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Bioactive Natural, Biocatalytic, and Semisynthetic Tobacco Cembranoids

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The two major Nicotiana tabacum tobacco cembranoids, (1 S,2 E,4 R,6 R,7 E,11 E)-2,7,11-cembratriene-4,6-diol (1) and its C-4 epimer, exhibit a wide range of interesting biological activities. Although the tumorigenesis inhibition activity of tobacco cembranoids have been known since the mid 1980's, only a limited number of investigations have targeted their optimization and structure-activity relationship. This study reports the isolation of the new (1 S,2 E,4 S,6 E,8 S,11 E)-2,6,11-cembratriene-8- O-methyl-4,8-diol (3) and the known (1 S,2 E,4 R,6 R,7 E,11 E)-2,7,11-cembratriene-4- O-methyl-4,6-diol (2) from fresh N. tabacum leaves. Cembranoid 2 showed good anti-migratory activity against prostate cancer cell lines, and was therefore subjected to microbial transformation and semisynthetic optimization studies. Biotransformation of 2 using the fungal strains Cunninghamella NRRL 5695 and Mucor ramannianus ATCC 9628 afforded new ( 4 and 5) and known ( 6 and 7) metabolites. Semisynthetic esterification, oxidation, epoxidation, and reaction with Lawesson's reagent afforded the new products 8- 14. Cembranoid 2 and its epoxidation product 9 showed potent anti-migratory activities against the highly metastatic human prostate cancer cell lines PC-3M-CT+ (spheroid disaggregation assay) and PC-3 (wound-healing assay).
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Introduction
!
Leaf and flower cuticular wax of most Nicotiana
species contain high amounts of cembranoids [1,
2]. Cembranoids are natural diterpenes with a
14-membered macrocyclic skeleton, an isopropyl
functionality at C-1 , and three symmetrically dis-
posed methyl groups at C- 4, C-8 , and C- 1 2 , com-
monly found in the plant and animal kingdoms
[3, 4]. Fermentation and biodegradation of cem-
branoids lead to formation of flavor compounds
having from 8 to 19 carbons [15]. The biosyn-
thesis of the cembrane skeleton originates from
the cyclization of geranylgeranyl pyrophosphate.
Cyclization via the antipode I and II pathways
leads to the 4Rand 4Sseries, respectively [6]. The
tobacco cembranoids (1S,2E,4R,6R,7E,11E)-
2,7,11-cembratriene-4,6-diol 1(l
"Fig. 1) and its
C-4 epimer, (1S,2E,4S,6R,7E,11E)-2,7,11-cembra-
triene-4,6-diol, were among the earliest reported
cembranoids [14]. The structure and absolute
configuration of 1was based on Xray crystallog-
raphy [1]. 4-O-Methyl ether cembranoids are
mainly found in Burley and Oriental tobacco,
while Virginia tobacco usually contains only mi-
nor amounts. This variation may be attributed to
both genetic and curing method differences.
(1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4-O-
methyl-4,6-diol (2)was reported as a constituent
of aged tobacco (l
"Fig. 1) [5].
Cembranoid 1and its C- 4 epimer are known
plant-growth and fungal spore germination in-
hibitors and insecticides, aldose reductase and
prostaglandin inhibitors [1,4, 7, 8], and anticancer
agents [9]. Recently, 1showed antiproliferative
and anti-invasive activity against the highly
metastatic human prostate PC-3M cancer cell line
[10]. Compound 1(1 µM) decreased the potency
of nicotine, i.e., increased the IC50 of nicotine, to
induce motility decrease in planarian, while it
had no effect on carbamylcholine [11]. Pretreat-
ment with the same dose of 1significantly re-
duced the withdrawal-like distress behaviors and
effects in planaria worm exposed to a 100 µM
dose of nicotine for 24 hours, which highlighted
the potential of the tobacco (4R)-cembranoids for
Abstract
!
The two major Nicotiana tabacum tobacco cem-
branoids, (1S,2E,4R,6R,7E,11E)-2,7,11-cembratri-
ene-4,6-diol (1) and its C-4 epimer, exhibit a wide
range of interesting biological activities. Although
the tumorigenesis inhibition activity of tobacco
cembranoids have been known since the mid
1980s, only a limited number of investigations
have targeted their optimization and structure-
activity relationship. This study reports the isola-
tion of the new (1S,2E,4S,6E,8S,11E)-2,6,11-cem-
bratriene-8-O-methyl-4,8-diol (3) and the known
(1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4-O-
methyl-4,6-diol (2) from fresh N. tabacum leaves.
Cembranoid 2showed good anti-migratory activ-
ity against prostate cancer cell lines, and was
therefore subjected to microbial transformation
and semisynthetic optimization studies. Biotrans-
formation of 2using the fungal strains Cunning-
hamella NRRL 5695 and Mucor ramannianus
ATCC 9628 afforded new (4and 5)andknown (6
and 7) metabolites. Semisynthetic esterification,
oxidation, epoxidation, and reaction with Lawes-
sonʼs reagent afforded the new products 814.
Cembranoid 2and its epoxidation product 9
showed potent anti-migratory activities against
the highly metastatic human prostate cancer cell
lines PC-3M-CT+(spheroid disaggregation assay)
and PC-3 (wound-healing assay).
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Bioactive Natural, Biocatalytic,
and Semisynthetic Tobacco Cembranoids
Authors Hany N. Baraka1,2, Mohammad A. Khanfar 1, John C. Williams 1, Emad M. El-Giar3, Khalid A. El Sayed1
Affiliations 1Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe,
Monroe, Louisiana, USA
2Current address: Department of Pharmacognosy, College of Pharmacy, Mansoura University, Mansoura, Egypt
3Department of Chemistry, University of Louisiana at Monroe, Monroe, Louisiana, USA
Key words
l
"antimigratory
l
"microbial transformation
l
"Nicotiana tabacum
l
"Solanaceae
l
"prostate cancer
l
"spheroid disaggregation
l
"woundhealing
received July 10, 2010
revised Sept. 19, 2010
accepted October 5, 2010
Bibliography
DOI http://dx.doi.org/
10.1055/s-0030-1250478
Published online November 3,
2010
Planta Med 2011; 77: 467476
© Georg Thieme Verl ag KG
Stuttgart · New Yo rk ·
ISSN 00320943
Correspondence
Dr. Khalid A. El Sayed
Department of Basic
Pharmaceutical Sciences
College of Pharmacy
University of Louisiana
at Monroe
1800 Bienville Drive
Monroe, LA 71201
USA
Phone: + 13183421725
Fax: +13183421737
elsayed@ulm.edu
467
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
treatment and investigation of tobacco abuse and dependence
[11].
Biocatalysis is the use of growing microbial cultures, their en-
zymes, or immobilized cells to optimize the bioactivity of a start-
ing material through induction of stereospecific reactions [10,
1214]. Microbial transformation of tobacco cembranoids gener-
ated several flavor-enhancers that were added to tobacco during
its fermentation [2, 4, 10, 15]. Biotransformation of 1using several
symbiotic marine bacteria resulted in hydroxylated analogues
which maintained similar anti-invasive activity compared to the
parent compound, but lacked cytotoxicity [10]. Biotransforma-
tion of (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol using
Mucor ramannianus ATCC 9628 and Cunninghamella elegans
ATCC 7929 afforded its (10S,11S)-epoxy analogue as the main
metabolite while biotransformation of (1S,2E,4S,6R,7E,11E)-
2,7,11-cembratriene-6-O-acetyl-4,6-diol using the marine sym-
biotic Bacillus megaterium strain MO31 afforded its (10R)-hy-
droxy analogue [16].
Several semisynthetic reactions have been reported for tobacco
cembranoids, including epoxidation of Δ7,8 and Δ11,12, allylic oxi-
dations around Δ11,12,oxidation of the C- 6 secondary alcohol to a
ketone group using CrO3/pyridine, and acid-catalyzed rearrange-
ments [17]. Only a limited number of structure-activity relation-
ship studies of tobacco cembranoids have been done with the
purpose to explore their anticancer activity [15, 17]. Acylation of
1and its C-4 epimer at C- 6 using acetic, propionic, butyric, valeric,
and benzoic acid anhydrides reduced the Epstein-Barr virus anti-
gen (EBA) formation inhibitory activity by increasing the IC50 val-
ues and cytotoxicity, suggesting the importance of the C- 6 hy-
droxy [15,18]. Cytotoxicity increase was parallel to the increase
of the acyl carbon number. Oxidation of the C-6 alcohol to the β-
hydroxyketones slightly reduced the activity. This may suggest
the importance of a hydrogen bond-forming pharmacophore at
C-6 . Saturation of macrocyclic double bonds by catalytic re-
duction decreased EBA induction, indicating the significance of
macrocyclic double bonds for EBA induction inhibitory activity
[18]. (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-11S,12S-epoxy-
4,6-diol was devoid of activity, suggesting the importance of
Δ11,12 [18]. This study adds insights to establish a preliminary
structure-activity relationship for the anti-migratory activity of
tobacco (4R)-cembranoids through the use of microbial and
semisynthetic optimizations of 2.
