Loss of tritium from coprostanone derived from [1,2(n)-3H]cholesterol or [7(n)-3H]cholesterol

After oral administration of a mixture of [1,2(n)-3H]cholesterol and [4-14C]cholesterol to a baboon, fecal coprostanone had a 46% lower 3H/14C ratio than the dose administered. Loss of 3H by enolization of the 3-ketone could account for the decrease in 3H/14C. If [7(n)-3H]cholesterol was administered instead of [1,2(n)-3H]cholesterol a 23% loss of 3H from coprostanone was found. Procedures requiring measurement of 3H-coprostanone derived from [1,2(n)-3H]- or [7(n)-3H]cholesterol could be seriously in error unless an appropriate correction for loss of 3H is made.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Synthesis of methyl [18-2H3], [17-2H2], [16-2H2], [14-2H2] and [12-2H2] cis-9-octadecenoates

Methyl [17-²H₂]oleate was prepared by stepwise reduction from 17-oxooleate in 24% yield. Methyl [18-²H₃], [16-²H₂], [14-²H₂] and [12-²H₂] oleates were synthesized from appropriately deuterated octylbromides by conversion to deuterated 7-hexadecyn-1-ols and chain extention to deuterated stearolates followed by semihydrogenation; overall yields were about 17%.     

Synthesis of [1,2-3H2]cholecalciferol and metabolism of [4-14C,1,2-3H2]- and [4-14C,1-3H]-cholecalciferol in rachitic rats and chicks

[1,2-(3)H(2)]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-(14)C,1-(3)H(2)]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-(14)C,1,2-(3)H(2)]cholecalciferol retained 36% of (3)H, the amount expected from its distribution between C-1 and C-2, the (3)H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The (3)H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal (3)H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its (3)H at C-1 is not a precursor of the intestinal (3)H-deficient substance P.     

D-homo isomers of 17-acetoxy-6-methylpregna-4,6-diene-3,20-dione

The three most readily accessible D-homo isomers of 17-acetoxy-6-methylpregna-4,6-diene-3,20-dione (Ib) have been prepared and characterized in detail. The 17-keto isomers IIb and IIIb were obtained by base-catalyzed rearrangement of Ia followed by reacetylation, and the 17a-ketone IVb by Lewis-Acid treatment of Ia.     

Improved and efficient synthesis of 1alpha-hydroxy-[6-(2)H] and 1alpha-hydroxy-[6,19,19-(2)H]vitamin D3 derivatives.

Improved and efficient procedures for deuterium-labeling at the 6,19,19 positions of 1alpha-hydroxyvitamin D3 derivatives via its sulfur dioxide-adduct by using a base-catalyzed H-D exchange reaction are described. Application of the known procedure using tBuOK/DMF-D2O, which is effective for labeling vitamin D3 derivatives, to 1alpha-hydroxy compounds gave only poor results because of isomerization and decomposition. We found that this procedure is improved by the use of iPrONa/iPrOD. During this study, we also found that the 6-monodeuterated product was selectively obtained when MeONa/CD3OD was employed instead of iPrONa/iPrOD. On the other hand, simple addition of 1,3-dimethyl-2-imidazolidinone as a co-solvent to the above conditions was effective for 1alpha,25-dihydroxy compounds. These improved procedures were successfully applied to the synthesis of 1alpha-hydroxy-[6,19,19-(2)H]vitamin D3 derivatives 4 and 1alpha-hydroxy-[6-(2)H]vitamin D3 derivatives 6 from the corresponding 1alpha-hydroxyvitamin D3 derivatives 1 via its sulfur dioxide-adducts 2, 3 and 5 in good over-all yield with high deuterium incorporation.     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

Synthesis and activity of 2-[4-(4-[3H]-2-cyanophenyl)piperazinyl]-N-(2,4,6-[3H]3-3-methylphenyl)acetamide: a selective dopamine D4 receptor agonist and radioligand

The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed.     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Synthesis of a [1,2-3H]-labeled pregnanolone.

A method is described for the synthesis and purification of 3 alpha-hydroxy-5 alpha-[1,2-3H]pregnan-20-one. [1,2-3H]progesterone (55 Ci/mmol) was incubated with a homogenate of rat brain tissue. The product was purified by Sephadex chromatography and thin-layer chromatography. The identity and purity of the product were established by successive recrystallizations and high-performance liquid chromatography. A 34% portion of the starting material was converted to 3 alpha-hydroxy-5 alpha-[1,2-3H]pregnan-20-one. The final radiopurity of 3 alpha-hydroxy-5 alpha-pregnan-20-one obtained from four independent preparations was 94% to 99%.     

The biological profile of a new synthetic progestagen--GR2-1159-(17-acetoxy-6,11 beta-dichloro-19-nor-4,6-pregnadiene-3,20-dione)

The biological activity of a new progestagen-GR2/1159-is reported. When tested by the oral, subcutaneous and intrauterine routes in the rabbit GR2/1159 had respectively 162, 191 and 226 times the activity of progesterone. High progestational activity has also been confirmed in a maintenance of pregnancy test in the rat and in the mouse deciduoma test. Compared with progesterone (= 1) GR2/1159 possessed anti-estrogenic activity of 73 subcutaneously in mice and of 114 and 79 by the subcutaneous and oral routes in rats. In the anti-gonadotrophic test GR2/1159 was 93 times as active as progesterone when administered subcutaneously to rats. GR2/1159 also showed weak adrenocorticoid activity in the rat. GR2/1159 was without estrogenic, androgenic, anabolic or anti-androgenic activity in rats and mice. Its biological profile in animals was thought to make it well suited for use as a continuous low-dose contraceptive preparation, but preliminary clinical investigation has not substantiated this.     

3-[Benzimidazo- and 3-[benzothiadiazoleimidazo-(1,2-c)quinazolin-5-yl]-2H-chromene-2-ones as potent antimicrobial agents

A series of 3-[benzimidazo(1,2-c)quinazolin-5-yl]-2H-chromene-2-one (6a-6f) and 3-[benzothiadiazole- imidazo(1,2-c)quinazolin-5-yl]-2H-chromene-2-one derivatives (7a-7f) that incorporate a variety of substituents at the 6- and/or 8-positions of the coumarin moieties have been synthesized utilizing cellulose sulfuric acid as an efficient catalyst under both conventional heating and microwave irradiation procedures. These analogs were evaluated for their antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes (Gram-positive bacteria), Escherichia Coli, Klebsiella pneumonia, Salmonella typhimurium (Gram-negative bacteria), and Aspergillus niger, Candida albicans, and Aspergillus flavus (Fungi). Two analogs, 6c (a 6,8-dichloro analog, MIC([SA]) = 2.5 μg/mL; MIC([ST]) = 2.5 μg/mL) and 7d (a 6,8-dibromo analog, MIC([ST]) = 2.5 μg/mL) were identified as potent antibacterial agents, and two analogs, 6b (a 6-bromo analog, MIC([AF]) = 10 μg/mL) and 6d (a 6,8-dibromo analog, MIC([AF]) = 15 μg/mL; MIC([CA]) = 15μg/mL), were identified as potent antifungal agents. Based on the MIC data, analogs 6b, 6c, 6d, and 7d were identified as the most potent antimicrobial agents in the series.