Biologically active metabolites from the marine isolate of the fungus myceliophthora lutea

Article abstract of DOI:10.1007/s10600-011-9940-2

The new compound isoacremine D (1) and acremine A (2) that was previously unknown for marine organisms were isolated from the marine isolate of the fungus Myceliophthora lutea. It was found that acremine A in CHCl₃ was converted through the action of light into spirocompounds called by us spiroacremines A (3) and B (4). The structures of the compounds were elucidated based on NMR spectroscopic and high-resolution mass spectrometric data. The absolute stereochemistry of the spiroacremines was determined by Mosher's method. It was shown that isoacremine D exhibited antimicrobial activity against Staphylococcus aureus at a concentration of 200 μg/mL and cytotoxic activity against embryos of the sea urchin Strongylocentrotus nudus. It was found that 1-4 exhibited cytotoxic activity against sea urchin sperm cells.

Full text of DOI:10.1007/s10600-011-9940-2

Chemistry of Natural Compounds, Vol. 47, No. 3, July, 2011 [Russian original No. 3, May-June, 2011]
BIOLOGICALLY ACTIVE METABOLITES FROM THE MARINE
ISOLATE OF THE FUNGUS Myceliophthora lutea
1*
2
1
1
O. F. Smetanina, A. N. Yurchenko, A. I. Kalinovskii, D. V. Berdyshev,
UDC 577.115:528.281
3
1
1
1
A. V. Gerasimenko, M. V. Pivkin, N. N. Slinkina, P. S. Dmitrenok,
1
1†
1
N. I. Menzorova, T. A. Kuznetsova, and Sh. Sh. Afiyatullov
The new compound isoacremine D (1) and acremine A (2) that was previously unknown for marine organisms
were isolated from the marine isolate of the fungus Myceliophthora lutea. It was found that acremine A in
CHCl was converted through the action of light into spirocompounds called by us spiroacremines A (3) and
3
B (4). The structures of the compounds were elucidated based on NMR spectroscopic and high-resolution
mass spectrometric data. The absolute stereochemistry of the spiroacremines was determined by Mosher’s
method. It was shown that isoacremine D exhibited antimicrobial activity against Staphylococcus aureus at
a concentration of 200 ꢀg/mL and cytotoxic activity against embryos of the sea urchin Strongylocentrotus
nudus. It was found that 1–4 exhibited cytotoxic activity against sea urchin sperm cells.
Keywords: acremines, marine isolate of the fungus Myceliophthora lutea, NMR data, 1-oxaspiro[4,5]decenes.
Facultative and obligate marine microscopic fungi are sources of biologically active secondary metabolites [1–3]. In
continuation of the search for producers of biologically active compounds among marine isolates of microscopic fungi, we
determined that the fungus Myceliophthora lutea Costantin [4], which was isolated from marine sediments of Sakhalin Bay
(Sea of Okhotsk), synthesizes compounds with antibacterial and cytotoxic activities. Little is known about secondary metabolites
from fungi of the genus Myceliophthora although these fungi are often encountered and can be isolated from various substrates
[5–8]. Herein we present data on the isolation and identification of known acremine A (2) [9] and previously unknown
compounds called by us isoacremine D (1) and spiroacremines A (3) and B (4). The last two compounds are apparently
artifacts of the isolation process.
The fungus was cultivated on solid medium for 21 d at 22°C in order to produce the metabolites. The dried EtOAc
extract of the biomass produced a CHCl extract, separation of which over silica gel and Sephadex LH-20 afforded pure 1 and
3
2 (Fig. 1) in addition to 3 and 4, which were not present in the starting total EtOAc extract according to TLC (Fig. 2).
The empirical formula of 1 was determined as C H O on the basis of the high-resolution mass spectrum and NMR
12 14
3
spectra. The PMR spectrum of 1 contained three 3H-singlets in the range 1.55–2.27 ppm, indicative of three tertiary methyls
in its structure. The DEPT and HSQC spectra of 1 confirmed the presence of three methyls (ꢁ 17.6, 31.0, 31.0 ppm) and also
C
indicated that three methine groups were present (ꢁ 101.2, 106.5, 113.4). The remaining six C atoms were classified as
C
quaternary (ꢁ 165.9, 153.0, 150.8, 128.6, 123.1, 69.7). Figure 1 shows the key HMBC-correlations of 1.
