Optimization of imidazo[4,5- b ]pyridine-based kinase inhibitors: Identification of a dual FLT3/aurora kinase inhibitor as an orally bioavailable preclinical development candidate for the treatment of acute myeloid leukemia

Article abstract of DOI:10.1021/jm300952s

Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin- 1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K_d = 7.5 nM, Aurora-B K _d = 48 nM), FLT3 kinase (K_d = 6.2 nM), and FLT3 mutants including FLT3-ITD (K_d = 38 nM) and FLT3(D835Y) (K_d = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children.

Full text of DOI:10.1021/jm300952s

Article
pubs.acs.org/jmc
Optimization of Imidazo[4,5‑b]pyridine-Based Kinase Inhibitors:
Identification of a Dual FLT3/Aurora Kinase Inhibitor as an Orally
Bioavailable Preclinical Development Candidate for the Treatment of
Acute Myeloid Leukemia
Vassilios Bavetsias,*,† Simon Crumpler,† Chongbo Sun,† Sian Avery,† Butrus Atrash,† Amir Faisal,†
Andrew S. Moore,†,∥ Magda Kosmopoulou,‡ Nathan Brown,† Peter W. Sheldrake,† Katherine Bush,†
Alan Henley,† Gary Box,† Melanie Valenti,† Alexis de Haven Brandon,† Florence I. Raynaud,†
Paul Workman,† Suzanne A. Eccles,† Richard Bayliss,‡,⊥ Spiros Linardopoulos,†,§ and Julian Blagg*,†
†Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold
Road, Sutton, Surrey, SM2 5NG, United Kingdom
‡Division of Structural Biology, Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB,
United Kingdom
§The Breakthrough Breast Cancer Research Centre, Division of Breast Cancer Research, The Institute of Cancer Research, Fulham
Road, London SW3 6JB, United Kingdom
S
* Supporting Information
ABSTRACT: Optimization of the imidazo[4,5-b]pyridine-based
series of Aurora kinase inhibitors led to the identification of 6-
chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-
pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor
of Aurora kinases (Aurora-A Kd = 7.5 nM, Aurora-B Kd = 48 nM),
FLT3 kinase (Kd = 6.2 nM), and FLT3 mutants including FLT3-
ITD (Kd = 38 nM) and FLT3(D835Y) (Kd = 14 nM). FLT3-ITD
causes constitutive FLT3 kinase activation and is detected in 20−
35% of adults and 15% of children with acute myeloid leukemia
(AML), conferring a poor prognosis in both age groups. In an in
vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4−11) following oral
administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase
inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical
development candidate for the treatment of human malignancies, in particular AML, in adults and children.
AML.19,20 Some inhibitors of Aurora kinases were reported
INTRODUCTION
■
to inhibit FLT3,18 and high expression of Aurora kinases has
Aurora kinases, a family of three serine-threonine kinases
been demonstrated in leukemia cell lines and patient
designated A, B, and C, play key and distinct roles in different
cohorts.21−24 A number of structurally diverse inhibitors of
stages of mitosis.1−3 At the early stages of mitosis, Aurora-A
Aurora kinase activity have been reported,18,25,26 including 1
forms a complex with TPX2 (targeting protein for Xklp2) that
[VX-680 (MK-0457)],27 2 (AZD1152),28 3 (PHA-
regulates centrosome maturation and mitotic spindle assem-
739358),29,30 and 4 (AMG 900)31 (Figure 1).
bly.4,5 Aurora-B forms complexes with the inner centromere
We have previously reported the discovery of 6 (Figure 2), a
novel, potent, and orally bioavailable inhibitor of Aurora kinases
protein (INCENP), survivin and borealin, thereby regulating
chromosome condensation, chromosome alignment, mitotic
checkpoint, and cytokinesis.6−9 Overexpression of Aurora-A
that inhibits growth of the SW620 human colon carcinoma
xenograft with concomitant biomarker modulation consistent
and -B has been reported in a wide range of human
with target engagement.32 However, the preclinical develop-
malignancies, including breast, colorectal, ovarian, glioma,
thyroid carcinoma, and seminoma.10−16 The function of
ment of this compound was limited by a narrow safety margin
against hERG (IC50 = 3.0 μM)32 and low human liver
Aurora-C during mitosis is less well understood. However,
high expression of Aurora-C has been reported in the testis.17,18
microsomal stability (86% metabolized after a 30 min
incubation, unpublished data). Herein, we report the evolution
In recent years, small-molecule targeting of the Aurora
kinases has become a common strategy for the discovery of new
molecularly targeted cancer therapeutics for the treatment of
solid tumors and hematological malignancies, including
Received: July 4, 2012
© XXXX American Chemical Society
A
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Figure 1. Inhibitors of Aurora kinases.
a
Scheme 2
a
Reagents and conditions: (a) NCS, CH3CN, 80 °C, 1 h; (b) NBS,
CH3CN, 80 °C, 1 h.
The benzaldehyde derivatives 9b−e were prepared as shown
in Scheme 3. Acetylation of 4-(piperazin-1-yl)benzaldehyde
afforded 9b, whereas 9c and 9d were obtained from 15 via a
Pd-catalyzed amination reaction34 followed by acid-mediated
acetal hydrolysis. Nucleophilic displacement of the F of
aldehyde 18 with (S)-1,2-dimethylpiperazine afforded the
corresponding 1,2-dimethylpiperazine derivative 9e.
Figure 2. Imidazo[4,5-b]pyridine-based Inhibitors of Aurora kinases.
of the medicinal chemistry program to identify orally
bioavailable dual FLT3/Aurora kinase inhibitors with higher
metabolic stability and wider therapeutic index against hERG
suitable for preclinical evaluation.
Access to imidazo[4,5-b]pyridine derivatives 20a−f (Table
2) was also gained by reacting 7 (Scheme 1) with the
appropriately substituted benzaldehyde in the presence of
Na2S2O4.32,33 3-Fluoro-4-(4-methylpiperazin-1-yl)-
benzaldehyde (19a) and 2-fluoro-4-(4-methylpiperazin-1-yl)-
benzaldehyde (19b) were prepared by reacting 1-methylpiper-
azine with 3,4-difluorobenzaldehyde and 2,4-difluorobenzalde-
hyde, respectively. 3-(4-Methylpiperazin-1-yl)benzaldehyde
(19f) was obtained from 1-bromo-3-(diethoxymethyl)benzene
via a Pd-catalyzed amination reaction followed by acid-
mediated acetal hydrolysis34 in a manner similar to that
described for synthesis of 9c and 9d (Scheme 3). Compounds
21a−i and 22a−e respectively presented in Tables 3 and 4
were readily accessed by reaction of 7 or 8 with the requisite
commercially available aldehyde in the presence of
Na2S2O4.32,33
CHEMISTRY
■
Compounds 10a−e shown in Table 1 were prepared from 7 or
832 by treatment with the appropriate benzaldehyde 9a−e in
the presence of Na2S2O4 (Scheme 1).32,33
a
Scheme 1
The synthesis of 1,3-dimethylpyrazole derivatives 27a−g
(Table 5) is shown in Scheme 4. The key 2-amino-3-
nitropyridine intermediates 26a−g, were obtained by nucleo-
philic displacement of the C-4 chloride of 12 with the
appropriate piperazine derivative. Boc-piperazines 24f and 24g
were prepared by a reductive alkylation of 1-Boc-piperazine
(23) with 5-formylpyrimidine and pyrazine-2-carbaldehyde,
respectively. The Boc-piperazine derivatives 24b and 24d were
obtained by a substitution reaction of 1-Boc-piperazine (23)
with 3-(chloromethyl)-1,2,4-oxadiazole and 3-(chloromethyl)-
1-methyl-1H-1,2,4-triazole, respectively (Scheme 4).
a
Reagents and conditions: (a) EtOH, 1 M aq Na2S2O4, heating.
2-Amino-4,5-dichloro-3-nitropyridine (12) and 2-amino-5-
bromo-4-chloro-3-nitropyridine (13), precursors for 7 and 8,
were synthesized as previously described32 or by halogenation
of 2-amino-4-chloro-3-nitropyridine (11) as shown in Scheme
2.
