Dopa Methyl Ester Research Articles

Diastereospecific Synthesis of Tetrahydroisoquinolines via Radical Cyclization: Application in the Synthesis of ent‐Tadalafil

AbstractAn enantioselective synthesis of 1‐substituted tetrahydroisoquinolines from L–Dopa methyl ester through intramolecular aryl radical cyclization is demonstrated. The strategy consists of bromination of (S)‐2‐amino‐3‐(2‐bromo‐4,5‐dimethoxyphenyl)propanoate followed by condensation with various aldehydes to afford bromoimidate ester. Aryl radicals generated from bromoimidate ester under the radical generating conditions (nBu3SnH/AIBN) cyclizes via 6‐endo mode to afford cis‐1‐substituted tetrahydroisoquinolines exclusively in 99% ee. The utility of this synthetic protocol is demonstrated in the synthesis of (6S, 12aS) Tadalafil (5 steps, 21%, 99% ee).magnified image

The novel R347g pathogenic mutation of aromatic amino acid decarboxylase provides additional molecular insights into enzyme catalysis and deficiency

We report here a clinical case of a patient with a novel mutation (Arg347→Gly) in the gene encoding aromatic amino acid decarboxylase (AADC) that is associated with AADC deficiency. The variant R347G in the purified recombinant form exhibits, similarly to the pathogenic mutation R347Q previously studied, a 475-fold drop of kcat compared to the wild-type enzyme. In attempting to unravel the reason(s) for this catalytic defect, we have carried out bioinformatics analyses of the crystal structure of AADC-carbidopa complex with the modelled catalytic loop (residues 328–339). Arg347 appears to interact with Phe103, as well as with both Leu333 and Asp345. We have then prepared and characterized the artificial F103L, R347K and D345A mutants. F103L, D345A and R347K exhibit about 13-, 97-, and 345-fold kcat decrease compared to the wild-type AADC, respectively. However, unlike F103L, the R347G, R347K and R347Q mutants as well as the D345A variant appear to be more defective in catalysis than in protein folding. Moreover, the latter mutants, unlike the wild-type protein and the F103L variant, share a peculiar binding mode of dopa methyl ester consisting of formation of a quinonoid intermediate. This finding strongly suggests that their catalytic defects are mainly due to a misplacement of the substrate at the active site. Taken together, our results highlight the importance of the Arg347-Leu333-Asp345 hydrogen-bonds network in the catalysis of AADC and reveal the molecular basis for the pathogenicity of the variants R347. Following the above results, a therapeutic treatment for patients bearing the mutation R347G is proposed.

Kinetic cooperativity of tyrosinase. A general mechanism.

Tyrosinase shows kinetic cooperativity in its action on o-diphenols, but not when it acts on monophenols, confirming that the slow step is the hydroxylation of monophenols to o-diphenols. This model can be generalised to a wide range of substrates; for example, type S(A) substrates, which give rise to a stable product as the o-quinone evolves by means of a first or pseudo first order reaction (α-methyl dopa, dopa methyl ester, dopamine, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, α-methyl-tyrosine, tyrosine methyl ester, tyramine, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid), type S(B) substrates, which include those whose o-quinone evolves with no clear stoichiometry (catechol, 4-methylcatechol, phenol and p-cresol) and, lastly, type S(C) substrates, which give rise to stable o-quinones (4-tert-butylcatechol/4-tert-butylphenol).

Changes in the mRNA Levels of α2A and α2C Adrenergic Receptors in Rat Models of Parkinson’s Disease and l-DOPA-Induced Dyskinesia

