2014

NPC Natural Product Communications Vol. 9

No. 11
Non-Competitive Inhibition of Acetylcholinesterase by 1559 - 1561
Bromotyrosine Alkaloids
Opeyemi J. Olatunjia, Akintayo L. Ogundajob, Ibrahim A. Oladosub, Kanokwan Changwichitc,
Kornkanok Ingkaninanc, Supreeya Yuenyongsawada and Anuchit Plubrukarna,*
a
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand
b
Organic/Medicinal Chemistry Unit, F 103 Research Laboratory, Department of Chemistry, University of Ibadan,
Ibadan, Nigeria
c
Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, and Center
of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand

anuchit.pl@psu.ac.th

Received: July 8th, 2014; Accepted: September 11th, 2014

Fifteen bromotyrosine-derived alkaloids were isolated from the sponge Pseudoceratina cf. purpurea. The acetylcholinesterase-inhibiting activity of all
the isolated compounds were examined; to purealidin Q, isoanomoian A, aplyzanzine A, and aplysamine 2 were active with IC50 values of 1.2, 70, 104, and
1.3 M, respectively. On the other hand, antiproliferative activity against MCF-7 cells of aerophobin 1 gave an IC50 value of 0.8 M. The Michaelis-Menten
plots of the active alkaloids indicated that all the four compounds inhibited acetylcholinesterase in a non-competitive manner. The structures of the active
compounds suggested that the N,N-dimethylaminopropyloxydibromotyramine moiety may play an important role in the enzyme-inhibiting activity,
presumably on the anionic and hydrophobic binding sites.

Keywords: Pseudoceratina cf. purpurea, Bromotyrosine alkaloids, Acetylcholinesterase inhibitors, Non-competitive inhibition.

Acetylcholinesterase (AChE) is one of the major targets in drug (10) [7], aplyzanzine A (11) [7,8], aplysamine 2 (12) [8].
development for neurodegenerative disorders that involve dementia purpureamine J (13) [9], and aerophobins 1 (14) and 2 (15) [10]
as a clinical marker, especially Alzheimer’s disease. Whereas it is (Figure 1). The configurations depicted here refer to the specific
agreeable that the etiology of the disease is multifactorial and might rotation reported for each compound, which was in a comparable
not be explainable through a single hypothesis, deficit in range of our measurements.
acetylcholine, the neurotransmitter that governs the cognitive
functions in the brains, has been proved to relate directly to memory All the isolated alkaloids were tested for their AChE-inhibiting
loss, the primary clinical symptom of Alzheimer’s disease [1]. This activity [11]; compounds, 6 and 10-12, were strongly to moderately
deficiency has been observed to result either from the decrease in active (Table 1). In addition, three other compounds, 1, 3, and 14,
acetylcholine production, or from an increase in AChE activity, showed very weak activity, showing 15-35% enzyme inhibition at
which also leads to the aggregation of amyloid beta proteins [1c]. the highest concentration of 0.1 mg/mL. The antiproliferative
Although having been considered merely a palliative treatment with activities [12] against MCF-7 and human fibroblast cells of all the
the efficacy lasting up to four years at most [1c,e], the uses of isolated compounds were also examined; compound 14 is the only
acetylcholinesterase inhibitors (AChE-I) to counteract the alkaloid that was active (Table 1).
neurotransmitter deficiency still have been one of the only two
successful medicinal approaches for the treatment of Alzheimer’s Table 1: AChE-inhibiting and cytotoxic activities of the isolated compounds.
disease. Along with memantine, an NMDA inhibitor, to date there Compounds AChE inhibition Cytotoxicity (IC50, μM)
have been only three AChE-Is; donepezil, galanthamine, and (IC50, μM)
MCF-7 Human fibroblast
rivastigmine, (tacrine, the first marketed AChE-I, was withdrawn in 6 1.2 - -
2013 due to its hepatotoxicity) that have been approved to be used 10 70 - -
11 107 - -
in patients diagnosed with mild-to-moderate and moderate-to-severe 12 1.3 - -
Alzheimer’s disease [1e]. 14 - 0.8 4.2

