U.S. patent number 3,656,906 [Application Number 05/028,015] was granted by the patent office on 1972-04-18 for method for detecting and quantitating the presence of cannabinoids and analogs thereof in biological materials and resulting products.
This patent grant is currently assigned to Arthur D. Little, Inc.. Invention is credited to Francis J. Bullock.
United States Patent |
3,656,906 |
Bullock |
April 18, 1972 |
METHOD FOR DETECTING AND QUANTITATING THE PRESENCE OF CANNABINOIDS
AND ANALOGS THEREOF IN BIOLOGICAL MATERIALS AND RESULTING
PRODUCTS
Abstract
A method for detecting and quantitating submicrogram quantities
of cannabinoids and their analogs in biological materials including
blood plasma and urine. Detection system is based on the
condensation of the cannabinoids with a polycarboxylic acid to give
a highly fluorescent derivative, the intensity of the fluorescence
being maximized in the pH range of 9-11.
Inventors: |
Bullock; Francis J. (Acton,
MA) |
Assignee: |
Arthur D. Little, Inc.
(Cambridge, MA)
|
Family
ID: |
21841070 |
Appl.
No.: |
05/028,015 |
Filed: |
April 13, 1970 |
Current U.S.
Class: |
549/26; 436/93;
436/901; 544/142; 544/146; 546/63; 546/90; 546/196; 546/202;
548/421; 548/525; 549/43; 549/279; 549/385; 549/390; 560/144;
568/743; 250/396R; 436/172; 544/126; 544/145; 544/150; 546/133;
546/200; 546/344; 548/430; 548/526; 549/277; 549/289; 560/141;
562/469; 436/92 |
Current CPC
Class: |
G01N
33/948 (20130101); Y10T 436/142222 (20150115); Y10T
436/141111 (20150115); Y10S 436/901 (20130101) |
Current International
Class: |
G01N
33/94 (20060101); G01n 031/22 () |
Field of
Search: |
;23/23B,230
;260/247.1,247.5B,247.7E,293.57,293.58,326.5B,327TH,332.3,340.9,343.2R |
Other References
irudayasamy, A. et al., Chem. Abstr. 71, 1969.
|
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Reese; R. M.
Claims
I claim:
1. A method of detecting the presence of a cannabinoid in a body
fluid, comprising the steps of treating an extract of a body fluid
containing a cannabinoid with a polycarboxylic acid in the presence
of an acid catalyst thereby to condense the cannabinoid with said
polycarboxylic acid to form a .delta.-lactone cannabinoid and then
converting the .delta.-lactone form to the free acid or salt form
which is intensely fluorescent and detecting said fluorescence as
indicative of the presence of said cannabinoid.
2. A method in accordance with claim 1 wherein said polycarboxylic
acid has the formula HOOC--CH.sub.2 --R--COOH, wherein R is
CH.sub.2,
3. A method in accordance with claim 1 wherein said cannabinoid is
represented by the formula ##SPC32##
wherein R.sub.1 is lower-alkyl;
R.sub.2 is alkyl, cycloalkyl-lower-alkyl or Alk-N.dbd.B where Alk
is lower alkylene, and N.dbd.B is di-lower-alkylamino,
N-morpholino, N-pyrrolidino or N-piperidino;
X is S and m is 1 or 2, n is 0 or 1 and m + n is 2 or 3;
X is
and m is 2 and n is 0;
X is
where R.sub.3 is hydrogen or lower alkyl and n is 0, 1, 2 or 3, m
is 0, 1, 2 or 3 and m + n is 2 or 3;
X is
where R.sub.4 is hydrogen or lower-alkyl and m is 0,1,2 or 3, n is
0,1,2 or 3 and m + n is 2 or 3;
X is
where R.sub.5 is hydrogen, alkyl, cycloalkyl-lower-alkyl,
lower-alkanoyl, cycloalkyl-lower-alkanoyl, lower-alkenyl,
lower-alkynyl, halo-lower-alkenyl, phenyl-lower-alkyl,
phenyl-lower-alkenyl, phenyl-lower-alkynyl, (the benzene ring of
the phenyl being substituted by 1 to 3 members of lower-alkyl,
lower-alkoxy, halo, nitro, lower-alkyl-mercapto, methylenedioxy,
trifluoromethyl) and m is 1 or 2, n is 1 or 2 and m + n is 2 or
3;
R.sub.6 is alkyl containing from one to 10 carbon atoms;
R.sub.7 is hydrogen or lower-alkanoyl; and
R.sub.8 is 1-(2-isopropyl-5-lower-alkyl-1-cyclohexenyl) having the
formula ##SPC33##
where R.sub.9 is lower-alkyl; 3-(1-R.sub.10
-4-isopropyl-1,2,5,6-tetrahydropyridyl) having the formula
##SPC34##
where R.sub.10 is hydrogen, lower-alkyl or phenyl-lower-alkyl;
4-(1-R.sub.10 -3-isopropyl-1,2,5,6-tetrahydropyridyl) having the
formula ##SPC35##
where R.sub.10 has the meaning given above; or
3-(2-isopropyl-1,4-ethano-1,4,5,6-tetrahydropyridyl) having the
formula ##SPC36##
Description
This invention relates to a method of detecting and quantitating
traces of cannabinoids and their analogs, including marihuana
constituents and certain of their derivatives, in biological
materials including blood plasma and urine and to the novel
compounds produced in the reactions which gives rise to the
test.
