Semisynthetic and biotransformation studies of (1S,2E,4S,6R,7E,11E)-2,7,11- cembratriene-4,6-diol

Article abstract of DOI:10.1021/np0704351

Tobacco-derived (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (1) and (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (2) were first shown to display potential antitumor-promoting activity in the mid-1980s. However, very little is currently understo

Full text of DOI:10.1021/np0704351

J. Nat. Prod. 2008, 71, 117–122
117
Semisynthetic and Biotransformation Studies of
(1S,2E,4S,6R,7E,11E)-2,7,11-Cembratriene-4,6-diol
Khalid A. El Sayed,* Surat Laphookhieo, Muhammad Yousaf, Justin A. Prestridge, Amit B. Shirode, Vikram B. Wali, and
Paul W. Sylvester
Department of Basic Pharmaceutical Sciences, College of Pharmacy, UniVersity of Louisiana at Monroe, 700 UniVersity AVenue, Monroe,
Louisiana 71209
ReceiVed August 16, 2007
Tobacco-derived (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (1) and (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-
4,6-diol (2) were first shown to display potential antitumor-promoting activity in the mid-1980s. However, very little
is currently understood regarding the structural activity relationships of tobacco cembranoids. The aim of this present
study was to explore antiproliferative activity of various derivatives of (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-
diol (1) using semisynthetic and biotransformation approaches. Derivatives of 1 include esterified, oxidized, halogenated,
and nitrogen- and sulfur-containing compounds (3–17). Biotransformation of 1 using Mucor ramannianus ATCC 9628
and Cunninghamella elegans ATCC 7929 afforded the known 10S,11S-epoxy analogue of 1 (4) as the main metabolite.
Biotransformation of the 6-O-acetyl analogue (3) using the marine symbiotic Bacillus megaterium strain MO31 afforded
(1S,2E,4S,6R,7E,11E,10R)-2,7,11-cembratriene-4,6,10-triol (18). (1S,2E,4S,6R,7E,11E,13R)-2,7,11-Cembratriene-4,6,13-
triol-6-O-acetate (6), (1S,2E,4S,6R,7E,11E,13S)-2,7,11-cembratriene-4,6,13-triol-6-O-acetate (7), the rearranged R-ketol
(1S,2E,4S,7Z,11E)-2,7,11-cembratrien-4-ol-6-one (11), and the secocembranoid 12 showed antiproliferative activity against
highly malignant +SA mammary epithelial cells with an IC50 range of 15–30 µM.
The leaf and flower cuticular wax of most Nicotiana species
contains high amounts of cembranoid diterpenes.1 They possess
14-membered macrocyclic rings substituted by an isopropyl residue
at C-1 and by three symmetrically disposed methyl groups at
positions C-4, C-8, and C-12. The two most abundant epimeric
tobacco cembranoids, (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-
4,6-diol (1) and (1S,2E,4R,6R,-7E,11E)-2,7,11-cembratriene-4,6-
diol (2), were among the first reported.1,2 The structure and absolute
stereochemistry of 2 was based on X-ray crystallography, while
the structure of 1 was based on chemical degradation and correlation
with 2,7,12(20)-cembratriene-4,6,11-triol, which was confirmed by
X-ray crystallography.1–4 Both compounds are key flavor ingredi-
ents in tobacco.1,2 Biodegradation of 1 and 2 during cure fermenta-
tion or flue curing of tobacco leaf produces a range of flavor
compounds having from 8 to 19 carbon skeletons.1,2,5
without any interaction with calmodulin. Unlike many anticancer
agents, 1 was not found to be cytotoxic even at very high doses
(100 µM).10 However, 1 did not inhibit the specific binding of 3H-
TPA to mouse epidermal particulate fraction even at doses up to 1
mM in Vitro and 10 mg in ViVo.10 Cembranoid 1 was found to
induce a dose-responsive inhibition of TPA-stimulated phospho-
rylation of the 47K-Da human platelet protein and displayed an
IC50 value of 100 µM.10 These results suggested that 1 does not
directly compete with or modulate TPA binding on the protein
kinase C (PKC) receptor.
Several semisynthetic reactions were reported for tobacco
cembranoids.11 These included oxidations of ∆7,8 and ∆11,12, allylic
oxidations around ∆11,12, oxidation of a C-6 secondary alcohol to
a keto group, and acid-catalyzed rearrangements.11 These reactions
were conducted to aid the structure elucidation or correlate different
compounds.11 Only a single structure–activity relationship (SAR)
study was carried out to optimize tobacco cembranoids’ antitumor-
promoting activity.10 This study highlighted the need for better
understanding of SAR through the use of additional semisynthetic
modifications. Therefore, the present study subjected the readily
available tobacco cembranoid 1 to semisynthetic and biotransfor-
mation methods in an attempt to further optimize anticancer activity.
Acylation of C-6 of 1 and 2 using acetic, propionic, butyric, valeric,
and benzoic acid anhydrides reduced the EBV early antigen
formation inhibitory activity and increased the cytotoxicity, sug-
gesting the importance of a free C-6 hydroxy for the activity.10
Cytotoxicity increased with the increase of acyl carbon number.10
Oxidation of the C-6 alcohol of 1 and 2 to ꢀ-hydroxyketones slightly
reduced the activity by increasing the IC50, which further confirmed
the importance of a hydrogen bonding donor pharmacophore at C-6.
