Group Title: Citrus Station mimeo report - Florida Citrus Experiment Station ; CES 64-7
Title: Physiologically active derivatives of citrus limonene
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 Material Information
Title: Physiologically active derivatives of citrus limonene
Series Title: Citrus Station mimeo report
Physical Description: 3 leaves : ; 28 cm.
Language: English
Creator: Newhall, W. F
Citrus Experiment Station (Lake Alfred, Fla.)
Florida Citrus Commission
Publisher: Florida Citrus Experiment Station :
Florida Citrus Commission
Place of Publication: Lake Alfred FL
Publication Date: 1963
Subject: Citrus fruit industry -- By-products -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
Bibliography: Includes bibliographical references (leaf 3).
Statement of Responsibility: William F. Newhall.
General Note: Caption title.
General Note: "October 8, 1963."
 Record Information
Bibliographic ID: UF00072420
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 75957257

Full Text

Citrus Station Mimeo Report CES 64-7
October 8, 1963

Physiologically Active Derivatives of Citrus Limonene

William F. Newhall
Florida Citrus Experiment Station
Lake Alfred, Florida

Among the naturally occurring amino alcohols (hydroxy-amines) are to be found
some important physiological substances, such as cholamine (CH2NH2CH20H) and choline
(HO(CH3)3NCH2CH20H). The acetyl derivative of choline (acetyl-choline) is present
in active muscle and possesses a powerful muscle-contracting effect. It is often
referred to as the hormone of the peristaltic intestine and a dilution of 1:1000
million is sufficient to renew contractions in the intestines of a surviving guinea-

The benzoic acid esters of many amino alcohols act as local anaesthetics and
have, therefore, become important as substitutes for cocaine. The most familiar of
these synthetic anaesthetics is of course novocaine. The cocaine substitutes
stovaine, panthesine and tutocaine are benzoate esters of complex amino alcohols.

Because of the physiological importance of amino alcohols, the preparation of
medicinals from citrus limonene has been directed toward the synthesis of these
nitrogen compounds and their derivatives.

At the Annual Citrus Processors' Meetings in 1959 and 1961 the preparation of
eight new amino alcohols from d-limonene was discussed. At that time the statement
was made that unfortunately none of these compounds showed any physiological activity
when tested on experimental animals. Since that time exhaustive biological screening
of these compounds by the Upjohn Company has shown that several possess enzyme in-
hibitory activity. These show the greatest activity in inhibiting the nonspecific
enzyme pseudocholinesterase which is present in the blood serum. This enzyme is
distinct and different from "true" cholinesterase which is present in the red blood
cells. Pseudocholinesterase is capable of not only splitting acetylcholine to
choline and acetic acid but can also hydrolyze other compounds such as tributyrin
and methyl butyrate. The study of cholinesterases, their inhibitors and their
function in the transmission of nerve impulses is more in the domain of the neuro-
physiologist and pharmacologist than in that of the chemist. Thus, only the
chemical preparation and relative physiological activities of these limonene
derivatives will be discussed.


The most active amino alcohols prepared from limonene were the tertiary amines
2-dimethylamino-8(l0)-p-menthen-l-ol (I) and 2-dimethylamino-l-p-menthanol (II)
prepared by the reaction of limonene-l,2-epoxide and p-menthane-l,2-epoxide, re-
spectively, with dimethylaminel,2.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
00o 10/8/63 WFN


Because of the previously mentioned physiological activity of many amino alcohol
esters, particularly benzoates, five ester derivatives of (I) or (II) were prepared
for testing. The preparation of these esters was accomplished by reaction of (I) or
(II) with the appropriate acid chloride. Normally the reaction of a tertiary hydroxyl
group, such as that at C1 in limonene, with an acid chloride would be expected to give
an abnormal 1-chloro derivative and not the desired ester. However, under carefully
controlled conditions, the desired esters, (III), (IV), (V), (VI), (VII) were obtained
in good yields.

Physiological Activity

These seven new limonene derivatives are listed in the accompanying Table to-
gether with their toxicities and their enzyme inhibitory test data. Complete re-
sults have not yet been obtained for compounds (VI) and (VII). Toxicities were
determined by intraperitoneal injection in mice.

These results show that hydrogenation of the 8(10) double bond in the isopro-
penyl side chain of limonene markedly reduces the toxicity. In amino alcohols (I)
and (II) such hydrogenation has little effect on the ability of these compounds to
inhibit pseudocholinesterase. Acetylation of the C1 hydroxyl in (I) to form (III)
has no appreciable effect on the activity but considerably decreases the toxicity.
This same decrease in toxicity on esterification of the C1 hydroxyl is evident in the
other amino alcohol esters (IV), (V) and (VII).

The benzoate ester (IV) is the most potent inhibitor of serum cholinesterase. A
concentration of one part per m lion of (IV) is sufficient to give a 50% inactivation
of this enzyme (IO0 = 3.0 x 101 M). Hydrogenation of the 8(10) double bond in (IV) to
give (V) causes a slight decrease in activity. The addition of an ortho methoxyl
substituent in the aromatic ring of (IV) to give (VI) also causes a slight decrease
in activity.

The last compound in Table 1 is neostigmine bromide which is included for
reference purposes. This drug is currently used in the treatment of atony of the
intestinal tract and bladder, myasthenia gravis and to relieve muscle spasms in
poliomyelitis. It is generally used as a standard for the evaluation of cholin-
esterase activity. Compound (IV), prepared from citrus limonene, has 1/5 of the
activity of neostigmine bromide. The intraperitoneal toxicity of neostigmine
bromide was unfortunately not available for comparison purposes.

The benzoate ester (IV) was also found to give 30% inhibition of 5-HTP-
decarboxylase enzyme of kidney at a concentration of 1 x 10-2 Molar. The net
result of this inhibition is a lowering of the level of Serotonin in the brain.
This causes a tranquilizing effect and induces a feeling of well being. This
type of drug is often used in the treatment of mental illness.


This work would not have been possible without the cooperation and assistance
of Dr. Jerome Korman, Head of the Biological Screening Laboratories of the Upjohn

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred Florida.
h00 10//63 WN

Table 1. Animal Test Data for Limonene Amino Alcohols and Amino Esters

Calc. Pseudo (serum) ChE
Compound LD50I.P. Inhibition at 10-3M




N (C H)

N (CH3)s






(150 3.0 x 10-M)

(Iso = 3.0 x 10-5M)

(150 =

2.0 x l-5M)


I o

(150 = 0.69 x 106M)

(Neostigmine Bromide)


Company. Dr. Margaret E. Greig and Miss Anna Gibbons of the Department of
Pharmacology (Upjohn Co.) deserve much credit for their discovery of the enzyme
inhibitory action of these limonene derivatives and for the test data which they
have contributed.

Literature Cited

1) W. F. Newhall, J. Org. Chem. 24, 1673 (1959).

2) W. F. Newhall, J. Org. Chem. (accepted for publication; tentatively
scheduled for the December issue, 1963.).

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
o00 10/8/63 WIN

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