Phytochemistry 57 (2001) 1277–1280
www.elsevier.com/locate/phytochem
Haplophytin-A and B: the alkaloidal constituents of
Haplophyllum acutifolium
Muhammad Shaiq Alia,*, Muhammad Kashif Perveza, Muhammad Saleema,
Rasool Bakhsh Tareenb
a
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
b
Department of Botany, Baluchistan University, Quetta (Baluchistan), Pakistan
Received 13 September 2000; received in revised form 30 November 2000
Dedicated to my PhD research supervisor, Professor Viqar Uddin Ahmad on his retirement
Abstract
During the phytochemical investigation of Haplophyllum acutifolium, two alkaloids named haplophytin-A (1) and B (2) have
been obtained. In addition, some known constituents: flindersine (3), kusunokinin (4), b-sitosterol, oleanolic acid, cholesterol and
hexadecanoic acid, have also been obtained. The known constituents have never been obtained from this source. The structures of
all the isolated constituents were elucidated by spectroscopic means. However, the structures of constituents 1 and 2 were further
confirmed through HMBC technique. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Haplophyllum acutifolium; Rutaceae; Haplophytin-A; Haplophytin-B; Structure elucidation; Spectroscopy
1. Introduction
The genus Haplophyllum belongs to the family Rutaceae having about 70 species distributed in the Mediterranean countries. Out of 70, seven are found in
Pakistan. Chemical literature survey reveals the presence of lignans (Gozler et al., 1996; Evcium et al., 1986;
Gozler and Gozler, 1984), lignan-glycosides (Gozler et
al., 1994), coumarins (Ulubelen et al., 1993), sterols,
flavonoids (Yuldasher et al., 1987), several classes of
alkaloids [furoquinoline (Gozler et al., 1996), quinoline
(Sheriha et al., 1987), pyranoquinoline (Ulubelen, 1985)
and tetrahydrofuroquinoline (Rozsa et al., 1986)] in
various species of the genus Haplophyllum. Several biologically active secondary metabolites have been detected
from various Haplophyllum species (Sheriha et al., 1987).
The presence of variety of biologically active chemical
constituents form the members of the genus Haplophyllum motivated us to start our phytochemical investigation on H. acutifolium. In the present communication,
we wish to describe the isolation and characterization of
two alkaloids named haplophytin-A and B, and several
* Corresponding author. Tel.: +92-21-496-8497; fax: +92-21-4963373.
E-mail address: shaiq303@super.net.pk (M.S. Ali).
known constituents [flindersine (3), kusunokinin (4), bsitosterol, oleanolic acid, cholesterol and hexadecanoic
acid] from the titled source.
2. Results and discussion
Haplophytin-A (1) was isolated from the methanol
soluble part of Haplophyllum acutifolium. Lack of optical rotation means that there is probably no chirality in
the molecule. The compound 1 gave positive response
with UV light (254 nm) and Dragendroff’s reagent confirming the presence of conjugated/aromatic system and
nitrogen atom, respectively. The presence of unsaturation was further determined through infra-red spectrum
which showed an absorption at 1500 cm1 (C¼C) together with other absorption bands at 3260 and 1660 cm1
due to the N–H and conjugated amide-carbonyl,
respectively. The molecular weight of 1 was confirmed
with the help of FDMS and found 257 a.m.u. The formula of the corresponding peak was depicted from HR–
EIMS as C15H15NO3.
The proton NMR of 1 showed the presence of three
quaternary methyl signals, out of them one appeared at
3.90 and the remaining two at 1.51 as a common
signal of six protons. The spectrum did not contain any
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M.S. Ali et al. / Phytochemistry 57 (2001) 1277–1280
methylene however, the same spectrum showed five
downfield methine signals at 7.26 (dd, J=8.8, 2.8 Hz,
H-6), 7.10 (d, J=8.8 Hz, H-7), 7.25 (dd, 8.8, 2.8 Hz, H8), 5.54 (d) and 6.76 (d). The two upfield methine signals
at 5.54 and 6.76 having the same magnitude of coupling constant (J=9.9 Hz) confirming the vicinal orientation attested for C-11 and C-12. The chemical shifts of
remaining three methine signals at 7.26, 7.10 and 7.25
concluded the presence of aromatic ring in the molecule.
The multiplicities of these three methine signals and
their corresponding coupling constants showed that
they are arranged in the manner that the H-6 and H-8
are ortho to H-7. The methoxyl moiety could be fixed
either at C-5 or C-8 which was partially confirmed
through HMBC experiment (Fig. 1) and finally, by
compression with the data of closely related published
compounds (Huffman and Hau, 1972).
