Discovery of a 2,4-disubstituted pyrrolo-[1,2-f][1,2,4]triazine inhibitor (BMS-754807) of insulin-like growth factor receptor (IGF-1R) kinase in clinical development

Article abstract of DOI:10.1021/jm900786r

This report describes the biological activity, characterization, and SAR leading to 9d (BMS-754807) a small molecule IGF-1R kinase inhibitor in clinical development. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900786r

7
360 J. Med. Chem. 2009, 52, 7360–7363
DOI: 10.1021/jm900786r
IGF-1R phosphorylates tyrosine residues on two major
substrates, IRS-1 and Shc, which subsequently signal
through the Ras/MAPK pathway and the PI3K/AKT/
Discovery of a 2,4-Disubstituted Pyrrolo-
1,2-f][1,2,4]triazine Inhibitor (BMS-754807)
[
4
,5
of Insulin-like Growth Factor Receptor (IGF-1R)
†
Kinase in Clinical Development
mTor pathway.
IGF-1R signaling affects cell proli-
feration, differentiation, migration, and regulation of the
apoptotic machinery. The crosstalk observed between
EGFR and IGF-1R signaling suggests the potential of
IGF-1R inhibitors in combination with other targeted
,
Mark D. Wittman,* Joan M. Carboni, Zheng Yang,
‡
^
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6
Francis Y. Lee, Melissa Antman, Ricardo Attar,
Praveen Balimane, Chiehying Chang, Cliff Chen,
agents, cytotoxics, and radiation therapy. IGF-1R activa-
tion has also been implicated in the development of resis-
#
ꢀ
^
^
‡
Lorell Discenza, David Frennesson,
Marco M. Gottardis, Ann Greer, Warren Hurlburt,
7
tance toward trastuzumab treatment in breast and lung
cancer.
8
^
^
^
‡
Walter Johnson, David R. Langley, Aixin Li,
§
^
Clinical proof of concept has been demonstrated with
monoclonal antibodies directed against the extracellular
ligand binding domain of the receptor. Small molecule IGF-
1R inhibitors are just beginning to be explored, including
‡
Jianqing Li, Peiying Liu, Harold Mastalerz,
‡
b
Arvind Mathur, Krista Menard, Karishma Patel,
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John Sack, Xiaopeng Sang, Mark Saulnier,
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Daniel Smith, Kevin Stefanski, George Trainor,
Upender Velaparthi, Guifen Zhang, Kurt Zimmermann,
and Dolatrai M. Vyas
9
,10
‡
‡
‡
OSI-906 (1)
inhibitor, BMS-754807 (9d), described herein.
and a novel pyrrolo[1,2-f][1,2,4]triazine based
‡
Screening a series of kinase-focused libraries identified
pyrazolopyrimidine 2 as a modest inhibitor of IGF-1R and
the closely related insulin receptor (IR). Several other library
members also had modest activity. Examination of their
structure revealed the aminopyrazole element was common
amongthe activemembers. X-ray crystal structures ofsomeof
the early analogues confirmed that the aminopyrazole forms a
donor/acceptor/donor triad with the hinge region (Met1052
and Glu1050; see Figure 4). Since the pyrazolopyrimidine
scaffold is a very common element in many ATP-competive
kinase inhibitors, we elected to transport the aminopyra-
zole binding motif onto the less common pyrrolo[1,2-f]-
‡
§
Discovery Chemistry and Macromolecular Structure and
Department of Chemical Synthesis and Bristol-Myers Squibb Co.,
^
5
Research Parkway, Wallingford, Connecticut 06492, Oncology
Drug Discovery and Metabolism and Pharmacokinetics and
#
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b
Macromolecular Structure and Pharmacuetics and Department
[
of Chemical Synthesis and Discovery Chemistry, Bristol-Myers
Squibb Co., Route 206 and Province Line Road, Princeton,
New Jersey 08540
Received June 2, 2009
Abstract: This report describes the biological activity, character-
ization, and SAR leading to 9d (BMS-754807) a small molecule
IGF-1R kinase inhibitor in clinical development.
