New derivatives of nonactic and homononactic acids from Bacillus pumilus derived from Breynia fruticosa

Article abstract of DOI:10.1002/cbdv.201300350

Six new nonactic and homononactic acid derivatives, ethyl homononactate (1), ethyl nonactate (2), homononactyl homononactate (6), ethyl homononactyl nonactate (7), ethyl homononactyl homononactate (8), and ethyl nonactyl nonactate (9), as well as four kno

Full text of DOI:10.1002/cbdv.201300350

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
New Derivatives of Nonactic and Homononactic Acids from Bacillus pumilus
Derived from Breynia fruticosa
by Li Hana), Peiyuan Huob)c), Huahong Chend), Songtao Lib), Yi Jiangd), Liya Lia), Lihua Xud),
Chenglin Jianga)d), and Xueshi Huang*a)b)
a) Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University,
Shenyang 110819, P. R. China (phone/fax: þ86-24-24245710; e-mail: huangxs@mail.neu.edu.cn)
b) Laboratory of Metabolic Disease Research and Drug Development, China Medical University,
Shenyang 110001, P. R. China
c) North China Pharmaceutical Group Corporation New Drug Research and Development Co., Ltd.,
Shijiazhuang 050015, P. R. China
d) Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, P. R. China
Six new nonactic and homononactic acid derivatives, ethyl homononactate (1), ethyl nonactate (2),
homononactyl homononactate (6), ethyl homononactyl nonactate (7), ethyl homononactyl homono-
nactate (8), and ethyl nonactyl nonactate (9), as well as four known compounds, homononactic acid (3),
nonactic acid (4), homononactyl nonactate (5), and bishomononactic acid (10), were isolated from
culture broth of Bacillus pumilus derived from Breynia fruticosa. The structures of new compounds were
elucidated by spectroscopic analysis and chemical methods. The optical purities of 1–6 were determined
by HPLC/MS after treatment with l-phenylalanine methyl ester. The dimeric compounds 5–9 showed
weak cytotoxic activities against five human cancer cell lines (IC50 19–100 mg/ml).
Introduction. – Endophytes from medicinal plants have been considered as a major
source for bioactive natural products for a long time [1]. Xishuangbanna, Yunnan, P. R.
China, possesses abundant medicinal plant resources because of its unique geo-
graphical and climatic characteristics. In a previous work, the co-authors at Yunnan
University had studied the diversity of microorganisms from medicinal plants collected
from Xishuangbanna, and more than 2,000 strains were isolated [2]. As a subsequent
work, searching for new bioactive metabolites from microbial sources associated with
Xishuangbanna medicinal plants, 87 strains were fermented in small amounts (100 ml)
with four culture broths, respectively, and the metabolites were assessed by the
chemical screening method. Bacillus pumilus (YIM 56368) was selected for inves-
tigations based on HTLC and HPLC analyses.
Bacillus pumilus derived from Breynia fruticosa (Euphorbiaceae), which is a Dai
medicinal plant, distributed in Xishuangbanna, used for treating dermatitis, eczema,
sore, and furuncle in local medicine. In this article, we describe the isolation and
structure elucidation of six new nonactic and homononactic acid derivatives from the
fermentation broth of B. pumilus, named ethyl homononactate (1), ethyl nonactate (2),
homononactyl homononactate (6), ethyl homononactyl nonactate (7), ethyl homo-
nonactyl homononactate (8), and ethyl nonactyl nonactate (9), along with the known
compounds homononactic acid (3) [3][4], nonactic acid (4) [3][4], homononactyl
ꢀ 2014 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1089
Scheme
a) 0.1n KOH, 95% EtOH, r.t. b) l-Phenylalanine methyl ester, N,N’-dicyclohexylcarbodiimide (DCC),
4-(dimethylamino)pyridine (DMAP), CH2Cl2, r.t.
nonactate (5) [5], and bishomononactic acid (10) [6] (Scheme). The structures of the
new compounds were determined on the basis of spectroscopic chemical methods.
Nonactic and homononactic acids were the building units of macrotetrolide
antibiotics, such as nonactin, monactin, dinactin, trinactin, etc. [4][7]. Although total
synthesis of the derivatives of homonoactic and nonactic acids had been accomplished
previously, the optical-rotation values of synthetic pure chiral molecules were very
small, just between 9–148 [6][8–10]. Thus, it was very difficult to confirm whether the
natural derivatives of them existed in optically pure or racemic form only on the basis
of optical-rotation values [3][11]. Furthermore, there was no report in the literature to
verify homonoactic and nonactic acid derivatives isolated from natural sources, as a
mixture of enantiomers. In the present work, ethyl homononactate (1), ethyl nonactate
(2), homononactic acid (3), nonactic acid (4), homononactyl nonactate (5), and
homononactyl homononactate (6) were confirmed as partially racemic mixtures by
treatment with l-phenylalanine methyl ester, and the derivatives 3a/3b, 4a/4b, 5a/5b,
and 6a/6b (Scheme, Table 1) were analyzed by HPLC/MS. Furthermore, biological
assays for the cytotoxic activities of these isolated compounds were performed.

