Two betaine-type alkaloids from Egyptian Pancratium maritimum

Pbytochemistry, Vol.31,No. 6, pp. 2139-2141,1992 Printedin Great Britain.

0031-9422/92 $5.00+0.00 0 1992PergamonPressLtd

TWO BETAINE-TYPE ALKALOIDS FROM EGYPTIAN PANCRATIUM MARZTIMUM AMINA H. ABOU-DONIA, ABDEL-AZIMABIB, AHMED SEIF EL DIN, ANTONIOEVIDENTE,* MOHSEN GABER and ANTONIO SCOPA? Department of Pharmacognosy, University of Alexandria, Alexandria, Egypt; *Dipartimento di ScienceChimico-Agrarie, Universita di Napoli “Federico II”, 80055 Portici, Italy; TDipartimento di Chimica, Universita della Basilicata, 85100 Potenxa, Italy (Received 24 July 1991)

Key Word In~x-P~~rari~ zefbetaine.

~ri~~~um;

Amaryllidace~; oxyphen~th~dinium

alkaloids; ungeremine;

Abstract-Two 2-oxyphenanthridinium alkaloids have been isolated from Egyptian Pancratium maritimum and identified as ungeremine and zefbetaine using spectroscopic and chemical methods. The identification of zefbetaine was supported by its partial synthesis from pseudolycorine and by comparison with its unnatural isomer, isozefbetaine, which was in turn prepared from stembergine.

INTRODUCTION Earlier investigation on the Egyptian Pancratium murjt~mum L. (Amaryilidaceae), resulted in the isolation of lycorine (!I) tazettine, pancracine, lycorenine, galanthamine, sickenbergine, homolycorine, haemanthidine, hippadine, demethylhomolycorine and trispheridine [l-3]. Recently, haemanthamine, pseudolycorine, 9-Odemethylhomolycorine and 11-hydroxyvittatine were also isolated and identified from Egyptian P. maritimum

c41. Thiswork describes the isolation and the identification of two 2-ox~henan~~d~ium betaine-type aikaloids: ungeremine (I) and zefbetaine (2), also known for their very interesting biological activity (i.e. cytotoxic and antibiotic [S-7] and plant growth regulator [S] activity). For the latter alkaloid (2), a corrected structure is reported, deduced from a more accurate spectroscopic and chemical analysis in contrast to the first isolation from Zephyrunthes~uuu [5]. The natural occurrence of ungeremine (1) was earlier reported only from Ungernia minor [S] (10 years after its synthesis [9]), and from Crinum americanurn [lo], C. asiaticum [ll J and Zephyranthes J%Iua ES). The isolation of these two oxyphen~thridin~um alkaloids from P. ~ritim~ may be of chemotaxonomic

importance because the complete aromatization of the phenanthridine moiety associated with the betaine nature of these two metabolites probably requires a special set of enzymes. RRSULTSAND D~U~ION The

crude

ethanolic

extract

of fresh

bulbs

of

P. maritimum was defatted, acidified, filtered and then

washed with ethyl ether. The residual aqueous solution was alkalized and extracted in succession with chloroform, ethyl acetate and n-butanoi. The residue left from the chloroform extracts, after removal of the precipitated lycorine, produced the crude compound 1 through two

successive column chromatography steps on alumina (eluent chloroform) and silica gel (eluent methanol). The orange needles of the recrystallized alkaloid 1 showed the same melting point [7,9] and the same R, values when co-chromatographed in two different TLC systems as an authentic sample of ungeremine obtained either by chemical [9] or microbiological [7] conversion of lycorine [see. Experimental). Its FAB mass spectrum showed a pseudomolecular ion while the UV spectrum exhibited at m/z 266 [Mm’, absorption maxima (see Experimental) typical for Zoxyph~nanthridinium betaine alkaloids [S, 7, 31. The signal pattern observed in the ‘H (Table 1) and i3C NMR spectra of I was identical to that of ungeremine [7]. A silica gel column eluted with a discontinuous chloroform-methanol gradient, was needed to purify the second beta&type alkaloid 2 present in the butanol extract. Its UV spectrum showed absorption maxima at 409,372 and 262 nm, again suggesting a 2-oxyphenanth~di~um betaine-type [S, 7, 91 structure for this alkaloid. Its ‘HNMR spectrum (Table 1) showed a signal pattern very close to that of compound 1; the only difference was a methoxy group resonating as a singlet at 64.10 instead of the singlet for the methylene (CHz-12) of the dioxole ring in 1. The i3C NMR spectra (Table 2) of 2 was again very similar to that of 1 [73; it differed from that of 1 in that the signal of CH,-12 was absent {in 1 at 6 105.2) and that a signal at 6 56.8 was present and was assigned to a methoxy group. Moreover, the unambiguous assignment of the signal at 6 111.6 and 108.2 to C-8 and C11, respectively, was made from a SFSD spectrum obtained irradiating H-l 1 at 6’7.94.The FAB mass spectrum of 2 gave a p~udomol~ular ion at m/z 268 [MH]‘. These results suggested that 2 had a similar structure to 1; a hydroxy and a methoxy group in 2 were attached to the aromatic A-ring instead of the dioxole ring in 1. In fact, 2 became red when its TLC was sprayed with Fast Red Salt B, a reagent for polyvalent phenols [12] and gave positive results when tested with 1% aqueous FeCI, [13, 141.

