Discovery and optimization of imidazo[1,2-a]pyridine inhibitors of insulin-like growth factor-1 receptor (IGF-1R)

Article abstract of DOI:10.1016/j.bmcl.2008.11.058

The optimization of imidazo[1,2-a]pyridine inhibitors as potent and selective inhibitors of IGF-1R is presented. Further optimization of oral exposure in mice is also discussed. Detailed selectivity, in vitro activity, and in vivo PK profiles of an optimi

Full text of DOI:10.1016/j.bmcl.2008.11.058

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1004–1008
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of imidazo[1,2-a]pyridine inhibitors of
insulin-like growth factor-1 receptor (IGF-1R)
a
a
a
a
Kyle A. Emmitte a, , Brian J. Wilson , Erich W. Baum , Holly K. Emerson , Kevin W. Kuntz ,
Kristen E. Nailor a, James M. Salovich a, Stephon C. Smith a, Mui Cheung c, Roseanne M. Gerding a,
Kirk L. Stevens a, David E. Uehling a, Robert A. Mook Jr. a, Ganesh S. Moorthy b, Scott H. Dickerson a,
Anne M. Hassell a, M. Anthony Leesnitzer a, Lisa M. Shewchuk a, Arthur Groy b, Jason L. Rowand b,
Kelly Anderson b, Charity L. Atkins b, Jingsong Yang b, Peter Sabbatini b, Rakesh Kumar b
*
a GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, USA
b GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
c GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The optimization of imidazo[1,2-a]pyridine inhibitors as potent and selective inhibitors of IGF-1R is pre-
sented. Further optimization of oral exposure in mice is also discussed. Detailed selectivity, in vitro activ-
ity, and in vivo PK profiles of an optimized compound is also highlighted.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 October 2008
Revised 13 November 2008
Accepted 17 November 2008
Available online 20 November 2008
Keywords:
Insulin-like growth factor-1 receptor
Insulin receptor
Kinase inhibitor
Cancer
Imidazo[1,2-a]pyridine
Insulin-like growth factor-1 receptor (IGF-1R) is a member of
the insulin receptor family of tyrosine kinases. IGF-1R is a tetra-
meric transmembrane-spanning protein with an intracellular ki-
nase domain. It plays an important role in several critical
signaling cascades. By activating both the Ras/Raf/MAPK and
PI3K/Akt pathways, IGF-1R is a key component in the proliferation,
survival, transformation, and metastasis of cancer cells.1 Monoclo-
nal antibodies specifically targeting IGF-1R are currently being
evaluated in clinical trials.2 In addition, several small molecule ap-
proaches to IGF-1R inhibition have also recently been disclosed.3
There are multiple examples demonstrating growth inhibition of
human tumor xenografts grown on mice through daily treatment
with small molecule IGF-1R inhibitors such as NVP-AEW541,4
NVP-ADW742,5 BMS-554417,6 BMS-539264,7 PQIP,8 and OSI-
906.9 Because of the high homology between IGF-1R and the clo-
sely related insulin receptor (IR), small molecule IGF-1R inhibitors
typically have activity against IR as well. Since IGF-1R is an attrac-
tive target for cancer therapy, a program focused on the discovery
of novel small molecule inhibitors of IGF-1R was initiated.
An ongoing effort directed toward multi-targeted kinase inhibi-
tors identified an imidazo[1,2-a]pyridine lead series as exemplified
by compound 1, which had modest IGF-1R activity in both the IGF-
1R enzyme10 and the cellular mechanistic assay11 (Table 1). An
important early discovery in this effort was that reversal of the
amide connectivity resulted in a significant increase in IGF-1R po-
tency to the template. For example, 2 showed an approximately
20-fold improvement in both the enzyme and cellular mechanistic
assays compared to 1. Encouraged by this potency, further efforts to
optimize this series were made in the context of this amide
orientation.
Synthesis of these imidazo[1,2-a]pyridine inhibitors began with
commercially available isophthalic monoester 3 (Scheme 1). Treat-
ment with oxalyl chloride and DMF yielded the corresponding acid
chloride, which upon exposure to 2,6-difluoroaniline afforded the
aryl amide 4. Subsequent treatment with the anion derived from
pyrimidine 5 produced ketone 6 in high yield. Generation of the
reactive a-bromoketone followed by immediate treatment with 2-
aminopyridine resulted in cyclization to afford the imidazo
[1,2-a]pyridine intermediate 7. Acid-catalyzed reaction of 7 with a
variety of anilines 8 at elevated temperatures afforded the target
inhibitors 9. This transformation was carried out in either
* Corresponding author. Tel.: +1 165 936 8401.
