Howard E. Smith

Structural and steric requirements for β-phenethylamines as agonists of the noradrenergic cyclic AMP generating system in the rat limbic forebrain

SummaryThe present studies were undertaken to assess the structural and steric requirements for β-phenethylamines as agonists of the noradrenergic cyclic AMP generating system in slices of the rat limbic forebrain. Significant agonist activity of β-phenethylamines requires a β-3,4-dihydroxyphenethylamine with a β-hydroxyl group in the R configuration. Thus, dopamine did not stimulate the system at concentrations up to 10−3 M. Moreover, β-hydroxyphenethylamines without a 3,4-catechol group (octopamine, phenylephrine, p-hydroxynorephedrine, metaraminol and methoxamine)-though exerting α-agonist activity in peripheral tissues-lack agonist activity in this particular cyclic AMP generating system. The effects of (R)-norepinephrine and (R)-isoproterenol at maximal concentrations were not additive. The results lend further support to the view that the cyclic AMP generating system in slices of the limbic forebrain is part of a norepinephrine receptor coupled adenylate cyclase system with a subpopulation of receptors that are β in nature.

Modification of the noradrenergic cyclic AMP generating system in the rat limbic forebrain by amphetamine: Role of its hydroxylated metabolites

SummaryThe cyclic AMP responses to norepinephrine (NE) in slices of the rat limbic forebrain after the administration of (S)-amphetamine and the role of its para- and β-hydroxylated metabolites were investigated. The chronic but not acute administration of (S)-amphetamine to rats causes a significant reduction in the sensitivity of the cyclic AMP generating system to NE without changing the basal level of the nucleotide. This change in the sensitivity of the system is not associated with a change in the EC50 value for NE but reflects mainly a decrease in the maximal response. After withdrawal of the drug, the cyclic AMP response to NE returned to control values within 4 days. In vitro, (S)-p-hydroxyamphetamine (POH) and all stereoisomers of p-hydroxynorephedrine (PHN) except (αS,βR)-PHN enhanced the cyclic AMP response to low concentrations of NE. Since (αS,βR)-PHN [like the other stereoisomers of PHN and (S)-POH] inhibited in a dose-dependent manner the high affinity uptake of 3H-NE into crude synaptosomal fractions of the limbic forebrain, the results might suggest that the presumably physiological enantiomer of PHN also exerts receptor blocking properties. The inhibition by (αS,βR)-PHN of the cocaine induced potentiation of the cyclic AMP response to NE supports this supposition. The results provide evidence that the hydroxylated metabolites of (S)-amphetamine, (S)-POH and (αS,βR)-PHN, modify the action of the parent drug on central noradrenergic function at the level of the NE receptor coupled adenylate cyclase system.

Determination of absolute configurations from vibrational raman optical activity: Trans-2,3-dimethylthiirane

Experimental and theoretical vibrational Raman optical activity (VROA) spectra of (2R,3R)-2,3-dimethylthiirane in the 200-1500 cm(-1) region are presented. The level of agreement obtained for the observed and predicted VROA signs suggests that the absolute configurations of chiral molecules can be determined confidently using VROA.

Facile Preparation of (S)-N-[(1-Ethyl-2-pyrrolidinyl)methyl]-2,3-dimethoxy-5-(tributyltin)benzamide from Isoremoxipride: The Precursor of [125I]- and [123I]Epidepride

Abstract [125I]Epidepride, (S)-(−)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[125I]iodo-2,3-dimethoxybenzamide ([125I]NCQ 219), is a new, extremly potent radioligand, useful in the study of the distribution of the dopamine D-2 receptors in the brain. Its synthesis requires radioiodination of the corresponding 5-(tributyltin) derivative. The aryltin precursor (TDP 526) can be conveniently prepared in high yield from isoremoxipride (FLB 457) by tetrakis(triphenylphosphine)palladium(0)-catalyzed stannylation using bis(tri-n-butyltin) in triethylamine. An improved method for the preparation of isoremoxipride from o-vanillin was developed.

Effect of fluorine on the circular dichroism of the benzene chromophore [1]

Abstract Examination of the electronic absorption and circular dichroism spectra of a number ofchiral benzene compounds of known absolute configurations in which there is a fluorineatom at a chiral center contiguous to the benzene ring gives the rotatory contributionof a fluorine atom to the 1 L b Cotton effects of the benzene chromophore in relation toother groups at the chiral center. These relative contributions may be used with thebenzene sector rule to establish the absolute configurations of benzene compounds inwhich one substituent at a contiguous chiral center is a fluorine atom. Thus, the R absolute configuration was assigned to (—)-α-fluoro-α-phenylpropionic acid.

