Indazolylpyrazolopyrimidine as highly potent B-Raf inhibitors with in vivo activity

Article abstract of DOI:10.1021/jm1007566

Novel indazolylpyrazolo[1,5-a]pyrimidine analogues have been prepared and found to be extremely potent type I B-Raf inhibitors. The lead compound shows good selectivity against a panel of 60 kinases, possesses a desirable pharmacokinetic profile, and demonstrates excellent in vivo antitumor efficacy in B-Raf mutant xenograft models.

Full text of DOI:10.1021/jm1007566

7874 J. Med. Chem. 2010, 53, 7874–7878
DOI: 10.1021/jm1007566
Indazolylpyrazolopyrimidine as Highly Potent B-Raf Inhibitors with in Vivo Activity
Xiaolun Wang,*,†,‡ Dan M. Berger,† Edward J. Salaski,† Nancy Torres,† Minu Dutia,† Cilien Hanna,† Yongbo Hu,†
Jeremy I. Levin,† Dennis Powell,† Donald Wojciechowicz,† Karen Collins,† Eileen Frommer,† and Judy Lucas†
†Pfizer Global Research and Development, 401 N. Middletown Road, Pearl River, New York 10965, United States , and
‡Pfizer Global Research and Development, 200 Cambridgepark Drive, Cambridge, Massachusetts 02140, United States
Received June 21, 2010
Novel indazolylpyrazolo[1,5-a]pyrimidine analogues have been prepared and found to be extremely
potent type I B-Raf inhibitors. The lead compound shows good selectivity against a panel of 60 kinases,
possesses a desirable pharmacokinetic profile, and demonstrates excellent in vivo antitumor efficacy in
B-Raf mutant xenograft models.
Introduction
headpiece was moved to the ortho position, the resulting 8o
still retained a moderate activity against B-Raf, while the
m-diazabicycloheptyl 8p showed a significant loss of potencies.
Mono-/polyfluorination on the various positions of phenyl
linker (8b, g, j-n) was beneficial for enzyme and cell potencies
except the meta fluorinated analogue (8g,m). An increase of
potency was observed when a fluorine atom is introduced in
the 7 position of the indazole ring (8q-t), which is consis-
tent with the SAR of the phenol series.4 It was found that
8l possessing two ortho fluorine atoms stood out with IC50
of 0.16 and 24 nM against B-Raf and A375 cell line,
respectively.
A binding model of 8l with active conformation B-Raf was
constructed (Figure 2).5 Compound 8l is well accommodated
in the ATP-binding pocket, and a key hydrogen bond is
formed between the pyridyl nitrogen atom and Cys532 in the
hinge range. The indazole moiety behaves as a hydrogen bond
donor to Glu501 and a hydrogen bond acceptor from Asp594,
mimicking well the phenol binding mode.6 One fluorine atom
occupies a small hydrophobic pocket defined by Ile463,
Gly464, and Val471, which explains the potency enhancement
brought by a small substituent ortho to the pyrazolopyrimi-
dine core. The second fluorine atom does not appear to have a
corresponding lipophilic pocket but may help to avoid en-
tropy loss upon complexation. The diazabicyclo[2.2.1]heptyl
group is partially solvent exposed. Therefore, variation in the
heterobicyclic moiety might be a viable approach for better
physicochemical properties. Another binding model was also
built for 8o with an ortho A1 substituent (Figure 3). The
phenyl linker of8o is moved to the bottom of the pocket, and a
hydrogen bond between the protonated aliphatic amine and
Ser465 in the G-loop is recognized. This new binding mode
may be relevant tothe single digit B-Raf IC50 of 8o, though the
protein has to make some movement to accommodate the
bulky ortho substituent.
