Synthesis, anticancer activity and potential application of diosgenin modified cancer chemotherapeutic agent cytarabine.

Diosgenin (DG), a steroidal saponin, is mainly found in yam tubers. DG and its derivatives displayed significant pharmacological activities against inflammatory, hyperlipidemia, and various cancers. DG was selected to modify the cancer chemotherapeutic agent cytarabine (Ara-C) due to its anti-tumor activities as well as lipophilicity. After characterization, the biomembrane affinity and the kinetic thermal processes of the obtained DG-Ara-C conjugate were evaluated by differential scanning calorimetry (DSC). Thin hydration method with sonication was applied to prepare the DG-Ara-C liposomes without cholesterol since the DG moiety has the similar basic structure with cholesterol with more advantages. Dynamic Light Scattering (DLS) analysis and cytotoxic analysis were employed to characterize the DG-Ara-C liposomes and investigate their biological activities, respectively. The results indicated that DG changed the biomembrane affinity of Ara-C and successfully replaced the cholesterol during the liposome preparation. The DG-Ara-C liposomes have an average particle size of around 116 nm with a narrow size distribution and revealed better anti-cancer activity against leukemia cells and solid tumor cells than that of free DG or Ara-C. Therefore, it can be concluded that DG displayed the potential application as an anti-cancer drug carrier to improve the bio-activities, since DG counted for a critical component in modulating the biomembrane affinity, preparation of liposome, and release of hydrophilic Ara-C from lipid vesicles.

Cytosine arabinoside prodrug designed to bind plasma serum albumin for drug delivery.

Rational design of anticancer prodrugs for efficient albumin binding can show distinct advantages in drug delivery in terms of high drug availability, long systemic circulation, potential targeting effect, and enhanced chemotherapy effect. In the present study, we reported a cytosine arabinoside (Ara-C) prodrug which could well formulate in solution and instantly transform into long-circulating nanocomplexes by hitchhiking blood-circulating albumin after i.v. administration. Specifically, Ara-C was conjugated with an albumin-binding maleimide derivative, the resulting Ara-C maleimide caproic acid conjugate (AM) was well formulated in aqueous solution, conferring high albumin-binding ability in vitro albumin-binding studies. Moreover, in vivo fluorescence images of sulfo-cyanine5 maleimide indirectly demonstrated that AM showed better accumulation in tumors, exhibiting superior tumor targeting ability and antitumor activity compared to Ara-C. Such a uniquely developed strategy, integrating high albumin-binding capability, has great potential to be applied in clinical cancer therapy.

Probing Synergistic Effects of DNA Methylation and 2'-ß-Fluorination on i-Motif Stability.

The possible role of DNA i-motif structures in telomere biology and in the transcriptional regulation of oncogene promoter regions is supported by several recent studies. Herein we investigate the effect of four cytidine nucleosides (and combinations thereof) on i-motif structure and stability, namely 2'-deoxycytidine (dC), 2'-deoxy-5-methyl-cytidine (5-Me-dC), 2'-deoxy-2'-fluoro-arabinocytidine (2'F-araC), and 2'-deoxy-2'-fluoro-5-methyl-arabinocytidine (5-Me-2'F-araC). The base pair 5-Me-2'F-araC:2'F-araC produced i-motifs with a pH1/2 ("pKa ") value that closely matches physiological pH (7.34±0.3). NMR analysis of the most stable telomeric sequence (HJ-2) at pH 7.0 indicated that the structure is stabilized by hybrid 5-Me-dC:2'F-araC hemiprotonated base pairs and therefore highlights the significance of the interplay between base and sugar modifications on the stability of i-motif structures.

A new class of inhibitors of the AraC family virulence regulator Vibrio cholerae ToxT.

Vibrio cholerae is responsible for the diarrheal disease cholera that infects millions of people worldwide. While vaccines protecting against cholera exist, and oral rehydration therapy is an effective treatment method, the disease will remain a global health threat until long-term solutions such as improved sanitation and access to clean water become widely available. Because of this, there is a pressing need for potent therapeutics that can either mitigate cholera symptoms, or act prophylactically to prevent the virulent effects of a cholera infection. Here we report the design, synthesis, and characterization of a set of compounds that bind and inhibit ToxT, the transcription factor that directly regulates the two primary V. cholerae virulence factors. Using the folded structure of the monounsaturated fatty acid observed in the X-ray structure of ToxT as a template, we designed ten novel compounds that inhibit the virulence cascade to a greater degree than any known inhibitor. Our findings provide a structural and functional basis for the development of viable antivirulence therapeutics that combat cholera and, potentially, other forms of bacterial pathogenic disease.

