An efficient and facile synthesis of deuterium-labeled anticancer agent bendamustine hydrochloride

Article abstract of DOI:10.1002/jlcr.3637

Bendamustine hydrochloride is an alkylating agent that was developed for the treatment of various human cancers. The stable isotope-labeled bendamustine was required to support clinic studies. An effective and operationally simple method for the synthesis of [D₆] bendamustine hydrochloride was developed using DCl as a catalyst and D₂O as a deuterium source. Under the present condition, regioselectively deuterated bendamustine hydrochloride with high deuterium incorporation is achieved.

Full text of DOI:10.1002/jlcr.3637

An efficient and facile synthesis of deuterium-labeled anticancer agent
bendamustine hydrochloride
Baomin Liu, Hui Qin, Yinsheng Zhang
Chia Tai Tianqing Pharmaceutical Group Co., LTD, Jiangsu Key Laboratory of Targeted
Antiviral Research, Nanjing, Jiangsu Province, China
*Correspondence to : Yinsheng Zhang, Chia Tai Tianqing Pharmaceutical Group Co., LTD,
Jiangsu Key Laboratory of Targeted Antiviral Research, 699-8 Xuanwu Ave. Building #9,
Nanjing, Jiangsu Province, China
Abstract: Bendamustine hydrochloride is an alkylating agent which was developed for the treatment
of various human cancers. The stable isotope-labeled bendamustine was required to support clinic
studies. An effective and operationally simple method for the synthesis of [D6] bendamustine
hydrochloride was developed using DCl as a catalyst and D2O as a deuterium source. Under the
present condition, regioselectively deuterated bendamustine hydrochloride with high deuterium
incorporation is achieved.
Keywords: bendamustine; deuterium-labeling; internal standard; H-D exchange
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Introduction
Bendamustine hydrochloride (Fig.1) is a water-soluble, bifunctional alkylating agent with a
benzimidazole ring that induces more long-lasting DNA double-strand breaks than other alkylating
drugs.1 It is reported that bendamustine had clinical activity against various human cancers including
non–Hodgkin’s lymphoma, multiple myeloma, chronic lymphocytic leukemia, breast cancer and small-
cell lung cancer.2 In addition, it is indicated that bendamustine is non–cross-resistant with alkylating
agents and other drugs in vitro and in the clinic.3, 4
Compounds labeled with stable isotopes are most commonly used as internal standards for the
quantification of drugs in biological samples.5 Stable isotope labeled compounds also played an
important role in the assessment of drug pharmacology to determine the pharmacokinetic profile or
mode of action of a drug substance.6 High quality information about the identity and quantification of
drug-related compounds in biological samples can be provided by a well-designed labeled compound,
which is extremely valuable at key decision points in drug development.
To our knowledge, there were a few procedures described in literature for the preparation of
deuterium-labeled bendamustine.7 For bendamustine, the abundance of the M+5 (362 amu)
isotopologues comes to 1.9%, considering the natural isotope distribution of 35Cl/37Cl and 12C/13C.
Therefore, a minimum of six deuterated sites for the internal standard appeared to be sufficient to
prevent unfavorable interferences between signals of non-deuterated and deuterated bendamustine in
MS.
Considering the structure of bendamustine, [D6]bendamustine hydrochloride
2 and
[D6]Bendamustine hydrochloride 3 were first designed as our target compounds. However, we failed to
synthesize [D6]bendamustine hydrochloride 2 due to the decomposition of the intermediate during the
H-D exchange reaction. At the same time, the route to prepare [D6]bendamustine hydrochloride 3 was
time-consuming and uncertain (11 steps starting from CD3NH2 and dihydro-2H-pyran-2,6(3H)-dione).
Fortunately, an effective and facile method was found to synthesize [D6]bendamustine hydrochloride 4
when we explored ways to synthesize [D6]bendamustine hydrochloride 2.
Experimental
General
Unlabeled intermediate (9) and authentic reference standards were supplied by Process Chemistry
Institute of Chia Tai Tianqing Pharmaceutical Group Co. All other reagents and solvents were
commercially available and used without further purification. NMR spectra were recorded on a Bruker
Avance 500 spectrometer. Chemical shifts (δ) in ppm are quoted relative to CDCl3, D2O or DMSO-d6.
