`
`my
`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
`
`Instimt fiir Pharmazie, Freie Universitit Berlin, K6nigin—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—ethy1)amino-3—1nethy1benzimidazoyl(2))butyric acid (bendamustin), an anti-
`neoplastic agent of the N-lost type (synthesized or isolated from crude bendamustin): 4-(6-((2-
`chloroethy1)(2-hydroxyethy1)amino)-3-methy1benzirnidazoy1(2))butyric acid (HP1), 4~(6gbis(2-
`hydroxyethy1)amino-3-methylbenzimidazoyl(2))butyric acid (HP2), ethyl-4-(6-bis(2-hydroxyethyl)
`amino-3-methylhenzimidazoy](2))butyrate (dihydroxyester), and ethyl-4-(6—bis(2-chloroethyl)
`amino-3-methylbenzimidazoyl(2))buty1ate (dichloroester). Furthermore, the so far unidentified side
`product 4—(7,8-dihydr0-6-(2-chloroethy]amino)—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 zur Stabilitiit von Bendamustin, einem Cytostatikum vom N-Lost-Typ, 1.
`Mitt: Synthese, Isolierung und Charaklerisierung von Vergleichssubstanzen
`
`Zusammcnfassung. Die folgendcn Verbindungen wurdcn als Verglcichssubstanzen fiir HPLC-
`analylische Untersuchungen von 4-(6-Bis(2-chlorcthyl)amino-3-methy1benzimidazoy1(2))butters§ure
`(Bendamustin), einem Antitumormittel des N-lost-Typs, synthetisiert oder aus Bendamustin-
`Rohstoff vor der Endreinigung isolicrt: (4-(6-((2-Chlorethyl)(2-hydroxyethyl)amino)—3-rnethylben—
`zimidazoyl(2))buttersiiure (I-IP1), 4-(6-Bis-(2-hydroxycthyl)amin0-3-methylbenzimidazoyl(2))butter-
`siiure (I-IP2), 4-(6-Bis(2-hydroxycthyl)am.ino-3-methylbenzimidazoyl(2))buttersiiureethylester
`(Dichlorester). Weiterhin konnte das bislang unbekannte Nebenprodukt 4—(7,8—Dihydro-6-(2-
`chlorethylamino)-3—methy1-1,4-thiazino[3,2-g]benzimidazoyl(2))buttersiiure (NPI), welches sich
`im letzten Schritt der Synthcse 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
`
`AGILA ET AL - EXHIBIT 1008
`
`AGILA ET AL - EXHIBIT 1008
`
`
`
`292
`
`R. Gust and R. Krauser
`
`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-chloroethyl)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 axyl 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 viva against several murine tumors [7—9]. In clinical
`tests,
`the
`cancerostatic effectiveness was confirmed for mammary carcinoma, lymphoma,
`and especially plasmocytoma [l0—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:
`tgo = 120h, 23°C:
`tgo = 9h; 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 Ozegowski et al. [14] in
`an eleven step sequence starting from 2,4-dinitrochlorobenzene. The crucial con-
`versions (Scheme 1) are the chlorination of ethyl 4-(6—bis(2-hydroxyethy1amino)-
`3-methylbenzimidazoyl(2))butyrate (dihydroxyester) with SOC12 affording ethyl
`4-(6-bis(2-chloroethyl)amino-3-methylbenzimidazoy1(2))butyrate (dicliloroester)
`and the subsequent ester cleavage with HCl to obtain 4~(6-bis(2-chloroethy1)—
`amino-3-methylbenzimidazoyl(2))butyric acid (bendamustin). Under the reaction
`conditions employed, bendamustin hydrolyzes, in small amounts to the hydroxy-
`chloro (HP1) 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.
`
`
`
`Investigations on Bendamustin
`
`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 esterification in ethanolic HCI. HP2 was obtained by quantitative hydrolysis of
`bendamustin in water as described by Werner et al. [15]. Since it was impossible to
`isolate I-[P1 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® (NPI, [13]).
