Process for the preparation of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl esters and formylated derivatives

Abstract

A process for preparing 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, in which 1 mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester is reacted with 2.0 to 2.2 moles of chloromethylenedimethyliminium chloride or more than 2.2 to 5 moles of chloromethylenedimethyliminium chloride to prepare a by-product in the form of 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester. The alkyl ester can be hydrolyzed to form 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid, which can be used in the pharmaceutical field.

Description

[Technical field]

[0001] The present invention relates to a process for the preparation of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl esters and their formylated derivatives.

[0002] By ester hydrolysis, 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester can be converted to 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid, which is known as an antitumor agent called "bendamustine."

[0003] EP 2468716(A1) discloses a method for preparing 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester. The reactant used is 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, the hydroxyethyl group of which can be converted to a chloroethyl group by reacting with oxalyl chloride in the presence of dimethylformamide to form 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester. Although the reaction can be carried out stoichiometrically, i.e. with a molar ratio of 2.0 moles of oxalyl chloride:1 mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, EP 2468716(A1) recommends using a stoichiometric excess, preferably corresponding to a molar ratio of 2.6 moles or more, in particular at least 3.0 moles of oxalyl chloride per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester. The in-situ reaction of oxalyl chloride with dimethylformamide used here is difficult to control with respect to the completeness of the reaction or undesirable interfering moisture in the reaction system. According to the teachings of EP 2468716(A1), the preferred and recommended superstoichiometric molar ratios give the desired 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester in good yield, but with an undesirable yellow coloration. This coloration is caused by an as yet unknown impurity and is difficult to remove, even by treatment with activated carbon. The intensity of the yellow coloration increases with the selected molar ratio of oxalyl chloride to 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester.

[0004] The object of the present invention is to provide an improved process for the preparation of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl esters starting from 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl esters.

[0005] This object can be achieved by a process for the preparation of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl esters by reacting 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl esters with chloromethylenedimethyliminium chloride having the formula ClCH=N(CH3)2Cl.

[0006] In a first embodiment, the preparation method according to the present invention can be carried out at a substantially stoichiometric molar ratio of chloromethylenedimethyliminium chloride in the range of 2.0-2.2 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, resulting in good yield of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester with almost no impurities and by-products.

[0007] However, in a second embodiment, the preparation method according to the invention can also be carried out with a superstoichiometric molar ratio of chloromethylenedimethyliminium chloride in the range of more than 2.2, for example 5 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester. However, from the viewpoint of obtaining 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester in high yield, it is not very preferable to select a superstoichiometric molar ratio.

[0008] Preferably, the 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester reacted with chloromethylenedimethyliminium chloride in the process according to the invention is 1-4 Alkyl esters, in particular 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester, in other words a preferred embodiment of the process according to the invention is the reaction of chloromethylenedimethyliminium chloride with 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C 1-4 The reaction is carried out using alkyl esters, in particular 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester. Thus, 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C 1-4 Alkyl esters, in particular 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester, are obtained as products of preferred embodiments of the process according to the invention.

[0009] Chloromethylenedimethyliminium chloride is commercially available, for example from Sigma-Aldrich. It is a solid.

[0010] The synthesis method according to the present invention comprises the steps of: T The reaction can be conveniently carried out in an anhydrous (dry) aprotic organic solvent having a (30) value in the range of 140-193 kJ / mol, or in a mixture of such solvents. Examples of such solvents include chlorinated organic solvents such as dichloromethane and chloroform, ethers such as dioxane and tetrahydrofuran, and acetonitrile.

[0011] The two reactants used in the synthesis method according to the invention, namely chloromethylenedimethyliminium chloride and 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, can be reacted with each other in solution or in suspension, in the latter case one or both reactants can be present in suspension. The two reactants can be added in any order, dissolved, suspended or as solids.

[0012] In the method according to the present invention, for example, it is possible to carry out the reaction at a ratio of 30 to 150 g of reactant (total amount of both reactants) per liter of organic solvent (mixture).

[0013] It is useful to maintain the temperature of the reaction mixture, for example in the range of 0 to 60° C., both during the addition of the reactants and during the reaction.

[0014] For example, the reaction time may be in the range of 30 to 360 minutes. It is preferred to mix, and in particular to stir, the reaction mixture.

[0015] After the reaction is complete, water can be added and a base can be added to adjust the pH to, for example, a value in the range of 7 to 10. Examples of bases that can be used include ammonia, alkali hydroxides, alkali carbonates, and alkali hydrogen carbonates.

