Discovery of pyrimidine benzimidazoles as Lck inhibitors: Part I

Article abstract of DOI:10.1016/j.bmcl.2008.08.104

A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src kinase family. Highly efficient parallel syntheses were devised to prepare analogues for SAR studies. A number of these 4-amino-6-benzimidazole-pyrimidines exhibited single-digit nanomolar IC₅₀s against Lck in biochemical and cellular assays. These 4-amino-6-benzimidazole-pyrimidines represent a new class of tyrosine kinase inhibitors.

Full text of DOI:10.1016/j.bmcl.2008.08.104

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5618–5621
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of pyrimidine benzimidazoles as Lck inhibitors: Part I
*
Guobao Zhang , Pingda Ren, Nathanael S. Gray, Taebo Sim, Yi Liu, Xia Wang, Jianwei Che, Shin-Shay Tian,
Mark L. Sandberg, Tracy A. Spalding, Russell Romeo, Maya Iskandar, Donald Chow, H. Martin Seidel,
,
*
Donald S. Karanewsky, Yun He
Genomics Institute of the Novartis Research Foundation (GNF), Medicinal Chemistry, 10715 John Jay Hopkins Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine
kinase (Lck), a member of the Src kinase family. Highly efficient parallel syntheses were devised to pre-
pare analogues for SAR studies. A number of these 4-amino-6-benzimidazole-pyrimidines exhibited sin-
Received 15 July 2008
Revised 22 August 2008
Accepted 27 August 2008
Available online 31 August 2008
gle-digit nanomolar IC50s against Lck in biochemical and cellular assays. These 4-amino-6-
benzimidazole-pyrimidines represent a new class of tyrosine kinase inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Lck inhibitor
Kinase inhibitor
Transplantation rejection
Autoimmune disease
Pyrimidine benzimidazoles
Lymphocyte-specific kinase (Lck) is a non-receptor protein tyro-
sine kinase in the Src-family with restricted expression in T cells
and natural killer (NK) cells.1 Lck plays a critical role in the initial
steps of T-cell receptor (TCR) signaling.2 Activation of TCR signaling
by Lck triggers a cascade of downstream signaling pathways, lead-
ing to the production of cytokines such as interleukin-2 (IL-2) and
ATP binding site of Lck. The N3 and adjacent NH of compound A
form bidentate hydrogen bonds to the backbone of Met319 in
the Lck hinge region. Phenyl ring II is positioned in the kinase spec-
ificity pocket.9 Also, based on our research result of this dihydropy-
rimido [4,5-d] pyrimidinone scaffold,10 the pyrimidine N1 of
compound A does not form any hydrogen bonds and can be re-
placed with a carbon without loss of protein kinase inhibitory
activity (data not shown). Based on this structural information,
we designed compound B, where the central ring is replaced with
a pseudo-ring constrained by an intramolecular hydrogen bond be-
tween the pyrimidine N and the benzimidazole NH group.11 The
4,6-pyrimidine substitution pattern allows retention of the biden-
tate hinge hydrogen bonds. This intramolecular hydrogen bond is
expected to position the phenyl ring II of compound B into the ki-
nase specificity pocket, as in compound A. This design strategy was
expected to allow scaffold B to mimic the binding conformation of
interferon-c.
3,4 Because of the restricted expression pattern of Lck,
selective inhibitors of Lck would be expected to exhibit improved
safety profiles over currently available immunosuppressive agents,
which possess non-lymphocyte-related toxicities. Therefore, there
have been extensive efforts to develop selective Lck inhibitors for
the treatment of autoimmune disease and organ transplant
rejection.5,6
A number of potent Lck inhibitors have been reported.7 In this
paper, we discuss the design, synthesis and preliminary struc-
ture–activity relationships (SAR) of a novel class of potent Lck
inhibitors based on 4-amino-6-benzimidazole-pyrimidines.
The design of this new class of Lck inhibitors was based on a
dihydropyrimido [4,5-d] pyrimidinone series of kinase inhibitors
originally reported by Hoffmann-La Roche8 (Fig. 1). Molecular
modeling studies on the binding mode of these dihydropyrimido
[4,5-d] pyrimidinones to Lck guided the selection of some impor-
tant features for our design (Fig. 2). Compound A binds to the
II R2
R2
II
H
N
N
3
N
N
N
I
I
H
H
N
N
N
O
N
N
N
1
R1
R1
* Corresponding authors. Tel.: +1 858 812 1614; fax: +1 858 812 1684 (G.Z.); tel.:
+86 21 3895 4910x2060; fax: +86 21 5079 0292 (Y.H.).
E-mail addresses: gzhang@gnf.org (G. Zhang), yun.he@roche.com (Y. He).
Present address: Roche R&D Center (China) Ltd, 720 Cai Lun Rd., Building 5,
Pudong, Shanghai 201203, China.
B
A
Figure 1. Rational design of pyrimidine benzimidazoles.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.104

G. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5618–5621
5619
Table 1
IC50 (lM) for inhibition of Lck kinase activity for 4a–c
Compound
Lck Lance
Ba/F3/Tel-Lck
4a
4b
4c
1.355
0.514
1.785
1.167
0.438
0.583
constitutive activation of its tyrosine kinase activity and conferred
cytokine-independent proliferation to the interleukin-3-depen-
dent Ba/F3 hematopoietic cell line.
Sixteen anilines with different substitution patterns were cho-
sen to react with compound 3. A selection of the compounds that
inhibited Lck kinase activity as measured using the Lck enzyme as-
say and Lck-dependent proliferation assay are shown in Table 1.
Most of the other compounds exhibited very weak or no Lck inhib-
itory activity and are not listed here. Analysis of the SAR revealed
that a 2,6-substitution pattern on aniline ring is important for
Lck inhibition. Although its Lck inhibitory activity is moderate,
compound 4b was the most promising compound to emerge from
this initial set of compounds and was chosen for further
optimization.
To optimize lead compound 4b, we further explored the SAR on
R1. A series of analogues 7a–i were synthesized following Scheme
2. Briefly, reacting 2-chlorobenzimidazole with 2-chloro-6-methy-
laniline under acid-catalyzed conditions gave compound 5 in
excellent yield. Compound 5 was then coupled to 4,6-dichloropy-
rimidine in the presence of NaH to provide compound 6. It is inter-
esting to note that use of DIEA as a base in this step led exclusively
to the other regioisomer as a result of the exo aniline NH of 5
attacking the chlorine on the pyrimidine. Compound 7a was ob-
tained by hydrogenation on Pd/C, and compounds 7b–i were ob-
tained by direct amination (7b–7d) with various amines or Pd-
catalyzed coupling (7e–7i) with various heteroaromatic amines.
Figure 2. Binding mode of compound 4b to Lck based on modeling studies. This
model was constructed by docking12 compound 4b into crystal structure of Lck
13
[PDB ID code: 1QPE]
based on the binding mode of structurally similar
compounds co-crystallized with c-Abl14 and c-Kit.15 Compound B overlaps with
4b very well and is shown as green and blue.
scaffold A but perhaps might allow for subtle conformational
changes that could influence kinase specificity and potency.
To simplify our exploration, we chose R1 = H, and compounds
4a–c were synthesized according to Scheme 1. First, coupling of
4,6-dichloropyrimidine and 2-chlorobenzimidazole (1) in the pres-
ence of NaH in DMF gave intermediate 2. Selective displacement of
the 4-Cl on the pyrimidine ring of 2 was readily achieved by reac-
tion with excess ammonia in isopropanol at 50 °C to give amino-
pyrimidine 3 in excellent yield. The 2-Cl on the benzimidazole ring
of 3 was rather inert to displacement by aniline under basic condi-
tions, even under Pd-catalyzed conditions. However, the reaction
was readily accomplished under acid–catalyzed conditions, giving
rise to analogues 4a–4c.
All analogues were tested for their ability to inhibit Lck enzyme
activity using an Lck Lance assay.16 Compounds that potently
inhibited Lck in the enzymatic assay were then tested to determine
their cellular activity using a cell proliferation assay in a TEL-Lck-
transformed Ba/F3 cell line.17 The TEL-Lck fusion construct in-
cludes the catalytic domain of Lck and the TEL oligomerization do-
main. TEL-induced oligomerization of TEL-Lck resulted in the
Cl
HN
HN
N
N
HN
a
b
HN
N
Cl
Cl
N
Cl
N
N
1
6
5
7f: R1
=
H
N
N
N
HN
N
c
N
Cl
R1
N
O
N
Cl
N
N
Cl
N
N
Cl
OH
7g: R1
7h: R1
=
=
N
H
HN
N
a
H2N
N
b
N
N
Cl
N
N
N
N
N
7
H
H
7a: R1=H
N
N
N
1
2
3
R1=morpholine
N
N
7b:
7c:
O
R1=NH-4-morpholino
Ar
7d: R1=NH(CH2)4NEt2
N
N
HN
7i: R1
=
H
H
H
4a: Ar = 2,4-diMe-6-Br-Ph
4b: Ar = 2-Cl-6-Me-Ph
4c: Ar = 2,4,6-triMe-Ph
N
N
7e: R1
=
H2N
N
N
N
c
N
N
N
N
O
N
O
Scheme 2. Synthesis of compounds 6–7i. Reagents and conditions: (a) 2-chloro-6-
methylaniline (1.5 equiv), CH3SO3H (2.0 equiv), N,N-dimethylimidazolinone, 90 °C,
2 h, 75%; (b) 4,6-dichloropyrimidine (2.5 equiv), NaH (1.5 equiv), DMF, 0–80 °C, 1 h,
80%; (c) i—For compound 7a, H2/Pd/C, 65%; ii—For compounds 7b–d, amine
(3.0 equiv), isopropanol, 50 °C, 1 h, 80–95%; iii—For compounds 7e–i, aniline
(1.2 equiv), Pd2dba3 (0.02 equiv), Xantphos (0.04 equiv), K3PO4 (2.0 equiv), dioxane,
90 °C, 12 h, 60–85%.
4
Scheme 1. Synthesis of compounds 4a–c. Reagents and conditions: (a). 4,6-
dichloropyrimidine (2.5 equiv), NaH (1.5 equiv), DMF, 0–25 °C, 12 h, 56%; (b). NH3
(excess), isopropanol, 50 °C, 12 h, 92%; (c). aniline (1.5 equiv), CH3SO3H (2.0 equiv),
N,N-dimethylimidazolinone, 90 °C, 2 h, 65–85%.

