Exploring the substituent effects on a novel series of C1′-dimethyl-aryl Δ8-tetrahydrocannabinol analogs

Article abstract of DOI:10.1016/j.bmc.2008.05.034

The synthesis and characterization of novel C1′-phenyl-substituted Δ⁸-THC analogs were previously reported by our laboratory. Within this small series of compounds, the C1′-dimethyl phenyl group was found to impart 13.5-fold selectivity for the CB2 receptor with a K_i 0.91 nM. The current study expands on the previous report by evaluating the effects of aromatic ring substitution on CB1 and CB2 receptor subtype binding and selectivity. The ring substituents synthesized in this study include aliphatic, halogen, nitrile, and acetamido functional groups. In addition, the isosteric replacement of the phenyl group by thiophene was evaluated. The anti-glioma activities of selected compounds were evaluated in vitro and compared to the lead compound 2.

Full text of DOI:10.1016/j.bmc.2008.05.034

Bioorganic & Medicinal Chemistry 16 (2008) 6489–6500
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploring the substituent effects on a novel series of C10-dimethyl-aryl
D
8-tetrahydrocannabinol analogs
*
Mathangi Krishnamurthy, Steven Gurley, Bob M. Moore II
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee-Memphis, 847 Monroe Avenue, Room 327D, Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
The synthesis and characterization of novel C10-phenyl-substituted
D
8-THC analogs were previously
Article history:
reported by our laboratory. Within this small series of compounds, the C10-dimethyl phenyl group was
found to impart 13.5-fold selectivity for the CB2 receptor with a Ki 0.91 nM. The current study expands
on the previous report by evaluating the effects of aromatic ring substitution on CB1 and CB2 receptor
subtype binding and selectivity. The ring substituents synthesized in this study include aliphatic, halo-
gen, nitrile, and acetamido functional groups. In addition, the isosteric replacement of the phenyl group
by thiophene was evaluated. The anti-glioma activities of selected compounds were evaluated in vitro
and compared to the lead compound 2.
Received 4 April 2008
Revised 13 May 2008
Accepted 15 May 2008
Available online 20 May 2008
Keywords:
D
8-THC analogs
C10-substituted aryl side chains
CB2 selective ligands
Glioma
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The hemp plant, Cannabis sativa, is the major source of tricyclic
terpenoids with a core benzopyran structure that are referred to as
classical cannabinoids. The natural product
8-THC (1, Fig. 1) are the prototypical members of this class of can-
nabinoids. They bind to the CB1 and CB2 cannabinoid receptors
and act as agonists or partial agonists.1 Compounds such as D8
D
9-THC, and its isomer
D
-
THC and its synthetic analogs have potential indication in the
treatment of diverse pathological conditions ranging from cancer,2
inflammation,3 glaucoma,4 epilepsy,5,6 and pain,7 thus kindling
extensive research in this field.
Efforts directed at elucidating the SAR of classical cannabinoids
have focused on the tricyclic ring structure and the C-3 alkyl side
chain. The different side chain modifications that have been re-
ported so far include branched chain alkyls,8,9 unsaturated al-
kyls,10–12
alkyls
containing
10,10-cyclic
functionality,13
adamantyl,14 and cycloalkyl side chains containing 10,10-dimethyl
and 10,10-dithiolane functionalities.15 These modifications have
been directed at improving binding affinities as well as the CB1/
Figure 1.
CB2 selectivity. We had previously reported a series of
D
8-THC
analogs with a phenyl side chain and with differing functionalities
at the 10 position including dimethyl, dithiolane, methylene, and
ketone groups. In this series, the analog with a 10,10-dimethyl func-
tionality (2) displayed greatly enhanced binding affinities for both
the CB1 and the CB2 receptor (CB1 Ki = 12.3 nM, CB2 Ki = 0.91 nM)
Ki = 25.0 nM) as well as 13-fold selectivity for the CB2 receptor
when compared to
D
8-THC.
The high CB2-binding affinity as well as modest CB2 selectivity
exhibited by compound 2 prompted us to further explore the SAR
of the phenyl-substituted side chain series of
D
8-THC analogs. The
when compared to
D
8-THC (CB1 Ki = 28.5 nM and CB2
objectives of this study include an improved understanding of the
functional group requirements of the ligand-binding pocket (LBP)
of the CB2 receptor as well as augmentation of CB2 affinity and
selectivity.
* Corresponding author. Tel.: +1 901 448 6085; fax: +1 901 448 6828.
E-mail address: bmoore@utmem.edu (B.M. Moore II).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.034

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M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
To probe potential hydrophobic, electrostatic, and hydrogen-
bonding interactions with the amino acid residues present in the
LBP of the receptor, substituents were introduced that include
methyl, ethyl, propyl, dimethyl, fluoro, chloro, bromo, nitrile, and
amide at the para, meta, and ortho-positions of the side chain phe-
nyl ring (3) or a combination thereof. The choice of substituents
not only enables the probing of different binding interactions with
the receptor but also is compatible with the existing synthetic
strategy. The effects of replacing the phenyl ring with the bioisos-
teric replacement 2-thiophene (4) on the binding affinities for the
CB1 and the CB2 receptor were investigated. Selected compounds
were also evaluated for cytotoxic effects on human glioma cell
lines.
Scheme 2. Reagents and conditions: (a) CuCN, DMF, reflux, 6 h; (b) BBr3, ꢀ78 °C,
12 h.
2. Results and discussion
2.1. Chemistry
The synthesis of the phenyl-substituted side chain analogs of
D
8-THC utilized 3,5-dimethoxy benzaldehyde as the starting mate-
rial. This was reacted with the appropriately substituted Grignard
reagents to afford the corresponding alcohols (Scheme 1).16 In
cases where the Grignard reagents were not commercially avail-
able, the Grignard reagents were prepared by reacting the substi-
tuted aromatic or heteroaromatic bromides with magnesium
turnings. Oxidation of the alcohols with PCC afforded the diphenyl
ketone intermediates 5–17, which were then reacted with di-
methyl zinc and titanium (IV) chloride to yield the corresponding
C10-dimethyl-substituted compounds 18–30. The 3,5-dimethoxy-
phenyl intermediates 18–30 were converted to resorcinols 31–43
using boron tribromide.17 Intermediates 28 and 29 were converted
to nitriles using copper (I) cyanide18 and then deprotected using
boron tribromide to yield resorcinols 44 and 45 (Scheme 2). Palla-
dium-catalyzed amination strategy19 was employed to effect the
conversion of 29 to the amine which was then reacted with acetyl
chloride to yield amide 46 (Scheme 3).
Scheme 3. Reagents and conditions: (a) Pd2(dba)2, DCPB, LiHMDS; (b) acetyl chl-
oride, pyr; (c) BBr3, ꢀ78 °C, 12 h.
this is an effective strategy for preparing (+)-cis-D
2,8-p-menthadi-
en-1-ol, only one half of the selenide is productively consumed.
To address this issue, trans-(R)-(+)-limonene oxide was obtained
from a 50:50 mixture of limonene oxide isomers (47) by Steiner’s
kinetic separation technique using aqueous pyrazole (Scheme
4).21 The trans-limonene oxide (48) was converted to the selenide
by reaction with diphenyl diselenide under reducing conditions
followed by oxidation using hydrogen peroxide.20 The selenoxide
was then decomposed in refluxing chloroform to yield (+)-
menthadien-1-ol (49). The
8-THC analogs 4 and 50–64 were then
obtained by reacting resorcinols 31–46 with (+)-
2,8-p-menthadi-
en-1-ol or (+)-
2-p-menthene-1,8-diol in the presence of p-toluene
D
2,8-p-
D
D
D
Synthesis of the
ring closure between the resorcinol and (+)-
8-diol or (+)-
D
8-THC generally employs an acid-catalyzed
2-p-menthene-1,
2,8-p-menthadien-1-ol. The later compound was
sulfonic acid (Scheme 5).
D
2.2. Receptor-binding assays
D
synthesized using the method of Rickards20 starting from trans-
(R)-(+)-limonene oxide. The published procedure utilized a 50:50
mixture of cis- and trans-(+)-limonene oxide in a trans-diaxial
epoxide ring opening utilizing sodium phenyl selenide. Although
Cell membranes from HEK293 EBNA cells transfected with the
human CB1 receptor or the human CB2 receptors were used in
the receptor-binding assays.15 The binding affinities of compounds
Scheme 1. Reagents and conditions: (a) 1—ArMgBr, THF, ꢀ20 °C, 6 h; 2—1 N HCl; (b) PCC, DCM, 12 h; (c) Me2Zn, TiCl4, DCM, ꢀ40 °C, 4 h; (d) BBr3, ꢀ78 °C, 12 h.

