Discovery and initial SAR of imidazoquinoxalines as inhibitors of the Src-family kinase p56Lck

Article abstract of DOI:10.1016/S0960-894X(02)00191-9

We have identified a novel series of 1,5-imidazoquinoxalines as inhibitors of Lck with excellent potency (IC₅₀s<5 nM) as well as good cellular activity against T-cell proliferation (IC₅₀s<1 μM). Structure-activity studies demonstrate the requirement for the core heterocycle in addition to an optimal 2,6-disubstituted aniline group.

Full text of DOI:10.1016/S0960-894X(02)00191-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1361–1364
Discovery and Initial SAR of Imidazoquinoxalines as Inhibitors
of the Src-Family Kinase p56Lck
Ping Chen,a,* Derek Norris,a Edwin J. Iwanowicz,a Steven H. Spergel,a James Lin,a
Henry H. Gu,a Zhongqi Shen,a John Wityak,a Tai-An Lin,d Suhong Pang,d
Henry F. De Fex,d Sidney Pitt,d Ding Ren Shen,d Arthur M. Doweyko,c
Donna A. Bassolino,c Jacques Y. Roberge,b Michael A. Poss,b Bang-Chi Chen,a
Gary L. Schievend and Joel C. Barrisha
aDepartment of Discovery Chemistry, Bristol Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543, USA
bDepartment of Early Discovery Chemistry, Bristol Myers Squibb Pharmaceutical Research Institute, PO Box 4000,
Princeton, NJ 08543, USA
cDepartment of Computer Aided Drug Design, Bristol Myers Squibb Pharmaceutical Research Institute, PO Box 4000,
Princeton, NJ 08543, USA
dDepartment of Immunology, Inflammation and Pulmonary Drug Discovery, Bristol Myers Squibb Pharmaceutical Research Institute,
PO Box 4000, Princeton, NJ 08543, USA
Received 22 January 2002; accepted 11 March 2002
Abstract—We have identified a novel series of 1,5-imidazoquinoxalines as inhibitors of Lck with excellent potency (IC50s<5 nM) as
well as good cellular activity against T-cell proliferation (IC50s<1 mM). Structure–activity studies demonstrate the requirement for
the core heterocycle in addition to an optimal 2,6-disubstituted aniline group. # 2002 Published by Elsevier Science Ltd.
As a member of the Src-family of non-receptor tyrosine
kinases, Lck is expressed predominantly in T cells and
natural killer cells. Lck plays essential roles in both T
cell development1 and activation,2 acting to initiate T
cell antigen receptor (TCR) signaling that leads to
cytokine production and T cell activation. Upon recog-
nition of an antigen by the TCR, Lck phosphorylates
the Immunoreceptor Tyrosine Activation Motif
(ITAM) sequences in the invariant chains of T cell
receptor, including those of the z-chain. Phosphoryla-
tion of the dual tyrosine residues of the z-chain ITAMs
allows binding of the Syk family tyrosine kinase ZAP-70
via its tandem SH2 domains.2c Activation of ZAP-70
via its phosphorylation by Lck and the combined action
of Lck, ZAP-70 and the related Src-family tyrosine
kinase Fyn drive downstream signaling events that ulti-
mately lead to T cell activation and proliferation. Inac-
tivation of the Lck gene by genetic ablation or over
expression of the dominant negative leads to early arrest
of thymocyte development prior to expression of CD4,
CD8, and the TCR.1 In addition, loss of expression of
Lck in Jurkat T cells results in the inability of these cells
to respond to anti-TCR antibodies and the absence of
Ca2+ mobilization required for T cell activation. Inhi-
bition of Lck is considered potentially useful for the
treatment of both chronic and acute T cell-mediated
autoimmune and inflammatory disorders such as rheu-
matoid arthritis, multiple sclerosis, transplant rejection
and delayed hypersensitivity (DTH) reactions. Several
classes of Lck inhibitors, both of the catalytic and SH2
domains, have recently been reported.3À11
*Corresponding author. Tel.: +1-609-252-5809; fax: +1-609-252-
6601; e-mail: ping.chen@bms.com
Figure 1. Activity for imidazoquinoxaline 1. Assay conditions descri-
bed in ref 13.
