New class of acetylcholinesterase inhibitors from the stem bark of Knema laurina and their structural insights

Article abstract of DOI:10.1016/j.bmcl.2011.04.065

Bioassay-guided extraction of the stem bark of Knema laurina showed the acetylcholinesterase (AChE) inhibitory activity of DCM and hexane fractions. Further repeated column chromatography of hexane and DCM fractions resulted in the isolation and purificat

Full text of DOI:10.1016/j.bmcl.2011.04.065

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4097–4103
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New class of acetylcholinesterase inhibitors from the stem bark of Knema
laurina and their structural insights
Muhammad Nadeem Akhtar a, Kok Wai Lam a,b, Faridah Abas a,c, Maulidiani a, Syahida Ahmad d,
Syed Adnan Ali Shah e, Atta-ur-Rahman f, M. Iqbal Choudhary f, Nordin Hj Lajis a,g,
⇑
a Laboratory of Natural Products, Institute of Bioscience, University Putra Malaysia, Malaysia
b UCSI University, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Malaysia
c Department of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, Malaysia
d Department of Biochemistry, Faculty of Biotechnology and Molecular Sciences, University Putra Malaysia, Malaysia
e Research Institute of Natural Products for Drug Discovery (RiND)—Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam,
Selangor Darul Ehsan, Malaysia
f H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
g Department of Chemistry, Faculty of Science, University Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioassay-guided extraction of the stem bark of Knema laurina showed the acetylcholinesterase (AChE)
inhibitory activity of DCM and hexane fractions. Further repeated column chromatography of hexane
and DCM fractions resulted in the isolation and purification of five alkenyl phenol and salicylic acid
derivatives. New compounds, (+)-2-hydroxy-6-(100-hydroxypentadec-80(E)-enyl)benzoic acid (1) and 3-
pentadec-100(Z)-enylphenol (2), along with known 3-heptadec-100(Z)-enylphenol (3), 2-hydroxy-6-(pen-
tadec-100(Z)-enyl)benzoic acid (4), and 2-hydroxy-6-(100(Z)-heptadecenyl)benzoic acid (5) were isolated
from the stem bark of this plant. Compounds (1–5) were tested for their acetylcholinesterase inhibitory
activity. The structures of these compounds were elucidated by the 1D and 2D NMR spectroscopy, mass
spectrometry and chemical derivatizations. Compound 5 showed strong acetylcholinesterase inhibitory
Received 4 January 2011
Revised 8 April 2011
Accepted 13 April 2011
Available online 5 May 2011
Keywords:
Knema laurina
Myristicaceae
Acetylcholinesterase inhibition
Alkyl resorcinol
Molecular docking
activity with IC50 of 0.573 0.0260
pound with an elongated side chain could possibly penetrate deep into the active site of the enzyme and
arrange itself through interaction, H-bonding, and hydrophobic contacts with some critical residues
along the complex geometry of the active gorge.
lM. Docking studies of compound 5 indicated that the phenolic com-
p–p
Ó 2011 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) was first reported by a German scien-
tist, Alois Alzheimer in 1907, and is the most common type a neu-
rodegenerative disease characterized by the death of nerve cells.
Neurodegenerative diseases are a group of disease conditions that
result from chronic breakdown and progressive functional or struc-
tural loss of the neurons in the central nervous system (CNS). AD
disease affects 25 million people worldwide in 2000 and it is
expected to increase to 114 million by 2050.1a AD in the early
stages is associated with a decline in cognitive function, particu-
larly short term memory. As the disease progresses, patients face
problems in reading, speaking and logical thinking, and long-term
memory. Recent reports on medicinal plants showed that about
63% of the low molecular drugs developed from 1981 to 2006 are
natural products. These reports suggested that natural products
have strong potential to be developed into biologically active com-
pounds with anti-AD activity. Only some natural sources such as
Ginkgo biloba and Huperzia serrata have been studied extensively
as natural therapeutic agents to treat AD patients.1b
Commercially available medicines such as tacrine, donepezil,
and the natural product-based rivastigmine are used for the treat-
ment of cognitive dysfunction and memory loss associated with
AD.1c These drugs have been known for their adverse effects
including gastrointestinal disturbances and problems associated
with bioavailability.1d,e A variety of plants exhibited AChE inhibi-
tory activity and are the potential candidate for further investiga-
tion towards the discovery of neurodegenerative disorders such
as AD.1f
The genus Knema comprises approximately 60 Southeast Asian
species.2a The methanolic extract of the stem bark of Knema laurina
exhibited neuroprotective and anti-inflammatory effect in cell-
based assay system of brain tissue.2b Some species of Knema are
also used in cancer therapy.2c The extract of the stem bark of Knem-
a furfuraceae Warb. is used in Thailand as a popular remedy for
sores, pimples, and cancer.2d,e The genus Knema also contains a
variety of natural compounds including alkyl and acyl resorcinol,
substituted stilbenes, lignans, flavonoids, isoflavonoids, flavan,2e,f
⇑
Corresponding author.
E-mail address: nhlajis@gmail.com (N.H. Lajis).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.065

4098
M. N. Akhtar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4097–4103
and phenylalkylphenol derivatives.2f Some species of Knema are
also distributed in tropical Africa, Asia, and Australasia, and used
in traditional medicine.2g ( )-Myristinins A and D, which were iso-
lated fromKnema elegens, exhibited the inhibition of DNA polymer-
ase b and cleaved DNA.2h,i Phytochemical analysis of K. laurina
revealed the presence of alkaloids, saponins, steroids, and triter-
penes in the leaves and bark,2j while resorcinols as well as anacar-
dic acids in its stem bark.2e,k Anacardic acid is also known to inhibit
glyceraladehyde-3-phosphate dehydrogenase.2l
methyl H-150 protons.
