Two cyclopeptide alkaloids from Zizyphus lotus

Phytochemisrry, Vol. 32,No. 6, pp. 1591-1594, 1993 Printedin GreatBritain.

TWO CYCLOPEPTIDE

0031-9422/93 $6.00+0.00 PergamonPressLtd

ALKALOIDS

FROM ZZZYPZ-ZUS LOTUS

KAMEL GHEDIRA, RACHID CHEMLI, BERNARD RICHARD,* JEAN-MARC NWLLARD,* and LOUISETTE LE MEN-OLIVIER*

MONIQUE

ZECHES*

Facultb de Pharmacie de Monastir, rue Avicenne, 5000 Monastir, Tunisia; *Fact&i de Pharmacic de Reims (U.A. au C.N.R.S. N ’ 492), 51 rue Cognaq-Jay,

51096 Reims Ckdex, France

(Received 27 July 1992)

Key Word Index-Zizyphus NMR

lotus; Rhamnaceae; root bark; cyclopeptide alkaloids; lotus&s-A

and D;

Ahstract-Cyclopeptide alkaloids were isolated from the root bark of Zizyphus lotus. The structures of two new compounds, lotusine A and lotusine D, were elucidated mainly to homo- and heteronuclear NMR techniques.

INTRODUCTION

Lotusine A showed a weak [M] + at m/z 5 18 in its mass spectrum suggesting the molecular formula C3,,H38N40.,, which was confirmed by ‘H and “C NMR spectra. The mass spectrometric fragmentation of 1 showed that this cyclopeptide alkaloid belonged to the amphibine subgroup [2,8,9], since the ions a at m/z 135 and b at m/z 96

Several cyclopeptide alkaloids have been already isolated from the plants of the genus Zizyphus [l]. Pharmacological investigations carried out on some of these species has shown sedative, analgesic, anti-inflammatory and hypoglycaemic activities [2-4]. Furthermore, cyclopeptides have exhibited antibacterial and antifungal properties [S]. Zizyphus lotus is a large shrub (or rarely a tree) distributed throughout Lybia to Morocco, Southern Europe (Spain, Sicily, Greece and Cyprus) and Arabia [6]. It is known in Tunisia as ‘Sedra’. The fruit, called ‘Nbag’, is described as having emollient properties. A mixture of dried ground leaves and fruits is applied topically in the treatment of boils, while the root bark is renowned for its antidiabetic activity [7]. From the root bark of Z. lotus, we have prepared different extracts for chemical and pharmacological investigations. From the alkaloidal extract, we have isolated several cyclopeptides and we have established the structures of two new alkaloids, lotusines A(1) and D(2), mainly by homo- and heteronuclear NMR techniques.

\--/ H

a nr/r135

c m/z86

b m/196

c’ m/z 12

RESULTSAND DISCUSSION The alkaloids were extracted by the classical acid-base method and yielded 1.33 g kg- ‘, alkaloid mixture (AM). Lotusine A(1) (1.56% AM) and lotusine D(2) (0.78%) were isolated by means of centrifugal TLC on silica gel, followed by preparative TLC. Compounds 1 and 2 gave a faint colour with Dragendorff’s reagent and showed many similar spectral properties. Their IR spectra were typical of peptide alkaloids and exhibited bands for -NH, -NH-CO, a conjugated cis-t,2-disubstituted C=C double bond and Ar-O-C. The W spectra of 1 and 2 displayed only strong end absorption (at 208 and 211 nm, respectively) and suggested a para-hydroxystyrylamine chromophore as found in 14-membered cyclopeptide alkaloids [2].

R d Me: m/z148 d’ H: nr/r134

Scheme

1591

R e Mu: m/r427 e’ H: mix413

1. Most significant mass spectral fragments pounds 1 and 2.

of com-

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K. GHEDIRA et al.

are typical of p-hydroxystyrylamine and of hydroxyproline units, respectively (Scheme 1). The ion c at m/z 86 suggested leucine or an isoleucine as a ring-bonded

Fig. 1. Most significant correlations observed in HMBC spectra of 1 and 2.

Table 1. ‘H and ‘VNMR

amino acid. Moreover, the presence of an N,N-dimethylphenylalanine unit as the terminal amino acid was deduced from the occurrence of intense peaks at m/z 148 (d), at m/z 427 (e, [M-C,H,]+) and at m/z 91 (f, [C,H,]‘). The ‘HNMR spectrum of 1 (Table 1) allowed the characteristic signals of the constitutive amino acids to be observed such an isoleucine unit by the presence of an ethyl group (t at 60.88, J=7.4 Hz), an hydroxyproline unit by the occurrence of protons H-8 (d at 64.26, J= 5.3 Hz) and H-9 (ddd at 65.52, J, =9.8 Hz; .I, = 7.2 Hz; J, = 5.3 Hz) as described in the case of amphibine D [lo]. Two olefinic protons H-l (d at 66.29, J = 7.8 Hz) and H-2 (dd at 66.75, J, = 10.6 Hz; J, = 7.8 Hz), as well as the H-3 (d at 66.51, J= 10.6 Hz), indicated the presence of a styrylamine moiety. Furthermore, a broad singlet (6H at 62.39) and a complex bulky signal (integrating for 9 protons) in the aromatic part suggested the presence of an NJ-dimthylphenylalanine group. The COSY spectrum of 1 allowed the assignment of all the protons except those of the aromatic region. It was

spectral data for compounds

13CNMR (75 MHz)

‘H NMR (300 MHz) Atom 1 2 3 4 5 6 7 8 9 10 11 12 12 13 13’ 14 15 16 16 17 18 19 19 20 20 21 22 23 23 24 25,25 26,26 27 28

1

1 and 2 (in CDCI,)

2

6.29 d (7.8) 6.75 dd (10.6, 7.8) 6.51 d (10.6)

6.34 d (7.7) 6.75 dd (10.5, 7.7) 6.57 d (10.5)

4.14 dd (8.6, 3.0) 6.70 d (8.6)

4.21 dd (8.5, 3.2) 6.75 d (8.5)

4.26 d (5.3) 5.52 ddd (9.8, 7.2, 5.3)

4.32 d (5.3) 5.44 ddd (9.8, 7.1, 5.3)

7.10 m 7.28 m 7.11 m 7.08 m

7.14 m 7.27 m 7.11 m 7.07 m

2.13-2.0 m 1.28 m 1.13 m 0.88 c (7.4) 0.62 d (7.0) 2.48 ddd (12.2, 7.2, 5.1) 2.13-2.0 m 4.21 dd (10.8, 8.0) 3.07-2.99 m

2.20 m 1.31 m 1.15 m

3.51 dd (9.1, 4.0) 3.07-2.99 m 2.90 dd (13.8, 4.0)

3.56 t (7.0) 2.90 dd (13.4, 7.0) 2.67 dd (13.4, 7.0)

7.19 (2H) m 7.07 (2H) m 7.15 m 2.39 (6H) br s

7.25 (2H) m 7.06 (2H) m 7.23 m 2.34 (3H) br s

t (7.3) 0.86 d (7.0) 2.44 ddd (12.2, 7.1, 5.2) 2.05 m 3.75 dd (11.3, 8.2) 2.59 m 0.90

Coupling constants (J in Hz) in parentheses

1

2

114.0 125.5

114.7 126.0

167.0 58.5

167.5 58.9

171.0 63.8 83.9

171.5 62.8 83.6

157.4 122.8 122.9 130.1 132.6 132.6 35.4 24.0

157.5 122.6 122.7 130.1 132.7 132.7 34.3 23.9

12.2 15.9 31.9

12.2 16.0 31.8

45.8

45.8

170.6 67.4 30.2

173.2 63.9 39.7

138.7 128.4 128.9 126.2 45.8

136.9 128.6 129.1 126.8 35.5

Cyclopeptide

alkaloids

thus possible to identify the protons of each of the three amino acids: isoleucine, hydroxyproline and a substituted N,N-dimethylalanine, and more especially to assign the overlapping signals for H-3 and H-6, H-19 and H-23 as well as H-15 and H-19’. The ‘jCNMR spectrum of 1 (Table 1) contains 30 carbon resonances, three of which correspond to three carbonyl groups. The assignments of all the carbons were deduced from literature data [l l] and confirmed the identities of the amino acids (isoleutine, hydroxyproline and N,N-dimethylphenylalanine), as well as the presence of the styrylamine unit. The latter assignments, as well as those of the ‘HNMR spectrum, were in agreement with the data from the HMQC [12] spectrum of 1, which also allowed correction of earlier chemical shift data concerning C-l, C-12, C-13, and C-14 in amphibine D. They were previously [11] given at 6122.7, 114.8, 132.4 and 130.1 instead of 6114.0, 122.9, 130.1 and 132.6 in 1. The points of attachment between the three constitutive amino acids and the styrylamine unit were deduced from the HMBC [13] spectrum. The most significant interresidue correlations are H-3, C-4; H-6, C-7; and H-20, C-21. The identification of the C-4, C-7 and C-21 resonances was ascertained by intraresidue HMBC correlations as C-4, H-5; C-7, H-8; C-21, H-22. Moreover, the combined data of HMQC and HMBC spectra permitted the assignment of the nine aromatic protons. The mass spectrum of 1otusineD (2) gave a [M] + at m/z 504 (14 mu less than that of I), as well as fragment ions a, b, c, d’, e’ and f suggesting structure 2 and molecular formula C,,HJ,N,O,. The ‘H NMR of 2 also supported the structure, showing an almost superimposable spectrum on that of 1 except for the following signals: H-22 at 63.56 (t, J=7.0Hz), H-23 at 62.90 (dd, 5,=13.4, J, =7.0 Hz) and H-23’ at 62.67 (dd, J, = 13.4, J2 =7.0 Hz). Furthermore, the signal at 62.34 (br s), integrating for three protons, corresponded to one methyl group instead of two as found in 1. The main differences between the 13C NMR spectra of 1 and 2 are in the changes for the C21, C-22, C-23 and C-28 resonance frequencies, in agreement with a structural change in the region of the phenylalanine residue. The interpretation of all protons (particularly those of the aromatic region), as well as the bonding sites between the styrylamine moiety, isoleucine, the hydroxyproline and the N-methyphenylalanine units, were deduced from combined analysis of the HMQC and HMBC spectra of 2. EXPERIMENTAL

