Pyrrolo[2,3-d]pyrimidines containing diverse N-7 substituents as potent inhibitors of Lck.

Article abstract of DOI:10.1016/S0960-894X(02)00195-6

A series of pyrrolo[2,3-d]pyrimidines was synthesized and evaluated as inhibitors of Lck. Lck accommodates a diverse set of substituents at N-7. Altering the substituent at N-7 provided compound 13, an orally available lck inhibitor which inhibited TCR me

Full text of DOI:10.1016/S0960-894X(02)00195-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1683–1686
Pyrrolo[2,3-d]pyrimidines Containing Diverse N-7 Substituents as
Potent Inhibitors ofLck
David J. Calderwood,* David N. Johnston,y Rainer Munschauer and Paul Rafferty
Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605-5314, USA
Received 30 November 2001; accepted 26 February 2002
Abstract—A series of pyrrolo[2,3-d]pyrimidines was synthesized and evaluated as inhibitors of Lck. Lck accommodates a diverse set
of substituents at N-7. Altering the substituent at N-7 provided compound 13, an orally available lck inhibitor which inhibited TCR
mediated IL-2production after oral dosing. # 2002 Elsevier Science Ltd. All rights reserved.
Lck, a src family tyrosine kinase expressed primarily in
T lymphocytes, provides a critical function during the
initial steps of T-cell receptor (TCR) signaling.1 A cas-
cade of downstream signaling pathways ultimately leads
to T-cell activation and the production of cytokines
such as interleukin-2(IL-)2 and IFN g.2,3 A selective
inhibitor of lck should prevent T-cell activation and
thus have broad application for the treatment of T-cell
dependent processes such as autoimmune and inflam-
matory diseases as well as allogeneic organ transplant
rejection.
lck (64-509). The closely related kinase src and two
receptor tyrosine kinases, kdr and tie-2served as
counterscreens.
Previous work had shown that the phenoxyphenyl moi-
ety (occupying the lipophilic pocket in lck) was respon-
sible for increased potency versus lck.9 We envisaged
improving solubility whilst maintaining potency by
retaining this group and modifying the N-7 position. It
was anticipated that additional binding interactions
could be gained at the N-7 position (sugar pocket in
ATP).
Strategies toward the design and synthesis of tyrosine
kinase inhibitors have been reviewed previously.4,5 Pyr-
rolo[2,3-d]pyrimidines as c-src kinase inhibitors have
also been described.6ꢀ8 Early work from our labora-
tories described the synthesis and SAR of a series of
pyrrolo[2,3-d]pyrimidines as lck inhibitors, exemplified
by compounds 1 and 2, containing a phenoxyphenyl
group at C-5 and alkyl/cycloalkyl substituents at N-7.9
This communication describes the synthesis and SAR of
a series of pyrrolo[2,3-d]pyrimidine analogues contain-
ing a variety of substituents at N-7. Since compound 2
did not exhibit the desired in vivo10 and physicochem-
ical profile our efforts focussed on improving these
aspects whilst maintaining lck potency.
Compound 2 is a potent inhibitor of IL-2production in
cellular assays with reasonable in vivo efficacy.10
Although 2 inhibits IL-2production (anti-CD3 mAb
stimulation in mice) after ip dosing (ED50=4 mg/kg),
activity is reduced (ED50=25 mg/kg) on po admini-
stration. The pharmacokinetic parameters after a single
(25 mg/kg) oral dose (10% EtOH, 10% DMSO, 80%
olive oil as vehicle) to female Balb/c mice are outlined in
Table 1.
Inhibitors were initially screened at 1 mM ATP against
a non-phosphorylated construct of human lck kinase,
*Corresponding author. Tel.: +1-508-688-8003; fax : +1-508-688-8100;
e-mail: david.calderwood@abbott.com
yCurrent address: Oxford Asymmetry, 151 Milton Park, Abingdon,
Oxon OX14 4SD, UK.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00195-6

