Imidazoquinoxaline Src-family kinase p56Lck inhibitors: SAR, QSAR, and the discovery of (S)-N-(2-chloro-6-methylphenyl)-2-(3-methyl-1- piperazinyl)imidazo-[1,5-a]pyrido[3,2-e]pyrazin-6-amine (BMS-279700) as a potent and orally active inhibitor with excellent in vivo antiinflammatory activity

Article abstract of DOI:10.1021/jm030217e

A series of novel anilino 5-azaimidazoquinoxaline analogues possessing potent in vitro activity against p56^Lck and T cell proliferation have been discovered. Subsequent SAR studies led to the identification of compound 4 (BMS-279700) as an orally active lead candidate that blocks the production of proinflammatory cytokines (IL-2 and TNFα) in vivo. In addition, an expanded set of imidazoquinoxalines provided several descriptive QSAR models highlighting the influence of significant steric and electronic features. The H-bonding (Met319) contribution to observed binding affinities within a tightly congeneric series was found to be significant.

Full text of DOI:10.1021/jm030217e

J . Med. Chem. 2004, 47, 4517-4529
4517
Im id a zoqu in oxa lin e Sr c-F a m ily Kin a se p 56Lck In h ibitor s: SAR, QSAR, a n d th e
Discover y of (S)-N-(2-Ch lor o-6-m eth ylp h en yl)-2-(3-m eth yl-1-p ip er a zin yl)im id a zo-
[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (BMS-279700) a s a P oten t a n d Or a lly Active
In h ibitor w ith Excellen t in Vivo An tiin fla m m a tor y Activity
Ping Chen,* Arthur M. Doweyko,* Derek Norris, Henry H. Gu, Steven H. Spergel, J agabundhu Das,
Robert V. Moquin, J ames Lin, J ohn Wityak, Edwin J . Iwanowicz, Kim W. McIntyre, David J . Shuster,
Kamelia Behnia, Saeho Chong, Henry de Fex, Suhong Pang, Sydney Pitt, Ding Ren Shen, Sara Thrall,
Paul Stanley, Octavian R. Kocy, Mark R. Witmer, Steven B. Kanner, Gary L. Schieven, and J oel C. Barrish
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New J ersey 08543-4000
Received May 7, 2003
A series of novel anilino 5-azaimidazoquinoxaline analogues possessing potent in vitro activity
against p56Lck and T cell proliferation have been discovered. Subsequent SAR studies led to
the identification of compound 4 (BMS-279700) as an orally active lead candidate that blocks
the production of proinflammatory cytokines (IL-2 and TNFR) in vivo. In addition, an expanded
set of imidazoquinoxalines provided several descriptive QSAR models highlighting the influence
of significant steric and electronic features. The H-bonding (Met319) contribution to observed
binding affinities within a tightly congeneric series was found to be significant.
In tr od u ction
70. Subsequent phosphorylation of ZAP-70 by Lck4,5
triggers a series of downstream cascade events that lead
T cell mediated immune responses play an important
role in the pathogenesis of many immunological disor-
ders, including but not limited to rheumatoid arthritis,
asthma, multiple sclerosis, inflammatory bowel disease,
systemic lupus erythematosus, psoriasis, and transplant
rejection. Immune responses in T cells are initiated by
the interaction of the T cell receptor (TCR) with an
antigen bound to a glycoprotein encoded by the major
histocompatibility complex (MHC). The engagement of
the TCR to the antigen bound MHC is followed by a
series of intracellular biochemical events, including an
increase in protein tyrosine and serine/threonine phos-
phorylation, phospholipid hydrolysis, and changes in
intracellular Ca2+ levels, that ultimately lead to T cell
proliferation and/or differentiation. Among all the in-
tracellular events that are required for T cell activation,
the earliest and perhaps most important one is the
increase in intracellular protein tyrosine phosphoryl-
ation due to the action of the nonreceptor tyrosine
kinase p56Lck (Lck).1
to mobilization of intracellular calcium (Ca2+ 6
) and
activation of protein kinase C (PKC), a serine/threonine-
specific kinase.7,8 The combination of both actions
results in the activation of several nuclear transcription
factors, including NF-AT and AP-1, and the transcrip-
tion of their corresponding genes, ultimately leading to
the generation of cytokines, such as IL-2, which drives
T cell proliferation. Studies with transgenic mice have
shown that T cells that lack Lck are severely impaired
in TCR tyrosine phosphorylation and are unable to be
activated via the TCR.9 Because Lck expression is
known to be restricted only to lymphoid cells and is
required for T cell signaling,10-13 a selective Lck inhibi-
tor is expected to be effective and, perhaps more
importantly, may offer an advantageous safety profile
relative to cyclosporine A or high doses of conventional
steroids for the treatment of chronic and/or acute T cell
mediated autoimmune and inflammatory disorders, and
for the prevention of transplant rejection.
As one of the nine known members of the Src family
of non-transmembrane protein tyrosine kinases that
share a high degree of homology within their ATP-
binding regions, Lck plays a critical role in the T cell
receptor (TCR) signal transduction pathway.1,2 Studies
have shown that the catalytic activity of Lck is regulated
by tyrosine phosphorylation at two sites: Tyr394 in the
catalytic domain and Tyr505 on the C-terminus.3 When
Tyr394 is phosphorylated and Tyr505 is dephosphoryl-
ated, the fully activated enzyme can phosphorylate
tyrosine residues within a special sequence called the
immunoreceptor tyrosine activation motif (ITAM) lo-
cated on the ú-chain of the TCR. This phosphorylation
creates a docking site for its downstream substrate ZAP-
Although knockout mice in which various Src-family
kinases have been deleted display a variety of defects,14
it has not been clearly defined in most cases whether
pharmacological inhibition of Src-family kinases in adult
animals would lead to similar effects, or whether at least
some phenotypes observed arise primarily due to a
requirement for specific Src-family kinases during
discreet stages of development. For example, Lck knock-
out mice have been reported to display partial retinal
detachment with infolding and rosette formation in the
photoreceptor sheet,15 whereas Fyn knockout mice
display defects in myelination of the forebrain,16 im-
paired long-term potentiation,17 and hypersensitivity to
ethanol.18 Selective inhibitors with potent in vivo activ-
ity are required to address the question of which of the
possible toxicities suggested by the phenotypes of the
* Authors to whom correspondence should be addressed. P.C.: tel
(609) 252-5809; fax (609) 252-6601; e-mail ping.chen@bms.com.
10.1021/jm030217e CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/27/2004

4518 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Chen et al.
F igu r e 1. Activity of imidazoquinoxalines 1-3.
F igu r e 2. Activity of compound 4 (BMS-279700).
Sch em e 1. Synthesis of Compound 10a
knockout mice would indeed be observed upon pharma-
cologic inhibition of Src-family kinases.
Several classes of Lck inhibitors, acting either at the
catalytic site19-25 or on the SH2 domains,26-29 have been
reported recently, some of which display good in vivo
activity in an IL-2 production model.24,25 Mechanistic
details of enzyme inhibition may be gleaned from
examination of the X-ray crystal structure of the cata-
lytic domain of Lck in its active autophosphorylated
state,30-32 shedding light on the design of novel inhibi-
tors. Our focus in regulating cytokine production via
inhibition of T cell activation prompted us to evaluate
the potential of Lck as an antiinflammatory therapeutic
target. Toward this end, novel anilinoimidazoquinoxa-
lines, represented by compound 1, were identified as Lck
inhibitors with modest potency (IC50 ) 170 nM) from a
high throughput screen of our compound collection.33
Subsequent structural modification of 1 led to the
discovery of 2, a highly potent Lck inhibitor (IC50 ) 2
nM) with >20-fold improvement of cellular activity
against T cell proliferation over the screening hit.
Unfortunately, similar to the initial hit 1, compound 2
suffered from poor aqueous solubility (<8 µM), hamper-
ing it from further in vivo evaluation. This low aqueous
solubility prompted a search for more water-soluble
analogues to allow for in vivo studies. It has been
reported in the literature that the addition of a “weakly
basic amine tail” at either the C6 or C7 positions of
various 4-anilinoquinazoline tyrosine kinase inhibitors
resulted in improved water solubility and cellular
activity.34-37 Moreover, a closer examination of the
binding model of 133 revealed that the 6- and 7-positions
of the fused phenyl ring point toward the open end of
the ATP binding site leading toward the solvent and
introduction of solubilizing groups at these positions
appeared to be a viable strategy. Accordingly, incorpora-
tion of a tether that bears a polar and weakly basic
tertiary amine moiety to the phenyl ring of 1 and 2 led
to compounds with enhanced aqueous solubility (>100
µM) and superior in vitro and cellular activities, as
demonstrated by compound 338 (Figure 1).
a
(a) NaHMDS, THF, 5 h. Yield: 40%. (b) K2CO3, DMA, reflux,
12 h. Yield: 83%. (c) POCl3, TEA, reflux, 4 h. Yield: 54%. (d)
2-Methyl-6-chloroaniline, NaHDMS, THF, rt, 2 h. Yield: 77-86%.
Sch em e 2. Synthesis of Compound 4 (BMS-279700)a
a
(a) 2-Methyl-6-chloroaniline, NaHDMS, THF, rt, 2 h. Yield:
98%. (b) (S)-(+)-2-Methylpiperazine (neat), 120 °C, 5 h. Yield: 90%.
tricyclic core, imidazo[1,5a]-pyrido[3,2-e]pyrazin-2-one
8, was achieved via an intramolecular cyclization of 7
using a procedure that was developed in our labora-
tory.39 Treatment of 8 with phosphorus oxychloride
yielded the chloroimidate 9, which was further coupled
with 2-methyl-6-chloroaniline in the presence of a base
(e.g. NaHMDS) to afford compound 10.
Introduction of amines at the C6 position of the
anilinoimidazo[1,5a]pyrido[3,2-e]pyrazine was accom-
plished by a two step sequence starting from dichloride
11, as illustrated by the synthesis of compound 4.
Reaction of 11, prepared from 2,6-dichloro-3-amino-
pyridine according to Scheme 1, with 2-chloro-6-methyl-
aniline yielded 12, which was further reacted with (S)-
3-methyl-piperazine to provide the desired compound
4. Interestingly, the chlorine at the C2 position is
significantly more reactive than the one at the C6
position, presumably due to the activation of the C2
carbon by the fused imidazole ring. This difference in
reactivity at the two sites allows for preferential sub-
stitution at the C2 position, and compound 12 was
obtained as the sole product (Scheme 2).
In parallel to the above approach, we also investigated
the replacement of the fused phenyl ring of 2 with
various heterocycles with the objective of increasing the
overall polarity of the molecule and thereby improving
its aqueous solubility. In this report, we describe our
effort in developing the structure-activity relationship
(SAR) around the tricyclic azaimidazoquinoxaline core,
which led to the discovery of compound 4 (BMS-279700),
a novel, potent and orally active Lck inhibitor with
excellent in vivo antiinflammatory activity (Figure 2).
Ch em istr y
The anilinoimidazo[1,5-a]pyrido[3,2-e]pyrazine ana-
logues were prepared as shown in Scheme 1 using 10
as a representative example. The construction of the

