R E S E A R C H

Drug Gives Insight into Mental Illness Studies with Lakeside's Ditran implicate acetylcholine or like chemical in lower brain activity

138TH ACS NATIONAL M E E T I N G

Medicinal Chemistry

A new, dual-action drug is giving med­ical science new insight, into mental illness. The drug: N-ethyl-3-piperidyl phenylcyclopentyl glycolate, brand-

maker. Clinically, Ditran has been used successfully as an antidepressant. The drug is not yet available commer­cially.

In early phases of its action, Ditran induces a temporary psychosis that can't be distinguished from the real thing. This means that, for the first time, symptoms resembling psychoses in man can be induced predictably in test animals. Chemicals can thus be rapidly screened for antipsychotic ac-named Ditran by Lakeside Labs, the

Acetylcholine May Be Involved in Brain Functions

tivity, say Dr. John H. Biel and co­workers at Lakeside. (Dr. Biel spoke at a symposium honoring Dr. Frederick F. Blicke, University of Michigan.)

Dr. Biel points out that no real progress was made in the treatment of epilepsy until chemicals were devel­oped to simulate the disease in ani­mals. This permitted large scale screening of compounds for anticon­vulsant activity.

Studies with Ditran and other piper-

Normal (in balance)

Chloroprom&zvrie type

chemicals block norepinephrine

Tranquilizers

Cause depressant center to dominate

Cause excitatory center to dominate

Monoamine oxidase inhibitors block metabolic destruction of norepinephrine

Psychic energizers

' Cause excitatory center to dominate

Ditran, some other anticholinergics may block acetylcholine

Antidepressants

50 C&EN SEPT. 19, I960

Lower brain Lower brain

idyl glycolate esters indicate that po­tent anticholinergic action is a pre­requisite for good central nervous sys­tem stimulation. This finding, Dr. Biel says, may implicate acetylcholine or a similar substance as one of the neu­rohormones acting to control some functions of the lower brain.

The body's sympathetic nervous sys­tem controls those functions having to do with physical activity involving skeletal muscles, action, and aggres­sion. Controlling this system is the neurohormone norepinephrine. The body's parasympathetic nervous sys­tem, controlled by acetylcholine, gov­erns the digestive processes, salivation, rest, and sleep. The two systems normally are in delicate balance.

Use of tranquilizers and psychic en-ergizers for treating mental illnesses has spurred research into how they act and how the brain functions. In­

vestigators in this area keep bumping into the same chemicals which are vitally concerned with the body's peripheral nervous system. This sug­gests that the brain, and especially the lower brain, is concerned with emotion and behavior; and it may be subject to controls similar to those for the rest of the body, instead of functioning in­dependently.

Dr. Biel points out that many of today's drugs used against mental ill­nesses started out as compounds aimed at vital chemicals of .the peripheral nervous system. Examples: antihista­mines, hypotensives, antiasthmatics, and the like.

Normal Balance. Apparently, the sympathetic (excitatory) center of the lower brain and the parasympathetic (depressant) are normally in balance. Upsetting the action of one permits the other to dominate.

Norepinephrine has been pinned down as the chemical which controls the sympathetic functions of the lower brain. Chemicals which block the ac­tion of norepinephrine—chloroproma-zine and other chemically related com­pounds—act as tranquilizers since they permit the depressant center to dom­inate. Monoamine oxidase inhibitors, which block metabolic destruction of norepinephrine, act as psychic ener-gizers (antidepressants), since they permit the excitatory material to ac­cumulate and tip the balance toward the sympathetic system.

But the material controlling the parasympathetic system of the lower brain hasn't been pinned down yet. Some investigators feel that serotonin is involved. But serotonin inhibitors don't produce the proper response (ex­citation) in tests. However, some an­ticholinergics—acetylcholine antago-

ath to Ditran Involves Unusual Chemistry

o or t

N-ethyl-S-chloropiperidinc Phenylcyclopentyl glycolic acid

N-ethyl-3-piper idyl phenylcyclopentyl glycolate (30%)

\ N-ethyl-2-pyrrolidylmethyl

\ phenylcyclopentyl glycolate (70%)

Ring contraction

under acid conditions

N-ethyl-3-piperidyl phenylcyclopentyl glycolate

\ Thermal ring expansion

S E P T . 19, I 9 6 0 C & E N 51

New Drug Licks Tough Fungus Infections Oxine ester's salicylate cures fungus infections of skin in 24 out of 25 cases

nists, for example,—show antidepres­sant activity.

