Synthesis and Absolute Configuration of Acanthodendrilline, a New Cytotoxic Bromotyrosine Alkaloid from the Thai Marine Sponge Acanthodendrilla sp.

Abstract

Acanthodendrilline (1), a new bromotyrosine alkaloid, was isolated from the Thai marine sponge Acanthodendrilla sp. The structure of 1 was fully characterized by spectroscopic analysis, in agreement with the synthesized compound used to resolve the single chiral center at C-11. Total synthesis of the enantiomers of 1 allowed for the comparison of specific rotation values and hence the determination of the absolute configuration as 11-S. Cytotoxicity evaluation revealed that (S)-1 exhibited approximately three-fold more potent cytotoxicity against the human non-small cell lung cancer H292 cell line than (R)-1.

Bromotyrosine alkaloids are a large group of secondary metabolites and commonly found in marine sponges.¹⁾ The remarkable diversities of their chemical structures, together with the wide range of bioactivities, including antimicrobial,^2–6) antiviral,⁷⁾ antiprotozoal,⁸⁾ cytotoxic,^3,4,9–12) anti-inflammatory,¹³⁾ and enzyme inhibitory activities,^14–16) make them highly attractive to both natural product chemists and molecular biologists. In the course of our search for bioactive secondary metabolites from Thai marine organisms, we recently reported the isolation of a series of bromotyrosine alkaloids from the Thai marine sponge Acanthodendrilla sp. along with homoaerothionin, a potent cholinesterase inhibitor¹⁷⁾ (Fig. 1). In addition, several bromotyrosine alkaloids isolated from this sponge, including verongiaquinol, aerothionin, 11-oxoaerothionin, and 11,19-dideoxyfistularin-3 (Fig. 1), were previously reported by other research groups as cytotoxic agents against several cancer cell lines.^3,4,12,18)

Fig. 1. Structures of Bioactive Bromotyrosine Alkaloids Isolated from the Thai Marine Sponge Acanthodendrilla sp.

Further investigation of the crude ethyl acetate (EtOAc) extract from this sponge resulted in the isolation of a new bromotyrosine alkaloid, acanthodendrilline (1, Fig. 2). In this work, we describe the structure elucidation of 1 based on spectroscopic techniques and synthetic protocols. The enantiomers of 1 were prepared from commercially available starting materials and used to determine the absolute configuration. We also report the cytotoxicity of the enantiomers to human non-small cell lung cancer H292 and normal human keratinocyte HaCaT cell lines.

Fig. 2. Structure of Acanthodendrilline (1)

Results and Discussion

The EtOAc extract of the Thai marine sponge Acanthodendrilla sp.¹⁷⁾ was repeatedly subjected to silica gel column chromatography, Sephadex^® LH-20 column chromatography, and preparative HPLC to afford 1 (Fig. 2).

