Structure-activity relationship study of EphB3 receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.09.010

A structure-activity relationship study for a 2-chloroanilide derivative of pyrazolo[1,5-a]pyridine revealed that increased EphB3 kinase inhibitory activity could be accomplished by retaining the 2-chloroanilide and introducing a phenyl or small electron donating substituents to the 5-position of the pyrazolo[1,5-a]pyridine. In addition, replacement of the pyrazolo[1,5-a]pyridine with imidazo[1,2-a]pyridine was well tolerated and resulted in enhanced mouse liver microsome stability. The structure-activity relationship for EphB3 inhibition of both heterocyclic series was similar. Kinase inhibitory activity was also demonstrated for representative analogs in cell culture. An analog (32, LDN-211904) was also profiled for inhibitory activity against a panel of 288 kinases and found to be quite selective for tyrosine kinases. Overall, these studies provide useful molecular probes for examining the in vitro, cellular and potentially in vivo kinase-dependent function of EphB3 receptor.

Full text of DOI:10.1016/j.bmcl.2009.09.010

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6122–6126
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of EphB3 receptor tyrosine
kinase inhibitors
Lixin Qiao a, Sungwoon Choi a, April Case a, Thomas G. Gainer a,b, Kathleen Seyb a, Marcie A. Glicksman a,
Donald C. Lo b, Ross L. Stein a, Gregory D. Cuny a,
*
a Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women’s Hospital and Harvard Medical School,
65 Landsdowne Street, Cambridge, MA 02139, USA
b Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, 4321 Medical Park Drive, Suite 200, Durham, NC 27704, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A
structure–activity relationship study for a 2-chloroanilide derivative of pyrazolo[1,5-a]pyridine
Received 3 August 2009
Revised 1 September 2009
Accepted 4 September 2009
Available online 9 September 2009
revealed that increased EphB3 kinase inhibitory activity could be accomplished by retaining the 2-chlo-
roanilide and introducing a phenyl or small electron donating substituents to the 5-position of the
pyrazolo[1,5-a]pyridine. In addition, replacement of the pyrazolo[1,5-a]pyridine with imidazo[1,2-a]pyr-
idine was well tolerated and resulted in enhanced mouse liver microsome stability. The structure–activ-
ity relationship for EphB3 inhibition of both heterocyclic series was similar. Kinase inhibitory activity was
also demonstrated for representative analogs in cell culture. An analog (32, LDN-211904) was also
profiled for inhibitory activity against a panel of 288 kinases and found to be quite selective for tyrosine
kinases. Overall, these studies provide useful molecular probes for examining the in vitro, cellular and
potentially in vivo kinase-dependent function of EphB3 receptor.
Keywords:
Structure–activity relationship
EphB3
Kinase
Inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
Microsomal stability
Erythropoietin-producing hepatocellular carcinoma (Eph) recep-
tors are highly conserved transmembrane proteins composed of
multiple domains that participate in an array of complex cell signal-
ing pathways. Sixteen Eph receptors have been identified in verte-
brates. They can be divided into two major classes (EphA and
EphB) based on sequence similarity in the extracellular domain
and binding characteristics. Mammals, including humans, have 14
Eph receptors (EphA1–EphA8, EphA10, EphB1–EphB4 and EphB6).1
The Eph receptors interact with cell surface ligands called Eph
receptor interacting proteins (ephrins).2 Currently, nine ephrins
are known and are divided into two major classes (ephrin A1–6
and ephrin B1–3). Humans have all but ephrin A6. Following bind-
ing of the Eph receptors to the ephrin ligands, which requires cell–
cell interactions, propagation of signaling occurs bi-directionally
into both the Eph receptor and the ephrin presenting cells.3 The
signaling events resulting from these interactions are important
in both neural development4 and during adulthood. For example,
the Eph receptors together with ephrins participate in axon guid-
ance by providing repulsive cues during axonal neurogenesis.
The EphB3 receptor subtype is expressed during embryonic
development and in discrete areas of the adult brain, including
the cerebellum and hippocampus. It co-localizes to brain regions
with high levels of ephrin B ligand expression.5 EphB3 receptor
expression also increases following central nervous system injury.
