Semisynthetic analogues of the marine cembranoid sarcophine as prostate and breast cancer migration inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.06.060

Sarcophine (1) is a bioactive cembranoid diterpene isolated from the Red Sea soft coral Sarcophyton glaucum. Previous semisynthesis attempts resulted in decreased or complete loss of 1's anticancer activity. Sarcophine and analogues showed antimigratory a

Full text of DOI:10.1016/j.bmc.2011.06.060

Bioorganic & Medicinal Chemistry 19 (2011) 4928–4934
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Semisynthetic analogues of the marine cembranoid sarcophine as prostate
and breast cancer migration inhibitors
Hossam M. Hassan a,b, Asmaa A. Sallam a, Rabab Mohammed b, Mohamed S. Hifnawy c,
Diaa T. A. Youssef d, Khalid A. El Sayed a,
⇑
a Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71201, USA
b Department of Pharmacognosy, College of Pharmacy, Beni-Suief University, Egypt
c Department of Pharmacognosy, College of Pharmacy, Nahda University, Egypt
d Department of Natural Products and Alternative Medicine, College of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Sarcophine (1) is a bioactive cembranoid diterpene isolated from the Red Sea soft coral Sarcophyton glau-
cum. Previous semisynthesis attempts resulted in decreased or complete loss of 1’s anticancer activity.
Sarcophine and analogues showed antimigratory activity against breast and prostate cancer cell lines.
This encouraged further semisynthestic optimizations to improve its activity and establish a preliminary
structure–activity relationship. Eight new and five known semisynthetic analogues were generated.
These compounds were evaluated for their ability to inhibit growth, proliferation, and migration of the
prostate and breast metastatic cancer cell lines PC-3 and MDA-MB-231, respectively. Most analogues
exhibited enhanced antimigratory activity.
Received 26 April 2011
Revised 15 June 2011
Accepted 21 June 2011
Available online 28 June 2011
Keywords:
Antimigratory
Breast cancer
Carbamoylation
Cembranoids
Etherification
Prostate cancer
Semisynthesis
Wound-healing
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
dose range of 0.4–10 l
M.5 This report pointed the critical impor-
tance of functional groups at carbons C-7/C-8 for the anticancer
activity. The antiproliferative effect of sarcophine was attributed
to its ability to increase the expression of cyclin dependant kinase
(CDK) inhibitor, p21, and to enhance the residual DNA repair with-
out affecting the DNA adduct formation in MCF-7 cells.6 Allylic oxi-
dation of sarcophine and its LiAlH4-reduced products showed
enhanced anticancer activity, especially those containing 1,2-pro-
panediol at C-15/C-16, suggesting that opening of the sarcophine
lactone greatly enhances the activity.7 This product, named sarco-
phine-diol, decreased cell growth and induced apoptosis through
caspase-dependent extrinsic pathway in A431 cells, which justified
its overall chemopreventive effects in mouse skin cancer models.8,9
Sarcophine, 16-deoxysarcophine, and 2-epi-16-deoxysarco-
phine (1–3) showed the most potent antimigratory activity out
of several other tested cembranoids against the highly metastatic
mouse melanoma B16B15b cell line.10 Oxymercuration–demercu-
ration of 1 produced rearranged and hydroxylated products that
displayed no antiproliferative effects, confirming the importance
of the macrocyclic double bonds for the activity.11 Bromination
of sarcophine improved the antiproliferative activity against the
highly malignant +SA mammary epithelial cells.11 The oxidation
product of 1, (+)-sarcophytoxin B, showed antiproliferative activity
Cembranoids are natural diterpenes possessing 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-12.1 They exhibit a wide range of biological activities
including neuroprotective, antimicrobial, and antitumor proper-
ties.1,2 Cembranoids are common secondary metabolites within
both the plant and animal kingdoms.1,2 Examples of plant-derived
cembranoids are those isolated from tobacco, pine oleoresin, olib-
anum (frankincense, Boswellia carteri and Boswellia sacra), and Cle-
ome species.1,2 The majority of natural cembranoids were isolated
from marine invertebrates including soft corals, octacorals, and
gorgonians.1,2
Sarcophine (1) is a bioactive cembranoid first isolated from the
Red Sea soft coral Sarcophyton glaucum by Kashman group in 1974
in up to 3% wet weight yield.3,4 In 1998, sarcophine and its metab-
olite 7a-hydroxy-D
8,(19)-deepoxysarcophine effectively suppressed
TPA-induced anchorage-independent JB6 cell transformation at
⇑
Corresponding author. Tel.: +1 318 342 1725; fax: +1 318 342 1737.