Synthesis of dual-action parthenolide prodrugs as potent anticancer agents.

Cancer stem cells are responsible for the failure of a large number of cancer treatments and the re-emergence of cancer in patients. Parthenolide is a potent anticancer sesquiterpene lactone that is also able to kill cancer stem cells. The main problem with this compound is its poor solubility in water. To solve this problem, medicinal chemists have tried to prepare amino-derivatives of parthenolide, however, most amino-derivatives have less potency than that of parthenolide. In this paper, we proposed a new approach to synthesize parthenolide derivatives with better solubility and higher potency. We prepared novel parthenolide derivatives through the aza-Michael addition of nitrogen-containing anticancer drug molecules (cytarabine and melphalan) to the α-methylene-γ-lactone group of parthenolide. Different types of catalysts were used to catalyze the aza-Michael addition. Among all the used catalysts, 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) was found to have the highest catalytic activity. In addition, we examined the effects of parthenolide-anticancer drug hybrids on the growth and proliferation of three cancer cell lines (MCF-7, LNcaP, Hep G2) and CHO. The parthenolide prodrugs showed potent cytotoxic property with IC50 values ranging from 0.2 to 5.2µM, higher than those of parthenolide and anticancer drugs (cytarabine and melphalan).

Nanoassemblies from amphiphilic cytarabine prodrug for leukemia targeted therapy.

The anti-leukemia effect of cytarabine (Ara-C) is severely restricted by its high hydrophilic properties and rapid plasma degradation. Herein, a novel amphiphilic small molecular prodrug of Ara-C was developed by coupling a short aliphatic chain, hexanoic acid (HA) to 4-NH2 of the parent drug. Based on the amphiphilic nature, the resulting bioconjugate (HA-Ara) could spontaneously self-assemble into stable spherical nanoassemblies (NAs) with an extremely high drug loading (∼71wt%). Moreover, folate receptor (FR)-targeting NAs with high grafting efficient folic acid - bovine serum albumin (FA-BSA) conjugate immobilized on the surface (NAs/FA-BSA) was prepared. The results of MTT assays on FR-positive K562 cells and FR-negative A549 cells demonstrated higher cytotoxicity of HA-Ara NAs than the native drug. Especially, the IC50 values revealed that NAs/FA-BSA was 3 and 2-fold effective than non-targeted NAs after 24 and 48h treatment with K562 cells, respectively indicating FR-mediated enhanced anti-tumor efficacy. In vitro cellular uptake, larger accumulation of HA-Ara NAs were observed in comparative with the free FITC and the results further confirmed the selective uptake of NAs/FA-BSA in folate receptor enriched cancer cells. Above all, self-assembled HA-Ara NAs exhibited potential superiority for Ara-C delivery and FA-modified NAs would be an excellent candidate for targeting leukemia therapy.

A novel radiochemical approach to 1-(2'-deoxy-2'-[(18) F]fluoro-ß-d-arabinofuranosyl)cytosine ((18) F-FAC).

(18) F-FAC (1-(2'-deoxy-2'-[(18) F]fluoro-ß-D-arabinofuranosyl)-cytosine) is an important 2'-fluoro-nucleoside-based positron emission tomography (PET) tracer that has been used for in vivo prediction of response to the widely used cancer chemotherapy drug gemcitabine. Previously reported synthetic routes to (18) F-FAC have relied on early introduction of the (18) F radiolabel prior to attachment to protected cytosine base. Considering the (18) F radiochemical half-life (110 min) and the technical challenges of multi-step syntheses on PET radiochemistry modular systems, late-stage radiofluorination is preferred for reproducible and reliable radiosynthesis with in vivo applications. Herein, we report the first late-stage radiosynthesis of (18) F-FAC. Cytidine derivatives with leaving groups at the 2'-position are particularly prone to undergo anhydro side-product formation upon heating because of their electron density at the 2-carbonyl pyrimidone oxygen. Our rationally developed fluorination precursor showed an improved reactivity-to-stability ratio at elevated temperatures. (18) F-FAC was obtained in radiochemical yields of 4.3-5.5% (n = 8, decay-corrected from end of bombardment), with purities ≥98% and specific activities ≥63 GBq/µmol. The synthesis time was 168 min.