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Table 1. Analysis of l-Phenylalanine Amide Derivatives of 3–6 Obtained from the Esters 1, 2, and 5
Compound
Derivatives
tR [min]
Peak area [%]
1
2
3
4
5
3a/3b
4a/4b
3a/3b
4a/4b
4.974/6.098
4.626/5.792
4.974/6.098
4.626/5.792
14 : 100
100 : 71
79 : 100
100 : 34
57: 100
5a/5b
3.949/4.679
3a/3b, 4a/4b
6a/6b
4.974/6.098, 4.626/5.792
6.345/6.959
48 : 100, 100 : 62
100 : 51
6
Results and Discussion. – Compound 1 was obtained as colorless oil, and its
molecular formula was deduced as C13H24O4 from HR-ESI-MS (m/z 267.1562 ([Mþ
Na]þ , C13H24NaOþ4 ; calc. 267.1572)). The IR spectrum revealed the presence of OH
(3329 cmꢀ1) and C¼O groups (1739 cmꢀ1). The 1H-NMR spectrum (Table 2) exhibited
one O-bearing CH2 signal (d(H) 4.10–4.19 (m), three CHꢀO signals (d(H) 4.10–4.16
(m), 3.98 (q-like, J¼6.6), 3.71–3.75 (m)), and one CH (d(H) 2.51 (quint.-like, J¼7.2 ) ,
four CH2, and three Me signals. The 13C-NMR spectrum of 1 showed all 13 signals
indicated by the molecular formula, including that of one ester C¼O group (d(C)
174.8). The HSQC spectrum allowed the assignment of all the H-atoms to the
corresponding C-atoms. The close similarity of NMR data indicated compound 1 to be
structurally related to homononactic acid (3). An additional EtO signal suggested 1
was ethyl ester derivative of 3, which was confirmed by HMBC between the signal at
d(H) 4.10–4.19 (m) and that at d(C) 174.8. Thus, the structure of 1 was elucidated as
ethyl homononactate. Homononactic acid (3), the precursor of 1, was also isolated
from the same strain. The absolute configuration of 3 ([a]2D0 ¼ ꢀ6.38 (c¼3.6, MeOH))
was inferred first by chemical derivatization and optical-purity analysis. Compound 3
was treated with l-phenylalanine methyl ester to yield two derivatives, 3a and 3b, with a
peak area ratio of 79 :100 determined by HPLC/MS. Compounds 3a and 3b were
isolated by preparative HPLC, and their structures were confirmed by 1H- and
13C-NMR, and mass spectra. Therefore, 3 was determined as a partially racemic
mixture composed of (þ)- and (ꢀ)-homononactic acid. By the same method, 1 ([a]2D0 ¼
þ28.9 (c¼0.95, MeOH)) was reacted with l-phenylalanine methyl ester after alkaline
hydrolysis, and two compounds, 3a and 3b, were detected by HPLC/MS with a peak
integral ratio of 14 :100. Thus, the structure of 1 was determined as a mixture composed
of (þ)- and (ꢀ)-ethyl homononactate.
Compound 2 was isolated as colorless oil, and its molecular formula was deduced as
C12H22O4 from the HR-ESI-MS (m/z 253.1418 ([MþNa]þ , C12H22NaO4þ ; calc.
1
253.1410)). The H- and 13C-NMR data indicated a close resemblance of 2 to the
known compound nonactic acid (4; Table 2). The only difference was the presence of
an additional EtO group in 2. The location of the EtO group at C(1) was confirmed by
HMBC between the signals for CH2O (d(H) 4.11–4.18 (m) and the C¼O C-atom (d(C)
174.8). To determine the absolute configurations of 2 and 4 ([a]2D0 ¼6.25 (c¼1.6,
MeOH)), their l-phenylalanine methyl ester derivatives were prepared and analyzed
by HPLC/MS. Two products 4a and 4b were detected respectively. Therefore, the
structure of 4 was established as a pair of enantiomers of (þ)- and (ꢀ)-nonactic acid

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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Table 2. 1H- and 13C-NMR Data of 1 and 2 in CDCl3. d in ppm, J in Hz.
