Howard M. Deutsch

Effects of methylphenidate analogues on phenethylamine substrates for the striatal dopamine transporter: potential as amphetamine antagonists?

Abstract : Methylphenidate (MPD) was found to inhibit competitively the striatal dopamine transporter (DAT) and bind at sites on the DAT in common with both cocaine (a non‐substrate site ligand) and amphetamine (a substrate site ligand). Some methylphenidate analogues modified on the aromatic ring and/or at the nitrogen were tested to determine whether the profile of inhibition could be altered. None was found to stimulate the release of dopamine in the time frame (≤60 s) of the experiments conducted, and each of the analogues tested was found to noncompetitively inhibit the transport of dopamine. It was found that halogenating the aromatic ring with chlorine (threo‐3,4‐dichloromethylphenidate hydrochloride ; compound 1) increased the affinity of MPD to inhibit the transport of dopamine. A derivative of MPD with simultaneous, single methyl group substitutions on the phenyl ring and at the nitrogen (threo‐N‐methyl‐4‐methylphenidate hydrochloride ; compound 2) bound at a site in common with MPD. A benzyl group positioned at the nitrogen (threo‐N‐benzylmethylphenidate hydrochloride ; compound 3) imparted properties to the inhibitor in which binding at substrate and non‐substrate sites could be distinguished. This analogue bound at a mutually interacting site with that of methylphenidate and had a Kint value of 4.29 μM. Furthermore, the N‐substituted analogues (compounds 2 and 3), although clearly inhibitors of dopamine transport, were found to attenuate dramatically the inhibition of dopamine transport by amphetamine, suggesting that the development of an antagonist for substrate analogue drugs of abuse may be possible.

Supercritical extraction of taxol from the bark of Taxus brevifolia

Abstract Taxol has been found to be an effective drug for the treatment of ovarian cancer. However, the only current source of this compound is from trees of the Taxus genus and methods need to be developed for its separation from the natural plant source. The extraction of taxol from the ground bark of Taxus brevifolia using supercritical carbon dioxide and carbon dioxide + ethanol mixtures was studied in this work. The extractions were preformed at 318 K and at pressures ranging from 18.07 to 25.79 MPa. It was demonstrated that supercritical carbon dioxide is able to extract taxol from the ground bark, especially at the higher pressures and with the addition of ethanol. The supercritical extractions of taxol were also found to be more selective than a conventional liquid ethanol extraction. Furthermore, a significant portion of the taxol present in the bark could be removed in the supercritical process.

Can stimulant binding and dopamine transport be differentiated? Studies with GBR 12783 derivatives.

Both 3- and 4-substituted GBR 12783 derivatives were synthesized in an effort to create site-directed cocaine antagonists. The potencies of these compounds to inhibit stimulant ([3H]WIN 35,428) binding and synaptosomal [3H]dopamine uptake were determined and compared with a large number of compounds assayed under identical experimental conditions. Three groups of compounds were identified which affected stimulant binding and dopamine transport to varying degrees. The 3-substituted GBR 12783 derivatives resembled the known non-amphetamine stimulant agents, in that they were approximately equipotent in inhibiting [3H]WIN 35,428 binding and [3H]dopamine transport. The 4-substituted GBR 12783 derivatives formed a second group that was approximately seven times more potent in the inhibition of [3H]dopamine uptake compared to [3H]WIN 35,428 binding. The third group, approximately one hundred-fold more potent at inhibiting [3H]dopamine uptake than stimulant binding, consisted mainly of substrates for the dopamine transporter. Although these GBR derivatives did not meet the criteria for potential cocaine antagonists, the results demonstrate that slight modifications of the molecular structure of a stimulant drug can differentially affect binding and transport. This finding holds great promise for the eventual development of a true pharmacological antagonist of cocaine.

