Patamawadee Silalai1, Suwichada Jaipea1, Jiraporn Tocharus2, Anan Athipornchai1, Apichart Suksamrarn3, Rungnapha Saeeng1,4. 1. Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand. 2. Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. 3. Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand. 4. The Research Unit in Synthetic Compounds and Synthetic Analogues from Natural Product for Drug Discovery (RSND), Burapha University, Chonburi 20131, Thailand.
Abstract
A novel series of 1,2,3-triazole-genipin analogues were designed, synthesized, and evaluated for neuroprotective activity, acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) inhibitory activity. The genipin analogues bearing bromoethyl- and diphenylhydroxy-triazole showed in vitro neuroprotective properties against H2O2 toxicity along with potent inhibitory activity on BuChE with IC50 values of 31.77 and 54.33 μM, respectively, compared with galantamine (IC50 = 34.05 μM). The molecular docking studies of these genipin analogues showed good binding energy and interact well with the key amino acids of BuChE via hydrogen-bonding and hydrophobic interactions. Triazole genipins might be promising lead compounds as anti-Alzheimer's agents.
A novel series of 1,2,3-triazole-genipin analogues were designed, synthesized, and evaluated for neuroprotective activity, acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) inhibitory activity. The genipin analogues bearing bromoethyl- and diphenylhydroxy-triazole showed in vitro neuroprotective properties against H2O2 toxicity along with potent inhibitory activity on BuChE with IC50 values of 31.77 and 54.33 μM, respectively, compared with galantamine (IC50 = 34.05 μM). The molecular docking studies of these genipin analogues showed good binding energy and interact well with the key amino acids of BuChE via hydrogen-bonding and hydrophobic interactions. Triazole genipins might be promising lead compounds as anti-Alzheimer's agents.
Alzheimer’s
disease (AD) is the most common form of neurodegenerative
disorder and the most prevalent cause of dementia, making it one of
the major public health problems.[1] A report
by the World Health Organization (WHO) showed that about 50 million
people are affected by dementia worldwide, and it is projected to
affect around 115.4 million people worldwide by 2050.[2] Nowadays, four common drugs for the treatment of Alzheimer’s
disease have been approved by the European and United States regulatory
authorities including tacrine,[3] memantine,[4] galantamine,[5] and
donepezil[6] (Figure ). The therapeutic drugs on the market are
not widely available since their efficacy is limited by diverse unpleasant
side effects. Thus, there is an urgent need for the development of
effective anti-Alzheimer’s agents with low side effects.
Figure 1
Examples of
drugs used for Alzheimer’s disease (AD) available
on the market.
Examples of
drugs used for Alzheimer’s disease (AD) available
on the market.AD is a multifactorial disease
commonly featuring neuronal cell
death and loss of cholinergic neurons due to a decrease in acetylcholine
availability at neuronal synapses.[7] From
a physiological point of view, the activity of acetylcholine in the
synapses can be diminished by the enzymes acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE).[8] Therefore,
one efficient approach to cure AD is to restore the level of acetylcholine
using AChE and BuChE inhibitors.[9] In normal
healthy brains, AChE plays an important role and BuChE is supportive
in the hydrolysis of acetylcholine.[10] As
AD progresses, the level of AChE in the brain declines by approximately
50% of normal values whereas BuChE progressively increases to 120%
of normal levels. Thus, the BuChE activity progressively increases
as the graveness of dementia increases but AChE activity diminishes.
Therefore, BuChE was examined as a key target for the treatment of
AD.[11,12] Hence, BuChE inhibitors with neuroprotection
potential may have a special therapeutic effect on AD.[13,14]Gardenia jasminoides Ellis
is a
flowering plant belonging to the gardenia genus in the Rubiaceae family.
The fruits are used as a therapeutic herb that is rich in biological
activity, such as inflammation, jaundice, and hepatic disorders.[15] Generally, this herb is used in herbal medicines
or functional food supplements displaying therapeutic effects on central
nervous system (CNS) diseases, including dementia, cerebral stroke,
and antioxidants with nonharmful and nontoxic side effects.[16] Geniposide, the main component in the fruit,
belongs to the class of iridoid glycoside and can be hydrolyzed into
genipin 1 by intestinal bacteria after ingestion (Figure ).[17]
Figure 2
Chemical structures of geniposide, genipin 1, and
derivatives.
Chemical structures of geniposide, genipin 1, and
derivatives.Pharmacokinetic studies have suggested
that genipin is the main
active compound and showed promising bioactivities as a strong neuroprotection
agent by inhibiting high-level lactate dehydrogenase (LDH) in the
blood, which causes amyloid-β (Aβ) peptide toxicity in
cultured neuronal cells.[18] Recently, Huang
et al.[19] reported that piperazine-genipin
analogues are dual AChE/Aβ1–42 aggregation
inhibitors, which repair the neuronal cell damage from amyloid-β
(Aβ) peptide toxicity by 22.3% (Figure ). These results led us to design and modify
the structure of genipin to explore the potential of its derivatives
as candidates for the treatment of AD.1,2,3-Triazole is a five-membered
heterocyclic compound containing
two carbon and three nitrogen atoms. 1,2,3-Triazole is found in abundance
in medicinal compounds.[20,21] The triazole ring displays
bond acceptor properties capable of forming significant interactions
with biomolecular targets through H-bonding, π–π
stacking, and dipole interactions. These scaffolds are commonly synthesized
through Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition between
alkynes and azides (CuAAC) by the concept of click.[22−26] Previously, some triazole derivatives were synthesized
as potent and highly selective BuChE inhibitors and neuroprotective
agents (Figure ).[27−30]
Figure 3
Some
examples of 1,2,3-triazole used as inhibitors of butyrylcholinesterase
(BuChE) and neuroprotective agents.
