Phytochemistry 146 (2018) 75e81

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Phytochemistry
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Unusual phenethylamine-containing alkaloids from Elaeocarpus

tectorius
Margret Chinonso Ezeoke a, Premanand Krishnan a, Dawn Su-Yin Sim b, Siew-Huah Lim b,
Yun-Yee Low b, Kam-Weng Chong b, Kuan-Hon Lim a, *
a
School of Pharmacy, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia
b
Department of Chemistry, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: From the leaves of Elaeocarpus tectorius (Lour.) Poir. four previously undescribed phenethylamine-
Received 6 October 2017 containing alkaloids were isolated, namely, tectoricine, possessing an unprecedented iso-
Received in revised form quinuclidinone ring system incorporating a phenethylamine moiety, tectoraline, representing a rare
29 November 2017
alkamide incorporating two phenethylamine moieties, and tectoramidines A and B, representing the first
Accepted 4 December 2017
naturally occurring trimeric and dimeric phenethylamine alkaloids incorporating an amidine function.
The structures of these alkaloids were established by detailed spectroscopic analysis. The absolute
configuration of tectoricine was determined by comparison of the experimental and calculated ECD
Keywords:
Elaeocarpus tectorius
spectra. Plausible biosynthetic pathways to the four alkaloids are proposed.
Elaeocarpaceae © 2017 Elsevier Ltd. All rights reserved.
Alkaloids
Phenethylamine
Isoquinuclidinone
Amidine
Formamidine

1. Introduction 1969c). Although there are ca. 30 species of Elaeocarpus native to

The genus Elaeocarpus L., which comprises ca. 360 species and
occurs widely in the tropical and subtropical regions of the world
(except for Africa), is the largest of the 12 genera in the Elaeo-
carpaceae family (Tang and Phengklai, 2007). Previous alkaloidal
investigations of Elaeocarpus plants have been limited to nine
species, namely, E. polydactylus Schltr. (Johns et al., 1968a, 1969a),
E. densiflorus Knuth. (Johns et al., 1968b, 1969b), E. dolichostylis
Schltr. (Johns et al., 1969c), E. sphaericus (Gaertn.) K. Schum. (Johns
et al., 1970, 1971; Zhou et al., 2011), E. kaniensis Schltr. (Hart et al.,
1971, 1972), E. ganitrus Roxb. (Ray et al., 1979), E. grandis F. Muell.
(Carroll et al., 2005; Katavic et al., 2006), E. habbemensis A.C.Sm.
(Katavic et al., 2007a) and E. fuscoides Knuth. (Katavic et al., 2007b).
With the exception of E. densiflorus, in which one indole alkaloid
was isolated, the other eight Elaeocarpus species were found to
contain indolizidine and pyrrolidine alkaloids that were postulated
to be derived from condensation of ornithine with a C8 or C12
polyketide chain (Carroll et al., 2005; Hart et al., 1971; Johns et al.,
* Corresponding author.
E-mail address: KuanHon.Lim@nottingham.edu.my (K.-H. Lim).
Peninsular Malaysia, to date there is no phytochemical report on
any of the Malaysian samples. Additionally, some of these species
are used in traditional medicine in Malaysia to treat headaches
(E. mastersii), fever (E. petiolatus), poultice sores (E. stipularis) and as
general tonic (E. floribundus and E. grandiflorus) (Aggarwal, 2001).
This has led to the search for alkaloid-containing Elaeocarpus spe-
cies occurring in Peninsular Malaysia. In this paper, the isolation
and structure elucidation of four unprecedented and unusual
phenethylamine-containing alkaloids, namely, tectoricine (1), tec-
toraline (2), and tectoramidines A and B (3 and 4) (Fig. 1), from the
leaves of Elaeocarpus tectorius (Lour.) Poir. were reported.