Position
1a)
2b)
d(H)
d(C)
d(H)
d(C)
1
2
3
4
5
6
7
174.8
45.3
81.0
28.7
30.6
76.6
40.6
174.8
45.3
81.1
28.7
30.5
77.2
42.6
2.51 (quint.-like, J¼7.2)
3.98 (q-like, J¼6.6)
1.97–2.01, 1.59–1.65 (2m)
1.97–2.01, 1.59–1.65 (2m)
4.10–4.16 (m)
1.72 (ddd, J¼14.4, 7.8, 4.2),
1.66 (ddd, J¼14.4, 7.2, 3.0)
3.71–3.75 (m)
2.52 (quint.-like, J¼7.2)
3.98 (q-like, J¼6.6)
1.98–2.03, 1.61–1.65 (2m)
1.97–2.02, 1.61–1.65 (2m)
4.11–4.18 (m)
1.74 (ddd, J¼13.3, 8.0, 4.6),
1.62–1.68 (m)
8
9
10
11
1’
70.4
29.9
10.1
13.4
60.4
14.1
3.96–4.06 (m)
65.2
23.1
13.5
1.50–1.54, 1.44–1.48 (2m)
0.93 (t-like, J¼7.2)
1.11 (d, J¼7.2)
1.21 (d, J¼6.3)
1.13 (d, J¼6.6)
4.10–4.19 (m)
4.11–4.18 (m)
1.27 (t, J¼7.0)
2.85 (br. s)
60.5
14.2
2’
OH
1.25 (t, J¼7.2)
2.78 (br. s)
b
a) At 600 and 150 MHz for 1H- and 13C-NMR, respectively. ) At 300 and 75 MHz for 1H- and 13C-NMR,
respectively.
with predominance of (þ)-nonactic acid. Compound 2 was also a mixture of (þ)- and
(ꢀ)-ethyl nonactate with predominance of (þ)-enantiomers.
The 1H- and 13C-NMR, and ESI-MS data of 5 were identical to those of the known
compound homononactyl nonactate which had been obtained from Streptomyces
globisporus as reported in [5]. Unfortunately, the authors had failed to determine the
relative and absolute configuration of homononactyl nonactate by alkaline hydrolysis.
The structure of related compound feigrisolide C was corrected later, and the absolute
configuration was identified as (2’S,3’S,6’R,8’R)-homononactoyl (2R,3R,6S,8S)-non-
actic acid by means of total synthesis [12][13]. By careful comparison of the 13C-NMR
data with those in [13], we could deduce the structure of 5 either as (2R,3R,6S,8S)-
homononactyl (2’S,3’S,6’R,8’R)-nonactate, or as an enantiomer mixture. A chemical
derivatization of 5 with l-phenylalanine methyl ester was conducted, and two products
5a and 5b were detected in a ratio of 57:100. Meanwhile, 5 was hydrolyzed and treated
with l-phenylalanine methyl ester. Four peaks for 3a/3b and 4a/4b were detected
with ratios of 48 :100 and 100 :62, respectively, by HPLC/MS. Thus, compound 5
was determined as a partially racemic mixture of (2R,3R,6S,8S)-homononactyl
(2’S,3’S,6’R,8’R)-nonactate.
Compound 6 was isolated as colorless oil, and its molecular formula, C22H38O7, was
deduced from HR-ESI-MS (m/z 413.2537 ([MꢀH]ꢀ , C22H37O7ꢀ ; calc. 413.2539)). The
IR spectrum indicated the presence of OH (3422 cmꢀ1) and C¼O (1729 cmꢀ1
)
functionalities. The 13C-NMR spectrum of 6 exhibited 22 C-atom signals, which were
assigned to four Me, eight CH2, and eight CH groups, and two quaternary C-atoms
(Table 3). The 1H- and 13C-NMR data of 6 suggested that there were two homononactic
acid residues in 6, a HMBC between the signal at d(H) 4.96 (quint.-like, J¼6.0) and
1
that at d(C) 174.6 confirmed the structure as homononactyl homononactate. The H-

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
and 13C-NMR, and MS data of 6 were identical to feigrisolide D reported in [12], which
was isolated from the culture broth of Streptomyces griseus, as well as feigrisolides
A–C, and the structure of feigrisolide D was proposed to be a macrodiolide.
Feigrisolides A–C were corrected to nonactic acid, homononactic acid, and homo-
nonactyl nonactate, respecitvely, by synthesis [13–15]. Although, no synthesis was
reported verifying the incorrect structure of feigrisolide D, we still could confirm that
feigrisolide D possessed the same structure as 6 according to identical spectral data.
The same derivatization method as in the case of 5 was applied to 6, and two
compounds 6a and 6b with a ratio of 100 :51 were identified by HPLC/MS. So, 6
was elucidated as a racemic mixture composed of (2R,3R,6S,8S)-homononactyl
(2’S,3’S,6’R,8’R)-homononactate and ent-6.
Table 3. 13C-NMR Data of 5–9. d in ppm.
Position
5a)c)
6b)c)
7b)c)
8b)c)
9b)d)
1
2
3
4
5
6
7
8
9
178.6
46.7
81.9
29.4
32.4
77.7
41.5
74.9
28.6
9.7
177.2
45.1
80.5
29.0
31.1
76.6
40.2
73.2
27.3
9.4
174.9
45.4
80.3
28.4
31.4
76.5
40.2
73.7
27.4
9.3
174.8
45.4
80.3
28.4
31.4
76.5
40.2
73.8
27.4
9.3
174.9
45.4
80.4
28.4
31.4
76.6
42.5
69.5
20.5
13.2
10
11
1’
2’
3’
4’
5’
6’
7’
8’
9’
10’
11’
1’’
2’’
13.8
176.5
47.5
82.0
29.7
32.2
77.9
46.3
66.1
24.3
14.2
13.4
174.6
45.5
80.9
28.8
30.5
77.0
40.6
70.3
29.9
10.1
13.8
13.1
174.3
45.5
80.8
28.6
30.6
76.9
43.0
65.1
23.3
13.5
13.5
174.4
45.5
80.9
28.6
30.6
76.9
40.8
70.3
30.0
10.1
13.5
60.3
14.2
174.2
45.4
80.9
28.6
30.6
77.2
42.9
65.1
23.3
13.2
60.3
14.2
60.3
14.2
a) In CD3OD. b) In CDCl3. c) At 75 MHz. d) At 150 MHz.
