Structure-based design of novel 2-amino-6-phenyl-pyrimido[5′, 4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-ones as potent and orally active inhibitors of lymphocyte specific kinase (Lck): Synthesis, SAR, and in vivo anti-inflammatory activity

Article abstract of DOI:10.1021/jm701095m

Lck, or lymphocyte specific kinase, is a cytoplasmic tyrosine kinase of the Src family expressed in T-cells and NK cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T-cell receptor-mediated signaling, leading to normal T-cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the structure-guided design, synthesis, structure-activity relationships, and pharmacological characterization of 2-amino-6-phenylpyrimido[5′,4′:5,6]pyrimido[1,2- a]benzimidazol-5(6H)-ones, a new class of compounds that are potent inhibitors of Lck. The most promising compound of this series, 6-(2,6-dimethylphenyl)-2- ((4-(4-methyl-1-piperazinyl)phenyl)amino)pyrimido[5′,4′:5,6] pyrimido-[1,2-a]benzimidazol-5(6H)-one (25), exhibits potent inhibition of Lck kinase activity. This activity translates into inhibition of in vitro cell-based assays and in vivo models of T-cell activation and arthritis, respectively.

Full text of DOI:10.1021/jm701095m

J. Med. Chem. 2008, 51, 1637–1648
1637
Structure-Based Design of Novel 2-Amino-6-phenyl-pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-
5(6H)-ones as Potent and Orally Active Inhibitors of Lymphocyte Specific Kinase (Lck): Synthesis,
SAR, and In Vivo Anti-Inflammatory Activity
Matthew W. Martin,*,† John Newcomb,‡ Joseph J. Nunes,† Christina Boucher,‡ Lilly Chai,‡ Linda F. Epstein,§ Theodore Faust,‡
Sylvia Flores,# Paul Gallant,‡ Anu Gore, Yan Gu,§ Faye Hsieh,| Xin Huang,§ Joseph L. Kim,§ Scot Middleton,#
Kurt Morgenstern,§ Antonio Oliveira-dos-Santos,‡ Vinod F. Patel,† David Powers,O Paul Rose,§ Yanyan Tudor,0
Susan M. Turci,‡ Andrew A. Welcher,0 Debra Zack,# Huilin Zhao,§ Li Zhu,# Xiaotian Zhu,§ Chiara Ghiron,4 Monika Ermann,4
David Johnston,4 and Carl-Gustaf Pierre Saluste4
Department of Medicinal Chemistry, Department of HTS and Molecular Pharmacology, and Department of Molecular Structure, Amgen Inc.,
One Kendall Square, Building 1000, Cambridge, Massachusetts 02139, Department of Inflammation, Department of Pharmaceutics,
Department of Pharmacokinetics and Drug Metabolism, Department of HTS and Molecular Pharmacology, and Department of Molecular
Sciences, Amgen Inc., One Amgen Center DriVe, Thousand Oaks, California 91320, and EVotec OAI, 151 Milton Park,
Abingdon, Oxon OX14 4SD, United Kingdom
ReceiVed September 4, 2007
Lck, or lymphocyte specific kinase, is a cytoplasmic tyrosine kinase of the Src family expressed in T-cells
and NK cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase
activity is critical for T-cell receptor-mediated signaling, leading to normal T-cell development and activation.
A small molecule inhibitor of Lck is expected to be useful in the treatment of T-cell-mediated autoimmune
and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the structure-guided
design, synthesis, structure–activity relationships, and pharmacological characterization of 2-amino-6-
phenylpyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-ones, a new class of compounds that are potent
inhibitors of Lck. The most promising compound of this series, 6-(2,6-dimethylphenyl)-2-((4-(4-methyl-1-
piperazinyl)phenyl)amino)pyrimido[5′,4′:5,6]pyrimido-[1,2-a]benzimidazol-5(6H)-one (25), exhibits potent
inhibition of Lck kinase activity. This activity translates into inhibition of in vitro cell-based assays and in
vivo models of T-cell activation and arthritis, respectively.
Introduction
activity display common phenotypes involving defects in T-cell
maturation and signaling.7–12 Taken together, these findings
suggest that a small molecule inhibitor of Lck kinase activity
might be a useful immunosuppressive agent for the treatment
of graft rejection and/or T-cell-mediated autoimmune diseases.
As part of our ongoing efforts to identify small molecule
inhibitors of Lck for the development of new therapeutic agents,
we recently disclosed a number of promising chemical series.13
In addition, several other groups have previously illustrated the
synthesis and characterization of Lck- or Src-family kinase
inhibitors.14–19 Potent and bioavailable Lck/Src inhibitors have
also been demonstrated to have efficacy in several in vivo
models of T-cell-dependent immune responses.13a,b,17f,18d,20
Screening of our in-house kinase preferred collection identi-
fied aminobenzimidazole triazines, such as N-phenyl-1-(4-(3,4,5-
trimethoxyphenylamino)-1,3,5-triazin-2-yl)-1H-benzo[d]imidazol-
2-amine and N-(2,6-dimethylphenyl)-1-(4-(3,4,5-trimethoxy-
phenylamino)-1,3,5-triazin-2-yl)-1H-benzo[d]imidazol-2-
amine (compounds 1 and 2, respectively, Figure 1), as potent
inhibitors of Lck. Unfortunately, these compounds were non-
selective kinase inhibitors, exhibiting, for example, equal, low-
nanomolar potency against KDR. A cocrystal structure of 1
bound to Lck revealed key interactions in the ATP-binding
pocket (Figure 2). Binding to Lck is anchored by three
hydrogen-bond interactions between the aminotriazine moiety
of compound 1 and the kinase linker region: Met319 H-bonds
T-cells play a pivotal role in the adaptive immune response
by acting either as potentiators (CD4+ T-helper cells) or
effectors (CD8+ cytolytic T-effector cells) of immune reactions.
The immune specificity of a particular T-cell is determined by
the T-cell receptor (TCRa) for antigen, or CD3 complex,
expressed on its surface.1 Signaling pathways used by the TCR
have been an intense area of research toward the discovery of
novel immunosuppressive agents. The goal of this research is
the development of improved therapies for graft rejection and
T-cell-mediated autoimmune disease. A number of protein
kinases have been shown to be important for TCR signal
transduction. The Src family of cytoplasmic tyrosine kinases
consists of nine members: Src, Lck, Fyn, Lyn, Hck, Fgr, Blk,
Yes, and Yrk.2,3 Among the Src family, Lck and Fyn have been
shown to have important roles in TCR signal transduction.4–6
TCR signals initiated by Lck lead to gene regulation events
resulting in cytokine release, proliferation, and survival of
antigen specific T-cells. Lck knockout mice and patients with
Lck mutations effecting protein expression and/or catalytic
* To whom correspondence should be addressed: Tel.: 617-444-5013.
Fax: 617-621-3908. E-mail: matmarti@amgen.com.
†
Department of Medicinal Chemistry.
Department of HTS and Molecular Pharmacology, Cambridge, MA.
Department of Molecular Structure.
‡
§
Department of Pharmaceutics.
Department of Pharmacokinetics and Drug Metabolism.
Department of Inflammation.
Department of HTS and Molecular Pharmacology, Thousand Oaks,
|
#
a Abbreviations; Lck, lymphocyte specific kinase; TCR, T-cell receptor;
IL-2, interleukin 2; PTK, protein tyrosine kinase; VEGFR-2, vascular
endothelial growth factor receptor-2; KDR, kinase insert domain receptor;
MAPK, mitogen-activated protein kinase; huMLR, human mixed lympho-
cyte reaction.
O
CA.
0
Department of Molecular Sciences.
Evotec OAI.
4
10.1021/jm701095m CCC: $40.75
2008 American Chemical Society
Published on Web 02/16/2008

1638 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Martin et al.
Figure 1. Structure and activity of aminobenzimidazole triazines 1
and 2.
Figure 3. Model of 2-amino-6-phenylpyrimido[5′,4′:5,6]pyrimido[1,2-
a]benzimidazol-5(6H)-one 3 bound to Lck.
Scheme 1. Retrosynthetic Analysis of 2-Amino-6-phenyl-
pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-ones
Figure 2. X-ray cocrystal structure aminobenzimidazole triazine 1
bound to Lck.
Table 1. Sequence Comparison at Gatekeeper Residue
kinase
Lck
KDR
p38R
gatekeeper residue
Thr316
Val916
Thr106
through the main chain NH and carbonyl to the triazine N3
(2.8 Å) and 2-amino NH (2.8 Å), respectively, while the main
chain carbonyl of Glu317 H-bonds to the CH in the 4-position
of the triazines (3.1 Å).21 This binding mode directs the aniline
substituent of the benzimidazole core into the hydrophobic
pocket adjoining the ATP-binding site in Lck. Interestingly, the
cocrystal structure revealed that the N5-triazine and 2-anili-
nobenzimidazole nitrogens were only 4.3 and 3.8 Å, respec-
tively, away from the γ-OH of the gatekeeper residue, Thr316.
which demonstrated inhibition of T-cell activation in in vitro
and in vivo biological assays as well as in models of rheumatoid
arthritis.