Compound 1, obtained as a colorless amorphous powder, was optically active [α]_D²⁵ +25.8 (c=0.1, MeOH). The appearance of the protonated molecular parent ion at m/z 450.9512 [M+H]⁺ (Calcd for 450.9504) in the high resolution (HR)-FAB-MS of 1 suggested that the molecular formula was C₁₄H₁₆Br₂N₂O₅. The ¹H-NMR, ¹³C-NMR, and heteronuclear multiple bond connectivity (HMBC) spectra of 1 (Table 1) indicated that 1 possessed a 3,5-dibromotyramine skeleton: signals assignable to two aromatic protons [δ_H 7.36 (s, H-2, 6)], six aromatic carbons [δ_C 138.3 (C-1), δ_C 133.1 (C-2, 6), δ_C 118.0 (C-3, 5), δ_C 150.6 (C-4)], and two methylenes [δ_H 2.75 (t, J=6.8 Hz, H₂-7)/δ_C 34.9 (C-7), δ_H 3.40 (td, J=6.8, 6.3 Hz, H₂-8)/δ_C 41.8 (C-8)] were observed. The presence of a methylcarbamate moiety was suggested by the typical chemical shifts of OCH₃ (δ_C 52.2) and C-9 (δ_C 157.0) in the ¹³C-NMR spectrum and confirmed by the HMBC correlation from the methoxy protons [δ_H 3.69 (s, OCH₃)] to C-9. The connectivity of the dibromotyramine and carbamate moieties was confirmed by the ¹H–¹H correlation spectroscopy (COSY) correlations of H₂-7/H₂-8/9-NH and the HMBC correlation from H₂-8 to C-9. In addition, a 5-methyl-2-oxazolidone moiety [δ_H 4.21 (d, J=5.1 Hz, H₂-10)/δ_C 71.9 (C-10), δ_H 5.04 (ddt, J=8.7, 5.9, 5.1 Hz, H-11)/δ_C 74.2 (C-11), δ_H 3.93 (dd, J=8.7, 5.9 Hz, H_a-12) and δ_H 3.83 (t, J=8.7 Hz, H_b-12)/δ_C 42.6 (C-12), δ_H 5.64 (br, 13-NH)/δ_C 159.3 (C-13)] was deduced from the ¹H–¹H COSY correlations of H₂-10/H-11/H_a,b-12 and the HMBC correlations from H-11, H_a,b-12, and 13-NH to C-13. The HMBC correlation from H₂-10 to C-4 suggested the connectivity of the dibromotyramine and 2-oxazolidone moieties. Selected ¹H–¹H COSY and HMBC correlations are illustrated in Fig. 3. Thus, 1 was elucidated as methyl-[2-(3,5-dibromo-4-{[2-oxo-1,3-oxazolidin-5-yl]methoxy}phenyl)ethyl]carbamate and named acanthodendrilline. Our spectroscopic studies depicted that 1 contained a stereogenic center at C-11. However, the absolute configuration of 1 remained ambiguous. Therefore, we decided to access the S and R enantiomers of 1 by chemical synthesis. Both synthetic enantiomers could serve as standards for the comparison of chemical and physical properties.

Table 1. ¹H- and ¹³C-NMR Data of 1 in CDCl₃

Position	δH (Multiplicity)	δC, Type	HMBC correlation from H to C
1		138.3 (C)
2, 6	7.36 (2H, s)	133.1 (CH)	C-2,6/C-3,5/C-4/C-7
3, 5		118.0 (C)
4		150.6 (C)
7	2.75 (2H, t, J=6.8)	34.9 (CH2)	C-1/C-2,6/C-8
8	3.40 (2H, td, J=6.8, 6.3)	41.8 (CH2)	C-1/C-7/C-9
9		157.0 (C)
10	4.21 (2H, d, J=5.1)	71.9 (CH2)	C-4/C-11/C-12
11	5.04 (1H, ddt, J=8.7, 5.9, 5.1)	74.2 (CH)	C-13
12	a 3.93 (1H, dd, J=8.7, 5.9)	42.6 (CH2)	C-10/C-11/C-13
	b 3.83 (1H, t, J=8.7)
13		159.3 (C)
9-NH	4.82 (1H, br)
13-NH	5.64 (1H, br)		C-11/C-12/C-13
OCH3	3.69 (3H, s)	52.2 (CH3)	C-9

Chemical shifts (δ) are expressed in ppm, and coupling constants (J) are presented in Hz.