There is a need for a qualitative and quantitative test for
detecting the presence of marihuana and of related compounds and
derivatives of marihuana in blood plasma and urine to determine
whether or not an individual has consumed one of the marihuana
related drugs in one form or another. Such a test must be reliable
and relatively easy to perform.
It is therefore a primary object of this invention to provide a
method of detecting and quantitating cannabinoids and particularly
of detecting and quantitating these compounds in biological
materials. It is another object to provide a method of the
character described which is reliable and relatively simple to
perform. It is another primary object of this invention to provide
novel compounds which are derivatives of cannabinoids. Other
objects of the invention will in part by obvious and will in part
be apparent hereinafter.
The invention accordingly comprises the several steps and the
relation of one or more of such steps with respect to each of the
others, and the composition of matter possessing the
characteristics, properties, and the relation of constituents which
are exemplified in the following detailed disclosure, and the scope
of the invention will be indicated in the claims .
The term "cannabinoid" is used hereinafter to designate compounds
with a cannabinoid structure and their analogs and it includes
those compounds which may be represented generally by formula I or
formula II ##SPC1##
wherein in formula I R.sub.1 is lower-alkyl,
R.sub.2 is alkyl, cycloalkyl-lower-alkyl or Alk-N.dbd.B where Alk
is lower alkylene, and N.dbd.B is di-lower-alkylamino,
N-morpholino, N-pyrrolidino or N-piperidino,
X is S and m is 1 or 2, n is 0 or 1 and m + n is 2 or 3, X is
and m is 2 and n is 0,
X is
where R.sub.3 is hydrogen or lower-alkyl and n is 0, 1, 2, or 3, m
is 0, 1, 2, or 3 and m + n is 2 or 3,
X is
where R.sub.4 is hydrogen or lower alkyl and m is 0, 1, 2 or 3, n
is , 1,2 or 3 and m + n is 2 or 3,
X is
where R.sub.5 is hydrogen, alkyl, cycloalkyl-lower-alkyl,
lower-alkanoyl, cycloalkyl-lower-alkanoyl, lower-alkenyl,
lower-alkynyl, halo-lower-alkenyl, phenyl-lower-alkyl,
phenyl-lower-alkenyl, phenyl-lower alkynyl, (the benzene ring of
the phenyl being substituted by 1 to 3 members of lower-alkyl,
lower-alkoxy, halo, nitro, lower-alkyl-mercapto, methylenedioxy,
trifluoromethyl) and m is 1 or 2, n is 1 or 2 and m + n is 2 or 3;
and wherein formula II
R.sub.6 is alkyl containing from one to 10 carbon atoms,
R.sub.7 is hydrogen or lower-alkanoyl,
R.sub.8 is 1-(2-isopropyl-5-lower-alkyl-1-cyclobexenyl) having the
formula ##SPC2##
where R.sub.9 is lower-alkyl; 3-(1-R.sub.10
-4-isopropyl-1,2,5,6-tetrahydropyridyl) having the formula
##SPC3##
where R.sub.10 is hydrogen, lower-alkyl, or phenyl-lower-alkyl;
4-(1-R.sub.10 -3-isopropyl-1,2,5,6-tetrahydropyridyl) having the
formula ##SPC4##
where R.sub.10 has the meaning given above; or
3-(2-isopropyl-1,4-ethano-1,4,5,6-tetrahydropyridyl) having the
formula ##SPC5##
As used herein, the term "lower-alkyl" means saturated, monovalent
aliphatic-radicals, including straight and branched-chain radicals
of from one to six carbon atoms, as illustrated by, but not limited
to methyl, ethyl, propyl, isopropyl, butyl, sec.- butyl, amyl,
hexyl, and the like.
