Shahidine, a novel and highly labile oxazoline from Aegle marmelos: the parent compound of aegeline and related amides

Article abstract of DOI:10.1016/j.tet.2008.11.088

A rare alkaloid, shahidine (1), having an unstable oxazoline core has been isolated as a major constituent from the fresh leaves of Aegle marmelos. It is moisture-sensitive, and found to be the parent compound of aegeline and other amides, however, it is

Full text of DOI:10.1016/j.tet.2008.11.088

Tetrahedron 65 (2009) 998–1004
Contents lists available at ScienceDirect
Tetrahedron
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Shahidine, a novel and highly labile oxazoline from Aegle marmelos: the parent
compound of aegeline and related amides
,
b
a
a
b
c
Shaheen Faizi a , Fatima Farooqi , Sadia Zikr-Ur-Rehman , Aneela Naz , Fatima Noor , Farheen Ansari ,
*
Aqeel Ahmad c, Shakeel Ahmed Khan c
a H.E.J Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
b Department of Chemistry, University of Karachi, Karachi-75270, Pakistan
c Department of Microbiology, University of Karachi, Karachi-75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2008
Received in revised form 10 November 2008
Accepted 27 November 2008
Available online 3 December 2008
A rare alkaloid, shahidine (1), having an unstable oxazoline core has been isolated as a major constituent
from the fresh leaves of Aegle marmelos. It is moisture-sensitive, and found to be the parent compound of
aegeline and other amides, however, it is stable in dimethyl sulfoxide. Its structure was established by
spectroscopic analysis. Biogenetically, oxazolines may be considered as the precursor of hydroxy amides
and oxazoles found in plants. Shahidine (1) showed activity against a few Gram-positive bacteria.
Ó 2008 Elsevier Ltd. All rights reserved.
Dedicated to the fond memory of Professor
Salimuzzaman Siddiqui FRS, (1897–1994)
the founding director of HEJ Research In-
stitute of Chemistry, University of Karachi,
Karachi
Keywords:
Aegle marmelos
Oxazoline
Amides
Antibacterial activity
1. Introduction
marmeline acetate (2) have been isolated from the leaves of A.
marmelos. Compound 1 has a rare oxazoline ring and has been
Aegle marmelos (Linn.) Correa, commonly known as Bael, be-
longs to the family Rutaceae, and is distributed throughout tropical
Asia and Africa.1–4 From a medicinal point of view, the Bael tree is
very important and every part of it is used in the Indian system of
medicine;1–4 indeed no drug has been longer or better known to
the people of the Indo-Pakistan subcontinent than Bael.4 The plant
is of very high value in treating cardiac disorders, dysentery, di-
arrhea, diabetes, fever, inflammation, and pain.2,5 The anticancer,
antihyperglycemic, anti-inflammatory, antipyretic, analgesic and
chemoprotective activities of the leaves of the plant have also been
studied.1–5
found to be the parent of aegeline (3)6–9 and related amides (4 and
5)7,10 (Figs. 1 and 2). Oxazoline (1) also possesses antibacterial
activity.
2. Results and discussion
The petroleum ether extract of the fresh leaves of A. marmelos,
on treatment with methanol afforded a methanol-soluble fraction,
which contained one major compound identified as optically
In previous phytochemical investigations on the leaves, alka-
loids, coumarins, flavonoids, steroids, triterpenes, and essential oils
were isolated.5–8 In our continuing search for bioactive compounds
from local medicinal plants, two new compounds shahidine (1) and
* Corresponding author. Tel.: þ92 21 4824912; fax: þ92 21 4819018/19.
E-mail address: shaheenfaizi@hotmail.com (S. Faizi).
Figure 1.
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.11.088

S. Faizi et al. / Tetrahedron 65 (2009) 998–1004
999
in CDCl3 (Table 1) showed a two-proton double doublet at
(J¼1.2, 8.2) and a three-proton multiplet centered at
with two doublets (J¼16.3) each one-proton at 7.40 and 6.68 for
d
7.47
d
7.35, along
d
the trans olefinic protons. These values, which resembled those for
the cinnamide group of 4 (Table 2), have been assigned to H-20,60;
H-30,40,50; H-70; and H-80, respectively, of 1. The HMQC plot has the
interaction points for the correlation of H-20,60 with 127.6 (C-20,60),
H-40 with 129.6 (C-40), H-30,50 with 128.8 (C-30,50), H-70 with 140.5
(C-70), and H-80 with 115.0 (C-80). In the 13C NMR spectrum C-10 and
C-90 resonated at 135.1 and 164.1, respectively. In this case, H-70 and
C-70 resonated at low frequency while H-80 appeared at high fre-
quency and its corresponding carbon as well as C-90 is shifted to
low frequency as compared to that of 4 (Table 2). On the other hand,
monosubstituted aromatic ring (A) protons and carbons have al-
most the same chemical shifts in both the compounds (Table 1, 2).
This indicated that some changes occurred at C-90, and shahidine
has not exactly the same cinnamide moiety as that of 4, instead it
has the conjugated styryl group. Important HMBC interactions of C-
90 with H-70 and H-80; C-10 with H-30, H-50 and H-80; and C-70 with
H-20 and H-60 supported the observations discussed above (Fig. 3).
