Microwave-Assisted Synthesis of Trazodone and Its Derivatives as New 5-HT1A Ligands: Binding and Docking Studies.

Trazodone, a well-known antidepressant drug widely used throughout the world, works as a 5-hydroxytryptamine (5-HT2) and α1-adrenergic receptor antagonist and a serotonin reuptake inhibitor. Our research aimed to develop a new method for the synthesis of trazodone and its derivatives. In the known methods of the synthesis of trazodone and its derivatives, organic and toxic solvents are used, and the synthesis time varies from several to several dozen hours. Our research shows that trazodone and its derivatives can be successfully obtained in the presence of potassium carbonate as a reaction medium in the microwave field in a few minutes. As a result of the research work, 17 derivatives of trazodone were obtained, including compounds that exhibit the characteristics of 5-HT1A receptor ligands. Molecular modeling studies were performed to understand the differences in the activity toward 5-HT1A and 5-HT2A receptors between ligand 10a (2-(6-(4-(3-chlorophenyl)piperazin-1-yl)hexyl)-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one) (5-HT1A Ki = 16 nM) and trazodone. The docking results indicate the lack of the binding of ligand 10a to 5-HT2AR, which is consistent with the in vitro studies. On the other hand, the docking results for the 5-HT1A receptor indicate two possible binding modes. Crystallographic studies support the hypothesis of an extended conformation.

A comparative solid-phase extraction study for the simultaneous determination of fluvoxamine, mianserin, doxepin, citalopram, paroxetine, and etoperidone in whole blood by capillary gas-liquid chromatography with nitrogen-phosphorus detection.

This paper reports a simple and reliable gas chromatographic method with nitrogen-phosphorus detection without derivatization for the simultaneous detection of fluvoxamine, mianserin, doxepin, citalopram, paroxetine, and etoperidone in whole blood as part of a systematic toxicological analysis (STA). All drugs were studied at concentration levels of 100-2000 ng/mL, except paroxetine for which it was necessary to study at concentration levels of 400-8000 ng/mL. A comparative and validation study using two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, was developed regarding their recovery, precision, sensitivity, and matrix purification efficiency. The Chem Elut columns, diatomaceous earth, are closely related to conventional liquid-liquid extraction. The Bond Elut Certify columns, more recently developed in the market, are mixed SPE (reversed-phase and cation exchange sorbent). Recoveries for the antidepressants using Chem Elut columns at 500 ng/mL (2000 ng/mL for paroxetine) were in the range 43-72% with intra- and interassay precisions of less than 10% and 16%, respectively. Limits of detection (LODs) and quantitation (LOQs) for fluvoxamine, mianserin, doxepin, citalopram, and etoperidone ranged from 18 to 236 ng/mL and 60 to 786 ng/mL, respectively. LOD and LOQ for paroxetine were 303 and 1009 ng/mL, respectively. Recoveries of these compounds using Bond Elut Certify columns at 500 ng/mL (2000 ng/mL for paroxetine) were in the range 52-83% with intra- and interassay precisions of less than 6% and 8%, respectively. LODs and LOQs for fluvoxamine, mianserin, doxepin, citalopram, and etoperidone ranged from 7 to 28 ng/mL and 23 to 93 ng/mL, respectively. LOD and LOQ for paroxetine were 113 and 376 ng/mL, respectively. An excellent linearity was observed with both procedures from the LOQs up to the upper studied concentration level. In general, higher recoveries, cleaner extracts, better sensitivity, better precision, and reduced solvent consumption and disposal were achieved for the screening of these antidepressants with the use of the mixed SPE Bond Elut Certify compared with Chem Elut columns. The application of these methods on a forensic case study is also presented.

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone.

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 +/- 3.1 pmole nmole(-1) min(-1). Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.

alpha(1)-Adrenoceptor antagonists. Rational design, synthesis and biological evaluation of new trazodone-like compounds.

