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nitrogen ring opened), and finally to nitrogen-free products.

Plant sources: steroidal sapogenins (Scheer and Kostic). — Because of
their practical importance as potential industrial soiirce material for the
partial synthesis of cortisone ajid allied compounds, a series of steroidal
sapogenins (A/b alio and normal, ring F, a and b config^iration) were studied
in respect to stereochemical relation of the corresponding dihydro-genins
and dihydro-pseudogenins. The significance of these dihydrogenins lies in
their oxidability to a number of pregnene derivatives (starting material for
progesterone, androgens, and corticolds); secondly their structures are
intimately related to the analogous "side chains" of tomatidine and
solasodine .

Plant sources: ergosterol (Wes and Kostic). - As an approach to the
synthesis of cortisone from ergosterol, A7,9(ii),22_ ergostatrienol and its
acetate were hydrogenated in order to determine the possibility of obtaining



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derivatives with an isolated A ^^ ■'-■'■ ^- or5°(9)_ double bond for the introduc-
tion of an 11-oxygen function. Platinum oxide and Raney nickel give largely
the A '-compound; copper chromium oxide under mild conditions, AT^22._ergosta-
dienol, A°''l^)22_ergostadienol, a8 (l^)-ergostenol, and one or more C^ -OH
epimers — the ratio depending upon time and tempera tiire . Finally, /^^o(3-'^■)>22_
ergostadienol coiold be obtained by the catalyst -induced isomerizatlon of
^ 7>22_ej.gostadlenol in a yield of 5^ percent.



Other studies of oxygen introduction at C-11, through addition of vari-
o\is agents to A^^ i ^9(ll)^22_gj.gQg^g^^g^j.g^gj^-L acetate, led to the discovery of
a novel skeleton rearrangement under the Influence of hydrogen chloride.
Evidence accumulated thus far points to the structure of an s-octahydroanthra-
cene for the chief rearrangement product.

Total synthesis towards cortisone (Fry and Miller) .—Synthetic routes to
hydrogenated phenanthrenes (with an oxj'-gen fimction in the position corre-
sponding to C-11 in the steroidal skeleton) are being explored, utilizing a
2,5-dihydroxycyclohexylacetic acid as starting material. From this compound
two of the principal building components, the necessary cyclohexenone deriva-
tive and a cyclohexane ethanol, can be constructed, which may be condensed to
give an immediate precursor to cortisone and closely allied (e.g., nor deriva-
tives) 11-oxygenated corticoid-like substances not found in nature.

Analytical and metabolic studies (Heftmann, Hayden, Earner and. Miller). — For
the ajialysis of cardio-active glycosides, the corresponding aglycones, emd
their acetates, a paper-chromatographic method has been developed. Seed ex-
tracts (up to 0.5 mg.) were prepared from some 'JO samples of various Strophan-
thus and (only partially identical) Apocynaceae species (plant material sup-
plied by the Plant Industry Sta., Beltsville, Md., Dept. Agric.) chromato-
graphed and rechromatographed after acetylation and after subsequent hydrolysis.
A number of seeds were foimd to contain sarmentogenin and several other known
cardiac aglycones. Several seed samples contain imknown glycosides ajid agly-
cones. Similarly, a chromatographic procedure for the identification of
steroidal sapogenins and their acetates was developed. In that method the
spots were made visible by spraying the paper with either blood or trichlo-
roacetic acid.

For the quantitative recovery of urinary corticosteroids, a continixous
solvent-extraction has been devised which permits simultaneous hydrolysis and
extraction. The extractor operating under vacuum can be used at room tempera-
ture with relatively high-boiling solvents, without undue exposure of the
liberated corticoids to heat or acid.

Two technicians of the Endocrinology Branch, National Cancer Institute,
are performing routine analyses on the urinary steroids excreted by cancer
patients iindergoing hormone therapy. This work, which is directed by the head
of this unit, involves analyses of 17-ketosterolds, corticosteroids, and preg-
nanediol. The methods are evaluated by duplicate and recovery experiments and
various refinements — e.g., fractionation of a- and B~hydroxy-17-ketosteroids.
Elimination of interfering chromogens is employed as needed.

Section on Carbohydrates

H. G. Fletcher, Jr., is chief of this section. A program of research
on methods for the synthesis of carbohydrates and carbohydrate derivatives has
been continued.



