Design, synthesis, and evaluation of genipin derivatives for the treatment of Alzheimer's Disease

Article abstract of DOI:10.1111/cbdd.13194

Twenty-two novel genipin derivatives have been designed, synthesized, and evaluated for their inhibitory activity against acetylcholinesterase (AChE). As a result, compound 13a bearing ligustrazine moiety displayed the most potent AChE inhibitory activity in this series with IC₅₀ value of 218?nm. Besides, MTT assay was performed to investigate the neuroprotection of these compounds against PC12 cells injured by Amyloid β-protein 1–42 (Aβ_1–42). Among them, 8a showed higher inhibition rate (%Inhibition?=?22.29) than the positive reference Donepezil (%Inhibition?=?17.65).

Full text of DOI:10.1111/cbdd.13194

MR. WEIJUN HUANG (Orcid ID : 0000-0003-2202-0574)
PROFESSOR JIAMING LI (Orcid ID : 0000-0003-2141-7561)
MR. PANHU ZHU (Orcid ID : 0000-0001-5440-4391)
Article type
: Research Article
Design, synthesis and evaluation of genipin derivatives for
the treatment of Alzheimer’s Disease
Weijun Huang, Yujun Wang, Jiaming Li*, Yanchun Zhang, Xiaodong Ma, Panhu
Zhu, Yang Zhang
(Department of Medicinal Chemistry, Anhui University of Chinese Medicine, 103 Meishan Road, Hefei
230031, PR China)
Abstract:
Twenty two novel genipin derivatives have been designed, synthesized and evaluated
for their inhibitory activity against acetylcholinesterase (AChE). As a result,
compound 13a bearing ligustrazine moiety displayed the most potent AChE inhibitory
activity in this serieswith IC50 value of 218 nM. Besides, MTT assay was performed
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to investigate the neuroprotection of these compounds against PC12 cells injured by
Amyloid β-protein 1-42 (Aβ1-42). Among them, 8a showed higher inhibition rate
(%Inhibition = 22.29) than the positive reference Donepezil (%Inhibition = 17.65).
Keywords: genipin derivatives; synthesis; Alzheimer’s disease;
acetylcholinesterase inhibitors; neuroprotection.
Introduction
Alzheimer’s disease (AD) is a progressive, age-related, neurodegenerative
disease that is characterized clinically by progressive memory loss, decline in
language skills, and other cognitive-related impairments.[1] Although the etiology of
AD is not well understood, several factors, such as β-amyloid (Aβ) aggregation and
deposition, hyper phosphorylation of tau protein, oxidative stress, neuroinflammation,
and low brain acetylcholine (ACh) levels, are thought to play critical roles in the
progressive dementia of this disease.[2-3] In accordance with judicial review in August
2007, National Institute for Health and Clinical Excellence (NICE) amended and
reissued its guidance that now states: “Donepezil, galantamine and rivastigmine are
recommended as options for the treatment of moderate Alzheimer’s disease only”.[4]
However, these drugs could merely result in modest and temporary benefit for
memory and cognitive function instead of delaying or stopping the progression of
neurodegeneration.
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Figure 1 The structure of genipin
The fruit of Gardenia jasminoides (Rubiaceae) is widely used as a traditional
Chinese medicine (TCM) in many Asian countries for its cholagogue, diuretic,
antiphlogistic, and antipyretic effects.[5] Geniposide, the main active component of
Gardenia jasminoides, belongs to the class of compounds called the iridoids, and can
be hydrolyzed to generate genipin (Figure 1) by β-glucosidase. Increasing evidence
suggests that geniposide and genipin can relieve the symptoms related to the
progression of AD. Yu et al. conducted a short-term memory assay on an Aβ
transgenic drosophila model and demonstrated that genipin and its glycoside
derivatives had a potential antagonistic effect against Alzheimer’s disease.[6]
However, combination of genipin with glycineproducesgardenia blue, thereby leading
to liver and kidney toxicity.[7] Another factor limiting the practical application of
genipin is its instability when exposed to medium containing nucleophiles.[8]
Figure 2 The structures of genipin derivatives
Suzuki and colleagues tried to address the issues of toxicity and instability by
alkylation at the C-1 hydroxyl group of genipin.[9] Despite the improved stability, the
neuritogenic activity was weakened, even for the most active compound
(1R)-1-isopropylgenipin (IPRG001, Figure 2). In addition to modification at the C-1
hydroxyl group, Wang et al. accomplished the stereoselective reduction of the double
bond between C7 and C8.[10] Both the resultant counterparts CHR20 and CHR21
(Figure 2) were more potent than corresponding C-1 R and S-isomers of IPRG001.
However, no obvious impact on chemical stability was achieved through reduction of
the double bond.[11] Luo et al. undertook the structural modifications at C1, C7, C8,
and C10 of genipin, and the neurotrophic effects of all derivatives were studied.[12]
Among them, Gardenamide A (Figure 2) exerted the best neuroprotective activity and
more favorable stability than genipin.
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Our group has been continuously dedicated to the development of the anti-AD
therapeutics on the basis of the bioactive component in TCM. Due to its advantage in
interfering with the progression of AD, genipin, in this research, was introduced as the
lead compound for the pursuit of anti-AD agents with structural novelty. Herein, a
pharmacophore-combination strategy was utilized via incorporating the substituted
arylmethyl peperazine moiety which can be regarded as a substitute for the benzyl
peperidine fragment of donepezil, to the C-8 position of genipin. Besides, with an
attempt to pursue more favorable chemical stability, the C-1 hydroxyl group was
structurally derivatized as well. Meanwhile, different replacements at the benzene ring
and O-substituents were introduced to investigate their impact on the activity or
stability (Figure 3).
Figure 3 Design of genipin derivatives via pharmacophore-combination strategy
C hemistry
Genipin, as the starting material, was treated with p-TsOH H2O in methanol and
isopropanol to afford the O-alkylated derivatives 2a-2b. Subsequent
methylsulfonylation transformed them into the key intermediates 3a-3b. Fragments
4-15 were prepared by condensation of substituted benzyl chlorides with anhydrous
piperazine. Ultimately, target compounds 4a-15a and 4b-13b were furnished as
hydrochloride salts after reaction of 3a-3b with 4-15 in the presence of K2CO3 and the
following treatment with hydrochloride in methanol.
Scheme 1 Synthetic route of the target compounds
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Biological evaluation
Anticholinesterase activity
Upon introducing Donepezil as the positive control, we first evaluated the AChE
inhibitory potency of the target compounds following a reported method.[13] As a
result (Table 1), 10a, 4a, 7a, 5a, 15a, 12a and 14a exhibited moderate to potent AChE
inhibitory activity comparable to Donepezil (IC50 = 244 nM) with IC50 values of
2642, 2325, 1907, 1779, 1087, 829 and 737 nM. In particular, compounds 13a and
13b, bearing ligustrazine substructure, demonstrated remarkable AChE inhibitory
activity comparable to Donepezil (IC50 = 244 nM) with IC50 values of 218 and 373
nM, respectively. Thus, the introduction of ligustrazine as the terminal aryl group
attached to the piperazinewas beneficial for boosting AChE inhibitory activity.
x
s
,
Table 1. In vitro inhibitory activity of genipin derivatives against AChE (
n = 3)
IC50(3 min)
nM
IC50(5 min)
nM
IC50(7 min) IC50(average)
Compound
SD
nM
2561
nM
2325
1919
2496
3.53
1.69
/
4a
5a
1592
1921
1824
1779
>10000
1587
>10000
1924
>10000
2210
>10000
1907
6a
3.12
/
7a
>10000
7881
>10000
7630
>10000
6762
>10000
7424
8a
5.87
1.83
/
9a
2852
2519
2556
2642
10a
11a
12a
13a
>10000
401
>10000
802
>10000
1283
>10000
829
4.42
0.85
121
253
281
218
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610
769
832
737
1.14
14a
15a
741
984
1535
1087
4.07
>10000
>10000
>10000
>10000
7631
>10000
>10000
>10000
>10000
8960
>10000
>10000
>10000
>10000
5376
>10000
>10000
>10000
>10000
7322
/
4b
/
5b
/
6b
/
7b
18.12
8b
>10000
>10000
>10000
9884
>10000
>10000
>10000
11520
422
>10000
>10000
>10000
8704
>10000
>10000
>10000
10036
373
/
9b
/
/
10b
11b
14.14
0.69
/
12b
294
403
13b
>10000
157
>10000
281
>10000
293
>10000
244
Genipin
Donepezil
0.75
Molecular Docking
Delighted by its remarkable AChE inhibitory activity, we further docked 13a into
TcAChE (PDB code: 1EVE) with the attempt to elucidate its possible binding mode
with the enzyme via utilizing MOE software. According to the results shown in Figure
4, the tetramethylpyrazine moiety of 13a occupied the peripheral anionic site (PAS)
and conferred potential hydrophobic interaction with residues Phe290, Phe331 and Trp
279. In addition, π-π stacking was formed between the piperazine and Tyr334. The
ester functionality of genipin template contacted with Phe288 via a hydrogen bond.
