Synthesis of New Tricyclic and Tetracyclic Fused Coumarin Sulfonate Derivatives and Their Inhibitory Effects on LPS-Induced Nitric Oxide and PGE2 Productions in RAW 264.7 Macrophages: Part 2

Article abstract of DOI:10.1002/ardp.201600243

The synthesis of a new series of 21 fused coumarin derivatives is described, and the biological evaluation of their in vitro antiinflammatory effects as inhibitors of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in RAW 264.7 macrophages. The target compounds 1a–u were first tested for cytotoxicity to determine a non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE₂ production would not be caused by cytotoxicity. Compounds 1f and 1p were the most active PGE₂ inhibitors with IC₅₀ values of 0.89 and 0.95 μM, respectively. Western blot and cell-free COX-2 screening showed that their effects were due to inhibition of both COX-2 protein expression and COX-2 enzyme activity. Their IC₅₀ values against the COX-2 enzyme were 0.67 and 0.85 μM, respectively, which is more potent than etoricoxib. The selectivity indexes of compounds 1f and 1p against COX-2 compared to COX-1 were 41.1 and 42.5, respectively. Compound 1f showed strong inhibitory effects at 5 μM concentration on COX-2 mRNA expression in LPS-induced RAW 264.7 macrophages. Moreover, the tricyclic compounds 1l and 1n as well as the tetracyclic analog 1u were the most potent NO inhibitors, with one-digit micromolar IC₅₀ values. They showed dose-dependent inhibition of inducible nitric oxide synthase (iNOS) protein expression. The tetracyclic derivative 1u was the most potent inhibitor of NO production. It also exhibited a strong inhibitory effect on iNOS mRNA expression in LPS-induced RAW 264.7 macrophages.

Full text of DOI:10.1002/ardp.201600243

RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
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Full Paper
Synthesis of New Tricyclic and Tetracyclic Fused Coumarin Sulfonate
Derivatives and Their Inhibitory Effects on LPS-Induced Nitric Oxide
and PGE2 Productions in RAW 264.7 Macrophages: Part 2
Mohammed I. El-Gamal1,2,3, Woo-Seok Lee4,5, Ji-Sun Shin4,5, Chang-Hyun Oh6,7, Kyung-Tae Lee4,5
Jungseung Choi8, Nohsun Myoung8, and Daejin Baek8
,
ꢀ
1
Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates
Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
Faculty of Pharmacy, Department of Medicinal Chemistry, University of Mansoura, Mansoura, Egypt
Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul, Republic of
Korea
Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul,
Republic of Korea
Center for Biomaterials, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea
DepartmentofBiomolecularScience,UniversityofScienceandTechnology(UST), Daejeon,RepublicofKorea
Department of Chemistry, Hanseo University, Seosan, Republic of Korea
2
3
4
5
6
7
8
The synthesis of a new series of 21 fused coumarin derivatives is described, and the biological evaluation
of their in vitro antiinflammatory effects as inhibitors of lipopolysaccharide (LPS)-induced nitric oxide
(NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophages. The target compounds 1a–u
were first tested for cytotoxicity to determine a non-toxic concentration for antiinflammatory screening,
so that the inhibitory effects against NO and PGE2 production would not be caused by cytotoxicity.
Compounds 1f and 1p were the most active PGE2 inhibitors with IC50 values of 0.89 and 0.95 mM,
respectively. Western blot and cell-free COX-2 screening showed that their effects were due to inhibition
of both COX-2 protein expression and COX-2 enzyme activity. Their IC50 values against the COX-2
enzyme were 0.67 and 0.85 mM, respectively, which is more potent than etoricoxib. The selectivity
indexes of compounds 1f and 1p against COX-2 compared to COX-1 were 41.1 and 42.5, respectively.
Compound 1f showed strong inhibitory effects at 5 mM concentration on COX-2 mRNA expression in
LPS-induced RAW 264.7 macrophages. Moreover, the tricyclic compounds 1l and 1n as well as the
tetracyclic analog 1u were the most potent NO inhibitors, with one-digit micromolar IC50 values. They
showed dose-dependent inhibition of inducible nitric oxide synthase (iNOS) protein expression. The
tetracyclic derivative 1u was the most potent inhibitor of NO production. It also exhibited a strong
inhibitory effect on iNOS mRNA expression in LPS-induced RAW 264.7 macrophages.
Keywords: Antiinflammatory / Coumarin sulfonate / COX-2 / NO / PGE2
Received: August 23, 2016; Revised: October 9, 2016; Accepted: October 11, 2016
DOI 10.1002/ardp.201600243
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
Inflammation is a biological symptom that occurs as a normal
response to some external factors such as edema or microbial
infection [1]. Mild and short-term inflammation is not
Correspondence: Dr. Daejin Baek, Department of Chemistry,
Hanseo University, Seosan 356-706, Republic of Korea.
E-mail: djbaek@hanseo.ac.kr
Fax: þ82-41-660-1119
ꢀAdditional correspondence: Dr. Kyung-Tae Lee,
E-mail: ktlee@khu.ac.kr
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considered as a pathological state. But exaggerated and
chronic inflammation requires treatment with antiinflamma-
tory therapeutic agents. The chronic inflammation is concom-
itant to several human diseases such as Alzheimer’s
disease [2], cancer [3], arthritis [4], inflammatory bowel
syndrome [5], and arteriosclerosis [6].
Several coumarin derivatives have been recently reported as
antiinflammatory agents [12–15]. Our group has published the
NO and PGE2 production inhibition effects of a series of tricyclic
cycloalkane-fused coumarin derivatives. A cycloheptane-fused
tricyclic coumarin compound possessing 4-(trifluoromethyl)
benzene sulfonate moiety showed high potency as inhibitor of
NO and PGE2 productions (Fig. 1). Its IC50 values as inhibitor of
NO and PGE2 productions in lipopolysaccharide (LPS)-induced
RAW 264.7 macrophages were 3.84mM and 6.45 nM, respec-
tively [16]. Its promising activity encouraged us to modify the
structure to produce new derivatives with modified ring fused
to the coumarin nucleus, with substitutions on the coumarin
nucleus, positional isomer, or a tetracyclic instead of tricyclic
structure as illustrated in Fig. 1, to explore the effects of these
structural modifications on the activity. Most of these new
derivatives (compounds 1a–n and 1q–t) were tested for
anticancer activity [17, 18]. Herein, the 21 derivatives were
tested for their inhibitory effects against NO and PGE2
productions in LPS-induced RAW 264.7 macrophage cells. The
most promising compounds were further considered at cellular
and molecular levels for more understanding of their redun-
dancy mechanisms of action.