Preparation of 17α-iodoethynylandrosta- and 17α-(2-iodoethenyl) androsta-4,6-dien-17β-ol-3-ones as active site-directed photoaffinity ligands for androgen-binding proteins ☆

Abstract Unsaturated analogues of androst-4-en-17β-ol-3-one, each with a 17α-iodoethynyl or 17α-(2-iodoethenyl) substituent, were prepared, and their relative binding affinities (RBAs) for androgen-binding protein (ABP) were compared with those of 5α-androstan-17β-ol-3-one, androst-4-en-17β-ol-3-one, androsta-4, 6-dien-17β-ol-3-one, and androsta-1,4,6-trien-17β-ol-3-one. These binding studies indicate that the iodine[125I] analogues of 17α-iodoethynyl and 17α-[(E)-2-iodoethenyl] derivatives of androsta-4,6-dien-17β-ol-3-one and androsta-1,4,6-trien-17β-ol-3-one will have RBAs at least twice as great as that of 5α-androstan-17β-ol-3-one. They can be prepared from 17α-ethynylandrosta-4-en-17β-ol-3-one, the final synthetic step using N-[125I]iodosuccinimide, and are potential radioiodinated, active site-directed photoaffinity ligands for ABP and testosterone-binding globulin.

[34] Synthesis of affinity labels for steroid-receptor proteins

Publisher Summary The general considerations underlying active-site-directed irreversible enzyme inhibition (affinity labeling of enzymes) have been applied to the study of steroid-binding proteins, restricting attention to cytoplasmic androgen and progestagen receptors. There are two requisites for the affinity labeling of a steroid receptor. The steroid must bind reversibly at the active site of the receptor and possess a functional group capable of forming a covalent bond with an amino acid residue either within (endo) or adjacent to (exo) the binding site. The first requisite seems relatively easy to fulfill. The progesterone receptor of chick oviduct forms a highly stable (k d ∼ 10 -10 ) complex with progesterone and competition studies show that other steroids form complexes somewhat less stable but with k d ∼ 10 -10 . Thus, in the absence of progesterone, the progesterone-binding protein should strongly bind other steroids. The second requisite is more difficult. The amino acids in the polypeptide chain at the binding site are not known. Cysteine may be one because it has been shown for both a rat prostate 5α-dihydrotestosterone receptor and the chick oviduct progesterone receptor that sulfhydryl groups are involved either in the binding of the steroid to the receptor or in the maintenance of the active structure of the protein. It is assumed that one or both of the oxygen functions of 5α-dihydrotestosterone (C-3 and C-17) and of progesterone (C-3 and C-20) is involved in binding. A reactive substituent at a position near one of these oxygen functions would probably be closer to the polypeptide chain in the steroid–receptor complex than a more remotely positioned substituent and consequently would be more likely to undergo reaction. Four general classes of chemical reactions that are used in attempts at affinity labeling apply here such as alkylation reactions, photochemical insertion reactions, disulfide bond formation, and mercaptide bond formation.

Determination of absolute configurations from vibrational circular dichroism: (+)-2-methylthiirane-3,3-d2

Experimental vibrational circular dichroism (VCD) spectra for the dextrorotatory enantiomer and theoretical VCD spectra obtained with localized molecular orbital theory using 6-31G* basis set for the (R) configuration of 2-methylthiirane-3,3-d(2) in the 700-1500 cm(-1) region are presented. The observed and predicted VCD signs are in very good agreement suggesting that the dextrorotatory enantiomer has the (R) configuration. This conclusion is also supported by the optical rotational data.

CNDO/S CI rotational strength calculations for (S)-1-methylindan

CNDO/S CI calculations on (S)-1-methylindan show erratic sign variation for the calculated rotational strengths for the four lowest energy Π → Π∗ transitions depending on the number of configuration interaction (CI) used. A tentative suggestion as to the choice of CI size is made through comparison with the experimental gas-phase circular dichroism.

Active-site-directed photoaffinity radioiodination of androgen-binding proteins.

Androgen-binding protein (ABP) in rat epididymal cytosol and sex hormone-binding globulin (SHBG) in rabbit serum and SHBG purified from human serum were active-site-directed photoaffinity radiolabeled with 17 alpha-[(E)-2-[125I]iodoethenyl]androstan-4,6-dien-17 beta-ol-3-one ([125I]1). The interaction of this compound with binding components in epididymal cytosol was dependent on exposure of the mixture to ultraviolet light and on the duration of exposure. Photolysis in the presence of [125I]1 and 5 alpha-dihydrotestosterone (5 alpha-DHT) resulted in a 40% inhibition of binding of [125I]1 to cytosolic components. These result indicate that, while [125I]1 interacted with 5 alpha-DHT binding sites, it also formed adducts with other sites. To characterize the labeled species, the photolysis mixture was subjected to electrophoresis under denaturing and reducing conditions. Autoradiography of the gel revealed that ABP and SHBG were labeled with [125I]1, but in cytosol and serum, higher and lower molecular weight components were also labeled. Purified SHBG was labeled, but no labeled contaminating protein was detected. The presence of 5 alpha-DHT completely inhibited [125I]1 photolabeling of human and rabbit SHBG and of ABP. However, in cytosol, the presence of 5 alpha-DHT also eliminated photolabeling to a component that may be albumin, but 5 alpha-DHT did not affect [125I]1 photolabeling of other contaminating proteins in cytosol. Thus, while [125I]1 is an effective photoaffinity radiolabel for ABP and SHBG, the observation that it also photolabels other proteins limits its practical use to the radiolabeling of purified ABP and SHBG preparations.