Studies on antibacterial and anti-HIV agents from Indonesianplants, marine sponges and fungi
Name of dispatched researcher（派遣研究者名）

IsmailInstructor（指導教官，研究指導者）

Subehan Ambo Lallo

Affiliation of Instructor（所属）

Faculty of Pharmacy, Hasanuddin
University

Host Collaborator（受入研究者）

Hiroyuki Morita

Abstract
An oxazinedione glycoside (1) was isolated from the methanol extract of Spongia sp. collected
in Indonesia. Its chemical structure was elucidated by combination of extensive NMR spectroscopy, which suggested that the isolate may be new compound. To further elucidate the structure,enough amounts of the marine sponge are now collecting. In contrast, two new tetracyclic quassinoids, picrajavanicins A (2) and M (3) were isolated from the CHCl3 soluble extract of Picrasma
javanica bark collected in Myanmar. The structure of 2 and 3 were elucidated using spectroscopic
techniques, including 1D and 2D NMR. The anti-HIV activity of the isolates were evaluated byinhibitory activity of Viral protein R (Vpr), which is an accessory gene of HIV-1 that plays important roles in viral pathogenesis, together with the antiprolifilative activity against a panel of fivedifferent human cancer cell lines. The CHCl3-soluble extract of P. javanica and 2 and 3 showed
mild anti-Vpr activity, in comparing with that of positive control, damnacanthal. In addition,
3 exhibited potent and selective antiproliferative activity against the human pancreatic cancer
PANC-1 cell line.
1. Introduction
Nowadays, many bacteria infections have become impossible to treat due to antibiotic resistance. Despite the emergency and a lot of effort to find new antibiotics, only two new antibiotics
have been approved since 2009. In addition, acquired immune deficiency syndrome (AIDS) also
remains a serious health problem worldwide. Human immunodeficiency virus (HIV) is a retrovirus that causes AIDS. According to UNAIDS, globally, there were 34.0 million people living withHIV and 1.7 million people died of AIDS at the end of 2011.
Indonesia is a rich country in biodiversity due to the location in the tropical region. Abundant of medicinal plants are distributed in thousand islands of Indonesia. However, the mostnatural resources in Indonesia have remained to be investigated. Therefore, it is assumed thatIndonesian natural resources such as medicinal plants, marine sponges, and fungi are goodsources useful for discovering new drug lead candidates. In this research, we aimed to isolate the− 111 −







O
5
4

O

2

N

5'

HO

MeO

O

O
O 18

11

1

9
10

2

4'

1'

3'

2'

OMe

OMe

6

O

5

3
4

12
19

CHO
MeO

14

H7

8

H 6 H

O

13
15

H
O

H

16

O

H
OH

OH
1

O

2

H

H
O

O

3

Fig. 1. Structures of oxazinedione derivative (1) and picrajavanicins A and M (2 and 3).

bioactive compoundsOfrom Spondia sp. collected in Indonesia. Simultaneously, we also performed
O
Picrasma javanica bark colthe isolation of bioactive
compounds fromOMea fraction
prepared fromOMe
O
O

O

O
lected in Myanmar. The studies ledOto isolation ofHthree compounds(1-3, Fig.1)
O

HO

N

MeO

MeO

O

2. Materials and Methods

O

O

O

2.1 General experimental
procedures
OH

O

OH

1 2D NMR spectra were
2
3 JNM-LA400 spectrometer
The 1H, 13C, andrecorded using a JEOL
13
Fig. 2. COSY
and key
HMBC(1H→C) (arrows) correlations
in Osaka,
compounds
1−3. as an(Japan Spectroscopic
Co.(bold
Ltd,lines)
Tokyo,
Japan)
withtetramethylsilane(Wako,
Japan)