Nitric oxide (NO) is an endothelial-derived relaxation factor
that causes vasodilation. Recent reports have highlighted the
significance of NO-releasing antiinflammatory agents as
cardioprotective and hypotensive agents [7–9]. However, NO
contributes to localized inflammation. During the chronic
inflammatory condition, the inducible nitric oxide synthase
(iNOS) secretes NO as an inflammatory mediator. It causes
vasodilation at the location of inflammation, and this causes
edema [10]. The cyclooxygenase-2 (COX-2) converts the
arachidonic acid into another inflammatory mediator,
PGE2 [11]. So inhibition of NO production through inhibition
of iNOS enzyme activity and/or protein expression can be a
useful avenue for treatment of inflammation. Similarly,
inhibition of PGE2 production via inhibition of COX-2 protein
expression and/or enzyme activity is another potential ap-
proach for inflammation therapy.
Figure 1. Structure of the lead compound [16] and structural modifications to the new derivatives.
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Scheme 1. Reagents and conditions: (i) diethyl carbonate, NaH, benzene, reflux, 90% (3a, n ¼ 1), 85% (3b, n ¼ 2); (ii) (substituted)
resorcinol, CF3COOH, conc. H2SO4, 0°C; rt, 3 h; (iii) appropriate sulfonyl chloride derivative, triethylamine, CH2Cl2, 0°C; rt, 1 h.
compound. Every tested compound will be examined at this
safe dose level for NO and PGE2 production inhibitory effect,
Results and discussion
Chemistry
and any inhibition recorded will not be due to cytotoxicity
against RAW 264.7 cells. The concentrations at which the cell
viability decreased by 20 or 50% (IC80 and IC50, respectively)
are depicted in Table 1. The results showed that most of the
compounds did not decrease the cell viability to 50% by using
up to 100 mM concentration. The IC80 values of the target
compounds were ꢁ10.97 mM, except for compound 1q. So it
was decided to exclude that compound from the next NO and
PGE2 production inhibition screening due to the recorded
cytotoxicity.
The target compounds 1a–u could be successfully prepared
utilizing the synthetic pathways illustrated in Schemes 1–3.
Refluxing cycloheptanone (2a) or cyclooctanone (2b) with
diethyl carbonate and sodium hydride in benzene yielded the
b-keto esters 3a,b as a mixture of keto and enol tauto-
mers [19]. Compounds 3a,b were interacted with (substituted)
resorcinol derivatives in the presence of conc. sulfuric and
trifluoroacetic acids to produce the fused cyclized phenolic
intermediates 4a–f [20]. They were subsequently reacted with
the appropriate sulfonyl chloride analog in the presence of
triethylamine to give the corresponding sulfonate target
compounds 1a–s. Compound 1t was synthesized by the same
procedure but using hydroquinone instead of resorcinol to
form the phenolic intermediate 5 (Scheme 2). The tetracyclic
derivative 1u was also synthesized by the same method but
starting with 2-indanone (6) at the first step to get the b-keto
ester 7 (Scheme 3). All the target compounds were purified by
normal phase column chromatography, and their structures
were confirmed by NMR and LC-MS analyses. Their purities
were confirmed by elemental microanalysis.
The target compounds inhibited LPS-induced NO and PGE2
production in RAW 264.7 macrophages
Based on the MTT cell viability assay, a single-dose concen-
tration was selected for each compound so that any inhibitory
effects on NO and/or PGE2 production would be due to direct
effect(s) on the NO and/or PGE2 releasing pathways, not
because of cytotoxicity. Every compound was tested at this
single-dose concentration for inhibitory effect of various
tested compounds on LPS-induced NO and PGE2 production in
RAW 264.7 macrophages. The inhibition percentage values
are summarized in Table 2.
Biological screening
The tricyclic para-fluorobenzenesulfonate compound 1l and
thetetracyclicanalog 1uwerethemostactive asNOproduction
inhibitors. Compound 1l possesses a cyclooctane ring fused to
the coumarin nucleus, and it was more active than the
corresponding less bulky cycloheptane analog. Moreover,
Cytotoxicity testing
Before conducting the NO and PGE2 production inhibition
screening, it was essential to carry out a cytotoxicity testing in
order to determine a safe and non-toxic concentration of each
Scheme 2. Reagents and conditions: (i) hydroquinone, CF3COOH, conc. H2SO4, 0°C; rt, 60 h; (ii) p-toluenesulfonyl chloride,
triethylamine, CH2Cl2, 0°C; rt, 1 h, 65%.
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Scheme 3. Reagents and conditions: (i) dimethyl carbonate, NaH, benzene, reflux, 3 h, 54%; (ii) resorcinol, CF3COOH, conc. H2SO4,
0°C; rt, 3h; (iii) p-toluenesulfonyl chloride, triethylamine, CH2Cl2, 0°C; rt, 1 h, 35%.
Table 1. Structures of the target compounds 1a–u and their IC50 and IC80 values against RAW 264.7 macrophage cell
viability (50 and 80% viability, respectively).
Compound no.
1a
R1
R2
n
IC50 (mM)a)
>100
IC80 (mM)b)
H
p-(tert-Butyl)
C6H4
1
19.89
1b
1c
1d
1e
1f
1g
1h
1i
H
OMe
OMe
OMe
OMe
OMe
Cl
Cl
Cl
H
p-(F)C6H4
Me
n-Pr
Ph
p-Tolyl
p-(CF3)C6H4
Me
1
1
1
1
1
1
1
1
1
2
>100
>100
>100
>100
>100
>100
89.63
>100
>100
>100
20.60
>100
50.91
12.06
35.65
20.55
30.31
42.95
13.39
26.93
Ph
1j
1k
p-Tolyl
p-(tert-Butyl)
C6H4
1l
H
p-(F)C6H4
2
2
2
2
2
2
2
2
>100
>100
>100
>100
>100
>100
>100
>100
>100
11.00
>100
>100
86.92
35.02
3.12
1m
1n
1o
1p
1q
1r
OMe
OMe
OMe
OMe
OMe
Cl
Me
n-Pr
Ph
p-Tolyl
p-(CF3)C6H4
Ph
15.33
10.97
40.56
1s
1t
Cl
p-Tolyl
1u
53.59
24.25
a) The concentration required to inhibit 50% of the cell viability. b) The concentration required to inhibit 20% of the cell viability.
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Table 2. Inhibitory effects of the target compounds 1a–u
on LPS-induced NO and PGE2 in RAW 264.7 macrophages.