E-mail address: kyle.a.emmitte@Vanderbilt.edu (K.A. Emmitte).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.058

K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1004–1008
1005
Table 1
Table 2
Early imidazo[1,2-a]pyridine leads
Aniline modifications
F
F
O
N
N
N
Y
N
N
X
H
F
N
F
N
N
R
N
NH
Compound
R
IGF-1R
IGF-1R
enzyme
IC50 (nM)
cellular
IC50 (nM)
a
a
N
10
9a
–NH2
440
28
>30,000
Compound
X
Y
IGF-1R enzyme IC50a (nM) IGF-1R cellular IC50a (nM)
H
N
1
2
C@O NH
NH C@O
180
10
5930
247
F
509
a
Data are n = 1.
H
N
O
*
9b
9c
9d
9e
9f
7
1
4
2
1
431
O
O
trifluoroethanol or isopropanol with either mild heating in a sealed
vessel or utilizing microwave irradiation.
H
N
177
174
344
N
Initial investigations into the structure–activity relationships of
the imidazo[1,2-a]pyrimidines focused on modification of the ani-
line attached to the pyrimidine ring, which binds the hinge of the
kinase (Table 2). Due to the potentially undesirable high molecular
weight of the inhibitors being generated, removal of the 2-aniline
was investigated. Disappointingly, this modification resulted in a
significant loss of potency (10 vs 2). Simple anilines (9a and 9b)
restored potency to moderate levels. Incorporation of anilines with
basic amine functionality further enhanced potency (9c–9f). For
example, the analog containing a 4-(1,40-bipiperidin-1-yl)aniline
group (9g) possessed an IC50 of less than 100 nM in the cellular
assay, a feature considered highly desirable. Cellular potency could
be further enhanced with the incorporation of a methoxy substitu-
ent at the 2-position of the aniline (9h).
N
O
N
H
N
H
N
OH
H
N
*
O
244
N
9g
2
2
87
38
N
H
N
N
Early analogs in this series were found to also inhibit Aurora B
kinase. Small molecule inhibitors of Aurora B have been shown
to inhibit cell division and compromise cell proliferation.12 Avoid-
ing the potential for masking or confusing the phenotype observed
from inhibiting IGF-1R was desirable. The 2-methoxy substituent
O
9h
N
H
N
N
a
Data are the average for n P 2, except for those with (*), which are n = 1.
O
O
F
O
O
a, b
OMe
HO
OMe
N
H
F
4
3
N
Cl
c
5
N
F
F
O
O
O
d
N
N
N
N
H
F
H
F
N
N
N
7
6
Cl
N
Cl
e
F
O
8
N
H2N
N
H
R
N
F
N
9
N
N
H
R
Scheme 1. Reagents and conditions: (a) (ClCO)2, DMF, CH2Cl2; (b) 2,6-difluoroaniline, pyridine, CH2Cl2 (84%, 2 steps); (c) LiN(SiMe3)2, THF (83%); (d) NBS, CH2Cl2, then
2-aminopyridine, dioxane, 60 °C (77%); (e) HCl or p-TSAꢀH2O, trifluoroethanol or isopropanol, 80–100 °C or 140–180 °C ( w) (50–90%).
l

1006
K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1004–1008
Table 3
Terminal amine SAR
NO2
F
NO2
F
OH
O
a
b
F
O
11
12
N
N
H
N
N
R
F
N
c
NO2
N
HO
N
N
H
O
13
O
Compound
R
IGF-1R enzyme IGF-1R cellular Mouse PO
d
DNAUCb
a
a
NO2
N
IC50 (nM)
IC50 (nM)
O
9h
16
2
2
38
41
239
O
N
14
276
N
N
O
R1
R2
R3 = NO2
R3 = NH2
R3
N
N
e
17
18
2
2
38
40
121
494
O
15
F
N
N
Scheme 2. Reagents and conditions: (a) K2CO3, MeI, DMF (92%); (b) 4-piperidinol,
K2CO3, DMSO (99%); (c) (ClCO)2, DMSO, NEt3, CH2Cl2, ꢁ78 °C to rt (97%); (d) NEt3,
AcOH, NaBH(OAc)3, HN(R1)R2, DCE or PhMe (60–80%); (e) hydrogenation or
NiCl2ꢀ6H2O, NaBH4, MeOH (50–90%).