Materials and Methods
!
General experimental procedures
Optical rotations were measured on a Rudolph Research Analyti-
cal Autopol III polarimeter. IR spectra were recorded on a Varian
800 FTIR. The NMR spectra were recorded on a JEOL ECLIPSE-
400 NMR spectrometer (1H:400MHz, 13C:100 MHz) using CDCl3
as solvent and TMS as the internal standard. HRMS experiments
were conducted at the University of Michigan on a Micromass
LCT spectrometer. TLC analyses were carried out on precoated sil-
ica gel 60 F254 500 µm TLC plates, using n-hexane-EtOAc or
CHCl3-MeOH solvent systems. Silica gel 60 (Natland International
Corporation, 63200 µm) or C- 1 8 reversed-phase silica gel (Ba-
kerbond octadecyl 40 µm; Mallinckrodt Baker, Inc.) were used
for column chromatography (CC). Generally, 1:100ratios of mix-
tures to be chromatographed versus the stationary phase were
used in all liquid chromatographic purifications. About 20
500 mL fractions were collected during early fractionation and
0.5-mL fractions in the final purifications.
Extraction and isolation
Fresh tobacco leaf powder (27.2 kg) (Custom Blends, Blend #28,
Batch Number TPTN15 001) containing Virginia, Oriental, and
Burley tobacco (1 : 1 : 1), was extracted three times with ethanol
(3 × 130 L) in percolators at room temperature. Taxonomic identi-
fication was provided by the vendor. The alcoholic extract was
concentrated, dr ied (1.05 kg), and subjected to vacuum liquid
chromatography (VLC) on silica gel (2 kg), and 500-mL fractions
were collected using a gradient n-hexane-EtOAc [10, 16]. Frac-
tions 69were combined to afford a crude cembranoid-contain-
ing fraction (100.0 g), which was further chromatographed on sil-
ica gel 60 (1 kg), and 500-mL fractions were collected using gra-
dient n-hexane-EtOAc, yielding fractions AC. Compound 3
(300 mg) was obtained from fraction A by repeated chromatogra-
phy on silica (Si) gel 60 (1 kg), and 100-mL fractions were col-
lected using a gradient n-hexane-EtOAc and CHCl3-MeOH. Com-
pound 2(1.60 g) was obtained from fraction C by flash chroma-
tography over Si gel 60 (1 kg), isocratic elution with CHCl3-MeOH
(9.5 : 0.5).
Microbial transformations
Nine growing fungal cultures were screened for their ability to
biotransform 2, including Cunninghamella verticillata ATCC
8986, C. elegans ATCC 7929, Lipomyces lipofer ATCC 32 371, Lipo-
myces starkeyi ATCC 58680, Botyris allii ATCC 9435, Mucor ra-
Fig. 1 The structure of natural, biocatalytic, and
semisynthetic tobacco cembranoids.
468
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
mannianus ATCC 9228, Rhizopus arrhizus ATCC 11145, Strepto-
myces griseus ATCC 19968, and Cunninghamella sp. NRRL 5695.
M. ramannianus ATCC 9228 and C. sp. NRRL 5695 were selected
for biotransformation scale-up [19]. Each organism was inocu-
lated in twenty 1-L flasks containing 250 mL compound me-
dium-α(20 g glucose and 5 g each of peptone, yeast extract, NaCl,
and Na2PO4in 1 L distilled H2O). After 48 h, a solution of 15 mg of
2in EtOH (2 mL) was added to each flask.After 8 days, the growth
medium was filtered, extracted with EtOAc (4 × 1 L), and the
EtOAc layer concentrated. Residue (1.95 g) obtained from C.sp.
NRRL 5695 was purified using Sephadex LH20 (CHCl3-MeOH,
9.5 : 0.5) and C- 1 8 silica gel (MeOHH2O, 1:2), affording 4(8 mg,
Rf0.33, CHCl3-MeOH, 9.5 : 0.5) and 5(10 mg, Rf0.36, CHCl3-
MeOH, 9.5 : 0.5). Similarly, M. ramannianus ATCC 9228 fermenta-
tion afforded 4(12 mg, Rf0.33, CHCl3-MeOH, 9.5 : 0.5), 6(11 mg,
Rf0.27, CHCl3-MeOH, 9.5 : 0.5), and 7(9 mg, Rf0.22, CHCl3-MeOH,
9.5 : 0.5).
Metal-catalyzed epoxidation of 2
About 50 mg of 2was dissolved in 2.5 mL toluene containing a
catalytic amount of vanadyl acetylacetonate, and the reaction
mixture was kept at 0°Cfor 30 min. A solution of 50 µL of t-butyl
hydroperoxide in 1.5 mL toluene was carefully added, and the re-
action was stirred for 3 h at room temperature (rt). The reaction
was quenched with ice water and extracted with EtOAc
(3 × 10 mL). The organic phase was washed with a saturated
aqueous solution of FeSO4and H2O and dried under reduced
pressure. The residue was subjected to Si gel 60 using n-hexane-
EtOAc, gradient elution, to afford 8(11 mg, Rf0.45, CHCl3-MeOH,
9.5 : 0.5) and 9(19 mg, Rf0.31, CHCl3-MeOH, 9.5 : 0.5).
Chromic acid oxidation of 2
About 200 mg of 2was dissolved in 10 mL of pyridine and mixed
with 100 mg of CrO3. The mixture was stirred at rt for 48 h. Water
(20 mL) was added and the mixture was extracted with EtOAc
(3 × 10 mL). The organic layer was dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was frac-
tionated over Si gel 60 using n-hexane-EtOAc gradient elution to
afford 12 (10 mg, Rf0.54, CHCl3-MeOH, 9.8 : 0.2), 11 (5 mg, Rf0.50,
CHCl3-MeOH 9.8 : 0.2), and 10 (25 mg, Rf0.45, CHCl3-MeOH,
9.8 : 0.2).
Reaction of 10 with Lawessonʼs reagent
A solution of 20 mg of 10 in 5 mL toluene was treated with 24 mg
of Lawessonʼs reagent and the reaction mixture was refluxed for
6 h. The reaction was monitored by TLC (n-hexane-EtOAc,
7.5 : 2.5). The react ion was stopped by adding cold H2O, extracted
with CHCl3(3 × 10 mL) and the organic layer evaporated under
vacuum. The residue was subjected to Si gel 60 CC, using n-hex-
ane-EtOAc gradient elution to afford 13 (7 mg, Rf0.54, hexane-
EtOAc, 1:1).
Epoxidation of 6-O-acetyl analogue of 2
A solution of 50 mg of 2in 0.5 mL pyridine was treated with 1 mL
acetic anhydride and the reaction mixture was stirred overnight.
The reaction mixture was quenched with 5 mL H2O and extracted
with CHCl3(3 × 10 mL). The organic layer was evaporated and the
residue was subjected to Si gel CC using CHCl3-MeOH gradient
elution to afford the expected 6-O-acetate analogue (35 mg). This
acetate was treated with a solution of 3-chloroperoxybenzoic ac-
id (m-CPBA, 40 mg) in 2 mL CH2Cl2in ice bath. The solution was
stirred at C for 1 h and monitored by TLC (CHCl3-MeOH 9:1).
The reaction was stopped using 5 mL 10% aqueous Na2SO3. The
organic and aqueous layers were separated. The aqueous layer
was twice extracted with 5 mL CHCl3. Combined organic layers
were washed with saturated aqueous NaHCO3, brine solution,
and H2O, dried over anhydrous Na2SO4and evaporated under re-
duced pressure. The residue was purified over Si gel 60 using
CHCl3-MeOH gradient elution to afford 14 (15 mg, Rf0.35,
CHCl3-MeOH, 9.5 : 0.5).
