Discovery of 2,4-bis-arylamino-1,3-pyrimidines as insulin-like growth factor-1 receptor (IGF-1R) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.02.075

The insulin-like growth factor-1 receptor (IGF-1R) plays an important role in the regulation of cell growth and differentiation, and in protection from apoptosis. IGF-1R has been shown to be an appealing target for the treatment of human cancer. Herein, w

Full text of DOI:10.1016/j.bmcl.2011.02.075

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2394–2399
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2,4-bis-arylamino-1,3-pyrimidines as insulin-like growth
factor-1 receptor (IGF-1R) inhibitors
John L. Buchanan a, , John R. Newcomb d, David P. Carney b, Stuart C. Chaffee a, Lilly Chai d, Rod Cupples h,
⇑
Linda F. Epstein c, Paul Gallant b, Yan Gu c, Jean-Christophe Harmange a, Kathy Hodge b, Brett E. Houk e,
Xin Huang c, Janan Jona g, Smriti Joseph g, H. Toni Jun d, Rakesh Kumar d, Chun Li e, John Lu d, Tom Menges e,
Michael J. Morrison b, Perry M. Novak a, Simon van der Plas a, Robert Radinsky d, Paul E. Rose c, Satin Sawant f,
Ji-Rong Sun d, Sekhar Surapaneni e, Susan M. Turci d, Keyang Xu e, Evelyn Yanez g, Huilin Zhao c,
Xiaotian Zhu c
a Department of Medicinal Chemistry, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA
b Department of HTS & Molecular Pharmacology, Amgen Inc., 360 Binney Street, Cambridge, MA 02142 and Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Department of Molecular Structure, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA
d Department of Oncology Research, Amgen Inc., 360 Binney Street, Cambridge, MA 02142 and Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
e Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
f Department of Toxicology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
g Department of Pharmaceutics, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
h Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The insulin-like growth factor-1 receptor (IGF-1R) plays an important role in the regulation of cell growth
and differentiation, and in protection from apoptosis. IGF-1R has been shown to be an appealing target for
the treatment of human cancer. Herein, we report the synthesis, structure–activity relationships (SAR),
X-ray cocrystal structure and in vivo tumor study results for a series of 2,4-bis-arylamino-1,3-pyrimidines.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 11 August 2010
Revised 16 February 2011
Accepted 17 February 2011
Available online 23 February 2011
Keywords:
Insulin-like growth factor-1 receptor
IGF-1R
Kinase
Pyrimidine
Calu-6 xenograft
X-ray cocrystal structure
The insulin-like growth factor-1 receptor (IGF-1R) belongs to
the insulin family of growth factors and receptors, and includes
the insulin receptor (InsR), insulin-like growth factors 1 and 2
(IGF-1 and IGF-2), and six insulin-like growth factor binding pro-
teins (IGFBP). Activation of IGF-1R by IGF-1 or IGF-2 leads to acti-
vation of both the Ras/Raf/MEK/ERK and PI3K/Akt/mTOR pathways,
promoting proliferation and inhibiting apoptosis.1 Conversely,
inhibition of IGF-1R results in decreased proliferation of tumor
cells and therefore represents an attractive oncology drug target.
To date, IGF-1R is well established as an oncology drug target with
a number of small molecules and antibodies currently undergoing
clinical trials, including ganitumab (AMG 479).2 The rationale for
targeting the IGF-1R pathway as well as early results from these
clinical trials have been well reviewed.3 In parallel with our
antibody efforts, we initiated a small molecule drug discovery pro-
gram. Herein we disclose the discovery of 2,4-bis-arylamino-1,3-
pyrimidines as IGF-1R inhibitors.