Some
examples of 1,2,3-triazole used as inhibitors of butyrylcholinesterase
(BuChE) and neuroprotective agents.Considering the work on genipin and 1,2,3-triazole mentioned above,
the linking of these two units might lead to hybrids with higher neuroprotective
activity than the parent genipin. Therefore, in this work, a new series
of 1,2,3-triazole-genipin analogues were designed and synthesized
and were focused on the biological evaluation as selective BuChE inhibitors
with neuroprotective properties. The molecular docking studies were
also explored for further understanding of enzyme inhibition (Figure ).
Figure 4
Design of novel 1,2,3-triazole-genipin
analogues.
Design of novel 1,2,3-triazole-genipin
analogues.
Results and Discussion
Chemistry
The synthetic route for
modification of genipin to 1,2,3-triazole-genipin analogues 8a and 8b is depicted in Scheme . The target compounds were synthesized via six- or seven-step reactions. Initially, genipin 1 was silylated to protect the hydroxy at C-10 by stirring
in tert-butyldimethylsilyl chloride (TBSCl) in pyridine
for 10 min to obtain 2. Compound 2 was further
reacted with imidazole and acetic anhydride (Ac2O) or tert-butyldiphenylsilyl chloride (TBDPSCl) for conversion
of the hydroxy of the hemiacetal to acetyl or silyl ether giving compounds 3a or 3b, respectively. Subsequently, deprotection
of tert-butyldimethylsilyl (TBS)-ether at the C-10
position of genipin was achieved by dropwise addition of HCOOH/H2O (9:1) at 0 °C and stirring for 6 h to obtain a crude
product 4a or 4b. Mesylation of the resulting
hydroxy group afforded the corresponding mesyl derivatives. No further
chromatography purification was necessary for the four steps. Azidation
of mesylate group to desired azido precursor 6a yielded
72% in five steps. Desilylation of the TBDPS group at C-1 of 6b was carried out using tetra-n-butylammonium
fluoride (TBAF) to obtain the hemiacetal analogue 7b in
43% yield in six steps. The azide–alkyne Huisgen cycloaddition
reaction was performed in the final step by mixing 6a or 7b with various alkynes using copper iodide to promote
reaction to give a series of C-10 triazole analogues 8a or 8b.
Scheme 1
Synthesis of the 10-Triazolylgenipin Analogues 8a and 8b
Reaction conditions:
(a) TBSCl,
pyridine, 10 min; (b) Ac2O or TBDPSCl, imidazole, dichloromethane
(DCM), 1 h; (c) HCOOH/H2O (9:1), tetrahydrofuran (THF),
0 °C to room temperature (rt), 6 h; (d) MsCl, Et3N,
DCM, 0 °C to rt, 24 h; (e) NaN3, dimethylformamide
(DMF), 0.5 h; (f) TBAF, DCM, 0.5 h; (g) alkyne, CuI, Et3N, CH3CN, rt.
Synthesis of the 10-Triazolylgenipin Analogues 8a and 8b
Reaction conditions:
(a) TBSCl,
pyridine, 10 min; (b) Ac2O or TBDPSCl, imidazole, dichloromethane
(DCM), 1 h; (c) HCOOH/H2O (9:1), tetrahydrofuran (THF),
0 °C to room temperature (rt), 6 h; (d) MsCl, Et3N,
DCM, 0 °C to rt, 24 h; (e) NaN3, dimethylformamide
(DMF), 0.5 h; (f) TBAF, DCM, 0.5 h; (g) alkyne, CuI, Et3N, CH3CN, rt.As shown in Scheme , the reactions of
different alkynes with azido genipin 6a and 7b were explored. Genipin analogues 8a and 8b bearing phenyl, benzyl ether, benzylamine, and
aliphatic, phthalimide, and alicyclic substituted triazoles with different
carbon chain lengths were successfully obtained in good to excellent
yields. It was observed that phenyl-substituted, benzyl ether, and
benzylamine afforded the corresponding products (8a-1–8a-8) in moderate to excellent yields (52–99%).
Different substituents on the aromatic ring (−OCH3, −F) in compounds 8a-2 and 8a-3 did not affect the yields in this transformation. A series of aliphatic
chains bearing bromine 8a-9, hydroxy group 8a-10 and 8a-11, silyl group (8a-12–8a-15), trityl 8a-16, and long-chain aliphatic
chains (8a-17–8a-19) were reacted
smoothly, affording the desired products in good to excellent yields
(66–92%). Moreover, the reaction proceeded in good to excellent
yields with alkynyl-phthalimides and hydroxy alicyclic alkyne (8a-20–8a-26).
Scheme 2
Scope of Alkyne for
Synthesis of the 1,2,3-Triazole-genipin Analogues 8a and 8b
Reaction conditions: 6a (0.3413 mmol) or 7b (0.3980 mmol), CuI (20 mol %),
Et3N (0.5 equiv), and alkyne (1.5 equiv) in CH3CN (1.0
mL) at rt (10 min to 24 h). % is yields of isolated products after
purification by column chromatography.