A broad multiplet appearing at dH
1.33–1.30, calculated for 8 protons (4 ꢁ CH2) was assigned to the
H-50 to H-60 and H-130 to H-140 protons. Another multiplet appear-
ing at dH 1.43–1.40, which accounted for 6 methylene protons
(3 ꢁ CH2) was assigned to the H-30 to H-40 and H-120 protons. These
assignments were further securitized with the help of COSY, HSQC,
HMBC, and 13C NMR spectra, as presented in Table 1. A prominent
signal at dH 2.99 (t, J = 7.5 Hz) was assigned to the benzylic (H-10)
proton, while two multiplets at dH 1.59 and 2.0 were assigned to
the H-20 and H-70, respectively. A broad multiplet appearing at dH
4.15 was assigned to the hydroxymethine (H-100) proton. This pro-
A preliminary cholinesterase inhibition assay using TLC bioau-
tography method was conducted on different fractions of the stem
bark of K. laurina.3a–c Based on the cholinesterase inhibiting activ-
ity of the fractions, hexane and DCM extracts were subjected to
column chromatography, which led to the isolation and identifica-
tion of compounds (1–5). The present article describes a new class
of natural acetylcholinesterase inhibitors isolated from this plant.
The bioactive hexane and DCM fractions were subjected to silica
gel column chromatography and high performance liquid chroma-
tography allowed the purification of compounds (1–5). Com-
pounds (2–5) were isolated from hexane, while 1 was from DCM
soluble part of the stem bark of K. laurina. Compound 1 was
obtained by the multiple column chromatography as a viscous li-
quid from the DCM fraction by using 50% ethyl acetate in hexane
as mobile phase. The IR spectrum showed absorption bands at
3616–3420, 2920, 2860, 1648, 1455, 986, and 786 cmꢀ1 suggesting
the presence of hydroxyl, acidic carbonyl, aromatic, and aliphatic
double bonds, respectively. The EI-MS showed the [M+] at m/z
362. The high resolution electron impact mass spectrum (HREI-
MS) of compound 1 showed the [M+] at m/z 362.2667, suggesting
the molecular formula as C22H34O4 (calcd 362.2669), which was
also supported by the 13C and 1H NMR spectra. Other important
fragments were at m/z 55, 77, 82, 91, 108, 134, 161, 121, 207,
326, 344, and 362. The presence of base a peak at m/z 108 and
prominent peak at m/z 91 resulting from the benzylic cleavage of
aromatic moiety followed by the loss of CO2 was suggestive of
the presence of alkyl resorcinol.4a–c An important peak at m/z
344 was indicative of the loss of H2O from the [M+].
ton appeared as a sharp doublet at dH 4.47 (J10 ,9 = 4.5 Hz)2d when
1H NMR was recorded in DMSO-d6. The characteristic signals,
0
0
0
0
which appeared as a doublet of triplet at dH 5.50 (dt, J8 ,9 = 14.5 Hz,
J70 = 6.5 Hz) and 5.35 (J9 ,8 = 14.5 Hz, J9 ,10 = 4.5 Hz) were assigned
0
0
0
0
,80
trans disposition for the H-80 and H-90 protons, respectively.4d The
chemical shift of the allylic carbon (C-70, dC 34.2) also supported
the trans disposition of double bond.4e Two downfield doublets
at dH 6.90 (d, J = 8.5 Hz) and 6.84 (d, J = 8.0 Hz) were attributed to
the H-3 and H-5 protons, respectively. The most downfield signal
at dH 7.32 (t, J = 8.0, 8.5 Hz) was assigned to the H-4 proton. The
alkenyl side chain was comparable with those previously isolated
carandols and anacardic acid from plants of same genus.2k
The H-80 proton (dH 5.50) showed COSY correlation with H-90
(dH 5.35), which was further extended to H-70 (dH 2.01) and H-100
(dH 4.15). The hydroxymethine proton H-100 (dH 4.15) showed
the HMBC correlations with C-90 (dC 132.7) and C-80 (dC 132.9)
carbon atoms. These observations suggested that the double bond
and hydroxyl group are adjacent to each other. The terminal
methyl (H-150) protons (dH 0.90) exhibited HMBC correlations with
the C-140 (dC 22.8) and C-130 (dC 31.9) carbons atoms. The aromatic
H-4 and H-5 (dH 7.32 and 6.84, respectively) showed the HMBC
correlations with C-6 (dC 147.6) and C-1 (dC 11.2). Furthermore,
the H-10 proton (dH 2.99) showed strong HMBC correlations with
the C-20 (dC 34.4), C-6 (dC 147.6) and C-1 (dC 111.2) carbon atoms.
The 1H and 13C NMR data are summarized in the Table 1 and
selected COSY and HMBC correlations are shown in Figure 1.
To establish the position of double bond, compound 1 was trea-
ted with dimethyl disulfide (DMDS) and I2.4f,g Analysis and the
The 1H NMR spectrum (in CDCl3) of compound 1 exhibited a
triplet at dH 0.90 (J = 7.5 Hz), which was assigned to the terminal
Table 1
1H and 13C NMR chemical shifts of compounds 1 and 2
Carbon No.
13C NMR (Ref.)
13C NMR 1
dH (multiplicity)
13C NMR 2
dH (multiplicity) 2
1
2
3
4
110.5
163.6
115.8
135.3
122.7
147.6
35.5
32.0
31.8
30.9
29.7
28.8
30.9
129.8
129.8
29.7
28.9
28.9
27.2!!
