Mirabolides A and B; new cytotoxic glycerides from the Red Sea sponge Theonella mirabilis

Article abstract of DOI:10.3390/md14080155

As a part of our continuing work to find out bioactive lead molecules from marine invertebrates, the CHCl₃ fraction of the organic extract of the Red Sea sponge Theonella mirabilis showed cytotoxic activity in our primary screen. Bioassay-guided purification of the active fractions of the sponge's extract resulted in the isolation of two new glycerides, mirabolides A and B (1 and 2), together with the reported 4-methylene sterols, conicasterol (3) and swinhosterol B (4). The structures of the compounds were assigned by interpretation of their 1D (¹H, ¹³C), 2D (COSY, HSQC, HMBC, ROESY) NMR spectral data and high-resolution mass determinations. Compounds 1-4 displayed marked cytotoxic activity against human breast adenocarcinoma cell line (MCF-7) with IC₅₀ values of 16.4, 5.18, 6.23 and 3.0 μg/mL, respectively, compared to 5.4 μg/mL observed by doxorubicin as reference drug.

Full text of DOI:10.3390/md14080155

marine drugs
Article
Mirabolides A and B; New Cytotoxic Glycerides from
the Red Sea Sponge Theonella mirabilis
Dina R. Abou-Hussein 1,2 and Diaa T. A. Youssef 1,
*
1
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University,
Jeddah 21589, Saudi Arabia; dabouhussein@kau.edu.sa or dina.abouhussein@pharma.cu.edu.eg
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo 11562, Egypt
Correspondence: dyoussef@kau.edu.sa; Tel.: +966-548-535-344
2
*
Academic Editor: Kirsten Benkendorff
Received: 15 May 2016; Accepted: 8 August 2016; Published: 18 August 2016
Abstract: As a part of our continuing work to find out bioactive lead molecules from marine
invertebrates, the CHCl3 fraction of the organic extract of the Red Sea sponge Theonella mirabilis
showed cytotoxic activity in our primary screen. Bioassay-guided purification of the active fractions
of the sponge’s extract resulted in the isolation of two new glycerides, mirabolides A and B (
together with the reported 4-methylene sterols, conicasterol ( ) and swinhosterol B ( ). The structures
of the compounds were assigned by interpretation of their 1D (1H, 13C), 2D (COSY, HSQC, HMBC,
ROESY) NMR spectral data and high-resolution mass determinations. Compounds displayed
marked cytotoxic activity against human breast adenocarcinoma cell line (MCF-7) with IC50 values
1 and 2),
3
4
1–4
of 16.4, 5.18, 6.23 and 3.0
reference drug.
µg/mL, respectively, compared to 5.4 µg/mL observed by doxorubicin as
Keywords: Red Sea sponge Theonella mirabilis; glycerides; Mirabolides A and B; 4-methylene sterols;
breast cancer cell line; cytotoxic activity
1. Introduction
Marine sponges are multicellular animals evolved from about 600 million years ago; they belong
to phylum Porifera and are considered the most primitive animals [
1]. More than 8000 species of
sponges have been described and are classified into three classes [ ]. Diverse classes of bioactive
2
metabolites were isolated from sponges and reported in the literature in the thousands; they proved
that sponges have been essential and important source of drug discovery [3].
Sponges of order Lithistida are characterized by a hard rocky skeleton formed by the interlocking
spicules of silica (desmas) [
metabolites such as polyketides, peptides (linear or cyclic), macrolides, alkaloids, lipids, steroids and
pigments [ ]. Genus Theonella is well-known producer of diverse bioactive constituents including
4,5]. They occur worldwide and are recognized for their different bioactive
4,6–8
4-methylene sterols [9], cyclic or linear peptides [10], and fatty acids [11,12].
Several metabolites isolated from the genus Theonella demonstrated several cytotoxic activities
viz. macrolides [13], cyclopeptides [14–16], cyclodepsipeptides [17,18], linear peptides [19], dimeric
macrolides [20], and alkaloids [21]. Certain depsipetides were proven to protect cells from
HIV infection [18]; others exhibited antifungal and antibacterial activities [22]. Glycolipids with
five-membered cyclitol were previously isolated from the Carribean sponge Pseudocertina crassa and
were known as crasserides [23]. Crasserides and isocrasserides were verified to be present in different
investigated species of marine sponges and then were distinctive to the phylum Porifera [24]. They
can be considered as natural feeding deterrents due of their exerted antifeedant activity on the fish
Carassius auratus [23].
Mar. Drugs 2016, 14, 155; doi:10.3390/md14080155
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2016, 14, 155
2 of 11
In the course of our ongoing quest to discover cytotoxic compounds from Red Sea marine lithistid
sponges [25 26], the crude extract of the sponge Theonella mirabilis showed cytotoxic activity with IC50
of 17 g/mL against human breast cancer cell line (MCF-7). Bioassay-directed fractionation of the
active fractions afforded four compounds including two new glycerides, mirabolides A and B ( and
and the known conicasterol ( ) [27] and swinhosterol B ( ) [28]. Herein, we report on the purification,
,
µ
1
2)
3
4
structure determination as well as the cytotoxic activities of these compounds.