The Ras-Raf-MEKa-ERK signal transduction pathway is
critical for cell survival, growth and proliferation.1 One of
the key components in this cascade, B-Raf, when mutated
(B-RafV600E) plays an important role in the development of
cancer.2 Thus, inhibition of mutant B-Raf offers a viable
means for treating cancer.3 Recently we disclosed a series
of type I pyrazolopyrimidine B-Raf inhibitors4 including 1a
(Figure1) having an indazole asphenolreplacement. While1a
exhibits much better microsomal stability than the corre-
sponding phenol analogue 1b, its cell potency against A375
bearing B-RafV600E is moderate. Therefore, further optimiza-
tion for the indazolylpyrazolopyrimidine series was initiated,
which eventually led to the discovery of compounds with
excellent antitumor efficacy in vivo against tumors driven by
the mutant B-Raf.
Results and Discussion
We started our optimization by exploring the influence of
substitutions to the phenyl linker. The synthesis of indazolyl-
pyrazolopyrimidine analogues (Table 1) is exemplified with
the preparation of 8l (Scheme 1). Aromatic nucleophilic
substitution of fluoroacetophenone 4l with commercially
available (1S,4S)-tert-butyl-2,5-diazabicyclo[2.2.1]heptane-
2-carboxylate followed by reaction with DMA-DMF afforded
enaminone 6l, which was then annulated with aminopyrazole
34 to give 7l. Deprotection of intermediate 7l followed by a
reductive amination with formaldehyde provided the final
product 8l. As reported previously,4 the introduction of a
small ortho group (R1) resulted in a significant B-Raf potency
enhancement (8a-d) while compounds with large ortho groups
such as trifluoromethyl (8e) and dimethylamino (8f) were
deleterious. Substitutions on the meta position (8g,h) or a large
naphthyl linker (8i) provided no advantage over the unsubsti-
tuted analogue. Interestingly when the diazabicycloheptyl
From our early work,4 we found that the diazabicyclo-
[2.2.1]heptyl headpiece is superior to other diazabicyclic
groups tested. To further investigate the headpiece SAR
especially for the cell activity, we examined several new sub-
stituents (Table 2). Compounds 15a and 15b were prepared by
a similar route used for lead 1a.4 The preparation (Scheme 2)
*To whom correspondence should be addressed. Phone: 617-665-5212.
Fax: 617-665-5682. E-mail: xxw104@gmail.com.
a Abbreviations: MAPK, mitogen-activated protein kinase; ERK,
extracellular signal-regulated kinase; MEK, mitogen-activated protein
kinase; SAR, structure-activity relationship.
r
pubs.acs.org/jmc
Published on Web 10/20/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7875
Scheme 1a
Figure 1. Indazolylpyrazolopyrimidine lead 1.
Table 1. Phenyl Linker Modifications of Indazolylpyrazolopyrimidines
B-Raf IC50 A375 cell
(nM)
compd
R1
Me
R2 R3 R4 R5 R6
IC50 (μM)
8a
8b
8c
8d
8e
8f
A1
A1
A1
A1
A1
A1
0.51
0.137
0.044
0.039
nd
F
Cl
0.23
<0.10
<0.32
1.4
Br
CF3
NMe2
nd
1.851
nd
7.3
1.9
8g
8h
8i
F A1
CF3 A1
2.0
1.0
0.273
25
a (a) 2, K2CO3, HMPA, 62%; (b) DMF-DMA, reflux, 90%; (c) TFA,
methanol, 87%; (d) 6 N HCl, 92%; (e) HCHO, NaBH(OAc)3, DMF, 85%.
Naphthyl
A1
A1
A1
A1
A1
A1
F
8j
F
F
F
F
0.61
0.38
0.16
9.6
0.107
0.087
0.024
1.253
0.070
0.250
1.397
0.068
0.188
0.033
0.017
8k
8l
F
F
F
8m
8n
8o
8p
8q
8r
8s
8t
F
F
F
F
F
A1
0.35
1.4
A1
61
A1
A1
A1
A1
F
F
F
F
0.50
<0.32
<0.1
0.11
Me
F
F
F
of analogues 15c-g started with an annulation between
aminopyrazole 3 and diethyl ethoxymethylenemalonate (9)
followed by a saponification to give carboxylic acid 12. De-
carboxylation and then chlorination of intermediate 12 gave
chloropyrazolopyrimidine 13. Suzuki coupling of intermediate
13 with boronic acid 10 followed by a reductive amination
provided the final products (15c-g). Compounds 15a and 15b
with a two-carbon bridge exhibited similar enzyme activity as
the corresponding diazabicyclo[2.2.1]heptyl analogues but
were less potent in the cell assay. Other heterobicyclic head-
pieces (15c-g) were also detrimental to the cell potency.
