RATIONAL APPROACHES TO DRUG STRUCTURE ALFRED BURGER University of Virginia, Charlottesville, Virginia
A DRUG is a chemical which exerts one or several biological activities of therapeutic value. It may he the ion of an element (ferrous, iodide, sodium) or more commonly an organic compound. It usually is foreign to the organism in which it acts, or an essential biocatalyst of that organism such as a vitamin. Also, there is no dividing line between drugs and compounds produced by the organism (hormones) which can, if necessary, be administered therapeutically in case of deficiency. All these substances have to reach the site of their biological action and there affect in some manner a cell chemical (receptor) or react with dissolved compounds and with enzymes. This article will attempt t o summarize some important facets of our present knowledge of transport in viva and mechanisms of drug action. It will point out difficulties in interpretation which meet suggestions for rational avenues to a better design of drug structure and recognition of drug action. PHARMACOLOGICAL ABSORP'I'ION. DISTRIBUTION, AND REMOVAL
A chemical must reach the site of its action. For this purpose i t should he soluble in isotonic aqueous solutions, and must have the proper ratio of solubilities between lipids and water in order to assure absorption across membrane barriers. The membranes of many living cells present to a drug a continuous outside layer of proteins. Bonded to them by hydrogen atoms, the polar groups of parallel layers of lipids lie below the protein surface, the lipophilic parts facing each other. Polyvalent cations and many organic polyfunctional compounds can be adsorbed a t these membranes in concentrations much above those in the surrounding medium, provided they fit sterieally to the protcin surface. While one cannot expect useful absorption of a cornpound which is utterly insoluble in water or in lipids, ionizable substances can be transported in the form of their soh~blesalts. Undissociated molecules pass cell membranes more easily than their ions, but ionic species are often bonded more readily by cell receptor chemicals. Substances which can he regarded as "detergents" are absorbed indiscriminately because of their low affinity for water molecules; they are ~ h e donto other surfaces from their aqueous solutions. A low degree of ionization is conducive to detergency. Highly ionized materials are adsorbed less readily and are either biologi-
cally inert (sulfonic, phosphonic acids, etc.) or, because they completely alter the physiological pH, chaotically toxic. Molecules which are well hydrated are adsorbed by specific cells and tissues subject to steric factors. Some weak acids such as phenols or barbituric acids are more active in the molecular than in the ionic form, but their ions also possess some activity. Hence, as ionization increases, biological activity will decrease only slowly because of the small but increasing contribution by the ions of the substance. Compounds which exist as switterions are usually biologically inert. Since ionization is frequently involved in chelation, substances requiring metallic ions for bonding to receptors must be ionized to some extent. A special case is presented by cationic compounds which can tautomerize to pseudobases (e. g., cotarnine, berberine). Their tautomeric form can combine v.ith receptors covalently rather than ionically. Many bioiogically active compounds contain intramolecular hydrogen bonds as determined by their infrared spectra. Among them are chloral hydrate, salicvlic acid and its derivatives. the antirheumatic 2,5-dihydroxyhenzoic acid, and the tetracycline antibiotics. Intermolecular association between nlolecules of such substances can be estimated thermodynamically from measurements of their entropy and free energy changes. It appears that even association between n~oleculesdoes not decrease appreciably their intramolecular chelation. This property will decrease the external (polar) field of the compound, allowing for more rapid diffusion and penetration (1). An application of present views of reactions a t the cell membrane is the unified theory of inflammation and stress by Eyring and Dougherty (2). These authors contrast the normal, resting or impermeable state of the cell membrane in which the lipoprotein molecules are orderly arranged with a less orderly permeable membrane state. If this disorganized state is maintained too long, the concentration gradient between inside and outside chemicals decreases and the membrane becomes sticky and falls prey to phagocytosis. The cellular enzymes become exposed and metabolize more rapidly, the metabolite concentration must increase, sodium ions are retained inside the cell, and the cell may swell and burst. Reactions such as equilibration of alkali ions across the membrane tend t o restore the impermeable state as a mechanism of cell survival. Inflammation may be regarded as a degeneration
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which increases the unstable permeable state of cell membranes. Hydrocortisone and other adrenal cortex hormones stabilize the impermeable state of the membrane and counteract retention of sodium ions. These hormones also diminish stress which is conceived as a "systemic response induced by the wave of cellular alterations identical with those which initiate inflammation." In stress one observes a similar stickiness of the endothelium, increased capillary permeability which can lead to shock, and the appearance of special stress lymphorytes which become phagocytes in the blood stream. When a biologically active drug is introduced into the body, the organism will tend to detoxify it. If the drug is deposited in tissues where its effect is not wanted, and if it is fed from such storage places to other more sensitive tissues in which its action is no longer desired, detoxifying mechanisms become therapeutically valuable. The oxidative biological detoxication by the liver of barbiturates stored in adipose tissue is an example for this case. In most instances, however, the length of action of a drug is to be extended rather than shortened. Obviously, a drug is useless if it is inactivated by premature biological conjugation or other metabolic effects before it reaches its site of action. Neither should it be detoxified or excreted after a ffeeting or short contact with its re11 receptors. A few compounds have become known which, although themselves biologically inactive, have a profound effect on the duration of action of a variety of other drugs. Among them are P-diethylaminoethyl 2,Zdiphenylvalerate (I), the corresponding 2,2-diphenylvaleric acid (11), 2-(@diethylaminoethoxy)3,5dichlorobiphenyl (111), and probably l-isopropyl-2-
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structural alterations of (I) and (11) cause complete loss of inhibitory action. True potentiators have also been found. They can make subeffective doses of various drugs effective. The best known of these potentiators is chlorpromazine (V), which itself has remarkable tranquilizing (ataractic)
properties in psychiatric disorders. The mechanism of its potentiating activity is unknown. As long as the metabolic fate of a drug is not known the question whether the compound itself or a metabolite represent the active material a t the site of action remains open. Usually the drug exerts its activity without first being altered in the organism. Some compounds, however, are metabolized to products to which the action is really due. Examples for this phenomenon are the in uiuo reduction of chloral to the actual hypnotic, trichloroethanol; the reduction of sulfonamide azo dyes t o sulfanilamide in the body; and the cyclization of chlorguanide to the active antimalarial metabolite, 2-amino-4,4-dimethyl-G-p-chloroanilino-3,4-dihydro-s-triazine. One can save the animal organism the effort of converting the "drug" to the active metabolite by administering the latter directly. This usually cuts down a lag period before the onset of action. The metabolic reartion which activates the compound may even have toxic consequences, as in the oxidation of acetanilide to p-hydroxyacetanilide. Here the administration of the metabolite or its suitable derivatives will protect the body from avoidable toxic side effects. Often it is simply more convenient to administer the inactive precursor if it has more desirable properties such as crystallizability, resistance to sterilization, etc. Or else, the in uiuo conversion may be difficult to imitate by laboratory methods, and its execution can be left to the animal with advantage. All metabolic alterations, whether desired or