Discovery and optimization of pyrazoline compounds as B-Raf inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.06.113

The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determin

Full text of DOI:10.1016/j.bmcl.2010.06.113

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4800–4804
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of pyrazoline compounds as B-Raf inhibitors
*
Matthew O. Duffey , Ruth Adams, Christopher Blackburn, Ryan W. Chau, Susan Chen, Katherine M. Galvin,
Khristofer Garcia, Alexandra E. Gould, Paul D. Greenspan, Sean Harrison, Shih-Chung Huang, Mi-Sook Kim,
Bheemashankar Kulkarni, Steven Langston, Jane X. Liu, Li-Ting Ma, Saurabh Menon, Masayuki Nagayoshi,
R. Scott Rowland, Tricia J. Vos, Tianlin Xu, Johnny J. Yang, Shaoxia Yu, Qin Zhang
Millennium Pharmaceuticals, Inc., Oncology, Medicinal Chemistry, 40 Landsdowne St., Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report.
Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent
and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected
inhibitors is also reported.
Received 11 May 2010
Revised 17 June 2010
Accepted 21 June 2010
Available online 25 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
B-Raf
B-Raf inhibitor
Pyrazoline
Raf, a serine/threonine protein kinase, is an integral part of the
MAP kinase signaling pathway and is involved in the Ras signaling
cascade of Raf-MEK-ERK.1 Uncoveringthe complex and intricaterole
of Raf pathway signaling in cancer is the continuing aim of intensive
research.2 Activating mutations of the Raf isoform B-Raf cause con-
stitutive activation of the MAP kinase pathway and uncontrolled
proliferation of tumor cells. Specific mutations of B-Raf are associ-
ated with various cancers includingnon-Hodgkin’s lymphoma, colo-
rectal adenocarcinoma, malignant melanoma, thyroid carcinoma,
non-small cell lung carcinoma, and adenocarcinoma of the lung.
Studies report that B-Raf mutations exist in up to 66% of malignant
melanomas with a single mutation (V600E) accounting for 80% of
these.3 Suppression of B-Raf (V600E) in human melanoma cells leads
to downregulation of the MAP kinase signaling pathway and apop-
tosis.4 Recently, treatment of B-Raf mutant melanoma patients with
a selective B-Raf inhibitor (PLX-4032) resulted in promising preli-
minary evidence of antitumor activity.5 Thus, inhibition of mutated
B-Raf could prove to be a useful therapeutic against melanoma as
well as a number of other cancers.
Optimization of 1, with respect to enzyme and cellular potency,
began with the synthesis of numerous analogues by parallel syn-
thesis (Fig. 1). The rapid synthesis of analogues via acylation of
the parent pyrazoline provided an opportunity for the screening
of a diverse set of compounds possessing replacements of the furan
moiety. We felt this was a natural starting point for analogue syn-
thesis as the furanoyl moiety could easily be replaced by a diverse
array of acylating agents. Implementation of this strategy led to the
identification of thiophene–pyridine as a furan replacement to give
2, a compound that showed a significant potency increase in the
in vitro assays.7
To better understand the activity of 2, an analysis of its binding
to B-Raf was performed (Fig. 2). Represented by white carbons, 2
An inhibitor (1) of B-Raf (V600E) was initially identified by
high-throughput screening of our compound library (Fig. 1). We
became interested in this initial hit due to the novelty of the pyr-
azoline scaffold as well as its biological activity. In vitro enzyme
and cell assays were employed to evaluate compound activity
against the target.6
* Corresponding author. Tel.: +1 617 551 7832; fax: +1 617 551 8907.
E-mail address: matthew.duffey@mpi.com (M.O. Duffey).