Moreover, the linker between genipin template and ligustrazine group was appropriate
in length for projecting the latter into the catalytic active site, where it interacted with
Gly118 via π-π stacking, and provided hydrophobic interaction with residues Trp84,
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His440 and Tyr121. Taken together, all the above interactions accounted for the
favorable enzymatic potency of 13a. As illustrated by the overlap of compound 13a
(green) with donepezil (red) in the binding pocket of AchE, the piperidine ring
fragment of donepezil and piperazine ring fragment of 13a were stacked together. Both
the phenyl fragment of donepezil and the tetramethylpyrazine fragment of 13a were
located at the PAS site, although they were oriented towards different directions.
Similarly, despite their different orientation, the indanone of donepezil and the iridoid
of 13a were both located at the CAS site.
Figure 4. The molecular model of the complex formed by compound 13a and
TcAChE (PDB code: 1EVE). (A) 3D representation of the ligand-enzyme binding
interactions; (B) 2D schematic representation of the hydrogen bonding and
hydrophobic interactions; (C) An overlap of compound 13a with donepezil in the
binding pocket of AChE
Neuroprotective activity
To investigate the neuroprotective activity of the target compounds, MTT assay
was used to identify the degree of PC12 cell death after treatment with Aβ1-42. As
shown in table 2, Aβ1-42 at the concentration of 5.5 μM could significantly reduce the
survival rate of PC12 cells (P < 0.001) by contrast with the normal group. On the
contrary, the reference Donepezil could remarkably suppress the cell damage resulted
from Aβ1-42 with the inhibition % being 17.65. Meanwhile, no significant difference
was observed between Aβ1-42 injury group and genipin-treated group.
Compound 8a at the concentration of 3.2, 10 and 32 μM could remarkably
suppress the cell damage with the respective inhibition % being 14.55, 19.20 and
22.29. In particular, at the concentration of both 10 and 32 μM, 8a treatment lead to a
comparable protection of cell damage to Donepezil.
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In addition to 8a, compound 8b also displayed acceptable efficacy in suppressing
the cell damage with the respective inhibition % being 10.84, 11.76 and 13.31 at the
concentration of 1, 3.2 and 10 μM.
Table 2. Genipin derivatives’ effect on protecting PC12 cells against Aβ1-42. n =
4, x s , P < 0.001 vs control group; *P < 0.05, **P < 0.01 vs Aβ1-42 group
Group
Control
Aβ1-42
8a
Conc.(μM)
MTT(570 nm OD)
0.745 0.03
0.422 0.01△
0.446 0.02
0.469 0.01*
0.484 0.02**
0.494 0.02**
0.457 0.01*
0.460 0.01*
0.465 0.02*
0.436 0.01
0.439 0.02
0.442 0.01
0.447 0.02
0.450 0.02
0.430 0.01
0.433 0.02
0.437 0.02
Inhibition rate of cell damage(%)
-
-
5.5
1
-
7.43
14.55
19.20
22.29
10.84
11.76
13.31
4.33
5.26
6.19
7.74
8.67
2.48
3.41
4.64
3.2
10
32
1
8b
3.2
10
32
1
13a
13b
3.2
10
32
1
3.2
10
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32
32
32
0.441 0.01
0.436 0.02
5.88
4.33
Genipin
Donepezil
0.479 0.01**
17.65
The cell morphology of each group is shown in Figure 5. Under an inverted
microscope, the cells in the normal group (Figure 5A) were dense, dendritic or
polygonal, and the cells were closely connected. Those normal PC12 cells had large
cell bodies and long neurites, which can form a network structure with light blue
fluorescence; in comparison with the normal group, cells of the injury group that
treated with Aβ1-42 became smaller. Besides, the damaged cells became ones with
shrunken neurites, almost disintegrated network structures and dim blue fluorescence.
As illustrated by the larger cell bodies harbouring stretched neurites and the
network structure with brighter blue fluorescence in 8a (Figure 5D) and 8b (Figure
5E)-treated groups than the model group (Figure 5B), both compounds featuring a
terminal benzene moiety could attenuate the cytotoxic effects resulted from Aβ1-42 in
PC12 cells. While compound 8a exhibited protective activity at the concentration of
32 μM, 8b was effective at the concentration as low as 10 μM.
Figure 5 PC12 cells changed under a light microscope at × 100 magnification. (A)
PC12 normal cells were shown. (B) PC12 cells were exposed to 5.5 μM Aβ1-42 for 0.5
h. There was a significant decrease in the cell number and blue fluorescence. (C
and E) 5.5 μM Aβ1-42 was added into PC12 cells for 0.5 h, and then (C) 32 μM
Donepezil or (D) 32 μM 8a or (E) 10 μM 8b was added into the above-processed
PC12 cells.
D
Structure-activity relationships (SARs)
With respect to the compounds bearing methyl as the O-substituent, replacement
at 3-position of the terminal benzene moiety was more beneficial for enzymatic
activity than substitution at the 4-position according to the biological data of 7a
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versus 6a and 14a versus 10a. Besides, methyloxy group substituting at the terminal
phenyl moiety was more favorable than methyl group, as illustrated by the superior
activity of 4a to that of 6a. When electron-withdrawing groups were introduced at the
terminal benzene ring, the improvement in activity was consistent with the
enhancement in electron-withdrawing property of the substituent, as exemplified by
compounds 9a, 10a and 12a, which possessed 4-choloro, 4-fluro and 4-nitro
replacement, respectively. As for the O-substituent at the genipin template, methyl
was preferable to isopropyl for the AChE inhibitory activity when comparing 12a
versus 12b and 7a versus 7b. Throughout the series, genipin derivatives bearing
ligustrazine as the terminal aryl group were identified to be the most two potent
compounds. In respect of neuroprotective activity, compounds harboring benzene as
the terminal aryl moiety has more acceptable efficacy than those with ligustrazine as
the terminal aryl group.
Conclusions
In summary, a series of novel genipin derivatives were designed, synthesized and
biologically evaluated for their potential for treating AD. A majority of them
displayed superior anti-AChE activity to that of genipin. In particular, 13a bearing
ligustrazine as the terminal aryl group displayed the most potent AChE inhibitory
activity throughout this series with IC50 value of 218 nM, which was comparable to
Donepezil. Besides, MTT assay was performed to investigate the neuroprotective
activity of some representative compounds in Aβ1-42-injuring PC12 cells. Among
them, 8a showed preferable neuroprotective efficacy (%Inhibition = 22.29) than the
positive reference Donepezil (%Inhibition = 17.65). Given the above experimental
results, compound 13a can be employed as lead for exploring AChE inhibitors, while
8a is valuable for further study to elucidate it mechanisms of neuroprotection.
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Experimental section
General information. All reagents and anhydrous solvents were obtained from
commercial sources and used without further purification unless noted otherwise.
Melting points were taken on a WRS-1B melting point apparatus, uncorrected and
reported in degrees Centigrade. 1H NMR spectra were recorded in CDCl3 and D2O on
a Bruker DRX-400 (300 MHz) using TMS as internal standard. 13C NMR were
recorded in CDCl3 and D2O on a Bruker 75 MHz spectrometer, using TMS as internal
standard.
Abbreviations used. CDCl3, Deuterochloroform; DMF, N, N-dimethylformamide;
ESI-MS, Electron spray ionization mass spectrum; h, hour; min, minute; IR, Infrared
absorption spectrum; mp, Melting point; NMR, Nuclear magnetic resonance; TLC,
Thin layer chromatography; DMSO, dimethylsulfoxide; δ(ppm), chemical shift.
p-TsOH H2O, para-toluenesulfonicacid monohydrate; iPrOH, isopropyl alcohol; PE,
Petroleum ether.
