WO2005111056A1

WO2005111056A1 - New esters of adenosine nucleosides and carboxylic acid

Info

Publication number: WO2005111056A1
Application number: PCT/PL2005/000031
Authority: WO
Inventors: Pawel Grieb; Juhani Vilpo; Zygmunt Kazimierczuk; Mariola Andrzejewska; Tomasz Kryczka
Original assignee: Fundacja Rozwoju Diagnostyki I Terapii
Priority date: 2004-05-19
Filing date: 2005-05-19
Publication date: 2005-11-24
Also published as: PL368093A1; PL201571B1; WO2005111056B1

Abstract

Novel esters of adenosine nucleosides and carboxylic acid, of formula 1, wherein R1 is hydrogen or chlorine atom, R2 is acylic group of carboxylic acid and an ester bond is located between carboxyl group of an acid and hydroxyl group in 5' position of 2'-deoxyadenosine and method of their preparation.

Description

New esters of adenosine nucleosides and carboxylic acid

FIELD OF THE INVENTION

The present invention relates to new esters of adenosine nucleosides and carboxylic acid. In the case of the present invention these are the esters of 2'-deoxyadenosine or its derivative, 2- chloro-2'-deoxyadenosine, and the carboxylic acid. Moreover, the present invention relates to the method of synthesis of the above-mentioned esters. BACKGROUND OF THE INVENTION

2'-Deoxyadenosine (dA) is one of DNA components. This nucleoside is synthesized in cells from adenosine or uptaken by cells by the membrane nucleoside transporter proteins. Inside cells the 2'-deoxyadenosine enters two different metabolic pathways. The first metabolic pathway is based on three-stage enzymatic phosphorylation which is controlled by the deoxyadenosine kinase enzyme (dAK) that catalyzes the first-step of this process; phosphorylation of dA is responsible for the incorporation of this nucleoside into DNA during the replication or repair of genetic material of cells. In the second metabolic pathway, dA is enzymatically deaminated, catalyzed by the adenosine deaminase enzyme (ADA) and due to this process the excess of 2'- deoxyadenosine is removed from cells. An efficient adenosine deaminase dependent removing of 2'-deoxyadenosine has particularly crucial importance in lymphocytes because the excess of 2'-deoxyadenosine causes death of these cells (R.F, Kefford, R.M. Fox, Purine deoxynucleoside toxicity in nondividing human lymphoid cells, Cancer Research 1982, 42, 324-30). However, the activity of the adenosine deaminase enzyme allows to keep a low concentration of 2'-deoxyadenosine inside them. Due to this, 2'-deoxyadenosine is neither toxic to organism nor to the neoplastic cells. No pharmacological properties of dA are known due to which this substance could be applied in medicine. 2-Chloro-2'-deoxyadenosine (2CdA, cladribine) is an analogue of 2'-deoxyadenosine which is resistant to the enzymatic deamination, but inside the lymphocytes it is a subject of phosphorylation catalyzed by the deoxycitidine kinase enzyme (dCK) and accumulation, and it is toxic to lymphocytes similarly to the excess of 2'-deoxyadenosine. Cladribine has been practically applied in the treatment of some leukemias, especially in the treatment of hairy cell leukemia, chronic lymphoblastoid B-cell leukemia and lymphomas of low malignant potential (J. Liliemark, The clinical pharmacokinetics of cladribine, Clinical Pharmacokinetics 1997, 32, 120-131). The carboxylic acid which forms the ester is 4-bis(2-chloroethyl)-p-aminobenzene-butyric acid, a cytotoxic drug known as chlorambucil. It is bifunctional alkylating agent toxic to cells . The alkylation process leads to DNA intrastrand and interstrand cross-links and to binding between DNA and proteins contained in nucleoproteins. Chlorambucil also interacts with other components of cell such as RNA, proteins and cell membranes. These interactions lead to apoptosis - the programmed cell death (A. Begleiter, M. Mowat, L.G. Israelis, J.B. Johnston, Chlorambucil in chronic lymphocytic leukemia: mechanism of action, Leukemia Lymphoma, 1996, 23, 187-201). Chlorambucil is currently used in the treatment of chronic lymphoblastoid leukemia, where it is used as the first-line drug, and ovarian cancer, and malignant lymphomas. The treatment of these neoplasms with chlorambucil is efficient only in some cases and its efficiency is time-limited i.e., till the resistance occurs, Therapeutic efficiency of chlorambucil cannot be increased with the use of higher doses of this drug because of side effects. Especially from the publication of M.S. Blumenreich, T.M. Woodcock, E.J. Sherrill, S.P. Richman, P.S. Gentile, B.E. Epremian, T.T. Kubota, J.C. Aliegra, A phase I trial of chlorambucil administered in short pulses in patients with advanced malignancies, Cancer Investigation, 1988, 6, 371-375, it is known that chlorambucil toxicity limits its doses and it is mainly due to its toxicity against central nervous system, expressed by the seizures. The only known chemical compounds of 2'-deoxyadenosine and chlorambucil are disclosed in publication of D. Florea-Wang, E. Haapala, J. Mattinen, K. Hakala, J. Vilpo, J. Hovinen, Reactions of the N,N -bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxyadenosine, Chemical Research in Toxicology, 2003, 16, 403-408; the adducts which are formed during incubation of 0,6 mM chlorambucil and 16.1 mM 2'- deoxyadenosine in the environment of the cacodylic acid buffer (pH 6.8). These derivatives are formed as a result of alkylation of nitrogen heteroatoms in purine ring of 2'-deoxyadenosine by chlorambucil. Thus, these compounds are structurally entirely different from 5'-O-ester of 2'-deoxyadenosine and chlorambucil, the chemical formula and the method of synthesis of which are the subjects of this invention.

