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.     

Identification of 2-hydroxymethyl-4-[5-(4-methoxyphenyl)-3-trifluoromethyl-pyrazol-1-yl]-N-propionylbenzenesulfonamide sodium as a potential COX-2 inhibitor for oral and parenteral administration

Synthesis of prodrugs of orally active COX-2 inhibitor 3 involving sulfamoyl (SO₂NH₂) and hydroxymethyl (CH₂OH) groups, and their biological evaluation are described. Of these prodrugs, the N-propionyl sulfonamide sodium 3k was found to be much superior to the parent compound 3 and other marketed COX-2 inhibitors in carrageenan induced rat paw edema model of inflammation due to highly elevated drug levels in systemic circulation. This prodrug has a potential both for oral as well as parenteral administration due to impressive analgesic activity, antipyretic potency, and extraordinary water solubility.     

Phosphoramidate and phosphate prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine: synthesis, anti-HIV activity and stability studies

A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phosphonate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity. The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cytotoxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and 7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phosphoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.     

Phosphoramidates of 2′-β-d-arabinouridine (AraU) as phosphate prodrugs; design,synthesis, in vitro activity and metabolism

2′-β-d-Arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.     

Synthesis of taxoids 5. Synthesis and evaluation of novel water-soluble prodrugs of a 3'-desphenyl-3'-cyclopropyl analogue of docetaxel.

A novel 3'-desphenyl-3'-cyclopropyl analogue of docetaxel was synthesized from 10-deacetyl-baccatin III. The cytotoxicity of the new taxoid was evaluated against several human tumor cell lines, and it had ca. 20 times stronger activity against human colon cancer cell lines (WiDr and Colon 320) than that of docetaxel. This taxoid was converted to its water-soluble prodrugs that have 2'-substituted amino acid derivatives with spacer. The prodrugs had good solubility in saline and showed more potent antitumor activity against B 16 melanoma in mice than that of docetaxel.     

Novel (E)-2-(aryl)-3-(4-methanesulfonylphenyl)acrylic ester prodrugs possessing a diazen-1-ium-1,2-diolate moiety: design, synthesis, cyclooxygenase inhibition, and nitric oxide release studies

A novel group of hybrid nitric oxide-releasing anti-inflammatory drugs (11) possessing a 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, or 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate, nitric oxide (.NO) donor moiety attached via a one-carbon methylene spacer to the carboxylic acid group of (E)-3-(4-methanesulfonylphenyl)-2-(phenyl)acrylic acids were synthesized. These ester prodrugs (11) all exhibited in vitro inhibitory activity against the cyclooxygenase-2 (COX-2) isozyme (IC(50)=0.94-31.6 microM range). All compounds released .NO upon incubation with phosphate buffer (PBS) at pH 7.4 (3.2-11.3% range). In comparison, the percentage of .NO released was significantly higher (48.6-75.3% range) when these hybrid ester prodrugs were incubated in the presence of rat serum. These incubation studies suggest that both .NO and the parent anti-inflammatory (E)-3-(4-methanesulfonylphenyl)-2-(phenyl)acrylic acid would be released upon in vivo cleavage by non-specific serum esterases. O(2)-[(E)-2-(4-Acetylaminophenyl)-3-(4-methanesulfonylphenyl)acryloyloxymethyl]-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (11f) is a moderately potent (IC(50)=0.94 microM) and selective (SI>104) COX-2 inhibitor that released 73% of the theoretical maximal release of two molecules of .NO/molecule of the parent hybrid ester prodrug upon incubation with rat serum. Hybrid ester .NO-donor prodrugs offer a potential drug design concept for the development of anti-inflammatory drugs that are devoid of adverse ulcerogenic and/or cardiovascular side effects.     

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.     

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.     

Amino acid prodrugs of NVR3-778: Design,synthesis and anti-HBV activity

A series of amino acid prodrugs of NVR3-778, a potent anti-HBV candidate currently under phase II clinical trial, were designed and synthesized as new anti-HBV agents. Except for 1e, all of them displayed roughly comparable anti-HBV activity (IC₅₀, 0.28–0.56 µM) to NVR3-778 (IC₅₀, 0.26 µM). Compound 1a, a l-valine ester prodrug of NVR3-778, was found to show significantly improved water solubility (0.7 mg/mL, pH 2) as we expected, and lower cytotoxicity (CC₅₀ > 10 µM) than NVR3-778 (CC₅₀, 4.81 µM). Moreover, 1a also exhibited acceptable PK properties and comparable in vivo efficacy in HBV DNA hydrodynamic mouse model to that of NVR3-778, suggesting it may serve as a promising lead compound for further anti-HBV drug discovery.     