High-resolution mass spectra were recorded on AB SCIEX Triple TOF 4600. TLC analysis and
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coloration was performed using UV light (254 nm) or phosphomolybdic acid. HPLC was obtained on
an Agilent HPLC using ZORBAX Eclipse XDB-C18, 150mm× 4.6mm, 5 μm column, and solvent
which contained water, acetonitrile and trifluoroacetic acid. Microwave reactions were carried using a
Discover SP-Microwave Synthesizer at max power of 250 W.
Ethyl 4-(5-amino-1-methyl-1H-benzo[d]imidazol-2-yl) butanoate (10)
A mixture of 4-(1-methyl-5-nitro-1H-benzoimidazol-2-yl)-butyric acid ethyl ester 9 (30.01 g, 103.02
mmol) and 10% palladium on charcoal (3.0 g) in methanol (400 mL) and ethyl acetate (200 mL) at
room temperature was stirred under hydrogen atmosphere for 16 h. The resulting mixture was filtered
and concentrated in vacuo to give compound 10 as a white solid (26.45 g, 98.3%) with 99.5% purity
(HPLC). 1H NMR (DMSO-d6, 500 MHz): δ 7.11 (d, J = 8.5 Hz, 1 H), 6.71 (d, J = 1.5 Hz, 1 H), 6.53
(m, 1 H), 4.66 (s, 2 H), 4.06 (q, J = 7 Hz, 2 H), 3.61 (s, 3 H), 2.79 (t, J = 7.5 Hz, 2 H), 2.44 (t, J = 7.5
13
Hz, 2 H), 1.98 (quintet, J = 7.5 Hz, 2 H), 1.18 (t, J = 7 Hz, 3 H); C NMR (DMSO-d6, 125 MHz): δ
173.14, 153.86, 144.09, 143.84, 128.95, 111.39, 109.80, 102.64, 60.25, 33.35, 29.69, 26.12, 22.76,
14.59; HRMS ESI (M+H) + m/z: 262.1544.
4-(5-amino-1-methyl-1H-benzo[d]imidazol-2-yl-4,6-d2)butanoic-2,2,4,4-d4 acid hydrochloride (11)
To a solution of compound 10 (1.02 g, 3.90 mmol) in D2O (15 mL) 35% DCl (1.64 g, 15.32 mmol) was
added in a 35 mL microwave reaction vial. The vial was sealed and heated to 180°C in the microwave
synthesis apparatus for 30 min. Then the mixture was evaporated to dryness in vacuo. To the residue
was added fresh D2O (15 mL) and another 35% DCl (1.01 g, 9.34 mmol). The resulting mixture was
heated again to 180°C in the microwave synthesis apparatus for 30 min. The operation was repeated
three times. The final residue was triturated with ethanol to afford compound 11 (1.01 g, 93.5%) as a
gray solid (HCl salt) with 95% purity (HPLC). 1HNMR (DMSO-d6, 500 MHz): δ 8.04(s, 1H), 3.98 (s,
3 H), 2.04 (s, 2 H); 13C NMR (DMSO-d6, 125 MHz): δ 174.13, 155.17, 131.66, 131.31, 130.37, 114.26,
31.78, 21.33; HRMS ESI (M+H) + m/z: 240.1482.
Ethyl 4-(5-amino-1-methyl-1H-benzo[d]imidazol-2-yl-4,6-d2)butanoate-2,2,4,4-d4 (12)
To a solution of compound 11 (7.51 g, 27.22 mmol) in ethanol-D (60 mL) thionyl chloride was added
dropwise (4.84 g, 40.67 mmol) at 0°C. The mixture was kept at 0°C for 30 min and warmed to 50°C in
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an oil bath for 4 h. The reaction was monitored by TLC. After the completion of the reaction, the mixture
was neutralized with saturated sodium bicarbonate solution and extracted with ethyl acetate (100 mL).
The organic phases were dried over Na2SO4 and concentrated under vacuum to give compound 12 (5.92
g, 81.3%) as a gay solid with 97% purity (HPLC). 1H NMR (DMSO-d6, 500 MHz): δ 7.13(s, 1 H), 4.85
(s, 2 H), 4.05 (q, J = 7 Hz, 2 H), 3.62 (s, 3 H), 1.95 (s, 2 H), 1.18 (t, J = 7 Hz, 3 H); 13C NMR(DMSO-
d6, 125 MHz)：δ 173.15, 153.73, 144.06, 143.37, 128.80, 109.80, 60.24, 29.75, 22.46, 14.59; HRMS
ESI (M+H) + m/z: 268.1929.