Synthesis of methyl 7-(hydroxymethyl)-1-isopropoxy-1,4a,5,
7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (2b) A mixture of genipin (2.0 g,
8.8 mmol), p-TsOH H2O (2.0 g, 10.5 mmol) iPrOH 20 mL were added to a round
bottom flask. The reaction mixture was heated to 80
for 1 h with stirring and
monitored by TLC (Ethyl acetate/PE = 1:1). The reaction was quenched by the
addition of saturated aqueous NaHCO3 and then filtered. The filtrate was extracted
with CH2Cl2 (3×50 mL), dried over anhydrous Na2SO4. The filtrate was evaporated in
vacuo and the residue was purified by chromatography on silica gel column with
(Ethyl acetate/PE = 1:4) as the elution. The resulting compound 2b (2.0 g) was
obtained as pale yellow oil in a 84.3% yield.
Synthesis of methyl 1-isopropoxy-7-(((methylthio)trioxidanyl)methyl)-1,4a,5,
7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (3b) To a 100 mL round bottom
flask, compound 2b (2.0 g, 7.5 mmol), dichloromethane (30 mL) and triethylamine
(1.6 mL, 11.6 mmol) were added, and the mixture was cooled to -10
.
Subsequently, methanesulfonyl chloride (0.9 g, 7.9 mmol) was added to the flask, and
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the mixture was stirred vigorously. The reaction was complete after 2 h. After
completion, the organic layer was washed with 1% aqueous HCl (3×20 mL), dried
over Na2SO4, and concentrated under reduced pressure. This procedure afforded
compound 3b (1.5 g) as oil in a 58.1% yield.
Synthesis of 1-(4-methylbenzyl) piperazine (6) Anhydrous piperazine (6.5 g, 75.6
mmol) was dissolved in CH2Cl2 (80 mL). para-methylbenzyl chloride (3.0 g, 21.4
mmol) in CH2Cl2 (40 mL) was added dropwise to the solution at 0 . The reaction
mixture was stirred for about 6 h at room temperature. Then the mixture was washed
with 5% K2CO3 solution (80 mL× 2) and water (80 mL× 4). The organic layer was
dried over anhydrous Na2SO4. The filtrate was evaporated in vacuo and the residue
was recrystallized from n-hexane to give the desired compound 6 (1.8 g) in a 44.3%
yield.
Synthesis of methyl
1-isopropoxy-7-((4-(4-methylbenzyl)piperazin-1-yl)methyl)-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (6b) A
mixture of compound 3b (1.5 g, 4.3 mmol), 1- (4-methylbenzyl) piperazine (1.6 g, 8.4
mmol), K2CO3 (1.2 g, 8.7 mmol), DMF 20 mL were added to a round bottom flask.
The reaction mixture was heated at room temperature for 4 h with stirring and
monitored by TLC (Ethyl acetate/PE = 1:1), then the solution was washed with H2O
(10 mL), and the resulting mixture was extracted with CH2Cl2 (2×30 mL). The
combined organic layers were washed with water and brine and dried (Na2SO4). The
crude product was purified by flash column chromatography on silica gel (Ethyl
acetate/PE = 1:4) to give yellow oil, then methanol (5 mL) was added to dissolve, the
pH was adjusted to 2 with concentrated hydrochloric acid, frozen, crystallized, filtered
and dried to provide the title compound as a white solid (1.0 g, yield 48.8%).
mp223.1-224.4
1H NMR (300 MHz, D2O) δ: 7.42 (s, 1H, 3-H), 7.28 (d, J = 8.0
Hz, 2H, ArH), 7.21 (d, J = 8.0 Hz, 2H, ArH), 6.28 (s, 1H, 7-H), 4.67 (d, J = 8.4 Hz,
1H, 1-H), 4.33 (s, 2H, CH2), 4.00 (d, J = 14.1 Hz, 1H, 10-Ha), 3.93 (d, J = 14.1 Hz,
1H, 10-Hb), 3.79 (s, 3H, COOCH3), 3.63 - 3.39 (m, 8H, piperazine-H), 3.14 (m, 1H,
CH), 2.77 (ddd, J = 11.1, 8.1, 2.7 Hz, 1H, 5-H), 2.52 (t, J = 8.1 Hz, 1H, 9-H), 2.22 (s,
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3H, CH3), 2.20 - 2.16 (m, 1H, 6-Ha), 2.04 (dd, J = 17.7, 9.4 Hz, 1H, 6-Hb), 1.13 (d, J
= 6.3 Hz, 3H, CH3), 1.02 (d, J = 6.3 Hz, 3H, CH3). 13C NMR (75 MHz, D2O) δ:
170.0, 153.4, 143.3, 141.3, 131.2, 130.0, 124.2, 110.3, 100.1, 95.6, 73.8, 60.2, 55.6,
51.8, 47.9, 47.0, 46.2, 38.8, 35.4, 23.7, 22.4, 20.9, 20.4; IR (KBr, cm-1) υ: 3031.5,
2972.9, 2945.6, 2873.2, 1712.3, 1630.8, 1436.1, 1384.2, 1371.4, 1284.7, 1242.9,
1157.0, 1103.4, 941.9, 849.0; ESI-Mass for C26H36N2O4: m/z 441.26 (M++H).
Synthesis of methyl 1-methoxy-7-((4-(4-methoxybenzyl)piperazin-1-yl)methyl)-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (4a)
Compound 4a according to procedure used for 6b synthesis. White solid, yield 53.6%,
mp 226.8-227.9 °C. 1H NMR (300 MHz, D2O) δ: 7.39 (s, 1H, 3-H), 7.32 (d, J = 8.1
Hz, 2H, ArH), 6.94 (d, J = 8.5 Hz, 2H, ArH), 6.18 (brs, 1H, 7-H), 5.11 (d, J = 2.7 Hz,
1H, 1-H), 4.31 (brs, 1H, ArCH2-Ha), 4.29 (s, 3H, ArOCH3), 4.02 - 3.82 (m, 1H,
ArCH2-Hb), 3.71 (s, 6H, COOCH3, OCH3), 3.65 – 3.47 (m, 8H, piperazine-H), 3.46
(s, 2H, 10-H), 3.41 (brs, 1H, 9-H), 3.30 (brs, 1H, 5-H), 3.18 - 2.98 (m, 1H, 6-Ha),
2.71 (dd, J = 17.6, 7.9 Hz, 1H, 6-Hb).13C NMR (75 MHz, D2O)δ: 169.9, 160.5, 151.8,
143.1, 132.9, 128.6, 119.7, 114.7, 111.3, 99.1, 59.9, 56.5, 55.4, 51.8, 47.8, 47.7, 45.8,
38.7, 33.0; IR (KBr, cm-1) υ: 2975.1, 2904.6, 2834.2, 1708.9, 1613.3, 1517.1, 1465.2,
1439.6, 1381.9, 1253.2, 1182.5, 1074.4, 1032.0, 848.9; ESI-Mass for C24H32N2O5:
m/z 429.25 (M++H).
Synthesis of methyl 1-methoxy-7-((4-(3-methoxybenzyl)piperazin-1-yl)methyl)-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (5a)
Compound 5a according to procedure used for 6b synthesis. White solid, yield 62.2%,
mp 226.3-227.4 °C. 1H NMR (300 MHz, D2O) δ: 7.47 (s, 1H, 3-H), 7.34 (t, J = 7.8
Hz, 1H, ArH), 7.02 (s, 1H, ArH), 6.99 (d, J=5.7 Hz, 2H, ArH), 6.22 (brs, 1H, 7-H),
4.54 (d, J = 8.4 Hz, 1H, 1-H), 4.34 (s, 3H, ArOCH3), 4.05 - 3.87 (m, 2H, ArCH2),
3.73 (s, 3H, COOCH3), 3.60 (s, 3H, OCH3), 3.59 - 3.47 (m, 8H, piperazine-H), 3.46
(s, 1H, 10-Ha), 3.31 (s, 1H, 10-Hb), 3.15 - 3.05 (m, 1H, 6-Ha), 2.79 (dd, J = 15.0, 8.6
Hz, 1H, 6-Hb), 2.57 (dd, J = 16.4, 7.7 Hz, 1H, 9-H), 2.17 - 1.95 (m, 1H, 5-H). 13C
NMR (75 MHz, D2O)δ: 170.0, 159.4, 153.1, 142.7, 130.8, 129.8, 128.8, 123.7, 116.7,
116.1, 110.4, 102.6, 60.2, 57.3, 55.4, 51.8, 48.0, 45.9, 40.6, 38.7, 35.1, 33.0. IR (KBr,
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cm-1) υ: 3430.4, 2976.8, 2945.4, 2838.6, 2621.8, 2514.3, 1707.5, 1630.5, 1585.7,
1492.8, 1438.3, 1369.3, 1269.5, 1178.3, 1162.5, 1080.1, 956.8, 892.5, 785.6, 698.5.