Regarding the effects on PGE2 production in LPS-induced
RAW 264.7 macrophages, several compounds showed near
100% inhibition at the tested concentration. The methoxy-
coumarin derivative 1e was more active than the corres-
ponding unsubstituted coumarin analog (81.85% PGE2
production inhibition at 5 mM concentration) [16]. And the
chlorocoumarin compounds 1j and 1r were also more active
than the corresponding unsubstituted derivatives (37.86 and
50.44% PGE2 inhibitions at 5 mM concentrations) [16]. So these
chloro or methoxy derivatives could be optimal for PGE2
production inhibitory activity of the compounds, and their
effects can be rationalized in the same way discussed above in
case of NO production inhibition. Some cycloheptane fused
derivatives such as 1b, 1d, and 1e showed higher activity than
the corresponding cyclooctane fused analogs 1l, 1n, and 1o,
while opposite result was noticed upon comparing the
activities of compounds 1a and 1k.
% Inhibitiona)
Compound no.
(tested
concentration)
NO production
PGE2 production
1a (10 mM)
1b (10 mM)
1c (10 mM)
1d (10 mM)
1e (5 mM)
1f (25 mM)
1g (10 mM)
1h (10 mM)
1i (25 mM)
1j (5 mM)
1k (10 mM)
1l (10 mM)
1m (50 mM)
1n (100 mM)
1o (10 mM)
1p (25 mM)
1q
23.24 ꢂ 0.89
34.10 ꢂ 0.86
74.17 ꢂ 1.08
45.83 ꢂ 0.33
53.32 ꢂ 1.73
70.99 ꢂ 1.72
78.78 ꢂ 0.62
48.33 ꢂ 0.77
59.96 ꢂ 1.32
59.32 ꢂ 0.98
26.97 ꢂ 0.68
91.18 ꢂ 1.45
63.66 ꢂ 0.99
86.93 ꢂ 0.48
46.12 ꢂ 0.56
65.65 ꢂ 1.08
NTb)
23.33 ꢂ 0.28
98.28 ꢂ 0.82
91.44 ꢂ 1.08
98.35 ꢂ 0.12
97.00 ꢂ 0.07
99.48 ꢂ 0.30
90.70 ꢂ 0.11
84.53 ꢂ 0.18
98.50 ꢂ 0.15
81.44 ꢂ 0.89
41.11 ꢂ 0.98
94.94 ꢂ 1.95
88.39 ꢂ 1.23
98.80 ꢂ 0.07
81.32 ꢂ 0.85
98.65 ꢂ 0.01
NTb)
The most active compounds against both NO and PGE2
productions were selected for further investigation at lower
doses. The six selected compounds 1f, 1i, 1l, 1n, 1p, and 1u
were tested at 1, 5, or 10 mM concentrations against NO
production, and at 0.01, 0.1, or 1 mM concentrations against
PGE2 production.
In case of LPS-induced NO production inhibition, the
cyclooctane-fused compounds 1l and 1n, as well as the
indene-fused tetracyclic derivative 1u showed the best results.
Compound 1p was more active than the corresponding
cycloheptane fused analog 1f. These reinforce the previously
mentioned assumption that the bulkier the ring fused to the
coumarin nucleus, the higher the NO inhibitory effect.
Concerning LPS-induced PGE2 production inhibition, the
highest activity was encountered with compounds 1f and 1p.
Both of them possess methoxycoumarin nucleus and para-
toluenesulfonate side chain. So both moieties could be
optimal for appropriate affinity at the receptor site.
1r (5 mM)
1s (5 mM)
1t (10 mM)
1u (10 mM)
L-NIL (20 mM)
NS398 (3 mM)
56.69 ꢂ 2.17
61.25 ꢂ 1.35
28.40 ꢂ 0.89
79.69 ꢂ 0.27
46.73
90.07 ꢂ 1.95
82.44 ꢂ 0.72
38.90 ꢂ 0.68
98.20 ꢂ 0.09
–
–
94.34
a) Values represent means ꢂ SD of three independent experi-
b)
ments. Not tested because of low IC80 value (Table 1).
compound 1u contains an indene motif fused with the
coumarin ring, so the bulkier ring fused to the coumarin
nucleus might be more favorable for NO inhibitory effect.
Compound 1e with methoxy group on the coumarin
nucleus was more active than the corresponding unsubsti-
tuted derivative previously reported by our group (0% NO
production inhibition at 20 mM concentration) [16]. Further-
more, the chlorocoumarin derivatives 1j, 1r, and 1s exhibited
higher activity than the corresponding unsubstituted couma-
rin analogs (37.45, 15.86, and 32.25% NO inhibitions at 20 mM
concentrations) [16]. So the chloro or methoxy groups on the
coumarin nucleus might contribute to higher affinity to the
receptor site through electronic and/or steric effects. More-
over, compound 1t was less active as NO production inhibitor
than the corresponding positional isomer reported in part 1 of
this study (37.45% NO production inhibition at 20 mM
concentration) [16]. So the site of substitution in compound
1t might not provide the proper orientation at the receptor
site, and hence deteriorated the affinity and potency. This
non-promising result of compound 1t discouraged us to
prepare more similar derivatives.
The IC50 values of compounds 1l, 1n, and 1u as inhibitors of
NO production, and the IC50 values of compounds 1f and 1p as
PGE2 production inhibitors were further calculated and
summarized in Table 3. The bulkiest tetracyclic compound 1u
was the most potent NO production inhibitor. It showed higher
Table 3. IC50 values of the most active compounds as
inhibitors of NO and PGE2 productions in LPS-induced
RAW 264.7 macrophages.
IC50 (mM)
Compound no.
NO production
PGE2 production
1f
1l
1n
1p
1u
NDa)
3.11
4.76
NDa)
2.95
0.89
NDa)
NDa)
0.95
NDa)
a) Not determined.
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potency than the previously reported tricyclic coumarin
derivatives (its IC50 value was found as 2.95mM while that
of the most potent previously reported lead compound
was 3.84 mM)[16]. Inaddition, themethoxycoumarinderivative
1n was more potent than the corresponding unsubstituted
coumarin derivative which showed IC50 value of 10 mM [16].
The para-fluorobenzenesulfonate compound 1l exhibited
superior potency compared to the corresponding para-(tri-
fluoromethyl)benzenesulfonate and para-toluenesulfonate
analogs (IC50 ¼ 75.32 and 40.69 mM, respectively). In addition,
compound 1f possessing cycloheptane-fused ring was slightly
more potent than the corresponding cyclooctane analog 1p as
PGE2 production inhibitors.
expression in a dose-dependent way. So the PGE2 inhibitors
and NO inhibitors could show their effects through inhibition
of COX-2 and iNOS protein expressions in a dose-dependent
manner.