However, it remains unclear if EphB3 is inhibitory to axonal regen-
eration or beneficial for axonal repair. For example, following adult
optic nerve injury, EphB3 receptor appears and coincides with ret-
inal ganglion cell axon sprouting and remodeling.6 However, after
spinal cord injury EphB3 expression increases and appears to con-
tribute to restricted axonal regeneration and sprouting.7 Increased
EphB3 receptor expression has also been documented in pancreatic
cancer cell lines,8 squamous cell carcinoma,9a and rhabdomyo-
sarcoma.9b
In addition to ligand binding domains, the Eph receptors have
an intracellular tyrosine kinase domain, although EphA10 and
EphB6 lack essential amino acid residues to enable catalysis. The
Eph receptor’s kinase activity is required for some, but not all, of
the signal transduction pathways involving Eph receptors.10
Engagement of the ephrin ligands with the Eph receptors ini-
tially results in receptor dimerization followed by autophosphory-
lation of tyrosine residues in the juxtamembrane region of the
receptor, which is located between the transmembrane and the ki-
nase domains. These phosphorylation events result in kinase acti-
vation by dissociation of the juxtamembrane segment from the
kinase domain.11 Once fully active, the kinase domain then can
bind and phosphorylate intracellular adaptor molecules perpetuat-
ing signaling.
Ligands that target different binding components of Eph recep-
tors could serve as useful molecular probes to help elucidate the
* Corresponding author. Tel.: +1 617 768 8640; fax: +1 617 768 8606.
E-mail address: gcuny@rics.bwh.harvard.edu (G.D. Cuny).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.010

L. Qiao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6122–6126
6123
X
N
Y
CO2Me
X
a
R
Cl
b
Y
Y
Z
R
Z
X
+
R
HN
N
N
N
NH2
O
N
_
MesitylSO3
8
5
X = CH or N
Y = CH or N
Z = CH or N
2
10
9
N
1
Cl
CO2H
Figure 1. EphB3 inhibitor identified by HTS. Also shown is the numbering system
for pyrazolo[1,5-a]pyridines.
c
d, e
Y
Z
HN
N
X
O
R
N
Y
Z
N
X
R
11
N
cellular biology and in vivo physiology of Eph receptors.12 These li-
gands could also be used to selectively modulate Eph receptor’s ki-
nase-dependent and independent functions.13 Utilizing a recently
developed high throughput screen (HTS) for EphB3 kinase activity,
the pyrazolo[1,5-a]pyridine derivative 1 (Fig. 1)14 was discovered
12
Scheme 3. Method C: (a) mesityl SO3NH2, DCM, 0 °C to rt, 30 min, 81%; (b)
HC„CCO2Me, K2CO3, DMF, 50 °C, 72 h, 14%; (c) LiOH, MeOH/H2O, rt, 2 h, then 1 N
HCl, 85–90%; (d) SOCl2, 80 °C, 1 h; (e) 2-ClPhNH2, pyridine, rt, 16 h.
as a moderately potent inhibitor (IC50 ꢀ1
lM). Herein, we describe
the results of a structure–activity relationship study to optimize
EphB3 kinase inhibition and to increase mouse liver microsome
stability. In addition, kinase inhibitory activity is also demon-
strated in cell culture and a select analog is profiled against a broad
panel of kinases.
Heterocycles 8 were converted to the N-amino derivatives 9 with
mesityl SO3NH2.16 Cycloaddition with methyl propiolate in the
presence of potassium carbonate gave 10 in low yield.17 Hydrolysis
of the esters yielded 11. Conversion of the carboxylic acids to the
corresponding acyl chlorides with thionyl chloride followed by
treatment with 2-chloroaniline in pyridine gave 12.
The synthesis of many pyrazolo[1,5-a]pyridine derivatives was
accomplished according to Scheme 1 (Method A). Pyrazolo[1,5-
a]pyridine-3-carboxylic acid, 2, was converted to the correspond-
ing acyl chloride and then treated with amines to give 3. Deriva-
tives incorporating other heterocycles in place of the
pyrazolo[1,5-a]pyridine were prepared in a similar manner, unless
otherwise noted. The amide could be reduced with LiAlH4 to give
amine 4.