Unusual Chemistry. Several sub­stituted glycolate esters of N-alkyl-3-hydroxypiperidines were found by Dr. Leo G. Abood of the University of Il­linois Medical School to be potent an­tidepressants. Dr. Biel and his group at Lakeside, pursuing this lead, syn­thesized and tested many such com­pounds. From this work came the finding that a compound must be a strong anticholinergic to stimulate the central nervous system. But not all an­ticholinergics are central stimulants, Dr. Biel says.

Synthesis of the top performer, N-e±hyl-3-piperidyl phenylcyclopentyl, glycolate, involves some unusual chem­istry. Reaction of 3-piperidyl halide with phenylcyclopentyl glycolic acid results in a ring contraction to 2-pyrrc-lidylmethyl ester. Such a contraction in esters of this type has been seen un­der alkaline conditions, but never under acid conditions, Dr. Bie! notes. Another surprise: Distillation of the 2-pyrrolidyl methyl glycolate results in a thermal ring expansion to the de­sired 3-piperidyl glycolate.

Ditran, when first administered, in­duces a psychotic state in the patient which lasts eight to 12 hours; the condition is indistinguishable from a genuine psychosis. Upon recovering, the patient is stimulated and his mood elevated for an extended length of time—in some cases for months, even a year. Clinical tests are still going on to learn how long the antidepressant action lasts.

Within five minutes after adminis­tration of 9-amino-l,2,3,4-tetrahydro-acridine (THA), a material which pre­vents metabolic destruction of acetyl­choline, all the psychotic and periph­eral symptoms induced by Ditran (a potent anticholinergic) are reversed. The implication of acetylcholine in this case is "more than circumstantial," Dr. Biel says. This finding adds to the growing pile of evidence that acetyl­choline or some similar material is act­ing as the counterpart to norepineph­rine in the lower brain.

Some other recent evidence: A structural modification of chloropro-mazine that shows increased anticho­linergic ability produces some types of excitation and psychotic activity. Thus, chemicals such as Ditran, which are capable of psychotominetic activ­ity, loom as important tools for de­veloping antidepressant and antipsy­chotic drugs, Dr. Biel says.

138TH ACS NATIONAL MEETING

Medicinal Chemistry

A series of new antifungal agents shows high activity against many fungi associated with stubborn skin infec­tions Top performer: salicylic acid salt of 8-quinolinyl benzoate, a chemi­cal modification of 8-hydroxy-quino-line (oxine).

Animal tests show the new com­pound to be less toxic than oxine, say Dr. Nathaniel Grier and Joseph A. Ramp of Metalsalts Corp. Skin tests (done elsewhere) on 200 human sub­jects show the material isn't irritating or sensitizing. And the new drug shows promise in preliminary clinical trials. It clears various fungus infec­tions in 24 out of 25 patients. (Dr. Grier spoke at a symposium honoring Dr. Frederick F. Blicke, University of Michigan.)

Fungus infections of skin are caused by dermatophytes. Various fungi are thought to cause "athlete's foot." Fungi are also responsible for "bar­ber's itch" and ringworm.

Treating fungus is a hard, slow .process. Until recently, treatment consisted of topical antifungal agents. Among the *nost popular is 8-hydroxy-quinoline and its derivatives. But ox­ine tends to be toxic and irritating, sometimes sensitizes the patient. Much effort has gone into chemically modifying oxine to minimize these shortcomings, and to broaden its activity.

From this and other work on anti­fungals have come general findings that a good antifungal agent should be lipophilic to penetrate the cell walls of fungi. And it should carry an electrical charge to inhibit or de­stroy intracellular enzymes of fungi.

Reaction Highly Selective. To gain these properties, Dr. Grier and his group attempted to synthesize car-boxylic acid salts of oxine's (and its derivatives') carboxylic acid esters. But no isolable salts are formed by

reacting the simplest aryl ester, 8-quinolinyl benzoate, with many or­ganic carboxylic acids. Nonesterified oxine, though, readily yields salts with the same acids.

Reason for this selectivity, Dr. Grier says, may be that salicylic acid and the ester form two types of properly oriented linkages. One is the result of an acid-base reaction, and the other forms a hydrogen bond between the two molecules.

