Studies toward the development of antiproliferative neoclerodanes from salvinorin A

Article abstract of DOI:10.1021/np5002048

The success rate for central nervous system (CNS) drug candidates in the clinic is relatively low compared to the industry average across other therapeutic areas. Penetration through the blood-brain barrier (BBB) to reach the therapeutic target is a major obstacle in development. The rapid CNS penetration of salvinorin A has suggested that the neoclerodane nucleus offers an excellent scaffold for developing antiproliferative compounds that enter the CNS. The Liebeskind-Srogl reaction was used as the main carbon-carbon bond-forming step toward the synthesis of quinone-containing salvinorin A analogues. Quinone-containing salvinorin A analogues were shown to have antiproliferative activity against the MCF7 breast cancer cell line, but show no significant activity at the κ-opioid receptors. In an in vitro model of BBB penetration, quinone-containing salvinorin A analogues were shown to passively diffuse across the cell monolayer. The analogues, however, are substrates of P-glycoprotein, and thus further modification of the molecules is needed to reduce the affinity for the efflux transporter.

Full text of DOI:10.1021/np5002048

Article
pubs.acs.org/jnp
Open Access on 07/30/2015
Studies toward the Development of Antiproliferative Neoclerodanes
from Salvinorin A
Tamara Vasiljevik,†,‡ Chad E. Groer,† Kurt Lehner,† Hernan Navarro,§ and Thomas E. Prisinzano*,†
†Department of Medicinal Chemistry, School of Pharmacy, The University of Kansas, Lawrence, Kansas 66045, United States
§Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: The success rate for central nervous system (CNS) drug candidates in
the clinic is relatively low compared to the industry average across other therapeutic
areas. Penetration through the blood−brain barrier (BBB) to reach the therapeutic
target is a major obstacle in development. The rapid CNS penetration of salvinorin A
has suggested that the neoclerodane nucleus offers an excellent scaffold for developing
antiproliferative compounds that enter the CNS. The Liebeskind−Srogl reaction was
used as the main carbon−carbon bond-forming step toward the synthesis of quinone-
containing salvinorin A analogues. Quinone-containing salvinorin A analogues were
shown to have antiproliferative activity against the MCF7 breast cancer cell line, but
show no significant activity at the κ-opioid receptors. In an in vitro model of BBB
penetration, quinone-containing salvinorin A analogues were shown to passively diffuse across the cell monolayer. The analogues,
however, are substrates of P-glycoprotein, and thus further modification of the molecules is needed to reduce the affinity for the
efflux transporter.
n 2010, it was estimated that approximately 688 000 people
third of the top-selling drugs in the world are natural products
Iin the United States are living with primary tumors of the or natural product derivatives.4 Natural products as well as
natural product derived entities have contributed to the
development of various anti-infectives, anticholesteromics,
and antitumor agents, most of which are still actively used in
the clinic.6 Because of the vast structural diversity that nature
has provided, it is safe to say that further investigation into
natural products and their derivatives can provide us with novel
drug entities and biological probes.
Nakijiquinones are marine sesquiterpene quinones that were
isolated from an Okinawan sponge of the Spongiidae family in
the early 1990s.7 These natural products possess three distinct
structural elements: a terpene core, an amino acid side chain,
and a central p-quinoid moiety (examples in Chart 1).7−12
Upon their isolation, this family of quinones was shown to be
the first naturally occurring inhibitors of the Her-2/Neu
receptor tyrosine kinase.8 Cytotoxicity assays in murine
leukemia and human epidermoid carcinoma cells demonstrated
IC50 values for several of the isolated quinones in the range 0.5
to 6 μg/mL, making them compounds of pronounced
interest.7,8,10
brain and central nervous system (CNS), 138 000 of which are
living with malignant tumors and 550 000 with nonmalignant
tumors.1 According to the Central Brain Tumor Registry of the
United States (CBTRUS), approximately 69 720 new cases of
primary tumors and nonmalignant brain and CNS tumors were
expected to be diagnosed in 2013 in the United States. One of
the most prevalent primary tumors in the CNS is gliomas.
Seventy percent of all CNS gliomas are malignant, and this type
of CNS cancer has been shown to be the most frequent and
lethal of the cancers originating in the CNS, with a high rate of
recurrence and mortality.2,3 Several therapeutic strategies are
available; however, they are not efficient for every patient with
recurrent glioblastoma, and prognosis remains uncertain. Thus,
new agents and novel diagnostic tools are necessary for the
improvement of the outcome for glioblastoma patients. The
discovery of novel biological probes could help us gain a better
understanding of this disease state, which could in turn assist in
faster diagnosis of affected patients, providing them with a
greater survival rate.
Recently, some clerodane diterpenes have also demonstrated
cytotoxicity against several cell lines.13−16 Therefore, it is
unclear whether the antiproliferative activity of the nakijiqui-
nones is due to the quinone, the terpene core, or their
combination. The terpene core of these molecules, however,
offers few options for structural manipulation and compound
optimization.
The use of natural products has been documented in many
ancient civilizations that were utilizing natural products in their
ethnomedicinal traditions and spiritual practices. They
represent secondary metabolites that are produced by various
organisms in response to external stimuli such as temperature,
growth, infection, and stress from competition.4 Natural
products have played a major role in the discovery and
development of drugs for the treatment of various human
diseases.5 That natural products are well represented in the
pharmaceutical industry is demonstrated by the fact that one-
Received: March 3, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chart 1. Examples of Nakijiquinones Isolated from an Okinawan Sponge of the Spongiidae Family and Salvinorin A
One clerodane that has not been explored for its cytotoxic
activity is salvinorin A.17−19 Previous studies have shown that
salvinorin A, a potent κ-opioid receptor (KOP) agonist, is able
to cross the blood−brain barrier (BBB) within 1 min of
intravenous or inhaled administration,20−22 although it is a
substrate for P-glycoprotein (P-gp).21 On the basis of its rapid
CNS penetration, the neoclerodane nucleus offers an excellent
scaffold for the development of CNS cancer probes that could
be used as potential diagnostic tools. In addition, the salvinorin
A nucleus offers additional opportunities for optimizing the
activity of such biological probes due to the various chemical
handles already present in the salvinorin A diterpene core.