not, depend on the animal species in which they proceed. A drug must not distribute itself indiscriminately in isonicotinyl hydraside (IV) (3). These compounds inhibit enzyme systems in liver microsomes where the tissues or else it will exert unwanted (toxic) acmany other drugs are degraded. They retard such di- tivity. No drug localizing in one tissue exclusively is verse reactions as the dealkylation of amiuopyrine, the known but the best chemotherapeutic agents approach deamination of amphetamine to phenylacetone, the de- that ideal. Localization is facilitated if a given paramethylation of codeine t o morphine, the nuclear sitic cell differs widely from the cells of the host and hydroxylation of acetanilide, and the biological con- thereby offers the drug sufficiently differing membranes jugation of morphine. I n doses devoid of sedative for absorption. Assuming that receptors are especially reactive activity, they prolong the action of hexobarbital by blocking it7 detoxifying side-chain oxidation. How- chemical groups of cell protein molecules, their acever, subeffective doses of hexobarhital are not raised cessibility will exert considerable influence on the acto a level of effectiveness by these action-prolonging tivity of a drug. If the receptor is engulfed in a fold substances. I n time, additional inhibitors of detoxify- of a protein surface, contact with the drug molecules ing enzymes are likely to be discovered although minor will he hindered sterically. Since the receptor is pre-
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sumahly biochemically essential to a given tissue, those cells or tissues containing the lowest concentration of receptor protein will be affected most by a widely distrihuted drug because all their specific and needed receptor activity will he blocked. Structural limitations of molecules which produce a given principal action are hard to delineate. I n restricted areas such as in homologous series one can follow gradual changes in physical properties and correlate them with stepmise changes in biological properties. As the molecular weight of the memhers of a homologous series increases, the compounds generally become less soluble, until one cannot dissolve enough of a higher member to produce minimum biological activity. Calculations based on solubilities and vapor pressures lead to values for the "thermodynamic activity" of a compound (4); this function increases slowly in homologous series, and when the value reaches one, a peak in biological activity mill he attained. This has been measured for various cytotoxic effects of alcohols, phenols, bases, etc. On occasion, the lowest homologue of a series of medium-sized molecules may be the most reactive compound, if solubility rapidly decreases for subsequent members. This is observed for I,l,l-trichloro-2,2-bis-(p-a1koxyphenyl)ethane where the methoxyl derivative is the most active insecticide. In other cases high initial biological activity decreases for the next fev homologues, but then the increase of biological intensity with ascending molecular weight reverses the curve and makes it climb. Of course, when solubility becomes too low and thermodynamic activity approaches unity, biological activity will reach its final peak for that series. As it drops off from this maximum, the higher membors often attain molecular weights and elongated shapes characteristic of typical surface-active agents. Thus not only the intensity but also the quality of biological a c t i ~ i t ywill change as homologous series are ascended. Even such a minor structural change as simple homology can alter the selective distribution of a drug in the tissues. A typical example is the pharmacodynamic difference between hexamethonium and decamethonium. Hexamethonium acts primarily at autonomic ganglia while decamethonium interferes preferably a t the myoneural endplate. There is considerable overlapping of these properties, however, and the dual point of attack is especially pronounced for intermediate homologues with seven, eight, and nine methylene groups. It is significant that at both locations, at the ganglia and the cholinergic endplate, the final mechanism of action seems to be the same (6). (CHdaN+(CHAN +(CHA n = 6 :hexamethonium n = 10:decamethonium
Structurally similar compounds may have analogous or mutually inhibitory biological activities. If the substrate of an enzyme happens to be a natural metabolite, inhibitors of the same enzyme are called metabolite
antagonists. The relation between such substances are eoverned bv the kinetics in those enzvme svstems mhich 'Ehe compo&ls affect. The classik stidies of Michaelis on kinetics of enzymatic reactions (6) apply to metabolites and antimetabolites as well. I n several homologous series, or series of closely related compounds, members mith a higher molecular weight sometimes have the opposite effect of those with lower weights. Intermediate memhers of such series may he metabolites or inhibitors. Well-known examples of these dual effectsare found among derivatives of phenethylamine or of phenylet,hanolamine mhich can raise or lower blood pressure depending on conditions; among some members of the methonium drugs which can depolarize nervous structures, or inhibit their depolarization; and among derivatives of p-aminobenzoic acid which can promote bacterial reproduction and a t other times inhibit it. This puzzling behavior has been studied kinetically by Ariens (7). Starting from the concept that enzyme and substrate (uiz., receptor and substrate or drug) form a complex reversibly, the term affinity was introduced to describe the (constant) tendency of drug or metabolite to combine with the receptor under given conditions. A potent drug sho~vsits effect at low concentrations, that is, it has a high "affinity." The maximum activity of a drug is measured by another constant, its intrinsic activity. This property determines the velocity of enzyme action or of growth, i. e., the measurable effect of a given amount of the drug-receptor complex. A substrate or metabolite is a substance which is used by its receptor; it has maximal intrinsic activity. Vice versa, inhibitors (antagonists) block a receptor but are not used by it; they do not react with it chemically, and therefore have zero intrinsic activity. Substances mith structures intermediate between mctabolite and drug have intermediate intrinsic activities, which are lower than that of the metabolite. I n the absence of the metabolite they take its place at the receptor, i. e . , they act like it. If metabolite is present in the system, they will compete with it for the receptor site, and thus lower total intrinsic activity; me then think of them as inhibitors. The effect of a compound mith intermediate (low) intrinsic activity will he positive (metabolite-like), negative (inhibitory), or zero depending on the amount of compound nith high intrinsic activity in the system. This latter compound is usually a metabolite, but may be a highly active drug as well. A mathematical expression of these theories may be obtained by extending the Michaelis-Menten equation of enzyme kinetics (8). It presupposes that receptors are available in small amounts, and metabolite or inhibitor is present in amounts large enough to cover all receptors. Well-known examples for this relation are the amounts of oxygen and hemoglobin in the formation of oxyhemoglobin. I n the equation:
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r, is the total concentration of receptors; a and P are the intrinsic activities of substances A and B (or is high, P low) ; [A] and [B] the concentrations of A and B; and K,, and K Bare the different affinities for the receptor. Examples illustrating these deductions have been given for sulfonamide drugs and for muscle-relaxant agents. I n the first example, PABA which has maximal (growth-promoting) intrinsic activity is contrasted with sulfanilamide whose intrinsic activity is zero. 2-Chloro-4-aminobenzoic acid has high growth-promoting activity, and acts like PABA; sulfanilamide analogues with zero activity act like sulfanilamide. However, 3-bromo-4-aminobenzoic acid and other derivatives with intermediate activities as determined from bacterial growth rates can act as growth promoters or inhibitors depending on the concentration of PABA in the cultures. A PABA concentration, constant for each derivative, can be found at which addition of the analogue with intermediate intrinsic activity in any concentration is without influence on growth mhats~ever. The second example is taken from analogues of decamethonium (R3N+(CH2),,N+R3;R = CH3) a11d suecinylcholine [(R3N+CHzCHzOCOCH&; R = CH,)].