2139

A. H. ABOU-DONIA et al.

2140

Table 2. 13C NMR data of zefbetaine (2) [chemical &values (ppm) from TMS] c

C

Rq+ R2

2

Rq= CH3s Rz=H

3

Rq=H,

1 2 3

104.5 d 162.4 s 117.6 d

9* 10*

3a

139.7s 28.4 t 57.1 t

113 1la* 1lb?

142.0 d 126.0 s

11ct Me0

4 5 7 7a*

=-EH2-

1

OH

Table

3. Nuclear

Overhauser

R2=CH3. R3=H

5

rzl=H,

R2+R3=-CH2-

6

R, =Ac,

7.94 (H-l 1) 7.78 (H-8) 4.10 (MeO)

Rz=H. R3=CH3

Table 1. ‘HNMR data of ungeremine (I), zefbetaine (2), and iso-zefbetaine (3) [chemical shifts are in S-values (ppm) from TMS] H

1

1 3 4 (2W

1.41 7.22 3.68 5.15 9.13

5 WI

2 d*

dt td

t

8t

br s 7.60 s

11t 12 (2H) Me0

1.95 s 6.34 s -

7

1.43 7.15 3.64 5.00 9.00 7.32 7.90 3.90

d* dt td

3

d dt td

s= sp

7.64 7.34 3.75 5.21 9.30 7.78 7.94

s

4.10 s

t br s

t br s s s

J (Hz): 1,3 = 3,4 = 1.8; 4,5 = 6.8. *Data recorded in CD,OD. t Attributed by comparison with the data reported [Iv and other structurally related alkaloids [18].

152.4s 108.2d 122.1 s 131.4 s 132.1 s 56.8 q

effects measured

1.19 7.35 3.76 5.22 9.24 7.61 8.07

d* dt rd t

br s s s

4.23 s

for lycorine

“These attributions may be reversed.

The location of the methoxy group on C-9 and the hydroxy on C-10, respectively, was deduced from a series of ‘H NOE difference spectra (Table 3). Results b and c indicated the spatial proximity between H-8 and the methoxy group, while result a located H-l 1 near to H-l. These data were corroborated by synthetic work. Pseudolycorine (4) [7, 151 was converted, in good yield, into 2 by oxidation with selenium dioxide. When the same

on zefbetaine

(2)

Observed

Irradiated

Rl=H,

111.6 d 157.1 s

8:

*, t Attributed by comparison with the resonance frequencies of the carbons of the corresponding ring of lycorine [17] and ungeremine [7], respectively. SAsslgned by evidence from a SFSD spectrum.

RP =CH3

4

shifts are in

a b C

7.64 (H-l) 4.10 (MeO) 7.78 (H-8)

reaction was performed on sternbergine (6), an alkaloid previously isolated from Sternbergia lutea Ker Gawl [ 161, the isomeric 2-oxyphenanthridinium alkaloid 3, which we called iso-zefbetaine, was obtained. It showed exchanged positions for the OH and Me0 groups on the A-ring, in comparison to 2. iso-Zefbetaine (3) and 2 showed different TLC behaviour on silica gel and reverse phase plates (see Experimental); their UV and ‘H NMR spectra (Table 1) were similar; the FAB mass spectrum of synthetic 3 showed the pseudomolecular ion at m/z 268 [MH]+. These results reveal that the structure of 2 is 4,5-dihydro2,1O-dihydroxy-9-methoxypyrrolo[3,2,1-de]phenanthridinium hydroxide, inner salt. Compound 2 corresponds to zefbetaine isolated from Zephyranthes fluon as reported by Ghosal et al. [SJ. However, the discussion on the partial synthesis reported in ref. [S] clearly identifies it with zefbetaine as described in this paper, but the structural formula shown in ref. [S] refers to iso-zefbetaine (3) in error. The authors strongly believe that Ghosal et al. [S] described the isolation and structural characterization of a zefbetaine which is identical to 2. In fact, 2 showed the same melting point (> 300”) and very similar UV and ‘H NMR (recorded in CD,OD, Table 1) data to those previously reported for zefbetaine c51. In conclusion, this paper reports the correct structure of natural zefbetaine 2 isolated from bulbs of P. maritimum L. Moreover, alkaloid 3 was prepared from the synthetic conversion of sternbergine (6) and named isozefbetaine. It is thought that compound 3 is not a naturally occurring alkaloid. EXPERIMENTAL Chemicals. Mps: uncorr.; UV spectra: MeOH, wise noted: ‘H and 13CNMR: CD,OD-CD,COZD,

