Fuscasins A-D, Cycloheptapeptides from the Marine Sponge Phakellia fusca

Article abstract of DOI:10.1021/acs.jnatprod.8b01033

Four new cycloheptapeptides, fuscasins A-D (1-4), were isolated from the marine sponge Phakellia fusca collected from the South China Sea. Their planar structures were fully characterized by spectroscopic methods, and the absolute configurations of amino acid residues were determined using the advanced Marfey's method. Structurally, 1 is a unique cycloheptapeptide with a backbone bearing a pyrrolidine-2,5-dione unit. Among the isolated compounds, 1 exhibited potent growth-inhibitory activity against HepG2 cells with an IC₅₀ value of 4.6 μM, whereas it did not show apparent inhibitory effects against the other five human cancer cell lines, MCF-7, HeLa, NCI-H460, PC9, and SW480. Encouragingly, 1 exhibited no cytotoxicity against nonmalignant cells even with a concentration up to 100 μM. These findings suggest that 1 may display a selective inhibitory effect on the growth of HepG2 cells.

Full text of DOI:10.1021/acs.jnatprod.8b01033

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Fuscasins A−D, Cycloheptapeptides from the Marine Sponge
Phakellia fusca
Ying Wu,†,⊥ Lei Liu,†,⊥ Hai-Feng Chen,‡ Wei-Hua Jiao,† Fan Sun,† Li-Yun Liu,† Hong-Rui Zhu,§
Shu-Ping Wang,*,† and Hou-Wen Lin*,†
†Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji
Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, People’s Republic of China
‡School of Pharmaceutical Sciences, Xiamen University, South Xiangan Road, Xiamen, Fujian 361102, People’s Republic of China
§School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
S
* Supporting Information
ABSTRACT: Four new cycloheptapeptides, fuscasins A−D (1−4), were isolated from the marine sponge Phakellia fusca
collected from the South China Sea. Their planar structures were fully characterized by spectroscopic methods, and the absolute
configurations of amino acid residues were determined using the advanced Marfey’s method. Structurally, 1 is a unique
cycloheptapeptide with a backbone bearing a pyrrolidine-2,5-dione unit. Among the isolated compounds, 1 exhibited potent
growth-inhibitory activity against HepG2 cells with an IC50 value of 4.6 μM, whereas it did not show apparent inhibitory effects
against the other five human cancer cell lines, MCF-7, HeLa, NCI-H460, PC9, and SW480. Encouragingly, 1 exhibited no
cytotoxicity against nonmalignant cells even with a concentration up to 100 μM. These findings suggest that 1 may display a
selective inhibitory effect on the growth of HepG2 cells.
ver since the discovery of spongouridine and spongothy-
Emidine, marine sponges have been in the spotlight of
natural product chemists for drug discovery.1 According to the
literature, 47% of marine natural products have been isolated
from sponges,2 and most have drug-like properties.3 Remark-
ably, sponge-derived cyclic peptides have been actively
investigated as biochemical tools and therapeutic agents, due
to their significant structural diversity, excellent stability, and
effective biological activity.4,5 Among these, the phakellistatins,
mainly isolated from the sponge genus Phakellia, represent
proline-rich compounds usually containing more than six or
seven amino acid residues.6 Since the isolation of phakellistatin
17a from Phakellia costata in the early 1990s, more than 20
phakellistatins have been reported in the past two decades. The
significant cytotoxicity of phakellistatins not only provides
important evidence for the role of proline residues but also has
stimulated the interest of synthetic chemists.7m,8 Phakellistatins
1−37a−c and phakellistatins 7−107g,h in this series have been
synthesized based on their strong cytotoxicity toward P388
murine leukemia cells.9
Herein, we report the isolation and characterization of four
new cycloheptapeptides containing proline residues, fuscasins
A−D (1−4), from Phakellia fusca collected from Yongxing
Island in the South China Sea. Their structures were deduced
by extensive LC-MS analyses, 1D and 2D NMR spectroscopy,
and characterization of degradation products. The cytotox-
icities of these compounds were measured against a number of
cancer cell lines (MCF7, HeLa, NCI-H460, PC9, HepG2, and
Received: December 7, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
SW480), and 1 exhibited selective cytotoxicity against HepG2
cells.
cycle simply accounted for 16 degrees of unsaturation, 1
should bear another ring. Further, an HMBC correlation of
Phe-Hα with the γ carbonyl carbon of Asp indicated that the γ
carbon of Asp was connected to the amino group of the Phe
residue. Based on the above evidence, 1 contains a pyrrolidine-
2,5-dione unit formed by a condensation between the Asp side
chain carboxyl group and the adjacent Phe nitrogen, which
results in a unique cycloheptapeptide backbone.10 The ΔδCβ‑Cγ
values of the Pro residue (5.2, 10.1, and 8.3 for Pro1, Pro2, and
Pro3, respectively) were indicative of one trans and two cis
geometries for the respective proline amide bonds in 1.11 The
absolute configurations of the amino acid units in 1 were
determined by the advanced Marfey’s method.12 The
hydrolysis products (6 N HCl, 110 °C, 12 h) of 1 were
subjected to L-FDLA derivatization and analyzed by UPLC-
ESI-QTOF MS, which showed that all amino acid residues
were L-configurations (Figure 3).
