Peptidomimetics derived from natural products.

Although much has been written in recent years about rational drug design, no drug has been designed de novo, that is, without using a natural substrate or inhibitor or screening lead as a starting point. Instead, as we have seen, medicinal chemists continue to depend upon serendipitous discovery of novel biological activities and novel chemical entities for structures on which to begin work. What rational drug design really means at present is rational drug discovery and rational optimization. These result from the application of modern structural and mechanistic biochemistry, and good synthetic chemistry, to obtain structures with the desired spectrum of biological activities. Traditionally, lead compounds were discovered in plant and animal extracts, and more recently in microorganisms and chemical libraries. These traditional approaches continue, but are augmented by advances in molecular biology, which now provide pure proteins in quantity for screening and structure determination, as well as for characterization by modern biophysical methods. Remarkably, x-ray and NMR methods can now provide the most important information needed to design new drugs, that is, the conformations of ligands bound to target proteins. Approaches to identifying possible ligands based only on the knowledge of the enzyme active site are being developed. Some of these, such as CAVEAT, have been recently reviewed. In spite of these impressive gains, de novo design of new drugs will not be achieved until we learn how to logically build specific inhibitors of a target enzyme knowing only the protein sequence of the enzyme or the amino acid sequence of the messenger substances. We have a long way to go, because by this very rigorous definition, even the successful design of a new nonpeptide drug beginning with enzyme-ligand NMR or x-ray structure constitutes rational optimization. However, as this article has illustrated, we have made great progress. Some of the current and futuristic approaches to drug design are shown in Fig. 8. Development of useful enzyme inhibitors, designed by knowing the enzyme catalytic mechanism or discovered by screening for natural inhibitors, is a very successful rational method. Discovery of receptor antagonists by screening protocols is also productive.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of preparation techniques on mixing of additives in intravenous fluids in nonrigid containers.

It has been observed that the procedures of some hospitals do not require any mixing after drugs are added to intravenous bags. Previous studies have shown that mixing often is incomplete in the absence of specific mixing procedures. It has been asserted that mixing is effected by normal handling of bags, and that mixing is sufficient if drug is added to "bottom up" bags, which are then inverted. In this study the extent to which mixing occurred on addition of KCl solution or water to intravenous bags as a function of bag and needle position, additive, speed of addition, and bag handling procedure was determined. Also determined was the extent to which mixing occurred spontaneously. Most important, we determined the effect of incomplete mixing on the concentration of drug actually delivered from the bags. It was concluded that mixing is critically dependent on many factors, and that an effective technique to achieve complete mixing is essential to assure homogenous solutions. The most effective method of mixing solutions in plastic infusion bags is to grasp the bag by its two ends and rapidly invert it twice. This "double inversion" mixing technique, a maneuver requiring only 2 seconds, assures complete mixing of intravenous solutions.

Darmstoff analogues. 3. Actions of choline esters of acetal phosphatidic acids on visceral smooth muscle.

A number of naturally occurring phospholipids, e.g. the acetal phosphatidic acid derivatives that comprise Darmstoff (1) and the phosphatidylcholine derivative platelet activating factor (PAF), cause contraction of certain visceral smooth muscles and cause platelet activation. Because the Darmstoff phosphatidic acids and PAF are structurally similar, it was of interest to compare the biological actions of choline esters of Darmstoff with those of PAF and of the parent Darmstoff phosphatidic acids. To this end, [(2-pentadecyl-1,3-dioxolan-4-yl)methyl]phosphocholine (3a), [[2-(cis-8-heptadecenyl)-1,3-dioxolan-4-yl]methyl]phosphocho line (3b), and [[2-(cis-8-pentadecenyl)-1,3-dioxolan-4-yl]methyl]phosphocho line (3c) were synthesized. Compounds 3a, 3b, 3c, and PAF caused dose-dependent relaxation of taenia coli strips. In contrast, the unesterified materials 1a and 1b, as well as lyso-PAF, caused contraction in taenia coli strips. Thus, the contractile effect of Darmstoff is reversed on esterification with choline. In preparations of whole trachea, both 1a and 3a had contractile effects similar to those of PAF.

Microsomal metabolism and covalent binding of [3H/14C]-bromobenzene. Evidence for quinones as reactive metabolites.

