Synthesis and SAR of novel imidazoquinoxaline-based Lck inhibitors: Improvement of cell potency

Article abstract of DOI:10.1016/S0960-894X(02)00677-7

A series of anilino(imidazoquinoxaline) analogues bearing solubilizing side chains at the 6- and 7-positions of the fused phenyl ring has been prepared and evaluated for inhibition against Lck enzyme and of T-cell proliferation. Significant improvement of the cellular activity was achieved over the initial lead, compound 2.

Full text of DOI:10.1016/S0960-894X(02)00677-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3153–3156
Synthesis and SAR of Novel Imidazoquinoxaline-Based Lck
Inhibitors: Improvement of Cell Potency
Ping Chen,a,* Edwin J. Iwanowicz,a,* Derek Norris,a Henry H. Gu,a James Lin,a
Robert V. Moquin,a Jagabandhu Das,a John Wityak,a Steven H. Spergel,a
Henry de Fex,b Suhong Pang,b Sydney Pitt,b Ding Ren Shen,b
Gary L. Schievenb and Joel C. Barrisha
aDiscovery Chemistry, Bristol Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ08543, USA
bImmunology, Inflammation, and Pulmonary Drug Discovery, Bristol Myers Squibb Pharmaceutical Research Institute, PO Box 4000,
Princeton, NJ08543, USA
Received 8 July 2002; accepted 24 July 2002
Abstract—A series of anilino(imidazoquinoxaline) analogues bearing solubilizing side chains at the 6- and 7-positions of the fused
phenyl ring has been prepared and evaluated for inhibition against Lck enzyme and of T-cell proliferation. Significant improvement
of the cellular activity was achieved over the initial lead, compound 2.
# 2002 Elsevier Science Ltd. All rights reserved.
The cytoplasmic, non-receptor Src-family tyrosine
kinase Lck plays a crucial role in T-cell antigen receptor
(TCR) phosphorylation, a process required for the acti-
vation of protein tyrosine kinase signal transduction
cascades and its down-stream events.1 Studies have
shown that T-cells that lack Lck are severely impaired
in TCR tyrosine phosphorylation and unable to be
activated via the TCR.2 Consequently, there has been a
continuous effort to develop small molecule inhibitors
against Lck for the treatment of T-cell mediated auto-
immune and inflammatory diseases.
quinoxaline core. Our primary objectives were to further
enhance the cellular activity and to improve the physical
properties of this series of inhibitors.
Chemistry
Our preliminary investigation focused on evaluating
various substituents on the phenyl ring of the imidazo-
quinoxaline core. As outlined in Scheme 1, two general
approaches were taken to prepare the key intermediate,
the imidazo[1,5-a]quinoxalin-4(5H)-one 4, using the
chemistry developed in this lab.4,5 Reaction of symme-
trical or unsymmetrical ortho-diaminobenzenes with
glyoxylate followed by selective alkylation with PMB-Cl
yielded the N-protected quinoxalin-2(1H)-one 3 as a
major product. In the cases where the unsymmetrical
diaminobenzenes were used, the desired regio-isomers
were usually obtained by silica gel chromatography
separation/purification. Subsequent reaction with Tos-
MIC/NaH followed by deprotection of PMB group
furnished desired imidazo-quinoxalinone 4 (Route A).
Alternatively, treatment of the substituted ortho-fluoro-
anilines with carbonyl-imidazole dimer5 in the presence
of a base gave intermediate 5. The intramolecular cycli-
zation of 5 was accomplished under the basic conditions
to generate 4 (Route B). Treatment of 4 with POCl3
High-throughput screening of the BMS compound col-
lection led to the discovery of 1, a structurally novel
inhibitor of Lck. Subsequent structure–activity rela-
tionship studies on the 1,5-imidazoquinoxaline core
have led to the identification of compound 2, a lead
analogue in this series with excellent inhibitory activity
against the isolated enzyme as well as in a T-cell pro-
liferation assay3 (Fig. 1).
In this paper, we describe our efforts in exploring
modifications of the fused phenyl ring of the imidazo-
*Corresponding authors. Tel.:+1-609-252-5809; fax:+1-609-252-
6601; e-mail: ping.chen@bms.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00677-7

