Carbon-13 and carbon-14 labeled dabigatran etexilate and tritium labeled dabigatran

Article abstract of DOI:10.1002/jlcr.3402

Dabigatran etexilate or pradaxa, a novel oral anticoagulant, is a reversible, competitive, direct thrombin inhibitor. It is used to prevent strokes in patients with atrial fibrillation and the formation of blood clots in the veins (deep venous thrombosis) in adults who have had an operation to replace a hip or a knee. Pradaxa is the only novel oral anticoagulant available with both proven superiority to warfarin and a specific reversal agent for use in rare emergency situations. The detailed description of the synthesis of carbon-13 and carbon-14 labeled dabigatran etexilate, and tritium labeled dabigatran is described. The synthesis of carbon-13 dabigatran etexilate was accomplished in eight steps and in 6% overall yield starting from aniline-¹³C₆. Ethyl bromoacetate-1-¹⁴C was the reagent of choice in the synthesis of carbon-14 labeled dabigatran etexilate in six steps and 17% overall yield. Tritium labeled dabigatran was prepared using either direct tritium incorporation under Crabtree's catalytic conditions or tritium-dehalogenation of a diiodo-precursor of dabigatran.

Full text of DOI:10.1002/jlcr.3402

Special issue article
Received 07 February 2016,
Revised 08 March 2016,
Accepted 28 March 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3402
Carbon-13 and carbon-14 labeled dabigatran
etexilate and tritium labeled dabigatran
a
b
b
a
*
*
Bachir Latli, Ralf Kiesling, Stefan Aßfalg, Max Chevliakov,
Matt Hrapchak,a Scot Campbell,a Nina Gonnella,a Carl A. Busaccaa and
Chris H. Senanayakea
Dabigatran etexilate or pradaxa, a novel oral anticoagulant, is a reversible, competitive, direct thrombin inhibitor. It is used
to prevent strokes in patients with atrial fibrillation and the formation of blood clots in the veins (deep venous thrombosis)
in adults who have had an operation to replace a hip or a knee. Pradaxa is the only novel oral anticoagulant available with
both proven superiority to warfarin and a specific reversal agent for use in rare emergency situations. The detailed
description of the synthesis of carbon-13 and carbon-14 labeled dabigatran etexilate, and tritium labeled dabigatran is
described. The synthesis of carbon-13 dabigatran etexilate was accomplished in eight steps and in 6% overall yield starting
from aniline-13C6. Ethyl bromoacetate-1-14C was the reagent of choice in the synthesis of carbon-14 labeled dabigatran
etexilate in six steps and 17% overall yield. Tritium labeled dabigatran was prepared using either direct tritium
incorporation under Crabtree’s catalytic conditions or tritium-dehalogenation of a diiodo-precursor of dabigatran.
Keywords: dabigatran etexilate; pradaxa; dabigatran; anticoagulant; carbon-13; carbon-14; tritium
the use of warfarin may be associated with an increased risk of
Introduction
serious hypoglycemic events among older people treated with
sulfonylureas.14 Therefore, research efforts were focused on the
The formation of blood clots in the blood vessels is a multistep
cascade that ends with the cleavage of prothrombin by the
prothrombinase complex and the generation of thrombin.
identification of other direct thrombin inhibitors. For example,
inhibition of the serine protease will prevent the conversion of
Thrombin then acts as a serine protease to convert fibrinogen
fibrinogen into fibrin during the coagulation cascade. A new
to fibrin.1,2 Cross-linkage of fibrin strands leads to the formation
class of non-peptidic inhibitors employing 1,2,5-trisubstituted
benzimidazole as the central scaffold were discovered based
on the X-ray crystal structure of the human α-thrombin complex
of hemostatic clots also known as thrombosis.3,4 These blood
clots migrate everywhere in the body in a process known as
thromboembolism, which causes life threatening conditions.5
Therefore, thrombin inhibition is a major therapeutic target for
several thromboembolic diseases.6 Current anticoagulant
therapies include heparin and vitamin K antagonists (coumarins
like warfarin and acenocoumarol). These drugs have been the
only oral anticoagulants available over the last 60 years.7,8
Warfarin was introduced in 1948 as a rodenticide. It exerts its
toxicity by causing rodents to hemorrhage to death.9 It was not
approved for medical use in human until a soldier tried
unsuccessfully to end his life with multiple doses of warfarin
but recovered fully after being treated with vitamin K.9 Warfarin
reached a special status when it was credited for saving the life
of US president Dwight Eisenhower after suffering a heart attack
in 1955.9 The use of both heparin and warfarin has been
hampered by numerous limitations and serious side effects.
Heparin, for example, needs to be administered parenterally,
formed with these benzamidazoles.15 From these series of
inhibitors, dabigatran (BIBR 953) (http://www.dabigatran.com)
was found to be the most favorable in vivo activity profile,
competitive, and direct thrombin inhibitor (factor IIa).15 Dabigatran
is a very polar compound and not suitable for oral administration
to patients. Because of its high polarity and low oral absorption, a
search for a prodrug culminated with the discovery of dabigatran
etexilate (BIBR 1048) (http://www.pradaxa.com), which exhibited
strong and long-lasting anticoagulant effects after oral
administration.16–23 Dabigatran etexilate was the first FDA-approved
alternative to warfarin and is now used to prevent strokes in patients
aChemical Development, Boehringer Ingelheim Pharmaceuticals, Inc., 900
Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-0368, USA
bDMPK, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65,
D-88397 Biberach, Germany
which is
a
major obstacle for chronic treatment of
thromboembolic diseases. Warfarin has delayed onset and offset
of action that often prolongs hospitalization and, thus increases
healthcare cost, requires frequent monitoring, and counseling
*Correspondence to: Bachir Latli, Chemical Development, Boehringer Ingelheim
Pharmaceuticals, Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-
0368, USA; and Ralf Kiesling, DMPK, Boehringer Ingelheim Pharma GmbH & Co.
