Pyrrolactams from Marine Sponge Stylissa massa Collected from Myanmar and Their Anti-Vpr Activities

Abstract

A new brominated pyrrolactam stylissaol A (1) together with four known analogues, 2-bromoaldisine, aldisine, spongiacidin D, and Z-hymenialdisine, were isolated from the EtOAc extract of marine sponge Stylissa massa collected in Myanmar. The absolute configuration at C-10 of 1 was determined as R by the electronic circular dichroism (ECD) data. Among the isolated compounds, 2-bromoaldisine showed anti-Viral Protein R (Vpr) activity against TREx-HeLa-Vpr cells with an effective dose of 10 µM and its potency was comparable to that of positive control damnacanthal.

Introduction

Viral Protein R (Vpr) is a small basic protein (14 kDa), and is well conserved in human immunodeficiency virus (HIV)-1, HIV-2, and simian immunodeficiency virus (SIV). Vpr was reported as a promising drug target for a comprehensive AIDS therapy.^1,2) In order to find Vpr inhibitors, we have established an assay system where we developed tetracycline regulated TREx-HeLa-Vpr cells and studied the inhibition effect of compounds on Vpr by monitoring the proliferation rate of cells.³⁾ By using this assay, we identified a number of natural small molecules including isopimarane diterpenoids,^3,4) quassinoids,⁵⁾ iridoid and bis-iridoid glucosides,⁶⁾ xanthone,⁷⁾ and triterpenoid⁷⁾ from medicinal plants of Myanmar as potent Vpr inhibitors.⁸⁾

Interestingly, as infectious microorganisms evolve and develop resistance to existing pharmaceuticals, marine sponges provide novel leads against bacterial, fungal, and viral diseases, although the availability of more sensitive and different bioassay systems to date will be needed for drug discovery against these diseases. Myanmar is a country with a rich source of not only medicinal plants, but also marine sponges. However, there is no report regarding natural products from sponges of Myanmar origin, except for an our previous study.⁹⁾ In our previous pioneer study, we identified two new pyrrolo-2-aminoimidazoles from a Myanmar marine sponge, Clathria prolifera.⁹⁾ In a continuous work, we found that an EtOAc extract of Stylissa massa collected in Myanmar exhibited potent Vpr inhibitory activity against the established cell line, TREx-HeLa-Vpr. S. massa is in a Demospongiae class and belongs to the family Scopalinidae. It is widely distributed in Andaman Sea Coral Coast, Indian Ocean, and South China Sea. A number of constituents such as cycloheptapeptides,¹⁰⁾ bromopyrrole alkaloids,¹¹⁾ and amphilectane diterpenes¹²⁾ have been discovered from S. massa and they were reported as antiproliferative, proteinkinase USP7, and nitric oxide (NO) inhibitors.^10–12) Herein, the isolation, structural elucidation, and the anti-Vpr activities of the active EtOAc extract and isolated chemical constituents are described.

Results and Discussion

Screening of the extracts of S. massa sponge collected in Myanmar against TREx-HeLa-Vpr cells revealed that the EtOAc extract possessed the anti-Vpr activity, with an effective dose of 10 µg/mL. Purification of the active EtOAc extract by a combination of various chromatographic techniques and analyses by spectroscopic techniques afforded five pyrrolactam alkaloids (1–5) including one new pyrrolactam alkaloid named stylissaol A (1), together with four related known ones, 2-bromoaldisine (2),¹³⁾ aldisine (3),¹³⁾ spongiacidin D (4),¹⁴⁾ and Z-hymenialdisine (5)^15,16) (Fig. 1).

