Novel pyrazolopyrimidines as highly potent B-Raf inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.058

A novel series of pyrazolo[1,5-a]pyrimidines bearing a 3-hydroxyphenyl group at C(3) and substituted tropanes at C(7) have been identified as potent B-Raf inhibitors. Exploration of alternative functional groups as a replacement for the C(3) phenol demons

Full text of DOI:10.1016/j.bmcl.2009.10.058

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6957–6961
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel pyrazolopyrimidines as highly potent B-Raf inhibitors
a
a
a
a
Martin J. Di Grandi a, , Dan M. Berger , Darrin W. Hopper , Chunchun Zhang , Minu Dutia ,
Alejandro L. Dunnick a, Nancy Torres a, Jeremy I. Levin a, George Diamantidis a, Christoph W. Zapf a,
Jonathan D. Bloom a, YongBo Hu b, Dennis Powell a, Donald Wojciechowicz c,
Karen Collins c, Eileen Frommer c
*
a Chemical Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
b Structural Biology and Computational Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
c Discovery Oncology, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrazolo[1,5-a]pyrimidines bearing a 3-hydroxyphenyl group at C(3) and substituted
tropanes at C(7) have been identified as potent B-Raf inhibitors. Exploration of alternative functional
groups as a replacement for the C(3) phenol demonstrated indazole to be an effective isostere. Several
compounds possessing substituted indazole residues, such as 4e, 4p, and 4r, potently inhibited cell pro-
liferation at submicromolar concentrations in the A375 and WM266 cell lines, and the latter two com-
pounds also exhibited good therapeutic indices in cells.
Received 22 September 2009
Revised 9 October 2009
Accepted 13 October 2009
Available online 28 October 2009
Keywords:
B-Raf
Ó 2009 Elsevier Ltd. All rights reserved.
Pyrazolopyrimidines
B-Raf inhibitors
Indazole
Phenol isostere
The Ras-MAP kinase pathway has been implicated in tumor
progression for a variety of human cancers. The Raf kinases, which
are components of this cascade, are serine/threonine kinases that
activate Mek1/2. Mutant B-Raf containing a V600E substitution
causes aberrant constitutive activation of this pathway and has
high occurrence in several human cancers.1 As such, chemothera-
peutic inhibition of mutant B-Raf offers a viable means for treating
cancer.2 Recently we disclosed the preliminary characterization of
pyrazolo[1,5-a]pyrimidine-3-carboxylates ( Fig. 1; 1) as B-Raf
inhibitors.3 Further enhancements to this class of compounds (2)
were able to boost the in vitro potency of these non-hinge region
binders but modest cellular potency remained an issue.4 Explora-
tion of substituents at the C(2) position provided analogs that
could form a hydrogen bond to the hinge region of B-Raf.5 In an
effort to further improve the potency and physical properties of
these compounds,4 we next explored C(2), C(3) disubstituted
pyrazolopyrimidines with smaller C(7) substituents to reduce
molecular weight. In this paper we describe a novel series of pyr-
azolopyrimidines incorporating a C(7) tropane moiety (Fig. 1; 3
and 4) to afford structurally unique compounds with improved po-
tency. Further modification around the core, based on the more
highly substituted 5, led to the preparation of fused tropanone ana-
logs 6. An important distinction to be noted is that while com-
pounds of series 2 bind the inactive conformation of B-Raf,
compounds 3–6 comprise a structural class that targets its active
conformation.6
The chemistry to prepare these analogs is shown in Scheme 1.
Commercially available tropanone 7 was converted in three steps
to enaminone 8.7 Condensation of this reagent with aminopyrazole
9a8 in hot AcOH gave pyrazolopyrimidine 10a.9 Selective function-
alization with N-iodosuccinimide yielded C(3) iodide 10b, which
upon exposure to a variety of boronic acids or esters afforded the
arylated products 3 (Table 1; R1 = CO2C2H5; R2 = OCH3; R3 = vari-
able). In some instances, these carbamates were further modified
to produce anisoles or phenols bearing different R1 groups on the
tropane. For instance phenols 3c–e (Table 1) were prepared from
their corresponding anisoles by exposure to BBr3. Trimethylsilyl
iodide (TMSI) was used to remove the N-carboethoxy group from
3a and 3b to yield products 3f and 3g and subsequent demethyla-
tion with BBr3 gave secondary aminophenols 3h and 3i. N-Ethyl
compound 3j was synthesized by alkylating 3g with C2H5I in the
presence of K2CO3. From the appropriate starting materials, phe-
nols 3k–m were synthesized in like manner followed by deprotec-
tion of the methoxy group with BBr3. Finally, acetamide 3n and
sulfonamide 3o were prepared from 3b in three steps: boron tri-
bromide to deprotect the anisole residue, followed by TMSI medi-
ated N-carboethoxy group cleavage, and subsequent treatment
with acetyl chloride (in the presence of triethylamine as base) to
provide 3n, or methanesulfonyl chloride to provide 3o.