General. ‘H and 13C NMR: CDCl, at 300 and 75 MHz respectively. Chemical shifts are given in 6 (ppm) with TMS as int. standard. EIMS: direct probe insert. TLC: silica gel K6F Whatman and centrifugal TLC was carried out on a Chromatotron apparatus (Harrison Research) (Merck). The alkaloids were on silica gel 60 PF,,, detected by spraying TLC plates with Dragendorffs reagent. Plant material. Collected in March 1992 in the region of Monastir (locality of Cherahil, Tunisia) and identified by

from Zizyphus

lotus

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Prof. M. A. Nabli, University of Tunis. Voucher specimens are kept in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Monastir. Extraction. Dried ground root bark (960 g) of Z. lotus (L.) Desf. was moistened with 50% NH,OH and percolated with 12 1 EtOAc. The percolate was extracted with 2% H,SO, until a Mayer’s test was negative, the acid layer sepd, made alkaline with NH,OH and extracted with CH&. The CH,Cl, soln was washed with H,O, dried (Na,SO,) and evapd in uncuo to give 1.28 g of alkaloid mixt. (AM; yield 1.33 g kg- ‘). Isolation. The AM was fractionated by centrifugal TLC (CTLC) on a 4 mm layer of silica gel and eluted in 30 ml frs with CHCl,-MeOH (49: 1) (frs l-8) and CHCl,MeOH (19: 1) (frs 9-12). Alkaloid 1 was in frs 2--3 (111 mg), 2 in fr. 5 (96 mg). Purification. Compound 1 was isolated from frs 2-3 by CTLC on a 2 mm layer of silica gel and elution in 10 ml frs with CHCI,-MeOH (99: 1) (frs I-XI) and CHCl,MeOH (99 : 2) (frs XII-XIV). Alkaloid 1 was purified by prep. TLC from fr. V (45 mg) using CHCl,-MeOH (19 : 1) as eluant. Compound 2 was isolated and purified from fr. 5 by prep. TLC and elution with CHCI,-MeOH (93 :7 and 19: 1). Lotusine A(1). [u]n--215” (CHCl,; ~1.0). UV nz:fH nm: 208,250 (sh). IR v~~~r3cm-’ 3390,2950,2790, 1640, 1615, 1220, 1040. MS m/z (rel. int.) 518 [M]’ (0.65), 517 (0.7), 475 (0.3), 427 (35), 322 (4), 279 (4), 274 (0.9) 229 (2), 209 (5), 203 (4), 190 (12), 186 (3), 181 (2), 167 (lo), 153 (15), 149 (93), 148 (lOO), 135 (20), 134 (47), 133 (59), 105 (31), 96 (12), 91 (69) 86 (25), 83 (65), 69 (59), 68 (41) 57 (100). ‘H and 13C NMR see Table 1. Lotusine D(i). [cr]n - 187’ (CHCl,; c 0.5). UV nzFH nm: 211. IR vzty3 cm -+ 3396, 2931, 1668, 1626, 1227, 1037. MS m/z (rel. int.): 504 [M]’ (0.4), 475 (0.12), 448 (0.038),414(61),413 (99), 385(8), 382(16), 371(66), 354(7), 300 (49) 272 (36), 256 (7), 229 (3) 209 (4), 201 (6), 186 (ll), 185 (ll), 174 (ll), 165 (22), 139 (24) 135 (lOO), 134 (lOO), 119 (45), 105 (20), 97 (20) 96 (13), 91 (lOO),86 (75), 84 (35), 77 (25), 69 (93), 68 (60), 65 (34), 57 (68). iH and 13C NMR, see Table 1.

Acknowledgements-This work was undertaken as part of a cooperative research programme between F.N.R.S. (Tunisia) and C.N.R.S. (France) on plants used as antidiabetic drugs in Tunisian traditional medicine. We are grateful to Prof. M. Jeddi, Faculty of Pharmacy, Monastir, for his interest in this work. We also thank A. Aissi, M. Ben Salah and A. Mahmoud for their technical contribution. This research programme was supported by the International Foundation for Science (IFS, G.A.N” F/1291-1).

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