2. Results and discussion
Tectoricine (1) was obtained from the leaves of E. tectorius as a
colorless oil, [a]D þ4 (c 0.05, CHCl3). The IR spectrum showed ab-
sorption bands due to a ketone carbonyl (1735 cm-1), an unsub-
stituted phenyl ring (701, 754 cm1) and a gem-dimethyl group
(1364, 1377 cm1). The HRESIMS data ([MþH]þ m/z 328.2284) of 1
established the molecular formula as C21H29NO2, requiring eight
degrees of unsaturation. The 13C NMR data of 1 (Table 1) indicated

https://doi.org/10.1016/j.phytochem.2017.12.003
0031-9422/© 2017 Elsevier Ltd. All rights reserved.
76 M.C. Ezeoke et al. / Phytochemistry 146 (2018) 75e81

Fig. 1. Structures of 16.

Table 1 to C-3, C-4 and C-8; Me-9 to C-3, C-8 and C-10; and Me-10 to C-3, C-
1
H and 13C NMR data for tectoricine (1).a 8 and C-9 in the HMBC spectrum (Fig. 2). Furthermore, the presence
Position dH (mult., J, Hz) dC of the gem-dimethyl group (Me-9C-8Me-10) was supported by
the observation of two absorption bands at 1364 and 1377 cm1 in
2 3.46 dd (6.9, 4.2) 58.9
3 54.8
the IR spectrum of 1.
4 215.4 Examination of the HSQC and COSY data of 1 (Fig. 2) revealed
5 2.32 m 45.5 three additional partial structures, namely, an NCH2CH2 fragment
2.37 m corresponding to NC-10 C-20 , an NCHCH2 fragment corresponding
6 3.14 t (3) 49.8
to NC-2C-12, and a CH2CH(N)CH2 fragment corresponding to C-
7a 1.40 dd (14, 3) 36.4
7b 2.12 dt (14, 3) 5C-6C-7. The NC-10 C-20 partial structure was established to be
8 33.4 part of the phenethylamino side chain based on the observed
9 1.12 s 25.7 HMBC correlations from H-10 and H-50 /70 to C-30 ; H-20 and H-60 to
10 0.87 s 29.6 C-40 /80 ; and H-40 /80 to C-20 , while the NC-2C-12 partial structure
11 0.71 s 10.7
12 2.03 dd (18.5, 4.1) 49.9
was determined to be associated with the acetonyl side chain based
2.39 m on the observed HMBC correlations from H-14 to C-12 and from H-
13 207.9 2 to C-13. The observed correlations from H-5 and H-11 to C-4 and
14 2.00 s 31.0 from H-9 to C-7 in the HMBC spectrum allowed assembly of the
10 2.70 m 55.2
cyclohexanone moiety by connecting the C-5C-6C-7 partial
2.99 m
20 2.65 m 35.9 structure to the ketonic C-4 and quaternary C-8 of the 2,3,3-
2.65 m trimethylpropanone partial structure. The observed correlations
30 140.4 from H-2 to C-10 and from H-10 to C-2 and C-6 indicated that C-2, C-
40 , 80 7.15 d (7.5) 128.9 6 and C-10 were attached to a tertiary N atom. Finally, the linkage
50 , 70 7.26 m 128.4
60 7.19 m 126.1
between C-2 and C-3, which completed the assembly of the iso-
a
quinuclidinone ring system of 1, was shown by the observed HMBC
Measured in CDCl3 at 600 and 150 MHz for 1H and 13
C NMR, respectively.
correlations from H-2 to C-4, C-6 and C-8; H-11 to C-2; and H-12 to
C-3. The proposed structure of 1 is consistent with the full HMBC
the presence of 19 discrete carbon signals, of which two are due to data (Fig. 2).
two pairs of equivalent aromatic methine carbons. In agreement The relative configurations at the three stereocenters in 1 (i.e., C-
with the HRESIMS data, the total number of carbons in 1 was 2, C-3 and C-6) were established based on the NOESY data (Fig. 3).
