Two new 14-membered cyclopeptide alkaloids from Zizyphus xylopyra

Article abstract of DOI:10.1080/14786419.2011.559641

The phytochemical investigation of the bark of Zizyphus xylopyra resulted in the isolation of two new 14-membered ring cyclopeptide alkaloids, xylopyrine-G and xylopyrine H. Their structures have been established by chemical and spectral evidences.

Full text of DOI:10.1080/14786419.2011.559641

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Two new 14-membered cyclopeptide
alkaloids from Zizyphus xylopyra
Manoj B. Pandey a , Sarita Singh b , Meenakshi Malhotra c , Vidya
B. Pandey b & Tryambak D. Singh b
a Department of Chemistry, S.G.R. Post Graduate College,
Purvanchal University, Dobhi 222149, Jaunpur, India
b Department of Medicinal Chemistry, Institute of Medical
Sciences, Banaras Hindu University, Varanasi 221005, India
c Department of Biomedical Engineering, Faculty of Medicine,
Duff Medical Building, 3775 University Street, McGill University,
Montréal, Quebec H3A 2B4, Canada
Published online: 14 Oct 2011.
To cite this article: Manoj B. Pandey , Sarita Singh , Meenakshi Malhotra , Vidya B. Pandey &
Tryambak D. Singh (2012): Two new 14-membered cyclopeptide alkaloids from Zizyphus xylopyra ,
Natural Product Research: Formerly Natural Product Letters, 26:9, 836-841
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Natural Product Research
Vol. 26, No. 9, May 2012, 836–841
Two new 14-membered cyclopeptide alkaloids from Zizyphus
xylopyra
Manoj B. Pandeya, Sarita Singhb, Meenakshi Malhotrac, Vidya B. Pandeyb and
Tryambak D. Singhb
*
aDepartment of Chemistry, S.G.R. Post Graduate College, Purvanchal University, Dobhi
222149, Jaunpur, India; bDepartment of Medicinal Chemistry, Institute of Medical Sciences,
Banaras Hindu University, Varanasi 221005, India; cDepartment of Biomedical Engineering,
Faculty of Medicine, Duff Medical Building, 3775 University Street, McGill University,
´
Montreal, Quebec H3A 2B4, Canada
(Received 30 August 2010; final version received 30 January 2011)
The phytochemical investigation of the bark of Zizyphus xylopyra resulted
in the isolation of two new 14-membered ring cyclopeptide alkaloids,
xylopyrine-G and xylopyrine H. Their structures have been established by
chemical and spectral evidences.
Keywords: Zizyphus xylopyra; rhamnaceae; cyclopeptide alkaloids;
xylopyrine-G; xylopyrine-H
1. Introduction
Zizyphus xylopyra Willd. (Rhamnaceae) is a large straggling shrub, distributed
throughout India (Council of Scientific and Industrial Research, 1976). In our
previous communication, we reported the isolation of cyclopeptide alkaloids,
xylopyrine-A, xylopyrine-B (A.K. Singh, M.B. Pandey, V.P. Singh, & V.B. Pandey,
2007), xylopyrine-C, scuitianine-C (A.K. Singh, M.B. Pandey, V.P. Singh, & V.B.
Pandey, 2008), xylopyrine-D, xylopyrine-E (M.B. Pandey, A.K. Singh, J.P. Singh,
V.P. Singh, & V.B. Pandey, 2008b), xylopyrine-F, nummularine-P and sativanine-H
(Pandey, J.P. Singh, A.K. Singh, & V.P. Singh, 2008a) from the above plant. Here,
we report the isolation and characterisation of two new cyclopeptide alkaloids,
xylopyrine-G and xylopyrine-H from the bark of Z. xylopyra.
2. Results and discussion
Chromatographic separation of crude base fraction of the bark of Z. xylopyra
followed by preparative TLC furnished the alkaloids, xylopyrine-G (1) and
xylopyrine-H (2).
Xylopyrine-G (1), C34H38N4O5 ([Mþ], 582.2848) and xylopyrine-H (2),
C32H36N4O4 ([Mþ], 540.2740) gave positive Dragendorff test for alkaloids (Shah,
Pandey, Eckhardt, & Miana, 1988). The IR spectra of 1 and 2 were typical for
*Corresponding author. Email: tryambak@yahoo.com; tryambak@bhu.ac.in
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.559641
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Natural Product Research
837
peptide alkaloids and showed strong bands characteristic of N–CH3 group,
secondary amido group of peptide linkage, styryl double bond and arylether
(Tschesche and Kaussmann, 1975) at ꢀmax values 2790, 1685 and 1635, 1609, 1235
and 1040 cmꢀ1, respectively. The UV spectrum exhibited typical strong end
absorption bands at ꢁmax 210 nm and shoulder at 250 nm characteristic of
styrylamine chromophore in the 14-membered ring containing cyclopeptide alkaloid
(Tschesche and Kaussmann, 1975).
The structure of the majority of the peptide alkaloids can largely be determined
by their High-Resolution Mass Spectrum (HRMS) analysis (Fehlhaber, 1968).
In view of this fact, the HRMS analysis of compounds 1 and 2 was applied to
elucidate their structures.
Like N-formylcyclopeptide alkaloid sativanine-K (Shah, Al-yahya, Devi, &
Pandey, 1987), alkaloid 1, which carried a N-formyl group on terminal amino acid,
showed an intense molecular ion peak in the mass spectrum at m/z 582. The cleavage
of the peptide bond took place between N-formylmonomethylleucine and phenyl-
alanine in 1, and the 14-membered ring system yielding the ion at m/z 427 and the
base peak at m/z 156. The ion m/z 156 further eliminates 2 ꢁ CO to give ions m/z 128
and m/z 100. The linkage of the side chain in 1 as N-formylmonomethylleucine with
a 14-membered ring bound phenylalanine can be deduced by the fragment at m/z 156
forming the base peak and ions m/z 243 and 215. The ꢂ-cleavage products of 2 gave
ion peaks at m/z 455 and the base peak at m/z 86. The ion m/z 455 further fragments
to give the ion m/z 427. The linkage of the side chain in 2 as N-monomethylvaline
with 14-membered ring-bound phenylalanine was established by the elementary
composition of ions m/z 427, 455, 215, 187 and 86. Further fragmentation of ion m/z
427 obtained from compounds 1 and 2 were identical to that of the reported alkaloid,
xylopyrine-C (3) (Singh et al., 2008) which fragments into ions m/z 412, 371, 308,
278, 250, 224, 135 (styrylamine), 131, 120 (phenylalanine) and 102. The elementary
composition of all the fragment ions was substantiated by HRMS.
Thus, compounds 1 and 2 differ from each other only in their terminal amino
acids. The identity of the ring-bound and terminal amino acids were proved to be
phenylalanine and N-formylmonomethylleucine in 1 and phenylalanine and
N-monomethylvaline in 2, respectively, by acid hydrolysis of 1 and 2 and co-paper
chromatographic comparison of the hydrolysate. Partial hydrolysis of compounds 1
and 2 furnished identical compound 4. Deformylation of compound 1 furnished
compound 5. Compound 5 on methylation gave methylated product as compound 6,
which was identical to the reported alkaloid, crenatine-A (Silva, Bhakuni, Sammes,
Pais, & Jarreau, 1974). Compound 2 on methylation gave a methylated compound
identical to integerrissine (7) (Lagarias, Goff, Klein, & Rapoport, 1979).
The structures of xylopyrine-G and xylopyrine-H were thus settled, respectively,
as 1 and 2 (Figure 1), which was further supported by 13C-NMR data.
3. Experimental
3.1. General
Melting points are uncorrected. UV spectra were measured on a Carry-14
spectrometer using spectral methanol. IR was recorded on Perkin–Elmer spectro-
photometer model 221 in KBr pellet. MS analyses were performed on Kratos MS-50