COX enzyme inhibition
Cell-free COX-2 enzyme assay was conducted for com-
pounds 1f and 1p. The IC50 values are summarized in
Table 4. The obtained results were compared to those of
etoricoxib, a selective COX-2 inhibitor [21]. Both com-
pounds showed submicromolar IC50 results, and they were
more potent than etoricoxib. So both compounds 1f and 1p
could inhibit the LPS-induced PGE2 production due to dual
effects: inhibition of COX-2 protein expression and COX-2
enzyme inhibition. The methoxycoumarin analog 1f was
more potent than the corresponding unsubstituted couma-
rin derivative as COX-2 inhibitor [17]. Both compounds
were further tested against COX-1 enzyme to investigate
their selectivity, the IC50 values are also shown in Table 4.
Compounds 1f and 1p were 41.1 and 42.5 times, respec-
tively, more selective toward COX-2 than COX-1. So PGE2
production inhibition could be due to selective inhibition of
COX-2 isozyme.
Western blotting
For the purpose of deep understanding of the molecular
mechanism of action of the target compounds, the most
promising compounds were tested for inhibitory effects on
COX-2 and iNOS protein expressions in LPS-induced RAW
264.7 macrophages by Western blot (Fig. 2). Compounds 1f
and 1p showed dose-dependent inhibition of the COX-2
expression, and compound 1p was more active. Moreover,
compounds 1l, 1n, and 1u inhibited the iNOS protein
Figure 2. Inhibitory effects of compounds 1f and 1p on LPS-induced COX-2 protein expression in RAW 264.7 macrophages (A), and
inhibitory effects of compounds 1l, 1n, and 1u on LPS-induced iNOS protein expression in RAW 264.7 macrophages (B). The cellular
lysates were prepared from the pretreated with/without the tested compounds for 1 h and then with LPS (10 ng/mL) for 24 h. Total
cellular proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and detected with specific iNOS and COX-2
antibodies. b-Actin was used as an internal control.
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Table 4. IC50 values of compounds 1f, 1p, and etoricoxib
against COX-2 and COX-1 enzymes in cell-free enzymatic
assays.
IC50 (mM)a)
Compound
COX-2
COX-1
1f
1p
Etoricoxib
0.67 ꢂ 0.08
0.85 ꢂ 0.12
1.10 ꢂ 0.09
27.52 ꢂ 0.76
36.15 ꢂ 1.17
–
a) Values represent means ꢂ SD of three independent
experiments.
Figure 4. Effect of compound 1f on COX-2 mRNA protein
expression in LPS-induced RAW 264.7 macrophages.
mRNA expression assay
The most potent NO production inhibitor 1u was tested for its
effect on iNOS mRNA expression in LPS-induced RAW 264.7
macrophages. Similarly, the most potent COX-2 inhibitor 1f
was tested for COX-2 mRNA expression inhibition. The results
are illustrated in Figs. 3 and 4. Compound 1u strongly
inhibited the iNOS mRNA expression, and compound 1f
inhibited COX-2 mRNA expression only at 5 mM.
was found that bulkier rings fused to the coumarin nucleus
were more favorable for activity. The three compounds,
especially the tetracyclic analog 1u, was more potent than
the previously reported tricyclic coumarin derivatives [16].
This study led to discovery of another two derivatives, 1f and
1p, as potential inhibitors of PGE2 production through
inhibition of both COX-2 protein expression and COX-2
enzymatic activity. Compound 1f also showed inhibitory
effect on COX-2 mRNA expression at 5 mM concentration.
Both compounds were more potent than etoricoxib. They
showed selective inhibition of COX-2 enzyme than COX-1.
And both of them possess methoxycoumarin and para-
toluenesulfonate moieties which could be favorable for
appropriate affinity and potency. The cycloheptane ring was
more suitable for PGE2 production inhibition than the
bulkier cyclooctane or indene rings. Compound 1t was less
active than the corresponding positional isomers, so its site
of substitution with the aryl sulfonate was improper. And
the chloro and methoxy substituents on the coumarin
nucleus generally enhanced the activity compared with
the corresponding unsubstituted coumarin derivatives.
Conclusion
We have designed and synthesized
a new series of
(substituted) fused coumarin derivatives as extension of
our previously reported series, and tested their biological
effects as inhibitors of LPS-induced NO and PGE2 productions
in RAW 264.7 macrophages. We could obtain three promis-
ing NO production inhibitors, 1l, 1n, and 1u, which worked
through inhibition of iNOS protein expression. Compound
1u exerted an additional iNOS mRNA expression inhibition. It
Experimental
Chemistry
General
The target compounds were purified by column chromatog-
raphy using silica gel (230–400 mesh, 0.040–0.063 mm) and
hexane/ethyl acetate (technical grade). After purification,
they were analyzed by 1H NMR and CMR using a Bruker
Avance 400 or 300 spectrometer. Mass spectra (MS) were
obtained by LC-MS analysis. All solvents and reagents were
commercially available and used as such.
1
Representative H NMR, 13C NMR, and MS spectra and the
InChI codes of the investigated compounds are provided as
Supporting Information.
Figure 3. Effect of compound 1u on iNOS mRNA protein
expression in LPS-induced RAW 264.7 macrophages.
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Synthesis of ethyl 2-oxocycloheptanecarboxylate (3a) and
ethyl 2-oxocyclooctanecarboxylate (3b)
They were synthesized through the procedure reported in the
100 MHz) d 161.5, 152.3, 148.4, 146.5, 139.7, 129.6, 119.2,
113.3, 107.0, 56.7, 38.8, 31.8, 28.4, 26.9, 25.5, 24.9; LC-MS:
339.0 (Mþþ1); elemental analysis (C16H18O6S): calculated C:
56.79%, H: 5.36%, S: 9.47%; found: C: 56.83%, H: 5.30%,
S: 9.60%.
literature [16, 17, 19].
Synthesis of the phenolic intermediates 4a–f
They were prepared through the procedure reported in the
literature [16–19].
