Discovery and optimization of N-acyl and N-aroylpyrazolines as B-Raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.06.110

A high throughput screen identified N-aroylpyrazoline 1 as a selective inhibitor of the V600E mutant of B-Raf kinase. Parallel synthesis of acyl, aroyl, and sulfonyl derivatives led to the identification of several potent inhibitors in both enzymatic and cellular (pERK) assays such as compound 42.

Full text of DOI:10.1016/j.bmcl.2010.06.110

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4795–4799
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of N-acyl and N-aroylpyrazolines as B-Raf
kinase inhibitors
*
Christopher Blackburn , Matthew O. Duffey, Alexandra E. Gould, Bheemashankar Kulkarni, Jane X. Liu,
Saurabh Menon, Masayuki Nagayoshi, Tricia J. Vos, Juliet Williams
Millennium Pharmaceuticals Inc., 40 Landsdowne St., Cambridge MA 01239, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A high throughput screen identified N-aroylpyrazoline 1 as a selective inhibitor of the V600E mutant of B-
Raf kinase. Parallel synthesis of acyl, aroyl, and sulfonyl derivatives led to the identification of several
potent inhibitors in both enzymatic and cellular (pERK) assays such as compound 42.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 11 May 2010
Revised 17 June 2010
Accepted 21 June 2010
Available online 25 June 2010
Keywords:
B-Raf inhibitor
Pyrazoline
Parallel synthesis
Raf, a serine/threonine protein kinase that is part of the Ras-Raf-
MEK-ERK signal transduction pathway1 regulating cellular growth
and proliferation is frequently mutated in many types of cancer.2
The most common activating mutation is a valine substitution by
glutamic acid at amino acid position V600E in the isoform B-Raf
resulting in the constitutive activation of the MAP kinase pathway
and uncontrolled proliferation of tumor cells. Conversely, suppres-
sion of B-Raf (V600E) in human melanoma cells leads to down-reg-
ulation of the MAP kinase signaling pathway and apoptosis.3
Pharmacological inhibition of the B-Raf mutant in melanoma pa-
tients by administration of a selective B-Raf inhibitor (PLX-4032)
also showed evidence of antitumor activity.4 Thus, there has been
considerable recent interest in developing small molecule inhibi-
tors of mutated B-Raf as therapeutic agents for melanoma and a
number of other cancers.5 Of particular note are sorafenib6 and a
triarylimidazole derivative.7 As part of our program to identify
selective inhibitors of B-Raf, we configured an HTS to assess the
effect of our screening compounds on the kinase activity of the mu-
tant V600E form of the protein.8 The HTS identified an N-aroylpy-
razoline inhibitor (1) of B-Raf (V600E), with an IC50 value of
200 nM (Fig. 1). The novelty of the pyrazoline scaffold, which has
not previously been associated with kinase inhibition, coupled
with the selectivity profile of compound 19 prompted us to use
automated parallel synthesis to prepare hundreds of analogs as
compound libraries. In this Letter we report the optimization of 1
to give several analogs with single-digit nM IC50 values and inhib-
itory activities in cells approaching 100 nM. In the accompanying
article10 we report on the further optimization of this series for po-
tency, physicochemical and PK properties.
Since there were few examples of compounds related to 1 in our
screening library, we prepared a diverse set of substitutions using
automated solution-phase synthesis initially investigating acyla-
tions and aroylations as shown in Scheme 1. Pyrazolines 2 were
prepared by condensation of the appropriate chalcone with hydra-
zine11,12 and isomer 2a subjected to a series of diimide-mediated
automated parallel acylations and aroylations.13 Focusing initially
on analogs derived from a diverse set of carboxylic acids, our initial
* Corresponding author. Tel.: +1 617 761 6811; fax: +1 617 551 8907.
E-mail address: blackburn@mpi.com (C. Blackburn).
Figure 1. Optimization of a high throughput screening hit for inhibitory activity of
V600E BRaf.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.110

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C. Blackburn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4795–4799
Scheme 1. Reagents and conditions: (i) hydrazine hydrate, EtOH, 80 °C, 3 h; (ii) RCOOH, EDCI, DCM, DMF.