Spheroid disaggregation assay
Spheroids were prepared from a single cell suspension of pros-
tate cancer cell lines [20, 21]. In brief, 5 × 104/mL cells in RPMI
1640 serum-free medium were placed on 96-well low-attach-
ment tissue culture plates. The plates were rocked on a gyrorota-
tory shaker in a CO2incubator at 37°C for 2 days, at the end of
which spheroids measuring 150300 µm in diameter (~ 4 × 104
cells/spheroid) were formed. A single spheroid was placed in the
center of each well of an extracellular matrix (ECM)-coated 24-
well microplate in 200 mL of serum-free medium. It was previ-
ously determined that 1 h is an appropriate time for spheroids
to begin adhering to an ECM [20, 21]. Thus t = 0 was set as 1 h
from initial plating, so that if the plate was not disturbed, the
spheroids would not move from their location at the time of plat-
ing. Spheroids were photographed digitally at t = 0, cultured at
37 °C for 48 h and re-photographed. The spheroids were fixed,
stained with Diff-Quik(Dade Behring) and examined under
light microscopy. The diameter of the area covered with cells mi-
grated from each spheroid center was measured in a microscope
calibrated with a stage and ocular micrometer. The radial dis-
tance of migration was calculated after subtraction of the mean
initial spheroidal diameter at t = 0. Val ue s shown represent the
average percent increase in the surface area of the spheroids.
Wound-healing assay (WHA)
PC-3 prostate cancer cells were cultured in RPMI 1640 medium
containing 10 mM HEPES, 4 mM L-glutamine, 10 % fetal bovine
serum, penicillin (100 IU/mL), and st reptomycin (50 µg/mL), and
grown in a 5% CO2atmosphere at 37°C. Cells were plated onto
sterile 24-well and allowed to recover for a conf luent cell mono-
layer formed in each well (> 90 % confluence). Wounds were in-
flicted to each cell monolayer using a sterile 200 µL pipette tip.
Media were removed, cell monolayers were washed once with
PBS, and fresh media containing test compounds were added to
each well to final concentrations of 50 µM. Test samples were
prepared in DMSO at different concentrations and added to the
plates, each in triplicate using the known potent anti-metastatic
4-mercaptoethyl-phenylmethylene hydantoin [22, 23] as a posi-
tive control at 50 µM and DMSO as a negative vehicle control.
The incubation was carried out for 24 h under serum-starved
conditions, after which media was removed and cells were fixed
and stained using Diff-Quikstaining. The number of cells mi-
grated on the scratched wound were counted under the micro-
scope in three or more randomly selected fields (magnification:
400 ×). Final results were expressed as mean ± SEM per 400 ×
field. All treatments, including the controls, were documented
photographically.
Isolates
(1S,2E,4S,6E,8S,11E)-2,6,11-cembratriene-8-O-methyl-4,8-diol (3):
Colorless oil; [α]
D25
+ 36.8 (c= 2.0, CHCl3); IR (CHCl3)νmax: 3596,
2946, 2856, 1462, 1373, 1098, 968 cm1; HREIMS: m/z=
469
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
320.2699 [M]+(calcd. for C21H36O2, 320.2715); 1Hand13CNMR:
see l
"Table 1.
(1S,2E,4R,6R,7E,11E,13R)-2,7,11-cembratriene-4-O-methyl-4,6,13-
triol (4): Colorless oil; [α]
D25
+38.3 (c= 0.24, CHCl3); IR (CHCl3)
νmax: 3597, 3428, 2929, 2856, 1462, 1375, 979 cm1; HRESIMS:
m/z= 359.2511 [M + Na]+(calcd. for C21H36O3Na+, 359.2562); 1H
and 13CNMR data: see l
"Table 1.
(1S,2E,4R,6R,7E,11S,12S)-2,7-cembradiene-4-O-methyl-11,12-ep-
oxy-4,8-diol (5): Colorless oil; [α]
D25
+ 53.3 (c= 0.06, CHCl3); IR
(neat) νmax: 3597, 2992, 2930, 2873, 1069, 981 cm1; HRESIMS
m/z= 359.2562 [M + Na]+(calcd. for C21H36O3Na+, 359.2562); 1H
and 13CNMR data: see l
"Table 1.
(1S,2E,4R,6R,7R,8R,11E)-2,11-cembradiene-4-O-methyl-7,8-ep-
oxy-4,8-diol (8): Colorless oil; [α]
D25
+8.5(c= 0.20, CHCl3); IR (neat)
νmax: 3533, 2987, 2930, 2872, 1462, 1374, 1074, 983 cm1;HRE-
SIMS: m/z= 359.2550 [M + Na]+(calcd. for C21H36O3Na+,
359.2562); 1Hand13CNMR data: see l
"Table 2.
(1S,2E,4R,6R,7S,8S,11E)-7,8-epoxy-2,11-cembradiene-4-O-methyl-
4,8-diol (9): Colorless oil; [α]
D25
+ 44.4 (c= 0.16, CHCl3); IR (neat)
νmax: 3578, 2985, 2928, 2872, 1462, 1375, 1073, 980 cm1;HRE-
SIMS: m/z= 359.2559 [M + Na]+(calcd. for C21H36O3Na+,
359.2562); 1Hand13CNMR data: see l
"Table 2.
(1S,2E,4R,7E,11E)-2,7,11-cembratriene-4-O-methyl-4-ol-6-one
(10): Yellowi sh oil; [α]
D25
+72.0 (c= 0.28, CHCl3); IR (neat) νmax:
2988, 2931, 2872, 1675, 1611, 1460, 1377, 983 cm1; HRESIMS:
m/z= 341.2442 [M + Na]+(calcd. for C21H34O2Na+, 341.2457); 1H
and 13CNMR data: see l
"Table 3.
(1S,2E,4R,7Z,11E)-2,7,11-cembratriene-4-O-methyl-4-ol-6-one
(11): Yellowi sh oil; [α]
D25
+ 105.0 (c= 0.24, CHCl3); IR (neat) νmax:
2987, 2930, 2872, 1675, 1611, 1461, 1369, 982 cm1; HRESIMS:
m/z= 341.2456 [M + Na]+(calcd. for C21H34O2Na+, 341.2457); 1H
and 13CNMR data: see l
"Table 3.
Table 1 13Cand1H NMR data of 35a.
Position 3 4 5
δC, mult. δH,(J,Hz) δC, mult. δH,(J,Hz) δC, mult. δH,(J,Hz)
1 47.3, CH 1.61, m 46.3, CH 1.68, m 47.1, CH 1.65, m
2 125.8, CH 5.23, dd (15.8, 8.8) 132.1, CH 5.23, dd (15.8, 9.0) 133.4, CH 5.17, dd (15.8, 8.0)
3 138.7, CH 5.32, d (15.8) 134.0, CH 5.07, d (15.8) 135.1, CH 5.22, d (15.8)
4 73.4, qC 75.5, qC 75.7, qC
5 48.6, CH22.53, ddd (13.2, 3.3, 1.8) 52.8, CH22.07, dd (13.9, 1.8) 52.6, CH22.07, dd (13.9, 1.8)
2.26, dd (13.2, 10.6) 1.77, dd (13.9, 3.7) 1.92, dd (13.9, 3.7)
6 127.1, CH 5.38, ddd (15.8, 10.6, 3.3) 63.5, CH 4.79, ddd (9.6, 3.7, 2.2) 64.2, CH 4.82, ddd (9.4, 3.7, 1.8)
7 139.2, CH 5.57, dd (15.8, 1.8) 133.0, CH 5.25, dq (9.6, 1.8) 133.3, CH 5.31, d (9.9)
8 77.9, qC 135.0, qC 133.0, qC
9 41.8, CH22.0, 2H, m 38.3, CH22.0, 2H, m 35.8, CH22.19, 2H, m
10 27.7, CH21.77, 2H, m 22.9, CH21.68, 2H, m 25.0, CH22.27, 2H, dt, (10.6, 2.5)
11 127.6, CH 5.25, m 128.3, CH 5.24, m 61.0, CH 2.82, dd (7.7, 2.5)
12 132.0, qC 133.1, qC 59.5, qC
13 37.3, CH22.20, m 1.93, m 77.5, CH 3.86, dd (11.0, 5.0) 36.6, CH22.19, 2H, m
14 22.1, CH21.42, 2H, m 30.0, CH21.58, 2H, m 28.3, CH21.46, 2H, m
15 32.9, CH 1.51, m 35.2, CH 1.54, m 33.8, CH 1.56, m
16 19.9, CH30.78, 3H, d (6.6) 20.2, CH30.84, 3H, d (6.3) 20.0, CH30.82, 3H, d (6.5)
17 20.7, CH30.82, 3H, d (6.6) 20.4, CH30.85, 3H, d (6.3) 20.6, CH30.86, 3H, d (6.5)
18 29.4, CH31.32, 3H, s 22.9, CH31.26, 3H, s 22.2, CH31.23, 3H, s
19 20.6, CH31.26, 3H, s 16.0, CH31.64, 3H, d (1.8) 16.2, CH31.78, 3H, s
20 13.9, CH31.46, 3H, s 15.1, CH31.50, 3H, s 16.3, CH31.19, 3H, s
21 50.0, CH33.14, 3H, s 50.2, CH33.12, 3H, s 50.3, CH33.14, 3H, s
aIn CDCl3, 400 MHz for 1H and 13C NMR. Coupling constants (J) are in Hz. Carbon multiplicities were determined by APT experiment s; qC = quaternary, CH = methine,
CH2= methylene, CH3= methyl carbons
Table 2 13Cand1H NMR data of 89a.