A rational design approach has been applied to synthesize a novel class of compounds with affinity for alpha(1) adrenergic receptors (AR). Molecular structures are characterized by a benzimidazolylpyridazinone or an imidazolylpyridazinone moiety, an original fragment in the field of the arylpiperazine compounds with alpha(1)-AR blocking properties. A 1.1 nM affinity toward alpha(1)-AR was found for compound 3, the most active of this series.

Evaluation of the absorption, excretion and metabolism of [14C] etoperidone in man.

1. The absorption, excretion and metabolism of 2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-4,5-diethyl-2,4-dihydro-3H-1,2,4- triazole-3-one hydrochloride (etoperidone HCl) was investigated in six healthy men. Subjects were tasted overnight before receiving a single oral dose of a 100 mg solution [14C] etoperidone HCl. 2. Plasma (0-48 h), urine (0-120 h) and faecal (0-120 h) samples were collected. The terminal half-life of the total radioactivity from plasma was 21.7 +/- 2.8h with an apparent clearance of 1.01 +/- 0.08 ml min(-1). Recoveries of total radioactivity in urine and faeces were 78.8 +/- 3.6% and 9.6 +/- 4.1% of the dose, respectively. 3. Etoperidone and 21 metabolites were isolated and identified in the plasma, urine and faecal extracts. Unchanged etoperidone accounted for <0.01% of the dose in all excreta samples. Nine metabolites were identified in the plasma extracts and 21 urinary metabolites were identified. Seven faecal metabolites were identified. 4. Five proposed pathways were used to describe the formation of the metabolites: alkyl oxidation, piperazinyl oxidation, N-dealkylation, phenyl hydroxylation and conjugation. Alkyl oxidation of etoperidone resulted in the formation of 2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-4-ethyl-2,4-dihydro-5- (1-hydroxyethyl)-3H-1,2,4-triazole-3-one. Piperazinyl oxidation of this metabolite leads to the formation of its N-oxide. N-dealkylation of the piperazinyl group led to the formation of 1-(3-chlorophenyl) piperazine and triazole propionic acid. Phenyl hydroxylation led to three important metabolites in the urine and faeces.

3-(omega-Aminoalkyl)-5,5-dialkyl(or spirocycloalkyl-1',5-)hydantoins as new 5-HT1A and 5-HT2A receptor ligands.

A series of new 3-(omega-aminoalkyl)-5,5-disubstituted hydantoins, containing 1-phenylpiperazine, 1-(o-methoxyphenyl)piperazine or 1,2,3,4-tetrahydroisoquinoline fragments, were synthesized by standard alkylation procedures and their 5-HT1A and 5-HT2A receptor affinities were determined. It has been shown that the investigated derivatives are recognized by 5-HT1A and 5-HT2A receptors due to the presence of a 1-arylpiperazine fragment however, the terminal hydantoin moiety plays an important role in stabilization of the receptor-ligand complex. It has also been found that the two 1-phenylpiperazine derivatives 32 and 36 are new selective 5-HT2A receptor ligands (Ki = 34 and 37 nM, respectively), whereas the derivative of 1-(o-methoxyphenyl)piperazine (38) is a new, highly potent 5-HT1A receptor ligand (Ki = 0.51 nM) with a moderate affinity for 5-HT2A receptors (Ki = 213 nM).

Anticholinergic, antihistaminic and cardiovascular effects of two new congeners of quipazine and trazodone.

Two recently reported analogues of quipazine (CAS 4774-24-7) and trazodone (CAS 19794-93-5) were found to be selective inhibitors of 5-hydroxytryptamine (5-HT) uptake, with an antidepressant profile similar to the atypical antidepressants. In the present work the anticholinergic, antihistamine and cardiovascular effects of the compounds were investigated in experimental animals and compared with imipramine and trazodone. Both compounds have demonstrated weak anticholinergic and antihistamine activities on the guinea-pig ileum and showed weak negative inotropic effects on the isolated rabbit heart as compared to imipramine. The marked bradycardia produced by imipramine and trazodone, and the extrasystoles produced by imipramine were not produced by either compound. Binding studies at 5-HT receptors indicated that the quipazine analogue, compound I, has moderate affinity for the selective 5-HT1D ligand while the trazodone analogue, compound II, binds to 5-HT2 receptors. Structural modifications of these compounds may offer new potent analogues with selective affinity at 5-HT receptor-subtypes.