SUMMARY OF THE INVENTION The main aspect of the present invention are novel esters of adenosine nucleosides and carboxylic acid, of formula 1 , wherein R1 is hydrogen or chlorine atom, R2 is acylic group of carboxylic acid and an ester bond is located between a carboxyl group of an acid and a hydroxyl group in 5' position of 2'-deoxyadenosine.

Formula 1. In the esters of the invention, the carboxylic acid is 4-bis(2- chloroethyl)aminobenzenebutyric acid. The nucleoside of 2'-deoxyadenosine type is 2'-deoxyadenosine or 2-chloro-2'- deoxyadenosine.

The novel esters of nucleoside, 2'-deoxyadenosine, and carboxylic acid, of formula 1 , wherein R1 is hydrogen or chlorine atom, and R2 is acylic group of carboxylic acid and an ester bond is located between carboxyl group of an acid and hydroxyl group in 5' position of 2'-deoxyadenosine are:

5'-0-[4-bis(2-chloroethyl)-p-aminobenzenobutyryl]-2'-deoxyadenosine; 5'-0-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl]-2-chloro-2'-deoxyadenosine. Another aspect of the invention is the method of synthesis of esters, of formula 1 , wherein R1 is hydrogen or halogen atom, and R2 is acylic group of carboxylic acid according to claim 1 or 2, where the regioselective reaction of esterification of nucleoside, 2'- deoxyadenosine, with carboxylic acid is performed in environment of mixture of triphenylphosphine and esters of azodicarboxylate acid in a polar organic solvent, at the temperature close to the room temperature; then, the resulting final product in a form of ester is isolated from the reaction mixture. We have found that these esters could be obtained with high efficiency. In the method of synthesis of the invention, the mixture of triphenylphosphine and esters of azodicarboxylate acid is used as the agent responsible for removing water in the reaction of esterification. The use of this method involves the regioselective synthesis of the esters of 2'- deoxyadenosine and 4-bis(2- chloroethyl)aminobenzenebutyric acid and due to this the esterification of -OH group undergoes preferentially in position 5' of 2'-deoxyadenosine or its derivative. In the method of synthesis of the invention, the regioselective reaction of esterification is performed in a polar aprotic organic solvent such as dimetyloformamid or pyridine within the temperature range 20-37°C. The final product is isolated from the reaction mixture by adsorption or high performance liquid chromatography. The results of research have shown that 5'-0 ester of [4-bis(2-chloroethyl)-p- aminobenzenebutyryl-2'-deoxyadenosine has much more beneficial pharmacological properties than 5'-0 ester [4- bis(2 -chloroethyl)-p -aminobenzenebutyryl-2-chloro-2'-deoxyadenosine. Investigations further showed that the mentioned 5'-0 ester of [4-bis(2-chloroethyl)-p- aminobenzenebutyryl-2'-deoxyadenosine possesses more beneficial pharmacological properties than chlorambucil with two regards. First, calculated on the base of experiments in animals, expressed in moles, acute toxicity of this ester after its intraperitoneal administration was lower than acute toxicity of chlorambucil. It means that the metabolism of the ester in organism is not the result of its total hydrolysis to chlorambucil before it is delivered to target cells because in such case toxic doses of ester and chlorambucil expressed in moles would be the same. Then, there is no doubt that at least a part of the ester is introduced to cells in a non-hydrolized form. Moreover, this ester in comparison to free chlorambucil does not induce seizures which are the symptoms of neurotoxicity. Second, cytotoxicity of the above-mentioned ester calculated on the base of experiments on the cell lines of various cancers, expressed in molar concentrations, indicates that it is more toxic to cancer cells than chlorambucil. The above observations allow to conclude that this ester will be better tolerated by the treated patients than chlorambucil. Thus, it can be expected that 5'-0 ester of [4-bis(2-chloroethyl)-p- aminobenzenebutyryl-