Fig. 3. Selected ¹H–¹H COSY () and HMBC () Correlations of 1

The four-step synthesis of (S)-1 and (R)-1 is shown in Fig. 4. Tyramine (2) was used as the starting material to prepare methyl-[2-(3,5-dibromo-4-hydroxyphenyl)ethyl]carbamate (4) in two steps. First, 2 was brominated by tetrabutylammonium tribromide to give 3,5-dibromotyramine¹⁹⁾ (3) in 63% yield. Subsequent acylation of 3 with a stoichiometric amount of methyl chloroformate at 0°C provided 4 in 56% yield. Commercially available (S)- and (R)-epichlorohydrins (5) were separately transformed in one step into the respective enantiomers 5-chloromethyl-2-oxazolidinone [(S)-6 and (R)-6, each ca. 40% yield] using potassium cyanate as the nucleophile to transform the oxirane ring into the oxazolidinone motif.²⁰⁾ Both (S)-6 and (R)-6 were finally coupled with 4 by base-mediated nucleophilic substitution to furnish (S)-1 (48% yield) and (R)-1 (37% yield), respectively.

Fig. 4. Synthetic Scheme of (S)-1 and (R)-1

The ¹H-NMR data of the synthetic enantiomers were identical to that of natural 1. Determination of the specific rotation of the enantiomers clarified that (S)-1 {[α]_D²⁵ +21.4 (c=0.1, MeOH)} and natural 1 {[α]_D²⁵ +25.8 (c=0.1, MeOH)} possessed almost identical optical rotation values, whereas the optical rotation of (R)-1 was the opposite {[α]_D²⁵ −19.7 (c=0.3, MeOH)}. Thus, the absolute configuration at C-11 of natural 1 was unambiguously determined as S. Interestingly, measurement of the circular dichroism (CD) spectra of 1 and the synthetic enantiomers yielded no characteristic absorptions.

The cytotoxicity of the synthetic enantiomers was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.²¹⁾ The results (Table 2) indicated that (S)-1 (IC₅₀ 58.5 µM) was approximately threefold more potent than (R)-1 (IC₅₀ 173.5 µM) against the H292 cell line. Interestingly, both enantiomers were not cytotoxic to normal HaCaT cell line (IC₅₀ >400 µM). Moreover, the acetylcholinesterase (AChE) inhibitory activity of the synthetic enantiomers was determined by the modified Ellman’s method.^22,23) Both (S)-1 and (R)-1 showed weak inhibitory activity toward AChE (23% and 25% enzyme inhibition at 100 µM, respectively).

Table 2. Cytotoxicity of Enantiomers of 1 to Cancer (H292) and Normal (HaCaT) Cells

Compound	IC50±S.D. (µM)
	H292	HaCaT
(S)-1	58.5±6.7	>400
(R)-1	173.5±24.7	>400
Cisplatin	7.5±0.6	4.6±0.9

Conclusion

Acanthodendrilline (1), a new bromotyrosine alkaloid, was isolated from the Thai marine sponge Acanthodendrilla sp. It is the first example of a bromotyrosine alkaloid that is composed of dibromotyramine, methylcarbamate, and 2-oxazolidone moieties in the molecule. The concise synthesis of the enantiomers of 1 was accomplished from commercial tyramine and (S)- and (R)-epichlorohydrins. By comparing the specific rotation values of natural 1 and the synthetic enantiomers, the stereocenter at C-11 of natural 1 was assigned the S configuration. Both enantiomers exhibited weak AChE inhibitory activity. Interestingly, cytotoxicity evaluation revealed that (S)-1 was approximately threefold more cytotoxic to the human non-small cell lung cancer H292 cell line than (R)-1. This biological information clearly emphasized the importance of the stereochemistry at C-11 of 1 as a key feature related to the cytotoxicity.

Experimental

General Experimental Procedures

¹H- and ¹³C-NMR spectra were measured on Bruker Avance DPX-300, JEOL JNM-AL 300, and JEOL JNM-AL 400 NMR spectrometers. The solvent signals served as the internal standard (CDCl₃: δ_H 7.26/δ_C 77.0). IR spectra were recorded on a Shimadzu IRAffinity-1 Fourier transform-infrared (FT-IR) spectrophotometer. HR-MS were acquired with a JEOL JMS 700 mass spectrometer. Optical rotation and circular dichroism measurements were carried out on a Horiba SEPA-200 polarimeter and a Jasco J-820 spectropolarimeter, respectively. HPLC was performed on a Shimadzu apparatus equipped with an LC-20AB binary pump, a Rheodyne 7125 injector port, and an SPD-20 A UV/Vis detector. Silica gel 60 F₂₅₄ aluminum sheets were used for TLC. Spots on TLC chromatograms were detected under UV light (254 nm) and by spraying with p-anisaldehyde reagent. Silica gel 60 (230–400 and 70–230 mesh) and Sephadex^® LH-20 were used for column chromatography.

