1. Introduction

The genus Gymnadenia (Orchidaceae), containing about 20 species, is mainly distributed in Asia and Europe. Gymnadenia conopsea R. Br., native to the provinces of Tibet, Xinjiang, Inner Mongolia, Sichuan, Qinghai, and Gansu of mainland China, is a very important traditional Tibetan medicinal plant. The dried tuber of this plant, known as "Wangla" (Chinese), has been traditionally used for treatment of cough, asthma, and other syndromes ^[1-3]. Besides phenanthrenes, stilbenes, cyclodipeptides, and cyclopentene derivatives, a small group of glycosyloxybenzyl 2-isobutylmalate derivatives have been reported previously from this plant, which showed neuroprotective effects on memory deficits and pathological changes in senescent mice [3-6]. The current study was begun to search for novel bioactive secondary metabolites from this plant, and has led to the isolation of a new ureido-substituted amino acid, conopsamide A (1) (Fig. 1). This paper deals with the isolation, structure elucidation and HDAC1 (Histone Deacetylase 1) inhibitory activity of the new compound.

2. Experimental 2.1. General experimental procedures

Optical rotations were measured on a P-2000 polarimeter. UV spectra were recorded on a V-650 spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR Microscope spectrometer (FT-IR Microscope Transmission). 1D NMR and 2D NMR spectra were obtained at 500 for ¹H and 125 MHz for ¹³C, respectively, on an Inova 500 MHz spectrometer with solvent peaks as references. ESIMS and HR-ESIMS data were obtained on an AccuToFCS JMST100CS spectrometer. Column chromatography (CC) was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Inc., China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). HPLC separation was performed on a system consisting of a Lab Alliance prep pump and a Model 500 absorbance detector using an YMC (250 mm×10 mm i.d.) semi-preparative column packed with C₁₈ silica gel (5 μm). TLC was conducted on precoated silica gel GF₂₅₄ plates. Spots were visualized under UV light (254 or 356 nm) or by spraying with 10% H₂SO₄ in 95% EtOH followed by heating.

2.2. Plant material

The tubers of G. conoposa were collected at Hezuo, Gansu Province, People's Republic of China, in September 2013. The plant identification was verified by Professor Lu Xuefeng, Northwest Institute of Plateau Biology, Chinese Academy of Sciences. A voucher specimen (No. 00769) is deposited at the Herbarium of College of Pharmaceutical Sciences, Qinghai University for Nationalities.

2.3. Extraction and isolation

The air-dried tubers of this plant (1.5 kg) were successively extracted with 5.0 L of 95% EtOH (48 h) and 5.0 L of 65% EtOH (2×48 h) at room temperature. The combined extracts were evaporated under reduced pressure to yield a residue (121 g). The residue was suspended in H₂O (600 mL) and then successively partitioned with petroleum ether (3×500 mL) and EtOAc (3×500 mL). The aqueous phase was applied to a HP-20 macroporous adsorbent resin column (20 cm×200 cm), and eluted successively with H₂O (3 L), 50% EtOH (10 L), and 95% EtOH (3 L) to yield three corresponding fractions A, B and C. After removing the solvent under reduced pressure, fraction B (0.3 kg) was separated by column chromatography (CC) over MCI gel CHP 20P (1 L), with successive elution using H₂O (2 L), 30% EtOH (2 L), 50% EtOH (2 L), 95% EtOH (1 L), and Me₂CO (1 L), to give fractions B1-B5. Fraction B2 (3.6 g) was fractioned by reversed-phase C-18 silica flash chromatography eluting with a step gradient from 20% to 80% MeOH in H₂O, to afford fractions (B21-B26) based on TLC analysis. Separation of B23 (135mg) was separated by semipreparative HPLC (RP₁₈, 5 μm, 222 nm, MeOH-H₂O, 10:90) to give 1 (16.0 mg).