Fig. 1. The Structures of Isolated Pyrrolactam Alkaloids (1–5) from S. massa Sponge

Compound 1 was obtained as an amorphous powder. The high resolution-electrospray ionization (HRESI)MS of 1 gave prominent molecular adduct ions at m/z 266.9731 [M + Na]⁺ and 268.9717 [M +2 + Na]⁺ with an intensity ratio of approx. 1 : 1, implying that 1 contains one bromine atom. Its molecular formula was thus determined as C₈H₉BrN₂O₂ from the HRESIMS in conjugation with ¹³C-NMR. The UV spectrum showed the absorption maximum at 272 nm, suggesting the presence of the pyrrole chromophore in conjugation with the carbonyl group. The Fourier transform (FT)IR spectrum of 1 showed absorption bands at 1680 and 1626 cm⁻¹ due to the presence of the carbonyl and amide groups, respectively. The ¹H-NMR spectrum of 1 showed signals corresponding to one olefinic proton [δ_H 6.20 (d, J = 2.5 Hz, H-2)], one oxygenated methine [δ_H 4.65 (m, H-10)], two methylene protons, two exchangeable NH protons [δ_H 7.72 (t, J = 4.5 Hz, NH-7), 11.89, s (NH-1)], and one exchangeable hydroxy proton [δ_H 5.12 (d, J = 5.7 Hz, OH-10)] (Table 1). The ¹³C-NMR spectrum of 1 in combination with ¹H-detected heteronuclear multiple quantum coherence (HMQC) spectrum displayed eight carbon signals, including one amide carbon [δ_C 162.1 (C-6)], four sp² olefinic carbons [δ_C 103.2 (C-3), 112.4 (C-2), 123.2 (C-5), 130.7 (C-4)], one oxygenated methine carbon [δ_C 65.0 (C-10)], and two methylene carbons [δ_C 35.7 (C-8), 36.0 (C-9)] (Table 1). Its ¹H- and ¹³C-NMR spectroscopic data were very similar to those of axinellin B, a pyrrolactam alkaloid isolated from the Chinese marine sponge Axinella sp.¹⁷⁾ The significant difference is the position of the bromo group. In axinellin B, an olefinic methine proton (δ_H 6.21) was appeared as singlet due to the presence of bromo group at C-2 of the pyrrole ring,¹⁸⁾ whereas the olefinic methine proton in 1 was appeared as doublet [δ_H 6.20 (d, J = 2.5 Hz)]. This appearance was consistent with that found in 3-bromoaldisine.¹⁸⁾ Thus, the bromo group was placed at C-3 on the pyrrole ring. The ¹H ¹H correlation spectroscopy (COSY) correlation between H-2 and NH-1 further supported this assumption (Fig. 2). The heteronuclear multiple bond connectivity (HMBC) correlations from NH-1 to C-2, from H-2 to C-3/C-4, H-10 to C-5/C-8, and from H-9 to C-6 assisted the connection between the pyrrole ring and lactam ring at C-4 and C-5, indicating that 1 is a pyrrolactam alkaloid. The absolute configuration at C-10 was determined on the basis of the comparison of the experimental and calculated electronic circular dichroism (ECD) data. The experimental ECD spectrum of 1 exhibited the negative Cotton effects (CE) at 280 nm and positive CE at 272 nm, which were in accordance with the calculated ECD data for the model compound of 1 with 10R configuration, using the time-dependent density functional theory (TDDFT) method¹⁹⁾ (Fig. 3). Thus, the absolute structure of 1 was elucidated as shown in Fig. 1 and 1 was named stylissaol A.

Table 1. ¹H (400 MHz) and ¹³C (100 MHz) NMR Spectroscopic Data of Stylissaol A (1) in DMSO-d₆ (δ in ppm and J Values in (Hz) in Parentheses)

Position	δH	δC
1	11.89, s	—
2	6.20, d (2.5)	112.4
3	—	103.2
4	—	130.7
5	—	123.2
6	—	162.1
7	7.72, t (4.5)	—
8	3.05, 3.30, both m	35.7
9	1.90, m	36.0
10	4.65, m	65.0
OH-10	5.12, d (5.7)	—

Fig. 2. ¹H ¹H COSY (Bold Lines) and Key HMBC (¹H–¹³C) Arrows of 1

Fig. 3. Experimental and Calculated Electronic Circular Dichroism (ECD) Data of 1 in MeOH

The structures of the known compounds 2–5 were elucidated by the analyses of their experimental NMR spectroscopic data as well as by comparing with the reported data.

