Synthesis and biological evaluation of a fatty acyl di-cytarabine prodrug

Abstract

Here, we developed a prodrug for acute myelogenous leukemia (AML) which could overcome the disadvantage of cytarabine such as short plasma half-life and rapid deamination to its inactive metabolite. The new drug-fatty acyl conjugate (Ara-R-Ara) has been synthesized from the hydrophilic anticancer drug cytarabine and hydrophobic fatty acyl suberoyl chloride via a hydrolyzable amido linkage. Suberoyl chloride has been conjugated to cytarabine to protect the NH₂ group from the enzymatic attachment to exhibit a longer blood retention half-life compared with the free drugs. A higher membrane permeability of Ara-R-Ara was obtained compared with pure cytarabine, about 15.4 times the pure drug sample in PAMPA permeability studies. In vitro cytotoxicity results showed that the new prodrug had a much lower IC₅₀ and a higher cell inhibition rate compared with the pure cytarabine for HL60 and K562 cells, indicating its effective therapy for leukemic cells.

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Introduction

Acute myeloid leukemia (AML) is a clonal disease characterized by the proliferation and accumulation of myeloid progenitor cells in the bone marrow, which ultimately leads to hematopoietic failure.¹ 1-β-D-Arabinofuranosylcytosine (cytarabine, Ara-C) (Fig. 1), a pyrimidine nucleoside analogue, is predominantly used in the treatment of both acute and chronic myeloblastic leukemias.^2,3 It is the cornerstone of induction therapy and consolidation therapy for AML. Cytarabine (Ara-C) as nucleoside analogues is inactive by itself and requires phosphorylation to the corresponding triphosphate (Ara-CTP) in vivo to exert its antineoplastic activity by inhibition of nucleic acid biosynthesis and is rapidly deaminated by cytidine deaminase (CDA). At a molecular level, cytarabine anticancer mechanisms rely on sequential conversion from monophosphate, diphosphate, to triphosphate forms. Cytarabine triphosphate replaces deoxycytidine during DNA replication, which leads to cell arrest.⁴ Failure to repair the DNA subsequently triggers apoptosis and blocks tumor growth. Unfortunately, Cytarabine, like all nucleoside analogues (such as gemcitabine, 5-fluorouracil, and fludarabine), suffers from several limitations. It has a low permeability in intestinal membrane and is rapidly deaminated to biologically inactive 1-β-D-arabinofuranosyluracil in intestinal and hepatic cells leading to a very low oral bioavailability (F = 20%).⁵ However, under intravenous or subcutaneous administration, it is also rapidly inactivated by systemic deamination which finally results in short plasma half-life.⁵ Consequently, cytarabine must be administered in continuous infusion or on a complex schedule to maintain plasma levels and longer effective concentration in clinic application. In addition, the use of high-dose cytarabine has adverse risk for patients. Therefore, extensive strategies to improve the treatment efficiency of cytarabine have been developed, including the chemical modifications and functionalization for the sustained release of this kind of drugs.^6–8

Fig. 1 Structure of cytarabine molecule.

Prodrug has been considered a chemistry-enabled drug delivery tool used to address shortcomings in bioavailability, efficacy, or safety profiles of otherwise promising candidates. For example, the highly hydrophilic drug gemcitabine has been modified into lipophilic prodrug in order to improve the pharmacokinetic behavior and the antitumor activity.⁹ Moreover, it has been reported that the modifications focused on 4-amino group can obviously increase the stability of gemcitabine and maintain its biological activity.^10,11 Therefore, it may protect cytarabine from deaminate and promote the concentration of active agent in the tumor cells on modifying the amino of cytarabine. In recent years, the efficacy of conjugation of antitumor drugs with fatty acid has been evaluated by other groups. In 1998, Eli Lilly patented the synthesis of lipophilic prodrug with saturated and monounsaturated, C18 or C20 long-chain to protect against the deamination of gemcitabine. A series of increasingly lipophilic prodrugs of gemcitabine are subsequently synthesized by linking the 4-amino group with valeroyl, heptanoyl, lauroyl and stearoyl linear acyl derivatives.¹² Many acyl derivatives of cytarabine such as 4-(N)-hexadecyl (NHAC) and 4-(N)-octadecyl (NOAC) 1-β-D-arabinofuranosylcytosine have also been synthesized.¹³ However, the reported prodrugs are mono-cytarabine derivatives and the drug loading contents of prodrug molecules have not been taken into consideration in the previous research. Drug loading content is certainly one of key issues for efficient anticancer treatment in drug delivery systems. It has reported that low drug loading content significantly limit the clinical application because repeated administration of a large amount of material into patients may induce systemic toxicity.^14,15 Therefore, a novel prodrug design reducing the introduction of material and avoiding an extra burden for the patients to excrete the carriers is necessary in the future study.

