5-Alkylresorcinol derivatives from the bryozoan Schizomavella mamillata: Isolation, synthesis, and antioxidant Activity

Article abstract of DOI:10.3390/md15110344

The chemical study of the bryozoan Schizomavella mamillata has led to the isolation of six new 5-alkylresorcinol derivatives, schizols A–F (1–6), whose structures were established by spectrocospic means. Schizol A (1) exhibits a (E)-6-phenylnon-5-enyl moi

Full text of DOI:10.3390/md15110344

marine drugs
Article
5-Alkylresorcinol Derivatives from the Bryozoan
Schizomavella mamillata: Isolation, Synthesis,
and Antioxidant Activity
María J. Ortega, Juan J. Pantoja, Carolina de los Reyes and Eva Zubía *
Departamento de Química Orgánica, Facultad de Ciencias del Mar y Ambientales, 11510 Puerto Real (Cádiz),
Spain; mariajesus.ortega@uca.es (M.J.O.); juanjose.pantoja@uca.es (J.J.P.); carolina.dereyes@uca.es (C.d.l.R.)
* Correspondence: eva.zubia@uca.es; Tel.: +34-956-016-021
Received: 11 October 2017; Accepted: 30 October 2017; Published: 2 November 2017
Abstract: The chemical study of the bryozoan Schizomavella mamillata has led to the isolation of six new
5-alkylresorcinol derivatives, schizols A–F (
means. Schizol A ( ) exhibits a (E)-6-phenylnon-5-enyl moiety linked to the C-5 of a resorcinol
ring, while in schizol B ( ) the substituent at C-5 contains an unusual 1,2-dihydrocyclobutabenzene
moiety. Schizols C ( ) and D ( ) have been characterized as the 1-sulfate derivatives of and
respectively, and schizols E ( ) and F ( ) are the corresponding 1,3-disulfates. Schizol A ( ) has been
1–6), whose structures were established by spectrocospic
1
2
3
4
1
2,
5
6
1
synthetized from 3,5-dimethoxybenzaldehyde through a sequence involving a Wittig reaction for the
construction of the C-10,C-20 bond and a Julia–Kocienski olefination for the synthesis of the C-50,C-60
0
double bond. In the ABTS (2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid)) antioxidant assay,
the natural compounds schizol A (
the Trolox standard.
1
) and schizol B (2) showed higher radical scavenging activity than
Keywords:
alkylresorcinols;
bryozoans;
Schizomavella;
1,2-dihydrocyclobutabenzene;
olefination; antioxidant
1. Introduction
Bryozoans are sessile aquatic invertebrates, with most of the known species living in the
marine environment [ ]. These filter-feeding organisms typically form colonies that thrive on
1
rocks near the coasts and coral reefs, but also on man-made structures such as piers and ships
hulls. The phylum Bryozoa has been much less studied from a natural products perspective than
other marine invertebrates such as sponges or coelenterates, and only about 220 new natural
products have been isolated from bryozoans [
alkaloids, mainly brominated alkaloids of diverse structural types that have been obtained from a few
bryozoan species of the families Vesiculariidae and Flustridae [ ]. However, the most renowned
bryozoan-derived compounds are the bryostatins, a large series of macrocyclic lactones among which
bryostatin-1 has reached clinical trials as an anticancer agent [ ] and more recently as a treatment
2]. The compounds more often described have been
3–8
9
for Alzheimer’s disease [10]. Interestingly, while bryostatins were first isolated from the bryozoan
Bugula neritina (family Bugulidae) [11], they were later shown to be produced by the symbiotic
bacterium “Candidatus Endobugula sertula” [12].
As a part of our research on bioactive natural products from marine invertebrates of the southern
coasts of Spain, we have studied samples of the bryozoan Schizomavella mamillata (Hincks, 1880),
which belongs to the family Bitectiporidae. Previous chemical studies of this family of bryozoans
have been limited to the species Pentapora fascialis, which was the source of pentaporins A–C [13].
These metabolites consist of two 5-alkylresorcinol-derived moieties linked through a disulfide bridge.
The present study of S. mamillata has led to the isolation of six unprecedented 5-alkylresorcinol
Mar. Drugs 2017, 15, 344; doi:10.3390/md15110344
www.mdpi.com/journal/marinedrugs

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derivatives, schizols A–F (
1
–
6
). In addition, we describe synthetic routes for the obtention of schizol
A (1 1–6) have also been tested in the
) and its geometrical isomer 10. The isolated compounds (
0
ABTS (2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid)) radical-scavenging assay to assess their
antioxidative properties.
2. Results and Discussion
2.1. Isolation and Structure Determination
Frozen specimens of S. mamillata were extracted with acetone/MeOH and, after evaporation of
the solvent, the residue was extracted with Et2O and then with n-butanol. The Et2O extract and the
n-butanol extract were separately subjected to column chromatography on silica gel. Further separation
of fractions eluted with hexane/Et2O (3:7, v/v), Et2O, and CHCl3/MeOH (7:3, v/v) by using SPE-C18
cartridges and HPLC led to the isolation of compounds 1–6 (Figure 1).
Figure 1. Chemical structures of schizols A–F (1–6).
Schizol A (
by HRESIMS(
1
) was obtained as a colorless oil whose molecular formula C21H26O2 was established
−
). The IR absorption at 3365 cm−1 indicated the presence of hydroxy groups in the
1
molecule. The H NMR spectrum (Table 1) exhibited a one-proton triplet at δH 6.07 (t, J = 2.2 Hz,
H-2) and a two-proton doublet at δH 6.13 (d, J = 2.1 Hz, H-4 and H-6) attributable to a symmetrical
1,3,5-trisubstituted aromatic ring. In addition, the presence of a monosubstituted benzene ring was
00
00
inferred from the signals at δH 7.29 (dd, J = 8.2 and 1.4 Hz, H-2 and H-6 ), 7.25 (dd, J = 8.0 and 7.4 Hz,
H-300 and H-500) and δH 7.17 (tt, J = 7.2 and 1.5 Hz, H-400), while a signal at δH 5.61 (t, J = 7.3 Hz, H-50)
was assigned to the proton of a trisubstituted double bond. The remaining signals of the 1H NMR
spectrum were due to six methylenes and a methyl group. The 1,3,5-trisubstituted ring was identified
as a 5-alkylresorcinol ring from the HMBC correlations of the aromatic protons H-2, H-4, and H-6 with
two phenolic carbons at δC 159.3 (C-1 and C-3) and the HMBC correlation of H-4 and H-6 with the
benzylic methylene carbon at δC 36.9 (C-10) (Figure 2). The trisubstituted double bond was located at
C-50,C-60 of the side chain on the basis of the COSY couplings observed from the benzylic methylene
protons H-10 (δH 2.48) through a sequence of three methylenes up to the olefinic methine (δH 5.61,
H-50). The HMBC correlation of the protons H-200 and H-600 (δH 7.29) of the monosubstituted benzene
ring with the olefinic carbon C-60 indicated that the phenyl ring was linked to C-60. The presence of a
propyl chain also linked to C-60 was indicated by the presence of a methyl group (δH 0.86) coupled in
the COSY spectrum with two methylene protons at δH 1.32 (H-8’) which were correlated in the HMBC
spectrum with the olefinic carbon C-60. On the other hand, the NOESY correlations of the olefinic