RESULTS AND DISCUSSION
■
Fuscasin A (1) was obtained as a yellowish, amorphous
powder and had a molecular formula of C39H53N7O8 based on
HRESIMS data (m/z 748.4034 [M + H]+) accounting for 17
degrees of unsaturation. The IR spectrum of 1 showed strong
absorption bands at 1714 and 1634 cm−1 (Figure S10),
1
revealing the presence of amide functionalities. The H NMR
spectrum showed three deshielded NH proton signals (δH
7.57, 7.90, and 9.13) and seven α-H (δH 3.88−4.71), indicating
a peptide structure bearing seven residues, while the 13C NMR
spectrum revealed the presence of eight carbonyls (δC 166.7−
173.6) and seven α-amino acid carbon resonances (δC 57.7−
61.0) (Table 1). Additionally, TOCSY experiments allowed
the assignment of seven spin systems, consisting of one Leu,
one Val, one Phe (absence of the NH proton), one Asp, and
three Pro residues. Amino acid side chains were assigned using
COSY, HMBC, and TOCSY NMR spectra whereby
connectivity was determined using HMBC and ROESY
analysis. HMBC correlations from Leu-NH to Val-CO, from
Val-Hα to Phe-CO, from Phe-Hα to Asp-CO, and from Asp-
Hα to Pro3-CO established the five amino acid sequence Pro3-
Asp-Phe-Val-Leu. ROESY correlations from Pro3-Hα and Pro1-
Hα to Pro2-Hα and from Pro1-Hδ to Leu-Hα resulted in the
final sequence of the heptapeptide being assigned as cyclo-
(Pro1-Pro2-Pro3-Asp-Phe-Val-Leu) (Figure 1). The assign-
ments were further confirmed by ESIMS/MS spectra, which
gave a series of “b” fragment ions at m/z 649, 503, 405, 308,
and 211, corresponding to the successive loss of Val, Phe, Asp,
Pro, and Pro (Figure 2). Accordingly, “y” fragment ions at m/z
635, 538, 441, 344, and 246 suggested the successive loss of
Leu, Pro, Pro, Pro, and Asp. Because 1 had 17 degrees of
unsaturation and seven amino acid residues and the macro-
Fuscasin B (2) was isolated as a yellowish, amorphous
powder, and HRESIMS gave an [M + H]+ ion at m/z
814.4574, indicating a molecular formula of C38H59N11O9 and
requiring 15 degrees of unsaturation. The peptidic nature of 2
was evident from the abundance of signals in the amide NH
region (δH 7.34−8.88), from the seven α-H (δH 3.68−4.36) in
1
the H NMR spectrum, and from the eight carbonyl carbons
(δC 170.4−172.8) attributable to amide functionalities in its
13C NMR spectrum (Table 2). TOCSY experiments revealed
seven side chain spin systems, consisting of Pro, Val, Asn, Arg,
Tyr, Ala, and Leu residues. In analogy to 1, the linkages and
assignments of the amino acid residues in 2 were established
by HMBC and ROESY NMR data (Table 2). Two fragments,
Ala-Leu and Pro-Val-Asn-Arg, were indicated by the HMBC
correlations from Leu-NH to Ala-CO, from Arg-NH to Asn-
CO, from Asn-NH to Val-CO, and from Val-NH to Pro-CO.
ROESY correlations from Leu-Hα to Pro-Hα, from Ala-NH to
Tyr-NH, and from Tyr-NH to Arg-NH resulted in the final
B
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. NMR Spectroscopic Data for Fuscasin A (1) at 600 MHz (1H) and 150 MHz (13C) in DMSO-d6
a
residue
Pro1
position
δC, type
δH, mult (J, Hz)
TOCSY
HMBC (H→C)
ROESY
CO
α
169.8, C
58.4, CH
29.2, CH2
4.40, m
Pro1-β, γ, δ
Pro2-α
Pro2-α
β
a: 2.14, m
Pro1-α, γ, δ
Pro1-α, γ, δ
Pro1-α, β, δ
Pro1-α, β, δ
Pro1-α, β, γ
Pro1-α, β, γ
Pro1-CO
b
b: 1.60 , dt (13.7, 7.0)
Pro1-CO
Pro1-Cα
b
γ
24.0, CH2
46.9, CH2
a: 1.75 , m
b: 1.67, m
Pro1-Cα, β
b
δ
a: 3.43,
m
Leu-α
Leu-α
b: 3.26, m
Pro1-Cγ
Pro2
CO
α
169.2, C
57.8, CH
31.1, CH2
4.65, m
a: 2.18, m
Pro2-β, γ, δ
Pro2-α, γ, δ
Pro2-α, γ, δ
Pro2-α, β, δ
Pro2-α, β, δ
Pro2-α, β, γ
Pro2-α, β, γ
Pro2-Cγ
Pro1-α, β, Pro3-α
β
Pro2-CO
Pro2-CO, Cδ
Pro2-Cδ
b
b: 1.83,
m
b
γ
21.0, CH2
46.4, CH2
a: 1.83,
b: 1.78,
m
m
m
m
b
b
δ
a: 3.46,
Pro2-Cα
b
b: 3.40,
Pro3
CO
α
171.5, C
57.9, CH
30.2, CH2
4.42, d (6.4)
a: 2.09, m
b: 2.02, m
Pro3-β, γ, δ
Pro3-α, γ, δ
Pro3-α, γ, δ
Pro3-α, β, δ
Pro3-α, β, δ
Pro3-α, β, γ
Pro3-α, β, γ
Pro3-Cβ, γ, δ
Pro3-CO
Pro2-α, Asp-NH
β
b
γ
21.9, CH2
46.4, CH2
a: 1.83,
b: 1.75,
m
m
m
m
b
b
δ
a: 3.46,
b
b: 3.40,
Asp
Phe
CO
α
173.6, C
48.6, CH
35.2, CH2
4.31, m
a: 2.98,
b: 2.57, dd (17.4, 6.0)
Asp-β, NH
Asp-α, NH
Asp-α, NH
Pro3-CO
Asp-CO, γ-CO
Asp-CO, Cα, γ-CO
b
β
m
Phe-α
Pro3-α
γ-CO
NH
CO
α
173.5, C
9.13, s
Asp-α, β
166.7, C
55.4, CH
34.0, CH2
4.71, m
a: 3.51, m
b: 2.99,
Phe-β
Phe-α
Phe-α
Asp-CO, γ-CO, Phe-CO, Cβ, 1
Phe-CO, Cα, 1−3, 5−6
Phe-CO, Cα, 1−3, 5−6
Phe-2, 6, Asp-β, Val-NH
Val-NH
β
b
m
1
137.1, C
2, 6
3, 5
4
CO
α
129.1, CH
128.5, CH
126.8, CH
169.6, C
61.0, CH
29.8, CH
19.2, CH3
19.0, CH3
7.43, d (7.5)
7.28, t (7.5)
7.20, t (7.8)
Phe-3−5
Phe-2, 4, 6
Phe-2, 3, 5, 6
Phe-Cβ, 2, 4, 6
Phe-C1, 3, 5
Phe-C2, 6
Phe-α
Val
3.88, t (8.6)
1.95, q (7.1)
0.73, d (6.7)
0.70, d (6.6)
7.57, d (9.1)
Val-β, γ, γ′, NH
Val-α, γ, γ′, NH
Val-α, β, NH
Val-α, β, NH
Val-α, β, γ, γ′
Phe-CO, Val-CO, Cβ, γ, γ′
Val-Cα, γ, γ′
Val-Cα, β, γ′
β
γ
γ′
Val-Cα, β, γ
NH
CO
α
Phe-α, β, Leu-NH
Pro1-δ
Leu
167.2, C
47.7, CH
40.9, CH2
4.69, m
Leu-β, γ, δ, δ′, NH
Leu-α, γ, δ, δ′, NH
Leu-α, γ, δ, δ′, NH
Leu-α, β, δ, δ′, NH
Leu-α, β, γ, NH
Leu-α, β, γ, NH
Leu-α, β, γ, δ, δ′
b
β
a: 1.60 , dt (13.7, 7.0)
Leu-CO, Cα, γ, δ, δ′
Leu-Cα, γ, δ, δ′
Leu-Cα, β, δ, δ′
Leu-Cβ, γ, δ′
Leu-Cβ, γ, δ
b: 1.34, m
1.51, m
0.88, d (3.9)
0.89, d (3.7)
7.90, d (9.0)
γ
24.1, CH
22.7, CH3
22.8, CH3
δ
δ′
NH
Val-CO
Val-NH
a
b
Sequential NOEs. Overlapping signals.