C
The position of methyl 1ꢂ (ꢁ 2.27 ppm) on C(6) was established by HMBC-correlations of C-1ꢂ with C-5
H
(ꢁ 153.0 ppm), C-6 (ꢁ 123.1), and C-7 (ꢁ 113.4). Singlets for H C-2ꢃ (ꢁ 31.0, ꢁ 1.55 ppm) and H C-3ꢃ (ꢁ 31.0, ꢁ 1.55)
C
C
C
3
C
H
3
C
H
were assigned to geminal methyls on the basis of mutual HMBC-correlations with each other and correlations of the methyl
protons with quaternary C-1ꢃ (ꢁ 69.7), which was bonded to oxygen.
C
______
Deceased.
†
1) Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, 690022, Vladivostok,
Prosp. 100-Letiya Vladivostoka, 159, Russian Federation, fax: (4232) 31 40 50, e-mail: smetof@rambler.ru; 2) Far-East
Federal University, 690091, Vladivostok, Sukhanova, 8, Russian Federation; 3) Institute of Chemistry, Far-East Branch, Russian
Academy of Sciences, 690022, Vladivostok, Prosp. 100-Letiya Vladivostoka, 159, Russian Federation, fax: (4232) 31 18 89,
e-mail: chem@online.ru. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 345–349, May–June, 2011. Original
article submitted October 28, 2010.
©
0009-3130/11/4703-0385 2011 Springer Science+Business Media, Inc.
385

TABLE 1. NMR Spectral Data for 1/Acremine D [9] (500 MHz, acetone-d , ꢁ, ppm, J/Hz)
6
C atom
DEPT
HMBC
COSY
NOE
ꢁÑ
ꢁH
2
3
3a
4
5
6
7
7a
1ꢃ
2ꢃ
165.9/165.3
101.2/99.6
128.6/135.1
106.5/104.7
153.0/150.0
123.1/122.3
113.4/111.8
150.8/144.3
69.7/77.8
C
CH
C
CH
C
C
CH
C
3a, 7a, 2
6.45/6.44 (ñ)
6.92/6.91 (ñ)
7
4, 2ꢃ, 3ꢃ, 1ꢃ-OH
3, 5, 6, 7a
7, 1ꢃꢃ
ꢃ
ꢃ
3, 2 , 3 , 5-OH
1ꢃꢃ, 3a, 5, 7a
7.15/7.15 (ñ)
2ꢃ, 3ꢃ
1ꢃꢃ, 3, 4
C
1ꢃ, 2
31.0/27.3
31.0/27.3
17.6/16.0
Me
Me
Me
1.55/1.54 (ñ)
1.55/1.54 (ñ)
2.27/2.26 (ñ)
4.25 (ñ)
3, 1ꢃ-OH
1ꢃ-OH
1ꢃ-OH
4, 7
2ꢃ, 3ꢃ
7
ꢃ
3
ꢃ
1 , 2
ꢃ
3, 1 -OH
5, 6, 7
1ꢃꢃ
1 -OH
5-OH
7, 2ꢃ, 3ꢃ
3, 2ꢃ, 3ꢃ, 1ꢃ-OH
4
ꢃ
ꢃ
ꢃ
3 , 1 , 2 , 2
ꢃ
7.93 (ñ)
4, 5, 6
+0.46
3
+0.05
+0.20
3
1''CH
HO
1''
CH
3'
+0.08
1'
H C
H C
+0.51
3
1'
3
1'
3
+0.04
A
+0.28
OMTPA
+0.25
2'
2
3
1O
+0.02
+0.05
O
OMTPA
3a
O
5
7a
H
-0 .0 1
-0 .2 9
B
H
7
9
4
-0 .2 1
O
O
-0 .0 7
7
5
H1''C'
OH
6
OH
4a, b
OH
H C 1'''
3
3
+0.11
-0 .0 1
1''
3a,b
1
Fig. 1
Fig. 2
Fig. 1. Key HMBC-correlations of 1.
