Δ9-tetrahydrocannabinol immunochemical studies: Haptens, monoclonal antibodies, and a convenient synthesis of radiolabeled Δ9-tetrahydrocannabinol

Article abstract of DOI:10.1021/jm050442r

Immunopharmacotherapy as an approach to combat drugs of abuse has become an active area of investigation. Marijuana is the most commonly used illicit drug in the U.S. The main active chemical in marijuana is Δ⁹- tetrahydrocannabinol (Δ⁹-THC); hence, monoclonal antibodies with high affinity and specificity for Δ⁹-tetrahydrocannabinol could be valuable immunopharmacotherapeutic intervention and diagnostic tools. We have synthesized immunoconjugates that induce an effective immune response to Δ⁹-THC and describe a convenient synthesis of radiolabeled Δ⁹-THC. We demonstrate the value and use of this probe to select anti-Δ⁹-THC antibodies that bind Δ⁹-THC with good affinity. The synthetic route to radiolabeled Δ⁹-THC has enabled the correct assessment of the affinity of these antibodies to their ligand and may facilitate future binding studies between Δ⁹- THC and its analogues and the cannabinoid receptors.

Full text of DOI:10.1021/jm050442r

J. Med. Chem. 2005, 48, 7389-7399
7389
∆9-Tetrahydrocannabinol Immunochemical Studies: Haptens, Monoclonal
Antibodies, and a Convenient Synthesis of Radiolabeled
∆9-Tetrahydrocannabinol
Longwu Qi,† Noboru Yamamoto,† Michael M. Meijler,† Laurence J. Altobell, III,† George F. Koob,‡
Peter Wirsching,† and Kim D. Janda*,†
Departments of Chemistry, Immunology, and Neuropharmacology, and The Skaggs Institute for Chemical Biology, The Scripps
Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received May 10, 2005
Immunopharmacotherapy as an approach to combat drugs of abuse has become an active area
of investigation. Marijuana is the most commonly used illicit drug in the U.S. The main active
chemical in marijuana is ∆9-tetrahydrocannabinol (∆9-THC); hence, monoclonal antibodies with
high affinity and specificity for ∆9-tetrahydrocannabinol could be valuable immunopharma-
cotherapeutic intervention and diagnostic tools. We have synthesized immunoconjugates that
induce an effective immune response to ∆9-THC and describe a convenient synthesis of
radiolabeled ∆9-THC. We demonstrate the value and use of this probe to select anti-∆9-THC
antibodies that bind ∆9-THC with good affinity. The synthetic route to radiolabeled ∆9-THC
has enabled the correct assessment of the affinity of these antibodies to their ligand and may
facilitate future binding studies between ∆9-THC and its analogues and the cannabinoid
receptors.
Introduction
ability of marijuana for medical use have heightened
societal awareness of the risks and possible benefits9
associated with this drug. This has further highlighted
the need to fully understand its mechanism of action
and the treatment of adverse effects.
Marijuana obtained from Cannabis sativa has been
used by humans for over 4000 years and continues to
be one of the most common recreational drugs among
substance abusers. The plant contains a mixture of
natural cannabinoids, and the study of these compounds
has intensified greatly since the early 1990s when the
chief cannabinoid receptor (CB1) and its endogenous
ligand, anandamide, were identified.1 The main psy-
choactive constituent of marijuana, (-)-∆9-tetrahydro-
cannabinol (∆9-THC), binds to the CB1 receptor and is
quickly metabolized principally to 11-hydroxy-∆9-THC,
9-carboxy-11-nor-∆9-THC, and 8â,11-dihydroxy-∆9-THC,
as well as their glucuronide conjugates.2,3 The structure
of ∆9-THC was first elucidated about 40 years ago by
Gaoni and Mechoulam,4 and total syntheses for many
of the natural cannabinoids have been reported.5,6
Endocannabinoid receptors have been recognized as
mediators of key physiological processes such as pain,
motility, memory, and appetite.2
Marijuana has been the most abused illicit substance
used in the U.S. for several decades.7,8 Understanding
changes in the use of marijuana over time is important
for a number of reasons. Marijuana use is associated
with impaired educational attainment, reduced work-
place productivity, and increased risk of use of other
substances. Marijuana use plays a major role in motor
vehicle crashes and has adverse effects on the respira-
tory and cardiovascular systems.7,8 The increasing
spread of marijuana use, especially among adolescents
and young adults, and the potential regulated avail-
Immunopharmacotherapy is based on the elicitation
or administration of antibodies that are capable of
binding the targeted drug before it can reach the brain.
This strategy has emerged as a powerful tool at the
interface of chemistry and biology. Immunopharmaco-
therapeutic studies have been reported to treat addic-
tion to cocaine, nicotine, PCP, and methamphetamine.10
In this study, we report our efforts to develop an
immunotherapeutic strategy to combat marijuana de-
pendence. Several different THC-based haptens were
designed and synthesized, and their immunogenic prop-
erties were studied in mice. Whereas several previously
reported immunoconjugates are based on conjugation
to the carrier protein through the phenol moiety of ∆9-
THC, our approach is based on conjugation through the
aliphatic side chain. Most of the immunoconjugates
showed good immunogenicity, and several monoclonal
antibodies (mAbs) with good affinity and specificity for
THC were isolated. The binding propertes of mAbs to
∆9-THC were assessed using radiolabeled ∆9-THC and
analogues. Radiolabeled ∆9-THC is not commercially
available perhaps because of a lack of demand, given
that past efforts to use it for detecting endogenous
cannabinoids were unsuccessful.11 Hence, one of our
aims was to synthesize the tritium-labeled compound,
based on literature procedures but without the use of
tritium gas that is subject to safety issues and regula-
tory complications. The syntheses of THC-based hap-
tens, generation of anti-THC antibodies, the synthesis
of radiolabeled ∆9-THC and analogues for the study of
antibody binding properties are presented herein.
* To whom correspondence should be addressed. Phone: (858) 784-
2516. Fax: (858) 784-2595. E-mail: kdjanda@scripps.edu.
† Departments of Chemistry and Immunology, and The Skaggs
Institute for Chemical Biology.
‡ Department of Neuropharmacology.
10.1021/jm050442r CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/14/2005

7390 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Qi et al.
Scheme 1. Synthesis of Haptens TCA, TCB, TCC, and
TCDa
Figure 1. Structures of ∆9-THC, ∆8-THC, and various hap-
tens.
Results and Discussion
1. Chemistry. 1.1. Hapten Design. Although ∆9-
THC is the major component of marijuana (Figure 1),
∆8-THC shows roughly similar bioactivities as ∆9-THC
and is thermodynamically stable.12 Therefore, we chose
to design haptens based on both THC isomers.
a Reagents and conditions: (a) triethyl 4-phosphonocrotonate,
NaH, 50%; (b) H2, Pd/C and Pd(OH)2, 85%; (c) LAH, 99%; (d) PPh3,
CBr4, 86%; (e) BBr3, 98%; (f) (s)-cis-verbenol, TsOH, 47%; (g) BF3/
OEt2, 71%; (h) OH(Me2)CCN, DBU, 86%; (i) NaOH, 94%; (j) H-â-
alanine-OEt, HBTU, 83%; (k) NaOH, 81%; (l) HCl, ZnCl2, 92%;
(m) KOtBu; (n) H-â-alanine-OtBu, HBTU, 86%; (o) TFA.
Previously reported THC-related haptens typically
contained short linkers attached to the phenol moiety.
In our design, the linker is conjugated to the aliphatic
side chain that is distant from the tricyclic core struc-
ture. A more specific immune response is expected due
to the intact THC core structure and the functional
groups in these haptens. Haptens TCA, TCB, TCC,
TCD, TCE, and TCF are shown in Figure 1. In a pre-
vious study, we had reported a beneficial linker effect
on antibody generation when â-alanine was incorpo-
rated in the hapten design.13 We continue to study the
potential generality of this phenomenon. With the new
amide functionality and increased linker length, the
haptens are expected to be more effective in generating
specific anti-THC antibodies. We also designed TCE and
TCF, which were modified from original THC structures
by using more “aromatic elements” and amide bonds for
increased immunogenicity.
1.2. Hapten Synthesis. The syntheses for TCA, TCB,
TCC, and TCD are shown in Scheme 1. Starting from
the commercially available 3, 5-dimethoxybenzaldehyde,
the aromatic moiety was prepared according to Huff-
man’s method14 of reacting 3,5-dimethoxybenzaldehyde
with triethyl-4-phosphonocrotonate to provide ester 1.
Catalytic hydrogenation followed by LiAlH4 reduction
gave alcohol 3 in good yield. Bromination followed by
demethylation gave bromide 5. Coupling of 5 with (s)-
cis-verbenol under TsOH provided compound 6, which
was cyclized to the brominated ∆8-THC analogue 7. The
bromide was then converted to give hapten TCC (9) by
cyanation and hydrolysis. TCC was coupled with â-ala-
nine ethyl ester to give TCD precursor 10, which was
hydrolyzed by NaOH to obtain TCD (11). TCA (13) was
prepared as a mixture with TCC by migration of the
double bond in TCC following published procedures.15,16
TCB (15) was prepared as a mixture with TCD from
compound 12, followed by purification. We decided to
prepare TCA and TCB based immunoconjugates as
mixtures incorporating TCC and TCD, respectively,
partly because of difficulties related to pure TCA and
TCB hapten preparation and partly because of our
experience in past immunization strategies that this
strategy may yield antibodies with specificity for ∆9-
THC as well as antibodies that will bind both isomers.
The latter could be advantageous because ∆8-THC,
which has roughly 75% of the bioactivity of ∆9-THC, is
present in low amounts in marijuana; furthermore,
since this compound is more stable than ∆9-THC, its
bioavailability may be significantly higher.
Haptens TCE and TCF were prepared as shown in
Scheme 2. Commercially available 3,5-dimethoxyben-
zaldehyde was reacted with ethanedithiol in the pres-
ence of BF3 etherate, after which demethylation yielded
compound 17, which was reacted with (s)-cis-verbenol
in the presence of TsOH to give diol 18. BF3 etherate
mediated ring closure, followed by phenol protection
with acetic anhydride, and deprotection of the aldehyde
moiety gave 21, which was converted to carboxylic acid
22 and coupled to â-alanine ethyl ester to provide
compound 23. The aromatic THC analogue was obtained

∆9-Tetrahydrocannabinol Studies
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7391
Scheme 2. Synthesis of Haptens TCE and TCFa
Scheme 3. Synthesis of Tritium Labeled ∆9-THC
Analogue 34ba
a Reagents and conditions: (a) ethanedithiol, BF3/Et2O, 99%;
(b) BBr3, 69%; (c) (s)-cis-verbenol, TsOH, 43%; (d) BF3/Et2O, 54%;
(e) Ac2O, Py, 75%; (f) AgNO3; (g) NaH2PO3, NaClO2, 2-methyl-2-
butene; (h) H-â-Ala-OEt, HBTU, Et3N, 24% (three steps); (i) sulfur,
200 °C, 49%; (j) LiOH, H2O, THF, 84%.
by the method of Tuchinda17 through treatment of 23
with sulfur at high temperature for 45 min. Removal of
protecting groups gave hapten TCE (25); this compound
was then coupled to â-alanine ethyl ester, followed by
deprotection to give hapten TCF (26).
a Reagents and conditions: (a) (i) Mg, THF, reflux; (ii) Li2CuCl4,
THF, -78 °C to room temp, 81%; (b) BBr3, CH2Cl2, -78 °C to room
temp, 84%; (c) Mg, THF, reflux -78 °C to room temp, 67%; (d)
Pd/C (10%), H2, AcOH, 54%; (e) (i) (PhSe)2, NaBH4, EtOH; (ii)
H2O2, THF; (iii) room temp, then reflux, 52% for three steps; (f)
BF3/Et2O, CH2Cl2, MgSO4, 0 °C, 29, 42%; (g) NaN3, DMF, NaI
(cat.), 60 °C, 92%; (h) THF/H2O, polymer-supported Ph3P; (i)
polymer-supported pyridine, Ac2O-(tritium-6), CH2Cl2, 80%; (j)
polymer-supported pyridine, Ac2O, CH2Cl2, 88% for two steps.