We sought to exploit the proximity and hydrogen-bonding
capacity of the gatekeeper threonine in Lck and take advantage
of sequence differences among structurally related kinases in
and around the ATP-binding pocket (Table 1). In particular,
we designed the 2-amino-6-phenylpyrimido[5′,4′:5,6]pyrimido[1,2-
a]benzimidazol-5(6H)-ones, exemplified by compound 3 (Figure
3), which possess a carbonyl oxygen that is well-positioned to
accept a hydrogen bond from the γ-OH of Thr316 in the ATP-
binding site of Lck (donor–acceptor distance is 2.4 Å, with an
acceptor–donor-hydrogen angle of about 20 degrees).22 We
hypothesized that such a new interaction would increase
selectivity over kinases with nonthreonine gatekeeper residues,
namely, KDR.23 Herein we describe the synthesis, structure-
–activity relationships, and pharmacological characterization of
2-amino-6-phenylpyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimida-
zol-5(6H)-ones, a new class of compounds with potent inhibition
of Lck. Lead optimization led to the discovery of compounds
Chemistry
Our strategy for the construction of the 2-amino-6-phenylpy-
rimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-ones de-
scribed in this paper is outlined in the retrosynthetic analysis
shown in Scheme 1. In this approach, we envisioned accessing
tetracycles 4 via the nucleophilic addition of anilines 5 to
2-substituted tetracyclic pyrimidines 6 (X ) Cl or SO2Me).
Tetracyclic pyrimidines 6, in turn, would be derived in two steps
via nucleophilic attack of anilines 9 on 2-chlorobenzimidazole
(10) followed by addition of the resulting 2-aminobenzimida-
zoles 7 to 2,4-disubstituted pyrimidine 8 (X ) Cl or SMe, Y )
Cl). This concise route would allow for rapid analoging as each
variation could be introduced by simply employing the desired
aniline.
The anilines required for the syntheses of the tetracycles
shown in Tables 1 and 2 were either obtained from commercial
sources or prepared via one of the methods shown in Scheme

Structure-Based Design of Lck Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1639
Scheme 2. Synthesis of Aniline Side Chainsa
a Reagents and conditions: (a) K2CO3, MeCN, reflux; (b) K2CO3, MeCN, 80 ºC, 57% (two steps); (c) H2, 10% Pd/C, EtOH, rt, 16 h, 100%; (d) 100 ºC,
49%.
Scheme 3. Synthesis of 2-Amino-6-phenylpyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-onesa
a Reagents and conditions: (a) reflux, 24 h, 79%; (b) (i-Pr)2NEt, toluene, 110 °C, 3 d, 60 %; (c) m-CPBA, CH2Cl2, rt, 20 h, 97%; (d) ArNH2, i-PrOH,
CF3CO2H, 80 °C, 5-16 h, 93%; (e) ArNH2, i-PrOH, 80 °C, 5 h, 58%.
2. The 4-alkoxy-substituted anilines 14 were synthesized from
the corresponding nitrophenols 11 via an alkylation with either
1-bromo-2-chloroethane or 1-bromo-3-chloropropane. The re-
sulting alkylchloride 12 then underwent nucleophilic displace-
ment with the desired amine (dimethylamine, morpholine, or
N-methylpiperazine) to afford p-alkoxynitrophenyl derivative
13. Catalytic hydrogenation with palladium on carbon afforded
the corresponding aniline 14. For 3-methoxy-4-(4-methylpip-
erazin-1-yl)aniline (17), 1-fluoro-2-methoxy-4-nitrobenzene (15)
was heated with N-methylpiperazine to afford 1-(2-methoxy-
4-nitrophenyl)-4-methylpiperazine (16). Reduction via catalytic
hydrogenation with palladium on carbon afforded the desired
aniline 17.
zimidazole 19 as the hydrochloride salt. Heating of 19 with ethyl
4-chloro-2-methylthiopyrimidine-5-carboxylate (20) in the pres-
ence of diisopropylethylamine afforded tetracycle 21. Activation
of the sulfide via oxidation with m-chloroperoxybenzoic acid
afforded sulfone 22, which could be displaced with a variety
of anilines to afford a series of tetracyclic pyrimidine derivatives
23. In general, heating a solution of the sulfone and aniline in
isopropanol with or without trifluoroacetic acid was used.24
Results and Discussion
Structure–Activity Relationships (SAR). All compounds
were screened for inhibitory activity against Lck in a homoge-
neous time-resolved fluorescent (HTRF) kinase assay. Selected
compounds were tested for cellular activity by measuring the
inhibition of T-cell receptor-induced IL-2 production in human
T-cells as well as for the inhibition of T-cell activation in a
human mixed lymphocyte reaction (huMLR).
The synthesis of the 2-amino-6-phenylpyrimido[5′,4′:5,6]py-
rimido[1,2-a]benzimidazol-5(6H)-ones is shown in Scheme 3.
A solution of 2-chlorobenzimidazole (10) and the desired aniline
18 were refluxed overnight to afford the desired 2-aminoben-

1640 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Table 2. SAR: Variations of the Aniline Side Chaina
Martin et al.
a IC50values are the means of two or more separate determinations, in duplicate; standard deviations were generally within (30% of the mean. b IL-2:
anti-CD3/CD28-induced T-cell IL-2 secretion assay. c huMLR: human mixed lymphocyte reaction. d Determined using ACD Labs 8.0. e SIF sol.: simulated
intestinal fluid solubility assay.
For purposes of determining kinase selectivity, the compounds
were also tested against related kinases Src, p38R, and KDR.
Mitogen-activated protein kinase (MAPK) p38R is a serine/
threonine kinase and a key signaling protein for multiple
processes that are activated in inflammation, such as the
biosynthesis of pro-inflammatory cytokines TNFR and IL-1ꢀ.25
The vascular endothelial growth factor receptor-2 (VEGFR-2;
also referred to as the kinase insert domain receptor or KDR)

Structure-Based Design of Lck Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1641
Table 3. SAR:Variations of the 6-Aryl Groupa
is a receptor protein tyrosine kinase involved in angiogenesis,
the formation of new vasculature from existing blood vessels.
Aberrant angiogenesis has been associated with a variety of
disorders including rheumatoid arthritis and cancer.26 Thus,
selectivity over p38R and KDR is important for proof of concept
studies.
Aminobenzimidazole triazines 1 and 2 exhibited excellent
enzyme potency on Lck (6 and 1 nM, respectively) but lacked
selectivity over KDR (7 and 4 nM). Our initial work focused
on the hypothesis that introducing a carbonyl group to engage
the Thr316 γ-OH in Lck (and clash with the Val916 γ-methyl
in KDR) would provide compounds with improved selectivity
while maintaining Lck potency. As shown in Table 2, compound
24, a closely related derivative of 3 (the inhibitor modeled to
examine the feasibility of this idea), served as a promising
starting point. While 24 did exhibit a 30-fold loss in potency
against Lck (relative to 2), we were encouraged by the 1000-
fold loss in potency against KDR. In addition, this change was
coupled with a surprising >100-fold loss in potency against
p38R (which also possesses a Thr gatekeeper) relative to
compound 2 (p38R IC50 ) 78 nM). With this promising result
in hand, we set out to explore whether altering the aniline side
chain could increase Lck potency while maintaining the high
levels of selectivity.
The crystal structure of compound 1 bound to Lck (Figure
2) as well as our model of 3 (Figure 3) revealed that substituents
on the aniline at C2 should be projected toward solvent. We
reasoned that the introduction of an ionizable group at the para-
position of this ring might improve cell permeability and
potency, as well as oral bioavailability. In addition, a slight boost
in enzyme potency might be gained via interactions with
residues along the solvent front.27 As shown in Table 2,
alterations to the aniline side chain were well-tolerated, with
all variations affording compounds that were potent inhibitors
of Lck and many exhibiting a marked improvement in solubility
(>10-fold increase relative to 24). The main area of differentia-
tion between these compounds was their cellular activity.
Replacing the methoxy groups with a tertiary amine such as
the N-methylpiperazine in 25 and 26 resulted in compounds
with a 3- to 5-fold increase in enzyme activity as well as a 10-
fold increase in cell potency (huMLR data; increased potency
presumably due to improved solubility and/or cell permeability).
Selectivity over KDR increased to well over 500-fold, with
>100-fold selectivity observed over p38R. Replacement of the
N-methylpiperazine with 2-(dimethylamino)ethoxy or 2-(dim-
ethylamino)propoxy groups (27 and 28) also improved cell
potency in the huMLR by 3- to 4-fold, though the potency
remained the same (relative to 24). Replacing the dimethylamino
group on the alkoxy side chain with a cyclic amine, such as
piperidine (compound 33), had little impact on potency or
selectivity.
a IC50 values are the means of two or more separate determinations, in
duplicate; standard deviations were generally within (30% of the mean.
b huMLR: human mixed lymphocyte reaction.
consistent with the aniline side chain pointing toward the solvent
front and having minimal interactions with the protein. Because
of its excellent enzyme and cellular potency and selectivity
profile, coupled with its ease of synthesis (4-(4-methylpiperazin-
1-yl)aniline is commercially available), we chose to conduct
subsequent SAR studies around 25.