Effects of etoperidone on sympathetic and pituitary-adrenal responses to diverse stressors in humans.

The effects of the psychotropic drug etoperidone on the response to laboratory stressors was investigated in a controlled study. Cardiovascular and hormonal (catecholamines, corticotropin, and cortisol) measurements were made in a group of young, healthy volunteers during a cold pressor test (CPT), a mental arithmetic test (MAT), and insulin-induced hypoglycaemia (ITT). One-week treatment with etoperidone (ETO) (150 mg/day, orally) reduced basal and stress-induced values of systolic and diastolic blood pressure (BP) on CPT, while it did not alter catecholamine output in response to the stressor. Cardiovascular response was also attenuated after ETO on MAT, in the absence of any hormone changes. Adrenocorticotropic hormone (ACTH) and cortisol secretions were markedly reduced on ITT after ETO, whereas catecholamine outflow and cardiovascular parameters were substantially unaffected. These findings on ITT suggest that the anti-serotoninergic and anti-alpha 1-adrenergic activities of ETO may be used in the pharmacological control of the potentially detrimental consequences of the stress response.

Side effects and the "blindability" of clinical drug trials.

A novel, simple approach to retrospective assessment of "blindability" was applied to data on outpatients in a controlled, double-blind clinical comparison of a putative antidepressant, etoperidone, and placebo. A "blind" evaluator proved capable of discriminating between the active drug and placebo on the basis of reported side effects alone, raising questions about the true blindness of the study.

Etoperidone, trazodone and MCPP: in vitro and in vivo identification of serotonin 5-HT1A (antagonistic) activity.

The Ki values for etoperidone, trazodone and MCPP (m-chlorophenylpiperazine dihydrochloride) at 5-HT1A sites (using rat cerebral cortical synaptosomes and [3H]8-OH-DPAT) were determined to be 20.2, 23.6 and 18.9 nM, respectively. In an effort to elucidate the functional nature of the interaction at 5-HT1A sites in vivo, the ability of each compound to elicit reciprocal forepaw treading (RFT) or to block the RFT induced by 8-OH-DPAT in reserpinized rats was tested. Specifically, 8-OH-DPAT (1.0 mg/kg SC)-challenged or non-challenged (control) reserpinized (1.0 mg/kg SC) rats were administered etoperidone, trazodone or MCPP (IP) and scored for the elicitation of RFT (indicative of 5-HT1A agonistic activity) or for block of RFT induced by 8-OH-DPAT (indicative of 5-HT1A antagonistic activity). Reference compounds confirmed the specificity of the test. We report that etoperidone, trazodone and MCPP inhibited 8-OH-DPAT-induced RFT (ID50 = 17.4, 23.8 and 13.4 mg/kg, respectively). Only marginal RFT was produced in non-challenged animals by etoperidone and trazodone at a high dose (40 mg/kg). Taken together, the results suggest a predominant antagonistic activity of etoperidone, trazodone and MCPP at 5-HT1A receptor sites in rat central nervous system. However, one cannot rule out the possibility that these compounds are weak partial agonists. This activity may be relevant to the antidepressant action of these compounds.

New hybrids of quipazine and trazodone as selective inhibitors of uptake of 5-hydroxytryptamine.