2. Results and Discussion
Compound 1 (Figure 1) was obtained as a white precipitate. Its molecular formula was assigned as
C40H78O8 as deduced by HRESIMS pseudomolecular ion peak at m/z 685.5625 [M − H]−, suggesting
two degrees of unsaturation. Combined 1D (1H and 13C) and 2D (COSY, HSQC, HMBC, ROESY)
NMR data allowed the assignment of a trisubstituted glycerol moiety attached to three subunits
including, a five-membered cyclitol moiety, an O-acyl chain (13-methylpentadecanoyl) and an O-alkyl
chain (14-methylpentadecanyl) (Figure 1 and Table 1). The signals at δH
5.17/69.3 (H-2/C-2) and 3.57,3.54/67.1 (H2-3/C-3) were assigned as a trisubstituted glycerol moiety
(Supplementary Materials, Figures S1 and S2) [23 24]. The signals at δH
δC 3.66/80.3 (H-10/C-10),
3.93/71.4 (H-20/C-20), 3.84/78.5 (H-30/C-30), 3.69/77.0 (H-40/C-40) and 3.90/77.2 (H-50/C-50) were
assigned as cyclopentane-1,2,3,4,5-pentaol [23 24]. The assignment of these signals was secured
/δC 3.78,3.73/67.7 (H2-1/C-1),
,
/
,
from COSY, and HSQC experiment (Supplementary Materials, Figures S3 and S4). The COSY
1
spectrum showed non-interrupted H–1H correlations from H-10 to H-50 through H-20, H-30 and
H-40 suggesting a five-membered cyclitol moiety [23
,
24]. The placement of this moiety at C-1 was
unambiguously secured from HMBC correlation of H2-1/C-10 (Figure 2). The configuration of
the cyclitol in moiety was assigned from ROESY experiment as well as by comparison of the
1H/13C NMR data as well as the coupling constants of H-10 H-50 with those in the literature [23
24].
1
→
,
The ROESY correlations between H-10 and H-20, H-20 and H-40 as well as between H-30 and
H-50 (Figure 2, Supplementary Materials, Figure S9) supported the configuration at the cyclitol
moiety which was in agreement with the reported one in crasserides [23]. The fatty acyl moiety
at C-2 was assigned as 13-methylpentadecanoyl based upon the signals at δH/δC 172.1 (C-1”),
2.33/32.5 (H2-2”/C-2”), 1.60/23.0 (H2-3”/C-3”), 1.25/20.8–35.1 (H2-4”-H2-11”/C-4”-C-11”), 1.06/35.2
(H2-12”/C-12”), 1.35/30.8 (H-13”/C-13”), 1.25/30.0 (H2-14”/C-14”), 0.88/12.2 (H3-15”/C-15”) and
0.83/17.7 (H3-16”/C-16”). Further COSY correlations and HMBC cross peaks (Figure 2) of H2-2”/C-1”
and H2-3”/C-1” supported this assignment. In addition, HMBC cross peaks of H2-12”/C-13”,
H3-15”/C-13”, H3-16”/C-13” and H2-12”/C-16” together with COSY correlations indicated the
presence of an isobutyl moiety at the end of the acyl chain. The linkage of the ester moiety at C-2 was
secured from HMBC cross peak of H-2/C-1” (Figure 2, Supplementary Materials, Figures S5–S8).

Mar. Drugs 2016, 14, 155
3 of 11
Figure 1. Structures of compounds 1–4.
Figure 2. Key COSY, HMBC and ROESY correlations of 1 and 2.

Mar. Drugs 2016, 14, 155
4 of 11
Table 1. NMR data of compound 1 (CDCl3, 600 & 150 MHz).
a
HMBC (H→C) b
Position
δC (mult.)
67.7 CH2
69.3 CH
67.1 CH2
80.3 CH
71.4 CH
78.5 CH
77.0 CH
77.2 CH
172.1 qC
32.5 CH2
23.0 CH2
20.8–35.1 CH2
35.2 CH2
30.8 CH
30.0 CH2
12.2 CH3
17.7 CH3
70.0 CH2
27.8 CH2
20.8–35.1 CH2
37.1 CH2
26.0 CH
δ (mult., J in Hz)
H
0
1
2
3
1
2
3
4
5
3.78, dd (10.8, 4.2) 3.73, dd (10.8, 6.6)
5.17, quin (5.4)
3.57, dd (10.8, 5.4) 3.54, dd (10.8, 4.8)
H-2, H2-3, H-1
H2-1, H2-3
H2-1, H-2, H2-1 , H2-2
000
000
0
0
0
0
0
0
0
0
0
3.66, t (6.0)
3.93, t (6.0)
3.84, t (6.0)
3.69, t (7.2)
3.90, t (6.6)
-
H2-1, H-2 , H-3 , H-4 , H-5
0 0 0
H-1 , H-3 , H-4 , H-5
0
0
0
0
0 0 0
H-1 , H-2 , H-4 , H-5
0 0 0
H-1 , H-2 , H-3 , H-5
H-1 , H-2 , H-3 , H-4
H-2, H-1 , H2-2”, H2-3”
0
0
0
0
1”
2”
3”
2.33, t (7.8)
1.60, quin (7.2)
1.25, m
H2-3”
H2-2”
4”–11”
12”
13”
14”
15”
16”
1.06, m
1.35, m
1.25, m
H2-11”, H-13”, H3-16”
H2-12”, H3-15”, H3-16”
H-13”, H3-15”, H3-16”
H-13”, H2-14”
0.88, t (6.6)
0.83, d (6.6)
3.45, dd (6.6, 2.4) 3.41, dd (6.6, 2.4)
1.53, quin (7.2)
1.25, m
H2-12”, H-13”, H2-14”
000
000
000
000
1
2
3
H2-3 , H2-2
000
000
H2-1 , H2-3
000
000
–12
000
000
000
000
000
13
14
1.15, m
1.50, m
H2-12 , H3-15 , H3-16
000
000
000
H2-13 , H3-15 , H3-16
000
000
000
H2-12 , H2-13 , H-14
,
000
15
20.7 CH3
20.7 CH3
0.85, d (6.6)
0.85, d (6.6)
000
H3-16
000
000
000
H2-12 , H2-13 , H-14
,
000
16
000
H3-15
a multiplicities were deduced from DEPT and multiplicity-edited HSQC; b HMBC correlations are from proton(s)
stated to the indicated carbons.
