Pyrrolo[2,3-d]pyrimidines containing an extended 5-substituent as potent and selective inhibitors of lck I

Article abstract of DOI:10.1016/S0960-894X(00)00441-8

Pyrrolo[2,3-d]pyrimidines containing a 5-(4-phenoxyphenyl) substituent are potent and selective inhibitors of lck in vitro; some compounds are selective for lck over src. Data are shown for two compounds demonstrating that they are potent and selective in

Full text of DOI:10.1016/S0960-894X(00)00441-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2167±2170
Pyrrolo[2,3-d]pyrimidines Containing an Extended 5-Substituent
as Potent and Selective Inhibitors of lck I
Lee D. Arnold,a David J. Calderwood,a Richard W. Dixon,a David N. Johnston,b
Joanne S. Kamens,a Rainer Munschauer,a Paul Ra€ertyb,* and Sheldon E. Ratnofskya
aBASF Bioresearch Corporation, 100 Research Drive, Worcester, MA 01605-5314, USA
bBASF Pharma, Pennyfoot Street, Nottingham, NG1 1GF, UK.
Received 2 June 2000; accepted 12 July 2000
AbstractÐPyrrolo[2,3-d]pyrimidines containing a 5-(4-phenoxyphenyl) substituent are potent and selective inhibitors of lck in vitro;
some compounds are selective for lck over src. Data are shown for two compounds demonstrating that they are potent and selective
inhibitors of IL2 production in cells. # 2000 Elsevier Science Ltd. All rights reserved.
lck, an src-family tyrosine kinase expressed primarily in
T lymphocytes, plays an essential role in the immune
response.1 A selective inhibitor of lck should speci®cally
inhibit T-cell activation and have use in the treatment of
autoimmune and in¯ammatory diseases and also in organ
transplantation. The src-family kinases src, lck, lyn, hck,
blk, fyn, fgr, yes, and yrk share a high level of homology
at the amino acid level (>70% identical in the kinase
domain) and have varying patterns of expression in the
body. A signi®cant challenge in the discovery of a lck
inhibitor is in achieving selectivity within this family.
Comparison of these structures suggests that there may
be more structural di€erence between inactive forms of
di€erent src-family kinases thus a€ording an increased
opportunity for obtaining selectivity.7
We screened inhibitors using two constructs of the
human lck kinase, lck (64±509), and lckcd.8 Using
phosphopeptide mapping and MS we determined that
lck (64±509) is in the inactive state (activation loop
Y394 not phosphorylated) while lckcd is in the active
state (activation loop Y394 is phosphorylated). Com-
pounds inhibited the inactive form of lck more potently
than the active form. The phosphorylation state of
Y394 was determined to be solely responsible for these
di€erences by studies using enzymatically dephos-
phorylated and phosphorylated versions of each of the
lck protein constructs (data not shown).
All src-family kinases have a similar domain structure,2
comprised of an Nt unique domain, an SH3 domain, an
SH2 domain, a tyrosine kinase catalytic domain and a
short Ct tail. Activity is regulated by tyrosine phos-
phorylation at two sites. Phosphorylation of a tyrosine
(Y505) in the Ct tail leads to down regulation by pro-
moting an intramolecular interaction between the Ct tail
and the SH2 domain. Phosphorylation of a tyrosine
(Y394) in the activation loop segment of the kinase
domain activates kinase activity.
Two crystal structures of the catalytic domain of lck in
its active state (Y394 phosphorylated) have been pub-
lished, one of the apoenzyme3 and the other complexed4
with AMP-PNP, staurosporine or PP2. The structure of
two src-family kinases, in their inactive states (Y394
unphosphorylated) has also been determined; namely src5
and hck6 complexed with AMP-PNP and hck7 with PP1.
Compounds were also screened against src, however,
the activation state of the src enzyme was not deter-
mined.
*Corresponding author. Tel.: +44-115-912-4260; fax: +44-115-912-
4187; e-mail: paul.ra€erty@basf-pharma.co.uk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00441-8

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L. D. Arnold et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2167±2170
Nanomolar inhibitors of lck, e.g., PP1 (1) and PP2 had
been described.9 These compounds were reported as
selective for src-family kinases over the non-receptor
tyrosine kinase ZAP70 and the receptor tyrosine kinases
JAK2 and EGFR.
