Article

Cite This: J. Nat. Prod. 2019, 82, 1503−1509 pubs.acs.org/jnp

Cytotoxic Trichothecene Macrolides Produced by the Endophytic

Myrothecium roridum
Li Shen,*,†,‡,§,⊥ Chun-Zhi Ai,¶ Yong-Chun Song,§ Feng-Wu Wang,§ Rui-Hua Jiao,§ Ai-Hua Zhang,§
Hui-Zi Man,# and Ren-Xiang Tan§
†
Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou 225001, People’s Republic of China
‡
Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases,
Yangzhou University, Yangzhou 225001, People’s Republic of China
§
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
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People’s Republic of China

⊥
Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of
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Veterinary Medicine, Yangzhou University, Yangzhou 225009, People’s Republic of China

¶
Institute for Advanced Study, Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong
Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518061, People’s Republic of China
#
Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian
116023, People’s Republic of China
*
S Supporting Information

ABSTRACT: Six new macrolides named myrothecines D−G

(1−4), 16-hydroxymytoxin B (5), and 14′-dehydrovertisporin
(6), including four 10,13-cyclotrichothecane derivatives, in
addition to 12 known compounds (7−18), were isolated from
three endophytic Myrothecium roridum, IFB-E008, IFB-E009,
and IFB-E012. The isolated compounds were characterized by
MS, NMR, CD, and single-crystal X-ray crystallography. The
isolated macrolides exhibited an antiproliferation effect against
chronic myeloid leukemia K562 and colorectal carcinoma
SW1116 cell lines. Compounds 1−6 were cytotoxic, with IC50
values ranging between 56 nM and 16 μM. Since slight
structural changes led to obvious activity differences, the CoMFA (comparative molecular field analysis) and CoMSIA
(comparative molecular similarity indices analysis) methods were then used to explore the 3D QSAR (three-dimensional
quantitative structure−activity relationship) of these macrolides. The result showed that the steric, electrostatic, hydrophobic,
and H-bond acceptor factors were involved in their cytotoxicity and provided an in-depth understanding of the structure−
activity relationships of these metabolites.