As used herein, the term "alkyl" means saturated, monovalent
aliphatic radicals, including straight and branched-chain radicals
of from one to 20 carbon atoms, as illustrated by, but not limited
to methyl, n-amyl, n-hexyl, 2-heptyl, n-heptyl, 3-methyl-2-octyl,
n-octyl, 2-nonyl, 2-tetradecyl, n-hexadecyl, 2-eicosanyl, and the
like.
As used herein, the term "lower-alkenyl" means monovalent,
aliphatic-radicals of from three to seven carbon atoms which
contain at least one double bond, and are either straight or
branched-chain, as illustrated by, but not limited to
1-(2-propenyl), 1-(3-methyl-2-propenyl),
1-(1,3-dimethyl-2-propenyl), 1-(2-hexenyl), and the like.
As used herein, the term "lower-alkynyl" means monovalent,
aliphatic-radicals, of from three to seven carbon atoms which
contain at least one triple bond, and are either straight or
branched, as illustrated by, but not limited to 1-(2-propynyl,
1-(1-methyl-2-propynyl), 1-(2-heptynyl), and the like.
As used herein, the term "cycloalkyl" means cyclic, saturated
aliphatic-radicals of from three to eight carbon atoms, as
illustrated by, but not limited to cyclopropyl, cyclobutyl,
2-methylcyclobutyl, cyclohexyl, 4-methylcyclohexyl, cyclooctyl, and
the like.
As used herein, the term "lower-alkanoyl" means saturated,
monovalent, aliphatic radicals, derived from a monocarboxylic acid,
including straight or branched-chain radicals of from one to six
carbon atoms, as illustrated by, but not limited to formyl, acetyl,
propionyl, a-methylpropionyl, butyryl, hexanoyl, and the like.
As used herein, the terms "phenyl-lower-alkyl,"
"phenyl-lower-alkenyl," and "phenyl-lower-alkynyl" means a
monovalent radical consisting of a phenyl nucleus bonded to the
rest of the molecule, respectively, through a divalent
lower-alkylene radical of from one to four carbon atoms as
illustrated by, but not limited to methylene, 1,1-ethylene,
1,2-ethylene, 1,3-propylene, 1,2-propylene, 1,4-butylene, and the
like, or through a divalent lower-alkenylene radical of from two to
four carbon atoms, as illustrated by, but not limited to
1,2-ethenylene, 1,3-(1-propenylene), 1,3-(1-butenylene),
1,4-(2-butenylene), and the like, or through a divalent
lower-alkynylene radical of from two to four carbon atoms, as
illustrated by, but not limited to 1,2-ethynylene, 1,3-propynylene,
1,3-(1-butynylene), and the like. Here and elsewhere throughout
this specification, it will be understood the benzene ring of
phenyl can bear any number and kind of substituents such as would
occur to the man skilled in organic chemistry. Solely for
illustration, and without limitation, such substituents include
lower-alkyl, lower-alkoxy, halo (chloro, bromo, iodo, or fluoro),
nitro, lower-alkylmercapto, and the like.
Exemplary of cannabinoids of formula I wherein X is S are
##SPC6##
1,2-dihydro-4,4-dimethyl-9-hydroxy-7-(1,2-dimethylheptyl)-4H-thieno-[2,3-c]
[1] benzopyran; ##SPC7##
1,3-dihydro-4,4-dimethyl-9-hydroxy-7-(1,2-dimethylheptyl)-4H-thieno-[3,4-c]
[1] benzopyran; and ##SPC8## 5,5-dimethyl-10-hydr
oxy-8-(1,2-dimethylheptyl)-1,2,4,5-tetrahydro-4H,5H-thiopyrano
[3,4-c] [1]benzopyran.
