Syntheses of isotopically labeled versions and major metabolites of CCR1 antagonist MLN3897

Article abstract of DOI:10.1002/jlcr.1771

MLN3897 citrate is a novel CCR1 antagonist that was taken into development for treatment of chronic inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. Non-clinical data had indicated extensive metabolism of MLN3897 via N-dealkylati

Full text of DOI:10.1002/jlcr.1771

Abstracts
Published online 13 May 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1771
10th International Symposium on the
Synthesis and Applications of Isotopes and
Isotopically Labelled Compounds—
Applications of Isotopes in Pharmacology,
Clinical and Medical Research
Session 16, Wednesday, June 17, 2009
SESSION CHAIRS: CHAD ELMOREa AND BRAD MAXWELLb
aAstraZeneca, USA
bBristol-Myers Squibb, USA
Abstract: Diverse topics, with a central theme are discussed. On the one hand, the preparation of an anti-epidermal growth factor
MAB, labelled with 177Lu is detailed. In addition, traditional syntheses of isotopically labelled small molecules are also highlighted.
Keywords: Epidermal growth factor; lutetium; MLN3897; C-14; Apixaban; Factor Xa; deuterium
LABELLING OF ANTI-EPIDERMAL GROWTH FACTOR MONOCLONAL ANTIBODY WITH 177LU:RADIO-
CHEMICAL AND BIOLOGICAL EVALUATION
DANA NICULAEa, VALERIA LUNGUa, RODICA ANGHELb, IULIANA GRUIAb, AND MARINA ILIESCUa
aHoria Hulubei National Institute for Physics and Nuclear Engineering, Bucharest Magurele, Romania
bAlexandru Trestioreanu Institute of Oncology, Bucharest, Romania
Abstract: This study aims to evaluate the synthesis parameters of 177Lu-anti-EGF-Mab using a macrocyclic ligand, DOTAM (2,20,200,2000-
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetamide) and its in vivo biodistribution in animal models. DOTAM-EGF was
synthesized in high yield at room temperature in 0.1 M NaHCO3 pH 8.2 using a mixture of DOTAM/anti-EGF-Mab in nonstoichiometric
ratio. The complex was then labeled with 177Lu (15 mCi 177LuCl3, pH 4) with more than 95% labeling efficiency. Biodistribution
evaluation revealed a major accumulation in periferal tumor tissues (up to 12.49% ID/g) at 24h after i.v. injection while the tumor mass
uptaked 7 times less activity. The liver, kidneys, and small intestine showed a moderate took up, the mean value was 3.4 ID/g in the first
two hours post injection following a decreasing curve up to 0.5% ID/g at 72h post injection. Based of the preliminary results of this
study 177Lu-(DOTAM)anti-EGF-Mab will be tested as a new radiopharmaceutical for epidermal malignancies.
Keywords: epidermal growth factor; lutetium-177; targeted radiotherapy
Introduction: One of the latest approaches in cancer therapy implies the use of a high linear energy transfer, nonpenetrating
radiation specifically delivered at cellular level. Receptor-mediated radiotherapy (also called targeted radiotherapy, TRT) relies on the
use of a receptor specific ligand to transport a radionuclide to tumor cells that overexpress the target receptor. The selection of the
ligand is based on its ability to selectively target cancer cells. The therapeutic radiation is delivered to tumors while minimizing
radiation exposure to normal tissues. In this concept, the radioligand should be rapidly cleared from circulation; the chelate should be
stable under physiological conditions; the tumor cells should express a sufficiently high density of receptors and a long biological
residence time of radioactivity is needed. The receptor internalization is the basic process that sustains the selectivity and therapeutic
efficacy of targeted radiotherapy while the radioactive metabolites should remain trapped in the cell. Researchers are investigating
both the agents that seek out specific tumor cells for treatment and the radioisotope payload that delivers the radiation.
The EGFR (Epidermal Growth Factor Receptor) is a validated anticancer target. The physiological roles of EGFR ensure epidermal renewal
and integrity. Alterations in the EGFR family are involved in the development of cancer leading to the stimulation of tumor growth,
protection from apoptosis, angiogenesis, and the formation of metastases[1]. Increased EGFR expression is an adverse prognostic feature of
J. Label Compd. Radiopharm 2010, 53 355–367
Copyright r 2010 John Wiley & Sons, Ltd.

Abstracts
many cancers. Epidermal growth factor receptor inhibitors have been developed in recent years as therapeutic molecules directed against
cancer with an overall dominating effect of inducing growth arrest and terminal differentiation of the keratinocytes in the basal layers.
A major decision to make for TRT is the radionuclide, where the nonpenetrating emissions are relevant for therapy. The suitability
of a radionuclide for TRT depends on its physical and chemical properties, its fate after antibody metabolism in vivo and the nature
of the radiation, such as low or high linear energy transfer. One radioisotope that may help create the first successful
radiopharmaceutical for solid tumors is lutetium-177 (Lu-177); it emits a low beta energy, which reduces radiation side effects and
produces a tissue-penetration range appropriate for small tumors.
Aim: Starting from the recently developed agents for targeted molecular imaging we tried to extrapolate the use of the epidermal
growth factor as biological carrier for targeted radiotherapy, aiming to develop a radiopharmaceutical for targeted radiotherapy based on
anti-epidermal growth factor antibody, as a carrier vehicle for non-penetrating radiation delivery. The epidermal growth factor monoclonal
antibodby (anti-EGF-Mab) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR[2,3]
.
We have labeled the EGF with beta emitter Lu-177 (half life 6.71 d, Ebmax496 keV, Eg 208 keV, short-range tissue penetration, mean
range 670 mm). Lu-177’s low beta energy together with long half-life and high specificity make it a promising isotope for
radiopharmaceutical treatment of solid tumors[4] and also for micro metastatic disease[5]
.
