Optimization of Marine Triterpene Sipholenols as Inhibitors of Breast Cancer Migration and Invasion

Article abstract of DOI:10.1002/cmdc.201200516

SipholenolA, a sipholane triterpene isolated from the Red Sea sponge Callyspongia siphonella, has the ability to reverse multidrug resistance in cancer cells that overexpress P-glycoprotein (P-gp). Here, the antimigratory activity of sipholenolA and analogues are reported against the highly metastatic human breast cancer cell line MDA-MB-231 in a wound-healing assay. SipholenolA and sipholenoneA were semisynthetically optimized using ligand-based strategies to generate structurally diverse analogues in an attempt to maximize their antimigratory activity. A total of 22 semisynthetic ester, ether, oxime, and carbamate analogues were generated and identified by extensive one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry analyses. SipholenolA 4β-4-chlorobenzoate and 19,20-anhydrosipholenolA 4β-4-chlorobenzoate esters were the most potent of all tested analogues in the wound-healing assay, with IC₅₀ values of 5.3 and 5.9μM, respectively. Generally, ester derivatives showed better antimigratory activities than the carbamate analogues. A KINOMEscan of 19,20-anhydrosipholenolA 4β-benzoate ester against 451 human protein kinases identified protein tyrosine kinase6 (PTK6) as a potential target. In breast tumor cells, PTK6 promotes growth factor signaling and migration, and as such the semisynthetic sipholanes were evaluated for their ability to inhibit PTK6 phosphorylation in vitro. The two analogues with the highest antimigratory activities, sipholenolA 4β-4-chlorobenzoate and 19,20-anhydrosipholenolA 4β-4-chlorobenzoate esters, also exhibited the most potent inhibition of PTK6 phosphorylation inhibition. None of the compounds exhibited cytotoxicity in a normal epithelial breast cell line. These derivatives were evaluated in an invitro invasion assay, where sipholenolA succinate potently inhibited MDA-MB-231 cell invasion at 10μM. These results highlight sipholane triterpenoids as novel antimigratory marine natural products with potential for further development as agents for the control of metastatic breast malignancies.

Full text of DOI:10.1002/cmdc.201200516

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DOI: 10.1002/cmdc.201200516
Optimization of Marine Triterpene Sipholenols as
Inhibitors of Breast Cancer Migration and Invasion
Ahmed I. Foudah, Sandeep Jain, Belnaser A. Busnena, and Khalid A. El Sayed*[a]
Sipholenol A, a sipholane triterpene isolated from the Red Sea
sponge Callyspongia siphonella, has the ability to reverse multi-
drug resistance in cancer cells that overexpress P-glycoprotein
(P-gp). Here, the antimigratory activity of sipholenol A and ana-
logues are reported against the highly metastatic human
breast cancer cell line MDA-MB-231 in a wound-healing assay.
Sipholenol A and sipholenone A were semisynthetically opti-
mized using ligand-based strategies to generate structurally di-
verse analogues in an attempt to maximize their antimigratory
activity. A total of 22 semisynthetic ester, ether, oxime, and car-
bamate analogues were generated and identified by extensive
one- and two-dimensional NMR spectroscopy and high-resolu-
tion mass spectrometry analyses. Sipholenol A 4b-4-chloroben-
zoate and 19,20-anhydrosipholenol A 4b-4-chlorobenzoate
esters were the most potent of all tested analogues in the
wound-healing assay, with IC50 values of 5.3 and 5.9 mm, re-
spectively. Generally, ester derivatives showed better antimigra-
tory activities than the carbamate analogues. A KINOMEscan of
19,20-anhydrosipholenol A 4b-benzoate ester against 451
human protein kinases identified protein tyrosine kinase 6
(PTK6) as a potential target. In breast tumor cells, PTK6 pro-
motes growth factor signaling and migration, and as such the
semisynthetic sipholanes were evaluated for their ability to in-
hibit PTK6 phosphorylation in vitro. The two analogues with
the highest antimigratory activities, sipholenol A 4b-4-chloro-
benzoate and 19,20-anhydrosipholenol A 4b-4-chlorobenzoate
esters, also exhibited the most potent inhibition of PTK6 phos-
phorylation inhibition. None of the compounds exhibited cyto-
toxicity in a normal epithelial breast cell line. These derivatives
were evaluated in an in vitro invasion assay, where sipholeno-
l A succinate potently inhibited MDA-MB-231 cell invasion at
10 mm. These results highlight sipholane triterpenoids as novel
antimigratory marine natural products with potential for fur-
ther development as agents for the control of metastatic
breast malignancies.
Introduction
Marine-derived natural products are among the important re-
sources for drug discovery and development.[1–3] In the last
three decades, thousands of new marine-derived compounds
have been discovered and shown to have potential anticancer
activities.[4,5] Marine-derived sipholane triterpenoids sipholeno-
l A (1) and sipholenone A (2) were first isolated from the Red
Sea sponge Callyspongia (Siphonochalina) siphonella by Kash-
man and co-workers.[6] Thirty sipholane triterpenoids have
been isolated so far, possessing four different skeletons,
namely, the sipholane, siphonellane, neviotane, and dahabi-
resistance (MDR) in human epidermoid cancer cells was investi-
gated.[9,10] Accordingly, 1 was reported to potentiate the cyto-
toxicity of several P-gp-substrate anticancer drugs, including
colchicine, vinblastine, and paclitaxel, but not non-P-gp sub-
strate drugs, such as cisplatin; compound 1 was also shown to
significantly reverse the MDR of P-gp-overexpressing human
epidermal carcinoma cell lines KB-C2 and KB-V1 in a concentra-
tion-dependent manner.[10,11] Furthermore, 1 showed no cyto-
toxicity towards numerous cell lines, regardless of their mem-
brane transporter status.[11–13] Sipholenol A (1) efficiently inhib-
ited the function of P-gp through direct interaction, and sipho-
lane triterpenoids were proposed as a novel class of potential
P-gp inhibitors for the reversal of MDR in P-gp-overexpressing
tumors.[12,13]
nane.[6–11] The sipholanes contain
a perhydrobenzoxepine
(rings A and B) and a [5,3,0]bicyclodecane system (rings C and
D), linked together through an ethylene bridge.[7,8] In recent
studies, the potential of sipholane triterpenoids as P-glycopro-
tein (P-gp) modulators and their ability to reverse multidrug
Breast cancer is the most common cancer and the second
leading cause of cancer-related death among women in the
USA.[14] Breast tumor kinase (Brk), also referred to as protein ty-
rosine kinase 6 (PTK6), is a member of the Src family of tyrosine
kinases and has been cloned from metastatic breast tumor
samples and cultured human melanocytes.[15] Brk is normally
expressed in the differentiating epithelial cells of the intestine,
skin, prostate, and oral cavity where it has been shown to pro-
mote cellular differentiation, apoptosis, and more recently, to
mediate migration/wound healing.[16] Overexpression of Brk
has been implicated as a mediator of cancer cell phenotypes,
[a] A. I. Foudah, Dr. S. Jain,+ B. A. Busnena, Dr. K. A. El Sayed
Department of Basic Pharmaceutical Sciences
College of Pharmacy, University of Louisiana at Monroe
1800 Bienville Dr., Monroe, Louisiana 71201 (USA)
E-mail: elsayed@ulm.edu
[+] Current address: Sun Pharmaceutical Advanced Research Company (SPARC)
Ltd, 17 B Mahal Industrial Estate, Mahakali Caves Rd Andheri (E), 400 093
Mumbai, (India).
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200516.
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including increased proliferation, survival, and migration. Brk is
overexpressed in up to 86% of invasive human breast tumors,
prostate and colon carcinomas.[17,18] Its expression levels in-
crease in association with the carcinoma content of breast
tumors, tumor grade, and invasiveness.[15–20] Inhibition of Brk
activity might provide a potentially novel approach to sensitize
tumor cells to other chemotherapeutics and prevent or inhibit
metastasis with enhanced therapeutic windows.
To the best of our knowledge, sipholanes 1 and 2 have
never been tested for their ability to inhibit the migration or
invasion of breast cancer cell lines. Structurally related polye-
poxysqualene-derived, cyclic ether triterpenoids (e.g., sodwa-
nones from the Indian Ocean
Bioactivity screening of our library of natural products for
the discovery of new potential antimigratory agents suggested
sipholanes 1 and 2 as inhibitors of the migration of the highly
metastatic MDA-MB-231 breast cancer cell line in a wound-
healing assay at mid-micromolar concentrations. The aim of
this study was to prepare different semisynthetic analogues of
1 and 2 with enhanced antimigratory and anti-invasive activity
against the highly metastatic MDA-MB-231 breast cancer cell
line, assess their PTK6 phosphorylation inhibitory activity as
a possible molecular target, and identify key pharmacophores
required for enhanced bioactivity.
sponge Axinella weltneri) were
reported to possess cytotoxicity
against human cancer cell
lines.[21] Several sodwanones
were reported to possess activi-
ty against various cancer cells.
For example, sodwanones G–I
were found to possess cytotox-
icity at 20 mm or less concentra-
tions against P-388 murine leu-
kemia, A-549 lung carcinoma,
HT-29 human colon carcinoma,
and MEL-28 human melanoma
cells.[21,22] Sodwanone A was
found to be cytotoxic in nano-
molar concentrations to IGROV-
ET human ovarian cancer cells
resistant to the cancer drug ET-
743, PANC1 human pancreatic
carcinoma cells, and LOVO-DOX
multidrug-resistant
human
colon adenocarcinoma cells.
Sodwanone A was also cytotoxic
to human lung cancer NSCLC-
N6 cells with an IC50 value of
12 mm.[23] Sodwanones G and S
also displayed cytotoxicity to
IGROV-ET, PANC1, and LOVO-
DOX cells with IC50 values of less
than 10 mm.[24] Recently, 3-epi-
sodwanone K-3 acetate was
found to be cytotoxic against
T47D breast cancer cells (IC50 =
22 mm), while sodwanone V was
cytotoxic to MDA-MB-231 breast
cancer cells (IC50 =23 mm). Sod-
wanone V also inhibited hypo-
xia-inducible factor 1 (HIF-1) ac-
tivation in both T47D breast and
PC3 prostate cancer cells, with
an IC50 value of 15 mm.[25] There-
fore, we hypothesized that si-
pholane triterpenoids could be
potential anticancer hits.
Scheme 1. Semisynthetic transformations of sipholenol A (1) and sipholenone A (2). Reagents and conditions:
a) p-toluenesulfonic acid, CHCl3, reflux, 3 h; b) acid anhydride, DMAP, anhyd CH2Cl2, reflux, 12 h; c) BnBr, DMF, NaH,
RT, 12 h; d) isocyanate, toluene, Et3N, reflux, 3 h; e) NH2OH, pyridine, EtOH, reflux, 4 h.
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paring their 1H and 13C NMR data with that reported in the
literature.[6]
Results and Discussion
Chemistry
Esterification
The sipholane triterpenoids 1 and 2 were subjected to semi-
synthetic modifications (Scheme 1) to generate structurally di-
verse analogues, to optimize their antimigratory activities, and
to establish preliminary structure–activity relationships (SARs).
The five different reactions types, including acid-catalyzed
elimination, esterification, etherification, carbamoylation and
oxime formation, were used to afford four known (3–6) and
several new (7–24) semisynthetic analogues of 1 and 2
(Table 1).
Several known and new esters of 1 were prepared using
acetic, succinic, benzoic and 4-methylbenzoic anhydrides, iso-
nicotinoyl, and 4-chlorobenzoyl chloride to assess the signifi-
cance of the C-4 hydroxy group and nearby space on the anti-
migratory activity of this class of compounds. Acetylation of
1 in pyridine was carried out to afford the corresponding mon-
oacetate (5) and diacetate (6) esters.[6] The new esters of 1 in-
clude the succinate (7), benzoate (8), isonicotinate (9), 4-meth-
ylbenzoate (10), and 4-chlorobenzoate (11) esters. In a similar
fashion, the acetate (12), succinate (13), benzoate (14), isonico-
tinate (15), 4-methylbenzoate (16), 4-chlorobenzoate (17)
esters of 19,20-anhydrosipholenol A (3) were also prepared.
The identification of derivatives 7–17 was based on extensive
spectral analyses.
Acid-catalyzed elimination
Sipholenol A (1) was converted into the known 19,20-anhydro-
sipholenol A (3) and 10,11,19,20-dianhydrosipholenol A (4), by
treatment with p-toluenesulfonic acid monohydrate (p-
TsOH·H2O) in chloroform.[3,6,7] The reaction aimed to eliminate
the tertiary alcohol groups at C-10 and C-19 and introduce ad-
ditional double bonds (D) at specific positions in the structure
of 1, to assess their effects on the antimigratory activity. The
major reaction product, 19,20-anhydrosipholenol A (3) was
formed due to the elimination of one molecule of water, which
The high-resolution mass spectrometry within (HRMS) of 7
showed an [M+Na]+ peak at m/z 599.3929, suggesting the
molecular formula C34H56O7Na and possible ester analogue of
sipholenol A. The H and 13C NMR data also showed the pres-
1
ence of peaks corresponding to two ester carbonyl carbons at
d values of 171.5 ppm and 176.5 ppm assigned to C-1’ and C-
4’, respectively. The esterification at the C-4 position was evi-
dent from the downfield shifting of the methine H-4 doublet
to a d value of 5.01 ppm (J=6.8 Hz) compared with its parent
resulted in the formation of
a
D19,20 bond. Similarly,
10,11,19,20-dianhydrosipholenol A (4) was a result of the loss
of two molecules of water with the formation of D10,11 and
D19,20 bonds. The identity of 3 and 4 was established by com-
3
1 (Dd= +1.27 ppm). This was further supported by a J-HMBC
correlation of H-4 with C-1’. The four-proton peak at 2.72 ppm
Table 1. Structures of sipholenol A (1) and sipholenone A (2) analogues.
