Two new N-acetyldopamine dimers, cicadamides A (1) and B (2), and 8 known compounds were isolated from the medicinal insect Periostracum cicadae. Their structures including absolute configurations were assigned by spectroscopic and computational methods. Biological activities of compounds 1, (+)-2, and (–)-2 toward human cancer cells BGC-823, HepG2, and A549 were evaluated.
Periostracum cicadae, the shell of the cicada Cryptotympana pustulata Fabricius that is cast-off during molting, is a commonly used traditional Chinese medicine for the treatment of throat soreness, hoarseness, itching, spasms, and other symptoms.1-4 Previous pharmacological studies showed that P. cicadae has anticonvulsant,5 anti-infective, antioxidation, antitumor, analgesic,6 antitussive, expectorant, antiasthmatic, and antipyretic effects.7 Previous chemical investigations led to the demonstration of N-acetyldopamine (NADA) derivatives,6,8-10 which are commonly found in many kinds of medicinal insects.11-15 During our efforts of searching for active nonpeptidal agents from medicinal insects, we initiated an investigation on P. cicadae, resulting in the isolation of 2 new NADA dimers and 8 known compounds (Figure 1). Their structures were elucidated on the basis of spectroscopic data. The absolute configurations of 1 and 2 were assigned by Electronic Circular Dichroism (ECD) computational methods. Biological activities of compounds 1, (+)-2, and (–)-2 toward human cancer cells BGC-823, HepG2, and A549 were evaluated.
Chemical structures of compounds 1 to 8.
Compound 1 was obtained as a yellowish gum. Its molecular formula was assigned as C26H32N2O11, with 12 degrees of unsaturation, based on its High Resolution Electrospray Ionization Mass Spectrometry (HRESIMS) data (m/z 549.2087 [M+H]+, calcd for C26H33N2O11, 549.2079). The 1H nuclear magnetic resonance (NMR) spectrum (Table 1 and Figure S1, Supplemental material) of 1 contains 2 typical ABX spin systems [δH 6.78 (d, J = 2.0 Hz, H-5), 6.75 (dd, J = 8.2, 2.0 Hz, H-7), and 6.87 (d, J = 8.2 Hz, H-8); δH 7.28 (d, J = 2.0 Hz, H-2′), 6.86 (d, J = 8.2 Hz, H-5′), and 6.98 (dd, J = 8.2, 2.0 Hz, H-6′)], suggesting the presence of 2 1,3,4-trisubstituted benzene rings.The 13C NMR and distortionless enhancement by polarization transfer (DEPT) spectra (Table 1 and Figure S2, Supplemental material) contain resonances for 26 carbons, including 2 methyl, 3 aliphatic methylene, 13 methine (6 olefinic), and 8 quaternary carbons (2 amide carbonyl groups, 4 oxygenated sp2 carbons). It is evident that there exists a hexose in 1 diagnosed by the presence of a group of carbon signals at δC 104.3, 78.4, 78.3, 74.9, 71.4, and 62.5. Apart from the signals accounting for a hexose, the remaining data prompted us to assume that 1 is likely a dopamine dimer derivative. Careful comparison revealed that NMR data of aglycone of 1 is in accordance with those of 4,6 allowing to identify the structure of 1's aglycone. The heteronuclear multiple bond correlations (HMBC, Figure 2 and Figure s5) of H-9/C-5, C-6, C-7, H-10/C-6, and H-7 [δH 6.75 (dd, J = 8.2, 2.0 Hz)], and H-2/C-8a further secure the position of the side chain at C-7. The relative configurations at C-2 and C-3 were also secured as trans by the coupling constant of J2,3 = 7.2 Hz.6 As for the sugar moiety, it was assigned as D-glucose by comparing the optical rotation of the hydrolysis product (sugar part) with that of the reference compound. This moiety is positioned at C-3' evidenced from the HMBC of H-1″/C-3′ and the rotational Overhauser effect spectroscopy cross peak caused by H-2′/H-1″. In addition, 1 was identified to be a β-D-glucose by the coupling constant of H-1″ (J = 7.4 Hz). To clarify the absolute configuration of 1, ECD calculations were carried out. It was found that the experimental ECD spectrum of 1 agrees well with the calculated ECD spectrum of 2R,3S-1 instead of that of 2S,3R-1 (Figure 3), thus the absolute configuration of 1 was assigned as 2R,3S and named cicadamide A. It is worth mentioning that most NADAs from the insects were characterized as racemates.11,15 In the present study, it is interesting that 1 was isolated as an optically active substance which was indicated by the optical rotation value, distinct Cotton effects, as well as chiral high-performance liquid chromatography (HPLC) analysis (data not shown).