The study aims to evaluate the synthesis parameters of the 177Lu-antiEGF-Mab using a macrocyclic ligand, DOTAM (2,20,200,2000-(1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetamide) and its in vivo biodistribution in normal and HRS1 tumor bearing animal models.
Materials and Methods: The labeling of anti-EGF-Mab with Lu-177
An indirect radiolabeling procedure was employed. DOTAM (Figure 1) was used as chelating agent for the metal, which forms an
in vivo stable complex with lutetium. The labeling procedure was optimized by varying the reaction parameters such as:
temperature, reaction time, molar ratio (DOTAM to mAb).
Figure 1. DOTAM (2,20,200,2000-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetamide).
DOTAM was attached to the mAb‘s structure in the inactive part not to alter the antibody biological specificity. The coupling of
DOTAM with EGF mAb was done in the first step and then the Lu was introduced in the construct.
First step: 1 mg EGF-mAb was added to 1.93 mg DOTAM in sodium carbonate buffer NaHCO3 0.1 M (pH = 8.2). After 24 h
incubation at room temperature the solution was dialyzed in acetate buffer (pH = 5.5). The pH of DOTAM-EGF conjugate was 5.5.
Second step: 14 mCi 177LuCl3 in 0.2 mL was added to the DOTAM-EGF conjugate. The probe was incubated at 451C for 1 h.
Radiolabeling yield and radiochemical purity of 177Lu-DOTAM–EGF were tested by PC (Whatman 1) and ascending instant thin-
layer chromatography (ITLC) with silicagel-coated fiberglass sheets 20 cm length (Polygram SIL G, Macherey-Nagel, Germany). The
Rf’s of free lutetium, lutetium chloride and 177Lu-DOTAM-EGF are presented in Table 1. The radiochemical purity was calculated by
subtraction of percents of impurities determined by both methods.
The probes intended for biological studies were diluted with saline (NaCl 0.9%) to 8 mL, according to the radioactive
concentration and biological concentration to be administered.
Table 1. Rf of the radiochemical species existing in the solution
Solvent
Method
177Lu-DOTAM-EGF
177LuCl3
177Lu free
Sodium citrate 0.1 M, pH = 5
Ammonium acetate 10%:Me-ol (30:70)
PC
ITLC
Rf = 0.62–0.70
Rf = 0.76–0.85
Rf = 1.0
—
—
Rf = 0.00–0.16
Biodistribution studies: The experimental model used was tumor bearing young male rats from Wistar line (rattus norvegicus
albinos variety, rodentia, mammalia), 200–250 g. The animals were kept in cages under ambient temperature and humidity,
receiving commercial ration and water ad libitum.
Studies regarding biodistribution of Lu-DOTAM-EGF were done using 3 animals/each time point. The animals were anesthetized with
a mixture (0.2mL/animal) containing 0.15 mL ketamine 10% and 0.05mL acepromazine (Calmivet), injection using an insulin type syringe
and needle in the peritoneal cavity. They received 177Lu-DOTAM-EGF by i.v. injection in the tail vein and then they were sacrificed at 2 h,
4h, 24h, 48h, 72h p.i., expressive tissues were removed and their radioactivity were measured. The results are expressed as %ID/organ.
Evaluation of the competitive binding of 177Lu-DOTAM-EGF to the EGFR: In the therapy protocol we propose to use stem cells as
support for the radiation therapy. In this context, we have tested the competitive binding of the radiolabelled antibody to its
receptors on a pancreatic cancer cell line and also to a mesenchimal stem cell line.
2 experiment plaques containing 4 ꢀ 105 pancreatic tumor cells (labeled P1 respectively P2) and 2 plaques containing 4ꢀ 105
mesenchimal stem cells (labeled P3 respectively P4) were prepared. To each probe 0.5 mL was added containing decreasing
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J. Label Compd. Radiopharm 2010, 53 355–367

Abstracts
concentrations of mAb (102, 10, 1, 10ꢁ1, 10ꢁ2, 10ꢁ3 mg EGF) and constant radioactivity (396.000cpm in 10mL) 177Lu-DOTAM-EGF. The
cells were incubated at 371C for 2 h (P1 and P3) and 4 h (P2 and P4). The cells were washed 2 times with 1 mL PBS and water. Cells were
detached and dissolved in 400 mL NaOH 2N/alveola, 30min, at 251C. The Eppendorf tubes were counted (Raytest, 177Lu calibrated).
Results and Discussion: Synthesis of 177Lu-DOTAM-EGF
The DOTAM-EGF complex was synthesized in high yield at room temperature in 0.1 M NaHCO3 pH 8.2, using a mixture of DOTAM/
anti-EGF-Mab in nonstoichiometric ratio. The complex was then labeled with 177Lu (15 mCi 177LuCl3, pH 4) with more than 95%
labeling efficiency; the specific activity was 1400 mCi/mg. The RCP of 177Lu-DOTAM-EGF was 95–96% (Figure 2).
50000
0
(a)
0
5
10
15
20
100000
0
0
5
10
15
20
(b)
Figure 2. Radiochromatograms of 177Lu-DOTAM-EGF using (a) PC and (b) ITLC.
25
2 h
4 h
24 h
48 h
72 h
20
15
10
5
0
72 h
24 h
2 h
Figure 3. Biodistribution of 177Lu-DOTAM-EGF in tumor bearing animal model.
Biodistribution of 177Lu-DOTAM-EGF: Biodistribution studies revealed important aspects of 177Lu-DOTAM-EGF in vivo behaviour (Figure 3).