Figure 1. Initial HTS hit and high-throughput chemistry optimization of 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.113

M. O. Duffey et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4800–4804
4801
was docked into a homology model of wild type B-Raf based on a
crystal structure of B-Raf bound sorafenib (PDB 1UWH)8 but mod-
ified to an active kinase conformation (DFG-in).9 In this model, the
nitrogen of the pyridine ring (region B) forms a hydrogen bond to
the hinge backbone NH of C532. In the back of the ATP pocket an
intramolecular hydrogen bond between the hydrogen of the phe-
nolic OH and the carbonyl of the pyrazoline amide helps organize
the phenol in a hydrophobic pocket (green surface). In addition, the
thiophene–pyridine extends under the P loop out towards solvent.
At this point we focused our efforts on a traditional medicinal
chemistry approach to optimize 2. Our goals were foremost to im-
prove potency and understand the SAR of B-Raf (V600E) inhibition.
The strategy devised to accomplish this task involved modification
of the three substituents of the central pyrazoline core. Initial
chemistry efforts that led to discovery of 2 from 1 demonstrated
that replacement of the furan ring with a biaryl system increased
potency.7 We wished to expand upon this discovery and evaluate
compounds that explored this area of the scaffold further (region
Figure 2. Proposed binding mode of 2.
Scheme 1. Synthetic scheme to construct substituted pyrazolines.
Table 1
SAR/optimization of region A
a
a
a
a
Ar
V600E IC50 (nM)
pERK IC50 (nM)
Ar
V600E IC50 (nM)
pERK IC50 (nM)
N
2
23
7
180 40
12
10
6
270 77
NH2
N
7
8
79
7
1500
210
13
14
6
5
110 54
82 38
Me
S
NH2
4
1
N
N
N
9
10
11
9
5
6
89 48
15
16
17
4
5
4
25
21
19
9
9
1
NH
NH
OH
140 120
390 200
NH
1
NH2
a
Standard deviations are reported for N P 3. Otherwise IC50 values are mean values of two determinations.

4802
M. O. Duffey et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4800–4804
Table 2
SAR/optimization of phenol
a
a
a
a
Ar
V600E IC50 (nM)
pERK IC50 (nM)
Ar
V600E IC50 (nM)
pERK IC50 (nM)
18
799
nd
21
22
13
7
1400
680
F
OH
F
HO
19
670 320
>25,000
80 29
OH
F
F
20
OH
a
5
1
23
11
1400
OH
Standard deviations are reported for N P 3. Otherwise IC50 values are mean values of two determinations.
Table 3
A). In addition, modification of the pyridine ring (region B) and the
phenol were also investigated.
ortho-Fluoride enhances potency
Scheme 1 shows the general route employed to synthesize the
desired pyrazoline analogues. The core pyrazoline functionality
(5) was made via a two-step process in which aldol condensation
between an appropriately substituted acetylpyridine (3) and a
2-hydroxybenzaldehyde (4)10 was followed by pyrazoline forma-
tion by addition of hydrazine.11 A standard amide coupling be-
tween 5 and an appropriate thiophene carboxylic acid then
completed the synthesis to provide general structure 6.12
Use of the route in Scheme 1 allowed for the construction of
numerous analogues that initially explored the effect of the outer
aryl ring of region A. While the discovery of 2 demonstrated that
an aromatic ring is beneficial at this site, further chemical manip-
ulation of this site confirmed this finding. In doing so, we made an
effort to choose aryl rings that contained solubilizing groups as the
a
a
Ar
X
V600E IC50 (nM)
pERK IC50 (nM)
20
2
F
H
5
23
1
7
80 29
180 40
N
24
9
N
F
H
3
9
55 19
89 48
N
aqueous solubility of 2 was so low (5.8 l
g/mL)13 we were unable to
formulate IV doses for pharmacokinetic experiments.