2'-deoxyadenosine will be used in therapy in the same - or even wider - range than chlorambucil and as the drug with a lower toxicity against organism, it will be better tolerated by the patients undergoing chemotherapy and, as the drug with a higher cytotoxicity to cancer cells, it will be more efficient in the treatment of cancer. Another aspects of the present invention are: the pharmaceutical composition indicated for the treatment of cancer diseases, in particular, of proliferation diseases of hematopoietic system, containing 5'-0-[4-bis(2-chloroethyl)-p- aminobenzenebutyryl-2'-deoxyadenosine as the active component at a dose necessary to exert the cytotoxic effect with at least one neutral pharmaceutically acceptable carrier, solvent or vehicle; and also the pharmaceutical composition indicated for treatment of cancer diseases, in particular, of proliferation diseases of hematopoietic system, containing 5'-0-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl-2-chloro-2'-deoxyadeno- sine as the active component at a dose necessary to exert the cytotoxic effect with at least one neutral pharmaceutically acceptable carrier, solvent or vehicle. The chlorambucil-deoxyadenosine esters being the subject of the invention can be used in the treatment as the pharmaceutical compositions indicated for parenteral or oral administration. Such compositions can be developed with the methods known in the art of preparation of drug forms. The chemical compounds of the present invention can be produced and administered in very different forms for parenteral and oral administration. Thus, the compounds according to the invention can be administered by the intravenous, intramuscular, intracutaneous, subcutaneous or intraperitoneal routes. For the parenteral administration of drug, there are dosage forms of drugs in a liquid form developed with the use of each chemical compound of the invention, i.e., 5'-0 ester of [4-bis(2-chloroethyl)-p- aminobenzenebutyryl-2'-deoxyadenosine or 5'-0 ester of [4- bis(2-chloroethyl)-p- aminobenzenebutyryI-2-chloro-2'-deoxyadenosine and sterile vehicle and the addition of water is beneficial. The above-mentioned compounds, considering the type of the pharmaceutically compatible carrier can be suspended or dissolved in vehicle. When the solution is produced, the active compound can be dissolved in the solution for injections and sterilized by filtration. The vials or ampoules are filled with the sterile solution obtained by this method and tightly closed. The preparation after filling the vial can be frozen and the solvent can be removed under vacuum. Then, such lyophilized powder is tightly closed in a vial and another vial containing water for injections is attached to the product to prepare the injection form of drug. It is obvious for specialists that the above-described dosage forms can contain new compounds as an active agent as well as pharmaceutically acceptable salt of each compound according to the invention. The present invention will be more easily understood on the ground of particular examples which are supplemented with attached figures (Fig. 1-5) aiming to illustrate the invention but not to limit its range.

