Discovery of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines as potent inhibitors of the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2008.12.110

Exploration of the SAR around a series of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines led to the discovery of novel pyrrolopyridine inhibitors of the IGF-1R tyrosine kinase. Several compounds demonstrated nanomolar potency in enzyme and cellular mechanis

Full text of DOI:10.1016/j.bmcl.2008.12.110

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3136–3140
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines as potent
inhibitors of the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase
a
a
b
a
a
Samarjit Patnaik a, , Kirk L. Stevens , Roseanne Gerding , Felix Deanda , J. Brad Shotwell , Jun Tang ,
Toshihiro Hamajima c, Hiroko Nakamura c, M. Anthony Leesnitzer a, Anne M. Hassell a,
Lisa M. Shewchuck b, Rakesh Kumar d, Huangshu Lei a, Stanley D. Chamberlain a
*
a GlaxoSmithKline, Oncology R&D, 5 Moore Drive, Research Triangle Park, NC 27709, USA
b GlaxoSmithKline, Computational and Structural Chemistry, 5 Moore Drive, Research Triangle Park, NC 27709, USA
c GlaxoSmithKline, K. K. Tsukuba Research Laboratories, 43 Wadai Tsukuba, Ibaraki 300-4247, Japan
d GlaxoSmithKline, Oncology R&D, 1250 S. Collegeville Road, Collegeville, PA 19426, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Exploration of the SAR around a series of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines led to the discov-
ery of novel pyrrolopyridine inhibitors of the IGF-1R tyrosine kinase. Several compounds demonstrated
nanomolar potency in enzyme and cellular mechanistic assays.
Received 22 October 2008
Revised 22 December 2008
Accepted 30 December 2008
Available online 6 January 2009
Ó 2009 Published by Elsevier Ltd.
Keywords:
IGF-1R
IGF-IR
Pyrrolopyridine
Kinase inhibitor
The overexpression or autoactivation of the insulin-like growth
factor-1 receptor (IGF-1R) tyrosine kinase has been associated with
various cancers.1 High plasma levels of IGF-1 and low levels of IGF
binding protein-3, which controls the amount of free circulating
IGF, are directly correlated with an increased risk for developing
colon, breast, prostate and lung cancer.2 Several antagonists of
the IGF-1 signaling pathway in the form of IGF-1R antibodies and
small molecules are currently being investigated in clinical trials.3
Structurally diverse preclinical small molecule inhibitors of IGF-1R
have also been reported and hold promise as useful antineoplastic
agents.4
During our efforts to generate leads for the inhibition of protein
kinases we identified 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines
with the general structure 5 as micromolar inhibitors of the IGF-1R
tyrosine kinase (Scheme 1). Given that our enzymatic assay was
comprised of a GST-fused kinase domain of IGF-1R,5 and that the
1H-pyrrolo[2,3-b]pyridine core6 was a known adenine mimic we
assumed that the inhibitor’s core was binding to the hinge region
of the kinase.7
The synthesis of these inhibitors originated with the known ver-
satile 5-bromo-3-iodo-1H-pyrrolo[2,3-b] pyridine intermediate 1,6
which was initially protected with para-toluenesulfonyl chloride
under phase transfer conditions to afford sulfonamide 2. Chemose-
lective Suzuki coupling of the iodide 2 with 3-aminophenyl boronic
acid was possible at 50 °C with dichlorobistriphenylphosphine pal-
ladium and 2N aqueous sodium carbonate in a 2:1 mixture of
dimethoxyethane and ethanol. This provided aniline 3 which could
be functionalized to the urea 4 via addition to various isocyanates.
A second Suzuki reaction in a microwave reactor at 120 °C with
bromoazaindole 4 was followed by base-mediated hydrolysis of
the tosyl group to yield a variety of 1H-pyrrolo[2,3-b]pyridines.
Table 1 lists the inhibitory activities against IGF-1R for a prelimin-
ary set of compounds from this series.
Inhibition near micromolar concentrations was observed with
unsubstituted phenyl rings at R1 and R2 (6) as well as with the
200-fluoro-400-trifluoromethyl substituted phenyl ring at R2 (7).
