The 14C, 13C, and 15N syntheses of a potent VEGFR-2 kinase inhibitor, Brivanib, and its prodrug, Brivanib Alaninate

Article abstract of DOI:10.1002/jlcr.1871

The interruption of tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) signaling by the binding of a small molecule inhibitor, for example, Brivanib, to VEGFR-2 kinase domain has been shown as an effective method of slowing angiogenes

Full text of DOI:10.1002/jlcr.1871

Research Article
Received 9 September 2010,
Accepted 7 December 2010
Published online 17 February 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1871
14
13
15
The C, C, and N syntheses of a potent
VEGFR-2 kinase inhibitor, Brivanib, and its
prodrug, Brivanib Alaninate
Ã
Scott B. Tran, Michael W. Lago, Yuan Tian, Sharon X. Gong, Indu Batra,
Alban J. Allentoff, Brad D. Maxwell, Samuel J. Bonacorsi, Marc Ogan,
J. Kent Rinehart, and Balu Balasubramanian
The interruption of tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) signaling by the binding of a
small molecule inhibitor, for example, Brivanib, to VEGFR-2 kinase domain has been shown as an effective method of
slowing angiogenesis and tumor progression. [14C]Brivanib, 13 and its prodrug [14C]Brivanib Alaninate, 15 were prepared
to support preclinical and clinical studies. Their respective stable isotope-labeled versions, [13C315N2]Brivanib, 21 and
[
13C135N2]Brivanib Alaninate, 28, were also prepared to support bioanalytical LC-MS analyses of clinical samples. All of the
four title compounds were synthetically derived from the respective isotopically labeled common pyrrolotriazinone
intermediate, 6 or 16. This labeled central core pyrrolotriazinone was also conveniently used to synthesize other structurally
related drug discovery candidates.
Keywords: carbon-14; carbon-13; nitrogen-15; Brivanib; Vegfr-2
preparations and characterizations of the four title isotopically
labeled molecules, 13, 15, 21, and 28 (Figure 1).
Introduction
Isoforms of signaling protein vascular endothelial growth factor
(VEGF) and their corresponding tyrosine kinase receptors
(vascular endothelial growth factor receptors (VEGFRs)) have
Experimental
General
been connected to angiogenesis that is essential for maintaining
the nutrients and oxygen needed to support solid tumor growth
and metastasis. In particularly, VEGFR-2, a tyrosine kinase
receptor, appears to account for most of the mitogenic and
chemotactic effects of VEGF on adult endothelial cells.1 Because
the process of solid tumor growth is angiogenesis dependent,
one of several strategies that has been developed is the
discovery of low molecular weight inhibitors of the specific
tyrosine kinase domain of VEGFR-2, suitable for chronic oral
administration and continual reduction of tumor angiogenic
growth. The drug candidate (R)-1-(4-(4-fluoro-2-methyl-1H-
indol-5-yloxy)-5-methylpyrrolo[1,2-f][1,2,4]triazin-6-yloxy)propan-2-
ol, Brivanib,2 has emerged as a potent and selective VEGFR-2
inhibitor, demonstrating promising preclinical in vivo antitumor
activity in human tumor xenograft models. The L-alanine ester
prodrug of Brivanib, Brivanib Alaninate, is currently under
evaluation in clinical trials for the treatment of solid tumors.
[14C]Brivanib, 13, and prodrug [14C]Brivanib Alaninate, 15, were
prepared to support preclinical and clinical metabolism and
pharmacokinetic studies. Their respective stable isotope-labeled
versions, [13C315N2]Brivanib, 21 and [13C315N2]Brivanib Alaninate, 28,
were also prepared to serve as internal standards for liquid
chromatography–mass spectrometry quantitative analyses of
parent and prodrug found in biological samples such as blood,
plasma, serum, urine, or tissues. We report here the details of the
Radioactivity was measured with a Wallac Model 1409 liquid
scintillation counter (Wallac-LKB instruments, Inc.). Counting
efficiency was determined by the channels ratio method. Mass
spectra were obtained with a Finnigan TSQ or a Finnigan LCQ
mass spectrometer. Proton NMR spectra were recorded on a
Bruker Advance Ultrashield 300 MHz or a Jeol EC1500 MHz. For
stable isotope labels, only carbon-13-enriched positions are
reported for 13C-NMR characterization purposes. UV and radio-
chemical purities were determined by High-Performance Liquid
Chromatography (HPLC) (Shimadzu SCL-10A VP/UV-1 detector
or Varian Prostar 330 PDA detector with an IN/US System b-Ram
radiometric flow detector with a 0.5 mL flow cell). TLC was
performed on 60 F254 silica gel plates (Merck). Flash chromato-
graphy was conducted on KP-Sil silica gel (Biotage). Radiola-
beled and stable isotope-labeled products were compared with
Department of Chemical Synthesis, Radiochemistry Group, Bristol-Myers Squibb
Pharmaceutical Research and Development, P.O. Box 4000, Princeton, NJ 08543,
USA
*Correspondence to: Scott B. Tran, Department of Chemical Synthesis, Radio-
chemistry Group, Bristol-Myers Squibb Pharmaceutical Research and Develop-
ment, P.O. Box 4000, Princeton, NJ 08543, USA.
E-mail: scott.tran@bms.com
J. Label Compd. Radiopharm 2011, 54 324–331
Copyright r 2011 John Wiley & Sons, Ltd.

