Rational design of 4,5-disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones as a novel class of inhibitors of epidermal growth factor receptor (EGF-R) and Her2(p185(erbB)) tyrosine kinases.

Article abstract of DOI:10.1016/S0960-894X(02)00364-5

A novel class of 4,5-disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones has been discovered as potent and selective inhibitors of the EGF-R tyrosine kinase family. These compounds selectively inhibit EGF-R kinase activity at low nanomolar concentration and tyrosine autophosphorylation in cells expressing EGF-R or Her2 (p185(erbB)). Structure-activity relationships (SARs) for this class of compounds are presented.

Full text of DOI:10.1016/S0960-894X(02)00364-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2153–2157
Rational Design of
4,5-Disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones
as a Novel Class of Inhibitors of Epidermal Growth Factor
Receptor (EGF-R) and Her2(p185erbB) Tyrosine Kinases
Li Sun,* Jean Cui, Congxin Liang, Yong Zhou, Asaad Nematalla, Xueyan Wang,
Hui Chen, Cho Tang and James Wei*
SUGEN, Inc., 230 East Grand Avenue, South San Francisco, CA 94080, USA
Received 12 March 2002; accepted 30 April 2002
Abstract—A novel class of 4,5-disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones has been discovered as potent and selective
inhibitors of the EGF-R tyrosine kinase family. These compounds selectively inhibit EGF-R kinase activity at low nanomolar
concentration and tyrosine autophosphorylation in cells expressing EGF-R or Her2 (p185erbB). Structure–activity relationships
(SARs) for this class of compounds are presented. # 2002 Elsevier Science Ltd. All rights reserved.
In the past decade, a numerous diverse small molecule
scaffolds have been discovered as selective ATP compe-
titive inhibitors against the tyrosine kinases.1ꢀ6 The
extensive SAR studies and co-crystallization of various
inhibitors within the catalytic domain of the kinases
(i.e., FGF-R1, Bcr-Abl, Src) provided rich information
on inhibitor–kinase interaction at the molecular level
which allowed further rational design of highly selective
tyrosine kinase inhibitors.1,2 Currently, at least 10 ATP
competitive tyrosine kinase inhibitors are on the market
or under clinical evaluations at different stages.7
core. The binding mode for these hybrid compounds is
proposed in this report. The synthesis of the target
molecules in Table 1 consists of three steps: (1) pre-
paration of the substituted 5,7-diazaindolinone core (4a,
4b, and 5 in Scheme 1) (2) preparation of substituted
pyrrol-2-carboxaldehyde (8, 9, 13, 19, 21, and 25 in Scheme
2) and (3) condensation of the substituted 5,7-diazaindoli-
none cores with the substituted pyrrol-2-carboxaldehydes
to afford the target molecules (Tables 1 and 2).
The 5,7-diazaindolinone core 4a or 4b was prepared via
an intermediate, 1 (Scheme 1), which was synthesized
according to a literature report.8 Chlorination of 1 with
phosphorus oxychloride yielded 2a which was methylated
to afford 2b. Amination of 2a or 2b with substituted
aniline catalyzed by silver triflate afforded 3a and 3b,
which were then oxidized with pyridinium bromide per-
bromide (PBPB) followed by reduction with zinc dust
to give, 4a or 4b. Direct oxidation of 2a with PBPB
followed by reduction with zinc dust afforded another
core 5.
Since overexpression of the EGF-R tyrosine kinase
family and their ligands have been implicated in many
disease indications such as tumors and psoriasis, specific
inhibitors of this tyrosine kinase family may demonstrate
therapeutic utility in the treatment of these diseases. In
our search for novel inhibitors of EGF-R/Her2
(p185erbB), we have designed 4,5-disubstituted-5,7-dihydro-
pyrrolo[2,3-d]pyrimidin-6-ones (5,7-diazaindolinones)
based on the SAR information from both indolinone
and quinazoline scaffolds. The 4-substituted pyrimidine
ring of this hybrid compound originated from quinazoline
while the five-membered lactam ring from indolin-2-one
3,5-Dimethyl-1H-pyrrole-2-carbaldehyde, for preparation
of 26d (Table 1), is commercially available and (5-formyl-
2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid, for synthe-
sizing 26b (Table 1), was synthesized according our
previous report.4 Other substituted pyrrol-2-carbox-
aldehydes were prepared using various methods depicted
*Corresponding authors. Fax: +1-650-837-3348; e-mail: connie-sun@
sugen.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00364-5

