Asymmetric Nitrogen-Containing Dimer from Aerial Parts of Mercurialis leiocarpa and Its Synthesis by Mimicking Generation Process through Radical Intermediates

Abstract

An asymmetric nitrogen-containing dimer, leiocarpanine A, was isolated from the aerial part of Mercurialis leiocarpa as a new compound. The new generation process of leiocarpanine A was estimated and a concise synthesis of leiocarpanine A could be detailed based on mimicking the generation process through the radical intermediates. In general, a lot of reaction step and organic reagents are required for the synthesis of asymmetric nitrogen-containing dimers. However, our new synthesis method enables a concise synthesis of asymmetric nitrogen-containing dimers through radical intermediates by only liquid-separation. This synthetic method provides a rapid and concise pathway to construct a library of nitrogen-containing dimers that might be useful for drug discovery. In addition, it is useful to elucidate the generation process of leiocarpanine A.

Introduction

Dye plants, such as Indigofera, Indigo, Persicaria, and Rubia plants, are not only used as dyes but also known to show several biological effects. For example, Indigo naturalis, a natural blue dye used in traditional Chinese medicine, was reported in 2018 to exhibit significant anti-inflammatory effects on ulcerative colitis.¹⁾ Indirubin, an indole dimer, was isolated as a major constituent from this plant.²⁾ Thus, natural dyes and nitrogen-containing dimers, such as indole dimers, are important for drug development. Among natural dye plants, Mercurialis leiocarpa (Euphorbiaceae), a perennial thriving in Asian forests, is one of the oldest dye plants. This plant had been used as a blue dye until indigo dye appeared in Japan. Indeed, the cut and dried stems of this plant turn deep blue. This color change may be caused by the conversion of hermidin into radical intermediate, cyanohermidin.³⁾ The aerial parts of M. leiocarpa have been reported to contain 2-oxo-3-pyrroline dimer isochrysohermidin,⁴⁾ which is different from indigo. As shown Fig. 1, it was suggested that isochrysohermidin was generated by dimerization, oxidation,^5,6) and rearrangement⁷⁾ from hermidin derived from nicotinic acid⁸⁾ via cyanohermidin,³⁾ after cutting and drying.⁹⁾ In the 1990s, Isochrysohermidin was synthesized by a unique method, namely, the [4 + 2] cycloaddition of ¹O₂^10–13) (Fig. 2). However, research of the construction of nitrogen-containing dimers and the elucidation of their biological activities has been insufficient. Therefore, we have focused our efforts on nitrogen-containing dimers from M. leiocarpa for the development of innovative drug seeds. In the present report, we discuss the isolation of the new asymmetric pyrrole dimer, leiocarpanine A (1), from M. leiocarpa. We also present the chemical elucidation, the estimation of the generation process, and the concise synthesis of 1.

Fig. 1. Plausible Generation Process of Isochrysohermidin

Fig. 2. Synthesis of Isochrysohermidin by [4 + 2] Cycloaddition of ¹O₂ across the Pyrrole, as Reported by Wasserman et al. and Boger et al.^10–13)

Results and Discussion

The dry aerial parts of M. leiocarpa were extracted with MeOH at 60°C for 3 h. The MeOH extract was partitioned between EtOAc and H₂O. The EtOAc fraction was subjected to normal- and reversed-phase column chromatography and HPLC to isolate new compound leiocarpanine A (1, 0.0049% from dry plants), together with four known nitrogen-containing compounds, plicatanin B (2, 0.00058%),¹⁴⁾ speranberculatine A (3, 0.00020%),¹⁵⁾ 5-hydroxy-4-methoxy-5-(methoxycarbonyl)-1-methyl-3-pyrrolin-2-one (4, 0.00041%),¹²⁾ and 3-methoxy-1-methylmaleimide (5, 0.00019%)¹⁶⁾ (Fig. 3). Leiocarpanine A (1) was obtained as yellow crystals. In the high resolution electrospray ionization (HRESI)MS measurement of 1, a pseudo-molecular ion peak [M + Na]⁺ was observed at m/z 363.0796 and the molecular formula was determined as C₁₄H₁₆N₂O₈ on the basis of the HRESIMS peak and the ¹H- and ¹³C-NMR data. The proton and carbon signals of 1 in the ¹H- and ¹³C-NMR spectra were similar to those of compounds 2 and 3 except for the signals around 2-position (Table 1). In addition, heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond connectivity (HMBC) experiments indicated it to be a pyrrole dimer with a methyl ester at 2-position. Finally, compound 1 was obtained as a monocrystal and Mo-Kα X-ray diffraction measurement clarified its chemical structure (Fig. 4). Based on these pieces of evidence, compound 1 was identified as an asymmetric pyrrole dimer and named leiocarpanine A (1). The isolation of pyrrole dimers has been limited to a few plants, such as Euphorbiaceae plant. Among them, 1 having an asymmetric pyrrole dimer structure is a rare compound derived from a medicinal plant.

