HU-446 and HU-465, Derivatives of the Non-psychoactive Cannabinoid Cannabidiol, Decrease the Activation of Encephalitogenic T Cells

Article abstract of DOI:10.1111/cbdd.12637

Cannabidiol (CBD), the non-psychoactive cannabinoid, has been previously shown by us to decrease peripheral inflammation and neuroinflammation in mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Here we have studied

Full text of DOI:10.1111/cbdd.12637

Chem Biol Drug Des 2016; 87: 143–153
Research Article
HU-446 and HU-465, Derivatives of the
Non-psychoactive Cannabinoid Cannabidiol,
Decrease the Activation of Encephalitogenic T Cells
ꢀ2
Abbreviations: APC, antigen-presenting cells; CBD, cannabid-
iol; IL-17, interleukin 17; MOG35-55, myelin oligodendrocyte
glycoprotein 35-55; TMOG, MOG35-55-specific T cells.
Ewa Kozela1,*,†, Christeene Haj2,†, Lumir Hanus ,
Mukesh Chourasia3, Avital Shurki2, Ana Juknat1,
Nathali Kaushansky4, Raphael Mechoulam2 and
Zvi Vogel1,4
Received 16 March 2015, revised 27 July 2015 and accepted
for publication 2 August 2015
1The Dr Miriam and Sheldon G. Adelson Center for the
Biology of Addictive Diseases, Sackler Faculty of Medicine,
Tel Aviv University, Tel Aviv 69978, Israel
2Institute for Drug Research, Faculty of Medicine, Hebrew
University of Jerusalem, Jerusalem 91120, Israel
3Department of Pharmacoinformatics, National Institute of
Pharmaceutical Education and Research, Hajipur 844102,
Bihar 844102, India
Endocannabinoids, as well as cannabinoids present in
Cannabis (e.g. in hashish or marijuana) and synthetic
cannabinoids, have been shown to exert potent anti-in-
flammatory activities in various experimental models of
inflammation and neuroinflammatory diseases (1,2). Multi-
ple sclerosis (MS) is one of the most common autoim-
mune neurodegenerative diseases with still missing
therapeutic solutions. The disease is driven by lineages
of self-reactive peripheral T cells falsely primed by anti-
gen-presenting cells (APC) to target components of the
CNS (in particular myelin) causing neuroinflammation,
neurodegeneration, and progressing disabilities (3). The
CB1 cannabinoid receptors (present mostly on neuronal
cells) and the CB2 receptors (present abundantly on
immune cells) were shown to mediate the neuroprotec-
tive and anti-inflammatory effects of the major psychoac-
4Neurobiology Department, Weizmann Institute of Science,
Rehovot 76100, Israel
*Corresponding author: Ewa Kozela,
ewa.kozela@weizmann.ac.il
†These authors equally contributed to this work.
Cannabidiol (CBD), the non-psychoactive cannabinoid,
has been previously shown by us to decrease periph-
eral inflammation and neuroinflammation in mouse
experimental autoimmune encephalomyelitis (EAE)
model of multiple sclerosis (MS). Here we have studied
the anti-inflammatory effects of newly synthesized
derivatives of natural (À)-CBD ((À)-8,9-dihydro-7-hy-
droxy-CBD; HU-446) and of synthetic (+)-CBD ((+)-8,9-
dihydro-7-hydroxy-CBD; HU-465) on activated myelin
tive
Cannabis-derived
CB1/CB2
ligand,
D-9-
tetrahydrocannabinol (THC) in the murine experimental
autoimmune encephalomyelitis (EAE) model of MS (4–6).
However, cannabinoids were found also to act via
mechanisms that do not involve CB1 or CB2 (7). For
example, cannabidiol (CBD), the major non-psychoactive
Cannabis constituent, has very low CB1/CB2 receptor
affinity (8) but shows very potent immunomodulatory and
anti-inflammatory properties (for reviews, see 9,10).
Recently, we reported that CBD administered systemi-
cally diminishes the CNS immune infiltration, microglial
activation, axonal damage, and motor disabilities in mye-
lin oligodendrocyte glycoprotein (MOG) 35-55-induced
EAE mice (11). These observations were confirmed by
several other groups (12–14) using various models of
EAE. However, we would like to state that the group of
Maresz et al. (5) using a model of homogenized spinal
cord-induced EAE did not find CBD to be effective in
ameliorating the disease symptoms (see also 15). CBD
at 5 mg/kg was also shown to reduce the symptoms of
oligodendrocyte
glycoprotein
(MOG)35-55-specific
mouse encephalitogenic T cells (TMOG) driving EAE/
MS-like pathologies. Binding assays followed by
molecular modeling revealed that HU-446 has negligi-
ble affinity toward the cannabinoid CB1 and CB2
receptors while HU-465 binds to both CB1 and CB2
receptors at the high nanomolar concentrations
(Ki = 76.7 Æ 5.8 nM and 12.1 Æ 2.3 nM, respectively).
Both, HU-446 and HU-465, at 5 and 10 lM (but not at
0.1 and 1 lM), inhibited the MOG35-55-induced prolif-
eration of autoreactive TMOG cells via CB1/CB2 recep-
tor independent mechanisms. Moreover, both HU-446
and HU-465, at 5 and 10 lM, inhibited the release of
IL-17, a key autoimmune cytokine, from MOG35-55-
stimulated TMOG cells. These results suggest that HU-
446 and HU-465 have anti-inflammatory potential in
inflammatory and autoimmune diseases.