Compd
R1
R2
Compd
R
Compd
R
1
5
6
7
8
9
10
11
18
20
21
H
a
a
b
c
d
e
f
H
H
a
3
H
a
b
c
d
e
f
22
23
24
H
c
g
12
13
14
15
16
17
19
H
H
H
H
H
H
H
H
g
g
h
i
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was assigned to methylene protons of succinate (H2-2’ and H2-
3’). Therefore, compound 7 was proved to be sipholenol A 4b-
succinate.
the 4b-O-benzyl-19,20-anhydrosipholenol A (19), based on its
HRMS spectrum, which showed an [MꢀH]+ peak at m/z
547.4120, suggesting the molecular formula C37H55O3. The
1
The HRMS spectrum of 8 showed an [M+Na]+ peak at m/z
603.4019, suggesting the molecular formula C37H56O5Na and
structures of 18 and 19 were confirmed by analyses of their H
and 13C NMR data. The upfield shift of the H-4 methine doublet
(d=3.40 ppm, J=6.6 Hz) and the downfield shift of the C-4
carbon atom peak (dC =84.6 ppm) indicated the selective
etherification of the C-4 secondary alcohol in 18 and 19. This
1
possible ester analogue of sipholenol A. The H and 13C NMR
data suggested benzoylation of 1 at C-4. The peak correspond-
ing to the methine proton H-4 was shifted downfield to a d
value of 5.19 ppm (d, J=6.8 Hz) in a similar fashion to 7, and
3
was further supported by the J-HMBC correlation of H-4 with
3
showed a J-HMBC correlation with C-1’ (dC =165.8 ppm). The
the benzylic methylene carbon C-1’ (dC =71.6 and 71.5 ppm, in
18 and 19, respectively).
peak corresponding to two aromatic methine protons at a d
value of 8.07 ppm (dd, J=7.7, 1.4 Hz) was assigned to H-3’ and
H-7’ based on the 3J-HMBC correlations with C-1’ (dC =
165.8 ppm) and C-5’ (dC =133.1 ppm). Therefore, compound 8
was found to be the 4b-benzoate ester of 1. Similarly, analysis
of the 1H and 13C NMR data of compounds 10 and 11 con-
firmed their identity as 4b-methylbenzoate and 4b-chloroben-
zoate esters of 1, respectively.
Carbamoylation
The HRMS spectrum of 20 showed an [M+Na]+ peak at m/z
632.4283, suggesting the molecular formula C38H59NO5Na and
possible carbamoyl analogue of sipholenol A. The 1H and
13C NMR data of 20 suggested the C-4 benzyl carbamoylation
of 1. The benzylic methylene protons H2-2’ (d=4.46 ppm)
showed 3J-HMBC correlations with C-4’/C-8’ (dC =127.6 ppm)
and the carbamate carbonyl C-1’ (dC =155.6 ppm). Thus, com-
pound 20 was determined to be the 4b-benzylcarbamate of 1.
The HRMS spectrum of 21 showed an [M+H]+ peak at m/z
674.4079, suggesting the molecular formula C38H60NO7S and
The HRMS spectrum of 9 showed an [M+H]+ peak at m/z
582.4157, suggesting the molecular formula C36H56NO5 and
possible ester analogue of sipholenol A. In a similar fashion,
1
the H and 13C NMR data of 9 confirmed the formation of isoni-
cotinate ester at C-4. The downfield peak at a d of 8.83 ppm
corresponding to aromatic methine protons was assigned to
H-4’ and H-6’ because they bear a heterocyclic nitrogen atom
N-5’, which caused a downfield shift. The remaining aromatic
methine proton peak at a d value of 7.86 ppm (2H, d, J=
4.4 Hz) was assigned to H-3’ and H-7’. Therefore, compound 9
was proved to be the 4b-isonicotinate ester of 1.
1
possible ester analogue of sipholenol A. The H and 13C NMR
data of 21 indicated 4b-methyl tosylcarbamoylation has oc-
curred. The benzylic methyl group at H3-8’ (d=2.44 ppm)
showed a 2J-HMBC correlation with the quaternary aromatic
carbon C-5’ (dC =135.0 ppm) and 3J-HMBC correlations with
the methine carbons C-4’ and C-6’ (dC =129.7 and 128.6 ppm,
respectively). Thus, compound 21 was determined to be the
4b-tosylcarbamate of 1.
The HRMS spectrum of 12 showed an [M+H]+ peak at m/z
501.3912, suggesting the molecular formula C32H53O4 and pos-
1
sible ester analogue of sipholenol A. The H and 13C NMR data
of 12 showed acetylation of 3 at C-4 position. The methyl sin-
glet H3-2’ (d=2.14 ppm) and methine doublet H-4 (d=
4.97 ppm, J=6.6 Hz) showed HMBC correlations with the car-
bonyl carbon C-1’ (dC =170.4 ppm). Therefore, compound 12
was proved to be 19,20-anhydrosipholenol A 4b-acetate. Simi-
Oxime formation
The HRMS spectrum of 22 showed an [M+H]+ peak at m/z
1
larly, H and 13C NMR data of 13–17 confirmed esterification of
490.3859, suggesting the molecular formula C30H52NO4 and
1
3 at C-4. The assignments of proton and carbon chemical
shifts of compounds 13–17 were carried out in a similar fash-
ion to those of compounds 7–11, respectively. Compounds
13–17 were found to be the 4b-succinate, 4b-benzoate, 4b-iso-
nicotinate, 4b-methylbenzoate, and 4b-chlorobenzoate esters
of 19,20-anhydrosipholenol A (3).
possible oxime analogue of 2. The H and 13C NMR data also
showed the upfield shift of the C-4 peak (dC =167.9 ppm), indi-
3
cating oxime formation. This was further supported by a J-
HMBC correlation between the methylene proton multiplets
H2-2 (d=1.21 and 1.90 ppm) with the quaternary carbon C-4
peak (dC =167.9 ppm).
The HRMS spectrum of 23 showed an [M+Na]+ peak at m/z
616.3967, suggesting the molecular formula C37H55NO5Na and
Etherification
1
possible oxime ester analogue of sipholenone A. The H and
Like semisynthetic sipholane esters (5–17), 4b-O-benzyl ethers
of 1 and 3 were prepared to test the significance of a free sec-
ondary alcohol group at C-4 on antimigratory activity. The
benzyl ethers were also prepared to explore whether an ether
(18 and 19) in this position imparts improved antimigratory ac-
tivity as compared with the ester analogues (8 and 14). Reac-
tion of 1 with benzyl bromide (BnBr) afforded the expected
4b-O-benzyl sipholenol A (18). The HRMS spectrum of 18
showed an [M+H]+ peak at m/z 567.4405, suggesting the mo-
lecular formula C37H59O4. The same reaction with 3 afforded
13C NMR data showed the downfield shift of the C-4 peak (dC =
176.0 ppm), indicating benzoylation of the C-4 oxime hydroxy
3
group of 22. This was further supported by the J-HMBC corre-
lation of the methylene proton H2-2 peaks (d=1.21 and
1.90 ppm) with the quaternary carbon C-4 peak (dC =
176.0 ppm). The peak corresponding to the two aromatic me-
thine protons at a d value of 8.01 ppm (dd, J=7.7, 1.4 Hz)
3
were assigned to H-3’ and H-7’ based on their J-HMBC correla-
tions with the C-1’ (dC =163.9 ppm) and C-5’ (dC =133.3 ppm)
peaks.
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The HRMS spectrum of 24 showed an [M+H]+ peak at m/z
580.4370, suggesting the molecular formula C37H58NO4 and
possible oxime ether analogue of 22. The structure of 24 was
confirmed by analysis of its 1H and 13C NMR data, which
3
showed J-HMBC correlations between the methylene proton
H2-2 multiplets (d=1.21 and 1.90 ppm) with the quaternary
carbon C-4 peak (dC =167.0 ppm). The assignment of the
benzyl moiety was achieved in a similar fashion as described
for 23.
The geometry of the oxime double bond in compounds 22–
24 was confirmed to be the Z-orientation based on NOESY
data. The peak corresponding to the aromatic H-3’ proton in
24 showed strong correlation with the H3-26 methyl singlet
proton peak in a NOESY experiment, indicating a close distance
range through space (<5 ꢁ). The distance between the aro-
matic H-3’ proton and the H3-26 protons in compound 24 was
calculated by using SYBYL version 8.0 to be 2.403 ꢁ, which is
in the range of NOE coupling (Figure S1 in the Supporting In-
formation). Thus, the same double bond geometry was also
assumed in compounds 22 and 23.
Biological activity
The antimigratory, anti-invasive, and cytotoxic activities of
compounds 1–24 were evaluated using a wound-healing
assay, a Cultrex invasion assay and an MTT assay, respectively.
The highly metastatic human breast cancer cell line MDA-MB-
231 was used to assess the antimigratory and anti-invasive ac-
tivities. These cells as well as the normal breast cell line
MCF10A were used to evaluate the cytotoxicity in an MTT
assay. Furthermore, Kinome profiling of analogue 14 was ap-
plied against a panel of 451 kinases. Additionally, a Z’-LYTE
kinase assay was used to evaluate the ability of compounds
1–24 to inhibit PTK6 phosphorylation.
Antimigratory (wound-healing) assay
The wound-healing assay[26,27] is a simple method for the study
of directional cell migration in vitro. The known antimigratory
lead 4-S-ethylphenylmethylene hydantoin (S-Ethyl) was used as
a positive control at a 50 mm dose.[20] Figure 1 shows the effect
of active analogues 11 and 17 on cell migration across the
wound inflicted in the MDA-MB231 cell monolayer compared
with the vehicle and positive controls. Generally, all sipholeno-
l A analogues, except 4 and 22, showed better antimigratory
activities than parent compound 1, which indicates the remov-
al of the hydrogen-bond donor capability of the free C-4 sec-
ondary alcohol group is beneficial for antimigratory activity
(Table 2). Of the diverse ester analogues tested, aromatic were
found to be more active than aliphatic esters. In contrast, car-
bamate analogues 20 and 21 were less active than the aromat-
ic esters. Furthermore, benzyl ethers 18 and 19 were less
active than benzoate esters 8 and 14, which illustrates the im-
portant role of the carbonyl group in 8 and 14 for enhanced
antimigratory activities. The activity of esters versus ether and
carbamate analogues suggests that esters fulfill the required
optimal distance between the side chain aromatic ring system
Figure 1. Antimigratory activity of 1, 2, 8–11 and 14–18 against the human
breast cancer cells MDA-MB-231. Error bars indicate the SD of n=3/dose.
DMSO was used as a vehicle control, while 4-S-ethylphenylmethylene hydan-
toin (S-Ethyl)[27] was used as a positive control at 50 mm.
and C-4 oxygen for maximal antimigratory activity. Shorter or
longer distances as represented by ethers or carbamates signif-
icantly decrease the antimigratory activity.
To assess the importance of para-substitution (C-5’) on the
activity of compounds 8 and 14, electron-donating (methyl)
and -withdrawing (chloro) groups were inserted in this position
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Table 2. IC50 values of analogues 1–24 determined using the wound-
healing assay (WHA) against MDA-MB-231 cells.
Compd
IC50 [mm]
Compd
IC50 [mm]
Compd
IC50 [mm]
1
2
3
4
5
6
7
8
37.5
28.1
35.3
46.7
16.8
20.3
18.0
7.9
9
10.6
11.8
5.3
16.0
21.0
7.2
17
18
19
20
21
22
23
24
5.9
12.2
13.4
13.9
18.0
52.7
28.5
31.4
10
11
12
13
14
15
16
9.5
12.3
to give compounds 10, 11, 16, and 17. 4-Chlorobenzoate
esters 11 and 17 were the most potent among all sipholanes
tested, with IC50 values of 5.3 and 5.9 mm, respectively, which
correlates well with their PTK6 phosphorylation inhibitory ac-
tivity. The presence of an electron-withdrawing substituent at
the para-position of the aromatic moiety (C-5’), as in com-
pounds 11 and 17, enhanced the antimigratory activity by
twofold when compared with an electron-donating substitu-
ent, as in compounds 10 and 16. Compounds 8, 10, 14, and
16 exhibited antimigratory activities with IC50 values of 7.9,
11.8, 7.2 and 12.3 mm, respectively, but with weaker inhibitory
activity against PTK6 phosphorylation, which could indicate
that this is not their only molecular target.
Figure 2. Anti-invasive activities of 5–7, 12 and 13 against the human breast
cancer cell line MDA-MB-231 using a Cultrex BME cell invasion assay kit.
Each concentration was run in triplicate, and the data are expressed as the
mean ꢁSEM. 4-S-Ethylphenylmethylene hydantoin (S-Ethyl)[27] was used as
a positive control at 50 mm.