Fig. 3. Structures of Nitrogen-Containing Compounds Isolated from Aerial Parts of M. leiocarpa

Table 1. ¹³C-NMR (150 MHz) and ¹H-NMR (600 MHz) Data for New Compound 1 and Known Compounds 2 and 3 in CDCl₃

Position	1		2		3
	δH	δC	δH	δC	δH	δC
2		86.8		169.6	5.22, s	81.4
3		168.4		156.7		170.2
4		95.8		99.6		95.9
5		169.8		164.9		169.3
6		169.0
1-CH3	2.80, s	24.3	3.05, s	24.1	2.97, s	26.1
3-OCH3	3.95, s	59.3	4.13, s	59.7	4.01, s	58.7
6-OCH3	3.90, s	54.5
1′
2′		171.0		169.6		171.5
3′		156.7		156.7		156.2
4′		100.4		99.6		100.8
5′		165.4		164.9		165.5
1′-CH3	3.03, s	24.1	3.05, s	24.1	3.02, s	24.1
3′-OCH3	4.11, s	59.3	4.13, s	59.7	4.10, s	59.2

Fig. 4. X-Ray Crystalline Structure and Key Correlations in HMBC Measurement of Leiocarpanine A (1)

Next, we tried to determine the generation process of 1. As shown in Fig. 5, we hypothesized that the generation process of asymmetric pyrrole dimer 1 was different from that of the symmetric pyrrole dimer, isochrysohermidin, displayed in Fig. 1. We predicted that the condensation reaction of cyanohermidin and radical receptor 3-methoxy-1-methyl-1H-pyrrole-2,5-dione (5) proceeded by radical addition to give 6. Certainly, there are several reports about radical addition with pyrrole-2,5-dione derivatives as a radical acceptor.^17–19) For example, Hsu and Sundén have shown about light-induced α-aminoalkyl radical addition to maleimides.¹⁹⁾ After the radical addition, the oxidation and benzylic-acid-type rearrangement of 6 was estimated to yield leiocarpanine A (1).

Fig. 5. Plausible Generation Process of Leiocarpanine A (1)

Furthermore, we attempted to synthesize asymmetric pyrrole dimer 1. In the synthesis of 1 using the methods shown in Fig. 2, many reaction steps and organic reagents were needed. Therefore, we tried to synthesize 1 by mimicking the generation process shown in Fig. 5. As starting material, we used 4-methoxy-1-methylpyridine-2,6-dione (8), which was easily obtained from 1,3-dimethyl acetonedicarboxylate (Fig. 6). Compound 8 was oxidized by potassium peroxodisulfate (K₂S₂O₈) in aqueous NaOH solution and the mixture was refluxed with aqueous H₂SO₄ solution to obtain a mixture of 4-methoxy-1-methylpyridine-2,3,6-(1H)-trione (9) and 3-methoxy-1-methyl-1H-pyrrole-2,5-dione (5). We considered that compound 5 was derived from intermediate 9 via 10 through hydroxide-ion-promoted benzylic-acid-type rearrangement and decarboxylation. The generation of compound 5 in the reaction mixture was confirmed by NMR measurement. Compounds 5 and 9 were used as a mixture in the next step without purification. The mixture was treated with sodium hydrosulfite (Na₂S₂O₄) aqueous solution and CHCl₃ (1 : 1 mixture, v/v) and stirred vigorously by bubbling with N₂ to produce hermidin from 9.²⁰⁾ Subsequently, the CHCl₃ layer of the reaction solution was recovered by separation. The yellow CHCl₃ layer was washed with small amount of water to remove slightly remaining Na₂S₂O₄. Next, water was added to the obtained CHCl₃ solution. The color of the water layer changed from yellow to blue. The blue water layer was recovered by separation as shown in Fig. 7 (Pictures A–C). The operation was repeated until the color of the water layer did not change anymore. This color change may be caused by the conversion of hermidin into cyanohermidin.³⁾ The obtained water layer was stirred or left to stand for 24 h at room temperature. Then, its color changed from blue to brown-red as shown in Fig. 7 (Picture D). It is considered that the color change is due to the disappearance of cyanohermidin by the radical addition to compound 5 or dimerization of cyanohermidine. Finally, the solvent in the brown-red solution was evaporated and the crude product was treated with triethylamine (Et₃N) in MeOH at room temperature to yield a mixture of 1 and isochrysohermidin.²¹⁾ Each compound was purified by normal- and reversed-phase column chromatography. Compound 1 was expected to obtain from 5 and cyanoherimidin via intermediate 7 because the positive and negative molecular ion peaks of 7 were detected in LC-MS analysis.

