Enzymatic Synthesis of 2-Chloropurine Arabinonucleosides with Chiral Amino Acid Amides at the C6 Position and an Evaluation of Antiproliferative Activity In Vitro

A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 μM) close to that of Nelarabine (IC50 = 3 μM) and was evaluated as active.


Introduction
The involvement of adenosine and its nucleotides into numerous biological processes has led to many works on the synthesis of structural analogues of adenosine. Agonists and antagonists of adenosine receptors [1,2] represent the broadest group of adenosine analogs obtained. Compounds with antitumor activity have been found among A 3 -type adenosine receptor agonists [3,4]. N 6 -Allyl, N 6 -isopropyl, and N 6 -propargyl analogs have shown to significantly increase the lifespans of mice experiencing mammary carcinoma. The short-chain adenosine analogues are more active in the treatment of animal carcinomas than in leukemia or sarcoma tumor cell systems [5].
Establishment of the structure of the adenylosuccinate (Figure 1) involved in the purine nucleotide cycle prompted researchers to synthesize a series of purine bases substituted at C6 by chiral amino acid residues [6][7][8][9]. Conjugates were synthesized both with amino acids [10,11] and with corresponding methyl esters [10,12]. Conjugates with amino acids have antimycobacterial activity, but no antitumor activity [6,7]. Interestingly, the arabinoside analogue of natural N 6 -isopentenyladenosine did not show anti-proliferative capacity on T24 human bladder carcinoma cells [13], while the N 6 -isopentenyladenosine riboside did.
Adenosine analogs lose their biological activity due to rapid degradation by intracellular adenosine deaminase (ADA). For example, the antiviral D-arabinofuranosyladenine (vidarabine or Ara-A) is rapidly deaminated in vivo to the less active D-arabinofuranosylhypoxantine capacity on T24 human bladder carcinoma cells [13], while the N 6 -isopentenyladenosine riboside did.
Adenosine analogs lose their biological activity due to rapid degradation by intracellular adenosine deaminase (ADA). For example, the antiviral D-arabinofuranosyladenine (vidarabine or Ara-A) is rapidly deaminated in vivo to the less active D-arabinofuranosylhypoxantine (Ara-H) [14]. Cordycepin (3′-deoxyadenosine) was found to be ineffective as an antibacterial agent due to metabolic degradation by ADA [15]. To enhance the biological activity of the compounds, it was necessary to increase the lifetime of the compound in the cell. Attempts have been made to synthesize ADA-resistant purine nucleosides. The introduction of a halogen atom in the C2 position of purine inhibits the action of intracellular adenosine deaminase or makes the nucleoside completely resistant to deamination [16]. The antitumor drugs Cladribine, Fludarabine, and Clofarabine ( Figure 1) are examples of such compounds. They are used in the treatment of oncohematological diseases [17][18][19].
Another way to make nucleosides resistant to the catabolic action of ADA is introducing various substituents at the C6 position of adenosine. However, no unambiguous relationship between the substituent structure and ADA resistance was found. The rate of hydrolysis of adenosines decreased in a series of C6 substituents: hydroxylamino, chlorine, bromine, iodine, methylamino, and ethylamino ( Figure 2). Deoxyadenosines with mercapto-, benzylamino-, and p-nitrobenzyl substituents at C6 were resistant to ADA [20][21][22]. To enhance the biological activity of the compounds, it was necessary to increase the lifetime of the compound in the cell. Attempts have been made to synthesize ADAresistant purine nucleosides. The introduction of a halogen atom in the C2 position of purine inhibits the action of intracellular adenosine deaminase or makes the nucleoside completely resistant to deamination [16]. The antitumor drugs Cladribine, Fludarabine, and Clofarabine ( Figure 1) are examples of such compounds. They are used in the treatment of oncohematological diseases [17][18][19].
Another way to make nucleosides resistant to the catabolic action of ADA is introducing various substituents at the C6 position of adenosine. However, no unambiguous relationship between the substituent structure and ADA resistance was found. The rate of hydrolysis of adenosines decreased in a series of C6 substituents: hydroxylamino, chlorine, bromine, iodine, methylamino, and ethylamino ( Figure 2). Deoxyadenosines with mercapto-, benzylamino-, and p-nitrobenzyl substituents at C6 were resistant to ADA [20][21][22]. ADA substrates (green frame) and non-substrates (red frame). Data from ref. [20,21].
On the other hand, ADA is a key intracellular enzyme that modifies the anticancer drug Nelarabine (9-β-D-arabinofuranosyl-6-O-methylguanine, Figure 1). A necessary step in the metabolism of Nelarabine is the demethoxylation of the purine base by ADA with the formation of 9-β-D-arabinofuranosyl guanine (Ara-G), which suppresses cell proliferation [23]. On the other hand, ADA is a key intracellular enzyme that modifies the anticancer drug Nelarabine (9-β-D-arabinofuranosyl-6-O-methylguanine, Figure 1). A necessary step in the metabolism of Nelarabine is the demethoxylation of the purine base by ADA with the formation of 9-β-D-arabinofuranosyl guanine (Ara-G), which suppresses cell proliferation [23].
It is difficult to find a clear correlation between the antimetabolic activity of nucleosides and the modification of the C2 and C6 purine positions or the carbohydrate residue. We decided to synthesize a number of 2-chloro-6-substituted arabinonucleosides using enzymatic synthesis. The next step was to study both their resistance to ADA and their antiproliferative activity, in order to draw conclusions about the relationship between the structure of the nucleoside, its activity against ADA, and its antiproliferative activity.

