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The tritiation of the beta-adrenergic catecholamines isoproterenol and hydroxybenzylisoproterenol is described.

d-amphetamine and tyramine are both amines which are active at the recently discovered trace amine-associated receptor 1 (TAAR1) and analogues of these compounds labelled with tritium would be useful reagents for neurochemistry investigators. This paper describes the tritiation and characterization of these substances. d-amphetamine was radiolabelled by a direct tritium catalytic method and characterized by proton decoupled tritium NMR. For tyramine, a bromination—catalytic tritium debromination approach worked best to afford a tritiated analogue at high specific activity.

The high specific activity tritiation of (+)-SKF-10,047 (1) and N,N-dimethyltryptamine (4) is described. [N-allyl-3H] (+)-SKF-10,047 (3) was prepared by Lindlar catalyst tritiation of (+)-N-propargylnormetazocine (2) and [N-methyl-3H] N,N-dimethyltryptamine (6) was synthesized by the alkylation of N-methyltryptamine (5) with [3H] methyl iodide. Both sigma-1 synthetic agonist 3 and endogenous agonist 6 have been useful in studying this receptor.

The tritiation of natural photoffinity ligands chlorpromazine and cytochalasin B is described.

The hormone melatonin acts at several G protein-linked receptors to cause a myriad of significant physiological effects. The tritiation of melatonin at high specific activity and characterization of it with tritium NMR for biological testing is reported.

Opiate receptor ligands lofentanil 1 and (-)-bremazocine 4 have been labeled with tritium at high specific activity, becoming useful tools to study subclasses of the opiate receptor family.

An efficient synthesis of [5,6-3H] (+/−) gamma vinyl GABA 5 from 4-aminohex-5-ynoic acid 3 is described.

[Vinylidene-3H] (−)-α-Kainic acid was prepared by means of a Wittig reaction with a protected ketone precursor and has proved useful as a tool to study neuroexcitatory amino acid receptors. Copyright © 2002 John Wiley & Sons, Ltd.

p-Aminoclonidine hydrochloride 1 was converted to m-dibromo-p-iodoclonidine 3 and the latter was used as a precursor to prepare [phenyl-3H] clonidine hydrochloride 4 at high specific activity via a selective catalytic dehalogenation with tritium. Copyright © 2001 John Wiley & Sons, Ltd.

Inositol-specific phospholipase Cs(PLCs) are a group of enzymes involved in the signal transduction pathway of many plasma membrane receptor mediated events. We developed a modified solid surface to capture [(3)H] PIP(2) onto the Basic FlashPlate(R) in order to monitor PLC activity. Our results clearly demonstrate the utility of [(3)H] PIP(2)-Coated Phospholipid FlashPlate(R) microtiter plates for assessing PLC activity for HTS of receptor-coupled functional assays. The results show that PLC activity can be measured easily from a variety of sources including purified recombinant enzyme preparations, crude HL60 cell lysates and permeabilized A431 human carcinoma cells. Moreover, this format provides a surface comparable to that used for classical solution based radiolabeled mixed phospholipid micelle studies and illustrates the feasibility of this assay for measuring PLC activation in a variety of different drug screening assays.

Two new photoreactive paclitaxel analogs bearing [3H2]-3-(4-benzoyl)phenylpropanoyl group as the photophore as well as radiolabeling unit at the 7 and 10 positions, respectively, are developed. These new photoreactive analogs showed excellent preliminary results on the photoaffinity labeling of tubulin and P-glycoprotein.

Methods are presented to tritiate the side chain of dopamine.

Serotonergic agonists buspirone 1 and (+/−)-DOB 4 have been labeled with tritium at high specific activity and have emerged as valuable tools to study the serotonin receptor family.

A new radiolabeled, bifunctional photoaffinity cross-linking reagent, N-succinimidyl p-benzoyl-[2,3-3H2]dihydrocinnamate, has been synthesized in high yield and with high specific activity. This reagent can be used to append the benzophenone photophore to amino groups of small molecules, such as O-aminoalkylinositol polyphosphates and polypeptides. The resulting tritiated photoaffinity labels can be purified and manipulated in ambient light and can be activated at 360 nm.