ꢂ
ꢂ
About 20% of the ID remains at the injection site, in the tail; fast blood clearance was observed (2–4 h);
Small liver uptake but important activity was found in the stomach and intestines; this activity washed out with time;
J. Label Compd. Radiopharm 2010, 53 355–367
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Abstracts
ꢂ
ꢂ
ꢂ
Other glands also show some uptake (up to 3% in pancreas and thyroid) at 2 h pi;
Excretion is done mainly by kidney which receive about 4% of the ID;
The tumor received not more than 3% ID but some regions of the tumor, like the tumor surface and skin around the melanoma received
4 and 12% of the ID respectively, which shows very good specificity of the product to the newly created and active tumor cells.
We observed important bone uptake, up to 9% ID, and we asked if it is free 177Lu as a radiochemical impurity or decomposition is
due to the in vivo metabolism process. We have also checked the bone uptake of lutetium chloride, as the free Lu specifically bonds
to this tissue. In the case of 177LuCl3, the radioactivity is accumulated up to 24 h followed by slow and constant lost. The bone
accumulation pattern after 177Lu-DOTA-EGF administration is similar to a pump, with alternative increasing and decreasing
segments. As the patterns of the bone uptake are different, we can conclude that the radioactivity accumulated in bones is released
continuously by the in vivo breakage of the radiolabeled antibody.
In vitro experiments: The preliminary in vitro experiments show that 177Lu-DOTAM-EGF binds to EGFR on the cell surface. The
binding stability and internalization depend on cell type (Figure 4).
80
2 h
4 h
70
60
50
40
30
20
10
0
0.001
0.01
0.1
1
10
100
(a)
[EGF] (micrograms)
60
50
40
30
20
10
0
2 h
4 h
0.001
0.01
0.1
1
10
100
(b)
[EGF] (micrograms)
Figure 4. Binding of 177Lu-DOTAM-EGF to: (a) pancreatic tumor cells and (b) mesenchimal stem cells at 2 and 4 h.
The bioaffinity of 177Lu-DOTAM-EGF to tumor cells is higher than the bioaffinity to stem cells at 2 h. At 4 h, the 177Lu-DOTAM-EGF
was internalized in cancer cells while the radiolabeled antibody was washed from stem cells.
In conclusion, the high accumulation of 177Lu-DOTAM-EGF in tumor surface and skin around the tumor (up to 12% ID) at 24 h after
i.v. injection shows a very good specificity of the product to the newly created and active tumor cells. The stem cells support
targeted radiotherapy since the stem cells should release 177Lu-DOTAM-EGF continuously up to 4 h after its in vivo
administration.177Lu-(DOTAM)-anti-EGF-Mab can be considered a potential new radiopharmaceutical for epithelial malignancies.
Acknowledgements: The research was supported by the National Authority for Scientific Research (ANCS) under contract PNII 71-
073 and Eureka E!3832.
The experiments comply with the current Romanian laws.
References
[1] P. J. Miettinen, J. E. Berger, J. Meneses, et al., Epithelial immaturity and multiorgan failure in mice lacking epidermal growth
factor receptor, Nature, 1995, 376, 337–341.
[2] Hens M, Vaidyanathan G, Welsh P, Zalutsky MR, Nucl Med Biol, 2009, 36, 17–28.
[3] J. H. Doroshow, New England Journal of Medicine, 2005, 353, 200–202.
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 355–367

Abstracts
[4] J. Baselga, Oncology Biotherapeutics, 2002, 2, 2–25.
[5] L. Sundberg, L. Orlova, A. Orlova, E. Blomquist, J. Carlsson V. Tolmachev, Cancer Biotherapy & Radio- pharmaceuticals 2004, 19, 195–204.
SYNTHESES OF ISOTOPICALLY LABELED VERSIONS AND MAJOR METABOLITES OF CCR1 ANTAGONIST
MLN3897
MIHAELA PLESESCU, SANDEEPRAJ PUSALKAR AND SHIMOGA R. PRAKASH
Department of Drug Metabolism and Pharmacokinetics, Millennium: The Takeda Oncology Company, 35 Landsdowne Street, Cambridge, MA 02139, USA
Abstract: MLN3897 citrate is a novel CCR1 antagonist that was taken into development for treatment of chronic inflammatory
diseases such as multiple sclerosis and rheumatoid arthritis. Non-clinical data had indicated extensive metabolism of MLN3897 via
N-dealkylation into two halves. The in vivo disposition of [14C]-MLN3897 in humans was therefore investigated with the [14C]-labeled
drug candidate in either the tricyclic (13) or the chlorophenyl (18) moiety. The use of two separate [14C]-radiolabels of MLN3897
facilitates identification and quantitation of metabolites resulting from each half of the molecule. The first preparation began with
commercially available [1-14C]-acetyl chloride via Friedel-Crafts acylation following the patented procedure. The second synthetic
route was designed to incorporate the radioactive label as late as possible. In this key step freshly generated [14C]-p-
chlorophenyllithium reacted with ketone 15 to provide a racemic mixture that was later resolved by preparative chiral HPLC.
Detailed syntheses of the two radiolabeled versions and significant metabolites will be described.
