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.     

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.     

Synthesis and antitumor activity of water-soluble duocarmycin B1 prodrugs

The water-soluble duocarmycin B1 prodrugs such as glycoside 3, phosphate 4 and carbamate 5 were synthesized for improving biological and pharmaceutical profiles of duocarmycin. Among these prodrugs, N-methylpiperazinylcarbamoyl derivative 5 exhibited potent antitumor activity against several human tumors in vivo.     

β-d-2'-α-F-2'-β-C-Methyl-6-O-substituted 3',5'-cyclic phosphate nucleotide prodrugs as inhibitors of hepatitis C virus replication: A structure-activity relationship study

The 3',5'-cyclic phosphate prodrug 9-[β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100μm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.     

Syntheses of cyclic prodrugs of RGD peptidomimetics with various macrocyclic ring sizes: evaluation of physicochemical, transport and antithrombic properties.

The objective of this work was to synthesize cyclic prodrugs 1a-d of RGD peptidomimetics 2a-d with various ring sizes (n[CH2] = 1, 3, 5 and 7) and to evaluate the effect of ring size on their transport, physicochemical, enzymatic stability, and antithrombic properties. The syntheses of cyclic prodrugs 1a-d were achieved by converging two key intermediates, Boc-Phe-O-CH2-OCO-OpNP (5) and H2N-(CH2)n-CO-Asp(OBzl)-OTce (8a-d), to give linear precursors Boc-Phe-O-CH2-OCO-HN-(CH2)n-CO-Asp(OBzl)-OTce (9a-d). The N- and C-terminus protecting groups were removed from 9a-d to give 10a-d. Linear precursors 10a-d were cyclized, and the remaining Bzl-protecting group was removed to produce cyclic prodrugs 1a-d in around 20% overall yield. The linear RGD peptidomimetics (2a-d) were synthesized using standard Boc-amino acid chemistry by solution-phase method. Increasing the ring size by adding methylene groups also increases the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics. The transport properties of cyclic prodrugs 1c and 1d were 2.6- and 4.4-fold better than those of parent compounds 2c and 2d, respectively. These results suggest that increasing the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics enhanced their transport properties. The hydrodynamic radii of the cyclic prodrugs were also smaller than those of their respective parent compounds, suggesting that the change in size may contribute to their transport properties. The chemical stability of the cyclic prodrugs was affected by the ring size, and the cyclic prodrug with the larger ring size (i.e. 1d) was more stable than the smaller one (i.e. 1a). All the cyclic prodrugs were more stable at pH 4 than at pH 7 and 10. Prodrug-to-drug conversion could be induced by isolated esterase as well as esterase found in human plasma. An increase in the length of methylene group (n[CH2] = 1, 3, 5, 7) enhanced the antithrombic activity of the prodrugs and the parent compounds. In summary, the ring size of cyclic prodrugs affected their transport, physicochemical, and antithrombic properties.     

Synthesis and Anti-HIV Activity of Alkyl Steroidal 3′-Azido-3′-deoxythymidin-5′-yl Phosphotriesters as Prodrugs of AZT

Abstract

Alkyl steroidal AZT 5′-monophosphate triesters are designed as lipophilic prodrugs of AZT to improve its therapeutic efficiency. We have synthesized four phosphotriesters of AZT, in one-pot, using phosphoramidite-phosphite triester methodology. This method afforded the desired prodrugs in high yields under mild conditions. The in vitro evaluation of anti-HIV activity of these prodrugs is also reported.     

Monoval-LdC: efficient prodrug of 2'-deoxy-beta-L-cytidine (L-dC), a potent and selective anti-HBV agent

In order to improve the oral bioavailability of LdC, valinyl esters were prepared as prodrugs. We report here the syntheses of the 3'-mono-, 5'-mono, and 3',5'-di-O-valinyl esters of LdC. The comparison of their ease of synthesis, their physicochemical properties, as well as their pharmacokinetic parameters in cynomologus monkeys has revealed 3'-mono-O-valinyl derivative as the most promising of the studied prodrugs. This compound is being developed as a new anti-HBV agent.     

