New Lycopodium Alkaloids from Huperzia serrata

• Abstract
• Materials and Methods
• References

Abstract

Three new lycopodium alkaloids, 11α-hydroxyphlegmariurine B (2), 7α-hydroxyphlegmariurine B (3) and 7α,11α-dihydroxyphlegmariurine B (4) along with a known compound, phlegmariurine B (1), were isolated from the herb Huperzia serrata. The structures of the above compounds were elucidated based on spectroscopic studies.

Huperzia serrata (Thunb.) Trev. is one of the most commonly used traditional Chinese herbal medicines for the treatment of contusion, strain, swelling and schirophrema [1]. Chemical and pharmacological studies showed that huperzine A, a lycopodium alkaloid isolated from this plant, was a potent acetylcholinesterase inhibitor [2], [3]. As part of our continuing interest in this species [4], [5], we have examined the CHCl₃ extract of dry whole plants (10 kg), to obtain a known compound, phlegmariurine B (1), and three new alkaloids, 11α-hydroxyphlegmariurine B (2), 7α-hydroxyphlegmariurine B (3) and 7α,11α-dihydroxyphlegmariurine B (4). In the present paper, we report on the isolation and structural elucidation of these compounds.[]

Compound 1 showed a molecular ion (EI-MS) at m/z 261 corresponding to the molecular formula C₁₆H₂₃NO_2. Comparison of EIMS, IR, ¹³C- and ¹H-NMR data firmly identified 1 as phlegmariurine B [6], [7]. The complete assignments (Tables [1] and 2) of ¹³C- and ¹H-NMR signals were established by using HMQC, DEPT and ¹H-¹H COSY spectra.

The molecular formula C₁₆H₂₃NO₃ for compound 2 was established by HREIMS. The IR spectrum showed the presence of a hydroxy group (ν _max 3455.9), a lactam group (ν _max 1685.5 ) and an α,β-unsaturated ketone group (ν _max 1619.1 and 1430.9). Comparison of HMQC spectrum with that of compound 1, indicated the most important difference was the absence of 11-CH₂ signals (δ_C: 28.8, δ_H: 2.64 and 2.73) and the presence of a methine group signal (δ_C: 67.8, δ_H: 4.58) in 2, which suggested 2 to be 11-hydroxyphlegmariurine B. The HMBC spectra (Fig. 1) validated the position of the hydroxy group and the assignments of C-1 (δ 52.0), C-9 (δ 49.3), C-12 (δ 175.0) and C-13 (δ 176.7). The relative stereostructure of 2 was defined by a NOESY experiment (Fig. 2). Significant NOEs were observed between H-11 and H-8exo, H-10β, H-14exo and H-7, and between H-15 and H-6β, which determined the relative positions of H-7, H-11 and H-15 as α, β and endo, respectively. Thus, the relative configuration was established as shown by structure 2.

Compound 3 was obtained as colorless prisms. The IR bands and EIMS fragments resembled those of 2, suggesting 3 to be also a hydroxylated phlegmariurine B. The ¹³C-NMR spectra showed a quaternary carbon at δ 79.3 rather than a methine at δ 41.1 (C-7 of 1), which indicated the hydroxy to be allocated to C-7. Furthermore, in contrast to those of 1, the increases of δ_C-6 and δ_C-8 and decreases of δ_C-11 and δ_C-5 of 3 were consistent with the induction effect and γ-gauche effect from 7-OH, respectively. In the NOESY spectra, obvious correlations were found between H-15 and H-6β, which elucidated the relative positions of H-15 and 7-OH were endo and α, respectively.

For compound 4, the IR bands at 3505.9 and 3399.9 cm^-1 revealed the presence of two hydroxy groups. Its ¹³C- and ¹H-NMR spectra data (Tab. [1] and 2) were similar to those of the corresponding segments, such as 10-CH₂ and 11-CH of 2, 6-CH₂, 7-C and 8-CH₂ of 3, and the others of 1 - 3, which suggested 4 to be 7,11-dihydroxyphlegmariurine B. According to the NMR theory of γ-gauche effect, its relative positions of 7-OH, 11-OH and 15-H should be same with that of 2 and 3, respectively, Therefore, compound 4 was assigned as 7α,11α-dihydroxyphlegmariurine B.