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
6491
Table 1
Binding affinities of the C10-substituted aryl 8D-THC analogs for the CB1 and CB2
receptorsa
Compound C10-Substituent
CB1 Kia (nM) CB2 Kia (nM)
Ratio CB1/CB2
1.14
D
8-THC
28.5 ( 3.30)
12.3 ( 0.61)
3.13 ( 0.37)
2.53 ( 0.54)
34.4 ( 2.84)
25.0 ( 4.80)
0.91 ( 0.08) 13.5
2
Phenyl
50
51
52
53
54
55
56
57
58
59
60
61
4
4-Methylphenyl
3-Methylphenyl
2-Methylphenyl
0.88 ( 0.05)
1.13 ( 0.02)
10.65 ( 1.27)
7.45 ( 0.38)
0.67 ( 0.05)
7.83 ( 0.79)
12.4 ( 0.24)
0.90 ( 0.02)
3.56
2.24
3.23
1.48
2.76
0.23
6.14
5.84
3,5-Dimethylphenyl 11.0 ( 1.67)
4-Ethylphenyl
4-Propylphenyl
4-Fluorophenyl
3-Fluorophenyl
4-Chlorophenyl
3-Chlorophenyl
4-Bromophenyl
3-Bromophenyl
2-Thiophene
1.85 ( 0.16)
1.77 ( 0.20)
76.1 ( 1.55)
5.26 ( 0.94)
18.8 ( 1.39)
2.80 ( 0.05)
5.03 ( 0.39)
1.59 ( 0.16)
1.08 ( 0.04)
9.25 ( 0.23)
2.72 ( 0.29)
1.68 ( 0.20) 11.2
3.54 ( 0.71)
1.54 ( 0.16)
0.54 ( 0.03)
0.27 ( 0.01)
2.53 ( 0.23)
0.91 ( 0.05)
13.6 ( 1.62)
0.79
3.26
2.94
4.0
3.66
2.98
1.01
Scheme 4. Reagents and conditions: (a) pyrazole, H2O, reflux, 5 h; (b) NaBH4, di-
phenyl diselenide, reflux, 2 h; (c) 1—H2O2, 20 °C 5 h; 2—reflux.
62
63
64
4-Cyanophenyl
3-Cyanophenyl
N-Acetamidophenyl 13.7 ( 1.88)
a
The Ki values for D8-THC and the C10 aryl analogs were obtained from three
independent experiments run in triplicate showing the standard error of the mean
in parentheses.
tion of the phenyl side chain, 61 (Br, Ki = 1.59 nM) > 59 (Cl,
Ki = 2.8 nM) > 57 (F, Ki = 5.26 nM), showed increase in CB1 recep-
tor-binding affinities with increasing size and decreased electro-
negativity of the halogen substituent. Interestingly, the meta-
substituted analogs exhibited 3- to 14-fold improvements in
CB1 receptor-binding affinity when compared to their para-
substituted counterparts as well as the lead compound
2
(Ki = 12.3 nM). These data show that halogen substitution in the
meta-position of the phenyl side chain is beneficial to enhancing
CB1 receptor affinity.
The CB2 receptor-binding affinities also increased with increas-
ing size and decreased electronegativity of the halogen substituent
in the para-position for compounds 60 (Br, Ki = 1.54 nM) > 58 (Cl,
Ki = 1.68 nM) > 56 (F, Ki = 12.4 nM). These compounds, however,
displayed a modest to high reduction in affinity for the CB2 recep-
tor when compared to compound 2 (Ki = 0.91 M). On the other
hand, introduction of halogens at the meta-position of the phenyl
side chain resulted in analogs with CB2 receptor affinities compa-
rable to or better than compound 2. These results suggest that the
presence of halogen substituents at the meta-position of the
phenyl side chain is favorable for both CB1- and CB2-binding
affinities.
Scheme 5. Reagents and conditions: (a)
ene-1,8-diol, p-TSA, C6H6, 80 °C.
D D
2,8-p-menthadien-1-ol or 2-p-menth-
4 and 50–64 were determined by measuring the displacement of
[3H]CP 55, 940 from the CB1 and CB2 receptor preparations by
Introduction of the nitrile and amide substituents also pro-
duced significant effects on both CB1 and CB2 receptor-binding
affinities. The presence of a nitrile group at the para-position of
the phenyl side chain resulted in a slight improvement in CB1
receptor-binding affinity (63, Ki = 9.25 nM), relative to the lead
compound 2. However, the meta substitution yielded almost a
5-fold improvement in CB1-binding affinity (63, Ki = 2.72) when
compared to 2. The improvement in CB1 receptor-binding affinity
with the introduction of a nitrile group is in agreement with pre-
increasing concentrations of the
specific binding was determined using 10
D
8-THC analogs (Table 1). Non-
lM WIN 55212-2.
The substituted phenyl analogs displayed a broad range of
binding affinities for the CB1 and CB2 receptors (Table 1). The
CB1-binding affinities ranged from 1.08 to 76.1 nM while the CB2
affinities ranged from 0.27 to 13.6 nM. The para- and the meta-
substituted compounds showed significantly different binding pro-
files, with the meta-substituted analogs generally displaying en-
hanced binding affinities for both the receptor subtypes when
compared to their para-substituted counterparts.
Introduction of halogen substituents fluoro, chloro, and bromo
at either the para or meta-positions of the phenyl side chain re-
sulted in considerable changes in CB1 and CB2 receptor-binding
affinities when compared to compound 2 (CB1 Ki = 12.3 nM and
CB2 Ki = 0.91 nM). In the para series, the CB1 affinities increased
with increasing size and decreased electronegativity of the halo-
gen substituent, thus following the order Br (60, Ki = 5.03 nM) > Cl
(58, Ki = 18.8 nM) > F (56, Ki = 76.1 nM). As with the para-substi-
tuted series, analogs with a halogen substituent at the meta-posi-
viously published reports on nitrile derivatives of
D A
8-THC.17
comparison of the CB2 receptor-binding affinities of the nitrile-
substituted analogs shows that these compounds either displayed
lower affinity (62, Ki = 2.53 nM) or comparable affinity (63,
Ki = 0.91 nM) to the lead compound 2 (Ki = 0.91 nM). Introduction
of the amide group into the phenyl ring (64) had no effect on the
CB1 affinity (Ki = 13.7 nM) relative to the parent compound 2,
however, this substitution resulted in a 13-fold decrease in the
CB2 receptor binding. The reduction in CB2-binding affinity for
the m-amide analog may be attributed to the presence of a bulky
substituent at the meta-position of the side chain, a conclusion

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M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
that is supported by the data obtained for the alkyl side chain
analogs.
phoma/leukemia32; furthermore, a decrease in tumor size and/or
cell growth inhibition has been reported in breast,25,33,34
uterus,35,36 and lung cancers,37,38 and in thyroid epithelioma.39 In
addition, cannabinoids manifest collateral effects by decreasing
the production of vascular epidermal growth factor and activation
of the vascular epidermal growth factor receptor 2 in vitro and in
vivo,40 inhibiting bi-dimensional capillary-like tube formation
and activity of matrix metalloprotease 241; and down-regulation
of the expression of tissue inhibitors of metalloproteinase 1 in
vitro.42
The substituted C10-phenyl- 8-THC analogs containing a halo-
D
gen, nitrile, or amide provide insights into potential electrostatic
and/or hydrogen bond interactions within the ligand-binding
receptor pockets. For the CB1 receptor, introduction of electron
withdrawing or hydrogen bond acceptors at either the para or
meta-position causes a significant change in CB1 binding. The
greatest impact on affinities is observed for meta substitution sug-
gesting the presence of favorable electrostatic interactions and/or
hydrogen-bond donors in the corresponding regions of the LBP of
the CB1 receptor. In the case of the CB2 receptor, introduction of
these substituents either resulted in decrease in CB2-binding affin-
ity with halogens and nitrile at the para-position, or meta analogs
with comparable or slightly higher CB2 affinity.
Introduction of a para-methyl phenyl side chain (50) resulted in
almost 4-fold improvement in CB1-binding affinity (Ki = 3.13 nM)
when compared to the lead compound 2 (Ki = 12.3 nM) while the
presence of a meta methyl group (51, Ki = 2.53 nM) produced a 5-
fold increase in CB1 affinity. The CB2-binding affinities of com-
pounds 50 and 51 were comparable to that of compound 2. On
the other hand, introduction of the ortho methyl group (52) caused
Glioblastoma multiforme (GBM) is the most common and
malignant of all the primary brain tumors with a median survival
for GBM patients of 3–12 months.43–45 Recent reports suggest
that chemotherapeutic intervention with
D
8-THC and synthetic
cannabinoids may offer a novel strategy for GBM therapy.30,31,46,47
The lead compound 2 was previously evaluated in the side flank
model of human glioma using the U87 cell line, wherein the tu-
mor load was decreased 75% relative to untreated controls.48 In
efforts to develop cannabinoid-based anti-neoplastic agents with
unique properties, selected analogs of 2 were screened against a
panel of human glioma cell lines U-87MG, T98G, LN-229,
DBTRG-O5MG, and MT310 (a primary tumor cell line). Com-
pounds were selected based on increasing ratios of CB1- to
CB2-binding affinities. Within this set of compounds the Kis ran-
ged from 1.08 to 76.1 nM for the CB1 and 0.27 to 12.4 nM for the
CB2. Increasing concentrations of compounds 4, 54, 55, 56, and 58
were assayed compared to the lead compound 2 (Table 2). A
quantitative analysis of the limited data set did not reveal a sig-
nificant correlation between the compound Ki and the EC50s.
Qualitatively, compound 4 manifested broad-spectrum cytotoxic-
ity across glioma cell lines (Fig. 2). The LN-229 glioma exhibited
the highest sensitivity to the C10 aryl analogs and the primary cell
line MT310 manifested reduced cytotoxicity. The high mortality,
short life-expectancy, and lack of efficacious drugs against GBM
a
significant decrease in both CB1 (Ki = 34.4 nM) and CB2
(Ki = 10.6 nM) binding affinities. The presence of the bulky methyl
group at the ortho-position will produce unfavorable steric interac-
tions with the dimethyl substituent at the C-10 position which, in
turn, would cause the side chain to adopt an unfavorable geometry
for receptor binding. Elongation of the alkyl substituent resulted in
analogs with enhanced CB1-binding affinities. The CB1-binding
affinities of the p-alkyl-substituted analogs followed the order p-
methyl (50, Ki = 3.13 nM) < p-ethyl (54, Ki = 1.85 nM) < p-propyl
(55, Ki = 1.77 nM). The rank order indicates that the LBP of the
CB1 receptor can tolerate bulky hydrophobic substituents, which
is in concurrence with previous reports that CB1 affinity is directly
linked to the length of the side chain. Specifically, the CB1 affinity
increases with increasing carbons from 3 to 10, with a seven or
eight carbon chain being optimal for activity.22 In the case of CB2
affinity, elongation of the alkyl substituent to ethyl (54,
Ki = 0.67 nM) resulted in increase in affinity while further elonga-
tion to a propyl group (55, Ki = 7.83 nM) led to an 8-fold decrease
in affinity, when compared to 2 (Ki = 0.91 nM). These results in
combination with the halogen and nitrile data suggest that the
CB2 receptor has lower steric tolerance as compared to the CB1
receptor. Introduction of 3,5-dimethyl functionality in the phenyl
side chain (53) decreased both CB1- and CB2-binding affinities
(CB1, Ki = 11.0 nM; CB2, Ki = 7.45 nM) relative to the meta- and
para-methyl analogs.
The introduction of the bioisosteric phenyl ring replacement
thiophene (62) led to 11-fold improvement in CB1 affinity
(Ki = 1.08 nM) and 3-fold improvement in CB2 affinity
(Ki = 0.27 nM) when compared to the lead compound 2. The en-
hanced affinity of the 2-thiophene ring may be related to reduced
steric interactions with the LBP. Alternatively, a reduction in the
rotational energy around the C10–C20 bond may allow the molecule
to adopt a favorable geometry to bind with the LBP. The most sig-
nificant finding is that compound 4 displays very high CB2 affinity
that is comparable to some of the most potent CB2 analogs re-
ported to date such as CP-55244 (CB2, Ki = 0.21 nM)23 and R-(+)-
WIN55212 (CB2, Ki = 0.28 nM).24
make our novel series of
D
8-THC analogs attractive leads for
anti-cancer drug development. Probably the most important
property of these compounds is the high probability for blood
brain barrier penetration. Unlike existing therapies, this property
should allow the drug to bath the CNS and facilitate treatment of
distant glioblastoma foci, the most common cause of reoccur-
rence and death in GBM patients.
3. Conclusions
In this study, we evaluated the functional group requirements
of the LBPs of the CB1 and CB2 cannabinoid receptors by intro-
ducing aromatic/heteroaromatic side chains. The substituents
methyl, ethyl, propyl, 3,5-dimethyl, fluoro, chloro, bromo, nitrile,
and amide were introduced in the phenyl ring. A classical bioisos-
teric replacement of the phenyl ring, thiophene, was also intro-
duced in the side chain. In general, the meta-substituted series
of compounds exhibited higher CB1 and CB2 affinities than their
para-substituted counterparts. Introduction of electron-withdraw-
ing substituents at the meta-position of the phenyl side chain
greatly enhanced CB1-binding affinities while the CB2 affinities
were equivalent or slightly enhanced when compared to the lead
compound 2. The introduction of aliphatic chains on the phenyl
side chain differentially affected CB1 and CB2 binding with re-
spect to the size/steric bulk of the substituent. Analog 4 contain-
ing the thiophene ring displayed very high CB2 and CB1-binding
affinities. The CB2-binding affinity of compound 4 is comparable
to some of the most potent CB2 ligands reported so far, such as
CP-55244 and R-(+)-WIN55212. A more thorough QSAR analysis
of analogs 2, 4, and 50–64 is presented in an accompanying
paper.
2.3. Cytotoxicity against human glioma cancer cells
Cannabinoids are gaining increasing attention as novel thera-
peutic agents for the treatment of a broad spectrum of cancer
types. Cannabinoids have now been shown to induce apoptosis
in prostate25–27 and skin carcinomas,28 glioma29–31 and lym-