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P. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3153–3156
Figure 1. Activity for imidazoquinoxoline 1 and 2.
generated the desired chloride 6. Analogues 7a–v were
prepared from 6 by displacement of the chloride with
the corresponding aniline in the presence of a base
(Scheme 1).
Scheme 2. (a) RR’NH, Pd(dba)2, BINAP, NaOt-Bu, Tol; (b) POCl3,
reflux; (c) R00-OH, NaH, DMSO.
Utilizing the appropriately substituted intermediate 4a–c
allowed for introduction of various polar groups on the
fused phenyl ring. For example, reaction of 6-bromo-
imidazoquinoxalin-4(5H)-one 4a with amines, in the
presence of palladium (0) catalyst, afforded substituted
anilines 8. Further reaction of 8 with POCl3 yielded the
desired imidazoquinolinechloride 6a. Similarly, the
6-bromo-7-fluoroimidazoquinoxalin-4(5H)-one 4b and
its regio-isomer 4c were sequentially treated with an
alkoxide, followed by a palladium (0)-catalyzed amina-
tion reaction affording 9 and 10. Subsequent chlorina-
tion provided the desired intermediate 6b and 6c
(Scheme 2). Finally, analogue 11a was obtained from 2
via removal of methyl groups with BBr3. Both 11b and
11c were obtained by bis-alkylation of 11a with dibro-
momethane and/or dibromoethane in the presence of a
base.
functionality is pointed toward solvent. Beneficial
effects are often noted, such as enhancement of cellular
activity as a result of improved cell permeability, as well
as improvement of other physical chemical properties,
for example aqueous solubility. Our objective was to
examine a number of analogues, with a variety of func-
tional residues appended to the fused phenyl moiety, to
establish the effects on inhibition of Lck and T-cell
proliferation in human PBLs.
Using a parallel synthesis approach, a large number of
differentially substituted phenyl analogues of 2 were
prepared and evaluated with the initial goal of identify-
ing the most appropriate site(s) for further elaboration.
Table 1 lists the structures and in vitro biological activ-
ities of selected anilino(imidazoquinoxaline) analogues
from this study.
Results and Discussion
Introduction of side chains bearing polar and/or weakly
basic amine-based functional groups into a bicyclo-het-
eroaryl scaffold is well documented for inhibitors6 of
protein tyrosine kinases. Such appendages often lead to
a minimal change in intrinsic potency, provided that the
side chains are properly positioned such that the polar
Replacement of the 2-chloro-6-methyl aniline of 2 with
2,6-di-methyl aniline resulted in no change in enzyme
activity, but led to a modest loss in cellular activity (2 vs
2a). The di-phenolic analogue 11a was significantly less
potent (loss of >13-fold in Lck inhibition). Both the
6,7-methylenedioxy (11b) and the 6,7-ethylenedioxy
analogues (11c) displayed decreased potency (2- to 5-fold)
against Lck as well as reduced cellular activity (3-fold)
in comparison to 2. Systematic exploration of substitu-
tion with a mono-methoxy moiety on the fused phenyl
revealed that substitution at 5-, 6-, and 7- positions was
allowed (11d–f and 11h), although increasing the steric
bulk at the 5-position appears detrimental to the
potency (11h vs 11i). However, substitution at the
8-position was not tolerated (11g). Introduction of an
electron-withdrawing group at the 5-, 6-, or 7-positions
(11j–p and 11s–v) also resulted in a significant loss of Lck
inhibitory activity. As a general trend, substitution at the
5-, 6- and 7-positions with an electron-donating group is
favored for the binding affinity (2, 2a, 11a–f, 11q–r)
whereas no substitution is favored at the 8-position.
While the initial study suggested that the 6- and 7-posi-
tions might be the suitable sites for further substitution,
none of the modifications led to any improvement in
cellular activity.
Scheme 1. PMB=p-MeO-benzyl: (a) ethyl glyocylate, toluene, reflux,
then PMB–Cl, NaH, THF; (b) Tos-MIC, NaH, then TfOH, TFA,
anisol; (c) see the above Scheme; (d) K2CO3, DMA or DBU, DMF; (e)
POCl3, reflux; (f) aniline, base, THF.
We next focused our attention on introducing polar
moieties to 6- and 7-positions, with the objective to

P. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3153–3156
Table 1. Inhibition of Lck (enzyme) and T-cell proliferation (human PBLs)
3155
No.
Syn. route
R1
R2
Lck IC50 (nM)3
T-cell prolif. IC50 (nM)3
2
2a
A
A
A
A
A
A
A
A
A
A
A
A
B
B
A
A
6,7-di-OMe
6,7-di-OMe
6,7-di-OH
6,7-OCH2O
6,7-O(CH2)2O
6-OMe
2-Cl, 6-Me
2,6-di-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-Cl, 6-Me
2,6-di-Me
2,6-di-Me
2-Cl, 6-Me
2-Cl, 6-Me
2-Cl, 6-Me
2, 6-di-Cl
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
2.4
4
4
10
3
8.7
280
9.4
240
100
70
26
15
26
24
100
7
670
1100
>9000
2000
2200
1400
2600
—
2300
—
—
—
11a
11b
11c
11d
11f
11g
11h
11i
7-OMe
8-OMe
5-OMe
5-BnO
5-NO2
5-NH2
6-F
6-Br
11j
11k
11l
—
—
—
—
11m
11n
11o
11pA
11q
11r
11s
11t
11u
11v
6-CO2Me
6-NO2
6-CN
—
A
A
B
A
A
A
6-NH2
1600
1400
—
—
—
6-NHAc
7-Br
7-NH2
7-NHAc
7-CONH2
3
14
21
11
30
—
Table 2. Inhibition of Lck (enzyme) and T-cell proliferation (human PBLs)
No. Syn. Route
R1
R2
R3
Lck enzymeb IC50 (nM)3 T-cell prolif.c IC50 (nM)3
12a
12b
12c
12d
12e
12f
12g
12h
12i
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
NMe2
NEt2
NHEt
NHCH2CH2NMe2
H
H
H
H
H
H
H
H
H
H
H
H
H
NEt2
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-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-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
5
2
10
6
7
3
4
3
1
2
9
3
9
9
10
9
6
11
8
2000
2600
2200
880
1300
760
780
380
240
270
530
340
540
1700
520
480
1000
1000
720
570
—
NHCH2CH2–morpholine
NHCH2CH2CH2–morpholine
Morpholine
Piperazine
N-Me-piperazine
N-Et piperazine
N-Formyl piperazine
3,5-di-Me-piperazine
N-Me-homopiperazine
H
12j
12k
12l
12m
12n
12o
12pB
12q
12r
12s
12t
12u
12v
12w
12x
12y
12z
Ba
H
H
H
H
NHCH2CH2NMe2
NHCH2CH2CH2NMe2
Morpholine
Piperazine
N-Me-piperazine
3,5-di-Me-piperazine
OMe
a
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
H
H
OCH2CH2–morpholine
3,5-di-Me-piperazine
13
36
18
2.4
5.4
1.7
5
OMe
OCH2CH2–morpholine
760
470
280
190
240
OMe
OMe
OMe
OMe
NHCH2CH2–morpholine 2-Cl, 6-Me
2-Cl, 6-Me
2-Cl, 6-F
NHCH2CH2NMe2
NHCH2CH2NMe2
aCompounds 6a–c (Scheme 2) were used as the intermediates.
bConcentration to inhibit by 50% the phosphorylation of an exogenous substrate by human Lck enzyme.
cConcentration to inhibit by 50% T-cell proliferation induced by anti-CD3/anti-CD28 co-stimulation in PBLs. CsA gave an IC50 of 115 nM in this
assay.

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P. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3153–3156
improve in vitro potency against T-cell proliferation.
Table 2 illustrates the results from some of the repre-
sentative analogues prepared in this series.
In summary, we have described a promising series of
anilinoimidazoquinoxalines, represented by 12y and 13,
with excellent enzyme activity (IC50 <5 nM). In parti-
cular, significant improvement of activity against T-cell
proliferation (IC50 <200 nM) was achieved from the
initial lead, compound 2, by incorporation of polar and
weakly basic amine-bearing side chains into the 7-posi-
tion of the fused phenyl ring of the imidazo-quinoxaline
core. Full characterization of these compounds in vivo
in pharmacokinetic and pharmacodynamic models will
be disclosed in due course.
Substitution with simple amines at either the 6- or 7-
position initially proved to be ineffective in achieving
this goal (11q, 12a–c, 11t, and 12n). Almost all analogues
displayed significantly reduced T-cell activity, even
though they all showed comparable inhibitory activity
against Lck as 2. Replacement of the simple amine of
11q with a side chain containing a basic amine moiety
resulted in a modest improvement of cell activity (12d).
Compounds 12d, 12f, and 12g are essentially equally
potent to 2 when examined for inhibition of T-cell pro-
liferation. A significant improvement in cellular activity
resulted from substitution at the 6-position with the bulky
secondary amines. All the piperazine substituted ana-
logues displayed excellent cellular potency (12h–l), with
a nearly 3-fold improvement for 12i over 2.
Acknowledgements
We would like to thank Dr. Bang-Chi Chen and Mr.
Mark S. Bednarz for providing assistance in the pre-
paration of key intermediate for the synthesis of ana-
logue 2, and the Discovery Analytical Science Group
for all the analytical support.
Substitution with amines at the 7-position displayed a
similar trend as that observed for the 6-position series,
although the magnitude of the improvement was less
significant. Overall, these analogues appear to be less
potent in the T-cell proliferation assay than their 6-
substituted counterparts (12g vs 12q, 12h vs 12r, 12i vs
12s, and 12l vs 12t).
References and Notes
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We also explored the effect of incorporation of a second
small, electron-donating group into the fused phenyl
ring of the imidazoquinoxaline core. Interestingly, the 6-
amino-7-methoxy di-substituted analogues were sig-
nificantly less potent (6-fold) than their corresponding
mono-amino-substituted analogues (12u vs 12d and 12v
vs 12l), whereas their regio-isomers, the 6-methoxy-7-
amino analogues (12w–z) showed similar or better
enzyme potency than the mono-amino substituted
counterparts. Most significantly, compound 12y dis-
played potent cellular activity (IC50 <200 nM!) in the
T-cell proliferation assay. Compound 13, prepared
according to a similar procedures outlined in Scheme 2,
represents the most cell potent analogue from this ser-
ies, as illustrated in Figure 2.
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Figure 2. Activity for imidazoquinoxaline 2 and 12.