Discovery of 4-amino-5,6-biaryl-furo[2,3-d]pyrimidines as inhibitors of Lck: Development of an expedient and divergent synthetic route and preliminary SAR

Article abstract of DOI:10.1016/j.bmcl.2007.01.057

4-Amino-5,6-biaryl-furo[2,3-d]pyrimidines were identified as potent non-selective inhibitors of Lck. A novel, divergent, and practical synthetic route was developed to access derivatives from bifunctional intermediates. Lead optimization was guided by X-r

Full text of DOI:10.1016/j.bmcl.2007.01.057

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2305–2309
Discovery of 4-amino-5,6-biaryl-furo[2,3-d]pyrimidines
as inhibitors of Lck: Development of an expedient
and divergent synthetic route and preliminary SAR
Erin F. DiMauro,a,* John Newcomb,b Joseph J. Nunes,a Jean E. Bemis,a
Christina Boucher,b John L. Buchanan,a William H. Buckner,a Alan Cheng,c
Theodore Faust,b Faye Hsieh,d Xin Huang,c Josie H. Lee,b Teresa L. Marshall,a
Matthew W. Martin,a David C. McGowan,a Stephen Schneider,b Susan M. Turci,b
Ryan D. Whitea and Xiaotian Zhuc
aDepartment of Medicinal Chemistry, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA
bDepartment of HTS and Molecular Pharmacology, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA
cDepartment of Molecular Structure, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA
dDepartment of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
Received 20 November 2006; revised 12 January 2007; accepted 18 January 2007
Available online 25 January 2007
Abstract—4-Amino-5,6-biaryl-furo[2,3-d]pyrimidines were identified as potent non-selective inhibitors of Lck. A novel, divergent,
and practical synthetic route was developed to access derivatives from bifunctional intermediates. Lead optimization was guided
by X-ray crystallographic data, and preliminary SAR led to the identification of compounds with improved cellular potency and
selectivity.
Ó 2007 Elsevier Ltd. All rights reserved.
The lymphocyte-specific kinase (Lck) is a member of the
Src family of cytoplasmic tyrosine kinases and is
expressed specifically in T cells and natural killer (NK)
cells. Genetic evidence from gene knockout mice and
human mutations demonstrates that Lck activity is crit-
ical for T cell receptor (TCR)-mediated signaling, lead-
ing to normal T cell development and activation.1
Selective inhibition of Lck is expected to offer a new
therapy for the treatment of graft rejection and/or T
cell-mediated autoimmune and inflammatory disease
such as rheumatoid arthritis, psoriasis, multiple sclero-
sis, atherosclerosis, and delayed-type hypersensitivity
reactions. In accordance with our ongoing efforts to
identify small molecule inhibitors of Lck for the devel-
opment of new therapeutic agents, we recently disclosed
two promising chemical series.2 Likewise, others have
shown that potent and bioavailable Lck antagonists
have in vivo inhibitory activities in models of T cell-de-
pendent immune responses.3
4-Amino-5,6-biaryl-furo[2,3-d]pyrimidines 1–3 were
identified as promising leads for the Lck program during
a high throughput screen of our kinase-preferred com-
pound collection (Table 1). This scaffold originated in
our Ack1 oncology program4 and has previously been
shown to possess inhibitory activity against other
kinases, including DDR2,5 EGFR,6 Tie-2,7 KDR,7
adenosine kinase,8 and Chk1.9 Furthermore, lead com-
pound 3 exhibited respectable pharmacokinetic proper-
ties in rats (Table 2). Herein we present an extension
of this chemical series toward the identification of sever-
al Lck inhibitors with promising cellular potency and
selectivity. Moreover, we disclose a novel, expedient,
and divergent synthetic route that allows for the intro-
duction of diversity at C4 and C6 from bifunctional
intermediates.
Keywords: Lymphocyte specific kinase (Lck); Anti-inflammatory;
Kinase inhibitors; Furopyrimidine.
A co-crystal structure of 3 and Lck revealed a rather
unusual binding mode (Fig. 1).10 Compound 3 occupies
the ATP binding site, making two H-bond contacts to
*
Corresponding author. Tel.: +1 617 444 5189; fax: +1 617 621
3907; e-mail: edimauro@amgen.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.057