25
12
F
H
7
10
6
6
85 38
200 77
Table 1 shows the results of these efforts. Replacement of the
2-pyridyl ring with an o-tolyl ring resulted in loss of activity (7)
while other heterocycles such as thiazole and pyrazole maintained
enzyme and cell potency (8, 9). p-Benzyl alcohol provided a com-
pound (10) with good enzyme and cell potency, as did the installa-
tion of benzyl amines (11–14). Significant increases in cell potency
were observed upon use of constrained amines such as the iso-
meric tetrahydroisoquinoline moieties (15, 16) and the isoindoline
functionality (17). We were unable to identify a reason for the dis-
crepancy between the enzyme and cell assays (permeability/solu-
bility were not the cause). As a result, our evaluation of potency
relied largely on the results of the cell assay as it gave us the ability
to distinguish between compounds that possessed good potency in
the enzyme assay.
Analogues were also prepared to explore SAR of the phenol ring
(Table 2). Removal of the OH (18) or placement of the OH in the
meta position (19) resulted in a loss of enzyme and cell activity,
suggesting that the intramolecular H-bonding of the C1–OH as pro-
posed by our homology model is essential for potency. With this
insight, we focused our efforts on substitution of the phenol to
NH2
26
13
F
H
8
6
41
9
110 54
N
27
14
F
H
3
5
37 10
82 38
NH2
1
28
15
F
H
3
4
21 10
25
9
NH
NH
29
16
F
H
3
5
34 11
21
9
1
30
17
F
H
2
4
60
19
NH
a
Standard deviations are reported for N P 3. Otherwise IC50 values are mean
values of two determinations.

M. O. Duffey et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4800–4804
4803
Table 4
SAR/optimization of region B
a
a
a
a
Ar
R
V600E IC50 (nM)
pERK IC50 (nM)
Ar
R
V600E IC50 (nM)
pERK IC50 (nM)
12
31
32
A
A
A
H
F
NH2
10
21 17
7
6
200 77
180 67
64 36
2
33
34
35
B
B
B
B
H
23
3
3
7
0.2
180 40
63 27
39 22
NHAc
CH2OH
NH2
3
31
6
a
Standard deviations are reported for N P 3. Otherwise IC50 values are mean values of two determinations.
Table 6
Table 5
Pharmacokinetic data for B-Raf (V600E) inhibitors15
Optimization of the three regions
a
a
a
Ar
X
pERK IC50
(nM)
CL (L/h/
kg)
AUC
(nM h)
%F
Ar
R
V600E IC50 (nM)
pERK IC50 (nM)
NH
17
30
H
F
19
60
1
3.5
0.9
890
8400
14
37
36
37
38
CH2OH
3
4
3
120
56
NH
12
25
H
F
200 77
85 38
3.9
1.6
4900
11,000
76
85
NH2
NH2
NH2
NH2
NH
18
8
N
39
F
180
0.7
26,000
89
a
Standard deviations are reported for N P 3. Otherwise IC50 values are mean
values of two determinations.
a
Standard deviations are reported for N P 3. Otherwise IC50 values are mean
values of two determinations.
determine the effect of further changes. We found that incorpora-
tion of fluoride in the ring (C-1) resulted in increased potency (20).
In turn, the subsequent transfer of the fluoride to other positions
on the ring resulted in varying degrees of decreased cell potency
(21–23).
for potency against the target (data not shown), we also observed
that substitution at C-5 of the pyridyl ring could provide a boost in
cell potency. We created analogues in two subseries, one contain-
ing the ortho benzylamine and the other containing the 2-pyridyl
group in region A as shown in Table 4. Addition of fluoride (31)
provided an analogue that was equipotent with 2, while addition
of the amino (32, 35), acylamino (33) and hydroxymethyl (34)
groups to this ring gave a significant increase in cell potency.
We attempted to combine the optimal modifications of these
three regions by using the same synthetic techniques described
previously. Representative compounds are shown in Table 5. By
inclusion of the C-1 fluorophenol and varying the outer aryl ring
of region A, we learned that the positive trends in cell potency
are additive in some cases while not in others. Combination of
the hydroxymethyl moiety in ring B with the bicyclic amine did
not provide an increase in cell potency (36). However, combining
the 3-aminopyridine in region B with the benzyl amine or pyridine
in region A provided very potent compounds in the cell assay,
including one of the most potent compound synthesized in this
study of B-Raf inhibitors (38).