therefore determined as 21, comprising four methyls, five methy- The rigid architecture of the isoquinuclidinone ring system
lenes, two aliphatic methines, five aromatic methines, two aliphatic required the configurations at C-3 and C-6 to be either 3S,6S or
quaternary carbons, one quaternary aromatic carbon and two ke- 3R,6R. This leaves only the relative configuration at C-2 to be
tone carbonyls. The presence of two pairs of equivalent methine
carbons at dC 128.4 and 128.9, a methine carbon at dC 126.1 and a
quaternary carbon at dC 140.4 suggested the presence of an
unsubstituted phenyl group. This was further supported by the 1H
NMR data (Table 1), which also showed signals due to an unsub-
stituted phenyl group (dH 7.15e7.26, 5H). In addition, the 1H NMR
data revealed the presence of four distinct 3H singlet signals at dH
0.71, 0.87, 1.12 and 2.00 due to four isolated methyl groups. The
three methyl signals at dH 0.71, 0.87 and 1.12 were determined to be
associated with a 2,3,3-trimethylpropanone fragment, corre-
sponding to the O¼C-4C-3(Me-11)C-8(Me-10)Me-9 partial
structure in 1, on the basis of the observed correlations from Me-11
Fig. 2. Selected COSY and HMBC correlations of 1.
 M.C. Ezeoke et al. / Phytochemistry 146 (2018) 75e81
Fig. 3. Selected NOESY correlations of 1.
considered. The NOEs observed for H-2/Me-9 and H-2/H-7b
required that H-2, H-7b and Me-9 to be directed into the same face
of the isoquinuclidinone ring system (the acetonyl group is ori-
ented away from Me-9), thus restricting the number of possible
structures of 1 to one enantiomeric pair, which corresponded to the
relative configuration 2S,3S,6S (or 2R,3R,6R). Additionally, since
both H-7b (dH 2.12) and Me-9 (dH 1.12) were equatorially b-ori-
ented, both H-7a (dH 1.40) and Me-10 (dH 0.87) must therefore be
axially a-oriented. This deduction was supported by the NOEs
observed for H-10/H-5a and H-10/H-7a. On the other hand, based
on the overall geometry of the isoquinuclidinone ring system of 1,
the Me-11 group is oriented in the anisotropic shielding zone of the
adjacent ketone at C-4, thus providing an explanation for the
notably shielded resonances observed for Me-11 (dH 0.71, dC 10.7) in
the 1H and 13C NMR spectra. Other NOEs observed are in complete
agreement with the structure and relative configuration of 1.
Finally, the absolute configuration of 1 was established as 2S,3S,6S
by comparing the experimental and calculated electronic circular
dichroism (ECD) spectra of 1 (Fig. 4).
Tectoricine (1) represents a new class of alkaloids in which the
phenethylamine N atom is incorporated into a previously unde-
scribed isoquinuclidinone ring system that is substituted with three
methyl groups (1,1,2-trisubstitution) and an acetonyl side chain.
Alkaloids that possess an isoquinuclidine moiety as a standalone
unit without being part of a larger fused-ring system are rare and
are to date only represented by mearsine (5) and grandisine B (6)
(Fig. 1) (Carroll et al., 2005; Robertson et al., 1984). The structure of
tectoricine (1) suggests that it is a monoterpenoid phenethylamine.
However, the monoterpenoid moiety in 1 appears to possess an
irregular monoterpene skeleton, which was mostly encountered in
monoterpenoids obtained from plants of the family Asteraceae (Liu
et al., 2012; Rivera et al., 2001). A plausible pathway to 1 starting
Fig. 4. Experimental and calculated ECD spectra of 1 in MeCN.
77
from two molecules of dimethylallyl pyrophosphate (DMAPP) is
presented in Fig. 5. Condensation of two molecules of DMAPP gives
chrysanthemyl diphosphate (CPP), which on diphosphate elimi-
nation furnishes a carbocation that undergoes a series of rear-
rangements. Trapping of the rearranged carbocation by a molecule
of water gives the allylic alcohol 7 (Poulter et al., 1974; Rivera et al.,
2001), which is regarded as a key intermediate to 1. A series of
oxidative transformations followed by cyclization then ensue to
give the cyclohexadione 8. This is then followed by a double
reductive amination of 8 with a molecule of phenethylamine
leading to the cyclic iminium ion 9. Nucleophilic attack of
acetoacetyl-CoA on 9 followed by hydrolysis give the b-keto acid 10,
which on subsequent decarboxylation gives the structure of tec-
toricine (1).