T richothecene fungal toxins with a macrocyclic dilactone

moiety are common bioactive secondary metabolites of
Myrothecium species,1−5 which have attracted attention as
potential anticancer drug leads.6,7 Myrothecium roridum IFB-
E008 and IFB-E009 are endophytic fungi residing in
Trachelospermum jasminoides (Lindl.) Lem., Apocynaceae. M.
roridum IFB-E012 is an endophytic fungus associated with
Artemisia annua L., Asteraceae. To characterize trichothecene
macrolides from endophytic M. roridum strains, fractionation
and isolation of trichothecenes from these three strains guided
by the 1H NMR spectra was carried out. Six new macrolides,
myrothecines D−G (1−4) (four 10,13-cyclotrichothecane
derivatives and named after myrothecines A−C1 in Scheme
S1), 16-hydroxymytoxin B (5), and 14′-dehydrovertisporin (6),
were characterized, in addition to 12 known compounds (7−
18). The new macrolides were cytotoxic against K562 and
SW1116 cell lines, with 14′-dehydrovertisporin (6) active at the
© 2019 American Chemical Society and
American Society of Pharmacognosy
nanomolar level (IC50 56 nM). A detailed three-dimensional
quantitative structure−activity relationship (3D QSAR) analysis
indicated that the cytotoxicity of these trichothecene macrolides
is dependent on steric, electrostatic, hydrophobic, and H-bond-
accepting factors. The possible biosynthetic pathways from
12,13-epoxytrichothec-9-ene macrolide to the 10,13-cyclo-
trichothecane derivative indicated that the endosymbiosis of
microorganisms with host plants may result in substantial
changes in metabolite profiles. This concept is of value in
diversifying secondary metabolites during the discovery process
leading to new drug leads.
Received: December 7, 2018
Published: May 22, 2019
1503 DOI: 10.1021/acs.jnatprod.8b01034
J. Nat. Prod. 2019, 82, 1503−1509
Journal of Natural Products
■
RESULTS AND DISCUSSION
Cultures of M. roridum strains IFB-E008, IFB-E009, and IFB-
E012 were extracted, and these extracts fractionated by a
combination of silica gel column chromatography, ODS column
chromatography, Sephadex LH-20 gel filtration, and HPLC. 1H
NMR was used to track trichothecene macrolides in the extracts
and subfractions thereof using the H-9′ signals around δ 6.5
(ddd, J ≈ 11, 9, 6 Hz) to detect the presence of the
trichothecenes. As a result, myrothecines D−G (1−4), 16-
hydroxymytoxin B (5), and 14′-dehydrovertisporin (6) were
obtained along with myrothecines A−C (7−9),1 12′-hydroxyr-
oridin E (10),8 roridin E (11),4 mytoxin B (12),5 roridin A
(13),9 verrucarin A (14),9 14′-hydroxymytoxin B (15),10 16-
hydroxyroridin E (16),10 16-hydroxyverrucarin J (17),6 and
trichoverritone (18).11
Structure Elucidation of New Macrolides. Myrothecine
D (1), isolated as colorless needle crystals, was ascertained to
have a molecular formula of C29H38O11 by high-resolution
electrospray ionization mass spectrometry (HR-ESIMS). The
1
H and 13C NMR data of 1 (Tables 1 and 2) were similar to
those of myrothecine C (9).1 However, the H-13′ singlet at δ
3.62 in the 1H NMR spectrum of 9 was replaced by a pair of
doublets (J = 5.0 Hz) at δ 3.75 and 5.36 in that of 1. This
observation, along with a hemiketal carbon resonance at δ 105.3,
showed the presence of a 2,3-dihydroxytetrahydrofuran moiety
in the molecule. This assignment was substantiated by the
single-crystal X-ray diffraction analysis of 1 (Figures 1, S10, and
S11).12 The 12′R-configuration of 1, identical to that of
myrothecine A (7), was determined from the Cotton effects at
234 (negative) and 252 nm (positive) in the circular dichroism
(CD) spectrum of 1, resulting respectively from π → π* and n →
π* transitions of the γ-chiral α,β-unsaturated ester chromo-
phore.1 Relative to the chirality of C-12′, the stereochemistry of
1 was convincingly determined as 2R, 4R, 5S, 6R, 9S, 10S, 11R,
12R, 6′S, 12′R, 13′S, and 14′S from its X-ray crystal structure.
Myrothecine E (2), obtained as colorless crystals, was
determined to have a molecular formula of C29H36O11 by HR-
1504
Article
ESIMS. Detailed interpretation of 1D and 2D NMR data of 2
(Tables 1 and 2) demonstrated that it was a 13′-epimer of
myrothecine C (9).1 This assumption was supported by the
NOE effect of H-13′ (δH 4.39) with H-7′ (δH 1.88), which was
not discerned in that of myrothecine C.1 However, the CD
spectrum of 2 (Figure S3) was identical to that of myrothecine
C, establishing its 2R,4R,5S,6R,9S,10S,11R,12R,6′S,12′R,13′S-
configurations.1
Myrothecine F (3), afforded as a white, amorphous powder,
was demonstrated to have a molecular formula of C29H38O10,
identical with that of myrothecine A (7)1 by HR-ESIMS
(569.2357 calcd for C29H38O10Na). The 1H and 13C NMR data
of 3 (Tables 1 and 2) showed that it was the 12′-epimer of 7.1
This was reinforced both by the CD spectrum of 3 (Figure S3),
which was different from that of 7,1 and by the upfield
movement of C-2′ and C-12′ signals by 3.1 and 3.3 ppm,
respectively, because of the increased γ-gauche effect from 12β-
OH.13 Accordingly, compound 3 possesses 2R, 4R, 5S, 6R, 9S,
10S, 11R, 12R, 6′S, 12′S-configurations.
Myrothecine G (4), isolated as colorless crystals, was deduced
to have a molecular formula of C31H40O11 by HR-ESIMS. The
1
H and 13C NMR data of 4 (Tables 1 and 2) were similar to
those of myrothecine A (7).1 However, a set of NMR signals
arising from an acetyl group at δH 2.01 as well as at δC 20.7 and
169.3 suggested a 12′-O-acetyl derivative of 7. This assumption
was confirmed by the HMBC correlations of C-15′ with H-12′,
which appeared at δH 4.96, shifting downfield by 0.99 ppm from
that of 7.1 Compound 4 was shown to share the same absolute
configuration (2R, 4R, 5S, 6R, 9S, 10S, 11R, 12R, 6′S, and 12′R)
as 7 according to its CD spectrum (Figure S3), which was
identical to that of myrothecine A (7).1
16-Hydroxymytoxin B (5, colorless crystals) was indicated to
have a molecular formula of C29H36O10 by HR-ESIMS. The 1H
and 13C NMR data of 5 (Tables 1 and 2) were close to those of
mytoxin B (12).4 Further scrutiny of the 1H and 13C NMR
spectra of 5 and 12 underscored the presence of the 16-
hydroxymethyl of 5, which resonated at δH 3.97 and δC 65.6.
This proposal was confirmed by the 2D NMR experimentations
(1H−1H COSY, NOESY, HMQC, and HMBC), which allowed
the exact assignment of all 1H and 13C NMR signals of 5.
Moreover, the absolute configuration of 5 was assigned as 2R,
4R, 5S, 6R, 11R, 12R, 6′S, and 12′R by its CD curve (Figure S3),
which was nearly identical to that of mytoxin B (12).1
14′-Dehydrovertisporin (6), isolated as colorless needle
crystals, was determined to have a molecular formula of
C29H34O10 by HR-ESIMS. The 2D NMR spectra of 6 allowed
the exact assignment of all 1H and 13C NMR signals (Tables 1
and 2). The presence of a pair of mutually coupled doublets (J =
4.1 Hz) at δH 3.03 and 2.81 ppm (assigned as H-13) and the
similar 1H and 13C NMR data of 6 from C/H-2 to C/H-16 to
those of mytoxin B (12) indicated the presence of the 12,13-
epoxytrichothec-9-ene moiety common to compounds 10 and
12.1 Moreover, the 1H and 13C NMR signals due to the diacyl
residue (from C/H-1′ to C/H-14′) of 6 as well as its CD curve
(Figure S3) were identical to those of myrothecine C (9),
indicating its 6′S, 12′R, 13′R-configurations.1 Thus, the absolute
stereochemistry of 6 was addressed to be 2R, 4R, 5S, 6R, 9S, 10S,
11R, 12R, 6′S, 12′R, and 13′R.
Bioactivity and 3D QSAR of Macrolides. All trichothe-
cene macrolides displayed in vitro cytotoxicity against human
chronic myeloid leukemia K562 and colorectal carcinoma
SW1116 cell lines. In particular, the new compounds 1−6 were
cytotoxic, with IC50 values ranging from 56 nM to 16 μM (Table
DOI: 10.1021/acs.jnatprod.8b01034
J. Nat. Prod. 2019, 82, 1503−1509
Journal of Natural Products Article

