Varitatin A, a Highly Modified Fatty Acid Amide from Penicillium variabile Cultured with a DNA Methyltransferase Inhibitor

Article abstract of DOI:10.1021/acs.jnatprod.5b00742

A new, highly modified fatty acid amide, varitatin A (1), was isolated from the fungus Penicillium variabile HXQ-H-1 cultivated with the DNA methyltransferase inhibitor 5-azacytidine. The structure including the absolute configuration of 1 was established by analysis of NMR and MS data, together with chemical degradation and Mosher's method based on MPA esters. Compound 1 showed cytotoxicity against HCT-116 cells with an IC₅₀ value of 2.8 μM and also inhibited the effects of protein tyrosine kinases.

Full text of DOI:10.1021/acs.jnatprod.5b00742

Note
pubs.acs.org/jnp
Varitatin A, a Highly Modified Fatty Acid Amide from Penicillium
variabile Cultured with a DNA Methyltransferase Inhibitor
Xueqian He, Zhenzhen Zhang, Yinghan Chen, Qian Che, Tianjiao Zhu, Qianqun Gu, and Dehai Li*
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China
*
S Supporting Information
ABSTRACT: A new, highly modified fatty acid amide,
varitatin A (1), was isolated from the fungus Penicillium
variabile HXQ-H-1 cultivated with the DNA methyltransferase
inhibitor 5-azacytidine. The structure including the absolute
configuration of 1 was established by analysis of NMR and MS
data, together with chemical degradation and Mosher’s
method based on MPA esters. Compound 1 showed
cytotoxicity against HCT-116 cells with an IC50 value of 2.8
μM and also inhibited the effects of protein tyrosine kinases.
arine-derived fungi are an excellent source of novel
M
molecules for drug discovery and have attracted great
1
attention of chemists and biologists. Given that most of the
fungal biosynthetic gene clusters are cryptic under classic
2
laboratory culture conditions, many efforts such as the
OSMAC approach, epigenetic modification, and genome
mining, have been devoted to activate cryptic pathways for
3
−5
exploration of new molecules.
Among them, epigenetic
manipulation has proved to be effective by inhibiting the
activities of histone deacetylase (HDAC) and DNA methyl-
6
,7
transferase (DNMT), which are involved in gene silencing.
During our efforts to tap the potential of marine-derived
fungi to produce diverse secondary metabolites, several HDAC
and DNMT inhibitors were tested on some selected fungal
strains. Among them, the Penicillium variabile strain HXQ-H-1
was found to be sensitive to 5-azacytidine, which led to the
appearance of new peaks in the HPLC-UV profile, indicating
that additional molecules were induced by the DNMT
inhibitor. The chemical investigation of the fermentation
extract led to the discovery of a new compound, named
varitatin A (1), which showed cytotoxicity against the HCT-
the resonances of three nonprotonated carbons including a
carbonyl (δ 165.9), a hemiketal (δ 95.1), and an oxygenated
(δ 75.0) one, seven methylenes, 14 methines including 10
C
C
C
3
olefinic and four sp methines, and two methyls (Table 1). The
planar structure of 1 was established by the 2D NMR data. A
hydrogenated chromene ring was deduced by the COSY
correlations (H-1/H-2/H-3, H-5/H-6/H-7/H-8) and the
HMBC correlations from H-1 and H-7 to C-9, from 9-OH to
C-4, C-8, and C-9, and from H-3 to C-4, C-5, and C-9. The
additional COSY correlations between H-5 and 5-OH and the
HMBC correlations from 4-OH to C-3, C-4, C-5, and C-9
located the two hydroxy groups. The fragment from C-1′ to C-
116 cell line (IC50 = 2.8 μM) and also inhibited the effects of
protein tyrosine kinases (PTKs). Herein, we describe the
isolation, structure elucidation, and bioactivity.