The changes in the mRNA levels of α(2A) and α(2C) adrenoceptors were investigated in unilateral 6-OHDA-lesioned rat model of Parkinson's disease and L: -DOPA-induced dyskinesia using in situ hybridization. In the untreated 6-OHDA-lesioned rats, α(2A) expression was elevated in the locus coeruleus (160 ± 8% and 142 ± 8% in lesioned and unlesioned sides compared to the comparable side in sham-operated rats). Following long-term (21 days, twice daily) treatment with L: -DOPA (25 mg/kg L: -DOPA methyl ester plus benserazide 6.25 mg/kg) in 6-OHDA-lesioned rats, levels of α(2A) adrenoceptor mRNA in the locus coeruleus were decreased, compared to the 6-OHDA-lesioned rats, returning to the levels of α(2A) mRNA in the sham-operated rats. α(2A) adrenoceptor expression was not changed in other brain regions in any treatment group. There was no change in α(2C) expression in the rostral or caudal striatum in which the highest density of α(2C) mRNA is present. In conclusion, the data presented in this study demonstrate an increase in α(2A) adrenoceptor mRNA in the locus coeruleus in the 6-OHDA-lesioned rat model of Parkinson's disease. In addition, the data show that repeated treatment with L: -DOPA in 6-OHDA-lesioned rats, which induces dyskinesia, restores α(2A) mRNA levels. These changes of α(2A) mRNA expression, observed in the locus coeruleus, might be of importance to basal ganglia transmission and motor function.

Flibanserin attenuates l-DOPA-sensitized contraversive circling in the unilaterally 6-hydroxydopamine-lesioned rat model of Parkinson’s disease

A central problem in the treatment of Parkinson's disease (PD) is the development of motor disturbances like L: -DOPA-induced dyskinesia (LID) after long-term treatment. Preclinical and clinical studies demonstrated that serotonin 5-HT(1A) receptor agonists attenuate this disabling motor side effect. The aim of this study was to investigate the ability of flibanserin compared to buspirone to attenuate L: -DOPA-sensitized contraversive circling in hemiparkinsonian rats, which is an animal model of LID. Both drugs have a preferential affinity for the serotonin 5-HT(1A) receptors. Buspirone was in comparison because it was expected to have an effect in this model. Unilaterally 6-hydroxydopamine lesioned rats were treated twice daily intraperitoneally (ip) with L: -DOPA methylester (12.5mg/kg) and benserazide (3.25mg/kg) for 21days (on days 1, 3, 5, 8, 11, 14, 17 and 21). On day 24, L: -DOPA-sensitized rats were treated ip 5min prior to administration of L: -DOPA methyl ester and benserazide with either saline (controls), 2.5, 5 and 10mg/kg buspirone or flibanserin. Acute administration of both flibanserin and buspirone, dose dependently, attenuated the increased contraversive circling. An almost complete inhibition of the turning response was observed at 5mg/kg buspirone and 10mg/kg flibanserin. The current preclinical findings further implicate the 5-HT(1A) receptor as a promising therapeutic target for the reduction of LID and predict a potential efficacy of flibanserin in the treatment of LID in PD.

Determination, purity assessment and chiral separation of levodopa methyl ester in bulk and formulation pharmaceuticals

A sensitive, reliable and reproducible HPLC method with electrochemical detection (HPLC-ECD) has been developed for the separation and quantification of levodopa methyl ester (LDME) and its impurities such as levodopa (l-DOPA), 3-methoxytyrosine (MTS) and l-tyrosine (TS) in bulk drug and pharmaceutical dosage form. The separation was performed on an LC18 column by isocratic elution with methanol-acetonitrile-50 mm potassium dihydrogen phosphate (8:2:90, v/v/v) containing 5 mm sodium 1-hexanesulfonate, 5 mm EDTA and 5 mm sodium chloride, adjusted with phosphoric acid to a pH of 3.2 as mobile phase. The correlation coefficients of linear regression for LDME, L-DOPA, MTS and TS were more than 0.999. The detection limits for L-DOPA, MTS and TS were 3.15, 2.04 and 2.88 ng/mL, respectively. The precision was checked in terms of F-test variance ratio using potentiometric titration as reference. The separation of dopa methyl ester enantiomers by chiral chromatography is also described. This method is capable of separating the two enantiomers with a selection of 1.4 and a resolution of 8.4. Both methods were found to be stable and useful in the quality control of the bulk material and formulations.

Instability of the apo form of aromatic L-amino acid decarboxylase in vivo and in vitro: implications for the involvement of the flexible loop that covers the active site.