Synthesis and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methyl-7-deazapurine nucleosides, their phosphoramidate prodrugs and 5'-triphosphates

Thirty novel α- and β-d-2'-deoxy-2'-fluoro-2'-C-methyl-7-deazapurine nucleoside analogs were synthesized and evaluated for in vitro antiviral activity. Several α- and β-7-deazapurine nucleoside analogs exhibited modest anti-HCV activity and cytotoxicity. Four synthesized 7-deazapurine nucleoside phosphoramidate prodrugs (18-21) showed no anti-HCV activity, whereas the nucleoside triphosphates (22-24) demonstrated potent inhibitory effects against both wild-type and S282T mutant HCV polymerases. Cellular pharmacology studies in Huh-7 cells revealed that the 5'-triphosphates were not formed at significant levels from either the nucleoside or the phosphoramidate prodrugs, indicating that insufficient phosphorylation was responsible for the lack of anti-HCV activity. Evaluation of anti-HIV-1 activity revealed that an unusual α-form of 7-carbomethoxyvinyl substituted nucleoside (10) had good anti-HIV-1 activity (EC(50)=0.71±0.25 μM; EC(90)=9.5±3.3 μM) with no observed cytotoxicity up to 100 μM in four different cell lines.     

Design and synthesis of vidarabine prodrugs as antiviral agents

5′-O-d- and l-amino acid derivatives and 5′-O-(d- and l-amino acid methyl ester phosphoramidate) derivatives of vidarabine (ara-A) were synthesized as vidarabine prodrugs. Some compounds were equi- or more potent in vitro than vidarabine against two pox viruses and their uptake by cultured cells was improved compared to the parent drug.     

Diazen-1-ium-1,2-diolated and nitrooxyethyl nitric oxide donor ester prodrugs of anti-inflammatory (E)-2-(aryl)-3-(4-methanesulfonylphenyl)acrylic acids: synthesis, cyclooxygenase inhibition, and nitric oxide release studies

A new group of hybrid nitric oxide-releasing anti-inflammatory drugs wherein an O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (11a-d), or 2-nitrooxyethyl (12a-d), (*)NO-donor moiety is attached directly to the carboxylic acid group of (E)-3-(4-methanesulfonylphenyl)-2-(phenyl)acrylic acids were synthesized. The 2-nitrooxyethyl ester prodrugs (12a-d) all exhibited in vitro inhibitory activity against the cyclooxygenase-2 (COX-2) isozyme (IC(50)=0.07-2.8 microM range). All compounds released a low amount of (*)NO upon incubation with phosphate buffer (PBS) at pH 7.4 (1.0-4.8% range). In comparison, the percentage (*)NO released was significantly higher (76.2-83.0% range) when the diazen-1-ium-1,2-diolate ester prodrugs were incubated in the presence of rat serum, or moderately higher (7.6-10.1% range) when the nitrooxyethyl ester prodrugs were incubated in the presence of L-cysteine. These incubation studies suggest that both (*)NO and the parent anti-inflammatory (E)-3-(4-methanesulfonylphenyl)-2-(phenyl)acrylic acid would be released upon in vivo cleavage by non-specific serum esterases in the case of the diazen-1-ium-1,2-diolate esters (11a-d), or interaction with systemic thiols in the case of the nitrate esters (12a-d). O(2)-Acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (E)-3-(4-methanesulfonylphenyl)-2-phenylacrylate (11a) released 83% of the theoretical maximal release of 2 molecules of (*)NO/molecule of the parent hybrid ester prodrug upon incubation with rat serum. Hybrid ester anti-inflammatory/(*)NO donor prodrugs offer a potential drug design concept targeted toward the development of anti-inflammatory drugs that are devoid of adverse ulcerogenic and/or cardiovascular effects.     

Diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of 5-(4-carboxymethylphenyl)-1-(4-methanesulfonylphenyl)-3-trifluoromethyl-1H-pyrazole and its aminosulfonyl analog: Synthesis,biological evaluation and nitric oxide release studies

A new class of hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrugs (NONO-coxibs) wherein an O²-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (13a–b), or O²-acetoxymethyl-1-(2-methylpyrrolidin-1-yl)diazen-1-ium-1,2-diolate (16a–b), NO-donor moiety was covalently coupled to the COOH group of 5-(4-carboxymethylphenyl)-1-(4-methane(amino)sulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (11a–b) was synthesized. The percentage of NO released from these diazen-1-ium-1,2-diolates was significantly higher (59.6–74.6% of the theoretical maximal release of 2 molecules of NO/molecule of the parent hybrid ester prodrug) upon incubation in the presence of rat serum, relative to incubation with phosphate buffer (PBS) at pH 7.4 (5.0–7.2% range). These incubation studies suggest that both NO and the AI compound would be released from the parent NONO-coxib upon in vivo cleavage by non-specific serum esterases. All compounds were weak inhibitors of the COX-1 isozyme (IC₅₀ = 8.1–65.2 μM range) and modest inhibitors of the COX-2 isozyme (IC₅₀ = 0.9–4.6 μM range). The most potent parent aminosulfonyl compound 11b exhibited AI activity that was about sixfold greater than that for aspirin and threefold greater than that for ibuprofen. The ester prodrugs 13b, 16b exhibited similar AI activity to that exhibited by the more potent parent acid 11b when the same oral μmol/kg dose was administered. These studies indicate hybrid ester AI/NO donor prodrugs of this type (NONO-coxibs) constitute a plausible drug design concept targeted toward the development of selective COX-2 inhibitory AI drugs that are devoid of adverse cardiovascular effects.     