22.8
14.2
111.2
163.7
115.9
135.0
122.6
147.6
36.7
—
—
155.7
115.5
145.2
121.2
129.6
112.7
36.0
—
6.67 (br, s)
—
6.90 (1H, d, J = 8.5 Hz)
7.32 (2H, t, J = 8.0, 8.5 Hz)
6.84 (1H, d, J = 8.0 Hz)
—
2.99 (2H, t, J = 7.5, 4.5 Hz)
1.59 (2H, m)
1.43 (2H, m)
1.40 (2H, m)
1.30 (m)
1.30 (m)
2.0–2.1 (2H, m)
5.50 (1H, dt, J8 ,9 = 14.5 Hz, J7 ,8 = 6.5 Hz)
5.35 (1H, dt, J9 ,8 = 14.5 Hz, J9 ,10 = 4.5 Hz)
6.75 (d, J = 7.5 Hz)
7.13 (t, J = 8.0, 7.5 Hz)
6.65 (br d)
2.56 (t, J = 7.5, each)
1.62 m
5
6
10
20
34.4
29.9
32.0
31.5
30
1.28–1.33 (br m)
40
29.4a
29.6a
28.7a
34.2
29.7a
29.5a
29.5a
28.5a
28.4a
27.2b
130.2
130.1
27.2b
27.4
30
60
70
80
132.9
132.7
74.4
37.3
32.2
31.9
22.8
14.7
0
0
0
0
90
2.02–2.03 (m, 4H)
5.37 (dt, J = 9.0,4.5 Hz)
5.35 (dt, 9.0, 4.5 Hz)
2.03 (m, 4H)
0
0
0
0
100
110
120
130
140
150
COOH
4.15 (1H, dd, J10 ,9 = 7.5 Hz, J10 ,11 = 4.5 Hz)
1.60 (2H, m)
1.43 (2H, m)
1.33 (2H, m)
1.30 (2H, m)
0
0
0
0
1.28–1.33 (br m)
22.6
14.2
0.90 (dd, J = 7.5 Hz)
0.91 (t. 3H, J = 7.0 Hz)
175.4
(Ref.) The data of has been taken from Ref. 2k. The data of compounds 1 and 2 are based on 1D and 2D NMR techniques.
a
Assignments are interchangeable.
Assignments are of overlapping signals.
b

M. N. Akhtar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4097–4103
4099
mass spectrum of the DMDS derived product showed a molecular
ion at m/z 412 along with important fragments ions at m/z 251 and
161, which confirmed the position of double at C-80 and C-90 as
shown in Scheme 1. All the spectroscopic data indicated that the
compound in hand is (+)-2-hydroxy-6-(100-hydroxypentadec-
80(E)-enyl)benzoic acid (1).
Compound 2 was isolated from the hexane soluble fraction of
the stem barks of K. laurina as a colorless oil. The mass spectrum
showed a molecular ion peak at m/z 302, and prominent peaks at
m/z 220, 300, 147, 134, and 108.5a The molecular formula
and H-6 protons, respectively, while
a
doublet at dH 6.75
(J = 7.5 Hz) and a triplets at dH 7.13 (J = 8.0 Hz, 7.5 Hz) were as-
signed to H-4 and H-5 protons, respectively. These assignments
were supported by the HSQC, COSY, and HMBC correlations. The
13C NMR downfield signal at dC 155.7 (C-1) indicated that only
one phenolic carbon is present in compound 2. Other 1H and 13C
NMR data are shown in Table 1.
The configuration of the double bond was assigned as cis based
on the diagnostic chemical shifts of the allylic methylene carbons
at dC 27.2 and 28.1.5b The characteristic fragment, which appeared
at m/z 108 (base peak) resulting from the benzylic cleavage indi-
cated the presence of phenolic moiety in compound 2.4a,5a Other
C21H34O was deduced from the HREI-MS at m/z 302.2604 (calcd
302.2610).
R1
R2
1'
1'
1'
2
R1
1
R2
2
OH
OMe
OH
15'
15'
10' 11'
10' 11'
10' 11'
3
2a
6
3
17'
R2
1
R1
R3
2
6
R1
OH
R2
R3
15'
1
COOH
OH
15'
1a
COOMe
OMe
OH
1'
1'
1'
1'
1'
4
OH
COOH
15'
15'
15'
10' 11'
4a
COOMe
OMe
10' 11'
10' 11'
10' 11'
10' 11'
COOH
COOH
COOMe
4b
5
p-BrPhCO
OH
17'
17'
5a
OMe
The 1H NMR spectrum showed a triplet at dH 0.91 (J = 7.5 Hz),
which was assigned to the terminal H-150 protons. A broad singlet
at dH 1.28–1.33, which accounted for 16 protons (8 ꢁ CH2), were
assigned to the H-30 to H-80 as well as to H-130 to H-140 methylene
protons. Other methylene protons appearing at dH 1.62, and 2.56
were attributed to the H-20 and H-10, respectively.
major fragment ion at m/z 206 and 41 were due to allylic cleavages
of C-80 and C-90 and C-120 and C-13 bonds (see Fig. 2).
The position of the double bond was further confirmed by the
bisthiomethylation followed by MS analysis, which revealed the
important fragments ions at m/z 396, 279, 117, 108, 107, 79, 67,
and 55.4g The key fragments at m/z 279 and 117 confirmed the po-
sition of double at C-100 and C-110 as shown in Scheme 2. On the
basis of these spectroscopic data and bisthiomethylation followed
by MS analysis compound 2 is assigned as 3-(100(Z)-pentadece-
nyl)phenol. Compound 2 was previously identified by GC–MS anal-
ysis as a mixture along with several cardanols.5c This Letter
represents the first isolation of pure compound along with confir-
mation of double bond from K. laurina. Compound 4 was also trea-
ted with dimethyl disulfide (DMDS) and I2,4f,g and respective
fragmentation ions were also observed in its MS. Other known
compounds were identified as 3-heptadec-100(Z)-enylphenol
(3),5d 2-hydroxy-6-pentadec-100(Z)-enylbenzoic acid (4),2k,5e and
2-hydroxy-6-heptadec-100(Z)-enylbenzoic acid (5),5c based on
extensive MS and 2D NMR techniques, as well as by comparison
with the previously published data.5f All compounds were tested
for their AChE inhibitory activity in vitro. Compound 5 exhibited
A multiplet at dH 2.02–203 was attributed to the H-90 and H-120
allylic methylene protons, while
a broad triplet at dH 5.37
0
0
0
0
0
0
0
0
(J10 ,11 = J11 ,10 = 9.5 Hz, J9 ,10 = J11 ,12 = 4.5 Hz) was assigned to the
olefinic H-100 and H-110.2f A triplet at dH 2.56 (J = 7.5 Hz) was due
the H-10 proton. There were four signals in the aromatic region.