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
6493
Table 2
The EC50 values of selected C10 aryl
D
8-THC analogsa
M)
Compound
U-87MG EC50
(l
T98G EC50
(l
M)
LN-229 EC50
(l
M)
DBTRG-O5MG EC50
(l
M)
MT310 EC50 (lM)
2
54
55
56
58
4
3.08 ( 0.30)
2.52 ( 0.26)
3.17 ( 0.31)
2.55 ( 0.43)
3.27 ( 0.17)
1.83 ( 0.06)
4.68 ( 0.45)
3.23 ( 0.47)
3.63 ( 0.51)
3.30 ( 0.73)
3.40 ( 0.32)
2.21 ( 0.2)
2.63 ( 0.32)
1.67 ( 0.21)
2.44 ( 0.45)
2.02 ( 0.29)
2.87 ( 0.21)
2.02 ( 0.33)
2.27 ( 0.18)
3.00 ( 0.23)
3.58 ( 0.22)
3.08 ( 0.24)
4.61 ( 0.17)
2.67 ( 0.04)
4.92 ( 1.08)
4.11 ( 0.18)
6.46 ( 0.62)
8.78 ( 0.80)
7.31 ( 0.53)
4.10 ( 0.24)
a
The EC50 values for the D8 -THC C10 aryl analogs were obtained from three independent experiments run in triplicate showing the standard error of the mean in
parentheses.
4.1. (p-Tolyl)-(3,5-dimethoxy-phenyl)-methanol
The starting material 3,5-dimethoxy benzaldehyde (3 g,
18.1 mmol) was dissolved in THF (30.5 mL) and the solution was
cooled to ꢀ20 °C. To the solution was added p-tolyl magnesium bro-
mide (21.7 mL, 1 M solution, 21.7 mmol) and the reaction mixture
was allowed to stir at ꢀ20 °C for 6 h. The reaction mixture was then
quenched with 1 N HCl, extracted with ether and the organic layer
was then dried over sodium sulfate and evaporated. The crude mix-
ture was then purified by column chromatography using ethyl ace-
tate/hexane (20:80) yielding alcohol as a white solid (3.73 g,
79.9%). Rf = 0.24 (ethyl acetate/hexane 20:80); 1H NMR (500 MHz,
CDCl3): d (ppm) 2.17 (s, 1H), 2.33 (s, 3H), 3.76 (s, 6H), 5.73 (s, 1H),
6.35 (t, J = 2.5 Hz, 1H), 6.55 (d, J = 2.5 Hz, 2H), 7.14 (d, J = 7.5 Hz,
2H), 7.26 (d, J = 8 Hz, 2H); MS: (ESI, Pos) m/z 281.0 [(M+23)+].
Utilizing the appropriately substituted Grignard reagents, the
following alcohols were similarly prepared.
Figure 2. Comparison of the EC50 values as a function of the CB1/CB2Ki ratio in five
human glioma cell lines.
4.2. (m-Tolyl)-(3,5-dimethoxy-phenyl)-methanol
Colorless oil (1.45 g, 31.1%). Rf = 0.26 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 2.17 (d, J = 3.5 Hz,
1H), 2.33 (s, 3H), 3.77 (s, 6H), 5.73 (d, J = 3.5 Hz, 1H), 6.36 (t,
J = 2.5 Hz, 1H), 6.56 (d, J = 2 Hz, 2H), 7.08 (d, J = 7.5 Hz, 1H), 7.19
(m, 3H); MS: (ESI, Pos) m/z 281.0 [(M+23)+].
Cannabinoids are gaining increasing attention as novel thera-
peutic agents for the treatment of a broad spectrum of cancer
types. The ability of
and the potential of related analogs to do the same make com-
pounds 4 and 50–64 a unique platform from which to develop
novel GBM therapies. To test the potential of the C10 aryl-substi-
tuted analogs, compounds 2, 4, 54, 55, 56, and 58 were screened
for cytotoxicity against five human glioma cell lines. The com-
pounds manifested broad-spectrum cytotoxicity against primary
and standard human glioma cell lines. The EC50s ranged from
D
8-THC to cross the blood–brain barrier
4.3. (o-Tolyl)-(3,5-dimethoxy-phenyl)-methanol
Colorless oil (4.47 g, 95.9%). Rf = 0.3 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 2.13 (d, J = 3.5 Hz,
1H), 2.29 (s, 3H), 3.75 (s, 6H), 5.93 (d, J = 3.5 Hz, 1H), 6.36 (t,
J = 2.5 Hz, 1H), 6.49 (d, J = 2 Hz, 2H), 7.14 (m, 2H), 7.21 (m, 2H),
7.46 (m, 1H); MS: (ESI, Pos) m/z 281.0 [(M+23)+].
1.67 to 8.78 lM with compound 4 exhibiting the highest efficacy.
In conclusion, we believe that the continued development of the
C10 aryl analogs will provide novel insights into the LBP of can-
nabinoid receptors and generate novel leads for anti-cancer drug
development.
4.4. (4-Fluoro-phenyl)-(3,5-dimethoxy-phenyl)-methanol
White solid (4.17 g, 88.1%). Rf = 0.18 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 2.27 (d, J = 3 Hz, 1H),
3.76 (s, 6H), 5.74 (d, J = 2 Hz, 1H), 6.37 (t, J = 2.5 Hz, 1H), 6.51 (d,
J = 2.5 Hz, 2H), 7.01 (m, 2H), 7.34 (m, 2H); MS: (ESI, Pos) m/z
285.2 [(M+23)+].
4. Experimental
All chemicals and reagents were purchased from Sigma–Aldrich
or Fisher Scientific Inc. Anhydrous solvents were prepared by dis-
tillation over sodium metal or calcium hydride prior to use. Reac-
tions were carried out under dry conditions under an argon
atmosphere. Silica Gel 60, 200–425 mesh was used for flash chro-
matography. Routine NMR spectra were obtained on a Varian Inova
500 MHz NMR and were consistent with the assigned structures.
High-resolution 1D and 2D spectra were obtained on a Varian Ino-
va 500 MHz NMR. All NMR were recorded in CDCl3 unless other-
wise specified. Routine mass spectra were determined on a
Bruker ESQUIRE Ion Trap LC/MS(n) system. Exact mass was deter-
mined at the University of Michigan mass spectrometry facility.
Thin-layer chromatography was performed on silica gel plates
(Merck TLC plates, silica gel 60, F254).
4.5. (3-Fluoro-phenyl)-(3,5-dimethoxy-phenyl)-methanol
White solid (3.95 g, 83.4%). Rf = 0.19 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 2.30 (d, J = 3.5 Hz,
1H), 3.77 (s, 6H), 5.73 (d, J = 3.5 Hz, 1H), 6.37 (t, J = 2 Hz, 1H),
6.52 (d, J = 2 Hz, 2H), 6.95 (m, 1H), 7.13 (m, 2H), 7.28 (m, 1H);
MS: (ESI, Pos) m/z 285.2 [(M+23)+].
4.6. (4-Chloro-phenyl)-(3,5-dimethoxy-phenyl)-methanol
White solid (4.23 g, 84.0%). Rf = 0.24 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 2.25 (d, J = 3.5 Hz, 1H),