Ethyl
4-(5-(bis(2-hydroxyethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl-4,6-d2)butanoate-
2,2,4,4-d4 (13)
To a solution of compound 12 (5.92 g, 22.14 mmol) in acetic acid (26.61 g, 443.2 mmol) and D2O (49.2
g, 2.46 mol) oxirane (7.72 g, 175.1 mmol) was added at 0°C. The mixture was kept at 0°C for 1 h and
warmed to 25 °C in an oil bath for 18 h. After the completion of the reaction, the mixture was neutralized
with saturated potassium carbonate solution and extracted with dichloromethane (3 × 50 mL). The
organic phases were dried over Na2SO4 and concentrated under vacuum to afford the crude product as
a yellow oil. To the crude product was added ethyl acetate and acetone (60 mL, v/v = 3:1), and after the
resulting mixture was sonicated in an ultrasonic apparatus for 30 min the white solid crashed out. The
product was filtered off and dried at 50°C under reduced pressure to give compound 13 as a white solid
(6.16 g, 78.3%) with 97% purity (HPLC). 1H NMR(CDCl3, 500MHz): δ 7.15(s, 1 H), 4.13 (q, J = 7 Hz,
2 H), 3.85 (t, J = 5 Hz, 4 H), 3.70 (s, 3 H), 3.57 (t, J = 5 Hz, 4 H), 2.13 (s, 2H), 1.26 (t, J = 7 Hz, 3 H);
13C NMR (CDCl3, 125MHz): δ 173.25, 153.99, 144.71, 142.67, 128.95, 109.30, 60.45, 60.41, 56.23,
29.79, 22.37, 14.23; HRMS ESI (M+H)+ m/z: 356.2494.
4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl-4,6-d2)butanoic-2,2,4,4-d4
acid hydrochloride (4)
To a solution of compound 13 (6.01 g, 16.91mmol) in dichloromethane (80 mL) thionyl chloride (19.3
g, 162.14 mmol) was added dropwise at 0°C. The mixture was kept at 0°C for 1 h and warmed to 25°C
in an oil bath for 9 h. The reaction was monitored by HPLC. After the completion of the reaction, the
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solvent was evaporated to afford the crude product 14 (7.90 g) as a black oil, which was used in the
next step without further purification.
A solution of the aforementioned product 14 (7.90 g) in 35% DCl (53.10 g, 496 mmol) was refluxed in
an oil bath for 2 h. The reaction was monitored by HPLC. After the completion of the reaction, the
solvent was concentrated under vacuum to afford the crude product as a black oil. The crude product
was added to D2O (28 mL) and stirred at room temperature to give a gray solid (6.32 g), which was
dissolved in 35% DCl (5 mL) and more D2O (18 mL) at 60°C. Activated carbon (1.02 g) was added
and stirring was continued for 30 min. The activated carbon was separated by filtration. The filtrate was
evaporated to dryness. Another 15 mL of D2O was added to the residue. The mixture was placed at 4°C
for crystallization for 24 h. The precipitate was separated by filtration and washed with D2O. The filter
cake was dried at 50°C under reduced pressure to give the titled compound 4 (2.05 g, 30.2%) as a white
solid with 99.1% purity (HPLC). 1H NMR(DMSO-d6, 500 MHz): δ 7.73 (s, 1 H), 3.91 (s, 1 H), 3.84 (t,
J = 6 Hz, 4 H), 3.79 (t, J = 6 Hz, 4 H), 1.99 (s, 2 H). 13C NMR(DMSO-d6, 125 MHz): δ 174.23, 152.21,
146.00, 132.09, 125.18, 113.81, 52.82, 41.48, 31.42, 21.70. HRMS ESI (M+H) + m/z: 364.1486.
Results and discussion
In order to get [D6]bendamustine hydrochloride, we first designed synthetic routes to prepare
[D6]bendamustine hydrochloride 2 (Scheme 1) and [D6]bendamustine hydrochloride 3. However, we
failed to obtain the labeled compound 8 due to the decomposition of compound 7 under several reported
H-D exchange conditions (DCl/D2O, Pd/Pt/D2O & [D]TFA/D2O).8, 9, 10 The nitro group of compound 7
was considered to be the trouble maker in this case. Thus, we attempted to use aniline compound 10 as
a starting material of the H-D exchange reaction to introduce 2 to 3 deuteriums into the benzene ring.
