Method for preparing 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester 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 method for preparing 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester and its formylated derivatives.

[0002] Ester hydrolysis can convert 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester to 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate. 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate is known as an antitumor agent called "bendamustine".

[0003] European Patent No. 2468716(A1) discloses a method for preparing 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester. The reactant used is 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester, and its hydroxyethyl group can be converted to a chloroethyl group by reacting it with oxalyl chloride in the presence of dimethylformamide to form 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester. The reaction can be carried out stoichiometrically, i.e., in a molar ratio of 2.0 moles of oxalyl chloride to 1 mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester. However, European Patent No. 2468716(A1) recommends preferably using a stoichiometric excess amount equivalent to 2.6 moles or more, particularly at least 3.0 moles of oxalyl chloride, per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester. The in-situ reaction of oxalyl chloride and dimethylformamide used here is difficult to control in terms of reaction integrity or undesirable interfering moisture in the reaction system. Using the above hyperstoichiometric molar ratio, which is recommended as preferred in accordance with the teachings of European Patent No. 2468716(A1), the desired 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester is obtained in good yield, but with an undesirable yellow coloration. This coloration is caused by an impurity that is still unknown and is difficult to remove even with 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]imidazole-2-yl]butyrate alkyl ester.

[0004] The object of the present invention is to provide an improved method for preparing 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester starting from 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester.

[0005] This objective can be achieved by a method for preparing 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester by reacting it with chloromethylenedimethyliminium chloride having the formula ClCH=N(CH3)2Cl.

[0006] In the first embodiment, the preparation method according to the present invention can be carried out in a substantially stoichiometric 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]imidazole-2-yl]butyrate alkyl ester, with good yield of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester and with little to no impurities and by-products.

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

[0008] Preferably, the alkyl 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate that reacts with chloromethylenedimethyliminium chloride in the method according to the present invention is C 1-4 Alkyl esters, particularly 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester, in other words, preferred embodiments of the method according to the present invention include chloromethylenedimethyliminium chloride and 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate C 1-4 This is carried out using alkyl esters, particularly 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester. Therefore, 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate C 1-4 Alkyl esters, particularly 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester, can be obtained as products of preferred embodiments of the method according to the present 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 is E T (30) This can be conveniently carried out in a non-protic anhydrous (dry) organic solvent having a value in the range of 140 to 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 present invention, namely chloromethylenedimethyliminium chloride and 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester, can react with each other in solution or suspension, in the latter case, one or both reactants may be present in the suspension. The two reactants can be added in any order, dissolved, suspended, or as a solid.

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

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

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

[0015] After the reaction is complete, water can be added, and the pH can be adjusted to a range of, for example, 7-10 by adding a base. Examples of bases that can be used include ammonia, alkali hydroxide, alkali carbonate, and alkali bicarbonate.

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

[0017] Alkyl 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate prepared according to the method of the present invention can be subjected to ester hydrolysis to form 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate or its hydrochloride salt, for example, in the same manner as the procedure known from European Patent No. 2468716(A1).

[0018] The preparation method according to the present invention has several advantages over the preparation method known from European Patent No. 2468716(A1). 1. Using chloromethylenedimethyliminium chloride as the chlorinating agent instead of oxalyl chloride / dimethylformamide is preferable in several respects from the standpoint of occupational safety. In addition to the advantage of avoiding the handling of hazardous substances, oxalyl chloride and dimethylformamide, it is possible to avoid the exothermic reaction and gas generation associated with the reaction between oxalyl chloride and dimethylformamide. 2. The use of chloromethylenedimethyliminium chloride allows for precise administration in adjusting the desired stoichiometric or hyperstoichiometric molar ratio between chloromethylenedimethyliminium chloride and 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester, and also allows for subsequent administration of chloromethylenedimethyliminium chloride as needed. 3. In particular, when the method according to the present invention is carried out in a substantially stoichiometric 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]imidazole-2-yl]butyrate alkyl ester, side reactions can be avoided or reduced. 4. The amount of base consumed when adjusting the pH value after the reaction is complete is reduced.

[0019] As described above, the preparation method according to the present invention in the second embodiment of the present invention can be carried out in a hyperstoichiometric molar ratio of chloromethylenedimethyliminium chloride in the range of more than 2.2 to, for example, 5 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester. In the second embodiment of the present invention, for example, 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate C 1-4When the reaction is carried out with alkyl esters, particularly 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]ethyl butyrate in a molar ratio of 2.6 to 5 moles of chloromethylenedimethyliminium chloride per mole, a yellow discoloration similar to the yellow discoloration already described at the beginning can be observed in the reaction system, and this discoloration increases with increasing stoichiometric excess of chloromethylenedimethyliminium chloride. The substance causing the yellow discoloration is a by-product of the method according to the present invention in the second embodiment of the present invention, and is 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]ethyl butyrate C 1-4 From the group of alkyl esters, a novel compound previously unknown was identified or isolated for the first time. Clearly, when the method according to the present invention is carried out in its second embodiment, regioselective formylation at the 4-position of the benzo[d]imidazole skeleton of the actual main method product may occur. The corresponding novel, previously unknown 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]ethyl butyrate was obtained and characterized after separation from the main method product, 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]ethyl butyrate. Thus, 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]ethyl butyrate is a method byproduct formed when the method according to the present invention is carried out in its second embodiment. Due to its structural relationship with 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate, ethyl 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate can also be expected to have an antitumor effect. Its aldehyde group, as a reactive functional group, can provide a starting point for various 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 complexes.

