Synthesis of three isotopically labeled versions and a metabolite of Aurora A kinase inhibitor

Article abstract of DOI:10.1002/jlcr.1607

Sodium ring-[¹⁴C]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H- pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoate (1A, MLN8054), an Aurora A kinase inhibitor, was synthesized from [¹⁴C]-cyanamide in two steps in an overall radiochemical yi

Full text of DOI:10.1002/jlcr.1607

Research Article
Received 8 January 2009,
Accepted 31 March 2009
Published online 3 July 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1607
Synthesis of three isotopically labeled
versions and a metabolite of Aurora A
kinase inhibitor
Ã
Yuexian Li and Shimoga R. Prakash
Sodium ring-[14C]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoate (1A, MLN8054),
an Aurora A kinase inhibitor, was synthesized from [14C]-cyanamide in two steps in an overall radiochemical yield of 7%. The
intermediate, [14C]-4-guanidinobenzoic acid, was prepared by coupling [14C]-cyanamide with 4-aminobenzoic acid. Sodium
carboxyl-[14C]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoate (1B) was synthe-
sized from carboxyl-[14C]-4-guanidinobenzoic acid in one step in a radiochemical yield of 35%. [D4,15N]-4-[[9-chloro-7-(2,6-
difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (1C) was synthesized from [15N2]-cyanamide and
[D4]-4-aminobenzoic acid in two steps in an overall yield of 37%. The major metabolite, b-acyl glucuronide of 4-[[9-chloro-7-
(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (14), was synthesized from D-glucuronic acid
in three steps in an overall yield of 1%. The key intermediate for synthesis of glucuronide was prepared by HATU
catalyzed coupling of 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid with allyl
glucuronate.
Keywords: sodium ring-[14C]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoate; sodium
carboxyl-[14C]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoate; [D4, 15N]-4-[[9-chloro-7-
(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid; b-acyl glucuronide of 4-[[9-chloro-7-(2,6-difluorophe-
nyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid; [14C]-4-guanidinobenzoic acid; [D4, 15N]-4-guanidinobenzoic acid
of the molecule (Scheme 1).4 Preclinical metabolism results
indicated that radiolabeling either portion of the molecule
Introduction
Aurora A kinase plays an essential role in the proper assembly
and function of the mitotic spindle.1,2 Inhibition of Aurora A
kinase represents an attractive modality for therapeutic inter-
vention of human cancers.3 The biological activity of the
would be adequate for the intended ADME studies in animals.
For expedient synthesis, we chose labeling guanidinobenzoic
acid 2 instead of benzoazepine 7 for both stable isotope and
radiolabeled versions (Scheme 1).
recently discovered Aurora
A
inhibitor, 4-[[9-chloro-7-
There are two possible approaches to radiolabel compound
2, either on the gualidine portion to prepare the ring-labeled
(2,6 - difluorophenyl) - 5H -pyrimido[5,4-d][2]benzazepin-2-yl]ami-
no]-benzoic acid (1, Figure 1),1–4 led to detailed investigations
on its disposition characteristics. The radiolabeled versions were
required to assist such investigations, especially in metabolite
profiling and whole body autoradiography studies in experi-
mental animals. The stable isotope labeled version was also
required as an internal standard for mass spectrometry-based
bio-analytical assays. The major metabolite, b-acyl glucuro-
nide of 4 - [[9 - chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]-
benzazepin-2-yl]amino]-benzoic acid, was required to support
the studies of inhibitory effects on transporters (PGP, BCRP, etc.)
and as a reference standard.5
1
or on carboxylic group to the carboxyl-labeled
1
(Scheme 1). We first chose to synthesize the ring-labeled
compound 1 either from [14C]-thiourea or [14C]-cyanamide. In
the unlabeled synthesis, the yield of aminoiminomethane-
sulfonic acid 4 from 5 and ethaneperoxoic acid was only
14%.6 But the reaction of cyanamide 6 and 3 resulted in a
reasonable yield.7 Therefore [14C]-cyanamide 6A was chosen
the radiolabeled precursor (Scheme 2). Reaction of 6A with
4-aminobenzoic acid 3 provided [14C]-4-guanidinobenzoic acid
Department of Drug Metabolism and Pharmacokinetics, Isotope Chemistry
Group, Millennium Pharmaceuticals, Inc., The Takeda Oncology Company,
35 Landsdowne Street, Cambridge, MA 02139, USA
Results and discussion
*Correspondence to: Yuexian Li, Department of Drug Metabolism and
Pharmacokinetics, Isotope Chemistry Group, Millennium Pharmaceuticals, Inc.,
The Takeda Oncology Company, 35 Landsdowne Street, Cambridge, MA 02139,
USA.