Screening of our kinase-preferred collection identified a number
of hits for our small molecule program. Among those hits were
many from the 2,4-bis-arylamino-1,3,5-triazine class of com-
pounds4 where several showed modest potency in our enzyme5
(double-digit nM) and cellular6 assays (ꢀ1
lM). More focused
screening identified triazines 1 and 2 as preferred compounds from
this series (Table 1). Replacement of the triazine core (X = N) in 1
and 2 with the pyrimidine7 core (X = CH) in 3 and 4 resulted in a
marked improvement in enzyme and cellular potency. This
improvement in potency can be explained by the pyrimidine
CH’s enhanced compatibility with the adjacent hydrophobic
⇑
Corresponding author. Tel.: +1 617 444 5031; fax: +1 617 621 3907.
E-mail address: john.buchanan@amgen.com (J.L. Buchanan).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.075

J. L. Buchanan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2394–2399
2395
Table 1
Initial assessment of screening hits 1 and 2 (IC50, nM)a
6
5
1
N
X
HN
N
O
R
2
4
O
O
X
R
IGF-1R IC50
41
huIGF-1R/3T3 IC50
175
N
N
1
2
3
4
5
N
N
H
N
13
409
19
N
H
Figure 1. Cocrystal structure of pyrimidine 8 in IGF-1R0P.
CH
CH
CH
2.5
2.2
48
N
H
making important hydrophobic interactions between its CH3 and
the quinoline bridging carbons (3.41 and 3.30 Å) and between its
CH2 and the quinoline nitrogen atom (3.35 Å). The sulfur atom
serves to position the CH3–CH2 carbons appropriately. Finally, the
morpholine group on the aniline ring is directed toward the sol-
vent front, while the adjacent methoxy group occupies a hydro-
phobic region formed by the side chains of Met1156 (3.20 Å) and
Met1142 (3.49 Å).
88
N
H
N
N
420
N
H
At this point, we decided to expand our examination of
the structure–activity relationships (SAR), focusing first on the
pyrimidine 4-position. Several bicyclic ring replacements were
examined, many of which displayed desirable enzyme and cellu-
lar potency as demonstrated by isoquinoline 9 and quinoline 10
(Table 2). Insertion of an additional nitrogen atom into quinoline
10 provided quinoxaline 11 which showed reduced potency. NH-
containing bicyclic ring replacements such as indazoles 12 and
13, benzimidazole 14 and indoline 15 resulted in significant
losses in potency. Attempts to replace the bicyclic ring with pyr-
idyl (16) and/or substituted phenyl (17) rings resulted in reduced
enzyme and cellular potency as compared to quinoline 3. The
loss in potency resulting from decreasing the lipophilicity of
the bicyclic ring (11–15) or from excising the outer ring (16 vs
9) demonstrated the importance of the hydrophobic interactions
between the side chain of Met1156 and the quinoline-type bicy-
clic rings.
We next turned our attention to the pyrimidine 2-position,
fixing the 4-position with 3-aminoquinoline while examining
the results of modifications to the trimethoxyaniline (Table 3).9
The removal of methoxy groups from compound 3, resulted in
losses in both enzyme and cellular potency as exemplified by
dimethoxy (18, 19) and monomethoxy (20, 21) arylamines.
Modification of the methoxy group of 21 to trifluoromethoxy re-
sulted in a significant loss in potency (22). 3-Chloro arylamine 23
displayed similar enzyme potency but slightly reduced cellular
potency when compared to 3-methoxy arylamine 21. Incorpora-
tion of a methoxy group at the 4-position of 23 afforded 24 which
exhibited improved cellular potency. Guided by the observation
that substituents at the 4-position of these arylamines were direc-
ted toward the solvent front, we decided to incorporate several
solubilizing substituents in lieu of the methoxy at the 4-position
toward improving cellular potency10 and decreasing O-demethyl-
ation.9 Toward this end, morpholine-containing compound 25 and
tethered piperidine analog 26 displayed improved cellular shifts
as compared to other 4-substituted arylamines (i.e., 20). Introduc-
tion of the 3-methoxy group to 25 provided 8 which resulted in
6
7
CH
CH
725
933
N
Me
N
O
a
IC50 values are means of two or more separate determinations, in duplicate.
environment (vide infra). Based on these results, additional
optimization efforts on 2,4-bis-arylamino-1,3-pyrimidines were
undertaken.