Similarly, the assignment of the alkyl moiety at C-3 was established by 1D and 2D NMR and was
identified as 14-methylpentadecanyl (Table 1 and Figure 2). The 1H and 13C NMR signals resonating at
000 000 000 000 000 000
δH/δC 3.45, 3.41/70.0 (H2-1 /C-1 ), 1.53/27.8 (H2-2 /C-2 ), 1.15/37.1 (H2-13 /C-13 ), 1.50/26.0
000
000
000
000
000
000
(H-14 /C-14 ), 0.85/20.7 (H3-15 /C-15 ), 0.85/20.7 (H3-16 /C-16 ) together with the methylene
cluster at 1.25/20.8–35.1 (H2-3000-H2-12000/C-3000-C-12000) supported this assignment. The two methyl
doublet signals at
δ
0.85 (H3-15000 and H3-16000) indicated the iso-branching of the fatty chain. This
was verified by COSY correlations and HMBC cross peaks (Table 1 and Figure 2) of H2-2000/C-1000,
H2-13000/C-12000, H3-15000/C-12000, H3-16000/C-12000, H3-15000/C-13000, H3-16000/C-13000, H3-15000/C-14000
and H3-16000/C-14000. Again, the placement of the alkyl moiety at C-3 was supported by HMBC cross
peak of H2-3/C-1000 (Figure 2, Supplementary Materials, Figures S5–S8), completing the structure of
1.
In the negative ESIMS of
presence of alkyl side chains at C-2 and C-3, respectively (Figure 3). The overlapping of the signals
of methylene cluster (4”–11” and 3000–12000) of both alkyl chains at δH
δC 1.25/20.8–35.1 and the lack
of clear COSY correlations on both side chains in this region, necessitated carrying out methanolysis
of and methylation of the liberated fatty acids followed by GLC-MS analysis of the hydrolysate
1, the presence of fragment ion peaks at m/z 254 and 240 supported the
/
1
for an accurate determination of the length and the position of branching of the acyl chain. The
obtained fatty methyl ester (methyl 13-methylpentadecanoate, m/z 270) was identified based on its
GLC retention time and mass fragmentation spectrum. Two relatively intense fragment peaks at m/z
213 and 241 indicated the favored α-cleavage with the respect of the tertiary carbon atom (C-13”)
carrying the methyl branch. Characteristic peaks at m/z 219 and 201 resulted from the loss of methanol
and water from the fragment at m/z 241.
To the best of our knowledge,
mirabolide A.
1 is reported here as a new natural product and was named

Mar. Drugs 2016, 14, 155
5 of 11
Figure 3. Key MS fragment ion peaks of 1 and 2.
Compound
2 (Figure 1) was obtained as a white precipitate. It showed a molecular formula
C42H80O8 as deduced from HRESIMS pseudomolecular ion peak at m/z 713.5933 [M + H]+,
suggesting three degrees of unsaturation. Investigation of the 1D (1H and 13C) and 2D (COSY,
HSQC and HMBC) NMR experiments allowed the assignment of a triglyceride skeleton (Table 2).
The signals at δH
/
δC 3.60/70.5 (H2-1/C-1), 5.22/71.5 (H-2/C-2) and 4.40, 4.16/63.9 (H2-3/C-3) were
24].
indicative for a trisubstituted glycerol moiety (Supplementary Materials, Figures S10 and S11) [23
,
Extensive study of the 1D and 2D NMR allowed the assignment of three fatty acyl chains as
4-hydroxy-2-methoxybutanoyl, 13-methylpentadecanoyl and 16-methylheptadecanoyl. The acyl
moiety at C-1 was assigned as 13-methylpentadecanoyl, based upon the signals δH/
δC at 175.1 (C-10),
2.30/35.0 (H2-20/C-20), 1.59/26.1 (H2-30/C-30), 1.28/28.2–31.1 (H2-40-H2-110/C-40-C-110), 1.10/38.2
(H2-120/C-120), 1.31/35.7 (H-130/C-130), 1.28/30.8 (H2-140/C-140), 0.88/11.8 (H3-150/C-150) and
0.85/19.6 (H3-160/C-160). The COSY correlations and HMBC cross peaks of H-20/C-10, H2-30/C-10
supported this assignment (Figure 2). Moreover, the correlations in COSY, HSQC and HMBC
experiments (Supplementary Materials, Figures S12–S17) of the two methyl groups at positions
130 and 140 indicated the presence of sec-butyl fragment at the end of the acyl chain. This was
secured from HMBC cross peaks of H2-120/C-130, H3-150/C-130/, H3-160/C-130 and H2-120/C-160
(Table 2 and Figure 2). Finally, the placement of the fatty acid at C-1 was secured from
HMBC correlation of H2-1/C-10 (Table 2 and Figure 2). Similarly, the acyl moiety at C-3 was
assigned as 16-methylheptadecanoyl. 1H and 13C NMR signals of this moiety showed resonating
signals at δH
(H2-4000-H2-14000/C-4000-C-14000), 1.19/40.2 (H2-15000/C-15000), 1.50/29.1 (H-16000/C-16000), 0.87/23.1
(H3-17000/C-17000) and 0.87/23.1 (H3-18000/C-18000). Two methyl doublets at 0.87 (H3-17000 and
/
δC 174.7 (C-1000), 2.30/35.1 (H2-2000/C-2000), 1.59/26.1 (H2-3000/C-3000), 1.28/28.2–31.1
δ
H3-18000) indicated the iso-branching of the fatty chain. The assignment was supported by COSY
and HMBC cross peaks (Table 2 and Figure 2) of H-2000/C-1000and H2-3000/C-1000, in addition to the
cross peaks of H2-15000/C-14000, H3-17000/C-14000, H3-17000/C-15000, H3-18000/C-15000, H3-17000/C-16000
and H3-18000/C-16000 (Table 2 and Figure 2). The placement of this moiety at C-3 was supported by
HMBC of H2-3/C-1000 (Table 2 and Figure 2). Finally, the signals at δH
/δC 171.6 (C-1”), 3.70/77.5
(H-2”/C-2”), 2.08, 2.22/29.1 (H2-3”/C-3”), 3.53, 3.67/68.6 (H2-4”/C-4”) and 3.20/52.4 (H3CO-2”) were
assigned as 4-hydroxy-2-methoxybutanoyl moiety attached to position 2 of the substituted glycerol.