Fluoride incorporation at C-1 of the phenol ring provided com-
pounds that generally were more potent than their non-fluorinated
counterparts in the cell assay. Table 3 shows the comparison be-
tween selected compounds with and without the C-1 fluoride on
the phenol. In general, a 2–3-fold increase in cell potency was ob-
served for the fluorinated analogues. While nitrogen-containing
heterocycles gave potent analogues (20, 24) so did benzylamines
substituted at the ortho, meta, and para positions (25–27). Ulti-
mately, the most potent compounds in the cell assay remained
the isomeric tetrahydroisoquinoline analogues (28, 29) while the
isoindoline decreased in potency with the addition of fluoride
(30). We also obtained selectivity data for 26. In a broad multikin-
ase binding screen, only 8 of 359 kinases screened were inhibited
greater than 50% in the presence of 1 l
M of 26.14
We also explored the effect changes in region B had upon activ-
ity (Table 4). While we learned that the 3-pyridyl ring was critical

4804
M. O. Duffey et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4800–4804
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.;
As significant potency gains had been made in the chemical ser-
Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.;
Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.;
Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949.
4. Hingorani, S. R.; Jacobetz, M. A.; Robertson, G. P.; Herlyn, M.; Tuveson, D. A.
Cancer Res. 2003, 63, 5198.
5. Flaherty, K.; Puzanov, I.; Sosman, J.; Kim, K.; Ribas, A.; McArthur, G.; Lee, R. J.;
Grippo, J. F.; Nolop, K.; Chapman, P. J. Clin. Oncol. 2009, 27, 15s.
6. (a) Raf activity was determined using a 384 well streptavidin coated flashplate.
Reactions were performed with biotinylated peptide substrate at 30 °C for 3 h
ies, an evaluation of the pharmacokinetic properties of the series
became a priority (Table 6). While insolubility of 2 prevented us
from formulating it for IV dosing, we were pleased to find that
modification of the scaffold improved solubility sufficiently to al-
low most compounds to be formulated for IV dosing. When dosing
in rats (IV and PO)15 in general, non-fluorinated analogues suffered
from high clearance or low bioavailability, or both. This, in turn, led
to low plasma exposures (17, 12). Fortuitously, the addition of fluo-
ride to the phenol ring led to compounds that possess higher bio-
availability and lower rates of clearance from plasma than their
non-fluorinated counterparts. This resulted in a marked increase
in plasma exposures (30, 25). In particular, the tetrahydroisoquin-
oline analogue should be highlighted (39). It is an example of a
fluorinated analogue that possesses low clearance and high bio-
availability resulting in the highest plasma exposure levels we ob-
served in this study. In contrast, region B analogues all showed
poor pk properties, with or without incorporation of the C-1 fluo-
rophenol (data not shown).
in a final volume of 30 lL. After addition of stop the reaction was incubated on
a flashplate for 2 h. The flashplates were read on a Topcount counter.; (b) The
whole cell ELISA assay utilized a human melanoma cell line (A375) possessing
the mutation BRaf V600E. A375 cells seeded overnight were incubated with Raf
inhibitors for 3 h. At the end of the incubation, the cells were fixed,
permeabilized, blocking buffer added and the plates were incubated
overnight. After the blocking buffer was discarded, the plates were incubated
with anti-phospho-ERK antibody for 1 h followed by treatment with anti-horse
radish peroxidase. Optical density was read at 650 nm for
tetramethylbenzidine as substrate.
7. Blackburn, C.; Duffey, M. O.; Gould, A. E.; Kulkarni, B.; Liu, J. X.; Menon, S.;
Nagayoshi, M.; Vos, T. J.; Williams, J. Bioorg. Med. Chem. Lett. 2010, 20, 4795.
8. Wan, P. T. C.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V.
M.Cancer Genome Project, Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford,
D.; Marais, R. Cell 2004, 116, 855.