ESI-Mass for C24H32N2O5: m/z 429.29 (M++H).
Synthesis of methyl 1-methoxy-7-((4-(4-methylbenzyl)piperazin-1-yl)methyl)-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (6a)
Compound 6a according to procedure used for 6b synthesis. White solid, yield 50.1%,
mp 231.3-232.4 °C. 1H NMR (300 MHz, D2O) δ: 7.40 (s, 1H, 3-H), 7.29 (d, J = 8.1
Hz, 2H, ArH), 7.23 (d, J = 8.1 Hz, 2H, ArH), 6.18 (brs, 1H, 7-H), 5.12 (d, J = 3.0 Hz,
1H, 1-H), 4.33 (s, 2H, CH2), 3.97 (d, J = 13.8 Hz, 1H, 10-Ha), 3.88 (d, J = 13.8 Hz,
1H, 10-Hb), 3.60 (s, 3H, COOCH3), 3.58 - 3.35 (m, 8H, piperazine-H), 3.30 (s, 3H,
OCH3), 3.09 (t, J = 8.1 Hz, 2H, 6-H), 2.76 - 2.68 (m, 1H, 9-H), 2.24 (s, 3H, CH3),
2.12 - 2.05 (m, 1H, 5-H).13C NMR (75 MHz, D2O) δ: 169.8, 151.7, 143.2, 141.3,
131.1, 130.0, 128.5, 124.3, 111.3, 99.1, 60.2, 56.4, 55.1, 51.8, 48.3, 47.8, 45.8, 38.7,
33.0, 20.3; IR (KBr, cm-1) υ: 2981.3, 2950.6, 1712.5, 1637.4, 1433.6, 1368.0, 1315.7,
1288.9, 1271.8, 1197.4, 1167.2, 1074.0, 1024.6, 954.1, 890.0; ESI-Mass for
C24H32N2O4: m/z 413.30 (M++H).
Synthesis of methyl 1-methoxy-7-((4-(3-methylbenzyl)piperazin-1-yl)methyl)-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (7a)
Compound 7a according to procedure used for 6b synthesis. White solid, yield 64.1%,
mp 230.5-231.6 °C. 1H NMR (300 MHz, D2O) δ: 7.48 (s, 1H, 3-H), 7.36 - 7.18 (m,
4H, ArH), 6.28 (brs, 1H, 7-H), 4.56 (d, J = 8.2 Hz, 1H, 1-H), 4.36 (s, 2H, CH2), 4.08
- 3.90 (m, 2H, 10-CH2), 3.63 (s, 3H, COOCH3), 3.61 - 3.50 (m, 8H, piperazine-H),
3.48 (s, 3H, OCH3), 3.12 (dd, J = 15.9, 7.5 Hz, 1H, 9-H), 2.81 (dd, J = 17.0, 8.5 Hz,
1H, 6-Ha), 2.64 - 2.49 (m, 1H, 6-Hb), 2.27 (s, 3H, CH3), 2.06 (dd, J = 16.5, 8.7 Hz,
1H, 5-H). 13C NMR (75 MHz, D2O)δ: 170.00, 153.10, 142.64, 139.77, 131.72,
131.23, 129.86, 129.33, 128.11, 127.31, 110.44, 102.57, 60.43, 57.30, 55.65, 51.82,
48.33, 47.94, 45.98, 38.75, 35.06, 20.33.IR (KBr, cm-1) υ: 2977.1, 2947.6, 2629.5,
1708.2, 1627.6, 1438.2, 1369.9, 1287.6, 1178.7, 1107.6, 957.8, 939.5, 890.1, 766.6,
700.3. ESI-Mass for C24H32N2O4: m/z 413.26 (M++H).
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Synthesis of methyl 7-((4-benzylpiperazin-1-yl)methyl)-1-methoxy-1,4a,5,
7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (8a) Compound
8a according to procedure used for 6b synthesis. White solid, yield 49.2%, mp
240.2-241.6 °C. 1H NMR (300 MHz, D2O) δ: 7.50 (s, 1H, 3-H), 7.44 - 7.40 (m, 5H,
ArH), 6.21 (brs, 1H, 7-H), 5.15 (d, J = 2.9 Hz, 1H, 1-H), 4.39 (s, 3H, COOCH3), 4.04
- 3.88 (m, 2H, CH2), 3.63 (s, 3H, OCH3), 3.60 - 3.52 (m, 8H, piperazine-H), 3.34 (s,
2H, 10-CH2), 3.17 - 3.07 (m, 2H, 6-CH2), 2.75 (dd, J = 17.4, 8.0 Hz, 1H, 9-H), 2.18 -
2.04 (m, 1H, 5-H). 13C NMR (75 MHz, D2O) δ: 169.9, 160.5, 151.8, 143.1, 132.9,
128.6, 119.7, 114.7, 111.3, 99.1, 59.9, 56.5, 55.4, 51.8, 47.8, 47.7, 45.8, 38.7, 33.0; IR
(KBr, cm-1) υ: 2989.5, 2948.6, 2880.1, 2830.8, 1708.8, 1628.0, 1436.1, 1287.1,
1270.2, 1172.6, 1073.4, 951.3, 753.6, 700.7; ESI-Mass for C23H30N2O4: m/z 399.29
(M++H).
Synthesis of methyl 7-((4-(4-chlorobenzyl)piperazin-1-yl)methyl)-1-methoxy-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (9a)
Compound 9a according to procedure used for 6b synthesis. White solid, yield 47.3%,
mp 230.1-231.2 °C. 1H NMR (300 MHz, D2O) δ: 7.50 (s, 1H, 3-H), 7.42 (dd, J =
14.1, 8.7 Hz, 4H, ArH), 6.27 (brs, 1H, 7-H), 4.58 (d, J = 8.3 Hz, 1H, 1-H), 4.38 (s,
2H, CH2), 4.05 - 3.90 (m, 2H, 10-CH2), 3.64 (s, 3H, COOCH3), 3.62 - 3.50 (m, 8H,
piperazine-H), 3.49 (s, 3H, OCH3), 3.13 (q, J = 8.3 Hz, 1H, 5-H), 2.82 (ddd, J = 11.1,
8.4, 2.4 Hz, 1H, 6-Ha), 2.61 (t, J = 7.5 Hz, 1H, 9-H), 2.08 (dd, J = 17.5, 9.1 Hz, 1H,
6-Hb ).13C NMR (75 MHz, D2O) δ: 170.06, 153.10, 142.59, 136.16, 132.73, 129.91,
129.47, 126.16, 110.46, 102.58, 59.62, 57.29, 55.67, 51.82, 48.39, 48.03, 45.97,
38.73, 35.09; IR (KBr, cm-1) υ: 2985.2, 2949.8, 2843.8, 1701.5, 1635.4, 1494.3,
1437.4, 1374.8, 1285.2, 1179.0, 1046.8, 1017.1, 938.7, 854.4; ESI-Mass for
C23H29ClN2O4: m/z 433.24 (M++H).