Imidazoquinoxaline Src-Family Kinase p56Lck Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4519
Ta ble 2. Inhibition of Lck (Enzyme) and T-Cell Proliferation
(Human PBLs)
F igu r e 3. Azaimidazoquinoxaline analogues 22, 23, and 24.
Ta ble 1. Inhibition of Lck (Enzyme) and T-Cell Proliferation
(Human PBLs)
a
Average of at least three determinations (n )3).
a
Average of three determinations (n )3). b For comparison, CsA
has an IC50 ) 115 nM against T cell proliferation in the same
assay.
SAR Discu ssion
Table 1 summarizes the structure-activity relation-
ships for the inhibition of Lck and T cell proliferation
from a series of analogues where the fused phenyl ring
of compound 13 is replaced by a fused heterocycle.
Incorporation of a nitrogen atom into the phenyl ring
of 13, a potent Lck inhibitor (IC50 ) 9 nM), resulted in
loss of intrinsic activity to various degrees, as illustrated
by 5-aza 10 (3-fold), 6-aza 14 (>100-fold), 7-aza 15 (8-
fold), and 8-aza analogues 16 and 17 (>10-fold). Re-
placement of the fused phenyl ring of 13 with other
heterocycles, such as N-methyl imidazole (18) or N-meth-
yl-C3-methyl pyrrole (19), essentially abolished Lck
activity, as did the 7-methoxy-6,8-pyrimidino analogue
20. As a comparison, 20 is >80-fold less active than its
carbocyclic counterpart, compound 21. Among the ana-
logues prepared in this series, the 5-aza analogue 10
showed the smallest reduction of activity (3-fold) from
the parent carbocycle, compound 13.
ity,33,38 we focused our attention on the SAR around the
6- and 7-positions of the fused pyridine ring of the
azaimidazoquinoxalines and substituted these positions
with various polar functionalities, including alcohols
and amines. To this end, compounds with general
structures 22, 23, and 24 were prepared and tested for
their respective inhibitory potency (Figure 3).
Substitution at the C6 position of 10 with a chloro (12)
or methoxy (22a ) group had a minimal effect on potency
(Table 2). Interestingly, incorporation of a small amino
group at this position led to a significant increase in
intrinsic potency, as demonstrated by compounds 22b
(30-fold) and 22c (15-fold). Unfortunately, this favorable
increase in potency did not translate into an improve-
ment in cellular activity. Compounds 22b and 22c are
essentially equipotent to 22a in a T cell proliferation
assay and are only slightly better than the parent
carbocycle, compound 13. In addition, introduction of a
polar N,N-dimethylaminoethylamino tether at the C6
Since our earlier SAR studies indicated that substitu-
tion at the C5 and C8 positions of the imidazoquinoxaline
core with small groups was detrimental to the activ-

4520 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Chen et al.
Ta ble 3. Inhibition of Lck (Enzyme) and T-Cell Proliferation
position of 10 led to a significant loss of enzyme activity
(22d ). Incorporation of a small heteroaromatic group
(e.g. imidazole) at this position led to not only a gain in
intrinsic potency but also an improvement in cellular
activity (22e). Further investigation using sterically
bulky secondary amines led to some interesting results.
While the morpholinyl (22f) and piperidinyl (22g)
analogues showed minimal improvement in enzyme and
cellular activities in comparison to the diethylamino
analogue 22c, compounds with heterocycles bearing an
additional basic nitrogen (22h -k ) all displayed mark-
edly enhanced cellular activity. Compound 22h is a
highly active Lck inhibitor that displays potent cellular
activity in blocking T cell proliferation (IC50 ) 230 nM)
(Table 2). It is conceivable that such an improvement
in cellular activity is most likely due to an improvement
in aqueous solubility as well as an enhancement in cell
permeability, a trend observed by other known PTK
inhibitors in the literature.34-37 Surprisingly, an ana-
logue with an aliphatic tether possessing a basic nitro-
gen was significantly less potent (>70-fold) than its
heterocyclic counterpart (22d vs 22h ), highlighting the
necessity for the conformational constraint imposed by
a heterocycle at this position (vide infra).
(Human PBLs)
Substitutions at the C6 position of 23 and C7 position
of 24 were also investigated (Table 2). In general, within
the C6-substituted series (i.e. 22 and 23), the 5-aza
analogues are consistently more potent than the 7-aza
analogues, as demonstrated by the activities of 22k and
23 (>10-fold).40 In addition, analogues derived from the
C7-substituted 6-aza scaffold 24 are significantly less
active, all of which displayed IC50 of greater than 100
nM (24a -c).
Encouraged by the significant improvement in cel-
lular activity by 22h , our attention focused on the 5-aza
series and substitution by various heterocycles at the
C6 position. The in vitro biological data from these
studies are summarized in Table 3.
Analogues with substituted pyrrolidine moieties (25-
30) showed similar enzyme and cellular potency in
comparison to 22h , with the exception of diol 28, which
displayed diminished potency in inhibiting T cell pro-
liferation. On the other hand, all piperazine analogues
(4, 22h , and 31-36) were consistently among the most
potent Lck inhibitors, both against the enzyme and in
blocking T cell proliferation. Unlike the N,N-dimethyl-
aminoethylamino analogue 22d of Table 2, analogues
bearing open-chained amino alcohol groups were potent
Lck inhibitors with good (37) to modest (36, 38-40)
cellular activity. However, due to the lack of a basic
amine moiety, these analogues suffered from reduced
solubility when compared with the piperazine ana-
logues, such as 4, 32, and 33 (Table 3).
a
b
Average of three determinations (n )3). Solubility in PG/
EtOH/IPM (50:45:5). c Ki (Lck) ) 0.55 ( 0.04 nM, Kd (Lck) ) 0.17
( 0.03 nM (n ) 3).
Ta ble 4. Selectivity Profile of Compound 4 (BMS-279700)
kinase
Syk
ZAP-70
PKA
PKC
CDK
IKK1,2
HER1,2
PDGF-R
VEGF-R
IC50 (nM)
kinase
IC50 (nM)
>10000
>1000
Lck
Src
Fyn
Lyn
Fgr
Yes
Hck
Blk
p38
4
4
5
>10000
>10000
>10000
>10000
>1000
0.5
2.8
4.7
23
27
380
Compound 4 was further profiled against a panel of
protein kinases for selectivity, and the results are shown
in Table 4. In general, compound 4 is highly selective
(>1000-10000-fold selectivity) vs kinases from other
families of serine/threonine (with the exception of p38,
a MAP kinase, where a 95-fold selectivity was observed)
and tyrosine kinases. Notably, it is significantly less
selective or nonselective in comparison to kinases within
the Src family.
>1000
>1000
In addition to the measurement of cellular activity
by the inhibition of T cell proliferation, inhibitory effects
on production of inflammatory cytokines, such as IL-2,

