Synthesis and spectroscopic characterization of cannabinolic acid

Article abstract of DOI:10.1055/s-2007-967129

Cannabinoids, the main constituents of the cannabis plant, are being increasingly studied for their medicinal properties. Cannabinolic acid (CBNA; 1) was synthesized from tetrahydrocannabinolic acid (THCA; 2), a major constituent of the cannabis plant, by aromatization using selenium dioxide mixed with trimethylsilyl polyphosphate as catalyst in chloroform. Purification was achieved by centrifugal partition chromatography, and the final product had a purity of over 96% by GC analysis. Spectroscopic data on CBNA such as ¹H-NMR and IR, and molar extinction coefficients, as well as chromatographic data are presented as useful references for further research on CBNA. The developed method allows production of CBNA on a preparative scale, making it available for further studies on its biological activities as well as use as a reference standard for analytical procedures. Georg Thieme Verlag KG Stuttgart.

Full text of DOI:10.1055/s-2007-967129

In this letter, we describe the production of CBNAby dehydro-
genation of THCAusing selenium dioxide mixed with trimethyl-
silyl polyphosphate (PPSE) as the catalyst in carbon tetrachloride
Synthesis and Spectroscopic
Characterization of Cannabinolic Acid
(see Fig.1) [10]. As a minor modification, we found that carbon
Krishna Prasad Bastola, Arno Hazekamp, Robert Verpoorte
tetrachloride could be replaced by the less toxic chloroform
without effects on the final transformation yield. Finally, the sig-
nificant amount of 26 mg of purified CBNAwas obtained in a sin-
gle experiment. The final product was highly pure (96% by GC
analysis), therefore rendering a quantified CBNAsolution suitable
Abstract
Cannabinoids, the main constituents of the cannabis plant, are for use as reference standard for analytical or biological studies.
being increasingly studied for their medicinal properties. Canna- Under the selected conditions for LC-MS analysis, isolated CBNA
binolic acid (CBNA; 1) was synthesized from tetrahydrocannabi- was mildly fragmented. Nevertheless, the highest intensity peak
nolic acid (THCA; 2), a major constituent of the cannabis plant, was the decarboxylated [M±CO ] product, indicating the relative
2
by aromatization using selenium dioxide mixed with trimethyl- instability of the carboxylic group.
silyl polyphosphate as catalyst in chloroform. Purification was
achieved by centrifugal partition chromatography, and the final Unfortunately, the conversion rate under the applied conditions
product had a purity of over 96% by GC analysis. Spectroscopic was only 10%. Reaction products other than the starting material
1
data on CBNAsuch as H-NMR and IR, and molar extinction coef- 2 or the desired product 1 were not further identified. However,
ficients, as well as chromatographic data are presented as useful the described method for dehydrogenation is relatively simple
references for further research on CBNA. The developed method and well described [10], and THCAis easy to obtain in large
allows production of CBNAon a preparative scale, making it
amounts from cannabis plant materials [7]. Purification can be
available for further studies on its biological activities as well as achieved by centrifugal partition chromatography (CPC), a tech-
use as a reference standard for analytical procedures.
nique which permits easy up-scaling. Therefore, the described
procedure allows the production of analytical grade CBNAon a
preparative scale.
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
1
Full spectroscopic data for compound 1 (UV, IR, H-NMR, MS) are
presented as Supporting Information, facilitating studies on the
The cannabis plant (Cannabis sativa L., Cannabaceae) is under in- role of CBNAas a component of cannabis products. The spectro-
tense study for its medicinal properties in a variety of illnesses scopic and chromatographic data were published in a systematic
such as multiple sclerosis, Gilles de la Tourette syndrome, chron- manner, complementing the data that were earlier obtained on
ic pain and wasting syndrome associated with AIDS/HIV and an- 17 natural cannabinoids [3].
orexia [1]. Although so far at least 489 compounds have been
273
identified in cannabis [2], most studies focus on the effects of
the cannabinoids. As a contribution to the study of the lesser Materials and Methods
known cannabinoids, we recently published the standardized
spectroscopic and chromatographic data of a variety of natural Chemicals and solvents: Selenium dioxide (SeO , purity > 98%, re-
2
cannabinoids [3]. In that study, data on cannabinolic acid agent grade), hexamethyldisiloxane (HMDSO, purity > 98%) and
(
CBNA; 1) was incomplete, due to unavailability of a calibrated phosphorus pentoxide (P O , purity > 97%) were purchased from
2 5
standard. Compound 1 is formed during storage and aging of Sigma-Aldrich (St. Louis, MO, USA). Organic solvents (analytical
plant samples by degradation of tetrahydrocannabinolic acid or HPLC reagent grade) were purchased from J.T. Baker (Deven-
(
THCA; 2), a major component of cannabis resin [4], [5]. The bio- ter, The Netherlands). Cannabinoid standards for THCAand CBN
logical activities of 1 have not been studied in detail, and analy- (purity ³ 98%) were produced and quantified as previously re-
tical study is complicated by the fact that the published spectro- ported [7], [8].
scopic data are incomplete. Although a full synthesis of the
closely related cannabinol (CBN; 3) has been described [6], the Synthesis: PPSE was prepared from P O and HMDSO [9]. Thus,
2
5
synthesis and/or preparative isolation of CBNA( 1) has not been HMDSO in chloroform (12% v/v) was refluxed for 30 minutes un-
reported.
der nitrogen gas, followed by addition of P O (50 mg/mL) and
2
5
additional refluxing for 2 hours. The clear chloroform phase, con-
taining PPSE, was separated from residual solid P O and trans-
2
5
Affiliation: Leiden University, Department of Pharmacognosy, Gorlaeus La- ferred to a reaction vessel. SeO (30 mg/mL final concentration)
2
boratories, Leiden, The Netherlands
and THCA(dissolved in chloroform, 50 mg/mL final concentra-
Correspondence: Arno Hazekamp ´ Leiden University ´ Department of Pharma- tion) were added, giving a molar ratio between SeO and sub-
2
cognosy ´ Gorlaeus Laboratories ´ Einsteinweg 55 ´ 2333CC, Leiden ´ The Nether-
lands ´ Phone: +31-71-527-4784 ´ Fax: +31-71-527-4511 ´ E-mail: ahazekamp@
rocketmail.com
strate of circa 2:1 [10]. The resultant mixture was mildly
refluxed for 6±8 hours to allow dehydrogenation of THCA. Sub-
sequently, the liquid phase containing the cannabinoids was sep-
Received: October 20, 2006 ´ Accepted: January 31, 2007
arated from the solid SeO . The liquid phase was evaporated
2
Bibliography: Planta Med 2007; 73: 273±275 ꢀ Georg Thieme Verlag KG Stutt- under vacuum and reconstituted in hexane, resulting in precipi-
gart ´ New York ´ DOI 10.1055/s-2007-967129 ´ Published online March 12,
tation of PPSE. The hexane fraction contained crude CBNA.
2
007 ´ ISSN 0032-0943