S. B. Tran et al.
H
N
H
N
O
N
(R)
F
N
O
N
O
O
(R)
N
F
N
O
O
HO
(S)
N
NH2
* = 14
C
=
14C
15, L-Alanine Ester Prodrug
13, Parent
[
14C]Brivanib Alaninate
[
14C]Brivanib
H
N
H
N
O
O
N
F
F
N
O
O
O
HO
N
N
N
N
O
NH2
* = 13C or 15N
28, L-Alanine Ester Prodrug
13C315N2]Brivanib Alaninate
* = 13C or 15N
21, Parent
[
13C315N2]Brivanib
[
Figure 1. Four isotopically labeled Brivanib molecules.
authentic standards when possible. All reagents and solvents were weighed into a dry 100 mL round bottom flask under
were ACS grade or better. nitrogen. THF (50 mL) was added and the solution was reduced
Specific activities were determined gravimetrically by dissolu- by rotovap to a volume of 13 mL resulting to a white
tion of the weighed sample (ca 0.5 mg) in a 10 mL volumetric suspension. The suspension was cooled to À51C and MeMgCl
flask and diluted to the mark with DMF. Aliquots of 25 and 50 mL (9.0 mL, 27.0 mmol, 3 M in THF) was added over a period of
of the solution were diluted in 10 mL Ecolite cocktail and 20 min. The reaction was stirred at 01C for 0.5 h and at 471C for
counted by liquid scintillation counting. HPLC methods 6 h with monitoring by TLC (40% EtOAc/hexane, product
described below were used for in process and final product Rf = 0.3) and HPLC (Method 1, product retention time = 9.62 min).
analyses. Co-injections with authentic samples were used when It was cooled to 01C, aqueous NH4Cl (1.6 g, 29.9 mmol, dissolved
possible. All HPLC purities were measurements of UV chemical in 15 mL water) was added slowly and extracted with EtOAc
and radiochemical purities.
(3 Â 20 mL). The combined organic extracts were washed with
Method 1: YMC-ODS-AQ C18, 3 mm, 4.6 Â 150 mm, detected at brine, dried over MgSO4, filtered, and concentrated to dryness.
230 nm. Mobile phase A: 0.05% acetic acid in water, B: 0.05% The crude product was purified by silica gel chromatography by
acetic acid in acetonitrile. Gradient: 0 min 5% B, 5 min 5% B, eluting with a gradient 25–100% EtOAc/Hexane then 5% MeOH/
18 min 95% B, 23 min 95% B, 25 min 5%B. Flowrate = 1.0 mL/min. EtOAc to give a white solid (865 mg, 92% yield). 1H NMR
Method 2: Zorbax SB-Aq, 3.5 mm, 4.6 Â 150 mm, detected at (300 MHz, DMSO-d6) d ppm 7.91 (1H, s), 7.83 (1 H, s), 4.82 (1 H,
237 nm. Mobile phase A: 10% Methanol, 90% water, 0.05% TFA, bs), 2.60 (3 H, s), 1.44 (6 H, m). MS ESI1 [M1H]1 = 208.0.
B: 10% Methanol, 90% acetonitrile, 0.05% TFA. Gradient: 0 min
20% B, 2 min 20%B, 47 min 25% B, 62 min 80% B, 63 min 80% B,
6-Hydroxy-5-methylpyrrolo[1,2-f][1,2,4]triazin-4(3H)-one-[14C], 8
70 min 80% B, 75 min 20% B. Flowrate = 2 mL/min. Method 3: Compound 7 (0.84 g, 4.0 mmol) was dissolved in THF (9.7 mL) in
YMC-ODS-AQ C18, 3 mm, 4.6 Â 150 mm, detected at 215 nm. a 100 mL round bottom flask. The solution was cooled to À81C
Mobile phase A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile. and charged with cold H2O2 (1.36 g, 40 mmol, 2.73 mL, 50
Gradient: 0 min 10% B, 5 min 10% B, 25 min 100% B, 30 min 10% weight% in water). Ice-cold MeSO3H (3.86 g, 40 mmol) diluted
B, 32 min 10%B. Flowrate = 1.0 mL/min.
with water (1.20 mL) was added dropwise over a period of
50 min and the solution was stirred at À81C for 2 h while being
monitored by TLC (50% EtOAc/hexane, product Rf = 0.3) and
HPLC (Method 1, product retention time = 7.69 min). The
6-(2-Hydroxypropan-2-yl)-5-methylpyrrolo[1,2-f][1,2,4]tria-
zin-4(3H)-one-[14C], 7
Ethyl 5-methyl-4-oxo-3,4-dihydropyrrolo[1,2-f][1,2,4]triazine-6- reaction was quenched slowly at 01C with a cold mixture of
carboxylate-[14C], 6 (345.7 mg, 1.54 mmol, 258.0 mCi/mg, SA =
NaHSO3 (58.5% SO2, 4.83 g, 44.2 mmol) and 28% NH3 (5.8 mL,
57.1 mCi/mmol, 89.2 mCi) and unlabeled 6 (660 mg, 3.0 mmol) 92.5 mmol) in water (10 mL). The reaction was extracted with
J. Label Compd. Radiopharm 2011, 54 324–331
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S. B. Tran et al.
ethyl acetate (4 Â 25 mL). After the layers were separated, the and concentrated to dryness. The crude product was purified by
combined organic extracts were washed with brine, dried over triturating in ether (4 mL) with stirring at rt for 1 h. The product
MgSO4, filtered, and concentrated in vacuo affording the desired was collected by vacuum filtration to give a white fluffy solid
crude product as a light-gray solid (0.70 g, quantitative yield). (0.94 g, 75% yield). 1H NMR (300 MHz, DMSO-d6) d ppm 11.37
This crude product was used in the next reaction without further (1 H, br. s.), 8.08 (1 H, s), 8.03 (1 H, s), 7.15 (1 H, d, J = 8.53 Hz),
1
purification. H NMR (300 MHz, DMSO-d6) d ppm 11.29 (1 H, br. 7.01 (1 H, t, J = 8.53 Hz), 6.25 (1 H, s), 2.41 (3 H, s), 2.38 (3 H, s),
s.), 8.80 (1 H, s), 7.61 (1 H, d, J = 4.02 Hz), 6.94 (1 H, s), 2.23 (3 H, s). 1.36 (9 H, s). MS ESI1 [M1H]1 = 397.3.
MS ESI1 [M1H]1 = 166.1.
(R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyr-
rolo[1,2-f][1,2,4]triazin-6-yloxy)propan-2-ol-[14C], 13
5-Methyl-4-oxo-3,4-dihydropyrrolo[1,2-f][1,2,4]triazin-6-yl
pivalate-[14C], 9
Sodium methoxide (25% in MeOH, 0.49 mL, 2.27 mmol) was
added slowly to a suspension of compound 12 (0.90 g,
Compound 8 (0.70 g, 4.23 mmol) was suspended in THF (8.0 mL).