Synthesis of methyl 7-((4-(4-fluorobenzyl)piperazin-1-yl)methyl)-1-methoxy-1,4a,
5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (10a)
Compound 10a according to procedure used for 6b synthesis. White solid, yield
43.3%, mp 237.4-238.6 °C. 1H NMR (300 MHz, D2O) δ: 7.51 (s, 1H, 3-H), 7.45 (dd,
J = 8.4, 5.4 Hz, 2H, ArH), 7.16 (t, J = 8.7 Hz, 2H, ArH), 6.24 (brs, 1H, 7-H), 4.59 (d,
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J = 8.4 Hz, 1H, 1-H), 4.38 (s, 2H, CH2), 4.02 (1H, d, J = 13.5 Hz, 10-Ha), 3.94 (1H,
d, J = 13.5 Hz, 10-Hb), 3.64 (s, 3H, COOCH3), 3.62 - 3.51 (m, 8H, piperazine-H),
3.49 (s, 3H, OCH3), 3.13 (q, J = 8.4 Hz, 1H, 5-H), 2.82 (ddd, J = 11.4, 8.7, 2.7 Hz,
1H, 9-H), 2.67 - 2.51 (m, 1H, 6-Ha), 2.09 (dd, J = 16.5, 8.4 Hz, 1H, 6-Hb). 13C NMR
(75 MHz, D2O) δ: 170.1, 162.1, 153.1, 142.5, 133.5, 133.4, 130.0, 123.6, 116.5,
116.2, 110.5, 102.6, 59.6, 57.3, 55.7, 51.8, 48.4, 47.9, 46.0, 38.7, 35.1; IR (KBr, cm-1)
υ: 2971.9, 2939.9, 2840.6, 1712.8, 1627.6, 1601.1, 1513.6, 1438.0, 1364.1, 1286.9,
1227.9, 1187.3, 1111.6, 1084.5, 947.7, 864.4; ESI-Mass for C23H29FN2O4: m/z
417.30 (M++H).
Synthesis of methyl 1-methoxy-7-((4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)
methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride
(11a) Compound 11a according to procedure used for 6b synthesis. White solid, yield
40.9%, mp 171.0-172.4 °C. 1H NMR (300 MHz, D2O)δ: 7.74 (brs, 2H, ArH), 7.64 (s,
1H, 3-H), 7.60 - 7.46 (m, 2H, ArH), 6.25 (brs, 1H, 7-H), 4.56 (d, J = 8.1 Hz, 1H,
1-H), 4.39 (s, 2H, CH2), 4.09 - 3.78 (m, 2H, 10-CH2), 3.61 (s, 3H, COOCH3), 3.58 -
3.50 (m, 8H, piperazine-H), 3.47 (s, 3H, OCH3), 3.14 (q, J = 8.3 Hz, 1H, 5-H), 2.80
(ddd, J = 11.1, 8.4, 2.4 Hz, 1H, 6-Ha), 2.59 (t, J = 7.5 Hz, 1H, 9-H), 2.06 (dd, J =
17.5, 9.1 Hz, 1H, 6-Hb ).13C NMR (75 MHz, D2O)δ: 170.1, 153.1, 142.6, 134.9,
131.0, 130.1, 129.9, 128.7, 127.9, 127.2, 121.9, 110.5, 102.6, 59.7, 57.3, 51.8, 50.9,
48.2, 46.0, 42.6, 35.3, 34.5. IR (KBr, cm-1) υ: 2983.6, 2952.7, 2883.1, 2847.2, 1708.9,
1628.2, 1458.6, 1440.9, 1329.8, 1265.5, 1157.5, 1121.7, 1077.7, 944.3, 819.6, 780.5,
700.9. ESI-Mass for C24H29F3N2O4: m/z 467.29 (M++H).
Synthesis of methyl 1-methoxy-7-((4-(4-nitrobenzyl)piperazin-1-yl)methyl)-1,4a,
5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (12a)
Compound 12a according to procedure used for 6b synthesis. White solid, yield
41.8%, mp 234.3-235.6 °C. 1H NMR (600 MHz, D2O) δ: 8.28 (d, J = 8.2 Hz, 2H,
ArH), 7.94 (brs, 2H, ArH), 7.40 (s, 1H, 3-H), 6.33 (s, 1H, 7-H), 5.73 (s, 1H, 1-H),
3.81 (brs, 1H, ArCH2-Ha), 3.61 (s, 3H, COOCH3), 3.58 - 3.42 (m, 8H, piperazine-H),
3.24 (s, 3H, OCH3), 3.08 (q, J = 7.8 Hz, 1H, 5-H), 2.97 - 2.88 (m, 1H, ArCH2-Hb),
2.73 (dd, J = 16.3, 8.1 Hz, 1H, 10-Ha), 2.48 (brs, 1H, 9-H), 2.10 (dd, J = 16.6, 7.3 Hz,
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1H, 10-Hb), 1.88 (s, 2H, 6-H). 13C NMR (75 MHz, D2O) δ: 169.96, 153.09, 148.74,
142.70, 134.87, 132.53, 129.86, 124.40, 110.44, 102.59, 59.19, 57.33, 55.67, 51.83,
48.41, 46.02, 38.78, 35.07, 20.41. IR (KBr, cm-1) υ: 2979.1, 2950.9, 2625.7, 2539.5,
1711.6, 1624.9, 1528.7, 1466.1, 1433.3, 1386.0, 1350.3, 1286.9, 1223.8, 1088.7,
959.9, 860.1. ESI-Mass for C23H29N3O6: m/z 444.27 (M++H).
Synthesis of methyl 1-methoxy-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)
piperazin-1-yl)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate
dihydrochloride (13a) Compound 13a according to procedure used for 6b synthesis.
White solid, yield 58.5%, mp 182.5-183.9 °C. 1H NMR (300 MHz, D2O)δ: 7.50 (s,
1H, 3-H), 6.28 (d, J = 17.9 Hz, 1H, 7-H), 4.74 (s, 2H, CH2), 4.59 (d, J = 8.3 Hz, 1H,
1-H), 4.13 - 3.95 (m, 2H, 10-CH2), 3.86 - 3.65 (m, 8H, piperazine-H), 3.62 (s, 3H,
COOCH3), 3.50 (s, 3H, OCH3), 3.34 (s, 1H, 9-H), 3.13 (q, J = 8.1 Hz, 1H, 5-H), 2.80
(ddd, J = 11.4, 8.4, 2.4 Hz, 1H, 6-Ha), 2.62 (s, 3H, CH3), 2.60 (s, 3H, CH3), 2.57 (s,
3H, CH3), 2.12 (dd, J = 17.1, 9.6 Hz, 1H, 6-Hb).13C NMR (75 MHz, D2O)δ: 170.1,
155.6, 153.1, 147.8, 144.3, 143.4, 129.8, 128.4, 110.5, 102.6, 57.3, 56.0, 51.8, 49.4,
48.4, 46.0, 38.8, 35.1, 33.0, 20.6, 17.4, 15.9. IR (KBr, cm-1) υ: 2997.5, 2951.2,
2873.4, 2843.1, 1701.7, 1631.0, 1436.7, 1392.6, 1290.9, 1179.7, 1078.7, 950.6, 891.0,
768.7, 722.6; ESI-Mass for C24H34N4O4: m/z 443.34 (M++H).
Synthesis of methyl 7-((4-(3-fluorobenzyl)piperazin-1-yl)methyl)-1-methoxy-1,4a,
5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride
(14a)Compound 14a according to procedure used for 6b synthesis. White solid, yield
41.9%, mp 236.0-237.4 °C. 1H NMR (300 MHz, D2O) δ: 7.48 (s, 1H, 3-H), 7.45 -
7.36 (m, 1H, ArH), 7.22 (d, J = 8.7 Hz, 2H, ArH), 7.17 (s, 1H, ArH), 6.25 (brs, 1H,
7-H), 4.56 (d, J = 8.3 Hz, 1H, 1-H), 4.37 (s, 2H, CH2), 4.05 - 3.87 (m, 2H, 10-CH2),
3.61 (s, 3H, COOCH3), 3.60 - 3.48 (m, 8H, piperazine-H), 3.46 (s, 3H, OCH3), 3.10
(q, J = 8.4 Hz, 1H, 5-H), 2.79 (ddd, J = 11.1, 8.7, 2.7 Hz, 1H, 6-Ha), 2.58 (t, J = 7.9
Hz, 1H, 9-H), 2.06 (dd, J = 17.1, 9.1 Hz, 1H, 6-Hb). 13C NMR (75 MHz, D2O)δ:
170.1, 164.2, 160.9, 153.1, 142.6, 131.4, 129.9, 127.1, 118.1, 117.6, 110.4, 102.6,
59.7, 57.3, 55.7, 51.8, 48.4, 48.1, 45.9, 38.7, 35.1. IR (KBr, cm-1) υ: 2940.7, 2627.1,
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1698.1, 1628.1, 1590.6, 1437.5, 1287.5, 1255.4, 1178.8, 1110.4, 965.9, 936.9, 892.9,