19
20
21
2
4
28
68
203
549
N
O
N
found in 9h imparted a level of selectivity over Aurora B not previ-
ously observed (Aurora B enzyme IC50 = 9 nM and 130 nM for 9g
and 9h, respectively). The anilino-pyrimidine moiety of the mole-
cule contacts the hinge of the kinase ATP binding site, and the
methoxy substituent is believed to sterically conflict with Tyr156
of Aurora B. The corresponding residue in IGF-1R is Leu1078, which
is smaller and able to accommodate the methoxy substituent.
Oral dosing of compound 9h (7.3 mg/kg) in mice13 resulted in
lower than desirable plasma levels (DNAUC = 239 ng h/mL/(mg/
kg)). In an effort to further improve the oral exposure of these mol-
ecules, a strategy focused on variation of the terminal piperidine
ring was initiated. The preferred synthetic route for the requisite
aniline intermediates began with methylation of commercially
available phenol 11 (Scheme 2). Addition of 4-piperidinol to 12
afforded the nitrobenzene intermediate 13. Swern oxidation14 of
alcohol 13 afforded ketone 14 which was subsequently reacted
with the appropriate secondary amine in the presence of sodium
triacetoxyborohydride to afford 4-aminopiperidines 15. The final
reduction of the nitro group to the corresponding anilines 15 was
accomplished using either a catalytic hydrogenation or a combina-
tion of nickel chloride and sodium borohydride.15 Alternatively,
commercially available or custom made 4-aminopiperidines could
be coupled with intermediate 12. The highlighted route was con-
sidered superior as chemical diversity was introduced later in the
synthetic sequence.
Variation of the terminal amine yielded some encouraging re-
sults (Table 3). In some cases the exposure was similar or inferior
to that observed with 9h (16, 17, 19, and 21); however, there were
clear examples of improved oral exposure (18, 20, and 22). In each
case the molecules with improved exposure were among those
with less basic terminal amine groups. Certainly, reducing the ba-
sicity of the amine did not guarantee improved oral exposure (16,
19, and 21), but it did appear to increase its probability. It is possi-
ble that in the more neutral environment of the gut, a higher pro-
portion of these amines are in their uncharged state leading to
better permeability and thus better absorption.16 It is unlikely that
the dramatic difference in oral exposure between 9h and 22 is due
to reduced metabolism as clearance in mice was low for both mol-
ecules (Cl = 13.4 and 13.6 mL/min/kg for 9h and 22, respectively).
O
3
5
65
36
216
N
N
N
N
O
22
1060
O
S
a
Data are the average for n P 2.
b
DNAUC units are ng h/mL/(mg/kg) determined from 0 to 6 h.
As expected, sulfonamide 22 was a potent dual inhibitor of both
IGF-1R and IR; however, the non-familial kinase activity also re-
mained fairly broad in nature (Table 4).17 Further optimization of
the selectivity profile was accomplished through additional modifi-
Table 4
Final optimization of selectivity
F
O
O
S
O
N
N
N
N
N
N
H
R2
O
F
R1
N
N
N
H
a
Kinase
IC50 (nM)
22
23
24
R
R
1 = H
2 = H
R1 = H
R2 = Et
R1 = OMe
R2 = Et
IGF-1R
IR
EGFR
ErbB4
ErbB2
B-Raf V600E
Aurora B
p-38
5
5
7
7
22
27
25
57
74
72
80
200
440
1600
1200
>10,000
>25,000
>10,000
> 2000
4500
11,000
>1500
>3000
140*
54
320
1300
1800*
>9500
710*
a
Aurora A
LCK
a
Data are the average for n P 2, except for those with (*), which are n = 1.

K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1004–1008
1007
Table 6
cations of the template. For example, incorporation of an ethyl group
PK profile of 24 in various species
at the 5-position of the aniline (23) resulted in a compound with
similar potency at the desired target (IGF-1R cellular IC50 = 66 nM).