Posi-
tion
8 9
δC, mult. δΗ(J,Hz) δC,
mult.
δΗ(J,Hz)
1 46.2, CH 1.69, m 46.9, CH 1.47, m
2 134.1, CH 5.27, dd
(15.8, 7.3)
134.6, CH 5.18, dd
(15.8, 7.3)
3 134.9, CH 5.32, d (15.8) 135.0, CH 5.24, d (15.8)
4 75.0, qC 76.8, qC
5 48.1, CH22.15, dd
(14.7, 1.5)
49.9, CH22.10, dd (15.0, 3.0)
1.83, dd
(14.7, 9.6)
1.82, dd (15.0, 5.1)
6 63.6, CH 4.55, ddd
(8.8, 3.7, 1.2)
64.8, CH 3.95, ddd
(8.2, 5.1, 3.0)
7 66.7, CH 2.98, d (4.0) 69.4, CH 2.82, d (9.1)
8 61.4, qC 63.5, qC
9 38.4, CH21.94, 2H, m 36.7, CH21.98, 2H, m
10 23.0, CH22.06, 2H, m 23.0, CH22.07, 2H, m
11 124.5, CH 4.98, m 123.7, CH 5.02, m
12 134.1, qC 134.6, qC
13 36.9, CH21.94, 2H, m 36.4, CH21.89, 2H, m
14 27.7, CH21.62, 2H, m 28.2, CH21.63, 2H, m
15 33.3, CH 1.73, m 33.0, CH 1.69, m
16 19.3, CH30.83, 3H, d (6.9) 19.7, CH30.82, 3H, d (6.6)
17 20.5, CH30.87, 3H, d (6.9) 21.0, CH30.87, 3H, d (6.6)
18 21.9, CH31.34, 3H, s 23.0, CH31.32, 3H, s
19 16.9, CH31.50, 3H, s 17.7, CH31.34, 3H, s
20 14.3, CH31.51, 3H, s 14.7, CH31.51, 3H, s
21 50.5, CH33.16, 3H, s 50.8, CH33.16, 3H, s
aIn CDCl3, 400 MHz for 1H and 13C NMR. Coupling constants (J) are in Hz. Carbon
multiplicities were determined by APT experiments ; qC = quaternary, CH = methine,
CH2= methylene, CH3= methyl carbons
470
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
(1S,2Z,4R,7E,11E)-2,7,11-cembratriene-4-O-methyl-4-ol-6-one
(12): Yellowi sh oil; [α]
D25
+ 91.6.0 (c= 0.12, CHCl3); IR (neat) νmax:
2988, 2930, 2872, 1674, 1611, 1461, 1369, 982 cm1; HRESIMS:
m/z= 341.2442 [M + Na]+(calcd. for C21H34O2Na+, 341.2457); 1H
and 13CNMR data: see l
"Table 3.
(1S,2R,3Z,5Z,7E,11E)-2,6-thia-3,5,7,11-cembratetraene (13): Ye l-
low oil; [α]
D25
+ 118.7 (c= 0.24, CHCl3); IR (CHCl3)νmax: 2927,
2855, 1457, 1379, 1111 cm1; HREIMS: m/z= 302.2070 [M]+
(calcd. for C20H30S, 302.2068); 1Hand13CNMR data: see l
"Table 4.
(1S,2E,4R,6R,7E,11E)-2,7-cembradiene-4-O-methyl-6-O-acetyl-
11,12-epoxy-4,8-diol (14): Yello wi sh oil; [α]
D25
+ 91.6.0 (c= 0.12,
CHCl3); IR (neat) νmax: 2988, 2933, 2873, 1725, 1462, 1372,
983 cm1; HREIMS: m/z= 378.2766 [M]+(calcd. for C23H38O4,
378.2770); 1Hand13C NMR data: see l
"Table 4.
Statistical analysis
The results were statistically evaluated by one-way ANOVA and
significance was derived from Newman-Keuls test. The difference
was considered statist ically significant when p < 0.05.
Supporting information
A plausible mechanism of transformation of the α-ketone 10 to
the thioether 13 by reaction with Lawessonʼs reagent is depicted
in the Supporting Information.
Results and Discussion
!
The known (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4-O-
methyl-4,6-diol (2, 0.0059%) [5] and the new (1S,2E,4S,6-
E,8S,11E)-2,6,11-cembratriene-8-O-methyl-4,8-diol (3, 0.0011%)
were isolated from Nicotiana tabacum leaf powder.
Table 3 13Cand1H NMR data of 1012a.
Position 10 11 12
δC, mult. δH,(J,Hz) δC, mult. δH,(J,Hz) δC, mult. δH,(J,Hz)
1 46.4, CH 1.64, m 46.1, CH 1.66,m 46.1, CH 1.62, m
2 131.8, CH 5.41, dd (15.8, 8.1) 133.2, CH 5.40, dd (15.8, 8.0) 133.7, CH 5.42, dd (8.8, 7.0)
3 133.2, CH 5.28, d (15.8) 135.3, CH 5.53, d (15.8) 135.4, CH 5.44, d (8.8)
4 77.1, qC 76.8, qC 76.3, qC
5 55.1, CH22.82, d (11.7) 55.9, CH22.85, d (11.7) 57.1, CH22.83, d (11.7)
2.45, d (11.7) 2.42, d (11.7) 2.46, d (11.7)
6 200.1, qC 199.5, qC 199.0, qC
7 127.4, CH 6.13, s 128.0, CH 6.15, s 126.6, CH 6.02, s
8 156.1, qc 156.9, qC 157.2, qC
9 40.3, CH22.10, 2H, m 31.1, CH22.09, m 3.47, dd (11.0, 9.9) 40.7, CH22.13, 2H, m
10 23.9, CH22.22, 2H, m 25.5, CH22.39, 2H, m 23.9, CH22.26, 2H, m
11 123.7, CH 5.00, m 124.7, CH 4.82, brd (9.5) 123.1, CH 4.89, dd (7.7, 7.7)
12 134.2, qC 133.5, qC 134.2, qC
13 37.1, CH22.11, 2H, m 36.5, CH22.01, 2H, m 37.0, CH22.01, 2H, m
14 27.6, CH21.44, 2H, m 27.5, CH21.39, 2H, m 27.6, CH21.39, 2H, m
15 32.8, CH 1.55, m 32.6, CH 1.88, m 33.3, CH 1.82, m
16 19.3, CH30.80, 3H, d (6.6) 20.1, CH30.83, 3H, d (6.6) 19.3, CH30.84, 3H, d (6.6)
17 20.5, CH30.83, 3H, d (6.6) 20.2, CH30.84, 3H, d (6.6) 20.2, CH30.86, 3H, d (6.6)
18 24.7, CH31.31, 3H, s 22.4, CH31.40, 3H, s 21.0, CH31.29, 3H, s
19 18.4, CH32.02, 3H, d (1.1) 24.3, CH31.81, 3H, s 18.7, CH32.13, 3H, d (1.1)
20 15.6, CH31.56, 3H, s 14.6, CH31.59, 3H, s 15.7, CH31.56, 3H, s
21 50.2, CH33.24, 3H, s 49.8, CH33.15, 3H, s 49.8, CH33.13, 3H, s
aIn CDCl3, 400 MHz for 1H and 13C NMR. Coupling constants (J) are in Hz. Carbon multiplicities were determined by APT experiments; qC = quaternary, CH = methine,
CH2= methylene, CH3= methyl carbons
Table 4 13Cand1H NMR data of 1314a.
