Monatsheftc fiir Chemic 128, 291-299 (1997)
`
`ii’
`Chemical Monthly
`@ Springer-Verlag 1997
`Primed in Austria
`
`Investigations on the Stability of Bendamustin,
`a Cytostatic Agent of the Nitrogen Mustard
`Type, 1. Synthesis, Isolation, and
`Characterization of Reference Substances
`
`R. Gust* and R. Krauser
`
`lnstitut fiir Pharmazie, Freie Universiuit Berlin, Kfinigin-Luise-StraBe 2 +4, D-14195 Berlin,
`Germany
`
`Summary. The following compounds were chosen as reference substances for HPLC investigations
`on 4-(6-bis(2-chloro-ethyl)an1ino-3-methylbenzimidazoyl(2))butyric acid (bendamustin). an anti-
`neoplastic agent of the N-lost type (synthesized or isolated from crude bendamustin): 4-(6-((2-
`chloroethyl)(2-hydroxyethyl)amin0)-3-mcthylbenzimidaz0yi(2))butyric acid (HP1), 4-(t'>bis(2-
`hydroxyethyl)amino-3-meLhylbenzimidazoyl(2))butyiic acid (HP2), ethyl-4-(6-bis(2-hydmxyethyl)
`amino-3-melhyibenzimidazoy1(2))butyrate (dihydroxyester). and ethyl-4—(6-bis(2-chloroethyl)
`amino-3-methylbenzimidaz0yi(2))butyrate (dichloroester). Rirthermore, the so far unidentified side
`product 4-(7,8-dihydro-6-(2-chlorocthylarnino)-3-rnethy1-1,4-thiazino[3.2-g]benzimidazoy1(2))-
`butyric acid (NPI), formed in the last step of the synthesis, was isolated and identified.
`
`Keywords. Bendamustin; Antineoplastic; Hydrolysis products: Reference substances: Spectro-
`
`scopic characterization.
`
`Untersuchungen iur Stabilitiit von Bendamustin, einem Cytoslatikum vom N-Lost-Typ, 1.
`Mitt: Synthese, Isolierung und Charakterisierung von Vergleichssubstanzen
`
`Zusammcnfassung. Die folgendcn Verbindungen wurdcn als Vergleichssubstanzen fiir HPLC-
`analytische Untersuchungen Von 4-(6-Bi.s'(2—chlorcthyl)arnino-3-methylbenzimidazoy1(2))bntters5ure
`(Bendamustin), einem Antitumormittel des N-lost-Typs, synthetisiert oder ans Bendamustim
`Rohstoff vor der Endreinigung isolicrt: (4-(6-((2-Chlorethyl)(2-hydroxyethy1)amino)-3-methylben-
`2.imidazoyl(2))buttersiiure (HP1), 4-(6-Bis-(2-hydroxycthyl)ami.no-3-methylbenzimidazoyl(2))butter—
`siiure (HP2), 4-(6-Bis(2-hydroxyetl1yl)amino-3-mcthylbenzimidazoyl(2))butters5iureethylester
`(Dichlorester). Weiterhin konnte das bislang unbekannte Nebenprodukt 4-(7,8-Dihydro-6-(2-
`chlorethylamino)-3-methyl-1,4-thiazino[3,2-g]benzimidazoyl(2))buttersiiu.re (NP1), welches sich
`im letzten Schritt der Synthesc bildet. isoliert und identifiziert werden.
`
`Introduction
`
`Substituted benzimidazoles are potent antagonists of amino acids and purines
`[1]. Depending on the substitution pattern, they inhibit the synthesis of proteins
`
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`and enzymes as well as the synthesis of nucleotides. Since tumor tissue needs
`high amounts of amino acids, benzimidazoles are suitable as antitumor agents.
`An enhancement of the tumor inhibiting properties can be achieved by combi-
`nation with the cytotoxic N-lost moiety [2—6]. Decisive for antitumor as
`well as toxic side effects of these compounds is the basicity of the N-lost
`group.