1.3. Design and Synthesis of Radiolabeled ∆9-
THC and Analogues. To assess the binding properties
of mAbs to ∆9-THC, we set out to synthesize radio-
labeled ∆9-THC and some closely related analogues. The
first radiolabeled analogue was designed on the basis
of the structure of TCA, and its synthesis is shown in
Scheme 3. Chloride 28 was prepared from the com-
mercially available reagents by a Grignard mediated
coupling reaction.18 However, we note that purification
of 28 from the starting materials was complicated;
hence, an alternative method was sought.19 3,5-Di-
methoxybenzaldehyde was reacted with the Grignard
reagent to give benzylic alcohol 27, which was dehy-
droxylated by catalytic hydrogenation to chloride 28.
Demethylation of 28 provided chloride 29 in very pure
form. Compound 30 was synthesized from (+)-limonene
oxide by a two-step approach.20 Acid-catalyzed coupling
between compounds 30 and 29 gave the desired product
with high purity after flash chromatography. The
chloride 31 was converted to azide 32, which was
reduced to primary amine 33 by a Staudinger reaction
using polymer-supported triphenylphosphine. A selec-
tive model acylation of the primary amine gave target
amide 34a, using polymer-supported pyridine as base
to facilitate its workup. Reaction of 33 with tritium-
labeled acetic anhydride afforded the tritium labeled
analogue 34b in good yield.
is not a good ∆9-THC mimic because poor binding was
observed with the antibodies tested. This was rather
surprising because the structure of 34b is highly
congruent to that of the TCB hapten used to elicit these
antibodies.
We next decided to concentrate our efforts on the
synthesis of [3H]-∆9-THC. Owing to the lack of avail-
ability of tritium as reducing agent, a synthesis with
high yield of radiolabel incorporation proved to be
difficult. With this second setback we decided to explore
a new synthetic approach that would require reduction
of a bromo-THC analogue to [3H]-∆9-THC with LiAlT4
or NaBT4.
3,5-Dimethoxybenzaldehyde was reacted with an in
situ generated Grignard reagent to give benzylic alcohol
35 (Scheme 4). Subsequent dehydroxylation by catalytic
hydrogenation was ineffective. After extensive experi-
ments, we found that a TMSCl/NaI combination effected
the transformation successfully.21 Product 36 was depro-
tected by BBr3 etherate, and the resulting primary
alcohol was converted to bromide 37 simultaneously.
Coupling of 37 with 30 under acidic conditions provided
the brominated ∆9-THC analogue 38. A model reduction
of the bromide with LiAlH4 or NaBH4 selectively de-
brominated 38 to give ∆9-THC without affecting the
internal double bond. This procedure was repeated
With the completion of a route to 34b, binding
analysis using equilibrium dialysis with several mAbs
that had shown good binding to ∆9-THC using competi-
tion ELISA was undertaken; however, we note that 34b

7392 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Qi et al.
Scheme 4. Synthesis of Tritium-Labeled ∆9-THC
Analogue 39ba
Figure 2. Binding curves for mAbs TCB 25G1, TCB 39H9,
and TCB 40B6 with ∆9-THC analogue 39b as ligand.
days later, the spleen was fused with a myeloma cell
line (X63-Ag8.653) to produce hybridomas according to
standard techniques22 with some modifications devel-
oped in our laboratory. The hybridomas were cloned into
96-well plates and screened against the respective BSA
conjugates by ELISA during the cloning process. Each
member of a final panel of 60 mAbs then was assessed
for binding and specificity to ∆9-THC by using competi-
tion ELISA. This assay yielded several good mAbs, of
which three were evaluated in more detail by equilib-
rium dialysis.
a Reagents and conditions: (a) Mg, THF, reflux -78 °C to room
temp, 93%; (b) TMSCl, MeCN, Et2O/H2O, room temp, 24 h, 88%;
(c) BBr3, CH2Cl2, -78 °C to room temp, 36 h, 80%; (d) BF3/Et2O,
CH2Cl2, MgSO4, 0 °C, 30, 38%; (e) LAH, THF, room temp or
NaBH4, DMSO 47%; (f) tritium-LAH, THF, room temp, 50% or
tritium-NaBH4, DMSO, room temp, 80%.
Table 1. Serum Antibody Titers and Relative Affinities for
∆9-THC and ∆8-THC
2.2. Monoclonal Antibody Binding Studies. We
used equilibrium dialysis, a powerful tool in the deter-
mination of physiologically relevant protein-ligand
dissociation constants,23 to study the affinity of mAbs
TCB25G1, TCB39H9, and TCB40B6 for [3H]-∆9-THC.
This technique is more quantitative than competition
ELISA. Since the results of this assay are obtained
under equilibrium conditions, the physiological inter-
action can be studied and low affinity interactions that
are not detectable with high reliability using other
methods can also be accomplished. The results of this
assay are shown in Figure 2. The mAbs TCB25G1,
TCB39H9, and TCB40B6 showed moderate to good
binding to ∆9-THC with Kd ) 0.23 ( 0.02, 0.71 ( 0.06,
and 1.41 ( 0.14 µM, respectively, a striking difference
to the results obtained from the assay with radiolabeled
compound 34b. This points to a relatively specific
interaction between the aliphatic side chain of ∆9-THC
and the selected mAbs. Hence, by employing [3H]-∆9-
THC, we were able to identify mAbs that have good
affinity for ∆9-THC. On the basis of ∆9-THC serum
concentrations that reach up to 0.8 µM after smoking,24
mAb TCB25G1 can be considered a good candidate to
bind ∆9-THC in vivo with potential physiological con-
sequences.
average
titer
∆9-THC affinity,
µM
∆8-THC affinity,
µM
hapten
TCA
TCB
TCC
TCD
TCE
TCF
1870
11600
2530
8800
25600
14400
39
48
72
51
20
48
85
53
>128
>128
>128
>128
successfully, using LiAlT4 and NaBT4, to give tritium-
labeled ∆9-THC analogue 39b. We note that the NaBT4-
mediated reduction was more efficient, with an increase
in yield (80% versus 50%) and resulting in a 17-fold
higher specific activity of the final product (4.0 Ci/mmol).
This increase in specific activity of the probe was crucial
in our endeavor to correctly assess the binding constants
of potential mAbs with high affinity for ∆9-THC.
2. Immunochemistry. 2.1. Murine Immunization.
Immunoconjugates of haptens conjugated to bovine
serum albumin (BSA) and keyhole limpet hemocyanin
(KLH) were prepared using standard EDC coupling
procedures and dialyzed against PBS, pH 7.4. The KLH
immunoconjugates were used to immunize mice (strain
129 GIX+) that were not older than 3 months of age.
The first injection (200 µL) contained the immunocon-
jugate (100 µg based on KLH) and RIBI adjuvant (50
µg) reconstituted in PBS. The injection was adminis-
tered ip. A second booster injection was given 2 weeks
later in a similar fashion. Bleeds of mice 7-10 days later
were used to assess antibody titers and specificities
using enzyme linked immunosorbent assay (ELISA) and
competition ELISA with ∆9-THC and ∆8-THC (Table 1).
On the basis of the titers and relative affinities of the
sera, we used mice that were immunized with TCB-
KLH for hybridoma production. A final tail vein iv
injection of KLH immunoconjugate (50 µg) in PBS (150
µL) was given 1 month after the final ip boost. Three
Conclusions
In this study, we synthesized several haptens based
on the ∆9-THC core and developed a new and convenient
route to radiolabeled ∆9-THC and related analogues. Six
THC-based immunoconjugates were prepared and used
for the immunization of mice, which in several cases
showed a strong immune response and a reasonable
degree of binding specificity for ∆9-THC. Our convenient
synthesis of tritium-labeled ∆9-THC allowed assessment
of binding constants for several mAbs that were elicited

∆9-Tetrahydrocannabinol Studies
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7393
1-(5-Bromopentyl)-3,5-dimethoxybenzene (4). To a so-
lution of 5-(3,5-dimethoxyphenyl)pentan-1-ol (717 mg, 3.20
mmol) in CH2Cl2 (11 mL) at 0 °C, were added PPh3 (1.43 g,
5.44 mmol) and CBr4 (1.59 g, 4.80 mmol). The mixture was
allowed to warm to room temperature. After being stirred for
14 h at room temperature, the solution was filtered by silica
gel and washed by CH2Cl2. After evaporation, the residue was
purified by chromatography on silica gel (EtOAc/hexane 1:10)
to afford the desired compound 4 as a yellow oil (793 mg, 2.76
mmol, 86%). 1H NMR (400 MHz, CDCl3): δ 1.42-1.54 (m, 2H),
1.60-1.70 (m, 2H), 1.83-1.93 (m,2H), 2.57 (t, J ) 7.6 Hz, 2H),
3.41 (t, J ) 6.8 Hz, 2H), 3.78 (s, 6H), 6.31 (t, J ) 2.4 Hz, 1H),
6.34 (d, J ) 2.4 Hz, 2H). 13C NMR (75.5 MHz, CDCl3): δ 27.9,
30.4, 32.8, 33.9, 36.1, 55.3, 97.6, 106.3, 144.5, 160.4. HRMS
(MALDI-FTMS) calcd for C13H20BrO2 (MH+) 287.0641, found
287.0637.
5-(5-Bromopentyl)benzene-1,3-diol (5). A solution of
1-(5-bromopentyl)-3,5-dimethoxybenzene (4, 762 mg, 2.65
mmol) and BBr3 (7.95 mL, 1 M solution in CH2Cl2) was stirred
for 45 min at -78 °C, after which the mixture was allowed to
warm to room temperature. After being stirred for 6 h, the
mixture was poured into saturated NaHCO3 and extracted four
times with CH2Cl2. The combined organic extracts were dried
over MgSO4 and evaporated. The residue was purified by
chromatography on silica gel, eluting with EtOAc/hexane (1:
1) to afford the desired compound 5 as a yellow oil (748 mg,
2.60 mmol, 98%). 1H NMR (300 MHz, CDCl3): δ 1.35-1.45
(m, 2H), 1.46-1.60 (m, 2H), 1.75-1.88 (m, 2H), 1.53 (pent, J
) 6.9 Hz, 2H), 2.43 (t, J ) 7.8 Hz, 2H), 3.50 (t, J ) 6.9 Hz,
2H), 6.15-6.18 (m, 1H), 6.23 (d, J ) 2.4 Hz, 2H). 13C NMR
(75.5 MHz, CDCl3): δ 27.8, 30.1, 32.6, 34.0, 35.5, 100.4, 108.2,
145.6, 156.1. HRMS (MALDI-FTMS) calcd for C11H16BrO2
(MH+) 259.0328, found 259.0331.
using the immunoconjugate TCB-KLH. The synthetic
strategy developed for [3H]-∆9-THC provides an efficient
route from readily available starting materials while
avoiding the use of tritium gas as a reducing agent. The
mAb TCB25G1 that we obtained using immunization
of mice with TCB-KLH showed good affinity for ∆9-THC
(Kd ) 0.23 µM) and we are currently studying the effects
of mAb TCB25G1 on the behavior of rats that are
treated with ∆9-THC.