Fig. 6. Concise Synthesis of Leiocarpanine A (1) from 8 by Only Liquid Separation and Plausible Synthetic Process of 1

Fig. 7. Relationship between Color Change of Mixed Solution and Structural Changes of Compounds in Synthesis of Leiocarpanine A (1)

Consequently, the above reaction proceeded in a mixture without purification by column chromatography except for the final step, and leiocarpanine A (1) as racemic compound was conveniently obtained in 0.3% yield from 8.

Conclusion

Leiocarpanine A (1) was isolated from the aerial parts of M. leiocarpa as a new compound and determined to be a nitrogen-containing asymmetric dimer on the basis of various physicochemical measurements, including NMR and X-ray crystal structure analysis. The new generation process of 1 was estimated and the concise synthesis of 1 could be realized by mimicking the generation process through radical intermediates. In general, multistep reactions and many organic reagents are required for the synthesis of 1. Our new synthesis enabled the concise synthesis of 1 by only liquid separation. This synthesis may be useful for the construction of asymmetric nitrogen-containing dimers. In addition, it is useful to elucidate the generation process of leiocarpanine A (1).

Experimental

General Experimental Procedures

The following instruments were used to obtain physical data: specific rotations, a Horiba SEPA-300 digital polarimeter (l = 5 cm); IR spectra, JASCO FT/IR-4600 Fourier Transform Infrared Spectrometer; ESIMS, Agilent Technologies Quadrupole LC/MS 6130; HRESIMS, SHIMADZU LCMS-IT-TOF; ¹H-NMR spectra, JEOL JNM-LA 500 (500 MHz,) and JNM-ECA 600 (600 MHz,) spectrometers; ¹³C-NMR spectra, JEOL JNM-LA 500 (125 MHz,) and JEOL JNM-ECA600 (150 MHz) spectrometers with tetramethylsilane as an internal standard; HPLC, a Shimadzu SPD-10AVP UV-VIS detector. YMC Triart C18 (250 × 4.6 and 250 × 10 mm i.d.) and YMC Triart PFP (250 × 4.6 and 250 × 10 mm i.d.) were used for analytical and preparative purposes. The following experimental materials were used for chromatography: normal-phase silica gel column chromatography (CC), silica gel BW-200 (Fuji Silysia Chemical, Ltd., Japan, 150–350 mesh); reversed-phase silica gel CC, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh); TLC, precoated TLC plates with silica gel 60F₂₅₄ (Merck, 0.25 mm) (ordinary phase) and silica gel RP-18 F_254S (Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC, precoated TLC plates with silica gel RP-18 WF_254S (Merck, 0.25 mm). Detection of compounds was achieved by UV irradiation and by spraying with 1% Ce(SO₄)₂–10% aqueous H₂SO₄ followed by heating.

Plant Material

The plant of Merucrialis leiocarpa was cultivated and collected in 2018 from the garden of medicinal plants, Kyoto Pharmaceutical University, Kyoto prefecture, Japan (KPU-2018-ML-1).