E-mail address: elsayed@ulm.edu (K.A. El Sayed).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.060

H. M. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 4928–4934
4929
equivalent to 1, suggesting that C-7/C-8 epoxide is not required for
activity.11 Attempts to insert sulfur functionalities into sarcophine
afforded the cis-oxathiolan-2-imine and the cyclic oxathiophos-
pholane-2-sulfide analogues, which inhibited +SA cells’ growth
better than 1.12 These analogues induced cell cycle arrest in G0/
G1 phase with concomitant decrease in the cell populations at S
and G2+M phases in the estrogen receptor alpha positive MCF-7
cells but had no effect on the estrogen-receptor negative MDA-
MB-231 breast cancer cells.6 These studies clearly highlighted the
importance of further optimization of C-7/C-8 epoxide functional-
ity of 1. Therefore, etherification and carbamoylation reactions at
C-7/C-8 of 1 were conducted to study the effect of using diverse
side chains at these positions on the antimigratory activity.
1985.13 This compound was selected as a scaffold to prepare di-
verse aliphatic, olefinic, and aromatic C-7-carbamates 10–15 by
reaction with their corresponding isocyanates (Scheme 1). The C-
7 ether and carbamate functionalities offer diverse hydrogen bond-
ing donor (HBD), acceptor (HBA) and
p-aromatic groups at this
critical position with diverse functionalized side chain to optimize
their binding affinities toward their plausible molecular target.
The HRTOFMS data of 5 suggested the molecular formula
C
23H36O4 and possible C-7 propyl ether formation. The 1H and
13C NMR data (Tables 1 and 3) further supported this conclusion.
The NMR data also showed the characteristic signals for the propyl
ether moiety. The b-orientation of C-7 hydroxy was based on the
coupling constant, splitting and chemical shift similarity of H-7
with that of the previously related methyl ether.13 Thus, 5 was
determined to be 7b-propyloxy-8a-hydroxy-deepoxysarcophine.
Similarly, compound 6 was determined to be 7b-butyloxy-8a-hy-
2. Results and discussion
droxy-deepoxysarcophine.
2.1. Semisynthesis of sarcophine analogues
The HRTOFMS data of 10 and 11 suggested possible butylcarba-
moylation of C-7. The 1H and 13C NMR data (Tables 1 and 3) further
supported this fact by showing signals for butylcarbamate and pen-
tylcarbamate moieties, respectively, and the downfield shift of C-7/
H-7 compared to their parent 9. Thus, compounds 10 and 11 were
Reaction of methanol, n-propanol, or n-butanol and p-toluene-
sulfonic acid with sarcophine (1) afforded the corresponding C-7-
ethers 4–6, along with the known 7b,8
phine (7),5,13 7-keto- 8(19)-deepoxysarcophine (8),11 and 7b-hy-
droxy-
8(19)-deepoxysarcophine (9) (Scheme 1).5,13 Compound 9
a-dihydroxy-deepoxysarco-
D
determined to be 7b-butylcarbamoyl-
D
8(19)-deepoxysarcophine
D
and 7b-pentylcarbamoyl-
D
8(19)-deepoxysarcophine, respectively.
was prepared in high-yield using the previously reported etherifi-
cation method using n-butanol.13 Compound 9 was the C-7 epimer
of the sarcophine’s bioconversion product, which was the most ac-
tive in suppressing the TPA-induced anchorage-independent JB6
cell transformation.5 The stereochemistry of 9 was based on the
fact that the epoxide opening in acidic condition should retain
the original configuration of the original epoxide.5,13 Compound 9
occurred as oil and had identical physical, chemical, and NMR
properties of the same compound reported by Czarkie, et al.
The HRTOFMS of 12 suggested the molecular formula
C
24H34NO4Cl and showed the M and M+2, 3:1, isotopic cluster
characteristic pattern for a monochlorinated compound. The 1H,
and 13C NMR data of 12 (Tables 2 and 3) confirmed the attachment
of a chloropropylcarbomyl moiety to C-7. Thus, compound 12 was
determined to be 7b-chloropropylcarbamoyl-D
8(19)-deepoxysarco-
phine. Similarly, compound 13 was determined to be 7b-cyclo-
hexylcarbamoyl-
D
8(19)-deepoxysarcophine.
O
H
HO
O
RO
HO
H
O
O
N
RNCO
p-TsOH, ROH
+
R
+
O
Et3N
Reflux
3h
rt, 1h
O
4-7
8
9
10-15
1
Scheme 1. Semisynthesis of sarcophine ether and carbamate analogues.