* Corresponding author.
E-mail address: digranm@pfizer.com (M.J. Di Grandi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.058

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M. J. Di Grandi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6957–6961
R
CH3
N
R1
N
H
N
H
N
CF3
CF3
N
N
O
O
N
X
7
N
Z
N
N
N
N
N
2
N
N
N
N
OH
3
N
X
CO2C2H5
R
Cl
3: X = Aryl, Z= H
4: X = Het, Z =H
5: X = 4-indazolyl, Z =CH3
6
1
2
Figure 1. The evolution of B-Raf inhibitors at Wyeth.
R1
N
d
N
N
N
N
X
Y
H2N
HN
4
N
O
OC2H5
O
OC2H5
N
N
N
9b
f
a, b, c
R1
N
CH3
O
O
OC2H5
O
N
CH3
d
N
N
7
8
H2N
HN
N
N
f
N
N
N
N
N
9a
N
N
R2
R
10a : R = H
10b : R = I
e
R3
3
Scheme 1. Reagents and conditions: (a) TosMic, KOtBu, À10 °C, 4 h; (b) MeMgBr, 5 °C, 3 h; (c) DMF-DMA, 100 °C, 3 d; (d) AcOH, 80–100 °C; (e) NIS, rt, overnight; (f)
Pd(PPh3)4, ArB(OR)2, aq Na2CO3, DME, 80 °C to 3; Pd(PPh3)4, HetB(OR)2, aq Na2CO3, DME, 80 °C to 4.
The final products 4 bearing C(3) heterocycles (Tables 2 and 3;
R1 = CO2C2H5; Y = heterocycles) were prepared by Suzuki coupling
of a heterocyclic boronic acid or ester with iodide 10b or by direct
condensation of 8 with the more elaborate aminopyrazoles 9b
(X = heterocycles).7,10 Subsequent reaction of these carbamates
afforded additional analogs. Thus, compound 4s (Table 3) was pre-
pared by TMSI induced deprotection of 4r. Similarly, reaction of 4e,
4p, 4t, or 4v with TMSI followed addition of the appropriate alkyl-
ating reagent in the presence of K2CO3 afforded compounds 4k,
4m, 4o, 4q, 4u, and 4w. Compound 4n was synthesized from the
free amine derived from 4e by reductive amination with acetone
in the presence of NaBH(OAc)3. Compounds 4g–j and 4l were
assembled by acylation of the free amine derived from 4e with
the appropriate reagent and 4f was derived from 4l by Boc depro-
tection with TFA.7
The preparation of the C(6) methyl analog 5 commenced with
the reaction of enaminone 11 with the aminopyrazole 9b (X = 4-
indazoyl) in hot AcOH to give the desired pyrazolopyrimidine
(Scheme 2). The fused analogs 6 (Table 4) required a slight varia-
tion of this scheme. Accordingly, tropanones 12a11 were treated
with dimethylformamide dimethylacetal to give the expected
enaminones 12b, which upon condensation with the fully substi-
tuted aminopyrazole 9b (X = 3-OH-4-Cl–C6H3) afforded the desired
fused compounds 6.
With in vitro IC50s <0.1 lM, all compounds presented in Table 1
are effective B-Raf inhibitors12,13 but typically the phenols were
17- to 87-fold more potent than their methyl ether counterparts
(compare 3a:3c; 3b:3d; 3f:3h; 3g:3i; 3j:3k). Additionally, consis-
tent with published data,6 incorporation of a chlorine atom ortho
to either the –OH or –OCH3 group generally increased in vitro po-
tency almost 10-fold (for instance, 3f:3g; 3h:3i), presumably due
to favorable hydrophobic interaction between this atom and the
binding pocket of the enzyme. Though chlorine could be replaced
with methyl (3d:3e) or fluorine (3k:3m) to give very potent ana-
logs, CN was far less active, apparently being too large (3k:3l).