9. We initially used
a homology model because no DFG-in structures were
We have discovered a series of novel inhibitors of mutant B-Raf
(V600E). The modular structure of the pyrazoline scaffold lent itself
well to analogue synthesis and we synthesized compounds that are
very potent in vitro. In particular, the discovery of the addition of
the C-1 fluoride to the phenol provided a significant increase in cell
potency. Furthermore, we have also identified fluorinated com-
pounds that possess outstanding pharmacokinetic properties and
in depth examination of these compounds in vivo activities is
warranted.
available. When these structures became available, they were found to be
nearly identical to the homology model. Initial attempts to dock 2 in the DFG-
out conformation proved to be unsatisfactory.
10. Annigeri, A. C.; Siddappa, S. Indian J. Chem. 1963, 1, 484.
11. Lange, J.; Coolen, H.; van Stuivenberg, H.; Dijksman, J.; Herremans, A.; Ronken,
E.; Keizer, H.; Tipker, K.; McCreary, A.; Veerman, W.; Wals, H.; Stork, B.;
Verveer, P.; den Hartog, A.; de Jong, N.; Adolfs, T.; Hoogendoorn, J.; Kruse, C. J.
Med. Chem. 2004, 47, 627.
12. Compounds were synthesized as racemic mixtures. The enantiomers of 2 were
separated via chiral HPLC revealing one enantiomer to be active (pERK IC50
280 nM) and the other to be inactive (pERK IC50: 14 M). Due to ease of
:
l
synthesis and time consuming methods required for HPLC separation we
evaluated all compounds as racemic mixtures.
13. Solubility was determined by a turbidimetric solubility assay which provides
the kinetic solubility of compounds in 50 mM potassium phosphate buffer at
pH 6.8.
References and notes
1. Robinson, M. J.; Cobb, M. H. Curr. Opin. Cell Biol. 1997, 9, 180.
2. (a) Heidorn, S. J.; Milagre, C.; Whittaker, S.; Nourry, A.; Niculesu-Davas, I.;
Dhomen, N.; Hussain, J.; Reis-Filho, J. S.; Springer, C. J.; Pritchard, C.; Marais, R.
Cell 2010, 140, 209; (b) Poulikakos, P. I.; Zhang, C.; Bollag, G.; Shokat, K. M.;
Rosen, N. Nature 2010, 464, 427; (c) Hatzivassiliou, G.; Song, K.; Yen, I.;
Brandhuber, B. J.; Anderson, D. J.; Alvarado, R.; Ludlam, M. J. C.; Stokoe, D.;
Gloor, S. L.; Vigers, G.; Morales, T.; Aliagas, I.; Liu, B.; Sideris, S.; Hoeflich, K. P.;
Jaiswal, B. S.; Seshagiri, S.; Koeppen, H.; Belvin, M.; Friedman, L. S.; Malek, S.
Nature 2010, 464, 431.
3. Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.;
Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.;
Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.;
Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.;
Busterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
14. Developed and marketed by Ambit Biosciences, KINOMEscan™ is
a
a
competition binding assay that quantitatively measures the ability of
compound to compete with an immobilized, active-site directed ligand (see:
http://www.kinomescan.com). Kinases inhibited >50% included DDR1(54%),
DDR2(60%), EGFR(L747-S752del P753S)(58%), EGFR(S752-I759del)(55%),
EPHB3(65%), KIT(D816 V)(95%), LCK(66%), and RET(M918T)(70%). B-Raf
(V600E) and WT B-Raf were both inhibited at 100%. IC50 data was not
generated in the Ambit assay. In-house enzyme assays revealed the related
compound 2 to be equipotent against B-Raf (V600E) (IC50: 23 nM), WT B-Raf
(IC50: 17 nM), and C-Raf (IC50: 27 nM).
15. Pharmacokinetic experiments were performed by dosing either intravenously
(0.5 mg/kg or 1 mg/kg) or orally (10 mg/kg) in male SD rats.