Antibacterial cannabinoids from Cannabis sativa: A structure-activity study

Article abstract of DOI:10.1021/np8002673

Marijuana (Cannabis sativa) has long been known to contain antibacterial cannabinoids, whose potential to address antibiotic resistance has not yet been investigated. All five major cannabinoids (cannabidiol (lb), cannabichromene (2), cannabigerol (3b), Δ⁹-tetrahydrocannabinol (4b), and cannabinol (5)) showed potent activity against a variety of methicillin- resistant Staphylococcus aureus (MRSA) strains of current clinical relevance. Activity was remarkably tolerant to the nature of the prenyl moiety, to its relative position compared to the n-pentyl moiety (abnormal cannabinoids), and to carboxylation of the resorcinyl moiety (pre-cannabinoids). Conversely, methylation and acetylation of the phenolic hydroxyls, esterification of the carboxylic group of pre-cannabinoids, and introduction of a second prenyl moiety were all detrimental for antibacterial activity. Taken together, these observations suggest that the prenyl moiety of cannabinoids serves mainly as a modulator of lipid affinity for the olivetol core, a per se poorly active antibacterial pharmacophore, while their high potency definitely suggests a specific, but yet elusive, mechanism of activity.

Full text of DOI:10.1021/np8002673

J. Nat. Prod. 2008, 71, 1427–1430
1427
Antibacterial Cannabinoids from Cannabis satiWa: A Structure-Activity Study
Giovanni Appendino,*,†,‡ Simon Gibbons,*, Anna Giana,†,‡ Alberto Pagani,†,‡ Gianpaolo Grassi,§ Michael Stavri,
Eileen Smith, and M. Mukhlesur Rahman
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, UniVersita` del Piemonte Orientale, Via BoVio 6,
28100 NoVara, Italy, Consorzio per lo Studio dei Metaboliti Secondari (CSMS), Viale S. Ignazio 13, 09123 Cagliari, Italy, Centre for
Pharmacognosy and Phytotherapy, The School of Pharmacy, UniVersity of London, 29-39 Brunswick Square, London WC1N 1AX, U.K., and
CRA-CIN Centro di Ricerca per le Colture Industriali, Sede distaccata di RoVigo, Via Amendola 82, 45100 RoVigo, Italy
ReceiVed May 1, 2008
Marijuana (Cannabis satiVa) has long been known to contain antibacterial cannabinoids, whose potential to address
antibiotic resistance has not yet been investigated. All five major cannabinoids (cannabidiol (1b), cannabichromene (2),
cannabigerol (3b), ∆9-tetrahydrocannabinol (4b), and cannabinol (5)) showed potent activity against a variety of
methicillin-resistant Staphylococcus aureus (MRSA) strains of current clinical relevance. Activity was remarkably tolerant
to the nature of the prenyl moiety, to its relative position compared to the n-pentyl moiety (abnormal cannabinoids),
and to carboxylation of the resorcinyl moiety (pre-cannabinoids). Conversely, methylation and acetylation of the phenolic
hydroxyls, esterification of the carboxylic group of pre-cannabinoids, and introduction of a second prenyl moiety were
all detrimental for antibacterial activity. Taken together, these observations suggest that the prenyl moiety of cannabinoids
serves mainly as a modulator of lipid affinity for the olivetol core, a per se poorly active antibacterial pharmacophore,
while their high potency definitely suggests a specific, but yet elusive, mechanism of activity.
Several studies have associated the abuse of marijuana (Cannabis
satiVa L. Cannabinaceae) with an increase in opportunistic infec-
tions,1 and inhalation of marijuana has indeed been shown to
interfere with the production of nitric oxide from pulmonary
macrophages, impairing the respiratory defense mechanisms against
pathogens and causing immunosuppression.2 The association of C.