Posi-
tion
13 14
δC, mult. δΗ(J,Hz) δC, mult. δΗ(J,Hz)
1 41.6, CH 2.17, m 47.0, CH 1.64, 1H, m
2 42.7, CH 2.75, dd
(7.3, 4.0)
133.7, CH 5.16, 1H, dd
(15.8,8.0)
3 115.3, CH 5.45, d (7.3) 137.9, CH 5.24, d (15.8)
4 131.9, qC 75.5, qC
5 122.7, CH 5.97, s 49.7, CH22.09, dd (13.9, 1.8)
1.84, m
6 132.3, qC 68.0, CH 5.83, dt(8.8, 1.6)
7 126.8, CH 5.56, s 129.4, CH 5.21, 1H, d (9.9)
8 137.8, qC 137.4, qC
9 31.4, CH21.93, m 2.74, m 35.9, CH21.84, 2H, m
10 25.4, CH21.95, m 2.03, m 25.1, CH 21.48, 2H, m
11 126.9, CH 5.18, dd
(7.7, 7.7)
61.1, CH 2.81, dd
(8.0, 1.5)
12 134.4, qC 59.5, qC
13 39.2, CH21.27, 2H, m 36.7, CH22.20, 2H, m
14 22.9, CH21.57, 2H, m 28.3, CH21.57, 2H, m
15 27.9, CH 2.00, m 33.9, CH 1.60, m
16 17.2, CH30.81, 3H, d (7.0) 18.9, CH30.79, 3H, d (7.0)
17 21.6, CH30.92, 3H, d (7.0) 20.6, CH30.83, 3H, d (7.0)
18 22.0, CH31.83, 3H, (1.4) 22.2, CH31.23, 3H, s
19 24.1, CH31.84, 3H, d (1.5) 16.1, CH31.87, 3H, d (1.2)
20 16.8, CH31.63, 3H, s 16.6, CH31.19, 3H, s
21 – – 50.4,CH33.13, 3H, s
CO – – 170.1, qC
CH3– – 21.6 2.00, 3H, s
aIn CDCl3, 400 MHz for 1H and 13C NMR. Coupling constants (J) are in Hz. Carbon
multiplicities were determined by APT experiments ; qC = quaternary, CH = methine,
CH2= methylene, CH3= methyl carbons
471
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
HREIMS analysis of 3displayed an [M]+at m/z 320.2699, suggest-
ing the molecular formula C21H36O2and four degrees of unsatu-
ration. The 1Hand13C NMR data of 3(l
"Table 1) showed resonan-
ces for an isopropyl group and three Edouble bonds. The me-
thoxy proton singlet at δH3.14 correlated with a methyl carbon
at δC50.0 in HTECOR, which was assigned to C-2 1 (l
"Table 1).
The methoxy singlet H3-21 showed a 3J-HMBC correlation with
the quaternary carbon C- 8 (δC77.9). The methyl singlet H3-19
(δH1.26) showed a 2J-HMBC correlation with the quaternar y oxy-
genated carbon C-8 and 3J-HMBC correlations with the olefinic
methine carbon C- 7 (δC139.2) and the methylene carbon C- 9 (δC
41.8). The olefinic doublet of doublet H-7 (δH5.57) showed a
COSY coupling with H-6 (δH5.38) and a 2J-HMBC correlation with
C-8 . The E-geometry of Δ6,7 was based on the large JH-6/H-7 val-
ue (15.8 Hz). The E-geometry of Δ2,3 and C- 1 1/ C- 1 2 double bond
was established similar to 1. The relative configuration of the chi-
ral centers C-4 and C-8 were assigned based on NOESY data. The
chiral center C-1 was assigned in S-configuration based on the
similarity of the chemical shift value and splitting pattern of H-1
and 13C chemical shift value of C-1 with those of the known 1and
2[2, 4, 7, 8, 10]. The β-oriented H-1 (δΗ1.61) showed a NOESY
correlation with the H3-18 singlet (δΗ1.32), suggesting a similar
stereo-orientation. The methyl singlet H3-18 also showed a
NOESY correlation with H-5a (δΗ2.53), indicating its β-orienta-
tion. Both H3-18 and H-5a showed NOESY correlations with H3-
19 (δΗ1.26), suggesting β-orientation for C- 1 9 . Molecular model-
ing study estimated a distance of 4.4 Aοbetween H3-18 and each
of H-1 and H3-19, while the estimated distance between H-5a
and H3-19 was 4.3 Aο, justifying the above-mentioned NOESY
correlations. Thus, 3was elucidated as (1S,2E,4S,6E,8S,11E)-
2,6,11-cembratriene-8-O-methyl-4,8-diol. The natural occur-
rence of 2and 3was confirmed through TLC analysis of freshly
extracted (CH2Cl2) tobacco. The closely related (1S,2E,4S,6-
E,8S,11E)-2,6,11-cembratriene-4,8-O-dimethyl-4,8-diol and five
other methyl ethers were previously reported as natural tobacco
constituents [5].
The methyl ether cembranoid 2displayed better anti-migratory
activity against PC-3 prostate cancer cells compared to the parent
alcohol 1and was therefore subjected to microbial and semisyn-
thetic optimization studies. Several fungi were screened for their
ability to biotransform 2, revealing that Cunninghamella sp. NRRL
5695 and Mucor ramannianus ATCC 9628 produced polar metab-
olites according to TLC analysis. Scale-up fermentation of 2using
C. sp. NRRL 5695 afforded new hydroxylated (4) and epoxidized
(5) metabolites. Scale-up fermentation of 2using M. ramannia-
nus ATCC 9628 afforded 4and the known O-demethylated
hydroxylated metabolites 6and 7[7, 8, 23, 24].
The HRESIMS data of 4showed a molecular ion peak [M + Na]+at
m/z 359.2511, suggesting the molecular formula C21H36O3and
four degrees of unsaturation. The NMR data (l
"Table 1) suggested
a monohydroxy derivative of the parent 2. The oxygenated meth-
ine proton signal at δΗ3.86 was assigned to H-13. This was based
on its COSY correlation with the methylene protons H2-14 (δΗ
1.58), which in turn was COSY-coupled with H-1 (δΗ1.68). The
methyl singlet H3-20 (δΗ1.50, s) showed a 3J-HMBC correlation
with C-13 (δC77.5). The relative configuration of C-1 3 was as-
signed based on NOESY data. The β-oriented H-1 showed a NO-
ESY correlation with H-13, suggesting a similar relative configu-
ration. Molecular modeling study showed a calculated distance of
3.9 Aοbetween both protons, justifying their NOESY correlation.
Thus, 4was elucidated as (1S,2E,4R,6R,7E,11E,13R)-2,7,11-cem-
bratriene-4-O-methyl-4,6,13-triol.
The HRESMS data of 5suggested the molecular formula C21H36O3
and four degrees of unsaturation. The NMR data (l
"Table 1)sug-
gested epoxidation of one double bond in 2. The oxirane methine
proton H-11 (δH2.82) was assigned based on its COSY coupling
with H2-10 (δH2.27) and 3J-HMBC correlations with C-9 (δC
35.8), C-1 3 (δC36.6), and C- 2 0 (δC16.3). The (11S)and(12S) con-
figuration was assigned based on comparison with published 13C
NMR and 1H NMR coupling constant data of related cembranoids
[25, 26]. Thus, 5was elucidated as (1S,2E,4R,6R,7E,11S,12S)-4-O-
methyl-11,12-epoxy-2,7-cembradiene-4,6-diol.
Metabolites 6and 7were determined to be (1S,2E,4R,6R,7E,
11E,10R)-2,7,11-cembratriene-4,6,10-triol and (1S,2E,4S,6R,7E,
11E,10R)-2,7,11-cembratriene-4,6,10-triol, respectively, based
on the published data [10,16].
Metal-catalyzed regioselective epoxidation of Δ7,8 of 2utilizing t-
butyl hydroperoxide and vanadyl acetylacetonate was attempted
to determine the effect of epoxidation on activity.
The HRESIMS data of 8suggested the molecular formula
C21H36O3and four degrees of unsaturation. The NMR data of 8
(l
"Table 2) confirmed 7,8-epoxidation. The oxirane proton dou-
blet H-7 (δΗ2.98) showed COSY coupling with the oxygenated
proton H-6 (δΗ4.55). The methyl singlet H3-19 (δH1.50) showed
3J-HMBC correlations with C-7 (δC66.7) and C- 9 (δC38.4). It also
showed a 2J-HMBC correlation with the quater nary carbon C- 8 (δC
61.4). The (7R,8R) configuration was determined based on com-
parison with 13C NMR and coupling constant data of related cem-
branoids [2527]. Thus, 8was elucidated as (1S,2E,4R,6R,7R,
8R,11E)-4-O-methyl-7,8-epoxy-2,11-cembradiene-4,6-diol.