A new method for the synthesis of highly deuterated or titriated leukotrienes was developed. The higher reactivity of a terminal alkene compared to a 1,2-disubstituted one permitted the selective deuteration or tritiation of the diyne 14,15,19,19,20,20-hexadehydro LTC 4 triester 1. After hydrolysis, LTC, was obtained in 36% overall yield. An average of seven deuterium or tritium atoms was incorporated and the specific activity of the tritiated LTC, was greater than 180 Ci/mmol. 1 was obtained from the addition of glutathione to 14,15,19,19,20,20-hexadehydro LTA 4 ethyl ester which was the product of a Wittig reaction between (3,8-nonanediyn-1-yl)triphenylphosphonium iodide and 5(S), 6(S), 7(E), 9(E) ethyl 5,6-epoxy-11-oxo-7,9-undecadienoate

Arachidonoyl ethanolamide-[1,2-14C] was prepared and evaluated as a substrate for anandamide amidase in a radioenzymatic assay that does not require a thin layer chromatography separation step. Using this substrate the release of ethanolamine-[1,2-14C] is linear for approximately thirty minutes. Anandamide amidase exhibits maximal activity between pH 8 and pH 9 with a steep decline in activity at pH values below 6 and above 10. Arachidonoyl ethanolamide-[1,2-14C] was used for the assay of anandamide amidase from 10 micrograms to 100 micrograms protein, from cow brain homogenate, in a 0.2 ml incubation mixture. When plotted as a rectangular hyperbola of the steady-state Michaelis-Menten equation, an approximate Km of 30 +/- 7 microM and a Vmax of 198 +/- 13 nmoles ethanolamine formed per hour per mg protein homogenate was obtained.

Gamma-aminobutyric acid was labelled with tritium by the tritium gas catalytic reduction of a convenient 4-aminotetrolic acid derivative. The purified product was characterized by HPLC and tritium NMR.

An improved and efficient one pot synthesis of 4-aminotetrolic acid 1 from protected propargyl amine 2 is described.

Methods are presented to synthesize and characterize [methylene-3H] haloperidol 2 and [N-methyl-3H]trifluoperazine 6.

Methods are presented to synthesize and characterize [N-methyl-3H] imipramine 3, [N-methyl-3H] 3-cyanoimipramine 6 and [N-methyl-3H] 2-nitroimipramine 9.

Methods are presented to synthesize and characterize [propyl-3H] trefentanil 3 and [propenyl-3H] naloxonazine 6.

MRS 1754 [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide] is a selective antagonist ligand of A(2B) adenosine receptors. This is the least well-defined adenosine receptor subtype, and A(2B) antagonists have potential as antiasthmatic drugs. For use as a radioligand, MRS 1754, a p-cyanoanilide xanthine derivative, was tritiated on the propyl groups in a two-step reaction using a p-carboxamido precursor, which was dehydrated to the cyano species using trifluoroacetic anhydride. [3H]MRS 1754 (150 Ci/mmol) bound to recombinant human A(2B) adenosine receptors in membranes of stably transfected HEK-293 cells. Specific binding was saturable, competitive, and followed a one-site model, with a K(D) value of 1.13 +/- 0.12 nM and a B(max) value of 10.9 +/- 0.6 pmol/mg protein. Specific binding utilizing 0.7 nM [3H]MRS 1754 was > 70% of total binding. The affinity calculated from association and dissociation binding constants was 1.22 nM (N = 4). Binding to membranes expressing rat and human A(1) and A(3) adenosine receptors was not significant, and binding in membranes of HEK-293 cells expressing human A(2A) receptors was of low affinity (K(D) > 50 nM). The effects of cations and chelators were explored. Specific binding was constant over a pH range of 4.5 to 6.5, with reduced binding at higher pH. The pharmacological profile in competition experiments with [3H]MRS 1754 was consistent with the structure-activity relationship for agonists and antagonists at A(2B) receptors. The K(i) values of XAC (xanthine amine congener) and CPX (8-cyclopentyl-1,3-dipropylxanthine) were 16 and 55 nM, respectively. NECA (5'-N-ethylcarboxamidoadenosine) competed for [3H]MRS 1754 binding with a K(i) of 570 nM, similar to its potency in functional assays. Thus, [3H]MRS 1754 is suitable as a selective, high-affinity radioligand for A(2B) receptors.