Tectoraline (2) was obtained from the leaves of E. tectorius as a
light yellowish oil, [a]D 21 (c 0.12, CHCl3). The IR spectrum showed
the presence of unsubstituted phenyl rings (701 and 751 cm-1), an
amide carbonyl (1650 cm1) and an NH group (3279 cm1). The
HRESIMS data ([MþNa]þ m/z 361.2232) of 2 established the mo-
lecular formula as C22H30N2O, requiring nine degrees of unsatura-
tion. The 13C NMR data of 2 (Table 2) indicated the presence of 18
discrete carbon signals, four of which are due to four pairs of
equivalent aromatic methine carbons. The total number of carbons
in 2 was therefore determined as 22 (comprising one methyl, seven
methylenes, one aliphatic methine, ten aromatic methines, two
quaternary aromatic carbons and one amide carbonyl), which
agrees with the molecular formula. The presence of four pairs of
equivalent methine resonances at dC 128.4, 128.5, 128.6 and 128.7,
two methine resonances at dC 126.3 and 126.4, and two quaternary
carbon resonances at dC 139.3 and 139.5, suggested the presence of
two unsubstituted phenyl groups with a similar chemical envi-
ronment. The presence of 10 aromatic signals in the 1H NMR
spectrum (dH 7.14e7.29) (Table 2) provided further support for this
suggestion. The 13C NMR spectrum also exhibited the presence of
an amide/ester carbonyl resonance at dC 172.0. However, since the
molecular formula contains only one O atom, the carbonyl reso-
nance was attributed to an amide function. In addition to the
methyl triplet observed at dH 0.85, the 1H NMR spectrum also
revealed the presence of a methylene group adjacent to a carbonyl
function, as inferred by the observation of a large Jgem value, i.e.,
16.0 Hz, for the pair of geminaly coupled H atoms.
The COSY and HSQC data revealed two CH2CH2N fragments
corresponding to the C-20 eC-10 eN and C-200 eC-100 eN partial
structures, and a CH2CH(N)CH2CH2CH3 fragment corresponding to
the C-2eC-3eC-4eC-5eC-6 partial structure in 2 (Fig. 6). The
presence of two phenethylamine moieties in 2 was readily estab-
lished by the HMBC data (Fig. 6), which connected C-20 to C-30 and
C-200 to C-300 , from the observed three-bond correlations from H-40 /
80 to C-20 ; H-20 to C-40 /80 ; H-10 to C-30 ; H-400 /800 to C-200 ; H-200 to C-
400 /800 ; and H-100 to C-300 . The attachment of the C-2eC-3eC-4eC-
5eC-6 partial structure to the amide carbonyl C-1 was revealed by
the HMBC correlation from H-3 to C-1. This is also consistent with
the large Jgem value observed for both H-2 (vide supra). On the other
hand, the amide function was established to be associated with the
PheC-20 eC-10 eN partial structure based on the observed correla-
tion from H-10 to C-1 in the HMBC spectrum. Finally, the PheC-
200 eC-100 eN partial structure was connected to C-3 based on the
observed correlation from H-100 to C-3. The proposed structure for
tectoraline (2) is therefore N-phenethyl-3-(phenethylamino)hex-
anamide and is entirely consistent with the full HMBC data (Fig. 6).
Tectoraline (2) represents a new alkamide that appears to be
derived from a molecule of 2-hexenoic acid and two molecules of
phenethylamine (Fig. 7).
Tectoramidine A (3) was obtained as a colorless oil. The IR
spectrum showed the presence of bands due to unsubstituted
78 M.C. Ezeoke et al. / Phytochemistry 146 (2018) 75e81

Fig. 5. A plausible biosynthetic pathway to 1.

