ISRN PharmaceuticsVolume 2011 (2011), Article ID 852619, 6 pagesdoi:10.5402/2011/852619

Research Article

Two New Cytotoxic Candidaspongiolides from an Indonesian Sponge

Agus Trianto,1,2 Idam Hermawan,¹ Toshimasa Suzuka,¹ and Junichi Tanaka¹

¹Department of Chemistry, Biology, and Marine Science, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa 903-0213, Japan²Department of Marine Sciences, Faculty of Fisheries and Marine Sciences, University of Diponegoro, Semarang, Central Java 50234, Indonesia

Received 2 May 2011; Accepted 27 May 2011

Academic Editors: T. Schotten and X.-W. Yang

Copyright © 2011 Agus Trianto et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Marine sponges have been recognized as potentially rich sources of various bioactive molecules. In our continuing search for new secondary metabolites from Indonesian marine invertebrates, we collected a sponge, whose extract showed cytotoxicity against cultured cells at 0.1 μg/mL. Purification of the extract yielded two new macrolides 2 and 3 along with known candidaspongiolide (1). The structures for compounds 2 and 3 were elucidated by spectral analysis (, , COSY, HMQC, HMBC) and by comparison of their NMR data with those of 1. Compounds 2 and 3 exhibited a little more potent cytotoxicity ( 4.7 and 19 ng/mL) than that ( 37 ng/mL) of candidaspongiolide (1) against NBT-T2 cells.

1. Introduction

Sponges, a group of sedentary organisms, cannot move and escape from predators. Most sponges are filter feeders pumping water to its body to obtain foods and oxygen and to expel wastes and may be threatened by microorganisms during filtering seawater rich in bacteria and fungi [1, 2]. In order to defend themselves against predators, pathogens and competitors, sponges may have developed to produce or accumulate secondary metabolites during their long evolution, such as feeding deterrent, antimicrobial, antifungal, and antifouling molecules. Interestingly, some of the compounds have also shown remarkable potency as drug candidates against various human diseases as discussed elsewhere [3–9].

In 1984, Schmitz and coworkers isolated tedanolide from the Caribbean marine sponge Tedania ignis [10]. Tedanolide is a unique 18-membered macrolide where lactonization occurs at a primary hydroxyl group instead of a common secondary one, and this class of macrolide has been reported to exhibit strong cytotoxicity at pico to nanomolar range [10, 11]. The unique structure in combination with promising biological activity leads tedanolide as an intriguing target for formal and total syntheses [12–14]. More recently, Meragelman and coworkers reported a macrolide named candidaspongiolide (1) related to tedanolide with modification at C-11 to C-15 from the marine sponge Candidaspongia sp. Candidaspongiolide exhibited potent cytotoxicity in NCI 60 cells panel with GI₅₀ of 14 ng/mL [15], protein synthesis inhibition, and apoptosis induction [16].

In our continuing search for potential drug leads from Indonesian marine invertebrates [17, 18], we obtained a sponge whose extract showed cytotoxicity at 0.1 μg/mL against NBT-T2 cells in a screening process. Purification of the extract provided candidaspongiolide (1) along with two new analogs 2 and 3, which are the subject of this paper.

2. Materials and Methods

2.1. Chemicals and Equipments

Methanol (MeOH) used for extraction was of technical grade. Reagent grade solvents were used for isolating compounds 1–3. Merck Si-60 (70–230 mesh) was used for silica gel column chromatography, while Merck Si-60 F₂₅₄ for analytical TLC. HPLC was performed either on a Waters 510 pump with a Waters 486 UV detector and a Shodex RI-101 or on a Hitachi L-6000 pump with a Hitachi L-4000 UV detector and a Shodex RI-101 using a Mightysil Si-60 (10 × 250 mm) column. Optical rotations were measured on a Jasco P-1010 polarimeter using a cell with 3.5 mm aperture. IR spectra were recorded on a Jasco FT/IR-6100 instrument, whereas HRESIMS was measured on a Jeol JMS-T100LP spectrometer using reserpine or sodium trifluoroacetate as an internal standard. Most of ¹H and ¹³C NMR spectra were measured in CDCl3, while those of compound 3 were measured in CD₃OD with TMS as an internal standard on a Jeol A500 and/or a Bruker AVANCE III-500 in CDCl₃. The ¹H and ¹³C chemical shifts were given in ppm, while coupling constants were in Hz.