Compound 7 was isolated as colorless oil, and its molecular formula, C23H40O7, was
determined by HR-ESI-MS (m/z 451.2680 ([MþNa]þ , C23H40NaO7þ ; calc. 451.2672)).
The 1H- and 13C-NMR spectra were very similar to those of 5, the spectra of 7 indicating
an additional EtO group (d(H) 4.12–4.18 (m, 2 H), 1.26 (t, J¼7.2, 3 H); d(C) 60.3,
14.2). The HMBC experiment confirmed that 7 was the ethyl ester of 5. Complete
assignments of the 1H- and 13C-NMR spectra of 7 were achieved (Tables 3 and 4) with
the aid of HSQC, HMBC, and NOESY experiments. Because of the lack of material,

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
we could not elucidate the optical purity of 7 by hydrolysis or derivatization. However,
based on comparison of the optical rotation value of 7 ([a]2D0 ¼ ꢀ12.53 (c¼0.76,
MeOH)) with that of 5 ([a]2D0 ¼ ꢀ7.63 (c¼3.10, MeOH)) of the same biogenetic
source, we could deduce that, like 5, 7 was a partial racemic mixture consisting of ethyl
(2R,3R,6S,8S)-homononactyl (2’S,3’S,6’R,8’R)-nonactate and ethyl (2S,3S,6R,8R)-ho-
mononactyl (2’R,3’R,6’S,8’S)-nonactate.
Compound 8 had the molecular formula C24H42O7, as deduced from HR-ESI-MS.
1
Analysis of H- and 13C-NMR data indicated a close structural relationship with 6,
except for another EtO group (d(H) 4.10–4.17 (m, 2 H), 1.27 (t, J¼7.2, 3 H); and d(C)
60.3, 14.2) in 8. HMBC from CH2(1’’) (d(H) 4.10–4.17) and Me(2’’) (d(H) 1.27) to
C(1’) (d(C) 174.4) evidenced the gross structure of 8 as an ethyl ester of 6. Both 6 and 8
were optically inactive; accordingly, 8 was also a racemic compound composed of ethyl
(2R,3R,6S,8S)-homononactyl (2’S,3’S,6’R,8’R)-homononactate and ent-8.
Compound 9 had the molecular formula C22H38O7 determined by HR-ESI-MS. The
1H- and 13C-NMR spectra were very similar to those of known dimeric nonactic acid
[16], with the exception of one more EtO group (d(H) 4.11–4.19 (m, 2 H); 1.26 (t, J¼
7.2, 3 H); d(C) 60.3, 14.2) in 9. All the spectral data suggested that 9 was the ethyl ester
of dimeric nonactic acid, which was confirmed by HMBCs. Complete assignments of
1
the H- and 13C-NMR spectra of 9 were achieved (Tables 3 and 4) with the aid of
HSQC, HMBC, and NOESY experiments. Hydrolysis or derivatization was not
conducted on 9 because of lack of material. By careful comparison of the 13C-NMR
data with those of feigrisolide C, which was obtained by total synthesis [15], all dimeric
compounds 5–9 exhibited the same configuration as feigrisolide C. All of them consist
of two parts of opposing optical rotations, i.e., (ꢀ)-homononactyl (þ)-nonactate (5),
(ꢀ)-homononactyl (þ)-homononactate (6), ethyl (ꢀ)-homononactyl (þ)-nonactate
(7), ethyl (ꢀ)-homononactyl (þ)-homononactate (8), ethyl (ꢀ)-nonactyl (þ)-non-
actate (9), and their enantiomers. The structure of 9, either an optically pure compound
or a racemic mixture, could not be determined since only the optical-rotation value
([a]2D0 ¼0 (c¼0.15, MeOH)) was available.
Compounds 1–10 were evaluated for their cytotoxic activities against five human
cancer cell lines, including human gastric carcinoma cell line (BGC-823), human
hepatocellular liver carcinoma cell line (HepG2), human large-cell lung carcinoma cell
line (H460), human cervix carcinoma cell line (HeLa), and human colon carcinoma
cell line (HCT116) with MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra-
zolium bromide) method [17]. Dimeric compounds 5–9 showed weak cytotoxic
activities (Table 5), whereas monomers 1–4 and 10 were inactive against the five cell
lines (IC50 >100 mg/ml).