Synthesis of Arabinonucleosides
Chemical modifications of purine are known to be more convenient to carry out in a nucleoside (where the N9 position of purine is protected by a carbohydrate residue) instead of a heterocyclic base [24,25]. The classical method for obtaining arabinonucleosides is the glycosylation of a modified purine base with a protected carbohydrate derivative [26][27][28][29] or complex ribose modifications [30,31]. Enzymatic methods for the synthesis of arabinosides [32][33][34] are very convenient due to the high selectivity and stereospecificity of enzymatic reactions.
We recently published an article describing the synthesis of purine ribosides 1a-12a (Scheme 1) modified with chiral amino acid amides at the C6 position of the purine [35]. We showed that derivatives with tyrosine, valine, and serine residues exhibit the properties of A 1 adenosine receptor partial agonists. The ribosides 1a-12a became the starting compounds for the enzymatic synthesis of arabinonucleosides (Scheme 1).
The synthesis of arabinosides was carried out using enzymatic transglycosylation with recombinant E. coli nucleoside phosphorylases [34,36]. Nucleosides substituted at the C6 position of purine are known to be good substrates for E. coli purine nucleoside phosphorylase (PNP) [37].
The transglycosylation reaction can be carried out in two ways: using synthetic arabinose 1-phosphate (1-P-Ara) (Scheme 1) [34] or 1-b-D-arabinofuranosyluracil (Ara-U) as an arabinose donor (Scheme 1, Table 1). In the first case, PNP is used (Scheme 1, black route). In the second case, PNP and uridine phosphorylase (UP) are used [38] (Scheme 1, azure route). Ara-U can be easily synthesized from uridine via 2,2 -anhydrouridine according to the method of I. Wempen [39]. Figure 3 shows the conversion of riboside 1a to arabinoside 1b. Ara-U turned out to be the best arabinose donor.
According to HPLC data, the concentration of the product in the reaction mixture reached 82%, with a fivefold excess of Ara-U (green trend line). The conversion of riboside to arabinoside was 90% in 5 days. Syntheses of nucleosides 1b-12b were carried out using Ara-U at its fivefold molar excess.
As a result of the optimization of enzymatic synthesis, the following conditions for obtaining arabinosides were chosen: the ratio of riboside substrates to Ara-U was 1:5; 0.80 units PNP per 1 mmol of substrate and 0.18 units UP per 1 µmol Ara-U; pH 7.0, 52 • C.
During the experiments, we found that the retention times of ribosides and arabinosides were very close (the HPLC profile of the reaction mixture for the synthesis of 6-N-[L-alanylamido]-2-chloro-9-β-D-arabinofuranosylpurine 2b is shown on Figure 4A, Table 2). In addition to the target arabinoside, the reaction mixture contains up to 8% of the starting riboside and a heterocyclic purine base. This made it difficult to isolate the target product. units PNP per 1 mmol of substrate and 0.18 units UP per 1 μmol Ara-U; pH 7.0, 52 °C.
During the experiments, we found that the retention times of ribosides and arabinosides were very close (the HPLC profile of the reaction mixture for the synthesis of 6-N-[L-alanylamido]-2-chloro-9-β-D-arabinofuranosylpurine 2b is shown on Figure 4A, Table 2). In addition to the target arabinoside, the reaction mixture contains up to 8% of the starting riboside and a heterocyclic purine base. This made it difficult to isolate the target product. Scheme 1. Possible pathways for the synthesis of arabinosides 1b-12b. Two approaches to carrying out the transglycosylation reaction: using synthetic arabinose 1-phosphate (1-P-Ara) (black route) [34] or 1-b-D-arabinofuranosyluracil (Ara-U) as an arabinose donor (azure route). Scheme 1. Possible pathways for the synthesis of arabinosides 1b-12b. Two approaches to carrying out the transglycosylation reaction: using synthetic arabinose 1-phosphate (1-P-Ara) (black route) [34] or 1-b-D-arabinofuranosyluracil (Ara-U) as an arabinose donor (azure route).   To decrease the number of components in the reaction mixture, we decided to employ the arsenolysis of ribosides. It is known that PNP catalyzes the reversible riboside phosphorolysis with the formation of α-D-ribose-1-phosphate.
