Synthesis and Evaluation of New Derivatives of Busulfan as an Anti-carcinogenic Drug against k562 Cancer Cells Using the AO / PI Method

2.1. Typical Procedure for Preparation of D-glucose Penta-acetate (2a)

A mixture of D-glucose (15.0g), acetic anhydride (70mL), sodium acetate (15.0g), and butyl acetate (150mL) was refluxed with stirring for one-half hours. Then, the reaction mixture was added to water (100mL), and the mixture was stirred to obtain neutral solution with a 3% sodium hydroxide. After the concentration of the organic layer, it afforded 62.0g (yield 95%) of penta-acetyl-Iβ-D-glucopyranose as crude crystals. The crude crystals contained 13% of penta-acetyl-Iβ-D-glucopyranose, and after recrystallization in ethanol, 49.7g of pure penta-acetyl-Iβ-D-glucopyranose was achieved (m.p= 132AβC, yield 77%). Spectral analysis of ¹H NMR: I' (ppm)6.6 (d, 1H, J = 3.6Hz), 5.80 (t, 1H, J = 9.8Hz), 5.33 (t, 1H, J= 9.8Hz,), 5.24 (dd, 1H, J = 10.5, 3.7Hz), 4.29 (dd, J = 12.2, 4.3Hz), 4.08-4.15 (m, 1H), 4.0 (dd, J = 12.3, 2.2Hz), 1.50-1.70 (3s, 5CH3).

3.1. General Method of Coupling Busulfan to Glucose Penta-acetate

The glucose penta-acetate obtained from the previous step (1.5 mol, 0.55 g) was dissolved in pyridine (2mL), and the solution was cooled to 0AβC. Drops of mesyl-chloride (2 mmol, 0.155 ml) were added and stirred for 6 h. Distilled water was added (5 mL) to the reaction mixture until the formation of a precipitate. The precipitate was dissolved by CH₂Cl₂ (12 ml), and the organic phase was extracted with salt water (2 x 6 ml). It was then purified by dry sodium sulfate and finally purified using ethanol. Ft-IR (KBr, I... cm^-1): 2958, 1753, 1371, 1232, 1059.

3.2. Investigation of Cell Viability

In this study, to evaluate the viability of cells with 4a compound, Acridine Orange / Propidium Iodide (AO / PI) method was performed using a fluorescence microscope. The viability of cells was expressed as a percentage. For this purpose, the sample was stained with AO/PI at a rate of 10 Aβ l / ml and incubated for a quarter at 37 Aβ C and 5% carbon dioxide. After incubation and washing twice with PBS solution, 10 I1/4L of propidium iodide was added and incubated for 5 minutes at 37 Aβ C and 5% carbon dioxide. In the last step, samples were again washed with PBS solution and centrifuged for 10 minutes at 1400 rpm to remove the supernatant. Then, 50 I1/4L was removed from the bottom of the cell. After placing the slide under a fluorescence microscope with a 40 lens, 5 microscopic fields were examined.

3.3. Evaluation of the Effect of a Resultant Substance (carbohydrate-bound busulfan) on the Proliferation and Survival rate of K562 Cancer Cells and PBMC Cells

In this study, the effects of the resultant substance on cancer cells and PBMC cells were investigated. Concentrations were shown in micrograms of substance X per mL. Cell proliferation was assessed by the MTT method using an Elisa reader.

Cell suspension with a concentration of 1A-10⁵ was prepared from PBMC cells and K562. Cells under optimal growth conditions in 3 groups of 10⁴ K562 cells and 10⁵ PBMC cells were cultured separately in 96 house culture plates in RPMI 1640 medium containing 15% FBS with different concentrations of compound 4a. One group was considered a control, and the other groups were incubated in the presence of different concentrations of Royal X for 72 hours. A total of 25 I1/4L of 5 mg / mL MTT solution (3-4,5-dimethylthiazole 2-yl 2,5-diphenyltetrazolium bromide) in PBS was added to each well and incubated for 4 hours under similar conditions. During this time, living, proliferating cells with MTT formed formazan. A total of 100 I1/4L of DMSO was added to each well. The color intensity was determined at 492 nm. RPMI 1640 culture medium was also used as a blank.

4.1. The Procedure of Synthesis of Busulfan and Acetylation of Glucose

The reaction of glucose with acetic anhydride in the presence of sodium acetate and pyridine was completed within 4 hours and produced glucose penta-acetate (Fig. 1).

In this study, busulfan drug was designed and synthesized according to the general reaction (Fig. 2) and was identified by different spectroscopic methods, which were used for the interpretation of each spectrum. In the FT-IR spectrum, this compound eliminated the tensile vibration of the OH functional groups, confirming the formation of the drug compound. In the ¹H NMR spectrum, a combination of three categories of peaks was observed. First (originally ternary) in the 1.92 ppm region corresponded to two methylene, second in the 4.29 ppm region corresponded to two methylene attached to oxygen atoms (originally ternary), and third in the 3.03 ppm region belonged to the methyl group. In the ¹³C NMR spectrum, this combination of three peaks was observed in the regions of 67.8, 36.4, and 24.4 ppm, confirming the formation of the desired composition. In the mass spectrum of this compound, the molecular ion peak of the compound at 247 m / z indicated the formation of the drug compound busulfan (Fig. 4). GC analysis diagram of this compound shows the Busulfan peak time of 3.9 min (Fig. 5).

