Hit to lead optimization of pyrazolo[1,5-a]pyrimidines as B-Raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.074

Our continued effort towards optimization of the pyrazolo[1,5-a]pyrimidine scaffold as B-Raf kinase inhibitors is described. Structure guided design was utilized to introduce kinase hinge region interacting groups in the 2-position of the scaffold. This s

Full text of DOI:10.1016/j.bmcl.2009.10.074

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6890–6892
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hit to lead optimization of pyrazolo[1,5-a]pyrimidines as B-Raf kinase inhibitors
a
a
a
a
a
Ariamala Gopalsamy a, , Greg Ciszewski , Mengxiao Shi , Dan Berger , Yongbo Hu , Frederick Lee ,
*
Larry Feldberg b, Eileen Frommer b, Steven Kim b, Karen Collins b, Donald Wojciechowicz b, Robert Mallon b
a Chemical and Screening Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
b Discovery Oncology, Wyeth Research, 401 N. 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:
Our continued effort towards optimization of the pyrazolo[1,5-a]pyrimidine scaffold as B-Raf kinase
inhibitors is described. Structure guided design was utilized to introduce kinase hinge region interacting
groups in the 2-position of the scaffold. This strategy led to the identification of lead compound 9 with
enhanced enzyme and cellular potency, while maintaining good selectivity over a number of kinases.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 14 September 2009
Revised 15 October 2009
Accepted 19 October 2009
Available online 22 October 2009
Keywords:
B-Raf inhibitor
Pyrazolo[1,5-a]pyrimidine
The RAS-RAF-MEK signal transduction pathway plays a key role
in tumor biology.1 A V600E mutation of the B-Raf isoform induces a
constitutive activation of this kinase in the ERK pathway that
increases cell proliferation and cell survival. Inhibitors of B-Raf
could be used in the treatment of melanomas, colorectal cancer,
and other Ras related human cancers.2 A number of small molecule
B-Raf inhibitors have been disclosed in the recent past including
our high through-put screen3 (HTS) effort that resulted in the iden-
tification of pyrazolo[1,5-a]pyrimidine-3-carboxylate 1 (Fig. 1).
As detailed in our previous Letter,4 variations of the amide lin-
ker and the benzamide group substituents of compound 1 resulted
in several sub-micromolar B-Raf inhibitors. Replacement of the es-
ter group with amides was also tolerated, with increased activity
for several amides bearing water solubilizing functionalities. How-
ever, from our proposed binding model of our HTS hit 1 with the B-
Raf protein, no specific polar interaction was observed between the
ester moiety and the protein. This observation prompted us to re-
move this metabolically labile moiety (microsomal stability—rat
T1/2: 8 min) and introduce groups in the 2-position of the pyrazol-
opyrimidine scaffold that could provide a potential hinge region
interaction with the kinase. In this communication we detail our
structure guided hit to lead optimization efforts on the pyrazol-
o[1,5-a]pyrimidine scaffold.
which in turn was prepared from methyl isonicotinate 2. Reaction
of 3-nitroacetophenone 5 with N,N-dimethylformamidedimethy-
lacetal followed by condensation with aminopyrazole 4 afforded
pyrazolopyrimidine 7. Reduction of the nitro group afforded the
key intermediate aniline 8 which was further derivatized to the
required amides by reacting with appropriate acid chlorides. Reac-
tion of the common intermediate amine 8 with various isocyanates
or triphosgene and appropriate amines generated urea analogs.
Synthetic analogs designed to identify optimal substituents on
the 2-position of the pyrazolopyrimidine scaffold were generated
starting from 3-substituted 5-aminopyrazoles 14 as shown in
Scheme 2. The 3-trifluoromethylbenzamide moiety was introduced
early on starting from 3-aminoacetophenone 15 which was con-
verted to enamine 17 and condensed with aminopyrazole 14 to
give N-(3-(2-morpholinopyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-
(trifluoromethyl)benzamide 18 (Scheme 2).
O
CF3
NH
The analogs designed to obtain structure–activity relationships
were synthesized by following the synthetic sequences shown in
Schemes 1–3.5 Pyridyl substituted aminopyrazole intermediate 4
was synthesized by condensation of the hydrazine with nitrile 3,
7
N
N
2
3
N
CO2Et
1
* Corresponding author.
E-mail address: gopalsa@wyeth.com (A. Gopalsamy).
Figure 1. B-Raf inhibitor hit 1 identified from a high through-put screen.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.074