[0020] In a second embodiment of the present invention, 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester, preferably 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate C 1-4When carrying out the method according to the present invention with alkyl esters, particularly 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]ethyl butyrate in a range of more than 2.2 to, for example, 5 moles of chloromethylenedimethyliminium chloride per mole, the molar ratio selected is the main method product (4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]alkyl butyrate, preferably 4-[5-[bis(2-chloroethyl)]amino]-1-methyl-1H-benzo[d]imidazole-2-yl]C1-4-alkyl butyrate, particularly 4-[5-[bis The ratio of (2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester to the method by-product (4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester, preferably 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate C1-4-alkyl ester, particularly 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester) can be controlled. In the method according to the first embodiment of the present invention, substantially no by-products are obtained, but a mixture of the main method product and the method by-product can be obtained in a molar ratio greater than 2.2 to, for example, 5. As the molar ratio increases, the relative proportion of method by-products rises to approximately 5 mol%, in addition to approximately 95 mol% of the main method product. Increasing the molar ratio of chloromethylenedimethyliminium chloride per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester to more than 5 moles is not recommended in the method according to the present invention in the second embodiment of the present invention.

[0021] The alkyl 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate obtained as a method byproduct of the method according to the present invention, as carried out in a second embodiment of the present invention, can be subjected to a further method step in the form of ester hydrolysis, for example, according to a procedure known from European Patent No. 2468716(A1) expressly referenced herein, to form 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate.

[0022] It is also possible to carry out the method according to a second embodiment of the present invention and ester hydrolyze the method byproduct together with the main method product. In other words, in this case, the product mixture consisting of the main method product and the method byproduct is not separated first, but similarly undergoes ester hydrolysis together, for example, based on a procedure known from European Patent No. 2468716(A1) expressly referenced herein. The resulting mixture of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyric acid and 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyric acid can then be separated using methods well known to organic chemists, such as crystallization and / or preparative chromatography.

[0023] As described above, the ester hydrolysis mentioned several times herein can be carried out according to the procedure known from European Patent No. 2468716(A1). For this purpose, it is preferable to mix the relevant isolated 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester, i.e., the corresponding formylated or non-formylated butyrate alkyl ester, or an unseparated mixture of both, with an acid. The acid used is preferably an inorganic acid, particularly 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 formylated and non-formylated esters by mixing the acid with the ester (mixture) or its solution in a suitable organic solvent. The resulting mixture is then stirred at a temperature preferably in the range of 10 to 80°C, more preferably in the range of 15 to 70°C, and even more preferably 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, and even more preferably 1 hour to 3 hours. After the reaction, any organic components, such as solvents or solvent residues, contained in the mixture can be removed. This can preferably be done by distillation of these organic components. Next, the acids contained in the mixture are removed from the hydrolyzed ester(s) by conventional means, preferably by distillation. The substituted butyric acid remains as a residue, i.e., formylated substituted butyric acid (4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyric acid) or non-formylated substituted butyric acid (4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyric acid) or an associated mixture of formylated substituted butyric acid and non-formylated substituted butyric acid.

[0024] 4-[5-[Bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid shows an antitumor effect against the same types of cancer as, for example, 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyric acid. This has been demonstrated in experiments as 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 pharmaceutically acceptable salts, such as chlorides, phosphates, sulfates, acetates, maleates, citrates or mesylates, show a cancer cell killing effect or a cancer cell growth inhibitory effect against, for example, skin cancer, kidney cancer, lung cancer, brain tumors and pancreatic cancer even in a low IC50 concentration range of 100 micromoles per liter. IC50 represents the "inhibitory concentration" at which 50% of cancer cells die.

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

[0026] Therefore, 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 an active ingredient, and its pharmaceutically acceptable salts can be used as listed below. 1. It is used in medicine, particularly in human medicine, specifically in cancer medicine. 2. It is used in the therapeutic treatment of cancer, particularly skin cancer, kidney cancer, lung cancer, brain tumors and pancreatic cancer, or for therapeutic treatment. 3. It may be used as a drug in combination with other drugs, and is used in or for therapeutic purposes in the treatment of cancer, particularly skin cancer, kidney cancer, lung cancer, brain tumors and pancreatic cancer. 4. It is used as a pharmaceutical product, particularly in or for the therapeutic treatment of cancer, especially skin cancer, kidney cancer, lung cancer, brain tumors and pancreatic cancer. 5. In the manufacture of pharmaceuticals, in particular for the therapeutic treatment of cancer, especially skin cancer, kidney cancer, lung cancer, brain tumors and pancreatic cancer, or used for the manufacture of pharmaceuticals.