The effects of additional minor changes around the central core
can be found in Table 1. Addition of a methyl group on the quin-
oline ring (5) resulted in a loss in both enzyme and cellular
potency relative to quinoline 3. Replacement of the 4-position
N–H with either N–Me (6) or O (7) resulted in a significant loss
in enzyme potency. These results can be explained by examining
the cocrystal structure of pyrimidine 8 (Table 3) bound to IGF-1R
(Fig. 1).
As shown in Figure 1, pyrimidine 8 (Table 3) binds to unphos-
phorylated IGF-1R (IGF-1R0P) in a U-shaped conformation.8 Key
interactions between pyrimidine 8 and IGF-1R include the typical
donor–acceptor–donor triad interaction with the hinge region.
Met1082 donates a backbone N–H to the pyrimidine N1 acceptor
(3.08 Å) while its carbonyl accepts the pyrimidine 2-position
N–H (3.00 Å). The carbonyl of Glu1080 accepts the pyrimidine
6-position C–H (2.92 Å). The pyrimidine 4-position N–H interacts
with Asp1153 from the DFG motif of the activation loop (3.07 Å).
Disruption of this interaction occurs in 5 resulting in a modest loss
in potency. The importance of this interaction is demonstrated
more markedly in the significant loss of potency seen in both
N–Me analog 6 and ether 7. The side chain of the gatekeeper
Met1079 is positioned adjacent to the pyrimidine 5-position
(3.63 Å), explaining the preference for pyrimidine (CH) over tri-
azine (N). Additionally, the side chain of Met1156 from the DFG
motif of the activation loop sits directly below the quinoline ring

2396
J. L. Buchanan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2394–2399
Table 2
Table 3
SAR of pyrimidine 4-position substituents (IC50, nM)a
SAR of pyrimidine 2-position substituents (IC50, nM)a
N
N
N
HN
O
N
O
R
R
N
N
H
R
IGF-1R IC50
2.5
huIGF-1R/3T3 IC50
19
O
HN
3
R
IGF-1R IC50
2.5
huIGF-1R/3T3 IC50
19
O
O
O
N
HN
3
9
N
H
18
19
20
18
20
55
507
O
N
O
HN
5.2
3.3
14
37
N
H
6453b
N
O
O
10
HN
N
H
17383
1044
N
N
O
O
11
12
58
448
N
H
HN
21
22
23
40
N
103
9219b
N
H
N
H
HN
H
N
198
41
F3CO
HN
N
13
14
15
34
5121
4221
490
N
H
3377
356
N
Cl
201
123
N
H
N
H
HN
24
25
26
8
36
22
19
2.4
3.8
Cl
O
N
H
N
H
HN
N
177
81
16
17
251
30
N
O
N
H
HN
1651b
N
H
OCF3
a
N
O
IC50 values are means of two or more separate determinations, in duplicate.
huIGF-1R/Rat1 proliferation IC50
b
.
HN
O
8.4
N
O
single digit nM enzyme and cellular potency. Introduction of the
3-methoxy group to 26 afforded 27, which displayed improved
enzyme potency with a slight improvement in cellular potency.
The improvements in potency demonstrated by compounds 8
and 27 supports the importance of the hydrophobic interaction
of the 3-methoxy group with the Met1156 and Met1142 side
chains as shown in the cocrystal structure of 8 and IGF-1R (Fig. 1).
Based on their in vitro profiles, several pyrimidines were
selected for rat pharmacokinetic (PK) studies.11 Pyrimidine
4-position analogs 4, 9 and 13 (Tables 1 and 2) were cleared at
rates near or greater than hepatic blood flow (2.4–4.7 L/h/kg).