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M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
3.76 (s, 6H), 5.72 (d, J = 3 Hz, 1H), 6.36 (t, J = 2.5 Hz, 1H), 6.50 (d,
J = 2.5 Hz, 2H), 7.3 (m, 4H); MS: (ESI, Pos) m/z 301.0 [(M+23)+].
4.13. (2-Thiophenyl)-(3,5-dimethoxy-phenyl)-methanol
Gray oil (2.52 g, 55.7%). Rf = 0.25 (ethyl acetate/hexane 20:80);
1H NMR (500 MHz, CDCl3): d (ppm) 2.38 (m, 1H), 3.78 (s, 6H),
5.99 (d, J = 4 Hz, 1H), 6.40 (t, J = 2 Hz, 1H), 6.62 (d, J = 2 Hz, 2H),
6.93 (m, 2H), 7.26 (m, 1H); MS: (ESI, Pos) m/z 273.0 [(M+23)+].
4.7. (3-Chloro-phenyl)-(3,5-dimethoxy-phenyl)-methanol
White solid (3.59 g, 71.3%). Rf = 0.27 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 2.22 (d, J = 3.5 Hz,
1H), 3.77 (s, 6H), 5.73 (d, J = 3.5 Hz, 1H), 6.38 (t, J = 2.5 Hz, 1H),
6.52 (d, J = 2.5 Hz, 2H), 7.25 (m, 3H), 7.39 (m, 1H); MS: (ESI, Pos)
m/z 301.0 [(M+23)+].
4.14. (p-Tolyl)-(3,5-dimethoxy-phenyl)-methanone (5)
(p-Tolyl)-(3,5-dimethoxy-phenyl)-methanol (3.02 g, 11.7 mmol)
was dissolved in CH2Cl2 (46 mL) and to that Celite (5.04 g) and
PCC (5.04 g, 23.4 mmol) were added. The reaction mixture was al-
lowed to stir at room temperature overnight, then diluted with
ether (100 mL), and filtered over a pad of silica. The filtrate was
evaporated and purified by column chromatography using ethyl
acetate/hexane (10:90) to afford the ketone as a white solid
(1.71 g, 57.0%). Rf = 0.27 (ethyl acetate/hexane 10:90); 1H NMR
(500 MHz, CDCl3): d (ppm) 2.44 (s, 3H), 3.83 (s, 6H), 5.99 (d,
J = 4 Hz, 1H), 6.40 (t, J = 2 Hz, 1H), 6.62 (d, J = 2 Hz, 2H), 6.93 (m,
2H), 7.26 (m, 2H); MS: (ESI, Pos) m/z 279.0 [(M+23)+].
4.8. (4-Bromo-phenyl)-(3,5-dimethoxy-phenyl)-methanol
The reaction of 1,4-dibromo-benzene (1 g, 4.24 mmol) with mag-
nesium turnings (0.15 g, 6.35 mmol) in ether (6 mL) followed by
reaction with 3,5-dimethoxy benzaldehyde (0.527 g, 3.18 mmol)
in THF (3 mL) yielded the alcohol (0.63 g, 61.3%) as a colorless oil.
Rf = 0.3 (ethyl acetate/hexane 20:80); 1H NMR (500 MHz, CDCl3): d
(ppm) 2.10 (br s, 1H), 3.69 (s, 6H), 5.64 (s, 1H), 6.30 (t, J = 2.5 Hz,
1H), 6.43 (d, J = 2.5 Hz, 2H), 7.19 (d, J = 8 Hz, 2H), 7.38 (d, J = 8.5 Hz,
2H); MS: (ESI, Pos) m/z 346.2 [(M+23)+].
Utilizing the appropriately substituted alcohols, the following
ketones were similarly prepared.
4.9. (3-Bromo-phenyl)-(3,5-dimethoxy-phenyl)-methanol
4.15. (m-Tolyl)-(3,5-dimethoxy-phenyl)-methanone (6)
White solid (0.87 g, 56.5%). Rf = 0.32 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 2.25 (br s, 1H), 3.79
(s, 6H), 5.73 (s, 1H), 6.40 (t, J = 2.5 Hz, 1H), 6.53 (d, J = 2.5 Hz,
2H), 7.21 (t, J = 8.0 Hz, 1H), 7.32 (d, 8 Hz, 1H), 7.42 (dt, J = 4, 8 Hz,
1H), 7.58 (m, 1H); MS: (ESI, Pos) m/z 346.2 [(M+23)+].
White solid (0.77 g, 59.7%). Rf = 0.29 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 2.42 (s, 3H), 3.83 (s,
6H), 6.67 (t, J = 2.5 Hz, 1H), 6.92 (d, J = 2 Hz, 2H), 7.37 (m, 2H), 7.58
(d, J = 7.5 Hz, 1H), 7.64 (s, 1H); MS: (ESI, Pos) m/z 279.2 [(M+23)+].
4.10. (3,5-Dimethoxy-phenyl)-(4-ethyl-phenyl)-methanol
4.16. (o-Tolyl)-(3,5-dimethoxy-phenyl)-methanone (7)
4-Bromo-ethyl benzene (3.72 mL, d = 1.34, 27 mmol) and mag-
nesium turnings (0.97 g, 40 mmol) were heated to reflux for 1 h
in THF (40 mL) then cooled to room temperature. The solution
was cooled to ꢀ20 °C and 3,5-dimethoxy benzaldehyde (3.32 g,
20 mmol) dissolved in THF (18 mL) cooled to ꢀ20 °C was added
and followed by stirring at that temperature for 6 h. The reaction
mixture was then quenched with 1 N HCl, extracted with ether,
and the organic layer was then dried over sodium sulfate and evap-
orated. The crude mixture was then purified by column chroma-
tography using ethyl acetate/hexane (20:80) to give the pure
alcohol as a white solid (4.95 g, 67.2%). Rf = 0.3 (ethyl acetate/hex-
ane 20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 1.143 (t, J = 7.5 Hz,
3H), 2.26 (br s, 1H), 2.55 (q, J = 7.5 Hz, 2H), 3.69 (s, 6H), 5.66 (s, 1H),
6.28 (t, J = 2.5 Hz, 1H), 6.48 (d, J = 2.5 Hz, 2H), 7.09 (d, J = 8.5 Hz,
2H), 7.21 (d, J = 9 Hz, 2H); MS: (ESI, Pos) m/z 295.1 [(M+23)+].
Utilizing the appropriately substituted bromo-benzene deriva-
tives the following alcohols were similarly prepared.
White solid (2.08 g, 67.8%). Rf = 0.31 (ethyl acetate/hexane
10:90). Mp 65–67 °C; 1H NMR (500 MHz, CDCl3): d (ppm) 2.34 (s,
3H), 3.81 (s, 6H), 6.67 (t, J = 2.5 Hz, 1H), 6.93 (d, J = 2 Hz, 2H),
7.24 (m, 1H), 7.30 (m, 2H), 7.38 (td, J = 1.5, 7.5 Hz, 1H); MS: (ESI,
Pos) m/z 279.1 [(M+23)+].
4.17. (3,5-Dimethyl-phenyl)-(3,5-dimethoxy-phenyl)-
methanone (8)
White solid (3.5 g, 86.9%); Rf = 0.35 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 2.37 (s, 6H), 3.83 (s,
6H), 6.67 (t, J = 2.5 Hz, 1H), 6.91 (d, J = 2.5 Hz, 2H), 7.22 (br s,
1H), 7.41 (br s, 2H); MS: (ESI, Pos) m/z 293.2 [(M+23)+].
4.18. (4-Ethyl-phenyl)-(3,5-dimethoxy-phenyl)-methanone (9)
White solid (3.67 g, 74.7%). Rf = 0.35 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 1.30 (t, J = 7.5 Hz,
3H), 2.75 (q, J = 7.5 Hz, 2H), 3.85 (s, 6H), 6.68 (t, J = 2.5 Hz, 1H),
6.93 (d, J = 2.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 9 Hz,
2H); MS: (ESI, Pos) m/z 293.2 [(M+23)+].
4.11. (3,5-Dimethoxy-phenyl)-(4-propyl-phenyl)-methanol
White solid (4.44 g, 61.8%). Rf = 0.31 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 0.97 (t, J = 7.5 Hz,
3H), 1.69 (m, 2H), 2.28 (br s, 1H), 2.67 (t, J = 7.5 Hz, 2H), 3.72 (s,
6H), 5.68 (s, 1H), 6.30 (t, J = 2.5 Hz, 1H), 6.52 (d, J = 2.5 Hz, 2H),
7.1 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 9 Hz, 2H); MS: (ESI, Pos) m/z
309.1 [(M+23)+].
4.19. (4-Propyl-phenyl)-(3,5-dimethoxy-phenyl)-methanone (10)
White solid (3.34 g, 75.9%). Rf = 0.36 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 0.97 (t, J = 7.5 Hz,
3H), 1.69 (m, 2H), 2.67 (t, J = 7.5 Hz, 2H), 3.83 (s, 6H), 6.67 (t,
J = 2.5 Hz, 1H), 6.91 (d, J = 2.5 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H),
7.75 (d, J = 9 Hz, 2H); MS: (ESI, Pos) m/z 307.2 [(M+23)+].
4.12. (3,5-Dimethoxy-phenyl)-(3,5-dimethyl-phenyl)-methanol
White solid (4.02 g, 54.6%). Rf = 0.33 (ethyl acetate/hexane
20:80); 1H NMR (500 MHz, CDCl3): d (ppm) 2.25 (br s, 1H), 2.37
(s, 6H), 3.76 (s, 6H), 5.69 (s, 1H), 6.36 (t, J = 2.5 Hz, 1H), 6.61 (d,
J = 2.5 Hz, 2H), 7.1 (s, 1H), 7.21 (s, 2H); MS: (ESI, Pos) m/z 295.1
[(M+23)+].
4.20. (4-Fluoro-phenyl)-(3,5-dimethoxy-phenyl)-methanone (11)
Colorless oil (2.71 g, 91.0%). Rf = 0.29 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 3.83 (s, 6H), 6.67 (t,

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
6495
J = 2.5 Hz, 1H), 6.88 (d, J = 2.5 Hz, 2H), 7.15 (m, 2H), 7.86 (m, 2H);
MS: (ESI, Pos) m/z 283.1 [(M+23)+].
as a yellow oil (1.11 g, 65.8%). Rf = 0.36 (ethyl acetate/hexane
5:95); 1H NMR (500 MHz, CDCl3): d (ppm) 1.63 (s, 6H), 2.30 (s,
3H), 3.73 (s, 6H), 6.29 (t, J = 2.5 Hz, 1H), 6.39 (d, J = 2.5 Hz, 2H),
7.06 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.5 Hz, 1H); MS: (ESI, Pos) m/z
293.3 [(M+23)+].
4.21. (3-Fluoro-phenyl)-(3,5-dimethoxy-phenyl)-methanone (12)
Colorless oil (2.81 g, 89.9%). Rf = 0.31 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 3.83 (s, 6H), 6.69
(t, J = 2 Hz, 1H), 6.90 (d, J = 2 Hz, 2H), 7.29 (m, 1H), 7.46 (m,
1H), 7.51 (m, 1H), 7.58 (m, 1H); MS: (ESI, Pos) m/z 283.2
[(M+23)+].
Utilizing appropriately substituted ketones, the following di-
methyl intermediates were similarly synthesized.
4.28. 1-[1-(m-Tolyl)-1-methyl-ethyl]-3,5-dimethoxy-benzene
(19)
4.22. (4-Chloro-phenyl)-(3,5-dimethoxy-phenyl)-methanone
(13)
Colorless oil (0.54 g, 75.3%). Rf = 0.35 (ethyl acetate/hexane
5:95); 1H NMR (500 MHz, CDCl3): d (ppm) 1.55 (s, 6H), 2.22 (s,
3H), 3.65 (s, 6H), 6.22 (t, J = 2 Hz, 1H), 6.32 (d, J = 2.5 Hz, 2H),
6.89 (m, 1H), 6.96 (m, 2H), 7.06 (t, J = 7.5 Hz, 1H); MS: (ESI, Pos)
m/z 293.2 [(M+23)+].
White solid (2.07 g, 74.8%). Rf = 0.29 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 3.83 (s, 6H), 6.68 (t,
J = 2 Hz, 1H), 6.88 (d, J = 2.5 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 7.77
(d, J = 8.5 Hz, 2H); MS: (ESI, Pos) m/z 299.0 [(M+23)+].
4.29. 1-[1-(o-Tolyl)-1-methyl-ethyl]-3,5-dimethoxy-benzene
(20)
4.23. (3-Chloro-phenyl)-(3,5-dimethoxy-phenyl)-methanone
(14)
Yellow oil (1.82 g, 82.9%). Rf = 0.35 (ethyl acetate/hexane 5:95);
1H NMR (500 MHz, CDCl3): d (ppm) 1.67 (s, 6H), 1.88 (s, 3H), 3.74
(s, 6H), 6.30 (t, J = 2 Hz, 1H), 6.34 (d, J = 2.5 Hz, 2H), 7.06 (d, J = 8 Hz,
1H), 7.22 (m, 2H), 7.54 (dd, J = 2, 8 Hz, 1H); MS: (ESI, Pos) m/z 293.2
[(M+23)+].
Yellow oil (2.22 g, 76.6%). Rf = 0.3 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 3.83 (s, 6H), 6.69
(t, J = 2.5 Hz, 1H), 6.89 (d, J = 2.5 Hz, 2H), 7.41 (t, J = 8 Hz, 1H),
7.55 (m, 1H), 7.67 (m, 1H), 7.79 (t, J = 2.5 Hz, 1H); MS: (ESI,
Pos) m/z 299.1 [(M+23)+].
4.30. 1-[1-(3,5-Dimethyl-phenyl)-1-methyl-ethyl]-3,5-
dimethoxy-benzene (21)
4.24. (4-Bromo-phenyl)-(3,5-dimethoxy-phenyl)-methanone
(15)
Yellow oil (2.17 g, 81.9%). Rf = 0.5 (ethyl acetate/hexane 6:94);
1H NMR (500 MHz, CDCl3): d (ppm) 1.62 (s, 6H), 2.26 (s, 6H),
3.73 (s, 6H), 6.29 (t, J = 2.5 Hz, 1H), 6.39 (d, J = 2.5 Hz, 2H), 6.80
(br s, 1H), 6.84 (br s, 2H); MS: (ESI, Pos) m/z 307.3 [(M+23)+].
White solid (3.3 g, 83.0%). Rf = 0.39 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 3.85 (s, 6H), 6.70 (t,
J = 2 Hz, 1H), 6.90 (d, J = 2.5 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.71
(d, J = 8.5 Hz, 2H); MS: (ESI, Pos) m/z 344.1 [(M+23)+].
4.31. 1-[1-(4-Ethyl-phenyl)-1-methyl-ethyl]-3,5-dimethoxy-
benzene (22)
4.25. (3-Bromo-phenyl)-(3,5-dimethoxy-phenyl)-methanone
(16)
Yellow oil (1.01 g, 78.3%). Rf = 0.45 (ethyl acetate/hexane 6:94);
1H NMR (500 MHz, CDCl3): d (ppm) 1.22 (t, J = 7.5 Hz, 3H), 1.64 (s,
6H), 2.60 (q, J = 7.5 Hz, 2H), 3.74 (s, 6H), 6.29 (t, J = 2.5 Hz, 1H), 6.40
(d, J = 2 Hz, 2H), 7.08 (d, J = 8 Hz, 2H), 7.14 (d, J = 8.5 Hz, 2H); MS:
(ESI, Pos) m/z 307.4 [(M+23)+].
White solid (0.72 g, 63.6%). Rf = 0.39 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 3.85 (s, 6H), 6.71 (t,
J = 2 Hz, 1H), 6.91 (d, J = 2.5 Hz, 2H), 7.38 (t, J = 8 Hz, 1H), 7.74
(m, 2H), 7.97 (br s, 1H); MS: (ESI, Pos) m/z 344.1 [(M+23)+].
4.32. 1-[1-(4-Propyl-phenyl)-1-methyl-ethyl]-3,5-dimethoxy-
benzene (23)
4.26. (2-Thiophenyl)-(3,5-dimethoxy-phenyl)-methanone (17)
Yellow solid (1.12 g, 49.6%). Rf = 0.21 (ethyl acetate/hexane
10:90); 1H NMR (500 MHz, CDCl3): d (ppm) 3.84 (s, 6H), 6.67 (t,
J = 2.5 Hz, 1H), 6.91 (d, J = 2.5 Hz, 2H), 7.16 (m, 1H), 7.70 (d,
J = 4 Hz, 1H), 7.72 (d, J = 4 Hz, 1H); MS: (ESI, Pos) m/z 270.9
[(M+23)+].
Yellow oil (0.3 g, 74.5%). Rf = 0.47 (ethyl acetate/hexane
6:94); 1H NMR (500 MHz, CDCl3): d (ppm) 0.93 (t, J = 7.5 Hz,
3H), 1.61 (m, 2H), 1.64 (s, 6H), 2.55 (t, J = 7.5 Hz, 2H), 3.73 (s,
6H), 6.29 (t, J = 2.5 Hz, 1H), 6.39 (d, J = 2.5 Hz, 2H), 7.06 (d,
J = 8 Hz, 2H), 7.13 (d, J = 8 Hz, 2H); MS: (ESI, Pos) m/z 321.2
[(M+23)+].
4.27. 1-[1-(p-Tolyl)-1-methyl-ethyl]-3,5-dimethoxy-benzene (18)
4.33. 1-[1-(4-Fluoro-phenyl)-1-methyl-ethyl]-3,5-dimethoxy-
benzene (24)
To methylene chloride (26.3 mL) in a flask cooled to ꢀ40 °C and
held at this temperature and titanium(IV) chloride (37.3 mL, 1 M
solution, 37.3 mmol) was added dropwise, followed by dimethyl
zinc (18.5 mL, 2 M solution, 37.3 mmol). The reaction mixture
was allowed to stir at that temperature for 15 min then a solution
of ketone 5 (1.6 g, 6.25 mmol) in methylene chloride (10 mL) was
added and the mixtusre was stirred at ꢀ40 °C for 2 h after which
it was allowed to warm to ꢀ10 °C for 2 h. The mixture was then
poured into ice and the aqueous layer was extracted with methy-
lene chloride. The organic layer was then evaporated under re-
duced pressure and the crude product was purified by column
chromatography using ethyl acetate/hexane (4:96) to afford 18
Yellow oil (1.52 g, 76.7%). Rf = 0.37 (ethyl acetate/hexane 6:94);
1H NMR (500 MHz, CDCl3): d (ppm) 1.63 (s, 6H), 3.73 (s, 6H), 6.30
(t, J = 2 Hz, 1H), 6.35 (d, J = 2.5 Hz, 2H), 6.93 (m, 2H), 7.19 (m, 2H);
MS: (ESI, Pos) m/z 297.3 [(M+23)+].
4.34. 1-[1-(3-Fluoro-phenyl)-1-methyl-ethyl]-3,5-dimethoxy-
benzene (25)
Yellow oil (1.66 g, 87.0%). Rf = 0.37 (ethyl acetate/hexane 6:94);
1H NMR (500 MHz, CDCl3): d (ppm) 1.68 (s, 6H), 3.77 (s, 6H), 6.35