Experimental Section
Chemistry. 1H and 13C NMR spectra were measured at
399.7 MHz on a Varian INOVA-400, 300 MHz on a Varian
MERCURY-300BB, 400 and 500 MHz on Bruker AMX-400 and
DRX-500 spectrometers. Chemical shifts (ppm) are reported
relative to internal TMS (1H, 0.00 ppm) and CDCl3 (13C, 77.0
ppm). HRMS and high-accurate spectra were measured using
MALDI and ESI-TOF techniques. Solvents were dried by
standard methods. Flash chromatography was performed on
silica gel 60 (230-400 mesh) and thin-layer chromatography
(TLC) on glass plates coated with a 0.25-mm layer of silica
gel 60 F-254.
5-(3,5-Dimethoxyphenyl)penta-2,4-dienoic Acid Ethyl
Ester (1). A mixture of NaH (50%, 3.5 g, 72.2 mmol) in THF
(60 mL) was stirred at 0 °C under Ar. To the mixture, triethyl
4-phosphonocrotonate (18 g, 72.2 mmol) in THF (40 mL) was
added dropwise in 20 min. After 15 h, 3,5-dimethoxybenzal-
dehyde (10 g, 60.2 mmol) in THF (30 mL) was added slowly
in 15 min at 0 °C. The mixture was allowed to warm to room
temperature. After being stirred for 12 h, the reaction mixture
was poured into water and extracted two times with Et2O. The
combined organic extracts were washed with water and then
saturated brine, dried over MgSO4, and evaporated. The
residue was purified by chromatography on silica gel, eluting
with EtOAc/hexane (1:10) to afford the desired compound 1
as yellow crystals (7.93 g, 30.3 mmol, 50%). 1H NMR (400 MHz,
CDCl3): δ 1.30 (t, J ) 7 Hz, 3H), 3,79 (s, 6H), 4.21 (q, J ) 7
Hz, 2H), 5.97 (d, J ) 15.6 Hz, 1H), 6.42 (t, J ) 2.2 Hz, 1H),
6.59 (d, J ) 2.3 Hz, 2H), 6.75-6.85 (m, 2H), 7.33-7.44 (m,
1H). 13C NMR (100 MHz, CDCl3): δ 14.2, 55.3, 60.3, 101.2,
105.0, 121.4, 126.6, 137.8, 140.2, 144.2, 160.9, 166.9. HRMS
(MALDI-FTMS) calcd for C15H19O4 (MH+) 263.1278, found
263.1274.
5-(3,5-Dimethoxyphenyl)pentanoic Acid Ethyl Ester
(2). A solution of 5-(3,5-dimethoxyphenyl)penta-2,4-dienoic
acid ethyl ester (7.93 g, 30.3 mmol) and 10% Pd/C (400 mg) in
MeOH (150 mL) was stirred under H2. After 3.5 h, 20% Pd-
(OH)2 (400 mg) was added and the mixture was stirred for 13
h. The mixture was filtered and evaporated, and the residue
was purified by chromatography on silica gel, with Et2O as
eluant, to afford the desired compound 2 as a pale-yellow oil
(6.86 g, 25.7 mmol, 85%). 1H NMR (400 MHz, CDCl3): δ 1.24
(t, J ) 7 Hz, 3H), 1.57-1.75 (m, 4H), 2.31 (t, J ) 7 Hz, 2H),
2.57 (t, J ) 7.1 Hz, 2H), 3.33 (s, 6H), 4.12 (q, J ) 7 Hz, 2H),
6.29-6.31 (m, 1H), 6.32-6.34 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 14.2, 24.5, 30.6, 34.1, 35.8, 55.1, 60.1, 97.6, 106.3,
144.5, 160.6, 173.6. HRMS (MALDI-FTMS) calcd for C15H22O4-
Na (M Na+) 289.141, found 289.1412.
5-(3,5-Dimethoxyphenyl)pentan-1-ol (3). To a mixture
of LiAlH4 (2.0 g, 52.5 mmol) in THF (175 mL), 5-(3,5-
dimethoxyphenyl)pentanoic acid ethyl ester 2 (14.0 g, 52.5
mmol) in THF (100 mL) was added by a dropping funnel over
40 min at 0 °C. After 2 h 40 min, H2O (2 mL), 2 N NaOH (2
mL), and H2O (6 mL) were added to the reaction mixture
successively. After the precipitate (aluminum hydroxide) was
removed by filtration, the clear solution was evaporated to
afford 3 as a colorless liquid (11.7 g, 52.0 mmol, 99%). 1H NMR
(400 MHz, CDCl3): δ 1.35-1.45 (m, 2H), 1.55-1.70 (m, 4H),
1.84-1.89 (m, 1H), 2.57 (t, J ) 7.8 Hz, 2H), 3.65 (t, J ) 6.6
Hz, 2H), 3.78 (s, 6H), 6.30 (t, J ) 2.1 Hz, 1H), 6.34 (d, J ) 2.1
Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 25.3, 31.0, 32.6, 36.2,
55.2, 62.8, 97.5, 106.4, 145.0, 160.6. HRMS (MALDI-FTMS)
calcd for C13H21O3 (MH+) 225.1485, found 225.1487.
5-(5-Bromopentyl)-2-[(1S-cis)-4,6,6-trimethylbicyclo-
[3.1.1]hept-3-en-2-yl]benzene-1,3-diol (6). To a solution of
5-(5-bromopentyl)benzene-1,3-diol (5, 4.5 g, 17.4 mmol) and
p-toluenesulfonic acid (331 mg, 1.74 mmol) in CHCl3 (100 mL)
at room temperature under Ar was added (s)-cis-verbenol (2.6
g, 17.4 mmol, 50% ee) in CHCl3 (50 mL). After being stirred
for 2.5 h, the reaction mixture was poured into saturated
NaHCO3 and extracted two times with CH2Cl2. The combined
organic extracts were dried over MgSO4 and evaporated. The
residue was purified by chromatography on silica gel, eluting
with EtOAc/hexane (1:9) to afford 6 as a yellow oil (3.23 g,
1
8.21 mmol, 47%). H NMR (400 MHz, CDCl3): δ 0.96 (s, 3H),
1.32 (s, 3H), 1.40-1.52 (m, 3H), 1.59 (pent, J ) 7.6 Hz, 2H),
1.84-1.91 (m, 5H), 2.16-2.20 (m, 1H), 2.25-2.33 (m, 2H), 2.45
(t, J ) 7.6 Hz, 2H), 3.40 (t, J ) 7.0 Hz, 2H), 3.90-3.93 (m,
1H), 5.70 (brs, 1H), 6.19 (s, 2H). 13C NMR (100 MHz, CDCl3):
δ 20.4, 23.7, 25.9, 27.8, 30.0, 32.6, 33.8, 35.2, 37.8, 40.7, 47.0,
47.8, 76.7, 108.2, 112.4, 116.4, 119.3, 134.7, 142.0, 142.4, 152.7,
155.5. HRMS (MALDI-FTMS) calcd for C21H30BrO2 (MH+)
393.1424, found 393.1428.
3-(5-Bromopentyl)-6,6,9-trimethyl-6a,7,10,10a-tetrahy-
dro-6H-benzo[c]chromen-1-ol (7). To a solution of 5-(5-
bromopentyl)-2-(4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-yl)-
benzene-1,3-diol (6, 3.23 g, 8.21 mmol) in CH2Cl2 (150 mL) at
0 °C under Ar was added BF3/Et2O (2.08 mL, 16.4 mmol), and
the mixture was allowed to warm to room temperature. After
being stirred for 2 h, the mixture was poured into saturated
NaHCO3 and extracted two times with CH2Cl2. The combined
organic extracts were dried over MgSO4 and evaporated. The
residue was purified by chromatography on silica gel, eluting
with CH2Cl2/hexane (1:1), to yield 7 as a yellow oil (2.29 g,
1
5.83 mmol, 71%). H NMR (400 MHz, CDCl3): δ 1.10 (s, 3H),
1.38 (s, 3H), 1.41-1.52 (m, 2H), 1.52-1.68 (m, 2H), 1.70 (brs,
3H), 1.76-1.96 (m, 5H), 2.06-2.22 (m, 1H), 2.45 (t, J ) 7.5
Hz, 1H), 2.64-2.76 (m, 1H), 3.20 (dd, J ) 16.5 Hz, 4.2 Hz,
1H), 3.39 (t, J ) 6.7 Hz, 2H), 5.02 (s, 1H), 5.42 (brd, J ) 3.9
Hz, 1H), 6.09 (d, J ) 1.2 Hz, 1H), 6.25 (d, J ) 1.2 Hz, 1H). 13
C
NMR (75.5 MHz, CDCl3): δ 18.6, 23.5, 27.6, 27.88, 27.9, 30.1,
31.6, 32.7, 33.9, 35.2, 36.0, 44.9, 76.7, 107.5, 109.9, 110.6, 119.2,
134.6, 141.8, 154.6, 154.7. HRMS (MALDI-FTMS) calcd for
C21H30BrO2 (MH+) 393.1424, found 393.1412.

7394 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Qi et al.
6-(1-Hydroxy-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-
6H-benzo[c]chromen-3-yl)hexanenitrile (8). To a solution
of 3-(5-bromopentyl)-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-
6H-benzo[c]chromen-1-ol (7, 2.16 g, 5.49 mmol) in acetonitrile
(60 mL) at room temperature under Ar were added DBU (2.51
g, 16.5 mmol) and acetone cyanohydrin (1.40 g, 16.5 mmol).
The reaction mixture was stirred for 17 h. After evaporation
of the solvent, the residue was partitioned between water and
EtOAc. The organic layer was washed with water and then
brine, dried over MgSO4, and evaporated. The residue was
purified by chromatography on silica gel, eluting with EtOAc/
hexane (1:3), to afford compound 8 as a yellow oil (1.61 g, 4.74
mmol, 86%). 1H NMR (400 MHz, CDCl3): δ 1.10 (s, 3H), 1.38
(s, 3H), 1.42-1.52 (m, 2H), 1.54-1.68 (m, 5H), 1.70 (br s, 3H),
1.74-1.90 (m, 2H), 2.10-2.20 (m, 1H), 2.33 (t, J ) 7.0 Hz, 2H),
2.47 (dt, J ) 2.8 and 7.6 Hz, 2H), 2.70 (dt, J ) 11.2 and 4.8
Hz, 1H), 3.19 (dd, J ) 16 and 4.4 Hz, 1H), 4.73 (s, 1H), 5.43
(brd, J ) 4.4 Hz, 1H), 6.10 (d, J ) 1.6 Hz, 1H), 6.25 (d, J ) 1.6
Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 17.1, 18.5, 23.5, 25.2,
27.5, 27.8, 28.2, 29.9, 30.3, 31.5, 34.9, 35.9, 44.8, 107.6, 109.9,
110.8, 119.3, 119.8, 125.5, 134.7, 141.6, 154.9. HRMS (MALDI-
FTMS) calcd for C22H29NO2Na (MNa+) 362.2096, found
362.2091.