In the present study we designed a new di-cytarabine prodrug with two moleculars cytarabine and one molecular suberoyl chloride chain via a hydrolyzable amido linkage (Ara-R-Ara). The Ara-R-Ara prodrug was synthesized by an effective method^10,11 and the fatty acid linkage is very important for the prodrug design. Firstly, suberoyl chloride has two reaction head groups and it is suitable as a linker to synthesize di-cytarabine prodrug. Secondly, suberoyl chloride can make the product (Ara-R-Ara) possess the proper polarity to penetrate the cell membrane.¹² The advantages of Ara-R-Ara prodrug are the following: (a) protecting the NH₂ group of cytarabine to avoid the deactivation by deamination (b) reducing the introduction of inactive material and markedly enhancing the drug loading (77.4% in theory) than mono-amide prodrugs. (c) Markedly increasing the lipophilicity of cytarabine thus enhancing the ability of the drug to penetrate the cell membrane. ¹H-NMR spectra and mass spectrometry (MS) were performed to characterize the prodrug synthesized successfully. To assess the feasibility of the prodrug for injection administration, hemolytic toxicity test was carried out. Parallel artificial membrane permeability assay (PAMPA) was used to measure the effective permeability and cytarabine prodrug cell proliferation inhibition was tested by in vitro cytotoxicity on HL60 cells and K564 cells. The results indicate that the prodrug molecule has a higher membrane permeability value, about 15.4 times of pure cytarabine and it has the much better anticancer efficiency to HL60 and K564 cells.

Experimental section

Materials

Cytarabine was purchased from Aladdine. Suberoyl chloride was purchased from J&K Chemical Ltd. Methanol, dichloromethane, triethylamine and acetic acid were purchased from Tianjin Fuyu Fine Chemical Co., Ltd, China. Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB Ltd.

Synthesis and purification of prodrug molecule Ara-R-Ara

The synthetic route of Ara-R-Ara was shown in Scheme 1. The mole ratio of cytarabine and suberoyl chloride and triethylamine is 1 : 0.5 : 1.2 in anhydrous DMF and the reaction mixture was kept under stirring at 0 °C for 4 h and room temperature for 24 h. Then the mixture was purified by chromatography on silica gel and Sephadex LH-20.

Scheme 1 The synthetic route of Ara-R-Ara.

¹H-NMR measurements. The obtained cytarabine fatty acyl derivative was characterized by ¹H-NMR. The ¹H-NMR data was recorded on a Bruker Avance 400 spectrometer operating at 400 MHz at room temperature. Ara-R-Ara was freshly dissolved in 0.5 mL DMSO-d₆ in NMR tubes. Chemical shifts were reported in ppm with respect to tetramethylsilane.

Mass spectrometry. Mass spectra were performed by the analytical and the mass spectrometry facilities in Drug Analysis Center at Shandong University on Agilent Technologies 1100 infinity HPLC, Applied Biosystems API4000.