Cannabinoids of the type illustrated by compounds III, IV and V are
the subject of U.S. Pat. application, Ser. No. 852,928 filed Aug.
25, 1969, in the names of Raj K. Razdan and Harry G. Pars and
assigned to the same assignee as the present application.
Exemplary of cannabinoids of formula I wherein X is
is ##SPC9##
5,5-dimethyl-1,4-ethano-10-hydroxy-81(1,2-dimethylheptyl)-1,2,3,4-tetrahydr
o-5H-[1]benzopyrano[3,4-c]pyridine.
Cannabinoids of the type illustrated by compound VI are the subject
of U.S. Pat. No. 3,493,579.
Exemplary of cannabinoids of formula I wherein X is
and R.sub.3 is hydrogen or lower alkyl are ##SPC10## ##SPC11##
1-hydroxy-3-pentyl-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]py
ran (VIII) and
1-hdyroxy-3-pentyl-6a,7,8,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]py
ran (IX).
Compounds VI and VII are well known as components of the active
ingredients of marihuana and compounds VIII and IX are the well
known .DELTA..sup.8 and .DELTA..sup.9 tetrahydrocannabinols,
respectively.
Exemplary of cannabinoids of formula I where X is
and R.sub.4 is hydrogen or lower alkyl are ##SPC12##
3-(2-dimethylaminoethyl)-1-hydroxy-6,6-dihexyl-9-methyl-7,8,9,10-tetrahydro
-6H-dibenzo[b,d]pyran (X) or
3-(2-dimethylaminoethyl)-1-hydroxy-6,6,9-trimethyl-7,8,9,10,11,12-hexahydr
o-6H-dibenzo[b,d]pyran (XI); ##SPC13##
4,4-dimethyl-9-hydroxy-7-(1,2-dimethylheptyl)-1,2,3,4-tetrahydrocyclopenta[
c][1]benzopyran (XII) or
4,4-di-(1-hexyl)-9-hydroxy-7-(1,2-dimethylheptyl)-1,2,3,4,12,13-hexahydroc
yclopenta [c][1]benzopyran (XIII).
Cannabinoids of the type illustrated by compounds X and XI are the
subject of U.S. Pat. application, Ser. No. 1,550, filed Jan. 8,
1970, in the names of Harry G. Pars and Felix E. Granchelli; and
those of the type illustrated by compounds XII and XIII are the
subject of U.S. Pat. application Ser. No. 642,192, filed May 2,
1967, in the names of Raj K. Razdan, Felix E. Granchelli and Harry
G. Pars. Both of these applications are assigned to the same
assignee as the present application.
Exemplary of cannabinoids of formula I wherein X is
are ##SPC14##
10-hydroxy-2,5,5,8-tetramethyl-1,2,3,4-tetrahydro-5H-[1]benzo-pyrano[3,4-d]
pyridine (XIV) or
10-hydroxy-2,5,5,8-tetramethyl-1,2,3,4,13,14-hexahydro-5H-[1]-benzopyrano[
3,4-d]pyridine (XV); ##SPC15##
10-hydroxy81(1,2-dimethylheptyl)-3,5,5-trimethyl-1,2,3,4-tetrahydro-5H-[1]b
enzopyrane[3,4-c]pyridine (XVI) or
10-hydroxy-8(1,2-dimethylheptyl)-3,5,5-trimethyl
1,2,3,4,13,14-hexahydro-5H-[1]benzopyrano[3,4-c]pyridine (XVII);
and ##SPC16##
2-benzyl-4-dimethyl-9-hydroxy-7-(1,2-dimethylheptyl)-1,2,3,4-tetrahydro[1]b
enzopyrano[3,4-c]pyrrole (XVIII) or
2-benzyl-4-dimethyl-9-hydroxy-7-(1,2-dimethylheptyl)-1,2,3,4,11,12-hexahyd
ro[1]benzopyrano[3,4-c]pyrrole (XIX).
Cannabinoids of the type illustrated by compounds XIV and XV are
disclosed in U.S. Pat. No. 3,429,889; cannabinoids of the type
illustrated by compounds XVI and XVII are the subject of U.S. Pat.
application Ser. No. 642,223, filed May 29, 1967, in the names of
Harry G. Pars, Felix E. Granchelli and Raj K. Razdan; and
cannabinoids of the type illustrated by compounds XVIII and XIX are
the subject of U.S. Pat. application, Ser. No. 842,690, filed July
17, 1969, in the names of Harry G. Pars and Raj K. Razdan. These
two applications are assigned to the same assignee as the present
application.