~ i6h -

One of the most valuable methods for the synthesis of ketoses is the
microbiological oxidation of sugar alcohols by Acetobacter suboxydans , an
organism vhich is used for this purpose at one step in the synthesis of vita-
min C. Unfortunately the orgemism is most selective in its action, converting
some sugar alcohols to the corresponding 2-keto sugars and leaving other sugar
alcohols linattacked. One facet of the program of the section entails study of
this specificity with the object of formulating, if possible, a generalization
relating the configuration of a s'ogar alcohol with its fate in cultures of A,
suboxydans . In the course of this work, it was found that the w-deoxy sugar
alcohol L-fucitol is oxidized by the organism to a 4-keto sugar, an lonexpected
£ind novel type of sugar in sharp contrast to the known ketoses which are all
2-ketoses. (Richtmyer and Stewart.)

During the past eighteen months, a variety of w-deoxy sugar alcohols
have been synthesized and some have already been subjected to the action of
A. suboxydans . Work along this line is continuing with the aforesaid objec-
tive of evolving a generalization regarding the mode of action of this useful
organism. (Zissis.)

While the above work deals primarily with the biochemical synthesis of
sijgars, a second phase of the program of the section is concerned with piirely
chemical methods of synthesis. The enhanced stability of the benzoic acid
derivatives of the sugars comjiared with other acyl derivatives strongly sug-
gests the utility of these derivatives in the synthesis of a variety of
relatively labile derivatives of the sugars which are of biological signifi-
cance: 0-glycosides, nucleosides, vitamin Bip, etc. Previous to the period
of this report, a study of the benzoyl derivatives of the sugars had led to
the evolution of a new method for the synthesis of aldopyranosides. Recently
this work has been extended to other aldopyranosides and also to a ketopyrano-
side. With this background, attention has been directed to the synthesis or
aldofuranose derivatives, and a process for the preparation of the two isomeric
D-xylofuranose tetrabenzoates has been developed. The program has now pro-
gressed to an attack upon the problem of the synthesis of ribofuranose deriva-
tives, since ribose occurs in biological systems largely if not wholly in the
firranose form. (Fletcher, Ness and Diehl.)

It has been known for many years that some monosaccharides are readily
converted with warm aqueous acid into anhydrides (called glycosans), while
other sugars are not affected. The structure of these anhydrides and the rela-
tionship between the structure of sugars and the ease of their conversion to
glycosans have been objects of Investigation in this section. The anhydrides
from D-altroheptulose, L-guloheptulose, and D-idoheptulose have now been shown
to be all 2,7-aiihyiro-B-heptiilopyranoses. An anhydride obtained from D-gluco-
D- gulo -heptose has been shown to be a 1,7-anhydro-B-heptopyranose, while evi-
dence has been obtained that the sugars D-gluco-D-ido-heptose and D-gulose
both give anhydrides of the 1,6-anhydro-pyranose type. The work on glycosans
carried out in this section has provided the means whereby groups in other
laboratories have been enabled to Identify D-altroheptulose (sedoheptiilose) as
an intermediate in photosynthesis and also in one enzymic path of the break-
down of D-glucose. (Pratt.)

As part of the work of the section, newly available chemical dgents are
regularly examined for their possible utility in carbo-hydrate synthesis. In
the last report period, lithium aluminiom hydride was shown to be valuable for
the synthesis of the 1,5-anhydro-glycitols such as styracltol. More recently
the value of this reagent in sugar chemistry has been explored through the
reduction of various sugar acids, an acetohalogenoaldofuranose and



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tetrabenzoyl-B-D-fructofuranosyl bromide. The latter case, incidentally, of-
fered the first opportimity for studying the stereochemistry of the replace-
ment of a tertiary halogen by hydrogen. (Fletcher, Ness and Diehl.)

This section has long been concerned with improvements in the methods
for the preparation of rare sugars so that these substances may be more
readily available for biological and medical experimentation. In the coiirse
of the past eighteen months, improvements were published for the preparation
of L-xylose, melibiose, styracitol, L-arabinose, and L-glucose. (Fletcher and
Diehl. )

Among Important byproducts of the above research activities may be men-
tioned new data on the identification of the sugars through the benzimidazoles
of the sugar acids.

Section on Analgesics

The over-all objective of the section (Nathan B. Eddy, Chief) is col-
laborative effort, chemical and pharmacological, in the preparation and
evaluation of new substances, potentially analgesic, leading to elucidation
of the relation of chemical structure to action and to possible clinical ap-
plications. A major function of the section is consultative, carried out
mainly by the chief of the section, in the field of analgesics and narcotics
generally, for other government agencies, for the National Research Council,
for pharmaceutical manufacturers in this field, for the World Health Organi-
zation, and for the National and International organizations for narcotics
control.