Dr. Grier notes that salicylic acid itself forms a chelate ring because of the proximity of donor and acceptor groups, a property that's reinforced in the salt that forms substituted salicylic acid. This promotes salt formation with the ester, compared to simple association of salicylic acids by inter-molecular hydrogen bonding.

But met a- and pam-hydroxybenzoic acids, being unable to form chelate rings, undergo molecular association preferentially. Thus, they fail to sup­ply the additional hydrogen bond needed to form a stable compound with 8-quinolinyl benzoate. In the case of hydroxy-substituted salicylic acids, presence of a second competi­tive hydrogen bond forming g r o u p -one that's unfavorably located with re­spect to the benzene ring's carboxylic acid radical—causes selective interfer­ence, Dr. Grier says.

High Activity, Low Toxicity. The new compounds show relatively high solubility in ether (thus the desired lipoid-like properties), ionize in water, and have excellent antifungal action, Dr. Grier says. Although benzoic and salicylic acids are weak antifungals, their use in chemically modifying ox­ine to yield the salicylic acid salt of 8-quinolinyl benzoate results in a very active compound with low toxicity (LD5o in rats is 4.4 grams per kilo­gram of body weight).

The high selectivity of 8-quinolinyl benzoate for salicylic acid may make it a useful model for studying the acid's unique antirheumatic properties. These aren't found in its isomers, or in hydroxysalicylic acids, according to Dr. Grier.

52 C & E N S E P T . 19, 1960

Large Inverse Isotope Effect Found Deuterium-hydrogen exchange gives new tool for research on reactions of weak acids

138TH ACS NATIONAL M E E T I N G

Organic Chemistry

One of the largest inverse secondary deuterium isotope effects observed to date is claimed by University of Cin­cinnati chemists. They find that sub­stituting deuterium for hydrogen in lithium borohydride methanolysis (in diglyme) increases the borohydride's reactivity about 1.6 times (kp /kn) . Usually, replacing an atom with one of its isotopes lowers reactivity.

The reaction, Dr. Raymond E. Dessy says, will help to study inverse secondary isotope effects more com­pletely. Hoped-for end result of this research: a better understanding of the hydrogen-yielding reaction be­tween a proton and a hydride ion. Understanding the mechanism of the proton plus hydride reaction should, in turn, help in profiling the properties of weak acids (compounds such as phe­nols, alcohols, and some hydrocar­bons), Dr. Dessy and his co-workers Edward Grannen and Yuzi Okazumi suggest.

An inverse secondary isotope effect was recently observed by others, using another system (solvolysis of a-sub-stituted methyl halides), Dr. Dessy says. But ICD/ICH found in that work is only 1.1. The Cincinnati group claims that its ratio is too large to account for by theories such as bond stiffening in the transition state.

One of Many Reactions. Hydroly­sis of lithium borohydride and its deuterated counterpart is only one of many reactions which tie in with his research on weak acids, explains Dr. Dessy. He defines weak acids as those compounds which fall somewhere be­tween the classical strong acids and neutral organic compounds; those having a dissociation constant (Ka) in the 1 0 1 0 to 10"30 range. The study is partly sponsored by the Air Force, the Petroleum Research Fund admin­istered by the American Chemical So­ciety, and Diamond Alkali.

Knowledge of the physical proper­ties of strong and moderately strong acids is extensive, he notes. This is true both experimentally and theoreti­cally. But understanding of very weak acids is incomplete, he adds.

The Cincinnati group uses two steps in its approach to the study of proper­ties of weak acids. First, they meas­ure how fast an acid can give up its proton to a base. Then, they deter­mine how variations in structures affect this donating ability. Reaction rates studied include those for phenols and aliphatic alcohols with borohydrides, and the rate at which a ccfmpound will exchange its acidic hydrogen for one in solution. Ultimately, the research should allow a quantitative measure of the effects of functionality on acidity of weak acids, Dr. Dessy believes.

The first reaction worked out is one between sodium borohydride and phenol in diglyme. After untangling a "kinetic jungle," Mr. Grannen found

that only one of borohydride's hydro­gens is removed under these condi­tions. This gives a much cleaner sys­tem to study, Dr. Dessy notes, than does the more commonly used aqueous system. Water knocks out &11 four hydrogens, with the last three coming off at rates too fast to measure.

Measuring rates of the reaction in diglyme, Dr. Dessy's team finds that the reaction doesn't obey second order kinetics. Instead, it's a third order re­action over-all; first order with respect to borohydride, and second order for phenol. Adding phenoxide ion to the reaction mixture reduces the observed rate, the trio says.