Exemplary of the cannabinoids of formula II wherein R.sub.7 is
hydrogen and R.sub.8 is
1-(2-isopropyl-5-lower-alkyl-1-cyclohex-enyl) is ##SPC17##
2-[1-(2-isopropyl-5-methyl-1-cyclohexenyl)
]-5-(1,2-dimethylheptyl)resorcinol.
Exemplary of the cannabinoids of formula II wherein R.sub.7 is
hydrogen and R.sub.8 is 3-(1-R.sub.10
-4-isopropyl-1,2,5,6-tetrahydropyridyl) is ##SPC18##
2-[3-(1-benzyl-4-isopropyl-1,2,5,6-tetrahydropyridyl)]-5-(1,2-dimethylhepty
l)resorcinol.
Exemplary of the cannabinoids of formula II wherein R.sub.7 is
hydrogen and R.sub.8 is 4-(1-R.sub.10
-3-isopropyl-1,2,5,6-tetrahydropyridyl) is ##SPC19##
2-[4-(1-benzyl-3-isopropyl-1,2,5,6-tetrahydropyridyl)]-5-methylresorcinol.
Exemplary of the cannabinoids of formula II wherein R.sub.7 is
hydrogen and R.sub.8 is
3-(2-isopropyl-1,4-ethano-1,4,5,6-tetrahydropyridyl) is
##SPC20##
2-[3-(2-isopropyl-1,4-ethano-1,4,5,6-tetrahydropyridyl)]-5-methylresorcinol
.
Cannabinoids of the type illustrated by compounds XX - XXIII are
the subject of U.S. Pat. application Ser. No. 811,701 filed Mar.
27, 1969, in the names of Raj K. Razdan, William R. Thompson, Felix
E. Granchelli and Harry G. Pars and assigned to the same assignee
as the present application.
The invention sought to be patented in one of its composition
aspects resides in the concept of a class of compounds represented
by formulas XXIV and XXV ##SPC21##
wherein R.sub.1, R.sub.2 and X have the same meanings as in the
compounds of formula I and R.sub.6 R.sub.7 and R.sub.8 have the
same meanings as in the compounds of formula II.
The invention sought to be patented in another of its composition
aspects resides in the concept of a class of compounds represented
by formulas XXVI and XXVII ##SPC22##
wherein R.sub.1, R.sub.2 and X have the same meanings as in the
compounds of formula I; R.sub.6, R.sub.7 and R.sub.8 have the same
meanings as in the compounds of formula II; and Z is a cation such
as H.sup..sup.+, Na,.sup.+ K.sup.+ and the like.
The invention sought to be patented in one method aspect resides in
reacting a cannabinoid of the class defined by formulas I and II
with a poycarboxylic acid of the general formula XXVIII
HOOC-CH.sub.2 -R.sub.11 -COOH, XXVIII
wherein R.sub.11 is CH.sub.2,
to produce the compounds of the general formulas XXIV and XXV by
the following reaction ##SPC23##
The invention sought to be patented in another method aspect
resides in converting the compounds of of formulas XXIV and XXV to
those of formulas XXVI and XXVII by the addition of an alkali to
raise the pH to at least 9. The formation of compounds of the
formulas XXVI and XXVII gives rise to a high degree of fluorescence
emmission at 470 nm when excited at 380 nm. The occurrence of this
fluorescence is indicative of the presence of a cannabinoid
structure and hence serves as the basis for the detection of
marihuana and marihuana derivatives as well as the cannabinoids and
their analogs as represented by general formulas I and II.
The cannabinoids in amounts from 0.6 micrograms and greater have
been detected by the method of this invention.
In reacting the cannabinoid with the polycarboxylic acid the
hydrogen ion is furnished by an acid catalyst such as
polyphosphoric acid, sulfuric acid or a mixture of sulfuric acid
and acetic acid. The reaction is preferably carried out at somewhat
elevated temperatures, typically in the range from about 80 to
90.degree. C. and for at least 20 minutes.
The polycarboxylic acids of formula XXVIII are illustrated by malic
acid, citric acid, isocitric acid, aconitic acid and succinic
acid.
