Exploring the Sponge Consortium Plakortis symbiotica-Xestospongia deweerdtae as a Potential Source of Antimicrobial Compounds and Probing the Pharmacophore for Antituberculosis Activity of Smenothiazole A by Diverted Total Synthesis

Article abstract of DOI:10.1021/acs.jnatprod.7b00300

Fractionation of the bioactive CHCl₃-MeOH (1:1) extracts obtained from two collections of the sponge consortium Plakortis symbiotica-Xestospongia deweerdtae from Puerto Rico provided two new plakinidone analogues, designated as plakinidone B (2) and plakinidone C (3), as well as the known plakinidone (1), plakortolide F (4), and smenothiazole A (5). The structures of 1-5 were characterized on the basis of 1D and 2D NMR spectroscopic, IR, UV, and HRMS analysis. The absolute configurations of plakinidones 2 and 3 were established through chemical correlation methods, VCD/ECD experiments, and spectroscopic data comparisons. When assayed in vitro against Mycobacterium tuberculosis H₃₇Rv, none of the plakinidones 1-3 displayed significant activity, whereas smenothiazole A (5) was the most active compound, exhibiting an MIC value of 4.1 μg/mL. Synthesis and subsequent biological screening of 8, a dechlorinated version of smenothiazole A, revealed that the chlorine atom in 5 is indispensable for anti-TB activity.

Full text of DOI:10.1021/acs.jnatprod.7b00300

Article
pubs.acs.org/jnp
Exploring the Sponge Consortium Plakortis symbiotica−Xestospongia
deweerdtae as a Potential Source of Antimicrobial Compounds and
Probing the Pharmacophore for Antituberculosis Activity of
Smenothiazole A by Diverted Total Synthesis
Carlos Jimenez-Romero,† Joanna E. Rode,‡ Yeiry M. Perez,† Scott G. Franzblau,§
́
́
and Abimael D. Rodríguez*,†
†Molecular Sciences Research Center, University of Puerto Rico, 1390 Ponce de Leon
́
Avenue, San Juan, Puerto Rico 00926, United
States
‡Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
§Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States
S
* Supporting Information
ABSTRACT: Fractionation of the bioactive CHCl3−MeOH
(1:1) extracts obtained from two collections of the sponge
consortium Plakortis symbiotica−Xestospongia deweerdtae from
Puerto Rico provided two new plakinidone analogues,
designated as plakinidone B (2) and plakinidone C (3), as
well as the known plakinidone (1), plakortolide F (4), and
smenothiazole A (5). The structures of 1−5 were charac-
terized on the basis of 1D and 2D NMR spectroscopic, IR, UV,
and HRMS analysis. The absolute configurations of
plakinidones 2 and 3 were established through chemical
correlation methods, VCD/ECD experiments, and spectro-
scopic data comparisons. When assayed in vitro against
Mycobacterium tuberculosis H37Rv, none of the plakinidones 1−3 displayed significant activity, whereas smenothiazole A (5)
was the most active compound, exhibiting an MIC value of 4.1 μg/mL. Synthesis and subsequent biological screening of 8, a
dechlorinated version of smenothiazole A, revealed that the chlorine atom in 5 is indispensable for anti-TB activity.
with CHCl3−MeOH. After solvent removal, the dark gum
obtained was suspended in H2O and extracted exhaustively
with n-hexane and CHCl3. In 2010, we reported on the results
of our chemical investigation of the n-hexane extract after
further fractionation, which led to the discovery of two strongly
biologically active five-membered-ring polyketide endoperox-
ides, each containing a conjugated triene along an unbranched
C16 alkyl side chain.2b As part of our ongoing effort to identify
novel metabolites from this sponge−sponge association,
hitherto we report the isolation of two new phenyl polyketide
lactones, plakinidones B (2) and C (3), along with the
previously described plakinidone (1)5,6 and plakortolide F
(4),2b,7 from the CHCl3 extract. Preliminary NMR (1H and
13C) and EIMS analyses of these lactones immediately revealed
their close structural relatedness and in particular the presence
in 1−4 of a terminal hydroxylated phenyl ring and of a five-
membered tetronic acid ring (i.e., 3-methyl-4-hydroxy-2(5H)-
furanone) in metabolites 1−3.8
or almost a decade, two groups of marine natural products
Fchemists have gradually scrutinized the chemical compo-
sition of the symbiotic two-sponge association Plakortis
halichondrioides−Xestospongia deweerdtae.1,2 These still-ongoing
investigations have led to the discovery of a variety of
structurally interesting cyclic polyketide peroxides with diverse
biological activities.1−3 Remarkably, by using a combination of
molecular and morphological data, Vicente and co-workers
recently demonstrated that the Plakortis species involved in this
unusual epizoic symbiosis is not P. halichondrioides but a new
sponge species, namely, Plakortis symbiotica.4
During a 2006 expeditionary voyage to Mona Island, located
in a strait between the Dominican Republic and Puerto Rico,
samples of a sponge consortium constituted by Plakortis
symbiotica (Vicente, Zea & Hill, 2016) and Xestospongia
deweerdtae (Lehnert & van Soest, 1999) specimens were
collected by members of our research team at a depth of 90−
100 ft. The extract of a small portion of the consortium
containing mainly P. symbiotica was found to be strongly toxic
to a series of human tumor cells and two pathogenic microbes
(Mycobacterium tuberculosis and Plasmodium falciparum); thus
the sponge was targeted for chemical analysis and extracted
Received: April 7, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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raphy afforded 926 mg (yield 0.23%) of plakinidone (1)5,6 and
110 mg (yield 0.028%) of plakortolide F (4)2b,7 as major
isolates whose IR, EIMS, [α]D, and 1H and 13C NMR data were
in agreement with published values, whereas plakinidones B (2)
(60 mg, yield 0.015%) and C (3) (16 mg, yield 0.004%) were
obtained in lower amounts. Interestingly, plakinidones B and C
are only the second and third members of the plakinidone
family of compounds to be found since the discovery of 1 in
1991 by Kushlan and Faulkner.5
The minor compound, plakinidone B (2), was obtained as a
yellowish optically active semisolid, [α]20 −10.6 (c 1.0,
D
MeOH). The molecular formula C24H36O4 was determined
by HRESIMS, from the peak at m/z 389.2694 [M + H]+. The
structural homology between plakinidone (1) and plakinidone
B (2) was evident from the HRMS data, which showed that
their molecular masses corresponded to molecular formulas
that differ from each other by a CH2 group. The IR absorption
bands at 3600−3100 and 1724 cm−1 suggested the presence in
2 of hydroxy and ester carbonyl groups, respectively. In the
NMR spectra, two singlet peaks at δH 1.39 and 1.65 (Table 1)
together with two peaks at δC 23.7 and 5.9 implied the presence
of two methyl groups. The only other clearly discernible proton
signals were attributable to a p-hydroxyphenyl group [δH 6.95
(d, J = 8.4 Hz) and 6.67 (d, J = 8.4 Hz)]. In addition, three
resolved sets of methylene protons at δH 2.48 (t, J = 7.4 Hz),
1.69 (t, J = 7.9 Hz), and 1.54 (m) together with nine
overlapped pairs of methylene protons at δH 1.28−1.20 (br m)
suggested that a straight dodecyl chain connected the terminal
ring subunits.
Furthermore, a bioactivity-directed fractionation based on
anti-TB activity of the aforementioned sponge conglomerate
collected in 2011 yielded the known hybrid peptide/polyketide
smenothiazole A (5). This compound, isolated recently by the
Costantino group from the Caribbean sponge Smenospongia
aurea, was fully characterized by 1D and 2D NMR
spectroscopic and HRMS analysis.9
RESULTS AND DISCUSSION
■
Extraction of the 2006 sponge collection with CHCl3−MeOH
followed by partitioning of the concentrated extract as
described above afforded the CHCl3 extract (8.0 g) used in
this investigation. In vitro antituberculosis screening of this
extract against Mycobacterium tuberculosis (Mtb) H37Rv at one
concentration (64 μg/mL) exhibited >90% inhibition.