Apparent mechanism for the whole reaction shapes up like this:

• Two moles of phenol are in equi­librium with protonated phenol (CGH5-OEL+) and phenoxide ion.

• Protonated phenol then reacts with sodium borohydride. Products are hydrogen and a complex of phenol with a sodium borohydride-derived positive ion (NaBH ; }+).

• Phenoxide ion reacts rapidly wi-th the NaBH:i +-phenol complex to form sodium phenoxyborohydride and phenol.

When deuterium is substituted for phenol's active hydrogen, says Dr. Dessy, k n / k D is 4, indicating that the slow step (protonated phenol plus borohydride) involves a hydrogen transfer. This result, he notes, is a normal isotope effect.

Applying this mechanism to various substituted phenols, Dr. Dessy and his group find (as they expected to) that electron withdrawing groups aid the reaction. An electron donor acts as ft brake. This kind of action is what accounts for lithium borodeuteride's reactivity being greater than the non-deuterated compound's. Borodeuter-

These Reactions Are Used to Study Weak Acid Properties

NaBH4 + Z-C0H5OH Diglyme

Sodium borohydride

Substituted phenol

H2 + NaBHsOCcHs-Z

Sodium phenoxyborohydride

R-H Acid (phenol, alcohol,

or hydrocarbon)

Excess D2O

Et3N,DMF R-D

Deuterated acid

SEPT. 19, I960 C&EN 53

Chlorocarbene Aids Synthesis Carbene intermediate reacts with alkyllithiums to give new olefins and cyclopropanes

Acidity Scale Developed for Some Weak Acids

Compound acidity

Cyclopentadiene 180°

m-CIC6H4C=CH 28

P-CIC6H4C=CH 4.9 C6H6C0CH3 3.3°

CHsOC^CH 2.0 P-CH30C6H4C^CH 1.0 ΟβΗδΟ^υπ 1.0 C^gC^^CH 0.058

Fluorene 0.033"

C6H5SO2CH3 0.0012°

CH3SO2CH3 0.00014°

ο Per hydrogen

ide ion is a stronger base than is boro-hydride ion, thanks to deuterium's electron withdrawing ability, Dr. Dessy explains.

Measuring Exchange Rate. The simplest expression of a strong acid's physical properties is its acidity con­stant; for a weak acid, ihe rate at which it will exchange its acidic hy­drogen for one in solution.

To learn how fast weak acids (such as acetylenic hydrocarbons) can trade an acidic hydrogen for one in solution, Mr. Okazumi uses a homogeneous solu­tion of weak acid, deuterium oxide (deuterium source), triethylamine as a catalyst, and dimethylformamide sol­vent. As the deuterated compound forms, it's tracked by infrared absorp­tion of the carbon-deuterium stretch or oxygen-hydrogen linkages which develop.

By varying the amounts of catalyst and deuterium oxide, a whole series of weak acids can be measured, Dr. Dessy says. These data can then be tied in with those for aliphatic alcohols whose dissociation constants are al­ready known, and which have been correlated with stronger acids. Then, he concludes, weak acids can be fitted into an acidity scale. Weak acids now being studied are hydrocarbons related to cyclopentadiene and triphenylmeth-ane, a series of nitro compounds, and aromatic and alicyclic ketones.

138TH ACS NATIONAL MEETING

Organic Chemistry

The reaction between methylene chlo­ride and alkyllithium compounds gives olefins and cyclopropanes having one carbon atom more than the lithium compound itself does. Key intermedi­

ate in the reaction: chlorocarbene, which reacts exclusively with the same organolithium that originally generates the carbene from methylene chloride (so long as there's no other nucleo-philic substrate present for the carbene to react with) .

Typical compounds made by the re­action, reports Dr. Gerhard L. Closs, University of Chicago:

• 1-Hexene and n-propylcyclopro-

Chlorocarbene adds a carbon to alkyllithiums, helps make new olefins and cyclopropanes

RLi + CH2CI2 - * :CHCI + RH + LiCI Alkyl- Methylene Chlorocarbene lithium chloride

Li

RLi + :CHCI — R-CH — R-CH + LiCI | Alkyl-

Cl carbene

The alkylcarbene rearranges to stable products R = n-amyl

C5H11—CH / \

C4H9—CH=Cn2 C3H;—CH—CH2

Yield 95% 5%

R = cyclohexyl

v" CH

y CH2 y CH3 >CH2

Yield 61% 2% 37%

R = f-butyl

Yield

CH3 H \ /

C=C

H3 CH3 13%

CH3

(CH3)3C-CH

I CH2 H \ /

C-C-H / \ CH3 CH3

18%

CH3

69%

54 C & E N S E P T . 19; 1960

Refative

Antibody, Ferritin Produce Specific Electron Stain

pane produced from n-amyllithium.