Key words: cannabidiol derivatives, encephalitogenic T cells,
HU-446, HU-465, IL-17, Th17
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12637
143

Kozela et al.
another autoimmune disease, a model of rheumatoid
arthritis in mice (16). Interestingly, however, CBD at
higher concentrations did not show this effect (16). CBD
also diminished, in our hands, the MOG35-55-induced
Synthesis of (À)-8,9-dihydro-7-hydroxy-CBD (6)
and (À)-8,9-dihydro-6-hydroxy-CBD (7)
(À)-CBD (1)
proliferation of autoreactive encephalitogenic
T
cells
(À)-CBD (1) (À)-CBD (1) whose structure and stereochem-
istry were described many years ago (22) was isolated
from hashish following the procedure described by (23) to
(TMOG) in culture (11) and the release of IL-17 cytokine
(17), the latter defining Th17 autoimmune pathogenic
phenotype (18). Indeed, IL-17 appears to be
a
key
give the desired compound as yellow crystals (Scheme 1).
1
mediator of EAE/MS neuroinflammation (19,20) and a
decrease in Th17 function delays the onset of EAE and
reduces its severity (21). Therefore, treatments targeting
these autoreactive T cells may offer promising tools in
ameliorating MS-like pathologies.
Yield 15%, melting point 62 °C. [a]20 = À56 in CHCl3. H
D
NMR, CDCl3, d (ppm): 0.90 (t, J = 7.5 Hz, 3H), 1.22–1.33
(m, 7H), 1.53–1.61 (m, 2H), 1.66 (s, 3H), 1.79 (s, 3H),
2.01–2.21 (br t, 2H), 2.39–2.46 (m, 3H), 3.91 (br s, 1H),
4.55 (s, 1H), 4.66 (s, 1H), 4.91–5.01 (br, 1H, OH), 5.57 (s,
1H), 5.95–6.05 (br, 1H, OH), 6.10–6.30 (br, 2H, ArH). MS
m/z: 314, 300, 285, 270, 260.
Here we describe the synthesis and the anti-inflammatory
effects of two new CBD derivatives, HU-446 ((À)-8,9-
dihydro-7-hydroxy-CBD,
CBD), which does not bind to CB1 and CB2, and HU-
465 ((+)-8,9-dihydro-7-hydroxy-CBD, derivative of
a
derivative of natural (À)-
(À)-8,9-Dihydro-CBD (2)
a
(À)-8,9-Dihydro-CBD (2) (À)-CBD (1) (544 mg, 1.73 mmol)
was hydrogenated in ethyl acetate (10 mL), over a PtO2
catalyst (21 mg), at 10 psi, for 2 min. The mixture was
synthetic (+)-CBD), which shows affinity toward both CB1
and CB2. Using TMOG/APC co-cultures, we evaluated
the effects of HU-466 and HU-465 on the proliferation
of
activated
TMOG
cells
and
on
MOG35-55-
induced secretion of IL-17 (Th17-like activity). The sup-
pressor effects of HU-446 and HU-465 were compared
to that of their prototype compound, CBD, and the
involvement of CB1 and CB2 receptors was investigated
using specific receptor antagonists. The results show that
HU-446 and HU-465 decrease the proliferation of
MOG35-55-stimulated TMOG cells and inhibit the release
of IL-17, a key autoimmune cytokine from these cells via
a mechanism that is not dependent on CB1 or CB2 acti-
vation.
Methods and Materials
Reagents
Chemicals and solvents were purchased from Biolab LTD
(Jerusalem, Israel), J.T.Baker (Center Valley, PA, USA),
Sigma-Aldrich (Rehovot, Israel), Acros (Yehud, Israel), Alfa
Aesar (Lancashire, UK), Merck (Darmstadt, Germany), and
Penta (Prague, Czech Republic) and were used without
further purification excluding dry diethyl ether and dichloro-
methane, which were refluxed over sodium and phospho-
rous pentoxide, respectively, and freshly distilled prior to
use.
1H-NMR spectra were obtained using a Bruker AMX
300 MHz apparatus using the deuterated chloroform
(CDCl3, d = 7.25 ppm) with tetramethylsilane (TMS) as
internal standard. Elemental analysis was performed using
a Perkin-Elmer (Boston, MA, USA) 2400 series II analyzer
at the Hebrew University Microanalysis Laboratory. Thin-
layer chromatography (TLC) was run on silica gel 60F254
plates (Merck). Column chromatography was performed
Scheme 1: Synthesis of HU-446. Reagents and conditions: (a)
PtO2/H2, ethyl acetate, 10 psi, room temperature, 2 min, yield
97.5%; (b) pyridine, acetic anhydride, room temperature,
overnight, yield 100%; (c) SeO2, ethanol, reflux, 4 h, yield 54%; (d)
NaBH4, ethanol, reflux, 1 h, to yield compound 6 (30%) and
compound 7 (60%).
ꢁ
on silica gel 60 A (Merck). Compounds were located using
a UV lamp at 254 nm.
144
Chem Biol Drug Des 2016; 87: 143–153

Anti-inflammatory Effects of Cannabidiol Derivatives
purified by silica gel column chromatography, using 1%
ether—petroleum ether as an eluent to obtain the desired
compound as brown oil. This material was previously
(À)-8,9-Dihydro-7-hydroxy-CBD (6) (HU-446)
Yield 30%, melting point 70–72 °C. [a]20 = À67.5 in
D
CHCl3; purity by HPLC (60% acetonitrile, 15% water and
reported by (24). Yield 97.5% [a]20 = À58 in CHCl3. 1H
25% methanol) 96.2%. H NMR CDCl3, d (ppm): 6.22 (2H,
1
D
NMR CDCl3, d (ppm): 0.90 (t, J = 7.5 Hz, 3H), 1.22–1.33
(m, 7H), 1.53–1.63 (m, 2H), 1.66 (s, 3H), 1.79 (s, 3H),
2.00–2.21 (br t, 2H), 2.40–2.46 (m, 3H), 3.87 (br s, 1H),
4.90–5.10 (br, 1H, OH), 5.57 (s, 1H), 5.95–6.05 (br, 1H,
OH), 6.10–6.30 (br, 2H, ArH). MS m/z: 316, 302, 287,
272, 262.
s, Ar), 5.69 (1H, s, olefin), 3.90 (1H, m, benzyl), 2.42 (1H,
t, allyl), 2.22 (2H, t, benzyl), 1.86 (2H, m), 1.62 (6H, s, allyl
CH3), 1.54 (2H, m), 1.29 (6H, m), 0.89 (9H, t, terminal
CH3). MS m/z: 548 (silylation), 506, 478, 458, 415, 392.