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proton H-50 with the aromatic protons H-200 and H-600 defined the E geometry of the double bond.
These data and the remaining NMR correlations led to the proposal of the structure 1 for schizol A.
1
Table 1. H and 13C NMR data of schizol A (1) and schizol B (2) a,b
.
1
2
Position
δC, Type
δ , m (J in Hz)
H
δC, Type
δ , m (J in Hz)
H
1
2
3
4
5
6
159.3, C
101.0, CH
159.3, C
107.9, CH
146.2, C
107.9, CH
36.9, CH2
32.1, CH2
30.6, CH2
29.4, CH2
129.9, CH
141.6, C
32.6, CH2
22.8, CH2
14.2, CH3
144.8, C
159.3, C
101.0, CH
159.3, C
108.0, CH
146.2, C
6.07, t (2.2)
6.13, d (2.1)
6.08, t (2.2)
6.14, d (2.2)
6.13, d (2.1)
2.48, t (7.6)
1.64, m
1.49, tt (7.7, 7.4)
2.21, dt (7.4, 7.4)
5.61, t (7.3)
108.0, CH
36.9, CH2
32.5, CH2
29.1, CH2
35.1, CH2
51.43, CH
51.37, CH
37.6, CH2
22.8, CH2
14.6, CH3
148.99 c, C
149.05 c, C
123.13 d, CH
127.9, CH
127.9, CH
123.07 d, CH
6.14, d (2.2)
2.45, t (7.5)
1.64, m
1.52, m
1.69, m
2.94, td (7.4, 2.1)
2.95, td (7.4, 2.1)
1.64, m
0
1
2
3
4
5
6
7
8
0
0
0
0
0
0
2.48, t (7.5)
1.32, tq (7.5, 7.5)
0.86, t (7.4)
0
1.48, m
0.97, t (7.4)
0
9
00
00
00
00
00
00
1
2
3
4
5
6
127.4, CH
129.2, CH
127.5, CH
129.2, CH
127.4, CH
7.29, dd (8.2, 1.4)
7.25, dd (8.0, 7.4)
7.17, tt (7.2, 1.5)
7.25, dd (8.0, 7.4)
7.29, dd (8.2, 1.4)
6.99, m
7.11, m
7.11, m
7.02, m
a
b
1H at 600 MHz, 13C at 150 MHz; Assignments aided by COSY, HSQC, HMBC, and NOESY experiments;
c,d
Assignments marked with the same letter in the same column may be interchanged.
Figure 2. Key COSY (bold bond) and HMBC (→) correlations observed for schizol A (1).
The molecular formula of schizol B (
2
), C21H26O2, was established by HRESIMS(
−
) and indicated
, showing the
that
2
was an isomer of schizol A ( ). The NMR spectra (Table 1) were related to those of 1
1
signals of a resorcinol ring bearing at C-5 an alkyl chain. The signals of a propyl moiety at the end of
the chain [δH 0.97 (3H, t, J = 7.4 Hz, H-90), δH 1.48 (2H, m, H-80), and δH 1.64 (2H, m, H-70)] were also
evident in the spectra of
of
2. However, significant differences were observed with respect to the spectra
1
, in particular the presence of signals due to a disubstituted benzene ring [δH 6.99 (1H, m, H-300),
δH 7.02 (1H, m, H-600), and δH 7.11 (2H, m, H-400 and H-500)], the absence of the signals due to the
double bond at C-50,C-60, and the presence of two methines at δC 51.43 (C-50)/δH 2.94 (td, J = 7.4 and
0
0
0
2.1 Hz, H-5 ) and δC 51.37 (C-6 )/δH 2.95 (td, J = 7.4 and 2.1 Hz, H-6 ). These methines were located at
C-50 and C-60 of the side chain since the protons at δH 2.94 (H-50) and δH 2.95 (H-60) were coupled in
the COSY spectrum with the methylene protons H-40 and H-70, respectively, and in the HMBC with
the methylene carbons C-30 and C-80, respectively (Figure 3). Moreover, the HMBC correlations of the
methine protons H-50 and H-60 with the aromatic carbons C-300 and C-600, respectively, indicated that

Mar. Drugs 2017, 15, 344
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the methines at C-50 and C-60 were linked to adjacent positions of the benzene ring. Further support
was obtained from the HMBC correlations of the methylene protons H-70 and H-40 with the aromatic
carbons C-100 and C-200, respectively.
Figure 3. Key COSY (bold bond) and HMBC (→) correlations observed for schizol B (2).
Because of the close chemical shifts of H-50 and H-60 it was not possible to obtain unambiguous
information on the relative configuration at C-50 and C-60 from the NOESY spectrum. The spectra
were also recorded in CD3COCD3, C5D5N, and CD3SOCD3 but no improvement of the resolution
of the signals of the methines at C-50 and C-60 was achieved. Interestingly, looking in literature
for NMR data of compounds encompassing a similar 1,2-dihydrocyclobutabenzene moiety we
found that the chemical shifts of the methines at C-1 and C-2 in trans-1,2-diethyl-6,7-dimethyl-1,2-
dihydrocyclobuta[a]naphthalene (δC 51.6 and 51.4, δH 3.06 and 3.18–3.25) differed significantly from
those of the cis isomer (δC 48.5 and 48.4, δH 3.64–3.72 and 3.51) [14]. The similarity of the chemical
shifts of C-50, C-60, H-50, and H-60 of compound
membered ring of trans-1,2-diethyl-6,7-dimethyl-1,2-dihydrocyclobuta[a]naphthalene supported a
2 with those reported for the methines of the four
0
0
trans relationship of H-5 and H-6 in compound
2. All these data led to the proposal of the structure
2
for schizol B.
Schizol C (
3
), whose molecular formula C21H26O5S was established by HRESIMS(
−
),
displayed NMR spectra (Table 2) similar to those of schizol A (
1
). Moreover, the COSY and HMBC
correlations confirmed that
3 possessed the same carbon framework as 1 and a double bond
at C-50,C-60 with the E geometry ascertained by the NOESY correlation between the methylene
protons H-40 (δH 2.22) and H-70 (δH 2.49). However, the H NMR spectrum of
2 exhibited three
1
resonances of meta-coupled aromatic protons [δH 6.64 (1H, br dd J = 1.8 and 1.5 Hz), δH 6.60 (1H, dd,
J = 2.2 and 2.2 Hz), and δH 6.45 (1H, br dd, J = 1.8 and 1.5 Hz)] which defined the 1,3,5-trisubstitution
pattern and indicated that the ring was not symmetrically substituted. In particular, the presence of a
hydroxy group and a sulfate group on the aromatic ring was deduced from the 13C NMR resonances at
δC 159.0 (C-3) and δC 154.7 (C-1), respectively, together with the molecular formula of
3, which differed
from that of
1 by a SO3 unit. The presence of a sulfate group in 3 was also consistent with the IR
absorptions at 1239 and 1053 cm−1. Moreover, O-sulfation of a phenolic hydroxy group has been
shown to cause deshielding of protons and carbons at the ortho and para positions and the shielding of
the carbon carrying the sulfate group [15]. Upon comparison with compound
H-2, H-4, and H-6 of exhibited higher chemical shifts (∆δH = +0.53, +0.32, and +0.51, respectively)
and also the carbons C-2, C-4, and C-6 (∆δC = +6.1, +5.0, and +5.7 ppm, respectively), while C-1
exhibited a lower chemical shift (∆δC 4.6). All these data strongly supported the presence of a
sulfate group in the resorcinol ring of schizol C (3).
The molecular formula of schizol D (
1, the aromatic protons
3
=
−
4
), C21H26O5S, was established by HRESIMS( ). The NMR
−
spectra (Table 2) showed the signals of two methines at δC 51.44 (C-50)/δH 2.94 (1H, td, J = 7.7 and
2.0 Hz, H-50) and δC 51.37 (C-60)/δH 2.95 (1H, td, J = 7.7 and 2.0 Hz, H-60) which indicated the
presence in
4 of a 1,2-dihydrocyclobutabenzene moiety similar to that described in compound 2.
On the other hand, the 13C NMR signals at δC 159.0 (C-3) and δC 154.6 (C-1) were attributable to two

