Benzylpiperazine: "A messy drug".

Designer drugs are synthetic structural analogues/congeners of controlled substances with slightly modified chemical structures intended to mimic the pharmacological effects of known drugs of abuse so as to evade drug classification. Benzylpiperazine (BZP), a piperazine derivative, elevates synaptic dopamine and serotonin levels producing stimulatory and hallucinogenic effects, respectively, similar to the well-known drug of abuse, methylenedioxymethamphetamine (MDMA). Furthermore, BZP augments the release of norepinephrine by inhibiting presynaptic autoreceptors, therefore, BZP is a "messy drug" due to its multifaceted regulation of synaptic monoamine neurotransmitters. Initially, pharmaceutical companies used BZP as a therapeutic drug for the treatment of various disease states, but due to its contraindications and abuse potential it was withdrawn from the market. BZP imparts predominately sympathomimetic effects accompanied by serious cardiovascular implications. Addictive properties of BZP include behavioral sensitization, cross sensitization, conditioned place preference and repeated self-administration. Additional testing of piperazine derived drugs is needed due to a scarcity of toxicological data and widely abuse worldwide.

In-vitro hydrolysis, permeability, and ocular uptake of prodrugs of N-[4-(benzoylamino)phenylsulfonyl]glycine, a novel aldose reductase inhibitor.

To enhance the ocular uptake of N-[4-(benzoylamino)phenylsulfonyl]glycine (BAPSG), two ester (methyl and isopropyl) prodrugs were synthesized and evaluated for their stability in various buffers (pH 1-9), hydrolysis in rabbit ocular tissues (cornea, conjunctiva, iris-ciliary body, lens, aqueous humor, and vitreous humor), transport across cornea and conjunctiva, and in-vivo uptake following topical administration. Over the pH range of 1-9, the rate constants for degradation ranged from 5.67 to 218.9 x 10(-3) h(-1) for the methyl ester and from 3.14 to 4.45 x 10(-3) h(-1) for the isopropyl ester. At all pH conditions, the isopropyl ester was more stable when compared with the methyl ester. A change in buffer concentration at pH 7.4 did not influence the stability of the prodrugs. The prodrugs were rapidly hydrolysed in the tissue homogenates, with the rate constants for hydrolysis ranging from 1.98 to 7.2x 10(-3) min(-1) for the methyl ester and 3.32 to 6.53 x 10(-3) min(-1) for the isopropyl ester. The in-vitro permeability of the methyl ester was less than the parent drug across cornea and conjunctiva. Isopropyl ester levels were not detectable in the receiver chamber even at the end of the 4-h transport study. Following topical administration of BAPSG and the two prodrugs at a dose of 60 microg/eye, the lowest levels were seen in vitreous humor for parent compound and its methyl ester. In general, the tissue uptake of methyl ester was less than BAPSG. Isopropyl ester levels were below detection limits in all the ocular tissues. Lipophilic ester prodrugs of BAPSG showed good aqueous solution stability in tissue homogenates. However, these prodrugs lacking the free carboxylate anion exhibited reduced in-vitro permeability and in-vivo uptake, suggesting the importance of free carboxylate anion in the delivery of BAPSG.

Chromatographic and mass spectral methods of identification for the side-chain and ring regioisomers of methylenedioxymethamphetamine.