Pyridoxine deficiency caused a decrease in the amount of aromatic L-amino acid decarboxylase (AADC) in PC12 cells to less than 5% of the control. The degree of the enzyme saturation with the coenzyme pyridoxal 5'-phosphate (PLP) was around 90% for both the control and the pyridoxine-deficient cells, contrary to earlier reports by others. Mathematical analysis of the result indicated that the AADC apoenzyme is degraded at least 20-fold faster than the holoenzyme in the cells. To determine the mechanism of the preferential degradation of the apoenzyme, in vitro model studies were carried out. AADC has a flexible loop that covers the active site. This loop was easily leaved by proteases at similar rates for both the holoenzyme and the apoenzyme. However, in the presence of the substrate analog, dopa methyl ester, the holoenzyme was not cleaved by proteases, while the apoenzyme was cleaved similarly. These results indicated that the ligand that forms a Schiff base (aldimine) with PLP is fixed to the active site and stabilizes the flexible loop. The structure of the rat AADC-dopa complex modeled on the crystal structure of pig AADC showed that the flexible loop can fit in the concave surface at the entrance of the active site, its aliphatic and aromatic residues forming hydrophobic interactions with the substrate catechol ring. It was postulated that the flexible loop of the holoenzyme is stabilized in vivo by taking a closed structure that holds the PLP-substrate aldimine, while the apoenzyme cannot bind the substrate and its flexible loop is easily cleaved, leading to the preferential degradation of the apoenzyme.

Identification of Drosophila melanogaster yellow-f and yellow-f2 proteins as dopachrome-conversion enzymes.

This study describes the identification of Drosophila yellow-f and yellow-f2 as dopachrome-conversion enzymes responsible for catalysing the conversion of dopachrome into 5,6-dihydroxyindole in the melanization pathway. Drosophila yellow -y gene and yellow -b, -c, -f and -f2 genes were expressed in an insect cell/baculovirus expression system and their corresponding recombinant proteins were screened for dopachrome-conversion enzyme activity. Among the yellow and yellow -related genes, the yellow -f and yellow -f2 genes were identified as the genes coding for Drosophila dopachrome-conversion enzyme based on the high activity of their recombinant proteins in catalysing the production of 5,6-dihydroxyindole from dopachrome. Both yellow-f and yellow-f2 are capable of mediating a decarboxylative structural rearrangement of dopachrome, as well as an isomerization/tautomerization of dopamine chrome and dopa methyl ester chrome. Northern hybridization revealed the transcription of yellow -f in larvae and pupae, but a high abundance of mRNA was observed in later larval and early pupal stages. In contrast, yellow-f2 transcripts were present at all stages, but high abundance of its mRNA was observed in later-stage pupae and adults. These data indicate that yellow-f and yellow-f2 complement each other during Drosophila development and that the yellow-f is involved in larval and pupal melanization, and yellow-f2 plays a major role in melanization reactions in Drosophila during later pupal and adult development. Results from this study provide the groundwork towards a better understanding of the physiological roles of the Drosophila yellow gene family.

New crown-carrier Cα,α-disubstituted glycines derived from α-methyl-( l)-DOPA

The side-chain catechol function of α-methyl-( l)-DOPA was utilized for the synthesis of a new series of crown-carrier C α,α-disubstituted glycines. During N-protection of α-methyl-( l)-DOPA methyl ester (H-Mdp-OMe) with Boc anhydride, the formation of N, O-di-Boc derivatives was observed. Selective aminolysis of the catechol tert-butyl carbonate group was achieved using pyrrolidine as nucleophile. Treatment of the resulting Boc-Mdp-OMe by Cs 2CO 3 in MeOH, followed by bis- O-alkylation with cyclization using various polyethyleneglycol ditosylates or catechol-derived bis-polyethyleneglycol ditosylates in DMF at 60°C, led to the derivatives Boc-[15-C-5]-Mdp-OMe, Boc-[18-C-6]-Mdp-OMe, Boc-[benzo-18-C-6]-Mdp-OMe, Boc-[benzo-24-C-8]-Mdp-OMe and then after saponification, to the corresponding N α-protected amino acids. The tripeptides Fmoc-Ala-[18-C-6]-Mdp-Ala-OMe and Boc-Aib-[18-C-6]-Mdp-Ala-OMe were prepared in solution by using Ala and Aib UNCAs for coupling at the N terminus of the [18-C-6]-Mdp residue and the EDC/HOAt method for coupling Ala at its C terminus.

Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedesaegypti

In this study we describe the purification and molecular cloning of a dopachrome conversion enzyme (DCE) from the yellow fever mosquito, Aedes aegypti. DCE catalyzes the conversion of l-dopachrome to 5,6-dihydroxyindole in the melanization pathway. Melanin biosynthesis is involved with crucial protective phenomena in mosquitoes, including egg chorion and cuticular tanning, wound healing, and the melanotic encapsulation immune response. The enzyme was purified to homogeneity by various chromatographic techniques from A. aegypti larvae and has a relative molecular mass of 51 kDa as-revealed by SDS-PAGE analysis. Physiochemical analysis of DCE revealed a pH optimum of 7.5–8.0 and substrate activity for l-dopachrome and aminochromes generated from dopa methyl ester, α-methyl dopa and dopamine. Trypsin digestion of the isolated DCE and subsequent reverse-phase separation resulted in the isolation of several polypeptide fragments, from which two partial internal amino acid sequences were obtained by Edman degradation. PCR amplification, using a degenerate primer based on one internal amino acid sequence and an oligo-dT primer, produced a 650 bp DNA fragment. Subsequent screening of an A. aegypti pupal cDNA library resulted in the isolation of a 1.6 kb clone containing coding sequence for both internal DCE amino acid sequences, thereby confirming the identity of the isolated gene product (pAaDce1) as DCE. Northern analysis revealed the constitutive expression of DCE message in developmental stages and adults, with the majority of transcript localized in the fat body and ovaries of adult females. AaDce1 mRNA increased in abundance above constitutive levels in adult females when a melanotic encapsulation immune response was initiated by the intrathoracic inoculation of Dirofilaria immitis microfilariae.

L-DOPA Cyclohexyl Ester Is a Novel Potent and Relatively Stable Competitive Antagonist Against l-DOPA Among Several l-DOPA Ester Compounds

We explored L-DOPA esters with chemically bulky structures to find a potent stable competitive antagonist against L-DOPA, compared to DOPA methyl ester (DOPA ME). In anesthetized rats, DOPA cyclohexyl ester (DOPA CHE), DOPA cyclopentyl ester (DOPA CPE) and DOPA cyclopentyldimethyl ester (DOPA CPDME) at 1 microgram microinjected into depressor sites of the nucleus tractus solitarii elicited or tended to elicit more marked antagonism against depressor responses to 60 ng L-DOPA, compared to DOPA ME. At 100 ng, DOPA CHE elicited the most potent antagonism. At 1 microgram, duration of the antagonistic activity of DOPA CHE was approximately three times longer than that of DOPA ME. During microdialysis of the nucleus accumbens, conversion from DOPA CHE at 1 microM perfused via probes to extracellular L-DOPA was the lowest among these compounds and less than one half of that from DOPA ME. Binding studies showed that the recognition site for L-DOPA differs from ionotropic glutamatergic, dopaminergic D1 and D2 receptors. We recently found that L-DOPA evoked by transient ischemia may act as a DOPA CHE-sensitive causal factor for glutamate release and resultant neuronal cell death. DOPA CHE is the most potent, relatively stable competitive antagonist against L-DOPA and is a useful mother compound to develop neuroprotective drugs.

Nicotine Alone and in Combination with [formula omitted]-DOPA Methyl Ester or the D2 Agonist N-0923 in MPTP-Induced Chronic Hemiparkinsonian Monkeys

Nicotine, the soluble methyl ester of l-DOPA, and the D2 agonist N-0923 were given alone and in combination im to five hemiparkinsonian monkeys. Daily nicotine in doses of 32–320 μg/kg for 6 days each, surprisingly, had slight effects on motor activity. When combined with N-0923, nicotine did not further enhance its effects. However, l-DOPA methyl ester plus nicotine produced greater contraversive circling than l-DOPA methyl ester plus 0.9% NaCl. Similar effects were obtained on significant motor movements of both the affected (contralateral) and normal (ipsilateral) arm and hand. The results indicate that nicotine is synergistic with l-DOPA methyl ester, but not with the postsynaptic D2 agonist N-0923.