Mar. Drugs 2017, 15, 344
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aromatic carbons bearing a hydroxy group and a sulfate group, respectively. These and the remaining
NMR data led to the conclusion that schizol D ( ) was the monosulfate derivative of compound
Moreover, the sulfation of the hydroxy group at C-1 was consistent with the higher 1H and 13C chemical
4
2.
shifts of the aromatic methines C-2, C-4, and C-6 in compound
4 with respect to the same methines in
compound 2.
1
Table 2. H and 13C NMR data of schizol C (3) and schizol D (4) a,b
.
3
4
Position
δC, Type
δ , m (J in Hz)
H
δC, Type
δ , m (J in Hz)
H
1
2
3
4
5
6
154.7, C
107.1, CH
159.0, C
112.9, CH2
145.8, C
113.6, CH
36.8, CH2
32.1, CH2
30.6, CH2
29.4, CH2
129.9, CH
141.6, C
32.6, CH2
22.8, CH2
14.2, CH3
144.7, C
154.6, C
107.1, CH
159.0, C
113.0, CH2
145.9, C
6.60, dd (2.2, 2.2)
6.60, dd (2.4, 2.0)
6.45, br dd (1.8, 1.5)
6.45, br dd (1.6, 1.6)
6.64, br dd (1.8, 1.5)
2.55, t (7.7)
113.6, CH
36.9, CH2
32.5, CH2
29.2, CH2
35.1, CH2
51.44, CH
51.37, CH
37.6, CH2
22.8, CH2
14.6, CH3
149.01 c, C
149.06 c, C
123.18 d, CH
127.95 e, CH
127.92 e, CH
123.07 d, CH
6.63, br dd (1.6, 1.6)
2.54, (t, 7.5)
1.66, m
1.52, m
1.69, m
2.94, td (7.7, 2.0)
2.95, td (7.7, 2.0)
1.64, m
0
1
2
3
4
5
6
7
8
0
1.66, m
0
1.48, tt (7.7, 7.3)
2.22, dt (7.3, 7.3)
5.61, t (7.3)
0
0
0
0
2.49, t (7.5)
1.33, tq (7.7, 7.3)
0.86, t (7.3)
0
1.49, m
0.97, t (7.5)
0
9
00
00
00
00
00
00
1
2
3
4
5
6
127.4, CH
129.2, CH
127.4, CH
129.2, CH
127.4, CH
7.29, dd (8.1, 1.3)
7.26, dd (8.1, 7.3)
7.16, tt (7.3, 1.3)
7.26, dd (8.1, 7.3)
7.29, dd (8.1, 1.3)
7.01, m
7.10, m
7.10, m
7.01, m
b
a 1H at 600 MHz, 13C at 150 MHz; Assignments aided by COSY, HSQC, HMBC, and NOESY experiments;
c–e
Assignments marked with the same letter in the same column may be interchanged.
Schizol E (
The NMR spectra were related to those of schizol A (
and carbons of the resorcinol ring (Table 3). In particular, upon comparison with
δC 154.2 (C-1 and C-3) in the 13C NMR spectrum of
was consistent with the O-sulfation of the
5
) possessed the molecular formula C21H26O8S2, that was established by HRESIMS(
) except for the chemical shifts of the protons
, the signal at
−
).
1
1
5
two hydroxy groups of the resorcinol ring. This proposal was supported by the chemical shifts of
H-2, H-4, and H-6 [δH 7.04 (3H, m)] which were significantly higher than the shifts of the same
protons in compound
groups explains the strong deshielding of carbons C-2 (δC 113.3), C-4 (δC 118.8), and C-6 (δC 118.8)
upon comparison with the same carbons in ∆δC = +12.3, +10.9, +10.9, respectively). These and the
remaining spectroscopic data led us to propose the structure 5 for schizol E.
The 1H and 13C NMR spectra of schizol F (
) were similar to those of schizol B (
signals of the resorcinol ring. These data, together with the molecular formula C21H26O8S2 established
by HRESIMS( ), indicated that was the disulfate derivative of . This proposal was fully confirmed
by conversion of 6 into 2 upon acid treatment of 6 that caused the hydrolysis of the sulfate esters.
From a structural point of view, compounds obtained from S. mamillata represent new
types of alkylresorcinol-based natural products. The distinctive feature of compounds , and
1 (∆δH = +0.97, +0.91, +0.91, respectively). Similarly, the presence of two sulfate
1
(
6
2) except for the
−
6
2
1–6
1,
3
5
is the presence of a phenyl branch on the side chain, an unprecedented characteristic among
the 5-alk(en)ylresorcinols previously described in the literature [16]. Most of the known natural
products displaying an alk(en)ylresorcinol-based structure have been isolated from several families
of higher plants and typically feature a linear side chain with length ranging from C5 to C29, that is

Mar. Drugs 2017, 15, 344
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saturated or contains up to four double bonds, and in some instances bearing hydroxy, acetoxy or
keto groups. Moreover, although a few 5-alkyl- and 5-alkenylresorcinol derivatives described from
plants also contain a phenyl ring, this is always located at the end of the alk(en)yl chain [17,18].
Two species of brown macroalgae and several bacteria and fungi have also been shown to contain
simple 5-alkylresorcinols similar to those of terrestrial plants [16,19].
1
Table 3. H and 13C NMR data of schizol E (5) and schizol F (6) a,b
.
5
6
Position
δC, Type
δ , m (J in Hz)
H
δC, Type
δ , m (J in Hz)
H
1
2
3
4
5
6
154.2, C
113.3, CH
154.2, C
118.8, CH
145.6, C
118.8, CH
36.8, CH2
32.1, CH2
30.7, CH2
29.4, CH2
129.9, CH
141.6, C
32.6, CH2
22.8, CH2
14.2, CH3
144.8, C
154.0, C
113.5, CH
154.0, C
119.1, CH2
145.8, C
7.04, m
7.04, m
7.11, m
7.01, br s
7.04, m
2.64, t (7.7)
1.70, m
119.1, CH
36.8, CH2
32.5, CH2
29.3, CH2
35.1, CH2
51.44, CH
51.37, CH
37.6, CH2
22.8, CH2
14.6, CH3
149.0 c, C
149.1 c, C
123.2 d, CH
128.0 e, CH
127.9 e, CH
123.1 d, CH
7.01, br s
2.64, t (7.7)
1.69, m
1.54, m
1.71, m
2.95, br t (7.3)
2.97, br t (7.3)
1.65, m
1.50, m
0.98, t (7.4)
0
1
2
3
4
5
6
7
8
0
0
1.51, m
2.24, dt (7.5, 7.3)
5.62, t (7.3)
0
0
0
0
2.50, t (7.7)
1.32, m
0.86, t (7.4)
0
0
9
00
00
00
00
00
00
1
2
3
4
5
6
127.5, CH
129.1, CH
127.4, CH
129.1, CH
127.5, CH
7.30, dd (8.1, 1.2)
7.25, dd (8.1, 7.5)
7.16, m
7.25, dd (8.1, 7.5)
7.30, dd (8.1, 1.2)
7.02, m
7.11, m
7.11, m
7.02, m
b
a 1H at 600 MHz, 13C at 150 MHz; Assignments aided by COSY, HSQC, HMBC, and NOESY experiments;
c–e
Assignments marked with the same letter in the same column may be interchanged.
On the other hand, compound 2 and its sulfated derivatives 4 and 6 are characterized by containing
a 1,2-dihydrocyclobutabenzene moiety, which is extremely unusual among natural products. To the
best of our knowledge the only previous data regard homoisoflavanones of the scillascillin type,
first isolated from the plant Scilla scilloides Druce [20], and then from other species of the family
Hyacinthaceae [21–23].
Previous reports on the isolation of alkylresorcinols from marine invertebrates are scarce. The first
account described eleven simple alkyl- and alkenylresorcinols, with side chains from C13 to C21, from a
sponge of the genus Haliclona [24]. More recently, the study of the bryozoan Pentapora fascialis yielded
three compounds exhibiting two 5-alkenylresorcinol residues, which contain two double bonds and a
sulfate function on a C15 side chain, and are linked through a disulfide bridge [13].
Bryozoans are known to host communities of microorganisms and consequently, the hypothesis
that many natural products isolated from these invertebrates may be truly produced by associated
microorganisms was proposed soon after the first chemical studies of bryozoans [25]. This has been
demonstrated for the bryostatins, which are produced by “Candidatus Endobugula sertula”, a symbiotic
bacterium of the bryozoan Bugula neritina [12]. In other instances, such as the tambjamines of Sessibugula
translucens, the microbial origin is supported by their structural resemblance to a compound isolated
from the marine bacterium Pseudoalteromonas tunicata [26]. With regard to the origin of compounds 1–6
isolated from S. mamillata, the possibility that they are actually produced by microorganisms cannot
be disregarded. In this context, it is worth noting that recent reports have described the isolation of a
series of 2,5-dialkylresorcinol derivatives from marine cyanobacteria [27,28].