Fig. 2. Differences in proton chemical shifts (ꢄꢁ = ꢁ – ꢁ ) of MTPA-esters of spiroacremines A (3a, b) and B (4a, b).
S
R
13
The resonances of C-2 and C-7a in the C NMR spectrum of 1 (ꢁ 165.9 and 150.8) indicated that these atoms were
C
bonded to oxygen. The positions of two hydroxyls on C-1ꢃ and C-5 were inferred from HMBC-correlations of the hydroxyl
protons with the corresponding C atoms (Fig. 1, Table 1). Coupling of HC-4 (ꢁ 6.92 ppm) and C-3 (101.2) in the
H
HMBC-spectrum of 1 in addition to NOE-coupling of HC-4 and HC-3 confirmed fully the structure shown in Fig. 1. Thus, 1
was 2-(1ꢃ-hydroxy-1ꢃ-methylethyl)-6-methylbenzofuran-5-ol.
A compound with this same formula, acremine D, was recently reported by Italian researchers [9]. The PMR spectra
13
of isoacremine D and acremine D coincided (Table 1). However, the C NMR spectra of 1 and acremine D were significantly
different. This fact and the different melting points of isoacremine D (260–262°C) and acremine D (142–145°C) were indicative
of a new compound.
+
The empirical formula of 2 (C H O ) was established by high-resolution mass spectrometry (observed [M] with
m/z 226.1221; calcd [M] 226.1205) and PMR and C NMR spectral data (Table 2). The similar resonances of protons and
C atoms in PMR and C NMR spectra of 2 and acremine A, which was isolated from the terrestrial fungus Acremonium
12 18
4
+
13
13
byssoides [9, 10], in addition to an x-ray structure analysis of 2 indicated that these two compounds were identical. Thus, 2
was 4S,6R-dihydroxy-3-[E-3ꢃ-hydroxy-3ꢃ-methylbut-1-enyl]-6-methylcyclohex-2-en-1-one.
Two new compounds, spiroacremines A (3) and B (4) formed upon storage of 2 in CHCl in the light for a week.
3
However, such conversions did not occur upon storage of 2 in CHCl in the dark. Figure 3 shows the probable pathway of the
3
transformations. Apparently a photochemical reaction that changed the configuration of the double bond occurred through the
action of light. This made further cyclization possible. Chloroform, which becomes acidic in light, was the proton donor
required for the cyclization. The hydroxyl oxygen of the side chain attacked the electron-poor enone C-5 to form the
spirocompounds (Fig. 3).
386

TABLE 2. NMR Spectral Data for 2 (500 MHz, ꢁ, ppm, J/Hz)
ꢁC
ꢁH
C atom
DEPT
methanol-d4
acetone-d6
methanol-d4
5.98 (s)
acetone-d6
1
2
3
4
5
203.0
123.0
161.3
67.0
201.4
123.3
160.3
66.7
C
CH
C
CH
CH2
5.92 (s)
4.62 (dd)
2.34 (dd)
2.07 (dd)
4.62 (m)
46.5
46.1
2.30 (dd, J = 4.5, J = 13.8)
2.10 (dd, J = 13.7, J = 6.6)
6
74.2
124.6
148.0
71.6
29.6
29.6
74.0
125.1
148.6
71.5
*
*
25.4
C
1
ꢃ
CH
CH
C
Me
Me
Me
6.42 (d)
6.66 (d)
6.45 (d, J = 16.0)
6.67 (d, J = 16.0)
2ꢃ
3
4
5
ꢃ
ꢃ
ꢃ
1.33 (s)
1.33 (s)
1.28 (s)
1.30 (s)
1.30 (s)
1.24 (s)
24.7
1
ꢃꢃ
______
*Signal overlapped by signal of acetone-d .
6
O
O
OH
HO
HO
O
OH
OH
OH
+
.
3
HO
HO
.
OH
O
h , H
O
OH
O
O
OH
HO
HO
2
O
OH
OH
OH
4
Fig. 3. Probable formation pathway of spiroacremines A and B.
