7-(2-Oxoalkoxy)coumarins: Synthesis and Anti-Inflammatory Activity of a Series of Substituted Coumarins

Article abstract of DOI:10.1002/jhet.2173

A series of 7-(2-oxoalkoxy)coumarins have been synthesized by conjugating substituted 7-hydroxycoumarins with different chloroketones. The anti-inflammatory properties of 7-(2-oxoalkoxy)coumarins were studied in LPS-induced inflammatory response in J774 macrophages. Western blot was used to determine the expression of iNOS and COX-2, NO was determined by measuring its metabolite nitrite by Griess reaction and IL-6 was measured by ELISA. Seventeen of the studied compounds inhibited NO and IL-6 production over 50% at 100 μM concentrations. IC₅₀ values of the best inhibitors were 21 μM/24 μM (NO/IL-6) for compound 12 and 30 μM/10 μM (NO/IL-6) for compound 20. The main result was that the substitution with 7-(2-oxoalkoxy) group improved the anti-inflammatory properties of most of the investigated 7-hydroxycoumarins.

Full text of DOI:10.1002/jhet.2173

Month 2014 7-(2-Oxoalkoxy)coumarins: Synthesis and Anti-Inflammatory Activity of a
Series of Substituted Coumarins
Juri Timonen,a Katriina Vuolteenaho,b Tiina Leppänen,b Riina Nieminen,b Eeva Moilanen,b Paula Aulaskari,a
a
*
and Janne Jänis
aDepartment of Chemistry, University of Eastern Finland, P.O. Box 111, FI-80101 Joensuu, Finland
bUniversity of Tampere, School of Medicine, Tampere University, Hospital, FI-33014 Tampere, Finland
*
E-mail: janne.janis@uef.fi
Additional Supporting Information may be found in the online version of this article.
Received April 28, 2013
DOI 10.1002/jhet.2173
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).
A series of 7-(2-oxoalkoxy)coumarins have been synthesized by conjugating substituted 7-hydroxycoumarins
with different chloroketones. The anti-inflammatory properties of 7-(2-oxoalkoxy)coumarins were studied
in LPS-induced inflammatory response in J774 macrophages. Western blot was used to determine the
expression of iNOS and COX-2, NO was determined by measuring its metabolite nitrite by Griess reaction
and IL-6 was measured by ELISA. Seventeen of the studied compounds inhibited NO and IL-6 production
over 50% at 100 μM concentrations. IC50 values of the best inhibitors were 21 μM/24 μM (NO/IL-6)
for compound 12 and 30 μM/10 μM (NO/IL-6) for compound 20. The main result was that the
substitution with 7-(2-oxoalkoxy) group improved the anti-inflammatory properties of most of the
investigated 7-hydroxycoumarins.
J. Heterocyclic Chem., 00, 00 (2014).
INTRODUCTION
Nitric oxide (NO) is a gaseous signaling molecule that
mediates various physiological and pathophysiological
responses in the human body, e.g., in the immune response
and inflammation, in cardiovascular system, and in
neurotransmission. In vivo, NO is synthesized from amino
acid L-arginine and molecular oxygen (O2) in a reaction
catalyzed by enzyme nitric oxide synthase (NOS). Three
different forms of NOS have been identified, namely
endothelial NOS (eNOS), neuronal NOS (nNOS), and
inducible NOS (iNOS) [14–18]. eNOS and nNOS are
calcium-dependent constitutive enzymes that are responsible,
in general, for the low physiological NO production. In
inflammation, pro-inflammatory cytokines and bacterial
products trigger iNOS expression in inflammatory and tissue
cells, and iNOS is responsible for prolonged production of
increased amounts of NO in the inflammatory focus
[16,17,19]. At high concentrations, NO has pro-inflammatory
and destructive properties, and selective iNOS inhibitors have
been proven anti-inflammatory in experimentally induced
arthritis and several other inflammatory models [20–22].
Coumarins are widely presented in plants, including
Orchidaceae, Rutaceae, and Umbelliferae, and both
synthetic and plant-derived coumarins are utilized in a
wide range of applications in chemical, pharmaceutical,
and agricultural industry [1–3]. Coumarins are also
important ingredients of plants used as anti-inflammatory
remedies in traditional medicine [4–6]. There are, however,
limited data on the anti-inflammatory effects of plant-
derived coumarins [7] and their synthetic derivatives [8].
7-Acetonyloxycoumarins or 7-(2-oxopropoxy)coumarins
(Fig. 1) are widely used as intermediates for synthetic
furocoumarins namely psoralens and angelicins [9–11].
Some applications for other 7-(2-oxoalkoxy)coumarins have
been developed such as the use of cyclic and acyclic
coumaryloxy-ketones as fluorogenic substrates in an assay
for monooxygenase enzyme activity [10]. Also, compounds
of this structure other than 7-acetonyloxycoumarins are used
as intermediates to furocoumarins [12] and coumarin-based
structures, geiparvarins [13].
© 2014 HeteroCorporation

J. Timonen, K. Vuolteenaho, T. Leppänen, R. Nieminen, E. Moilanen, P. Aulaskari, and J. Jänis
Vol 000
presence of K2CO3 and acetone as solvent. Usually, the
mixture turned slightly brownish-yellow within reaction.
The potassium carbonate bound the hydrogen chloride
formed in conjugation by forming potassium chloride,
which is presented as a very fine powder. To prevent the
dissolution of inorganic salts during isolation, the acetone
used was dried with drying agent before using.
Compound 23 containing phenacyl instead of acetyl
group was prepared in the same manner as compounds
1–22. The only exception was the use of phenacyl chloride
in molar ratio 1:1 to coumarin rather than excess of
chloride. The reason was the difficulties on the removal
Figure 1. Structure of studied 7-(2-oxoalkoxy)coumarins 1–24.
Our previous study considering 7-hydroxycoumarins [23]
showed potency of this scaffold as an inhibitor of several
inflammatory processes. A few studies have been carried
out using 7-oxycoumarins [24] but other oxygen-linked
substituents at 7-position have not yet been published. Plant
derived coumarins, i.e., 3-(1′-1′-dimethyl-allyl)-6-hydroxy-
7-methoxy-coumarin isolated from Ruta graveolens L.
and sesquiterpene coumarins extracted from roots of
Ferula Fukanensis and oxycoumarin derivatives, have
been shown to inhibit NO production and iNOS
expression [7,24–26]. Because these 7-oxygen-substituted
coumarins (7-methoxycoumarins) have been found to
inhibit NO production, we were interested to investigate
effects of 2-oxoalkoxygroup at the same position. These ear-
lier studies encouraged us to investigate the effects of replac-
ing 7-OH by 7-(2-oxoalkoxy). In this study, we synthesized
twenty-four 7-(2-oxoalkoxy)coumarin derivatives and investi-
gated their effects on the expression of inflammatory enzymes
iNOS and COX-2, and on the production of inflammatory
mediators NO and IL-6 in activated macrophages.
