Hybrids of aurantiamide acetate and isopropylated genipin as potential anti-inflammatory agents: The design, synthesis, and biological evaluation

Article abstract of DOI:10.1111/cbdd.13809

A novel series of hybrids designed on the basis of aurantiamide acetate and isopropylated genipin were synthesized and biologically evaluated as anti-inflammatory agents. Among them, compound 7o exhibited the best inhibitory activity against TNF-α secretion (IC₅₀?=?16.90?μM) and was selected for further in vitro and in vivo functional study. The results demonstrated that 7o was capable of suppressing the expression of LPS-induced iNOS and COX-2, as well as reducing the production of NO at the concentration of 5?μM, which may be resulted from its regulation of NF-κB signaling and MAPK signaling. Moreover, compound 7o exhibited favorable in vivo anti-inflammatory activity with an inhibition rate of 53.32% against xylene-induced ear swelling in mice at the dose of 5?mg/kg.

Full text of DOI:10.1111/cbdd.13809

PROFESSOR JIAMING LI (Orcid ID : 0000-0003-2141-7561)
Article type
: Research Article
Hybrids of aurantiamide acetate and isopropylated genipin as potential anti-inflammatory
agents: the design, synthesis and biological evaluation
Hongwei Wang a, Sufan Gao a, Jiaming Li a, b, *, Xiaodong Ma a, b, **, Wandong Liu a, Shihu Qian a
a College of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012, China;
b Deparment of Medicinal Chemistry, Anhui Academy of Chinese Medicine, Hefei 230012, China
* Corresponding author.
** Corresponding author.
Tel.: +8613705694971; e-mail address: lijiaming2017@sina.com (J. Li)
Tel.: +8618056545906; e-mail address: o-omaxiaodong@163.com (X. Ma)
ABSTRACT
A novel series of hybrids designed on the basis of aurantiamide acetate and isopropylated
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through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.13809
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genipin were synthesized and biologically evaluated as anti-inflammatory agents. Among them,
compound 7o exhibited the best inhibitory activity against TNF-α secretion (IC50 = 16.90 μM), and
was selected for further in vitro and in vivo functional study. The results demonstrated that 7o was
capable of suppressing the expression of LPS-induced iNOS and COX-2, as well as reducing the
production of NO at the concentration of 5 μM, which may be resulted from its regulation of
NF-κB signaling and MAPK signaling. Moreover, compound 7o exhibited favourable in vivo
anti-inflammatory activity with an inhibition rate of 53.32% against xylene-induced ear swelling
in mice at the dose of 5 mg/kg.
Keywords: Anti-inflammatory, Aurantiamide acetate, Synthesis, NF-κB, MAPK
1. Introduction
As a fundamental defense mechanism of host protection against injury or infection, the
inflammatory process also results in tissue damage and causes a myriad of diseases such as
arthritis, atherosclerosis and diabetes[1-2]. Mounting researches showed that Nuclear factor kappa B
(NF-κB) and Mitogen-activated protein kinase (MAPK) signaling pathways play vital roles in
inflammatory diseases[3-7]. NF-κB paticipates in regulating the expression of downstream targets
including COX-2, iNOS, as well as inducing the generation of pivotal inflammatory factors such
as TNF-α, IL-6[8-10]. MAPK is a serine/threonine protein kinase which is mediating the production
of TNF-α, NO and other inflammatory factors, thereby being crucial for the initiation and
progression of inflammation as well[11]
.
Aurantiamide acetate (1, Fig. 1) originating from medicinal plants exhibits a broad spectrum
of biological activities, including antioxidant, anti-inflammatory and neurotrophic activities.
Among these, the anti-inflammatory effect stems from its capability to modulate the NF-κB and
MAPK pathways, leading to dose-dependent suppression of NO and TNF-α production, as well as
iNOS and COX-2 expression[12]. Throughout the past decades, structural modification of 1 has
predominantly focused on the terminal phenyl moiety and the phenylalanine residue (2, Fig. 1)[13]
.
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Upon modifying the phenyl of the phenylalanine residue and unmasking its carboxylic
functionality, Sircar[14] and co-workers discovered a series of N-benzoylphenylalanine derivatives.
Compound 3 (Fig. 1), as a representative, has entered the clinical research owing to its excellent
anti-inflammatory activity and drug-like properties. As illustrated by the chemical structure of 3,
the α-amino acid residue is crucial for activity, and unmasking its carboxylic functionality does
not dramatically weaken the activity.
R2
O
O
O
O
H
H
N
N
R1
N
O
CH3
N
O
CH3
H
H
O
O
1
2
MeO
COOCH3
H
OMe
Cl
O
O
OH
H
N
HO
OH
H
O
Cl
3
4
Figure 1 The structures of compounds 1-4
Genipin (4, Fig. 1) is the main biologically active component of Geniposide with diverse
pharmacological activities, including anti-thrombotic, anti-tumor, anti-inflammatory and
hepatoprotective activities. Recent studies have shown that Genipin could significantly
downregulate LPS-induced expression of inflammation-related enzymes and suppress the
production of pro-inflammatory factors by regulating NF-κB and MAPK signaling pathways[15-17]
.
The hydroxyl at the C-3 position and the hemiacetal at the C-10 position in Genipin are the main
reasons for its poor stability, low lipid solubility and limited pharmacological activity in
physiological solution. Therefore, we further optimized the structure of Genipin according to the
report of Li Qingshan et al.[18]
.
As stated above, both 1 and 4 may serve as an anti-inflammatory agent by the capability to
interfere with NF-κB and MAPK signaling pathways. Hence, a series of aurantiamide
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acetate-isopropylated genipin hybrids have recently designed via combing their key
pharmacophores. Firstly, according to the chemical structure of 3, the phenylalanine carboxylic
functionality of 1 was unmasked for providing a site for derivatization (Fig. 3). Then, the primary
alcoholic hydroxyl group was replaced by an amino group for generating amide after condensation
with the phenylalanine derivative, which was more stable than the ester functionality.
Additionally, we also investigated whether incorporation of a trans-carbon-carbon double bond
between the terminal phenyl and the amide group had an impact on activity[19-23]
.
Herein, we communicate the synthesis and biological evaluation of these aurantiamide
acetate-genipin hybrids as potential anti-inflammatory agents. Their anti-inflammatory activity
was evaluated by lipopolysaccharide (LPS)-induced cytokine production in RAW264.7 cells.
Furthermore, the most potent compound 7o was used to illustrate the preliminary mechanism of
anti-inflammatory action.