789.9. ESI-Mass for C23H29FN2O4: m/z 417.27 (M++H).
Synthesis of methyl 1-methoxy-7-((4-(pyridin-3-ylmethyl)piperazin-1-yl)
methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride
(15a) Compound 15a according to procedure used for 6b synthesis. White solid, yield
48.0%, mp 197.1-198.3 °C. 1H NMR (300 MHz, D2O) δ: 8.98 (s, 1H, Pyridine-H),
8.84 (d, J = 5.7 Hz, 1H, Pyridine-H), 8.74 (d, J = 8.2 Hz, 1H, Pyridine-H), 8.11 (t, J =
7.8 Hz, 1H, Pyridine-H), 7.45 (s, 1H, 3-H), 6.23 (d, J = 18.1 Hz, 1H, 7-H), 4.55 (d, J
= 8.3 Hz, 1H, 1-H), 3.97 (brs, 2H, CH2), 3.83 - 3.61 (m, 8H, piperazine-H), 3.59 (s,
3H, COOCH3), 3.46 (s, 2H, 10-CH2), 3.30 (s, 3H, OCH3), 3.09 (t, J = 8.1 Hz, 1H,
9-H), 2.79 (ddd, J = 11.1, 8.7, 2.7 Hz, 1H, 5-H), 2.70 - 2.56 (m, 1H, 6-Ha), 2.04 (dd,
J = 17.1, 9.4 Hz, 1H, 6-Hb). 13C NMR (75 MHz, D2O) δ: 169.8, 153.1, 151.7, 149.7,
143.4, 129.8, 129.1, 128.2, 110.4, 102.5, 99.1, 57.3, 56.48, 56.0, 55.6, 55.1, 51.8,
48.6, 46.0, 38.7, 35.0, 32.9. IR (KBr, cm-1) υ: 2978.7, 2956.3, 2824.1, 1701.8, 1633.2,
1532.7, 1436.2, 1460.2, 1379.3, 1289.1, 1179.3, 1083.5, 946.7, 891.0, 686.1;
ESI-Mass for C22H29N3O4: m/z 400.22 (M++H).
Synthesis of methyl 1-isopropoxy-7-((4-(4-methoxybenzyl)piperazin-1-yl)
methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride
(4b) Compound 4b according to procedure used for 6b synthesis. White solid, yield
52.2%, mp 225.3-226.5 °C. 1H NMR (300 MHz, D2O) δ: 7.45 (s, 1H, 3-H), 7.38 (d, J
= 8.7 Hz, 2H, ArH), 6.97 (d, J = 8.7 Hz, 2H, ArH), 6.32 (s, 1H, 7-H), 4.68 (d, J = 8.4
Hz, 1H, 1-H), 4.35 (s, 2H, CH2), 4.02 (dd, J = 12.7, 6.3 Hz, 2H, 10-CH2), 3.94 - 3.85
(m, 1H, CH), 3.75 (s, 3H, COOCH3), 3.62 (s, 3H, OCH3), 3.61 - 3.45 (m, 8H,
piperazine-H), 3.12 (q, J = 8.4 Hz, 1H, 6-Ha), 2.82 (ddd, J = 11.4, 8.7, 2.7 Hz, 1H,
5-H), 2.55 (t, J = 8.0 Hz, 1H, 9-H), 2.07 (dd, J = 17.4, 9.4 Hz, 1H, 6-Hb), 1.17 (d, J =
6.0 Hz, 3H, CH3), 1.07 (d, J = 6.0 Hz, 3H, CH3). 13C NMR (75 MHz, D2O) δ: 170.0,
160.5, 153.4, 143.1, 132.9, 130.0, 119.7, 114.8, 110.3, 100.1, 73.8, 72.9, 60.0, 55.6,
55.4, 51.8, 48.3, 47.8, 46.2, 38.8, 35.4, 33.8, 22.4, 20.9; IR (KBr, cm-1) υ: 2972.1,
2830.7, 1712.0, 1631.0, 1518.0, 1437.8, 1382.9, 1251.4, 1157.9, 1104.3, 945.2, 857.9;
ESI-Mass for C26H36N2O5: m/z 457.21 (M++H).
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Synthesis of methyl 1-isopropoxy-7-((4-(3-methoxybenzyl)piperazin-1-yl)
methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride
(5b) Compound 5b according to procedure used for 6b synthesis. White solid, yield
60.6%, mp 208.0-209.2 °C. 1H NMR (300 MHz, D2O) δ: 7.44 (s, 1H, 3-H), 7.36 (t, J
= 8.3 Hz, 1H, ArH), 7.07 - 6.97 (m, 3H, ArH), 6.26 (s, 1H, 7-H), 4.67 (d, J = 8.4 Hz,
1H, 1-H), 4.38 (s, 2H, CH2), 4.03 (dd, J = 13.5, 7.8 Hz, 2H, 10-CH2), 3.96 - 3.85 (m,
1H, CH), 3.74 (s, 3H, COOCH3), 3.66 (s, 3H, OCH3), 3.61 - 3.47 (m, 8H,
piperazine-H), 3.11 (q, J = 8.4 Hz, 1H, 6-Ha), 2.81 (ddd, J = 11.4, 8.4, 2.7 Hz, 1H,
5-H), 2.55 (t, J = 7.5 Hz, 1H, 9-H), 2.06 (dd, J = 17.6, 9.5 Hz, 1H, 6-Hb), 1.16 (d, J =
6.0 Hz, 3H, CH3), 1.06 (d, J = 6.0 Hz, 3H, CH3). 13C NMR (75 MHz, D2O) δ: 170.0,
159.4, 153.4, 143.3, 130.8, 129.9, 128.8, 123.8, 116.6, 116.2, 110.3, 100.1, 73.8, 60.3,
55.6, 55.5, 51.8, 48.3, 48.1, 46.2, 38.8, 35.4, 23.7, 22.4, 20.9; IR (KBr, cm-1) υ:
2973.2, 2947.8, 2883.6, 2831.9, 1706.6, 1629.8, 1371.3, 1437.1, 1270.1, 1160.3,
1101.6, 940.3, 888.5, 787.1, 697.6; ESI-Mass for C26H36N2O5: m/z 457.16 (M++H).
Synthesis of methyl
1-isopropoxy-7-((4-(3-methylbenzyl)piperazin-1-yl)methyl)-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (7b)
Compound 7b according to procedure used for 6b synthesis. White solid, yield
62.8%, mp 204.1-205.4 °C. 1H NMR (300 MHz, D2O) δ: 7.46 (s, 1H, 3-H), 7.37 -
7.17 (m, 4H, ArH), 6.32 (s, 1H, 7-H), 4.68 (d, J = 8.4 Hz, 1H, 1-H), 4.36 (s, 2H,
CH2), 4.03 (dd, J = 12.3, 5.7 Hz, 2H, 10-CH2), 3.98 - 3.85 (m, 1H, CH), 3.62 (s, 3H,
COOCH3), 3.61 - 3.40 (m, 8H, piperazine-H), 3.12 (q, J = 8.4 Hz, 1H, 5-H), 2.82
(ddd, J = 11.1, 8.4, 2.4 Hz, 1H, 6-Ha), 2.56 (t, J = 7.9 Hz, 1H, 9-H), 2.27 (s, 3H,
CH3), 2.08 (dd, J = 17.5, 9.3 Hz, 1H, 6-Hb), 1.17 (d, J = 6.0 Hz, 3H, CH3), 1.07 (d, J
= 6.0 Hz, 3H, CH3). 13C NMR (75 MHz, D2O) δ: 170.0, 153.4, 143.1, 139.8, 131.7,
131.2, 130.0, 129.4, 128.1, 127.3, 110.3, 100.1, 73.8, 60.5, 55.6, 51.8, 48.3, 48.0,
46.2, 38.8, 35.4, 22.4, 21.0, 20.4; IR (KBr, cm-1) υ: 2972.9, 2879.6, 2847.2, 1709.2,
1624.7, 1438.9, 1383.6, 1288.3, 1161.8, 1102.4, 942.5, 893.8, 767.1, 703.8; ESI-Mass
for C26H36N2O4: m/z 441.26 (M++H).