The assignment of this moiety was deduced from HMBC cross peaks of H-2”/C-1”, H2-3”/C-1” and

Mar. Drugs 2016, 14, 155
6 of 11
H3CO-2”/C-2” (Figure 2, Supplementary Materials, Figures S14–S17). Evidence for the length of
the two fatty acyl chains attached to C-1 and C-3 was provided by ESIMS fragmentation pattern
of compound
2 and by GLC-MS analysis of the methyl esters produced after methanolysis. In the
positive mode, fragment ion peaks at m/z 256 and 284 were evident for the presence of alkyl side
chains at C-1 and C-3, respectively (Figure 3). GLC-MS analysis of the methylated fatty acids obtained
after methanolysis and methylation of
2 was shown to comprise three methyl esters identified as
methyl-2,4-dimethoxybutanoate (m/z 162), methyl-13-methyl-pentadecanoate (m/z 270) and methyl
16-methylheptadecanoate (m/z 298), corresponding to the 0three fatty acyl chains attached to C-2, C-1
and C-3, respectively. Branching of methyl at position C-13 of methyl-13-methyl-pentadecanoate was
indicated by the mass fragmentation pattern as in 1.
Table 2. NMR data of compound 2 (CD3OD, 600 & 150 MHz).
a
HMBC (H→C) b
H-2, H2-3
Position
δC (mult.)
70.5 CH2
71.5 CH
63.9 CH2
175.1 qC
35.0 CH2
26.1 CH2
28.2–31.1 CH2
δ (mult., J in Hz)
H
1
2
3
1
2
3
3.60, m
5.22, m
H2-1, H2-3
H2-1, H-2
4.40, dd (12.0, 3.0) 4.16, dd (12.0, 7.2)
0
0
0
0
0
0
-
H2-1, H2-2 , H2-3
0
0
0
2.30, m
1.59, m
1.28, m
H2-3 , H2-4
0
H2-2 , H2-4
0
4 –11
0
0
0
H2-11 , H-13 , H2-14 ,
0
12
38.2 CH2
35.7 CH
30.8 CH2
1.10, m
1.31, m
1.28, m
0
H3-16
0
0
0
0
13
H2-12 , H3-15 , H3-16
0
0
0
H2-12 , H-13 , H3-15 ,
0
14
0
H3-16
0
0
0
0
15
16
11.8 CH3
19.6 CH3
171.6 qC
77.5 CH
29.1 CH2
68.6 CH2
52.4 CH3
174.7 qC
35.1 CH2
26.1 CH2
28.2–31.1 CH2
0.88, t (6.6
0.85, d (6.6)
-
3.70, brd (3.0)
2.08, m; 2.22, m
3.53, m; 3.67, m
3.20, s
H-13 , H2-14
0
0
0
H2-12 , H-13 , H2-14
H-2”, H2-3”, H2-4”
1”
2”
3”
MeO-2”, H2-3”, H2-4”
H-2”, H2-4”
000
4
H- 2”, H2-3”, OCH3-2”
H-2”
MeO-2”
000
000
000
1
2
3
4
-
H2-3, H2-2 , H2-3
000
000
000
000
2.30, m
1.59, m
1.28, m
H2-3 , H2-4
H2-2 , H2-4
000
000
000
000
–14
000
000
000
H2-14 , H-16 , H3-17
H3-18
,
000
15
40.2 CH2
1.19, m
000
000
000
000
000
000
000
000
16
17
18
29.1 CH
23.1 CH3
23.1 CH3
1.50, m
H3-17 , H3-18
000
000
000
0.87, d (6.6)
0.87, d (6.6)
H2-14 , H2-15 , H-16
000
H2-14 , H2-15 , H-16
a multiplicities were deduced from DEPT and multiplicity-edited HSQC; b HMBC correlations are from proton(s)
stated to the indicated carbons.
To the best of our knowledge,
mirabolide B.
The structures of compounds
2
is reported here as a new natural product and was named
3
and (Figure 1) were assigned by interpretation of the 1D and
4
2D NMR data and MS as well as by comparison with literature data. Thus, compounds
identified as conicasterol [27], and swinhosterol B [28], respectively.
3 and 4 were
Compounds
cell line (MCF-7). Compound
to doxorubicin (IC50 = 5.4
1
–
4
were evaluated for their cytotoxic activity against human breast adenocarcinoma
was the most active compound with IC50 value of 3.0 g/mL compared
g/mL). Compounds 1–3 were less active with IC50 values of 16.4, 5.18 and
4
µ
µ
6.23 µg/mL, respectively.

Mar. Drugs 2016, 14, 155
7 of 11
3. Materials and Methods
3.1. General Experimental Procedure
Optical rotations were recorded by a P800 series digital automatic high-speed polarimeter
(A. KRÜSS Optronic, Hamburg, Germany). UV spectra were recorded on a Hitachi 300 spectrometer
(Hitachi High-Technologies Corporation, Kyoto, Japan). IR spectral analysis were carried on Nicolet
™
iS 10 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA); ESIMS spectra were obtained with a
™
LCQ DECA mass spectrometer (ThermoFinnigan, Bremen, Germany) coupled to an Agilent 1100 HPLC
system equipped with a photodiode array detector. Mass spectral data were obtained with a Micromass
Q-tof equipped with lockspray mass spectrometer using Leucine Enkaphalin at m/z 556.2771 [M + H]+
as a reference mass. NMR spectra were obtained in CD3OD or CDCl3 on Bruker Avance DRX 600 MHz
spectrometer (Bruker, Rheinstetten, Germany) at 600 MHz for 1H NMR and 150 MHz for 13C NMR.