This was also evident when the dehydration and rearranged products
of 1, were found to be almost inactive.10 In this study, semisynthetic
carbamoylation of C-6 in 1 was conducted to add more hydrogen
bonding donor and acceptor pharmacophores at this key position,
which may enhance the binding affinity of the products toward its
target protein(s). Similarly, biotransformation and semisynthetic
hydroxylation studies were carried out to test the effect of possible
additional allylic hydroxylation. Halogen substitution in the mac-
Tobacco cembranoids block the expression of the behavioral
sensitization to nicotine and inhibit neuronal acetylcholine receptors
in rats, suggesting a possible use of these compounds for the
treatment of nicotine addiction.6 Tobacco cembranoids also show
insecticidal, prostaglandin, plant growth, and fungal spore germina-
tion inhibitory activities.1,2,5,7
Cembranoids 1 and 2 from cigarette smoke condensate were
reported as antitumor-promoter ingredients in 1985.8 Later, 1 and
2 were reported to inhibit the induction of Epstein–Barr virus (EBV)
early antigen by the phorbol ester 12-O-tetradecanoylphorbol-13-
acetate (TPA) in lymphoblastoid Raji cells.9 Their activity level
was similar to that displayed by retinoic acid, an active form of
vitamin A.9 Cembranoids 1 and 2 were also found to inhibit
the induction of ornithine decarboxylase (OD) activity by TPA in
the mouse epidermis.10 Cembranoids 1 and 2 also inhibited the
promotional effects of TPA on skin tumor formation initiated by
7,12-dimethylbenz[a]anthracene (DMBA) in mice.10 In these
initiation-promotion experiments, 1 was much more active than
its epimer 2. Application of 3.3 µM 1 for 40 min prior to TPA
treatment resulted in a 53% reduction in the incidence of papillomas
in mice.10 A dose of 50 µM 1 was able to inhibit 28.4% of 32Pi
incorporation into phospholipids of TPA-stimulated HeLa cells
* To whom correspondence should be addressed. Tel: 318-342-1725.
Fax: 318-342-1737. E-mail: elsayed@ulm.edu.
10.1021/np0704351 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/05/2008

118 Journal of Natural Products, 2008, Vol. 71, No. 1
El Sayed et al.
rocyle aimed at the examination of the effect of electron-
withdrawing groups (+σ) like Cl or Br on the anticancer activity.
Catalytic reduction of 1 resulted in a totally inactive hexahydro
analogue.10 Hence, macrocyclic double bonds proved essential for
tobacco cembranoid EBV early antigen formation induction inhibi-
tory activity.10 The 11S,12S-epoxy analogue of 1 was devoid of
activity, indicating the importance of ∆11,12 for activity.10 C-11 or
C-12 hydroperoxide analogues of 1 were also found to be much
less active and more cytotoxic than the parent compound, further
confirming the significance of ∆11,12 for the activity.10 Reaction
of NH4SCN/SbCl3 with the C-7/C-8 oxirane group in the related
marine cembranoid sarcophine afforded the ꢀ-hydroxy thiocyanate
and 1,3-oxathiolan-2-imine with enhanced antiproliferative activ-
ity.12 The same reaction was conducted using 4 as a starting material
to test whether the activity would also be increased and to test
whether the CdNH group in the 1,3-oxathiolan-2-imine will be
able to replace the ∆11,12 pharmacophore. In addition, studies were
conducted to determine if antiproliferative activity could be
increased through enhancing the binding affinity toward the target
protein of 1, since sulfur and oxygen may act as hydrogen bonding
acceptors while the NH (in 1,3-oxathiolan-2-imine) and C-11
hydroxy group (in ꢀ-hydroxy thiocyanate) may act as hydrogen
bonding donors. Condensation reaction of NH4SCN with R-ketol
(1S,2E,4S,7E,11E)-2,7,11-cembratrien-4-ol-6-one (10) was also
conducted to prepare the R,ꢀ-unsaturated imine hydrothiocyanate
to replace the C-6 hydroxy with this polar group and determine
the effect of this modification on anticancer activity as compared
to the parent compound.
(4 and 5, respectively) in a 2:1 ratio.13 Reaction of 3 with SeO2 in
1,4-dioxane, without H2O2, resulted in the known (1S,2E,4S,-
6R,7E,11E,13R)-2,7,11-cembratriene-4,6,13-triol-6-O-acetate (6)
and (1S,2E,4S,6R,7E,11E,13S)-2,7,11-cembratriene-4,6,13-triol-6-
O-acetate (7).14 Identification of these compounds was based on
extensive analysis of their NMR data and comparison with
literature.11,13,14
The epoxide (1S,2E,4S,6R,7E,11S,12S)-2,7-cembradiene-11,12-
epoxy-4,6-diol-6-O-acetate (4) was reacted with NH4SCN/SbCl3
to afford ꢀ-hydroxy thiocyanate (8) and 1,3-oxathiolan-2-imine (9)
in 23.1% combined yields. The HRESMS of 8 showed a molecular
ion peak at m/z 466.2341 [M + Na]+, suggesting the molecular
formula C23H37NO4S. The IR spectrum of 8 showed a characteristic
1
band for a SCN group at 2152 cm-1. The H and 13C data of 8
(Tables 1 and 2) further suggested the replacement of the C-11/
C-12 oxirane with a ꢀ-hydroxy thiocyanate functionality. The
quaternary carbon at δ 111.9 was assigned the thiocyanate carbon
C-21. The quaternary carbon at δ 62.8 was assigned C-12. This
was based on its 2J-HMBC correlation with the methyl singlet H3-
20 (δ 1.51). Similarly, the oxymethine proton doublet at δ 3.43
was assigned H-11. This was also based on the 3J-HMBC
correlation between the methyl singlet H3-20 and the oxygenated
methine carbon C-11 (δ 70.3). Proton H-11 shows a COSY
correlation with H2-10 (δ 1.58). The location of the thiocyanate
group on C-12 rather than C-11 was also based on the carbon
chemical shift of the quaternary C-12. The relative stereochemistry
of C-11 and C-12 was established by extensive study of NOESY
spectral data. The ꢀ-oriented H3-18 (δ 1.31) showed a strong
NOESY correlation with H-11, which in turn showed a NOESY
correlation with H3-20, suggesting a similar orientation.
Results and Discussion
The HRESMS data of 9 suggested a molecular formula identical
High amounts of tobacco cembranoids 1 and 2 were isolated
from fresh Nicotiana tabacum leaf powder. Their identification was
based on extensive analyses of NMR data and comparison with
the literature.1–3,11
Allylic hydroxylation of 1 using SeO2 was unsuccessful.
Therefore, the more stable 6-O-acetate (3) was prepared from 1
using acetic anhydride/pyridine and was subsequently used for
oxidations with SeO2 under different conditions. Reaction of 3 with
SeO2 in 1,4-dioxane at room temperature in the presence of 30%
H2O2 produced the 11S,12S- and 11R,12R-epoxide analogues of 3
1
to 8. The H and 13C NMR spectral data of 9 (Tables 1 and 2)
suggested the formation of a 1,3-oxathiolane ring at C-11/C-12.
The methyl singlet H3-20 (δ 1.37) showed a 2J-HMBC correlation
with C-12 (δ 64.0) and a 3J-HMBC correlation with C-11 (δ 91.4).
