Kinetics of hydrolysis in vitro of nornitrogen mustard,a metabolite of phosphoramide mustard and cyclophosphamide

A gas chromatographic (GC-ECD) method was developed for the determination of nornitrogen mustard (NOR) and its hydrolysis products. The method was based on derivatization by heptafluorobutyric anhydride. The structures of the derivatives of NOR were established by GC-MS. The method was used to characterize the rate of transformation of NOR and phosphoramide mustard (PAM), important metabolites of cyclophosphamide, into secondary products in vitro at 37° C and pH 7.4. The rate of disappearance of NOR had a half-life of 20 min. The half-life of appearance of N-(2-chloroethyl)-N-(2-hydroxy-ethyl)amine (NOR-OH) was 19 min. While most NOR appeared to be converted to NOR-OH, the yield of N,N-bis(2-hydroxyethyl)amine (NOR-OH-OH) was a small fraction of the starting material. The disappearance of NOR, when PAM was used as a starting material, had a half-life of 19 min; in these experiments NOR-OH and NOR-OH-OH were relatively much more abundant compared to when NOR was used as the starting material.Abbreviations NOR nornitrogen mustard - NOR-OH N-(2-chloroethyl)-N-(2-hydroxyethyl)amine - NOR-OH-OH, N N-bisN,-(2-hydroxyethyl)amine - PAM phosphoramide mustard - GC gaschromatography - GC-ECD gas chromatography-electron capture detection - GC-MS gas chromatography-mass spectroscopy - HFBA heptafluorobutyric anhydride     

Synthesis of deuterium-labeled analogs of cyclophosphamide and its metabolites.

Convenient syntheses are described of d4 analogs of cyclophosphamide and some of its metabolites, potential standards for the quantitative analysis of the drug and its metabolites in human body fluids by stable isotope dilution-mass spectrometry. Base-catalyzed H-D exchange on N-nitrosobis(2-hydroxyethyl)amine gave N-nitrosobis(1,1-dideuterio-2-hydroxyethyl)amine from which bis(2-chloro-1,1-dideuterioethyl)amine (nor-HN2-d4) was readily obtained. Established synthetic routes were then used to convert nor-HN2-d4 into d4 analogs of cyclophosphamide [2-[bis(2-chlorethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide], 4-ketocyclophosphamide [2[BIS(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorin-4-one 2-oxide], and carboxyphosphamide [2-carboxyethyl N-N-bis(2-chloroethyl)phosphorodiamidate], and these analogs were used in a preliminary investigation into the quantitation of the appropriate components in human plasma and urine. Also prepared were d4 analogs of phosphoramide mustard [N,N-bis(2-chloroethyl)phosphorodiamidic acid (cyclohexylammonium salt)] and 3-(2-chloroethyl)oxazolidone and the methyl and trideuteriomethyl esters of phosphoramide mustard.     

Some studies of the active intermediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide

Evidence is presented in support of the following metabolic pathways, in the liver, of the antitumour agent cyclophosphamide 2-[bis(2-chloroethyl)amino]-tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide. The drug is first converted, presumably by the mixed-function oxidases, into 4-hydroxycyclophosphamide which may then break down by elimination of acrolein from its tautomeric form, aldophosphamide, to yield phosphoramide mustard [N,N-bis(2-chloroethyl)phosphorodiamidic acid], a known cytotoxic agent. In competition with this process is the enzymic conversion of 4-hydroxycyclophosphamide (by dehydrogenation) and aldophosphamide (by oxidation) into the known in vivo metabolites of cyclophosphamide, 4-ketocyclophosphamide and carboxyphosphamide, respectively, each of which has low cytotoxicity.4-Hydroxycyclophosphamide, which was too unstable to allow identification directly by conventional procedures, was trapped by reaction with ethanol. The resulting two, apparently isomeric, ethyl derivatives, (1) were amenable to mass spectrometry, (2) yielded acrolein 2,4-dinitrophenylhydrazone on treatment with acidic 2,4-dinitrophenylhydrazine, (3) were hydrolysed in water (pH 4.3), each isomer apparently regenerating 4-hydroxycyclophosphamide, (4) were highly toxic to Walker tumour cells in culture.Phosphoramide mustard was also isolated after in vitro metabolism of cyclophosphamide. On the basis of a bioassay involving Walker tumour cells in whole animals it appeared that, of the known metabolites of cyclophosphamide, only phosphoramide mustard possesses the cytoxicity and biological half-life appropriate to the active antitumour metabolite.Four other metabolites of low cytotoxicity were isolated and identified, namely, 4-ketocyclophosphamide, carboxyphosphamide, 2-(2-chloroethylamino)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide, and 3-hydroxypropyl-N,N-bis(2-chloroethyl)phosphorodiamidate.The significance of metabolic detoxification processes in relation to the selective cytotoxicity of cyclophosphamide towards tumour cells in vivo is discussed.The metabolic activation of isophosphamide appears to follow a pathway similar to that of cyclophosphamide.     