Molecular mass calculated for C21H32O3: 332.2391 and
found 332.2401.
(À)-8,9-Dihydro-CBD-diacetate (3)
(À)-8,9-Dihydro-6-hydroxy-CBD (7)
Compound 2 (534 mg, 1.689 mmol) was dissolved in pyri-
dine (3 mL) and acetic anhydride (3 mL), and the solution
was stirred overnight at room temperature. This solution
was then poured onto ice-cold water (50 mL) and
extracted three times with ether. The combined organic
extract was washed successively with 1 M HCl, aqueous
sodium bicarbonate, and 10% aqueous sodium chloride,
dried over anhydrous MgSO4, filtered, and evaporated to
dryness, to give the desired compound as a yellow oil.
Yield 100%, 1H NMR CDCl3, d (ppm): 0.90 (t, J = 7.5 Hz,
3H), 1.28–1.33 (m, 7H), 1.53–1.63 (m, 2H), 1.66 (s, 3H),
1.79 (s, 3H), 2.00–2.21 (br t, 2H), 2.30–2.07 (6H, br s),
2.40–2.46 (m, 3H), 3.87 (br s, 1H), 4.90–5.10 (br, 1H,
OH), 5.57 (s, 1H), 5.95–6.05 (br, 1H, OH), 6.10–6.30 (br,
2H, ArH). MS m/z: 400, 386, 371, 356, 346.
Yield 60%, melting point 74–76 °C. [a]20 = À69.1 in
D
CHCl3; purity by HPLC (60% acetonitrile, 15% water and
1
25% methanol) 95%. H NMR CDCl3, d (ppm): 6.22 (2H, s,
Ar), 5.69 (1H, s, olefin), 3.90 (1H, m, benzyl), 3.69 (1H, t),
2.42 (1H, t, allyl), 1.828 (3H, s), 1.62 (6H, s, allyl CH3), 1.54
(2H, m), 1.29 (6H, m), 0.89 (9H, t, terminal CH3). MS m/z:
548 (silylation), 506, 478, 458, 415, 392. Molecular mass
calculated for C21H32O3: 332.2391 and found 332.2412.
Synthesis of (+)-8,9-dihydro-7-hydroxy-CBD (14)
and (+)-8,9-dihydro-6-hydroxy-CBD (15)
(+)-8,9-Dihydro-CBD (10)
Basic aluminum oxide (156 mg) was added to dry dichlor-
omethane (150 mL) (Scheme 2). To this suspension,
BF3Ádiethyletherate (2.3 mL) was added under nitrogen.
The mixture was stirred for 15 min at room temperature
under N2 atmosphere. Compound 8, (À)-L-piperitenol
(950 mg, 6.25 mmol) and compound 9, olivetol (145 mg,
6.25 mmol) in dry dichloromethane (50 mL) were added,
and the reaction mixture was quenched within 10 seconds
with 10% aqueous solution of sodium bicarbonate
(50 mL). The organic phase was separated, and the aque-
ous layer was further extracted with dichloromethane. The
combined dichloromethane solution was extracted with
water, 10% aqueous sodium chloride, dried on anhydrous
Na2SO4, and evaporated to give red oil. This oil was puri-
fied by silica gel column chromatography, using 2% ether—
petroleum ether as an eluant to give compound 10 as a
(À)-8,9-Dihydro-7-hydroxy-CBD-diacetate (4) and
(À)-8,9-dihydro-6-hydroxy-CBD-diacetate (5)
Compound 3 (677 mg, 1.69 mmol) was dissolved in etha-
nol (4 mL), and selenium oxide (564 mg, 5.08 mmol) was
added. The mixture was refluxed for 4 h and monitored by
TLC. The ethanol was removed under reduced pressure,
and the residue was mixed with water and extracted sev-
eral times with ether. The combined ether extract was
dried over MgSO4, filtered and evaporated to dryness. The
residue was chromatographed on silica gel (with 20%
ether—in petroleum ether) to give the mixture of 4,5, with
a yield of 54%.
red oil. Yield 60% [a]20 = +60 in CHCl3. 1H NMR CDCl3,
D
(À)-8,9-Dihydro-7-hydroxy-CBD (6) and (À)-8,9-
dihydro-6-hydroxy-CBD (7)
d (ppm): 0.90 (t, J = 7.5 Hz, 3H), 1.22–1.33 (m, 7H),
1.53–1.63 (m, 2H), 1.66 (s, 3H), 1.79 (s, 3H), 2.00–2.21
(br t, 2H), 2.40–2.46 (m, 3H), 3.87 (br s, 1H), 4.90–5.10
(br, 1H, OH), 5.57 (s, 1H), 5.95–6.05 (br, 1H, OH), 6.10–
6.30 (br, 2H, ArH). MS m/z: 316, 302, 287, 272, 262.
Mixture 4,5 (380 mg, 0.91 mmol) was dissolved in ethanol
(125 mL), and NaBH4 (56 mg, 1.505 mmol) was added
and the reaction refluxed for 1 h. The ethanol was
removed under reduced pressure, the residue was diluted
with water (180 mL), and the solution extracted several
times with ether. The combined organic extract was
washed with 10% aqueous sodium chloride, dried on
MgSO4 and filtered. Removal of the organic solvent under
Compounds 14 (HU-465) and 15 were prepared from
compound 10 by the same procedure as reported above
for 6 and 7 and were obtained as white solids.
reduced pressure yielded
a residue that was chro-
(+)-8,9-Dihydro-7-hydroxy-CBD (14) (HU-465)
matographed on silica gel (30% ether—in petroleum ether)
and dried to give compound 6 (HU-446) and compound 7
as white solids.