The exchangeable NH singlet at δ 7.20 showed a 2J-HMBC
correlation with the imine quaternary carbon C-21 (δ 187.5),
confirming the 1,3-oxathiolane skeleton. The relative stereochem-
istry of C-11 and C-12 was deduced by a NOESY experiment. The
ꢀ-oriented methyl singlets H3-18 (δ 1.31) and H3-20 (δ 1.37)

(1S,2E,4S,6R,7E,11E)-2,7,11-Cembratriene-4,6-diol
Journal of Natural Products, 2008, Vol. 71, No. 1 119
Table 1. 1H NMR Data of 8, 9, 11, 13, and 14a
8
9
11
13
14
position
δH
δH
δH
δH
δH
1
2
3
5
1.88, m
1.76, m
1.61, m
5.57, dd (8.4, 15.4)
5.44, d (15.4)
2.72, dd (5.4, 14.6)
2.42, dd (2.9, 14.6)
1.70, m
1.70, m
5.75, dd (9.5, 15.4)
5.64, d (15.4)
2.07, dd (3.6, 16.8)
1.97, dd (5.4, 16.8)
5.56, m
5.52, dd (9.1, 15.0)
5.35, d (15.0)
2.12, dd (5.4, 14.6)
1.94, dd (2.9, 14.6)
5.66, m
5.52, dd (9.1, 15.4)
5.41, d (15.4)
2.20, dd (5.8, 14.3)
1.80, dd (2.5, 14.3)
4.60, m
5.52, dd (9.1, 15.4)
5.42, d (15.4)
2.22, dd (5.8, 14.3)
1.80, dd (2.5, 14.3)
4.60, m
6
7
9
5.55, d (15.0)
2.30, 2H, m
1.58, 2H, m
3.43, d (10.2)
1.71, m 1.39, m
1.85, m 1.35, m
1.40, m
0.79, 3H, d (6.6)
0.90, 3H, d (6.6)
1.31, 3H, s
5.35, d (15.0)
2.25, m; 1.82, m;
1.45, 2H, m
4.44, dd (3.6, 11)
1.35, 2H, m
1.73, 2H, m
1.65, m
0.85, 3H, d (6.6)
0.88, 3H, d (6.6)
1.31, 3H, s
6.03, s
5.57, br d (9.5)
2.00, 2H, m
5.56, br d (9.8)
1.85, 2H, m
3.65, m; 2.02, m
2.22, m; 2.02, m
4.96, dd (6.2, 6.2)
2.10, m 1.90, m
1.49, 2H, m
10
11
13
14
15
16
17
18
19
20
Ac-6
NH
2.33, m 1.95, m
4.46, dd (6.2, 10.2)
2.29, m 1.90, m
1.65, m 1.45, m
1.59, m
0.83, 3H, d (6.6)
0.86, 3H, d (6.6)
1.28, 3H, s
2.24, m 1.82, m
4.31, dd (6.2, 9.8)
2.15, m 1.75, m
1.65, m 1.35, m
1.60, m
0.85, 3H, d (6.6)
0.88, 3H, d (6.6)
1.26, 3H, s
1.51, m
0.79, 3H, d (6.6)
0.82, 3H, d (6.6)
1.27, 3H, s
1.84, 3H, d (1.4)
1.47, 3H, s
1.65, 3H, s
1.51, 3H, s
2.02, 3H, s
1.72, 3H, s
1.37, 3H, s
2.00, 3H, s
7.20, s
1.63, 3H, s
4.99, s; 5.18, s
1.62, 3H, s
5.00, s; 5.15, s
a In CDCl3, 400 MHz. Coupling constants (J) are in Hz.
Table 2. 13C NMR Data of Compounds 8, 9, 11, 13, and 14a
is predicted to undergo a cis ring closure to form the stable 1,3-
oxathiolan-2-imine (9), which represents a strained ring system.12,15
The thiocyanohydrin intermediates of oxirane conversion with
thiocyanate have been particularly difficult to isolate until the regio-
and stereoselective synthesis of trans intermediates was reported.15–18
It is worth noting that this reaction resulted in cis thiocyanohydrin
and 1,3-oxathiolan-2-imine intermediates similar to sarcophine.12
This selectivity could be attributed to the similarity of the
cembranoid macrocycle ring size and conformation.12
8
9
11
δC
13
δC
14
δC
position
δC
δC
1
48.6, CH
50.4, CH
46.9, CH
48.6, CH
48.8, CH
2
3
132.5, CH 128.4, CH 131.2, CH 128.6, CH 128.4, CH
134.9, CH 140.1, CH 136.1, CH 140.3, CH 140.0, CH
4
5
6
72.6, qC
47.2, CH2 46.6, CH2 54.1, CH2 46.8, CH2 46.8, CH2
68.3, CH 70.1, CH 202.2, qC 68.3, CH 68.4, CH
72.5, qC
72.6, qC
74.1, qC
74.2, qC
7
8
9
123.5, CH 123.5, CH 126.5, CH 128.3, CH 128.3, CH
141.1, qC 139.7, qC 160.3, qC 136.7, qC 136.9, qC
35.4, CH2 34.6, CH2 31.5, CH2 37.0, CH2 36.1, CH2
23.2, CH2 25.9, CH2 25.7, CH2 29.4, CH2 29.4, CH2
Overnight reflux of 1 with CrO3 in pyridine afforded the R-ketol
(1S,2E,4S,7E,11E)-2,7,11-cembratrien-4-ol-6-one (10).3,5 In an at-
tempt to prepare the R,ꢀ-unsaturated imine hydrothiocyanate,
R-ketol 10 was reacted with NH4SCN in toluene.19 Although the
reaction did not proceed as planned, two major products, 11 and
the known secocembranoid 12, were isolated.3,5,11
10
11
12
13
14
15
16
17
18
19
20
21
70.3, CH
62.8, qC
91.4, CH 123.5, CH
64.0, qC 134.7, qC 148.5, qC 148.0, qC
56.8, CH
64.1, CH
30.3, CH2 33.8, CH2 36.2, CH2 35.6, CH2 34.7, CH2
27.5, CH2 27.3, CH2 29.0, CH2 28.8, CH2 28.6, CH2
The HRESMS of 11 showed a molecular ion peak at m/z
327.2288 [M + Na]+, suggesting the molecular formula C20H32O2.
