Synthesis of novel paclitaxel prodrugs designed for bioreductive activation in hypoxic tumour tissue.

The syntheses and preliminary evaluation of the first potential bioreductive paclitaxel prodrugs are described. These prodrugs were designed as potential candidates in more selective chemotherapy by targeting hypoxic tumour tissue. Aromatic nitro and azide groups were used as the bioreductive trigger. Generation of paclitaxel occurs after reduction and subsequent 1,6-elimination or 1,8-elimination. All prodrugs are stable in buffer and indeed give paclitaxel after chemical reduction of the aromatic nitro or azide functionality. In aerobic cytotoxicity assays several prodrugs exhibit diminished cytotoxicity. These compounds are interesting candidates for further biological evaluation.     

Modulating paclitaxel bioavailability for targeting prostate cancer

Four novel water-soluble peptide-paclitaxel conjugates were designed and synthesized as prostate-specific antigen (PSA)-activated prodrugs for prostate cancer therapy. These prodrugs were composed of a peptide, HSSKLQ or SSKYQ, each of which is selectively cleavable by PSA; a self-immolative linker, either para-aminobenzyl alcohol (PABS) or ethylene diamine (EDA); and the parent drug, paclitaxel. Introduction of a PABA or EDA linker between the peptide and paclitaxel in prodrugs 2-5 resulted in products with an increased rate of hydrolysis by PSA. The stability of prodrugs 2 and 3, with the PABA linker, was poor in the serum-containing medium because of the weak carbonate bond between the PABA and paclitaxel; however, this disadvantage was overcome by introducing a carbamate bond using an EDA linker in prodrugs 4 and 5. Thus, the incorporation of an EDA linker increased both the stability and PSA-mediated activation of these prodrugs. The cytotoxicity of each prodrug, as compared to paclitaxel, was determined against a variety of cell lines, including the PSA-secreting CWR22Rv1 prostate cancer cell line. The EDA-derived prodrug of paclitaxel 5 was stable and capable of being efficiently converted to an active drug that killed cells specifically in the presence of PSA, suggesting that this prodrug and similarly designed PSA-cleavable prodrugs may have potential as prostate cancer-specific therapeutic agents.     

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC > DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.     

Reductively activated disulfide prodrugs of paclitaxel

A series of unsymmetrical polar disulfide prodrugs 2–5 of paclitaxel were designed and synthesized as reductively activated prodrugs. These compounds behaved as prodrugs in vitro on L2987 lung carcinoma cells. In vivo evaluation in mice demonstrated that the mutual prodrug 5 with captopril exhibited significant regressions and cures.     

New Taxol (paclitaxel) prodrugs designed for ADEPT and PMT strategies in cancer chemotherapy

Two new glucuronide paclitaxel prodrugs have been synthesized. Linked to the 2'-OH of the drug by a carbonate function, they include a self-immolative spacer bearing an arylnitro or arylamino group between the drug and the glucuronic acid residue. Both prodrugs were well detoxified and easily cleaved in the presence of beta-D-glucuronidase with fast removal of the spacer, releasing paclitaxel. The arylamino spacer-containing prodrug, more stable than the corresponding nitro analogue, was selected for further studies.     

Paclitaxel esters of malic acid as prodrugs with improved water solubility

The synthesis of paclitaxel esters of malic acid is described. These compounds were found to have improved water solubility and are stable in solution at neutral pH. The C2' modified compounds behave as prodrugs, that is, paclitaxel is generated upon exposure to human plasma, whereas the C7 modified derivatives do not. 2'-Malyl paclitaxel sodium salt demonstrated enhanced antitumour activity and less toxicity in a P388 murine leukaemia in vivo model when compared to paclitaxel.     