Successive reversed- and normal-phase silica gel chromatog-
The 13C NMR spectrum of 2 (Table 1) showed 24 signals,
including those for an ester carbonyl [δC 177.8 (C)], eight
1
Table 1. 13C NMR and H NMR for Plakinidone B (2) and Plakinidone C (3) in CD3OD
2
3
a
b
a
b
position
δC, type
δH, mult (J in Hz)
position
δC, type
δH, mult (J in Hz)
1, 5
2, 4
3
130.2 (2 × CH)
116.0 (2 × CH)
156.2, C
6.95, d (8.4)
6.67, d (8.4)
1
116.5, CH
146.0, C
6.58, d (2.0)
2
3
144.0, C
6
134.9, C
4
116.2, CH
120.6, CH
135.7, C
6.64, d (8.0)
7
36.0, CH2
33.0, CH2
30.6, CH2
30.6, CH2
30.6, CH2
30.6, CH2
30.6, CH2
30.6, CH2
30.6, CH2
30.2, CH2
24.1, CH2
37.5, CH2
85.0, C
2.48, t (7.4)
5
6.46, dd (8.0, 2.0)
8
1.54, m
6
9
1.28−1.20, br m
1.28−1.20, br m
1.28−1.20, br m
1.28−1.20, br m
1.28−1.20, br m
1.28−1.20, br m
1.28−1.20, br m
1.28−1.20, br m
1.28−1.20, br m
1.69, t (7.9)
7
36.3, CH2
33.2, CH2
27.6, CH2
37.9, CH2
33.8, CH2
37.9, CH2
27.9, CH2
30.8, CH2
24.1, CH2
37.4, CH2
85.0, C
2.43, t (7.6)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
8
1.51, m
9
1.25−1.20, br m
1.28, m/1.07, m
1.36, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1.28, m/1.07, m
1.25−1.20, br m
1.25−1.20, br m
1.25−1.20, br m/1.10, br m
1.72, m
180.2, C
179.8, C
96.0, C
96.2, C
177.8, C
177.6, C
5.9, CH3
1.65, s
1.39, s
5.9, CH3
23.7, CH3
20.1, CH3
1.64, s
23.7, CH3
1.39, s
0.82, d (6.5)
a
b
Data (δ) measured at 125 MHz; CH3, CH2, CH, and C multiplicities were determined by HSQC and DEPT-135 NMR experiments. Data (δ)
measured at 500 MHz. J values are omitted if the signals overlapped as multiplets. The overlapped signals were assigned from HSQC and HMBC
spectra without designating multiplicity.
B
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olefinic carbons [δC 180.2 (C), 156.2 (C), 134.9 (C), 130.2 (2
× CH), 116.0 (2 × CH), and 96.0 (C)], and one esterified
carbon [δC 85.0 (C)]. These carbon signals were subsequently
assigned to a p-hydroxylphenyl ring and a tetronic acid moiety
through COSY, HSQC, and HMBC experiments (Figure 1).
the nearly identical specific rotation data between compounds 1
and 2 ([α]20 −13.1 (c 0.61, MeOH) versus [α]20 −10.6 (c
1.0, MeOH), respectively) suggest that plakinidone B possesses
the same absolute configuration at its only stereogenic center as
plakinidone.6b,12 Therefore, we propose that natural plakini-
done B is accurately represented by structure 2.
D
D
The least abundant compound, plakinidone C (3), was
isolated as a yellowish oil. Its molecular formula was C23H34O5
based on the HREIMS data, which has one oxygen more than
that of 1. The UV spectrum showed similar absorptions to
plakinidones at 226 and 256 nm. Comparison of its NMR
spectra with those of plakinidone (1) indicated that the 1H and
13C NMR data of 3 (Table 1) were strikingly similar except for
the substitution pattern of the aromatic ring.5 The signal for H-
2 in plakinidone [δH 6.67 (d, J = 8.4 Hz)] was no longer
present, and δC at C-2 (116.0) was deshielded to δC 146.0,
leading to the assignment of a C-2 hydroxy group. The
molecular formula and the key HMBC correlations of H-1 to
C-2/C-3/C-5/C-6 and H-4 to C-2/C-3/C-5/C-6 also con-
firmed the structure of 3. The configuration of (11S,17R)-3,
suggested by [α]D data comparisons between 1 and 3, was
confirmed from a chemical correlation study wherein the
hydroxylation of plakinidone (1) with 2-iodoxybenzoic acid
(IBX) in dimethylformamide (DMF) led to 3 in 53% yield
(Scheme 2) based on TLC, NMR, and [α]D data.
As previously remarked, our investigation of the compounds
responsible for the antitubercular activities of the active extracts
led us to the isolation of 3.5 mg (yield 0.005%) of previously
known smenothiazole A (5).9 The stereostructural elucidation
of compound 5 was determined by de novo analysis of its NMR
data and, later, by comparing its NMR data with published data
(Table S6). Although 5 has displayed substantial cytotoxicity,
the original isolate was not assayed for anti-TB activity. As it
happens with many marine natural products, the lack of an
adequate supply of material has impeded research into the
biological potential of the smenothiazoles.13 While we
attempted to solve the supply issue by total synthesis, all of
our efforts to prepare 5 in a practical manner capable of
providing an adequate supply of material for biological
screening were unsuccessful. Thus, after some consideration,
we embarked on a diverted total synthesis of smenothiazole A
wherein the chlorine atom in 5 was to be “edited out”
chemically through truncation, thus reducing the structural
complexity of the natural product. Thus, the aim of this study
was to identify a probable pharmacophore for antituberculosis
activity in smenothiazole A (5) and to deliver improved
compounds for biological evaluation.14
Figure 1. Key COSY (bold) and HMBC (→) correlations for
plakinidone B (2).
Inspection of the NMR spectra for 2, when compared to those
of compound 1, revealed minor differences for the alkyl chain,
suggesting that 2 has the same five-membered-ring lactone
joined to a terminal p-hydroxyphenyl ring through a slightly
modified hydrocarbon chain. The slight spectral differences
observed could be accounted for by the replacement of the
branched C10 alkyl chain moiety in 1 by an unbranched C12
alkyl chain in 2.
With the planar structure of plakinidone B (2) confidently
established, we undertook an investigation to establish its
absolute configuration unambiguously. Given the high suscept-
ibility of tetronic acid derivatives for air oxidation, we prepared
two stable derivatives from a sample of naturally occurring
plakinidone B (Scheme 1).6b Thus, the natural product was
Scheme 1. Preparation of Dimethylated Derivatives 6 and 7
treated with CH2N2 to furnish in 85% yield a 1:1.3 mixture of
dimethylated adducts 6 and 7, which were easy to separate
using conventional chromatographic methods. The absolute
configuration was assigned by vibrational and electronic circular
dichroism (VCD and ECD) measurements of dimethylated
derivative 610 in combination with density functional theory
calculations at the B3LYP/aug-cc-pVDZ/PCM(MeCN) level
of theory (Gaussian09 package of programs; see Figures S1−S9
and Tables S1−S5 in the Supporting Information).11 Our
combined efforts and the agreement between the experimental
and calculated VCD/ECD spectra of 6 revealed that the
absolute configuration of plakinidone B is (19R). Furthermore,
Our retrosynthetic analysis of the total synthesis of dechloro-
smenothiazole A (8) is delineated in Scheme 3. We envisioned
the disconnection of the secondary amide bond to give
fragments 9 and 12. The peptidic fragment 9 could be prepared
from commercially available Fmoc-Val-OH (10) and 2-
pyrrolidin-2-ylthiazole (11). The pivotal polyketide segment
12 was envisioned from key C-18/C-19 and C-14/C-16
Scheme 2. Semisynthesis of Plakinidone C (3) from Plakinidone (1)
C
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Scheme 3. Retrosynthetic Analysis of Dechloro-smenothiazole A (8)
Scheme 4. Synthesis of Primary Amines 9
Scheme 5. Synthesis of Key Polyketide Segment 12
double-bond disconnections. The precursor to intermediate
ketone 13 will be β-hydroxy aldehyde 14, which in turn could
be generated from commercially available benzyl cyanide (15)
and allyl bromide (16).