Ocular and systemic responses to water soluble material derived from Cannabis sativa (marihuana)

A water soluble material, isolated from Cannabis sativa, has been tested in albino and pigmented rabbits and rhesus monkeys for both ocular and systemic effects. Intravenous administration produced a dose-related fall in intraocular pressure in both albino and pigmented rabbits with concentrations as low as 0.005 mg/animal being effective, but no response was found in monkeys. High concentrations (0.2 to 1 mg/animal) induced a hypertensive phase in intraocular pressure prior to the ocular hypotension; higher concentrations (2 or 5 mg/animal) also induced antidiuresis and general relaxation. Tachyphylaxis was found to repeated daily injections. Alpha and beta-adrenergic antagonists caused some reduction of the hypertensive phase but had no effect on the hypotensive phase. Superior cervical ganglionectomy did not influence the time course of the intraocular pressure response. Indomethacin inhibited the hypertensive intraocular pressure phase but was ineffective against the hypotensive phase. Systemic blood pressure was unchanged following intravenous administration of 0.2 mg material/animal. Aqueous tumor protein concentration was increased at both 1 and 6 hours after intravenous administration, becoming greater at the later time. Aqueous humor turnover rate was substantially reduced reaching a minimum 8.75 hours after administration. Topical administration was ineffective in eyes when the epithelium was removed in rabbits with and without pretreatment with aspirin. Neither gastric nor suppository administration of large quantities (10 mg or greater) of material had any influence on intraocular pressure.

Isolation of Ocular Hypotensive Agents From Cannabis sativa

Abstract: Recent work in our laboratories has shown that a hydrophilic fraction from Cannabis sativa (marihuana) has extremely potent intraocular pressure (IOP)‐lowering activity as measured in albino rabbits when delivered by intravenous injection. A crude extract reduced IOP by 50–60 per cent (to the episcleral venous pressure) at dosage levels of about 500 μg/animal. Fractionation of this material by solvent extraction, high‐performance liquid chromatography, and gel filtration chromatography has produced samples with high activity at 50 μg/animal. The active material has been shown to be noncannabinoid and of high molecular weight.

Chemical ecology ofReticulitermes flavipes (Kollar) andR. virginicus (Banks) (Rhinotermitidae) : Chemistry of the soldier cephalic secretions.

Reticulitermes flavipes andR. virginicus have been examined for the presence and possible defense functions of soldiers specific secretions. The cephalic extracts for soldiers of both species contained the identical two major sesquiterpenes which were absent from other castes. The sesquiterpenes have been identified as γ1-cadinene (I) and the corresponding aldehyde (II) by high-resolution nuclear magnetic resonance spectrometry using homonuclear proton decoupling and by high-resolution mass spectrometry. When groups of termite soldiers were exposed to foraging parties of the sympatric native fire ant,Solenopsis geminata, the termites utilized only mechanical defenses. No evidence was obtained to indicate that the ants had been sprayed or coated with either an irritant or toxicant, and there was no evidence that an alarm had been promulgated.

Synthesis and pharmacology of irreversible affinity labels as potential cocaine antagonists: aryl 1,4-dialkylpiperazines related to GBR-12783

As part of a program aimed at designing irreversible antagonists of the stimulant and reinforcing properties of cocaine, derivatives of GBR-12783 containing electrophilic substituents were synthesized. GBR-12783, a potent and selective inhibitor of both stimulant binding and dopamine transport, was modified to incorporate either isothiocyanate or maleimido groups at the meta- or para-positions in one phenyl ring of the geminal diphenyl portion of the molecule. The effect of these compounds, as well as their respective amino- or nitro-substituted precursors, on stimulant binding to rat striatal tissue was studied using the [3H]methylphenidate radioreceptor assay. Under the assay conditions used, the compounds were found to have IC50s (nM) ranging from 11.9 (m-nitro) to 1677 (p-maleimido); the parent compound, GBR-12783, had an IC50 of 12.0. Using a washout technique (repeated washing with 100 mM KCl) which completely removed the tightly bound, but reversible GBR-12783, both the m- and p-isothiocyanate compounds were found to irreversibly inhibit binding of [3H]methylphenidate to the stimulant recognition site. The m-maleimido derivative also irreversibly inhibited binding, albeit with lower efficacy than was observed with the isothiocyanate compounds. Neither the p-maleimido, nor the amino or nitro intermediates, were capable of irreversible inhibition.