In the presence of arsenates in the active site of PNP, the intermediate α-D-ribose-1-arsenate is formed from the riboside. Being extremely unstable, it rapidly hydrolyzes to ribose and inorganic arsenate, making the reaction irreversible (Scheme 2) [40][41][42].     To decrease the number of components in the reaction mixture, we decided to employ the arsenolysis of ribosides. It is known that PNP catalyzes the reversible riboside phosphorolysis with the formation of α-D-ribose-1-phosphate.
In the presence of arsenates in the active site of PNP, the intermediate α-D-ribose-1arsenate is formed from the riboside. Being extremely unstable, it rapidly hydrolyzes to ribose and inorganic arsenate, making the reaction irreversible (Scheme 2) [40][41][42]. According to the HPLC data, the number of components in the reaction mixture decreases from six to four when using arsenolysis ( Figure 4B, Table 2), and the isolation of target products is facilitated to a significant degree. We used the usual column chromatography instead of preparative HPLC to isolate the target nucleosides.
The efficiency of arabinoside synthesis does not change depending on whether the arsenate was added at the beginning of the synthesis or upon reaching the equilibrium state. For example, when arsenate was added at the beginning of the synthesis of methionine arabinoside 10b in the first 10 h, the equilibrium shifted towards the formation of arabinoside ( Figure 5). However, over time (24 h), both reactions achieved the same equilibrium state. The use of arsenate helps to obtain a higher conversion of the riboside in a shorter period of time. Previously, we showed that the competitive arsenolysis reaction removes ribose from the reaction sphere [43]. When a catalytic amount of sodium arsenate (up to 0.5 mM) is added, arabinose arsenate is essentially not formed in the reaction mixture. The modified arabinose nucleosides hardly undergo arsenolysis (<0.5% in 24 h at 50 °C). As a result, the formation of arabinosides in the reaction mixture prevails. According to the HPLC data, the number of components in the reaction mixture decreases from six to four when using arsenolysis ( Figure 4B, Table 2), and the isolation of target products is facilitated to a significant degree. We used the usual column chromatography instead of preparative HPLC to isolate the target nucleosides.
The efficiency of arabinoside synthesis does not change depending on whether the arsenate was added at the beginning of the synthesis or upon reaching the equilibrium state. For example, when arsenate was added at the beginning of the synthesis of methionine arabinoside 10b in the first 10 h, the equilibrium shifted towards the formation of arabinoside ( Figure 5). However, over time (24 h), both reactions achieved the same equilibrium state. The use of arsenate helps to obtain a higher conversion of the riboside in a shorter period of time.
Previously, we showed that the competitive arsenolysis reaction removes ribose from the reaction sphere [43]. When a catalytic amount of sodium arsenate (up to 0.5 mM) is added, arabinose arsenate is essentially not formed in the reaction mixture. The modified arabinose nucleosides hardly undergo arsenolysis (<0.5% in 24 h at 50 • C). As a result, the formation of arabinosides in the reaction mixture prevails. The conditions of enzymatic reactions for the production of nucleosides 1b-12b are shown in Table 3. The physicochemical characteristics of all the synthesized arabinosides are given in Table 4. NMR spectra and HPLC-profiles are provided in the Supporting Information ( Figure S1-S61).  The conditions of enzymatic reactions for the production of nucleosides 1b-12b are shown in Table 3. The physicochemical characteristics of all the synthesized arabinosides are given in Table 4. NMR spectra and HPLC-profiles are provided in the Supporting Information (Figures S1-S61).  To establish the structure-activity relationship in the biological experiments, we synthesized six nucleosides: 9-β-D-arabinofuranosyl-2-amino-6-(N α -glycinyl)-purine (13), 9-β-Darabinofuranosyl-2-amino-6-(N α -glycinylamido)-purine (14) [25] and 16 [44] (Scheme SI-1 in Supporting Information.), 17, and 18 [45] were synthesized according to previously developed procedures. NMR spectra and HPLC-profiles are provided in the Supporting Information (Figures S62-S74).