838
M.B. Pandey et al.
12„
13„
21„
10
22„
14
4
1
O
11
7
20
12
13
23
2
9
O
3
O
O
8
HN
21
22
18„
17„
6
HN
O
NH
O
5
16
N
H
19
O
15
NH2
4
NH
R1
18
17
N
R2
R3
R1
R2
R3
1: CH2CH(CH3)2
2: CH(CH3)2
CH3
CH3
CH3
CH3
CH3
CH3
CHO
H
3: CH2Ph
H
5: CH2CH(CH3)2
6: CH2CH(CH3)2
7: CH(CH3)2
H
CH3
CH3
Figure 1. Structure of compounds 1–7.
mass spectrometer operating at 70 eV with the evaporation of sample in the ion
source at 200ꢂC. 13C-NMR spectra were taken on 125 MHz Brucker WH90
spectrometer. TLC was performed on silica gel G (Merck); paper chromatography
was carried out on Whatman No. 1 paper; column chromatography was performed
on silica gel columns (BDH, 60–120 mesh); solvents used for TLC: CHCl3–MeOH
(8 : 1) (solvent A), (2 : 1) (solvent B) and for paper chromatography: n-BuOH–
HOAc–H2O (4 : 1 : 5) (solvent C) and spots on paper chromatograms were visualised
by ninhydrin reagent.
3.2. Plant material
The plant Z. xylopyra was collected from Mirzapur District, UP, India and identified
by Prof. N.K. Dubey, Department of Botany, Banaras Hindu University, Varanasi.
A specimen sample no. 222 is preserved in the department. The aerial bark was used
in this study.
3.3. Extraction and isolation
Dried aerial bark (4 kg) was powdered and extracted repeatedly with a mixture
of C6H6–NH4OH–MeOH (100 : 1 : 1). The combined extract was concentrated under
reduced pressure and extracted thoroughly with 7% aqueous citric acid. The acidic