1
1
7′ was constructed by the COSY correlations from H-2′ to H-
7′ and HMBC correlations from H-2′ and H-3′ to C-1′. The
The P. variabile strain was cultured at 20 °C under static
conditions with the presence of 0.2 mM 5-azacytidine. The
EtOAc extract of the fementation (15 L) was injected to
repeated silica gel, Sephadex LH20, and preparative HPLC,
yielding the new compound 1 (100 mg).
fatty acid chain was further connected to the chromene ring via
a nitrogen atom, evidenced by the COSY correlation between
NH (δ 7.08) and H-2 and the HMBC correlations from NH
H
to C-2 and C-1′. Finally, the gross structure was constructed by
attaching the chlorine atom to C-6 with the consideration of
Varitatin A (1) was isolated as a colorless oil, and the
molecular formula was determined as C H ClNO based on
2
6
38
5
the protonated peak at m/z 480.2520 in the HRESIMS
spectrum. The H and C NMR data of compound 1 displayed
Received: August 18, 2015
1
13
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00742
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
1
13
Table 1. H (500 MHz) and C (150 MHz) NMR Data for 1
H-1β and H-3β. The vicinal coupling constant between H-5
3
in Acetone-d6
and H-6 ( J
= 8.0 Hz) indicated their anti orientation
5−6
16
(
Figure 2). However, the absolute configuration of C-14′
varitatin A
cannot be determined by NMR data.
position
1
δC, type
δH (J in Hz)
63.2, CH2
3.66, dd (10.2, 5.2), H-1α
3
.57, dd (10.7, 10.2), H-1β
2
3
44.1, CH
34.6, CH2
4.15−4.08, m
2.52, dd (12.1, 4.5), H-3α
1
.76, dd (12.5, 12.1), H-3β
4
5
6
7
8
9
75.0, C
74.7, CH
63.1, CH
129.5, CH
130.5, CH
95.1, C
4.12, dd (8.0, 7.7)
4.67, ddd (8.0, 2.1, 1.6)
5.72, dd (10.2, 2.1)
5.56, dd (10.2, 1.6)
Figure 2. Key NOESY correlations of 1.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
′
′
′
′
′
′
′
′
165.9, C
124.6, CH
141.0, CH
129.3, CH
140.2, CH
131.1, CH
139.5, CH
33.4, CH2
29.3, CH2
30.0, CH2
33.0, CH2
137.1, CH
129.3, CH
39.2, CH
30.5, CH2
12.1, CH3
20.9, CH3
5.98, d (15.0)
7.16, dd (15.0, 11.4)
6.25, dd (14.8, 11.4)
6.56, dd (14.8, 10.8)
6.18, dd (15.1, 10.8)
5.93, dt (15.1, 6.9)
2.14, q (6.9)
The absolute configuration of the chromene ring was
8
RS
determined by making the MPA esters of 1. The Δδ values
between 2a and 2b (R- and S-MPA esters of 1 on 5-OH,
respectively) were negative for H-5/6/7 and positive for H-1/
2
/3/8 (Figure 3), which indicated the 5R configuration. Then
the 2S, 4S, 5R, 6R, 9S absolute configuration of the bicyclic core
moiety of 1 was deduced and also confirmed by the proton
spectra of 3a and 3b (Figure 3), which were obtained during
the formation of 2a and 2b. For the two sets of proton signals
for an ethanol group linked to a stereogenic carbon with the S
configuration, Minale and co-workers have proved that those in
the R-MTPA derivatives are closer than those in the S-MTPA
′
1.48−1.34, m
1.48−1.34, m
2.00, q (6.8)
0′
1′
2′
3′
4′
5′
6′
7′
5.38, dd (15.3, 6.8)
5.27, dd (15.3, 7.6)
1.98−1.92, m
1.33−1.20, m
0.83, t (7.4)
9
ester, which agreed with the data for 3a and 3b.
To determine the stand-alone stereogenic center C-14′ in the
fatty acid chain, compound 1 was oxidatively digested by
RuCl −NaIO to form 2-methylbutyric acid, which was further
0.94, d (6.7)
3
4
NH
7.08, d (7.6)
esterified by 2-bromo-1-(4-bromophenyl)ethanone to generate
4
5
9
-OH
3.75, s
2
(
-(4-bromophenyl)-2-oxoethyl 2-methylbutanoate (BOMB)
-OH
-OH
4.76, d (7.7)
10−12
4).