sequence of the heptapeptide being assigned as cyclo-(Pro-Val-
Asn-Arg-Tyr-Ala-Leu) (Figure 1), which was subsequently
confirmed using MS/MS experiments (Figure S19). The
ΔδCβ‑Cγ value of the Pro residue (9.4) was indicative of a cis
geometry for the proline amide bond in 2.11 The absolute
configurations of the amino acid residues in 2 were determined
as L-configurations (Figure S20) by advanced Marfey’s
method.12
Fuscasin C (3) was isolated as a yellowish, amorphous
powder, and HRESIMS data (m/z 776.3983 [M + H]+)
supported a molecular formula of C40H53N7O9, requiring 18
degrees of unsaturation. The 1H and 13C NMR data (Table 3)
C
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Key COSY/TOCSY, HMBC, and ROESY correlations of 1−4.
Figure 2. MS/MS fragmentation pattern of 1. The dashed lines through the structures indicate the “y” and “b” fragments (y shown in red, b shown
in blue). Arrows represent direction of sequential fragmentation after initial ring opening during positive-mode MS/MS, and the numbers indicate
the corresponding m/z value (n.d.: not detected).
revealed typical peptide characteristic signals: five deshielded
NH protons (δH 7.46−9.12), seven α-H (δH 3.43−4.59), and
seven amide carbonyls (δC 169.7−171.6). Additionally,
TOCSY experiments revealed seven side chain spin systems,
consisting of one Leu, two Ala, two Pro, and two Tyr residues.
The amino acid side chains were assigned using COSY,
HMBC, and TOCSY NMR spectra whereby connectivity was
delineated using HMBC, ROESY, and ESIMS/MS analysis.
HMBC correlations from Ala1-NH to Pro1-CO, from Leu-NH
to Ala1-CO, and from Tyr1-NH to Leu-CO established the four
amino acid sequence Pro1-Ala1-Leu-Tyr1. HMBC correlations
from Tyr2-NH to Pro2-CO and from Ala2-NH to Tyr2-CO
established the three amino acid sequence Pro2-Tyr2-Ala2.
ROESY correlations from Tyr1-Hα to Pro2-Hα and from Pro1-
Hδ to Ala2-Hα resulted in the final sequence of the
heptapeptide being assigned as cyclo-(Pro1-Ala1-Leu-Tyr1-
D
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 3. Advanced Marfey’s analysis of compound 1 by LC-MS. Selective ion monitoring channels were set up as m/z 410 (a), m/z 426 (b), m/z
460 (c), m/z 428 (d), and m/z 412 (e). Marfey’s derivatives of 1 are shown in red peaks, and standards in green.
Pro2-Tyr2-Ala2) (Figure 1), which was subsequently confirmed
using MS/MS experiments (Figure S31). The ΔδCβ‑Cγ values
of the Pro residues (2.4 and 9.4 for Pro1 and Pro2, respectively)
were indicative of a trans and a cis geometry for the respective
proline amide bonds in 3.11 The absolute configurations of the
amino acid residues in 3 were determined as L-configurations
(Figure S32) via the advanced Marfey’s method.12
bonds in 4.11 The amino acid residues in 4 were determined as
L-form amino acids (Figure S44) using the advanced Marfey’s
method.12
The cytotoxicities of 1−4 were tested against six human
cancer cell lines, MCF-7, HeLa, NCI-H460, PC9, HepG2, and
SW480. As a result, 1 exhibited notable cytotoxicity against
HepG2 cells with an IC50 value of 4.6 μM (Figure S47), and
the remaining isolated compounds proved to be inactive at
concentrations up to 20 μM (Figure S48). Compound 1
showed no cytotoxicity against the nonmalignant rat
cardiomyoblast H9C2 cell line even at concentrations up to
100 μM (Figure S49).
In conclusion, fuscasins A−D (1−4) are new cyclo-
heptapeptides from the sponge genus Phakellia related to the
phakellistatin family, which emphasize the unique structural
features and wide range of biological properties of cyclo-
peptides. Structurally, phakellistatins commonly consist of
seven amino acid residues, including at least one proline
residue. Compounds 1−4 are in line with this structural
feature. Here we report a new cycloheptapeptide, fuscasin A,
which incorporates a pyrrolidine-2,5-dione into the peptide
backbone. This enriches the structural diversity of the
phakellistatin family. Some phakellistatins show cancer cell
cytotoxicities, but there is no obvious regularity based on the
structures. Surprisingly, 1 exhibits growth-inhibitory activity
selectively against the human cancer cell line HepG2 and
shows no cytotoxicity against nonmalignant cells, which
suggests that it has potential to be a targeted antitumor drug.