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2.2. Synthesis of Schizol A (1)
After the isolation of schizols A–F (
1
–
6), we turned our attention to the synthesis of these
metabolites. Herein, we describe a synthetic route for the obtention of schizol A (1). Extensive work of
synthesis of 5-alkylresorcinols from plants has been reported [16]. The critical step in the synthesis of
alkylresorcinols is always the formation of the C-C bond between the aromatic ring and the alkyl side
chain. For this purpose, several approaches including Suzuki [29] and Sonogashira [19] couplings or
Grignard reagents [30] have been employed. However, the most common procedure entails a Wittig
reaction using 3,5-dimethoxybenzaldehyde and the corresponding phosphonium ylide as starting
materials [31,32]. Differing from other syntheses of alkylresorcinols, the obtention of schizol A (1)
posed an additional key step, namely, the formation of the trisubstituted double bond.
Scheme 1 outlines a retrosynthetic protocol aimed at the target molecule. The natural product
1
can be prepared from key C-10/C-20 and C-50/C-60 double-bond disconnections such as Wittig and
Julia–Koscienski olefination, respectively, that will allow the formation of the carbon skeleton from the
commercially available compounds 3,5-dimethoxybenzaldehyde, butane-1,4-diol, and butyrophenone.
Scheme 1. Retrosynthetic analysis of schizol A (1).
Our synthetic strategy is shown in Scheme 2. The synthesis starts with the Wittig reaction between
3,5-dimethoxybenzaldehyde and the alkyl phosphonium salt
(see Supplementary Materials pages S7 and S8). This reaction led to a mixture of the isomeric alkene
compounds
80 (30:70) in 60% yield [33]. This isomer distribution was not of consequence, since
arrival at alcohol was founded on subsequent catalytic hydrogenation. Thus, the treatment of
80
with 10% Pd-C/H2 (1 atm) led almost quantitatively to the removal of the benzyl protecting group and
7, easily prepared from butane-1,4-diol
8/
9
8/
the concomitant alkene reduction, providing the alcohol
9 which accounted for the left-hand fragment
of the target molecule 1.
At this point, the coupling with butyrophenone would allow us to obtain the complete carbon
skeleton of was carried out by
. In particular, the installation of the double bond at C50,C60 of
a Julia–Kocienski olefination [34 35]. Thus, alcohol was treated with 1-phenyl-1H-tetrazol-5-thiol
in a Mitsunobu reaction yielding in good yield the corresponding sulfide 10, which was oxidized
using (NH4)6Mo7O24 4H2O and H2O2 30% to obtain the olefinating reagent 11 36 37]. With the
sulfone 11 in hand and some experimentation, we defined better conditions for the coupling with
butyrophenone, including the preformation of the deprotonated sulfone at
78 ◦C with LiHMDS in a
1
1
,
9
·
[ ,
−
mixture of THF/HMPA (9:1) and the subsequent addition of an excess of butyrophenone solution in
THF. Under these conditions an inseparable mixture (1:4) of the E-alkene 12 and its Z isomer 120 was
obtained. Although a better E-selectivity was expected for this reaction [38] our results are in line with
the data recently reported for Julia reactions using aromatic ketones [39]. Alternatively, the formation

Mar. Drugs 2017, 15, 344
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0
0
of the double bond at C5 ,C6 was accomplished by a Wittig reaction that yielded compound 120 as the
only isomer (Scheme 3).
◦
Scheme 2. Reagents and conditions: (
a
) LiHMDS, THF,
7
d
,
−
78 C, 2 h, 60%; (
b
) H2, Pd/C, AcOEt, 15 h,
96%; (
c
) PPh3, DEAD, HSPT, 0 ◦C, THF, 24 h, quant.; (
) (NH4)6Mo7
·
4H2O/H2O2, 30%, EtOH, 0 ◦C,
◦
20 h, 97%; (
24 h, 75%.
e) LiHMDS, THF/HMPA (9:1), butyrophenone,
−
78 C to rt, 24 h, 32%; (
f
) EtSNa, DMF,
∆
,
◦
Scheme 3. Reagents and conditions: (
a
) PPh3, CBr4, CH2Cl2, 0 C to rt, 2 h, 84%; (
b) PPh3/
∆
, overnight,
◦
99%; (c) LiHMDS, THF, butyrophenone, −78 C to rt, 2 h, 28%.
For the O-demethylation of compounds 12 and 120 we first attempted standard Lewis acid
conditions such as BBr3 in CH2Cl2 [40] or the treatment with an excess of Py HCl [41]. Both procedures
led, in moderate to good yields, to an E/Z mixture of the alkene at C-50,C-60 ( and 10) together with
·
1
some amounts of the E/Z alkenes at C-60,C-70, formed by acid-catalyzed double bond isomerization.
To prevent the isomerization of the double bond installed in compounds 12 and 120 we turned
our attention to demethylation procedures under basic conditions. In particular, the treatment of a
mixture 1:4 of the alkenes 12
/
120 with EtSNa in DMF [42] allowed the obtention of the demethylated
was
compounds 1
1
/
10 in 75% yield, which were separated by HPLC. The synthetic compound
identical in all respects to the natural metabolite schizol A (1).
Therefore, we have achieved the synthesis of the new natural product schizol A (1) and its Z
isomer 10. Key synthetic strategies include two C-C bond formation reactions to directly construct the
linear skeleton and the route can be easily applied to the synthesis of analogs with different substitution
pattern, chain length, or double bond stereochemistry. Furthermore, the synthesis herein described
does not need expensive starting materials or reagents nor harsh conditions for most steps.