32.6, CH
32.8, CH
32.2, CH
33.3, CH
33.3, CH
20.7, CH3 19.4, CH3 20.0, CH3 19.3, CH3 19.3, CH3
21.1, CH3 21.0, CH3 20.2, CH3 21.0, CH3 20.9, CH3
29.7, CH3 32.0, CH3 30.0, CH3 32.5, CH3 32.4, CH3
15.9, CH3 15.9, CH3 25.0, CH3 15.6, CH3 15.6, CH3
27.2, CH3 25.4, CH3 15.0, CH3 113.3, CH3 113.5, CH3
111.9, qC 187.5, qC
1
The H and 13C NMR data of 11 (Tables 1 and 2) were similar
to the parent R-ketol 10 except for the segment C-7-C-9-C-19.20,21
The methyl carbon C-19 (δ 25.0) of 11 was downfield shifted,
compared with that of 10 (δ 15.7).20,21 The methylene carbon at δ
31.5 was assigned C-9. This was based on the 3J-HMBC correlation
with the H3-19 methyl doublet (δ 1.84). Carbon C-9 showed a
HETCOR correlation with proton multiplets at δ 3.65 and 2.02
(H-9a and H9b). The downfield shifting of proton H-9a may be
due to its location in the deshielding cone of ∆7,8. The double bond
geometry of ∆7,8 was assigned as Z on the basis of the 13C NMR
chemical shift of the C-19 methyl and the strong NOESY correlation
between H-7 (δ 6.03) and H3-19.3,5,11,21 Therefore, compound 11
was identified as (1S,2E,4S,7Z,11E)-2,7,11-cembratrien-4-ol-6-one.
Ac-6 170.3, qC 169.3, qC
21.4, CH3 21.3, CH3
a In CDCl3, 100 MHz. Carbon multiplicities were determined by
DEPT135° or APT experiments. qC ) quaternary, CH ) methine, CH2
) methylene, CH3 ) methyl carbons.
showed strong NOESY correlations with H-11 (δ 4.44), suggesting
a similar stereo-orientation.
Reactions of epoxides with NH4SCN usually afford the ꢀ-hy-
droxy thiocyanate intermediates followed by the end products
thiiranes.15 Although this reaction mechanism was proposed in the
1950s, the thiocyanohydrin intermediate was not isolated until
recently.15–17 Cyclic epoxides yield trans ꢀ-hydroxy thiocyanates
under reasonably mild conditions and with proper catalysts.16–18
These reactions are known to proceed with high regioselectivity,
almost yielding anti-Markovnikov products.16–18 However in sar-
cophine and (1S,2E,4S,6R,7E,11S,12S)-2,7-cembradiene-11,12-
epoxy-4,6-diol-6-O-acetate (4), where the epoxide is attached to a
14-membered macrocycle, sulfur attack was exclusively on the more
substituted carbon as per Markovnikov’s rule, suggesting a car-
bocation intermediate followed by a thermodynamically stable
product.12,15 The syn addition of the nucleophile resulted in the
cis ꢀ-hydroxy thiocyanate 8.12 The cis thiocyanohydrin intermediate
Halogenations of 1 using N-bromosuccinimide (NBS) or N-
chlorosuccinimide (NCS) in 10% aqueous acetone at room tem-
perature afforded the halogenated cembranoids 13 and 14,
respectively.
Compound 13 showed the molecular ion peak at m/z 407.1564
[M + Na]+, suggesting the molecular formula C20H33BrO2. The
mass spectrum of 13 also showed typical M and M + 2, 1:1,
isotopic clusters characteristic for monobrominated compounds. The
1H and 13C NMR data of 13 (Tables 1 and 2) showed a different
C-11-C-12-C-20 segment compared to those of the starting
material 1. The 1H NMR spectrum of 13 showed signals for
brominated and exocyclic methylene protons at δ 4.46 (H-11), 5.18
(H-20a), and 4.99 (H-20b), replacing the olefinic H-11 and the
methyl H3-20 in 1, respectively. Proton H-11 showed a COSY

120 Journal of Natural Products, 2008, Vol. 71, No. 1
El Sayed et al.
a
1
Table 3. 13C and H NMR Data of Compounds 15–17
15
16
17
position
δC
δH
δC
δH
δC
δH
1
2
3
4
46.4, CH
127.9, CH
137.2, CH
72.4, qC
1.60, m
5.29, dd (15.8, 9.2)
5.31, d (15.8)
46.4, CH
127.9, CH
137.4, CH
72.4, qC
1.60, m
5.30, dd (15.4, 9.2)
5.32, d (15.4)
46.4, CH
127.9, CH
137.3, CH
72.4, qC
1.60, m
5.30, dd (15.4, 8.8)
5.31, d (15.4)
5
6
7
8
50.8, CH2
69.7, CH
126.8, CH
139.4, qC
38.9, CH2
23.2, CH2
124.4, CH
133.6, qC
36.7, CH2
27.9, CH2
33.0, CH
19.5, CH3
20.8, CH3
29.8, CH3
16.3, CH3
14.9, CH3
155.7, qC
42.8, CH2
44.4, CH2
1.95, 2H, m
5.40, dd (10.5, 9.9)
5.21, d (10.2)
50.9, CH2
69.1, CH
127.1, CH
139.1, qC
39.0, CH2
23.3, CH2
124.50, CH
133.5, qC
36.7, CH2
27.9, CH2
33.0, CH
19.5, CH3
20.8, CH3
29.8, CH3
16.3, CH3
14.9, CH3
155.8, qC
36.0, CH2
15.3, CH3
1.95, 2H, m
5.40, dd (9.9, 9.2)
5.19, d (9.9)
50.9, CH2
69.5, CH
1.99, 2H, m
5.45, dd (9.9, 9.1)
5.21, d (9.9)
127.0, CH
139.3, qC
39.0, CH2
23.3, CH2
124.5, CH
133.5, qC
36.7, CH2
27.9, CH2
33.0, CH
19.5, CH3
20.8, CH3
29.8, CH3
16.9, CH3
1490., CH3
156.0, qC
45.2, CH2
138.5, qC
127.6, CH
128.8, CH
127.6, CH
9
2.20, m 2.05, m
2.20, m 2.10, m
4.98, dd (5.9, 5.2)
2.25, m 2.10, m
2.10, m
4.99, dd (5.4, 5.2)
2.25, m 2.05, m
2.30, m 2.10, m
4.99, dd (5.2, 5.2)
10
11
12
13
14
15
16
17
18
19
20
1′
2′
3′
4′, 8′
5′, 7′
6′
2.05, m 1.90, m
1.65, m 1.30, m
1.55, m
0.77, d (6.6)
0.80, d (6.6)
1.38, s
2.05, 2H, m
1.60, m 1.35, m
1.55, m
0.77, d (6.6)
0.80, d (6.6)
1.38, s
2.05, m 1.90, m
1.65, m 1.25, m
1.55, m
0.77, d (6.6)
0.81, d (6.6)
1.39, s
1.49, s
1.72, s
1.49, s
1.72, s
1.49, s
1.73, s
3.59, brt (4.8)
3.50, brdt (5.9, 4.8)
3.20, dt (7.0, 4.3)
1.11, t (7.4)
4.31, brs
7.26, 2H, m
7.32, 2H, m
7.27, d (8.4)
4.95, brs
-NH
5.09 brt, (5.9)
4.60, brt (4.4)
a In CDCl3, 400 MHz for 1H and 100 MHz for 13C NMR. Coupling constants (J) are in Hz. Carbon multiplicities were determined by DEPT135° or
APT experiments. qC ) quaternary, CH ) methine, CH2 ) methylene, CH3 ) methyl carbons.