2.27 mmol) in acetonitrile (1.8 mL) under nitrogen at 101C. The
The suspension was cooled to 01C, N,N-diisopropylethylamine
mixture was stirred at rt for 1 h. The progress of the reaction was
(0.66 g, 5.08 mmol) was added followed by the addition of
monitored by HPLC (Method 1) to indicate complete formation
trimethylacetyl chloride (0.61 g, 5.08 mmol) over 30 min. The
of the intermediary alcohol, 4-(4-fluoro-2-methyl-1H-indol-5-
(Rt = 15.70 min).
reaction was stirred at 01C for 30 min and at rt for 2 h while
yloxy)-5-methylpyrrolo[1,2-f][1,2,4]triazin-6-ol
being monitored by TLC (50% EtOAc/hexane, product Rf = 0.6)
and HPLC (Method 1, product retention time = 17.03 min). The
reaction was cooled to 01C, water (20 mL) was added, and the
solution was extracted with EtOAc (3 Â 25 mL). The combined
organic extracts were washed with brine, dried over MgSO4,
filtered, and concentrated to dryness. The crude product was
purified by silica gel flash chromatography eluting with 5-100%
EtOAc/hexane to afford a white solid (0.49 g, 46% yield). 1H NMR
(300 MHz, CDCl3) d ppm 9.82 (1 H, br. s.), 7.50 (1 H, s), 7.47 (1 H,
d, J = 3.89 Hz), 2.39 (3 H, s), 1.38 (9 H). MS ESI1 [M1H]1 = 250.2.
Water (4.6 mL) was added to the reaction followed by (R)-(1)-
propylene oxide (233.2 mg, 4.01 mmol). The mixture was stirred
at 281C for 24 h. The suspension was cooled to 101C, filtered to
collect the solid, and the filtrate was rinsed with water/
acetonitrile (80:20, 2 mL). The crude solid was recrystallized in
acetone/water (1:1, 8 mL) at 501C. The product was collected by
vacuum filtration. The mother liquor was also collected and
purified by flash chromatography eluting with 10–60% EtOAc/
hexane then 3% MeOH/CH2Cl2 to afford a combined 0.62 g (74%
yield) of a white solid. TLC (50% EtOAc/hexane, product Rf = 0.2)
and HPLC (Method 1, product retention time = 16.60 min). 1H
NMR (300 MHz, DMSO-d6) d ppm 11.34 (1 H, br. s.), 7.90 (2 H, d,
J = 6.27 Hz), 7.14 (1 H, d, J = 8.78 Hz), 7.04À6.92 (1H, m), 6.24 (1 H,
4-Chloro-5-methylpyrrolo[1,2-f][1,2,4]triazin-6-yl pivalate-
[
14C], 10
Compound 9 (0.78 g, 3.13 mmol) was suspended in acetonitrile s), 4.90 (1 H, d, J = 4.77 Hz), 4.08À3.95 (1 H, m), 3.93À3.81 (2 H,
(9.5 mL). N,N-diisopropylethylamine (0.47 g, 3.6 mmol) was m), 2.45À2.36 (6 H, m), 1.18 (3 H, m). MS ESI1 [M1H]1 = 371.2.
added under nitrogen to the suspension followed by POCl3
(S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-
pyrrolo[1,2-f][1,2,4]triazin-6-yloxy)propan-2-yl) 2-(benzyloxy-
carbonylamino)propanoate-[14C], 14
(1.3 g, 8.48 mmol). The cloudy white suspension turned to a clear
solution. The reaction was stirred at 821C for 3 h and at 601C for
17 h while being monitored by TLC (30% EtOAc/hexane, product
Rf = 0.8) and HPLC (Method 1, product retention time = 20.09 -
min). The reaction was warmed to rt and the solvent was
removed by rotovap. The residue was washed with K2HPO4
(3.3 g, 18.8 mmol) dissolved in water (30 mL) and extracted with
CH2Cl2 (3 Â 20 mL). The combined organic extracts were washed
with brine, dried over MgSO4, filtered, and concentrated to
dryness. The crude product was dried under vacuum to give
1.05 g (quantitative yield) as an off-white solid. The crude
product was used in the next reaction without further
Compound 13 (0.62 g, 1.7 mmol) was dissolved in THF/DMF (4:1)
(6 mL) under nitrogen. The solution was cooled to 01C and
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
(0.55 g, 2.89 mmol), 4-dimethylaminopyridine (8 mg, 0.07 mmol),
and Cbz-L-alanine (0.56 g, 2.91 mmol) were added. The mixture
was stirred at 01C for 4.5 h while monitoring by TLC (50% EtOAc/
hexane, product Rf = 0.7) and HPLC (Method 1, product retention
time = 20.09 min). The reaction mixture was cooled to 101C,
washed with water (5 mL) and extracted with EtOAc (3 Â 20 mL).
The combined organic extracts were washed with brine, dried
over Na2SO4, filtered, and concentrated to dryness. The crude
product was purified by silica gel flash chromatography eluting
with 0–50% EtOAc/hexane to afford a white solid (0.92 g, 96%
yield). 1H NMR (300 MHz, CDCl3) d ppm 8.01 (1 H, br. s.), 7.84
(1 H, s), 7.40 (1 H, s), 7.35À7.26 (5 H, m), 7.08 (1 H, d, J = 8.80 Hz),
6.99 (1 H, t, J = 7.14 Hz), 6.34 (1 H, s), 5.30À5.20 (2 H, m), 5.11
(2 H, m), 4.42 (1 H, m), 4.15 (2 H, m), 2.46À2.45 (6 H, m),
1.45–1.38 (6 H, dd, J = 7.15, 26.94 Hz). MS ESI1 [M1H]1 = 576.2.
1
purification. H NMR (300 MHz, CDCl3) d ppm 7.51 (1 H, s), 7.48
(1 H, d, J = 3.89 Hz), 2.40 (3 H, s), 1.38 (9 H). MS ESI1 [M1H]1 =
268.2.