Table 1
B-Raf inhibitory activity of selected compounds from a diverse screening library of N-acyl and N-aroylpyrazolines
R
N
O
N
OH
N
R
V600E IC50 (nM)
pERK IC50 (nM)
R
V600E IC50 (nM)
110 11
pERK IC50 (nM)
9500 1000
CF3
3
4
83
8
8000 2200
12
13
F
N
F
>10,000
>10,000
NDa
260
42
5
>10,000
S
N
N
N
5
6
ND
ND
14
15
6
8
8700 1000
F
>10,000
28
>10,000
N
N
O
N
Ph
NO2
O
O
Ph
7
8
156 10
>10,000
ND
ND
16
17
130
2
ND
O
Br
7.9 0.9
1400 650
CF3
F
F
EtO
9
>10,000
ND
18
25.7 1.7
3500 1000
O
CF3
F
HN
CF3
10
11
660 20
200 31
ND
19
20
22.6 3.0
24.2 1.0
2000 600
1500 500
CF3
S
F
3800 900
a
Not determined.
library of 184 compounds included aliphatic, alicyclic, substituted
benzoic, heteroaromatic, biaryl, and bicyclic examples. Compounds
were tested for their ability to inhibit the phosphorylation of a bio-
tinylated peptide by B-Raf V600E.8 Inhibition of cellular activity

C. Blackburn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4795–4799
4797
Table 2
Optimization of B-Raf inhibitory activity of N-aroyl pyrazolines
R
N
N
OH
N
R
V600E IC50 (nM)
16.0 3.0
pERK IC50 (nM)
1700 150
R
V600E IC50 (nM)
6.5 0.15
pERK IC50 (nM)
Ph
N
N
O
O
O
21
22
33
34
710 100
S
O
N
Ph
HN
O
58.0 4.0
2400 15
41.5 11.5
1100 200
O
OMe
O
O
23
24
18.0 1.0
3700 30
35
36
10.7 3.4
240 40
O
O
O
N
N
O
O
N
H
N
Ph
O
S
8.0 3.0
1100 100
10.3 0.6
410 200
N
H
O
N
O
O
Ph
O
O
25
26
27
8.0 2.0
5.6 0.3
6.7 1.3
810 300
970 300
930 400
37
38
39
7.0 0.1
9.1 0.7
6.9 0.7
350 100
61 25
N
N
N
H
O
O
N
N
O
O
N
S
O
S
920 175
OMe
O
O
N
S
O
OMe
28
15.4 5.6
460 180
600 250
40
216 55
NDa
ND
Cl
Cl
O
O
N
N
O
O
O
O
29
30
8.6 1.6
41
42
140
9
O
S
S
O
N
14.3 1.6
130
5
18.7 7.1
180 40
HO
(continued on next page)

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C. Blackburn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4795–4799
Table 2 (continued)
R
V600E IC50 (nM)
37.9 4.2
pERK IC50 (nM)
1300 40
R
V600E IC50 (nM)
2000 50
pERK IC50 (nM)
>10,000
N
N
Ph
O
S
O
O
31
32
43
44
S
S
O
NC
N
8.1 2.8
940 110
450 15
ND
S
S
N
O
O
a
Not determined.
was assessed by determining the decrease in phosphorylation of
the Raf kinase substrate pERK in A375 cells.14
compounds with inhibitory activity in cells approaching 100 nM.
Although phenyl-thiazole 31 showed modest enzyme and cellular
potencies, incremental improvements resulted from incorporation
of pyridyl nitrogens (compounds 32 and 33). Phenylpyrrole 34
proved to be slightly superior to furan 21 in cells even though
the enzymatic activity was lower. Several potent pyrazoles were
identified such as 35 and 36, the more active with the fused diox-
ane ring system and fusion of an alicyclic ring onto the pyrazole
was also beneficial as in compound 37. A tricyclic compound 38
was also identified that showed the highest cellular potency
among this series of library compounds. In the thiophene series,
a fused analog 39 with promising properties was identified but iso-
meric chlorophenyl derivatives 40 and 41 were not very active.
Pyridyl derivative 42, however, showed significant improvements
compared to the corresponding compound 13 with the pyridyl
and thiophene rings transposed.
