Synthesis and in vitro evaluation of [18F]BMS-754807: A potential PET ligand for IGF-1R

Article abstract of DOI:10.1016/j.bmcl.2013.05.026

Radiosynthesis and in vitro evaluation of [¹⁸F](S)-1-(4-((5- cyclopropyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f][1,2,4]triazin-2-yl) -N-(6-fluoropyridin-3-yl)-2-methylpyrrolidine-2-carboxamide ([ ¹⁸F]BMS-754807 or [¹⁸F]1) a specific IGF-1R inhibitor was performed. [¹⁸F]1 demonstrated specific binding in vitro to human cancer tissues. Synthesis of reference standard 1 and corresponding bromo derivative (1a), the precursor for radiolabeling were achieved from 2,4-dichloropyrrolo[2,1-f][1,2,4]triazine (4) in three steps with 50% overall yield. The radioproduct was obtained in 8% yield by reacting 1a with [ ¹⁸F]TBAF in DMSO at 170 C at high radiochemical purity and specific activity (1-2 Ci/μmol, N = 10). The proof of concept of IGF-IR imaging with [¹⁸F]1 was demonstrated by in vitro autoradiography studies using pathologically identified surgically removed grade IV glioblastoma, breast cancer and pancreatic tumor tissues. These studies indicate that [ ¹⁸F]1 can be a potential PET tracer for monitoring IGF-1R.

Full text of DOI:10.1016/j.bmcl.2013.05.026

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4191–4194
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of [18F]BMS-754807: A potential PET
ligand for IGF-1R
Vattoly J. Majo a, Victoria Arango a,b, Norman R. Simpson b, Jaya Prabhakaran a, Suham A. Kassir b,
Mark D. Underwood a,b, Mihran Bakalian b, Peter Canoll c, J. John Mann a,b,d, J. S. Dileep Kumar a,b,
⇑
a Division of Molecular Imaging and Neuropathology, Department of Psychiatry, Columbia University College of Physicians and Surgeons, NY, USA
b New York State Psychiatric Institute, NY, USA
c Department of Pathology, College of Physicians and Surgeons, NY, USA
d Department of Radiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
a r t i c l e i n f o
a b s t r a c t
Radiosynthesis and in vitro evaluation of [18F](S)-1-(4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)pyrrol-
o[2,1-f][1,2,4]triazin-2-yl)-N-(6-fluoropyridin-3-yl)-2-methylpyrrolidine-2-carboxamide ([18F]BMS-
754807 or [18F]1) a specific IGF-1R inhibitor was performed. [18F]1 demonstrated specific binding
in vitro to human cancer tissues. Synthesis of reference standard 1 and corresponding bromo derivative
(1a), the precursor for radiolabeling were achieved from 2,4-dichloropyrrolo[2,1-f][1,2,4]triazine (4) in
three steps with 50% overall yield. The radioproduct was obtained in 8% yield by reacting 1a with
Article history:
Received 28 March 2013
Revised 30 April 2013
Accepted 7 May 2013
Available online 16 May 2013
Keywords:
IGF-1R
Radiotracer
PET
[
18F]TBAF in DMSO at 170 °C at high radiochemical purity and specific activity (1–2 Ci/
lmol, N = 10).
The proof of concept of IGF-IR imaging with [18F]1 was demonstrated by in vitro autoradiography studies
using pathologically identified surgically removed grade IV glioblastoma, breast cancer and pancreatic
tumor tissues. These studies indicate that [18F]1 can be a potential PET tracer for monitoring IGF-1R.
Ó 2013 Elsevier Ltd. All rights reserved.