Imidazoquinoxaline Src-Family Kinase p56Lck Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4521
The aqueous solubility of compound 4 was deter-
mined. As expected, in water, the HCl salt of compound
4 is significantly more soluble than the free base (Table
5).
The enhancement (>10-fold) of intrinsic potency
displayed by C6-substituted 5-aza analogues, repre-
sented by 4 and 22h , over the nonsubstituted 5-aza
analogue 10 is consistent with our proposed binding
model established using the published coordinates of an
activated Lck kinase domain bound to ANP (a nonhy-
drolyzable ATP mimic). Figure 4 illustrates the proposed
binding mode of compound 22h in the ATP pocket of
Lck. Favorable hydrophobic interactions between the
2,6-disubstituted aniline moiety (which adopts a pre-
ferred out-of-plane orientation in comparison to the
tricyclic system) and the angular hydrophobic pocket of
the enzyme are likely. In addition, two critical hydrogen
bonding interactions (N3-nitrogen of imidazole to the NH
of Met319 and the NH of the aniline to the OH of
Thr316) appear to be probable. This hydrogen-bonding
motif is common to this class of inhibitors. Perhaps
equally important is the presence of a third ionic
hydrogen-bonding interaction between the NH of the
piperazine of 22h and the carboxylate of Asp326, which
may play a significant role in the enhancement in the
binding affinity of these analogues (Figure 4).
F igu r e 4. Inhibitor 22h (protonated at piperazine NH)
modeled into the ATP-binding site of Lck illustrating key
interactions between the ligand and binding site: (1) deep
hydrophobic pocket occupied by the angular di-ortho-substi-
tuted aniline ring, (2) hydrogen bond between Thr316 and
aniline NH, and (3) hydrogen bond between Met319 and
imidazolyl N, and (4) a putative electrostatic interaction
between protonated piperazine and Asp326.
Ta ble 5. Solubility of 5-Azaimidazoquinoxaline Analogue 4
(Free Base and HCl Salt)
On the basis of its promising in vitro and cellular
activities, compound 4 was further evaluated in vivo.
The pharmacokinetic properties of 4 were obtained from
studies in mice, rats, and dogs following intravenous
and oral administration (Table 6). In general, compound
4 had a more favorable phamacokinetic profile in dogs
than in rodents. For example, in mice and rats, com-
pound 4 displayed a higher clearance rate (86 and 109
mL/min/kg, respectively) and a relatively short half-life
(3.2 and 2.4 h, respectively). This trend is also reflected
in the observed order of oral bioavailabilities in three
species (19%, 20%, and 77%, respectively). In addition,
compound 4 has a favorable protein binding profile:
91.12% ((0.81) for mouse and 84.82% ((1.41) for
human, indicating that a relatively high free fraction
can be achieved upon in vivo dosing.
To evaluate the in vivo efficacy of the 5-azaimidazo-
quinoxaline chemotype, compound 4 was tested in a
mouse anti-CD3-stimulated IL-2 production model. In
this model, compound 4 displayed a 50% reduction of
serum IL-2 when dosed orally at 20 mpk (data not
shown). At 50 mpk, a 78% reduction of serum IL-2 levels
was achieved (Figure 5).
compd
water (mg/mL)
4 (BMS-279700)
4‚HCl
0.003
9.2
by the 5-azaimidazoquinoxaline analogues were also
examined. In T cells, compound 4 displayed potent
inhibitory activity against IL-2 production (IC50 ) 200
nM) when challenged by anti-CD3/anti-CD28 costimu-
lation. This is consistent with the mechanism of action
of an Lck inhibitor since inhibition of Lck blocks TCR
signaling, resulting in the reduction of IL-2 production
and, ultimately, leading to T cell inactivation. Interest-
ingly, compound 4 also inhibits LPS-induced tumor
necrosis factor R (TNFR) production in human PBLs
(IC50 ) 295 nM). This inhibition of TNFR may be due
to its ability to inhibit the Src-family kinases expressed
in monocytes. LPS has been reported to activate the Src-
family kinases Hck, Fgr, and Lyn in monocytes,41 and
manipulation of levels of active Hck in a murine
macrophage cell line and in transgenic mice indicates
that Hck can regulate LPS-induced TNFR production
in murine macrophages.42,43 Inhibition of p38 kinase
may also potentially contribute to the inhibition of
TNFR production by the compound.
As one of the major proinflammatory cytokines, TNFR
plays a critical role in both acute and chronic inflam-
matory responses and is often implicated in various
Ta ble 6. Pharmacokinetic Properties of Compound 4 (BMS-279700)
mouse (n ) 3)
rat (n ) 3)
dog (n ) 2)
iv dose
po dose
10
iv dose
po dose
10
iv dose
10
po dose
10
dosea (mg/kg)
AUC-iv (µM h)
Vss (L/kg)
10
10
4.75 ( 0.36
14.5 ( 1.4
86 ( 6
3.75 ( 0.18
21 ( 1
9.97 ( 1.16
22 ( 3
Cl (mL/min/kg)
109 ( 5
41 ( 5
6 ( 1
t
1/2 (h)
3.2 ( 1.4
2.4 ( 0.1
AUC-po (µM h)
MRT (h)
F (%)
0.90 ( 0.17
6.1 ( 1.4
19
0.62 ( 0.28
4.1 ( 0.2
20
6.84 ( 2.69
9 ( 1
77
a
Compound 4 was dosed as a solution in PEG300:water (3:1).

4522 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Chen et al.
F igu r e 5. Inhibition of anti-CD3/anti-CD28-induced IL-2
production in mouse by compound 4 (BMS-279700).
F igu r e 7. Lck model derived from published structures25-27
and homology modeling based on the Hck structure.4 Com-
pound 2 was docked into the ATP binding site.
F igu r e 6. Inhibition of LPS-induced TNF-R production in
mouse. Compound 4 (BMS-279700) was dosed orally.
inflammatory autoimmune diseases.44-51 Studies have
shown that treatment of RA patients with monoclonal
antibodies or soluble TNF receptor to antagonize TNFR
resulted in significant clinical efficacy.52-57 The promis-
ing clinical data from anti-TNFR biologics led to FDA
approval of Enbrel (Etanercept), Remicade, and Humira
as antiarthritic therapies. In addition, small molecule
drugs that inhibit the formation of TNFR have also been
disclosed recently.58 Since compound 4 displayed in vitro
activity against LPS-induced TNFR production in hu-
man PBLs (vide supra), it was further tested in a mouse
LPS-induced TNFR production model for in vivo efficacy.
Consistent with the in vitro result, compound 4 sup-
pressed serum TNFR production in mice in a dose-
dependent fashion (Figure 6), demonstrating that com-
pound 4 could be potentially useful as an oral agent in
treating inflammatory diseases associated with elevated
TNFR levels.
F igu r e 8. Imidazoquinoxaline scaffold used in QSAR analyses
1 (R1 ) di-ortho substituted, R2 ) R3 ) H) and 2 (R1 ) mono-,
di-, and trisubstituted; R2 ) H, OCH3; R3 ) H, OCH3).
based on the active, autophosphorylated form of Lck.
Inspection of the inhibitor binding motif suggested
several possibly critical interactions, namely, (1) a deep
pocket, hydrophobic interaction involving the pendant
di-ortho-substituted aniline ring; (2) a hydrogen bond
between Thr316 OH (acceptor) and the anilino NH
(donor); and (3) a hydrogen bond between Met319
backbone NH (donor) and the imidazolyl N (acceptor).
These interactions were considered critical to the overall
activity of the imidazoquinoxaline series (as illustrated
in Figure 4).
Mod elin g Discu ssion
Two QSAR analyses were conducted in order to
ascertain the relative contributions of key features
within the imidazoquinoxaline series. The descriptor
sets included typical Hansch parameters (F, R, π, MR,
and Vm ) molecular volume) along with indicator
variables as appropriate. The first analysis was limited
to those compounds containing ortho-disubstituted
anilines (R1, Figure 8, where R2 ) R3 ) H) and
comprised a total of 19 analogues with an IC50 range33
of 9 nM to 12.5 µM. Two equations (eqs 1, 2) emerged
with modest correlation (r2 ) 0.65-0.68) which were
statistically indistinguishable. While both indicated a
negative effect on binding affinity due to large ortho
substitutents (MRo), eq 1 suggests that the mathemati-
cal counterpoint comes from positive effects due to the
electron-withdrawing nature of such groups (F, R),
whereas eq 2 achieves an equivalent result with hydro-
phobicity (π).
Mod elin g
In an effort to ascertain the structural and/or molec-
ular properties associated with potent inhibition, quan-
titative structure-affinity correlations were determined
for selected members of the imidazoquinoxaline series
using several multiple linear regression approaches
wherein classical Hansch descriptors served to highlight
significant structural/electronic features potentially cor-
related with observed Lck binding affinity. In addition,
it was of interest to examine the possibly significant role
of hydrogen bonding between the ligand imidazolyl N
and backbone NH (Met319).
Some structural details of kinase inhibitor binding
have been published which provided the basis for the
construction of an Lck binding site model illustrated in
Figure 7.30-32 This model of the catalytic domain is

Imidazoquinoxaline Src-Family Kinase p56Lck Inhibitors
QSAR An a lysis 1.
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4523
pIC50 ) -0.95 MRo + 0.65 F + 2.62 R + 3.16 (1)
r2 ) 0.65
pIC50 ) -0.92 MRo + 1.75 π2 - 0.43 π + 0.862 (2)
r2 ) 0.68
F ) 9.24
s ) 0.61
N ) 19
F ) 10.8
s ) 0.58
N ) 19
A second QSAR analysis focused on an expanded set
of imidazoquinoxalines (N ) 114, IC50 range of 1 nM to
12.5 µM), wherein mono-, di-, and trisubstituted anilines
(Figure 8, R1) were included, along with OCH3 substit-
uents at positions 6 and 7 (Figure 8, R2, R3). The
descriptor set was augmented with indicator variables
accounting for specific substitution patterns, e.g., oMe
) o-methyl aniline. A step-up multiple linear regression
approach yielded a series of plausible equations (eqs
3-7, all correlated with pIC50 (pIC50 ) -log IC50)).
Values in parentheses refer to positions on the aniline
ring. Bolded descriptors are newly added in the step-
up regression analysis sequence.
F igu r e 9. Hydrogen bond donor locations are illustrated
herein as determined using a grid-based analysis.10 Compound
2 is shown with contours (outer -2.0 kcal/mol; inner -4.0 kcal/
mol) developed using an NH3+ probe at a grid spacing of 0.25
Å. The upper left contour represents the location for water in
the SAM1 hydrogen bond modeling procedure.
Ta ble 7. Imidazoquinoxaline Hydrogen-Bonding Analysis Data
Set (Hydrogen Bond Enthalpies, HB Hf, Determined Using
SAM1)
QSAR An a lysis 2.
+1.37 F(2) + 1.66 π(6) - 0.14 MR(6) + 2.81 F(6) +
1.82 oMe - 0.69 (3)
r2 ) 0.65
r(cv)2 ) 0.60
F ) 39.4
HB Hf
+1.34 F(2) + 1.96 π(6) - 0.13 MR(6) + 2.46 F(6) +
1.83 oMe - 0.01 Vm - 0.20 (4)
compound
R1
R2
R3 A B C pIC50 (µM) (kcal/mol)
22b
2
21
N(CH3)2
H
N
3.00
2.70
2.52
2.22
2.15
2.06
2.05
1.85
1.80
1.69
1.68
1.60
1.59
1.52
1.52
1.11
1.10
1.05
-3.56
-3.60
-3.44
-3.39
-3.45
-3.50
-3.33
-3.27
-3.17
-2.90
-3.50
-3.09
-3.14
-3.33
-3.12
-3.16
-2.98
-3.23
OCH3
OCH3
Ph
NH2
H
OCH3
r2 ) 0.69
r(cv)2 ) 0.64
F ) 39.2
H
H
H
-1.17 F(3) + 0.66 π(6) + 2.67 F(6) +1.59 oMe -
0.01 Vm + 0.80 oCl - 0.07 (5)
OCH3
H
H
H
COOCH3
r2 ) 0.71
r(cv)2 ) 0.66
F ) 43.3
NO2
Cl
H
Cl
NH2
H
H
Cl
F
H
H
H
F
H
-1.17 F(3) + 0.64 π(6) + 1.53 F(6) +1.35 oMe -
0.01 Vm + 0.62 oCl + 0.80 d iMeO + 0.06 (6)
12
N
N
H
Cl
H
H
H
r2 ) 0.75
r(cv)2 ) 0.71
F ) 45.4
10
15
N
Br
+ 0.39 π(2) - 0.91 R(4) +0.96 oMe -0.02 Vm +
0.64 oCl + 0.71 diMeO + 0.94 d iOr th o - 0.20 (7)
16
H
N
r2 ) 0.76
r(cv)2 ) 0.71
F ) 46.7
calculated to have an optimum size and electronic
property according to eq 1.
QSAR analysis 2 clearly points out the significant
contributions to activity brought about by specific sub-
stituents, namely, the presence of a OCH3 at positions
6 and 7 (diMeO), the di-ortho substitution (diOrtho), the
o-methyl and o-chloro (oMe and oCl, respectively). These
indicator variables are set to 1 when the group is
present and 0 when absent. The ortho-substitution effect
is consistent with the proposed binding model, since
such substitution of the aniline would place the ring into
the out-of-plane conformation required for the aniline
ring to enter the deep hydrophobic pocket. The apparent
preference for an o-chloro and o-methyl pattern is
consistent with QSAR analysis 1, wherein these two
moieties are among the set of di-ortho substituents
The possibility that 6,7-di-OCH3 substitution yielded
congeners with enhanced Lck binding affinities because
of a modulating effect on the proposed hydrogen bond
between imidazolyl N and Met319 was examined next.
In order to explore the possible correlation of hydrogen
bond strength to observed binding, we needed a model
method to estimate the hydrogen bond enthalpy be-
tween a water molecule acting as donor and a hetero-
atom (imidazolyl N) acting as an acceptor. To this end,
the semiempirical method, SAM1,61 was used to geom-
etry optimize a modeled system consisting of the imi-
dazoquinoxaline core and water (see Figure 9 and Table
7), affording an estimate for the enthalpies of complexed
and free species. The differences provided the hydrogen