Table 1
Inhibition of NO production and iNOS expression by tested coumarins in
J774 macrophages stimulated with LPS 100 ng/mL for 24 h.
Inhibition of NO
production (%)
Inhibition of iNOS
protein expression (%)
Compound
10 μM
100 μM
10 μM
100 μM
1
2
3
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
12 6.4
À1 6.7
0 4.7
20 7.1
À5 5.6
16 5.4
38 3.5
42 4.1
À1 1.8
2 1.5
27 3.6
49 2.6
28 2.0
23 3.4
17 3.3
2 2.0
14 3.2
31 6.5
49 2.0
47 5.2
49 3.2
13 4.2
25 4.7
50 3.3
22 5.1
19 0.4
58 1.3
41 1.9
51 2.6
94 0.3
98 0.4
74 0.5
À3 2.8
89 0.4
94 0.3
71 1.2
74 1.4
66 1.5
43 1.6
73 1.4
95 1.2
90 1.6
76 2.8
88 0.7
48 3.4
65 0.5
11 7.3
11 3.3
À40 1.4
24 5.0
25 3.1
À15 7.4
À6 12.8
À9 11.1
24 2.4
18 4.6
47 7.2
32 4.4
15 7.2
17 8.5
À5 4.7
À5 3.3
16 6.5
33 4.2
58 5.5
38 7.2
36 5.1
46 1.9
46 4.5
16 7.0
42 2.4
À5 5.1
68 1.2
64 7.3
34 7.0
92 1.1
97 0.8
42 4.9
36 1.6
97 0.6
97 0.4
69 1.5
66 4.7
40 2.0
25 2.7
70 1.2
93 0.7
99 0.1
72 3.9
85 2.2
63 4.8
88 0.7
RESULTS AND DISCUSSION
Syntheses. To alter the structure of previously studied 7-
hydroxycoumarins, we combined the coumarin scaffold with
2-oxoalkoxy group (Scheme 1). Twenty-two of synthesized
compounds were substituted with the aid of chloroacetone
and two compounds with phenacylchloride or 1-
chloropinacolone. The method we used is a known
substitution reaction of phenolic hydroxyl group. It was
reported first time in the literature with phenol [27] and β-
naphtol [28] and more recently with dihydroxynaphtalen [29].
Compounds 1–22 were synthesized by reaction of
suitable 7-hydroxycoumarin and chloroacetone in the
Mean SEM, n = 4.
Scheme 1. Preparation of 7-(2-oxoalkoxy) coumarins 1–24. Reagents and conditions: (a) Me2CO, K2CO3, reflux 14–18 h.
Journal of Heterocyclic Chemistry
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7-(2-Oxoalkoxy)coumarins: Synthesis and Anti-Inflammatory Activity of a Series of
Substituted Coumarins
Pharmacology. To study the anti-inflammatory effects of
7-(2-oxoalkoxy)coumarins, we measured their effects on the
expression of inflammatory enzymes iNOS and COX-2
(which produce nitric oxide and prostaglandins, respectively)
and on the production of inflammatory mediators nitric
oxide (NO) and interleukin-6 (IL-6) in macrophages. As an
inflammatory stimulus lipopolysaccharide (LPS) was used
to activate the macrophages through a toll-like-receptor 4
(TLR4) pathway. In the primary testing, two concentrations
of the 7-(2-oxoalkoxy)coumarins, i.e., 10 and 100 μM,
were used.
The compounds were dissolved as a 100 mM stock
solution in DMSO for the cell culture experiments.
Compound 5 was excluded from the studies because it
was not soluble in DMSO at desired concentrations. In
all experiments (including controls), the final DMSO
concentration was adjusted to be 0.1%, which was shown
not to affect the measured parameters. The pharmacologi-
cal studies on the newly synthesized compounds were
started by cytotoxicity testing. For these purposes, XTT
test was used. The test measures cells’ mitochondrial
dehydrogenase activity that only occurs in viable cells.
Triton-X (0.1%) was used as a positive control of cell death.
of the solid starting chloride traces from crude product.
The formed crude product was almost pale powder. To
provide tert-butyl group to oxoalkoxy tail in the compound
24, 1-chloropinacolone was used. Equimolar amounts of
starting materials were used to prevent the formation of
scarcely separable liquid mixture of product and high
boiling chloropinacolone. In addition to compounds 23
and 24, compounds 15 and 18 were also prepared with
equimolar amounts of starting materials.
Most of the compounds could be dried successfully
by evaporating the solvent in rotary evaporator, but
compound 18 did contain impurities unable to be
removed by this method. Crude products were slightly
brownish moist powder except compound 18, which
was dark brown sticky oil. After recrystallization from
ethanol all products were pale crystalline powders. To get
a solid product from compound 18 the reaction mixture
was evaporated and kept at open air until mostly precipitated.
After several careful recrystallizations from ethanol the
product formed crystals. Yields of recrystallized products
vary largely depending on the substitution but were
generally good when compared with the yields reported in
the literature [30–32].
Figure 2. Dose-dependent effects of compounds 12 and 20 on iNOS and NO. Effects of compounds 12 (A and B) and 20 (C and D) on NO production
(A and C) and iNOS expression (B and D) in J774 macrophages stimulated with LPS 100 ng/mL for 24 h. Results are expressed as mean SEM, n = 4.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. Timonen, K. Vuolteenaho, T. Leppänen, R. Nieminen, E. Moilanen, P. Aulaskari, and J. Jänis
Vol 000
None of the compounds (100 μM) showed cytotoxic effects
Seven compounds (8, 9, 12, 13, 19, 20, and 22) inhibited
when tested in similar conditions where the experiments
for anti-inflammatory activities were carried out.
Inhibition of inducible nitric oxide synthase expression and
nitric oxide production. In inflammation, NO is principally
formed in a reaction catalyzed by iNOS, which is an
inducible enzyme. Unstimulated cells did not produce NO
or express iNOS protein at detectable levels, whereas LPS
enhanced both considerably. Most of the compounds
significantly inhibited LPS-induced NO production and
iNOS expression when used at 100μM concentrations.