Melting point and solubility. The melting point of Ara-R-Ara prodrug was measured by automatic melting point apparatus (RY-1, Tianjing Analysis, China). In order to assess the saturation solubility of the newly synthesized cytarabine derivative, the solubility experiments were conducted with a magnetic stirrer (RCT Basic, IKA, Staufen, Germany). Excess pure Ara-R-Ara powder was dispersed in aqueous solution. The temperature and stirring rate were set at 37.0 ± 0.5 °C and 100 rpm. After stirring 72 h, 1 mL samples were withdrawn and centrifuged at 14 000 rpm for 10 min with Zonkia HC-2062 high speed centrifuge (Anhui USTC Zonkia Scientific Instruments Co., Ltd.), then the supernatants were filtered using 0.22 μm microporous membrane filter before ultraviolet spectrophotometrically analysis. The experiment was carried out in triplicate.

Hemolytic activity. The RBC suspension was obtained as the well-known reported procedure for hemolytic studies.¹⁶ New Zealand White rabbits blood was centrifuged at 4000 rpm for 10 min and resuspended in normal saline solution (0.9% NaCl solution) to obtain the red blood cells suspension (RBCs 2%). Negative control (producing no hemolysis) was prepared with 2.0 mL of RBCs dispersed in 8.0 mL normal saline solution and 2.0 mL of RBCs dispersed in a in 8.0 mL distilled water as a positive control (producing 100% hemolysis). One milliliter of adequately diluted plain Ara-C or Ara-R-Ara solution was incubated with 2 mL RBCs suspension and 1 mL normal saline at 37.0 ± 1.0 °C for 2 h, then the solutions were centrifugation at 4000 rpm for 15 min. The supernatant was collected and measured spectrophotometrically at 541 nm which is the typical absorbance of haemoglobin (Hb) released from RBCs, using normal saline as blank. The formulations were taken in separate tubes in such amount that the resultant final concentration of Ara-C was equivalent in all the cases so as to facilitate the comparison of the extent to hemolysis. The degree of hemolysis was determined for each sample using the following equation.
where A_samples refers to the ultraviolet absorption of Ara-C and Ara-R-Ara at 541 nm. A_{negative control} and A_{positive control} refers to negative control and positive control at 541 nm, respectively.

Cell culture. Human leukemic cell lines K562 and HL60 were kindly supplied by Immunopharmacology Institute of Shandong University and Shandong Analysis and Test Center which were respectively used in the treatment chronic granulocytic leukemic cell line and acute myeloblastic leukemic cell line. The culture medium contains RPMI-1640 supplemented 10% fetal bovine serum (GIBCO, Vitrogen Corporation), 100 units per mL penicillin, and 100 μg mL⁻¹ streptomycin. The cells were cultivated in the culture medium and incubated in a humidified (37 °C, 5% CO₂) incubator (SANYO), grown in 30 mL culture flasks (Falcon BD).

In vitro cytotoxicity. Cytotoxicity of cytarabine and Ara-R-Ara prodrug were evaluated by measuring the cell viability using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. K562 and HL60 cells were placed into 96-well plates with the cell density being 1.0 × 10⁴ cells per 100 μL per well in medium with different sample concentration. After cultivated for 24 h and 48 h, each well was added 10 μL of 5% MTT reagents (Dojindo, Kumamoto, Japan) and incubated at 37 °C for 4 h. Then the medium was removed and 150 μL of DMSO was added to each well, the optical density of cells was determined at 490 nm using a microplate reader (Enspire instruments, Perkin Elmer, America). All experiments were performed in triplicate to determine the mean values and SDs.

Parallel artificial membrane permeability assay (PAMPA). Scheme 2 showed the schematic representation of PAMPA experiment. PAMPA studies were carried out using the following protocol. The donor and acceptor compartments are two 96-well filter plates with artificial membrane coating on. And artificial membrane solution consisted of 1.5% (w/v) L-α-phosphatidylcholine (PC) and 0.5% (w/v) stearic acid (SA) in 1, 7-octadiene. The donor solutions containing 250 μL of 20 mM test compounds and the acceptor plate was filled with 350 μL PBS (pH 7.4). Then the sample was incubated at 30 °C for 16 hours. Finally, samples were collected from the receiver and donor wells and analyzed by UV spectrophotometry. The permeability coefficient (P_am) representing the transport ability through the membrane was calculated by the following equations.¹⁷
where V_dn (mL) is the volume of the donor compartment, V_ac (mL) is the volume of the acceptor compartment, S (cm²) is the membrane area (0.24 cm²), t (s) is the incubation time, C_dn (μM L⁻¹) is the concentration of drug in the donor compartment, C_ac (μM L⁻¹) is the concentration of drug in the acceptor compartment, C_equilibrium is theoretical equilibrium concentration.

Scheme 2 Schematic representation of PAMPA experiment.

Data analysis was as follows. Three replicates of each experiment were used. Student's T test was used to evaluate the significance of difference (SPSS statistics 17.0). A minimum P value of 0.05 was fixed as the significance level.

Results and discussion

Chemical structure of prodrug molecule

The yield of the cytarabine derivative was 37.68% and the desired compound was obtained as white powder. ¹H NMR spectrum of Ara-R-Ara sample (DMSO-d₆, δ, ppm) (Fig. 2): 10.78 (s, 1H, –NH), 8.05 (d, 1H, J = 7.60 Hz, 6-H) 7.21 (d, 1H, 1′-H), 6.06 (d, 1H, J = 7.60 Hz, 5-H), 5.48 (br, 2H, 2′-OH, 3′-OH), 5.05 (t, 1H, J = 5.1 Hz, 5′-OH), 4.03–4.09 (m, 1H, 2′-H), 3.90–3.95 (m, 1H, 4′-H), 3.79–3.86 (m, 1H, 3′-H), 3.61 (t, 2H, J = 2.8 Hz, 5′-H), 2.39 (t, 4H, alpha-H), 1.43–1.61 (m, 4H, beta-H), 1.21–1.35 (m, 8H, gamma-H). The analysis of ¹H NMR spectrum for Ara-R-Ara prodrug has been put in ESI.†

Fig. 2 ¹H-NMR spectra of prodrug Ara-R-Ara. The integral area values of peaks have been given under peak.

ESI-MS for C₂₆H₃₆N₆O₁₂ (M + H⁺) is 625.3 (Fig. 3).

Fig. 3 Mass spectra of prodrug Ara-R-Ara.

Melting point and solubility

Table 1 showed the saturation solubility and melting points of pure cytarabine and Ara-R-Ara in water. The melting point of Ara-R-Ara prodrug was in the range of 87–89 °C and the saturation solubility of pure Ara-R-Ara was 761.48 μg mL⁻¹. Compared with pure cytarabine, the solubility of the Ara-R-Ara was decreased due to the hydrophobic suberoyl chloride part.

Table 1 Melting points and saturation solubility of cytarabine and Ara-R-Ara

Samples	Melting points (°C)	Saturation solubility (mg mL^−1)
Cytarabine	212–214	437.4 ± 7.11
Ara-R-Ara	87–89	0.7615 ± 0.044

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Hemolytic toxicity

Taking into account the possibility of the i.v. administration, the hemolytic potential was evaluated using red blood cell (RBC) hemolysis measurement. Once cell membrane was damaged, haemoglobin (Hb) releases from the red blood cell. It is a simple and widely method to study molecular–membrane interaction by measuring haemoglobin (Hb) release and a good indicator of drugs' toxicity in the in vivo conditions. The data obtained in the measurement also gives a qualitative indication of potential of intravenous injection. Fig. 4 summarized the results of the hemolytic toxicity assay of cytarabine and the prodrug Ara-R-Ara after 2 h incubation at concentrations from 5 × 10⁻⁴ to 4 × 10⁻³ mol L⁻¹. In the study, the hemolytic rate of cytarabine and Ara-R-Ara didn't change with their concentrations and the prodrug Ara-R-Ara solution showed merely 2–5% larger than that of cytarabine. It has been reported that hemolysis may be caused by direct disruption of the membrane through solubilization of membrane lipids or by the intercalation of the compounds into the membrane changing the permeability of membrane.¹⁸ Compared with cytarabine, the Ara-R-Ara showed higher lipophilic property by inducing the hydrocarbon chain. Thereby, it was easier to intercalate the membrane and enlarged the permeability of membrane, resulting in higher hemolytic rate. However, the hemolytic rates of the prodrug Ara-R-Ara were slightly larger than 5% which was the criteria in the biological function test of biomaterial followed the Standard Practice for Assessment of Hemolytic Properties of Materials (ASTM F756-2008), indicating its unsecurity for intravenous administration in drug delivery system.

Fig. 4 Dependence of erythrocyte hemolysis on samples' concentration incubated at 37.0 ± 0.5 °C for 2 hours. Each point represents the mean value of three independent experiments ±SD (error bars).

In vitro cytotoxicity

The cytotoxicity of prodrug Ara-R-Ara against acute and chronic myeloblastic leukemias HL60 cells and K562 cells were subsequently assessed using standard MTT assay (Fig. 5). It should be noted that the concentrations of Ara-C is double of Ara-R-Ara concentration in each control group in order to keep the same concentration active component of Ara-C. Obviously, Ara-R-Ara showed significant cytotoxicities to the two investigated cell lines compared with the contrast group. The cells incubated with Ara-R-Ara are collapsed and shrinkage, and the number of cells was significantly reduced. The Ara-R-Ara samples had no distinct difference compared with the Ara-C except the slight reduced cells number.

Fig. 5 Phase contrast microscope of HL60 cells and K562 cells after incubation of different samples.

In order to quantitatively investigate the cytotoxicity of the samples, we calculated cell inhibition rates which were shown in Fig. 6 and Table 2. As depicted in Fig. 6 Ara-R-Ara showed similar cytotoxicity to both HL60 and K562 cells when compared with free Ara-C. For HL60 cells, Ara-R-Ara showed a higher cytotoxicity than Ara-C when C < 100 μM incubated 24 h (Fig. 6a), and a remarkable higher cytotoxicity in all the doses tested incubated 48 h (Fig. 6b). Ara-R-Ara was able to inhibit the growth by approximately 45.30% and 92.04% at a concentration of 10 μM after incubation for 24 h and 48 h, and they were remarkable higher than 19.76% and 34.76% of Ara-C at the same concentration. It could be concluded that the prodrug Ara-R-Ara molecules took action faster and showed more durable cytotoxicity effect compared with cytarabine. With the increase of concentration, the cytarabine also showed high cytotoxicity as same as prodrug Ara-R-Ara. From an overall perspective, prodrug Ara-R-Ara and Ara-C were both more sensitive to HL60 than K562 cells, and prodrug Ara-R-Ara showed better inhibitive effect on K562 cells. Cell inhibition rates for K562 cells were shown in Fig. 6c and d. There was no significant difference between cytarabine and Ara-R-Ara solution when incubated for 24 h (Fig. 6c), however, Ara-R-Ara prodrug showed a higher cytotoxicity than Ara-C when incubated for 48 h (Fig. 6d).

Fig. 6 Cell inhibition rates of different treatment groups to HL60 cells and K562 cells following 24 h and 48 h incubation. All the experiments were performed in triplicate.

Table 2 IC₅₀ of K562 cells and HL60 cells incubated with Ara-C and Ara-R-Ara (μM)

Cell lines	Ara-C		Ara-R-Ara
	24 h	48 h	24 h	48 h
K562	>1000	60.05	349.46	32.78
HL60	52.70	8.837	63.70	0.8329