5620
G. Zhang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5618–5621
Table 2
Table 3
IC50
(l
M) for inhibition of Lck kinase activity for 6-7i
Kinase selectivity profiling of compound 7f and 7g (IC50 lM)
Compound
Lck Lance
Ba/F3/Tel-Lck
Compound
7f
7g
6
>2.5
2.09
>2.5
>2.5
0.789
0.014
0.031
0.025
0.005
0.119
5.704
N/A
>10
Lck Lance
0.031
0.034
0.215
0.147
1.012
5.165
0.025
0.007
0.03
0.005
0.356
2.345
7a
7b
7c
7d
7e
7f
7g
7h
7i
Ba/F3/Tel-Lck
Ba/F3/Tel-Lyn
Ba/F3/Tel-Src
Ba/F3/Tel-KDR
Ba/F3/Tel-InsR
>10
5.036
0.017
0.034
0.007
0.013
0.359
not form hydrogen bond to the hinge, and the R-group would inter-
fere with the hydrogen bond between N1 and the hinge. This also
explains why compounds 7b and 7c have even weaker inhibition
compared to compound 7a.
Based on this conformational analysis, we designed compounds
7e and 7f. Computational analysis shows conformer III has lower
energy than the other three conformers, which are also energy
The inhibition data for compounds 6–7i are shown in Table 2. It
is not surprising to see that compounds 6, 7a and 7b all lost po-
tency compared with compound 4b, because they all lack the
hydrogen-bond-donating NH group required for strong Lck hinge
binding according to our proposed binding mode. It is also interest-
ing to note that compound 6 is less potent than compound 7a. It
can be rationalized that the electron withdrawing effect of –Cl
makes its adjacent pyrimidine nitrogen less basic and therefore
weakens the hydrogen bonding of that nitrogen with the Lck hinge
region.
From the perspective of electronic effects, the amino group in 7c
and 7d should make N1 more basic than that in 4b. Both 7c and 7d
are expected to form stronger hydrogen bonds and therefore have
better inhibitory activity on Lck. However, compounds 7c and 7d
both have much weaker potency compared to compound 4b. From
the binding mode, the substitution on the amino group of the
pyrimidine is exposed to solvent and should be well-tolerated. To
rationalize these data, we performed a conformational analysis of
these compounds (Fig. 3). In order for compound 7c and 7d to form
bidentate hydrogen bonds to the Lck hinge region, they must as-
sume conformation I as shown in Figure 3. However, a relatively
larger R group would introduce a steric clash with Ha, which would
make conformation II more favorable. In conformation II, Hb can-
minima. Conformer V is significantly lower in energy (DE = 7.74 k-
cal/mol) than conformer VI. These energy favorable conformers III
and V are ideally suited for hydrogen bonding to the hinge region.
The Lck inhibitory activity of compounds 7e and 7f confirmed
our conformational analysis. Both compounds are very potent in
both enzyme and cellular assays, and the enzyme data correlate
very well with the cellular data for both compounds. Compound
7e is about twice as potent as compound 7f, suggesting that the
solubility-enhancing morpholine group is better positioned meta
relative to the NH group.
With this observation in mind, we further synthesized com-
pounds 7g–i, all of which have the water solubility-enhancing
group bearing a tertiary nitrogen meta to the NH. Also, the pyridine
ring of these compounds was changed to a pyrimidine ring, a sim-
ilar modification to what can be found in the Src-family kinase
inhibitor dasatinib. Both compounds 7g and 7h exhibit biochemi-
cal and cellular potency comparable to the most potent Lck inhib-
itors reported in the literature. Compound 7i is 25-fold less potent
than compound 7h. This is probably due to the fact that the methyl
group could have a steric clash with the hinge region of Lck.
The kinase selectivity profiles of compounds 7f and 7g are
shown in Table 3. Although both compounds are quite potent
inhibitors of other Src-family kinases such as Lyn and Src, they
exhibited good selectivity over non-Src-family kinases KDR and
InsR. Compound 7f also showed moderate selectivity over the
Src-family kinases Lyn and Src.
1
N
1
N
HN
HN
N
N
Cl
Cl
Hb
R
N
N
N
N
N
R
N
Hb
Ha
Ha
In summary, a promising new structural class of potent Lck
inhibitors was discovered through rational design. Synthesis of
these structures proceeds through an efficient scheme that is ame-
nable to rapid analogue synthesis using widely available diversity
elements. These Lck inhibitors represent a new class of tyrosine ki-
nase inhibitors that are promising candidates for further
optimization.
II
I
R2
HN
HN
N
N
N
N
Cl
Cl
Hb
N
N
N
N
N
N
N
Hb
Ha
ΔE = 1.95 kcal/mol
Ha
N
References and notes
IV
III
R2
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ΔE = 3.16 kcal/mol
ΔE = 8.95 kcal/mol
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N
2
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Hb
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Hb
ΔE = 7.74 kcal/mol
Ha
Ha
VI
V
R2
Figure 3. Conformation analysis of compounds 8a–i.