2.2. Sponge

Specimens of the sponge tagged K09-02 was collected by hand using SCUBA at 15–25 m depth at Kupang, West Timor, East Nusa Tenggara, Indonesia on August 2009. By comparing underwater images of our specimen with that of the specimen of NCI group [15], it is likely to be the same sponge. The specimen was kept frozen until extraction. The sponge K09-02 may be an endemic species to this region. The colonies are grey in color and stand.

2.3. Extraction and Isolation

After cutting into small pieces, the sponge (653 g, wet) was soaked in MeOH for 24 h for three times. Then, the solution was concentrated under vacuum to obtain a crude extract. The methanolic extract (17.0 g) was triturated with ethyl acetate (EtOAc) to provide a lipophilic fraction (2.7 g), which killed NBT-T2 cells at 0.1 μg/mL. This fraction was subjected to a silica gel column eluting with stepwise gradient solvents (hexane : EtOAc = 2 : 1, 1 : 1, 1 : 2, 0 : 1, EtOAc : MeOH = 10 : 1) to afford ten fractions. Fraction 5 (126.0 mg) was purified by repetitive Si-60 HPLC using hexane-EtOAc mixtures to provide candidaspongiolide 1 (15.4 mg). Fraction 6 (70.8 mg) was also purified by Si-60 HPLC using a solvent system hexane : EtOAc = 2 : 1 to afford compound 2 (9.8 mg). Fraction 9 (107.1 mg) was also subjected to repetitive Si-60 HPLC using hexane : EtOAc = 1 : 6, EtOAc : CH₂Cl₂ : MeOH = 20 : 20 : 1, and EtOAc : CH₂Cl₂ : MeOH = 10 : 20 : 1 as solvent systems sequentially, to give compound 3 (21.8 mg). Isolation of these compounds was guided by cytotoxicity testing and NMR spectra.

2.4. Compound 1

Colorless glass, +69 (c 0.55, MeOH). IR (neat) 3419, 2925, 2854, 1742, 1715, 1456, 1372, 1234, 1086 cm⁻¹. ¹H and ¹³C NMR; see Tables 1 and 2. HR-ESIMS [M+Na]⁺  m/z 945.55514, 959.57079, 973.58884 (calcd for C₅₀H₈₂Na 945.55459 (Δ +0.58 ppm), C₅₁H₈₄Na 959.57024 (+0.57 ppm), and C₅₂H₈₆Na 973.58589 (+3.0 ppm)).

Table 1: ¹³C NMR data for compounds 1, 2, and 3.

C no.

