Synthesis of a potential bendamustine deschloro dimer impurity

Abstract

Bendamustine deschloro dimer was considered as a potential impurity in bendamustine hydrochloride resulting from the hydrolysis of bendamustine followed by intermolecular esterification. An efficient synthesis of bendamustine deschloro dimer was achieved from 4-(5-amino-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate involving nine sequential steps including benzyl-protection/deprotection of the amine and carboxylic acid groups, saponification, ring-opening reaction of oxirane as well as Fischer/Steglish esterfication and so on. The target bendamustine deschloro dimer was obtained using a high-performance liquid chromatography in a purity of 95.63%.

Bendamustine deschloro dimer, a potential impurity of bendamustine hydrochloride, has been efficiently synthesized from compound 1 in nine steps which involve two key intermediate compounds 4 and 6.

Introduction

Bendamustine (BM) hydrochloride is a bifunctional alkylating agent used to treat patients with chronic lymphoytic leukaemia. It features an active nitrogen mustard moiety (mechlorethamine unit) together with a benzimidazole ring and a butanoic acid residue.^1–4 Its mechlorethamine moiety is highly active in forming covalent bonds with DNA, causing DNA damage and leading to cell death.^5,6

During the synthesis and storage of active pharmaceutical ingredients (APIs), impurities including organic/inorganic species as well as residual solvents may exist that have a great influence on the quality and safety of the pharmaceuticals. ⁷ Owing to the presence of the intrinsically reactive nitrogen mustard moiety, BM is so moisture-sensitive that hydrolysis of mechlorethamine unit can easily occur to generate hydrolyzed species, namely monohydroxy and dihydroxy derivatives (a and b), ⁸ which can be further coupled to each other via an ester linkage, giving rise to BM dimer impurities, namely BM1 dimer and BM deschloro dimer (Scheme 1). According to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) regulations, it is mandatory to identify and control the level of impurities before final approval for commercialization of a pharmaceutical. ⁹

Scheme 1.

Plausible formation mechanism of BM deschloro dimer and BM1 dimer.

In this context, we report the synthesis of BM deschloro dimer, starting from ethyl 4-(5-amino-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate (compound 1), which was efficiently obtained in nine reaction steps (Scheme 2).

Scheme 2.

The synthetic route to BM deschloro dimer.

Results and discussion

4-(5-Amino-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate (compound 1) was first converted into mono-benzyl (4)- and bis-benzyl (6)-protected intermediate in different methods. Compound 4 was prepared via the reductive amination reaction involving a one-pot reaction of benzaldehyde in the presence of sodium cyanoborohydride (NaBH₃CN), ¹⁰ which was subsequently reacted with oxirane to give compound 2. Compound 2 underwent saponification to afford compound 3, which had its carboxylic acid group protected by the Bn protecting group through a Fischer esterification reaction (benzyl alcohol/H₂SO₄), leading to the key intermediate of 4 with a hydroxyl reactive site. By contrast, the bis-benzyl-protected intermediate (5) was obtained through a direct N-benzylation reaction using excess benzyl bromide under basic conditions ((iPr)₂NEt/CH₃CN). ¹¹ Upon saponification, compound 5 was transformed into another key intermediate (6) bearing the naked carboxylic acid group. The two key intermediates (4 and 6) were then coupled to each other via an ester linkage, that is, the carboxylic acid group of compound 6 reacted with the hydroxyl group of compound 4 under Steglish esterification condition (DCC/DMAP: DCC = dicyclohexyl-carbodiimide; DMAP = N,N-dimethylaminopyrdine) to produce the dimer of compound 7. ¹² Finally, the four Bn protecting groups in compound 7 were completely and cleanly removed in a one-pot reaction through a catalytic hydrogenolysis (Pd/C, H₂) under a mild condition. Finally, the amino groups reacted with oxirane via the ring-opening polymerization (ROP) reaction and generating the target BM deschloro dimer.

Conclusion

In brief, a convenient and efficient synthetic route was developed towards the potential BM deschloro dimer impurity of BM hydrochloride. The key strategy of the current synthesis lies in employing benzyl groups to mask the reactive amine and carboxylic acid groups in the two key intermediates. These can be cleanly removed in a one-pot manner by mild hydrogenolysis after coupling of two intermediates. Our current work offers a facile method to gain high-quality BM deschloro dimer impurity reference for quality control of BM hydrochloride although BM deschloro dimer was not found as an impurity in bendamustine hydrochloride.

Experiment

All chemicals and solvents were commercially available and used without further purification. Flash column chromatography and thin-layer chromatography (TLC) inspections were performed on Aladdin silica gel (300–400 mesh) and silica gel plates (GF254), respectively. ¹H NMR spectra were recorded on Bruker AV-300 and DRX-500 spectrometers. Electrospray ionization mass spectra (ESI-MS) were obtained on Agilent 6540 UHD Accurate-Mass Q-TOF ESI-MS and Finigan MAT SSQ710ESI spectrometers. High-performance liquid chromatography (HPLC) analysis was conducted on a Shimadzu LC-10ATvp/10ADvp.