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E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2305–2309
Table 1. Lead compounds 1–3 identified from high throughput
screening of Ack1 inhibitors (IC50, lM)a
might improve cell permeability and potency, and oral
bioavailability.
Our original synthetic route, depicted in Scheme 1,
makes use of minor modifications to established proce-
dures.4,11 Although this route was sufficient, we sought
an alternative strategy that would allow for later-stage
variation of both the amine head group (R1) at C4
and the aryl group (R2) at C6.
R
Ack1
0.523
Lck
1.21
MLRb
2.13
IL-2c
—
1
2
0.024
0.181
0.122
0.081
8.95
1.71
2.41
0.69
The modified route (Scheme 2) utilizes a more reproduc-
ible and scalable method for the construction of the
5-phenylfuropyrimidinone 6.9,12 Regioselective iodination
of 6, followed by reaction with POCl3, affords the key
4-chloro-6-iodo intermediate 14 in 57% yield over two
steps. Compound 14 can be reacted with amines in the
presence of base to generate penultimate 4-amino-6-iodo
compounds 16 for scanning R2 at C6 via Suzuki chemis-
try. Alternatively, iodide 13 can be reacted under Suzuki
conditions with boronates to afford the C6-substituted
pyrimidinones 15. Intermediates 15 are used for scanning
R1 at C4 via a two-step chlorination-SNAr sequence,
which avoids purification of the intermediate C4-chloride
and affords final compounds 10 in 50–60% overall yield
from 15.
3
a Values are means of two or more separate determinations run in
duplicate.
b huMLR, human mixed lymphocyte reaction.
c IL-2: Anti-CD3/CD28-induced T cell IL-2 secretion assay.
Table 2. Rat pharmacokinetic properties of 3a
ivb
poc
CL
Vss
t1/2
887 mL/h/kg
2767 mL/kg
2.37 h
%F
Cmax
15
180 ng/mL
1037 ng h/mL
AUC0–inf
a n = 3 animals per study.
Selected SAR from our C6 scan is collected in Tables 3
and 4. As hypothesized, the introduction of ionizable,
solubilizing groups at the para-position of the C6 phenyl
led to an improvement in cellular potency over the par-
ent compounds 2 and 3. These derivatives also exhibited
a 2 to 20-fold increase in potency at the enzyme level.
Modeling suggests that this potency improvement is
due to charge-charge interactions between the tertiary
amines and the carboxylic acid side chain of Asp326
(Fig. 2).13 In general, compounds containing the
(S)-N-methyl-THF head group at C4 were equipotent
on Ack1 and Lck (17, 18, 20, and 21). In this series, selec-
tivity over Ack1 was marginal at best (e.g., 19; five-fold
b Dosed intravenously at 1 mg/kg in DMSO to male Sprague–Dawley
rats.
c Dosed orally at 5 mg/kg as a suspension in 2% HPMC/1% Tween80/
97% water to male Sprague–Dawley rats.
Figure 1. X-Ray co-crystal structure of 3 (green) and Lck. Oxygen
atoms, red; nitrogen atoms, blue.
the hinge region: the pyrimidine N1 binds to the NH of
˚
˚
Met319 (3.3 A), and the carbonyl of Glu317 (3.6 A)
accepts the CH in the 2-position. The two phenyl rings
are aligned edge-to-face, while the ethylpiperazine
makes conformation-stabilizing van der Waals interac-
tions with several residues, approaching but not entering
the hydrophobic pocket. Since the phenyl at C6 is pro-
jected toward solvent, we reasoned that the introduction
of an ionizable group at the para-position of this ring
Scheme 1. Reagents and conditions: (a) malononitrile, NEt3, DMF,
0 °C ! rt, 70%; (b) HCO2H, Ac2O, reflux, 61%; (c) POCl3, benzene,
reflux, 90%; (d) R1NH2, DIPEA, i-PrOH, reflux, 70–92%; (e) NBS,
KOAc, 80%; (f) R2B(Pin), 5% Pd(PPh3)4, K2CO3, DME/H2O, 75 °C,
30–60%.

E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2305–2309
2307
Table 4. Select SAR: variations at C6 with N-ethyl-piperazine head
group at C4 (IC50, lM)a
R
Ack1
0.017
Lck
MLRb
0.227
IL-2c
—
22
23
24
25
26
27
28
0.011
0.097
0.091
0.085
0.148
0.098
0.586
0.042
0.027
0.022
0.014
0.009
0.022
3.084
0.138
0.121
0.360
0.227
3.223
—
—
—
0.468
0.430
2.634
Scheme 2. Reagents and conditions: (a) bromo(nitro)methane, Me2N-
HÆHCl, KF, xylenes, reflux, 80%; (b) pyrimidine-4,6-diol, DBU, EtOH,
reflux, 70%; (c) NIS, CH3CN/DME, reflux, 85%; (d) POCl3, NEt3,
DCE, 80 °C, 67%; (e) R2B(Pin), 10% Pd(dppf)Cl2, K2CO3, DMF/H2O,
80 °C, 60–73%. (f) R1NH2, DIPEA, i-PrOH, reflux, 70–90%; (g) 1—
POCl3, reflux; 2—R1NH2, CH3CN/CH2Cl2, reflux, 50–60% over two
steps.
a Values are means of two or more separate determinations run in
duplicate.
b huMLR, human mixed lymphocyte reaction.
c IL-2: Anti-CD3/CD28-induced T cell IL-2 secretion assay.
Table 3. Select SAR: variations at C6 with (S)-N-methyl-THF head
group at C4 (IC50, lM)a
R
Ack1
0.011
Lck
MLRb
0.464
IL-2c
0.119
17
18
19
0.006
0.016
0.038
0.011
0.008
0.670
0.825
0.435
0.193
Figure 2. Model of 17 and Lck based on the co-crystal structure of 3
and Lck (Fig. 1).
20
21
0.007
0.013
0.006
0.011
0.337
0.437
0.293
2.420
selective; up to 30-fold selectivity over Ack1 was
observed with some compounds (e.g., 28).
Based on the X-ray co-crystal structure of 3 and Lck
(Fig. 1), we speculated that a hydrophobic substituent
at C4 may be better tolerated than an ionizable head
group. We therefore sought to reduce polarity and size
by fixing the phenoxyethylpyrrolidine at C6 and examin-
ing lipophilic residues at C4 (Table 5). Initial results are
promising as 29 exhibits 24-fold selectivity over Ack1
and 32 demonstrates significant size-reduction without
a substantial loss in cellular potency.
a Values are means of two or more separate determinations run in
duplicate.
b huMLR, human mixed lymphocyte reaction.
c IL-2: Anti-CD3/CD28-induced T cell IL-2 secretion assay.
selective). With the N-ethyl-piperazine head group, how-
ever, a variety of R groups imparted some degree of
selectivity over Ack1 (23, 24, and 25; up to four-fold).
Phenoxyethylamines (e.g., 26, 27, and 28) proved most
Compounds 27 and 32, which demonstrated good cellu-
lar potency and moderate selectivity over Ack1, were