Yield 30%, melting point 71–73 °C. [a]20 = +69 in CHCl3;
D
HPLC (60% acetonitrile, 15% water and 25% methanol)
Chem Biol Drug Des 2016; 87: 143–153
145

Kozela et al.
These optimized geometries were submitted to the LIGPREP
module at the physiological pH range of 7.4 Æ 2 and then
served as initial conformations for an exhaustive conforma-
tional search with CONFGEN module (26). Fifteen and 22
possible conformations were obtained for HU-446 and
HU-465, respectively. All these conformations were used
later in the docking process.
Protein preparation
The sequences of mouse CB1 and CB2 were obtained from
UniProt (Accession No. P47746 and P34972). The crystal
structures of mouse CB1 and CB2 are not available. Using
homology modeling, we constructed, therefore, models
based on the crystal structure of bovine rhodopsin (PDB ID:
1F88). The loops of these 3D models were iteratively refined
using MODELLER 9.14 (27). The quality of the modeled struc-
tures was cross-validated with Ramachandran plots (RAM-
PAGE) (28) and by comparison with available models from
literature (29,30). The models chosen had < 3% residues
outside the allowed regions of Ramachandran plot. Further-
more, the residues located outside the allowed regions were
also away from the active site, hence, not expected to con-
tribute significantly to the ligand–receptor interaction. The
€
protein preparation wizard of SCHRODINGER SUIT 2014.3 was
used to prepare the modeled proteins. OPLS2005 force field
was applied with charges of non-polar hydrogens summed
€
into heavy atoms. Sitemap of SCHRODINGER SUIT 2014.3 was
used to identify the plausible active sites within the modeled
receptors (31). Based on the position and the cavity size,
potential ligand-binding sites were identified in each one of
the modeled receptors.
Scheme 2: Synthesis of HU-465. Reagents and conditions: (a)
BF3 diethyletherate, Al2O3, dichloromethane, yield 60%; (b)
pyridine, acetic anhydride, room temperature, overnight, yield
100%; (c) SeO2, ethanol, reflux, 4 h, yield 54%; (d) NaBH4, ethanol,
reflux, 1 h, to yield compound 14 (30%) and compound 15 (60%).
Docking protocol
The rigid docking protocol of GLIDE 4.5 has been employed
96.8%. 1H NMR CDCl3, d (ppm): 6.22 (2H, s, Ar), 5.69
(1H, s, olefin), 3.90 (1H, m, benzyl), 2.42 (1H, t, allyl), 2.22
(2H, t, benzyl), 1.86 (2H, m), 1.62 (6H, s, allyl CH3), 1.54
(2H, m), 1.29 (6H, m), 0.89 (9H, t, terminal CH3). MS m/z:
548 (silylation), 506, 478, 458, 415, 392. Molecular mass
calculated for C21H32O3: 332.22391 and found 332.2401.
ꢁ
(32). A 20 A cubical grid was generated from the centroid of
three residues, known to be important for binding, namely,
F201, F279, and W357 within CB1 (29) and F117, W194,
and W258 within CB2 (33). The default standard precision
docking settings were initially used with all possible confor-
mations of the ligands obtained by the exhaustive search,
generating 10 different conformers of bound ligand for each
initial ligand conformation. The 10 lowest energy ligand–re-
ceptor complexes obtained from all the different standard
precision results were used as initial structures for a more
refined search, using the extra precision module of ‘GLIDE’.
Again ten different binding poses of the ligands within the two
receptors were calculated for each ligand. The lowest energy
conformations of docked ligand–receptor complexes which
are considered as the best docking poses are expected to
have the highest propensity to bind at the active site and
were used to understand the binding.
(+)-8,9-Dihydro-6-hydroxy-CBD (15)
Yield 60%, melting point 73–75 °C. [a]20 = +68.5 in
D
CHCl3; HPLC (60% acetonitrile, 15% water and 25%
methanol) 95.5%. 1H NMR CDCl3, d (ppm): 6.22 (2H, s,
Ar), 5.69 (1H, s, olefin), 3.90 (1H, m, benzyl), 3.69 (1H, t),
2.42 (1H, t, allyl), 1.83 (3H, s), 1.62 (6H, s, allyl CH3), 1.54
(2H, m), 1.29 (6H, m), 0.89 (9H, t, terminal CH3). MS m/z:
548 (silylation), 506, 478, 458, 415, 392. Molecular mass
calculated for C21H32O3: 332.2391 and found 332.2412.
Molecular modeling studies
Ligand preparation
Reagents for biological assays
HU-446 and HU-465 were first optimized at the B3LYP/6-
311 + +G** level of calculation using JAGUAR 8.5 (25).
Lyophilized MOG35-55 peptide (MEVGWYRSPFSRVVH-
LYRNGK) purchased from GenScript (Piscataway, NJ,
146
Chem Biol Drug Des 2016; 87: 143–153

Anti-inflammatory Effects of Cannabidiol Derivatives
USA) was reconstituted in sterile PBS and the stock solu-
tion stored in aliquots at À20 °C. (À)-CBD was obtained
from the National Institute on Drug Abuse (Baltimore, MD,
USA). The CB1 receptor antagonist, SR141716, was
obtained from Tocris (Ellisville, MO, USA). The CB2 antag-
onist, AM630, was purchased from Cayman Chemicals
(Ann Arbor, MI, USA). [3H] CP-55,940 and unlabeled CP-
55,940 were purchased from Perkin Elmer and Cayman
Chemical (Tallinn, Estonia), respectively. (À)-CBD, HU-446,
HU-465, and SR141716 were dissolved and applied in
ethanol while AM630 was applied in DMSO. The final con-
centrations of ethanol or DMSO in the various experiments
did not exceed 0.1% and had no effect on the results.
Fetal calf serum and other tissue culture reagents were
obtained from Biological Industries (Kibbutz Beit HaEmek,
Israel).