Synthesis of cytarabine lipid drug conjugate for treatment of meningeal leukemia: development, characterization and in vitro cell line studies.

Cytarabine (Cyt) used in the treatment of meningeal leukemia is associated with drawbacks like non selectivity to tumor cells, very short half-life and inability to cross blood brain barrier (BBB) due to its hydrophilic nature. Therefore, stable lipid drug conjugate (LDC) of Cyt with stearic acid was prepared. LDC was characterized by NMR, FTIR, DSC and XRD studies. Polysorbate 80 stabilized nanoparticles of this LDC (LDC-NP) were prepared using solvent injection method and characterized for size, zeta potential and loading efficiency. The LDC-NPs were loaded with appreciable amount (considering hydrophilic nature of drug, prior to conjugation) of drug conjugate (58.39 +/- 4.69%). The prepared LDC-NPs had smooth surface, particle size of 136.80 +/- 3.24 nm, were non-aggregated and had almost spherical and uniform shapes. In vitro release pattern showed initial fast release (14.89 +/- 0.056% in 1 h) followed by sustained release up to 72 h (76.26 +/- 0.156%). The blank stearic acid nanoparticles showed no significant cytotoxic effect on leukemic EL-4 cells and LDC-NPs were more cytotoxic than Cyt solution at 48 h. The lyophilized LDC-NPs were found to be physically stable with respect to size and zeta potential at refrigerated condition for 90 days. These results suggest that Polysorbate 80 stabilized LDC-NPs can be explored for treatment of meningeal leukemia owing to their ability of providing sustained drug release, stability and improved cytotoxicity in leukemic EL-4 cell line.

A facile whole-cell biocatalytic approach to regioselective synthesis of monoacylated 1-ß-D-arabinofuranosylcytosine: influence of organic solvents.

The lyophilized Pseudomonas fluorescens cell was an efficient alternative catalyst to enzymes for highly regioselective acylation of a polar nucleoside, 1-ß-D-arabinofuranosylcytosine (ara-C). The cells showed an evident solvent dependence in the reaction. Among the tested solvents except for acetonitrile-pyridine, catalytic activity of the cells clearly increased with increasing the polarity of the organic solvents used. Among all the tested solvents both pure and binary, the best results were observed in isopropyl ether-pyridine system, in which the catalyst also showed good thermal and operational stabilities. For the biocataylsis in isopropyl ether-pyridine, the optimal isopropyl ether concentration, water content, acyl donor/ara-C ratio, biocatalyst dosage and reaction temperature were 30% (v/v), 4%, 45, 50mg/mL and 30 °C, respectively, under which the initial rate, yield and 5'-regioselectivity were 2.93 mM/h, 77.1% and 97.3%, respectively. The bacterial cells exhibited comparable 5'-regioselectivity to the expensive immobilized enzyme, which could also have environmental and cost advantages.

Synthesis of n-squalenoyl cytarabine and evaluation of its affinity with phospholipid bilayers and monolayers.

Cytarabine (1-ß-D-arabinofuranosylcytosine, Ara-C), a pyrimidine nucleoside analogue, is an attractive therapeutic agent for the treatment of both acute and chronic myeloblastic leukemias. 1,1',2-tris-nor-Squalene acid (squaleneCOOH) has been conjugated to cytarabine with the formation of the squalenoyl-cytarabine prodrug, in order to improve the drug lipophilicity and, consequently, the affinity towards the environment of biological membranes, as well as of lipophilic carriers. The interaction of cytarabine and its prodrug with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and monolayers has been studied by the differential scanning calorimetry and the Langmuir-Blodgett techniques. The interaction has been evaluated considering the effect of the compounds on the DMPC MLV and monolayers behaviour. The aim was to have information on the interaction of the drug and the prodrug with the biological membranes and on the possibility to use liposomes as carriers for the prodrug. The results showed an improved affinity of the prodrug with MLV and monolayers with respect to the free drug.