Conclusions. – Six new derivatives of homonoactic and nonactic acids were isolated
from the culture broth of B. fruticosa and identified. The enantiomeric excesses of ethyl
homononactate (1), ethyl nonactate (2), homononactic acid (3), nonactic acid (4),
homononactyl nonactate (5), and homononactyl homononactate (6) were revealed by
chemical derivatization and HPLC/MS analysis. Dimeric compounds 5–9 showed weak
cytotoxic activities compared to the monomer compounds 1–4 and 10.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1095
Table 5. Cytotoxic (IC50 [mg/ml]) Activities of Compounds 5–9
Compound
H460
HeLa
27.8
HCT116
BGC-823
HepG-2
5
6
7
8
19.1
61.6
43.2
78.8
69.6
0.57
29.8
73.2
71.4
32.2
>100
>100
>100
>100
0.86
58.4
67.9
>100
>100
>100
0.29
87.5
65.8
97.7
>100
>100
0.80
9
>100
0.56
Adriamycin
This work was funded by the National Natural Science Foundation of China (No. 20602041,
312700040, and U0932601) and the Basic Scientific Research Fund of Northeastern University, China (No.
N120820002).
Experimental Part
General. Agilent 1290-6420 Triple Quadrupole LC/MS system with a Agilent SB-C18 column (2.1ꢁ
50 mm, 1.8 mm) was used to analyze the l-phenylalanine methyl ester derivatives. Column chromato-
graphy (CC): silica gel (SiO2; 100–200 mesh and 200–300 mesh; Qingdao Marine Chemical Ltd.,
Qingdao, P. R. China), Sephadex LH-20 (GE Healthcare Bio-scienses AB, SE-Uppsala), and YMC*GEL
ODS-A (S-50 mm, 12 nm; YMC Co., Ltd., Kyoto, Japan). HPLC: Waters 600-2487 chromatograph with a
YMC pack ODS-A column (30ꢁ250 mm, S-15 mm, 12 nm; flow rate, 10 ml/min), with MeOH/H2O
70 :30. Optical rotations: SGW-1 automatic polarimeter (Shanghai Precision & Scientific Instrument Co.,
Ltd., Shanghai, P. R. China). IR Spectra: Nicolet Avatar 330 FT-IR spectrometer (KBr); ˜n in cmꢀ1. 1D-
and 2D-NMR spectra: Bruker ARX-300 or Bruker AV-600 spectrometers; d in ppm rel. to Me4Si as
internal standard, J in Hz. ESI-MS: Thermo Finnigan LCQ mass spectrometer; in m/z. HR-ESI-MS:
Waters LCT Premier XE TOF mass spectrometer; in m/z. MTT assay was conducted on a microplate
reader (KHB ST-360, SH Kehua Laboratory System Co., Ltd., Shanghai, P. R. China).
Microbiological Material. The producing organism was isolated from a plant sample (Breynia
fruticosa) collected from Xishuangbanna, Yunnan Province, P. R. China, in October 2009. The strain was
identified as Bacillus pumilus by Y. J. based on morphological characteristics and 16S rRNA gene
sequences. The strain (No. YIM 56368) was deposited with the Yunnan Institute of Microbiology,
Yunnan University, P. R. China.
Fermentation, Extraction, and Isolation. A slant culture of the strain was inoculated into 500-ml
Erlenmeyer flasks containing 100 ml of seed medium composed of yeast extract (4 glꢀ1), glucose
(4 glꢀ1), malt extract (5 glꢀ1), multiple vitamins soln. (1.0 ml), and trace-element soln. (1.0 ml), at
pH 7.2, with no adjustment and cultured for 2 d at 288 on a rotary shaker at 180 rpm. This seed culture
was used to inoculate the fermetation medium with 10% volume. The fermentation was carried out in a
500-ml Erlenmeyer flask containing 100 ml of fermentation medium containing soybean meal (10 glꢀ1),
peptone (2 glꢀ1), glucose (20 glꢀ1), soluble starch (5 glꢀ1), yeast extract (2 glꢀ1), NaCl (4 glꢀ1),
K2HPO4 (0.5 glꢀ1), MgSO4 ·7 H2O (0.5 glꢀ1), CaCO3 (2 glꢀ1), at pH 7.8, with no adjustment, and
incubated for 7 d at 288 on a rotary shaker at 180 rpm.
The completed fermentation broth (80 l) was separated into filtrate and mycelium by centrifugation.
