Pseudoalteromone B: a novel 15C compound from a marine bacterium Pseudoalteromonas sp. CGH2XX.

A novel 15C compound, pseudoalteromone B (1), possessing a novel carbon skeleton, was obtained from a marine bacterium Pseudoalteromonas sp. CGH2XX. This bacterium was originally isolated from a cultured-type octocoral Lobophytum crassum, that was growing in cultivating tanks equipped with a flow-through sea water system. The structure of 1 was established by spectroscopic methods. Pseudoalteromone B (1) displayed a modestly inhibitory effect on the release of elastase by human neutrophils.

1. Introduction

Marine bacteria belonging to the genus Pseudoalteromonas sp. (family Pseudoalteromonadaceae) have proven to be not only an important source of various antibiotics, but have also played an interesting role in marine ecology [1,2,3,4]. In the continuing research aimed at the discovery of new natural substances from marine microorganisms, an organic extract of the bacterium identified as Pseudoalteromonas sp. CGH2XX, which was originally isolated from a cultured-type octocoral Lobophytum crassum (family Alcyonacea), exhibited significant cytotoxicity toward the HL-60 (human acute promyelocytic leukemia) and CCRF-CEM (human T cell acute lymphoblastic leukemia) tumor cells (IC50 = 0.9, 1.2 µg/mL) and displayed a significant inhibitory effect (inhibition rate 45.1%) on the release of elastase by human neutrophils at a concentration of 10 µg/mL. We isolated a novel 15C compound, pseudoalteromone B (1) (Figure 1), from this microorganism. The structure of 1 was established by spectroscopic methods and this compound displayed a modestly inhibitory effect on the release of elastase by human neutrophils.

Figure 1

The structure of pseudoalteromone B (1).

2. Results and Discussion

Pseudoalteromone B (1) was isolated as an oil and had the molecular formula C15H26O3, as determined by HRESIMS (C15H26O3 + Na, m/z found 277.1779, calculated 277.1780) indicating three degrees of unsaturation. The IR absorption bands at 3502 and 1706 cm−1 were characteristic for the hydroxy and ketone groups.

The 1H and 13C NMR data of 1 (Table 1) showed the presence of 15 carbon signals, which were identified by the assistance of a DEPT spectrum as four methyls, six sp3 methylenes, an sp2 methine, an sp3 quaternary carbon, and three sp2 quaternary carbons including two ketone carbonyls. The 1H NMR spectrum of 1 showed a signal of olefinic proton (δH 5.09, 1H, tq, J = 7.2, 1.2 Hz, H-7), two acetyl methyls (δH 2.18, 3H, s, H3-1; 2.12, 3H, s, H3-13), a vinyl methyl (δH 1.58, 3H, br s, H3-15), a tertiary methyl attaching at an oxygenated quaternary carbon (δH 1.22, 3H, s, H3-14) and six pairs of methylene protons (δH 2.65, 1H, d, J = 17.2 Hz; 2.58, 1H, d, J = 17.2 Hz, H2-3; 2.37, 2H, t, J = 7.2 Hz, H2-11; 2.04, 2H, m, H2-6; 1.96, 2H, t, J = 7.2 Hz, H2-9; 1.66, 2H, quintet, J = 7.2 Hz, H2-10; 1.51, 2H, m, H2-5).

Table 1

1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data for 1.

Position	δH (J in Hz)	δC, Mult.
1	2.18 s	31.9, CH3
2		211.0, qC
3a/b	2.58 d (17.2); 2.65 d (17.2)	52.3, CH2
4		71.5, qC
5	1.51 m	41.9, CH2
6	2.04 m	22.5, CH2
7	5.09 tq (7.2, 1.2)	124.8, CH
8		134.6, qC
9	1.96 t (7.2)	38.8, CH2
10	1.66 quintet (7.2)	21.8, CH2
11	2.37 t (7.2)	43.0, CH2
12		209.1, qC
13	2.12 s	29.9, CH3
14	1.22 s	26.7, CH3
15	1.58 br s	15.7, CH3

The constitution of the carbon skeleton of 1 was elucidated initially by the 1H–1H COSY and HMBC correlations of 1 (Figure 2), it was possible to establish the separate spin systems that map out the proton sequences from H2-5/H2-6/H-7 and H2-9/H2-10/H2-11. These data, together with the HMBC correlations between H3-1/C-2, C-3; H2-3/C-2, C-4, C-5; H2-5/C-4, C-6; H-7/C-9; H2-9/C-7, C-8, C-10, C-11; H2-10/C-8, C-9, C-11, C-12; H2-11/C-9, C-10, C-12; and H3-13/C-11, C-12, permitted elucidation of the main straight carbon skeleton. The vinyl methyl at C-8 was confirmed by the HMBC correlations between H-7, H2-9/C-15; and H3-15/C-7, C-8, C-9; and further supported by an allylic coupling between H-7 and H3-15 (J = 1.2 Hz). Based on these data, together with the HMBC correlations between H3-14/C-3, C-4, C-5 and H2-3, H2-5/C-14, the planar structure of 1 was established.

Figure 2

The 1H–1H COSY and selective HMBC correlations (protons→quaternary carbons) of 1.