In-vitro transdermal penetration of cytarabine and its N4-alkylamide derivatives.

OBJECTIVES: The aim of this study was to synthesise and determine the transdermal penetration of cytarabine alkylamide derivatives and assess the correlation of flux with physicochemical properties. METHODS: The alkylamide derivatives of cytarabine were synthesised by acylation at the N4-amino group by the mixed anhydride method. The in-vitro permeation studies were performed using the Franz diffusion cell methodology. Furthermore, partition coefficients (n-octanol-water) and aqueous solubility of the N4-alkylamide derivatives of cytarabine were determined in order to obtain information about their lipophilicity and hydrophilicity. KEY FINDINGS: The N4-alkylamides of cytarabine (acetyl, butanoyl, hexanoyl, octanoyl, and decanoyl derivatives) showed decreased hydrophilicity and increased lipophilicity. The log D values of the alkylamides were higher than that of the parent compound and increased linearly as the alkyl chain lengthened. N4-hexanoyl-4-amino-1-[(2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl] pyrimidin-2-one) showed the highest median steady-state flux (J(ss)) of 89.0 nmol/cm(2) per h in the series, which shows a high statistical difference with the parent compound flux value (3.70 nmol/cm(2) per h). CONCLUSIONS: The prodrug approach appears to be a promising strategy for the enhancement of transdermal penetration of cytarabine.

Synthesis and transdermal permeation of novel N4-methoxypoly(ethylene glycol) carbamates of cytarabine.

BACKGROUND: Cytarabine is a deoxycytidine analogue commonly used in the treatment of hematological malignant diseases. Its clinical utility, however, is severely limited by its short plasma half-life because of the catabolic action of nucleoside deaminases. METHOD: In this study, N(4)-carbamate derivatives of cytarabine (1) were synthesized and evaluated for transdermal penetration because this mode of administration may circumvent its limitations. The synthesis of these compounds was achieved in a two-step process. First, the methoxypoly(ethylene glycol) was activated by p-nitrophenyl chloroformate. Second, the activated intermediates were reacted with cytarabine in the presence of N-hydroxysuccinamide to give the N(4)-methoxypoly(ethylene glycol) carbamate derivatives. The transdermal flux values of the N(4)-carbamates of cytarabine were determined in vitro by Franz diffusion cell methodology. Aqueous solubility and log D (pH 7.4) values were determined and assessed for correlation with transdermal flux values. RESULTS: The synthesized carbamates, particularly, (9)-(13), showed increased solubility in both aqueous and lipid media. Log D values decreased as the oxyethylene chain lengthened. CONCLUSION: Although none of the derivatives showed significantly higher transdermal penetration than cytarabine (1), it should be mentioned that the mean for cytarabine N(4)-methoxyethyleneoxycarbamate (8) was 10 times higher and the median was 2 times higher.

Efficient synthesis of 5'-O-laurate of 1-beta-D-arabinofuranosylcytosine via highly regioselective enzymatic acylation in binary solvent mixtures.

Regioselective enzymatic acylations of 1-beta-D-arabinofuranosylcytosine (ara-C) with vinyl laurate (VL) in binary organic solvents were explored for the preparation of 5'-O-laurate of ara-C. Among the nine kinds of enzymes, Novozym 435 showed the highest regioselectivity (>99.9%) towards the 5'-OH of ara-C. This lipase showed higher catalytic activity in hexane-pyridine than in other tested solvent mixtures. The most suitable VL to ara-C molar ratio, initial water activity, and reaction temperature were shown to be 15:1, 0.07, and 50 degrees C, respectively, under which the initial reaction rate and the maximum substrate conversion were as high as 84.0 mmol L(-1) h(-1) and 98.1%, respectively. The product of Novozym 435-catalyzed acylation was characterized by (13)C NMR and confirmed to be 5'-O-laurate of ara-C.

Transdermal penetration of cytarabine and its 5'-O alkyl ester derivatives.