Therefore, we hypothesized that a combination of the
salvinorin A nucleus and the nakijiquinone p-quinoid moiety
will result in a novel scaffold that is expected to pass the BBB
and have antiproliferative activity (Figure 1). As a first step, we
evaluated a series of quinone-containing analogues similar in
structure to the quinone moiety in the nakijiquinones. It was
envisioned that these modifications would provide an initial
proof of concept before embarking on a more complex
synthetic undertaking. The resulting molecules showed no
activity at the KOP, but exhibited antiproliferative activity
against cancer cell lines. Finally, the salvinorin A quinones are
predicted to rapidly cross the BBB by passive diffusion and are
an important starting point for the development of novel
probes for studying CNS cancers.
Figure 1. Design strategy.
RESULTS AND DISCUSSION
■
Chemistry. The use of the Liebeskind−Srogl reaction as the
main carbon−carbon bond cross-coupling step was based on
the recent observation that the mild coupling conditions of this
B
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a
Scheme 1
a
Reagents and conditions: (a) NaIO4, RuCl3·3H2O, CCl4/CH3CN/H2O; (b) PhSH, CDMT, NMM, THF; (c) CuTC, Pd(dba)2, RB(OH)2,
P(OEt)3, THF; (d) (R)-(+)-2-methyl CBS reagent, borane-dimethyl sulfide complex, toluene, −78 to −30 °C; (e) Et3SiH, BF3·OEt2, CH2Cl2, rt; (f)
CAN, CH3CN, rt.
a
Scheme 2
a
Reagents and conditions: (a) H2O2, H2SO4, MeOH; (b) K2CO3, MeI, acetone; (c) n-BuLi, B(OMe)3, THF, −78 °C to rt; (d) Br2, CH2Cl2, 0 °C;
(e) n-BuLi, trimethyborate, THF, −78 °C.
reaction, which utilize bis(dibenzylideneacetone)palladium(0),
copper(I) thiophene carboxylate, and triethylphosphite at
ambient temperature, are well tolerated by the salvinorin A
scaffold.23 As described previously, salvinorin A was isolated
from Salvia divinorum leaves and subjected to oxidative
degradation utilizing sodium periodate and ruthenium(III)
chloride to yield the corresponding carboxylic acid in 74% yield.
The resulting acid was then esterified with thiophenol to yield
thioester 3 in 60% yield (Scheme 1).21 Thioester 3 was used as
the key intermediate for the synthesis of our targeted quinones.
The reaction of 3 with several different boronic acids yielded
aromatic ketones 4−6 in 48%, 79%, and 90% yield, respectively.
Ketones 4−6 were reduced using Corey−Bakshi−Shibata
(CBS) conditions to yield benzylic alcohols 7−9 in 64−90%
yield. Deoxygenation of 7−9 was accomplished using
trimethylsilane and boron trifluoride diethyl etherate to give
methylene analogues 10−12. Finally, oxidation of 10−12 with
ceric ammonium nitrate (CAN) gave quinones 13−15 in 28−
72% yield.
Several of the necessary boronic acids were prepared
following published reports.24,25 2,3,4,5-Tetramethoxyphenyl-
boronic acid was synthesized following and slightly modifying
conditions proposed by Tremblay and co-workers (Scheme
2).24 Oxidation of 2,3,4-trimethoxybenzaldehyde (16) with
hydrogen peroxide afforded phenol 17 in 84% yield.
Methylation of 17 with iodomethane under basic conditions
gave 1,2,3,4-tetramethoxybenzene (18) in 67% yield. Treat-
ment of 18 with n-butyllithium followed by trimethylborate
C
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gave boronic acid 19 in 52% yield. 2,4,5-Trimethoxyphenylbor-
onic acid was prepared following conditions proposed by
Sutherland and co-workers.25 The reaction of 1,2,4-trimethox-
ybenzene 20 with bromine gave bromobenzene 21 in 96%
yield. Treatment of 21 with n-butyllithium followed by
trimethylborate afforded boronic acid 22 in 38% yield.
Biological Testing. Two natural product scaffolds were
utilized to afford analogues in which the C-12 furanyl moiety of
salvinorin A was replaced with functionalized p-benzoquinone
groups, which were tested for activity at KOP as well as
antiproliferative properties. Analogues 13−15 were subjected to
a calcium mobilization assay, which is a functional assay that
will determine their activity at the KOP (Table 1). Salvinorin A
before a conclusion about an optimal substitution pattern for
the quinone ring can be reached.
Salvinorin A-derived analogues were tested in an in vitro
model of BBB penetration (Table 3). The MDCK-MDR1 cell
Table 3. In Vitro Model of Brain Penetration in MDCK-
a
MDR1 Cells (% Transport )
pretreatment
b
c
cmpd
vehicle
verapamil
caffeine
prazosin
salvinorin A
14
85.9 2.4###
73.0 17.8
6.7 1.8
35.5 2.1***
54.2 3.4**
47.2 11.9**
38.4 5.3**
21.2 3.2
5.0 1.5
15
9.7 3.4
Table 1. KOP Activity Using a Calcium Mobilization Assay
a
% transport equals ratio of basolateral to apical concentration of test
a
b
cmpd
EC50 (nM)
Emax
103
27
b
compound. Data are mean
SEM; n = 3−5. One-way ANOVA:
F(4,13) = 93.33, p < 0.0001; Bonferroni post-test ###p < 0.001 versus all
Salvinorin A
1.7 0.6
2
4
c
13
14
15
2500 900
other compounds after vehicle pretreatment. **p < 0.01, ***p <
0.001 versus vehicle pretreatment, Student’s t test.
c
d
>10 000
ND
c
d
>10 000
ND
a
EC50 = concentration for 50% maximal response; mean SEM, n =
line (Madin-Darby canine kidney cells stably transfected with
MDR1; MDR1, multidrug resistance gene 1) has been useful in
determining whether compounds may passively diffuse across a
cell monolayer and whether they may be substrates for P-gP, an
important efflux transporter in the BBB and gene product of
MDR1.27,28 Caffeine, which is not a P-gp substrate, shows high
transport from the apical to the basolateral side of the cell
monolayer with and without P-gp inhibition by verapamil
(73.0% and 85.9% transport, respectively),29 indicative of
passive diffusion and limited efflux by P-gp. In contrast,
prazosin, a known P-gp substrate,30 shows significantly less
transport from the apical to basolateral side of the membrane.