Materials and Methods

All melting points were determined on a Fisher-Johns melting point apparatus and were uncorrected. CD spectra were taken on a Jasco J-715 spectropolarimeter. IR spectra were recorded on a Nicolet Magna 750 FTIR (KBr) spectrophotometer. EIMS and HREIMS data were obtained with MAT-95 and MAT-711 mass spectrometers. NMR spectra were recorded on a Bruker AM X 400 instrument. Silica gel (200 - 300, 400 mesh) was used for column chromatography and precoated plates of silica gel for TLC. Optical rotations were measured using a P.E.241 MC polarimeter in CHCl₃ or MeOH. Fresh whole plants of Huperzia serrata (Thunb) Trev. were collected in Zhejiang Province, China in August 1997 and identified by Dr. Xiao-Qiang Ma of this Institute. A voucher (No. 97 - 63) was deposited in the Herbarium of this institute.

The total crude alkaloids from 10 kg of Huperzia serrata were obtained as previously described [4], and chromatographed over silica gel (1 kg) with gradient eluants (CHCl₃, 1000 ml; 1 - 4 % methanol in CHCl₃, each 1500 ml) to afford fr.1 - 5. Fr. 3 (2.0 g) was chromatographed on a silicia gel column eluting with EtOAc /acetone (2 : 1, 1200 ml), collected every 100 ml volume, to furnish 3 fr. s.: fr.3.1 - 3.4, fr. 3.5 - 3.7, and fr.3.8 - 3.12. They were concentrated and dissolved with 10 ml acetone, respectively. Fr. 3.5 - 3.7 yielded crude crystals, recrystallized twice with acetone resulting in phlegmariurine B (1, 750 mg); the concentrated mother liquid (235 mg) was subjected to silicia gel (20 g) column chromatography with CHCl₃/CH₃OH (15 : 1, 480 ml), collected with 10 ml tubes and detected using TLC (silica gel HSGF₂₅₄, CHCl₃/CH₃OH 12 : 1, iodine vapor for detection); yielding 3 (12 mg, R_f: 0.43,), 2 (28 mg, R_f: 0.40), and 4 (8 mg, R_f: 0.30).

11α-Hydroxyphlegmariurine B (2): Colorless prisms from Me₂CO-CH₃OH, m. p. 246 - 248 °C. CD (c 5.6 × 10^-4, MeOH): [Θ]₂₀₂ - 1.1 × 10⁴, [Θ]₂₂₃+ 7.8 × 10⁴, [Θ]₂₅₃ - 8.2 × 10⁴. [α]²⁵ _D: -1.2° (MeOH, c 0.67). IR: ν _max (cm^-1) = 3455.9 (OH), 2908.2, 1685.5, 1619.9, 1430.9, 1230.4, 1020.2. EIMS: m/z (rel. int.) = 277 (M⁺, 100), 260 (6), 249 (7), 234 (9), 233 (8), 232 (10), 220 (26), 219 (43), 208 (8), 206 (6), 194 (11), 192 (23), 190 (13), 166 (10), 165 (22), 163 (54), 151 (54), 150 (24). HR-EIMS : C₁₆H₂₃NO₃, found: 277.1658 (M⁺), cald: 277.1678. ¹H-NMR (CD₃OD, 400 MHz): see Table [2]. ¹³C-NMR (CD₃OD, 100 MHz): see Table [1].

7α-Hydroxyphlegmariurine B (3): Colorless prisms from Me₂CO, m. p. 214 - 216 °C. CD (c 9.4 × 10^-4, MeOH): [Θ]₂₀₃ - 5.1 × 10⁴, [Θ]₂₂₄ + 3.6 × 10⁴, [Θ]₂₅₈ - 1.2 × 10⁴. [α]²⁵ _D: -2.2^o (MeOH, c 0.17). IR: ν _max (cm^-1) = 3423.1 (OH), 2923.6, 1697.1, 1616.1, 1483.0, 1197.6, 1035.6. EI-MS: m/z (rel. int.) = 277 (M⁺, 69), 260 (5), 249 (12), 234 (4), 225 (21), 192 (17), 185 (21), 165 (19), 164 (16), 150 (39), 149 (64), 71 (65), 70 (24), 69 (52), 57 (100), 55 (67). HR-EIMS : C₁₆H₂₃NO₃, found: 277.1696 (M⁺), cald: 277.1678. ¹H-NMR (CDCl₃, 400 MHz): see Table [2]. ¹³C-NMR (CDCl₃, 100 MHz): see Table [1].

7α,11α-Dihydroxyphlegmariurine B (4): Colorless prisms from Me₂CO-MeOH, m. p. 265 - 267 °C; CD (c 4.3 × 10^-4, MeOH): [Θ]₂₀₃ - 5.1 × 10⁴, [Θ]₂₂₃ + 5.2 × 10⁴, [Θ]₂₅₂ -3.1 × 10⁴. [α]²⁵ _D: -1.7^o (MeOH, c 0.42). IR: ν _max (cm^-1) = 3505.9 (OH), 3399.9 (OH), 2923.6, 1697.1, 1604.5, 1486.9, 1344.2, 1037.5. EI-MS: m/z (rel. int.) = 293 (M⁺, 23), 276 (10), 275 (42), 260 (5), 249 (12), 234 (4), 225 (21), 192 (17), 185 (21), 165 (19), 164 (16), 150 (39), 149 (64), 71 (63), 70 (18), 69 (53), 57 (100), 55 (57); HR-EIMS: C₁₆H₂₃NO₃, found: 293.1619 (M⁺), cald: 293.1626; ¹H-NMR (CD₃OD, 400 MHz): see Table [2]. ¹³C-NMR (CD₃OD, 100 MHz): see Table [1].

References

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• 2 Liu J -S, Zhu Y -L, Yu C -M, Zhou Y -Z, Han Y -Y, Wu F -W, Qui B -F. The structures of huperzine A and B, two new alkaloids exhibiting marked anticholinesterase activity.  Canadian Journal of Chemistry.. 1986;  64 837-9
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• 4 Zhu D -Y, Jiang S -H, Huang M -F, Lin L -Z, Cordell G A. Huperserratinine from Huperzia serrata .  Phytochemistry. 1994;  36 1069-72
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• 5 Zhu D -Y, Huang M -F, Wang B -D, Kong X -M, Yang Y -Q. The structures of huperzine E and F.  Chinese Journal of Applied & Environmental Biology.. 1996;  2 352-5
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• 6 Tong S -H, Xiang G -Q. Studies on the alkaloids of Phlegmariurus fordii (Baker) Ching.  Acta Botanica Sinica.. 1984;  26 411-5
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• 7 Yuan S -Q, Feng L, Gu G -M. Studies on the alkaloids of Huperzia serrata (II).  Chinese Traditional and Herbal Drugs (Zhongcaoyao). 1994;  25 453-4
  PubMedSearch in Google Scholar

Prof. Da-Yuan Zhu

State Key Laboratory of Drug Research

Shanghai Institute of Materia Medica

Shanghai Institutes for Biological Sciences

Chinese Academy of Sciences

294 Taiyuan Road

Shanghai 200031

P.R.China

Email: dyzhu@mail.shcnc.ac.cn

Fax: +86-21-64370269

References

• 1 Jiangsu New Medical C ollege. The Dictionary of Traditional Chinese Medicine. Shanghai Science and Technology Press 1985: p. 1138
  Search in Google Scholar
• 2 Liu J -S, Zhu Y -L, Yu C -M, Zhou Y -Z, Han Y -Y, Wu F -W, Qui B -F. The structures of huperzine A and B, two new alkaloids exhibiting marked anticholinesterase activity.  Canadian Journal of Chemistry.. 1986;  64 837-9
  CrossrefPubMedSearch in Google Scholar
• 3 Tang X -C, Han Y -F, Chen X -P, Zhu X -D. Effects of huperzine A on learning and retrieval process of discrimination performance in rats.  Acta Pharmacological Sinica.. 1986;  7 501-11
  PubMedSearch in Google Scholar
• 4 Zhu D -Y, Jiang S -H, Huang M -F, Lin L -Z, Cordell G A. Huperserratinine from Huperzia serrata .  Phytochemistry. 1994;  36 1069-72
  CrossrefPubMedSearch in Google Scholar
• 5 Zhu D -Y, Huang M -F, Wang B -D, Kong X -M, Yang Y -Q. The structures of huperzine E and F.  Chinese Journal of Applied & Environmental Biology.. 1996;  2 352-5
  PubMedSearch in Google Scholar
• 6 Tong S -H, Xiang G -Q. Studies on the alkaloids of Phlegmariurus fordii (Baker) Ching.  Acta Botanica Sinica.. 1984;  26 411-5
  PubMedSearch in Google Scholar
• 7 Yuan S -Q, Feng L, Gu G -M. Studies on the alkaloids of Huperzia serrata (II).  Chinese Traditional and Herbal Drugs (Zhongcaoyao). 1994;  25 453-4
  PubMedSearch in Google Scholar

Prof. Da-Yuan Zhu

State Key Laboratory of Drug Research

Shanghai Institute of Materia Medica

Shanghai Institutes for Biological Sciences

Chinese Academy of Sciences

294 Taiyuan Road

Shanghai 200031

P.R.China

Email: dyzhu@mail.shcnc.ac.cn

Fax: +86-21-64370269