The purpose of this study was to synthesize and determine the in vitro transdermal penetration of cytarabine and its 5'-alkyl esters and to establish a correlation, if any, with selected physicochemical properties. The n-alkyl esters were synthesized by acylation of cytarabine (1) at its pharmacophoric 5'-OH. The transdermal flux values of (1) and its esters were determined in vitro using Franz diffusion cell methodology. Aqueous solubility and log D (pH 7.4) values were determined and assessed for correlation to transdermal flux. An inverse relation was observed between the water solubility (Sw) and log D values. Of all esters, (4) exhibited the highest flux value of 22.2 nmol x cm(-2) x h(-1), which is significantly different to that of the parent drug cytarabine (3.70 nmol x cm(-)2 x h(-1)). No trend was found between water solubility and flux values.

Special feature of mixed phosphotriester derivatives of cytarabine.

Despite the unquestionable therapeutic interest of bis(SATE) pronucleotides, a presystemic metabolism preventing the delivery of the prodrugs in target cancer cells or tumours may constitute a limitation to the in vivo development of such derivatives. In order to overcome these drawbacks several strategies have been envisaged and we report herein the application of the S-acyl-2-thioethyl (SATE) phenyl pronucleotide approach to the well-known cytotoxic nucleoside cytosine-1-beta-D-arabinofuranoside (cytarabine, araC). We describe modifications of the SATE moieties with the introduction of polar groups on the acyl residue, in order to study how these changes affect antitumoral activity and metabolic stability. Two different synthetic pathways were explored and lead to obtain the corresponding mixed derivatives in satisfactory yields. Cytotoxicity was studied in murine leukaemia cells L1210 as well as in cells derived from solid human tumours (Messa and MCF7). Biological evaluation of these compounds in cell culture experiments with nucleoside analogue-sensitive and resistant cell lines showed that the modified compounds were active at higher concentrations than unmodified cytarabine, yet were much able to partially reverse resistance due to deficient nucleoside transport or activation. These results can be correlated with an incomplete decomposition mechanism into the corresponding 5'-mononucleotide.

Discovery of a new template for anticancer agents: 2'-deoxy-2'-fluoro-4'-selenoarabinofuranosyl-cytosine (2'-F-4'-seleno-ara-C).

The first synthesis of 2'-deoxy-2'-fluoro-4'-selenoarabinofuranosyl pyrimidines as potent anticancer agents was accomplished using the DAST fluorination as a key step. It was first revealed that selenium atom participated in the DAST fluorination of 4'-selenonucleosides and that conformational bias induced by bulky selenium acted as a decisive factor in the DAST fluorination. Among compounds tested, 2'-F-4'-seleno-ara-C (4a) exhibited highly potent anticancer activity in all cancer cell lines tested and was more potent than ara-C (1).

Synthesis of radiolabeled cytarabine conjugates.

N4-Modified, novel Ara-C conjugate capable of radiolabeling with gamma ray-emitting ((99m)Tc) as well as positron emitting ((18)F) radionuclides, that is, N4-hydrazine derivative was synthesized. The radiolabeling of N4-(hydrazinonicotinyl)-1-beta-arabinofuranosyl cytosine (HAra-C) with (99m)Tc was performed with over 95% labeling yield. To label HAra-C with (18)F, 4-fluoro((18)F)-benzaldehyde was synthesized from 4-formyl-N,N,N-trimethylanilinium triflate in 30% radiochemical yield; it quantitatively formed hydrazone derivative with HAra-C within 45min. The radiolabeled conjugates were analyzed by radio-UV-RP-HPLC. The cold precursors were characterized by (1)H, (13)C NMR. Additionally, HAra-C was evaluated for cytotoxicity in lung adenocarcinoma (H441) cells and found to be comparable in cell killing efficiency to that of Ara-C. Uptake of (99m)Tc-HAra-C in cultures of H441 cells and sensitive pancreatic cancer cells (MIAPaCa-2) was inhibited by nucleoside transporter inhibitor nitrobenzylthioinosine. The results suggest that (99m)Tc-labeled HAra-C is a substrate for the membrane nucleoside transporters, and that it may be used in molecular imaging of nucleoside transporter expression for the verification of potential anticancer efficacy of nucleoside drugs, such as Ara-C and gemcitabine.

Synthesis and evaluation of anti-tumor activities of N4 fatty acyl amino acid derivatives of 1-beta-arabinofuranosylcytosine.