Conopsamide A (1): White amorphous; UV (MeOH) λ_max (log ε)₂₀₁ (3.68), 222 (3.79), 270 (2.21) nm; IR vmax 3332 (broad), 2928, 1694, 1668, 1591, 1537, 1513, 1447, 1392, 1233, 1167, 1102, 1012, 952, 821 cm^-1; ¹H NMR (MeOH-d₄, 500 MHz) data, see Table 1; ¹³C NMR (MeOH-d₄, 125 MHz) data, see Table 1; (+)-ESIMS m/z 318 [M + Na]⁺ and m/z 334 [M + K]⁺; (-) HR-ESIMS 294.1461 [M-H]^-(calcd. for C₁₄H₂₀N₃O₄, 294.1459).

2.4. Acid hydrolysis of 1 and Marfey's analysis of the hydrolyzates

Conopsamide A (1 mg) was hydrolyzed in 1.0 μL of 6 mol/L HCl at 120 ℃ for 12 h. Excess aqueous HCl was removed under vacuum. The dry material was suspended in 100 μL of H₂O, and then treated with 1% FDAA in acetone (100 μL) and 1 mol/L NaHCO₃ in 60 μL of water at 40 ℃ for 1 h. After cooling to room temperature, the mixture was neutralized with 2 mol/L HCl (30 μL) and filtrated. The standard FDAA-amino acids were prepared in the same manner using the corresponding _L-and _D-amino acids (0.8 and 1.1 mg). The FDAA-amino acid derivatives from the hydrolysate were compared with standard FDAA-amino acids using HPLC analysis: ZORBAX SB-C₁₈ (4.6 mm×150 mm, 5 μm); flow rate, 1.0 mL/min; UV detection at 340 nm; column temperature, 20 ℃; mobile phase, a linear gradient of 9:1 50 μmol/L triethylammonium phosphate (TEAP) buffer (pH 3)/CH₃CN to 1:9 TEAP/CH₃CN over 90 min. The FDAA derivatives of amino acids liberated from 1 showed a peak at 31.013 min, matching the retention time of FDAA _D-ornithine (31.0=min; 32.246 min for _L-ornithine).

2.5. HDAC1 inhibition screen of 1

HDAC1 (Cisbio Bioassays No. NP_0049=.2) inhibitory activity was measured using 2 ng/μL HDAC1, 0.2 μmol/L H3(1-21) K9 incubated for 60 min at 37 ℃ with a range of concentrations of compound 1. Reaction was stopped by addition 5 μL SA-XL665 and 2 ng H₃K9me0 antibody. Then, fluorescence signal was measured on a fluorescence microplate reader with an emission wavelength of 665 nm. All dilutions were prepared in a buffer containing 50 mmol/L Tris-HCl, pH 8.0; 137 mmol/L NaCl; 2.7 mmol/L KCl; 1 mmol/L MgCl₂; and 0.01% Tween 20.

3. Results and discussion

Compound 1 was obtained as a white amorphous, [α]_D²⁰-38.7 (c 0.10, MeOH). The IR spectrum of 1 showed absorption bands for hydroxyl and amino (3332 cm^-1), carbonyl (1694 and 1668 cm^-1), and aromatic ring (1591 and 1513 cm^-1) functionalities. The positive mode ESIMS of 1 exhibited a quasimolecular ion peak at m/z 318 [M + Na]⁺ and 334 [M + K]⁺, and the molecular formula C₁₄H₂₁N₃O₄ was determined by HR-ESIMS at m/z 294.1461 [M-H]^-× (calcd. for C₁₄H₂₀N₃O₄, 294.1459), combined with the NMR data (Table 1). The ¹H NMR spectrum of 1 in MeOH-d₄ showed characteristic signals attributable to 4-substitued benzyl alcohol at δ_H 7.09 (d, 2H, J=8.0 Hz, H-2″, 6″), 6.69 (d, 2H, J=8.0 Hz, H-3″, 5″), and 4.33 (s, 2H, H₂-7″) and an N-methyl singlet at δ_H 2.73 (3H, s, H₃-1″′). In addition, it showed signals assignable to one nitrogenbearing methine at δ_H 3.47 (1H, t, J=5.5 Hz, H-2), one nitrogenbearing methylene at δ_H 3.19, 3.15 (dt, each 1H, J=13.5, 6.5 Hz, H-5a, 5b), and two methylene at δ_H 1.84, 1.73 (m, each 1H, H-3a, 3b), and 1.57 (m, 2H, H₂-4). The ¹³C NMR spectrum displayed 13 carbon resonances corresponding to the above units and one carbonyl carbon at dC 161.6 (C-1′) (Table 1). The six degrees of unsaturation inherent in the molecular formula of 1, coupled with data showing the presence of one carbonyl group, indicated that 1 must possess another carbonyl carbon (C-1) instead of one ring, since one of the carbon signal was absent in the ¹³C NMR spectrum. Therefore, the above spectroscopic data, coupled with the consideration of chemical shifts of the proton and carbon resonances, indicate that 1 is an amino acid with a 4-hydroxybenzyl and N-methyl moieties, of which the structure was further elucidated by comprehensive analysis of 2D NMR data.

