Absolute stereochemistry of pentacecilides, new inhibitors of lipid droplet formation in mouse macrophages, produced by Penicillium cecidicola FKI-3765-1

Article abstract of DOI:10.1038/ja.2010.39

The structure of a new pentacecilide congener, pentacecilide D, produced by Penicillium cecidicola FKI-3765-1 was elucidated by various NMR experiments. The absolute stereochemistry of pentacecilides was elucidated by using the modified Mosher method for

Full text of DOI:10.1038/ja.2010.39

The Journal of Antibiotics (2010) 63, 315–318
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ORIGINAL ARTICLE
Absolute stereochemistry of pentacecilides, new
inhibitors of lipid droplet formation in mouse
macrophages, produced by Penicillium cecidicola
FKI-3765-1
Hiroyuki Yamazaki, Narihiro Ugaki, Daisuke Matsuda and Hiroshi Tomoda
The structure of a new pentacecilide congener, pentacecilide D, produced by Penicillium cecidicola FKI-3765-1 was elucidated
by various NMR experiments. The absolute stereochemistry of pentacecilides was elucidated by using the modified Mosher
method for pentacecilide C. The inhibitory activity of all pentacecilides against lipid droplet formation and acyl-CoA:cholesterol
acyltransferase isozymes was compared.
The Journal of Antibiotics (2010) 63, 315–318; doi:10.1038/ja.2010.39; published online 23 April 2010
Keywords: absolute stereochemistry; acyl-CoA:cholesterol acyltransferase; fungal metabolite; lipid droplet formation; Penicillium
cecidicola; pentacecilides D
INTRODUCTION
CH3CN containing 0.050 % trifluoroacetic acid (250mlꢀ2 tubes for
As we reported previously, pentacecilides A to C (Figure 1) were each solvent). Pentacecilides A and B were present in the first tube of
isolated from the whole culture of Penicillium cecidicola FKI-3765-1 as 100% CH3CN. The first tube of 70% CH3CN containing pentaceci-
inhibitors of lipid droplet formation in mouse macrophages.1,2 From lides C and D was concentrated in vacuo to dryness to give a
the biochemical study,1 pentacecilides inhibited lipid droplet forma- red–brown material. The material (172mg) was finally purified by
tion in mouse macrophages, probably due to the blockade of preparative HPLC (column, PEGASIL ODS, 20ꢀ250 mm; Senshu
acyl-CoA:cholesterol acyltransferase (ACAT) activity. Furthermore, Scientific, Tokyo, Japan; solvent, 55% CH3CN containing 0.050%
pentacecilides appeared as dual inhibitors of ACAT1 and ACAT2 trifluoroacetic acid; detection, UVat 210 nm; flow rate, 8.0 mlminꢁ1).
isozymes.1,3–7 The structures including the relative stereochemistry Under these conditions, pentacecilides C and D were eluted as peaks
were also elucidated;2 however, the indication of the relative stereo- with retention times of 15.8 and 25.8min, respectively. The respective
chemistry at C-2 was revised8,9 because erroneous structures of fractions were concentrated in vacuo to dryness to give pure penta-
pentacecilides had been shown in our previous reports.1,2 Further cecilides C (56.6mg) and D (5.1mg) as white crystals.
precise analysis of metabolites in the whole culture led to the discovery
of a new congener named pentacecilide D (Figure 1). In this study, the Structural elucidation of pentacecilide D
structure of the new pentacecilide congener was elucidated. Further- The physicochemical properties of pentacecilide D are summarized in
more, the absolute stereochemistry of pentacecilides was determined Table 1. Pentacecilide D showed UV absorption maxima at 219, 276
by using the modified Mosher method10 for pentacecilide C.
and 310 nm, similar to pentacecilides A to C.2 IR absorption at 3426
and 1712–1617cmꢁ1 suggested the presence of hydroxy and carbonyl
groups in the structure.
