Biochemical activities of trimetoquinol analogs at human beta(1)- and beta(3)-adrenergic receptors.

The biochemical activities of trimetoquinol (TMQ) analogs were evaluated at the human beta(1)- and beta(3)-adrenergic receptor (AR) subtypes expressed in Chinese hamster ovary cells. In radioligand binding assays, the 1-benzyl iodine-substituted analogs exhibited higher binding affinities at both beta(1)- and beta(3)-AR subtype as compared to TMQ. In cAMP accumulation assays, these analogs exhibited high potencies at both beta(1)- and beta(3)-AR. The 3',5'-diiodo-4'-amino analog of TMQ was the most potent beta(3)-AR agonist, 17-fold more potent at the beta(3)-AR versus the beta(1)-AR. Masking of the 6,7-dihydroxy group of the catechol ring of 3',5'-diiodo-4'-acetamido analog of TMQ, a potent beta(1)- and beta(3)-AR agonist, abolished activity at both beta-AR subtypes. Furthermore, substitution of a strong electron withdrawing group such as the trifluoromethyl moiety at the 1-benzyl ring of TMQ dramatically decreased potency at beta(1)- and beta(3)-AR compared to TMQ. Replacement of the 1-benzyl ring of TMQ with a naphthalene ring did not alter affinity but reduced potency of resulting 1-naphthylmethyl and 2-naphthylmethyl analogs at beta(1)- and beta(3)-AR compared to TMQ. Our results define the structural and electronic properties of substituents on TMQ necessary for potent activation of beta(1)- and beta(3)-AR and suggest that further modifications of the 1-benzyl iodine-substituted analogs may yield potent beta(3)-AR agonists.

Synthesis and human beta-adrenoceptor activity of 1-(3,5-diiodo-4- methoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-6-ol derivatives in vitro.

Trimetoquinol (1, TMQ) is a potent nonselective beta-adrenergic receptor (AR) agonist and a thromboxane A(2)/prostaglandin endoperoxide (TP) receptor antagonist, while 3',5'-diiodo-TMQ (2) exhibits beta(3)-AR selectivity. In search of selective beta(3)-AR agonists as potential drugs for the treatment of human obesity and type II diabetes mellitus, a series of 1-(3, 5-diiodo-4-methoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-6-ols has been prepared and evaluated for their biological activities at human beta(1)-, beta(2)-, and beta(3)-ARs expressed in Chinese hamster ovary (CHO) cells. The compounds have been synthesized by the Bischler-Napieralski cyclization of corresponding amides followed by NaBH(4) reduction, and the halogens in the aromatic ring A were introduced by direct halogenation of protected compound 11. Whereas halogen substitution in ring A reduced either potency or intrinsic activity on beta(3)-AR, the non-halogen-substituted compounds 8 and 10 were potent, selective, nearly full agonists for beta(3)-AR.

Biochemical and functional characterization of 1-benzyl substituted trimetoquinol affinity analogs on rat and human beta-adrenoceptors.

The site of interaction for the 1-(3',4',5'-trimethoxybenzyl) group of trimetoquinol (TMQ) with beta-adrenoceptors (beta-ARs) is important for the rational design of highly potent and beta3-AR-selective analogs. 1-Benzyl ring-substituted TMQ analogs were evaluated for binding affinities and biochemical activities (cyclic AMP accumulations) in Chinese hamster ovary (CHO) cells expressing the rat and human beta3-AR, and for functional activities on isolated rat tissues. Binding affinities (K1 approximately 0.055 to 1.5 microM) for the rat beta3-AR and potencies for adenylyl cyclase activation (K(act) approximately 0.43 to 2;5 nM) of the 3'-monoiodo or 3',5'-diiodo derivatives with 4'-isothiocyanato-, 4'-amino, 4'-acetamido, or 4'-alpha-haloacetamido substitutions were higher than those of (-)-isoproterenol, and comparable to those of BRL 37344 [(+/-)-(R*,R*-[4-[2-[[2-(3-chlorophenyl)-2-hydroxy-ethyl]amino]propyl]ph enoxy]-acetic acid sodium]. A similar rank order of binding affinities (K(i) approximately 0.11 to 2.5 microM) and potencies (K(act) approximately 0.45 to 9.5 nM) was obtained for TMQ analogs on the human beta3-AR. The 4'-acetamido and 4'-alpha-chloroacetamido analogs of 3',5'-diiodoTMQ were more potent than (-)-isoproterenol in rat atria (beta1-AR) and rat trachea (beta2-AR) and exhibited partial agonist activities, whereas full agonist activities were observed in rat esophageal smooth muscle (EC50 approximately 2-8 nM, beta3-AR). 4'-alpha-Chloroacetamido-3',5'-diiodoTMQ-mediated chronotropic responses in atria were sustained and resistant to washout. Further, the 4'-alpha-chloroacetamido and 4'-alpha-bromoacetamido analogs of 3',5'-diiodoTMQ demonstrated significant concentration-dependent irreversible binding to the rat beta3-AR. Reversible beta-AR agonists such as (-)-isoproterenol, BRL 37344, and 4'-acetamido-3',5'-diiodoTMQ or nucleophilic 1-amino acids (lysine, glutathione, cysteine) did not protect against this irreversible binding. Thus, the lipophilic 1-benzyl ring of TMQ analogs interacts with a hydrophobic region of the beta-AR that may represent an exo-site or an allosteric binding site.