correlation with H2-10 (δ 2.33 and 1.95) and a 2J-HMBC correlation
to C-10. The exomethylene protons H2-20 showed a J-HMBC
The 1H and 13C NMR data of 17 (Table 3) indicated a
monosubstituted benzene moiety replacing the methyl C-3′ in 16.
The broad methylene singlet H2-2′ (δ 4.31) showed a J-HMBC
correlation with the aromatic proton multiplet H-4′ and H-8′ (δ
7.26). The latter protons showed a COSY coupling with H-5′ and
H-7′ multiplet (δ 7.32), which in turn showed a COSY coupling
with the H-6′ doublet (δ 7.27).
3
3
correlation to C-11 (δ 56.8). The relative stereochemistry assign-
ment at C-11 was aided by NOESY data. The proton doublet of
doublets H-5a (δ 2.20) showed a strong NOESY correlation with
H-6, suggesting its ꢀ-orientation. The R-oriented H-5b (δ 1.80)
showed a strong NOESY correlation with H-10a (δ 2.33), which
in turn showed a NOESY correlation with H-11, suggesting similar
stereo-orientation. Therefore, compound 13 was identified as
(1S,2E,4S,6R,7E,11S)-2,7,12(20)-cembratriene-11-bromo-4,6-
diol.
Biocatalysis of 1 using Mucor ramannianus ATCC 9628 and
Cunninghamella elegans ATCC 7929 afforded the known (1S,2E,
4S,6R,7E,11S,12S)-2,7-cembradiene-11,12-epoxy-4,6-diol-6-O-ac-
etate (4) as the main metabolite, an observation that was consistent
with previous fermentation studies using plant cell cultures.22
Biocatalysis of the cembranoid analogue (3) using the marine
symbiotic Bacillus megaterium strain MO31, isolated form the Red
Sea sponge Negombata magnifica, afforded the known (1S,2E,4S,6R,
7E,11E,10R)-2,7,11-cembratriene-4,6,10-triol (18) in 30% yield.5,14
Antiproliferative Activity. The effect of various concentrations
of the cembranoids 1-18 was studied on the proliferation of the
highly malignant mice +SA mammary epithelial cells.25 Cembra-
noid 1 and analogues 6, 7, 11, and 12 show potent antiproliferative
activity against malignant +SA mammary epithelial cells at a 15–40
µM dose compared to their respective vehicle controls (Figure 1).
This is a relevant activity compared with the positive drug control
δ-tocotrienol, which showed IC50 7 µM under the same assay
conditions.25 Anticancer activity was enhanced by the use of allylic
oxidation at C-13 (Figure 1). Cembranoid 1 and its derivatives were
found to be cytostatic, but not cytotoxic, to the neoplastic mammary
epithelial cells grown in culture. The most active analogues were
(1S,2E,4S,6R,7E,11E,13S)-2,7,11-cembratriene-4,6,13-triol-6-O-
acetate (7) and (1S,2E,4S,6R,7E,11E,13R)-2,7,11-cembratriene-
4,6,13-triol-6-O-acetate (6), with IC50 20 and 30 µM, respectively
(Figure 1). This clearly indicates that the C-13 hydroxy group
enhances the binding affinity and the activity. The 13S configuration
as in 7 was much more active than the 13R, suggesting that
ꢀ-hydroxylation is more favorable for the activity. The microbial
metabolite (1S,2E,4S,6R,7E,11E,10R)-2,7,11-cembratriene-4,6,10-
triol (18) had no affect on mammary tumor cell growth, indicating
The HRESMS data of 14 suggested a molecular formula similar
1
to that of 13 with the replacement of Br with Cl. The H and 13C
NMR data of 14 (Tables 1 and 2) further supported this conclusion.
The chlorinated carbon C-11 (δ 64.1) was assigned on the basis of
its 3J-HMBC correlation with H2-20. The relative stereochemistry
of C-11 was assigned in a similar fashion to that of 13. Therefore,
compound 14 was identified as (1S,2E,4S,6R,7E,11S)-2,7,12(20)-
cembratriene-11-chloro-4,6-diol.
Reflux of 1 in toluene with chlororethyl, ethyl, and benzyl
isocyantes in the presence of a catalytic amount of triethylamine
afforded the C-6 carbamates 15-17, respectively.
The HRMS data analysis of 15 revealed the molecular formula
C23H38ClNO3 with the M and M + 2, 3:1, isotopic cluster
characteristic pattern for a monochlorinated compound. The down-
field shifting of the C-6 carbon chemical shift in 15 (+3.6 ppm)
compared with that of the starting material 1 suggested carbam-
oylation at this position.5,11 1H and 13C NMR data (Table 3) further
supported this fact. The carbonyl carbon at δ 155.7 was assigned
C-1′. This was based on its 3J-HMBC correlation with the
methylene triplet H2-2′ (δ 3.59), which in turn showed COSY
coupling with the H2-3′ doublet of triplets (δ 3.50).