RESULTS
Isolation of pentacecilide D
The molecular formula was determined to be C25H32O7 on the basis
The 13-day-old whole culture (1000g) was extracted with 2.0 l of of HRESI-TOF-MS measurement, indicating that pentacecilide D was
acetone. After the acetone extracts were filtered and concentrated smaller than pentacecilide C.2 The 13C NMR spectrum (in CDCl3)
to remove acetone, the aqueous solution was extracted with ethyl acetate. showed 25 resolved signals, which were classified into five methyl
The extracts were dried over Na2SO4 and concentrated in vacuo to carbons, four methylene carbons, two sp3 methine carbons, one
dryness to yield a red–brown material (1.7g). The material was sp2 methine carbon, three oxygenated sp3 methine carbons, two sp3
dissolved in 30% CH3CN, applied to an octadecyl silyl (ODS) column quaternary carbons, one oxygenated sp3 quaternary carbon, three sp2
chromatography (100g), and eluted stepwise with 30, 50, 70, 100% quaternary carbons, two oxygenated sp2 quaternary carbons and two
Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan
Correspondence: Professor H Tomoda, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
E-mail: tomodah@pharm.kitasato-u.ac.jp
Received 23 February 2010; revised 26 March 2010; accepted 2 April 2010; published online 23 April 2010

Absolute stereochemistry of pentacecilides
H Yamazaki et al
316
Table 2 1H and 13C NMR chemical shifts of pentacecilide D
9’
H3C
O
O
Pentacecilide D
3’
7’
OH
No.
qC
q
H (J in Hz)
1’
5’
1
44.9
1.60 m
2.38 m
4.64 m
—
11
15
CH3
Pentacecilide
R1
-H
R2
-H
1
9
R1
O
O
2
3
4
5
6
69.9
214.8
47.6
A
B
C
D
12
CH3
H
-OAc
-OAc
-OH
-H
3
—
5
7
-OH
-OH
46.4
1.80 m
1.86 m
2.18 m
R2
H
26.6
H3C
CH3
13 14
7
71.8
79.2
4.13 dd (10.0, 3.0)
Figure 1 Structures of pentacecilides A–D.
8
—
9
43.4
2.21 m
10
11
12
13
14
15
1¢
2¢
3¢
4¢
5¢
36.0
—
Table 1 Physicochemical properties of pentacecilide D
21.5
2.52 m
20.9
1.23 s
Pentacecilide D
20.9
1.20 s
Appearance
White crystaline solid
444
25.4
1.19 s
Molecular weight
Molecular formula
23.9
1.54 s
C25H32O7
110.9
139.4
103.6
162.4
103.6
159.1
31.9
—
—
HRESI-TOF-MS (m/z)
Calcd:
—
445.2226 (M+H)+
445.2216 (M+H)+
—
6.34 s
Found:
6¢
7¢
—
UV (MeOH) lmax, nm (e)
219 (34800), 276 (24700), 310 (11800)
ꢁ51.41 (c 0.10, CHCl3)
2.71 dd (17.0, 11.0)
2.84 dd (17.0, 3.5)
4.64 m
26
[a]D
IR (KBr) nmax (cmꢁ1
)
3426, 1712, 1666, 1617, 1473
8¢
9¢
10¢
4¢-OH
74.7
20.9
1.56 d (7.0)
—
170.0
carbonyl carbons by analysis of the DEPT and HSQC spectra. The 1H
NMR spectrum (in CDCl3) displayed 30 proton signals, one of which
was suggested to be a hydroxy proton (d 11.09). Taking the molecular
formula into consideration, the presence of two hydroxy protons was
suggested. The connectivity of proton and carbon atoms was esta-
11.09 s
blished by the 13C-1H HSQC spectrum (Table 2). These spectral data calculated Dd values (Dd¼dSꢁdR) of H2-11, H3-12, H-5¢ and H-8¢
from NMR and physicochemical properties showed that pentacecilide were positive, whereas those (Dd¼dSꢁdR) of H2-1, H-2, H-5 H2-6,
D has the same skeleton as pentacecilide C.2 Comparison of the H H-9, H3-13, H3-14, H3-15 and H3-17 were negative, indicating that
1
NMR spectra between pentacecilides C and D indicated that the the absolute stereochemistry of C-7 was elucidated to be 7S. Accord-
methyl proton signals (C-17, d 2.17) derived from the acetyl group in ingly, the absolute stereochemistry of the seven chiral centers in penta-
pentacecilide C disappeared from pentacecilide D. In addition, the cecilide C was concluded to be 2R5R7S8R9R10S8¢S, as shown in
chemical shift of C-2 (d 69.9) and the molecular formula showed the Figure 1. It is plausible that all pentacecilides are biosynthesized by the
presence of a hydroxy group at C-2. These data led to the conclusion same pathway; therefore, the absolute stereochemistry of the other
that the structure of pentacecilide D was 2-deacetyl pentacecilide C pentacecilides was deduced to be the same as that of pentacecilide C
(Figure 1). The structure satisfied the degree of unsaturation and the (Figure 1).
molecular formula.