0960-894X/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(02)00191-9

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P. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1361–1364
High throughput screening of our compound collection
resulted in the identification of the 1,5-imidazoquinoxa-
line 1 (Fig. 1) as a relatively potent and selective Lck
inhibitor. Although anilino-substituted quinoxalines are
known inhibitors of tyrosine kinases,12 fusion of an
imidazo group to the quinoxaline ring represented a
novel tyrosine kinase inhibitor motif. In addition, com-
Table 1.
Compd
R1 or R2
X
Lck IC50 (nM)13a
1
2-Br
Cyclohexyl
Cyclopropyl
n-Propyl
n-Hexyl
Benzyl
3-Cl-phenethyl
3-Pyridyl
4-Pyridyl
2-Br
NH
170
3170
3170
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
naa
na
na
na
na
na
na
na
O
2680
>12,500
>12,500
4390
820
>12,500
3700
>12,500
>12,500
>12,500
60
>12,500
810
pound 1 displays modest potency against anti-CD3/
13b
anti-CD28 induced PBLproliferation
and shows
encouraging selectivity against non-Src family Tyr and
Ser/Thr kinases.13c This report describes our pre-
liminary efforts toward optimizing the enzymatic and
cellular activity of this lead.
2-Br
S
—
2,6-di-Me
2-Cl
2-Br
2-Me
2,6-di-Cl
N-Me
–NHCO—
–NHCO—
–NHCO—
1,2-Imidazoquinoxaline 2, the regioisomer of 1, was first
prepared, as a 1:1 mixture, by reaction of the precursor
urea 3 with POCl3 at reflux (Fig. 2). Compound 2 dis-
plays poor activity against Lck, indicating that the N-2
nitrogen of the imidazole ring plays an important role in
enzyme binding, presumably as a hydrogen bond
acceptor (vide infra).
7g
aNot applicable.
Alternatively, intermediate 4 could be obtained from
1-arylimidazole-5-carboxylates 816 via a base-catalyzed
intramolacular cyclization process (Scheme 2).
Approximately 160 compounds were prepared in this
effort using parallel synthesis and led to the following
SAR: (1) Replacement of the 2-bromoaniline of 1 with
aliphatic and arylalkyl amines (6a–f) resulted in sub-
stantial losses in activity against Lck. (2) Both oxygen
(7a) and sulfur (7b) linkers were significantly less
potent than the amine linker. (3) Biaryl analogue (7c)
and N-methyl analogue (7d) displayed poor activity
suggesting that the aniline N-H is important for activity.
Interestingly, while the amide 7e displays similar potency
to 1, 7f and 7g are substantially less active. Finally, a
quick survey of heterocyclic replacements for the aniline
phenyl group reveals a significant difference in potency
between nitrogen regioisomers (6g vs 6h) (Table 1).
Figure 2. Preparation and activity of 1,2-imidazo regioisomer.
Having confirmed that the regioisomeric 1,5-imidazo-
quinoxaline is the more potent scaffold of the two, we
next examined replacement of the bromoaniline moiety
by other aromatic and non-aromatic amines. The
requisite compounds were readily prepared by reaction
of the 2-chloroimidazoquinoxaline intermediate 5 with
the appropriate amines, anilines (where X=NH), phe-
nols (where X=O) and thiophenols (where X=S)
(Scheme 1). The intermediate 5 was obtained from
reaction of imidazoquinoxalinone 4 with POCl3. Pre-
cursor 4 in turn was prepared by methods recently
reported from these labs.14,15
Modification of the substitution on the aniline phenyl
ring led to a dramatic change in potency (Table 2).
Removal of the 2-bromo-substituent of 1 causes a 5-fold
loss in activity (7h). Exchange of 2-fluoro for 2-bromo
(7i) results in a 2-fold loss of activity whereas replacing
Table 2.
Compd
R2
X
Lck IC50 (nM)13a
1
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
2-Br
H
2-F
3-F
2-Cl
2-OMe
2-NO2
4-Br
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
170
890
390
1330
60
5520
>12,500
>12,500
240
Scheme 1.
2-Cl, 4-Me
2-Cl, 4,6-di-Me
2,4,6-tri-Me
2,6-di-Me
2,6-di-Br
2,6-di-Cl
2,6-di-F
2-Cl, 6-Me
2,6-di-Et
30
40
16
50
9
360
9
1690
7u
7v
7w
Scheme 2.

P. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1361–1364
1363
Table 3.
No.
R1
R2
R3
R4
IC50 (nM)13a
1
7k
7r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Me
H
H
H
H
H
H
H
H
H
H
H
2-Br
2-Cl
2,6-di-Me
2-Cl, 6-Me
2-Br
170
60
16
9
Scheme 3.
7s
12a
12b
12c
>12,500
30
2-bromo with 2-chloro (7k) led to a 3-fold increase in
potency. However, replacement of the bromine with
electron-donating (7l) or electron-withdrawing (7m)
groups yielded compounds with >10-fold reduction in
activity. As a general trend, ortho substitution is much
more favored over meta (7i vs 7j) or para (1 vs 7n). Most
significantly, when both ortho positions of the aniline
are substituted by small groups, the potency increased
by more than an order of magnitude, as demonstrated
by three 2,6-disubstituted analogues 7r, 7t and 7v. The
2,6-dibromo substituted analogue (7s) displays a 3-fold
reduction of potency compared to its dichloro counter-
part (7t). Surprisingly, the difluoro analogue (7u)
showed a 40-fold loss of activity, indicating the impor-
tance of steric bulk at both ortho positions. On the other
hand, there also appears to be a size limit for such
ortho-substitution. The 2,6-diethyl analogue (7w) is
more than 100-fold less active than its dimethyl coun-
terpart 7r. When the substituent is small in size, 2,4,6-
trisubstitution is tolerable, as evidenced by the activity
of compounds 7p and 7q.
H
Me
Me
CH2OH
CHO
CHN=OH
CHOHMe
CHOHi-Pr
(CH2)3OH
2-Cl
2-Br
180
7.4
6.2
10
4.3
53
238
69
665
2.4
2
12d 7,8-di-MeO
12e
12f
12g 7,8-di-MeO
12h 7,8-di-MeO
12i
12j
12k 7,8-di-MeO C(=O)NHEt
12l 7,8-di-MeO
12m 7,8-di-MeO
2-Cl, 6-Me
2-Cl, 6-Me
2-Cl, 6-Me
2-Cl, 6-Me
2-Cl, 6-Me
2-Cl, 6-Me
2-Cl, 6-Me
2-Cl, 6-Me
2, 6-di-Me
2-Cl, 6-Me
7,8-di-MeO
7,8-di-MeO
7,8-di-MeO
7,8-di-MeO
H
H
enables the phenyl ring to adopt a preferred orientation
wherein the ring is skewed out of plane with respect to
the tricyclic system. This conformation permits the ring
to occupy an angular hydrophobic pocket common to
many tyrosine kinases. QSAR analyses of phenyl ring
substitution patterns correlated small size, di-ortho sub-
stitution and electron-withdrawing properties to higher
observed Lck affinities, underscoring the choice of 2-
chloro-6-methyl substitution in the present series.20 In
addition, two critical hydrogen bonding interactions are
thought to be involved: the nitrogen of the imidazole
ring acting as an acceptor to backbone Met 319 N-H
and the N-H of the aniline acting as a donor to the Thr
316 hydroxyl oxygen.
Both 7r and 7v display improved potency against anti-
13b
CD3/anti-CD28 induced PBLproliferation
with
IC50’s of 3000 and 7000 nM, respectively. Since 7r and
7s represent the most potent aniline analogues prepared
in this series, the 2-chloro-6-methyl and 2,6-dimethyl
anilines were used in our subsequent SAR studies.
In summary, we have identified novel Lck inhibitors
with a 1,5-imidazoquinoxaline template, as represented
by 1, using high throughput screening of our in-house
compound collection. Optimization of 1 led to com-
pound 12m with excellent enzymatic activity against
Lck (IC50=2 nM) and good potency blocking T cell
proliferation13b (IC50=0.67 mM). Structure–activity
studies demonstrate the requirement for the core het-
erocyclic scaffold in addition to an optimal 2,6-di-
substituted aniline moiety. Further characterization of
12m and related SAR will be reported in due course.
Next, we turned our attention to the substitution on the
imidazole ring. Compounds 12a–k were prepared by
methods analogous to those described earlier14,15 and by
an automated synthetic approach using a solid-phase
support17 as outlined in Scheme 3.