The HRMS data of 16 suggested the molecular formula
1
C23H39NO3. The H and 13C NMR data (Table 3) were similar to
those of 15, with the replacement of the terminal chlorinated
methylene C-3′ with a methyl group. The methyl triplet H3-3′ (δ
1.11) was assigned on the basis of its COSY coupling with the
H2-2′ doublet of triplets (δ 3.20).

(1S,2E,4S,6R,7E,11E)-2,7,11-Cembratriene-4,6-diol
Journal of Natural Products, 2008, Vol. 71, No. 1 121
was stirred at room temperature for 3 h. Water (10 mL) was then added,
and the product was extracted with EtOAc. The EtOAc extract was
dried over anhydrous Na2SO4 and concentrated under reduced pressure.
The crude product was purified by CC on Si gel 60 (EtOAc/n-hexane,
2:3, isocratic) to give compounds 4 (42 mg, 42.2%) and 5 (20 mg,
20.1%).
Preparation of Compounds 6 and 7. To a solution of 3 (35.2 mg)
in dioxane (2 mL) was added SeO2 (11 mg), and the solution was stirred
at room temperature for 5 h. Water (10 mL) was then added, and the
product was extracted with EtOAc. The EtOAc extract was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The crude
product was purified by CC on Si gel 60 (EtOAc/n-hexane, 2:3,
isocratic) to give compounds 6 (5.0 mg, 13.5%) and 7 (3.4 mg, 9.2%).
Preparation of Compounds 8 and 9. Compound 4 (34 mg) was
dissolved in 5 mL of anhydrous CH3CN. To this solution were added
19 mg of NH4SCN and 10 mg of SbCl3, and the mixture was stirred
under reflux for 4 h. Water (10 mL) was then added, and the product
was extracted with EtOAc. The EtOAc extract was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The crude product
was purified by CC (EtOAc/n-hexane, 2:3, isocratic) to give compounds
8 (3.7 mg, 9.4%) and 9 (5.4 mg, 13.7%).
Preparation of Compounds 10–12. Compound 1 (113.2 mg) was
dissolved in 5 mL of pyridine. About 56.5 mg of CrO3 was added, and
the mixture was stirred at room temperature for 24 h. Water (10 mL)
was then added, and the product was extracted with EtOAc. EtOAc
extract was dried over anhydrous Na2SO4 and concentrated under
reduced pressure to give 10 (84 mg). Compound 10 (84 mg) was
dissolved in toluene (3 mL), and then 50 mg of NH4SCN was added
and the mixture was stirred with reflux for 5 h. Water (10 mL) was
then added, and the product was extracted with EtOAc. The EtOAc
extract was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The crude product was purified by CC on Si gel
(EtOAc/hexane, 2:3, isocratic) to give 11 (10.3 mg, 12.2%) and 12
(6.6 mg, 7.8%).
Preparation of 13 and 14. A solution of 25 mg of NBS or 36 mg
of NCS in 1 mL of 10% aqueous acetone was slowly added to 2 mL
of a solution of 1 (80 or 30.9 mg) in 10% aqueous acetone. The reaction
mixture was stirred at room temperature for 15 min (NBS) or 18 h
(NCS). Each reaction was stopped by adding water (10 mL), and the
mixture was extracted with CHCl3 (2 × 10 mL). The CHCl3 layer was
dried over anhydrous NaSO4 and evaporated under vacuum to give a
crude product. This crude product was chromatographed by CC on Si
gel 60 using elution with n-hexane/EtOAc (1:1) to afford 13 (70.5 mg;
70%) or 14 (9.3 mg; 27%).
Preparation of Carbamates 15–17. To solutions of 1 (22.3, 73.3,
or 88.8 mg) in toluene (2 mL) was added 59 µL of 2-chloroethyl
isocyanate or 47 µL of ethyl isocyanate or 36 µL of benzyl isocyanate,
respectively, and the solutions were separately mixed with 10 µL of
Et3N. Each solution was separately stirred and refluxed for 3 h. Water
(10 mL) was then added, and the product of each reaction mixture was
extracted with EtOAc. Each EtOAc extract was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. Crude products were
then purified by CC on Si gel 60 (EtOAc/n-hexane, 2:8 or 1:9) to give
compounds 15, 15.7 mg, 52%; 1d, 49.6 mg, 55%; and 17, 64.0 mg,
50%, respectively.
Symbiotic Bacteria Culture and Isolation. About 2 g of frozen
N. magnifica voucher, which was collected and kept frozen in sterile
bags, was macerated overnight in 0.5 L of Instant Ocean solution, and
then the mixture was vacuum filter sterilized. Another 2 g of the sponge
aseptically was blended in a sterile blender with 18 mL of the sponge
Instant Ocean solution. Ten mL 1/10, 1/102, 1/103, 1/104, and 1/105
serial dilutions were made using the previously prepared Instant Ocean
solution. About 100 µL of each concentration was inoculated on the
top of sterile marine agar plates. Plates were incubated for 72 h at 28
°C. Symmetric fine colonies were separated and reinoculated on marine
agar media. Pure cultures were subjected to PCR analysis, DNA
extraction (MO Bio Laboratories, Inc., Carlsbad, CA), and finally 16S
rRNA sequencing.23 Obtained alignments were subjected to nucleotide
blast queries. Identity was based on >99% matching.
Biocatalysis. Biocatalytic studies were conducted as described
elsewhere.24 Thirty growing fungal and marine bacterial cultures were
used for screening of 1 and 3.24 M. ramannianus ATCC 9628 and C.
elegans ATCC 7929 were selected for biocatalysis scale-up of 1, while
the marine symbiotic B. megaterium strain MO31 was selected for the
scale-up of 3. Each of these organisms was inoculated in ten 1000 mL
Figure 1. Effects of various doses of 1 and active analogues on
malignant +SA mammary epithelial cell proliferation.
that C-10 hydroxylation reduces anticancer bioactivity, unlike C-13
hydroxylation.
The cis ꢀ-hydroxy thiocyanate (8) and the 1,3-oxathiolan-2-imine
(9) analogues were devoid of activity, indicating that ∆11,12 is
essential for activity and cannot be replaced with heterocyclic SP2,
SP, or heteroatom-containing functional groups. The carbamates
15–17 were also inactive, suggesting the importance of the free
C-6 hydroxy group. This is supported by the reduced activity of
C-6-O-acetate (3), compared with 1. Most likely 3 acts as a prodrug,
which is activated by enzymatic acetate hydrolysis with the
formation of free 1. This conclusion was based on the rapid
enzymatic acetate hydrolysis by most microbial species during
biocatalytic screening, while 3 was stable for 14 days in the substrate
control (tested compound in blank media without microorganisms).