KG, Birkendorfer Strasse 65, D-88397 Biberach, Germany.
about drug and food interactions. There is
a risk of
thromboembolism and bleeding as well despite careful
E-mail: bachir.latli@boehringer-ingelheim.com; ralf.kiesling@boehringer-ingelheim.
individualized dose adjustment.10–13 A recent study found that com
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B. Latli et al.
with atrial fibrillation (AFib) and the formation of blood clots in the brominated at the para-position using tetrabutylammonium
veins (deep venous thrombosis) in adults who have had an tribromide in 71% yield.27 The resulting 4-bromoaniline [13C6]-(2)
operation to replace a hip or a knee.24 Dabigatran etexilate also was then converted in 54% yield to 4-aminobenzonitrile [13C6]-
known as pradaxa is the only novel oral anticoagulant (NOAC) with (3) using copper cyanide in pyridine at 170°C.28 Nucleophilic
a specific reversal agent for use in rare emergency situations.25 substitution of ethyl bromoacetate (4) gave [13C6]-(5) in 91% yield.
Following oral administration, dabigatran etexilate is rapidly Ester hydrolysis to [13C6]-(6), activation with carbonyldiimidazole
converted to its active form by human carboxylesterase enzymes (CDI) followed by condensation with (7)15,29 gave [13C6]-(8) in
as shown in Scheme 1.26 In this manuscript, we report the synthesis 76% yield over three steps. Pinner reaction30 followed by
of this drug labeled with carbon-13, carbon-14, and tritium.
treatment of the resulting ethylamidate with ammonium
carbonate gave [13C6]-(9) in 69% yield. Reaction with n-hexyl
chloroformate (10) in acetone gave [13C6]-dabigatran etexilate
[13C6]-(11) in 38% yield after silica gel chromatography purification.
Finally, the methanesulfonate salt was obtained in 84% yield from
the crystallization of dabigatran etexilate and methanesulfonic
acid in isopropanol.
Results and discussion
Synthesis of carbon-13 and carbon-14 labeled dabigatran
etexilate
The route described in Scheme 2 was used to prepare
carbon-14 labeled dabigatran as well. The commercially
Carbon-13 dabigatran etexilate was prepared in eight steps and in
6% overall yield, Scheme 2. Aniline-13C6 was regioselectively
Scheme 1. The hydrolysis of dabigatran etexilate (Pradaxa) to dabigatran.
Scheme 2. Synthesis of [13C6]-Pradaxa.
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B. Latli et al.
available ethylbromoacetate-1-14C ([14C]-(4)) was reacted with and purified by HPLC to more than 98% radiochemical purity.
4-aminobenzonitrile (3) and the product [14C]-(5) was used as The specific activity was calculated to be 43 Ci/mmol, Scheme 4.
described before to obtain [14C]-dabigatran etexilate in five
Dabigatran is highly insoluble in most organic solvents. For
steps and in 17% overall yield with a specific activity of NMR studies, we used either pyridine-d5 or DMSO-d6 with a drop
9.6 mCi/mmol (13.2 μCi/mg) and chemical and radiochemical of TFA to obtain homogenous solutions. Tritium NMR indicated
purities of more than 98% as determined by HPLC, Scheme 3. possibly a mixture of tritium labeled dabigatran species. Tritium
This labeled material was a valuable tool in drug metabolism was incorporated mainly at position 3. Both di tritiation and
and disposition studies.31,32
mono tritiation at position 3 and the presence of a third
unidentified species at 8.2 ppm were observed, which might
have resulted from dabigatran degradation, Figures 1 and 2.
This tritiated compound was not very stable for long-term
storage. Excessive degradation was observed after one month
of storage at À80 °C, and we needed to purify this material
frequently. To replenish our stock of tritiated dabigatran, we
opted to prepare a specifically tritium-labeled dabigatran in the
hope of improving the stability. A newly reported work from
Chirik’s group and Merck using iron catalyst would have been
Synthesis of tritium labeled dabigatran
Using Crabtree’s catalyst
The first approach to prepare tritium labeled dabigatran was the
direct exposure of Boc-protected dabigatran (12) to tritium gas in
the presence of Crabtree’s catalyst in methylene chloride.33 The
product was then hydrolyzed using TFA and NaOH respectively,
Scheme 3. Synthesis of [14C]-Pradaxa.
Scheme 4. Direct tritiation of Boc-dabigatran (12) using Crabtree’s catalyst. Asterisks indicate expected tritium positions in the molecule.
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B. Latli et al.
Figure 1. 1H NMR of unlabeled dabigatran in pyridine-d5-TFA showing the aromatic regions.
Figure 2. 3H NMR (1H-decoupled) of [3H]-dabigatran in pyridine-d5-TFA.
another way of introducing tritium atoms on dabigatran on tritium labeled dabigatran with specific activity of 30 Ci/mmol
different locations on the molecule.34
and radiochemical purity of 98% after HPLC purification,
Figures 3 and 4.
Specific tritium labeling of dabigatran
The iodide precursor (19) was prepared in six steps and 6%
overall yield (Scheme 5). 4-Aminobenzonitrile (3) was subjected
to iodination using iodine and silver.35 However, the alkylation
with ethyl bromoacetate failed to give the desired product using
the conditions described in the synthesis of carbon-13 and
carbon-14 dabigatran etexilate. This is probably due to steric
congestions. Sodium hydride was used instead as a base to
convert 4-cyano-2,6-diiodoaniline (14) to (15) in 80% yield. The
sequence described in Scheme 3 and Scheme 4 was then
followed to prepare the iodide precursor (19). This diiodo-
derivative was exposed to carrier-free tritium gas in the presence
of Pd/C in methanol to give the tritium labeled [3H2]-(12).