Extraction and Isolation

The EtOAc extract (67.81 g) previously prepared from the Thai marine sponge Acanthodendrilla sp.¹⁷⁾ was fractionated with the following chromatographic steps: SiO₂ (70% EtOAc in n-hexane; then 100% EtOAc), SiO₂ (50% EtOAc in n-hexane), Sephadex LH-20 (MeOH), SiO₂ (4% MeOH in CH₂Cl₂), and C₁₈ RP-HPLC (LiChrospher^® 100 RP-18, 10 µm, 250×10 mm, 65% MeOH in H₂O, flow rate 3.0 mL/min, UV detector 230 nm), to yield 1 (43.1 mg, 0.0006% yield of sponge wet weight).

Acanthodendrilline (1)

Colorless amorphous powder. ¹H-NMR (300 MHz) and ¹³C-NMR (75 MHz), see Table 1. IR (KBr) cm⁻¹: 3329, 2950, 1751, 1705, 1541, 1466, 1449, 1256, 1094, 739. UV λ_max (MeOH) nm (log ε): 214 (4.37). HR-FAB-MS m/z: 450.9512 [M+H]⁺ (Calcd for C₁₄H₁₇Br₂N₂O₅: 450.9504). [α]_D²⁵ +25.8 (c=0.1, MeOH).

4-(2-Aminoethyl)-2,6-dibromophenol (3)

Tetrabutylammonium tribromide [(n-Bu)₄NBr₃, 14.1 g, 29.9 mmol] was added to a solution of tyramine (2.0 g, 14.6 mmol) in CH₂Cl₂ (105 mL) and MeOH (70 mL). The reaction mixture was stirred at room temperature for 30 min. After the solvent was removed in vacuo, the residue was suspended in EtOAc–CHCl₃ (1 : 1), and the precipitate was filtered off and washed with CH₂Cl₂. Compound 3 was obtained as a pale yellow powder (2.71 g, 63%). ¹H-NMR (300 MHz, CD₃OD) δ: 7.43 (2H, s), 3.14 (2H, t, J=7.7 Hz), 2.86 (2H, t, J=7.7 Hz).¹⁹⁾ HR-electron ionization (EI)-MS m/z 292.9045 [M]⁺ (Calcd for C₈H₉Br₂NO: 292.9051).

Methyl-[2-(3,5-dibromo-4-hydroxyphenyl)ethyl]carbamate (4)

Methyl chloroformate (ClCO₂CH₃, 47.7 mL, 0.59 mmol) was added to a solution of 3 (151 mg, 0.51 mmol) in tetrahydrofuran (THF) (2 mL), H₂O (2 mL), and NaHCO₃ (135 mg, 1.61 mmol) at 0°C. After stirring for 1.5 h, the reaction mixture was diluted with EtOAc (20 mL) and then extracted with 1 N NaOH aq. (2×10 mL). The combined alkaline solution was acidified with 1 N HCl aq. and then extracted with CHCl₃ (3×10 mL). The combined extract was washed with brine (10 mL), dried, and concentrated in vacuo to give a residue (103 mg). Chromatography on a silica gel column with n-hexane–EtOAc (2 : 1) as the eluent gave 4 (100.3 mg, 56%) as a colorless powder. ¹H-NMR (400 MHz, CDCl₃) δ: 7.28 (2H, s), 4.87 (1H, br s), 3.67 (3H, s), 3.38 (2H, q, J=6.8 Hz), 2.71 (2H, t, J=6.8 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ: 157.0 (s), 148.1 (s), 133.3 (s), 132.1 (d), 109.9 (s), 52.2 (q), 42.0 (t), 34.7 (t). HR-EI-MS m/z 350.9104 [M]⁺ (Calcd for C₁₀H₁₁Br₂NO₃: 350.9106).