satiVa with a decreased protection against bacterial infections is
paradoxical, since this plant has long been known to contain
powerful antibacterial agents.3 Thus, preparations from C. satiVa
were investigated extensively in the 1950s as highly active topical
antiseptic agents for the oral cavity and the skin and as antituber-
cular agents.3 Unfortunately, most of these investigations were done
at a time when the phytochemistry of Cannabis was still in its
infancy, and the remarkable antibacterial profile of the plant could
not be related to any single, structurally defined and specific
constituent. Evidence that pre-cannabidiol (1a) is a powerful plant
antibiotic was, nevertheless, obtained,4 and more recent investiga-
tions have demonstrated, to various degrees, antibacterial activity
for the nonpsychotropic cannabinoids cannabichromene (CBC, 2),5
class of antibacterial has been introduced in the last 30 years.9
cannabigerol (CBG, 3b),6 and cannabidiol (1b),7 as well as for the
Despite the excellent antibacterial activity of many plant secondary
psychotropic agent ∆9-tetrahydrocannabinol (THC, 4b).7 These
metabolites10 and the ability of some of them to modify the
observations, and the inactivity of several noncannabinoid con-
resistance associated with MDR strains11 and efflux pumps,12 plants
stituents of C. satiVa as antibacterial agents, suggest that cannab-
are still a substantially untapped source of antimicrobial agents.
inoids and their precursors are the most likely antibacterial agents
These considerations, as well as the observation that cross-
present in C. satiVa preparations.8 However, differences in bacterial
resistance to microbial and plant antibacterial agents is rare,10 make
strains and end-points make it difficult to compare the data reported
C. satiVa a potential source of compounds to address antibiotic
in these scattered studies, and the overall value of C. satiVa as an
resistance, one of the most urgent issues in antimicrobial therapy.
antibacterial agent is therefore not easy to assess.
To obtain structure-activity data and define a possible microbio-
There are currently considerable challenges with the treatment
cidal cannabinoid pharmacophore, we investigated the antibacterial
of infections caused by strains of clinically relevant bacteria that
profile of the five major cannabinoids, of their alkylation and
show multidrug-resistance (MDR), such as methicillin-resistant
acylation products, and of a selection of their carboxylic precursors
Staphylococcus aureus (MRSA) and the recently emerged and
(pre-cannabinoids) and synthetic positional isomers (abnormal
extremely drug-resistant Mycobacterium tuberculosis XDR-TB.
cannabinoids).
New antibacterials are therefore urgently needed, but only one new
Results and Discussion
* To whom correspondence should be addressed. Tel: +39 0321
The antibacterial cannabinoid chemotype is poorly defined, as
is the molecular mechanism of its activity. Since many simple
phenols show antimicrobial properties, it does not seem unreason-
able to assume that the resorcinol moiety of cannabinoids serves
as the antibacterial pharmacophore, with the alkyl, terpenoid, and
carboxylic appendices modulating its activity. To gain insight into
373744 (G.A.); +44 207 753 5913 (S.G.). Fax: +39 0321 375621 (G.A.);
+44 207 753 5909 (S.G.). E-mail: appendino@pharm.unipmn.it (G.A.);
simon.gibbons@pharmacy.ac.uk (S.G.).
† Universita` del Piemonte Orientale.
‡ Consorzio per lo Studio dei Metaboliti Secondari.
University of London.
§ Centro Ricerca Colture Industriali.
10.1021/np8002673 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/06/2008

1428 Journal of Natural Products, 2008, Vol. 71, No. 8
Appendino et al.
properties. However, in light of the potent activity of the monophe-
nols CBC (2), THC (4b), and CBN (5), it was surprising that
monomethylation of the diphenols CBD (1b) and CBG (3b) was
so poorly tolerated in terms of antibacterial activity.
Cannabinoids are the products of thermal degradation of their
corresponding carboxylic acids (pre-cannabinoids).17 Investigation
of the antibacterial profile of the carboxylated versions of CBD,
CBG, and THC (compounds 1a, 3a, and 4a, respectively) showed
a substantial maintenance of activity. On the other hand, methylation
of the carboxylic group (compounds 1f and 3f, respectively) caused
a marked decrease of potency, as did esterification with phenethyl
alcohol (compounds 1g and 3g, respectively). This operation is
associated with a potentiation of the antibacterial properties of
phenolic acids, as exemplified by phenethyl caffeate (CAPE), the
major antibacterial from propolis, compared to caffeic acid.20
Remarkably, the synthetic abnormal cannabinoids abn-CBD (6)21
and abn-CBG (7)22 showed antibacterial activity comparable to,
although slightly less potent than, their corresponding natural
products, while olivetol (10) showed modest activity against all
six strains, with MICs of 64-128 µg/mL, and resorcinol (11) did
not exhibit any activity even at 256 µg/mL. Thus, the pentyl chain
and the monoterpene moiety greatly enhance the activity of
resorcinol.
