NPC
2008 Vol. 3 No. 12 1977 - 1980
Natural Product Communications
A Polar Cannabinoid from Cannabis sativa var. Carma Giovanni Appendinoa*, Anna Gianaa, Simon Gibbonsb, Massimo Maffeic, Giorgio Gnavic, Gianpaolo Grassid and Olov Sternere* a
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Via Bovio 6, 28100 Novara, Italy
b
Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK c
Dipartimento di Biologia Vegetale e Centro di Eccellenza CEBIOVEM, Università di Torino, Viale Mattioli 25, 10125 Torino, Italy d
CRA-CIN Centro di Ricerca per le Colture Industriali, Sede Distaccata di Rovigo, Via Amendola 82, 45100 Rovigo, Italy e
Department of Organic Chemistry, Lund University, P.O. Box 124, 221 00 Lund, Sweden
[email protected];
[email protected] Received: July 29th, 2008; Accepted: October 15th, 2008
The aerial parts of Cannabis sativa var. Carma afforded a novel polar cannabinoid whose structure was established as rac-6’,7’-dihydro,6’,7’-dihydroxycannabigerol (carmagerol, 1) on the basis of spectroscopic data and semisynthesis from cannabigerol (2a). The dihydroxylation of the !-double bond was detrimental to the anti-bacterial activity. Keywords: Cannabis sativa, Cannabinaceae, carmagerol, cannabigerol, antibacterial activity.
The successful development of Sativex, a combination of Cannabis extracts, for the management of multiple sclerosis and cancer pain [1] has rekindled interest in the phytochemistry of Cannabis sativa L. Over 70 natural cannabinoids are known [2], most of them characterized in the 1960s and 70s in the wake of the identification of "9tetrahydrocannabinol (THC) as the psychotropic constituent of marijuana [3]. Recently, a series of very apolar terpenyl esters of pre-cannabinoids was reported from a THC-rich chemotype of marijuana [4], suggesting that the earlier investigations, focused on a defined range of polarity, might have missed minor compounds with higher or lower polarity than the major cannabinoids. We report here the isolation of the novel polar cannabinoid 1 from the Carma variety of hemp. This hemp is named after the Piedmontese town of Carmagnola, where the cultivation of the celebrated pest- and stress resistant homonymous fibre hemp thrived for centuries [5].
9'
HO 8'
10'
7'
3'
5' 6'
OH
4'
OH
1' 2'
2
1
HO
6
4
1
4''
2''
5
3
1''
3''
5''
OR
RO R 2a H 2b Ac
The minor (0.0045% isolation yield) cannabinoid 1 was named carmagerol because of its plant origin and its structrual relationship with cannabigerol (CBG, 2a). It was obtained from the polar fraction of an acetone extract from the aerial parts of Carma hemp. Its isolation involved filtration over RP-18 silica gel to eliminate fats and pigments, partition between aqueous methanol and light petroleum to remove most of CBG (2a), its major cannabinoid, and gravity
1978 Natural Product Communications Vol. 3 (12) 2008
column chromatography on silica gel. The 1H NMR spectrum of a fraction more polar than those containing the prenylated flavonoids cannflavins [6] showed a series of signals in the aliphatic region that suggested the presence of a compound with a gross cannabinoid structure. A pure sample was eventually obtained after preparative HPLC on silica gel. Carmagerol (1) was obtained as an optically inactive, colorless gum, and had molecular formula C21H34O4 (MS). Its 1H NMR spectrum was similar to that of CBG (2a) [7], except for the replacement of the terminal olefinic double bond with two oxygenated functions, as evidenced by the observation of an oxymethine resonance at # 3.35, and by the upfield shift of the allylic geminal methyls, resonating as sharp singlets at # 1.19 and 1.15. While the chemical shift of the oxymethine was compatible with the epoxidation of the !-double bond of the geranyl moiety of CBG, the 13C chemical shift of the corresponding carbon (# 78.2) and of that of the adjacent quaternary oxygenated carbon (# 73.2) showed that, in accordance with the molecular formula, the terminal double bond of the geranyl residue had undergone dihydroxylation and not epoxidation. To confirm the structure elucidation of carmagerol and obtain further amounts of the compound necessary to investigate its biological profile, a semisynthesis from CBG was undertaken. Racemic dihydroxylation with the Upjohn protocol gave a complex mixture, but, after acetylation, the asymmetric version of the reaction with AD-mix-! [8] afforded, in excellent yield, a compound identical, apart from the optical rotation, to the natural product. A compound with the gross formula of carmagerol was mentioned in a study on the mammal metabolism of cannabigerol. The major metabolic pathway was the hydroxylation of the allylic methyls, but epoxidation of the !-double bond was also observed, without, however, detecting its hydrolysis product, namely carmagerol [9]. The racemic nature of carmagerol is puzzling, since biological oxidations are generally enantioselective. On the other hand, auto-oxidation of geranylated phenols shows a strong bias toward the proximal, and not the terminal, double bond [10], and carmagerol was clearly detectable by HPLC in crude extracts of Carma hemps (see Experimental), in accordance with its natural origin.