Table 1. 1H NMR Data of Compounds 1−6 (δ in ppm, J in Hz)

1a 2b 3c 4d 5b 6b
no. δH δH δH δH δH δH
2 3.94, d (4.0) 3.89, d (4.0) 3.88, d (4.2) 4.00, d (4.1) 3.72, d (5.3) 3.71, d (5.2)
3 2.24, m 2.11, m 2.15, m 2.38, br dd (15.6, 8.0) 2.56, dd (15.4, 8.1) 2.55, dd (8.1, 15.4)
1.83, m 1.91, m 1.88, m 1.84, m 2.04, m 2.02, m
4 5.25, dd (7.9, 2.4) 5.29, dd (8.0, 3.0) 5.12, dd (8.0, 3.0) 5.19, dd (7.9, 2.2) 5.86, m 5.88, dd (6, 8.1)
7 2.18, m 2.17, m 2.15, m 2.15, m 1.95, m 1.92, m
1.58, m 1.57, m 1.61, m 1.55, td (14.6, 5.4) 1.85, m 1.91, m
8 1.81, m 1.79, m 1.80, m 1.78, m 2.13, m 2.00−2.08, m
1.42, br dd (14.5, 4.9) 1.35, m 1.35, br dd (14.3, 5.4) 1.40, m 1.94, m 2.00−2.08, m
10 2.21, m 2.11, m 2.10, m 2.16, m 5.65, dd (4.6) 5.41, d (3.8)
11 3.58, d (3.5) 3.58, d (3.5) 3.57, d (3.8) 3.62, d (3.6) 3.80, (d, 5.0) 3.75, d (5.2)
13 1.93, dd (13.7, 12.0) 1.85, m 1.85, m 1.93, m 3.03, d (4.0) 3.03, d (4.1)
1.55, m 1.57, m 1.57, m 1.47, dd (13.9, 5.4) 2.80, d (4.0) 2.81, d (4.1)
14 1.15, s 1.13, s 1.15, s 1.10, s 0.82, s 0.85, s
15 4.56, d (11.5) 4.52, d (11.5) 4.52, d (11.6) 4.55, d (11.7) 4.21, d (12.5) 4.21, br d (12.6)
3.64, d (11.5) 3.62, d (11.5) 3.54, d (11.6) 3.65, d (11.7) 3.93, d (12.5) 4.20, br d (12.6)
16 1.23, s 1.18, s 1.18, s 1.25, s 3.97, m 1.71, s
3.97, m
2′ 5.99, br s 6.23, s 6.06, br s 6.00, br s 5.86, br s 6.21, s
4′ 3.52, br d (13.4) 3.60, br d (14.0) 3.86, br d (13.4) 3.57, d (13.3) 3.55, d (12.9) 3.63, br d (12.9)
2.64, ddd (13.3,12.5,6.6) 2.43, m 2.30, m 2.53, m 2.76, m 2.46, dd (12.6, 5.7)
5′ 4.14, td (11.5, 2.7) 4.13, td (12.5, 2.5) 4.14, t (11.0) 4.23, t (11.7, 2.3) 3.93, m 4.10, t (11.0)
4.01, br dd (11.0, 6.2) 3.92, m 3.91, br dd (11.2, 6.1) 4.06, dd (11.0, 5.9) 4.03, m 3.91, dd (10.8, 5.5)
7′ 1.92, m 2.02, m 1.97, m 2.05, m 2.30, ddd (13.6,13.6, 7.8) 2.02, m
1.65, m 1.88, m 1.90, m 1.70, m 1.57, td (13.6, 3.0) 2.00, m
8′ 2.74, m 2.71, m 2.75, m 2.83, m 2.76, m 2.12, td (4.8, 13.9)
2.32, m 2.38, m 1.58, m 1.70, m 1.67, m 1.78, td (3.5, 13.9)
9′ 6.55, ddd (11.6, 9.2, 6.3) 6.57, m 6.51, m 6.42, m 6.53, m 6.61, m
10 5.89, d (11.6) 5.87, dd (11.5, 1.0) 5.80, dd (11.6, 2.3) 5.78, dd (11.6, 2.1) 5.78, dd (11.6, 1.8) 5.83, dd (1.8, 11.6)
12′ 4.26, br s 4.61, s 4.46, br s 4.96, br s 4.02, s 4.65, s
13′ 3.75, d (5.0) 4.39, s 3.81, s
14′ 5.36, d (5.0) 2.25, s 2.30, s 2.21, s
16′ 2.01, s
a
Recorded at a Bruker DRX500 in CD3OD. bRecorded at a Bruker DRX500 in acetone-d6. cRecorded at a Bruker DPX300 in acetone-d6.
d
Recorded at a Bruker DRX500 in CDCl3.