1
171.3 qC
171.3 qC
171.3 qC
171.3 qC

2
70.7 CH
70.8 CH
70.8 CH
72.9 CH

3
83.4 CH
83.3 CH
83.1 CH
84.7 CH

4
47.8 CH
47.7 CH
47.9 CH
49.5 CH

5
214.6 qC
214.9 qC
216.2 qC
217.6 qC

6
48.5 CH
48.3 CH
49.8 CH
51.1 CH

7
80.1 CH
80.1 CH
79.2 CH
79.9 CH

8
131.6 qC
132.0 qC
136.2 qC
139.0 qC

9
132.0 CH
131.7 CH
129.4 CH
129.8 CH

10
46.2 CH
46.1 CH
45.9 CH
46.5 CH

11
211.5 qC
211.7 qC
211.9 qC
212.7 qC

12
42.6 CH₂
42.7 CH₂
42.5 CH₂
44.3 CH₂

13
68.9 CH
68.8 CH
69.0 CH
69.3 CH

14
81.6 qC
81.6 qC
81.6 qC
85.0 qC

15
210.8 qC
211.0 qC
211.2 qC
216.5 qC

16
46.9 CH
47.0 CH
46.9 CH
46.4 CH

17
77.8 CH
77.7 CH
77.7 CH
78.4 CH

18
62.7 qC
62.7 qC
62.6 qC
63.9 qC

19
67.1 CH
67.0 CH
67.0 CH
67.4CH

20
31.1 CH
31.1 CH
30.9 CH
32.4 CH

21
129.7 CH
129.4 CH
129.6 CH
131.6 CH

22
125.5 CH
125.5 CH
125.4 CH
126.2 CH

23
13.5 CH₃
13.4 CH₃
13.3 CH₃
13.3 CH₃

24
14.6 CH₃
14.5 CH₃
14.4 CH₃
15.3 CH₃

25
14.7 CH₃
14.5 CH₃
15.0 CH₃
15.6 CH₃

26
10.8 CH₃
10.7 CH₃
10.2 CH₃
10.5 CH₃

27
16.3 CH₃
16.2 CH₃
16.4 CH₃
15.7 CH₃

28
63.5 CH₂
63.5 CH₂
63.5 CH₂
65.7 CH₂

29
63.2 CH₂
63.2 CH₂
62.8 CH₂
64.8 CH₂

30
11.1 CH₃
11.0 CH₃
10.9 CH₃
11.5 CH₃

31
18.6 CH₃
18.4 CH₃
18.3 CH₃
18.7 CH₃

32
60.3 CH₃
60.3 CH₃
60.3 CH₃
60.3 CH₃

33
169.5 qC
169.9 qC
—
—

34
21.6 CH₃
21.5 CH₃
—
—

35
173.5 qC
173.7 qC
173.6 qC
—

36
34.2 CH₂
34.1 CH₂
34.0 CH₂
—

37
29.0 CH₂
29.0 CH₂
29.1 CH₂
—

easured in CDCl₃. easured in CD₃OD.

Table 2: ¹H NMR data for compounds 1, 2, and 3 (J in Hz).

C no.

1
—
—
—
—

2
3.96 dd (1.0, 7.3)
3.96 dd (1.3, 7.5)
3.98 dd (1.3, 7.8)
3.76 d (2.2)

3
3.64 dd (1.3, 7.8)
3.67 dd (1.3, 8.0)
3.67 dd (1.3, 8.4)
3.81 dd (2.2, 9.8)

4
3.12 m
3.13 dd (8.0, 7.1)
3.10 dq (8.4, 7.3)
3.16 dq (9.8, 7.1)

5
—
—
—
—

6
3.18 dq (10.7, 7.3)
3.22 dq (10.7, 6.8)
3.04 dq (9.8, 6.8)
3.16 dq (10.0, 7.1)

7
5.39 d (10.7)
5.41 d (10.7)
4.12 d (10.0)
4.03 d (10.0)

8
—
—
—
—

9
5.60 d (9.3)
5.62 d (9.6)
5.48 d (10.5)
5.33 d (9.3)

10
3.38 dq (9.3, 6.8)
3.41 dq (9.6, 7.0)
3.49 dq (10.5, 7.1)
3.36 dq (9.3, 6.8)

11
—
—
—
—

12
2.66 dd (9.8, 16.1)
2.69 dd (9.8, 16.1)
2.72 dd (9.8, 16.1)
2.75 dd (9.5, 17.6)

2.49 dd (2.4, 16.1)
2.49 dd (2.5, 16.1)
2.51 dd (2.0, 16.1)
2.23 dd (2.0, 17.6)

13
4.40 m
4.42 m
4.39 dt (2.0, 9.8)
4.44 dd (2.9, 9.5)

14
—
—
—
—

15
—
—
—
—

16
4.02 dt (3.9, 10.9)
4.03 ddd (3.9, 10.5, 11.5)
4.09 dt (3.9, 11.0)
4.08 ddd (3.9, 10.8, 11.4)

17
3.12 m
3.13 m
3.20 dd (11.0)
3.20 d (10.7)

18
—
—
—
—

19
2.56 d (9.3)
2.59 d (9.3)
2.58 d (9.8)
2.62 d (9.3)

20
2.44 m
2.47 m
2.47 m
2.48 m

21
5.23 dt (1.5, 10.7)
5.25 ddd (0.7, 10.2, 10.9)
5.24 dt (1.5, 10.5)
5.31 m

22
5.48 dq (10.7, 6.8)
5.51 dq (10.9, 6.8)
5.49 dq (10.5, 6.8)
5.51 dq (10.7, 6.8)