Phosphor imaging
Insulin-like growth factors (IGFs or IGF-I and IGF-II) are growth
hormones that have high sequence homology to insulin and func-
tion as regulators of cellular proliferation, apoptosis, energy
metabolism and various organ-specific functions.1,2 The functions
of IGFs are mediated through two tyrosine kinase receptors IGF-
1R, IGF-2R and IGF binding proteins (IGF-BP).3,4 Overexpression
of IGF-1R has been found in many cancers affecting multiple as-
pects of malignancy and metastases. These include mesenchymal,
epithelial and hematopoietic classes of a wide variety of tumors/
cancers.5–12 In addition, a role in brain development and normal
brain functions, alterations of IGF-1R are also found in Alzheimer’s
Disease, traumatic brain injury, Amyotrophic Lateral Sclerosis,
Fredreich Ataxia and aging.13–18 IGF-1R overexpression has also
been reported in brain tumors such as glioblastoma, neuroblas-
toma, astrocytomas, meningiomas and medulloblastoma.19–22 The
biological roles of IGF-1R in malignancy have been demonstrated
by preclinical, epidermological and clinical studies.6–8,23,24 IGF-IR
is a potential therapeutic target and there are several active clinical
trials evaluating anti IGF-1R effects in various diseases.6–8 These
agents include antibodies for IGF-I, monoclonal antibodies for
IGF-1R, small molecule targeting tyrosine kinase receptor
inhibitors (TKRIs) and ligand binding antibodies and antisense
oligonucleotides. Although clinical trials with at least six monoclo-
nal antibody based IGF-1R therapy are still under preclinical and
phases I–III clinical evaluation, many clinical trials based on anti-
body based IGF-1R therapy reported negative results and safety
concerns.25–30 There is a growing interest in the development of
small molecule TKRIs inhibitors of IGF-1R.10,24 Several small mole-
cules based on TKRIs are in various phases of clinical investigations
(phases I–III).7–11 The identification of noninvasive biomarkers
would enable detection and quantification of IGF-1R in vivo to
monitor target occupancy and treatment responses and accelerate
development of medications that target IGF.
To date, five major classes of ligands have been considered for
IGF-1R imaging including proteins, antibodies, peptides, affibodies
and small molecule TKRIs.23 SPECT ligands based on I125 analogues
of IGF-I were not successful in in vivo imaging IGF-I due to high
protein binding and in vivo deiodination.23 In vivo imaging of
IGF-1R with 111In-IGF1(E3R) in a mouse breast cancer xenograft tu-
mor model shows good tumor contrast, a strong linear correlation
with in vitro and in vivo IGF-1R expression level and tracer up-
take.23 R1507, a monoclonal antibody in human was radiolabeled
with PET and SPECT isotopes (111In, 89Zr and 125I) and tested in tu-
mor model.31 Higher tumor uptake of 111In-R1507 and 89Zr-R1507
was observed in comparison to that of 125I-R1507.32 111In-R1507
SPECT has also been utilized to predict the response to anti-IGF-
1R therapy in human bone sarcoma xenografts.33 Although anti-
body based imaging is successful in mice models, its translation
⇑
Corresponding author. Fax: +1 212 543 1054.
E-mail address: kumardi@nyspi.columbia.edu (J.S. Dileep Kumar).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.026

4192
V. J. Majo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4191–4194
to human study is not practical as the imaging probe requires days
for equilibration, and offer poor target/nontarget contrast due to
the expression of IGF-1R in normal tissues. There have been unsuc-
cessful attempts to image IGF-1R antibodies with quantum dots
using fluorescent techniques.23,34,35 IGF-1R based PNA has been re-
cently radiolabeled with 64Cu and 99mTc and tested in IGF-1R over-
exchange reaction with cold BMS-754807 under microwave condi-
tions using [18F]KF/K222. However, the bromo precursor 1a did not
undergo radiofluorination using [18F]KF/K222 under thermal or