Two broad singlets at dH 6.65 and 6.67 were assigned to the H-2
H
COOH
H
7'
5'
13`
14`
9'
1
15`
HO
H
6
11`
1'
H
10`
3'
4'
2'
8'
6'
12`
H
OH
4
H
most potent AChE inhibitory activity with IC50 0.573 0.0260
(Table 2).
lM
Figure 1. Important COSY (
compound 1.
) and HMBC (
) correlations of

4100
M. N. Akhtar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4097–4103
Table 2
COOH
9'
15'
Acetylcholinesterase inhibitory activity of compounds
1–5 and their IC50 values
HO
HO
(CH2)5
8'
OH
Compounds
IC50 (lM)
CH3S2CH3, I2, -CO2
1
1a
2
2a
3
4
4a
4b
3.182 1.097
342.06 9.00
17.224 2.231
178.0 3.226
13.114 0.550
2.172 2.025
358.23 5.441
NA
CH3
S
15'
OH
m/ z = 412
9'
(CH2)5
H3C
8'
S
5
0.573 0.0260
303.21 2.77
0.250 0.001
5a
CH3
CH3
S
CH
S
Tacrinea
15'
HO
HC
The IC50 values are the mean standard deviations of
three independent experiments. The inhibitory effects
are represented as compounds concd (lM) giving 50%
(CH2)6
8'
9'
OH
m/ z = 161
m/ z = 251
inhibition on AChE activity (IC50).
-CH3SCH2,
-H2O
a
Tacrine was used as a positive control.
15'
HO
(CH2)6
pounds 1, 4, and 5, while compounds 2 and 3, which lacked
carboxyl moiety showed slightly a low activity (Table 2), thus sug-
gesting that the acidic group is essential for good AChE activity.
The activity dramatically decreased when the acidic and the phe-
nolic OH groups were methylated as displayed by compounds 1a,
2a, 4a, and 5a, indicating the importance of the acidic and phenolic
moieties (Table 2).5g,h This decrease of activity may be related to
the loss of hydrophilicity of the aromatic terminal in this class of
compounds. The brief data of compounds are presented in the Ref-
erences Section.6a–i
Among the salicylic acid group, 5 displayed the strongest AChE
inhibition activity. This compound is fourfold stronger than com-
pound 4, which has shorter alkenyl side chain by two carbons.
Thus, a longer alkenyl side chain in the salicylic acid analogues
seemed to increase the activity. The presence of polar hydroxyl
group slightly decreased the activity, as shown by compound 1,
thus suggesting that hydrophilicity of this moiety may play an
important role binding. Similar trend was also observed in the phe-
nol group. Compound 3, which contains C17 alkenyl side chain
exhibited stronger activity than 2, (with C15 alkenyl side chain),
although only slightly. Smaller difference in activity between the
two analogues may be due to lower potency of this group of
compounds. Acetylcholinesterase inhibition was performed using
Ellman’s protocol.8a
Structural insights: The catalytic site of Torpedo californica ace-
tylcholinesterase (TcAChE) comprises five important regions,
which include the peripheral anionic site (PAS), the esteractic site
(CT), the oxyanion hole (OH), the acyl pocket and the anionic sub-
site (AS).7a PAS is located at the surface of the gorge, and consists of
Tyr70, Asp72, Tyr121, Tyr334, and Trp279 residues. Several studies
revealed that the binding of ligand in PAS region could possibly
change the conformation of the active center allosterically,7b while
recent discovery showed that thioflavin T could act as substrate
inhibitor by blocking the entry of acetylcholine (ACh) or departure
of any products.7c Deep bottom into the narrow gorge, where the
active site of the enzyme resides, there are four interesting sites
including the CT (Ser200, His440, and Glu327), OH (Gly118,
Gly119, and Ala201), acyl pocket (Phe288 and Phe290) and AS
(Trp84, Phe330, and Glu199), which are responsible for the hydro-
m/ z = 83
m/ z = 185
Scheme 1. The mass spectrometric fragmentations of bisthiomethylated product of
compound 1.
m/ z 41
m/ z 108
m/ z 206
9'
10' 11'
Figure 2. Main mass fragmentations of compound 2.
12'
1'
HO
1
3
13'
15'
H
H
1
HO
C
C
15'
(CH2)8
10'
11'
CH3S2CH3,
I2
-CO2
H3C
CH3
13'
S
C
H
S
C
H
HO
(CH2)8
15'
14'
12'
m/ z = 396
H3C
S
CH3
S
HO
HC
CH
15'
(CH2)8
14'
m/ z = 117
-CH3SH
m/ z = 279
15'
14'
m/ z = 69
lysis of ACh into acetate and choline. The docking methodology as
9a–c
mentioned in Ref.
.
Scheme 2. The MS fragmentations of bisthiomethylation adducts of compound 2.
This conserved residue of Trp84 in the anionic subsite showed
some interesting details, where it formed a stacking interac-
p–p
tion between the acridine and the inhibitor. The positively charged
quarternary group of decamethonium was opposed to the indole
group of the residue. The studies revealed that Trp84 served as
Compounds containing salicylic acid moiety seemed to demon-
strate strong AChE inhibitory activity as represented by com-

M. N. Akhtar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4097–4103
4101
Figure 3. Binding interactions of compound 5 in the catalytic site of TcAChE (green
stick = compound 5). The most important residues involved in the interactions with
5 including the peripheral anionic site (PAS) and anionic subsite (choline-binding
subsite).
an important molecular recognition especially for the discovery of
new inhibitors.