3). Furthermore, the structural diversity of this collection of
compounds enabled insight into the structure−activity relation-
ships. Briefly, the 12′,13′-epoxide group contributes to the
activity, and removing this decreases cytotoxicity. The 12′-O-
acetyl group in compound 4 enhanced bioactivity compared to
compound 7. The introduction of 16-OH into roridin E (11)
and mytoxin B (12) (compared to compound 16 and compound
5, respectively) reduced the activity remarkably. The cytotox-
icity was shown to be chirality-dependent by the difference in
bioactivity between roridin E (11) and epiisororidin E (21), as
well as between myrothecines A (7) and F (3). The CoMFA
(comparative molecular field analysis) and CoMSIA (com-
parative molecular similarity indices analysis) approaches were
coapplied for the subsequent 3D QSAR study,14,15 which would
shed light on the optimization for drug leads derived from this
type of compound. Vertisporin (19),16 12′-episatratoxin H
(20),17 epiisororidin E (21),18 and 16-hydroxyverrucarin A
(22),7 isolated by us from Myrothecium sp. Z16 (not reported),
were also evaluated for their cytotoxicity for the 3D QSAR study.
For K562 and SW1116 cell-based data sets, the CoMFA and
CoMSIA 3D QSAR models were generated on the basis of the
training sets of 17 compounds (Table S2). The CoMFA and
CoMSIA models have good predictabilities (Figure 2), and the
pIC50’s for both K562 and SW1116 cells are shown in Table S3.
1505
The 3D contour maps for the CoMFA and CoMSIA models of
K562 cells are shown in Figure 3 and indicate the relationship
between the structural features and bioactivity. Because of its
strong activity (IC50 values to K562 and SW1116 cell lines: 10
and 30 nM), the contours of steric, electrostatic, hydrophobic,
and H-bond-accepting factors were aligned for epiisororidin E
(21). In the steric contour map (Figure 3a), a large green plot is
located around the region of C-6′, C-8′, and C-12′ of the
macrocyclic ring of 21, suggesting that the activity would be
increased if this area is bulky. Compounds with an expanded
ring, such as compounds 13, 14, 17, 18, 21, and 22, show higher
activities. In the electrostatic contour map (Figure 3b), the blue
contour near the C-1′ and C-11′ positions indicates that the
electropositivity around this region would enhance activity, and
the red plot at the 12,13-epoxide suggests that an electronegative
group at the position is desirable for bioactivity. The yellow
contours around C-1′ and C-3′ (Figure 3c) indicate that the
hydrophobic groups around this position increase the activity,
while the gray plot near the C-6′ demonstrates the positive
dependence of activity upon the hydrophilicity near this region,
as shown with vertisporin (19) > 14′-dehydrovertisporin (6)
and 12′-episatratoxin H (20) > 16-hydroxymytoxin B (5). The
gray block over the C-5, C-6, and C-4 regions also reveals a
positive dependence of activity upon the hydrophilicity; for
DOI: 10.1021/acs.jnatprod.8b01034
J. Nat. Prod. 2019, 82, 1503−1509
Journal of Natural Products Article

Table 2. 13C NMR Data of Compounds 1−6 (δ in ppm)