Carboxylic acid 2 was also allowed to react with (PhO)2P(O)N3
to generated the corresponding acyl azide, which upon heating in
the presence of benzyl alcohol underwent a Curtius rearrangement
followed by alcohol addition to the intermediate isocyanate to pro-
duce 5 (Scheme 2, Method B).15 Deprotection of the benzyl carba-
mate in the presence of hydrogen (1 atm) and 10% Pd/C yielded
amine 6, which upon treatment of 2-chlorobenzoyl chloride gave 7.
The synthesis of substituted pyrazolo[1,5-a]pyridine and deriv-
atives that incorporate additional nitrogen atoms into the pyrazol-
The synthesis of 5-amino substituted pyrazolo[1,5-a]pyridine
derivatives is outlined in Scheme 4 (Method D). 4-Chloropyridini-
um hydrochloride, 13, was treated with an amine to generate 14,
via a nucleophilic aromatic substitution. Conversion of 14 to the
N-aminopyridine 15 with 2,4-(NO2)2PhONH2 followed by cycload-
dition with N-(2-chlorophenyl)-2-propynamide18 produced 16.
The synthesis of imidazo[1,2-a]pyridine derivatives was accom-
plished according to Scheme 5 (Method E). 2-Amino-5-bromopyr-
idine, 17, was coupled with phenyl boronic acid to give 18. 5-
Bromo-2-nitropyridine, 19, was treated with piperidine to give
20, which was subsequently hydrogenated in the presence of 10%
Pd/C to yield 21. The 2-aminopyridines 18 or 21 were heated at re-
flux in toluene with N,N-dimethylformamide dimethyl acetal
(DMF-DMA) and then treated with 2-bromo-N-arylacetamides
(prepared by coupling anilines with 2-bromoacetyl chloride) to
give 22.19 b-Ketoamide 23 was chlorinated with sulfuryl chloride
yielding 24, which was used without purification. Heating 24 in
the presence of 17 and sodium bicarbonate gave the 2-methyl imi-
dazo[1,2-a]pyridine derivative 25.
o[1,5-a]pyridine is illustrated in Scheme
3
(Method C).
R1
N
R1
R2
N
CO2H
R2
O
c
a, b
R1
R2
N
R2
R1
N
Cl
N
N
N
N
N
N
N
a
b
2
3
4
N
• HCl
+
N
Scheme 1. Method A: Reagents and conditions: (a) (C@O)2Cl2, DCM, cat. DMF, 0 °C
NH2
to rt, 2 h; (b) R1R2NH, DCM, DIPEA, rt, 18 h; (c) LiAlH4, THF,
D, 2 h.
2,4-(NO2)2PhO_
13
14
15
Cl
Bn
O
Cl
c
NH2
HN
N
R2
N
O
O
O
d
NH
NH
a, b
c
R1
2
N
N
N
N
N
N
N
5
6
7
16
Scheme 2. Method B: (a) (PhO)2P(O)N3, THF, DIPEA, rt, 16 h; (b) BnOH,
D
, 12 h, 80%
Scheme 4. Method D: (a) HNR1R2, H2O, MW, 130 °C, 30 min, 90%; (b) 2,4-
for two steps; (c) H2 (1 atm), 10% Pd/C, MeOH/EtOAc (1:1), 45 min; (d) 2-Cl-
PhC(O)Cl, DCM, DIPEA, rt, 16 h, 36% over two steps.
(NO2)2PhONH2, CH3CN,
72 h.