1H (600 MHz) and 13C Nuclear Magnetic Resonance (150 MHz) Data of 1 in Methanol-d4 (Δ in ppm, J in Hz).
No.
δH
δC
No.
δH
δC
2
4.73 (d, 7.2)
77.6 d
4′
149.0 s
3
5.74 (d, 7.2)
78.1 d
5′
6.86 (d, 8.2)
117.0 d
4a
143.6 s
6′
6.98 (dd, 8.2, 2.0)
124.2 d
5
6.78 (d, 2.0)
118.1 d
1″
4.74 (d, 7.4)
104.3 d
6
134.4 s
2″
3.50 (m)
74.9 d
7
6.75 (dd, 8.2, 2.0)
123.1 d
3″
3.47 (m)
78.4 d
8
6.87 (d, 8.2)
118.2 d
4″
3.40 (m)
71.4 d
8a
143.0 s
5″
3.43 (m)
78.3 d
9
2.71 (t, 7.5)
35.8 t
6″
3.93 (dd, 12.0, 2.2)3.71 (dd, 12.0, 5.6)
62.5 t
10
3.35 (overlap)
42.2 t
3a
173.3 s
1′
128.8 s
3b
1.91 (s)
22.6 q
2′
7.28 (d, 2.0)
118.0 d
10a
173.3 s
3′
146.5 s
10b
1.88 (s)
22.5 q
Correlational spectroscopy (—) and key heteronuclear multiple bond correlations () and rotational Overhauser effect spectroscopy () correlations of 1 and 2.
Comparison between the calculated ECD spectrum for (2R,3S)-1 with the experimental spectrum of 1 in MeOH at B3LYP/6-31G (d,p) level. σ = 0.3 eV; shift = 10 nm.
Compound 2 was obtained as a yellowish gum. Its molecular formula was assigned as C18H17NO7, with 11 degrees of unsaturation, based on its HRESIMS data (m/z 360.1082 [M+H]+, calcd for C18H18NO7, 360.1078). The 1H NMR spectrum of 1 (Table 2, Figure S8, Supplementary Material) contains 2 typical ABX spin systems [δH 6.99 (d, J = 8.3 Hz, H-5), 7.54 (dd, J = 8.3, 2.5 Hz, H-6), and 7.55 (d, J = 2.5 Hz, H-8); δH 6.86 (d, J = 1.6 Hz, H-2′), 6.77 (d, J = 8.0 Hz, H-5′), and 6.76 (dd, J = 8.0, 1.6 Hz, H-6′)], suggesting the presence of 2 1,3,4-trisubstituted benzene rings. The 13C NMR and DEPT spectra (Table 2, Figure S9, Supplementary Material) contain resonances for 18 carbons, including 1 methyl, 1 aliphatic methylene, 8 methine (including 2 oxygenated aliphatic and 6 olefinic), and 8 quaternary carbons. The NMR data of 2 are quite similar to those of 3, indicating that they are analogs. The main difference is a side chain at C-7. An HMBC of H-10/C-9 (δC 198.6) (Figure 2, Figure S12) indicates the presence of fragment A (Figure 1), which is positioned at C-7 by the observation of HMBC of H-10/C-7 (δC 129.6) and H-6, H-8/C-9, C-4a (δC 148.8). In addition, the benzodioxane ring was determined by HMBC of H-2/C-8a (δC 144.6) and H-3/C-4a. The relative configurations at C-2 and C-3 were determined as trans by the J2,3 coupling constant (7.3 Hz).6 Because NADAs are commonly racemic, we purposely examined the optical purity by chiral HPLC and found that 2 is a racemic mixture. Subsequent separation of 2 on chiral HPLC afforded 2 enantiomers, (+)-2 and (–)-2. Their absolute configurations were determined to be 2S,3R for (+)-2 and 2R,3S for (−)-2 by ECD calculations followed by comparison with the experimental ECD spectra (Figure 4). Thus, the structure of 2 was identified and named (+)-cicadamide B and (−)-cicadamide B, respectively.
1H (600 MHz) and 13C Nuclear Magnetic Resonance (150 MHz) Data of 2 and 5 in Methanol-d4 (Δ in ppm, J in Hz).
No.