`For derivatives of the 2-(bis(2-chloroethy1)aminomethyl)benzimidazole type, in
`which the CH2 group prevents the influence of the heteroaromatic ring system on
`the basicity, strong toxic side effects have been predicted [4]. Therefore, for
`optimization of the pharmacological effects, the N-lost moiety was introduced in
`the 6—position of the benzimidazole ring. Additionally, position 3 was substituted
`with alkyl or aryl groups and position 2 with hydrophilic residues, e.g. aliphatic
`carboxylic acids. Among these compounds, bendamustin was found to be the most
`active one in vivo against several murine tumors [7-9]. In clinical
`tests,
`the
`cancerostatic effectiveness was confirmed for mammary carcinoma, lymphoma,
`and especially plasmocytoma [10—12]. Today, bendamustin (Ribomustin®) is a
`widely used chemotherapeutic agent, either alone or — more often - in combination
`with other antineoplastics,
`in the treatment of hematologic diseases and
`metastasized breast cancer.
`
`Bendamustin is administered iv using a 0.9% NaCl solution. However, it
`must be considered that bendamustin hydrolyzes in water similar to other
`N-lost compounds. Recently, Maas et al. [13] have reported about the stability
`of the market drug product in aqueous NaCl solution (0.25 mg/ml, 0.9% NaCl
`solution; 4°C:
`:99 = 12%, 23°C:
`tgo = 9b; determined by the decrease of the
`bendarnistin peak in HPLC).
`In addition to the characteristic bendamustin
`peak,
`the chromatograms exhibited further peaks which were empirically
`assigned, since no crystalline reference substances were available. In this paper
`we describe the synthesis or isolation as well as the characterization of the
`most
`important reference substances for the HPLC investigations of benda-
`mustin.
`‘
`
`Results and Discussion
`
`Synthesis or isolation of bendamustin derivatives
`
`The first synthesis of bendamustin has been performed by Ozegowslci et al. [14] in
`an eleven step sequence starting from 2,4—dinitroch1orobenzene. The crucial con-
`versions (Scheme l) are the chlorination of ethyl 4-(6-bis(2—hydroxyethylamino)-
`3-methylbenzimidazoyl(2))butyrate (dihydroxyester) with SOCI2 affording ethyl
`4-(6—bis(2-chloroethyl)amino-3-methylbenzimidazoyl(2))butyrate (dicliloroester)
`and the subsequent ester cleavage with HCl
`to obtain 4—(6-bis(2-chloroethyl)-
`amino-3-methy1benzimidazoy1(2))butyric acid (bendamustin). Under the reaction
`conditions employed, bendamustin hydrolyzes_ in small amounts to the hydroxy-
`chloro (HPI) and the dihydroxy derivative (HP2). For_the HPLC analytical in-
`vestigation of the drug substance and the market drug product Ribomustin®, the
`dihydroxyester, the dichloroester, and both hydrolysis products were chosen as
`suitable reference substances.
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`Investigations on Bendamustin
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`293
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`Whereas the dihydroxyester and bendamustin were made available by courtesy
`of the Ribosepharm company, we synthesized the dichloroester from bendamustin
`by esteiification in ethanolic HC1. HP2 was obtained by quantitative hydrolysis of
`bendamustin in water as described by Werner et al. [15]. Since it was impossible to
`isolate HPI by fractional crystallization from an aqueous solution of bendamustin,
`MPLC on RP 18 was used for the separation. In this connection it was also possible
`to isolate the impurity detected by Maas er al. in Ribomustin® (NP1, [13]).
`
`Characterization of bendamustin and its derivatives
`
`Bendamustin and its derivatives were characterized by their elemental analyses,
`NlVIR, and mass spectra (Tables 1 and 2).
`The ‘H NMR spectra exhibit a six spin system of the form AA’BB’CC’ for the
`butyric acid moieties with signals at 6 = 2.92—3.22(CH°), 6 = 2.09-2.15 (CH5),
`and 6 = 2.45—2.53(CH7). The spin systems were approximatively interpreted
`following first order rules (see Table 2). It must be mentioned that - with exception
`of the dihydroxyester — the compounds were measured as their hydrochlorides. The
`positive charge leads to a low field shift for CH3, H“, H“, and CH“. Characteristic
`for bendamustin and its derivatives are the signals of the methylene groups CH‘/C
`and CH3”). These protons afford an AA’BB’ system consisting of 12 badly
`~ resolved lines which again was interpreted using first order rules.
