Novel 3-alkoxy-1H-pyrazolo[3,4-d]pyrimidines as EGFR and erbB2 receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.12.035

Novel 4-anilino-1H-pyrazolo[3,4-d]pyrimidines have been synthesized and evaluated in vitro for erbB2 and EGFR kinase inhibition. A representative compound displaying oral bioavailability in rat and dog illustrates the potential of this series to provide o

Full text of DOI:10.1016/j.bmcl.2007.12.035

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 959–962
Novel 3-alkoxy-1H-pyrazolo[3,4-d]pyrimidines as EGFR
and erbB2 receptor tyrosine kinase inhibitors
Richard Ducray,a,* Peter Ballard,b Bernard C. Barlaam,a Mark D. Hickinson,b
Jason G. Kettle,b Donald J. Ogilvieb and Catherine B. Trigwellb
aAstraZeneca, Centre de Recherches, Z.I. la Pompelle, BP1050, 51689 Reims Cedex 2, France
bCancer and Infection Research, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
Received 24 October 2007; revised 13 December 2007; accepted 16 December 2007
Available online 23 December 2007
Abstract—Novel 4-anilino-1H-pyrazolo[3,4-d]pyrimidines have been synthesized and evaluated in vitro for erbB2 and EGFR kinase
inhibition. A representative compound displaying oral bioavailability in rat and dog illustrates the potential of this series to provide
orally active erbB2 inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The erbB2 receptor tyrosine kinase (also known as
HER2 or neu) belongs to the erbB family which also in-
cludes the epidermal growth factor receptor (EGFR),
erbB3 and erbB4.1 Overexpression of EGFR and erbB2
has been implicated in the development of various types
of cancers. Since the introduction of the monoclonal
antibody trastuzumab (HerceptinTM) and the dual
tion (structure 6, Fig. 2) were so far unknown. In this
paper, we describe the synthesis and in vitro evaluation
of 3-alkoxy-4-anilino-1H-pyrazolo[3,4-d]pyrimidines as
erbB2 inhibitors.
The synthesis of compounds 12a–q (Scheme 1) starts
with the known pyrazole 810 obtained by reaction of
hydrazine with 2-dicyanomethylene-1,3-dioxolane 7.11
Reaction of 8 with DMF dimethyl acetal provided ami-
dine 9 which underwent a cyclo-condensation with the
appropriate aniline in acetic acid presumably via a Dim-
roth rearrangement12 to give intermediates 10a–i. The
hydroxyl group was then transformed into a chloride
or a mesylate, which was displaced by a range of pri-
mary and secondary amines providing the desired prod-
ucts.13 Compounds 16a–c have been obtained through
the same synthetic route starting from dicyanoketene
trimethylene acetal 13.14
EGFR/erbB2 kinase inhibitor lapatinib (1, TykerbTM
)
in clinical practice, inhibition of the erbB2 pathway
has been an increasingly attractive approach for the
treatment of advanced metastatic breast cancer.1,2 Other
erbB2 kinase inhibitors are in ongoing clinical trials such
as the dual EGFR/erbB2 inhibitors AEE7883 (2) and
BMS-5996264 (3), the irreversible dual inhibitors HKI-
2725 and BIBW-2992,6 and the erbB2 selective inhibitor
CP-7247147 (4) (See Fig. 1).