To assess the effect of the orientation of the hydroxyl group on
B-Raf inhibitory activity, the meta hydroxyl pyrazoline 2b was con-
verted to a series of acyl and aroyl derivatives. Of the approxi-
mately 100 compounds prepared, the only compound to exhibit
any significant enzymatic activity was the analog of lead com-
pound 42, which was some 35-fold less active in the enzymatic as-
say with no cellular activity.10 Lastly we prepared a library of
sulfonyl derivatives by reaction of 2a with one hundred sulfonyl
chlorides in pyridine solution. The inputs included aliphatic, ben-
zylic, and arylsulfonyl halides with a diverse range of substituents
including heteroaryl derivatives yet most of the compounds
showed no significant B-Raf V600E inhibitory activity. Sulfonyl
analog 43 of the lead compound 42 from the carboxamide series
Over half the compounds from the first library exhibited IC50
values <1 l
M in the enzymatic assay for B-Raf V600E activity;15
general SAR trends are illustrated by the compounds listed in Table
1. Small acyl groups such as cyclopropyl were tolerated, (com-
pound 3), but these derivatives showed little cellular activity. In
the case of analogs derived from aryl carboxylic acids, a wide range
of activity was observed largely determined by conformational ef-
fects. Thus, while numerous active compounds were identified, 1-
naphthyl derivative 4 and related quinolines (not shown) were
found to be inactive. Similarly, compounds 5 and 6 with large
ortho-substituents, showed no inhibitory activity of B-Raf V600E,
while aryl ether 7, with the phenoxy group meta to the carbonyl,
showed improved enzyme inhibitory activity in comparison to
the original hit 1. The potencies of five isomeric fluoro-trifluoro-
methyl benzoic acid derivatives (8–12) that were represented in
the library further illustrate this steric effect. While compounds 8
and 9 with substituents ortho to the carbonyl group were inactive,
the other three isomers showed sub-lM activity in the enzyme as-
say with the more potent compounds 11 and 12 having the larger
trifluoromethyl group in the meta (or 30)-position. Compounds 11
and 12 are comparable to 1 in enzyme inhibitory activity albeit
considerably less active in the cellular assay. Interestingly, com-
pounds 13 to 15 with larger substituents meta to the carbonyl, also
showed comparable or improved potency compared to the original
hit (1) but cellular activity was again negligible in each case.
Substituted furans and benzofurans also featured in the library.
Derivatives 16 to 18, for example, all had improved activities com-
pared to unsubstituted furan 1, bromo derivative 17 being partic-
ularly noteworthy with an IC50 in the enzyme assay of 8 nM
accompanied by the best cellular activity observed for members
of the first library. Pyrrole carboxylic acid derivative 19 and thio-
phene compound 20 were also found to be superior to 1 in enzyme
inhibitory activity without attendant improvements in cellular
activity. In summary, the first library identified a large number of
compounds with improved B-Raf V600E inhibitory activity, ten of
which are listed in Table 1. However, only two compounds, furan
17 and thiophene 20 showed minor improvements to cellular
activity compared to the original hit.
had an IC50 value of 2
lM in the enzymatic assay and showed no
cellular activity; only compound 44, had sub-lM activity (Table 2).
In conclusion, we have used automated parallel solution-phase
synthesis to prepare over four hundred pyrazoline derivatives that
were screened in enzyme and whole cell assays measuring the
inhibition of B-Raf V600E. We have discovered a series of novel
and selective16 inhibitors of mutant B-Raf (V600E) many of which
show low nM activity in the enzymatic assay. Of the three com-
pounds identified with promising cellular activities (30, 38, and
42), arylthiophene 42 was judged to be the most attractive ana-
logue from this series of chiral compounds17 for further optimiza-
tion. Docking into a homology model of the active conformation of
wild-type B-Raf suggests that the phenol of compound 42 forms an
intramolecular hydrogen bond to the carbonyl oxygen with the
nitrogen of the 3-pyridyl ring forming a hydrogen bond to the
backbone N–H of C532 in the hinge region. This binding mode
and optimization of the series for cellular activity and PK proper-
ties are described further in the accompanying Letter.10
We next focused our attention on improving cellular activity by
preparing targeted libraries encompassing a selection of five-mem-
bered heterocyclic carboxylic acids with additional ring substitu-
tions or fusions (Table 2). 5-Phenyl furan 21 showed 16 nM
enzymatic and 1.7 lM cellular activity but substitutions in the
phenyl ring (e.g., compounds 22 and 23) did not lead to further
improvements in cellular activity. Phenyl-substituted isoxazoles
24 and 25 also showed high inhibitory activities and several addi-
tional analogs (26–30) showed promising enzyme and cell poten-
cies with the 4-methoxy (28) or fused piperonyl (29) derivatives
having the best combinations of enzyme and cellular activities.
