New secondary metabolites from Allium victorialis

Article abstract of DOI:10.1002/hlca.201200581

Allumines A and B (1 and 2, resp.), two new steroidal alkaloids, and a new cyclopentene derivative, 3, were isolated from the CHCl₃-soluble fraction of the whole plant of Allium victorialis. Their structures were elucidated by spectroscopic techniques, including 1D- and 2D-NMR spectroscopy. Copyright

Full text of DOI:10.1002/hlca.201200581

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Helvetica Chimica Acta – Vol. 96 (2013)
New Secondary Metabolites from Allium victorialis
by Sadia Khana), Itrat Fatimab), Mehdi Hussain Kazmia), and Abdul Malik*b)
a) Department of Applied Chemistry, University of Karachi, Karachi-75270, Pakistan
b) International Centre for Chemical and Biological Sciences, HEJ Research Institute of Chemistry,
University of Karachi, Karachi-75270, Pakistan (phone: þ 92-21-4824926;
e-mail: abdul.malik@iccs.edu)
Allumines A and B (1 and 2, resp.), two new steroidal alkaloids, and a new cyclopentene derivative,
3, were isolated from the CHCl3-soluble fraction of the whole plant of Allium victorialis. Their structures
were elucidated by spectroscopic techniques, including 1D- and 2D-NMR spectroscopy.
Introduction. – The plant family Alliaceae comprises of 21 genera. The largest
genus of this family is Allium with ca. 600 species distributed in Asia, Europe, and
North West America. In Pakistan, 41 species of this genus have so far been discovered
[1]. Various Allium species are used for the treatment of a variety of ailments [2 – 5].
One of the species is Allium victorialis, a shrub occurring in Europe, temperate Asia to
Japan, and North West America. It grows in northern mountainous regions of Pakistan
[1]. It is used by local population as antithrombotic [6] and antiscorbutic agent [1], and
to treat profuse menstruation and cold. Previously, a cyclopentane derivative has been
reported by us from this plant [7]. The chemotaxonomic and ethnopharmacological
importance of the genus Allium prompted us to carry out further phytochemical studies
on A. victorialis. As a result, we herein report two new steroidal alkaloids, named
allumines A and B (1 and 2, resp.), along with a new cyclopent-1-enecarboxylate 3.
Results and Discussion. – The EtOH extract of the whole plant was partitioned into
fractions soluble in hexane, CHCl3, AcOEt, BuOH, and H2O. Column chromatography
of the CHCl3-soluble fraction provided compounds 1 – 3 as described in the Exper. Part
(Fig. 1).
Allumine A (1) was obtained as a colorless gummy solid. The molecular formula
C36H53NO3 was deduced from HR-EI-MS with Mþ peak at m/z 547.4025. The IR
spectrum exhibited the absorption bands of OH/NH (3450 – 3330 cmꢀ1) and ester C¼O
groups (1700 cmꢀ1), C¼C bond (1660 cmꢀ1), and aromatic moiety (1602, 1540, and
1500 cmꢀ1). In the EI-MS, the fragment-ion peak at m/z 398 resulted from the loss of a
hydroxy-dimethyl-benzoyl moiety. The base peak at m/z 98 originated from a bond
cleavage between C(20) and C(22) of 20,26-epiminocholestane [8], and a further
intense peak at m/z 126 was due to bond cleavage between C(17) and C(20). These data
indicated that the compound 1 was a dihydroverazine-type alkaloid [9]. The 13C-NMR
and DEPT spectra (Table 1) showed 36 signals for six Me, eleven CH2, and eleven CH
groups, and eight quaternary C-atoms. The ester C¼O resonated at d(C) 170.0, while
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 96 (2013)
1177
Fig. 1. Structures of allumines A and B (1 and 2, resp.), and compound 3
the olefinic C-atom signal appeared at d(C) 137.9 and 122.1. The CHꢀO C-atom
resonated at d(C) 79.0, and the signals of four Me groups of the steroidal nucleus were
detected at d(C) 11.9, 13.6, 19.3, and 19.6, respectively. Two aromatic Me signals
1
appeared together as a singlet at d(C) 16.9. In the H-NMR spectrum, signals for
Me(18) and Me(19) groups of a ꢂnormalꢃ steroid ring system with a C¼C bond were
observed as singlets at d(H) 0.71 and 1.02, and a vinyl H-atom at C(6) resonated at
d(H) 5.24. In addition, two Me signals appeared as doublets at d(H) 0.90 (J ¼ 6.5) and
0.81 (J ¼ 6.5), which were attributed to Me(21) and Me(27), respectively. The CHꢀO
H-atom resonated as a multiplet at d(H) 3.18 – 3.20, and the w1/2 value of 23 Hz
confirmed its axial orientation. The relative configuration of the Me-substituted
piperidine moiety was determined as (22R,25S) by NOEs (Fig. 2).