We also explored the effect of varying the substitution on
the aniline pointing into the kinase specificity pocket. As shown
in Table 3, 2,6-disubstitution affords the most potent compounds
(25, 36, and 37). This is consistent with observations from
previous groups,16a,17a as well as our own in-house studies,13a
which demonstrate that a 2,6-disubstituted aniline is the optimal
substitution pattern. To avoid steric interactions, the two ortho-
groups position the aniline orthogonal to the tetracyclic core,
allowing it to easily fill the hydrophobic pocket. Compounds
with monosubstitution in either the para- (38) or meta-positions
(39 and 40) showed reduced potency (15- to 50-fold) relative
to 25. The loss of enzyme potency was less significant (3-fold)
when the compound contained a 2,5-disubstituted aniline (41),
but this was combined with a 20-fold loss in cell potency.
Introduction of larger groups, such as the p-butoxy in 42,
resulted in an almost complete loss of potency against Lck (>4
µM). Because none of these alterations offered any improve-
ment, we chose to further explore the properties of 25.
Structural Confirmation. To confirm the binding elements
responsible for the observed potency and selectivity of this series
of compounds, we obtained a cocrystal structure of Lck with
2-amino-6-phenylpyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimida-
zol-5(6H)-one 25 (Figure 4). Consistent with findings from our
previous work,13a the aniline side chain of 25 extends to the
mouth of the ATP-binding pocket, with the N-methylpiperazine
moiety fully exposed to solvent. The 6-phenyl group is rotated
80 degrees from the tetracyclic plane due to the presence of
We also investigated the impact of combining substituted
anilines with variations on the alkoxy side chain. 3-Fluoro-
substituted aniline (31) showed a slight improvement in KDR
but with similar levels of Lck enzyme and cell potency relative
to 24. 3,5-Dimethoxy derivatives 30 and 35 showed improved
enzyme and cellular potency while maintaining excellent
selectivity over KDR and p38R (>500-fold for both). Replacing
the N-methylpiperazine with a morpholine (32) had little impact
on enzyme and cell potency, but a slight decrease in selectivity
over KDR (relative to compound 30) was observed. Overall,
changes to the aniline side chain had minimal impact in terms
of potency and selectivity. This is consistent with previously
observed results in related series of compounds13a and is

1642 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Martin et al.
Table 4. Kinase Selectivity Profile of Compound 25
a
a
kinase
IC50 (µM)
kinase
IC50 (µM)
Lck
Src
Lyn
Syk
ZAP-70
Tyk2
JAK3
Itk
Btk
c-Met
Tie-2
0.007
0.042
0.021
0.20
1.5
IGFR
FGFR
KDR
2.4
1.6
5.1
c-Kit
0.56
0.54
>40
7.3
>10
>100
>10
>100
b-Raf
JNK2
JNK3
p38R
ERK1
CDK5
MSK1
1.2
0.15
>20
0.41
>10
0.75
a IC50 values are the means of two or more separate determinations, in
duplicate; standard deviations were generally within (30% of the mean.
Table 5. Cellular Profile of Compound 25
Figure 4. X-ray cocrystal structure of 2-amino-6-phenylpyrimido[5′,4′:
5,6]pyrimido[1,2-a]benzimidazol-5(6H)-one 25 bound to Lck.
cell assaya
b
IC50 (µM)
IL-2
0.46
0.53
1.2
0.047
1.6
T-cell prolif.
huWB IL-2
huMLR
TCR ꢁ-chain
PMA/iono
JKT
the 2,6-substituents, which appropriately orients the aromatic
ring to enter the deep hydrophobic pocket adjoining the ATP-
binding site. Hydrogen bond interactions between the inhibitor
25 and the linker region of the protein are similar to those
observed with compound 1 and described above. Additionally,
a novel, fourth hydrogen bond is observed. As we hypothesized,
the carbonyl of the tetracycle 25 accepts a hydrogen bond from
the γ-OH of Thr316 (2.7 Å). This interaction with the threonine
gatekeeper of Lck is most likely responsible for the observed
selectivity over KDR, which possesses a valine at this position.
Although p38R also contains a threonine side-chain in the
gatekeeper position, the most common conformation observed
for this threonine in p38R structures available through the
Protein Data Bank positions the γ-OH away from the ATP-
binding site where it mediates a hydrogen bond between main
chain atoms from strands ꢀ4 and ꢀ5.28 This side-chain confor-
mation positions the γ-methyl of Thr106 near the ATP-binding
pocket, which would yield an unfavorable interaction with the
carbonyl of 25. However, two structures of apo p38R have been
reported in which the Thr106 side chain adopts a conformation
similar to that seen in the Lck structures presented here.29
Selectivity of this series of compounds over p38R may instead
result from the presence of Ile84 on strand ꢀ4 of p38R, which
places a δ-methyl group in close proximity to the tetracycle
carbonyl oxygen (∼2.5 Å; model not shown). The equivalent
residue in all Src-family kinases is a valine, with the γ-methyl
of this valine (301) in Lck residing 3.5 Å away from the
carbonyl oxygen of 25.
Kinase Selectivity Profile. To evaluate tetracyclic pyrimidine
25 in an in vivo setting, it was critical to understand the
selectivity profile of this molecule. To that end, compound 25
was assayed against an extended panel of tyrosine and serine/
threonine kinases (Table 4).30 Within the protein tyrosine kinase
(PTK) group, tetracycle 25 was selective (>100-fold) over
representatives from the VEGFR, FGFR, InsR, Tie, and Met
families, with a slight decrease in selectivity (20- to 50-fold)
over members of the Tec, Syk, and Jak families. Greater
selectivity was seen over kinases outside of the PTK group
(CMGC and AGC), as 25 demonstrated >1000-fold selectivity
over MAPK, CDK, and RSK family representatives. As
expected, due to the close homology of Lck to other members
of the Src family, little selectivity was observed against Src or
Lyn.
5.8
0.49
a IL-2: anti-CD3/CD28-induced T-cell IL-2 secretion assay; T-cell prolif.:
anti-CD3/CD28-induced T-cell proliferation assay; huWB IL-2: anti-CD3/
CD28-induced T-cell IL-2 secretion assay using human whole blood;
huMLR: human mixed lymphocyte reaction; TCR ꢁ-chain: T-cell receptor
ꢁ-chain phosphorylation assay; PMA/iono: anti-CD3/CD28-induced T-cell
IL-2 secretion assay stimulated with phorbol myristic acid and calcium
ionophore; JKT: Jurkat proliferation/survival assay. See Experimental
Section for a description of each assay. b IC50 values are the means of two
or more separate determinations, in duplicate; standard deviations were
generally within (30% of the mean.
Table 6. Pharmacokinetic Parameters for Compound 25 following IV
Dose in Sprague–Dawley Ratsa
doseb
(mg/kg)
AUC0f∞
(ng h/mL)
CL
(L/h/kg)
Vss
t1/2
(h)
(L/kg)
2
1153 ( 137
1.8 ( 0.3
7.5 ( 1.4
3.12 ( 0.04
a n ) 3 animals per study. b Dosed as a solution in DMSO.
2) and also inhibited subsequent T-cell proliferation (T-cell
prolif.) in the same human T -cells. When the IL-2 secretion
assay was performed using human whole blood (huWB IL-2),
there was only a 3-fold shift in potency. Tetracyclic pyrimidine
25 also inhibited a human mixed lymphocyte reaction (huMLR)
with a 10-fold increase in potency as compared to the other in
vitro cell assays utilizing purified human cells. This shift in
potency is most likely due to the longer duration of the MLR
assay as compared to the IL-2 secretion and proliferation assays.
This compound also displayed inhibition of a mechanism-based
biochemical cell assay probing Lck-dependent TCR ꢁ-chain
phosphorylation (TCR ꢁ-chain). As expected, the compound
showed a 10-fold reduction in potency when IL-2 was induced
in a receptor-independent fashion by stimulating with phorbol
ester and calcium ionophore (PMA/iono). In addition, the
compound exhibited a similar level of potency when tested in
a general proliferation assay using the human T-cell line, Jurkat
(JKT), suggesting the possibility of some off-target kinase
activity. With this promising in vitro data, we set out to examine
the in vivo properties of 25.