Table 1
1H NMR data of compounds 5, 6, 10, 11a
Position
dH (J in Hz)
5
6
10
11
2
3
5
6
7
9
5.56, dq (10.2, 1.8)
4.94, dq (10.3, 1.1)
5.56, dq (10.2, 1.8)
4.94, dq (10.3, 1.1)
2.40, ddd (13.8, 13.1, 3.3); 2.16, m
2.09, m; 1.58, m
3.41, dd (9.5, 9.5)
2.35, m; 1.95, m
2.28, m; 2.25, m
5.07, dd (6.6, 6.6)
2.13, m; 2.08, m
2.56, m; 2.20, m
1.82, s
1.86, s
1.10, s
1.63, s
3.30, t (6.2)
1.48, m
1.35, m
0.90, t (7.4)
5.60, dq (10.2, 2.2)
5.00, d (10.3)
2.44, ddd (13.8, 12.8, 3.8); 2.16, m
2.06, m; 1.41, m
4.64, d, (10.2)
2.34, m; 2.02, m
2.23, m; 2.18, m
5.10, dd (7.0, 7.0)
2.19, m; 2.12, m
2.71, m; 1.91, m
1.83, s
5.60, dq (10.2, 2.2)
5.00, d (10.3)
2.44, ddd (13.8, 12.8, 3.8); 2.16, m
2.06, m; 1.41, m
4.64, d, (10.2)
2.34, m; 2.02, m
2.23, m; 2.18, m
5.10, dd (7.0, 7.0)
2.19, m; 2.12, m
2.71, m; 1.91, m
1.83, s
2.40, ddd (13.8, 13.1, 3.3); 2.16, m
2.09, m; 1.58, m
3.41, dd (9.5, 9.5)
2.35, m; 1.95, m
2.28, m; 2.25, m
5.07, dd (6.6, 6.6)
2.13, m; 2.08, m
2.56, m; 2.20, m
1.82, s
1.86, s
1.10, s
1.63, s
3.26, t (6.2)
10
11
13
14
17
18
19
20
10
1.88, s
5.92, br s; 4.87, br s
1.61, s
1.88, s
5.92, br s; 4.87, br s
1.61, s
20
1.47, m
0.90, t (7.7)
3.23, m
1.80, m
1.31, m
0.88, t (7.3)
3.12, m, 3.25, m
1.28, m
1.44, m
1.60, m
0.87, t (6.9)
30
40
50
60
a
In CDCl3, 400 MHz. Coupling constants (J) are in Hz.

4930
H. M. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 4928–4934
Table 2
1H NMR data of compounds 12–15a
Position
dH (J in Hz)
12
13
14
15
2
3
5
6
7
9
5.60, dq (10.2, 2.2)
5.00, d (10.3)
2.44, ddd (13.8, 12.8, 3.8); 2.16, m
2.06, m; 1.41, m
4.64, d (10.2)
2.34, m; 2.02, m
2.23, m; 2.18, m
5.10, dd (7.0, 7.0)
2.19, m; 2.12, m
2.71, m; 1.91, m
1.83, s
1.88, s
5.92, br s; 4.87, br s
1.61, s
3.56, t (3.7)
1.97, m
5.60, dq (10.2, 2.2)
5.00, d (10.3)
2.44, ddd (13.8, 12.8, 3.8); 2.16, m
2.06, m; 1.41, m
4.54, d (10.2)
2.34, m; 2.02, m
2.23, m; 2.18, m
5.10, dd (7.0, 7.0)
2.19, m; 2.12, m
2.71, m; 1.91, m
1.83, s
5.60, dq (10.2, 2.2)
5.00, d (10.3)
2.44, ddd (13.8, 12.8, 3.8); 2.16, m
2.06, m; 1.41, m
4.54, d (10.2)
2.34, m; 2.02, m
2.23, m; 2.18, m
5.10, dd (7.0, 7.0)
2.19, m; 2.12, m
2.71, m; 1.91, m
1.83, s
1.88, s
5.92, br s; 4.87, br s
1.61, s
4.41, 2H, ddd (5.2, 1.5, 1.5)
5.94, m
5.21, m; 5.29, dd (1.8, 1.8)
5.60, dq (10.2, 2.2)
5.00, d (10.3)
2.44, ddd (13.8, 12.8, 3.8); 2.16, m
2.06, m; 1.41, m
4.54, d (10.2)
2.34, m; 2.02, m
2.23, m; 2.18, m
5.10, dd (7.0, 7.0)
2.19, m; 2.12, m
2.71, m; 1.91, m
1.83, s
10
11
13
14
17
18
19
20
20
1.88, s
5.92, br s; 4.87, br s
1.61, s
1.88, s
5.92, br s; 4.87, br s
1.61, s
3.40, m
30
1.64, m; 1.39 m
1.21, m; 1.11 m
1.33, m
1.21, m; 1.11, m
1.64, m; 1.39, m
7.04, dd (7.3, 1.8)
7.13, m
7.12, m
7.13, m
7.04, dd (7.3, 1.8)
40
3.3, t (6.2)
50
60
3.87, ddd (5.5, 1.5, 1.5)
5.87, m
5.12, m, 5.20, m
70
80
90
100
110
120
3.88, ddd (5.5, 1.5, 1.5)
5.84, m
5.11, m; 5.20, m
a
In CDCl3, 400 MHz. Coupling constants (J) are in Hz.