Glutathione conjugation with phosphoramide mustard and cyclophosphamide. A mechanistic study using tandem mass spectrometry.

The conjugations of cyclophosphamide and of phosphoramide mustard with glutathione are shown to be catalyzed by hepatic cytosolic glutathione-S-transferases. Cyclophosphamide conjugation is also catalyzed by microsomal glutathione-S-transferases, both in intact microsomes and after solubilization and immobilization. Deuterium isotope labels are used to test whether chloride is directly displaced by glutathione in the enzyme-catalyzed conjugations, or whether conjugation takes place via symmetrical cyclic aziridinium ions. Tandem mass spectrometry with high energy collisional activation is shown to provide reliable analysis of the isotope-labeling patterns in the conjugated products. This experiment leads to the conclusion that the aziridinium ion is opened in the conjugation of phosphoramide mustard in both the enzyme-catalyzed and the chemical reactions. Cyclophosphamide, on the other hand, is shown to be conjugated through direct displacement of chloride.     

Pathways of decomposition of propylbenzilylcholine mustard in neutral and alkaline solution

High-voltage electrophoresis has been used to follow the decomposition of propylbenzilylcholine mustard (PrBCM) in aqueous solution. Dilute solutions of PrBCM in 10 mM phosphate buffer, pH 7.5, or Krebs-Henseleit solution allowed to stand for 1 h at room temperature (22-24 degrees C) contain mainly the aziridinium ion derivative. At pH 7.5 the concentration of this ion declines slowly, giving rise first to the N-hydroxyethyl derivative and then ultimately, following hydrolysis of the ester bond, to NN-bis(2-hydroxyethyl)propylamine and benzilic acid. In contrast, in 5 mM NaOH the ester bond undergoes rapid hydrolysis, so that the major species present after 15 min at room temperature is the N-hydroxyethylaziridinium ion. This ion then undergoes slow reaction with hydroxy ion to yield the same final decomposition product, NN-bis(2-hydroxyethyl)propylamine, as is observed at pH 7.5.     

Aporphines. 50. Kinetics of solvolysis of N-(2-chloroethyl)norapomorphine, an irreversible dopamine receptor antagonist

The rates and mechanism of solvolysis of (-)-N-(2-chloroethyl)norapomorphine (NCA, 1c) in aqueous solution have been examined by reversed-phase liquid chromatography (HPLC) to follow the levels of starting material and products. The first-order rate constants for aziridinium ion formation at 25 and 37 degrees C at pH 7.0 are 0.024 and 0.096 min-1, respectively. Determination of the first-order rate constant for the disappearance of NCA as a function of pH has allowed the calculation of an approximate pKa of 6.3 for the tertiary amine, while the influence of reaction conditions (e.g., pH, buffer salt and concentration, and added nucleophiles) on product distribution support the view that NCA solvolysis proceeds through an intermediate aziridinium ion. Application of the HPLC procedure allowed us to observe simultaneously the loss of NCA and the appearance of an intermediate and multiple products at trace levels; it also permitted the facile isolation and subsequent identification of small amounts of hydrolysis products. At pH 7, maximum aziridinium concentration is reached only after 10 min at 37 degrees C and at 25 degrees C after 1 h. Increased temperatures and pH facilitate the rate of aziridinium ion formation, as well as of non-dopamine antagonist solvolysis products. The significance of these findings, including the ease with which buffer ions add to the intermediate ion, are discussed in relation to the use of NCA and its tritiated isomer, [3H]NCA, in dopamine receptor studies.     