Pharmacokinetic Profile. The pharmacokinetic properties of
tetracyclic pyrimidine 25 were assessed in the rat following both
intravenous (i.v.) and oral (p.o.) dosing (Tables 6 and 7,
respectively). The compound displayed a moderate clearance
(1.8 L/h/kg) and high volume of distribution (Vss ) 7.5 L/kg),
resulting in a half-life of 3.12 h after i.v. dosing. When dosed
Cellular Activity. To further characterize the cellular proper-
ties of tetracycle 25, it was subjected to a series of functional
cell assays. As shown in Table 5, compound 25 exhibited good
potency in the T-cell receptor-induced IL-2 secretion assay (IL-

Structure-Based Design of Lck Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1643
Table 7. Pharmacokinetic Parameters for Compound 25 following Oral
Dose in Sprague–Dawley Ratsa
doseb
Cmax
tmax
AUC0f∞
bioavailability,
(mg/kg)
(ng/mL)
(h)
(ng h/mL)
F (%)
5
82 ( 8
3.0 ( 1.0
862 ( 34
17
a n ) 2 animals per study. b Dosed as an aqueous suspension in 1%
Tween 80 in 2% HPMC.
Figure 6. Effect of compound 25 on adjuvant-induced arthritis in
the rat. Arthritis was induced by administration of Mycobacterium
tuberculosis H37Ra suspended in paraffin oil and injected into the
base of the tail (0.5 mg/rat) on day 0 to male Lewis rats (∼250 g,
obtained from CRL, Andover, MA). Compound 25 or vehicle was
administered orally, once daily from day 9 to day 17. Paw volume
was measured daily from day 9 through day 18. Values represent
the mean ( standard error for N ) 8 animals per group. *Indicates
P < 0.05 vs vehicle control by Mann–Whitney U-test. From this
experiment, the ED50 of 25 was estimated as 24 mg/kg (AUC, 2.7
µM · h; Cmax, 0.7 µM).
Figure 5. Effect of compound25 on anti-CD3-induced IL-2 production
in mice. BALB/c mice were dosed 1 h prior to challenge with compound
p.o. (eight per group), as described in the Experimental Section. Mice
were then challenged i.v. with antimouse CD3ꢀ monoclonal antibody.
A total of 90 min after the challenge, IL-2 levels in serum were
determined by ELISA. Data points represent the mean IL-2 levels/
group ( the standard error. P values were determined vs vehicle control
by Mann–Whitney U-test. *Indicates P e 0.05. From this experiment,
the ED50 of compound 25 was estimated at 26 mg/kg.
p.o. (5 mpk administered as an aqueous suspension in 1% Tween
80 in 2% HPMC), compound 25 had a tmax of 3.0 h, with a
relatively low bioavailability (17%), but was still a suitable tool
for further proof of concept studies.
Pharmacodynamic Profile. On the basis of its PK and
selectivity profile, tetracyclic pyrimidine 25 was tested in a short-
term T-cell receptor dependent mouse model. A single dose of
25 was administered at 3, 10, and 30 mg/kg orally to mice and
evaluated for its ability to inhibit anti-CD3-mediated IL-2
secretion in serum (Figure 5). The compound showed a dose-
dependent inhibition of IL-2 production with an ED50 estimated
at 26 mg/kg.
Figure 7. Effect of compound 25 on collagen-induced arthritis in
the rat. Arthritis was induced by intradermal injection of Porcine
type II collagen emulsified in IFA to female Lewis rats. Animals
were assigned to a treatment group at disease onset (study day 0),
which occurred 10–12 days following immunization. Compound 25
or vehicle was administered orally, once daily for 7 days. Paw
diameter was measured daily from day 0 through day 7. Values
represent the mean ( standard error for N ) 8 animals per group.
*Indicates P < 0.05 vs vehicle control by Mann–Whitney U-test.
From this experiment, the ED50 of 25 was estimated as 3.2 mg/kg
(AUC, 3.3 µM · h; Cmax, 0.5 µM).
In Vivo Arthritis Models. Based on its promising activity
in the in vivo mouse IL-2 model, compound 25 was tested in
two in vivo disease models of arthritis. In the adjuvant-induced
arthritis model, arthritis was induced by injection of a suspension
of Mycobacterium tuberculosis H37Ra in paraffin oil (0.5 mg/
rat) into the base of the tail of male Lewis rats, and the disease
was allowed to progress for 9 days. A once daily dose of 25
was administered orally at 10, 30, and 60 mg/kg from day 9 to
day 17. Paw volume was measured daily from day 9 through
day 18. The compound showed a dose-dependent inhibition of
arthritis, with an ED50 estimated at 24 mg/kg (Figure 6). Based
on the measured plasma levels from the three dose groups, the
exposure of 25 at the ED50 was estimated to be 2.7 µM·h (Cmax
≈ 0.7 µM). In a separate study, arthritis was induced by
intradermal injection of Porcine type II collagen emulsified in
IFA to female Lewis rats. Disease onset occurred 10–12 days
following immunization. Compound 25 was administered orally
at 0.3, 1, 3, and 10 mg/kg once daily for 7 days. The anti-
inflammatory effect of 25 was determined by measuring paw
diameter daily for 7 days. Compound 25 showed a dose-
dependent inhibition of arthritis with an ED50 estimated at 3.2
mg/kg (Figure 7). Based on the measured plasma levels from
the three dose groups, the exposure of 25 at the ED50 was
estimated to be 3.3 µM·h (Cmax ≈ 0.5 µM).
Conclusions
The novel 2-amino-6-phenylpyrimido[5′,4′:5,6]pyrimido[1,2-
a]benzimidazol-5(6H)-ones have been shown to be potent and
selective inhibitors of Lck. Our investigation of this series of
compounds combined with structural analysis has identified the
key components necessary for kinase inhibition and selectivity.
Optimization efforts led to tetracyclic pyrimidine 25, which
exhibits potent and selective inhibition of Lck kinase activity.
This activity translates into mechanism-based inhibition of cell-

1644 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Martin et al.
over anhydrous magnesium sulfate prior to addition) was added
dropwise. The mixture was stirred for 15 h, dimethylsulphoxide
(1.5 mL) was added, and the mixture was stirred for 1 h. Water
(10 mL) was added and the organic layer was separated and washed
with water, saturated aqueous sodium bicarbonate solution (twice),
and brine. The solvent was removed under reduced pressure to give
6-(2,6-dimethylphenyl)-2-methanesulphonyl-benzo[4,5]imidazolo[1,2-
a]pyrimido[5,4-e]pyrimidin-5-one (22a; 2.10 g, 97%) as a yellow
solid. 1H NMR (400 MHz, D2O) δ ppm 2.1 (s, 6 H), 3.5 (s, 3 H),
7.2–7.5 (m, 5 H), 7.6 (m, 1 H), 8.6 (m, 1 H), 9.6 (s, 1 H). MS
(ESI, pos. ion) m/z 420 (M + 1).
6-(2,6-Dimethylphenyl)-2-((4-(4-methyl-1-piperazinyl)phen-
yl)amino)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-
one (25). A sealed tube was charged with 6-(2,6-dimethylphenyl)-
2-methanesulphonyl-benzo[4,5]imidazolo[1,2-a]pyrimido[5,4-e]py-
rimidin-5-one (22a; 3.8 g, 9.0 mmol), 4-(4-methylpiperazin-1-
yl)aniline (1.8 g, 10.0 mmol), and isopropanol (75 mL). The tube
was flushed with nitrogen for 2 min, fitted with a screw-cap lid,
and heated at 85 °C for 15 h. The reaction mixture was cooled to
room temperature and concentrated. The green residue was
partitioned between dichloromethane and 1 M aqueous sodium
hydroxide solution. The aqueous phase was separated and extracted
with dichloromethane. The combined organic layers were dried over
anhydrous magnesium sulfate, filtered, and concentrated to give a
dark green oil. This material was purified via column chromatog-
raphy on silica gel (eluting with 10% methanol-dichloromethane)
to afford 6-(2,6-dimethylphenyl)-2-((4-(4-methyl-1-piperazinyl)phe-
nyl)amino)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-
one (25; 2.76 g, 58%) as a yellow solid. 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.05 (s, 6 H), 2.25 (s, 3 H), 2.45–2.54 (m, 4 H),
3.10–3.18 (m, 2 H), 3.18–3.26 (m, 2 H), 7.00 (d, J ) 8.4 Hz, 1 H),
7.08 (d, J ) 8.2 Hz, 1 H), 7.20–7.29 (m, 2 H), 7.30–7.43 (m, 3 H),
7.49–7.65 (m, 3 H), 8.36 (d, J ) 7.7 Hz, 1 H), 9.08 (s, 1 H), 10.55
(s, 1 H). MS (ESI, pos. ion) m/z: 531 (M + 1). HRMS ([M +
H]+) calcd, 531.26153; found, 531.26176. Anal. (C31H30N8 · 1.5
H2O) C, H, N.
based T-cell activation in vitro and in vivo and disease
modification of T-cell mediated arthritis.