Table 3
13C NMR Data of Compounds 5, 6, and 10–15a
Position
dC
5
6
10
11
12
13
14
15
1
2
3
4
5
6
7
8
163.0, qC
79.2, CH
121.0, CH
144.3, qC
32.7, CH2
26.9, CH2
72.8, CH
78.9, qC
36.5, CH2
19.6, CH2
125.6, CH
134.2, qC
35.6, CH2
26.6, CH2
122.8, qC
174.9, qC
9.0, CH3
163.0, qC
79.2, CH
121.0, CH
144.3, qC
32.7, CH2
26.9, CH2
72.9, CH
78.9, qC
36.5, CH2
19.6, CH2
125.6, CH
134.2, qC
35.6, CH2
26.6, CH2
122.8, qC
174.9, qC
9.1, CH3
16.1, CH3
19.1, CH3
15.8, CH3
60.6 CH2
31.7, CH2
23.5, CH2
14.1, CH3
162.8, qC
79.0, CH
162.8, qC
79.0, CH
162.8, qC
79.0, CH
162.8, qC
79.0, CH
121.5, CH
1437, qC
36.1, CH2
32.2, CH2
72.9, CH
154.6, qC
33.9, CH2
30.0, CH2
125.9, CH
135.1, qC
36.9, CH2
26.4, CH2
123.0, qC
174.8, qC
9.0, CH3
16.7, CH3
110.6, CH2
16.1, CH3
155.0, qC
49.6, CH
29.6, CH2
25.6, CH2
30.4, CH2
25.5, CH2
29.8, CH2
162.8, qC
79.0, CH
162.8, qC
79.0, CH
121.5, CH
1437, qC
36.1, CH2
32.2, CH2
72.9, CH
154.6, qC
33.9, CH2
30.0, CH2
125.9, CH
135.1, qC
36.9, CH2
26.4, CH2
123.0, qC
174.8, qC
9.0, CH3
16.7, CH3
110.6, CH2
16.1, CH3
155.1, qC
43.1, CH2
34.4, CH2
20.0, CH2
13.8, CH3
121.5, CH
1437, qC
36.1, CH2
32.2, CH2
72.9, CH
154.6, qC
33.9, CH2
30.0, CH2
125.9, CH2
135.1, qC
36.9, CH2
26.4, CH2
123.0, qC
174.8, qC
9.0, CH3
16.7, CH3
110.6, CH2
16.1, CH3
154.0, qC
43.1, CH2
34.4, CH2
25.5, CH2
22.5, CH2
14.0, CH3
121.5, CH
1437, qC
36.1, CH2
32.2, CH2
72.9, CH
154.6, qC
33.9, CH2
30.0, CH2
125.9, CH
135.1, qC
36.9, CH2
26.4, CH2
123.0, qC
174.8, qC
9.0, CH3
16.7, CH3
110.6, CH2
16.1, CH3
155.4, qC
42.4, CH2
32.5, CH2
38.3, CH2
121.5, CH
1437, qC
36.1, CH2
32.2, CH2
72.9, CH
154.6, qC
33.9, CH2
30.0, CH2
125.9, CH
135.1, qC
36.9, CH2
26.4, CH2
123.0, qC
174.8, qC
9.0, CH3
16.7, CH3
110.6, CH2
16.1, CH3
154.2, qC
46.1, CH2
134.8, CH
118.0, CH2
154.3, qC
43.1, CH2
134.6, CH
117.1, CH2
154.3, qC
43.1, CH2
134.3, CH
116.2, CH2
121.5, CH
1437, qC
36.1, CH2
32.2, CH2
72.9, CH
154.6, qC
33.9, CH2
30.0, CH2
125.9, CH
135.1, qC
36.9, CH2
26.4, CH2
123.0, qC
174.8, qC
9.0, CH3
16.7, CH3
110.6, CH2
16.1, CH3
154.1, qC
138.0, qC
118.6, CH
129.2, CH
122.0, CH
129.2, CH
118.6, CH
9
10
11
12
13
14
15
16
17
18
19
20
10
16.1, CH3
19.1, CH3
15.8, CH3
62.5, CH2
23.8, CH2
11.0, CH3
20
30
40
50
60
70
80
90
100
110
120
a
In CDCl3, 100 MHz. Carbon multiplicities were determined by APT experiments, C = quaternary, CH = methine, CH2 = methylene, CH3 = methyl carbons.