1a 2b 3c 4d 5b 6b
no. δC, type δC, type δC, type δC, type δC, type δC, type
2 84.6, CH 81.6, CH 81.6, CH 80.8, CH 79.6, CH 79.6, CH
3 41.8, CH2 40.8, CH2 41.4, CH2 40.4, CH2 35.6, CH2 35.5, CH2
4 82.9, CH 79.3, CH 79.4, CH 79.2, CH 74.6, CH 74.6, CH
5 53.2, C 52.0, C 51.9, C 51.3, C 50.4, C 50.4, C
6 45.8, C 44.7, C 44.5, C 44.0, C 44.2, C 43.8, C
7 30.6, CH2 28.8, CH2 29.0, CH2 28.2, CH2 20.9, CH2 21.1, CH2
8 32.6, CH2 31.9, CH2 31.9, CH2 31.3, CH2 23.6, CH2 28.1, CH2
9 74.8, C 72.9, C 72.9, C 73.6, C 143.5, C 139.5, C
10 46.0, CH 45.0, CH 45.1, CH 44.2, CH 119.2, CH 120.5, CH
11 71.6, CH 70.0, CH 70.1, CH 69.0, CH 67.7, CH 67.9, CH
12 78.7, C 77.4, C 77.5, C 78.1, C 66.0, C 66.0, C
13 30.0, CH2 30.3, CH2 30.6, CH2 28.2, CH2 47.6, CH2 47.6, CH2
14 11.8, CH3 10.9, CH3 11.0, CH3 10.3, CH3 8.30, CH3 8.30, CH3
15 75.2, CH2 74.2, CH2 73.9, CH2 73.0, CH2 64.8, CH2 65.0, CH2
16 28.4, CH3 28.0, CH3 27.9, CH3 28.1, CH3 65.6, CH2 23.2, CH3
1′ 168.1, C 166.0, C 167.2, C 165.3, C 167.1, C 166.5, C
2′ 119.9, CH 121.5, CH 114.2, CH 120.1, CH 117.1, CH 120.4, CH
3′ 154.8, C 149.0, C 156.8, C 149.6, C 156.6, C 150.0, C
4′ 27.9, CH2 26.2, CH2 31.4, CH2 25.9, CH2 26.4, CH2 26.1, CH2
5′ 66.0, CH2 64.4, CH2 63.9, CH2 63.2, CH2 63.9, CH2 65.6, CH2
6′ 85.9, C 82.4, C 87.8, C 85.7, C 88.7, C 73.3, C
7′ 30.8, CH2 29.0, CH2 25.0, CH2 29.5, CH2 28.5, CH2 22.5, CH2
8′ 23.8, CH2 22.5, CH2 21.2, CH2 21.1, CH2 22.7, CH2 22.6, CH2
9′ 149.9, CH 148.4, CH 148.5, CH 147.9, CH 150.4, CH 150.7, CH
10′ 123.1, CH 122.3, CH 122.1, CH 120.9, CH 121.6, CH 121.7, CH
11′ 168.6, C 166.9, C 167.1, C 165.8, C 166.6, C 166.7, C
12′ 85.3, CH 81.1, CH 74.0, CH 77.3, CH 78.1, CH 84.6, CH
13′ 80.3, CH 76.8, CH 209.6, C 211.6, C 212.3, C 73.2, CH
14′ 105.3, CH 175.6, C 25.3, CH3 28.0, CH3 29.0, CH3 175.9, C
15′ 169.3, C
16′ 20.7, CH3
a
Recorded at a Bruker DRX500 in CD3OD. bRecorded at a Bruker DRX500 in acetone-d6. cRecorded at a Bruker DPX300 in acetone-d6.
d
Recorded at a Bruker DRX500 in CDCl3.

cells (Figure 4). Figure 4a shows the steric contour maps with
the yellow contour near C-1′ and C-3′, suggesting that reduction
of bulky groups at the position would increase the activity, and
the green contour near C-6′, C-2, and C-5 indicated that bulky
groups at this position would increase the activity. The
electrostatic contour maps are displayed in Figure 4b, in
which blue contour near the C-13′, C-3′, and C-9′ positions
suggests that the activity would be enhanced by the electro-
positivity. The theoretical prediction regarding the steric,
electrostatic, hydrophobic, and H-bond-accepting factors agrees
well with the experimental data, underpinning its guiding value
in the drug lead optimization starting from this collection of
macrolides.

Figure 1. X-ray crystal structure of myrothecine D (1, five H2O
molecules omitted for clarity).
example, the cytotoxicity of 16-hydroxyverrucarin A (22) is
higher than that of roridin A (13), 12′-hydroxyroridin E (10),
and 16-hydroxyroridin E (16). In the CoMSIA acceptor contour
map (Figure 3d), the magenta contours located at the ester
carbonyl C-1′ and C11′ indicate the hydrogen bond acceptor
would increase the activity, while the red at the C-16 position
illustrates that the activity would be weakened by a H-bond-
accepting substituent. The steric and electrostatic contour maps
were generated for vertisporin (19) with the model of SW1116
■
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
measured on an XT-4 apparatus and are uncorrected. Optical rotation
was determined in MeOH on a WXG-4 disc polarimeter, and IR spectra
in KBr disks on a Nexus 870 FT-IR spectrometer. The UV spectra were
recorded on a Hitachi U-3000 spectrophotometer. NMR spectra were
acquired on Bruker DRX-500 and DPX-300 NMR spectrometers using
solvent signals as internal standards. HR-ESIMS were taken on a
Mariner Mass 5304 instrument. CD spectra were recorded on a Jasco J-
810 circular dichroism spectrometer. The ELISA plate reader was from
Sunrise, USA. HPLC were performed with an Apollo C18 (250 × 4.6
mm, 5 μm) column, a Hitachi pump L-7100, and a UV detector L-7400.