COOCH3
H
O
O
H
N
O
N
O
CH3
H
H
O
HO
OH
COOCH3
H
H
O
H
O
O
N
N
H
O
COOCH3
H
H
O
H
O
O
N
N
n
R
H
n=0 or 1
R=H; CH3; OCH3; F; Cl; CF3; NO2; OH; dioxole
O
Figure 2
Design rationale of target compounds
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2. Results and discussion
2.1. Chemistry
The synthetic route of target compounds 7a-7o was outlined in Scheme 1. Genipin, as the starting
material was subjected to O-alkylation to afford 2. Its hydroxyl functionality was converted to
azide after methylsulfonylation and the further treatment with NaN3. Afterwards, reduction of the
azide group furnished the key intermediate 5. The key fragment 8a-o was prepared via
transformation of 6a-o to corresponding acyl chlorides and subsequent condensation with
L-phenylalanine in the presence of NaOH. Finally, condensation of 5 with 8a-o provided 7a-7o as
the target compounds.
COOCH3
COOCH3
COOCH3
COOCH3
H
H
H
H
H
H
H
i
ii
iii
O
O
O
O
O
O
O
H
H3CO2SO
HO
HO
N3
OH
2
3
4
Genipin(1)
COOCH3
COOCH3
H
H
v
iv
O
H
O
O
O
O
N
N
H
n
H
H
R
H2N
O
5
7a~o
O
O
vi
OH
n
OH
R
N
H
n
R
O
6a~o
8a~o
Compounds
R
a
b
c
d
e
f
g
h
i
j
k
l
n=0
n=1
H
p-CH3 m-CH3 p-OCH3 m-OCH3 p-Cl
m-Cl
p-F
m-F p-NO2 p-CF3 dioxole
m
n
o
m-F m-OCH3 p-OH,m-OCH3
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Reagents and conditions: (i) p-TsOH·H2O, iso-PrOH, reflux; (ii) CH3SO2Cl, Et3N, DCM, rt; (iii) NaN3, DMF,
rt; (iv) SnCl2, MeOH, rt; (v) 8a~o, HOBT, EDCl, Et3N, DCM, rt; (vi) SOCl2, reflux.
Scheme 1 The synthetic route of target compounds
2.2. The inhibitory effect on TNF-α secretion
Since TNF-α is an important pro-inflammatory cytokine, we first investigated the inhibitory
activity of the target compounds against LPS-induced TNF-α secretion in RAW264.7 cells with
Genipin and Aurantiamide acetate as reference compounds. Tables 1 demonstrated that positive
control groups and Aurantiamide acetate analogues were the ability to reduce TNF-α production.
Among them, 7d ， 7h ， 7i ， 7o performed better than Aurantiamide acetate in inhibiting
LPS-induced TNF-α expression. Particularly, compound 7o exerted the most potent inhibitory
activity against TNF-α secretion throughout this series (IC50= 16.90±1.23 μM). Thus, it was
selected for further in vitro and in vivo biological evaluation.
Table 1 Inhibitory effect of Target compounds on LPS induced TNF-α secretion in RAW264.7
Compound
IC50（μM）
39.11±2.17
51.59±3.66
57.68±3.84
18.44±1.26**
24.13±1.58**
Compound
IC50（μM）
28.16±1.59
34.97±1.54
25.12±1.40**
27.83±1.44
86.04±1.94
7a
7b
7c
7d
7e
7j
7k
7l
7m
7n
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7f
7g
7h
7i
31.45±1.86
25.73±1.37**
18.14±1.13**
19.58±1.29**
7o
4
16.90±1.23**
98.83±5.97
23.65±1.37
1
Compared with Aurantiamide**p＜0.01，Values are expressed as the mean ± SD (n=5).
2.2 Compound 7o inhibits inflammatory mediators in RAW264.7 cells
Considering the essential role played by NO in the inflammation, as well as its close correlation
with the expression of COX-2 and iNOS, we then investigated the effect of 7o on LPS-induced
NO production in RAW264.7 cells. As shown in Fig. 3, compound 7o was capable to inhibit the
NO generation in a concentration-dependent manner. At the concentration as low as 5 μM, it
remarkably inhibited the NO production. As shown in Fig. 3, the impact of 7o on iNOS and
COX-2 expressions in RAW264.7 was then detected by western blot assay. Compound 7o
dramatically downregulated the expressions of both iNOS and COX-2 at the concentration of 5
μM. Besides, the downregulation was dose-dependent. These results illustrated that compound 7o
could reduce LPS-induced inflammatory response in RAW264.7 cells.
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B
A
C
D
Figure 3 Compound 7o reduced NO production in RAW264.7 cells
Fig. 3 Effects of 7o on LPS-induced iNOS and COX-2 expression in RAW264.7 cells. The cells were
pre-treated with different concentrations of 7o for 2h and then were stimulated with LPS (200 ng/mL) or without
LPS for 8 h. Bay 11-7082 is the NF-κB inhibitor. Representative bands were shown in (A), Quantitative analysis
of iNOS expressions in (B), Quantitative analysis of COX-2 expressions in (C). The results were presented as
mean ± SD (n = 3).*p < 0.05, **p < 0.01.
2.3 Compound 7o suppresses NF-κB activation in RAW264.7 cells.
As an important pathway regulating the production of proinflammatory cytokines, NF-κB
signaling can be activated by LPS-stimulation, which leads to the phosphorylation and degradation
of IκB to NF-κB p65. Then p65 translocates to the nucleus and interacts with target promoters to
cause the release of inflammatory factors such as TNF-α. As shown in Fig. 4, the levels of IκB,
phos-IκB, phos-p65 were detected by western blot assay to evaluate the suppressive effect of 7o
on NF-κB signaling after LPS-stimulation. Taken together, 7o was capable of modulating NF-κB
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signaling.
Fig.
4
Compound 7o inhibited LPS-induced NF-κB activation in RAW264.7 cells. RAW264.7 cells were co-incubated
with 7o (5, 10, 20 μM) and LPS (200 ng/mL) for 2 h. Representative bands were shown in (A); The levels of
IκB expression (B) and the phosphorylation levels of IκB (C) were normalized to β-actin; Phosphorylation levels
of p65 (D) were normalized to control. The results were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.
2.4 Compound 7o inhibits MAPK signaling activation in RAW264.7 cells.
MAPKs are a family of signal transduction proteins, including p38, ERK, and JNK, and play
crucial roles in the expression of iNOS and COX-2. Besides, they can induce the production of
NO and TNF-α. To determine the effect of compound 7o on the LPS-induced activation of
MAPKs, the expressions of phos-ERK, phos-JNK, and phos-p38 were examined. As was
expected, compound 7o inhibited p38 activation in the MAPK signaling pathway in a
concentration-dependent manner, but had no obvious effect on phosphorylation of JNK or ERK
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(Fig. 5). Therefore, the anti-inflammatory activity of compound 7o was speculated to have a
relationship with its negative effects on p38 activation.
Fig. 5 Compound 7o inhibited LPS-induced MAPK-signalling activation in RAW264.7 cells. Representative
bands were shown in (A). Phosphorylation levels of p38 (B), JNK (C) and ERK1/2 (D) were normalized to
control. The results were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.
2.5 Anti-inflammatory activity in vivo
Table 2 The effect of different compounds on xylene-induced ear swelling in mice.