[
1,2,4]triazine scaffold. This provided compounds that re-
tained the IGF-1R potency of the screening lead. Some
preliminary SAR was generated by varying the C-2 amine
and alkyl substitution of the pyrazole. These efforts
identified the C-2 pyrrolidine and the C-4 cyclopropylamino-
pyrazole (Figure 1, 3) as preferred groups for further optimi-
zation.
Oncology drug discovery efforts have advanced a number
of small molecule kinase inhibitors into clinical investigation.
Among previously unexploited targets, small molecule inhi-
a
bitors of insulin-like growth factor (IGF-1R ) are advancing
into clinical research. The interest in IGF-1R stems from
The general synthetic route used to prepare these molecules
is illustrated in Scheme 1. Starting from the dichloropyrrolo-
triazine 4, treatment with 5-cyclopropyl-3-aminopyrazole
resulted in the preferential displacement of the C-4 chloride.
Microwave heating of 5 with the potassium salt of (S)-proline
provided acid 6. Amides 8a-g were prepared using standard
amide formation conditions. Significant racemization was
observed in the formation of amides 8f and 8g due to the
low nucleophilicity of the heterocyclic amines. Individual
enantiomers were chromatographically separated and tested.
To block this racemization, the (S)-2-methyl group was
installed. Coupling of the chloride 5 with the potassium salt
of (S)-2-methylproline under microwave heating provided the
acid 7 from which the corresponding amides 9a-d were
prepared.
1
the early recognition of the mitogenic properties of insulin.
These observations and the efficacy data for an IGF-1R
2
3
specific antibody and epidemiological findings have posi-
tioned IGF-1R among the emerging cell signaling pathways
currently being explored.
IGF-IR plays an important role in tumor cell growth and
survival. Upon stimulation with the ligand IGF-1 or IGF-2,
†
Coordinates of 9d cocrystallized with IGF-1R have been submitted
to the RCSB Protein Data Bank (PDB code 3I81).
*To whom correspondence should be addressed. Phone: (203)
6
77-6318. Fax: 203-677-7702. E-mail: mark.wittman@bms.com.
a
Abbreviations: IGF-1R, insulin-like growth factor receptor I; SAR,
structure-activity relationship; IGF-1, insulin-like growth factor 1;
IGF-2, insulin-like growth factor 2; IRS-1, insulin receptor substrate
1
; Shc, Src homology containing adapter protein; MAPK, mitogen-
The primary amide 8a has good IGF-1R potency and
cell activity in the IGF-Sal derived cell line (see Table 1).
11
activated protein kinase; PI3K, phosphatidylinositol 3-kinase; mTor,
mammial target of rapamycin; EGFR, epidermal growth factor recep-
tor; IR, insulin receptor; CDK2E, cyclin-dependent kinase 2E; NMP,
Previous compounds with modest IGF-1R potency and
strong CDK2E inhibition also exhibit an antiprolifera-
tive response in the IGF-Sal cell line, so it could not be
conclusively determined that CDK2E inhibition was not
contributing to the cellular (antiproliferative) potency observed
for 8a.
1-methyl-2-pyrrolidinone; PyBOP, (benzotriazol-1-yloxy)tripyrroli-
dinophosphonium hexafluorophosphate; HOBT, 1-hydroxybenzotria-
zole; PEG400, poly(ethylene glycol) 400; pIGF-1R, phospho-IGF-1R;
pAKT, phospho-AKT; PAMPA, parallel artificial membrane perme-
ability; PK, pharmacokinetics; CYP, cytochrome P-450; PD, pharma-
codynamic.