Development of novel water-soluble photocleavable protective group and its application for design of photoresponsive paclitaxel prodrugs

A novel coumarin-based highly water-soluble photocleavable protective group was designed and synthesized, and then this photosensitive protecting group was used to design paclitaxel prodrugs. These novel paclitaxel conjugates demonstrated excellent water solubility, over 100mgmL(-1). Thus, the use of a detergent in the formulation can be omitted completely, even at high doses. Phototaxel 11 released the parent drug, paclitaxel, quickly and efficiently by minimal tissue-damaging 365nm UV light irradiation at low power, while laser activation at 355nm led to extensive decomposition of the prodrug. The carbamate-type prodrug, phototaxel 11, was stable in the dark prior to activation, whereas carbonate-type phototaxel 9 demonstrated poor stability under aqueous conditions. For such prodrugs, tumor-tissue targeting after administration could be achieved by selective light delivery, similar to that used in photodynamic therapy. In addition, newly designed coumarin derivative 8 can be applied in organic chemistry as a photosensitive protective group and for the design of caged compounds.     

Synthesis and preclinical characterization of a paclitaxel prodrug with improved antitumor activity and water solubility

The development of novel chemotherapy strategies based on prodrugs remains a major challenge for effective treatment of malignancies. We tested the hypothesis that this can be achieved by a prodrug of paclitaxel where one biologically active center, represented by the C7 hydroxyl group, was blocked by a dihydroxypropyl side chain which can be hydrolytically cleaved by a pH-dependent, slow-release mechanism. The prodrug was synthesized by condensation of solketal chloroformate with the C7 hydroxyl group of paclitaxel followed by a ring-opening reaction to the dihydroxyl derivative. The cytotoxicity of the prodrug was similar to paclitaxel, when tested in vitro against a variety of human tumor cell lines. In vitro cell cycle analysis indicated that concentrations within the micromolar range of both drug and prodrug are required to induce sufficient G2M arrest. The hydrophilic paclitaxel prodrug proved to be more than 50-fold more water soluble than the parental drug and effectively converted to paclitaxel by pH dependent hydrolysis. Importantly, the prodrug could be used at a 3-fold higher maximum tolerated dose (MTD) and revealed a markedly improved antitumor activity in mice compared to paclitaxel. Taken together, our results demonstrate, that a hydrolytically activated paclitaxel prodrug exhibits greater water solubility and superior antitumor activity than the parental drug.     

O-N intramolecular acyl migration reaction in the development of prodrugs and the synthesis of difficult sequence-containing bioactive peptides

N-Ointramolecular acyl migration in Ser- or Thr-containing peptides is a well-known side reaction in peptide chemistry. It results in the mutual conversion of ester and amide bonds. Our medicinal chemistry study focused on the fact that the O-acyl product can be readily converted to the original N-acyl form under neutral or slightly basic conditions in an aqueous buffer and the liberated ionized amino group enhances the water solubility of O-acyl products. Because of this, we have developed a novel class of "O-N intramolecular acyl migration"-type water-soluble prodrugs of HIV-1 protease inhibitors. These prodrugs released the parent drugs via a simple chemical mechanism with no side reaction. In this study, we applied this strategy to important cancer chemotherapeutic agents, paclitaxel and its derivatives, to develop water-soluble taxoid prodrugs, and found that these prodrugs, 2'-O-isoform of taxoids, showed promising results with higher water solubility and proper kinetics in their parent drug formation by a simple pH-dependent chemical mechanism with O-N intramolecular acyl migration. These results suggest that this strategy would be useful in toxicology and medical economics. After the successful application of O-N intramolecular acyl migration in medicinal chemistry, this concept was recently used in peptide chemistry for the synthesis of "difficult sequence-containing peptides." The strategy was based on hydrophilic O-acyl isopeptide synthesis followed by the O-N intramolecular acyl migration reaction, leading to the desired peptide. In a model study with small, difficult sequence-containing peptides, synthesized "O-acyl isopeptides" not only improved the solubility in various media and efficiently performed the high performance liquid chromatography purification, but also altered the nature of the difficult sequence during SPPS, resulting in the efficient synthesis of O-acyl isopeptides with no complications. The subsequent O-N intramolecular acyl migration of purified O-acyl isopeptides afforded the desired peptides as precipitates with high yield and purity. Further study of the synthesis of a larger difficult sequence-containing peptide, Alzheimer's disease-related peptide (A beta 1-42), surprisingly showed that only one insertion of the O-acyl group drastically improved the unfavorable nature of the difficult sequence in A beta 1-42, and achieved efficient synthesis of 26-O-acyl isoA beta 1-42 and subsequent complete conversion to A beta 1-42 via the O-N intramolecular acyl migration reaction of 26-O-acyl isoA beta 1-42. This suggests that our new method based on O-N intramolecular acyl migration is an important method for the synthesis of difficult sequence-containing bioactive peptides.     