[0016] When the reaction is carried out in a water-immiscible solvent, the 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester formed in the process according to the invention is present in the organic phase after the addition of water and pH adjustment. Alternatively, the 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester formed can be converted to a water-immiscible organic phase after the addition of a water-immiscible solvent such as dichloromethane, from which it can be recovered and purified by conventional methods well known to organic chemists. Examples of such methods include crystallization, precipitation using anti-solvents, and preparative chromatography.

[0017] The 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl esters prepared according to the process of the invention can undergo ester hydrolysis to form 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid or its hydrochloride, for example, analogously to the procedure known from EP 2 468 716 A1.

[0018] The process according to the invention has several advantages over the process known from EP 2 468 716 A1. 1. The use of chloromethylenedimethyliminium chloride as a chlorinating agent instead of oxalyl chloride / dimethylformamide is preferable in several respects from the viewpoint of occupational safety. In addition to the advantage of avoiding the handling of the hazardous substances oxalyl chloride and dimethylformamide, the exothermic reaction and gas evolution associated with the reaction of oxalyl chloride with dimethylformamide can be avoided. 2. The use of chloromethylene dimethyl iminium chloride allows for precise dosing with respect to adjustment of the desired stoichiometric or superstoichiometric molar ratio between chloromethylene dimethyl iminium chloride and 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, as well as subsequent dosing of chloromethylene dimethyl iminium chloride if necessary. 3. In particular, side reactions can be avoided or reduced when the process according to the invention is carried out at a substantially stoichiometric molar ratio in the range of 2.0 to 2.2 moles of chloromethylenedimethyliminium chloride per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester. 4. Less base consumption when adjusting the pH value after the reaction is completed.

[0019] As mentioned above, the preparation method according to the invention in the second embodiment of the invention can be carried out with a superstoichiometric molar ratio of chloromethylenedimethyliminium chloride in the range of more than 2.2, for example 5 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester. In the second embodiment of the method according to the invention, for example, 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C 1-4When carried out with a molar ratio of chloromethylenedimethyliminium chloride in the range of 2.6 to 5 moles per mole of alkyl ester, in particular 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester, a yellow coloration similar to that already mentioned at the beginning can be observed in the reaction system, which coloration increases with increasing stoichiometric excess of chloromethylenedimethyliminium chloride. The substance causing the yellow coloration is a process by-product of the process according to the invention in the second embodiment of the invention, 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C 1-4 From the group of alkyl esters, it has been identified or isolated for the first time as a new, hitherto unknown compound. Apparently, when carrying out the method according to the invention in its second embodiment, regioselective formylation at the 4-position of the benzo[d]imidazole skeleton of the actual main process product can occur. The corresponding, hitherto unknown, novel 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester was obtained and characterized after separation from the main process product 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester. Thus, 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester is a process by-product formed when carrying out the method according to the invention in its second embodiment. Due to its structural relationship with 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid, 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester is also expected to have antitumor effects. As a reactive functional group, its aldehyde group can provide a starting point for a variety of intramolecular and intermolecular derivatization reactions, and therefore can also provide a starting point for the preparation of antitumor compounds, such as the formation of antibody conjugates.

[0020] In a second embodiment of the present invention, 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl esters are provided, preferably 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C 1-4The molar ratios selected when carrying out the process according to the invention in the range of more than 2.2, for example 5, moles of chloromethylenedimethyliminium chloride per mole of alkyl ester, in particular 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester, are such that the main process product (4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, preferably 4-[5-[bis(2-chloroethyl)]amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C1-4-alkyl ester, in particular 4-[5-[bis The ratio of the main process product (4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester) to the process by-product (4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, preferably 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C1-4-alkyl ester, in particular 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester) can be controlled. In the process according to the first embodiment of the invention, substantially no by-products are obtained, but a mixture of the main process product and the process by-products can be obtained in a molar ratio in the range of more than 2.2, for example 5. With increasing molar ratio, the relative proportion of process by-products rises to about 5 mol %, in addition to about 95 mol % of the main process product. Increasing the molar ratio of chloromethylenedimethyliminium chloride to more than 5 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester is not recommended for the process according to the invention in the second embodiment of the invention.

[0021] The 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester obtained as a process by-product of the process according to the invention carried out in the second embodiment of the invention can be subjected to a further process step in the form of ester hydrolysis to form 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid, for example according to the procedure known from EP 2 468 716 A1, which is expressly referred to herein.

[0022] It is also possible to carry out the process according to the second embodiment of the invention and to ester hydrolyze the process by-product together with the main process product. In other words, in this case, the product mixture consisting of the main process product and the process by-product is not first separated, but is also subjected to ester hydrolysis together, for example based on the procedure known from EP 2468716 A1, which is expressly referred to herein. The resulting mixture of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid and 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid can then be separated using methods well known to organic chemists, such as crystallization and / or preparative chromatographic methods.