`The H0—CH2-CH2 and Cl—CH2—CH2— side chains allow an unequivocal
`assignment of bendamustin and its derivatives using the signals of Cl-IA/C and
`CH3") which are shifted about 0.10-0.15 ppm to lower field in the spectra of
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`R. Gust and R. Krauser
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`Table 2. ‘H NMR data of bendamuslin and its derivatives (250 MHZ, methanol-d4. TMS)
`
`N
`
`"c
`
`Dihydmxyester
`
`R‘ R3 R’
`01-1
`011
`B1
`
`Dichlorocstcr
`
`c1
`
`c1
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`121
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`Bendnmustin
`
`c1
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`Cl
`
`11
`
`3.70 (1, 31 = 5.s11z,cI13f°)
`3.84 (1, 31= 6.0 Hz, CH‘/C)
`
`3.75 (1.31 = 5.91-1z, C113/D)
`3.37 (1. 31 = 5.711z,c11*/C)
`
`11131
`
`c1
`
`11132
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`3
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`011
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`N121
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`n‘-ca;-cH"2\
`\>—t:H§-c11§-cu;-coon“
`R2-1:112-cflg’
`A
`11:
`H’
`H3
`
`Cl-I‘/CH3/CH“/CH“
`CI-1°‘/CH“ /CH*
`H‘ /H“/H‘
`N»-CH3
`-—CH; — CH3
`(ppm)
`(ppm)
`(ppm)
`(ppm)
`(ppm)
`3.51 (1.31 = 6.2Hz.,CH”"D)
`2.09 (q1u'n.31 = 7.4112, CH4’)
`6.97 (11.31 = 2.011z, 11°)
`3.72 (1.31 = 5.7 Hz,CI-I"/C)
`2.45 (1.31 = 6.91-lz,CI-17)
`6.88, 6.91 (dd. 31 = 2.4 11z,s.9 Hz, H3)
`2.92 (1, 31 = 7.8 Hz,CI-I“)
`7.26 (11. 31 .—. 8.7 Hz,I-I‘)
`2.11 (qu1n,31 = 7.o11z,c113)
`6.93 (11, 31 = 2.411z, H‘)
`2.51 (1. 31: 6.9 I-Iz,CI-IV)
`7.10, 7.12 (dd. 31= 2.4 Hz, 9.4 Hz, H”)
`3.13 (1, 31 = 3.0 Hz,CH“)
`7.65 (11.31 .— 9.4 Hz,[-I‘)
`2.14 (quin, 31 —. 6.9 Hz, CH?)
`6.94 (d, 31 .—. 2.3 Hz, 11°)
`253 (1.31 = 6.9 Hz. CH7)
`7.13, 7.16 (dc1,31 .— 2.3 Hz, 9.2 Hz,113) 3.97 (s)
`3.22 (1, 31 —. s.011z, CH“)
`7.67 (11, 31 = 9.2 Hz, H‘)
`2.15 (br, CH3)
`6.98 (11.31 = 1.711z,11=)
`2.53 (1, 31 = 6.511z, cm)
`7.15. 7.13 (dd, 31 = 1.7 Hz, 9.2Hz, 11") 3.97 (s)
`3.22 (1, 31 — 7.6!-Iz, CH")
`7.64 (11, 31 = 9.211z, 11-)
`2.12 (qui11,31 = 7.1 Hz, CH3)
`6.90 (11.31 = 2.2 Hz, 11‘)
`2.51 (1, 31 = 6.7Hz, cm)
`7.11. 7.15 (1111,31 = 2.3 Hz, 9.3 Hz. 1-13) 3.94 (1)
`3.19 (1, 31 = 3.o11z, CH“)
`7.57 (11, 31 = 9.3 Hz, 11*)
`2.09 (quin,31 = 7.01-1z. CH3)
`7.12 (31 = 9.2 Hz, 113)
`3.17 —3.23 (111, 211)
`2.51 (1. 31 = 6.6 Hz, CH7)
`7.40 (31 = 9.2Hz, 11*‘)
`3.78-3.87 (111, 611)
`3.20 (1,31 =7.1 Hz, CH“)
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`1.20 (1. 31 = 7.1 Hz,CH2)
`4.04 (q, 31 —. 7.1 Hz, cH,)
`
`3.93 (s)
`
`1.16 (1, 31 = 7.1Hz,CH2)
`4.00 (q,31 ~.= 7.111z,cH,)
`
`3.96 (1)
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`FRESENIUS KABI 1006-OOO5
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`11
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`011
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`3.65 (1.31 = s.s11z,c11°)
`3.86 (1.31 = 6.0 Hz. CH‘)
`3.73-3.76 (111. 411. C1-13/C)
`01-1 H 3.62 (1.31 —. 5.9112, C11“/'3)
`3.76 (1.31 = 5.9112. C113/C)
`
`

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`