By comparing the HPLC traces on a chiral-phase
5.34, s
column of 4 (t = 9.6 min) with the BOMBs derived from
R
commercially available (S)-(+)-2-methylbutyric acid (5: t =
R
the chemical shift of CH-6 (δH 4.67 and δ 63.1) and the
molecular formula.
C
1
9
0.4 min) and the racemic-2-methylbutyric acid (t = 10.4 and
.6 min) (Figure 4), the absolute configuration of C-14′ was
R
The four double bonds in the fatty acid chain were all
assigned the E geometry on the basis of the coupling constants
determined as R.
The cytotoxicity of compound 1 was evaluated against HCT-
16 cells, showing an IC50 value of 2.8 μM. In addition, the
3
3
3
3
(
J2′‑3′ = 15.0 Hz, J
= 14.8 Hz, J
= 15.1, and J
=
4′‑5′
6′‑7′
12′‑13′
1
15.3 Hz) and the NOESY correlations (Figure 1). The relative
inhibitory activities against a panel of PTKs were also screened
in vitro using the enzyme-linked immunosorbent assay
13
(
ELISA), indicating that compound 1 could inhibit the
activity of PDGFR-β and ErbB4 kinases (inhibitory rates of
0% and 40% at a concentration of 1 μM, respectively) (Table
5
S1 in the Supporting Information).
A literature survey showed that the most similar structure to
compound 1 is scyphostatin, which was isolated from the
14
fungus Trichopeziza mollissima. Although the biogenetically
related analogues such as the dankastatins, gymnastatins, and
isariotins were discovered from various fungi including
Figure 1. Selected 2D NMR correlations of 1.
15
16
Gymnascella dankaliensis, Isaria tenuipes, Arachniotus puncta-
1
7
18
configuration of the bicyclic core was determined based on the
NOESY correlations and coupling constants. The NOESY
cross-peaks of 9-OH/H-1β/H-3β/4-OH/H-6 suggested that
these groups were on the same face of the chromene ring and
the axial orientation of H-1β and H-3β. The coupling constant
tus, Ramaria madagascariensis, and Gibellula formosana (with
19
the presence of a DNA methyltransferase inhibitor RG-108),
it is the first time that this kind of highly modified fatty acid
amide was isolated from a Penicillum strain.
3
between H-1β and H-2 ( J = 10.7 Hz) and between H-2 and
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
obtained on a JASCO-1020 digital polarimeter. UV spectra were
1
β‑2
■
3
H-3β ( J2
= 12.5 Hz) suggested that H-2 was axial and
−3β
oriented toward the other face of the chromene ring opposite
B
DOI: 10.1021/acs.jnatprod.5b00742
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
RS
Figure 3. (a) Δδ values of 2a and 2b. (b) Proton spectra of 3a and 3b.
Figure 4. (a) Formation of compounds 4, (S)-5, and racemic-BOMB. (i. RuCl /NaIO ; ii. p-bromophenacyl bromide−KF−DMF). (b) HPLC
3
4
analysis on a chiral Daicel Chiralpack IC column (detected at 254 nm).
recorded on a Waters 2487, while the ECD spectrum was measured on
a JASCO J-815 spectropolarimeter. IR spectra were taken on a Nicolet
azacytidine. The flasks were cultured under static conditions at 20 °C
for 3 weeks.
