A carbamate-based approach to primaquine prodrugs: antimalarial activity, chemical stability and enzymatic activation

O-Alkyl and O-aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O-alkyl and O-aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O-aryl carbamates being ca. 10(6)-10(10) more reactive than their O-alkyl counterparts. In human plasma O-alkyl carbamates were stable, whereas in contrast their O-aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O-aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O-aryl and O-alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei, Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n-hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O-alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.     

Synthesis and evaluation of esters and carbamates to identify critical functional groups for esterase-specific metabolism

In an effort to develop novel prodrugs for viral directed enzyme prodrug therapy (VDEPT) approaches to chemotherapy, eleven esters and carbamates of o-nitrophenol, p-nitrophenol, and beta-naphthol were synthesized and characterized as substrates for rabbit (rCE) and human liver (hCE1) carboxylesterases. All of the esters of o-, p-nitrophenols, and beta-naphthols showed moderate hydrolysis by both rCE and hCE1. Esters of beta-naphthols exhibited higher hydrolysis rates compared to esters of p-nitrophenols by rCE. Of the carbamates, 4-benzyl-piperazine-1-carboxylic acid 2-nitrophenol showed preferential hydrolysis by rCE compared to hCE1 with a V(max) of 54.4 micromoles/min/mg, and a K(m) value of 1071 microM. Substrate metabolism by a specific CE or inhibition of CEs by each compound depended on several factors, including the types of functional groups and linking moieties.     

Synthesis and in vitro/in vivo evaluation of novel oral N-alkyl- and N,N-dialkyl-carbamate esters of entacapone

Entacapone has a relatively low oral bioavailability which may, in part, be due to its low aqueous solubility at low pH and/or its hydrophilic character at neutral pH. Various novel N-alkyl and N,N-dialkyl carbamate esters of entacapone were synthesized as possible prodrugs of entacapone in order to increase its aqueous solubility at an acidic pH and to increase its lipophilicity at neutral pH. Oral bioavailability of entacapone and selected carbamate esters were investigated in rats. Both N-alkyl and N,N-dialkyl carbamate esters were relatively stable against chemical hydrolysis at pH 7.4 (t1/2 = 14.9-20.7 h), but hydrolyzed rapidly (t1/2 = 0.8-2.7 h) in human serum. However, in contrast to N-alkyl carbamates, N,N-dialkyl carbamates did not release entacapone in in vitro enzymatic hydrolysis (human serum) studies. N-Alkyl carbamates, 2a-c, showed increased aqueous solubility at pH 7.4, of which 2a and 2c also show increased aqueous solubility at pH 5.0, compared to entacapone. In addition to increased aqueous solubility, 2c showed increased lipophilicity at pH 7.4. However, two N-alkyl carbamates of entacapone did not increase the oral bioavailability of the parent drug in rats. Thus, it can be concluded that the relatively low lipophilicity of entacapone is not the cause of its low bioavailability.     

Structure-activity relationship study of human liver microsomes-catalyzed hydrolysis rate of ester prodrugs of MENT by comparative molecular field analysis (CoMFA)

A series of MENT esters (3-71) was designed, prepared and tested to study the structure-activity relationship (SAR) of the hydrolysis rate with human liver microsomes of these prodrugs. Compounds were obtained covering a wide range of metabolic stability. The results are useful for the proper selection of prodrugs for different pharmaceutical formulations to deliver the potent and prostate-sparing androgen MENT. The MENT esters can especially be administered for male hormone replacement therapy and male contraception. Comparative molecular field analysis (CoMFA) was applied to a dataset of 28 esters, for which ED50 values could be obtained. The CoMFA model where the electrostatic and H-bond molecular fields were combined turned out to be most predictive. Despite the limited size of the dataset, CoMFA can help to rationalize the SAR of the ester hydrolysis rate of ester prodrugs of MENT.     