The kinase selectivity profile of 8l was evaluated against a
panel of 60 protein kinases.7 Only 10 kinases including B-Raf
and C-Raf showed higher than 70% activity inhibition at
Figure 2. Docked model of compound 8l in the active site of B-Raf.
1 μM 8l. Compound 8l exhibits greater than 340-fold selectivity
for non-Raf kinases, and the selectivity for C-Raf (IC50 =
8.5 nM) is 30-fold. The high selectivity of 8l minimizes the
possibility of any undesired off-target effects.

7876 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Wang et al.
Scheme 2a
Figure 3. Docked model of compound 8o in the active site of B-Raf
(green line represents 8l).
Table 2. Headpiece Modifications of Indazolylpyrazolopyrimidines
B-Raf
IC50 (nM)
A375 cell
IC50 (μM)
compd
R1
F
R3
15a
15b
15c
15d
15e
15f
15g
A2
A2
A3
A3
A4
A5
A6
0.71
0.41
10
0.185
0.118
0.945
0.268
0.078
9.475
0.304
a Reagents and conditions: (a) 9, HOAc, reflux, 55%; (b) 2.5 N NaOH,
EtOH, reflux, 4.5 h, 85%; (c) Dowtherm, 245 °C, 90%; (d) POCl3, N,N-
diethylaniline, 100°C; (e) 10, cat. PdCl2(dppf), Na2CO3, DME, microwave,
110 °C, 1 h, 43-50%; (f) RaRbNH, NaBH(OAc)3, HOAc, DMF, room
temp, 16 h, 27-58%.
F
F
1.2
0.52
2.3
F
0.77
In an early metabolite identification study with 8b, signifi-
cant amounts of desmethylation (16a, B-Raf kinase IC50
e
0.32 nM; A375 IC50 = 0.117 μM) and N-oxidation (17a,
B-Raf kinase IC50 = 0.59 nM;A375IC50 = 2.153 μM), on the
alkylamino nitrogen were observed (see Figure 4). Therefore,
a survey of the N-alkyl group was performed to test if bulky
substituents can block these metabolic pathways. Surprisingly
analogues (18a-d) with large alkyl groups are more labile
than the parent methyl compound (Table 3), though all of
them showed excellent enzyme activity consistent with what
the model predicts. Nevertheless, it was found that 8l with two
ortho fluorine atoms was quite stable in human and nude
mouse microsomes and gave very small amounts of these
Figure 4. Metabolites from 8b and 8l.
metabolites (16b, B-Raf kinase IC50 e 0.32 nM; A375 IC50
0.020 μM; 17b, B-Raf kinase IC50 = 17 nM; A375 IC50
0.229 μM).
=
=
Moreover, it was also found that 8l stays in tumor tissue for
a long period. The postdose (po 25 mg/kg) tumor concentra-
tions of 8l8 were 6.6 and 6.1 μM after 6 and 24 h, respectively,
while the plasma concentration of 8l was 0.06 μM after 24 h. A
pharmacodynamic study in A375 xenograft mouse model
indicated complete ERK phosphorylation inhibition even
after 24 h (iv, 50 mg/kg, data not shown). Repeat oral dosing
in the A375 mouse resulted in excellent tumor growth inhibi-
tion and good suppression of ERK phosphorylation in tumor
tissue at 10 mg/kg q.d. (Figure 6). Even at a dose of 7.5 mg/kg
q.d. a 39% tumor growth inhibition (TGI) was observed
A biomarker study showed that 1b, 8b, and 8l significantly
decrease the phosphorylation of downstream ERK kinase in
cell culture (Figure 5), which correlated well with the inhibi-
tion ofcellular proliferation. This result strongly supports that
the antitumor activity of these compounds comes from the
inhibition of the Raf signaling pathway.