Regarding the relative stereochemistry of pentacecilide D, NOESY Biological properties
experiments were carried out showing coupling constants in 1H NMR Inhibition of CE synthesis in macrophages and ACAT1- and ACAT2-
similar to pentacecilide C.2,8,9 Accordingly, the relative stereochemistry CHO cells. In the previous study, pentacecilides inhibited cholesteryl
of pentacecilide D was elucidated as shown in Figure 1.
ester (CE) synthesis in mouse macrophages and ACAT1- and ACAT2-
CHO cells, strongly suggesting that they were dual inhibitors of
ACAT1 and ACAT2 isozymes.1 Pentacecilide D and semisynthetic
Absolute stereochemistry of pentacecilides
To elucidate the absolute stereochemistry of pentacecilides, the (S)- 3S*-hydroxy pentacecilide A (3) were evaluated in these three cell-
(1) and (R)- a-methoxy-a-(trifluoromethyl)phenylacetyl (MTPA) based assays, and the IC50 values of CE synthesis were compared.
esters (2) were prepared by treatment of pentacecilide C with (R)- Pentacecilide A is the most potent inhibitor of CE synthesis in the
(ꢁ)- and (S)-(+)- MTPA chloride in the presence of 4-(dimethyl- three assays, followed by pentacecilide B.1 Unfortunately, pentaceci-
amino) pyridine and triethylamine (in CH2Cl2, at room temperature, lides D and 3 showed almost no inhibitory activity at 22.5–24.2mM.
80.6 and 61.5% yield, respectively) (Scheme 1). The 1H NMR spectra
of 1 and 2 were completely assigned. As shown in Figure 2, the ACAT1- and ACAT2-CHO cells, even at 20.6–24.3 mM.
No pentacecilides showed any cytotoxic effects on macrophages and
The Journal of Antibiotics

Absolute stereochemistry of pentacecilides
H Yamazaki et al
317
H3C
O
O
H3C
O
O
OH
OR3
4’
4’
(R)-(-)-/(S)-(+)-MTPA chloride
DMAP,TEA, CH2Cl2
CH3
CH3
AcO
O
O
O
AcO
O
r.t, 10h
H
H
CH3
OH
CH3
OR2
7
7
H
H
CH3
H3C
H3C
CH3
1: R2, R2 = (S)-(+)-MTPA
2: R2, R3 = (R)-(-)-MTPA
Pentacecilide C
H3C
O
O
O
H3C
O
OH
OH
NaBH4, CH3OH
r.t, 12h
CH3
H
CH3
O
O
H
CH3
CH3
3
3
O
HO
H
H
CH3
H3C
CH3
H3C
Pentacecilide A
3
Scheme 1 Semisynthetic preparation of 1–3.
0.00
H3C
Table 3 Effects of pentacecilides on cholesteryl ester synthesis
in macrophages, and ACAT1- and ACAT2-CHO cells
O
O
8’
+0.01
IC50 (mM)
0.00
OMTPA
Pentacecilide
Macrophages
ACAT1-CHO
ACAT2-CHO
+0.04
5’
A
B
C
D
3
3.65
4.76
1.09
10.8
0.69
3.97
+0.07
-0.08
11
15CH3
-0.02
-0.02
420.6
422.5
424.2
420.6
422.5
424.2
420.6
422.5
19.0
-0.01
17AcO
9
O
2
1
-0.05
+0.03
H
CH3
-0.05
12
5
-0.04
6
O
-0.03
-0.03
H
OMTPA
13
conclusion was supported by evidence that all pentacecilides have
negative optical rotations.2
14CH3
-0.20
H3C
-0.12
In a cell-based assay using mouse macrophages, ACAT1- and
ACAT2-CHO cells,11–13 pentacecilides A and B showed more potent
inhibition of CE synthesis than pentacecilides C and D1 (Table 3).
Furthermore, 3 showed no inhibition of CE synthesis in mouse
macrophages, and ACAT1- and ACAT2-CHO cells, even at 24.2mM
(Table 3). Taking the structural differences into consideration
MTPA plane
Figure 2 Absolute stereochemistry determination. Dd values (Dd (in p.p.m.)¼
dSꢁdR) obtained for di-Mosher esters 1 and 2.