Scope of Alkyne for
Synthesis of the 1,2,3-Triazole-genipin Analogues 8a and 8b
Reaction conditions: 6a (0.3413 mmol) or 7b (0.3980 mmol), CuI (20 mol %),
Et3N (0.5 equiv), and alkyne (1.5 equiv) in CH3CN (1.0
mL) at rt (10 min to 24 h). % is yields of isolated products after
purification by column chromatography.For
a series of hemiacetal triazole genipin analogues 8b,
phenyl-substituted with the electron-withdrawing group fluorine 8b-3 were more favorable for conversions than the electron-donating
group methoxy 8b-2. Triazoles bearing diphenyl or aliphatic
chains provided the desired genipin analogues (8b-4–8b-6) in 46–78% yields. Additionally, the reaction
of the long-chain aliphatic substituted triazole delivered the corresponding
products (8b-7–8b-9) in moderate
yields. The triazole substituted with phthalimide gave products 8b-10 and 8b-11 in 68 and 80% yields, respectively.Finally, hydroxy alicyclic reacted smoothly to furnish corresponding
triazole products 8b-12 and 8b-13 in excellent
yields.Based on the above experimental results, the hemiacetal
products 8b gave products with lower yields and stability
than acetoxy-substituted
analogues 8a.
Biological Evaluations
Neuroprotective Effects of 1,2,3-Triazole-genipin
Analogues on the H2O2 Induced Decrease in Cell
Viability
The protective effect of 1,2,3-triazole-genipin
analogues on the H2O2-induced cell viability
was studied. As shown in Table , 250 μM H2O2 significantly reduced
cell viability (43.0 ± 0.9%). During treatment with our synthetic
compounds, 14 analogues exhibited significant neuroprotective activity
with a level up to 70% of cell viability at 0.075 μM and some
analogues showed better activity than the parent genipin 1 (78.0%). In the series of acetoxy analogues 8a, compound 8a-2 with substituted p-methoxy and 8a-3 with p-fluoro substituent on aryltriazole
exhibited neuroprotective activity with 72.5 and 70.9% cell viability
at 0.075 μM, respectively. Both the electron-withdrawing and
electron-donating groups on aryltriazole showed similar results. Triazole
genipin analogue 8a-5 with benzyl ether (79.7% of cell
viability) and 3,4-OCH38a-7 (78.1% of cell
viability) showed better cell viabilities than the analogues 4-OCH38a-6 (62.7% of cell viability) and benzylamine 8a-8 (50.8% of cell viability). When the benzyl ether substituent
(8a-5–8a-8) was replaced by an alkyl
chain (8a-9–8a-19), the neuroprotective
activity was significantly increased. Substituents such as Br and
OH aryltriazoles on compounds 8a-10, 8a-11, and 8a-14 showed superior neuroprotective activity,
while compounds 8a-20–8a-24 containing
substituted phthalimide displayed moderate neuroprotective activity.
The exception was 8a-20, which showed remarkable cell
viability of up to 78.5% at 0.075 μM. Furthermore, hydroxy-hexacyclic
compound 8a-26 showed better activity than the pentacyclic
compound 8a-25 at 0.075 μM, which indicated the
effect of substituted groups on the neuroprotective activity.
Table 1
Neuroprotective Effects of 1,2,3-Triazole-genipin
Analogues
cell
viability (%)a/recovery of cell viability
(%)b
compounds
0.075 μM
0.15 μM
0.3 μM
0.6 μM
control
100
H2O2 (250 μM)
43.0 ± 0.9
genipin 1
78.0 ± 0.5 (35.0)
71.4 ± 0.5 (28.4)
57.8 ± 0.7 (14.8)
64.4 ± 0.5 (21.4)
8a-1
NAc
NAc
NAc
NAc
8a-2
72.5 ± 0.6 (29.5)
66.6 ± 0.7 (23.6)
60.1 ± 1.0 (17.1)
54.2 ± 0.8 (11.2)
8a-3
70.9 ± 1.5
(27.9)
64.9 ± 1.2 (21.9)
58.6 ±
0.9 (15.6)
52.3 ± 0.9 (9.3)
8a-4
NAc
NAc
NAc
NAc
8a-5
79.7 ± 0.4 (36.7)
73.0 ± 0.2 (30.0)
67.1 ± 0.2 (24.1)
60.6 ± 0.4 (17.6)
8a-6
62.7 ± 0.3 (19.7)
65.9 ± 0.7
(22.9)
67.6 ± 0.5 (24.6)
69.5 ±
0.2 (26.5)
8a-7
78.1 ± 0.4 (35.1)
71.7 ± 0.06 (28.7)
65.3 ± 0.21 (22.3)
59.0 ± 0.21 (16.0)
8a-8
50.8 ± 0.9
(7.8)
50.8 ± 0.6 (7.8)
52.9 ±
0.5 (9.9)
51.2 ± 0.8 (8.2)
8a-9
NAc
NAc
NAc
NAc
8a-10
80.9 ± 0.4 (37.9)d
74.8 ± 0.6 (31.8)
68.9 ± 0.5 (25.9)
61.9 ± 0.2 (18.9)
8a-11
83.5 ±
2.5 (40.5)d
77.1 ±
2.1 (34.1)
71.2 ± 2.8 (28.2)
65.3
± 0.29 (22.3)
8a-12
49.6 ± 0.6 (6.6)
48.5 ± 0.8 (5.5)
47.8 ± 0.3 (4.8)
53.2 ± 0.5 (10.2)
8a-13
74.2 ± 0.4
(31.2)
68.6 ± 0.15 (25.6)
62.8 ± 0.2 (19.8)
56.6 ±
0.2 (13.6)
8a-14
78.8 ± 0.4 (35.8)
72.7 ± 0.4 (29.7)
66.6 ± 0.1 (23.6)
60.8 ± 0.8 (17.8)
8a-15
NAc
NAc
NAc
NAc
8a-16
NAc
NAc
NAc
NAc
8a-17
56.1 ± 0.5
(13.1)
62.5 ± 0.4 (19.5)
68.1 ±