B
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a
Scheme 3
a
Reagents and conditions: (a) TFA, CH2Cl2, rt, 1.5 h; (b) CH2Cl2, acetyl chloride, iPr2NEt, 0 °C to rt; (c) (i) toluene, Pd2(dba)3, BINAP, NaOtBu,
100 °C, (ii) 1 M aq HCl; (d) MeOH/THF, 33% HCHO in water, Na(OAc)3BH, rt, 18 h; (e) (i) TFA/CH2Cl2, (ii) iPr2NEt, DMSO, 120 °C, 2 h.
analogue of 6 where R2 = p-methoxyphenyl (i.e., 6-bromo-2-(4-
a
Scheme 4
methoxyphenyl)-7-[4-(5-methyl-isoxazol-3-ylmethyl)-pipera-
zin-1-yl]-3H-imidazo[4,5-b]pyridine),32 the pKa of the piper-
azine nitrogen bearing the 5-methylisoxazol-3-yl substituent
was measured as 5.39 and the acidic pKa of the imidazopyridine
N3 proton was 9.51, consistent with a strong hydrogen bond
donor.37 The pKa of the N-Me piperazine nitrogen in 6 was
calculated as 8.50.38,39
In pharmacophore models for hERG blockade, it is reported
that a basic nitrogen in lipophilic molecules can increase hERG
inhibitory potency via π-cation stacking.40−42 As a consequence,
one of the most common strategies for designing out hERG
affinity involves the reduction of the pKa and/or the
introduction of steric bulk or shielding around the basic
a
nitrogen.41 The concomitant reduction of both pKa and clogP
Reagents and conditions: (a) for 24b,d, CH2Cl2, 3-(chloromethyl)-
1,2,4-oxadiazole or 3-(chloromethyl)-1-methyl-1H-1,2,4-triazole, Et3N,
often leads to a reduction in hERG activity.41 It has been
50 °C; for 24f,g, CH2Cl2, 5-formylpyrimidine or pyrazine-2-
suggested that a 1 log unit reduction in clogP leads to 0.8 log
carbaldehyde, NaBH(OAc)3, AcOH; (b) TFA, CH2Cl2, rt; (c)
unit reduction in hERG activity.41 In a recent study by Waring
iPrOH, heating; (d) 1,3-dimethyl-1H-pyrazole-4-carbaldehyde,
and Johnstone, the relationship between hERG and log P for
acids, bases, neutrals, and zwitterions was investigated.43 In that
study, it was reported that a higher log P is associated with an
increasing potential for hERG affinity, with basic compounds
being more likely to have hERG liabilities than neutrals.43 With
this in mind, our approaches to lower hERG affinity involved
modulation of the pKa of the N-Me piperazine nitrogen,
introduction of steric bulk around this nitrogen, and
replacement of the R2 (4-methylpiperazin-1-yl)phenyl sub-
stituent with a weakly basic or neutral five-membered
heteroaromatic moiety. The latter approach could potentially
lead to a neutral molecule of lower molecular mass and clogP
with the potential to reduce both hERG affinity and
susceptibility to oxidative metabolism.44,45
Replacement of the N-methylpiperazine moiety in 6 with a
morpholine ring (compound 10a) was beneficial in reducing
affinity for hERG, consistent with our hypothesis that hERG
affinity is driven by the presence of a piperazine basic center.
However, this change was detrimental to human liver
microsomal stability (99% of parent compound was metabo-
lized following 30 min incubation, Table 1). Tactics to
introduce steric bulk and lower the pKa of the piperazine
nitrogen by acetylation (nonbasic, compound 10b) or
introduction of a pendant methoxyethyl side chain (compound
10d, calculated pKa = 8.0638,39) did not lower hERG affinity.
The introduction of steric bulk around the N-methylpiperazine
nitrogen (compounds 10c and 10e, Table 1) resulted in
EtOH, 1 M aq Na2S2O4, 80 °C.
RESULTS AND DISCUSSION
■
Compound 6 and its 6-Cl counterpart 5 (Figure 2) served as
starting points for our investigation aimed at identifying orally
bioavailable inhibitors of Aurora kinases suitable for preclinical
evaluation. The crystal structure of 6 bound to Aurora-A32 and
the existing structure−activity relationship in this series32,35
provided us with a clear understanding of the interactions of
this class of compounds with Aurora kinases. The N4 pyridine
hydrogen bond acceptor and the N3 imidazole hydrogen bond
donor of the imidazo[4,5-b]pyridine scaffold form hydrogen
bonds to Ala213 in the hinge region of the kinase.32 The R2 (4-
methylpiperazin-1-yl)phenyl substituent points into the solvent
accessible area with the lipophilic phenyl ring residing in close
proximity to Gly216. The R3 5-methylisoxazol-3-yl group
makes contact with the Gly-rich loop, and in vitro compound
profiling and subsequent Free−Wilson analysis36 identified p-
chlorophenyl, and 5-methylisoxazol-3-yl as the R3 substituents
most frequently observed in highly active compounds. The
incorporation of a Br or Cl substituent at the C6 position
results in a significant increase in enzyme inhibitory activity
with 6-Br/Cl derivatives >8-fold more potent relative to their
unsubstituted counterparts.32,35 Selected physicochemical
properties for this class of compounds were also studied. The
measured log D7.4 for compound 6 is 2.58,37 and for the
C
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d e
,
Table 1. N-Methylpiperazine Modifications
a
b
c
Results are mean values for samples run in triplicate. From ref 32. MLM/HLM: Percentage of parent compound metabolized after a 30 min
d
incubation. For both Aurora-A IC50 and SW620 human colon cancer cell GI50 determinations, the results are mean values of two independent
determinations or mean ( SD) for n > 2 unless specified otherwise. n.d. = not determined.
e
retention of the Aurora-A, and tumor cell growth inhibitory
activity but failed to reduce hERG inhibitory potency.
Analogues 10c,e displayed affinities for hERG similar to
those observed with the parent compounds 5 and 6 (Table
1). In addition, the high susceptibility to human liver
metabolism remained a concern, with HLM stability values
for compounds 10b−e similar to those for the parent
compounds 5 and 6 (Table 1).
In a further attempt to lower the pKa of the N-Me piperazine
nitrogen in 5, a fluoro substituent was introduced on the phenyl
ring ortho or meta to the piperazine (Table 2, compounds 20a,
20b, respectively). The o-F substituted analogue 20a
(piperazine N-Me nitrogen calculated pKa = 7.9838,39) was
less potent in inhibiting hERG compared to 5; however, its
human liver microsomal stability remained low (68%
metabolized after a 30 min incubation). The m-F substituted
derivative 20b (piperazine N-Me nitrogen calculated pKa =
8.5038,39) displayed a similar affinity for hERG compared to the
parent compound 5. The m-F substituted analogue 20b was a
less potent inhibitor of Aurora-A compared to its o-regioisomer
20a, a trend consistent with previously reported SAR in this
series.35
tively) displayed similar hERG inhibitory activities to 5 (Tables
1 and 2). The moderately basic morpholino derivative 20e
exhibited low affinity for hERG, but was rapidly metabolized in
mouse and human liver microsomes. However, the introduction
of the N-methyl piperazine moiety at the m-position
(compound 20f, Table 2) resulted in a desirable outcome in
relation to both hERG inhibitory activity and MLM/HLM
metabolic stability. Compound 20f inhibited hERG with an
IC50 value of 9.50 μM (Table 2; see also general experimental)
and displayed high human liver microsomal stability (24%
metabolized after a 30 min incubation versus 89% for
compound 5 and 86% for compound 6).