Figure 5. Interatomic contacts (yellow) of the ligand with the residues in the active
Based on docking studies of compound 5 (Fig. 3), a phenolic
compound with an elongated side chain could possibly penetrate
deep into the active site and arrange itself along the complex
geometry of the active gorge. Early insight reveals the flexibility
of the long alkyl chain could demonstrate a similar arrangement
as decamethonium. The alkyl linker is well placed along the nar-
row aromatic gorge starting from the surface until the anionic ac-
tive site where Trp84 residue is located (Fig. 4). According to the
biological results, compounds (1–5) showed potential inhibition
against AChE, which could be due to the presence of hydroxyl
group at C-2 in the phenyl ring. This is further strengthened by
the docking results, the importance of this hydroxyl group which
binds to the carbonyl oxygen of Gly117 through hydrogen bonding
at the distance of 2.977 Å and could help to position the phenyl
site based on Van der Waals radii generated by Chimera software.
the interactions between quaternary nitrogen of endrophonium7d
and decamethonium and the stacking of acridine with indole of
Trp84. Interestingly the hydroxyl group is also involved in a short
contact with Tyr130 at the distance of 2.142 Å. In another example,
their inhibition are totally diminished once the hydroxyl group is
methylated, preventing the free hydrogen to participate in the
above hydrogen bonding. However, none of the 10 best poses gen-
erated by docking of compound 5, make any important binding in
the active site of CT, OH and acyl pocket possibly due to the bulk-
iness of the whole structure. Compound 5 could not be considered
as a bivalent ligand due to its inability to occupy the ES region.7e
The occupancy of alkyl group of 5 in the peripheral site could act
as an anchor while permitting the penetration of the alkyl chain
and the aromatic residues in the gorge. These hydrophobic interac-
tions between C150, 160, and 170 of 5 with PAS residues Trp279,
Trp70 and Tyr121 are made accessible due the presence of cis (Z)
configured double bond moiety at C-100. This double bond could
be directing the alkyl group to twist towards PAS at the surface
of the gorge. The whole molecule eventually appears to be in
L-shaped (Fig. 5). Short contact is also observed between H-15
and the aromatic Trp 279 at distance of 2.313 Å. Overall, the mech-
anism of inhibition for compound 5 is based on its capability of
blocking the path leading to the active site while docking studies
ring to involve in a p–p stacking interaction with Trp84. This type
of interaction is common in some of the AChE inhibitors along with
showed that compound 5 interacted on the basis of
p–p interac-
tion, H-bonding, and hydrophobic contacts with some critical res-
idues in the active site. Failure to occupy the esteractic site
explains its incapability to match the inhibition activity demon-
strated by tacrine. However, the potential of this compound should
not be undermined especially since it could serve as a potential
candidate in combating AChE related diseases.
In conclusion, a new potential class of AChE inhibitor has been
isolated from a plant, K. laurina, and identified based spectrometric
techniques. Molecular docking experiment suggested a unique
structural alignment of the substrate to suit the active gorge site.
Alkenylsalicylic acid may provide a new template for further struc-
tural modification into a new drug.
Acknowledgments
This project was supported by the Ministry of Science,
Technology and Innovation (MOSTI) Science Fund Scheme (vote
no. 5450290) and Research Management Center, University Putra
Malaysia under RUGS research Grant No. 91972.
Figure 4. Molecular interactions of compound 5 with the residues in the gorge of
the catalytic site of TcAChE (in green space filled structure).

4102
M. N. Akhtar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4097–4103
MS, 390.3254 (calcd 390.3248 for C24H38O4), 362, 344, 326, 330, 245, 175, 161,
References and notes
121, 91, 81, 55. IR mmax (CHCl3) cmꢀ1; 3205, 2824, 1650, 1580, 1450. 1H NMR
(500 MHz, DMSO-d6): d 7.30 (t, J = 8.0 Hz, 8.5Hz, H-4), 6.90 (d, J = 8.5 Hz, H-3),
1. (a) Wimo, A.; Winblad, B.; Aguero-Torres, H.; von Strauss, E. Alzheimer Dis. Assoc.
Disord. 2003, 17, 63; (b) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461; (c)
Oh, M. H.; Houghton, P. J.; Whang, W. K.; Cho, J. H. Phytomedicine 2004, 11, 544;
(d) Schulz, V. Phytomedicine 2003, 74; (e) Melzer, D. BMJ 1998, 316, 762; (f)
Mukherjeea, P. K. V.; Kumarb, M.; Malb, H. P. J. Phytomedicine 2007, 14, 289.
2. (a) Pinto, M. M. M.; Kijjoa, A.; Mondranondra, I.-N.; Gutierrez, A. B.; Herz, W.
Phytochemistry 1990, 29, 1985; (b) Hake, I.; Schonenberger, S.; Neumann, J.;
Franke, K.; Paulsen-Merker, K.; Reymann, K.; Ismail, G.; Bin din, L.; Said, I. M.;
Latiff, A.; Wessjohann, L.; Zipp, F.; Ullrich, O. J. Neuroimmunol. 2008, 91; (c)
Graham, J. G.; Quinn, M. L. F. D. S. F. N. R. J. Ethnopharmacol. 2000, 73, 347; (d)
Zeng, I.; Gu, Z.-M.; Fang, X.-P.; McLaughlin, J. L. J. Nat. Prod. 1994, 57, 376; (e)
Kijjoa, A.; Gonzalez, M. J. T. G.; Pinto, M. M. M.; Monanondra, I.-O.; Herz, W.
Planta Med. 1991, 57, 575; (f) Gonzalez, M. J. T. G.; Pinto, M. M. M.; Kijjioa, A.;
Anantachoke, C.; Herz, W. Phytochemistry 1993, 32, 433; (g) Zahir, A.; Jossang, A.;
Bodo, B.; Hadi, H. A.; Schaller, H.; Sevenet, T. J. Nat. Prod. 1993, 56, 1634; (h)
Deng, J.-Z.; Starck, S. R.; Li, S.; Hecht, S. M. J. Nat. Prod. 2005, 68, 1625; (i)
Maloney, D. J.; Deng, J.-Z.; Starck, S. R.; Gao, Z.; Sidney, M.; Hecht, S. M. J. Am.