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2.3. Antioxidant Activity
We have assessed the antioxidant properties of schizols A–F (
1–
6) by using the ABTS assay [43].
The results are shown in Table 4, which also includes the activity of Trolox, a synthetic analogue of
-tocopherol that was measured in the same assay. Schizols A ( ) and B ( ) exhibited higher activity
than the Trolox standard. Schizols C ( ) and D ( ) were less active, displaying activity 34% and 46%
α
1
2
3
4
that of the Trolox, respectively, and schizols E (5) and F (6) were inactive.
Table 4. Antioxidant activities of compounds 1–4 in the ABTS assay a.
Compound
Trolox
1
2
3
4
EC50 (µM ± SD, n = 3)
10.43 ± 0.06
6.24 ± 0.03
7.66 ± 0.24
30.25
±
0.10 22.59
±
0.53
ABTS: 2,20-azinobis(3-ethylbenzothiazoline-6-sulphonic acid).
a
Previous reports on the antioxidant activity of 5-alk(en)ylresorcinols have described that
5-n-heptadecyl- and 5-n-heptadecenylresorcinols prevent Fe2+-induced peroxidation of fatty acids
and phospholipids in liposomal membranes [44] while 5-n-pentadecylresorcinol protect erythrocyte
membranes against H2O2-induced oxidation [45]. In addition, a series of 5-alkylresorcinol homologues
possessing linear chains C15 to C23 have been shown to reduce the oxidative DNA damage induced by
H2O2 on HT29 cells and inhibit the copper-mediated oxidation of human low-density lipoproteins [46].
However, in DPPH radical scavenging assays these long chain 5-n-alkylresorcinols were less active than
ferulic acid [46] and showed EC50 values significantly higher than those of Trolox and tocopherols [47].
On the other hand, 5-(tridec-40,70-dienyl)resorcinol has been reported to exhibit significant activity
in the DPPH and in the lipid peroxidation assays [48]. Herein we provide data on the activity of
the isolated resorcinol derivatives in the ABTS assay, which as preliminary findings indicate the
antioxidant properties of schizols A (1) and B (2).
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Easton, MD, USA). UV-Vis data
were obtained on a Varian Cary 50Bio (Varian Inc., Palo Alto, CA, USA) or on a VWR UV-1600PC
(VWR, Radnor, PA, USA). IR spectra were recorded on a Perkin-Elmer FT-IR Spectrum Two spectrometer
(Perkin Elmer, Boston, MA, USA). 1H and 13C NMR spectra were recorded on an Agilent 600, 500 or
400 spectrometer (Agilent Technologies, Santa Clara, CA, USA) using CD3OD or CDCl3 as solvent.
Chemical shifts were referenced using the corresponding solvent signals (δH 3.30 and δC 49.0 for CD3OD;
δH 7.26 and δC 77.0 for CDCl3). COSY, HSQC, HMBC, and NOESY experiments were performed
using standard Agilent pulse sequences. High resolution mass spectra (HRMS) were obtained on a
Waters XEVO G2-S Mass spectrometer (Waters, Milford, MA, USA). All reactions were carried out under
an inert nitrogen atmosphere with dry solvents, using anhydrous conditions unless otherwise stated.
Dry Et2O, CH2Cl2, toluene, and THF were obtained by passing these solvents through activated alumina
columns under argon atmosphere. DMF and MeOH were purchased in anhydre grade. Reagents were
purchased at the highest commercial quality and used without further purification. Reaction yields refer to
chromatographically and spectroscopically (1H NMR) homogeneous material. Column chromatography
was carried out on Merck Silica gel 60 (70–230 mesh) (Merck, Darmstadt, Germany). SPE separations
were performed on Supelco DSC18 cartridges (500 mg/3 mL) (Supelco, Bellefonte, PA, USA).
HPLC separations were performed on a LaChrom-Hitachi apparatus using a differential refractometer
RI-71 or a UV detector L-7400 (Merck, Darmstadt, Germany) working at 254 nm. LiChrospher Si-60
(
(
250 × 10 mm, 10
Phenomenex, Torrance, CA, USA) columns were used for separations in normal phase (NP-HPLC).
m) (Merck, Darmstadt, Germany) and Kromasil 100-5C18
µ
m) (Merck, Darmstadt, Germany) and Luna Si (2) (250 × 4.6 mm, 5
µm)
LiChrospher 100 RP-18 (250 × 10 mm, 10
µ

Mar. Drugs 2017, 15, 344
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(250 × 4 mm, 5
µm) (Hichrom, Berkshire, UK) columns were used for separations in reversed phase mode
(RP-HPLC). All solvents were of HPLC grade. ABTS (2,20-azinobis(3-ethylbenzothiazoline-6-sulphonic
acid)) diammonium salt and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were
purchased from Sigma (St. Louis, MO, USA).
3.2. Biological Material
Specimens of Schizomavella mamillata, Hincks 1880 (class Gymnolaemata, order Cheilostomatida,
family Bitectiporidae) were collected off the coasts of Cadiz, Spain (36◦18’6.869” N, 6◦8’55.37” W)
and immediately frozen. A voucher specimen (B-Sm-0609) is deposited at the Marine Natural
Products Laboratory, Faculty of Marine and Environmental Sciences, University of Cadiz, Puerto Real
(Cadiz), Spain.
3.3. Extraction and Isolation
Frozen samples of S. mamillata were extracted with acetone/MeOH (1:1, v/v, 2.7 L) at room
temperature. The solvent was evaporated under reduced pressure and the aqueous residue was
extracted with Et2O (50 mL
dried over MgSO4, and evaporated under reduced pressure to yield 441 mg of extract. The n-butanol
×
4) and then with n-butanol (100 mL
×
2). The Et2O layers were combined,
layers were combined and evaporated under reduced pressure to yield 758 mg of extract. The Et2O
extract was subjected to silica gel column chromatography (18
×
2.5 cm) using as eluents hexanes/Et2O
(7:3, v/v, 70 mL), hexanes/Et2O (1:1, v/v, 150 mL), hexanes/Et2O (3:7, v/v, 50 mL), Et2O (50 mL),
CHCl3/MeOH (8:2, v/v, 50 mL), CHCl3/MeOH (7:3, v/v, 75 mL), CHCl3/MeOH (1:1, v/v, 100 mL),
and finally MeOH (50 mL). The n-butanol extract was also subjected to column chromatography using
as eluents CHCl3/MeOH (85:15, v/v, 300 mL), CHCl3/MeOH (8:2, v/v, 200 mL), CHCl3/MeOH (7:3,
v/v, 300 mL), CHCl3/MeOH (1:1, v/v, 200 mL), and finally MeOH (200 mL). The fractions of the
Et2O extract that had been eluted with hexanes/Et2O (3:7, v/v) and with Et2O were combined and
evaporated to dryness yielding a mixture (119.1 mg) that was suspended in MeOH/H2O (9:1, v/v,
2 mL) and transferred onto two SPE-C18 cartridges preconditioned with MeOH/H2O (9:1, v/v, 1 mL).
Each cartridge was eluted with 10 mL of MeOH/H2O (9:1, v/v). The obtained solution was evaporated
to dryness under reduced pressure and subjected again to separation onto two SPE-C18 cartridges as
described above using MeOH/H2O (8:2, v/v). The resulting solution was evaporated under reduced
pressure yielding a mixture (43.3 mg) that was subjected to repeated RP-HPLC separations using
as eluents MeOH/H2O (8:2, v/v) and CH3CN/H2O (55:45, v/v), to yield compounds
1 (9.0 mg)
and (6.1 mg). The fractions of the Et2O extract and of the n-butanol extract that had been eluted
2
with CHCl3/MeOH (7:3, v/v) (58.8 mg and 131.2 mg, respectively) were combined, dissolved in
MeOH/H2O (7:3, v/v, 3 mL), and transferred onto three SPE-C18 cartridges preconditioned with
MeOH/H2O (7:3, v/v, 1 mL). Each cartridge was eluted with 10 mL of MeOH/H2O (7:3, v/v).
After evaporation of the solvent, the resulting mixture (159.8 mg) was subjected to repeated RP-HPLC
using as eluents MeOH/H2O + 0.1% HCOOH (7:3, v/v), MeOH/H2O (6:4, v/v), and CH3CN/H2O
(4:6, v/v), to yield compound
3 (17.6 mg), compound 4 (18.3 mg) and a mixture of compounds 5 and 6
(28.6 mg). This mixture proved to be difficult to separate and after NP-HPLC using CHCl3/MeOH
(8:2, v/v) and repeated RP-HPLC using MeOH/H2O (6:4, v/v) some amounts of pure compounds
(1.0 mg) and 6 (3.8 mg) were obtained.
5
Schizol A (
1
): colorless oil; UV (MeOH)
λ
(log
ε
) 203 (4.59), 246 (3.56) nm; IR (film)
υ
3365, 2929,
max
max
1599, 1462, 1159, 697 cm−1; 1H NMR (CD3OD, 600 MHz) Table 1; 13C NMR (CD3OD, 150 MHz) Table 1;
HRESIMS m/z 309.1859 [M − H]− (calcd. for C21H25O2, 309.1855).
Schizol B (
2
): colorless oil; [α]2D5 +32.0 (c 0.07, MeOH); UV (MeOH)
λ
max
(log ε) 204 (4.67), 266 (3.41),
272 (3.43) nm; IR (film)
Table 1; 13C NMR (CD3OD, 150 MHz) Table 1; HRESIMS m/z 309.1861 [M
C21H25O2, 309.1855).
υ
max 3360, 2925, 1598, 1454, 1149, 995, 741 cm−1; 1H NMR (CD3OD, 600 MHz)
−
H]− (calcd. for