internal standard. Open column chromatography was performed with silica gel 60N, spherical,
neutral, 40 − 50 μ m, (Kanto Chemical Co., Inc., Japan). Analytical TLC was carried out on precoated silica gel 60F254 (0.25 or 0.50 mm thickness, Merck). Analytical TLC was carried out on
precoated silica gel 60F254 (0.25 or 0.50 mm thickness, Merck). The cell lines A549, HeLa, PSN-1,
and MDA-MB-231 were available and maintained in our laboratory. Cell culture ﬂasks and 96well plates were from Corning (NY, USA).
2.2 Sponge material
The marine sponges were collected from the sea of Baranglompo in Indonesia and identified
by Ms. Anna Manuputty in Research Center for Oceanography, LIPI (Indonesia Institute of Sciences) as Spongia sp. A voucher sample (SS-07-009) is preserved at the Bipharmaca ResearchCenter of Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia.
2.3 Extraction and isolation of compounds from Spongia sp.
Marine Spongia sp. (100 g) were sliced and extracted with MeOH (200 mL) under sonicationfor 3 h. The extraction was repeated three times and then the combined extract was concentrated
under reduced pressure and lyophilized to give a crude extract (3.2 g). The crude extract (3.2 g)
was subjected to a silica gel open column chromatography eluted with n-hexane–EtOAc (80:20
to 0:100) followed by methanol elution to obtain 13 fractions. Fraction 13 (0.6 g) was chromatgraphed on a silica gel open column eluted with EtOAc–methanol(15:1 to 1:1) to give five further

subfractions. The fraction 13-3 (7.5 mg) was furher separated by normal-phase preparative TLC
with EtOAc–methanol–ammonia (15:1:0.1) to give compound 1 (0.6 mg).

− 112 −

2.4 Isolation of compounds from P. javanica
Fraction 7 (3.7 g), previously fractionated from the CHCl3-soluble extract of the dried barkof P. javanica, was rechromatographed on a silica gel column chromatograph with n-hexane −CHCl3 − EtOAc (1:1:1) to give four subfractions [7-1: 131 mg; 7-2: 534 mg; 7-3: 1.80 g; 7-4: 550
mg]. Purification of subfraction 7-2 using Cosmosil 75C18-OPN with MeCN − acetone − MeOH −H2O (1:1:1:1) followed by a normal-phase preparative TLC with toluene − CHCl3 − EtOAc (1:1:1)
afforded picrajavanicin A (2, 18 mg).
Subfraction 7-4 (1.10 g), previously fractionated from the CHCl3-soluble extract of the driedbark of P. javanica, was purified by chromatography on Sephadex LH20 with MeOH followed bynormal-phase preparative TLC with CHCl3–EtOA–propan-2-ol (2:2:0.5) (development four times)
to afford picrajavanicin M (3, 15 mg).
2.5 Anti-Vpr assay
The established cell line, TREx-HeLa-Vpr (6,000 cells/well, 150 m L), was seeded in 48-wellplates and incubated in a-minimal essential medium (a-MEM, Wako), supplemented with 10%
fetal bovine serum (FBS, Nichirei Bioscience), 1% antibiotic antimycotic solution (Sigma-Aldrich),
5mg/mL of blasticidin (Invitrogen), and 50 m g/mL of zeocin, at 37°C under a 5% CO2 and 95% air
atmosphere, for 24 h. Since the expression of Vpr is regulated by tetracycline, the tetracyclinetreated cells were designed to express Vpr by the addition of 50 m L of tetracycline (10 m g/mL). After 24 h incubation, 50 m L portions of various samples at different concentrations (1.25, 2.5, 5 m g/
mL or m M) were added to the tetracycline-treated cells, and the wells without samples were used
as controls. After 48 h incubation, 50 m L of 10% WST-8 cell counting kit (Dojindo; Kumamoto,Japan) solution was added to the wells. After 2 h incubation, the absorbance at 450 nm was measured, and the cell viability was calculated from the mean values of data from three wells, by thefollowing equation.
(%) Cell viability = 100 × [{Abs(test samples) － Abs(blank)}/{Abs(control) － Abs(blank)}]
The inhibitory activity of the tested sample was obtained by comparing the number of viable
cells treated with both tetracycline and sample to the number of viable cells treated withtetracycline without sample.
2.6 In vitro antiproliferative activity
The in vitro antiproliferative activity of 2 and 3 against the A549 (human lung cancer), HeLa
(human cervix cancer), PANC-1 and PSN-1 (human pancreatic cancer), MDA-MB-231 (humanbreast cancer), and TIG-3 (normal human primary fibroblast) cell lines was evaluated by theprocedure as described previously [1]. Briefly, each cell line was seeded in 96-well plates (2 ×103 per well) and cultured either in a-MEM or DMEM at 37°C under a 5% CO2 and 95% air
atmosphere for 24 h. After the cells were washed with PBS (Nissui Pharmaceuticals), serial
dilutions of the samples to be tested were added. After 72 h incubation, the cells were washedwith PBS, and 100 μL of a-MEM or DMEM containing 10% WST-8 cell counting kit solution− 113 −