The popular drug of abuse 3,4-methylenedioxymethamphetamine (MDMA) is one of a total of 10 regioisomeric 2,3- and 3,4-methylenedioxyphenethylamines of MW 193 that yields regioisomeric fragment ions with equivalent mass (m/z 58 and 135/136) in the electron-impact (EI) mass spectrum. Thus, these 10 methylenedioxyphenethylamines are uniquely isomeric; they have the same molecular weight and equivalent major fragments in their mass spectra. The specific identification of one of these compounds (i.e., Ecstasy or 3,4-MDMA) in a forensic drug sample depends upon the analyst's ability to eliminate the other regioisomers as possible interfering or coeluting substances. This study reports the synthesis, chemical properties, spectral characterization, and chromatographic analysis of these 10 unique regioisomers. The ten 2,3- and 3,4-regioisomers of MDMA are synthesized from commercially available precursor chemicals. In the EI mass spectra, the side-chain regioisomers show some variation in the relative intensity of the major ions, with the exception of only one or two minor ions that might be considered side-chain specific fragments. The position of substitution for the methylenedioxy ring is not easily determined by mass spectral techniques, and the ultimate identification of any one of these amines with the elimination of the other nine must depend heavily upon chromatographic methods. The chromatographic separation of these 10 uniquely regioisomeric amines are studied using reversed-phase liquid chromatographic methods with gradient elution and gas chromatographic techniques with temperature program optimization.

Rabbit corneal and conjunctival permeability of the novel aldose reductase inhibitors: N-[[4-(benzoylamino)phenyl] sulphonyl]glycines and N-benzoyl-N-phenylglycines.

Corneal and conjunctival permeability has been investigated for novel aldose reductase inhibitors (ARIs) of the N{[4-(benzoylamino)phenyl]sulphonyl}glycine (benzoylaminophenylsulphonylglycine) and N-benzoyl-N-phenylglycine (benzoylphenylglycine) series, compounds developed for prevention of cataract formation in diabetic subjects. Six benzoylaminophenylsulphonylglycines were synthesized with modifications either of the phenyl group or of the glycine structure and three benzoylphenylglycines were synthesized with modification in the phenyl group of the benzoyl moiety. Transport of ARIs in the mucosal to serosal direction was evaluated across rabbit cornea and conjunctiva bathed in glutathione-bicarbonate Ringer's solution maintained at pH 7.4 and 37 degrees C. The permeability coefficients of the novel ARIs across cornea and conjunctiva ranged from 1.87 to 8.95 x 10(-6) cm s(-1) and from 4.6 to 19.15 x 10(-6) cm s(-1), respectively. The ratio of corneal to conjunctival permeability ranged from 0.12 to 0.79. The calculated log partition coefficient (log P) values for the ARIs were in the range 0.84 to 2.78. The log distribution coefficients (log D) were in the range -2.87 to -0.89. There was no apparent relationship between log P or log D and the permeability coefficients of the ARIs for either tissue. Cornea was more resistant to ARI transport than was conjunctiva. Substitution of a phenyl group for hydrogen in the glycine methylene group reduced the permeability coefficient. Permeability coefficients were different for different stereoisomers. Compared with the permeability coefficient of benzoylaminophenylsulphonylglycine, that of 4-fluorobenzoylaminophenylsulphonylglycine was lower in the cornea but similar in the conjunctiva. In both tissues, the permeability coefficient of 2-nitrobenzoylaminophenylsulphonylglycine was less than that of 4-nitrobenzoylaminophenylsulphonylglycine. There was no significant difference between the permeability coefficients of 3-nitro- and 4-nitrobenzoylphenylglycines through either tissue and the permeability coefficients of these compounds were greater than that of the more lipophilic 4-methylbenzoylphenylglycine. The lack of dependence of the permeability coefficients on log P or log D and the different permeabilities of stereoisomers imply the existence of specialized transport processes for the ARIs tested in this study.

Development of a gas chromatographic-mass spectrometric drug screening method for the N-dealkylated metabolites of fentanyl, sufentanil, and alfentanil.

A sensitive, specific urinary assay for fentanyl, sufentanil, and alfentanil based on their N-dealkylated metabolites is described. Norfentanyl, norsufentanil-noralfentanil, and 2H5-norfentanyl are synthesized and characterized by standard analytical techniques. Derivatization of these secondary amines to yield the pentafluorobenzamides produces stable products with good gas chromatographic properties and unique, high-mass fragments in their mass spectra. These properties are utilized to develop a drug screening procedure based on gas chromatography-mass spectrometry to detect these major metabolites in human urine. The metabolites are isolated from urine samples by a liquid-liquid extraction procedure. The method allows for detection of metabolite concentrations as low as 0.3 ng/mL.