DISCUSSION
As described above, from various spectral analyses, the structure (Figure 1), the presence of a hydroxy group at C-7 is unfavorable
of a new congener, pentacecilide D, was elucidated to be 2-deacetyl for inhibiting lipid droplet formation and ACAT activity. Regarding
pentacecilide C with the same relative stereochemistry as shown in the C-3 position, 3 with a hydroxy group at C-3 showed weaker
Figure 1.
inhibitory activity than pentacecilide A, indicating that the presence of
To determine the absolute stereochemistry of pentacecilides, a carbonyl group at C-3 is important for activity. Regarding the C-2
the modified Mosher method10 was adapted to pentacecilide C. position, introduction of acethoxy and hydroxy groups resulted
Calculation of the Dd values led to the clear conclusion that penta- in decreased activity. Namely, no modification at this position is
cecilide C has the absolute stereochemistry of 2R5R7S8R9R10S8¢S preferable for inhibitory activity. Among these pentacecilides, it was
(Figure 1). Furthermore, assuming that all pentacecilides are generated concluded that pentacecilide A has the most favorable structure for
through the same biosynthetic pathway, pentacecilides A, B and D showing potent inhibitory activity against lipid droplet formation
have the same absolute stereochemistry as pentacecilide C. This and ACAT isozymes. Regarding the structure–activity relationships of
The Journal of Antibiotics

Absolute stereochemistry of pentacecilides
H Yamazaki et al
318
the hydroxy group at C-4¢, synthesis of acyl derivatives at this position Preparation of 3S*-Hydroxy-pentacecilide A
To a solution of pentacecilide A (2.9mg, 0.0070 mmol) in CH3OH (500ml),
is now in progress.
NaBH4 (3.3mg, 0.087mmol) was added, and the resulting solution was stirred
at room temperature. After 10 h, the reaction mixture was diluted with H2O
and the aqueous phase was extracted with EtOAc. The organic layer was
recovered, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to
dryness. The product was purified by preparative HPLC (column; PEGASIL
ODS, 20ꢀ250 mm; Senshu Scientific; solvent, 80% CH3CN; detection, UV at
210 nm; flow rate, 8.0 mlminꢁ1) to give 3 (2.5mg, 0.0060mmol, 85.7%) as a
white powder.
3S*-Hydroxy-pentacecilide A (3): 1H NMR (400MHz, CDCl3) d 0.86
(s, 3H), 1.02 (s, 3H), 1.09 (s, 3H), 1.26 (m, 1H), 1.29 (s, 3H), 1.46, 1.56
(m, 2H), 1.55 (d, 3H, J¼6.0 Hz), 1.66, 1.80 (m, 2H), 1.70 (m, 2H), 1.76
(m, 1H), 2.00, 2.08 (m, 2H), 2.40 (m, 2H), 2.68, 2.85 (dd, 2H, J¼17.0, 11.0Hz,
J¼17.0, 3.5 Hz), 3.26 (dd, J¼10.0, 4.0 Hz, 1H), 4.63 (m, 1H), 6.26 (s, 1H), 11.0
(s, 1H); HRESI-TOF-MS (m/z) found: 437.2290, calcd: 437.2304 [M+Na]+ for
C25H34NaO5.
METHODS
Materials
(S)-(+)- and (R)-(ꢁ)-MTPA chlorides, and sodium borohydride (NaBH4)
were purchased from Wako Pure Chemical Industries (Osaka, Japan). Acetic
anhydride (Ac2O) and 4-(dimethylamino) pyridine were purchased from Kanto
Chemical (Tokyo, Japan). Triethylamine was purchased from Nacalai Tesque
(Kyoto, Japan).
General experimental procedures
Fungal strain P. cecidicola FKI-3765-1 was used for the production of penta-
cecilides.1,2 Pentacecilide D was from 13-day-old culture broth fermented as
reported previously.1 For determination of the amounts of pentacecilide D in
culture broths, samples (ethyl acetate extracts) dissolved in methanol were
analyzed by the HP1100 system (Hewlett-Packard, Palo Alto, CA, USA) under
the following conditions: column, Symmetry (2.1ꢀ150 mm; Waters, Milford,
MA, USA); flow rate, 0.2 mlminꢁ1; mobile phase, a 20-min linear gradient
from 60% CH3CN to 100% CH3CN containing 0.050% H3PO4; detection, UV
at 210 nm. Under these conditions, pentacecilide D was eluted with a retention
time of 3.59min, respectively.
Biological assays
An assay for the synthesis of CE, triacylglycerol and phospholipids by mouse
macrophages was carried out according to the method described previously.11
An assay for ACAT1 and ACAT2 activities in ACAT1- and ACAT2-CHO cells
was carried out by our established method.12,13
SSC-ODS-7515-12 (Senshu Scientific) was used for ODS column chromato-
graphy. HPLC was carried out using the L-6200 system (Hitachi, Tokyo, Japan).