Subsequently, our chemical efforts were focused on the
introduction of neutral or weakly basic R2 heteroaryl
substituents. To this end, the Aurora-A and tumor cell growth
inhibitory effect of replacing the R2 (4-methylpiperazin-1-
yl)phenyl moiety in 5 and 6 with a range of five-membered
heteroaromatic rings was studied (Table 3). With the exception
of 1-methyl-1H-imidazol-5-yl and 1-methyl-1H-pyrazol-4-yl
derivatives 21a and 21i, all analogues presented in Table 3
were considerably less potent inhibitors of Aurora-A compared
to 5 or 6 (Table 1). In particular, the incorporation of a 1-
methyl-1H-imidazol-2-yl, 2,5-dimethyloxazol-4-yl, 5-methyli-
soxazol-3-yl, and 1,2,3-thiadiazol-4-yl as an R2 substituent
(compounds 21b, 21d, 21e, 21f, respectively) resulted in a
significant drop in Aurora-A inhibitory potency. To unequiv-
The positional effect of the basic nitrogen on human
microsomal stability and hERG affinity was next investigated
(Table 2, compounds 20c−f). The m-CH2NMe2 and m-
OCH2CH2NMe2 analogues (compounds 20d, 20c, respec-
D
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b
Table 2. Basic Nitrogen Positional Effect and pKa
Modulation
Table 3. R2 Modifications
c
a
b
Results are mean values for samples run in triplicate. MLM/HLM:
Percentage of parent compound metabolized after a 30 min
c
incubation. For both Aurora-A IC50 and SW620 human colon cancer
cell GI50 determinations, the results are mean values of two
independent determinations or mean ( SD) for n > 2 unless specified
otherwise.
a
b
Results are mean values for samples run in triplicate. Results are
mean values of two independent determinations or mean ( SD) for n
> 2 unless specified otherwise.
ocally establish the binding mode of these 2-heteroaryl-based
analogues to Aurora-A, compound 21a was cocrystallized with
the catalytic domain of Aurora-A (residues 122−403). The
crystal structure of 21a bound to Aurora-A was determined to a
resolution of 2.5 Å (Figure 3, Table S1, Supporting
Information), and shows that 21a occupies the ATP-binding
site in a mode similar to that described for 6.32 As was observed
for 6, the isoxazole group of 21a interacts with the Gly-rich
loop of Aurora-A between residues 141 and 146; in addition,
the side chain of Lys143 makes an additional contact, although
the significance of this is unclear. As expected, the R2 imidazole
ring of 21a sits close to Gly216, in an equivalent position to the
phenyl ring of 6, with the methyl substituent pointing away
from the hinge to avoid steric clashes with the main chain
Ala213 and Pro214. The Aurora-A affinity observed for
compounds 21a (IC50 = 0.015 μM), 21c (IC50 = 0.068 μM),
and 21i (IC50 = 0.045 μM) is consistent with a preferred,
sterically restricted, lipophilic interaction with Ala213, which is
satisfied by the C4−H of the imidazole ring of 21a (Figure 3)
Figure 3. 2.5 Å resolution crystal structure of compound 21a bound to
Aurora-A (PDB code 4B0G). The color scheme used is nitrogen, blue;
oxygen, red; bromine, brown; carbon in Aurora-A, white; carbon in
21a, green. The final 2mFo-DFc electron density map surrounding
21a is shown as a gray wire-mesh, contoured at 1.0 σ.
but is precluded in 21d (IC50 = 2.2 μM) owing to the presence
of either a polar nitrogen atom or more sterically demanding
methyl moiety in the same vector.
E
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d
Table 4. R2-Pyrazole Modifications
a
b
Results are mean values for samples run in triplicate. MLM/HLM: Percentage of parent compound metabolized after a 30 min incubation.
Calculated log P.46 For both Aurora-A IC50 and SW620 human colon cancer cell GI50 determinations, the results are mean values of two
c
d
independent determinations or mean ( SD) for n > 2 unless specified otherwise.
The two most promising compounds (i.e., 21a and 21i,
Table 3) were subject to additional in vitro profiling which
revealed the 1-methyl-1H-imidazol-5-yl derivative 21a to be an
inhibitor of hERG (94% inhibition at 10 μM). The 1-methyl-
1H-pyrazol-4-yl analogue 21i was also an inhibitor of hERG
(67% at 10 μM), but exhibited high stability in both MLM
(20% metabolized after a 30 min incubation) and HLM (10%
metabolized after a 30 min incubation). The tPSA and the
clogP for 21i were calculated as 80.42 and 2.37, respectively.46
On the basis of these findings, 21i was selected as a scaffold for
an optimization study aimed at increasing the Aurora-A/tumor
cell growth inhibitory potencies and reducing hERG affinity.
Replacement of the N1-methyl in the R2 pyrazole subsituent
of 21i with an ethyl, isopropyl, or difluoroethyl substituent
(compounds 22a, 22b, 22c, respectively, Table 4) gave no
Aurora-A or tumor cell growth inhibitory benefit, although high
stability to human liver microsomal metabolism was maintained
(Table 4). On the other hand, the introduction of an additional
methyl group at the 3-position of the pyrazole ring in 21i
(compound 22d, Table 4) resulted in an enhancement to
Aurora-A inhibition and SW620 cell growth inhibitory potency.
In addition, the inhibitory activity of 22d against hERG was
determined as 48% at 10 μM (IC50 = 6.3 μM, Table 4).
Subsequent introduction of a C3-cyclopropyl group, a bulkier
and more lipophilic substituent, led to a marginal increase in
Aurora-A inhibitory potency (22e vs 22d, Table 4). However,
compound 22e inhibited hERG with an IC50 value of 2.5 μM
(Table 4). On balance, the data presented in Table 4 point to
1,3-dimethyl-1H-pyrazol-4-yl as an optimal R2 substituent.
Having identified 1,3-dimethyl-1H-pyrazol-4-yl as a preferred
R2 substituent, our efforts focused on exploring the R3
substituent. Our aim was to further improve the in vitro
profile, in particular to reduce hERG affinity, and to
subsequently progress the most promising analogues to in
vivo PK characterization and evaluation in a human tumor
xenograft model. The R3 5-methylisoxazol-3-yl group in 22d
(Table 4) was replaced with a range of five- and six-membered
aromatic heterocycles and the p-chlorophenyl previously
identified by Free−Wilson analysis as a preferred substituent
(Table 5). The 4-methyl-1,2,5-oxadiazole derivative 27a was a
more potent inhibitor of Aurora-A and SW620 tumor cell
growth relative to 22d but highly susceptible to metabolism, in
particular in mouse liver microsomes. On the other hand, the
1,2,4-oxadiazole analogues 27b and 27c exhibited lower
Aurora-A and SW620 tumor cell inhibitory potency relative
to 22d. Both 27b and 27c displayed similar inhibitory activities
against hERG (IC50 values of 11.0 and 9.5 μM, respectively;
Table 5). The 1,2,4-triazole analogue 27d displayed consid-
erably lower Aurora-A and SW620 tumor cell inhibitory
potencies relative to 22d, though its affinity for hERG was
significantly reduced (14% inhibition at 10 μM). The
introduction of the R3 p-chlorophenyl substituent was,
however, beneficial versus inhibition of SW620 tumor cell
growth (compound 27e (CCT241736), GI50 = 0.283 μM). In
addition, 27e inhibited Aurora-A with an IC50 value 0.038 μM,
F
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d
Table 5. R3-Isoxazole Replacements
assessed by profiling 27e in a 442-kinase panel (containing
386 nonmutant kinases) at a concentration of 1 μM using the
KINOMEScan technology (see Table S2, Supporting Informa-
tion).48−50 The S(10) selectivity score, which is calculated by
dividing the number of nonmutant kinases for which >90%
competition of control ligand is observed (this is measured as
<10% of control) by the total number of nonmutant kinases
tested, was determined as 0.057, i.e., 22 hits from the 386
nonmutant kinases tested. As expected, Aurora-A, -B, and -C
were potently inhibited with percent control values of 3.4, 1,
and 16, respectively, at 1 μM. Consistent with previous data on
this imidazo[4,5-b]pyridine series,51 screening also revealed
greater than 94% competition for wild-type FLT3 kinase and
clinically relevant FLT3-resistant mutants including FLT3-ITD
and FLT3(D835Y) which was confirmed by Kd determination
(Table 6). Taken together, these data indicate that 27e is a
potent dual inhibitor of FLT3 and Aurora kinases with few off-
target kinase activities across the kinome (Figure 4).