Experimental Section
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. The
synthesis of anilines 14a–h has been previously reported.13a 4-(3-
Piperidin-1-yl-propoxy)-phenylamine (14i) was prepared as previ-
ously reported.31 4-(2-(Dimethylamino)ethoxy)-3-methoxyaniline
was obtained from Evotec OAI, Oxon, U.K. Anhydrous solvents
were obtained from Aldrich and used directly. All reactions
involving air- or moisture-sensitive reagents were performed under
a nitrogen or argon atmosphere. All final compounds were purified
to >95% purity, as determined by high-performance liquid chro-
matography (HPLC). Purity was measured using Agilent 1100
Series high performance liquid chromatography (HPLC) systems
with UV detection at 254 nm (System A: Agilent Zorbax Eclipse
XDB-C8 4.6 × 150 mm, 5 µm, 5 to 100% CH3CN in H2O, with
0.1% TFA, for 15 min at 1.5 mL/min; System B: Zorbax SB-C8,
4.6 × 75 mm, 10 to 90% CH3CN in H2O, with 0.1% formic acid,
for 12 min at 1.0 mL/min). Silica gel columns were performed using
either glass columns packed with silica gel (200–400 mesh, Aldrich
1
Chemical) or prepacked silica gel cartridges (Biotage). H NMR
spectra were recorded on a Bruker AV-400 (400 MHz) spectrometer
or a Varian 400 MHz spectrometer at ambient temperature. All
observed protons are reported as parts per million (ppm) downfield
from tetramethylsilane (TMS) or other internal reference in the
appropriate solvent indicated. Data are reported as follows: chemical
shift, multiplicity (s ) singlet, d ) doublet, t ) triplet, q ) quartet,
br ) broad, m ) multiplet), coupling constants, and number of
protons. Low-resolution mass spectral (MS) data were determined
on an Agilent 1100 Series LCMS with UV detection at 254 nm
and a low resonance electrospray mode (ESI). High resolution mass
spectra were obtained on a high resonance electrospray time-of-
flight mass spectrometer. Combustion analysis was performed by
Galbraith Laboratories, Knoxville, TN, and were within 0.4% of
calculated, unless otherwise noted.
2-((4-((3-(Dimethylamino)propyl)oxy)phenyl)amino)-6-(2,6-
dimethylphenyl)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimida-
zol-5(6H)-one (28). A sealed tube was charged with 6-(2,6-
dimethylphenyl)-2-methanesulphonyl-benzo[4,5]imidazolo[1,2-a]py-
rimido[5,4-e]pyrimidin-5-one (22a; 1.0 g, 2.4 mmol), 4-(3-
(dimethylamino)propoxy)aniline (0.47 g, 2.6 mmol), and isopropanol
(30 mL). Trifluoroacetic acid (0.60 g, 4.8 mmol) was added, and
the tube was flushed with nitrogen for 2 min, fitted with a screw-
cap lid, and heated at 85 °C for 15 h. The reaction mixture was
cooled to room temperature and concentrated. The green residue
was partitioned between dichloromethane (30 mL) and 1 M aq
sodium hydroxide solution (10 mL). The aqueous phase was
separated and extracted with dichloromethane. The combined
organic layers were dried over anhydrous magnesium sulfate,
filtered, and concentrated to give a yellow solid. This material was
purified via column chromatography on silica gel (eluting with 5%
methanol-dichloromethane) to afford 2-((4-((3-(dimethylamino)-
propyl)oxy)phenyl)amino)-6-(2,6-dimethylphenyl)pyrimido[5′,4′:
5,6]pyrimido[1,2-a]benzimidazol-5(6H)-one (28; 1.2 g, 93%) as a
N-(2,6-Dimethylphenyl)-1H-benzo[d]imidazol-2-amine Hy-
drochloride (19a). A mixture of 2-chlorobenzimidazole (0.500 g,
3.28 mmol) and 2,6-dimethylaniline (2.94 g, 3.00 mL, 24.2 mmol)
was heated at 100 °C for 24 h. The mixture was cooled to room
temperature, and the solvent was removed under reduced pressure
to afford an oily residue. Trituration with ethyl acetate afforded
N-(2,6-dimethylphenyl)-1H-benzo[d]imidazol-2-amine hydrochlo-
ride (19a; 0.707 g, 79%) as a dark purple solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 2.24 (s, 6 H), 7.13 – 7.56 (m, 7 H), 10.78
(s, 1 H), 12.73 (s, 2 H). MS (ESI, pos. ion) m/z 238 (M + 1).
6-(2,6-Dimethylphenyl)-2-methylthio-benzo[4,5]imidazolo-
[1,2-a]pyrimido[5,6-e]pyrimidin-5-one (21a). A mixture of ethyl
4-chloro-2-methylthiopyrimidine-5-carboxylate (20; 12.5 g, 54
mmol), N-(2,6-dimethylphenyl)-1H-benzo[d]imidazol-2-amine hy-
drochloride (19a; 13.5 g, 49 mmol), and N,N-diisopropylethylamine
(17.6 mL, 102 mmol) in toluene (30 mL) was refluxed for 3 days.
The solvent was removed under reduced pressure and the residue
was partitioned between dichloromethane and 2 N HCl (aq). The
organic phase was washed with additional 2 N HCl (aq) and then
brine. The resulting suspension was then dissolved completely by
heating and concentrated to 100 mL. The resulting suspension was
filtered, and the solids were washed with dichloromethane and dried
toafford6-(2,6-dimethylphenyl)-2-methylthio-benzo[4,5]imidazolo[1,2-
a]pyrimido[5,6-e]pyrimidin-5-one (21a; 12.5 g, 60%) as a white
1
yellow solid. H NMR (400 MHz, DMSO-d6) δ ppm 1.84–1.95
(m, 2 H), 2.06 (s, 6 H), 2.18 (s, 6 H), 2.34–2.45 (m, 2 H), 3.93–4.13
(m, 2 H), 6.96–7.03 (m, 1 H), 7.08 (d, J ) 6.3 Hz, 1 H), 7.25 (s,
1 H), 7.27 (s, 1 H), 7.30–7.41 (m, 1 H), 7.32–7.36 (m, 2 H), 7.54
(d, J ) 7.1 Hz, 1 H), 7.58–7.71 (m, 2 H), 8.32 (d, J ) 9.3 Hz, 1
H), 9.09 (s, 1 H), 10.60 (s, 1 H). MS (ESI, pos. ion) m/z: 534 (M
+ 1). HRMS ([M + H]+) calcd, 534.26120; found, 534.26178.
Anal. (C31H31N7O2) C, H, N.
1
amorphous solid. H NMR (400 MHz, D2O) δ ppm 2.3 (s, 6 H),
The following compounds were synthesized according to the
general procedure detailed above for compound 25:
6-(2,6-Dimethylphenyl)-2-((3-(methoxy)-4-(4-methyl-1-piper-
3.0 (s, 3 H), 7.2–7.3 (m, 2 H), 7.5–7.7 (m, 3 H), 7.8 (m, 1 H), 8.75
(m, 1 H), 9.5 (s, 1 H). MS (ESI, pos. ion) m/z 388 (M + 1).
6-(2,6-Dimethylphenyl)-2-methanesulphonyl-benzo[4,5]-
imidazolo[1,2-a]pyrimido[5,4-e]pyrimidin-5-one (22a). 6-(2,6-
Dimethylphenyl)-2-methylthio-benzo[4,5]imidazolo[1,2-a]pyrimi-
do[5,6-e]pyrimidin-5-one (21a; 2.00 g, 5.16 mmol) was suspended
in chloroform (50 mL), and a solution of m-chloroperoxybenzoic
acid (70%, 2.79 g, 11.3 mmol) in chloroform (50 mL, predried
azinyl)phenyl)amino)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benz-
1
imidazol-5(6H)-one (26). H NMR (400 MHz, DMSO-d6) δ ppm
2.06 (s, 6 H), 2.24 (s, 3 H), 2.44–2.56 (m, 4 H), 2.92–3.11 (m, 4
H), 3.80 (s, 3 H), 6.90–7.04 (m, 1 H), 7.14–7.24 (m, 1 H), 7.25 (s,
1 H), 7.27 (s, 1 H), 7.30–7.43 (m, 4 H), 7.55 (d, J ) 8.2 Hz, 1 H),

Structure-Based Design of Lck Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1645
8.33–8.40 (m, 1 H), 9.11 (s, 1 H), 10.60 (s, 1 H). MS (ESI, pos.
ion) m/z: 561 (M + 1). HRMS ([M + H]+) calcd, 561.27210; found,
561.27267. Anal. (C32H32N8O2 ·2 H2O) C, H, N.
Anal. (C29H23Cl3N8O·H2O) H. C: calcd, 55.83; found, 55.25; N:
calcd, 17.96; found, 16.72.
6-(4-Methylphenyl)-2-((4-(4-methyl-1-piperazinyl)phenyl)-
6-(2,6-Dimethylphenyl)-2-((3-(methoxy)-4-((2-(4-methyl-1-
piperazinyl)ethyl)oxy)phenyl)amino)pyrimido[5′,4′:5,6]pyrim-
ido[1,2-a]benzimidazol-5(6H)-one (29). 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.06 (s, 6 H), 2.17 (s, 3 H), 2.30–2.43 (m, 4 H),
2.48–2.59 (m, 4 H), 2.64–2.77 (m, 2 H), 3.79 (s, 3 H), 4.02–4.17
(m, 2 H), 7.01–7.20 (m, 2 H), 7.25 (s, 1 H), 7.27 (s, 1 H), 7.31–7.44
(m, 4 H), 7.55 (d, J ) 7.7 Hz, 1 H), 8.37 (d, J ) 6.7 Hz, 1 H),
9.11 (s, 1 H), 10.59 (s, 1 H). MS (ESI, pos. ion) m/z: 605 (M + 1).