4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrro-
lo[1,2-f][1,2,4]triazin-6-yl pivalate-[14C], 12
Compounds 10 (1.0 g, 3.88 mmol) and 11 (0.71 g, 4.27 mmol)
were dissolved in acetonitrile (10.0 mL) under nitrogen. 1,4-
Diazabicyclo[2.2.2]octane (0.48 g, 4.27 mmol) was added slowly
to the solution. The reaction became a suspension. It was stirred
at 481C for 1.5 h. The reaction was monitored by TLC (50%
EtOAc/hexane, product Rf = 0.5) and HPLC (Method 1, product
retention time = 21.70 min). The acetonitrile was removed by
(S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methyl-
pyrrolo[1,2-f][1,2,4]triazin-6-yloxy)propan-2-yl) 2-aminopro-
panoate-[14C], 15
rotovap. The mixture was washed with water (20 mL) and To a solution of compound 14 (0.31 g, 0.54 mmol) in EtOAc
extracted with CH2Cl2 (3 Â 25 mL). The combined organic (3 mL) was added K2CO3 (50 mg, 0.36 mmol). The mixture was
extracts were washed with brine, dried over MgSO4, filtered, placed under vacuum at 170 mBar for 5 min and nitrogen (g)
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S. B. Tran et al.
was introduced. Wet (50%) palladium (5%) on activated carbon brine, dried over Na2SO4, filtered, and rotovapped to dryness.
(150 mg) was quickly added to the mixture. The mixture was MeOH (7 mL) was used to triturate the crude product. The pure
placed under vacuum and purged with hydrogen (g). This step product (578 mg) was collected by vacuum filtration. The
was repeated three times. The mixture was reacted under mother liquor was also collected, concentrated, and chromato-
hydrogen (g) for 2 h while monitoring by HPLC (Method 2, graphed on silica gel eluting with 25% EtOAc/hexane to give an
product retention time = 45.83 min) and TLC (50% EtOAc/ additional 95 mg of white solid (673 mg, 66% yield). TLC (40%
Hexane, product Rf = 0.7 or 20% iPA/EtOAc, product Rf = 0.2). EtOAc/Hexane, product Rf = 0.5). 1H NMR (500 MHz, CDCl3) d
The reaction was filtered through a bed of celite and rinsed with ppm 8.15 (1 H, t, J = 5.50 Hz), 8.04 (1 H, s), 7.89 (1 H, q,
EtOAc (50 mL). The EtOAc was removed by rotovap to dryness J = 8.24 Hz), 7.10 (1 H, d, J = 8.25 Hz), 6.98 (1 H, t, J = 7.15 Hz), 6.35
and the crude product was purified by flash chromatography. It (1 H, s), 4.38 (2 H, dq, J = 3.14 Hz), 2.86 (3 H, dm, J = 129.2 Hz), (3
was incrementally eluted with 10% EtOAc/hexane to EtOAc and H, s), 1.40 (3 H, t, J = 7.14 Hz). 13C NMR (CDCl3) d ppm 164.84 (d,
then with 10% iPA/EtOAc. The pure fractions were collected and J = 83.93 Hz), 119.12 (t, J = 56.17 Hz), 116.83 (m), 12.02 (d,
rotovapped to dryness to give the product as a white foam J = 48.32 Hz). MS ESI1 [M1H]1 = 374.9.
(206 mg, radiochemical purity = 92.71%). This material was
further purified by recrystallization. In a 25 mL pear shaped- 2-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-(methyl-13C)-
methyl(5,6-13C2,1,8-15C2)pyrrolo[1,2-f][1,2,4]triazin-6-yl)pro-
flask was added the impure material (206 mg, 83% yield, with a
radiochemical purity = 92.71%) and EtOAc (1.1 mL). The clear
pan-2(13C)-ol, 19
solution was heated slowly to 501C and heptane (2.1 mL) was
Compound 18 (665 mg, 1.78 mmol) was dissolved in THF/
added over 15 min. Cloudiness was observed at the end of the
toluene (1:1) (10 mL). To the solution was added LiCl (1.03 g,
addition, but gradually all the solid dissolved. It was seeded with
pure unlabeled 15 (0.5 mg). The solution was cooled slowly.
It was stirred at rt for 16 h. The crystallized product was collected
by vacuum filtration as a white solid (114.5 mg, 56% yield,
24.2 mmol) and it was cooled to 01C under nitrogen. To the
mixture was slowly added CH3MgBr (1.4 M in toluene/THF,
75:25) (8.07 mL, 5.76 mmol). The reaction was stirred at 01C for
1 h and at 151C for 3 h. The reaction was cooled to 01C and an
ice-cold 15% NH4Cl (30 mL) was added. The aqueous layer was
SA = 23.24 mCi/mg, 10.30 mCi/mmol, chemical purity = 99.20%,
radiochemical purity = 99.23%). 1H NMR (300 MHz, CDCl3) d ppm
extracted with EtOAc (4 Â 30 mL). The combined organic
7.95 (1 H, br. s.), 7.78 (1 H, s), 7.35 (1 H, s), 7.08 (1 H, d,
extracts were washed with brine, dried over Na2SO4, filtered,
J = 8.80 Hz), 6.90 (1 H, m), 6.27 (1 H, s), 5.32À5.20 (1 H, m), 4.04
(2 H, m), 3.56 (1 H, m), 2.47 (3 H, s), 2.39 (3 H, s), 1.64 (2 H, br. s.),
and rotovapped to dryness. The crude product was chromato-
graphed on silica gel eluting with 10–100% EtOAc/hexane to
1.36 (6 H, m). MS ESI1 [M1H]1 = 442.2.
afford 360 mg (56% yield) of a white solid. TLC (1% MeOH/
CH2Cl2, product Rf = 0.5) and HPLC (Method 3, product, retention
Ethyl 4-chloro-5-(methyl-13C)methyl(5,6-13C2,1,8-15C2)pyrro-
1
time = 19.64 min). H NMR (500 MHz, CDCl3) d ppm 8.01 (1 H, s),
lo[1,2-f][1,2,4]triazine-6-(carbonyl-13C)carboxylate, 17
7.85À7.81 (1 H, m), 7.68 (1 H, m), 7.09 (1 H, d, J = 8.24 Hz), 6.97
Compound 165 (0.60 g, 2.64 mmol) was suspended in dry
(1 H, q, J = 6.60 Hz), 6.34 (1 H, s), 5.23 (1 H, m), 2.72 (3 H, m), 2.46
toluene (15 mL) under a nitrogen atmosphere. To the flask
(3 H, s), 1.71 (6 H, q, J = 3.85 Hz). 13C NMR (CDCl3) d ppm 134.14
(t, J = 55.95 Hz), 112.85 (q, J = 50.87 Hz), 70.67 (d, J = 53.41 Hz),
12.27 (t, J = 40.69 Hz). MS ESI1 [M1H]1 = 361.16.