Pretreatment with verapamil, however, significantly increases
the transport of prazosin to the basolateral side, indicating that
the control compound prazosin passively diffuses across the
monolayer and is actively effluxed by P-gp. Salvinorin A and the
two benzoquinoids 14 and 15 show similar results to prazosin,
suggesting that they passively diffuse across the cell monolayer
and are actively effluxed by P-gp, consistent with salvinorin A
being a known P-gp substrate.21,31 Therefore, these benzoqui-
noids are predicted to rely heavily on passive diffusion to enter
the brain, similar to salvinorin A.20−22 Since the short duration
of action of salvinorin A is due in part to its affinity for the P-gp,
manipulation of the salvinorin A scaffold may be necessary to
reduce efflux, in order to pursue novel cancer therapies and/or
diagnostic probes based on this approach. Alternatively, a
targeting strategy may be utilized to direct the compounds to
cancer cells.
b
2. Emax = % stimulation compared to (−)-U-69,593; mean SEM n =
c
2. EC50 could not be calculated because no activity was observed.
d
ND = not determined.
showed full efficacy and potency (EC50 of 1.7
0.6 nM),
consistent with previous observations.26 Analogues 13−15
showed little to no agonist activity at the KOP, which is
consistent with observed SAR trends, wherein replacing the
furan ring of salvinorin A generally reduces affinity, efficacy, and
potency for the KOP.26
To determine whether the salvinorin A-derived analogues
exhibited antiproliferative activity, their effects on the growth of
the MCF7 breast cancer cell line were tested (Table 2). The
Table 2. Antiproliferative Activity in MCF7 Breast Cancer
Cell Line
a
b
cmpd
GDA
IC50 (μM)
% inhibition
81
85 0.3
0.04 0.02
3
1,4-BQ
2.57 0.81
c
d
salvinorin A
>100
ND
77
13
14
15
6.54 1.57
2.70 0.91
5.84 0.50
8
3
4
86
83
a
IC50 = concentration for 50% growth inhibition. Data are mean
b
SEM, n = 2−5. % growth inhibition compared to DMSO-treated
c
cells. Data are mean SEM, n = 2−5. IC50 could not be calculated
d
because no inhibition occurred. ND = not determined.
The similarity between two natural products, the plant-
derived diterpenoid salvinorin A and the marine sponge derived
nakijiquinone family of natural products, prompted an idea that
a combination of the two scaffolds may yield biological probes
that could have potential in CNS-related cancers. Several
compounds were synthesized with similarities to both scaffolds,
i.e., salvinorin A analogues that possess a C-12 quinone instead
of a furanyl moiety. These benzoquinoids exhibited negligible
activity at the KOP receptors, but exhibited moderate
antiproliferative activity due to the quinonoid moiety. Finally,
the salvinorin A-derived benzoquinoids are predicted to
passively diffuse across a membrane, but are subject to active
efflux by P-gp. These promising results are indicative of the
possibility for use of these salvinorin A-derived compounds as
positive controls geldanamycin (GDA) and 1,4-benzoquinone
(1,4-BQ) inhibited over 80% of cell growth compared to
DMSO-treated controls, with potencies of 0.04 and 2.57 μM,
respectively. Similarly, salvinorin A-derived analogues, 13−15,
show antiproliferative efficacy, inhibiting between 77% and 86%
of cell growth, with potencies of 6.54, 2.70, and 5.84 μM,
respectively. Salvinorin A did not show any detectable
antiproliferative effects, suggesting that the activity of analogues
13−15 is due primarily to the quinone moiety. Similar results
were obtained from an additional breast cancer cell line
(SKBr3; data not shown). Additional information is needed
D
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equiv, 0.21 mmol) in MeCN (7 mL) at ambient temperature. The
reaction was stirred until completion (TLC monitoring), CH2Cl2 (20
mL) was added, and the resulting mixture was washed with H2O and
brine, dried (Na2SO4), and concentrated under reduced pressure. The
resulting residue was purified by flash chromatography on silica gel
using mixtures of EtOAc/n-hexanes.
biological probes in the study of certain brain cancers. This
represents the first report of salvinorin A-derived compounds
that exhibit antiproliferative activity.
EXPERIMENTAL SECTION
■
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-(2,5-
dimethoxybenzoyl)-6a,10b-dimethyl-4,10-dioxododecahydro-
1H-benzo[f ]isochromene-7-carboxylate (4). Compound 4 was
synthesized from compound 3 using general procedure A and
commercially available 2,5-dimethoxyphenylboronic acid to afford
0.250 g (48% yield) of a white solid, mp = 97−100 °C. TLC system:
45% EtOAc/55% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 7.36 (d, J
= 3.2 Hz, 1H), 7.10 (dd, J = 9.0, 3.2 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H),
5.95 (t, J = 8.1 Hz, 1H), 5.13−5.03 (m, 1H), 3.90 (s, 3H), 3.80 (s,
3H), 3.71 (s, 4H), 2.78−2.66 (m, 2H), 2.31−2.22 (m, 2H), 2.19−2.07
(m, 7H), 1.80−1.60 (m, 3H), 1.54 (td, J = 13.4, 3.8 Hz, 1H), 1.42 (s,
3H), 1.40−1.32 (m, 1H), 1.07 (s, 3H); 13C NMR (126 MHz, CDCl3)
δ 201.84, 196.68, 171.61, 169.78, 153.78, 153.42, 124.04, 122.28,
114.45, 113.45, 79.12, 74.86, 65.02, 56.20, 55.84, 53.31, 51.96, 49.45,
42.04, 38.04, 37.88, 35.58, 30.75, 20.64, 18.31, 16.68, 16.05; HRMS
(m/z) [M + Na]+ calcd for C28H34NaO10 553.2050, found 553.2063.