[0023] As mentioned above, the ester hydrolysis mentioned several times in this specification can be carried out according to the procedure known from EP 2468716 (A1). For this purpose, it is preferred to mix the relevant isolated 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, i.e. the corresponding formyl or non-formyl butyric acid alkyl ester, or a non-separated mixture of both, with an acid. The acid used is preferably an inorganic acid, in particular hydrochloric acid. The acid is usually used as a concentrated acid, for example concentrated hydrochloric acid. The acid can be added to the mixture of formyl and non-formyl esters by mixing the acid with the ester (mixture) or its solution in a suitable organic solvent. The resulting mixture is then stirred, preferably at a temperature in the range of 10 to 80°C, more preferably at a temperature in the range of 15 to 70°C, even more preferably at a temperature in the range of 20 to 60°C. The reaction time is preferably 30 minutes to 6 hours, more preferably 1 hour to 4 hours, even more preferably 1 hour to 3 hours. After the reaction, any organic components contained in the mixture, such as the solvent or solvent residues, can be removed. This can be preferably done by distilling these organic components. The acid contained in the mixture is then removed from the hydrolyzed ester(s) by conventional means, preferably by distillation. The substituted butyric acid remains as a residue, i.e., formyl substituted butyric acid (4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid) or non-formyl substituted butyric acid (4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid) or an associated mixture of formyl substituted butyric acid and non-formyl substituted butyric acid.

[0024] 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid, for example, exhibits antitumor effects against the same types of cancer as 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid. This was demonstrated in experiments such as those shown in Example 3 below. 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid and its pharma- ceutically acceptable salts, such as chloride, phosphate, sulfate, acetate, maleate, citrate or mesylate, exhibit cancer cell killing or cancer cell growth inhibitory effects against, for example, skin, kidney, lung, brain and pancreatic cancers, even in the low IC50 concentration range of 100 micromoles per liter. IC50 represents the "inhibitory concentration" at which 50% of cancer cells are killed.

[0025] The aldehyde and carboxyl groups of 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid, as reactive functional groups, can provide starting points for a variety of intramolecular and intermolecular derivatization reactions and therefore also for the preparation of antitumor active compounds, e.g., for the formation of antibody conjugates.

[0026] Thus, 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid, more precisely 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid functioning as the active ingredient, and its pharma- ceutically acceptable salts can be used as listed below. 1. Used in medicine, especially human medicine, specifically cancer medicine. 2. Used in or for the therapeutic treatment of cancer, in particular skin cancer, kidney cancer, lung cancer, brain cancer and pancreatic cancer. 3. As a medicine, optionally in combination with other medicines, especially as a medicine in or for the therapeutic treatment of cancer, especially skin cancer, kidney cancer, lung cancer, brain cancer and pancreatic cancer. 4. For use as a medicine, in particular as a medicine in or for the therapeutic treatment of cancer, in particular skin cancer, kidney cancer, lung cancer, brain cancer and pancreatic cancer. 5. For use in or for the manufacture of medicinal products, especially medicinal products for the therapeutic treatment of cancer, especially skin cancer, kidney cancer, lung cancer, brain cancer and pancreatic cancer.

[0027] Example 1 (reaction of chloromethylenedimethyliminium chloride with 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester in a molar ratio of 2.0:1): 3.5 g of chloromethylenedimethyliminium chloride and 55.8 g of dichloromethane were cooled to 5°C while stirring in a flask. To this suspension, 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester dissolved in 28.7 g of dichloromethane was added in small portions at 1°C, then heated to 40°C and stirred under reflux for 9 hours. It was then cooled to 10°C, 10 mL of water was added, the pH value was adjusted to 8-10 with 10 wt% aqueous potassium carbonate solution, stirred for 10 minutes, and the two-phase system was transferred to a separatory funnel. The dichloromethane phase was separated. 46 mL of dichloromethane was added to the aqueous phase and the dichloromethane phase was separated by shaking. The combined dichloromethane phase was washed with 15 mL of concentrated saline, and the dichloromethane was removed under vacuum by applying heat to a rotary evaporator. The residue was recrystallized from 10 mL of ethyl acetate.

[0028] This gave 4.92 g of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester (89% yield based on 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester used).