The ADA Substrate Specificity
The resistance of the obtained compounds to the action of E. coli adenosine deaminase was evaluated (Scheme 3). All arabinonucleosides 1-18 were resistant to bacterial ADA.

The ADA Substrate Specificity
The resistance of the obtained compounds to the action of E. coli adenosine deaminase was evaluated (Scheme 3). All arabinonucleosides 1-18 were resistant to bacterial ADA.

Scheme 3.
Testing the substrate specificity of E. coli ADA towards the synthesized nucleosides.
The inhibition of adenosine deaminase by the obtained compounds was studied. Adenosine was used as a control compound for ADA testing. Then, adenosine was deaminated in the presence of synthesized nucleosides. The synthesized compounds at 0.1 mM concentration had little effect on the enzymatic activity of ADA. These reduced the rate of adenosine deamination by less than 10%. Nucleosides 4b, 15, and 16 reduced the rate of adenosine deamination under the action of ADA by 23%, 63%, and 54%, respectively. The effect of 2-chloradenosine (15), 2-chloro-9-β-D-arabinofuranosyladenine (16), and L-seryl arabinofuranoside 4b on ADA activity is shown in Figure 6. The inhibition of adenosine deaminase by the obtained compounds was studied. Adenosine was used as a control compound for ADA testing. Then, adenosine was deaminated in the presence of synthesized nucleosides. The synthesized compounds at 0.1 mM concentration had little effect on the enzymatic activity of ADA. These reduced the rate of adenosine deamination by less than 10%. Nucleosides 4b, 15, and 16 reduced the rate of adenosine deamination under the action of ADA by 23%, 63%, and 54%, respectively. The effect of 2-chloradenosine (15), 2-chloro-9-β-D-arabinofuranosyladenine (16), and L-seryl arabinofuranoside 4b on ADA activity is shown in Figure 6.
2-Cloroadenosine appeared to be the best inhibitor among the tested compounds. When ribose was replaced by arabinose (compound 16), Ki increased by 1.7 times ( Table 5). The introduction of the serine fragment at the C6 position of purine (compound 4b) increased Ki 9.2 times. The compounds 15, 16, and 4b are competitive inhibitors. Lineweaver-Burk plots for the adenosine deamination at various concentrations of 15, 16, and compound 4b are presented in Figure S75.  2-Cloroadenosine appeared to be the best inhibitor among the tested compounds. When ribose was replaced by arabinose (compound 16), Ki increased by 1.7 times ( Table  5). The introduction of the serine fragment at the C6 position of purine (compound 4b) increased Ki 9.2 times. The compounds 15, 16, and 4b are competitive inhibitors. Lineweaver-Burk plots for the adenosine deamination at various concentrations of 15, 16, and compound 4b are presented in Figure S75.