Extraction and Isolation

Aerial part of Merucrialis leiocarpa (3 kg) were chopped. The mixture was soaked in MeOH for 3 h at 60°C. Evaporation of the filtrate under reduced pressure provided the MeOH extract (640.9 g, 21.4%). The extract was partitioned between EtOAc and H₂O (1 : 1, v/v) to obtained EtOAc fraction (90.1 g, 3.0%) and aqueous phase. The EtOAc-soluble fraction was subjected to normal phase silica gel column chromatography [n-hexane-CHCl₃ (1 : 1→1 : 3, v/v)→CHCl₃→CHCl₃-EtAc (1 : 1, v/v)→EtAc→EtAc-MeOH (1 : 1, v/v)→MeOH ] to give 9 fractions. Fraction 3 (14.579 g) was further separated by reversed phase silica gel column chromatography [MeOH–H₂O (10 : 90→20 : 80→30 : 70→40 : 60→50 : 50→60 : 40→70 : 30→80 : 20→90 : 10)→MeOH] to give 14 fractions. Fraction 3-2 (65.2 mg) was separated by HPLC {mobile phase: CH₃CN-H₂O–CH₃COOH (20 : 79.7 : 0.3, v/v) [YMC-Triart PFP (250 × 10 mm i.d.)]} to give 3-methoxy-1-methylmaleimide (5, 5.8 mg, 0.00019%). Fraction 3-3 (133.6 mg) was separated by HPLC {mobile phase: CH₃CN–H₂O–CH₃COOH (12 : 87.7 : 0.3, v/v) [YMC-Triart PFP (250 × 10 mm i.d.)]} to give plicatanin B (2, 21.5 mg, 0.00058%). Fraction 5 (11.338 g) was separated by reversed phase silica gel column chromatography [MeOH–H₂O (10 : 90→20 : 80→40 : 60→60 : 40→70 : 30→80 : 20→90 : 10)→MeOH] to give 15 fractions. Fraction 5-2 (174.9 mg) was separated by HPLC {mobile phase: MeOH–H₂O–CH₃COOH (12 : 87.7 : 0.3, v/v) [YMC-Triart PFP (250 × 10 mm i.d.)]} to give 5-hydroxy-4-methoxy-5-(methoxycarbonyl)-1-methyl-3-pyrrolin-2-one (4, 12.4 mg, 0.00041%). Fraction 5-3 (442.7 mg) was separated by HPLC {mobile phase: MeOH–H₂O–CH₃COOH (23 : 76.7 : 0.3, v/v) [YMC-Triart C18 (250 × 10 mm i.d.)]} to give speranberculatine A (3, 6.0 mg, 0.00020%), leiocarpanine A (1, 148.4 mg, 0.0049%).

Leiocarpanine A (1)

Yellow crystal [Obtained as racemic compound]; mp 115.1–116.3°C; IR (attenuated total reflectance (ATR)) v_max 3403, 2954, 1756, 1694 cm⁻¹; For ¹H-NMR (CDCl₃, 600 MHz) and ¹³C-NMR (CDCl₃, 150 MHz) spectroscopic data, see Table 1 (The axially chiral was not confirmed in the NMR data); High resolution ESI-MS: m/z 364.0796 [M + Na]⁺ (Calcd for C₁₄H₁₆N₂O₈: 364.0799); ESI-MS: m/z 341.1 [M + H]⁺, 363.1 [M + Na]⁺, 703.2 [2M + Na]⁺.

Plicatanin B (2)

Yellow oil; IR (ATR) v_max 2954, 1777, 1705 cm⁻¹; For ¹H-NMR (CDCl₃, 600 MHz) and ¹³C-NMR (CDCl₃, 150 MHz) spectroscopic data, see Table 1; ESI-MS: m/z 281.1 [M + H]⁺, 303.1 [M + Na]⁺, 583.2 [2M + Na]⁺.

Speranberculatine A (3)

Yellow oil [obtained as racemic compound]; IR (ATR) v_max 3287, 2953, 1698 cm⁻¹; For ¹H-NMR (CDCl₃, 600 MHz) and ¹³C-NMR (CDCl₃, 150 MHz) spectroscopic data, see Table 1; ESI-MS m/z 283.1 [M + H]⁺, 305.1 [M + Na]⁺, 587.1 [2M + Na]⁺.

5-Hydroxy-4-methoxy-5-(methoxycarbonyl)-1-methyl-3-pyrrolin-2-one (4)

Colorless crystal [obtained as racemic compound]; IR (ATR) v_max 3246, 2954, 1748, 1682 cm⁻¹; ¹H-NMR (CDCl₃, 600 MHz) δ: 2.75 (s, 3H), 3.84 (s, 3H), 3.87 (s, 3H), 5.11 (s, 1H); ¹³C-NMR (CDCl₃, 150 MHz) δ: 23.6, 54.4, 58.8, 87.1, 94.2, 169.6, 170.6, 171.7; ESI-MS m/z 202.1 [M + H]⁺, 224.1 [M + Na]⁺, 425.1 [2M + Na]⁺.