Conversion of N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) to an aziridinium ion and its interaction with muscarinic receptors in various tissues.

A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an aziridinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4 and 37 degrees, 4-DAMP mustard released an equivalent amount of chloride. The release of chloride was consistent with a first-order process having a half-time of 5.7 min. The aziridinium ion reached a peak concentration at 32 min, corresponding to 75% of the initial concentration of 4-DAMP mustard. When homogenates of rat brain, heart, and submaxillary gland were incubated with 4-DAMP mustard (9 nM) for 1 hr, washed extensively, and then assayed for muscarinic receptor binding properties, a 56% decrease in the binding capacity of N-[3H]methylscopolamine in the heart and brain and a 71% decrease in the gland were observed, without a significant change in the dissociation constants. The affinity of 4-DAMP mustard and its transformation products for muscarinic receptors was determined in competitive binding experiments with N-[3H] methylscopolamine, and the results show that the aziridinium ion of 4-DAMP mustard was the most potent form, compared with the parent 2-chloroethylamine (4-DAMP mustard) and the alcoholic hydrolysis product. The rates of receptor alkylation by 4-DAMP mustard were measured in the rat heart and gland. Virtually no alkylation (less than 1%) occurred in the heart at a 4-DAMP mustard concentration of 1.6 nM, after 30 min, whereas almost 50% alkylation was observed in the gland under the same conditions. Almost complete alkylation of receptors in the gland could be achieved at a 4-DAMP mustard concentration of 200 nM, after 1 hr. Treatment of the isolated rat ileum with 4-DAMP mustard caused an irreversible blockade of contractions elicited by the muscarinic agonist oxotremorine-M, and this blockade persisted after extensive washing. The results presented here show that 4-DAMP mustard forms an aziridinium ion that binds irreversibly to muscarinic receptors and exhibits selectivity for M3, compared with M2 muscarinic receptors.     

Kinetics of solvolysis and muscarinic actions of an N-methyl-N-(2-bromoethyl)amino analogue of oxotremorine

An N-methyl-N-(2-bromoethyl)amino analogue (2) of oxotremorine cyclized in phosphate buffer to an aziridinium ion (3). The first-order rate constants (k1) for the cyclization reaction at 22 and 37 degrees C (pH 7.3) were 0.14 and 0.85 min-1, respectively. Determination of k1 as a function of pH gave a pKa value of 5.6 for 2. The rate constants (k2) for the hydrolysis of 3 at 22 and 37 degrees C (pH 7.3) were 0.0083 and 0.040 min-1, respectively. Compound 3 was 3-fold more active than oxotremorine as a muscarinic agonist on the guinea pig ileum, whereas its nicotinic actions, as estimated on the frog rectus, were quite weak. Because of its greater rate of cyclization and the higher peak concentrations of the aziridinium ion which ensue, 2 may offer advantages over its (2-chloroethyl)amino analogue (1) as an alkylating ligand for muscarinic receptors.     

Alkylation of guanosine by phosphoramide mustard, chloromethine hydrochloride and chlorambucil

Guanosine was reacted in vitro with phosphoramide mustard, chloromethine hydrochloride, and chlorambucil. The products were isolated by HPLC and characterized by UV and fluorescence spectroscopy, and C-8 tritium exchange. The primary products were 7-alkylguanosines according to such evidence. Phosphoramide mustard had 1/10 of the apparent alkylation activity of two other mustards. The primary 7-alkylguanosines were unstable at pH 7.4 and 37 degrees; t1/2 were 3 min. for chloromethine hydrochloride, 2.7 hrs for chlorambucil and 3.0 hrs for phosphoramide mustard. Both dechlorination at the unbound arm of the mustard and imidazole ring opening og guanosine appeared to account for such instability.     