Group
Blank
Dose(mg/kg)
Swelling degree (mg)
0.12±0.66
Inhibition (%)
-
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Model
-
5
14.74±1.28
6.88±1.33**
7.84±2.29**
4.92±0.59**
7o
53.32
46.81
66.65
Aurantiamide acetate
Dexamethasone
5
0.5
* p<0.05, ** p<0.01 vs Model.
swelling degree (mg) = MR - ML.
Inhibition (%) = ((MR-ML) model-(MR-ML) treated)/(MR-ML) model * 100%.
Values are expressed as the mean ± SD (n=6).
The in vivo anti-inflammatory activity of compound 7o was further determined by the allergic
contact dermatitis model. The results were expressed as percent inhibition of mouse ear swelling
over the untreated control group and were shown in Table 2. Based on the data, compound 7o
showed better anti-inflammatory activity than aurantiamide acetate at the dose of 5 mg/kg. In
other words, compound 7o could inhibit mouse ear swelling and show good anti-inflammatory
activity.
3. Conclusions
In conclusion, a novel series of hybrids designed on the basis of aurantiamide acetate and
isopropylated genipin were synthesized and biologically evaluated as anti-inflammatory agents.
Among these compounds, compound 7o exhibited the best inhibitory activity against TNF-α
secretion (IC50=16.90 μM). Then the mechanisms of action of compound 7o were investigated in
further studies. The results demonstrated that 7o was capable of suppressing the LPS-induced
expression of LPS-induced iNOS and COX-2, as well as the production of NO, which may be
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resulted from its regulation of NF-κB and MAPK signaling pathways. Moreover, compound 7o
also showed great anti-inflammatory activity in vivo, and might serve as promising lead candidates
for anti-inflammatory agent development.
4. Experimental section
4.1. Chemistry
Melting points were determined on SGWX-4 melting point apparatus (Precision Science Inc.,
Shanghai, China), which were uncorrected. NMR spectra were measured at 400 or 600 MHz for
1H NMR, and 100 or 150 MHz for 13C NMR, respectively, on Bruker DRX-400(Bruker Inc, CH)
in DMSO-d6, using TMS as internal standard. ESI-MS data was collected on an LCQ Advantage
MAX spectrometer (Finnigan Inc. USA). Column chromatography was performed with silica gel
(200 mesh, Qingdao Marine Chemical Inc., Qingdao, China). TLC was carried out on glass
pre-coated silica gel GF 254 plates (Qingdao Marine Chemical Inc.). Unless otherwise noted, all
chemicals were purchased from commercially available sources.
4.2. General procedure for preparation of compound (5)
4.2.1
methyl
(4aS,7aS)-7-(hydroxymethyl)-1-isopropoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate
(2)
In a three-neck flask, Genipin (2.0 g, 8.84 mmol), p-Toluenesulfonic acid monohydrate (2.0
g, 10.51 mmol) and Isopropyl alcohol (20 ml) were added. The reaction mixture was heated to 80
℃ for 2 h with stirring (TLC, PE/EA=4:1). After the reaction was completed, water (100 ml) was
added. The reaction solution was extracted with dichloromethane (3 × 50 mL) and combined
organic layers, dried with sodium sulfate and evaporated in vacuo to afford a yellow oil. The oil
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was purified by chromatography (PE/EA=4:1) to give the title compound as white solid. Yield
84.4%, m.p. 89.5 ℃-92.7 ℃. 1H NMR (400 MHz, DMSO-d6) δ: 7.46 (s, 1H, =CH), 5.76-5.68 (m,
1H, =CH), 4.71 (d, J = 7.8 Hz, 1H, CH), 4.10 (d, J = 15.0 Hz, 1H, CH2OH), 3.99-3.92 (m, 2H),
3.61 (s, 3H, COOCH3), 2.98 (d, J = 7.8 Hz, 1H, CH2), 2.70-2.64 (m, 1H, CH), 2.43 (t, J = 7.8 Hz,
1H, CH2), 2.05-1.95 (m, 1H, CH), 1.16 (d, J = 6.0 Hz, 3H, CHCH3), 1.09 (d, J = 6.0 Hz, 3H,
CHCH3); 13C NMR (101 MHz, DMSO-d6) δ: 170.08, 155.23, 147.84, 128.62, 113.38, 102.78,
74.54, 62.66, 54.04, 48.83, 41.21, 38.64, 26.43, 24.73; ESI-MS for C14H20O5 m/z: 268.87 [M+H]+.
4.2.2
methyl
(4aS,7aS)-1-isopropoxy-7-(((methylsulfonyl)oxy)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-
4-carboxylate (3)
Compound 2 (2.0 g, 7.45 mmol), Et3N (1.6 mL, 11.51 mmol) was dissolved in DCM (30
mL), Methanesulfonyl chloride (0.9 g, 7.86 mmol) was added dropwise under ice-cooling. After
the addition was completed, the reaction mixture was continued to stir for 1 h. After the reaction
was completed, DCM (50 ml) was added, then washed with water (20 mL×3), dried with sodium
sulfate and evaporated in vacuo to afford a yellow oil (2.5g). Yield 96.9%. No purification for the
next reaction.
4.2.3
methyl
(4aS,7aS)-7-(azidomethyl)-1-isopropoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate
(4)
Compound 3 (2.0 g, 7.45 mmol), NaN3 (2.4 g, 37.25 mmol) was dissolved in DMF (30 mL),
The reaction mixture was stirred at room temperature for 3 h. After the reaction was completed,
the reaction solution was poured into 100 mL of ice water, then extracted with ether (50 mL×3)
and combined organic layers, dried with sodium sulfate and evaporated in vacuo to afford
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Compound 4. No purification for the next reaction.
4.2.4
methyl
(4aS,7aS)-7-(aminomethyl)-1-isopropoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate
(5)
In the flask, compound 4 was dissolved in Methanol (20 mL), Stannous chloride dihydrate
(2.05 g, 10.83 mmol) was added. The mixture was stirred at room temperature for 3 h. After the
reaction was completed, the reaction solution was poured into 100 mL of ice water and diluted
with 5% NaOH (liq.) was slowly added dropwise to pH = 8, then extracted with Ethyl acetate (50
mL×3) and combined organic layers, dried with sodium sulfate and evaporated in vacuo. The
residues were purified by chromatography (DCM/Methanol=20:1) to give the title compound as
brown oil (1.43 g). Yield 74.1%, 1H NMR (400 MHz, DMSO-d6) δ: 7.44 (s, 1H, =CH), 5.68-5.62
(m, 1H, =CH), 4.69 (d, J = 7.8 Hz, 1H, CH), 3.99-3.93 (m, 1H, CHCH3), 3.61 (s, 3H, COOCH3),
3.34 (d, J = 16.8 Hz, 1H, CH2NH2), 3.18 (d, J = 16.8 Hz, 1H, CH2NH2), 2.97 (q, J = 7.8 Hz, 1H,
CH2), 2.69-2.58 (m, 1H, CH), 2.45 (t, J = 7.8 Hz, 1H, CH2), 2.15 (s, 2H, CH2NH2), 1.98-1.93 (m,
13
1H, CH), 1.16 (d, J = 6.0 Hz, 3H, CHCH3), 1.09 (d, J = 6.0 Hz, 3H, CHCH3); C NMR (101
MHz, DMSO-d6) δ: 170.06, 155.16, 149.18, 127.90, 113.38, 102.96, 74.53, 53.97, 49.18, 44.59,
41.28, 38.65, 26.38, 24.65; ESI-MS for C14H21NO4 m/z: 267.94 [M+H]+.