Pyrimidine 2-position analogs 19, 24 and 8 (Table 3) were also
cleared at rates near or greater than hepatic blood flow (2.9–
9.5 L/h/kg). Compound 27 displayed some improvement, but
was cleared at a rate of 1.9 L/h/kg. In contrast, lower clearance
rates and generally improved PK properties were observed with
trimethoxyaniline-containing quinolines 10 and 3 and tethered
piperidine 26 (Table 4). Compound 3 exhibited a low clearance
rate (0.4 L/h/kg) and acceptable oral bioavailability (33%)
HN
O
27
36
O
N
a
IC50 values are means of two or more separate determinations, in duplicate.
huIGF-1R/Rat1 proliferation IC50
b
.
Table 4
Rat pharmacokinetics for selected compoundsa
iv/po dose (mg/kg)
CL (L/h/kg)
Vss (L/kg)
T1/2 (h)
F (%)
10
3
26
1/5
1/5
2/5
1.2
0.4
0.6
1.1
0.2
9.5
1.3
1.7
12.1
16
33
9
a
Dosed as a solution in DMSO (iv) or as a suspension (po) in 1% Tween 80 and 1%
HPMC in water.

J. L. Buchanan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2394–2399
2397
Table 5
Table 7
Mouse pharmacokinetics for selected compoundsa
Kinase selectivity data for compounds 3 and 26
a
a
iv/po dose (mg/kg)
CL (L/h/kg)
Vss (L/kg)
T1/2 (h)
F (%)
Kinase
3 IC50
(l
M)
Sel. ratio
26 IC50
(l
M)
Sel. ratio
10
3
26
1/5
1/5
12/30
6.4
1.6
5.9
1.8
0.3
28.1
2.3
1.7
5.3
56
63
30
IGF-1R
InsR
0.0025
0.0030
>5
0.711
2.11
0.853
1.58
0.094
0.746
1
1
0.019
0.024
>5
19.5
8.99
2.29
4.53
1.37
4.13
1
1
AurA1
CDK2
cMet
EGFR
FGFR
KDR
>2000
284
844
341
632
38
>263
1026
473
121
238
72
a
Dosed as a solution in DMSO (iv) or as a suspension (po) in 20% captisol in PBS
for 3 and 10. Dosed in aqueous propylene glycol (iv) or as a suspension in 20%
captisol in PBS (pH 5.73) for 26.
Tie2
298
217
a
IC50 values are means of two or more separate determinations, in duplicate.
compared to compound 10. Compound 26 also displayed a low
clearance rate (0.6 L/h/kg) but much lower bioavailability (9%).
In contrast to compounds 3 and 10, compound 26 had a very
large volume of distribution (9.5 L/kg) and
(T1/2 = 12.1 h).
a
long half-life
compound 26 inhibited autophosphorylation of IGF-1R ꢀ80% com-
pared to controls at the efficacious dose (100 mg/kg) but not at the
lower dose (30 mg/kg). In addition, compound 26 presented no sig-
nificant effect on blood glucose levels following insulin and glucose
challenges up to 200 mg/kg (data not shown).17b
Select kinase selectivity data for compounds 3 and 26 can be
found in Table 7. Compound 26 displayed good selectivity com-
pared to a number of kinases involved in tumor cell growth and
differentiation. All compounds tested were equipotent with re-
spect to IGF-1R versus InsR.18,19
In preparation for a mouse Calu-6 tumor xenograft study,
trimethoxyaniline-containing quinolines 10 and 3 as well as
tethered piperidine 26 were evaluated in mouse PK studies
(Table 5). Oral bioavailability was observed for all three com-
pounds, with compound 3 displaying an acceptable clearance
rate but a low volume of distribution. Compound 26 displayed
a high clearance rate (5.9 L/h/kg) and a very large volume of dis-
tribution (28.1 L/kg) which in turn led to an extended half-life
(T1/2 = 5.3 h).