(Dojindo; Kumamoto, Japan) was added to the wells. After 2 h incubation, the absorbance at
450 nm was measured. The different concentrations of the serial dilutions of the tested samples
were 100 － 3.125 m g/mL for the crude extract, 100 － 3.125 mM for the isolated compounds, and
10 － 0.3125 mM for the positive control, respectively. Cell viability was calculated from the meanvalues of data from three wells by using the following equation, and the antiproliferative activity
was expressed as the IC50 (50% inhibitory concentration) value.
(%) Cell viability = 100 × [{Abs(test samples) － Abs(blank)}/{Abs(control) － Abs(blank)}]
Results
The oxazinedione glycoside (1) was isolated as a white powder. Its molecular formula was determined to be C10H13NO6 by HRFABMS, in conjunction with an NMR analysis data. The 1H NMR
spectrum (Table 1) showed signals of an olefinic proton [dH 7.80, d (J = 1.2, 1.2 Hz, H-4)], an anomeric proton [dH 6.26, t (J = 6.58 Hz, H-1′)], two oxygenated methine protons [dH 4.38, m (H-3′), 3.88, dd (J4= 6.7, 3.2 Hz, H-4′)], an oxygenated methylene protons with geminal coupling as a methine proton [dH 3.78, 3.71, both dd (J = 12.0, 3.2 Hz, H2-5′)], a methylene proton [dH 2.22, m (H2-2′)], and a4

tertiary methyl proton [dH 1.86, d (J = 1.2 Hz, H3-7)]. The 13C NMR (Table 1) and HMQC spectra
showed 10 carbon signals, including two carbonyl carbons (dC 166.6, 152.5), an oleofinic carbon (dC
138.3), a quaternary oleofinic carbon (dC 111.6), two oxygenated methine carbons (dC 88.9, 72.4),
an anomeric carbon (dC 86.4), an oxigenated methylene carbon (dC 63.0), a methylene carbone (dC41.3), and a methyl carbon (dC 12.6).
The 1H − 1H COSY and HMBC spectra (Fig. 2) revealed the connectivities between H-1′ (dH
4

6.26, t), H-3′ (dH 4.38, m), H-4′ (3.88, dd), H2-5′ (dH 3.78, 3.71, both dd), and H2-2′ (dH 2.22, m) and4
 C-1′ (dC 86.4), C-3′ (dC 72.4), C-5′ (dC 63.0), and C-2′ (dC 41.3), suggesting the presC-4′(dC 88.9),

ence of a pentose moiety. This also confirmed that the pentose moiety is C-2′ deoxy type. HowTable/Figures
ever,
the HMBC correlations from H-4 (d 7.80, d) to C-6 (d 166.6), C-2 (d 152.5), and C-5 (d
C

H
4

C

Table 1. 1H NMR Spectroscopic Data (600 MHz, CDCl3) of oxazinedione glycoside (1) (δ in ppm and J
Values in (Hz) in Parentheses)1

δH

position
2
4

δC
152.5

7.80, d (1.2)

138.3

2.31, m
5

111.6

6

166.6

7

1.86, d (1.2)

12.6

1'

6.26, t (6.58)

86.4

2'

2.22, m41.3

3'4.38, m72.4

4'

3.88, dd (6.7, 3.2)

88.9

5'

3.78, dd (12.0, 3.2)

63.0

3.71, dd (12.0, 3.2)

− 114 −

C

3'

4

2'

H 6 H

H
OH

OH
1

2

H
3

Fig. 1. Structures of oxazinedione derivative (1) and picrajavanicins A and M (2 and 3).

O
OMe

OMe O
O
O
O
HO

N

O

O

H

O

MeO

MeO

O
O

O

O

OH
1

O

OH
2

3

Fig. 2. COSY (bold lines) and key HMBC (1H→13C) (arrows) correlations in compounds 1−3.