0.1 (25.1)
74.4 ± 0.6 (31.4)
8a-18
52.1 ± 0.5 (9.1)
58.2 ± 0.3 (15.2)
60.8 ± 0.6 (17.8)
61.2 ± 0.8 (18.2)
8a-19
57.1 ± 0.3 (14.1)
63.2 ± 0.3
(20.2)
69.2 ± 0.4 (26.2)
75.3 ±
0.5 (32.3)
8a-20
78.5 ± 0.4 (35.5)
71.6 ± 0.1 (28.6)
66.4 ± 0.1 (23.4)
60.1 ± 0.1 (17.1)
8a-21
53.5 ± 0.6
(10.5)
54.2 ± 1.1 (11.2)
56.8 ±
1.3 (13.8)
58.2 ± 1.7 (15.2)
8a-22
53.2 ± 2.5 (10.2)
54.9 ± 2.7 (11.9)
60.8 ± 1.6 (17.8)
65.5 ± 2.7 (22.5)
8a-23
50.9 ± 0.4 (7.9)
52.9 ± 0.7
(9.9)
61.6 ± 0.5 (18.6)
66.1 ±
0.8 (23.1)
8a-24
50.7 ± 0.5 (7.7)
51.7 ± 0.7 (8.7)
60.5 ± 0.7 (17.5)
65.9 ± 1.1 (22.9)
8a-25
52.3 ± 0.8
(9.3)
58.1 ± 1.1 (15.1)
60.2 ±
1.2 (17.2)
63.4 ± 0.2 (20.4)
8a-26
75.1 ± 0.9 (32.1)
69.2 ± 1.1 (26.2)
63.3 ± 1.0 (20.3)
57.1 ± 1.2 (14.1)
8b-1
NAc
NAc
NAc
NAc
8b-2
54.9 ± 2.5 (11.9)
63.0
± 2.6 (20.0)
75.2 ± 2.3 (32.2)
78.6 ± 1.9 (35.6)
8b-3
50.9 ± 1.2 (7.9)
52.6 ± 1.6
(9.6)
55.9 ± 1.1 (12.9)
59.9 ±
0.7 (16.9)
8b-4
73.0 ± 3.1 (30.0)
80.9 ± 3.0 (37.9)d
72.7 ± 2.6 (29.7)
69.2 ± 1.7 (26.2)
8b-5
NAc
NAc
NAc
NAc
8b-6
NAc
NAc
NAc
NAc
8b-7
51.6 ± 1.4 (8.6)
53.2 ± 1.7 (10.2)
53.7 ± 2.5 (10.7)
65.9 ± 1.1 (22.9)
8b-8
52.1 ± 0.5 (9.1)
58.2 ± 0.3
(15.2)
60.8 ± 0.6 (17.8)
61.2 ±
0.8 (18.2)
8b-9
NAc
NAc
NAc
NAc
8b-10
52.0 ± 1.8 (9.0)
70.9 ± 1.3 (27.9)
73.6 ± 1.6 (30.6)
77.2 ± 1.8 (34.2)
8b-11
70.1 ± 1.5
(27.1)
72.7 ± 1.5 (29.7)
75.5 ±
0.1 (32.5)
72.2 ± 0.9 (29.2)
8b-12
75.0 ± 2.5 (32.0)
78.3 ± 1.8 (35.3)
78.2 ± 0.9 (35.2)
77.2 ± 0.9 (34.2)
8b-13
72.5 ± 2.5 (29.5)
79.9 ± 0.5
(36.9)
79.2 ± 1.0 (36.2)
74.1 ±
1.7 (31.1)
Cell viability (%): the cell viability
in control was taken as 100%; all data were expressed as mean ±
standard deviation (SD) (n = 3).
Recovery of cell viability (%):
the difference value between the cell viability of compound-treated
cells and that of H2O2-treated cells.
NA: not active.
Bold values highlight the most potent
activity.
Cell viability (%): the cell viability
in control was taken as 100%; all data were expressed as mean ±
standard deviation (SD) (n = 3).Recovery of cell viability (%):
the difference value between the cell viability of compound-treated
cells and that of H2O2-treated cells.NA: not active.Bold values highlight the most potent
activity.For the series
of hemiacetal triazole genipin analogues 8b, compounds
bearing electron-donating 4-OCH3 aryl (compound 8b-2, 78.6%) exhibited remarkably higher neuroprotective activity
than the electron-withdrawing p-fluoroaryl (compound 8b-3, 59.9%) at the same concentration of 0.6 μM. When
the concentration was reduced to 0.075–0.3 μM, the cell
viability decreased. The activity results of 8a-2 and 8b-2 demonstrated that a substituted group at the C-1 position
showed a significant difference between the neuroprotective potencies.
In addition, at a concentration of 0.15 μM, compound 8b-4 exhibited significant neuroprotective effects and the cell viability
was up to 80.9%. When the concentration was reduced to 0.075 μM,
the cell viability decreased to 73.0%. Therefore, a concentration
of 0.15 μM was suitable for treatment by the synthetic compounds.
For the replacement of long-chain aliphatic ether groups on triazole
with different carbon chain lengths, both 8b-7 and 8b-8 decreased the cell viability compared with an aromatic
substituted triazole (8b-2 and 8b-4). On
replacing alkyl with phthalimide, compounds 8b-10 and 8b-11 exhibited strong neuroprotective activities of 77.2
and 75.5%, respectively. Moreover, compounds 8b-12 and 8b-13 bearing a hydroxyl cyclic group increased cell viability
with percentages of 79.9 and 77.3%, respectively, at 0.15 μM,
which might be due to its favorable conformation that allows these
scaffolds to fit within the active site of the enzyme.The overall
results indicated that some 1,2,3-triazole-genipin
analogues (8a-5, 8a-7, 8a-10, 8a-11, 8a-14, 8a-20, 8b-2, 8b-4, 8b-12, and 8b-13) at concentrations of 0.075–0.6 μM significantly improved
the cell viability rate of H2O2-treated neuronal
cells by up to 78% (Figure ). Compound 8a-11 showed the highest protective
capability (83.5% of cell viability) at 0.075 μM recovering
the neuronal cell damage from H2O2 toxicity
with 40.5%. Compounds 8a-10 and 8b-4 displayed
similar protective ability (80.9% of cell viability) recovering the
neuronal cell damage by H2O2 toxicity with 37.9%.