Synthesis, biological evaluation and kinetic studies of glyceride prodrugs of diclofenac

The prodrugs (glyceride derivatives) 3a and 3b of diclofenac were prepared by reacting 1, 2, 3-trihydroxy propane-1,3-dipalmitate/stearate with the acid chloride of diclofenac as potential prodrugs to reduce the gastrointestinal toxicity associated with them. These prodrugs were evaluated for their ulcerogenicity, anti-inflammatory and analgesic activity. It was found that the prodrugs were significantly less irritating to the gastric mucosa as indicated by severity index of 0.86, 0.78 compared to 1.6 of diclofenac. The prodrugs 3a and 3b showed better anti-inflammatory and analgesic activity than the parent drugs. The hydrolysis of prodrugs 3a and 3b were studied at pH 3, 4, 5 and 7.4. The HPLC analysis showed that the prodrugs were resistant to hydrolysis at pH 3, 4 and 5 indicating that they did not hydrolyze in acidic environment, whereas at pH 7.4 the prodrugs readily released the parent drug in significant quantities. The plasma levels of diclofenac were also analyzed by HPLC in rats after single oral dose of the prodrugs. The results indicated that the parent drugs were readily released. The concentration of diclofenac during the study was found higher in animals treated with prodrugs 3a and 3b compared with animals treated with diclofenac. The concentration of diclofenac was found to be 38.59, 33.6 and 30.36 microg/ml in animals treated with prodrugs 3a, 3b and diclofenac respectively. In conclusion, all these studies indicated that the glyceride prodrugs of diclofenac might be considered as potential biolabile prodrugs of diclofenac.     

Tyrosine and glycine derivatives as potential prodrugs: design,synthesis, and pharmacological evaluation of amide derivatives of mefenamic acid

This study deals with the synthesis, pharmacological activity, and kinetic studies of mefenamic acid (MA) prodrugs of tyrosine and glycine. The synthesis involved a series of protection and deprotection reactions. The hydrolysis of these prodrugs in the intestine was confirmed by hydrolysis kinetics studies in simulated gastric fluid, simulated intestinal fluid, and 80% plasma. The prodrugs were also evaluated for analgesic, anti-inflammatory, and ulcerogenic activities. The glycine prodrug showed maximum analgesic activity of 86%, and both tyrosine and glycine prodrugs showed better anti-inflammatory activity of 74% and 81%, respectively, when compared to the 40% of MA. Further, the prodrugs showed fewer gastric ulcers compared to MA; tyrosine and glycine prodrugs had an average ulcer index of 9.1 and 4.5, respectively, while an average ulcer index of 24.2 was observed with MA. These findings suggest that both prodrugs are better in action as compared to MA, and are advantageous in having fewer gastrointestinal side effects.     

Synthesis, hydrolysis studies and phamacodynamic profiles of amide prodrugs of dexibuprofen with amino acids

The present investigation deals with the synthesis of novel prodrugs of dexibuprofen with amino acids with an aim to achieve potent anti-inflammatory activity and less gastrointestinal toxicity. Structures of synthesized compounds were confirmed by spectral and elemental analyses. In vitro hydrolytic studies in simulated intestinal fluid, 80% plasma and rat faecal matter showed satisfactory release of dexibuprofen due to enzymatic cleavage. The synthesized prodrugs were evaluated for anti-inflammatory activity, analgesia, ulcerogenicity and histopathology. The anti-inflammatory activity of dexibuprofen was 43.3% whereas an improved value of 73.4, 77.3, 72.8 and 64.5% was observed for the synthesized prodrugs. The percentage analgesia of the prodrugs increased, whereas a decrease in the mean ulcer index values than dexibuprofen was observed. The histopathological studies revealed less ulceration in the gastric region when treated with prodrugs. Thus, the prodrugs were proved to be better in action as compared with the parent drug.     