The composition and method aspects of this invention are
illustrated by the following examples which are meant to be
illustrative and not limiting.
EXAMPLE 1
1-Hydroxy-3-pentyl-6a,7,8,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyr
an-2-acrylic Acid .delta.-lactone
Blood plasma containing .DELTA..sup.9 tetrahydrocannabinol
(1-hydroxy-3-pentyl-6a,7,8,10a-tetrahydro-6,6,9-trimethyl-6H,
dibenzo[b,d]pyran) was prepared by the following procedure. A
concentrated solution of the .DELTA..sup.9 THC (prepared according
to the procedure disclosed by D. Petrzilka, W. Haefliger and C.
Sikemeier in Helvetica Chemica Acta, 52: 1102 (1969)) was made by
dissolving 1 milligram of the .DELTA..sup.9 THC in 1 milliliter of
absolute ethanol. To 0.2 milliliters of this solution was then
added 20 milliliters of human blood plasma. This blood plasma
containing the THC was then diluted with additional fresh plasma to
the desired concentration which in the following examples was 0.3
micrograms or more of THC/milliliter of plasma.
Two milliliters of the THC-containing plasma (equivalent to 0.6
micrograms of .DELTA..sup.9 THC was diluted with an equal volume of
pH 5 citrate buffer and the mixture was saturated with sodium
chloride. It was then extracted twice with 5-milliliter portions of
hexane containing 1.5% ethanol. The combined hexane layers were
washed once by shaking with 5 milliliters of 0.1 normal sodium
hydroxide solution. The hexane layer was separated and evaporated
to dryness in a 15-milliliter graduated, stoppered conical
centrifuge tube. 0.5 milliliter of an ethanolic solution of malic
acid (1 mg/ml) was added and the ethanol evaporated by warming the
tube in a water bath at 90.degree.. The refluxing ethanol was
allowed to wash the contents of the tube into the tip. After
evaporation of the ethanol, 0.5 ml of a polyphosphoric acid mixture
(prepared by mixing polyphosphoric acid and 85% phosphoric acid in
a 2:1 volume ratio) was added; the tube was stoppered and heated at
90.degree. for 20 minutes. The mixture was diluted with 1
milliliter of distilled water and extracted with 10 milliliters of
hexane. The hexane layer was washed once by shaking with 5
milliliters of pH 5 citrate buffer. The aqueous layer was removed
and there remained in the hexane layer the product
1-hydroxy-3-pentyl-6a,7,8,10a-tetrahydro-6,6,9-trimethyl-6-H-dibenzo[b,d]p
yran-2-acrylic acid .delta.-lactone of the formula ##SPC24##
The formula for the resulting lactone is written in this manner
since at least a part, if not all, of the .DELTA..sup.9 THC is
probably converted to the .DELTA..sup.8 THC along with the
formation of the .delta.-lactone in the polyphosphoric acid at
90.degree. C.
EXAMPLE 2
1-Hydroxy-3-pentyl-6a,7,8,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyr
an-2-sodium Acrylate
One milliliter of pH (0.2 molar) carbonate buffer was added to the
hexane layer of Example 1 and it was shaken with the hexane
solution for 30 minutes. The hexane layer was then aspirated off
and discarded. The fluorescence of the buffer layer was then read
using an excitation of 380 nm. The presence of ##SPC25##
was indicated by a fluorescence emission at 470 nm which was
significantly higher than that obtained by carrying 2 milliliters
of control plasma through the procedures of Examples 1 and 2.
The production of the fluorescent acrylate (or the corresponding
acrylic acid) is indicative of the presence of the cannabinoid
structure as represented by formula I. It will be seen that this
method of THC detection was capable of detecting the presence of as
little as 0.6 micrograms of the THC.
EXAMPLE 3
1-Hydroxy-3-pentyl-6a,7,10, 10a-tetrahydro
6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-acrylic acid
.delta.-lactone
The procedure of Example 1 was repeated by substituting a
concentrated solution of .DELTA..sup.8 THC
(1-hydroxy-3-pentyl-6a,7,10,10
a-tetra-hydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) for the
.DELTA..sup.9 THC Example 1. This resulted in the formation of the
compound of the formula ##SPC26##
EXAMPLE 4
1-Hydroxy-3-pentyl-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]py
ran-2-sodium Acrylate
The product lactone of Example 3 was subjected to the procedure
detailed in Example 2. An intense fluorescence at the excitation of
380 nm was noted.