4.3. General procedure for preparation of compound (8a-8o)
A solution of substituted benzoic acid or substituted benzoenoic acid (16.38 mmol), SOCl2
(15 mL) were refluxed for 5 h, then evaporated in vacuo to afford corresponding acyl chloride,
L-phenylalanine (2.25 g, 13.65 mmol) was dissolved in 2 M NaOH (20 mL), and corresponding
acyl chloride was slowly added dropwise under ice-cooling. After the dropwise addition, the
reaction mixture was continued to stir for 2 h(DCM/Methanol=5:1). After the reaction was
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completed, the reaction solution was diluted with 2M HCl (liq.) was slowly added dropwise to pH
= 5, then filtrated and dried to give the title compound 8a-8o.
4.4. General procedure for preparation of compound (7a-7o)
To a solution of 8a-8o (2 mmol), EDC·HCl (0.422 g, 2.2 mmol), HOBt (0.297 g, 2.2 mmol)
and 5 (0.564 g, 2 mmol.) in DMF (20 mL), was added Et3N (0.83 mL). The reaction mixture was
stirred at room temperature for 2 h and monitored by TLC (DCM/Methanol=10:1). After the
reaction was completed, the reaction mixture was added 10% citric acid (30 mL) and filtered. The
residue was purified via silica using methanol/DCM = 1:20 to provide the target compound 7a-7o.
4.4.1
methyl
(4aS,7aS)-7-((2-benzamido-3-phenylpropanamido)methyl)-1-isopropoxy-1,4a,5,7a-tetrahydrocycl
1
openta[c]pyran-4-carboxylate (7a). white soild, yield, 45.2%, m.p. 143.5℃-145.6 ℃; H NMR
(400 MHz, DMSO-d6) δ 8.54 (s, 1H, ArCONH), 8.22 (s, 1H, CONH), 7.77 (d, J = 7.8 Hz, 2H,
ArH), 7.49-7.44 (m, 2H, =CH, ArH), 7.40 (t, J = 7.8 Hz, 2H, ArH), 7.32 (d, J = 7.8 Hz, 2H, ArH),
7.22 (t, J = 7.8 Hz, 2H, ArH), 7.13 (t, J = 7.8 Hz, 1H, ArH), 5.59-5.47 (m, 1H, =CH),
4.76-4.66(m, 2H, OCHO, NHCHCO), 4.04-3.91(m, 2H, CCH2NH), 3.75-3.64 (m, 1H,
CH(CH3)2), 3.60 (s, 3H, COOCH3), 3.10-3.04 (m, 1H, ArCH2CH), 3.02-2.94 (m, 2H, ArCH2CH,
CH2), 2.67-2.57 (m, 1H, CH), 2.42 (t, J =7.8 Hz, 1H, CH2), 2.03-1.93 (m, 1H, CH), 1.20-1.08 (m,
6H, CH(CH3)2);13C NMR (101 MHz, DMSO-d6) δ: 174.13, 170.06, 169.34, 155.30, 144.05,
141.54, 137.14, 134.33, 132.25, 131.22, 131.13, 130.51, 129.30, 129.28, 111.29, 102.78, 74.78,
58.14, 54.09, 49.41, 41.74, 41.21, 40.45, 38.59, 26.47, 24.80; ESI-MS for C30H34N2O6 m/z:
518.99 [M+H]+.
4.4.2
methyl
(4aS,7aS)-1-isopropoxy-7-((2-(4-methylbenzamido)-3-phenylpropanamido)methyl)-1,4a,5,7a-tetra
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1
hydrocyclopenta[c]pyran-4-carboxylate (7b). white soild, yield, 40.8%, m.p. 153.2-155.4 ℃; H
NMR (600 MHz, DMSO-d6) δ 8.44 (s, 1H, ArCONH), 8.19 (s, 1H, CONH), 7.68 (d, J = 7.8 Hz,
2H, ArH), 7.45(s, 1H, =CH), 7.31 (d, J = 7.8 Hz, 2H, ArH), 7.24-7.17 (m, 4H, ArH), 7.12 (t, J =
7.2 Hz, 1H, ArH), 5.58-5.50 (m, 1H, =CH), 4.75-4.65 (m, 2H, OCHO, NHCHCO), 4.05-3.90 (m,
2H, CCH2NH), 3.75-3.63 (m, 1H, CH(CH3)2), 3.60 (s, 3H, COOCH3), 3.09-3.03 (m, 1H,
ArCH2CH), 3.02-2.94 (m, 2H, ArCH2CH, CH2), 2.68-2.56 (m, 1H, CH), 2.42 (t, J = 7.8 Hz, 1H,
CH2), 2.30 (s, 3H, ArCH3), 2.04-1.92 (m, 1H, CH), 1.20-1.08 (m, 6H, CH(CH3)2); 13C NMR (150
MHz, DMSO-d6) δ: 174.21, 170.06, 169.18, 155.29, 144.20, 144.06, 141.56, 134.34, 132.26,
131.74, 131.11, 130.53, 129.30, 129.25, 113.29, 102.79, 74.78, 58.08, 54.08, 49.41, 41.73, 41.21,
40.46, 38.60, 26.47, 24.80, 24.03; ESI-MS for C31H36N2O6 m/z: 533.12 [M+H]+.
4.4.3
methyl
(4aS,7aS)-1-isopropoxy-7-((2-(3-methylbenzamido)-3-phenylpropanamido)methyl)-1,4a,5,7a-tetra
1
hydrocyclopenta[c]pyran-4-carboxylate (7c). white soild, yield, 49.1%, m.p. 132.3-134.7 ℃; H
NMR (600 MHz, DMSO-d6) δ 8.47 (s, 1H, ArCONH), 8.19 (s, 1H, CONH), 7.62-7.51 (m, 2H,
ArH), 7.45 (s, 1H, =CH), 7.34-7.25 (m, 4H, ArH,), 7.22 (t, J = 7.2 Hz, 2H, ArH), 7.12 (t, J = 7.2
Hz, 1H, ArH), 5.58-5.48 (m, 1H, =CH), 4.76-4.65 (m, 2H, OCHO, NHCHCO), 4.05-3.90 (m, 2H,
CCH2NH), 3.75-3.63 (m, 1H, CH(CH3)2), 3.60 (s, 3H, COOCH3), 3.10-3.03 (m, 1H, ArCH2CH),
3.02-2.92 (m, 2H, ArCH2CH, CH2), 2.68-2.56 (m, 1H, CH), 2.42 (t, J = 7.8 Hz, 1H, CH2), 2.30 (s,
3H, ArCH3), 2.03-1.93 (m, 1H, CH), 1.22-1.07 (m, 6H, CH(CH3)2); 13C NMR (150 MHz,
DMSO-d6) δ: 174.14, 170.05, 169.44, 155.29, 144.05, 141.52, 140.45, 137.14, 134.86, 132.25,
131.12, 131.10, 131.05, 129.32, 129.27, 127.64, 113.28, 102.80, 74.79, 58.06, 54.07, 49.39, 41.74,
41.21, 40.45, 38.60, 26.47, 24.79, 23.99; ESI-MS for C31H36N2O6 m/z: 533.01 [M+H]+.