As parts of our search for new drugs and medicinal entities from
Thai marine invertebrates, we found that extracts from the sponge
Pseudoceratina cf. purpurea, collected from Koh-Ha Islets,
Thailand, showed a strong AChE-inhibiting activity (>90%
inhibition at 0.1 mg/mL). Chemical investigation of the sponge led
to the isolation of 15 bromotyrosine alkaloids. The structure
determination was performed by means of UV, IR, and NMR
spectroscopy and MS, to show that they are aeroplysinin (1) [2],
araplysillins I (2) and II (3) [3], purealidins B (4), J (5), Q (6), and R
(7) [4], fistularin 3 (8) [5], hemifistularin 3 (9) [6], isoanomoian A
The four AChE-inhibiting alkaloids were subjected to enzyme-
inhibiting kinetics study. Determination of a Michaelis-Menten plot
for each compound was performed to show that the Vmax of AChE
decreased drastically upon exposure to each tested sample
(Figure 2). Such a change in Vmax with no significant change in Km
indicated that all the active alkaloids inhibited AChE in a non-
competitive manner.
Although limited to only 15 compounds in the dataset, comparison
of the chemical structures of all the isolated compounds
1560 Natural Product Communications Vol. 9 (11) 2014
Figure 1: Chemical structures of compounds 1-15.
Figure 2: Michaelis-Menten plots of AChE inhibition by compounds 6 (a), 10
(b), 11 (c), and 12 (d).
demonstrated an interesting structural feature. The active alkaloids
indicated the specific necessity of the N,N-dimethylaminopropyloxy
terminal extended as a para substitution from the dibromotyramine
residue. Among the four active compounds, the pre-existing
quaternary ammonium functionality of 6 and 12 seemingly
outweighed the tertiary amino group of 10 and 11 up to 50-100
folds; however, the buffered condition (pH 8) might have to be
taken into consideration that all four may interact with the enzyme
Olatunji et al.
in a rather comparable state. Nonetheless, it is clear that the
electronic alteration of the amino group toward an N-oxide
(compound 13) diminishes the activity. On the other hand,
changing the structural features on the other end of either active
compound, from the spiro-isoxazole unit (6) to the non-isoxazole
bromotyramine with α-keto oxime (12) and amine (10 and 11) does
not cast a strong effect on the potency of the enzyme inhibition.
Although more structural variation in the dataset is needed for a
more concrete conclusion, among the four active alkaloids, having
an oxime (12) or related functionality (as an isoxazole of 6) may
exert a superiority over the amino group of 10 and 11.
The presence of the N,N-dimethylaminopropyloxy-
dibromotyramine subunit in the active alkaloids also allows the
prediction of the binding loci on the enzyme. It is very well known
that the active sites of AChE are located deep in the core of the
enzyme molecule, with the catalytic triad at the bottom of the
binding gorge, the anionic site for the quaternary ammonium
inhibitors lying nearby the triad, and the hydrophobic binding areas
of aromatic amino acids lining the gorge wall [1b,c]. The
dimethylamino terminal of the active alkaloids is proposed to bind
to the anionic site, whereas the dibromotyramine subunit binds to
the allosteric site of the aromatic-lining gorge, and hence the non-
competitive inhibition. The importance of the specific chain length
of the propyloxy linker has been reported [13]. Compared with
compounds 2-4, the lack of activity of the three compounds may be
attributed either to the change in the length of the linkers, or to the
substitution pattern in the terminal nitrogens, or both.
Bromotyrosine alkaloids are among the groups of marine natural
products that have been shown to exhibit a wide range of biological
activities. However, their AChE-inhibiting activity and the mode of
enzyme inhibition have never been tapped, and are reported here for
the first time. Despite the limited compound dataset, the results
indicated the significance of the N,N-dimethylaminopropyloxy-
dibromotyramine subunit. Extended studies, particularly on
enzyme-binding simulation, may allow the identification of possible
pharmacophores, and open up an opportunity for the development
of new AChE-Is based on the structures of the bromotyrosines.
Experimental
General: Unless stated otherwise, all the chemicals and solvents
were of reagent-grade, and were used as purchased. HPLC was
performed on a Waters 600E system controller, equipped with a
Rheodyne 7125 injector port and a Waters 484 tunable UV detector.
Animal material: The sponge Pseudoceratina cf. purpurea (family
Pseudoceratiniidae) was collected at a depth of 15-20 m from Koh-
Ha Islets, Thailand (7° 25' N, 98° 53' E) in February, 2010. The
specimen was kept in an ice-chest upon surfacing, and at -20° once
it arrived in the lab. The taxonomic identification was kindly
supported by Dr Sumaitt Putchakarn, Institute of Marine Science,
Burapha University, Chonburi, Thailand. A voucher specimen
(AP10-008-10K) was lodged at the Department of Pharmacognosy
and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Prince of Songkla University.
Extraction and Isolation: The extract (EtOAc/CH3OH 1:1) of the
sponge (1.2 kg wet weight) was consecutively partitioned to yield
n-hexane-, C2Cl4-, CHCl3-, and n-BuOH-extracts (4.5, 1.7, 1.4, and
3.5 g, respective). The CHCl3-extract, inhibiting AChE activity by
more than 90% at 0.1 mg/mL, was fractionated over silica gel
(CH3OH/CH2Cl2 1:9), Sephadex LH20 (CH3OH), and RP-C18
HPLC (VertiSep®, 10 m, 10×250 mm; 0.1% TFA/CH3OH 2:3)
Bromotyrosine alkaloids as acetylcholinesterase inhibitors Natural Product Communications Vol. 9 (11) 2014 1561