intrinsic (depolarizing) activity. Homologues with every R = CPHLare inactive. Intermediate activity is exhibited by homologues in which some R are methyl, some are ethyl. Such compounds are either depolarizine (muscle contractinei. or thev inhibit denolarizati& '(they are muscleu'~elaxantsj depending on the amount of contracting (maximally depolarizing) substance present in the experiment (frog abdominal muscle). The possibility cannot be overlooked that some dissociation constants of enzyme-metabolite complexes may profoundly alter the relation of antagonist to metabolite in multiple-stage enzyme systems. For example, riboflavin is distributed in rat liver as riboflavin, riboflavin phosphoric acid, and riboflavin-adenine dinucleotide; the amount of the dinucleotide is 82 per cent, and it appears passible that this compound does not dissociate readily in uiuo. Thus, the greatest amount of the vitamin may he immobilized in the dinucleotide and not he available for displacement reactions with riboflavin antagonists. The modified Michaelis-Menten equation could then be applied only to the 18 per cent of available riboflavin and riboflavin phosphoric acid, and the displacement of riboflavin from the dinucleotide by the antagonist will have to be treated separately. The observations discussed in the preceding paragraphs apply to cells susceptible to a given drug; a more complex behavior is exhibited by cells which have acquired tolerance (resistance) to a drug. A chemical which has initially inhibited a vital funct,ion gradually may lose this ability until zero (intrinsic) activity appears. Indeed, it may even reverse its effect and assnme the role of a metabolite for the cell species it or-
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dinarily inhibits. I n the field of chemotherapy, two explanations have been offered for this phenomenon. One is that versatile microbial cells can adapt themselves by suitable mutations to the new environment of the drug and metabolize the toxic agent in lieu of the usual nutrient or synthetic intermediate, or that they synthesize excessive amounts of nutrient as a defense against the drug. The second explanation for microbial resistance postulates that all but a very small percentage of genetically different cells are inhibited. The small number of resistant uninhibited cells survive and become the parent cells of a strain mhich is now drug resistant. Relations of drug and metabolite can be applied readily to this metabolically adaptable surviving mutant. Tolerance, which is resistance of tissues to drugs, has not yet been explained adequately. Mutation of cells (adaptive enzyme formation) within a tissue is not a common procedure--except perhaps as an explanation of malignancy-and the existence of genetically different, drug-resistant tissue cells has not been demonstrated convincingly. An excellent discussion of mechanisms of resistance has been given by Martin (9). A little learning is a. dsng'rous thing Drink deep, or taste not the Pierian Spring; There ehn.llnw intnxirntn hrsin. ~ ~ - drmehta ~ ~ ~ -- ~ - . t,he - ~ ~, . -~ ~ -~~~ ~~ -~ And drinking largely sobers us again. Pope, "Essay on Criticism," I, 215 ~
At the receptor sites, chemicals may act in more than one may. They may stimulate the receptor (react with it chemically) or decrease its activity by blocking access to it by steric factors. A substance, whether produced in the body or whether foreign, seldom exerts only one sperific effect. This has been called biological relativity (9). Few chemicals behave otherwise. Fluorocitrate ions appear to he specific inhibitors of aconitase, and of no other enzyme. Vice versa, there are some enzymes of extreme specificity. Urease has been found to catalyze no simple reaction other than the conversion of urea to ammonium carbonate but it does inhibit other substances, for example, trypsin (10). The interaction of a natural or artificial substrate with a receptor enzyme may be initiated, like all other organic reactions, by free radical or ionic mechanisms. Free radicals are encountered in many enzymatically catalyzed oxidations and reductions; antioxidants act predominantly by free radical mechanisms, breaking oxidation or polymerization chain reactions. Applications of this concept have been made in the protection against irradiation damage, by 2-aminoethanethiol; the damage appears to be caused by formation of free hydroxyl radicals from water under the influence of radiant energy, which sets in motion depolymerization reactions of nucleic acids. The relation of ionization to mode of action of drugs has been studied extensively (11). The antibacterial action of many aminoacridines and several other heteroaro-
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matic amines is proportional to their cationic disssociation. It is enhanced if the amino group is located in a nuclear position which permits tautomeric shifts to imino forms, and hence, to increased resonance. It is also aided if the molecule presents sufficient aromatic rings (a flat area) t o assist in the adsorption of the drug by van der Waals forces. The cations are the active species of the drug and compete with hydrogen ions a t the receptor surfaces. Throughout, ionization fartors influencing absorption and final reaction with receptors have to be balanced; echinoderm eggs are inhibited by salicylate ions but penetrated more easily by nndissociated salicylic acid (1B) ; barbiturates penetrate Arbacia eggs in the molecular form but anesthetize them better as ions (18). I n an extension of these examples, thiamine is phosphorylated and the resulting anions become highly adsorbable a t complementary surfaces. Receptor sites themselves can be ionized. Assuming that typical functional groups of polypeptides participate in receptor mechanisms, the pK values of the corresponding monomers may apply to the reactive sites. Nothing is known about receptor ionization in the interior of cells, but for those located at outside surfaces, the pK. could be stndied by measuring their response to drugs over a range of hydrogen ion concentrations, provided that the cell is unaffected by these changes, and the ionization of the drug does not change within the pH range studied. An interesting hypothesis for the explanation of the general mode of action of certain drugs has been summarized by Brody (14). I t is based on their ahility to "uncouple" phosphorylation from oxidative processes. The normal oxidative metabolism of living cells and of certain isolated cell components (mitochondria) leads to liberation of phosphate ions which are utilized in the synthesis of high-energy phosphate bonds. Various drugs can depress this process without simultaneously decreasing the oxygen consumption of the system. Phosphorylation is dependent on the presence of metallic ions, especially magnesium. Some uncoupling agents studied, such as dinitrophenol, stimulate oxygen consumpt,ion but are insensitive to changes in magnesinm ion concentration. Others, such as the metal-chelating antibiotics of the tetracycline group, do not increase oxygen consumption, and their uncoupling activity is reversed by magnesium ions. Regardless of its ultimate fine mechanism with the receptor chemicals, a drug must be able to approach the molecular surface of these chemicals, and must fit to this surfare. The amount of interaction, its rate, and even the quality of its activity are determined by steric fit. The surfaces of apoenzymes, coenzymes, and of bonding ions are involved in this phenomenon. Unfortunately, an understanding of cavitations and other spatial peculiarities of high-molecular apoenzymes is almost completely lacking. Speculations about such surface shapes cent,er around known shapes of inhibitory