4524 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Chen et al.
with excellent in vivo antiinflammatory efficacy in mice
(inhibition of IL-2 and TNFR). Compound 4 and the
other members of this series should be of value to help
define the benefits of Src-family kinase inhibition in
animal models of disease, as well as to investigate
possible toxicities that may arise from inhibition of these
kinases. In addition, using tools of molecular modeling,
the structural factors underlying p56Lck binding affinity
were identified. Multiple regression analysis (QSAR)
highlighted the importance of the di-ortho-substituted
aniline and the use of small, electron-withdrawing
substituents to ensure an out-of-plane ring orientation
F igu r e 10. Correlation of observed Lck binding affinities
(actual pIC50) with SAM1 calculated hydrogen bond enthal-
pies.
and best fit for the deep, hydrophobic pocket of p56Lck
.
Furthermore, a significant role of the putative hydrogen
bond between inhibitor (imidazolyl N) and protein
(Met319, NH) was proposed on the basis of the correla-
tion observed between calculated H-bond enthalpies and
binding affinities for a selected set of imidazoquinoxa-
lines.
bond enthalpy between water and the imidazoquinoxa-
line (the enthalpy, Hf, for water was determined as
-57.04 kcal/mol and was used as a constant in these
calculations).
The 18 imidazoquinoxalines of Table 7 were selected
on the basis of their orientations in the Lck binding site
model, with care being taken to limit the selection to
those molecules with relatively small R1/R2 groups in
order to avoid additional obfuscating interactions with
the site. SAM159 estimates of hydrogen bond enthalpies
appeared to correlate well with observed Lck binding
affinities (r2 ) 0.53), especially when considering the
underlying assumption that no other significant inter-
actions were introduced in this series. These compari-
sons are given in Table 7 and illustrated in Figure 10.
The observation of a significant correlation between
hydrogen bonding in a series of kinase inhibitors and
their observed binding affinities is not without prece-
dence. Such an inference was made in the case of
pyridyl-containing p38 kinase inhibitors wherein the
relationship focused on the electrostatic potential at the
pyridyl nitrogen,60,61 and for a series of flavonoid inhibi-
tors of p56Lck where a quantum chemical parameter set
defining hydrogen bonding was correlated with activ-
ity.62 Interestingly, in a study aimed at defining the
determinants of ligand binding to cAMP-dependent
protein kinase, theoretical models based on continuum
electrostatics and surface area dependent nonpolar
terms suggested that hydrogen bonding represents a
negligible contribution to binding.63 In contrast, the
present study clearly demonstrates a non-negligible
contribution to binding. It would appear that further
investigation into the role of hydrogen bonding in
protein-ligand interactions may provide some needed
illumination and that generalizations regarding the
secondary role of intermolecular hydrogen bonds in
binding sites may be premature.
Exp er im en ta l Section
Ch em istr y. All solvents and reagents were obtained from
commercial sources and used without further purification
unless indicated otherwise. Melting points were obtained from
a Mel-Temp 3.0 or Fisher-J ohns melting point apparatus and
are uncorrected. 1H NMR and 13C NMR spectra were obtained
on a J EOL 400 MHz CPF-270 spectrometer operating at 270
or 67.5 MHz, respectively, and chemical shifts are reported in
parts per million (δ) from the tetramethysilane resonance in
the indicated solvent. High-resolution mass spectra were taken
with a Micromass LCT spectrometer using ESI-TDF ioniza-
tion. All new compounds were homogeneous by thin-layer
chromatography (TLC) and reversed-phase HPLC (>98%).
Flash chromatography was carried out on E. Merck Kieselgel
60 silica gel (230-400 mesh). Preparative HPLC was per-
formed on a YMC OD S-10 50 × 500 mm column eluting with
a mixture of solvents A and B using a gradient elusion (solvent
A, 10% MeOH-90% H2O-0.1% TFA; solvent B, 90% MeOH-
10% H2O-0.1% TFA; UV 254 or 210 nm). Analytical HPLC
was performed on a YMC OD S-5 4.6 × 50 mm column eluting
with a mixture of solvents A and B using a gradient elution
(solvent A, 10% MeOH-90% H2O-0.1% TFA; solvent B, 90%
MeOH-10% H2O-0.1% TFA; UV 254 or 210 nm).
N-(2-Ch lor o-3-p yr id in yl)-1H -im id a zole-4-ca r b oxa m -
id e (7). To 3-amino-2-chloropyridine (2.87 g, 22.3 mmol) in
THF (13 mL), cooled in a -10 °C bath, was added sodium bis-
(trimethylsilyl)amide (51 mL, 51.0 mmol, 1.0 M in THF). The
mixture was stirred for 1 h, and a suspension of imidazole
carbonyl dimer39 (2 g, 10.6 mmol) in THF (20 mL) was added
and let warm to room temperature. The mixture was stirred
for 2 h, and acetic acid was added. The reaction mixture was
concentrated in vacuo followed by the addition of water and
saturated sodium bicarbonate. The solid was collected by
filtration, washed by water and hexane, and dried under high
1
vacuum to give 7 as a light beige solid (Yield: 40%). H NMR
(400 MHz, CDCl3): δ 12.13 (bs, 1H), 9.65 (s, 1H), 8.93 (dd, J
) 1.71 Hz, J ) 8.12 Hz, 1H), 8.09 (dd, J ) 1.71 Hz, J ) 4.49
Hz, 1H), 7.74 (s, 1H), 7.63 (s, 1H), 7.29 (dd, J ) 4.49 Hz, J )
8.12 Hz, 1H).
Con clu sion s
Im id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin e-6(5H )-on e (8). A
mixture of 7 (1.89 g, 8.49 mmol) and potassium carbonate (3.5
g, 25.5 mmol) in dimethylacetamide (40 mL) was heated to
reflux for 6 h. The reaction mixture was then concentrated in
vacuo followed by the addition of water and saturated am-
monium chloride. The solid was collected by filtration, washed
with water, and dried under high vacuum to give 8 as a light
beige solid (Yield: 83%). Mp: 295-300 °C. 1H NMR (400 MHz,
DMSO-d6): δ 11.49 (s, 1H), 8.88 (s, 1H), 8.23 (dd, J ) 1.37
Hz, J ) 4.69 Hz, 1H), 7.89 (s, 1H), 7.67 (dd, J ) 1.37, J )
8.06, 1H), 7.45 (dd, J ) 4.57, J ) 8.06, 1H). MS (ESI): m/z )
187 (M + 1). HR-MS (FAB) for C9H7N4O (MH+): calcd
187.0620, found 187.0650.
A series of novel anilino 5-azaimidazoquinoxaline
analogues possessing potent in vitro inhibitory activity
against p56Lck have been identified. Incorporation of
polar, small heterocycles at the C6 position of the
5-azaimidazoquinoxaline tricyclic scaffold led to com-
pounds with enhanced intrinsic potency as well as
improved activities in blocking human T cell prolifera-
tion. These modifications resulted in enhanced physi-
cochemical properties for this class of compounds. From
this series, compound 4 (BMS-279700) was identified
as the lead candidate and was shown to be orally active