The secondary metabolites of S. massa native to the marine biosphere of Myanmar are not different from those found in China, India, Japan and Australia. Pyrrolactam derivatives found in S. massa reportedly possessed various biological activities such as anti-cancer,^18,20,21) antibacterial,²²⁾ anti-inflammatory,²³⁾ and neuroprotective activities.¹³⁾ In the present study, we evaluated the Vpr inhibitory activities of the isolated brominated pyrrolactams (1–2, 4, 5) and pyrrolactam (3) against Vpr-transfected and tetracycline regulated HeLa (TREx-HeLa-Vpr) cells. Out of the tested compounds, 2-bromoaldisine (2) was the strongest inhibitor exhibiting anti-Vpr activity at 10 µM [Cell proliferation (%): 134] and its potency was comparable to that of the positive control damnacanthal [Cell proliferation (%): 158]. In contrast, non-brominated pyrrolactam, aldisine (3), did not display the Vpr activity. Thus, the bromine substituent at C-2 of the pyrrole ring may be important for activity. However, even though bromine is present at C-2, the additional amino-imidazolinone or the hydantoin ring linking to the pyrrolactams led cytotoxicity to TREx-HeLa-Vpr cells [Cell proliferation (%) at 10 µM: 22 (4), 86 (5)] (Fig. 4).

Fig. 4. The Vpr Inhibition Effects of the Ethyl Acetate Extract, the Isolated Compounds (1–5), and the Positive Control Damnacanthal in TREx-HeLa-Vpr Cells

Conclusion

Biochemical studies of Myanmar marine sponge, S. massa was investigated for the first time. The EtOAc extract was found to be active against TREx-HeLa-Vpr cells with an effective dose of 10 µg/mL. The isolation of the active EtOAc extract afforded a new brominated pyrrolactam, stylissaol A (1), together with four known analogues. The 2-bromoaldisine (2) was identified as the most active Vpr inhibitor. The position of bromine group at C-2 of the pyrrole ring might be important for brominated pyrrolactam in inhibiting the effect of Vpr where favoring the proliferation of TREx-HeLa-Vpr cells.

Experimental

General Experimental Procedures

Optical rotations were recorded on a Jasco P2100 polarimeter. CD measurements were carried out on a Jasco J-805 spectropolarimeter. UV spectra were measured on a NANODROP 2000c spectrophotometer (Thermo Scientific, MA, U.S.A.). FTIR spectra were recorded as KBr pellets on a Jasco FT/IR-460 Plus spectrometer. NMR spectra were recorded at 400 MHz (¹H-NMR) and 100 MHz (¹³C-NMR) on a Jeol JNM-ECX400 spectrometer. The chemical shifts were calibrated to the residual proton and carbon resonances of the deuterated solvent used for each compound measurement (δ_H 2.50 ppm and δ_C 39.5 ppm for dimethyl sulfoxide (DMSO)-d₆). The HRESIMS was recorded on a Shimadzu LCMS-IT-TOF spectrometer. Open column chromatography was performed with Diaion HP-20, normal-phase silica gel (silica gel 60N, spherical, neutral, 40–50 µm, Kanto Chemical Co., Inc., Japan) and Cosmosil 75C18-OPN (Nacalai Tesque Inc., Kyoto, Japan). TLC was performed on precoated silica gel 60F₂₅₄ and RP-18 F₂₅₄ plates (Merck, 0.25 or 0.50 mm thickness). An SH-1200 Microplate Reader^® (Corona, Hitachinaka, Japan) was used to measure the absorbance in the bioassays. The cell line, TREx-HeLa-Vpr was maintained in our laboratory. Cell culture flasks and 48-well plates were purchased from Corning Inc. (Corning, NY, U.S.A.).

Animal Material

The marine sponge, S. massa, was collected from Than-kyune-nge Island, Kawthaung, Tanintharyi Region, Myanmar in March 2017. The sponge was identified by Lecturer Dr. Aung Aung Aye (Department of Marine Sciences, Myeik University) and was air dried. A voucher sample (No. KS-3) is maintained at Department of Marine Sciences, Myeik University, Myanmar.