Fuscasin D (4) was isolated as a yellowish, amorphous
powder. The HRESIMS data showed an [M + H]+ ion at m/z
812.4567, which was suggestive of a molecular formula of
C41H61N7O10, establishing 15 degrees of unsaturation. The 1H
and 13C NMR data (Table 4) showed typical peptide
characteristic signals: five deshielded NH protons (δH 7.17−
9.03), seven α-H (δH 3.99−4.58), and seven amide carbonyls
(δC 168.8−172.1). Detailed analysis of the 2D NMR (HSQC,
TOCSY, and HMBC) spectra of 4 allowed the assignment of
seven spin systems, including those of one Tyr, one Asp, two
Pro, and three Leu residues. The peptide sequence and
connectivity of amino acid residues were determined by
HMBC, ROESY, and ESIMS/MS analysis. HMBC correlations
from Tyr-NH to Asp-CO, from Leu2-NH to Tyr-CO, and from
Leu3-NH to Leu2-CO established the four amino acid
sequence Asp-Tyr-Leu2-Leu3. HMBC correlations from Leu1-
NH to Pro1-CO established the two amino acid sequence Pro1-
Leu1. ROESY correlations from Pro2-Hα to Asp-Hα, from Asp-
NH and Pro2-Hδ to Leu1-Hα, and from Leu1-NH to Leu3-Hα
resulted in the final sequence of the heptapeptide being
assigned as cyclo-(Pro1-Leu1-Pro2-Asp-Tyr-Leu2-Leu3) (Figure
1), which was subsequently confirmed using MS/MS experi-
ments (Figure S43). The ΔδCβ‑Cγ values of the Pro residues
(9.2 and 4.2 for Pro1 and Pro2, respectively) were indicative of
a cis and a trans geometry for the respective proline amide
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a PerkinElmer model 341 polarimeter with a 10 cm
E
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. NMR Spectroscopic Data for Fuscasin B (2) at 600 MHz (1H) and 150 MHz (13C) in DMSO-d6
a
residue
Pro
position
δC, type
δH, mult (J, Hz)
TOCSY
HMBC (H→C)
ROESY
CO
α
170.4, C
60.4, CH
30.8, CH2
4.26, m
a: 2.34, dd (12.2, 6.4)
b: 1.97, m
a: 1.85, dt (13.6, 6.6)
b: 1.48, m
a: 3.43, m
Pro-β, γ, δ
Pro-CO, Cβ, γ, δ
Pro-CO, Cα
Pro-CO
Leu-α, β, δ′
β
Pro-α, γ, δ
Pro-α, γ, δ
Pro-α, β, δ
Pro-α, β, δ
Pro-α, β, γ
Pro-α, β, γ
γ
21.5, CH2
46.1, CH2
δ
b: 3.31, d (11.2)
Val
CO
α
170.7, C
62.6, CH
29.5, CH
19.4, CH3
19.5, CH3
3.71, t (8.4)
Val-β, γ, γ′, NH
Val-α, γ, γ′, NH
Val-α, β, NH
Val-α, β, NH
Val-α, β, γ, γ′
Val-CO, β, γ, γ′
Asn-α
Val-NH
β
2.13, dq (14.6, 6.8)
0.91, d (6.5)
0.93, d (7.0)
8.12, d (8.0)
γ
Val-α, β, γ′
Val-α, β, γ
Pro-CO
γ′
NH
CO
α
Asn-α, NH, Leu-α
Asn
172.8, C
49.6, CH
36.3, CH2
4.32, m
a: 3.18, m
b: 3.06, dd (14.0, 3.4)
Asn-β, NH
Asn-α, NH
Asn-α, NH
Val-α, NH, Arg-NH
Arg-α, NH
Arg-α
β
Asn-γ-CO
Asn-CO
γ-CO
NH
γ-NH2
171.22
7.62, d (5.7)
a: 7.82, s
b: 7.34, d (6.6)
Asn-α, β
Val-CO
Val-NH, γ-NH2
Val-NH
Arg
CO
α
170.8, C
55.4, CH
27.2, CH2
3.68, p (4.1)
Arg-β, γ, δ, NH
Arg-α, γ, δ
Arg-α, γ, δ
Arg-α, β, δ
Arg-α, β, δ
Arg-α, β, γ
Arg-α, β, γ
Arg-CO
Asn-β, Leu-δ′
β
a: 1.49, m
b
b: 1.47,
m
γ
23.7, CH2
40.4, CH2
157.5, C
a: 1.24, m
b: 0.98, m
b
δ
a: 2.87 , d (17.8)
b
b: 2.87, d (17.8)
guanidine
NH
CO
α
8.83, d (8.9)
Arg-α, β, γ, δ
Asn-CO
Tyr-NH, Asn-α, β
Tyr-β, 2, 6
Tyr
171.9, C
55.3, CH
36.0, CH2
4.27, m
a: 3.00, dd (14.0, 3.4)
b: 2.74, t (13.0)
Tyr-β
Tyr-α
Tyr-α
β
Tyr-CO
Tyr-Cα, 1, 2, 6
1
128.5, C
2, 6
3,5
4
129.8, CH
115.0, CH
155.9, C
6.89, d (8.2)
6.62, d (8.0)
Tyr-3, 5
Tyr-2, 6
Tyr-Cβ, 2, 4, 6
Tyr-C1, 3−5
NH
CO
α
8.04, d (9.5)
Tyr-α, β
Ala-α, NH, Tyr-2, 6
Leu-NH, Tyr-NH
Tyr-NH
Ala
172.3, C
48.1, CH
16.2, CH3
4.36, t (17.8)
1.07, d (6.4)
7.34, d (6.6)
Ala-β, NH
Ala-α, NH
Ala-α, β
Ala-CO, Cβ
Ala-CO, Cα
β
NH
CO
α
Leu
170.9, C
51.4, CH
38.2, CH2
4.15, m
a: 1.58, ddd (14.6, 11.9 3.7)
b: 1.21, m
Leu-β, γ, δ, δ′, NH
Leu-α, γ, δ, δ′, NH
Leu-α, γ, δ, δ′, NH
Leu-α, β, δ, δ′, NH
Leu-α, β, γ, NH
Leu-α, β, γ, NH
Leu-α, β, γ, δ, δ′
Pro-α, Val-NH
Pro-α
β
γ
24.3, CH
23.4, CH3
20.7, CH3
1.75, m
δ
0.87, d (6.6)
0.78, d (6.4)
8.88, d (4.7)
Leu-Cβ, γ, δ′
Leu-Cβ, γ, δ
Ala-CO
δ′
NH
Pro-α, Arg-α
Ala-α
a
b
Sequential NOEs. Overlapping signals.
length cell at room temperature. The UV spectra were recorded on a
Hitachi U-3010 spectrophotometer, and the IR spectra were collected
on a Bruker Tensor 27 FT-IR instrument with an ATR accessory.