Imidazoquinoxaline Src-Family Kinase p56Lck Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4525
6-Ch lor oim id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin e (9). To 8
(1.37 g, 7.36 mmol) was added phosphorus oxychloride (20 mL),
and the mixture was heated to reflux for 12 h. The reaction
mixture was concentrated in vacuo, and the residue was cooled
in an ice bath. Water was added to the residue and neutralized
with saturated sodium bicarbonate. The solid was collected
by filtration and purified using flash column chromatography
on silica gel to yield 9 as a light yellow solid (Yield: 54%).
CDCl3): δ 8.91 (bs, 1H), 8.64 (s, 1H), 8.49 (d, J ) 5.33 Hz,
1H), 7.66 (d, J ) 5.33 Hz, 1H), 7.39 (m, 1H), 7.25-7.35 (m,
3H), 6.80 (bs, 1H), 2.36 (s, 3H). MS (ESI): m/z ) 310+ (M+
+
1). HR-MS (ESI-TDF) for C16H13ClN5 (M+ + H): calcd 310.0859,
found 310.0852.
N-(2-Ch lor o-6-m eth ylp h en yl)im id a zo[1,5-a ]p yr id o[2,3-
e]p yr a zin e-4-a m in e (16). Compound 16 was prepared from
imidazo[1,5-a]pyrido[2,3-e]pyrazine-6(5H)-one64 using a method
analogous to the preparation of 10. 1H NMR (400 MHz, DMSO-
d6): δ 9.08 (bs, 1H), 8.59 (d, J ) 8.12 Hz, 1H), 8.36 (d, J )
4.27 Hz, 1H), 7.89 (bs, 1H), 7.35-7.40 (m, 1H), 7.33 (dd, J )
8.12 Hz, J ) 4.27 Hz, 1H), 7.17-7.28 (m, 2H), 2.30 (s, 3H).
1
Mp: 194-195 °C. H NMR (400 MHz, DMSO-d6): δ 9.28 (s,
1H), 8.70 (dd, J ) 4.70 Hz, J ) 1.48 Hz, 1H), 8.36 (dd, J )
1.48 Hz, J ) 8.10 Hz, 1H), 8.07 (s, 1H), 7.74 (dd, J ) 4.70 Hz,
J ) 8.10 Hz, 1H). MS (ESI): m/z ) 205 (M + 1). HR-MS (ESI-
TDF) for C9H6ClN4 (MH+): calcd 205.0281, found 205.0278.
MS (ESI): m/z ) 310+ (M + 1). HR-MS (ESI-TDF) for C16H13
-
ClN5 (MH+): calcd 310.0859, found 310.0848.
N-(2-Ch lor o-6-m eth yp h en yl)im id a zo[1,5a ]p yr id o[3,2-
e]p yr a zin -6-a m in e (10). To a solution of 2-chloro-6-methyl-
aniline (12.5 mg, 0.088 mmol) in THF (0.4 mL) was added a
solution of sodium bis(trimethylsilyl)amide (0.22 mL, 0.22
mmol, 1.0 M in THF), and the reaction mixture was heated to
reflux for 0.5 h. The reaction mixture was cooled to room
temperature,and compound 9 (18 mg, 0.088 mmol) in THF (0.8
mL) was added. The mixture was again heated to reflux for
0.5 h and then cooled to room temperature and quenched with
acetic acid. The reaction mixture was concentrated in vacuo
followed by addition of water and saturated sodium bicarbon-
ate. The solid was collected by filtration, washed with water
and hexane, and dried under high vacuum to give 23.4 mg of
N-(2,6-Dim eth ylph en yl)im idazo[1,5-a ]pyr ido[2,3-e]pyr a-
zin e-4-a m in e (17). Compound 17 was prepared from imidazo-
[1,5-a]pyrido[2,3-e]pyrazine-6(5H)-one64 using a method analo-
1
gous to the preparation of 10. H NMR (400 MHz, DMSO-d6):
δ 9.10 (bs, 1H), 8.60 (d, J ) 8.12 Hz, 1H), 8.42 (d, J ) 4.30
Hz, 1H), 7.89 (bs, 1H), 7.35-7.40 (m, 1H), 7.33 (dd, J ) 8.12
Hz, J ) 4.30 Hz, 1H), 7.19-7.29 (m, 2H), 2.32 (s, 6H). MS
(ESI): m/z ) 290+ (M + 1). HR-MS (ESI-TDF) for C17H16N5
(MH+): calcd 290.1406, found 290.1410.
(2-Ch lor o-6-m eth ylp h en yl)(8-m eth oxy-im id a zo[1,5-a ]-
qu in oxa lin -4-yl)a m in e (21). Compound 21 was prepared
according to a procedure described in the literature.28,29 Mp:
1
1
10 as a pale yellow solid (Yield: 86%). Mp: 189-192 °C. H
202-204 °C. H NMR (400 MHz, DMSO-d6): δ 9.30 (bs, 1H),
NMR (400 MHz, DMSO-d6): δ 9.89 (bs, 1H), 9.30 (s, 1H), 8.72
(dd, J ) 4.70 Hz, J ) 1.48 Hz, 1H), 8.40 (dd, J ) 1.48 Hz, J )
8.10 Hz, 1H), 8.07 (s, 1H), 7.74 (dd, J ) 4.70 Hz, J ) 8.10 Hz,
1H), 7.25-7.43 (m, 3H), 2.25 (s, 3H). MS (ESI): m/z ) 310 (M
+ 1). HR-MS (ESI-TDF) for C16H12ClN5 (MH+): calcd 310.0789,
found 310.0792.
9.10 (s, 1H), 8.11 (d, J ) 8.86 Hz, 1H), 7.44 (d, J ) 7.49 Hz,
1H), 7.25-7.38 (m, 2H), 6.87 (d, J ) 8.86 Hz, 1H), 6.83 (s,
1H), 3.77 (s, 3H), 2.24 (s, 3H).
N-(2-Ch lor o-6-m eth ylp h en yl)-2-m eth oxyim id a zo[1,5-a ]-
p yr id o[3,2-e]p yr a zin -6-a m in e (22a ). Compound 22a was
prepared from 12 by a route analogous to that used for the
preparation of 20. Mp: 261-262 °C. 1H NMR (400 MHz,
DMSO-d6): δ 9.32 (s, 1H), 8.98 (s, 1H), 8.03 (bs, 1H), 7.76 (d,
J ) 8.70 Hz, 1H), 7.43 (m, 1H), 7.27-7.35 (m, 2H), 6.87 (d, J
) 8.70 Hz, 1H), 4.00 (s, 3H), 2.25 (s, 3H). MS (ESI): m/z )
340+ (M + 1). HR-MS (ESI-TDF) for C17H15ClN5O (MH+):
calcd 340.0965, found 340.0965.
2,6-Dich lor oim id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin e (11).
Compound 11 was prepared from 2,6-dichloro-3-amino-pyri-
dine using a method analogous to the preparation of 9. 1H
NMR (400 MHz, DMSO-d6): δ 9.25 (s, 1H), 8.39 (d, J ) 8.56
Hz, 1H), 8.09 (s, 1H), 7.81 (d, J ) 8.56 Hz, 1H).
2-Ch lor o-N-(2-ch lor o-6-m eth yl-p h en yl)im id a zo[1,5-a ]-
p yr id o[3,2-e]p yr a zin -6-a m in e (12). Compound 12 was pre-
pared from 11 (142 mg, 0.59 mmol), 2-chloro-6-methylaniline
(0.073 mL, 0.59 mmol), and sodium bis(trimethylsilyl)amide
(1.43 mL, 1.43 mmol, 1.0 M solution) in THF (10 mL) using a
method analogous to the preparation of 10 (Yield: 98%). Mp:
N6-(2-Ch lor o-6-m eth ylp h en yl)-N2,N2-d im eth ylim id a zo-
[1,5-a ]p yr id o[3,2-e]p yr a zin e-2,6-d ia m in e (22b). Compound
22b (>30 mg) was prepared in >80% yield from 12 (30 mg)
1
by a route analogous to that used for the preparation of 4. H
NMR (400 MHz, DMSO-d6): δ 9.07 (bs, 1H), 8.95 (s, 1H), 8.01
(bs, 1H), 7.64 (d, J ) 9.00 Hz, 1H), 7.48 (m, 1H), 7.30-7.47
(m, 2H), 6.80 (d, J ) 9.00 Hz, 1H), 3.18 (s, 6H), 2.30 (s, 3H).
1
185-187 °C. H NMR (400 MHz, DMSO-d6): δ 9.90 (bs, 1H),
8.96 (s, 1H), 8.18 (m 1H), 7.76 (d, J ) 8.32 Hz, 1H), 7.33-7.45
MS (ESI): m/z ) 353+ (M + 1). HR-MS (ESI-TDF) for C18H17
-
(m, 2H), 7.24-7.31 (m, 2H), 2.23 (s, 3H).
ClN6 (MH+): calcd 353.1282, found 353.1289.
(S)-N-(2-Ch lor o-6-m eth ylp h en yl)-2-[3-m eth yl-1-p ip er -
a zin yl)im id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (4) ()
BMS-279700, F r ee Ba se). The mixture of compound 12 (300
mg, 0.87 mmol) and (S)-3-methyl-1-piperazine (5 mL) was
heated to 120 °C for 5 h. After cooling to room temperature,
water and saturated sodium bicarbonate were added. The solid
was collected by filtration, washed with water and hexane, and
dried under high vacuum to give 4 as an off-white solid
N6-(2-Ch lor o-6-m et h ylp h en yl)-N2,N2-d iet h ylim id a zo-
[1,5-a ]p yr id o[3,2-e]p yr a zin e-2,6-d ia m in e (22c). Compound
22c (>30 mg) was prepared in >80% yield from 12 (30 mg) by
a route analogous to that used for the preparation of 4. 1H
NMR (400 MHz, DMSO-d6): δ 8.98 (bs, 1H), 8.83 (s, 1H), 8.01
(bs, 1H), 7.64 (d, J ) 9.05 Hz, 1H), 7.48 (m, 1H), 7.30-7.47
(m, 2H), 6.69 (d, J ) 9.05 Hz, 1H), 3.58 (m, 4H), 2.25 (s, 3H),
1.15 (t, J ) 6.96 Hz, 6H). MS (ESI): m/z ) 381+ (M + 1). HR-
MS (ESI-TDF) for C20H22ClN6 (MH+): calcd 381.1595, found
381.1561.
1
(Yield: 90%). Mp: 165-168 °C. H NMR (400 MHz, DMSO-
d6): δ 9.00 (bs, 1H), 8.87 (s, 1H), 7.89 (bs, 1H), 7.52 (d, J )
8.98 Hz, 1H), 7.36 (dd, J ) 1.22 Hz, 1H), 7.18-7.26 (m, 2H),
6.83 (d, J ) 8.98 Hz, 1H), 4.17 (m, 2H), 2.90 (m, 1H), 2.58-
2.75 (m, 3H), 2.34 (m, 1H), 2.22 (m, 1H), 2.17 (s, 1H), 0.99 (d,
J ) 6.26 Hz, 3H). MS (ESI): m/z ) 408+ (M + 1). HR-MS (ESI-
TDF) for C21H22ClN7 (MH+): calcd 408.1703, found 408.1710.
N6-(2-Ch lor o-6-m et h ylp h en yl)-N2-[2-(d im et h yla m in o-
et h yl)]-N2-m et h ylim id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin e-
2,6-d ia m in e (22d ). Compound 22d (>30 mg) was prepared
in >80% yield from 12 (30 mg) by a route analogous to that
1
N-(2-Ch lor o-6-m eth ylp h en yl)im id a zo[1,5-a ]p yr id o[4,3-
e]p yr a zin e-4-a m in e (14). Compound 14 was prepared from
imidazo[1,5-a]pyrido[4,3-e]pyrazine-6(5H)-one64 using a method
analogous to the preparation of 10. Mp: 200-202 °C. 1H NMR
(400 MHz, DMSO-d6): δ 9.90 (s, 1H), 9.46 (s, 1H), 9.31 (s, 1H),
8.37 (d, J ) 5.26 Hz, 1H), 8.23 (s, 1H), 7.23-7.55 (m, 4H),
2.25 (s, 3H). MS (ESI): m/z ) 310+ (M + 1). HR-MS (ESI-
TDF) for C16H13ClN5 (MH+): calcd 310.0859, found 310.0865.
N-(2-Ch lor o-6-m eth ylp h en yl)im id a zo[1,5-a ]p yr id o[3,4-
e]p yr a zin e-4-a m in e (15). Compound 15 was prepared from
imidazo[1,5-a]pyrido[3,4-e]pyrazine-6(5H)-one64 using a method
analogous to the preparation of 10. 1H NMR (400 MHz,
used for the preparation of 4. H NMR (400 MHz, CDCl3): δ
8.80 (s, 1H), 7.70 (d, J ) 8.87 Hz, 1H), 7.34 (m, 1H), 7.18-
7.24 (m, 3H), 6.62 (d, J ) 8.87 Hz, 1H), 6.45 (bs, 1H), 3.78 (t,
J ) 7.28 Hz, 2H), 3.15 (s, 3H), 2.58 (t, J ) 7.28 Hz, 1H), 2.34
(s, 6H), 2.32 (s, 3H).
N-(2-Ch lor o-6-m eth ylph en yl)-2-(4-m or ph olin o)im idazo-
[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (22f). Compound 22f
(>30 mg) was prepared in >80% yield from 12 (30 mg) by a
route analogous to that used for the preparation of 4. Mp:
1
222-224 °C. H NMR (400 MHz, DMSO-d6): δ 9.12 (bs, 1H),
8.95 (s, 1H), 7.97 (bs, 1H), 7.63 (d, J ) 8.95 Hz, 1H), 7.43 (d,
J ) 7.87 Hz, 1H), 7.25-7.33 (m, 2H), 6.93 (d, J ) 8.95 Hz,

4526 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Chen et al.
1H), 3.74 (m, 4H), 3.56 (m, 4H), 2.24 (s, 3H). MS (ESI): m/z )
395+ (M + 1). HR-MS (ESI-TDF) for C20H20ClN6O (MH+):
calcd 395.1387, found 395.1392.
7.72 Hz, 1H), 6.55 (d, J ) 8.82 Hz, 1H), 5.00 (bs, 1H), 4.42
(bs, 1H),3.50-3.63 (m, 3H), 3.42 (m, 1H), 2.24 (s, 3H), 2.05
(m, 1H), 1.94 (m, 1H).
N-(2-Ch lor o-6-m eth ylph en yl)-2-(1-piper idin yl)im idazo-
[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (22g). Compound 22g
(>30 mg) was prepared in >80% yield from 12 (30 mg) by a
route analogous to that used for the preparation of 4. Mp:
(S)-1-[6-[(2-Ch lor o-6-m eth ylp h en yl)a m in o]im id a zo[1,5-
a ]p yr id o[3,2-e]p yr a zin e-2-]-3-p yr r olid in ol (26). Compound
25 (>30 mg) was prepared in >80% yield from 12 (30 mg)
using a method analogous to the preparation of 4. 1H NMR
(400 MHz, DMSO-d6): δ 8.99 (s, 1H), 8.84 (bs, 1H), 7.95 (bs,
1H), 7.58 (d, J ) 8.82 Hz, 1H), 7.42 (d, J ) 7.72 Hz, 1H), 7.31
(d, J ) 6.90 Hz, 1H), 7.29 (d, J ) 7.72 Hz, 1H), 7.25 (d, J )
7.72 Hz, 1H), 6.55 (d, J ) 8.82 Hz, 1H), 5.00 (bs, 1H), 4.42
(bs, 1H),3.50-3.63 (m, 3H), 3.42 (m, 1H), 2.24 (s, 3H), 2.05
(m, 1H), 1.94 (m, 1H).
1
257-259 °C. H NMR (400 MHz, DMSO-d6): δ 8.93 (bs, 1H),
8.79 (s, 1H), 7.84 (bs, 1H), 7.46 (d, J ) 8.98 Hz, 1H), 7.31 (d,
J ) 7.70 Hz, 1H), 7.13-7.22 (m, 2H), 6.79 (d, J ) 8.98 Hz,
1H), 3.46-3.57 (m, 4H), 1.40-1.60 (m, 6H). MS (ESI): m/z )
393+ (M + 1). HR-MS (ESI-TDF) for C21H22ClN7 (MH+): calcd
393.1595, found 393.1592.
N-(2-Ch lor o-6-m eth ylph en yl)-2-(1-piper azin yl)im idazo-
[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (22h ). Compound 22h
(>30 mg) was prepared in >80% yield from 12 (30 mg) by a
route analogous to that used for the preparation of 4. Mp:
(S)-2-(3-Amino-1-pyrrolidinyl)-N-(2-chloro-6-methylphen-
yl)im id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (29). Com-
pound 29 (>30 mg) was prepared in >80% yield from 12 (30
mg) using a method analogous to the preparation of 4. 1H NMR
(400 MHz, DMSO-d6): δ 9.10 (bs, 1H), 8.90 (s, 1H), 8.03 (bs,
1H), 7.67 (d, J ) 8.78 Hz, 1H), 7.48 (d, J ) 7.88 Hz, 1H), 7.38
(d, J ) 6.92 Hz, 1H), 7.36 (d, J ) 7.64 Hz, 1H), 7.32 (d, J )
7.64 Hz, 1H), 3.42-3.92 (m, 5H), 2.30 (s, 3H), 2.29 (m, 1H),
2.04 (m, 1H).
(R)-2-(3-Amino-1-pyrrolidinyl)-N-(2-chloro-6-methylphen-
yl)im id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (30). Com-
pound 30 (>30 mg) was prepared in >80% yield from 12 (30
mg) using a method analogous to the preparation of 4. 1H NMR
(400 MHz, DMSO-d6): δ 9.10 (bs, 1H), 8.90 (s, 1H), 8.03 (bs,
1H), 7.67 (d, J ) 8.78 Hz, 1H), 7.48 (d, J ) 7.88 Hz, 1H), 7.38
(d, J ) 6.92 Hz, 1H), 7.36 (d, J ) 7.64 Hz, 1H), 7.32 (d, J )
7.64 Hz, 1H), 3.42-3.92 (m, 5H), 2.30 (s, 3H), 2.29 (m, 1H),
2.04 (m, 1H).
1
191-193 °C. H NMR (400 MHz, DMSO-d6): δ 8.91 (bs, 1H),
8.74 (s, 1H), 7.78 (bs, 1H), 7.42 (d, J ) 8.82 Hz, 1H), 7.24 (dd,
J ) 6.65 Hz, J ) 1.09 Hz, 1H), 7.15 (d, J ) 6.65 Hz, 1H), 7.10
(dd, AB pattern, J ) 7.66 Hz, ∆ν ) 14.15, 1H), 6.72 (d, J )
8.92 Hz, 1H), 3.34-3.37 (m, 4H), 2.64-2.67 (m, 4H), 2.07 (s,
3H). MS (ESI): m/z ) 394+ (M + 1). HR-MS (ESI-TDF) for
C
20H21ClN7 (MH+): calcd 394.1547, found 394.1552.
N-(2-Ch lor o-6-m et h ylp h en yl)-2-(4-m et h yl-1-p ip er a zi-
n yl)im idazo[1,5-a ]pyr ido[3,2-e]pyr azin -6-am in e (22i). Com-
pound 22i (>30 mg) was prepared in >80% yield from 12 (30
mg) by a route analogous to that used for the preparation of
4. Mp: 232-233 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.15
(s, 1H), 8.99 (s, 1H), 8.02 (bs, 1H), 7.66 (d, J ) 8.97 Hz, 1H),
7.48 (d, J ) 7.57 Hz, 1H), 7.31-7.39 (m, 2H), 6.98 (d, J ) 8.97
Hz, 1H), 3.65 (m, 4H), 2.56 (s, 3H), 2.49 (m, 4H), 2.29 (s, 3H).
(R)-N-(2-Ch lor o-6-m eth ylp h en yl)-2-[3-m eth yl-1-p ip er -
a zin yl)im id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (31).
Compound 31 (>30 mg) was prepared in >80% yield from 12
(30 mg) using a method analogous to the preparation of 4.
MS (ESI): m/z ) 408+ (M + 1). HR-MS (ESI-TDF) for C21H23
-
ClN7 (MH+): calcd 408.1703, found 408.1693.
N-(2-Ch lor o-6-m eth ylp h en yl)-2-(h exa h yd r o-1H-1,4-d i-
a z e p i n -1-y l)i m i d a z o [1,5-a ]p y r i d o [3,2-e]p y r a z i n -6-
a m in e (22j). Compound 22j (>30 mg) was prepared in >80%
yield from 12 (30 mg) by a route analogous to that used for
the preparation of 4. Mp: 148-149 °C. 1H NMR (400 MHz,
DMSO-d6): δ 8.92 (bs, 1H), 8.80 (s, 1H), 7.87 (s, 1H), 7.49 (d,
