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

Article abstract of DOI:10.1016/S0960-894X(00)00442-X

Pyrrolo[2,3-d]pyrimidines containing a 5-(4-phenoxyphenyl) substituent are novel, potent and selective inhibitors of lck in vitro. Exploration of C-6 position of the pyrrolo[2,3-d]pyrimidine and the terminal phenyl group structure-activity relationship (SAR) is detailed. Compound 1 is orally active in animal models. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00442-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2171±2174
Pyrrolo[2,3-d]pyrimidines Containing an Extended 5-Substituent
as Potent and Selective Inhibitors of lck II
Andrew F. Burchat,a David J. Calderwood,a Gavin C. Hirst,a,* Nicholas J. Holman,b
David N. Johnston,b Rainer Munschauer,a Paul Ra€ertyb,y and Gerald B. Tometzkib
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 novel, potent and selective inhibitors of lck
in vitro. Exploration of C-6 position of the pyrrolo[2,3-d]pyrimidine and the terminal phenyl group structure±activity relationship
(SAR) is detailed. Compound 1 is orally active in animal models. # 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 Productive T-cell activation is characterized by
the appearance of a hyperphosphorylated z-chain and by
phosphorylation and catalytic activation of the syk family
ZAP-70 kinase by lck.2,3 Selective inhibition of lck
function therefore represents an attractive target for thera-
peutic intervention in the treatment of autoimmune and
in¯ammatory diseases and also in organ transplantation.
the pyrrolo[2,3-d]pyrimidine in an attempt to identify
more potent and selective inhibitors of lck.
Two crystal structures of the catalytic domain of lck in
its active state (Y394 phosphorylated) have been pub-
lished.5,6 The structure of two src-family kinases, in
their inactive states (Y394 unphosphorylated) has also
been determined; namely src7 and hck.8,9 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 opportu-
nity for obtaining selectivity.9
The previous paper in this journal4 detailed our discovery
of a novel series of pyrrolo[2,3-d]pyrimidines containing a
5-(4-phenoxyphenyl) substituent, typi®ed by compound
1, as potent and selective inhibitors of lck in vitro.
We screened inhibitors using two forms of the lck
kinase, lck (64±509) and lckcd. As detailed in the pre-
ceding paper, we determined that lck (64±509) is in the
inactive state while lckcd is in the active state. Com-
pounds inhibited the inactive form of lck more potently
than the active form. The phosphorylation state of
Y394 is responsible for these di€erences.
The proposed binding mode of 1 to lck (64±509) in our
homology model delineated in the preceding paper sug-
gests that the hydrophobic pocket in lck (64±509) is
occupied by the phenoxyphenyl moiety of 1 and results
in increased potency against lck (64±509) and also
selectivity against lckcd. This is one of the regions we hoped
to exploit.
This paper documents our initial explorations centered
around the terminal phenyl ring and the 6-position of
Results and Discussion
*Corresponding author. Tel.: +1-508-849-2651; fax: +1-508-831-3590;
e-mail: hirstg@basf.com
yCurrent address: BASF Bioresearch Corporation, 100 Research
Drive, Worcester, MA 01605-5314, USA.
The structural requirement at the 6-position of the pyr-
rolo[2,3-d]pyrimidine was probed with a diverse set of
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00442-X