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pH 7.5, 0.05% Tween-20, 1% BSA, and the mixture was incubated at room
temperature for 90 min. Ten microliters of 22.5 mM EDTA were added to stop
the reaction followed by 45 lL of detection buffer [20 mM Tris/HCl, pH 7.5,
150 mM NaCl, 0.5% Tween-20, 0.1% BSA, 0.6 nM Lance Europium-labeled anti-
phosphotyrosine antibody, 220 nM Allophycocyanin-Streptavidin conjugate
(Perkin-Elmer, Waltham MA)]. The plates were incubated for 1 h in the dark
then read on an Analyst (Molecular Devices, Sunnyvale CA) or ViewLux
(Perkin-Elmer, Waltham MA) plate reader using the HTRF method.
17. (a) Melnick, J. S.; Janes, J.; Kim, S.; Chang, J. Y.; Sipes, D. G.; Gunderson, D.;
Jarnes, L.; Matzen, J. T.; Garcia, M. E.; Hood, T. L.; Beigi, R.; Xia, G.; Harig, R. A.;
Asatryan, H.; Yan, S. F.; Zhou, Y.; Gu, X. J.; Saadat, A.; Zhou, V.; King, F. J.; Shaw,
C. M.; Su, A. I.; Downs, R.; Gray, N. S.; Schultz, P. G.; Warmuth, M.; Caldwell, J. S.
Proc. Natl. Acad. Sci. 2006, 103, 3153; b In a typical assay, transformed Ba/F3
cells were plated in 384-well plates (25,000 cells per well) and incubated with
serial dilutions of inhibitor or DMSO for 48 h. Luciferase expression was used
as a measure of cell proliferation/survival and was evaluated with the Bright-
Glo Luciferase Assay System (Promega, Madison, WI).
16. (a) Hemmila, I. J. Biomol. Screen. 1999, 4, 303; b In a typical assay, 15
lL of assay
buffer (50 mM Tris/HCl, pH 7.5, 0.05% Tween-20, 10 M ATP, 20 mM MnCl2,
l