In the NOESY experiment of 1, a correlation between H-7 with H2-9, as well as the lack of correlation between H-7 and H3-15, reflected the E-configuration of C-7/8 double bond. Furthermore, by comparison of the rotation value of 1 ([α]23D −20 (c 0.03, CHCl3)) with that of a known synthetic compound, (S)-4-hydroxy-4-methyl-6-phenylhexan-2-one (2) ([α]25D −14.5 (c 1.1, CHCl3)) (Figure 3) [5], the absolute configuration for the C-4 chiral center of 1 was determined as S form as that of 2. Based on the above findings, the structure of 1 was determined unambiguously.

Figure 3

The structure of (S)-4-hydroxy-4-methyl-6-phenylhexan-2-one (2).

The in vitro cytotoxicity of pseudoalteromone B (1) toward HCT116 (human colorectal carcinoma), K-562 (human chronic myelogenous leukemia), HL-60 (human acute promyelocytic leukemia), CCRF-CEM (human T cell acute lymphoblastic leukemia), T-47D (human breast ductal carcinoma), and MDA-MB-231 (human breast adenocarcinoma) cells was tested. Unfortunately, the new compound 1 described herein is not active toward the above cells (all IC50 values > 20 µg/mL). The in vitro anti-inflammatory effect of 1 was tested. Pseudoalteromone B (1) displayed a modestly inhibitory effect (inhibition rate 20.7%) on the release of elastase by human neutrophils at a concentration of 10 µg/mL.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were measured on a Jasco P-1020 polarimeter. IR spectra were recorded on a Jasco FT/IR-4100 infrared spectrophotometer. The NMR spectra were recorded on a Varian Mercury Plus 400 FT-NMR at 400 MHz for 1H and 100 MHz for 13C, in CDCl3, respectively. Proton chemical shifts were referenced to the residual CHCl3 signal (δH 7.26 ppm). 13C NMR spectra were referenced to the center peak of CDCl3 at δC 77.1 ppm. ESIMS and HRESIMS data were recorded on a Bruker APEX II mass spectrometer. Silica gel (Merck, 230–400 mesh) and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography. TLC was carried out on precoated Kieselgel 60 F254 (0.25 mm, Merck); spots were visualized by spraying with 10% H2SO4 solution followed by heating.

3.2. Marine Bacteria Isolation, Culture Conditions and Extract Preparation

A marine bacterium number CGH2XX was isolated from soft coral Lobophytum crassum that was growing in cultivating tanks equipped with a flow-through sea water system [4]. The bacterium strain CGH2XX was 98.3% identical with Pseudoalteromonas sp. H02P24-23 (Genebank accession no. HQ161380) on the basis of 16S rDNA gene sequence. The marine bacterium was cultured in 2.5 L flasks containing 1 L M1 broth (not containing agar) with 80% seawater. Flasks were incubated at 25 °C on a rotatory shaker at 120 rpm. After five days of incubation, extraction of the culture broth (10.0 L) with ethyl acetate (EtOAc, 2 × 10.0 L) yielded 1.71 g of crude extract. The extracts obtained were stored at −20 °C.

3.3. Separation

Crude extract was separated on Sephadex LH-20 and eluted using a mixture of dichloromethane and methanol (1:1) to yield 17 fractions. Fraction 6 was selected for further study and purified by silica gel, using a mixture of n-hexane and EtOAc (2:1) as a mobile phase to afford compound 1 (4.2 mg).

Pseudoalteromone B (1): colorless oil; [α]23D −20 (c 0.03, CHCl3); IR (neat) νmax 3502, 1706 cm−1; 1H (CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMR data, see Table 1; ESIMS: m/z 277 (M + Na)+; HRESIMS: m/z 277.1779 (calcd for C15H26O3 + Na, 277.1780).

3.4. Cytotoxicity Testing

The cytotoxicity of compound 1 was assayed with a modification of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method. Cytotoxicity assays were carried out according to previously described procedures [6,7,8].

3.5. Elastase Release by Human Neutrophils

Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate [9,10,11].

4. Conclusions

In a previous study [4], an ubiquinone derivative, pseudoalteromone A, was isolated from Pseudoalteromonas sp. CGH2XX, and this compound was found to be cytotoxic toward MOLT-4 (human acute lymphoblastic leukemia) and T-47D (human breast ductal carcinoma) cells (IC50 = 3.8, 4.0 µg/mL) and displayed moderately inhibitory effects on the generation of superoxide anion and the release of elastase (inhibition rates 38.0, 20.2%) by human neutrophils at a concentration of 10 µg/mL [12]. However, as described in the beginning of this communication, the organic extract of Pseudoalteromonas sp. CGH2XX showed significant cytotoxicity and anti-inflammatory activity. At this stage, the results showed that pseudoalteromone B (1) displayed a modestly anti-inflammatory activity and this compound was not cytotoxic toward HCT116, K-562, HL-60, CCRF-CEM, T-47D and MDA-MB-231 cells. We suggested that the other active components exist in the other fractions. The possible activity for pseudoalteromone B (1) will be studied if we can get enough material from Pseudoalteromonas sp. CGH2XX. Furthermore, to the best of our knowledge, compounds pseudoalteromones A and B, were the first two compounds from the marine bacterium belonging to the genus Pseudoalteromonas associated with octocorals.