Table 2
1
H and 13C NMR data for tectoraline (2), tectoramidine A (3) and tectoramidine B (4).a

Position 2 3 4

dH (mult., J, Hz) dC dH (mult., J, Hz) dC dH (mult., J, Hz) dC

2 2.12 dd (16.0, 7.2) 38.7 3.86 br s 74.8
2.35 dd (16.0, 3.4)
3 2.76 m 54.7
4 1.34 m 35.7 163.0 8.13 s 161.1
5 1.19 m 19.0 3.02 br s 55.6
6 0.85 t (7.3) 14.0
10 3.41 m 39.9 3.49 m 48.2 3.56 t (7)k 39.2m
20 2.73 t (7.0) 35.6 3.06 t (6.3) 35.8 2.848 t (7)l 35.4n
30 139.3d 138.2 138.1
40 , 80 7.14 d (7.5)b 128.6e 7.19 m 129.1 7.20 m 128.6
50 , 70 7.29 t (7.5)c 128.4f 7.30 m 129.4 7.31 m 128.7
60 7.22 t (7.5) 126.3g 7.24 mh 127.0i 7.24 m 126.7
100 2.75 m 47.0 4.16 t (6) 46.8 3.57 t (7)k 40.8m
2.85 dt (11.2, 6.7)
200 2.65 t (6.7) 36.2 3.04 t (6) 33.2 2.852 t (7)l 35.5n
300 139.5d 137.3 138.1
400 , 800 7.18 d (7.5)b 128.7e 7.29 m 128.89j 7.20 m 128.6
500 , 700 7.30 t (7.5)c 128.5f 7.23 m 128.91j 7.31 m 128.7
600 7.22 t (7.5) 126.4g 7.24 mh 126.7i 7.24 m 126.7
1000 2.34 t (7.3) 56.1
2000 2.45 t (7.3) 34.8
3000 138.4
4000 , 8000 7.03 d (7.3) 128.5j
5000 , 7000 7.30 m 128.6j
6000 7.22 mh 127.2i
CONH 172.0
NH 8.06 br s 7.50 br s
a
Measured in CDCl3 at 600 and 150 MHz for 1H and 13C NMR, respectively.
beo
Signals are interchangeable within the same column.

phenyl rings (701 and 752 cm-1) and an amidine C¼N (1687 cm1). molecular formula as C27H31N3, requiring 14 degrees of unsatura-
The HRESIMS data ([MþH]þ m/z 398.2593) of 3 established the tion. The 13C NMR data of 3 (Table 2) indicated the presence of 21
 M.C. Ezeoke et al. / Phytochemistry 146 (2018) 75e81
Fig. 6. Selected COSY and HMBC correlations of 2.
Fig. 7. A plausible biosynthetic pathway to 2.
discrete carbon signals, six of which are due to six pairs of equiv-
alent aromatic methine carbons. The total number of carbons in 3
was therefore determined as 27 (comprising eight methylenes, 15
aromatic methines, three quaternary aromatic carbons and one
amidine carbon), which agrees with the molecular formula. The
presence of six pairs of equivalent methine resonances at dC 128.5,
128.6, 128.89, 128.91, 129.1 and 129.4, three methine resonances at
dC 126.7, 127.0 and 127.2, and three quaternary carbon resonances
at dC 137.3, 138.2 and 138.4, revealed the presence of three
unsubstituted phenyl groups. The 13C NMR spectrum also showed
the presence of the amidine N¼CN resonance at dC 163.0.
The COSY and HSQC data of 3 revealed the presence of three
CH2CH2N fragments corresponding to the three phenethylamine
moieties in 3, i.e., PheC-20 eC-10 eN, PheC-200 eC-100 eN, and PheC-
2000 eC-1000 eN (Fig. 8). This was confirmed by the HMBC data (Fig. 8),
which showed three-bond correlations from H-10 to C-30 ; H-100 to C-
300 ; H-1000 to C-3000 ; H-40 /80 to C-20 ; H-400 /800 to C-200 ; and H-4000 /8000 to
C-2000 . By subtracting the three phenethylamine moieties (C24H27
Fig. 8. Selected COSY and HMBC correlations of 3 and 4.