Table 6. Kd Values for Compound 27e
kinase
Kd (nM)
Aurora-A
7.5
48
Aurora-B
FLT3
6.2
11
FLT3(D835H)
FLT3(D835Y)
FLT3-ITD
14
38
FLT3(K663Q)
FLT3(N841I)
FLT3(R834Q)
5.1
16
a
b
Results are mean values for samples run in triplicate. MLM/HLM:
percentage of parent compound metabolized after a 30 min
110
incubation. Calculated log P46 For both Aurora-A IC50 and
SW620 human colon cancer cell GI50 determinations, the results are
mean values of two independent determinations or mean ( SD) for n
> 2 unless specified otherwise.
c
d
Selected examples from the imidazo[4,5-b]pyridine series
including compound 6, which we recently reported as a dual
FLT3/Aurora inhibitor,51 were also tested against FLT3 and
FLT3-ITD. All compounds tested displayed potent affinity for
both forms of FLT3 kinase (Table 7), indicating that FLT3
binding is a common feature for this series of imidazo[4,5-
b]pyridines. FLT3 is a trans-membrane kinase of the class III
receptor tyrosine kinase (RTK) family. Binding of FLT3-ligand
(FL) to its receptor leads to dimerization, autophosphorylation,
and subsequent activation of downstream signaling pathways,
playing a role in the survival and proliferation of leukemic
cells.52 High levels of FLT3 expression have been found in
AML blasts, and two major classes of activating mutations, i.e.
internal-tandem duplications (ITDs) and tyrosine kinase
domain (TKD) point mutations, have been identified in
AML patients.52,53 ITDs are detected in 20−25% of AML
patients, and TKD point mutations in 5−10% of AML
patients.52,53 Internal tandem duplication of the FLT3 gene
(FLT3-ITD) results in constitutive FLT3 kinase activation.54
Meshinchi et al. reported that FLT3-ITD is detected in 20−
35% of adults and 15% of children with AML, conferring a poor
prognosis in both age groups.55 Over the past decade FLT3
inhibition has emerged as a therapeutic strategy of interest for
the treatment of AML, and a number of small-molecule
inhibitors have been evaluated in clinical trials.53,56
was highly stable in mouse and human microsomes (34% and
10% metabolized after a 30 min incubation respectively), and
displayed low affinity for hERG (IC50 > 25 μM). Finally, two
more polar six-membered heteroaromatic substituents were
explored, i.e., pyrimid-5-yl and pyrazin-2-yl (27f and 27g,
respectively); both derivatives exhibited low affinity for hERG
(32% and 23% inhibition at 10 μM respectively) but also low
stability in both mouse and human liver microsomes. Although
a trend to lower hERG inhibition was observed with less
lipophilic compounds (compare compounds 22b,c in Table 4
with compounds 27d,f,g in Table 5), structural modifications
that increase log P could also interestingly reduce affinity for
hERG, as illustrated for compound 27e (clogP = 4.81,47 hERG
IC50 > 25 μM) compared to 22d (clogP = 2.34, hERG IC50
=
6.3 μM). Considering the impact of these R3 modifications
(Table 5) on MLM stability, all four R3 five-membered
heteroaromatic-substituted compounds, 27a (clogP = 2.34),
27b (clogP = 1.45), 27c (clogP = 1.72), and 27d (clogP =
1.21), were more susceptible to mouse liver metabolism
compared with 22d despite displaying lower or equal clogP
(22d, clogP = 2.34). A similar trend was observed with the R3
six-membered heteroaromatic compounds 27f (clogP = 1.64)
and 27g (clogP = 1.64), both being significantly more
susceptible to mouse liver metabolism compared with p-
chlorophenyl derivative 27e. On the basis of a desirable set of
in vitro properties, compound 27e was selected for in depth in
vitro and in vivo characterization. Kinase selectivity was
Consistent with dual FLT3/Aurora inhibitory activity,
compound 27e displayed antiproliferative activity in a range
of human tumor cell lines, including HCT116 human colon
carcinoma (GI50 = 0.300 μM) and the human FLT3-ITD
positive AML cell lines MOLM-13 (GI50 = 0.104 μM) and
MV4−11 (GI50 = 0.291 μM). In Hela cervical cancer cells, 27e
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inhibition of FLT3 signaling (FLT3 phosphorylation, data not
shown).
The inhibition of cytochrome P450 isoforms by 27e was also
investigated. Compound 27e did not significantly inhibit the
major cytochrome P450 isoforms (CYP1A2, CYP2A6,
CYP2C9, CYP2C19, CYP2D6, CYP3A4); all IC50 values
were greater than 10 μM. Compound 27e performed well in
the Caco-2 assay, giving a permeability of 18.6 × 10−6 cm/s
with no efflux. The desirable set of in vitro properties led to the
in vivo evaluation of 27e. The mouse plasma protein binding
for 27e was determined as 97.3%, and the PK profile of 27e in
mouse revealed a highly orally bioavailable compound (F =
100%) with moderate clearance (0.058 L/h, 48 mL/min/kg)
and Vd (0.066 L, 3.3 L/kg) (Table 8). Pharmacokinetic
Table 8. PK Parameters in Mouse and Rat for Compound
27e (iv dosing, 5 mg/kg; oral dosing, 5 mg/kg)
T1/2 (iv)
(h)
Cl (iv)
(L/h)
AUCinf (iv)
(h nmol/L)
F (po)
(%)
Vd (L)
mouse
rat
0.84
4.62
0.058
0.057
3753
0.066
0.369
100
79
39853
evaluation in rats also showed high oral bioavailability (79%),
low clearance (0.057 L/h, 4.57 mL/min/kg), and moderate
volume of distribution (0.369 L, 1.79 L/kg) (Table 8).
The encouraging in vivo pharmacokinetic profile coupled
with dual FLT3/Aurora inhibitory activity prompted us to
evaluate 27e in a human AML subcutaneous xenograft model.
As shown in Figure 5, 27e strongly inhibited the growth of
MV4−11 human tumor xenografts in a dose-dependent
manner with no observed toxicity as defined by body weight
loss and general condition. When therapy was discontinued
after 11 days, tumors were undetectable in mice treated with an
oral dose of 100 mg/kg po b.i.d. 27e and had decreased to 42%
of the initial volume in those treated with 50 mg/kg b.i.d.
Control mice were culled on day 18 from the start of therapy
when the mean tumor volume had increased by over 500%. In
contrast, individual mice were culled when tumors progressed
to this stage as follows: days 28 and 31 at 50 mg/kg and days
46 and 56 at 100 mg/kg. Three out of five mice in each
treatment group (60%) did not develop progressively growing
tumors at the time the study was terminated on day 60,
indicative of long-term growth control. As a result of this potent
in vivo antitumor effect, tumors from treated animals were too
small to provide material for a pharmacokinetic/pharmacody-
namic analysis.
Figure 4. TREEspot interaction map48 depicting the selectivity profile
for 27e. Significant off-target inhibition was observed for FLT1, JAK2,
RET, and PDGFRB with percent control values of 0.3, 1.3, 1.8, and 4
at 1 μM respectively (Supporting Information, Table S2).
Table 7. FLT3 and FLT3-ITD Kd Values (nM)
compd
FLT3
FLT3-ITD
A 4-day PK/PD study (27e po at 50 and 100 mg/kg b.i.d.)
showed clear inhibition of both histone H-3 phosphorylation
and Stat5 phosphorylation (a direct downstream target of
FLT3 kinase) at 2 h after the final dose, consistent with dual
inhibition of Aurora and FLT3 kinases in the tumor (Figure
6).51,59,60 In addition, average free drug concentrations in
plasma samples obtained 2 h after the final dose (222 and 488
nM for the 50 and 100 mg/kg dosing schedules, respectively;
Figure 6) significantly exceed Kd values of 27e against the
relevant kinases, i.e., Aurora-A (Kd = 7.5 nM), Aurora-B (Kd =
48 nM), FLT3 (Kd = 6.2 nM), FLT3-ITD (Kd = 38 nM). These
findings demonstrate that 27e significantly inhibits the growth
of a clinically relevant FLT3-ITD-positive AML human tumor
xenograft model in vivo, with biomarker modulation and free
drug exposure consistent with dual FLT3 and Aurora kinase
target inhibition.
6
1.2
14
4.9
62
66
26
10
14
26
15
21a
21i
20f
22d
27b
27c
27f
12
5.4
2.4
4.4
5.6
5.9
inhibited both the autophosphorylation of Aurora-A at T288 (a
biomarker for Aurora-A inhibition: IC50 = 0.030 μM) and
histone H3 phosphorylation at S10 (a biomarker for Aurora-B
inhibition: IC50 = 0.148 μM),57,58 consistent with potent
cellular activity versus both Aurora-A and -B. Compound 27e
also inhibited Aurora-A in MOLM-13 cells with concomitant
H
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Figure 5. Oral efficacy of 27e against MV4−11 human FLT3-ITD positive AML tumor xenografts in athymic mice (dosing interval day 0−11; twice
daily for 7 days, and once daily for a further 4 days): (A) relative tumor volumes SEM and (B) mouse body weights. N = 5 per group.
dimethyl-1H-pyrazol-4-yl as an optimal R2 substituent.