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-[(2,5-
dimethoxyphenyl)(hydroxy)methyl]-6a,10b-dimethyl-4,10-
dioxododecahydro-1H-benzo[f ]isochromene-7-carboxylate
(7). Compound 7 was synthesized from compound 4 using general
procedure B to afford 0.31 g (90% yield) of a white solid, mp = 125−
General Experimental Procedures. Unless otherwise indicated,
all reagents were purchased from commercial sources and were used
without further purification. Melting points were determined on a
Thomas-Hoover capillary melting apparatus. NMR spectra were
recorded on a Bruker DRX-400 with a qnp probe or a Bruker AV-
500 with a cryoprobe using δ values in ppm (TMS as internal
1
standard) and J (Hz) assignments of H resonance coupling. HRMS
data were collected on either an LCT Premier (Waters Corp., Milford,
MA, USA) TOF mass spectrometer or an Agilent 6890 N gas
chromatograph in conjunction with a Quarto Micro GC mass
spectrometer (Micromass Ltd., Manchester, UK). TLC was performed
on 0.25 mm Analtech GHLF silica gel plates using EtOAc/n-hexanes,
in 1:1 v/v ratio, as the solvent unless otherwise noted. Spots on TLC
were visualized by UV (254 or 365 nm), if applicable, and
phosphomolybdic acid in EtOH. Column chromatography was
performed with silica gel (40−63 μm particle size) from Sorbent
Technologies (Atlanta, GA, USA). Analytical HPLC was carried out
on an Agilent 1100 Series capillary HPLC system with diode array
detection at 254 nm on an Agilent Eclipse XDB-C18 column (250 × 10
mm, 5 μm) with isocratic elution in CH3CN/H2O (3:2, v/v) unless
otherwise specified.
1
128 °C. H NMR (500 MHz, CDCl3) δ 6.96 (dd, J = 11.1, 2.5 Hz,
General Procedure A: Liebskind−Srogl Coupling Reaction.
Thioester 3 (1 equiv), appropriate boronic acid (3 equiv),
bis(dibenzylideneacetone)palladium(0) (5 mol %), and copper(I)
thiophene carboxylate (1.5 equiv) were placed in a 100 mL round-
bottom flask and flushed twice with argon. Anhydrous THF was added
immediately followed by triethylphosphite (20 mol %, color change
from red to green with a brown tint). The reaction was allowed to stir
at ambient temperature and upon completion (TLC monitoring) was
diluted with Et2O (30 mL). The organic portion was washed with
saturated NaHCO3 and brine, dried (Na2SO4), and concentrated
under reduced pressure. The resulting residue was purified by flash
chromatography on silica gel using mixtures of EtOAc/n-hexanes.
General Procedure B: CBS Reduction. The corresponding
aromatic ketone (1 equiv) was placed in a round-bottom flask and
flushed three times with argon. A solution of (R)-(+)-2-methyl-CBS-
oxazaborolidine (1 M in toluene, 1 equiv) was added, the temperature
was cooled to −78 °C, and the solution was allowed to stir for 5 min.
A solution of borane-dimethyl sulfide complex (2 M in diethyl ether, 1
equiv) was added dropwise, and the mixture was warmed to −30 °C
and allowed to stir until completion (TLC monitoring). Upon
completion, MeOH (1 mL), H2O (0.5 mL), and Et2O (5 mL) were
added, and the mixture was warmed to room temperature over a
period of 1 h. H2O (20 mL) and Et2O (20 mL) were added, and the
two layers separated. The aqueous layer was washed with two
additional portions of Et2O, the combined organic layers were washed
with brine and dried over Na2SO4, and the solvent was removed under
reduced pressure. The resulting residue was purified by flash
chromatography on silica gel using mixtures of EtOAc/n-hexanes.
General Procedure C: Deoxygenation Procedure. The
corresponding alcohol (1 equiv, 0.21 mmol) was placed in a round-
bottom flask and flushed with argon. CH2Cl2 (10 mL) was added, and
the mixture was cooled to 0 °C. Triethylsilane (2 equiv) was added,
and the mixture was stirred for 2 min. Boron trifluoride diethyl
etherate (2 equiv) was added dropwise, and the resulting mixture was
allowed to stir at 0 °C until completion (TLC monitoring). The
reaction was quenched with H2O, and the mixture was warmed to
ambient temperature. The mixture was extracted with EtOAc (3 × 20
mL), dried (Na2SO4), and concentrated to dryness under reduced
pressure. The resulting residue was purified by flash chromatography
on silica gel using mixtures of EtOAc/n-hexanes.
1H), 6.85−6.70 (m, 2H), 5.27 (dd, J = 5.2, 3.1 Hz, 1H), 5.12 (dd, J =
12.4, 7.5 Hz, 1H), 3.78−3.76 (m, 5H), 3.72 (d, J = 4.7 Hz, 3H), 2.80−
2.70 (m, 2H), 2.35−2.19 (m, 2H), 2.19−2.09 (m, 5H), 2.02 (dd, J =
11.5, 3.3 Hz, 1H), 1.87 (dd, J = 13.2, 6.1 Hz, 1H), 1.75 (dt, J = 10.1,
2.9 Hz, 1H), 1.60−1.52 (m, 3H), 1.30 (s, 3H), 1.06 (d, J = 6.0 Hz,
3H); 13C NMR (126 MHz, CDCl3) δ 202.16, 172.13, 171.67, 169.60,
153.82, 150.10, 127.29, 113.79, 113.21, 111.26, 78.80, 74.92, 70.54,
64.28, 55.92, 55.72, 53.44, 51.94, 50.56, 42.13, 38.10, 34.85, 34.81,
30.91, 20.60, 18.18, 16.15, 15.21; HRMS (m/z) [M + Na]+ calcd for
C28H36NaO10 555.2206; found 555.2217.