Compound 8l also possesses a favorable pharmacokinetic
profile in nude mice. It has moderate half-life (3.5 h) and
clearance (23 (mL/min)/kg) and good exposure (AUCinf =
4000 h ng/mL, po 10 mg/kg) and bioavailability (54%).
3

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7877
Table 3. N-Alkyl Variation of Indazolylpyrazolopyrimidine Analogues
B-Raf
A375
t1/2 (min)
t1/2 (min)
compd
R7
IC50 (nM) IC50 (μM) (human) (nude mice)
8b
8l
0.23
0.16
0.044
0.024
0.056
0.055
0.089
0.078
17
>30
11
5
>30
>30
nd
17
Me
Et
18a
18b
18c
18d
<0.32
<0.32
<0.32
<0.32
i-Pr
i-Bu
3
4
cyclobutyl
3
5
Figure 6. A375 xenograft growth inhibition. Compound 8l was
orally dosed at 10 mg/kg once a day. Each arm had n = 10, and
tumors were measured with calipers at the designated times. Error
bars represent standard deviation, and the results are statistically
significant. A375 tumors were harvested from treated and non-
treated animals 6 h after an initial oral dose of 10 mg/kg 8l. Tumor
lysates were made and probed by immunoblotting for the presence
of pERK and total ERK.
decoupling. All reactions were carried out in air-dried glassware
under a nitrogen atmosphere unless otherwise noted. Unless
otherwise noted, reagents were obtained from commercial
sources and were used without further purification. All the final
products were >95% pure as determined by HPLC.
Figure 5. Biomarker (pERK) inhibition in A375 cell line.
7-(2,6-Difluoro-4-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]-
heptan-2-yl)phenyl)-3-(1H-indazol-4-yl)-2-(pyridin-4-yl)pyrazolo-
[1,5-r]pyrimidine (8l). Step 1. To a solution of 1-(2,4,6-trifluor-
ophenyl)ethanone (4l) (3.9 g, 22.5 mmol) in 30 mL of hexam-
ethylphosphoramide, (1S,4S)-tert-butyl 2,5-diazabicyclo[2.2.1]-
heptane-2-carboxylate (2) (3.0g,15mmol) andpotassiumcarbonate
(6.2 g, 45 mmol) were added. This solution was stirred at room
temperature for 4 days. The mixture was then diluted with 200 mL
of diethyl ether and was washed with 200 mL of water. The
aqueous solution was extracted twice with diethyl ether. The
combined organic layer was washed with water three times, then
dried over anhydrous sodium sulfate, and concentrated. The
residue was purified by silica gel chromatography (isopropanol,
hexanes) to give (1S,4S)-tert-butyl 5-(4-acetyl-3,5-difluorophenyl)-
2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (5l) (3.3 g, 62%)
(data not shown).9 Although 8l appeared well tolerated, we are
aware that this series of compounds can promote the MAPK
pathway in certain B-Raf wild type cell lines,10 leading to some
undesired consequences. This observation has been reported
for other B-Raf inhibitors,3a,c and an inhibitor induced dimer-
ization/activation mechanism11 has been proposed to explain it.
Conclusions
In summary, a novel series of selective B-Raf inhibitors is
disclosed. The potency of indazolylpyrazolopyrimidine ana-
logues was optimized through fine-tuning of the substituents
on the phenyl linker. The introduction of two ortho fluorine
atoms led to the identification of an extremely potent com-
pound 8l, which also demonstrated potent antitumor activity
in the B-Raf mutant xenograft mouse model.12
1
as an off-white solid. H NMR (400 MHz, CDCl3, mixture of
rotamers): δ 6.02 (d, J=12.4 Hz, 2H), 4.68 (br s, 0.54H), 4.54 (br
s, 0.46H), 4.37 (br s, 1H), 3.54-3.14 (m, 4H), 2.52 (t, J=2.8 Hz,
3H), 2.04-1.92 (m, 2H), 1.47 (br s, 4.1H), 1.43 (br s, 4.9H).