Experimental
Materials and methods
1H NMR and 13C NMR spectra acquisition of non-radio-labeled
compounds were performed using Bruker 400 MHz. The full proton
assignment of dabigatran was completed by COSY, ROESY, HMQC,
HMBC, and INEPT. 3H NMR was run using double encapsulation on
400 MHz Bruker NMR instrument and 5 mm PADUX 3H-1H/D Z-GRD
Z136359/0001 probe. Liquid scintillation counting was accomplished
using a Beckman LS6500TA and UltimaGoldTM cocktail (PerkinElmer,
Boston, MA). Flash chromatography purifications were performed using
silica gel (LiChroprep® Si 60 [40–63 μm], Merck Millipore, Germany) or
Removal of the protecting groups using TFA, then NaOH gave disposable silica gel columns from Silicycle® (Quebec City, Quebec,
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B. Latli et al.
Scheme 5. Synthesis of specifically tritium labeled dabigatran.
Figure 3. 1H NMR of unlabeled dabigatran in DMSO-d6-TFA showing the aromatic regions.
Canada). HPLC analysis was performed on an Agilent 1200 instrument.
MeCN (0.1%TFA) to 5% water in 1.7 min, hold to 2 min at 5% water, flow
HPLC conditions: flow rate 1 mL/min, injection volume 10 μL; (a) Mobile 2.5 mL/min; column: Agilent Zorbax SB-C18 (3.5 μm, 4.6 × 30 mm), using
Phase gradient: 95:5 to 5:95 (MeCN/H2O with 0.1% TFA) over 20 min,
Waters Acquity™ Ultra Performance LC (Milford, MA, USA). The
column: Halo C8 (2.7 μm, 4.6 × 150 mm); (b) same mobile phase, column: radiochemical purity was measured using a radio-HPLC detector β-Ram
Agilent Zorbax SB-C18 (5 μm, 4.6 × 150 mm); (c) Mobile phase: 10–100% model 3 or model 4 with a 500 μL cell size (LabLogic systems, Inc. Brandon,
B (H2O with 0.1% TFA, B: MeCN) over 30 min, column: Agilent Zorbax FL, USA) connected to Agilent HPLC instrument using IN-FLOW™ 2:1 liquid
C18 (5 μm, 4.6 × 250 mm). LCMS were acquired using a fast medium scintillation (LabLogic systems, Inc. Brandon, FL). Ethylbromoacetate-1-14C
polar method: run time 2.0 min, gradient 95% water (0.1% TFA) and 5%
(3.7 GBq, 100 mCi, 25 mCi/mmol) was purchased from American
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B. Latli et al.
Figure 4. 3H NMR (1H-decouple) of [3H2]-dabigatran in DMSO-d6-TFA.
Radiolabeled Chemicals, Inc., (St. Louis, MO, USA). Aniline-13C6, 99 atom %
1H), 6.84(m, JC-H = 159.5 Hz, 1H), 6.45(m, JC-H = 159.5 Hz, 1H), 4.10(q,
13C was obtained from Isotec (Sigma-Aldrich) (St. Louis, MO, USA). J = 7.1 Hz, 2H), 4.00(m, 2H), 1.20(t, J = 7.1 Hz, 3H). 13C NMR (CDCl3) δ:
Crabtree’s catalyst was obtained from Strem (Newburyport, MA, USA). The 170.5, 151.8 (dt, J = 9.1, 60 Hz), 133.3(dt, J = 6.1, 59.4 Hz), 112.1(dt, J = 6,
rest of the reagents were obtained from Sigma-Aldrich Company.
60.2 Hz), 96.5(dt, J = 9.1, 59.4 Hz), 60.5, 43.9, 14.1.
(4-Cyanophenyl-1,2,3,4,5,6-13C6)glycine, [13C6]-(6)
Synthesis of [13C6]-dabigatran etexilate
A suspension of [13C6]-(5) (1.5 g, 7.0 mmol) in methanol (7 mL) was stirred
with sodium hydroxide (7.7 mL, 1 N) for 4 h at room temperature. The
product was precipitated by addition of hydrochloric acid (7.7 mL, 1 N),
collected on a suction filter, washed with isopropanol (1 mL × 2), and
dried to give 1.25 g of product in 97% yield. HPLCa: tR = 6.81 min; LCMS
m/z: 183.1 (MH+). 1H NMR (CDCl3) δ: 12.71 (m, 1H), 7.67(m, JC-
H = 162.4 Hz, 1H), 7.25(m, JC-H = 162.4 Hz, 1H), 6.91(s, 1H), 6.84 (m, JC-
H = 160 Hz, 1H), 6.43 (m, JC-H = 160 Hz, 1H), 3.90 (m, 2H). 13C NMR (CDCl3)
δ: 172.0, 151.8 (dt, J = 9.2, 59.0 Hz), 133.2(dt, J = 6.1, 59.6 Hz), 112.0(dt,
J = 6.0, 60.5 Hz), 96.3(dt, J = 9.0, 59.4 Hz), 43.8.
4-Bromoaniline-1,2,3,4,5,6-13C6, [13C6]-(2)
A solution of tetrabutylammoniumtribromide (9.73 g, 20.2 mmol) in
CHCl3 (75 mL) was added to a mixture of aniline-13C6 (2.0 g, 20.2 mmol)
in CHCl3 (75 mL). After stirring for 3 min, an aqueous 10% solution of
Na2S2O3 (108 mL) was added. The reaction suspension was neutralized
with a saturated solution of Na2CO3 (100 mL) and then extracted with
chloroform (100 mL × 3). The combined extracts were dried over MgSO4,
filtered, and evaporated to leave an oily residue. This crude compound
was purified by silica gel chromatography using diethyl
ether/petroleum ether (33/66) as eluent to give 2.54 g of [13C6]-(2) in
Ethyl 3-(2-(((4-cyanophenyl-1,2,3,4,5,6-13C6)amino)methyl)-1-methyl-
N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido)propanoate,
[13C6]-(8)
71% yield. HPLCa: tR = 5.32 min; LCMS m/z: 179.6 (MH+). H NMR (CDCl3)
1
δ: 7.32(m, JC-H = 164 Hz, 1H), 6.90(m, JC-H = 164 Hz, 1H), 6.69(m, JC-
H = 160, Hz, 1H), 6.30(m, JC-H = 160 Hz, 1H). 13C NMR (CDCl3) δ: 148.0(dt,
J = 7.8, 59 Hz), 131.2(dt, J = 6.5, 63 Hz), 115.7(dt, J = 6.5, 63 Hz), 105.8(dt,
J = 9.4, 63 Hz).