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-(2,5-
dimethoxybenzyl)-6a,10b-dimethyl-4,10-dioxododecahydro-
1H-benzo[f ]isochromene-7-carboxylate (10). Compound 10 was
synthesized from compound 7 using general procedure C to afford
0.11 g (36% yield) of a white solid, mp = 96−98 °C. TLC system: 40%
EtOAc/60% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 6.79−6.73 (m,
3H), 5.12 (dd, J = 11.6, 8.4 Hz, 1H), 4.76 (dq, J = 11.3, 5.6 Hz, 1H),
3.76 (s, 3H), 3.75 (s, 3H), 3.72 (s, 3H), 2.91 (dd, J = 5.8, 2.7 Hz, 2H),
2.73 (dd, J = 11.7, 5.2 Hz, 1H), 2.33−2.23 (m, 3H), 2.17 (s, 3H),
2.14−2.06 (m, 2H), 1.92−1.83 (m, 1H), 1.79−1.71 (m, 1H), 1.59−
1.49 (m, 2H), 1.33 (s, 3H), 1.08 (s, 3H); 13C NMR (126 MHz,
CDCl3) δ 202.11, 171.61, 171.58, 169.88, 153.31, 151.83, 125.67,
117.47, 112.57, 111.30, 75.05, 64.20, 55.79, 55.73, 53.58, 51.96, 51.23,
42.25, 42.15, 38.22, 37.22, 35.08, 30.81, 20.60, 18.16, 16.31, 15.18;
HRMS (m/z) [M + Na]+ calcd for C28H36NaO9 539.2257; found
539.2269.
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-[(3,6-
dioxocyclohexa-1,4-dien-1-yl)methyl]-6a,10b-dimethyl-4,10-
dioxododecahydro-1H-benzo[f ]isochromene-7-carboxylate
(13). Compound 13 was synthesized from compound 10 using general
procedure D to afford 0.04 g (39% yield) of a yellow solid, mp = dec at
212−214 °C. 1H NMR (500 MHz, CDCl3) δ 6.80−6.72 (m, 2H), 6.70
(dd, J = 2.3, 1.2 Hz, 1H), 5.14 (dd, J = 11.5, 8.5 Hz, 1H), 4.69 (dddd, J
= 11.9, 9.6, 4.9, 3.2 Hz, 1H), 3.73 (s, 3H), 2.81−2.70 (m, 2H), 2.60−
2.50 (m, 1H), 2.39 (dd, J = 13.3, 4.8 Hz, 1H), 2.34−2.25 (m, 2H),
2.18 (s, 3H), 2.12 (d, J = 3.7 Hz, 1H), 1.97 (dd, J = 11.5, 3.1 Hz, 1H),
1.81−1.74 (m, 1H), 1.65−1.57 (m, 1H), 1.53 (s, 1H), 1.35 (s, 3H),
1.32−1.22 (m, 1H), 1.09 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
201.91, 187.17, 187.10, 171.50, 170.74, 170.00, 143.61, 136.61, 136.59,
135.20, 75.11, 75.03, 64.04, 53.60, 52.00, 51.41, 43.04, 42.09, 38.14,
36.78, 35.26, 30.75, 20.59, 18.12, 16.37, 15.17; HRMS (m/z) [M +
General Procedure D: Quinone Formation. An aqueous
solution of ceric(IV) ammonium nitrate (2−3 equiv, 0.42 mmol, 1.4
mL) was added dropwise to a solution of salvinorin A derivative (1
K]+ calcd for C26H30KO9 525.1527, found 525.1528; HPLC tR
=
6.424; purity = 99.2%.
E
dx.doi.org/10.1021/np5002048 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-6a,10b-di-
methyl-4,10-dioxo-2-(2,3,4,5-tetramethoxybenzoyl)-
dodecahydro-1H-benzo[f ]isochromene-7-carboxylate (5).
Compound 5 was synthesized from compound 3 using general
procedure A and 2,3,4,5-tetramethoxyphenylboronic acid24 to afford
0.57 g (79% yield) of an amorphous solid. TLC system: 45% EtOAc/
55% n-hexanes. 1H NMR (500 MHz, CDCl3) δ 7.06 (s, 1H), 5.88 (t, J
= 8.2 Hz, 1H), 5.13−5.04 (m, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.90 (s,
3H), 3.86 (s, 3H), 3.72 (s, 3H), 2.71 (ddd, J = 12.1, 8.3, 2.5 Hz, 2H),
2.28 (td, J = 9.5, 2.2 Hz, 2H), 2.18−2.17 (m, 3H), 2.14−2.13 (m, 3H),
2.12 (s, 1H), 1.76 (dt, J = 13.4, 3.2 Hz, 1H), 1.72−1.58 (m, 2H), 1.43
(s, 3H). 1.07 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 201.81, 196.30,
171.58, 171.37, 169.80, 149.46, 148.77, 148.54, 146.62, 122.46, 107.23,
78.79, 74.89, 64.92, 62.02, 61.25, 56.16, 53.38, 51.97, 49.51, 42.07,
38.38, 37.91, 35.55, 30.95, 30.71, 20.60, 18.29, 16.51, 16.05; HRMS
(m/z) [M + Na]+ calcd for C30H38NaO12 613.2261, found 613.2289.
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-
(hydroxy(2,3,4,5-tetramethoxyphenyl)methyl)-6a,10b-dimeth-
yl-4,10-dioxododecahydro-1H-benzo[f ]isochromene-7-car-
boxylate (8). Compound 8 was synthesized from compound 5 using
general procedure B to afford 0.78 g (89% yield) of a white solid, mp =
104−107 °C. TLC system: 55% EtOAc/44% n-hexanes. 1H NMR
(500 MHz, CDCl3) δ 6.65 (s, 1H), 5.26 (dd, J = 4.7, 2.9 Hz, 1H), 5.11
(dd, J = 12.2, 7.7 Hz, 1H), 4.73 (ddd, J = 11.5, 5.8, 3.0 Hz, 1H), 3.93
(s, 3H), 3.90 (s, 3H), 3.83 (d, J = 5.1 Hz, 6H), 3.72 (s, 3H), 2.96 (s,
1H), 2.88 (s, 1H), 2.74 (dd, J = 12.7, 4.1 Hz, 1H), 2.65 (d, J = 4.9 Hz,
1H), 2.31−2.22 (m, 2H), 2.17 (s, 2H), 2.12 (s, 3H), 1.97 (td, J = 13.2,
12.6, 7.0 Hz, 2H), 1.81−1.74 (m, 1H), 1.31 (s, 3H), 1.06 (s, 3H); 13C
NMR (126 MHz, CDCl3) δ 202.18, 171.89, 171.55, 169.53, 149.68,
146.51, 144.04, 142.65, 125.54, 104.67, 79.77, 74.90, 70.07, 64.23,
61.07, 61.05, 60.99, 56.33, 53.51, 51.91, 50.64, 42.19, 38.14, 34.81,
34.62, 30.82, 20.50, 18.12, 16.15, 15.14; HRMS (m/z) [M + Na]+
calcd for C30H40NaO12 615.2418, found 615.2435.
synthesized from compound 3 using general procedure A and 2,4,5-
trimethoxyphenylboronic acid25 to afford 1.01 g (90% yield) of a white
solid, mp = 103−106 °C. TLC system: 60% EtOAc/40% n-hexanes.