columns to yield 6 (15 mg), 12 (28 mg), 1 (18 mg), 13 (11 mg), 14
(5 mg), 8 (17 mg), 9 (5 mg), and 7 (5 mg). The n-BuOH-extract
was also active against AChE at 0.1 mg/mL (> 90% inhibition).
Fractionation over silica gel (H2O/CH3OH/CH2Cl2 1:4:15),
Sephadex LH20 (CH3OH), and RP-C18 HPLC (VertiSep®, 10 m,
10×250 mm; 0.1% TFA/CH3OH 1:1) columns yielded 11 (10 mg),
10 (7 mg), 4 (10 mg), 5 (4 mg), 15 (16 mg), and 2 (24 mg). The
CCl4-extract was fractionated over Sephadex LH20 (CH3OH) and
by RP-C18 HPLC (VertiSep®, 10 m, 10×250 mm ; CH3CN/i-
PrOH/H2O 9:8:3) to yield 3 (20 mg).
AChE inhibitory activity determination: The enzyme inhibiting
activity was determined using Ellman’s method [11]. To a solution
containing 3 mM DTNB (125 L), 15 mM acetylthiocholine iodide
(25 L), and Tris buffer (pH 8.0, 50 L) was added a solution of the
test samples (10-1-10-5 mg/mL; 25 L) and 0.28 U/mL AChE (25
L) in Tris buffer. The developing yellow solution was measured
at 405 nm every 5 s for 2 min. Enzyme activity was calculated
from the reaction velocity and expressed as IC50, with galanthamine
as standard drug (IC50 0.59 M). The Michaelis-Menten plots for
compounds 6, 10, 11, and 12 were prepared in the same manner at
the IC50 value of each test sample. The reaction velocities upon
varying the concentrations of acetylthiocholine iodide (25-10000
M) were compared with that of a blank solution (Tris buffer).
Antiproliferative activity determination: The SRB method [12] was
used for the antiproliferative activity against MCF-7 breast
carcinoma and fibroblast cells. The monolayer culture of each cell
line was treated with a serial dilution of each compound in EMEM
medium. After an incubation period (7 d), the cells were fixed with
TCA and stained with SRB solution in acetic acid. The dye was
dissolved in Tris, and the developed pink color was measured at 429
nm. The activity was reported as IC50s, and camptothecin was the
standard drug (IC50s 1.2×10-3 and 459.3×10-3 M against MCF-7
and fibroblast cells, respectively).
Acknowledgments – The investigation was supported by the
Thailand Research Fund (RSA5480030) and by the Higher
Education Commission through the National Research University
Program (PHA540539S). O.J.O. thanks Graduate School, Prince of
Songkla University, and the National Research University Program,
for his financial support. K.C. and K.I. acknowledge the supporting
grant from the Center of Excellence for Innovation in Chemistry
(PERCH-CIC), Office of the Higher Education Commission,
Ministry of Education.

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