As outlined in Scheme 3, our synthesis commenced from
coupling of commercially available Fmoc-Val-OH (10) and 2-
pyrrolidin-2-ylthiazole (11) in the presence of peptide coupling
reagents (ethylene dichloride (EDC) and hydroxybenzotriazole
(HOBt))15 to afford protected intermediate 17 in 90% yield.
Removal of the Fmoc protecting group with 25% morpholine
in tetrahydrofuran (THF) furnished primary amines 9 in 95%
yield as a 1:1 mixture of epimers (Scheme 4).
With the epimeric mixture of amines 9 in hand, we took aim
at the synthesis of key polyketide segment 12. As shown in
Scheme 5, benzyl cyanide (15) was coupled with allyl bromide
(16) through a Barbier-type reaction aided by Zn/AlCl3 to
afford the desired β,γ-unsaturated ketone (not shown) in 93%
yield.16 The known ketone16 was treated with NaBH4 in
MeOH to give racemic homoallylic alcohol 18, also a known
compound, in 78% yield.17 The one-pot ozonolysis−Wittig
D
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olefination18,19 of 18 yielded the desired α,β-unsaturated ester
19 (through intermediate 14) as a mixture of E- and Z-isomers
(E:Z, 18:1). Fortunately, the two isomers could be separated by
flash chromatography over silica gel easily, and pure E-19 was
obtained in 63% yield. Compound E-19 was then immediately
reduced with diisobutylaluminum hydride (DIBAL-H) in
CH2Cl2 to furnish alcohol 20 in 76% yield, which was then
protected regioselectively with tert-butyldimethylsilyl chloride
(TBDMS-Cl) in the presence of imidazole to give 21 in 88%
yield. Alcohol 21 was later oxidized to ketone 22 with Dess-
Martin periodinane in 78% yield.20 Treatment of ketone 22
using the Peterson olefination methodology21 followed by
exposure of the intermediate tertiary alcohol (not shown) to
catalytic amounts of TsOH·H2O in MeOH afforded the desired
olefin 23 in 57% yield over two steps. To conclude, a two-step
oxidation sequence of allylic alcohol 23 with Dess-Martin
periodinane in CH2Cl2 followed by exposure to sodium chlorite
furnished the desired polyketide portion 12 in 45% yield.22
Finally, following a standard protocol, key segments 9 and 12
were coupled to afford 8 in 78% yield as a 1:1 mixture of
epimers at C-4 (Scheme 6). A side-by-side comparison of our
13C NMR spectroscopic data for 8 (mixture of epimers) with
those for natural smenothiazole A (5) revealed excellent
agreement (Figure S10).23
Table 2. In Vitro Antituberculosis Activity for Pure Isolated
Compounds 1−5 and Synthetic Compound 8
a
b
compound
plakinidone (1)
MABA MIC (μg/mL)
>128
>128
>128
plakinidone B (2)
plakinidone C (3)
c
plakortolide F (4)
59.0
smenothiazole A (5)
dechloro-smenothiazole A (8)
4.1
d
>128
e
Ctrl+
0.1
a
b
c
Mtb H37Rv strain. Values are means of three experiments. Value
d
e
taken from ref 2b. Mixture of two epimers. Rifampicin (RMP) was
used as the positive control.
peptide/polyketide as a new lead compound endowed with
high activity toward Mtb H37Rv, exhibiting an MIC value of 4.1
μg/mL. Interestingly, the lack of in vitro activity for synthetic
derivative 8 suggests that the presence of an electron-
withdrawing group, such as a chlorine atom, at the C-19
position of smenothiazole A (5) is essential for anti-TB activity.
These preliminary results validate our contention that the vinyl
chloride functionality of 5 could represent a new pharmaco-
phore for antituberculosis activity and thus should be taken into
consideration by research groups that are exploring the utility
of future peptide/polyketide-based compounds as effective anti-
TB drugs.26,27
Scheme 6. Synthesis of Dechloro-smenothiazole A (8) from
the Coupling of Segments 9 and 12
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Rudolph Research Analytical Autopol IV. The UV
spectra were recorded on a Shimadzu UV-2401 PC UV−visible
spectrometer. The ECD spectra in MeCN were measured in a quartz
cell with a path length of 0.1 cm between 300 and 180 nm at room
temperature (rt) on a JASCO J-815 circular dichroism spectrometer.
The VCD spectra were measured in CD3CN on a ChiralIR-2X FT-
VCD spectrometer (BioTools Inc.). The IR spectra were measured on
a Bruker FT-IR spectrometer. The NMR spectra were recorded on a
Bruker DRX-500 FT-NMR or a Bruker Ascend 700 FT-NMR
spectrometer. CDCl3 was used both as a solvent and as an internal
reference at δH 7.26 and δC 77.0. The HRESIMS data were obtained at
the Mass Spectrometry Laboratory of the School of Chemical
Sciences, University of Illinois, using a VG 70-VSE (EI+, 70 eV).
TLC was carried out on precoated silica gel 60 GF254 or RP-18 silica
gel 60 GF254 (Analtech). Gravity column chromatography was
performed with normal- and reversed-phase (RP-18) silica gel (35−
75 mesh, Analtech). The plates were analyzed under UV light, treated
with I2 vapors, or sprayed with phosphomolybdic acid solution in
EtOH. HPLC was carried out on an Agilent 1260 Infinity equipped
with an Agilent 1260 photodiode array detector using HPLC-grade
solvents. All chemical reagents were purchased from Sigma-Aldrich
Co.
In Vitro Inhibition of Mycobacterium tuberculosis
H37RV. Pure isolated natural products 1−5 and the synthetic
mixture of epimers 8 were analyzed using a serial dilution from
128 to 1 μg/mL against Mtb H37RV in order to compare the
effects of each of the compounds and to select the most
promising active substance, using rifampicin (RMP) as a
positive control. The percentage inhibition at each concen-
tration was used to determine the MIC (the lowest compound
concentration that prevents visible growth of the bacterium) of
each compound in a broth microdilution Alamar blue assay
(MABA).24 As shown in Table 2, neither compounds 1−4 nor
8 showed significant effects on Mtb H37Rv growth.25 On the
other hand, smenothiazole A (5) gave the best (lowest) MIC
value (4.1 μg/mL). Compound 5 was then assessed for
potential cytotoxicity to human cells through the use of
cultured Vero cells, giving an IC50 value of >128 μg/mL. Given
its good MIC and low cytotoxicity, 5 is a potential candidate for
efficacy studies in mice.
Sponge Material (First Collection), Extraction, and Isolation.