The empirical formula of 3 was determined from high-resolution mass spectral and PMR and C NMR spectral data.
13
1
13
An analysis of H and C DEPT spectra of 3 proved that C-7 was a ketone (ꢁ 211.2 ppm); that three quaternary C atoms were
C
bonded to oxygen functional groups at C-2 (ꢁ 90.0), C-5 (ꢁ 95.7), and C-8 (ꢁ 76.5); that two methylenes occurred at C-6
C
C
C
(ꢁ 49.2) and C-9 (ꢁ 44.6); that a single hydroxymethylene was present at C-10 (ꢁ 70.4); and that three methyls were
C
C
C
situated at C-1ꢃꢃꢃ (ꢁ 25.9, ꢁ 1.39), C-1ꢃ (ꢁ 28.6, ꢁ 1.31), and C-1ꢃꢃ (ꢁ 29.9, ꢁ 1.30). The spectral data indicated that 3
C
H
C
H
C
H
had a single double bond at C-3 (ꢁ 138.4, ꢁ 5.88) and C-4 (ꢁ 128.9, ꢁ 5.61) and were consistent with a bicyclic structure
C
H
C
H
1
1
for the compound (Table 3). The H– H COSY NMR spectrum enabled two spin–spin systems to be identified. These
corresponded to C-1ꢃ/C-3/C-4 and C-1ꢃꢃꢃ/C-9/C-10 groupings in 3. HMBC-correlations of the H C-1ꢃꢃꢃ protons with oxygenated
3
quaternary C-8, methylene C-9, and carbonyl C-7 indicated that C-8 was bonded to C-7, C-9, and C-1ꢃꢃꢃ. HMBC-correlations
of H C-6 with C-5, C-7, and C-10 determined that C-6 was bonded to C-7 and C-5 of cyclohexane ring B in 3. Correlations
2
of proton HC-3 (ꢁ 5.88) with C-5 (ꢁ 95.7) in addition to cross peaks between HC-6ꢅ (ꢁ 2.37) and C-8 (ꢁ 76.5) and
H
C
H
C
protons H C-6 and C-4 confirmed that 3 contained a spirocyclic system. The SSCC between HC-3 and HC-4 (J = 5.9 Hz)
2
3,4
was typical of a cis-double bond in a five-membered ring [11] (ring A). The relative configurations of the asymmetric C atoms
in 3 were determined based on NOESY experiments and SSCC of the corresponding protons.
Proton HC-3 (ꢁ 5.88) showed NOESY cross peaks with both HC-4 (ꢁ 5.61) and methyl protons of C-1ꢃꢃ (ꢁ 1.31)
H
H
H
and C-1ꢃ (ꢁ 1.30) whereas H-4 had cross peaks with HC-6ꢆ (ꢁ 2.95) and H-10 (ꢁ 4.0) (Table 3). These protons, in turn,
H
H
H
were coupled to each other and with protons of the C-1ꢃꢃꢃ methyl (ꢁ 1.39). These data and the SSCC of HC-10 and HC-9
H
confirmed that the six-membered ring in 3 had a pseudo-chair conformation in which HC-6ꢆ (ꢁ 2.95), HC-10, and H C-1ꢃꢃꢃ
H
3
were axial. The absolute configuration of 3 was established by Mosher’s method [12]. Esterification of 3 with (S)- and (R)-
MTPA-Cl at the C-10 hydroxyl formed (S)- and (R)-MTPA esters 3a and 3b, respectively. The differences in the chemical
shifts (ꢄꢁ = ꢁ – ꢁ ) in proton spectra of the esters (Fig. 2) were consistent with the 10S-configuration. Therefore, C-5 and C-
S
R
8 should have had the S- and R-configurations, respectively, taking into account the previously found NOESY-couplings in 3
(Fig. 2). Thus, 3 was (5S,8R,10S)-8,10-dihydroxy-2,2,8-trimethyl-1-oxaspiro[4,5]dec-3-en-7-one.