A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6890–6892
6891
H
O
O
N
H
N
NH2
H
N
N
CN
a
b
CF3
O
CF3
O
N
N
O
a
2
3
4
N
N
N
N
N
N
N
N
NO2
NO2
N
NO2
N
N
Cl
19
20
d
NH
c
R
O
N
O
N
Scheme 3. Reagents and conditions: (a) RNH2, NMP or pyridine, microwave oven,
170 °C, 1–2 h.
6
5
N
7
H
NH2
N
CF3
Table 1
e
f
SAR for 2-substituted pyrazolo[1,5-a]pyrimidine derivatives
O
N
N
N
H
N
N
N
N
CF3
N
N
O
9
8
N
N
Scheme 1. Reagents and conditions: (a) CH3CN, t-BuOK, toluene, reflux, 18 h, 52%;
(b) NH2NH2, EtOH, reflux, 18 h, 65%; (c) DMF–DMA, reflux, 18 h, 79%; (d) 4, HOAc,
80 °C, 18 h, 81%; (e) Fe, HOAc, 80 °C, 3 h, 72%; (f) pyridine, 3-CF3PhCOCl, CH2Cl2, rt,
15 h, 71%.
W
N
a
Compds
W
B-Raf kinase IC50
(l
M)
21
22
23
24
25
9
26
27
28
18
3-Trifluoromethylphenyl
4-Methoxyphenyl
Thiophen-2-yl
1H-Imidazol-5-yl
1-Methyl-1H-imidazol-5-yl
4-Pyridyl
3-Pyridyl
Piperazin-1-yl
4-Methylpiperazin-1-yl
Morpholino
>10
>10
>10
1.6
0.10
0.032
>10
6.2
O
O
HO
S
S
O
a
b
EtO
S
S
NC
OEt
NC
NC
NC
12
11
10
>10
0.099
c
N
d
HN
H2N
N
O
a
N
O
Values are means of two or more experiments.
S
e
14
13
led to a substantial loss in activity (compounds 21–22). Of the het-
eroaromatic groups explored (compounds 23–26 and 9), only the
4-pyridinyl analog 9 improved the enzyme potency significantly.
The corresponding 3-pyridyl analog 26 was not favorable.
This observation was readily explained by our predicted
binding pose for these analogs (Fig. 2), wherein a 4-pyridyl group
is ideally positioned for the nitrogen atom to form hydrogen bond
to Cys531 in the hinge region. This was further confirmed by the
similar activity displayed by analogs 25 and 18 which can maintain
NH2
H
N
f
CF3
O
O
15
16
O
CF3
H
H
N
N
CF3
O
O
g
N
O
N
N
O
N
N
17
18
Scheme 2. Reagents and conditions: (a) K2CO3, CS2 MeI, DMF, rt 8 h, 85%; (b) NaOH/
THF, rt, 6 h, 30%; (c) morpholine, Et3N, MeOH, rt, 8 h, 80%; (d) hydrazine, EtOH,
reflux, 18 h, 100%; (e) 3-CF3PhCOCl, pyridine, CH2Cl2, 18 h, 100%; (f) DMF–DMA,
100 °C 18 h, 100%; (g) 14, HOAc, 80 °C, 18 h, 80%.
Analogs designed to introduce polar water solubilizing groups
(compound 20) were prepared starting from 2-chloropyridinyl
pyrazolopyrimidine intermediate 19 under microwave condition
using various amines as shown in Scheme 3.
Our SAR effort commenced with the placement of a variety of
aromatic and heterocyclic rings in the 2-position of the pyrazolo-
pyrimidine scaffold designed to reach out to the hinge region of
the ATP binding pocket. While exploring this region, the 3-trifluo-
romethylbenzamide portion of the molecule was held constant. As
seen from the representative examples in Table 1, evaluation of a
number of substituted phenyl rings explored at the 2-position
Figure 2. Predicted binding model of compound 9. The pyridine nitrogen of the
compound forms a hydrogen bond to the hinge region residue Cys531. The two
hydrogen bonds from amide linker to Glu500 and Asp593 are maintained.