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Synthesis of methyl 7-((4-benzylpiperazin-1-yl)methyl)-1-isopropoxy-1,4a,5,
7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (8b)Compound
8b according to procedure used for 6b synthesis. White solid, yield 48.6%, mp
214.3-215.7 °C. 1H NMR (300 MHz, D2O) δ: 7.47 (s, 1H, 3-H), 7.42 - 7.40 (m, 5H,
ArH), 6.32 (s, 1H, 7-H), 4.73 (d, J = 6.0 Hz, 1H, 1-H), 4.41 (s, 2H, CH2), 4.02 (dd, J
= 11.7, 6.3 Hz, 2H, 10-CH2), 3.99 - 3.94 (m, 1H, CH), 3.63 (s, 3H, COOCH3), 3.61 -
3.46 (m, 8H, piperazine-H), 3.13 (q, J = 8.5 Hz, 1H, 6-Ha), 2.82 (ddd, J = 11.4, 8.7,
2.7 Hz, 1H, 5-H), 2.56 (t, J = 7.7 Hz, 1H, 9-H), 2.09 (dd, J = 17.4, 9.3 Hz, 1H, 6-Hb),
1.17 (d, J = 6.2 Hz, 3H, CH3), 1.07 (d, J = 6.2 Hz, 3H, CH3). 13C NMR (75 MHz,
D2O) δ: 170.0, 153.4, 143.1, 131.3, 130.7, 130.0, 129.5, 127.4, 110.3, 100.1, 73.8,
60.5, 55.6, 51.9, 48.3, 48.0, 46.2, 38.8, 35.4, 22.4, 21.0; IR (KBr, cm-1) υ: 2976.4,
2923.7, 2830.1, 1716.0, 1633.0, 1457.8, 1440.1, 1380.9, 1294.7, 1156.4, 1107.0,
952.0, 937.2, 891.3, 751.2, 701.1; ESI-Mass for C25H34N2O4: m/z 427.15 (M++H).
Synthesis of methyl 7-((4-(4-chlorobenzyl)piperazin-1-yl)methyl)-1-isopropoxy-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (9b)
Compound 9b according to procedure used for 6b synthesis. White solid, yield
44.3%, mp 238.1-239.3 °C. 1H NMR (300 MHz, D2O) δ: 7.48 (s, 1H, 3-H), 7.41 (q, J
= 8.5 Hz, 4H, ArH), 6.30 (brs, 1H, 7-H), 5.28 (s, 1H, 1-H), 4.37 (s, 2H, CH2), 4.06 -
4.00 (m, 1H, CH), 3.98 - 3.86 (m, 2H, 10-CH2), 3.63 (s, 3H, COOCH3), 3.60 - 3.37
(m, 8H, piperazine-H), 3.12 (q, J = 8.4 Hz, 1H, 5-H), 2.82 (dd, J = 15.1, 8.7 Hz, 1H,
6-Ha), 2.54 (t, J = 7.6 Hz, 1H, 9-H), 2.09 (dd, J = 17.3, 9.0 Hz, 1H, 6-Hb), 1.16 (d, J
= 6.2 Hz, 3H, CH3), 1.06 (d, J = 6.0 Hz, 3H, CH3). 13C NMR (75 MHz, D2O) δ:
170.1, 153.4, 143.1, 136.1, 132.7, 130.1, 129.5, 126.2, 110.3, 100.1, 73.8, 59.6, 55.6,
51.8, 48.4, 48.1, 46.2, 38.8, 35.5, 22.3, 20.9; IR (KBr, cm-1) υ: 2972.6, 2880.2,
2863.3, 1710.2, 1630.2, 1438.1, 1385.0, 1288.6, 1162.4, 1088.7, 935.6, 853.0;
ESI-Mass for C25H33ClN2O4: m/z 461.26 (M++H).
Synthesis of methyl 7-((4-(4-fluorobenzyl)piperazin-1-yl)methyl)-1-isopropoxy-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (10b)
Compound 10b according to procedure used for 6b synthesis. White solid, yield
42.9%, mp 239.5-240.8 °C. 1H NMR (300 MHz, D2O) δ: 7.49 (s, 1H, 3-H), 7.44 (dd,
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J = 9.0, 5.4 Hz, 2H, ArH), 7.16 (t, J = 8.8 Hz, 2H, ArH), 6.27 (brs, 1H, 7-H), 5.30
(brs, 1H, 1-H), 4.40 (s, 2H, CH2), 4.07 - 4.00 (m, 2H, 10-CH2), 3.98 - 3.87 (m, 1H,
CH), 3.63 (s, 3H, COOCH3), 3.62 - 3.42 (m, 8H, piperazine-H), 3.13 (q, J = 8.8 Hz,
1H, 5-H), 2.83 (dd, J = 16.1, 7.2 Hz, 1H, 6-Ha), 2.56 (t, J = 8.0 Hz, 1H, 9-H), 2.10
(dd, J = 17.8, 9.6 Hz, 1H, 6-Hb), 1.17 (d, J = 6.2 Hz, 3H, CH3), 1.07 (dd, J = 6.2 Hz,
3H, CH3). 13C NMR (75 MHz, D2O) δ: 170.1, 165.4, 162.1, 153.4, 143.2, 133.6,
133.5, 130.0, 116.5, 116.2, 110.3, 100.1, 73.8, 59.6, 55.6, 51.8, 48.0, 46.2, 38.8, 35.4,
33.8, 22.4, 20.9; IR (KBr, cm-1) υ: 2975.9, 2931.8, 2884.5, 2826.1, 1710.4, 1631.8,
1514.7, 1437.3, 1385.9, 1289.3, 1226.9, 1166.9, 1105.2, 936.0, 888.7, 859.7;
ESI-Mass for C25H33FN2O4: m/z 445.27 (M++H).
Synthesis of methyl
1-isopropoxy-7-((4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)
methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride
(11b) Compound 11b according to procedure used for 6b synthesis. White solid, yield
40.1%, mp 226.2-227.5 °C. 1H NMR (300 MHz, D2O) δ: 7.75 (d, J = 8.1 Hz, 2H,
ArH), 7.61 (d, J = 8.0 Hz, 2H, ArH), 7.49 (s, 1H, 3-H), 6.32 (s, 1H, 7-H), 4.75 (d, J =
6.0 Hz, 1H, 1-H), 4.47 (s, 2H, CH2), 4.02 (dd, J = 14.7, 6.3 Hz, 2H, 10-CH2), 3.98 -
3.85 (m, 1H, CH), 3.63 (s, 3H, COOCH3), 3.61 - 3.46 (m, 8H, piperazine-H), 3.14 (q,
J = 8.7 Hz, 1H, 5-H), 2.83 (ddd, J = 11.1, 8.1, 2.4 Hz, 1H, 6-Ha), 2.56 (t, J = 7.7 Hz,
1H, 9-H), 2.10 (dd, J = 17.5, 9.4 Hz, 1H, 6-Hb), 1.17 (d, J = 6.2 Hz, 3H, CH3), 1.07
(d, J = 6.2 Hz, 3H, CH3). 13C NMR (75 MHz, D2O) δ: 170.1, 153.4, 143.2, 131.8,
130.0, 126.4, 126.3, 125.6, 122.0, 110.3, 100.1, 73.8, 64.2, 59.7, 55.6, 51.8, 48.3,
46.2, 38.8, 35.5, 23.7, 22.3, 20.9; IR (KBr, cm-1) υ: 2975.3, 2907.8, 1705.6, 1635.1,
1439.0, 1386.7, 1327.4, 1290.4, 1271.5, 1166.7, 1068.7, 938.1, 861.0; ESI-Mass for
C26H33F3N2O4: m/z 495.16 (M++H).