Subsequent property refinement by R3 substituent modification
led to 27e, an orally bioavailable, dual FLT3/Aurora kinase
inhibitor with high selectivity within the tested kinome.
Compound 27e also potently inhibits mutant FLT3 kinases,
including FLT3-ITD, which causes constitutive FLT3 kinase
activation and is detected in 20−35% of adults and 15% of
children with AML, conferring a poor prognosis in both age
groups. In an in vivo setting, 27e strongly inhibited the growth
of a FLT3-ITD-positive AML human tumor xenograft with
biomarker modulation and plasma free drug exposures
consistent with dual FLT3 and Aurora kinase target inhibition.
Compound 27e, an orally bioavailable dual FLT3/Aurora
kinase inhibitor, has been selected as a preclinical development
candidate for the treatment of human malignancies, with
particular relevance for the treatment of AML in adults and
children who become resistant to existing therapies.61
EXPERIMENTAL SECTION
■
Aurora Kinase Assays. Aurora kinase IC50 values were
determined as previously described.32,62
Kinase Selectivity Profiling. Kinase profiling was performed
using the KINOMEScan technology, and Kd determinations were
performed by KINOMEscan, a Division of DiscoveRx Corp., San
Diego, CA (www.kinomescan.com).
Figure 6. PK/PD study in mice bearing MV4−11 human FLT3-ITD-
positive AML tumor xenografts treated with 27e (50 and 100 mg/kg
Cell Viability Assay. GI50 values (50% cell growth inhibitory
po, b.i.d., for 4 days). (A) Total plasma and tumor concentrations and
free plasma concentrations; all samples were taken 2 h after the final
dose. (B) Inhibition of histone H-3 phosphorylation at S10 and
concentration) were determined as previously described.32,62
Determination of Cellular IC50 Values of 27e for Aurora-A
and -B Inhibition. Myc-tagged Aurora-A was transfected in Hela cells
inhibition of Stat5 phosphorylation at Y694. Tumor samples were
using Lipofectamine LTX in 24 well plates. Twenty-four hours after
obtained 2 h after the final dose. Total histone H3, total Stat5, and
transfection, cells were treated with different concentrations of 27e for
GAPDH were used as loading controls.
2 h. Cells were then lysed in 2× LDS sample buffer. Proteins from
different samples were resolved by 4−12% Bis-Tris NuPage
(Invitrogen) gels and analyzed by Western blotting using P-histone
H3 (S10) and P-Aurora-A (T288) antibodies. The bands for P-histone
H3 and P-Aurora-A were quantified using Image J software, and IC50
values were calculated using Graphpad Prism.
CONCLUSION
■
Compound 6 and its 6-Cl counterpart 5 were used as starting
points for medicinal chemistry optimization aimed at the
identification of an orally bioavailable preclinical development
candidate. In the course of this work we paid particular
attention to minimization of hERG inhibition and human liver
microsomal instability. We found that the switch of the N-
methylpiperazine moiety in compound 5 to the m-position was
beneficial in reducing affinity for hERG and significantly
increasing the human liver microsomal stability. Replacement of
the R2 (4-methylpiperazin-1-yl)phenyl substituent with five-
membered heteroaromatics led to the identification of 1,3-
Cocrystallization of Aurora-A with Ligand. Wild-type Aurora-A
catalytic domain (residues 122−403) was expressed and purified as
previously described.5 Cocrystals with 21a were produced using 0.1 M
sodium acetate pH 4.5, 0.2 M (NH4)2SO4, 25% PEG 4000/2000
MME as crystallization buffer. Structures were solved by molecular
replacement using Aurora-A (PDB code 1MQ4) as a model. Ligand
fitting and model rebuilding was carried out using Coot63 and
refinement was carried out using Phenix.64 Coordinates and structure
factors have been deposited in the Protein Data Bank with accession
code 4B0G.
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Mouse Liver Microsomal Stability. Compounds (10 μM) were
incubated with male CD1 mouse liver microsomes (1 mg mL−1)
protein in the presence of NADPH (1 mM), UDPGA (2.5 mM), and
MgCl2 (3 mM) in phosphate-buffered saline (10 mM) at 37 °C.
Incubations were conducted for 0 and 30 min. Control incubations
were generated by the omission of NADPH and UDPGA from the
incubation reaction. The percentage compound remaining was
determined after analysis by LC−MS.
Human Liver Microsomal Stability. Compounds (10 μM) were
incubated with mixed gender pooled human liver microsomes (1 mg
mL−1) protein in the presence of NADPH (1 mM), UDPGA (2.5
mM), and MgCl2 (3 mM) in phosphate-buffered saline (10 mM) at 37
°C. Incubations were conducted for 0 and 30 min. Control incubations
were generated by the omission of NADPH and UDPGA from the
incubation reaction. The percentage compound remaining was
determined after analysis by LC−MS.
Inhibition of Cytochrome P450 Isoforms. Inhibition of human
liver CYP isozymes was assessed in human liver microsomes (pool of
50 individuals) as previously described65 with the following
modifications: microsomal protein concentration 0.5 mg/mL,
incubation time 10 min, mephenytoin as the CYP2C19 substrate,
and metabolite detection by LC−MSMS ESI+ on an Agilent 1290
Infinity Series LC system with 6410 triple quadrupole mass
spectrometer (4-hydroxytolbutamide, hydroxymephenytoin) or
Waters Acquity UPLC connected to a QTRAP 4000 (Applied
Biosystems).
hERG Inhibition. All hERG percentage inhibitions at 10 μM
compound concentration were determined by Millipore in a high-
throughput cell-based electrophysiology assay for inhibition of hERG
tail current,66 and values are reported as a mean of multiple
determinations. 0.3% DMSO aqueous vehicle negative control gave
7−16% inhibition. Cisapride (1 μM) positive control gave 96−104%
inhibition. hERG IC50 values were determined by Millipore,66 and the
hERG IC50 for compound 27e was also determined by Cyprotex plc.67
The hERG IC50 value for compound 20f (Table 2) was determined by
Cyprotex plc measuring hERG tail currents by whole-cell voltage
clamping,67 and in this assay, the hERG IC50 value for compound 5
was determined as 4.3 μM.
were implanted subcutaneously with 107 FLT3-ITD-positive MV4−11
human leukemia cells. When the tumor xenografts were well-
established (10 days after implantation, mean tumor volumes of at
least 100 mm3), animals were treated with either vehicle (10% DMSO,
20% PEG 400, 5% Tween 80 and 65% water) or 27e administered
orally at two doses, 50 and 100 mg/kg (n = 5 per group). Dosing was
twice daily for 7 days, and once daily for a further 4 days.
PK/PD study. A 4-day PK/PD study was performed by oral
administration of vehicle as above or 50 and 100 mg/kg of 27e twice
daily in athymic mice bearing well-established MV4−11 AML tumor
xenografts (17 days after implantation). Plasma and tumor samples
were collected 2 and 6 h after the final doses.
Chemistry. Commercially available starting materials, reagents, and
dry solvents were used as supplied. Flash column chromatography was
performed using Merck silica gel 60 (0.025−0.04 mm). Column
chromatography was also performed on a FlashMaster personal unit
using ISOLUTE flash silica columns or a Biotage SP1 purification
system using Biotage flash silica cartridges. Preparative TLC was
performed on Analtech or Merck plates. Ion exchange chromatog-
raphy was performed using acidic ISOLUTE Flash SCX-II cartridges.