microwave conditions (Scheme 1). However, the radioproduct
was obtained in 8% yield by treating bromo precursor 1a with
[
18F]TBAF in DMSO at 170 °C in high radiochemical purity (>95%)
expressing breast cancer xenografts in mice.36,37
A
few
and specific activity (1–2 Ci/
fluorination via the displacement of 2-bromo pyridyl system with-
out using catalyst is widely reported in literature.47 18F]TBAF was
l
mol).49 Nucleophilic aromatic radio-
radioligands based on IGF-1R selected antibody have been labeled
with 111In and show modest tumor uptake and tumor to blood ra-
tio.38 Recently, [18F]FDG and [18F]FLT have been employed to ac-
cess response to IGF-1R inhibitors in preclinical and human
subjects.39–41
[
in situ generated from freshly prepared tetrabutylammonium
bicarbonate by azeotropic distillation with [18F]fluoride. Tetrabu-
tylammonium bicarbonate in turn was freshly prepared by bub-
bling carbon dioxide from dry ice into tetrabutylammonium
hydroxide (4 mL, 13% by weight in water, 0.5 M) for 4 h at room
temperature. The radioproduct was stable in 10% ethanol–saline
solution for over 6 h. The experimental partition coefficient (logP)
of [18F]1 was measured as 2.8, indicating that the radioligand has
adequate lipophilicity for brain imaging.50
Some of the ligands listed above show promise for imaging IGF-
1R, but do not cross the blood brain barrier (BBB) and hence are
limited to use in imaging studies outside the brain. To quantify
binding to IGF-1R in brain requires development of selective non-
peptide PET ligands. High affinity, lipophilic TKRIs of IGF-1R are po-
tential candidates for developing such imaging agents. We have
screened a large number of TKRIs for this purpose and identified
BMS-754807 as a small molecule dual inhibitor of IGF-1R/insulin
receptor (IR), that has been clinical evaluation, as a lead ligand
for imaging using PET.42–44 BMS-754807 (1) is an orally bioavail-
able, potent and reversible small molecule inhibitor of the IGF-
1R/IR family kinases (Ki <2 nmol/L), which is currently in phase II
clinical trial for the treatment of a variety of human cancers.42–46
The selectivity of compound 1 over other kinases, presence of met-
abolically stable fluorine in the 2-substituted pyridine ring, which
is amenable for radiolabeling using nucleophilic displacement with
After optimizing the synthesize of [18F]1, the proof of concept of
IGF-1R imaging with the radiotracer was performed in surgically
removed and pathologically identified grade IV-glioblastoma,
breast cancer and pancreatic tumor using phosphor imager autora-
diography (Fig. 1).51–53 Pathologically identified frozen sections of
glioblastoma IV, pancreatic tumor and breast cancer were used
for phosphor image studies. The slide mounted tumor sections
were brought to room temperature and incubated with [18F]1
(0.1 nM) for 1 h.54 The adjacent sections were incubated with
1 lM of GSK1838705A, a specific IGF-1R ligand to determine non-
[
18F]fluoride47 and a calculated lipophilicity (clogP) 3.5, prompted
specific binding. After incubation, sections were washed, dried and
exposed to phosphor imaging screen. As evident from Figure 1,
there is specific binding of [18F]1 in the three tumor/cancer tissues
tested. Grade IV gliobalstoma shows higher tracer uptake and spe-
cific binding in comparison to pancreatic tumor and breast cancer
tissues. The binding rations of total verses nonspecific binding ob-
tained are 5.25 (N = 9), 1.92 (N = 4) and 1.7 (N = 4) for grade IV glio-
blastoma, pancreatic tumor and breast cancer, respectively.
In summary, we successfully synthesized [18F]1, a potential
imaging agent for IFG-1R. The total time required for radiosynthe-
sis was 60 min from EOS in 8% yield with excellent chemical and
radiochemical purities and specific activity. Autoradiography stud-
ies by phosphor imaging indicate that [18F]1 binds to IGF-1R of
us to choose it as a candidate radiotracer for imaging IGF-1R with
PET.