HRMS ([M + H]+) calcd, 605.29831; found, 605.29925. Anal.
(C34H36N8O3 ·7 H2O) C. H: calcd, 6.90; found, 5.96; N: calcd, 15.33;
found, 14.25.
2-((3,5-Dimethoxy)-4-((2-(4-methyl-1-piperazinyl)ethyl)oxy)-
phenyl)amino)-6-(2,6-dimethylphenyl)pyrimido[5′,4′:5,6]py-
rimido[1,2-a]benzimidazol-5(6H)-one (30). 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.06 (s, 6 H), 2.17 (s, 3 H), 2.29–2.41 (m, 4 H),
2.46–2.55 (m, 4 H), 2.60–2.69 (m, 2 H), 3.80 (s, 6 H), 3.93–4.04
(m, 2 H), 6.98–7.06 (m, 1 H), 7.18–7.23 (m, 1 H), 7.26 (s, 1 H),
7.28 (s, 1 H), 7.31–7.40 (m, 3 H), 7.52–7.59 (m, 1 H), 8.42 (s, 1
H), 9.14 (s, 1 H), 10.60 (s, 1 H). MS (ESI, pos. ion) m/z: 635 (M
+ 1). HRMS ([M + H]+) calcd, 635.30888; found, 635.30868.
Anal. (C35H38N8O4 ·1.5 H2O) C, H. N: calcd, 16.93; found:16.24.
6-(2,6-Dimethylphenyl)-2-((3-fluoro-4-((2-(4-methyl-1-piper-
azinyl)ethyl)oxy)phenyl)amino)pyrimido[5′,4′:5,6]pyrimido-
[1,2-a]benzimidazol-5(6H)-one (31). 1H NMR (400 MHz, DMSO-
d6) δ ppm 2.06 (s, 6 H), 2.16 (s, 3 H), 2.29–2.38 (m, 4 H), 2.47–2.57
(m, 4 H), 2.68–2.78 (m, 2 H), 4.07–4.29 (m, 2 H), 7.23–7.49 (m,
7 H), 7.56 (d, J ) 8.8 Hz, 1 H), 7.67–7.87 (m, 1 H), 8.38 (d, J )
9.7 Hz, 1 H), 9.14 (s, 1 H), 10.74 (s, 1 H). MS (ESI, pos. ion) m/z:
593 (M + 1). HRMS ([M + H]+) calcd, 593.27833; found,
593.27937. Anal. (C33H33FN8O2 ·1 CH3OH) C, H, N.
amino)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-
1
one (38). H NMR (400 MHz, DMSO-d6) δ ppm 2.42 (s, 3 H),
2.50 (s, 3 H), 2.52–2.65 (m, 4 H), 2.80–3.08 (m, 4 H), 6.98–7.18
(m, 3 H), 7.19–7.29 (m, 1 H), 7.30–7.41 (m, 4 H), 7.46–7.69 (m,
3 H), 8.34 (d, J ) 7.3 Hz, 1 H), 9.04 (s, 1 H), 10.54 (s, 1 H). MS
(ESI, pos. ion) m/z: 517 (M + 1). HPLC purity: 94% (system A),
95% (system B). HRMS ([M + H]+) calcd, 517.24588; found,
517.24490.
6-(3-Methylphenyl)-2-((4-(4-methyl-1-piperazinyl)phenyl)-
amino)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-
one (39). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.21–2.29 (m, 3
H), 2.40 (s, 3 H), 2.45–2.57 (m, 4 H), 3.05–3.27 (m, 4 H), 6.91–7.14
(m, 2 H), 7.19–7.39 (m, 5 H), 7.43–7.67 (m, 4 H), 8.34 (d, J ) 8.4
Hz, 1 H), 9.06 (s, 1 H), 10.51 (s, 1 H). MS (ESI, pos. ion) m/z:
517 (M + 1). HPLC purity: 96% (system A), 96% (system B).
HRMS ([M + H]+) calcd, 517.24588; found, 517.24633.
6-(3-(Methoxy)phenyl)-2-((4-(4-methyl-1-piperazinyl)phen-
yl)amino)pyrimido-[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-
1
one (40). H NMR (400 MHz, DMSO-d6) δ ppm 2.25 (s, 3 H),
2.44–2.55 (m, 4 H), 3.07–3.24 (m, 4 H), 3.79 (s, 3 H), 6.88–7.13
(m, 5 H), 7.20–7.39 (m, 2 H), 7.44–7.69 (m, 4 H), 8.34 (d, J ) 7.5
Hz, 1 H), 9.06 (s, 1 H), 10.51 (s, 1 H). MS (ESI, pos. ion) m/z:
533 (M + 1). HPLC purity: 98% (system A), 99% (system B).
HRMS ([M + H]+) calcd, 533.24080; found, 533.24237.
6-(5-Chloro-2-(methoxy)phenyl)-2-((4-(4-methyl-1-pipera-
zinyl)phenyl)amino)-pyrimido[5′,4′:5,6]pyrimido[1,2-a]-benz-
imidazol-5(6H)-one (41). 1H NMR (400 MHz, DMSO-d6) δ ppm
2.25 (s, 3 H), 2.44–2.58 (m, 4 H), 3.05–3.27 (m, 4 H), 3.74 (s, 3
H), 6.93–7.15 (m, 2 H), 7.19–7.45 (m, 4 H), 7.48–7.69 (m, 4 H),
8.33 (d, J ) 7.9 Hz, 1 H), 9.07 (s, 1 H), 10.57 (s, 1 H). MS (ESI,
pos. ion) m/z: 567 (M + 1). HPLC purity: 96% (system A), 94%
(system B). HRMS ([M + H]+) calcd, 567.20183; found, 567.20314.
6-(4-(Butoxy)phenyl)-2-((4-(4-methyl-1-piperazinyl)phenyl)-
amino)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-
6-(2,6-Dimethylphenyl)-2-((3-(methoxy)-4-((3-(4-methyl-1-
piperazinyl)propyl)oxy)phenyl)amino)pyrimido[5′,4′:5,6]py-
rimido[1,2-a]benzimidazol-5(6H)-one (34). 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.82–1.95 (m, 2 H), 2.06 (s, 6 H), 2.15 (s, 3 H),
2.24–2.48 (m, 10 H), 3.79 (s, 3 H), 3.94–4.11 (m, 2 H), 6.96–7.12
(m, 1 H), 7.13–7.24 (m, 1 H), 7.24–7.29 (m, 2 H), 7.30–7.44 (m,
4 H), 7.55 (d, J ) 7.8 Hz, 1 H), 8.36 (d, J ) 9.4 Hz, 1 H), 9.11 (s,
1 H), 10.58 (s, 1 H). MS (ESI, pos. ion) m/z: 619 (M + 1). HRMS
1
one (42). H NMR (400 MHz, DMSO-d6) δ ppm 0.97 (t, J ) 7.4
Hz, 3 H), 1.40–1.58 (m, 2 H), 1.66–1.84 (m, 2 H), 2.26 (s, 3 H),
2.43–2.59 (m, 4 H), 3.03–3.27 (m, 4 H), 4.07 (t, J ) 6.5 Hz, 2 H),
6.92–7.14 (m, 4 H), 7.18–7.42 (m, 4 H), 7.43–7.69 (m, 3 H), 8.33
(d, J ) 6.8 Hz, 1 H), 9.03 (s, 1 H), 10.49 (s, 1 H). MS (ESI, pos.
ion) m/z: 575 (M + 1). HPLC purity: 98% (system A), 100%
(system B). HRMS ([M + H]+) calcd, 575.28775; found, 575.28829.
The following compounds were synthesized according to the
general procedure detailed above for compound 28:
([M
+
H]+) calcd, 619.31396; found, 619.31449. Anal.
(C35H38N8O3 ·1.5 H2O) C, H, N.
2-((3,5-Dimethoxy)-4-((3-(4-methyl-1-piperazinyl)propyl)-
oxy)phenyl)amino)-6-(2,6-dimethylphenyl)pyrimido[5′,4′:5,6]-
pyrimido[1,2-a]benzimidazol-5(6H)-one (35). 1H NMR (400
MHz, DMSO-d6) δ ppm 1.74–1.88 (m, 2 H), 2.06 (s, 6 H), 2.25
(s, 3 H), 2.36–2.60 (m, 10 H), 3.79 (s, 6 H), 3.86–4.01 (m, 2 H),
7.01 (s, 1 H), 7.15–7.24 (m, 1 H), 7.24–7.30 (m, 2 H), 7.30–7.43
(m, 3 H), 7.56 (d, J ) 8.7 Hz, 1 H), 8.39 (s, 1 H), 9.14 (s, 1 H),
10.60 (s, 1 H). MS (ESI, pos. ion) m/z: 649 (M + 1). HRMS ([M
+ H]+) calcd, 649.32453; found, 649.32443. Anal. (C36H40N8O4 ·2.5
H2O) C, H, N: calcd, 16.15; found, 15.48.