1
13
NEXUS 470 spectrophotometer as KBr disks. H NMR, C NMR,
DEPT, and 2D NMR spectra were recorded on an Agilent 500 MHz
DD2 spectrometer. HRESIMS and ESIMS data were obtained using a
Thermo Scientific LTQ Orbitrap XL mass spectrometer. Column
chromatography (CC) was performed on silica gel (100−400 mesh,
Qingdao Marine Chemical Factory), Sephadex LH-20 (Amersham
Biosciences), and ODS resin (50 mm, Merck). Preparative HPLC
collection used a C18 column (YMC-Pack ODS-A, 10 × 250 mm, 5
μm, 3 mL/min). Chiral-phase HPLC used a Daicel Chiralpack IC
column (4.6 × 250 mm, 5 μm, 1 mL/min). (S)-(+)-2-Methylbutyric
acid and racemic-2-methylbutyric acid were supplied by Acros Organics
and Aladdin Industrial Corporation, respectively.
Fungal Material. The fungal strain HXQ-H-1 was isolated from
the mangrove rhizosphere soil collected on the coast of Fujian
Province (China) and identified as Penicillium variabile based on
sequencing of the ITS region (GenBank no. KT429657) with 100%
similarity to P. variabile. The strain was deposited at the Key
Laboratory of Marine Drugs, the Ministry of Education of China,
School of Medicine and Pharmacy, Ocean University of China,
Qingdao, People’s Republic of China.
Extraction and Purification. The whole fermentation broth (15
L) was filtered through cheesecloth to separate the supernatant from
the mycelia. The supernatant was extracted with EtOAc (3 × 15 L),
and the mycelia were macerated and extracted with acetone (3 × 5 L).
All extracts were evaporated under reduced pressure to give a crude
gum (10.0 g). The extract was separated by VLC on silica gel using
CH Cl −MeOH (20:1) to give five fractions (fractions 1 to 5).
2
2
Fraction 3 was further separated on a Sephadex LH-20 column with
MeOH to provide five subfractions (fractions 3-1 to 3-5). Fraction 3-3
was separated by MPLC and then semipreparative HPLC eluted with
MeOH−H O (80:20), to obtain compound 1 (100.0 mg, t = 30.5
2
R
min).
Varitatin A (1): colorless oil; [α]24 −22.0 (c 1.0, MeOH); UV
D
(MeOH) λmax (log ε) 300 (4.01) nm; ECD (1 mM MeOH) λmax (Δε)
259 (+1.0), 284 (+0.5), 293 (+0.7), 307 (+0.2), 312 (+0.3), 326
(−0.2), 369 (+0.2) nm; IR (KBr) ν 3296, 2958, 2926, 2854, 1657,
max
−1
1
13
1605, 1369, 1279, 1161, 1045, 1003 cm ; H and C NMR data,
Table 1; HRESIMS m/z 480.2520 [M + H] (calcd for
+
35
C H ClNO , 480.2511).
26
39
5
Cytotoxicity Assay. The cytotoxic activity of 1 was evaluated
using the HCT-116 cell line (human colon cancer) by using the SRB
Fermentation. Erlenmeyer flasks (1 L) containing 300 mL
fermentation media were directly inoculated with spores. The media
contained maltose (20 g), mannitol (20 g), glucose (10 g), sodium
glutamate (10 g), yeast extract (3 g), corn syrup (1 g), KH PO (0.5
2
0
method, with doxorubicin (IC50 = 0.2 μM) as the positive control.
Preparation of MPA Esters Derived from 1 (2a/2b and 3a/
3b). The sample of varitatin A (1) (0.5 mg each) was treated with (R)-
or (S)-MPA (0.4 mg) with DCC (0.5 mg) and DMAP (0.3 mg) in dry
CH Cl (0.5 mL). The reaction was stirred 3 h at 0 °C. The solvent
2
4
g), and MgSO ·7H O (0.3 g) dissolved in 1 L of naturally collected
4
2
seawater (Huiquan Bay, Yellow Sea) in the presence of 0.2 mM 5-
2
2
C
DOI: 10.1021/acs.jnatprod.5b00742
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
was removed in vacuo, and the productions were individually purified
7.4 Hz, 3H); HRESIMS m/z 299.0277 [M + H]+ (calcd for
by RP-HPLC eluting with MeOH−H O (85:15) to obtain the (R)-
C H BrO , 299.0277).
2
13 16
3
MPA ester (2a/3a) and (S)-MPA ester (2b/3b), respectively.