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-3-yl)hexanoylami-
no]propionic acid ethyl ester (10, 80 mg, 0.180 mmol) in THF/
H2O (3 mL/1 mL) at 60 °C under Ar was added LiOH/H2O (15
mg, 0.360 mmol). After being stirred for 1 h, the reaction
mixture was poured into water and extracted two times with
EtOAc. The organic layer was washed with saturated brine,
dried over MgSO4, and evaporated. The residue was purified
by chromatography on silica gel, eluting with MeOH/CH2Cl2
(1:9), to afford the desired compound 11 (TCD) as a yellow oil
1
(60.9 mg, 0.146 mmol, 81%). H NMR (400 MHz, CDCl3): δ
1.09 (s, 3H), 1.20-1.36 (m, 2H), 1.37 (s, 3H), 1.45-1.66 (m,
5H), 1.68 (s, 3H), 1.72-1.87 (m, 2H), 2.08-2.20 (m, 1H), 2.16
(t, J ) 7.4 Hz, 2H), 2.39 (t, J ) 7.4 Hz, 2H), 2.59 (t, J ) 5.6
Hz, 2H), 2.64-2.75 (m, 1H), 3.27 (brd, J ) 16.8 Hz, 1H), 3.45-
3.60 (m, 2H), 5.41 (brd, J ) 2.8 Hz, 1H), 6.12 (brs, 1H), 6.20
(brs, 1H), 6.39 (brs, 1H). 13C NMR (100 MHz, CDCl3): δ 18.4,
23.5, 25.4, 27.5, 27.9, 28.5, 29.7, 30.3, 31.6, 35.0, 35.3, 35.9,
36.3, 44.9, 76.8, 107.8, 109.3, 110.9, 119.2, 134.8, 141.8, 154.5,
155.7, 174.9. HRMS (MALDI-FTMS) calcd for C25H35NO5Na
(MNa+) 452.2407, found 452.2400.
6-(9-Chloro-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-
hexahydro-6H-benzo[c]chromen-3-yl)hexanoic Acid (12).
To a solution of 6-(1-hydroxy-6,6,9-trimethyl-6a,7,10,10a-tet-
rahydro-6H-benzo[c]chromen-3-yl)hexanoic acid (400 mg, 1.12
mmol) and ZnCl2 (80 mg, 0.587 mmol) in toluene at -10 °C
(40 mL) was bubbled gasous HCl for 2 h. The mixture was
stirred for 1.5 h after stopping the bubbling of HCl. The
reaction mixture was poured into water and extracted two
times with EtOAc. The organic layer was washed with
saturated brine, dried over MgSO4, and evaporated. The
residue was purified by chromatography on silica gel, eluting
with EtOAc/hexane (1:1), to afford chloride 12 as a yellow oil
(409 mg, 1.03 mmol, 92%). 1H NMR (400 MHz, CDCl3): δ 1.13
(s, 3H), 1.30-1.50 (m, 3H), 1.39 (s, 3H), 1.54-1.70 (m, 8H),
1.67 (s, 3H), 1.71-1.79 (m, 2H), 2.14-2.22 (m, 1H), 2.35 (t, J
) 7.4 Hz, 2H), 2.44 (dd, J ) 6.8 Hz, 8.4 Hz, 2H), 3.05 (dt, J )
2.8 Hz, 11.2 Hz, 1H), 3.44 (dt, J ) 14 Hz, 2.8 Hz, 1H), 6.08 (d,
J ) 1.6 Hz, 1H), 6.24 (d, J ) 1.6 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ 19.1, 24.2, 24.4, 27.6, 28.5, 30.3, 31.3, 33.9, 34.2,
35.0, 42.0, 44.7, 48.7, 72.7, 76.9, 107.7, 109.1, 109.9, 142.3,
154.6, 155.0, 179.8. HRMS (MALDI-FTMS) calcd for C22H32-
ClO4 (MH+) 395.1984, found 395.1990.
6-(1-Hydroxy-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-
6H-benzo[c]chromen-3-yl)hexanoic Acid (9, TCC). 6-(1-
Hydroxy-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]-
chromen-3-yl)hexanenitrile (8, 1.06 g, 2.65 mmol) in a solution
of 50% aqueous MeOH (25 mL) containing 15% NaOH was
refluxed for 4 h. The reaction mixture was acidified with 1 N
HCl and extracted two times with EtOAc. The combined
extracts were washed with saturated brine, dried over MgSO4,
and evaporated. The residue was purified by chromatogra-
phyon silica gel, eluting with EtOAc/hexane (1:1), to afford the
desired compound 9 (TCC) as a yellow amorphous solid (895
1
mg, 2.50 mmol, 94%). H NMR (400 MHz, CDCl3): δ 1.10 (s,
3H), 1.32-1.42 (m, 2H), 1.37 (s, 3H), 1.53-1.72 (m, 5H), 1.70
(brs, 3H), 1.74-1.90 (m, 2H), 2.06-2.20 (m, 1H), 2.35 (t, J )
7.3 Hz, 2H), 2.41-2.45 (m, 2H), 2.69 (dt, J ) 4.8 and 10.8 Hz,
1H), 3.19 (dd, J ) 4.2 and 16.2 Hz, 1H), 4.55-4.95 (br, 1H),
5.43 (d, J ) 4.0 Hz, 1H), 6.09 (d, J ) 1.6 Hz, 1H), 6.26 (d, J )
1.6 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 18.4, 23.5, 24.4,
27.5, 27.8, 28.6, 30.4, 31.5, 33.9, 35.1, 35.9, 44.8, 76.7, 107.7,
109.9, 110.7, 119.3, 134.7, 142.1, 154.7, 154.9, 180.0. HRMS
(MALDI-FTMS) calcd for C22H30O4Na (MNa+) 381.2036, found
381.2040.
6-(1-Hydroxy-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-6H-
benzo[c]chromen-3-yl)hexanoic Acid (13, TCA). To a
solution of KOtBu (44 mg, 0.391 mmol) in benzene (4 mL) at
4 °C under Ar was added 6-(9-chloro-1-hydroxy-6,6,9-trimethyl-
6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)hexano-
ic acid (12, 52 mg, 0.112 mmol) in benzene (2 mL). After being
stirred for 30 min, the mixture was warmed to 65 °C for 20
min, cooled, and bubbled with CO2 for 30 min. The reaction
mixture was poured into water and extracted two times with
EtOAc. The organic extract was washed with saturated brine,
dried over MgSO4, and evaporated. The desired compound 13
(TCA) was obtained as a 1:1 mixture with 9 (TCC), in the form
3-[6-(1-Hydroxy-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-
6H-benzo[c]chromen-3-yl)hexanoylamino]propionic Acid
Ethyl Ester (10). To a mixture of 6-(1-hydroxy-6,6,9-trimeth-
yl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-3-yl)hexanoic acid
(9, 237 mg, 0.661 mmol), Et3N (0.1 mL, 0.728 mmol), and
HBTU (276 mg, 0.728 mmol) in DMF (12 mL) at room
temperature under Ar was added H-â-alanine ethyl ester
hydrochloride (112 mg, 0.728 mmol). After being stirred for 2
h, the reaction mixture was poured into water and extracted
two times with Et2O/EtOAc (1:1). The organic layer was
washed with saturated brine, dried over MgSO4, and evapo-
rated. The residue was purified by chromatography on silica
gel, eluting with EtOAc/hexane (1:1), to afford 10 as a yellow
oil (244 mg, 0.550 mmol, 83%). 1H NMR (400 MHz, CDCl3): δ
1.10 (s, 3H), 1.27 (t, J ) 7.2 Hz, 3H), 1.24-1.35 (m, 2H), 1.37
(s, 3H), 1.47-1.68 (m, 5H), 1.69 (s, 3H), 1.72-1.87 (m, 2H),
2.06-2.20 (m, 1H), 2.16 (t, J ) 7.2 Hz, 2H), 2.41 (t, J ) 7.8
Hz, 2H), 2.54 (t, J ) 6 Hz, 2H), 2.70 (dt, J ) 4.4 and 10.8 Hz,
1H), 3.28 (dd, J ) 4.4 and 16 Hz, 1H), 3.52 (q, J ) 6.0 Hz,
2H), 4.16 (q, J ) 7.2 Hz, 2H), 5.41 (brd, J ) 4.0 Hz, 1H), 6.16
(brs, 1H), 6.19 (brs, 1H), 6.27 (brt, J ) 6.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3): δ 14.1, 18.4, 23.5, 25.4, 27.5, 27.9, 28.6,
30.4, 31.6, 33.9, 34.8, 35.1, 35.9, 36.6, 44.9, 60.8, 76.6, 107.7,
109.2, 110.7, 119.1, 134.9, 141.8, 154.6, 155.7, 172.9, 173.7.
HRMS (MALDI-FTMS) calcd for C27H40NO5 (MH+) 458.2902,
found 458.2092.
1
of a brown oil (41 mg, 96%). H NMR (400 MHz, CDCl3): δ
1.09 (s, 3H), 1.30-1.43 (m, 4H), 1.41 (s, 3H), 1.50-1.70 (m,
5H), 1.67 (brs, 3H), 1.86-1.95 (m, 1H), 2.12-2.18 (m, 1H), 2.34
(t, J ) 7.6 Hz, 2H), 2.44 (t, J ) 7.6 Hz, 2H), 3.15-3.22 (m,
1H), 6.13 (brs, 1H), 6.24 (brs, 1H), 6.32 (brs, 1H). 13C NMR
(100 MHz, CDCl3): δ 19.2, 23.3, 24.5, 25.0, 27.5, 28.5, 29.7,
30.4, 31.1, 33.6, 35.0, 45.7, 77.3, 107.6, 109.2, 109.9, 123.8,
134.2, 142.2, 154.3, 154.7, 180.0. HRMS (MALDI-FTMS) calcd
for C22H30O4Na (MNa+) 381.2036, found 381.2036.
3-[6-(9-Chloro-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,
10a-hexahydro-6H-benzo[c]chromen-3-yl)hexanoylami-
no]propionic Acid tert-Butyl Ester (14). To a mixture of
6-(9-chloro-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahy-
dro-6H-benzo[c]chromen-3-yl)hexanoic acid (12, 200 mg, 0.506
mmol), Et3N (0.18 mL, 1.27 mmol), and HBTU (231 mg, 0.608
mmol) in DMF (6 mL) at room temperature under Ar was
added H-â-alanine tert-butyl ester hydrochloride (114 mg,
0.608 mmol). After being stirred for 1 h, the reaction mixture
was poured into water and extracted two times with Et2O/
EtOAc (1:1). The organic extract was washed with saturated
3-[6-(1-Hydroxy-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-
6H-benzo[c]chromen-3-yl)hexanoylamino]propionic Acid
(11, TCD). To a solution of 3-[6-(1-hydroxy-6,6,9-trimethyl-

∆9-Tetrahydrocannabinol Studies
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7395
brine, dried over MgSO4, and evaporated. The residue was
purified by chromatography on silica gel, eluting with EtOAc/
hexane (1:1), to afford 14 as a yellow oil (229 mg, 0.438 mmol,
2), 5.51 (s, 1H, PhCH), 6.26 (t, J ) 2.4 Hz, 1H, Ph), 6.59 (d, J
) 2.4 Hz, 2H, Ph). GC-MS 214 (M+).