D, 16 h, 81%; (c) HC„CC(O)NH-2-ClPh, K2CO3, DMF, 50 °C,

6124
L. Qiao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6122–6126
Table 1
IC50 determinations for inhibiting EphB3 phosphorylation of BTK-peptide
R3
R2
R1
d
N
HN
N
O
Y
R1
R2
X
4
17 R1 = NH2, R2 = Br
18 R1 = NH2, R2 = Ph
19 R1 = NO2, R2 = Br
N
R2
N
a
b
N
N
7
22
Cmpd
X
Y
R1
R2
Method
IC50 (lM)
20 R1 = NO2, R2
21 R1 = NH2, R2
=
=
c
1
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NMe
NH
C@O
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
2-Cl-Ph
Ph
2-F-Ph
3-Cl-Ph
4-Cl-Ph
2,3-Cl2-Ph
2-OMe-Ph
2-CF3-Ph
2-CN-Ph
2-MeSO2Ph
2-Cl-Ph
CH2-2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
—
A
A
A
A
A
A
A
A
A
A
A
B
A
C
C
C
C
C
C
C
C
C
C
D
D
D
C
1.0
NA
N
34
35
36
37
38
39
40
41
42
43
44
7
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
ꢀ20
NA
Cl
Cl
NA
O
O
HN
ꢀ20
f
O
NA
N
H
Me
Br
>20
>20
NA
NA
NA
>20
>20
ꢀ15
0.26
ꢀ15
NA
N
R
Me
N
23 R = H
24 R = Cl
e
25
Scheme 5. Method E: (a) PhB(OH)2, Pd(PPh3)4, Na2CO3, CH3CN, H2O, 90 °C, 16 h,
98%; (b) piperidine, DMSO, n-Bu4NI, K2CO3, 95 °C, 16 h, 84%; (c) H2 (1 atm), 10% Pd/
C, EtOH/MeOH, rt, 16 h, 100%; (d) Me2NCH(OMe)2, toluene,
BrCH2C(O)NH-2-R-Ph (where R = Cl or OMe), MeOH,
0 °C to rt; (f) 17, toluene, NaHCO3, 120 °C, 3 d, 10%.
CH2
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
4-Me
5-Me
6-Me
7-Me
4-Cl
5-Cl
6-Cl
5-OMe
5-Ph
5-NMe2
5-Pyrr
5-Morph
5-Pip
5-OBn
D
,
6 h then
D, 16 h, 17–45%; (e) SO2Cl2,
ꢀ20
2.0
The synthesis of several other 5-substituted imidazo[1,2-a]pyr-
idine derivatives was accomplished according to the method out-
lined in Scheme 6 (Method F). A Pd-mediated coupling of boronic
ester 26 with 17 followed by hydrogenation of the alkene yielded
27. Amine 17 was also converted to intermediate 28,20 which
was not isolated, but subjected to halogen metal exchange with
n-BuLi followed by addition of N-Boc-4-piperidone to give 29 after
an acidic work-up. Both 27 and 29 were converted to 30 and 31,
respectively, utilizing the procedure described in Method E.19
Finally, removal of the carbamate protecting groups from 30 and
31 with 30% TFA in dichloromethane produced amines 32 and
33, respectively.
>20
0.19
0.066
0.077
0.20
0.24
0.063
ꢀ20
NA: not active at 20
piperidinyl.
lM; Pyrr = N-pyrrolidinyl; Morph = N-morpholinyl; Pip = N-
the BTK-peptide in the presence of varying test compound concen-
trations. Progress curves for production of phospho-BTK were
linear for at least 30 min, allowing the calculation of observed
velocities. IC50 values were then determined using a two parameter
fit.14
Evaluation of EphB3 kinase inhibitory activity for the various
compounds was accessed by measuring 33P incorporation into
Boc
The 2-chloro substituent on the aniline ring was necessary for
kinase inhibitory activity (Table 1). Removal of this group, replac-
ing it with other electron withdrawing or donating substituents, or
transposing it to the 3- or 4-positions were all detrimental. In addi-
tion, methylation of the amide (43), insertion of a methylene to
give a benzylamine derivative (44), inverting the amide functional
group (7) or reduction of the amide to an amine (45) all resulted in
diminished EphB3 kinase inhibitory activity.
Interestingly, addition of a methyl substituent to the 4-, 6-, or 7-
positions of the pyrazolo[1,5-a]pyridine was also detrimental.
However, placement of the methyl substituent in the 5-position
(47) resulted in increased activity. Similarly, introduction of a chlo-
rine to the 5-position (51) was also tolerated, but not at the 4- or 6-
positions. Based on these results, other substituents were exam-
ined at the 5-position of the pyrazolo[1,5-a]pyridine. Introduction
of a phenyl (54), dimethylamino (55) or N-piperidinyl (58) further
increased activity resulting in IC50 values <100 nM. However, a lar-
ger benzyloxy (59) substituent was not tolerated at the 5-position.