MTT cytotoxicity assay
The MTT assay[30,31] allows for the measurement of cell viability
and populations in a quantitative colorimetric fashion by utiliz-
ing cellular ability to reduce the MTT reagent to insoluble
purple formazan dye. The cytotoxic effects of compounds 1–
24 were evaluated in MDA-MB-231 (malignant) and MCF10A
(normal) cells. Cells were treated with test compound under
the same conditions to those used for the wound-healing
assay before the addition of MTT (for full details, see the Exper-
imental Section), thus allowing the valid comparison of cyto-
toxicity and migration inhibition. Against MDA-MB-231 cells, all
compounds were found to be noncytotoxic at 20 mm. The cy-
totoxicity of the most active analogues in the wound-healing
assay was also assessed in normal mammary epithelial cells
(Figure 3). All active analogues were nontoxic at concentrations
equal their IC50 values in the wound-healing assay, suggesting
Invasion assay
The Cultrex basement membrane extract (BME) cell invasion
assay[28] is an accelerated in vitro screening process for com-
pounds that influence cell migration through extracellular ma-
trices, which is a fundamental function of cellular processes
such as angiogenesis, embryonic development, immune re-
sponse, and metastasis of cancer cells.[29] The anti-invasive ac-
tivities of compounds 1–24 were evaluated in the BME cell in-
vasion assay using highly metastatic MDA-MB-231 human
breast cancer cells (Figure 2). In
a preliminary screening at two
different doses (10 and 20 mm),
sipholenol A
succinate
(7)
showed the most potent activity,
allowing only 52% and 34% in-
vasion at the two concentra-
tions, respectively. In addition,
compounds 5, 6, 12, and 13
showed more than 50% inhibi-
tion of invasion at 20 mm, which
suggests the importance of a car-
bonyl group especially in the ali-
phatic ester analogues of 1.
Moreover, derivative 7 highlight-
ed the significance of increased
side chain length and an addi-
tional carboxy carbonyl group
for enhancement of the anti-in-
vasion activity.
Figure 3. Cytotoxic activity of 1, 2, 8–11 and 14–20 against the normal human mammary epithelium cells
MCF10A. Error bars indicate the SD of n=3/dose.
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to inhibit PTK6 phosphorylation
in vitro. Table 3 shows the per-
centage of inhibition at a single
dose concentration (25 mm) for
compounds 1–24; the results of
those compounds with the most
positive inhibitory effects are
also shown in Figure 5a. Based
on this initial assay, analogues
18 and 19 were identified as the
most potent inhibitors of PTK6
phosphorylation, with IC50 values
of 15.1 and 12.3 mm, respectively
(Figure 5b). In contrast, parent
compounds 1 and 2 showed
weak inhibition even at high
concentrations, which is consis-
tent with their moderate antimi-
gratory activities.
Conclusions
Aromatic sipholenol A esters
showed improved antimigratory
activity over analogous ether
and carbamate derivatives. p-
Chloro substitution in the aro-
matic C-4 esters significantly im-
proved the activity of these
compounds. In most cases, the
antimigratory activity is paral-
leled with their ability to inhibit
PTK6 phosphorylation, and in all
Figure 4. Kinome dendrogram of the selectivity profiling of compound 14 at 10 mm. PTK6 is colored blue, and
other target kinases are colored red. Dendrogram was generated using TREEspot Software tool with 35% cutoff.
Dendrogram reprinted with permission from KINOMEscan.
selectivity towards malignant cells. Ester and ether analogues
5–18 did not show any effect on P-gp accumulation or expres-
sion in P-gp-expressing cell lines (data not shown).
Table 3. PTK6 inhibitory activity of sipholane analogues 1–24 in Z-Lyte
kinase assay.[a]
Compd
I [%]
Compd
I [%]
Compd
I [%]
KINOMEscan kinase assay
1
2
3
4
5
6
7
8
ꢀ12.0
ꢀ4.2
ꢀ17.5
ꢀ6.4
ꢀ9.0
ꢀ6.8
ꢀ9.0
30.2
9
ꢀ22.4
25.8
58.8
ꢀ15.3
ꢀ12.9
38.6
ꢀ1.5
29.3
17
18
19
20
21
22
23
24
67.2
95.5
89.6
ꢀ6.5
ꢀ10.2
ꢀ3.0
ꢀ6.0
ꢀ4.8
10
11
12
13
14
15
16
The KINOMEscan screening platform employs a novel and pro-
prietary active site-directed competition binding assay to
quantitatively measure interactions between test compound
and a diverse kinase panel. Analogue 14 was screened against
451 human kinases using an ATP site competition binding
assay at a concentration of 10 mm. In the KINOMEscan panel,
only three kinases showed greater than 65% displacement
from an immobilized ligand by 14, which indicate the selectivi-
ty of this compound as a kinase inhibitor with selectivity score
(S35) less than 0.01 (Figure 4). The S35 value is calculated by di-
viding the number of kinases with less than 35% binding com-
pared with a control by the total number of tested kinases.
[a] % Inhibition (I) of phosphorylation was determined at a compound
concentration of 25 mm.
cases, no cytotoxicity to normal cells was observed. Aliphatic
polar esters, such as compound 7, showed better activity in
the invasion assay compared with aromatic esters. These re-
sults demonstrate the potential of the marine natural products
sipholanes as a new scaffold for the design of novel PTK6 in-
hibitors and their potential for development for use to control
metastatic breast cancer.
Z’-LYTE PTK6 kinase assay
A Z’-LYTE kinase assay[32] was used to assess the ability of the
natural products 1 and 2, and semisynthetic analogues 3–24
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General procedure A:[6] A solution of sipholenol A (1 equiv) in
CHCl3 (5 mL/equiv) was treated with p-TsOH·H2O (0.1 equiv), and
the reaction mixture was refluxed for 3 h. Water (5 mL) was added
with stirring, and the CHCl3 layer was separated, dried with anhy-
drous Na2SO4, filtered and concentrated to obtain a residue, which
was purified by column chromatography (silica gel 60) with
n-hexane/EtOAc gradient elution to afford compounds 3 and 4.
General procedure B:[33] 1. Using acid chlorides: A solution of si-
pholenol A (1 equiv) in anhydrous CH2Cl2 (10 mL/equiv) was treat-
ed with Et3N (5 equiv) and acid chloride (2 equiv), and the reaction
mixture was heated at reflux overnight. The reaction was then par-
titioned with water (10 mL), the organic phase was collected,
washed with 1n HCl (3ꢂ2 mL), dried over anhydrous Na2SO4, fil-
tered and concentrated in vacuo to obtain a residue, which was
purified by column chromatography (silica gel 60) with n-hexane/
EtOAc gradient elution to afford esters 9, 11, 15, and 17.
2. Using acid anhydrides: A solution of sipholenol A (1 equiv) in an-
hydrous CH2Cl2 or pyridine (10 mL/equiv) was treated with 4-dime-
thylaminopyridine (DMAP, 1 equiv) and acid anhydride (2 equiv),
and the reaction mixture was heated at reflux overnight. Water
(5 mL) was added to stop the reaction, and the resulting mixture
was partitioned to collect the organic phase, which was dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The residue
obtained was purified by column chromatography (silica gel 60)
with CHCl3/MeOH (95:5) isocratic elution to afford esters 5–8, 10,
12–14, and 16.
General procedure C (etherification): A solution of sipholenol A
(0.1 mmol) in dry DMF (1 mL/equiv) was treated with excess NaH
(10 equiv, 60% dispersion in mineral oil) and BnBr (1 equiv). Excess
NaH was used to increase the reaction yield. The reaction mixture
was stirred overnight at RT, and later for 15 min at 08C at the end
of reaction. Gradually, 0.2n HCl (5 mL) was added, and the vapors
were allowed to subside. The resulting aqueous phase was extract-
ed with CHCl3 (3ꢂ6 mL), and the combined organic phase was
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue obtained was purified by column chromatography
(silica gel 60) with n-hexane/EtOAc gradient elution to afford
benzyl ethers 18 and 19.
Figure 5. PTK6 phosphorylation inhibition of a) compounds 8, 10, 11, 14
and 16–19 at a concentration of 25 mm, and b) compounds 18 and 19 at
various concentrations. Results were obtained using a Z’-LYTE assay kit; error
bars indicate the SD of n=3/dose; staurosporine was used as a positive con-
trol.
Experimental Section
Chemistry
General procedure D (carbamoylation):[34] A solution of siphole-
nol A (1 equiv) in toluene (2 mL/equiv) was treated with the appro-
priate isocyanate (3 equiv) and Et3N (10 mL, 0.05 mmol, 0.5 equiv).
The reaction was heated at refluxed with stirring for 3 h. Water
(10 mL) was then added, and the mixture was extracted with
EtOAc (3ꢂ10 mL). The combined organic phase was dried over an-
hydrous Na2SO4, filtered and concentrated in vacuo. The residue
was purified by column chromatography (silica gel 60) with CHCl3/
MeOH (95:5) isocratic elution to afford carbamates 20 and 21.
25
General: Optical rotations (½aꢂD ) were measured on an analytical
Autopol III polarimeter (Rudolph Research, Hackettstown, NJ, USA).
IR spectra were recorded on a Varian 800 FT-IR spectrophotometer
(Agilent Technologies, Santa Clara, CA, USA). H and 13C NMR spec-
1
tra were recorded in CDCl3, using tetramethylsilane (TMS) as an in-
ternal standard, on a JEOL Eclipse-400 NMR spectrometer operat-
ing at 400 MHz (1H) and 100 MHz (13C). High-resolution mass spec-
trometry (HRMS) experiments were conducted at Louisiana State
University on a 6200-TOF LC-MS (Agilent) equipped with multi-
mode source: electrospray ionization (ESI) or atmospheric-pressure
chemical ionization (APCI). Analytical HPLC analyses were per-
formed on a DIONEX Summit III system using a Phenomenex Luna
250ꢂ4.6 mm, RP-C18 column, and isocratic elution (100% MeOH)
with UV detection set at 210–235 nm to verify the purity of each si-
pholenol and (Z)-5-(4-[ethylthio]benzylidene)-imidazolidine-2,4-
dione (S-Ethyl). A minimum of 98% HPLC purity for each com-
pound was verified. TLC analysis was carried out on precoated
silica gel 60 F254 500 mm TLC plates (EMD Chemicals), using variable
proportions of n-hexane/EtOAc as a mobile phase. 1% Vanillin in
concd H2SO4 was used as a visualizing reagent. For column chro-
matography, silica gel 60 (63–200 mm; Natland Intl, Research Trian-
gle Park, NC, USA) was used.
General procedure E: A solution of sipholenone A (1 equiv) in pyri-
dine/EtOH (1:1, 2 mL/equiv) was treated with NH2OH·HCl (1 equiv),
and the solution was heated at reflux for 4 h. Water (10 mL) was
added, and the mixture was extracted with EtOAc (3ꢂ10 mL). The
combined organic phase was dried over anhydrous Na2SO4, filtered
and concentrated in vacuo. The residue was purified by column
chromatography (silica gel 60) with CHCl3/MeOH (95:5) isocratic
elution to afford oxime 22.
19,20-Anhydrosipholenol A (3) and 10,11,19,20-dianhydrosipho-
lenol A (4): Prepared according to the general procedure A from
1
(150 mg, 0.31 mmol), CHCl3 (5 mL), p-TsOH·H2O (6 mg,
0.031 mmol). Purification by flash chromatography (n-hexane/
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EtOAc, 80:20) afforded 3 (120 mg, 83.2%) and 4 (5 mg, 3.6%).
CH, C-4’/C-6’), 129.6 (2C, CH, C-3’/C-7’), 130.6 (qC, C-2’), 133.1 (CH,
C-5’), 143.2 (qC, C-15), 165.8 ppm (qC, C-1’); IR (CHCl3): nmax =3454,
2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cmꢀ1; HRMS-ESI: m/z
[M+Na]+ calcd for C37H56O5Na: 603.4020, found: 603.4019.