unless other3: 1, unless

2141

Alkaloids from Pancratium maritimum otherwise noted, 270 or 300 MHz and at 67.93 or 75.47 MHz, respectively, using TMS as int. standard. The NOE experiments were performed using the spectral subtraction technique according to a Bruker standard microprog~. FABMS were recorded on samples dissolved in glycerol-thioglycerol on a Cu probe tip and inserted into the source at lo-’ Torr pressure of Xe. The sample was bombarded with Xe atoms of 9.5 kv energy and the spectra were recorded on UV paper. Analytical TLC: silica gel plates (Merck, Kieselgel 60 F,,,, 0.25 mm) or on reverse phase plates (Whatman, Stratocrom SIF,,, C-18,0.20 mm); the spots were visualized by exposure to I, or UV radiation or by spraying with a 10% Echtrosaltz (Fast Red Salt B: 1-ammo-2-methoxy-~ nitrobenzenediazotate naphthalendisulphonate [12]) in Ha0 or 1% aq. FeCl, [13,14] followed by exposure to NH, vapour. CC: neutral alumina (BDH, Brockmann grade I) or on silica gel (Merck, Kieselgel 60, 0.063-0.20 mm). Solvent systems: (A) n-BuOH-HOAc-Ha0 (12: 3: 5); (B) H,O-MeCN (1: 1); (C) H,O-EtOH (1.5: 1). Plant ~terial. Fresh bulbs of Pu~rot~~m muritimum L. were collected from sandy hills on the northern coastal strip of Egypt @him), during the flowering and fruit-producing stages in August 1985. The plant was identified by Prof. Dr N. Al-Hadidy, University of Cairo, Egypt. A voucher sample is deposited in the Department of Pharmacognosy, Faculty of Pharmacy, University of Alexandria, Egypt. Extraction and puri$cation. Fresh bulbs (8 kg) of P. maritimum L. were minced and exhaustively extracted with cold 95% EtOH; the combined extracts were coned under red. press., and then defatted with petrol (40-60”), acidified at pH 2 with 2 M tartaric acid, fiitered, and then washed with E&O. The acidic aq. phase was rendered alkaline (pH 10-12) with NH,OH and extracted with CHCl,, EtOAc and n-BuOH. The combined CHCI, extracts were washed with H,O, dried over Na,SO,, then coned to a small vol. where lycorine was precipitated. The crude alkaloid residue leR after the removal of the solvent was frac~onated over a column of neutral alumina. Elution was obtained using C,H,, CHCI, and MeOH with increasing polarity. The CHCl, eluate was further chromatographed over silica gel column, using CHCl, and MeOH for elution. The most polar fraction, eluted with MeOH resulted in the isolation of alkaloid 1 (IOmg) as brilliant orange needles which were recrystallized from*MeOH-C,H,; mp 268-270” (refs [7] and [9], mp 265-275” decomp. and 260-270“ decomp., resp.); UV &, nm: 360, 258 (0.1 M HQ); 408, 262 (0.1 M NaOH). ‘HNMR: Table 1; FABMS, m/z: 266 [MH]‘; 1 had the same R, values when cochromatog~ph~ as an authentic sample of ungeremine by TLC on silica gel (eluent A) and on reverse phase (eluents B and C) plates. The residue 1eR from the n-BuOH extract was fractionated over a silica gel column using CHCl,-MeOH gradients; the fractions eluted with 40% MeOH in CHCI, yielded alkaloid 2 (4 mg), which was obtained as an amorphous pale yellow deposit from MeOH-Et,0 mixture: mp >300” (charring at 273”) (ref. PI, mp> 30@?; UV Amaxrun: 409,262,224; ‘H and 13CNMR: Tables 1 and 2, respectively; FABMS, m/z.: 268 [MH]+. Conversion of pseudolycorine (4) into zejbetaine (2). Pseudolycorine (4,3 mg) in EtOH (1 ml) was oxidized with SeOz. according to the procedure reported in ref. [9] to convert lycorine (5) into ungeremine (1). The reaction was complete after 3 hr and the mixture was then filtered to remove the Se metal precipitated. Evapn of the solvent gave an amo~hous yellow solid (2.7 mg). Synthetic 2 showed the same behaviour as compared with authentic zefbetaine, even when co-chromatographed, by TLC

on silica gel (eluent A) and on reverse phase (eluents B and C). Its spectroscopic properties (UV, ‘H NMR and FABMS) coincided with those of natural zefbetaine isolated from P. muritimum. Conoersion of st~~bergine (6) into iso-ze~betu~~ (3). Sternbergine (6,3 mg) was transformed into betaine 3 according to the procedure used for the preparation of 2 from 4. The amorphous yellow solid obtained (2.8 mg) had a different mobility as compared with 2, even when co-chromatography, by TLC on silica gel (eluent A) and on reverse phase (eluents B and C). UV I,,, nm (logs): 370 (3.60), 285 (sh), 274 (sh), 264 (4.10); ‘H NMR: Table 1; FABMS, m/z: 268 [MH]‘. ~o~~Ia~u~e. Ungeremine, 4,S-d~ydro-2-hydroxy[l,3]~oxolo[4,5-~]pyrrolo[3,2,1-delphenanth~dinium hydroxide, inner salt; zefbetaine, 4,5-dihydro-2,10-dihydroxy-9-methoxyp~rolo[3,~1-delphenanth~dinium hydroxide, inner salt. Acknowledgements-This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research. Mass spectral data were provided by ‘Servizio di Spettrometria di Massa de1 CNR-Universita di Napoh’. The assistance of the staff is gratefully acknowledge.

RBPRRENCES 1. Ahmed, Z., Rizk, A. and Hamouda, F. (1964) Lloydfa 27,115.

2. El Tohamy, D. (1988) Master Degree Thesis. Faculty of Pharmacy, University of Assiut, Egypt. 3. Ah, A. A., Mesbah, M. K. and Mohamed, M. H. (1984) Bull. Pharm. Sci. Assiut University 7,351. 4. Gaber, M. (1990) Master Degree Thesis. Faculty of Pharmacy, University of Alexandria, Egypt. 5. Ghosal, S., Singh, S. K. and Srivastava, R. S. (1986) Pkytochemistry 25, 1975.

6. Ghosal, S., Singh, S. K., Kumar, Y., Unnikrishnan, S. and Chattopadhyay, S. (1988) PZanta Med. 54, 114. 7. Evidente, A., Randazzo, G., Surico,G., Lavermicocca, P. and Arrigoni, 0. (1985) J. Nat. Prod. 48, 564. 8. Normatov, M., Abduazimov, Kh. A. and Yunusov, S. Yu. (1965) Uzbeksk. Khim. Zh. 9,25 [through Chem. Abstr. (1965) 63, 7061f]. 9. Fales, H. M., Wamhoff, E. W. and Wildman, W. C. (1955) J. Am. Chem. Sot. 77, 5885. 10. Ali, A. A., El Sayed, H. M., Abclallah, 0. M. and Steglich, W. (1986) Phytochemistry 25,2399. 11. Ghosal, S., Kumar, Y., Singh, S. K. and Kumar, A. (1986) J. Chem. Res. Synop. 112. 12. Heinrich, P. and Schuler, W. (1947) Hefv. Chim. Acta 30,886. 13. Kirchner, J. G. (1978) in Technique of Chemistry-Thin Layer Chromatography (Weissberger, A., ed.), Vol. XIV, 2nd Edn, pp. 193-264. John Wiley, New Y&k. 14. Krebs, K. G., Heusser, D. and Wimmer, H. (1969) in 77rinLayer Chromat~raphy-A ~~rutory ~a~book (Sthal, E., ed.), pp. 854-909. Springer, Berlin. 15. Ghosal, S., Saini, S. K. and Razdan, S. (1985) Pkytochemistry 24,214l.

16. Evidente, A., Iasiello, I. and Randazzo, G. (1984) .I. Not. Prod. 47, 1003. 17. Evidente, A., Cicala, M. R., Giudicianni, I., Randazzo, G. and Riccio, R. (1983) Phytochem~rry 22,581. 18, Ghosal, S., Saini, K. S. and Frahm, A. W. (1983) Phytochemistry 22, 230.5.