With respect to the tropane moiety, a diverse set of substituents
on nitrogen was tolerated (3d, 3i, 3k, 3n, 3o) and with the excep-
tion of the surprisingly potent amine 3g (compared to analogs 3b,
3j), all tropane derivatives with comparable substitution on the
C(3) aryl ring were essentially equipotent.
A depiction of 3d docked into the DFG-in conformation of the
active site of B-Raf is shown in Figure 2. The phenolic OH group
forms two hydrogen bonds, one to the Glu501 residue, and one
to the NH group of Asp594. Additionally, the C(3) pyridine residue

M. J. Di Grandi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6957–6961
6959
Table 1
Table 2
In vitro and cell-based IC50 data for C(3) m-anisoles and phenols16
In vitro and cell-based IC50 data for phenol replacements
R1
N
O
OC2H5
N
N
N
N
N
N
N
N
N
X
R2
Number
X
B-Raf (
l
M) A375 (
l
M) WM266-4 (
lM)
R3
F
F
3p
0.302
0.151
nd
nd
Number
R1
R2
R3
B-Raf
M)
A375
M)
WM266-4
(lM)
(
l
(
l
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
H
H
H
H
C2H5
C2H5
C2H5
C2H5
COCH3
SO2CH3
OCH3
OCH3
OH
OH
OH
OCH3
OCH3
OH
OH
OCH3
OH
H
Cl
H
Cl
CH3
H
Cl
H
Cl
Cl
Cl
CN
F
0.087
0.0541
0.001
9
8
nda
nd
>10
0.24
0.12
0.23
8.4
3q
nd
nd
N
<0.002
<0.0003
0.0708
0.0085
0.004
0.0004
0.0435
0.0005
0.0579
0.003
nd
0.57
6.4
N
O
2.6
3.5
2.25
0.44
2.2
0.32
>10
0.76
0.9
0.22
0.09
5.6
0.23
>10
0.45
0.26
0.09
4a
4b
0.036
>1.0
7.5
nd
>10.0
nd
N
H
OH
OH
OH
OH
Cl
Cl
0.0003
<0.002
0.27
N
H
a
nd = not determined.
is hydrogen bonded to the hinge region Cys532.14 Hydrophobic
binding interactions of the tropane moiety and the chlorophenol
group with the enzyme appear to contribute significantly to the
overall binding affinity of this compound.
O
4c
>1.0
>1.0
nd
nd
nd
nd
N
H
H
N
Subsequent screening against the B-Raf mutant (V600E) human
melanoma cell lines A375 and WM26615 showed the anisoles were
4d
O
only weakly active in both (A375 IC50s >2.2
>3.5 M) and thus were dropped from further consideration. On
the other hand, the phenols had IC50s <1 M against both cell lines
lM; WM266 IC50s
N
H
l
l
(the A375 inhibitory activity of analog 3h is the sole exception).
Most noteworthy were compounds 3c, 3d, 3i, 3k, and 3o, which
displayed excellent potencies (IC50s <0.24 lM) in the WM266 cell
4e
0.002
0.38
0.3
N
N
H
line. While possessing exceptional inhibitory activity in both enzy-
matic and cell-based assays, the phenol moiety was of concern gi-
ven the known metabolic liabilities of this functional group.
Therefore, attention was focused on the development of an appro-
priate isostere.
Early attempts to replace the phenol focused on H-bond accep-
tor groups, such as compounds 3p and 3q (Table 2). However these
were substantially less active B-Raf inhibitors and therefore we
sought to incorporate functional groups that more closely resem-
bled the donor properties of a phenol. The data for indoles 4a
and 4b clearly indicated a preference by B-Raf for the 6-regioisom-
er and though 4a was not as potent as 3c, this was the first isostere
with good enzyme potency. Unfortunately, this activity did not
was reversed for the indazoles as the 4-indazole analog was at
least 10-fold more active in vitro and in cells (data not shown).
As shown in Figure 3, the C(3) indazole residue of 4e can form
two hydrogen bonds to Glu501 and Asp594 in a similar manner
to that proposed for the phenol of 3d in Figure 2.
Having identified the 4-indazoyl group as a novel replacement
for the C(3) phenol, further optimization was pursued (Table 3).