1506 DOI: 10.1021/acs.jnatprod.8b01034

J. Nat. Prod. 2019, 82, 1503−1509
Journal of Natural Products Article

Table 3. Cytotoxicity of Compounds 1−22 Expressed in IC50 MeOH−H2O (50:50, 1.2 mL/min) to give 3 (8 mg, tR = 15.5 min). The
Values (μM) HPLC separation of Fr-3-3-3 using MeOH−H2O (60:40, 1.0 mL/min)
afforded 4 (6 mg, tR = 14.2 min). The dried extract (19.8 g) derived
compound K562 SW1116 from IFB-E008 was first fractionated as described elsewhere.1 Fr-5 was
myrothecine D (1) 8.20 0.57 purified by silica gel column chromatography with a CHCl3−MeOH
myrothecine E (2) 15.98 11.61 gradient (100:0 → 100:16) followed by gel filtration over Sephadex
myrothecine F (3) 0.97 10.62 LH-20 with CHCl3−MeOH (1:1) to yield 1 (4.7 mg). The dried
extract derived from IFB-E009 was separated as outlined earlier.1 The
myrothecine G (4) 1.53 4.25
obtained Fr-2 was chromatographed over a silica gel column with
16-hydroxymytoxin B (5) 2.87 0.18
CHCl3−MeOH (100:1 → 100:8), and the afforded subfraction Fr-2-3
14′-dehydrovertisporin (6) 0.056 0.20 was repeatedly purified over a Sephadex LH-20 column using CHCl3−
myrothecine A (7) 31.59 22.71 MeOH (1:1) to provided 6 (6 mg).
myrothecine B (8) 26.69 23.13 Myrothecine D (1): colorless needles; mp 195−197 °C; [α]25 D −35.5
myrothecine C (9) 15.95 21.07 (c 0.28 CH3OH); UV (MeOH) λmax (log ε) 213 (4.21) nm; CD
12′-hydroxyroridin E (10) 3.15 7.92 (CH3OH) λmax (Δε) 234 (−4.30), 252 (+0.36) nm; IR (KBr) νmax
roridin E (11) 0.54 0.039 3408, 2962, 2926, 1714, 1657, 1635, 1413, 1397, 1253, 1191, 1061,
mytoxin B (12) 0.0013 0.0015 1024, 996 cm−1; 1H and 13C NMR, see Tables 1 and 2; HR-ESIMS m/z
roridin A (13) 0.0071 0.21 585.2305 [M + Na]+ (calcd for C29H38O11Na, 585.2306).
verrucarin A (14) 0.0030 0.0018 Myrothecine E (2): colorless crystals; mp 258−260 °C; [α]25 D +47.1

