Substituted 5,7-Diphenyl-pyrrolo[2,3d]pyrimidines: Potent inhibitors of the tyrosine kinase c-Src

Article abstract of DOI:10.1016/S0960-894X(00)00131-1

5,7-Diphenyl-pyrrolo[2,3d]pyrimidines represent a new class of highly potent inhibitors of the tyrosine kinase c-Src (IC₅₀ < 50 nM) with specificity against a panel of different tyrosine kinases. The substitution pattern on the two phenyl rings

Full text of DOI:10.1016/S0960-894X(00)00131-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 945±949
Substituted 5,7-Diphenyl-pyrrolo[2,3d]pyrimidines:
Potent Inhibitors of the Tyrosine Kinase c-Src
Martin Missbach, a,* Eva Altmann, a Leo Widler, a Mira Susa, a
Elisabeth Buchdunger, b Helmut Mett, b Thomas Meyer b and Jonathan Green a
aNovartis Pharma AG, Therapeutic Areas Arthritis & Bone Metabolism, CH-4002 Basel, Switzerland
bNovartis Pharma AG, Department of Oncology, CH-4002 Basel, Switzerland
Received 2 February 1999; accepted 22 February 2000
AbstractÐ5,7-Diphenyl-pyrrolo[2,3d]pyrimidines represent a new class of highly potent inhibitors of the tyrosine kinase c-Src
(IC50 <50 nM) with speci®city against a panel of di€erent tyrosine kinases. The substitution pattern on the two phenyl rings
determines potency and speci®city and provides a means to modulate cellular activity. # 2000 Elsevier Science Ltd. All rights
reserved.
Chemistry and methods
Introduction
Compelling evidence indicates that the protein tyrosine
kinase c-Src plays a unique and crucial role in osteo-
clastic bone resorption.1 4 Mutant mice with a dis-
rupted Src gene have functionally inactive osteoclasts in
the absence of any other overt pathological signs. c-Src
may be responsible for the phosphorylation of cytoske-
letal and/or docking proteins involved in the transloca-
tion and exocytosis of vesicles during the resorptive
process. Inhibitors of c-Src may be useful for the treat-
ment of diseases characterized by excessive bone
resorption such as osteoporosis and tumor-induced
hypercalcemia. In addition, c-Src inhibitors could also
be bene®cial in the treatment of c-Src overexpressing
tumors, for example colon carcinoma.
The synthesis of a few 5,7-diphenyl-pyrrolo[2,3d]pyr-
imidines had been described earlier,7 but without any
data on kinase inhibition. Our substituted 5,7-diphenyl-
pyrrolo[2,3d]pyrimidines have been prepared as outlined
by a generic example8 in Scheme 1.
The ®rst two steps to form the 2-amino-3-cyano-pyrrol
usually gave yields between 50 and 80%. The sub-
sequent 3-step cyclization to the pyrrolo[2,3d]pyr-
imidine resulted in purer products than the alternative
one pot reaction with formamide at high temperature
and produced good yields of 65±85% over all three
steps. 5,7-Diphenyl-pyrrolo[2,3d]pyrimidines with basic
side chains were obtained by conversion of the hydroxy-
methyl intermediate into the corresponding chloride,
followed by treatment with various amines (neat or in a
polar solvent). The proposed binding mode of CGP
62464 (compared to that of ATP) based on the pub-
lished X-ray structure of human Src9 is shown in Figure 2.
According to this model, the 5,7-diphenyl-pyrrolo-
[2,3d]pyrimidines bind at the ATP binding site in an
orientation similar to that of ATP, making use of the
same two hydrogen-bonds to and from the backbone of
the enzyme (Met 341 and Glu 339). The 5-phenyl locks
into a rather small pocket, which is not used by ATP
itself. Similar interactions, as well as the existence of a
hydrophobic pocket, have been proposed for the bind-
ing mode of EGF-receptor inhibitors.10
We started our investigation of substituted 5,7-diphenyl-
pyrrolo[2,3d]pyrimidines as potential c-Src inhibitors
based on the ®nding that the closely related pyrrazolo-
pyrimidine CGP 191 inhibited c-Src phosphorylation
with an IC50 of 0.5 mM. The pyrrolopyrimidine analogue
CGP 62464 proved to be ®ve times more potent
(IC50=0.1 mM).5 We therefore concentrated our e€orts
on optimising the 5,7-diphenyl-pyrrolo[2,3d]pyrimidine
class with the aim to identify potent and speci®c com-
pounds with good cellular activity.6
*Corresponding author. Fax: +41-61-6966071; e-mail: martin.
missbach@pharma.novartis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00131-1