4-(5-amino-1-methyl-1H-(4, 6-D2)benzo[d]imidazol-2-yl) butanoic acid was expected as a desired
labeled compound based on the reported method.8 However, deuteriums were introduced not only into
the expected positon of the benzene ring, but also into the benzyl position and alpha position of the
carboxylic acid group. Thus, compound 11 with six deuteriums incorporated was obtained. With the
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labeled compound 11 in hand we were able to successfully synthesize [D6]bendamustine hydrochloride
4 via the route depicted in Scheme 2.
Reagents and conditions: (i) CD3NH2.HCl, AcONa, DMF; (ii) Na2S, NaHCO3, H2O, reflux; (iii) a) D2O,
35% DCl, MW, 150-180 °C, 30 min or b) Pd/Pt/D2O MW,150-180 °C, 30 min or c) [D]TFA/D2O, MW,
150-180 °C, 30 min.
Briefly, unlabeled aniline compound 10 was prepared by reduction of nitro compound 9 with 10% Pd/C
catalyst in 98% yield. This compound was then treated with D2O and 35%DCl under microwave heating
condition to give deuterium-labeled compound 11 in 94% yield after 3 exchange cycles. The acid 11
was converted to the ethyl ester 12 in 82% yield using SOCl2 and ethanol. Double N-alkylation of 12
with ethylene oxide offered the compound 13 in 78% yield. Double chlorination of 13 with SOCl2 gave
the dichloride 14, which was immediately hydrolyzed with a D2O solution of DCl to give the desired
compound 4 ([D6]bendamustine hydrochloride) in 32% yield. It should be noted that compound 14 was
very unstable in aqueous media,11 and the crude material should be hydrolyzed to form an HCl salt as
soon as possible.
Reagents and conditions: (i) H2, 10% Pd/C, MeOH/Ethylacetate, rt, 16 h, 98.3%; (ii) D2O, 35% DCl,
MW, 180 °C, 30 min, three cycles, 93.5%; (iii) SOCl2, EtOD, 50 °C, 4 h, 81.3%; (iv) Ethylene oxide,
AcOH, D2O, 25 °C, 78.3%; (v) SOCl2, 9 h; (vi) 35% DCl, D2O, reflux, 30.2%.
The 1H NMR spectroscopic analysis of the compound 11 subjected to the H-D exchange condition for
one cycle revealed that protons ortho to aniline nitrogen and methylene protons αto the benzimidazole
had undergone a complete H-D exchange. In addition, to some extent, also the methylene protons α to
the carboxyl function had also been replaced by deuteriums (~50%, determined by 1HNMR, Figure 1).
After three cycles all the protons mentioned above had undergone an almost complete H-D exchange
(deuterium enrichment >98%, Figure 2 and Figure 3) to give the compound 11. The structure of the
final target, [D6]bendamustine hydrochloride 4, was confirmed by 1H & 13C NMR and HRMS (Figure
4).
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Conclusion
In summary, we have developed an efficient method for the preparation of deuterium-labeled
bendamustine hydrochloride 4. The key labeled intermediate 11 with six deuteriums was prepared using
direct H-D exchange reaction with a catalyst of DCl and a deuterium source of D2O. This efficient
labeling method allowed us to quickly access the qualified deuterium-labeled bendamustine
hydrochloride 4 as an internal standard.
Acknowledgements
The authors would like to thank Xushi Liu, Yuhan Li and Lu Zhang for their support necessary to
successfully complete this project.
Conflict of interest
The authors confirm that this article content has no conflicts of interest.
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Figure 1. Bendamustine hydrochloride and its deuterium-labeled compounds 1-4
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Figure 2. 1H NMR-spectra of compound 11 after three H-D exchange cycles
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Figure 3. HRMS of compound 11 after three H-D exchange cycles
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Figure 4. HRMS of compound 4
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Scheme 1. Synthetic route of [D6]bendamustine hydrochloride 2.
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Scheme 2. Synthesis of [D6]bendamustine hydrochloride 4.
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Graphic Abstract
An efficient and facile synthesis of deuterium-labeled anticancer agent
bendamustine hydrochloride
Baomin Liu, Hui Qin, Yinsheng Zhang
The stable isotope-labeled bendamustine was required to support clinic studies. An effective and
operationally simple method for the synthesis of [D6]bendamustine hydrochloride was developed
using DCl as a catalyst and D2O as a deuterium source. Under the present condition, regioselectively
deuterated bendamustine hydrochloride with high deuterium incorporation is achieved
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