® Methylenecyclohexane, bicyclo(4,-1,0) heptane, and 1-methylcyclohex-ene-1 from cyclohexyllithium.

• 1,1-Dimethylcyclopropane, 2-methyl-2-butene, and 2-methyl-l-bu-tene from f-butyllithium.

Formation of cyclopropanes and the ratio of olefins to cyclopropanes pro­duced in these reactions parallel the product distribution found when alkyl-carbenes are made by other methods, Dr. Closs says. Alkylcarbene forma­tion via base-catalyzed pyrolysis of tosyl hydrazones, for instance, shows the same reaction pattern, the Chicago chemist notes. Dr. Closs then began working to identify the reaction prod­ucts, followed this with attempts to peg the reaction mechanisms involved.

Reaction Mechanism Shown. Dr. Closs outlines the probable reaction mechanism this way: Alkyllithium and methylene chloride generate chloro-carbene. The lithium compound then adds (in a nucleophilic addition) to the highly reactive carbene, forming alkylchloromethyllithium. This mole­cule is unstable, looses lithium chlo­ride, and thus forms an alkylcarbene intermediate, Dr. Closs theorizes.

Just how the carbene rearranges to a stable product depends on the na­ture of the alkyl group. The Chicago chemist lists these three paths: Hy­drogen migration from the neighboring carbon atom forms terminal olefins; cyclopropanes are made by an intra­molecular insertion into a y-carbon-hy-drogen bond; and finally, alkyl migra­tion makes a nonterminal olefin.

Of the three, hydrogen migration seems to be the preferred process, al­though all three types of rearrange­ments have been observed, says Dr. Closs. Alkyl migration appears the least likely. In fact, he finds alkyl mi­gration only when the /^-carbon atom carries no hydrogen. Case in point: £-butylcarbene, which rearranges to 2-methyl-2-butene partly by alkyl migra­tion.

Dr. Closs points out that two of the products made can't come out of a simple rearrangement of their corre­sponding alkylcarbenes. The two, 1-methyl-cyclohexene-1 and 2-methyl-l-butene, are probably products of sec­ondary rearrangements. But their method of formation isn't clear yet. The same products have been found in other carbenoid rearrangements, he notes.

138TH ACS NATIONAL MEETING

Biological Chemistry

Work by Dr. S. J. Singer and Dr. Anita F. Schick of Yale University has produced a specific "s ta in" that expands the scope of electron microscopy. Because most macromolecules have similar electron-scattering power, the electron micro­scope can't detect or distinguish between specific proteins or other large molecules within cells. The Yale scientists use anti­bodies to pinpoint specific macromole. cules. But for general purposes, anti­body molecules themselves don't scatter electrons enough to be seen with the electron microscope, so Dr. Singer and Dr. Schick attach to the antibody a mole­cule of ferritin—a protein containing up to 23% iron. They do this in a two-stage

reaction, using either m-xylylene diiso-cyanate or toluene-2,4-diisocyanate. The antibody and ferritin molecules are linked through stable, covalent ureido bonds. The high iron content makes single ferritin molecules readily visible in electron micrographs.

At the Virus Laboratory of the Univer­sity of California, Dr. Singer made these electron micrographs of tobacco mosaic virus (TMV), using a ferritin conjugate with antibody specific for TMV. In the upper picture, separate TMV molecules are visible as rod-like objects 3000 A. long. Each small black dot (55 A. across) is due to the ferric hydroxide inner core of a single ferritin molecule. The lower mi­crograph of TMV is made with a ferritin conjugate of nonantibody 7-globulin. No specific attachment to TMV is appar­ent. Similarly, the ferritin conjugate with TMV antibody does not attach itself to other viruses, Dr. Singer says. In both pictures, the large black spots are poly­styrene spheres 2600 A. in diameter.

S E P T . 19, 1 9 6 0 C & E N 55