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker.

The objective of this work was to synthesize the cyclic prodrugs 1 and 2 of [Leu5]-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, using an (acyloxy)alkoxy linker. The cyclic prodrugs 1 and 2 were synthesized via a convergent method using the (acyloxy)alkoxy promoiety that connected the C- and N-terminus of the peptides. The key intermediates were compounds 6a and 9a for cyclic prodrug 1 and compounds 6b and 9b for cyclic prodrug 2. The key intermediates 6a and 9a (or 6b and 9b) were coupled to give compound 10a (or 10b). The N- and C-terminus protecting groups were removed from 10a and 10b to give compounds 11a and 11b, respectively, which were then treated with HBTU to give 1 and 2 in 40% and 53% yields, respectively. The cyclic prodrugs 1 and 2 exhibited Stokes-Einstein molecular radii similar to those of [Leu5]-enkephalin and DADLE; however, the cyclic prodrugs were shown to be significantly more lipophilic than the corresponding opioid peptides, as determined by partitioning experiments using immobilized artificial membrane (IAM) column chromatography. In addition, the cyclic prodrugs exhibit stable solution conformations, which reduce their hydrogen bonding potentials. Based on these physicochemical characteristics, the cyclic prodrugs 1 and 2 should have exhibited better transcellular flux across the Caco-2 cell monolayer than [Leu5]-enkephalin and DADLE, respectively. However, the cyclic prodrugs 1 and 2 were shown in separate studies to be substrates for P-glycoprotein, which significantly reduced their ability to permeate across Caco-2 cell monolayers. When P-glycoprotein was inhibited, the permeability characteristics of prodrugs 1 and 2 were consistent with their physicochemical properties.     

The effect of conformation on the membrane permeation of coumarinic acid- and phenylpropionic acid-based cyclic prodrugs of opioid peptides.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the coumarinic-based (3 and 4) and the phenylpropionic acid-based (5 and 6) cyclic prodrugs were more able to permeate the cell monolayers than were the corresponding opioid peptides, [Leu5]-enkephalin (1, H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In an attempt to explain the increased permeation of the cyclic prodrugs, we have determined the possible conformations of these cyclic prodrugs in solution, using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as molecular dynamic studies indicate that cyclic prodrug 4 exhibits two major conformers (A and B) in solution. Conformer A exhibited a type I beta-turn at Tyr1-D-Ala2-Gly3-Phe4. The presence of a turn was supported by ROE cross-peaks between the NH of D-Ala2 and the NH of Gly3 and between the NH of Gly3 and the NH of Phe4. Conformer B of cyclic prodrug 4 consisted of type II beta-turns at the same positions. The type II turn was stabilized by hydrogen bonding, thus forming a more compact structure, whereas the type I turn did not exhibit similar intramolecular hydrogen bonding. Spectroscopic data for compounds 3, 5 and 6 are consistent with the conclusion that these cyclic prodrugs have solution structures similar to those observed with cyclic prodrug 4. The increased lipophilicity and well-defined secondary structures in cyclic prodrugs 3-6, but not in the linear peptides 1 and 2, could both contribute to the enhanced ability of these prodrugs to permeate membranes.     

Sensitive liquid chromatographic assay for the basic DNA intercalator (N,N-dimethylaminoethyl)-9-amino-5-methylacridine-4-carboxamide and its nitroarylmethyl quaternary prodrugs in biological samples

Nitroarylmethyl quaternary (NMQ) ammonium salts of the basic DNA intercalator AMAC (N,N-dimethylaminoethyl-9-amino-5-methylacridine-4-carboxamide) are of interest as anticancer prodrugs. A sensitive HPLC assay has been developed for quantitation of AMAC and its NMQ prodrugs in cultured cells, plasma and tissue. Recovery of the prodrugs, without conversion to AMAC, was achieved using extraction in alkaline acetonitrile followed by immediate reneutralisation. Reversed-phase HPLC with fluorescence detection gave a detection limit of 3 fmol for AMAC, with linearity to 20 nmol (using diode array absorbance at high concentrations). This assay was used to measure cellular uptake, and hypoxic metabolism to AMAC, of three NMQ-AMAC prodrugs.     