Based on its cellular potency and oral bioavailability in the
mouse, compound 3 was chosen to be evaluated in a mouse
Calu-6 tumor xenograft model.12 Upon dosing up to 100 mg/kg
b.i.d. for 35 days, compound 3 showed no effect on tumor growth
(Table 6) nor was there any change in blood glucose levels or body
weight (data not shown). Terminal PK studies revealed a low
The relative ease of synthesis of the 2,4-bis-arylamino-1,3-
pyrimidines7 is illustrated in Scheme 1.20 The sequential addition
of amines occurs first at the pyrimidine 4-position followed by
the pyrimidine 2-position. For example, 3-aminoquinoline was
added to a solution of 2,4-dichloropyrimidine in the presence of
Hunig’s base in i-PrOH and the mixture was heated overnight,
giving the intermediate chloropyrimidine 28. Following purifica-
tion, the addition of 3,4,5-trimethoxyaniline in DMSO at 100 °C
led to aminoquinoline 3. Alkylation of piperidine with 1-bromo-
3-chloropropane provided the intermediate chloride which was
used in the alkylation of 4-nitrophenol giving 29. Reduction of
the nitro group in 29 afforded amine 30 which was then added
to the intermediate chloropyrimidine 28 as described earlier to
give 26. Finally, morpholine was coupled to the commercially
available bromide 31 using standard conditions.21 Reduction of
the nitro group in 32 provided amine 33 which could be added
to intermediate chloropyrimidine 28 to afford 8.
In summary, we have discovered a series of potent, orally bio-
available IGF-1R inhibitors. Pyrimidine 26 displayed tumor
growth inhibition in vivo in a mouse Calu-6 tumor xenograft
model with comparable activity to mAb391. An X-ray cocrystal
structure was obtained and revealed key binding elements of this
series of compounds and provided suggestions for further optimi-
zation. Results from these optimization efforts will be reported in
due course.
plasma concentration of
3 (unbound <200 nM at 100 mg/kg
b.i.d.). In addition, compound 3 had poor tumor distribution
(AUC tumor/AUC plasma <0.2).13
Anticipating that the high Vss and long T1/2 displayed by com-
pound 26 would result in greater tumor exposure, compound 26
was also evaluated in the mouse Calu-6 tumor xenograft model.
Upon dosing up to 100 mg/kg b.i.d. for 28 days, compound 26
displayed 56% tumor growth inhibition (TGI), comparable to
49% TGI demonstrated by the commercial IGF-1R antibody
mAb39114,15 (Table 6). In this Letter, a 200 mg/kg b.i.d. dose
was not tolerated.16 There was no change in blood glucose levels
for either dose of compound 26 or for mAb391 (data not show-
n).17a Terminal PK studies demonstrated much higher tumor
exposure at 100 mg/kg as well as a large tumor/plasma ratio
(AUC tumor/AUC plasma >80) in contrast to that seen for 3 (Table
6).13
Tumors were assessed for levels of IGF-1R phosphorylation by a
semi-quantitative western blot analysis that showed that
Table 6
In vivo antitumor activity against Calu-6 tumor xenografts implanted subcutaneously in mice
Cmax plasma (
lM)
Cmax plasma (fu)c
(
lM)
Cmax tumor (
lM)
AUC tumor (h ng/mL)
AUC tumor/AUC plasma
TGI %
3
3
26
26
30 mg/kga
100 mg/kga
30 mg/kga
100 mg/kga
5.34
13.4
0.18
2.22
0.0748
0.188
0.0018
0.0189
0.525
2.72
1.36
989
5174
13,271
0.129
0.180
11.6
0
0
0
142.9
1,118,319
81.9
56d
49e
mAb391
300 l
gb
a
b
c
b.i.d.
Twice per week.