These three analogues evidenced the most significant protection in
reducing H2O2-induced neurotoxicity in neuroblastoma
cells.
Figure 5
Neuroprotective effect of 1,2,3-triazole-genipin analogues on survival
of H2O2-treated neurons, Compounds 1, 8a-5, 8a-7, 8a-10, 8a-11, 8a-14, 8a-20, 8b-2, 8b-4, 8b-12, and 8b-13 significantly
exhibited the neuroprotective effect with >78% cell viability.
Neuroprotective effect of 1,2,3-triazole-genipin analogues on survival
of H2O2-treated neurons, Compounds 1, 8a-5, 8a-7, 8a-10, 8a-11, 8a-14, 8a-20, 8b-2, 8b-4, 8b-12, and 8b-13 significantly
exhibited the neuroprotective effect with >78% cell viability.
Cholinesterase Inhibition
Assay
The inhibitory activity of the newly synthesized 1,2,3-triazole-genipin
analogues (8a and 8b) was evaluated against
electric eel-derived AChE (eeAChE) and equine serum-derived BuChE
(eqBuChE)[31] and compared with galantamine,
a reference drug with IC50 values 12.7 and 34 μM,
respectively. As shown in Table , the results of inhibitory activity for butyrylcholinesterase
(BuChE) were superior to acetylcholinesterase (AChE). All synthetic
analogues demonstrated inhibition of AChE less than 50%, and hence
further IC50 measurement was not carried out. Almost all
synthesized compounds gave higher inhibitory activities against BuChE
than the natural product geniposide and genipin 1 indicating
that the introduction of the triazole ring greatly influenced the
inhibitory behavior of genipin.
Table 2
ChE Inhibitory Activity
of 1,2,3-Triazole-genipin
Analoguesa
AChE
inhibitory activity
BuChE
inhibitory activity
compounds
inhibition (%)b
IC50 (μM ± SD)b
inhibition (%)b
IC50 (μM ± SD)b
geniposide
0.73 ± 0.11
NAc
2.09 ± 0.18
NAc
genipin 1
4.94 ± 0.05
NAc
2.42 ± 0.18
NAc
8a-1
1.31 ± 0.08
NAc
47.75 ± 0.29
NAc
8a-2
9.28 ± 0.14
NAc
34.97 ± 0.26
NAc
8a-3
7.89 ± 0.05
NAc
33.37 ± 0.18
NAc
8a-4
20.89 ± 0.22
NAc
27.14 ± 0.09
NAc
8a-5
0.30 ± 0.31
NAc
39.26 ± 0.18
NAc
8a-6
6.03 ± 0.15
NAc
42.91 ± 0.20
NAc
8a-7
20.35 ± 0.06
NAc
35.99 ± 0.29
NAc
8a-8
36.60 ± 0.42
NAc
41.96 ± 0.12
NAc
8a-9
9.39 ± 0.11
NAc
1.21 ± 0.18
NAc
8a-10
9.27 ± 0.19
NAc
97.34 ± 0.18
31.77 ± 0.17
8a-11
0.26 ± 0.15
NAc
35.48 ± 0.59
NAc
8a-12
1.16 ± 0.14
NAc
1.79 ± 0.17
NAc
8a-13
0.43 ± 0.19
NAc
9.03 ± 0.30
NAc
8a-14
1.33 ± 0.08
NAc
27.54 ± 0.20
NAc
8a-15
0.28 ± 0.21
NAc
22.32 ± 0.52
NAc
8a-16
8.00 ± 0.79
NAc
10.70 ± 0.15
NAc
8a-17
8.12 ± 0.22
NAc
37.35 ± 0.61
NAc
8a-18
12.11 ± 0.30
NAc
23.68 ± 0.17
NAc
8a-19
44.70 ± 0.19
NAc
25.56 ± 0.05
NAc
8a-20
5.52 ± 0.32
NAc
27.29 ± 0.17
NAc
8a-21
6.62 ± 0.36
NAc
78.64 ± 0.10
274 ± 3.9
8a-22
19.46 ± 0.20
NAc
99.85 ± 0.13
203 ± 1.7
8a-23
0.13 ± 0.29
NAc
71.55 ± 0.22
419 ± 4.4
8a-24
0.88 ± 0.30
NAc
47.55 ± 0.13
NAc
8a-25
0.62 ± 0.24
NAc
40.70 ± 0.23
NAc
8a-26
0.48 ± 0.17
NAc
42.73 ± 0.18
NAc
8b-1
8.19 ± 0.11
NAc
67.51 ± 0.16
537 ± 2.0
8b-2
4.41 ± 0.03
NAc
98.69 ± 0.24
109.1 ± 0.61
8b-3
7.53 ± 0.18
NAc
84.64 ± 0.12
281 ± 2.7
8b-4
11.11 ± 0.88
NAc
99.72 ± 0.18
54.3 ± 0.34
8b-5
2.30 ± 0.14
NAc
24.94 ± 0.23
NAc
8b-6
16.44 ± 0.25
NAc
35.42 ± 0.20
NAc
8b-7
1.70 ± 0.09
NAc
42.57 ± 0.16
NAc
8b-8
0.49 ± 0.22
NAc
25.04 ± 0.23
NAc
8b-9
2.73 ± 0.43
NAc
31.51 ± 0.14
NAc
8b-10
1.06 ± 0.18
NAc
42.30 ± 0.64
NAc
8b-11
0.51 ± 0.09
NAc
81.22 ± 0.38
289. ± 1.0
8b-12
0.97 ± 0.13
NAc
27.26 ± 0.27
NAc
8b-13
6.14 ± 0.12
NAc
32.03 ± 0.74
NAc
galantamine
98.52 ± 0.12
12.67 ± 0.07
96.21 ± 0.18
34.05 ± 0.32
The most potent compound is given
in bold.