4.4.4
methyl
(4aS,7aS)-1-isopropoxy-7-((2-(4-methoxybenzamido)-3-phenylpropanamido)methyl)-1,4a,5,7a-tet
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rahydrocyclopenta[c]pyran-4-carboxylate (7d). white soild, yield, 50.6%, m.p. 143.3-145.6 ℃; 1H
NMR (600 MHz, DMSO-d6) δ 8.39 (s, 1H, ArCONH), 8.19 (s, 1H, CONH), 7.76 (d, J = 7.8 Hz,
2H, ArH), 7.45 (s, 1H, =CH), 7.31 (d, J = 7.8 Hz, 2H, ArH), 7.21 (t, J = 7.8, 2H, ArH), 7.11 (t, J
= 7.8 Hz, 1H, ArH), 6.93 (d, J = 7.8 Hz, 2H, ArH), 5.56-5.50 (m, 1H, =CH), 4.75-4.62 (m, 2H,
OCHO, NHCHCO), 4.04-3.90 (m, 2H, CCH2NH), 3.75 (s, 3H, ArOCH3), 3.74-3.63 (m, 1H,
CH(CH3)2), 3.60 (s, 3H, COOCH3), 3.08-3.02 (m, 1H, ArCH2CH), 3.01-2.92 (m, 2H,
ArCH2CH, CH2), 2.67-2.56 (m, 1H, CH), 2.41 (t, J = 7.8 Hz, 1H, CH2), 1.99-1.95 (m, 1H, CH),
,
1.20-1.07 (m, 6H, CH(CH3)2); 13C NMR (150 MHz, DMSO-d6) δ: 174.30, 170.06, 168.80, 164.48,
155.29, 144.08, 141.61, 132.36, 132.25, 131.11, 129.34, 129.27, 129.24, 116.42, 113.28, 102.79,
74.78, 58.41, 58.09, 54.08, 49.40, 41.72, 41.20, 40.45, 38.59, 26.47, 24.80; ESI-MS for
C31H36N2O7 m/z: 549.12 [M+H]+.
4.4.5
methyl
(4aS,7aS)-1-isopropoxy-7-((2-(3-methoxybenzamido)-3-phenylpropanamido)methyl)-1,4a,5,7a-tet
1
rahydrocyclopenta[c]pyran-4-carboxylate (7e). white soild, yield, 51.8%, m.p. 107.8-110.5 ℃; H
NMR (600 MHz, DMSO-d6) δ 8.53 (s, 1H, ArCONH), 8.21 (s, 1H, CONH), 7.45 (s, 1H, =CH),
7.36-7.29 (m, 5H, ArH), 7.22 (t, J = 7.2 Hz, 2H, ArH), 7.12 (t, J = 7.2 Hz, 1H, ArH), 7.03 (d, J =
7.2 Hz, 1H, ArH), 5.53-5.47 (m, 1H, =CH), 4.76-4.63 (m, 2H, OCHO, NHCHCO), 4.05-3.90 (m,
2H, CCH2NH), 3.75 (s, 3H, ArOCH3), 3.73-3.62 (m, 1H, CH(CH3)2), 3.60 (s, 3H, COOCH3),
3.10-3.04 (m, 1H, ArCH2CH), 3.02-2.93 (m, 2H, ArCH2CH, CH2), 2.68-2.56 (m, 1H, CH), 2.42
13
(t, J = 7.8 Hz, 1H, CH2), 2.03-1.98 (m, 1H, CH), 1.22-1.07 (m, 6H, CH(CH3)2); C NMR (150
MHz, DMSO-d6) δ: 174.10, 170.05, 169.10, 162.09, 155.30, 144.04, 141.52, 138.60, 132.35,
132.26, 131.12, 129.32, 129.28, 122.76, 120.18, 115.68, 113.29, 102.78, 74.78, 58.31, 58.14,
54.07, 49.40, 41.74, 41.20, 40.45, 38.59, 26.46, 24.79; ESI-MS for C31H36N2O7 m/z: 549.14
[M+H]+.
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4.4.6
methyl
(4aS,7aS)-7-((2-(4-chlorobenzamido)-3-phenylpropanamido)methyl)-1-isopropoxy-1,4a,5,7a-tetra
1
hydrocyclopenta[c]pyran-4-carboxylate (7f). white soild, yield, 48.9%, m.p. 167.5-169.6 ℃; H
NMR (600 MHz, DMSO-d6) δ 8.65 (s, 1H, ArCONH), 8.22 (s, 1H, CONH), 7.82-7.72 (m, 2H,
ArH), 7.49-7.39 (m, 3H, ArH), 7.31-7.25 (m, 2H, ArH), 7.22-7.16 (m, 2H, ArH), 7.13-7.06 (m,
1H, ArH), 5.55-5.45 (m, 1H, =CH), 4.72-4.62 (m, 2H, OCHO, NHCHCO), 4.98-3.88 (m, 2H,
CCH2NH), 3.73-3.65 (m, 1H, CH(CH3)2), 3.58 (s, 3H, COOCH3), 3.07-3.01 (m, 1H, ArCH2CH),
2.98-2.91 (m, 2H, ArCH2CH, CH2), 2.63-2.55 (m, 1H, CH), 2.42-2.36(m, 1H, CH2), 1.99-1.91
13
(m, 1H, CH), 1.15-1.09 (m, 6H, CH(CH3)2); C NMR (150 MHz, DMSO-d6) δ: 173.99, 170.05,
168.72, 155.30, 144.02, 141.46, 139.16, 135.85, 132.45, 132.22, 131.32, 131.13, 129.33, 129.30,
113.28, 102.77, 74.77, 58.19, 54.09, 49.40, 41.74, 41.20, 40.44, 38.59, 26.47, 24.79; ESI-MS for
C30H33ClN2O6 m/z: 553.06 [M+H]+.