To explore structure±activity relationships (SARs) and
to improve synthetic accessibility, N7 alkyl and
cycloalkyl substituted analogues12 of 3 were synthesised
(Table 2). Compounds 4 to 9 in Table 2 are potent
inhibitors of lck (64±509) with reduced potency against
lckcd; they are selective for both forms of lck over kdr
and tie-2. All these compounds demonstrate selectivity
for lck (64±509) over src; however, only compounds 3,
6, 7, 8, and 9, show greater than 30-fold selectivity for
lckcd over src, which in view of the uncertain activation
state of the src enzyme used is the most stringent selec-
tivity measure. All the compounds are competitive with
ATP.
This, and the following report, describe pyrrolo[2,3-
d]pyrimidines containing an extended substituent in the
5-position which are potent inhibitors of lck, with some
compounds being selective over src.10
Results and Discussion
From a library of 4-arylaminoquinazolines, the 4-phenox-
phenylquinazoline 2 was identi®ed as a potent inhibitor
of lck (64±509) (Table 1). We reasoned that the phenoxy-
phenyl moiety of 2 occupies a hydrophobic pocket,
present in a number of kinases, which is not accessed by
ATP.11 Also, we considered that PP1 and ATP may
bind to lck in a similar manner (i.e., the 4-NH2 group
and N5 of PP1 form the H-donor/H-acceptor system)
the t-butyl group occupies the sugar pocket and the
5-(4-tolyl) group is positioned in the hydrophobic
pocket. Replacement of the methyl substituent in PP1
by a phenoxy group and conversion of the pyrazole into
a pyrrole ring ought lead to a novel lck inhibitor 4, with
increased potency over PP1 by virtue of improved
occupation of the hydrophobic pocket. Evaluation of 4
(Table 1) con®rmed that it is a potent inhibitor of lck
(64±509), being at least 75-fold more potent than PP1.
The 3-hydroxycyclopentyl analogue 8 is the most potent
inhibitor of lck of this set of compounds. The hydroxy-
cyclopentyl analogues 7, 8, and 9 are mixtures of stereo-
isomers; no separation or resolution was attempted.16
Varying the position of the phenoxy substituent, as in
compounds 10 and 11, led to a large loss of potency
against both forms of lck.
The activity of 1, 6, and 8 against a range of kinases and
in cellular assays13 is in Tables 3 and 4. They are selective
for lck over a range of receptor, non-receptor tyrosine
kinases and seronine/threonine kinases. The cellular
pro®le of 6 and 8 compared with PP1 is in Table 4. The
pyrrolopyrimidines 6 and 8 are potent inhibitors of IL2
production in Jurkat cells stimulated with anti-CD3
antibody, being at least 100-fold more potent than PP1.
The increased potency of 8 in vitro is mirrored by its high
potency in cells. Both 6 and 8 display remarkable cellular
selectivity. Their potencies for inhibition of IL2 pro-
duction are in the micromolar range when the cells are
stimulated with phorbol 12-myristyl 13-acetate (PMA)
in the presence of ionophore. Such stimulation bypasses
lck indicating minimal inhibition of PKC or down-
stream kinases.
Modelling
Compared to PP1, 4 showed selectivity for lck (64-509)
and lckcd (300- and 30-fold, respectively) over the closely
related enzyme src and demonstrated at least 100-fold
selectivity for lck over the human receptor tyrosine
kinases kdr and tie-2. The 4-phenoxy group confers
both improved potency for lck and selectivity over src.
The pyrrolo[2,3-d]pyrimidine analogue of PP2, 3, is
equipotent to PP1, further supporting the crucial role of
the phenoxy group in inhibition of lck.
A homology model of lck based on Kuriyan's inactive
hck (PDB accession code 1AD5) structure6 was con-
structed in Quanta using the program Modeler. The
pyrrolopyrimidine ring of 6 (the structure of 6 was built
in Quanta and minimized in Mopac) was overlaid with
the adenine ring of AMP-PNP and the system mini-
mised using Charmm. An excellent ®t of 6 into the ATP
binding site of lck was observed, the phenoxyphenyl
group having near optimal occupation of the hydro-
phobic pocket. The 4-NH2 group makes a hydrogen
bond donor contact with the backbone carbonyl of
Glu317 and the N-3 of the pyrimidine ring accepts a
hydrogen bond from the backbone NH of Met319. The
cyclopentyl ring is in the space occupied by the ribose
ring of AMP-PNP.