Example 1. 5'-0-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl]-2'-deoxyadenosine (dA - CLB). In the 50 ml flask 377 mg (1.5 mmol) of 2'-deoxyadenosine, 456 mg (1.5 mmol) of 4-bis(2- chloroethyl)-p-aminobenzenebutanoic acid and 10 mi of dry pyridine were placed. The solvent was evaporated to remove water from the sample. This action was repeated with a new portion of pyridine - 10 ml. Then, 12 ml of pyridine was added and stirred at room temperature for about 5 min and triphenylphosphine (525 mg, 2 mmol) and diisopropyl azodicarboxylate (405 mg, 2 mmol) were added portionwise and the sample was stirred for another 30 min. After that, the sample was evaporated to an oil. To remove the remaining pyridine, the sample was thrice evaporated with toluene (3 x 15 ml). The residue was applied onto a silica gel column (3 x 25 cm, silica gel 60, Merck) and chromatographed with methylene chloride (400 ml) and, then, with the mixture of methylene chloride - methanol (95:5). The fractions containing product were evaporized to a dry foam. 452 mg of the compound was obtained. Yield 56%. TLC: Rf (MeOH/CHC)₃): 0.44. 1H NMR (D₆Me₂SO, δ(ppm)): 1.74-2.80 (2m, H-2' and H-2" and -CH2-CH₂-CH2-), 3.68 (bs, N-CH2-CH2-), 4.00 (m, H-4'), 4.18 and 4.25 (2m, H-5' and H-5"), 4.47 (m, H-3'), 5.50. (bs, OH-3'), 6.36 (t, H-1'), 6.98 and 6.64 (2d, H-phenyl), 7.29 (s, NH₂), 8.15 and 8.30 (2s, H -2 and H-8). ESI - TOF mass enumerated for C25H32CI2N6O4 (M+Na⁺) 559.1598. Found: 559.1582. Elemental analysis: calculated for C25H32CI2N6O4 (537.45): C, 53.64; H, 5.63; N, 15.64. Found: C 53.37; H, 5.55; N, 15.54.

Example 2. 5'-O-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl]-2-chloro-2'-deoxyadenosine (2- CdA - CLB).

In the 50 ml flask, 428 mg (1.5 mmol) of 2-chloro-2'-deoxyadenosine, 456 mg (1.5 mmol) of 4- bis(2-chloroethylo)-p-aminobenzenebutanoic acid and 10 ml of dry pyridine were placed. The solvent was evaporated to remove water from the sample. This action was repeated with a new portion of pyridine - 10 ml. Then, 12 ml of pyridine was added and stirred at room temperature for about 5 min. Triphenylphosphine (525 mg, 2 mmol) and diisopropyl azodicarboxylate (405 mg, 2 mmol) were then added portionwise and the sample was stirred for another 90 min. Then, the sample was evaporated to oil. To remove the remaining pyridine the sample was thrice evaporated with toluene (3 x 15 ml), The residue was applied onto a silica gel column (3 x 25 cm, silica gel 60, Merck) and chromatographed with chloride methylene chloride ( 400 ml) and, then, with the mixture of methylene chloride - methanol (95:5). The fractions containing the product were evaporated to a solid foam.

420 mg of the compound was obtained. Yield 49%, TLC: Rf (MeOH/ CHCI3): 0.49. 1H NMR (D₆Me₂SO, δ(ppm)): 1.73-2.72 (2m, H-2' and H-2" and -CH2-CH2-CH2-), 3.68 (bs, N-CH2-CH2-), 4.00 (m, H-4'), 4.16 and 4.23 (2m, H-5' and H-5"), 4.46 (m, H- 3'), 5.50 (bs, OH-3'), 6.36 (t, H-1'), 6.99 and 6.64 (2d, H-phenyl), 7.83 (bs, NH₂), 8.31 (s, H-8). ESI-TOF mass of C25H31CI3N6O4 (M+H⁺) - Calculated: 5711389. Found: 571.1376. Elemental analysis: calculated for C25H31CI3N6O4 (571.90): C50.41 ; H, 5.11 ; N, 14.69. Found: C, 50.29; H, 5.15; N, 14.52.