Keywords: [14C]-MLN3897; [1-14C]-acetyl chloride; [14C]-p-chlorophenyllithium; [D6]-MLN3897; MLN3897 metabolites
Introduction: MLN3897 citrate, (S)-4-(4-chloro-phenyl)-1-f3-[7-(1-hydroxy-1-methyl-ethyl)-11H-10-oxa-1-aza-dibenzo[a,d]cyclohepten-(5E)-
ylidene]-propylg-3,3-dimethyl-piperidin-4-ol, is an orally active, small molecule antagonist of C-C Chemokine Receptor-1 (CCR1)[1–3]. CCR1
is found on the surface of various cells in the immune system and is involved in a number of inflammatory diseases. Non-clinical data had
indicated that MLN3897 is extensively metabolized, initially by an oxidative N-dealkylation process into two halves. Labeling the tricyclic
(13) or the chlorophenyl (18) moiety with carbon-14 was required to address in vivo disposition of MLN3897. The use of two labeled
versions of MLN3897 facilitates identification and quantitation of metabolites resulting from each half of the molecule. The stable isotope
labeled version of MLN3897 (7) was also required as an internal standard for mass spectrometry based bio-analytical assays.
Results and Discussion: Synthesis of [D6]-MLN3897 (7)
Based on the synthesis of unlabeled compound reported earlier[4,5], [D6]-MLN3897 was prepared from inexpensive deuterated
iodomethane. Since MLN3897 contains a chlorine atom, a labeled version that has at least 5 amu higher than the unlabeled version is
required to ensure complete separation of labeled and unlabeled molecular ion clusters during mass spectrometric assays. The synthesis
commenced from commercially available N-Boc-piperidone (1) which was treated with a slight excess of deuterated iodomethane leading
to a mixture of mono-, bis- and tri-alkylated products. The desired bis-alkylated product (2) was isolated by trituration with petroleum
ether, then condensed with a substituted organometallic reagent. Subsequent Boc-deprotection and classical resolution from
L-(1)-tartaric acid provided the [D6]-(S)-4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol (4) with high enantiomeric purity (499%). The
tricyclic bromide 6 was elaborated from commercially available 7H-furo[3,4-b]-pyridin-5-one in 5 steps[4,5]. Final coupling with the bromide
provided [D6]-MLN3897 (7) with 498% isotopic abundance (Scheme 1).
Cl
Cl
O
O
Br
CD3
CD3
2.5 eq. CD3I
Cl
TFA, CH2Cl2
OH
OH
CD3
CD3
CD3
CD3
nBuLi, THF, -78 o
C
NaH, THF
33%
N
N
57%
Boc
Boc
N
H
N
Boc
1
2
4
3
D3C
HO
CD3
Br
N
HO
Cl
Cl
N
CD3
O
NH
L-(+)-tartaric acid
6
OH
CD3
MEK/H2O, 9/1, reflux
99.8% ee
(32% over 2 steps)
K2CO3, ACN, H2O
87%
OH
N
O
5
7
Scheme 1. Synthesis of [D6]-MLN3897.
J. Label Compd. Radiopharm 2010, 53 355–367
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Abstracts
Synthesis of tricyclic [14C]-MLN3897 (13): [14C]-MLN3897 was labeled on the tricyclic portion of the molecule in three
synthetic steps according to the patented methodology as shown in Scheme 2[4,5]. Initial treatment of 8 with phosphorous
tribromide at ꢁ151C afforded the (E)-isomer of the 3-bromopropylidene derivative (9) in 98% isomeric purity after flash
chromatographic purification. The homoallylic bromide was susbsequently acylated with [1-14C]-acetyl chloride under Friedel-
Crafts conditions to provide (E)-1-[5-(3-bromopropylidene)-5,11-dihydro-10-oxa-1-aza dibenzo[a,d]cyclohepten-7-yl]-[1-14C]-
ethanone (10) in 80% yield. Grignard addition of methyl magnesium bromide completed the synthesis of 11, which
was subsequently alkylated with the chiral piperidinol (12). The desired product 13 was isolated by flash chromato-
graphic purification. Final treatment with citric acid and recrystallization from acetone provided tricyclic [14C]-MLN3897 as the
citrate salt.
Br
Br
O
14C
OH
0.84 eq. CH314COCl
PBr3, CH2Cl2
- 15 o
69%
C
N
N
AlCl3, CH2Cl2
-20 o
80%
O
N
O
O
C
10
9
8
HO
Br
Cl
N
N
OH
14C
OH
K2CO3, ACN, H2O
14C
CH3MgBr
THF, 0 oC
72%
HO
Cl
N
O
NH
O
12
13
62%
11
Scheme 2. Synthesis of tricyclic [14C]-MLN3897.
Synthesis of chlorophenyl [14C]-MLN3897 (18): The radiolabeling of the northern part of MLN3897 molecule was designed to
incorporate the carbon-14 label at a later stage in the synthetic sequence. In order to minimize the radiochemical steps we
explored the coupling of the keto-alcohol 15 with a labeled aryl halide (Table 1). This would create a new chiral center that can also
be resolved by utilization of chiral HPLC methods. The key precursor 15 required for the labeling step was prepared
from commercial N-Boc-piperidone (1) which was subsequently bis-alkylated, deprotected, and condensed with the tricyclic
bromide 6.
Initial condensation reactions of ketone 15 were attempted with commercially available p-chlorophenyl magnesium bromide.
Unfortunately no product was detected in an ethereal solvent (Et2O or THF) either at room temperature or under reflux conditions
for a prolonged period of time (1 to 5 days)[6]. It was believed that the ‘‘bulky’’ ketone 15 may require additional time and large
excess of Grignard reagent for advancing the reaction. This led us to the development of another methodology that is based on the
use of p-chlorophenyllithium. Attempts made at reactions of p-chlorophenyllithium with ketone 15 are summarized in Table 1.
Addition of slight excess of ketone 15 to aryllithium species (generated from p-chloroiodobenzene and t-butyllithium in the
presence of LiBr salt) did not result in formation of the desired product. Use of two equivalents of p-chlorophenyllithium at ꢁ781C
Table 1. Optimization of chlorophenyl [14C]-MLN3897 synthesis
Ketone (eq.)
p-Cl-I-benzene (eq.)