1. The metabolism and covalent binding of [3H/14C]bromobenzene has been investigated using liver microsomes from untreated and phenobarbital (PB)-pretreated rats. A model has been developed to relate the observed 3H/14C ratios in the covalently bound residues to the type of metabolite (epoxide versus quinone) responsible for their formation. 2. With control microsomes metabolism was linear for 60 minutes, but with PB microsomes the time course showed a short-lived burst of rapid metabolism followed by a long phase with an overall rate comparable to control. With both types of microsomes covalent binding was synchronous with metabolism. 3. The normalized 3H/14C ratios of recovered substrate and water-soluble metabolites was 1.0, whereas that of the covalently bound material was only 0.5. Such extensive loss of tritium implies that a considerable portion of the covalent binding arises from bromobenzene metabolites more highly oxidized than an epoxide (e.g. quinones). 4. The normalized 3H/14C ratios for bromobenzene metabolites covalently bound to liver proteins in vivo (total and microsomal) was the same as with microsomes in vitro (0.5). However, for the lung and kidney the 3H/14C ratios were considerably higher (0.71 and 0.62), indicating that differences between tissues in vivo may be greater than between liver microsomes in vitro and in vivo.

Tetracyclic pyridazines as potential psychopharmacological agents.

Since the Z isomer of chlorprothixene (1) is far more active than its E counterpart, it was of interest to develop a stereoselective synthesis for this class of compounds. Insertion of a benzenesulfonamido group at the peri position of a chlorprothixene precursor did affect the stereochemistry of side-chain olefin formation, but after hydrolysis attempted removal of the resulting amine led to a Widman-Stoermer cyclization to afford the corresponding tetracyclic pyridazine-containing compound (4). Since this material displayed encouraging activity in neurotransmitter uptake inhibition studies, compounds in which the sulfur bridge was replaced with an ethano bridge similar to that found in imipramine (8) and with sulfur removed (7) were also prepared. These, together with the corresponding peri amino compounds (3, 5, and 6), were tested as neurotransmitter-uptake The two bridged arylamines 3 and 6 displayed potent and selective inhibition of norepinephrine uptake both when tested in vitro and after in vivo administration. The pyridazine-containing compounds exhibited reasonable activity in vitro, but the activity was lost when they were administered in vivo. None of the compounds displayed significant ability to interfere with spiroperidol binding.

Chemical models for toxic metabolites of bromobenzene derivatives. Relative toxicity toward isolated hepatocytes.

In the rat the hepatotoxicity of bromobenzene is greatly enhanced by the introduction of a cyano group adjacent to the bromine (i.e. o-bromobenzonitrile). Epoxide metabolites of these aryl halides are believed to be the actual toxic species, but epoxides of the latter compound also possess a second site of chemical reactivity not found in epoxides of bromobenzene, i.e. a Michael acceptor group formally related to acrylonitrile. Because these epoxides have never been isolated or synthesized for direct evaluation of their toxicity we have determined the toxicity toward isolated rat hepatocytes of a series of cyclohexene and cyclohexadiene derivatives containing epoxide and/or alpha, beta-unsaturated nitrile functional groups. Simple epoxides and unsaturated nitriles were much less toxic than bromobenzene itself, even if both groups were present in the same molecule. However, alpha, beta-unsaturated epoxides were found to be 2-3 times more toxic than bromobenzene, and at least 10 times more toxic than their saturated analogs, which is consistent with their relatively greater chemical reactivity. It is unlikely that Michael acceptor metabolites account for the increased toxicity of o-bromobenzonitrile relative to bromobenzene.

Toxicity-distribution relationships among 3-alkylfurans in mouse liver and kidney.

The present study was designed to extend previous observations regarding toxicity of furans and related compounds to liver and kidney. It was desired to test a series of homologous 3- alkylfurans , where changes in lipophilic character might be related to changes in toxicity. Additionally, it was desired to measure distribution of toxins to the target organs to ascertain whether organ selectivity might be determined by the concentrations attained in the target organs by the toxins. A synthesis for 3- ethylfuran and 3- pentylfuran was devised, and the toxicity of these, in addition to 3- methylfuran and furan itself, to mouse liver and kidney at 2.6 mmol/kg was determined. 2- Furamide and 2- ethylfuran were used as examples of substances known to be toxic to liver and kidney, respectively. 3- Methylthiophene was also included to determine whether results with furans extend to the closely related thiophenes . Histopathological examination of both organs was done, and quantitative estimates of liver toxicity were obtained from plasma levels of glutamate-pyruvate transaminase. Renal urine concentrating ability and plasma urea nitrogen levels were useful as quantitative indices of nephrotoxicity. It was found that both 3-ethyl and 3- pentylfuran exhibited pronounced toxicity to the kidney, and that both also caused moderate liver damage. Furan caused serious damage to the liver and produced somewhat lesser effects on the kidney. Equimolar doses of 3- methylfuran did not significantly damage either organ. Among the 3-alkyl furans, there is an impression that the more volatile compounds damage lung (3- methylfuran is reported to be a potent lung toxin), with liver and kidney toxicity increasing with molecular weight, and that compounds found in higher concentration produce greater damage in liver and kidney. However, among compounds other than alkyl furans, there is no obvious correlation between toxicity and organ concentration of toxin.