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molecules from which the complementary ("homologous") receptor surface of the apoenzyme might be deduced. A study of this type has been made for acetylcholinesterase (AChE) (16). This enzyme and its congeners not only hydrolyze the neurohormone, acetylcholine, but also many structurally related esters although at greatly differing rates. If AChE is preoccupied with (inhibited by) such an ester, hormonal acetylcholine may not reach the catalytic sites of the enzyme. I t may then react at its neuroreceptors producing striking pharmacological symptoms. Each catalytic unit of AChE is assumed to have t ~ v o reactive sites, an anionic site which binds cations, and an esteratic site (H-G:) which contains a basic (G:) and an acidic group (H-) necessary for activity. Evidence for these last two groups is not as yet conclusive. The characteristic strncture of a compound t o be bonded to these sites is a region of high electron density (-CO-, -0CO-) situated near a tertiary amine center a t a distance of two n~ethyleuegroups. This distance a t extended configuration of the two-carbon chain is 4.4 A. I n one of the best-knonn inhibitors of AChE, physostigmine, the ester group is para $0 an aromatic amino nitrogen,at a distance of 5.5 A. If these distances in the substrates are to be matched by corresponding distances between the anionic and esteratic sites of the enzyme, the enzyme matrix must be fixed. The role of solvent molecules or metal atoms such as magnesium which participate in the bonding of substrate to enzyme is to provide positive charges t o offset the negative charge of the high electron density substrate, and of the esteratic site, H - 4 : , if this concept is correct. Acetylcholinesterase is highly activated by magnesium ions. Borate or citrate which can chelate magnesium lower the activity of the enzyme.
EB
X-Mg-Esterstic
Subatrate
site
Enzyme
Using purified AChE from Brasilian eel, Electrophorcts electrieus (Linnaeus), Friess and McCarville (16) measured the kinetics of dissociation of six substrates ( V XI) from the enzyme and tried to deduce additional
Ti;"
(CH&N+CH2CH2N
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larger, more hindered diisopropyl phosphoryl group, displacement is much less likely. However, in the dialkyl phosphorylated enzyme, the anionic site can still bind ammonium structures although only with o n e sixtieth the affinity of the normal enzyme. These two features, an ammonium group and a nucleophilic -NOH function, have been combined in quaternary salts of several hydroxamic acids and oximes in the information about the enzyme surface. The assump- pyridine series (IQ), especially 2-pyridinealdoxime metion was made that the quaternary ammonium nit,ro- thiodide (XII). This compound can attach itself to gen of (VI), (VII), (X), and (XI) rather than the tertiary nitrogen would serve as point of attachment to the anionic site of the enzyme. The tertiary heterocyclic nitrogen atoms in (VI), (VII), (VIII), and (IX), the dimethylamino nitrogen in (X), and the chlorine atom in (XI) may be regarded as highly localized the anionic site of the enzyme by means of its quatersources of electron density which should satisfy the r e nary ammonium gronp, and this gives the -NOH function, placed and held a t approximately the right disquirements of the esteratic site of the enzyme. Measurements of the basicity constants (Ka)of (VI) tance, the opportunity to dephosphorylate the enzyme and (VII) indicated about 25 per cent higher basicity, (2'4. :GP(O) (OR), + HOHeH-G+ P(0) (OR)?+ OHi. e., electron localization, for (VII). The dissociation constant for AChE and (VI) was 2.3 X 10W8,for AChE and (VII) 1.6 X Thus. (VII) is the more effective inhibitor of the two. since the nitrogen of the piperidino compound (VII) takes up less space than that The hydrogen ion needed to restore the H-G+ of the pyrrolidino derivative (VI) it may be conrluded function must come from the solvent since methylthat the esteratic site is a cavitation of the enzyme pyridinium ions cannot supply it. I n practice, acetylsurface which can accommodate the narrower piperidin0 nitrogen better than the wider pyrrolidino nitro- cholinesterase is reactivated effectively after being gen. However, the additionaimass and shape of the severely inhibited by dialkyl fluorophosphates. The vast difference in rate of reactivation between samples piperidine ring may also influence the reaction. Comparisons between the quaternary compound (VI) inhibited by the reagents where R is ethyl versus isoand the ditertiary amine (VIII) (and by the same propyl has been attributed to steric hindrance exerted token between (VII) and (IX)) showed that a quater- by the isopropyl groups; they shield the near-by anionic nary nitrogen is not necessary to face the anionic site site from the approaching reactivator. Although the interaction of the detoxifying enzyme, since (VIII) and (IX) are still much more potent inhibitors of AChE than prostigmine, although from AChE, and its substrates and inhibitors has thus been two to four times weaker than (VI) and (VII), respec- clarified in part, nothing is known about the reaction tively. The simpler compound (X) is still about twice mechanism of acetylcholine with the other three proa s effective as prostigmine, even though its activity has teins with ~vhichit can react directly. They are the dropped to 3.5 times below (VI), and 5.2 times below enzyme choline acetylase a t which it may be formed (VII). Even P-trimethylammoniumethyl chloride is from choline and 8-acetyl coenzyme-A, the storage prostill 25 per cent more potent than prostigmine although tein where acetylcholine is kept in a hound inactive state, and the all-important receptor protein, presumless inhibitory than physostigmine. Further evidence for the presence and nature of the ably the same at nerve endings and synapses in many two reactive sites in AChE mas deduced by studying locations in the body. According to Nachmansohn the reactivation of the highly inhibited enzyme with (21) acetylcholine is involved in altering the permeahydroxylamine derivatives (17, 18). The esteratic bility of the membrane of nerve cells for and during site (H-G:) was inhibited with dialkyl fluorophos- conduction of an electrical impulse which in turn is phates, (RO),P(O)F. The dialkyl phosphoryl group re- caused by a sudden influx of sodium ions into the cell places the acidic hydrogen of the receptor, attarhing it- from its environment, and an outflow of potassium ions in the opposite direction. It has been post,ulated that self to the basic position, :G. the hormone unfolds the proteinogenons receptor, so H-G: (RO)2P(0)F-:GP(O) (OR)z HF that carboxyl groups face each other in the matrix and The dialkyl phosphoryl group (RO),P(O) should be alkali ions can pass by rapidly. Although this is only removable from the enzyme site, G, by means of a bi- one of several hypothetical explanations, it undermolecular nucleophilic displacement reaction, and the scores the possibility that a drug may react with several enzyme could thus be reactivated. This can be ac- proteins or enzymes by diverse mechanisms due to difcomplished by such nucleophilic reagents as hydroxyl- ferent shapes of their reactive surface^. Few other enzymes whose coenzymes are not known amine if the alkyls are methyl or ethyl, but for the