(±)-Phenoxybenzamine is a ligand capable of irreversibly alkylating the alpha-1 adrenergic receptor. Previous attempts to radiolabel it have achieved only low specific activities which impeded their use in neurochemical investigations. (±)-[Phenoxy-3H(N)] Phenoxybenzamine.HCL (1) has been prepared in our laboratory by the catalytic reductive aryl debromination of an appropriate brominated precursor with tritium gas. 3H NMR showed exclusive radiolabeling on the phenoxy ring and the radioligand had a specific activity of 32.4 Ci/mmol.

[3H] Histamine.2HCl will continue to play a key role in establishing the neurotransmitter status of histamine.2HCl. To more consistently achieve high specific activity in the preparation of the radioligand and incorporate as much tritium into the stable side chain positions as possible, we explored the use of alternative tritiation precursors. Imidazole 4-acetonitrile (5) and 2,5-diiodoimidazole-4-acetonitrile (6) were catalytically reduced with tritium gas and the radiolabeling specificity of the resulting [3H] histamine.2HCl produced was confirmed by 3H NMR.

Strychnine (1a) was reduced with tritium gas via heterogeneous catalysis to yield [21, 22-3H] dihydrostrychnine (2b) at 31 Ci/mmol and via homogeneous catalysis to yield 2b at 50 Ci/mmol. [2-3H] Strychnine (1b) at 25 Ci/mmol was prepared by the catalytic reductive tritiation of 2-iodostrychnine (4). Other polyhalo strychnine analogues were synthesized in an attempt to prepare [3H] strychnine at even higher specific activity.

Cast skins of the Florida indigo snake (Drymarchon corais) yielded up to 8% chloroform: methanol-extractable lipid, which was found to contain methyl ketones (20%), free secondary alcohols (15%), free primary alcohols (30%), free cholesterol (15%), free fatty acids (5%), and hydrocarbons (5%). The hydrocarbons appeared to be contaminants, because gas chromatography revealed a distribution characteristic of petroleum hydrocarbons. The methyl ketones were predominantly monounsaturated, with double bonds almost exclusively in the ω7 position. The structures of the secondary alcohols corresponded with the methyl ketones in regard to chain length distribution, location of the oxygen function in the 2 position, and the proportion and position of unsaturation. The primary alcohols were also predominantly straight, odd-carbon, unsaturated compounds, with ω7 double bonds, but with chain lengths principally of 29 and 31 carbon atoms. The free fatty acids were mainly even-carbon monounsaturated compounds of 16–20 carbon atoms with double bonds mainly in the Δ9-position. Inspection of the lipid structures obtained from the Indigo snake suggest a biogenetic relationship whereby palmitic and palmitoleic acids are extended in chain length mainly to 32 and 34 carbonatom fatty acids. Retention or introduction of an oxygen function in the 3 position, followed by decarboxylation, could then yield structures corresponding with the methyl ketones and the related secondary alcohols. Insertion of an oxygen atom between carbons 2 and 3 of the methyl ketones, followed by loss of the two carbon atoms thereby isolated from the chain, would produce the series of odd-carbon primary alcohols that were observed.

Bei der Bromierung von (I) erhalt man ein Gemisch aus Dibromid (IIa) und Monobromid (IIb), das in die Chloride (III) umgewandelt wird.

The synthesis of the title compounds (1c and its 2H isomer 1b) from N-(2-hydroxyethyl)norapomorphine was carried out by ring bromination, followed by chlorination to the 2-chloroethyl compound 6. Further reduction with 2H2 or 3H2 and Pd/C gave 1b or 1c. Radiochemically pure (97%) 1c was obtained with a specific activity of 16.3 Ci/mmol. The purity of 1c was determined by LC, HPLC, UV, and NMR. [3H]NCA was shown to label the D2 receptor; however, the D2 signal appears to be only a small portion of the total signal, which may include binding to other dopamine receptor subtypes (D1 and D3).

Conversion of arachidonic acid to prostaglandin E 2 by acetone powders of sheep seminal vesicles was irreversibly inhibited by low concentrations of eicosa-5,8,11,14-tetraynoic acid. This compound also inhibited the hydroxylation of linoleic and linolenic acids by the tissue preparation, thereby preventing the elimination of the inhibitory effects of these polyethylenic acids. The tetraacetylenic acid can therefore limit prostaglandin biosynthesis both by its direct effect on prostaglandin synthetase and by preventing the inactivation of endogenous inhibitors.