Fig. 2. NOE Correlations (H $ H) of piperdine moiety of 1
Basic hydrolysis of 1 provided 4-hydroxy-3,5-dimethylbenzoic acid and a base
which gave a positive digitonine test, characteristic of a genuine steroid with a 3b-OH
group [10]. Its physical and NMR spectral data showed complete agreement with those
of oblonginine [11] which is a 22-epimer of veramiline [10]. Thus, compound 1 is a 3-O-
benzoyl derivative of oblonginine. The presence of 4-hydroxy-3,5-dimethylbenzoyloxy
group at C(3) was concluded by the downfield shift of C(3) in 13C-NMR spectrum
compared to that of oblonginine, and also by HMBC experiment showing 3J correlation

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Helvetica Chimica Acta – Vol. 96 (2013)
Table 1. 1H- and 13C-NMR Data (500 and 125 MHz, resp.; CDCl3) of Compounds 1 and 2. Atom
numbering as indicated in Fig. 1; d in ppm, J in Hz.
Position
1
2
d(H)
d(C)
d(H)
d(C)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1’
2’,6’
3’,5’
4’
7’
1.48 – 1.50 (m)
1.51 – 1.52 (m)
3.18 – 3.20 (m)
1.58 – 1.61 (m)
–
5.24 (br. s)
1.61 – 1.62 (m)
1.88 – 1.89 (m)
1.06 – 1.08 (m)
–
0.80 – 0.81 (m)
0.98 – 0.99 (m)
–
1.98 – 2.00 (m)
0.92 – 0.93 (m)
1.87 – 1.88 (m)
1.15 – 1.17 (m)
0.71 (s)
38.5
29.7
79.0
39.0
137.9
122.1
31.9
32.9
52.7
35.0
22.6
39.6
42.0
56.6
24.1
28.0
54.1
11.9
19.3
39.5
13.6
59.8
24.6
33.2
30.9
54.2
19.6
124.4
129.6
130.0
160.7
170.0
16.9
1.48 – 1.49 (m)
1.50 – 1.52 (m)
3.17 – 3.18 (m)
1.57 – 1.58 (m)
–
38.2
29.2
79.2
39.3
138.1
122.3
32.0
33.1
52.5
35.2
22.6
40.0
42.6
56.9
24.3
28.4
54.4
11.9
19.5
39.8
13.8
60.0
24.6
33.8
31.2
54.4
19.7
12.4
129.7
130.0
162.1
170.1
16.9
102.3
75.4
79.1
71.2
78.5
61.8
5.24 (br. s)
1.61 – 1.63 (m)
1.87 – 1.88 (m)
1.07 – 1.08 (m)
–
0.79 – 0.81 (m)
0.98 – 0.99 (m)
–
1.96 – 1.97 (m)
0.91 – 0.93 (m)
1.86 – 1.87 (m)
1.15 – 1.17 (m)
0.73 (s)
1.02 (s)
1.03 (s)
0.96 – 0.98 (m)
0.90 (d, J ¼ 6.5)
2.50 (dt, J ¼ 10.8, 2.0)
1.44 – 1.45 (m)
1.86 – 1.88 (m)
1.41 – 1.43 (m)
2.24 – 2.26 (m)
0.81 (d, J ¼ 6.5)
–
8.08 (s)
–
–
–
0.96 – 0.97 (m)
0.90 (d, J ¼ 6.5)
2.50 (dt, J ¼ 11.0, 2.2)
1.46 – 1.47 (m)
1.88 – 1.89 (m)
1.41 – 1.42 (m)
2.25 – 2.26 (m)
0.83 (d, J ¼ 6.5)
–
8.08 (s)
–
–
–
MeꢀC(3’,5’)
2.01 (s)
2.02 (s)
1’’
2’’
3’’
4’’
5’’
6’’
5.85 (d, J ¼ 7.5)
4.35 – 4.36 (m)
4.21 – 4.23 (m)
4.36 – 4.38 (m)
4.38 – 4.39 (m)
4.51 (dd, J ¼ 2.0, 11.0), 4.60 (dd, J ¼ 6.1, 11.0)
of HꢀC(3) with the C¼O C-atom of the ester resonating at d(C) 170.0. The structure of
allumine 1 was, therefore, deduced as 3b-O-(4-hydroxy-3,5-dimethylbenzoyl)oblongi-
nine (¼(3b,17b)-17-{(1S)-1-[(2R,5S)-5-methylpiperidin-2-yl]ethyl}androst-5-en-3-yl 4-
hydroxy-3,5-dimethylbenzoate).
