Pyrazolo[3,4-d]pyrimidines containing an extended 3-substituent as potent inhibitors of Lck -- a selectivity insight.

Article abstract of DOI:10.1016/S0960-894X(02)00196-8

A series of para-substituted 3-phenyl pyrazolopyrimidines was synthesized and evaluated as inhibitors of lck. The nature of the substitution affected enzyme selectivity and potency for lck, src, kdr, and tie-2. The para-phenoxyphenyl analogue 2 is an orally active lck inhibitor with a bioavailability of 69% and exhibits an extended duration of action in animal models of T cell inhibition.

Full text of DOI:10.1016/S0960-894X(02)00196-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1687–1690
Pyrazolo[3,4-d]pyrimidines Containing an Extended 3-Substituent
as Potent Inhibitors of Lck — a Selectivity Insight
Andrew F. Burchat, David J. Calderwood, Michael M. Friedman, Gavin C. Hirst,*
Biqin Li, Paul Rafferty, Kurt Ritter and Barbara S. Skinner
Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605-5314, USA
Received 30 November 2001; accepted 26 February 2002
Abstract—A series of para-substituted 3-phenyl pyrazolopyrimidines was synthesized and evaluated as inhibitors of lck. The nature
of the substitution affected enzyme selectivity and potency for lck, src, kdr, and tie-2. The para-phenoxyphenyl analogue 2 is an
orally active lck inhibitor with a bioavailability of 69% and exhibits an extended duration of action in animal models of T cell
inhibition. # 2002 Elsevier Science Ltd. All rights reserved.
The src family tyrosine kinase lck expressed primarily in
T lymphocytes,1,2 plays an essential role in the immune
response.3 Antigen-induced T cell activation is char-
acterized by the appearance of a lck-driven hyperphos-
phorylated TcR z-chain, a critical event necessary for
recruitment of the syk family kinase ZAP-70 to the sig-
naling complex.4 Genetic data validate lck as an immu-
The preceding publication in this journal8 described
SARof a series of pyrrolo[2,3- d]pyrimidines containing
substituents at N-7. Compound 1 was identified as an
orally active lck inhibitor that inhibits T-cell receptor
(TCR) stimulated (a-CD3 mAb) IL-2 production in
mice after oral administration with an ED50=2.5 mg/
kg.
nosuppressive molecular target as
a
SCID-like
phenotype was observed in mice rendered lck deficient
by targeted gene disruption.5 Lck À/Àmice are incap-
able of rejecting either MHC-incompatible or minor
incompatible skin grafts despite the presence of periph-
eral T cells.6 A selective inhibitor of lck should inhibit
T-cell activation and have broad application for the
treatment of autoimmune and inflammatory diseases
and also organ transplant rejection.7
Although compound 1 demonstrated sub-optimal phar-
macokinetic characteristics such as a very high volume
of distribution and high plasma clearance, it represented
a key compound for further optimization. We also
showed that occupying the lipophilic pocket in lck with
the phenoxyphenyl moiety resulted in increased potency
versus lck.9 Our results also indicated that an appended
solubilizing heterocycle in the ribose pocket, such as the
N-methyl piperazine in 1, facilitated oral dosing.8
This paper describes our studies towards modifying the
pyrrolopyrimidine core and the phenoxyphenyl motif of
1 to optimize the in vivo profile. In order to reduce the
volume of distribution of 1 by lowering its lipophilicity,
we elected to probe the corresponding pyrazolo[3,4-
d]pyrimidine, compound 2.10 These compounds have
measured LogD values at pH 7.4 of 4.5 and 3.04,
respectively.
We screened inhibitors against a non-phosphorylated
construct of human lck kinase,9 in an HTRF format11,12
at a physiologically relevant13 ATP concentration of
1 mM. The closely related kinase src, and two receptor
tyrosine kinases (RTKs), kdr and tie-2, served as
*Corresponding author. Tel.: +1-508-849-2651; fax: +1-508-831-
3590; e-mail: gavin.hirst@abbott.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00196-8