The latter was subsequently crossed with a range of additionally
substituted phenyl rings at R1. While 30,40-dimethoxy substitution
at R1 (8) decreased potency, a moderate improvement was made
with 30-fluoro substitution (9). Anilines at the 30- or 40-positions
(10, 11) improved the enzyme IC50 to more acceptable levels. The
homologated congener 40-benzylamine 12 proved to be 4-fold
more potent than the simple aniline 11. The potency imparted by
the 40-benzylamine substituent was also observed with the
* Corresponding author. Present address: NIH Chemical Genomics Center, 9800
Medical Center Dr, Rockville, MD 20850, USA. Tel.: +1 919 4836229; fax: +1 919
4836053.
E-mail addresses: patnaiks@mail.nih.gov, samarjitpatnaik@gmail.com (S.
Patnaik).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.12.110

S. Patnaik et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3136–3140
3137
NH2
I
I
Br
5
a
b
Br
Br
3
N
N
N
N
N
N
O
S
O
S
H
O
1
2
O
3
H
N
H
N
H
N
H
N
R2
R2
O
d, e
Br
c
O
R1
N
N
N
N
O
S
H
O
5
4
Scheme 1. (a) TsCl, 5 M NaOH, Bu4NSO4, CH2Cl2, 95%; (b) 3-aminophenylboronic acid, Pd(PPh3)2Cl2, DME/EtOH (2:1), 2M Na2CO3, 50 °C, 90%; (c) R2NCO, THF, 60–90%; (d)
R1B(OH)2, DMF, microwave, Pd(PPh3)2Cl2, 2M Na2CO3, 120 °C; (e) 5 M NaOH, MeOH, 50–90% over 2 steps.
300-chloro phenyl ring at R2 as well (compare 18 to 17). N-Methyl
piperidine carboxamides at the 30- and 40-positions (13 and 15)
had moderate levels of potency. However, the corresponding
unconstrained amides 14 and 16 attained sub-100 nM potencies.
Having discovered groups at R1 (12, 14, 16), which imparted
nanomolar potencies to the chemical series, we further explored
the chemical space at R2. In particular, installation of the ortho-
phenoxy substituent in R2 led to the identification of the very po-
tent derivative 19. This substructure, when crossed with other
optimal groups at R1, further increased the enzymatic activity to
single digit nanomolar potencies as exemplified by compounds
20 and 21. Of particular note was an improvement in kinase selec-
tivity as well. For example, in a preliminary panel of 36 kinases
benzyl amine 12 showed >10-fold selectivity against 24 kinases
with significant co-activity against IKK1, IKK2, JNK1, JNK3, LYN,
and SYK with IC50s measured at 32, 79, 79, 250, 40, and 80 nM
respectively. >10-fold selectivity against these six kinases was ob-
served with the related congener 19. The same trend was exhibited
by 20 and 21. However the ortho-phenoxy substructure could not
achieve selectivity against the closely related insulin receptor (IR)
tyrosine kinase. Having discovered the selectivity imparted by
the ortho-biphenyl ether for IGF-1R we decided to explore what
other groups would be tolerated at R1. At this time we also devel-
oped an assay to measure the cellular activity (phospho IGF-1R
Cellular IC50 in Tables 2 and 3) of these inhibitors as well.8
With the R2 biphenyl ether held constant, we expanded the SAR
around R1. This effort revealed that the above-mentioned enzyme
potent derivative 20 was a moderate inhibitor in the cellular
assay.9 The unsubstituted phenyl ring (22) also had mediocre cell
potency (Table 2). On the other hand, the 30-dimethyl carboxamide
23 was a potent IGF-1R inhibitor in the enzyme and cellular assay.
The cellular potency for the isosteric 30-dimethyl sulfonamide 24
was reduced by about 17-fold. A similar trend was observed for
the same substituents at the 40-position (compare 25 to 26). Car-
boxylic acid derivatives, whether directly attached to the phenyl
ring at R1 (27) or removed from it (28), had poor cellular activities.
Unsubstituted heterocycles at R1, for example, furan 29 or imidaz-
ole 30, also affected the cellular potency adversely. All derivatives
were essentially equipotent against IR in comparable enzymatic
and cellular assays.