NMR spectra were referenced to the residual protonated solvent signals (CH3OH: 3.30 ppm for 1H
1
and 49.0 ppm for 13C, CHCl3: 7.25 ppm for H and 77.23 ppm for 13C). Silica gel (Sigma-Aldrich,
Darmstadt, Germany, 70–230 Mesh) and silica gel (Sigma-Aldrich, Darmstadt, Germany, 23–400 Mesh)
were used for isolation of compounds on vacuum liquid chromatography and column chromatography,
respectively. The HPLC separation was performed on a RP18, 250 × 10 mm, 5
µm Cosmosil ARII
column (Nacalai Inc., San Diego, CA, USA). Trace Ultra GC system (Thermo Scientific, Surrey, UK) was
used for GC analysis. Fatty acid methyl esters were separated on 70% (cyanopropyl-polysil phenylene
◦
◦
siloxane) capillary column; injection temperature was set at 200 C and detector temperature at 250 C
(MSD). The flow rate of the carrier, Helium, was 1.5 mL/min. The column temperature was 80 ◦C
for 2 min then increased to 230 ◦C by the rate of 3 ◦C/min. For TLC, pre-coated silica gel 60 F-254
plates (Merck, Germany) were used, solvent systems were CHCl3 (100%), CHCl3/MeOH (95:5) and
CHCl3/MeOH (85:15), spots were visualized by p-anisaldehyde/sulphuric acid reagent. Doxorubicin
(Sigma-Aldrich, Darmstadt, Germany) was used as positive cytotoxic control.
3.2. Animal Material
The sponge was collected off Sharm El-Sheikh in the Red Sea at a depth of 10–14 m in July 2013.
Identification of the sponge was kindly provided by Prof. Rob van Soest. The sponge consists of
a group of thick-walled tubes with bluish green color in life, beige-yellow in preserved condition.
The individual tubes measure up to 6 cm high and 2 cm in diameter, with a terminal oscule which
measures up to 3 mm in preserved condition. The surface is optically smooth. The consistency is
hard but toughly compressible. This species belongs to a group of “soft” lithistids. The skeleton is
mainly a desma reticulation but in more than half the outer part of the tubes, the desmas are absent
and the skeleton is there formed by bundles of strongyloxeas (strongyles with tapering endings)
measuring 384–462
with acanthomicrorhabd microscleres measuring 9–13
throughout the choanosome. In the inner parts of the tubes, single desmas gradually form a loose
reticulation. The desmas are peculiar in being calthrops-like and having four bifurcate cladi with
×
5–12
µ
m. The surface membrane lacks the usual phyllotriaenes but is crowded
×
2–3 m, and these are densely distributed
µ
trilophose sharply pointed endings. The cladomes measure 240–270
µm. The primary cladi measure
up to 100–120 20 m, while the secondary cladi measure 30–35 15 µ
×
µ
×
m. The specimen is assigned
to the widespread Indo-Pacific species Theonella aff. mirabilis, because it conforms closely to its original
description from Micronesia. In view of the great distance from the type locality, some doubt of
conspecificity is here expressed. A voucher fragment was deposited in the Naturalis Biodiversity
Center at Leiden under registration number ZMA Por. 17590. Another voucher was deposited at the
Department of Natural Products at King Abdulaziz University under the code #DY-60.
3.3. Extraction and Isolation
The freeze-dried sponge (750 g) was extracted with a mixture of CH2Cl2/MeOH (1:1)
(3 × 2000 mL) at room temperature. The combined extracts were concentrated under vacuum to

Mar. Drugs 2016, 14, 155
8 of 11
afford 11.0 g of dried extract. The extract was partitioned between CHCl3 and H2O and the
organic layer was evaporated under vacuum to afford 6.0 g of dried residue. The residue was
then chromatographed on SiO2 VLC using n-hexane/EtOAc/MeOH gradient to give five main
fractions (A–E). Fraction B (eluted by n-hexane/EtOAc mixture (70:30, 1.2 g) was purified on silica gel
column using n-hexane/CHCl3 gradient to give several fractions. The active fraction was purified on
C18-reversed phase semipreparative HPLC using 70% ACN to give compound
3 (4.9 mg). Fraction C
which was eluted by n-hexane/EtOAc (60:40–20:80, 570 mg) was purified on silica gel column using
n-hexane/CHCl3 gradient and final HPLC purification of the active fraction on C18-reversed phase
semipreparative HPLC using 70% ACN to yield compound
4 (14 mg). Fraction D was eluted with
EtOAc/MeOH (80:20–60:40, 930 mg) was subjected to SiO2 column using CHCl3/MeOH gradient
to give four fractions. The cytotoxic fraction was subjected to final HPLC purification on C18
semipreparative column using 60% ACN to afford compound
1 (5.8 mg). Finally, fraction E was
eluted with EtOAc/MeOH mixtures (40:60, 280 mg) was chromatographed over silica gel column
using CHCl3/MeOH gradient to give several fractions. The cytotoxic fraction was purified on HPLC
using 60% ACN to give compound 2 (3.3 mg).
3.4. Spectral Data of the Compounds
Mirabolide A (
1
). White precipitate; [α]2D5 +0.36◦ (c 0.025, CH3OH); UV (MeOH)
λ
292 nm; IR:
max
ν
max 3350, 1735 cm−1; HRESIMS m/z 685.5625 (calcd. for C40H77O8, 685.5624 [M − H]−); NMR data:
see Table 1.