+
+
368
have been analyzed by the method of substrate specificity. A typical experiment in this direction is a study of eight animal and plant phosphatases which did not hydrolyze nine simple trialkyl and triaryl phosphates. Dialkyl phosphates could be hydrolyzed slowly by these enzymes, monalkyl phosphates more rapidly. To explain this behavior, one can postulate the need of a bond between acidic groups of the substrate ester and the enzyme for interaction. If the alkyl or aryl groups of a triester contain polar groups (NO*, choline), enzymat,ic cleavage becomes possible (22). Steric fitting of drugs to receptor sites a t which coenzymes act is more readily accomplished by imitating the structure of the coenzyme, or arranging some of its functional features suitably spaced in the new molecule. The drug may act like the coenzyme, or as its structurally inhibitory antagonist. These activities may overlap in the same cell systcm under different conditions, or may depend on the species. The same reasoning may be applied to all enzymatic substrates and all the primary and intermediary products of amino acid, carbohydrate and fat metabolism, vitamins, and hormones. I t applies also to all artificial replicas of known drugs by whatever route they have been discovered. As an example for this postulate, a readily appreciated antagonism of cytotoxic agents may be given. Alloxan (XIII) is a specific toxic drug for the insulinogenic Islets of Langerhans, and has been used to induce experimental diabetes in laboratory animals. Pretreatment with barbituric acid (XIV) protects animals from the diabetogenic effects of alloxan (83). I t should be noted that no structural analogue of these compounds is known which is needed for the normal metabolic function of the islet tissue.
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more tuberculostatic in the presence of cupric ions. A different situation exists for 8-hydroxyquinoxaline; this compound is not tuberculostatic but becomes highly so on addition of 5 X lo-= molar cupric ion solutions. However, when 8-hydroxyquinoxaline is added to an isoniazid-resistant rulture, the activity of isoniazid is restored. The explanation of this phenomenon may be that both isoniazid and 8-hydroxyquinoxaline are needed to displace metabolites from the coordinationsphere of metal ions of some enzyme system essential for the tubercle bacilli. Another recent example of the effect of metallic ions roncerns the ability of mitochondria to catalyze the oxidation of metabolites of the carbohydrate utilization cycles. They participate in these oxidation processes only as long as they retain their compact structure but lose this ability when they swell. The swelling can be inhibited by ATP, probably by complexing disturbing calcium ions. Ethylenediaminetetraacetic acid and other chelating agents also inhibit the swelling of liver cell mitochondria and enhance their oxygen consumption (25). A nontoxic chelating agent can replace an ATP function in this case. One may even be tempted to forecast the key enzymes under attack by certain drugs, if activation of the drng by metallic ions has become known. The natural tetracycline antibiotics have a pronounced affinity for various metallic ions but their activity against bacterial enzymes is suppressed most markedly by magnesium and/oreiron ions. These cations are most often essential for many microbial metal-requiring enzymes. One can conclude that those ions suppressing antibiotic activity must be of greatest importance to the bacterial enzymes affected (86). 0 0 Metallic ions may affect the metabolism of acids, salts, and other metallic ions. Ascorbic acid autoxidizes much faster than usual in the preaence of cupric ions; here the mechanism of antoxidation is altered because in the presence of copper the mono-anion is ratedetermining while this species plays a minor role if cupric ions are absent (11). Plasma copper, which occurs to The function of metallic ions in bonding a compound the greatest part in or-globulin, has been shown to moto an enzyme surface may explain the observation that bilize iron from tissues, causing an increase in plasma compounds which are capable of chelating metals are iron. Copper deficiency actually impairs utilization usually more biologically active in their presence, or of iron in the biosynthesis of hemine (87). The most even become active only if metallic ions are available. important effect of metallic ions, however, and probErlenmeyer (24) believes that these compounds enter ably of nonmetallic and even organic ions capable of into the coordination sphere of the metal ions of enzymes chelation, is their shapedetermining function for and there occupy those locations at which normally polypeptides and proteins. The twisting and folding some similar metabolite molecule is admitted. The polypeptide chains are stabilized in definite spatial similarity of substrate and antagonist may thus be a matrixes which stamp them as enzymatic and antigenic coordination similarity. entities. The recognized need of many biologically Some interesting observations may amplify this rela- specific enzymes for metallic ions supports this view tionship. Certain strains of tubercle bacilli which are (9,$8). originally sensitive to inhibition by isoniazid may develop resistance to this drug after exposure for from 16 BIOISOSTERISM to 21 days. Addition of cupric ions restores the original In restricted series of closely related compounds, sensitivity to a large extent although cupric ions alone careful strnctural manipulation often emphasizes one are not bacteriostatic. Apparently, isoniazid becomes biological property at the expense of side actions or
VOLUME 33, NO. 8, AUGUST, 1956
vice versa. This can be done by slight alterations of the carbon skeleton, changes in functional groups, masking or unmasking of polar groups, steric alterations, etc. Studies in this field, with emphasis on spatial relations, have been headed bioisosterism. The term isosterism had been coined originally to express the similarity of physical properties of simple molecules such as carbon monoxide and nitrogen. It \\-asthen expanded to larger molecules whose peripheral lavers of electrons can be considered identical (29). ~bmpoundsdiffering only by one characteristic chosen on the basis of the so-called hydride displacement rule have been compared most often. Grimm had shown that the combination of an element in the first period of t,he periodic system with one atom of hydrogen leads to a group similar to the element one position to the left in over-all behavior ("pseudoatoms"). Thus, CH is equivalent to X, CHPto 0 or NH, etc. If such groups appear in otherwise structurally similar molecules, the respective compounds often exhibit similar biological properties. Indeed, many of their physical properties are similar, tsoo, except for solvolytic and dipole characteristics. A further extension of this concept was achieved by comparison of aromat,ic systems such as benzene and thiophene, or pyridine and thiazole in which one sulfur at,om takes the place of a resonating -CH=CHgroup. The parent systems themselves, and many comparable derivatives in these series, show a striking similarity of many important physical properties. They also overlap considerably in their effects on the same enzyme systems. Thus thiazole-5-carboxylic acid exhibits niacin-like behavior, and the thiophene nucleus may replace benzene in many compounds without radical change in biological properties. An example for the application of these ideas may be seen in the field of antihistaminic and antispasmodic drugs, for which bhe general formula:
369
ture is considered important, the field of isosterism is widened considerably. For example, ethyl l-methyl-4phenylpiperidine-4-carboxylate (XV) is very similar as an analgesic and antispasmodic to its isomer, l-methyl4-phenyl-4-piperidinol propionate (XVI) although the
ester functions have been exchanged, and hydrolysis yields entirely different products (50). Similarly, crotonohetaine, (CH3j3N+CH2CH=CHCOzCH:,, possesses cholinergic activity of the same order as acetylcholine although the cationic head is located in the acid moiety (31), and replacement of the acetate ester group in the anticholinergic diethylaminoethyl diphenylacetate methiodide (XVII) by a phosphinate ester in diethylaminoethyl diphenylphosphinate methiodide (XVIII) yields a compound ~ ~ - i typical th anticholinergic properties (52). (C~H~)2CHC0,CHsCH~N+(CH,)(C,H,),.I(XVII) (C~H,),POaCHsCHaN+(CHa)(C2HH)x.I(XVIII) Position isomers, compounds containing different halogens or other groups of equivalent character, provide additional examples of bioisosteres. The high thyromimetic potency of 3,3'-diiodo-5-hromothyronine (XIX) which compares favorably with that of 3,3',5triiodothyronine (XX) may be mentioned here (53).
NHI (XIX): R = Br (XX): R = I
Bases with different heterocyclic amino groups (VI-IX) have been compared above. Such compounds must have similar molecular weights, over-all electrical fields, and above all reasonably similar molecular shapes. One cannot expect that two substances differing even has been derived. Here A and B usually stand for by minor structural alterations only will exert exactly cyclic systems such as benzene, thiophene, furan, thi- the same biological activities on critical examination. -N-, or -4-; However, if one can achieve a gradual change of hiazole, pyridine, etc.; Y is =CH-, and Z is an aliphatic or simple cyclic tertiary amino ological behavior and follow it accurately a t each step group (often -N(CH&) or quaternary ammonium of minor structural alteration, one is hound to enhance function. Such type formulas have been constructed one property, suppress another, and ultimately arrive for central nervous system depressants (sedatives, a t a drug suitable for therapy. Shortcuts t o this dishypnotics, anticonvulsants, ataractics), for cholinergic concertingly tedious process have not been found, and and anticholinergic drugs, for adrenergic and sympath- this is probably responsible for the still prevailing opinion that new useful drugs will be discovered most olytic agents, and for various other drugs. Type formulas emphasize primarily the importance easily by more or less empirical procedures. An interesting observation about the effect of one of similarity of molecular size and shape as a guide in recognizing potentially similar or antagonistic biological ether oxygen atom (54) has been made by Friedman. action in related structures. They also usually take The table gives a few examples which show that omisinto account similar locations of high and low electron sion of an ether oxygen changes biological activity much densities expressed by suitable functional groups (ester, less than replacement by an isosteric methylene group. halide, amide, etc.). If this aspect of molecular struc- This anomalous behavior is not readily explained. In
JOURNAL OF CHEMICAL EDUCATION
usually been discovered only after the over-all action of the drug has been recognized and its importance warranted a detailed biochemical inhibition analysis. I n only few cases has planning on biochemical grounds terminated in the successful choice of a useful, selectively toxic drug without considerable additional structural variation. Although many isotopically labeled metabolites have been prepared for study of cellular metabolism, the essential requirements of most cell species and especially their minimum biosynthetic ability remains obscure. The role of pyridoxal antagonists (@) offers an instructive example of the difficulties confronting biochemical planning of drug stucture. Pyridoxal-5-phosphoric acid is the coenzyme of a t least five aniino acid decarboxylases and transaminases, and is associated with certain reactions of purines, the cleavage of thioethers, and several other reactions ( 4 , 4 5 ) . The simple structure of the vitamin has made possible wide structural variation with the hope of attaining a pyridoxal OUTLOOK antagonist which, a t least in the microbiological field, In designing a drug structure, the application of the might he chemotherapeutically interesting. Among observations on biological antagonism offer the best the many analogous compounds tested, some (XXI and hope of rational success. If a proposed structure is XXII) compete with pyridoxal for ATP in the formaanalogous to that of an essential metabolite, one expects tion of pyridoxal-5-phosphoric acid, while others that the relative specificity of the agent mill provide (XXIII and XXIV) compete with pyridoxald-phosspecificity of action, that is, low toxicity. If the drug phoric acid for a catalytic site a t the apoenzyme. The were used in toxic concentrations, the metabolite mould best-studied antagonist, deoxypyridoxine (XXII), probe available immediately as a specific antidote. De- duces typical vitamin Bsdeficiency, and in biochemical tails of designing an antimetabolite are still obscured by experiments undoubtedly antagonizes the vitamin. unknown variations in the accessibility of receptor sites, Interference by and the possibility exists that receptors for the same (XXI), ( S X I I ) substrate may vary slightly in different anatomical loca- HOCH,,/OH CHO tions. Finally, a polyfunctional antimetabolite could affect different enzyme systems. Slight stereochemical f N Interference bv pH i Enzyme or structural changes may alter considerably the biO=POCHq, OH (XXIII), ( X X N ) ological role of a compound. Patient variation of at least a reasonable number of structures is still the only answer to this question. A drug may also interfere with biosynthetic steps leading to a substrate, either by inhibiting an enzyme unspecifically or by competing for i t with a synthetic intermediate. The point and mode of interference has the case of methonium ions (examples 3-5 in the table) where the distance for a linear molecule at maximum extension appears to determine the extent of depolarizing activity in nervous structures, the activity of the ether structures implies that they are not maximally extended. It may he that the unshared electrons on the oxygen atoms of these compounds exert an attraction on the cationic centers two carbon atoms away. Example 8 illustrates the experience that morpholino derivatives are generally less active than the corresponding piperidino or diethylamino compounds. Similar biological activities in the same range of potency are not infrequently found in ketones (RCOR') and the corresponding esters (RC02R1), while the homologous ketones (RCOCH2R1)differ quantitatively in pharmacological behavior. Attempts to test this effect of omitting oxygen from phosphonic acid analogues of metabolically active carbohydrate phosphoric acids have remained inconclusive (35).