N-(2-hydroxypropyl)methacrylamide copolymers of a glutathione (GSH)-activated glyoxalase i inhibitor and DNA alkylating agent: synthesis, reaction kinetics with GSH, and in vitro antitumor activities

The incorporation of anticancer prodrugs into polyacrylamide conjugates has been shown to improve tumor targeting via the so-called "enhanced permeability and retention" effect. This strategy has now been expanded to include two different classes of glutathione (GSH)-activated antitumor agents prepared by radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-methacryloyloxy-methyl-2-cyclohexenone (7) and/or with S-(N-4-chlorophenyl-N-hydroxycarbamoyl-thioethyl)methacrylamide (8), followed by treatment with 3-chloroperoxybenzoic acid, to give the HPMA copolymers of 7 and the 8-sulfoxide, respectively. In aqueous-buffered solution at pH 6.5, GSH reacts rapidly with poly-HPMA-8-sulfoxide (k approximately 2.3 mM(-1) min(-1)) to give S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (1), a tight-binding transition state analogue inhibitor of the antitumor target enzyme glyoxalase I (K(i) = 46 nM), or with poly-HPMA-7 (k approximately 0.02 mM(-1) min(-1)) to give the electrophilic antitumor agent 3-glutathio-2-methylenecyclohexenone (4). Indeed, B16 melanotic melanoma in culture is inhibited by poly-HPMA-8-sulfoxide and by poly-HPMA-7 with IC(50) values of 168 +/- 8 and 284 +/- 5 microM, respectively. These values are significantly greater than those of the unpolymerized prodrugs suggesting that the cytotoxicity of the polymer prodrugs might be limited by slow cellular uptake via pinocytosis. This prodrug strategy should be applicable to a range of different GSH-based antitumor agents.     

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     

Discovery of 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs for delivering nucleoside HCV NS5B polymerase inhibitors

Our HCV research program investigated novel 2′-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5′-phosphoramidate prodrug of 2′-deoxy-2′-α-bromo-β-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5′-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs. Compounds that produced high TP concentrations in hepatocytes were tested in dog liver biopsy studies. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrug 14, which provided 100-fold higher TP concentrations in dog liver in comparison to 1 (4 and 24 h after 5 mg/kg oral dose).     

Water-soluble phosphate prodrugs of pleuromutilin analogues with potent in vivo antibacterial activity against Gram-positive pathogens

A phosphate prodrug strategy was investigated to address the problem of poor aqueous solubility of pleuromutilin analogues. Water-soluble phosphate prodrugs 6a, 6b and 6c of pleuromutilin analogues were designed and synthesized. Three compounds all exhibited excellent aqueous solubility (>50 mg/mL) at near-neutral pH and sufficient stability in buffer solution. In particular, the phenol pleuromutilin prodrug 6c displayed favourable pharmacokinetic profiles and comparable potency with vancomycin against MSSA and MRSA strains in vivo.     

Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 2. Synthesis and characterization of a novel imide-type prodrug for improving oral absorption

As an alternative to the previously reported solid dispersion formulation for enhancing the oral absorption of thiazolo[5,4-b]pyridine 1, we investigated novel N-acyl imide prodrugs of 1 as RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors. Introducing N-acyl promoieties at the benzanilide position gave chemically stable imides. N-tert-Butoxycarbonyl (Boc) introduced imide 6 was a promising prodrug, which was converted to the active compound 1 after its oral administration in mice. Cocrystals of 6 with AcOH (6b) possessed good physicochemical properties with moderate thermodynamic solubility (19μg/mL). This crystalline prodrug 6b was rapidly and enzymatically converted into 1 after its oral absorption in mice, rats, dogs, and monkeys. Prodrug 6b showed in vivo antitumor regressive efficacy (T/C=-6.4%) in an A375 melanoma xenograft model in rats. Hence, we selected 6b as a promising candidate and are performing further studies. Herein, we report the design, synthesis, and characterization of novel imide-type prodrugs.     

Synthesis and anti-HCV activity of a series of β-d-2′-deoxy-2′-dibromo nucleosides and their corresponding phosphoramidate prodrugs

Several β-d-2′-deoxy-2′-substituted nucleoside analogs have displayed potent and selective anti-HCV activities and some of them have reached human clinical trials. In that regard, we report herein the synthesis of a series of 2′-deoxy,2′-dibromo substituted U, C, G and A nucleosides 10a–d and their corresponding phosphoramidate prodrugs 13a–d. The synthesized nucleosides 10a–d and prodrugs 13a–d were evaluated for their inhibitory activity against HCV as well as cellular toxicity. The results showed that the most potent compound was prodrug 13a, which exhibited micromolar inhibitory activity (EC₅₀?=?1.5?±?0.8?µM) with no observed toxicity. In addition, molecular modeling and free energy perturbation calculations for the 5′-triphosphate formed from 13a and related 2′-modified nucleotides are discussed.     

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.