However, this small change improved selectivity against multiple
kinases although potency against the ErbB family kinases and LCK
remain similar to 22. Addition of a methoxy substituent at the 4-po-
sition of the inner phenyl ring further enhances selectivity (24).
Although the enzyme potency of 24 against IGF-1R
(IC50 = 27 nM) is lower than earlier compounds such as compound
22 (IC50 = 5 nM), cellular potency was actually improved (IGF-1R
cellular IC50 = 22 nM). Compound 24 was confirmed to be an
ATP-competitive inhibitor; however, kinetic studies showed that
both the association and disassociation rates for this compound
PO dose mg/kg
Cl mL/min/kg
VSS L/kg
PO DNAUCa
F (%)
Rat
Dog
Monkey
3.0
7.2
2.1
5.8
8.7
13.9
7.5
4.7
9.8
1220
870
1070
63
47
97
a
DNAUC units are ng h/mL/(mg/kg).
kinase, indicative of an ‘inactive-like’ conformation of the protein.
It is possible that this binding mode contributes to the aforemen-
tioned slow kinetics seen with this inhibitor. This type of binding
is a characteristic of type II kinase inhibitors and similar to those
observed with marketed drugs such as imatinib, sorafenib, and
lapatinib.20
Compound 24 was further profiled in additional cellular assays
(Table 5). Not surprisingly, it was an equipotent inhibitor of both
IGF-1R and IR phosphorylation in the mechanistic assays. A broad-
er panel of tumor cell lines representing various tumor types were
treated with 24 in order to measure the compound’s ability to in-
hibit cellular proliferation.21 The potency was excellent in a num-
ber of cell lines representing multiple tumor types, such as
myeloma, sarcoma and colon. Certain cell lines such as A549 lung
showed no response to treatment with 24. Interestingly, 24
showed good selectivity towards normal HFF cell line when com-
pared to most tumor cell lines.
Oral exposure of 24 in mice was similar to 22
(DNAUC = 1150 ng h/mL/(mg/kg)). Additional pharmacokinetic
properties of 24 were evaluated in multiple species (Table 6).22
The molecule proved to be a low clearance compound with good
to excellent oral bioavailability. It is worth noting that the encour-
aging oral exposure observed with 24, given its rather large molec-
ular weight (MW = 851), certainly places it outside the range that is
considered desirable (6500).23 Given the structural requirements
necessary to achieve sufficient potency and selectivity within this
chemical series, significant optimization was necessary in order
to identify 24, which demonstrated a considerably better pharma-
cological profile than would be predicted based on its large molec-
ular weight.
with IGF-1R and IR were quite slow (t > 180 min). As the standard
½
enzyme assay does not include a pre-incubation of enzyme and
inhibitor and is only one hour in length, the potency of 24 in this
assay was therefore underestimated. Assays which included pre-
incubation of enzyme and compound were developed to more
accurately reflect inhibitor potency.18 The apparent Ki values for
24 in these assays were 1.6 0.1 nM and 1.3 0.1 nM against
IGF-1R and IR, respectively. Apparent Ki values for 22 were much
closer to the enzyme IC50 values, 5.2 0.8 nM and 2.2 0.3 nM
against IGF-1R and IR, respectively.
A crystal structure of 24 in an IR double mutant (C981S,
D1132N) was solved to 2.2 Å (Fig. 1).19 As expected, the anilino-
pyrimidine contacts the b-strand, with the piperazino-piperidine
oriented in the direction of the solvent exposed area. The 2,6-diflu-
oroamide head group is oriented deep into the back pocket of the
In conclusion, a highly selective, potent inhibitor of IGF-1R and
IR was discovered in an imidazo[1,2-a]pyrimidine chemical series.
Although inhibition of IR has the potential for metabolic alteration,
recent evidence points to a role for IR signaling in cancer, suggest-
ing a potential therapeutic advantage of inhibiting both IGF-1R and
IR.24 Should an IGF-1R inhibitor with substantial selectivity over IR
ultimately prove necessary, strategies directed toward the design
of inhibitors that bind outside of the ATP binding site may be re-
quired. Optimization of mouse oral exposure was used as a tool
in the imidazo[1,2-a]pyrimidine chemical series for the ultimate
identification of 24, a compound with good oral exposure in multi-
ple pre-clinical species. Further communications regarding the bio-
logical profile of 24 both in vitro and in vivo will be forthcoming in
the near future.