Inhibition % and
IC50 values represent the concentration of inhibitor required
to decrease
enzyme activity by 50% and are the mean of three independent experiments,
each performed in triplicate (SD = standard deviation).
NA = no activity. Compounds defined
as “no activity” means that the percent inhibition is
less than 50% at a concentration of 10.0 mM in the assay conditions.
AChE from electric eel., BuChE from horse serum.
The most potent compound is given
in bold.Inhibition % and
IC50 values represent the concentration of inhibitor required
to decrease
enzyme activity by 50% and are the mean of three independent experiments,
each performed in triplicate (SD = standard deviation).NA = no activity. Compounds defined
as “no activity” means that the percent inhibition is
less than 50% at a concentration of 10.0 mM in the assay conditions.
AChE from electric eel., BuChE from horse serum.The series of acetoxy analogues 8a with a diverse
range of substituents on the triazole ring resulting in different
activities were indicated by both the % inhibition of BuChE and IC50 values. Genipin analogue 8a-1 bearing phenyl
triazole exhibited moderate inhibitory activity against BuChE, and
replacement with 4-OMe and 4-F aryltriazole (8a-2 and 8a-3) resulted in the loss of activity.Di- and triphenyl,
benzyl ether, and benzylamine triazole genipin 8a-4–8a-9 showed no improved activity
compared to phenyl triazole genipin 8a-1–8a-3. When triazoles were substituted with an alkyl chain
(8a-10–8a-19), they exhibited low
to high inhibitory activity. Surprisingly, genipin analogue 8a-10 with a bromoethyltriazole scaffold exhibited the most
potent inhibitory activity with an IC50 value of 31.8 μM
better than galantamine (IC50 value: 34.1 μM). In
contrast, triazolgenipin containing long-chain aliphatic groups showed
lower activity than the compound comprising a bromoethyl group (8a-10). Furthermore, the replacement of the long-chain alkyl
group with phthalimide scaffolds (8a-21–8a-23) leads to significant improvement in BuChE inhibitory
potencies and exhibited IC50 values of 273.9, 203.4, and
418.5 μM, respectively. Hydroxy-cyclic compounds such as 8a-25 and 8a-26 showed no significant change
in inhibitory activity.The series of hemiacetal triazole genipin
analogues 8b exhibited promising inhibitory potential
against BuChE. The behavior
of phenyl substitution in the triazole ring (8b-1–8b-4) showed relatively more than 50% inhibitory potential
against BuChE. Among all investigated compounds, the diphenylhydroxy
analogue 8b-4 displayed the most potent inhibitory potential
against BuChE with an IC50 of 54.3 μM. While alkyl-chain-substituted
compounds 8b-5–8b-9 and the hydroxy-cyclic
analogues 8b-12 and 8b-13 showed less inhibitory
activities. Changing to phthalimide groups at triazoles (8b-10 and 8b-11) increased the inhibitory activity but less
than compound 8b-4. Triazole genipin 8a-10 with a bromoethyl group showed the best BuChE inhibitory activity
(IC50 = 31.8 μM) and selectivity toward BuChE, surpassing
that of the control galantamine (IC50 = 34.1 μM),
while 8b-4 with a diphenylhydroxy group showed comparable
activity to galantamine.Compared with the report of neuroprotective
activity of piperazine-genipin
analogues (Figure ) by Huang et al.,[19] triazole genipin
analogues 8a-11 in this work showed neuroprotective capability
(83.5% of cell viability at 0.075 μM) higher than piperazine
analogues in the previous report (22.29% at 32 μM). Moreover,
triazole genipin analogues showed selective BuChE activity better
than galantamine while piperazine analogues exhibited inhibitory potential
against anti-AChE.
Kinetic Study for the
Inhibition of BuChE
To gain further insights into the inhibitory
mechanism of 1,2,3-triazole-genipin
analogues, the kinetic behavior of the most active compounds 8a-10 and 8b-4 was investigated using Ellman’s
method reference. The inhibition model and inhibition constant Ki were obtained from plots between 1/velocity versus 1/substrate produced with five different concentrations
of the substrate butylthiocholine iodide (0.3125, 0.625, 1.25, 2.5,
and 5.0 mM). The results showed that the plots of 1/vversus 1/[S] gave straight lines with different
slopes but the same x-intercept points. This graphical
presentation of Lineweaver–Burk plots indicated that the selected
compounds were a noncompetitive enzyme inhibitor and the inhibition
constants (KI, KIS) are nearly identical. The inhibition constants
(KI, KIS) for compounds 8a-10 and 8b-4 were
estimated to be 0.03 and 0.1 mM, respectively (Figure ).
Figure 6
Lineweaver–Burk plot for the inhibition
of BuChE by compounds 8a-10 and 8b-4 at
different concentrations of
substrate.
Lineweaver–Burk plot for the inhibition
of BuChE by compounds 8a-10 and 8b-4 at
different concentrations of
substrate.