A behavioral comparison of Nexus, cathinone, BDB, and MDA.

The effects of 4-bromo-2,5-dimethoxyphenethylamine (Nexus), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyphenyl-2-butanamine (BDB), and cathinone were studied in the newly hatched chicken and compared to the effects of d-amphetamine and three hallucinogens in the same species. Cathinone, a psychomotor stimulant in man (6), produced effects that were qualitatively similar to effects seen after administration of d-amphetamine (i.e., distress vocalization, wing extension, inability to stand, and loss of righting reflex). BDB, a compound with unknown activity in man, and two known hallucinogens, Nexus (5) and MDA (1), produced effects in the chicken that are common to both stimulants and hallucinogens in this species. For example, both MDA and BDB produced abnormal body posture that was identical to that reported after administration of hallucinogens such as lysergic acid diethylamide (LSD) and harmine (11). Nexus, on the other hand, produced rigid penguin-like posture, an effect seen in the chicken after administration of another hallucinogen, mescaline (12). BDB also produced bursting forward movements, an effect commonly observed after LSD and harmine. Our findings suggest that the young chicken can be used as an alternative, nonmammalian, model for predicting classification of new compounds.

Structure-activity relationships of BDB and its monomethyl and dimethyl derivatives.

The purpose of the present study was to evaluate the behavioral effects of 3,4-methylenedioxyphenyl-2-butanamine (BDB), N-methyl BDB (MBDB), and N,N-dimethyl BDB (MMBDB) in the newly hatched chicken. The primary amine, BDB, produced effects that are commonly seen in the chicken after administration of both hallucinogens and psychomotor stimulants (i.e., distress vocalization, tremor, and wing extension). It also produced abnormal body posture and bursting forward locomotion, effects elicited only by hallucinogens. Loss of righting reflex also occurred at the highest (16 mg/kg) dose of BDB, and this effect is typical of d-amphetamine but has not been reported for hallucinogens. The monomethylated derivative of BDB, MBDB, was less potent than BDB, and the N,N-dimethyl analogue of BDB, MMBDB, had no effect on behavior at the doses tested.

Behavioral and developmental effects of two 3,4-methylenedioxymethamphetamine (MDMA) derivatives.

The effects of 3,4-methylenedioxymethamphetamine (MDMA or 'ecstacy') and two structurally related compounds, N-methyl-1-(3,4-methylenedioxyphenyl)-1-ethanamine (MDM1EA) and N-methyl-1-(3,4-methylenedioxyphenyl)-3-butanamine (HMDMA) were examined in two preparations: (i) a drug discrimination procedure in MDMA-trained rats and (ii) the chicken embryo, for determination of the direct effects of these compounds on the developing organism. The highest doses of MDM1EA and HMDMA partially substituted for MDMA, whereas higher (30-60 mg/kg) doses of HMDMA evoked clonic seizures in a separate group of rats. In chicken embryos MDMA had no effect on body, brain or liver weight, while the highest dose of MDM1EA decreased body weight and the 2 lowest doses of HMDMA increased body weight. All doses of HMDMA decreased liver weight (expressed as % body weight) when compared with contemporaneous water-treated controls. Taken together, the results of these experiments suggest that structurally related compounds share some stimulus properties with MDMA and may therefore share abuse liability. Furthermore, both MDMA-related compounds produced adverse effects on the developing organism, whereas MDMA did not.

Effects of designer drugs on the chicken embryo and 1-day-old chicken.