Figure 1. X-ray crystal of 24 bound to IR double mutant.
Table 5
Profile of 24 in cellular assays
a
Target
IC50 (nM)
Mechanistic assays (receptor autophosphorylation)
IGF-1R
IR
22
19
References and notes
a
Cell line
Tumor type
IC50 (nM)
1. (a) LeRoitho, D.; Roberts, C. T., Jr. Cancer Lett. 2003, 195, 127; (b) Baserga, R.;
Peruzzi, F.; Reiss, K. Int. J. Cancer 2003, 107, 873; (c) Bohula, E. A.; Playford, M.
P.; Macaulay, V. M. Anticancer Drugs 2003, 14, 669.
2. (a) Hartog, H.; Wesseling, J.; Boezen, H. M.; van der Graaf, W. T. A. Eur. J. Cancer
2007, 43, 1895; (b) Sachdev, D.; Yee, D. Mol. Cancer Ther. 2007, 6, 1.
3. (a) Sarma, P. K. S.; Tandon, R.; Gupta, P.; Dastidar, S. G.; Ray, A.; Das, B.; Cliffe, I. A.
Expert Opin. Ther. Patents 2007, 17, 25; (b) Garcia-Echeverria, C. IDrugs 2006, 9,
415; (c) Yuan, J. S. P.; Macaulay, V. M. Expert Opin. Ther. Targets 2008, 12 5, 589.
4. (a) Piao, W.; Wang, Y.; Adachi, Y.; Yamamoto, H.; Li, R.; Imsumran, A.; Li, H.;
Maehata, T.; Ii, M.; Arimura, Y.; Lee, C.-T.; Shinomura, Y.; Carbone, D. P.; Imai, K.
Mol. Cancer Ther. 2008, 7, 1483; (b) Maiso, P.; Ocio, E. M.; Garayoa, M.; Montero,
Proliferation assays
NCI-H929
LP-1
TC-71
SK-ES
COLO205
A549
HFF
Multiple myeloma
Multiple myeloma
Ewing’s sarcoma
Ewing’s sarcoma
Colon
81
104
35
61
124
Lung
>20,000
>20,000
Normal foreskin fibroblast
a
Data are the average for n P 2.

1008
K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1004–1008
J. C.; Hofmann, F.; Garcia-Echeverria, C.; Zimmermann, J.; Pandiella, A.; San
13. Low dose mouse PK studies were conducted with a 1% DMSO, 10% solution in
water formulation. Composite sampling was used with three mice per time
point. AUC was determined from 0 to 6 h.
14. Swern, D.; Mancuso, A. J.; Huang, S. J. Org. Chem. 1978, 43, 2480.
15. Nose, A.; Kudo, T. Chem. Pharm. Bull. 1981, 29, 1159.
Miguel, J. F. Br. J. Haemetol. 2008, 141, 470; (c) Manara, M. C.; Landuzzi, L.;
Nanni, P.; Nicoletti, G.; Zambelli, D.; Lollini, P. L.; Nanni, C.; Hofmann, F.;
Garcia-Echeverria, C.; Picci, P.; Scotlandi, K. Clin. Cancer Res. 2007, 13, 1322; (d)
Tanno, B.; Mancini, C.; Vitali, R.; Mancuso, M.; McDowell, H. P.; Dominici, C.;
Raschella, G. Clin. Cancer Res. 2006, 12, 6772; (e) Scotlandi, K.; Manara, M. C.;
Nicoletti, G.; Lollini, P.-L.; Lukas, S.; Benini, S.; Croci, S.; Perdichizzi, S.; Zambelli,
D.; Serra, M.; Garcia-Echeverria, C.; Hofmann, F.; Picci, P. Cancer Res. 2005, 65,
3868; (f) Garcia-Echeverria, C.; Pearson, M. A.; Marti, A.; Meyer, T.; Mestan, J.;
Zimmerman, J.; Gao, J.; Brueggen, J.; Capraro, H.-G.; Cozens, R.; Evans, D. B.;
Fabbro, D.; Furet, P.; Porta, D. G.; Liebetanz, J.; Martiny-Baron, G.; Ruetz, S.;
Hofmann, F. Cancer Cell 2004, 5, 231.