79
N3, corresponding to 12 degrees of unsaturation) and the amidine
carbon from the molecular formula of 3, only two methylene
groups (dC 55.6, dH 3.02; dC 74.8, dH 3.86) and one degree of unsa-
turation remained to be considered. Therefore, it could be deduced
that to complete the construction of the entire structure of 3, for-
mation of another ring involving the two methylene groups (C-2
and C-5), the amidine function (C-4) and the three phenethylamine
moieties was required. The PheC-200 eC-100 eN fragment was
deduced to be connected to C-2 (dC 74.8, dH 3.86) and C-4 (dC 163.0)
as a result of the correlations from H-100 to C-2 and C-4; and from H-
2 to C-4, while the attachment of the PheC-2000 eC-1000 eN fragment
to C-2 and C-5 (dC 55.6, dH 3.02) was deduced from the correlations
from H-1000 to C-2 and C-5; H-2 to C-1000 ; and H-5 to C-1000 and C-2.
Finally, the linkage between C-4 and C-5, which completed the
assembly of the imidazolidinyl ring, was shown by the observed
HMBC correlation from H-5 to C-4. Other HMBC correlations (Fig. 8)
are entirely consistent with the structure of tectoramidine A (3).
Additionally, the structure of 3 is consistent with the observation
that the signal of H-100 was notably deshielded compared to that of
H-10 (dH 4.16 versus dH 3.29, respectively) in the 1H NMR spectrum.
This observation can be rationalized by the fact that C-100 is
attached to the electron-deficient N-3 of the amidine function,
while C-10 is attached to the electron-rich N-1. Tectoramidine A (3)
represents the first naturally occurring trimeric phenethylamine
alkaloid incorporating a rare trisubstituted amidine function with
an exocyclic C¼N as a partial structure.
Tectoramidine B (4) was obtained as a colorless oil. As in the case
for 3, the IR spectrum of 4 showed the presence of unsubstituted
phenyl rings (698 and 748 cm1) and an amidine C¼N (1644 cm1).
The HRESIMS data ([MþH]þ m/z 253.1700) of 4 established the
molecular formula as C17H20N2, requiring nine degrees of unsatu-
ration. The 1H NMR of 4 (Table 2) revealed the presence of two pairs
of mutually coupled triplets at dH 2.848/2.852 and 3.56/3.57 (each
80 M.C. Ezeoke et al. / Phytochemistry 146 (2018) 75e81
Fig. 9. A plausible biosynthetic pathway to 3 and 4.
pair integrated for 4H), suggesting the presence of two closely
comparable phenethylamine moieties in 4. This was further sup-
ported by the presence of three clusters of aromatic resonances at
dH 7.20 (4H), 7.24 (2H) and 7.31 (4H) in the 1H NMR. The 1H NMR
also showed a 1H singlet at dH 8.13, attributed to an amidine
N¼CHN moiety. In addition to the resonances due to the two
phenethyl units, the 13C NMR (Table 2) showed a resonance at dC
161.1, which correlated to dH 8.13 in the HSQC spectrum of 4.
Therefore, the resonance at dC 161.1 was deduced to be attributed to
the amidine N¼CHN moiety. This deduction was further
confirmed by the observed three-bond correlations from H-10 /100 to
dC 161.1 and from dH 8.13 to C-10 /100 in the HMBC spectrum of 4
(Fig. 8). These HMBC correlations also established that the amidine
N¼CHN is bridging the two phenethyl units. To the best of our
knowledge, tectoramidine B (4) represents the first dimeric phe-
nethylamine alkaloid incorporating a formamidine core from
plants.