The HRTOFMS, 1H, and 13C NMR data of 14 (Tables 2 and 3) sug-
gested the molecular formula C32H43N3O6, and the possibility of
reaction of three molecules of allylisocyanate with the C-7 second-
ary alcohol in 9 to form trimeric carbamates. The previous conclu-
sion was supported by the presence of three non-symmetric allyl
groups along with three chemically non-equivalent carbamoyl

H. M. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 4928–4934
4931
carbonyl carbons (C-10, C-50, and C-90, dC 154.2, 154.3, and 154.4,
respectively). This was further supported via the 3J-HMBC correla-
tions of H2-20 (dH 4.41) with C-10, C-40, and C-50, H2-60 (dH 3.87) with
C-50, C-80, and C-90, and H2-100 (dH 3.88) with C-90 and C-120. Thus,
14 was determined to be 7b-allyl-(allyl-(allylcarbamoyl)-carbam-
and migration through, the basement membrane and extracellular
matrix surrounding tumor epithelium, mediated through integrins
and proteases including urokinase form of plasminogen activator,
matrix metalloproteinases and cathepsins; 4-intravasation of the
tumor cells into the blood vessels; 5-adhesion of the circulating tu-
mor cells to the endothelial cell lining mediated by selectins, inte-
grins and members of the immunoglobulin superfamily; 6-
invasion of the tumor cells through the endothelial cell layer and
surrounding basement membrane and target organ tissue; and 7-
development of secondary tumor foci at the target organ site.14
The wound-healing assay (WHA) is a simple method for the study
of directional cell migration in vitro.14 The scratched monolayer
heals in a characteristic manner; therefore, this assay is widely
used to study cell migration rates, cell polarization, and matrix
remodeling studies.15–17 WHA proved to be useful as a proxy for
metastasis, angiogenesis and other pathophysiological and physio-
logical processes.18–21 Therefore, WHA was used in this study to as-
sess and rank the antimigratory potential of semisynthetic
sarcophine derivatives.
oyl)-carbamate-D
8(19)-deepoxysarcophine.
The HRTOFMS of 15 suggested the molecular formula C27H33NO4
and possible C-7 phenylcarbamoylation. The 1H and 13C NMR data
(Tables 2 and 3) further supported this conclusion. Thus, 15 was
proved to be 7b-phenylcarbamoyl-D
8(19)-deepoxysarcophine.
2.2. Biological activity and preliminary structure–activity
relationship
Metastasis is the process by which cancer cells leave the pri-
mary tumor to form secondary tumors at distant sites, causing
the morbidity and mortality of most cancer patients.14 Metastasis
results from a complex interconnected molecular cascade via sev-
eral adhesive interactions and invasive processes and responses to
chemotactic stimuli.14 The main elements of metastasis include
1-angiogenesis; 2-disaggregation of tumor cells from the primary
tumor mass, mediated by cadherins and catenins; 3-invasion of,
All cembranoids were evaluated for their ability to inhibit the
proliferation and migration of the human metastatic prostate can-
cer PC-3 and breast cancer MDA-MB-231 cell lines using MTT and
Figure 1. Relative number of migrated MDA-MB-231 cells after 24 h treatment with vehicle control and 50
l
M dose of 6 in wound-healing assay. Magnification = 100ꢀ.
120.00
100.00
80.00
60.00
40.00
20.00
0.00
PC-3
MDA-MB-231
DMSO PMH*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Concentration (50 μM)
Figure 2. Effect of 50
lM dose of each of 1–15 on the percent of migration on the highly metastatic PC-3 and MDA-MB-231 cells in wound healing assay. A 200 lM dose of
4-hydroxyphenylmethylene hydantoin (PMH) was used a positive drug control.12,13

4932
H. M. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 4928–4934
wound-healing assays (Figs. 1 and 2).22–24 Most compounds showed
no effect on the proliferation of PC-3 or MDA-MB-231 cells up to a
showed better activity compared to olefinic or aromatic carba-
mates. Butyloxy, butyl or hexylcarbamate functionalities were
optimal for enhanced antimigratory activity. Inclusion of halogens
in the side chain as represented by the carbamate 12 remarkably
reduced the activity. Cembranoids can be potential scaffolds for
the design of potent antimetastatic leads.