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L. Sun et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2153–2157
Table 1. Inhibition of biochemical kinases
ID
R1
R2
R3
R4
R5
Biochemical kinase inhibition
(tyrosine phosphorylation)
IC50, mMa
EGF-R PDGF-R VEGF-R2
(Flk-1/KDR)
26a
26b CH3
No substitution at the C-3 position of the core
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Ethynyl-phenylamino
4-Cl-2-F-phenylamino
Indan-5-amino
>20
0.18
0.20
0.041
0.0047
0.029
0.022
0.0065
>20
5.31
0.17
0.0028
0.21
0.41
0.013
0.0012
0.0059
0.00045
0.018
0.45
>20
>20
>20
>100
>20
>20
>100
>100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
5.62
>20
>20
3.47
16.5
>20
8.92
>20
>20
5.82
10.1
>20
>20
>20
>20
>20
0.3
1.63
0.36
>20
0.04
3.46
2.05
2.1
CH2CH2COOH
26c CH3 CONH(CH2)2N(C2H5)2
CH3
CH3
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
26d CH3
26e CH3
26f CH3
H
H
H
CH3
CH2CH2COOH
COOH
26g
H
H
H
H
CONHCH2CH2N(CH2CH2)2O
CONHCH2CH2N(CH2CH2)2O
CH3
26h CH3
26i CH3
26j CH3
26k CH3
26l CH3
H
H
H
CH3
CONHCH2CH2N(CH2CH2)2O
CON(CH2CH2)2O
CON(CH2CH2)2NCH3
CON(CH2CH2)2NCH3
H
26m
H
26n CH3 CON(CH2CH2)2NCH3
26o CH3
26p CH3
26q CH3
26r CH3
26s CH3
26t CH3
28a CH3
28b CH3
28c CH3
28d CH3
H
H
H
H
H
H
H
H
H
H
H
CON(CH2CH2)2NC2H5
CONH(CH2)2N (C2H5)2
CON[CH2CH(CH3)] N
2
CONHCH2CH(OH)CH2N (C2H5)2
CONHCH2CH2N(CH2CH2)2O
CONH(CH2)2N (C2H5)2
CON(CH2CH2)2O
CON(CH2CH2)2O
CON(CH2CH2)2O
CONHCH2CH2N(CH2CH2)2O
CON(CH2CH2)2O
H
H
H
H
H
H
1.41
>20
>20
>10
>100
>20
1-Benzyl-1H-indol-5-amino 0.0045
1-Benzyl-1H-indazol-5-amino 0.0018
1-Benzyl-1H-indol-5-amino 0.0011
3.47
>20
29
CH3
Piperidine-1-yl
>20
aIC50 values were determined by at least two separate tests and are reported as mean values.
Scheme 1. Synthesis of substituted 5,7-diazaindolinone cores.
in Scheme 2. Aldehyde 9 was prepared starting from
commercially available 6 followed by Vilsmeier, hydro-
lysis, and amidation with selected amines (Scheme 2).
Aldehyde 13 was synthesized from commercially avail-
able 10 by hydrolytic formylation with triethyl ortho-
formate in the presence of trifluoroacetic acid at ꢀ5 ꢁC
to room temperature followed by base hydrolysis of the
ethyl ester and an amidation reaction (Scheme 2). Alde-
hyde 19 was synthesized from commercially available 14
via Wittig condensation followed by hydrogenation,
deprotection of the Boc group, hydrolysis of the ethyl
ester, decarboxylation, and formylation. Furthermore,
hydrolysis of 14 followed by amidation, as described for
the preparation of 9, afforded aldehyde 21. Starting