The 1H NMR spectrum also displayed a three-proton singlet at
Figure 2.
inactive amide (4).7,10 A review of the literature disclosed it to be an
artifact of the isolation procedure and aegeline (3) was suggested as
its precursor,7 which is not comprehensible. In fact methanol being
a poor nucleophile cannot replace the hydroxy group of aegeline,
which is a weak leaving group. This reaction would require strong
acidic conditions while the process employed for isolation of 4 was
mild.7 To solve the discrepancy, the petroleum ether extract of the
leaves of A. marmelos was reinvestigated. Thus TLC and co-TLC of 4
with the extract showed that the latter possessed no trace of amide
4; instead it contained another major compound, having a very
close Rf (0.18, silica gel, CHCl3) value to that of 4 (0.16, silica gel,
CHCl3), which was separated through PTLC and isolated as a yellow
amorphous powder and identified as shahidine (1). It was obtained
in large amounts along with marmeline acetate (2)9 and aegeline
(3)6 through flash column chromatography of the ethyl acetate
soluble fraction of the petroleum ether extract. Oxazoline (1) was
found to be highly labile and unstable as on contact with water,
methanol, ethanol, and glacial acetic acid, it yielded aegeline (3),6
d
3.80 for the aromatic methoxy group and two one-proton dou-
blets (J¼8.7) at
7.27 and 6.91 for H-200,600 and H-300,500 of the p-
substituted phenyl ring (B), respectively. The HMQC spectrum
showed the one-bond correlation of H-200,600 with C-200,600 (
127.5),
and H-300,500 with C-300,500
114.2), while the HMBC spectrum
showed the long-range correlation of 400-OCH3, 200,600, and 300,500
protons with C-400at 159.8. In the broad band 13C NMR spectrum
C-100 resonated at
132.7. These NMR data, which are consistent
d
d
(d
d
d
with the presence of a disubstituted aromatic ring with a p-
methoxy group in the molecule, are almost identical with those of
the corresponding protons and carbons for 4. Ten out of the eleven
double equivalents present in the molecule were thus accounted
for.
amides 4,7,10 5,7 and 6,6 respectively (Fig. 2).
25
Shahidine (1) [
a
]
þ98.33 (c 0.015, CHCl3) has the molecular
D
The 1H NMR spectrum also exhibited two double doublets at
formula C18H17NO2, as determined by HREIMS (m/z calcd:
279.1259; found: 279.1267) indicating eleven double bond equiv-
alents. The UV spectrum of 1 displayed absorption maxima at 201,
278, and 340 nm, indicating the presence of a conjugated or aro-
matic system. As its molecular formula has a CH3OH unit less than
that of amide 4, it was initially thought that it bears a double bond
between C-1 and C-2, as is present in styrylamides isolated from
d
3.91 (J¼8.0, 15.2) and 4.35 (J¼9.8, 15.2) ascribed to methylene
protons at C-1, which has correlation with dC 62.4 in the HMQC plot.
The high frequency shift of the 1H and 13C NMR
values of this
d
methylene unit as compared to those of 4 (Table 2) indicated its
attachment to the nitrogen of an imino (C]N) moiety. This was
supported by the absence of any NH resonance and the appearance
of methylene protons as double doublets instead of doublet of
double doublets as observed in the case of 4. The 1H NMR spectrum
Piper guayranum and Amyris plumieri.11,12 However, the 1H and 13
NMR data did not support this hypothesis (Table 1) as no trans
C
olefinic proton doublets at d
7.65 and 6.22 were observed in the 1H
further displayed a resonance for the oxy-methine proton at
d 5.51
NMR spectrum of shahidine; instead, the 1H NMR spectrum of 1 run
(dd, J¼8.0, 9.8, H-2), correlated with dC 80.8 (C-2) in the HMQC
spectrum. The C-1 and C-2 protons together formed the ABC pat-
1
tern of the spin system. The H–1H COSY-45ꢀ measurements
showed through-bond linkage of H-1a with H-1b; and H-2 with H-
1a and H-1b (Fig. 3). The high frequency shift of H-2 as compared to
that of 4, absence of an aliphatic methoxy signal, and the large
coupling constant of the methylene protons of C-1 as well as the
molecular formula, suggested that C-1 and C-2 were part of a ring,
which was likely to be an oxazoline located between the C-80 and
C-100 positions, constructing the 2,5-disubstituted oxazoline nu-
cleus13,14 for the basic skeleton of 1. In the case of 1, oxazoline ring
numbering is based on that of 4 for the sake of comparison and
clarity. The linkage of C-100 and C-2 was shown by the three-bond
couplings of methylene protons with C-100, H-2 with C-200,600 and
H-200,600 with C-2 in the HMBC plot (Fig. 3). Another crucial long-range
coupling was observed for C-90 with methylene protons H-1a,b and
H-2. The NOESY spectrum showed a through-space interaction of
the methoxy group with the aromatic protons of ring B (Fig. 3). The
absolute configuration of shahidine was proposed to be 5S on
the basis of similarities in specific rotation (þ98.33) with synthetic
(þ162.3)13a and natural (oxytriphine, þ116.0)13b (þ)-(5S)-2,5-
diphenyl-2-oxazoline. On the basis of these facts, it was concluded
Table 1
1H (400 MHz) and 13C (100 MHz) NMR for shahidine* (1) in CDCl3
(d
ppm; J Hz)
1
H/C
HMQC
HMBC
1Hꢁ H
dC
dH (pattern, J)
C/H
COSY-45ꢀ
1a
1b
2
10
62.4
d
4.35 (dd, 9.8, 15.2)
3.91 (dd, 8.0, 15.2)
5.51 (dd, 8.0, 9.8)
d
d
H-1b, H-2
H-1a, H-2
H-1a,b
d
H-30, H-50
H-20, H-60
d
H-80
H-70
d
d
H-200, H-600
H-80, H-30, H-50
d
80.8
135.1
20,60
30,50
40
127.6 7.47 (dd, 1.2, 8.2)
128.8 7.30–7.37 m
129.6 7.30–7.37 m
140.5 7.40 (d, 16.3)
115.0 6.68 (d, 16.3)
H-40
H-20, H-60
70
H-20, H-60
d
80
90
164.1
132.7
d
H-1a,b, H-70, H-80, H-2
H-1a,b, H-300, H-500
100
d
d
200,600
300,500
400
127.5 7.27 (d, 8.7)
114.2 6.91 (d, 8.7)
H-2
d
H-300, H-500
H-200, H-600
d
159.8
d
H-200, H-600, H-300, H-500, –OCH3
–OCH3 55.2
3.80 s
d
d
*Numbering as that of 4 for the sake of comparison.