NO production and iNOS expression by more than 85%
when used at 100 μM concentrations as compared with the
cells treated with LPS alone. The compounds 12 and 20
appeared to be the most potent inhibitors of iNOS protein
expression with more than 97% and 47% inhibition at
100 and 10 μM concentrations, respectively (Table 1).
A highly selective iNOS inhibitor 1400 W [33] was
used as a positive control compound. 1400 W (1000 μM)
inhibited NO production in LPS-stimulated cells by more
than 95%, but as an iNOS inhibitor, it did not affect
iNOS protein expression. Because NF-κB is an important
transcription factor for iNOS expression, we used
pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-κB
activation [34] as a positive control for inhibition of
iNOS expression. PDTC (100μM) inhibited iNOS
expression by 97%.
The two most potent compounds (12 and 20) were
selected for the further studies. To investigate dose-
dependency, the compounds were added into to cell culture
in 1–100 μM concentrations. Both of the compounds
inhibited NO production in a dose-dependent manner with
IC50 values of 21 μM (compound 12) and 30 μM
(compound 20), respectively, and a similar effect was seen
on iNOS expression (Fig. 2). iNOS mRNA was measured
by quantitative RT-PCR after 8 h incubation with a
combination of LPS and the compound 12 or 20. The
Figure 3. The effects of compounds 12 and 20 on iNOS mRNA
expression. Effects measured with J774 macrophages stimulated with
LPS 100 ng/mL for 8 h. Results are expressed as mean SEM, n = 4.
Table 2
Inhibition of IL-6 production and COX-2 expression by tested coumarins in J774 macrophages stimulated with LPS (100 ng/mL) for 24 h.
Inhibition of IL-6 production (%)
Inhibition of COX-2 protein expression (%)
Compound
10 μM
100 μM
10 μM
100 μM
1
2
3
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4 3.6
8 6.0
8 3.7
21 3.1
28 1.3
7 5.0
22 2.8
38 2.1
36 1.7
68 0.9
71 0.7
40 2.1
93 0.1
82 0.5
71 2.0
39 4.1
90 0.4
73 0.7
80 1.2
78 0.7
60 0.9
46 2.4
74 0.6
90 1.0
89 0.7
76 2.0
82 1.8
56 3.0
70 0.4
À19 7.6
4 5.6
À37 12
12 5.7
57 3.2
41 6.2
65 1.9
3 9.7
29 5.9
À11 9.3
35 6.9
À7 8.6
3 8.7
7 11.5
18 1.4
4 4.9
29 7.8
55 2.2
29 6.7
23 7.9
7 3.5
À9 5.3
20 5.8
À2 10.8
À8 17.4
À4 13.5
13 3.0
17 3.6
20 6.3
33 4.3
34 1.3
17 1.5
13 3.8
47 4.5
62 1.2
43 2.5
43 1.3
15 3.9
À1 4.8
27 1.7
27 2.8
33 4.6
46 4.1
51 4.8
21 13.3
18 0.6
67 5.2
65 5.9
33 4.4
33 1.9
57 1.6
26 4.8
49 4.3
59 2.7
70 3.0
30 6.3
63 2.7
58 4.7
37 15.2
12 5.6
19 3.6
À1 8.8
44 5.2
Mean SEM, n = 4.
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7-(2-Oxoalkoxy)coumarins: Synthesis and Anti-Inflammatory Activity of a Series of
Substituted Coumarins
iNOS mRNA levels were very low in resting cells, but they
increased significantly when the cells were exposed to
LPS. Compounds 12 and 20 had a clear inhibitory effect
on iNOS mRNA levels (Fig. 3) which is consistent with
their inhibitory effects of iNOS expression and NO
production.
The effects of 7-(2-oxoalkoxy)coumarins were also
tested on LPS-stimulated COX-2 expression in activated
J774 macrophages. Nine of the tested compounds (3, 6,
8, 9, 12, 15, 16, 18, and 19) inhibited the COX-2
expression by more than 50% when used at a concentration
of 100 μM (Table 2). Dexamethasone (10 μM) was used
as a control compound and inhibited COX-2 expression
by 87%.
We also tested the effects of 7-(2-oxoalkoxy)coumarins
on the LPS-induced production of pro-inflammatory
cytokine IL-6, by measuring its concentration in the cell
culture medium by immunoassay. IL-6 production in
resting cells was very low, but it was significantly
enhanced when the cells were activated by adding LPS.
With the higher concentration used (100 μM), seven of
the studied compounds (8, 9, 12, 14, 19, 20, and 22)
inhibited IL-6 production by more than 80%, and only
one compound (1) had an inhibitory effect smaller than
30%. With the lower concentration used (10 μM), nine
compounds inhibited IL-6 production by more than 30%
(Table 2). Dexamethasone (10 μM) was used as a control
compound [23,35], and it inhibited IL-6 production by
74%. Dose–response studies showed IC50 values of 24 μM
for compound 12 and 10 μM for compound 20 on IL-6
production (Fig. 4).
Structure-activity study.
Our earlier study on 7-
hydroxycoumarins contained 17 similar structures as the
present study, namely 1–4, 6–10, 13, 14, 16–19, 21, and 22.
Comparison of the results showed over 90% inhibition of
iNOS expression with compounds 8, 9, and 19 from both
series, while derivative 13 showed over 90% activity only
as 7-(2-oxoalkoxy)coumarin and derivative 21 only as 7-
hydroxycoumarin. Results on NO production were similar:
compounds 8 and 9 from both series inhibited NO
production by more than 90% while compounds 13 and 19
were effective only as 7-(2-oxoalkoxy)coumarins. Generally,
acetonyloxylation of 7-hydroxycoumarins affected more on
NO production than on iNOS expression.
Instead of acetonyloxy group the substitution with 2-oxo-2-
phenylethoxy or 3,3-dimethyl-2-oxobutoxy improved inhibition
Figure 4. Dose-dependent effects of compounds 12 and 20 on IL-6 and COX-2. Effects of compounds 12 (A and B) and 20 (C and D) on IL-6 production
(A and C) and COX-2 expression (B and D) in J774 macrophages stimulated with LPS 100 ng/mL for 24 h. Results are expressed as mean SEM, n = 4.