r
pubs.acs.org/jmc
Published on Web 09/24/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7361
Table 1. SAR of Proline Amide
1
R
2
R
a
a
b
IGF-1R
CDK2E
IGF-Sal
8
8
8
8
8
8
8
9
9
9
9
a
b
c
d
e
f
H (S)
H (R)
H (S)
H (S)
H (S)
H (S)
H (S)
NH
NH
2
41
199
13
40
717
2
2
24
47
310
133
426
2
NHPh
NH(4-Me-Ph)
NMePh
322
1422
200
40
2-NH-thiazole
3-NH-pyridine
69
39
67
24
1
g
a
b
c
d
9
577
329
1432
897
1035
Me (S) 2-NH-thiazole
Me (S) 3-NH-pyridine
Me (S) 2-pyrazine
4
6
3
Me (S) 3-NH-6-F-pyridine
2
7
a
All data reported are for stereochemically pure enantiomers and
IC50 values reported in nM and are determined with ATP concentration
1
2 b
3
of K
m
for each kinase.
Cell activity measured by [ H]thymidine incor-
poration after 72 h of exposure. The IGF-Sal cell line is derived from
spontaneous tumors in transgenic mice that overexpress a constitutively
activated IGF-1R and as such is very sensitive to IGF-1R inhibitors relative to
Figure 1. New IGF-1R lead chemotypes.
1
1
a
inhibitors of other kianses.
Scheme 1. General Synthetic Scheme
a
Table 2. Mouse PK/Efficacy Screen
2
1
R
13
b
c
R
% TGI
MED
Δ wt (g)
9
9
9
9
a
b
c
Me
Me
Me
Me
2-NH-thiazole
3-NH-pyridine
2-pyrazine
74
76
12.5
25
0.3
1.9
113
102
6.25
6.25
-3.6
0.2
d
3-NH-6-F-pyridine
a
b
Compounds dosed orally bid in 80/20 PEG400/water. Minimum
effective dose in mg/kg, the lowest dose at which an efficacious result is
achieved. Change in body weight between the treated and untreated
c
groups at 25 mg/kg dose. Weight loss of >1 g represents a 5% loss in
total body weight over 5 days and was generally considered indicative of
the maximum tolerated dose.
analogue 9b was also investigated. The 6-fluoro-3-pyridyl
analogue 9d provides the optimal balance of potency, cell
activity, and selectivity.
Compound 9d inhibits the phosphorylation of IGF-1R
IC50 = 13nM) and the downstream targets Akt (IC50 =
(
2
2nM) and MAPK (IC50 = 13nM) in the IGF-Sal cell line
with IC50 consistent with the antiproliferative IC50 (7 nM) in
this cell line. These data provide confidence that the observed
antiproliferative effect is due to IGF-1R inhibition.
In a traditional medicinal chemistry approach, exposure
data are collected and together with cellular potency decisions
are made on which analogues to advance to efficacy testing.
To accelerate the lead optimization of this pyrrolotriazine
series, an alternative strategy was developed. Since the trans-
a
(
2
a) 5-Cyclopropyl-3-aminopyrazole, EtN(i-Pr) , i-PrOH; (b) (S)-
proline or (S)-2-methylproline, KO-t-Bu, NMP, 170 ꢀC, microwave;
c) PyBOP, HOBT, amine; (d) SOCl
(
2
, DMF, 50 ꢀC; (e) 3-amino-
-fluoropyridine, 50-25 ꢀC, 20 h (71% for two steps).
1
1
6
genic-derived IGF-Sal tumor model has a very rapid tumor
doubling time (2 days), it was possible to conduct an oral PK/
efficacy screen in mice. By inclusion of doses of 6, 12, and
25 mg/kg b.i.d., an efficacy and tolerability readout could by
obtained within 5 days from the initiation of the experiment.
Guided by cellular potency, in vitro permeability, and meta-
bolic stability data, in vivo active compounds were quickly
identified (see Table 2). Among the compounds of interest
were 9a-d. Compounds 9c and 9d achieved complete tumor
The enantiomeric amide 8b, prepared using (R)-proline, is
-fold less potent vs IGF-1R with a similar loss in cell potency.