We recently published the discovery of 5-alkoxy-4-anili-
no-quinazolines (e.g., compound 5, Fig. 2) as selective
erbB2 inhibitors.8 In the course of these studies, we were
interested in utilizing 1H-pyrazolo[3,4-d]pyrimidine as
the central core to replace the well-known 4-anilino-qui-
nazoline scaffold. Although 4-anilino-1H-pyrazolo[3,4-
d]pyrimidines incorporating either an amino or an aryl
group at the 3 position have been described as kinase
inhibitors,9 such compounds with a 3-alkoxy substitu-
Compounds listed in Tables 1 and 2 were tested in erbB2
and EGFR isolated enzyme kinase assays and in a cellu-
lar erbB2 autophosphorylation assay in an MCF-7 cell
line engineered to overexpress the erbB2 receptor (Clone
24). Table 1 summarizes the SAR of different anilines at
C4. Our most potent compound 12a, which incorporates
a fluoro-benzyloxy group, showed excellent activity in
the kinase and cellular assays. Whereas most of these
compounds displayed good activity against erbB2
(IC50 1–17 nM), EGFR activity could be modulated
by the para substitution (R2) of the aniline. As shown
with compound 12f, a 2-methylpyridin-5-yl ether was
Keywords: Pyrazolopyrimidines; erbB2; Receptor tyrosine kinase.
*
Corresponding author. Tel.: +33 3 26 61 69 03; fax: +33 3 26 61 68
42; e-mail: richard.ducray@astrazeneca.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.035

960
R. Ducray et al. / Bioorg. Med. Chem. Lett. 18 (2008) 959–962
N
SO2Me
HN
H
F
N
O
N
HN
F
O
O
O
N
O
Cl
N
H
N
N
N
N
H
HN
O
N
O
3
N
OMe
1
N
HN
N
HN
N
N
N
H
N
N
4
2
Figure 1. Representative erbB2 kinase inhibitors in clinical trials.
R1
R
N
Cl
HN
N
O
O
O
O
R2
HO
CN
HO
N
N
NC
O
CN
O
a
c
N
N
HN
N
HN
N
O
N
O
R
X
4
H
H
3
5
N
6
N
10a-i
2 N
8: X= NH2
9: X= N=(CH)NMe2
7
N
b
N
H
6
5
7
1
R
R
Figure 2. Quinazoline and pyrazolopyrimidine based erbB2 inhibitors.
HN
N
O
HN
N
R1R2N
O
N
Y
f
N
d or e
N
N
found to significantly remove EGFR activity compared
to the pyridin-2-yl-methyl and fluorobenzyl groups. This
effect is even more pronounced with a fluorine on the
meta position (R1) whereas a meta-chloro maintains
the EGFR activity (see 12g and 12c, respectively). The
optimal meta substitution for erbB2 activity was found
to be chloro or methyl, the chloro derivatives being gen-
erally more active in the cell based assay (compare 12a
with 12d and 12b with 12e for example). The methoxy
derivatives 12h and 12i were found to be less active than
their methyl counterparts.
N
N
H
H
12a-q
11a-c, e, f: Y= Cl
Y= OMs
11d, g-i:
HO
b
O
N
NC
O
CN
O
CN
X
a
N
H
14: X= NH2
13
15: X= N=(CH)NMe2
Influence of the side chain on the C3 position is illus-
trated in Table 2. We carried out this exploration using
the 4-pyridin-2-yl-methoxy-3-chloroaniline at the C4
position because it provided the best pharmacokinetic
properties. We also postulated that the superior
in vitro activity of the lipophilic 3-fluorobenzyl deriva-
tives would not translate into a better in vivo efficacy be-
cause of their higher protein binding.15 Furthermore,
their lower aqueous solubility (data not shown) might
increase the risk of a limited oral absorption. Several
amino-ethoxy (12b, 12j–q) or amino-propoxy side chains
(16a–c) provided good kinase inhibition with cellular
IC50 values typically ranging from 0.1 to 1.0 lM. As
illustrated by compound 10b, a basic amine is not neces-
sary for the erbB2 activity.
c, d, f
R
R1R2N
HN
O
N
N
16a-c
N
H
N
Scheme 1. Synthesis of compounds 12a–q and 16a–c. Reagents and
conditions: (a) hydrazine hydrate, water, room temp, 30 min. (b) DMF
dimethylacetal, acetonitrile, 50 ꢁC, 1 h; (c) aniline, AcOH, microwave
irradiation at 150 ꢁC for 2 min. (d) SOCl2, cat. DMF, 80 ꢁC, 2 h. (e)
MsCl, pyridine, room temp, 1 h. (f) R1R2NH, KI (when Y@Cl),
DMA, microwave irradiation at 150 ꢁC for 5 min. For intermediates
10 and 11, the letter refers to the definitions in Table 1.