Mouse plasma protein binding (3 = 98.6%; 26 = 99.15%).
p = 0.046.
p = 0.033.
d
e

2398
J. L. Buchanan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2394–2399
N
N
N
a
b
N
+
HN
O
N
O
NH
Cl
N
NH
Cl
N
Cl
H2N
3
28
N
N
O
NH2
N
NO2
O
c,d
e
f
HN
O
N
NH
NH
26
N
O
N
N
29
30
N
N
NH2
NO2
NO2
Br
HN
N
NH
g
h
i
8
O
O
O
28
N
N
O
O
N
O
N
O
31
32
33
Scheme 1. Synthesis of 2,4-bis-arylamino-1,3-pyrimidines 3, 26, and 8. Reagents and conditions: (a) i-Pr2NEt, i-PrOH, 100 °C, 12 h, 40%; (b) 3,4,5-trimethoxyaniline, DMSO,
100 °C, 65%; (c) 1-bromo-3-chloropropane, THF, rt, 67%; (d) p-nitrophenol, K2CO3, DMF; (e) H2, Pd/C, rt, 86% (two steps); (f) 28, DMSO, 100 °C, 33%; (g) morpholine, Pd2(dba)3,
NaOt-Bu, BINAP, PhCH3, 80 °C, 3 h, 66%; (h) H2, Pd/C, EtOH, rt; (i) 28, DMSO, 100 °C.
6. (a) Human tumor cell lines or a rat fibroblast cell line were plated out in flat-
Acknowledgments
well plates in complete medium and allowed to adhere overnight. The cells
were then starved in medium containing 0.1% bovine serum albumin (BSA)
The authors thank Nick Lydon, David Armistead, Rick Kendall,
Murray Robinson, Dave Lacey, Joe Kim and Vinod Patel for their
support of this research program. We thank Jean Bemis for provid-
ing compound 17, Lucian DiPietro for providing compounds 1 and
2, Pedro Beltran for help with statistics and Erin DiMauro and Mar-
garet Chu-Moyer for helpful discussions.
overnight, pre-incubated for 1 h with or without dilutions of compound, then
activated overnight with 50 ng/mL insulin-like growth factor (IGF-1).
Proliferation was determined by the level of 3H-thymidine incorporation into
DNA. IC50’s were determined by comparing the level of thymidine
incorporation found in the presence of compound compared to controls. (b)
Compounds were routinely assessed in an assay measuring IGF-1-induced
auto-phosphorylation of IGF-1Rb and displayed good correlation with the
proliferation results.
7. (a) Armistead, D. M.; Bemis, J. E.; DiPietro, L. V.; Geuns-Meyer, S. D.; Habgood,
G. J.; Kim, J. L.; Nunes, J. J.; Patel, V. F.; Toledo-Sherman, L. M. WO 01/60816.; (b)
Buchanan, J. L.; Chaffee, S.; Harmange, J.-C.; Novak, P. M.; Zhu, X. WO 03/
018022.; (c) Harmange, J.-C.; Booker, S.; Buchanan, J. L.; Chaffee, S.; Novak, P.
M.; Van Der Plas, S.; Zhu, X. WO 03/018021.
8. The kinase domain of human IGF-1R (res 988 to 1286) with an N-terminal GST
tag was expressed in insect cells and purified by immobilized glutathione
affinity, anion exchange, and size exclusion chromatographies. The coordinates
for the X-ray co-crystal structure of IGF-1R and 8 have been deposited in the
PDB. The RCSB ID code is RCSB063987 and the PDB ID code is 3QQU.
9. Incubation of 3 with rat or human liver microsomes in the presence of NADPH
identified O-demethylation as a major route of metabolism.
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Gupta, P.; Dastidar, S. G.; Ray, A.; Das, B.; Cliffe, I. A. Expert Opin. Ther. Patents
2007, 17, 25; (r) Garcia-Echeverria, C. IDrugs 2006, 9, 415; (s) Hofmann, F.;
Garcia-Echeverria, C. Drug Discovery Today 2005, 10, 1041; (t) Zhang, H.; Yee, D.
Expert Opin. Invest. Drugs 2004, 13, 1569.