The structure of 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimi-
do[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (1) provides
two potential sites for labeling, either on the top pyrimidinimine
benzoic acid portion or the bottom benzoazepine portion
E-mail: Yuexian.Li@mpi.com
J. Label Compd. Radiopharm 2009, 52 355–359
Copyright r 2009 John Wiley & Sons, Ltd.

Y. Li and S. R. Prakash
2A in a yield of 43%. Ring-[14C]-4-[[9-chloro-7-(2,6-difluorophe-
nyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoate (1A) 5 amu higher than the unlabeled version is required to ensure
Because 1 contains a chlorine atom, a labeled version that is
was prepared by K2CO3-mediated coupling of 2A with 7 in complete separation of labeled and unlabeled molecular ion
methanol (16%).4
clusters during mass spectrometric assays of 1. To increase the
The solid C-14-labeled compound 1A was found to be molecular weight, labeling of the pyrimidinimine benzoic acid
unstable, but its solution of ethanol/water (or methanol/water) portion of the molecule or benzoazepine portion was required.
was stable. For a better stability in solid phase, carboxyl-[14C]-4- Labeling the 4-guanidinobenzoic acid 2 with deuterium and
[[9 - chloro - 7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzaze- nitrogen-15 was chosen. Similar to compound 1A, compound 1C
pin-2-yl]amino]-benzoate (1B) was synthesized from commercially was synthesized from [15N]-cyanamide 6B and [D4]-4-amino-
available carboxyl-[14C]-4-guanidinobenzoic acid in a yield of benzoic acid 3A (Scheme 3).
35% (Scheme 2). The solid compound 1B was found to be much
more stable than the solid compound 1A.
To chemically synthesize acyl glucuronides, several strategies
have been utilized. Mesmaeker et al. employed an approach of a
fully protected glucuronic acid that included ten-step synthesis
with several protections and deprotections.8 Juteau et al.
utilized a Mitsunobu reaction of allyl glucuronate with carboxylic
acids.9 But the ratio of b/a was only from 5/1 to 2/1.10 Stachulski
et al. reported a more selective method for the synthesis of this
type of compound by b-acylation of allyl glucuronate with
carboxylic acids catalyzed by HATU (o-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate).10 There-
fore, Stachulski’s method was chosen for the synthesis of
b-acyl glucuronide 14, (Scheme 4). The protected allyl glucur-
onate 12 was prepared from D-glucuronic acid 11 and allyl
bromide.10 HATU-mediated coupling of 12 with 1 provided the
key intermediate compound 13. But no product was found
when only acetonitrile was used as the solvent.10 To dissolve the
highly insoluble compound 1, the co-solvent of acetonitrile and
N-methylmorpholine was utilized for the coupling reaction.
b-Acyl glucuronide of 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyr-
imido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (14) was
prepared by de-protection of 13 using Pd(PPh3)4 in conjunction
with pyrrolidine in THF.10
O
OH
HN
N
N
N
Cl
F
F
1
Figure 1. The structure of MLN8054.
O
O
O
HO3S
HN
OH
+
S
OH
NH2
OH
NH2
H2N
H2N
3
4
5
HN
HN
N
N
O
NH2
HN
OH
2
+
H2N CN
6
N
Cl
H2N
3
F
F
NMe2
O
O
1
NMe2
NMe2
O
O
N
Cl
N
Cl
+
O
F
F
F
F
10
9
7
8
Scheme 1.
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J. Label Compd. Radiopharm 2009, 52 355–359

Y. Li and S. R. Prakash
O
NMe2
X
O
ONa
O
OH
X
O
OH
HN
N
Cl
Y
N
F
F
N
HCl/H2O
H2N 14CN
+
7
6A
HN
NH2
Y
NH2
N
Cl
.HCl
1. K2CO3/CH3OH
2. HCl
3. NaOH
NH
F
F
3
2A, X = 12C, Y = 14
2B, X = 14C, Y = 12
C
C
1A, X = 12C, Y = 14
C
1B, X = 14C, Y = 12
C
Scheme 2.