NMR experiments were conducted on an Agilent 600 MHz NMR
instrument, where chemical shifts (δ) are referenced to the DMSO-d6
residual solvent signal (δH 2.50, δC 39.52). HRESIMS and ESIMS/
MS spectra were acquired with a Waters Xevo G2-XS QTOF
spectrometer. UPLC-MS spectra were obtained using a Waters
Acquity UPLC system equipped with a Waters XevoG2-XS QTOF
spectrometer and a C18 Acquity UPLC HSST3 column (Waters, 2.1
× 100 mm, 1.8 μm). Preparative medium-pressure liquid chromatog-
raphy (MPLC) was carried out on an Interchim PuriFlash 450
F
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 3. NMR Spectroscopic Data for Fuscasin C (3) at 600 MHz (1H) and 150 MHz (13C) in DMSO-d6
a
residue
Pro1
position
δC, type
δH, mult (J, Hz)
TOCSY
HMBC (H→C)
ROESY
CO
α
171.3, C
62.5, CH
28.6, CH2
4.13, m
Pro1-β, γ, δ
Pro1-CO, Cβ
Pro1-Cδ
Pro1-CO, Cβ
Pro1-Cα
Ala2-NH
Ala1-NH
β
a: 2.21, m
b: 1.77, m
a: 2.03, m
b: 1.86, m
a: 3.72, t (8.6)
b: 3.68, m
Pro1-α, γ, δ
Pro1-α, γ, δ
Pro1-α, β, δ
Pro1-α, β, δ
Pro1-α, β, γ
Pro1-α, β, γ
γ
25.0, CH2
46.6, CH2
δ
Ala2-α
Ala1
Leu
CO
α
171.22, C
48.5, CH
16.8, CH3
b
4.18,
m
Ala1-β, NH
Ala1-α, NH
Ala1-α, β
Ala1-CO, Cβ
Leu-α
Leu-NH
Leu-NH, Pro1-β
Tyr1-NH, Ala1-α
β
1.30, d (7.3)
7.70, d (7.2)
Ala1-CO, Cα
NH
CO
α
Pro1-CO, Ala1-Cα, β
171.16, C
50.8, CH
40.1, CH2
4.38, dt (10.7, 5.0)
a: 1.68, m
b: 1.25, m
Leu-β, γ, δ, δ′, NH
Leu-α, γ, δ, δ′, NH
Leu-α, γ, δ, δ′, NH
Leu-α, β, δ, δ′, NH
Leu-α, β, γ, NH
Leu-α, β, γ, NH
Leu-α, β, γ, δ, δ′
Leu-CO
Leu-Cδ
β
Leu-CO, Cγ, δ
Leu-Cδ, δ′
Leu-Cβ, γ, δ′
Leu-Cβ, γ, δ
Leu-Cα, Ala1-CO
γ
24.4, CH
21.8, CH3
23.2, CH3
1.56, m
b
δ
0.93, d (6.5)
δ′
NH
CO
α
0.87, d (6.7)
7.46, d (6.2)
Tyr1-α, NH, Ala1-β, NH
Tyr1
170.1, C
54.0, CH
35.6, CH2
b
4.16,
m
Tyr1-β
Tyr1-α
Tyr1-α
Leu-NH, Tyr1-2, 6, Pro2-α
Tyr1-2, 6, NH
Tyr1-2,6,NH
β
a: 2.88, dd (14.0, 6.1)
b: 2.76, m
Tyr1-CO, Cα, 1, 2, 6
Tyr1-CO, Cα, 1, 2, 6
1
126.0, C
2, 6
3,5
4
NH
CO
α
129.7, CH
115.3, CH
156.1, C
6.94, d (8.1)
6.69, d (8.2)
Tyr1-3, 5
Tyr1-2, 6
Tyr1-Cβ, 2−6
Tyr1-α, β, NH
Tyr1-C1, 3, 4, 5
9.12, d (4.1)
Tyr1-α, β
Leu-CO
Leu-α, NH, Tyr1-β, 2, 6
Tyr1-α
Pro2
169.7, C
60.0, CH
29.9, CH2
3.43, d (7.8)
a: 1.64, m
Pro2-β, γ, δ
Pro2-α, γ, δ
Pro2-α, γ, δ
Pro2-α, β, δ
Pro2-α, β, δ
Pro2-α, β, γ
Pro2-α, β, γ
Pro2-CO, Cβ, γ, δ
Pro2-CO, Cδ
Pro2-CO, Cα
Pro2-Cα
β
b
b: 0.93, d (6.5)
b
γ
20.5, CH2
45.6, CH2
a: 1.31, d (6.8)
b: 0.52, m
b
δ
a: 3.05,
m
Tyr2-α
b: 2.71, m
Tyr2
CO
α
170.3, C
56.5, CH
36.5, CH2
4.08, m
a: 3.06,
b: 2.73, d (3.3)
Tyr2-β
Tyr2-α
Tyr2-α
Tyr2-CO
Tyr2-C1, 2, 6
Tyr2-CO, Cα, 1, 2, 6
Pro2-δ, Tyr2-2, 6
Tyr2-2, 6
b
β
m
1
127.1, C
2, 6
3,5
4
NH
CO
α
129.3, CH
114.9, CH
155.9, C
6.89, d (8.1)
6.64, d (8.3)
Tyr2-3, 5
Tyr2-2, 6
Tyr2-Cβ, 2−6
Tyr2-α, β, NH
Tyr2-C1, 3, 4, 5
7.95, d (7.8)
Tyr2-α, β
Tyr2-Cα, β, Pro2-CO
Ala2-NH, Tyr2-2, 6
Pro1-δ
Ala2
171.6, C
46.8, CH
17.1, CH3
4.59, p (7.2)
Ala2-β, NH
Ala2-α, NH
Ala2-α, β
Ala2-CO, Cβ
b
β
1.31, d (6.8)
Ala2-CO, Cα
NH
7.74, d (7.4)
Ala2-CO, Tyr2-CO
Tyr2-β, NH, Pro1-α
a
b
Sequential NOEs. Overlapping signals.
instrument. Semipreparative reversed-phased HPLC (RP-HPLC) was
performed using a Waters XBridge C18 column (Waters, 10 × 250
mm, 5 μm) connected to a Waters autopurification system consisting
of a binary gradient module (Waters 2545), system fluidics organizer
(Waters SFO), photodiode array detector (Waters 2998), mass
spectrometer (Waters Acquity QDa), and sample manager (Waters
2767) with a Masslynx data handling system. Column chromatog-
raphy was performed on ODS (15 μm, YMC Co.) and Sephadex LH-
20 (18−110 μm, Pharmacia Co.). HPLC-grade CH3CN and MeOH
were purchased from Merck KGaA, and HPLC-grade H2O was
obtained by filtration using a Milli-Q Direct water purification system.
Deuterated NMR solvents were purchased from Cambridge Isotope
Laboratories. 1-Fluoro-2,4-dinitrophenyl-5-L-/D-leucinamide (L-/D-
FDLA) and all amino acid standards were purchased from Sigma-
Aldrich Chemical Corporation.