It is envisaged that both tectoramidines A and B (3 and 4), which
appear to be structurally related, share a common biosynthetic
origin. A plausible pathway to the tectoramidines starting from
phenethylamine (11) and the iminium ion of N-methylphenethyl-
amine (12) is presented in Fig. 9. Nucleophilic addition of 11 onto
the iminium ion 12 gives the dimeric aminoacetal 13, which on
subsequent dehydrogenation/oxidation furnishes tectoramidine B
(4). Alternatively, intermolecular and intramolecular coupling re-
actions (nucleophilic substitution) involving 13 and oxalic acid or
an oxalate ester (including oxalyl-CoA (Malathi et al., 1970)) gives
the symmetrical imidazolidinyl ring intermediate 14. Finally,
reductive amination of 14 with another molecule of phenethyl-
amine (11) followed by ketone reduction furnishes tectoramidine A
(3).
3. Conclusions
This is the first report on the phytochemical investigation of
alkaloids from Elaeocarpus tectorius (Lour.) Poir. Four unprece-
dented and unusual phenethylamine-containing alkaloids (1e4)
were isolated and identified from the leaf crude extract. Tectoricine
(1) possesses a previously undescribed isoquinuclidinone ring
system that incorporates a phenethylamine moiety, while tec-
toraline (2) is an alkamide that incorporates two phenethylamine
moieties. Tectoramidines A and B (3 and 4) on the other hand are
trimeric and dimeric phenethylamine alkaloids incorporating an
amidine function. With respect to alkaloid content, only
indolizidine, pyrrolidine and indole alkaloids were previously re-
ported from nine Elaeocarpus species. Therefore, compounds 1e4,
which display phenethylamine as a common structural feature,
represent another distinct class of alkaloids from Elaeocarpus. Un-
fortunately, the lack of compounds 1e4 due to poor isolation yields
and decomposition has precluded biological evaluation.
4. Experimental
4.1. General experimental procedures
Optical rotations were determined on a JASCO P-1020 automatic
digital polarimeter. UV spectra were obtained on a Perkin Elmer
Lambda 25 UVeVis spectrophotometer. IR spectra were recorded
on a Perkin Elmer Spectrum RX1 FT-IR and 400 FT-IR/FT-FIR spec-
trometers. ECD spectra were obtained on a J-815 circular dichroism
spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 using
TMS as internal standard on a Bruker 600 MHz NMR spectrometer.
HRESIMS were obtained on a JEOL Accu TOF-DART mass
spectrometer.
4.2. Plant material
The leaves of Elaeocarpus tectorius (Lour.) Poir. (synonym:
Elaeocarpus robustus Roxb.) (Elaeocarpaceae) were collected in June
2014 from the sides of a public road running through the Berembun
Forest Reserve, Negeri Sembilan, Malaysia (GPS coordinates
2.83559 N, 102.05371 E), and the plant was identified by K. T. Yong
(Institute of Biological Sciences, University of Malaya, Malaysia).
Herbarium voucher specimens (KLU49072) were deposited at the
Herbarium, University of Malaya.
4.3. Extraction and isolation
The dried leaves (15 kg) of E. tectorius were extracted with 95%
EtOH (15 L x 4) at room temperature overnight. The concentrated
ethanolic extract was added into 3% tartaric acid solution with
vigorous stirring. The acidic solution was then filtered through
kieselguhr to remove the insoluble substances. Concentrated NH3
solution was then added to the acidic filtrate until pH 10 was
achieved. The liberated alkaloids were exhaustively extracted with
CHCl3, washed with water, dried over anhydrous Na2SO4, and
concentrated to afford a basic crude alkaloid mixture (5.1 g). The
crude alkaloid mixture was initially fractionated by column
 M.C. Ezeoke et al. / Phytochemistry 146 (2018) 75e81
chromatography (silica gel 60, CHCl3-MeOH, with increasing pro-
portions of MeOH) followed by further fractionation of the partially
resolved fractions using column chromatography or centrifugal
preparative thin layer chromatography (Chromatotron). The sol-
vent systems used for centrifugal preparative thin layer chroma-
tography were CHCl3-hexane (NH3-saturated), CHCl3-MeOH (NH3-
saturated), CH2Cl2 (NH3-saturated) and CH2Cl2-MeOH (NH3-satu-
rated). The yields (mg kg1) of the alkaloids obtained were as fol-
lows: 1 (0.20), 2 (0.20), 3 (0.33) and 4 (0.13).