O
D
NMe2
O
OH
D
D
O
OH
O
OH
HN
N
Cl
D
D
D
D
D
15N
D
D
D
D
15N
F
F
HCl/H2O
H215N C15
6B
N
+
7
HN
15NH2
NH2
3A
N
Cl
.HCl
1. K2CO3/CD3OD
2. HCl
15NH
2C
F
F
1C
Scheme 3.
performed on an Agilent 1100 Series LC/MSD and a Thermo-
Finnigan LCQ mass spectrometers. Column chromatography
was performed on silica gel (230À400 mesh) supplied by
SiliCycle, Canada.
Experimental
General
All commercial reagents were used as supplied unless
otherwise noted. [14C]-Cyanamide was purchased from
American Radiolabeled Chemicals, USA. Carboxyl-[14C]-4-guani-
dinobenzoic acid was purchased from PerkinElmer Life Sciences,
USA. [15N2]-Cyanamide was purchased from Aldrich, USA. [D4]-4-
Aminobenzoic acid was purchased from C/D/N Isotopes,
Canada. Radioactivity was quantified by liquid scintillation
[
14C]-4-Guanidinobenzoic acid (2A)7
Aqueous [14C]-cyanamide solution (50%, 108 mCi, 2.5 mmol,
43 mCi/mmol) was added to a mixture of concentrated HCl
(37%, 0.15 mL, 1.82 mmol) and 4-aminobenzoic acid (312 mg,
2.27 mmol) at 851C. Another portion of concentrated HCl (37%,
0.04 mL, 0.454 mmol) was added. After stirring for 4 h at 851C,
the mixture was allowed to cool to room temperature. The
solvents were removed by evaporation. The resulting solid was
counting using
a
Beckman LS6500 counter. Purities
were determined by HPLC (Agilent 1100) on Luna C18(2)
column (5 mm, 4.5 Â 150 mm) eluted at 1 mL/min with A
(0.1% formic acid in 99% water and 1% CH3CN) and B (0.1%
formic acid in 5% water and 95% CH3CN). Elution was 0% B
for 5 min, 0–100% B over 15 min, and 100% B holding for 5 min.
UV detection was at 254 nm. Radioactive detection was with an
IN/US b-Ram RHPLC detector using an Ultima Flo M scintillant
at 3 mL/min. NMR spectra were recorded on either a Varian
purified by preparative HPLC on
a Luna column (5 mm
250 Â 21.2 mm) using a gradient of 0.1% HCOOH in water and
0.1% HCOOH in acetonitrile. Product containing fractions were
combined and dried under vacuum to yield 46 mCi (43%,
43 mCi/mmol) product. HPLC: chemical purity 95% (UV at
254 nm); radiochemical purity, 96%. LC-MS/MS: m/z 182 (M11),
180.
600 or
a 300 MHz spectrometer. LC-MS analyses were
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Y. Li and S. R. Prakash
OH
O
NH
N
N
OH
11
O
N
Cl
allyl bromide/DMF
fluoride-polymer
O
O
OH
OH
F
F
O
1
O
OH
OH
HO
OH
HO
OH
HATU/CH3CN
N-methylmorpholine
12
OH
O
O
O
O
OH
OH
O
OH
OH
O
O
OH
OH
O
1. (Ph3P)4Pd
pyrrolidine/THF
2. prep-HPLC
O
NH
NH
N
N
N
N
N
Cl
N
F
Cl
F
F
F
14
13
Scheme 4.
Sodium ring-[14C]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyri-
added to a dry flask. Methanol (6.8 mL) and K2CO3 (553 mg,
4.0 mmol) were added under nitrogen. The resulting mixture
was heated at 601C under nitrogen overnight. After cooling to
room temperature, water (2 mL) and 5 M HCl (1.4 mL) were
added. The resulting solid was collected by filtration, dissolved
in methanol (80 mL) and mixed with 1 N NaOH (1 eq). The
solvents were removed. The resulting solid was crystallized from
ethanol and dried to give 39.9 mCi (57 mCi/mmol, 35%) of the
title compound. HPLC: 99.0% (UV at 300 nm); radiochemical
purity, 98.4%. LC-MS/MS: m/z 479 (M11), 481. 1H-NMR (CD3OD):
d8.58 (NH, s), 8.38 (1H, d), 7.95 (2H, d), 7.78 (4H, m), 7.50 (1H, m),
7.28 (1H, s), 7.05 (2H, t), 3.90 (2H, s).