fNy ,
Comparison of Ether end Nonether Structures (31) Comnound (reference)
No.
Test Phenol coefficient versus Slaph. aweus EDin me./ke. for musc&r trpirdysis Ganglionic blockade Curaremimetie activity Curaremimetic PDw.
" 4 6 2-Imidazolyl-CHXCrHs (41) 7 CrHsXCH&H%NHt (4% 8 RCH&H*N~X
X = O
X
=
CH,
X absent
..
Effect on blood pressure
Effect on blood pres8ure Aotihia%aminio, antispasmodic activity
0.3 Pressor Depressor L o x activity
0.01 Weak depressor Weak depressor Higher activity
0.25 Strong depressor Strong depressor Higher activity
VOLUME 33, NO. 8, AUGUST. 1956
371
disorders resembling those seen in schizophrenia has tempted some investigators to postulate that complex indole derivatives, perhaps a faulty metabolite of 5-hydroxytryptamine or of adrenochrome, might be the natural etiological agents of the disease (48). While this But if animals receive a vitamin Ba deficient diet plus idea still lacks experimental basis, it introduces the deoxypyridoxine, a confusing picture develops: the thought that naturally occurring antimetabolites may tissues are not depleted of vitamin Be in the early stages cause physiological disorders. This is really only a new of the experiment, and the activity of pyridoxal-5- version of the long-known observation that toxic prodphosphoric acid-catalyzed enzymes remains essentially ucts arising or remaining in the body by metabolic unaltered. Obviously, some reaction not yet recognized aberrations cause symptoms associated with various disorders. A chemically plausible example in the field deeply affects the relation of vitamin and antagonist. As the biochemist will reveal additional mechanisms of plant diseases has been given for the extracellular of syntheses in cells and tissues, both in uitro and in the toxin of the pathogenic bacterium, Pseudomonas taban', untold more complex and interlacing in vivo environ- which causes wildfire disease of tobacco and certain ment, more intelligent guesses concerning specific other crops (49). The toxin ( X X V ) antagonizes antagonistic structures will become possible. Modest methionine, although not in tobacco, but in the unicelinitial successes of hona fide planning of reasonably lular algae, Chlorella vulgaris. The toxic effects of a useful drugs on the basis of information about metabo- synthetic derivative of methionine, methionine sullites hare been scored with antagonists of pteroylglu- foximine (XXVI) can also he overcome by methionine, tamic acid, i. e., Aminopterin and Methotrexate which and (XXVI) in turn can produce the typical lesions of have been used to prolong life in lenkemia patients (46). wildfire disease on tobacco leaves. The sparsity of such practical results does not mean that planning based on metabolite antagonism has not been highly informative. Inhibition of virtually every enzyme system involved and production of many experimental metabolite deficiencies has been realized with structurally inhibitory analognes of diverse meFor years, vitamins, essential fatty acids, and indistabolites. Finally, when an experimental drug has been pensable amino acids have been added to the diet or recognized as an antagonist of a given metabolite by biochemical experiments, a few and often slight struc- administered therapeutically to abolish or prevent defitural modifications have led to t,herapeutically useful ciency symptoms associated with the metabolic lack of agents. This has been the case for the antileukemia these compounds. It appears now that, a t least in drug, 6-mereaptopurine, which inhibits incorporation of some cases, the mechanism of action of these substances normal metabolic purines into nucleot,ides (47), and may be the cancellation of toxic effects of natural anthus retards malignancieb. The tuherculostatic p- tagonists to which some symptoms of disease can be aininosalicylic acid was developed as an antagonist to attributed. Since metabolites balance each other's salicylic and p-aminobenzoic acids which stimulate cer- action in a natural chemical trend toward homeostasis, tain essential fnnctions of acid-fast bacilli. Many use- checking a pathogenic chemical within the body should ful ant,icholinergic drugs have been designed to rontain be most effect,ive by a combination of essential metabocholine-like moieties as well as groups which by their lites. One of the tasks of medicinal chemistry is therebulk wonld hinder the approach of acetylcholine to the fore to round out the knowledge of such essential suhstances and to make them available, singly and in comslightly blocked receptor site. In some cases the contorted polycyclic or polyfunc- binat,ion, for therapeutic use. tional stnieture of a chance drug presents almost iuLITERATURE CITED superable obstacles to rationally interpreting it as a (1) DAVIES, M., Chemistry & Industry, 1953,614. metabolite antagonist. This is the case for the morH., AXD T.F. DOUGHERTY, Am. Scienlut, 43, 457 phine alkaloids for which antagonistic metaholites are (2) EYEING, ,. not yet known although cholinergic and adrenergic J. R., AND B. B. BRODIE, J. Pharmwl. Ezp. Therap., "moieties" can be scanned in their molecules. The 115,68(1955). (4) JAN& G. J., Quart. Revs. (Londm),9.229 (1955). stnictures of the tranquilizing drug, reserpine, and of ~. (5j BURGER, A.,k ~amaco,10,47 (i955). the halucinant, lysergic acid diethylamide, contain J. B.. AND P. K. STUMPF. "Outlines of Enzvme ~, NEILANDS. indolylethylamine fragments, and these compounds 16) chemistry," John Wiley & Sons, inc., New York, ld55. have been shown to influence the metabolic pathways (7) ARI~NS, E. J., Arch. i n l m . phammdynamie, 99,32 (1954). of the t.issue constituent, 5-hydroxytryptarnine. HowARIENS AND A. M. SIMONIS, Arch. inlern. phamacodynamie, 99,175(1954). ARIENSAND W.M. DE GROOT, ever, the complexity of their structures would not have Arch. intern. phamacodynamie, 99,193(1954). encouraged anyone to predict t,his relationship if the (8) MICHAELIS. . L... A N D M. L. MENTEN,Biochpm. 2.. . 49.. 333 . . action of these drugs on the central nervous system had (1913). not already been known. (9) MARTIX, G. J., "Riological Antagonism," The Blakiston The abi1it.y of lysergic acid diethylamide to produce Co., Philadelphia, 1951 ; J. CHEW.E ~ n c .33, , 204 (1956).