Studies on novel, stable and potent competitive antagonists against L-DOPA

DOPA has been proposed to be a neurotransmitter of the primary baroreceptor afferents terminating in the NTS and a neuromodulator in striata of rats. Pre- and post-synaptic recognition sites for DOPA itself may exist. DOPA methyl ester (DOPA ester), a potent competitive antagonist for DOPA in continuously superfused slices or within a several min when microinjected in the NTS has some limitation in in vivo use because of its unstableness as a prodrug for DOPA. We have explored to find stable and potent antagonists for DOPA. At first, we tried to clarify whether or not newly synthesized DOPA esters with bulky structure such as DOPA cyclohexyl ester (CHE), DOPA cyclopentyl ester (CPE) and DOPA cyclopentyldimethyl ester (CPDME) antagonize depressor responses to DOPA microinjected into the NTS, and to what degree these analogues 1 microM perfused via probes are converted to DOPA during striatal microdialysis in urethane-anesthetized rats. Then, we analyzed mode of antagonism of some candidate in the NTS system. During striatal microdialysis, DOPA ester 1 microM increased extracellular levels of DOPA to an 100 fold higher level at a peak of the 1st 20 min sample after the perfusion. Conversion ratio of CHE, CPE and CPDME, compared to DOPA ester, was less than 1/5 at the peak of the 3rd sample, 1/3.3 at the peak of the 2nd, and 1/2.7 at the peak of the 3rd, respectively. In the NTS, all analogues 1 microgram microinjected abolished depressor responses to DOPA 60 ng. Time required to recover a base was 20 min for CHE and 10 min for DOPA ester, CPE and CPDME. At 100 ng, CHE inhibited peak depressor responses to DOPA 60 ng by 53%, CPE by 40%, and CPDME by 24%, compared to DOPA ester by 22%. Then, DOPA 10-300 ng microinjected produced dose-dependent hypotension. DOPA ester 300 ng, microinjected 1 min previously, shifted a dose-response curve for DOPA 30-300 ng to the right without reduction of the maximum response, showing a competitive mode of antagonism. CHE 30 ng competitively antagonized depressor responses to DOPA 18-300 ng without reduction of the maximum response. CHE 100 ng further shifted the dose-response curve for DOPA 18-100 ng to the right with 25% reduction of the maximum response. Antagonistic activity of CHE 100 ng was equipotent with DOPA ester 300 ng. CHE is a competitive antagonist including partially some noncompetitive components. A possible component might be related to NMDA receptors, since high concentrations of CHE displaced selective binding of [3H]-MK-801, an antagonist. In conclusion, CHE is suitable for the purpose.

L-DOPA Cyclohexyl Ester Is a Novel Stable and Potent Competitive Antagonist Against L-DOPA, as Compared to L-DOPA Methyl Ester

We explore stable potent competitive antagonists against L-DOPA. In anesthetized rats, DOPA cyclohexyl ester (DOPA CHE) (30-100ng) microinjected in depressor sites of the nucleus tractus solitarii dose-dependently shifted the dose-response-curve for L-DOPA (18-300 ng) to the right, with DOPA CHE (100 ng)-induced slight reduction of the maximum response. DOPA methyl ester (DOPA ME) at 100 ng also produced competitive antagonism. Antagonistic activity of DOPA CHE (100 ng) was similar to that of DOPA ME (300 ng). DOPA CHE is suitable for the purpose of screening.