Characterization data were as reported previously.[6]
4b-O-Acetylsipholenol A (5) and 4,19b-O-diacetylsipholenol A (6):
Prepared according to procedure B from 1 (50 mg, 0.1 mmol), an-
hydrous pyridine (0.5 mL), and Ac2O (20 mL, 0.21 mmol). Purification
4b-O-Isonicotinoylsipholenol A (9): Prepared according to proce-
dure B from 1 (30 mg, 0.063 mmol), anhydrous CH2Cl2 (10 mL), Et3N
(43.5 mL, 0.06 mmol) and isonicotinoyl chloride hydrochloride
(22.4 mg, 0.12 mmol). Purification by flash chromatography (n-
hexane/EtOAc, 70:30) afforded 9 as an amorphous solid (24 mg,
65.5%): ½aꢂ2D5 =ꢀ59.6 (c=0.72, CHCl3); 1H NMR (400 MHz, CDCl3):
d=0.76 (1H, m, H-11), 0.90 (3H, s, H-31), 1.04 (6H, s, H-24/H-30),
1.12 (3H, s, H-27), 1.16 (1H, m, H-12a), 1.24 (6H, s, H-26/H-29), 1.26
(3H, s, H-25), 1.31 (1H, m, H-2a), 1.45 (1H, m, H-8a), 1.52 (1H, m, H-
12b), 1.54 (1H, m, H-2b), 1.57 (1H, m, H-14), 1.60 (1H, m, H-20),
1.62 (2H, m, H-9), 1.66 (1H, m, H-13a), 1.68 (1H, m, H-21a), 1.73
(3H, s, H-28), 1.77 (1H, m, H-18), 1.78 (1H, m, H-17a), 1.82 (1H, m,
H-8b), 1.87 (1H, m, H-21b), 1.95 (1H, m, H-13b), 1.97 (1H, m, H-3a),
1.98 (1H, m, H-17b), 2.13 (1H, m, H-3b), 2.41 (1H, m, H-22), 3.47
(1H, dd, J=11.7, 4.0 Hz, H-7), 5.21 (1H, d, J=6.6 Hz, H-4), 5.43 (1H,
m, H-16), 7.86 (2H, d, J=4.4 Hz, H-3’/H-7’), 8.83 ppm (2H, br s, H-
4’/H-6’); 13C NMR (100 MHz, CDCl3): d=13.2 (CH3, C-24), 21.8 (CH3,
C-26), 23.2 (CH2, C-3), 24.8 (CH2, C-17), 24.9 (CH2, C-21), 25.7 (CH3,
C-29), 26.7 (CH2, C-8), 26.8 (CH2, C-12), 29.1 (CH3, C-25), 29.5 (CH3,
C-31), 30.1 (2C, CH3, C-27/C-28), 31.7 (CH3, C-30), 33.8 (CH2, C-13),
35.1 (CH2, C-2), 35.4 (qC, C-23), 37.2 (CH2, C-20), 39.5 (CH2, C-9),
42.7 (qC, C-1), 48.9 (CH, C-18), 52.8 (CH, C-22), 56.5 (CH, C-11), 57.4
(CH, C-14), 72.4 (qC, C-10), 77.3 (CH, C-7), 77.8 (qC, C-5), 80.2 (CH,
C-4), 82.2 (qC, C-19), 121.5 (CH, C-16), 122.9 (2C, CH, C-3’/C-7’),
137.9 (qC, C-2’), 143.0 (qC, C-15), 150.8 (2C, CH, C-4’/C-6’),
164.3 ppm (qC, C-1’); IR (CHCl3): nmax =3432, 2987, 2952, 2931,
2859, 1724, 1287, 1122, 910 cmꢀ1; HRMS-ESI: m/z [M+H]+ calcd for
C36H56NO5: 582.4153, found: 582.4157.
by flash chromatography (n-hexane/EtOAc, 80:20) afforded
(40 mg, 73.5%) and 6 (13 mg, 22.1%). Characterization data were
as reported previously.[6]
5
4b-O-Succinylsipholenol A (7): Prepared according to procedure B
from 1 (288 mg, 0.6 mmol), anhydrous CH2Cl2 (10 mL), DMAP
(72 mg, 0.5 mmol) and succinic anhydride (60 mg, 1.2 mmol). Purifi-
cation by HPLC was performed on a Phenomenex Gemini-NX C18
column (250ꢂ4.6 mm, 5 mm) at l210 nm, using a flow rate of
1 mLminꢀ1 and H2O/CH3CN (4:6) as a mobile phase (isocratic elu-
tion) to afford 7 (tR =7.5 min) as a colorless oil (167 mg, 47.9%):
½aꢂ2D5 =ꢀ41.5 (c=0.57, CHCl3); 1H NMR (400 MHz, CDCl3): d=0.75
(1H, m, H-11), 0.98 (3H, s, H-24), 1.02 (3H, s, H-31), 1.08 (3H, s, H-
30), 1.10 (3H, s, H-27), 1.13 (3H, s, H-26), 1.18 (3H, s, H-25), 1.18
(1H, m, H-12a), 1.24 (1H, m, H-2a), 1.25 (3H, s, H-29), 1.37 (1H, m,
H-8a), 1.43 (1H, m, H-2b), 1.48 (1H, m, H-9a), 1.52 (1H, m, H-12b),
1.57 (1H, m, H-20a), 1.62 (1H, m, H-14), 1.63 (1H, m, H-9b), 1.70
(2H, m, H-13a/H-20b), 1.74 (3H, s, H-28), 1.75 (1H, m, H-8b), 1.78
(2H, m, H-3a/H-17a), 1.80 (2H, m, H-18/H-21a), 1.95 (1H, m, H-21b),
1.98 (1H, m, H-3b), 2.01 (1H, m, H-13b), 2.02 (1H, m, H-17b), 2.46
(1H, m, H-22), 2.72 (4H, m, H-2’/H-3’), 3.34 (1H, dd, J=11.9, 4.0 Hz,
H-7), 5.01 (1H, d, J=6.8 Hz, H-4), 5.45 ppm (1H, m, H-16); 13C NMR
(100 MHz, CDCl3): d=12.9 (CH3, C-24), 21.6 (CH3, C-26), 23.1 (CH2,
C-3), 24.8 (CH2, C-17), 25.0 (CH2, C-21), 25.6 (CH3, C-29), 26.6 (CH2,
C-8), 26.9 (CH2, C-12), 28.9 (CH2, C-2’), 29.1 (CH3, C-25), 29.3 (CH2, C-
3’), 29.6 (CH3, C-31), 30.0 (CH3, C-27), 30.3 (CH3, C-28), 31.8 (CH3, C-
30), 33.7 (CH2, C-13), 35.0 (CH2, C-2), 35.5 (qC, C-23), 37.1 (CH2, C-
20), 39.2 (CH2, C-9), 42.6 (qC, C-1), 48.9 (CH, C-18), 52.8 (CH, C-22),
56.0 (CH, C-11), 57.5 (CH, C-14), 72.5 (qC, C-10), 77.0 (CH, C-7), 77.4
(qC, C-5), 79.7 (CH, C-4), 82.5 (qC, C-19), 121.4 (CH, C-16), 143.2 (qC,
C-15), 171.5 (qC, C-1’), 176.5 ppm (qC, C-4’); IR (CHCl3): nmax =3169,
2987, 2949, 2864, 1730, 1717, 1364, 1162, 1080, 909 cmꢀ1; HRMS-
ESI: m/z [M+Na]+ calcd for C34H56O7Na: 599.3924, found:
599.3929.
4b-O-4’-Methylbenzoylsipholenol A (10): Prepared according to
procedure
B from 1 (25 mg, 0.052 mmol), anhydrous CH2Cl2
(10 mL), DMAP (6 mg, 0.05 mmol) and 4-methylbenzoic anhydride
(23.8 mg, 0.1 mmol). Purification by flash chromatography (CHCl3/
MeOH, 95:5) afforded 10 as an amorphous solid (30 mg, 98.5%):
½aꢂ2D5 =ꢀ57.6 (c=0.36, CHCl3); 1H NMR (400 MHz, CDCl3): d=0.76
(1H, m, H-11), 0.90 (3H, s, H-31), 1.04 (6H, s, H-24/H-30), 1.12 (3H,
s, H-27), 1.16 (1H, m, H-12a), 1.24 (6H, s, H-26/H-29), 1.26 (3H, s, H-
25), 1.38 (1H, m, H-2a), 1.45 (1H, m, H-8a), 1.52 (1H, m, H-12b),
1.56 (1H, m, H-2b), 1.57 (1H, m, H-14), 1.60 (1H, m, H-20), 1.64 (2H,
m, H-9), 1.66 (1H, m, H-13a), 1.68 (1H, m, H-21a), 1.73 (3H, s, H-28),
1.77 (1H, m, H-18), 1.78 (1H, m, H-17a), 1.82 (1H, m, H-8b), 1.87
(1H, m, H-21b), 1.94 (1H, m, H-3a), 1.95 (1H, m, H-13b), 1.98 (1H,
m, H-17b), 2.12 (1H, m, H-3b), 2.41 (1H, m, H-22), 2.44 (3H, m, H-
8’), 3.55 (1H, dd, J=11.3, 3.6 Hz, H-7), 5.19 (1H, d, J=6.6 Hz, H-4),
5.44 (1H, dd, J=9.2, 5.1 Hz, H-16), 7.26 (2H, d, J=8.2 Hz, H-4’/H-6’),
7.98 ppm (2H, d, J=8.2 Hz, H-3’/H-7’); 13C NMR (100 MHz, CDCl3):
d=13.2 (CH3, C-24), 21.3 (CH3, C-8’), 21.8 (CH3, C-26), 23.2 (CH2, C-
3), 24.8 (CH2, C-17), 24.9 (CH2, C-21), 25.7 (CH3, C-29), 26.7 (CH2, C-
8), 26.8 (CH2, C-12), 29.1 (CH3, C-25), 29.5 (CH3, C-31), 30.1 (2C, CH3,
C-27/C-28), 31.7 (CH3, C-30), 33.8 (CH2, C-13), 35.1 (CH2, C-2), 35.4
(qC, C-23), 37.2 (CH2, C-20), 39.5 (CH2, C-9), 42.7 (qC, C-1), 48.9 (CH,
C-18), 52.8 (CH, C-22), 56.5 (CH, C-11), 57.4 (CH, C-14), 72.4 (qC, C-
10), 77.3 (CH, C-7), 77.8 (qC, C-5), 80.2 (CH, C-4), 82.2 (qC, C-19),
121.4 (CH, C-16), 127.8 (qC, C-5’), 129.6 (2C, CH, C-4’/C-6’), 130.2
(2C, CH, C-3’/C-7’), 143.2 (qC, C-15), 143.8 (qC, C-2’), 165.8 ppm (qC,
C-1’); IR (CHCl3): nmax =3454, 2987, 2950, 2930, 2858, 1713, 1282,
4b-O-Benzoylsipholenol A (8): Prepared according to procedure B
from 1 (25 mg, 0.052 mmol), anhydrous CH2Cl2 (10 mL), DMAP
(6 mg, 0.05 mmol) and benzoyl anhydride (23.8 mg, 0.1 mmol). Pu-
rification by flash chromatography (CHCl3/MeOH, 95:5) afforded 8
as an amorphous solid (30 mg, 98.5%): ½aꢂ2D5 =ꢀ59.6 (c=0.96,
1
CHCl3); H NMR (400 MHz, CDCl3): d=0.76 (1H, m, H-11), 0.90 (3H,
s, H-31), 1.04 (6H, s, H-24/H-30), 1.12 (3H, s, H-27), 1.16 (1H, m, H-
12a), 1.24 (6H, s, H-26/H-29), 1.26 (3H, s, H-25), 1.38 (1H, m, H-2a),
1.45 (1H, m, H-8a), 1.52 (1H, m, H-12b), 1.56 (1H, m, H-2b), 1.57
(1H, m, H-14), 1.60 (1H, m, H-20), 1.64 (2H, m, H-9), 1.66 (1H, m, H-
13a), 1.68 (1H, m, H-21a), 1.73 (3H, s, H-28), 1.77 (1H, m, H-18),
1.78 (1H, m, H-17a), 1.82 (1H, m, H-8b), 1.87 (1H, m, H-21b), 1.94
(1H, m, H-3a), 1.95 (1H, m, H-13b), 1.98 (1H, m, H-17b), 2.12 (1H,
m, H-3b), 2.41 (1H, m, H-22), 3.55 (1H, dd, J=11.3, 3.6 Hz, H-7),
5.19 (1H, d, J=6.6 Hz, H-4), 5.44 (1H, dd, J=9.2, 5.1 Hz, H-16), 7.48
(2H, dd, J=7.7, 7.7 Hz, H-4’/H-6’), 7.60 (1H, m, H-5’), 8.07 ppm (2H,
dd, J=7.7, 1.4 Hz, H-3’/H-7’); 13C NMR (100 MHz, CDCl3): d=13.2
(CH3, C-24), 21.8 (CH3, C-26), 23.2 (CH2, C-3), 24.8 (CH2, C-17), 24.9
(CH2, C-21), 25.7 (CH3, C-29), 26.7 (CH2, C-8), 26.8 (CH2, C-12), 29.1
(CH3, C-25), 29.5 (CH3, C-31), 30.1 (2C, CH3, C-27/C-28), 31.7 (CH3, C-
30), 33.8 (CH2, C-13), 35.1 (CH2, C-2), 35.4 (qC, C-23), 37.2 (CH2, C-
20), 39.5 (CH2, C-9), 42.7 (qC, C-1), 48.9 (CH, C-18), 52.8 (CH, C-22),
56.5 (CH, C-11), 57.4 (CH, C-14), 72.4 (qC, C-10), 77.3 (CH, C-7), 77.8
(qC, C-5), 80.2 (CH, C-4), 82.2 (qC, C-19), 121.4 (CH, C-16), 128.6 (2C,
1114, 910 cmꢀ1
;
HRMS-ESI: m/z [M+H]+ calcd for C38H59O5:
595.4357, found: 595.4324.