5-[1,3]Dithiolan-2-yl-2-(4,6,6-trimethylbicyclo[3.1.1]hept-
3-en-2-yl)benzene-1,3-diol (18). To a mixture of 17 (2.0 g,
9.40 mmol) and CSA (racemic) (281 mg, 0.94 mmol) in CHCl3
(100 mL) at room temperature under Ar was added (s)-cis-
verbenol (1.43 g, 9.40 mmol, 50% ee) in CHCl3 (50 mL). After
being stirred for 2 h, the reaction mixture was poured into
saturated NaHCO3 and extracted two times with CH2Cl2. The
CH2Cl2 layer was dried over MgSO4 and evaporated. The
residue was purified by chromatography on silica gel, eluting
with EtOAc/hexane (2:8), to afford the desired diol 18 as a
yellow oil (1.39 g, 4.00 mmol, 43%). 1H NMR (400 MHz,
CDCl3): δ 0.95 (s, 3H, -Me), 1.35 (s, 3H, -Me), 1.45-1.48 (m,
2H), 1.85 (dd, J ) 1.6 and 2.4 Hz, 3H), 2.15-2.20 (m, 1H),
2.22-2.34 (m, 2H), 3.28-3.36 (m, 2H), 3.42-3.50 (m, 2H),
3.89-3.93 (m, 1H), 5.49 (s, 1H), 5.68 (brs, 1H), 6.53 (s, 2H).
13C NMR (100 MHz, CDCl3): δ 20.4, 23.7, 25.9, 27.8, 37.9, 40.0,
40.7, 46.8, 47.8, 55.6, 107.7, 114.8, 116.0, 140.3, 153.0, 155.1.
HRMS (MALDI-FTMS) calcd for C19H25O2S2 (M+ + H) 349.1296,
found 349.1296.
1
86%). H NMR (400 MHz, CDCl3): δ 1.13 (s, 3H), 1.23-1.35
(m, 4H), 1.38 (s, 3H), 1.40-1.50 (m, 1H), 1.46 (s, 9H), 1.50-
1.70 (m, 5H), 1.65 (s, 3H), 1.70-1.84 (m, 1H), 2.10-2.22 (m,
1H), 2.15 (t, J ) 7.4 Hz, 2H), 2.40 (t, J ) 7.8 Hz, 2H), 2.46 (t,
J ) 5.8 Hz, 2H), 3.55 (brd, J ) 14 Hz, 1H), 6.14 (brs, 1H),
6.18 (brs, 1H), 6.18-6.25 (m, 1H). 13C NMR (100 MHz,
CDCl3): δ 19.1, 24.2, 25.5, 27.7, 28.1, 28.6, 30.4, 31.4, 34.2,
34.98, 35.04, 36.7, 42.1, 44.6, 48.7, 72.7, 76.7, 81.3, 107.7, 109.0,
109.3, 142.1, 154.9, 155.4, 172.3, 173.5.
3-[6-(1-Hydroxy-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-
6H-benzo[c]chromen-3-yl)hexanoylamino]propionic Acid
(15, TCB). To a solution of 3-[6-(9-chloro-1-hydroxy-6,6,9-
trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-
yl)-hexanoylamino]propionic acid tert-butyl ester (14, 200 mg,
0.383 mmol) in CH2Cl2 (1.7 mL) at 0 °C was added TFA (5
mL) which was precooled at 0 °C, and the mixture was stirred
for 3 h at room temperature. The mixture was coevaporated
with toluene (three times) to give 3-[6-(9-chloro-1-hydroxy-
6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-
3-yl)-hexanoylamino]propionic acid, which was used for the
next reaction without further purification.
3-[1,3]Dithiolan-2-yl-6,6,9-trimethyl-6a,7,10,10a-tetrahy-
dro-6H-benzo[c]chromen-1-ol (19). To a solution of diol 18
(1.0 g, 2.87 mmol) in CH2Cl2 (70 mL) at 0 °C under Ar was
added BF3/Et2O (0.73 mL, 5.74 mmol). The mixture was
allowed to warm to room temperature and stirred for 1 h, after
which the mixture was poured into saturated NaHCO3 and
extracted two times with CH2Cl2. The organic layer was dried
over MgSO4 and evaporated. The residue was purified by
chromatography on silica gel, eluting with EtOAc/hexane (1:
9), to afford the desired target product 19 as a yellow oil (541
To a solution of KOtBu (71 mg, 0.630 mmol) in benzene (5
mL) at 4 °C under Ar was added 3-[6-(9-chloro-1-hydroxy-6,6,9-
trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl-
)hexanoylamino]propionic (58.7 mg, 0.126 mmol) in benzene
(1 mL). After being stirred for 30 min, the mixture was warmed
to 65 °C for 20 min, cooled, and bubbled with CO2 for 30 min.
The reaction mixture was poured into water and extracted two
times with EtOAc. The organic extract was washed with
saturated brine, dried over MgSO4, and evaporated. The
desired compound 15 (TCB) was obtained as a 1:1 mixture with
1
mg, 1.55 mmol, 54%). H NMR (400 MHz, CDCl3): δ 1.09 (s,
3H), 1.37 (s, 3H), 1.69 (brs, 3H), 1.71-1.88 (m, 4H), 2.07-
2.18 (m, 1H), 2.69 (dt, J ) 4.8 and 10.8 Hz, 1H), 3.14-3.30
(dd, J ) 4.4 and 6.8 Hz, 1H), 3.25-3.35 (m, 2H), 3.40-3.49
(m, 2H), 5.08 (s, 1H), 5.42 (d, J ) 4.0 Hz, 1H), 5.49 (s, 1H),
6.47 (d, J ) 2.0 Hz, 1H), 6.56 (d, J ) 2.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3): δ 18.5, 23.5, 27.5, 27.8, 31.6, 35.6, 30.96,
40.0, 44.6, 55.6, 77.3, 106.5, 109.8, 113.1, 119.2, 134.7, 140.0,
154.9, 155.1. HRMS (MALDI-FTMS) calcd for C19H25O2S2 (M+
+ H) 349.1296, found 349.1295.
1
11 (TCD), as a brown oil (41 mg, 72%). H NMR (400 MHz,
CDCl3): δ 1.09 (s, 3H), 1.20-1.36 (m, 2H), 1.37 (s, 3H), 1.45-
1.66 (m, 5H), 1.68 (s, 3H), 1.72-1.87 (m, 2H), 2.08-2.20 (m,
1H), 2.16 (t, J ) 7.4 Hz, 2H), 2.39 (t, J ) 7.4 Hz, 2H), 2.59 (t,
J ) 5.6 Hz, 2H), 2.64-2.75 (m, 1H), 3.27 (brd, J ) 16.8 Hz,
1H), 3.45-3.60 (m, 2H), 5.41 (brd, J ) 2.8 Hz, 1H), 6.12 (brs,
1H), 6.20 (brs, 1H), 6.39 (brs, 1H). 13C NMR (100 MHz, CDCl3)
δ 18.9, 23.3, 24.6, 25.0, 27.5, 28.3, 29.2, 29.8, 30.6, 31.2, 33.9,
34.7, 35.8, 45.8, 77.4, 107.8, 109.1, 110.7, 123.5, 134.5, 141.9,
154.1, 154.4, 175.2. HRMS (MALDI-FTMS) calcd for C25H35-
NO4Na (MNa+) 452.2407, found 452.2407.
Acetic Acid 3-[1,3]Dithiolan-2-yl-6,6,9-trimethyl-6a,7,
10,10a-tetrahydro-6H-benzo[c]chromen-1-yl Ester (20).
To a solution of 19 (510 mg, 1.55 mmol) in pyridine (4.0 mL)
was added acetic anhydride (4.0 mL) at room temperature.
After being stirred overnight, the reaction mixture was
concentrated under reduced pressure. The residue was purified
by chromatography on silica gel, eluting with CH2Cl2/hexane
(1:3), to afford the desired product 20 (456 mg, 1.17 mmol,
75%). 1H NMR (400 MHz, CDCl3): δ 1.09 (s, 3H), 1.37 (s, 3H),
1.68 (brs, 3H), 1.70-1.95 (m, 4H), 2.08-2.18 (m, 1H), 2.28 (s,
3H, Ac), 2.60 (dt, J ) 4.8 Hz, 10.8 Hz, 1H), 2.71 (dd, J ) 4.4
Hz, 16.8 Hz, 1H), 3.25-3.35 (m, 2H), 3.40-3.48 (m, 2H), 5.43
(brd, J ) 3.0 Hz, 1H), 5.52 (s, 1H), 6.75 (d, J ) 2.0 Hz, 1H),
6.88 (d, J ) 2.0 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 18.5,
21.2, 23.5, 27.3, 27.6, 31.9, 35.8, 39.9, 40.0, 44.4, 55.3, 77.1,
113.6, 114.8, 118.5, 119.7, 133.7,140.6, 149.8, 154.7, 168.7.
HRMS (MALDI-FTMS) calcd for C21H27O3S2 (M+ + Na)
413.1215, found 413.1206.
2-(3,5-Dimethoxyphenyl)[1,3]dithiolane (16). To 2.8 g
(16.8 mmol) of 3,5-dimethoxybenzaldehyde and 2.1 mL (25.2
mmol) of ethanedithiol in 80 mL of dry CH2Cl2 under N2 was
added 0.64 mL (5.05 mmol) of BF3/Et2O, and the mixture was
stirred for 15 h. Then the reaction was quenched with
saturated Na2CO3, and the appropriate layer was extracted
with CH2Cl2 twice. The combined organic extracts were
washed with brine and dried over MgSO4. After concentration,
the residue was purified by column chromatography on silica
gel, eluting with EtOAc/hexane (1:9), to afford 16 as a yellow
1
oil (4.09 g, 16.6 mmol, 99%). H NMR (400 MHz, CDCl3): δ
3.30-3.38 (m, 2H), 3.44-3.52 (m, 2H), 3.79 (s, 6H), 5.58 (s,
1H), 6.36 (t, J ) 2.4 Hz, 1H), 6.69 (d, J ) 2.4 Hz, 2H) ppm.
13C NMR (100 MHz, CDCl3) δ 40.1, 55.3, 56.3, 100.0, 105.8,
142.7, 160.7 ppm. ESI-MS 259 (MH+), 281 (MNa+).
3-[(1-Acetoxy-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-
6H-benzo[c]chromene-3-carbonyl)amino]propionic Acid
Ethyl Ester (23). To a stirred solution of dithiolan 20 (794
mg, 2.03 mmol) in ethanol (50 mL) and water (5 mL) at room
temperature was added AgNO3 (1.04 g, 6.10 mmol). After 20
h, the reaction mixture was filtered through a pad of Celite.