Chemical Laboratories. Derivatives of isomethadone {'May). — Reduction
of the keto group of dl isomethadone and its optical isomers with lithiimi
aliuniniam hydride produced only one of the two possible diastereoisomeric
alcohols (designated a-dl, a-1, and a-d-isomethadols) in excellent yield.
When reduction was effected with sodium in propanol, the other possible
diastereoisomer (B-form) resulted predominantly, although appreciable
amounts of the a-isomers were also obtained. Each of alcohols was acylated
with acetic anhydride -pyridine (acetylderivatives).

Experiments in the methadone series (May and Perrine). — ^An attempt to
methylate a-dl-methadol [ (CH3)2 NCH ( CH3 ) 2CH2CPH2 CHOHC2H5] sulfate with
dimethyl sulfate in boiling ethyl acetate gave, instead of the 0-methyl
derivative, an alcohol isomeric with a-dl-methadol . The isomeric alcohol
was proved to be 6-dimethylamino-3,^-diphenyl-4-heptanol, by a Hofmann degra-
dation and hydrogenation of the resultant non-nitrogenous olefin to 3,^-
diphenyl-4-heptanol, identical with that synthesized from a-ethyldesoxybenzoin
and propyllithium. A by-product of the reaction which formed the isomeric
alcohol was a desoxy compound, whose ultraviolet absorption spectrum, chemical
properties and analytical data were compatible with those of a-ethyl-a'-(2-
dimethylaminopropyl)stilbene. The isomeric alcohol had only very weak anal-
gesic action, indicating that asymmetry in the position of the phenyl rings is
a disadvantageous modification of the methadone structirre.

The reaction of me thane sulfonyl or p-toluene-sulfonyl chloride and
a-dl-methadol led to the methochloride of a desoxymethadol as the only isolable
material. This was proved to be either l,5-dimethyl-3,3-diphenyl-2ethyl-
pyrollidine or l,2,6-trimethyl-it-,l4-diphenylpiperidine by Hofmann degradation



- 166 -

to a product still containing a basic nitrogen. Hydrogenation of the Hofmann
degradation product gave 6-dimethylamino-4,4-diphenylheptane or an isomer
thereof. An attempt is being made to synthesize this product for final proof.

Synthesis of potential analgesics from 2-phenylcyclohexanone (May and
MTirphy). — This readily available starting material caji be converted to simple
nitrogenous compounds having several structiiral features in common with such
potent analgesics as morphine^ dromoran, methadone, and demerol. Alkylation
of the ketone gave 2- ( 2-dimethylami noethyl ) -2-phenylcyclohexanone whose C-0
group was reduced to CH2 and to CHOH. Acetylation of the latter was also
effected. Cyclization of the aminoketone led to a phenylmorphan in which the
nitrogen is heterocyclic as in morphine and demerol. The attempt to convert
the phenylmorphan derivative to a hydrophenanthrene isomer is proceeding
satisfactorily. The phenylcyclohexanone derivatives so far tested have not
shown analgesic action. They are, however, only early steps in the intended
build-up on the basic molecxile.

The 0-acetyl derivative of d-dromoran was prepared £ind showed distinct
morphine-like effects in mice and cats, although the d-dromoran itself is
inactive.

The study of demethylation of amines with potassimn ferricyanlde is
continuing. (Perrine.)

The study of the reactions of 9-viny] -acridine is continuing.
( Perrine. )

Wolfe-Kishner reduction and mercaptol deoxygenation of dihydrocodeinone
was completed. One product, ethylmercaptodihydrothebainone, showed moderate
analgesic effect about half that of codeine but only one-foi.ui'th that of
dihydrothebainone . ( Perrine . )

A program has been undertaken to utilize 2,2-diphenyl-4~dimethyl-
aminovaleronitrile in the synthesis of potential analgesic agents, beginning
with reduction to the corresponding aldehyde (a known reaction which we have
developed to give rapid and very satisfactory conversion) and subsequent
substitution of the nitrile (or aldehyde) part of the molecule by mercapto,
sulfonyl, keto, pyrimidyl, etc., groups. (Perrine.)

Work has continued throughout the period on the elucidation of struc-
ture of the morphine derivative, metopon. Effort has been directed toward
synthesis of 3,^,5-'triethyl-5,6-dimethoxytetrahydronaphthalene, the novel
degradation product derived from dihydi'othebaine. Two approaches to this
synthesis have failed along the way; two new approaches remain to be under-
taken. (Sargent.)

