7-pyrrolidinyl- and 7-piperidinyl-5-aryl-pyrrolo[2,3-d]pyrimidines - Potent inhibitors of the tyrosine kinase c-Src

Article abstract of DOI:10.1016/S0960-894X(01)00080-4

7-Heterocyclyl-5-aryl-pyrrolo[2,3-d]pyrimidines represent a new class of highly potent and selective inhibitors of the tyrosine kinase pp60^c-Src.

Full text of DOI:10.1016/S0960-894X(01)00080-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 853–856
7-Pyrrolidinyl- and 7-Piperidinyl-5-aryl-pyrrolo[2,3-d]-
pyrimidines—Potent Inhibitors of the Tyrosine Kinase c-Src
Eva Altmann,* Martin Missbach, Jonathan Green, Mira Susa,
Hans-Achim Wagenknecht and Leo Widler
Novartis Pharma Research, Arthritis and Bone Metabolism Therapeutic Area, CH-4002 Basel, Switzerland
Received 15 December 2000; revised 25 January 2001; accepted 2 February 2001
Abstract—7-Heterocyclyl-5-aryl-pyrrolo[2,3-d]pyrimidines represent a new class of highly potent and selective inhibitors of the
tyrosine kinase pp60c-Src. # 2001 Elsevier Science Ltd. All rights reserved.
In the preceding paper1 we have described a series of
novel 7-substituted 5-aryl-pyrrolo[2,3-d]pyrimidines as
potent inhibitors of the tyrosine kinase pp60c-Src (c-Src).
c-Src is an essential enzyme for osteoclastic bone
resorption2,3 and synthetic c-Src inhibitors have been
shown to interfere with this process both in vitro4 and in
vivo.5 We report herein on a second optimization strat-
egy for our lead compound 1 (5,7-diphenyl-pyrrolo[2,3-
d]pyrimidine), which involves replacement of the N7-
phenyl moiety by different types of heterocycles. In
analogy to the strategy described previously1 these het-
erocyclic N7-substituents are assumed to occupy the
pocket which is usually utilized by the ribose moiety of
ATP. Substitution of polar heterocyclic moieties for the
N7-phenyl substituent in 1 might result in additional
interactions with polar amino acids within this sugar
pocket that are not possible with 1 (Fig. 1).
rolidine or piperidine as well as analogues that are
derived from 2-ethoxycarbonyl or 2-hydroxymethyl
pyrrolidine, respectively.
A representative synthesis of N7-heterocyclyl-5-aryl-
pyrrolo-pyrimidines is outlined in Scheme 1. The pre-
paration of 4-amino-5-aryl-7H-pyrrolo[2,3-d]pyrimidine
7 was already described in the preceding paper.1
Attachment of the N-Boc-protected heterocycle6 6 to 7
was achieved via mesylate displacement in the presence
of potassium carbonate and 18-crown-6 in DMF at
55 ꢀC.7 Removal of the Boc protecting group with 4 N
HCl in Et2O gave 4k which was subsequentely alkylated
with methyl bromoacetate. Treatment of ester 4n with
ammonia in MeOH resulted in amide 4o.
As illustrated by the data summarized in Table 1, for 2-
substituted pyrrolidine derivatives with trans stereo-
chemistry the N-Boc protected compounds (2a, b and
3a, b) were always slightly more active than the corre-
sponding free amines (2c, d and 3c, d). In contrast, the
We have investigated four different types of heterocycles
attached to the N7-position of the pyrrolo-pyrimidine
moiety. These include N-substituted derivatives of pyr-
Figure 1.
*Corresponding author. Fax: +41-61-6966071; e-mail: eva.altmann@pharma.novartis.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00080-4