Chem. Soc. 2005, 127, 4140; (j) Ahmad, W. Y.; Tee, S. K.; Din, L. B.; In A Scientific
Journey through Borneo Tawau Hills Park, Sabah Ismail, G., Ed.; Palahniuk
Publications: Kuala Lumpur; 1995, p 139.; (k) Gonzales, M. J.; DeOliveira, C.;
Fernandes, J.; Kijjoa, A.; Herz, W. Phytochemistry 1996, 43, 1333; (l) Pereira, J. M.;
Severino, R. P.; Vieira, P. C.; Fernandes, J. B.; Silva, M. F. G. F.; Zottis, A.; Adriano,
D.; Andricopulo, A. D.; Oliva, G.; Corrêa, A. G. Bioorg. Med. Chem. 2008, 16, 8889.
3. (a) Marston, A.; Kissling, J.; Hostettmann, K. Phytochem. Anal. 2002, 13, 51; (b)
Tanaka, S.; Yoichi, S.; Ao, L.; Matumoto, M.; Morimoto, K.; Akimoto, N.; Honda,
G.; Tabata, M.; Oshima, T.; Masuda, T.; bin Asmawi, M. Z.; Ismail, Z.; Yusof, S. M.;
Din, L. B.; Said, I. M. Phytother. Res. 2001, 15, 681; (c) Rhee, I. K.; Meent, M. V.;
Ingkaninan, K.; Verpoorte, R. J. Chromatogr. 2001, 15, 217.
4. (a) Spencer, G. F.; Tjarks, L. W.; Kleiman, R. J. Nat. Prod. 1980, 43, 724; (b) Kubo,
I.; Nihel, K.-I.; Kazuo, K. J. Agric. Food Chem. 2003, 51, 7624; (c) Du, Y.; Oshima, R.;
Yamauchi, Y.; Kumanotani, J.; Miyakoshi, T. Phytochemistry 1986, 25, 2218; (d)
Atta-ur-Rahman; Zareen, S.; Choudhary, M. I.; Akhtar, M. N.; Ngounou, F. N.
Phytochemistry 2009, 69, 2400; (e) Cheng, M.-J.; Lee, S.-J.; Chang, Y.-Y.; Wu, S.-H.;
Tsai, I.-L.; Jayaprakasam, B.; Chen, I.-S. Phytochemistry 2003, 63, 603; (f)
Carballeira, N. M.; Cruz, M. Chem. Lipids 1996, 84, 81; (g) Francis, G. W.;
Veland, K. J. Chromatogr. 1981, 219, 379.
5. (a) Franke, K.; Masaoud, M.; Schmidt, J. Planta Med. 2001, 67, 477; (b) Kato, M. J.;
Lopes, L. M. X.; Fo, H. F. P.; Yoishid, M.; Gottlieb, O. R. Phytochemistry 1985, 24,
533; (c) Liua, Y.; Abreu, P. J. M. J. Braz. Chem. Soc. 2006, 17, 527; (d) Kubo, I.; Kim,
M.; Naya, K.; Komatsu, S.; Yamagiwa, Y.; Ohashi, K.; Sakamoto, Y.; Hirakawa, S.;
Kamikawa, T. Chem. Lett. 1987, 1101; (e) Lee, J. S.; Cho, Y. S.; Park, E. J.; Kim, J.;
Oh, W. K.; Lee, H. S.; Ahn, J. S. J. Nat. Prod. 1998, 61, 867; (f) Itokawa, H.; Totsuka,
N.; Nakahara, K.; Takeya, K.; Lepoittevin, J.-P.; Asakawa, Y. Chem. Pharm. Bull.
1987, 35, 3016; (g) Green, I. R.; Tocoli, F. E.; Lee, S. H.; Niheib, K.-I.; Kubob, I.
Bioorg. Med. Chem. 2007, 15, 6236; (h) Lund, A.-K.; Lemmich, J.; Andsersen, A.;
Olsen, C. E. Phytochemistry 1997, 44, 689.
6. (a) The stem bark of K. laurina (6.2 kg) was collected from the Pasir Raja Forest
Reserve, Trengganu, Malaysia, in May 2008. The voucher specimen SK 1540/08
was deposited at the Herbarium of the Institute of Bioscience (IBS), University
Putra Malaysia. The plant was identified by the Resident Botanist of IBS. The
stem barks were cleaned, cut into small pieces and left under the shade for
5 days at room temperature. The dry pieces were ground to powder and then
steeped in distilled methanol (10 L) at room temperature for 2 days. The
methanol extract was then filtered off and the filtrate was concentrated under
reduced pressure. This procedure was repeated three times and the extracts
were combined to yield 90.1 g of crude methanolic extract. The combined
extract was then fractionated between water and organic solvents (sequentially:
hexane; dichloromethane; ethyl acetate and finally butanol) to yield the hexane
(20.2 g), DCM (3.64 g), EtOAc (7.41 g), BuOH (6.90 g) and aqueous (45.8 g)
soluble fractions. The hexane soluble fraction was subjected to silica gel column
chromatography (230–400 mesh, ASTM, Merck) using the gradient solvent
system hexane/EtOAc (8:2 to 6.5:3.5) to yield seven sub-fractions (kl-1 to kl-7).