16. Avdeef, A. Curr. Top. Med. Chem. 2001, 1, 271.
17. Other kinases were screened using conditions and assays optimized for each.
18. A filter binding assay was used for apparent Ki determinations. IGF-1R and IR
kinase domains were activated by pre-incubating at 500 nM in assay buffer A
[50 mM Hepes, pH 7.2, 10 mM MgCl2, 0.2 mg/ml BSA, and 1ꢂ phosphatase
inhibitor cocktail II (Sigma–Aldrich)] with 1 mM ATP for 30 min. Reactions
typically contained (in 40
Hollister, CA), and 7.5
Marlboro, MA; 1ꢂ Km and 1.5ꢂ Km concentration, respectively, for both
IGF1R and IR) and
33P]ATP (100–500 cpm/pmol, 10 mCi/mL in 10 mM
lL) 0.1 nM activated enzyme, 40
lM ATP (Teknova,
5. Mitsiades, C. S.; Mitsiades, N. S.; McMullan, C. J.; Poulaki, V.; Shringarpure, R.;
Akiyama, M.; Hideshima, T.; Chauhan, D.; Joseph, M.; Libermann, T. A.; Garcia-
Echeverria, C.; Pearson, M. A.; Hofmann, F.; Anderson, K. C.; Kung, A. L. Cancer
Cell 2004, 5, 221.
6. Haluska, P.; Carboni, J. M.; Loegering, D. A.; Lee, F. Y.; Wittman, M.; Saulnier, M.
G.; Frennesson, D. B.; Kalli, K. R.; Conover, C. A.; Attar, R. M.; Kaufmann, S. H.;
Gottardis, M.; Erlichman, C. Cancer Res. 2006, 66, 362.
lM peptide substrate (21st Century Biochemicals,
[c-
Tricine, Perkin-Elmer). The peptide substrate had the same sequence as that
used for the time-resolved fluorescence assay, but without the biotin moiety.
Inhibitor dilutions were prepared in DMSO and dispensed (1
lL/well) into the
wells of 96-well, half-area nonbinding surface (NBS) plates (Corning,
a
7. (a) Zimmermann, K.; Wittman, M. D.; Saulnier, M. G.; Velaparthi, U.; Langley, D.
R.; Sang, X.; Frennesson, D.; Carboni, J.; Li, A.; Greer, A.; Gottardis, M.; Attar, R.
M.; Yang, Z.; Balimane, P.; Discenza, L. N.; Vyas, D. Bioorg. Med. Chem. Lett. 2008,
18, 4075; (b) Wittman, M.; Carboni, J.; Attar, R.; Balasubramanian, B.; Balimane,
P.; Brassil, P.; Beaulieu, F.; Chang, C.; Clarke, W.; Dell, J.; Eummer, J.;
Frennesson, D.; Gottardis, M.; Greer, A.; Hansel, S.; Hurlburt, W.; Jacobson,
B.; Krishnananthan, S.; Lee, F. Y.; Li, A.; Lin, T.-A.; Liu, P.; Ouellet, C.; Sang, X.;
Saulnier, M. G.; Stoffan, K.; Sun, Y.; Velaparthi, U.; Wong, H.; Yang, Z.;
Zimmermann, K.; Zoeckler, M.; Vyas, D. J. Med. Chem. 2005, 48, 5639.
8. Ji, Q-S.; Mulvihill, M. J.; Rosenfeld-Franklin, M.; Cooke, A.; Feng, L.; Mak, G.;
O’Connor, M.; Yao, Y.; Pirritt, C.; Buck, E.; Eyzaguirre, A.; Arnold, L. D.; Gibson,
N. W.; Pachter, J. A. Mol. Cancer Ther. 2007, 6, 2158.
9. Rosenfeld-Franklin, M.; Cooke, A.; Pirritt, C.; Landfair, D.; Silva, S.; Turton, R.;
Feng, L.; Mulvihill, M. J.; Buck, E.; Pachter, J. A.; Ji, Q.-S.; Wild, R. Abstract of
Papers, AACR-NCI-EORTC International Conference: Molecular Targets and
Cancer Therapeutics, San Francisco, CA; American Association for Cancer
Research: Philadelphia, PA, National Cancer Institute: Bethesda, MD, and
European Organisation for Research and Treatment of Cancer: Brussels,
Belgium, 2007; Abstract B244.