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M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
(t, J = 2 Hz, 1H), 6.41 (d, J = 2.5 Hz, 2H), 6.87 (td, J = 3, 8.5 Hz, 1H),
6.97 (dt, J = 2, 11 Hz, 1H), 7.04 (d, J = 8 Hz, 1H), 7.24 (m, 1H); MS:
(ESI, Pos) m/z 297.2 [(M+23)+].
4.41. 3-[1-(3,5-Dimethoxy-phenyl)-1-methyl-ethyl]-
benzonitrile (45)
Colorless oil. Rf = 0.23 (ethyl acetate/hexane 8:92); 1H NMR
(500 MHz, CDCl3): d (ppm) 1.67 (s, 6H), 3.80 (s, 6H), 6.31 (t,
J = 2 Hz, 1H), 6.35 (d, J = 2.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 1H), 7.48
(m, 2H), 7.56 (m, 1H); MS: (ESI, Pos) m/z 304.3 [(M+23)+].
4.35. 1-[1-(4-Chloro-phenyl)-1-methyl-ethyl]-3,5-dimethoxy-
benzene (26)
Yellow oil (1.63 g, 88.6%). Rf = 0.42 (ethyl acetate/hexane 6:94);
1H NMR (500 MHz, CDCl3): d (ppm) 1.62 (s, 6H), 3.73 (s, 6H), 6.30
(t, J = 2 Hz, 1H), 6.35 (d, J = 2 Hz, 2H), 7.16 (d, J = 8.5 Hz, 2H), 7.21
(d, J = 9 Hz, 3H); MS: (ESI, Pos) m/z 313.1 [(M+23)+].
4.42. 3-[1-(3,5-Dimethoxy-phenyl)-1-methyl-ethyl]-phenyl
amine
Into an oven-dried flask were added Pd2(dba)3 (0.016 g,
0.018 mmol) and 2-dicyclohexylphosphinobiphenyl (0.015 g,
0.043 mmol) and the flask was evacuated and filled with argon.
To this were added bromide 29 (1.2 g, 3.58 mmol) and LiHMDS
(4.3 mL, 1 M solution, 4.3 mmol) and the reaction mixture was
stirred in a preheated oil bath at 65 °C for 15 h. The mixture
was cooled to room temperature and then reacted with 1 M HCl
(5 mL) for 5 min. The solution was then neutralized with 1 M
NaOH and the aqueous phase was extracted with CH2Cl2, washed
with water and brine, and then dried over sodium sulfate. The or-
ganic layer was evaporated and the product was obtained as a
light brown oil (0.75 g, 77.2%) by column chromatography using
ethyl acetate/hexane (15:85). Rf = 0.21 (ethyl acetate/hexane
15:85); 1H NMR (500 MHz, CDCl3): d (ppm) 1.61 (s, 6H), 3.21
(br s, 1H), 3.73 (s, 6H), 6.29 (t, J = 2 Hz, 1H), 6.40 (d, J = 2.5 Hz,
2H), 6.51 (m, 1H), 6.54 (t, J = 2.5 Hz, 1H), 6.67 (m, 1H), 7.05 (t,
J = 7.5 Hz, 1H); MS: (ESI, Pos) m/z 294.2 [(M+23)+] and 272.2
[(M+1)+].
4.36. 1-[1-(3-Chloro-phenyl)-1-methyl-ethyl]-3,5-dimethoxy-
benzene (27)
Colorless oil (1.85 g, 88.0%). Rf = 0.42 (ethyl acetate/hexane
6:94); 1H NMR (500 MHz, CDCl3): d (ppm) 1.63 (s, 6H), 3.73 (s,
6H), 6.30 (t, J = 2 Hz, 1H), 6.36 (d, J = 2 Hz, 2H), 7.08 (dt, J = 1.5,
7.5 Hz, 1H), 7.16 (m, 2H), 7.23 (t, J = 2 Hz, 1H); MS: (ESI, Pos) m/z
313.1 [(M+23)+].
4.37. 1-[1-(4-Bromo-phenyl)-1-methyl-ethyl]-3,5-dimethoxy-
benzene (28)
Colorless oil (2.37 g, 82.3%). Rf = 0.43 (ethyl acetate/hexane
6:94); 1H NMR (500 MHz, CDCl3): d (ppm) 1.66 (s, 6H), 3.79 (s,
6H), 6.32 (t, J = 2 Hz, 1H), 6.38 (d, J = 2.5 Hz, 2H), 7.12 (d,
J = 8.5 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H); MS: (ESI, Pos) m/z 358.2
[(M+23)+].
4.38. 1-[1-(3-Bromo-phenyl)-1-methyl-ethyl]-3,5-dimethoxy-
benzene (29)
4.43. N-{3-[1-(3,5-Dimethoxy-phenyl)-1-methyl-ethyl]-
phenyl}acetamide (46)
Colorless oil (2.34 g, 89.7%). Rf = 0.39 (ethyl acetate/hexane
5:95); 1H NMR (500 MHz, CDCl3): d (ppm) 1.68 (s, 6H), 3.77 (s,
6H), 6.33 (t, J = 2 Hz, 1H), 6.38 (d, J = 2.5 Hz, 2H), 7.15 (m, 2H),
7.33 (m, 1H), 7.42 (m, 1H); MS: (ESI, Pos) m/z 358.1 [(M+23)+].
Acetyl chloride (0.08 mL, d = 1.10, 1.21 mmol) and pyridine
(0.15 mL) were added into a solution of 3-[1-(3,5-Dimethoxy-phe-
nyl)-1-methyl-ethyl]-phenyl amine (0.3 g, 1.10 mmol) in CH2Cl2
(1.53 mL) cooled to 0 °C. The reaction mixture was then allowed
to stir at 0 °C for 2 h. The mixture was washed with 2 N HCl, water,
and brine and dried over sodium sulfate. The organic layer was
then evaporated and the amide was obtained by column chroma-
tography using ethyl acetate/hexane (30:70) as colorless oil
(0.24 g, 69.3%). Rf = 0.39 (ethyl acetate/hexane 50:50); 1H NMR
(500 MHz, CDCl3): d (ppm) 1.63(s, 6H), 2.13 (s, 3H), 3.74 (s, 6H),
6.30 (t, J = 2 Hz, 1H), 6.38 (d, J = 2.5 Hz, 2H), 7.01 (d, J = 7.5 Hz,
1H), 7.12 (s, 1H), 7.13 (br s, 1H), 7.23 (t, J = 8 Hz, 1H), 7.53 (d,
J = 7.5 Hz, 1H); MS: (ESI, Pos) m/z 336.2 [(M+23)+].
4.39. 2-[1-(3,5-Dimethoxy-phenyl)-1-methyl-ethyl]-thiophene
(30)
Colorless oil (0.69 g, 65.3%). Rf = 0.31 (ethyl acetate/hexane
5:95); 1H NMR (500 MHz, CDCl3): d (ppm) 1.74 (s, 6H), 3.74 (s,
6H), 6.31 (t, J = 2.5 Hz, 1H), 6.47 (d, J = 2 Hz, 2H), 6.83 (dd, J = 1.5,
3.5 Hz, 1H), 6.90 (m, 1H), 7.13 (dd, J = 1.5, 5 Hz, 1H); MS: (ESI,
Pos) m/z 285.2 [(M+23)+].
4.40. 4-[1-(3,5-Dimethoxy-phenyl)-1-methyl-ethyl]-
benzonitrile (44)
4.44. 5-(1-Methyl-1-p-tolyl-ethyl)-benzene-1,3-diol (31)
A solution of compound 18 (0.85 g, 3.15 mmol) in methylene
chloride (128 mL) was cooled to ꢀ78 °C to which was added
BBr3 (8.06 mL, 1 M solution, 8.06 mmol). The reaction mixture
was allowed to stir at ꢀ78 °C for 3 h then warmed to room tem-
perature and stirred overnight. The reaction was then quenched
with methanol (20 mL) and extracted with ether. The ethereal
layer was washed with bicarbonate, water, and brine and dried
over sodium sulfate. The organic layer was evaporated and com-
pound 31 was obtained by column chromatography using ethyl
acetate/hexane (30:70) as a white solid (0.71 g, 93.2%). Rf = 0.25
Bromide 28 (0.57 g, 1.7 mmol) and copper (I) cyanide (0.40 g,
4.48 mmol) in DMF (4.1 mL) were heated to reflux for 6 h. The
mixture was then poured into a solution of ethylene diamine
(3 mL) in water (10 mL). The crude product was extracted by re-
peated washing with ethyl acetate. The organic layer was washed
with water and brine and then dried over sodium sulfate. The sol-
vent was removed under reduced pressure and purified via col-
umn chromatography using ethyl acetate/hexane (8:92) to yield
a colorless oil (0.42 g, 87.8%). Rf = 0.21 (ethyl acetate/hexane
8:92); 1H NMR (500 MHz, CDCl3): d (ppm) 1.67 (s, 6H), 3.76 (s,
6H), 6.32 (t, J = 2 Hz, 1H), 6.38 (d, J = 2.5 Hz, 2H), 7.35 (d,
J = 8.5 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H); MS: (ESI, Pos) m/z 304.3
[(M+23)+].
(ethyl acetate/hexane 30:70); 1H NMR (500 MHz, CDCl3):
d
(ppm) 1.60 (s, 6H), 2.31 (s, 3H), 4.69 (s, 2H), 6.16 (t, J = 2.5 Hz,
1H), 6.27 (d, J = 2.5 Hz, 2H), 7.07 (d, J = 8 Hz, 2H), 7.13 (d,
J = 8.5 Hz, 2H); MS: (ESI, Neg) m/z 241.2 [(Mꢀ1)ꢀ].
Utilizing the same procedure, the following resorcinols were
synthesized.
Utilizing appropriately substituted bromide, the following di-
methyl intermediate was similarly synthesized.