372
JOURNAL OF CHEMICAL EDUCATION
(10) TAUBER.H., ASD I. S. KLEINER,J. Gen. Physiol., 15, 155 (1931). (11) ALBERT,A,, Pharmacol. Revs., 4, 136 (1952). (12) SMITH,H., Am. J . Physiol., 72,347 (1925). (13) CLOWES,G. H., A. K. KELTCH,AND M. E. KMHL, J . Pharmacol. Ezp. The~ap.,68,312(1940). (14) BRODY,T . XI., Pharmacol. Revs., 7, 335 (1955). (15) NACHMANSOHN, D., a m I. B. WILSON,Advances in Enzymol., 12,253 (1951). J. Am. C h a . Sac., (16) FRIESS, S. L.,a m W. J. MCCARVILLE, 76, 13G3, 2260 (1954). (17) WILSON,I. B.,S. GINSBURG, AND E. K. MEISLICH,J. Am. Chern. Soe., 77,4286 (1955). F.. Brit. J. Phamacol.. 10.356 (19551. (18) HOBBIGER. (19) WILSON,I: B.', AND S. GINSBURG,'A&.Biochem: Biophys., 54, 560 (1955). N GIZSBURG,Riochim. et Biophys. Ada, 18, 168 (20) W I L ~ O ASD (195.5) - -- ,. (21) P~ACHMANSOHS, D.,"Principles fo~.testing drugs during grou,th," in "Biochemistry of the Developing Nervous Academic Press, Ine., System," ed ted by H. WAEL~CH, New Y o ~ k ,1955. E..AYD J. RIEBL. Chem. Ber.. 88. 1556 (19551. (221 BAXMAN. , MARTINEZ, c., Am. J. ~h&iol.,182,26'7 (1955). ERLENMEI-EE,H., A N D H. REY-BELLET,Helv. Chim. Ada, 37, 234 (1954). RAAFLAUB, J., Helv. Chim. Ada, 38, 27 (1955). WEINBERG,E. D., J. Pharmacol. Ezp. Thwap., 115, 61 (1955). WINTROBE, >I. M., G. E. CARTWRIGHT, AND C. J. GUBLEN, J . hrutrition. 50. 395 (19531.
(30) FRIEDMAN, H.L.,"Influence of isosteric replacement upon biological activity," Symposium on Chemical-Biological Correlation, National Research Council Publication 206, Washington, D. C., 1950, p. 295. (31) BERGEL,F., Chemistq & Industru, 1951,928. J. Am. Chem. Soe., 76, 5891 (32) SMITH,B. E.,AND A. BCT~GER, (1953). (33) GEMMILL,C. L., J. J. ANDERSON, A N D A. BURGER, J . Am. Chem. Soe., 78,in press (1956). (34) FRIEDMAN, H. L., "Some bio-isoateric anomalies," paper presented a t the 126th Meeting of the bmerican Chemical Society, New York, 1954. (35) GRIFFIN, B. S., AKD A. BURGER,J. Am. Chem. Soc., 78, 2336 (1956>. , . ALBERT, A., "Selective Toxicity," John Wi1e.v & Sons, New l'ark, 1951,p. 52. BERGER,F. M., J . Pharmaeol. Ezp. Thcrap., 93,470(1948). KUNKEL,A. M., A. H. OIKEMUS, AND J. H. WILLS, Federation Pme., 11,365 (1952). FTJSCO,R.. 8. ~ ~ I A V A R E LG. L I PALAZZO. , A N D D. BOVET. G~z;. ehim. ital., 78,951 (1948). THESLEFF,S., AND K. R. UBSA, .I. Phannacol. E r p . Therap., 111. 99 (19541. ~ ~ ~ , ~ SCHOLZ, C. R., Ind. Eny-. Chem., 37,120(1945). LANDS,A. M., National Rcsearrh Council Publication 206, Washington, D. C., 1950, p. 73. UMBREIT,W. W., Am. 3.Clzn. Nutrition, 3, 291 (1955). SEBRELL,W. H., JR.,AND R. 8. HARRIS,"The vitamins," Academic Press, Inc., New Yok, 1954, p. 220. SNELL.E. E.. Phusiol. Revs.. 33. 509 11953). BURCHENAL. J. H:. Am. .I. ~ l i n . ' i V x t & m 3. 311 11!l55\. HITCHINGS, G.H . , ' A ~ J. . Clin. ~ u t r i l i o n :3; 321 (1955). , . (29) For revie< see B ~ G E R A,, , " ~ k d i c i ~ a l ~ h e r n i s t r y , " VI, al. LEA, A. J., J. Mental Sci., 101,538 (1955). Intersrienre Publishers, Inr., New York, 1951, Chap. IV. YOLLEY, D. D., Am. J . Clin. Nutrition, 3,303 (1935). \
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