23
1.59 dd (1.5, 6.8)
1.62 dd (1.2, 6.8)
1.62 dd (1.5, 6.8)
1.62 dd (1.7, 6.8)

24
1.18 d (6.8)
1.21 d (7.1)
1.21 d (7.3)
1.23 d (7.1)

25
1.13 d (7.3)
1.16 d (6.8)
1.28 d (6.8)
1.26 d (7.1)

26
1.54 brd (1.0)
1.59 d (0.8)
1.63 s
1.65 d (1.5)

27
1.07 d (6.8)
1.09 d (7.0)
1.10 d (7.1)
1.03 d (6.8)

28
4.44 d (11.7)
4.46 d (11.5)
4.45 d (11.5)
3.75 d (10.5)

4.19 d (11.7)
4.22 d (11.5)
4.21 d (11.5)
3.76 d (10.5)

29
4.17 dd (3.7, 9.8)
4.20 dd (3.7, 10.2)
4.24 dd (3.0, 9.5)
4.35 dd (3.9, 10.5)

4.10 dd (10.2, 10.9)
4.12 dd (10.2, 11.2)
4.08 m
3.91 dd (10.5, 11.4)

30
1.38 s
1.42 s
1.42 s
1.35 s

31
1.11 d (6.4)
1.13 d (6.3)
1.14 d (6.6)
1.11 d (6.6)

32
3.28 s
3.31 s
3.30 s
3.39 s

33
—
—
—
—

34
2.01 s
2.04 s
—
—

35
—
—
—
—

36
2.24 t (7.6)
2.27 t (7.6)
2.27 t (11.8)

37
1.53 brs
1.59 brs
1.25 brs

OH-2
2.85 d (7.3)
2.92 d (7.5)
2.99 d (7.8)
2.92 d (7.5)

OH-13
—

4.71 s

easured in CDCl₃. easured in CD₃OD.

2.5. Compound 2

Colorless glass, +72 (c 0.75, MeOH). IR (neat) 3421, 2925, 2854, 1748, 1715, 1456 cm⁻¹. ¹H and ¹³C NMR; see Tables 1 and 2. HR-ESIMS [M+Na]⁺  m/z 903.54964, 917.56023, 931.57576, 945.60036 and 959.61056 (calcd for C₄₈H₈₀Na 903.54403 (Δ +6.2 ppm), C₄₉H₈₂Na 917.55968 (+0.60 ppm), C₅₀H₈₄Na 931.57533 (+0.46 ppm), C₅₁H₈₆Na 945.59098 (+9.9 ppm), and C₅₂H₈₈Na 959.60663 (+4.1 ppm)).

2.6. Compound 3

Yellow glass, +97 (c 0.35, MeOH). IR (neat) 3418, 2925, 2854, 1715, 1457, 1373, 1244, 1084, 995 cm⁻¹. ¹H and ¹³C NMR; see Tables 1 and 2. HR-ESIMS [M+Na]⁺  m/z 665.31522 (calcd for C₃₂H₅₀Na 665.31436 (+1.3 ppm)).

2.7. Acetylation

Compound 1 (0.2 mg) was dissolved in pyridine (50 μL) and acetic anhydride (50 μL). The mixture was stirred for three days under a nitrogen atmosphere at room temperature. After removal of excess reagents with nitrogen flow and vacuum, the reaction product 4 was checked with ¹H NMR and ESIMS. Compound 2 was similarly treated to give 4.