[0029] Example 2 (reaction of chloromethylenedimethyliminium chloride with 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester in a 4.0:1 molar ratio, followed by ester hydrolysis and product recovery): 7.0 g of chloromethylenedimethyliminium chloride and 55.8 g of dichloromethane were cooled to 5°C while stirring in a flask. To this suspension, a 1°C cold solution of 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester in 28.7 g of dichloromethane was added in small portions, then heated to 40°C and stirred under reflux for 9 hours. It was then cooled to 10°C, 10 mL of water was added, the pH value was adjusted to 8-10 with 10 wt% aqueous potassium carbonate solution, stirred for 10 minutes, and the two-phase system was transferred to a separatory funnel. The dichloromethane phase was separated. 46 mL of dichloromethane was added to the aqueous phase and the dichloromethane phase was separated by shaking. The combined dichloromethane phase was washed with 15 mL of concentrated saline. The washed dichloromethane phase was mixed with 5.78 g of 32 wt.% hydrochloric acid and the dichloromethane was removed under vacuum by applying heat to the rotary evaporator. An additional 23.8 mL of 32 wt.% hydrochloric acid was added and stirring was continued for 90 min at an internal temperature of 40° C. After cooling to 20° C., 0.58 g of activated carbon was added and stirred for 20 min, then the activated carbon was filtered off and rinsed with 24.8 mL of water. The solution was then concentrated to 16.0 g at a bath temperature of 45° C. and a vacuum of 15 mbar, a mixture of 19.75 g of water and 2.87 g of acetone was added and cooled to 5° C. within 60 min. The resulting suspension was stirred for 90 min at an internal temperature of 5° C., filtered and washed with 23 mL of cold water and 18 mL of ethyl acetate. After drying under vacuum, 3.58 g of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid was obtained as the major product (70% yield based on 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester used).

[0030] The acetone-based / water-based mother liquor obtained during filtration was stripped of acetone under vacuum and the pH value was adjusted to 3-4 using 0.1 M NaOH. The resulting weakly acidic solution was treated by reversed-phase chromatography (stationary phase: DuPont™ AmberChrom™ CG161M chromatography resin). As eluent, an acetonitrile gradient was used, increasing from 0 to 50 wt.% with 0.1 wt.% aqueous trifluoroacetic acid. The organic solvent was removed from the eluent in vacuum and then dried by lyophilization. Ethyl acetate was added to the lyophilized material and it was recrystallized therefrom.

[0031] 0.25 g of 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid (4.5% yield based on 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester used) was obtained as a by-product in the form of a yellow solid.

[0032] UV-VIS, HRMS, 1 H-NMR, 13 The yellow solid was identified as 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid by C-NMR spectroscopy.

[0033] UV-VIS in acetonitrile / water containing 0.1% HCOOH (nm): 249 (peak, broad), 295 (shoulder), 405 (peak, broad).

[0034] HRMS:C 17 H 21 O3N3Cl2, 386.10298 (delta m=-0.76 ppm)

[0035] NMR data (in CDCl3):

[0036] [Table 1]

[0037] Example 3 Selected cell lines were incubated with different concentrations of 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid (concentration range 1.5 μmol-5 mmol / L) at 37 °C for 72 h. Cells were then incubated with MTS solution (Promega) for 1 h and the effect of 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid on cell proliferation was measured colorimetrically at 490 nm. Cell proliferation was expressed as a percentage of the corresponding control (0.5 wt% dimethyl sulfoxide in water). Respective IC50 values ​​were calculated based on this data.

[0038] [Table 2]

Claims

1. A process for preparing 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester, comprising reacting 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester with chloromethylenedimethyliminium chloride.

2. The 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester is １－４ The method of claim 1 , wherein the alkyl ester is selected from alkyl esters.

3. 3. The method according to claim 1 or 2, wherein said 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester is used.

4. The process according to any one of claims 1 to 3, wherein a molar ratio of chloromethylenedimethyliminium chloride in the range of 2.0 to 2.2 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester is used.

5. 2. The process of claim 1, wherein a molar ratio of chloromethylenedimethyliminium chloride in the range of greater than 2.2 to 5 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester is used.

6. 6. 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid alkyl ester obtained as a by-product of the process according to claim 5.

7. 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C １－４ 3. The process of claim 2, wherein a molar ratio of chloromethylenedimethyliminium chloride in the range of greater than 2.2 to 5 moles per mole of alkyl ester is used.

8. 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid C obtained as a by-product of the process according to claim 7. １－４ Alkyl esters.

9. 4. The process of claim 3, wherein a molar ratio of chloromethylenedimethyliminium chloride in the range of greater than 2.2 to 5 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester is used.

10. 10. 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid ethyl ester obtained as a by-product of the process according to claim 9.

11. 10. The process of any one of claims 5, 7 or 9, wherein the process by-product obtained by the process of any one of claims 5, 7 or 9 undergoes subsequent ester hydrolysis to form 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid.

12. 12. The process of claim 11 , wherein the process by-products undergo ester hydrolysis along with the main process product.

13. 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid obtained as a product of the process according to claim 11 or 12.

14. 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid or a pharma- ceutically acceptable salt thereof for use in medicine.

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