Fresh specimens of the sponge Plakortis symbiotica (Vicente, Zea &
Hill, 2016) (phylum Porifera; class Homoscleromorpha; order
Homosclerophorida; family Plakinidae) in association with Xestospon-
gia deweerdtae (Lehnert & van Soest, 1999) (phylum Porifera; class
Demospongiae; subclass Heteroscleromorpha; order Haplosclerida;
family Petrosiidae) were collected by hand using scuba at depths of
90−100 ft off Mona Island, Puerto Rico, in July 2006.4 A voucher
specimen (No. IM06-09) is stored at the Chemistry Department of
́
the University of Puerto Rico, Rio Piedras Campus. In prior reports
CONCLUSION
■
with these symbiotic sponges we originally classified the species
P. symbiotica as P. halichondrioides.2 The specimens were frozen and
lyophilized prior to extraction. The dry specimens (395 g) were cut
into small pieces and blended in a mixture of CHCl3−MeOH (1:1)
(11 × 1 L). After filtration, the crude extract was concentrated and
A significant finding of this study was the discovery of the
second and third examples of a rare class of natural products,
namely, the plakinidones.5,6 Screening of the antituberculosis
activity of smenothiazole A (5) identified this interesting hybrid
E
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stored under vacuum to yield a dark gum (100 g), which was
suspended in H2O (2 L) and extracted with n-hexane (3 × 2 L) and
CHCl3 (3 × 2 L). Concentration of the CHCl3 extract under reduced
pressure yielded 8.0 g of a dark brown oil, which was chromatographed
over RP-18 silica gel (100 g) using mixtures of MeOH−H2O of
increasing polarity (70−100%). A total of 10 fractions (I−X) were
generated on the basis of TLC and 1H NMR analyses. Initial
purification of fraction IV(D) (228 mg) by column chromatography
(silica gel, 5.0 g) using a gradient mixture of CHCl3−MeOH (90:10 →
80:20) afforded five subfractions, denoted as D(1)−D(5). Purification
of subfraction D(1) (30 mg) through a short plug of silica gel (1.0 g)
eluting with CHCl3−MeOH (95:5) afforded plakinidone C (3) (16
mg, yield 0.004%). Careful scrutiny of combined spectroscopic data
revealed that fractions V(E) and VI(F) consisted of known
plakinidone (1) (926 mg, yield 0.23%).5,6 Purification of fraction VII
(700 mg) by silica gel (16.0 g) column chromatography using a
gradient mixture of CHCl3−MeOH (100:0 → 90:10) afforded known
cyclic peroxide plakortolide F (4) (110 mg, yield 0.028%).2b,7 Further
purification of fraction VIII(H) (760 mg) by RP-18 silica gel (20 g)
column chromatography in MeOH−H2O (9:1) afforded five
subfractions, denoted as H(1)−H(5). Subfraction H(3) (100 mg)
was purified by RP-18 silica gel (4.0 g) column chromatography in
MeOH−H2O (8:2) to afford plakinidone B (2) (60 mg, yield 0.015%).
Interestingly, no traces of known smenothiazole A (5)9 were detected
in any of the extracts obtained using animal material stemming from
this collection.
to afford pure smenothiazole A (5) (tR = 22.4 min, 3.5 mg, yield
0.005%).9
A cursory NMR- and chromatography-based dereplication analysis
of a small portion (100 mg) of the gross CHCl3 extract (13.1 g) led us
to select four compounds as being potentially of interest for isolation
regarding their putative novelty. However, after gathering non-
quantified amounts suitable for full structure elucidation and
identification, the compounds were quickly found to be previously
extracted plakinidone (1), plakinidone B (2), plakinidone C (3), and
plakortolide F (4).
Smenothiazole A (5): colorless oil; [α]20 −45 (c 0.8, CHCl3)
D
(specific rotation data were not provided for the original isolate);9 UV
(MeOH) λmax 206 nm (log ε 4.35); IR (film) νmax 3330, 2963, 2929,
1
1724, 1628, 1497, 1431, 1321, 1178, 1077, 734, 702 cm−1; H NMR
(500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Table S6;
LREIMS m/z 485 (26), 304 (43), 233 (66), 182 (45), 154 (27), 91
(100); HREISMS m/z 486.1987 [M + H]+ (calcd for C26H33ClN3O2S,
486.1982).
Methylation of Plakinidone B (2). To a solution of plakinidone
B (35 mg, 0.090 mmol) in CHCl3 (5 mL) was added a solution of
excess diazomethane in ether (25 mL), and the resulting mixture was
stirred at 25 °C for 24 h. The reaction mixture was concentrated in
vacuo, and the oily residue left over was chromatographed by silica gel
column chromatography (1.0 g) eluting with mixtures of CHCl3−n-
hexanes of increasing polarity (8:2; 9:1; 10:0) to afford pure
compounds 6 (14 mg, 37% yield) and 7 (18.2 mg, 48% yield) as
Plakinidone (1): [α]20 −12 (c 0.6, MeOH); lit.6b [α]20 −13.1 (c
colorless oils; minor isomer 6 [α]20 −12 (c 0.9, CHCl3); UV (1.7 ×
D
D
D
10−4 M, MeCN) λmax (ε) 195 (41 200), 226 (16 700), 278 (1650), 285
(1400) nm; ECD (1.7 × 10−4 M, MeCN), λmax (Δε) 191 (−1.78), 199
(−1.1), 226 (+0.31) nm; IR (film) νmax 2919, 2849, 1742, 1665, 1511,
0.6, MeOH).
Plakinidone B (2): yellowish semisolid; [α]20 −10.6 (c 1.0,
D
MeOH); UV (MeOH) λmax (log ε) 225 (4.74), 256 (4.48) nm; IR
(film) νmax 3600−3100 (br), 2926, 2854, 1878 (weak), 1724, 1661,
1515, 1456, 1374, 1228, 1105, 828, 765 cm−1; 1H NMR (CD3OD, 500
MHz) and 13C NMR (CD3OD, 125 MHz), see Table 1; LREIMS m/z
388 (3), 372 (4), 318 (28), 304 (18), 260 (6), 246 (6), 149 (3), 133
(7), 120 (12), 107 (100); HRESIMS m/z 389.2694 [M + H]+ (calcd
for C24H37O4, 389.2692).
1
1465, 1388, 1315, 1246, 1036, 979, 814 cm−1; H NMR (CDCl3, 500
MHz) δ 7.09 (2H, d, J = 8.4 Hz, H-1), 6.82 (2H, d, J = 8.4 Hz, H-2),
4.10 (3H, s, OCH3, H3-25), 3.78 (3H, s, OCH3, H3-26), 2.53 (2H, t, J
= 7.6 Hz, H2-7), 1.72/1.60 (2H, m, H2-18), 2.00 (3H, s, H3-23), 1.57
(2H, m, H2-8), 1.38 (3H, s, H3-24), 1.29−1.23 (18H, br envelope, H2-
9 through H2-17); 13C NMR (CDCl3, 125 MHz) δ 176.0 (C, C-20),
174.3 (C, C-22), 157.5 (C, C-3), 135.0 (C, C-6), 129.2 (2 × CH, C-
1), 113.6 (2 × CH, C-2), 96.0 (C, C-21), 83.1 (C, C-19), 58.9 (OCH3,
C-25), 55.2 (OCH3, C-26), 36.8 (CH2, C-18), 35.0 (CH2, C-7), 31.7
(CH2, C-8), 29.6−29.3 (8 × CH2, C-9 through C-16), 23.5 (CH3, C-
24), 23.0 (CH2, C-17), 8.5 (CH3, C-23); LREIMS m/z 416 (6), 177
Plakinidone C (3): yellowish oil; [α]20 −4.7 (c 0.6, MeOH); UV
D
(MeOH) λmax (log ε) 201 (4.18), 226 (3.99), 256 (3.61) nm; IR
(film) νmax 3290 (br), 2928, 2856, 1720, 1660, 1519, 1454, 1284, 1112,
949, 758 cm−1; 1H NMR (CD3OD, 500 MHz) and 13C NMR
(CD3OD, 125 MHz), see Table 1; LREIMS m/z 390 (86), 306, 123
(100); HREIMS m/z 390.2400 [M]+ (calcd for C23H34O5, 390.2407).