LiBr (eq.)
Solvent
Temp.(1C)
Rxn time (h)
Yield (%)
1.2
1.2
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2.35
2.35
2
2
2
2
2
2
Ether
THF
Ether
Toluene
Toluene
Toluene
Toluene
Ether/Toluene 1/1
ꢁ78
ꢁ78
ꢁ78
ꢁ78
ꢁ50
ꢁ40
ꢁ10
ꢁ30
6
6
6
6
6
6
6
6
0
0
11
14
30
17–33
31(impurities)
impurities
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J. Label Compd. Radiopharm 2010, 53 355–367

Abstracts
for 6 h provided the product in 11% yield[7]. Slight improvement was noted with the use of non-polar solvent toluene[8]. The best
yield was observed when this reaction was conducted at ꢁ401C. Many impurities were also formed when using solvents of
intermediate polarity (toluene/diethyl ether mixture).
Scheme 3 illustrates the final synthetic route to chlorophenyl [14C]-MLN3897. Using the best conditions described above,
2 equivalents of freshly generated [14C]-p-chlorophenyllithium (from p-chloroiodo[U-14C]benzene 16 and tert-butyllithium) reacted
with 15 to provide a racemic mixture 17. The desired (S)-isomer 18 isolated by preparative chiral HPLC in 499% enantiomeric excess
(ee) was converted to the citrate salt.
Br
*
I
HO
O
*
*
*
*
2 eq.
Cl
O
O
N
*
N
O
1. NaOtBu, CH3I, THF
38%
16
6
OH
LiBr, tBuLi, Toluene, -20 oC
15-18% (based on labeded precursor)
2. TFA, CH2Cl2
K2CO3, ACN, H2O
77%
N
N
H
Boc
N
1
14
O
15
HO
*
*
HO
*
*
*
*
*
*
Preparative chiral HPLC
50%
Cl
Cl
N
N
*
*
*
*
Chiralpak AD column:
10 µm, 20 x 250 mm
Heptane/i-Propanol/MeOH
9/0.5/0.5
OH
OH
N
N
O
O
* = 14
C
17
18
Scheme 3. Synthesis of chlorophenyl [14C]-MLN3897.
Syntheses of major MLN3897 metabolites: Apart from MLN3897, the predominant metabolites in systemic circulation
were the N-oxides of MLN3897 (M28) and of the carboxylic acid (M11), carboxylic acid (M19), and 4-(4-chlorophenyl)-4-hydroxy-3,
3-dimethylpiperidin-2-one (M44)[9]. These metabolites (M28, M11, M19 and M44) were synthesized to provide positive
identification. The labeled internal standard of M28 was also prepared to assist in monitoring of this metabolite in
clinical trials.
Metabolite M28 (22): For the preparation of M28 (as N-oxide of MLN3897) we chose selective oxidation of N-pyridine with
selenium dioxide or hydrogen peroxide. Unfortunately in both cases we also formed significant impurities that were difficult to
remove during chromatography. The total synthesis of target compound 22A involved overnight treatment of 2-[(5E)-5-(3-
bromopropylidene)-5,11-dihydro[1]benzoxepino[3,4-b]pyridin-7-yl]propan-2-ol (20A) with m-chloroperbenzoic acid (77%) in
methylene chloride followed by alkylation with chiral piperidinol 12 as described in the last step for the preparation of tricyclic
[14C]-MLN3897 (13).
M28 is a pharmacologically active metabolite and a labeled internal standard was required for monitoring and quantitation in
clinical trials (Scheme 4). Commercially available [13C2]-acetyl chloride was used for the Friedel-Crafts acylation reaction of bromide 9,
followed by the Grignard reaction with 13CD3MgBr as previously described for the radiosynthesis of tricyclic-[14C]-MLN3897.
Treatment of 20B with m-chloroperbenzoic acid (77%) in methylene chloride followed by alkylation with 12 afforded the internal
standard of M28 (22B).
J. Label Compd. Radiopharm 2010, 53 355–367
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Abstracts
Br
Br
Br
*
OH
O
*
*CX3
*
*
* CH3*COCl
*CX3MgBr
THF, 0 oC
47%
AlCl3, CH2Cl2, 0 oC
56%
N
N
N
O
O
O
20A: *=12C, X=H
20B: *=13C, X=D
19: *=13C
9
HO
Br
Cl
N
*
OH
*CX3
*
K2CO3, ACN, H2O
HO
mCPBA
*
OH
*
CH2Cl2
99%
Cl
N+
*CX3
NH
12
28-46%
O
-O
N+
-O
O
21A: *=12C, X=H
21B: *=13C, X=D
22A: *=12C, X=H
22B: *=13C, X=D
Scheme 4. Synthesis of M28 and its internal standard.
Metabolites M11 and M19 (27 and 28): Tricyclic ketone 23 was treated with carbethoxy methylene triphenyl phosphorane
under Wittig conditions to form the ester of (2E/Z)-[1]benzoxepino[3,4-b]pyridin-5(11H)-ylideneacetic acid (24) in a ratio of 3:1.
The ester was saponified to give carboxylic acid (24). Treatment of the acid chloride with trimethylsilyldiazomethane followed by the
thermal treatment in the presence of ethanol and 2,4,6-collidine gave the ethyl ester of a homologated carboxylic acid (25)[10]. After
saponification, the acid was subjected to Friedel-Crafts acylation and Grignard reagent as described earlier. The mixture of (E)- and
(Z)-isomers of the carboxylic acid 27 was carefully separated by reverse phase chromatography to give M19. A small portion of the
(E)-isomer was treated with m-chloroperbenzoic acid to form metabolite M11 (28) (Scheme 5).