Toxicity-distribution relationships among 3-alkylfurans in the mouse lung.

The present study was designed to investigate the extent to which a homologous series of 3-alkylfurans, 3-methylfuran, 3-ethylfuran, and 3-pentylfuran, may cause lung injury in mice in order to determine whether the chemical properties of these compounds are related to their toxic potential. The pulmonary concentration and pneumotoxicity of these and various other furan derivatives were also measured to determine if a correlation existed between the magnitude of pneumotoxicity produced by these toxins and their concentration in the lung. Along with the 3-alkylfurans, the other furan derivatives investigated were furan, 2-ethylfuran, 2-furamide, and 3-methylthiophene. The absence or presence of lung damage was evaluated by light microscopy. The quantitative index used to assess and rank the compounds as pneumotoxins was the incorporation of [14C]thymidine into DNA. The results obtained with the 3-alkylfurans showed that 3-methylfuran and 3-ethylfuran were toxic to the lung whereas 3-pentylfuran did not produce pneumotoxicity. 2-Ethylfuran, furan, and 2-furamide also caused lung damage but 3-methylthiophene did not. All the compounds, with the exception of furan, reach the lung in comparable concentrations; therefore, there does not seem to be a correlation between pneumotoxicity and concentration of the toxin in the lung. From these studies, it is also apparent that the pneumotoxicity of the 3-alkylfurans extends beyond the methyl group but that the toxicity decreases with increasing chain length.

Synthesis and bacterial metabolism of cis- and trans-2-alkyl analogues of sodium cyclamate.

Sodium cyclamate is an effective artificial sweetner, which has been banned from the U.SD. market because of alleged carcinogenic properties. It appears that cyclohexylamine, liberated from cyclamate as a result of bacterial mtabolism, is the proximate carcinogen. In an effort to elucidate the extent to which analogues of cyclamate would enter into the bacterial metabolic pathway, as well as any stereochemical requirements which might exist, several 2-alkaly analogues of sodium cyclamate were prepared. It was found that trans-N-(2-methylcyclohexyl)sulfamate (trans-2a) and trans-N-(2-ethylcyclohexyl)sulfamate were hydrolyzed by freshly collected fecal suspensions from rats fed cyclamate, but not from control rats, at the same rate as cyclamate itself. trans-N-(2-Isopropylcyclohexyl)sulfamate (trans-2c) was not hydrolyzed at all. Surprisingly, two of the analogous cis compounds (cis-2a and cis-2c, respectively) were hydrolyzed by fecal suspensions from control, as well as from cyclamate-fed, rats. Moreover, cis-2a was hydrolyzed by incubating it in medium only. Thus, it is apparent that stereochemical influences on the chemical properties of these compounds are substantial. These results do not appear to point the way toward a safe, nonmetabolizable sweetening agent.

Toxicity of ortho-substituted bromobenzenes to isolated hepatocytes: comparison to in vivo results.

In this study, freshly prepared isolated rat hepatocytes were exposed to various concentrations of bromobenzene and six ortho-substituted bromobenzenes. Toxicity was estimated by trypan blue dye exclusion and release of GPT and K+ over a 0- to 4-hr time course. In all cases a close correlation among these three indices was observed. The rate of response depended on the exposure level and the nature of the o-substituent. The relative toxicity followed the order C2H5 greater than CH3 greater than or equal to CF3 greater than Br much greater than H greater than OCH3 much greater than CN. This order does not correlate well with the in vivo toxicity data previously obtained in this laboratory (Toranzo E. G., Gillesse, T., Mendenhall, M., Traiger, G.J., Riley, P.G., Hanzlik, R.P., and Wiley, R.A. (1977). Toxicol. Appl. Pharmacol. 40, 415-425.); in fact an almost inverse relationship is indicated. Several possible reasons are proposed in order to rationalize the discrepancy between these two results. The results emphasize the need for caution when using isolated hepatocytes to screen new compounds for purposes of determining their potential in vivo toxicity, at least until the differences between hepatocytes in vivo and hepatocytes in vitro are more thoroughly understood.

Acetal phosphatidic acids: novel platelet aggregating agents.