116.6) and from H3-7 (dH 1.86, d) to C-4 (dC 138.3), C-5, and C-6 indicated the presence of a heterocycle oxazinedione moiety as the partial structure of 1. The HMBC spectrum also displayedcorrelations from H-1′ on the pentose moiety to C-2 and C-4 on the oxazinedione moiety. These
results suggest that the pentose moiety directly linked to nitrogen atom at 3 positon of the oxazinedione moiety, as well as the confirmation of the strcture of aglycone part of 1 to be 5-methyl-2,3dihydro-6H-1,3-oxazine-2,6-dione.
Remaining relative configuration of sugar moiety of 1 assigned on the basis of 2D NOESY
experiment, which showed the NOESY correlations between H-1′ and H-4′ and between H-3′ andH2-5′, whereas not represented any correlations between H-1′, H-4, and H-5. These data suggested that the configuration of the pentose moiety is β at the anomeric proton (Fig.1). Further acidhydrolysis of 1 is now under way.
Picrajavanicin A (2) was obtained as a pale yellow amorphous solid, and its molecular formula was determined as C21H24O7 from the HREIMS and 13C NMR data. Spectroscopic analysis
revealed UV absorption bands at 264 nm due to a conjugated enone system. The IR spectrum−1displayed absorption bands at 1730, 1702, and 1666 cm , indicative of the presence of d -lactone
and a, β -unsaturated carbonyl functionalities. The 1H NMR data (Table 2) displayed signals corresponding to an olefinic proton [d H 5.58, dd (J = 5.7, 2.3 Hz, H-3)], an oxymethine [d H 4.33, t (J =3.0 Hz, H-7 β )], three methines [d H 2.30, m (H-5a), 2.97, dd (J = 11.1, 7.3 Hz, H-14 β ), 3.06, s (H9a)], three methylenes, two methoxy groups [d H 3.61, s (MeO-2), 4.00, s (MeO-12)], two tertiary
methyls [d H 1.16, s (H3-19), 1.56, s (H3-18)], and an aldehyde group [d H 10.27, s (CHO-13)]. The
13

C NMR data (Table 2) included 21 signals for four olefinic carbons [d C 110.4 (C-3), 129.8 (C-13), 148.9 (C-2), 158.5 (C-12)], an oxymethine [d C 81.7 (C-7)], three methines [d C 35.8 (C-5), 37.9
(C-14), 48.0 (C-9)], three methylene carbons [d C 26.8 (C-4), 29.0 (C-6), 32.1 (C-15)], two methoxy
groups [d C 55.0 (MeO-2), 60.8 (MeO-12)], two tertiary methyl groups [d C 11.4 (C-18), 22.1 (C-19)],
two quaternary carbons [d C 37.3 (C-8), 45.8 (C-10)], an aldehyde carbon [d C 191.7 (CHO-13)], twocarbonyls [d C 194.5 (C-11), 196.9 (C-1)], and one lactone carbonyl carbon [d C168.5 (C-16)]. These
data suggested that 1 is a tetracyclic quassinoid similar to javanicin F [2], which was reported by
Koike et al. However, there were significant differences found, including the absence of a tertiary
methyl group and the presence of an aldehyde group in 1. The 1H － 13C HMBC correlations (Fig. 2)of an aldehyde proton (d H 10.27, s) to C-13 (d C 129.8) and of H-14 [d H 2.97, dd (J = 11.1, 7.3 Hz)] to− 115 −



Table 2.1H NMR Spectroscopic Data (600 MHz, CDCl3) of Picrajavanicins A and M (2, 3) (δ in ppm and J
Values in (Hz) in Parentheses)2

δH

position

3

δC

δH
196.9

196.8

2

148.9

149.1

3/3α

5.58, dd (5.7, 2.3)

110.4

4/4α

2.21, m26.8

4β2.31, m5α2.30, m

35.8
29.0

6/6α

1.80, dt (14.6, 3.0)

6β2.02, td (14.6, 2.3)

7/7β

4.33, t (3.0)

8

5.56, d (2.5)

3.06, s

10

112.8
68.2

4.35, m1.99, m46.0a

2.42, dd (14.9, 3.7)

24.6

1.97, dd (14.9, 1.9)
81.7

4.33, t (1.9)

37.3

9/9α

48.0

81.6
37.1

3.01, s

45.8

46.0a
47.0

11

194.5

190.4

12

158.5

148.2

13

129.8

137.9

14/14β

2.97, dd (11.1, 7.3)

37.9

2.43, dd (11.6, 7.2)

46.5

15/15α2.46, dd (18.7, 11.1)

32.1

2.63, dd (18.8, 11.6)

31.5

15β

3.05, dd (18.7, 7.3)

3.00, dd (18.8, 7.2)

16

168.5

17

11.4

169.0
12.8

18

1.56, s22.1

1.55, s22.2

19

1.16, s55.0

1.22, s

55.2

60.8

3.67, s59.3

1.89, s

15.4

MeO-2

3.61, sMeO-12

4.00, s3.63, s

Me-13
a

δC

1

CHO-13
10.27, s
191.7
Overlapping resonances within the same column.