Targeted brain delivery of 17 beta-estradiol via nasally administered water soluble prodrugs

The utility of the nasal route for the systemic delivery of 17beta-estradiol was studied using watersoluble prodrugs of 17beta-estradiol. This delivery method was examined to determine if it will result in preferential delivery to the brain. Several alkyl prodrugs of 17beta-estradiol were prepared and their physicochemical properties were determined. In vitro hydrolysis rate constants in buffer, rat plasma, and rat brain homogenate were determined by high-performance liquid chromatography. In vivo nasal experiments were carried out on rats. Levels of 17beta-estradiol in plasma and cerebral spinal fluid (CSF) were determined with radioimunoassay using a gamma counter. The study revealed that the aqueous solubilities of the prodrugs were several orders of magnitude greater than 17beta-estradiol with relatively fast in vitro conversion in rat plasma. Absorption was fast following nasal delivery of the prodrugs with high bioavailability. CSF 17beta-estradiol concentration was higher following nasal delivery of the prodrugs compared to an equivalent intravenous dose. It was determined that water-soluble prodrugs of 17beta-estradiol can be administered nasally. These prodrugs are capable of producing high levels of estradiol in the CSF and as a result may have a significant value in the treatment of Alzheimer's disease.     

Effect of gabapentin derivates on mechanical allodynia-like behaviour in a rat model of chronic sciatic constriction injury

A series of mutual prodrugs derived from gabapentin, pregabalin, memantine, venlafaxine were synthesized and their pharmacological properties to treat neuropathic pain were investigated in a rat model of chronic sciatic nerve constriction injury (CCI). In vivo evaluation demonstrated that the mutual prodrugs 2002413A, 2002823A composed of two gabapentins, 2002414 composed of gabapentin and pregabalin were effective in reversal tactile allodynia in CCI rats. The prodrugs 2002413A, 2002414 had no significant influence on the rotarod activity. The result suggest that the prodrugs may be possible candidates for further development.     

Synthesis,hydrolysis studies and phamacodynamic profiles of amide prodrugs of dexibuprofen with amino acids

The present investigation deals with the synthesis of novel prodrugs of dexibuprofen with amino acids with an aim to achieve potent anti-inflammatory activity and less gastrointestinal toxicity. Structures of synthesized compounds were confirmed by spectral and elemental analyses. In vitro hydrolytic studies in simulated intestinal fluid, 80% plasma and rat faecal matter showed satisfactory release of dexibuprofen due to enzymatic cleavage. The synthesized prodrugs were evaluated for anti-inflammatory activity, analgesia, ulcerogenicity and histopathology. The anti-inflammatory activity of dexibuprofen was 43.3% whereas an improved value of 73.4, 77.3, 72.8 and 64.5% was observed for the synthesized prodrugs. The percentage analgesia of the prodrugs increased, whereas a decrease in the mean ulcer index values than dexibuprofen was observed. The histopathological studies revealed less ulceration in the gastric region when treated with prodrugs. Thus, the prodrugs were proved to be better in action as compared with the parent drug.     

Simple mono-derivatisation of the aryl moiety of D4A and DDA-based phosphoramidate prodrugs significantly enhances their anti-HIV potency in cell culture.

Simple mono-derivatisation of the aryl moiety of some phosphoramidate pronucleotide derivatives of d4A and ddA served to increase the lipophilicity of these membrane-soluble prodrugs. A concomitant and significant enhancement of potency against HIV-1 and HIV-2 in vitro was observed for the ddA- and d4A-based prodrugs compared to the original underivatised prodrugs.     