Chemically stable, lipophilic prodrugs of phosphoramide mustard as potential anticancer agents

Benzyl phosphoramide mustard (3), 2,4-difluorobenzyl phosphoramide mustard (4), and methyl phosphoramide mustard (5) were examined as lipophilic, chemically stable prodrugs of phosphoramide mustard (2). These phosphorodiamidic esters are designed to undergo biotransformation by hepatic microsomal enzymes to produce 2. The rate of formation of alkylating species, viz., 2, from these prodrugs and their in vitro cytotoxicity toward mouse embryo Balb/c 3T3 cells were comparable to or better than that of cyclophosphamide (1). Preliminary antitumor screening against L1210 leukemia in mice, however, suggests that these prodrugs are devoid of any significant antitumor activity in vivo.     

31P NMR and chloride ion kinetics of alkylating monoester phosphoramidates

31P NMR spectroscopy was used to study the solvolysis kinetics of a novel series of alkylating monoester phosphoramidates (4a-d) under model physiologic conditions. Halide ion kinetics were used to determine the rate of aziridinium ion formation. The solvolysis rates showed the expected dependence upon substitution at the reactive nitrogen; comparison of 4a with phosphoramide mustard (1a) indicated that replacement of the amino group by alkoxy decreased the solvolysis rate by approximately 10-fold. The rate of conversion of starting compound (4a-d) to solvolysis product was essentially equal to the rate of halide ion release, suggesting that the aziridinium ion is a short-lived intermediate. 1H NMR and 31P NMR kinetics experiments performed in the absence and presence of trapping agent (dimethyldithiocarbamate) confirmed that the aziridinium ion was too short-lived to be observed via NMR. These compounds were also tested for cytotoxicity against L1210 leukemia and B16 melanoma cells in vitro; the monoalkylators 4c and 4d showed no activity, 4a was weakly cytotoxic, and 4b was comparable in activity to phosphoramide mustard.     

N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine, the putative major DNA adduct of cyclophosphamide in vitro and in vivo in the rat

The anti-cancer agent, cyclophosphamide, metabolises to the cytotoxic alkylating agent phosphoramide mustard, which can be dephosphoramidated to give nornitrogen mustard. A rat liver mitochondrial supernatant system was used to study the binding of [chloroethyl 3H]cyclophosphamide to DNA. The reacted DNA was acid-hydrolysed and one major adduct was identified using Sephadex G-10 chromatography, followed by HPLC, using reversed-phase or ion-exchange systems. Further studies, using [14C]guanine as reaction substrate for [chloroethyl 3H]cyclophosphamide, phosphoramide mustard or nornitrogen mustard, demonstrated the main adduct from each reaction had identical chromatographic properties in these systems. The radiolabelled ratio in the [3H]cyclophosphamide-[14C]guanine reaction demonstrated a monoadducted product. From this evidence and from 1H NMR data, the common adduct was putatively identified as a hydroxylated nornitrogen mustard adduct (N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine). In in vivo studies, rats were injected intraperitoneally with 2.775 MBq [3H]cyclophosphamide. Total organ [3H] content and DNA binding levels were ascertained. Maximal levels of [3H] binding to DNA were seen between 1-4 hr with the highest binding levels observed in the bladder. The in vivo adduct was shown, using various HPLC systems, to co-chromatograph with the in vitro adduct and thus the main in vivo adduct was putatively identified as N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine.     