Beta-adrenoceptor subtype activities of trimetoquinol derivatives: biochemical studies on human beta-adrenoceptors expressed in chinese hamster ovary cells.

The beta-adrenoceptor activities of trimetoquinol (TMQ) isomers and selected derivatives were evaluated on human beta-adrenoceptor subtypes expressed in Chinese hamster ovary cells. In cAMP accumulation assays, (-)-TMQ was 214-, 281-, and 776-fold more potent than (+)-TMQ at stimulating beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes, respectively. In radioligand binding assays, (-)-TMQ exhibited 123-, 331-, and 5-fold greater affinity than (+)-TMQ for beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes, respectively. (-)-TMQ and (+/-)-TMQ activated the human beta(3)-adrenoceptor with an 8.2- and 3.4-fold greater efficacy, respectively, than the reference beta-adrenoceptor agonist (-)-isoproterenol (efficacy = 1). The 3',5'-diiodo analogs of TMQ were partial agonists of the beta(2)-adrenoceptor relative to (-)-isoproterenol, and their potencies were 5- to 10-fold higher at the beta(3)-adrenoceptor as compared with beta(1)-adrenoceptors. Modification of the catechol (6,7-dihydroxy) nucleus, such as replacement of the 7-hydroxy group with a chloro group (7-chloroTMQ), ring fluorination (8-fluoro and 5,8-difluoro analogs), or preparation of bioisosteric tetrahydrothiazolopyridine (THP) derivatives of TMQ yielded compounds that displayed partial agonist activity (relative to (-)-isoproterenol) or were inactive at the beta(2)-adrenoceptor and exhibited beta(3)-adrenoceptor-selective stimulation compared with the beta(1)-adrenoceptor. Furthermore, the 3',5'-diiodo-4'-methoxybenzylTHP derivative of TMQ was 65-fold more potent than the corresponding 3',4',5'-trimethoxybenzylTHP at the human beta(3)-adrenoceptor. Our results indicate that 6, 7-dihydroxy-catechol-modified and 1-benzyl halogen-substituted derivatives of TMQ represent promising leads for the development of beta(3)-adrenoceptor-selective agonists.

2-Amino-4-benzyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridines: novel selective beta3-adrenoceptor agonists.

Trimetoquinol (TMQ, 7) is a potent nonselective beta-adrenoceptor (AR) agonist. Replacement of the catechol moiety of TMQ with a 2-aminothiazole group resulted in novel thiazolopyridine derivatives 9-11 which have been synthesized and evaluated for biological activity on human beta1-, beta2-, and beta3-AR. The Bischler-Napieralski reaction has been employed as a novel approach to construct the 2-amino-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine ring system. Although in radioligand binding studies analogues 9 and 10 did not show selectivity toward beta3-AR, they exhibited a high degree of selective beta3-AR agonist activity in functional assays. Moreover, the beta3-AR agonist activity of the 2-aminothiazole derivatives is abolished by N-acetylation (analogue 11) or ring opening (analogue 25). This illustrates the importance of the intact 2-amino-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine ring for beta3-AR activity.

Iodinated analogs of trimetoquinol as highly potent and selective beta 2-adrenoceptor ligands.