A series of C(3) indazoles (4f–o) was prepared to test the effect
of substitution on the tropane N on B-Raf potency. While tolerance
to a variety of functional groups at this position was similar to the
anisole/phenol series, there was a much greater range of IC50 val-
ues. For instance, equipotent compounds were observed in the ani-
sole/phenol series regardless of whether R1 was CO2C2H5 or C2H5
(vide supra). In the indazole series, the N-carboethoxy group was
characteristically 10–25 times more potent than the ethyl analog
(4e:4m; 4p:4q; 4t:4u; 4v:4w). Also, unlike the case in the phenol
series, methansulfonamide 4g and acetamide 4j were several fold
less active than the heterocyclic carboethoxy analog 4e. Other
nitrogen substituents such as proline amide 4f and ureas 4h, 4i
also afforded effective B-Raf inhibitors but there was a distinct
translate in the cell-based assays (IC50 >7 lM in both A375 and
WM266 cell lines). As these analogs were substantially less active
B-Raf inhibitors than phenol 3c, attention was drawn to a surrogate
that could function as both an H-bond donor and acceptor. Phenol
replacements 6-indolin-2-one 4c and 4-imidazolone 4d were sig-
nificantly less active. Fortunately, 4-indazole 4e was comparable
in potency to phenol 3c in the primary assay (IC50 = 0.002
0.001 M) and the cell based potency of the original phenol was
maintained (0.30 M in WM266 vs 0.24
M for 3c).17 Surprisingly,
the preference for the 6-regioisomer observed in the indole series
lM vs
l
l
l

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M. J. Di Grandi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6957–6961
Table 3
Table 4
In vitro and cell-based IC50 data for indazoles 4
In vitro and cell-based IC50 data for 5 and 6
R1
N
O
OC2H5
R
CH3
N
N
N
N
N
N
H3C
N
N
N
N
N
N
N
N
OH
N
N
N
Cl
N
H
X
H
5
6
Number
R1
X
B-Raf IC50
A375 IC50
WM266-4 IC50
M)
(
lM)
(lM)
(l
Number
R
B-Raf IC50
A375 IC50
M)
WM266-4 IC50
(lM)
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
CO2C2H5
H-Pro-CO
SO2CH3
CON(CH3)2
CONHC2H5
COCH3
CH2CH2OH
Boc-Pro-CO
C2H5
HC(CH3)2
CH2COCH3
CO2C2H5
C2H5
CO2C2H5
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
F
0.002
0.38
5.94
0.95
3.00
2.81
2.54
3.44
3.40
2.90
1.96
nd
0.58
1.09
0.62
nd
2.50
1.70
1.00
1.60
0.30
1.86
0.57
2.14
1.52
1.80
1.37
6.17
2.40
1.75
nd
0.28
0.86
0.30
nd
2.30
2.90
0.67
0.84
(l
M)
(l
0.0021
0.0049
0.0064
0.0067
0.0091
0.0228
0.0394
0.0466
0.0670
0.0759
0.0004
0.0044
0.0004
0.0038
0.0044
0.0450
0.0007
0.0170
5
—
0.0084
0.0016
0.0006
0.0008
1.22
2.30
1.70
1.00
1.87
1.10
0.75
0.60
6a
6b
6c
CO2CH3
CH2OH
SO2C6H5
Based on the anisole/phenol series precedent, a new set of func-
tionalized C(7) indazoles (4p–w) was prepared in the tropane ser-
ies.7 As expected, the 7-Cl (4p), 7-F (4r), and 7-CH3 (4v) ethyl
carbamates were sub-nanomolar enzyme inhibitors and each was
more potent than the parent 4e. Substitution with CF3 at C(7) of
the indazole also gave a very potent compound (4t) but it was sev-
eral fold less active than the other C(7) substituted indazoles
examined. In contrast to the potency of phenol 3k
F
CO2C2H5
C2H5
CO2C2H5
C2H5
CF3
CF3
CH3
CH3
(IC50 = 0.0005
(IC50 = 0.0044
l
l
M), the similarly substituted N-ethyl tropane 4q
M) was only modestly potent in the enzyme assay.
Based on the observed trend in the phenol series (vide supra) that
demonstrated analogs with in vitro IC50s <0.001 M possessed
submicromolar cellular potency, the observed efficacy of 4q in cells
(IC50s >1 M) was not surprising. For substituted indazoles, the
aforementioned trend with respect to A375 and WM266 activity
drop in potency for amide 4l and basic amines 4m–o. Nonetheless,
despite excellent in vitro potency, for this series of indazoles with
differentially substituted tropanes (4e–o), only compounds 4e and
l
l
4g were appreciably active in cells (IC50 <1 lM).