The broad-band spectrum of 1 displayed 15 carbon
signal which were sorted out into three methyls, five
methines and remaining seven quaternary carbons. The
chemical shift of methoxyl moiety ( 55.7) inferred the
direct attachment of this moiety to aromatic ring. The
position of this methoxyl moiety at C-5 was determined
through NOE experiment. Upon irradiation at 1.51
(H-14, H-15), a 33 and 16% NOE effect of the signals of
H-12 and OMe, respectively, was observed. This 16%
NOE effect would not have been observed if the methoxyl moiety had been situated at C-8. The other two
quaternary methyls (C-14 and C-15) appeared at 28.0,
found to be attached with a quaternary carbon connected to the electron withdrawing hetero-atom and
thus assigned to C-13 ( 79.0). The most downfield signal at 162.4 was due to the conjugated amide whereas
the second quaternary downfield signal at 155.0 was
due to the carbon at which methoxyl moieties is
attached (C-5). The two methine signals of the ring
having the oxygen as an hetero-atom appeared at
126.2 and 117.5 due to C-11 and C-12, respectively. The
remaining three aromatic methine signals were observed
in the carbon spectrum at their usual positions ( 103.5,
120.5 and 117.4).
With the help of spectral evidences given earlier, the
structure of the discussed compound was assigned as 1
Fig. 1. HMBC connectivities.
and named haplophytin-A which is a new addition in
the alkaloidal constituents of Haplophyllum acutifolium.
Although similar types of compounds are already
reported in the literature (Huffman and Hau, 1972) but
having no methoxyl at C-5.
Haplophytin-B (2) was obtained from the same
extract with the elution of 45% ethyl acetate in hexane.
It showed positive UV response confirming the presence
of conjugated/aromatic system in the molecule. The
presence of unsaturation was supported by the absorption at 1500 cm1 (C¼C) in the IR spectrum which also
showed an intense absorption band at 3320 cm1 due to
the hydroxyl function in the molecule. The molecular
mass of 2 was determined through FDMS and found
347 a.m.u. The formula of this peak was observed as
C18H21NO6 with the aid of HRMS.
The proton spectrum of 2 exhibited four methyl resonances, out of them two at 4.47 and 4.10 due to the
methoxyl moieties and their chemical shifts suggested
direct attachment to the aromatic system. The remaining two quaternary methyl signals appeared at 1.29
and 1.25 (H-40 and H-50 ). The molecule contains only
one methylene appeared as two separate sets of doubledoublets at 4.13 (J=9.9, 8.0 Hz, Ha-0 1) and 3.81
(J=8.0, 2.5 Hz, Hb-0 1). The same spectrum showed four
downfield methine signals at 8.01 (d, J=9.4 Hz, H-5),
7.34 (d, J=9.4 Hz, H-6), 7.73 (d, J=2.8 Hz, H-a) and
7.30 (d, J=2.8 Hz, H-b) and an upfield carbinylic proton at 4.49 (H-0 2) as a double-doublet (J=9.9, 2.5 Hz).
The signals at 8.01 and 7.34 and their same coupling
constant confirmed their ortho arrangement and thus
assigned to H-5 and H-6, respectively. The methine
signal at 7.73 showed the presence of an electron
withdrawing hetero-atom in the vicinity and attested
for H-a. With the help of coupling constant of H-a
(J=2.8 Hz) the H-b was located in the spectrum at
7.30.
The broad-band spectrum of haplophytin-B exhibited
18 carbon signals which were resolved into four methyls,
one methylene, five methines and remaining eight quaternary carbon atoms. Out of four methyl signals, two
were at 60.0 and 61.9 due to the methoxyl moieties in
the molecule and their positions were fixed through
HMBC experiment (Fig. 2) and literature values (Bessonova and Yunusov, 1986; Rasulova et al., 1987). The
remaining two methyl signals appeared at 25.0 and
26.9 due to the C-40 and C-50 , respectively. The only one
methylene (C-10 ) present in the molecule resonated at
72.5. An upfield methine signal resonated at 77.8 corresponded to the carbinylic carbon. The molecule contains four downfield methine signals at 119.5 (C-5),
114.0 (C-6), 144.3 (C-a) and 106.5 (C-b). The most
downfield methine signal at 144.3 was due to the direct
attachment to an hetero-atom (oxygen). A hydroxyl
function attached to the quaternary carbon at which
two quaternary methyls are attached appeared at 72.8.
M.S. Ali et al. / Phytochemistry 57 (2001) 1277–1280
1279
Fig. 2. HMBC connectivities.
Three more quaternary carbons resonated at 153.9
(C-4), 142.1 (C-8) and 142.9 (C-7) were due to the two
methoxyl and a side chain containing carbons, respectively. The position of side chain at C-7 was determined
through HMBC connectivities (Fig. 2) and n.O.e measurements. Upon irradiation at 4.10 (C-8 OMe), a 29
and 13% NOE effect of the signals of H-20 and H-10 ,
respectively, was observed suggested the attachment of
side chain at C-3. Such a type of skeleton is already
reported in literature with either different side chains or
sugar units (Bessonova and Yunusov, 1986; Rasulova et
al., 1987) and therefore, the previously discussed compound is assigned as 2 and named haplophytin-B. This
compound is also a new addition in the alkaloidal constituents of H. acutifolium.