L-DOPAの圧受容器第一次求心性感覚神経の一伝達物質としてのさらなる証明

L-DOPA is proposed to be a neurotransmitter of the primary baroreceptor afferents terminating in NITS, whereas GABA inhibits baroreflex within NTS. Thus, if our proposal is truly the case, GABA should tonically inhibit pre and/or post-synaptic DOPAergic functions in NTS. Muscimol 10 and 30 μM, locally perfused via probes, inhibited DOPA release by 50 and 60% during microdialysis of NTS in anesthetized rats, with a peak 2 hr after perfusion. Muscimol (10 μM)-induced inhibition was antagonized by noneffectivel0 μM bicuculline. Bicuculline 30 μM alone increased DOPA release in a similar time course. By microinjections into depressor sites of NTS, DOPA 10-60 ng or GABA 3-300 ng elicited dose-dependent hypotension or hypertension. Cardiodepressor responses to DOPA 30 ng were dose-dependently reduced by prior injections of GABA 3-30 ng, also by nipecotic acid 100 ng, but inversely potentiated by bicuculline 10 ng, when respective regulatory response to GABA-related drug returned to control. Meanwhile, DOPA methyl ester, a competitive DOPA antagonist, did not displace [3H]-GABA binding in brain membrane preparations. When a tonic function of endogenously released DOPA was antagonized by this antagonist 1 μg microinjected 1 min previously, cardiopressor responses to GABA 300 ng were reduced by a half. DOPA 10 nM evoked GABA by 40% in the first 20 min sample. Under inhibition of central AADC by perfusion of NSD-1015 200 μM, DOPA 1-100 nM concentration-dependently evoked GABA by 35-60%. DOPA (10 nM)-induced GABA release was not mimicked by dopamine or D-DOPA 10 nM, but was abolished by Ca2+ deprivation or perfusion of tetrodotoxin 1μM. GABA seems to inhibit tonically via activation of GABAA receptors presynaptic DOPA release and postsynaptic depressor and bradycardic responses to DOPA and to induce hypertension and tachycardia in part via inhibition of tonic postsynaptic cardiodepressor function of endogenously released DOPA within NTS. DOPA seems to evoke by itself endogenous GABA from GABAergic neurons as a compensatory mechanism of baroreflex within NTS. These findings further support the idea that DOPA is a neurotransmitter of the primary baroreceptor afferents terminating in rat NTS.

Partial Purification and Characterization of DOPA Quinone Imine Conversion Factor from Larval Hemolymph of the Silkworm,Bombyx mori

DOPA quinone imine conversion factor was partially purified from larval hemolymph of the silkworm, Bombyx mori, using a novel assay. The factor was labile at 4°C below pH 5 and above pH 11, and at pH 8.5 above 55°C. Activity of the factor was maximal at pH 7.5–9. The factor catalyzed the decolorizations of L-DOPA methyl ester and α-methyl DOPA methyl ester chromes besides DOPA quinone imine. HPLC analysis indicated that the factor-catalyzed decolorization reaction of DOPA quinone imine resulted in the accumulation of dihydroxyindole. Based on spectral changes of α-methyl DOPA methyl ester chrome, it was suggested that the factor catalyzed the intramolecular oxidoreduction of DOPA quinone imine. During the oxidation of L-DOPA with endogenous phenoloxidase, the factor facilitated the formation of melanochrome.

Chronic Treatment with Levodopa and/or Selegiline Does Not Affect Behavioral Recovery Induced by Fetal Ventral Mesencephalic Grafts in Unilaterally 6-Hydroxydopamine-Lesioned Rats

It has been suggested that levodopa ( l-dopa), a dopamine precursor used to treat Parkinson's disease, may be toxic to grafted fetal neuroblasts; if so, the use of the monoamine oxidase B inhibitor selegiline might prevent such toxicity. We randomly assigned 30 unilaterally 6-hydroxydopamine-lesioned male Sprague-Dawley rats, whose lesions were verified with low-dose apomorphine-induced rotations, to one of five treatment groups: (i) l -dopa methyl ester (125 mg/kg/day) with benserazide (a peripheral decarboxylase inhibitor; 25 mg/kg/day), (ii) l-dopa methyl ester with benserazide and selegiline ( l-deprenyl; 0.5 mg/kg/day), (iii) selegiline only, (iv) and (v) vehicle (ascorbate in normal saline) only. After 2 weeks of twice-daily ip injections, the rats received fetal ventral mesencephalic grafts into the lesioned striatum; one vehicle group received sham grafts. Drug therapy was continued for 2 1/2 months post grafting. At 1 month after grafting, and every 2 weeks thereafter, the rats were tested using low-dose apomorphine-induced rotation. A 70% decrease in rotations among all grafted groups, relative to the shams, was found. No statistical differences among groups receiving various drug therapies were seen in behavior or in counts or dimensions of tyrosine hydroxylase-positive cells. We therefore conclude that, in the unilaterally lesioned rat model of Parkinson's disease, there is no adverse effect of l-dopa nor any significant effect of selegiline, either alone or coadministered with l-dopa, on behavioral recovery induced by fetal ventral mesencephalic grafts.