Synthesis,characterization and pharmacological evaluation of certain enzymatically cleavable NSAIDs amide prodrugs

The presence of free carboxylic acid group in majority of non-steroidal anti-inflammatory drug (NSAIDs) is responsible from GI irritation. Coupling of the appropriate NSAIDs (diclofenac, naproxen, dexibuprofen and meclofenamic acid) 1–4, respectively with the appropriate amino acid ester 5 using dicyclohexylcarbodiimide afforded prodrugs 6–13. The structures of the prodrugs were verified based on spectral data. Chemical hydrolysis studies performed in three different non enzymatic buffer solutions at pH 1.2, 5.5 and 7.4, as well as in 80% human plasma and 10% rat liver homogenate using HPLC indicate no conversion of prodrugs to their respective NSAID in the studied buffers, while they underwent a reasonable plasma and rat liver homogenate hydrolysis. Furthermore, ulcerogenicity of prodrugs 9 and 12 were studied and results revealed no gastro-ulcerogenic effects.     

Comparative chemical and biological hydrolytic stability of homologous esters and isosteres

Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability. In this paper, we describe a comparative chemical and biological stability of homologous esters and isosteres in base media as well as in rat plasma and rat liver microsomes. Our results provided evidence for the hydrolytic structure lability relationship and demonstrated that the hydrolytic stability in plasma and liver microsome might depend on carboxylesterase activity. Molecular modelling studies were performed in order to understand the experimental data. Taken together, the data could be useful to design bioactive compounds or prodrugs based on the correct choice of the ester subunit, addressing compounds with higher or lower metabolic lability.     

Pharmacokinetic studies of naproxen amides of some amino acid esters with promising colorectal cancer chemopreventive activity

Naproxen (nap) is belonging to Non-steriodal anti-inflammatory drugs (NSAIDs) group of drugs that characterized by their free carboxylic group. The therapeutic activity of nap is usually accompanied by GI untoward side effects. Recently synthesized naproxen amides of some amino acid esters prodrugs to mask the free carboxylic group were reported. Those prodrugs showed a promising colorectal cancer chemopreventive activity. The current study aims to investigate the fate and hydrolysis of the prodrugs kinetically in different pH conditions, simulated gastric and intestinal fluids with pHs of 1.2, 5.5 and 7.4 in vitro at 37 °C. The effect of enzymes on the hydrolysis of prodrugs was also studied through incubation of these prodrugs at 37 °C in human plasma and rat liver homogenates. The pharmacokinetic parameters of selected prodrugs and the liberated nap were studied after oral and intraperitoneal administration in male wistar rats. The results showed the hydrolysis of naproxen amides of amino acid esters to nap through two steps first by degradation of the ester moiety to form the amide of nap with amino acid and the second was through the degradation of the amide link to liberate nap. The two reactions were followed and studied kinetically where K₁ and K₂ (rate constants of degradation) is reported. The hydrolysis of prodrugs was faster in liver homogenates than in plasma. The relative bioavailability of the liberated nap in vivo was higher in case of prodrug containing ethyl glycinate moiety than that occupied l-valine ethyl ester moiety. Each of nap. prodrugs containing ethyl glycinate and l-valine ethyl ester moieties appears promising in liberating nap, decreasing direct GI side effect and consequently their colorectal cancer chemopreventive activity.     

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.     

Dimeric L-dopa derivatives as potential prodrugs

A series of dimeric derivatives (+)-1, and (+)-2, and (+)-3a-d of L-Dopa diacetyl esters was synthesized and evaluated as potential L-Dopa prodrugs with improved physicochemical properties. All the new compounds showed chemical stability in aqueous buffer solutions (pH 1.3 and 7.4). A relatively slow release of L-Dopa in human plasma was observed.     

Species differences for stereoselective hydrolysis of propranolol prodrugs in plasma and liver