was added phosphorus oxychloride (0.61 g, 3.96 mmol) and N,N-
diisopropylethyl amine (0.44 g, 3.43 mmol). The mixture was
heated to 1101C for 18 h as the reaction became clear and
brownish. The reaction was monitored by TLC (50% EtOAc/
Hexane, product Rf = 0.75). Upon completion, the reaction was
rotovapped to dryness. To the crude solid was added EtOAc
(30 mL), cooled to 01C, washed with ice-cold K2HPO4 (1 M) until
pH reached 8, then extracted further with EtOAc (3 Â 20 mL).
The combined organic extracts were washed with brine, dried
over Na2SO4, filtered, and concentrated to dryness. The crude
product was dried under vacuum for 16 h to give an off-white
solid (677 mg, quantitative yield). This product was used crude
in the next reaction without purification. 1H NMR (300 MHz,
CDCl3) d ppm 8.23 (1 H, t, J = 5.60 Hz), 8.12 (1 H, q, J = 6.28 Hz),
4.38 (2 H, m, dq, J = 3.14 Hz), 2.87 (3 H, dm, J = 130 Hz), 1.40 (3 H,
t, J = 7.12 Hz). 13C NMR (CDCl3) d ppm 164.69 (m), 121À118 (m),
12.39 (m). MS ESI1 [M1H]1 = 246.2.
4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-(methyl-13C)-
methyl(5,6-13C2,1,8-15C2)pyrrolo[1,2-f][1,2,4]triazin-6-ol, 20
Hydrogen peroxide (46.91 mg, 1.38 mmol) (50%) at 01C was
added to cold CH2Cl2 (2.0 mL) followed by BF3OEt2 (2.38 g,
16.74 mmol). The solution was stirred at 01C for 20 min. To a
separate flask containing compound 19 (355 mg, 0.99 mmol)
dissolved in CH2Cl2 (8.0 mL) and cooled to 151C was added over
20 min the above prepared solution. At the end of the addition,
the reaction became cloudy. The reaction was stirred at À151C
for 1 h while being monitored by TLC (50% EtOAc/hexane or 1%
MeOH/CH2Cl2, both, product Rf = 0.5) and HPLC (Method 3,
product, retention time = 18.49 min). The reaction was then
washed with ice-cold Na2SO3 (10%) (25 mL) and ethanolamine
(33%) (25 mL) and extracted with EtOAc (4 Â 34 mL). The
combined organic extracts were washed with brine, dried over
Na2SO4, filtered, and concentrated to dryness. The crude
product was purified by silica gel flash chromatography eluting
with 10–60% EtOAc/hexane to afford a white solid (212.9 mg,
Ethyl 4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-(methyl-13C)-
methyl(5,6-13C2,1,8-15C2)pyrrolo[1,2-f][1,2,4]triazine-6-(car-
bonyl-13C)carboxylate, 18
Compound 17 (674 mg, 2.74 mmol) was suspended in DMF 68% yield). 1H NMR (500 MHz, CDCl3) d ppm 8.01 (1 H, s),
(10 mL), K2CO3 (1.13 g, 8.22 mmol) and 4-fluoro-2-methyl-1H- 7.87–7.83 (1 H, q, J = 8.25 Hz), 7.43 (1 H, t, J = 4.95 Hz), 7.08 (1 H,
indol-5-ol (452 mg, 2.74 mmol) were added. The reaction was d, J = 8.79 Hz), 6.98 (1 H, t, J = 8.25 Hz), 6.33 (1 H, s), 2.55 (3 H, dm,
stirred at rt for 21 h. The reaction was cooled in an ice bath, J = 128.1 Hz), 2.45 (3 H, s), 2.37 (1.5 H, m). 13C NMR (CDCl3) d ppm
washed with ice-cold water (30 mL) and extracted with EtOAc 144.48 (dd, J = 7.63, 66.11 Hz), 101.12 (m), 8.38 (d, J = 48.32 Hz).
(3 Â 50 mL). The combined organic extracts were washed with MS ESI1 [M1H]1 = 317.9.
J. Label Compd. Radiopharm 2011, 54 324–331
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org

S. B. Tran et al.
(R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-(methyl-13C)-
methyl(5,6-13C2,1,8-15C2)pyrrolo[1,2-f][1,2,4]triazin-6-yloxy)-
propan-2-ol, 21
6-(2-Hydroxy(13C)propan-2-yl)-5-(methyl-13C)methyl(5,6-
13C2,1,8-15C2)pyrrolo[1,2-f][1,2,4]triazin-4(3H)-one, 22
Prepared using same procedure as in preparation of 7. Product
22 (off-white solid, 637 mg, 77% yield). 1H NMR (500 MHz,
DMSO-d6) d ppm 11.27 (1 H, br. s.), 7.64 (1 H, dd, J = 8.80, 6.05
Hz), 7.28 (1 H, t, J = 6.60 Hz), 4.82 (1 H, br. s.), 2.52 (3 H, dm,
J = 130.0 Hz), 1.40À1.48 (6 H, m). 13C NMR (DMSO-d6) d ppm
132.69 (t, J = 52.92 Hz), 117.42 (t, J = 54.18 Hz), 68.39 (d,
J = 54.18 Hz), 11.54 (d, J = 45.36 Hz). MS ESI1 [M1H]1 = 214.19.