4.4.7
methyl
(4aS,7aS)-7-((2-(3-chlorobenzamido)-3-phenylpropanamido)methyl)-1-isopropoxy-1,4a,5,7a-tetra
1
hydrocyclopenta[c]pyran-4-carboxylate (7g). white soild, yield, 47.7%, m.p. 136.2-137.6 ℃; H
NMR (600 MHz, DMSO-d6) δ 8.72 (s, 1H, ArCONH), 8.26 (s, 1H, CONH), 7.83 (s, 1H, ArH),
7.72 (d, J = 7.2 Hz, 1H, ArH), 7.54 (d, J = 7.2 Hz, 1H, ArH), 7.48-7.40 (m, 2H, ArH), 7.34-7.27
(m, 2H, ArH), 7.25-7.18 (m, 2H, ArH), 7.15-7.09 (m, 1H, ArH), 5.56-5.50 (m, 1H, =CH),
4.77-4.65 (m, 2H, OCHO, NHCHCO), 4.05-3.89 (m, 2H, CCH2NH), 3.77-3.64 (m, 1H,
CH(CH3)2), 3.59 (s, 3H, COOCH3), 3.12-3.05 (m, 1H, ArCH2CH), 3.02-2.91 (m, 2H, ArCH2CH,
CH2), 2.66-2.55 (m, 1H, CH), 2.44-2.38 (m, 1H, CH2), 2.04-1.93 (m, 1H, CH), 1.26-1.06 (m, 6H,
13
CH(CH3)2); C NMR (150 MHz, DMSO-d6) δ: 173.90, 170.05, 167.94, 155.29, 144.01, 141.43,
139.11, 136.10, 134.16, 133.25, 132.21, 131.14, 130.35, 129.33, 129.32, 129.28, 113.28, 102.77,
74.78, 58.20, 54.07, 49.41, 41.76, 41.21, 40.41, 38.59, 26.46, 24.79; ESI-MS for C30H33ClN2O6
m/z: 553.07 [M+H]+.
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4.4.8
methyl
(4aS,7aS)-7-((2-(4-fluorobenzamido)-3-phenylpropanamido)methyl)-1-isopropoxy-1,4a,5,7a-tetra
1
hydrocyclopenta[c]pyran-4-carboxylate (7h). white soild, yield, 47.2%, m.p. 112.8-114.7 ℃; H
NMR (600 MHz, DMSO-d6) δ 8.64 (s, 1H, ArCONH), 8.29 (s, 1H, CONH), 7.83 (t, J = 7.2 Hz,
2H, ArH), 7.44 (s, 1H, =CH), 7.30 (d, J = 7.2 Hz, 2H, ArH), 7.26-7.15 (m, 4H, ArH), 7.10 (t, J =
7.2 Hz, 1H, ArH), 5.56 -5.46 (m, 1H, =CH), 4.75-4.61 (m, 2H, OCHO, NHCHCO), 4.04-3.88 (m,
2H, CCH2NH), 3.73-3.61 (m, 1H, CH(CH3)2), 3.58 (s, 3H, COOCH3), 3.07-3.01 (m, 1H,
ArCH2CH), 2.99-2.90 (m, 2H, ArCH2CH, CH2), 2.63-2.54 (m, 1H, CH), 2.39 (t, J = 7.2 Hz, 1H,
13
CH2), 2.01-1.92 (m, 1H, CH), 1.20-1.06 (m, 6H, CH(CH3)2); C NMR (150 MHz, DMSO-d6) δ:
174.12, 170.06, 168.25, 166.95 (JC-F = 247.2Hz), 155.35, 144.06, 141.53, 133.53, 133.18 (JC-F
=
8.8Hz), 132.24, 131.14, 129.29, 129.19 (JC-F = 3.3Hz), 118.16 (JC-F = 21.8Hz), 113.22, 102.79,
74.79, 58.22, 54.11, 49.33, 41.74, 41.15, 40.42, 38.62, 26.48, 24.79; ESI-MS for C30H33FN2O6
m/z: 537.01 [M+H]+.
4.4.9
methyl
(4aS,7aS)-7-((2-(3-fluorobenzamido)-3-phenylpropanamido)methyl)-1-isopropoxy-1,4a,5,7a-tetra
1
hydrocyclopenta[c]pyran-4-carboxylate (7i). white soild, yield, 46.9%, m.p. 121.1-123.7 ℃; H
NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H, ArCONH), 8.32 (s, 1H, CONH), 7.67 (d, J = 7.8 Hz,
1H, ArH), 7.64-7.59 (m, 1H, ArH), 7.54-7.46 (m, 2H, ArH, =CH), 7.39-7.32 (m, 3H, ArH), 7.26
(d, J = 8.3 Hz, 2H, ArH), 7.20-7.13 (m, 1H, ArH), 5.64-5.50 (m, 1H, =CH), 4.83-4.64 (m, 2H,
OCHO, NHCHCO), 4.11-3.93 (m, 2H, CCH2NH), 3.82-3.70 (m, 1H, CH(CH3)2), 3.64 (s, 3H,
COOCH3), 3.19-3.10 (m, 1H, ArCH2CH), 3.06-2.96 (m, 2H,ArCH2CH, CH2), 2.72-2.60(m, 1H,
CH), 2.46 (t, J = 7.7 Hz, 1H, CH2), 2.07-1.97 (m, 1H, CH), 1.24-1.04 (m, 6H, CH(CH3)2).13C
NMR (101 MHz, DMSO-d6) δ 171.33, 167.44, 165.36, 163.51, 161.08, 152.69, 140.11(JC-F
=
259.3Hz), 136.79, 130.74(JC-F = 8.0Hz), 129.61, 128.53, 126.69, 124.10(JC-F = 2.9Hz), 118.72,
118.51, 114.67(JC-F = 22.8Hz), 110.65, 100.16, 72.16, 55.61, 51.47, 46.77, 37.81, 35.98, 23.85,
22.17; ESI-MS for C30H33FN2O6 m/z: 537.13 [M+H]+.
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4.4.10
methyl
(4aS,7aS)-1-isopropoxy-7-((2-(4-nitrobenzamido)-3-phenylpropanamido)methyl)-1,4a,5,7a-tetrah
1
ydrocyclopenta[c]pyran-4-carboxylate (7j). white soild, yield, 52.8%, m.p. 151.1-154.4 ℃; H
NMR (600 MHz, DMSO-d6) δ 8.95 (s, 1H, ArCONH), 8.31 (s, 1H, CONH), 8.25 (d, J = 8.4 Hz,
2H, ArH), 7.99 (d, J = 8.4 Hz, 2H, ArH), 7.45 (s, 1H, =CH), 7.31 (d, J = 7.2 Hz, 2H, ArH), 7.22
(t, J = 7.2 Hz, 2H, ArH), 7.12 (t, J = 7.2 Hz, 1H, ArH), 5.58-5.53 (m, 1H, =CH), 4.76-4.69 (m,
2H, OCHO, NHCHCO), 4.06-3.91 (m, 2H, CCH2NH), 3.77-3.64 (m, 1H, CH(CH3)2), 3.60 (s, 3H,
COOCH3), 3.13-3.07 (m, 1H, ArCH2CH), 3.02-2.93 (m, 2H, ArCH2CH, CH2), 2.66-2.58 (m, 1H,
13
CH), 2.42 (t, J = 7.8 Hz, 1H, CH2), 2.04-1.92 (m, 1H, CH), 1.14 (m, 6H, CH(CH3)2); C NMR
(150 MHz, DMSO-d6) δ: 173.74, 170.05, 167.71, 155.30, 152.11, 143.98, 142.74, 141.32, 132.23,
132.01, 131.16, 129.35, 129.31, 126.47, 113.28, 102.78, 74.78, 58.32, 54.07, 49.41, 41.78, 41.20,
40.48, 38.61, 26.47, 24.79; ESI-MS for C30H33N3O8 m/z: 564.13 [M+H]+.