Sponge Material. The marine sponge Phakellia fusca was
collected off Yongxing Island in the South China Sea in May 2017
and was identified by Prof. Jin-He Li (Institute of Oceanology,
G
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 4. NMR Spectroscopic Data for Fuscasin D (4) at 600 MHz (1H) and 150 MHz (13C) in DMSO-d6
a
residue
Pro1
position
δC, type
δH, mult (J, Hz)
TOCSY
HMBC (H→C)
ROESY
CO
α
171.07, C
60.5, CH
31.2, CH2
4.21, m
a: 2.33, dd (12.1, 6.3)
Pro1-β, γ, δ
Pro1-Cβ, γ, δ
Pro1-CO, Cδ
Pro1-CO, Cα, δ
Pro1-Cα
Leu1-α, NH
β
Pro1-α, γ, δ
Pro1-α, γ, δ
Pro1-α, β, δ
Pro1-α, β, δ
Pro1-α, β, γ
Pro1-α, β, γ
b
b: 2.06,
m
m
m
b
γ
22.0, CH2
46.7, CH2
a: 1.84,
b: 1.51,
b
δ
a: 3.39, m
b: 3.31, m
Pro1-Cβ
Leu1
CO
α
170.2, C
49.7, CH
38.4, CH2
4.58, m
Leu1-β, γ, δ, δ′, NH
Leu1-α, γ, δ, δ′, NH
Leu1-α, γ, δ, δ′, NH
Leu1-α, β, δ, δ′, NH
Leu1-α, β, γ, NH
Leu1-α, β, γ, NH
Leu1-α, β, γ, δ, δ′
Pro2-δ, Pro1-α
b
β
a: 2.07 , m
Leu1-CO
Leu1-Cγ
b: 1.69, m
1.72, m
0.87, d (5.8)
0.98, d (6.0)
9.03, d (8.1)
γ
24.8, CH
21.3, CH3
23.1, CH3
δ
Leu1-Cβ, γ, δ′
Leu1-Cβ, γ, δ
Pro1-CO
δ′
NH
CO
α
Pro2-α, Leu2-α
Pro2
171.13, C
60.8, CH
29.1, CH2
b
4.04,
m
Pro2-β, γ, δ
Pro2-α, γ, δ
Pro2-α, γ, δ
Pro2-α, β, δ
Pro2-α, β, δ
Pro2-α, β, γ
Pro2-α, β, γ
Leu1-NH, Asp-α, NH
b
β
a: 2.06,
b: 1.75,
m
m
Pro2-CO, Cα
Pro2-Cγ,δ
b
γ
24.2, CH2
47.5, CH2
a: 1.94, m
b
b: 1.83,
a: 4.02,
m
m
Pro2-Cα
b
δ
Leu1-α
Leu1-α
b: 3.65, m
Asp
Tyr
CO
α
171.6, C
50.4, CH
36.4, CH2
4.28, m
a: 2.56, m
b: 2.56, m
Asp-β, NH
Asp-α, NH
Asp-α, NH
Asp-CO
Pro2-α
β
Pro2-CO
Asp-CO, γ-CO
γ-CO
NH
CO
α
172.1
8.05, brd
Asp-α, β
Pro2-α
168.8, C
57.7, CH
36.9, CH2
4.18, m
a: 2.81, m
b: 2.67, m
Tyr-β
Tyr-α
Tyr-α
Tyr-CO, Cβ
Tyr-CO, Cα, 1, 2, 6
Tyr-Cα, 1, 2, 6
Leu2-α, NH
β
1
127.1, C
2, 6
3, 5
4
129.9, CH
115.2, CH
156.2, C
6.98, d (8.1)
6.65, d (8.0)
Tyr-3, 5
Tyr-2, 6
Tyr-Cβ, 2, 4, 6
Tyr-Cβ, 1, 3−5
NH
CO
α
7.67, brd
Tyr-α, β
Asp-CO
Leu2
172.0, C
50.9, CH
41.8, CH2
4.35, dt (6.4, 5.2)
a: 1.36, m
b: 1.36, m
Leu2-β, γ, δ, δ′, NH
Leu2-α, γ, δ, δ′, NH
Leu2-α, γ, δ, δ′, NH
Leu2-α, β, δ, δ′, NH
Leu2-α, β, γ, NH
Leu2-α, β, γ, NH
Leu2-α, β, γ, δ, δ′
Leu2-CO, Cβ, γ
Leu2-Cγ
Leu2-Cγ
Leu2-Cβ
Leu2-Cβ, γ, δ′
Leu2-Cβ, γ, δ
Tyr-CO
Leu3-NH, Tyr-α
β
γ
23.9, CH
22.7, CH3
22.8, CH3
1.37, m
b
δ
0.82, d (5.4)
b
δ′
NH
CO
α
0.82 , d (5.4)
7.17, d (7.0)
Tyr-α, Leu3-NH
Leu3
170.4, C
51.4, CH
38.3, CH2
3.99, m
Leu3-β, γ, δ, δ′, NH
Leu3-α, γ, δ, δ′, NH
Leu3-α, γ, δ, δ′, NH
Leu3-α, β, δ, δ′, NH
Leu3-α, β, γ, NH
Leu3-α, β, γ, NH
Leu3-α, β, γ, δ, δ′
Leu3-CO
Leu3-Cα
Leu3-CO
Leu1-NH
b
β
a: 1.52,
m
b: 1.29, m
b
γ
24.1, CH
20.7, CH3
23.4, CH3
1.76, m
δ
0.79, d (6.6)
0.89, d (6.7)
8.56, brs
Leu3-Cβ, γ, δ′
Leu3-Cβ, γ, δ
Leu2-CO
δ′
NH
Leu2-α, Leu2-NH
a
b
Sequential NOEs. Overlapping signals.
Chinese Academy of Sciences, People’s Republic of China). A
voucher sample (No. 2017051502) was deposited at the Marine
Drugs Research Center, Ren Ji Hospital, School of Medicine,
Shanghai Jiao Tong University, China.
H
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Extraction and Isolation. The sponge (4 kg, dry wt) was minced
and exhaustively extracted with 95% EtOH, and combined EtOH
extracts were evaporated to dryness under vacuum. The residue was
then partitioned between H2O and EtOAc, and the organic layer was
further partitioned between 90% MeOH and petroleum ether. The
remaining fraction after removal of the petroleum ether soluble extract
(64.7 g) was concentrated under vacuum to yield 59.6 g and then was
subjected to column chromatography on Sephadex LH-20 with
CH2Cl2/MeOH (1:1, v/v) as eluent, affording four fractions (Fr.1−
4). Fr.2 was fractionated using automated reversed-phase flash
chromatography with a linear gradient from 10% aqueous MeOH
to 100% MeOH over 300 min (flow rate 30.0 mL/min, UV detection
at 200 nm) to afford 15 subfractions (Fr.2.A−O). Fr.2.G was
separated by preparative reversed-phase HPLC (Waters XBridge C18,
5 μm, 19 × 250 mm, linear gradient, from 5% aqueous CH3CN (0.1%
formic acid) to 100% CH3CN over 150 min, 20.0 mL/min) to yield
10 fractions. The fifth fraction was further purified by an automatic
purification system guided by mass spectrometry using semi-
preparative RP-HPLC (Waters XBridge C18, 5 μm, 10 × 250 mm,
5.0 mL/min) eluting with 25% aqueous CH3CN (0.1% formic acid)
to yield fuscasin B (2, 1.2 mg, tR 15.9 min) and fuscasin C (3, 2.8 mg,
tR 17.3 min). Similarly, Fr.2.I.8 and Fr.2.J.3 were purified by the MS-
guided isolation method mentioned above, eluting with 50−60%
aqueous MeOH (0.1% formic acid) to yield fuscasin D (4, 3.7 mg, tR
26.0 min) and fuscasin A (1, 9.1 mg, tR 24.7 min), respectively.
min; L-FDLA-L-Arg 10.03 min, D-FDLA-L-Arg 9.53 min; and L-FDLA-
L-Asp 12.48 min, D-FDLA-L-Asp 13.19 min.