2-Methoxy-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]-
chromen-3-yl propane-1-sulfonate (1d)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 10:1 v/v, then switching to hexane/ethyl
acetate 6:1 v/v); yield: 95%; 1H NMR (CDCl3, 400 MHz) d 7.29 (s,
1H), 7.16 (s, 1H), 3.96 (s, 3H), 3.35–3.31 (m, 2H), 2.94–2.89 (m,
4H), 2.08–2.00 (m, 2H), 1.95–1.91 (m, 2H), 1.73–1.67 (m, 2H),
1.64–1.58 (m, 2H), 1.13 (t, 3H, J ¼ 7.5 Hz); 13C NMR (CDCl3,
100 MHz) d 161.6, 152.5, 148.5, 146.5, 139.8, 129.4, 119.0,
113.1, 107.0, 56.7, 31.8, 28.4, 26.9, 25.5, 24.9, 17.4, 12.9; LC-
MS: 367.0 (Mþþ1); elemental analysis (C18H22O6S): calculated
C: 59.00%, H: 6.05%, S: 8.75%; found: C: 58.65%, H: 6.31%, S:
8.53%.
Synthesis of the target sulfonate compounds 1a–s
To a solution of the appropriate hydroxyl compound 4a–f
(0.2 mmol) in dry methylene chloride (10 mL), triethylamine
(0.033 mL, 0.4 mmol) was added at 0°C. A solution of the
appropriate sulfonyl chloride derivative (0.22 mmol) in dry
methylene chloride (5 mL) was added dropwise to the
reaction mixture at the same temperature. The reaction
mixture was stirred at room temperature for 1 h. The reaction
mixture was washed with saline (3 ꢃ 10 mL) and dried using
anhydrous magnesium sulfate.
6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl
4-(tert-butyl)benzenesulfonate (1a)
2-Methoxy-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]-
chromen-3-yl benzenesulfonate (1e)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 20:1 v/v, then switching to hexane/ethyl
acetate 15:1 v/v); yield: 94%; 1H NMR (CDCl3, 400 MHz) d 7.79
(d, 2H, J ¼ 8.5 Hz), 7.64 (d, 1H, J ¼ 8.9 Hz), 7.56 (d, 2H,
J ¼ 8.5 Hz), 7.08 (dd, 1H, J ¼ 8.8 Hz, 2.1 Hz), 6.90 (d, 1H,
J ¼ 2.2 Hz), 2.91 (dt, 4H, J ¼ 10.6 Hz, 9.8 Hz), 1.90 (q, 2H,
J ¼ 6.0 Hz), 1.69–1.59 (m, 4H), 1.36 (s, 9H); 13C NMR (CDCl3,
100 MHz) d 161.5, 158.8, 152.9, 152.8, 150.7, 132.2, 128.9,
128.3, 126.4, 125.1, 118.8, 118.5, 110.7, 35.4, 31.9, 31.0, 28.2,
26.8, 25.5, 24.9; LC-MS: 427.2 (Mþþ1); elemental analysis
(C24H26O5S): calculated C: 67.58%, H: 6.14%, S: 7.52%; found:
C: 67.80%, H: 6.03%, S: 7.57%.
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 7:1 v/v, then switching to hexane/ethyl
acetate 5:1 v/v); yield: 92%; 1H NMR (CDCl3, 300 MHz) d
7.92–7.89 (m, 2H), 7.73–7.68 (m, 1H), 7.58–7.53 (m, 3H), 7.04 (d,
2H, J ¼ 3.0 Hz), 3.70 (s, 3H), 2.91–2.88 (m, 4H), 1.90 (q, 2H,
J ¼ 6.0 Hz), 1.70–1.59 (m, 4H); 13C NMR (CDCl3, 75 MHz) d 161.7,
152.5, 148.9, 146.2, 140.0, 136.0, 134.4, 129.4, 129.1, 128.5,
119.1, 112.6, 106.8, 56.4, 31.8, 28.4, 26.9, 25.5, 24.9; LC-MS:
401.0 (Mþþ1); elemental analysis (C21H20O6S): calculated C:
62.99%, H: 5.03%, S: 8.01%; found: C: 62.71%, H: 4.90%, S:
8.12%.
2-Methoxy-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]-
chromen-3-yl 4-methylbenzenesulfonate (1f)
6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl
4-fluorobenzenesulfonate (1b)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 10:1 v/v, then switching to hexane/ethyl
acetate 6:1 v/v); yield: 96%; 1H NMR (CDCl3, 300 MHz) d 7.80–
7.74 (m, 2H), 7.36–7.25 (m, 2H), 7.06–6.94 (m, 2H), 3.76 (s, 3H),
2.91–2.88 (m, 4H), 2.48 (s, 3H), 1.89 (t, 2H, J ¼ 6.0 Hz), 1.68–1.61
(m, 4H); 13C NMR (CDCl3, 75 MHz) d 162.3, 161.7, 152.6, 149.0,
146.2, 145.7, 140.2, 133.0, 129.8, 129.4, 128.5, 119.0, 112.4,
106.9, 56.5, 31.9, 28.4, 26.9, 25.5, 24.9, 21.8; LC-MS: 415.0
(Mþþ1); elemental analysis (C22H22O6S): calculated C: 63.75%,
H: 5.35%, S: 7.74%; found: C: 63.54%, H: 5.43%, S: 7.81%.
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 30:1 v/v, then switching to hexane/ethyl
acetate 25:1 v/v); yield: 90%; 1H NMR (CDCl3, 400 MHz) d 7.91–
7.88 (m, 2H), 7.64 (d, 1H, J ¼ 8.8 Hz), 7.26–7.22 (m, 2H), 7.03
(dd, 1H, J ¼ 8.8 Hz, 2.3 Hz), 6.89 (d, 1H, J ¼ 2.2 Hz), 2.94–2.88
(m, 4H), 1.90 (q, 2H, J ¼ 6.0 Hz), 1.69–1.60 (m, 4H); 13C NMR
(CDCl3, 100 MHz) d 161.4, 152.8, 152.7, 150.4, 131.4, 131.3,
129.1 (d, JCF ¼ 9.12 Hz), 125.3, 119.0, 118.3, 117.0, 116.8, 110.7,
31.8, 28.2, 26.8, 25.4, 24.8; elemental analysis (C20H17FO5S):
calculated C: 61.85%, H: 4.41%, S: 8.25%; found: C: 61.60%, H:
4.50%, S: 8.35%.