Mirabolide B (
max 3240, 1740 cm−1; HRESIMS m/z 713.5933 (calcd. for C42H81O8, 713.5931 [M + H]+); NMR data:
see Table 2.
Conicasterol (
3450, 1712 cm−1; ESIMS: m/z 413.3 [M + H]+, C29H49O.1H NMR (600 MHz, CDCl3): δH 5.07
). White precipitate; [α]D25
−
0.52 (c 0.025, CH3OH); UV (MeOH)
λ
292 nm; IR:
◦
2
max
ν
3
). White precipitate; [α]2D5 +79◦ (c 0.1, CHCl3); UV (MeOH)
λ
235 nm; IR:
max
ν
max
(d, J = 1.2, H-29a), 4.63 (d, J = 0.6 Hz, H-29b), 4.02 (dd, J = 10.8, 4.8, H-3), 2.48 (ddd, H-15a), 2.25
(m, H2-7), 1.95 (dt, H-12b), 1.78 (brd, J = 14.4, H-5), 1.75 (m, H-1a), 1.75 (dd, J = 10.2, 2.4, H-9),
1.74 (m, H-15b), 1.64 (m, H-11a), 1.58 (m, H-16a), 1.49 (m, H-11b), 1.52 (m, H-25), 1.48 (m, H-6b),
1.45 (m, H-20), 1.39 (m, H-6a), 1.38 (m, H-16b), 1.35 (m, H-1b), 1.33 (m, H-22a), 1.23 (m, H2-2), 1.22
(m, H-23a), 1.22 (m, H-24), 1.14 (m, H-12a), 1.13 (m, H-17), 1.13 (m, H-22b), 1.10 (m, H-23b), 0.92
(
d, J = 6.6, H3-21), 0.85 (d, J = 6.6, H3-27), 0.83 (s, H3-18), 0.80 (d, J = 7.2, H3-26), 0.78 (d, J = 6.6, H3-28),
0.58 (s, H3-19); 13C NMR (150 MHz, CDCl3): δC 153.1 (C-4), 142.9 (C-14), 125.6 (C-8), 102.7 (C-29), 73.3
(C-3), 56.8 (C-17), 49.4 (C-5), 49.2 (C-9), 42.7 (C-13), 39.9 (C-10), 37.3 (C-24), 36.7 (C-12), 34.5 (C-1), 32.3
(C-20), 31.9 (C-22), 30.1 (C-25), 29.3 (C-23), 27.1 (C-2), 27.0 (C-15), 25.7 (C-6), 24.6 (C-7), 22.7 (C-16), 20.2
(C-11), 19.0 (C-27), 18.2 (C-21), 18.1 (C-26), 15.3 (C-18), 14.1 (C-28), 13.1 (C-19).
Swinhosterol B (
4
). White precipitate; [α]D25
−
50◦ (c 0.1, CHCl3); UV (MeOH)
λ
max
280 nm; IR:
ν
max 3460, 1735, 1710 cm−1; ESIMS: m/z 445.4 [M + H]+, C29H49O3.1H NMR (600 MHz, CDCl3): δH
5.16 (brs, H-29b), 4.67 (brs, H-29a), 4.0 (dd, J = 11.7, 5.9, H-3), 2.41 (m, H2-7), 2.35 (m, H-15b), 2.29
(m, H-5), 2.20 (m, H-16b), 2.12 (m, H-9), 2.10 (m, H-2b), 2.04 (m, H-15a), 1.97 (m, H-17), 1.97 (m, H-6b),
1.82 (m, H-6a), 1.77 (m, H-1b), 1.71 (m, H-11b), 1.63 (m, H-12b), 1.53 (m, H-25), 1.51 (m, H-20), 1.48
(m, H-1a), 1.47 (m, H-16a), 1.43 (m, H-22a), 1.41 (m, H-12a), 1.37 (m, H-22b), 1.29 (m, H-2a), 1.25 (m,
H-23b), 1.23 (m, H-24), 1.19 (m, H-23a), 1.08 (d, J = 6.6, H3-21), 0.87 (d, J = 6.6, H3-27), 0.82 (d, J = 6.6,
H3-26), 0.81 (d, J = 5.1, H3-28), 0.81 (m, H-11a); 13C NMR (150 MHz, CDCl3): δC 224.9 (C-14), 211.2
(C-8), 150.9 (C-4), 104.2 (C-29), 72.9 (C-3), 62.5 (C-9), 52.5 (C-13), 48.4 (C-5), 46.6 (C-17), 44.3 (C-10), 41.6
(C-7), 38.9 (C-24), 37.9 (C-15), 37.3 (C-12), 36.5 (C-1), 34.4 (C-20), 32.4 (C-25), 32.3 (C-22), 32.2 (C-2), 30.6
(C-23), 26.0 (C-6), 23.7 (C-16), 20.2 (C-27), 18.5 (C-18), 18.2 (C-26), 18.1 (C-21), 18.0 (C-11), 15.4 (C-28),
13.0 (C-19).

Mar. Drugs 2016, 14, 155
9 of 11
3.5. Methanolysis of Compounds 1 and 2 and Methylation of Fatty Acids
Compounds 1 and 2 (3 mg, each) were separately refluxed with 5% methanolic KOH (5 mL) for
2 h [29]. The reaction product was diluted with water and extracted with CHCl3. The fatty acids were
liberated from the aqueous layer after acidification with 1 N HCl followed by extraction with CHCl3,
then were methylated using CH2N2 and subjected to GC-MS analysis.
3.6. Evaluation of the Cytotoxic Activities of Compounds 1–4
The cytotoxic activity of compounds
HTB-22 ) were evaluated using sulforhodamine assay [30]. Cells were grown 24 h in 96-well plates
1–4
against breast adenocarcinoma cell line (MCF-7, ATCC®
™
before addition of samples, then incubated for 48 h after treatment with tested compounds. The dose
of the compound that reduced survival to 50% (IC50) was calculated from the log dose response curve.