(5S)-5-(Chloromethyl)-1,3-oxazolidin-2-one [(S)-6]

(S)-Epichlorohydrin [(S)-5, 390 µL, 4.99 mmol] and MgSO₄ (1.2 g, 10 mmol) were added to a solution of potassium cyanate (KOCN, 811 mg, 10 mmol) in H₂O (5 mL). After stirring at 100°C for 5 h, the reaction mixture was diluted with H₂O (5 mL) and then extracted with EtOAc (3×20 mL). The combined extract was washed with brine (20 mL), dried, and concentrated in vacuo to give a residue (304 mg). Chromatography on a silica gel column with n-hexane–EtOAc (1 : 2) as the eluent gave (S)-6 (275.7 mg, 41%) as a colorless powder. ¹H-NMR (300 MHz, CDCl₃) δ: 6.37 (1H, br s, NH), 4.92–4.81 (1H, m), 3.76 (1H, td, J=9.2, 0.7 Hz), 3.72–3.70 (2H, m), 3.54 (1H, ddd, J=9.2, 5.9, 0.9 Hz).²⁰⁾ HR-EI-MS m/z 135.0087 [M]⁺ (Calcd for C₄H₆ClNO₂: 135.0087). [α]_D²⁵ +10.0 (c=1.0, CHCl₃).

(5R)-5-(Chloromethyl)-1,3-oxazolidin-2-one [(R)-6]

(R)-Epichlorohydrin [(R)-5, 390 µL, 4.99 mmol] and MgSO₄ (1.2 g, 10 mmol) were added to a solution of KOCN (811 mg, 10 mmol) in H₂O (5 mL). After stirring at 100°C for 5 h, the reaction mixture was diluted with H₂O (5 mL) and then extracted with EtOAc (3×20 mL). The combined extract was washed with brine (20 mL), dried, and concentrated in vacuo to give a residue (320 mg). Chromatography on a silica gel column with n-hexane–EtOAc (1 : 2) as the eluent gave (R)-6 (272.8 mg, 40%) as a colorless powder. ¹H-NMR (300 MHz, CDCl₃) δ: 6.37 (1H, br s, NH), 4.92–4.81 (1H, m), 3.76 (1H, td, J=9.2, 0.7 Hz), 3.72–3.70 (2H, m), 3.54 (1H, ddd, J=9.2, 5.9, 0.9 Hz).²⁰⁾ HR-EI-MS m/z 135.0086 [M]⁺ (Calcd for C₄H₆ClNO₂: 135.0087). [α]_D²⁵ −9.9 (c=1.0, CHCl₃).

Methyl-[2-(3,5-dibromo-4-{[(5S)-2-oxo-1,3-oxazolidin-5-yl]methoxy}phenyl)ethyl]carbamate [(S)-1]

(S)-6 (21.7 mg, 160 µmol) in N,N-dimethylformamide (DMF) (4.0 mL) was added to a solution of 4 (43.5 mg, 123 µmol). K₂CO₃ (204 mg, 1.48 mmol) was added, and the reaction mixture was stirred at 140°C for 45 min. The mixture was filtered, and the filtrate was diluted with H₂O (5 mL) and then extracted with CHCl₃ (3×30 mL). The combined extract was washed with brine (20 mL), dried, and concentrated in vacuo to give a residue (60 mg). Chromatography on a silica gel column with n-hexane–EtOAc (1 : 2–1 : 1) as the eluent gave (S)-1 (26.8 mg, 48%) as a colorless powder. ¹H-NMR (400 MHz, CDCl₃) δ: 7.35 (2H, s), 5.79 (1H, br), 5.02 (1H, ddt, J=8.7, 6.2, 5.0 Hz), 4.82 (1H, br), 4.20 (2H, d, J=5.0 Hz), 3.91 (1H, dd, J=8.7, 6.2 Hz), 3.82 (1H, t, J=8.7 Hz), 3.67 (3H, s), 3.39 (2H, q, J=6.7 Hz), 2.74 (2H, t, J=6.7 Hz). HR-FAB-MS m/z 450.9498 [M+H]⁺ (Calcd for C₁₄H₁₇Br₂N₂O₅: 450.9504). [α]_D²⁵ +21.4 (c=0.1, MeOH).