an aldehyde carbonyl carbon (d C 191.7) suggested that an aldehyde group is located at C-13. The
relative configuration of 2 was assigned on the basis of a 2D NOESY NMR experiment. The NOESY
correlations between H-5 and H-9 suggested a orientations of these protons, whereas thosebetween H-7 and H-14/H3-19 and between H3-18 and H3-19 suggested that they are b -oriented.
Hence, the structure of 2 was assigned as shown, and was assigned the trivial name as picrajavanicin A (Fig. 1) [3]. To the best of our knowledge, this is the first report of the substitution by
an aldehyde group at C-13 in a compound bearing the des-4-methylated picrasane skeleton.
Compound 3 was obtained as amorphous solids. The molecular formula, C21H26O7, and the1

H and 13C NMR spectroscopic data (Tables 2) of 3 were similar to those of picrajavanicin L. The

COSY and HMBC correlations (Fig. 2) confirmed that 3 is a C-4 isomer of picrajavanicin L. The
significant difference was the a orientation of the C-4 hydroxy group, which was established fromthe
NOESY correlations between H-4 (d H 4.35, m) and H3-18 (d H 1.55, s). With respect to the ab
solute structure of picrajavanicin L, the absolute configuration at C-4 in 3 was deduced to be S.
Accordingly, the structure of 3 was established, and it was named picrajavanicin M (Fig. 1) [4].

− 116 −

Since the CHCl3-soluble extract of the P. javanica bark exhibited anti-Vpr activity at 5 m g/
mL concentration, the anti-Vpr activity of 2 and 3 was evaluated against TREx-HeLa-Vpr cells.
Damnacanthal [5], a natural product that is a potent inhibitor of Vpr-induced cell death, wasused as a positive control in the present study. 2 and 3 showed the mild anti-Vpr activity (i.e. no.
of cell viability treated with 1.25-5 mM of 2 and 3 is lesser than that of treated with damnacanthal). However, 2 exhibited no inhibitory activity against any of the cancer cells used (IC50 values >10 m M) [3]. In contrast, 3 exhibited potent and selective antiproliferative activity against the human pancreatic cancer PANC-1 cell line with an IC50 value of 7.37 m M [4].
Discussion
The isolation of the secondary metabolites from a methanolic extract of Spongia sp. gave theoxazinedione glycoside (1). Although further careful strcutre elucidaton of 1 is requried due toless amount of this compound, 1 was likely to be the new compound, 3-(2-deoxy- β -D-erythropentofuranosyl)-5-methyl-2,3-dihydro-6H-1,3-oxazine-2,6-dione. Further structure elucidation
and bioactvity of 1 would provide new insight into the marine natural products.
In contrast, further separation of the fraction previosuly prepared from the CHCl3-soluble
extract of the dried bark of P. javanica led to the isolation of picrajavanicins A (2) and M (3). 3
had potent and selective antiproliferative activity against the human pancreatic cancer PANC1 cell line. Interestingly, most of previously isolated picrajavanicins A−L [3, 4] and quassinoids
such as javanicins B [6], F [2], and I [7], picrasin A [8], and 2′-isopicrasin A [8] exhibited potent
and selective activity against PANC-1 cells. Structure comparison of 2 and 3 with the other qussinoids indicated that the substitution at C-4 by carbonyl, hydroxy or methyl group was important
for the antiproliferative activity. The presence of one of these functional groups at C-4 resulted in
the increased potency [picrajavanicin K > 2′-isopicrasin A > picrasin A > 2 > picrajavanicin L > javanicin F (IC50 > 100 mM)]. Furthermore, the presence of –CH2OH at C-13 has been shown to increase
the activity [picrajavanicin H > javanicin F (IC50 > 100 mM); 2 > picrajavanicin B (IC50 > 100 mM)].
These structure−activity relationships (SAR) suggested that the substituents at C-4 and C-13 arethe critical factors for antiprolieferative activities of this class of quassinoids. However, 2 and 3
exhibited mild anti-Vpr activity in comparing with that of positive control, damnacanthal. To further clarify SAR of qussinoids regarding anti-Vpr activity, further isolations of qussinoids having
anti-Vpr activity investigation will be necessary.
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Ngwe, H.; Asakawa, Y.; Abe, I.; Morita, H. Tetrahedron 2016, 72, 746−752.
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