[
13C6]-(6) (1.25 g, 6.8 mmol) was added to a mixture of CDI (1.2 g,
7.3 mmol) in THF (10 mL) at room temperature and stirred for 1 h. N-[3-
Amino-4-(methylamino)benzoyl-N-2-pyridinyl-ß-alanine ethyl ester (7)
(2.33 g, 6.8 mmol) was added at 40 °C and the reaction mixture heated
to reflux for 2 h. Then, the solvent was stripped off, and the residue
was heated to reflux in acetic acid (6.4 mL) for another 1 h. The crude
product was precipitated by addition of water (24 mL). The solid was
filtered, dried, and recrystallized from isopropanol (10 mL) to give 2.6 g
of product in 78% yield. LCMS m/z: 489.5 (MH+).
4-Aminobenzonitrile-1,2,3,4,5,6-13C6, [13C6]-(3)
4-Bromoaniline-1,2,3,4,5,6-13C6 ([13C6]-(2)) (2.54 g, 14.3 mmol) was stirred
with copper (I) cyanide (1.3 g, 14.6 mmol) and pyridine (1.2 g, 15.2 mmol))
at 170 °C. After stirring at this temperature for 2 h and concentration in
vacuo, the crude oil was purified by silica gel chromatography using
diethyl ether/petroleum ether (1/1) as eluent to give 963 mg of product
in 54% yield. HPLCa: tR = 7.40 min; LCMS m/z: 125.1 (MH+). 1H NMR
(CDCl3) δ: 7.58(m, JC-H = 164.7 Hz, 1H), 7.18(m, JC-H = 164.7 Hz, 1H), 6.80
(m, JC-H = 160.5, Hz, 1H), 6.40(m, JC-H = 160.5, Hz, 1H), 6.13 (s, 2H). 13C
NMR (CDCl3) δ: 152.9(dt, J = 10.0, 58.4 Hz), 133.2(dt, J = 6.5, 59.4 Hz),
113.2(dt, J = 6.3, 60.0 Hz), 95.4(dt, J = 8.9, 59.4 Hz).
Ethyl 3-(2-(((4-carbamimidoylphenyl-1,2,3,4,5,6-13C6)amino)methyl)-
1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido)
propanoate, [13C6]-(9)
A suspension of [13C6]-(8) (2.6 g, 5.3 mmol) in ethanol (14 mL) was cooled
to À10 °C and saturated with HCl gas. The reaction mixture was kept for
1 h at À10 °C and then it was allowed to stand at room temperature
Ethyl (4-cyanophenyl-1,2,3,4,5,6-13C6)glycinate, [13C6]-(5)
To a mixture of [13C6]-(3) (963 mg, 7.8 mmol) and diisopropylethyl amine overnight. The volatile components of the reaction were evaporated,
(2.01 g, 15.56 mmol) stirred at 100 °C was added ethylbromoacetate (4)
(1.43 g, 8.54 mmol). After two hours, the reaction was cooled to 60 °C,
and the product was precipitated by addition of water (8 mL) and
and the dark residue was dissolved in ethanol (14 mL) and treated at
+5 °C with ammonium hydroxide solution (12 %, 9.6 mL). After stirring
for 3.5 h at room temperature, water (10 mL) was added, and most of
hydrochloric acid (1.86 mL, 6 N). The yellowish crystals were collected the solvents were evaporated under vacuum at 40 °C. The remaining
on a suction filter, washed with water and diisopropyl ether, and dried solid was suspended in water (15 mL) and stirred at room temperature
in a vacuum oven to a constant mass at 50 °C to give 1.48 g of [13C6]-
overnight. The solid was filtered and while still moist recrystallized from
1
(5) in 91 % yield. HPLCa: tR = 10.15 min; LCMS m/z: 211.2 (MH+). H NMR acetone/methyl tert-butyl ether (MTBE) (2/1) to give 2.03 g of product
(CDCl3) δ: 7.67(m, JC-H = 161.5 Hz, 1H), 7.26(m, JC-H = 161.5 Hz, 1H), 7.00(s, in 69% yield. LCMS m/z: 506.5 (MH+).
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B. Latli et al.
Ethyl (E)-3-(2-(((4-(N’-((hexylloxy)carbonyl)carbamimidoyl)phenyl- Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-methyl-N-
1,2,3,4,5,6-13C6)amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-ben- (pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido-2-14C)propa-
zo[d]imidazole-5-carboxamido)propanoate, [13C6]-(11)
noate, [14C]-(9)
To a stirred solution of [13C6]-(9) (1.0 g, 1.8 mmol) in water (2.8 mL) and
acetone (7 mL), potassium carbonate (734 mg, 5.3 mmol) was added at
0 °C. n-Hexyl chloroformate (10) (0.37 g, 2.2 mmol) was dissolved in
acetone (1 mL) and added slowly. The reaction was stirred for 1 h at room
temperature. The product was precipitated by addition of water (7 mL),
A suspension of [14C]-(8) (1.96 g, 4.1 mmol) in ethanol (11 mL) stirred at
À10 °C was saturated with HCl gas. The reaction mixture was kept for
1 h at À10 °C and then it was allowed to stand at room temperature
overnight. The volatile components of the reaction were evaporated,
and the dark remainder was dissolved in ethanol (10 mL), cooled to
filtered, and purified by silica gel chromatography using +5 °C, and treated with a solution of ammonium hydroxide (12%, 7 mL).
dichloromethane/ethanol (9/1) as eluent. Crystallization from ethyl After stirring for 3.5 h at room temperature, water (7.5 mL) was added,
acetate gave 428 mg of product in 38% yield. LCMS m/z: 634.7 (MH+).
and most of the solvents were evaporated under vacuum at 40 °C. The
remaining solid was suspended in water (15 mL) and stirred at room
temperature overnight. The solid was filtered, and the still moist solid
was recrystallized from acetone/MTBE (2/1) to give 1.53 g (36 mCi) of
the desired product in 68% yield.