The acrylate salt ##SPC27##
or of example 2 may easily be converted to the acrylic acid form by
reaction with a suitable acid such as hydrochloric acid.
EXAMPLE 5
1-Hydroxy-3-pentyl-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-acrylic
Acid .delta.-lactone
5 micrograms of cannabinol in 5 microliters of ethanol was added to
a 15-milliliter conical centrifuge tube with 500 micrograms of
malic acid and 0.5 milliliter of ethanol. The solvent was removed
by warming at 60.degree. C. and then 0.5 milliliter of the
polyphosphoric acid mixture of Example 1 was added. The tube was
stoppered and the contents heated at 90.degree. C. for 20 minutes.
After the contents were cooled, 1 milliliter of water was added and
the mixture was extracted with 7 milliliter of hexane. The hexane
layer was removed and washed with 5 milliliters of pH 5 citrate
buffer to form the product having the formula ##SPC28##
EXAMPLE 6
1-Hydroxy-3-pentyl-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2sodium
acrylate
A 6-milliliter aliquot of the hexane layer of Example 5 treated
with the pH 5 citrate buffer was then transferred to a dry
centrifuge tube and shaken for 30 minutes with 2 milliliters of pH
10 buffer. The fluorescence of the aqueous phase was recorded
(.lambda. excitation 380 nm, .lambda. emission 470 nm) and was
significantly higher than that of a reagent blank carried through
the procedures of Example 5 and this example. The intensity of the
fluorescence was about one-third that obtained with an equivalent
amount of .DELTA..sup.9 THC. The acrylate salt formed had the
formula ##SPC29##
EXAMPLE 7
2,4-Dihydroxy-5-(6-isopropenyl-3-methyl-2-cyclohexenyl)-6-pentyl-phenylacry
lic Acid 2-.delta.-lactone
5 micrograms of cannabidiol in 5 microliters of ethanol was added
to a 15-milliliter conical centrifuge tube with 500 micrograms of
malic acid in 0.5 milliliter of ethanol. The solvent was removed by
warming at 60.degree. C. and then 0.5 milliliter of the
polyphosphoric acid mixture of Example 1 was added. The tube was
stoppered and the contents were heated at 90.degree. C. for 20
minutes. After the contents were cooled, 1 milliter of water was
added and the mixture was extracted with 7 milliliters of hexane.
The hexane layer was removed and washed with 5 milliliters of pH 5
citrate buffer to form the product having the formula ##SPC30##
EXAMPLE 8
2,4-Dihydroxy-5-(6-isopropenyl-3-methyl-2-cyclohexenyl)-6-pentyl-phenyl-1-s
odium Acrylate
A 6-milliliter aliquot of the hexane layer of Example 7 was
transferred to a dry centrifuge tube and shaken for 30 minutes with
2 milliliters of pH 10 buffer. The fluorescence of the aqueous
phase was recorded (.lambda. excitation 380 nm, .lambda. emission
470 nm) and was significantly higher than that of a reagent blank
carried through the same procedure. The intensity of the
fluorescence obtained with the cannabidiol was about 3 times that
obtained with .DELTA..sup.9 THC and about 10 times that obtained
with cannabinol. The intensely fluorescent compound can be
represented by the formula ##SPC31##
It will be seen from the above description and from the examples
that there is here provided a method of identifying the presence of
a cannabinoid, and that the novel reactions disclosed may be used
as a test for the presence of a cannabinoid, e.g., one or more of
the active ingredients of marihuana, in a body fluid. The novel
acrylate salts of the cannabinoids as represented by formulas XXVI
and XXVII have utility as standards in a test procedure for
identifying cannabinoids and the novel .delta.-lactones of formulas
XXIV and XXV have utility as intermediates in the preparation of
compounds of the formulas XXVI and XXVII, respectively.
It will thus be seen that the objects set forth above, among those
made apparent from the preceding description, are efficiently
attained, and, since certain changes may be made in carrying out
the above method and in the compositions set forth without
departing from the scope of the invention, it is intended that all
matter contained in the above description shall be interpreted as
illustrative and not in a limiting sense.
* * * * *