Table 1
IGF-1R enzyme assay results for 6–21 (values represent an average of P2 individual
measurements)
H
N
H
N
R2
O
R1
3
5
N
N
H
Compound R1
R2
IGF-1R enzyme
IC50 (nM)
3'
2''
4'
2'
3''
4''
6
749
F
F
F
F
7
8
1262
CF3
O
O
2198
605
CF3
CF3
CF3
9
F
10
242
H2N
H2N
F
11
200
CF3
A co-crystal of IR (insulin receptor) in complex with 19 offered
insight into the binding mode of these inhibitors.10 Derived from
(continued on next page)

3138
S. Patnaik et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3136–3140
Table 2
Table 1 (continued)
IGF-1R enzyme and mechanistic cell assay results for biphenyl ether urea containing
compounds 20, 22–30 (values represent an average of P2 individual measurements)
Compound R1
R2
IGF-1R enzyme
IC50 (nM)
H
F
F
F
F
F
N
H
O
N
H2N
12
50
335
67
O
R1
3
5
CF3
CF3
CF3
CF3
N
N
N
H
N
13
Compound R1
IGF-1R enzyme Phospho IGF-1R
IC50 (nM)
cellular IC50 (nM)
O
O
N
H
N
20
22
4
415
14
N
H
N
O
O
3'
4'
2'
127
693
15
286
36
N
N
4'
O
N
23
24
21
57
68
3'
16
O
N
H
N
CF3
Cl
1116
N
S
O
O
17
1015
20
O
25
26
27
31
128
20,793
1041
N
Cl
H2N
18
O
O
S
nd
N
H2N
O
O
19
14
2"
2"
HO
184
O
O
O
20
4
N
H
28
29
30
210
251
233
3623
1264
7463
HO
O
N
N
H
N
H
N
O
21
4
2"
O
nd, not determined.
this crystal structure, a structural model of IGF-1R in complex with
19 is illustrated in Figure 1. Given that the sequence identity
between the kinase domains of IR and IGF-1R is roughly 80%, IR
was a reasonable surrogate for IGF-1R, and we concluded that
the binding mode of these inhibitors would be nearly identical
within the ATP-binding site of both kinases. A frontal view of the
IGF-1R ATP-binding site (Fig. 1A) clearly shows that the 1H-pyrrol-
o[2,3-b]pyridine core forms two H-bond interactions at the hinge
region, one with the backbone carbonyl of Glu1050 and the second
with the backbone amino group of Met1052. The C3 phenyl at the
1H-pyrrolo[2,3-b]pyridine core lies at the inner hydrophobic re-
gion where it interacts with Val983, Lys1003, Met1049, Met1112,
and Phe1124.11 Near the DFG motif, the urea lies just past the acti-
vation loop, where the urea hydrogens interact with one of the car-
boxylate oxygens of Glu1020 (Fig. 1B). This, interaction directs the
biphenyl ether deeper into the back-pocket region, where it lies be-
low the
aC-helix in contact with Val1023, Met1024, Phe1027,
Val1032, Leu1096, Phe1101, His1103, and Ile1121 (Fig. 1B).

S. Patnaik et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3136–3140
3139
Table 3
IGF-1R enzyme and cellular assay results for 23, 31–35 (values represent an average
of P2 individual measurements)
N
O
H
N
H
3'
N
R2
O
3
5
N
N
H
Compound R2
IGF-1R enzyme
IC50 (nM)
Phospho IGF-1R
cellular IC50 (nM)
O
2''
23
21
68
3''
4''
HN
O
31
32
39
407
517
132
Figure 1. Structural model of IGF-1R (carbons in gray) in complex with 19 (carbons
in green). (A) Frontal view of the ATP-binding site. (B) Cross-sectional view of the
ATP-binding site and back-pocket region. Intermolecular H-bond interactions are
highlighted with yellow lines.
33
64
385
O
group to meta (34) or para (35) positions also led to significant
losses of potency with respect to 23. This work implied that the
conformation adapted by the 200-phenoxy phenyl group at R2 was
optimal in making the appropriate contacts with the hydrophobic
residues at the back pocket of the enzyme, as minor deviations
away from the structure reduced the effectiveness of this binding
mode.
34
35
212
961
2039
3071
O
In summary, SAR exploration around the 1H-pyrrolo[2,3-b]pyr-
idine core led to the discovery of potent inhibitors of the IGF-1R
tyrosine kinase. These compounds bind to the enzyme such that
the 200-phenoxy ether accesses a unique back pocket in the enzyme.
Subtle changes to this pharmacophore led to loss of activity under-
scoring its unique nature. Additional development within this
series of compounds will be discussed in future communications.
The additional hydrophobic contacts created by the biphenyl
ether derivatives over those with a single phenyl may account in
part for the greater potency seen with the former over the latter.
Also, such a deep back-pocket binding mode may account for the
IGF-1R selectivity associated with the biphenyl ether derivatives.
Finally, the C5 phenyl (R1) occupies the outer hydrophobic region
with the ionizable primary amine exposed to solvent. At this posi-
tion, the presence of water solubilizing groups is well tolerated.