387

TABLE 3. NMR Spectral Data for 3 (500 MHz, CD OD, ꢁ, ppm, J/Hz)
3
C atom
DEPT
NOESY
HMBC
ꢁÑ
ꢁH
2
3
4
5
6
90.0
138.4
128.9
95.7
C
CH
CH
5.88 (d, J = 5.9)
5.61 (d, J = 5.9)
ꢃ
ꢃꢃ
4, 1 , 1
2, 5, 1ꢃ, 1ꢃꢃ
2, 5
3, 6ꢆ, 10
C
7, 4, 5, 10
7, 4, 5, 8, 10
49.2
2.95 (d, J = 14.4)
2.37 (d, J = 14.4)
4, 10, 1ꢃꢃꢃ
1ꢃꢃ
ꢆ-CH2
ꢅ-CH2
C
7
8
9
211.2
76.5
44.6
C
5, 10, 1ꢃꢃꢃ, 7, 8
2.04 (dd, J = 12.6, J = 4.7)
2.13 (d, J = 12.0)
4.00 (dd, J = 11.4, J = 4.7)
1.31 (s)
1ꢃꢃꢃ
ꢆ-CH2
ꢅ-CH2
CH
Me
Me
ꢃꢃꢃ
5, 10, 1 , 7, 8
10
4, 5, 8, 9
1ꢃꢃ, 3, 2
1ꢃ, 3, 2
9, 7, 8
70.4
28.6
29.9
25.9
1ꢃꢃꢃ, 4, 6ꢆ
3
3, 6
ꢃ
1
ꢅ
1.30 (s)
1.39 (s)
1ꢃꢃ
1ꢃꢃꢃ
10, 6ꢆ, 9ꢆ
Me
TABLE 4. NMR Spectral Data for 4 (500 MHz, CD OD, ꢁ, ppm, J/Hz)
3
C atom
DEPT
NOESY
HMBC
ꢁC
ꢁH
2
3
4
5
6
90.0
138.3
128.4
96.3
C
CH
CH
5.93 (d, J = 6.0)
5.83 (d, J = 6.0)
ꢃ
1
4, 2, 5, 1, 1ꢃꢃ
1ꢃ, 9ꢅ, 6ꢅ
3, 2, 5
C
47.7
2.38 (d, J = 13.7)
3.19 (d, J = 13.7)
1ꢃꢃ, 1ꢃꢃꢃ, 10
1ꢃꢃ, 4
7, 4, 5, 8, 10
7, 4, 5, 8, 10
ꢆ-CH2
ꢅ-CH2
C
7
8
9
209.9
76.5
42.5
C
2.13 (dd, J = 14.4, J = 3.7)
1.92 (dd, J = 14.4, J = 6.1)
3.73 (dd, J = 3.6, J = 6.1)
1.28 (s)
1ꢃꢃꢃ, 10
1ꢃꢃꢃ, 4, 10
1ꢃꢃꢃ, 9ꢅ, 6ꢆ, 9ꢆ
3, 4
ꢃꢃꢃ
5, 10, 1 , 7, 8
ꢆ-CH2
5, 10, 1ꢃꢃꢃ, 7, 8
4, 5, 6, 8, 9
3, 2
ꢅ
-CH2
CH
Me
Me
Me
10
1ꢃ
1ꢃꢃ
1ꢃꢃꢃ
74.4
29.2
29.7
24.2
1.29 (s)
1.26 (s)
ꢆ ꢅ
6 , 6
3, 2
9, 7, 8
9ꢅ, 6ꢆ, 9ꢆ, 10
Compound 4 had molecular formula C H O , which was confirmed by high-resolution electron-impact mass
12 18
4
13
1
1
spectrometry. An analysis of PMR and C NMR spectra, H– H COSY, HSQC, and HMBC spectra for 4 (Table 4) and a
comparison of them with the data for 3 indicated that 4 had the same spiro[4,5]decene skeleton. The differences in chemical
shifts of protons in the (S)- and (R)-MTPA esters (4a and 4b) proved the absolute configuration of C-10 as S. The 1D
NOE-correlations of HC-10 in 4b with protons of the C-8 methyl and with HC-6ꢆ confirmed that these protons and H C-1ꢃꢃꢃ
3
were axial and that C-8 had the R-configuration in 4. A 1D NOE-correlation of H-4 with HC-9ꢅ in 4a and 4b indicated that
C-5 had the R-configuration. Thus, it was found that 4 was (5R,8R,10S)-8,10-dihydroxy-2,2,8-trimethyl-1-oxaspiro[4,5]dec-
3-en-7-one.