Kinetics and mechanism of degradation of epothilone-D: an experimental anticancer agent

The objective of this study was to investigate the stability and the degradation pathway of epothilone-D (Epo-D), an experimental anticancer agent. In pH range 4-9, Epo-D displayed pH-independent stability and the highest stability was observed at pH 1.5-2 where its thiazole group is protonated. Increasing the pH >9 or <1.5 resulted in an increase in the degradation rate. Epo-D contains an ester group that can be hydrolyzed. The formation of the hydrolytic product was confirmed by the nuclear magnetic resonance (NMR), fast atom bombardment mass spectroscopy and liquid chromatography/mass spectroscopy/mass spectroscopy techniques. The largely sigmoidal pH-rate profile is not consistent with the normal pH dependency of ester hydrolysis involving an addition/elimination mechanism. Hence, a hydrolysis mechanism through a carbonium ion was suggested. At pH 4 and 7.4, no buffer catalysis was observed (0.01, 0.02, and 0.05 M buffers) and no significant deuterium kinetic solvent isotope effect was noted. The degradation was very sensitive to changes in the dielectric constant of the solvents as significant enhancement in the stability was observed in buffer-acetonitrile and 0.1 M (SBE)7m-beta-cyclodextrin solutions compared with just buffer, suggesting that the rate-determining step in the degradation pathway involved formation of a polar transition state. Mass spectral analysis of the reaction run in 18O water was consistent with incorporation of the 18O in the alcohol hydroxyl rather than the carboxylate group. These observations strongly support the carbonium ion mechanism for the hydrolysis of Epo-D in the pH range 4-9. A pKa value of 2.86 for Epo-D was estimated from the fit of the pH-rate profile. This number was confirmed independently by the changes in ultraviolet absorbance of Epo-D as a function of pH (pKa 3.1) determined at 25 degrees C and the same ionic strength.     

Hydrolysis of l-phenylalanine mustard (melphalan). II. Further observations on the effects of pH,chloride ions and buffers on the rate of reaction

The effects of pH (3.7—13), ionic strength, buffer composition (acetate, phosphate and borate) and buffer concentration (15–200 mM) on the rate of degradation of melphalan in the presence 0.3 M chloride at 50 ± 0.1 °C were investigated using high-performance liquid chromatography. In addition, the data published in the literature for the degradation of phosphoramide mustard have been compared with those of melphalan, placing emphasis on mechanisms of hydrolysis and the effects of pH and chloride. In the presence of chloride, the degradation rate of melphalan was influenced by pH and buffer composition but not by ionic strength. These effects were not seen in the absence of added chloride and have been explained in terms of competition between chloride and other nucleophiles such as the hydroxide ion, water and buffer components for the active intermediate of the alkylating agent. These results help to explain differences in reported values for the rates of hydrolysis of various alkylating agents in the presence of chloride.     

Spiromustine analogues. Relationships between structure, plasma stability, and antitumor activity

Spiromustine is a hydantoin-containing nitrogen mustard currently in Phase I clinical trial. Since the in vitro plasma half-life of this compound (6.4 min, 37 degrees C, pH 7.4) appeared to be influenced by the hydantoin ring, analogues containing 2-5 methylene spacer groups between this ring and the nitrogen mustard moiety were prepared and evaluated for hydrolytic stability and antitumor activity. Stability correlated with structure and pKa values. The proximity of the hydantoin ring to the mustard function was a stabilizing factor. Activity against murine P-388 leukemia was demonstrated and a gradual decrease in this activity was observed as the hydrolytic instability increased. A relationship between analogue structure and a mass spectral rearrangement ion was identified.     

The hydrolysis and alkylation activities of S-(2-haloethyl)-L-cysteine analogs--evidence for extended half-life