13C NMR (75 MHz, CDCl3): δ 193.1, 163.3 (dd, J = 252.0,
10.5 Hz), 154.0, 150.1 (t, J=14.7 Hz), 105.9 (t, J=15.6 Hz), 95.2
(d, J=29.2 Hz), 80.1, 57.9/57.4, 57.0/56.8, 56.5/56.1, 52.2/51.8,
37.7/37.2, 32.5 (t, J=3.9 Hz), 28.4. HRMS [(M þ H)þ] calcd for
C18H22F2N2O3 353.1670, found 353.1676.
Step 2. A mixture of 5l (3.3 g, 9.4 mmol) and 30 mL of 1,
1-dimethoxy-N,N-dimethylmethanamine was refluxed for 35 h. The
reaction mixture was concentrated and the residue was purified by
silica gel chromatography (isopropanol, dichloromethane) to give
(1S,4S)-tert-butyl 5-(4-((E)-3-(dimethylamino)acryloyl)-3,5-
difluorophenyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (6l)
Experimental Section
Chemistry. 1H NMR spectra were recorded at 400 MHz with
a Bruker DPX-400 spectrometer (unless otherwise noted) at
ambient temperature. 13C NMR spectra were recorded at 100
MHz (unless otherwise noted) at ambient temperature. Chemical
shifts were reported in parts per million relative to CDCl3
(1H, δ 7.24), CD3OD (1H, δ 3.31), or DMSO-d6 (1H, δ 2.50).
Data for 1H NMR are reported as follows: chemical shift,
integration, multiplicity (s = singlet, d = doublet, t = triplet,
q=quartet, m=multiplet, br=broad), and coupling constants.
All 13C NMR spectra were recorded with complete proton

7878 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Wang et al.
(3.5 g, 90%) as an oil. 1H NMR (400 MHz, CDCl3, mixture of
rotamers): δ 7.53 (br, 2H), 6.03 (d, J = 11.2 Hz, 2H), 4.64 (br s,
0.53H), 4.51 (br s, 0.47H), 4.32 (br s, 1H), 3.67-3.33 (m, 4H), 3.10
(s, 3H), 2.87 (s, 3H), 2.01-1.88 (m, 2H), 1.46 (br s, 4.2H), 1.43 (br s,
4.8H). MS [(M þ H)þ] calcd for C21H28F2N3O3 408.2, found 408.2.
Step 3. To a solution of 2,2,2-trifluoroacetic acid (0.54 mL) in
18 mL of methanol, 6l (1.1 g, 2.6 mmol) and 4-(1H-indazol-4-yl)-
3-(pyridin-4-yl)-1H-pyrazol-5-amine (3) (0.72 g, 2.6 mmol) were
added. This solution was stirred at room temperature for 7 days
(not optimized). The mixture was basified with methanolic
ammonia, taken up with silica gel, and purified by silica gel
chromatography (isopropanol, dichloromethane) to give
(1S,4S)-tert-butyl-5-(4-(3-(1H-indazol-4-yl)-2-(pyridin-4-yl)-
pyrazolo[1,5-R]pyrimidin-7-yl)-3,5-difluorophenyl)-2,5-diaza-
bicyclo[2.2.1]heptane-2-carboxylate (7l) (1.4 g, 87%) as a yellow
solid. 1H NMR (400 MHz, CDCl3, mixture of rotamers): δ 8.54
(d, J = 4.4 Hz, 1H), 8.46 (d, J = 6.0 Hz, 2H), 7.72, (s, 1H),
7.56-7.45 (m, 4H), 7.28 (d, J=2.4 Hz, 1H), 6.99 (d, J=4.0 Hz,
1H), 6.28 (d, J = 11.6 Hz, 2H), 4.73 (br s, 0.54H), 4.59 (br s,
0.46H), 4.45 (br s, 1H), 3.67-3.22 (m, 4H), 2.10-1.98 (m, 2H),
1.50 (br s, 4.2H), 1.46 (br s, 4.8H). MS [(M þ H)þ] calcd for
C34H30F2N8O2 621.3, found 621.3.