O
EtO
CO2Et
HO2C
O
1.
P(C6H5)3
1. Oxalyl chloride, CH2Cl2
Toluene
2. LiOH, THF/H2O
2. TMSCHN2, ACN/THF, TEA
3. 2,4,6-collidine, EtOH
O
O
N
N
O
N
24
25
23
O
O
O
HO
HO
OH
HO
OH
O
mCPBA, CH2Cl2
1. CH3MgBr, THF
1. CH3COCl, AlCl3, CH2Cl2
2. LiOH, THF/H2O
2. Preparative HPLC
separation
O
N
O
N+
O-
O
N
27
26
28
Scheme 5. Synthesis of M11 and M19.
Metabolite M44 (34): The structure of M44 was proposed to be a lactam resulting from N-dealkylation of MLN3897 and oxidation
(Scheme 6). The synthesis of (Z)-b-aminoacrylonitrile (30) should be accessible from malonitrile (29) and LAH, but our attempts gave
mixtures of (Z)- and (E)-30 along with small amounts of 2-aminonicotinonitrile[11]. The separation of (E)- and (Z)-isomers was
attempted by column chromatography, but the recovered product decomposed quickly when exposed to air at room temperature.
A solution of 2-bromo-2-methylpropanoylbromide was added to a well-stirred 2-phase system composed of K2CO3 in water and 30
in CH2Cl2 to give (Z)-2-bromo-N-(2-cyanovinyl)-2-methylpropanamide (31). Treatment of 31 with a solution of ethylmagnesium
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J. Label Compd. Radiopharm 2010, 53 355–367

Abstracts
bromide under reflux conditions followed by quenching in a mixture of ice and aqueous NH4Cl provided 3,3-dimethylpyridine-
2,4(1H,3H)-dione (32)[12]. Hydrogenation of the double bond followed by coupling of 33 with aryllithium gave the final product 4-(4-
chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-2-one (34) which was isolated after reversed phase chromatographic purification.
Br
O
NH
Br
EtMgBr, THF
22%
LiAlH4
N
N
O
N
N
NH2
Br
THF, ether
10%
K2CO3, CH2Cl2, H2O
20%
30
31
29
Cl
NH
H
1.
Br
H
N
O
N
O
H2
Cl
O
nBuLi, THF
HO
2. Preparative HPLC
7%
10% Pd/C, EtOH
91%
O
O
33
32
Scheme 6. Synthesis of M44.
34
Conclusions: In support of ADME studies conducted in laboratory animals and humans we synthesized [14C]-MLN3897 citrate by
two separate routes allowing incorporation of a 14C label in two different positions in the molecule. A stable isotope labeled version
of MLN3897 was also prepared for use as an internal standard in bioanalytical assays.
In the preparation of C-14 labeled tricyclic MLN3897 using the method described by Carson et al., [1-14C]-acetyl chloride was used
as the starting material. The synthesis was completed in three radiochemical steps with an overall yield of 36% and radiochemical
purity of 99%. Chlorophenyl [14C]-MLN3897 was synthesized in one radiochemical step. Although the method was short, observed
low yield was probably a result of introduction of the label at a sterically demanding site.
The predominant metabolites, M11, M19, M28 and M44 were synthesized with high chemical purity in order to provide positive
identification.
Acknowledgements: The authors would like to thank the Inflammation Medicinal Chemistry and Process Chemistry groups at
Millennium Pharmaceuticals for assistance with this project. We would also like to acknowledge the Radiochemistry group at
Girindus America, Inc. for completing the GMP syntheses of [14C]-MLN3897.
References
[1] K. G. Carson B. D. Jaffee, G. C. B. Harriman, Annual Reports in Medicinal Chemistry 2004, 39, 149–158.
[2] J.-F. Cheng, R. Jack, Mol Divers 2008, 12, 17–23.
[3] J. F. Pease, R. Horuk, Expert Opin Ther Patents 2009, 19(1), 39–58.
[4] K. G. Carson, G. C. B. Harriman, PCT Int Appl 2004; WO 2004/043965.
[5] J. R. Luly, Y. Nakasato, E. Ohshima, C. B. Harriman, K. G. Carson, S. Ghosh, A. M. Elder, K. M. Mattia, Pat Appl Publ 2005; US 2005/
0070549.
[6] U. Holzgrabe, B. Piening, R. Haller, Arch Pharm 1990, 323(3), 177–180.
[7] I. Fellows, T. A. Harrow, R. Honeyman, J Label Cmpd Radiopharm 1978, 449–461.
[8] V. Lecomte, E. Stephan, F. Le Bideau, G. Jaouen, Tetrahedron 2003, 59, 2169–2176.
[9] S. Pusalkar, M. Plesescu, S. K. Balani, S. R. Prakash, (unpublished).
[10] T. Aoyama, T. Shioiri, Tetrahedron Lett 1980, 21, 4461–4462.
[11] S. Rayat, M. Qian, R. Glaser, Chem Res Toxicol 2005, 18, 1211–1218.
[12] J. Bergman, A. Brynolf, E. Vuorinen, Tetrahedron 1986, 42(13): 3689–3696.