Compound 20 (205 mg, 0.65mmol) was dissolved in ethanol
(3mL) in a pressure tube. To the solution was added triethylamine
(1 drop), lithium chloride (109.5mg, 2.58mmol), followed by (R)-
(1)-propylene oxide (233.2mg, 4.01mmol). The mixture was
heated to 701C for 3 h while monitoring by TLC (50% EtOAc/
hexane, product Rf = 0.3) and HPLC (Method 3, product, retention
time= 19.41 min). The reaction was cooled and rotovapped to
dryness. To the residue was added water (20 mL) and extracted
6-Hydroxy-5-(methyl-13C)methyl(5,6-13C2,1,8-15C2)pyrro-
with EtOAc (4 Â 30mL). The combined organic extracts were lo[1,2-f][1,2,4]triazin-4(3H)-one, 23
washed with brine, dried over Na2SO4, filtered, and concentrated
Prepared using same procedure as in preparation of 8. Product
to dryness. The crude product was purified on silica gel flash
chromatography eluting with 10–60% EtOAc/hexane to afford a
23 (light yellow solid, 535 mg, 97% yield). 1H NMR (500 MHz,
DMSO-d6) d ppm 11.38 (0.5 H, br. s.), 11.20 (0.5 H, br. s.), 8.80 (1
H, s), 7.60 (1 H, m), 6.94 (1 H, t, J = 5.0 Hz), 2.23 (3 H, dm,
J = 130.0 Hz). 13C NMR (DMSO-d6) d ppm 143.41 (dd, J = 8.82,
64.26 Hz), 107.05À106.18 (m), 8.27 (d, J = 49.14 Hz). MS ESI1 [M1
H]1 = 171.12.
1
white solid (140mg, 58% yield). Product HPLC purity= 98.0%. H
NMR (500MHz, CDCl3) d ppm 8.02 (1 H, s), 7.87À7.83 (1 H, q,
J = 8.25 Hz), 7.42 (1 H, t, J = 4.95 Hz), 7.09 (1 H, d, J = 8.80 Hz), 6.98
(1 H, t, J = 8.25Hz), 6.34 (1 H, s), 4.26 (1 H, m), 3.97 (1 H, m), 3.85 (1
H, m), 2.50 (3 H, dm, J = 128.9 Hz), 1.32 (3 H, d, J = 6.60 Hz). 13C
NMR (CDCl3) d ppm 148.05 (dd, J = 7.63, 68.67 Hz), 101.37 (m), 8.67
(dd, J = 17.8, 48.32 Hz). MS ESI1 [M1H]1 = 375.9, mass isotopic
distribution m/z 375.9 = 94.1%, 374.9 = 5.3%, 373.9 = 0.6%, 370.9 =
undetectable.
5-(Methyl-13C)methyl-4-oxo-3,4-dihydro(5,6-13C2,1,8-15C2)-
pyrrolo[1,2-f][1,2,4]triazin-6-yl pivalate, 24
Prepared using same procedure as in preparation of 9. Product
1
24 (white solid, 487 mg, 61% yield). H NMR (500 MHz, CDCl3) d
ppm 10.16 (0.5 H, br. s.), 9.98 (0.5 H, br. s.), 7.49À7.47 (2 H, m),
2.39 (3 H, dm, J = 130.0 Hz), 1.37 (9 H, s). 13C NMR (CDCl3) d ppm
137.20 (d, J = 76.44 Hz), 113.16À112.59 (m), 8.63 (d, J = 48.41 Hz).
(S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-(methyl-13C)
methyl(5,6-13C2,1,8-15C2)pyrrolo[1,2-f][1,2,4]triazin-6-yloxy)pro-
pan-2-yl) 2-(benzyloxycarbonylamino)propanoate, 27
Prepared using same procedure as in preparation of 14. Product
27 (off-white foam, 45.5 mg, 92% yield). 1H NMR (500 MHz,
CDCl3) d ppm 8.01 (1 H, s), 7.85 (1 H, m), 7.40 (1 H, m), 7.35À7.26
(5 H, m), 7.10 (1 H, d, J = 8.79 Hz), 6.99 (1 H, t, J = 7.14 Hz), 6.34
(1 H, s), 5.25À5.20 (2 H, m), 5.11 (2 H, s), 4.42 (1 H, m), 4.15 (2 H,
m), 2.45 (3 H, s), 2.10 (3 H, dm, J = 128.0 Hz), 1.45 (3 H, m). 13C
NMR (CDCl3) d ppm 148.01 (dd, J = 7.63, 68.67 Hz), 101.45 (m),
8.55 (dd, J = 17.8, 48.32 Hz). MS ESI1 [M1H]1 = 580.9.
4-Chloro-5-(methyl-13C)methyl(5,6-13C2,1,8-15C2)pyrrolo[1,2-
f][1,2,4]triazin-6-yl pivalate, 25
Prepared using same procedure as in preparation of 10. Product
1
25 (white solid, 239 mg, 89% yield). H NMR (500 MHz, CDCl3) d
ppm 8.16–8.11 (1 H, m), 8.0 (1 H, m), 2.49 (3 H, dm, J = 128.1 Hz),
1.41 (9 H, s). 13C NMR (CDCl3) d ppm 140.09 (d, J = 76.86 Hz),
107.15À106.18 (m), 9.14 (d, J = 49.14 Hz). MS ESI1 [M1H]1
273.08.
=
(S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-(methyl-13C)
methyl(5,6-13C2,1,8-15C2)pyrrolo[1,2-f][1,2,4]triazin-6-yloxy)pro-
pan-2-yl) 2-aminopropanoate, 28
4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-(methyl-13C)methyl-
(5,6-13C2,1,8-15C2)pyrrolo[1,2-f][1,2,4]triazin-6-yl pivalate, 26
To a solution of compound 27 (45.5 mg, 0.08 mmol) in EtOAc
(0.9 mL) under nitrogen was added ground K2CO3 (6.8 mg,
0.05 mmol) in a 2 mL V shaped vial. Wet (50%) palladium (5%) on
activated carbon (6.80 mg) was quickly added to the mixture.