The culture filtrate was condensed to 10 l, and then partitioned in H2O/AcOEt to yield 32.0 g of a dried
AcOEt extract and an aq. residue. The AcOEt extract was subjected to CC (SiO2; CH2Cl2/MeOH 50 :1–
1:1). The eluents were combined to nine fractions, Frs. 1–9, on the basis of TLC analysis. Fr. 4 (3.48 g)
was subjected to CC (Sephadex LH-20; MeOH) to give ten fractions, Frs. 4.1–4.10. Fr. 4.3 was submitted
to CC (Sephadex LH-20; MeOH) and further separated by CC (ODS; MeOH/H2O 70 :30) to yield 1
(23.0 mg), 2 (13.0 mg), and 9 (5.2 mg). Fr. 5 (0.94 g) was purified by CC (Sephadex LH-20; MeOH) to
give six fractions, Frs. 5.1–5.6. Fr. 5.2 was separated by CC (ODS; MeOH/H2O 70 :30) to yield 7 (5.5 mg)
and 8 (6.5 mg). Fr. 6 (2.40 g) was subjected to by CC (Sephadex LH-20; MeOH) to afford 16 fractions,

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Frs. 6.1–6.16. Fr. 6.4 was purified by CC (Sephadex LH-20; MeOH) to afford five subfractions,
Frs. 6.4.1–6.4.5. Fr. 6.4.1 was separated by CC (ODS; MeOH/H2O 80 :20), and then further purified by
CC (SiO2; petroleum ether/AcOEt 5 :3) to furnish 5 (46.0 mg) and 6 (22.0 mg). Fr. 6.4.3 was separated by
CC (ODS; MeOH/H2O 40 :60) to give 3 (350 mg) and 10 (9.5 mg). Fr. 7 (1.03 g) was submitted to CC
(Sephadex LH-20, MeOH) to afford eleven fractions, Frs. 7.1–7.11. Fr. 7.4 was subjected to CC (ODS;
MeOH/H2O 40 :60), and then further purified by CC (SiO2; CH2Cl2/MeOH 40 :1) to yield 4 (80 mg).
Ethyl Homononactate (¼ Ethyl (2S)-2-{(2S,5R)-Tetrahydro-5-[(2R)-2-hydroxybutyl]furan-2-yl}pro-
panoate; 1): Colorless oil. [a]2D0 ¼ þ28.9 (c¼0.95, MeOH). IR (KBr): 3329, 2926, 2854, 1739, 1454,
1260, 1093, 1029. 1H- and 13C-NMR: see Table 2. ESI-MS: 267 ([MþNa]þ ), 511 ([2MþNa]þ ), 245
([MþH]þ ). HR-ESI-MS: 267.1562 ([MþNa]þ , C13H24NaOþ4 ; calc. 267.1572).
Ethyl Nonactate (¼ Ethyl (2S)-2-{(2S,5R)-Tetrahydro-5-[(2R)-2-hydroxypropyl]furan-2-yl}propa-
noate; 2): Colorless oil. [a]2D0 ¼ þ9.2 (c¼0.55, MeOH). IR (KBr): 3449, 2972, 2935, 1735, 1461, 1376,
1191, 1092, 1060. 1H- and 13C-NMR: see Table 2. ESI-MS: 231 ([MþH]þ ), 253 ([MþNa]þ ), 483 ([2Mþ
Na]þ ), 269 ([MþK]þ ). HR-ESI-MS: 253.1418 ([MþNa]þ , C12H22NaOþ4 ; calc. 253.1410).
Homononactyl Nonactate (¼ rac-(2R)-2-{(2R,5S)-Tetrahydro-5-[(2S)-2-{[(2S)-2-{(2S,5R)-tetrahy-
dro-5-[(2R)-2-hydroxypropyl]furan-2-yl}propanoyl]oxy}butyl]furan-2-yl}propanoic Acid; 5): Colorless
oil. [a]2D0 ¼ ꢀ7.63 (c¼3.10, MeOH). 1H- and 13C-NMR: see Tables 3 and 4. ESI-MS: 401 ([MþH]þ ), 418
([MþNH4]þ ), 423 ([MþNa]þ ), 818 ([2MþNH4]þ ), 823 ([MþNa]þ ), 399 ([MꢀH]ꢀ ), 799 ([2Mꢀ
H]ꢀ ).
Homononactyl Homononactate (¼ rac-(2R)-2-{(2R,5S)-Tetrahydro-5-[(2S)-2-{[(2S)-2-{(2S,5R)-5-
[(2R)-tetrahydro-2-hydroxybutyl]furan-2-yl}propanoyl]oxy}butyl]furan-2-yl}propanoic Acid; 6): Color-
1
less oil. [a]2D0 ¼0 (c¼0.80, MeOH). IR (KBr): 3422, 2968, 2936, 1729, 1462, 1383, 1197, 1064. H- and
13C-NMR: see Tables 3 and 4. ESI-MS: 415 ([MþH]þ ), 437 ([MþNa]þ ), 413 ([MꢀH]ꢀ ). HR-ESI-MS
413.2537 ([MꢀH]ꢀ , C22H37Oꢀ7 ; calc. 413.2539).
Ethyl Homononactyl Nonactate (¼(2S)-1-{(2S,5R)-5-[(2R)-1-Ethoxy-1-oxopropan-2-yl]tetrahydro-
furan-2-yl}butan-2-yl (2S)-2-{(2S,5R)-Tetrahydro-5-[(2R)-2-hydroxypropyl]furan-2-yl}propanoate; 7):
Colorless oil. [a]2D0 ¼ ꢀ12.53 (c¼0.76, MeOH). IR (KBr): 3461, 2971, 2937, 1733, 1460, 1377, 1191,
1
1091, 1062. H- and 13C-NMR: see Tables 3 and 4. ESI-MS: 429 ([MþH]þ ), 446 ([MþNH4]þ ), 451
([MþNa]þ ), 874 ([2MþNH4]þ ). HR-ESI-MS: 451.2680 ([MþNa]þ , C23H40NaOþ7 ; calc. 451.2672).