Extraction and Isolation

The dried powder of S. massa (1 kg) were percolated in MeOH (5L × 90 min × 3) at room temperature, and the solvent was evaporated under reduced pressure to give 100 g of a MeOH extract. The MeOH extract (100 g) was subjected to Diaion HP-20 resin and eluted with MeOH (10 L) after washing the resin with water (15 L) to remove salt. Evaporation of MeOH yielded 50 g of a MeOH extract. The MeOH extract (49 g) was dissolved in 500 mL of H₂O and partitioned with the 1 L of n-hexane three times to yield n-hexane-soluble extract (15 g) and an aqueous portion. The aqueous portion was further partitioned with EtOAc (1 L × 3) and n-BuOH (300 mL × 3), successively, and evaporation of the solvents afforded EtOAc extract (3 g), n-BuOH extract (8 g), and aqueous extract (20 g), respectively. The screening of the extracts revealed that the EtOAc extract was active and it was subjected for further chemical isolation and identification.

The EtOAc extract (3 g) was subjected to silica gel column chromatography, eluted with 1 L each of CHCl₃–MeOH (30 : 1, 20 : 1, 15 : 1, 10 : 1, 5 : 1, 3 : 1) to give six fractions [fr. 1 (388 mg), fr. 2 (385 mg), fr. 3 (127 mg), fr. 4 (350 mg), fr. 5 (650 mg), fr. 6 (700 mg)]. Recrystallization of fr. 1 (388 mg) in MeOH afforded 2 (100 mg) as colorless crystals. Compound 3 (50 mg) was directly obtained as precipitated form from fr. 2 (385 mg). Fraction 3 (127 mg) was rechromatographed on Cosmosil 75 C18-OPN using an isocratic solvent system of MeOH–H₂O (2 : 3) to afford three subfractions [3-1: 100 mg; 3-2 : 65 mg; 3-3 : 45 mg]. Purification of subfraction 3 : 1 (100 mg) by normal-phase column chromatography using CHCl₃–MeOH (7 : 1) solvent system gave precipitate of 4 (30 mg). Compound 5 (4 mg, Rf = 0.51) was obtained by purification of subfraction 3 : 2 (65 mg) by reverse-phase TLC using a MeCN–H₂O (1 : 2) solvent system. Purification of subfraction 3 : 3 (45 mg) by normal phase preparative TLC using a CHCl₃–MeOH (10 : 1) solvent afforded 1 (2 mg, Rf = 0.30).

Stylissaol A (1): amorphous powder; [α]_D¹⁸ −18.3 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 210 (2.5), 230 (2.7), 272 nm (2.9); FTIR (KBr) ν_max 3278, 2926, 1680, 1626, 1483, 1438, 1025 cm⁻¹; CD (c 4.08 × 10⁻⁴ M, MeOH) [θ]₂₈₀ −16509.41, [θ]₂₇₂ +7736.14; ¹H- and ¹³C-NMR data, see Table 1; HRESIMS m/z 266.9731 [M + Na]⁺, 268.9717 [M +2 + Na]⁺; (Calcd for C₈H₉BrN₂NaO₂, 266.9745).

ECD Calculation

The eight conformers with low-energy of 1 were produced by the DFT/TDDFT calculations, Merck Molecular Force Field (MMFF94) using Winmostar program. All DFT/TDDFT calculations were conducted using the Gaussian 16 program. Firstly, each low-energy conformer obtained by MMFF calculations was subjected to geometry optimization by the DFT method at the B3LYP/6-31G(d) level. Each optimized conformer was then subjected to a frequency calculation at the B3LYP/6-31G(d) level in order to estimate the thermal free energy (ΔG) and check for the presence of imaginary frequencies. On the basis of the estimated thermal energy, the abundance ratio of each conformer was calculated by the Boltzmann distribution. Finally, three conformers of 1 (in total, occupying approximately 99%) were selected for ECD calculations. The ECD spectra of all conformers were calculated using the TDDFT method at the B3LYP/6-31G(d) level with PCM in methanol, and the weighted-average spectra were compared with the experimental ECD spectra recorded in methanol.

In Vitro Anti-Vpr Activity

We used the cell line TREx-HeLa-Vpr, which was established in our lab.³⁾ The in vitro anti-Vpr activities of the extracts and the isolated compounds were determined according to the procedure described previously.^3,5,8)

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grants JP20H00490, JP20KK0173, JP19H04649, 19K23794, 20K07111).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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