(26), 149 (100), 130 (22), 121 (32), 98 (26); major isomer 7 [α]20
D
Plakortolide F (4): [α]20D +5.2 (c 0.6, CHCl3); lit.2b [α]20 +5.7 (c
+22 (c 1.0, CHCl3); UV (MeOH) λmax (log ε) 201 (4.00), 224 (3.87),
264 (4.08) nm; IR (film) νmax 2924, 2853, 1701, 1610, 1512, 1476,
D
1.1, CHCl3).
1
1394, 1367, 1246, 1197, 1037, 978, 819 cm−1; H NMR (CDCl3, 500
Sponge Material (Second Collection), Extraction, and
Isolation. Fresh specimens of the sponge consortium P. symbiotica−
X. deweerdtae were collected by hand using scuba at depths of 90 ft off
Mona Island, Puerto Rico, in June 2011. A voucher specimen (No.
IM11-04) is stored at the Chemistry Department of the University of
MHz) δ 7.09 (2H, d, J = 8.4 Hz, H-1), 6.82 (2H, d, J = 8.4 Hz, H-2),
4.00 (3H, s, OCH3, H3-25), 3.78 (3H, s, OCH3, H3-26), 2.53 (2H, t, J
= 7.6 Hz, H2-7), 1.73 (2H, m, H2-18), 1.58 (3H, s, H3-23), 1.56 (2H,
m, H2-8), 1.39 (3H, s, H3-24), 1.29−1.23 (18H, br envelope, H2-9
through H2-17); 13C NMR (CDCl3, 125 MHz) δ 201.0 (C, C-20),
179.3 (C, C-22), 157.5 (C, C-3), 135.0 (C, C-6), 129.2 (2 × CH, C-
1), 113.6 (2 × CH, C-2), 93.0 (C, C-21), 87.4 (C, C-19), 55.6 (OCH3,
C-25), 55.2 (OCH3, C-26), 36.3 (CH2, C-18), 35.0 (CH2, C-7), 31.7
(CH2, C-8), 29.6−29.2 (8 × CH2, C-9 through C-16), 23.0 (CH2, C-
17), 22.0 (CH3, C-24), 3.9 (CH3, C-23); LREIMS m/z 416 (30), 177
(11), 155 (21), 149 (36), 142 (59), 135 (14), 121 (100).
́
Puerto Rico, Rio Piedras Campus. Freeze-dried specimens (1.59 kg)
were cut into small pieces and blended in a mixture of CHCl3−MeOH
(1:1) (36 L). After filtration, the extract was concentrated and stored
under vacuum to yield a dark gum (323 g), which was suspended in
H2O and extracted with n-hexane (16 L). Concentration under
reduced pressure yielded the n-hexane extract (69.0 g) as a dark brown
oil, a portion of which (16.3 g) was chromatographed over silica gel
(250 g) using mixtures of n-hexane−acetone of increasing polarity (0−
100%). A total of 12 fractions (I−XII) were generated on the basis of
TLC and 1H NMR analysis. Further purification of fraction VI (1.3 g)
by silica gel (30.0 g) column chromatography in CHCl3−EtOAc
(100:0; 95:5; 90:10) afforded six subfractions, denoted as A−F.
Fraction F (700 mg) was purified by column chromatography over
silica gel (10.0 g), eluting with an isocratic mixture of CHCl3−EtOAc
(9:1), to give four subfractions, denoted as F1−F4. Subfraction F4
(287 mg) was purified sequentially by column chromatography over
cyano silica gel (4.0 g) using isocratic n-hexanes−isopropyl alcohol
(96:4) as mobile phase followed by purification of subfraction F4b (25
mg) by chiral-phase HPLC (stationary phase, (R,R)-Whelk-O 1 10
μm, 150 × 4.6 mm i.d. (Regis Technologies, Inc.); mobile phase,
MeOH−H2O (85:15); flow rate, 1 mL/min; detection, UV 254 nm)
Semisynthesis of Plakinidone C (3). IBX (15 mg, 0.030 mmol)
was added in the dark to a solution of plakinidone (1) (10 mg, 0.027
mmol) in DMF (3 mL). After stirring the reaction mixture for 4 h at
25 °C the red-colored solution was treated with L-ascorbic acid (0.25
mL, 1 M in H2O) and left stirring until the color turned light yellow
(∼4 h). After extracting with EtOAc (3 × 15 mL) the organic layer
was concentrated to give an oil, which was passed through a short plug
of silica gel (0.7 g) using a mixture of CHCl3−MeOH (4.9:0.1) to
yield plakinidone C (3) (5.5 mg, 53% yield). Upon careful
1
comparison, the overall spectroscopic (IR, UV, EIMS, H and 13C
NMR), TLC, and specific rotation data ([α]20D −4.8 (c 0.8, MeOH))
of synthetic 3 and the natural product were in excellent agreement.
Compound 17. To a solution of 2-pyrrolidin-2-ylthiazole (11, 325
mg, 2.11 mmol, 1 equiv) in dry CH2Cl2 (25 mL) were added Fmoc-
F
DOI: 10.1021/acs.jnatprod.7b00300
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Val-OH (10, 832 mg, 1 equiv), EDC (406 mg, 1 equiv), and HOBt
(285 mg, 1 equiv), and the mixture was stirred at rt for 48 h. The
reaction mixture was then concentrated to dryness, basified with
saturated aqueous NaHCO3 (50 mL), and extracted with EtOAc (3 ×
50 mL). The crude product obtained after solvent removal was
purified by silica gel (5 g) column chromatography using CHCl3 to
afford 17 (900 mg, 90%) as a white semisolid, which was used in the
following step without further purification.
85:15) to afford esters Z-19 (25 mg, 3.5%) and E-19 (450 mg, 63%) as
clear, colorless oils: minor isomer Z-19 IR (film) νmax 3444 (br), 3086,
3062, 3028, 2981, 2928, 1710, 1647, 1603, 1496, 1454, 1373, 1228,
1132, 1081, 907, 859, 746, 701 cm−1; 1H NMR (CDCl3, 700 MHz) δ
7.30−7.22 (5H, m), 6.06 (1H, dt, J = 7.8, 1.4 Hz), 4.19 (2H, q, J = 7.1
Hz), 3.94 (1H, quintet, J = 6.7 Hz), 2.80 (1H, d, J = 6.5 Hz), 2.63 (1H,
q br, J = 7.8 Hz), 2.49 (1H, br s, exchangeable), 1.94 (3H, s), 1.29
(3H, t, J = 7.1 Hz); 13C NMR (CDCl3, 176 MHz) δ 168.4, 138.4,
138.3, 129.9, 129.4, 128.5, 126.4, 72.3, 60.4, 44.0, 36.4, 20.6, 14.2;
LRESIMS m/z 271.6 [M + Na]+, 249.6 [M + H]+, 231.6 [M + H −
H2O]+; major isomer E-19 IR (film) νmax 3454 (br), 3062, 3028, 2982,
2933, 1707, 1649, 1603, 1496, 1454, 1368, 1276, 1125, 1038, 918, 867,
Compound 9. To a solution of 17 (890 mg, 1.87 mmol) in THF
(5 mL) was added 25% morpholine in THF (10 mL). After stirring for
8 h at 25 °C the mixture was concentrated under reduced pressure to
give a crude material that was purified by silica gel (5 g) column
chromatography with 95:5 CHCl3−MeOH to afford compound 9
(449 mg, 95%) as a 1:1 mixture of epimers as a colorless foam: IR
(film) νmax 3373, 3302, 3081, 2960, 2874, 1643, 1499, 1430, 1364,
1
740, 701 cm−1; H NMR (CDCl3, 500 MHz) δ 7.30−7.21 (5H, m),
6.86 (1H, dt, J = 7.4, 1.4 Hz), 4.18 (2H, q, J = 7.1 Hz), 3.97 (1H,
quintet), 2.83 (1H, dd, J = 13.4, 4.9 Hz), 2.73 (1H, dd, J = 13.4, 8.1
Hz), 2.39 (2H, t, J = 7.1 Hz), 2.07 (1H, br s, exchangeable), 1.84 (3H,
s), 1.29 (3H, t, J = 7.1 Hz); 13C NMR (CDCl3, 125 MHz) δ 167.9,
137.7, 137.9, 129.8, 129.5, 128.5, 126.5, 71.7, 60.5, 43.5, 35.8, 14.2,
12.6; LRESIMS m/z 271.6 [M + Na]+, 249.6 [M + H]+, 231.6 [M + H
− H2O]+.