Effect of the acyl groups on O-->N acyl migration in the water-soluble prodrugs of HIV-1 protease inhibitor

To improve the low water-solubility of HIV-1 protease inhibitors KNI-272, -279 and -727, we previously reported the water-soluble prodrugs of these inhibitors based on O-->N intramolecular acyl migration reaction. These prodrugs were rapidly converted to the corresponding parent drugs under physiological conditions. To understand the steric and electrostatic effects of O-acyl moiety on the migration rate, we examined several types of prodrug. A remarkably slow migration was observed in the benzoyl-type prodrugs, and Hammett plot of migration rate constants of p-substituted benzoyl-type prodrugs gave a linear free energy relationship.     

Mutual prodrugs containing bio-cleavable and drug releasable disulfide linkers

We report herein the design and synthesis of several representative examples of novel mutual prodrugs containing nine distinct types of self-immolative drug-releasable disulfide linkers with urethane, ester, carbonate, or imide linkages between the linker and any two amine/amide/urea (primary or secondary) or carboxyl or hydroxyl (including phenolic)-containing drugs. We also report drug release profiles of a few representative mutual prodrugs in biological fluids such as simulated gastric fluid and human plasma. We also propose plausible mechanisms of drug release from these mutual prodrugs. We have also conducted a few mechanistic studies based on suggested sulfhydryl-assisted cleavage of mutual prodrugs and characterized a few important metabolites to give support to the proposed mechanism of drug release from the reported mutual prodrugs.     

Flurbiprofen derivatives in Alzheimer's disease: synthesis, pharmacokinetic and biological assessment of lipoamino acid prodrugs

Flurbiprofen (FLU) lipophilic prodrugs with lipoamino acids (LAA) 6a- e were synthesized for brain delivery. Chemical and plasmatic stability of prodrugs 6a- e as well as pharmacokinetic distribution studies for the prodrugs 6b and 6d were carried out. FLU prodrugs 6a- e were compared to the parent drug for their ability to inhibit binding of [F-18]FDDNP to in vitro formed beta-amyloid protein (Abeta fibrils). FLU-LAA conjugates showed a typical prodrug stability profile, being stable in PBS at pH 7.4 and releasing the active drug in plasma. Compound 6d yielded a slow accumulation of FLU in the brain. In the in vitro inhibition assay, all prodrugs except for the prodrug with the longest alkyl side chain ( 6e) were effective as inhibitors of [F-18]FDDNP binding to Abeta fibrils with EC50 values in the 10-300 nM range. The different brain accumulation kinetics shown by FLU and its LAA conjugate 6d suggested a possible slow-releasing activity of FLU by these prodrugs in the brain or a differential pharmacological effect deserving further, detailed studies on their biodistribution and pharmacological profile.     

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers.

In an attempt to improve the membrane permeabilities of opioid peptides, we have synthesized cyclic prodrugs of [Leu5]-enkephalin and DADLE using a coumarinic acid or a phenylpropionic acid linker. The synthesis of the coumarinic acid- and phenylpropionic acid-based cyclic prodrugs followed similar strategies. Key intermediates were the compounds with the C-terminal amino acids of opioid peptides (L-Leu, [Leu5]-enkephalin; D-Leu, DADLE) attached to the phenol hydroxyl group and the remaining amino acids of the peptide linked via the N-terminal amino acid (L-Tyr) attached to the carboxylic acid groups of the prodrug moieties (coumarinic acid or propionic acid). Cyclization of these linear precursors gave the cyclic prodrugs in 30-50% yields. These cyclic prodrugs exhibited excellent transcellular permeation characteristics across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. To correlate the cellular permeabilities of these cyclic prodrugs with their physicochemical properties, we calculated their Stokes-Einstein molecular radii from their diffusion coefficients which were determined by NMR and we determined their membrane interaction potentials using immobilized artificial membrane (IAM) column chromatography. The cyclic prodrugs exhibited molecular radii similar to those of the parent compounds, [Leu5]-enkephalin and DADLE. However, these cyclic prodrugs were shown to have much higher membrane interaction potentials than their corresponding opioid peptides. Therefore, the enhanced cellular permeation of the cyclic prodrugs is apparently due to the alteration of their lipophilicity and hydrogen bonding potential, but not their molecular sizes.