L. Sun et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2153–2157
2155
Scheme 2. Synthesis of functionalized pyrrole aldehydes.
from commercially available 22, aldehyde 25 was
synthesized following the same procedure for the
preparation of 9.
The substitution pattern on the pyrrole ring (at the C-3
position of the core) has a dramatic impact on the inhi-
bitory potency against the EGF-R kinase. Compounds
with amide or acid functionality at the C-50 position of
the pyrrole ring have been found to be more potent than
the compounds with the same substituent at the C-40
position of the pyrrole ring (26b vs 26e, 26c vs 26p, 26n
vs 26l). This observation is opposite to the SAR results
on indolin-2-ones as VEGF-R inhibitors (data not
shown), which implied 5,7-diazaindolinones might have
different binding mode from indolin-2-ones. Further
modification of the side chain at the C-50 position with
different alkylaminoalkylamides, in general, retained the
potency against the EGF-R kinase (e.g., 26h, 26l, 26p,
26r, and 26q) but could change the kinase selectivity
profile. Compounds 26p and 26r have also displayed
inhibitory activity against the Flk-1 kinase in the sub-
micromolar concentration range. Compound 26k (with
morpholine amide), which does not have a terminal basic
amine moiety at the C-50 position, has shown decreased
potency against the EGF-R kinase when compared to 26l
(with a basic N-methyl piperazine amide). All of these
results indicated that a terminal basic amino functionality
of the 5,7-diazaindoliones might be important for inhi-
bitory potency against the EGF-R kinase.
The final compounds (Table 1) were synthesized by
condensation of substituted pyrrol-2-carboxaldehyde (8,
9, 13, 19, 21, and 25) and the substituted 5,7-diaza-
indolinone (4a, 4b, and 5) using either one of the methods
depicted in Scheme 3.
Compounds were evaluated for their inhibitory activity
towards tyrosine phosphorylation for the EGF-R,
VEGF-R2 (Flk-1/KDR), and PDGF-Rb kinases
according to previously reported methods.6 The results
for these compounds are summarized in Tables 1 and 2.
Initial SAR studies indicated that substitution at the
C-3 position of the core is essential for inhibitory activ-
ity against the EGF-R kinase. The core molecule with-
out substitution at the C-3 position (26a in Table 1) is
inactive against any kinases in the study (IC50>20 mM)
whereas condensation of the core with substituted pyr-
role-2-carboxaldehyde at the C-3 position afforded
compound 26d (Table 1), which was observed to be
active against the EGF-R kinase (IC50=0.041 mM).
In addition, the proton at the N-1 position was
found to be essential for the EGF-R kinase inhibitory
activity, as demonstrated by comparison of 26d to 26i or
26h to 26j.
Modification at the C-4 position of the core by repla-
cing 3-chloro-4-fluoroaniline with other aryl or aliphatic

2156
L. Sun et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2153–2157
Table 2. Inhibition of cellular kinases
ID
R1
R2
R3
R4
R5
Cellular assays IC50, mMa
Tyrosine phosphorylation 3T3 Proliferation
EGF-R
Her-2
EGF
Her2
26d CH3
26f CH3
26h CH3
26k CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
COOH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Cl-4-F-phenylamino
3-Ethynyl-phenylamino
4-Cl-2-F-phenylamino
Indan-5-amino
0.3
>5
0.04
0.04
0.1
0.2
0.2
0.2
0.04
0.5
5
0.3
>5
0.1
0.4
0.2
0.1
0.2
0.5
0.04
NTb
>5
0.1
0.1
0.04
7.82
>50
2.05
0.44
0.75
7.50
1.29
2.58
0.62
NT
2.50
49.3
1.24
1.21
0.37
0.35
0.37
0.51
0.50
NT
CONHCH2CH2N(CH2CH2)2O
CON(CH2CH2)2O
CON(CH2CH2)2NCH3
CON(CH2CH2)2NC2H5
CONHCH2CH2N (C2H5)2
26l
CH3
26o CH3
26p CH3
26q CH3
CON[CH2CH(CH3)] N
2
26s
26t
CH3
CH3
CONHCH2CH2N(CH2CH2)2O
CONHCH2CH2N (C2H5)2
CON(CH2CH2)2O
CON(CH2CH2)2O
CON(CH2CH2)2O
CONHCH2CH2N(CH2CH2)2O
28a CH3
28b CH3
28c CH3
28d CH3
4.32
NT
NT
1.95
2.78
NT
NT
1.56
1-Benzyl-1H-indol-5-amino
1-Benzyl-1H-indazol-5-amino
1-Benzyl-1H-indol-5-amino
0.1
0.1
0.2
aIC50 values were determined by at least two separate tests and are reported as mean values.
bNT=not tested.
Scheme 3. Synthesis of the target molecules: 5,7-diazaindolinones.
amines revealed that 1-benzyl-1H-indol-5-ylamino or
1-benzyl-1H-indazol-5-ylamine substitution dramati-
cally enhanced the inhibitory activity against the EGF-
R kinase (28b or 28c vs 26k and 28d vs 26h). Saturated
piperidine substitution at the C-4 position of the core
abolished the kinase inhibitory activity (29). These SAR
results indicated that arylamino substitution at the C-4
position is essential for kinase inhibitory activity. Inter-
estingly, kinase potency and selectivity could be drama-
tically altered by a subtle change in the substitution
pattern on the aniline side chain at the C-4 position of
the core. In this respect, 26p is a very potent and selec-
tive inhibitor of EGF-R whereas 26t is a selective inhi-
bitor of the Flk-1 kinase. This SAR trend is similar to
the one observed for the 4-phenylaminoquinazoline
scaffold,9 which implies the similar binding mode to
quinazoline scaffold.
A few compounds have also been assessed for their ability
to inhibit EGF-R or Her2 (p185erbB) autophosphorylation
in A431 or SK-OV-3 cells, respectively, as well as ligand
induced BrdU incorporation in 3T3 cells (Table 2). In
general, most of the compounds showed submicromolar
inhibitory activity against autophosphorylation of both