2-Methoxy-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]-
chromen-3-yl 4-(trifluoromethyl)benzenesulfonate (1g)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 10:1 v/v, then switching to hexane/ethyl
acetate 6:1 v/v); yield: 87%; 1H NMR (CDCl3, 400 MHz) d 8.05 (d,
2H, J ¼ 7.7 Hz), 7.83 (d, 2H, J ¼ 7.6 Hz), 7.16 (s, 1H), 7.04 (s, 1H),
3.64 (s, 3H), 2.89 (brs, 4H), 1.91 (d, 2H, J ¼ 4.0 Hz), 1.69–1.61 (m,
4H); 13C NMR (CDCl3, 100 MHz) d 161.5, 152.3, 148.4, 146.3,
139.4, 136.0, 135.7, 129.7, 129.1, 126.2, 126.1, 119.3, 112.8,
2-Methoxy-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]-
chromen-3-yl methanesulfonate (1c)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 10:1 v/v, then switching to hexane/ethyl
acetate 3.5:1 v/v); yield: 85%; 1H NMR (CDCl3, 400 MHz) d 7.30
(s, 1H), 7.17 (s, 1H), 3.79 (s, 3H), 3.25 (s, 3H), 2.94–2.90 (m, 4H),
1.95–1.90 (m, 2H), 1.73–1.62 (m, 4H); 13C NMR (CDCl3,
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Antiinflammatory Coumarin Derivatives
Archiv der Pharmazie
106.7, 56.1, 31.8, 28.4, 26.9, 25.4, 24.8; LC-MS: 469.1 (Mþþ1);
elemental analysis (C22H19F3O6S): calculated C: 56.41%, H:
4.09%, S: 6.84%; found: C: 56.27%, H: 4.22%, S: 6.60%.
6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-
3-yl 4-fluorobenzenesulfonate (1l)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 30:1 v/v); yield: 89%; 1H NMR (CDCl3,
400 MHz) d 7.92–7.89 (m, 2H), 7.58 (d, 1H, J ¼ 8.8 Hz), 7.24–
7.22 (m, 2H), 7.05 (dd, 1H, J ¼ 2.2 Hz, 8.7 Hz), 6.88 (d, 1H,
J ¼ 2.2 Hz), 2.97 (t, 2H, J ¼ 6.5 Hz), 2.81 (t, 2H, J ¼ 6.1 Hz),
1.81–1.73 (m, 4H), 1.53–1.44 (m, 4H); 13C NMR (CDCl3,
100 MHz) d 160.8, 153.0, 150.2, 149.5, 131.4, 131.3, 127.3 (d,
JCF ¼ 9.6 Hz), 125.5, 118.5, 118.4, 117.0, 116.8, 110.8, 29.6,
29.1, 26.9, 26.6, 26.4, 25.9; elemental analysis (C21H19FO5S):
calculated C: 62.68%, H: 4.76%, S: 7.97%; found: C: 62.73%,
H: 4.93%, S: 7.67%.
2-Chloro-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]-
chromen-3-yl methanesulfonate (1h)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 10:1 v/v, then switching to hexane/ethyl
acetate 3.5:1 v/v); yield: 85%; 1H NMR (CDCl3, 300 MHz) d 7.76
(s, 1H), 7.43 (s, 1H), 3.30 (s, 3H), 2.92–2.90 (m, 4H), 1.93–1.91
(m, 2H), 1.70–1.61 (m, 4H); 13C NMR (CDCl3, 75 MHz) d 162.3,
151.6, 151.3, 145.7, 130.4, 125.6, 122.6, 120.0, 113.0, 39.1, 31.8,
28.3, 27.0, 25.4, 24.8; LC-MS: 343.1 (Mþþ1); elemental analysis
(C15H15ClO5S): calculated C: 52.56%, H: 4.41%, S: 9.35%;
found: C: 52.18%, H: 4.62%, S: 9.57%.
2-Methoxy-6-oxo-7,8,9,10,11,12-hexahydro-6H-
cycloocta[c]chromen-3-yl methanesulfonate (1m)
2-Chloro-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]-
chromen-3-yl benzenesulfonate (1i)
It was purified by flash column chromatography (silica
gel, hexane/ethyl acetate 10:1 v/v, then switching to
hexane/ethyl acetate 3.5:1 v/v); yield: 85%; 1H NMR (CDCl3,
300 MHz) d 7.30 (s, 1H), 7.11 (s, 1H), 3.96 (s, 3H), 3.26 (s, 3H),
2.98 (t, 2H, J ¼ 6.0 Hz), 2.82 (t, 2H, J ¼ 6.0 Hz), 1.84–1.74
(m, 4H), 1.53–1.44 (m, 4H); 13C NMR (CDCl3, 75 MHz) d 161.0,
149.0, 148.5, 146.7, 139.6, 127.9, 118.7, 113.4, 107.1, 56.7,
38.8, 29.7, 29.1, 27.0, 26.8, 26.6, 25.9; LC-MS: 353.0 (Mþþ1);
elemental analysis (C17H20O6S): calculated C: 57.94%, H:
5.72%, S: 9.10%; found: C: 57.69%, H: 5.87%, S: 8.90%.
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 13:1 v/v, then switching to hexane/ethyl
acetate 10:1 v/v); yield: 85%; 1H NMR (CDCl3, 300 MHz) d 7.94
(d, 2H, J ¼ 6.0 Hz), 7.86–7.66 (m, 4H), 7.18 (s, 1H), 2.91–2.89 (m,
4H), 1.92–1.90 (m, 2H), 1.66–1.62 (m, 6H); 13C NMR (CDCl3,
75 MHz) d 161.0, 151.8, 151.1, 146.2, 135.4, 134.9, 130.2, 129.5,
129.4, 128.6, 125.5, 123.4, 119.7, 112.5, 31.8, 28.3, 27.0, 25.4,
24.8; LC-MS: 405.0 (Mþþ1); elemental analysis (C20H17ClO5S):
calculated C: 59.33%, H: 4.23%, S: 7.92%; found: C: 59.20%, H:
4.40%, S: 7.74%.
2-Methoxy-6-oxo-7,8,9,10,11,12-hexahydro-6H-
cycloocta[c]chromen-3-yl propane-1-sulfonate (1n)
It was purified by flash column chromatography (silica
gel, hexane/ethyl acetate 10:1 v/v, then switching to
hexane/ethyl acetate 6:1 v/v); yield: 91%; 1H NMR (CDCl3,
400 MHz) d 7.29 (s, 1H), 7.10 (s, 1H), 3.95 (s, 3H), 3.34 (t, 2H,
J ¼ 7.7 Hz), 2.97 (t, 2H, J ¼ 6.4 Hz), 2.80 (t, 2H, J ¼ 6.0 Hz),
2.10–1.97 (m, 2H), 1.86–1.73 (m, 4H), 1.53–1.44 (m, 4H), 1.12
(t, 3H, J ¼ 6.9 Hz); 13C NMR (CDCl3, 100 MHz) d 161.0, 149.2,
146.6, 139.6, 127.6, 121.8, 118.4, 113.1, 107.1, 56.7, 53.6,
29.7, 29.0, 26.9, 26.7, 26.5, 25.8, 17.4, 12.9; LC-MS: 381.0
(Mþþ1); elemental analysis (C19H24O6S): calculated C:
59.98%, H: 6.36%, S: 8.43%; found: C: 59.67%, H: 6.62%,
S: 8.30%.