1684
D.J.Calderwood et al./ Bioorg.Med.Chem.Lett.12 (2002) 1683–1686
Table 1. Pharmacokinetic parameters for 2
The low peak plasma concentrations could be indicative
of either poor absorption and/or extensive first pass
metabolism. The aqueous solubility of 2 (0.44 mg/mL)
precluded dosing in optimal aqueous vehicles for
chronic studies and prevented further advancement of
this series of compounds. Efforts were undertaken to
improve the physicochemical properties of the com-
pounds specifically the goal of improving the aqueous
solubility and in vivo profile of this class of inhibitor. A
variety of strategies were adopted to address solubility,
including incorporation of alcohols, ethers, basic amines
and low molecular weight heterocycles into our tem-
plate.11 Table 2highlights certain aspects of the SAR
derived from this strategy. Kinase Homogeneous Time
Resolved Fluorescence (HTRF) data are shown at 1
mM ATP where noted.
Cmax
(nM)
Tmax
(h)
AUC
(ng h/mL)
T1/2
(h)
28
0.25
34.3
7.7a
aTerminal half-life.
Table 2. Inhibition of lck (64–509), src, kdr and tie-2for compounds
2–13 (IC50 mM) by HTRF a method
R
lck
src
kdr
tie-2
0.10
0.56
4.44
9.07
2
3b
4
All compounds are potent, ATP competitive (data not
shown) inhibitors of lck.
0.0420.32NT
0.121.1N2 T
5.15
Incorporation of heteroatoms such as oxygen and
nitrogen into the carbocyclic framework in general had
little effect on lck activity as shown in compounds 3, 4,
7, 8, and 9. Although aminoethyl analogue (6) allowed
for easy salt formation, activity versus lck dropped
9-fold. The hydroxyethyl analogue (5) was equipotent
but solubility was only marginally improved (data not
shown). Selectivity for the receptor tyrosine kinases kdr
(where shown) and tie-2is >40-fold. However, src
selctivity is generally <10-fold although the impli-
cations of this lack of selectivity are currently unclear.
11.62
5
6
0.13
1.47
NT
NT
16.71
0.89
32.34
>50
7b
8b
9
0.046
0.24
0.17
0.39
2.36
1.78
NT
NT
NT
6.62
NT
21.38
We realized that a carbocyclic framework directly
attached to N-7 was a motif that was beneficial for lck
activity and therefore elected to append ‘solubilizing
substituents’ to this motif. A cyclohexyl motif (as
opposed to the cyclopentyl moiety in 2) was chosen to
avoid inherent stereochemical issues. Compounds 10–13
were inhibitors of lck with the trans diastereoisomers 11
and 13 being more potent than the corresponding cis
diastereoisomers. The src family and csk data (HTRF
data at 1 mM ATP) for compounds 12 and 13 are
highlighted in Table 3.
10
0.525.46
NT
.16 12
11
12
09.08820.2 NT
0.24
NT
1.19
10.74
5.85
Table 3. Src family and csk profilea for 12 and 13 (IC50 mM)
13
0.015
0.0421.19
5
0.2
blk
csk
fyn
lck
lyn
12
13
0.37
0.046
4.27
0.041
2.03
0.059
0.24
0.015
0.43
0.029
aMean of two experiments perfomed with seven concentrations of test
compound.
bRacemic mixture.
aMean of two experiments performed with seven concentrations of test
compound.

D.J.Calderwood et al./ Bioorg.Med.Chem.Lett.12 (2002) 1683–1686
Table 4. Pharmacokinetic parameters for 13
Oral dosing, 10 mg/kg IV dosing, 5 mg/kg
1685
Cmax
(nM)
Tmax
(h)
AUC
(ng h/mL)
T1=2
(h)
Vz
(L/kg)
Cl
(L/h/kg)
F
(%)
113
4
661
14.3
140
5.3
54
The overall src family selectivity for 13 is low and only
ꢁ3-fold selectivity is shown for the negative regulatory
enzyme, csk. Compound 13 is inactive (IC50>20 mM, 1
mM ATP) against a panel of other kinases including
ZAP-70, cMet, IGFR, INSR, PKC, cdc2and PDGFR.
Not withstanding the observed src family selectivity
profile, compound 13 was further profiled primarily due
to the aqueous solubility of the corresponding trim-
aleate salt, which allowed for effective in vivo screening.
Compound 13 is a potent inhibitor of IL-2production
in Jurkat cells stimulated with anti-CD3 antibody
(IC50=25 nM). In a whole blood assay, it inhibits IL-2
production with an IC50=70 nM. Significantly, com-
pound 13 (trimaleate salt) inhibits TCR stimulated
(anti-CD3 mAb) IL-2production in mice (po admini-
stration, water vehicle) with an ED50 of 2.5 mg/kg,
10-fold more potent than 2 and utilizing a tolerable
dosing regimen. Compound 13 inhibited antigen specific
T-Cell immune response. After administering 13 for 3
days (po, q.d.) during the in vivo priming phase, inhi-
bition of IFN-g production was seen upon subsequent
antigen-specific (KLH) challenge of lymphocytes from
the draining lymph nodes in vitro (ED50=2.2 mg/kg).
The pharmacokinetic parameters after a single (10 mg/
kg) oral dose (water vehicle) and 5 mg/kg iv dose to
male Sprague–Dawley rats for 13 are outlined in Table 4.
Scheme 1. Reagents and conditions: (a) ROTs, NaH, DMF, 95 ꢂC,
30–60%; (b) TFA, CH2Cl2, 0 ꢂC, 90–95%.
As seen in Table 4, compound 13 has improved expo-
sure and a long half-life after oral dosing. The volume
of distribution is very high with clearance slightly above
hepatic blood flow. Bioavailability of 13 amounted to
54% with maximum plasma levels of 113 nM (above the
whole blood assay IC50 for inhibition of IL-2) at a dose
of 10 mg/kg po.
Further profiling of compounds related to 13 in T-cell
dependent disease models will be reported subsequently.
Two routes were used to prepare the pyrrolo[2,3-d]pyri-
midines described above. Scheme 1 starts from a pre-
viously described intermediate (14)9 and involves
nucleophilic displacement of the appropriate tert-
butoxycarbonyl protected tosylate (7, 8 and 9) followed
by deprotection. The tosylate starting materials were
prepared by standard methods. This sequence allowed
access to 3, 4, 7, 8, and 9. Compound 5 was prepared
from the same intermediate by alkylation with ethylene
carbonate (NaOH, DMF) in 47% yield. Compound 6
was obtained by alkylation of bromoethylphthalimide
(NaH, DMF) followed by hydrazinolysis (17% over
two steps). Compounds 10–13 were prepared by a con-
vergent route (Scheme 2) which allows access to a more
structurally diverse set of analogues.12,13 Thus, Mitsu-
nobu coupling of 1514 with the ketal-alcohol 16 (readily
Scheme 2. Reagents and conditions: (a) Ph3P, DEAD, THF, 70%;
(b) 4-phenoxyphenylboronic acid, Pd(PPh3)4, Na2CO3, DME, H2O,
75%; (c) NH4OH, dioxan, Parr pressure vessel, 120 ꢂC, 80%; (d) ace-
tone, 5 N HCl (aq), 92%; (e) amine, Na(OAc)3BH, ClCH2CH2Cl,
AcOH, 65–72%.
prepared by NaBH4 reduction of the commercially
available ketone) followed by a Suzuki coupling, ami-
nation and deprotection protocol furnished the inter-
mediate ketone 18.
Reductive amination with the appropriate amine pro-
vided a 3:1 mixture of cis and trans diastereoisomers,
which were easily separated by flash silica gel column
chromatography.