The values were the results of three determinations. Doxorubicin was used as reference drug and
showed an IC50 value of 5.4 µg/mL.
4. Conclusions
Column chromatographic fractionation of the organic extract of the Red Sea sponge
Theonella mirabilis afforded four compounds (
1–4) including two sterols and two glycerides.
Compounds and are new glycerides and are reported here for the first time. The structures
1
2
of the isolated compounds were determined by careful examination of their 1D and 2D NMR, and
HRESIMS spectral data. All isolated compounds demonstrated significant cytotoxic activity against
human breast adenocarcinoma cell line (MCF-7) with IC50 values ranging from 3.0 to 16.4 µg/mL.
Supplementary Materials: The followings are available online at www.mdpi.com/1660-3397/14/8/155/s1,
Figure S1: 1H NMR Spectrum of
1
(CDCl3), Figure S2: 13C NMR Spectrum of
1
(CDCl3), Figure S3: COSY Spectrum
(CDCl3), Figure S5: HMBC Spectrum of (CDCl3), Figure S6: Partial
“Expansion A” (CDCl3), Figure S7: Partial HMBC Spectrum of “Expansion B” (CDCl3),
“Expansion C” (CDCl3), Figure S9: ROESY Spectrum of (CDCl3), Figure
(CD3OD), Figure S12: COSY Spectrum
(CD3OD), Figure S14: HMBC Spectrum of (CD3OD), Figure S15:
“Expansion A” (CD3OD), Figure S16: Partial HMBC Spectrum of “Expansion B”
(CD3OD, Figure S17: Partial HMBC Spectrum of 2 “Expansion C” (CD3OD).
of
1
(CDCl3), Figure S4: HSQC Spectrum of
1
1
HMBC Spectrum of
1
1
Figure S8: Partial HMBC Spectrum of
1
1
1
S10: H NMR Spectrum of
2
(CD3OD), Figure S11: 13C NMR Spectrum of
2
of
2
(CD3OD), Figure S13: HSQC Spectrum of
2
2
Partial HMBC Spectrum of
2
2
Acknowledgments: The authors were grateful to the Deanship of Scientific Research, King Abdulaziz University,
Jeddah, Kingdom of Saudi Arabia for the support of this work, under grant number 166-599-D1435. Our thanks
also to Rob van Soest from Naturalis Biodiversity Center, Department of Marine Zoology, for taxonomic
identification of the sponge.
Author Contributions: D.R.A.-H. and D.T.A.Y. designed experiments; D.T.A.Y. collected the sponge specimen;
D.R.A.-H. performed experiments; D.R.A.-H. and D.T.A.Y. analyzed the data; D.R.A.-H. wrote the manuscript;
D.T.A.Y. revised and edited the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Müller, W.E.G.; Brümmer, F.; Batel, R.; Müller, I.M.; Schröder, H.C. Molecular biodiversity. Case study:
Porifera (sponges). Naturwissenschaften 2003, 90, 103–120. [PubMed]
2.
Van Soest, R.W.M.; Boury-Esnault, N.; Hooper, J.N.A.; Rützler, K.; de Voogd, N.J.; de Glasby, A.B.; Hajdu, E.;
Pisera, A.B.; Manconi, R.; Schoenberg, C.; et al. World Porifera Database 2006. Available online: http:
//www.marinespecies.org/poriferaon2016-04-03 (accessed on 16 June 2016).
Blunt, J.W.; Copp, B.R.; Hu, W.-P.; Munro, M.H.G.; Northcote, P.T.; Prinsep, M.R. Marine natural products.
Nat. Prod. Rep. 2008, 25, 35–94. [CrossRef] [PubMed]
Bewley, C.A.; Faulkner, D.J. Lithistid sponges: Star performers or hosts to the stars. Angew. Chem. Int. Ed.
1998, 37, 2162–2178. [CrossRef]
Hooper, J.N.A.; van Soest, R.W.M. Systema Porifers: A Guide to the Classification of Sponges; Kluwer Academic:
New York, NY, USA, 2002; Volume 1.
3.
4.
5.

Mar. Drugs 2016, 14, 155
10 of 11
6.
D’auria, M.V.; Zampella, A.; Zollo, F. The chemistry of Lithistid sponges: A spectacular source of new
metabolites. In Studies in Natural Products Chemistry, Volume 26: Bioactive Natural Products; Attar-ur-Rahman,
Ed.; Elsevier: Amsterdam, The Netherlands, 2002; Volume 26, pp. 1175–1258.
7.
8.
Fusetani, N.; Matsunga, S. Bioactive sponge peptides. Chem. Rev. 1993, 93, 1793–1806. [CrossRef]
Winder, P.L.; Pomponi, S.A.; Wright, A.E. Natural products from the Lithistida: A Review of the literature
since 2000. Mar. Drugs 2011, 9, 2643–2682. [CrossRef] [PubMed]
9.
Zhang, H.-J.; Yi, Y.-H.; Lin, H.-W. Oxygenated 4-methylidene sterols from the South China Sea sponge
Theonella swinhoei. Helv. Chim. Acta 2010, 93, 1120–1126. [CrossRef]
10. Buchingham, J. Dictionary of Natural Products on DVD v.20:1; CRC Press Taylor and Francis Group: Boca Raton,
FL, USA, 2011.
11. Angawi, R.F.; Calcinai, B.; Cerrano, C.; Dien, H.A.; Fattorusso, E.; Scala, F.; Taglialatela-Scafati, O.
Dehydroconicasterol and aurantoic acid, a chlorinated polyene derivative, from the Indonesian sponge
Theonella swinhoei. J. Nat. Prod. 2009, 72, 2195–2198. [CrossRef] [PubMed]
12. Ratnayake, A.S.; Davis, R.A.; Harper, M.K.; Veltri, C.A.; Andjelic, C.D.; Barrows, L.R.; Ireland, C.M.
Aurantosides G, H, and I: Three new tetramic acid glycosides from a Papua New Guinea Theonella swinhoei.