Further assessment of compound 12k has been carried
out in anti-proliferative cellular assays (Table 3). Very
good activity was seen against the BT474C cell line,
which is sensitive to inhibition of either erbB2 or EGFR
(unstimulated assay). The moderate activity of 12k in
the EGF stimulated KB cell proliferation assay shows
that 12k is less selective against EGFR compared to
the quinazoline 5.16
Pharmacokinetic properties of 12k were evaluated in rat
and dog and the results are shown in Table 4. Accept-
able oral bioavailability is observed, associated with a
moderate half-life. The limitation in bioavailability can
be explained by a moderate to high plasma clearance,
respectively, 55% and 130% of the hepatic blood flow
in rat and dog. Indeed good intestinal absorption was

R. Ducray et al. / Bioorg. Med. Chem. Lett. 18 (2008) 959–962
961
Table 1. Modification of the aniline
R1
O
R2
N
HN
N
O
HO
N
N
N
H
Compound
R1
R2
Kinase inhibitiona
Clone 24a
erbB2
EGFR
12a
12b
12c
12d
12e
12f
12g
12h
12i
Cl
Cl
3-Fluorobenzyl
CH2-(2-pyridyl)
2-Me-pyridin-5-yl
3-Fluorobenzyl
CH2-(2-pyridyl)
2-Me-pyridin-5-yl
2-Me-pyridin-5-yl
3-Fluorobenzyl
CH2-(2-pyridyl)
<0.001
0.005
<0.001
<0.001
0.005
0.011
0.005
0.003
0.017
<0.001
0.013
0.007
<0.001
0.018
0.216
1.43
0.019
0.135
0.184
0.107
0.695
0.415
—
Cl
Me
Me
Me
F
OMe
OMe
0.013
0.104
0.613
1.48
a IC50 (lM). Values are means of at least two experiments.
Table 2. Modification of the C3 side chain
HO
Cl
10b: R=
O
O
N
R1R2N
R1R2N
HN
N
R
12b, j-q: R=
O
N
N
N
H
O
16a-c: R=
Compound
NR1R2
Kinase inhibitiona
Clone 24a
erbB2
EGFR
12b
12j
4-OH-piperidinyl
NHMe
0.005
0.003
0.001
0.013
0.017
0.005
0.003
0.008
0.008
<0.001
0.028
0.001
0.016
0.016
0.009
0.014
0.135
1.0
12k
12l
N-Me-piperazinyl
N-Ac-piperazinyl
Pyrrolidinyl
0.166
0.129
0.818
0.128
0.148
0.082
—
<0.001
0.002
0.003
12m
12n
12o
12p
12q
10b
16a
16b
16c
Piperazinyl
Oxazepanyl
<0.001
0.004
4-OMe-piperidinyl
Morpholinyl
—
<0.001
0.004
—
Morpholinyl
4-OH-piperidinyl
N-Me-piperazinyl
<0.001
0.006
0.004
0.440
—
—
a IC50 (lM). Values are means of at least two experiments.
Table 4. Rat and dog pharmacokinetic parameters for 12k
Species Plasma protein Vdss Cl Half-life F%
binding (%free) (l/kg) (ml/min/kg) (h)
Table 3. Cellular anti-proliferative data for 12k and 5
Compound
BT474Ca
KB (EGF)a
Rata
Dogb
10
21
4.5
9.0
41
46
1.4
2.4
23
30
12k
5
0.017
0.041
0.22
2.60
a IC50 (lM). Values are means of two experiments.
a Female rats dosed at 0.4 mg/kg i.v. and 1 mg/kg p.o.
b Mean values for male and female beagle dogs dosed at 0.2 mg/kg i.v.
and 0.4 mg/kg p.o.
anticipated from the MDCK-MDR1 assay in which 12k
showed good permeability (Papp A-B
9.5 · 10À6 cm sÀ1) and limited efflux (efflux ratio 3.6).