Two new congeners, 4-(chloropropyl)-1-(2-quinolyl)piperazine- and 2-[3-[4-[2-(quinolyl)]-1-piperazinyl]propyl]-1,2,4-triazolo] 4,3-a]pyridin-3(2H)-one, of trazodone were synthesized and found to be potent and selective inhibitors of synaptosomal uptake of 5-hydroxytryptamine [5-HT, serotonin; IC50 = norepinephrine greater than 5 microM, 5-HT = 210-890 nM], with minimal effects in antagonizing (-)-apomorphine-induced climbing behavior and suppression of spontaneous locomotor activity in mice (ED50 greater than 50 mg/kg). The two compounds behaved like atypical antidepressants, since they weakly antagonized reserpine-induced hypothermia. The acute toxicity studies have shown that these compounds were less lethal when compared with imipramine or quipazine. Furthermore, chronic treatments (20 mg/kg, daily for 10 and 21 days) significantly decreased the isoprenaline-induced increase in cyclic AMP in the rat brain cortex, suggesting desensitization of beta-adrenoceptors. These findings point to the effects of these compounds as potential antidepressants dealing with specific serotonergic mechanisms.

High-performance liquid chromatographic assay with ultraviolet detection for the determination of etoperidone and two active metabolites, 5-(1-hydroxyethyl) etoperidone and 1-(3-chlorophenyl)piperazine, in plasma.

A selective and sensitive high-performance liquid chromatographic assay with ultraviolet detection for the determination of the antidepressant drug etoperidone and two active metabolites in plasma is described. The drug, metabolites and internal standard are isolated from plasma using a two-step liquid-liquid extraction procedure. The resulting sample is chromatographed on a C18 column (10 cm x 2.1 mm I.D.) with ultraviolet detection at 254 nm. Standard curves are linear for each compound over the concentration range 2-1000 ng/ml. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from the true value and the relative standard deviation (inter-run), are less than or equal to 10% at all concentrations except the minimum quantification limit. Using an automated injector and computerized data acquisition, eighty samples can be routinely processed in one day. The assay has been successfully used for the analysis of plasma samples from pharmacokinetic studies in mice, rats, dogs and humans.

Blockade of conditioned avoidance responding by trazodone, etoperidone, and MCPP.

Phenylpiperazines, such as meta-chlorophenylpiperazine (MCPP) a serotonin agonist, have recently been reported to block conditioned avoidance responding (CAR) in the rat, which is an indication of possible antipsychotic utility. Since MCPP is a major metabolite of both antidepressant drugs trazodone (TZ) and etoperidone (ET), both were examined for activity in blocking CAR in a single-trial lever press task in Fisher 344 rats. Both TZ and ET produced dose-related falls in CAR with ED50 values (95% confidence limits) of 13.3 (9.6, 18.5) and 10.4 (8.5, 13.2) mg/kg IP, respectively. In contrast, MCPP had an ED50 value of 2.5 (1.8, 3.6) mg/kg IP TZ, ET, and MCPP were also examined for the production of catalepsy and the blockade of amphetamine-induced stereotypy to determine whether each was acting to block CAR via a dopaminergic mechanism of action. None, however, was found highly active. On the other hand, the serotonin receptor blocker metergoline (1.0 mg/kg IP) significantly reduced the CAR block produced by each, suggesting a serotonergic mechanism of action. Since TZ and ET are both less potent than MCPP, the data also suggest TZ and ET may block CAR via formation of MCPP.

Inhibitory effects of etoperidone on the spontaneous activity of brainstem neurones and their excitatory responses to some putative transmitters.

In this microiontophoretic study delta 2,1,2,4-triazolin-5-one [1,3-(4-m-chlorophenyl-1-piperazinyl) propyl]-3,4-diethyl hydrochloride (etoperidone, ET, Staff) was applied on rat brainstem (medullary-pontine) reticular neurones to verify its effects on the spontaneous firing and neuronal responses to administrations of 5-hydroxytryptamine (5HT), noradrenaline (norepinephrine, NA), acetylcholine (ACh) and gamma-aminobutyric acid (GABA). ET was able to depress the spontaneous firing by a dose-dependent (for a current intensity range of 40-70 nA) local anaesthetic-like mechanism. At 75 nA a reduction in the amplitude of the action potentials occurred, partially due to a non-specific effect of ET. Repeated administrations of ET caused a progressive neuronal desensitization to the inhibition (tachyphylaxis). All the excitatory neuronal responses to ACh, 5HT and NA (interpreted respectively as nicotinic cholinergic, alpha-noradrenergic, 5HT3-serotonergic) were blocked by ET, while the inhibitory responses to 5HT, NA and GABA were not affected. The analysis of the results leads to postulate for ET a postsynaptic mechanism of action.