J ) 8.97 Hz, 1H), 7.35 (dd, J ) 7.72 Hz, J ) 1.03 Hz, 1H),
7.25 (d, J ) 7.72 Hz, 1H), 7.21 (dd, AB pattern, J ) 7.72 Hz,
∆ν ) 14.51, 1H), 6.67 (d, J ) 8.97 Hz, 1H), 3.70 (m, 2H), 3.66
(m, 2H), 2.82 (m, 2H), 2.58 (m, 2H), 2.18 (s, 3H), 1.78 (m, 2H).
1
Mp: 162-164 °C. H NMR (400 MHz, DMSO-d6): δ 9.00 (bs,
1H), 8.87 (s, 1H), 7.89 (bs, 1H), 7.52 (d, J ) 8.98 Hz, 1H), 7.36
(dd, J ) 1.22 Hz, 1H), 7.18-7.26 (m, 2H), 6.83 (d, J ) 8.98
Hz, 1H), 4.17 (m, 2H), 2.90 (m, 1H), 2.58-2.75 (m, 3H), 2.34
(m, 1H), 2.22 (m, 1H), 2.17 (s, 1H), 0.99 (d, J ) 6.26 Hz, 3H).
MS (ESI): m/z ) 408+ (M + 1). HR-MS (ESI-TDF) for C21H22
-
ClN7 (MH+): calcd 408.1703, found 408.1706.
(R)-4-[6-[(2-Ch lor o-6-m eth ylph en yl)am in o]im idazo[1,5-
a ]p yr id o[3,2-e]p yr a zin -2-yl]-2-p ip er a zin e Meth a n ol (32).
Compound 32 (>30 mg) was prepared in >80% yield from 12
(30 mg) using a method analogous to the preparation of 4.
MS (ESI): m/z ) 408+ (M + 1). HR-MS (ESI-TDF) for C21H23
-
ClN7 (MH+): calcd 408.1704, found 408.1717.
1
N-(2-Ch lor o-6-m eth ylp h en yl)-2-(3,5-d im eth yl-1-p ip er -
a zin yl)im id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin -6-a m in e (22k ).
Compound 22k (>30 mg) was prepared in >80% yield from
12 (30 mg) by a route analogous to that used for the prepara-
Mp: 177-179 °C. H NMR (400 MHz, DMSO-d6): δ 8.99 (bs,
1H), 8.83 (s, 1H), 7.87 (bs, 1H), 7.51 (d, J ) 9.01 Hz, 1H), 7.33
(d, J ) 7.71 Hz, 1H), 7.16-7.24 (m, 2H), 6.80 (d, J ) 9.01 Hz,
1H), 4.62 (m, 1H), 4.17 (m, 2H), 3.25-3.42 (m, 2H), 2.92 (m,
1H), 2.76 (m, 1H), 2.63 (m, 2H), 2.15 (s, 3H). MS (ESI): m/z )
424+ (M + 1). HR-MS (ESI-TDF) for C21H23ClN7O (MH+):
calcd 424.1654, found 424.1670.
1
tion of 4. Mp:249-251 °C. H NMR (400 MHz, DMSO-d6): δ
8.83 (s, 1H), 7.72 (d, J ) 8.92 Hz, 1H), 7.35 (dd, J ) 1.86 Hz,
J ) 7.46 Hz, 1H), 7.18-7.24 (m, 3H), 6.77 (d, J ) 8.92 Hz,
1H), 6.58 (bs, 1H), 4.25 (dd, J ) 2.23 Hz, J ) 12.54 Hz, 2H),
2.99 (m, 2H), 2.51 (t, J ) 12.54 Hz, 2H), 2.32 (s, 3H), 1.21 (s,
3H), 1.19 (s, 3H). MS (ESI): m/z ) 422+ (M + 1). HR-MS (ESI-
TDF) for C22H25ClN7 (MH+): calcd 422.1860, found 422.1863.
N-(2-Ch lor o-6-m eth ylp h en yl)-8-(3,5-d im eth yl-1-p ip er -
a zin yl)im id a zo[1,5-a ]p yr id o[3,4-e]p yr a zin -6-a m in e (23).
Compound 23 was prepared from 4,6-dichloro-3-pyridine car-
boxylic acid65 or 2-chloro-4-amino-5-nitropyridine66 by a route
(S)-4-[6-[(2-Ch lor o-6-m eth ylp h en yl)a m in o]im id a zo[1,5-
a ]p yr id o[3,2-e]p yr a zin -2-yl]-2-p ip er a zin e Meth a n ol (33).
Compound 33 (>30 mg) was prepared in >80% yield from 12
(30 mg) using a method analogous to the preparation of 4.
1
Mp: 182-184 °C. H NMR (400 MHz, DMSO-d6): δ 8.99 (bs,
1H), 8.83 (s, 1H), 7.87 (bs, 1H), 7.51 (d, J ) 9.01 Hz, 1H), 7.33
(d, J ) 7.71 Hz, 1H), 7.16-7.24 (m, 2H), 6.80 (d, J ) 9.01 Hz,
1H), 4.62 (m, 1H), 4.17 (m, 2H), 3.25-3.42 (m, 2H), 2.92 (m,
1H), 2.76 (m, 1H), 2.63 (m, 2H), 2.15 (s, 3H). MS (ESI): m/z )
424+ (M + 1). HR-MS (ESI-TDF) for C21H23ClN7O (MH+):
calcd 424.1654, found 424.1659.
1
analogous to that used for the preparation of 4. H NMR (400
MHz, DMSO-d6): δ 9.31 (s, 1H), 9.15 (bs, 1H), 8.26 (bs, 1H),
8.05 (s, 1H), 7.54 (s, 1H), 7.49 (d, J ) 7.74 Hz, 1H), 7.39 (d, J
) 6.60 Hz, 1H), 7.35 (d, J ) 7.64 Hz, 1H), 7.32 (d, J ) 7.64
Hz, 1H), 4.27 (d, J ) 12.04 Hz, 2H), 2.86 (m, 2H), 2.35 (m,
2H), 2.30 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H).
(R)-1-[6-[(2-Ch lor o-6-m eth ylph en yl)am in o]im idazo[1,5-
a ]p yr id o[3,2-e]p yr a zin e-2-]-3-p yr r olid in ol (25). Compound
25 (>30 mg) was prepared in >80% yield from 12 (30 mg)
using a method analogous to the preparation of 4. 1H NMR
(400 MHz, DMSO-d6): δ 8.99 (s, 1H), 8.84 (bs, 1H), 7.95 (bs,
1H), 7.58 (d, J ) 8.82 Hz, 1H), 7.42 (d, J ) 7.72 Hz, 1H), 7.31
(d, J ) 6.90 Hz, 1H), 7.29 (d, J ) 7.72 Hz, 1H), 7.25 (d, J )
4-[6-[2-Ch lor o-6-m eth ylp h en yl)a m in o]im id a zo[1,5-a ]-
pyr ido[3,2-e]pyr azin -2-yl]-1-piper azin e Eth an ol (34). Com-
pound 34 (>30 mg) was prepared in >80% yield from 12 (30
mg) using a method analogous to the preparation of 4. Mp:
235.5-236.5 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.01 (bs,
1H), 8.86 (s, 1H), 7.89 (bs, 1H), 7.53 (d, J ) 8.97 Hz, 1H), 7.35
(dd, J ) 7.84 Hz, J ) 1.22 Hz, 1H), 7.25 (d, J ) 7.76 Hz, 1H),
7.21 (dd, J ) 7.76 Hz, ∆ν ) 13.99, 1H), 6.84 (d, J ) 8.97 Hz,
1H), 4.38 (t, J ) 5.34 Hz, 1H), 3.46-3.53 (m, 6H), 2.47 (m,
4H), 2.37 (m, 2H), 2.16 (s, 3H). MS (ESI): m/z ) 438+ (M +