Journal of Heterocyclic Chemistry
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J. Timonen, K. Vuolteenaho, T. Leppänen, R. Nieminen, E. Moilanen, P. Aulaskari, and J. Jänis
Vol 000
Table 3
The differences in the inhibition activities between 7-(2-oxoalkoxy)coumarins that improved activity in either iNOS/NO or IL-6 inhibition over 7-
hydroxycoumarinsa at 100 μM concentration. Positive value means improved activity of 7-(2-oxoalkoxy)coumarin over 7-hydroxycoumarin.
Difference in inhibition of NO
production (percent unit)
Difference in inhibition of iNOS
expression (percent unit)
Difference in inhibition of IL-6
production (percent unit)
Compound
4
8
9
À13
2
À6
À2
6
À48
32
À19
À34
0
0
0
1
26
2
1
À8
À18
À22
26
6
6
7
8
10
13
14
16
18
19
22
23
24
15
À11
10
7
5
22
17
1
À6
35
12
26
aValues of 7-hydroxycoumarins are presented in the supporting information.
activities remarkably. Compounds 23 and 24 were compared
to 7-hydroxy-4-methylcoumarin. Substitution with 3,3-
dimethyl-2-oxobutoxy increased activities more: the iNOS
inhibition was almost 90% compared to 62% inhibition
activity of 7-hydroxy-4-methylcoumarin.
Inhibition of IL-6 production was also improved by
substitution with acetonyloxy group. Compounds 14 and
22 gained the major effect as the IL-6 inhibition of
hydroxycoumarins were approximately 50% but
acetonyloxylation rose them above 80%. The efficiency
of the 7-(2-oxoalkoxy)coumarins to inhibit COX-2
expression is enhanced in the case of 13 out of the 17
comparable structures, with an average between 40% and
60% inhibition of LPS-induced COX-2 expression, while
none of the 7-hydroxycoumarins were able to show over
30% inhibition.
Generally, the substitution of hydroxyl by 2-oxoalkoxy
group improved the anti-inflammatory activity of some of
the previously studied 7-hydroxycoumarins. In Table 3,
there are twelve 7-(2-oxoalkoxy)coumarins that have
improved activity over respective 7-hydroxycoumarin in
either iNOS/NO or IL-6 inhibition at 100 μM concentra-
tion and nine of them showed meaningful increase in the
inhibition activity (difference over 5 percentage units
compared with the respective 7-hydroxycoumarin). Results
of the relevant 7-hydroxycoumarins are presented in
supporting information and all values can be found from
our previous paper [23].
CONCLUSIONS
Twenty-four substituted 7-(2-oxoalkoxy)coumarins were
synthesized according to commonly known procedure. Ten
of the coumarins (3, 4, 7, 8, 12, 15, 17, 18, 21, and 22)
synthesized in the present study are novel compounds.
In the study, 7-(2-oxoalkoxy)coumarins inhibited iNOS
expression and NO production as well as IL-6 production
and COX-2 expression in a dose-dependent manner. The
most promising compounds were 8, 9, 12, 19, 20, and
22, i.e., 3,4,5-trimethyl, 3-ethyl-4-methyl, 3-ethyl-4,5-
dimethyl, 4-phenyl, 8-methyl-4-phenyl, and 8-methoxy-4-
phenyl derivatives. These compounds inhibited iNOS
expression, and the production of IL-6 and NO by more than
80% at the concentration of 100 μM, without cytotoxicity.
The structure-activity elucidation points out two major
properties of the most active coumarins. Lipophilic
substituents, namely methyl or ethyl (compounds 8, 9,
12, and 13), at position 3 increased the activity whereas
the 3-fluorine hindered the activity rather than activated
the compounds 7 and 17. The 4-phenyl substitution
was also a clear enhancer of the inhibition activity. Within
4-phenyl compounds, the fluorine at the position 2 of the
All of the six most active compounds (8, 9, 12, 19, 20, and
22) contain either 4-phenyl or 3-alkyl substituent. In addition
to these compounds, the compound 13, namely 7-
acetonyloxy-3-ethyl-4,8-dimethylcoumarin inhibited iNOS
expression over 95% and NO production by almost 95%,
and the IL-6 production was reduced by 73%, which is
good but below the 80% level. This was observed also
by experiments with 7-hydroxycoumarins. Further, two
out of three 3-alkylated compounds (6 and 16) lacking
excellent anti-inflammatory activity contain substituent
at position 8.
From the four tested 4-phenyl compounds, three (19, 20,
and 22) are regarded as very effective anti-inflammatory
compounds. There is also minor decreasing effect of the 8-
substituent on the activity: the 8-methoxy substituted
compound (22) is less active than the other two promising
compounds (19 and 20). Within 4-phenyl compounds, the
fluorine at the position 2 of the phenyl ring (compound 21)
as well as the 3-fluorine in the compounds 7 and 17
decreases the activity remarkably.
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7-(2-Oxoalkoxy)coumarins: Synthesis and Anti-Inflammatory Activity of a Series of
Substituted Coumarins
1 h at room temperature and incubated overnight at 4°Cwith
COX-2, iNOS or actin (loading control) antibodies in TBS/T
containing 5% nonfat milk. Thereafter, the membrane was
washed four times in TBS/T for 5 min, incubated with a
secondary antibody coupled to horseradish peroxidase in the
blocking solution for 0.5 h at room temperature, and washed
four times with TBS/T for 5 min. The bound antibody was
detected and quantitated by using a SuperSignal West Pico
chemiluminescent substrate (Pierce, Cheshire, UK) and an
ImageQuant LAS 4000 mini imaging system (GE Healthcare
Bio-Sciences, AB, Uppsala, Sweden). The chemiluminescent
signal was quantified with ImageQuant TL 7.0 Image Analysis
Software [37].
phenyl ring (compound 21) decreased the activity remarkably.
The activity order of different 2-oxoalkoxy substituents seems
to be 3,3-dimethyl-2-oxobutoxy > 2-oxo-2-phenylethoxy
2-oxopropoxy and substitution of 7-hydroxycoumarins
to 7-(2-oxoalkoxy)coumarins seemed to improve the
inhibitory activities.
EXPERIMENTAL
Reagents.