Various replacements of the pyrazolo[1,5-a]pyridine were next
examined (Table 2). Most of the heterocycles, including indoles (60
and 61), a quinoline (62), a 1,2-benzisoxazole (63), an indazole (64)
and pyrazolo[1,5-a]pyridines incorporating an additional nitrogen
atom into the heterocycle (65–68) did not inhibit EphB3 kinase
N
Boc
N
a, b
O
Me
Me
B
O
Me
Me
N
NH2
27
26
Br
Boc
N
OH
Me
d
c
Me
Si
17
N
N
Me Si
Me
N
NH2
29
28
Cl
30 R1 = Boc, R2 = H
R1
HN
N
e
31 R1 = Boc, R2 = OH
32 R1 = H, R2 = H
27 or 29
R2
N
O
f
33 R1 = H, R2 = OH
N
Scheme 6. Method F: (a) 17, Pd(PPh3)4, Na2CO3, CH3CN, H2O, 90 °C, 6 h; (b) 10% Pd/
C, H2 (1 atm), EtOH, 16 h, 67% for two steps; (c) n-BuLi (2 equiv), (Me2SiClCH2)2,
THF, À78 °C, 1 h; (d) n-BuLi (1 equiv), N-Boc-4-piperidone, À78 °C to rt, 18 h, 58%;
(e) DMF-DMA, 85 °C, toluene 8 h, then BrCH2C(O)NH-2-Cl-Ph, MeOH, 85 °C, 18 h,
26–30%; (f) 30% TFA in DCM, 1 h, rt, 82%.
activity at concentrations <15 lM. However, replacement of the

L. Qiao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6122–6126
6125
Table 2
Table 4
IC50 determinations for inhibiting EphB3 phosphorylation of BTK-peptide
Mouse liver microsome stability half-life and clearance values
Compound
t1/2 (min)
CLint
261 18
1.4
51 2.1
167 4.2
361 64
26 0.80
71 2.0
(
l
L/min/mg protein)
Cl
1
5.3
348
27
8.3
3.8
53
32a
54
58
69
70
71
4
HN
O
R
Cmpd
R
Method
IC50 (lM)
20
a
60
61
62
63
64
65
66
67
68
69
3-Indolyl
N-Methyl-3indolyl
4-Quinolinyl
3-(1,2-Benzisoxazolyl)
NA
NA
NA
NA
>20
>20
NA
a
A
A
Oxylate salt.
C
3-(1H-indazolyl)
Cb
C
R1
R2
R3
R4
R5
2.5
Cb
C
ꢀ15
2.0
1.5
1.0
0.5
0.0
C
NA
Cb
0.46
a
Prepared by EDCI mediated coupling of indole-3-carboxylic acid with 2-
chloroaniline.
b
Method C, steps d and e; NA: not active at 20
l
M.
N
N
R3
=
R1
=
R2
N
=
N
N
N
N
N
N
N
D EB3 1 32 33 34 35 37 58 61 71 72
N
Figure 2. Inhibition assay of EphB3-induced autophosphorylation in HEK293 cells.
D = DMSO, EB3 = ephrinB3, [compound] = 10
salt.
R4
=
R5
=
lM, N = 3. Note: 32 was the oxylate
N
N
N
pyrazolo[1,5-a]pyridine with an imidazo[1,2-a]pyridine (69) re-
tained activity with an IC50 = 0.46 M.
itory activity was eroded with further introduction of a hydroxyl
group onto the 4-piperidinyl ring (33).