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Schizol C (
3
): colorless oil; UV (MeOH)
λ
(log
ε
) 204 (4.78), 243 (3.86) nm; IR (film)
υ
3428,
max
max
2929, 1590, 1457, 1239, 1053, 988, 697 cm−1 1H NMR (CD3OD, 600 MHz) Table 2; 13C NMR
;
(CD3OD, 150 MHz) Table 2; HRESIMS m/z 389.1429 [M − H]− (calcd. for C21H25O5S, 389.1423).
Schizol D (
265 (3.32), 272 (3.37) nm; IR (film)
(CD3OD, 600 MHz) Table 2; 13C NMR (CD3OD, 150 MHz) Table 2; HRESIMS m/z 389.1424 [M
4
): colorless oil; [α]2D5 +23.7 (c 0.09, MeOH); UV (MeOH)
λ
max
(log ε) 202 (4.52),
υ
3443, 2925, 1591, 1455, 1240, 1054, 989, 740 cm−1; 1H NMR
max
−
H]−
(calcd. for C21H25O5S, 389.1423).
Schizol E (
5
): colorless oil; UV (MeOH)
λ
(log
ε
) 202 (4.68), 245 (3.19) nm; IR (film)
υ
max
max
3445, 2927, 1614, 1591, 1454, 1236, 1058, 981 cm−1
;
1H NMR (CD3OD, 600 MHz) Table 3;
13C NMR (CD3OD, 150 MHz) Table 3; HRESIMS m/z 491.0794 [M
C21H24O8S2Na, 491.0810).
−
2H + Na]− (calcd. for
Schizol F (
6
): colorless oil; [α]2D5 +16.2 (c 0.09, MeOH); UV (MeOH)
λ
(log ε) 202 (4.68) nm;
max
1
IR (film)
υ
3445, 2925, 1615, 1590, 1455, 1236, 1061, 981 cm−1; H NMR (CD3OD, 600 MHz)
max
Table 3; 13C NMR (CD3OD, 150 MHz) Table 3; HRESIMS m/z 491.0814 [M
−
2H + Na]− (calcd. for
C21H24O8S2Na, 491.0810).
3.4. Hydrolysis of Schizol F (6)
◦
Compound
6
(2.8 mg) was dissolved in HC1 1 M (1 mL) and heated at 90 C for 5 min.
After cooling at rt, the pH was adjusted to 5 with NaOH 1 M and the solution was extracted with
EtOAc (2 2 mL). The organic layers were combined, washed with brine (2 mL), dried over anhydrous
×
MgSO4 and the solvent evaporated. The residue was purified by HPLC (MeOH/H2O 8:2, v/v) to yield
compound 2 (1.0 mg).
3.5. Synthesis of Schizol A (1)
3.5.1. Synthesis of Compounds 8/80
A flame dried flask equipped with a stir bar under nitrogen atmosphere was charged with a
solution of 560 mg of
7
(1.11 mmol) in 5 mL of dry THF and 1.5 mL of LiN(SiMe3)2 1 M in THF
◦
under an atmosphere of nitrogen. After 300 of stirring at rt, the mixture was cooled to
−
78 C
and 1.0 mmol of 3,5-dimethoxybenzaldehyde in 1 mL of THF was added dropwise. After 2 h at
rt the reaction was quenched with 5 mL of a saturated solution of NH4Cl and extracted with EtOAc
(
3 × 5 mL). The organic phase was dried over anhydrous MgSO4 and the solvent concentrated in
vacuo yielding an oily residue that was purified by CC (hexanes/Et2O 9:1) to yield compounds 8/80
(187 mg, 0.60 mmol, 60%). For the characterization of these alkenes, a mixture of 8/80 was subjected to
HPLC (hexanes/EtOAc 98:2).
Compound
8
(isomer E): IR (film)
υ
3030, 2936, 2839, 1591, 1455, 1204, 1151, 736, 697 cm−1
;
max
1H NMR (400 MHz, CDCl3)
δ
7.36–7.26 (m, 5H, H300-H700), 6.50 (d, J = 2.4 Hz, 2H, H20 and H60),
6.33 (d, J = 15.8 Hz, 1H, H1), 6.21 (dt, J = 15.8, 6.7 Hz, 1H, H2), 6.29 (t, J = 2.2 Hz, 1H, H40), 4.52 (s, 2H,
H100), 3.80 (s, 6H, -OMe), 3.53 (t, J = 6.4 Hz, 2H, H5), 2.32 (m, 2H, H3), 1.81 (quint., J = 6.5 Hz, 2H,
H4); 13C NMR (see Supplementary Materials page S9); HRESIMS m/z 313.1805 [M + H]+ (calcd. for
C20H25O3, 313.1804).
Compound 80 (isomer Z): IR (film)
υ
3004, 2935, 2854, 1590, 1454, 1204, 1155, 736, 697 cm−1
;
max
1H NMR (400 MHz, CDCl3) 7.33 (m, 2H, H400 and H600), 7.30 (m, 2H, H300 and H700), 7.28 (m, 1H,
δ
H500), 6.46 (d, J = 2.1 Hz, 2H, H20 and H60), 6.39 (d, J = 11.7 Hz, 1H, H1), 6.38 (t, J = 2.1 Hz, 1H, H40),
5.68 (dt, J = 11.7, 7.3 Hz, 1H, H2), 4.49 (s, 2H, H100), 3.78 (s, 6H, -OMe), 3.52 (t, J = 6.5 Hz, 2H, H5),
2.47 (m, 2H, H3), 1.79 (quint., J = 6.7 Hz, 2H, H4); 13C NMR (see Supplementary Materials page S9);
HRESIMS m/z 313.1812 [M + H]+ (calcd. for C20H25O3, 313.1804).