N
O
OC2H5
X
N
O
O
OC2H5
N
O
OC2H5
H2N
N
H
9b
N
N
O
H3C
N
N
a, b, c
N
d
C2H5
N
(X = 4-indazoyl)
N
CH3 C2H5
N
N
H
7
11
5
N
X
R
CH3
N
R
N
H2N
CH3
N
H
N
9b
N
N
N
O
d
N
(X = 3-OH-4-Cl-C6H3)
X
OH
Cl
12a : X = H2
12b : X = HCN(CH3)2
e
6
Scheme 2. Reagents and conditions: (a) TosMic, KOtBu, À10 °C, 4 h; (b) EtMgBr, 5 °C, 3 h; (c) diethylformamide dimethylacetal, reflux, 48 h; (d) AcOH, 80–100 °C; (e) DMF-
DMA, 100 °C, overnight.

M. J. Di Grandi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6957–6961
6961
detailed description of our efforts to further improve the potency
and properties of this series of B-Raf inhibitors will be reported
in due course.
Acknowledgments
We thank Drs. Tarek Mansour and Robert Abraham for their
support of this work. We also thank the members of the Wyeth
Chemical Technologies group for analytical and spectral
determination.
References and notes
1. Wan, P. T. C.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.;
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Martelli, A. M.; Lunghi, P.; Bonati, A.; Cervello, M.; Lee, J. T.; Steelman, L. S. Curr.
Opin. Invest. Drugs 2008, 9, 614.
Figure 2. Docking model of 3d bound to the active conformation of B-Raf.
3. Gopalsamy, A.; Ciszewski, G.; Hu, Y.; Lee, F.; Feldberg, L.; Frommer, E.; Kim, S.;
Collins, K.; Wojciechowicz, D.; Mallon, R. Bioorg. Med. Chem. Lett 2009. 19, 6519.
4. Berger, D.; Torres, N.; Dutia, M.; Powell, D.; Ciszewski, G.; Gopalamy, A.; Levin,
J. I.; Kim, K.-H.; Xu, W.; Wilhelm, J.; Hu, Y.; Collins, K.; Feldberg, L.; Kim, S.;
Frommer, E.; Wojciechowicz, D.; Mallon, R. Bioorg. Med. Chem. Lett. 2009, 19,
6519.
5. Shi, M.; Gopalsamy, A.; Berger, D. M.; Dutia, M.; Hu, Y.; Lee, F.; Feldberg, L.;
Frommer, E.; Kim, S.; Collins, K.; Wojciechowicz, D.; Mallon, R. Abstracts of
Papers, 235th ACS National Meeting, New Orleans, LA, United States, April 6–
10, 2008, MEDI-081.
6. Takle, A. K.; Brown, M. J. B.; Davies, S.; Dean, D. K.; Francis, G.; Gaiba, A.; Hird, A.
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Bioorg. Med. Chem. Lett. 2006, 16, 378.
7. For experimental details describing the preparation of all compounds
presented in this paper, see: (a) Levin, J. I.; Hopper, D. W.; Torres, N.; Dutia,
M. D.; Berger, D. M.; Wang, X.; Di Grandi, M. J.; Zhang, C.; Dunnick, A. L. PCT Int.
Appl. 2009, WO 2009108838; (b) Levin, J. I.; Diamantidis, G.; Bloom, J. D.; Zapf,
C. W. PCT Int. Appl. 2009, WO 2009108827.
8. Prepared in two steps from methyl isonicotinate: (1) KOtBu, CH3CN; (2)
H2NNH2; see Ref. 7a.
9. This reaction yielded a mixture of regioisomers (typically 4:1) of which 10a
was isolated as the major component.
10. For Het = 4-indazoyl, the aminopyrazole was prepared in several steps from
the known methyl 1H-indazole-4-carboxylate; see: Batt, D. G.; Petraitis, J. J.;
Houghton, G. C.; Modi, D. P.; Cain, G. A.; Corjay, M. H.; Mousa, S. A.; Bouchard,
P. J.; Forsythe, M. S.; Harlow, P. P.; Barbera, F. A.; Spitz, S. M.; Wexler, R. R.;
Jadhav, P. K. J. Med. Chem. 2000, 43, 41. and Ref. 7 for details.