Corning, NY). Reaction mix containing ATP and peptide was added to the
plate and reactions were initiated by addition of enzyme mix. Reactions were
quenched after 240 min by addition of 1% H3PO4. For compound 24, reaction
conditions were altered slightly due to slow inhibition kinetics. Final enzyme
concentration was increased to 0.5 nM. Reaction mix containing enzyme and
ATP was added and incubated with inhibitor for 6 h to allow the inhibitor–
enzyme binding to reach equilibrium. Reactions were initiated by the
addition of the reaction mix containing ATP and peptide substrate. The
reactions were quenched after 120 min. After quenching, all reactions were
filtered through MAPHN0B50 filter plates (Millipore), washed, and the
product was quantified by liquid scintillation counting using MicroScint-20
(Perkin-Elmer) in a MicroBeta-1450 plate-reader (Perkin-Elmer). IC50 values
were determined using a two-parameter fit (Hill coefficient and IC50) using
GraFit software (Erithacus, Surrey, UK). The apparent Ki* values were
calculated from the IC50 values using the Cheng–Prusoff relationship for
ATP competitive inhibition.
19. IR protein was expressed and purified as previously described (Li, S.; Covino, N.
D.; Stein, E. G.; Till, J. H.; Hubbard, S. R. J. Biol. Chem. 2003, 278, 26007). Protein
at 10 mg/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:1P14 as a starting model.
10. Enzyme assays: Baculovirus-expressed GST-tagged proteins encoding the
intracellular domain of IGF-1R (amino acids 957–1367) and IR (amino acids
979–1382) were used for determination of IC50s. Kinases were activated by
pre-incubating the enzyme (2.7 lM final concentration) in 50 mM Hepes, pH
7.5, 10 mM MgCl2, 0.1 mg/mL BSA, 2 mM ATP (all chemical reagents from
Sigma–Aldrich, St. Louis, MO, unless otherwise noted). Compounds were
diluted in dimethyl sulfoxide (DMSO) and dispensed into the assay plates
20. (a) Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2, 358; (b) Wood, E. R.; Truesdale, A.
R.; McDonald, O. B.; Yuan, D.; Hassell, A.; Dickerson, S. H.; Ellis, B.; Pennisi, C.;
Horne, E.; Lackey, K.; Alligood, K.; Rusnak, D. W.; Gilmer, T. M.; Shewchuk, L.
Cancer Res. 2004, 64, 6652.
(100 nL/well). Kinase reactions contained (in 10
lL volume) 50 mM Hepes, pH
7.5, 3 mM DTT, 0.1 mg/mL BSA, 1 mM CHAPS, 10 mM MgCl2, 10
500 nM substrate peptide (biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2;
QPC, Hopkinton, MA) and 0.5 nM activated enzyme. Reactions were stopped
l
M ATP,
21. Cell proliferation assays: Cells were seeded in 96-well dishes, incubated
overnight at 37 °C, and treated with various concentrations of inhibitors for
72 h. Cell proliferation was quantified using the CellTiter-Glo Luminescent Cell
Viability Assay (Promega, Madison, WI), following the manufacturer’s
recommendations. IC50 values were determined from using a 4-parameter
curve fit software package (XLfit4).
after 1 h at room temperature with 33 lM EDTA. Peptide phosphorylation was
measured by time resolved fluorescence with 7 nM streptavidin-SurelightTM
allophycocyanin (Perkin-Elmer, Waltham, MA) and 1 nM Europium-
conjugated phospho-tyrosine antibodies (Perkin-Elmer). Plates were read in
a Victor X5 or Viewlux 1430 ultra HTS microplate imager (Perkin-Elmer).
11. Cellular mechanistic assays: NIH-3T3-hIGF-1R and NIH-3T3-hIR cell lines were
seeded in culture medium in collagen-coated 96-well tissue culture plates (BD
Biosciences, Franklin Lakes, NJ). After 24 h, cells were treated with DMSO or
various concentrations of inhibitors and stimulated 2 h later with 30 ng/ml
22. PK studies were conducted as a solution in 1.0% DMSO, 20% CaptisolÒ in
saline (IV) or water (PO). The PO study in dogs was run as
a partial
suspension. IV doses ranged from 0.6 to 1.3 mg/kg, and PO doses ranged
from 2.1 to 7.2 mg/kg depending on species. Rats were male Sprague–
Dawleys. Dogs were male beagles. Monkeys were male cynomolgus.