2
5
δH
δC
δH
δC
1
127.6 s
2
4.76 (d, 7.3)
78.2 d
7.38 (d, 2.0)
115.6 d
3
5.80 (d, 7.3)
78.8 d
146.8 s
4
152.9 s
4a
148.8 s
5
6.99 (d, 8.3)
118.3 d
7.41 (dd, 8.3, 2.0)
127.6 d
6
7.54 (dd, 8.3, 2.5)
123.3 d
6.85 (d, 8.3)
116.0 d
7
129.6 s
193.2 s
8
7.55 (d, 2.5)
117.8 d
5.32 (s)
67.1 t
8a
144.6 s
9
198.6 s
172.3 s
10
4.82 (s)
66.1 t
2.17 (s)
20.5 q
1′
128.1 s
2′
6.86 (d, 1.6)
115.6 d
3′
146.6 s
4′
147.4 s
5′
6.77 (d, 8.0)
116.2 d
6′
6.76 (dd, 8.0, 1.6)
120.7 d
3a
173.3 s
3b
1.89 (s)
22.6 q
Comparison between the calculated ECD spectrum for (2R,3S)-2 with the experimental spectra of (+)-2 and (–)-2 in MeOH at B3LYP/6-31G (d,p) level. σ = 0.3 eV; shift = 4 nm.
Eight known compounds were identified as trans-2-(3′,4′-dihydroxy-phenyl)-3-acetylamino-7-(N-acetyl-2″-aminoethyl)-1,4-benzodioxane (3),6trans-2-(3′,4′-dihydroxy-phenyl)-3-acetylamino-6-(N-acetyl-2″-aminoethyl)-1,4-benzodioxane (4),6N-acetylarterenone (5),16 cyclo(L-4-hydroxypro-L-Phe) (6),17 3-hydroxy-9-methoxypterocarpan (7),18 3-hydroxy-8,9-methylenedioxypterocarpan (8),19 4-hydroxy-3-methoxy-benzoic acid (9),20 and 4-hydroxybenzaldehyde (10),21 respectively, by comparison of their NMR data with the literature. In addition, the 1H and 13C NMR data of 5 (Table 2, Figure S14 and S15, Supplementary Material) were assigned for the first time.
The new isolates 1, (+)-2, and (–)-2 were evaluated for their cytotoxic activity against 3 human cancer cells (BGC-823, HepG2, and A549). Unfortunately, none of them exhibits biological activities in these assays.
Experimental
General
Optical rotations were recorded on an Anton Paar MCP 100 Polarimeter. UV spectra were recorded on a UV/VIS spectrophotometer. Circular dichroism spectra were measured on a Chirascan instrument. The NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer, with trimethyl silane as an internal standard. Electronspray ionization mass spectrometry and HRESIMS were measured on an AutoSpec Premier P776 spectrometer. RP-18 silica gel (40-60 μm; Daiso Co., Japan), YMC GEL ODS-A-HG (12 nm S-50 µm; Japan), MCI gel CHP 20P (75-150 μm, Mitsubishi Chemical Industries, Tokyo, Japan), and Sephadex LH-20 (Amersham Pharmacia, Uppsala, Sweden) were used for column chromatography (CC). Semipreparative HPLC was carried out using a SEP LC-52 with an MWD UV detector (Separation Technology Co. Ltd., Beijing, PR China) equipped with a YMC-Pack ODS-A column (250 mm ×10 mm, i.d. 5 µm); all the flow rate was 3 mL/min unless otherwise indicated. Analytic HPLC was conducted using an Agilent 1260 liquid chromatograph equipped with a Daicel Chiralpak IC column (250 mm × 4.6 mm, i.d. 5 µm).
Insect Material
The dried P. cicadae was purchased from Changdu Zhenxin Traditional Chinese Herbal Medicine Co. Ltd., Changsha (Hunan), PR China, in April 2018. The material was identified by Prof. De-Po Yang at the College of Pharmacy, Zhongshan University, and a voucher specimen (CHYX-0633) is deposited at the School of Pharmaceutical Sciences, Shenzhen University Health Science Center, PR China.