Pharmacological research: 1. The comparison of acute toxicity of 5'-0-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl]-2'- deoxyadenosine and toxicity of 4-bis(2- chloroethyl)aminobenzenebutyric acid (chlorambucil). Both tested substances were dissolved in DMSO and, then, administered intraperitoneally to BALB/c female mice aged 2.5 months and of body weight of 18.6-19.6 g. 5'-0-[4-bis(2- chloroethyl)-p-aminobenzenebutyryl]-2'-deoxyadenosine was administered at doses of 48.8, 78.1 or 125 mg per kg of body weight and chlorambucil was administered at doses of 31.3, 50.0 or 80.0 mg per kg of body weight, using five animals for each of the doses used. After the administration of the lowest dose of 5'-0-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl]-2'- deoxyadenosine (48.8 mg per kg of body weight), all animals showed dejection but they survived the 14-days observation period. After the administration of the mean dose (78.1 mg per kg of body weight) all animals showed dejection, decreased mobility in four of them and one animal died within 24 hours after the administration of the tested substance. After the administration of the highest dose (125 mg per kg of body weight), all animals showed dejection and decreased mobility and four of them died, but three of them died within 24 hours and one died within 48 hours after the administration of the tested substance. The median lethal intraperitoneal LD50 dose for 5'-0-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl]-2'-deoxyadenosine (ester of chlorambucil and 2'-deoxyadenosine) calculated on the base of the above results with the regression logarithmic-probit method was 100.8 mg per kg of body weight and its 95% confidential interval was 91.0-111 ,7 mg per kg of body weight. After the administration of the lowest dose of chlorambucil (31.3 mg per kg of body weight) all animals showed dejection, decreased mobility, tremors, seizures and muscle tonicity but all of them survived the 14-days observation period. After the administration of the mean dose (50.0 mg per kg of body weight), four animals died and after the administration of the highest dose (80.0 mg per kg of body weight) all 5 animals died within 24 hours after the administration of the tested substance. The median lethal intraperitoneal dose LD50 for chlorambucil calculated on the base of the above results with the regression logarithmic-probit method was 48.3 mg per kg of body weight and its 95% confidential interval was 46.4-50.3 mg per kg of body weight. Because the molecular mass of chlorambucil is 304.2 and the molecular mass of the ester of chlorambucil and 2'-deoxyadenosine is 537.45, LD50 expressed in micromole per kg of body weight are 159 x 10-³ for chlorambucil and 194 x 10-³ for the ester of chlorambucil and 2'- deoxyadenosine, These results mean that at least a part of dose of ester after its intraperitoneal administration does not undergo immediate hydrolysis in the organism to chlorambucil but affects the target cells in non-hydrolyzed form. If 5^,-0-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl]-2'- deoxyadenosine after the intraperitoneal administration was immediately hydrolyzed to chlorambucil, its toxicity and toxicity of free chlorambucil expressed in micromole would be the same. Thus, it must be concluded that at least a part of the ester dose is delivered to the target cells and is hydrolyzed inside them with the release of free chlorambucil.

2. Cytotoxicity to some hematological cancer cell lines.

Cell cultures of acute promyelocytic leukemia HL60, acute lymphoblastoid T-cell leukemia MOLT4 and chronic myeloid leukemia K562 were exposed to various concentrations of chlorambucil or 5'-O-[4-bis(2-chloroethyl)-p-aminobenzenobutyryl]-2'-deoxyadenosine (ester of chlorambucil and 2'-deoxyadenosine) for 48 and 96 hours. Cell viability was measured with the MTT test. Briefly, 10 μl of 5 mg/ml MTT stock solution (Sigma, USA) was added to the 100 μl cell cultures and the cells were incubated for another 4 hours at 37°C. Then, 100 μl of 10% SDS in 10 mmol/L HCI was added and the cells were incubated at 37°C for further 16 hours. The absorbance of the product in each culture well was measured on a PowerWaveXS (BioTec, USA) microplate reader at 570 nm and the reference measure was performed at 690 nm. For each cell line, each concentration and both incubation periods, the experiments were performed 4 times and mean values were calculated. In all the experiments the ester of chlorambucil and 2'- deoxyadenosine (dA-CLB) was more cytotoxic to cancer cells than free chlorambucil. The results of these experiments are shown in Figs. 1-5.

Description of the figures:

Fig.1 shows the viability of cells of acute promyelocytic leukemia HL60 exposed to 5'-0-[4-bis(2- chloroethyl)-p-aminobenzenobutyryl]-2'-deoxyadenosine (dA-CLB) and, for comparison, chlorambucil (CLB) after 48 hours. Fig.2 shows the viability of cells of acute promyelocytic leukemia HL60 exposed to 5'-0-[4-bis(2- chloroethyl)-p-aminobenzenobutyryl]-2'-deoxyadenosine (dA-CLB) and, for comparison, chlorambucil (CLB) after 96 hours. Fig.3 shows the viability of cells of acute lymphoblastoid T-cell leukemia MOLT4 exposed to 5'-0- [4-bis(2-chloroethyl)-p-aminobenzenobutyryl]-2'-deoxyadenosine (dA-CLB) and, for comparison, chlorambucil (CLB) after 48 hours. Fig.4 shows the viability of cells of acute lymphoblastoid T-cell leukemia MOLT4 exposed to 5'-0- [4-bis(2-chloroethyl)-p-aminobenzenobutyrylj-2'-deoxyadenosine (dA-CLB), and, for comparison, chlorambucil (CLB) after 96 hours.