The reagents were obtained as following: goat
polyclonal anti-mouse COX-2, rabbit polyclonal anti-mouse
iNOS, rabbit polyclonal anti-mouse actin, horseradish
peroxidase conjugated donkey polyclonal anti-goat IgG, and
goat polyclonal anti-rabbit IgG antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, USA). All other reagents
were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
RNA extraction and quantitative reverse transcription
polymerase chain reaction.
monolayers were rapidly washed with ice-cold phosphate-
buffered saline, and cells were homogenized using
After 8 h incubation, cell
a
QIAshredder (QIAGEN, Valencia, CA, USA). RNA extraction
was carried out with the use of an RNeasy kit for isolation of
total RNA (QIAGEN). Total RNA (100 ng) was reverse-
transcribed to cDNA using TaqMan Reverse Transcription
reagents and random hexamers (Applied Biosystems, Foster
City, CA, USA). cDNA obtained from the RT reaction (amount
corresponding to approximately 2.5 ng of the total RNA) was
subjected to PCR using a TaqMan Universal PCR Master Mix
and an ABI PRISM 7000 Sequence detection system (Applied
Biosystems). The primer and probe sequences and concentrations
were optimized according to the manufacturer’s guidelines in the
TaqMan Universal PCR Master Mix Protocol part number
4304449 revision C, and were: 5′-CCTGGTACGGGCATTGCT-
3′,and 5′-GCTCATGCGGCCTCCTT-3′ (forward and reverse mouse
iNOS primers, respectively, both 300 nM); 5′-CAGCAGCGGCT
CCATGACTCCC-3′ (mouse iNOS probe, 150 nM, containing 6-
FAM as 5′-reporter dye and TAMRA as 3′-quencher); 5′-GCAT
GGCCTTCCGTGTTC-3′ and 5′-GATGTCATCATACTTGG
CAGGTTT-3′ [forward and reverse mouse glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) primers, respectively,
both 300 nM]; and 5′-TCGTGGATCTGACGTGCCGCC-3′
(mouse GAPDH probe, 150 nM, containing 6-FAM as 5′-
reporter dye and TAMRA as 3′-quencher). The PCR
reaction parameters were as follows: incubation at 50°C for
2 min, incubation at 95°C for 10 min, and, thereafter,
40 cycles of denaturation at 95°C for 15 s and annealing
and extension at 60°C for 1 min. Each sample was
determined in duplicate.
A standard curve method was used to determine the
relative mRNA levels, as described in the Applied
Biosystems User Bulletin: a standard curve for each gene
was created using RNA isolated from LPS-stimulated J774
macrophages. Isolated RNA was reverse-transcribed and the
dilution series of cDNA ranging from 1 pg to 10 ng were
subjected to real-time PCR. The obtained threshold cycle
values were plotted against the dilution factor to create a
standard curve. Relative mRNA levels in the test samples
were then calculated from the standard curve. When calculat-
ing the results the iNOS mRNA levels were first normalized
against GAPDH [37].
Cell culture.
J774 murine macrophages (American Type
Culture Collection, Rockville, MD) were cultured in Dulbecco’s
modified Eagle’s medium with Ultraglutamine 1. The culture
medium was supplemented with 10% heat-inactivated fetal
bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin,
and 250 ng/mL amphotericin B (all from Gibco, Paisley,
Scotland). Cells were seeded on 24-well plates, and the cell
monolayers were grown for 72 h to confluency before the
experiments were started, and the compounds of interest were
added in fresh culture medium.
XTT-test.
Proliferation Kit II, which measures cells’ ability to metabolize
sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis-(4-
Cell viability was tested by using Cell
methoxy-6-nitro) benzene sulphonic acid hydrate (XTT) to
formazan by mitochondrial dehydrogenases that only occur in
viable cells (Boehringer Mannheim, Indianapolis, IN, USA).
Nitrite assays. At indicated time points, the culture medium
was collected for nitrite measurement, which was used as a
measure of NO production. Culture medium (100 μL) was
incubated with 100 μL of Griess reagent [36] (0.1%
napthalethylenediamine dihydrochloride, 1% sulfanilamine, and
2.5% H3PO4), and the absorbance was measured at 540 nm.
The concentration of nitrite was calculated with sodium nitrite
as the standard.
Preparation of cell lysates for western blotting. At indicated
time points, cells were rapidly washed with ice-cold phosphate-
buffered saline. The cells were resuspended in a lysis buffer
containing 1% Triton X, 50 mM NaCl, 10 mM Tris-base pH 7.4,
5 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride, 1 mM
sodiumorthovanadate, 20 μM leupeptin, 50μg/mL aprotinin,
5 mM NaF, 2 mM sodiumpyrophosphate, and 10 μM N-octyl-β-D-
glucopyranoside. After incubation for 15 min on ice, the lysates
were centrifuged (13,500 g, 10 min). The protein content of the
supernatants was measured by the Coomassie blue method.
Western blot analysis.
After boiling for 5 min, equal
aliquots of protein (20 μg) were loaded onto a 10% SDS-
polyacrylamide electrophoresis gel and electrophoresed for 1 h
at 120 V in a buffer containing 95 mM Tris–HCl, 960 mM
glycine, and 0.5% SDS. After electrophoresis, the proteins were
transferred to
a
Hybond enhanced chemiluminescence
Enzyme-linked immunosorbent assay.
Culture medium
nitrocellulose membrane (Amersham, Buckinghamshire, UK)
with a semi-dry blotter at 2.5 mA/cm2 for 60 min. After transfer,
the membrane was blocked in TBS/T (20 mM Tris-base pH 7.6,
150 mM NaCl, 0.1% Tween-20) containing 5% nonfat milk for
samples were kept at À20°C until assayed. The concentration of
mouse IL-6 (DuoSet® ELISA, R&D Systems Europe Ltd,
Abindgon, UK) was determined by ELISA according to the
manufacturer’s instructions.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. Timonen, K. Vuolteenaho, T. Leppänen, R. Nieminen, E. Moilanen, P. Aulaskari, and J. Jänis
Vol 000
Statistics.
The results are expressed as the mean SEM.
J= 2.8 Hz, 3H, 4-Me), 4.98 (s, 2H, OCH2COCH3), 7.02 (m, 2H,
Statistical significances of the differences were calculated by analyses
of variance, supported by the Dunnett’s multiple comparisons test.
Differences were considered significant at p < 0.05 and shown as
* = p < 0.05, **= p < 0.01, and *** = p < 0.001.
6-H and 8-H), 7.67 (d, J = 9.2 Hz, 5-H); 13C-NMR (DMSO-d6) δ:
11.58, 27.75, 73.88, 103.10, 114.16, 114.21, 114.72, 128.11,
133.33, 141.48, 152.81, 161.62, 204.74; HRMS (ESI-FTICR)
Calcd for C13H12FO4 (M+H+): 251.0714; found, 251.0712; Anal.