Preparation of ethyl 4-(5-(benzyl(2-hydroxyethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate (2)

A mixture of ethyl 4-(5-amino-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate (compound 1) (5.0 g, 19.1 mmol), benzaldehyde (2.4 g, 22.6 mmol) and 20 drops of acetic acid in 150 mL of methanol was stirred at room temperature for 0.5 h. Upon addition of NaBH₃CN (1.5 g, 23.9 mmol), the above reaction further continued for 1.5 h. The reaction mixture was poured into 100 mL of water, and extracted with dichloromethane. The combined organic extracts were washed with saturated brine and dried over anhydrous Na₂SO₄. After evaporation of solvent under reduced pressure, the resultant oily liquid was dissolved into a mixture of water (60 mL) and acetic acid (60 mL), and cooled to 0°C. 11.5 mL of oxirane was added dropwise into the solution, and the resultant solution was gradually warmed to room temperature and stirred overnight. The reaction pH was adjusted to 8–9 and extracted using dichloromehane. The organic extracts were combined, washed with saturated brine and dried over anhydrous Na₂SO_4. Compound 2 was obtained as off-white solid (6.5 g, 87% yield in two steps); ¹H NMR (300 MHz, CDCl3) δ 7.22 (m, 6H), 7.08 (s, 1H), 7.11 (s, 1H), 6.89 (m, 1H), 4.55 (s, 2H), 4.11 (m, 2H), 3.79 (t, 2H, J = 5.52 Hz), 3.67 (s, 3H), 3.54 (t, 3H, J = 5.61 Hz), 2.89 (t, 2H, J = 7.59 Hz), 2.47 (t, 2H, J = 6.93 Hz), 2.17 (m, 3H),1.24 (t, 3H, J = 7.14 Hz); MS ESI m/z calcd for C₂₃H₃₀N₃O₃⁺ [M + H]⁺: 396.2; found 396.2.

Preparation of 4-(5-(benzyl(2-hydroxyethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid (3)

Compound 2 (7.0 g, 17.7 mmol) was added into 60 mL of NaOH solution (10 wt%), and stirred at room temperature overnight. The resultant clear solution was concentrated to 15 mL under the reduced pressure followed by adjusting pH to 5–6 with acetic acid. The precipitates were filtered by suction and dried at 50°C under vacuum. Compound 3 was obtained as white solid (4.3 g, 66%); 1H NMR (300 MHz, dimethyl sulfoxide (DMSO)) δ 7.25 (m, 6H), 6.81 (s, 1H), 6.73 (d, 1H, J = 8.76 Hz), 4.61 (m, 4H), 3.50 (d, 2H, J = 5.82 Hz), 4.58 (s, 2H), 2.78 (t, 2H, J = 7.23 Hz), 2.33 (t, 2H, J = 7.05 Hz), 1.94 (m, 2H); MS (ESI) m/z calcd for C₂₁H₂₆N₃O₃⁺ [M + H]⁺: 368.2; found 368.2.

Preparation of benzyl 4-(5-(benzyl(2-hydroxyethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate (4)

Compound 3 (1.8 g, 4.9 mmol) was suspended in 15 mL of benzyl alcohol at room temperature. After slow addition of 25 drops of concentrated sulfuric acid (98 wt%), the reaction was heated to 80°C and stirred for 2 h. After cooled to room temperature, the reaction was neutralized with NaOH solution (10 wt%) and extracted with ethyl acetate. The combined extracts were washed with saturated brine and dried over anhydrous Na₂SO₄. After solvent evaporation under the reduced pressure, compound 4 was isolated as white solid (1.5 g, 48.7%) by flash chromatography (CH₂Cl₂/CH₃OH = 50/1, v/v); ¹ H NMR (500 MHz, CDCl3) δ 7.37 (m, 5H), 7.28 (m, 2H), 7.23 (m, 4H), 7.12 (m, 1H), 6.87 (m, 1H), 5.31 (s, 1H), 5.11 (s, 2H), 4.59 (s, 2H), 4.08 (brs, 2H), 3.82 (t, 2H, J = 5.7Hz), 3.65 (s, 3H), 3.59 (m, 2H, J = 5.7 Hz), 2.96 (t, 2H, J = 7.65 Hz), 2.55 (t, 2H, J = 6.75 Hz), 2.17 (m, 2H); MS (ESI) m/z calcd for C₂₈H₃₂N₃O₃⁺ [M + H]⁺: 458.2; found 458.2.