100 lg/mL streptomycin, 100 U/mL penicillin, 2 mM L-glu-
tamine, and 50 lM b-mercaptoethanol. HU-446 and HU-
465 at final doses of 0.1, 1, 5, and 10 lM were added to
the cells together with the MOG35-55 peptide at 1 or
2.5 lg/mL. The cells were then incubated for 72 h at
37 °C in 5% CO2 humidified air (11,36). Cell proliferation
was measured by pulsing the cells with [3H] thymidine
(0.5 lCi/well; Perkin Elmer) for the last 16 h of the incuba-
tion period, and the cells were then harvested and
counted using a Matrix 96 Direct b counter (Packard Instr.,
Meriden, CT, USA). The proliferative responses of the
TMOG cells in each well were converted to percent values
with MOG35-55 effect expressed as 100%. Percent values
are calculated from stimulation index (SI) values which are
the fold changes in mean counts per minutes (cpm) of
MOG35-55 cultures over mean cpm of cultures without
MOG (spontaneous proliferation). Statistical analysis was
performed on percent values.
Cannabinoid CB1 and CB2 receptor binding assays
The binding methods used here were described previously
for CB1 by (34) and for CB2 by (35). In brief, for CB1
receptor binding assay, synaptosomal membranes from
rat brains were used. Sabra male rats weighing ~200 g
were decapitated, and their brains, without the brain stem,
were quickly removed. Synaptosomal membranes were
prepared from the brains by centrifugation and sucrose
density gradient ultracentrifugation after their homogeniza-
tion. The CB2 receptor binding assays were performed
using crude membranes obtained from Chinese hamster
ovary (CHO) cells stably transfected with the human CB2
cDNA.
Enzyme-linked immunosorbent assay (ELISA)
Culture and assay conditions were described previously
(17). In brief, TMOG cells were cultured in 24-well plates
(0.25 9 106 cells/well) together with non-irradiated splenic
APC (5 9 106 cells/well). HU-446, HU-465, and (À)-CBD
at final concentrations of 5 or 10 lM or their vehicles were
added to the cells just before the addition of MOG35-55
at 5 lM. After 24 h incubation, the cell-conditioned media
was collected, spun down (800 9 g for 10 min), and ana-
lyzed for IL-17A (from here on defined as IL-17) concentra-
tion by ELISA according to the manufacturer instructions
(R&D Systems, Minneapolis, MN, USA).
The high affinity CB1/CB2 receptor ligand [3H] CP-
55,940, with a dissociation constant of 2 nM for CB1
and CB2, was incubated for 90 min at 30 °C with
synaptosomal membranes for CB1, or with membranes
of transfected cells for CB2 in the presence of the tested
CBD derivatives. Bound and free radioligand were sepa-
rated by centrifugation (13 000 9 g for 6 min). Non-
specific binding, which was determined with 50 nM unla-
beled CP-55,940, was subtracted. Binding experiments
were repeated 2–3 times and each point performed in
triplicate. The Ki values were determined using the
GraphPad Prism program (La Jolla, CA, USA) and the
Cheng–Prusoff equation.
Statistical analysis
Data are expressed as the mean Æ SEM of 2–4 indepen-
dent experiments and analyzed for statistical significance
using one-way analysis of variance (ANOVA), followed by
Newman-Keul’s post hoc test. p < 0.05 was considered
significant. GraphPad Prism program was used for statisti-
cal analysis of the data.
Results
Syntheses of (+)- and (À)-8,9-dihydro-7-hydroxy-
CBD (HU-446 and HU-465)
Encephalitogenic T-cell proliferation
The CBD derivatives in the natural (À) series were synthe-
sized from (À)-CBD which was extracted from hashish as
described by (22) (see Scheme 1). The compounds in the
unnatural (+) series were prepared from synthetic dihydro-
(+)-CBD (10), obtained via condensation between (À)-L-
piperitenol and olivetol in the presence of boron trifluoride
etherate on alumina as catalyst (Scheme 2) as described
previously (37).
MOG35-55-reactive line of T cells (TMOG) raised from
MOG35-55 immunized mice was obtained and maintained
as previously described (11,17). To assay the effect of
cannabinoids on cell proliferation, these TMOG cells were
cultured in 96-well plates (1.25 9 104 cells per well)
together with splenic antigen-presenting cells (APC;
5 9 105 cells/well). APC were irradiated (25 Gy) before
co-culturing to prevent them from proliferation. The co-cul-
tured cells (TMOG/APC) were maintained in 0.2 mL RPMI
containing 2.5% fetal calf serum, supplemented with
(À)-CBD (1) was hydrogenated with PtO2 in ethyl acetate
to give 8-dihydro-(À)-CBD derivative (2). The two respec-
Chem Biol Drug Des 2016; 87: 143–153
147

Kozela et al.
Table 1: Affinity of HU-446 and HU-465 for the CB1 and CB2
receptors. The table presents the Ki of HU-446 and HU-465 as
measured by competition with binding of [3H]-CP-55,940 to rat
synaptosomes (CB1) or to CB2-transfected CHO cell membranes.
The Ki values are presented as mean Æ SEM from three experi-
ments performed in triplicates. The affinities of (À)-CBD and (+)-
CBD to CB1 and CB2 are provided according to previous studies
Binding of HU-446 and HU-465 to the cannabinoid
CB1 and CB2 receptors
The compound HU-446 (6) which was derived from natural
(À)-CBD binds very weakly to the cannabinoid receptors
CB1 (Ki value ~1 lM) and CB2 (Ki value >10 lM) (Table 1).
By contrast, its stereoisomer HU-465 (14), the derivative of
synthetic (+)-CBD, behaves as a high affinity ligand of the
CB2 receptor (Ki value of 12.1 nM) as well as of the CB1
receptor (Ki = 76.7 nM) although its affinity to CB1 is
around six times lower than to CB2 (Table 1).