Table 1. Inhibition of lck (64±509), lckcd, src, kdr and tie-2 for
compounds 1±4 (IC50s, 5 mM ATP)
Compound
lck (64±509)
lckcd
src
kdr
tie-2
1
2
3
4
0.151
<0.008
0.22
0.25
NT
NT
0.014
0.17
NT
0.15
0.61
1.6
7
Comparison of the crystal structures of active and
inactive src family kinases suggest that the hydrophobic
pocket accessed by the phenoxyphenyl moiety of 6 in
our model is subject to dramatic changes in size and
2.72
2.68
2.29
17.87
7
2.63
0.002

L. D. Arnold et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2167±2170
2169
Table 2. Inhibition of lck (64±509), lckCD, src, kdr and tie-2 for compounds 4±11 (IC50 values)
lck(64-509)
lckcd
src
kdr
tie-2
Compound
R1
PhO
5 mM ATP
1 mM ATP
5 mM ATP
1 mM ATP
5 mM ATP
5 mM ATP
5 mM ATP
4
5
6
7
8
9
10
11
t-Butyl
i-Propyl
4
4
4
4
4
4
3
2
0.002
<0.001
<0.001
<0.001
<0.001
<0.002
0.14
0.075
0.011
0.016
0.24
0.005
0.023
1.18
0.0138
0.01
NT
NT
0.61
0.048
0.07
2.29
0.43
1.57
1
0.085
0.27
7.1
2.68
0.43
1.98
0.6
0.0583
0.15
3.4
Cyclopentyl
2-OH-Cyclopentyl
3-OH-Cyclopentyl
3,4-Di-OH-cyclopentyl
Cyclopentyl
0.002
<0.008
<0.001
0.005
1.2
1.07
1.32
0.544
0.317
>10
NT
0.04
0.088
0.084
>1
Cyclopentyl
0.39
NT
NT
NT
>50
4.9
Table 3. Kinase activity of 6 and 8
Compound Zap70 EGFR PKC CDC2/CyB lck (64±509)
from blk, Ser323 to Cys). However, there are di€erences
between lck and src in the hydrophobic binding pocket,
the nature and structure of which are likely due to the
relative orientation of the N and C lobes and the activation
state of the protein. Rationalisation of selectivity awaits a
crystal structure comparison with bound inhibitor.
6
8
>50
>50
3.2
NT
>33
>33
>50
>50
<0.001
<0.001
Maintaining the H-bond donor/acceptor interactions
for 10 and 11 with Glu317 and Met319, respectively, in
the lck homology model prevents the phenoxyphenyl
moiety from making optimum use of the hydrophobic
pocket. Alternatively the 4-NH2 and the pyrimidine N3
cannot make productive H-bond contacts with the pro-
tein if the phenoxyphenyl group is docked into the
hydrophobic pocket.
Table 4. Cellular activity of compounds 1, 6, and 8
Compound
Jurkat cell
antiCD3
Jurkat cell
PMA
MLR
1
6
8
0.1±1.2
<0.001±0.04
<0.001
NT
1.5
3
0.3±0.4
0.2±0.8
0.028
character. Helix aC of phosphorylated lck (PDB acces-
sion code 3LCK) is rotated and drawn inward relative
to the same structural element of unphosphorylated
hck. This conformational change results in a binding
pocket approximately 20% smaller in the phosphoryl-
ated enzyme. The reduced potency of compounds 6 to 9
for lckcd compared to lck (64±509) in Table 2 is particu-
larly evident at 1 mM ATP and may be a result of the
reduced volume of the hydrophobic pocket in the active
form of lck. We propose that the increased potency of 8
against both forms of lck is due to a hydrogen bond
between the 3-hydroxyl on the cyclopentyl ring of 8 with
either Ser343 or Asp345; the 2-hydroxyl in 7 appears to
be too far away to make a similar contact.
Synthesis
Two routes were used to prepare the pyrrolopyrimidine
compounds described in this paper. Scheme 1, starting
The proposed binding mode of 6 to lck (64±509) in our
homology model (Fig. 1) is virtually identical to that
described by Kuriyan6 for PP1 bound to inactive hck
(the 5-(4-tolyl) group of PP1 only partially ®lling the
hydrophobic pocket) and to that described by Zhu5 of
PP2 bound to active lck. Currently we have no struc-
tural explanation for the apparent selectivity of 6 (or 4,
7, 8, and 9) for lck over src. The residues in the ribose
binding pocket of src family kinases are identical (apart
Figure 1. Model of 6 (yellow) and PP1 bound to lck (64±509).

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L. D. Arnold et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2167±2170
active and inactive forms of lck in vitro, some com-
pounds show selectivity over src. Compounds 6 and 8
are potent inhibitors of IL2 synthesis in cells and inhibit
the MLR. They also show cellular selectivity by virtue of
the lack of inhibition of IL2 release from Jurkat cells when
stimulated with PMA in the presence of ionophore.