Water soluble marihuana-derived material: Pharmacological actions in rabbit and primate

Further studies have been made with water soluble marihuana-derived material (MDM). Neither adrenergic, cholinergic, aldosterone, dopamine or serotonin antagonism affected the fall in intraocular pressure induced by MDM. Partial blockade was obtained with galactose, glucose, or mannose, but not arabinose, when the latter were given at intravenous concentrations of 1 gm/animal and MDM was given at 25 micrograms animal, suggesting that these sugars may be involved at the active site of the MDM glycoproteins. Dexamethasone was without effect on either intravenous or intravitreal MDM indicating that the MDM effect is not a non-specific response to a protein. A similar plant glycoprotein, larch arabinogalactan, at 200 micrograms/animal was without effect on intraocular pressure. Aqueous humor flow rate was increased 3 hours after MDM administration, a period corresponding to the intraocular pressure increase caused by MDM, and fell to 20% of control values when the fall in intraocular pressure occurred. Blood flow through the iris was increased at both one and six hours after intravenous MDM injection indicating a vasodilation which could contribute to the initial increase in intraocular pressure. Intravitreal injection of MDM in rabbit and rhesus monkey caused a fall in intraocular pressure only after a 24 hour delay: the unilateral response indicated that systemic metabolism was not required for activity and the delay was likely caused by the diffusion time to the ciliary processes from the mid-vitreal injection site. The changes in beta-receptors, adenylate cyclase and carbonic anhydrase in the ciliary processes are minimal indicating a possible vascular mechanism of action of MDM.

Synthesis and pharmacology of site specific cocaine abuse treatment agents: a new synthetic methodology for methylphenidate analogs based on the Blaise reaction

In order to make new analogs of the dopamine (DA) uptake inhibitor methylphenidate, a synthetic methodology based on the Blaise reaction was developed. The reaction between alpha-bromophenylacetic acid esters, zinc and alpha-cyano-omega-mesylates gave stable primary enamines. After reduction of the enamines with cyanoborohydride, the amines could be cyclized to methylphenidate analogs in which the amine ring size and aromatic ring were varied. These compounds were tested for inhibitory potency against [(3)H]WIN 35,428 binding to the cocaine recognition site and [(3)H]DA uptake using rat striatal tissue. When the heterocyclic ring size was varied, the six-membered ring of methylphenidate appeared to be the optimum ring size. When the aryl ring was varied the 4-trifluoromethylphenyl analog was less potent than methylphenidate, the beta-naphthyl congener was considerably more potent, whereas the alpha-naphthyl congener was less potent. Most of the compounds tested had ratios of uptake to binding inhibition (discrimination ratio) that were similar to cocaine and were therefore not lead compounds for the development of cocaine antagonists.

Conformational analysis of methylphenidate: comparison of molecular orbital and molecular mechanics methods

SummaryMethylphenidate (MP) binds to the cocaine binding site on the dopamine transporter and inhibits reuptake of dopamine, but does not appear to have the same abuse potential as cocaine. This study, part of a comprehensive effort to identify a drug treatment for cocaine abuse, investigates the effect of choice of calculation technique and of solvent model on the conformational potential energy surface (PES) of MP and a rigid methylphenidate (RMP) analogue which exhibits the same dopamine transporter binding affinity as MP. Conformational analysis was carried out by the AM1 and AM1/SM5.4 semiempirical molecular orbital methods, a molecular mechanics method (Tripos force field with the dielectric set equal to that of vacuum or water) and the HF/6-31G* molecular orbital method in vacuum phase. Although all three methods differ somewhat in the local details of the PES, the general trends are the same for neutral and protonated MP. In vacuum phase, protonation has a distinctive effect in decreasing the regions of space available to the local conformational minima. Solvent has little effect on the PES of the neutral molecule and tends to stabilize the protonated species. The random search (RS) conformational analysis technique using the Tripos force field was found to be capable of locating the minima found by the molecular orbital methods using systematic grid search. This suggests that the RS/Tripos force field/vacuum phase protocol is a reasonable choice for locating the local minima of MP. However, the Tripos force field gave significantly larger phenyl ring rotational barriers than the molecular orbital methods for MP and RMP. For both the neutral and protonated cases, all three methods found the phenyl ring rotational barriers for the RMP conformers/invertamers (denoted as cte, tte, and cta) to be: cte, tte> MP > cta. Solvation has negligible effect on the phenyl ring rotational barrier of RMP. The B3LYP/6-31G* density functional method was used to calculate the phenyl ring rotational barrier for neutral MP and gave results very similar to those of the HF/6-31G* method.