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
6497
4.45. 5-(1-Methyl-1-m-tolyl-ethyl)-benzene-1,3-diol (32)
4.53. 5-(1-(3-Chloro-phenyl)-1-methyl-ethyl)-benzene-1,3-diol
(40)
Colorless oil (0.33 g, 94.4%). Rf = 0.28 (ethyl acetate/hexane
30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.61 (s, 6H), 2.31 (s,
3H), 4.80 (s, 2H), 6.17 (t, J = 2.5 Hz, 1H), 6.27 (d, J = 2.5 Hz, 2H),
6.99 (d, J = 7.5 Hz, 1H), 7.03 (m, 2H), 7.16 (t, J = 8 Hz, 1H); MS:
(ESI, Neg) m/z 241.2 [(Mꢀ1)ꢀ].
Cream colored solid (0.94 g, 93.7%). Rf = 0.31 (ethyl acetate/hex-
ane 30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.60 (s, 6H), 4.89 (s,
2H), 6.19 (t, J = 2.5 Hz, 1H), 6.25 (d, J = 2.5 Hz, 2H), 7.08 (dt, J = 1.5,
7.5 Hz, 1H), 7.19 (m, 3H); MS: (ESI, Neg) m/z 261.2 [(Mꢀ1)ꢀ].
4.46. 5-(1-Methyl-1-o-tolyl-ethyl)-benzene-1,3-diol (33)
4.54. 5-(1-(4-Bromo-phenyl)-1-methyl-ethyl)-benzene-1,3-diol
(41)
Colorless oil (0.32 g, 93.2%). Rf = 0.26 (ethyl acetate/hexane
30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.64 (s, 6H), 1.98 (s,
3H), 4.59 (s, 2H), 6.18 (t, J = 2 Hz, 1H), 6.23 (d, J = 2.5 Hz, 2H),
7.08 (d, J = 8 Hz, 1H), 7.20 (m, 2H), 7.55 (dd, J = 2, 8 Hz, 1H); MS:
(ESI, Neg) m/z 241.2 [(Mꢀ1)ꢀ].
White solid (0.39 g, 92.5%). Rf = 0.33 (ethyl acetate/hexane
30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.62 (s, 6H), 4.61 (s,
2H), 6.20 (t, J = 2.5 Hz, 1H), 6.26 (d, J = 2.5 Hz, 2H), 7.12 (d, J = 8.5 Hz,
2H), 7.39 (d, J = 8.5 Hz, 2H); MS: (ESI, Neg) m/z 306.2 [(Mꢀ1)ꢀ].
4.47. 5-(1-(3,5-Dimethyl-phenyl)-1-methyl-ethyl)-benzene-1,3-
diol (34)
4.55. 5-(1-(3-Bromo-phenyl)-1-methyl-ethyl)-benzene-1,3-diol
(42)
White solid (1.3 g, 83.4%). Rf = 0.33 (ethyl acetate/hexane
30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.62 (s, 6H), 2.23 (s,
6H), 4.60 (s, 2H), 6.19 (t, J = 2 Hz, 1H), 6.29 (d, J = 2.5 Hz, 2H),
6.86 (m, 3H); MS: (ESI, Neg) m/z 255.3 [(Mꢀ1)ꢀ].
Yellow oil (0.49 g, 92.5%). Rf = 0.34 (ethyl acetate/hexane
30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.63 (s, 6H), 4.62 (s,
2H), 6.20 (t, J = 2.5 Hz, 1H), 6.27 (d, J = 2.5 Hz, 2H), 7.16 (d,
J = 4 Hz, 2H), 7.33 (m, 1H), 7.40 (m, 1H); MS: (ESI, Neg) m/z 306.2
[(Mꢀ1)ꢀ].
4.48. 5-(1-(4-Ethyl-phenyl)-1-methyl-ethyl)-benzene-1,3-diol
(35)
4.56. 5-(1-Methyl-1-thiophen-2-yl-ethyl)-benzene-1,3-diol (43)
Colorless oil (1 g, 65.2%). Rf = 0.32 (ethyl acetate/hexane 30:70);
1H NMR (500 MHz, CDCl3): d (ppm) 1.25 (t, J = 7.5 Hz, 3H), 1.63 (s,
6H), 2.65 (q, J = 7.5 Hz, 2H), 4.59 (s, 2H), 6.19 (t, J = 2.5 Hz, 1H), 6.30
(d, J = 2.5 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H);
MS: (ESI, Neg) m/z 255.3 [(Mꢀ1)ꢀ].
White solid (0.87 g, 95.3%). Rf = 0.3 (ethyl acetate/hexane 30:70);
1H NMR (500 MHz, CDCl3): d (ppm) 1.60 (s, 6H), 4.65 (s, 2H), 6.18
(t, J = 2 Hz, 1H), 6.24 (d, J = 2.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 7.23
(d, J = 8.5 Hz, 2H); MS: (ESI, Neg) m/z 233.1 [(Mꢀ1)ꢀ].
4.57. 4-[1-(3,5-Dihydroxy-phenyl)-1-methyl-ethyl]-
benzonitrile (44)
4.49. 5-(1-(4-Propyl-phenyl)-1-methyl-ethyl)-benzene-1,3-diol
(36)
Colorless oil (0.19 g, 54.1%). Rf = 0.21 (ethyl acetate/hexane
30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.65 (s, 6H), 4.73 (s,
2H), 6.22 (t, J = 2.5 Hz, 1H), 6.26 (d, J = 2.5 Hz, 2H), 7.35 (d,
J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H); MS: (ESI, Neg) m/z 252.3
[(Mꢀ1)ꢀ].
Colorless oil (0.68 g, 68.2%). Rf = 0.34 (ethyl acetate/hexane
30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 0.97 (t, J = 7.5 Hz,
3H), 1.28 (t, J = 7.5 Hz, 2H), 1.63 (s, 6H), 2.57 (m, 2H), 4.64 (s,
2H), 6.19 (t, J = 2 Hz, 1H), 6.30 (d, J = 2.5 Hz, 2H), 7.09 (d,
J = 8.5 Hz, 2H), 7.16 (d, J = 8.5 Hz, 2H); MS: (ESI, Neg) m/z 269.3
[(Mꢀ1)ꢀ].
4.58. 3-[1-(3,5-Dihydroxy-phenyl)-1-methyl-ethyl]-
benzonitrile (45)
4.50. 5-(1-(4-Fluoro-phenyl)-1-methyl-ethyl)-benzene-1,3-diol
(37)
Colorless oil (0.28 g, 97.2%). Rf = 0.22 (ethyl acetate/hexane
30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.65 (s, 6H), 4.68 (s,
2H), 6.22 (t, J = 2 Hz, 1H), 6.26 (d, J = 2.5 Hz, 2H), 7.38 (t,
J = 7.5 Hz, 2H), 7.49 (tt, J = 1.5, 7.5 Hz, 2H), 7.56 (t, J = 1.5 Hz, 1H);
MS: (ESI, Neg) m/z 252.3 [(Mꢀ1)ꢀ].
Cream colored solid (0.89 g, 96.2%). Rf = 0.29 (ethyl acetate/hex-
ane 30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.63 (s, 6H), 4.63 (s,
2H), 6.20 (t, J = 2.5 Hz, 1H), 6.274 (d, J = 2.5 Hz, 2H), 6.97 (m, 2H),
7.203 (m, 2H); MS: (ESI, Neg) m/z 245.1 [(Mꢀ1)ꢀ].
4.59. N-{3-[1-(3,5-dihydroxy-phenyl)-1-methyl-ethyl]-phenyl}-
acetamide (46)
4.51. 5-(1-(3-Fluoro-phenyl)-1-methyl-ethyl)-benzene-1,3-diol
(38)
Light brown oil (0.18 g, 79.1%). Rf = 0.15 (ethyl acetate/hexane
50:50); 1H NMR (500 MHz, DMSO): d (ppm) 1.51 (s, 6H), 1.99 (s,
3H), 5.99 (t, J = 2 Hz, 1H), 6.04 (d, J = 2.5 Hz, 2H), 6.89 (d,
J = 7.5 Hz, 1H), 7.18 (t, J = 8 Hz, 1H), 7.29 (s, 1H), 7.53 (d,
J = 7.5 Hz, 1H), 9.01 (s, 2H), 9.83 (s, 1H); MS: (ESI, Pos) m/z 308.2
[(M+23)+].
Cream colored solid (1.01 g, 94.5%). Rf = 0.28 (ethyl acetate/hex-
ane 30:70); 1H NMR (500 MHz, CDCl3): d (ppm) 1.63 (s, 6H), 4.60 (s,
2H), 6.20 (t, J = 2.5 Hz, 1H), 6.28 (d, J = 2.5 Hz, 2H), 6.94 (m, 3H),
7.23 (m, 1H); MS: (ESI, Neg) m/z 245.1 [(Mꢀ1)ꢀ].
4.52. 5-(1-(4-Chloro-phenyl)-1-methyl-ethyl)-benzene-1,3-diol
(39)
4.60. trans-(R)-(+)-Limonene oxide (48)
White solid (0.87 g, 95.3%). Rf = 0.3 (ethyl acetate/hexane 30:70);
1H NMR (500 MHz, CDCl3): d (ppm) 1.60 (s, 6H), 4.65 (s, 2H), 6.18 (t,
J = 2.5 Hz, 1H), 6.24 (d, J = 2.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 7.23
(d, J = 8.5 Hz, 2H); MS: (ESI, Neg) m/z 261.2 [(Mꢀ1)ꢀ].
The kinetic separation of cis/trans(+)-limonene oxide (53.8 mL,
d = 0.93, 328 mmol) was carried out as described by Steiner et
al.,21 using pyrazole (3.72 g, 54.7 mmol) and de-ionized water
(177 mL). Yield 21.2 g, (84.8%); 1H NMR (500 MHz, CDCl3): d

