Synthesis and immunological activity of water-soluble thalidomide prodrugs

A series of new water-soluble thalidomide prodrugs was prepared. All compounds were derivatized on the nitrogen of the glutarimide ring. Esters of natural amino acids and succinic acid derivatives have been introduced by reaction with the hydroxymethyl thalidomide 2. Nicotinic acid derivatives were prepared from halomethyl derivatives. Additionally, a methoxymethyl derivative and a carboxymethyl derivative were prepared directly from thalidomide. Most compounds showed a very large increase in water solubility compared to thalidomide itself (0.012mg/mL). The amorphous hydrochlorides of the N-methylalanine ester 8, valine ester 9, and glycylglycine ester 10, respectively, were the most soluble compounds showing solubility greater than 300mg/mL, which equals an increase greater than 15,000-fold. The lipophilicity of the prodrugs has been determined by their HPLC capacity factors k'. The stability of selected compounds was determined. The hydrolysis rates follow pseudo-first order kinetics. In order to assess the immunological activity, the prodrugs were tested using tumor necrosis factor-alpha and interleukin-2 inhibition assays. Selected compounds were additionally investigated on their abililty to inhibit the local Shwartzman reaction, an assay to determine the vascular permeability. The prodrugs retained high effectiveness in the inhibition of TNF-alpha release. Our results indicated that the more stable prodrugs exhibited higher activity in the immunological assays. Some compounds showed higher activity than thalidomide itself, suggesting a high affine binding to the pharmacophore. In conclusion, the prodrugs exhibited high water solubility and high activity and might therefore be used in therapeutic applications.     

Water soluble prodrugs of the antitumor agent 3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one

Fourteen prodrugs of the antitumor agent 3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one (1) were prepared to improve its water solubility and potency. These prodrugs include alpha-amino acid (1a-1h), aliphatic amino acid (1i-1l), phosphoramidate (1m), and phosphate (1n) derivatives. All of the prodrugs showed improved water solubility. A number of the amino acid prodrugs (1a, 1b, 1d-1f, 1h, 1j, and 1k) exhibited more potent antitumor activity compared to the parent compound (1). The phosphate prodrug 1n also offered a potent antitumor activity, but the phosphoramidate 1m did not show any antitumor activity in vivo. None of the prodrugs exhibited significant toxicities in mice. These results indicate that the design and preparation of the amino acid prodrugs (1a, 1b, 1d-1f, 1h, 1j, and 1k) and phosphate prodrug (1n) are beneficial for enhancing the antitumor activity of 1. The similar approaches may be used to improve water solubility and bioactivity of other poorly soluble aromatic amines.     

Synthesis and evaluation of water-soluble paclitaxel prodrugs

A series of water-soluble 2'-paclitaxel prodrugs were synthesized by attaching paclitaxel to polyethylene glycol (PEG) through amino acid spacers. The prodrugs showed highly improved water solubility, enhanced in vitro cytotoxicity and in vivo antitumor activity compared with the native drug, paclitaxel.     

Effect of hydroxypropyl beta cyclodextrin complexation on aqueous solubility,stability, and corneal permeation of acyl ester prodrugs of ganciclovir

The purpose of the study was to investigate the effect of hydroxypropyl beta cyclodextrin (HPβCD) on aqueous solubility, stability, and in vitro corneal permeation of acyl ester prodrugs of ganciclovir (GCV). Aqueous solubility and stability of acyl ester prodrugs of Ganciclovir (GCV) were evaluated in pH 7.4 isotonic phosphate buffer solution (IPBS) in the presence and absence of HPβCD. Butyryl cholinesterase-mediated enzymatic hydrolysis of the GCV prodrugs was studied using various percentage w/v HPβCD. In vitro corneal permeation of GCV and its prodrugs (with and without 5% HPβCD) across isolated rabbit cornea was studied using side-by-side diffusion cells. HPβCD-prodrug complexation was of the A_L type with values for complexation constants ranging between 12 and 108 M⁻¹. Considerable improvement in chemical and enzymatic stability of the GCV prodrugs was observed in the presence of HPβCD. The stabilizing effect of HPβCD was found to depend on the degree of complexation and the degradation rate of prodrug within the complex. Five percent w/v HPβCD was found to enhance the corneal permeation of only the most lipophilic prodrug GCV dibutyrate (2.5-fold compared with 0% HPβCD). All other prodrugs showed little or no difference in transport in the presence of 5% w/v HPβCD. Agitation in the donor chamber largely influenced the transport kinetics of GCV dibutyrate across cornea. Results indicate the presence of an unstirred aqueous diffusion layer at the corneal surface that restricts the transport of the highly lipophilic GCV dibutyrate prodrug. HPβCD improves corneal permeation by solubilizing the hydrophobic prodrug and delivering it across the mucin layer at the corneal surface.     

Monodisperse oligoethylene glycols modified Propofol prodrugs

The low water solubility of Propofol resulted in complicated formulation and adverse effects during its clinical application. To improve its water solubility and maintain its anesthetic effects, Propofol prodrugs with monodisperse oligoethylene glycols as solubility enhancer were designed and synthesized. Monodisperse oligoethylene glycols enable the concise manipulation of water solubility, biocompatibility and anesthetic effects. Through the physicochemical and biological assay, a few water soluble prodrugs of Propofol were identified as promising anesthetic to overcome the drawbacks associated with Propofol.     

Glycosyl and polyalcoholic prodrugs of lonidamine

Polyhydric alcohol derivatives of the anticancer agent lonidamine (LND) have been synthesized. The increased water solubility showed by prodrugs 4, 7, and 25 together with their logP values (2.19, 2.55, and 2.54, respectively) and chemical stability might be beneficial for prodrugs absorption after oral administration. Moreover, the new prodrugs undergo enzymatic hydrolysis in plasma and release LND demonstrating that they are promising candidates for in vivo investigations.     

Synthesis, stability and in vitro dermal evaluation of aminocarbonyloxymethyl esters as prodrugs of carboxylic acid agents

Aminocarbonyloxymethyl esters based on (S)-amino acid carriers were synthesised and evaluated as potential prodrugs of carboxylic acid agents. In addition, the compounds were evaluated as topical prodrugs with the aim of improving the dermal delivery of two non-steroidal anti-inflammatory agents: naproxen and flufenamic acid. The lipophilicities of these compounds were determined and their hydrolyses in aqueous solutions and in human plasma were examined. Compounds containing a secondary carbamate group were hydrolysed at pH 7.4 by two different routes: (i) direct nucleophilic attack at the ester carbonyl carbon leading to the release of the parent carboxylic acid and (ii) intramolecular rearrangement involving an O-->N acyl migration, leading to the formation of the corresponding amide. The rearrangement pathway is highly dependent on the size of the carboxylic acid and amino acid substituents, being eliminated when the amino acid is valine or leucine. In contrast, compounds decomposed in plasma exclusively through ester hydrolysis, most releasing the parent carboxylic acid quantitatively with half-lives shorter than 5 min. The permeation of selected prodrugs across excised postmortem human skin was studied in vitro. All prodrugs evaluated exhibited a lower flux than the corresponding parent carboxylic acid. The poor skin permeation observed for compounds is most probably due to their low aqueous solubility and high partition coefficient.     

Synthesis of novel water soluble benzylazolium prodrugs of lipophilic azole antifungals

Water soluble N-benzyltriazolium or N-benzylimidazolium salt type prodrugs of several highly lipophilic triazole or imidazole antifungals have been synthesized. They were designed to undergo an enzymatic activation followed by a self-cleavage to release a parent drug. The prodrugs such as 16 had enough chemical stability and water solubility for parenteral use and were rapidly and quantitatively converted to the active substance in human plasma.     

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.     

Pyrrolo[2,1-c][1,4]benzodiazepine-beta-glucuronide prodrugs with a potential for selective therapy of solid tumors by PMT and ADEPT strategies

Pyrrolo[2,1-c][1,4]benzodiazepine-beta-glucuronide prodrugs 15a-b, with a potential for selective therapy of solid tumors by PMT and ADEPT have been designed, synthesized and evaluated for selective cytotoxicity in the presence of the enzyme beta-glucuronidase. The prodrugs have been found to possess reduced cytotoxicity compared to the parent moieties, and are excellent substrates for the enzyme, exhibiting cytotoxicity selectively in the presence of the enzyme. Enhanced water solubility and improved stability are the other important outcomes upon modifying these molecules as their prodrugs.     