The HRESIMS and NMR data of 9(l
"Table 2)were similar to those
of 8except C-6 , C- 7, and C- 8 and the proton chemical shift of H3-
19. This variation suggested that 9is the 7,8-diastereoisomer of 8.
This was further confirmed by comparing the carbon chemical
shift and Jvalues of C- 6 , C- 7 , and C- 8 in 9with related cembranoids
with (7S,8S)-configuration [26, 27]. Thus, 9was elucidated as
(1S,2E,4R,6S,7S,8S,11E)-7,8-epoxy-2,11-cembradiene-4-O-meth-
yl-4,6-diol.
To test the significance of the C- 6 secondary alcohol moiety with
respect to anti-migratory activity, chromic acid oxidation of 2
was attempted, yielding the new α-keto-cembranoids 1012.
The HRESMS and NMR (l
"Table 3) data of 10 suggested a ketone
moiety at C- 6 , as was evident from the α,β-unsaturated ketone
carbon at δC200.1 (C-6) and the downfield shift of the α-proton
singlet H-7 (δH6.13). Proton H-7 showed a 2J-HMBC correlation
with C-6 and 3J-HMBC correlations with C-1 9 (δC18.7) and C- 9
(δC40.7). Thus, 10 was elucidated as (1S,2E,4R,7E,11E)-2,7,11-
cembratriene-4-O-methyl-4-ol-6-one.
The HRESMS for 11 suggested a C21H34O2molecular formula, five
degrees of unsaturation, while the NMR data (l
"Table 3)were
nearly identical to those of 10 except for the segment C- 7 - C- 8 - C-
9-C-19. The methyl carbon C- 1 9 (δC24.3) was downfield shifted
(Δδ+ 5.9), compared to that of 10. The methyl singlet H3-19 (δH
1.81) showed 3J-HMBC correlations with C-7 (δC128.0) and C- 9
(δC31.1). The proton H-9a (δH3.47) was downfield shifted, possi-
bly due to its location in the deshielding cone of Δ7,8.TheZ-geom-
etry of Δ7,8 was based on the strong NOESY correlation of H-7 (δΗ
6.15) with H3-19 and the chemical shift value of C-1 9 [2, 4, 10, 24].
Thus, 11 was elucidated as (1S,2E,4R,7Z,11E)-2,7,11-cembra-
triene-4-O-methyl-4-ol-6-one.
Similarly, 12 was determined to be a geometrical isomer of 10
and 11, based on its HRESIMS and NMR (l
"Table 3) data. The
main difference between 12 versus 10 and 11 was in the coupling
pattern of the Δ2,3 protons and the slight downfield shifting of C- 5
472
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
(Δδ+ 2.0 and + 1.1, respectively). The proton doublet of doublet
H-2 (δH5.42, J= 8.8 and 7.0) showed COSY coupling with H-1
(δH1.62) and with the proton doublet H-3 (δH5.41). The small
coupling between H-2 and H-3 (8.8 Hz) suggested cis-coupling
[2, 4, 24]. Thus, 12 was elucidated as (1S,2Z,4R,7E,11E)-2,7,11-
cembratriene-4-O-methyl-4-ol-6-one.
Lawessonʼs reagent is used for thionation of ketone and carbonyl
containing compounds as well as syntheses of thiols and hetero-
cyclic compounds [28]. However, this reagent is also known to in-
duce unexpected reactions, resulting in unusual products [28].
Reflux of 10 with Lawessonʼs reagent in toluene for 6 h afforded
a less polar product 13. The HREIMS data of 13 showed a mo-
lecular ion peak [M]+at m/z 302.2070, revealing the molecular
formula C20H30S and six degrees of unsaturation. The NMR data
for 13 (l
"Table 4) suggested a 15-thiabicyclo[9.3.1]pentadeca-
tetraene system with the elimination of C- 4 methoxy. The narrow
methyl doublet H3-18 (δH1.83) showed 3J-HMBC correlations
with the olefinic methine carbons C- 3 (δC115.3) and C- 5 (δC
122.7). Proton H-3 showed a COSY coupling with the downfield
shifted H-2 (δH2.75) and a 3J-HMBC correlation with C- 1 (δC
41.6). Proton H-2 showed COSY coupling with H-1 and 3J-HMBC
correlations with C-4 (δC131.9), C-14 (δC22.9), and C- 1 5 (δC27.9)
and with the quaternar y olefinic carbon C- 6 (δC132.3), via the
sulfur atom, confirming the CH-2-SC- 6 connectivity. The relative
configuration of C-2 was assigned based on NOESY data. The β-
oriented H-1 showed a strong NOESY correlation with H-2, sug-
gesting a β-orientation for H-2. The calculated distance between
both protons in SYBYL 8.1 was 3.02 A°, justifying this NOESY cor-
relation. Both protons H-3 and H-5 showed NOESY correlations
with H3-18, suggesting Z-geometry for Δ3,4 and Δ5,6 systems.
Thus, 13 was elucidated as (1S,2R,3Z,5Z,7E,11E)-2,6-thia-
3,5,7,11-cembratetraene. A possible reaction mechanism for the
formation of 13 includes formation of thioketone 10a,followed
by thio-enol tautomerism and intramolecular cyclization with
the loss of methanol (Scheme 1S, Supporting Information). The
driving force for the ring closure is the formation of a stable thia-
cyclohexane structure.
In an effort to determine the activity of C- 6 acetoxylated 11,12-
epoxy cembranoids, 2was acetylated and epoxidized to afford
14. The HREIMS and NMR (l
"Table 4) data of 14 confirmed C- 6
acetylation and 1112-epoxidation. NMR data were similar to
those of 5with additional acetyl resonances at δC170.1 and 21.6
and δΗ2.00 (3H, s). The downfield shift of C- 6 and H-6 (Δδ +3.8
and + 1.01, respectively) compared to those of 5indicated C-6
acetylation. The (11S,12S)-epoxide configuration was based on
comparison with 5and literature data [25, 26]. Thus, 14 was elu-
cidated as (1S,2E,4R,6R,7E,11S,12S)-2,7-cembradiene-4-O-meth-
yl-6-O-acetyl-11,12-epoxy-4,6-diol.
The spheroid disaggregation model is based on disaggregation of
prostate cancer cell spheroids and radial migration of released
cells on an extracellular matrix (ECM) [2830]. Primary tumor
cells are generally released in clumps in vivo and attach to a favor-
able ECM. Subsequently, attached clumps are released radially to
migrate in all directions [20,2931]. Therefore, the spheroid dis-
aggregation model is closer to in situ tumor metastasis than any
other linear invasion assays. Studies have shown that calcitonin
(CT) can significantly enhance tumor growth and metastasis of
PC cell lines [20, 22]. These effects may be mediated by the dis-
ruption of cell-cell adhesion. To avoid repeated addition of exog-
enous CT during long assay incubation period, PC-3MCT+ cells
were used because they secrete large amounts of CT and have cal-
citonin receptors (CTR) [20].
The anti-migratory activity of cembranoids 214 against the
highly metastatic human prostate cancer cell lines PC-3M- CT+was
evaluated in the spheroid disaggregation assay (l
"Figs. 24). At
50 µM, 310 and 1214 showed better anti-migratory activity
compared to the vehicle control (p < 0.05). Compound 11 showed
no significant difference versus the vehicle control (DMSO,
p < 0.05, l
"Fig. 2), indicating the preference of E- over Z-geometry
of Δ7,8 for the activity, while 2and 9were cytotoxic at this dose. The
activity of 2and 9was therefore assessed at doses from 10 to
1000 nM (l
"Fig. 3). The lowest tested dose of 2and 9(10 nM)
showed no activity while 100, 500, and 1000 nM showed a typical
dose-response effect versus vehicle control (l
"Fig. 3). A dose of
1000 nM of 2decreased the disaggregation and cell migration of
PC-3M-CT+ spheroids by 65 %, while the same dose of 9induced
75% reduction. The known antimetastatic leads 4-hydroxyphe-
nylmethylene hydantoin and 4-mercaptoethylphenylmethylene
hydantoin were inactive at this dose range [22, 23], only showing
activity at 50200 µM. Therefore, cembranoids 2and 9could be
Fig. 2 Anti-migratory activity of a 50-µM dose of
38and 1014 against PC-3M-CT+cells using the
spheroid disaggregation assay.
473
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
appropriate anti-migratory hits for future optimizations. Hydrox-
ylation, epoxidation, acetylation, or oxidation at C-6 of 2de-
creased activity, with only the 7S,8S-epoxide analogue 9main-
taining potent activity, indicating the possibility of using 7S,8S-
oxirane to replace the EΔ7,8 system without a signif icant decrease
in activity.