1H NMR (500 MHz, CDCl3) δ 7.47 (s, 1H), 6.47 (s, 1H), 5.94 (dd, J
= 8.8, 7.3 Hz, 1H), 5.07 (ddd, J = 10.8, 9.3, 1.0 Hz, 1H), 3.96 (s, 3H),
3.94 (s, 3H), 3.87 (s, 3H), 3.70 (s, 3H), 2.78 (dd, J = 13.7, 8.8 Hz,
1H), 2.72−2.65 (m, 1H), 2.30−2.22 (m, 2H), 2.21−2.07 (m, 6H),
1.74 (dt, J = 12.9, 3.1 Hz, 1H), 1.65 (ddd, J = 14.8, 11.6, 3.3 Hz, 1H),
1.57−1.49 (m, 1H), 1.42 (s, 3H), 1.35−1.27 (m, 1H), 1.06 (s, 3H);
13C NMR (126 MHz, CDCl3) δ 201.89, 171.94, 171.63, 169.81,
155.64, 155.37, 143.68, 118.18, 114.77, 112.83, 96.12, 78.93, 74.89,
65.22, 56.30, 56.26, 56.20, 53.30, 51.95, 49.16, 42.07, 38.37, 37.88,
35.62, 30.76, 20.66, 18.35, 16.95, 16.00; HRMS (m/z) [M + K]+ calcd
for C29H36KO11 599.1895, found 599.1903.
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-
(hydroxy(2,4,5-trimethoxyphenyl)methyl)-6a,10b-dimethyl-
4,10-dioxododecahydro-1H-benzo[f ]isochromene-7-carboxy-
late (9). Compound 9 was synthesized from compound 6 using
general procedure B to afford 0.78 g (64% yield) of a white solid, mp =
133−136 °C. TLC system: 60% EtOAc/40% n-hexanes. 1H NMR
(500 MHz, CDCl3) δ 6.92 (s, 1H), 6.49 (s, 1H), 5.28 (dd, J = 5.0, 2.8
Hz, 1H), 5.15−5.05 (m, 1H), 4.75 (ddd, J = 11.6, 5.8, 3.0 Hz, 1H),
3.90 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.71 (s, 3H), 2.74 (dd, J =
12.7, 4.2 Hz, 1H), 2.63 (d, J = 5.2 Hz, 1H), 2.32−2.19 (m, 2H), 2.14
(d, J = 19.1 Hz, 5H), 2.02−1.94 (m, 1H), 1.91 (dd, J = 13.2, 5.8 Hz,
1H), 1.80−1.72 (m, 1H), 1.58−1.50 (m, 2H), 1.30 (s, 3H), 1.05 (s,
3H); 13C NMR (126 MHz, CDCl3) δ 202.19, 172.04, 171.63, 169.55,
150.16, 149.20, 143.24, 117.56, 111.01, 96.89, 79.36, 74.93, 69.90,
64.31, 56.73, 56.12, 56.02, 53.56, 51.94, 50.67, 42.23, 38.20, 34.85,
34.74, 30.89, 20.55, 18.18, 16.18, 15.19; HRMS (m/z) [M + K]+ calcd
for C29H38KO11 601.2051, found 601.2029.
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-6a,10b-di-
methyl-4,10-dioxo-2-(2,4,5-trimethoxybenzyl)dodecahydro-
1H-benzo[f ]isochromene-7-carboxylate (12). Compound 12 was
synthesized from compound 9 using general procedure C to afford
0.42 g (56% yield) of a white solid, mp = 108−111 °C. TLC system:
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-6a,10b-di-
methyl-4,10-dioxo-2-(2,3,4,5-tetramethoxybenzyl)-
dodecahydro-1H-benzo[f ]isochromene-7-carboxylate (11).
Compound 11 was synthesized from compound 8 using general
procedure C to afford 0.42 g (55% yield) of a white solid, mp = 91−94
1
55% EtOAc/45% n-hexanes. H NMR (500 MHz, CDCl3) δ 6.70 (s,
1H), 6.51 (s, 1H), 5.15−5.01 (m, 1H), 4.71 (dd, J = 11.4, 5.5 Hz, 1H),
3.89 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.71 (s, 3H), 2.96−2.80 (m,
2H), 2.76−2.67 (m, 1H), 2.32−2.21 (m, 3H), 2.17 (s, 3H), 2.07 (d, J
= 11.7 Hz, 2H), 1.85−1.71 (m, 2H), 1.57−1.46 (m, 2H), 1.32 (s, 3H),
1.20 (dd, J = 13.5, 11.6 Hz, 1H), 1.07 (s, 3H); 13C NMR (126 MHz,
CDCl3) δ 202.13, 171.65, 171.60, 169.89, 151.77, 148.48, 142.77,
115.84, 115.16, 97.43, 77.47, 75.07, 64.24, 56.62, 56.18, 56.15, 53.60,
51.95, 51.20, 42.18, 41.92, 38.23, 36.07, 35.02, 30.80, 20.60, 18.15,
16.32, 15.20; HRMS (m/z) [M + K]+ calcd for C29H38KO10 585.2102,
found 585.2084.
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-[(4-me-
thoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)methyl]-6a,10b-di-
methyl-4,10-dioxododecahydro-1H-benzo[f ]isochromene-7-
carboxylate (15). Compound 15 was synthesized from compound
12 using general procedure D to afford 0.50 g (72% yield) of a yellow
solid, mp = 127−130 °C. TLC system: 60% EtOAc/40% n-hexanes.