Subfractions kl-3 (1.20 g) and kl-4 (0.09 g) were separately subjected to
repeated column chromatography (230–400 mesh) by eluting it gradiently
using hexane/EtOAc (9:5 to 5.5:4.5) as solvent system, to yield compounds 2
0
0
0
0
6.87 (d, J = 8.0 Hz, H-5), 5.50 (dt, J8
, , = 14.5,
= 14.5, 6.5 Hz, H-80), 5.36 (dt, J9
9
8
4.5 Hz, H-90), 4.47 (d, J10 ,9 = 4.5 Hz), 3.79 (s, 3H, OMe), 3.72 (s, 3H, COOMe), 2.56
(t, J = 7.5 Hz, C-10), 1.99 (m, 2H, C-70), 1.50 (m, 2H, C-110), 1.42–1.40 (br m, 6H,
3 ꢁ CH2, C-30 to C-40 and C-120), 1.31–129 (br m, 8H, 4 ꢁ CH2, C-5 to C-60 and C-
130 to C-140), 0.89 (t, J = 7.5 Hz, 3H, C-150).; (d) Bisthiomethylation of compound 1:
Compound 1 (3.0 mg) was dissolved in dimethyl disulfide (3 ml) in a 25 ml
single necked round bottom flask and iodine (10 mg) was added. The reaction
mixture was kept at 50–60 °C for overnight. The reaction mixture was diluted
with 5 ml diethyl ether and excess iodine was decomposed with 5% sodium
thiosulfate solution. The diethyl ether layer was separated and dried over
sodium sulfate anhydrous and subjected to GC–MS analysis, which showed the
peaks at m/z = 412, 396, 394, 251, 185, 161, 108, and 83 (Scheme 1).; (e) 3-
0
0
(pentadecen-100(Z)-yl)phenol (2): Colorless oil; UVMk eOH nm (log
e): 277 (2.8), 272
(2.6): 222 (3.0); IR
m
max (CHCl3) cmꢀ1, 3418–3220,m2ax928, 2809, 1610, 1455, 890.
EI-MS m/z (rel. int.): 302 [M+], HREI-MS, m/z 302.2604 (calcd 302.2610 for
C
21H34O), 302, 276, 220, 206, 175, 161, 147, 133, 121, 108 (100%), 77, 43. 1H and
13C NMR CDCl3 (125 MHz) (Table 2).; (f) 3-Methoxypentadec-(100(Z)-enyl)-
benzene (2a): Compound 2 (10 mg) was dissolved in diethyl ether at room
temperature, and into it few drops of diazomethane was added. After drying the
sample in a fume hood the compound 2b was obtained as colorless oil. EI-MS m/
z (rel. int.): 316 [M+], 302, 276, 220, 206, 175, 161, 147, 133, 123, 121, 108
(100%), 77, 55. IR m
max (CHCl3) cmꢀ1: 3252–3160, 2930–2936, 1620, 1456, 1289,
780. 1H NMR (500 MHz, CDCl3): d 7.13 (t, J = 8.0, 7.5 Hz, H-5), (d, 6.66 (br d, IH,
C-2), 6.75 (d, J = 7.5 Hz, H-4), 6.65, (br d, 1H, H-6), 3.73 (s, 3H, OMe), 2.55 (t, 7.5,
each, H-10), 2.02 (m, 4H, H-90 and H-120), 1.62 (m, 2H, H-20), 1.27–1.30 (br m,
16H, 8 ꢁ CH2, H-30 to C-80 and H-130 to H-140), 0.90 (t, 3H, J = 7.5, H-150).; (g)
Bisthiomethylation of compound 2: Dimethyl disulfide (DMDS) adduct was
prepared by taking 5 mg of compound 2 in a 25 ml round bottle flask containing
10 ml hexane. About 1.5 ml dimethyl disulfide was added into it followed by
20 mg of iodine. The reaction mixture was stirred at 50–60 °C for overnight. The
diethyl ether layer was separated and dried over sodium sulfate anhydrous and
subjected to GC–MS analysis. Fragmentation peaks observed include m/z = 396,
279, 117, 108, 107, 69, and 55.; (h) 2-Hydroxy-6-(pentadec-100(Z)-enyl)benzoic
acid (4): Colorless oil, IR m
max (CHCl3) cmꢀ1; 3510–3228, 2920, 2860, 1650, 1609,
1580, 1450. El-MS m/z (rel. int.): 346 [M+], C22H34O3, (55), 328 (29), 320 (15),
310 (22), 302 (34), 285 (10), 257 (10), 227 (11), 175 (20), 161 (24), 152 (50), 147
(42), 134 (37), 120 (10), 108 (50), 91 (100); 1H NMR (500 MHz, CDC13): d 7.36 (t,
J = 8.0, 8.5 Hz, each, H-4), 6.87 (dd, J = 8.0 Hz, H-3), 6.76 (dd, J = 8.5 Hz, H-5), 5.35
(centre of AB system of H-100 and H-110 J10
,
= J11
,
= 9.0 Hz,
0
0
0
0
11
10
0
0
0
0
J9
,
10
= J11
,
12
= 6.5 Hz), 2.98 (d, J = 7.5 Hz, C-10), 2.01 (m, 4H, C-90 and C-120),
1.60 (m, 2H, C-20), 1.41–1.40 (br m, 8H, 4 ꢁ CH2, C-30 to H-40 and C-130 to C-140),
1.30–1.29 (br m, 8H, 4 ꢁ CH2, C-50 to C-80), 0.91 (t, J = 7.5 Hz, 3H, C-150).; (i) 2-
Hydroxy-6-(pentadec-100(Z)-enyl)benzoic acid methyl ester (4a): Compound
4
(10 mg) was treated with ethereal solution of diazomethane and methylated
compound 4a was purified by evaporation of solution and passing through silica
gel funnel. The EI-MS showed m/z 374.2 [M+], C24H38O3, [M+], (55), 328 (29), 320
(15), 310 (22), 302 (34), 285 (10), 257 (10), 227 (11), 175 (20), 161 (24), 152
(50), 147 (42), 134 (37), 121 (10). 1H NMR (500 MHz CDC13): d 7.36 (t, J = 8.0,
8.5 Hz, H-4), 6.87 (dd, J = 8.5 Hz, H-3), 6.76 (dd, J = 8.0 Hz, H-5), 5.35 (centre of
AB system of H-100, J10 ,11 = J11 ,10 = 9.0 Hz, J9 ,10 = J11 ,12 = 6.5 Hz, H-100 and H-
110), 3.80 (s, 3H, OMe), 3.74 (s, 3H, COOMe), 2.98 (d, J = 7.5 Hz, 2H, C-10), 2.01 (m,
4H, C-90 and C-120), 1.60 (m, 2H, C-20), 1.41–1.39 (br m, 8H, 4 ꢁ CH2, C-30 to H-40,
C-130 to C-140), 1.31–1.29 (br m, 8H, 4 ꢁ CH2, C-50 to C-80), 0.90 (t, J = 7.5 Hz, 3H,
C-150).