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The percentage of surviving cells was calculated as the absorbance ratio of treated to untreated cells. The inhibitory concentration 50% (IC₅₀) of the treatments was determined from the dose–response curve. Table 2 showed the IC₅₀ for each agent added alone to HL-60 cells and K562 cells. It was shown that Ara-R-Ara showed lower IC₅₀ values compared with free cytarabine in the two cell lines. In Table 2, HL60 cells were more sensitive to pure cytarabine compared with K562 cells. After 48 h incubation with K562 cells, the IC₅₀ values decreased from 349.46 μM to 32.78 μM for Ara-R-Ara obviously below the pure cytarabine, meaning the highest anti-tumor activity. And there was no significant difference between the IC₅₀ values of two groups for HL60 cells in 24 h, whereas in 48 h the IC₅₀ values of Ara-R-Ara were smaller than that of cytarabine, which meaning the anticancer effect of Ara-R-Ara were better than Ara-C for the same cell line.

The enhancement of cytotoxicity of Ara-R-Ara seems due to the effect of increased hydrocarbon chain from suberoyl chloride chain molecules and improved affinity of Ara-R-Ara with the membranes which makes cytarabine molecules intaked by cells more easily. On the other hands, higher expression of cytidine deaminase (CDA) in AML cells results in a certain degree inactivation of cytarabine by rapid deamination, but it has less impact on the prodrug Ara-R-Ara because NH₂ has been protected.

PAMPA permeability studies

Due to ethical and economical considerations associated with in vivo tests, an in vitro approach is the preferred approach for assessing the rate and extent of absorption of a compound through cell membrane. Parallel artificial membrane permeability assay (PAMPA) as a high-throughput screening (HTS) tool for compounds is one of the most interesting techniques which is able to measure the relative passive transcellular diffusion of compounds with reasonable accuracy.^19,20 The 96-well plate format has been demonstrated to be suitable for the rapid determination of passive transport.²¹ PAMPA used for the assessment of gastrointestinal absorption, and blood–brain barrier permeability and skin have been reported.^22,23 The permeability coefficient P_am which is used to reflect the passive diffusion behavior of gastrointestinal absorption were calculated and the results are shown in Table 3. The two samples contain the same mole of cytarabine in order to compare. These results suggest that Ara-R-Ara had better membrane permeability compared with pure cytarabine which was about 15.4 times of pure drug sample. It has been reported that the degree of transport enhancement depends on the physicochemical properties of the drug (primarily lipophilicity). Modification of –NH₂ group increases the lipotropism of the molecule, which results in easier across to the cell membrane.

Table 3 C and P_am values for the cytarabine and Ara-R-Ara

Samples	C (×10^3 mol L^−1)	Pam (×10^6 cm s^−1)
Cytarabine	1.506	1.15 ± 0.38
Ara-R-Ara	0.753	17.71 ± 1.14

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Conclusions

A new di-cytarabine prodrug (Ara-R-Ara) was successfully synthesized. The chemical structure of the derivative was confirmed using ¹H-NMR and MS. The derivative was characterized by melting point and solubility. The first important advantage of di-cytarabine prodrug is the high drug loading content which is the key issue for efficient anticancer treatment in drug delivery systems. Secondly, the introduction of fatty acid linkage can markedly increase the lipophilicity of cytarabine, which can increase the bioavailability of cytarabine. The PAMPA assay results indicate that the permeability of Ara-R-Ara prodrug is about 15.4 times of the cytarabine solution. The results of hemolytic toxicity studies demonstrate that the Ara-R-Ara prodrug is unsuitable for intravenous administration in drug delivery system. The Ara-R-Ara cytotoxicity was evaluated for the inhibition of HL60 and K562 cells, the data indicated that the prodrug molecules markedly increased the cytotoxicity of cytarabine. It can be concluded that it is possible using structure modification to improve the anti-leukemia activity of cytarabine molecule. The synthesized prodrug molecule showed a bright prospect for the further application of cytarabine. Further optimization is on-going to produce more effective prodrugs with improved cell proliferation inhibition.

Acknowledgements

We gratefully acknowledge financial support from National Natural Science Foundation of China (NSFC, 21373126) and China–Australia Centre for Health Sciences Research (CACHSR).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra17255h

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