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4b-O-4’-Chlorobenzoylsipholenol A (11): Prepared according to
1.38 (1H, m, H-8a), 1.47 (1H, m, H-9a), 1.48 (1H, m, H-2b), 1.52 (1H,
m, H-12b), 1.62 (1H, m, H-9b), 1.67 (1H, m, H-21a), 1.67 (3H, s, H-
29), 1.68 (1H, m, H-14), 1.75 (3H, s, H-28), 1.76 (1H, m, H-8b), 1.78
(1H, m, H-3a), 1.86 (1H, m, H-17a), 1.98 (1H, m, H-3b), 2.06 (1H, m,
H-21b), 2.07 (1H, m, H-17b), 2.08 (1H, m, H-22), 2.16 (1H, m, H-
13a), 2.32 (1H, m, H-18), 2.42 (1H, m, H-13b), 2.73 (2H, m, H-2’),
2.73 (2H, m, H-3’), 3.34 (1H, dd, J=11.7, 4.0 Hz, H-7), 5.01 (1H, d,
J=6.6 Hz, H-4), 5.27 (1H, br s, H-20), 5.48 ppm (1H, m, H-16);
13C NMR (100 MHz, CDCl3): d=13.0 (CH3, C-24), 15.5 (CH3, C-29),
21.5 (CH3, C-26), 23.1 (CH2, C-3), 26.5 (CH2, C-12), 26.6 (CH2, C-8),
26.9 (CH2, C-17), 29.0 (CH2, C-3’), 29.1 (CH3, C-25), 29.2 (CH2, C-2’),
29.6 (CH3, C-31), 30.0 (CH3, C-27), 30.2 (CH3, C-28), 31.3 (CH3, C-30),
33.3 (CH2, C-21), 34.0 (CH2, C-13), 35.0 (CH2, C-2), 35.3 (qC, C-23),
39.2 (CH2, C-9), 42.6 (qC, C-1), 45.2 (CH, C-18), 56.0 (CH, C-11), 56.4
(CH, C-22), 57.1 (CH, C-14), 72.5 (qC, C-10), 77.0 (CH, C-7), 77.8 (qC,
C-5), 79.8 (CH, C-4), 121.7 (CH, C-16), 123.5 (CH, C-20), 143.0 (qC, C-
15), 146.7 (qC, C-19), 171.4 (qC, C-1’), 177.2 ppm (qC, C-4’); IR
(CHCl3): nmax =3165, 2987, 2929, 2857, 1729, 1717, 1364, 1164,
procedure
B from 1 (25 mg, 0.052 mmol), anhydrous CH2Cl2
(10 mL), DMAP (6 mg, 0.05 mmol) and 4-chlorobenzoyl anhydride
(23.8 mg, 0.12 mmol). Purification by flash chromatography (CHCl3/
MeOH, 95:5) gave 11 as an amorphous solid (30 mg, 98.5%):
½aꢂ2D5 =ꢀ61.3 (c=0.52, CHCl3); 1H NMR (400 MHz, CDCl3): d=0.76
(1H, m, H-11), 0.90 (3H, s, H-31), 1.04 (6H, s, H-24/H-30), 1.12 (3H,
s, H-27), 1.16 (1H, m, H-12a), 1.24 (6H, s, H-26/H-29), 1.26 (3H, s, H-
25), 1.38 (1H, m, H-2a), 1.45 (1H, m, H-8a), 1.52 (1H, m, H-12b),
1.56 (1H, m, H-2b), 1.57 (1H, m, H-14), 1.60 (1H, m, H-20), 1.64 (2H,
m, H-9), 1.66 (1H, m, H-13a), 1.68 (1H, m, H-21a), 1.73 (3H, s, H-28),
1.77 (1H, m, H-18), 1.78 (1H, m, H-17a), 1.82 (1H, m, H-8b), 1.87
(1H, m, H-21b), 1.94 (1H, m, H-3a), 1.95 (1H, m, H-13b), 1.98 (1H,
m, H-17b), 2.12 (1H, m, H-3b), 2.41 (1H, m, H-22), 3.55 (1H, dd, J=
11.3, 3.6 Hz, H-7), 5.19 (1H, d, J=6.6 Hz, H-4), 5.44 (1H, dd, J=9.2,
5.1 Hz, H-16), 7.44 (2H, d, J=8.6 Hz, H-4’/H-6’), 7.97 ppm (2H, d,
J=8.6 Hz, H-3’/H-7’); 13C NMR (100 MHz, CDCl3): d=13.2 (CH3, C-
24), 21.8 (CH3, C-26), 23.2 (CH2, C-3), 24.8 (CH2, C-17), 24.9 (CH2, C-
21), 25.7 (CH3, C-29), 26.7 (CH2, C-8), 26.8 (CH2, C-12), 29.1 (CH3, C-
25), 29.5 (CH3, C-31), 30.1 (2C, CH3, C-27/C-28), 31.7 (CH3, C-30),
33.8 (CH2, C-13), 35.1 (CH2, C-2), 35.4 (qC, C-23), 37.2 (CH2, C-20),
39.5 (CH2, C-9), 42.7 (qC, C-1), 48.9 (CH, C-18), 52.8 (CH, C-22), 56.5
(CH, C-11), 57.4 (CH, C-14), 72.4 (qC, C-10), 77.3 (CH, C-7), 77.8 (qC,
C-5), 80.2 (CH, C-4), 82.2 (qC, C-19), 121.4 (CH, C-16), 128.9 (qC, C-
5’), 129.0 (2C, CH, C-4’/C-6’), 130.9 (2C, CH, C-3’/C-7’), 139.6 (qC, C-
2’), 143.2 (qC, C-15), 164.9 ppm (qC, C-1’); IR (CHCl3): nmax =3454,
2987, 2950, 2930, 2858, 1713, 1282, 1114, 910 cmꢀ1; HRMS-ESI: m/z
[M+Na]+ calcd for C37H55ClO5Na: 637.3636, found: 637.3685.
1080, 909 cmꢀ1
;
HRMS-ESI: m/z [M+H]+ calcd for C34H55O6:
559.3993, found: 559.3969.
4b-O-Benzoyl-19,20-anhydrosipholenol A (14): Prepared accord-
ing to procedure B using 3 (20 mg, 0.044 mmol), anhydrous CH2Cl2
(10 mL), DMAP (5.3 mg, 0.05 mmol) and benzoic anhydride (30 mg,
0.12 mmol). Purification by flash chromatography (n-hexane/EtOAc,
70:30) gave 14 as a colorless oil (10.4 mg, 42.4%): ½aꢂ2D5 =ꢀ25.9
1
(c=0.21, CHCl3); H NMR (400 MHz, CDCl3): d=0.76 (1H, m, H-11),
0.89 (3H, s, H-31), 1.04 (6H, s, H-24/H-30), 1.11 (3H, s, H-27), 1.18
(1H, m, H-12a), 1.24 (3H, s, H-26), 1.27 (3H, s, H-25), 1.40 (1H, m,
H-2a), 1.45 (1H, m, H-8a), 1.50 (1H, m, H-9a), 1.52 (1H, m, H-12b),
1.56 (1H, m, H-2b), 1.66 (2H, m, H-14/H-21a), 1.66 (3H, s, H-29),
1.68 (1H, m, H-9b), 1.74 (3H, s, H-28), 1.82 (1H, m, H-8b), 1.84 (1H,
m, H-17a), 1.96 (1H, m, H-3a), 1.98 (1H, m, H-21b), 2.01 (1H, m, H-
22), 2.02 (1H, m, H-13a), 2.03 (1H, m, H-17b), 2.12 (1H, m, H-3b),
2.26 (2H, m, H-13b/H-18), 3.55 (1H, dd, J=11.9, 4.2 Hz, H-7), 5.20
(1H, d, J=6.6 Hz, H-4), 5.23 (1H, br s, H-20), 5.47 (1H, m, H-16),
7.50 (2H, dd, J=7.7, 7.7 Hz, H-4’/H-6’), 7.61 (1H, dd, J=7.3, 7.3 Hz,
H-5’), 8.07 ppm (2H, dd, J=7.9, 1.1 Hz, H-3’/H-7’); 13C NMR
(100 MHz, CDCl3): d=13.2 (CH3, C-24), 15.4 (CH3, C-29), 21.8 (CH3,
C-26), 23.2 (CH2, C-3), 26.3 (CH2, C-12), 26.7 (CH2, C-8), 26.9 (CH2, C-
17), 29.1 (CH3, C-25), 29.2 (CH3, C-31), 30.2 (CH3, C-28), 30.8 (CH3, C-
27), 31.2 (CH3, C-30), 33.3 (CH2, C-21), 34.0 (CH2, C-13), 35.1 (CH2, C-
2), 35.2 (qC, C-23), 39.5 (CH2, C-9), 42.7 (qC, C-1), 45.2 (CH, C-18),
56.3 (CH, C-22), 56.5 (CH, C-11), 57.1 (CH, C-14), 72.4 (qC, C-10),
77.3 (CH, C-7), 77.8 (qC, C-5), 80.1 (CH, C-4), 121.8 (CH, C-16), 123.4
(CH, C-20), 128.6 (2C, CH, C-4’/C-6’), 129.5 (2C, CH, C-3’/C-7’), 130.5
(qC, C-2’), 133.2 (CH, C-5’), 143.0 (qC, C-15), 146.5 (qC, C-19),
165.8 ppm (qC, C-1’); IR (CHCl3): nmax =2985, 2929, 2857, 1713,
1451, 1364, 1280, 1114, 909 cmꢀ1; HRMS-ESI: m/z [M+H]+ calcd for
C37H55O4: 63.4095, found: 563.4095.
4b-O-Acetyl-19,20-anhydrosipholenol A (12): Prepared according
to procedure B from 3 (30 mg, 0.065 mmol), anhydrous CH2Cl2
(3 mL), DMAP (8 mg, 0.06 mmol) and Ac2O (12.3 mL, 0.18 mmol).
Purification by flash chromatography (n-hexane/EtOAc, 80:20) gave
12 as a colorless oil (22 mg, 67.2%): ½aꢂ2D5 =ꢀ4.6 (c=0.69, CHCl3);
1H NMR (400 MHz, CDCl3): d=0.74 (1H, m, H-11), 0.98 (3H, s, H-24),
1.01 (3H, s, H-31), 1.08 (6H, s, H-27/H-30), 1.13 (3H, s, H-26), 1.18
(3H, s, H-25), 1.19 (1H, m, H-12a), 1.26 (1H, m, H-2a), 1.37 (1H, m,
H-8a), 1.46 (1H, m, H-2b), 1.53 (1H, m, H-12b), 1.60 (1H, m, H-9),
1.66 (3H, s, H-29), 1.68 (2H, m, H-14/H-21a), 1.74 (1H, m, H-8b),
1.75 (3H, s, H-28), 1.76 (1H, m, H-3a), 1.84 (1H, m, H-17a), 1.94 (1H,
m, H-3b), 2.05 (1H, m, H-21b), 2.06 (1H, m, H-17b), 2.07 (1H, m, H-
22), 2.14 (3H, s, H-2’), 2.14 (1H, m, H-13a), 2.31 (1H, m, H-18), 2.41
(1H, m, H-13b), 3.34 (1H, dd, J=11.9, 4.2 Hz, H-7), 4.97 (1H, d, J=
6.6 Hz, H-4), 5.27 (1H, br s, H-20), 5.48 ppm (1H, m, H-16); 13C NMR
(100 MHz, CDCl3): d=12.9 (CH3, C-24), 15.4 (CH3, C-29), 21.4 (CH3,
C-2’), 21.6 (CH3, C-26), 23.0 (CH2, C-3), 26.5 (CH2, C-12), 26.6 (CH2, C-
8), 26.9 (CH2, C-17), 29.1 (CH3, C-25), 29.4 (CH3, C-31), 30.1 (CH3, C-
27), 30.3 (CH3, C-28), 31.3 (CH3, C-30), 33.3 (CH2, C-21), 34.0 (CH2, C-
13), 35.0 (CH2, C-2), 35.3 (qC, C-23), 39.2 (CH2, C-9), 42.6 (qC, C-1),
45.2 (CH, C-18), 56.0 (CH, C-11), 56.4 (CH, C-22), 57.1 (CH, C-14),
72.3 (qC, C-10), 76.9 (CH, C-7), 77.3 (qC, C-5), 79.2 (CH, C-4), 121.6
(CH, C-16), 123.4 (CH, C-20), 143.0 (qC, C-15), 146.7 (qC, C-19),
170.4 ppm (qC, C-1’); IR (CHCl3): nmax =2986, 2930, 2858, 1728,
1461, 1365, 1262, 1080, 910 cmꢀ1; HRMS-ESI: m/z [M+H]+ calcd
for C32H53O4: 501.3938, found: 501.3912.