Allumine B (2) was obtained as a colorless gummy solid. Its molecular formula was
deduced as C42H63NO8 by a quasimolecular-ion peak at m/z 708.4475 in negative-ion

Helvetica Chimica Acta – Vol. 96 (2013)
1179
mode HR-FAB-MS. The IR spectrum was similar to that of 1. The 13C-NMR and
DEPT spectra showed 42 signals for six Me, twelve CH2, and 16 CH groups, and eight
quaternary C-atoms. They showed close similarities to those of 1 except additional
peaks due to a hexose moiety (anomeric C-atom at d(C) 102.3, and further signals of
CHꢀO and CH2O C-atoms in the range of 79.1 – 61.8). The 1H-NMR spectrum was also
similar to that of compound 1 with an additional signal of an anomeric H-atom of the
hexose moiety as a doublet at d(H) 5.85 (J ¼ 7.5), and further signals of CHꢀO and
CH2O H-atoms in the range of 4.60 – 4.21. The larger value of coupling constant of the
anomeric H-atom allowed us to assign b-configuration to the hexose moiety. The EI-
MS showed the base peak at m/z 98, and intense peaks at m/z 398 and 126 were
common to compound 1, revealing the presence of hexose moiety in the aromatic ester.
Basic hydrolysis and extraction with CHCl3 furnished oblonginine and glycoside of an
aromatic compound, which, on subsequent acid hydrolysis, provided 4-hydroxy-3,5-
dimethylbenzoic acid, and the glycone which could be identified as d-glucose by sign of
its optical rotation and co-TLC with an authentic sample. The anomeric H-atom
3
resonating at d(H) 5.85 showed J correlation with C(4’) in HMBC spectrum of 2,
confirming the presence of the glucose moiety at C(4’). Allumine B (2) is therefore, 3b-
O-[4-(b-d-glucopyranosyloxy)-3,5-dimethylbenzoyl]oblonginine (¼(3b,17b)-17-{(1S)-
1-[(2R,5S)-5-methylpiperidin-2-yl]ethyl}androst-5-en-3-yl 4-(b-d-glucopyranosyloxy)-
3,5-dimethylbenzoate).
Compound 3 was obtained as a white crystalline solid. Its HR-EI-MS showed Mþ
peak at m/z 636.4542 consistent with the molecular formula C44H60O3. The IR spectrum
evidenced the presence of a conjugated C¼O group (1665 cmꢀ1), conjugated olefinic
C¼C bond (1625 cmꢀ1), and aromatic moieties (1540, 1500 cmꢀ1). The 13C-NMR and
DEPT spectra showed 44 signals for one Me, 22 CH2, and 13 CH groups, and eight
quaternary C-atoms (Table 2). The most downfield signal at d(C) 164.0 was assigned to
the ester C¼O group, while the signals of a disubstituted cyclopentene moiety were
observed at d(C) 37.6, 32.0, and 23.2. The signals of olefinic C-atom appeared at d(C)
155.6 and 123.4. The signals between d(C) 155.4 and 115.1 were due to aromatic C-
Table 2. 1H- and 13C-NMR Data (500 and 125 MHz, resp.; CDCl3) of Compound 3. Atom numbering as
indicated in Fig. 1; d in ppm, J in Hz.