BMS-754807 (1) and the corresponding bromo precursor (1a)
for radiosynthesis, were prepared based on modification of a pub-
lished procedure.45 Preferential displacement of the C-4 chloride in
compound 4 with 5-cyclopropyl-1H-pyrazol-3-amine (5) followed
by displacement at C-2 position with (S)-2-methylpyrrolidine-
2-carboxylic acid gave the corresponding carboxylic acid 7 in two
steps with 65% yield. The carboxylic acid was then condensed with
6-bromopyridin-3-amine or 6-fluoropyridin-3-amine to yield the
precursor 1a or the standard 1 in 78–70% yield (Scheme 1).48
Radiosynthesis of [18F]BMS-754807 was initially optimized by
H
O
N
N
Cl
H
OH
N
N
HN
N
H
N
+
DIPEA
N
H2N
N
N
Cl
2-propanol, rt
NMP, KOtBu, 180 ºC
N
N
Cl
4
5
6
H
N
H
N
N
N
N
HN
HN
1. SOCl2, DMF
N
X
HO
O
N
O
2.
N
H
N
X
N
N
N
N
N
N
1 X = F
H2N
7
1a X= Br
H
N
N
18F]TBAF
DMSO, 170 o
[
18F]F-, MW
or Heat
HN
[
N
18F
O
1
1a
N
C
N
H
N
N
N
[
18F]BMS754807 ([18F]1)
Scheme 1. Synthesis and radiosynthesis of BMS-754807 and [18F]BMS-754807.

V. J. Majo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4191–4194
4193
Figure 1. Phosphor image of [18F]1 in surgically removed cancer tissues. Column 1: total binding of [18F]1 in glioblastoma IV sections; column 2: nonspecific binding of [18F]1
in glioblastoma IV sections; column 3: total binding of [18F]1 in pancreatic tumor sections; column 4: nonspecific binding of [18F]1 in pancreatic tumor sections; column 5:
total binding of [18F]1 in breast cancer tissues; column 6: nonspecific binding of [18F]1 in breast cancer tissues.
27. Press release from Pfizer: Pfizer discontinuesa phase III trail of figitumumab in
surgically removed postmortem human glioblastoma grade IV,
non-small cell lung cancer (NSCLC). Press release 12/29/2009 and 3/11/2010.
breast cancer and pancreatic tumor tissues. [18F]BMS-754807 is
28. Jassem, J.; Langer, C. J.; Karp, D. D.; Mok, T.; Benner, R. J.; Green, S. J.; Park, K.;
the first small molecule radioligand that showed promise for
imaging IGF-1R in tumor/cancer. The results indicate that
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30. Weroha, S. J.; Haluska, P. IGF-1 receptor inhibitors in clinical trials-early
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[
18F]BMS-754807 can be
a potential PET imaging agent for
in vivo monitoring IGF-1R.
31. Zhang, Y.; Cai, W. Am. J. Nucl. Med. Mol. Imaging 2012, 2(2), 259.
32. Cornelissen, B.; McLarty, K.; Kersemans, V.; Reilly, R. M. Nucl. Med. Biol. 2008,
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Acknowledgments
33. Heskamp, S.; van Laarhoven, H. W.; Molkenboer-Kuenen, J. D.; Franssen, G. M.;
Versleijen-Jonkers, Y. M.; Oyen, W. J.; Van der Graaf, W. T.; Boerman, O. C. J.
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University Medical Center, New York for providing tumor tissues.
