-fold loss in cellular activity. Other
substituents were not well tolerated, including Me-, MeO- and
F3C- (10–12, 20 and 21). Overall, the trends observed within each
series correlated closely to each other. A comparison between the
3-methoxy pyrazolopyridine and pyrrolopyridine hinge-binders
revealed that the former is consistently more active in both the
enzymatic and cellular assays regardless of the substitution on
the central ring, which corroborates our earlier studies.5
While both chloro and fluoro were well-tolerated in the 2- and
6-positions of the central phenyl ring, 2,6-difluoro was the optimal
combination for cellular activity across both series (1–4, 13, 14).
The 2-fluoro substitution was only 2–3-fold less potent than 2,6-
The X-ray crystal structure of 1 in complex with B-Raf reveals
that the kinase adopts the DFG-in conformation, but that the pro-
pyl sulfonamide tail occupies the small lipophilic pocket formed by

S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5533–5537
5535
an outward shift of the
a
C-helix.5 Few kinases are known to
Table 4
B-RafV600E activity of 3-alkoxy pyrazolopyridines 55-63
achieve this peculiar binding mode,9,10 which likely contributes
to the outstanding selectivity of these inhibitors for the Raf ki-
nases.5 To further elucidate the nature of this particular binding
mode this group was modified on both hinge-binding cores. Table
2 summarizes these results.
F
O
O
H
N
S
N
N
H
O
F
HN
Decreasing (22) or increasing (34) the length of the alkyl group
resulted in a loss of potency versus 1 or 3. In contrast, 3-fluoropro-
pyl led to a slight increase in activity over propyl (23 vs 1 and 35 vs
3). The bulkier iso-butyl group (24 and 36) slightly improved po-
tency with the 3-methoxy pyrazolopyridine hinge-binder, but not
with pyrrolopyridine. Other alkyl and fluorinated alkyl groups re-
sulted in a loss of potency with both cores (25, 26, 37–40), while
a benzyl group resulted in a significant loss of potency (27). Sulfa-
mides were also prepared (28–33 and 41–43) and, for good cellular
activity, disubstituted amine inputs were determined to be essen-
tial. In particular, N-pyrrolidylsulfamide 33 was superior within
this sub-series and equipotent to the lead compound 1. Again,
the trends observed within the two series correlated closely to
each other, and a comparison between the two hinge-binding
groups revealed that 3-methoxy pyrazolopyridine still consistently
produced more active inhibitors in the enzymatic and cellular
assays.
The superior potency observed for the 3-methoxy pyrazolopyri-
dine hinge-binder may be explained by the X-ray crystal structure
of 1 in which the methoxy group makes Van der Waals contacts
with several residues of the kinase binding domain, including
Ile463, Trp531, Ser535, Ser536, and Phe583.5 Based on this obser-
vation, attempts were made to duplicate these interactions with a
3-substituent on the pyrrolopyridine. The enzyme and cellular
activities of the resulting compounds were compared to the corre-
sponding analogs in the pyrazolopyridine series and these data are
provided in Table 3.11
Small, lipophilic substituents at the 3-position improved the po-
tency of the pyrrolopyridine series (50–52 vs 3) as expected, but
groups larger than methyl lost cellular activity (53 and 54). The
SAR for the pyrazolopyridine hinge-binder was similar (44–49). Be-
tween the two hinge-binding groups, the potency of analogs bear-
ing the same 3-substituent were within two-fold of each other,
with the exception of 3-ethyl. Attempts to prepare 3-methoxy
substituted pyrrolopyridine, the direct analog of 1, failed and were
attributed to insufficient chemical stability. Because the desired le-
vel of potency with the pyrrolopyridine hinge-binder could not be
achieved, this group was not pursued further.
N
R
O
a
a
R
Compd
B-Raf IC50 (nM)
pERK IC50 (nM)
Me
Et
CH2(CH3)2
CHCH2F
CH2CF3
1
55
56
57
58
4.8
2.4
3.6
2.5
5.3
19
12
20
26
28
59
3.5
76
60
61
4.2
6.9
43
62
O
O
CHCH2OCH3
CHCH2CH2OH
62
63
6.3
5.6
70
74
a
Values are means of at least two experiments.
B-RafV600E, additional 3-alkoxy groups were installed on this core
(Table 4). Analogs were prepared that incorporated both lipophilic
(55–59) and polar groups (60–63). While 3-ethoxy (55) slightly
improved the enzymatic and cellular activity compared to com-
pound 1, all other analogs in this series were similarly potent
and within a narrow window. The flat SAR within the 3-alkoxy ser-
ies correlated with the observation from X-ray crystallography
that, while the methoxy group itself makes several contacts with
the kinase binding domain, it is directed away from the pocket to-
ward a solvent-exposed region.5
The SAR of the sulfonamide tail of 1 was further explored with a
series of aryl and heteroaryl sulfonamides (Table 5). The X-ray
crystal structure of 1 suggested that the lipophilic pocket occupied
by the propyl group would not likely accommodate an aromatic
ring; surprisingly, phenyl sulfonamide 64 was equipotent to 1.