Compound CB1 (Ki)
CB2 (Ki)
>10 lM
Characteristics
HU-446
HU-465
~1 lM
Derivative of the
natural (À)-CBD
76.7 Æ 5.8 nM 12.1 Æ 0.3 nM Derivative of the
synthetic (+)-CBD
Molecular modeling studies
(À)-CBD
(+)-CBD
>10 lM
842 Æ 36 nM
>10 lM
203 Æ 16 nM
See (40)
See (40)
We carried out molecular modeling studies to provide a
possible explanation for the reduced binding affinity of HU-
446 compared with HU-465 to both CB1 and CB2. We
demonstrate, in agreement with earlier studies (41), that
the binding site cavity of CB1 receptor is very large in size
and volume and generates two different subpockets where
the ligands are allowed to bind (Figure 1A). CB2 receptor,
on the other hand, has an overall smaller biding cavity
generating only one ligand-binding pocket (Figure 1B)
(29,30). However, the single binding pocket in CB2 is lar-
ger than any of the two binding pockets in CB1 if consid-
ered separately. The binding pose of the ligands seems to
be characteristic of the receptor. The lowest binding pose
of the two ligands is almost parallel to the Z-axis of CB1
but perpendicular to the Z-axis of CB2 (Figure 1C,D,
respectively). Furthermore, in CB1, the bicyclic rings of the
tive (À) and (+) dihydro-CBD derivatives (2, 10) were con-
verted into their diethyl acetates (3, 11) by pyridine and
acetic anhydride. The allylic hydroxylation reaction by sele-
nium oxide that was widely investigated (38,39) has been
used for the oxidation of the cyclohexenyl ring system,
yielding mixtures of the allylic alcohols (4, 5) and (12, 13),
respectively.
The mixtures (4, 5) and (12, 13) were reacted with sodium
borohydride in ethanol to give the compounds (6) and (14)
in 30% yield and the compounds (7) and (15) in 60% yield.
They were purified by silica gel chromatography.
B
A
Figure 1: Visualization of HU-446 and HU-
465 docking into CB1 and CB2 receptors.
The access channel of the studied ligands
dictated by the hydrophobic surface area in
the modeled structure of (A) mouse CB1
and (B) mouse CB2. The binding pocket of
(C) mouse CB1 and (D) mouse CB2. The
lowest binding energy pose of HU-446
(cyan) and HU-465 (purple) is shown in all
panels. Gray fonts represent residues
forming hydrophobic interactions with the
ligands. Residues forming hydrogen bonds
with the ligands are highlighted by respective
ligand color. Receptor orientation is identical
in all panels.
D
C
148
Chem Biol Drug Des 2016; 87: 143–153

Anti-inflammatory Effects of Cannabidiol Derivatives
two ligands are buried deeper in the transmembrane
domains 3, 4, 5, and 6 compared to the corresponding
ligands in CB2, which are closer to the extracellular
domain of the cell membrane (Figure 1A,B). The aliphatic
chain is turning deep into the binding pocket in all cases.
The smaller binding pocket size of CB1 results with overall
more interactions compared with CB2. The hydrophobic
lining of the CB1 binding site with both HU-446 and HU-
465 includes F201, V205, I248, V365, V368, Y276, F279,
V283, L361, M364, L288, L360, W367 and that of CB2
includes F117, I164, G193, W194, F197, I198, A261,
L262, M265, and W258.
The hydrophobic lining of the CB1 binding site shares
common interactions for both HU-446 and HU-465. How-
ever, due to slight differences in the relative positioning
and orientation of the two ligands, HU-465 seems to enjoy
better hydrophobic interactions, which are responsible for
the difference in the binding. In particular, HU-465 benefits
from p–p interaction with W357 which does not exist when
HU-446 is bound due to a different positioning of its 8,9-
dihydro ring in the binding pocket relative to W357. H-
bonds seem to contribute to the difference in the binding
affinity in an opposite manner in this case. HU-446 has
three H-bonds (with the backbone of Y276, V283, and
W357, Figure 1C) compared with only two for HU-465
(with the backbone of V283 and side chain of T284, Fig-
ure 1C). Yet, overall the hydrophobic interactions dominate
the affinity of the two ligands to CB1 (see also Figure S1).
Figure 2: HU-446 inhibits MOG35-55-induced TMOG cell
proliferation. MOG35-55-induced TMOG proliferation was
determined by [3H] thymidine incorporation assay. TMOG/APC co-
cultures were exposed to (A) 1 lg/mL or (B) 2.5 lg/mL of
MOG35-55. HU-446 was added just prior to MOG35-55.
SR141716 and AM630 were applied 30 min before MOG35-55.
Each experiment (n = 3) was carried out in triplicates. Data are
expressed as mean percent values Æ SEM, and the MOG35-55
effect is expressed as 100%. (A) One-way ANOVA F(6, 11) = 37.4,
p < 0.001; (B) one-way ANOVA F(6, 11) = 57.6, p < 0.001.
Newman–Keul’s post hoc test. Symbols: ***p < 0.001 versus
MOG35-55 alone treated cells; ns—non-significant.
Analyzing the interactions within the CB2 suggests that
mainly the H-bonding interactions are responsible for the
difference in the binding in this case, while the hydrophobic
contributions are analogous for the two ligands. HU-465
seems to have better H-bonding network in CB2 compared
with HU-446. In particular, the various hydroxyl groups of
HU-465 form two H-bonds with the side chain groups of
T114 and S285, whereas the corresponding hydroxyl
groups of HU-446 form only one H-bond with the side chain
of K109. These binding modes explain the higher affinity of
HU-465 compared with that of HU-446 to both receptors.