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3.5.2. Synthesis of Compound 9
A total of 62 mg of Pd/C was added to alkenes 8/80 (2 mmol) dissolved in 5 mL of EtOAc.
The reaction mixture was subjected to a hydrogen atmosphere (1 atm) for 15 h. The catalyst was
removed by filtration and the resulting solution was taken to dryness yielding 430 mg (1.92 mmol,
96%) of compound 9 as a colorless oil.
Compound
(400 MHz, CDCl3)
9
: IR (film)
6.33 (d, J = 2.2 Hz, 2H, H20 and H60), 6.29 (t, J = 2.2 Hz, 1H, H40), 3.76 (s, 6H,
υ
max 3359, 3026, 2935, 2858, 1596, 1462, 1205, 1150, 1057, 829, 695 cm−1; 1H NMR
δ
-OMe), 3.61 (t, J = 6.6 Hz, 2H, H1), 2.55 (t, J = 7.7 Hz, 2H, H5), 1.63 (m, 2H, H4), 1.58 (m, 2H, H2),
1.39 (m, 2H, H3); 13C NMR (see Supplementary Materials page S9); HRESIMS m/z 225.1502 [M + H]+
(calcd. for C13H21O3: 225.1491).
3.5.3. Synthesis of Compound 10
To 200 mg of
9 (0,89 mmol) in 10 mL of dry THF and under an inert atmosphere, 280 mg of PPh3
(1.07 mmol) and 239 mg of 1-phenyl-1H-tetrazol-5-thiol (1.34 mmol) were added at rt. The reaction
◦
mixture was cooled to 0 C with an ice water bath and diethyl azodicarboxylate (DEAD) 40% in toluene
(520
µL, 1.16 mmol) was added. After 24h at rt, the solvent was evaporated and the residue purified
by CC (hexanes/EtOAc (9:1 → 75:25)), yielding 10 quantitatively.
1
Compound 10: IR (film)
υ
max
2932, 2855, 1594, 1460, 1427, 1203, 1149, 760, 693 cm−1; H NMR
(400 MHz, CDCl3)
δ
7.58–7.52 (m, 5H, Ph), 6.33 (d, J = 2.3 Hz, 2H, H20 and H60), 6.29 (t, J = 2.3 Hz, 1H,
H40), 3.77 (s, 6H, -OMe), 3.38 (t, J = 7.4 Hz, 2H, H1), 2.56 (t, J = 7.6 Hz, 2H, H5), 1.86 (quint, J = 7.5 Hz
,
2H, H2), 1.66 (m, 2H, H4), 1.49 (m, 2H, H3); 13C NMR (see Supplementary Materials page S9);
HRESIMS m/z 385.1703 [M + H]+ (calcd. for C20H25SO2N4, 385.1698).
3.5.4. Synthesis of Compound 11
◦
To a solution of 10 (277 mg, 0.72 mmol) in 10 mL of EtOH at 0 C a mixture of (NH4)6Mo7O24
·
4H2O
(89 mg, 0.072 mmol) and H2O2 30% (0.85 mL, 8.5 mmol) was added. After stirring for 20 h at rt,
the reaction mixture was quenched by addition of H2O (10 mL) and extracted with CH2Cl2 (3 × 20 mL).
The organic layers were separated, dried over anhydrous MgSO4, and the solvent evaporated at
reduced pressure to yield 292 mg (0.70 mmoles, 97%) of 11.
1
Compound 11: IR (film)
υ
2925, 2853, 1594, 1460, 1340, 1150, 1050, 762, 689 cm−1; H NMR
max
(400 MHz, CDCl3)
δ
7.70–7.57 (m, 5H, Ph), 6.32 (d, J = 2.3 Hz, 2H, H20 and H60), 6.31 (t, J = 2.3 Hz, 1H,
H4 ), 3.78 (s, 6H, -OMe), 3.72 (m, 2H, H1), 2.57 (t, J = 7.6 Hz, 2H, H5), 1.98 (m, 2H, H2), 1.66 (m, 2H, H4),
1.53 (m, 2H, H3); 13C NMR (see Supplementary Materials page S9); HRESIMS m/z 417.1604 [M + H]+
(calcd. for C20H25SO4N4: 417.1597).
0
3.5.5. Synthesis of Compounds 12/120 (Julia–Kocienski Reaction)
A total of 95 mg of 11 (0.228 mmol) was disolved in 3 mL of THF/HMPA 9:1 under nitrogen
atmosphere. The solution was cooled to
added. After 30’ at
78 ◦C, butyrophenone (50
reaction was stirred for another 30’ at this temperature. Then, the reaction was stirred for 24 h at rt,
quenched with NH4Cl (10 mL), and extracted with EtOAc (3 20 mL). The combined organic phases
were washed with brine (2 20 mL) and dried over anhydrous MgSO4. The solvent was evaporated
−
78 ◦C and LiHMDS 1M in THF (350
µ
L, 0.350 mmol) was
−
µL, 0.350 mmol) in 1 mL of THF was added and the
×
×
under reduced pressure and the residue purified by CC (hexanes/Et2O, 98:2) to yield 25 mg of an
inseparable mixture of 12/120 (32%) in a 1:4 ratio that was used in the next reaction step.
3.5.6. Synthesis of Compounds 1/10
To 75 mg of EtSNa (0.092 mmol) under nitrogen atmosphere and stirring, a solution of 12
(0.089 mmol) in 1 mL of DMF was added. The reaction mixture was refluxed for 24 h. After cooling to
/
120