The reaction vial was placed under vacuum and flushed with
hydrogen. This step was repeated three times. It was charged
with hydrogen to 20 psi for 2.5 h. The reaction was monitored by
HPLC (Method 2, product retention time = 45.83 min) and TLC
(50% EtOAc/Hexane, product Rf = 0.7). The reaction was filtered
through a syringe filter (0.45 mm nylon membrane) then rinsed
with EtOAc (2 mL). The solvent was removed by rotovap leaving
Prepared using same procedure as in preparation of 12. Product
26 (white solid, 335 mg, 82% yield). 1H NMR (500 MHz, DMSO-d6)
d ppm 11.36 (1 H, s), 8.08À8.00 (2 H, m), 7.16 (1 H, d, J = 10.0 Hz),
7.01 (1 H, t, J = 10.0 Hz), 6.25 (1 H, s), 2.41 (3 H, s), 2.37 (3 H, dm,
J = 129.0 Hz), 1.35 (9 H, s). 13C NMR (DMSO-d6) d ppm 138.07 (d,
J = 69.30 Hz), 104.84 (m), 8.51 (d, J = 50.40 Hz). MS ESI1 [M1H]1 =
402.0.
Results and discussion
a solid. The solid product was dried under vacuum for 18 h to Synthesis of [14C]Brivanib, 13 was completed, via a ring
give 29.6 mg of glassy solid (84% yield). HPLC chemical cyclization reaction, by reacting 1.0–1.1 eq [14C]HC(OEt)3 with
purity = 96.50%. 1H NMR (500 MHz, DMSO-d6) d ppm 11.35 (1 ethyl 1-amino-5-carbamoyl-4-methyl-1H-pyrrole-3-carboxylate,
H, br. s.), 8.05–7.95 (2 H, m), 7.14 (1 H, d, J = 8.25 Hz), 6.98 (1 H, t, 5,3 to give [14C]ethyl 5-methyl-4-oxo-3,4-dihydropyrrolo[1,2-
J = 7.97 Hz), 6.24 (1 H, s), 5.19 (1 H, td, J = 6.60, 3.85 Hz), f][1,2,4]triazine-6-carboxylate, 6, in 38% yield by conventional
4.35À4.10 (2 H, m), 3.50À3.40 (1 H, m), 2.45À2.20 (6 H, m), heating or in 76% yield by microwave at 1501C and 200 W
1.40À1.34 (3 H, d, J = 6.59 Hz), 1.24 (3 H, d, J = 7.14 Hz).13C NMR power.4 The ethyl ester at the C-6 position was converted to an
(DMSO-d6) d ppm 147.01 (m), 101.45 (m), 8.29 (m). MS ESI1 [M1 hydroxyl group, 8 via a sequence of reduction, oxidation, and
H]1 = 447.19, mass isotopic distribution m/z 447.19 = 94.8%, Baeyer–Villiger rearrangement. Subsequent chemical steps,
446.19 = 5.1%, 442.19 = undetectable.
involving alcohol protection with trimethylacetyl chloride,
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Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 324–331

S. B. Tran et al.
chlorination with phosphorus oxychloride, coupling with 4- selective O-alkylation with (R)-(1)-propylene oxide preferably
fluoro-2-methyl-1H-indol-5-ol, 11, afforded pyrrolotriazine-aryl at the site of the unhindered carbon of the epoxide to produce
ether, 12 in an overall yield of 23% from 6. The pivaloyl group 17.2 mCi of the desired parent drug, 13, with a 98.1%
was removed by deprotecting with NaOMe followed by radiochemical purity in an overall yield of 34% (Scheme 1).
O
1,1-Dimethoxy- N,N-
dimethylmethanamine
O
O
N
O
O
O
O
1) DMF, NaH, 0˚C - rt, 30 min
2) 0.15 M NH2Cl, Ether/DMF,
rt, 25 h
1) ACOH, rt, 20 h
2) 100˚C, 5 h
NH2
O
O
p-TsOH (cat.)
80˚C, 1 h
OEt
NH
H2N
NH2
+
OEt
NH2
.HCl
4
40%
1
98%
55%
2
3
1.0 - 1.1 equi. [14C]HC(OEt)3
DMA, p-TsOH (cat.)
O
O
O
THF, MeMgCl,
-5 - 45˚C, 6h
O
Conventional heating or
NH
NH
microwave heating4
O
NH2
NH2
N
N
N
O
*
N
HO
N
*
92%
O
38 - 76%
7
5
* = 14
6
C
THF, DIEA,
trimethylacetyl
chloride
O
1) THF, 50% H2O2,
MSA, -10 - 0˚C
O
Cl
POCl3, DIEA,
ACN, reflux,
17 h
O
O
O
NH
HO
NH
N
2) NaHSO3 (60% SO2),
NH4OH
N
O
N
*
N
N
46% over 2 steps
N
9
*
N
*
8
10
H
N
H
1) NaOMe, ACN, 10˚C
2) Water/ACN
N
4-Fluoro-2-methyl-
1H-indol-5-ol, 11
O
28oC, 24 h
O
F
DABCO,
O
O
(R)-(+)-propylene oxide
N
ACN, 50˚C, 1.5 h
F
O
HO
N
N
N
74%
*
N
N
*
75% over 2 steps
13
12
H
N
H
N
O
CBz-L-alanine
EDAC . HCl, DMAP,
EtOAc/DMF,
F
1) Condition:
O
N
N
See Table 1, Run 4
83%
F
O
O
N
N
0oC - rt, 5 h
O
N
*
O
N
O
2) Crystallization,
*
EtOAc/Heptane, 50˚C
56%
NH2
96%
O
14
15
NHCBz
14
Scheme 1. Synthesis of parent drug candidate [14C]Brivanib, 13 and prodrug [14C]Brivanib Alaninate, 15.
Table 1. Investigation of balloon hydrogenation to remove CBz group
Run Unlabeled Compound 14( mg) 5% Pd/C, wet 50/50 (mg) K2CO3 (mg) EtOAc (mL) Time (h) HPLC Yield (%) of 15
1
2
3
4
5
300
300
300
300
300
36
36a
250
150
100
50
50
50
50
50
3
3
3
3
3
32
32
6
2
1.5
15
15
42
77
49
aRun 2, 10% Pd/C was used.