the microbiocidal cannabinoid pharmacophore, we have investigated
how the nature of the terpenoid moiety, its relative position
compared to the n-pentyl group, and the effect of carboxylation of
the resorcinyl moiety are translated biologically, assaying the major
cannabinoids and a selection of their precursors and regioisomeric
analogues against drug-resistant bacteria of clinical relevance.
Within these, we have selected a panel of clinically relevant
Staphylococcus aureus strains that includes the (in)famous EMRSA-
15, one of the main epidemic methicillin-resistant strains,13 and
SA-1199B, a multidrug-resistant strain that overexpresses the NorA
efflux mechanism, the best characterized antibiotic efflux pump in
this species.14 SA-1199B also possesses a gyrase mutation that, in
addition to NorA, confers a high level of resistance to certain
fluoroquinolones. A macrolide-resistant strain (RN4220),15 a tet-
racycline-resistant line overexpressing the TetK efflux pump
(XU212),16 and a standard laboratory strain (ATCC25923) com-
pleted the bacterial panel.
∆9-Tetrahydrocannabinol (THC, 4b), cannabidiol (CBD, 1b),
cannabigerol (CBG, 3b), cannabichromene (CBC, 2), and cannab-
inol (CBN, 5) are the five most common cannabinoids.17 They could
be obtained in high purity (>98%) by isolation from strains of C.
satiVa producing a single major cannabinoid (THC, CBD, CBG),
by total synthesis (CBC),6 or by semisynthesis (CBN).18 Their
antimicrobial properties are listed in Table 1. All compounds
showed potent antibacterial activity, with MIC values in the 0.5-2
µg/mL range. Activity was exceptional against some of these strains,
in particular the multidrug-resistant (MDR) SA-1199B, which has
a high level of resistance to certain fluoroquinolones. Also
noteworthy is the potent activity demonstrated against EMRSA-
15 and EMRSA-16, the major epidemic methicillin-resistant S.
aureus strains occurring in U.K. hospitals.13,19 These activities
compare highly favorably with the standard antibiotics for these
strains. The potent activity against strains possessing the NorA and
TetK efflux transporters suggests that cannabinoids are not sub-
strates for the most common resistance mechanisms to current
antibacterial agents, making them attractive antibacterial leads.
Given their nonpsychotropic profiles, CBD (1b) and CBG (3b)
seemed especially promising, and were selected for further
structure-activity studies. Thus, acetylation and methylation of their
phenolic hydroxyls (compounds 1c-e and 3c-e, respectively) were
both detrimental for activity (MIC >100 µg/mL), in accordance
with the essential role of the phenolic hydroxyls in the antibacterial
Taken together, these observations show that the cannabinoid
antibacterial chemotype is remarkably tolerant to structural modi-
fication of the terpenoid moiety and its positional relationship with
the n-pentyl chain, suggesting that these residues serve mainly as
modulators of lipid affinity, and therefore cellular bioavailability.
This view was substantiated by the marked decrease of activity
observed when the antibacterial activity of CBG (3b) was compared
to that of its polar analogue carmagerol (8).23 The results against
the resistant strains confirm this suggestion, and it is likely that the
increased hydrophilicity caused by the addition of two hydroxyls
greatly reduces the cellular bioavailability by substantially reducing
membrane permeability. Conversely, the addition of a further prenyl
moiety, as in the bis-prenylated cannabinoid 9,21 while increasing
membrane solubility, may result in poorer aqueous solubility and
therefore a lower intracellular concentration, similarly leading to a
substantial loss of activity. A single unfunctionalized terpenyl
moiety seems therefore ideal in terms of lipophilicity balance for
the antibacterial activity of olivetol derivatives. The great potency
of cannabinoids suggests a specific interaction with a bacterial
target, whose identity is, however, still elusive.