4.4.11 methyl (4aS,7aS)-1-isopropoxy-7-((3-phenyl-2-(4-(trifluoromethyl)benzamido)propanami
do)methyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate(7k). white soild, yield, 47.8
1
%, m.p. 148.3-151.1 ℃; H NMR (400 MHz, DMSO-d6) δ 8.89 (s, 1H, ArCONH), 8.34 (
s, 1H, CONH), 8.00 (d, J = 8.1 Hz, 2H, ArH), 7.83 (d, J = 8.1 Hz, 2H, ArH), 7.49 (s,
1H, =CH), 7.35 (d, J =7.6 Hz, 2H, ArH), 7.29-7.23 (m, 2H, ArH), 7.20-7.12 (m, 1H, Ar
H), 5.63-5.57 (m, 1H, =CH), 4.83-4.71 (m, 2H, OCHO, NHCHCO), 4.13-3.94 (m, 2H, C
CH2NH), 3.84-3.70 (m, 1H, CH(CH3)2), 3.64 (s, 3H, COOCH3), 3.18-3.10 (m, 1H, ArCH2
CH), 3.07-2.96 (m, 2H, ArCH2CH, CH2), 2.73-2.59 (m, 1H, CH), 2.46 (t, J = 7.9 Hz, 1
H, CH2), 2.10-1.96 (m, 1H, CH), 1.26-1.12 (m, 6H, CH(CH3)2). 13C NMR (101 MHz, D
MSO-d6) δ 171.23, 167.44, 165.54, 152.69, 141.38, 138.77, 131.61(JC-F = 32.3Hz),129.62, 1
28.79, 128.53, 126.71, 125.66(JC-F = 3.2Hz), 121.69(JC-F = 274.2 Hz), 110.65, 100.15, 72.1
6, 55.62, 51.46, 46.77, 38.57, 37.83, 35.98, 23.85, 22.17; ESI-MS for C31H33F3N2O6 m/z:
585.26 [M+H]-.
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4.4.12
methyl
(4aS,7aS)-7-((2-(benzo[d][1,3]dioxole-5-carboxamido)-3-phenylpropanamido)methyl)-1-isopropo
xy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (7l). white soild, yield, 55.2%, m.p.
1
167.8-169.2 ℃; H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H, ArCONH), 8.25 (s, 1H, CONH),
7.54-7.46 (m, 1H, ArH), 7.42 (d, J = 8.1 Hz, 1H, =CH), 7.38-7.30 (m, 3H, ArH), 7.29-7.21 (m,
2H, ArH), 7.21-7.11 (m, 1H, ArH), 7.00-6.90 (m, 1H, ArH), 6.09 (s, 2H,OCH2O), 5.59-5.55 (m,
1H, =CH), 4.82-4.72 (m, 1H, NHCHCO), 4.72-4.62 (m, 1H, OCHO), 4.13-3.91 (m, 2H,
CCH2NH), 3.85-3.74 (m, 1H, CH(CH3)2), 3.64 (s, 3H, COOCH3), 3.19-2.92 (m, 3H, ArCH2CH,
CH2), 2.77-2.58 (m, 1H, CH), 2.47-2.41 (m, 1H, CH2), 2.09-1.94 (m, 1H, CH), 1.29-1.12 (m, 6H,
13
CH(CH3)2). C NMR (101 MHz, DMSO-d6) δ 171.60, 167.45, 165.80, 152.69, 150.16, 147.62,
141.43, 138.95, 129.62, 128.51, 126.65, 122.97, 110.65, 108.20, 107.94, 102.09, 100.16, 72.17,
55.57, 51.48, 46.76, 38.57, 37.81, 35.97, 23.85, 22.17; ESI-MS for C31H34N2O8 m/z: 563.14
[M+H]+.
4.4.13
methyl
(4aS,7aS)-7-((2-((E)-3-(4-fluorophenyl)acrylamido)-3-phenylpropanamido)methyl)-1-isopropoxy-
1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (7m). white soild, yield, 40.9%, m.p.
1
178.6-180.4 ℃; H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H, ArCONH), 8.29 (s, 1H, CONH),
7.64-7.56 (m, 2H, ArH), 7.49 (s, 1H, =CH), 7.36 (d, J = 15.8 Hz, 1H, ArCH=CH), 7.30-7.22 (m,
6H, ArH), 7.21-7.16 (m, 1H, ArH), 6.66 (d, J = 15.9 Hz, 1H, ArCH=CH), 5.49-5.44(m, 1H,
=CH), 4.77-4.63 (m, 2H, NHCHCO, OCHO), 4.11 -3.83 (m, 2H, CCH2NH), 3.81-3.69 (m, 1H,
CH(CH3)2), 3.63 (s, 3H, COOCH3), 3.09-2.93 (m, 2H, ArCH2CH), 2.89-2.79(m, 1H, CH2),
2.71-2.56 (m, 1H, CH), 2.42 (t, J = 7.8 Hz, 1H, CH2), 2.04-1.92 (m, 1H, CH), 1.28-1.15 (m, 6H,
13
CH(CH3)2). C NMR (101 MHz, DMSO-d6) δ 171.26, 166.26(JC-F = 236.67Hz), 161.90, 152.70,
141.30, 138.35, 138.16, 131.96(JC-F = 3.1Hz), 130.16(JC-F = 8.3Hz), 129.62, 128.55, 126.73,
122.34, 116.37(JC-F = 23.3Hz), 110.64, 100.03, 72.14, 56.49, 54.73, 51.48, 46.69, 38.55, 36.00,
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23.86, 22.15; ESI-MS for C32H35FN2O6 m/z: 563.17 [M+H]+.
4.4.14
methyl
(4aS,7aS)-1-isopropoxy-7-((2-((E)-3-(4-methoxyphenyl)acrylamido)-3-phenylpropanamido)methy
l)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (7n). white soild, yield, 38.3%, m.p.
