(±)-Cryptamides A–D, Four Pairs of Novel Dopamine Enantiomer Trimers from the Periostracum Cicadae

Four pairs of novel dopamine enantiomer trimers, (±)-cryptamides A–D (1–4), and 10 pairs of previously described dopamine enantiomer dimers (5–14) were isolated from the Periostracum cicadae, the cast-off shell of the insect Cryptotympana pustulata. Aside from being pairs of enantiomers, the eight trimers were also elucidated to be regioisomers, most likely resulting from their mechanism of formation, [4 + 2] cycloaddition. The discovery of dopamine trimers is rarely reported when it comes to natural products derived from insects.

Hereby, we report the isolation and structural elucidation of four novel enantiomeric dopamine trimer pairs (1-4) and 10 enantiomeric dopamine dimer pairs (Figure 1), the latter of which are already described in the literature [2,10,11,[13][14][15]. A detailed comparison revealed that the novel four pairs of compounds were also regioisomers. Moreover, the anti-inflammatory effects of new compounds toward a lipopolysaccharide (LPS)induced RAW264.7 macrophage model were evaluated. Among them, (±)-cryptamide B, (±)-cryptamide C, and (−)-cryptamide D can effectively reduce the levels of nitric oxide.

General Experimental Procedures
The Jasco P-1020 polarimeter (Jasco, Easton, MD, USA) was used to acquire optical rotations. UV spectra were acquired on an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Experimental ECD spectra in MeOH were acquired in a quartz cuvette of 1 mm optical path length on a Chirascan V100 spectropolarimeter (Applied Photophysics Ltd., London, UK). A Waters Xevo G2 QTOF mass spectrometer with a Synapt G2 HDMS quadrupole time-of-flight (TOF) mass spectrometer (Waters, Milford, MA, USA) was used to measure the HR-ESI-MS data. Both 1D and 2D-NMR spectra of the isolated compounds were obtained on a Bruker AVANCE III 500 MHz spectrometer (Bruker, Billerica, MA, USA). For semi-preparative, high-performance liquid chromatography (HPLC), a SEP SP-5030 Binary HPLC pump, equipped with a Sep UV300 photodiode array detector (SEP Corporation, Beijing, China), was used to isolate and purify the compounds. The chiral resolution was carried out using the Shimadzu Prominence HPLC system with SPD-M20A series Prominence HPLC UV-Vis detectors (Shimadzu, Tokyo, Japan) equipped with a Chiral-INA (250 × 4.6 mm i.d., 5 μm) column (Guangzhou FLM Scientific Instrument Co., Guangzhou, China). An LC/MS analysis was carried out using an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a 6130 series ESI mass spectrometer, using an analytical Waters column (2.1 × 50 mm, 1.7 μm). Cosmosil RP-C18 silica gel (Nacalai Tesque, Inc., Kyoto, Japan), MCI gel CHP 20P (75-150 μm, Mitsubishi Chemical Industries, Tokyo, Japan), HW-40F (Tosoh Corporation, Tokyo, Japan), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography (CC). Silica gel HSGF254 plates were used for thin-layer chromatography (TLC). Spots on the TLC were detected under UV light or by using iodine.

Plant Material
The dried Periostracum cicadae was purchased from Zhengzhou medicine Co., Ltd., Zhengzhou (Henan), PR China, in September 2020, and was identified by Professor Guo Tao of Henan University of Traditional Chinese Medicine as the skin shell shed from the insect Cryptotympana pustulata Fabricius.

General Experimental Procedures
The Jasco P-1020 polarimeter (Jasco, Easton, MD, USA) was used to acquire optical rotations. UV spectra were acquired on an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Experimental ECD spectra in MeOH were acquired in a quartz cuvette of 1 mm optical path length on a Chirascan V100 spectropolarimeter (Applied Photophysics Ltd., London, UK). A Waters Xevo G2 QTOF mass spectrometer with a Synapt G2 HDMS quadrupole time-of-flight (TOF) mass spectrometer (Waters, Milford, MA, USA) was used to measure the HR-ESI-MS data. Both 1D and 2D-NMR spectra of the isolated compounds were obtained on a Bruker AVANCE III 500 MHz spectrometer (Bruker, Billerica, MA, USA). For semi-preparative, high-performance liquid chromatography (HPLC), a SEP SP-5030 Binary HPLC pump, equipped with a Sep UV300 photodiode array detector (SEP Corporation, Beijing, China), was used to isolate and purify the compounds. The chiral resolution was carried out using the Shimadzu Prominence HPLC system with SPD-M20A series Prominence HPLC UV-Vis detectors (Shimadzu, Tokyo, Japan) equipped with a Chiral-INA (250 × 4.6 mm i.d., 5 µm) column (Guangzhou FLM Scientific Instrument Co., Guangzhou, China). An LC/MS analysis was carried out using an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a 6130 series ESI mass spectrometer, using an analytical Waters column (2.1 × 50 mm, 1.7 µm). Cosmosil RP-C18 silica gel (Nacalai Tesque, Inc., Kyoto, Japan), MCI gel CHP 20P (75-150 µm, Mitsubishi Chemical Industries, Tokyo, Japan), HW-40F (Tosoh Corporation, Tokyo, Japan), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography (CC). Silica gel HSGF254 plates were used for thin-layer chromatography (TLC). Spots on the TLC were detected under UV light or by using iodine.

Plant Material
The dried Periostracum cicadae was purchased from Zhengzhou medicine Co., Ltd., Zhengzhou (Henan), PR China, in September 2020, and was identified by Professor Guo Tao of Henan University of Traditional Chinese Medicine as the skin shell shed from the insect Cryptotympana pustulata Fabricius.

Extraction and Isolation
The powder of the dried Periostracum cicadae (5 kg) was extracted with 70% ethanol for 72 h each time, for a total of three times. The crude extract (631 g) was obtained through concentrating the extract under pressure. This extract was loaded onto a macroporous resin column and eluted with MeOH-H 2 O (10:90-100:0, v/v, gradient system) to provide five fractions (A-E).