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A. F. Burchat et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1687–1690
counterscreens. Inhibitors were progressed for profiling
in cellular settings and ultimately in vivo. Data are
shown for the inhibition of a-CD3 mAb induced IL-2
production in human whole blood and for inhibition of
TCRstimulated ( a-CD3 mAb) IL-2 production in mice
after oral dosing. In lieu of extensive pharmacokinetic
profiling we probed the pharmacodynamic duration of
action by measurement of the inhibition of IL-2 pro-
duction 8 h and 18 h after oral dosing. This approach
was designed to identify those compounds with the most
advantageous pharmacodynamic coverage as a harbin-
ger of good pharmacokinetics.
atom (shown as R1). This latter modification also sim-
plified our synthetic approaches vide infra. Combina-
tion of these two concepts led to the benzamide 5 and
the carbamate 6, both of which showed increases in
selectivity against the two RTKs when compared to the
benchmark of compound 2. In the first instance, this
was primarily due to a reduction in potency for kdr and
tie-2 and in the latter, because of an enhancement in
potency for lck.
In accord with the close catalytic site homology,14 src
inhibition closely mirrored potency against lck. These
results showed that the oxygen bridge in 2 could be
substituted by linkers of varying lengths and differing
functionalities resulting in enhancement of both potency
for lck and selectivity against the receptor tyrosine
kinases. Further modification of the linker utilizing ani-
line 21 in Scheme 1 as our building block allowed for
rapid exploration of the generality of this observation.
These analogues are exemplified in Tables 3 and 4. The
most striking results were obtained for the amide 5, the
carbamate 6, the urea 7 and the sulfonamide 8 in Table
3 as these highlight remarkable selectivity switches
across the kinases on linker modification. Amide 5, and
in particular carbamate 6, exhibited high selectivity for
lck over kdr and tie-2. Conversely, the urea linker in 7
enhanced kdr potency to such an extent that this resul-
ted in a pluripotent compound. Also surprising was the
identification of the tie-2 selective sulfonamide 8, a 137
nM inhibitor of tie-2 with greater than 20- and 300-fold
selectivity over the src family kinases and kdr, respec-
tively. Although a fuller understanding of the structural
basis of these observations awaits the identification of
ligand:protein co-crystal structures, these two com-
The pyrazolopyrimidine 2 and its pyrrolopyrimidine
congener 1 in Table 1 exhibited comparable in vitro
potency for lck showing the functionally silent nature of
the CH to N interchange and suggested no major bind-
ing contribution from N2 in compound 2.
This observation supports our working model that the
key beneficial protein interactions of compound 1 are
N3 and the 4-NH2 making contacts with Glu317 and
Met319 of lck. A critical hallmark is also that the lipo-
philic phenoxyphenyl group accessed the hydrophobic
cavity which is not exploited by ATP.9
As shown in Table 2, compound 2 was a potent inhi-
bitor of IL-2 production in vivo with an ED50 of 1.5
mg/kg and greater than 18 h duration of action when
dosed at ED90.
The data for compound 2 showed that there was value
in prosecuting the pyrazolopyrimidine series further. To
explore potential selectivity enhancements we studied
the role of the atom linking the two phenyl groups.
Atomic substitution in the ‘linker’ is forgiving as the
aniline 3 is indistinguishable from the ether 2 in our
kinase panel. This enabled utilization of a parallel
chemistry approach for exploration of the role of the
tether. In addition, molecular modeling studies sug-
gested that a productive contact with the side-chain
hydroxyl group of Thr316 could be achieved by instal-
lation of a methoxy residue juxtaposed to the linker
Table 1. Inhibition of lck, src, kdr, and tie-2 for 1 and 2 (IC50 mM)a
lck
src
kdr
tie-2
1
2
0.015
0.040
0.042
0.035
1.19
5.32
0.25
0.75
aMean of two experiments performed with seven concentrations of test
compound.
Table 2. Mouse in vivo data for 2
a
a
ED50 (mg/kg)
ED90 (mg/kg)
Inhibition (%)b
8 h
84
18 h
49
Scheme 1. Reagents and conditions: (a) formamide, 180 ꢀC; (b) NIS,
DMF, 50 ꢀC; 84% two steps; (c) Ph3P, DEAD, THF; (d) acetone, 5 N
HCl (aq), 50%; (e) N-methyl piperazine, Na(OAc)3BH, DCE, AcOH,
17%; (f) Pd(PPh3)4, Na2CO3, DME, H20, 80 ꢀC, 77%; (g) TFA, DCM
74%.
1.5
6
aOral dosing, measured 2.5 h. after dosing.
bAfter dosing at ED90
.