2172
A. F. Burchat et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2171±2174
functional groups (Table 1).10 Compounds 1±11 in
Table 1 are potent inhibitors of lck (64±509) exhibiting
eroded potency against lckcd. With the exception of
compounds 7 and 9, they are selective for lck (64±509)
over kdr and tie-2. All compounds demonstrate selectivity
for lck (64±509) and lckcd over src and are competitive
with ATP (data not shown).
be seen from compounds 19±23, 25, and 27, installation
of functionality at the 2-position negatively impacts
potency for lck (64±509). A similar trend is seen for the
parent src-family member although the e€ect against
tie-2 and kdr is insigni®cant. Substitution at the 4-posi-
tion has a variable e€ect on lck (64±509) potency with
the 4-CN (24) and 4-COOH (28) exhibiting signi®cant
erosion; the latter example being greater than 100-fold
less potent than its parent 13. Analysis of this set of
compounds indicates that modi®cations in the terminal
ring do not impact tie-2 potency more than 11-fold. In
contrast, signi®cant perturbation is seen for kdr inhibition.
Although full kinase inhibition data were not generated
for all compounds, some general trends are worthy of
note. Within electronically similar functional groups,
there is a steric component at C-6 as evidenced by
comparison of the primary alcohol 6 to the sterically
more demanding secondary alcohol 7; the latter being
25-fold less potent against lck (64±509) than the former.
This trend is also seen for the 6-H analogue 1 compared
to the less active 6-methyl inhibitor 4. This stringency
does not manifest itself for the receptor tyrosine kinases
kdr or tie-2.
Within this set of compounds there were no examples
with an improved pro®le over compound 1, hence a
more detailed in vitro pro®ling of compound 1 was
warranted. Table 3 depicts inhibition data against three
further src-family members, blk, fyn, and lyn. Addi-
tionally, the datum for the negative-regulatory tyrosine
kinase, csk11 is shown. All data are at 1 mM ATP. Aside
from src itself, the highest selectivity (greater than 25-
fold) within the src members was observed against lyn;
less selectivity was seen against the other representatives
blk and fyn. We did not generate counterscreen data
against the remainder of the src family. Elucidation of
a structural explanation for the diversity of selectivity
within the src family awaits a co-crystal structure deter-
mination. Greater than 300-fold selectivity was seen for
lck (64±509) against csk.
The installation of electron withdrawing (e.g., 2 and 5),
electron donating (4), acidic (10) or basic functionalities
(9) does not improve potency for lck (64±509) over the
reference compound 1. The morpholinomethyl analogue
12 shows a signi®cant drop in potency and is the least
potent inhibitor of lck of this set of compounds. Com-
pound 12 exhibits no selectivity over the two receptor
tyrosine kinases kdr and tie-2. In summary, irrespective
of the nature, modi®cation at the 6-position results in a
reduction in lck in vitro potency.
Compound 1 is a potent inhibitor of IL-2 production in
cells4 and has progressed to in vivo settings. Compound
1 inhibits T-cell receptor stimulated (a-CD3 mAb) IL-2
production in mice at low doses (ED50=4 mg/kg) after
ip administration. However, ecacy is greatly reduced
after oral administration (ED50=25 mg/kg) which is
Modi®cations to the terminal phenyl ring were explored
in an e€ort to access bene®cial protein contacts in the
hydrophobic pocket. For historical reasons, analogues
were made in the N-7 cyclopentyl, tert-butyl and also
the isopropyl series; data are shown in Table 2. As can
Table 1. Inhibition of lck (64±509), lckCD, src, kdr and tie-2 for compounds 1±12 (IC50 values, mM)
lck (64±509)
lckcd
1 mM ATP
src
kdr
tie-2
Compound
R1
5 mM ATP
1 mM ATP
5 mM ATP
1 mM ATP
5 mM ATP
5 mM ATP
1
2
3
4
5
6
7
8
H
Cl
Br
Me
<0.001
0.011
0.03
0.016
0.38
0.075
NT
0.069
0.29
NT
0.002
0.06
2.29
NT
0.91
NT
NT
NT
0.93
NT
1.07
8.1
11.7
NT
15.2
18.3
NT
>50
>50
NT
1.57
16.9
1.60
3.35
3.2
1.08
1.86
4.24
0.677
9.82
9.11
3.68
1.98
23.4
11.1
1.81
0.50
0.36
0.61
2.26
0.13
0.41
0.58
1.02
0.26
NT
CN
0.004
0.017
0.47
>50
>50
NT
CH2OH
CH(OH)CH3
CH2OMe
CH2NH2
COOH
0.31
NT
NT
NT
9
0.144
0.020
0.030
1.49
3.09
1.06
26.2
NT
25.1
33.5
43.2
NT
>50
>50
>50
NT
10
11
12
CONH2
CH2 (morpholino)
NT
NT