Calcd for C13H11FO4: C, 62.40; H, 4.43; found: C, 61.96; H, 4.67.
Syntheses. Chemicals were used as received if else has not
been mentioned. 7-Hydroxycoumarins were synthesized
according to the literature [23]. Melting points were measured
with Gallenkamp Melting Point Apparatus (Gallenkamp,
Loughborough, UK) and are uncorrected. NMR spectra were
measured either with Bruker Avance 250 (250 MHz for 1H,
62.9 MHz for 13C; Bruker BioSpin AG, Fällanden, Swizerland)
or with Bruker Avance 400 FT NMR (400 MHz for 1H,
100.6 MHz for 13C; Bruker BioSpin AG, Fällanden,
Swizerland). IR spectra were measured with Nicolet Avatar 320
FTIR (Thermo Electron Scientific Instruments, LLC, Madison,
WI, USA). Combustion analysis was done using vario MICRO
cube (Elementar Analysensysteme GmbH, Hanau, Germany)
Mass spectra were measured with 12-T APEX-Qe FT-ICR
instrument (Bruker Daltonik GmbH, Bremen, Germany).
7-Acetonyloxy-3,4,5-trimethylcoumarin (8).
White crystals.
Yield 67%; mp 184–185°C; IR (KBr, cmÀ1): 3073 (ν C–H, aro-
matic), 1744 (ν C═O, lactone), 1689 (ν C═O, R–C(O)–R), 1605
(ν C═C, lactone), 1476 (das C–H, CH═CRCH3), 1385 (ds C–H,
CH═CRCH3), 1358 (ds C–H, C(O)–CH3), 1348 (ds C–H, C(O)–
CH3), 1186 (ν O–C, lactone), 1107 (ν O–C, O–CH2), 1087 (r C–
1
C–C, CH2–CO–CH3), 860 (γ C–H, aromatic); H-NMR (DMSO-
d6) δ: 2.07 (s, 3H, 3-Me), 2.18 (s, 3H, OCH2COCH3), 2.60
(s, 3H, 4-Me), 4.98 (s, 2H, OCH2COCH3), 6.68 (s, 1H, 8-H), 7.77
(s, 1H, 6-H); 13C-NMR (DMSO-d6) δ: 14.53, 21.24, 22.75, 27.93,
74.58, 109.73, 110.62, 111.19, 120.84, 143.05, 149.34, 154.3,
156.98, 162.22, 204.47; HRMS (ESI-FTICR) Calcd for C15H17O4
(M+H+): 261.1121; found, 261.1121; Anal. Calcd for C15H16O4:
C, 69.22; H, 6.20; found: C, 68.75; H, 6.20.
7-Acetonyloxy-3-ethyl-4-methylcoumarin (9).
Light yellow
General method for synthesis of 7-acetonyloxycoumarins 1–
24. 15 mmol of substituted 7-hydroxycoumarin was dissolved in
400 mL of dry acetone and 21 g (138 mmol) of potassium carbonate
was added. The chloroacetone (6.66 g, 72 mmol) was added, and
the mixture was refluxed for 24 h. After refluxing, the solution was
filtered, and acetone was removed with rotary evaporator. The
products were purified by recrystallization from ethanol.
crystals. Yield 72%; mp 140°C; IR (KBr, cmÀ1): 3070 (ν C–H,
aromatic), 1728 (ν C═O, lactone), 1697 (ν C═O, R–C(O)–R), 1612
(ν C═C, lactone), 1462 (d C–H, R′C–CH2CH3), 1393 (ds C–H, R–
CH2–CH3), 1366 (ds C–H, C(O)–CH3), 1231 (ν O–C, lactone),
1146 (ν O–C, O–CH2), 1072 (r C–C–C, CH2–CO–CH3), 856 (γ C–
H, aromatic), 814 (γ C–H, aromatic), 779 (r C–(CH2)n–C, R–CH2–
CH3); 1H-NMR (DMSO-d6) δ: 1.03 (t, 3H, J=7Hz, CH2CH3), 2.17
(s, 3H, OCH2COCH3), 2.37 (s, 3H, 4-Me), 2.50 (q, 2H, J=7.5Hz,
CH2CH3), 4.96 (s, 2H, OCH2COCH3), 6.92 (m, 2H, 6-H and 8-H),
7.67 (d, 1H, J=8.25Hz, 5-H); 13C-NMR (DMSO-d6) δ: 14.55,
15.93, 27.76, 51.78, 73.83, 102.61, 113.84, 115.5, 125.45, 127.89,
148.04, 154.55, 161.45, 162.35, 204.88; HRMS (ESI-FTICR) Calcd
for C15H17O4 (M+H+): 261.1121; found, 261.1121; Anal. Calcd for
C15H16O4: C, 69.22; H, 6.20; found: C, 68.81; H, 6.13.
7-Acetonyloxy-5-methylcoumarin (3).
White crystals. Yield
68%; mp 127–128°C; IR (KBr, cmÀ1): 3070 (ν C–H, aromatic),
1717 (ν C═O, lactone), 1711 (ν C═O, R–C(O)–R), 1606
(ν C═C, lactone), 1440 (das C–H, Ar–CH3), 1387 (ds C–H,
Ar–CH3), 1358 (ds C–H, C(O)–CH3), 1204 (ν O–C, lactone),
1123 (ν O–C, O–CH2), 1079 (r C–C–C, CH2–CO–CH3), 855
(γ C–H, aromatic), 829 (γ C–H, RCH═CHR′); 1H-NMR (DMSO-
d6) δ: 2.17 (s, 3H, OCH2COCH3), 2.46 (s, 3H, 5-Me), 4.96 (s,
2H, OCH2COCH3), 6.28 (d, 1H, J = 9.75 Hz, 3-H), 6.81 (m, 2H,
6-H and 8-H), 8.08 (d, J = 9.5 Hz, 4-H); 13C-NMR (DMSO-d6) δ:
17.91, 26.06, 72.05, 99.09, 111.41, 111.83, 113.69, 137.94,
141.46, 155.75, 160.16, 160.42, 203.08; HRMS (ESI-FTICR)
Calcd for C13H13O4 (M+H+): 233.0808; found, 233.0807; Anal.
Calcd for C13H12O4: C, 66.71; H, 6.37; found: C, 66.26; H, 5.15.
7-Acetonyloxy-4-propylcoumarin (10).
Light yellow crystals.