The SSCC for protons on C-9 and C-10 [(14.4; 3.7), (14.4; 6.1) and (3.6; 6.1)] indicated that the cyclohexane ring of
spiroacremine B was slightly distorted (Table 4). Introduction of a bulky substituent in the 10-position during preparation of
the MTPA esters caused ring B to adopt a pseudo-chair conformation. This was evident in the SSCC for the C-9 and C-10
protons (see Experimental).
Compound 1 exhibited antimicrobial activity against Staphylococcus aureus at a concentration of 200 ꢀg/mL. The
activity of 1 at a concentration of 50 ꢀg/mL on developing sea urchin embryos was such that only 35–50% of them passed the
blastula stage. Compounds 2–4 at the studied concentrations did not exhibit antimicrobial activity against Gram-positive and
Gram-negative bacteria and did not show cytotoxic activity against developing sea urchin embryos. The fertilizing ability of
sea urchin spermatozoids decreased by 50% upon incubation with 1–4 at concentrations of 40, 50, 15, and 30 ꢀg/mL, respectively.
388

EXPERIMENTAL
Melting points were determined on a Leica VMTG instrument (Germany). Optical rotation was measured on a
Perkin–Elmer Model 343 polarimeter (Germany). UV spectra were taken in MeOH on a CECIL, CE 7250, 7000
spectrophotometer (England). IR spectra were recorded in CHCl on a Bruker OPUS Vector-22 spectrophotometer. PMR and
3
13
C NMR spectra were measured on a Bruker DRX-500 (500 and 125.6 MHz) spectrometer with TMS internal standard.
High-resolution electron-impact mass spectra were taken on anAMD 604 S spectrometer (Germany). Column chromatography
was carried out over silica gel L (40/100 ꢀm, Chemapol, Czechoslovakia) and Sephadex LH-20 (Pharmacia Biotech, Sweden).
Silica-gel plates (4.5 ꢇ 6.0 cm, 5–17 ꢀm, Sorbfil, Russia) were used for TLC.
Cultivation of Fungus. The fungus Myceliophthora lutea was isolated from marine sediments of Sakhalin Bay (Sea
of Okhotsk, 32 m depth) and cultivated for three weeks on standing at 22°C in six 1-L flasks, each of which contained medium
consisting of wort (50 mL), agar (5 g), and seawater (200 mL).
Extraction and Isolation of 1–4. Fungal mycelium was extracted with EtOAc (2.5 L). The solvent was evaporated.
The solid (2.5 g) was treated with H O:EtOH (4:1, 200 mL). The resulting suspension was extracted successively with hexane
2
(200 mL ꢇ 2), CHCl (200 mL ꢇ 2), and n-BuOH (100 mL ꢇ 2). The CHCl extract was evaporated. The solid (900 mg) was
3
3
chromatographed over a column (20 ꢇ 2 cm) of silica gel using hexane:EtOAc (95:5, 90:10, 85:15, 80:20). Gel filtration over
Sephadex LH-20 in CHCl afforded pure 1 (5 mg), 2 (57 mg), 3 (15 mg), and 4 (9 mg). Compounds 3 and 4 were not present
3
in the starting EtOAc extract and were formed during the isolation process. This was confirmed experimentally.
Conversion of Acremine A (2) to Spiroacremines A (3) and B (4). Acremine A (16 mg) was dissolved in CHCl
3
(5 mL) and left on a table. Two compounds in addition to acremine A were observed in the solution after a week according to
TLC. The solution was evaporated. The resulting mixture was separated using column chromatography over silica gel and
hexane:EtOAc (95:5–80:20) to afford 1 (8 mg), 3 (5 mg), and 4 (3 mg). Longer storage (about one month) of acremine A in
CHCl solution transformed it completely to the spiroacremines.