Additional analogs indicated that ortho and meta fluorines were
well tolerated (65–67), while para fluoro resulted in a significant
loss of potency (68). Pyridine (69–70) and imidazole (71) sulfona-
mides lost cellular activity, possibly due to their polarity, while
lipophilic heterocycles retained good cellular activity (72–73).
Due to the superiority of the 3-methoxy substitution on the
pyrazolopyridine towards providing cell-potent inhibitors of
Table 3
B-RafV600E activity of 3-substituted pyrazolo- and pyrrolopyridines 44–54
F
F
O
O
S
O O
H
N
H
N
S
N
N
N
N
H
H
O
F
O
F
HN
HN
N
1, 44-49
3, 50-54
R
R
a
a
a
a
R
Compd
B-Raf IC50 (nM)
pERK IC50 (nM)
Compd
B-Raf IC50 (nM)
pERK IC50 (nM)
OMe
H
Br
1
44
45
46
47
48
49
4.8
17
4.0
1.8
12
19
62
44
36
86
—
3
—
—
38
3.4
2.2
16
19
6.2
94
39
60
62
290
230
50
51
52
53
54
I
Me
Et
CF3
9.2
5.8
84
280
a
Values are means of at least two experiments.

5536
S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5533–5537
Table 5
B-RafV600E activity of aryl- and heteroaryl sulfonamides 64-73
F
O
O
H
N
S
N
N
R
H
O
F
HN
N
OMe
a
a
R
Compd
B-Raf IC50 (nM)
pERK IC50 (nM)
64
1.6
17
9.1
12
20
65
66
67
1.8
0.7
1.0
F
F
F
F
68
46
340
F
N
69
70
16
280
83
Figure 2. Overlay of the X-ray crystal structures of compounds 1 (blue) and 64
(orange). Surfaces are shaded correspondingly to the inhibitor. Both the propyl and
phenyl groups occupy a pocket that is enlarged by a shift of the aC-helix, while the
DFG sequence resides in its active conformation (DFG-in) (3.2 Å resolution).
N
0.5
71
72
73
320
0.2
0.7
>10,000
30
N
N
Table 6
Kinase selectivity of compounds 1, 33 and 66a,b
S
Compd
Aur A
FGR
FLT3
LCK
PTK6
SRC
SRMS
O
1
33
66
1
47
52
88
101
98
52
85
77
56
90
96
83
105
101
22
52
77
94
105
98
66
a
Values are means of at least two experiments.
a
Percent inhibition at 1 lM concentration of test compound.
Values are means of at least two experiments.
b
A comparison of the X-ray crystal structures of compounds 1
and 64 led to a rationale for the unexpected potency of the aryl sul-
fonamide series (Fig. 2).12 While the
C-helix is similarly posi-
tioned in both structures, the orientation of Phe595 from the
DFG sequence is shifted. This movement enlarges the pocket by
ꢀ1 Å, enough to accommodate a phenyl group as well as the sub-
stitution of ortho and meta fluorines.
Given the importance of the propyl sulfonamide for the selec-
tive inhibition of B-RafV600E, the kinase selectivity profile of two di-
verse, cell potent sulfonamide analogs was obtained and compared
to that of compound 1 (Table 6). Each compound was screened
F
a
F
H
N
NH2
HO
N
N
NH2
NO2
75
O
F
O
F
HN
HN
N
N
76
a, b
OMe
OMe
74
F
O
O
S
H
N
c
N
N
Ar
against a panel of 65 kinases at 1 lM and those with >40% inhibi-
H
O
F
tion are presented in Table 6. The selectivity profile for sulfamide
33 and aryl sulfonamide 66 was similar to lead compound 1
although they demonstrated increased activity against AuroraA,
FLT3, LCK and SRC. Nonetheless, the overall effects of these propyl
sulfonamide alternatives were minimal, and these compounds are
still very selective inhibitors of B-RafV600E. These data, coupled
with the crystal structure of compound 64, suggest that selectivity
HN
64-73
N
OMe
Scheme 1. Reagents and conditions: (a) EDCI, HOBt, DMF, 25 °C, 69%; (b)
SnCl2Á2H2O, EtOAc, 77 °C, 84%; (c) ClSO2Ar, 4:1 DCM/pyridine, 15–58%.
for this kinase via a shift of the
broad range of sulfonamide substitutions.
a
C-helix can be achieved for a
coupled to commercially available 2,6-difluoro-3-nitrobenzoic acid
75, and the product was reduced to aniline 76 with tin chloride.
Aryl sulfonamides were then prepared in the final step with the
appropriate sulfonyl chloride in a 4:1 mixture of DCM and pyri-
dine. This route allowed for rapid and efficient preparation and
screening of this class of B-RafV600E inhibitors.