Cytotoxicity Assay. The cytotoxicity assay was performed
according to a previous method.13 The Cell Counting Kit-8 (CCK-
8) method was used for in vitro evaluation of the cytotoxicities of
compounds 1−4 against human cancer cell lines MCF-7, HeLa, NCI-
H460, PC9, HepG2, and SW480 and nonmalignant cells (rat
cardiomyoblast cell line H9C2). The MCF-7, HeLa, HepG2, and
H9C2 cells were cultured at 37 °C in DMEM, while NCI-H460, PC9,
and SW480 cell lines were grown in RPMI 1640. The medium was
supplemented with 10% fetal bovine serum and antibiotics. The cell
lines cited above (3 × 103 cells/well) were treated with test
compounds for 72 h, and then 10 μL of CCK-8 solution was added.
After 1 h of incubation at 37 °C (5% CO2), the optical density (OD)
was recorded at 450 nm by a microplate reader (SpectraMax 190,
Molecular Devices). The half-maximal inhibitory concentration
(IC50) was calculated by fitting the data with a log (inhibitor) values
response model of GraphPad Prism 5.0 software. Cisplatin was used
as the positive control against cancer cell lines MCF-7, HeLa, NCI-
H460, PC9, HepG2, and SW480, with IC50 values of 4.4, 4.8, 3.2, 2.9,
4.2, and 3.8 μM, respectively.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b01033.
Fuscasin A (1): yellowish, amorphous powder; [α]25 −97.8 (c
D
0.90, MeOH); IR (ATR) νmax 3279, 3061, 2956, 2931, 2874, 1714,
1634, 1523, 1438, 1371, 1318, 1242, 1158, 1043, 1026, 922, 823, 751,
1
701 cm−1; H and 13C NMR data, Table 1; ESIMS/MS data, Figure
1D and 2D NMR, HRESIMS, UV, IR spectra, and
cytotoxicity data of 1−4; HRESIMS/MS spectra and the
advanced Marfey’s analysis of 2−4 (PDF)
2; HRESIMS m/z 748.4034 [M + H]+ (calcd for C39H54N7O8,
748.4034).
Fuscasin B (2): yellowish, amorphous powder; [α]25 −47.2 (c
D
0.12, MeOH); UV (MeOH) λmax (log ε) 225 (3.85), 275 (3.59) nm;
IR (ATR) νmax 3586, 3289, 3183, 2957, 2926, 2852, 2363, 1634, 1575,
1556, 1539, 1515, 1446, 1158, 1373, 1346, 1260, 1240, 1170, 1084
cm−1; 1H and 13C NMR data, Table 2; ESIMS/MS data, Figure S19;
HRESIMS m/z 814.4574 [M + H]+ (calcd for C38H60N11O9,
814.4575).
AUTHOR INFORMATION
Corresponding Authors
*E-mail: shupingwang2007@163.com (S. P. Wang).
*E-mail: franklin67@126.com (H. W. Lin).
■
Fuscasin C (3): yellowish, amorphous powder; [α]25 −64.8 (c
ORCID
D
0.25, MeOH); UV (MeOH) λmax (log ε) 225 (4.06), 277 (3.45) nm;
IR (ATR) νmax 3302, 2926, 2872, 1626, 1514, 1450, 1372, 1346, 1237,
1186, 1169, 1099, 1043, 881, 827 cm−1; 1H and 13C NMR data, Table
3; ESIMS/MS data, Figure S31; HRESIMS m/z 776.3983 [M + H]+
(calcd for C40H54N7O9, 776.3983).
Hai-Feng Chen: 0000-0002-8181-7893
Wei-Hua Jiao: 0000-0003-4835-4775
Hou-Wen Lin: 0000-0002-7097-0876
Author Contributions
⊥Y. Wu and L. Liu contributed equally to this work.
Fuscasin D (4): yellowish, amorphous powder; [α]25 −65.4 (c
D
0.32, MeOH); UV (MeOH) λmax (log ε) 225 (3.90), 278 (3.24) nm;
IR (ATR) νmax 3280, 2954, 2921, 2852, 2322, 1631, 1515, 1442, 1389,
Notes
The authors declare no competing financial interest.
1
1366, 1230, 1202, 1174, 1096, 1044, 920, 800 cm−1; H and 13C
NMR data, Table 4; ESIMS/MS data, Figure S43; HRESIMS m/z
ACKNOWLEDGMENTS
812.4567 [M + H]+ (calcd for C41H62N7O10, 812.4558).
■
This project is financially supported by National Key Research
and Development Program of China (No. 2018YFC0310900)
and National Natural Science Foundation of China (Nos.
81402844, 21502113, U1605221, 20720160117, 41576130,
and 81502936). The authors are grateful to Engineer Yi-zhen
Yan (Ren Ji Hospital) for his devotion in sponge sampling.
Absolute Configuration Assignments. Compounds 1−4 (0.1
mg each) were hydrolyzed with stirring in 6 N HCl (200 μL) at 110
°C for 12 h. The residual HCl fumes were removed under a N2
stream. Acid hydrolysates (suspended in 50 μL of H2O) were treated
with 1 M NaHCO3 (20 μL) and then with L-FDLA (100 μL of a 10
mg/mL solution in acetone), and the mixture was stirred at 37 °C for
1 h. The reaction was quenched with 1 N HCl (20 μL) and then
diluted with MeOH for subsequent analysis. Authentic standards of L-
Pro, L-Ala, L-Val, L-Phe, L-Leu, L-Tyr, L-Arg, and L-Asp were treated
with L-FDLA and D-FDLA as described above and yielded the L-
FDLA -L-amino acids and D-FDLA -L-amino acids standards. Marfey’s
derivatives of 1−4 were analyzed by UPLC-HRMS (Acquity UPLC
HSS T3, 2.1 × 100 mm, 1.8 μm, 0.4 mL/min), and their retention
times were compared with those from the authentic standard
derivatives. Retention times for the derivatized amino acid standards
are as follows: L-FDLA-L-Pro 14.33 min, D-FDLA-L-Pro 15.95 min; L-
FDLA-L-Ala 14.19 min, D-FDLA-L-Ala 16.42 min; L-FDLA-L-Val
16.06 min, D-FDLA-L-Val 18.28 min; L-FDLA-L-Phe 17.24 min, D-
FDLA-L-Phe 18.71 min; L-FDLA-L-Leu 17.15 min, D-FDLA-L-Leu
19.12 min; L-FDLA-L-Tyr (di) 20.04 min, D-FDLA-L-Tyr (di) 22.21
REFERENCES
■
(1) (a) Bergmann, W.; Feeney, R. J. J. Am. Chem. Soc. 1950, 72,
2809−2810. (b) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M.