3
Isoacremine D (1), C H O , white crystals, mp 260–262°C (hexane:EtOAc, 9:1). UV spectrum (EtOH, ꢈ ,
12 14
3
max
–1
nm): 203, 249, 295 (log ꢉ 4.22, 3.96, 3.60). IR spectrum (CHCl , , cm ): 3605, 2928, 1737, 1602. High-resolution mass
spectrum (EI): found: m/z 206.0949 [M] ; calcd: 206.0943. Table 1 presents NMR spectral data for 1.
3
+
20
Acremine A (2), C H O , white crystals, mp 121–123°C (hexane:EtOAc, 9:1), [ꢆ] +12.1° (c 0.31, EtOH).
12 18
4
D
–1
UV spectrum (EtOH, ꢈ , nm): 281 (log ꢉ 4.05). IR spectrum (CHCl , , cm ): 3467, 3330, 1681, 1639, 1598. High-
resolution mass spectrum (EI): found: m/z 226.1221 [M] ; calcd: 226.1205. Table 2 presents NMR spectral data for 2.
max
3
+
20
Spiroacremine A (3), C H O , white crystals, mp 128–131°C (hexane:EtOAc, 8:1), [ꢆ] +18° (c 0.3, EtOH).
12 18
4
D
–1
UV spectrum (EtOH, ꢈ , nm): 217, 281 (log ꢉ 2.92, 2.10). IR spectrum (CHCl , , cm ): 3571, 3494, 2979, 2932, 1721,
1602. High-resolution mass spectrum (EI): found: m/z 226.1209 [M] ; calcd: 226.1205. Table 3 presents NMR spectral data
for 3.
max
3
+
Preparation of (S)- and (R)-MTPA (methoxy trifluoromethyl phenyl acetic acid) Esters of Spiroacremine A
(3a and 3b). A solution of 3 (2 mg) in Py (200 ꢀL) was treated with several crystals of 4-dimethylaminopyridine and
(R)-MTPA-Cl (20 ꢀL). The resulting mixture was left for 1 h at room temperature. The solvent was evaporated. The solid was
chromatographed over silica gel using hexane:EtOAc (95:5) to afford the (S)-MTPA ester 3a (3 mg). The (R)-MTPA ester 3b
was prepared in the same manner using (S)-MTPA-Cl.
(S)-MTPA Ester (3a). PMR spectrum (CD OD, ꢁ, ppm, J/Hz): 5.89 (1H, d, J = 5.7, H-3), 5.66 (1H, d, J = 5.7,
3
H-4), 5.49 (1H, dd, J = 5.4, 11.6, H-10), 3.49 (3H, s, OMe), 3.14 (1H, d, J = 14.7, H-6ꢆ), 2.31 (1H, d, J = 14.7, H-6ꢅ), 2.15
(1H, dd, J = 5.1, 12.0, H-9ꢆ), 2.11 (1H, t, J = 12.0, H-9ꢅ), 1.48 (3H, s, Me-1ꢃꢃꢃ), 1.23 (3H, s, Me-1ꢃ), 1.03 (3H, s, Me-1ꢃꢃ),
+
7.39-7.57 (5H, m, Ph). Mass spectrum (EI): m/z 442 [M] .
(R)-MTPA Ester (3b). PMR spectrum (CD OD, ꢁ, ppm, J/Hz): 5.38 (1H, d, J = 5.7, H-3), 5.38 (1H, m, J = 5.7,
3
H-4), 5.49 (1H, dd, J = 4.8, 12.0, H-10), 3.58 (3H, s, OMe), 3.12 (1H, d, J = 14.7, H-6ꢆ), 2.26 (1H, d, J = 14.7, H-6ꢅ), 2.22
(1H, dd, J = 4.8, 11.7, H-9ꢆ), 2.32 (1H, t, J = 12.0, H-9ꢅ), 1.49 (3H, s, Me-1ꢃꢃꢃ), 1.15 (3H, s, Me-1ꢃ), 0.83 (3H, s, Me-1ꢃꢃ),
+
7.38–7.49 (5H, m, Ph). Mass spectrum (EI): m/z 442 [M] .
20
Spiroacremine B (4), C H O , white crystals, mp 142–145°C (hexane:EtOAc, 8:1), [ꢆ] +4.2° (c 0.24, EtOH).