1000
S. Faizi et al. / Tetrahedron 65 (2009) 998–1004
Table 2
NMR data for amides (3–6,
d ppm; J Hz)
H/C
3 CDCl3
500 MHz
dH (J)
4 CDCl3
125 MHz 500 MHz
dC dH (J)
4 CDCl3
4 (CD3)2SO
400 MHz
dH (J)
4 C6D6
400 MHz
dH (J)
5 CDCl3
400 MHz
dH (J)
6 CDCl3
500 MHz
dH (J)
1a
1b
3.45 (ddd, 4.9, 7.9, 13.1) 46.0
3.33 (ddd, 4.0, 7.0, 13.5) 3.31–3.33 m
3.81 (ddd, 4.0, 8.8, 13.5) 3.42 (ddd, 4.6, 5.9, 14.5) 4.07 (ddd, 4.0, 8.6, 13.6) 3.77–3.80 m
3.47 (ddd, 4.0, 8.5, 13.6) 3.43–3.46 m
3.76 (t, 6.0)
3.76 (t, 6.0)
3.80 (ddd, 3.4, 9.4, 13.1)
d
2
4.85 (dd, 3.4, 7.9)
d
81.8
135.6
127.8
128.8
129.6
141.1
120.8
165.9
131.1
128.0
114.21
159.7
55.0
d
4.27 (dd, 4.0, 8.8)
d
4.23 (dd, 4.6, 3.2)
d
4.32 (dd, 4.0, 8.6)
d
4.37 (dd, 3.9, 8.8) 5.85 (t, 6.0)
10
d
d
20,60
7.48 (dd, 2.0, 7.9)
7.33–7.36 m
7.33–7.36 m
7.63 (d, 15.6)
6.38 (d, 15.6)
d
7.49 (dd, 2.1, 7.8)
7.36–7.39 m
7.36–7.39 m
7.62 (d, 15.6)
6.41 (d, 15.6)
d
7.53 (dd, 1.9, 8.4)
7.36–7.39 m
7.36–7.39 m
7.39 (d, 15.9)
6.69 (d, 15.9)
d
7.32 (dd, 3.0, 7.9)
6.95–7.18 m
6.95–7.18 m
8.03 (d, 15.5)
6.04 (d, 15.5)
d
7.47–7.50 m
7.32–7.35 m
7.32–7.35 m
7.63 (d, 15.5)
6.38 (d, 15.5)
d
7.49 (dd, 2.0,7.8)
7.33–7.36 m
7.33–7.36 m
7.61 (d, 15.6)
6.32 (d, 15.6)
d
30,50
40
70
80
90
100
d
d
d
d
d
d
200,600
7.29 (d, 8.7)
6.88 (d, 8.7)
d
7.24 (d, 8.6)
6.91 (d, 8.6)
d
7.23 (d, 8.6)
6.92 (d, 8.6)
d
**
7.30 (d, 8.4)
6.88 (d, 8.4)
d
7.30 (d, 8.6)
6.89 (d, 8.6)
d
300,500
6.91 (d, 8.6)
d
400
400–OCH3
2-OCH2-CH3
2-OCH2-CH3
2-OCH3
2-OCOCH3
N–H
3.81 s
d
3.80 s
d
3.74 s
d
3.43 s
d
3.77 s
3.79 s
d
3.38–3.41 m
1.37 (t, 7.2)
d
d
d
d
d
d
d
d
56.8
d
3.24 s
d
3.11 s
d
3.16 s
d
d
d
d
2.08 s
6.12
*
(dd, 4.9, 9.4)
d
6.01
*
(dd, 4.0, 7.0)
8.16
*
(t, 5.9)
5.69
*
(br m)
6.05
*
(br s)
5.78 (t, 6.0)
*
*Exchangeable with D2O.
**Signal obscured in the solvent peak.
often found in natural products, and have also been discovered in
the plant kingdom.15,17,18
Shahidine (1) bearing an oxazoline ring converted readily to
aegeline (3) on keeping at room temperature. This indicated that 1
is the parent compound of aegeline (3). It may be conjectured that
the oxazoline 1 transformed to its hydrated product (3) through the
incorporation of a water molecule in the oxazoline core and si-
multaneous opening of the ring. The p-methoxy group on ring B is
the driving force for the reaction (Scheme 2). Similarly methanol,
ethanol, and acetic acid (glacial), added readily across the oxazoline
ring of shahidine (1), furnishing amides 4, 5, and 6, respectively. It is
important to note in this context that shahidine (1), a masked
amide, is inert toward aerial oxidation (auto-oxidation), and did not
Figure 3.
that shahidine has the structure (þ)-(5S)-2(trans-styryl)-5-(400-
methoxyphenyl)-2-oxazoline (1), which was also corroborated by
the diagnostic mass spectral fragments i and j observed at m/z
134.0346 (C8H6O2) and 143.0755 (C10H9N). Other mass fragments
appeared at m/z 91 (C7H7), 103.0542 (C8H7), 115.0535 (C9H7),
149.0798 (C9H11NO), 116 (C9H8), 151 (C9H11O2), 117 (C8H5O), 121
(C8H9O), and 150.0592 (C8H8NO2) (Scheme 1, fragments a–h, k),
respectively, also substantiated the structure (1). The conjugated
imino group in 1 may be responsible for the yellow color of 1 and
that of the plant extracts containing it, like Schiff bases, which are
usually yellow or orange in color.