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48. BMS-754807 (1) and (S)-N-(6-bromopyridin-3-yl)-1-(4-((5-cyclopropyl-1H-
pyrazol-3-yl)amino)pyrrolo[2,1-f][1,2,4]triaz-in-2-yl)-2-methylpyrroli-dine-2-
carboxamide: (S)-1-(4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)pyrrolo
[2,1-f][1,2,4]tria-zin-2-yl)-2-methylpyrrolidine-2-carboxylic acid (0.220 g,
0.6 mmol) in N,N-dimethylformamide (3 mL) was cooled to 5 °C in an ice
bath. Thionyl chloride (0.057 ml, 0.78 mmol) was then added dropwise. The
reaction mixture was stirred at 5–20 °C for 1 h. 6-Fluoropyridin-3-amine
(0.201 mg, 1.8 mmol) or 6-bromopyridin-3-amine (0.311 g, 1.80 mmol) in
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1 mL of DMF was then added in one portion and the reaction mixture was
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stirred at rt for 24 h. The reaction mixture was then poured into 0.5 N NaHCO3
solution (20 mL). The resulting suspension was then stirred at rt for 1 h. The
suspension was then extracted with EtOAc (3 Â 50 mL), dried over MgSO4,
concentrated and column chromatographed using 50:50 EtOAc/hexane to yield
1 (192 mg, 70%) or 1a as a colorless solid (244 mg, 78%). The spectral data for 1
were identical to that reported for reported compound45 1a: 1H NMR
(400 MHz, CDCl3) d 8.98 (s, 1H), 8.27 (d, J = 3.1 Hz, 1H), 7.96–7.76 (m, 2H),
7.48–7.31 (m, 2H), 6.56 (ddd, J = 23.2, 4.8, 2.2 Hz, 2H), 6.19 (d, J = 3.6 Hz, 1H),
3.89–3.67 (m, 2H), 2.60 (td, J = 9.9, 8.5, 5.0 Hz, 1H), 2.20–1.88 (m, 2H), 1.75 (s,
3H), 1.72–1.67 (m, 1H), 1.34–1.19 (m, 1H), 1.02–0.86 (m, 2H), 0.81–0.58 (m,
2H); HRMS Calcd For C23H25BrN9O (MH+): 522.1365. Found: 522.1376.
54. In vitro phosphor image with [18F]1: the in vitro experiments were performed
using pathologically identified surgically removed glioblastoma grade IV,
pancreatic cancer and breast cancer tissues obtained from Tissue bank of
Herbert Irving Comprehensive Cancer Center, Columbia University. The frozen
tumor tissues obtained were sectioned (20 um) were collected onto glass
slides. The sections were allowed to dry and stored at À80 °C until use. The
phosphor image autoradiography studies were described previously51–53 and
were used for the phosphor image studies. Four sections each for total and
nonspecific binding were assayed. The slides mounted sections were brought
to room temperature and were incubated with 50 mM Tris–HCl, 5 mM of KCl
and 150 mM of NaCl at pH 7.4 containing [18F]1 (0.1 nM) for 1 h. The adjacent
49. Radiosynthesis of [18F]1: 1–2 mg of 1a in 0.5 mL DMSO was added to a freshly
prepared and dried [18F]TBAF. The reaction mixture was heated in a sealed vial
for 30 min at 170 °C. The crude product was directly injected into a semi
preparative RP-HPLC (phenomenex C18, 10 Â 250 mm, 10) and eluted with
acetonitrile: 0.1 M ammonium formate solution (45:55) at a flow rate of
10 mL/min. The precursor eluted after 10–11 min during the HPLC analysis.
The product fraction with a retention time of 7–8 min based on c-detector was
collected, diluted with 50 mL of deionized water, and passed through a C-18
Sep-PakÒ cartridge. Reconstitution of the product in 1 mL of absolute ethanol
afforded [18F]1 (8 2% yield, based on [18F]FÀ at EOS). A portion of the ethanol
solution was analyzed by analytical RP-HPLC (Phenomenex, Prodigy ODS(3)
sections were incubated with 1 lM of GSK1838705A for 1 h to determine non
specific binding. After the incubation, sections were washed with ice-cold
buffer containing 50 mM Tris–buffer (pH 7.4) at 4 °C and briefly dipped in ice-
cold water to remove salts. Slides were quickly dried under a stream of cold air
and exposed to a phosphor-imaging screen (Packard, ST screen wrapped in
Mylar film) with high- and low-activity standards (American Radiolabeled
Chemicals) for 60 min. Screens are scanned with a Packard Cyclone phosphor-
imaging system and analyzed with OptiQuant Acquisition and Analysis
software (Packard).
4.6 Â 250 mm, 5
l; mobile phase: acetonitrile/0.1 M ammonium formate; 45–
55, flow rate: 2 mL/min, retention time: 7 min, wavelength: 254 nm) to
determine the specific activity and radiochemical purity. The ethanol solution
of [18F]1 was used for further studies.
50. Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle, S. Appl. Radiat. Isot. 2001,
54, 203.