L. Sun et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2153–2157
2157
EGF-R and Her2 (p185erbB) in cells. Compound 28d
Acknowledgements
showed some selectivity against autophosphorylation of
Her2 (p185erbB) over the EGF-R kinase. It should be
noted, however, there is no direct correlation between
biochemical and cellular results (Table 2). For example,
26k was 60-fold less potent than 26l in the biochemical
assay, but has been found to be equally potent, if not
more potent, against EGF-R stimulated BrdU incor-
poration in 3T3 cells and 25-fold more potent against
EGF-R autophosphorylation in cells. This might be the
result of factors such as chemical stability, solubility,
cell membrane permeability, and in vitro assay condi-
tions. Of particular interest, most of the compounds have
been found to show submicromolar inhibitory activity
against Her-2 (p185erbB) autophosphorylation in cells.
The authors would like to thank Dr. Gerald McMahon
and Dr. Ken Lipson for their valuable input and dis-
cussion.
References and Notes
1. Sun, L.; McMahon, G. Drug Discov. Today 2000, 5, 344.
2. Traxler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195.
3. Fry, D. W.; Kraker, A. J.; McMichael, A.; Ambroso, L. A.;
Nelson, J. M.; Leopold, W. R.; Connors, R. W.; Bridges, A. J.
Science 1994, 265, 1093.
4. Sun, L.; Tran, N.; Tang, T.; App, H.; Hirth, P.; McMahon,
G.; Tang, C. J. Med. Chem. 1998, 41, 2588.
5. Sun, L.; Tran, N.; Liang, C.; Tang, F.; Rice, A.; Schreck,
R.; Waltz, K.; Shawver, L. K.; McMahon, G.; Tang, C. J.
Med. Chem. 1999, 42, 5120.
6. Sun, L.; Tran, N.; Liang, C.; Hubbard, S.; Tang, F.; Lip-
son, K.; Schreck, R.; Zhou, Y.; McMahon, G.; Tang, C. J.
Med. Chem. 2000, 43, 2655.
7. Morin, M. J. Oncogene 2000, 19, 6574.
8. Davoll, J. J. Chem. Soc. 1960, 131.
9. Hennequin, L. F.; Thomas, A. P.; Johnstone, C.; Stokes,
E. S. E.; Ple, P. A.; Lohmann, J.-J. M.; Ogilvie, D. J.; Dukes,
M.; Wedge, S. R.; Curwen, J. O.; Kendrew, J.; Brempt, C. L. D.
J. Med. Chem. 1999, 42, 5369.
In conclusion, we have discovered 5,7-diazaindolinones
as a novel class of potent and selective inhibitors of
EGF-R and Her2 (p185erbB) tyrosine kinases. These
compounds exhibited low nanomolar IC50 values against
the EGF-R kinase without inhibiting PDGF-R and other
related kinases. Of particular interest, these compounds
have been found to show inhibitory activity toward not
only the EGF-R but also Her2 (p185erbB) kinases in
cells. Moreover, 26q has been found to exhibit efficacy in
tumor-bearing mice.10 This later finding suggests that these
substituted 5,7-diazaindoliones may be developed for the
treatment of human cancers that require both EGF-R
and Her2 (p185erbB) kinase for growth and survival.
10. Christensen, J. G.; Schreck, R. E.; Chan, E.; Wang, X.;
Yang, C.; Liu, L.; Cui, J.; Sun, L.; Wei, J.; Cherrington, J. M.;
Mendel, D. B. Clin. Cancer Res. 2001, 7, 4230.