Synthesis of methyl 1-isopropoxy-7-((4-(4-nitrobenzyl)piperazin-1-yl)methyl)-1,
4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate dihydrochloride (12b)
Compound 12b according to procedure used for 6b synthesis. White solid, yield
38.9%, mp 240.6-241.8 °C. 1H NMR (300 MHz, D2O) δ: 8.25 (d, J = 8.8 Hz, 2H,
ArH), 7.70 (d, J = 8.8 Hz, 2H, ArH), 7.48 (s, 1H, 3-H), 6.28 (brs, 1H, 7-H), 5.30 (d, J
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= 2.1 Hz, 1H, 1-H), 4.53 (s, 2H, CH2), 4.07 - 4.00 (m, 2H, 10-CH2), 3.98 - 3.90 (m,
1H, CH), 3.63 (s, 3H, COOCH3), 3.62 - 3.46 (m, 8H, piperazine-H), 3.14 (q, J = 8.5
Hz, 1H, 5-H), 2.83 (dd, J = 16.1, 10.0 Hz, 1H, 6-Ha), 2.57 (t, J = 8.1 Hz, 1H, 9-H),
2.10 (dd, J = 17.5, 9.4 Hz, 1H, 6-Hb), 1.17 (d, J = 6.3 Hz, 3H, CH3), 1.07 (dd, J = 6.3
Hz, 3H, CH3). 13C NMR (75 MHz, D2O) δ: 170.1, 153.4, 148.8, 143.2, 135.0, 132.5,
130.0, 124.4, 110.3, 100.1, 73.9, 59.2, 55.6, 51.8, 48.5, 46.2, 38.8, 35.5, 33.9, 22.4,
20.9; IR (KBr, cm-1) υ: 2968.7, 2956.4, 2887.9, 2821.8, 1700.2, 1635.1, 1522.3,
1439.7, 1385.5, 1347.6, 1297.1, 1269.4, 1160.8, 1111.6, 938.7, 858.9; ESI-Mass for
C25H33N3O6: m/z 472.24 (M++H).
Synthesis of methyl 1-isopropoxy-7-((4-((3,5,6-trimethylpyrazin-2-yl)methyl)
piperazin-1-yl)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate
dihydrochloride (13b) Compound 13b according to procedure used for 6b synthesis.
White solid, yield 57.2%, mp 185.3-186.7 °C. 1H NMR (300 MHz, D2O) δ: 7.49 (s,
1H, 3-H), 6.35 (s, 1H, 7-H), 5.32 (brs, 1H, 1-H), 4.78 - 4.73 (m, 1H, CH), 4.17 - 4.03
(m, 2H, CH2), 4.02 - 3.87 (m, 2H, 10-CH2), 3.84 - 3.64 (m, 8H, piperazine-H), 3.62
(s, 3H, COOCH3), 3.2 - 3.0 (m, 2H, CH2), 2.84 (ddd, J = 11.4, 8.4, 2.4 Hz, 1H, 5-H),
2.61 (s, 3H, CH3), 2.60 (s, 3H, CH3), 2.56 (s, 3H, CH3), 2.11 (dd, J = 17.5, 9.2 Hz,
1H, 9-H), 1.17 (d, J = 6.2 Hz, 3H, CH3), 1.07 (dd, J = 6.2 Hz, 3H, CH3). 13C NMR
(75 MHz, D2O) δ: 170.1, 155.3, 153.5, 148.0, 144.2, 143.7, 143.3, 129.9, 110.3,
100.2, 73.9, 56.1, 55.7, 51.8, 49.4, 48.4, 46.2, 38.9, 35.5, 22.4, 21.0, 20.6, 17.6, 16.1.
IR (KBr, cm-1) υ: 2975.4, 2898.8, 1705.9, 1630.2, 1533.8, 1436.6, 1386.4, 1285.7,
1264.9, 1163.4, 1103.5, 1003.7, 949.1, 888.5, 761.9; ESI-Mass for C26H38N4O4: m/z
471.28 (M++H).
In vitro AChE inhibition assay
Acetylcholinesterase (AChE, E.C.3.1.1.7, from the rat striatum), 5,5′-dithiobis-
(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATCI) and donepezil
hydrochloride were purchased from Sigma Aldrich. The inhibitory activities against
AChE of genipin and target compounds were performed according to the method
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developed by Ellman et al, using donepezil as the reference compound.[13]As shown
in Figure6, without AChE inhibitor, the entire enzymatic reaction is very rapid, a
linear relationship within 10 min, so the IC50 calculation was selected in the third,
fifth and seventh minute time point. All the in vitro assays were carried out in 0.05
mol/L KH2PO4/K2HPO4 buffer (pH 7.2) using a Shimadzu UV-2550 UV Vis
Spectrophotometer. The concentration of ATCI was 5 mmol/L, the concentration of
DTNB was 1 mmol/L, the test compounds were dissolved in PBS solvent and used
doubling dilutions method (Concentrations are 2.00×10-5, 1.00×10-5, 5.00×10-6,
2.50×10-6, 1.25×10-6, 6.25×10-7, 3.13×10-7, 1.56×10-7, 7.81×10-8, 3.91×10-8, 1.95×10-8,
9.77×10-9 mol/L respectively). 100 μL of the test compounds and 20 μL of enzyme
were added to the 96-well plate by pipetting, the mixture was gently shaken and
incubated for 10 min in a 37 °C constant temperature oven, then 40 μL of ATCI was
added and continued to incubate for 20 min, followed by the addition of 40 μL
DTNB. Activity was determined by measuring the increase in absorbance at 412 nm.
For determining the blank value, 20 µL buffer replaced the AChE solution. Each
concentration was assayed three times and 50% inhibitory concentration (IC50) was
calculated graphically from inhibition curves.
Figure 6 The enzymatic reaction curve without AChE inhibitor (x-min y-OD)
Neuroprotective activity was evaluated by MTT assay
PC12 cells in logarithmicphase were seeded on the 96-well plate with one well
10000 PC12 cells and cultured for 24 h. Then the original culture medium was removed
and was replaced by the low serum culture medium with nerve growth factor (NGF)
(Concentration of 50 μg/L). Continue to incubate for about 96 h. On the
above-mentioned 96-well plate, Aβ1-42 was added to induce PC12 cells injury. The
final concentration of Aβ1-42 was 25 μg/ml. After 30 min, test compounds of different
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concentrations were added. The final concentrations were 0, 3.2 10-7, 1.0 10-6, 3.2
10-6, 1.0 10-5, 3.2 10-5, 1.0 10-4 and 3.2 10-4 mol/L respectively. The 96-well
plate was then put into incubator to further culture 72 h. Cell viability was measured by
quantitative MTT colorimetric assay as described previously. Then, 20 µL of MTT
solution (5 mg/mL) was added to 200 µL culture medium of each well. Four hours
later the medium was removed, and the resultant formazan product was dissolved by
addition of 200 µL of DMSO. The absorbance of MTT was measured at 570 nm with
a microplate reader (ELX 800; Bio-TEK instruments, Inc.). Inhibition of PC12 cells
injury in percent (I %) was calculated using the following equation: I(%)= [(Atest
-
AAβ1-42)/(Acontrol - AAβ1-42)]×100(%). The results were expressed as percentage of cell
viability (%), assuming that of control cells as 100%.
Morphological observation of PC12 cells
After the damage induced by Aβ1-42, rhodamine 123 was added in 24-well plate
(final concentration was 20 µmol/L). After 30 min of incubation, the culture medium
in the 24-well plate was carefully removed, PBS was added to rinse the 24-well plate
for 1-2 times, and then paraformaldehyde was added for 15 min. Fluorescence
microscopy was applied to photo and record the background fluorescence intensity to
decide whether further rinse should be adopted.
Statistical Analysis.
Biological results are reported as means standard error. Statistical analysis was
performed by using one-way analysis of variance. Pvalue of less than 0.05 was
considered to be statistically significant.
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Author Contributions
All authors have given approval to the final version of the manuscript.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
This study was supported by grants from the National innovative drug incubation base project
(2012ZX09401066) Anhui Education Department Science key project (KJ2016A414).
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Figure 1 The structure of genipin
Figure 2 The structures of genipin derivatives
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Figure 3 Design of genipin derivatives via pharmacophore-combination strategy
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Scheme 1 Synthetic route of the target compounds
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Figure 4. The molecular model of the complex formed by compound 13a and
TcAChE (PDB code: 1EVE). (A) 3D representation of the ligand-enzyme binding
interactions; (B) 2D schematic representation of the hydrogen bonding and
hydrophobic interactions; (C) An overlap of compound 13a with donepezil in the
binding pocket of AChE
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