Natural Product Research
839
solution was basified with ammonia and extracted with CHCl3. The CHCl3 extract
was evaporated to dryness which gave a mixture of crude alkaloids (4 g). The crude
alkaloidal fraction was chromatographed over SiO2 gel column eluting with a
mixture of CHCl3 and MeOH. The eluants collected from CHCl3–MeOH (25 : 1)
and (20 : 1) were purified separately by preparative TLC with solvents A and B,
which furnished the compounds xylopyrine-G (31 mg) (1) and xylopyrine-H (26 mg)
(2), respectively.
3.3.1. Xylopyrine-G (1)
Compound 1 crystallised from MeOH as colourless granules, m.p. 231–233ꢂC, Rf
25
0.25 (solvent A), 0.35 (solvent B), ½ꢂꢃD ¼ 230 (c, 0.32, CHCl3). It showed UV
(MeOH) ꢁmax, nm (log "): 210 (4.30), 250 (3.10); IR (KBr) ꢀmax cmꢀ1: 3410 (–NH),
2965–2860 (ꢀCH), 2790 (–OCH3), 1680, 1635 (sec. amide), 1610 (–C¼C–), 1240,
1040 (aryl ether); 125 MHz 13C-NMR (CDCl3 þ CD3OD) ꢃ: 121.4 (C-1), 125-5 (C-2),
169.7 (C-4), 53.9 (C-5), 171.7 (C-7), 55.2 (C-8), 80.8 (C-9), 155.8 (C-11), 121.5 (C-12),
121.5 (C-120), 130.4 (C-13), 130.4 (C-130), 133.3 (C-14), 38.9 (C-15), 135.6 (C-16),
127.8 (C-17), 127.8 (C-170), 128.7 (C-18), 128.7 (C-180), 126.1 (C-19), 134.6 (C-20),
129.0 (C-21), 129.0 (C-210), 130.2 (C-22), 130.2 (C-220), 127.5 (C-23), terminal amino
acid N-formylmonomethylleucine [172.4, 74.6, 25.6, 30.3, 19.2, 20.2, 171.8, 41.6];
HRMS, m/z: 582.2848 (C34H38N4O5), 427.1898 (C26H25N3O3), 412.1782
(C26H24N2O3), 371.1764 (C24H23N2O2), 308.1160 (C18H16N2O2), 278.1178
(C18H16NO2), 250.1233 (C17H16NO), 243.0770 (C13H11N2O3), 224.1078
(C15H14NO), 215.0836 (C12H11N2O2), 156.1026 (C8H14NO2), 135.0684 (C8H9NO),
131.0498 (C9H7O), 128.1078 (C7H14NO), 120.0814 (C8H10N), 102.0548 (C8H6) and
100.1128 (C6H14N).
3.3.2. Deformylation of xylopyrine-G (1)
Compound 1 (7 mg) was deformylated by treatment with 0.5 N HCl in MeOH at
room temperature for a period of 48 h. Usual work-up and crystallisation from
MeOH gave a colourless crystalline solid of compound 5 (4 mg) m.p. 185–187ꢂC;
MS, m/z: 554.2896 ([M]þ C33H38N4O4).
3.3.3. Methylation of compound 5
Compound 5 (8 mg) was treated with HCHO and NaBH4 by slow addition and
checking the reaction product by TLC. On usual work-up and crystallisation from
MeOH, it furnished the N-methylated product 6 as colourless granules (6 mg), m.p.
221–223ꢂC; MS, m/z: 568.3052 ([M]þ, C34H40N4O4). It was identical to crenatine-A
(Silva et al., 1974) (mmp, co-TLC and super-imposable IR) on comparison with
authentic sample.
3.3.4. Xylopyrine-H (2)
Compound 2, crystallised from MeOH as colourless granules, m.p. 216–218ꢂC, Rf
0.45 (solvent A), 0.62 (solvent B); [ꢂ]25 ¼ 280 (c, 0.28, CHCl3). It showed UV
(MeOH) ꢁmax, nm (log "): 215 (4.10), 248 (3.20); IR (KBr) ꢀmax, cmꢀ1: 3440 (–NH),
2955–2840 (–CH), 2785 (–OCH3), 1678, 1630 (sec. amide), 1610 (–C¼C–), 1240, 1040