Shahidine (1), which is the main constituent of petroleum ether,
dichloromethane, chloroform, dry acetone, and ethyl acetate ex-
tracts of the leaves, belongs to a rare class of 2-oxazoline natural
product, which are rarely found in plants.15,16a Careful sifting of the
literature revealed that oxytriphine was the first 2-oxazoline nat-
ural constituent isolated from the plant kingdom.13b On the other
hand the 2-oxazoline ring is a component of numerous compounds
isolated from microorganisms.15,16a It is important to mention that
about 80% of plants live in close symbiotic relationship with sym-
biotic fungi (ectomycorrhiza, endophytes), which can produce the
natural products isolated from plants.16b There are several natural
cyclic hepta- and octapeptides, e.g., ascidiacyclamide bearing the 2-
oxazoline ring, which impart conformational rigidity to these
compounds.16a It is important to mention that while compounds
bearing the oxazoline core are rare in Nature,13,15 the oxazole ring is
Scheme 1. Mass fragmentation of shahidine (1).

S. Faizi et al. / Tetrahedron 65 (2009) 998–1004
1001
It is the first instance of isolation of an oxazoline-bearing
compound from A. marmelos, which, contained both oxazoles and
cinnamides as natural products.6–10,26,27 Compounds such as oxa-
zoline 1 can be considered the precursor of oxazole-type alkaloids,
e.g., annuloline.17 Crow and Hodgkin in 1963 suggested that the
oxazole nucleus could well arise from a type of common precursor,
which produced the amides aegeline (3) and O-methyl tyramine-N-
methylcinnamide.17
24
Compound 2 (gum, [
a]
þ52.71) was obtained from fraction
D
sixteen of flash column chromatography. It has a composition
C24H27O4N (MW 393) corresponding to twelve degrees of unsatu-
ration as derived from the HREIMS spectrum, which showed a peak
at m/z 334.1801, due to the loss of an acetyl group (MþꢂC2H3O2).
The IR spectrum displayed peaks at 3465 (N–H), 1743 (acetoxy
carbonyl), and 1661 (conjugated –C]Cꢂ) cmꢂ1, while its UV
spectrum showed maxima at 220, 227, and 275 nm.
Its 1H NMR spectrum (CDCl3, Table 3) displayed the same cin-
namoyl moiety as that of compound 4 (Table 2), the main difference
between the benzylic methine proton in compounds 2 and 4 was
the presence of a high frequency chemical shift value (d 5.87) of H-2
in the case of 2, which is comparable to that for amide 6 (Table 2).
The HMBC spectrum confirmed the placement of an acetyl group at
C-2 showing important long-range coupling of H-2 and N–H with
the carbonyl carbon of the acetyl group at (
the 1H NMR spectrum of 2 exhibited a two-proton doublet at
(J¼6.5, H-1%), one-proton triplet at 5.45 (J¼6.5, H-2%) and two
closely spaced three-proton singlets at 1.77 and 1.71 due to the
d
173.5). Furthermore,
d
4.48
Scheme 2. Formation of amides (3–6) on addition of H2O, CH3OH, C2H5OH, and
CH3COOH, respectively, to compound (1).
d
d
gem dimethyl groups indicating the presence of a prenyloxy chain.
The HMQC plot illustrated the correlation of H-1% with dC 64.8
(C-1%) and H-2% with dC 119.5. That the prenyloxy chain was linked
at C-400 was confirmed with the important 3J correlation of H-1% in
the HMBC spectrum. It was also supported by the NOESY plot,
which showed a through-space interaction of H-1% with H-300,500. In
light of above experimental details the structure of 2 was de-
termined to be N-[2-acetoxy-2-[400-(3%,3%-dimethylallyloxy)-
phenyl]ethylcinnamide (marmeline acetate), which was
substantiated by the diagnostic fragment ions in the mass spectrum
particularly at m/z 334.1801 (MþꢂC2H3O2) and 324.1231
(C19H18O4N). It is important to note that compound 2, which was
produce oxidized products on exposure to light and oxygen. It is
also stable in dimethyl sulfoxide (DMSO) solution as manifested by
the periodic 1H NMR spectra run in DMSO d6; however, in chloro-
form it readily transformed into aegeline (3). Probably the slightly
acidic nature of chloroform facilitates the attack of atmospheric
water on the oxazoline ring of 1. While kept in refrigerator 1 con-
verted into 3, within a few months. It seems it is sensitive to
moisture, but, resistant to oxidation.