Ethyl Homononactyl Homononactate (¼(2S)-1-{(2S,5R)-5-[(2R)-1-Ethoxy-1-oxopropan-2-yl]tetra-
hydrofuran-2-yl}butan-2-yl (2S)-2-{(2S,5R)-Tetrahydro-5-[(2R)-2-hydroxybutyl]furan-2-yl}propanoate;
8): Colorless oil. [a]2D0 ¼0 (c¼0.25, MeOH). IR (KBr): 3449, 2969, 2932, 1732, 1459, 1388, 1193, 1066. 1H-
and 13C-NMR: see Tables 3 and 4. ESI-MS: 443 ([MþH]þ ), 460 ([MþNH4]þ ), 465 ([MþNa]þ ). HR-
ESI-MS: 465.2831 ([MþNa]þ , C24H42NaO7þ ; calc. 465.2823).
Ethyl Nonactyl Nonactate (¼(2S)-1-{(2S,5R)-5-[(2R)-1-Ethoxy-1-oxopropan-2-yl]tetrahydrofuran-
2-yl}propan-2-yl (2S)-2-{(2S,5R)-Tetrahydro-5-[(2R)-2-hydroxypropyl]furan-2-yl}propanoate; 9): Col-
orless oil. [a]2D0 ¼0 (c¼0.15, MeOH). IR (KBr): 3448, 2973, 1734, 1459, 1377, 1192, 1090, 1059. 1H- and
13C-NMR: see Tables 3 and 4. ESI-MS: 415 ([MþH]þ ), 437 ([MþNa]þ ). HR-ESI-MS: 415.2702 ([Mþ
H]þ , C22H39Oþ7 ; calc. 415.2696).
Bishomononactic Acid (¼ rac-(2S)-2-{(2S,5R)-Tetrahydro-5-[(2S)-2-hydroxy-3-methylbutyl]furan-
1
2-yl}propanoic Acid; 10): Colorless oil. H-NMR (300 MHz, CD3OD): 3.96–4.00 (m, 1 H); 3.86–3.90
(m, 1 H); 3.39–3.45 (m, 1 H); 2.32–2.37 (m, 1 H); 1.87–1.96 (m, 2 H); 1.36–1.65 (m, 5 H); 1.01 (d, J¼
6.9, 3 H); 0.80 (d, J¼7.2, 6 H). 13C-NMR (75 MHz, CD3OD): 181.6; 82.0; 78.0; 74.6; 47.1; 42.3; 35.3; 32.5;
29.6; 19.2; 17.9; 14.0. ESI-MS: 253 ([MþNa]þ ), 229 ([MꢀH]ꢀ , 459 ([2MꢀH]ꢀ ). The spectral
properties were identical to those reported in [6].
Synthesis of 3a and 3b. Homononactic acid (3; 18 mg) and l-phenylalanine methyl ester (25 mg)
were dissolved in 5 ml of CH2Cl2, then 25 mg of N,N’-dicyclohexylcarbodiimide (DCC) and 16 mg of 4-
(dimethylamino)pyridine (DMAP) were added, and the mixture was stirred at r.t. for 48 h for the
preparation of homonoactic amide derivatives. Completion of the reaction was monitored by TLC. HCl
(1n, 10 ml) was added with vigorous stirring to quench the reaction. The org. layer was separated, and the
aq. layer was extracted with CH2Cl2 (2ꢁ10 ml). The combined org. extracts were washed with brine
(30 ml) and dried (Na2SO4). After filtration and evaporation in vacuo, the residue was purified by prep.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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HPLC (YMC Pack ODS-A (30ꢁ250 mm, S-15 mm, 12 nm); 70% aq. MeOH soln.; flow rate, 10 ml/min).
Collected fractions were evaporated under reduced pressure to afford 3a (8.5 mg) and 3b (15 mg).
Data of 3a. White powder. 1H-NMR (300 MHz, CDCl3): 7.20–7.31 (m, 3 H); 7.08–7.14 (m, 2 H); 4.90
(q-like, J¼5.7, 1 H); 4.05–4.08 (m, 1 H); 3.74–3.82 (m, 1 H); 3.70 (s, 3 H); 3.63–3.70 (m, 1 H); 3.04–
3.18 (m, 2 H); 2.33 (qunint.-like, J¼7.3, 1 H); 1.92–2.02 (m, 2 H); 1.64 (t, J¼5.9, 2 H); 1.56–1.60 (m,
2 H); 1.45 (quint.-like, J¼6.7, 2 H); 1.13 (d, J¼7.0, 3 H); 0.92 (t, J¼7.3, 3 H). 13C-NMR (75 MHz,
CDCl3): 173.8; 172.2; 136.2; 129.3 (2 C); 128.4 (2 C); 126.9; 80.8; 76.9; 70.3; 53.0; 52.1; 45.8; 41.6; 38.1;
30.6; 30.3; 29.7; 14.3; 9.9. ESI-MS: 378 ([MþH]þ ), 400 ([MþNa]þ ).