1-Beta-D-arabinofuranosylcytosine (Ara-C, Cytarabine) is one of the drugs used for acute nonlymphocytic leukemia (ANLL). However, the bioavailability of Ara-C is relatively low due to its low lipophilicity. In order to improve the lipophilicity and bioavailability of Ara-C, a series of N(4) derivatives of Ara-C, i.e., (fatty acid)-(amino acid)-Ara-C analogues, were prepared. The 15 derivatives synthesized were characterized by their melting points, optical rotations and partition coefficients. It was found that the Ara-C derivatives synthesized in this study were more lipophilic than Ara-C as determined by their partition coefficients. Their in vitro cytotoxicity and in vivo anti-tumor activity were determined and compared with that of Ara-C. It was found that the derivatives were more active than Ara-C in Hela cells, but not in HL-60 cells. The in vivo results showed that some of the derivatives were more effective than Ara-C in mice bearing S(180) tumor while others showed a decreased activity in comparison with Ara-C.

Radiosynthesis of F-18 labeled cytidine analog 2'-fluoro-5-iodo-l-beta-d-arabinofuranosylcytosine ([(18)F]FIAC).

We reported the synthesis of 2'-deoxy-2'-[(18)F]fluoro-5-iodo-1-beta-d-arabinofuranosyl-5-iodo-cytosine ([(18)F]FIAC) with 15-20% radiochemical yield (decay corrected) in 3.5h. 2-deoxy-2-[(18)F]fluoro-1,3,5-tri-O-benzoyl-alpha-d-arabinofuranose was prepared following literature procedures with some modifications (yield>70%). The (18)F-fluorosugar was converted to 1-bromo-(18)F-fluorosugar, and then coupled with 5-iodocytocine silyl ether. A mixture of acetonitrile (ACN) and 1,2-dichloroethane (DCE) were employed to achieve optimum radiochemical yield and acceptable beta-anomer selectivity (alpha/beta=1/3). After hydrolyzed with sodium methoxide, the crude product was purified using HPLC to afford the beta-[(18)F]FIAC with high radiochemical purity (>or=98%).

Synthesis, transport and pharmacokinetics of 5'-amino acid ester prodrugs of 1-beta-D-arabinofuranosylcytosine.

Cytarabine (1-beta-d-arabinofuranosylcytosine, ara-C, 1) suffers from low oral bioavailability due to low intestinal membrane permeability and poor metabolic stability, and intravenous infusion is usually adopted as the clinical standard dosing administration. To develop an oral alternative for 1 and utilize the intestinal oligopeptide transporter 1 (PepT1), a series of 5'-amino acid ester derivatives of 1 was synthesized to clarify which modification was the most suitable to increase the oral bioavailability of 1. Their apical-to-basolateral permeability across Caco-2 cells and the antiproliferative activity with HL-60 cells were screened. 5'-Valyl prodrug 2 demonstrated the highest permeability and was selected for further study. Glycylsarcosine (gly-sar, a typical substrate of PepT1) uptake by Caco-2 cells can be inhibited by 2 in a concentration-dependent manner, and IC(50) for 2 was 2.18 +/- 0.12 mM. The uptake of 2 was markedly increased in the long-term leptin-treated Caco-2 cells compared with the control Caco-2 cells, and was significantly inhibited by the excess of gly-sar, but not by l-valine. A dose-proportional pharmacokinetics was observed in rats when 5, 15, 30 mg/kg doses of 2 (calculated as 1) were orally administered. The oral absolute bioavailability of 1 was 60.0% and 21.8% after 2 and 1 were orally administered to rats 30 mg/kg, respectively. Following oral administration of 15 mg/kg, the absorption and bioactivation of 2 were extensive and rapid, over 98% of prodrug hydrolysis occurring before appearance in the portal vein. The in vivo dispositions of 1-beta-D-arabinofuranosyluracil (ara-U), a deaminated product of 1, were investigated. Oral administration of 2 resulted in an increased 1/ara-U ratio (2.76) in the blood, much higher than that (1.25) after 1 orally taken. Overall, these results demonstrated that the PepT1-mediated absorption of 2 and the increased metabolic stability resulted in a dramatic increase in the oral bioavailability of 1 in rats and further corroborated the thought that prodrug design strategy targeting intestinal PepT1 was an important and promising strategy to improve oral bioavailability of poorly absorbed drugs.