1
1265, 1203, 1134, 1055, 890, 830, 729 cm−1; H NMR (CDCl3, 500
MHz) δ 7.47 (1H, d, J = 3.3 Hz)/7.46 (1H, d, J = 3.3 Hz); 7.03 (1H,
d, J = 3.3 Hz)/7.02 (1H, d, J = 3.3 Hz); 5.35 (1H, dd, J = 7.9, 2.4 Hz)/
5.29 (1H, dd, J = 7.5, 2.0 Hz); 3.39−3.54 (4H, m); 3.21 (1H, d, J = 4.9
Hz)/3.12 (1H, d, J = 6.3 Hz); 2.17 (1H, m)/2.02 (1H, m); 1.98−1.86
(4H, m); 1.77−1.69 (4H, m); 0.82 (3H, d, J = 6.8 Hz)/0.79 (3H, d, J
= 6.8 Hz); 0.74 (3H, d, J = 6.8 Hz)/0.74 (3H, d, J = 6.8 Hz); 13C
NMR (CDCl3, 125 MHz) δ 173.9/173.7, 172.0/171.5, 141.7/141.6,
118.4/118.2, 58.2/58.0, 57.7/57.6, 46.5/46.3, 31.3/31.2, 31.1/30.9,
24.0/23.7, 19.7/19.3, 16.8/16.0; LRESIMS m/z 254.6 [M + H]+;
HREISMS m/z 254.1329 [M + H]+ (calcd for C12H20N3OS,
254.1327).
Compound 20. A solution of ethyl ester E-19 (600 mg, 2.42
mmol, 1 equiv) in dry CH2Cl2 (5.0 mL) was cooled to −78 °C and
treated with 1.0 M DIBAL-H in THF (7.0 mL, 3 equiv) dropwise over
a period of 5 min. The resulting solution was stirred at −78 °C for
another 20 min, slowly warmed to rt, and then stirred for an additional
20 h. The reaction was then carefully quenched in sequence with
MeOH (0.5 mL), 10% NaOH (15 mL), and H2O (35 mL). After
extraction with EtOAc (3 × 40 mL) the combined organic layer was
dried (MgSO4) and concentrated to leave an oily residue, which was
purified by column chromatography over silica gel (4.5 g, n-hexanes−
EtOAc, 7:3; 6:4; 1:1) to afford pure diol 20 (380 mg, 76%) as a
colorless oil: IR (film) νmax 3446 (br), 3062, 3028, 2918, 2861, 1603,
1-Phenylpent-4-en-2-ol (18). To a suspension of zinc powder
(1.2 g, 18.8 mmol, 4 equiv) in dry THF (6 mL) kept at 0 °C under a
N2 atmosphere was added allyl bromide (16, 555 mg, 4.69 mmol, 1
equiv). After stirring for 3 h at 0−25 °C, benzyl cyanide (15, 535 mg,
4.69 mmol, 1 equiv) in dry THF (1 mL) was added dropwise. After
stirring for 1.5 h the mixture was cooled to 0 °C, and then AlCl3 (248
mg, 1.88 mmol, 0.4 equiv) was slowly added (exothermic!). The
mixture was stirred for the next 1.5 h while allowing the temperature
to rise to rt. After adding H2O (15 mL), the reaction mixture was
stirred for another 1.5 h, more H2O was added (60 mL), and the
mixture was extracted with EtOAc (3 × 75 mL). The combined
organic layer was dried (MgSO4), and the solvent removed under
reduced pressure. The yellow oil obtained was identified as known 1-
phenylpent-4-en-2-one (700 mg, 93%),16 which was used in the
following step without further purification. To a solution of the latter
compound (700 mg, 4.37 mmol, 1 equiv) in MeOH (10 mL) at 0 °C
was added NaBH4 (200 mg, 5.25 mmol, 1.2 equiv). The reaction
mixture was stirred at 0 °C for 3 h and then concentrated under
reduced pressure, diluted with H2O (25 mL), and extracted with
CHCl3 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4, and the solvent was removed under reduced
pressure. The crude was purified by flash silica gel column
chromatography (5 g, 95:5 n-Hex−EtOAc) to give homoallylic
1
1496, 1453, 1356, 1230, 1046, 1006, 896, 863, 744, 701 cm−1; H
NMR (CDCl3, 700 MHz) δ 7.29−7.20 (5H, m), 5.47 (1H, t, J = 7.7
Hz), 3.94 (2H, s), 3.85 (1H, m), 2.75 (2H, m), 2.22 (2H, m), 1.63
(3H, s); 13C NMR (CDCl3, 176 MHz) δ 138.5, 137.3, 129.3, 128.3,
126.2, 121.3, 72.4, 68.1, 43.3, 34.6, 13.9; LRESIMS m/z 171.1 [M + H
− 2H2O]+, 229.1 [M + Na]+, 245.1 [M + K]+.
Compound 21. To a solution of diol 20 (590 mg, 2.86 mmol, 1
equiv), imidazole (253 mg, 1.3 equiv), and 4-dimethylaminopyridine
(DMAP) (35 mg, 0.1 equiv) in CH2Cl2 (6 mL) kept at 0 °C was
added 1.0 M tert-butyldimethylsilyl chloride in THF (3.72 mL, 1.3
equiv) over 5 min. The mixture was stirred for 5 h at 0 °C and later
quenched with saturated NH4Cl (30 mL). After the aqueous layer was
extracted with EtOAc (3 × 30 mL) the combined organic layer was
washed with H2O (30 mL) and brine (30 mL), dried over MgSO4, and
concentrated. The crude product was purified by column chromatog-
raphy over silica gel (3.5 g, n-hexanes−EtOAc, 1:0; 95:5; 90:10) to
yield silyl ether 21 (810 mg, 88%) as a colorless oil: IR (film) νmax
3442 (br), 3063, 3028, 2929, 2857, 1603, 1496, 1471, 1362, 1254,
1
1
alcohol 18 (554 mg, 78%) as a colorless oil. The H and 13C NMR
1071, 1006, 838, 776, 743, 700, 668 cm−1; H NMR (CDCl3, 700
spectroscopic properties were in agreement with data published in the
MHz) δ 7.33−7.25 (5H, m), 5.53 (1H, t, J = 7.4 Hz), 4.07 (2H, s),
3.88 (1H, m), 2.85 (1H, dd, J = 13.6, 4.7 Hz), 2.72 (1H, dd, J = 13.6,
8.0 Hz), 2.29 (2H, m), 1.65 (3H, s), 0.94 (9H, s), 0.10 (6H, s); 13C
NMR (CDCl3, 176 MHz) δ 138.6, 137.6, 129.4, 128.4, 126.3, 119.6,
72.5, 68.3, 43.2, 34.9, 25.9, 18.4, 13.7, −5.3; LRESIMS m/z 359.2 [M
+ K]+, 343.2 [M + Na]+.