4.3.1. Tectoricine (1)
Colorless oil; ½a25
D þ4 (c 0.05, CHCl3); UV (EtOH) lmax (log ε) 227
(3.77) nm; ECD (c 0.06 mM, MeCN), lmax (Dε): 212 (1.43), 226
(0.59), 243 (0.97), 261 (0.60), 276 (0.83), 306 (1.21) nm; IR
(dry film) nmax 1735 (ketone), 1364, 1377 (gem-dimethyl), 701, 754
(unsubstituted phenyl) cm1; 1H and 13C NMR data, see Table 1;
HRESIMS m/z 328.2284 [M þ H]þ (calcd for C21H29NO2 þ H,
328.2277).
4.3.2. Tectoraline (2)
Light yellowish oil; ½a25
D 21 (c 0.12, CHCl3); UV (EtOH) lmax (log
ε) 227 (3.85) nm; IR (dry film) nmax 3279 (NH), 1650 (amide), 701,
751 (unsubstituted phenyl) cm1; 1H and 13C NMR data, see Table 2;
HRESIMS m/z 361.2232 [M þ Na]þ (calcd for C22H30N2O þ Na,
361.2256).
4.3.3. Tectoramidine A (3)
Colorless oil; ½a25
D 0 (c 0.25, CHCl3); UV (EtOH) lmax (log ε) 229
(3.79) nm; IR (dry film) nmax 1687 (C¼N), 701, 752 (unsubstituted
phenyl) cm1; 1H and 13C NMR data, see Table 2; HRESIMS m/z
398.2593 [M þ H]þ (calcd for C27H31N3 þ H, 398.2596).
4.3.4. Tectoramidine B (4)
Colorless oil; ½a25
D 0 (c 0.15, CHCl3); IR (dry film) nmax 3324 (NH),
1644 (C¼N), 698, 748 (unsubstituted phenyl) cm1; 1H and 13C
NMR data, see Table 2; HRESIMS m/z 253.1700 [M þ H]þ (calcd for
C17H20N2 þ H, 253.1705).
4.4. Computational method for ECD calculations for tectoricine (1)
The conformations of tectoricine (1) were obtained by
Spartan'14 software (Wavefunction, Inc., Irvine, CA, USA) using the
MMFF94 force field. Conformers occurring within a 5 kcal/mol
energy window from the global minimum were then imported into
the Gaussian 09 software (Gaussian 09, Revision C.01, Gaussian,
Inc., Wallingford, CT, USA) for DFT-level geometry optimization and
frequency calculation using the M06-2X functional with basis set of
6-31þG(d,p). TDDFT ECD calculations were performed at the
B3LYP/6-311þþG(d,p) level with the optimized conformers using a
PCM solvation model for MeCN. The ECD curve for each optimized
conformer was weighted by Boltzmann distribution after UV
correction and the overall ECD curves were produced by the
SpecDis 1.64 software (Bruhn et al., 2015). Optical rotation calcu-
lations at the wavelength of 589.3 nm were performed with the
optimized conformers at the B3LYP/6-311þþG(d,p) computational
level using a PCM solvation model for CHCl3.
Conflict of interest
The authors have declared that there is no conflict of interest.
Acknowledgments
The authors thank Professor J.F.F. Weber (Atta-ur-Rahman
Institute for Natural Product Discovery, Universiti Teknologi MARA
81
(UiTM), Puncak Alam Campus) for help with the circular dichroism
measurements. This research did not receive any specific grant
from funding agencies in the public, commercial, or not-for-profit
sectors.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at https://doi.org/10.1016/j.phytochem.2017.12.
003. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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