mido[5,4-d][2]benzazepin-2-yl]amino]-benzoate (1A)4
[14C]-4-Guanidinobenzoic acid (2A) (41 mCi, 0.942 mmol, 43 mCi/
mmol) and compound 7 (309 mg, 0.856 mmol) were added to a dry
flask. Methanol (3.2 mL) and K2CO3 (260 mg, 1.88 mmol) were added
under nitrogen. The resulting mixture was heated at 601C under
nitrogen overnight. After cooling to room temperature, water
(0.9 mL) and 5 M HCl (0.64 mL) were added. The resulting solid was
collected by centrifuge. The impure product was purified by
preparative HPLC on a Luna column (5 mm, 250 Â 21.2 mm) using a
gradient of 0.1% HCOOH in water and 0.1% HCOOH in acetonitrile.
Product containing fractions was combined and dried under
vacuum. 1 N NaOH (1 eq) was added to the resulting solid dissolved
in methanol. The solvents were removed to give 6.6 mCi (43 mCi/
mmol, 16%) of the title compound. HPLC: 99.0% (UV at 300 nm);
radiochemical purity, 99.2%. LC-MS/MS: m/z 479 (M11), 481.
1H-NMR (CD3OD): d 8.58 (NH, s), 8.38 (1H, d), 7.95 (2H, d), 7.80 (4H,
m), 7.50 (1H, m), 7.30 (1H, s), 7.05 (2H, t), 4.00 (2H, s).
[D4,15N]-4-guanidinobenzoic acid (2C)7
Concentrated DCl in D2O (35%, 0.14 mL, 1.82 mmol) was added
to [D4]-4-aminobenzoic acid (282 mg, 2.0 mmol). [15N2]-cyana-
mide solution in D2O (50%, 3.0 mmol) was added into the
reaction mixture at 851C. A further concentrated DCl in D2O
(35%, 0.04 mL, 0.454 mmol) was added. After stirring for 4 h at
851C, the mixture was allowed to cool to room temperature. The
solvents were removed by evaporation. Acetonitrile was added
to the resulting solid. The mixture was filtered and washed with
acetonitrile and acetone. The resulting solid was dried under
Sodium carboxyl-[14C]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H-
pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoate (1B)4
Carboxyl-[14C]-4-guanidinobenzoic acid (2B) (114 mCi, 2.0 mmol,
57 mCi/mmol) and compound 7 (656 mg, 1.82 mmol) were
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J. Label Compd. Radiopharm 2009, 52 355–359

Y. Li and S. R. Prakash
vacuum to yield 380 mg (80%) product. HPLC: 96% (UV at (1H, s), 7.00 (2H, s), 5.70 (1H, s), 4.78 (1H, s), 3.90 (1H, s), 3.82 (2H, d),
1
254 nm). LC-MS/MS: m/z 186 (M11). H-NMR ((CD3)2SO): d13.00 3.50 (2H, d).
(COOH, s), 10.43 (1H, s), 7.78 (4H, s).
Conclusion
[D4,15N]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-
d][2]benzazepin-2-yl]amino]-benzoic acid (1C)4
In summary, practical methods for the preparation of carbon-14
and stable isotope-labeled Aurora A inhibitor, 4-[[9-chloro-7-
(2,6 - difluorophenyl) - 5H-pyrimido[5,4-d][2]benzazepin-2-yl]ami-
no]-benzoic acid, were developed. The key labeled intermediate
[D4,15N]-4-guanidinobenzoic acid (2C) (346, 1.38 mmol) and
compound 7 (454 mg, 1.26 mmol) were added to a dry flask.
[D4]-Methanol (4.7 mL) and K2CO3 (382 mg, 2.77 mmol) were
product, 4-guanidinobenzoic acid, was synthesized from cyana-
added under nitrogen. The resulting mixture was heated at 601C
under nitrogen overnight. After cooling to room temperature,
D2O (1.4 mL) and 5 M DCl in D2O (0.9 mL) were added. The
mide and 4-aminobenzoic acid in HCl aqueous solution.
A convenient method for the synthesis of the b-acyl glucur-
onide, a common drug metabolite, was developed. The key
mixture was filtered and washed with methanol and water. The
intermediate product was prepared by the HATU-mediated
resulting solid was crystallized from [D4]-Methanol. The purified
solid was dried to give 394 mg (58%) of the title compound.
coupling of 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-
d][2]benzazepin-2-yl]amino]-benzoic acid with allyl glucuronate.