It is important to mention that in the literature, 2-methoxy
amide (4) was reported to be formed from 2-hydroxy amide,
aegeline (3).7 However, the present investigation revealed that
shahidine (1) is in fact the parent compound of amides 3–5 claimed
as natural products.6–10 This was strengthened by these observa-
tions: (a) Chatterjee et al., and Marcano et al., obtained aegeline
from A. marmelos and Zanthoxylum occumarense as an optically
inactive compound.6,19 Moreover, amide (4) was found to be opti-
cally inactive in the present studies, as would be expected from the
proposed mechanism depicted in Scheme 2. (b) When A. marmelos
leaves were extracted separately with methanol and ethanol by
Manandhar et al., amide 4 and amide 5 were obtained from the
methanol and ethanol extracts, respectively.7
Table 3
1H (400 MHz) and 13C (100 MHz) NMR for marmeline acetate (2) in CDCl3
J Hz)
(d ppm;
1
H/C
HMQC
HMBC
1Hꢁ H
dC
dH (pattern, J)
C/H
COSY-45ꢀ
1
2
10
44.6
74.0
3.75–3.82 m
5.87 (t, 6.5)
d
H-2, N–H
H-2, N–H
H-1
H-200, H-600, H-1
134.7
128.1
130.5
131.9
141.6
120.1
166.0
128.2
128.8
114.7
159.0
H-80
d
d
It is noteworthy that in the case of shahidine (1), hydrolysis of
the C–N bond of the oxazoline ring did not take place, and there-
fore, no ester of the b-amino alcohol group was obtained. This may
20,60
30,50
40
7.44–7.48 m
7.30–7.34 m
7.30–7.34 m
7.59 (d, 15.6)
6.23 (d, 15.6)
d
H-30, H-50
H-20, H-60
d
H-80
H-70
d
d
d
H-80
70
be due to two reasons: (a) hydrolysis of 1, which has an oxazoline
core requires acidic conditions.20–22 (b) The methoxy group of B
ring facilitated the cleavage of the C–O bond instead of fission of the
C–N bond of the oxazoline ring in 1, and it is the key factor.
It is concluded that due to the sensitivity and lability of the oxa-
zolinering,theisolationofnaturalproductsbearingthis groupisvery
difficult. This was evident by the fact that only two such compounds
have been obtained from the plant kingdom, i.e., oxytriphine13b and
shahidine (1). Reinvestigation of plants belonging to the genera,
Aeglopsis,18a Aegle,18a Amyris,18a Fagara,23 Halfordia,17,18a Lolium,18a
Micromelum,18a Oxytropis,13b and Zanthoxylum,19,24,25 which con-
tained amides, hydroxy amides, and oxazoles and employing dry
solvents, and careful rapid experimental procedures may lead to the
isolation of parent compounds bearing an oxazoline core.
80
d
90
H-80, H-1
H-2, N–H
H-300, H-500, H-2
d
H-1%, H-300, H-500,
H-200, H-600
d
100
d
d
200,600
300,500
400
7.26 (d, 8.5)
6.87 (d, 8.5)
d
H-300, H-500
H-200, H-600
d
1%
2%
3%
4%-CH3
5%-CH3
OCOCH3
OCOCH3
ꢂNH
64.8
119. 5
128.2
18.1
25.7
18.1
4.48 (d, 6.5)
5.45 (t, 6.5)
d
H-2%
H-1%
H1%, 5%-CH3
H-1%, 5%-CH3
H-2%, 5%-CH3
H-2%, 4%-CH3
d
1.71 br s
1.77 br s
2.15 s
d
d
d
173.5
d
d
H-2, N–H
d
d
5.85
*
(dd, 3.5,6.5)
H-1
*Exchangeable with D2O.

1002
S. Faizi et al. / Tetrahedron 65 (2009) 998–1004
previously known as a synthetic compound,9 is now found in Na-
ture for the first time. Its 13C NMR data, which were not reported
earlier are included in Table 3.
A plausible biogenetic pathway for the formation of 1 and 2 is
illustrated in Scheme 3. Enzymatic condensation of cinnamic acid
Finnigan MAT-112 and JMS HX-110 spectrometers. 1H NMR spectra
were measured using CDCl3, (CD3)2SO or C6D6 on a Bruker Aspect
AM-400 and a -500 spectrometer at proton frequencies of 400 and
500 MHz. Standard Bruker acquisition and processing software was
used for the 1D (DEPT) and the 2D-COSY experiments. Assignments
of proton chemical shifts are based on COSY-45ꢀ, NOESY and HMQC
with tyramine may provide the key intermediate (7), b-hydroxyl-
ation of which can lead to the formation of the hydroxy amide (8).
Internal cyclization of 8 followed by methylation can produce sha-
hidine (1). The formation of dihydroxy amide intermediate (8) was
supported by the isolation of marmeline acetate (2) from the plant.
spectroscopy. Chemical shifts are in parts per million (d) and cou-
pling constants (J) are in hertz. 13C NMR spectra (Broad Band
decoupled and DEPT) were recorded at (100 and 125 MHz) in CDCl3,
spectral assignments of which have been made partly through DEPT,
HMQC, and HMBC spectra and partly through comparison with the
reported values of similar compounds. E. Merck Kieselgel 60 GF254
precoated cards (0.2 mm thickness) and glass plates were used for
analytical (thin layer) and preparative (thick layer) chromatography,
respectively, while for flash column chromatography (Model
Aldrich) silica gel 9385 (E. Merck) was used.
On the otherhand alkylation of amide 7 followed by b-hydroxylation
can give marmeline (10), which can also be biosynthesized by al-
kylation of amide 8. Enzymatic acetylation of 10 can ultimately yield
marmeline acetate (2). The intermediates 7 and 8–10 have been
isolated from Piper steerni28 and A. marmelos,8,9 respectively.
The versatility of oxazolines29 to serve as precursors to a variety
of functional groups amplifies the importance of shahidine (1),
which is present abundantly in the fresh leaves of A. marmelos.