1H NMR spectra were recorded on a Bruker Avance-500. Samples
were prepared as solutions in a deuterated solvent and referenced to
the appropriate internal nondeuterated solvent peak or tetramethylsi-
lane. Chemical shifts were recorded in ppm (δ) downfield of
tetramethylsilane. LC−MS analysis was performed on a Waters LCT
with a Waters Alliance 2795 separations module and Waters 2487
dual-wavelength absorbance detector coupled to a Waters/Micromass
LCT time-of-flight mass spectrometer with ESI source. Analytical
separation was carried out at 30 °C either on a Merck Chromolith
SpeedROD column (RP-18e, 50 × 4.6 mm) using a flow rate of 2 mL/
min in a 3.5 min gradient elution with detection at 254 nm or on a
Merck Purospher STAR column (RP-18e, 30 × 4 mm) using a flow
rate of 1.5 mL/min in a 3.5 min gradient elution with detection at 254
nm. The mobile phase was a mixture of methanol (solvent A) and
water (solvent B) both containing formic acid at 0.1%. Gradient
elution was as follows: 1:9 (A/B) to 9:1 (A/B) over 2.25 min, 9:1 (A/
B) for 0.75 min, and then reversion back to 1:9 (A/B) over 0.3 min,
and finally 1:9 (A/B) for 0.2 min.
Physicochemical Properties. log D and pKa measurements were
performed by Pharmorphix Solid State Services, Member of the Sigma-
Aldrich Group, Cambridge, UK.
LC−HRMS analysis was performed on an Agilent 1200 series
HPLC and diode array detector coupled to a 6520 quadrupole-time of
flight mass spectrometer with dual multimode APCI/ESI source.
Analytical separation was carried out at 30 °C on a Merck Purospher
STAR column (RP-18e, 30 × 4 mm) using a flow rate of 1.5 mL/min
in a 4 min gradient elution with detection at 254 nm. The mobile
phase was a mixture of methanol (solvent A) and water (solvent B)
both containing formic acid at 0.1%. Gradient elution was as follows:
1:9 (A/B) to 9:1 (A/B) over 2.5 min, 9:1 (A/B) for 1 min, and then
reversion back to 1:9 (A/B) over 0.3 min, finally 1:9 (A/B) for 0.2
min. The following references masses were used for HRMS analysis:
In Vivo Mouse PK (Compound 27e). Mice (female Balb/C)
were dosed po or iv with 27e (5 mg kg−1) in 10% DMSO, 5% Tween
20 in saline. After administration, mice were sacrificed at 5, 15, and 30
min and 1, 2, 4, 6, and 24 h. Blood was removed by cardiac puncture
and centrifuged to obtain plasma samples. Plasma samples (100 μL)
were added to the analytical internal standard (Olomoucine; IS),
followed by protein precipitation with 300 μL of methanol. Following
centrifugation (1200g, 30 min, 4 °C), the resulting supernatants were
analyzed for 27e levels by LC−MS using a reverse-phase Acquity
UPLC C18 (Waters, 50 × 2.1 mm) analytical column and positive ion
mode ESI MRM on an Agilent 1200 liquid chromatography system
coupled to a 6410 triple quadrupole mass spectrometer (Agilent Ltd.).
In Vivo Rat PK (Compound 27e). Rats (female Sprague−
Dawley) were dosed po or iv with 27e (5 mg kg−1) in 10% DMSO, 5%
Tween 80, 20% PEG400 in water (po) or 10% DMSO, 5% Tween 20,
85% saline, 0.3% 1 M HCl (iv). Blood was removed by serial tail vein
bleed at 15 and 30 min and 2, 4, 6, and 24 h and centrifuged to obtain
plasma samples. Plasma samples (50 μL) were added to the analytical
internal standard (Olomoucine; IS), followed by protein precipitation
with 150 μL of methanol. Following centrifugation (1200g, 30 min, 4
°C), the resulting supernatants were analyzed for 27e levels by LC−
MS using a reverse-phase Acquity UPLC C18 BEH (Waters, 50 × 2.1
mm) analytical column and positive ion mode ESI MRM on an
Acquity H-Class UPLC coupled to Xevo TQ-S triple quadrupole mass
spectrometer (Waters Ltd.).
caffeine, [M
+
H]+ 195.087 652; hexakis(1H,1H,3H-
tetrafluoropentoxy)phosphazene, [M + H]+ 922.009 798); and
hexakis(2,2-difluoroethoxy)phosphazene, [M + H]+ 622.028 96, or
reserpine, [M + H]+ 609.280 657.
Analytical HPLC analysis was performed on a Thermo-Finnigan
Surveyor HPLC system or an Agilent Technologies 1200 series HPLC
system at 30 °C using a Phenomenex Gemini C18 column (5 μm, 50 ×
4.6 mm) and 10 min gradient of 10→90% MeOH/0.1% formic acid,
visualizing at 254, 309, or 350 nm. The purity of final compounds was
determined by analytical HPLC as described above and is ≥95% unless
specified otherwise.
4-(4-Ethylpiperazin-1-yl)benzaldehyde (9c). To a mixture of 4-
bromobenzylaldehyde diethyl acetal (0.518 g, 2.0 mmol) and
anhydrous toluene (4 mL) was added 1-ethylpiperazine (0.274 g,
2.4 mmol) followed by Pd2(dba)3 (0.018 g, 0.02 mmol), racemic
BINAP (0.037 g, 0.06 mmol), and NaOtBu (0.326 g, 3.40 mmol). The
reaction mixture was placed into an oil bath preheated to 100 °C,
stirred at this temperature for 5 h under argon, and then allowed to
cool to room temperature. Aqueous HCl (1 M, 10 mL) was added, the
mixture was vigorously stirred for 2.5 h, and then the pH was adjusted
to 13 with aqueous NaOH and extracted with ethyl acetate (3 × 50
Human Tumor Xenograft Efficacy Study. Procedures involving
animals were carried out within guidelines set out by The Institute of
Cancer Research’s Animal Ethics Committee and in compliance with
national guidelines.68 Female adult CrTacNCr-Fox1(nu) athymic mice
J
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1
mL). The combined organics were dried (Na2SO4) and concentrated
in vacuo, and the residue was absorbed on silica gel and placed on a 20
g ISOLUTE column. Elution with a gradient of methanol (from 0 to
4%) in ethyl acetate/dichloromethane (v/v, 1:1) afforded the title
compound as an oil (0.115 g, 26%). 1H NMR (500 MHz, DMSO-d6):
1.04 (t, J = 7.2 Hz, 3H, CH2CH3), 2.37 (q, J = 7.2 Hz, 2H, CH2CH3),
2.54 (m, 4H, piperazine C−H), 3.39 (t, J = 5.0 Hz, 4H, piperazine C−
H), 7.05 (d, J = 8.9 Hz, 2H) and 7.71 (d, J = 8.9 Hz, 2H) (2,6-C6H4
and 3,5-C6H4), 9.72 (s, 1H, CHO). LC−MS (ESI, m/z): tR = 0.85
min; 219 (M + H)+.
after trituration with diethyl ether (0.070 g, 54%). H NMR (500
MHz, DMSO-d6): 2.24 (s, 3H, N−CH3), 2.40 (s, 3H, isoxazole 5-
CH3), 2.62 (br s, 4H, piperazine C−H), 3.24 (poorly resolved t, 4H,
piperazine C−H), 3.59 (s, 2H, N−CH2-isoxazole), 3.69 (br s, 4H,
piperazine C−H), 6.25 (s, 1H, 4-H isoxazole), 7.06 (dd, J = 1.8, 8.3
Hz, 1H, PhH), 7.35 (t, J = 8.3 Hz, 1H, PhH), 7.61 (d, J = 7.8 Hz, 1H,
PhH), 7.72 (s, 1H, PhH), 8.10 (s, 1H, imidazo[4,5-b]pyridine 5-H),
13.40 (br s, 1H, imidazo[4,5-b]pyridine N−H). LC−MS (ESI, m/z):
tR = 1.53 min; 507, 509 (M + H)+, Cl isotopic pattern. HRMS: found
507.2380, calcd for C26H32ClN8O (M + H)+ 507.2382.
3-((4-(6-Chloro-2-(4-(4-ethylpiperazin-1-yl)phenyl)-3H-imidazo-
[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (10c).