2-Chloro-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]-
chromen-3-yl 4-methylbenzenesulfonate (1j)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 13:1 v/v, then switching to hexane/ethyl
acetate 10:1 v/v); yield: 86%; 1H NMR (CDCl3, 300 MHz) d 7.81
(d, 2H, J ¼ 9.0 Hz), 7.66 (s, 1H), 7.37 (d, 2H, J ¼ 9.0 Hz), 7.15 (s,
1H), 2.89 (q, 4H, J ¼ 6.0 Hz), 2.48 (s, 3H), 1.95–1.87 (m, 2H),
1.71–1.57 (m, 4H); 13C NMR (CDCl3, 75 MHz) d 161.0, 151.8,
151.0, 146.4, 146.3, 132.4, 130.1, 129.9, 128.6, 128.5, 125.5,
123.5, 119.6, 112.4, 31.8, 28.3, 27.0, 25.4, 24.8, 21.8; LC-MS:
418.9 (Mþþ1); elemental analysis (C21H19ClO5S): calculated C:
60.21%, H: 4.57%, S: 7.65%; found: C: 60.14%, H: 4.82%, S:
7.48%.
2-Methoxy-6-oxo-7,8,9,10,11,12-hexahydro-6H-
6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-
3-yl 4-(tert-butyl)benzenesulfonate (1k)
cycloocta[c]chromen-3-yl benzenesulfonate (1o)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 10:1 v/v, then switching to hexane/
ethyl acetate 5:1 v/v); yield: 87%; 1H NMR (CDCl3, 400 MHz) d
7.93 (d, 2H, J ¼ 7.5 Hz), 7.71 (t, 1H, J ¼ 7.5 Hz), 7.59–7.55 (m,
2H), 7.00 (brs, 2H), 3.72 (s, 3H), 2.94 (t, 2H, J ¼ 6.3 Hz), 2.81 (t,
2H, J ¼ 5.9 Hz), 1.81–1.73 (m, 4H), 1.53–1.44 (m, 4H);
13C NMR (CDCl3, 100 MHz) d 161.1, 149.2, 149.0, 146.4,
139.9, 134.4, 129.3, 129.1, 128.9, 128.5, 127.7, 118.5, 112.6,
107.0, 56.4, 29.7, 29.0, 26.9, 26.7, 26.5, 25.9; LC-MS: 415.0
(Mþþ1); elemental analysis (C22H22O6S): calculated C:
63.75%, H: 5.35%, S: 7.74%; found: C: 63.52%, H: 5.58%,
S: 7.47%.
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 20:1 v/v, then switching to hexane/ethyl
acetate 15:1 v/v); yield: 95%; 1H NMR (CDCl3, 400 MHz) d 7.81
(d, 2H, J ¼ 8.5 Hz), 7.59–7.56 (m, 3H), 7.07 (dd, 1H, J ¼ 8.8 Hz,
2.2 Hz), 6.91 (d, 1H, J ¼ 2.2 Hz), 2.96 (t, 2H, J ¼ 6.4 Hz), 2.79
(t, 2H, J ¼ 6.0 Hz), 1.83–1.69 (m, 4H), 1.52–1.43 (m, 4H), 1.36
(s, 9H); 13C NMR (CDCl3, 75 MHz) d 160.9, 158.8, 153.0, 150.5,
149.7, 132.2, 128.3, 127.1, 126.5, 125.4, 118.6, 118.3, 110.7,
35.4, 31.0, 29.7, 29.1, 26.9, 26.6, 26.4, 25.9; elemental analysis
(C25H28O5S): calculated C: 68.16%, H: 6.41%, S: 7.28%; found:
C: 67.84%, H: 6.60%, S: 7.13%.
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M. I. El-Gamal et al.
Archiv der Pharmazie
2-Methoxy-6-oxo-7,8,9,10,11,12-hexahydro-6H-
6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-2-yl
4-methylbenzenesulfonate (1t)
It was synthesized by the same method used for synthesis
cycloocta[c]chromen-3-yl 4-methylbenzenesulfonate (1p)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 10:1 v/v, then switching to hexane/ethyl
acetate 5:1 v/v); yield: 90%; 1H NMR (CDCl3, 400 MHz) d 7.80 (d,
2H, J ¼ 8.0 Hz), 7.68–7.66 (m, 1H), 7.36–7.33 (m, 2H), 7.02 (s,
1H), 6.94 (s, 1H), 3.76 (s, 3H), 2.95 (t, 2H, J ¼ 6.0 Hz), 2.81 (t, 2H,
J ¼ 5.6 Hz), 2.48 (s, 3H), 1.81–1.73 (m, 4H), 1.53–1.44 (m, 4H);
13C NMR (CDCl3, 100 MHz) d 161.1, 149.3, 145.6, 140.0, 133.1,
129.8, 129.5, 127.5, 122.0, 118.4, 112.4, 107.0, 56.5, 29.7, 29.0,
26.9, 26.7, 26.5, 25.9, 21.8; LC-MS: 429.2 (Mþþ1); elemental
analysis (C23H24O6S): calculated C: 64.47%, H: 5.65%, S: 7.48%;
found: C: 64.35%, H: 5.76%, S: 7.61%.
of compounds 1a–s starting with compound
5 and
p-toluenesulfonyl chloride. Compound 1t was purified by
column chromatography (silica gel, hexane/ethyl acetate
10:1 v/v, then switching to hexane/ethyl acetate 7:1 v/v);
yield: 65%; 1H NMR (DMSO-d6, 400 MHz) d 7.93 (d, 1H,
J ¼ 18.4 Hz), 7.79 (d, 2H, J ¼ 15.0 Hz), 7.49 (d, 2H, J ¼ 15.0
Hz), 7.10 (s, 1H), 6.99 (d, 1H, J ¼ 9.0 Hz), 2.98–2.76 (m, 4H),
2.44 (s, 3H), 1.86–1.79 (m, 2H), 1.61–1.46 (m, 4H); 13C NMR
(DMSO-d6, 100 MHz) d 161.0, 153.6, 152.7, 150.4, 146.7,
131.5, 130.8, 128.9, 127.0, 126.8, 119.1, 119.0, 110.8, 31.8,
27.8, 26.6, 25.4, 21.8, 21.5; elemental analysis (C21H20O5S):
calculated C: 65.61%, H: 5.24%, S: 8.34%; found: C: 65.43%,
H: 5.45%, S: 8.20%.