Yield 70%; mp 89–90°C; IR (KBr, cmÀ1): 3075 (ν C–H, aromatic),
1728 (ν C═O, lactone), 1713 (ν C═O, R–C(O)–R), 1612 (ν C═C,
lactone), 1373 (ds C–H, R–(CH2)2–CH3), 1350 (ds C–H, C(O)–
CH3), 1207 (ν O–C, lactone), 1146 (ν O–C, O–CH2), 1069 (r C–
C–C, CH2–CO–CH3), 852 (γ C–H, aromatic), 845 (γ C–H, RR′
C═CHR″), 791 (γ C–H, aromatic), 756 (r C–(CH2)n–C, R–(CH2)
2–CH3); 1H-NMR (DMSO-d6) δ: 0.98 (t, 3H, J=7.5Hz,
CH2CH2CH3), 1.64 (sxt, 2H, J=7.5Hz, CH2CH2CH3), 2.18
(s, 3H, OCH2COCH3), 2.74 (t, 2H, CH2CH2CH3), 5.00 (s, 2H,
OCH2COCH3), 6.17 (s, 1H, 3-H), 6.96 (m, 2H, 6-H and 8-H), 7.73
(d, 1H, J=9.75Hz, 6-H); 13C-NMR (DMSO-d6) δ: 13.57, 21.23,
26.07, 28.37, 72.18, 101.44, 110.31, 112.40, 112.51, 126.11,
154.81, 156.65, 160.17, 160.65, 203.11; HRMS (ESI-FTICR) Calcd
for C15H17O4 (M+H+): 261.1121; found, 261.1120; Anal. Calcd for
C15H16O4: C, 69.22; H, 6.20; found: C, 69.11; H, 6.16.
7-Acetonyloxy-4,5-dimethylcoumarin (4).
White crystals.
Yield 77%; mp 179–180°C; IR (KBr, cmÀ1): 3066 (ν C–H, aro-
matic), 1713 (ν C═O, lactone), 1694 (ν C═O, R–C(O)–R),
1612 (ν C═C, lactone), 1454 (das C–H, CH═CRCH3), 1440
(das C–H, CH═CRCH3), 1369 (ds C–H, CH═CRCH3), 1358
(ds C–H, C(O)–CH3), 1206 (ν O–C, lactone), 1113 (ν O–C, O–
CH2), 1070 (r C–C–C, CH2–CO–CH3), 860 (γ C–H, aromatic),
844 (γ C–H, RR′C═CHR″); 1H-NMR (DMSO-d6) δ: 2.18
(s, 3H, OCH2COCH3), 2.35 (s, 3H, 4-Me), 2.60 (s, 3H, 5-Me),
4.99 (s, 2H, OCH2COCH3), 6.15 (s, 1H, 3-H), 6.72 (s, 1H, 8-
H), 6.81 (s, 1H, 6-H); 13C-NMR (DMSO-d6) δ: 21.24, 23.82,
26.24, 72.90, 107.56, 108.85, 109.76, 112.91, 143.01, 153.93,
154.55, 159.41, 202.65; HRMS (ESI-FTICR) Calcd for
C14H15O4 (M+H+): 247.0965; found, 247.0963; Anal. Calcd for
C14H14O4: C, 68.28; H, 5.73; found: C, 68.51; H, 5.86.
7-Acetonyloxy-3-ethyl-4,5-dimethylcoumarin (12).
White
crystals. Yield 47%; mp 166–167°C; IR (KBr, cmÀ1): 3070 (ν
C–H, aromatic), 1719 (ν C═O, lactone), 1693 (ν C═O, R–C
(O)–R), 1619 (ν C═C, lactone), 1452 (d C–H, R–CH2–CH3),
1389 (ds C–H, RR′C═CR″–CH3), 1351 (ds C–H, C(O)–CH3),
1190 (ν O–C, lactone), 1114 (ν O–C, O–CH2), 1067 (r C–C–C,
CH2–CO–CH3), 856 (γ C–H, aromatic), 783 (r C–C–C, C–
CH2–CH3); 1H-NMR (CDCl3-d3) δ: 1.13 (t, 3H, J = 7.5 Hz,
CH2CH3), 2.29 (s, 3H, OCH2COCH3), 2.36 (s, 3H, 5-Me), 2.67
(m, 5H, CH2CH3 & 5-Me), 4,67 (s, 2H, OCH2COCH3), 6.35
(s, 1H, 6-H), 6.78 (s, 1H, 8-H); 13C-NMR (CDCl3-d3) δ: 12.87,
7-Acetonyloxy-3-fluoro-4-methylcoumarin
(7).
White
crystals. Yield 36%; mp 162–163°C; IR (KBr, cmÀ1): 3095 (ν C–
H, aromatic), 1721 (ν C═O, R–C(O)–R), 1618 (ν C═C, lactone),
1365 (ds C–H, C(O)–CH3), 1121 (ν O–C, O–CH2), 1076 (r C–C–
C, CH2–CO–CH3), 857 (γ C–H, aromatic), 834 (γ C–H, aromatic);
1H-NMR (DMSO-d6) δ: 2.17 (s, 3H, OCH2COCH3), 2.34 (d,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2014
7-(2-Oxoalkoxy)coumarins: Synthesis and Anti-Inflammatory Activity of a Series of
Substituted Coumarins
19.42, 20.64, 21.76, 26.73, 73.56, 108.17, 109.37, 111.19,
126.47, 141.34, 147.13, 153.74, 155.44, 161.37, 203.24; HRMS
(ESI-FTICR) Calcd for C16H19O4 (M+H+): 275.1278; found,
275.1278; Anal. Calcd for C16H18O4: C, 70.06, H 6.61: found:
C, 69.88; H, 6.56.
δ: 0.99 (t, 3H, J = 7.5 Hz, CH2CH2CH3), 1.55 (sxt, 2H, J = 7.5 Hz,
CH2CH2CH3), 2.21 (s, 3H, OCH2COCH3), 2.75 (t, 2H,
J = 7.5Hz, CH2CH2CH3), 3.89 (s, 3H, 8-OMe), 5.06 (s, 2H,
OCH2COCH3), 6.21 (s, 1H, 3-H), 6.98 (d, 1H, J = 9.0Hz, 6-H),
7.48 (d, 1H, J = 9.0 Hz, 5-H); 13C-NMR (DMSO-d6) δ: 13.56,
21.24, 26.08, 32.75, 60.61, 72.47, 109.58, 110.62, 113.56,
119.81, 135.30, 147.33, 153.39, 156.77, 159.76, 203.32; HRMS
(ESI-FTICR) Calcd for C16H19O5 (M+H+): 291.1227; found,
291.1226; Anal. Calcd for C16H18O5: C, 66.20; H, 6.25; found: C,
65.86; H, 6.24.