NMR spectroscopic study of cyclodextrin inclusion complexes with A-007 prodrugs

One- and two-dimensional NMR spectroscopy was used to demonstrate the formation of inclusion cyclodextrin complexes with several A-007 prodrugs. These complexes are comprised from the encapsulation of the two phenol moieties of the A-007 prodrugs within the cyclodextrin cavity. Considering the size of the two phenol moieties of the A-007 prodrugs compared to the sizes of alpha-, beta-, and gamma-cyclodextrin cavities, we observed complementary binding of the A-007 prodrug with only beta-cyclodextrin, which was also demonstrated spectroscopically. The beta-cyclodextrin inclusion complexes increased the prodrug solubility and modified the prodrug half-life in water. Therefore, beta-cyclodextrin inclusion complexes can be used as an essential form of A-007 prodrug delivery.     

Tripartate poly(ethylene glycol) prodrugs of the open lactone form of camptothecin

Two PEG prodrugs utilizing conjugation of PEG through the C-21 acid functionality as well as the C-17 OH group of CPT hydroxy-amide open forms were synthesized and characterized. Both of these open lactone tripartate prodrugs were shown to be water soluble and highly effective in MX-1 mouse xenograph studies. Indirect evidence implies that the initial ester or carbonate bond breaking is esterase mediated in the first step of the cascade of CPT release.     

Pharmacokinetics of salicylate ester prodrugs of cyclic HPMPC in dogs

A series of aryl ester prodrugs of cyclic HPMPC have been synthesized and their physicochemical properties, pharmacokinetics and metabolism have been evaluated. Chemical stability was dependent on the orientation of the exo-cyclic ligand; the equatorial isomers were 5.4 to 9.4 fold more reactive than the axial isomers. The oral bioavailability of cyclic HPMPC from the aryl ester prodrugs ranged from 11.2% for o-pentylphenyl cyclic HPMPC to 46.3% for butylsalicylyl cyclic HPMPC. Cyclic HPMPC was the major metabolite observed for all the salicylyl ester prodrugs. Cidofovir accounted for 2 to 12% of the total plasma AUC for butyl-, cyclohexyl- and phenethyl-salicylyl esters of cyclic HPMPC. Intact prodrug or the corresponding monosalicylyl esters of cidofovir each accounted for less than 10% of the total AUC for salicylyl ester prodrugs.     

N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH), a powerful coupling reagent for bioconjugation

Prodrugs are increasingly used as delivery vehicles for pharmaceutical agents that present solubility and/or pharmacokinetic/metabolic issues. In the course of the development of prodrugs for the antitumoral agent thiocoraline, standard coupling reagents and procedures failed to provide the desired target derivatives because of the lack of reactivity of its quinolinic alcohol. In contrast, the use of N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH) resulted in good yields of ester prodrugs of thiocoraline and could be applicable to other drugs with similar limitations.     

Upregulation of p53 through induction of MDM2 degradation: Amino acid prodrugs of anthraquinone analogs

Previously, we reported a class of MDM2-MDM4 dimerization inhibitors that upregulate p53 and showed potent anticancer activity in animal models. However, water solubility hinders their further development. Herein we describe our effort to develop a prodrug approach that overcomes the solubility problem. The prodrug of BW-AQ-238, a potent anthraquinone analog, was made by esterification of the hydroxyl group with various natural amino acids. Cytotoxicity of these compounds toward Hela and EU-1 cells, their aqueous solubility, and the release kinetics of these prodrugs in buffer and in the presence of hydrolytic enzymes were studied. The results demonstrate that the amino acid prodrug approach significantly improved the water solubility while maintaining the potency of the parent drug.     