Preparation of ethyl 4-(5-(dibenzylamino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate (5)

A mixture of compound 1 (2.0 g, 7.7 mmol), benzyl bromide (4.6 g, 26.9 mmol), N,N-diisoproylethylamine (2.6 g, 20.1 mmol) in 100 mL of acetonitrile was stirred at room temperature for 24 h. The solvent was evaporated to dryness under vacuum, and the residue was subjected to flash chromatography (CH₂Cl₂/CH₃OH = 80/1, v/v) to give compound 5 (2.8 g, 82.9%); ¹H NMR (300 MHz, CDCl3) δ 7.24 (m, 10H), 7.08 (s, 1H), 7.06 (s, 1H), 6.81 (m, 1H), 4.65 (s, 4H), 4.11 (m, 3H), 3.65 (s, 3H), 2.87 (t, 2H, J = 7.56 Hz), 2.46 (t, 2H, J = 6.96 Hz), 2.16 (m, 2H), 1.23 (t, 3H, J = 7.11 Hz); MS (ESI) m/z calcd for C₂₈H₃₂N₃O₂⁺ [M + H]⁺: 442.2; found 442.2.

Preparation of 4-(5-(dibenzylamino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid (6)

Compound 5 (2.2 g, 4.5 mmol) was dissolved into 30 mL of methanol, and the reaction was heated to 80°C for 2 h after addition of 30 mL of 10 wt% NaOH solution. After cooled to room temperature, the white solid was precipitated from the solution when the pH was adjusted to 5–6 with acetic acid. The white precipitates were collected by suction and dried under vacuum at 50°C to give compound 6 (1.8 g, 87.8%); ¹H NMR (300 MHz, CDCl3) δ 7.26 (m, 10H), 7.07 (m, 1H), 6.82 (m, 1H), 4.63 (s, 4H), 3.61 (s, 3H), 2.96 (t, 2H, J = 6.91 Hz), 2.44 (t, 2H, J = 6.27 Hz), 2.11 (t, 2H, J = 6.33 Hz); MS (ESI) m/z calcd for C₂₆H₂₈N₃O₂⁺ [M + H]⁺: 414.2; found 414.2.

Preparation of benzyl 4-(5-(benzyl(2-((4-(5-(dibenzylamino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoyl)oxy)ethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate (7)

A mixture of compound 4 (1.5 g, 3.3 mmol), compound 6 (2.0 g, 4.8 mmol) and 4-dimethylaminopyrdine (DMAP) (4.0 g, 32.7 mmol) was stirred in 150 mL of dichloromethane at room temperature for 0.5 h followed by addition of dicyclohexyl-carbodiimide (DCC) (1.4 g, 6.8 mmol). The resultant clear solution was further stirred overnight. After removal of the white precipitate by filtration, the filtrate was evaporated to dryness under reduced pressure, and the residue was purified by flash chromatography to afford compound 7 (1.8 g, 64.5%); ¹H NMR (300 MHz, CDCl3) δ 7.24–7.35 (m, 20H), 7.05–7.10 (m, 4H), 6.82 (d, 2H, J = 8.8 Hz), 5.11 (s, 2H), 4.66 (s, 4H), 4.58 (s, 2H), 4.30 (s, 2H), 3.69 (s, 2H), 3.55 (d, 6H, J = 17.7 Hz), 2.82 (dt, 4H, J = 27.6, 7.35 Hz), 2.53 (t, 2H, J = 6.5 Hz), 2.41 (t, 2H, J = 6.5 Hz), 2.08–2.18 (m, 4H); MS (ESI) m/z calcd for C₅₄H₅₇N₆O₄⁺ [M + H]⁺: 853.4; found 853.4.

Preparation of BM deschloro dimer

Compound 7 (1.8 g, 2.1 mmol) was dissolved into the mixture of water (50 mL) and acetic acid (50 mL). Upon addition of 5 wt% Pd/C (0.36 g), the reaction was subjected to hydrogenolysis (1 atm H₂, 25°C) overnight. The Pd/C catalyst was removed by filtration, and the filtrate was cooled to 5°C in an ice water bath. 10.0 mL of oxirane was then added into the solution, and the reaction was naturally warmed to room temperature and reacted overnight. BM deschloro dimer was afforded by preparative HPLC (1.0 g, 78% yield in two steps); ¹H NMR (300 MHz, D₂O) δ 7.18 (s, 1H), 7.12 (s, 1H), 6.91 (t, 2H, J = 10.0 Hz), 6.86 (d, 2H, J = 10.0 Hz), 4.22- (d, 2H, J = 5.0 Hz), 3.8–3.64 (m, 6H), 3.58 (t, 2H, J = 5.0 Hz), 3.50–3.38 (6H, m), 3.31 (3H, s), 3.29 (3H, s), 2.53 (2H, t, J = 8.0 Hz), 2.40 (2H, t, J = 6.5 Hz), 2.36 (2H, t, J = 8.0 Hz), 2.17 (2H, t, J = 7.5 Hz), 1.90–1.72 (4H, m); MS (ESI) m/z calcd for C₃₂H₄₅N₆O₇⁺ [M + H]⁺: 625.3; found 625.3

Supplemental Material

Supporting_Information_7 – Supplemental material for Synthesis of a potential bendamustine deschloro dimer impurity

Supplemental material, Supporting_Information_7 for Synthesis of a potential bendamustine deschloro dimer impurity by Jie Yuan and Hai-Bin Zhu in Journal of Chemical Research