3-Methoxy-1-methylmaleimide (5)

Yellow crystal; IR (ATR) v_max 2949, 1714 cm⁻¹; ¹H-NMR (CDCl₃, 600 MHz) δ: 3.01 (s, 3H), 3.94 (s, 3H), 5.42 (s, 1H); ¹³C-NMR (CDCl₃, 150 MHz) δ: 23.5, 58.9, 96.2, 161.1, 165.7, 170.3; ESI-MS m/z 142.1 [M + H]⁺.

Preparation of Reaction Starting Material, 4-Methoxy-1-methylpyridine-2,6-dione (8)

To a solution of 1,3-dimethyl acetonedicarboxylate (47 mL) and trimethyl orthoformate (48 mL) in MeOH (50 mL) was added concentrated sulphuric acid (H₂SO₄, 1.6 mL). The solution was left for 12 h in the refrigerator (4°C), then heated at 65°C for 6 h. Next, the solvent was removed and the cooled solution was diluted with ether (l.0 L). The solution was washed with aqueous saturated NaHCO₃ (3 × 500 mL), dried over Na₂SO₄, and removed the organic solvent to give the crude dimethyl 2-methoxypropene-1,3-dicarboxylate. To a solution of the crude dimethyl 2-methoxypropene-1,3-dicarboxylate (53.0 g) was added an aqueous solution of methylamine (30 mL, 40% (w/v)) at 0°C and the solution was stirred for 2 h. The solution was left for 12 h in the refrigerator (4°C). Then. the color of the solution turned from blue to red brownish. Next, the solvent including methylamine was removed and toluene (100 mL) was added and removed again. The obtained product was refluxed in sodium methoxylate solution (50 mL, 28% (w/w)) and MeOH (210 mL) at 65°C for 2 h. The solvent was removed and water (500 mL) was added. The aqueous solution was washed with diethyl ether (3 × 200 mL). Then, acetic acid (38 mL) was added to the aqueous solution. Finally, the aqueous solution was extracted with CHCl₃ (3 × 500 mL). The CHCl₃ extract was dried over Na₂SO₄, filtered and the solvent was removed to give crude 4-methoxy-1-methylpyridine-2,6(1H,3H)-dione (33.78 g). The crude compound was purified by medium pressure liquid chromatography [CHCl₃] to obtain 4-methoxy-1-methylpyridine-2,6(1H,3H)-dione (8, 27.82 g) as white powder.⁶⁾

4-Methoxy-1-methylpyridine-2,6-dione (8)

Colorless crystal; ¹H-NMR (CDCl₃, 600 MHz) δ: 3.21 (s, 3H), 3.44 (s, 2H), 3.77 (s, 3H), 5.44 (s, 1H); ¹³C-NMR (CDCl₃, 150 MHz) δ: 26.1, 36.5, 56.2, 93.7, 166.7, 167.2, 168.3; ESI-MS m/z 178.2 [M + Na]⁺.

Synthesis of Leiocarpanine A (1)