Ethyl (E)-3-(2-(((4-(N’-((hexylloxy)carbonyl)carbamimidoyl)phenyl-
1,2,3,4,5,6-13C6)amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-ben-
zo[d]imidazole-5-carboxamido)propanoate-methane sulfonate
([13C6]-pradaxa)
Ethyl (E)-3-(2-(((4-(N’-((hexylloxy)carbonyl)carbamimidoyl)phenyl)-
amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-
carboxamido-2-14C)propanoate ([14C]-dabigatran etexilate), ([14C]-11)
[
13C6]-(11) (428 mg, 0.68 mmol) was dissolved in isopropanol (3.8 mL).
Methanesulfonic acid (65 mg, 0.68 mmol) was added at 35 °C, and the
reaction was cooled to 0 °C. Crystallization of the final product was
initiated by addition of some seed crystals. The precipitate was collected
on a suction filter, washed with small amounts of isopropanol and
diisopropyl ether, and dried to a constant mass at 45 °C under reduced
pressure to give 0.437 g (0.60 mmol) of [13C6]-pradaxa in 88% yield. HPLC:
98.9% with isotopic enrichment of > 98.0 % as indicated by MS. LCMS m/
z: 634.7 (MH+). The material was obtained as a salt of methanesulfonic
acid (1:1). Melting point: 171–174 °C, non-labeled reference: 172–174 °C.
1H NMR (DMSO-d6) δ: 11.8(brs, 1H), 10.6(brs, 1H), 9.99(br s, 1H), 8.38(dd,
1H), 7.66(dm, J = Hz, 2H), 7.59(dt, 1H), 7.55(dd, 1H), 7.47(d, 1H), 7.42(d,
1H), 7.17(d, 1H), 7.12(d, 1H), 6.90(d, 1H), 6.86(m, 2H), 4.69(m, 2H), 4.25(q,
2H), 4.22(q, 2H), 3.98(q, 2H), 3.78(s, 3H), 2.68(t, 2H), 2.30(s, 3H, CH3SO3),
1.68(m, 2H), 1.29–1.38(m, 6H), 1.12(t, 3H), 0.88(t, 3H). 13C NMR (DMSO-
d6) δ: 170.9, 170.2, 163.3, 155.9, 154.0(t), 153.7, 153.1, 148.6, 140.5,
137.8, 137.1, 131.3(t), 129.5, 122.8, 122.0, 121.2, 119.3, 112.3(m), 11.6(m),
103.5, 66.9, 59.9, 44.3, 39.7 (CH3SO3H), 39.5, 33.0, 30.7, 29.9, 27.9, 24.7,
21.9, 13.9, 13.8.
To a stirred solution of [14C]-(9) (1.53 g, 2.8 mmol) in water (5.5 mL) and
acetone (11 mL), potassium carbonate (1.13 g, 8.15 mmol) was added.
The mixture was cooled in an ice-bath, and n-hexyl chloroformate (10)
(0.57 g, 3.46 mmol) in acetone (1.6 mL) was added slowly. After stirring
for 1 h at room temperature, the crude product was precipitated by
addition of water (11 mL). The solid was filtered then purified by silica
gel chromatography using dichloromethane/ethanol (9/1) as eluent.
The solid was recrystallized from ethyl acetate to give 0.95 g (20 mCi) in
55% yield.
Ethyl (E)-3-(2-(((4-(N’-((hexylloxy)carbonyl)carbamimidoyl)phenyl)-
amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-
carboxamido-2-14C)propanoate-methane sulfonate ([14C]-pradaxa)
[
14C]-(11) (0.95 g, 1.52 mmol) was dissolved in isopropanol (8.5 mL) at
60 °C. The solution was cooled to 35 °C, and methanesulfonic acid
(146 mg, 1.52 mmol) was added. The resulting was cooled to 0 °C and
seeded by addition of some seed crystals. The precipitate was collected
on a suction filter, washed with small amounts of isopropanol and
diisopropyl ether, and dried to constant mass at 45 °C under reduced
pressure to give 925 mg (16.6 mCi) in 84% yield. The specific activity
was 9.6 mCi/mmol or 13.2 μCi/mg. Radiochemical purity by HPLC:
98.35%, UV (254 nm): 98.3%. The material was obtained as a salt of
methanesulfonic acid (1:1). Melting point: [14C]Pradaxa:170–173 °C, non-
labeled reference:172–174 °C. 1H NMR (DMSO-d6) δ: 11.83(brs, 1H),
10.42(brs, 1H), 9.92(brs, 1H), 8.38(dd, 1H), 7.66(d, 2H), 7.55(dt, 1H), 7.51
(t, 1H), 7.47(d, 1H), 7.42(d, 1H), 7.16(dd, 1H), 7.12(dd, 1H), 6.91(d, 1H),
6.86(d, 2H), 4.67(d, 2H), 4.22(m, 2H), 3.98(q, 2H), 3.77(s, 3H), 2.68(t, 2H),
2.23(s, 3H, CH3SO3), 2.23(s, 2H), 1.67(m, 2H), 1.29–1.37(m, 6H), 1.12(t,
3H), 0.88(t, 3H).