THE SYNTHESES AND IN VITRO METABOLISM OF [14C]APIXABAN, AN INHIBITOR OF BLOOD
COAGULATION FXA
BRAD D. MAXWELLa, SCOTT B. TRANa, SHIANG-YUAN CHENa, DONGLU ZHANGb, BANG-CHI CHENc, HUIPING ZHANGc AND
SAMUEL J. BONACORSI, JRa
aRadiochemistry-Discovery Chemical Synthesis,
bPharmaceutical Candidate Optimization,
cDiscovery Chemical Synthesis
Bristol-Myers Squibb Company, P. O. Box 4000, Princeton, New Jersey 08543, USA
J. Label Compd. Radiopharm 2010, 53 355–367
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

Abstracts
Abstract: Apixaban is a potent inhibitor of blood coagulation Factor Xa in the late stages of development. Two isotopologues of
[14C]Apixaban were synthesized for in vitro and in vivo metabolism studies. A nine step synthesis of [14C]Apixaban, 10, with the label
in the central lactam ring was completed in 14% overall yield. A second synthesis of [14C]Apixaban, 14, with the C14 label in the
outer lactam ring was completed in three steps in a 9% overall yield. No significant differences were observed between the
metabolite profiles of 10 and 14, [14C]Apixaban, in both rat and human hepatocyte or microsomal incubations.
Keywords: Apixaban; Factor Xa; Thrombotic diseases; microsomes; hepatocytes; ADME
Introduction: Thrombotic diseases are the leading cause of death among people living in developed countries. Although the oral
anticoagulant Warfarin is effective in both venous and arterial thrombosis, it suffers from a narrow therapeutic index, slow onset of
therapeutic effects, several drug and dietary interactions, the need for monitoring and dose adjustments. Thus, the discovery and
development of a highly potent, selective, efficacious and orally bioavailable inhibitor of blood coagulation Factor Xa as a
replacement for Warfarin would be extremely valuable[1]. Apixaban represents such an agent and the preparation of [14C] Apixaban
was required for various preclinical and clinical studies. This paper describes the syntheses, purifications, analyses and
characterizations of [14C]Apixaban and its use in microsomal and hepatocyte in vitro metabolism studies.
Results and Discussion: Two isotopologues of [14C]Apixaban, were synthesized. The first isotopologue, [14C]10, synthesis is shown
in Scheme 1. 4-Nitroaniline was reacted with [1-14C]5-Bromovaleryl chloride under basic conditions to generate the nitro lactam 2 in
quantitative yield. Product 2 was reacted with PCl5 to produce the a,a- dichlorinated lactam, 3 in 79% yield. Product 3 was reacted with
Li2CO3 at 1051C to produce the a,b-unsaturated lactam, 4 in 96% yield. Product 4 was reacted with Ethyl 2-chloro-2-(2-(4-
methoxyphenyl)hydrazono)acetate 5 synthesized by the Bristol-Myers Squibb Process Chemistry Group to generate the pyrazole ring
system product 6, in 67% yield. The aromatic nitro group was reduced to aniline 7 with hydrogen gas and 5% Pd on Al2O3 in 99% yield.
O
Cl
O
N
O
N
Br
PCl5
PhCl
NO2
Cl
*
Cl
*
*
NO2
NO2
K2CO3, H2O
THF, PhCl
TBAB, KOH
53˚C
79%
H2N
1
2
3
* = 14
C
100%
Cl
EtO2C
N
O
Li2CO3
LiCl
H
Et3N
N
*
Toluene
N
Cl
+
NO2
N
DMF
105˚C
96%
N
*
N
67%
CH3O
CO2Et
4
5
O
NO2
6
CH3O
EtO2C
EtO2C
N
O
H2
N
Cl
N
8
N
7
*
5% Pd/Al2O3
*
Cl
N
N
O
NMP, 52˚C
99%
O
O
NMP
42%
NH
NH2
Cl
CH3O
CH3O
H2NOC
N
EtO2C
N
HCONH2
DMF
1. CH(OEt)3
TFA, NMP
N
N
*
*
N
N
CH(OMe)3
TFA
NaOMe in MeOH
84%
O
O
2. 21% NaOEt
in EtOH
N
N
9
78%
O
14C]Apixaban
O
10
CH3O
CH3O
[
Scheme 1 – Synthesis of 10, [14C]Apixaban.
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Abstracts
The second lactam ring was prepared in three steps by reacting aniline product 7 with Chlorovaleryl chloride to generate amide 8
in 42% yield followed by a base promoted lactam ring formation in 78% yield. The final step involved the conversion of ethyl ester 9
to the primary amide with formamide to generate [14C]Apixaban, 10, in 84% yield with the Carbon-14 label located in the central
lactam ring system. The overall yield for the nine step synthesis was 14%. The product was 99.27% radiochemically pure and 99.89%
chemically pure. MS and 1H-NMR matched those expected for [14C]Apixaban. The Specific Activity was measured gravimetrically at
56.36 mCi/mmol or 122.1 mCi/mg.
A separate synthesis of [14C]Apixaban with the carbon-14 label in the outer lactam ring system was completed and is shown in
Scheme 2. In this synthesis, compound 7 was reacted with [1-14C]Chlorovaleryl chloride to generate amide 11 in 54% yield. Product
11 was converted with Potassium ethoxide to the penultimate lactam 12 in 35% yield after purification by preparative HPLC.
The penultimate ester was converted to the amide, [14C]Apixaban, 13, with ammonia in 47% yield. The three step sequence was
completed in 9% overall yield. The final product was 100% Radiochemically pure. MS and 1H-NMR matched those expected for the
labeled product. The specific activity of [14C]Apixaban, 13, was measured at 49.46 mCi/mmol or 107.7 mCi/mg. The specific activity of
a portion of the product was reduced to 2.49 mCi/mmol or 5.4 mCi/mg with unlabeled clinical grade Apixaban for use in the Human
ADME Clinical Study[2]
.