946
M. Missbach et al. / Bioorg. Med. Chem. Lett. 10 (2000) 945±949
Figure 1.
Results and Discussion
leads to a compound mainly present in its carbonyl
tautomeric form) or chlorine as well as N-methylation,
which prevents this interaction, resulted in a dramatic
loss of potency (compounds 1b±1d, Table 1). Further-
more, the substitution pattern on the two phenyl rings
proved to be important, for potency as well as speci®-
city. Only limited variations were tolerated at the phenyl
ring in the 5 position (compounds 1e±1i). A large sub-
stituent in 40 position (1i) or substitution in 20 position
(1h) lead to decreased activity, which can be rationalised
based on the size of the hydrophobic pocket. At the
phenyl ring in position 7 even large substituents in the
30- or 40-position (but not in the 20-position; compounds
1k and 1o) lead to potent c-Src inhibitors. This ®nding
can again be explained by the proposed binding mode
since substituents in 30- and 40-position reach the surface
of the enzyme, whereas substituents in 20-position would
disturb the enzyme structure. By adding the aminoethyloxy
All the 5,7-diphenyl-pyrrolo[2,3d]pyrimidines synthe-
sised were tested for c-Src inhibition in a liquid phase
tyrosine phosphorylation assay with chicken c-Src and
the synthetic substrate poly-Glu-Tyr (4:1).11 Inhibitors
with an IC50 below 0.5 mM were tested further for spe-
ci®city in a panel of di€erent in vitro kinase enzyme
assays including receptor (EGF-R, VEGF-receptor
Kdr) and non-receptor tyrosine kinases (v-Abl), as well
as a serine/threonine kinase implicated in cell cycling
(Cdc2).
The SAR developed with 5,7-diphenyl-pyrrolo[2,3d]pyr-
imidines (Table 1) can be summarized as follows: The
two hydrogen bonds indicated in Figure 2 are important
for activity. Replacement of the amino-substituent in
the 4-position of the pyrimidine ring by hydroxy (which
Scheme 1. Synthesis of 5,7-diphenyl-pyrrolo[2,3d]pyrimidines.8 (A) NaHCO3, EtOH, 50±80 ꢀC; (B) malonodinitrile, NaOEt, EtOH, 50±80 ꢀC,
(A+B: 50±80%); (C) triethylorthoformiate, 80±120 ꢀC, acidic work up; (D) NH3, MeOH, rt; (E) cat. NaOEt, EtOH, re¯ux; then 2 N NaOH/THF
overnight (C±E: 65±85%); (F): chlorenamine, THF, rt; (G) R1R2NH, EtOH, 100 ꢀC, sealed tube (75±95%); (H) R1R2NH (excess, no solvent), 70±
120 ꢀC (80±95%).