Active drug metabolites. An overview of their relevance in clinical pharmacokinetics.

This review underlines the importance of considering in the overall evaluation of drug effect and efficacy not only the kinetics and activities of the administered drug, but also those of the chemical species (metabolites) which are formed in the body. The circumstances in which a role for active drug metabolites may be suspected are described, and a number of specific examples are given. Four different categories are described: drugs which are inactive precursors of active metabolites (e.g. DOPA and cyclophosphamide); active metabolites which contribute to the duration of action of the parent compound (e.g. hexamethylmelamine and clobazam); active metabolites showing a mechanism of action different from that of the parent compound (e.g. buspirone and 1-pyrimidinyl piperazine; fenfluramine and norfenfluramine); and active metabolites showing an antagonistic effect on the activity of the parent drug (e.g. trazodone and m-chlorophenyl-piperazine; aspirin and salicylate).

Gas chromatographic-mass spectrometric method for trazodone and a deuterated analogue in plasma.

A plasma assay method for trazodone and a 2H4 analogue is described which uses gas chromatography--electron-impact selected-ion monitoring mass spectrometry. Etoperidone is used as an internal standard. The analytes are extracted from basic medium into n-butyl chloride, then back extracted into aqueous 0.1 M hydrochloric acid. The aqueous layer is made basic and re-extracted with n-butyl chloride. The solvent is reduced under nitrogen at 35 degrees C and the residue is redissolved in toluene for gas chromatographic--mass spectrometric analysis. The ions monitored are m/z 231, 235, and 225 for trazodone, [2H4] trazodone and etoperidone, respectively. Quantitation is in the range 40-1000 ng/ml with acceptable precision and accuracy. The method is suitable for biopharmaceutical studies.

Changed plasma prolactin levels after etoperidone and placebo.

A neuroendocrinological study was carried out by evaluating plasma prolactin levels after etoperidone i.m. (100 mg) and placebo. Fourteen male inpatients (mean age: 35.36 +/- 11.7 years) with chronic schizophrenia were selected for the study, whose aim was to improve interpretation of the pharmacological activity of etoperidone. The results suggest that etoperidone plays the role of an atypical psychotropic drug since it does not affect prolactin levels. In addition, the drug is devoid of anticholinergic effects, which facilitates its prospective clinical use for medium-term and long-term therapy.

A comparative pharmacological study of trazodone, etoperidone and 1-(m-chlorophenyl)piperazine.

Since 1-(m-chlorophenyl)piperazine (mCPP) is a metabolite of trazodone (TRZ) and etoperidone (ETO), two atypical antidepressants, a pharmacological study was undertaken to establish the possible contribution of mCPP to the effects of the parent compounds. Behavioral effects of mCPP in rats consist in head shakes and other signs of serotoninergic stimulation; subtoxic doses also produce clonic convulsions and prostration. TRZ and ETO produce sedation and signs of alpha-adrenergic blockade; subtoxic doses produce tremors, clonic convulsions and prostration. Peripheral effects of norepinephrine (NE) and serotonin (5-HT) in rats are potentiated by mCPP and inhibited by TRZ and ETO. 5-hydroxytriptophan (5-HTP)-induced head twitches in mice are inhibited by TRZ and ETO and unaffected by mCPP. At similar doses mCPP, TRZ and ETO inhibit some nociceptive responses in rats and mice.

[3H]tetrahydrotrazodone binding. Association with serotonin binding sites.

High (17 nM) and low (603 nM) affinity binding sites for [3H]tetrahydrotrazodone ([3H] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [3H]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [3H] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [3H]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors.