A New Epi-Lycorine from Crinum Macowanii Baker (Amaryllidaceae)

Sciences, TechnologiesSciences, Technologies et Développement, & Développement, Volume 15, ISSN pp31-40, 1029 -Septembre 2225 2014 http://www.univ-douala.com/sdt/

A new epi -lycorine from Crinum macowanii Baker (Amaryllidaceae)

Kouam a, Marie Fomani a, Flavien Aristide Alfred Toze a, Jean Duplex Wansi a, Alain François Kamdem Waffo a,b,d* , Neil Anthony Koorbanalli b, Dulcie Mulholland b,d , Neil Crouch b,C , Augustin Ephrem Nkengfack e

a Department of Chemistry, Faculty of Science, University of Douala P.O. Box 24157 Douala, Cameroon b School of Chemistry, University of KwaZulu-Natal, Durban 4041, South Africa c Ethnobotany Unit, National Botanical Institute, P.O. Box 52099, Berea Road 4007, Durban, South Africa d School of Biomedical and Molecular Sciences, University of Surrey, Guildford,GU2 7XH, London, United Kingdom e Department of Organic Chemistry, Faculty of Science, University of Yaoundé I P.O. Box 812 Yaoundé, Cameroon Corresponding author. Tel.:+237 97 35 27 38. E-mail address: [email protected] (Alain F. Kamdem Waffo) Received : April 2014 Revised: May 2014 Accepted: May 2014 Available online: June 2014 Abstract

An isomer of lycorine, the (11b, 11c) epi-lycorine has been isolated from the bulbs of Crinum macowanii, along with the known alklaloids, lycorine 2, 1-O-acetyllycorine 3 and crinine 4 and the acetophenone derivatives: 4’ -hydroxy-3’ -methoxyacetophenone 5 and 3’ -hydroxy-4’ - methoxyacetophenone 6. Lycorine is already reported in the literature while (11b, 11c) epi-lycorine 1 is not yet reported. 4-hydroxy-3- methoxyacetophenone 5 and 3-hydroxy-4-methoxyacetophenone 6 are isolated for the first time in the Amaryllidaceae family.

Résumé Des bulbes de Crinum macowanii ont été isolés un isomère de la lycorine, la (11b, 11c) epi-lycorine 1 avec des alcaloides connus : lycorine 2, 1-O- acétyllycorine 3 et crinine 4, et deux dervivés de l’acétophénone : la 4’ -hydroxy-3’ -méthoxyacétophénone 5 et la 3’ -hydroxy-4’ - méthoxyacétophénone 6. La lycorine 2 a déjà été decrite dans la litterature tandis que la (11b, 11c) epi-lycorine 1 n’a pas encore été publiée. La 4 - hydroxy-3-méthoxyacétophénone 5 et la 3-hydroxy-4-méthoxyacétophénone 6 sont isolées pour la première fois dans la famille des Amaryllidaceae.

1. Introduction aquatic purpose. In addition, huge numbers of them are traded for traditional medicines. 4The genus Crinum represents an important sector in Amaryllidaceae family (90 genera and about 1310 species) Crinum macowanii , a member of this family of plants genus (Benson, 1970) with wide geographical distribution has found extensive use in traditional medicine for the throughout the tropics, subtropics and warm temperate treatment of various illnesses such as oedema, ‘heart regions of the world (Mabberly 1990). This genus is related to disease’, rheumatic fever, cancer and skin diseases (Duncan a group of mostly southern African endemic genera, et al., 1999; Elgorashi et al. 2001; Elgorashi et al. 2002; constituting the tribe Amaryllideae (Meerow et al., 1998). Elgorashi et al., 2003). Botanically, Crinum are perennial herbaceous plants with Phytochemical investigations have resulted in isolation of giant fleshy bulbs larger in stature than most other species of several diverse classes of compounds and have been Amaryllidaceae. They can grow from 1-5 feet in height focused predominantly on alkaloids. Phenolics prevail among depending on the species and produce a neck or a pseudo the non-alkaloidal constituents identified (Tram et al., 2002) stem made up of the sheathing old leaves. Flowers usually A recent investigation of Crinum macowanii by Nair et al. appear in May, June or August (Hutchinson, 1964). (2000) resulted in the isolation of eleven alkaloids, lycorine, Worldwide, Crinum have a great economic value as cherylline, crinine, krepowine, powelline, buphanidrine, ornamentals due to their showy flowers and it's also used for crinamidine, undulatine, 1-epideacetylbowdensine, 4a- dehydroxycrinamibine and macowine. Lycorine and crinine *Corresponding author: Alain F. Kamdem Waffo, Tel.:+237 97 are the alkaloids commonly found in the Amaryllidaceae 35 27 38. E-mail address: [email protected]. family.

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2. Results and discussion crinine 4 (Chetty, 2001), 4-hydroxy-3-methoxyacetophenone 5, 3-hydroxy-4-methoxyacetophenone 6 and the The chopped bulbs of Crinum macowanii were extracted methanol/chloroform extract yielded a lycorine isomer, which using ethanol and the conventional acid-base wash normally we named (11b, 11c) epi -lycorine 1 (Fig. 1). (11b, 11c) epi - employed for the Amaryllidaceae bulbs. The basic extract lycorine 1 is readily soluble in methanol unlike lycorine 2, yielded lycorine 2 (Likhitwitayawuid et al., 1993, Nivan 2004, which is partially soluble in methanol. No compounds of Refaat et al , 2012), 1-O-acetyllycorine 3 (Chetty, 2001), interest were found in the acidic extract.

OH OH OH H H 1 2 AcO HO 3 HO H H H H 11 11b H O 10 3a O O 11a H 11c 4 N N N O O 9 7a O 8 7 5 2 1 3

O OH O

O H OH N OCH O 3 OCH OH 3 4 5 6

Figure 1: Structures of isolated compounds 1-6

Coumpound 1 was assigned a molecular formula of H-4 resonance was also seen to be coupled with both the H-5 C16 H17 NO 4 on the basis of HREIMS data (molecular ion at proton resonances. The H-11b proton resonance showed m/z 287.1449). The mass spectrum of (11b, 11c) epi-lycorine HMBC correlations to C-3a, C-7a, C-11a, C-11, C-1 and C- 1 is very similar to that of lycorine 2 with twin peaks at m/z 11c and the H-7α proton resonance showed HMBC 227 and 226 resulting from the migration of the double bond correlations to C-7a, C-11a, C-8, and C-11c. These between positions 11c and 3a, followed by a retro Diels Alder correlations supported the assignments of H-8 and H-11. fragmentation and then loss of a proton at C-7 (Kinstle et al, Furthermore, H-11 showed an NOE interaction with H-1. 1966). The proton resonances of H-3, H-4, H-5, H-7α, H -8, H-11b The 1H NMR spectrum of (11b, 11c) epi -lycorine 1 shows two and H-11c are all deshielded, compared to their aromatic proton singlets at δ 6.85 (H -8) and δ 6.99 (H -11), a corresponding proton resonances in lycorine. Since there is methylenedioxy doublet proton resonance at δ 5.99 ( J = 2.0 very little deviation in the 13 C resonances, this indicates that Hz), a broad olefinic singlet resonance at δ 5.79 (H -3), a the stereochemistry at H-11b and H-11c is reversed. In broad singlet at δ 4.55 (H -1), a doublet at δ 4.50 ( J= 14.0 Hz) compound 1 there is a large trans coupling constant between characteristic of the H-7α proton resonance, overlapping H11b and H1 ( J: 11.72 Hz), while in lycorine it is a small cis resonances for H-2 (δ 4.20) and H -7β at δ 4.23, two doublets coupling constant ( J: 5.30Hz). H-11b is now alpha and H-11c at δ 2.98 and δ 3.91 ( J = 11.5 Hz) attributed to H-11b and H- is now beta. In addition, since the irradiation of the H-1 proton 11c, two multiplets at δ 3.52 and 3.74 (H -5endo and H-5exo) resonance showed a positive NOE effect for the H-11b and overlapping resonances at δ 2.85 for the H -4endo and H- resonance and the H-11 resonance only and not for the H-2 4exo protons. COSY correlation could be seen for H-11c with resonance, this indicated that both H-11b and H-1 must be H-11b, H-11b with H-1, H-1 with H-2 and H-2 with H-3. The alpha and that H-2 must be beta. Therefore the

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stereochemistry of H-1 and H-2 were also reversed. indicating that H-11c was beta. The optical rotation of (11b, Irradiation of the H-11b resonance only showed a positive 11c) epi -lycorine 1 was +67°, compared to -68° in lycorine 2 effect for the H-1 resonance and not for the H-11c resonance, reported in the literature.

Table 1 : Comparison of the 13 C and 1H NMR data of lycorine 2 and epi -lycorine 1

Pos. Lycorine 1H epi -lycorine 1H Lycorine 13 C epi -lycorine 13 C HMBC Correlations

1 4.50 ( br s ) 4.55 ( br s ) 73.10 70.00 C-2, C-11b,

C-11a, C-11c and C-3

2 4.20 ( br s ) 4.23 ( br s ) 71.90 71.80 C-1, C-11b, C-3

and C-3a

3 5.58 (br s) 5.79 ( br s ) 119.20 123.30

3a 143.60 137.40

4 2.65 ( m ) 2.85 ( m ) 29.30 30.20

5 2.48 (dd , 17.58, 8.72 3.52 ( m ) 54.70 55.20

Hz)

3.38 ( m) 3.74 ( m )

7 α 3.60 (d, 14.28 Hz) 4.50 ( d, 14.00 Hz) 57.70 54.40 C-7a, C-11a, C-8,

and C-11c

7β 4.16 (d, 14.28 Hz) 4.23 *

7a 130.40 130.40

8 6.67 ( s ) 6.85 ( s ) 108.20 108.70

9 148.20 148.20

10 147.70 149.80

11 6.91 6.99 ( s ) 106.00 106.40

11a 129.70 125.40

11b 2.70 ( dd , 5.30 and 2.98 ( dd , 11.53 and 41.30 38.40 C-3a, C-7a, C-11a,

11.36 Hz) 11.72 Hz) C-11, C-1 and C-11c

11c 2.93 ( d, 11.36 Hz) 3.91 ( d, 11.53 Hz) 62.50 62.30 C-1, C-11b, C-11a,

C-3a, C-4 and C-3

OCH 2O 5.94 ( s) 5.99 ( s) 102.40 102.90

* resonance overlaps with H-2.

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3. Experimental methoxyacetophenone 5 and 3’ -hydroxy-4’ - 3.1. General Experimental Procedures methoxyacetophenone 6 were identified from their 1H NMR spectra and (11b, 11c) ep i-lycorine 1 was identified using NMR spectra were recorded in CDCl 3 or CD 3OD on a Varian NMR, mass spectroscopy, optical rotation, UV, IR and 400 MHz spectrometer. EIMS data were recorded on an physical characteristics. Agilent MS 5973 instrument connected to a GC 6890. MeOH Ultraviolet absorption spectra were recorded on a Varian Epi -lycorine 1, 15 mg, white crystalline, mp: 272 °C, ν max DMS 300 UV visible spectrophotometer and Infrared spectra KBr -1 were recorded using a Nicolet Impact 400D Fourier nm (log ε): 210 (4.22), 237 (3.43), 292 (3.43). IR ν max cm : Transform Infrared spectrometer. 3306, 3172 (O-H stretching); 1502 (aromatic C=C stretching); 1248 (C-N stretching); 1021 (C-O stretching); 929 (C-O-C 22 stretching). [α] D + 67° (c = 0.012). EIMS 70 eV, m/z (rel. 3.2. Plant Material and Extraction int.): 287 (21), 268 (18), 250 (12), 227 (63), 226 (100). 1H and Bulbs of Crinum macowanii (2.5 kg wet mass) were collected 13 C NMR data are tabulated in table 1. in Kloof, KwaZulu-Natal and a voucher specimen retained at

the National Botanical Institute, KwaZulu-Natal, South Africa (N.Crouch 950, NH). The material was dried, chopped into Acknowledgements small pieces and extracted with 95% ethanol on a labcon We wish to thank the University of KwaZulu-Natal and the shaker. The ethanol was removed under reduced pressure, National Research Foundation (NRF) for financial aid. A.F. 100 mL of water was added to it and the extract was acidified Kamdem Waffo gratefully acknowledges a postdoctoral to pH 4 using 4M HCl. The resultant extract was extracted fellowship from the NRF. We thank Mr. Delip Jagjivan for the three times with 200 mL portions of chloroform. The running of NMR spectra, Mr. Bret Parel and Mr. Tommy van chloroform was then removed yielding the acidic extract (42.5 der Merwe for GC-Ms and HRMS, respectively. g). The aqueous phase was then basified to pH 9 using 4M ammonium hydroxide and extracted with chloroform as in the acidic extract to yield 74.6 g of basic extract. The aqueous References phase was further extracted three times with a 1:1 mixture of chloroform and methanol to yield 126.8 g of Benson, L. 1970. Plant Classification. Oxford and I.B.H chloroform/methanol extract. publishing Co., New Delhi, Bombay, 793-797. Chetty, J. 2001. Extractives from the Amaryllidaceae: Brunsvigia radulosa and Cyrtanthus brevifloru s.

3.3. Chromatography Unpublished Ph.D. Dissertation, University of Natal, All three extracts were chromatographed using silica gel Durban, South Africa 33-37. (Merck 9385) as the stationery phase and varying proportions Duncan, A.C., Jäger, A.K., Van Staden, J. 1999. Screening of of hexane, dichloromethane, ethyl acetate and methanol. The Zulu medicinal plants for an-giotensin converting acidic extract yielded only fatty acids, which were not enzyme (ACE) inhibitors. Journal of characterized further. Elution of the basic extract with 20% Ethnopharmacology , 68(1) : 63-70. dichloromethane in hexane yielded 1-O-acetyllycorine 3 (10 Elgorashi, E.E., Drewes, S.E., Van Staden, J. 2001. Alkaloids mg), lycorine 2 (70 mg), eluted with 60% dichloromethane in from Crinum macowanii . Biochemical Systematics hexane, crinine 4 (6 mg), eluted with 100% dichloromethane and Ecology 29(7) : 749-750. and 4’ -hydroxy-3’ -methoxyacetophenone 5 (4 mg) and 3’ - Elgorashi, E.E., Drewes, S.E., Van Staden, J. 2002. Organ- hydroxy-4’ -methoxyacetophenone 6 (7 mg), which were elutd to-organ and seasonal variation in alkaloids from with 1% methanol in dichloromethane. Elution of the Crinum macowanii . Fitoterapia 73(6) : 490 - 495. chloroform/methanol extract with 2% methanol in dichloromethane yielded the (11b, 11c) epi -lycorine 1 (15 Elgorashi, E.E., Drewes, S.E., Morris, C., Van Staden, J. mg). Lycorine 2, 1-O-acetyllycorine 3 and crinine 4 were 2003 Variation among three Crinum pecies in alkaloid identified by a comparison of their chromatographic and content. Biochemical Systematics and Ecology, 31(6) spectroscopic properties (TLC, IR, MS, 1H and 13 C NMR) with : 601-615. those of authentic samples obtained from other plant sources Hutchinson, J. 1964. The Genera of flowering plants. The in this laboratory. The acetophenones, 4’ -hydroxy-3’ - claredon press, Oxford, Vol I, 43. 36 Kouam et al., Sciences, Technologies et Développement (Septembre 2014), Volume 16, 33-37 Sciences, Technologies & Développement, ISSN 1029 - 2225

Kinstle, T.H., Wildman, W.C. and Brown, C.L. 1966. Mass Alkaloids from Crinum macowanii. Phytochemistry 54 : spectra of Amaryllidaceae alkaloids. The structure of 945-950. narcissidine. Tetrahedron Letters 7: 4659-4666. Nivan, M. 2004. The chemical investigation of the Likhitwitayawuid, K., Angerhofer, C.K., Chai, H., Pezzuto, Amaryllidaceae an Hyacinthaceae. Unpublished Ph. J.M., Cordell, G.A. 1993. Cytotoxic and antimalarial D thesis, University of KwaZulu-Natal, Durban South alkaloids from the bulbs of Crinum amabile . Journal of Africa Natural Products 56 : 1331-1338. Refaat J., Kamel S. M., Ramadan M. A. and Ali A. A. 2012. Mabberly, D.J. 1990. The plant book. Cambridge University Crinum, an endless source of bioactive principles: A Press. review. Part 1. Crinum alkaloids: lycorine-type Meerow, A.W. and Snijman, D.A. 1998. Families and Genera alkaloids. International Journal of Pharmaceutical of Vascular Plants. Springer-Verlag, Berlin, Vol. III : Sciences and Research 3(7) : 1883-1890 83 –110. Tram, N.T., Titorenkova, T., Bankova, V., Handjieva, N., Nair, J.J., Machocho, A.K., Campbell, W.E., Brun, R., Popov, S.S. 2002. Crinum L. Amaryllidaceae. Viladomat, F., Codina, C. and Bastida, J. 2000. Fitoterapia, 73(3) : 183 –208.

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