UV spectra were recorded on a spectrophotometer (8453 UV-Visible spectro-
photometer; Agilent Technologies, Santa Clara, CA, USA). IR spectra were
recorded on a Fourier transform infrared spectrometer (FT-710; Horiba, Kyoto,
Japan). Optical rotations were measured with a digital polarimeter (DIP-1000;
JASCO, Tokyo, Japan). ESI-TOF-MS and HRESI-TOF-MS spectra were recorded
on a mass spectrometer (JMS-T100LP; JEOL, Tokyo, Japan). Various NMR
spectra were measured with a spectrometer (XL-400; Varian, Palo Alto, CA, USA).
ACKNOWLEDGEMENTS
This study was supported in part by a Sasakawa Scientific Research Grant
(to HY) from The Japan Science Society. We express our thanks to Ms N Sato
for NMR experiments, and Dr K Nagai and Ms A Nakagawa for measuring
mass spectra.
Preparation of the (S)-(+)- and (R)-(ꢁ)-MTPA ester derivatives
of pentacecilide C
1
2
3
4
Yamazaki, H. et al. Pentacecilides, new inhibitors for lipid droplet formation in mouse
macrophages produced by Penicillium cecidicola FKI-3765-1:I. Taxonomy, fermenta-
tion, isolation and biological properties. J. Antibiot. 62, 195–200 (2009).
To a solution of pentacecilide C (5.0mg, 0.010 mmol) in CH2Cl2 (500ml),
(R)-(ꢁ)-MTPA chloride (16.5 mg, 0.065 mmol), 4-(dimethylamino) pyridine
(5.3mg, 0.043 mmol) and triethylamine (10ml, 0.073mmol) were added. The
reaction mixture was stirred at room temperature. After 10h, the reaction
mixture was diluted with 1.0 N HCl and the aqueous phase was extracted with
EtOAc. The organic layer was recovered, dried over anhydrous Na2SO4, filtered
and concentrated in vacuo to dryness. The product was purified by preparative
HPLC (column; PEGASIL ODS, 20ꢀ250 mm; Senshu Scientific; solvent, 80%
CH3CN; detection, UV at 210nm; flow rate, 8.0 mlminꢁ1) to give 1 (7.40 mg,
0.0081 mmol, 80.6%) as a white powder. Similarly, 2 (4.63 mg, 0.0050 mmol,
61.5%) was obtained using (S)-(+)-MTPA chloride.
¯
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& Tomoda, H. Pentacecilides, new inhibitors for lipid
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5
6
7, 4¢-(S)-(+)-a-methoxy-a-(trifluoromethyl)phenylacetoxy pentacecilide C
(1): 1H NMR (400 MHz, CDCl3) d 1.05 (s, 3H), 1.06 (s, 3H), 1.10 (s, 3H), 1.36
(s, 3H), 1.52 (d, 3H, J¼7.0 Hz), 1.68, 2.28 (m, 2H), 1.85, 2.14 (m, 2H), 2.17
(s, 3H), 2.57 (m, 2H), 2.74, 2.82 (dd, 2H, J¼17.0, 11.0Hz, J¼17.0, 3.5Hz), 3.52
(s, 3H), 3.73 (s, 3H), 4.58 (m, 1H), 5.54 (m, 1H), 5.56 (m, 1H), 6.35 (s, 1H),
7.26 (m, 3H), 7.44 (m, 3H), 7.52 (m, 2H), 7.75 (m, 2H); HRESI-TOF-MS
(m/z) found: 941.2935, calcd: 941.2948 [M+Na]+ for C47H48 F8NaO12.
7, 4¢-(R)-(ꢁ)-a-methoxy-a-(trifluoromethyl)phenylacetoxy pentacecilide C
(2): 1H NMR (400 MHz, CDCl3) d 1.14 (s, 3H), 1.17 (s, 3H), 1.30 (s, 3H), 1.33
(s, 3H), 1.53 (d, 3H, J¼7.0Hz), 1.87, 2.16 (m, 2H), 2.31 (m, 2H), 2.17 (s, 3H),
2.50 (m, 2H), 2.74, 2.82 (dd, 2H, J¼17.0, 11.0 Hz, J¼17.0, 3.5 Hz), 3.53 (s, 3H),
3.76 (s, 3H), 4.57 (m, 1H), 5.60 (m, 1H), 5.61 (m, 1H), 6.31 (s, 1H), 7.32 (m,
3H), 7.44 (m, 3H), 7.52 (m, 2H), 7.74 (m, 2H); HRESI-TOF-MS (m/z) found:
941.2980, calcd: 941.2948 [M+Na]+ for C47H48 F8NaO12.
7
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The Journal of Antibiotics