l
Several of the SAR findings established for the pyrazolo[1,5-
a]pyridine derivatives were next examined for the imidazo[1,2-
a]pyridine series (Table 3). Introduction of substituents, such as
bromo (25), phenyl (70) and N-piperidinyl (71), at the 5-position
again resulted in a significant increase in EphB3 inhibitory activity
with IC50 values <100 nM, whereas replacing the 2-chloroanilide
with a 2-methoxyanilide (72) or introduction of a methyl substitu-
ent at the 2-position of the imidazo[1,2-a]pyridine (73) abolished
inhibitory activity. The 4-piperidinyl analog (32) retained signifi-
cant EphB3 inhibitory activity, while also offering greater aqueous
solubility due to the presence of a basic amine. However, the inhib-
Next, several derivatives were assessed for in vitro metabolic
stability in pooled mouse liver microsomes.14,21 The results of
these studies are shown in Table 4. Both pyrazolo[1,5-a]pyridine
and imidazo[1,2-a]pyridine analogs either without substitution
or with an amino group attached through a N–C bond at the 5-po-
sition of the heterocycle (1, 58, 69, and 71) demonstrated poor sta-
bility (t1/2 6 20 min). However, introduction of
a substituent
through a C–C bond at the 5-position (54 and 70) resulted in in-
creased stability, with 32 demonstrating the best stability with a
t1/2 of 348 min and a CLint of 4 lL/min/mg protein.
A subset of analogs was also evaluated for inhibition of EphB3
receptor autophosphorylation in HEK293 cells following stimula-
tion with ephrinB3 (Fig. 2).14 Several compounds (34, 35, 37, 61,
and 72) that were inactive or weakly active in the in vitro biochem-
ical kinase assay were found to be inactive or weakly active in this
cell-based assay. In contrast, derivatives (1, 32, 33, 58, and 71) that
were active in the biochemical assay again demonstrated potent
activity in cells.
Table 3
IC50 determinations for inhibiting EphB3 phosphorylation of BTK-peptide
R1
HN
O
R3
Finally, 32 was profiled for functional inhibitory activity against
N
R2
a panel of 288 kinases at 5 l
M.22 The results demonstrated that
N
this compound was quite selective for tyrosine kinases (Table S1
Cmpd
R1
R2
R3
Method
IC50 (lM)
and Fig. S1).14 The only noted exceptions were the three serine/
threonine kinases p38a, p38b, and Qik. In addition, the compound
25
70
71
72
73
32b
33
Cl
Cl
Cl
OMe
Cl
H
H
H
H
Me
H
H
Br
E
E
E
E
E
F
F
0.173
Ph
Pip
Ph
Ph
R4
ꢀ0.060a
0.087
>20
only showed moderate selectivity among the tyrosine kinases and
little selectivity verses other EphA and EphB subtypes, except for
EphA6 and EphA7.
>20
0.079
0.228
In conclusion, a structure–activity relationship study of the pyr-
azolo[1,5-a]pyridine 1, identified as an EphB3 kinase inhibitor uti-
lizing a recently developed HTS assay, revealed that increased
inhibitory activity could be accomplished by retaining the 2-chlo-
roanilide and introducing a phenyl or small electron donating sub-
stituent to the 5-position of the pyrazolo[1,5-a]pyridine. In
addition, replacement of the pyrazolo[1,5-a]pyridine with an imi-
dazo[1,2-a]pyridine was well tolerated and the resulting struc-
Cl
Cl
R5
Pip = N-piperidinyl.
a
An accurate IC50 value was difficult to obtain due to poor compound solubility
at higher concentrations.
b
Oxylate salt.
OH
R4
=
R5
=
HN
HN

6126
L. Qiao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6122–6126
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However, the imidazo[1,2-a]pyridines offered enhanced in vitro
metabolic stability as assessed in mouse liver microsomes. In par-
ticular, 32 (LDN-211904) was a potent EphB3 inhibitor, displaying
excellent liver microsome stability and had enhanced aqueous sol-
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EphB3 inhibitory activity was also demonstrated for representative
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activity against a panel of kinases and found to be quite selective
for tyrosine kinases. These studies provide useful molecular probes
for examining the in vitro and cellular kinase-dependent function
of EphB3. Continued optimization of this compound series to fur-
ther increase selectivity could afford additional analogs useful for
exploring the in vivo pharmacology of EphB3 kinase inhibition in
various animal disease models where EphB3 receptor expression
occurs, either endogenously or as a result of injury or disease.
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Acknowledgments
The authors thank the Harvard NeuroDiscovery Center for
financial support. This work was also supported in part by an
NIH grant (U24 NS049339). We also would like to thank Dr.
Bing-Cheng Wang (Case Western Reserve University School of
Medicine, Cleveland, OH) for the HEK293 cells expressing wild-
type or kinase-inactive EphB3.
14. See supplementary data for details.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.09.010.
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