1H NMR (500 MHz, CDCl3) δ 6.63 (t, J = 1.1 Hz, 1H), 5.93 (s, 1H),
1
°C. TLC system: 45% EtOAc/55% n-hexanes. H NMR (500 MHz,
CDCl3) δ 6.45 (s, 1H), 5.12 (dd, J = 11.5, 8.4 Hz, 1H), 4.71 (dq, J =
11.3, 5.5 Hz, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.80 (d, J = 9.0 Hz, 6H),
3.72 (s, 3H), 2.88 (qd, J = 13.8, 5.9 Hz, 2H), 2.77−2.70 (m, 1H),
2.33−2.22 (m, 3H), 2.16 (s, 3H), 2.10 (d, J = 3.9 Hz, 2H), 1.87 (dd, J
= 11.6, 3.1 Hz, 1H), 1.80−1.72 (m, 1H), 1.59−1.49 (m, 2H), 1.34 (s,
3H), 1.30−1.24 (m, 1H), 1.08 (s, 3H); 13C NMR (126 MHz, CDCl3)
δ 202.14, 171.58, 171.49, 169.85, 149.22, 146.94, 145.69, 141.92,
123.97, 108.53, 77.69, 75.05, 64.12, 61.15, 61.06, 61.04, 56.23, 53.57,
51.97, 51.28, 42.28, 42.14, 38.20, 36.72, 35.07, 30.78, 20.58, 18.17,
16.33, 15.18; HRMS (m/z) [M + Na]+ calcd for C30H40NaO11
599.2468, found 599.2459.
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-2-[(4,5-di-
methoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)methyl]-6a,10b-di-
methyl-4,10-dioxododecahydro-1H-benzo[f ]isochromene-7-
carboxylate (14). Compound 14 was synthesized from compound
11 using general procedure D to afford 0.11 g (28% yield) of an
orange solid, mp = 102−104 °C. TLC system: 55% EtOAc/45% n-
hexanes. 1H NMR (500 MHz, CDCl3) δ 6.55−6.48 (m, 1H), 5.14 (dd,
J = 11.7, 8.3 Hz, 1H), 4.68 (dddd, J = 11.7, 9.5, 4.7, 3.2 Hz, 1H), 4.02
(s, 3H), 4.00 (s, 3H), 3.73 (s, 3H), 2.80−2.70 (m, 2H), 2.54 (ddd, J =
14.6, 9.3, 1.1 Hz, 1H), 2.38 (dd, J = 13.3, 4.9 Hz, 1H), 2.34−2.26 (m,
2H), 2.18 (s, 3H), 2.14−2.09 (m, 2H), 2.00−1.93 (m, 1H), 1.81−1.75
(m, 1H), 1.60−1.51 (m, 2H), 1.34 (s, 3H), 1.30−1.21 (m, 1H), 1.09
(s, 3H); 13C NMR (126 MHz, CDCl3) δ 201.92, 183.85, 183.80,
171.51, 170.80, 169.98, 145.04, 144.87, 141.74, 133.24, 75.19, 75.02,
64.01, 61.31, 61.24, 53.58, 52.00, 51.38, 42.97, 42.08, 38.12, 36.43,
35.25, 30.74, 20.59, 18.11, 16.36, 15.17; HRMS (m/z) [M + Na]+
calcd for C28H34NaO11 569.1999, found 569.1960; HPLC tR = 6.959;
purity = 98.1%.
5.14 (dd, J = 11.6, 8.4 Hz, 1H), 4.70 (dddd, J = 14.6, 8.6, 4.8, 3.3 Hz,
1H), 3.83 (s, 3H), 3.73 (s, 3H), 2.83−2.71 (m, 2H), 2.56 (ddd, J =
14.4, 9.2, 1.0 Hz, 1H), 2.38 (dd, J = 13.3, 4.9 Hz, 1H), 2.34−2.28 (m,
2H), 2.18 (s, 4H), 2.11 (d, J = 4.1 Hz, 2H), 2.00−1.94 (m, 1H), 1.82−
1.75 (m, 1H), 1.54 (d, J = 12.9 Hz, 1H), 1.35 (s, 3H), 1.27 (t, J = 12.5
Hz, 1H), 1.09 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 201.90,
187.00, 181.77, 171.52, 170.77, 169.98, 158.75, 144.44, 133.15, 107.66,
75.35, 75.02, 64.04, 56.31, 53.59, 52.00, 51.40, 43.01, 42.08, 38.14,
36.65, 35.26, 30.76, 20.60, 18.11, 16.37, 15.17; HRMS (m/z) [M +
K]+ calcd for C27H32KO10 555.1633, found 555.1678; HPLC tR
=
6.049; purity = 95.4%.
Calcium Mobilization Assay. The assay was performed as
described previously.23,32 Briefly, CHO cells stably expressing KOP R-
Gαq16 were maintained in F-12 media with 10% FBS, 1% penicillin
and streptomycin, and 0.2% normocin (Life Technologies, Carlsbad,
CA, USA) at 37 °C and 5% CO2. Cells were plated at 30 000 cells/well
(2S,4aR,6aR,7R,9S,10aS,10bR)-Methyl 9-Acetoxy-6a,10b-di-
methyl-4,10-dioxo-2-(2,4,5-trimethoxybenzoyl)dodecahydro-
1H-benzo[f ]isochromene-7-carboxylate (6). Compound 6 was
F
dx.doi.org/10.1021/np5002048 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
in the same media in a black Costar 96-well optical bottom plate and
incubated at 37 °C overnight. The media was replaced with fluorescent
calcium dye (Calcium 5 dye, Molecular Devices, Sunnyvale, CA, USA)
in 225 μL of assay buffer (HBSS buffer containing 20 mM HEPES,
0.25% BSA, 1% DSMO, and 10 μM probenecid (Sigma, St. Louis,
MO, USA)), and cells were incubated for 1 h at 37 °C. Stock solutions
(10 mM) were prepared for all compounds in DMSO. Stocks were
used to make fresh serial dilutions in DMSO for each experiment and
diluted to 10× in assay buffer. Cells were stimulated with 25 μL of 10×
compounds using the Flexstation 3 plate-reader, and the change in
fluorescence over baseline was recorded for 60 s. Maximum change
over baseline was determined from two independent experiments
performed on different days (n = 2). For each experiment, 10-point
dose−response curves spanning at least seven log units were generated
in duplicate (two wells per dose per compound) for each compound.