[0027] Example 1 (reaction of chloromethylenedimethyliminium chloride with 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate 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 in a flask while stirring. To this suspension, a 1°C cold solution of 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester dissolved in 28.7 g of dichloromethane was gradually added, and the mixture was then heated to 40°C and stirred under reflux for 9 hours. Next, the mixture was cooled to 10°C, 10 mL of water was added, and the pH was adjusted to 8-10 with 10 wt% potassium carbonate aqueous solution. The mixture was 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 solution, 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 yielded 4.92 g of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester (89% yield based on 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester used).

[0029] Example 2 (chloromethylenedimethyliminium chloride and 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester are reacted in a molar ratio of 4.0:1, followed by ester hydrolysis and product recovery): 7.0 g of chloromethylenedimethyliminium chloride and 55.8 g of dichloromethane were cooled to 5°C in a flask while stirring. To this suspension, a 1°C cold solution of 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester dissolved in 28.7 g of dichloromethane was gradually added, and the mixture was then heated to 40°C and stirred under reflux for 9 hours. Next, the mixture was cooled to 10°C, 10 mL of water was added, and the pH was adjusted to 8-10 with 10 wt% potassium carbonate aqueous solution. The mixture was 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 physiological saline. The washed dichloromethane phase was mixed with 5.78 g of 32 wt% hydrochloric acid, and the dichloromethane was removed under vacuum by heating in a rotary evaporator. Further 23.8 mL of 32 wt% hydrochloric acid was added, and stirring was continued at an internal temperature of 40°C for 90 minutes. After cooling to 20°C, 0.58 g of activated carbon was added and stirred for 20 minutes. The activated carbon was then filtered out, and the mixture was rinsed with 24.8 mL of water. Next, the solution was 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 the mixture was cooled to 5°C within 60 minutes. The resulting suspension was stirred at an internal temperature of 5°C for 90 minutes, filtered, and washed with 23 mL of cold water and 18 mL of ethyl acetate. After vacuum drying, 3.58 g of 4-[5-[bis(2-chloroethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate was obtained as the main product (70% yield based on 5.0 g of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester used).

[0030] Acetone was removed under vacuum from the acetone-based / water-based mother liquor obtained during filtration, and the pH value was adjusted to 3 - 4 using 0.1 M NaOH. The resulting weakly acidic solution was treated by reverse phase chromatography (stationary phase: DuPont™ AmberChrom™ CG161M chromatography resin). As the eluent, an aqueous solution of 0.1 wt% trifluoroacetic acid with an acetonitrile gradient increasing from 0 to 50 wt% was used. The organic solvent was removed from the eluent under vacuum and then dried by freeze drying. Ethyl acetate was added to the freeze-dried substance and 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 ethyl 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazol-2-yl]butyrate used) was obtained as a by-product in the form of a yellow solid.

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

[0033] UV-VIS (nm) in acetonitrile / water containing 0.1% HCOOH: 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 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate at different concentrations (concentration range 1.5 μmol to 5 mmol / L) at 37°C for 72 hours. Next, the cells were incubated with MTS solution (Promega) for 1 hour, and the effect of 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate on cell proliferation was measured by colorimetric analysis at 490 nm. Cell proliferation was expressed as a percentage of the corresponding control (0.5 wt% dimethyl sulfoxide in water). Based on this data, the respective IC50 values ​​were calculated.

[0038] [Table 2]

Claims

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

2. The aforementioned 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester is C １－４ The method according to claim 1, selected from alkyl esters.

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

4. The method according to claim 1 or 2, using 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]imidazole-2-yl]butyrate alkyl ester.

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

6. The method according to 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]imidazole-2-yl]butyrate alkyl ester is used, and 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester is obtained as a by-product.

7. 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate C １－４ The method according to claim 2, wherein a molar ratio of chloromethylenedimethyliminium chloride in the range of more than 2.2 to 5 moles per mole of alkyl ester is used.

8. 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate C １－４ Using a molar ratio of chloromethylenedimethyliminium chloride in the range of greater than 2.2 to 5 moles per mole of alkyl ester, 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate C １－４ The method according to claim 2, wherein an alkyl ester is obtained as a by-product.

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

10. The method according to claim 3, wherein a molar ratio of chloromethylenedimethyliminium chloride in the range of more than 2.2 to 5 moles per mole of 4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate is used, and 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate ethyl ester is obtained as a by-product.

11. The method according to claim 5, wherein the by-product 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate alkyl ester obtained by the method according to claim 5 undergoes subsequent ester hydrolysis to form 4-[5-[bis(2-chloroethyl)amino]-4-formyl-1-methyl-1H-benzo[d]imidazole-2-yl]butyrate.

12. The method according to claim 11, wherein the by-product undergoes ester hydrolysis together with the main method product.

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