A series of S-(2-haloethyl)-L-cysteine derivatives, which are analogs of the proposed glutathione half-mustard metabolites of dihaloethanes, were synthesized and studied with respect to their hydrolysis and alkylation rates in aqueous solution. The trend of relative hydrolysis rates, Br greater than Cl much greater than F, paralleled their respective leaving group abilities; however, a dramatic rate increase was seen at pH 8 versus pH's 6 or 4. Hydrolysis of S-(2-chloroethyl)-L-cysteine analogs, where the ionizable groups were blocked (carboxyl esterified and/or N-acetylated), revealed that the amine moiety was responsible for the increased hydrolysis of mustard gas (beta, beta'-dichlorodiethyl sulfide) gave similar results with S-(2-chloroethyl)-L-cysteine, a finding which is consistent with the reaction intermediate being a highly charged species. The alkylation rates with 4-(p-nitrobenzyl)-pyridine were not affected by blocking the ionizable groups. A mechanism of internal cyclization is proposed to explain the accelerated alkaline hydrolysis rates noted with S-(2-haloethyl)-L-cysteines but not with the N-acetylated analogs (mercapturic acids). This scheme proposes the formation of 3-(thiomorpholine)-carboxylic acid as an alternative pathway to the generally accepted hydrolysis reaction. This compound and not S-(2-hydroxyethyl)-L-cysteine was the identified product following pH 10 hydrolysis. Increased hydrolysis half-time of amine-blocked cysteine analogs versus parent cysteine analogs may exist with S-(2-haloethyl)-glutathione derivatives which may explain the substantial nucleic acid alkylation seen with S-(2-haloethyl) derivatives of glutathione.     

N-nitrosotolazoline: decomposition studies of a typical N-nitrosoimidazoline

N-nitrosotolazoline ( N-nitroso-2-benzylimidazoline), a N-nitrosated drug typical of N-nitrosoimidazolines, reacts readily with aqueous acid, nitrous acid, or N-acetylcysteine to produce highly electrophilic diazonium ions capable of alkylating cellular nucleophiles. The kinetics and mechanism of the acidic hydrolytic decomposition of N-nitrosotolazoline have been determined in mineral acids and buffers. The mechanism of decomposition in acidic buffer is proposed to involve the rapid reversible protonation of the imino nitrogen atom followed by slow general base-catalyzed addition of H2O to the 2-carbon of the imidazoline ring to give a tetrahedral intermediate, which is also a alpha-hydroxynitrosamine. Rapid decomposition of this species gives rise to the diazonium from which the products are derived by nucleophilic attack, elimination, and rearrangement. The proposed mechanism is supported by the observations of general acid catalysis, a negligible deuterium solvent kinetic isotope effect ( kH/kD = 1.15) and delta S = -34 eu. In phosphate buffer at 30 degrees C, the half-lives of N-nitrosotolazoline range from 5 min at pH 3.5 to 4 h at pH 6. The main reaction product of the hydrolytic decomposition is N-(2-hydroxyethyl)phenylacetamide. This and other products are consistent with the formation of a reactive diazonium ion intermediate. N-nitrosotolazoline nitrosates 50 times more rapidly than tolazoline and results in a set of products derived from reactive diazonium ions but different from those produced from the hydrolytic decomposition of the substrate. N-acetylcysteine increases the decomposition rate of N-nitrosotolazoline by 25 times at pH 7 and results in both N-denitrosation and induced decomposition to produce electrophiles. These data suggest that N-nitrosotolazoline shares the chemical properties of many known direct-acting mutagens and carcinogens.     

Teratogenicity of cyclophosphamide metabolites: Phosphoramide mustard,acrolein, and 4-ketocyclophosphamide in rat embryos cultured in vitro

Phosphoramide mustard, acrolein, and 4-ketocyclosphosphamide, known stable metabolic products of bioactivated cyclophosphamide, were tested for their teratogenicity against rat embryos grown in vitro from Day 10 to Day 11 of gestation. Results indicate that phosphoramide mustard is the teratogenic metabolite of cyclophosphamide, since the effects of phosphoramide mustard exactly parallel those of bioactivated cyclophosphamide. These effects are reductions in total embryo protein content, crown-rump length and number of somites, characteristic malformations, and pattern of cell necrosis. Acrolein at a dose equimolar to a dose of bioactivated cyclophosphamide which produced malformations in 100% of treated embryos had no effect on any of the parameters measured. An equimolar dose of 4-ketocyclophosphamide had no effect on total embryo protein content or crown-rump length and number of somites, but consistently produced some malformed embryos. The kinds of malformations observed, however, are not seen in embryos treated with phospharamide mustard or bioactivated cyclophosphamide.     