Imidazoquinoxaline Src-Family Kinase p56Lck Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4527
1). HR-MS (ESI-TDF) for C22H25N7ClO (MH+): calcd 438.1809,
found 438.1812.
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.
N-(2-Ch lor o-6-m eth ylp h en yl)-2-[4-[2-(d im eth yla m in o)-
eth yl]-1-p ip er a zin yl]im id a zo[1,5-a ]p yr id o[3,2-e]p yr a zin -
6-a m in e (35). Compound 35 (>30 mg) was prepared in >80%
yield from 12 (30 mg) using a method analogous to the
preparation of 4. Mp: 177-179 °C. 1H NMR (400 MHz, DMSO-
d6): δ 9.01 (bs, 1H), 8.86 (s, 1H), 7.89 (bs, 1H), 7.53 (d, J )
8.97 Hz, 1H), 7.36 (dd, J ) 7.84 Hz, J ) 1.22 Hz, 1H), 7.25 (d,
J ) 7.76 Hz, 1H), 7.20 (dd, J ) 7.76 Hz, ∆ν ) 13.99, 1H), 6.84
(d, J ) 8.97 Hz, 1H), 3.51 (m, 4H), 2.40-2.50 (m, 4H), 2.32-
2.37 (m, 4H), 2.16 (s, 3H), 2.09 (s, 6H). MS (ESI): m/z ) 465+
(M + 1). HR-MS (ESI-TDF) for C24H30ClN8 (MH+): calcd
465.2282, found 465.2286.
In h ibition of Ser u m In ter leu k in -2 (IL-2) in Mou se.
BALB/c female mice, 8-10 weeks of age (Harlan Teklad,
Indianapolis, IN), were dosed by oral gavage (po) with either
vehicle alone (propylene glycol-water, 50:50, v/v) or compound
4 dissolved in vehicle, or cyclosporin A (CsA, Neoral; Novartis,
East Hanover, NJ ). One hour later, mice were injected
intravenously (iv) with 500 µg/kg purified anti-CD3 antibody
(clone 145-2C11; American Type Culture Collection, Rockville,
MD). The mice were bled 1.5 h after anti-CD3 injection. Levels
of IL-2 in the serum were determined by ELISA assay
according to manufacturer’s instructions (R&D Systems, Min-
neapolis, MN).
In h ibition of Ser u m Tu m or Necr osis F a ctor r (TNF r)
in Mou se. C57BL/6 female mice, 8-10 weeks of age (Harlan
Teklad, Indianapolis, IN), were dosed by oral gavage (po) with
either vehicle alone (propylene glycol-water, 50:50, v/v) or
compound 4 dissolved in vehicle. One hour later, mice were
injected intravenously (iv) with 500 µg/kg lipopolysaccharide
(LPS, Escherichia coli 0111:B4; Sigma, St. Louis, MO). Mice
were bled 1 h after LPS injection. Levels of TNFR in the serum
were determined by ELISA assay according to manufacturer’s
instructions (R&D Systems, Minneapolis, MN).
(R)-3-[[6-[(2-Ch lor o-6-m et h ylp h en yl)a m in o]im id a zo-
[1,5-a ]p yr id o[3,2-e]p yr a zin -2-yl]m et h yla m in o]-1,2-p r o-
p a n ed iol (37). Compound 37 (>30 mg) was prepared in >80%
yield from 12 (30 mg) using a method analogous to the
preparation of 4. Mp: 187-189 °C. 1H NMR (400 MHz, DMSO-
d6): δ 8.84 (bs, 1H), 8.72 (s, 1H), 7.79 (bs, 1H), 7.41 (d, J )
8.92 Hz, 1H), 7.26 (d, J ) 7.69 Hz, 1H), 7.16 (d, J ) 6.75 Hz,
1H), 7.11 (dd, J ) 7.69 Hz, 1H, ∆ν ) 14.32, 1H), 6.57 (d, J )
8.92 Hz, 1H), 4.63 (d, J ) 6.07 Hz, 1H), 4.51 (t, J ) 5.61 Hz,
1H), 3.60-3.70 (m, 2H), 3.20 (m, 3H), 3.00 (s, 3H), 2.08 (s, 3H).
MS (ESI): m/z ) 413+ (M + 1). HR-MS (ESI-TDF) for C20H22
-
ClN6O2 (MH+): calcd 413.1493, found 413.1497.
Mod elin g Meth od s. Lck Mod el. An initial homology
model was developed using the published structure of the
Hck-ANP-PNP complex67 and the Modeler module within
Insight.68 Minor adjustments to the ATP binding site residues
were made consistent with observed SAR in the imidazoqui-
noxaline series, as well as incorporating more recently pub-
lished Lck structural details.30
Mu ltip le Lin ea r Regr ession . Regression analyses were
conducted using the QSAR module within the Cerius2 pro-
gram.69 Structures were built within Sybyl70 and imported into
Cerius2 as SD files.
Hyd r ogen Bon d En th a lp y. The potential strength of the
hydrogen bond to the imidazolyl nitrogen was determined by
modeling a water molecule as a donor (see illustration in Table
6). For each imidazoquinoxaline analogue, three systems were
geometry optimized/energy minimized using SAM1:61 the
ligand, the water-complexed ligand, and water. The difference
in calculated heats of formation provided an estimate for the
hydrogen bond strength in units of kcal/mol.
(S)-3-[[6-[(2-Ch lor o-6-m et h ylp h en yl)a m in o]im id a zo-
[1,5-a ]p yr id o[3,2-e]p yr a zin -2-yl]m et h yla m in o]-1,2-p r o-
p a n ed iol (38). Compound 38 (>30 mg) was prepared in >80%
yield from 12 (30 mg) using a method analogous to the
preparation of 4. Mp: 190-192 °C. 1H NMR (400 MHz, DMSO-
d6): δ 8.84 (bs, 1H), 8.72 (s, 1H), 7.79 (bs, 1H), 7.41 (d, J )
8.92 Hz, 1H), 7.26 (d, J ) 7.69 Hz, 1H), 7.16 (d, J ) 6.75 Hz,
1H), 7.11 (dd, J ) 7.69 Hz, ∆ν ) 14.32, 1H), 6.57 (d, J ) 8.92
Hz, 1H), 4.63 (d, J ) 6.07 Hz, 1H), 4.51 (t, J ) 5.61 Hz, 1H),
3.60-3.70 (m, 2H), 3.20 (m, 3H), 3.00 (s, 3H), 2.08 (s, 3H). MS
(ESI): m/z ) 413+ (M + 1). HR-MS (ESI-TDF) for C20H22
-
ClN6O2 (MH+): calcd 413.1493, found 413.1496.
2-[[6-(2-Ch lor o-6-m eth ylp h en yl)a m in o]im id a zo[1,5-a ]-
pyr ido[3,2-e]pyr azin -2-yl]m eth ylam in o]eth an ol (39). Com-
pound 39 (>30 mg) was prepared in >80% yield from 12 (30
mg) using a method analogous to the preparation of 4. Mp:
1
195-197 °C. H NMR (400 MHz, CDCl3): δ 8.78 (s, 1H), 7.73
(d, J ) 8.96 Hz, 1H), 7.35 (m, 1H), 7.18-7.24 (m, 3H), 7.67 (d,
J ) 8.96 Hz, 1H), 3.96 (m, 2H), 3.85 (m, 2H), 3.19 (s, 3H),
3.00 (bs, 1H), 2.32 (s, 3H).
Ack n ow led gm en t. We would like to thank the
Discovery Analytical Science Group for analytical sup-
port including the high-resolution mass spectra (HR-
MS). The authors are grateful for the initial Lck
homology model provided by Dr. Donna Bassolino, and
the grid-contouring program71 and helpful suggestions
provided by Dr. Andrew Pudzianowski.
2-[[6-(2-Ch lor o-6-m eth ylp h en yl)a m in o]im id a zo[1,5-a ]-
p yr id o[3,2-e]p yr a zin -2-yl]a m in o]eth a n ol (40). Compound
40 (>30 mg) was prepared in >80% yield from 12 (30 mg)
using a method analogous to the preparation of 4. Mp: 259-
1
261 °C. H NMR (400 MHz, DMSO-d6): δ 8.88 (bs, 1H), 8.75
(s, 1H), 7.85 (bs, 1H). 7.39 (d, J ) 8.76 Hz, 1H), 7.36 (d, J )
7.68 Hz, 1H), 7.26 (d, J ) 6.75 Hz, 1H), 7.18 (dd, J ) 7.68 Hz,
∆ν ) 14.48, 1H), 6.87 (m, 1H), 6.54 (d, J ) 8.76 Hz, 1H), 4.70
(bs, 1H), 3.54 (m, 2H), 3.40 (m, 2H), 2.17 (s, 3H). MS (ESI):
m/z ) 369+ (M + 1). HR-MS (ESI-TDF) for C18H18N6ClO
(MH+): calcd 369.1230, found 369.1236.
Refer en ces
(1) Isakov, N.; Wange, R. L.; Samolson, L. E. The role of tyrosine
kinases and phosphotyrosine-containing recognition motifs in
regulation of the T cell-antigen receptor-mediated signal trans-
duction pathway. J . Leukocyte Biol. 1994, 55, 265-271.
(2) Bolen, J . B.; Brugge, J . S. Leukocyte protein tyrosine kinases:
potential targets for drug discovery. Annu. Rev. Immunol. 1997,
15, 371-404.
(3) J ohnson, L. N.; Noble, M. E.; Owen, D. J . Active and inactive
protein kinases: structural basis for regulation. Cell 1996, 85
(2), 149-158.
(4) Neumeister, E. N.; Zhu, Y.; Richard, S.; Terhorst, C.; Chan, A.
C.; Shaw, A. S. Binding of ZAP-70 to phosphorylated T-cell
receptor ú and η enhances its autophosphorylation and generates
specific binding sites for SH2 domain-containing proteins. Mol.
Cell. Biol. 1995, 15 (6), 3171-3178.
(5) Chan, A. C.; Dalton, M.; J ohnson, R.; Kong, G. H.; Wang, T.;
Thoma, R.; Kurosaki, T. Activation of ZAP-70 kinase activity
by phosphorylation of tyrosine 493 is required for lymphocyte
antigen receptor function. EMBO J . 1995, 14 (11), 2499-2508.
(6) Trobridge, P. A.; Levin, S. D. Lck plays a critical role in Ca2+
mobilization and CD28 co-stimulation in mature primary T cells.
Eur. J . Immunol. 2001, 31, 3567-3579.
Lck En zym e Assa y. Recombinant Lck expressed as a His-
tagged protein in insect cells using a baculovirus expression
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 concentration
of 1 µM ATP, 3.3 µCi/mL [33P] g-ATP, and 0.1 mg/mL acid
denatured enolase, and was stopped after 10 min by the
addition of 10% trichloroacetic acid, 100 mM sodium pyro-
phosphate followed by 2 mg/mL bovine serum albumin. The
labeled enolase protein substrate was precipitated at 4 °C,
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T Cell (Hu m a n P BL) P r olifer a tion Assa y. A 96-well
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the antibody was allowed to bind, and the plate was washed.
Normal human peripheral blood T cells were added to the wells

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