To a mixture of 5-chloro-4-(4-((5-methylisoxazol-3-yl)methyl)-
piperazin-1-yl)-3-nitropyridin-2-amine32 (0.052 g, 0.15 mmol) and
EtOH (7.0 mL) was added 4-(4-ethylpiperazin-1-yl)benzaldehyde
(0.046 g, 0.21 mmol) followed by a freshly prepared aqueous solution
of Na2S2O4 (1 M, 0.60 mL, 0.60 mmol). The reaction mixture was
stirred at 80 °C for 18 h, allowed to cool to room temperature, and
concentrated in vacuo. The residue was absorbed on silica gel and
placed on a 10 g ISOLUTE silica column which was eluted first with a
gradient of methanol (0 to 5%) in ethyl acetate/dichloromethane (v:v,
1:1) and then 6% methanol in chloroform. Fractions containing the
product were combined and concentrated in vacuo, and the resulting
solid residue was triturated with diethyl ether. The title compound was
isolated by filtration as a white solid and washed with diethyl ether,
3-((4-(6-Bromo-2-(1-methyl-1H-imidazol-5-yl)-3H-imidazo[4,5-
b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (21a). To a
mixture of 5-bromo-4-(4-((5-methylisoxazol-3-yl)methyl)piperazin-1-
yl)-3-nitropyridin-2-amine32 (0.1 g, 0.25 mmol) and EtOH (15 mL)
was added 1-methyl-1H-imidazole-5-carbaldehyde (30 mg, 0.32
mmol) followed by a freshly prepared aqueous solution of Na2S2O4
(1 M, 1 mL, 1 mmol). The reaction mixture was heated at reflux for 24
h, allowed to cool to room temperature, and concentrated in vacuo.
The residue was taken up in chloroform and a 10% aqueous
bicarbonate solution. The aqueous layer was further extracted with
dichloromethane, and the combined organic solutions were dried and
concentrated. Ether was added to the residue and a pale white powder
precipitated; this was filtered and dried (70 mg). The product was
purified on an SCX ion-exchange column to provide the title
compound as an off-white powder after trituration with ether (60
1
1
water, and finally diethyl ether (0.012 g, 15%). H NMR (500 MHz,
mg, 53%). H NMR (500 MHz, DMSO-d6): 2.39 (s, 3H, isoxazole 5-
DMSO-d6): 1.04 (t, J = 6.8 Hz, 3H, CH2CH3), 2.37 (q, obscured by
isoxazole 5-CH3 peak, CH2CH3), 2.40 (s, 3H, isoxazole 5-CH3), 2.62
(br s, 4H, piperazine C−H), 3.27 (br s, 4H, piperazine C−H), 3.59 (s,
2H, NCH2isoxazole), 3.67 (br s, 4H, piperazine C−H), 6.25 (s, 1H,
isoxazole 4-H), 7.05 (d, J = 8.9 Hz, 2H) and 8.02 (d, J = 8.9 Hz, 2H)
(2,6-C6H4 and 3,5-C6H4), 8.04 (s, 1H, imidazo[4,5-b]pyridine 5-H),
13.22 (br s, 1H, imidazo[4,5-b]pyridine N−H). LC−MS (ESI, m/z):
tR = 1.70 min; 521, 523 (M + H)+, Cl isotopic pattern. HRMS: found
521.2532, calcd for C27H34ClN8O (M + H)+ 521.2539.
3-(4-Methylpiperazin-1-yl)benzaldehyde (19f). To a solution of 3-
bromobenzylaldehyde diethyl acetal (0.518 g, 2.0 mmol) and
anhydrous toluene (4 mL) was added 1-methylpiperazine (0.240 g,
2.4 mmol) followed by Pd2(dba)3 (0.018 g, 0.02 mmol), racemic
BINAP (0.037 g, 0.06 mmol), and NaOtBu (0.326 g, 3.4 mmol). The
reaction mixture was placed into an oil bath preheated to 100 °C,
stirred at this temperature for 18 h under argon, and then allowed to
cool to room temperature. Aqueous HCl (1 M, 10 mL) was added, the
mixture was vigorously stirred for 2.5 h, the pH was adjusted to 13
with 6 M aqueous NaOH, and the mixture was extracted with ethyl
acetate (3 × 30 mL). The combined organics were dried (Na2SO4),
and concentrated in vacuo, and the residue was absorbed on silica gel
and placed on a 10 g ISOLUTE column. Elution with ethyl acetate/
CH2Cl2 (v/v, 4:1) and then a gradient of methanol (from 3 to 7%) in
ethyl acetate afforded the title compound as a yellow oil (0.170 g,
42%). 1H NMR (500 MHz, DMSO-d6): 2.22 (s, 3H, N-Me), 2.46 (t, J
= 5.0 Hz, 4H, piperazine C−H), 3.21 (t, J = 5.1 Hz, 4H, piperazine C−
H), 7.28 (m, 2H, PhH), 7.41 (m, 2H, PhH), 9.94 (s, 1H, CHO). LC−
MS (ESI, m/z): tR = 0.86 min; 205 (M + H)+.
CH3), 2.63 (br s, 4H, piperazine NCH2), 3.59 (s, 2H, N-CH2-
isoxazole), 3.65 (br s, 4H, piperazine NCH2), 4.05 (s, 3H, N−CH3),
6.23 (s, 1H, 4-H isoxazole), 7.73 (s, 1H imidazole-CH), 7.86 (s, 1H,
imidazole-CH), 8.21 (s, 1H, imidazo[4,5-b]pyridine 5-H), 13.30 (br s,
1H, imidazo[4,5-b]pyridine N−H). LC−MS (ESI, m/z): tR = 1.72
min; 457, 459 (M + H)+, Br isotopic pattern. HRMS: found 457.1094,
calcd for C19H22BrN8O (M + H)+ 457.1091.
3-((4-(6-Chloro-2-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]-
pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (21i). To a
mixture of 5-chloro-4-(4-((5-methylisoxazol-3-yl)methyl)piperazin-1-
yl)-3-nitropyridin-2-amine (0.06 g, 0.17 mmol) and EtOH (10 mL)
was added 1-methyl-1H-pyrazole-4-carbaldehyde (0.023 g, 0.21 mmol)
followed by a freshly prepared aqueous solution of Na2S2O4 (1 M, 0.45
mL, 0.45 mmol). The reaction mixture was heated at reflux for 24 h,
allowed to cool to room temperature, and concentrated in vacuo. The
residue was taken up in chloroform and 10% bicarbonate. The aqueous
layer was further extracted with dichloromethane, and the combined
organic solutions were dried and concentrated in vacuo. The crude
product was purified by silica column chromatography eluting with 2−
10% methanol in dichloromethane. The pure fractions provided 38 mg
1
of title compound (54%). H NMR (500 MHz, DMSO-d6): 2.40 (s,
3H, isoxazole 5-CH3), 2.61 (br s, 4H, piperazine NCH2), 3.59 (s, 2H,
N−CH2-isoxazole), 3.62 (br s, 4H, piperazine NCH2), 3.92 (s, 3H,
NCH3), 6.24 (s, 1H, 4-H isoxazole), 8.03 (s, 1H, pyrazole-H), 8.04 (s,
1H, H pyrazole-H), 8.32 (s, 1H, imidazo[4,5-b]pyridine 5-H), 13.20
(br s, 1H, imidazo[4,5-b]pyridine N−H). LC−MS (ESI, m/z): tR
=
2.35 min; 413, 415 (M + H)+, Cl isotopic pattern. HRMS: found
413.1599, calcd for C19H22ClN8O (M + H)+ 413.1599.