Mar. Drugs 2017, 15, 344
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0 ◦C, a 5% aqueous HCl solution was added, the aqueous layer was extracted with EtOAc, and the
combined organic layers were dried over anhydrous MgSO4 and evaporated in vacuo. The residue
was purified by CC (hexanes/Et2O, 3:7) to yield 21 mg of a mixture of 1/10 (75%) that was subjected
to HPLC in reversed phase mode (CH3CN/H2O, 55:45) to yield pure
1 (7 mg, 0.023 mmol, 25%) and
10 (14 mg, 0.045 mmol, 49%). Compound 1 was identical in all respects to the natural schizol A.
Compound 10: IR (film) 3348, 3077, 2956, 2929, 2857, 1598, 1492, 1462, 1303, 1150, 700 cm−1
1H NMR (600 MHz, CDCl3)
υ
;
max
δ
7.30 (brt, J = 7.6 Hz, 2H, H300 and H500), 7.09 (dd, J = 8.1, 1.2 Hz, 2H,
H2000 and H600), 7.21 (tt, J = 7.4, 1.3 Hz, 1H, H400), 6.06 (brs, 3H, H2, H4 and H6), 5.41 (t, J = 7.4 Hz, 1H,
0
0
0
H5 ), 2.33 (t, J = 7.7, 2H, H1 ), 2.30 (t, J = 7.5, 2H, H7 ), 1.92 (td, J = 7.4, 7.4, 2H, H4 ), 1.47 (quint, J = 7.6
,
2H, H20), 1.33 (quint, J = 7.5, 2H, H30), 1.28 (sext, J = 7.4, 2H, H80), 0.85 (t, J = 7.4 Hz, 3H, H90);
13C NMR (see Supplementary Materials page S10); HRESIMS m/z 311.2018 [M + H]+ (calcd. for
C21H27O2, 311.2011).
3.5.7. Synthesis of Compound 13
To a solution of 1.12 g of
9 (5.0 mmol) and 1.57 g of PPh3 (6.0 mmol) in 15 mL of CH2Cl2 at
0 ◦C, 1.82 g of CBr4 (15.5 mmol) was added. The resulting mixture was stirred at rt for 2 h and then
concentrated under reduced pressure to give a residue that was purified by CC (hexanes/Et2O 9:1) to
yield Compound 13 (1.19 g, 4.20 mmol, 84%) as a colorless oil.
1
Compound 13: H NMR (400 MHz, CDCl3)
δ
6.35 (d, J = 2.3 Hz, 2H, H20 and H60), 6.31 (t, J = 2.3 Hz
,
1H, H40), 3.79 (s, 6H, -OMe), 3.41 (t, J = 7.0 Hz, 2H, H1), 2.58 (t, J = 7.3 Hz, 2H, H5), 1.89 (m, 2H,
H2), 1.65 (m, 2H, H4), 1.49 (m, 2H, H3); 13C NMR (see Supplementary Materials page S10);
HRESIMS m/z 287.0680 [M + H]+ (calcd. for C13H20O279Br, 287.06474), m/z 289.0645 [M + H]+
(calcd. for C13H20O281Br, 289.0626).
3.5.8. Synthesis of Compound 14
A total of 288 mg of 13 (1.00 mmol) and 262 mg of PPh3 (1.00 mmol) was heated overnight in an
oven at 100 ◦C yielding 542 mg of 14 (0.99 mmol, 99%) as an amorphous white solid.
Compound 14: δ
1H NMR (400 MHz, CDCl3) 7.79 (m, 6H, H200 and H600), 7.75 (m, 3H, H400),
7.66 (m, 6H, H300 and H500), 6.23 (m, 2H, H20 and H60), 6.22 (m, 1H, H40), 3.71 (s, 6H, OMe),
3.70 (m, 2H, H1), 2.46 (t, J = 7.3 Hz, 2H, H5), 1.64 (m, 2H, H3), 1.58 (m, 2H, H2), 1.56 (m, 2H, H4);
13C NMR (see Supplementary Materials page S10); HRESIMS m/z 469.2307 [M
C31H34O2P, 469.2296).
−
Br]+ (calcd. for
3.5.9. Synthesis of Compound 120 (Wittig Reaction)
A flame dried flask equiped with a stir bar under nitrogen atmosphere was charged with a
suspension of 14 (1.0 mmol) in 5 mL of dry THF and 1.5 mL of LiN(SiMe3)2 1M in THF under an
atmosphere of nitrogen. After 30’ of stirring at rt, the mixture was cooled to
of butyrophenone in 1 mL of THF was added dropwise. After 2 h at rt, the reaction was quenched
with 5 mL of a saturated solution of NH4Cl and extracted with EtOAc (3 5 mL). The organic phase
was dried under anhydrous MgSO4 and the solvent concentrated in vacuo yielding an oily residue
−
78 ◦C and 1.0 mmol
×
that was purified by CC (hexanes/Et2O 95:5
0.28 mmol, 28%).
→
1:1) to yield compound 120 as a colorless oil (94 mg,
Compound 120: IR (film)
υ
max 3056, 2956, 2933, 2859, 1596, 1461, 1205, 1150, 830, 701 cm−1; 1H NMR
7.33 (tt, J = 7.3, 1.2 Hz, 2H, H300 and H500), 7.24 (tt, J = 7.3, 1.2 Hz, 1H, H400),
(400 MHz, CDCl3)
δ
7.14 (m, 2H, H200 and H600), 6.32 (d, J = 1.8 Hz, 2H, H4 and H6), 6.31 (t, J = 1.8 Hz, 1H, H2),
5.43 (t, J = 7.3 Hz, 1H, H50), 3.78 (s, 6H, -OCH3), 2.49 (0t, J = 7.6 Hz, 2H, H10), 2.32 (t, J = 7.3 Hz, 2H,
0
0
0
0
H7 ),1.98 (m, 2H, H4 ), 1.57 (m, 2H, H2 ), 1.38 (m, 2H, H3 ), 1.33 (sext, J = 7.3, 2H, H8 ), 0.88 (t, J = 7.3 Hz
,

Mar. Drugs 2017, 15, 344
14 of 17
3H, H90); 13C NMR (see Supplementary Materials page S10); HRESIMS m/z 339.2326 [M + H]+
(calcd. for C23H31O2, 339.2324).
3.6. Antioxidant Assay
Antioxidant activity was determined by the ABTS free radical decolorization assay developed
by Re et al. [42], with slight modifications. In brief, a solution of the radical cation ABTS+• was
prepared by mixing (1:1, v/v) a solution of ABTS diammonium salt (7 mM) and a solution of potassium
persulfate (2.45 mM) in H2O. The mixture was kept in the dark at room temperature for 12–18 h
before use. Then the solution was diluted with EtOH to an absorbance of 0.70
solutions of Trolox (standard) and of the tested compounds were prepared in EtOH. For the assay,
100 L of the Trolox solution or 100 L of tested compound solution were mixed with 2 mL of the
ABTS+• solution and the absorbance at 734 nm was measured after 6 min. Controls were prepared
by adding 100
L of EtOH to 2 mL of ABTS+• solution. All of the determinations were carried
±
0.02 at 734 nm. Stock
µ
µ
µ
out in triplicate. The percentage of inhibition of the absorbance was calculated by the following
equation: % Inhibition = [(A0 − A1)/A0] × 100, where A0 expresses the absorbance of control and A1
the absorbance of the tested compound.
4. Conclusions
Marine organisms continue to be a valuable source of bioactive natural products with unique
structural features. In this study of the bryozoan S. mamillata we have isolated two new types of
alkylresorcinol-derived metabolites, characterized by possessing either a phenyl branch on the side
chain (compounds 1, 3, and 5) or a 1,2-dihydrocyclobutabenzene moiety (compounds 2, 4, and 6),
the latter structural feature being very rarely found among natural products. Within each group,
the isolated compounds differed by the sulfation degree of the phenolic hydroxy groups. We have
achieved the synthesis of schizol A (
olefination reactions to directly construct the carbon skeleton. Schizols A (
activity in preliminary antioxidant assays and further studies in this and other bioactivity fields would
be of interest. In this context, the synthetic route developed for schizol A ( ) is flexible enough to
1
) and its Z isomer 10 by a synthetic strategy that includes two
1
) and B (2) have shown
1
access analogs with different substitution patterns, chain lengths, or double bond stereochemistry that
will allow us to advance the study of the biological properties of this type of phenolic compounds.
Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/15/11/344/s1:
1
1
1
Figure S1: H and 13C NMR spectra of
1
; Figure S2: H and 13C NMR spectra of
2
; Figure S3: H and 13C NMR
1
1
1
spectra of
13C NMR spectra of
data of 8, 8’, 9, 10 and 11; Page S10: 13C NMR data of 1’, 12’, 13, and 14.
3
; Figure S4: H and 13C NMR spectra of
4
; Figure S5: H and 13C NMR spectra of
5
; Figure S6: H and
6
; Pages S7 and S8: Full experimental details for the chemical synthesis of 7
; Page S9: 13C NMR
Acknowledgments: This research was supported by a grant from Junta de Andalucía, Spain (FQM-169).
J.J.P. acknowledges an FPU fellowship from the Ministerio de Educacion (Spain).
Author Contributions: M.J.O. and E.Z. conceived and designed the experiments; M.J.O., J.J.P., C.d.l.R. and E.Z.
performed the experiments; M.J.O. and E.Z. analyzed the data and wrote the paper, which was revised and
approved by all the authors.
Conflicts of Interest: The authors declare no conflict of interest.
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