Step 4. A solution of 7l (1.4 g, 2.3 mmol) in 6 N HCl (19 mL of
concentrated HCl and 19 mL of methanol) was stirred for 1 h. The
mixture was concentrated, basified with methanolic ammonia,
taken up with silica gel, and purified by silica gel chromatography
(ammonia, methanol, dichloromethane) to give 7-(4-((1S,4S)-2,5-
diazabicyclo[2.2.1]heptan-2-yl)-2,6-difluorophenyl)-3-(1H-indazol-
4-yl)-2-(pyridin-4-yl)pyrazolo[1,5-R]pyrimidine (16b) (1.1 g, 92%)
as a yellow solid. 1H NMR (400 MHz, CD3OD): δ 8.55 (d, J=
4.4 Hz, 1H), 8.40-8.37 (m, 2H), 7.64-7.47 (m, 5H), 7.26 (dd,
J=7.2, 0.8 Hz, 1H), 7.17 (d, J=4.4 Hz, 1H), 6.48 (d, J=12.0 Hz,
2H), 4.61 (br s, 1H), 4.03 (br s, 1H), 3.65 (dd, J = 9.8, 2.2 1H),
3.32-3.23 (m, 1H), 3.17-3.09 (m, 2H), 2.09 (d, J= 9.6 Hz, 1H),
1.93 (d, J=10.0 Hz, 1H). MS [(M þ H)þ] calcd for C29H22F2N8
521.2, found 521.3.
Note Added after ASAP Publication. This paper was pub-
lished on the web on October 20, 2010 with an error in Figure 1.
The revised version was published on November 4, 2010.
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(8) VSS =4.7 L/kg.
(9) Another efficacy study with Colo205 model also showed great
tumor growth inhibition. See Supporting Information for details.
(10) Data are not shown and will be present elsewhere.
Step 5. To a solution of 16b (1.1 g, 2.1 mmol) in 30 mL of
DMF, formaldehyde (0.47 mL, 6.3 mmol) and sodium triace-
toxyhydroborate (1.3 g, 6.3 mmol) were added. This solution
was stirred at room temperature for 2 h and then concentrated
on rotavapor. The residue was stirred with 15 mL of 7 N meth-
anolic ammonia overnight. The solution was concentrated
and purified with silica gel chromatography (methanol, dic-
1
hloromethane) to give 8l (0.95 g, 85%) as a yellow solid. H
NMR (400 MHz, DMSO-d6): δ 13.21 (s, 1H), 8.63 (d, J =
4.4 Hz, 1H), 8.51-8.46 (m, 2H), 7.59 (d, J=8.4 Hz, 1H), 7.54
(s, 1H), 7.44 (dd, J=8.4, 6.8 Hz, 1H), 7.40-7.36 (m, 2H), 7.32
(d, J=4.4 Hz, 1H), 7.19 (d, J=7.2 Hz, 1H), 6.58 (d, J=7.2 Hz,
2H), 4.54 (s, 1H), 4.35 (s, 1H), 3.37-3.31 (m, 2H), 2.83 (dd, J=
9.2, 1.6 Hz, 1H), 2.56-2.50 (m, 1H), 2.32 (s, 3H), 1.92 (d, J=
9.6 Hz, 1H), 1.78 (d, J = 9.2 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6): δ 161.0 (dd, J= 243.9, 9.7 Hz), 150.1 (t, J =14.8
Hz), 149.8, 149.6, 149.5, 147.3, 140.10, 140.06, 137.1, 133.5,
126.0, 123.5, 122.4, 122.3, 122.2, 112.2, 109.4, 107.8, 94.8 (d,
J=27.2 Hz), 93.7 (t, J=20.2 Hz), 62.1, 59.4, 58.3, 52.4, 40.7,
35.2. HRMS [(M þ H)þ] calcd for C30H24F2N8 535.2165,
found 535.2158.
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Acknowledgment. We thank Drs. Tarek Mansour, Robert
Abraham, John Ellingboe, Derek Cole, Ariamala Gopalsamy,
and Suvit Thaisrivongs for their support of this work.
Supporting Information Available: Synthesis details for com-
pounds, spectroscopic data, assay protocols, and kinase selectivity
profile of 8l. This material is available free of charge via the
Internet at http://pubs.acs.org.
(12) Compound 8l is inefficacious in tumor cell lines with wild type
B-Raf.