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.     

Synthesis and evaluation of novel amidate prodrugs of PMEA and PMPA

Some novel amidate prodrugs of PMEA and PMPA have been synthesised and tested in vitro for their biological activity. Compound 5 in particular showed greatly enhanced antiviral potency compared with the parent nucleotide analogue. In vitro enzymatic studies and structure-activity relationships indicate that the degradation mechanism of such prodrugs may be the same as that described for the phosphoramidate triesters of nucleotide analogues.     

Synthesis and DNA cleavage reaction characteristics of enediyne prodrugs activated via an allylic rearrangement by base or UV irradiation

A number of enediyne prodrugs 1-5 possessing an (E)-3-hydroxy-4-(2'-hydroxy-1'-phenylethylidene)cyclodeca-1,5-diyne scaffold have been synthesized via the Sonogashira coupling and an intramolecular Nozaki-Hiyama-Kishi reaction as the key steps. Upon incubation with enediyne prodrugs 4 and 5 possessing a free hydroxymethyl group on the exocyclic double bond, circular supercoiled DNA (Form I) underwent single strand cleavage into circular relaxed DNA (Form II) in buffer solution at pH 8.5, while the silylated analogs 1-3 showed very weak DNA cleavage activity. Alternatively, the silylated analogs 1-3 could be activated by UV irradiation via a photochemical alkene isomerization followed by an allylic rearrangement to form the putative epoxy enediyne, resulting in efficient DNA cleavage similar to the level observed with the prodrugs 4 and 5.     

Comparison of anti-HBV activity of beta-D- and beta-L-DDA-5'monophosphate prodrugs and effectiveness in combination with lamivudine.

We have investigated the effects of several beta-D-ddA 5'-monophospate (beta-D-ddAMP), and their corresponding beta-L-enantiomers prodrugs against HBV replication. All ddAMP prodrugs inhibited HBV replication in a dose-dependent manner.     

Simultaneous determination of 3'-azido-2',3'-dideoxyuridine and novel prodrugs in rat plasma by liquid chromatography

3'-Azido-2',3'-dideoxyuridine (AZDU) is a nucleoside analog structurally similar to zidovudine (AZT) with proven activity against human immunodeficiency virus (HIV). The purpose of this study was to develop and validate a high-performance liquid chromatographic (HPLC) method to quantitatively determine AZDU and its novel prodrugs in rat plasma simultaneously. A reversed-phase gradient elution HPLC method was developed to quantitate AZDU and its prodrugs, N3-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (I), 5'-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (II), 5'-O-valinyl-3'-azido-2',3'-dideoxyuridine hydrochloride (III) and 5'-O-phenylalanyl-3'-azido-2',3'-dideoxyuridine hydrochloride (IV), in rat plasma. AZDU and its prodrugs were analyzed using an octadecyl silane column with a mobile phase consisting of 0.04 microM sodium acetate buffer, pH 5.0, and acetonitrile, running in a segmented gradient manner at a flow rate of 2 ml/min. Acetonitrile was increased from 10 to 50% during the first 8 min by 5% per min, followed by 10% per min until it reached 90% acetonitrile. 3'-Azido-2',3'-dideoxy-5-ethyluridine (CS-85) was used as an internal standard (25 microg/ml). Compounds were detected by UV absorption at 261 nm. Extraction recoveries for all compounds were greater than 80%. Retention times of AZDU, CS-85, prodrugs I, II, III and IV were 3.3, 5.2, 9.1, 8.8, 6.3 and 7.3 min, respectively. Calibration plots were linear over the range of 0.25-100 microg/ml for AZDU and prodrugs II, III, and IV and 0.5-100 microg/ml for prodrug I. The limit of quantitation was 0.25 microg/ml for prodrugs II, III and IV and 0.5 microg/ml for prodrug I. The intra- and inter-day variations were less than 10% and accuracies were greater than 90%. This method is rapid, sensitive and reproducible for the determination of AZDU and prodrugs in rat plasma.     