6-(2,6-Dimethylphenyl)-2-((3,4,5-trimethoxyphenyl)amino)-
pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-one (24). 1H
NMR (400 MHz, DMSO-d6) δ ppm 2.06 (s, 6 H), 3.73 (s, 3 H),
3.81 (s, 6 H), 7.02 (s, 1 H), 7.21 (s, 1 H), 7.26 (s, 1 H), 7.28 (s, 1
H), 7.32–7.40 (m, 3 H), 7.56 (d, J ) 8.2 Hz, 1 H), 8.38–8.47 (m,
1 H), 9.14 (s, 1 H), 10.60 (s, 1 H). MS (ESI, pos. ion) m/z: 523 (M
+ 1). HRMS ([M + H]+) calcd, 523.20883; found, 523.20942.
Anal.(C29H26N6O4 ·2.5 H2O) C, N. H: calcd, 5.51; found, 4.75.
2-((4-((2-(Dimethylamino)ethyl)oxy)phenyl)amino)-6-(2,6-
dimethylphenyl)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-
5(6H)-one (27). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.06 (s, 6
H), 2.20 – 2.30 (m, 6 H), 2.67 (s, 2 H), 3.99 – 4.20 (m, 2 H), 6.97
– 7.05 (m, 1 H), 7.10 (d, J ) 7.8 Hz, 1 H), 7.25 (s, 1 H), 7.27 (s,
1 H), 7.31 – 7.37 (m, 2 H), 7.36 – 7.41 (m, 1 H), 7.54 (d, J ) 6.8
Hz, 1 H), 7.58 – 7.74 (m, 2 H), 8.33 (d, J ) 8.5 Hz, 1 H), 9.10 (s,
1 H), 10.61 (s, 1 H). MS (ESI, pos. ion) m/z: 523 (M + 1). HRMS
6-(2,6-Dichlorophenyl)-2-((4-(4-methyl-1-piperazinyl)phen-
yl)amino)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-5(6H)-
1
one (36). H NMR (400 MHz, DMSO-d6) δ ppm 2.27 (s, 3 H),
2.48–2.58 (m, 4 H), 3.15 (d, J ) 4.1 Hz, 2 H), 3.19–3.28 (m, 2 H),
7.00 (d, J ) 8.4 Hz, 1 H), 7.05–7.14 (m, 1 H), 7.29 (t, J ) 7.3 Hz,
1 H), 7.34–7.49 (m, 2 H), 7.52–7.64 (m, 2 H), 7.64–7.71 (m, 1 H),
7.75–7.83 (m, 2 H), 8.34 (d, J ) 7.8 Hz, 1 H), 9.11 (s, 1 H), 10.70
(s, 1 H). MS (ESI, pos. ion) m/z: 571 (M + 1). HPLC purity: 100%
(system A), 99% (system B). HRMS ([M + H]+) calcd, 571.15229;
found, 571.15223.
([M
+
H]+) calcd: 523.20883 found: 523.20942. Anal.
(C30H29N7O2 · H2O) C, H, N.
2-((4-(4-Methyl-1-piperazinyl)phenyl)amino)-6-(2,4,6-tri-
chlorophenyl)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-
5(6H)-one (37). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.25 (s, 3
H), 2.43–2.50 (m, 4 H), 3.09–3.18 (m, 2 H), 3.18–3.28 (m, 2 H),
7.00 (d, J ) 8.9 Hz, 1 H), 7.09 (d, J ) 8.8 Hz, 1 H), 7.23–7.48 (m,
2 H), 7.52–7.67 (m, 3 H), 8.06 (s, 2 H), 8.33 (d, J ) 7.6 Hz, 1 H),
9.05–9.19 (m, 1 H), 10.71 (s, 1 H). MS (ESI, pos. ion) m/z: 605
(M + 1). HRMS ([M + H]+) calcd, 605.11332; found, 605.11312.
2-((3,5-Dimethoxy)-4-((2-(4-morpholinyl)ethyl)oxy)phenyl)-
amino)-6-(2,6-dimethylphenyl)pyrimido[5′,4′:5,6]pyrimido-
[1,2-a]benzimidazol-5(6H)-one (32). 1H NMR (400 MHz, DMSO-
d6) δ ppm 2.06 (s, 6 H), 2.46–2.52 (m, 4 H), 2.62–2.70 (m, 2 H),
3.55–3.62 (m, 4 H), 3.80 (s, 6 H), 3.95–4.06 (m, 2 H), 6.98–7.06
(m, 1 H), 7.17–7.24 (m, 1 H), 7.26 (s, 1 H), 7.28 (s, 1 H), 7.32–7.40
(m, 3 H), 7.56 (d, J ) 8.4 Hz, 1 H), 8.37–8.46 (m, 1 H), 9.14 (s,

1646 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Martin et al.
1 H), 10.61 (s, 1 H). MS (ESI, pos. ion) m/z: 622 (M + 1). HRMS
acid (PMA) and calcium ionophore were used in combination to
induce IL-2 secretion from purified T-cells. Potential inhibitor
compounds were tested for inhibition of this response, as described
above for anti-CD3 and anti-CD28 antibodies. Human whole-blood
anti-CD3/anti-CD28-induced IL-2 secretion assays were run in a
similar fashion as described above using whole blood from normal
volunteers diluted 50% in tissue culture medium prior to stimulation.
TCR ꢀ-Chain Phosphorylation Assay. Jurkat cells (1.25 × 106/
well) were seeded in a 96-well plate (round-bottom) in 150 µL of
RPMI + 10% FCS and then preincubated with 50 µL compound
or without compound (50 µL medium) at 37 °C for 30 min. For
anti-CD3 stimulation, cells were cooled on ice for 15 min and then
incubated with 10 µL of anti-CD3 mAb (UCHT-1) at a final
concentration of 1.2 µg/mL for 30 min. A total of 20 µL of GAM-
IgG1 was added to cells at 30 µg/mL final and incubated for 20
min on ice. Cells were transferred to a 37 °C water bath and
stimulated for 1 min. Cells were spun at speed 1000 rpm in the
centrifuge for 5 min. Supernatant was aspirated and cells were lysed
in 30 µL of cold lysis buffer (1% Triton X-100 in NaCl/HEPES)
with phosphatase/protease inhibitors for 30 min on ice. Lysates were
spun at 3000 rpm in the centrifuge for 5 min to remove nuclei
before 25 µL of supernatant was transferred to new tubes for the
Igen ꢁ-chain phosphorylation detection assay.
A master mix was made containing (vol. per sample tube) 21
µL of Igen dilution buffer (PBS, 1% BSA, 0.1% tween-20; Igen/
BioVeris; Gaithersburg, MD), 4 µL of the capture antibody,
antihuman CD247 mAb, clone G3 (BD Coulter, San Diego, CA),
coupled with Dynabeads M-280, 200 µL of the detection antibody,
ruthenylated p-CD3-ꢁ (415.9A; Santa Cruz Biotech., Santa Cruz,
CA) at 1 to 16000 dilution in Igen dilution buffer. A total of 25
µL per sample lysate was transferred from the lysate plate to a
tube including the negative and positive controls. Sample tubes were
shaken at room temperature overnight. Tubes then were placed in
a carousel of Igen tube analyzer and analyzed. The signal-to-noise
ratio was generally around 15–20.
The procedure of coupling the capture antibody, antihuman
CD247 mAb, clone G3 to Dynabeads M-280 was based on the
protocol provided by Dynal Biotech. The procedure of p-CD3-ꢁ
ruthenylation is based on the protocol provided by Bio Veris.
Pharmacokinetic Studies. Sprague–Dawley rats were admin-
istered a solution of compound in DMSO at the indicated doses
i.v. For oral dosing, a suspension of the compound in 2%
hydroxypropyl methylcellulose with 1% Tween-80 was adminis-
tered. Samples were taken at the indicated times and analyzed for
parent compound by LC-MS.
Anti-CD3-Induced IL-2 Production in Mice. Twelve week old
(20 g) BALB/c mice were dosed 1 h prior to challenge with
compound p.o. (eight per group) at the indicated doses in 2%
hydroxypropyl methylcellulose with 1% Tween-80. Mice were then
challenged i.v. with antimouse CD3ꢀ monoclonal antibody (145.2C11,
BD PharMingen, San Diego, CA; 3 µg/mouse) diluted in PBS. A
total of 90 min after anti-CD3 challenge, blood was collected via
cardiac puncture. IL-2 levels were measured in serum by ELISA
(BioSource, Camarillo, CA). Data points represent the mean IL-2
levels/group ( the standard error. P values were determined versus
vehicle control by Mann–Whitney U-test.
([M
+
H]+) calcd, 622.27724; found, 622.27962. Anal.
(C34H35N7O5 ·0.5 H2O) C, H, N.