Species differences and substrate specificities for the stereoselective hydrolysis of fifteen O-acyl propranolol (PL) prodrugs were investigated in pH 7.4 Tris-HCl buffer and rat and dog plasma and liver subfractions. The (R)-isomers were preferentially converted to propranolol (PL) in both rat and dog plasma with the exception of isovaleryl-PL in rat plasma, although the hydrolytic activities of prodrugs in rat plasma were 5–119-fold greater than those in dog plasma. The prodrugs with promoieties (C(=O)CH(R)CH₃) based on propionic acid showed marked preference for hydrolysis of the (R)-enantiomers in plasma from both species (R/S ratio 2.5–18.2). On the other hand, the hepatic hydrolytic activities of prodrugs were greater in dog than rat, especially in cytosolic fractions. The hydrolytic activity was predominantly located in microsomes of the liver in rat, while the cytosol also contributed to hepatic hydrolysis in dog. Hepatic microsomal hydrolysis in dog showed a preference for the (R)-isomers except acetyl- and propionyl-PL. Interestingly, in rat liver all types of prodrugs with substituents of small carbon number showed (S)-preference for hydrolysis. The hydrolyses of (R)- and (S)-isomers of straight chain acyl esters in rat liver microsomes were linearly and parabolically related with the carbon number of substituents, respectively, while these relationships were linear for both isomers in dogs. Chirality 9:661–666, 1997. © 1997 Wiley-Liss, Inc.     

Synthesis and biological evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for use in antibody-directed enzyme prodrug therapy

The design, synthesis and evaluation of four novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) prodrugs (1a,b and 2a,b; ) for potential use in carboxypeptidase G2 (CPG2)-based antibody-directed enzyme prodrug therapy (ADEPT) is reported. Although all four prodrugs were shown to be less cytotoxic than the released parent PBDs 3 and 4, the urea prodrugs 1b and 2b were found to be too unstable for use in ADEPT, whereas carbamates 1a and 2a are both stable in an aqueous environment and are good substrates for CPG2.     

Identification of human,rat and mouse hydrolyzing enzymes bioconverting amino acid ester prodrug of ketoprofen

Alkyl ester prodrugs are well known to be bioconverted by carboxylesterases, particularly in rodents’ by first-pass metabolism in the systemic circulation and liver. However, the bioconversion of structurally more complex esters with polar functional groups is less well understood, especially in humans. Therefore, it is not clear if ester prodrugs can be utilized for targeted drug delivery. In the present study a brain-targeted ester prodrug (1) of ketoprofen, utilizing the l-type amino acid transporter 1 (LAT1) was prepared and the enzymes involved in its metabolism in human plasma and liver S9 subcellular fraction as well as rat brain S9 fraction were identified. Furthermore, species differences among mouse, rat and human plasma and liver S9 fraction were compared. The results showed that bioconversion of the ester prodrug was much faster in mouse plasma compared to human, while it’s half-life in rat plasma was closer to the one of human. Moreover, both rodent species showed more efficient bioconversion in the liver S9 fractions compared to human and relatively efficient bioconversion in the brain S9 fractions. More specifically, butyrylcholinesterase (BChE) and paraoxygenase 1 (PON1) were the main hydrolyzing enzymes of the prodrug 1 in human plasma, while carboxylesterases 1 and 2 (CES1 and CES2) as well as PONs were the main bioconverting enzymes in human liver S9 fractions. In rat brain S9 fraction, acetylcholinesterase (AChE) was hydrolyzing the prodrug 1, although also other unidentified metal-and pH-dependent enzyme(s) were recognized to be participating to the total bioconversion of the compound 1 in the brain.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

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

The utility of the nasal route for the systemic delivery of 17β-estradiol was studied using watersoluble prodrugs of 17β-estradiol. This delivery method was examined to determine if it will result in preferential delivery to the brain. Several alkyl prodrugs of 17β-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 17β-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 17β-estradiol with relatively fast in vitro conversion in rat plasma. Absorption was fast following nasal delivery of the prodrugs with high bioavailability. CSF 17β-estradiol concentration was higher following nasal delivery of the prodrugs compared to an equivalent intravenous dose. It was determined that water-soluble prodrugs of 17β-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. Published: March 25, 2002.     