Substitution at the 4-position (R3) of the imidazole ring
with a small alkyl group is tolerable (7k vs 12b) whereas
substitution with a larger phenyl residue (12a) led to a
complete loss in potency. Similarly, potency remains
unchanged when a small alkyl group was placed at the
2-position (R2) of the imidazole (1 vs 12c). In addition,
small polar groups are also tolerable at this position
(12e–g vs 12h–k). Finally, incorporation of small, elec-
tron-rich functional groups at the open positions of the
fused phenyl ring18 further enhances potency against the
enzyme (12l, 12m vs 7r, 7s), suggesting the potential of
improvement in activity via substitution at this portion
of the molecule. Follow-up SAR at the fused phenyl
ring is the subject of a subsequent report (Table 3).
Our SAR study is consistent with the binding model
which was developed based on the published coordi-
nates of activated Lck kinase domain bound to ANP
(phosphoaminophosponic acid-adenylate ester, a non-
hydrolyzable ATP mimic).19 As shown in Figure 3, we
believe that the 2,6-disubstitution of the aniline of 1
Figure 3. Inhibitor 1 modeled into the ATP-binding site of Lck illus-
trating hydrogen bond interactions between Thr 316 and Met 319 and
the ligand. The ortho-disubstituted aniline occupies a deep hydro-
phobic pocket.

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P. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1361–1364
Cardozo, M.; Hickey, E. R.; Ingraham, R.; Jakes, S.; Kabce-
Acknowledgements
nell, A.; Kirrane, T.; Lukas, S.; Patel, U.; Proudfoot, J.;
Sharma, R.; Tong, L.; Moss, N. J. Med. Chem. 1999, 42, 1757.
12. Aventis Pharmaceuticals, PCT Int. Appl., 1998, WO
9854156. Chem. Abstract: 1998:794991.
We would like to thank Mr. Peng Guo and Mr. Mark S.
Bednarz for providing assistance in the preparation of
key intermediates, Dr. J. Z. Gougoutas, Mr. J. DiMarco
and Mrs. M. F. Malley for X-ray crystallographic ana-
lysis support, and the Discovery Analytical Science
Group for all the other analytical support.
13. (a) Lck Enzyme Assay: Recombinant Lck expressed as a
His-tagged protein in insect cells using a baculovirus expres-
sion system and purified by nickel affinity chromatography is
incubated in kinase buffer (20 mM MOPS, pH7, 10 mM
MgCl2) in the presence of the test compound. The reaction
was initiated by the addition of substrates to the final con-
centration of 1 mM ATP, 3.3 mCi/mL[ 33P] g-APT, and 0.1 mg/
mLacid denatured enolase, and was stopped after 10 min by
the addition of 10% trichloro-acetic acid, 100 mM sodium
pyrophosphate followed by 2 mg/mLbovine serum albumin.
The labeled enolase protein substrate was precipitated at 4 ꢀC,
harvested onto Packard Unifilter plates and counted in a
Topcount scintilla-tion counter. (b) PBL Proliferation Assay:
A 96-well plate was coated with a monoclonal antibody to
CD3 (G19–4), the antibody was allowed to bind, and the plate
was washed. Normal human peripheral blood T-cells were
added to the wells along with test compound plus anti-CD28
(E.3) antibody. After 3 days, the [3H]-thymidine was added to
the cells, and after further incubation, the cells were harvested
and counted in a scintillation counter. (c) Preliminary study
showed that compound 1 displays selectivity against other
non-Src-family Tyr and Ser/Thr kinases (unpublished data).
14. Chen, P.; Barrish, J. C.; Iwanowicz, E.; Lin, J.; Bednarz,
M. S.; Chen, B.-C. Tetrahedron Lett. 2001, 42, 4293.
15. Norris, D.; Chen, P.; Barrish, J. C.; Das, J.; Moquin,
R. V.; Chen, B.-C.; Guo, P. Tetrahedron Lett. 2001, 42, 4297.
16. Chen, B.-C.; Bednarz, M. S.; Zhao, R.; Sundeen, J. E.;
Chen, P.; Shen, Z.; Skoumbourdis, A. P.; Barrish, J. C. Tet-
rahedron Lett. 2000, 41, 5453.