Moreover, compounds bearing an oxazoline core are also very
important as they are used as chiral auxiliaries in asymmetric
synthesis.30 To examine the antibacterial activity of the leaves, its
various extracts and pure compounds were evaluated against
Gram-positive and Gram-negative organisms. In preliminary ex-
periments, all the extracts and compounds 1, 3, and 4 showed good
activity against Gram-positive bacteria, while aegeline (3) also
inhibited the growth of a few Gram-negative organisms (Table 4).
Amide 4 was also evaluated for any anticancer activity in NCI, NIH,
Bethesda (USA). It was tested against three NCI cancer cell lines,31
MCF7 (breast), NCI-h460 (lung), and SF-268 (CNS) and found to be
inactive as an anticancer agent.
3.2. Plant material
The leaves of A. marmelos were collected from the Karachi
University Campus in January 1999 and October 2005. The plant
was authenticated by Dr. Sherwali at the Department of Botany,
University of Karachi, and the voucher specimen (No. 68509 KUH)
was deposited in the same department.
3.3. Extraction and isolation
Fresh, undried, and uncrushed leaves (1.00 kg) of A. marmelos
werecutintosmallpiecesandkeptinpetroleumether(2ꢁ5 l)fortwo
days. The extracts were filtered and combined together and the two
layers formed in the extract due to the presence of plant water were
separated. The upper layer was evaporated under vacuum to give
a dark and gummy residue, which was treated with methanol,
affording a greenish brown methanol-soluble fraction and an in-
soluble white powder. The former fractionwas dried with anhydrous
Na2SO4, treated with charcoal and filtered, and the filtrate (37.5 mg)
wassubjectedtopreparativethinlayerchromatography(PTLC)(silica
gel, CHCl3), affording six bands of which band three showed a single
UV and iodine-active spot on TLC (silica gel, CHCl3, Rf¼0.16). It was
3. Experimental
3.1. General experimental procedures
IR (in CHCl3) and UV (in MeOH) spectraweremeasured on JASCO-
A-302 and Hitachi-U-3200 spectrophotometers, respectively, while
optical rotation was measured with a Schmidt and Haansch Polar-
tronic-D instrument. EI and HREI mass spectra were recorded on
Scheme 3. Proposed biogenetic pathway for the formation of (1) and (2) in the fresh leaves of A. marmelos.

S. Faizi et al. / Tetrahedron 65 (2009) 998–1004
1003
Table 4
Preliminary antibacterial activity of different extracts and pure compounds from A. marmelos leaves
Samples (20 mg/ml)
Shahidine
(1)
Aegeline
(3)
Compound
(4)
AGLP (pet.
ether extract)
AGLDC
(dichloromethane
extract)
AGLC (chloroform
extract)
AGLM
(methanol
extract)
Gram-positive bacteria
Staphylococcus aureus ATCC
Staphylococcus aureus AB188
Staphylococcus epidermidis
Staphylococcus saprophyticus
Staphylococcus fecalis
MRSA-1
MRSA-2
MRSA-3
Corynebacterium xerosis
C. hoffmanii
C. diphtheriae
Bacillus cereus
Bacillus subtilis ATCC
B. thuringiensis
Micrococcus luteus ATCC
Micrococcus luteus WT
8
9
7
8
9
8
9
9
11
10
9
8
9
9
9
9
7
7
8
ND
d
9
ND
ND
7
8
8
8
7
8
d
9
9
9
ND
9
7
d
12
8
8
9
9
9
7
7
11
15
d
d
11
9
9
8
9
10
11
14
10
9
10
9
d
ND
d
7
9
7
7
8
d
ND
d
7
ND
ND
d
7
12
d
d
d
d
10
9
11
8
10
8
9
ND
ND
8
7
d
d
9
9
8
8
d
7
8
d
8
9
9
7
d
9
9
8
Gram-negative bacteria
Proteus mirabilis
Proteus vulgaris
d
d
ND
d
d
d
d
d
d
d
d
d
d
16
8
8
d
d
d
d
d
d
9
d
ND
11
9
d
d
d
d
d
8
d
ND
12
d
d
9
12
15
d
d
d
d
d
d
d
d
d
12
d
d
d
d
d
d
d
d
d
d
d
d
d
d
20
20
d
d
d
d
d
d
d
d
d
d
d
Salmonella paratyphi ATCC
Salmonella paratyphi A
Salmonella paratyphi B
Esherichia coli MDR
Esherichia coli WT
Pseudomonas aeruginosa PAO286
Pseudomonas aeruginosa
Shigella flexneri
7
8
7
ND
7
7
d
Shigella boydii
Shigella dysenteriae
Klebsiella pneumoniae
d
8
7
9
10
d
d¼No zone of inhibition; ND¼not done.
identified as N-[2-methoxy-2-(4-methoxyphenyl)ethyl]cinnamide7
(4, 28.0 mg) through spectroscopic studies.