The Influence of the Synthesized Nucleosides on U937 Cell Survival
The activity of the synthesized compounds was tested on the U937 cell line. Atrians (9-β-D-arabinofuranosyl-6-O-methylguanine, Nelarabine, GlaxoSmithKline) was used as a reference drug. Atrians is used to treat recurrent and stable T-cell acute lymphoblastic leukemia.
The activity of the compounds was evaluated using IC50. The IC50 is commonly used to compare the effects of cytotoxic compounds on cell lines. This index determines both the effect on cell death and proliferation during incubation.

The Influence of the Synthesized Nucleosides on U937 Cell Survival
The activity of the synthesized compounds was tested on the U937 cell line. Atrians (9-β-D-arabinofuranosyl-6-O-methylguanine, Nelarabine, GlaxoSmithKline) was used as a reference drug. Atrians is used to treat recurrent and stable T-cell acute lymphoblastic leukemia.
The activity of the compounds was evaluated using IC 50 . The IC 50 is commonly used to compare the effects of cytotoxic compounds on cell lines. This index determines both the effect on cell death and proliferation during incubation.
9-β-D-Arabinofuranosyl-2-chloro-6-(N α -L-serinylamido)-purine 4b has shown inhibitory activity comparable to Nelarabine. Dose-response curves for Nelarabine (IC 50 = 3 µM) and compound 4b (IC 50 = 16 µM) are shown in Figure 7. For both compounds, the effect depended on the concentration in the range of 0.1-50 µM. Among all the studied compounds, only nucleoside 4b and, to a lesser extent, 2-chloroadenosine 15 exhibited antiproliferative activity. The mechanism of antiproliferative activity is not entirely clear, since compound 4b is not a substrate for bacterial ADA, while compound 15 is a competitive ADA inhibitor.
In addition, the chlorine/fluorine atom at the C2 position of purine prevents nucle- Among all the studied compounds, only nucleoside 4b and, to a lesser extent, 2chloroadenosine 15 exhibited antiproliferative activity. The mechanism of antiproliferative activity is not entirely clear, since compound 4b is not a substrate for bacterial ADA, while compound 15 is a competitive ADA inhibitor.
In addition, the chlorine/fluorine atom at the C2 position of purine prevents nucleosides from binding to the ADA active site. Testing of Cl-Nelarabine and F-Nelarabine showed the absence of both the ADA-substrate and antitumor properties (IC 50 > 50 µM).
Both glycine arabinofuranosides of 2-chloroadenine 1b and 2-aminoadenine 14 are non-substrates of bacterial ADA. The introduction of an amino acid amide at the C6 position of purine makes nucleosides resistant to adenosine deaminase.
A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine 1b-12b did not exhibit antiproliferative activity (IC 50 over 50 µM) ( Table 6). A correlation was found between the lack of ADA substrate properties of the arabinosides synthesized and low anti-tumor activity against human T-lymphoblastic leukemia cells. ADA cannot replace the amino acid residue at position C6 with a hydroxyl to form an analog of the active Ara-G molecule. It can be concluded that the antiproliferative effect of compound 4b does not depend on the action of intracellular ADA. Thus, the mechanism of antiproliferative activity of the serine analog 4b differs from that of Nelarabine. The antiproliferative activity of the compounds synthesized was tested on other tumor cell lines: LS174T human Caucasian colon adenocarcinoma, SKOV3 ovarian cancer cells, MCF7 breast cancer cells, and A549 non-small-cell lung cancer cells. None of the compounds tested affected cell viability: survival was >80% (within concentrations up to 50.00 µM).

General Procedures
All chemicals and solvents were of laboratory grade, were obtained from commercial suppliers, and were used without further purification. The method of producing of recombinant E. coli phosphorylases was described above. The following recombinant E. coli [36] enzymes were used in the present study: UP with specific activity 100 units per mg of protein, 9 mg per mL; PNP 52 units per mg, 15 mg per mL. Ara-U was synthesized as described in [34]. 1-P-Ara was synthesized as described in [46]. Analytical HPLC was performed using NMR spectra were recorded using a Bruker Avance II 700 spectrometer (Bruker BioSpin, Rheinstetten, Germany) in DMSO-d 6 at 30 • C. Chemical shifts in ppm (δ) were measured relative to the residual solvent signals as internal standards (2.50). Coupling constants (J) were measured in Hz.
Liquid chromatography mass spectrometry was performed using an Agilent 6210 TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA). UV-Spectra were recorded using a Hitachi U-2900 spectrophotometer (Tokyo, Japan).
The optical rotations were measured for purified samples using the digital polarimeter JASCO model DIP-370 (cylindrical glass cell 3.5 mm D × 50 mm) (Tokyo, Japan).