The wound-healing assay is a commonly used model for studying
cell migration [3234]. The scratched monolayer heals the
wound in a characteristic manner (l
"Fig. 5); therefore, this assay
is widely used to study cell migration rates, cell polarization, and
matrix remodeling studies. It can be used for validation of small
molecule leads that affect cell migration and for detailed cell bio-
logical studies.
The anti-migratory activity of cembranoids 114 against the
highly metastatic human prostate cancer cell lines PC-3 was eval-
uated in the wound-healing assay (l
"Figs. 5 and 6). 4-Mercapto-
ethylphenylmethylene hydantoin was used as a positive control
[22]. Results of the wound-healing assay were parallel to those
of the spheroid assay and suggest 2and 9as the most active cem-
branoids. A 50-µM dose of 2was more active than 1and the pos-
itive drug control (l
"Fig. 6). This indicates that the 4-O-methyla-
tion of 1enhances the activity. Cembranoids 4and 6were the
least active, which further highlights the fact that C- 1 0 or C- 1 3 -
hydroxylation will reduce activity. Compounds 5and 10 were
the next most active, suggesting the minimal adverse effects of
C-6 oxidation to ketone or 11S,12S-epoxidation on the activity.
The poor activity of 12 suggests the preference of Eover the Z-ge-
ometry of Δ2,3.
In conclusion, new and known 4-O-methylcembranoids were
isolated from N. tabacum leaves. Cembranoid 2showed potent
anti-migratory activity against prostate cancer cell lines, and
therefore was subjected to microbial and semisynthetic optimi-
zations. All analogues were generally less active than its parent
2. Hydroxylation of 2reduced its activity. (7R,8R)and(11S,12S)-
epoxidation of 2did not significantly reduce the activity. E- was
preferred over Z-geometry of Δ7,8 and Δ2,3 for the activity. Cem-
branoids 2and 9are potential anti-migratory hit compounds ap-
propriate for future optimization aiming at their therapeutic use
in the control and prevention of metastatic prostate cancer.
Acknowledgements
!
Dr. G. Shah, University of Louisiana-Monroe, is acknowledged for
spheroid disaggregation assay assistance. The laboratory of K. El
Sayed was supported by Philip Morris USA, Inc. and Philip Morris
International between 2005 and 2007.
References
1Wahlberg I, Enzell CR. To b ac co cembranoids. Beitrage zur Tabakfor-
schung International 1984; 12: 93104
2Eklund AM, Hatanaka SI, Wahlberg I. Progress in the chemistry of or-
ganic natural products. Wien, New Yor k: Springer Verl ag; 1992: 143
293
3Dauben WG, Thiessen WE, Resnick PR. Cembrene, a 14-membered ring
diterpene hydrocarbon. J Am Chem Soc 1962; 84: 20152016
4Wahlberg I, Eklund AM. Tr en ds in flavourresearch. Oxford: Elsevier Sci-
ence B.V.; 1994: 449462
Fig. 4 Spheroid disaggregation model for assessment of anti-migratory
activity against PC-3M-CT+cells. aand c: spheroid at t = 0 for vehicle control
and active cembranoid treatment, respectively. band d: spheroid after 24 h
for vehicle control and active cembranoid treatment, respectively.
Fig. 3 Anti-migratory and dose-response ef fect of
10 nM-1 µM dose range of 2and 9against PC-3M-C T+
cells using the spheroid disaggregation assay.
474
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
5Bylov A, Brummer U, Hass W, Seehofer F, Heemann VZ. New cembra-
noids from tobacco. Naturfo rsch 1983; 38c: 515516
6Pham NB, Butler MS, Quinn RJ. Naturally occurring cembranes from an
Australian Sarcophyton species. J Nat Prod 2002; 65: 11471150
7Olsson E, Berg JE, Wahlberg I. Eight new cembranoids from tobacco-
structural elucidation and conformational studies. Tetrahedron 1993;
49: 49754992
8Olsson E, Holth A, Kumlin E, Bohlin L, Wahlberg I. Structure-related in-
hibiting activity of some tobacco cembranoids on the prostaglandin
synthesis in vitro. Planta Med 1993; 59: 293295
9Saito Y, Takazawa H, Konishi S, Yoshi da D, Mizusaki S. Identification of
cembratriene-4,6-diol as antitumor-promoting agent from cigarette
smoke condensate. Carcinogenesis 1985; 6: 11891194
10 El Sayed KA, Laphookhieo S, Baraka HN, Yousaf M, Hebert A, Bagaley D,
Rainey FA, Muralidharan A, Thomas S, Shah GV. Biocatalytic and semi-
synthetic optimization of the anti-invasive tobacco (1S,2E,4R,
6R,7E,11E)-2,7,11-cembratriene-4,6-diol. Bioorg Med Chem 2008; 16:
28862893
11 Pagan OR, Rowlands AL, Fattore AL, Coudron T, Urban KR, Bidja AH, Ete-
rovic VA. A cembranoid from tobacco prevents the expression of nico-
tine-induced withdrawal behavior in planarian worms. Eur J Pharma-
col 2009; 615: 118124
12 Smith RV, Rosazza JP. Microbial models of mammalian metabolism.
Aromatic hydroxylation. Arch Biochem Biophys 1974; 161: 551558
13 Clark AM, McChesney JD, Hufford CD. The use of microorganisms for the
study of drug metabolism. Med Res Rev 1985; 5: 231251
14 Thayer AN. Enzymes at work. Chem Eng News 2006; 84: 1425
15 El Sayed KA, Hamann MT, Waddl ing CA, Jensen C, Lee SK, Dunstan CA,
Pezzuto JM. Structurally novel bioconversion products of the marine
natural product sarcophine effectively inhibit JB6 cell transformation.
J Org Chem 1998; 63: 74497455
16 El Sayed KA, Laphookhieo S, Yousaf M, Prestridge JA, Shirode AB, Wa li VB,
Sylvester PW. Semisynthetic and biotransformation studies of
(1S,2E,4S,6R,7E,-11E)-2,7,11-cembratriene-4,6-diol. J Nat Prod 2008;
71: 117122
17 Wahlberg I, Eklund AM. Cembranoids, pseudopteranoids and cubita-
noids of natural occurrence. In: Herz W, Kirby GW, Moore RE, Steglich
W, Tamm CH, editors. Progress in the chemistry of organic natural
products, Vol. 59. Wien, New York: Springer-Verlag; 1992: 141294
18 Saito Y, Tsujin o Y, Kaneko H, Yo sh id a D, Mizusaki S. Inhibitory ef fects of
cembratriene-4,6-diol derivatives on the induction of Epstein-Barr vi-
rus early antigen by 12-O-tetradecanoylphorbol-13-acetate. Agric Biol
Chem 1987; 51: 941943
19 El Sayed KA, Sylvester PW. Biocatalytic and semisynthetic studies of the
anticancer tobacco cembranoids. Expert Opin Investig Drugs 2007; 16:
877887
20 Thomas S, Chigurupati S, Anbalagan M, Shah GV. Calcitonin increases
tumorigenicity of prostate cancer cells: evidence for the role of protein
kinase A and urokinase-type plasminogen receptor. Mol Endocrinol
2006; 20: 18941911
21 Thomas S, Chiriva-Internati M, Shah GV. Calcitonin receptor-stimulated
migration of prostate cancer cells is mediated by urokinase receptor-
integrin signaling. Clin Exp Metastasis 2007; 24: 363377
22 Shah GV, Muralidharan A, Thomas S, Gokulgandhi M, Mudit M, Khanfar
M, El Sayed KA. Identification of a small molecule(s) class to enhance
cell-cell adhesion and attenuate prostate tumor growth and metasta-
sis. Mol Cancer Ther 2009; 8: 509520
23 Mudit M, Khanfar M, Muralidharan A, Thomas S, Shah GV, El Sayed KA.
Discovery, design, and synthesis of anti-metastatic lead phenylmethy-
lene hydantoins inspired by marine natural products. Bioorg Med
Chem 2009; 17: 17311738
Fig. 5 Wound-healing assay model for assessment
of anti-migratory activity against PC-3 cells. Vehicle
control (DMSO) and active cembranoid treatment,
50 µM of 2.Reading was taken after 24 h of treat-
ment. X = 100.
Fig. 6 Anti-migratory activit y of a 50 µM dose of
214 against PC-3 cells using wound-healing assay.
50 µM 4-Mercaptoethylphenylmethylene hydantoin
(S-ethyl-PMH) was used as a positive drug control.
DMSO was used as a vehicle control.