These results are further supported energetically by the
scoring values obtained, having higher absolute value score
for HU-465 compared with that of HU-446 (see Table S1).
p < 0.001; data not shown). We, then, evaluated the effects
of HU-446, the CBD derivative with negligible affinity toward
CB1 and CB2, on the MOG35-55-induced proliferation of
TMOG cells (Figure 2). The results presented in Figure 2A
show the inhibitory effect of HU-446 on TMOG stimulated by
1 lg/mL of MOG35-55 (presented as 100%). Addition of
5 lM of HU446 to TMOG/APC cells stimulated with 1 lg/mL
of MOG35-55 resulted in the inhibition of TMOG proliferation
by 75% (p < 0.001); 10 lM of HU-446 inhibited TMOG prolif-
eration by 90% (p < 0.001). Similar inhibition was observed
when the TMOG cells were stimulated with 2.5 lg/mL of
MOG35-55 (Figure 2B). In this case, 5 lM of HU-446 inhib-
ited the proliferation by 73% (p < 0.001) and 10 lM of HU-
446 by 89% (p < 0.001). Lower doses of HU-446 (e.g. 0.1
and 1 lM) had no effect on TMOG proliferation induced by
either 1 or 2.5 lg/mL MOG35-55.
Inhibition of MOG35-55-induced TMOG proliferation
by HU-446 and HU-465
TMOG cells were co-cultured with irradiated APC cells in the
presence of MOG35-55 at two doses, 1 and 2.5 lg/mL.
These concentrations were chosen following previous
reports (11,36). Application of MOG35-55 increased the
proliferation of the encephalitogenic cells as measured
using [3H] thymidine incorporation. MOG35-55 at 1 lg/mL
induced a 9.4 Æ 2.4-fold increase (p < 0.01) in TMOG prolif-
The inhibitory effect of HU-446 does not seem to be medi-
ated by either CB1 or CB2 receptors. We found that nei-
ther SR141617 (a CB1 receptor antagonist) at 1 lM nor
AM630 (a CB2 receptor antagonist) at 0.1 lM affected the
HU-446 (5 lM) inhibition of T-cell proliferation induced by
MOG35-55 (Figure 2A,B). The doses of CB1 and CB2
receptor antagonists were chosen based on our previous
experiments (11,17).
eration, and MOG35-55 at 2.5 lg/mL produced
a
13.0 Æ 2.1-fold increase in TMOG proliferation (p < 0.001
versus non-stimulated cells; ANOVA F(2, 15) = 11.4;
Chem Biol Drug Des 2016; 87: 143–153
149

Kozela et al.
Figure 4: HU-446 and HU465 decrease IL-17 release from
MOG35-55-stimulated encephalitogenic T cells co-cultured with
APC. TMOG/APC co-cultures were stimulated for 24 h with 5 lg/mL
of MOG35-55 in the presence or absence of HU-446, HU-465 or
(À)-CBD, 5 or 10 lM. Cell free media was subjected to ELISA for IL-
17. MOG35-55-induced IL-17 release is expressed as 100%. Data
were analyzed by one-way ANOVA followed by Newman–Keul’s post
hoc test. ANOVA F(7, 16) = 59.7, p < 0.01. Symbols: +++p < 0.001
versus non-MOG35-55, control TMOG/APC; ***p < 0.001 versus
MOG35-55-stimulated TMOG/APC; ##p < 0.01 and ###p < 0.001
versus MOG35-55+ HU-446 at 5 lM.
Figure 3: HU-465 inhibits MOG35-55-induced TMOG cell
proliferation. Experiments were carried out as described in
Figure 1. (A) 1 lg/mL MOG35-55 (one-way ANOVA F(6, 13) = 6.8,
p < 0.01) and (B) 2.5 lg/mL MOG35-55 (one-way ANOVA F(6,
13) = 6.4, p < 0.01). Newman–Keul’s post hoc test. Symbols:
*p < 0.05 versus MOG35-55 alone treated cells; ns—non-
significant.
465 decreased IL-17 secretion from stimulated TMOG/APC
by 76% (p < 0.001) and by 84% (p < 0.001) at the doses
of 5 and 10 lM, respectively. In parallel, we compared the
new cannabinoid derivatives to their prototype, CBD. We
found that (À)-CBD was somewhat more efficient than
HU-446 and HU-465 and that at the doses of 5 and 10 lM
it suppressed IL-17 release by 87% and 90%, respectively.
Next, we studied the effects of HU-465, the (+)-CBD deriva-
tive that interacts with both CB1 and CB2 receptors, on the
MOG35-55-induced TMOG proliferation. Addition of 5 and
10 lM of HU-465 to TMOG/APC, co-cultures stimulated with
1 lg/mL of MOG35-55 decreased TMOG proliferation by
65% (p < 0.05) and 73% (p < 0.05), respectively (Fig-
ure 3A,B). Similar inhibition was induced by 5 and 10 lM
HU-465 when the T cells were stimulated with 2.5 lg/mL
of MOG35-55 (64% and 71%, p < 0.05, respectively). Simi-
larly to HU-446, low doses of HU465 (0.1 and 1 lM) had no
effect on MOG35-55-induced TMOG proliferation induced by
either 1 or 2.5 lg/mL of MOG35-55 (Figure 3A,B). More-
over, as found with HU-446, neither SR141617 nor AM630
affected the HU-465 inhibition of TMOG proliferation.
As indicated by our previous studies, IL-17 was released
only by TMOG cells that were co-cultured with APC in the
presence of MOG35-55. We did not detect IL-17 produc-
tion and release (using quantitative PCR and ELISA,
respectively) in TMOG or APC cultured separately in either
the presence or absence of MOG35-55 (17).
Discussion
The present study describes the synthesis and the anti-in-
flammatory activities of two isomeric CBD derivatives, HU-
446 and HU-465 ((À)- and (+)-8,9-dihydro-7-hydroxy-CBD,
respectively). HU-446 is a derivative of the natural (À)-CBD
(isolated from hashish) with negligible affinity toward either
CB1 or CB2, and HU-465 is a derivative of synthetic (+)-
CBD, capable of interacting with both receptors. Indeed,
ligand–receptor docking studies demonstrated favorable
hydrophobic interactions with CB1 binding pocket and
stronger H-bonding interactions with CB2 binding pocket
for HU-465 in comparison with HU-446. Both HU-446 and
HU-465 decrease the in vitro activation of encephalito-
genic T cells by MOG35-55 including their proliferation
and IL-17 cytokine release. However, they appear to be
slightly less efficient than the (À)-CBD.