J. Nat. Prod. 2005, 68, 104–107. [CrossRef] [PubMed]
13. Piel, J. Bacterial symbionts: Prospects for the sustainable production of invertebrate-derived pharmaceuticals.
Curr. Med. Chem. 2006, 13, 39–50. [CrossRef] [PubMed]
14. Kabayashi, J.; Itagaki, F.; Shigemori, H.; Ishibashi, M.; Takahashi, K.; Ogura, M.; Nagasawa, S.; Nakamura, T.;
Hirota, H.; Ohta, T.; et al. Keramamides B–D, novel peptides from the Okinawan marine sponge Theonella sp.
J. Am. Chem. Soc. 1991, 113, 7812–7813. [CrossRef]
15. Itagaki, F.; Shigemori, H.; Ishibashi, M.; Nakamura, T.; Sasaki, T.; Kabayashi, J. Keramamide F, a new
thiazole-containing peptide from the Okinawan marine sponge Theonella sp. J. Org. Chem. 1992, 57,
5540–5542. [CrossRef]
16. Chill, L.; Kashman, Y.; Schleyer, M. Oriamide, a new cytotoxic cyclic peptide containing a novel amino acid
from the marine sponge Theonella sp. Tetrahedron 1997, 53, 16147–16152. [CrossRef]
17. Kobayashi, M.; Lee, N.K.; Shibuya, H.; Momose, T.; Kitagawa, I. Marine natural products. XXVI. Biologically
active tridecapeptide lactones from the Okinawan marine sponge Theonella swinhoei (Theonellidae). (2).
Structures of theonellapeptolides Ia, Ib, Ic, and Ie. Chem. Pharm. Bull. 1991, 39, 1177–1184. [CrossRef]
[PubMed]
18. Ford, P.W.; Gustafson, K.R.; McKee, T.C.; Shigematsu, N.; Maurizi, L.K.; Pannell, L.K.; Williams, D.E.;
de Silva, E.D.; Lassota, P.; Allen, T.M.; et al. Papuamides A–D, HIV-inhibitory and cytotoxic depsipeptides
from the sponges Theonella mirabilis and Theonella swinhoei collected in Papua New Guinea. J. Am. Chem. Soc.
1999, 121, 5899–5909. [CrossRef]
19. Fusetani, N.; Warabi, K.; Nogata, Y.; Nakao, Y.; Matsunaga, S. Koshikamide A1, a new cytotoxic linear
peptide isolated from a marine sponge, Theonella sp. Tetrahedron Lett. 1999, 40, 4687–4690. [CrossRef]
20. Carmely, S.; Kashman, Y. Structure of swinholide-A, a new macrolide from the marine sponge
Theonella swinhoei. Tetrahedron Lett. 1985, 26, 511–514. [CrossRef]
21. Kobayashi, J.; Murayama, T.; Ohizumi, Y.; Sasaki, T.; Ohta, T.; Nozoe, S. Theonelladins A–D, novel
antineoplastic pyridine alkaloids from the Okinawan marin sponge Theonella swinhoei. Tetrahedron Lett.
1989, 30, 4833–4836. [CrossRef]
22. Tsuda, M.; Shimbo, K.; Kubota, T.; Mikami, Y.; Kobayashi, J. Two theonellapeptolide congeners from marine
sponge Theonella sp. Tetrahedron 1999, 55, 10305–10314. [CrossRef]
23. Costantino, V.; Fattorusso, E.; Mangoni, A. Isolation of five-membered cyclitol glycolipids, crasserides:
Unique glycerides from the sponge Pseudoceratina crassa. J. Org. Chem. 1993, 58, 186–191. [CrossRef]
24. Costantino, V.; Fattorusso, E.; Imperatore, C.; Mangoni, A. Glycolipids from sponges. 11. Isocrasserides,
novel glycolipids with a five-membered cyclitol widely distributed in marine sponges. J. Nat. Prod. 2002, 65,
883–886. [CrossRef] [PubMed]
25. Youssef, D.T.A.; Shaala, L.A.; Mohamed, G.A.; Badr, J.M.; Bamanie, F.H.; Ibrahim, S.R.M. Theonellamide G, a
potent antifungal and cytotoxic bicyclic glycopeptide from the Red Sea marine sponge Theonella swinhoei.
Mar. Drugs 2014, 12, 1911–1923. [CrossRef] [PubMed]
26. Youssef, D.T.A.; Mooberry, S.L. Hurghadolide A and swinholide I, potent actin-microfilament disrupters
from the Red Sea sponge Theonella swinhoei. J. Nat. Prod. 2006, 69, 154–157. [CrossRef] [PubMed]

Mar. Drugs 2016, 14, 155
11 of 11
27. Sugo, Y.; Inouye, Y.; Nakayama, N. Structures of nine oxygenated 4-methylene sterols from Hachijo marine
sponge Theonella swinhoei. Steroids 1995, 60, 738–742. [CrossRef]
28. Umeyama, A.; Shoji, N.; Enoki, M.; Arihara, S. Swinhosterol A–C, 4-methylene secosteroids from the marine
sponge Theonella swinhoei. J. Nat. Prod. 1997, 60, 296–298. [CrossRef]
29. Vogel, A.I. A Text Book of Practical Organic Chemistry, 3rd ed.; Longmans and Green Co., Ltd.: London,
UK, 1975.
30. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahom, J.; Vistica, D.; Warren, T.; Bokesch, H.; Kenney, S.;
Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82,
1107–1112. [CrossRef] [PubMed]
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).