With an understanding of the binding mode of the biphenyl
ether we decided to elaborate on the SAR around this substructure
(Table 3). The C5 30-dimethyl benzamide substituent was chosen
for comparison as it was found to impart high potency in our cel-
lular assay (e.g., 23, Table 2).12 The biphenyl aniline isostere 31 lost
about 6-fold cellular potency compared to 23, while maintaining
enzyme activity. The homologated 200-benzyl ether 32 and the di-
rectly attached 200-biphenyl derivative 33 had reduced activity in
both enzyme and cellular assays. The movement of the phenoxy
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7. For a recent discussion of crystal structures of 1H-pyrrolo[2,3-b]pyridines bound
to protein kinases, see: Tsai, J. et al Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3041.
8. Cell assay. NIH-3T3 cells overexpressing human IGF-1R were plated in 96-well
plates (10,000 cells/well) in culture media containing 10% fetal bovine serum
and incubated at 37° C in a 5% CO2 incubator. Twenty four hours post-plating,
cells were treated with different concentrations of test compounds ranging
from 30 lM to 1.5 nM. Two hours after compound addition, cells were
stimulated with human IGF-1 (30 ng/ml). Cell lysates were analyzed for
phosphorylated receptors using dissociation enhanced lanthanide fluor-
immuno assay (DELFIA) with anti-IGF-1R (MAB391, R&D Systems,
Minneapolis, MN) capture antibody and europium-labeled anti-pTyr antibody
(Eu-N1 PT66, Perkin-Elmer, Waltham, MA) for detection. The fluorescence
signal for cells treated with compounds was expressed as percent relative to
100% stimulation (IGF-1 stimulated signal). Concentration of test compound
that inhibited 50% of ligand-induced receptor phosphorylation (IC50) was
determined by 4 parameter fit of data using XLfit (value of no cell control was
subtracted from all samples for background).
5. IGF-1R enzyme assay. GST-rTEV-IGF-1R(957-1367) containing amino acid
residues 957–1367 was purified from a baculovirus expression system in Sf9
cells using Glutathione Sepharose 4FF column chromatography followed by
Sephadex-200 size exclusion column chromatography. Assays were performed
in 384-well (Greiner, Catalog No. 784076) microtiter plates. Reaction buffer
(50 mM Hepes buffer, pH 7.5; 10 mM MgCl2; 3 mM DTT; 1 mM CHAPS; 0.1 mg/
9. The cellular potencies of 19 and 21, reported in Table 1, were not determined.
10. IR protein was expressed and purified as previously described in Hubbard, S. R.
et al. Nature, 1994, 372, 746. Protein at 10 mg s/ml was complexed with a 3-
fold molar excess of inhibitor for 1 h prior to crystallization. Crystals were
grown by hanging drop vapor diffusion at 22° from 0.1 M MOPS, pH 7.0, 1.0 M
trisodium citrate. Crystals were flash frozen in PFO prior to data collection. The
structure was solved by molecular replacement using PDB:1IRK as a starting
model and refined to an Rfactor of 20% at 2.6 Å using REFMAC. Crystallographic
data for the structure in Figure 1 have been deposited at PDB:3ETA.
11. For kinase ATP binding pocket nomenclature, see: Vulpetti, A.; Bosotti, R. Il
Farmaco 2004, 59, 759.
ml BSA) for peptide phosphorylation (10
concentrations, 500 nM biotinylated peptide substrate; 10
purified, activated hIGF-1R (0.5 nM). Activation of GST-rTEV-IGF-1R(957-
1367) was achieved by a 4-min incubation of hIGF-1R (2.7 M final) with
l
l
volume) contained, in final
lM
ATP; and
l
2 mM ATP in 50 mM Hepes, 20 mM MgCl2, 0.1 mg/ml BSA, at room
temperature. Compounds, titrated in DMSO, were evaluated at eleven
concentrations ranging from 50
1 h at room temperature and were stopped by a 5-
33 mM). further addition of detection reagents (for final 7 nM
l
M to 0.2 nM. Reactions were incubated for
ll addition of EDTA (to
A
5 ll
Streptavidin–APC (Perkin-Elmer #CR130-150), 1 nM Europium-labeled anti-
phosphotyrosine monoclonal antibody (Perkin-Elmer #AD0067), added in
reaction buffer (without DTT), was required for signal generation. After 30
12. Compound 23 inhibited cellular proliferation in a COLO205 cell line with EC50
at 274 nM (average of 4 measurements).