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E. Altmann et al. / Bioorg. Med. Chem. Lett. 11 (2001) 853–856
Scheme 1. (a) K2CO3, 18-crown-6, DMF, 55 ꢀC, 51%; (b) 4 N HCl/Et2O, rt, 70%; (c) BrCH2CO2CH3, DMF, DIEA, 70%; (d) NH3, MeOH, sealed
tube, 60 ꢀC, 83%.
Table 1. Inhibition of c-Src enzyme activity and cellular activity of inhibitors 2 and 3
Compd
R1
R2
R3
c-Src (enzyme)a IC50 (mM)
c-Src (cell)b IC50 (mM)
2a
2b
2c
2d
3a
3b
3c
3d
H
OH
H
OH
H
OH
H
OH
CH2CO2CH2CH3
CH2CO2CH2CH3
CH2CO2CH2CH3
CH2CO2CH2CH3
CH2OH
CO2t-Bu
CO2t-Bu
H
0.41
n.d.c
0.70
n.d.c
n.d.c
n.d.c
3.3
0.035
0.60
0.10
H
CO2t-Bu
CO2t-Bu
H
0.13
CH2OH
CH2OH
CH2OH
0.024
0.30
0.095
n.d.c
>5
H
2e
2f
2g
2h
3e
3f
H
OH
H
OH
H
OH
H
OH
CH2CO2CH2CH3
CH2CO2CH2CH3
CH2CO2CH2CH3
CH2CO2CH2CH3
CH2OH
CO2t-Bu
CO2t-Bu
H
5
n.d.c
n.d.c
>5
0.30
0.015
0.005
2
0.35
0.13
0.05
H
3.3
CO2t-Bu
CO2t-Bu
H
n.d.c
n.d.c
n.d.
>5
CH2OH
CH2OH
CH2OH
3g
3h
H
aInhibition of c-Src enzyme activity in the liquid-phase tyrosine phosphorylation assay, c-Src concentration: 830 ng/mL, IC50 values are the mean of
2 experiments carried out in duplicate, individual data points in each experiment were within a 3-fold range with each other.
bInhibition of c-Src mediated phosphorylation of Fak in IC8.1 fibroblasts.5
cNot determined.
free amines (2g, h and 3g, h) proved to be more potent
inhibitors of c-Src in the cis-pyrrolidine series. However,
despite the fact that nanomolar potency was achieved
for enzyme inhibition in some cases, none of the pyrro-
lidine derived analogues exhibited submicromolar inhi-
bition of c-Src in cells.
substituent at this position is not required for low
nanomolar activity.
Methylation of the 4-OH or 3-OH substituent on the 5-
phenyl ring as in compounds 4e, f and 4i, k proved to be
less favorable for c-Src inhibition, resulting in a 10–50-
fold loss in potency in comparison with 4c, d and 4g, h,
respectively. These 2-unsubstituted analogues were then
further modified by alkylation at the pyrrolidine nitro-
gen with bromoacetic acid methyl ester. These N-sub-
In order to investigate the role of the substituent at the
2-position of the pyrrolidine ring we prepared the race-
mic derivatives 4a–d (Table 2). Rather surprisingly, a