No significant activity against P450 enzymes
(IC50 > 10 lM on five isoforms) was observed with 12k.
a

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R. Ducray et al. / Bioorg. Med. Chem. Lett. 18 (2008) 959–962
F.; Shen, R.; Shi, X.; Tsou, H.-R.; Wang, Y.-F.; Wissner,
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6. Solca, F.; Baum, A.; Himmelsbach, F.; Amelsberg, A.;
Adolf, G. Eur J Cancer – Suppl. 2006, 4(12) Abst 567. See
also Ref. 1a.
In conclusion, we have discovered a new class of erbB2
tyrosine kinase inhibitors based on the 1H-pyrazolo[3,4-
d]pyrimidine scaffold. Encouraging results with com-
pound 12k highlight the potential of this series to pro-
vide potent, orally active erbB2 kinase inhibitors.
7. (a) Jani, J. P.; Finn, R. S.; Campbell, M.; Coleman, K. G.;
Connell, R. D.; Currier, N.; Emerson, E. O.; Floyd, E.;
Harriman, S.; Kath, J. C.; Morris, J.; Moyer, J. D.;
Pustilnik, L. R.; Rafidi, K.; Ralston, S.; Rossi, A. M. K.;
Steyn, S. J.; Wagner, L.; Winter, S. M.; Bhattacharya, S.
K. Cancer Res. 2007, 67, 9887; For a related series of
selective erbB2 inhibitors, see also (b) Lippa, B.; Kauff-
man, G. S.; Arcari, J.; Kwan, T.; Chen, J.; Hungerford,
W.; Bhattacharya, S.; Zhao, X.; Williams, C.; Xiao, J.;
Pustilnik, L.; Su, C.; Moyer, J. D.; Ma, L.; Campbell, M.;
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8. Ballard, P.; Bradbury, R. H.; Hennequin, L. F. A.;
Hickinson, D. M.; Johnson, P. D.; Kettle, J. G.; Kli-
nowska, T.; Morgentin, R.; Ogilvie, D. J.; Olivier, A.
Bioorg. Med. Chem. Lett. 2005, 15, 4226.
Acknowledgments
We thank Rob Bradbury, Jamie Scott and Teresa Kli-
nowska for helpful discussions and the following people
for their contribution to the synthesis of the compounds
or their evaluation in the different assays described here-
in: Georges Pasquet, Michel Vautier, Patrice Koza,
´
Anne-Laurence Fontaine, Melissa Jactel, Robin Smith,
Sara Davenport, Sarah Beck, John Vincent, Rowena
Callis, Vikki Flemington, Christian Delvare and Del-
phine Dorison.
9. (a) Traxler, P.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.;
Mett, H.; Buchdunger, E.; Meyer, T.; Mueller, M.; Furet,
P. J. Med. Chem. 1997, 40, 3601; see also (b) Wu, T. Y. H.;
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11. Neidlein, R.; Kikelj, D. Synthesis 1988, 981.
12. For a similar transformation, see: Ugarkar, B. G.;
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13. For experimental procedures and description of kinase
and clone 24 assays, see: Barlaam, B.C., Ducray, R.,
Kettle, J.G. PCT Int. Appl. WO2006064196.
14. Middleton, W. J.; Engelhardt, V. A. J. Am. Chem. Soc.
1958, 80, 2788.
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15. Replacement of the 2-pyridyl group in compound 12k by a
3-fluorophenyl led to a significant decrease of the unbound
fraction in rat plasma (from 10% to 0.9% free drug).
16. IC50 values of 5 in erbB2 and EGFR kinase inhibition
assays are, respectively, 0.002 and 0.14 lM (see Ref.
8).
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