Interestingly, although the R-ketol (1S,2E,4S,7E,11E)-2,7,11-
cembratrien-4-ol-6-one (10) was almost devoid of antiproliferative
activity, its geometrical isomer (1S,2E,4S,7Z,11E)-2,7,11-cem-
bratrien-4-ol-6-one (11) was found to be a potent antiproliferative
agent, suggesting the possible selectivity of certain double-bond
geometry. Although this compound was no more active than its
parent cembranoid (1) at higher doses, the secocembranoid 12
showed potent antiproliferative activity with an IC50 of 15 µM
(Figure 1). This activity indicated that the 14-membered macrocycle
is not essential for antiproliferative activity.
Experimental Section
General Experimental Procedures. The NMR data were acquired
in CDCl3, on a JEOL Eclipse NMR spectrometer operating at 400 MHz
1
for H and 100 MHz for 13C. Optical rotations were measured on a
Rudolph Research Analytical Autopol III polarimeter. IR spectra were
recorded on a Varian 800 FT-IR Scimitar series. The HRESMS
experiments were conducted at the University of Michigan on a
Micromass LCT spectrometer. TLC analyses were carried out on
precoated silica gel 60 F254 500 µm TLC plates, using the developing
system n-hexane/EtOAc (1:1) or CHCl3/MeOH (9.5:0.5). For CC, Si
gel 60 (particle size 63–200 µm) or Bakerbond octadecyl (C18), 40
µm, was used.
Extraction and Isolation. Fresh tobacco leaf powder (Custom
Blends, New York, 27.2 kg) was extracted with n-hexane (45 L) in
percolators three times at room temperature. The n-hexane extract was
concentrated under vacuum, and the dried extract (1050 g) was vacuum
liquid chromatographed on silica gel (200–300 mesh, 2 kg, Natland
International Corporation) using a gradient of n-hexane/EtOAc to yield
a crude cembranoid-containing fraction (64.0 g), which was further
chromatographed on normal-phase and finally on reversed-phase silica
gel (MeOH/H2O, 2:3, isocratic) to give 1 (17.9 g) and 2 (3.6 g). The
identity of 1 was confirmed by extensive NMR analysis and comparison
with literature.1,2,5,11
Epoxidation of 1. To a solution of 2 (100 mg) in dioxane (3 mL)
were added SeO2 (20 mg) and 30% H2O2 (100 µL), and the solution

122 Journal of Natural Products, 2008, Vol. 71, No. 1
El Sayed et al.
flasks each containing 250 mL of compound medium R (for fungi) or
marine broth (for bacteria).24 After 72 h, compounds 1 and 3 were
added into their respective flasks (15 mg/flask). After 14 days, the
growth medium was filtered and extracted with EtOAc (4 × 1000 mL).
The EtOAc layer was then concentrated under vacuum. Residues
obtained from biocatalysis of 1 or 3 were purified on a silica gel 60
column, followed by reversed-phase Si gel medium-pressure liquid
chromatography (MPLC) to yield (1S,2E,4S,6R,7E,11S,12S)-2,7-cem-
bradiene-11,12-epoxy-4,6-diol-6-O-acetate (4) as the main metabolite
(38 mg, Rf 0.46, CHCl3/MeOH, 9:1), along with other unstable minor
metabolites. B. megaterium MO31 afforded the known (1S,2E,
4S,6R,7E,11E,10R)-2,7,11-cembratriene-4,6,10-triol (18) (46 mg, Rf
0.31, CHCl3/MeOH, 9:1).
responsibility of the authors and do not necessarily represent the official
views of the NIH. The American Foundation for Pharmaceutical
Education is acknowledged for fellowship support to J.A.P.
References and Notes
(1) Wahlberg, I.; Enzell, C. R. Beitr. Tabakforsch. Int. 1984, 12, 93–104.
(2) Wahlberg, I.; Eklund, A. M. Trends in FlaVour Research; Maarse,
H.; van der, Heji, Eds.; Elsevier Science: Oxford, UK, 1994; pp 449–
462.
(3) Eklund, A.-M.; Hatanaka, S.-I.; Wahlberg, I. Progress in the Chemistry
of Organic Natural Products; Springer-Verlag Wien: New York, 1992;
pp 143–293.
(4) Dauben, W. G.; Thiessen, W. E.; Resnick, P. R. J. Am. Chem. Soc.
1962, 84, 2015–2016.
(5) Wahlberg, I.; Olsson, E.; Berg, J. E. Progress in FlaVour Precursor
Studies Proceedings of International Conference; Schreier, P., Win-
terhalter, P., Eds.; Allured Publishing Corporation: Carol Stream, IL,
1993; pp 83–95.
(6) Ferchmin, P. A.; Hao, J.; Perez, D.; Penzo, M.; Maldonado, H. M.;
Gonzalez, M. T.; Rodriguez, A. D.; Jean, D. V. J. Neurosci. Res. 2005,
82, 631–641.
(7) Olsson, E.; Holth, A.; Kumlin, E.; Bohlin, L.; Wahlberg, I. Planta
Med. 1993, 59, 293–295.
(8) Saito, Y.; Takayawa, H.; Konishi, S.; Yoshida, D.; Mizusaki, S.
Carcinogenesis 1985, 6, 1189–1194.
(9) Saito, Y.; Nishino, H.; Yoshida, D.; Mizusaki, S.; Ohnishi, A. Oncology
1988, 45, 122–126.
(10) Saito, Y.; Tsujino, Y.; Kaneko, H.; Yoshida, D.; Mizusaki, S. Agric.
Biol. Chem. 1987, 51, 941–943.
(11) Wahlberg, I.; Eklund, A. M. In Progress in the Chemistry of Organic
Natural Products; Herz, W., Kirby, G. W., Moore, R. E., Steglich,
W., Tamm, C. H., Eds.; Springer-Verlag, Wien: New York, 1992;
Vol. 59, pp 142–293.