840
M.B. Pandey et al.
(aryl ether); 125 MHz 13C-NMR (CDCl3 þ CD3OD) ꢃ: 121.8 (C-1), 125.8 (C-2),
170.0 (C-4), 54.1 (C-5), 172.0 (C-7), 55.1 (C-8), 80.3 (C-9), 156.0 (C-11), 121.8 (C-12),
121.8 (C-120), 130.7 (C-13), 130.7 (C-130), 134.0 (C-14), 39.0 (C-15), 135.0 (C-16),
128.0 (C-17), 128.0 (C-170), 128.3 (C-18), 128.3 (C-180), 126.5 (C-19), 133.9 (C-20),
130.2 (C-21), 130.2 (C-210), 129.8 (C-22), 129.8 (C-220), 127.1 (C-23), terminal
amino acid N-monomethylvaline [171.9, 56.6, 30.6, 18.8, 18.6, 41.8]; HRMS, m/
z: 540.2740 (C32H36N4O4), 455.1850 (C27H25N3O4), 427.1896 (C26H25N3O3),
412.1784 (C26H24N2O3), 371.1776 (C24H23N2O2), 308.1159 (C18H16N2O3),
278.1176 (C18H16NO2), 250.1235 (C17H16NO), 224.1076 (C15H14NO), 215.0824
(C12H11N2O2), 187.0875 (C11H11N2O), 135.0682 (C8H9NO), 131.0496 (C9H7O),
120.0812 (C8H10N), 102.0842 (C8H6), and 86.0970 (C5H12N).
3.3.5. Methylation of xylopyrine-H (2)
Compound 2 (8 mg) was treated with HCHO and NaBH4 by slow addition and
checking the reaction mixture by TLC. On usual work-up and crystallisation
from MeOH, it furnished the N-methylated product 7 as colourless granules, m.p.
282–284ꢂC; MS, m/z: 554.2898 ([M]þ, C33H35N4O4). It was identical to alkaloid
integerrissine (Lagarias et al., 1979) (mmp, co-TLC and super-imposable IR) on
comparison with an authentic sample.
3.3.6. Hydrolysis of xylopyrine-G (1) and xylopyrine-H (2)
Compounds 1 and 2 (6 mg each) were hydrolysed separately with 6 N HCl (1 mL) by
heating in a sealed tube on oil bath at 120ꢂC for 24 h. The hydrolysates were
examined by paper chromatography (solvent C) and the spots visualised by spraying
with ninhydrin. Compound 1 showed two spots on paper chromatogram for
phenylalanine and monomethylleucine, whereas compound 2 exhibited spots for
phenylalanine and monomethylvaline, identified by comparison with authentic
samples.
3.3.7. Partial hydrolysis of xylopyrine-A (1), xylopyrine-B (2) and
xylopyrine-C (3)
The partial hydrolysis of compounds 1 (6 mg), 2 (7 mg) and 3 (7 mg) was done
by heating with 4 mL of a mixture of conc. HCl–HOAc–H2O (1 : 1 : 1) separately on
water bath for 5 h. On usual work-up, they furnished identical compound 4 as a
colourless amorphous solid; MS, m/z: 407.1824 ([M]þ, C23H25N3O4). Compound 4
on hydrolysis with 6 N HCl in a sealed tube for 20 h a 120ꢂC on oil bath gave
phenylalanine (co-PC with authentic sample).
Acknowledgements
The authors thank Prof G. Rucker, ex-Director, Pharmazentisches Institut der Universitat
¨
¨
Bonn, Germany for the spectral analysis.

Natural Product Research
841
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