50
lines. Only compounds 6, 10, and 13 showed antiproliferative activ-
ity against MDA-MB-231 cells, with IC50 values 20, 7 and 45 M,
respectively. The antimigratory activity of all compounds was as-
sessed in the wound-healing assay at a 50 M dose, except com-
lM dose, indicating the lack of cytotoxicity toward these cell
l
l
pounds 6, 10, and 13 which were tested at lower concentrations
(Fig. 3) against MDA-MB-231 and PC-3 cells, to avoid false positive
activity due to their possible cytotoxicity. The activity was com-
3. Experimental
3.1. General experimental procedures
pared to a 200 lM dose of the antimetastatic marine natural product
lead 4-hydroxyphenylmethylene hydantoin (PMH).25,26 Compound
13 showed potent antimigratory activity against the prostate cancer
PC-3 cell line, followed by 14 and 15, which showed moderate activ-
ity (Figs. 1 and 2 and Table 4). Cembranoids 3, 5, 10, and 13–15 were
Optical rotations were measured on a Rudolph Research Analyt-
ical Autopol III polarimeter. IR spectra were recorded on a Varian
800 FT-IR spectrophotometer. The 1H and 13C NMR spectra were
recorded in CDCl3, using TMS as internal standard, on a JEOL
Eclipse NMR spectrometer operating at 400 MHz for 1H and
100 MHz for 13C. The HREIMS experiments were conducted at Lou-
isiana State University on Agilent 6200-TOF LCMS. TLC analysis
more active against PC-3 at 50
ine natural product PMH. The IC50 values of 13–15 were 15.53, 24.28,
and 16.29 M, respectively (Table 4). On the other hand, a 50
lM than a 200 lM of the known mar-
l
lM
dose of 8 showed a potent antimigratory activity against the estro-
gen negative breast cancer MDA-MB-231 cell line, followed by 7,
2, 9, 14, 5, 11, and 15, respectively, with better activities than the
was carried on precoated Si gel 60 F254 500 lm TLC plates (EMD
Chemicals), using isocratic n-hexane-EtOAc (1:1) as a developing
system. For column chromatography, Si gel 60 (Natland, 63–
200 lm) and n-hexane-EtOAc (9.5:0.5) with increasing polarity to
200 l
M dose of the PMH positive control,25,26 while 3, 4ß and 12
showed moderate activity but better than the parent compound 1,
which showed a very limited activity (Fig. 2). This was consistent
with literature which limited the activity of 1 only to the estrogen
positive breast cancer cell lines.6 The IC50 of the most active antimi-
n-hexane-EtOAc (7:3) systems were used. A purity of >95% was
established for each of 1–15 as assessed by TLC, HPLC, and/or 1H
NMR spectroscopy.
gratory analogues 6, 10, and 13 were 30.02, 4.83, and 17.23 lM,
3.2. Biological material
respectively, against MDA-MB-231 cells (Table 4). To avoid the cor-
relation of the antimigratory activity with possible cytotoxic activ-
ity, compounds 6, 10, and 13 were retested at different doses
lower than their antiproliferative IC50 values (Fig. 3). A dose of 5
The soft coral S. glaucum was collected by SCUBA June 2003
from Hurghada, at the Egyptian Red Sea coast. A voucher specimen
(03RS24) was deposited in the Department of Basic Pharmaceutical
Sciences, College of Pharmacy, University of Louisiana at Monroe.
The frozen soft coral (680 g) was extracted four times with isopro-
panol. The extract (86 g) was then concentrated under vacuum and
chromatographed on silica gel using n-hexane/EtOAc to yield 1.5 g
and 30
comparable to that produced by a 200
affecting the cell shape or viability, while compound 6 showed no
l
M of 10 and 13, respectively, induced antimigratory effects
l
M dose of PMH without
significant activity up to 30
lM.
18
O
O
O
O
O
O
O
R
H
H
H
RO
HO
O
15
7
3
O
O
1
19
17
9
13
R
R
2
3
-H
20
β
4
5
6
7
CH3
a
8
1
α-H
b
H
O
O
H
H
H
R
1'
O
N
O
N
1'
1'
1'
4'
2'
4'
3'
2'
6'
O
O
a
b
c
d
R
O
9
OH
c
O
NH
9'
10
11
12
13
14
15
H
N
H
N
H
N
N
O
O
O
3'
10'
O
1'
O
1'
1'
5'
d
1'
Cl
N
2'
6'
12'
2'
e
5'
5'
2'
8'
O
O
f
O
e
f
g4'
h
g
h
Acid catalyzed conversion of the epoxide moiety of 1 to second-
ary alcohol or ketone at C-7 with a
8(19) exomethylene system sig-
nificantly improved the antimigratory activity. Modification of C-7,
including etherification and carbamoylation, enhanced the activity
and demonstrated its significant contribution to the binding at the
plausible molecular target(s). Saturated ethers and carbamates
of sarcophine (1), which was further recrystallized from EtOAc.
Further chromatographic separation of the extract afforded the less
polar 16-deoxysarcophine (2, 1.5 g, Rf 0.52 n-hexane/EtOAc 7:3)
and 2-epi-16-deoxysarcophine (3, 4.1 g, Rf 0.50 n-hexane/EtOAc
7:3). Identity was confirmed via spectral analysis and comparison
with literature.3,4,6,10
D

H. M. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 4928–4934
4933
160
140
120
100
80
60
40
20
0
DMSO
PMH 6 (5 μM) 6 (10 μM) 6 (30 μM) 10 (1 μM) 10 (2 μM) 10 (5 μM) 13 (5 μM) 13 (10 μM) 13 (30 μM)
Figure 3. Effect of different doses of 6, 10 and 13 on the percent of migration on the highly metastatic MDA-MB-231 in wound healing assay. A 200
lM dose of 4-
hydroxyphenylmethylene hydantoin (PMH) was used a positive drug control.12,13
999 cmꢂ1
;
1H and 13C NMR, see Tables 1 and 3; HRTOFMS m/z
Table 4
IC50 values of selected sarcophine analogues against the highly metastatic MDA-MB-
438.2615 [M+Na]+ (calcd for C25H37NO4Na, 438.2620).