Appendino et al.
While cannabigerol (2a) is a potent antibacterial agent, especially against the so called super-bugs (IC50 = 1 $g/mL against methicillin-resistant Staphylococcus aureus SA1199B) [11], the activity of carmagerol (1) was modest (IC50 = 32 $g/mL), showing that dihydroxylation of the !-double bond is detrimental to antibiotic activity, an important observation that points to the existence of strict structure-activity relationships within the antimicrobial cannabinoid chemotype. The characterization of a novel polar cannabinoid from Cannabis sativa suggests that, despite studies spanning almost 50 years and the identification of over 500 different constituents [2], modern phytochemical techniques can still lead to the isolation of new minor compounds missed by earlier studies and worth investigation in terms of bioactivity.
Experimental Plant material: Cannabis sativa var. Carma came from greenhouse cultivation at CRA-CIN, Rovigo (Italy), where a voucher specimen is kept, and was collected in November 2006. The isolation and manipulation of all cannabinoids was in accordance with their legal status (Licence SP/101 of the Ministero della Salute, Rome, Italy). Isolation of 1: The powdered plant material (500 g) was distributed as a thin layer on cardboard, and heated at 120°C for 2 h in a ventilated oven to affect decarboxylation, and then extracted with acetone (ratio solvent: plant material 3:1, x 3). The residue (20.5 g) was dissolved in methanol, adsorbed onto a pad of RP-18 silica gel (100 g), and washed with methanol (400 mL). The yellowish filtrate was evaporated and then partitioned between 10% aq. methanol (150 mL) and light petroleum (150 mL). The lower phase was separated, washed again with light petroleum (100 mL), and evaporated The residue (5.1 g) was purified by gravity column chromatograpy on silica gel (50 g), using a light petroleum-EtOAc gradient. Fractions eluted with light petroleum-EtOAc 4:6 were further purified by prep HPLC on a silica gel column (250 x 21.2 mm Chromasyl column) using light petroleum-EtOAc 3:7 as eluant to give 21 mg (0.0042%) 1a as a colorless foam.
Cannabinoids from Cannabis sativa
Natural Product Communications Vol. 3 (12) 2008 1979
Carmagerol nabigerol, 1)
petroleum-EtOAc 7:3, and filtered over silica gel (10 mL). The filtrate was evaporated, dissolved in THF (10 mL), and treated with pyrrolidine (3 mL, 24 mmol, 12 mol. equiv.). After heating at 50°C for 16 h, the reaction was cooled to room temp. and worked up by partition between 2N H2SO4 and EtOAc. The organic phase was separated, washed with brine, dried (Na2SO4), evaporated, and the residue purified by gravity column chromatography on silica gel (10 g, light petroleum-EtOAc 5:5 as eluant) to afford 700 mg 1a (overall 63% from 2a) as a colorless foam. [%]D = +51 (c 0.8, MeOH).
(6’,7’-Dihydro-6’-7’-dihydroxycan-
Colorless gum. Rf : 0.35 (light petroleum-EtOAc 4:6). IR (KBr): 3293, 3199, 1703, 1601, 1425, 1372, 1155, 1029, 833 cm-1. 1 H NMR (500 MHz, CDCl3): 0.89 (3H, t, J = 7.0 Hz, H-5’’), 1.15 (3H, s, H-10’), 1.19 (3H, s, H-8’), 1.29 (2H, m, H-3’’a,b), 1.31 (2H, m, H-4’’a,b), 1.44 (1H, m, H-5’a), 1.56 (2H, m, H-2’’a,b), 1.60 (1H, m, H-5’b), 1.82 (3H, s, H-9’), 2.13 (1H, m, H-4’a), 2.27 (1H, m, H-4’b), 2.44 (2H, t, J = 7.8 Hz, H-1’’a,b), 3.35 (1H, dd, J = 10.2, 2.1 Hz, H-6’), 3.38 (2H, t, J = 7.2 Hz, H-1’), 5.32 (1H, br t, J = 7.2 Hz, H-2’), 6.24 (2H, s, H-4 and H-6). 13 C NMR (125 MHz, CDCl3): 14.0 (t, C-5’’), 16.1 (q, C-9’), 22.2 (t, C-1’), 22.5 (t, C-4’’), 23.3 (q, C-10’), 26.3 (q, C-8’), 29.4 (t, C-5’), 30.8 (t, C-2’’), 31.4 (t, C-3’’), 35.5 (t, C-1’’), 36.9 (t, C-4’), 73.2 (s, C-7’), 78.2 (d, C-6’), 108.2 (d, C-4 and C-6), 110.9 (s, C-2), 122.8 (d, C-2’), 139.4 (s, C-3’), 146.2 (s, C-5), 154.8 (s, C-1 and C-3). CI-EIMS: m/z [M]+ 373 (M + Na)+, (C21H34O4 + 23)+. Synthesis of carmagerol (1) from cannabigerol (2a): To a stirred soln of AD-mix-% (3.5 g) in tert-ButOHwater (1:1, 10 mL), N-methylmorpholine oxide (875 mg, 7.5 mmol, 3 mol. equiv.), methansulfonamide (250 mg, 2.5 mmol, 1 mol. equiv.), and a soln of cannabigerol diacetate (2b, 1.0 g, 2.5 mmol; obtained from the treatment of 2a (1 g) with excess Ac2O (10 mL) in pyridine (10 mL) in tert-ButOH (7 mL) were added. After stirring at room temp. overnight, the reaction was worked up by the addition of sat. Na2SO3, and stirred at room temp. for 30 min. EtOAc was then added, and the reaction mixture was extracted with EtOAc. The organic phase was washed with brine, dried with Na2SO4, and evaporated. The residue was taken up in light