Synthesis and evaluation of photolabile insulin prodrugs

We have developed two photolabile insulin prodrugs, insulin-2P and insulin-3P. These prodrugs were synthesized by protecting GlyA1 (N(alphaA1)), and one or both of the PheB1 (N(alphaB1)) and LysB29 (N(epsilonB29)) amino groups in insulin using 5'-(alpha-methyl-nitro-piperonyl)oxy-carbonyl as the protecting group. These insulin prodrugs were efficiently activated by exposure to longwave UV light to produce insulin quantitatively. Using 2-deoxyglucose uptake assays, both di- and tri-protected compounds were less active than native insulin in the protected state, and showed comparable activity to native insulin upon photoactivation.     

Monoval-LdC: Efficient Prodrug of 2′-Deoxy-β-L-cytidine (L-dC), A Potent and Selective Anti-HBV Agent

Abstract

In order to improve the oral bioavailability of LdC, valinyl esters were prepared as prodrugs. We report here the syntheses of the 3′-mono-, 5′-mono, and 3′,5′-di-O-valinyl esters of LdC. The comparison of their ease of synthesis, their physicochemical properties, as well as their pharmacokinetic parameters in cynomologus monkeys has revealed 3′-mono-O-valinyl derivative as the most promising of the studied prodrugs. This compound is being developed as a new anti-HBV agent.     

5'-O-Palmitoyl- and 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine--novel lipophilic analogues of 5'-fluoro-2'-deoxyuridine: synthesis, incorporation into liposomes and preliminary biological results

5'-O-palmitoyl- and 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine were prepared by the reaction of 5-fluoro-2'-deoxyuridine in dimethylacetamide with palmitic acid chloride. The incorporation of the synthesized prodrugs into liposomes composed of egg phosphatidylcholine/stearylamine/cholesterol/alpha-tocopherol at a molar ratio of 10:1:2:0.05 was nearly quantitative; homogeneous bilayer vesicles (75 nm diameter) were obtained. Preliminary tolerance studies revealed that the prodrug-liposome preparations are about 20-60 times more toxic than the parent drug. The prodrugs incorporated into liposomes were 10 to 30 times more active against murine colon 38 carcinoma compared to the free drug. In comparison to the administration of the prodrugs in peanut oil the liposomal preparations seem to exert improved effects and represent a valuable drug delivery system for parenteral applications.     

5-氟尿嘧啶前体药物研究进展

5-氟尿嘧啶(5-fluorouracil,5-FU)是目前临床广泛应用的抗癌药物之一,抗瘤谱广,但其毒副作用大,治疗剂量与中毒剂量接近,且半衰期短,口服吸收不稳定,严重限制了其临床应用。5-氟尿嘧啶前体药物能够增强抗肿瘤活性和靶向性,同时降低5-氟尿嘧啶的毒副作用。文章综述了近年来5-氟尿嘧啶前药及其靶向抗肿瘤活性研究进展。     

Are 5'-O-carbamate-2',3'-dideoxythiacytidine new anti-HIV and anti-HBV nucleoside drugs or prodrugs?

In contrast to 5'-O-carbonate 3TC derivatives (23, 24), which are clearly 3TC prodrugs, the corresponding 3TC carbamates (15-21 and 25), found to be very stable compounds with respect to enzymatic hydrolysis (cellular lysates and culture cell media) and still active on both HIV-1 and HBV infected cells, may not be 3TC prodrugs. The antiviral properties as well as the mechanism of action of 3TC analogues have been studied and evaluated.