12 18
4
D
–1
UV spectrum (EtOH, ꢈ , nm): 215, 281 (log ꢉ 3.37, 2.39). IR spectrum (CHCl , , cm ): 3600, 3500, 2977, 2929, 1719,
max
3
+
1602. High-resolution mass spectrum (EI): found: m/z 226.1195 [M] ; calcd: 226.1205. Table 4 presents NMR spectral data
for 4.
389

Preparation of (S)- and (R)-MTPA Esters of Spiroacremine B (4a and 4b). A solution of 4 (2 mg) in Py (200 ꢀL)
was treated with several crystals of 4-dimethylaminopyridine and (R)-MTPA-Cl (20 ꢀL). The resulting mixture was left
for 1 h at room temperature. The solvent was evaporated. The solid was chromatographed over silica gel using hexane:EtOAc
(93:7) to afford the (S)-MTPA ester 4a (3 mg). The (R)-MTPA ester 4b was prepared in the same manner using (S)-MTPA-Cl.
(S)-MTPA Ester (4a). PMR spectrum (CD OD, ꢁ, ppm, J/Hz): 5.98 (1H, d, J = 6.0, H-3), 5.99 (1H, d, J = 6.0,
3
H-4), 5.42 (1H, dd, J = 5.3, 11.5, H-10), 3.49 (3H, s, OMe), 3.13 (1H, d, J = 15.8, H-6ꢅ), 2.71 (1H, d, J = 15.8, H-6ꢆ), 2.50
(1H, t, J = 12.0, H-9ꢅ), 2.36 (1H, dd, J = 12.5, 5.4, H-9ꢆ), 1.66 (3H, s, Me-1ꢃꢃꢃ), 1.20 (3H, s, Me-1ꢃ), 1.30 (3H, s, Me-1ꢃꢃ),
+
7.39–7.57 (5H, m, Ph). Mass spectrum (EI): m/z 442 [M] .
(R)-MTPA Ester (4b). PMR spectrum (CD OD, ꢁ, ppm, J/Hz): 5.93 (1H, d, J = 5.9, H-3), 5.74 (1H, m, J = 5.9,
3
H-4), 5.14 (1H, dd, J = 4.1, 12.8, H-10), 3.49 (3H, s, OMe), 2.51 (1H, t, J = 12.4, H-9ꢅ), 2.65 (1H, dd, J = 12.1, 4.1, H-9ꢆ),
1.57 (3H, s, Me-1ꢃꢃꢃ), 0.74 (3H, s, Me-1ꢃ), 1.26 (3H, s, Me-1ꢃꢃ), 7.39–7.57 (5H, m, Ph). Resonances for H-6ꢆ and H-6ꢅ
+
overlapped with those of the solvent. Mass spectrum (EI): m/z 442 [M] .
Crystallographic data for 2 are deposited at the Institute of Chemistry, FEB, RAS. Copies of these data can be
obtained at the address: Russian Federation, 690022, Vladivostok, Prosp. 100-Letiya Vladivostoka, 159; fax: (4232) 31 18 89;
e-mail: pumalych@ich.dvo.ru.
Determination of Biological Activity. Antimicrobial activity of the isolated compounds was determined against
Gram-positive bacteria Bacillus subtilis (KMM 430) and Staphylococcus aureus (ATCC 21027), Gram-negative bacteria
Pseudomonas aeruginosa (KMM 433) and Escherichia coli (ATCC 15034), and yeast fungus Candida albicans (KMM 455)
by the literature method [13].
Gametes of the sea urchin Strongylocentrotus nudus were used for the test of inhibition of fertilizing ability of
spermatozoids and the test of fertilized eggs. The usual procedure was used for this [14].
ACKNOWLEDGMENT
The work was supported financially by the Russian Foundation for Basic Research (Projects Nos. 09-04-00388 and
08-04-00289), the RAS Presidium (Molecular and Cellular Biology Program), the RF FederalAgency for Science and Innovation
(State Contract No. 02.518.11.7169), and a Grant of the RF President (Project NSh-3531.2010.4).
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