6892
A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6890–6892
Table 2
Table 3
SAR for benzamide region of pyrazolo[1,5-a]pyrimidine derivatives
SAR for pyridine region modifications
R4
H
R3
N
CF3
O
N
L
N
N
Y
N
a
N
N
Compds
Y
B-Raf kinase IC50 (lM)
N
9
35
36
37
38
39
40
41
42
H
0.032
0.053
0.16
0.13
0.068
0.11
N
–NHCH2CH2N(CH3)2
a
–NHCH2CH2–N-morpholinyl
–NHCH2CH2CH2–N-morpholinyl
–NHCH2CH2–N-pyrrolidinyl
–NHCH2CH2CH2–N-pyrrolidinyl
–NHCH2CH2CH2–N-(2-oxo-pyrrolidinyl) 0.11
–NHCH2CH2CH2–N-(1H-imidazolyl) 0.066
–NHCH2CH2–N-(4-hydroxy-piperidinyl) 0.066
Compound
R3
R4
L
B-Raf IC50
(lM)
29
30
9
31
32
33
34
Cl
H
H
H
Cl
Cl
H
Cl
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–
–NH–CO–NH–
–NH–CO–NH–
1.1
4.0
OCH3
CF3
Cl
CF3
CF3
CF3
0.032
0.66
0.060
0.025
0.040
a
Values are means of two or more experiments.
a
Values are means of two or more experiments.
Table 4
Cell growth inhibition data
this hydrogen bond interaction with Cys531 due to the position of
the ring nitrogen in the imidazole of 25 and the oxygen of the mor-
pholine analog 18. Piperizine analog 27, which has a hydrogen
bond donor instead of acceptor, was not tolerated. Analog 28,
which has an N-methylpiperazine moiety, was not favorable due
to steric hindrance posed by the methyl group.
a
a
Tumor cell lines
Compound 1 IC50
(
lM)
Compound 9 IC50 (lM)
A375
3.8
0.28
0.4
0.35
2.6
3.1
3.5
LoVo
HT29
3.87
7.0
CaCo-2
BXPC3
WM-266-4
6.6
3.25
6.2
From our earlier optimization work, we have determined that
the 3-trifluoromethyl substituted benzamide was preferred and
was predicted to occupy a hydrophobic pocket created by Ile512,
His513 and Ile571. The amide itself was involved in two hydrogen
bonds to Glu500 and Asp593. In order to confirm that the 2-substi-
tuted pyrazolopyrimidines like 9 still retain these key interactions,
we varied the substituents on the phenyl ring as shown by analogs
29–32 in Table 2. The SAR observed earlier for the HTS hit 1 was
found to be reproducible for the 2-(40-pyridyl)pyrazolo[1,5-
a]pyrimidine scaffold. The flexibility shown by the linker to accom-
modate urea in the place of amide was found to be valid as shown
by analogs 33–34. A number of polar groups placed in the adjacent
carbon atom of the pyridyl moiety were very well tolerated leading
to a number of potent analogs as shown in Table 3. Docking studies
indicated that these groups are ideally positioned to reach out to
the solvent exposed region of the binding pocket.
a
Values are means of two or more experiments.
hinge region interacting groups. This structure guided hit to lead
optimization led to significant improvement in both enzyme and
cellular activity while maintaining good selectivity over a number
of kinases. Our continued effort in the area towards lead optimiza-
tion will be communicated in the future.
Acknowledgements
The authors thank Dr. John Ellingboe for his support of this
work. We also thank Dr. Girija Krishnamurthy for KD determination
and Wyeth Chemical Technologies group for analytical data.
Compound 9 was further profiled for its ability to inhibit prolif-
eration in a variety of tumor cell lines. As seen from Table 4, the in-
creased potency observed in the enzyme inhibition resulted in
improvement in cellular growth inhibition. Compound 9, in spite
of invoking a hinge interaction in the ATP binding pocket of B-
References and notes
1. Garnett, M. J.; Marais, R. Cancer Cell 2004, 6, 313.
Raf enzyme, was found to be highly selective (IC50: >50
against a number of kinases including CDK1, CDK2, PKB , PKC
PKCb, IKKb, JNK1, ERK2, P38 , MK2, PKA, ROCK1, CK1 , Src, Fyn,
Abl1, GCK, CHK1, RSK, PLK1, IGFR1, LCK, p70S6K, PI3K , m-TOR,
lM)
2. 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.;
Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.;
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.
a
a,
a
c
a
Tpl2 and PDK1. Employing fluorescence spectroscopy techniques,
compound 9 exhibited a single digit nanomolar KD from the
changes in the endogenous tryptophan fluorescence of the enzyme
upon inhibitor binding at the emission and excitation wavelengths
of 465 nm and 295 nm, respectively. Compound 9 showed good
permeability (PAMPA: 2.51 ꢀ 10ꢁ6 m/s @ pH 7.4) and microsomal
stability (rat T1/2: >30 min; nude mouse T1/2: 24 min).
3. Mallon, R.; Feldberg, L. R.; Kim, S. C.; Collins, K.; Wojciechowicz, D.; Hollander, I.;
Kovacs, E. D.; Kohler, C. Anal. Biochem. 2001, 294, 48.
4. 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, 2735.
5. For experimental details, see: Gopalsamy, A.; Ciszewski, G. M.; Shi, M.; Berger, D.
M.; Torres, N.; Levin, J. I.; Powell, D. W. U.S. Pat. Appl. Publ., 2007; Chem. Abstr.
2007, 147, 386006.
In summary, the pyrazolopyrimidine scaffold identified as a
B-Raf inhibitor from HTS has been modified to incorporate kinase