4b-O-Isonicotinoyl-19,20-anhydrosipholenol A (15): Prepared ac-
cording to procedure B using 3 (18 mg, 0.065 mmol), anhydrous
CH2Cl2 (10 mL), Et3N (27.2 mL, 0.032 mmol) and isonicotinoyl chlo-
ride hydrochloride (14 mg, 0.2 mmol). Purification by flash chroma-
tography (n-hexane/EtOAc, 70:30) gave 15 as a colorless oil (6 mg,
27.1%): ½aꢂ2D5 =ꢀ24.3 (c=0.25, CHCl3); 1H NMR (400 MHz, CDCl3):
d=0.76 (1H, m, H-11), 0.89 (3H, s, H-31), 1.04 (6H, s, H-24/H-30),
1.11 (3H, s, H-27), 1.18 (1H, m, H-12a), 1.24 (3H, s, H-26), 1.27 (3H,
s, H-25), 1.35 (1H, m, H-2a), 1.45 (1H, m, H-8a), 1.50 (1H, m, H-9a),
1.52 (1H, m, H-12b), 1.54 (1H, m, H-2b), 1.66 (3H, s, H-29), 1.66
(2H, m, H-14/H-21a), 1.68 (1H, m, H-9b), 1.74 (3H, s, H-28), 1.82
(1H, m, H-8b), 1.84 (1H, m, H-17a), 1.95 (1H, m, H-3a), 1.98 (1H, m,
H-21b), 2.01 (1H, m, H-22), 2.02 (1H, m, H-13a), 2.03 (1H, m, H-
4b-O-Succinyl-19,20-anhydrosipholenol A (13): Prepared accord-
ing to procedure B from 3 (25 mg, 0.055 mmol), anhydrous CH2Cl2
(10 mL), DMAP (6.7 mg, 0.06 mmol) and succinic anhydride (22 mg,
0.16 mmol). Purification by flash chromatography (CHCl3/MeOH,
95:5) gave 13 as a colorless oil (19.6 mg, 64.4%): ½aꢂ2D5 =ꢀ4.3 (c=
1
0.49, CHCl3); H NMR (400 MHz, CDCl3): d=0.74 (1H, m, H-11), 0.99
(3H, s, H-24), 1.01 (3H, s, H-31), 1.09 (6H, s, H-27/H-30), 1.13 (3H, s,
H-26), 1.19 (1H, m, H-12a), 1.19 (3H, s, H-25), 1.27 (1H, m, H-2a),
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17b), 2.12 (1H, m, H-3b), 2.26 (2H, m, H-13b/H-18), 3.47 (1H, dd,
J=11.7, 4.0 Hz, H-7), 5.23 (1H, d, J=7.0 Hz, H-4), 5.25 (1H, br s, H-
20), 5.47 (1H, m, H-16), 7.86 (2H, d, J=4.0 Hz, H-3’/H-7’), 8.85 ppm
(2H, br s, H-4’/H-6’); 13C NMR (100 MHz, CDCl3): d=13.2 (CH3, C-24),
15.4 (CH3, C-29), 21.8 (CH3, C-26), 23.2 (CH2, C-3), 26.3 (CH2, C-12),
26.7 (CH2, C-8), 26.9 (CH2, C-17), 29.1 (CH3, C-25), 29.2 (CH3, C-31),
30.8 (CH3, C-27), 30.2 (CH3, C-28), 31.2 (CH3, C-30), 33.3 (CH2, C-21),
34.0 (CH2, C-13), 35.1 (CH2, C-2), 35.2 (qC, C-23), 39.5 (CH2, C-9),
42.7 (qC, C-1), 45.2 (CH, C-18), 56.3 (CH, C-22), 56.5 (CH, C-11), 57.1
(CH, C-14), 72.4 (qC, C-10), 77.3 (CH, C-7), 77.8 (qC, C-5), 80.1 (CH,
C-4), 121.8 (CH, C-16), 122.9 (2C, CH, C-3’/C-7’), 123.4 (CH, C-20),
137.8 (qC, C-2’), 142.9 (qC, C-15), 146.6 (qC, C-19), 150.8 (2C, CH, C-
4’/C-6’), 164.4 ppm (qC, C-1’); IR (CHCl3): nmax =2985, 2953, 2929,
2856, 1724, 1286, 1123 cmꢀ1; HRMS-ESI: m/z [M+H]+ calcd for
C36H54NO4, 564.4047, found: 564.4052.
3), 26.3 (CH2, C-12), 26.7 (CH2, C-8), 26.9 (CH2, C-17), 29.1 (CH3, C-
25), 29.2 (CH3, C-31), 30.2 (CH3, C-28), 30.8 (CH3, C-27), 31.2 (CH3, C-
30), 33.3 (CH2, C-21), 34.0 (CH2, C-13), 35.1 (CH2, C-2), 35.2 (qC, C-
23), 39.5 (CH2, C-9), 42.7 (qC, C-1), 45.2 (CH, C-18), 56.3 (CH, C-22),
56.5 (CH, C-11), 57.1 (CH, C-14), 72.4 (qC, C-10), 77.3 (CH, C-7), 77.8
(qC, C-5), 80.1 (CH, C-4), 121.8 (CH, C-16), 123.4 (CH, C-20), 128.9
(qC, C-5’), 129.0 (2C, CH, C-4’/C-6’), 130.9 (2C, CH, C-3’/C-7’), 139.6
(qC, C-2’), 143.0 (qC, C-15), 146.5 (qC, C-19), 164.9 ppm (qC, C-1’);
IR (CHCl3): nmax =2985, 2929, 2857, 1713, 1451, 1364, 1280, 1114,
909 cmꢀ1; HRMS-ESI: m/z [M+H]+ calcd for C37H54ClO4: 597.3711,
found: 597.3711.
4b-O-Benzylsipholenol A (18): Prepared according to procedure C
using 1 (48 mg, 0.1 mmol), dry DMF (1 mL), NaH (10 equiv) and
BnBr (12 mL, 1 equiv). Purification by flash chromatography (n-
hexane/EtOAc, 90:10) gave 18 as an amorphous solid (36 mg,
63.1%): ½aꢂ2D5 =ꢀ48.8 (c=0.53, CHCl3); 1H NMR (400 MHz, CDCl3):
d=0.78 (1H, m, H-11), 0.98 (3H, s, H-24), 1.04 (3H, s, H-31), 1.06
(3H, s, H-27), 1.09 (3H, s, H-30), 1.11 (3H, s, H-25), 1.18 (1H, m, H-
12a), 1.25 (3H, s, H-29), 1.27 (3H, s, H-26), 1.36 (1H, m, H-8a), 1.43
(2H, m, H-2), 1.50 (1H, m, H-9a), 1.51 (1H, m, H-12b), 1.57 (1H, m,
H-9b), 1.61 (1H, m, H-14), 1.62 (1H, m, H-20a), 1.71 (1H, m, H-8b),
1.74 (1H, m, H-13a), 1.75 (3H, s, H-28), 1.78 (2H, m, H-17a/H-20b),
1.80 (2H, m, H-3a/H-18), 1.82 (1H, m, H-21a), 1.92 (1H, m, H-3b),
1.94 (1H, m, H-21b), 1.97 (1H, m, H-13b), 2.01 (1H, m, H-17b), 2.48
(1H, m, H-22), 3.40 (1H, d, J=6.6 Hz, H-4), 3.50 (1H, dd, J=11.7,
4.0 Hz, H-7), 4.31 (1H, d, J=11.9 Hz, H-1’b), 4.55 (1H, d, J=11.9 Hz,
H-1’a), 5.45 (1H, dd, J=9.1, 5.1 Hz, H-16), 7.29 (1H, m, H-5’), 7.35
(2H, m, H-4’/H-6’), 7.36 ppm (2H, m, H-3’/H-7’); 13C NMR (100 MHz,
CDCl3): d=13.1 (CH3, C-24), 21.7 (CH2, C-3), 22.2 (CH3, C-26), 24.8
(CH2, C-17), 25.1 (CH2, C-21), 25.7 (CH3, C-29), 26.6 (CH2, C-8), 26.9
(CH2, C-12), 29.5 (CH3, C-31), 29.6 (CH3, C-25), 29.9 (CH3, C-27), 30.2
(CH3, C-28), 31.7 (CH3, C-30), 34.0 (CH2, C-13), 34.1 (CH2, C-2), 35.5
(qC, C-23), 37.4 (CH2, C-20), 39.2 (CH2, C-9), 42.5 (qC, C-1), 48.9 (CH,
C-18), 52.8 (CH, C-22), 55.9 (CH, C-11), 57.6 (CH, C-14), 71.6 (CH2, C-
1’), 72.5 (qC, C-10), 76.5 (CH, C-7), 78.6 (qC, C-5), 82.2 (qC, C-19),
84.6 (CH, C-4), 121.3 (CH, C-16), 127.6 (3C, CH, C-3’/C-5’/C-7’), 128.4
(2C, CH, C-4’/C-6’), 139.2 (qC, C-2’), 143.2 ppm (qC, C-15); IR
(CHCl3): nmax =3454, 2987, 2949, 2863, 1463, 1455, 1376, 1363,
4b-O-4’-Methylbenzoyl-19,20-anhydrosipholenol A (16): Prepared
according to procedure B using 3 (20 mg, 0.044 mmol), anhydrous
CH2Cl2 (10 mL), DMAP (5.3 mg, 0.05 mmol) and 4-methylbenzoic
anhydride (30 mg, 0.18 mmol). Purification by flash chromatogra-
phy (n-hexane/EtOAc, 95:5!80:20) gave 14 as a colorless oil
1
(10.4 mg, 42.4%): ½aꢂ2D5 =ꢀ23.9 (c=0.36, CHCl3); H NMR (400 MHz,
CDCl3): d=0.76 (1H, m, H-11), 0.89 (3H, s, H-31), 1.04 (6H, s, H-24/
H-30), 1.11 (3H, s, H-27), 1.18 (1H, m, H-12a), 1.24 (3H, s, H-26),
1.27 (3H, s, H-25), 1.40 (1H, m, H-2a), 1.45 (1H, m, H-8a), 1.50 (1H,
m, H-9a), 1.52 (1H, m, H-12b), 1.56 (1H, m, H-2b), 1.57 (1H, m, H-
14), 1.66 (2H, m, H-13a/H-21a), 1.66 (3H, s, H-29), 1.68 (1H, m, H-
9b), 1.74 (3H, s, H-28), 1.78 (1H, m, H-17a), 1.82 (1H, m, H-8b), 1.95
(1H, m, H-13b), 1.96 (1H, m, H-3a), 1.98 (2H, m, H-17b/H-21b), 2.01
(1H, m, H-22), 2.03 (1H, m, H-18), 2.12 (1H, m, H-3b), 2.44 (3H, m,
H-8’), 3.55 (1H, dd, J=11.9, 4.2 Hz, H-7), 5.20 (1H, d, J=6.6 Hz, H-
4), 5.23 (1H, br s, H-20), 5.47 (1H, m, H-16), 7.26 (2H, d, J=7.8 Hz,
H-4’/H-6’), 7.98 ppm (2H, d, J=7.8 Hz, H-3’/H-7’); 13C NMR
(100 MHz, CDCl3): d=13.2 (CH3, C-24), 15.4 (CH3, C-29), 21.3 (CH3,
C-8’), 21.8 (CH3, C-26), 23.2 (CH2, C-3), 26.3 (CH2, C-12), 26.7 (CH2, C-
8), 26.9 (CH2, C-17), 29.1 (CH3, C-25), 29.2 (CH3, C-31), 30.2 (CH3, C-
28), 30.8 (CH3, C-27), 31.2 (CH3, C-30), 33.3 (CH2, C-21), 34.0 (CH2, C-
13), 35.1 (CH2, C-2), 35.2 (qC, C-23), 39.5 (CH2, C-9), 42.7 (qC, C-1),
45.2 (CH, C-18), 56.3 (CH, C-22), 56.5 (CH, C-11), 57.1 (CH, C-14),
72.4 (qC, C-10), 77.3 (CH, C-7), 77.8 (qC, C-5), 80.1 (CH, C-4), 121.8
(CH, C-16), 123.4 (CH, C-20), 127.8 (qC, C-5’), 129.6 (2C, CH, C-4’/C-
6’), 130.2 (2C, CH, C-3’/C-7’), 143.0 (qC, C-15), 143.8 (qC, C-2’), 146.5
(qC, C-19), 165.8 ppm (qC, C-1’); IR (CHCl3): nmax =2985, 2929, 2857,
1713, 1451, 1364, 1280, 1114, 909 cmꢀ1; HRMS-ESI: m/z [M+H]+
calcd for C38H57O4: 577.4251, found: 577.4272.
1082, 909 cmꢀ1
;
HRMS-ESI: m/z [M+H]+ calcd for C37H59O4:
567.4413, found: 567.4405.