3.4. Characterization of compounds
In another working, the original yellow-colored petroleum ether
extract (32.5 mg) was subjected directly to preparative thin layer
chromatography, (silica gel, CHCl3) affording three bands. Band two
showed a single spot on TLC (silica gel, chloroform, Rf¼0.18), and
3.4.1. 2-(trans-Styryl)-5-(4-methoxyphenyl)-D
2-oxazoline:
shahidine (1)
Yellow and amorphous; [
(MeOH) lmax nm (
24
a]
þ98.33 (c 0.015, CHCl3); UV
D
3
): 201 (83,700), 278 (11,559), 340 (3236); IR
was characterized as a new
D
2-oxazoline alkaloid (shahidine, 1,
(CHCl3) nmax: 2881 (m) (aliphatic –CH), 1661 (s) (C]N stretching in
conjugation with C]C), 1613 (s) (>C]C< conjugated with aro-
matic ring), 1513 (m), 1611 (s) (benzene stretchings), 1170 (s) (C–O
stretching), 825 (m) (para-disubstituted benzene) cmꢂ1; FAB (þve):
280 m/z [Mþþ1] (C18H18NO2); FAB (ꢂve): 278 m/z [Mþꢂ1]
(C18H16NO2); HREIMS m/z: 279.1267 [Mþ] (calculated for
C18H17NO2, 279.1259), 150.0592 (C8H8NO2, fragment k), 149.0798
(C9H11NO, fragment d), 143.0755 (C10H9N, fragment j), 134.0346
(C8H6O2, fragment i), 121.0668 (C8H9O, fragment h), 115.0535 (C9H7,
fragment c), 103.0542 (C8H7, fragment b); EIMS m/z (relative in-
tensity, %): 279 (Mþ, C18H17NO2, 38.06), 151 (C9H11O2, fragment f,
69.88), 149 (C9H11NO, fragment d, 37.65), 143 (C10H9N, fragment j,
100), 134 (C8H6O2, fragment i, 55.83), 121 (C8H9O, fragment h,
57.09), 117 (C8H5O, fragment g, 15.99), 116 (C9H8, fragment e, 51.79),
115 (C9H7, fragment c, 87.76), 103 (C8H7, fragment b, 61.55), 91
(C7H7, fragment a, 55.78); 1H and 13C NMR data, see Table 1.
24.1 mg, 0.519%, dry wt basis) through spectroscopic studies (UV,
IR, Mass, 1D, and 2D NMR). Shahidine (1), on treatment with water,
methanol, ethanol and glacial acetic acid, afforded compounds 3,6
4,7 5,7 and 6,6 respectively.
To obtain 1 in large quantities, fresh, undried, and uncrushed
leaves (2.00 kg) of A. marmelos were also percolated in petroleum
ether (2ꢁ11 l) for 2 days. The golden yellow extract on evaporation
yielded a residue, which was taken up in ethyl acetate (300 ml). In
the ethyl acetate fraction, white powder deposited, which was fil-
tered and the filtrate was treated with Na2SO4 anhydrous and
charcoal and filtered. The filtrate thus obtained was evaporated
under reduced pressure furnishing a residue (4.93 g), which
showed one major and a few minor spots on TLC (silica gel, CHCl3).
This residue was subjected to flash column chromatography
[Aldrich model; column size: 1000 ml, silica gel (180 g); petroleum
ether and ethyl acetate, in order of increasing polarity by 5%]
affording 46 fractions. The volume eluted with each system was
0.25 l. Fraction sixteen (P.E/E.A, 7.5:2.5) exhibiting a single UV ac-
tive spot on TLC (silica gel, CHCl3, Rf¼0.21) was identified by
spectral studies as a new natural product, marmeline acetate9 (2,
115 mg). Eluate 17 (P.E/E.A, 7.0:3.0) and 25 (P.E/E.A, 1:1) showed
single spots on TLC (silica gel, CHCl3, Rf¼0.18) and (silica gel, CHCl3,
Rf¼0.09), respectively, and characterized as shahidine (1, 365 mg)
and aegeline (3, 207 mg), respectively, through spectroscopic
studies (UV, IR, EIMS, and 1H NMR).
3.4.2. N-[2-Acetoxy-2-[40-(300,300-dimethylallyloxy)]phenyl]-
ethylcinnamide: marmeline acetate (2)
24
Colorless gum; [
nm (
a]
þ52.71 (c 0.019, CHCl3); UV (MeOH) lmax
D
3
): 220 (17,779), 227 (17,370), 275 (16,988); IR (CHCl3) nmax:
3465 (m) (N–H), 1661 (s) (C]O), 1545 (m) cmꢂ1; HREIMS m/z:
334.1801 (calculated for MþꢂC2H3O2, C22H24O2N, 334.1806),
324.1231 (C19H18 NO4); EIMS m/z: 334 (MþꢂC2H3O2), 324
(MþꢂC5H9), 308 (MþꢂC5H9O), 289, 278, 262, 247, 232 (C13H14O3N);
1H and 13C NMR data, see Table 3.

1004
S. Faizi et al. / Tetrahedron 65 (2009) 998–1004
3.4.3. N-[2-Hydroxy-2-(4-methoxyphenyl)ethyl]-3-phenyl-2-
From a stock solution of 10 mg/ml; 10 ml, i.e., 100 mg of the sample
propenamide: aegeline (3)
was applied to a disc. The discs were air dried and then placed on
a lawn of the test organism. The plates were incubated at 37 ꢀC for
24 h and then zones of inhibition were observed around each discs,
measured, and recorded (Table 4).