After the filtrate was dried and concentrated, the crude
reaction mixture was used in the next reaction without
5-[1,3]Dithiolan-2-ylbenzene-1,3-diol (17). A solution of
2-(3,5-dimethoxyphenyl)[1,3]dithiolane (16, 6.4 g, 26.4 mmol)
and BBr3 (66 mL, 1 M solution in CH2Cl2) was stirred for 2 h
at -78 °C, after which the mixture was allowed to warm to
room temperature. After being stirred for 2 h, the mixture was
cooled to 0 °C and BBr3 (20 mL, 1 M solution in CH2Cl2) was
added again. The mixture was allowed to warm to room
temperature and stirred overnight. Then the mixture was
poured into saturated NaHCO3 and extracted four times with
CH2Cl2. The organic layer was dried over MgSO4 and evapo-
rated. The residue was purified by chromatography on silica
gel, eluting with EtOAc/hexane (1:1), to give 17 as a brown
oil (3.9 g, 18.2 mmol, 69%). 1H NMR (400 MHz, CDCl3): δ
3.29-3.37 (m, 2H), 3.42-3.50 (m, 2H), 5.02 (brs, 2H, OH ×
t
purification. The crude aldehyde was dissolved in BuOH (30
mL) and water (8 mL). Sodium dihydrogen phosphate (122 mg,
1.02 mmol), a 2 M THF solution of 2-methyl-2-butene (1.5 mL,
3.0 mmol), and sodium chlorite (188 mg, 2.03 mmol) were
subsequently added at room temperature. The mixture was
stirred for 5 h, and the reaction was then quenched with 1 N
HCl. The aqueous organic phases were washed with brine,

7396 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Qi et al.
dried over MgSO4, filtered, and evaporated to dryness. The
crude carboxylic acid was dissolved in DMF (20 mL). Triethyl-
amine (0.4 mL, 2.84 mmol), â-alanine ethyl ester hydrochloride
(217 mg, 1.42 mmol), and HBTU (539 mg, 1.42 mmol) were
subsequently added. After the mixture was stirred overnight
at room temperature, the reaction was quenched with water.
The mixture was then extracted with Et2O/EtOAc (1:1, two
times), and the combined extracts were washed with water
and then brine, dried over MgSO4, filtered, and evaporated to
dryness. The resulting crude product was purified by chroma-
tography on silica gel, eluting with EtOAc/hexane (1:2 to 1:1),
to afford 23 as a white amorphous solid (206 mg, 0.480 mmol,
24% yield in three steps). 1H NMR (400 MHz, CDCl3): δ 1.09
(s, 3H, Me), 1.27 (t, J ) 7.2 Hz, Et), 1.39 (s, 3H, Me), 1.64 (s,
3H, Me), 1.55-1.95 (m, 3H), 2.09-2.24 (m, 1H), 2.31 (s, 3H,
Ac), 2.60 (t, J ) 6.0 Hz, 2H, CH2CO2), 2.60-2.68 (m, 1H), 2.69-
2.77 (m, 1H), 3.68 (q, J ) 6.0 Hz, 2H, NCH2), 4.16 (q, J ) 7.2
Hz, 2H, Et), 5.41-5.46 (m, 1H, olefinic), 6.75 (brt, J ) 6.0 Hz,
1H, amide), 7.01 (d, J ) 1.6 Hz, 1H, Ph), 7.07 (d, J ) 1.6 Hz,
1H, Ph). 13C NMR (100 MHz, CDCl3): δ 14.14, 18.43, 21.16,
23.50, 27.26, 27.58, 32.09, 33.90, 35.21, 35.71, 44.36, 60.80,
77.47, 113.18, 113.89, 119.75, 122.54, 133.62, 133.91, 150.11,
154.99, 166.05, 168.73, 172.83. HRMS (MALDI-FTMS) calcd
for C24H32NO6 (M+ + H) 430.2224, found 430.2216.
amide), 6.89 (d, J ) 1.6 Hz, 1H), 7.09 (dd, J ) 1.2 and 7.6 Hz,
1H), 7.12 (d, J ) 7.6 Hz, 1H), 7.47 (t, J ) 6.0 Hz, 1H, amide),
8.43 (brs, 1H), 9.37 (s, 1H, OH). HRMS (MALDI-FTMS) calcd
for C25H30N2O6 (M+ + H) 455.2177, found 455.2181.
3-{3-[(1-Hydroxy-6,6,9-trimethyl-6H-benzo[c]chromene-
3-carbonyl)amino]propionylamino}propionic Acid (26,
TCF). To a stirred solution of the ethyl ester (21.6 mg, 0.0475
mmol) in THF (4 mL) and water (1 mL) was added lithium
hydroxide monohydrate (4.0 mg, 0.095 mmol). After being
stirred for 2.5 h at 50 °C, the mixture was concentrated under
reduced pressure. Purification of the residue by column
chromatography on silica gel, eluting with MeOH/CH2Cl2 (2:
8), gave the title product as a yellow crystalline solid (15.5
1
mg, 0.037 mmol, 78%). H NMR (400 MHz, CD3OD): δ 1.56
(s, 6H, Me × 2), 2.36 (s, 3H, Me), 2.39-2.51 (m, 2H), 3.45 (t,
J ) 7.2 Hz, 4H, CH2CO2, CH2CON), 3.61 (t, J ) 6.8 Hz, 4H,
NCH2 × 2), 6.83 (d, J ) 2.0 Hz, 1H), 6.97 (d, J ) 2.0 Hz, 1H),
7.11 (brd, J ) 8.0 Hz, 1H), 7.18 (d, J ) 8.0 Hz, 1H), 8.44 (brs,
1H). 13C NMR (100 MHz, CDCl3): δ 21.73, 27.66, 30.87, 36.91,
37.22, 37.70, 78.67, 109.14, 109.74, 123.63, 128.57, 128.96,
129.59, 135.68, 137.85, 138.59, 156.05, 156.99. HRMS (MALDI-
FTMS) calcd for C23H27N2O6 (M+ + H) 427.1864, found
427.1860.
6-Chloro-1-(3,5-dimethoxyphenyl)hexan-1-ol (27). To
an oven-dried flask equipped with an addition funnel, con-
denser, and stirring bar were added Mg turnings (406.5 mg,
16.7 mmol) under Ar at room temperature. A solution of
1-bromo-5-chloropentane (2 mL, 15.2 mmol) in dry THF (20
mL) was added dropwise via the addition funnel (after the first
several drops of the solution were added, the flask was heated
with hot gun to reflux; the rest was added dropwise). The
mixture was stirred at room temperature for 30 min, then
heated to reflux for an additional 1 h. After the Grignard
reagent was cooled to room temperature, it was transferred
into another flask containing 3,5-dimethoxybenzaldehyde
(1.486 g, 8.94 mmol) in dry THF (30 mL) via cannula at -78
°C under Ar over 30 min. The temperature was gradually
raised to room temperature (30 min), and the mixture was
stirred for 1 h. The reaction was quenched by dropwise
addition of a saturated aqueous NH4Cl solution (20 mL). The
crude suspension was diluted with EtOAc (20 mL) and brine
(10 mL) and then separated. The aqueous phase was extracted
with EtOAc (3 × 20 mL). The combined organic phases were
dried over MgSO4, and after filtration the solvent was removed
in vacuo. The crude product was purified by column chroma-
tography (silica, EtOAc/hexane 1:4 to 3:7) to give the product
3-[(1-Acetoxy-6,6,9-trimethyl-6H-benzo[c]chromene-3-
carbonyl)amino]propionic Acid Ethyl Ester (24). The
method of Tuchinda was employed. The olefinic compound (23,
79 mg, 0.184 mmol) was heated with sulfur (24 mg, 0.736
mmol) at 180-200 °C for 45 min. After the mixture was cooled
to room temperature, concentration of the mixture and puri-
fication by preparative TLC (silica, eluant EtOAc/hexane, 4:6)
gave the title compound 24 as a pale-yellow oil (38.3 mg, 0.09
1
mmol, 49%). H NMR (400 MHz, CD3OD): δ 1.25 (t, J ) 7.2
Hz, 3H, Et), 1.58 (s, 6H, Me × 2), 2.34 (s, 3H), 2.39 (s, 3H),
2.65 (t, J ) 6.8 Hz, 2H, CH2CO2), 3.63 (t, J ) 6.8 Hz, 2H, NH2),
4.15 (q, J ) 7.2 Hz, 2H, Et), 7.12 (brd, J ) 8.0 Hz, 1H), 7.21
(d, J ) 2.0 Hz, 1H), 7.26 (d, J ) 8.0 Hz, 1H), 7.31 (d, J ) 2.0
Hz, 1H), 7.87 (brs, 1H). HRMS (MALDI-FTMS) calcd for
C24H28NO6 (M+ + H) 426.1911, found. 426.1910.
3-[(1-Hydroxy-6,6,9-trimethyl-6H-benzo[c]chromene-3-
carbonyl)amino]propionic Acid (25, TCE). To a stirred
solution of ethyl ester 24 (38.3 mg, 0.090 mmol) in THF (6
mL) and water (2 mL) was added lithium hydroxide monohy-
drate (11 mg, 0.270 mmol). After 2 h at 50 °C, the mixture
was concentrated in vacuo. Purification of the residue by
column chromatography on silica gel, eluting with MeOH/CH2-
Cl2 (3:7), gave the title product as a yellow crystalline solid
1
(27, 1.64 g, 67%). H NMR (CDCl3, 400 MHz): δ 6.49 (d, J )
1
(27 mg, 0.0753 mmol, 84%). H NMR (400 MHz, CD3OD): δ
2.23 Hz, 2H), 6.37 (t, J ) 2.26 Hz, 1H), 4.60 (t, J ) 6. 55 Hz,
1 H), 3.79 (s, 6 H), 3.51 (t, J ) 6.67 Hz, 2 H), 1.76 (m, 4 H),
1.69 (m, 2 H), 1.45 (m, 2H). 13C NMR (100 MHz, CDCl3):
δ160.89, 147.36, 103.72, 99.34, 74.56, 55.34, 45.02, 38.70,
32.48, 26.73, 25.07. HRMS (ESI-TOF high-acc) calcd [M + H]+
for C14H21ClO3 273.1252, found 273.1259.
1.56 (s, 6H, Me × 2), 2.36 (s, 3H, Me), 2.54 (t, J ) 6.8 Hz, 2H,
CH2CO2), 3.60 (t, J ) 6.8 Hz, 2H, NCH2), 6.84-6.87 (m, 1H),
6.94-6.97 (m, 1H), 7.11 (brd, J ) 8.0 Hz, 1H), 7.18 (d, J ) 8.0
Hz, 1H), 8.43 (brs, 1H). 13C NMR (100 MHz, CDCl3): δ 14.0,
21.5, 27.1, 33.9, 35.1, 35.7, 60.9, 77.5, 107.9, 108.9, 114.2, 122.3,
127.0, 127.6, 128.5, 133.5, 136.8, 137.1, 154.7, 155.3, 167.6,
172.1, 172.5. HRMS (MALDI-FTMS) calcd for C20H22NO5 (M+
+ Na) 378.1312, found 378.1311.
1-(6-Chlorohexyl)-3,5-dimethoxybenzene (28, Method
B). To a flask containing 10% Pd/C (66.6 mg) was added glacial
acetic acid (16 mL). The mixture was degassed, and the
benzylic alcohol 27 (1.309 g, 4.8 mmol) was added. The mixture
was stirred overnight under a H2 atmosphere at room tem-
perature. Upon completion the mixture was diluted with ether
(30 mL) and water (10 mL) and the catalyst was removed by
filtration through Celite. The organic layer was separated and
diluted with water (10 mL) and neutralized by portionwise
addition of solid NaHCO3. The aqueous phase was extracted
with ether (3 × 20 mL). The organic layers were dried over
MgSO4, and after filtration the solvent was removed in vacuo.