`
`Characterization of bendamustin and its derivatives
`
`Bendamustin and its derivatives were characterized by their elemental analyses,
`NMR, 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/D. 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 C1-C1-I2—CI-12- side chains allow an unequivocal
`assignment of bendamustin and its derivatives using the signals of CHM‘: and
`CH5“) which are shifted about 0.10-0.15 ppm to lower field in the spectra of
`
`
`
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`Table 2. ‘H NMR data of bendamuslin and its derivatives (250 MHz, met.hanol—d4, TMS)
`
`E3
`
`
`
`n1_cH$_cHzé\~ 111:
`112-cré-cHf,,’
`11°
`
`\>—c1-13-c1-12-cH‘a'-cou1=13
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`Dichlorocstet
`
`C1
`
`C1
`
`E1
`
`3.70 (1, 31 = s.3Hz,CH°“°)
`3.34 (1,31= 6.0 Hz, CH’-/C)
`
`Bendamustin
`
`c1
`
`c1
`
`H 3.75 (1, 31 = s.9H2,cI1BID)
`3.87 (1,31 = 5.7112, CI-WC)
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`5
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`S63
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`3.73 (s)
`
`1.20 (1. 31 = ‘7.1!-[z,CH;)
`4.04 (q, 31 —. 7.1 Hz, C11,)
`
`3.93 (3)
`
`1.16 (1, 31 = 7.1 Hz, CH2)
`4.00 (q, 31 2 7.1 Hz, CH,)
`
`3.96 (1)
`
`CH‘/CHBICHC/CH”
`CH“/CH” /CI-I’
`H‘ /H"/H‘
`N—CH3
`-—CH2 — CH3
`R‘ R’ R’ (pm)
`(mm)
`(mm)
`(ppm)
`(ppm)
`
`Dihydmxyestet OH 011
`B1
`3.51 (1,141 = 6.2Hz,CHBm)
`2.09 (q-4111,31 = 7.4112, CH")
`6.97 (d,3J = 2.0112, 11=)
`3.72 (1,31 = 5.7 Hz,CH"/C)
`2.45 (1,31 = 6.91-lz,C[-l")
`6.88,6.91 (dd, 31 .—. 2.4 Hz, 8.91-12,1-I")
`2.92 (1,31 = 7.8 Hz,CI-I‘)
`7.26 (11.31 = 3.7 112,11‘)
`2.11 (qu1n,31 = 7.0112, CH5)
`6.93 (1131 = 2.4112,:-1°)
`2.51 (1.31: 6.9 Hz,CI-IV)
`7.1o,7.12 (1111, 31= 2.4Hz,9.4H2, H”)
`3.18 (1, 31 = 3.0112, CH“)
`7.65 (1131 .— 9.4 Hz,l-I")
`2.14 (qu1n,31 —. 6.9 Hz,cH¥’)
`6.94 (11,511 .—. 2.3 Hz, 11°)
`2.53 (1, 31 = 6.9112, cm)
`7.13, 7.16 (dd, 31 .— 2.3 Hz, 9.2 Hz, 11“) 3.97 (s)
`3.22 (1.31 -. 8.0 Hz, CH“)
`7.67 (11, 31 = 9.2 Hz. H“)
`2.15 (br, CH1‘)
`6.98 (11.31 = 1.7 Hz, 11°)
`2.53 (1, 31 = 6.5 Hz, cm)
`7.15, 7.13 (1111, 31 = 1.7 1-12, 9.2 Hz, 11") 3.97 (s)
`3.22 (1, 31 — 7.6 Hz, CH")
`7.64 (11, 31 = 9.2 Hz, 11')
`2.12 (quin,31 = 7.1 Hz, C11“)
`6.90 (11.31 = 2.2 Hz, H‘)
`2.51 (1, 31 = 6.7Hz, CH?)