1
Data of 3b. White powder. H-NMR (300 MHz, CDCl3): 7.15–7.23 (m, 3 H); 7.03 (d, J¼7.3, 2 H);
4.82 (q-like, J¼6.2, 1 H); 4.02–4.06 (m, 1 H); 3.68–3.76 (m, 1 H); 3.65 (s, 3 H); 3.48–3.52 (m, 1 H); 3.11
(dd, J¼13.5, 5.9, 1 H); 3.01 (dd, J¼13.5, 5.9, 1 H); 2.23 (quint.-like, J¼7.3, 1 H); 1.84–1.93 (m, 2 H);
1.31–1.65 (m, 6 H); 1.04 (d, J¼7.0, 3 H); 0.82 (t, J¼7.3, 3 H). 13C-NMR (75 MHz, CDCl3): 173.8; 172.3;
136.2; 129.2 (2 C); 128.4 (2 C); 126.9; 80.8; 76.8; 69.9; 53.2; 52.2; 45.8; 41.6; 37.9; 30.4; 30.2; 29.8; 13.6; 9.9.
ESI-MS: 378 ([MþH]þ ), 400 ([MþNa]þ ).
Preparation of l-Phenylalanine Amide Derivatives of 3–6 for HPLC Analysis. In an entirely
analogous way as that for the preparation of 3a and 3b, with the exception of purification by prep. HPLC,
amide derivatives of 3 (2.0 mg), 4 (1.9 mg), 5 (2.5 mg), and 6 (2.5 mg) were obtained for HPLC/MS
analysis. Derivatives of 3–6 were dissolved in 5 ml of MeOH, resp., and further diluted 1,000 times with
MeOH/H2O 1:1 for HPLC analysis.
Preparation of Amide Derivatives of 1, 2, and 5 for HPLC Analysis. To a soln. of 1 (2 mg) in 95% aq.
EtOH (2 ml), 0.1n KOH (0.5 ml) was added. After stirring at r.t. for 24 h, the mixture was adjusted to
pH 7–8 with 0.1n HCl soln. and concentrated in vacuo. The residue was worked up by dilution with H2O
and extraction with CH2Cl2. The org. phase was washed with brine, dried (Na2SO4), and evaporated. The
residue was taken up in CH2Cl2 (2 ml) and treated with an excess of l-phenylalanine methyl ester (5 mg),
DCC (3 mg), and DMAP (1.5 mg). After stirring for 48 h at r.t., the mixture was diluted with H2O and
extracted with CH2Cl2. The org. phase was washed sequentially with 1n HCl and brine, dried (Na2SO4),
and evaporated to afford the amide of 1.
In an entirely analogous way, the amides of 2 and 5 (2 and 2.6 mg, resp.) were prepared. Amides of 1,
2, and 5 were dissolved in 5 ml of MeOH, and further diluted 1,000 times with MeOH/H2O 1:1 for HPLC
analysis, resp.
HPLC/MS Analysis of 3a/3b, 4a/4b, 5a/5b, and 6a/6b. Agilent 1290-6420 Triple Quadrupole LC/MS
system with a Agilent SB-C18 column (2.1ꢁ50 mm, 1.8 mm) was used to analyze the l-phenylalanine
methyl ester derivatives. A positive SIM (selected ion monitoring) mode was used to analyze 3a/3b (m/z
400 ([MþNa]þ )), 4a/4b (m/z 386 ([MþNa]þ )), 5a/5b (m/z 584 ([MþNa]þ )), and 6a/6b (m/z 598
([MþNa]þ )). The instrument was operated in positive-ion electrospray ionization mode. The TQ (triple
quadrupole)-MS conditions utilized were a gas temp. of 3308, gas flow of 10 l/min, nebulizer pressure of
35 psi, cap. voltage of 4000 V, and cell accelerator voltage of 1 V. The mobile-phase solvent composition
was 50% A (0.1% HCOOH in H2O) and 50% B (MeOH) for 3a/3b, 55% A and 45% B for 4a/4b, and
32% A with 68% B for 5a/5b and 6a/6b. The flow rate was 0.4 ml/min, and the column was heated to 308.
Cytotoxicity Assays. An MTTassay procedure was used to determine the cytotoxic activities of 1–10
against human gastric carcinoma (BGC-823), human hepatocellular liver carcinoma (HepG2), human
large-cell lung carcinoma (H460), human colon carcinoma (HCT116), and human cervix carcinoma cell
lines (HeLa) [17]. The IC50 value was defined as the 50% reduction of absorbance in the control assay.
Adriamycin was used as a positive control.
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Received November 8, 2013