0
0
0
0
0
0
0
0
7. (a) Wiesner, J.; Kriz, Z.; Kuca, K.; Jun, D.; Koca, J. J. Enzyme Inhib. Med. Chem. 2007,
22, 417; (b) Radic, Z.; Duran, R.; Vellom, D. C.; Li, Y.; Cervenansky, C.; Taylor, P. J.
Biol. Chem. 1994, 269, 11233; (c) Harel, M.; Sonoda, L. K.; Silman, I.; Sussman, J.
L.; Rosenberry, T. L. J. Am. Chem. Soc. 2008, 130, 7856; (d) Leonetti, F.; Catto, M.;
Nicolotti, O.; Pisani, L.; Cappa, A.; Stefanachi, A.; Carotti, A. Bioorg. Med. Chem.
2008, 16, 7450; (e) Holzgrabe, U.; Kapkova, P.; Alptuzun, V.; Scheiber, J.;
Kugelmann, E. Exp. Opin. Ther. Targets 2007, 11, 161.
8. In vitro cholinesterase inhibition assay: Acetylcholinesterase inhibiting activity
was measured by the spectrophotometric method of Ellman et al.8a Electric eel
AChE (type VI-S, electric eel, Sigma Chemical Co. code: C2888), were used as
sources of the cholinesterases. Acetylthiocholine iodide (Sigma Chemical Co.
[ATCI] code: A5751) were used as substrates of the reaction, and 5,5-dithiobis-
(2-nitrobenzoic) acid (DTNB) (Sigma Chemical Co. Code: D21200, 99%) were
used for the measurement of the cholinesterase activity. In this procedure;
(45.5 mg) and
3 (57.0 mg), respectively. Using the same elution system,
subfraction kl-5 (1.80 g) was subjected to repeated silica gel column
chromatography (230–400 mesh) to obtained compounds 4 (90.4 mg) and 5
(21.0 mg). The semi pure compounds (2–5) were purified by HPLC [mobile
phase: ACN/H2O (40:60 to 45:55; UV: 254 nm; flow rate: 4.00 ml/min; column
200
ll
of 0.15 mM sodium phosphate buffer pH 7.4 with 10
ll of DTNB
(0.15 mM), 20
l
l of test compound solution, and 20 l of acetylcholinesterase
l
XterraÒ Prep MS C18 OBD™ (19 ꢁ 150 mm, 5
lm)], which led to the purification
(final concentration 0.037 U/ml in 0.1 M phosphate buffer solution) were mixed
and incubated for 10 minutes at 25 °C. The reaction was then started by the
addition of 20 ll of acetylthiocholine iodide (0.25 mM, substrate). The final
volume of the assay in each well was 270 y monitoring the formation of the
yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with
of 5 (tR: 11.2 min), 4 (tR: 11.7 min), 3 (tR: 16.3 min) and 2 (tR: 16.8 min).; (b)
Physical and spectroscopic data of compounds: (a) (+)-2-hydroxy-6-(100-
hydroxypentadec-80(E)-enyl)benzoic acid (1): colorless oil, ½a 2D5
ꢂ
+23.2, CHCl3).
UVMk eOH nm (log
e): 306 (3.9), 288 (3.3), 272 (2.3), 227 (1.8). IR mmax (CHCl3)
max
cmꢀ1; 3616–3420, 2920, 2860, 1648, 1455, 786. El-MS m/z (rel. int.): 362 [M+],
HREI-MS: 362.2667 (362.2669 calcd for C22H34O4), 344, 326 (29), 330 (15), (22),
302 (34), 285 (10), 257 (10), 227 (11), 175 (20), 161 (24), 152 (50), 147 (42), 134
(37), 120 (10), 108 (50), 91 (100), 82 (19), 55 (37). 1H and 13C NMR data (Table
2).; (c) (+)-2-Methoxy-6-(100-hydroxypentadec-80(E)-enyl)benzoic acid methyl
thiocholine released by the enzymatic hydrolysis of acetylthiocholine at
wavelength of 412 nm at 25 °C monitored for 3 min. Test compounds were
dissolved in analytical grade DMSO. The same volume 270 l was used for
negative control by the addition of 20 of buffer instead of sample. All
reactions were carried out in triplicate. The concentration used in M,
a
l
l
l
l
ester (1a): In
a 25 ml round bottomed flask, compound 1 (5.0 mg) was
percentage inhibition was calculated in Microsoft excel and IC50 were
calculated by using graph Pad software. (a) Ellman, G. L.; Courtney, K. D.;
Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88.
dissolved in CHCl3 and few drops of diazomethane were added. After drying
compound 1a was obtained. Colorless oil, El-MS m/z (rel. int.): 390 [M+], HREI-

M. N. Akhtar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4097–4103
4103
9. Computational study: Protein crystal structure with PDB code 1ACL9a was
obtained from the protein data bank followed by docking studies using
GOLD4.14.9b The ligands were constructed and energy minimized using MOPAC
program with MMFF94 force field calculation. The binding site was defined
based on the cavity detection by GOLD centered on the reference ligand and the
atoms selection was restricted to solvent-accessible surface. All other
redocking of the crystal ligand (decamethonium) into the active site. Finally, the
ligand was superimposed on the reference crystal structure with a RMSD of
1.8 Å (good pose <2.0 Å) which justified the ability of the software to produce
correct solution. Ten top ranked poses results were saved and evaluated. The
highest GOLD fitness score was chosen as a resultant coordinate. (a) Harel, M.;
Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.; Hirth, C.; Axelsen, P. H.;
Silman, I.; Sussman, J. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9031; (b) Verdonk,
M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Proteins 2003, 52,
609; (c) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997,
267, 727.
parameters were set up according to the standard protocols of GOLD. The
standard docking procedure includes the use of genetic algorithm9c to explore
the full range of ligand conformational flexibility with the partial flexibility of
protein while the scoring function includes the terms for hydrogen-bonding,
vdW, and intramolecular energies. Validity of the docking was justified from the