14′-hydroxymytoxin B (15) 0.92 0.55
16-hydroxyroridin E (16) 7.13 9.06
16-hydroxyverrucarin J (17) 0.0064 0.16
trichoverritone (18) 0.0012 0.0031
vertisporin (19) 0.0011 0.0015
12′-episatratoxin H (20) 0.51 2.27
epiisororidin E (21) 0.0010 0.033
16-hydroxyverrucarin A (22) 0.0017 0.012
5-fluorouracil (control) 32.92 76.92
Silica gel (200−300 mesh) for column chromatography and silica GF254
for TLC were produced by Qingdao Marine Chemical Company,
China. Sephadex LH-20 was purchased from Pharmacia Biotech,
Sweden. ODS silica gel was from Nacalai Tesque, Kyoto, Japan. All
chemicals used in the study were of analytical grade.
Endophytic Strains. The M. roridum IFB-E008 and IFB-E009 were
separated from T. jasminoides (Apocynaceae), and IFB-E012 from A.
annua (Asteraceae). The three fungal strains were cultured on PDA
medium on Petri dishes for morphologic observation. The spore masses
(colony) look viscous and green when young and turn hard and black
while aging. Conspicuous sporodochia were formed on densely
compacted branching conidiophores, with the ultimate branches
being phialides. Phialides were cylindrical with conically tapering tips
and undifferentiated collarettes. Conidia were olive-brown, single-
celled, and cylindrical with both ends rounded ((5.2−7.3) × 2.0 μm).
The identification of these M. roridum strains was reinforced by their
18S rDNA sequences, which showed a 99% similarity to those
accessible at the BLASTN of M. roridum. The sequences of M. roridum
IFB-E008, IFB-E009, and IFB-E012 have been deposited as EF211124,
EF211125, and DQ102373 in GenBank, respectively. The live cultures
of the three M. roridum strains were kept at the Institute of Functional
Biomolecules, Nanjing University (China).
Isolation of New Macrolides (1−6). The endophytic strains were
cultured as detailed previously,1 and the obtained biomasses were
extracted exhaustively with EtOAc or MeOH, respectively. The dried
extract derived from IFB-E012 was fractionated into six parts (Fr-1−Fr-
6), and Fr-3 was subjected to further column chromatography over
silica gel with a CHCl3−MeOH gradient (100:0 → 100:16). The
macrolide-containing subfraction traced by 1H NMR was further
separated through gel filtration over Sephadex LH-20 with CHCl3−
MeOH (1:1) followed by ODS column chromatography eluting with
H2O−MeOH gradient (100:0 → 80:20) and purified by semi-
preparative HPLC using MeOH−H2O (50:50, 1 mL/min) to yield 2
(6 mg, tR = 6 min). Gel filtration of the subfraction Fr-3-3 over
Sephadex LH-20 with CHCl3−MeOH (1:1) followed by silica gel
column chromatography with CHCl3−MeOH gradient (100:0 →
100:16) gave Fr-3-3-1, Fr-3-3-2, and Fr-3-3-3. Fr-3-3-1 was further
purified by HPLC using MeOH−H2O (47:53, 1.2 mL/min) to afford 5
(10 mg, tR = 32 min). Fr-3-3-2 was further purified by HPLC using
(c 0.40 CH3OH); UV (MeOH) λmax (log ε) 219 (4.62) nm; CD
(CH3OH) λmax (Δε) 234 (−3.06), 249 (+0.57), 270 (−0.20) nm; IR
(KBr) νmax 3393, 2961, 2928, 1713, 1659, 1636, 1414, 1249, 1180,
1156, 1056, 1024, 774 cm−1; 1H and 13C NMR data, see Tables 1 and 2;
HR-ESIMS m/z 583.2151 [M + Na]+ (calcd for C29H36O11Na,
583.2150).
Myrothecine F (3): white powder; mp 160−163 °C; [α]25 D −15.0 (c
0.53 CH3OH); UV (MeOH) λmax (log ε) 219 (4.13) nm; CD
(CH3OH) λmax (Δε) 235 (−5.60), 255 (−0.06), 296 (−4.02) nm; IR
(KBr) νmax 3445, 2958, 2919, 1713, 1692, 1650, 1414, 1385, 1250,
1182, 1154, 1100, 1057, 1022, 819, 744 cm−1; 1H and 13C NMR data,
see Tables 1 and 2; HR-ESIMS m/z 569.2353 [M + Na]+ (calcd for
C29H38O10Na, 569.2357).
Myrothecine G (4): colorless crystals; mp 250−252 °C; [α]25 D +9.1 (c
0.33 CH3OH); UV (MeOH) λmax (log ε) 220 (4.20) nm; CD
(CH3OH) λmax (Δε) 230 (−4.97), 248 (+1.20), 293 (−0.41) nm; IR
(KBr) νmax 3500, 2960, 2922, 2852, 1748, 1717, 1661, 1413, 1227,
1181, 1154, 1061, 1923, 753 cm−1; 1H and 13C NMR data, see Tables 1
and 2; HR-ESIMS m/z 611.2468 [M + Na]+ (calcd for C31H40O11Na,
611.2463).
16-Hydroxymytoxin B (5): colorless crystals; mp 195−198 °C; [α]25 D
+30.1 (c 1.26 CH3OH); UV (MeOH) λmax (log ε) 220 (4.18) nm; CD
(CH3OH) λmax (Δε) 232 (−1.50), 248 (+3.75), 289 (−1.45) nm; IR
(KBr) νmax 3408, 2962, 1714, 1640, 1413, 1384, 1250, 1231, 1179,
1083, 1056, 1026, 1006, 971, 818, 772 cm−1; 1H and 13C NMR data, see
Tables 1 and 2; HR-ESIMS m/z 567.2197 [M + Na]+ (calcd for
C29H36O10Na, 567.2201).
14′-Dehydrovertisporin (6): colorless crystals; mp 197−199 °C;
D −25 (c 0.20 CH3OH); UV (MeOH) λmax (log ε) 215 (4.40) nm;
[α]25
CD (CH3OH) λmax (Δε) 232 (−2.82), 248 (+1.66) nm; IR (KBr) νmax
3436, 2966, 1794, 1713,1636, 1437, 1411, 1248, 1185, 1092, 993, 967,
818 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HR-ESIMS m/z
565.2047 [M + Na]+ (calcd for C29H34O10Na, 565.2044).
Single-Crystal X-ray Diffraction. Crystal structure determination
of 1 was carried out on a Bruker SMART APEX CCD diffractometer
equipped with graphite-monochromated Mo Kα (λ = 0.710 73)
radiation with Lorentz polarization and absorption corrections for a
crystal of 1 (0.35 mm × 0.20 mm × 0.21 mm). The intensities were
collected at 293 K using the ω-scan mode with variable scan speed. A
total of 3440 reflections were collected in the range of θ = 1.17−25.97°,
of which 1679 were independent, which were used in the structure
solution and refinements. The structure was solved by direct methods
and refined on F2 by full-matrix least-squares methods using SHELX-
97. All the non-hydrogen atoms were refined anisotropically. All the
hydrogen atoms were placed in calculated positions and were assigned
fixed isotropic thermal parameters at 1.2 times the equivalent isotropic
U of the atoms to which they are attached and allowed to ride on their
respective parent atoms. The contributions of these hydrogen atoms
were included in the structure-factor calculations. The refinement gave
the final R1 = 0.1021 with w = [σ2(Fo)2 + (0.1(max(0,Fo2) + 2Fc2)/