1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 muM for human platelets and 0.25-0.5 muM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.3 PGAP-induced irreversible aggregation of [(14)C]-5-hydroxytryptamine ([(14)C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0+/-2.4% (s.e.) of the platelet [(14)C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [(14)C]-5-HT-labelled sheep platelets was dose-dependent.4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A(2) inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E(1) (PGE(1)); these agents abolished the second, but not the first phase of ADP-induced aggregation.5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 muM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 muM.6 It is concluded that the acetal phosphatidic acids induce platelet aggregation per se by direct action at the platelet membrane, and that the acetal function is of primary importance in their potent platelet-stimulating activity. Moreover, as the acetal phosphatidic acids are the major components of the smooth muscle-contracting acidic phospholipid tissue extract ;Darmstoff' (Vogt, 1949), their potent platelet-aggregating properties may be of physiological or pathological significance.

The effect of nephrotoxic furans on urinary N-acetylglucosaminidase levels in mice.

The effects of several potentially nephrotoxic furans on urinary levels of N-acetylglucosaminidase (NAG) were examined to determine whether this test might serve as a useful quantitative index of nephrotoxicity for this series of compounds. Whereas others have found that nephrotoxins such as sodium salicylate and biphenyl cause increases in urinary NAG, we observed that these furans, which were shown to be nephrotoxic by histological procedures, caused significant decreases in urinary levels of the enzyme. This effect was dose-related in the one case examined.

Darmstoff analogues. 2. Ring and side-chain effects on smooth-muscle contraction.

2-cis-delta 8-Heptadecenyl-4-(hydroxymethyl)-1,3-dioxolane monosodium phosphate (1b) has been shown to be present as a major component of Darmstoff in mammalian intestine and to be a potent inducer for contraction of intestinal smooth muscle. The analogous 2-pentadecyl material 1a, also found abundantly in the intestine, is inactive. In this study, synthesis of phosphorylated hydroxymethyl-1,4-dioxanes, -tetrahydrofurans, -cyclopentanes, and -oxathiolanes bearing both oleyl and palmityl side chains is reported. Of these, 2-(hydroxymethyl)-5-cis-delta 8-heptadecenyl-1,4-dioxane monosodium phosphate (2b) exhibits about 12% of the activity of 1b. Its pentadecyl analogue 2a, like 1a, is totally inactive, as are all other compounds prepared. The results indicate that Darmstoff-like compounds exhibit specific chemical requirements for activity and that where activity is encountered, the side-chain specificity noted in 1a and 1b can be preserved.

Solution conformations of muscarine and some analogue.

Proton magnetic spectra have been recorded for muscarine and two biologically active cyclopentane analogues. In order to observe homonuclear intramolecular nuclear Overhauser effects, the -N+(CH3)3 signal was irradiated and increases in integrated intensities for other key signals in the molecule were observed. The results indicate that the quaternary side chain in these compounds is in an extended conformation in aqueous solution.

Hypotensive phospholipids. Some stable analogues of Darmstoff.

2-Pentadecyl-4-hydroxymethyl-1,3-dioxolane monosodium phosphate (1) has been shown to exhibit bradycardia and hypotension in rats. It is, however, unstable in aqueous solution. In this study, the stable analogues of 1, 2-pentadecyl-5-hydroxymethyltetrahydrofuran monosodium phosphate (7), and 1-hydroxymethyl-3-pentadecylcyclopentane monosodium phosphate (17) were prepared. These substances exhibited cardiovascular effects similar both qualitively and quantitatively to those elicited by 1 and were stable in solution for about 24 and at least 72 h, respectively.

Novel analogs of tricyclic psychopharmacological agents.

The synthesis of several novel analogs of amitriptyline and chlorprothixene, in a number of which the position of the side-chain nitrogen atom rigidly fixed with respect to the tricyclic nucleus, is described. The compounds were evaluated for antidepressant-like activity in the Dopa and serotonin interaction tests and for potential antipsychotic activity in the methamphetamine interaction test. 5-(3-Dimethylaminocyclohex-1-enyl)-5H-dibenzo[a,d]cycloheptene (12) was about equipotent with imipramine in the Dopa and methamphetamine tests, and 3-chloro-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5-spiro-6'-3'-methyl-3'-azabicyclo[3.1.0]hexane (23) also displayed marked activity in the same tests. Prototype compounds for other ring systems, 3-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene tropane (16) and 5-(3-dimethylaminocycloheptylidene)-10,11-dihydro-5H-dibenzo[a,d]cycloheptene (18), were less active.