6-(2,6-Dimethylphenyl)-2-((4-((3-(1-piperidinyl)propyl)oxy)-
phenyl)amino)pyrimido[5′,4′:5,6]pyrimido[1,2-a]benzimidazol-
5(6H)-one (33). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.33–1.43
(m, 2 H), 1.43–1.55 (m, 4 H), 1.83–1.97 (m, 2 H), 2.06 (s, 6 H),
2.26–2.47 (m, 6 H), 3.97–4.14 (m, 2 H), 6.95–7.03 (m, 1 H), 7.08
(d, J ) 7.4 Hz, 1 H), 7.23–7.29 (m, 2 H), 7.30–7.43 (m, 3 H), 7.54
(d, J ) 7.5 Hz, 1 H), 7.58–7.71 (m, 2 H), 8.28–8.39 (m, 1 H), 9.09
(s, 1 H), 10.60 (s, 1 H). MS (ESI, pos. ion) m/z: 574 (M + 1).
HRMS ([M + H]+) calcd, 574.29250; found, 574.29376. Anal.
(C34H35N7O2 ·H2O) C, H. N: calcd, 16.57; found, 15.83.
Lck Kinase Assay. The Lck HTRF kinase assay involved ATP-
dependent phosphorylation of a biotinylated substrate peptide of
gastrin in the presence or absence of inhibitor compound. The final
concentration of gastrin was 1.2 µM. The final concentration of
ATP was 0.5 µM (Km app ) 0.6 ( 0.1 µM) and the final
concentration of Lck (a GST-kinase domain fusion (AA 225–509))
was 250 pM. Buffer conditions were as follows: 50 mM HEPES,
pH ) 7.5, 50 mM NaCl, 20 mM MgCl, 5 mM MnCl, 2 mM DTT,
0.05% BSA. The assay was quenched and stopped with 160 µL of
detection reagent. Detection reagents were as follows: buffer was
made of 50 mM Tris, pH ) 7.5, 100 mM NaCl, 3 mM EDTA,
0.05% BSA, 0.1% Tween20. Prior to reading, StreptaVidin allo-
phycocyanin (SA-APC) was added at a final concentration in the
assay of 0.0004 mg/mL, along with europilated antiphosphotyrosine
Ab (Eu-anti-PY) at a final concentration of 0.025 nM. The assay
plate was read in a Discovery fluorescence plate reader with
excitation at 320 nm and emission at 615 and 655 nm.
Assays for other kinases were done in a similar way as described
above, varying the concentrations of enzyme, peptide substrate, and
ATP added to the reaction, depending on the specific activity of
the kinase and measured Km’s for the substrates.
Human Mixed Lymphocyte Reaction (huMLR). This assay
tests the potency of T-cell activation inhibitors in an in vitro model
of allogeneic T-cell stimulation. Human peripheral blood lympho-
cytes (hPBL; 2 × 105/well) were incubated with mitomycin
C-treated B lymphoblastoid cells (JY cell line (ATCC, Rockville,
MD); 1 × 105/well) as allogeneic stimulators in the presence or
absence of dilutions of potential inhibitor compound in 96-well
round-bottom tissue culture plates. These cultures were incubated
at 37 °C in 5% CO2 for 6 days total. The proliferative response of
the hPBL was measured by 3H-thymidine incorporation overnight
between days 5 and 6 after initiation of culture. Cells were harvested
3
onto glass fiber filters and H-thymidine incorporation into DNA
was analyzed by liquid scintillation counter.
Jurkat Proliferation/Survival Assay. This assay tests the
general antiproliferative/cytotoxic effect of compounds on the Jurkat
human T-cell line (ATCC, Rockville, MD). Jurkat cells (1 × 105/
well) were plated in 96-well flat-bottom tissue culture plates with
or without compound dilutions and cultured for 72 h at 37 °C in
5% CO2. Viable cell number was determined during the last 4 h of
culture by adding 10 µL/well WST-8 dye (Alexis; San Diego, CA).
WST-8 dye conversion relied on active mitochondrial electron
transport for reduction of the tetrazolium dye. The dye conversion
was read by OD at 450–600 nM.
Anti-CD3/CD28-Induced T-Cell IL-2 Secretion and Pro-
liferation Assay. This assay tests the potency of T-cell receptor
(TCR; CD3) and CD28 signaling pathway inhibitors in human
T-cells. T-cells were purified from human peripheral blood lym-
phocytes (hPBL) and preincubated with or without compound prior
to stimulation with a combination of an anti-CD3 and an anti-CD28
antibody in 96-well tissue culture plates (1 × 105T cells/well). Cells
were cultured for ∼20 h at 37 °C in 5% CO2, then secreted IL-2 in
the supernatants was quantified by cytokine ELISA (Pierce/
Endogen, St. Louis, MO). The cells remaining in the wells were
then pulsed with 3H-thymidine overnight to assess the T-cell
proliferative response. Cells were harvested onto glass fiber filters
and 3H-thymidine incorporation into DNA was analyzed by liquid
scintillation counter. For comparison purposes, phorbol myristic
Adjuvant-Induced Arthritis in Rats. Arthritis was induced on
day 0 by injection of Mycobacterium tuberculosis H37Ra suspended
in paraffin oil (0.5 mg/rat) into the base of the tail of male Lewis
rats (∼250 g, obtained from CRL, Andover, MA). The compound
or vehicle (2% hydroxypropyl methylcellulose with 1% Tween-
80) was administered orally, once daily from day 9 to day 17. Paw
volume was measured daily from day 9 through day 18. Values
represent the mean ( standard error for N ) 8 animals per group.
*P < 0.05 versus vehicle control by Mann–Whitney U-test.
Collagen-Induced Arthritis in Rats. Arthritis was induced by
intradermal injection of Porcine type II collagen emulsified in IFA
to female Lewis rats. Animals were assigned to treatment group at
disease onset (study day 0), which occurred 10–12 days following
immunization. The compound or vehicle (2% hydroxypropyl
methylcellulose with 1% Tween-80) was administered orally, once

Structure-Based Design of Lck Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1647
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) 8 animals per group. *P < 0.05 versus vehicle control by
Mann–Whitney U-test.
SIF Solubility Assay. The solubility media was fasted state
simulated intestinal fluid (SIF), pH 6.8, containing 5 mM sodium
taurochol, 1.5 mM lecithin, 2.9 mM KH2PO4, and 0.22 M KCl.
The Symyx solubility system (Santa Clara, CA) consisted of a liquid
handling robot and an Agilent 1100 HPLC. An experiment template
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calculated and dispensed into the 4 mL vial for each compound by
the liquid handling robot to obtain a stock concentration of 500
µg/mL. From each of the 24 compound stock solutions, volumes
of 200, 250, 250, and 250 µL were robotically transferred to an
array of four 1 mL glass vials on a 96-well plate: one vial for the
calibration standard and the other three vials for solubility samples.
After evaporation of the stock solvent using a centrifugal evaporator,
500 µL of the solubility media was added to the corresponding
sample vials on the 96-well plate for each compound to give a
calculated concentration of 250 µg/mL. A stir bar was added to
each vial using a 96-well stir bar dispenser, and the vials were sealed
with a 96-well cap mat. The samples were equilibrated by stirring
at 100 rpm for 24 h and subsequently left standing unstirred for an
additional 24–48 h at room temperature. After equilibration, 500
µL of 50/50 CH3OH/DME was added to the corresponding standard
vials on the same plate for each compound to give a calibration
concentration of 200 µg/mL. Immediately before solubility mea-
surement by HPLC, the sealed 96-well plate was centrifuged at
1650 rpm for 10 min using a plate centrifuge to allow separation
of the compound supernatant from undissolved solid. The solubility
samples and the calibration standards in 1 mL vials on a 96-well
plate were injected directly onto the HPLC column after centrifug-
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for retention and resolution of both polar and nonpolar compounds.
The injection volume was 2 µL, with a detection wavelength of 220
nm. For each compound, its solubility was quantified through individual
standard peak area calibration at a concentration equal to the solubility
screening criterion (i.e., 200 µg/mL).
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DiMauro, E. F.; Newcomb, J.; Nunes, J. J.; Bemis, J. E.; Boucher,
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D. C.; Schneider, S.; Turci, S. M.; White, R. D.; Zhu, X. Discovery
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J.; Nunes, J. J.; Bemis, J. E.; Boucher, C.; Chai, L.; Chaffee, S. C.;
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Henkle, B.; Hsieh, F.; Huang, X.; Kim, J. L.; Lee, J. H.; Martin, M. W.;
McGowan, D. C.; Metz, D.; Mohn, D.; Morgenstern, K. A.; Oliveira-
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Acknowledgment. We thank our colleagues Alan Cheng for
the model of compound 3 in Lck; Matthew Plant for the p38R
kinase assay data; Isaac Marx for the resynthesis of 1 and 19a;
and Maggie Reed for the SIF solubility assay.
Supporting Information Available: Elemental analysis and
HPLC purity data for the compounds synthesized and data collection
and refinement statistics for the X-ray structures of compounds 1
and 25 bound to Lck. This material is available free of charge via
the Internet at http://pubs.acs.org.
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