10. This strategy to improve cellular potency has been previously reported, for
example: Martin, M. W.; Newcomb, J.; Junes, J. J.; Boucher, C.; Chai, L.; Epstein,
L. F.; Faust, T.; Flores, S.; Gallant, P.; Gore, A.; Gu, Y.; Hsieh, F.; Huang, X.; Kim, J.
L.; Middleton, S.; Morgenstern, K.; Oliveira-dos-Santos, A.; Patel, V. F.; Powers,
D.; Rose, P.; Tudor, Y.; Turci, S. M.; Welcher, A. A.; Zack, D.; Zhao, H.; Zhu, L.;
Zhu, X.; Ghiron, C.; Ermann, M.; Johnston, D.; Saluste, C.-G. P. J. Med. Chem.
2008, 51, 1637.
11. (a) Male Sprague–Dawley rats were administered a solution of compound in
DMSO at the indicated doses iv For oral dosing, a suspension of the compound
in 1% Tween 80 and 1% HPMC in water was administered. Samples were
collected over a 12–24 h period and analyzed for parent compound by LC–MS.
(b) In vitro clearance (RLM and MLM) was not predictive of in vivo clearance,
thus compounds were chosen for PK studies based on their enzyme and
cellular potency profiles.
12. Calu-6 cell lines were obtained originally from ATCC and were maintained in
RPMI 1640 10% FBS, 1 Â NEAA, 2 mM
L-glutamine (Gibco/BRL, Grand Island,
NW). CD1 mice within approximately 4–8 weeks were challenged with
subcutaneous injections of Calu-6 (human NSCLC) cells (5 Â 106 cells per
mouse). After 1 day, animals began continuous daily treatment with
compound administered po, b.i.d at the indicated dose levels in 1% Tween 80
and 1% HPMC in water (3) or in 20% Captisol in PBS at pH 3.5 (26). Tumor
volumes as established by caliper measurements were recorded twice per
week, along with body weights as an index of toxicity. Blood glucose levels
were recorded before dosing and once per week. Data are expressed as mean
4. Armistead, D. M.; Bemis, J. E.; Buchanan, J. L.; DiPietro, L. V.; Elbaum, D.;
Habgood, G. J.; Kim, J. L.; Marshall, T. L.; Geuns-Meyer, S. D.; Novak, P. M.;
Nunes, J. J.; Patel, V. F.; Toledo-Sherman, L. M.; Zhu, X. WO 01/25220.
5. All compounds were screened in a homogeneous time-resolved fluorescence
(HTRF) kinase assay at the apparent Km of ATP with respect to 1 lM of peptide
substrate.
values plus or minus standard errors as
a function of time. Statistical
significance of observed differences was evaluated by repeated measures

J. L. Buchanan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2394–2399
2399
analysis of variance (RMANOVA) followed by Scheffe post hoc testing for
multiple comparisons.
insulin by modulating blood glucose. It is possible that at the 100 mg/kg dose
of compound 26, the level of insulin receptor inhibition is not complete enough
to result in metabolic effects.
13. IC50’s were determined for
3 (0.110 lM) and 26 (0.69 lM) in a Calu-6
proliferation assay.6,12 Proliferation assays are performed under serum-
starved conditions, hindering the typical calculation of target coverage. The
total tumor concentration for 3 was 25-fold higher than the cellular IC50 at the
high dose (100 mg/kg). The total tumor concentration for 26 at the efficacious
dose (100 mg/kg) was 207-fold higher than the Calu-6 cellular IC50, while only
2-fold higher at the lower dose (30 mg/kg). Using mouse plasma protein
binding to estimate free concentration in tumors, the efficacious dose of 26
18. Recent data suggests that the inhibition of both IGF-1R and InsR may be
desirable. See for example: (a) Pollak, M. Nat. Rev. Cancer 2008, 8, 915; (b)
Zhang, H.; Fagan, D. H.; Zeng, X.; Freeman, K. T.; Sachdev, D.; Yee, D. Oncogene
2010, 29, 2517; (c) Buck, E.; Gokhale, P. C.; Koujak, S.; Brown, E.; Eyzaguirre, A.;
Tao, N.; Rosenfeld-Franklin, M.; Lerner, L.; Chiu, M. I.; Wild, R.; Epstein, D.;
Pachter, J. A.; Miglarese, M. R. Mol. Cancer Ther. 2010, 9, 1349.