J. Label Compd. Radiopharm 2011, 54 324–331
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org

S. B. Tran et al.
Radiochemical stability of 13 as a solid at a specific activity of 94.1%. A molecular ion of 374.9 Da, corresponding to the
24.3 mC/mg (10.3 mCi/mmol) was shown to decrease by 0.7% empirical formula 13C2C17H19F15N2N2O3, was 5.3% (Scheme 2).
after 3 years of storage at À781C.
Subsequently, a second lot of 215 mg at 98.0% chemical
The pharmaceutic and pharmacokinetic properties of the parent purity was produced using the later established Process
were improved by preparing the Alaninate prodrug. Balloon Research and Development (PR&D) synthetic route6 (Scheme 3).
hydrogenation of the penultimate, 14, removed the CBz group and The removal of the CBz group on a small scale required
generated the prodrug, Brivanib Alaninate. Results of various high pressure hydrogenation using this route. The [13C315N2]pro-
conditions for CBz group deprotection are summarized in Table 1. drug penultimate, 27 (45 mg, 0.08mmol), dissolved in 0.9 mL
Reaction conditions shown in Run 4 were applied in the radioactive EtOAc, conditioned with ground K2CO3 (6.8mg), Pd-C 5%
synthesis to produce an 83% yield of the desired product. (6.8mg) was hydrogenated at 20psi for 2.5 h to give 29.6mg,
Recrystallization of the final product gave 2.7 mCi of [14C]Brivanib 84% yield of the prodrug [13C315N2]Brivanib Alaninate, 28 at 96.5%
Alaninate, 15, with a chemical purity of 99.2%, a specific activity of chemical purity. This represents a reduction of 70% less 5% Pd-C
23.2 mCi/mg (10.3 mCi/mmol) and a radiochemical purity of 99.2%. than in the previous balloon hydrogenation (Scheme 4). Both
The stable isotope-labeled central core pyrrolotriazinone 16, [13C315N2]Brivanib, 21 and [13C135N2]Brivanib Alaninate, 28 had
prepared from suitably labeled materials using the same undetectable levels of [M10] unlabeled products by LC-MS and
route as in the [14C]synthesis, was reported by Ogan et al.5 were suitable for use as internal standards to support bioanaly-
Initially, the medicinal chemistry synthetic route was used to tical LC-MS analyses of biological samples.
produce 140 mg of [13C315N2]Brivanib, 21, with a chemical
purity of 98.6%. Mass spectrometric analysis of [13C315N2]Brivanib
Conclusion
was performed using positive ion electrospray on a Q-Tof
Ultima mass spectrometer giving a molecular ion [M1H]1 at
375.9 Da. This is consistent with the molecular formula
13C3C16H19F15N2N2O3. Isotopic purity was calculated to be
The synthesis of [14C]Brivanib was completed to produce
17.2 mCi of the desired parent drug, 13 with 98.1%
radiochemical purity. Balloon hydrogenation of the penultimate,
a
H
N
O
O
Cl
LiCl,
POCl3, DIEA,
toluene, 110˚C,
18 h
O
O
NH
11, DMF,
K2CO3, rt
F
THF/Toluene (1:1),
CH3MgBr
O
O
O
O
N
N
N
N
N
N
N
N
56%
66%
98%
13C or 15N
18
17
16
H
N
H
N
H
N
O
N
O
N
O
Et3N, LiCl,
EtOH,
(R)-(+)-propylene oxide
50% H2O2,
BF3OEt2,
CH2Cl2,
F
F
F
N
N
HO
O
N
N
N
N
N
HO
OH
68%
58%
20
19
21
Scheme 2. Synthesis of stable isotope labeled parent drug candidate [13C315N2]Brivanib, 21 prepared by the medicinal chemistry route.
1) THF, 50% H2O2,
MSA, -10 - 0˚C
O
N
O
O
O
THF, DIEA,
trimethylacetyl
chloride
O
O
THF, MeMgCl,
-5 - 45˚C, 6 h
NH
NH
HO
NH
NH
2) NaHSO3,
NH4OH
PivO
N
N
HO
N
N
N
N
N
77%
61%
97%
13C or 15N
23
24
22
16
H
N
H
N
POCl3,
DIEA,
ACN,
Cl
1) NaOMe, ACN, 10oC
O
11, DABCO,
ACN, 50˚C,
1.5 h
2) Water : ACN (2:1)
28oC, 24 h,
(R)-(+)-propylene oxide
O
N
F
reflux
N
PivO
F
N
PivO
N
N
N
82%
89%
N
O
N
N
25
OH
26
71% over 2 steps
21
Scheme 3. Synthesis of stable isotope-labeled parent drug candidate [13C315N2]Brivanib, 21 prepared by the PR&D route.
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Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 324–331

S. B. Tran et al.
H
H
H
N
N
N
O
N
CBz-L-alanine
EDAC . HCl,
DMAP,
O
O
N
F
F
F
N
N
N
K2CO3, Pd-C 5%,
H2, 20 psi, 2.5 h
EtOAc / DMF,
0˚C - rt, 5 h
O
O
O
O
N
O
O
N
N
N
OH
O
O
O
92%
84%
21
HN
27
28
NH2
13C or 15N
Scheme 4. Synthesis of stable isotope labeled prodrug candidate [13C315N2]Brivanib Alaninate, 28.
14 to generate the final prodrug Brivanib Alaninate was
References
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radioactive synthesis to produce 2.7 mCi of [14C]Brivanib
Alaninate, 15 with a specific activity of 23.2 mCi/mg (10.3 mCi/
mmol) and a radiochemical purity of 99.2%.
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215 mg at 98.0% chemical purity was produced using the
later established PR&D synthetic route. A small scale high-
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[13C135N2]Brivanib Alaninate, 28. The stable isotope-labeled
products had undetectable levels of [M10] unlabeled products
by LC-MS.
Acknowledgements
We gratefully acknowledge John Thornton, Paul Lobben, Lori
Spangler, and Zhongping Shi of the Bristol-Myers Squibb
Process Research & Development, Rulin Zhao of Chemical
Synthesis, and Donna Wei, Wen-Ching Han, Rajeev Bhide, and
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with intermediates and synthetic methodologies. We also thank
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for completing the analytical mass spectroscopy analysis of
intermediates and final products.
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J. Label Compd. Radiopharm 2011, 54 324–331
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org