In vivo release of endogenous dopamine from rat caudate nucleus by beta-phenylethylamine and alpha,alpha,-dideutero-beta-phenylethylamine

The release of endogenous dopamine (DA) has been measured in the rat striatum following the intracardial administration of various doses of beta-phenylethylamine (PEA) or alpha,alpha-dideutero-beta-phenylethylamine (deuterated PEA). The release was significantly increased for a period of approximately 15 minutes by a dose of 25 mg/kg PEA. Both the dose required to stimulate DA release and the duration of the effect were in good agreement with previously reported behavioral and locomotor effects of administered PEA. When the animals were given 25 mg/kg of deuterated PEA, the increase in DA release was both longer lasting and significantly greater in magnitude than that observed in response to the non-deuterated amine. The results of these experiments provide direct evidence that DA release is stimulated by amounts of PEA known to cause behavioral effects and locomotor activity in rats, and suggest that these effects are likely to be mediated, at least in part, by DA.     

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.     

In vitro metabolism of opioid tetrapeptide agonists in various tissues and subcellular fractions from rats

The metabolism of three mu-selective opioid tetrapeptide agonists, Tyr-D-Arg-Phe-Nva-NH(2) (TArPN), Tyr-D-Arg-Phe-Phe-NH(2) (TArPP), and Tyr-D-Ala-Phe-Phe-NH(2) (TAPP), was investigated in different rat tissues. High metabolic activity (<20% peptide remaining after 30 min) was found against the three peptides in the kidney homogenate and against TArPN in spleen homogenate. Low metabolic activity (>80% peptide remaining after 30 min) was found for all peptides in brain homogenate and plasma, and for TArPN and TArPP in blood. The other tissue homogenates, prepared from the small and large intestine, liver and lung, all exhibited intermediate metabolic activity (20-80% peptide remaining after 30 min) against the peptides. In all tissues investigated, the tetrapeptides were metabolized at the C-terminal amide by deamidation.A further in depth metabolic investigation was performed in subcellular fractions isolated from three tissues (small intestine, liver and kidney). In the liver, the deamidation was predominantly localized to the mitochondrial/lysosomal fraction, while hydrolysis at the N-terminal Tyr residue was the major metabolic pathway in the microsomal/brush-border membrane fraction from the kidney and small intestine.     

Prodrugs of 4'-demethyl-4-deoxypodophyllotoxin: synthesis and evaluation of the antitumor activity

A series of prodrugs of 4'-demethyl-4-deoxypodophyllotoxin (DDPT) including carbamates (3-8), a carbonate (9) and water-soluble amino acid derivatives (10-17) were prepared and tested for their antitumor activity. The carbamate 6 (2-hydroxyethylcarbamoyl-DDPT), carbonate 9 (2-chloroethyloxycarbonyl-DDPT), and most of amino acid prodrugs (12-17) showed enhanced antitumor activity.     

Preparation,characterization, and in vitro efficacy of O-carboxymethyl chitosan conjugate of melphalan

A series of melphalan-O-carboxymethyl chitosan (Mel-OCM-chitosan) conjugates with different spacers were prepared and structurally characterized. All conjugates showed satisfactory water-solubility (160-217 times of Mel solubility). In vitro drug release behaviors by both chemical and enzymatic hydrolysis were investigated. The prodrugs released Mel rapidly within papain and lysosomal enzymes of about 40–75%, while released only about 4–5% in buffer and plasma, which suggested that the conjugates have good plasma stability and the hydrolysis in both papain and lysosomes occurs mostly via enzymolysis. It was found that the spacers have important effect on the drug content, water solubility, drug release properties and cytotoxicity of Mel-OCM-chitosan conjugates. Cytotoxicity studies by MTT assay demonstrated that these conjugates had 52–70% of cytotoxicity against RPMI8226 cells in vitro as compared with free Mel, indicating the conjugates did not lose anti-cancer activity of Mel. Overall these studies indicated Mel-OCM-chitosan conjugates as potential prodrugs for cancer treatment.     

Diazeniumdiolated carbamates: a novel class of nitric oxide donors

We report an indirect method for synthesis of previously inaccessible diazeniumdiolated carbamates. Synthesis involves use of previously reported triisopropylsilyloxymethylated isopropylamine diazeniumdiolate (TOM-ylated IPA/NO). These novel diazeniumdiolated carbamate prodrugs upon activation release nitric oxide (NO) similar to their secondary amine counterparts. They are also efficient sources of intracellular NO. These prodrugs may have potential applications as therapeutic NO-donors.