11. (a) Sawa, M.; Imaeda, Y.; Hiratake, J.; Fujii, R.; Umeshita, R.; Watanabe, M.;
Kondo, H.; Oda, J. Bioorg. Med. Chem. Lett. 1998, 8, 647; (b) Dennis, N.; Katritzky,
A. R.; Matsuo, T.; Parton, S. K.; Takeuchi, Y. J. Chem. Soc., Perkin Trans. 1 1974,
746.
Figure 3. Compound 4e docked in the active conformation of B-Raf, showing the
network of H-bonds made by the indazole group.
generally held true. Thus, both the 7-Cl (4p) and 7-F (4r) analogs
were potent in both cell lines (IC50s 60.3
analogs) but the methyl 4v analog was only nominally active
M in A375). Not surprisingly, the less active analogs 4t,
lM in WM266 for both
12. For a description of the assays used in this Letter, see Ref. 3.
13. As a preliminary measure of selectivity, compounds were screened for IC50s in
(IC50 ꢀ1
l
a 22-kinase panel and the most potent analogs (IC50 <0.01 lM) described
herein demonstrated good to excellent selectivity. For instance, the very potent
4u, and 4w had modest cell potency.
Another structural modification that was explored was based
on the observation that N-carboethoxy tropane 5 was identified
as a potent B-Raf inhibitor. Though approximately fourfold less po-
tent than 4e, the presence of the C(6) methyl group raised the pos-
sibility of fusing the tropane to the pyrimidine ring of the core. The
three analogs shown (6a–c) in Table 4 were more potent than B-
Raf inhibitor 5. However, the cellular activity of these analogs
was modest.
B-Raf inhibitor 4r was more than 200-fold selective against all the kinases
screened except one (p38a), where it exhibited 19-fold selectivity.
14. A model built from 2FB8(PDB) was used; see: 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.; Wang, L.; Karreth, F.; Lougheed, J. C.; Lee, J.;
Chau, D.; Stout, T. J.; May, E. W.; Contractor, R. G.; Smalley, K. S. M.; Herlyn, M.;
Morrissey, M. M.; Tuveson, D. A.; Huang, P. Cancer Res. 2006, 66, 11100.
15. Caco-2 (WT) cells were used as a control. All described compounds with an
IC50 <1 lM in either A375 or WM266 cells had IC50s >3.6 lM in Caco-2
cells and therapeutic indices (control/functional activity) ranging from 5.8
to 37 for A375 cells and 9.7 to 110 for WM266 cells (data not shown). The
In conclusion, a novel series of pyrazolo[1,5-a]pyrimidines
bearing a 3-hydroxyphenyl group at C(3) and substituted tropanes
at C(7) has been identified as potent B-Raf inhibitors. However, the
potential metabolic soft spot posed by the phenol led to the iden-
tification of indazole as an effective isostere. Several compounds
possessing substituted indazoles, such as 4e, 4p, and 4r, potently
inhibited cell proliferation at submicromolar concentrations in
the A375 and WM266 cell lines, and the latter two compounds also
exhibited good therapeutic indices in cells.18 Fusing the tropane
moiety to C(6)–C(7) of the pyrimidine core led to novel, potent
B-Raf inhibitors that unfortunately lacked good efficacy in cells. A
lone exception to this was compound 4e, which had a Caco-2 IC50 = 0.8
and therapeutic index of 2.1 and 2.7 for A375 and WM266 cells,
respectively.
lM
a
16. All IC50 values reported in this Letter are a mean of at least two separate
determinations with typical variation <30% between replicate runs.
17. For a report that discloses the use of an indazole as a phenol isostere, see:
Bamborough, P.; Angell, R. M.; Bhamra, I.; Brown, D.; Bull, J.; Christopher, J. A.;
Cooper, A. W. J.; Fazal, L. H.; Giordano, I.; Hind, L.; Patel, V. K.; Ranshaw, L. E.;
Sims, M. J.; Skone, P. A.; Smith, K. J.; Vickerstaff, E.; Washington, M. Bioorg. Med.
Chem. Lett. 2007, 17, 4363.
18. Compound 4p had a therapeutic index of 9.4 and 19.3 for A375 and WM266
cells, respectively, and compound 4r had indices of 5.8 and 12, respectively;
see Ref. 15.