Sodium ring-[14C]-4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimi-
HPLC: 96% (UV at 254 nm). LC-MS/MS: m/z 483 (M11), 485.
1H-NMR (CD3OD): d12.50 (COOH, s), 10.28 (NH, s), 8.78 (1H, d),
do[5,4-d][2]benzazepin-2-yl]amino]-benzoate was prepared from
[14C]-cyanamide in two steps in an overall yield of 7%. Sodium
8.30 (1H, d), 7.90 (1H, m), 7.58 (1H, m), 7.38 (1H, s), 7.18 (2H, m),
4.90 (1H, s), 3.90 (1H, s).
carboxyl - [14C] - 4 -[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido-
[5,4-d][2]benzazepin-2-yl]amino]-benzoate was synthesized
D-allyl glucuronate 1210
from carboxyl-[14C]-4-guanidinobenzoic acid in one step in
a
yield of 35%. [D4,15N]-4-[[9-chloro-7-(2,6-difluorophenyl)-
D-glucuronic acid 11 (4.0 g, 20.6 mmol) in DMF (40 mL) was
added allyl bromide (2.0 mL, 23 mmol) and polymer-supported
fluoride (8.5 g, 22 mmol). The resulting mixture was stirred at
521C under nitrogen overnight. The reaction mixture was cooled
to room temperature and filtered. The filtrate was evaporated.
The resulting residue was purified by chromatography on silica
gel (heptanes/dichloromethane/ethanol) to yield 1.64 g (34%)
product. This product was used in the next step without further
characterization.
5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid was
synthesized from [15N2]-cyanamide and [D4]-4-aminobenzoic
acid in two steps in an overall yield of 37%. b-Acyl glucuronide
of 4-[[9-chloro - 7 - (2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benza-
zepin-2-yl]amino]-benzoic acid was synthesized from D-glucuro-
nic acid in three steps.
Acknowledgement
Thanks are due to the Process Research Department and the
Medicinal Chemistry Department of Millennium Pharmaceuticals
for the provision of unlabeled intermediates. Thanks are also
due to Mark Milton of the Analytical Chemistry Department for
providing 600 MHz NMR spectra of labeled compounds.
b-Allyl glucuronide of 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-
pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid 13
4-[[9-Chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benza-
zepin-2-yl]amino]-benzoic acid 1 (2.95 g, 6.18 mmol), D-allyl
glucuronate 12 (1.448 g, 6.18 mmol), and HATU (2.82 g,
7.42 mmol) were stirred in acetonitrile (32 mL) with N-methyl-
morpholine (32 mL) under nitrogen for 2 h. The reaction was
quenched by adding Amberlyst A-15 (H1 form, 24 g). After
evaporation, the residue was purified by chromatography on
silica gel (dichloromethane/ethanol) to get the product (385 mg,
9%). HPLC: 96% (UV at 254 nm). LC-MS/MS: m/z 693 (M11),
695. 1H-NMR ((CD3)2SO): d 10.40 (1H, s), 8.80 (1H, s), 8.38
(1H, d), 8.20 (4H, s), 7.90 (1H, d), 7.60 (1H, m), 7.38 (1H, s), 7.20
(2H, t), 5.92 (1H, m), 5.65 (1H, d), 5.50 (1H, m), 5.38 (1H, m), 5.12
(1H, d), 4.62 (1H, d), 4.02 (1H, d), 3.78 (1H, s), 3.40 (2H, m), 3.16
(1H, s).
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To a solution of compound 13 (83 mg, 0.12 mmol) in THF (2 mL)
at 01C was added Pd(PPh3)4 (14 mg, 0.12 mmol) followed by
pyrrollidine (0.01 mL, 0.12 mmol). The reaction solution was
stirred half hour at 01C. After evaporation, the residue was
purified by preparatory HPLC on a C18(2) Luna column (water/
acetonitrile/0.1% HCOOH) to get the product (28 mg, 36%).
HPLC: 99% (UV at 254 nm). LC-MS/MS: m/z 653 (M11), 655.
1H-NMR (15% CD3CN/85% D2O): d10.39 (NH, s), 8.70 (1H, s), 8.20
(1H, d), 8.00 (2H, s), 7.80 (2H, m), 7.65 (1H, m), 7.45 (1H, m), 7.40
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