Given the availability of C. satiVa strains producing high
concentrations of nonpsychotropic cannabinoids, this plant repre-
sents an interesting source of antibacterial agents to address the
problem of multidrug resistance in MRSA and other pathogenic
bacteria. This issue has enormous clinical implications, since MRSA

Antibacterial Cannabinoids from Cannabis satiVa
Journal of Natural Products, 2008, Vol. 71, No. 8 1429
Table 1. MIC (µg/mL) Values of Cannabinoids and Their Analogues toward Various Drug-Resistant Strains of Staphylococcus
aureusa,b
compound
SA-1199B
RN-4220
XU212
ATCC25923
EMRSA-15
EMRSA-16
1a
1b
2
3a
2
1
2
2
1
2
2
2
1
1
4
2
0.5
2
2
1
2
2
2
1
2
4
4
4
3b
3f
4a
4b
1
64
8
2
1
1
1
64
8
1
1
1
2
1
c
c
c
c
4
1
1
4
1
1
8
2
1
4
0.5
c
5
6
7
8
10
1
2
1
1
1
1
1
16
128
1
0.25
0.25
0.125
1
2
1
c
32
64
128
>128
0.125
>128
0.5
16
64
4
>128
128
128
32
64
32
0.25
0.25
0.25
32
64
1
64
0.25
0.25
16
64
0.5
>128
0.125
32
norfloxacin
erythromycin
tetracycline
oxacillin
a Compounds 1c-g, 3c-e, 3g, and 9 exhibited MIC values of >128 µg/mL for all organisms in which they were evaluated. b Compound 11
exhibited MIC values of >256 µg/mL for all organisms in which they were evaluated. c Not tested.
chemotype) and 3b (561 mg, 0.56%, from the CBG chemotype),
precipitated from hot hexane to obtain white powders. Crude THC (3.2
g, 3.2%, from the THC chemotype) was obtained as a greenish oil,
part of which (400 mg) was further purified by RP-18 flash chroma-
tography with methanol-water (1:1) as eluant, affording 4b as a
colorless oil (315 mg).
is spreading throughout the world and, in the United States, currently
accounts for more deaths each year than AIDS.24 Although the use
of cannabinoids as systemic antibacterial agents awaits rigorous
clinical trials and an assessment of the extent of their inactivation
by serum,25 their topical application to reduce skin colonization
by MRSA seems promising, since MRSA resistant to mupirocin,
the standard antibiotic for this indication, are being detected at a
threatening rate.26 Furthermore, since the cannabinoid anti-infective
chemotype seems remarkably tolerant to modifications in the prenyl
moiety, semipurified mixtures of cannabinoids could also be used
as cheap and biodegradable antibacterial agents for cosmetics and
toiletries, providing an alternative to the substantially much less
potent synthetic preservatives, many of which are currently
questioned for their suboptimal safety and environmental profile.27
Isolation of Pre-cannabinoids (1a, 3a, 4a). The powdered plant
material (100 g) was extracted with acetone (ratio solvent to plant
material 5:1, ×3). After removal of the solvent, the residue (7.7 g for
the CBD chemotype, 4.9 g for the CBG chemotype, 7.9 g for the THC
chemotype) was fractionated by vacuum chromatography on RP-18
silica gel (ratio stationary phase to extract 5:1) using methanol-water
(75:25) as eluant. Fractions of 100 mL were taken, and those containing
pre-cannabinoids were pooled, concentrated to ca. half-volume at 30
°C, saturated with NaCl, and extracted with EtOAc. After removal of
the solvent, the residue was further purified by gravity column
chromatography on silica gel (ratio stationary phase to crude compound
5:1) using a petroleum ether-EtOAc gradient (from 8:2 to 5:5) to afford
1.59 g (1.6%) of 1a from the CBD chemotype, 0.93 g (0.93%) of 3a
from the CBG chemotype, and 2.1 g (2.1%) of 4a from the THC
chemotype. All pre-cannabinoids were obtained as white foams that
resisted crystallization.
Experimental Section
General Experimental Procedures. IR spectra were obtained on a
1
Shimadzu DR 8001 spectrophotometer. H NMR (300 MHz) and 13C
NMR (75 MHz) spectra were obtained at room temperature with a JEOL
Eclipse spectrometer. The spectra were recorded in CDCl3, and the
solvent signals (7.26 and 77.0 ppm, respectively) were used as reference.