4b-O-Benzyl-19,20-anhydrosipholenol A (19): Prepared according
to procedure C from 3 (46 mg, 0.1 mmol), dry DMF (1 mL), NaH
(10 equiv) and BnBr (12 mL, 1 equiv). Purification by flash chroma-
tography (n-hexane/EtOAc, 70:30) gave 19 as a colorless oil
(14 mg, 25.4%): ½aꢂ2D5 = +2.4 (c=0.38, CHCl3); 1H NMR (400 MHz,
CDCl3): d=0.78 (1H, m, H-11), 0.98 (3H, s, H-24), 1.02 (3H, s, H-31),
1.05 (3H, s, H-27), 1.09 (3H, s, H-30), 1.11 (3H, s, H-25), 1.21 (1H, m,
H-12a), 1.26 (3H, s, H-26), 1.36 (1H, m, H-8a), 1.46 (2H, m, H-2),
1.48 (1H, m, H-9a), 1.51 (1H, m, H-12b), 1.56 (1H, m, H-9b), 1.67
(3H, s, H-29), 1.70 (2H, m, H-14/H-21a), 1.72 (1H, m, H-8b), 1.75
(3H, s, H-28), 1.80 (1H, m, H-3a), 1.85 (1H, m, H-17a), 1.94 (1H, m,
H-3b), 2.02 (1H, m, H-21b), 2.05 (1H, m, H-17b), 2.06 (1H, m, H-22),
2.14 (1H, m, H-13a), 2.32 (1H, m, H-18), 2.42 (1H, m, H-13b), 3.40
(1H, d, J=6.6 Hz, H-4), 3.50 (1H, dd, J=11.9, 4.2 Hz, H-7), 4.31 (1H,
d, J=11.7 Hz, H-1’a), 4.56 (1H, d, J=11.7 Hz, H-1’b), 5.29 (1H, br s,
H-20), 5.48 (1H, m, H-16), 7.30 (1H, m, H-5’), 7.35 (2H, m, H-4’/H-
6’), 7.37 ppm (2H, m, H-3’/H-7’); 13C NMR (100 MHz, CDCl3): d=13.2
(CH3, C-24), 15.4 (CH3, C-29), 21.7 (CH2, C-3), 22.2 (CH3, C-26), 26.4
(CH2, C-12), 26.6 (CH2, C-8), 26.9 (CH2, C-17), 29.5 (CH3, C-31), 29.6
(CH3, C-25), 30.0 (CH3, C-27), 30.2 (CH3, C-28), 31.3 (CH3, C-30), 33.4
(CH2, C-21), 34.0 (CH2, C-13), 34.1 (CH2, C-2), 35.5 (qC, C-23), 39.2
(CH2, C-9), 42.4 (qC, C-1), 45.3 (CH, C-18), 56.0 (CH, C-11), 56.4 (CH,
4b-O-4’-Chlorobenzoyl-19,20-anhydrosipholenol A (17): Prepared
according to procedure B starting with 3 (25 mg, 0.052 mmol), an-
hydrous CH2Cl2 (10 mL), DMAP (6 mg, 0.05 mmol) and 4-chloroben-
zoic anhydride (23.8 mg, 0.16 mmol). Purification by flash chroma-
tography (n-hexane/EtOAc, 95:5) gave 11 as an amorphous solid
(30 mg, 98.5%): ½aꢂ2D5 =ꢀ26.8 (c=0.27, CHCl3); 1H NMR (400 MHz,
CDCl3): d=0.76 (1H, m, H-11), 0.89 (3H, s, H-31), 1.04 (6H, s, H-24/
H-30), 1.11 (3H, s, H-27), 1.18 (1H, m, H-12a), 1.24 (3H, s, H-26),
1.27 (3H, s, H-25), 1.40 (1H, m, H-2a), 1.45 (1H, m, H-8a), 1.50 (1H,
m, H-9a), 1.52 (1H, m, H-12b), 1.56 (1H, m, H-2b), 1.66 (3H, s, H-
29), 1.66 (2H, m, H-14/H-21a), 1.68 (1H, m, H-9b), 1.74 (3H, s, H-
28), 1.82 (1H, m, H-8b), 1.84 (1H, m, H-17a), 1.96 (1H, m, H-3a),
1.98 (1H, m, H-21b), 2.01 (1H, m, H-22), 2.02 (1H, m, H-13a), 2.03
(1H, m, H-17b), 2.12 (1H, m, H-3b), 2.26 (2H, m, H-13b/H-18), 3.55
(1H, dd, J=11.9, 4.2 Hz, H-7), 5.20 (1H, d, J=6.6 Hz, H-4), 5.23 (1H,
br s, H-20), 5.47 (1H, m, H-16), 7.44 (2H, d, J=8.6 Hz, H-4’/H-6’),
7.97 ppm (2H, d, J=8.6 Hz, H-3’/H-7’); 13C NMR (100 MHz, CDCl3):
d=13.2 (CH3, C-24), 15.4 (CH3, C-29), 21.8 (CH3, C-26), 23.2 (CH2, C-
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C-22), 57.1 (CH, C-14), 71.5 (CH2, C-1’), 72.5 (qC, C-10), 76.5 (CH, C-
7), 78.5 (qC, C-5), 84.6 (CH, C-4), 121.6 (CH, C-16), 123.5 (CH, C-20),
127.5 (CH, C-5’), 127.6 (2C, CH, C-3’/C-7’), 128.4 (2C, CH, C-4’/C-6’),
139.2 (qC, C-2’), 143.0 (qC, C-15), 146.5 ppm (qC, C-19); IR (CHCl3):
1757, 1355, 1152, 1088, 890 cmꢀ1; HRMS-ESI: m/z [M+H]+ calcd for
C38H60NO7S: 674.4090, found: 674.4079.
Sipholenone A-4Z-oxime (22): Prepared according to procedure E
nmax =2986, 2929, 2857, 1463, 1455, 1378, 1363, 1084, 908 cmꢀ1
;
from
2 (25 mg, 0.05 mmol), pyridine/EtOH (1:1, 2 mL) and
HRMS-ESI: m/z [M-H]+ calcd for C37H55O3: 547.4145, found:
NH2OH·HCl (3.5 mg, 0.10 mmol). Purification by flash chromatogra-
phy (CHCl3/MeOH, 95:5) gave 21 as a colorless oil (16 mg, 65%):
½aꢂ2D5 =ꢀ14.8 (c=0.33, CHCl3); 1H NMR (400 MHz, CDCl3): d=0.71
(1H, m, H-11), 0.85 (3H, s, H-31), 1.04 (3H, s, H-30), 1.06 (3H, s, H-
24), 1.13 (3H, s, H-27), 1.15 (1H, m, H-12a), 1.21 (1H, m, H-2a), 1.24
(6H, s, H-26/H-29), 1.25 (3H, s, H-25), 1.47 (1H, m, H-8a), 1.57 (1H,
m, H-20), 1.61 (1H, m, H-14), 1.62 (1H, m, H-12b), 1.66 (2H, m, H-9),
1.73 (1H, m, H-13a), 1.73 (3H, s, H-28), 1.77 (1H, m, H-18), 1.79 (2H,
m, H-17a/H-21a), 1.82 (1H, m, H-17b), 1.90 (1H, m, H-2b), 1.94 (2H,
m, H-3a/H-8b), 1.98 (1H, m, H-21b), 1.99 (1H, m, H-13b), 2.17 (1H,
m, H-3b), 2.45 (1H, m, H-22), 2.96 (1H, dd, J=12.3, 3.6 Hz, H-7),
5.44 ppm (1H, dd, J=9.2, 5.1 Hz, H-16); 13C NMR (100 MHz, CDCl3):
d=12.6 (CH3, C-24), 14.2 (CH3, C-25), 22.7 (CH3, C-26), 24.8 (CH2, C-
17), 24.9 (CH2, C-21), 25.6 (CH3, C-29), 26.7 (CH2, C-8), 26.8 (CH2, C-
12), 29.6 (CH3, C-31), 30.0 (CH3, C-27), 30.2 (CH3, C-28), 31.7 (CH3, C-
30), 33.5 (CH2, C-3), 33.8 (CH2, C-13), 35.5 (qC, C-23), 37.2 (CH2, C-
20), 39.1 (CH2, C-9), 39.3 (CH2, C-2), 41.8 (qC, C-1), 48.9 (CH, C-18),
52.8 (CH, C-22), 56.5 (CH, C-11), 57.5 (CH, C-14), 72.4 (qC, C-10),
77.8 (qC, C-5), 78.7 (CH, C-7), 82.1 (qC, C-19), 121.4 (CH, C-16),
143.1 (qC, C-15), 167.9 ppm (qC, C-4); IR (CHCl3): nmax =3456, 3328,
2986, 2935, 2861, 1665, 1363, 1082, 953 cmꢀ1; HRMS-ESI: m/z [M+
H]+ calcd for C30H52NO4: 490.3890, found: 490.3859.
547.4120.
4b-O-Benzylcarbamoylsipholenol A (20): Prepared according to
procedure D from 1 (30 mg, 0.052 mmol), toluene (2 mL), Et3N
(10 mL, 0.05 mmol) and benzyl isocyanate (25 mg, 0.16 mmol). Pu-
rification by flash chromatography (CHCl3/MeOH, 95:5) gave 20 as
a colorless oil (12 mg, 40%): ½aꢂ2D5 =ꢀ29.4 (c=0.48, CHCl3); H NMR
1
(400 MHz, CDCl3): d=0.76 (1H, m, H-11), 0.90 (3H, s, H-31), 1.04
(6H, s, H-24/H-30), 1.12 (3H, s, H-27), 1.16 (1H, m, H-12a), 1.24 (6H,
s, H-26/H-29), 1.26 (3H, s, H-25), 1.38 (1H, m, H-2a), 1.45 (1H, m, H-
8a), 1.52 (1H, m, H-12b), 1.56 (1H, m, H-2b), 1.57 (1H, m, H-14),
1.60 (2H, m, H-20), 1.64 (2H, m, H-9), 1.66 (1H, m, H-13a), 1.68 (1H,
m, H-21a), 1.73 (3H, s, H-28), 1.77 (1H, m, H-18), 1.78 (1H, m, H-
17a), 1.82 (1H, m, H-8b), 1.87 (1H, m, H-21b), 1.94 (1H, m, H-3a),
1.95 (1H, m, H-13b), 1.98 (1H, m, H-17b), 2.12 (1H, m, H-3b), 2.41
(1H, m, H-22), 3.55 (1H, dd, J=11.3, 3.6 Hz, H-7), 4.46 (2H, d, J=
6.2 Hz, H-2’), 5.19 (1H, d, J=6.6 Hz, H-4), 5.44 (1H, dd, J=9.2,
5.1 Hz, H-16), 7.30 (2H, d, J=6.9 Hz, H-4’/H-8’), 7.32 (1H, dd, J=5.8,
5.8 Hz, H-6’), 7.34 ppm (2H, dd, J=5.8, 5.8 Hz, H-5’/H-7’); 13C NMR
(100 MHz, CDCl3): d=13.1 (CH3, C-24), 21.8 (CH3, C-26), 23.2 (CH2,
C-3), 24.8 (CH2, C-17), 24.9 (CH2, C-21), 25.7 (CH3, C-29), 26.7 (CH2,
C-8), 26.9 (CH2, C-12), 29.1 (CH3, C-25), 29.5 (CH3, C-31), 29.9 (CH3,
C-27), 30.2 (CH3, C-28), 31.7 (CH3, C-30), 33.8 (CH2, C-13), 35.1 (CH2,
C-2), 35.5 (qC, C-23), 37.4 (CH2, C-20), 39.5 (CH2, C-9), 42.7 (qC, C-1),
45.4 (CH2, C-2’), 48.9 (CH, C-18), 52.8 (CH, C-22), 56.5 (CH, C-11),
57.4 (CH, C-14), 72.4 (qC, C-10), 77.3 (CH, C-7), 77.8 (qC, C-5), 80.2
(CH, C-4), 82.2 (qC, C-19), 121.4 (CH, C-16), 127.6 (2C, CH, C-4’/C-8’),
129.4 (2C, CH, C-5’/C-7’), 136.7 (CH, C-6’), 139.1 (qC, C-3’), 143.2
(qC, C-15), 155.6 ppm (qC, C-1’); IR (CHCl3): nmax =3454, 2987, 2950,
2927, 1745, 1603, 1443, 1097 cmꢀ1; HRMS-ESI: m/z [M+Na]+ calcd
for C38H59NO5Na: 632.4285, found: 632.4283.