Docking
Study of BuChE
The molecular
docking simulation study of the most potent compounds 8a-10 and 8b-4 was performed to understand the inhibition
mechanism within the active site of the target enzymes BuChE (PDB
code: 4BDS)
using AutoDock 4.2 software.Analog 8a-10 showed
a good fit in the pocket site of the enzyme by interaction with important
amino acid residues and exhibited a binding free energy of −9.77
kcal/mol with BuChE (Table ). Molecular docking of 8a-10 showed three hydrogen
bonds of the ester unit at the C4-position of the iridoid moiety with
the residues His438 (catalytic subsite) and Ser198 of the CAS along
with the interaction of the acetoxy group at the C1-position. The
carbonyl group of acetoxy formed three hydrogen bonds with the Trp82
(a key residue in the CAS of BuChE), Trp430, and Tyr440 moieties.
Furthermore, the triazole group also formed a hydrogen-bond interaction
with the Tyr332 and showed remarkable ionic interaction with Asp70
residue in the PAS region (Figure ). These interaction behaviors indicated the potential
of 8a-10 to inhibit BuChE. The molecular docking studies
of 8b-4 also showed preferential interaction with the
active site of BuChE with a binding energy of −9.74 kcal/mol
(Table ). The iridoid
core of 8b-4 is mostly surrounded by residues of the
CAS pocket while diphenyl moiety is oriented toward the PAS pocket.
The carbonyl group of the iridoid formed a hydrogen-bond interaction
with the Trp82, a key residue in the CAS of BuChE. Meanwhile, the
diphenylhydroxy group of 8b-4 showed the same binding
orientation within the active site of the target enzyme via two π–π interactions with Tyr332 (a key residue
in the PAS of BuChE) (Figure ). Furthermore, hydrogen bonds and a π–π
interaction between the triazole moiety and His438, Ser198, and Phe329
of the CAS were also observed (Figure ). The modification of introducing a substituted triazole
to genipin led to analogues 8a-10 and 8b-4, which increased the potential interaction of the molecule with
Trp82 and Tyr332, the important active site of BuChE.
Table 3
Molecular Docking Analysis of BuChE
with 1,2,3-Triazole-genipin Analogues 8a-10 and 8b-4a
intermolecular
hydrogen bonding
compounds
binding energy (kcal/mol)
amino acid interaction
distance (Å)
intermolecular π–π interaction
8a-10
–9.77
Trp82
2.10
Asp70
Ser198
1.92
Ser198
2.01
Tyr332
2.98
Trp430
2.22
His438
2.03
Tyr440
1.84
8b-4
–9.74
Trp82
2.34
Tyr332, Trp231, Phe329
Tyr440
2.11
Trp430
2.25
Ser198
2.06
Ser198
2.66
The binding energies were evaluated
using AutoDock 4.2 software.
Figure 7
Proposed binding mode
of compound 8a-10 in the active
site of BuChE (PDB code: 4BDS).
Figure 8
Proposed binding mode
of compound 8b-4 in the active
site of BuChE (PDB code: 4BDS). (A) The protein structure is shown as a ribbon,
and 1,2,3-triazole-genipin 8b-4 is shown as a stick model.
(B) Two-dimensional (2D) interaction molecular docking diagrams. Hydrogen
bonds and π–π interactions are shown as green and
pink dotted lines, respectively.
Proposed binding mode
of compound 8a-10 in the active
site of BuChE (PDB code: 4BDS).Proposed binding mode
of compound 8b-4 in the active
site of BuChE (PDB code: 4BDS). (A) The protein structure is shown as a ribbon,
and 1,2,3-triazole-genipin 8b-4 is shown as a stick model.
(B) Two-dimensional (2D) interaction molecular docking diagrams. Hydrogen
bonds and π–π interactions are shown as green and
pink dotted lines, respectively.The binding energies were evaluated
using AutoDock 4.2 software.
Conclusions
In summary, a novel series
of 1,2,3-triazole-genipin analogues 8 were successfully
designed and synthesized as efficient
multitarget agents for the treatment of AD. Among the synthesized
compounds, analogues 8a-10 and 8b-4 were
found as the most active inhibitors with IC50 values of
31.8 and 54.3 μM, respectively. These two analogues also showed
inhibitory activity of BuChE selectively over AChE and showed better
activity than the standard drug galantamine. Moreover, compounds 8a-10 and 8b-4 were able to rescue the cells
from the toxicity induced by H2O2. Molecular
docking studies of these two compounds confirmed their preferable
binding with BuChE and showed interactions with key amino acid residues.
Therefore, 1,2,3-triazole-genipin analogues 8a-10 and 8b-4 have the potential for the treatment of neurodegenerative
diseases.
Experimental Section
All chemicals
were purchased from commercial sources and used without
further purification. Proton NMR spectra were recorded using a BRUKER
AVANC (400 MHz) spectrometer. All spectra were recorded in CDCl3 solvent, and chemical shifts are reported as δ values
in parts per million (ppm) relative to tetramethylsilane (δ
0.00), CDCl3 (δ 7.26) as internal standard. Carbon
NMR spectra were recorded on a BRUKER AVANC (100 MHz) spectrometer.
All spectra were recorded in CDCl3 solvent, and chemical
shifts are reported as δ values in parts per million (ppm) relative
to CDCl3 (δ 77.0) as the internal standard. High-resolution
mass spectra (HRMS) were recorded at Naresuan University. Analytical
thin-layer chromatography (TLC) was conducted on precoated TLC plates;
silica gel 60F-254 [E. Merck, Darmstadt, Germany]. Silica gel columns
for open-column chromatography utilized silica gel 60 PF254 [E. Merck,
Darmstadt, Germany]. Melting points were measured using a melting
point apparatus (Griffin) and are uncorrected. Genipin as a starting
material (CAS No. 6902-77-8) was purchased from commercial sources
and used without further purification.