Synthesis of [14C]-dabigatran etexilate
Ethyl (4-cyanophenyl)glycinate-1-14C, [14C]-(5)
To
a mixture of 4-aminobenzonitrile (3) (945 mg, 8 mmol) and
isopropylethyl amine (2.1 g, 16 mmol) stirred at 100 °C was added
ethylbromoacetate-1-14C (672 mg, 4 mmol, 3.7 GBq or 100 mCi) diluted
with non-labeled ethylbromoacetate (668 mg, 4 mmol). After stirring at
this temperature for 4 h, the reaction was cooled to 60 °C, and the
product was precipitated by addition of water (8 mL) and hydrochloric
acid (1.36 mL, 6 N). The yellowish crystals were collected on a suction
filter, washed with water and diisopropyl ether, and dried in a vacuum
oven to a constant mass at 50 °C to give 1.25 g (76 mCi) in 76% yield.
(4-Cyanophenyl)glycine-1-14C, [14C]-(6)
Synthesis of tritium labeled dabigatran
A suspension of [14C]-(5) (1.2 g, 5.9 mmol) in methanol (6 mL) was stirred
with sodium hydroxide (6.54 mL, 1 N) for 4 h at room temperature. The
product was then precipitated by addition of hydrochloric acid
(6.54 mL, 1 N), collected on a suction filter, and washed with isopropanol
(1 mL × 2) to give 1.03 g (74.5 mCi) of product in 98% yield.
Using the Crabtree’s catalyst
Compound (12) (5.4 mg, 9 μmol) and Crabtree’s catalyst (6.96 mg,
8.64 μmol) were dissolved in dichloromethane (0.8 mL). The solution
was degassed three times at the high vacuum manifold and stirred under
an atmosphere of tritium gas (7.7 Ci) for 2.5 h at room temperature. The
maximum pressure at room temperature was 883 mbar. The solvent
was removed in vacuo, and labile tritium was exchanged by adding
methanol (1 mL), stirring the solution, and removing the solvent again
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-1-methyl-N-(pyridin-
2-yl)-1H-benzo[d]imidazole-5-carboxamido-2-14C)propanoate,
[
14C]-(8)
[
14C]-(6) (1.0 g, 5.7 mmol) was added to a suspension of CDI (993 mg, under vacuo. This process was repeated three times before the residue
6.13 mmol) in THF (9 mL) at room temperature. After stirring for 1 h, (7) was dried. Without further analysis, the crude product was dissolved in
(1.96 g, 5.73 mmol) was added at 40 °C, and the reaction mixture was TFA (0.25 mL). Immediately, a red-colored solution was formed and
heated to reflux for 2 h. The solvent was evaporated, and the residue stirred for 1 h at ambient temperature. All volatile components were
was heated in acetic acid (5.4 mL) for 1 h to reflux. The crude product removed in vacuo. The residue was suspended in an aqueous solution
was precipitated by addition of water (20 mL), filtered, and recrystallized of 6.5 N NaOH (0.5 mL). To improve the solubility, THF (0.1 mL) was
from isopropanol to give 1.96 g (53 mCi) of product in 71% yield.
added, and the orange solution was stirred at ambient conditions for
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B. Latli et al.
40 min. The crude mixture was neutralized with 50% HOAc (0.8 mL), and
MeCN (0.1 mL) was added. The activity of the crude product was
determined to be 372 mCi (13.8 GBq). The radiochemical purity of the
crude product was 47% using the following HPLC system: Macherey &
Nagel Nucleodur Pyramid C18 (5 μm, 4.6 × 150 mm); gradient A: 10 mM
NH4OAc, B: MeCN; 0 min 5% B; 2 min 5% B; 12 min 95% B; 16.5 min
95% B; 17 min 5% B; 254 nm; flow rate: 1.0 mL/min; 30 °C. A batch of
refluxed at 120 °C for 1 h. After cooling to room temperature, acetic acid
was removed under reduced pressure and the crude product was
purified on SiO2 cartridge (120 g) using CombiFlash system and eluting
with DCM (CH2Cl2): MeOH gradient from 100:0 to 90:10 to give 0.8 g of
product, which was recrystallized from EtOH to give 0.55 g of the desired
product as an off-white solid in 24% yield. HPLCb : tR = 9.80 min; LCMS m/
1
z: 735.3 (MH+). H NMR (400 MHz, CDCl3) δ: 8.40(dd, J = 5.0, 1.32 Hz, 1H),
110 mCi (4.1 GBq) of this crude material was purified using the following 8.04(s, 2H), 7.76(s, 1H), 7.33 (m, 2H), 7.16(d, J = 8.4 Hz, 1H), 6.98(m, 1H),
HPLC conditions: Macherey & Nagel Nucleodur Pyramid C18 (5 μm,
8 × 150 mm); solvents A: 25 mM NH4OAc; B: MeCN; 0 min 12.6% B;
8 min 12.6% B; 8.5 min 95% B; 13 min 95% B; 13.5 min 12.6% B; 254 nm
and 230 nm; 3.1 mL/min; 25 °C. The desired product (30 mCi) eluted at
6.70(d, J = 8.0 Hz, 1H), 5.17(t, J = 5.6 Hz, 1H), 4.73(d, J = 5.7 Hz, 2H), 4.43(t,
J = 7.2 Hz, 2H), 4.07(q, J = 7.2 Hz, 2H), 3.69(s, 3H), 2.82(t, J = 7.2 Hz, 2H),
1.22(t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 171.8, 171.0, 156.4,
153.2, 152.0, 148.9, 143.6, 141.6, 137.4, 137.2, 130.3, 124.1, 122.4, 121.2,
6.5 min (co-eluted with unlabeled standard). After evaporation of the 120.9, 115.7, 108.9, 108.5, 88.6, 60.5, 45.2, 44.7, 33.4, 30.1, 14.2.
solvents, the residue was dissolved in 12 mM NH4OAc/acetonitrile = 1/1.