(12) Sawant, S.; Youssef, D.; Mayer, A.; Sylvester, P.; Wali, V.; Arant,
M.; El Sayed, K. Chem. Pharm. Bull. 2006, 58, 1119–1123.
(13) Behr, D.; Wahlberg, I.; Nishida, T.; Enzell, C. R.; Berg, J. E.; Pilotti,
A. M. Acta Chem. Scand. Ser. B: Org. Chem. Biochem. 1980, B34,
195–202.
(14) Forsblom, I.; Berg, J. E.; Wahlberg, I. Acta Chem. Scand. 1993, 47,
80–88.
(15) (a) Van Tamelen, E. E. J. Am. Chem. Soc. 1951, 73, 3444–3448. (b)
Sharghi, H.; Nasseri, M. A.; Niknam, K. J. Org. Chem. 2001, 66,
7287–7293.
(16) Tamami, B.; Mahdavi, H. Tetrahedron Lett. 2002, 43, 6225–6228.
(17) Yadav, J. S.; Reddy, B. V. S.; Reddy, S. Tetrahedron Lett. 2004, 45,
1291–1293.
(18) Akhrem, A. A.; Istomina, Z. I.; Turuta, A. M.; Kogan, G. A. B. Acad.
Sci. USSR CH+ 1973, 22, 82–87.
(19) Kabas, G. J. Org. Chem. 1967, 32, 218–220.
(20) Iwagawa, T.; Nakamura, S.; Okamura, H.; Nakatami, M. Bull. Chem.
Soc. Jpn. 1996, 69, 3543–3549.
Compound 8: colorless oil; [R]25 +11.2 (c 0.26, CHCl3); IR νmax
D
(neat) 3440, 2957, 2928, 2871, 2152, 1725, 1458 cm-1; H and 13C
1
NMR see Tables 1 and 2, respectively; HRESMS m/z 446.2341 [M +
Na]+ (calcd for C23H37NO4SNa, 446.2341).
Compound 9: colorless oil; [R]25 +25.6 (c 0.22, CHCl3); IR νmax
D
(neat) 3437, 2958, 2928, 2856, 1727, 1602, 1485 cm-1; H and 13C
1
NMR see Tables 1 and 2, respectively; HRESMS m/z 446.2343 [M +
Na]+ (calcd for C23H37NO4SNa, 446.2341).
Compound 11: colorless oil; [R]25D +57.8 (c 0.69, CHCl3); IR νmax
1
(neat) 3455, 2957, 2928, 2871, 1669, 1606 cm-1; H and 13C NMR
see Tables 1 and 2, respectively; HRESMS m/z 327.2288 [M + Na]+
(calcd for C20H32O2Na, 327.2300).
Compound 13: yellowish oil; [R]25D +27.8 (c 0.47, CHCl3); IR νmax
(neat) 3482, 2955, 29298, 2870, 16429, 1457 cm-1; 1H and 13C NMR
see Tables 1 and 2, respectively; HRESMS m/z 407.1564 [M + Na]+
(calcd for C20H33BrO2Na, 407.1562).
Compound 14: yellowish oil; [R]25 +43.8 (c 0.062, CHCl3); IR
D
ν
max (neat) 3502, 2956, 2927, 2856, 1602, 1462 cm-1; 1H and 13C NMR
see Tables 1 and 2, respectively; HRESMS m/z 363.2049 [M + Na]+
(calcd for C20H33ClO2Na, 363.2067).
Compound 15: colorless oil; [R]25D +59.8 (c 0.54, CHCl3); IR νmax
(neat) 3450, 2995, 2928, 1713, 1605, 1509, 1457, 1371, 1305, 1258
1
cm-1; H and 13C NMR see Table 3; HRESMS m/z 434.2433 (calcd
for C23H38ClNO3Na, 434.2438).
Compound 16: colorless oil; [R]25D +54.1 (c 0.10, CHCl3); IR νmax
(neat) 3452, 2928, 2855, 1711, 1508, 1459, 1380, 1139, 1099 cm-1
;
1H and 13C NMR see Table 3; HRESMS m/z 400.2838 (calcd for
C23H39NO3Na, 400.2828).
Compound 17: yellowish oil; [R]25D +56.4 (c 0.41, CHCl3); IR νmax
(neat) 3450, 2995, 2928, 1714, 1603, 1509, 1455, 1366, 1261, 1128
1
cm-1; H and 13C NMR see Table 3; HRESMS m/z 462.2973 [M +
Na]+ (calcd for C28H41 NO3Na a, 462.2984).
Antiproliferative Assay. The antiproliferative effects of semisyn-
thetic derivatives were tested in culture on the highly malignant +SA
mouse mammary epithelial cell line maintained on serum-free media
and containing 10 ng/mL EGF and 10 µg/mL insulin as mitogens, as
described previously in detail.25 Cells were plated at a density of 5 ×
104 cells/well (6 wells/group) in 24-well culture plates and fed media
containing various concentrations (0.01–1000 µM) of each compound.
After a 4-day culture period, viable +SA cell number was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) colorimetric assay as described previously.25
(21) Wahlberg, I.; Forsblom, I.; Vogt, C.; Eklund, A.-M.; Nishida, T.;
Enzell, C. R.; Berg, J. E. J. Org. Chem. 1985, 5, 4527–4538.
(22) El Sayed, K. A.; Sylvester, P. W. Expt. Opin. InVest. Drugs 2007, 16,
877–887.
(23) (a) Weisburg, W. G.; Barns, S. M.; Pelletier, D. A.; Lane, D. J. J.
Bacteriol. 1991, 173, 697–703. (b) Reysenbach, A.-L.; Giver, L. J.;
Wickham, G. S.; Pace, N. R. Appl. EnViron. Microbiol. 1992, 58,
3417–3418.
(24) El Sayed, K. A.; Hamann, M. T.; Waddling, C. A.; Jensen, C.; Lee,
S. K.; Dunstan, C. A.; Pezzuto, J. M. J. Org. Chem. 1998, 63, 7449–
7455.
(25) McIntyre, B. S.; Briski, K. P.; Gapor, A.; Sylvester, P. W. PSEBM
2000, 224, 292–301.
Acknowledgment. This publication was made possible by the
support of Philip Morris USA Inc. and Philip Morris International as
well as the NIH grant no. P20RR16456 from the BRIN Program of
the National Center for Research Resources. Its contents are solely the
NP0704351