EtO2C
N
EtO2C
N
O
*
Cl
N
N
Cl
N
N
O
*
K2CO3,
THF
54%
O
O
NH
NH2
12
11
Cl
CH3O
CH3O
EtO2C
N
H2NOC
N
NH3
KOEt
THF
N
1,2-Propanediol
N
N
N
35%
47%
O
O
N
N
*
*
13
O
O
14
CH3O
CH3O
[
14C]Apixaban
Scheme 2 – Synthesis of 13, [14C]Apixaban.
The in vitro biotransformation profiles of both labels, 10 and 13, in cryopreserved hepatocytes and liver microsomes from
rats and humans were determined. At the completion of a 4–hr incubation, 83% and 92% of cells remained viable for rat
and human hepatocytes, respectively. After 4-hr incubation, 44.1% and 51.0% of 7-Ethylcoumarin (EC) and 42.1% and
97.8% of 7-Hydroxycoumarin (HC) were metabolized by rat and human hepatocytes, respectively. In addition, significant
amounts of 7-HC sulfate and 7-HC glucuronide were also observed. Thus, both hepatocyte preparations used in this
study were enzymatically active for the 4-hr incubation. After 1-hr incubation, 71.2% and 90.2% of 7-EC was metabolized
by rat and human liver microsomes, respectively. In addition, significant amount of 7-HC, a metabolite of 7-EC, was
observed in 1-hr incubation samples. Thus, both microsomal preparations used in this study were enzymatically active for the 1-
hr incubation.
The 1-, or 4-hr microsomal or hepatocyte incubation mixtures of [14C]Apixaban (both 10 and 13, at 1 and 10 mM), were extracted
with ice-cold acetonitrile. The extraction recoveries of the total radioactivity from the incubation mixtures were quantitative for rat
and human hepatocyte and microsomes for 13 and 10. [14C]Apixaban, both 10 and 13, appeared to be relatively stable in
hepatocyte incubation media and phosphate buffer at 10 mM. Greater than 97% of [14C]Apixaban remained unchanged after a 1- or
4-hr incubation control. No detectable degradation products were observed in the 1 mM negative control incubations of both
13 and 10.
J. Label Compd. Radiopharm 2010, 53 355–367
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Abstracts
Apixaban
Compound 13
180
160
140
120
100
80
M7
M3
M6
M2
60
M4
40
M1
20
0
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Min
Apixaban
Compound 10
180
160
140
120
100
80
M7
M3
M6
60
M2
M4
40
M1
20
0
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Min
Figure 1. HPLC Radio-Chromatograms of 4–Hour Human Hepatocyte Incubations with 10 mM [14C]Apixaban, Compounds 10 and 13.
The metabolism of Apixaban in rat and human hepatocyte or microsomal incubations was very limited, with 492% and 95% of
the total radioactivity being attributed to Apixaban after a 1- or 4-hr incubation. Six metabolites, M1-M4 and M7, were tentatively
identified in hepatocytes and 5 metabolites, M2-M4 and M7 were tentatively identified in microsomal incubations. These in vitro
metabolites were similar to the in vivo metabolites[2,3] M2 was identified as the O-desmethyl metabolite. M4 and M7 were
hydroxylated metabolites. M1 was a sulfate conjugate of M2. M3 was a ring opened metabolite and M6 was the amide hydrolyzed
metabolite. There were no significant differences observed between the metabolite profiles of both 10 and 13 of [14C]Apixaban
from both rat or human hepatocyte or microsomal incubations, see Figures 1 and 2. Metabolite profiles were similar for 1 and 10 mM
incubations. Similar metabolic profiles of 10 and 13 supported the use of 13 for Human ADME studies and that experiments with
compound 13 adequately assessed the metabolic profile of Apixaban.
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J. Label Compd. Radiopharm 2010, 53 355–367

Abstracts
Apixaban
240
220
200
180
160
140
120
100
80
Compound 13
M7
M2
M6
M3
M4
60
40
20
0
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Min
Apixaban
240
220
200
180
160
140
120
100
80
Compound 10
M7
M2
M6
M3
60
40
20
0
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Min
Figure 2. HPLC Radio-Chromatograms of 1–Hour Human Liver Microsomal Incubations with 10 mM [14C]Apixaban, Compounds 10 and 13.
Conclusion: Two separate syntheses of isotopologues of [14C]Apixaban, 10 and 13, were completed for various in vitro and in vivo
studies. There were no significant differences observed between the metabolite profiles of 10 and 13 [14C]Apixaban in both rat or
human hepatocyte or microsomal incubations.
Acknowledgements: The authors would like to thank members of the Bristol-Myers Squibb Radiochemistry Group for helpful
suggestions and members of the Process Chemistry Group at Bristol-Myers Squibb for providing valuable intermediates, 5 and 7,
and unlabeled Apixaban.
References
[1] D. J. P. Pinto, M. J. Orwat, S. Koch, K. A. Rossi, R. S. Alexander, A. Smallwood, P. C. Wong, A. R. Rendina, J. M. Luettgen,
R. M. Knabb, K. He, B. Xin, R. R. Wexler, P. Y. S. Lam, J. Med. Chem. 2007, 50, 5339–5356, DOI : 10.1021/jm070245n and references
therein.
[2] N. Raghavan, C. E. Frost, Z. Yu, K. He, H. Zhang, W. G. Humphreys, D. Pinto, S. Chen, S. Bonacorsi, P. C. Wong and D. Zhang, Drug
Metab. Dispos. 2009, 37, 74–81, DOI: 10.1124/dmd.108.023143.
[3] D. Zhang, N. Raghavan, L. Wang and W. G. Humphreys, Drug Metab. Dispos. Manuscript in preparation.
J. Label Compd. Radiopharm 2010, 53 355–367
Copyright r 2010 John Wiley & Sons, Ltd.
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