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E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2305–2309
Table 5. Select SAR: variations at the C4 amine with phenoxyethyl-
pyrrolidine at C6 (IC50, lM)a
Acknowledgments
We are grateful to our colleagues Linda Epstein, Yan
Gu, Paul Rose, and Huilin Zhao for protein expression
and purification, and Antonio Oliveira-dos-Santos for
assistance with the biological assays. We thank Dr.
Mark Schwinden of Synexis, Inc. for the multi-gram
scale synthesis of 13 and 14.
R
Ack1
0.332
Lck
MLRb
2.452
IL-2c
—
29
30
0.014
References and notes
0.001
0.650
0.078
0.005
0.083
0.036
1.672
2.376
0.533
5.719
3.257
0.199
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32
a Values are means of two or more separate determinations run in
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Table 6. Kinase Selectivity Summary for 27 and 32 (IC50, lM)a
Kinase
27
32
Lck
Src
0.009
0.045
0.098
0.397
0.960
0.992
3.096
3.146
0.036
0.914
0.078
1.029
1.426
3.272
0.537
1.493
4.891
>8.333
Ack1
EGFR
Jak3
ZAP70
KDR
IGFR
Jak2
>8.333
>25.000
Tie-2
a Values are means of two or more separate determinations run in
duplicate.
further profiled for inhibitory activity against a panel of
receptor and non-receptor tyrosine kinases. Selectivity
was generally good (>100-fold) to excellent (>1000-fold)
over a range of enzymes (Table 6). As anticipated based
on the close sequence homology among members of the
Src family, compounds 27 and 32 exhibit modest selec-
tivity over Src.
In summary, 4-amino-5,6-biaryl-furo[2,3-d]pyrimidines
1–3 were identified as potent, non-selective inhibitors
of Lck. A novel and expeditious synthetic route
allowed for rapid diversification of the core scaffold
with focus on exploring SAR at the C4-amine head
group and the C6-aryl group. This focused explora-
tion was guided by X-ray crystallographic data and
has led to the identification of compounds (27 and
32) with acceptable cellular potency and selectivity
profiles. Lessons learned from the 4-amino-5,6-biaryl-
furo[2,3-d]pyrimidines were invaluable in advancing
a
subsequent, closely related 2,3-diarylfuro[2,3-b]
pyridine-4-amine series, which we introduce in this
issue.14

E. F. DiMauro et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2305–2309
2309
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and size exclusion chromatographies. The protein was
then phosphorylated by incubation with of 5 mM
Mg2+.ATP for 10 min at room temperature. Phosphory-
lated Lck was further purified by anion exchange chro-
matography and concentrated to 10 mg/ml. The
coordinates for the X-ray co-crystal structure of Lck and
3 have been deposited in the PDB. The RCSB ID code is
RCSB041056 and the PDB ID code is 2OF4.
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(Chemical Computing Group, Montreal, version 2006.08)
based on the co-crystal structure of 3 and Lck. The
alternate favorable rotamer of Asp326 used in the model
was generated using MOE Rotamer Explorer. Graphics
generated in PyMol (Delano Scientific, South San Fran-
cisco, version 0.99).
14. Martin, M. W., Newcomb, J.; Nunes, J. J.; Bemis, J. E.;
McGowan, D. C.; White, R. D.; Buchanan, J. L.;
DiMauro, E. F.; Boucher, C.; Faust, T.; Hsieh, F.;
Huang, X.; Lee, J. H.; Schneider, S.; Turci, S. M.; Zhu,
X. Bioorg. Med. Chem. Lett., in press, doi:10.1016/
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