A. F. Burchat et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1687–1690
Table 3. Inhibition of lck, src, kdr and tie-2 for 3–8 (IC50 mM)a
Table 5. Human whole blood and murine in vivo data
1689
Whole blood
Mouse in vivo data
IC50 (mM)
aED50 (mg/kg) aED90 (mg/kg)
Inhibitionb
8 h
18 h
3
5
6
11
13
15
0.135
0.050
0.002
0.038
0.008
0.006
2.5
9
3
5
2
8.5
10.7
23
14
30
12.5
12.5
95
37
51
63
98
48
<10
<10
14
<10
22
<10
aOral dosing measured 2.5 h after dosing.
bAfter dosing at ED90
.
R
R1
lck
src
kdr
tie-2 kdr/lck tie-2/lck
NHPh
COPh
H
H
3 0.040 0.065
4 0.110 0.427 >50
3.81 0.606
95
5.73 >450
4.68 >500
15
52
50
130
Table 6. Pharmacokinetic parameters for 2
NHCOPh
NHCO2Bn
NHCONHPh OMe 7 0.209 0.310
OMe 5 0.093 0.189 >50
OMe 6 0.004 0.0125 34.2 0.521 8550
Cmax (mmol/L) Tmax (h) Vd (l/kg) Clp (l/kg) T1/2 (h) F (%)
0.62 1.5 9.6 1.2 5.2 69
0.263 0.933
1.25 4.4
NHSO2Ph OMe 8 3.120 3.910 >50
0.137 >16
0.044
aMean of two experiments performed with seven concentrations of test
compound.
As a strategy to further increase potency in the extended
amide 11, we elected to reduce the entropic options
available to the linker. This tactic led to the synthesis of
the two regioisomeric geminal dimethyl analogues 12
and 13 in addition to the two amides incorporating
cyclized extended motifs, exemplified in 14 and 15. In
striking contrast to the unadorned aminomethyl ana-
logue 10, that showed no inhibition of kdr at 50 mM, it
is interesting to note the increased potency of the ami-
nomethyl indole 16 for kdr.
Table 4. Inhibition of lck, src, kdr, and tie-2 for 9–16 (IC50 mM)a
Table 5 shows the human whole blood potency and in
vivo data for a selection of these compounds. The
alignment of the in vitro lck potency with the whole
blood data suggested that the compounds achieved
effective cellular penetration and that plasma protein
binding was not effecting potency. All the compounds
were potent inhibitors of IL-2 production in vivo, pos-
sessing an ED50 of less than 10 mg/kg after oral dosing.
Although two of the compounds exhibited greater than
90% inhibition of IL-2 production at 8 h, they por-
trayed minimal activity at 18 h. As these compounds
showed efficacy at 2.5 h, we ascribed their low efficacy at
18 h to be a function of poor pharmacokinetics. In
contrast, compound 2 had sufficient activity at 18 h to
warrant full pharmacokinetic profiling.
R
R1
lck
src
kdr
NT
tie-2
NHSO2NHPh
NHCH2Ph
NHCOCH2CH2Ph
NHCOCMe2CH2Ph
NHCOCH2CMe2Ph
OMe
9
0.167 0.463
0.888
1.13
2.45
1.44
0.91
OMe 10 0.055 0.068 >50
OMe 11 0.028 0.155 >50
OMe 12 0.092 0.46
OMe 13 0.031 0.115
>50
34.6
OMe 14 0.019 0.027 >50
0.275
The pharmacokinetic parameters for compound 2 after
a single (5 mg/kg) oral and intravenous dose to male
Sprague–Dawley rats are detailed in Table 6.
NHCO(benzfuran-2-yl) OMe 15 0.010 0.043 >50
NHCH2(indol-2-yl) 16 0.050 0.526
1.02
0.435 0.203
H
aMean of two experiments performed with seven concentrations of test
compound.
The 5 mg/kg dose generated Cmax plasma levels 23-fold
higher than the whole blood IC50 for IL-2 inhibition
(data not shown). This, in combination with the high
bioavailability and acceptable elimination half-life,
account for the extended pharmacodynamic efficacy
observed. Compound 2 showed a 14-fold reduction in
volume of distribution and a 4-fold drop in plasma
clearance compared to compound 1.
pounds suggest a possible starting point for the design
of selective kdr and tie-2 inhibitors.
Emphasis was then placed on exploration of the exten-
ded amides exemplified by the phenethyl amide 11.
The increased degrees of freedom compared to the
benzamide 5 do not translate into a reduction in lck
potency as 11 is 3-fold more potent than 5.
Utilizing an analogous modeling approach to our earlier
paper9 Figure 1 depicts an array of germane hydrogen

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A. F. Burchat et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1687–1690
In summary, the nature of the Ph-X-Ph linker in these
tyrosine kinase inhibitors defines the potency and, per-
haps more interestingly, the selectivity for lck, src, kdr
and tie-2. A sulfonamide linker as in 8 drives tie-2
potency, whereas a urea, such as 7 introduces kdr
activity. Ethers, ketones, carbamates and amides
engender lck and src activity. In line with its lower
LogD compared to compound 1, the ether-linked pyr-
azolopyrimidine 2 exhibits an enhanced pharmacoki-
netic profile and also portrays prolonged activity in
animal models of T cell activation after oral adminis-
tration.
Acknowledgements
We are grateful to K. Dixon, B. McRae and N. Bohan-
Broderick for generating the in vivo data for the com-
pounds in Tables 2 and 5 and to R. Dixon for the
modeling of compound 11.
Figure 1. Model of 11 bound to lck.
bond interactions that orient compound 11 within the
protein. The donor–acceptor pair of the 4-NH2 and N5
make contact with the main chain carbonyl of Glu317
and Met319 of lck, respectively. The methoxy group is
within hydrogen bond donating distance of Thr316 and
N2 has no interactions with the protein. The solvent
exposed region is characterized by contacts between the
internal piperazine nitrogen and the conserved side-
chain acid of Asp326. Productive contacts to the aspar-
tic acid 382 in the DFG motif of lck are established
from the amide linker. An X-ray crystallographic ana-
lysis of a representative set of these inhibitors bound to
lck will be the subject of future publications.
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