In summary, thorough structure–activity relationships were
developed around the 3-methoxy pyrazolopyridine and pyrrolo-
pyridine series of B-RafV600E inhibitors. Although the pyrrolopyri-
Pyrrolo- and 3-alkoxy pyrazolopyridines were generally pre-
pared via an amide bond coupling between the hinge-binding core
anilines and the fully elaborated benzoic acids, and these synthetic
methods have been described elsewhere.5,13 The aryl sulfonamide
subseries (compounds 64–73) was an exception to this general
synthetic route and utilized the incorporation of the sulfonamide
in the final step (Scheme 1). Pyrazolopyridine-5-amine 745 was

S. Wenglowsky et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5533–5537
5537
W. C.; Wen, Z.; Willis, B. S.; Woessner, R. D.; Wu, W.-I.; Young, W. B.; Grina, J.
ACS Med. Chem. Lett. 2011, 2, 342.
6. Choo, E. F.; Afflerbaugh, L.; Alicke, B.; Boggs, J.; Dinkel, D.; Gould, S.; Grina, J.;
West, K.; Menghrajani, K.; Ran, Y.; Rudolph, J.; Wenglowsky, S. Xenobiotica,
accepted for publication.
dine hinge-binder is a commonly utilized motif in kinase inhibitor
development, 3-methoxy pyrazolopyridine consistently produced
more active inhibitors in the enzymatic and cellular assays. Fur-
thermore, attempts to discover equipotent pyrrolopyridines by
incorporating substituents at the 3-position were unsuccessful.
Based on these results, 3-alkoxypyrazolopyridines were further ex-
plored and as a class were determined to be broadly active.
Lastly, it was discovered that the small pocket formed by an
7. Pyrrolopyridine has been utilized as a hinge-binder for inhibitors of several
kinases including, AKT: (a) Blake, J. F.; Kallan, N. C.; Xiao, D.; Xu, R.; Bencsik, J.
R.; Skelton, N. J.; Spencer, K. L.; Mitchell, I. S.; Woessner, R. D.; Gloor, S. L.;
Risom, T.; Gross, S. D.; Martinson, M.; Morales, T. H.; Vigers, G. P. A.;
Brandhuber, B. J. Bioorg. Med. Chem. Lett. 2010, 20, 5607; Aurora: (b) Medina,
J. R.; Grant, S. W.; Axten, J. M.; Miller, W. H.; Donatelli, C. A.; Hardwicke, M. A.;
Oleykowski, C. A.; Liao, Q.; Plant, R.; Xiang, H. Bioorg. Med. Chem. Lett. 2010, 20,
2552; Bcr-Abl: (c) Deng, X.; Lim, S. M.; Zhang, J.; Gray, N. S. Bioorg. Med. Chem.
Lett. 2010, 20, 4196; B-Raf: (d) Tang, J.; Hamajima, T.; Nakano, M.; Sato, H.;
Dickerson, S. H.; Lackey, K. E. Bioorg. Med. Chem. Lett. 2008, 18, 461; IKK2: (e)
Liddle, J.; Bamborough, P.; Barker, M. D.; Campos, S.; Cousins, R. P. C.; Cutler, G.
C.; Hobbs, H.; Holmes, D. S.; Ioannou, C.; Mellor, G. W.; Morse, M. A.; Payne, J. J.;
Pritchard, J. M.; Smith, K. J.; Tape, D. T.; Whitworth, C.; Williamson, R. A. Bioorg.
Med. Chem. Lett. 2009, 19, 2504; Met: (f) Williams, D. K.; Chen, X.-T.; Tarby, C.;
Kaltenbach, R.; Cai, Z.-W.; Tokarski, J. S.; An, Y.; Sack, J. S.; Wautlet, B.; Gullo-
Brown, J.; Henley, B. J.; Jeyaseelan, R.; Kellar, K.; Manne, V.; Trainor, G. L.;
Lombardo, L. J.; Fargnoli, J.; Borzilleri, R. M. Bioorg. Med. Chem. Lett. 2010, 20,
2998; ROCK: (g) Henderson, A. J.; Hadden, M.; Guo, C.; Douglas, N.; Decornez,
H.; Hellberg, M. R.; Rusinko, A.; McLaughlin, M.; Sharif, N.; Drace, C.; Patil, R.
Bioorg. Med. Chem. Lett. 2010, 20, 1137; SGK: (h) Hammonda, M.; Washburn, D.
J.; Hoang, T. H.; Manns, S.; Frazee, J. S.; Nakamura, N.; Patterson, J. R.; Trizna,
W.; Wu, C.; Azzarano, L. M.; Nagilla, R.; Nord, M.; Trejo, R.; Head, M. H.; Zhao,
B.; Smallwood, A. M.; Hightower, K.; Laping, N. J.; Schnackenberg, C. G.;
Thompson, S. K. Bioorg. Med. Chem. Lett. 2009, 19, 4441.
outward shift of the aC-helix can expand to accommodate sulfon-
amides as large as a substituted phenyl, and that this group still
maintained excellent selectivity toward B-RafV600E. This observa-
tion provides additional scope to pursue alternative groups that
may achieve this particular B-Raf binding mode, but possess dis-
tinct properties from a sulfonamide. These efforts will be reported
in due course.
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