Analysis of carmagerol in Cannabis sativa var. Carma: A 5 g sample of plant material was extracted with acetone (3 x 30 mL) by stirring at room temp. (10 min. for each extraction). The pooled extracts were evaporated, and a portion of the residue (100 mg) was dissolved in 1 mL methanol and filtered in a Pasteur pipette over Celite (50 mg). The filtration pad was washed with 0.5 mL methanol, and the pooled filtrates were diluted with 0.15 mL water. After washing twice with light petroleum (2 x 2.5 mL) to remove most of the apolar cannabinoids, the lower methanol phase was evaporated, taken up in 200 mL methanol, and analyzed by HPLC (C-18 column, detection at 210 nm). The following conditions were employed: Solvent A: 0.5% orthophosphoric acid in water; Solvent B: acetonitrile. Gradient: from 0 to 8 min, 60% A, 40% B; from 9 to 14 min, 50% A, 50% B, from 15 to 24 min 10% A, 90% B, from 25 to 30 min 1% A and 99% B. The Rt of 1 was 13.8 min, and its concentration was in the range of 56-98 mg/Kg depending on the sample analyzed Acknowledgments – We are grateful to Dr Lucia Maxia (Università del Piemonte Orientale, Faculty of Pharmacy) for her help in the isolation of carmagerol.
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Perez J, Ribera MV. (2008) Managing neuropathic pain with Sativex: a review of pros and cons. Expert Opinions in Pharmacotherapy, 9, 1189-1195.
[2]
El-Sohly MA, Slade D. (2005) Chemical constituents from Marijuana: The complex mixture of natural cannabinoids. Life Sciences, 78, 539-548.
[3]
Mechoulam R, Gaoni Y (1964) Isolation, structure and partial synthesis of an active constituent of hashish. Journal of American Chemical Society, 86, 1646-1647.
[4]
Ahmed SA, Ross SA, Slade D, Radwan MM, Zulfiquar F, Elsohly MA. (2008) Cannabinoid ester constituents from high-potency Cannabis sativa. Journal of Natural Products, 71, 536-542.
[5]
The name of the famous French revolutionary song “la Carmagnole” was in fact inspired by the trade of this variety of hemp, characterized by very long fibers and insuperable for the manufacture of naval ropes, from Carmagnola and Marseille. See: http://architettura.supereva.com/image/festival/1998/en/bonino.htm
1980 Natural Product Communications Vol. 3 (12) 2008
Appendino et al.
[6]
Minassi A, Giana A, Ech Chahad A, Appendino G. (2008) A regiodivergent synthesis of ring C prenylflavones. Organic Letters, 10, 2257-2270.
[7]
Choi YH, Hazekamp A, Peltenburg-Looman AMG, Frédérich M, Erkelens C, Lefeber AWM, Verpoorte R. (2004) NMR assignments of the major cannabinoids and cannabiflavonoids isolated from flowers of Cannabis sativa. Phytochemical Analysis, 15, 345-354.
[8]
For a related example, see: Vidari G, Di Rosa A, Castronovo F, Zanoni G. (2000) Enantioselective synthesis of each stereoisomer of the pyranoid linalool oxides: the geraniol route. Tetrahedron Asymmetry, 11, 981-989.
[9]
Harvey, D, Brown NK. (1990) In vitro metabolism of cannabigerol in several mammalian species. Biomedical & Environmental Mass Spectrometry, 19, 545-553.
[10]
A series of cannabigerol derivatives epoxidized on the proximal double bond have recently been reported (Radwan MM, Ross SA, Slade D, Ahmed SA, Zulfiquar F, Elsohly MA. (2008) Isolation and characterization of new Cannabis constituents from a high potency variety, Planta Medica, 74, 267-272.
[11]
Appendino G, Gibbons S, Giana A, Pagani A, Grassi G, Stavri M, Smith E, Mukhlesur Rahman M. (2008) Antibacterial cannabinoids from Cannabis sativa. A structure-activity study. Journal of Natural Products, 71, 1427-1430.