A. F. Burchat et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2171±2174
2173
Table 2. Inhibition of lck (64±509), lckcd, src, kdr and tie-2 for compounds 13±28 (IC50 values, mM)
lck (64±509)
lckcd
5 mM ATP
src
kdr
tie-2
Compound
R2
R1
5 mM ATP
1 mM ATP
1mM ATP
5 mM ATP
5 mM ATP
5 mM ATP
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
i-Propyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
i-Propyl
Cyclopent
Cyclopent
i-Propyl
i-Propyl
i-Propyl
i-Propyl
i-Propyl
i-Propyl
i-Propyl
H
H
4-OMe
4-OH
<0.001
0.002
0.011
0.075
NT
0.01
0.014
0.07
0.017
0.019
0.015
0.15
0.02
0.33
0.038
NT
NT
NT
NT
NT
NT
NT
9.12
7.1
3.13
4.13
2.53
24.8
10.01
16.04
NT
0.048
0.61
NT
0.25
0.075
0.085
0.554
0.068
4.0
0.208
0.77
3.10
0.224
0.45
26.1
6.9
0.43
2.29
1.31
2.0
0.90
0.40
3.3
0.50
0.09
0.90
0.161
0.129
0.144
0.029
5.06
6.89
0.43
2.68
1.22
1.8
0.90
1.5
0.40
0.60
0.56
1.90
0.239
0.97
0.162
0.246
0.677
0.43
<0.001
<0.001
<0.003
<0.003
0.005
0.006
0.008
<0.003
<0.003
0.040
0.03
4-NH2
3-NH2
2-NH2
2-OMe
2-OH
2-NO2
2-CN
4-CN
2-CH2OH
4-CH2OH
2-COOH
4-COOH
0.003
NT
1.01
2.11
0.633
0.79
0.459
5.55
0.529
0.751
15.8
NT
NT
NT
NT
<0.003
<0.003
0.942
0.131
1.31
NT
NT
Table 3. Inhibition of lyn, blk, fyn and csk (IC50 values, mM)
Compound
lck (64±509)
0.016
blk
fyn
lyn
csk
1
0.066
0.126
0.42
5.18
presumed to re¯ect poor intestinal absorption in the
latter regimen. Inhibition of antigen speci®c T-cell
immune responses was also seen for compound 1. After
administration of compound 1 twice daily (100 mg/kg
po) for 3 days during the in vivo priming phase, a 70%
inhibition of IFNg production was seen upon sub-
sequent antigen-speci®c (KLH) challenge of lympho-
cytes from the draining lymph nodes in vitro. Further in
vivo characterization of compound 1 and analogues will
be published subsequently.
Scheme 1. Reagents and conditions: (1) NBS, DMF, rt, 16 h, 94%; (2)
Zn(CN)2, P(2-furyl)3, Pd(dba)3, NMP, 90ꢀC, 18 h, 47%; (3) 1,4-dioxane,
2 N aq NaOH, 2 N aq KOH, 100ꢀC, 5 days, 52%; (4) n-Bu3SnCHCH2,
Pd(PPh3)4, toluene, 100 ꢀC, 18h, 95%; (5) NaIO4, OsO4, 1,4-dioxane,
H2O, rt, 1 h, 63%; (6) morpholine, NaHB(OAc)3, DCE, rt, 18 h, 51%.
additional route by lithium halogen exchange is shown
in Scheme 2 for compounds 4, 6, 8, and 10.
Synthesis
C-6 analogues
Terminal phenyl analogues 13±28
Two routes were used to prepare the pyrrolopyrimidine
compounds described in this paper. Scheme 1 starts
from compound 1 and exempli®es compounds 3, 5, 11,
and 12. Initial installation of bromine at C-6 allowed
manipulation to the cyanide 5 and primary amide 11
utilizing palladium catalysis. Stille coupling and
Lemieux±Johnson oxidation presented the aldehyde
allowing access to the aminomethyl derivative 12. Other
analogues are easily accessible by known routes. An
A general route to representative compounds from
Table 2 is shown in Scheme 3. Compound 29 was pre-
pared in a manner analogous to that detailed for com-
pound 1. Demethylation unmasked the phenol 30 and
subsequent reaction with an appropriate aryl ¯uoride
generated many of the compounds in Table 2 after suit-
able further functional group interconversion. This is
exempli®ed for carboxylic acid 28.

2174
A. F. Burchat et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2171±2174
In summary, a novel series of pyrrolo[2,3-d]pyrimidines
is described, many of which are selective for lck (64±509)
over src. Compound 1 exhibits modest selectivity across
a panel of src-family kinases and portrays oral activity
in models of T-cell activation. E€orts are underway to
maximize selectivity for lck against src-family members
and also to enhance oral activity.
References and Notes
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Scheme 2. Reagents and conditions: (1) NBS, DMF, rt, 24 h, 95%; (2)
n-BuLi, THF, 65 ꢀC, 30 min; (3) (i) CH3I; (ii) (CH2O)n; (iii)
ClCH2OMe, (iv) CO2; (4) 30% NH4OH, 1,4-dioxane, 120 ꢀC, 18 h.
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Scheme 3. Reagents and conditions: (1) BBr3, CH2Cl2; (2) 4-
FC6H4CN, K2CO3, DMF, 120 ꢀC, 4 h; (3) KOH, 1,4-dioxane, 120 ꢀC,
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