The NMR and MS data obtained for the racemic-2-(4-
bromophenyl)-2-oxoethyl 2-methylbutanoate mixture were the same
as those for compound 5.
1
(
R)-MPA Ester (2a): colorless oil; H NMR (500 MHz, acetone) δH
7
1
1
.53−7.28 (m, 5H), 7.16 (dd, J = 14.9, 11.3 Hz, 1H), 6.56 (dd, J =
4.9, 10.6 Hz, 1H), 6.26 (dd, J = 14.8, 11.3 Hz, 1H), 6.18 (dd, J = 15.2,
0.7 Hz, 1H), 5.95 (d, J = 14.9 Hz, 1H), 5.96−5.89 (m, 1H), 5.72 (dd,
ASSOCIATED CONTENT
Supporting Information
■
J = 10.2, 2.1 Hz, 1H), 5.69 (d, J = 8.6 Hz, 1H), 5.63 (dd, J = 10.2, 1.9
Hz, 1H), 5.37 (dd, J = 14.3, 7.7 Hz, 1H), 5.27 (dd, J = 15.3, 7.7 Hz,
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00742.
1
3
2
H), 5.01 (s, 1H), 4.74 (dt, J = 8.6, 2.0 Hz, 1H), 4.25−4.15 (m, 1H),
.70 (dd, J = 10.2, 4.7 Hz, 1H), 3.59 (t, J = 10.9 Hz, 1H), 3.49 (s, 3H),
.15 (q, J = 7.0 Hz, 2H), 2.01 (dd, J = 13.6, 6.8 Hz, 2H), 1.98−1.92
(
1
m, 1H), 1.85 (dd, J = 13.8, 6.5 Hz, 1H), 1.80 (t, J = 12.9 Hz, 1H),
.50−1.34 (m, 4H), 1.33−1.20 (m, 2H), 0.94 (d, J = 6.7 Hz, 3H), 0.84
The MS and NMR spectra for compounds 1, 2a, 2b, 3a,
3b, 5, and the racemic-BOMB and the IR spectrum and
+
(
t, J = 7.4 Hz, 3H); HRESIMS m/z 650.2852 [M + Na] (calcd for
proposed biosynthetic pathway of 1 (PDF)
35
46
C H ClNO Na, 650.2855).
35
7
1
(
S)-MPA Ester (2b): colorless oil; H NMR (500 MHz, acetone) δH
AUTHOR INFORMATION
Corresponding Author
Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail:
7
1
1
.51−7.27 (m, 5H), 7.21 (dd, J = 14.9, 11.1 Hz, 1H), 6.59 (dd, J =
4.8, 10.8 Hz, 1H), 6.29 (dd, J = 14.8, 11.2 Hz, 1H), 6.20 (dd, J = 15.0,
0.4 Hz, 1H), 5.97 (d, J = 14.8 Hz, 1H), 5.97−5.90 (m, 1H), 5.78 (dd,
■
*
J = 10.2, 2.0 Hz, 1H), 5.73 (d, J = 8.7 Hz, 1H), 5.63 (dd, J = 10.2, 2.1
Hz, 1H), 5.37 (dd, J = 14.6, 7.8 Hz, 1H), 5.27 (dd, J = 15.2, 7.6 Hz,
dehaili@ouc.edu.cn.
Notes
1
3
2
H), 4.93 (s, 1H), 4.85 (dt, J = 8.5, 2.0 Hz, 1H), 4.10−4.05 (m, 1H),
.63 (dd, J = 9.2, 4.8 Hz, 1H), 3.49 (t, J = 10.7 Hz, 1H), 3.40 (s, 3H),
.16 (q, J = 6.6 Hz, 2H), 2.01 (dd, J = 13.4, 6.5 Hz, 2H), 1.98−1.93
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(
1
m, 1H), 1.55 (t, J = 12.8 Hz, 1H), 1.48−1.35 (m, 4H), 1.30 (m, 1H),
■
.29−1.21 (m, 2H), 0.94 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.4 Hz, 3H);
This work was financially supported by the National Natural
Science Fundation of China (21542001 and 21372208), the
Shandong Provincial Natural Science Fund for Distinguished
Young Scholars (JQ201422), the Program for New Century
Excellent Talents in University (NCET-12-0499), the National
High Technology Research and Development Program of
China (2013AA092901), and NSFC-Shandong Joint Fund for
Marine Science Research Centers (U1406402).