Extraction and Isolation
The dried P. cicadae powders (5 kg) were extracted with MeOH at room temperature (45 L × 3 × 24 h), and the residue was again extracted with EtOH/H2O (4:1) (45 L × 2 × 24 h). The combined extracts were concentrated under reduced pressure to afford a crude extract (136.7 g). This extract was divided into 10 parts (Fr. A-J) by using an MCI gel CHP 20P column (35 cm × 7 cm) eluted with gradient aqueous MeOH (10:90-100:0). Fraction F (8.8 g) was gel filtrated over Sephadex LH-20 (MeOH) column to yield Fr.F.1 to Fr.F.3. Fraction F.2 (3.7 g) was submitted to RP-18 CC (aqueous MeOH, 20:80-100:0) to produce 3 fractions (Fr.F.2.1-Fr.F.2.3). Fraction F.2.2 (3.3 g) was gel filtrated over Sephadex LH-20 (MeOH) column to give Fr.F.2.2.1 and Fr.F.2.2.2. Fraction F.2.2.2 (3.2 g) was further submitted to RP-18 CC (CH3CN/H2O, 10:90) to obtain Fr.F.2.2.2.1 to Fr.F.2.2.2.3. Fraction F.2.2.2.2 (30.0 mg) was again chromatographed over semipreparative HPLC (CH3CN/H2O, 18:82) to afford compound 1 (2.0 mg, tR = 15.6 min). Fraction F.3 (1.2 g) was divided into 6 portions (Fr.F.3.1-Fr.F.3.6) by RP-18 CC (MeOH/H2O, 20:80-100:0). Fraction F.3.2 (18 mg) was finally passed through semipreparative HPLC (MeOH/H2O, 25:75) to afford compound 10 (4.0 mg, tR = 21.4 min). Fraction F.3.4 (145.0 mg) was gel filtrated over Sephadex LH-20 (MeOH) to give Fr.F.3.4.1 to Fr.F.3.4.5. Of these, Fr.F.3.4.3 (90.0 mg) underwent semipreparative HPLC (MeOH/H2O, 35:65) to give compound 5 (7.0 mg, tR = 12.0 min). Fraction F.3.4.4 (15.0 mg) underwent semipreparative HPLC (MeOH/H2O, 32:68) to afford compounds 2 (1.1 mg, tR = 11.6 min) and 9 (2.0 mg, tR = 23.7 min). Compound 2 is a racemate which was submitted to semipreparative HPLC on a chiral phase (n-hexane/ethanol, 72:28, flow rate 1.0 mL/min) to afford (+)-2 (1.2 mg, tR = 2.1 min) and (–)-2 (2.3 mg, tR = 14.2 min), respectively. Fraction E (4.8 g) was separated on Sephadex LH-20 (MeOH) to give Fr.E.1 to Fr.E.4, where Fr.E.3 (0.4 g) was subjected to RP-18 CC (MeOH/H2O, 25:75-100:0) to produce 4 portions (Fr.E.3.1-Fr.E.3.4). Fraction E.3.1 (130.0 mg) underwent semipreparative HPLC (MeOH/H2O, 22:78) to give Fr.E.3.1.1 to Fr.E.3.1.5. Among them, Fr.E.3.1.1 (60.0 mg) was further submitted to semipreparative HPLC (MeOH/H2O, 20:80) to give compounds 3 (15.0 mg, tR = 25.9 min) and 4 (10.0 mg, tR = 30.7 min). Compound 6 (1.0 mg, tR = 31.6 min) was purified by semipreparative HPLC (CH3CN/H2O, 10:90). Fraction I (1.1 g) was separated on Sephadex LH-20 (MeOH) to give Fr.I.1 to Fr.I.7, where Fr.I.6 (55.0 mg) was subjected to semipreparative HPLC (MeOH/H2O, 65:35) to yield compound 7 (3.0 mg, tR = 16.5 min). Compound 8 (20.0 mg, tR = 7.9 min) was purified by semipreparative HPLC (MeOH/H2O, 70:30).
HRESIMS m/z 360.1082 [M+H]+ (calcd for C18H18NO7, 360.1078).
Acid Hydrolysis of 1
A solution of 1 (0.8 mg) in 6 N HCl was stirred at 60°C for 1.5 hours. After cooling, the mixtures were extracted with EtOAc. The aqueous layer was concentrated in vacuo followed by thin-layer chromatography examination and optical rotation measurement. The optical rotation of the glucose of 1 is as follows: [α]D25 +43.0 (c 0.054, H2O). By comparing the optical rotation with D-glucose: [α]D25 +42.3 (c 0.106, H2O), the glucose in compound 1 was determined to be D-configuration.
Supplemental Material
Supplementary Information - Supplemental material for Cicadamides A and B, N-Acetyldopamine Dimers From the Insect Periostracum cicadae
Supplemental material, Supplementary Information, for Cicadamides A and B, N-Acetyldopamine Dimers From the Insect Periostracum cicadae by Huan Liu, Yong-Ming Yan, Li Liao, Shao-Xiang Wang, Yi Zhang, and Yong-Xian Cheng in Natural Product Communications
Footnotes
Acknowledgments
We are indebted to the Instrumental Analysis Center of Shenzhen University (Xili Campus) for NMR data measurement.
Author note
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was financially supported by Shenzhen Government’s Plan of Science and Technology (JCYJ20170412110504956) and National Natural Science Fund for Distinguished Young Scholars (81525026).
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Supplementary Material
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) correlations of 1 and 2.