231 (breast) and PC-3 (prostate) cancer cell linesa
8(19)-deepoxysarcophine (11)
L of penty-
max (CHCl3)
MDA-MB-231
Compound IC50
PC-3
3.3.6. 7b-Pentylcarbamoyl-
D
Compound 11 was prepared in 98% yield using 3.5
l
(lM)
Compound
IC50 (lM)
lisocyanate. Colorless oil; ½a D25
ꢁ
+31.1 (c 0.27, CHCl3); IR m
6
10
13
30.02
4.83
17.23
13
14
15
15.53
24.28
16.29
3690, 3481, 3446, 3054, 2986, 2874, 1747, 1695, 1645, 1513, 1422,
1258, 1166, 896 cmꢂ1 1H and 13C NMR, see Tables 1 and 3;
;
HRTOFMS m/z 452.2773 [M+Na]+ (calcd for C26H39NO4Na,
a
The values are the mean SD. Each experiment was conducted in triplicate.
452.2776).
3.3. Chemical reactions
3.3.7. 7b-Chloropropylcarbamoyl-
D
8(19)-deepoxysarcophine (12)
L of chloro-
+34 (c 0.1, CHCl3); IR mmax
3.3.1. Etherification of 13,10
Compound 12 was prepared in 58% yield using 3.7
l
propylisocyanate. Colorless oil; ½a D25
ꢁ
About 100 mg of sarcophine dissolved in 2.5 mL of each of
MeOH, n-propanol, and n-butanol and each stirred for 1 h at rt with
p-toluenesulfonic acid (5 mg) (Scheme 1). After neutralization with
NaHCO3, organic solvent was evaporated under vacuum and CHCl3
(5 mL) was added to each reaction mixture. Each solution was then
washed with water (5 mL), dried over anhydrous Na2SO4 and evap-
orated. Each residue was subjected to repeated column chromatog-
(CHCl3) 3689, 3447, 3054, 2927, 2855, 1748, 1720, 1510, 1422,
1216, 989 cmꢂ1
;
1H and 13C NMR, see Tables 2 and 3; HRTOFMS
m/z 470.1862 [M+Cl]+ (calcd for C24H34NO4Cl2, 470.1864)
3.3.8. 7b-Cyclohexylcarbamoyl-D
8(19)-deepoxysarcophine (13)
Compound 13 was prepared in 98% yield using 3.6 lL of cyclo-
hexylisocyanate afforded 13 in a 98% yield. Colorless oil; ½a D25
ꢁ
+42.0
raphy to afford compounds 4–93,10
.
(c 0.2, CHCl3); IR mmax (CHCl3) 3689, 3434, 3055, 2930, 2856, 1746,
1716, 1506, 1452, 1216,1096, 989 cmꢂ1
bles
and 3; HRTOFMS m/z 464.2771 [M+Na]+ (calcd for
27H39NO4Na, 464.2776).
;
1H and 13C NMR, see Ta-
3.3.2. Carbamoylation of 927–29
2
Compound 9 (10 mg) in toluene (2 mL) was reacted with vari-
C
ous isocyanates and catalytic amount of triethylamine (Et3N,
10 l
L) (Scheme 1).27–29 Each solution was separately refluxed for
3.3.9. 7b-Allyl(allyl(allylcarbamoyl)carbamoyl)-D8(19)
deepoxysarcophine (14)
-
3 h. Water (10 mL) was added and each reaction mixture was ex-
tracted with EtOAc (3 ꢀ 10 mL). Each EtOAc extract was dried over
anhydrous Na2SO4, concentrated under vacuum and subjected to
repeated column chromatography to afford 10–15.
Compound 14 was prepared in 98% yield using 5.2
lL of allylis-
ocyanate. Colorless oil; ½a D25
ꢁ
+40.5 (c 0.13, CHCl3); IR mmax (CHCl3)
3688, 3445, 3056, 3020, 2928, 2856, 1747, 1700, 1509, 1216, 1096,
;
993 cmꢂ1 1H and 13C NMR, see Tables 2 and 3; HRTOFMS m/z
3.3.3. 7b-Propyloxy-8a-hydroxy-deepoxysarcophine (5)
588.3045 [M+Na]+ (calcd for C32H43N3O6Na, 588.3049).