The present study was conducted to examine the effects of d-amphetamine and the designer drugs 3,4-methylenedioxymethamphetamine (MDMA), N-methyl-3,4-methylenedioxyphenyl-3-butamine (HMDMA), 3,4-methylenedioxyphenyl-2-butanamine (BDB), 3,4-methylenedioxyphenyl-1-ethanamine (MDM1EA) in the chick embryo and the young chicken. HMDMA and MDM1EA had no effect on motility on day 14 of embryogenesis, while MDMA, BDB, and d-amphetamine decreased embryonic motility at one or more doses. On day 1 posthatch, chickens were challenged with cumulative injections of water or the same drug that they had received in ova. With the exception of MDM1EA, all of the drugs produced effects such as distress vocalization, wing extension, tremor, flat body posture, bursting forward movements, loss of righting reflex, and convulsant-like kicking. Pretreatment with drug in ova resulted in tolerance to certain drug effects and supersensitivity to other drug effects. Furthermore, BDB significantly decreased hatchability, MDM1EA decreased body weight, and HMDMA decreased liver weight. Further studies are needed to determine the mechanism(s) of toxicity in this species.

Glutamine-supported motility of adult filarial parasites in vitro and the effect of glutamine antimetabolites.

The survival in culture of adult female Brugia pahangi, Acanthocheilonema viteae, and Onchocerca volvulus and adult male Onchocerca gibsoni was assessed by measuring parasite motility. Survival of all species was maximal in a nutritionally complex medium (RPMI-1640). All species survived for up to 48 hr in a simpler medium in which the only energy source was 10 mM glutamine; motility in this medium was dependent upon pH. For the species of Onchocerca, motility was maintained better in the presence of glutamine as the sole energy source than in glucose-only medium. Motility of B. pahangi incubated in 10 mM succinate was equivalent to that seen with 10 mM glutamine, but no other tricarboxylic acid intermediate supported this parasite in vitro. Antimycin A (1 microM) and potassium cyanide (KCN, 100 microM) paralyzed B. pahangi incubated in 10 mM glutamine, an effect antagonized by glucose. KCN at 10 or 100 microM was effective also against Onchocerca gutturosa in glutamine-only medium. Several glutamine antimetabolites reduced motility of B. pahangi by 72 hr. This inhibition was prevented by 2 mM glutamine. However, the inhibition of motility in the species of Onchocerca caused by these compounds was attenuated only partially by glutamine. These data demonstrate that, under certain conditions, filarial nematodes can utilize non-sugar substrates as energy sources. The differential sensitivity seen among these organisms to mitochondrial toxins and glutamine antimetabolites may be related to the extent to which they can use these alternative substrates to generate energy.

Enzyme selectivity analyses of arylsulfonylamino acid aldose reductase inhibitors.

Arylsulfonylamino acids, displaying a wide range of inhibitory activities versus rat lens aldose reductase (RLAR), were analyzed for enzyme selectivity in several test systems. These RLAR inhibitors were found not to produce significant inhibition of genetically-linked reductases (aldehyde reductase, ALR), catalytically similar reductases (Pachysolen tannophilus xylose reductase, PTXR), functionally distinct oxidoreductases (glutathione reductase, GR, lactate dehydrogenase, LDH, and gamma-transaminase, GABA-T), and thymidylate synthase (TS). These data suggest that aldose reductase differs significantly from other oxidoreductases in its inhibitor binding domain(s). Furthermore, the aldose reductase selectivity demonstrated by the arylsulfonylamino acids suggests that these compounds may not inhibit other key metabolic transformations in various cell types and that they may function as selective probes for studies of the relationship between aldose reductase mediated biochemical changes and the pathologies of chronic diabetes.

GC-MS identification of amine-solvent condensation products formed during analysis of drugs of abuse.

The use of methanol or ethanol as the injection solvent for the gas chromatographic-mass spectral (GC-MS) analysis of low molecular weight amine drugs of abuse results in the formation of additional components in the sample. Primary amines, such as amphetamine, 3,4-methylenedioxyamphetamine, and phenethylamine, yield imines upon injection as methanol or ethanol solutions. In methanol, the imine formed has a mass that is 12 mass units higher than the parent compound. In ethanol, the products formed have 26 additional mass units. Secondary amines appear to undergo methylation under similar conditions with methanol as the injection solvent. These products are absent from the analysis of equivalent amine samples dissolved in chloroform.

Comparison of the catalytic and inhibitory properties of Pachysolen tannophilus xylose reductase to rat lens aldose reductase.