2.8. Compound 4   from   1

¹H NMR: δ 5.65 dd (J = 2.7, 9.5 Hz), 5.52 m, 5.51 d (J = 10.1 Hz), 5.36 m, 5.34 dd (J = 2.2, 9.1 Hz), 5.28 dt (J = 2.3, 10.5 Hz), 5.07 d (J = 11.0 Hz), 4.79 d (J = 6.6 Hz), 4.54 dd (J = 2.0, 11.3 Hz), 4.25 dd (J = 2.3, 8.1 Hz), 4.13 dd (J = 6.3, 11.5 Hz), 3.43 s (3H), 3.34 dq (J = 10.0, 6.7 Hz), 3.29 dq (J = 10.5, 7.0 Hz), 3.01 dd (J = 9.2, 18.4 Hz), 2.88 d (J = 9.3 Hz), 2.60 dd (J = 2.6, 18.4 Hz), 2.35–2.4 m, 2.32 dd (J = 1.9, 9.3 Hz), 2.21 s (3H), 2.10 s (3H), 2.09 s (3H), 2.02 s (3H), 1.69 d (J = 1.1 Hz, 3H), 1.66 dd (J = 1.7, 6.8 Hz, 3H), 1.44 d (J = 7.1 Hz, 3H), 1.37 s (3H), 1.17 d (J = 7.1 Hz, 3H), 1.08 d (J = 6.6 Hz, 3H), 0.90 t (J = 6.1 Hz, 3H). HR-ESIMS m/z 1072.60225, 1086.60997, 1099.62465 (calcd for ¹²CH₈₈Na 1072.59019 (+11.24 ppm), ¹²CH₉₀Na 1086.60584 (+3.8 ppm), and C₅₈H₉₂Na 1099.61814 (+5.9 ppm)).

2.9. Compound 4 from 2

¹H NMR: δ 5.65 dd (J = 2.6, 9.8 Hz), 5.52 m, 5.51 d (J = 10.2 Hz), 5.37 m, 5.34 dd (J = 1.7, 9.1 Hz), 5.28 dt (J = 1.7, 10.5 Hz), 5.07 d (J = 10.9 Hz), 4.78 d (J = 6.3 Hz), 4.54 dd (J = 3.1, 11.7 Hz), 4.25 dd (J = 2.3, 8.1 Hz), 4.13 dd (J = 6.5, 11.3 Hz), 3.43 s (3H), 3.34 dq (J = 10.1, 5.7 Hz), 3.29 dq (J = 10.5, 7.2 Hz), 3.01 dd (J = 9.4, 18.6 Hz), 2.88 d (J = 9.2 Hz), 2.60 dd (J = 2.5, 18.6 Hz), 2.35–2.4 m, 2.32 (J = 1.9, 9.3 Hz), 2.22 s (3H), 2.10 s (3H), 2.09 s (3H), 2.02 s (3H), 1.70 d (J = 1.1 Hz, 3H), 1.62 dd (J = 1.7, 6.8 Hz, 3H), 1.44 d (J = 7.1 Hz, 3H), 1.37 s (3H), 1.17 d (J = 7.1 Hz, 3H), 1.08 d (J = 6.6 Hz, 3H), 0.90 t (J = 6.1 Hz, 3H). HR-ESIMS [M+Na⁺] m/z 1071.58258, 1085.60199, 1099.61165 (calcd for C₅₆H₈₈Na 1071.58629 (−4 ppm), C₅₇H₉₀Na 1085.60194 (−.5 ppm), and C₅₈H₉₂Na 1099.61759 (−5.9 ppm)).

2.10. Screening Process

NBT-T2 cells were purchased from Riken and used for cytotoxicity testing. NBT-T2 is a cell line derived from chemically induced rat bladder carcinoma cells [19]. The sponge extract was tested at 0.1, 1, and 10 μg/mL in triplicate, while fractions were done at 0.01, 0.1, and 1 μg/mL. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with Sigma antibiotic-antimycotic, Biowest fetal bovine serum, Gibco MEM nonessential amino acid in a Falcon 24-well plate or 48-well plate. After adding the extract or a fraction, cells were incubated for 24 h under 5% CO₂ at 36°C [16]. Then, the cells were observed under a microscope to evaluate viability of cells whether the fractions were cytotoxic or not.

2.11. MTT Assay

Cultured cells were inoculated to a 96-well plate with approximate cell density of  cells/mL in DMEM. After 24 h incubation, a series of DMSO solution of compounds 1–3 were applied to each well and the final concentrations were adjusted as 0, 1, 12.5, 25, 37.5, 50, 62.5, to 75 ng/mL. Cells were incubated for another 24 h, and the media were replaced with 20 μL of 5 g/mL MTT solution in PBS and incubated for 3.5 h. After removal of PBS solution, an amount of 150 μL of DMSO was added to each well and the cells were reincubated for 15 min prior to measurement with a Tecan microplate reader at 590 nm with reference filter at 620 nm [20, 21].