Compound 5 was prepared in 15% yield using the reaction condi-
tions described above. White powder; ½a D25
ꢁ
+15.5 (c 0.53, CHCl3); IR
3.3.10. 7b-Phenylcarbamoyl-
D
8(19)-deepoxysarcophine (15)
L of phen-
+51.0 (c 0.13, CHCl3); IR mmax
m
max (CHCl3) 3689, 3565, 3055, 2927, 2875, 1747, 1680, 1455, 1387,
1096, 999 cmꢂ1; 1H and 13C NMR, see Tables 1 and 3; HRTOFMS m/z
Compound 15 was prepared in 68% yield using 3.7
l
ylisocyanate. Colorless oil; ½a D25
ꢁ
399.2506 [M+Na]+ (calcd for C23H36O4Na, 399.2511).
(CHCl3) 3687, 3600, 3425, 3054, 2986, 2928, 2855, 1746, 1703,
1597, 1525, 1443, 1209, 1095, 896 cmꢂ1
bles
and 3; HRTOFMS m/z 458.2302 [M+Na]+ (calcd for
27H33NO4Na 458.2307).
;
1H and 13C NMR, see Ta-
3.3.4. 7b-Butyoxy-8a-hydroxy-deepoxysarcophine (6)
Compound 6 was prepared in 20% yield. White powder; ½a D25
ꢁ
2
C
+13.7 (c 0.57, CHCl3); IR mmax (CHCl3) 3689, 3055, 2959, 2932,
2872, 1747, 1681, 1422, 1096, 989, 896 cmꢂ1
see Tables 1 and 3; HRTOFMS m/z 413.2662 [M+Na]+ (calcd for
24H38O4Na, 413.2667).
;
1H and 13C NMR,
4. Biological assays
4.1. Cell culture
C
3.3.5. 7b-Butylcarbamoyl-
D
8(19)-deepoxysarcophine (10)
L of butylis-
+33.4 (c 0.23, CHCl3); IR mmax (CHCl3)
Compound 10 was prepared in 98% yield using 3.1
l
Prostate and breast cancer cell lines PC-3 and MDA-MB-231
were purchased from ATCC. Cells were grown in 10% fetal bovine
serum (FBS) and RPMI 1640 (GIBCO-Invitrogen) supplemented
ocyanate. Colorless oil; ½a D25
ꢁ
3689, 3565, 3055, 2927, 2875, 1747, 1680, 1455, 1387, 1096,

4934
H. M. Hassan et al. / Bioorg. Med. Chem. 19 (2011) 4928–4934
with 2 mmol/L glutamine, 100
streptomycin at 37 °C under 5% CO2.
l
g/mL penicillin G, and 100
lg/mL
include MOL files and InChiKeys of the most important compounds
described in this article.
4.1.1. Proliferation assay
References and notes
The antiproliferative effects of the isolated compounds were
tested in culture on malignant PC-3 and MDA-MB-231 epithelial cell
lines using MTT kit (TACS, Trevigen, Inc.). After passing the cells for
3–4 times, growing cells were incubated in a 96-well plate at a den-
sity of 8 ꢀ 103 cells per well, and allowed to attach for 24 h. Com-
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scribed.28,29 The number of cells per well was calculated against a
standard curve prepared by plating various concentration of cells.
Growth curves were determined to ensure that cells used in exper-
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reduction of MTT is catalyzed by mitochondrial dehydrogenase en-
zymes and is therefore a measure for cell viability.28,29
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PC-3 and MDA-MB-231 cells were cultured and plated onto ster-
ile 24-well and allowed to form a confluent cell monolayer in each
well (>90% confluence) as previously reported.29 Wounds were then
inflicted to each cell monolayer using a sterile 200 lL pipette tip.
Test compounds were prepared in DMSO at different concentrations
and added to the plates, each in triplicate using PMH as positive con-
trol and DMSO as a vehicle control.25,26 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 Quick stain-
ing (Dade Behring Diagnostics). The number of cells migrated on the
scratched wound were counted under the microscope in three or
more randomly selected fields (magnification: 400ꢀ). Final results
are expressed as mean SEM per 400ꢀ field. All treatments, includ-
ing the controls, were documented photographically.
15. Finder, I. J. Nat. Rev. Cancer 2003, 3, 453.
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1105.
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Acknowledgments
The Louisiana Board of Regents is acknowledged for supporting
the new NMR facility (LEQSF(2010-11)-ENH-TR-41). The Egyptian
government is acknowledged for the fellowship support of H.H.
28. Hassan, H. M.; Khanfar, M. A.; Elnagar, A. Y.; Mohammed, R.; Shaala, L. A.;
Youssef, D. T. A.; Hifnawy, M. S.; El Sayed, K. A. J. Nat. Prod. 2010, 73, 848.
29. Hassan, H. M.; Khanfar, M. A.; Elnagar, A. Y.; Mohammed, R.; Shaala, L. A.;
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1122.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.060. These data