Sipholenone A-4Z-N-benzoyloxime (23): Prepared according to
procedure B from 22 (25 mg, 0.052 mmol), anhydrous CH2Cl2
(10 mL), DMAP (6 mg, <0.05 mmol) and benzoic anhydride
(23.8 mg, <0.11 mmol). Purification by flash chromatography
(n-hexane/EtOAc, 70:30) afforded 23 as a colorless oil (58.5%):
½aꢂ2D5 =ꢀ19.6 (c=0.26, CHCl3); 1H NMR (400 MHz, CDCl3): d=0.71
(1H, m, H-11), 0.85 (3H, s, H-31), 1.04 (3H, s, H-30), 1.06 (3H, s, H-
24), 1.13 (3H, s, H-27), 1.15 (1H, m, H-12a), 1.21 (1H, m, H-2a), 1.24
(6H, s, H-26/H-29), 1.25 (3H, s, H-25), 1.47 (1H, m, H-8a), 1.57 (1H,
m, H-20), 1.61 (1H, m, H-14), 1.62 (1H, m, H-12b), 1.66 (2H, m, H-9),
1.73 (1H, m, H-13a), 1.73 (3H, s, H-28), 1.77 (1H, m, H-18), 1.79 (1H,
m, H-17a), 1.79 (1H, m, H-21a), 1.82 (1H, m, H-17b), 1.90 (1H, m, H-
2b), 1.94 (2H, m, H-3a/H-8b), 1.98 (1H, m, H-21b), 1.99 (1H, m, H-
13b), 2.17 (1H, m, H-3b), 2.45 (1H, m, H-22), 2.96 (1H, dd, J=12.3,
3.6 Hz, H-7), 5.44 (1H, dd, J=9.2, 5.1 Hz, H-16), 7.42 (2H, dd, J=
7.7, 7.7 Hz, H-4’/H-6’), 7.56 (1H, m, H-5’), 8.01 ppm (2H, dd, J=7.7,
1.4 Hz, H-3’/H-7’); 13C NMR (100 MHz, CDCl3): d=12.6 (CH3, C-24),
14.2 (CH3, C-25), 22.7 (CH3, C-26), 24.8 (CH2, C-17), 24.9 (CH2, C-21),
25.6 (CH3, C-29), 26.7 (CH2, C-8), 26.8 (CH2, C-12), 29.6 (CH3, C-31),
30.0 (CH3, C-27), 30.2 (CH3, C-28), 31.7 (CH3, C-30), 33.5 (CH2, C-3),
33.8 (CH2, C-13), 35.5 (qC, C-23), 37.2 (CH2, C-20), 39.1 (CH2, C-9),
39.3 (CH2, C-2), 41.8 (qC, C-1), 48.9 (CH, C-18), 52.8 (CH, C-22), 55.6
(CH, C-11), 57.5 (CH, C-14), 72.4 (qC, C-10), 77.8 (qC, C-5), 78.7 (CH,
C-7), 82.1 (qC, C-19), 121.4 (CH, C-16), 128.6 (2C, CH, C-4’/C-6’),
129.3 (qC, C-2’), 129.4 (2C, CH, C-3’/C-7’), 133.3 (CH, C-5’), 142.9
4b-O-Tosylcarbamoylsipholenol A (21): Prepared according to
procedure D from 1 (30 mg, 0.052 mmol), toluene (2 mL), Et3N
(10 mL, 0.05 mmol) and p-toluenesulfonyl isocyanate (34 mg,
0.16 mmol). Purification by flash chromatography (CHCl3/MeOH,
95:5) gave 21 as a colorless oil (18 mg, 60%): ½aꢂ2D5 =ꢀ27.7 (c=
1
0.41, CHCl3); H NMR (400 MHz, CDCl3): d=0.72 (1H, m, H-11), 0.92
(3H, s, H-31), 1.04 (6H, s, H-24/H-30), 1.13 (3H, s, H-27), 1.16 (1H,
m, H-12a), 1.23 (6H, s, H-26/H-29), 1.26 (3H, s, H-25), 1.31 (1H, m,
H-2a), 1.45 (1H, m, H-8a), 1.54 (2H, m, H-2b/H-12b), 1.57 (1H, m, H-
14), 1.60 (2H, m, H-20), 1.62 (2H, m, H-9), 1.68 (2H, m, H-13a/H-
21a), 1.73 (3H, s, H-28), 1.77 (1H, m, H-18), 1.78 (1H, m, H-17a),
1.82 (1H, m, H-8b), 1.94 (1H, m, H-21b), 1.97 (2H, m, H-3a/H-17b),
1.98 (1H, m, H-13b), 2.13 (1H, m, H-3b), 2.43 (1H, m, H-22), 2.44
(3H, s, H-8’), 3.47 (1H, dd, J=11.7, 4.0 Hz, H-7), 5.21 (1H, d, J=
6.6 Hz, H-4), 5.43 (1H, m, H-16), 7.33 (2H, d, J=8.4 Hz, H-4’/H-6’),
7.90 ppm (2H, d, J=8.4 Hz, H-3’/H-7’); 13C NMR (100 MHz, CDCl3):
d=13.1 (CH3, C-24), 21.7 (CH3, C-8’), 21.8 (CH3, C-26), 23.2 (CH2, C-
3), 24.8 (CH2, C-17), 24.9 (CH2, C-21), 25.7 (CH3, C-29), 26.7 (CH2, C-
8), 26.9 (CH2, C-12), 29.1 (CH3, C-25), 29.5 (CH3, C-31), 29.9 (CH3, C-
27), 30.2 (CH3, C-28), 31.7 (CH3, C-30), 33.8 (CH2, C-13), 35.2 (CH2, C-
2), 35.5 (qC, C-23), 37.3 (CH2, C-20), 39.5 (CH2, C-9), 42.7 (qC, C-1),
48.8 (CH, C-18), 52.7 (CH, C-22), 56.5 (CH, C-11), 57.4 (CH, C-14),
72.4 (qC, C-10), 77.4 (qC, C-5), 77.6 (CH, C-7), 81.4 (CH, C-4), 82.1
(qC, C-19), 121.5 (CH, C-16), 128.5 (CH, C-3’), 128.6 (CH, C-6’), 129.7
(2C, CH, C-4’/C-7’), 135.0 (qC, C-5’), 143.0 (qC, C-15), 145.1 (qC, C-
2’), 150.8 ppm (qC, C-1’); IR (CHCl3): nmax =3454, 2987, 2950, 2858,
(qC, C-15), 163.9 (qC, C-1’), 176.0 ppm (qC, C-4); IR (CHCl3): nmax
=
;
3454, 2987, 2950, 2930, 2858, 1713, 1659, 1282, 1114, 945 cmꢀ1
HRMS-ESI: m/z [M+Na]+ calcd for C37H55NO5Na: 616.3972, found:
616.3967.
Sipholenone A-4Z-N-benzyloxime (24): Prepared according to
procedure C from 22 (25 mg, 0.052 mmol), dry DMF (1 mL), NaH
(10 equiv) and BnBr (12 mL, 0.006 mmol). Purification by flash chro-
matography (n-hexane/EtOAc, 90:10) gave 24 as a colorless oil
(43.1%): ½aꢂ2D5 =ꢀ17.9 (c=0.21, CHCl3); 1H NMR (400 MHz, CDCl3):
d=0.71 (1H, m, H-11), 0.85 (3H, s, H-31), 1.04 (3H, s, H-30), 1.06
(3H, s, H-24), 1.13 (3H, s, H-27), 1.15 (1H, m, H-12a), 1.21 (1H, m,
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H-2a), 1.24 (6H, s, H-26/H-29), 1.25 (3H, s, H-25), 1.47 (1H, m, H-8a),
1.57 (1H, m, H-20), 1.61 (1H, m, H-14), 1.62 (1H, m, H-12b), 1.66
(2H, m, H-9), 1.73 (1H, m, H-13a), 1.73 (3H, s, H-28), 1.77 (1H, m,
H-18), 1.79 (2H, m, H-17a/H-21a), 1.82 (1H, m, H-17b), 1.90 (1H, m,
H-2b), 1.94 (2H, m, H-3a/H-8b), 1.98 (1H, m, H-21b), 1.99 (1H, m,
H-13b), 2.17 (1H, m, H-3b), 2.45 (1H, m, H-22), 2.96 (1H, dd, J=
12.3, 3.6 Hz, H-7), 5.02 (1H, d, J=11.9 Hz, H-1’b), 5.11 (1H, d, J=
11.9 Hz, H-1’a), 5.44 (1H, dd, J=9.2, 5.1 Hz, H-16), 7.29 (1H, m, H-
5’), 7.35 (2H, m, H-4’/H-6’), 7.36 ppm (2H, m, H-3’/H-7’); 13C NMR
(100 MHz, CDCl3): d=12.6 (CH3, C-24), 14.2 (CH3, C-25), 22.7 (CH3,
C-26), 24.8 (CH2, C-17), 24.9 (CH2, C-21), 25.6 (CH3, C-29), 26.7 (CH2,
C-8), 26.8 (CH2, C-12), 29.6 (CH3, C-31), 30.0 (CH3, C-27), 30.2 (CH3,
C-28), 31.7 (CH3, C-30), 33.5 (CH2, C-3), 33.8 (CH2, C-13), 35.5 (qC, C-
23), 37.2 (CH2, C-20), 39.1 (CH2, C-9), 39.3 (CH2, C-2), 41.8 (qC, C-1),
48.9 (CH, C-18), 52.8 (CH, C-22), 55.6 (CH, C-11), 57.5 (CH, C-14),
72.4 (qC, C-10), 75.6 (CH2, C-1’), 77.8 (qC, C-5), 78.7 (CH, C-7), 82.1
(qC, C-19), 121.3 (CH, C-16), 127.6 (2C, CH, C-3’/C-7’), 128.2 (CH, C-
5’), 128.5 (2C, CH, C-4’/C-6’), 138.3 (qC, C-2’), 143.1 (qC, C-15),
167.0 ppm (qC, C-4); IR (CHCl3): nmax =3454, 2987, 2949, 2863, 1665,
1463, 1455, 1376, 1363, 1082, 947 cmꢀ1; HRMS-ESI: m/z [M+H]+
calcd for C37H58NO4: 580.4366, found: 580.4370.
cells in the wound of the treatment group/number of cells in the
wound for vehicle control]ꢂ100) per 40ꢂ field. IC50 values (mm) are
the doses that produced 50% migration and were calculated from
nonlinear fitting of the dose–response curve using GraphPad Prism
5.04 (GraphPad Software, La Jolla, CA, USA).
Cultrex basement membrane extract (BME) cell invasion assay:[28]
BME (~50 mL) was added to each well of the top chamber. After
overnight incubation at 378C in 5% CO2, MDA-MB-231 cells
(50000/50 mL) in 0.5% FBS RPMI medium were added to each well
of the top chamber. RPMI 1640 medium (150 mL) was then added
to the lower chamber; this media contained 10% FBS and penicil-
lin/streptomycin, as well as fibronectin (1 mLmLꢀ1) and N-formyl-
Met-Leu-Phe (10 nm) as chemoattractants. The test compound sol-
utions were prepared at six-times the desired concentration, and
an aliquot (10 mL) was added to each well of the top chamber.
Cells were incubated at 378C in 5% CO2, which allowed for cell in-
vasion from the top to the lower chamber. After 24 h, the top and
bottom chambers were aspirated and washed with washing buffer
supplied with the assay kit. Cell dissociation solution/calcein-AM
solution (~100 mL) was added to the bottom chamber, and cells
were incubated at 378C under 5% CO2 for 1 h. The cells internalize
calcein-AM, and the intracellular esterases cleave the acetomethy-
lester (AM) moiety to generate free calcein. Fluorescence of the
samples was determined at lexcitation 485 nm and lemission 528 nm
using an ELISA plate reader (BioTek, Winooski, VT, USA). The
number of cells that invaded through the BME coat was calculated
using a standard curve.
Biology
Cell culture: Metastatic human mammary epithelial breast cancer
(MDA-MB-231) and normal (MCF10A) cell lines were purchased
from ATCC (Manassas, VA, USA). Cells were grown in RPMI 1640
medium (GIBCO-Invitrogen, Grand Island, NY, USA) with 10% fetal
bovine serum (FBS) and supplemented with l-glutamine
(2 mmolLꢀ1), penicillin G (100 mgmLꢀ1), and streptomycin
(100 mgmLꢀ1) at 378C under 5% CO2.
MTT cytotoxicity assay: The cytotoxic effects of test compounds
were evaluated in MDA-MB-231 and MCF10A cells using an MTT
assay kit (TACS, Trevigen Inc., Gaithersburg, MD, USA). Cells in ex-
ponential growth were plated in a 96-well plate at a density of
20ꢂ103 cells per well and allowed to attach for 24 h at 378C in 5%
CO2. Complete growth medium was then replaced with RPMI 1640
or DMEM/F-12 serum-free medium (100 mL) containing various
doses (5, 10, 20, and 40 mm) of test compound and incubation re-
sumed at 378C in 5% CO2 for 24 h. Cells were then treated with
MTT solution (20 mL/well) and re-incubated for 4 h. The color reac-
tion was stopped by addition of solubilization/stop solution
(DMSO; 100 mL/well), and incubation at 378C was continued to
ensure complete dissolution of the formazan product. Absorbance
of the samples was determined at l=570 nm using an ELISA plate
reader (BioTek).
Preparation of solutions for cell-based assays: A stock solution of
each analogue was prepared by dissolving the compound in
DMSO at a concentration of 50 mm for all assays. An aliquot
(~2 mL) of each stock solution was transferred to serum-free
medium (998 mL) to obtain a solution with a concentration of
100 mm and 0.2% DMSO. Serial dilutions were then conducted to
obtain the desired concentrations for each assay. The vehicle con-
trol was prepared as follows: for the MTT assay: DMSO (2 mL) was
added to serum-free media (998 mL); for the wound-healing assay:
DMSO (3 mL) was added to serum-free media (1497 mL) containing
0.5% FBS.
Phosphorylation inhibition assay: A Z’-LYTE kinase assay-Tyr 1 pep-
tide kit (Invitrogen) was used to assess the ability of sipholenol A
and its analogues to inhibit protein tyrosine kinase 6 (PTK6) phos-
phorylation. Briefly, 10 mL/well reactions were set up in 384-well
plates containing kinase buffer, 150 mm ATP, 2 mm Z’-LYTE-Tyr 1 pep-
tide substrate, 5000 ngmLꢀ1 PTK6, and test compound (inhibitor).
After 1 h of incubation at RT, 5 mL development solution containing
site-specific protease was added to each well. Incubation was con-
tinued for 1 h. The reaction was then stopped, and the fluorescent
signal ratio of 445 nm (coumarin)/520 nm (fluorescein) was deter-
mined on an FLx800 plate reader (BioTek), which reflects the pep-
tide substrate cleavage status and/or the kinase inhibitory activity
in the reaction.
Antimigratory (wound-healing) assay:[26] MDA-MB-231 cells were cul-
tured in the RPMI 1640 medium containing 10 mm 4-(2-hydrox-
yethyl)-1-piperazineethanesulfonic acid (HEPES), l-glutamine
(4 mm), 10% FBS, penicillin (100 IUmLꢀ1), and streptomycin
(50 mgmLꢀ1), and incubated in 5% CO2 at 378C. Cells were plated
onto sterile 24-well plates and allowed to form a confluent mono-
layer in each well (>95% confluence). Wounds were then inflicted
to each cell monolayer using a sterile 200 mL pipette tip. Media
were removed, cells were washed twice with phosphate-buffered
saline (PBS), and then test compound was added in fresh
RPMI 1640 media (same as culture media but with only 0.5% FBS).
Four non-cytotoxic concentrations of test compound were pre-
pared in serum-free media containing 0.5% FBS. Each concentra-
tion was tested in triplicate. Incubation was carried out for 24 h in
5% CO2 at 378C, after which time, the cell medium was removed,
and cells were fixed and stained using a DiffQuick staining kit
(Dade Behring Diagnostics, Aguada, Puerto Rico). The number of
cells migrated across the wound was counted under the micro-
scope in three or more randomly selected fields (magnification:
40ꢂ). Final results are expressed as the % migration ([number of
Acknowledgements
The Saudi Arabian Government is greatly acknowledged for pro-
viding the fellowship support to A.F.
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 497 – 510 509

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Published online on February 12, 2013
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ChemMedChem 2013, 8, 497 – 510 510