H. G.; Prinsep, M. R. Nat. Prod. Rep. 2017, 34, 235−294.
(2) Miller, J. H.; Field, J. J.; Kanakkanthara, A.; Owen, J. G.; Singh,
A. J.; Northcote, P. T. J. Nat. Prod. 2018, 81, 691−702.
(3) Andersen, R. J. Biochem. Pharmacol. 2017, 139, 3−14.
(4) Molinski, T. F. Org. Biomol. Chem. 2018, 16, 21−29.
(5) Mehbub, M. F.; Perkins, M. V.; Zhang, W.; Franco, C. M. M.
Biotechnol. Adv. 2016, 34, 473−491.
(6) Fang, W. Y.; Dahiya, R.; Qin, H. L.; Mourya, R.; Maharaj, S.
Mar. Drugs 2016, 14, 194−215.
I
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(7) (a) Pettit, G. R.; Cichacz, Z.; Barkoczy, J.; Dorsaz, A. C.; Herald,
D. L.; Williams, M. D.; Doubek, D. L.; Schmidt, J. M.; Tackett, L. P.;
Brune, D. C. J. Nat. Prod. 1993, 56, 260−267. (b) Pettit, G. R.; Tan,
R.; Williams, M. D.; Tackett, L.; Schmidt, J. M.; Cerny, R. L.; Hooper,
J. N. A. Bioorg. Med. Chem. Lett. 1993, 3, 2869−2874. (c) Pettit, G.
R.; Tan, R.; Herald, D. L.; Williams, M. D.; Cerny, R. L. J. Org. Chem.
1994, 59, 1593−1595. (d) Pettit, G. R.; Xu, J.-P.; Cichacz, Z. A.;
Williams, M. D.; Chapuis, J.-C.; Cerny, R. L. Bioorg. Med. Chem. Lett.
1994, 4, 2677−2682. (e) Pettit, G. R.; Xu, J. P.; Cichacz, Z.; Schmidt,
J. M.; Dorsaz, A. C.; Boyd, M. R.; Cerny, R. L. Heterocycles 1995, 40,
501−506. (f) Pettit, G. R.; Xu, J. P.; Cichacz, Z. A.; Williams, M. D.;
Dorsaz, A. C.; Brune, D. C.; Boyd, M. R.; Cerny, R. L. Bioorg. Med.
Chem. Lett. 1994, 4, 2091−2096. (g) Pettit, G. R.; Xu, J.-P.; Dorsaz,
A.-C.; Williams, M. D.; Boyd, M. R.; Cerny, R. L. Bioorg. Med. Chem.
Lett. 1995, 5, 1339−1344. (h) Pettit, G. R.; Tan, R.; Ichihara, Y.;
Williams, M. D.; Doubek, D. L.; Tackett, L. P.; Schmidt, J. M. J. Nat.
Prod. 1995, 58, 961−965. (i) Pettit, G. R.; Tan, R. Bioorg. Med. Chem.
Lett. 2003, 13, 685−688. (j) Li, W. L.; Yi, Y. H.; Wu, H. M.; Xu, Q.
Z.; Tang, H. F.; Zhou, D. Z.; Lin, H. W.; Wang, Z. H. J. Nat. Prod.
2003, 66, 146−148. (k) Pettit, G. R.; Tan, R. J. Nat. Prod. 2005, 68,
60−63. (l) Zhang, H. J.; Yi, Y. H.; Yang, G. J.; Hu, M. Y.; Cao, G. D.;
Yang, F.; Lin, H. W. J. Nat. Prod. 2010, 73, 650−655. (m) Pelay-
Gimeno, M.; Meli, A.; Tulla-Puche, J.; Albericio, F. J. Med. Chem.
2013, 56, 9780−9788. (n) Kwon, O. S.; Kim, C. K.; Byun, W. S.; Oh,
J.; Lee, Y. J.; Lee, H. S.; Sim, C. J.; Oh, D. C.; Lee, S. K.; Oh, K. B.;
Shin, J. J. Nat. Prod. 2018, 81, 1426−1434.
(8) (a) Pettit, G. R.; Rhodes, M. R.; Tan, R. J. Nat. Prod. 1999, 62,
409−414. (b) Pettit, G. R.; Toki, B. E.; Xu, J. P.; Brune, D. C. J. Nat.
Prod. 2000, 63, 22−28. (c) Pettit, G. R.; Lippert, J. W., III; Taylor, S.
R.; Tan, R.; Williams, M. D. J. Nat. Prod. 2001, 64, 883−891.
(d) Napolitano, A.; Rodriquez, M.; Bruno, I.; Marzocco, S.; Autore,
G.; Riccio, R.; Gomez-Paloma, L. Tetrahedron 2003, 59, 10203−
10211. (e) Napolitano, A.; Bruno, I.; Riccio, R.; Gomez-Paloma, L.
Tetrahedron 2005, 61, 6808−6815. (f) Ali, L.; Musharraf, S. G.;
Shaheen, F. J. Nat. Prod. 2008, 71, 1059−1062.
(9) Meli, A.; Tedesco, C.; Della Sala, G.; Schettini, R.; Albericio, F.;
De Riccardis, F.; Izzo, I. Mar. Drugs 2017, 15, 78−93.
(10) Tian, J. M.; Shen, Y. H.; Yang, X. Y.; Liang, S.; Tang, J.; Shan,
L.; Zhang, W. D. Org. Lett. 2009, 11, 1131−1133.
(11) (a) Siemion, I. Z.; Wieland, T.; Pook, K. H. Angew. Chem., Int.
Ed. Engl. 1975, 14, 702−703. (b) Zhan, K. X.; Jiao, W. H.; Yang, F.;
Li, J.; Wang, S. P.; Li, Y. S.; Han, B. N.; Lin, H. W. J. Nat. Prod. 2014,
77, 2678−2684.
(12) (a) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada,
K. Anal. Chem. 1997, 69, 3346−3352. (b) Fujii, K.; Ikai, Y.; Mayumi,
T.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem. 1997, 69, 5146−
5151. (c) Ding, L. J.; Yuan, W.; Liao, X. J.; Han, B. N.; Wang, S. P.;
Li, Z. Y.; Xu, S. H.; Zhang, W.; Lin, H. W. J. Nat. Prod. 2016, 79,
2045−2052.
(13) Liu, L.; Wu, W.; Li, J.; Jiao, W. H.; Liu, L. Y.; Tang, J.; Liu, L.;
Sun, F.; Han, B. N.; Lin, H. W. Biomed. Pharmacother. 2018, 100,
417−425.
J
DOI: 10.1021/acs.jnatprod.8b01033
J. Nat. Prod. XXXX, XXX, XXX−XXX