1
183.3-185.0℃; H NMR (400 MHz, DMSO-d6) δ 8.33-8.24 (m, 2H,ArCONH,CONH), 7.52-7.46
(m, 3H, ArH, =CH), 7.37-7.22 (m, 5H, ArH, ArCH=CH), 7.21-7.15 (m, 1H, ArH), 7.01-6.94 (m,
2H, ArH), 6.56 (d, J = 15.8 Hz, 1H, ArCH=CH), 5.46-5.41 (m, 1H, =CH), 4.81-4.63 (m, 2H,
NHCHCO, OCHO), 4.19-3.89 (m, 2H, CCH2NH), 3.78 (s, 3H, OCH3), 3.77-3.69 (m, 1H,
CH(CH3)2), 3.63 (s, 3H, COOCH3), 3.10-2.92 (m, 2H, ArCH2CH), 2.92-2.79 (m, 1H, CH2),
2.70-2.56 (m, 1H, CH), 2.42 (t, J = 7.8 Hz, 1H, CH2), 2.10-1.89 (m, 1H, CH), 1.26-1.14 (m, 6H,
13
CH(CH3)2). C NMR (101 MHz, DMSO-d6) δ 171.37, 167.44, 165.49, 160.78, 152.69, 141.33,
139.07, 138.43, 129.62, 129.54, 128.54, 127.86, 126.67, 119.95, 114.84, 110.64, 100.18, 72.14,
55.71, 54.71, 51.48, 46.69, 38.56, 35.99, 23.86, 22.16; ESI-MS for C33H38N2O7 m/z: 575.26
[M+H]+.
4.4.15
methyl
(4aS,7aS)-7-((2-((E)-3-(4-hydroxy-3-methoxyphenyl)acrylamido)-3-phenylpropanamido)methyl)-
1-isopropoxy-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate (7o). white soild, yield,
1
35.6%, m.p. 88.6-92.1℃; H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H, OH), 8.33-8.12 (m, 2H,
ArCONH, CONH), 7.49 (s, 1H, =CH), 7.32-7.22 (m, 5H, ArH, CH=CH), 7.21-7.14 (m, 1H,
ArH), 7.10 (s, 1H, ArH), 6.97 (d, J = 8.2 Hz, 1H, ArH), 6.80-6.75 (m, 1H, ArH), 6.54 (d, J = 15.7
Hz, 1H, ArCH=CH), 5.54-5.42 (m, 1H, =CH), 4.80-4.59 (m, 2H, NHCHCO, OCHO), 4.08-3.90
(m, 2H, CCH2NH), 3.80 (s, 3H, OCH3), 3.78-3.70 (m, 1H, CH(CH3)2), 3.63 (s, 3H, COOCH3),
3.11-2.93 (m, 2H, ArCH2CH), 2.90-2.78 (m, 1H, CH2), 2.70-2.57 (m, 1H, CH), 2.42 (t, J = 7.9
Hz, 1H, CH2), 2.10-1.91 (m, 1H, CH), 1.31-1.09 (m, 6H,CH(CH3)2). 13C NMR (101 MHz,
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DMSO-d6) δ 171.40, 167.44, 165.65, 152.69, 148.76, 148.24, 141.33, 139.74, 138.43, 129.61,
128.54, 126.67, 122.05, 119.18, 116.08, 111.11, 110.64, 100.18, 72.15, 55.92, 54.66, 51.47, 46.70,
39.05, 38.53, 35.99, 23.86, 22.16; ESI-MS for C33H38N2O8 m/z: 590.94 [M+H]+.
4.5 Cell Culture
RAW264.7 cells (ATCC, Rockville, MD, USA) were maintained in RPMI 1640 medium
supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 10% FBS Cells were
grown at 37 ℃ and 5% CO2 in humidified air.
4.6 Assay for TNF-α inhibition
TNF-α production was measured by Enzyme-Linked Immuno-Sorbent Assay (ELISA). In
brief, RAW264.7 macrophage cells (1×105 cells/well) were plated in 96-well plates, until the 90%
cells fusion, then cells were starved by being cultured in serum free medium for another 2 h to
eliminate FBS influence. The cells were then treated with compounds for 2 h before exposure to
200 ng/ml LPS for 8 h. The culture medium was used to assay the cytokine production with mouse
ELISA kit (TNF-α; BOSTER Biological Technology co.ltd, China) according to manufacturer's
instructions.
4.7 Assay for NO inhibition
NO production was quantified by nitrite accumulation in the culture medium using the Griess
reaction. Briefly, RAW264.7 cells were pre-treated with vehicle or 7o (0-20uM dose range) for 2
h, and then stimulated with or without LPS (200 ng/ml) for 8 h. At the indicated time points, the
cells were harvested and washed, and cell lysates were prepared. The lysates were mixed with
Griess reagent for 30 min measured at 540 nm. The cells treated with diluent were used as
negative controls for the background levels of nitrite production, while sodium nitrite at different
concentrations was used as the positive controls for the establishment of a standard curve.
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4.8 Western blotting
The cell in 96-well plates were treated as described above and then stimulated with LPS
(200ng/mL) for 24h. The cells were harvested and lysed in an extraction lysis buffer (Beyotime
Biotechnology, Shanghai, China) containing protease inhibitors. The protein concentration was
determined using a BCA protein assay kit (Thermo Scientific, 23227). The whole cell lysates were
separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to
a nitrocellulose membrane. Each membrane was incubated with Tris-buffered saline (pH 7.6,
containing 0.05% Tween-20 and 5% non-fat milk). The nitrocellulose membrane was incubated
with the primary antibody against p-JNK1/2, JNK1/2, p-p38, p38, p-ERK1/2, ERK1/2, IκBa,
NF-κB p65, COX-2, iNOS or β-Actin (Abcam, Cambridge, UK). Immunoreactive bands were
detected by incubating with horseradish peroxidase-conjugated secondary antibodies, and
visualised using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA).
4.9 Animals
The title compound was evaluated for its anti-inflammatory activity using xylene-induced ear
swelling in mice. In brief, Dexamethasone was administered orally at a dose level of 0.5 mg/kg
and other compounds were administered orally at a dose level of 5 mg/kg(suspended in 0.5% Na
CMC given p. o.) every day for a total of six days. DNFB (10 µL of 1% (w/v)) was given for
sensitization and challenged with DNFB on the right ear 6d later. Round ear piece of 9mm
diameter was cut from both right and left ear 24 h after challenge, and weighted separately. Degree
of ear swelling was expressed as the weight difference of right and left ear piece and was
determined using the following formula:
Ear swelling = right ear weight - left ear weight
Inhibition% = (average swelling degree of the model control group - average swelling degree
of the administration group) / average swelling degree of the model control group × 100%.
4.10 Statistical analysis
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Results are expressed as mean standard deviation (SD) and were analysis statistically with
analysis of variance (ANOVE), and the Tukey method were assessed differences between groups.
A value of p < 0.05 is considered to be statistically significant.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
The authors thank the support of the National Health Commission of "Major New Drug Creation" Scientific and
Technological Major Project (No. 2018zx09739- 001).
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