Ethyl 3-(2-(((4-carbamimidoyl-2,6-diiodophenyl)amino)methyl)-1-
methyl-N-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido)-
propanoate (18). Compound (17) (0.5 g, 0.68 mmol) was dissolved in
EtOH (30 mL) and cooled to 0 °C in an ice-water bath. Hydrogen
chloride gas was bubbled through this solution for 5 min. The reaction
mixture was left stirring in a cooling bath for 8 h. HPLC analysis
indicated complete reaction. The reaction mixture was concentrated
providing 0.5 g (75%, 80% pure). HPLCb : tR = 8.36 min; LCMS m/z:
781.2 (MH+). To a solution of this crude material in EtOH (30 mL),
ammonium carbonate (746 mg, 7.69 mmol) was added as a solid at
room temperature. The reaction was stirred at 45 °C for 5 h. HPLC
analysis indicated complete reaction. The reaction was concentrated
under reduced pressure, and crude reaction mixture was purified on
SiO2 cartridge (40 g) using CombiFlash system eluting with DCM:MeOH
gradient from 90:10 to 80:20 to give 300 mg of product as a slightly
yellow solid in 60% yield. HPLCb: tR = 7.48 min; LCMS m/z: 752.1 (MH+).
1H NMR (400 MHz, CDCl3) δ: 8.35(m, 3H), 7.83(m, 1H), 7.66(m, 2H), 7.43
(d, J = 8.4 Hz, 1H), 7.20(m, 1H), 7.11(d, J = 8.0 Hz, 1H), 4.95(br s, 2H),
4.24(t, J = 7.0 Hz, 2H), 3.98(q, J = 7.0 Hz, 2H), 3.95(3H, s, 3H), 2.72(t,
J = 6.9 Hz, 2H), 1.32(t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ:
171.0, 170.3, 162.3, 155.9, 153.4, 153.2, 148.6, 140.8, 139.6, 137.8,
137.1, 129.3, 122.8, 122.7, 122.0, 121.2, 119.5, 109.6, 89.7, 60.0, 44.3,
44.0, 33.0, 30.0, 13.9.
Radiochemical purity: >98 % (HPLC). The specific activity of 43 Ci/mmol
(1.59 TBq/mmol) was calculated by measuring the activity of the product
and by comparing the UV signal areas of the product solution and of the
solution with known concentration. Storage Concentration: 1.0 mCi/mL
(37 MBq/mL) 12 mM NH4OAc/acetonitrile = 1/1
Using iodine-tritium exchange
4-Amino-3,5-diiodobenzonitrile (14). 4-Aminobenzonitrile (3) (5.0 g,
42.3 mmol) was added to a mixture of iodine (21.3 g, 84.65 mmol) and
silver sulfate (26.4 g, 84.6 mmol) in EtOH (350 mL) at room temperature.
The mixture was stirred for 2.5 h, filtered through a fine fritted funnel,
and washed with EtOAc (300 mL). The filtrate was concentrated, and
the residue was dissolved in CH2Cl2 (300 mL), washed with 1 N NaOH
(300 mL) water (300 mL), dried over MgSO4, filtered, and concentrated
to a solid. Crystallization from hot EtOH (75 mL) gave 12.45 g of an off
white solid in 79.5% yield. 1H (CDCl3) δ: 7.87(s, 2H), 5.18(br s, 2H). 13C
NMR (CDCl3) δ: 149.9, 142.5, 120.9, 103.5, 79.1. Identical to the literature.35
Ethyl (4-cyano-2,6-diiodophenyl)glycinate (15). A solution of (14)
(4.6 g, 12.4 mmol) in DMF (30 mL) was cooled to 0 °C in an ice-bath.
NaH (0.55 g, 13.7 mmol, 60% in mineral oil dispersion) was added portion
wise. After stirring at 0 °C for 15 min, ethyl bromoacetate (4) (2.70 g,
16.17 mmol) in DMF (5 mL) was added dropwise. The reaction was
allowed to warm up to room temperature over 1 h and stirred for 4 h.
The reaction was quenched by slow addition of water (20 mL). EtOAc
(100 mL), and water (50 mL) were then added. The organic layer was
separated, washed with ice-water (40 mL × 2), dried over MgSO4, and
concentrated under reduced pressure to give 6.0 g of slightly yellow
solid. Crystallization from EtOH gave 4 g of (15) in 83% yield as an off-
white solid. HPLCb : tR = 13.89 min; LCMS m/z: 457.3 (MH+). 1H NMR
(400 MHz, CDCl3) δ: 8.01(s, 2H), 5.19(t, J = 5.0 Hz, 1H), 4.27(q, J = 7.2 Hz,
2H), 4.25(d, J = 5.0 Hz, 2H), 1.32(t, J = 7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ: 169.9, 152.9, 143.6, 115.9, 106.9, 85.9, 61.9, 49.8, 14.2
Ethyl 3-(2-(((4-(N-(tert-butoxycarbonyl)carbamimidoyl)-2,6-diiodo
phenyl) amino)methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]-
imidazole-5-carboxamido)propanoate (19). To a solution of (18)
(60 mg, 0.08 mmol) and Boc2O (38.7 mg, 0.176 mmol) in MeOH (1.5 mL)
was added Et3N (24.5 mg, 0.24 mmol) at room temperature. The reaction
was heated to 40 °C and stirred for 4 h. The reaction was concentrated
under reduced pressure, and the residue was purified on SiO2 cartridge
(12 g) using CombiFlash system eluting with DCM:MeOH gradient from
100:0 to 95:5 to give 50 mg of slightly yellow solid in 91% yield. HPLCb:
tR = 9.10 min; LCMS m/z: 852.1 (MH+). 1H NMR (400 MHz, CDCl3) δ: 8.41
(4-Cyano-2,6-diiodophenyl)glycine (16). To a solution of (15) (3.5 g, (d, J = 3.2 Hz, 1H), 8.25(s, 2H), 7.71(s, 1H), 7.31(m, 2H), 7.11(d, J = 8.4 Hz,
7.68 mmol) in a mixture of MeOH/THF (1:1, 80 mL), a solution of LiOH
(38.4 mL, 1.0 M in water) was added dropwise at room temperature.