Stability studies of some glycolamide ester prodrugs of niflumic acid in aqueous buffers and human plasma by HPLC with UV detection

Glycolamide esters (compounds 1-17) of 2-(3-trifluoromethyl-phenylamino)nicotinic acid (niflumic acid, CAS 4394-00-7) have been synthesized and evaluated as possible prodrugs. In-vitro hydrolysis studies were conducted at selected pH values (1.2, 3.5, 4.8, 7.4 and 7.8) and in human plasma at 37 +/- 0.5 degree C using HPLC with UV detection. The aqueous (pH 7.4 and 7.8) and enzymatic rates of hydrolysis were substantially affected by the nature of promoieties in this series. The compounds showed good chemical stability in the buffers of low pH values (1.2, 3.5 and 4.8) and appreciable hydrolysis under alkaline conditions and in human plasma. They exhibited long hydrolytic half-lives of 7-46 h in aqueous buffer solutions (pH 7.4 and 7.8) and 14-21 min in human plasma, respectively. It was observed that N,N-disubstituted and cyclic glycolamide derivatives showed 2 fold more hydrolysis in the alkaline pH than monosubstituted derivatives, whereas the piperidino and thiomorpholino derivatives did not undergo chemical hydrolysis. The compounds contain two possible sites for hydrolysis with an increased hydrolytic susceptibility at the terminal aliphatic carbonyl site in aqueous buffers and human plasma solutions. They were found to be cleaved at two hydrolytic carbonyls, namely the nicotinyl (2-5 % in enzymatic hydrolysis) and the aliphatic site (7-55 % and 70-85 % in buffer and plasma hydrolysis, respectively) as revealed by HPLC analysis. The glycolamide ester prodrugs of niflumic acid underwent chemical and enzymatic hydrolysis to release mainly the metabolite 2-(3-trifluoromethyl-phenylamino) nicotinic acid carboxymethyl ester (III) and not the parent drug 2-(3-trifluoromethyl-phenylamino)nicotinic acid. The structure of the metabolite was confirmed by liquid chromatography-mass spectroscopy (LCMS).     

The hydrolysis and alkylation activities of S-(2-haloethyl)- -cysteine analogs—Evidence for extended half-life

A series of S-(2-haloethyl)- -cysteine derivatives, which are analogs of the proposed glutathione half-mustard metabolites of dihaloethanes, were synthesized and studied with respect to their hydrolysis and alkylation rates in aqueous solution. The trend of relative hydrolysis rates, Br > Cl F, paralleled their respective leaving group abilities; however, a dramatic rate increase was seen at pH 8 versus pH's 6 or 4. Hydrolysis of S-(2-chloroethyl)- -cysteine analogs, where the ionizable groups were blocked (carboxyl esterified and/or N-acetylated), revealed that the amine moiety was responsible for the increased hydrolysis rate with alkaline conditions. Compounds which were previously shown to inhibit the hydrolysis of mustard gas (β,β′-dichlorodiethyl sulfide) gave similar results with S-(2-chloroethyl)- -cysteine, a finding which is consistent with the reaction intermediate being a highly charged species. The alkylation rates with 4-(p-nitrobenzyl)-pyridine were not affected by blocking the ionizable groups. A mechanism of internal cyclization is proposed to explain the accelerated alkaline hydrolysis rates noted with S-(2-haloethyl)- -cysteines but not with the N-acetylated analogs (mercapturic acids). This scheme proposes the formation of 3-(thiomorpholine)-carboxylic acid as an alternative pathway to the generally accepted hydrolysis reaction. This compound and not S-(2-hydroxyethyl)- -cysteine was the identified product following pH 10 hydrolysis. Increased hydrolysis half-time of amine-blocked cysteine analogs versus parent cysteine analogs may exist with S-(2-haloethyl)-glutathione derivatives which may explain the substantial nucleic acid alkylation seen with S-(2-haloethyl) derivatives of glutathione.