To an ice cold water (640 mL) containing NaOH (35.7 g) was added 4-methoxy-1-methylpyridine-2,6(1H,3H)-dione (8, 27.82 g). Potassium peroxodisulfate (K₂S₂O₈, 59.3 g) was added to the solution and stirried for 15 min. Furthermore, the solution kept for 48 h in the refrigerator (4°C). H₂SO₄ (96% (w/w), 53 mL) was added dropwise and the solution was refluxed for 1 h. Next, water (300 mL) was added the solution and extracted with CHCl₃ (3 × 500 mL). The CHCl₃ fraction was dried over Na₂SO₄ and the solvent was removed to give a mixture (15.37 g) including compounds 9 and 5. The mixture (3.24 g) was treated with sodium hydrosulfite (Na₂S₂O₄, 50.0 g) aqueous solution (250 mL) and CHCl₃ (250 mL) [1 : 1 mixture, v/v] and stirred vigorously by bubbling with N₂ for 20 min. Subsequently, the CHCl₃ layer of the reaction solution was recovered by separation. The yellow CHCl₃ layer was washed with small amount of water to remove slightly remaining Na₂S₂O₄. Next, water (100 mL) was added to the obtained CHCl₃ solution. The color of the water layer changed from yellow to blue. The blue water layer was recovered by separation. The operation was repeated three times until the color of the water layer did not change anymore. The obtained water layer was stirred or left to stand for 24 h at room temperature. Then, its color changed from blue to brown-red. The solvent in the brown-red solution was evaporated and the deep red crude product (530 mg) containing compound 7 and chrysohermidin was obtained. CHCl₃ (200 mL) was added to the solid and was left for 30 min. The reddish CHCl₃ solution was filtrated and the solvent was removed to yield deep reddish solid (300.0 mg) containing chrysohermidin and comopound 7. Next, To the deep reddish solid (234.5 mg) in MeOH (35 mL) was added triethylamine (0.5 mL). The solution was stirred for 3 h and the solvent was removed. The residue was dissolved in aqueous saturated NH₄Cl. The aqueous solution was extracted with CHCl₃ (3 × 100 mL). The solvent was the CHCl₃ solution was removed and was subjected to medium pressure liquid chromatography [the mobile phase: CH₃CN (mobile phase A) and H₂O (mobile phase B); Starting with 5% (A) for 10 min, a liner gradient was followed to 25% (A) at 110 min] to yield the mixture (28.4 mg) of isochrysohermidin and leiocarpanine A (1). The crude compound (28.4 mg) was further separated by medium pressure liquid chromatography [The mobile phase: CHCl₃ (mobile phase A) and CH₃CN (mobile phase B); Starting with 100% (A), a liner gradient was followed to 100% (B) at 120 min, continuing for 10 min] to yield isochrysohermidin (7.0 mg, 0.3%) and leiocarpanine A (1, 5.9 mg, 0.3%).

Isochrysohermidin

White powder; IR (ATR) v_max 3403, 2956, 1756, 1681 cm⁻¹; ¹H-NMR (CDCl₃, 600 MHz) δ: 2.85 (s, 6H), 3.83 (s, 6H), 3.98 (s, 6H), 6.84 (s, 2H); ¹³C-NMR (CDCl₃, 150 MHz) δ: 24.8, 53.6, 59.0, 87.7, 95.6, 167.2, 169.8, 173.2; ESI-MS m/z 423.1 [M + Na]⁺, 439.1 [M + K]⁺.

LC-MS Analysis of Intermediate, Chrysohermidin and Compound (7)

We performed LC-MS analysis by using an LCMS-8040 tandem quadrupole mass spectrometer and a Nexera UHPLC system. The instruments were controlled by LabSolutions LCMS software. The Nexera UHPLC system used in the analysis was composed of a system controller (CBM-20 A), two pumps (LC-20 A), an autosampler (SIL-20AC), a column heater (CTO-20AC) and a degasser (DGU-20A3R). The mobile phase consisted of CH₃CN [mobile phase A (Wako Pure Chemical Corporation, LC-MS grade)] and H₂O/HCOOH 0.2 : 99.8 [mobile phase B (Wako Pure Chemical Corporation, LC-MS grade)]. Starting with 0% (B), a liner gradient was followed to 10% (A) at 10 min, then increasing to 23% (A) at 60 min, further increasing to 100% (A) at 65 min.²²⁾ Flow rate was set to 1.0 mL/min. Column temperature was 25°C. We used a YMC-Triart PFP (250 × 4.6 mm i.d.) column to separate the sample. The sample was dissolved in CH₃CN to make a 10.0 mg/mL. The injection volume was 10 µL. UV detection was conducted at 254 nm. The LCMS-8040 tandem quadrupole mass spectrometer was operated in the positive and negative mode with an ESI source. The operating parameters were optimized as follows: nebulizer gas flow (3 L/min), drying gas flow (15 L/min), desolvation line (DL) temperature (250°C) and heat block (HB) temperature (400°C). Their positive and negative molecular ion peaks of chrysohermidine and intermediate 7 were detected in 38.79 min.

Acknowledgments

We thank Ms. Junko Tsukioka (assistant professor, Kyoto Pharmaceutical University) for supplying materials. This research was supported in part by a Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan-Supported Program for the Strategic Research Foundation at Private Universities 2015–2019. This work was supported by JSPS KAKENHI Grant Number 17K08354 (S.N.).

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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