In addition to 1 and 2, six more known constituents
have been detected which have never been reported so
far from our investigated source and their data are given
in Section 3. The new constituents, haplophytin-A and
B were found totally inactive against various tested
bacteria and fungi.
3. Experimental
3.1. General
3.3. Extraction and isolation
The 1H and 13C NMR spectra were recorded at 500,
400, 300 and 75,100, 125 MHz, respectively, on Brüker
AM-500, AM-400 and AM-300 in CDCl3.
3.2. Collection and identification
The plant material was collected from Quetta (Pakistan) in May 1999 and identified by Dr. R.B. Tareen,
Department of Botany, Baluchistan University, Quetta
(Baluchistan), where the voucher specimen has been
deposited in the herbarium.
The collected plant material (15 kg) was dried under
shade and chopped into small pieces. The dried and
chopped material (8 kg) was soaked in MeOH (14 l) for
a period of 10 days. The percolation was repeated three
times. The MeOH was evaporated under reduced temperature (28 C) and the gummy material thus obtained
(276 g) was subjected to silica gel column chromatography using hexane, hexane–ethyl acetate, ethyl acetate, ethyl acetate–methanol and finally, pure methanol
as mobile phase.
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M.S. Ali et al. / Phytochemistry 57 (2001) 1277–1280
The fraction obtained with 15% ethyl acetate in hexane showed a strong UV-active spot on TLC with some
minor impurities which on washing with methanol yielded 1 as a white powder (13 mg). The same UV active
spot on spraying with Dragendroff’s reagent changed
into an orange spot confirming the presence of nitrogen
in the molecule.
Haplophytin-A (1): m.p.: 209–210.5 C; [a]D: 0 (CHCl3,
c 0.506 ); UV lmax CHCl3 nm (log "): 244 (7.92); IR
(CHCl3): 3260 (N–H), 1660 (C¼O), 1500 (C¼C) cm1;
EI–MS: m/z 257 [M+], 242 [M–Me]+ (100%), 199, 121,
106; FDMS: m/z 257; HR–MS: m/z 257.9968 (C15H15
NO3 requires m/z 257.10518); 1H NMR (CDCl3, 400
MHz): 7.26 (1H, dd, J=8.8, 2.8 Hz, H-6), 7.25 (H, dd,
J=8.8, 2.8 Hz, H-8), 7.10 (1H, d, J=8.8 Hz,H-7), 6.76
(1H, d, J=9.9 Hz, H-12), 5.54 (1H, d, J=9.9 Hz, H-11),
3.90 (3H, s, OMe) and 1.51 (6H, s, H-14, 15); 13C NMR
(CDCl3, 100 MHz): 162.4 (C-2), 115.7 (C-3), 156.0 (C-4),
155.0 (C-5), 103.5 (C-6), 120.5 (C-7), 117.4 (C-8), 132.9
(C-9), 106.5 (C-10), 126.2 (C-11), 117.5 (C-12), 79.0 (C13), 28.0 (C-14, 15) and 55.7 (OMe); HMBC: see Fig. 1.
Elution with 45% ethyl acetate in hexane from the
same column afforded another UV and Dragendroff’s
reagent positive sample as a dirty white powder (16.5
mg) which on washing with a 1:1 mixture of chloroform
and hexane yielded 2.
Haplophytin-B (2): mp: 151.5–153 C; [a]D: +63.6
(MeOH, c 0.33 ); UV lmax CD3OD nm (log "): 251
(5.16); IR (KBr): 3320 (OH), 1500 (C=C) cm1; EI–
MS: m/z 347 [M]+, 287, 227 [M–side chain+] (100%),
216; FDMS: m/z 347; HR–MS: m/z 347.13711
(C18H21N O6 requires m/z 347.13687); 1H NMR
(CD3OD, 500 MHz): 8.01 (1H, d, J=9.4 Hz, H-5),
7.73 (1H, d, J=2.8 Hz, H-a), 7.34 (1H, d, J=9.4 Hz, H6), 7.30 (1H, d, J=2.8 Hz, H-b), 4.49 (1H, dd, J=9.9,
2.5 Hz, H-0 2), 4.47 (3H, s, C-4 OMe), 4.13 (1H, ds,
J=9.9, 8.0 Hz, Ha-0 1), 4.10 (3H, s, C-8 OMe), 3.81 (1H,
ds, J=8.0, 2.5 Hz, Hb-0 1), 1.29 (3H, s, H-0 4), 1.25 (3H,
s, H-0 5); 13C NMR (CD3OD, 125 M Hz): 165.8 (C-2),
115.9 (C-3), 153.9 (C-4), 119.5 (C-5), 114.0 (C-6), 142.9
(C-7), 142.1 (C-8), 159.0 (C-9), 103.3 (C-10), 144.3 (Ca), 106.5 (C-b), 61.9 (C-8 OMe), 60.0 (C-4 OMe), 72.5
(C-0 1), 77.8 (C-0 2), 72.8 (C-0 3), 25.0 and 26.9 (C-0 4, C-0 5);
HMBC: see Fig. 2.
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