Role of Tyrosine Hydroxylase in the Blood of the Ghost Crab DcΥPode PlatΥTarsis H. Milne Edwards, 1852 (Decapoda, Brachyura)

The blood of Ocypode platytarsis contains a single fraction of tyrosine hydroxylase (E.C.1.14.16.2), which is capable of converting the monophenol into O-diphenol and 3 fractions of phenoloxidase (E.C.1.14.18.1), which convert O-diphenol into quinone. The phenoloxidases do not show any monophenoloxidase activity. The phenoloxidase fractions exist as proenzyme and can be activated by sodium dodecyl sulphate. Tyrosine hydroxylase may be involved in the synthesis of O-diphenol. The stored O-diphenol may be oxidised into melanin during immunological response, after the activation of prophenoloxidase. Thin layer chromatographic analysis reveals that there is an accumulation of dopa methyl ester in the reaction medium containing the enzyme sample, tyrosine methyl ester and phenylthiourea (PTU), a specific inhibitor of phenoloxidase. In the absence of PTU, the reaction medium becomes brown and there is no accumulation of O-diphenol.

Intravenous boluses and continuous infusions of L‐DOPA methyl ester in fluctuating patients with Parkinson's disease

In six patients with Parkinson's disease exhibiting severe "on-off" phenomena, a 200-mg intravenous bolus of either L-DOPA or of its methyl ester were equally effective in reversing motor deficits, although the duration of action of the methyl ester was shorter. There were no marked differences in pharmacokinetic parameters for L-DOPA plasma levels after administration of L-DOPA and the methyl ester. In three patients, optimal infusion rates for the maintenance of mobility were established for L-DOPA and L-DOPA methyl ester. Both drugs were able to maintain patients "on" throughout a 12-h infusion period. However, on average the optimal infusion rate of L-DOPA methyl ester was 2.7 times greater than that for L-DOPA. There was no marked difference in the plasma levels of L-DOPA achieved, but 3-O-methyl DOPA levels increased more after infusion of L-DOPA methyl ester than after infusion of L-DOPA itself. The half-life of elimination and volume of distribution of L-DOPA formed from the methyl ester were markedly increased compared with values obtained after either an intravenous bolus of methyl ester or after an intravenous infusion of L-DOPA itself. An intravenous bolus of L-DOPA methyl ester produces an equivalent magnitude of clinical response to the same dose of L-DOPA. However, higher optimal infusion rates of methyl ester than L-DOPA are required to produce continuous effect. The pharmacokinetic handling of L-DOPA methyl ester given by intravenous infusion may differ from that of L-DOPA when given by the same route.

Oxidative coupling of dopa with resorcinol and phloroglucinol: Isolation of adducts with an unusual tetrahydromethanobenzofuro[2,3-d]azocine skeleton

Under biomimetic conditions, both phloroglucinol and resorcinol cause significant inhibition of the oxidative conversion of dopa to dopachrome, a key step in the biosynthesis of melanin pigments. The major product (λmax = 377 nm) from oxidation of dopa in the presence of phloroglucinol was characterized as 2, containing the unusual tetrahydromethanobenzofuroazocine ring system. Another product, formed in somewhat smaller amount, was formulated as 3, arising from coupling of phloroglucinol with 2 dopaquinone derived units. Analogous cross coupling adducts 4 and 5 were obtained by reaction of dopa or dopa methylester with resorcinol, respectively. These results suggest the trapping of labile dopaquinone, generated in situ from oxidation of dopa, by the reactive phenolic compounds, leading to the novel adducts 2, 4 and 5.