6498
M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
(ppm) 1.31 (s, 3H), 1.39 (m, 2H), 1.69 (s, 3H), 1.71 (m, 2H), 1.86 (m,
2H), 2.06 (m, 1H), 2.98 (m, 1H), 4.66 (s, 2H); MS: (ESI, Pos) m/z
175.2 [(M+23)+].
4.65. 3-[1-(4-Fluoro-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (56)
Light pink foam (0.59 g, 45.5%). Rf = 0.37 (methylene chloride/
hexane, 1:1); 1H NMR (500 MHz, CDCl3): d (ppm) 1.04 (s, CH3,
3H), 1.30 (s, CH3, 3H), 1.51 (m, CH3, 6H), 1.62 (s, CH3, 3H), 1.75
(m, CH2 and CH, 3H), 2.07 (m, CH2, 1H), 2.61 (m, CH, 1H), 3.09
(dd, J = 4.5, 15 Hz, CH2, 1H), 4.49 (s, OH, 1H), 5.35 (d, J = 4.5 Hz,
@CH, 1H), 5.90 (d, J = 2 Hz, ArH, 1H), 6.32 (d, J = 2 Hz, ArH, 1H),
6.86 (m, ArH, 2H), 7.12 (m, ArH, 2H); MS: (ESI, Neg) m/z 379.5
[(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for C25H29FO2: 380.2152, experi-
mental 380.2152.
4.61. (+)-(1S,4R)-p-Mentha-2,8-dien-1-ol (49)
trans-(R)-(+)-Limonene oxide (48, 5.1 g, 33.5 mmol) was con-
verted to (+)-(1S,4R)-p-mentha-2,8-dien-1-ol, utilizing the method
of Richards and Watson.20 Yield 1.58 g (31.6%) as a colorless oil. a2D3
+53.8° (CHCl3); 1H NMR (500 MHz, CDCl3): d (ppm) 1.22 (s, 3H),
1.46 (br s, 1H), 1.59 (m, 2H), 1.67 (s, 3H), 1.76 (m, 2H), 2.67 (m,
1H), 4.59 (m, 1H), 4.71 (t, J = 1.5 Hz, 1H), 5.54 (dd, J = 3, 7 Hz,
1H), 5.64 (dd, J = 1.5, 7 Hz, 1H); MS: (ESI, Pos) m/z 151.2
[(Mꢀ1)ꢀ].
4.66. 3-[1-(3-Fluoro-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (57)
4.62. 6,6,9-Trimethyl-3-(1-methyl-1-p-tolyl-ethyl)-6a,7,10,10a-
tetrahydro-6H-benzo[c]chromen-1-ol (50)
White foam (0.36 g, 24.4%). Rf = 0.37 (methylene chloride/hex-
ane, 1:1); 1H NMR (500 MHz, CDCl3): d (ppm) 1.06 (s, CH3, 3H),
1.21 (s, CH3, 3H), 1.53 (m, CH3, 6H), 1.66 (s, CH3, 3H), 1.76 (m,
CH2 and CH, 3H), 2.17 (m, CH2, 1H), 2.52 (m, CH, 1H), 3.08 (dd,
J = 4.5, 15 Hz, CH2, 1H), 4.36 (s, OH, 1H), 5.41 (d, J = 4.5 Hz, @CH,
1H), 5.82 (d, J = 2 Hz, ArH, 1H), 6.22 (d, J = 2 Hz, ArH, 1H), 6.67
(td, J = 2.5, 8.5 Hz, ArH, 1H), 6.88 (m, ArH, 1H), 7.02 (m,
ArH, 1H), 7.12 (m, ArH, 1H); MS: (ESI, Neg) m/z 379.5 [(Mꢀ1)ꢀ].
Resorcinol 31 (0.63 g, 2.60 mmol), (+)-D
2-p-menthene-1,8-diol
(0.44 g, 2.60 mmol), and p-toluene sulfonic acid (0.02 g,
0.11 mmol) were dissolved in benzene (21.7 mL) and the reaction
mixture was placed in a preheated oil bath at 80 °C and allowed
to stir at that temperature for 6 h. The mixture was then cooled, di-
luted with ether, and washed with bicarbonate, water, and brine.
The ethereal layer was then separated, dried over sodium sulfate,
and evaporated. The product was purified by column chromatogra-
phy using methylene chloride/hexane (40:60) yielding a white
foam (0.545 g, 55.6%). Rf = 0.33 (methylene chloride/hexane, 1:1);
1H NMR (500 MHz, CDCl3): d (ppm) 1.11 (s, CH3, 3H), 1.37 (s,
CH3, 3H), 1.59 (m, CH3, 6H), 1.69 (s, CH3, 3H), 1.81 (m, CH2 and
CH, 3H), 2.14 (m, CH2, 1H), 2.31 (s, CH3, 3H), 2.67 (m, CH, 1H),
3.16 (dd, J = 4.5, 15 Hz, CH2, 1H), 4.53 (s, OH, 1H), 5.42 (d,
J = 4.5 Hz, @CH, 1H), 5.98 (d, J = 2 Hz, ArH, 1H), 6.43 (d, J = 2 Hz,
ArH, 1H), 7.07 (d, J = 8 Hz, ArH, 2H), 7.143 (d, J = 8 Hz, ArH, 2H);
MS: (ESI, Neg) m/z 375.4 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for
HR-EI-MS m/z calcd for
C25H29FO2: 380.2152, experimental
380.2152.
4.67. 3-[1-(4-Chloro-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (58)
White foam (0.63 g, 50.4%). Rf = 0.41 (methylene chloride/hex-
ane, 1:1); 1H NMR (500 MHz, CDCl3): d (ppm) 1.04 (s, CH3, 3H),
1.30 (s, CH3, 3H), 1.51 (m, CH3, 6H), 1.62 (s, CH3, 3H), 1.75 (m,
CH2 and CH, 3H), 2.07 (m, CH2, 1H), 2.61 (m, CH, 1H), 3.09 (dd,
J = 4.5, 15 Hz, CH2, 1H), 4.49 (s, OH, 1H), 5.35 (d, J = 5 Hz, @CH,
1H), 5.89 (d, J = 2 Hz, ArH, 1H), 6.30 (d, J = 2 Hz, ArH, 1H), 7.09 (d,
J = 8.5 Hz, ArH, 2H), 7.15 (d, J = 9 Hz, ArH, 2H); MS: (ESI, Neg) m/z
395.9 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for C25H29ClO2: 396.1856,
experimental 396.1856.
C26H32O2: 376.2402, experimental 376.2391.
Utilizing the same procedure, the following analogs were syn-
thesized using the appropriately substituted resorcinols.
4.63. 6,6,9-Trimethyl-3-(1-methyl-1-m-tolyl-ethyl)-6a,7,10,10a-
tetrahydro-6H-benzo[c]chromen-1-ol (51)
4.68. 3-[1-(3-Chloro-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (59)
Yellow oil (0.139 g, 28.8%). Rf = 0.34 (methylene chloride/hexane,
1:1); 1H NMR (500 MHz, CDCl3): d (ppm) 1.12 (s, CH3, 3H), 1.38 (s,
CH3, 3H), 1.59 (m, CH3, 6H), 1.69 (s, CH3, 3H), 1.82 (m, CH2 and CH,
3H), 2.14 (m, CH2, 1H), 2.31 (s, CH3, 3H), 2.68 (m, CH, 1H), 3.16 (dd,
J = 4.5, 15 Hz, CH2, 1H), 4.53 (s, OH, 1H), 5.42 (d, J = 4.5 Hz, @CH,
1H), 5.98 (d, J = 2 Hz, ArH, 1H), 6.43 (d, J = 2 Hz, ArH, 1H), 6.98 (d,
J = 7 Hz, ArH, 1H), 7.07 (m, ArH, 2H), 7.15 (t, J = 7.5 Hz, ArH, 1H);
MS: (ESI, Neg) m/z 375.5 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for
White foam (0.66 g, 50.6%). Rf = 0.37 (methylene chloride/hex-
ane, 1:1); 1H NMR (500 MHz, CDCl3): d (ppm) 1.04 (s, CH3, 3H),
1.30 (s, CH3, 3H), 1.51 (m, CH3, 6H), 1.62 (s, CH3, 3H), 1.75 (m,
CH2 and CH, 3H), 2.07 (m, CH2, 1H), 2.61 (m, CH, 1H), 3.09 (m,
CH2, 1H), 4.51 (s, OH, 1H), 5.35 (d, J = 4.5, 15 Hz, @CH, 1H), 5.89
(d, J = 1.5 Hz, ArH, 1H), 6.31 (d, J = 1.5 Hz, ArH, 1H), 7.03 (dt, J = 2,
7.5 Hz, ArH, 1H), 7.07 (m, ArH, 2H), 7.17 (t, J = 2 Hz, ArH, 1H);
MS: (ESI, Neg) m/z 395.9 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for
C26H32O2: 376.2402, experimental 376.2387.
C25H29ClO2: 396.1856, experimental 396.1869.
4.64. 6,6,9-Trimethyl-3-(1-methyl-1-o-tolyl-ethyl)-6a,7,10,10a-
tetrahydro-6H-benzo[c]chromen-1-ol (52)
4.69. 3-[1-(4-Bromo-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (60)
White foam (0.13 g, 27.9%). Rf = 0.33 (methylene chloride/hex-
ane, 1:1); 1H NMR (500 MHz, CDCl3): d (ppm) 1.11 (s, CH3, 3H),
1.37 (s, CH3, 3H), 1.61 (m, CH3, 6H), 1.69 (s, CH3, 3H), 1.82 (m,
CH2 and CH, 3H), 1.88 (s, CH3, 3H), 2.12 (m, CH2, 1H), 2.67 (m,
CH, 1H), 3.16 (dd, J = 4.5 Hz, 15 Hz, CH2, 1H), 4.52 (s, OH, 1H),
5.42 (d, J = 4.5 Hz, @CH, 1H), 5.90 (d, J = 2 Hz, ArH, 1H), 6.38 (d,
J = 2 Hz, ArH, 1H), 7.05 (d, J = 7.5 Hz, ArH, 1H), 7.19 (m, ArH, 2H),
7.50 (dd, J = 1, 7.5 Hz, ArH, 1H); MS: (ESI, Neg) m/z 375.4
[(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for C26H32O2: 376.2402, experimen-
tal 376.2413.
White foam (0.13 g, 25.2%). Rf = 0.34 (methylene chloride/hex-
ane, 40:60); 1H NMR (500 MHz, CDCl3): d (ppm) 1.11 (s, CH3,
3H), 1.37 (s, CH3, 3H), 1.58 (m, CH3, 6H), 1.69 (s, CH3, 3H), 1.82
(m, CH2 and CH, 3H), 2.15 (m, CH2, 1H), 2.68 (m, CH, 1H), 3.17
(dd, J = 4.5, 15 Hz, CH2, 1H), 4.57 (s, OH, 1H), 5.42 (d, J = 4.5 Hz,
@CH, 1H), 5.97 (d, J = 2 Hz, ArH, 1H), 6.37 (d, J = 2 Hz, ArH, 1H),
7.12 (d, J = 8.5 Hz, ArH, 2H), 7.36 (d, J = 8.5 Hz, ArH, 2H); MS: (ESI,
Neg) m/z 440.4 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for C25H29BrO2:
440.1351, experimental 440.1358.