Fig. 1 Chemical structures ofcannabinolic
acid (1), tetrahydrocannabinolic acid (2)
and cannabinol (3). The formation of 1 by
dehydrogenation ofring A of 2 is indicated.
Isolation and characterization: Purified CBNA( 1) (26 mg) was ob- tion of isolated CBNAin ethanol solution was performed by
1
tained by fractionation of the crude synthesis sample by centri- quantitative H-NMR [8].
fugal partition chromatography, using hexane/methanol/water,
5
:3:2 (v/v/v) with 0.1% formic acid [7]. The eluent was moni- Cannabinolic acid (1): greenish oil; R = 0.25, silica gel 60 F254,
f
tored at the maximal absorption wavelength for CBNAof 261
nm. Fractions containing CBNAwere detected by LC-D AD -MS.
The purified compound was positively identified by comparing (4.30), 324 (4.11); IR (KBr): nmax = 2925, 1620, 1260 cm ; H-
MeOH/H O/acetic acid (19:1:0.05); Rf = 0.54, RP-18 F254,
2
CHCl /MeOH (19:1); UV (EtOH): l
(log e) = 261 (4.70), 298
3
max
±
1
1
retention times in HPLC and GC [3] and spectroscopic data NMR (CDCl , 300 MHz): d = 8.40 (1H, s, H-10), 7.11 (2H, dd,
3
(
HPLC-DAD-MS) to literature data [3], [11], [12] (Figs. 1S±3S, J = 12.31, 8.58 Hz, H-7, H-8), 6.40 (1H, s, H-4), 2.96 (2H, t,
1
Supporting Information). Aquantitative H-NMR method was J = 7.78 Hz, H-1¢), 2.38 (3H, s, H-11), 2.15 (2H, m, H-2¢), 1.60 (6H,
used to prepare a quantified ethanolic solution of CBNA[8]. The
purity of isolated 1 was determined by GC analysis at a concen- 5¢); APCI-MS: m/z = 355.2 [M + H ], 337.2 [M±H O], 311.2 [M±
s, H-6a, H-6b), 1.32 (4H, m, H-3¢, H-4¢), 0.83 (3H, t, J = 6.91 Hz, H-
+
2
tration of 1 mg/mL (5 mL injected). The quantified solution was CO2].
used to measure the molar extinction coefficients of CBNAin
the range of 200±400 nm and to measure the infrared (IR) spec-
trum in FT-IR (Fig. 4 S, Supporting Information) [3].
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