19. Clinical results for two advanced small molecule dual IGF-1R/InsR inhibitors in
the clinic should help define the importance of InsR inhibition. See for
example: (a) Wittman, M. D.; Carboni, J. M.; Yang, Z.; Lee, F. Y.; Antman, M.;
Attar, R.; Balimane, P.; Chang, C.; Chen, C.; Discenza, L.; Frennesson, D.;
Gottardis, M. M.; Greer, A.; Hurlburt, W.; Johnson, W.; Langley, D. R.; Li, A.; Li,
J.; Liu, P.; Mastalerz, J.; Mathur, A.; Menard, K.; Patel, K.; Sack, J.; Sang, X.;
Saulnier, M.; Smith, D.; Stefanski, K.; Trainor, G.; Velaparthi, U.; Zhang, G.;
Zimmermann, K.; Vyas, D. M. J. Med. Chem. 2009, 52, 7360; (b) Carboni, J. M.;
Wittman, M.; Yang, Z.; Lee, F.; Greer, A.; Hurlburt, W.; Hillerman, S.; Cao, C.;
Cantor, G. H.; Dell-John, J.; Chen, C.; Discenza, L.; Menard, K.; Li, A.; Trainor, G.;
Vyas, D.; Kramer, R.; Attar, R. M.; Gottardis, M. M. Mol. Cancer Ther. 2009, 8,
3341; (c) Mulvihill, M. J.; Cooke, A.; Rosenfeld-Franklin, M.; Buck, E.; Foreman,
K.; Landfair, D.; O’Connor, M.; Pirritt, C.; Sun, Y.; Yao, Y.; Arnold, L. D.; Gibson,
N. W.; Ji, Q.-S. Future Med. Chem. 2009, 1, 1153.
(100 mg/kg b.i.d.) was the only dose which achieved a concentration (1.2
lM)
higher (1.8-fold) than the IC50 (0.69
assay.
lM) determined in the Calu-6 proliferation
14. (a) Hailey, J.; Maxwell, E.; Koukouras, K.; Bishop, W. R.; Pachter, J. A.; Wang, Y.
Mol. Cancer Ther. 2002, 1, 1349; (b) 300 g was chosen based on the maximal
l
effect shown in an engineered IGF-1R-dependent 32D tumor xenograft model
(Refs. 2b and 15). Data not shown.
15. Peruzzi, F.; Prisco, M.; Dews, M.; Salomoni, P.; Grassilli, E.; Romano, G.;
Calabretta, B.; Baserga, R. Mol. Cell. Biol. 1999, 19, 7203.
16. Higher doses (200 mg/kg b.i.d.) were tolerated in an engineered IGF-1R-
dependent 32D tumor xenograft study (Refs. 2b and 15), but were not tolerated
in the longer term Calu-6 xenograft study. Data not shown.
17. (a) Blood glucose levels were checked at day 0, 9 and 21 within the Calu-6
xenograft study. No significant changes in blood glucose levels outside of the
normal range were observed. (b) An insulin challenge test was done within the
Calu-6 xenograft at day 17 of dosing. No significant differences were noted (at
either the 30 or 100 mg/kg doses) in the ability of the animals to respond to
20. All new compounds were characterized by mass spectroscopy and 1H NMR and
were determined to be of >95% purity by reverse phase HPLC.
21. Wolfe, J. P.; Wagaw, S.; Buchwald, S. L. J. Am. Chem. Soc. 1996, 118,
7215.