White crystalline solid; [
nm ( ): 201 (53,994), 217 (49,401), 222 (50,094), 275 (51,866); IR
a]
24 0 (c 0.010, CHCl3); UV (MeOH) lmax
D
3
(CHCl3) nmax: 3405 (m) (N–H), 3355 (m) (O–H br), 2910 (w) (ali-
phatic), 2852 (w) (aryl ether), 1665 (s) (conjugated C]C), 1692 (m)
(conjugated C]O), 1625 (s) (trans conjugation), 1512 (m) (amide II;
N–H bending), 1460 (m), 1575 (w) (benzene stretching), 1507 (w)
(C]C aryl C–H vibration), 1577 (m) (aryl C–H vibrations), 980 (m)
5. Anticancer activity
Compound (4) was tested for anticancer activity against three
NCI cancer cell lines, MCF7 (breast), NCI-H460 (lung), and SF-268
(CNS), in National Cancer Institute, National Institute of Health,
Bethesda, USA.31
(CH]CH trans), 818 (m) (para-disubstituted benzene) cmꢂ1
;
HREIMS m/z: 297.1365 [Mþ] (calcd for C18H19NO3, 297.1360),
280.1285, (C18H18NO2, MþꢂOH), 279.1283 (C18H17NO2, MþꢂH2O),
160.0777 (C10H10NO), 150.0677 (C9H10O2), 137.0617 (C8H9O2),
131.0532 (C9H7O), 103.0556 (C8H7); EIMS m/z: 297 (Mþ,
C18H19NO3), 220 (C12H14NO3), 207 (C11H13NO3), 194 (C10H12NO3),
190 (C11H12NO2), 166 (C9H12NO2), 151 (C9H11O2), 150, 148, 146
(C9H8NO), 136, 135, 107 (C7H7O), 103 (C8H7), 90 (C7H6), 77 (C6H5); dC
(125 MHz, CDCl3) 167.0, 159.3, 141.7, 134.6, 133.8, 129.8, 128.8, 127.8,
127.1, 120.0, 114.0, 73.4, 55.3, 47.6; 1H NMR data, see Table 2.
Acknowledgements
We thank Dr. Gordon Cragg and Dr. Narayanan (National Cancer
Institute, National Institute of Health, Bethesda, USA) for carrying
out the anticancer activity. One of the authors (S.Z.-U.-R.) ac-
knowledges the enabling role of the Higher Education Commission
Islamabad, Pakistan and appreciates its financial support through
Merit Scholarship Scheme for Ph.D. studies in Science and Tech-
nology (200 Scholarships).
3.4.4. N-[2-Methoxy-2-(4-methoxyphenyl)ethyl]-3-phenyl-2-
propenamide (4)
Colorless and gummy; [
nm ( ): 217 (19,965), 222 (22,915), 274 (24,559); IR (CHCl3) nmax
a]
D
24 0 (c 0.012, CHCl3); UV (MeOH) lmax
3
:
References and notes
3405 (m) (N–H), 2952 (m) (aliphatic-CH2), 1663 (s) (amide I; C]O
stretching), 1626 (s) (>C]C< conjugated with aromatic ring), 1630
(s) (trans olefinic bond), 1502 (m) (C]C), 1512 (m) (amide II; N–H
bending),1443 (w), 1500 (m) (benzene stretchings), 1112 (m) (C–O),
1. Kala, C. P. Indian J. Trad. Knowledge 2006, 5, 537–540.
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3. Sondhi, N.; Bhardwaj, R.; Kaur, S.; Kumar, N.; Singh, B. Plant Growth Regulate.
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825 (m) (para-disubstituted benzene), 981 (s) (–HC]CH–) cmꢂ1
;
Peak matching m/z: 311.1517 (Mþ, calcd for C19H21NO3, 311.1521),
164.0792 (MþꢂC9H9NO), 135.0382 (MþꢂC10H10NO2); EIMS m/z:
311 (Mþ), 280 (MþꢂOCH3), 164 (C10H12O2), 163 (C10H11O2), 152
(C9H12O2), 151 (MþꢂC10H10NO), 135 (C9H11O), 103 (C8H7), 77
(C6H5); 1H and 13C NMR data, see Table 2.
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White powder; UV (MeOH) lmax nm (3): 215 (12,028), 224
(12,030), 272 (12,581); IR (CHCl3) nmax: 3401 (s) (N–H), 2950 (m)
(aliphatic-CH2), 1664 (m) (amide I; C]O stretching), 1619 (m)
(>C]C< conjugated with aromatic ring), 1510 (w) (amide II; N–H
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m/z: 325 (Mþ, C20H23NO3), 280 (MþꢂOC2H5), 179 (C11H15O2), 165
(C10H13O2), 103 (C8H7), 77 (C6H5); dC (125 MHz, CDCl3) 165.7, 159.4,
141.1, 134.8, 131.9, 129.6, 128.7, 128.0, 127.8, 120.7, 114.0, 79.9, 64.1,
55.2, 45.6, 27.9; 1H NMR data, see Table 2.
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White powder; UV (MeOH) lmax nm (3): 222 (10,181), 273 (10,324),
280 (9806); IR (CHCl3) nmax: 3345 (s) (N–H), 1742 (s) (O–C]O), 1641
(s) (NH–C]O) cmꢂ1
;
EIMS m/z: 339 (Mþ, C20H21NO4), 265
(MþꢂC2H3O2ꢂCH3), 192 (C11H12O3), 160 (MþꢂC10H11O3), 149
(C9H9O2), 137 (C8H9O2), 131 (C9H7O), 103 (C8H7), 77 (C6H5); dC
(100 MHz, CDCl3) 170.5, 165.9, 159.7, 141.5, 134.7, 129.8, 129.7, 128.8,
127.9,127.8,120.2,114.0, 74.3, 55.2, 44.4, 21.2; 1H NMR data, see Table 2.
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4. Antimicrobial activity
4.1. Antibacterial activity
31. http://www.dtp.nic.gov/branches/btb/hfa.html; http://www.dtp.nic.nih.nih.gov/
docs/aids/anti-hiv-screening.html; http://www.dtp.nic.nih.gov/btb/ivclsp.html.
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Antibacterial activity was determined by the disc diffusion
method of Baur et al.32 using Nutrient Agar Medium. For this pur-
pose, sterile discs of 6 mm diameter filter paper were prepared.