Enzymatic Reactions
The reaction conditions of arabinosides synthesis (ratio of reagents, PNP amount, reaction time, and conversion of the base into nucleoside) are provided in Table 1. Riboside (1a-12a), KH 2 PO 4 , and Ara-U were dissolved in water at 40-50 • C, the pH was adjusted up to 7.0, and Na 2 HAsO 4 was added to the reaction mixture.
The enzymes (PNP, UP) were added and the reaction mixtures were incubated at 50 • C. The reaction progress was monitored by HPLC. When conversion reached the highest value, the reaction was terminated by addition of ethanol (50%, v/v). The reaction mixtures were evaporated up to 5 mL, and the desired products 1b-12b were isolated by reversed-phase column chromatography (silica gel C18, Merck), column 100 × 20 mm (the elution conditions are shown in Table 1). The physicochemical and spectral properties of nucleosides are reported in Table 3. The NMR spectra and HPLC of nucleosides are provided in the Supporting Information.

The ADA Substrate Specificity
The substrate specificity of E. coli adenosine deaminase toward arabinosides 1b-12b was determined using a previously published method [47]. According to the HPLC data, the obtained nucleosides do not undergo deamination in vitro.

Biological Assay
For the experiments, all compounds were dissolved in DMSO and then brought to the desired concentration with an RPMI 1640 nutrient medium. The final concentration of DMSO in the samples did not exceed 0.1% and did not affect cell growth.
The antiproliferative activity of the compounds synthesized was tested on tumor cell lines: U937, LS174T, SKOV3, MCF7, and A549 cancer cells. The cells lines were kindly provided by the Tumor Strain Bank of the N. N. Blokhin Cancer Research Center. The cells were grown in RPMI1640 medium containing 2 mM glutamine and 10% fetal calf (FCS) serum at 37 • C and 5% CO 2 .
Influence on cell growth was evaluated according to the cell survival rate in the presence of compounds in the studied concentrations. Cells were seeded in 96-well flatbottom microplates at a seeding density of 8000 cells/well. The test compounds were added to the wells in a volume of 20 µL. The total incubation volume was 200 µL. The incubation time with nucleosides was 48 h (96 h for U937). At the end of the incubation, the MTT reagent was added to the cells and the cells were incubated for 2 h.
The formed formazan crystals were dissolved in 100 µL of DMSO at 37 • C for 20 min. The absorbance of DMSO solutions was measured on an optical counter for multiwell plates at λ = 540 nm.
Cell survival for the corresponding concentration was calculated using the following formula: Ti/C × 100%, where Ti is the OD after incubation with the test agents and C is the OD of parallel control samples (without test compounds). A compound was considered active if the concentration of it that caused growth inhibition by 50% (IC 50 calculated from the dose-response curve) was equal to or less than IC 50 for Nelarabine. The measurement error did not exceed 5%.
The results were expressed as averages for 4 parallel measurements in 2-3 experiments as cell survival in % (experiment/control) ×100.
Statistical processing of the obtained results was carried out using the Microsoft Excel 2010 program with the method of variational statistics with the determination of the arithmetic mean and the error of the mean (M ± m). The differences between values were considered significant at p < 0.05.
Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used in the enzymatic synthesis to simplify the composition of the reaction mixtures. In the presence of arsenates in the active site of PNP, the unstable intermediate α-D-ribose-1arsenate was formed from the riboside, but arabinose arsenate was essentially not formed in the reaction mixture. The modified arabinose nucleosides hardly undergo arsenolysis, and the formation of arabinosides in the reaction mixture prevails.
The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all compounds, the serine derivative exhibited an activity level (IC 50 = 16 µM) close to that of Nelarabine (IC 50 = 3 µM) and was evaluated as active.