Aminoacridines have been reported recently to antagonize some of the
effects of morphine. To test this action with respect to analgesic effect,
9-aminoacridine and l,2,3,4-tetrahydro-9-aminoacridine were synthesized.
Neither was antagonistic to the analgesic effect of morphine in mice; the
latter was convulsant and very toxic. (Sargent.)

To elucidate the structure of several new acridine antimalarials
(first prepared in this laboratory), the synthesis of the four unknown
ethylacridines was undertaken. Three have been prepared and the fourth is
well under way. (Sargent.)



- 167 -

A new and simple method has been devised for the preparation of the
powerful analgesic dehydrodesoxymorphine-D ("Desomorphine") . This consists
in conversion of dihydrocodeine to its tosyl ester, reduction with lithium
aluminum hydride, and subsequent demethylation. (Small. )

A number of new types of morphine derivatives has been prepared for
analgesic testing, by introduction of hydroxyl groups with osmic acid into
such narcotics as morphine, heroin, and neopine. (Small,)

For police and control purposes, specific identification tests were
developed for the new synthetic drugs demerol, nisentil, methadone, and
dromoran. The tests will distinguish these drugs from each other, from the
narcotics of the opium and coca group, and from various medicinals not con-
trolled by Federal law. (Small.)

In connection with determination of the configuration of the basic
portion of the cocaine molecule, a study of the stereochemistry of the re-
lated bases tropine axid pseudotropine has been carried out. The most im-
portant result of this has been isolation of two isomeric W-substituted
tropines which owe their isomerism to a pseudoasymmetric nitrogen atom, a
hitherto unreported phenomenon. (Findlay. )

Research on new analytical methods has resulted in a Dumas nitrogen
procedure which is applicable to organic compounds heretofore resisting all
analytical methods. In service capacity, 1^4-, 200 analytical determinations
were made. This includes over I7OO lactic acid analyses by Mr. Remsburg for
a continuing project of the National Microbiological Institute. (Alford. )

Studies on the catabolism of simino acids center around the twofold
oxidation of tryptophan and tyrosine. By exchange of iodo groups with
hydroxyl, three new hydroxyhistidines have been prepared. Application of a
similar method to imidazolepropionic acid is expected to be usefiil in
identification of the labile isomer of fonnylisoglutamine isolated, by
Silverman, Tabor, and associates, from folic acid deficient animals.
Synthesis of possible metabolites of hydroxyproline, by controlled oxidation
of siii table derivatives, and from urocanic acid, is in progress. (Witkop
and Beiler, )

Pharmacological Laboratories (Eddy and Leimbach). Evaluation of many
compounds for analgesic and toxic effects was completed, using the assay
method devised in this laboratory. These included dithienylbutenyl- and
dithienylbutylamines, a large number of methadone derivatives (mainly alco-
hols and acylated alcohols derived from methadone, isomethadone . and their
optical isomers), new morphine derivatives, phenyl eye lohexanone derivatives,
and a number of other diphenyl compoxonds. The unsaturated dithienyl com-
pounds were more effective than the saturated, and, in some instances, ex-
hibited analgesic activity approximating that of morphine. All of the
dithienyls showed remarkably weak analgesic effect when administered orally
and a very much wider difference in analgesic effectiveness than in toxicity
with oral and subcutaneous administration. Among morphine derivatives the
difference in effectiveness by the two routes of administration is not at all
uniform. In some instances the oral dose is 100 times the subcutaneous, in
other cases only about twice as great. Comparisons in the morphine series is
being extended to see if the differences can be related to any chemical
config\iration.



- 168 -

The alcohols derived from the methadones and isomethadones, with one
exception^ were much weaker analgesics than the ketones; the acetylated com-
poxonds, on the other hand, were all more effective than the parent ketone.
The alcohols and acetylated alcohols derived from d-methadone were as effective
or more effective orally than subcutaneously and were relatively long acting.
This was not true of the compounds derived from d-isomethadone. Throughout,
isomethadone and its derivatives are less toxic and less analgesic thsin corre-
sponding members of the methadone series.

Work along three lines having to do with the general phenomena of
analgesia is under way: (a) acute tolerance to analgesic effect, (h) the
ability of various agents to antagonize or potentiate the effect of different
types of potent analgesics, (c) variation in analgesic effect with season and
different lots of mice.

In collaboration with Drs. Brodie and Axelrod of the National Heart
Institute, a beginning was made on metabolic studies designed to throw light
upon differences in effect with route of administration and upon differences
in speed and degree of effectiveness in different species.