5
The change in stereochemistry did not impact kinase selectiv-
ity. Substitution of the (S)-amide increased the potency and
selectivity for IGF-1R (it is noted that all of the analogues
described also inhibit the related IR with equal potency).
Aromatic and heterocylic amides improve the potency for
IGF-1R and the kinase selectivity (8c-g). The importance of
the secondary amide is noted with the lack of activityobserved
for the N-methylanalogue 8e. The pyridylamide8ghas similar
IGF-1R potency to the thiazole analogue 8f with improved
cell activity and kinase selectivity vs CDK2E and other
kinases. The corresponding (S)-2-methyl analogues 9a and
1
3
growth inhibition at the 6.25 mg/kg dose. Compound 9c
showed signs of toxicityat the 25 mg/kgdosewith greater than
2 g of weight loss observed (>10% body weight loss over
5 days). Compound 9d was identified with strong efficacy in
the IGF-Sal tumor model with minimal weight loss at the
upper dose and was chosen for further characterization.
A dose response study was conducted in the IGF-Sal tumor
model, and 9d was found to be active at doses from 3 mg/kg
upward. Tumor samples were collected 2 h after final dose and
9b retain potency with improved kinase selectivity and cell
activity in comparison to the proline derivates 8f and 8g.
Optimization of the pyridyl substitution of the 3-pyridyl

7
362 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Wittman et al.
Figure 2. Dose response and target inhibition in the IGF-Sal tumor
model following oral dosing of 9d.
Figure 3. Rh41 efficacy for 9d. Compound was administered on
a twice daily schedule for 14 days represented by the diamonds.
The compound was administered as a solution in PEG400/water
Figure 4. X-ray crystal structure of 9d cocrystallized with IGF-1R
(RCSB Protein Data Bank PDB code 3I81).
a
(
80:20 v/v) vehicle.
Table 3. Average Clearance and Bioavailability among Species
CLtot ((mL/min)/kg)
F (%)
1
4
analyzed for inhibition of pIGF-1R and pAKT. Inhibition
of pIGF-1R and pAKT showed a dose response that corre-
lated with % TGI (see Figure 2). Efficacy was also observed
in the IGF-1R dependent rhabdomyosarcoma model (see
mouse
rat
dog
113
20
6
30
59
8
3.5
41
monkey
a
1
5
Figure 3), Rh41, and in other xenograft models.
An optimized synthesis of 9d is illustrated in Scheme 1. The
S)-2-methylproline acid 7 could be activated with thionyl
Clearances were determined after iv administration. Compound was
administered as a solution in PEG400/water (80:20 v/v).
(
permeability and potency of the compound. The species-
dependent clearances and oral bioavailabilities make the
prediction of the human dose challenging. However, even
with the “worst case” scenario of human PK being similar to
monkey (high clearance, low bioavailability), the projected
human efficacious dose was still reasonable (<500 mg/day).
A full complete discussion of the PK/efficacy relationship will
be reported elsewhere.
Compound 9d was run through a battery of preclinical
safety assays (Table 4). No significant ion channel activity,
CYP inhibition, hepatotoxicity (>50 μM), or genotoxicity
(Ames negative) was observed, and broad based pharmaco-
logical profiling did not reveal any off-target activities.
chloride to provide the cyclic urea 10 which was directly
reacted with 3-amino-6-fluoropyridine to provide 9d in 71%
yield from acid 7.
The crystal structure of 9d cocrystallized with the kinase
domain of IGF-1R is shown in Figure 4. Key features include
the donor/acceptor/donor hydrogen bond triad with Met1052
and Glu1050 within the hinge region of the kinase. The
cyclopropyl group occupies the shallow “gatekeeper” region
of the kinase, with the fluoropyridylamide extending into the
sugar pocket. In this crystal structure R1127, D1128, and
I1129 are not well-defined, indicating flexibility in the P-loop
of the kinase.