The chemical shifts (δ) are given in ppm, and the coupling constants
(J) in Hz. Silica gel 60 (70-230 mesh) and Lichroprep RP-18 (25-40
mesh) were used for gravity column chromatography. Reactions were
monitored by TLC on Merck 60 F254 (0.25 mm) plates and were
visualized by UV inspection and/or staining with 5% H2SO4 in ethanol
and heating. Organic phases were dried with Na2SO4 before evaporation.
All known cannabinoids were identified according to their physical and
spectroscopic data.28 Semisynthetic cannabinoids 1c-f, and 3c-f were
prepared and identified according to their corresponding literature
Synthesis of CBC (2) and CBN (5). CBG (2) was synthesized from
olivetol,6 and CBN was prepared from THC (6) by aromatization with
sulfur.18
Mitsunobu Esterification of Pre-cannabinoids (synthesis of 3g
as an example). To a cooled (ice bath) solution of 3a (360 mg, 1.1
mmol) in dry CH2Cl2 (4 mL) were added sequentially phenethyl alcohol
(92 µL, 0.76 mmol, 0.75 molar equiv), triphenylphosphine (TPP) (220
mg, 0.84 mmol, 0.80 molar equiv), and diisopropyldiazodicarboxylate
(DIAD) (228 µL, 1.1 mmol, 1 molar equiv). At the end of the addition,
the cooling bath was removed, and the reaction was stirred at room
temperature. After 16 h, the reaction was worked up by evaporation,
and the residue was dissolved in toluene and cooled at 4 °C overnight
to remove most of the TPPO-dihydroDIAD adduct. The filtrate was
evaporated and purified by gravity column chromatography on silica
gel (10 g, petroleum ether as eluant) to afford 126 mg (32%) of 3g.
Under the same reaction conditions, the yield of 1g from 1a was 26%.
references.22,29,30 Synthetic [abnormal (6,21 76) and polyprenyl (9)21
]
cannabinoids were synthesized and characterized according to the
literature.
Plant Material. The three strains of Cannabis satiVa used for the
isolation of THC, CBD, and CBG came from greenhouse cultivation
at CRA-CIN, Rovigo (Italy), where voucher specimens are kept for
each of them, and were collected in September 2006. The isolation
and manipulation of all cannabinoids were done in accordance with
their legal status (License SP/101 of the Ministero della Salute, Rome,
Italy).
Isolation of Cannabinoids (1b, 3b, 4b). The powdered plant
material (100 g) was distributed in a thin layer on cardboard and heated
at 120 °C for 2 h in a ventilated oven to affect decarboxylation, then
extracted with acetone (ratio solvent to plant material 3:1, ×3). The
residue (6.5 g for the CBD chemotype, 4.1 g for the CBG chemotype,
7.4 g for the THC chemotype) was purified by gravity column
chromatography on silica gel (ratio stationary phase to extract 6:1) using
a petroleum ether-ether gradient. Fractions eluted with petroleum
ether-ether (9:1) afforded 1b (628 mg, 0.63%, from the CBD
Pre-cannabigerol Phenethyl Ester (3g): colorless foam; IR νKBr
max
3746, 3513, 3313, 1715, 1589, 1421, 1274, 1164, 980, 804, 690 cm-1
;
1H NMR (300 MHz, CDCl3) δ 12.08 (1H, s), 7.25 (5H, m), 6.02 (1H,
s), 5.98 (1H, s), 5,25 (1H, br t, J ) 7.0 Hz), 5.01 (1H, br t, J ) 6.5
Hz), 4.56 (2H, t, J ) 6.6 Hz), 3.40 (2H, d, J ) 7.3 Hz), 3.1 (2H, t, J
) 6.6 Hz), 2.7 (2H, t, J ) 6.6 Hz), 2.05 (4H, m), 1.79 (3H, s), 1.65
(3H, s), 1.57 (3H, s), 1.24 (6H, m), 0.88 (3H, t, J ) 7.1 Hz); 13C NMR
(75 MHz, CDCl3) δ 172.1 (s), 162.7 (s), 159.5 (s), 148.8 (s), 139.1 (s),
137.4 (d), 132.1 (s), 128.8 (d), 126.8 (d), 125.9 (d), 121.5 (d), 111.5
(s), 110.8 (s), 65.8 (t), 39.8 (t), 36.6 (t), 35.0 (t), 32.0 (t), 31.5 (t), 26.5
(t), 25.8 (q), 22.2 (t), 17.8 (q), 16.3 (q), 14.2 (q); CIMS m/z [M + H]
465 [C30H40O4 + H].