The reaction was stirred at room temperature for 5 h. The reaction was
1H), 6.98(m, 1H), 6.70(d, J = 8.0 Hz, 1H), 4.61(s, 2H), 4.43(t, J = 7.2 Hz, 2H),
4.07(q, J = 7.2 Hz, 2H), 3.62(s, 3H), 2.81(t, J = 7.2 Hz, 2H), 1.55(s, 9H), 1.22
(t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 171.7, 171.2, 164.3, 164.0,
acidified with 1.0 M aqueous HCl till pH = 3–4 and extracted with EtOAc 156.2, 152.6, 151.6, 148.9, 141.4, 137.4, 139.4, 137.3, 132.3, 129.9, 123.9,
(80 mL × 2). The combined extracts were dried over MgSO4, filtered, and 122.5, 121.0, 109.0, 90.9, 79.9, 60.6, 53.5, 45.0, 44.7, 33.4, 30.4, 28.2, 14.2.
concentrated under reduced pressure to give 3.0 g of slightly yellow solid
Ethyl 3-(2-(((4-(N-(tert-butoxycarbonyl)carbamimidoyl) phenyl-2,6-
3H2)amino) methyl)-1-methyl-N-(pyridin-2-yl)-1H-benzo[d]imida-
zole-5-carboxamido)propanoate ([3H2]-(12)). Unlabeled hydroge-
nation was performed using (19) (8.0 mg, 3.0 μmol), Pd/C catalyst (8 mg),
MeOH (0.8 mL) under 14 psi of H2 for 20 h. HPLC analysis indicated complete
reaction.
in 91% yield. This crude material was used in the next step without any
further purification. HPLCb: tR = 11.28 min; LCMS m/z: 429.2 (MH+). 1H
NMR (400 MHz, CDCl3) δ: 8.17(s, 2H), 5.90 (br.s, 1H), 4.02(m, 2H). 13C
NMR (100 MHz, DMSO-d6) δ: 171.1, 152.9, 143.3, 116.5, 104.6, 85.8, 49.0.
Ethyl 3-(2-(((4-cyano-2,6-diiodophenyl)amino)methyl)-1-methyl-N-
(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamido)propanoate
(17). To a solution of (16) (1.5 g, 3.51 mmol) in THF (30 mL), CDI (0.57 g,
Thus, (8.0 mg, 3.0 μmol) was transferred to a two-necked flask fitted
with septa, followed by Pd/C (10%, 10 mg). The flask was attached to a
3.51 mmol) was added at room temperature. The reaction was refluxed tritium manifold and evacuated. Methanol (1 mL) was added, and the
for 1 h. The diamine derivative (7) (1.1 g, 3.17 mmol) was added to the
mixture was cooled to À190 °C. The mixture was evacuated. This cycle
reaction mixture, and the reaction was refluxed overnight. After cooling was repeated twice before tritium gas was introduced until the pressure
to room temperature, the reaction mixture was concentrated under
reduced pressure. The residue was dissolved in acetic acid (20 mL) and
reached 1.2 atm. The reaction was stirred at ambient temperature for
16 h. Excess tritium was removed, and the solution was evaporated to
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J. Label Compd. Radiopharm 2016

B. Latli et al.
dryness and reconstituted in methanol. Analysis by HPLC showed 75%
product co-eluted with unlabeled material. HPLCc, tR = 11.57 min (75%).
A total of 1.5 Ci of crude material was isolated.
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(0.2 mL) in DCM (1 mL) and stirred for 1 h in order to remove the Boc
protecting group. The reaction was concentrated in vacuo. The residue
was treated with 2 N NaOH (0.5 mL) in MeOH (0.5 mL) and THF (0.5 mL)
at room temperature with stirring for 2 h to hydrolyze the ethyl ester.
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XDB-C18. The desired fractions were combined and lyophilized to give
a white solid, which was diluted with 20% water/EtOH (5 mL) to give
[3H]-BIBR 953 as a solution; 18.3 mCi with a specific activity of 38.4 Ci/
mmol; and radiochemical purity: 98%, tR = 3.87 min. HPLC conditions:
gradient 10% to 95% MeCN (water), 0.1% TFA in 15 min, column: Zorbax
XDB-C18 (5.0 μm, 4.6 × 150 mm), co-eluted with unlabeled sample. This
material showed similar stability issues seen with the tritium labeled
product obtained using Crabtree’s catalyst. The purity of the stored
material in 1:1 ethanol: water dropped to 80% after three months of
storage at À80 °C.
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Conclusion
Dabigatran etexilate was the first FDA-approved alternative to
warfarin. It is used to prevent strokes in patients with atrial
fibrillation (AFib) and the formation of blood clots in the veins
in adults who have had an operation to replace a hip or a knee.
Dabigatran etexilate is the only NOAC with a specific reversal
agent for use in rare emergency situations. Dabigatran etexilate
and dabigatran labeled with carbon-13, carbon-14, and with
tritium were prepared to enable critical metabolism,
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The carbon-13 labeled dabigatran etexilate was prepared in
eight steps and 6% overall yield starting from aniline-13C6.
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Carbon-14 labeled dabigatran etexilate was obtained in six steps
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incorporation under Crabtree’s catalytic conditions or tritium-
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seen using Crabtree’s catalyst.
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Acknowledgements
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J. Label Compd. Radiopharm 2016
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