literature.17
Compound 19. Through a stirred solution of homoallylic alcohol
18 (550 mg, 3.39 mmol) and NaHCO3 (spatula tipful) in CH2Cl2 (15
mL) kept at −78 °C was bubbled a stream of ozone. Ozone treatment
was terminated when the reaction mixture maintained a blue color for
1 min. Thereafter, it was replaced by N2, and the solution stirred until
the color dissipated (∼2 h). To the clear mixture was added
dimethylsulfide (2.5 mL), and the stirring continued for 36 h while the
temperature ranged from −78 °C to rt. The crude β-hydroxy aldehyde
14 obtained after solvent removal was stored under vacuum for over
12 h (610 mg, 86%), which was then used in the next reaction without
further purification. To the latter intermediate (600 mg, 2.86 mmol, 1
equiv) in dry CH2Cl2 (15 mL) was added phosphorus ylide
(carbethoxyethylidene)triphenylphosphorane (1.45 g, 4.0 mmol, 1.4
equiv). After the resulting solution was stirred at 25 °C for 48 h, silica
gel (∼0.3 g) was added and the solvent rotoevaporated. The crude
obtained was chromatographed over silica gel (10 g) and eluted with
mixtures of n-Hex−EtOAc of increasing polarity (95:5, 90:10, and
Compound 22. To a stirred solution of 21 (765.3 mg, 2.39 mmol,
1 equiv) in CH2Cl2 (10 mL) kept at 0 °C were added Dess-Martin
periodinane (1.12 g, 2.63 mmol, 1.1 equiv) and NaHCO3 (402 mg,
4.78 mmol, 2 equiv). The mixture was stirred for 2 h, diluted with
saturated Na2S2O3 (25 mL), and extracted with EtOAc (3 × 25 mL).
The combined organic layer was washed with saturated NaHCO3 (25
mL) and brine (25 mL), dried (Na2SO4), filtered, and concentrated.
The crude product was purified by column chromatography over silica
gel (3 g, n-hexane−EtOAc, 1:0; 98:2; 96:4) to give ketone 22 (589 mg,
78%) as a colorless oil: IR (film) νmax 2954, 2930, 2857, 1717, 1700,
1
1685, 1456, 1254, 1098, 838, 779, 743, 702 cm−1; H NMR (CDCl3,
700 MHz) δ 7.35−7.23 (5H, m), 5.64 (1H, t, J = 7.2 Hz), 4.07 (2H,
s), 3.74 (2H, s), 3.22 (2H, d, J = 7.2 Hz), 1.58 (3H, s), 0.94 (9H, s),
G
DOI: 10.1021/acs.jnatprod.7b00300
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Journal of Natural Products
Article
0.10 (6H, s); 13C NMR (CDCl3, 176 MHz) δ 206.4, 138.6, 134.2,
129.4, 128.7, 127.0, 115.4, 68.6, 49.5, 41.3, 25.9, 18.4, 13.7, −5.3;
LRESIMS m/z 357.2 [M + K]+, 341.2 [M + Na]+; HREISMS m/z
341.1915 [M + Na]+ (calcd for C19H30O2SiNa, 341.1913).
1076, 897, 738, 701 cm−1; H NMR (CDCl3, 700 MHz) δ 7.70/7.67
(1H, d, J = 3.2 Hz, H-1); 7.28 (2H, m, H-23/H-25); 7.23/7.16 (1H, d,
J = 3.3 Hz, H-2); 7.20 (1H, m, H-24); 7.17 (2H, m, H-22/H-26);
6.41/6.33 (1H, dt, J = 8.1 Hz, H-16); 6.23/5.93 (NH, d, J = 9.1 Hz);
5.52/5.48 (1H, dd, J = 8.0, 2.8 Hz, H-4); 4.85 (1H, br s, H-19); 4.84
(1H, br s, H-19′); 4.77/4.72 (1H, dd, J = 8.7, 5.8 Hz, H-9); 3.87/3.78
(2H, m, H-7); 3.35 (2H, br s, H-20); 2.78 (2H, d, J = 7.0 Hz, H-17);
2.38 (1H, m, H-10); 2.27/2.10 (2H, m, H-5/H-5′), 2.15/2.04 (2H, m,
H-6); 1.76/1.66 (3H, s, H-15); 1.01/0.98 (3H, d, J = 6.7 Hz, H-12);
0.91/0.79 (3H, d, J = 6.7 Hz, H-11); 13C NMR (CDCl3, 176 MHz) δ
171.9/171.4 (C, C-3), 171.7/170.9 (C, C-8), 169.1/169.0 (C, C-13),
145.9/145.8 (C, C-18), 142.3 (CH, C-1), 139.1/139.0 (C, C-21),
133.6/133.3 (CH, C-16), 132.2/132.0 (C, C-14), 128.9 (CH, C-
22,26), 128.4 (CH, C-23,25), 126.3/126.2 (CH, C-24), 118.8/118.7
(CH, C-2), 112.9/112.8 (CH2, C-19), 58.6/58.4 (CH, C-4), 55.9/55.3
(CH, C-9), 47.4/47.2 (CH2, C-7), 43.2 (CH2, C-20), 34.3 (CH2, C-
17), 31.9/31.5 (CH2, C-5), 31.6/31.2 (CH, C-10), 24.5/23.9 (CH2, C-
6), 19.8/19.6 (CH3, C-12), 18.0/17.5 (CH3, C-11), 12.7/12.6 (CH3,
C-15); LRESIMS m/z 490.1 [M + K]+, 474.2 [M + Na]+, 452.2 [M +
H]+; HREISMS m/z 452.2372 [M + H]+ (calcd for C26H34N3O2S,
452.2372).
1
Compound 23. A cooled (0 °C) solution of ketone 22 (338.9 mg,
1.07 mmol, 1 equiv) in THF (3 mL) kept under a N2 atmosphere was
treated with 1.0 M TMSCH2−MgCl in Et2O (7.46 mL, 7 equiv) and
stirred at 0 °C for 1 h. Thereafter, the solution was stirred for 5 h while
allowing the temperature to rise to rt. After quenching with H2O (5
mL) the mixture was diluted with EtOAc (30 mL), and the organic
layer was washed with saturated NH4Cl (3 × 20 mL), dried (MgSO4),
and concentrated to afford the intermediate tertiary alcohol (structure
not shown), which was used in the next step without further
purification. A mixture of the latter intermediate (346 mg, 0.852
mmol) and p-TsOH·H2O (8.5 mg, 0.0426 mmol) in MeOH (2 mL)
was stirred for 30 min at 25 °C. The reaction mixture was diluted with
saturated NaHCO3 (25 mL) and extracted with EtOAc (2 × 25 mL).
The combined organic layer was washed with brine (15 mL), dried
over MgSO4, and concentrated, and the residue left over was passed
through a short plug of silica gel (1.0 g, n-hexanes−EtOAc, 1:0 → 8:2)
to furnish pure 23 (97.5 mg, 57% over two steps) as a colorless oil: IR
(film) νmax 3447 (br), 3064, 3028, 2916, 2859, 1696, 1645, 1602, 1495,
1453, 1434, 1248, 1064, 1017, 1002, 894, 862, 838, 740, 700 cm−1; 1H
NMR (CDCl3, 700 MHz) δ 7.28−7.21 (5H, m), 5.43 (1H, t, J = 7.4
Hz), 4.85 (1H, s), 4.78 (1H, s), 4.00 (2H, d, J = 5.6 Hz), 3.35 (2H, s),
2.71 (2H, d, J = 7.4 Hz), 1.58 (3H, s); 13C NMR (CDCl3, 176 MHz) δ
147.6, 139.6, 136.3, 129.0, 128.3, 126.1, 123.3, 111.9, 68.8, 43.1, 33.8,
13.6; LRESIMS m/z 241.3 [M + K]+, 225.5 [M + Na]+, 185.5 [M + H
− H2O]+.