M. Missbach et al. / Bioorg. Med. Chem. Lett. 10 (2000) 945±949
947
side chain both speci®city and solubility (data not
shown) could be improved while retaining high potency
(compounds 1p±1r; 1t).
The cellular potency of compounds 1a, 1e, 1l and 1p was
in the micromolar range (Table 2). But for compounds
1q and 1r with comparable potency in the liquid phase
assay but with polar 40-substituents at the 7 phenyl ring
inhibition of c-Src in intact cells below 1 mM could be
achieved. Increasing the length of the side chain by one
carbon (1t) resulted in a strong c-Src inhibitor in the
liquid phase enzyme assay and in intact cells that had a
favorable speci®city pro®le against other kinases. For
the amide 1s similar potency in the liquid phase assay
but a clear drop in cellular activity was observed, which
indicated the importance of a free amino group in the
side chain. In intact cells, good speci®city was found
with these inhibitors against all kinases investigated,
Inhibition of cellular phosphorylation was investigated
by measuring the c-Src mediated phosphorylation of the
120 kDa protein FAK in IC8.1 ®broblasts transfected
with chicken c-Src (Western blot analysis).12 For speci-
®city determination the compounds were tested in the
same cellular system for the inhibition of the c-Src
deactivating kinase Csk. In separate experiments the
inhibition of receptor autophosphorylation in intact
cells after stimulation with the corresponding growth
factor was measured (cellular ELISA; Table 2).13
Table 1. Potency and speci®city of 5,7-diphenyl-pyrrolo[2,3d]pyrimidines
Compound
R1
R2
R3
c-Src
(mM)
EGF-R
(mM)
Kdr
(mM)
v-Abl
(mM)
Cdc-2
(mM)
mp
(ꢀC)
1a
1b
1c
1d
1e
1f
H
H
H
NH2
Cl
0.1
8.0
0.5
nd
0.5
nd
0.02
nd
54.0
nd
200
H
155±157
255
120
H
H
OH
10.0
>10.0
nd
nd
nd
nd
nd
>10.0
43.0
H
H
NHCH3
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
31.0
0.5
0.7
0.35
2.65
1.3
1.1
0.6
0.7
0.8
6.4
3.4
1.4
0.3
0.8
0.3
>10.0
0.06
0.6
30-OCH3
30-Cl
40-OCH3
20-CH3
40-COOEt
H
H
0.05
0.2
>1.0
1.0
159±160
173±175
187±188
181±182
158±160
174±175
165
H
H
>10.0
>10.0
90.0
1g
1h
1i
0.15
6.0
0.2
0.4
H
nd
nd
H
10.0
0.3
1.1
>10.0
3.4
0.2
>10.0
>10.0
47.0
1k
1l
20-CH2OH
nd
nd
H
30-CH2OH
0.06
0.02
0.06
5.0
1m
1n
1o
1p
1q
1r
1s
1t
H
40-CH2OH
0.6
0.07
0.05
nd
30.0
33.0
207±209
151±152
170
30-OCH3
30-CH2OH
>1.0
nd
H
20-CH2NH(CH2)2OH
30-CH2NH(CH2)2OH
40-CH2NH(CH2)2OH
40-CH2NH(CH2)2OH
40-CH2CONH-(CH2)2OH
40-CH2CH2N(CH3)-(CH2)2OH
>10.0
>10.0
82.5
H
0.01
0.03
0.03
0.02
0.03
3.6
nd
265±Dec.
120±122
126±128
148±150
104±106
H
nd
1.0
30-OCH3
30-OCH3
30-OCH3
>1.0
>1.0
>1.0
0.23
0.22
0.3
>10.0
>10.0
>10.0
Table 2. Cellular potency and speci®city of selected 5,7-diphenyl-pyrrolo[2,3d]pyrimidines:
Compound
R1
R2
c-Src Enz.
(mM)]
c-Src Cell12
Csk Cell12
EGF-R Cell13
PDGF-R Cell13
(mM)
(mM)
(mM)
(mM)
1a
1e
1l
H
30-OCH3
H
H
0.1
3.5
3.3
>5.0
>5.0
>5.0
5.0
3.0
>10.0
12.0
nd
nd
2.5
10.0
nd
H
0.05
0.06
0.01
0.03
0.03
0.02
0.03
30-CH2OH
>5.0
4.0
1p
1q
1r
1s
1t
H
30-CH2NH(CH2)2OH
40-CH2NH(CH2)2OH
40-CH2NH(CH2)2OH
40-CH2CONH-(CH2)2OH
40-CH2CH2N(CH3)-(CH2)2OH
H
0.7
0.5
4.3
nd
nd
30-OCH3
30-OCH3
30-OCH3
>5.0
>5.0
>5.0
6.4
0.6
nd
3.9
0.2
nd
10.0
0.3

948
M. Missbach et al. / Bioorg. Med. Chem. Lett. 10 (2000) 945±949
Figure 2.
References and Notes
except for the PDGF-receptor, which was inhibited at
concentrations comparable to those necessary for c-Src
inhibition.
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11(suppl 1), T393.
Conclusions
Our optimization strategy resulted in highly potent and
remarkably selective inhibitors of the tyrosine kinase c-
Src in vitro. They exhibit potent inhibition of c-Src
mediated phosphorylation of intracellular substrates in
intact cells. Polar substituents at the phenyl ring in
position
7 of 5,7-diphenyl-pyrrolo[2,3d]pyrimidines
6. Missbach, M.; Green, J.; Susa M.; Jeschke, M.; Buchdunger,
E.; Mett, H.; Meyer, Th. 215th ACS National Meeting, Dallas,
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inhibitors: Hollis Showalter, H. D.; Kraker, A. J. Pharmacol.
Ther. 1997, 76, 55±71.
increase the potency of c-Src inhibition in the enzyme
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Patel, R. B. Indian J. Chem., Sect. B 1988, 27B, 778.
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pounds synthezised were characterized by melting point, TLC/
Acknowledgements
We are grateful to Dr. T. Hall for initiating the c-Src
inhibitor project.
1
HPLC; H NMR, MS (M++1).

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949
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