E. Altmann et al. / Bioorg. Med. Chem. Lett. 11 (2001) 853–856
855
Table 2. Inhibition of c-Src enzyme activity, cellular activity, and selectivity profile of inhibitors 4
Compd
R1
R2
c-Srca (enzyme)
c-Srcb (cell)
EGF-Rc (enzyme)
v-Abld (enzyme)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4k
4l
4m
4n
4o
H
H
CO2t-Bu
H
CO2t-Bu
H
CO2t-Bu
H
CO2t-Bu
H
CO2t-Bu
H
CH2CO2CH3
CH2CONH2
CH2CO2CH3
CH2CONH2
0.18
0.23
0.008
0.037
0.40
>5
n.d.e
0.40
2.7
8.3
13.4
n.d.e
2.67
n.d.e
n.d.e
2.2
1.53
2.1
1.12
0.74
0.4
n.d.e
n.d.e
n.d.e
2.8
n.d.e
n.d.e
0.058
0.29
2.4
4-OH
4-OH
4-OMe
4-OMe
3-OH
3-OH
3-OMe
3-OMe
3-OH
3-OH
3-OMe
3-OMe
0.4
0.30
n.d.e
3.1
0.005
0.006
0.27
0.053
0.003
0.004
0.050
0.038
0.80
4.4
>5
0.7
0.4
1.4
0.054
0.15
0.59
0.7
>5
>5
0.83
0.44
aInhibition of c-Src enzyme activity in the liquid-phase tyrosine phosphorylation assay, c-Src concentration: 830 ng/mL, IC50 values are the mean of
2 experiments carried out in duplicate, individual data points in each experiment were within a 3-fold range with each other.
bInhibition of c-Src mediated phosphorylation of Fak in IC8.1 fibroblasts.5
cInhibition of epidermal growth factor receptor (EGF-R) tyrosine kinase enzyme activity.
dInhibition of v-Abl tyrosine kinase enzyme activity.
eNot determined.
Table 3. Inhibition of c-Src enzyme activity, cellular activity, and selectivity profile of inhibitors 5
Compd
R1
R2
c-Srca (enzyme)
c-Srcb (cell)
EGF-Rc (enzyme)
v-Abld (enzyme)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
OH
OH
OH
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
CH2CO2CH3
CH2CONH2
CH2CON(CH3)2
CH2CH2OH
CH2CO2CH3
CH2CONH2
CH2CON(CH3)2
CH2CH2OH
<0.001
0.001
0.003
0.001
0.084
0.022
0.067
0.027
0.006
0.015
0.047
0.4
0.8
0.5
0.6
2.8
0.7
>5
1.4
0.6
0.29
0.5
0.8
0.17
0.5
0.056
0.21
0.12
0.098
1
0.55
0.51
1.03
0.36
0.26
0.50
2.54
1.47
0.35
0.75
0.9
0.295
0.31
0.93
(CH2)2N(CH3)(CH2)2OH
(CH2)2NH(CH2)2OCH3
(CH2)2N(CH3)(CH2)2OCH3
5k
5l
aInhibition of c-Src enzyme activity in the liquid-phase tyrosine phosphorylation assay, c-Src concentration: 830 ng/mL, IC50 values are the mean of
2 experiments carried out in duplicate, individual data points in each experiment were within a 3-fold range with each other.
bInhibition of c-Src mediated phosphorylation of Fak in IC8.1 fibroblasts.5
cInhibition of epidermal growth factor receptor (EGF-R) tyrosine kinase enzyme activity.
dInhibition of v-Abl tyrosine kinase enzyme activity.
stituents were designed to interact with amino acid side
chains that are located in the vicinity of the triphos-
phate binding pocket. From these derivatives, those
bearing a 3-OH substituent on the 5-phenyl ring are
nanomolar c-Src inhibitors with a favorable selectivity
profile. In the view of the interesting results obtained
with the pyrrolidine derivatives it appeared mandatory
to investigate the activity of the corresponding piper-
idinyl-derivatives, particularly in view of the fact that
these compounds are achiral.
loss of potency for c-Src inhibition compared to the 3-
OH analogues 5a–5d and are generally not potent in the
cellular assay. This finding is consistent with what we
observed for the corresponding pyrrolidine derivatives
(4g, h!4i, k). Finally attachment of a large substituent
incorporating a basic nitrogen at the piperidine nitrogen
led to compounds 5i–l, which apart from high potency
for c-Src inhibition exhibit cellular activity with sub-
micromolar IC50 values in combination with a favorable
selectivity profile.8,9
Table 3 summarizes the c-Src inhibitory activity of the
N7-piperidinyl analogues 5. Out of this series, 5a–d,
bearing a 3-OH substituent on the 5-phenyl ring, are
low nanomolar inhibitors of c-Src, which also exhibit
good cellular activity, inhibition of cellular phosphor-
ylation of Fak with submicromolar IC50s. In addition,
5a–d possess a remarkable selectivity profile with regard
to the inhibition of EGF-R- and v-Abl-kinases.
The above summarized optimization strategy resulted in
novel, extremly potent and remarkably selective inhibi-
tors of the tyrosine kinase c-Src.
Acknowledgements
The authors thank Dr. H. Mett for providing the speci-
ficity assays and B. Fluckiger, D. Hurzeler, H. Jeker, R.
Klein, N.-H. Luong-Nguyen, M. Schaublin and Y. Sel-
tenmeyer for their excellent technical assistance.
We therefore investigated the O-methylated analogues
5e–h. These 3-methoxy derivatives show a 22- to 84-fold

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E. Altmann et al. / Bioorg. Med. Chem. Lett. 11 (2001) 853–856
(Table 1) is described in Baker, G. L.; Fritschel, S. J.; Stille,
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8. IC50 values for inhibition of lck, another src kinase family
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exception (5i, IC50=7.8 mM).
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