HU-446 and HU-465 decrease IL-17 secretion from
MOG35-55-stimulated TMOG/APC
Addition of MOG35-55 (5 lg/mL) for 24 h to TMOG/APC
co-cultures induced a vast secretion of IL-17A (measured
by ELISA) reaching the level of 99 Æ 27 pg/mL (p < 0.001;
assigned as 100% activation). A much smaller amount of
IL-17 (11 Æ 4 pg/mL) was detected in the culture media of
non-stimulated TMOG/APC, amounting to 11% of the IL-17
detected following incubation with MOG35-55. Figure 4
shows that treatment of stimulated TMOG/APC with either
HU-446 or HU-465 markedly decreased the MOG35-55-
induced IL-17 secretion. HU-446 at the dose of 5 lM
decreased IL-17 secretion by 50% (p < 0.001), while
10 lM of HU-446 decreased it by 81% (p < 0.001). HU-
CBD and THC are the main cannabinoids present in Can-
nabis preparations (e.g. marijuana and hashish). THC
serves as an agonist of CB1 (and is therefore psychoac-
150
Chem Biol Drug Des 2016; 87: 143–153

Anti-inflammatory Effects of Cannabidiol Derivatives
tive) as well as of CB2. CBD, due to its lack of activity at
neuronal CB1 receptors, is devoid of the psychocognitive
effects accompanying the use of THC. There is compelling
evidence that CB2 receptor activation is protective in vari-
ous types of inflammation (42,43). However, the psychoac-
tive effects of THC and other compounds with CB1 or
CB1/CB2 activity limit their possible therapeutic applica-
tions. Interestingly, despite negligible affinity toward the
CB2 receptor, CBD has been shown to exert very potent
anti-inflammatory activities in various models of peripheral
and CNS inflammation (9,10). Therefore, there is consider-
able interest in the development of either CB2 ligands or
CBD-like compounds that lack psychoactive properties
while exerting potent immunoregulatory effects.
tives of (+)-CBD enantiomers show that while this family of
compounds exhibits moderate binding affinity to CB1
in vitro, it displays poor CB1-like profile when examined
in vivo (40,50). Briefly, (+)-CBD derivatives lack typical
THC-like centrally mediated effects including sedation or
hypothermia (51), suggesting that (+)-CBD derivatives,
such as HU-465, may be much better tolerated when
applied systemically than THC-like cannabinoids.
Altogether, our findings show that the newly synthesized
(À)-CBD analog with negligible CB1/CB2 activity (HU-446)
and the CB1/CB2 moderately active (+)-CBD derivative
(HU-465) are immunomodulators and are effective in ame-
liorating excessed inflammation induced by encephalito-
genic
T cell, the latter driving autoimmune MS-like
We have recently shown that CBD ameliorates EAE pro-
gression in mice via decreasing neuroinflammation in vivo
and that CBD decreases the inflammatory induction of the
Th17 phenotype (11,17). This Th17 phenotype, defined by
the induction of IL-17 release, is the main subtype of mem-
ory T cells causing EAE/MS-like neuroinflammation and
neuronal dysfunction (19,20). Adoptive transfer of these
encephalitogenic T cells to healthy animals results in rapid
and severe EAE clinical symptoms (44,45). Our present
work shows that two newly synthesized CBD derivatives,
HU-446 and HU-465, significantly decrease encephalito-
genic TMOG cells proliferation and prevent them from
releasing neurotoxic IL-17 (Th17 activity) in the presence of
MOG35-55 self-antigen. Both, HU-446 and HU-465,
decrease TMOG proliferation in CB1/CB2 independent man-
ner, despite the fact that HU-465 is a potent CB1 and CB2
agonist. Indeed, a number of reports show that cannabi-
noids, including CB2 ligands, exert immunomodulatory
effects via mechanisms that do not involve the classic
cannabinoid receptors (7,46,47). Accordingly, in our hands,
even THC, a prototype CB1/CB2 ligand, diminishes micro-
glial and encephalitogenic T-cell activations (including IL-17
release) via a CB1/CB2 independent mechanism (17,47).
Moreover, the finding that the anti-inflammatory effects of
cannabinoids are preserved in CB2 knockout mice strongly
supports the existence of additional, still unknown receptor
or non-receptor target(s) that mediate these effects (48).
Due to the high hydrophobicity of the cannabinoids, it has
been suggested that some of their activities may involve
changes in membrane structure and integrity (e.g. lipid raft
re-organization) followed by impaired signaling (49). At this
stage, we cannot rule out that the effect observed here
could be due, at least in part, to the hydrophobicity of the
tested cannabinoids. Indeed, CBD, which is somewhat
more hydrophobic than HU-446 and HU-465, is also more
efficient in inhibiting of T-cell proliferation and Th17 activa-
tion. Other options, like cannabinoid interactions with
PPARc, are also possible (see 7 and references within).
pathologies.
Acknowledgments
TMOG cell line was kindly provided by the laboratory of Pro-
fessor Avraham Ben-Nun from the Immunology Depart-
ment at the Weizmann Institute of Science, Rehovot,
Israel. This work was supported by the Dr Miriam and
Sheldon G. Adelson Medical Research Foundation. A.J. is
supported by the Israeli Ministry for Absorption in Science.
Authors’ Contributions
C.H. synthesized the HU-446 and HU-465 compounds.
L.H. performed the binding assays. A.S and M.C. per-
formed the molecular modeling studies. N.K. and E.K. pro-
vided the know-how in TMOG cell line establishing and
culturing. E.K. performed the biological experiments and
wrote the article. All authors contributed to discussions of
the article and to reviewing and editing the manuscript
before submission.
Competing Interest
The authors state no conflict of interest.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
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Figure S1. The binding pocket of mouse CB1 with A.
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Table S1. Experimental Ki values along with calculated
Glide scores (in kcal/mol) of the studied agonists in mouse
CB1 and CB2 receptors.
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