In vitro solubility,stability and permeability of novel quercetin–amino acid conjugates

In order to discover a quercetin prodrug with improved bioavailability, we synthesized nine quercetin–amion acid conjugates and estimated their pharmacokinetic properties including water solubility, stability against chemical or enzymatic hydrolysis, and cell permeability. Among the synthesized quercetin prodrugs, quercetin–glutamic acid conjugate Qu-E (4g/5g) showed remarkable increases in water solubility, stability, and cell permeability compared with quercetin, which warrants further development as a quercetin prodrug.     

Synthesis and biological activities of a pH-dependently activated water-soluble prodrug of a novel hexacyclic camptothecin analog

CH0793076 (1) is a novel hexacyclic camptothecin analog showing potent antitumor activity in various human caner xenograft models. To improve the water solubility of 1, water-soluble prodrugs were designed to generate an active drug 1 nonenzymatically, thus expected to show less interpatient PK variability than CPT-11. Among the prodrugs synthesized, 4c (TP300, hydrochloride) having a glycylsarcosyl ester at the C-20 position of 1 is highly water-soluble (>10 mg/ml), stable below pH 4 and rapidly generates 1 at physiological pH in vitro. The rapid (ca. <1 min) generation of 1 after incubation of TP300 with plasma (mouse, rat, dog and monkey) was also demonstrated. TP300 showed a broader antitumor spectrum and more potent antitumor activity than CPT-11 in various human cancer xenograft models.     

Potential of Piperazinylalkylester Prodrugs of 6-Methoxy-2-Naphthylacetic Acid (6-MNA) for Percutaneous Drug Delivery

Piperazinylalkyl ester prodrugs (4a–5d) of 6-methoxy-2-naphthylacetic acid (6-MNA) (1) were synthesized and evaluated in vitro for the purpose of percutaneous drug delivery. These ionizable prodrugs exhibited varying aqueous solubilities and lipophilicities depending on the pH of the medium. The prodrugs (4a–5c) showed higher aqueous solubility and similar lipophilicity at pH 5.0 and lower aqueous solubility and higher lipophilicity at pH 7.4 in comparison to 6-MNA. The chemical and enzymatic hydrolyses of the prodrugs was investigated in aqueous buffer solutions (pH 5.0 and 7.4) and in 80% human serum (pH 7.4) at 37°C. The prodrugs showed moderate chemical stability (t_1/2 = 6–60 h) but got readily hydrolyzed enzymatically to 6-MNA with half-life ranging from 10–60 min. In the in vitro permeation study using rat skin, the flux of 6-MNA and the prodrugs was determined in aqueous buffers of pH 5.0 and 7.4. The prodrug (5b) showed 7.9- and 11.2-fold enhancement in skin permeation compared to 6-MNA (1) at pH 5.0 and 7.4, respectively. It was concluded that the parent NSAIDs having favorable pharmacokinetic and pharmacodynamic properties coupled with increased skin permeability of their prodrugs could give better options for the treatment of rheumatic diseases.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0240-6) contains supplementary material, which is available to authorized users.KEY WORDS: 6-MNA, NSAID, piperazinylalkylester, prodrug, skin permeation     

Cyclosporin A prodrugs: design, synthesis and biophysical properties.

The cyclic undecapeptide cyclosporin A (CsA) has a remarkable spectrum of diverse biological activities, including anti-inflammatory, antifungal, antiparasitic as well as immunosuppressive activities. However, the low water solubility of this drug is a serious problem causing undesirable pharmacological properties such as erratic oral absorption. In order to overcome this problem, the design and synthesis of water-soluble prodrugs of CsA are described. Using the OH-MeBmt-1-group as attachment site, we investigate dipeptide systems exhibiting differential tendencies for intramolecular cyclization [diketopiperazine (DKP) formation] for tailoring the chemoreversible release of the parent CsA. In modulating the chemical and structural features of the dipeptide esters (N-alkylation, side chains, C-terminal Pro), we find conversion rates at physiological conditions ranging from minutes to several days. Together with their thermodynamic stability in the solid state and strongly enhanced solubility in water, these chemoreversible CsA prodrugs represent versatile candidates for therapeutical use.