Reported data are for 0–24 h.
human IGF-1 or 3
l
g/mL bovine insulin (Sigma–Aldrich) for 15 min. Cells were
23. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Freeney, P. J. Adv. Drug Deliv. Rev.
1997, 23, 3.
lysed in RIPA buffer (Roche Diagnostics, Indianapolis, IN) and lysates were
transferred to wells of 96-well assay plates (MaxiSorpTM, NalgeNunc, Rochester,
NY) coated with either anti-IGF-1R (R&D Systems, Minneapolis, MN) or anti-
IRb (Santa Cruz Biotechnology, Santa Cruz, CA). Plates were incubated at 4 °C,
overnight, washed and incubated with Eu-labeled phospho-tyrosine antibody
PT66 (Perkin-Elmer, Waltham, MA) for the detection of phosphorylated IGF-1R
and IR. Total IGF-1R and IR were detected with anti-IGF-1Rb (Santa Cruz
Biotechnology, Santa Cruz, CA) or anti-IR (Lab Vision/Thermo Fisher Scientific,
Fremont, CA), respectively. Secondary antibodies used were Eu-labeled goat
anti-rabbit IgG (for IGF-1R) and Eu-labeled goat anti-mouse IgG (for IR)
24. (a) Belfiore, A. Curr. Pharm. Des. 2007, 13, 671; (b) Denley, A.; Carroll, J.
M.; Brierley, G. V.; Cosgrove, L.; Wallace, J.; Forbes, B.; Roberts, C. T., Jr.
Mol. Cell Biol. 2007, 27, 3569; (c) Jones, H. E.; Gee, J. M. W.; Barrow, D.;
Tonge, D.; Holloway, B.; Nicholson, R. I. Br. J. Cancer 2006, 95, 172–180;
(d) Denley, A.; Wallace, J. C.; Cosgrove, L. J.; Forbes, B. E. Horm. Met. Res.
2003, 35, 778; (e) Vella, V.; Pandini, G.; Sciacca, L.; Mineo, R.; Vigneri, R.;
Pezzino, V.; Belfiore, A. J. Clin. Endocrinol. Metab. 2002, 87, 245; (f) Kalli,
K.; Falowo, O. I.; Bale, L. K.; Zschunke, M. A.; Roche, P. C.; Conover, C. A.
Endocrinology 2002, 143, 3259; (g) Sciacca, L.; Mineo, R.; Pandini, G.;
Murabito, A.; Vigneri, R.; Belfiore, A. Oncogene 2002, 21, 8240; (h) Pandini,
G.; Frasca, F.; Mineo, R.; Sciacca, L.; Vigneri, R.; Belfiore, A. J. Biol. Chem.
2002, 277, 39684; (i) Sciacca, L.; Costantino, A.; Pandini, G.; Mineo, R.;
Frasca, F.; Scalia, P.; Sbraccia, P.; Goldfine, I. D.; Vigneri, R.; Belfiore, A.
Oncogene 1999, 18, 2471; (j) Frasca, F.; Pandini, G.; Scalia, P.; Sciacca, L.;
Mineo, R.; Costantino, A.; Goldfine, I. D.; Belfiore, A.; Vigneri, R. Mol. Cell
Biol. 1999, 19, 3278; (k) Pandini, G.; Vigneri, R.; Costantino, A.; Frasca, F.;
Ippolito, A.; Fujita-Yamaguchi, Y.; Siddle, K.; Goldfine, I. D.; Belfiore, A.
Clin. Cancer Res. 1999, 5, 1935.
(Perkin-Elmer). Eu-fluorescence was quantified using
a Victor Multilabel
Counter (Perkin-Elmer; excitation 340 nm/emission 615 nm). The ratio of
phosphorylated kinase to total kinase was determined, and concentration–
response curves were plotted. IC50 values were determined from inhibition
curves using XLfit4 software (Guildford, UK).
12. (a) Girdler, F.; Gascoigne, K. E.; Eyers, P. A.; Hartmuth, S.; Crafter, C.; Foote, K.
M.; Keen, N. J.; Taylor, S. S. J. Cell Sci. 2006, 119, 3664; (b) Carvajal, R. D.; Tse, A.;
Schwartz, G. K. Clin. Cancer Res. 2006, 12, 6869; (c) Keen, N.; Taylor, S. Nat. Rev.
Cancer 2004, 4, 927.