1686
D.J.Calderwood et al./ Bioorg.Med.Chem.Lett.12 (2002) 1683–1686
3. Van Oers, N. S.; Kileen, N.; Weiss, A. J.Exp.Med. 1996,
183, 1053.
Conclusion
4. Traxler, P.; Furet, P. Pharmacol.Ther. 1999, 82, 195.
5. Toledo, L. M.; Lydon, N. B.; Elbaum, D. Curr.Med.
Chem. 1999, 6, 775.
6. Altmann, E; Missbach, M.; Green, J.; Susa, M.; Wagen-
knecht, H.; Widler, L. Bioorg.Med.Chem.Lett. 2001, 11, 853.
7. Missbach, M.; Altmann, E.; Widler, L.; Susa, M.; Buch-
dunger, E.; Mett, H.; Meyer, T.; Green, J. Bioorg.Med.Chem.
Lett. 2000, 10, 945.
Pyrrolo[2,3-d]pyrimidines with a variety of substituents
at N-7 are shown to be potent inhibitors of lck. One of
these inhibitors, compound 13, inhibits IL-2production
in cellular and whole blood assays. Significantly, it is
bioavailable and shows oral efficacy in mice after dosing
in water vehicle. Further efforts to improve overall src
family selectivity and more detailed in vivo analysis of
compounds related to 13 will be disclosed in due
course.
8. Widler, L.; Green, J.; Missbach, M.; Susa, M.; Altmann, E.
Bioorg.Med.Chem.Lett. 2001, 11, 849.
9. Arnold, L. D.; Calderwood, D. J.; Dixon, R.; Johnston,
D. N.; Kamens, J. S.; Munschauer, R.; Rafferty, P.; Rat-
nofsky, S. E. Bioorg.Med.Chem.Lett. 2000, 10, 2167.
10. Burchat, A.; Calderwood, D. J.; Hirst, G. C.; Holman,
N. J.; Johnston, D. N.; Munschauer, R.; Rafferty, P.;
Tometzki, G. B. Bioorg.Med.Chem.Lett. 2000, 10, 2171.
11. See for example Chan, H. O.; Stewart, B. H. Drug Discov.
Technol. 1996, 1, 461.
Acknowledgements
We are grateful to Dr. D. McCormick, Dr. D. Hick-
man, B. Bettencourt, and S. Warriner for generating
pharmacokinetic data on 2 and 13.
12. Arnold L. D.; Calderwood, D. J.; Johnston, D. N.;
Munchauser, R.; Rafferty, P.; Twigger, H. US Patent
6,001,839, 1998; Chem.Abstr . 1998, 129, 275922.
13. Arnold, L. D.; Calderwood, D. J.; Mazdiyasni, H.; Hirst,
G. C.; Deng, B. B. WO 200017202 Chem.Abstr. 2000, 132,
251159.
14. Drach, J. C.; Kern, E. R.; Nassiri, M. R.; Pudlo, J. S.;
Townsend, L. B.; Wotring, L. L. J.Med.Chem. 1990, 33,
1984.
References and Notes
1. Hanke, J. H.; Pollok, B. A.; Changelian, P. S. Inflamm.
Res. 1995, 44, 357.
2. Weil, R.; Veillette, A. Curr.Top.Microbiol.Immunol. 1996,
205, 63.