The dose range for salvinorin A was chosen based on literature
precedent26 and to provide clear lower and upper asymptotes for the
semilog plots of the dose−response curves. The dose range for test
compounds began at the highest concentration allowed by solubility
(10 μM). Data from each experiment were analyzed separately using
nonlinear regression (GraphPad Prism 5.0, San Diego, CA, USA) to
determine the potency (EC50) and efficacy (Emax) values.
not shown).30,37 Transepithelial electrical resistance was randomly
monitored in monolayers prior to the assay to ensure development of
resistance and monolayer integrity using chopstick-style electrodes
(EVOM, World Precision Instruments). Each study was conducted
with one or two independent samples of each compound and controls.
Studies were repeated two or three times, and data were averaged to
produce mean and SEM; therefore, each value represents data from n
= 3−5 independent measurements.
LC/MS Methods. Sample preparation: 20 μL of media was mixed
with 180 μL of 0.1% HCO2H in MeCN. Samples were centrifuged,
and 100 μL of supernatant was mixed with 300 μL of 50:50 MeOH/
H2O in 96-well plates. Sample analysis was conducted in positive
electrospray mode using an Applied Biosystems API 5000 triple
quadrupole mass spectrometer (Framingham, MA, USA) interfaced to
a Waters Acquity UPLC system (Waters Corporation, Milford, MA,
USA). Chromatography was accomplished using a Phenomenex Luna
C18 column (50 mm × 2 mm i.d., 5 μm particle size) fitted with a
guard cartridge. Injection volumes were 20 μL. Two mobile phase
solutions were used: (A) 0.1% HCO2H with 10 mM ammonium
formate in H2O; (B) 0.1% HCO2H with 10 mM ammonium formate
in MeOH. The flow rate was 0.5 mL/min. Initial chromatographic
conditions were 90% A held for 1 min, decreasing to 5% A over 2.5
min, and held for 1 min before returning to initial conditions.
Compound 14 was monitored with a multiple reaction monitoring
(MRM) transition of 547.18 → 487.00 with DP = 186, CE = 17, and
CXP = 20. Compound 15 was monitored with an MRM transition of
517.35 → 457.20 with DP = 201, CE = 17, and CXP = 18. Salvinorin
A was monitored with an MRM transition of 433.19 → 373.30 with
DP = 141, CE = 15, and CXP = 30. Prazosin was monitored with an
MRM transition of 384.19 → 247.00 with DP = 186, CE = 39, and
CXP = 18. Caffeine was monitored with an MRM transition of 195.32
→ 138.00 with DP = 86, CE = 25, and CXP = 22. Mass spectrometer
parameters were as follows: CUR = 10, GS1 = 40, GS2 = 60, IS =
2000, TEM = 650 °C, CAD = 5.
Antiproliferation Assay.33 MCF7 and SKBr3 cells were
maintained in Advanced DMEM/F12 (1:1; Life Technologies)
supplemented with L-glutamine (2 mM), streptomycin (500 μg/
mL), penicillin (100 units/mL), and 10% FBS at 37 °C and 5% CO2.
Cells were grown to confluence, seeded (2000 cells/well, 100 μL total
media) in clear, flat-bottom 96-well plates, and allowed to attach
overnight. Stock solutions (10 mM) were prepared for all compounds
in DMSO. Stocks were used to make fresh dilutions in DMSO for each
experiment. Six-point dose−response curves were generated in
duplicate (two wells per dose per compound) using the following
dose ranges: GDA, 1 nM to 1 μM; all test compounds, 100 nM to 100
μM. Compounds were added (1% DMSO final concentration), and
cells were incubated at 37 °C for 72 h. After 72 h, cell growth was
determined using an MTS/PMS cell proliferation kit (Promega) per
the manufacturer’s instructions. Cells incubated in 1% DMSO were
used as controls (i.e., 0% inhibition), and the relative growth of cells
incubated with each compound concentration was normalized to
DMSO-treated controls. Data from at least two independent
experiments performed on different days (n ≥ 2) were analyzed by
nonlinear regression using GraphPad Prism 5.0 to generate IC50 and %
inhibition values.
In Vitro Predictive Model of BBB Penetration. MDCK-MDR1
cells (Madin-Darby canine kidney cells stably transfected with MDR1,
which codes for P-gp) obtained from The Netherlands Cancer
Institute were maintained in DMEM/F12 (Life Technologies) media
with 10% FBS and antibiotics at 37 °C and 5% CO2. MDCK-MDR1
cells were plated (40 000 cells/well) on semipermeable membranes in
a 24-well Transwell system with a 0.4 μm membrane pore size, and
media was changed the next day. Cells were grown to confluence
(generally 3 to 4 days after plating), and growth media was replaced
with transport media (Hanks’ balanced salt solution supplemented
with 25 mM glucose and 25 mM HEPES) containing vehicle or
verapamil (100 μM) for 15 min at 37 °C. Transport media was
replaced with fresh transport media containing 10 μM test compound
(and vehicle or verapamil) on the apical chamber and incubated at 37
°C for 60 min. The concentration of DMSO was maintained at 1%
throughout the experiment to facilitate solubility and minimize
aggregation. Samples (20 μL) from the apical and basolateral
chambers were collected and analyzed by LC/MS for the presence
of test compound (LC/MS details below). The difference in peak
areas between the apical and basolateral sides with account of volume
differences in each side was used to determine transport across a
monolayer.34−36 Caffeine, which penetrates the BBB efficiently, was
used as a positive control for membrane permeability. Prazosin was
used as a control for P-gp-mediated transport, and 5-(4-chlorophenyl)-
1-(2,4-dichlorophenyl)-N-{[4-(methanesulfonamidomethyl)-
cyclohexyl]methyl}-4-methylpyrazole-3-carboxamide was used as a
negative control for transport across MDCK-MDR1 monolayers (data
ASSOCIATED CONTENT
■
S
* Supporting Information
Supporting data including HPLC, H NMR, and 13C NMR
1
spectra of compounds 13−15. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 785-864-3267. Fax: 785-864-5326. E-mail: prisinza@
ku.edu.
Present Address
‡Department of Chemistry and Biochemistry, University of
Notre Dame, Notre Dame, Indiana 46556, United States
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank J. Hall and Dr. B. S. J. Blagg for their
technical assistance. This work was supported in part by a grant
from the National Institute on Drug Abuse (DA018151).
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