2-Methoxy-6-oxo-7,8,9,10,11,12-hexahydro-6H-
cycloocta[c]chromen-3-yl 4-(trifluoromethyl)-
benzenesulfonate (1q)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 10:1 v/v, then switching to hexane/ethyl
acetate 6:1 v/v); yield: 85%; 1H NMR (CDCl3, 400 MHz) d 8.07 (d,
2H, J ¼ 8.1 Hz), 7.84 (d, 2H, J ¼ 8.2 Hz), 7.14 (s, 1H), 6.99 (s, 1H),
3.65 (s, 3H), 2.94 (t, 2H, J ¼ 6.0 Hz), 2.81 (t, 2H, J ¼ 5.8 Hz), 1.81–
1.74 (m, 4H), 1.53–1.44 (m, 4H); 13C NMR (CDCl3, 100 MHz) d
160.9, 149.0, 148.5, 146.4, 139.7, 139.4, 136.1, 135.7, 129.1,
128.0, 126.2, 118.8, 112.8, 106.8, 56.2, 29.6, 29.0, 26.9, 26.7,
26.6, 25.8; LC-MS: 483.1 (Mþþ1); elemental analysis
(C23H21F3O6S): calculated C: 57.26%, H: 4.39%, S: 6.64%;
found: C: 56.98%, H: 4.61%, S: 6.47%.
Methyl 2-oxo-2,3-dihydro-1H-indene-1-carboxylate (7)
It was synthesized by the same procedure utilized for the
synthesis of compounds 2a,b starting with 2-indanone. It was
purified by column chromatography using hexane/dichloro-
methane 15:1 v/v, then 10:1 v/v. It exists as a mixture of keto
1
and enol tautomers. Yield: 54%; H NMR (CDCl3, 300 MHz) d
11.00 (brs, 1H), 7.59 (d, 1H, J ¼ 9.0 Hz), 7.30–7.24 (m, 2H), 7.10
(dt, 1H, J ¼ 15.0 Hz, 9.0 Hz, 3.0 Hz), 3.96 (s, 3H), 3.57 (s, 2H);
13C NMR (CDCl3, 75 MHz) d 180.8, 169.3, 139.5, 133.1, 127.1,
123.8, 123.6, 120.2, 105.2, 105.2, 51.5, 37.7.
3-Hydroxyindeno[1,2-c]chromen-6(11H)-one (8)
It was synthesized by the same procedure utilized for the
synthesis of compounds 4a–f starting with compound 7 and
resorcinol. It was used in the next step as such with no further
purification.
2-Chloro-6-oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]-
chromen-3-yl benzenesulfonate (1r)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 13:1 v/v, then switching to hexane/ethyl
acetate 6:1 v/v); yield: 81%; 1H NMR (CDCl3, 400 MHz) d 8.39 (d,
2H, J ¼ 7.5 Hz), 7.97 (t, 1H, J ¼ 7.4 Hz), 7.75–7.70 (m, 2H), 7.05
(brs, 2H), 2.92 (t, 2H, J ¼ 6.3 Hz), 2.79 (t, 2H, J ¼ 5.9 Hz), 1.79–
1.71 (m, 4H), 1.50–1.41 (m, 4H); LC-MS: 419.0 (Mþþ1);
elemental analysis (C21H19ClO5S): calculated C: 60.21%, H:
4.57%, S: 7.65%; found: C: 59.90%, H: 4.86%, S: 7.55%.
6-Oxo-6,11-dihydroindeno[1,2-c]chromen-3-yl 4-
methylbenzenesulfonate (1u)
It was synthesized through the same procedure utilized for
synthesis of compounds 1a–t through reaction of compound 8
and tosyl chloride in the presence of triethylamine. It was
purified by column chromatography using hexane/ethyl
acetate 10:1 v/v. Yield: 35%; 1H NMR (CDCl3, 400 MHz) d
7.77 (dd, 3H, J ¼ 19.6 Hz, 8.0 Hz), 7.66 (d, 1H, J ¼ 8.2 Hz), 7.40–
7.36 (m, 3H), 7.33–7.28 (m, 3H), 6.81 (d, 1H, J ¼ 8.0 Hz), 4.28 (s,
2H), 2.46 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 150.0, 147.7,
145.5, 142.0, 134.6, 132.0, 131.8, 130.1, 129.9, 129.7, 129.4,
129.1, 128.5, 128.1, 127.1, 123.3, 121.2, 121.0, 118.5, 40.1, 21.8;
LC-MS: 405.0 (Mþþ1); elemental analysis (C23H16O5S): calcu-
lated C: 68.31%, H: 3.99%, S: 7.93%; found: C: 68.05%, H:
4.07%, S: 8.02%.
2-Chloro-6-oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]-
chromen-3-yl 4-methylbenzenesulfonate (1s)
It was purified by flash column chromatography (silica gel,
hexane/ethyl acetate 13:1 v/v, then switching to hexane/ethyl
acetate 7:1 v/v); yield: 80%; 1H NMR (CDCl3, 400 MHz) d 8.26 (d,
2H, J ¼ 7.9 Hz), 7.94–7.92 (m, 1H), 7.56–7.53 (m, 2H), 7.28 (s, 1H),
7.03 (s, 1H), 2.93 (t, 2H, J ¼ 5.9 Hz), 2.79 (t, 2H, J ¼ 5.6 Hz), 2.48 (s,
3H), 1.78–1.70 (m, 4H), 1.50–1.41 (m, 4H); LC-MS: 433.0 (Mþþ1);
elemental analysis (C22H21ClO5S): calculated C: 61.04%, H:
4.89%, S: 7.41%; found: C: 60.87%, H: 5.10%, S: 7.24%.
Biological evaluation
Cell culture, sample treatment, nitrite determination,
PGE2 assay, and MTT assay
Synthesis of 2-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]-
chromen-6(7H)-one (5)
It was prepared utilizing the same method reported in the
literature, similar to synthesis of compounds 4a–f starting
with hydroquinone [20].
Cell culture and sample treatment, nitrite determination,
PGE2 assay, and MTT assay for cell viability have been carried
out following the reported procedures [16, 22].
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–11
Antiinflammatory Coumarin Derivatives
Archiv der Pharmazie
Western blot
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This work was supported by 2016 research program of Hanseo
University in Korea.
The authors have declared no conflicts of interest.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
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