Position
d(H)
d(C)
Position
d(H)
d(C)
1
2
3
4
5
6
1’
2’,6’
3’,5’
4’
1’’
2’’,6’’
3’’,5’’
–
–
123.4
155.6
37.6
23.2
32.0
164.0
125.6
134.1
130.1
125.9
150.0
119.5
129.2
4’’
7’’
8’’
1’’’
2’’’,6’’’
3’’’,5’’’
4’’’
7’’’
8’’’
9’’’ – 21’’’
22’’’
23’’’
24’’’
–
137.2
37.3
70.5
155.4
115.1
128.7
133.2
35.0
31.8
29.6
31.9
22.6
1.27 – 1.28 (m)
3.63 (t, J ¼ 6.0)
–
6.74 (d, J ¼ 8.5)
7.15 (d, J ¼ 8.5)
–
1.24 (br. s)
1.24 (br. s)
1.24 (br. s)
1.24 (br. s)
1.24 (br. s)
0.85 (t, J ¼ 7.0)
1.27 – 1.28 (m)
1.54 – 1.56 (m)
1.22 – 1.23 (m)
–
–
7.64 – 7.69 (m)
7.91 – 7.95(m)
7.89 – 7.90 (m)
–
7.07 (d, J ¼ 9.0)
7.52 (d, J ¼ 9.0)
14.1

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Helvetica Chimica Acta – Vol. 96 (2013)
atoms, while CH2ꢀO C-atom signal was observed at d(C) 70.5. An octadecyl moiety
was evident by the terminal Me resonance at d(C) 14.1, and signals of 17 CH2 groups in
the range of 35.0 – 22.6. In the 1H-NMR spectrum, The H-atoms of two para-
substituted phenyl rings formed two AA’BB’ systems resonating at d(H) 7.07 (d, J ¼ 9.0,
2 H), 7.52 (d, J ¼ 9.0, 2 H), 6.74 (d, J ¼ 8.5, 2 H), and 7.15 (d, J ¼ 8.5, 2 H), respectively.
The signals of octadecyl moiety were observed at d(H) 1.24 (br. s, 17 CH2), and a triplet
of terminal Me group at d(H) 0.85 (J ¼ 7.0 ) . The CH2ꢀO H-atoms resonated at d(H)
3.63 (t, J ¼ 6.0). The assignments of various signals were accomplished with the help of
1H,1H-COSY and HMQC experiments, and supported by HMBC features, in which
HꢀC(8’’) exhibited 2J correlation with C(7’’) (d(C) 37.3), as well as 3J correlations with
2
C(4’’) (d(C) 137.2) and C(1’’’) (155.4) (Fig. 3). The HꢀC(5) ((d(H) 1.22) showed J
correlation with C(1) (d(C) 123.4) and C(4) (23.2), as well as 3J correlations with C(2)
(d(C) 155.6) and C(3) (37.6). Further HMBC features were in complete agreement
with the assigned structure of compound 3 as 4-{[2-(4-octadecylphenyl)oxy)]ethyl}-
phenyl 2-phenylcyclopent-1-ene-1-carboxylate (Fig. 3).
Fig. 3. Key HMBCs (H ! C) of compound 3
Experimental Part
General. Arachidonic acid and sodium citrate were purchased from the Sigma Chemical Co. (St.
Louis, Mo. USA). All other chemicals used were of the highest purity grade available. TLC: Silica-gel 60
F254 plates (SiO2; E. Merck, D-Darmstadt). Column chromatography (CC): SiO2 (250 – 400 mesh; E.
Merck, D-Darmstadt). Optical rotations: Jasco DIP-360 digital polarimeter. IR Spectra: Jasco 302-A
spectrophotometer; in KBr; ˜n in cmꢀ1. NMR Spectra: Bruker 500 MHz instrument; d in ppm rel. to Me4Si
as internal standard, J in Hz. EI- and HR-FAB-MS: Jeol JMS-HX-110 and JMS-DA-500 mass
spectrometers with glycerol as matrix; in m/z (rel. %).
Plant Material. The whole plant material of Allium victorialis L. was collected from northern areas of
Pakistan in 2004 and identified by Dr. Surraiya Khatoon, Plant Taxonomist, Department of Botany,
University of Karachi, Karachi, Pakistan, where a voucher specimen has been deposited with the
herbarium (voucher specimen No. 202/KUH).
Extraction and Isolation. The freshly collected whole plants of A. victorialis (20 kg) were shade-
dried, ground, and extracted with EtOH (3 ꢁ 40 l, 10 d each) at r.t. The combined EtOH extract was
evaporated under reduced pressure to yield a residue (800 g) which was suspended in H2O (1.0 l), and
successively extracted with hexane (80 g), CHCl3 (170 g), AcOEt (220 g), and BuOH (150 g). The
CHCl3-soluble fraction (80 g) was subjected to CC (SiO2; hexane, hexane/CHCl3, CHCl3, and CHCl3/
MeOH in increasing order of polarity) to obtain 20 subfractions. The subfraction which eluted with
hexane/CHCl3 3.0 :7.0 gave one major spot on TLC. It was purified by prep. TLC (hexane/CHCl3 3.5 :6.5)
to furnish compound 3 (16 mg). The subfraction which eluted with CHCl3/MeOH 9.8 :0.2 was
rechromatographed and eluted with same solvent system to obtain allumine A (1; 18 mg). The
subfraction which eluted with CHCl3/MeOH 9.5 :0.5 was rechromatographed and eluted with CHCl3/
MeOH 9.0 :1.0 to provide allumine B (2; 15 mg).