The residue was purified by column chromatography (silica,
EtOAc/hexane 1:100 to 1:4) to give product 28 as a light-yellow
3-{3-[(1-Hydroxy-6,6,9-trimethyl-6H-benzo[c]chromene-
3-carbonyl)amino]propionylamino}propionic Acid Eth-
yl Ester. The carboxylic acid (TCE, 27 mg, 0.0753 mmol) was
dissolved in DMF (3.7 mL). Triethylamine (0.052 mL, 0.377
mmol), â-alanine ethyl ester hydrochloride (23 mg, 0.151
mmol), and HBTU (57 mg, 0.151 mmol) were subsequently
added. After 3.5 h at room temperature, the reaction was
quenched with water. The mixture was then extracted with
Et2O/EtOAc (1:1, two times), and combined organic phases
were washed with water and then brine, dried over MgSO4,
filtered, and evaporated to dryness. The resulting crude was
purified by chromatography on silica gel, eluting with EtOAc/
hexane (1:2 to 1:1) to afford, in order of elution, the desired
title ethyl ester as a yellow solid (21.6 mg, 0.0475 mmol, 63%).
1H NMR (400 MHz, CD3OD): δ 1.19 (t, J ) 7.2 Hz, 3H, Et),
1.57 (s, 6H, Me × 2), 2.37 (s, 3H, Me), 2.47-2.60 (m, 2H, CH2
× 2), 3.50 (q, J ) 6.0 Hz, 2H, NCH2), 3.73 (q, J ) 6.0 Hz, 2H,
PhCONCH2), 4.07 (q, J ) 7.2 Hz, 2H, Et), 6.80 (t, J ) 6.0 Hz,
1
oil (666 mg, 54% yield). H NMR (CDCl3, 400 MHz): δ 6.41
(d, J ) 2.20 Hz, 2 H), 6.37 (t, J ) 2.19 Hz, 1 H), 3.81 (s, 6 H),
3.55 (t, J ) 6.78 Hz, 2 H), 2.61 (t, J ) 7.69 Hz, 2 H), 1.80
(quint, J ) 7.08 Hz, 2 H), 1.68 (quint, J ) 7.57 Hz, 2 H), 1.50
(quint, J ) 7.41 Hz, 2 H), 1.40 (quint, J ) 7.50 Hz, 2 H). 13C
NMR (125 MHz, CDCl3): δ 160.48, 144.62, 106.12, 97.27,

∆9-Tetrahydrocannabinol Studies
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7397
54.74, 44.70, 35.83, 32.31, 30.79, 28.25, 26.47. HRMS (ESI-
TOF high-acc): calcd [M + H]+ for C14H21ClO2 257.1303, found
257.1288.
to 66 °C and stirred for 12 h. The mixture was then diluted
with CH2Cl2 (20 mL), after which the solid was removed by
filtration and the solvent was evaporated in vacuo. The residue
was filtered through a short pad of silica gel with ether as
eluent. The crude product was purified with column chroma-
tography (silica, Et2O/hexane 1:4) to give product 32 as a
colorless oil (27.3 mg, 92% yield). 1H NMR (CDCl3, 400 MHz):
δ 6.30 (m, 1 H), 6.27 (d, J ) 1.34 Hz, 1 H), 6.14 (d, J ) 1.40
Hz, 1 H), 4.78 (s, 1 H, OH), 3.25 (t, J ) 6.94 Hz, 2 H), 3.19-
3.21 (m, 1 H), 2.45 (t, J ) 7.73 Hz, 2 H), 2.15-2.19 (m, 2 H),
1.89-1.95 (m, 1 H), 1.68-1.72 (m, 4 H), 1.53-1.62 (m, 4 H),
1.31-1.42 (m, 8 H), 1.10 (s, 3 H). 13C NMR (125 MHz, CDCl3):
δ 154.80, 154.18, 142.38, 134.48, 123.61, 110.06, 109.13,
107.49, 51.43, 45.76, 35.29, 33.53, 31.14, 30.70, 28.72, 28.69,
27.55, 26.55, 24.99, 23.37, 19.25. HRMS (ESI-TOF high-acc):
calcd [M + H]+ for C22H31N3O2 370.2489, found 370.2478.
3-(6-Aminohexyl)-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-
6H-benzo[c]chromen-1-ol (33). The azide (32, 25.3 mg, 0.068
mmol) was treated with polymer supported Ph3P (27.3 mg, 3
mmol/g, 0.082 mmol) in THF/H2O (10:1.5, 0.1 mL) at room
temperature, and the mixture was shaken overnight. The resin
was filtered out and washed sequentially by MeOH (2 × 2 mL),
CH2Cl2 (2 × 2 mL), and THF (2 × 2 mL), and the organic
mixture was dried over Na2SO4. After filtration the solvent
was removed under reduced pressure. The crude product (24.0
mg) was not further purified for the next step. LC-MS
analysis (HP-1100, using a flow rate of 0.75 mL/min in a
gradient of 25-99% acetonitrile in water (0.5% formic acid)
in 8 min) showed only one peak at 3.983 min, [M + H]+: 344.2.
1H NMR (CD3OD, 500 MHz): δ 6.38 (br s, 1 H), 6.14 (d, J )
1.42 Hz, 1 H), 6.04 (d, J ) 1.48 Hz, 1 H), 3.12 (d, J ) 10.88
Hz, 1 H), 2.60 (t, J ) 7.11 Hz, 2 H), 2.38 (t, J ) 7.59 Hz, 2 H),
2.11 (m, 2 H), 1.86-1.94 (m, 1 H), 1.60-1.70 (m, 4 H), 1.50-
1.59 (m, 4 H), 1.40-1.46 (m, 2 H), 1.28-1.36 (m, 6 H), 1.01 (s,
3 H).
N-[6-(1-Hydroxy-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-
6H-benzo[c]chromen-3-yl)hexyl]acetamide (34a and 34b).
The amine (33, 20.3 mg, 0.059 mmol) was treated with polymer
supported pyridine (5.48 mmol/g, 29.0 mg, 0.159 mmol) in CH2-
Cl2 (0.1 mL) and shaken for 30 min. Then Ac2O (8.9 uL, 0.094
mmol) was added and shaking was continued for 4 h. The resin
was removed by filtration and washed with CH2Cl2 (2 × 2 mL),
MeOH (2 × 2 mL), and CH2Cl2 (2 × 2 mL) sequentially. The
solution was concentrated in vacuo, and the residue was
purified using PTLC, eluting with EtOAc/hexane (1:4) to give
product 34a (20.1 mg, 88% for two steps). 1H NMR (CDCl3,
400 MHz): δ 6.37 (br, 1 H), 6.22 (br, 1 H), 6.19 (d, J ) 1.31
Hz, 1 H), 5.53 (br, 1 H), 3.18-3.30 (m, 3 H), 2.45 (dt, J ) 7.78,
2.43 Hz, 2 H), 2.13-2.20 (m, 2 H), 2.00 (s, 3 H), 1.86-1.96 (m,
1 H), 1.62-1.73 (m, 6 H), 1.43-1.50 (m, 4 H), 1.41 (s, 3 H),
1.30-1.36 (m, 2 H), 1.09 (s, 3 H). 13C NMR (125 MHz, CDCl3):
δ 170.38, 154.79, 154.70, 142.09, 133.97, 123.98, 109.66,
109.16, 107.51, 45.79, 39.46, 34.78, 33.64, 31.18, 30.11, 29.21,
28.07, 27.58, 26.23, 25.03, 23.39, 23.36, 19.27. HRMS (ESI-
TOF high-acc): calcd [M + H]+ for C24H35NO3 386.2690, found
386.2691.
5-(6-Chlorohexyl)benzene-1,3-diol (29). To a solution of
the chloride (28, 565.2 mg, 2.2 mmol) in dry CH2Cl2 (13 mL)
at -78 °C was added dropwise BBr3 (1 M in CH2Cl2, 6.6 mL,
6.6 mmol) over 15 min, after which the mixture was warmed
to room temperature during 35 min. The mixture was stirred
for 3 h at room temperature. The reaction was quenched with
a saturated aqueous NaHCO3 solution and extracted with
DCM (3 × 20 mL). The organic layers were dried over MgSO4
and filtered. The solvent was removed under reduced pressure.
The residue was purified by column chromatography (silica,
EtOAc/hexane 1:4 to 2:3) to give diol 29 as a colorless oil (424
mg, 84%). 1H NMR (CDCl3, 500 MHz): δ 6.26 (d, J ) 1.81 Hz,
2 H), 6.19 (broad, 1 H), 3.53 (t, J ) 6.69 Hz, 2 H), 2.49 (t, J )
7.68 Hz, 2 H), 1.76 (quint, J ) 7.12 Hz, 2 H), 1.58 (quint, J )
7.63 Hz, 2 H), 1.45 (quint, J ) 7.56 Hz, 2 H), 1.34 (m, 2 H).
13C NMR (125 MHz, CDCl3): δ 156.48, 145.83, 108.10, 100.27,
45.15, 35.59, 32.47, 30.74, 28.37, 26.64. HRMS (ESI-TOF high-
acc): calcd [M - H]- for C12H17ClO2 227.0844, found 227.0844.
4-Isopropenyl-1-methylcyclohex-2-enol (30). To a solu-
tion of PhSeSePh (5.49 g, 17.59 mmol) in dry EtOH (17 mL)
was added NaBH4 (1.32 g, 34.86 mmol) under Ar. The mixture
was stirred, and after it turned colorless, a solution of (+)-
limonene oxide (2.72 mL, 16.60 mmol) in EtOH (6.5 mL) was
added dropwise. The mixture was stirred and heated to reflux
for 2 h under Ar, after which the reaction was quenched with
1 M HCl and the aqueous layer was extracted with EtOAc (3
× 30 mL). The organic extract was washed with a saturated
NaHCO3 solution, water, and brine, dried over MgSO4, filtered,
and concentrated in vacuo. The residue was dissolved in THF
(200 mL), and 35% H2O2 (14.22 mL, 166.0 mmol) was added
dropwise at 0 °C. The mixture was stirred at room temperature
for 1 h and then heated to reflux for 2 h. The mixture was
stirred overnight at room temperature. The reaction was
quenched with water, and the aqueous layer was extracted
with EtOAc (3 × 200 mL). The organic extract was washed
with water and brine, dried over MgSO4, filtered, and concen-
trated in vacuo. The residue was purified over silica gel, eluting
with EtOAc/hexane (1:10 to 2:3) to provide 30 as a light-yellow
1
oil (1.30 g, 52%). H NMR (CDCl3, 500 MHz): δ 5.71 (ddd, J
) 10.01, 2.26, 1.28 Hz, 1 H), 5.65 (ddd, J ) 10.01, 2.28, 0.97
Hz, 1 H), 4.78 (m, 1 H), 4.75 (m, 1 H), 2.66 (m, 1 H), 1.75-
1.85 (m, 2 H), 1.74 (m, 3 H), 1.58-1.65 (m, 2 H), 1.29 (s, 3 H).
13C NMR (125 MHz, CDCl3): δ 148.14, 133.99, 132.14, 110.59,
67.43, 43.49, 36.78, 29.44, 24.92, 20.81. HRMS (ESI-TOF high-
acc): calcd [M + H]+ for C10H16O 153.1274, found 153.1269.