475
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
24 Wahlberg I, Olsson E, Berg JE. Progress in Flavour Precursor Studies Pro-
ceeding International Conference. Carol Stream: Allured Publishing
Corporation; 1993: 8395
25 Forsblom I, Berg JE, Wahl ber g I. Toba cc o chemistry. 75. Two new cem-
bratriene triols from tobacco. Acta Chem Scand 1993; 47: 8088
26 Wahlberg I, Nishida T, Enzell C, Berg JE, Pilotti AM. Tob acco chemistry.
51. New cembranic diterpenoids from Greek tobacco. Acta Chem Scand
1980; 34: 195202
27 Wahlberg I, Eklund AM, Vogt C, Enzell RC, Berg JE. Tobacco chemistry. 65.
Two new 7,8-epoxycembranoids from tobacco. Acta Chem Scand 1986;
40: 855860
28 Jesberger M, Davis TP, Barner L. Applications of Lawessonʼs reagent in
organic and organometallic syntheses. Synthes is 2003; 13: 19291958
29 Shah GV, Noble MJ, Austenfeld M, Weigel J, Deftos LJ, Mebust WK, Winston
K. Presence of calcitonin-like immunoreactivity (iC T) in human pros-
tate gland: evidence for iCT secretion by cultured prostate cells. Pros-
tate 1992; 21: 8797
30 Alroy I, Yard en Y. The ErbB signaling network in embryogenesis and on-
cogenesis: signal diversification through combinatorial ligand-recep-
tor interactions. FEBS Lett 1997; 410: 8386
31 Chien J, Ren Y, Wang YQ, Bordelon W, Thompson E, Davis R, Rayford W,
Shah G. Calcitonin is a prostate epithelium-derived growth stimulatory
peptide. Mol Cell Endocrinol 2001; 181: 6979
32 Lu KV, Jong KA, Rajasekaran AK, Cloughesy TF, Mischel PS. Upregulati on
of tissue inhibitor of metalloproteinases (TIMP)-2 promotes matrix
metalloproteinase (MMP)-2 activation and cell invasion in a human
glioblastoma cell line. Lab Invest 2004; 84: 820
33 Herren B, Garton KJ, Coats S, Bowen-Pope DF, Ross R, Raines EW.
ADAM15 overexpression in NIH3T3 cells enhances cell-cell interac-
tions. Exp Cell Res 2001; 271: 152160
34 Cascone I, Giraudo E, Caccavari F, Napione L, Bertotti E, Collard JG, Serini
G, Bussolino F. Temporal and spatial modulation of Rho GTPases during
in vitro formation of capillary va scular network: Adherens junctions
and myosin light chain as targets of Rac1 and RhoA. J Biol Chem 2003;
278: 50 70250 713
476
Baraka HN et al. Bioactive Natural, Biocatalytic, Planta Med 2011; 77: 467476
Original Papers
... The α-CBT and its hydroxylated mammalian metabolites identified as potent antagonists for VEGFR2, potently suppressing the in vivo angiogenesis of TNBC in a nude mouse xenograft model [17]. The tobacco cembranoid β-CBT and its 4-O-methyl ether showed potent anti-invasive activity in spheroid disaggregation assay against the human PC cell line PC-3M cells at sub-μM doses, while it was non-significantly cytotoxic even at 50 μM treatment dose [18,19]. Earlier, β-CBT at 50 nM treatment dose reversed the calcitonin-stimulated reduction in transepithelial resistance and enhanced the paracellular permeability in PC-3M cells [18]. ...
... The α-CBT and its hydroxylated mammalian metabolites identified as potent antagonists for VEGFR2, potently suppressing the in vivo angiogenesis of TNBC in a nude mouse xenograft model [17]. The tobacco cembranoid β-CBT and its 4-O-methyl ether showed potent antiinvasive activity in spheroid disaggregation assay against the human PC cell line PC-3M cells at sub-µM doses, while it was non-significantly cytotoxic even at 50 µM treatment dose [18,19]. Earlier, β-CBT at 50 nM treatment dose reversed the calcitonin-stimulated reduction in transepithelial resistance and enhanced the paracellular permeability in PC-3M cells [18]. ...
... Furthermore, the 13 C NMR chemical shift data further confirmed the β-CBT identity via the C-6 methine carbon resonating at δ C 67.5. Thin layer chromatography (TLC): R f value 0.42 (Si gel, n-hexane-EtOAc 1:1) [16,17,19,28]. ...
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The methylethers 4-O,8-O-dimethyl-(1S, 2E, 4R, 6E, 8S, 11E)-2,6,11-cembratriene-4,8-diol (9), 4-O-methyl-(1S, 2E, 4R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (4) und 4-O,6-O-methyl-(1S, 2E, 4R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (8) were identified as new natural products from tobacco. They were isolated from processed tobacco as well as from leaf surface gum of fresh tobacco. The spectral data of all six methylethers of (1S, 2E, 4S, 6R, 7E, 11E)- and (1S, 2E, 4R, 6R, 7E, 11E)-2,7,11-cembratrienen-4,6-diol are given.
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The discovery of the first diterpenoids of the cembrane type in tobacco dates back to the early 1960's. Since then some forty tobacco cembranoids have been en- countered. Most of these have a hydroxyl substituent at C-4 and are commonly divided into two series: those having a 4R- and those having a 4S~configuration. Additional oxygenation is found at C-6, C-7, C-8, C-11 or C-t2. These compounds, which are present in the gummy ex- udate of the tobacco leaf and flower, are susceptible to biodegradation thus accounting for the presence of the large number of odoriferous norcembranoids in tobacco. They are also reported to include representatives having growth inhibiting and insect resistance proper- ties. A considerable insight into the biological transformations of the tobacco cembranoids has been obtained by isolation and determination of the stereostructures of new compounds and by biomimetic experiments. The latter have involved singlet oxygen reactions, epoxidations and acid- and base-induced rearrangements. The results obtained sUpport the importance of the (!S,2E,4R,6R,7 E, tiE)- ond (!S,2E,4S,6R,7 E, tiE)-2,7, tt-cembratriene-4, 6-diols, the major tobacco cembranoids, as key metabolites in the biogenesis of the other cembranic compounds. An account of these biogenetic reactions will be given and the isolation of a few new cembranoids will be reported.
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Eight new cembranoids have been isolated from an extract of flowers of Greek tobacco. They have been identified as (1S,2E,4S,R,7E,10R,11E)-2,7,11-cembratriene-4,6,10-triol (1), the corresponding (10S)-, (4R*)- and (4R*,10S*)-diastereomers (2–4), (1S*,2E,4R*,6R*,7E,10S*,11Z)-2,7,11-cembratriene-4,6,10-triol (5), (1S,2E,4S,6R,7E,11E)-4,6-dihydroxy-2,7,11-cembratrien-10-one (6), the corresponding (4R*)-epimer (7) and (1S*,2E,4S*,7E,10S,11E)-4,10-dihydroxy-2,7,11-cembratrien-6-one (8) with the aid of chemical and spectral methods, 2D-NMR techniques being particularly helpful. The crystal structure of the diacetate 11 has been determined and is described. The solution conformations of triols 1 and 3 have been studied by using NMR methods in conjunction with molecular mechanics calculations (MM3). The biogenesis of the new compounds is discussed.
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Aus griechischem Tabak wurden die Verbindungen (lS,2E,4R,6R,7E,l 1S, 12S)- l 1,12-Epoxy-2,7-cembradien-4,6-diol (Ia), (1S,2E,4R,6E,8R,1 1S,12E)- (II) und (1S,2E,4S,6E,8R,1 lS,12E)-8,1 1-Epoxy-2,6,12-cembratrienol-(4) (III) sowie (1S,2E,- 4R,6E,8R,1 1S)-8,1 l-Epoxy-2,6,l 2(20)-cembratrienol-(4) (IV) isoliert.
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CHEMISTS HAVE BEEN PRACTICING ORGANIC CHEMISTRY for hundreds of years; microbes have been at it even longer. Microbial and other enzymes are superbly enantio-, chemo-, and regioselective across a diverse range of reactions under mild conditions of pH, temperature, and pressure. Why, then, has it taken chemists so long to put aside a dislike of "bugs" and use their enzyme catalysts? The question is especially pertinent when it comes to making pharmaceuticals. When scientists at GlaxoSmithKline, AstraZeneca, and Pfizer examined 128 syntheses from their own companies, they found that as many as half of the drug compounds made by their process R&D groups are not only chiral but also contain an average of two chiral centers each ( Org. Biomol Chem. 2006 ,4,2337). And to meet regulatory requirements, enantiomeric purities of 99.5% were found to be necessary. "When it comes to wanting selectivities of 98% or higher, you are probably bound to a bioprocess, because getting beyond ...