6-Chloro-2-(1,3-dimethylpyrazol-4-yl)-7-(4-(5-methylisoxazol-3-
ylmethyl)piperazin-1-yl)-3H-imidazo[4,5-b]pyridine (22d). To a
mixture of 1,3-dimethylpyrazole-4-aldehyde (27.3 mg, 0.20 mmol)
and 2-amino-5-chloro-4-(4-(5-methylisoxazol-3-yl)methylpiperazin-1-
yl)-3-nitropyridine (70.5 mg, 0.20 mmol) in ethanol (1.4 mL) was
added freshly prepared 1 M aqueous Na2S2O4 solution (0.7 mL, 0.7
mmol). The reaction was stirred and heated at 75 °C for 19.5 h. The
reaction was cooled, 5 M ammonia solution (0.4 mL) was added, and
the mixture was stirred for 15 min. Most of the ethanol was evaporated
and water (2 mL) was added. The product was extracted into ethyl
acetate (8, 4, 4 mL), and the combined extracts were washed with
brine, dried (Na2SO4), and evaporated to leave a cream solid. This was
triturated with ether (2 mL) and the solid further washed with ether (1
mL) to leave the product (58 mg, 67%). 1H NMR (500 MHz, DMSO-
d6): 2.41 (s, 3H, CH3), 2.52 (s, 3H, CH3), 2.63 (m, 4H, piperazine C−
H), 3.59 (s, 2H, CH2), 3.69 (m, 4H, piperazine C−H), 3.85 (s, 3H,
3-((4-(6-Chloro-2-(3-(4-methylpiperazin-1-yl)phenyl)-3H-
imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxa-
zole (20f). To a mixture of 5-chloro-4-(4-((5-methylisoxazol-3-
yl)methyl)piperazin-1-yl)-3-nitropyridin-2-amine32 (0.090 g, 0.26
mmol) and EtOH (20 mL) was added 3-(4-methylpiperazin-1-
yl)benzaldehyde (0.057 g, 0.28 mmol) followed by a freshly prepared
aqueous solution of Na2S2O4 (1 M, 0.76 mL, 0.76 mmol). The
reaction mixture was stirred at 80 °C for 24 h, allowed to cool to room
temperature, and concentrated in vacuo. The residue was partitioned
between chloroform and 5% aqueous sodium hydrogen carbonate.
The two layers were separated, and the organic layer was dried
(Na2SO4) and concentrated in vacuo. The crude product was applied
to an SCX ion-exchange column (5 g, 25 mL) which was eluted with
10% methanol in chloroform followed by 1 M ammonia in methanol.
Fractions containing the pure product were combined and
concentrated in vacuo. The title compound was obtained as a powder
K
dx.doi.org/10.1021/jm300952s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
N−CH3), 6.24 (s, 1H, heteroaryl H), 8.03 (s, 1H, heteroaryl H), 8.19
(s, 1H, heteroaryl H), 12.98 (s, 1H, NH). LC−MS (ESI, m/z): tR =
1.78 min; 427, 429 (M + H)+, Cl isotope pattern. HRMS: found
427.1756, calcd for C20H24ClN8O (M + H)+ 427.1756.
Notes
The authors declare the following competing financial
interest(s): Please note that all authors who are, or have
been, employed by The Institute of Cancer Research are
subject to a “Rewards to Inventors Scheme” which may reward
contributors to a programme that is subsequently licensed. Paul
Workman was a founder and shareholder in Piramed Pharma
and is a founder and shareholder in Chroma Therapeutics. Paul
Workman is a former employee of AstraZeneca. Julian Blagg is
former employee and shareholder of Pfizer Inc.
5-Chloro-4-(4-(4-chlorobenzyl)piperazin-1-yl)-3-nitropyridin-2-
amine (26e). To a mixture of 2-amino-4,5-dichloro-3-nitropyridine
(0.152 g, 0.73 mmol) and 2-propanol (22 mL) was added 1-(4-
chlorobenzyl)piperazine (0.165 g, 0.78 mmol) followed by diisopro-
pylethylamine (0.17 mL, 0.97 mmol). The reaction mixture was heated
at 45 °C for 18 h, allowed to cool to room temperature, and diluted
with 2-propanol (5 mL). The precipitate was collected by filtration,
washed with 2-propanol, and diethyl ether. The title compound was
1
thus obtained as a yellow solid (0.215 g, 77%). H NMR (500 MHz,
ACKNOWLEDGMENTS
DMSO-d6): 2.48 (br s, obscured by DMSO peak, 4H, piperazine C−
H), 3.06 (br t, J = 4.3 Hz, 4H, piperazine C−H), 3.52 (s, 2H,
NCH2C6H4Cl), 6.95 (s, 2H, NH2), 7.35 (d, J = 8.5 Hz, 2H) and 7.38
(d, J = 8.5 Hz, 2H) (3,5-ArH and 2,6-ArH), 8.06 (s, 1H, 6-H). LC−
MS (ESI, m/z): tR = 1.70 min; 382, 384, 386 (M + H)+, Cl2 isotopic
pattern.
■
This work was supported by Cancer Research UK [CUK] grant
numbers C309/A8274 and C309/A11566. A.S.M. was
supported by Cancer Research UK (Grant number C1178/
A10294) and in part by a New Investigator Scholarship
awarded by the Haematology Society of Australia and New
Zealand. S.L. is also supported by Breakthrough Breast Cancer.
R.B. is a Royal Society University Research Fellow and
acknowledges the support of Breakthrough Breast Cancer
Project Grant AURA 05/06 and infrastructural support for
Structural Biology at the ICR from Cancer Research UK. We
acknowledge NHS funding to the NIHR Biomedical Research
Centre at The Institute of Cancer Research and the Royal
Marsden NHS Foundation Trust. We also thank Dr. Amin
Mirza, Mr. Meirion Richards, and Dr. Maggie Liu for their
assistance with NMR, mass spectrometry, and HPLC.
6-Chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-
1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e). To a mixture of 5-
chloro-4-(4-(4-chlorobenzyl)piperazin-1-yl)-3-nitropyridin-2-amine
(0.076 g, 0.20 mmol) and EtOH (4.0 mL) was added 1,3-dimethyl-
1H-pyrazole-4-carbaldehyde (0.027 g, 0.22 mmol) followed by a
freshly prepared aqueous solution of Na2S2O4 (1 M, 0.85 mL, 0.85
mmol). The reaction mixture was stirred at 80 °C for 24 h, allowed to
cool to room temperature, and concentrated in vacuo, and the residue
was absorbed on silica gel and placed on a 10 g ISOLUTE silica
column. Elution with ethyl acetate/dichloromethane (v/v, 1:1) and
then 4% methanol in ethyl acetate/dichloromethane (v/v; 1:1)
afforded the title compound as a white solid after trituration with
diethyl ether (0.023 g, 25%). 1H NMR (500 MHz, DMSO-d6): 2.51 (s,
obscured by solvent peak, pyrazole 3-CH3), 2.57 (br s, 4H, piperazine
C−H), 3.54 (s, 2H, N-CH2C6H4Cl), 3.68 (br s, 4H, piperazine C−H),
3.84 (s, 3H, pyrazole N-Me), 7.37 (d, J = 8.5 Hz, 2H) and 7.40 (d, J =
8.5 Hz, 2H) (C6H4Cl), 8.02 (s, 1H), and 8.18 (s, 1H) (pyrazole 5-H,
and imidazo[4,5-b]pyridine 5-H), 12.95 (br s, 1H, imidazo[4,5-
b]pyridine N−H). LC−MS (ESI, m/z): tR = 1.97 min; 456, 458, 460
(M + H)+, Cl2 isotopic pattern. HRMS: found 456.1457, calcd for
C22H24Cl2N7 (M + H)+ 456.1465.
ABBREVIATIONS USED:
■
AML, acute myeloid leukemia; AUC, area under the curve; Boc,
tert-butoxycarbonyl; DIPEA, N,N-diisopropylethylamine; ESI,
electrospray ionization; FLT3, FMS-like tyrosine kinase 3;
hERG, the human ether-a-go-go related gene; HPLC, high-
performance liquid chromatography; HLM, human liver
microsomes; HRMS, high-resolution mass spectrometry; inh,
inhibition; LC, liquid chromatography; MLM, mouse liver
microsomes; NBS, N-bromosuccinimide; NCS, N-chlorosucci-
nimide; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium(0);
PPB, plasma protein binding; SAR, structure−activity relation-
ship; TFA, trifluoroacetic acid.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for compounds 11−13, 9b,d,e, 10a−
e, 17, 19a,b, 20a-e, 21b−h, 22a−c,e, 24b,d,f,g, 25b,d,f,g, 26a−
d, 26f,g, 27a−d, 27f,g; summary of crystallographic analysis of
compound 21a (Table S1); and kinase selectivity profiling of
compound 27e (Table S2). This material is available free of
charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*V.B.: telephone, +44 (0) 20 86438901, ext 4601; e-mail;
vassilios.bavetsias@icr.ac.uk. J.B.: telephone, +44(0)20
87224051; e-mail: julian.blagg@icr.ac.uk.
Present Addresses
∥Queensland Children’s Medical Research Institute, Royal
Children’s Hospital, Herston Rd, Herston, QLD 4029,
Australia.
⊥University of Leicester, Department of Biochemistry,
Lancaster Road, Leicester LE1 9HN, United Kingdom.
L
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