17. This portion of the work will be published elsewhere.
18. (a) Similar observation has been reported in other tyrosine
kinase inhibitors Bridges, A. J.; Zhou, H.; Cody, D. R.;
Rewcastle, G. W.; McMichael, A.; Showalter, H. D. H.; Fry,
D. W.; Kraker, A. J.; Denny, W. A. J. Med. Chem. 1996, 39,
267. (b) Hennequin, L. F.; Thomas, A. P.; Johnstone, C.;
Stokes, E. S. E.; Ple, P. A.; Lohmann, J.-J. M.; Ogilvie, D. J.;
Dukes, M.; Wedge, S. R.; Curwen, J. O.; Kendrew, J.; van der
Brempt, C. L. J. Med. Chem. 1999, 42, 5369.
References and Notes
1. (a) Molina, T. J.; Kishihara, K.; Siderovski, D. P.; van
Ewijk, W.; Narendran, A.; Timms, E.; Wakeham, A.; Paige,
C. J.; Hartman, K.-U.; Veillette, A.; Davison, D.; Mark, T. W.
Nature 1992, 357, 161. (b) Levin, D. S.; Anderson, S. J.; For-
bushm, K. A.; Perlmutter, R. M. EMBO J. 1993, 12, 1671.
2. (a) Straus, D. B.; Weiss, A. Cell 1992, 70, 585. (b) Chan,
A. C.; Desai, D. M.; Weiss, A. C. Ann. Rev. Immunol. 1994,
12, 555. (c) Weiss, A.; Littman, D. Cell 1994, 76, 263. (d)
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. (e) van Oers, N. S. C., et al.
Immunity 1996, 5, 429.
3. Myers, M. R.; Setzer, N. N.; Spada, A. P.; Zulli, A. L.;
Hsu, C.-Y.; Zilberstein, A.; Johnson, S. E.; Hook, L. E.;
Jacoski, M. V. Bioorg. Med. Chem. Lett. 1997, 7, 417.
4. Padmanabha, R.; Shu, Y. Z.; Cook, L. S.; Veitch, J. A.;
Donovan, M.; Lowe, S.; Huang, S.; Pirnik, D.; Manly, S. P.
Bioorg. Med. Chem. Lett. 1998, 8, 569.
5. Bullington, J. L.; Cameron, J. C.; Davis, J. E.; Dodd, J. H.;
Harris, C. A.; Henry, A. R.; Pellegrino-Gensey, J. L.; Rupert,
K. C.; Siekierka, J. J. Bioorg. Med. Chem. Lett. 1998, 8, 2489.
6. Kraker, A. J.; Hartl, B. G.; Amar, A. M.; Bravian, M. R.;
Showalter, H. D. H.; Moore, C. W. Biochem. Pharmacol.
2000, 60, 885.
7. Arnold, L. D.; Calderwood, D. J.; Dixon, R. W.; Johnston,
D. N.; Kamens, J. S.; Munschauer, R.; Rafferty, P.; Rat-
nofsky, S. E. Bioorg. Med. Chem. Lett. 2000, 10, 2167.
8. Burchat, A. F.; 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.
9. Hajduk, P. J.; Zhou, M. M.; Fesik, S. W. Bioorg. Med.
Chem. Lett. 1999, 9, 2403.
19. (a) Two crystal structures of the catalytic domain of Lck
(Y394 was phosphorylated) have been published. See: Yama-
guchi, H.; Hendrickson, W. A. Nature 1996, 384, 484. (b) Zhu,
X.; Kim, J. L.; Rose, P. E.; Stover, D. R.; Toledo, L. M.;
Zhao, H.; Morgenstern, K. A. Structure 1999, 7, 651 Protein
Data Bank (1QPC pdb, Research Collaboratory for Structural
Bioinformatics).
10. Llinas-Brunet, M.; Beaulieu, P. L.; Cameron, D. R. J.
Med. Chem. 2000, 42, 722.
11. (a) Lee, T. R.; Lawrence, D. S. J. Med. Chem. 2000, 4,
1173. (b) Beaulieu, P. L.; Cameron, D. R.; Ferland, J.-M.;
Gauthier, J.; Ghiro, E.; Gillard, J.; Gorys, V.; Poirier, M.;
Rancourt, J.; Wernic, D.; Llinas-Brunet, M.; Betageri, R.;
20. Unpublished results.