A series of trimetoquinol (1, TMQ) analogs were designed and synthesized based on the lead compound 2, a diiodinated analog of trimetoquinol which exhibits improved selectivity for beta 2-versus beta 1-adrenoceptors (AR). To determine the influence of 1-benzyl substituents of trimetoquinol on beta 2-AR binding affinity and selectivity, we replaced and/or removed the 3'-, 4'-, and 5'-methoxy substituents of trimetoquinol. Replacement of the 4'-methoxy group of 2 with an amino (21c) or acetamido (15) moiety did not significantly alter beta 2-AR and thromboxane A2/prostaglandin H2 (TP) receptor affinity. Substitution with a 4'-hydroxy (18) or -iodo (21b) group did not significantly alter beta 2-AR affinity, but greatly reduced TP receptor affinity (380- and 1200-fold, respectively). Further, the beta 2-AR can accommodate larger substituents such as a benzamide at the 4'-position (26b). Other monoiodo derivatives (24, 26a) have similar or slightly lower affinity to both beta 2-AR and TP receptor compared to their diiodo analogs. Interestingly, removal of the 4'-substituent of 3',5'-diiodo analogs increased beta 2-AR affinity with little or no effect on beta 1-AR and TP binding. Thus, analog 21a displayed highly potent (pKi 9.52) and selective (beta 2/beta 1 = 600) binding affinity for beta 2-AR. On the other hand, trifluoromethyl substituents at the 3'- and 5'-positions (27) essentially abolished binding affinity at beta 2-AR and TP receptors. The differential binding effects of the aforementioned trimetoquinol modifications on the receptor systems may reflect differences in the binding pocket that interacts with the benzyl portion of trimetoquinol analogs. Thus, manipulation of the 1-benzyl moiety of trimetoquinol (1) has resulted in analogs that exhibit potent beta 2-AR binding affinity and significantly lower beta 1-AR and TP receptor affinities.

[Blood phospholipases in burn shock]. / Fosfolipazy plazmy krovi pri ozhogovom shoke.

The activity of blood plasma phospholipases A and C has been observed during burn shock periods in patients with severe burns affecting more than 20% of the body surface. It was experimentally demonstrated that the enzyme plasma activity increased 30 minutes after burning. Simultaneously, intensification of lipid peroxidation took place. Phospholipases are suggested to liberate from the damaged tissues with their following activation by free-radical products.

[Importance of cooling after death for restoration of metabolism during resuscitation]. / Znachenie postmortal'nogo okhlazhdeniia dlia vosstanovleniia metabolizma pri ozhivleniia.

Incorporation of labelled metabolites into proteins, RNA and DNA was completely inhibited in rabbit tissues under conditions of prolonged hypothermia (10 degrees). The incorporation of labelled metabolites into all the biopolymers studied was restored after subsequent warming up to 38 degrees. Within 60-90 min after death of the animals due to acute anoxia, the labelled metabolites did not incorporate into protein, RNA and DNA. Artificial postmortal hypothermia (20 degrees) increased (by about 2.5-3-fold) the period of viability, during which reanimation is possible. The hypothermia enables subsequent restoration of the anabolic processes from the zero level.

[Protein biosynthesis of the xenogeneic heart during perfusion with the aid of a donor]. / Biosintez belkov ksenogennogo serdtsa pri perfuzii s pomoshch'iu donora

Biosynthesis of sarcoplasmic and contractile proteins was studied in cat heart, extracorporeally connected to the rabbit circulation. Biosynthesis of sarcoplasmic proteins was inhibited by 34-40% in ventriculus dexter of the perfused xenogenic heart. Heteroperfusion caused an increased incorporation of 35S-methionine into total proteins of the right auricle and of the interventricular septa and also into contractile proteins of both myocardium ventricles of rabbit-donor. The heteroperfusion did not affect the penetration of the labelled amino acid into various parts of perfused and donor hearts.

Protein degradation to low-molecular compounds after death and during reanimation.

The process of protein degradation to amino acids and peptides in rabbits following death and during reanimation in terms of the effects of artificial postmortem cooling on that process has been studied. Protein degradation was judged by increase of low-molecular nitrogenous compounds in serum and in organs by increase in soluble radioactivity with time in animals the proteins of which had been marked in vivo with radioisotopes. It has been found that immediately after death resulting from acute anoxia the processes of protein degradation to amino acids as well as synthesis stops in liver, skeletal and cardiac muscles, spleen, brain and spinal cord. Similar phenomenon takes place in the case of deep hypothermy. During reanimation the process of protein degradation to low-molecular compounds in organs restores.

Restoration of nucleic acid biosynthesis after clinical death and factors stimulating the process in vivo.

The biosynthesis of RNA and DNA falls almost to zero in 60 min after the death of rabbits from anoxia, in all the organs of the body. Rapid artificial cooling of the rabbits to 20 degrees C undertaken within 10 min after death preserved nucleic acid biosynthesis and permitted restoration of life 3-4 h after death, with recovery beginning in 60 min. During the reanimation the addition of ATP to the blood stimulated the restoration of RNA biosynthesis in the spinal cord to a considerable extent; the addition of cocarboxylase to the blood promoted cardiac RNA biosynthesis as well as cardiac and pancreatic DNA biosynthesis during recovery.