+
35
HRESIMS m/z 650.2854 [M + Na] (calcd for C H ClNO Na,
35
46
7
6
50.2855).
1
(R)-MPA Ester (3a): colorless oil; H NMR (500 MHz, acetone) δH
7.46−7.32 (m, 5H), 7.12 (dd, J = 14.9, 11.3 Hz, 1H), 6.83 (dd, J =
10.2, 2.8 Hz, 1H), 6.57 (dd, J = 14.8, 10.8 Hz, 1H), 6.26 (dd, J = 14.8,
11.3 Hz, 1H), 6.20 (dd, J = 15.1, 10.7 Hz, 1H), 5.99 (dd, J = 10.2, 1.8
Hz, 1H), 5.98−5.91 (m, 1H), 5.88 (d, J = 15.0 Hz, 1H), 5.37 (dd, J =
1
4
4.5, 7.5 Hz, 1H), 5.27 (dd, J = 15.3, 7.7 Hz, 1H), 4.95−4.90 (m, 1H),
.86 (s, 1H), 4.37−4.29 (m, 1H), 4.23 (dd, J = 11.0, 6.6 Hz, 1H), 4.17
(
(
dd, J = 11.0, 4.9 Hz, 1H), 3.95 (d, J = 5.8 Hz, 1H), 3.37 (s, 3H), 2.22
dd, J = 14.6, 7.9 Hz, 1H), 2.15 (q, J = 6.8 Hz, 2H), 2.09 (dd, J = 11.4,
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2
1
.0 Hz, 1H), 2.01 (dd, J = 13.7, 6.8 Hz, 2H), 1.98−1.91 (m, 1H),
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+
(
t, J = 7.4 Hz, 3H); ESIMS m/z 650.48 [M + Na] .
Preparation of 2-(4-Bromophenyl)-2-oxoethyl 2-Methylbu-
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tanoate. Varitatin A (1) (5 mg) was dissolved in MeCN−CCl −H O
4
2
(
2:2:3, 0.7 mL), and then NaIO (20 mg) and RuCl (1 mg) were
4
3
added. After stirring overnight at room temperature (rt), the product
was filtered through a small plug of silica eluting with CH Cl (10
2
2
mL), then removed in vacuo. The mixture was further dissolved in
DMF (0.5 mL) and treated with 2-bromo-1-(4-bromophenyl)-
ethanone (20 mg) and KF (10 mg) at rt for 3 h. The product was
purified by RP-HPLC eluting with MeOH−H O (70:30), to obtain 2-
2
(4-bromophenyl)-2-oxoethyl 2-methylbutanoate (4). Similarly, the
(S)-(5) and racemic-BOMB were obtained from the commercial (S)-
(+)-2-methylbutyric acid and racemic-2-methylbutyric acid.
(
S)-2-(4-Bromophenyl)-2-oxoethyl 2-methylbutanoate (5): white
1
powder; H NMR (500 MHz, CDCl ) δ 7.77 (d, J = 8.5 Hz, 2H), 7.63
3
(
1
d, J = 8.5 Hz, 2H), 5.27 (s, 2H), 2.58−2.52 (m, 1H), 1.86−1.69 (m,
H), 1.60−1.48 (m, 1H), 1.22 (dd, J = 14.5, 6.6 Hz, 3H), 0.98 (t, J =
D
DOI: 10.1021/acs.jnatprod.5b00742
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
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7
(
1
(
1
(
5
(
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Cancer Inst. 1990, 82, 1107−1112.
E
DOI: 10.1021/acs.jnatprod.5b00742
J. Nat. Prod. XXXX, XXX, XXX−XXX