`R. Gust and R. Krauser
`
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`Fig. 1. “C NMR spectra (CPD, DEPT) of bendamustin (A. B) and NP! (C. D); methanol-d4. TMS
`
`bendamustin and its dichloroester. The spectrum of HPI exhibits the signals of
`both the HO—CH2-CH2-N and the C1-CH2-CH2—N group: the chemical shifts of
`CH‘ and CH3 are unchanged compared with bendamustin, whereas CH‘: and CH”
`are high field shifted as it was found for HP2. Together with the results from
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`name 3. “C NMR data“ of bcndamustin and NP1 (62.9 MHz. methanol-cl... ppm, ms)
`
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`C16
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`175.8
`33.5
`23.0
`25.6
`31.7
`54.7
`41.6
`133.3
`126.5
`114.5
`114.4
`148.0
`96.1
`152.9
`Bcndamustin
`
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`
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`
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`
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`142.3 114.7 109.8 126.6 130.0 25.0 51.5 42.0 56.2 31.6 25.5 23.5 33.4103.5153.1NP]. 175.9
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`R. Gust and R. Krauser
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`elemental analyses (Table 1) and mass spectrometry, the chemical structures of
`bendamustin,
`its dihydroxyester, and its dichloroester as well as those of the
`hydrolysis products HP] and HP2 were confirmed.
`The side product NP] is formed in the last step of the synthesis (ester cleavage
`with HCl). The isolation from the crude product was achieved using MPLC. For
`the structural analysis, the 13C NMR spectra of bendamustin and NP1 were used
`(Fig. l).
`The assignment of the peaks were performed via a DEPT experiment which
`allows the distinction of signal multiplicities [16]. Fig. 1A shows the decoupled 13C
`NMR spectrum of bendamustin together with the corresponding DEPT experiment.
`Together with an estimation of chemical shifts using an increment system [17], all
`signals of bendamustin could be assigned (Table 3).
`In the “C spectrum of NP] the signals of C10 to C16 exhibit shift values
`comparable to those of bendamustin;
`therefore, an intact butyric acid moiety
`as well as the presence of an N-CH3 group and at least one Cl—CH2—CH2—N
`side chain can be assumed. Among the aromatic C atoms, especially the signal
`of C2 is changed. Besides a low field shift of 7.4 ppm,
`the DEPT experi-
`ment indicates its conversion into a quaternary C—atom. Furthermore, the signals
`at 6 = 25.0 and 51.5 can be identified in the DEPI‘ experiment as CH2 groups. The
`resonance positions are very similar to those found in 1,4-thiazine (6 = 28.3 and
`47.9); therefore, cyclization of an N—CH2-CH2 chain via an S bridge to C2 can be
`assumed. This reduces the multiplicity of the aromatic protons in the ‘H NMR
`spectrum to an AB system with a coupling constant of 9.2 Hz (see Table 2). The
`proposed structure is also in accordance with the elemental analysis and the results
`of mass spectrometry (Table 1).