Fig.5 shows the viability of cells of chronic myeloid leukemia K562 exposed to 5'-0-[4-bis(2- chloroethyl)-p-aminobenzenobutyryl]-2'-deoxyadenosine (dA-CLB) and, for comparison, chlorambucil (CLB) after 96 hours.

3. Toxicity to some in vitro cancer cell lines . Cancer cell lines cultures were exposed to various concentrations of chlorambucil or 5'- 0-[4-bis(2-chloroethyl)-p-aminobenzenobutyryl]-2'-deoxyadenosine (ester of chlorambucil and 2'- deoxyadenosine) for 72 hours. Cell lines of acute myeloid leukemia KG-1, acute lymphoblastoid T-cell leukemia CEM and MOLT-3, hairy cell leukemia Jok-1 and acute lymphoblastoid B-cell leukemia BALL were used in the experiments. The viability of cells in cultures was measured with Trypan blue exclusion test. Using 3-5 various doses of each of the substances tested, the dose- response curves were calculated for each cell line tested. For each cell line and tested substances concentrations, the experiments were performed 6 times and the results were expressed as mean values. According to the dose-response curves the IDβo (drug concentration inhibiting the viability of cells by 80%) was calculated. The results are shown in Tablel . The values of IDso of the ester of chlorambucil and 2'-deoxyadenosine expressed in micromoles were lower than IDβo of chlorambucil and this difference varied from 1.4-fold for CEM cell line to 3-fold for Jok-1, KG-1 and MOLT-3 cell lines. Table l.

Claims

Claims 1. Novel esters of adenosine nucleosides and carboxylic acid, of formula 1, wherein Ri is hydrogen or chlorine atom, R₂ is acylic group of carboxylic acid and an ester bond is located between carboxyl group of an acid and hydroxyl group in 5' position of 2'-deoxyadenosine.

Formula 1.

2. Esters according to claim 1 wherein an acylic group of carboxylic acid is 4-bis(2-chloroethyl)-p- amino-benzenobutyryl.

3. Esters according to claim 1 wherein the nucleoside is 2'-deoxyadenosine.

4. Esters according to claim 1 wherein the nucleoside is 2-chloro-2'-deoxyadenosine.

5. An ester according to claim 1 which is 5'-0-[4-bis(2-chloroethyl)-p-aminobenzenobutyryl]-2'- deoxyadenosine.

6. An ester according to claim 1 which is 5'-0-[4-bis(2-chloroethyl)-p-aminobenzenebutyryl]-2- chloro-2'-deoxyadenosine.

7. A method of preparation of the esters of formula 1 wherein Ri is hydrogen or halogen atom, R2 is an acylic group of carboxylic acid according to claim 1 or claim 2, wherein the regioselective esterification reaction of 2'deoxyadenosine or its derivative with carboxylic acid is carried out in the environment of mixture of triphenylphosphine and esters of azodicarboxylate acid in a polar organic solvent at a temperature close to the room temperature, then the resulting final product in a form of ester is isolated from the reaction mixture,

8. A method of preparation of the esters of formula 1 according to claim 7 wherein the regioselective esterification reaction is carried out in a polar aprotic organic solvent such as dimetyloformamide or pyridine.

9. A method of preparation of esters according to claim 7 wherein the esterification reaction is carried out at the temperature of 20-37°C.

10.A method of preparation of esters according to claim 7 wherein the product is isolated from the reaction mixture by adsorption or high performance liquid chromatography.

11. A pharmaceutical composition indicated for the treatment of neoplastic diseases, especially, proliferative diseases of hematopoietic system, wherein 5'-0-[4-bis(2-chloroethyl)-p- aminobenzenobutyryl]-2'-deoxyadenosine as an active compound at a dose necessary to exert the cytotoxic effect with at least one neutral pharmaceutically acceptable carrier, solvent or vehicle is included,

12. A pharmaceutical composition indicated for the treatment of neoplastic diseases, especially proliferative diseases of hematopoietic system, wherein 5'-0-[4-bis(2-chloroethyl)-p- aminobenzenebutyryl]-2-chloro-2'-deoxyadenosine as an active compound at a dose necessary to exert the cytotoxic effect with at least one neutral pharmaceutically acceptable carrier, diluent or vehicle is included.