3-(6-Chlorohexyl)-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-
6H-benzo[c]chromen-1-ol (31). A suspension of chloride 29
(248 mg, 1.08 mmol), dienol 30 (181.43 mg, 1.192 mmol), and
anhydrous MgSO4 (812.2 mg) in dry CH2Cl2 (14 mL) was cooled
to -3 °C, and BF3/Et2O (68.3 µL, 0.539 mmol) was added
dropwise under Ar. The mixture was stirred for 2.5 h at 0 °C,
and then 1.8 g of anhydrous NaHCO3 was added. The mixture
was warmed to room temperature, stirred vigorously for 30
min, and filtered through Florisil, after which the filtrate was
evaporated under reduced pressure. The residue was purified
with flash chromatography (silica gel, Et2O/hexane 0:100 to
1:9) to give product 31 (164 mg, 42%) as a light-yellow oil. 1H
NMR (CDCl3, 500 MHz): δ 6.30 (m, 1 H), 6.27 (m, 1 H), 6.14
(d, J ) 1.45 Hz, 1H), 4.76 (b, 1H), 3.53 (t, J ) 6.79 Hz, 2 H),
3.21 (d, J ) 11.94 Hz, 1 H), 2.46 (t, J ) 7.70 Hz, 2 H), 2.18 (m,
2 H), 1.93 (m, 1 H), 1.77 (m, 2 H), 1.69 (m, 4 H), 1.58 (m, 2 H),
1.42 (m, 6 H), 1.34 (m, 2 H), 1.09 (s, 3 H). 13C NMR (125 MHz,
CDCl3): δ 154.82, 154.18, 142.41, 134.48, 123.63, 110.08,
109.13, 107.49, 45.78, 45.12, 35.30, 33.55, 32.53, 31.15, 30.69,
28.44, 27.56, 26.72, 25.00, 23.35, 19.26. HRMS (ESI-TOF high-
acc): calcd [M - H]- for C22H31ClO2 361.194, found 361.1926.
The isotopic analogue 34b was synthesized the same way
with use of tritium-labeled Ac2O (47.5 mCi in toluene),
providing the product (13.4 mg, 80%) with low radiochemical
yield (0.347 mCi, 0.73%).
1-(3,5-Dimethoxyphenyl)-5-phenoxypentan-1-ol (35).
To a flame-dried flask containing Mg (530.5 mg, 21.82 mmol)
was added THF (22 mL), and a solution of 4-phenoxybutyl
bromide (5.257 g, 21.82 mmol) was added dropwise while the
suspension was kept mildly refluxing for 20 min. Then the
mixture was heated to reflux for 1 h. After cooling to room
temperature, the solution of Grignard reagent was transferred
to a solution of 3,5-dimethoxybenzaldehyde (2.133 g, 12.84
mmol) in THF (43 mL) at -78 °C during 30 min. The mixture
was then warmed to room temperature and stirred for 1 h.
The reaction was quenched by dropwise addition of saturated
aqueous NH4Cl (20 mL), and the mixture was diluted with
EtOAc (40 mL) and brine (20 mL). The organic layer was
separated, and the aqueous phase was extracted with EtOAc
3-(6-Azidohexyl)-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-
6H-benzo[c]chromen-1-ol (32). A suspension of chloride 31
(29.3 mg, 0.08 mmol) in DMF (27 mL) was treated with NaN3
(7.8 mg, 0.12 mmol) and a catalytic amount of NaI (0.4 mg)
under Ar at room temperature. The mixture was then warmed

7398 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Qi et al.
(3 × 50 mL). The combined organic layers were washed with
brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified (silica gel, EtOAc/hexane 1:4 to 2:3) to
6,6,9-Trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-ben-
zo[c]chromen-1-ol (∆9-THC, 39a). To a solution of bromide
38 (4.7 mg, 0.015 mmol) in dry THF (0.5 mL) was added
dropwise LAH (1 M in THF, 30 µL) at room temperature under
Ar. The mixture was stirred for 1 h. The reaction was
monitored by TLC and quenched with EtOAc (1 mL) and
aqueous NaOH solution (0.5 mL, 1 M). The mixture was
filtered through a short pad of Celite and anhydrous Na2SO4,
and the filtrate was concentrated. The residue was purified
by PTLC (EtOAc/hexane 1:4) to give ∆9-THC (39a, 2.2 mg,
47%). 1H NMR (CDCl3, 600 MHz): δ 6.31 (m, 1 H), 6.28 (m, 1
H), 6.15 (m, 1 H), 4.73 (s, 1 H), 3.21 (d, J ) 3.21 Hz, 1 H), 2.46
(m, 2 H), 2.18 (d, J ) 5.14 Hz, 2 H), 1.92 (m, 1 H), 1.70-1.77
(m, 2 H), 1.69 (s, 3 H), 1.58 (m, 5 H), 1.40-1.48 (m, 2 H), 1.25-
1.35 (m, 2 H), 1.10 (s, 3 H), 0.89 (t, J ) 6.53 Hz, 3 H). 13C
NMR (100 MHz, CDCl3): δ 154.76, 154.11, 152.90, 142.81,
134.44, 123.65, 110.08, 107.49, 45.76, 35.45, 33.53, 31.49,
30.65, 27.56, 24.99, 23.37, 22.53, 19.25, 14.01. HRMS (ESI-
TOF high-acc): calcd [M + H]+ for C21H30O2 315.2318, found
315.2315.
1
give 35 (3.77 g, 93%) as a colorless oil. H NMR (CDCl3, 400
MHz): δ 7.26-7.31 (m, 2 H), 6.94 (t, J ) 7.34 Hz, 1 H), 6.89
(dd, J ) 8.66, 0.87 Hz, 2 H), 6.52 (d, J ) 2.27 Hz, 2 H), 6.39
(t, J ) 2.28 Hz, 1 H), 4.65 (dd, J ) 7.43, 5.63 Hz, 1 H), 3.96 (t,
J ) 6.46 Hz, 2 H), 3.80 (s, 6 H), 1.72-1.91 (m, 5 H), 1.63 (m,
1 H), 1.50 (m, 1 H). 13C NMR (125 MHz, CDCl3): δ 160.85,
158.95, 147.35, 129.38, 120.49, 114.43, 103.71, 99.37, 67.57,
55.31, 38.60, 29.12, 22.40. HRMS (MALDI-FTMS high-acc):
calcd [M + Na]+ for C19H24O4 339.1567, found 339.1566.
1-(3,5-Dimethoxyphenyl)-5-phenoxypentane
(36).
TMSCl (3.33 mL, 26.22 mmol) and NaI (3.943 g, 26.30 mmol)
in MeCN (1.8 mL) were stirred at room temperature for 15
min. To this mixture was added a solution of compound 35
(1.3813 g, 4.37 mmol) in Et2O/hexane (3 mL /2.3 mL), and the
mixture was stirred at room temperature for 24 h under Ar.
The reaction was quenched with H2O, and the appropriate
layer was extracted with Et2O (3 × 10 mL). The combined
organic layers were washed with saturated aqueous Na2S2O3
solution and brine. The solution was then dried over MgSO4,
filtered, and concentrated under reduced pressure to give the
reduced product 36 (1.16 g, 88%) as a light-yellow oil. The
product was used in the next step without further purification.
1H NMR (CDCl3, 400 MHz): δ 7.29 (d, J ) 8.69 Hz, 1 H), 7.28
(d, J ) 8.56 Hz, 1 H), 6.92 (m, 3 H), 6.36 (d, J ) 2.23 Hz, 2 H),
6.31 (t, J ) 2.25 Hz, 1 H), 3.96 (t, J ) 6.52 Hz, 2 H), 3.79 (s,
6 H), 2.60 (t, J ) 7.69 Hz, 2 H), 1.83 (quint, J ) 6.64 Hz, 2 H),
1.70 (quint, J ) 7.65 Hz, 2 H), 1.52 (m, 2H). 13C NMR (125
MHz, CDCl3): δ 160.70, 159.04, 144.96, 129.38, 120.47, 114.47,
106.47, 97.64, 67.70, 55.23, 36.19, 31.01, 29.17, 25.76. HRMS
(ESI-TOF high-acc): calcd [M + H]+ for C19H24O3: 301.1798,
found 301.1793.
39b. The tritium-labeled analogue 39b was prepared using
the following procedures.
(a) By LAH. Bromide 38 (1.64 mg, 4.16 µmol) in dry THF
was treated with regular LAH (1.0 M in THF, 4.2 µL) and
stirred for 30 min. Then tritium-labeled LAH (80 Ci/mmol, 100
mCi) was added and stirring was continued at room temper-
ature for 1 h. Regular LAH (1.0 M in THF, 1.0 µL, 1.0 µmol)
was added, and the mixture was stirred for 10 min. After
workup and preparative thin-layer chromatography (PTLC)
(EtOAc/hexane 1:9), product 39b was obtained (0.65 mg, 2.08
µmol, 50% yield, 0.23 Ci/mmol, 0.48 mCi, radiochemical yield
1.9%).
(b) By NaBH4. Bromo-THC (1.01 mg, 2.57 µmol) in DMSO
was treated with tritium-labeled NaBH4 (100 mCi, 80 Ci/mmol)
at room temperature, and the mixture was stirred for 2.5 h,
after which regular NaBH4 (0.1 mg, 2.64 µmol) was added to
complete the reaction. The mixture was extracted with with
1:4 ether/hexane (×3) and filtered through a short pad of silica
gel (EtOAc/hexane 1:9). The solvent was removed in vacuo to
give product 39b (one spot on TLC, 0.65 mg, 2.056 µmol, 80%
yield, 8.0 mCi, 4 Ci/mmol, radiochemical yield 32%).
5-(5-Bromopentyl)benzene-1,3-diol (37). To a solution of
phenoxyether 36 (835.1 mg, 2.78 mmol) in CH2Cl2 (6 mL) at
-78 °C was added 13.9 mL of a 1 M BBr3 solution in CH2Cl2.
The mixture was slowly warmed to room temperature and
stirred for 36 h. The reaction was quenched by careful addition
of a saturated aqueous NaHCO3 solution at 0 °C. The mixture
was extracted with ether (3 × 30 mL), and the combined
organic layers were washed with saturated aqueous NaHCO3
solution (3 × 20 mL), followed by brine (3 × 20 mL), then dried
over MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (silica gel, EtOAc/
hexane 1:4 to 2:3) to give product 37 (1.236 g, 80%) as a light-
brown oil. 1H NMR (CDCl3, 400 MHz): δ 6.27 (d, J ) 1.9 Hz,
2 H), 6.21 (s, 1 H), 3.36 (t, J ) 6.8 Hz, 2 H), 2.43 (t, J ) 7.6
Hz, 2 H), 1.81 (quint, J ) 7.13 Hz, 2 H), 1.52 (quint, J ) 7.54
Hz, 2 H), 1.39 (quint, J ) 7.32,2 H). 13C NMR (100 MHz,
CDCl3): δ 156.11, 145.68, 108.20, 100.39, 35.43, 33.98, 32.50,
29.97, 27.67. HRMS (ESI-TOF high-acc): calcd [M + H]+ for
C11H15BrO2 259.0328, found 259.0327.
Acknowledgment. This research was supported by
the National Institutes of Health Grant DA15700 and
by The Skaggs Institute for Chemical Biology. The
authors thank Diane Kubitz and Dr. Andrew P. Brogan
for excellent technical assistance, and Dr. R. Mechoulam
for helpful advice.
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