1430 Journal of Natural Products, 2008, Vol. 71, No. 8
Appendino et al.
schaften 1958, 45, 188. (c) Krejci, Z. Pharmazie 1958, 13, 155–156.
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Pre-cannabidiol Phenethyl Ester (1g): colorless oil; IR (KBr) νmax
3587, 3517, 3423, 3027, 1642, 1499, 1425, 1274, 1172, 1143, 980,
894 cm-1; 1HNMR (300 MHz, CDCl3) δ 12.13 (1H, s), 6.23 (5H, m),
6.48 (1H, s), 6.19 (1H, s), 5,55 (1H, s), 4.52 (3H, m), 4.4 (1H, s), 4.08
(1H, br s), 3.08 (2H, t, J ) 7.0 Hz), 2.7 (2H, m), 2.11 (1H, m), 1.78
(3H, s), 1.71 (3H, s), 1.5 (4H, m), 1.28 (6H, m), 0.88 (3H, t, J ) 6.9
Hz); 13C NMR (75 MHz, CDCl3) δ 172.2 (s), 171.5 (s), 163.5 (s), 160.0
(s), 148.8 (s), 147.0 (s), 145.9 (s), 140.2 (s), 137.4 (s), 128.7 (d), 126.7
(d), 124.0 (d), 114.4 (t), 112.3 (d), 105.8 (s), 65.6 (t), 46.6 (d), 39.1
(t), 37.0 (d), 31.9 (d), 31.5 (t), 27.8 (t), 25.3 (q), 22.6 (t), 21.9 (t), 18.5
(q), 14.1 (q); CIMS m/z [M + H] 463 [C30H38O4 + H].
Bacterial Strains and Chemicals. A standard S. aureus strain
(ATCC 25923) and a clinical isolate (XU212), which possesses the
TetK efflux pump and is also a MRSA strain, were obtained from E.
Udo.16 Strain RN4220, which has the MsrA macrolide efflux pump,
was provided by J. Cove.30 EMRSA-1513 and EMRSA-1619 were
obtained from Paul Stapleton. Strain SA-1199B, which overexpresses
the NorA MDR efflux pump, was the gift of Professor Glenn Kaatz.14
Tetracycline, norfloxacin, erythromycin, and oxacillin were obtained
from Sigma Chemical Co. Oxacillin was used in place of methicillin
as recommended by the NCCLS. Mueller-Hinton broth (MHB; Oxoid)
was adjusted to contain 20 mg/L Ca2+ and 10 mg/L Mg2+
.
Antibacterial Assays. Overnight cultures of each strain were made
up in 0.9% saline to an inoculum density of 5 × 105 cfu by comparison
with a MacFarland standard. Tetracycline and oxacillin were dissolved
directly in MHB, whereas norfloxacin and erythromycin were dissolved
in DMSO and then diluted in MHB to give a starting concentration of
512 µg/mL. Using Nunc 96-well microtiter plates, 125 µL of MHB
was dispensed into wells 1-11. Then, 125 µL of the test compound or
the appropriate antibiotic was dispensed into well 1 and serially diluted
across the plate, leaving well 11 empty for the growth control. The
final volume was dispensed into well 12, which being free of MHB or
inoculum served as the sterile control. Finally, the bacterial inoculum
(125 µL) was added to wells 1-11, and the plate was incubated at 37
°C for 18 h. A DMSO control (3.125%) was also included. All MICs
were determined in duplicate. The MIC was determined as the lowest
concentration at which no growth was observed. A methanolic solution
(5 mg/mL) of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliium
bromide (MTT; Lancaster) was used to detect bacterial growth by a
color change from yellow to blue.
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