M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
6499
4.70. 3-[1-(3-Bromo-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (61)
J = 7.5 Hz, CH3, 3H), 1.11 (s, CH3, 3H), 1.37(s, CH3, 3H), 1.59 (m,
CH3, 6H), 1.63 (m, CH2, 2H), 1.69 (s, CH3, 3H), 1.83 (m, CH2 and
CH, 3H), 2.14 (m, CH2, 1H), 2.54 (t, J = 7.5 Hz, CH2, 2H), 2.68 (m,
CH, 1H), 3.16 (dd, J = 4.5, 15 Hz, CH2, 1H), 4.54 (s, OH, 1H), 5.42
(d, J = 4.5 Hz, @CH, 1H), 5.98 (d, J = 2 Hz, ArH, 1H), 6.44 (d,
J = 2 Hz, ArH, 1H), 7.06 (d, J = 8.0 Hz, ArH, 2H), 7.15(d, J = 8.5 Hz,
ArH, 2H); MS: (ESI, Neg) m/z 403.2 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd
for C28H36O2: 404.2715, experimental 404.2734.
White foam (0.14 g, 21.3%). Rf = 0.34 (methylene chloride/hex-
ane, 40:60); 1H NMR (500 MHz, CDCl3): d (ppm) 1.12 (s, CH3,
3H), 1.38 (s, CH3, 3H), 1.58 (m, CH3, 6H), 1.69 (s, CH3, 3H),
1.83(m, CH2 and CH, 3H), 2.15 (m, CH2, 1H), 2.68 (m, CH, 1H),
3.16 (dd, J = 4.5, 15 Hz, CH2, 1H), 4.59 (s, OH, 1H), 5.42 (d,
J = 4.5 Hz, @CH, 1H), 5.96 (d, J = 2 Hz, ArH, 1H), 6.38 (d, J = 2 Hz,
ArH, 1H), 7.14 (m, ArH, 2H), 7.30 (m, ArH, 1H), 7.4 (m, ArH, 1H);
MS: (ESI, Neg) m/z 440.4 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for
4.75. 3-[1-(4-Cyano-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (62)
C25H29BrO2: 440.1351, experimental 440.1367.
Cream colored foam (0.08 g, 40.2%). Rf = 0.2 (methylene chlo-
ride/hexane, 50:50); 1H NMR (500 MHz, CDCl3): d (ppm) 1.11 (s,
CH3, 3H), 1.37 (s, CH3, 3H), 1.61 (m, CH3, 6H), 1.69 (s, CH3, 3H),
1.83 (m, CH2 and CH, 3H), 2.14 (m, CH2, 1H), 2.68 (m, CH, 1H),
3.16 (dd, J = 4.5, 15 Hz, CH2, 1H), 4.54 (s, OH, 1H), 5.42 (d,
J = 4.5 Hz, @CH, 1H), 5.98 (d, J = 2 Hz, ArH, 1H), 6.34 (d, J = 2 Hz,
ArH, 1H), 7.35 (d, J = 8.0 Hz, ArH, 2H), 7.55(d, J = 8.5 Hz, ArH, 2H);
MS: (ESI, Neg) m/z 386.2 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for
4.71. 6,6,9-Trimethyl-3-(1-methyl-1-thiophen-2-yl-ethyl)-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (4)
White foam (0.12 g, 21.9%). Rf = 0.37 (methylene chloride/hex-
ane, 1:1); 1H NMR (500 MHz, CDCl3): d (ppm) 1.04 (s, CH3, 3H),
1.30 (s, CH3, 3H), 1.63 (m, CH3, 9H), 1.74 (m, CH2 and CH, 3H),
2.07 (m, CH2, 1H), 2.61 (m, CH, 1H), 3.10 (dd, J = 4.5, 15 Hz, CH2,
1H), 4.50 (s, OH, 1H), 5.35 (d, J = 4.5 Hz, @CH, 1H), 6.04 (d,
J = 2 Hz, ArH, 1H), 6.38 (d, J = 2 Hz, ArH, 1H), 6.76 (dd, J = 1.5 Hz,
3.5 Hz, ArH, 1H), 6.84 (m, ArH, 1H), 7.07 (dd, J = 1 Hz, 5 Hz, ArH,
1H); MS: (ESI, Neg) m/z 367.4 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for
C
26H29NO2: 387.2198, experimental 387.2202.
4.76. 3-[1-(3-Cyano-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (63)
C23H28O2S: 368.1810, experimental 368.1818.
Cream colored foam (0.04 g, 12.1%). Rf = 0.21(methylene chlo-
ride/hexane, 50:50); 1H NMR (500 MHz, CDCl3): d (ppm) 1.11 (s,
CH3, 3H), 1.37(s, CH3, 3H), 1.61 (m, CH3, 6H), 1.69 (s, CH3, 3H),
1.80 (m, CH2 and CH, 3H), 2.16 (m, CH2, 1H), 2.68 (m, CH, 1H),
3.16 (dd, J = 4.5, 15 Hz, CH2, 1H), 4.63 (s, OH, 1H), 5.42 (d,
J = 4.5 Hz, @CH, 1H), 5.96 (d, J = 2 Hz, ArH, 1H), 6.33 (d, J = 2 Hz,
ArH, 1H), 7.35 (t, J = 7.5 Hz, ArH, 1H), 7.46 (m, ArH, 2H), 7.54 (m,
ArH, 1H); MS: (ESI, Neg) m/z 386.2 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd
for C26H29NO2: 387.2198, experimental 387.2203.
4.72. 3-[1-(4-Ethyl-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (54)
Resorcinol 35 (0.4 g, 1.56 mmol), (+)-D
2,8-p-mentha-2,8-dien-1-
ol (49, 0.29 g, 1.95 mmol) and p-toluene sulfonic acid (0.055 g,
0.29 mmol) in benzene (13 mL) were dissolved in benzene
(18.3 mL) and the reaction mixture was placed in a preheated oil
bathat80 °C and allowedtostirfor 6 h. Themixturewas thencooled,
diluted with ether, and washed with bicarbonate, water, and brine.
The ethereal layer was then separated, dried over sodium sulfate,
and evaporated. The product was purified by column chromatogra-
phy using methylene chloride/hexane (40:60) yielding a white foam
(0.13 g, 25.2%). Rf = 0.29 (methylene chloride/hexane, 40:60); 1H
NMR (500 MHz, CDCl3): d (ppm) 1.11 (s, CH3, 3H), 1.22 (t,
J = 7.5 Hz, CH3, 3H), 1.37(s, CH3, 3H), 1.59 (m, CH3, 6H), 1.69 (s, CH3,
3H), 1.82(m, CH2 and CH, 3H), 2.15 (m, CH2, 1H), 2.62 (q, J = 7.5 Hz,
CH2, 2H), 2.68 (m, CH, 1H), 3.17 (dd, J = 4.5, 15 Hz, CH2, 1H), 4.55 (s,
OH, 1H), 5.42 (d, J = 4.5 Hz, @CH, 1H), 5.98 (d, J = 2 Hz, ArH, 1H),
6.43 (d, J = 2 Hz, ArH, 1H), 7.09 (d, J = 8.0 Hz, ArH, 2H), 7.16 (d,
J = 8.5 Hz, ArH, 2H); MS: (ESI, Neg) m/z 389.4 [(Mꢀ1)ꢀ]. HR-EI-MS
m/z calcd for C27H34O2: 390.2559, experimental 390.2570.
4.77. N-{3-[1-(1-hydroxy-6,6,9-trimethyl)-6a,7,10,10a-
tetrahydro-6H-benzo[c]chromen-3-yl-1-methyl-ethyl]-
phenyl}acetamide (64)
Yellow oil (0.1 g, 24.6%). Rf = 0.35 (ether/methylene chloride,
8:92); 1H NMR (500 MHz, CDCl3): d (ppm) 1.11 (s, CH3, 3H), 1.37(s,
CH3, 3H), 1.56 (m, CH3, 6H), 1.67 (s, CH3, 3H), 1.84 (m, CH2 and CH,
3H), 2.03 (s, –NHCOCH3, 3H), 2.16 (m, CH2, 1H), 2.67 (m, CH, 1H),
3.21 (dd, J = 4.5, 15 Hz, CH2, 1H), 5.41 (d, J = 4.5 Hz, @CH, 1H), 5.91
(d, J = 2 Hz, 1H), 6.40 (d, J = 2 Hz, 1H), 7.01 (d, 8 Hz, ArH, 1H), 7.11
(s, ArH, 1H). 7.18 (t, J = 8 Hz, ArH, 1H), 7.32 (br s, OH, 1H), 7.41 (d,
J = 8 Hz, ArH, 1H), 8.09 (s, -NHCOCH3, 1H); MS: (ESI, Neg) m/z
418.5 [(Mꢀ1)ꢀ]. HPLC (retention time: 2.75 min).
4.73. 3-[1-(3,5-Dimethyl-phenyl)-1-methyl-ethyl]-6,6,9-
trimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (53)
4.78. Receptor-binding assays
Cream colored foam (0.20 g, 33.1%). Rf = 0.34 (methylene chlo-
ride/hexane, 40:60); 1H NMR (500 MHz, CDCl3): d (ppm) 1.12 (s,
CH3, 3H), 1.38(s, CH3, 3H), 1.58 (m, CH3, 6H), 1.69 (s, CH3, 3H),
1.80 (m, CH2 and CH, 3H), 2.14 (m, CH2, 1H), 2.27 (s, CH3, 6H),
2.67 (m, CH, 1H), 3.18 (dd, J = 4.5, 15 Hz, CH2, 1H), 4.54 (s, OH
1H), 5.41 (d, J = 4.5 Hz, @CH, 1H), 5.97 (d, J = 2 Hz, ArH, 1H), 6.42
(d, J = 2 Hz, ArH, 1H), 6.81 (s, ArH, 1H), 6.86 (s, ArH, 2H); MS:
(ESI, Neg) m/z 389.3 [(Mꢀ1)ꢀ]. HR-EI-MS m/z calcd for C27H34O2,
390.2559: experimental 390.2560.
Cell membranes from HEK293 cells transfected with the human
CB1 receptor (Bmax: 1.7 pmol/mg protein, Kd for [3H]CP 55,940
binding: 186 pM) and membranes from CHO-K1 cells transfected
with the human CB2 receptor (Bmax: 3.3 pmol/mg protein, Kd for
[3H]CP 55,940 binding: 0.12 nM) were purchased from Perkin-El-
mer Life Sciences, Inc. [3H]CP 55,940 having a specific activity of
120 Ci/mmol was obtained from Perkin-Elmer Life Sciences, Inc.
All other chemicals and reagents were obtained from Sigma–Al-
drich. The assays were carried out in 96-well plates obtained from
Millipore, Inc. fitted with glass fiber filters (hydrophilic, GFC filters)
4.74. 3-[1-(4-Propyl-phenyl)-1-methyl-ethyl]-6,6,9-trimethyl-
having a pore size of 1.2 lm. The filters were soaked with 0.05%
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (55)
polyethyleneimine solution and washed five times with deionized
water prior to carrying out the assays. The filtrations were carried
out on a 96-well vacuum manifold (Millipore Inc.), the filters
punched out with a pipette tip directly into scintillation vials at
Cream colored foam (0.30 g, 50.1%). Rf = 0.35 (methylene chlo-
ride/hexane, 40:60); 1H NMR (500 MHz, CDCl3): d (ppm) 0.94 (t,

6500
M. Krishnamurthy et al. / Bioorg. Med. Chem. 16 (2008) 6489–6500
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Binding protocol for the CB1 receptor: 8 lg of membranes
(20
l
l
L of a 1:8 dilution in incubation buffer) was incubated with
5
L of drug solution (10ꢀ4–10ꢀ12 M) and 5
l
L of 5.4 nM [3H]CP
55,940 in a total volume of 200
lL for 90 min at 30 °C. Non-specific
binding was determined using 10
lM WIN55, 212-2 (Ki = 20 nM).
The membranes were filtered and the filters washed seven times
with 0.2 mL ice-cold incubation buffer, and allowed to air-dry un-
der vacuum.
Binding protocol for the CB2 receptor: 15.3 lg of membranes
(20
l
l
L of a 1:20 dilution in incubation buffer) was incubated with
5
L of drug solution (10ꢀ4–10ꢀ12 M) and 5
l
L of 10 nM [3H]CP
55,940 in a total volume of 200
lL for 90 min at 30 °C. Non-specific
binding was determined using 10
lM WIN55,212-2 (Ki = 4.4 nM).
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Acknowledgment
42. Blázquez, C.; Carracedo, A.; Salazar, M.; Lorente, M.; Egia, A.; González-Feria, L.;
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WethankDr. ValeryKukekov,UniversityofTennesseeHealthSci-
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glioma cell line. This research was supported by the College of Phar-
macy, University of Tennessee Health Science Center.
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