References and Notes
1. Qian, D.; Weiss, A. Curr. Opin. Cell Biol. 1997, 9, 205.
2. Sicheri, F.; Kuriyan, F. Curr. Opin. Cell Biol. 1997, 7, 777.
3. Yamaguchi, H.; Hendrickson, W. A. Nature 1996, 384, 484.
4. Zhu, X.; Kim, J. L.; Newcomb, J. R.; Rose, P. E.; Stover,
D. R.; Toledo, L. M.; Zhao, H.; Morgenstern, K. A. Structure
1999, 7, 651.
Scheme 1. Reagents and conditions: (1) pyridinium perbromide, acetic
acid; (2) cyclopropylamine, toluene, <15 ꢀC then rt, 20 h, 38%; (3)
malononitrile, methanol, KOH, water, <10 ꢀC then 65 ꢀC, 1 h, 53%;
(4) formamide, ammonia (gas), 200 ꢀC, 2.5 h, 31%.
5. Xu, W.; Harrison, S. C.; Eck, M. J. Nature 1997, 385, 595.
6. Sicheri, F.; Moare®, I.; Kuriyan, J. Nature 1997, 385, 602.
7. Schindler, T.; Sicheri, F.; Pico, A.; Gazit, A.; Levitzki, A.;
Kuriyan, F. J. Mol. Cell 1999, 3, 639.
8. lck (64±509) contains amino acids 64±509 of human lck
with amino acids MVL added to the Nt. The lckcd construct
contains amino acids 234±509 of human lck amino acids
MHHHHHHLVPRGS added to the Nt. All proteins were
puri®ed from insect cells. IC50 values were determined by an
enzyme linked immunosorbent (ELISA) phosphorylation
assay using the universal tyrosine kinase substrate Poly
(Glu,Tyr) 4:1 at 5 mM ATP.14
9. Hanke, J. H.; Gardner, J. P.; Dow, R. L.; Changelian, P.
S.; Brissette, W. H.; Weringer, E. J.; Pollok, B. A.; Connelly,
P. A. J. Biol. Chem. 1996, 271, 695.
10. For recent review on tyrosine kinase inhibitors, see Trax-
ler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195. See also Alt-
man, E.; Widler, L.; Missbach, M. PCT Int. Appl. WO
9728161, 1997. Chem. Abstr. 1997, 127, 220666.
11. Furet, P.; Caravatti, G.; Lydon, N.; Priestle, J.; Sowadski,
J.; Trinks, U.; Traxler, P. J. Comput.-Aided Mol. Des. 1995, 9,
465.
12. Calderwood, D. J.; Johnston, D. J.; Ra€erty, P.; Twigger,
H. L.; Munschauer, R. M.; Arnold, L. D. US Patent
6,001,839, 1999. Chem. Abstr. 1998 129, 275922.
13. Cellular assays were performed using standard protocols
and supernatants were tested by ELISA for cytokine levels.15
14. Farley, K.; Mett, H.; McGlynn, E.; Murray, B.; Lydon, B.
B. Anal. Biochem. 1992, 203, 151.
Scheme 2. Reagents and conditions: (1) pyridinium perbromide, acetic
acid; (2) potassium phthalimide, DMF, rt, 18 h; (3) malononitrile,
NaOMe, 35 ꢀC for 1 h; rt for 18 h, 47% overall; (4) triethyl ortho-
formate, 140 ꢀC, 1 h, 100% (5) ammonia (gas), methanol, <30 ꢀC then
rt for 18 h, 68%; (6) NaOEt, ethanol, re¯ux, 1 h, 83%; (7) 3,4-epoxy-
cyclopentene, Pd(PPh3)4, 0±25 ꢀC (exclude light), 65%; (8) H2, Pd/C,
EtOH (100%).
15. Coligan, J. E.; Kruisbeek, A. M.; Margulies, D. H.; She-
vach, E. M.; Strober, W. In Current Protocols in Immunology;
John Wiley & Sons: New York, 1998.
16. 7 is a racemic mixture of (1S,2R)- and (1R,2S)-trans-2-
(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2-d]pyrimidin-7-yl)
cyclopentanol. 8 is a racemic mixture of (1S,3R)- and (1R,3S)-
cis-3-(4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2-d]pyrim-
idin-7-yl)cyclopentanol. 9 is a mixture of cis and trans stereo-
isomers, namely c-4 and t-4-(4-amino-5-(4-phenoxyphenyl)-
7H-pyrrolo[3,2-d]pyrimidin-7-yl)cyclopentan-r-1,c-2-diol.
from 4-phenoxyacetophenone, is a four-step route,
exempli®ed for 6.
The more versatile route is shown in Scheme 2 for 8.
In summary, novel pyrrolopyrimidine compounds con-
taining a 5-(4-phenoxyphenyl) substituent are described,
some of which are potent and selective inhibitors of