The catalytic and inhibitory profiles of xylose reductase isolated from the yeast Pachysolen tannophilus (PTXR) are compared to those of aldose reductase (AR) obtained from rat lens. While both PTXR and rat lens AR are NADPH-specific enzymes and have an affinity for a variety of substrates such as D-xylose, D,L-glyceraldehyde, and 4-nitrobenzaldehyde, the enzymes differ in their substrate affinity profiles. Also, PTXR is not inhibited by standard inhibitors of AR thus supporting a hypothesis that this enzyme may not possess the inhibitor binding site found in rat lens AR.

Studies on aldose reductase inhibitors from fungi. I. Citrinin and related benzopyran derivatives.

The fungal metabolites, citrinin (4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6- oxo-3H-2-benzopyran-7-carboxylic acid) and DHMI (3,4-dihydro-6-methoxy-3,7-dimethyl-1H-2-benzopyran-8-ol), as well as certain synthetic derivatives, have been evaluated for aldose reductase inhibitory activity using a rat lens enzyme preparation. Citrinin and its reduction product, dihydrocitrinin, were found to have significant activity (IC50 approximately 10 microM), whereas the other compounds were 3-10 times less potent. Kinetic studies showed that citrinin was not an irreversible inhibitor of the enzyme, as might be expected of a quinone methide. Spectroscopic (NMR) evidence is presented for the existence of citrinin predominantly in the form of its hemi-acetal in aqueous solutions, suggesting that it is this benzo[c]pyran derivative which interacts with the enzyme, rather than the quinone methide form.

Relative structure-inhibition analyses of the N-benzoyl and N-(phenylsulfonyl) amino acid aldose reductase inhibitors.

A number of N-benzoyl amino acids were synthesized and tested to compare structure-inhibition relationships with the isosteric N-(phenylsulfonyl) amino acid (PS-amino acid) aldose reductase inhibitors. Inhibition analyses with these series reveals that their kinetic mechanisms of inhibition are similar, but that significant differences in structure-inhibition relationships exist. For example, while the PS-alanines and PS-2-phenylglycines produce enantioselective inhibition (S greater than R), no consistent pattern of enantioselectivity is observed with the isosteric N-benzoylalanines and 2-phenylglycines. Also, N-methyl and N-phenyl substitution in the PS-amino acid series does not substantially alter inhibitory activity, while similar substitutions in the N-benzoyl series (particularly N-phenyl) results in a significant increase in inhibitory activity. Proton NMR analysis of the N-benzoylsarcosines reveals that these compounds exist as a mixture of rotamers in solutions including the enzyme assay buffer and that the preferred conformer is one in which the carboxymethyl moiety is trans to the aromatic ring. Similar analyses with the N-benzoyl-N-phenylglycines demonstrate that these derivatives exist exclusively in the trans rotameric conformation in solution. No such N-substituent effects on conformation were observed in the PS-amino acid series. These results suggest that the differences in structure-inhibition trends between these structurally related series may result from the effect of substituents on preferred conformation.

Methods for the analysis of 1-(3,4-methylenedioxyphenyl)-2-butanamine and N-methyl-1-(3,4-methylenedioxyphenyl)-2-propanamine (MDMA).

The infrared and mass spectra of N-methyl-1-(3,4-methylenedioxyphenyl)-2-propanamine (MDMA) and 1-(3,4-methylenedioxyphenyl)-2-butanamine are quite similar. These two compounds differ only in the position of substitution of a single methyl group. MDMA is a controlled street drug known as Ecstasy, while the isomeric butanamine is a member of a new class of potential psychotherapeutic agents called entactogens. These two compounds produce similar mass spectral fragmentation patterns including a common base peak at m/z 58. Reversed-phase liquid chromatographic (RPLC) methods consisting of a C18 stationary phase and an aqueous scidic mobile phase were used to separate these two compounds. Thus, LC methods can be used to differentiate MDMA from the isomeric butanamine for forensic analysis.