The acetylated carbohydrate was dissolved into dichloromethane, and the reaction mixture was cooled at 5 AβC. Mesyl chloride (3 mL) was then added drop-wise and stirred for 12 h. Finally, by adding 10 mL of distilled water, a precipitate was formed and dissolved in DMF solvent. The organic phase was extracted and washed twice with distilled water and dried with sodium sulfate. The process of galactose acetylation and the release of anomeric position using imidazole and reaction with busulfan are shown in Fig. (6). Moreover, the processes of lactose acetylation and deacetylation of the anomeric position using imidazole and reaction with Busulfan are shown in Figs. (2 and 3), respectively.

The samples of carbohydrates, consisting of glucose, galactose, xylose, fructose and lactose, were acetylated using Ac₂O/AcONa under 50-60AβC and anomeric configuration of acetylated carbohydrates using imidazole as a new catalyst was investigated. Busulfan coupling was performed at the anomeric position of sugar to prepare new drug derivatives. Their anti-carcinogenic properties were studied by AO / PI method (Fig. 6). In Ft-IR spectra, peaks at 1730 cm^-1 showed a carbonyl group and peaks at 1000 cm^-1 were related to the aliphatic group. Absorption peaks of esters appeared in the range of 1000-1300 cm^-1 due to the acetylation of galactose while organizing carbonyl in position 2 showed the effect of the neighboring group, so the existing quitter group took apart from carbon no.1 and organized stable carbocation. In this mode, there was a possibility of attacking carbon anomeric by the nucleophilic agent. The absorption band at 1379 cm^-1 was related to the symmetrical curvature of methyl CO-CH₃, and absorption at 1221cm^-1 was related to the neighbor C-O vibration of an ester carbonyl group. The absorption band at 1100 cm^-1 was due to the ester tension of the alcoholic part of an ester.

4.2. Evaluation of Anti-Cancer Efficacity Using AO/PI Cell Viability Survival Method

Microliters of cell suspension containing 100 cells were mixed with one microliter of staining solution and stirred slowly and thoroughly. Each sample was stained with color immediately before microscopic evaluation, and the samples were examined under a microscope, and the cells were counted. A microliter of the sample mixture was placed on a slide. At least 100 cells were counted with a fluorescein filter and a 60x object lens magnification. Depending on the cell type, different magnifications could be selected to distinguish the nucleus structure. Live and healthy cells would be uniformly green. Early apoptotic cells turn green and contain bright green spots in the nucleus due to chromatin condensation and nuclear fragmentation. Secondary (late-maturing) apoptotic cells absorb propidium iodide and are orange in color, except that, unlike necrotic cells, these cells often have dense fragmented nuclei. Orange-colored necrotic cells have a structure similar to healthy cells and do not have a dense nucleus. All cells that emit all or part of the orange light are considered dead, and all cells that emit only green light are considered alive.

4.3. Results of AO/PI Cell Viability Survival for Compound 4a

A total of 100 living cells were taken to determine primary apoptosis, delayed apoptosis, and necrosis. In the dual staining method of AO/PI, acridine penetrates all cells and causes the nucleus of cells to turn green. Propidium iodide only enters cells whose membranes are not healthy and causes a red color in their nuclei. So, green cells present healthy cells, whereas red cells present unhealthy cells. Cells that are light green with dense, fragmented chromatin are primary to apoptosis. Cells that are red with dense, fragmented chromatin are secondary to apoptosis, and those with normal, swollen, red nuclei are necrotic. Furthermore, the effective dose of the drug was determined to be 0.02 micrograms. The details of the cancer cell are given in Table 1 and Figs. (7-10).

The K562 cell line derived from a CML patient was examined for characteristics of B and T lymphocytes and cell lines. K562 lacks the B markers of immunoglobulins, Epstein-Barr virus (EBV) genome and associated nuclear antigen, and receptors for EBV.

4.4. Cell Line Description

K562 cells were established as the first human immortalized myelogenous leukemia line. This cell line was derived from a 45-year-old female chronic myelogenous leukemia patient in blast crisis. They can be used as a highly sensitive target for the in vitro natural killer assay. Recently, studies have shown the K562 blasts are multipotential, hematopoietic malignant cells that spontaneously differentiate into recognizable progenitors of the granulocyte, erythrocyte, and monocytic series.

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Busulfan acts non-specifically in the cell cycle by inhibiting DNA activity by alkylation and binding to the DNA strand. Alkylating agents usually bind an alkyl group to the number seven nitrogen atom in the guanine nucleotide in a DNA molecule (Fig. 11). A new derivative of busulfan di-alkylene agent binds to two different nitrogen atoms, thus causing cross-linking in DNA. DNA deformation inhibits tumor growth by preventing cell replication. The mechanism of action is through the alkylation of guanine adenine. Guanine-guanine cross-links occur through an SN₂ reaction in which the guanine N₇ nucleus attacks adjacent carbons to leave the mesylate group. This type of damage is not always repaired by cellular apparatus, thus causing cellular apoptosis.