The proton and proton-bearing carbon resonances in the NMR spectra of 1 were unambiguously assigned by the HSQC experiment. In the ¹H-¹H COSY spectrum of 1, vicinal coupling correlations of H-2″′ (H-6″′)↔H-3″′ (H-5″′) (Fig. 2, thick lines) and HMBC correlation from H₂-7″′ to C-2″′ (C-6″′) (Fig. 2, arrows), confirmed the presence of the 4-hydroxybenzyl. The ¹H-¹H COSY correlations of H-2↔H₂-3 and H₂-3↔H₂-4, and H₂-4↔H₂-5 and the HMBC correlations from H₂-3 to C-1 and C-5, from H₂-4 to C-2 (Fig. 2, arrows), together with chemical shifts of the proton and carbon resonances, indicated the presence of an ornithinyl group. Finally, the connection of the 4-hydroxybenzyl, ornithinyl, and methyl via an ureido linkage was unambiguously established by HMBC correlations from H₂-5 to C-1′, from N-methyl (H₃-1″) to C-1′ and C-7″, and from H₂-7″ to C-1″′ and C-1″′ (Fig. 2, arrows), completing the full planar structure of conopsamide A (1).

The absolute configurations of the a-amino residue in conopsamide A was determined by application of the Marfey's method [7]. Hydrolysis of conopsamide A (1) using 6 mol/L HCl yielded the free amino acid. The hydrolysis products were derivatized using the Marfey's reagent, (1-fluoro-2, 4-dinitrophenyl)-5-_L-alanine amide (FDAA), and analyzed by reversed-phase HPLC. Peaks in the chromatogram were identified by comparing the retention times with those of the FDAA derivatives of authentic amino acids. Marfey's reagent derivative of the amino acid liberated from 1 showed peaks matching D-ornithine (Fig. S7 in Supporting information). Therefore, the structure of 1 was determined as (-)-R-2-amino-5-{[3-(4-hydroxybenzyl)-3-methyl] ureido}pentanoic acid, and named conopsamide A.

In the in vitro bioassays performed in this study, conopsamide A (1) showed moderate inhibitory activity against HDAC1 with an IC₅₀ of 5.0 μmol/L. Other assays assessed inhibitory activity against the release of glucuronidase in rat polymorphonuclear leukocytes (PMN) induced by PAF [8]; cytotoxicity against several human cancer cell lines [9]; and inhibitory activity against protein tyrosine phosphatase 1B (PTP1B) [10]. However, conopsamide A (1) were inactive at a concentration of 10 × 10^-6 mol/L in each assay.

4. Conclusion

A new ureido-substituted amino acid, conopsamide A (1), was isolated from the ethanolic extract of G. conopsea tubers, and it showed moderate inhibitory activity against HDAC1. It is well known that over expression of HDCA promotes the deacetylation of histone in cancer cells, and enhances the attractiveness between DNA and histone by regaining the positive charges of histone. HDAC inhibition prevents the removal of acetyl groups from histones, causing their accumulation in cell nuclei and resulting in several downstream effects including apoptosis, differentiation, and reduced proliferation of cancer cells [11, 12]. The new structure with inhibitory activity against HDAC1 provides a framework for derivatives synthesis and further in deep biological evaluation.

Acknowledgments

Financial support from the National Natural Science Foundation of China (No. 81522050), Beijing outstanding talents cultivation fund (No. 2013D009008000002) and the Natural Science Foundation of Qinghai Province (No. 2014-ZJ-923) is acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.08.005.