Inhibitory activity and mechanism of inhibition of the N-[[(4-benzoylamino)phenyl]sulfonyl]amino acid aldose reductase inhibitors.

A series of substituted N-[[(4-benzoylamino)phenyl]sulfonyl]amino acids (BAPS-amino acids) were synthesized by established methods, and the stereochemistry of the products was confirmed by HPLC analysis after chiral derivatization. When tested against aldose reductase (alditol:NADP+ oxidoreductase; EC 1.1.1.21; ALR2) isolated from rat lens, all of the BAPS-amino acids were determined to be significantly more inhibitory than the corresponding N-(phenylsulfonyl)amino acids. Structure-inhibition and enzyme kinetic analyses suggest that the BAPS-amino acids inhibit ALR2 by a mechanism similar to the N-(phenylsulfonyl)amino acids. However, multiple inhibition analyses indicate that the increased inhibitory activity of the BAPS-amino acids is a result of interaction with multiple sites present on ALR2. Enzyme specificity studies with several of the BAPS-amino acids demonstrated that these compounds do not produce significant inhibition of other nucleotide-requiring enzymes including aldehyde reductase (alcohol: NADP+ oxidoreductase; EC 1.1.1.2; ALR1).

Diastereoisomeric derivatization and liquid chromatographic analysis of N-(phenylsulfonyl)-2-phenylglycine aldose reductase inhibitors.

A series of (S)- and (R)-N-(phenylsulfonyl)-2-phenylglycines are synthesized as potential inhibitors of the enzyme aldose reductase. In vitro analysis of these compounds reveals that the S-enantiomers are more potent than the corresponding R-enantiomers and that the difference in potencies between enantiomeric pairs is dependent on the nature of the ring substituent. To ensure that the enantioselectivity observed does not reflect varying degrees of racemization during the synthesis of the N-(phenylsulfonyl)-2-phenylglycines, the enantiomeric purity of these products is determined by HPLC after chiral derivatization. Each 2-phenylglycine inhibitor is derivatized with R-alpha-methylbenzylamine, and the resulting diastereomers are analyzed using reversed and normal achiral stationary phases. Reversed-phase methods with C18 or phenyl stationary phases and solvent mixtures of acetonitrile or methanol in water do not provide satisfactory resolution of the diastereomers. However, normal-phase analyses with a silica stationary phase and mixtures of methanol, ethanol, or acetonitrile in chloroform provide good separations with relatively short analysis times. The normal-phase analyses demonstrate that a single diastereomeric amide forms from each N-(phenylsulfonyl)-2-phenylglycine product, establishing that these compounds do not racemize during synthesis.

Liquid chromatographic and mass spectral analysis of the anabolic 17-hydroxy steroid esters.

The liquid chromatographic separation of the common anabolic steroid esters is accomplished in an isocratic reversed-phase system. These anabolic steroids are now controlled substances in many states in the U.S. due to their potential for abuse and misuse. The esters are designed to be very lipophilic materials for slow release from intramuscular injection sites. With a C18 stationary phase material and a mobile phase of methanol-water (85:15), the more lipophilic esters such as testosterone decanoate show a retention time of about 50 min. These esters of varying lipophilicity are well resolved by reversed-phase liquid chromatography, and this technique is very useful for the identification of these compounds in forensic samples.

Liquid chromatographic and spectral analysis of the 17-hydroxy anabolic steroids.

The liquid chromatographic properties of various 17-hydroxy anabolic steroids are examined under reversed-phase conditions. These anabolic steroids are now listed as controlled drugs in many states due to their abuse potential in athletics, body building, and other areas. These nonesterified steroids are separated on a C18 stationary phase with a 70% methanol in water mobile phase. In a few cases, two compounds display very similar retention properties. However, dual-wavelength detection at 254 and 280 nm allows for their differentiation. Reversed-phase retention parallels steroid lipophilicity based on hydroxyl and methyl group substituents. Also, those steroids containing a dienone substructure are more polar than steroids containing an enone moiety.