Compound 12. A 25 °C mixture of allyl alcohol 23 (57.5 mg,
0.284 mmol, 1 equiv), Dess-Martin periodinane (157 mg, 1.3 equiv),
and NaHCO3 (48 mg, 2 equiv) in dry CH2Cl2 (2 mL) was stirred for 1
h. The reaction mixture was then diluted with saturated Na2S2O3 (20
mL) and extracted with EtOAc (3 × 20 mL), and the combined
extracts were washed with saturated NaHCO3 (20 mL) and brine (20
mL), dried (MgSO4), and concentrated to dryness. The crude product
obtained was purified by silica gel (1.0 g) using n-hexanes−EtOAc
(9:1) to afford the desired aldehyde intermediate (51.4 mg), which
was used without delay in the next step. A solution of the latter
aldehyde (51.4 mg, 0.257 mmol, 1 equiv) in t-BuOH (0.5 mL) was
treated sequentially with 2 M 2-methyl-2-butene in THF (3.0 mL,
6.168 mmol), NaClO2 (70 mg, 3 equiv), and NaH2PO4 (123.3 mg, 4
equiv) dissolved in H2O (0.5 mL). The reaction mixture was stirred at
25 °C for 3 h, during which time all of the starting material had been
consumed to give the desired carboxylic acid as the major product
along with two slightly more polar byproducts. Upon solvent removal,
the residue left over was diluted with saturated NH4Cl (1 mL) and
treated with 1 M HCl (0.5 mL, pH ≈ 3.0). Following dilution with
H2O (15 mL) and extraction with EtOAc (3 × 15 mL), the combined
organic layer was concentrated and the residue obtained passed
through a short plug of silica gel (0.7 g) with n-hexanes−EtOAc (4:1)
to afford carboxylic acid 12 (25 mg, 45% over two steps) as a colorless
oil: IR (film) νmax 3028 (br), 2928 (br), 2666, 2549, 1690, 1646, 1495,
1453, 1421, 1286, 1132, 1075, 900, 736, 700 cm−1; 1H NMR (CDCl3,
500 MHz) δ 7.30 (2H, t, J = 7.5 Hz), 7.26 (1H, t, J = 7.5 Hz), 7.19
(2H, t, J = 7.5 Hz), 6.95 (1H, t, J = 7.6 Hz), 4.87 (2H, br s), 3.37 (2H,
s), 2.84 (2H, d, J = 7.6 Hz), 1.76 (3H, s); 13C NMR (CDCl3, 125
MHz) δ 173.0, 145.5, 141.9, 138.9, 128.9, 128.4, 128.3, 126.3, 113.1,
43.3, 34.6, 11.9; LRESIMS m/z 215.5 [M − H]+; HREISMS m/z
215.1072 [M − H]+ (calcd for C14H15O2, 215.1072).
VCD Measurements. The VCD and IR spectra of 6 dissolved in
CD3CN were measured using the ChiralIR-2X FT-VCD spectrometer
from BioTools Inc. The spectra were collected in the 2000−1000 cm−1
range at a resolution of 4 cm−1. The spectrometer was equipped with
dual sources and dual ZnSe photoelastic modulators optimized at 1400
cm−1. A solution with a concentration of ca. 0.14 M was measured in a
BaF2 cell with a path length of 99.7 μm. The final spectrum was
averaged from 9 blocks, each of 2048 interferometric scans (1 block
accumulated for 40 min). Baseline correction using the spectrum of
the relevant solvent obtained under the same conditions was
performed.
ECD Measurements. The ECD and UV spectra of 6 in MeCN
(∼1.7 × 10−4 M) were recorded in a quartz cell with a path length of
0.1 cm between 300 and 180 nm at 25 °C on a Jasco J-815
spectrometer. The spectrum was recorded using a 100 nm/min
scanning speed, a step size of 0.2 nm, a bandwidth of 1 nm, a response
time of 0.5 s, and an accumulation of 10 scans. The spectra were
background corrected using respective solvents recorded under the
same conditions.
Evaluation of Antituberculosis Activity. In vitro antitubercu-
losis screenings of compounds 1−5 and 8 against the pathogenic
microbe Mycobacterium tuberculosis H37Rv were performed as
previously described.28 Rifampicin was used as positive control during
the assay.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00300.
Computational results of 6 (Scheme S1, Figures S1−S9,
and Tables S1−S5), Figure S10, Table S6, H, 13C, and
1
2D NMR spectra of 2 and 3, and underwater photograph
of the sponge−sponge consortium (PDF)
AUTHOR INFORMATION
■
Dechloro-smenothiazole A (8). A mixture of 9 (14.3 mg, 0.056
mmol, 1 equiv), carboxylic acid 12 (12.2 mg, 1 equiv), EDC (11.8 mg,
1.1 equiv), and HOBt (8.3 mg, 1.1 equiv) in dry CH2Cl2 (3 mL) was
stirred at 25 °C for 12 h. The reaction mixture was concentrated to
dryness, basified with saturated NaHCO3 (15 mL), extracted with
EtOAc (3 × 15 mL), and concentrated under reduced pressure to
leave a residue, which was passed through a short plug of silica gel (0.7
g) with CHCl3−n-hexanes (9:1) to afford 8 (19.8 mg, 78% yield) as a
1:1 mixture of epimers as a colorless oil: IR (film) νmax 3325, 3082,
3027, 2961, 2929, 1782, 1718, 1635, 1496, 1430, 1321, 1249, 1176,
Corresponding Author
*Tel: +1 (787) 523-5320. Fax: +1 (787) 522-2150. E-mail:
abimael.rodriguez1@upr.edu.
ORCID
Abimael D. Rodríguez: 0000-0001-7748-0856
Notes
The authors declare no competing financial interest.
H
DOI: 10.1021/acs.jnatprod.7b00300
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
appears to be insensitive to the stereocenter remote from the furanone
ring. For further validation of these arguments, see refs 1a and 2a.
(13) Amazingly, with only 225 μg of pure material in hand, the Italian
group correctly established the molecular structure of smenothiazole A
(5) and also assessed its antitumor properties in vitro.
(14) (a) Prinz, H. Chem. Biol. 2008, 15, 207−208. (b) Maier, M. E.
Org. Biomol. Chem. 2015, 13, 5302−5343. (c) Wach, J.-Y.; Gadermann,
K. Synlett 2012, 23, 163−170.
ACKNOWLEDGMENTS
■
Financial support to C.J.-R. was provided by the IFARHU-
SENACYT Program of the Republic of Panama. We thank J.
Vicente, J. Asencio, and the crew of the R/V Sultana for
providing logistic support during collection of the sponge
specimens. We are particularly indebted to J. Vicente
(University of Maryland Center for Environmental Science)
for helpful discussions and for identification of the sponges. Dr.
M. Jawiczuk from the Institute of Organic Chemistry, Polish
Academy of Sciences, is acknowledged for providing the ECD
measurements. The calculations were performed at the
Interdisciplinary Centre for Mathematical and Computational
Modeling of the University of Warsaw (G19-4) and the PL-
Grid Infrastructure. We are grateful to Y. Wang and B. Wan
(ITR-UIC) for technical assistance during anti-TB and
cytotoxicity assays. This research was supported by the NIH
Grant 1SC1GM086271-01A1 awarded to A.D.R.
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I
DOI: 10.1021/acs.jnatprod.7b00300
J. Nat. Prod. XXXX, XXX, XXX−XXX