1507 DOI: 10.1021/acs.jnatprod.8b01034

J. Nat. Prod. 2019, 82, 1503−1509
Journal of Natural Products
Figure 2. Graphs of the experimental versus predicted pIC50 values of compounds in training and test sets for CoMFA and CoMSIA models. (a) and
(b) represent activities in K562 cells, (c) and (d) denote activities in SW1116 cells. Triangles and circles represent compounds in the training and test
sets, respectively.
3)2]−1.12 Crystallographic and experimental data for myrothecine D (1)
are listed in Table S1.
Cytotoxicity Assay. The in vitro cytotoxicity against K562 and
SW1116 cell lines was evaluated as described earlier.1
3D QSAR. 3D QSAR models were developed using the data set of 22
trichothecene macrolides. The data set was divided into a training set of
17 and a test set of five compounds, respectively. The activity (i.e., IC50
value) of these macrocylic compounds against K562 and SW1116 cells
were converted to the corresponding pIC50’s (= −log IC50’s) (Table
S3). All the compounds’ partial charges were calculated using the
Gasteiger−Huckel method, and their optimal geometry was done using
the Tripos force field with a distance-dependent dielectric function and
an energy convergence criterion of 0.05 kcal/mol Å using 100
iterations, which were done using the SYBYL6.9 program (Tripos Inc.).
It is a crucial step to perform the alignment of molecules in the 3D
QSAR study. In order to obtain a consistent alignment, the lowest
energy conformation of the most active compound in the series was
used as the template; therefore, compounds epiisororidin E (21) and
vertisporin(19) were selected as the templates of SW1116 and K562
cells, respectively. The aligned compound results are similar and are
shown inFigure S2.
1508
Article
CoMFA and CoMSIA are considered be the most reliable methods
for the 3D QSAR study. The Tripos Sybyl 6.9 program was used to
perform CoMFA and CoMSIA modelings. Since the experimental
K562 and SW1116 activities varied significantly, different test and
training sets were used to develop the 3D QSAR models. The CoMFA
models were developed with steric and electrostatic fields, while the
CoMSIA models also explored the impacts of more fields such as
hydrophobic, hydrogen bond donor, and hydrogen bond acceptor in
addition to steric and electrostatic fields. The partial least-squares
analysis method was used to derive the 3D QSAR models, describing
the discrimination between independent variables (the descriptors of
CoMFA and CoMSIA) and dependent variables (the pIC50 values).
The leave-one-out cross-validation was carried out to obtain the
optimal number of components and the highest correlation coefficient,
which was further used to obtain the final 3D QSAR model. The
robustness of the developed models was evaluated with various
parameters such as non-cross-validation correlation coefficient, stand-
ard error of estimate, and Fischer ratio value, and the predictive ability
was further tested by compounds in the test set.
DOI: 10.1021/acs.jnatprod.8b01034
J. Nat. Prod. 2019, 82, 1503−1509
Journal of Natural Products
Figure 3. Contour maps derived from 3D QSAR model of K562 cells.
(a) The green and yellow represent the favorable and unfavorable steric
fields, respectively. (b) The blue and red denote electropositive and
electronegative fields, respectively. (c) The yellow and gray indicate the
favorable and unfavorable hydrophobic fields, respectively. (d) The
magenta and red describe the favorable and unfavorable hydrogen bond
acceptor fields, respectively. Epiisororidin E (21) was overlaid in each
plot.
Figure 4. Contour maps generated from the 3D QSAR model of
SW1116. (a) The favorable and unfavorable steric fields are denoted by
green and yellow plots, respectively. (b) The blue and red represent
electropositive and electronegative fields, respectively. Vertisporin (19)
was overlaid in each map.
■
*
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.8b01034.
CD and NMR spectra of 1−6, structures of compounds
13−22, crystallographic and experimental data for 1, X-
ray molecular structure and X-ray packing diagram of 1,
observed and predicted pIC50’s for 1−22, statistical
results of CoMFA and CoMSIA models, alignment of the
1509
Article
trichothecene macrolide data set, and the proposed
biosynthetic pathway (PDF)
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: shenli@yzu.edu.cn. Tel: +86-514-87992233.
ORCID
Li Shen: 0000-0003-1947-5236
Ren-Xiang Tan: 0000-0001-6532-6261
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The work was cofinanced by the National Natural Science
Foundation of China (21372191, 81121062, 21132004, and
21072092).
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DOI: 10.1021/acs.jnatprod.8b01034
J. Nat. Prod. 2019, 82, 1503−1509