We also identified ortho-hydroxy derivative 30, one of the few
References and notes
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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.
3. Hingorani, S. R.; Jacobetz, M. A.; Robertson, G. P.; Herlyn, M.; Tuveson, D. A.
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activated by addition of a solution of EDCI (110
by a Tecan liquid handler. A solution of 2 (100
l
mol) in DMF (1 mL) delivered
mol) in a mixture of DMF
l
(0.5 mL) and dichloromethane (0.5 mL) was then added to each vessel and
agitated for 24 h. Each reaction mixture was then partitioned between
dichloromethane and water with the solvents dispensed and mixed by the
liquid handler. The organic phase from each extraction was collected and
evaporated from deep well plates to give the crude products that were purified
by preparative reverse phase HPLC using mass-directed fraction collection
conducted on an Agilent 1100 series LC/MSD instrument using a Waters
SunFire C18 5
lm Prep OBD column (19 ꢀ 150 mm). The compounds were
eluted with a water–MeCN gradient (0.1% formic acid) optimized by the
A2Prep Agilent software. All compounds described herein were further
characterized by 1H NMR spectroscopy and mass spectrometry.
7. King, A. J.; Patrick, D. R.; Batorsky, R. S.; Ho, M. L.; Do, H. T.; Zhang, S. Y.; Kumar,
R.; Rusnak, D. W.; Takle, A. K.; Wilson, D. M.; Hugger, E. Cancer Res. 2006, 66,
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14. Inhibition of Raf kinase activity in whole cells was assessed by determining the
decrease in phosphorylation of the Raf kinase substrate pERK. This whole cell
ELISA assay utilized
a human melanoma cell line (A375) possessing the
8. The Raf enzyme inhibition assay was conducted in a Flash PlateÒ format in
mutation B-Raf V600E. A375 cells seeded overnight were incubated with Raf
inhibitors for 3 h at 37 °C. 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 the substrate
tetramethybenzidine.
streptavidin coated 384 well plates monitoring the phosphorylation by 33P ATP
(0.5 lCi/reaction) of a biotinylated surrogate peptide at 4 lM concentration by
20 nM Raf mutant V600E at pH 7.5 buffered by 50 mM HEPES in the presence of
10 mM DTT and 50 mM MnCl2 at 30 °C. After 3 h the reaction was stopped by
addition of 100 mM EDTA and the reaction mixture transferred to a Flash
PlateÒ, incubated for 2 h and then read on a Topcount analyzer.
9. Pyrazoline 1 was found to have inhibitory IC50 values >10
l
M for a panel of
15. In general, inhibitory IC50 values for the wild-type (WT) enzyme B-Raf were
found to be comparable (within twofold) to those determined for the mutant
V600E. Compounds 20 (Table 1) and 42 (Table 2), for example, showed IC50
values of 17 nM and 18 nM, respectively, for inhibition of the WT enzyme.
16. Compounds 14, 17, 18, 19, and 20, for example, were found to have inhibitory
kinases that included CAMKII, CDK1, CDK2E, CHK1, CHK2, CKII, KDR2, FLT3,
CSF1R, KIT2, LCK2, PKA2, and FGFR1.
10. Duffey, M. O.; Adams, R.; Blackburn, C.; Chau, R.; Chen, S.; Galvin, K.; Garcia, K.;
Gould, A. E.; Greenspan, P.; Harrison, S.; Huang, S.-C.; Kim, M.-S.; Kulkarni, B.;
Langston, S.; Liu, J. X.; Ma, L.; Menon, S.; Nagayoshi, M.; Rowland, S.; Vos, T. J;
Xu, T.; Yang, J. J.; Yu, S.; Zhang, Q. Accompanying article.
IC50 values >10 lM for a panel of kinases that included KDR2, FLT3 and CSF1R,
KIT2, AKT2, and FGFR1.
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.
17. Separation of compound 42 into its component enantiomers could be effected
by chiral chromatography on
methanol, 33% water containing 0.1% TFA. The early eluting isomer showed
IC50 values of 1.2 and 14 M in the enzymatic and cellular assays, respectively.
a Chirobiotic V column eluting with 67%
l
12. Abdel-Latif, N. A.; Sabry, N. M.; Mohamed, A. M.; Abdulla, M. M. Monatsh. Chem.
2007, 138, 715.
The later eluting isomer was considerably more active (15 nM in the enzymatic
assay and 280 nM in the cellular assay).