Methyl-[2-(3,5-dibromo-4-{[(5R)-2-oxo-1,3-oxazolidin-5-yl]methoxy}phenyl)-ethyl]carbamate [(R)-1]

(R)-6 (21.7 mg, 160 µmol) in DMF (4.0 mL) was added to a solution of 4 (43.5 mg, 123 µmol). K₂CO₃ (204 mg, 1.48 mmol) was added, and the reaction mixture was stirred at 140°C for 1 h. The reaction mixture was filtered, and the filtrate was diluted with H₂O (5 mL) and then extracted with chloroform (3×30 mL). The combined extract was washed with brine (20 mL), dried, and concentrated in vacuo to give a residue (60 mg). Chromatography on a silica gel column with n-hexane–EtOAc (1 : 1) as the eluent gave (R)-1 (20.5 mg, 37%) as a colorless solid. ¹H-NMR (400 MHz, CDCl₃) δ: 7.35 (2H, s), 5.75 (1H, br), 5.02 (1H, ddt, J=8.7, 6.1, 5.1 Hz), 4.81 (1H, br), 4.20 (2H, d, J=5.1 Hz), 3.91 (1H, dd, J=8.7, 6.1 Hz), 3.82 (1H, t, J=8.7 Hz), 3.67 (3H, s), 3.39 (2H, q, J=6.6 Hz), 2.74 (2H, t, J=6.6 Hz). HR-FAB-MS m/z 450.9507 [M+H]⁺ (Calcd for C₁₄H₁₇Br₂N₂O₅: 450.9504). [α]_D²⁵ −19.7 (c=0.3, MeOH).

Cytotoxicity

Cytotoxicity to the human non-small cell lung cancer NCI-H292 cell line (ATC C, Manassas, VA, U.S.A.) and the normal human keratinocyte HaCaT cell line (Cell Lines Service, Eppelheim, Germany) was investigated by performing the MTT assay.²¹⁾ H292 cells and HaCaT cells were seeded onto 96-well plates at the densities of 2.5×10³ and 1×10⁴ cells/well, respectively, and were treated with various concentrations of samples dissolved in the culture medium containing not more than 0.5% dimethyl sulfoxide (DMSO) for 72 h. The detailed experimental procedure was described in our previous study.¹⁷⁾ Cell viability was calculated with respect to non-treated control cells. IC₅₀ was determined using GraphPad Prism (GraphPad Software, U.S.A.).

Acetylcholinesterase Inhibitory Activity

Inhibitory activity toward acetylcholinesterase from electric eel (Electrophorus electricus) was determined by the modified Ellman’s method.^22,23) Details of the experimental procedure were described in our previous report.¹⁷⁾

Acknowledgments

Financial support was provided by the Royal Golden Jubilee Ph.D. Program Grant No. PHD/0276/2552 (N.Si.), by the Meiji Pharmaceutical University Asia/Africa Center for Drug Discovery (MPU-AACDD), and by the Thailand Research Fund (A.P.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials. ¹H-NMR, ¹³C-NMR, ¹H–¹H COSY, HSQC, and HMBC spectra, and HR-FAB-MS of natural 1, and ¹H-NMR spectra of synthetic (S)-1 and (R)-1 are available.

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