Synthesis
of Compound 2
To a rapidly stirring solution
of genipin 1 (2.00 g,
8.850 mmol) in pyridine (10.0 mL) was added tert-butyldimethylsilyl chloride (2.00 g, 13.275 mmol) at room temperature.
The reaction mixture was stirred at room temperature for 10 min. After
TLC showed that the reaction was complete, the mixture was diluted
with EtOAc (30 mL), quenched with saturated NH4Cl solution,
and then extracted with EtOAc. The mixture was quenched with CuSO4·5H2O to remove the pyridine and extracted
with EtOAc and washed with brine, the combined organic layers were
dried over anhydrous Na2SO4, and the solvent
was removed by rotary evaporation to obtain the crude product of compound 2.
Synthesis of Compound 3
To a solution of crude product compound 2 (8.85 mmol)
in DCM (20 mL), imidazole (1.8 g, 26.55 mmol) was added and stirred
for 10 min, then acetic anhydride (2.5 mL, 26.55 mmol) or tert-butyldiphenylsilyl chloride (6.9 mL, 26.55 mmol) was
added to the mixture, and stirred at room temperature for further
1 h. After TLC indicated that the reaction was complete, the reaction
mixture was diluted with DCM (10 mL) and quenched with cold-saturated
NaHCO3, extracted with DCM and washed with brine, then
dried over with Na2SO4 anhydrous, and concentrated
in vacuo to obtain crude product 3a and 3b.
Synthesis of Compound 4
To a stirred solution of compound 3a or 3b (8.85 mmol) in THF (20 mL) was added HCOOH/H2O (9:1)
(40 mL) dropwise at 0 °C and stirred for 6 h. After TLC showed
that the reaction was complete, the reaction mixture was diluted with
EtOAc (30 mL) and quenched with cold-saturated NaHCO3 and
the mixture was extracted with EtOAc. The combined organic extracts
were washed with brine solution, dried (Na2SO4), filtered, and concentrated to obtain a crude product of compound 4a or 4b.
Synthesis
of Compounds 5 and 6
To a solution
of crude products 4a and 4b (8.85 mmol)
in DCM (50 mL), Et3N
(1.8 mL, 13.28 mmol) was added and stirred for 30 min. Then, methanesulfonyl
chloride (1.0 mL, 13.28 mmol) was added to the reaction mixture at
0°C and stirred at room temperature for 24 h. After TLC showed
that the reaction was complete, the reaction mixture was diluted with
DCM (30 mL) and cold-saturated NaHCO3. The reaction mixture
was extracted with DCM, washed with water, then dried over anhydrous
Na2SO4, filtered, and evaporated in
vacuo to obtain the mesylate crude product (5a, 5b). The mesylate of crude product (5a, 5b) was dissolved in DMF (30 mL), and NaN3 (0.863 g, 13.28 mmol) was added at 0 °C. The reaction was stirred
at room temperature, and stirring was continued for 30 min. After
TLC showed that the reaction was complete, the reaction mixture was
diluted with EtOAc (30 mL) and quenched with cooled water. The reaction
mixture was extracted with EtOAc, washed with brine, then dried over
anhydrous Na2SO4, filtered, and evaporated in vacuo to obtain the crude product of 6a and 6b. The crude product 6a was purified by column
chromatography (10% EtOAc/n-hexane) to afford 6a in 72% in five steps.
Synthesis
of Compound 7b
To a stirred solution of compound 6b (8.85 mmol) in
DCM (30 mL), TBAF (2.5 g, 9.735 mmol) was added at 0 °C and stirred
for 30 min. After TLC showed that the reaction was complete, the reaction
mixture was diluted with DCM (30 mL) and quenched with cold-saturated
NH4Cl. The reaction mixture was extracted with DCM, washed
with H2O and brine, followed by drying over with Na2SO4 anhydrous, and concentrated in vacuo to obtain crude product and purified by column chromatography (10%
EtOAc/n-hexane) to afford compound 7b in 43% in six steps.
General Procedure for the
Preparation of 1,2,3-Triazole-genipin
Analogues (8a), (8b)
To the solution
of compound 6a (100 mg, 0.3413 mmol) or compound 7b (100 mg, 0.3980 mmol) in CH3CN (1.0 mL) were
added CuI (20 mol %), Et3N (0.5 equiv), and alkyne (1.5
equiv). After TLC indicated that the reaction was complete, the reaction
mixture was diluted with EtOAc (2 mL), quenched with cooled water,
and extracted with EtOAc (3 × 30 mL). The reaction mixture was
diluted with water (15 mL) and extracted with EtOAc (3 × 15 mL).
The combined organic extracts were washed with brine solution, dried
(Na2SO4), filtered, and concentrated. The resulting
crude product was purified by column chromatography to obtain 1,2,3-triazole-genipin
analogues 8a and 8b.
Authors: L Bartorelli; C Giraldi; M Saccardo; S Cammarata; G Bottini; A M Fasanaro; A Trequattrini Journal: Curr Med Res Opin Date: 2005-11 Impact factor: 2.580
Authors: Mohammad A Kamal; Peter Klein; Weiming Luo; Yazhou Li; Harold W Holloway; David Tweedie; Nigel H Greig Journal: Neurochem Res Date: 2007-11-06 Impact factor: 3.996
Figure 1
Figure 2
Figure 3
Figure 4
Scheme 1
Scheme 2
Figure 5
Figure 6
Figure 7