`
`Experimental
`
`‘H NMR spectra (250 MHZ) and "C NMR spectra (62.9 MHZ): PFI‘ NMR spektrometer WM 250
`(Bruker); mass spectra: Finnigan MAT 95A, PILISI-FAB in a methanol/glycerol matrix; MPLC:
`Gradientenforrner Labomat VS200, MPLC pump MD-80/100, Labocol Roto-Ultral [20, Fa. Kronlab;
`FPGC-ODS4-S-120-S-15/30 MPLC precolumn (26 x 313 mm) und MPLC main column
`(37 x 539 mm).
`
`Separation of bendamustin analogs by MPLC
`
`Crude bendamustin (150 mg) was dissolved in 2 ml of a mixture of methanol/1 N HCl (1:1), loaded
`onto the MPLC column, and eluted with a mixture of methanollwater (1:1) which was adjusted to
`pH = 3 with 1 N l-lCl. From the separated fractions, the methanol was removed in vacuo and the
`resulting aqueous solutions were freeze dried. The derivatives eluted from the column in the foll-
`owing series: HP2. HPI, NP1, bendamustin, and dichloroester.
`
`Syntheses
`
`._._
`
`The syntheses of bendamustin and its hydrolysis product HP2 were carried out according to 029-
`gowslci at al. [4] and Werner et al. [15].
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`Investigations on Bendamustin
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`299
`
`4-(6-Bis(2-chlomethyl)amino-3-methylbenzimidazayl(2))ethyl butyrate (dichlamester)
`
`Bendamustin (1 g, 2.48 mmol) was dissolved in 20 ml of et.hanol and treated with gaseous HCl for 20
`min. After 4h stirring at room temp.. the solvent was evaporated and the crude dichlorester was
`recrystallized from ethanol to give a white powder; yield: 890 mg (85%).
`For analytical data see Tables 1-3.
`
`Acknowledgements
`
`This work was supported by the Fonds der Chemischen Industrie and the Ribosefarm company. The
`technical assistance of S. Bollwein and M. Kamau is gratefully aclmowledged. R.G. thanks Werner
`Roth for helpful discussions and critical reading of the manuscript.
`
`References
`
`[1] Kiihnau J (1956) Arch Exp Path Pharmacol 228: 87
`[2] Knobloch W (1958) Chem Ber 91: 2557
`[3] I-lirschberg W. Gellhom A, Gump WS (1957) Cancer Res 17: 904
`[4] Ozegowski W, Krebs D (1971) Zbl Pharm 110: 1013
`[5] Rintelen K, Knobloch W (1958) Acta Biol Med German 1: 109
`[6] Ultmann IE, Thompson HG, 1-Iirschberg E, Zeidenweber J, Gellhom A (1959) Cancer Res 19:
`719
`
`[7] Anger G, Hesse P, K'o'h1er P, Baufeld H (1967) Dtsch Gesundheitsw 22: 1099
`[8] Jungstand W, Gutsche W (1967) Monatsber Dtsch Akad Wiss 9: 335
`[9] Schnabel R, Jungstand W, Gutsche W, Grimm H, Fritsch S (1967) Acta Biol Med German 19:
`543
`
`[10] Hesse P, Iaschke E, Anger A (1972) Dtsch Gesundheitsw 27: 2058
`[11] Brockmann B, Geschke E, Schmidt UM, Ebeling K (1991) Geburtsh u Frauenheilk 51: 383
`[12] Herold M, Anger G, I-Ifiche D. Kiistner D (1987) Med Klin 82: 345
`[13] Maas B, Huber C, Kramer I (1994) Pharmazie 49: 775
`[14] Ozegowski W, Krebs D (1963) J Prakt Chem IV, 20: 178
`[15] Werner W, Letsch G, l11n W (1987) Pharmazie 42: 272
`[16] Doddrell DM, Pegg DT, Bendall MR (1982) J Magn Reson 48: 323
`[17] Kalinowski l-IO, Berger S, Braun S (1984) “C-NMR-Spelctroskopie. Thicme, Stuttgart
`
`Received August 26, 1996. Accepted (revised) October 21, I996.
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`FRESENIUS KABI 1006-0009