Helvetica Chimica Acta – Vol. 96 (2013)
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Allumine A (¼(3b,17b)-17-{(1S)-1-[(2R,5S)-5-Methylpiperidin-2-yl]ethyl}androst-5-en-3-yl 4-Hy-
droxy-3,5-dimethylbenzoate ¼ 3b-O-(4-Hydroxy-3,5-dimethylbenzoyl)oblonginine; 1). Colorless gummy
solid. [a]2D0 ¼ ꢀ53 (c ¼ 0.02, CHCl3). IR (KBr): 3450 – 3330, 1700, 1660, 1602, 1540, 1500. 13C- and
1H-NMR: see Table 1. EI-MS: 547 (9, Mþ), 398 (20), 126 (28), 98 (100). HR-EI-MS: 547.4025 (Mþ,
C36H53NOþ3 ; calc. 547.4029).
Allumine B (¼(3b,17b)-17-{(1S)-1-[(2R,5S)-5-Methylpiperidin-2-yl]ethyl}androst-5-en-3-yl 4-(b-d-
Glucopyranosyloxy)-3,5-dimethylbenzoate ¼ 3b-O-[4-(b-d-Glucopyranosyloxy)-3,5-dimethylbenzoyl]-
oblonginine; 2). Colorless gummy solid. [a]2D0 ¼ ꢀ60 (c ¼ 0.03, CHCl3). IR (KBr): 3450 – 3330, 1700,
1
1660, 1602, 1540, 1500. 13C- and H-NMR: see Table 1. EI-MS: 546 (7, [M ꢀ 162 ꢀ H]þ), 398 (18), 126
(30), 98 (100). HR-FAB-MS (neg.): 708.4475 ([M ꢀ H]ꢀ, C42H62NO8ꢀ ; calc. 708.4479).
4-{[2-(4-Octadecylphenyl)oxy)]ethyl}phenyl 2-Phenylcyclopent-1-ene-1-carboxylate (3). White crys-
talline solid. M.p. 82 – 838. IR (KBr): 1665, 1625, 1540, 1500. 13C- and 1H-NMR: see Table 2. HR-EI-MS:
636.4542 (Mþ, C44H60Oþ3 ; calc. 636.4545).
Alkaline Hydrolysis of 1. The compound 1 (8 mg) was added to a soln. of 4% NaOH (2 ml), MeOH
(6 ml), and H2O (1.5 ml). The suspension was warmed gently on a steam-bath, until a vigorous
exothermic reaction started, and in 10 min the entire solid had dissolved. The soln. was then heated under
reflux for further 5 min. H2O (3 ml) was then added, the MeOH was removed in vacuo and repeatedly
extracted with CHCl3. The residue from the org. phase crystallized from acetone/hexane, m.p. 2208,
[a]2D3 ¼ ꢀ40.3 (c ¼ 0.14; CHCl3). Its physical and spectral data were in complete agreement with those
reported in literature for oblonginine [11].
The alkaline soln. and H2O washings were combined and acidified to pH 2 with 0.1n HCl, and the
resulting precipitate was filtered and crystallized from benzene, m.p. 2268. Its physical and spectral data
corresponded to those reported in literature for 4-hydroxy-3,5-dimethylbenzoic acid [12].
Hydrolysis of Compound 2. Alkaline hydrolysis of compound 2 was carried out as described for
compound 1 to obtain oblonginine. The basic soln. was acidified with 0.1n HCl to pH 2 and freeze-dried.
The residue was subjected to acid hydrolysis by refluxing with 10% aq. HCl for 3 h at 1008. On cooling,
the aq. fraction was extracted with CHCl3. The CHCl3 fraction was repeatedly washed with H2O, dried
(Na2SO4), and freed of solvent. The residue crystallized from benzene to furnish 4-hydroxy-3,5-
dimethylbenzoic acid. The aq. phase was neutralized with Ag2CO3 and concentrated. The sugar was
identified as d-glucose through co-TLC with an authentic sample and sign of its optical rotation ([a]D23
þ51).
¼
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Received October 18, 2012