The Survey of Chemotherapeutic Agents (Hionter). The Survey Office of
Chemotherapeutic Agents serves primarily as liaison between the Chemical-
Biological Coordination Center of the National Research Council and various
laboratories of the National Institutes of Health. From July I95I through
December 1952, 1,55^ compounds were processed. These were obtained primarily
from the Chemical-Biological Coordination Center, further from laboratories of
the National Institutes of Health, academic and industrial laboratories, and
•from miscellaneous private sources. Laboratories of the National Institutes
of Health cooperated in the testing for toxicity, analgesia effect, tumor-
necrotising effect, experimental malaria, schistosomiasis, and fumicidal,
insect, and snail control. The results of ^k2 tests were commionicated to this
office.

NIAMD Editorial Committee (Charles I. Wright, Chairman). The Editorial
Board of NIAMD, a scientific-administrative service attached to the Laboratory
of Chemistry, received and recorded 373 manuscripts (ranging from books to ab-
stracts) since July 1951- Each manuscript was usually assigned to two or more
members of the Board, including the chairman, for review. When no board mem-
ber q\;ialif led, the chairman sought and received aid from members of the other
Institutes or occasionally from persons In the National Bureau of Standards,
Carnegie Institution of Washington, or Naval Hospital Research Laboratories.
The evaluation of each reviewer's comments was made by the chairman, espe-
cially when a manuscript was not recommended for publication or severely
criticized. Such a manuscript reqiilred consultation with the author and a
second or third reading before final approval or rejection.

LABORATORY OF BIOCHEMESTRy AND NUTRITION

The Laboratory of Biochemistry and Nutrition (James M. Himdley, Chief)
is organized to -conduct both fundamental and applied research in the broad
fields of biochemistry and physiology. Within the Laboratory, the Section on
Fractionation and Isolation searches for and isolates chemical substances
from natiiral sources such as vitamins, growth factors, or other substances of
biological importajice. The Section on Enzymes studies the fundamental chemi-
cal processes by which an organism derives energy from its feed, and isolates
and studies the enzymes and substrates involved. The Section on Endocrinology



- l69 -
studies the influence of the endocrine glands on metabolic processes aaid
varioxis disease states, as well as the interrelations between the central
nervous system and the activity of the endocrine glands. The Section on
Biochemistry and Physiology of Nutrition studies the actions, uses, and bio-
chemistry of vitamins and eunino acids and the role of nutrition in various
disease processes, and searches for new vitamins and other substances of
possible medicinal importance. Studies are being made on the growth, develop-
ment, and nutritional status of human beings. The overall program of the
laboratory is planned to emphasize research bearing on arthritis and allied
diseases, peptic iilcer, and diseases of the liver and blood-forming organs.

Section on Biochemistry and Physiology of nutrition (James M. Himdley, Chief)

Nutritional Requirements. A nutritionally complete purified diet for
the guinea pig has been developed which will maintain norual growth and
physiological functioning. With this diet, deficiencies of pantothenic acid,
choline, folic acid, niacin, pyridoxine, thiamine, and riboflavin have been
produced in the guinea pig. A piurified diet has been developed which permits
young rabbits to grow as well as on a commercial feed. The rabbit, like the
guinea pig, requires a fairly high level of potassium in its diet. The rab-
bit appears to be the only non-ruminant studied that requires no dietary
source of thiamine or riboflavin. Investigations are under way on the still
unidentified vitamins that are required by the chick. The presence of such
substances in natural foods has been confirmed, and improved methods of assay
therefore have been developed. A vitamin Bip deficiency can be produced in
chicks fed purified diets that are high in fat, protein, or fiber and by the
use of low siilfur diets. When the citrovorum factor is fed to chicks, it is
not as active for growth as pteroyl- glutamic acid, but it is" as active when
injected. (Briggs, Reid, and Wooley. )

The Interrelation of Various Dietary and Physiological Factors.
Previous work in this laboratory showed that a diet consisting of rice, salts,
and vitamins can be improved by the addition of lysine and threonine. When
very low levels of certain antibiotics are added to this diet, rats grow as
well as they do on a 15 percent casein diet. The rice diet contains only 6.5
percent protein. The addition of small amounts of avireomycin to a purified
diet containing minimal amounts of folic acid increases the chick's require-
ment for that vitamin. This is in contrast to the folic-acid sparing effect
of aureomycin seen under these circumstances in the rat. (Briggs and Pecora. )

Previous work from this laboratory showed that large amoimts of vitamin
C or a variety of antibiotics prevented the development of deficiency signs
and permitted almost normal growth in rats deprived of certain of the B



Online LibraryNational Institutes of Health (U.S.)Annual report : National Institutes of Health (Volume 1952) → online text (page 21 of 29)