`7.11. 7.15 (11131 = 2.31-12, 9.3 Hz, 1-1”) 3.94 (s)
`3.19 (1, 31 = 8.0Hz, CH“)
`757 (d,3J = 9.3 Hz, H‘)
`2.09 (quin,31 = 7.0112, CH5)
`7.12 (31 = 9.2 Hz, H”)
`3.17-3.23 (m, 211)
`2.51 (1. 31 = 6.6 Hz. cm)
`7.40 (31 -_- 9.2 Hz, 11*)
`3.78-3.87 (m, 6H)
`320 (131 = 7.1112, CH“)::_
`
`011 H 3.65 (1,31 = 5.81-Iz, CH”)
`3.86 (1, 31 = 6.0 Hz, CH‘)
`3.73- 3.76 (m, 411. CH3/C)
`3.62 (1.31 = 5.9 Hz, CHBID)
`3.76 (1,31 = 5.9 Hz, CH“/C)
`
`OH OH 11
`
`HP1
`
`Cl
`
`1192
`
`NP1
`
`
`
`296
`
`R. Gust and R. Krauser
`
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`Fig. 1. “C NMR spectra (CPD, DEPT) of bendamustin (A, B) and NPI (C. D): methanol-d4, TMS
`
`bendarnustin and its dichloroester. The spectrum of HP1 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 CHC and CH”
`are high field shifted as it was found for HP2. Together with the results from
`
`
`
`5 §W
`
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`N
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`g‘C
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`L63
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`Table 3. “C NMR data‘ of bendamusljn and NPI (62.9 MHz. methanol—d4, ppm, TMS)
`
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`C11
`
`
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`C1
`
`C2
`
`C3
`
`C4
`
`C5
`
`C6
`
`C7
`
`C8
`
`C9
`
`C10
`
`C12
`
`C13
`
`C14
`
`C15
`
`C16
`
`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
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`NP1
`153.1
`103.5
`142.8
`114.7
`109.8
`126.6
`130.0
`25.0
`51.5
`42.0
`56.2
`31.6
`25.5
`23.5
`33.4
`175.9
`
`
`
`
`298
`
`R. Gust and R. Krauser
`
`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 HP1 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. 1).
`The assignment of the peaks were performed via a DEPT experiment which
`allows the distinction of signal multiplicities [16]. Fig. 1A shows the decoupled “C
`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 NP1 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 C1-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 DEPT 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 1-12 (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:
`Gradientenformer Labomat VS200, MPLC pump MD-80/100, Labocol Roto-UltraIl20, 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 analog: by MPLC
`
`Crude bendamustin (150 mg) was dissolved in 2 ml of a mixture of methanol/l N HCl (1:1), loaded
`onto the MPLC column, and eluted with a mixture of methanol/water (1:1) which was adjusted to
`pH = 3 with 1 N HCl. From the separated fractions, the methanol was removed in vdcuo and the
`resulting aqueous solutions were freeze dried. The derivatives eluted from the column in the foll-
`owing series: HP2, I-[P1, NP1, bendamustin, and dichloroester.
`
`Syntheses
`
`._._
`
`The syntheses of bendamustin and its hydrolysis product HP2 were carried out according to 0ze-
`gowski et al. [4] and Werner et al. [15].
`
`
`
`Investigations on Bendarnustin
`
`299
`
`4-(6-Bis(2—chloroethyl)amino-3—methylbenzimidazoyl(2))erhyl butyrate (dichlomester)
`
`Bendamustin (1 g, 2.48 mmol) was dissolved in 20 ml of ethanol 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. Komau is gratefully acknowledged. R.G. thanks Werner
`Roth for helpful discussions and critical reading of the manuscript.
`
`References
`
`[1] Kiihnau J (1956) Arch Exp Path Pharrnaeol 228: 87
`[2] Knobloch W (1958) Chem Ber 91: 2557
`[3] I-Iirschberg W. Gellhorn A, Gurnp WS (1957) Cancer Res 17: 904
`[4] Ozegowski W, Krebs D (1971) Zbl Pharm 110: 1013
`[S] Rintelen K, Knobloch W (1958) Acta Biol Med German 1: 109
`[6] Ultmann JE, Thompson HG, I-Iirschberg E, Zeidenweber J, Gellhom A (1959) Cancer Res 19:
`719
`
`[7] Anger G, Hesse P, Kfihler P, Baufeld H (1967) Dtsch Gesundheitsw 22: 1099
`[8] Jungstand W, Gutsche W (1967) Monatsber Dtsch Akad W158 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
`[ll] Brockmann B, Geschke E, Schmidt UM, Ebeling K (1991) Geburtsh u Frauenheilk 51: 383
`[12] Herold M, Anger G, Htiche D, Kiistner D (1987) Med Klin 82: 345
`[13] Maas B. Huber C, Kréimer I (1994) Pharmazie 49: 775
`[14] Ozegowski W, Krebs D (1963) J Prakt Chem IV, 20: 178
`[15] Werner W, Letsch G, Ihn W (1987) Pharmazie 42: 272
`[16] Doddrell DM, Pegg DT, Bendall MR (1982) J Magn Reson 48: 323
`[17] Kalinowski HO, Berger S, Braun S (1984) 13C—NMR-Spektroskopie. Thieme, Stuttgart
`
`Received August 26, 1996. Accepted (revised) October 21, J 996.