The kinase selectivity of 9d was evaluated by screening
against a number of in-house kinases. In addition to IGF-1R
and IR, in vitro kinase activity was noted for AurB, Axl, Met,
Ron, and TrkA/B although the functional effects of inhibiting
these kinases in cells were much less pronounced than the
Table 4. Ion Channel and CYP Inhibition IC50
ion channel
IC50 (μM)
CYP inhibition
IC50 (μM)
hERG
L-type Ca
>10
>10
>10
CYP3A4 (BzR)
CYP3A4 (BFC)
CYP2C19
>40
7
2þ
1
5
effect observed for inhibition of IGF-1R.
þ
Na
11
23
The protein binding for 9d ranges from of 98.5% in mouse
plasma to 95.9% in human plasma. This compound is highly
permeable in the PAMPA assay (387-713 nm/s, pH 7.4) and
in Caco-2 cells (141-161 nm/s), suggesting the compound is
well-absorbed. Moderate to good bioavailability in rats and
dogs is observed where clearance is moderate to low (Table 3).
However, in mouse and monkey, high clearance contributes
to poor bioavailability. Despite the high clearance and low
bioavailability in mice, a sufficient concentration of 9d could
be achieved to provide efficacy presumably due the high
CYP2C9
CYP2D6
CYP1A2
>40
>40
In summary, the pyrrolo[1,2-f][1,2,4]triazine chemotype was
optimized for IGF-1R potency, selectivity, and safety to pro-
vide the clinical candidate 9d. In preclinical animal safety
studies elevation of glucose and insulin levels was observed at
doses that exceeded an efficacious exposure presumably due to
concomitant inhibition of the insulin receptor. Initial human

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7363
PK indicates that human clearances are low and good bioavail-
growth-inhibitory and proapoptotic effects of gefitinib (Iressa) in
human breast cancer cells. Breast Cancer Res. 2005, 7, R465–R474.
8) Morgillo, F.; Woo, J. K.; Kim, E. S.; Hong, W. K.; Lee, H. Y.
Heterodimerization of insulin-like growth factor receptor/
epidermal growth factor receptor and induction of survivin expres-
sion counteract the antitumor action of erlotinib. Cancer Res. 2006,
15
ability has been observed. The full biological profile, PK/PD
studies, and further clinical studies of 9d will be presented in due
course.
(
(
6
6, 10100–10111.
Acknowledgment. The authors thank Gordon Todderud
and the Lead Evaluation group at BMS for generating the
kinase data presented in Table 1 and the Lead Profiling Depart-
ment at BMS for generating the data presented in Table 4.
9) Arnold, L. D.; Cesario, C.; Coate, H.; Crew, A. P.; Dong, H.;
Foreman, K.; Honda, A.; Laufer, R.; Li, A.-H.; Mulvihill, K. M.;
Mulvihill, M. J.; Nigro, A.; Panicker, B.; Steinig, A. G.; Sun, Y.;
Weng, Q.; Werner, D. S.; Wyle, M. J.; Zhang, T. Patent Applica-
tion WO2005/097800, 2005.
(
10) Volk, B.; Buck, E.; Coate, H.; Cooke, A.; Dong, H.; Eyzaguirre, A.;
Feng, L.; Foreman, K.; Rosenfeld-Franklin, M.; Kleinberg, A.;
Landfair, D.; Mak, G.; Nigro, A.; O’Connor, M.; Pirritt, C.; Silva,
S.; Siu, K.; Steinig, A.; Stolz, K.; Tavares-Greco, P.; Turton, R.;
Werner, D.; Yao, Y.; Arnold, L.; Gibson, N. W.; Pachter, J. A.;
Wild, R.; Ji, Q.-S.; Mulvihill, M. J. OSI - 906: A Novel, Potent, and
Selective First-in-Class Small Molecule Insulin-like Growth Factor
Supporting Information Available: Procedure for the prepara-
tion of 9d, analytical data, and procedures for in vitro and in
vivo assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
Receptor (IGF-1R) Inhibitor in Phase I Clinical Trials. Abstracts
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