3. Results and Discussion

As an EtOAc soluble portion of a methanolic extract of the sponge K09-02 showed potent cytotoxicity against cultured NBT-T2 cells, the portion was separated repetitively on a silica gel column followed by Si-60 HPLC affording three compounds 1, 2, and 3 as shown in Figure 1.

Figure 1: Structures of compounds (1)–(4).

By inspecting ¹H and ¹³C NMR spectra of compound 1 together with database search (Tables 1 and 2, Figures  S1 and  S2 (Supplementary Materials available online at doi:10.5402/2011/852619)), we could readily identify that it is a member of candidaspongiolide, a series of 18-membered cytotoxic macrolide retaining one of fatty acid moieties from C₁₄ to C₁₈ at C-28 [15]. HR-ESIMS of our material exhibited molecular-related ions at m/z 945.55514, 959.57079, 973.58884 [M+Na]⁺ indicating that compound 1 is candidaspongiolide esterified with the homologs of three saturated fatty acids (palmitic, margaric, and stearic acids).

Compound 2 was obtained as a colorless glass with +72. After elucidation of its ¹H and ¹³C NMR spectra, compound 2 was found to be an analog of 1. However, the ¹³C NMR spectrum showed two carbonyl carbons at 171.3 q (C-1) and 173.1 (C-35) instead of three in 1 (Table 1, Figure  S3). As the signals for an acetoxy group ( 2.04 s, 21.5 q) in 1 are missing in 2, it was suggested that 2 is a deacetyl derivative of 1. The lack of the acetyl group is in a good agreement with ¹H NMR spectrum and COSY analysis showing that H-7 proton signal ( 4.12 d, J = 10.0 Hz) in 2 shifted to higher field than that ( 5.41 d, J = 10.7 Hz) in 1 (Table 2). HR-ESIMS of 2 showed a series of sodiated ions [M+Na]⁺ at m/z 903.54964, 917.56023, 931.57576, 945.60036, and 959.61056 corresponding to the presence of C₁₆ to C₂₀ esters. For structural confirmation, compound 2 was acetylated to give tetraacetate 4, which showed signals identical with 4 obtained from 1 (Figure  S4). Compound 4 exhibited four acetyl signals at 2.22 s, 2.10 s, 2.09 s, and 2.02 s and molecular-related ions corresponding to macrolide esters with C₁₆ to C₁₈ fatty acids.

Compound 3 was isolated as a yellowish glass with +97. Its molecular formula was established as C₃₂H₅₀O₁₃ by observing a molecular-related ion at m/z 665.31522 [M+Na]⁺ in HR-ESIMS. ¹H and ¹³C NMR spectra (Tables 1 and 2, Figures  S5 and  S6) revealed that compound 3 has a similar macrolide structure to that of compound 1 except for the lack of a fatty acid ester moiety and an acetate found in 1. Higher field chemical shifts observed for H-7 ( 4.03) and H-28 ( 3.75) indicated that 3 is devoid of acyl groups. Close similarity of ¹H and ¹³C NMR data of 3 to 1 (Table 2) indicated that the macrolide core structure of compound 3 is identical to compound 1.

All of natural compounds 1–3 exhibited potent cytotoxicity, IC₅₀ 37, 4.7, and 19 ng/mL, against NBT-T2 cells. The result is not in good agreement with those reported by Meragelman and coworkers, that is, candidaspongiolide (1) showed stronger growth inhibition (GI50 14 ng/mL) than the core compound (42 ng/mL) [15]. Additionally Paul et al. paperd the importance of a linear carbon chain on the cytotoxicity in the case of amphidinol [22]. The difference may be explained either by the number of cell lines or by different sensitivity of NBT-T2 cells.

Acknowledgments

The authors appreciate Professor Rob van Soest, University of Amsterdam, for showing a sponge image and suggestions on the sponge identification and Mr. T. Iha for mass measurements. they thank PharmaMar S. A. for financial support.

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