7-Acetonyloxy-5-methyl-4-propylcoumarin (14).
White
crystals. Yield 78%; mp 150–152°C; IR (KBr, cmÀ1): 3062 (ν
C–H, aromatic), 1724 (ν C═O, lactone), 1709 (ν C═O, R–C
(O)–R), 1605 (ν C═C, lactone), 1385 (ds C–H, R–(CH2)2–
CH3), 1373 (ds C–H, C(O)–CH3), 1204 (ν O–C, lactone), 1123
(ν O–C, O–CH2), 1061 (r C–C–C, CH2–CO–CH3), 870 (γ C–H,
aromatic), 829 (γ C–H, RR′C═CHR″), 752 (r C–C–C, R–(CH2)
2–CH3); 1H-NMR (DMSO-d6) δ: 0.95 (t, 3H, J = 7.5 Hz,
CH2CH2CH3), 1.55 (sxt, 2H, J = 7.5 Hz, CH2CH2CH3), 2.19
(s, 3H, OCH2COCH3), 2.34 (s, 3H, 5-Me), 2.98 (t, 2H,
J = 7.5 Hz, CH2CH2CH3), 5.02 (s, 2H, OCH2COCH3), 6.11
(s, 1H, 3-H), 6.70 (s, 1H, 8-H), 6.81 (s, 1H, 6-H); 13C-NMR
(DMSO-d6) δ: 13.60, 21.19, 22.43, 26.13, 37.57, 72.77, 106.76,
108.81, 109.91, 112.35, 142.87, 154.90, 155.41, 157.58,
159.57, 202.28; HRMS (ESI-FTICR) Calcd for C16H19O4 (M
+H+): 275.1278; found, 275.1278; Anal. Calcd for C16H18O4:
C, 70.06; H, 6.61; found: C, 69.65; H, 6.54.
7-Acetonyloxy-4-(2-fluorophenyl)coumarin (21).
White
crystals. Yield 62%; Mp 174–175°C; IR (KBr, cmÀ1): 3072 (ν
C–H, aromatic), 1730 (ν C═O, lactone), 1713 (ν C═O, R–C
(O)–R), 1617 (ν C═C, lactone), 1384 (ds C–H, C(O)–CH3),
1209 (ν O–C, lactone), 1159 (ν C–F, Ph–F), 1126 (ν O–C, O–
CH2), 1074 (r C–C–C, CH2–CO–CH3), 834 (γ C–H, RR′
1
C═CHR″), 821 (γ C–H, aromatic), 754 (γ C–H, Ph); H-NMR
(DMSO-d6) δ: 2.28 (s, 3H, OCH2COCH3), 5.02 (s, 2H,
OCH2COCH3), 6.36 (s, 1H, 3-H), 6.90 (dd, 1H, J1 = 8.75 Hz,
J2 = 2.5 Hz, 5-H), 7.11 (m, 2H, 6-H and 8-H), 7.41 (m, 4H, 4-
(2-FPh)); 13C-NMR (DMSO-d6) δ: 26.06, 72.25, 101.55,
111.84, 112.91, 115.82, 122.23, 122.47, 125.08, 127.48,
130.73, 132.02, 149.81, 154.81, 159.63, 161.10, 202.93; HRMS
(ESI-FTICR) Calcd for C18H14O4F (M+H+): 313.0871; found,
313.0871; Anal. Calcd for C18H13O4F: C, 69.23; H, 4.20; found:
C, 68.91; H, 4.39.
7-Acetonyloxy-5-methyl-4-isopropylcoumarin (15).
White
crystals. Yield 52%; Decomposed over 250°C; IR (KBr, cmÀ1):
3075 (ν C–H, aromatic), 1724 (ν C═O, lactone), 1609 (ν C═C,
lactone), 1443 (das C–H, R–CH–(CH3)2), 1396 (ds C–H, Ar–
CH3), 1362 (ds C–H, C(O)–CH3), 1331 (d C–H, R–CH–(CH3)
2), 1200 (ν O–C, lactone), 1173 (r C–C–C, R–CH–(CH3)2),
1142 (r C–C–C, R–CH–(CH3)2), 1115 (ν O–C, O–CH2), 1076
(r C–C–C, CH2–CO–CH3), 872 (γ C–H, aromatic), 818 (γ C–H,
7-Acetonyloxy-8-methoxy-4-phenylcoumarin (22).
White needles.
Yield 64%; Mp 155–156°C; IR (KBr, cmÀ1): 3098 (ν C–H, Ph),
3059 (ν C–H, aromatic), 1721 (ν C═O, lactone), 1454 (das,s C–H,
Ar–O–CH3). 1373 (ds C–H, C(O)–CH3), 1111 (ν O–C, O–CH2),
1065 (r C–C–C, CH2–CO–CH3), 1015 (ν C–O, Ar–O–CH3), 841
(γ C–H, RR′C═CHR″), 818 (γ C–H, aromatic), 760 (γ C–H, Ph),
698 (γ C–H, Ph); 1H-NMR (DMSO-d6) δ: 2.18 (s, 3H,
OCH2COCH3), 3.82 (s, 3H, 8-OMe), 5.04 (s, 2H, OCH2COCH3),
6.21 (s, 1H, 3-H), 6.95 (d, 1H, J = 9.25 Hz, 5-H), 7.07 (d, 1H,
J = 9 Hz, 6-H), 7.55 (m, 5H, 4-Ph); 13C-NMR (DMSO-d6) δ:
26.06, 60.65, 72.46, 109.71, 111.77, 113.11, 121.55, 128.35,
128.74, 129.59, 134.83, 135.54, 147.70, 153.64, 155.18, 159.49,
203.11; HRMS (ESI-FTICR) Calcd for C19H17O5 (M+H+):
325.1071; found, 325.1072; Anal. Calcd for C19H16O5: C, 70.36;
H, 4.97; found: C, 69.89; H, 5.11.
1
RR′C═CHR″); H-NMR (DMSO-d6) δ: 1.30 (d, 6H, J = 6.8 Hz,
CH(CH3)2), 2.31 (s, 3H, OCH2COCH3), 2.78 (m, 1H, CH(CH3)
2) 4.62 (s, 2H OCH2COCH3), 6.03 (s, 1H, 3-H), 6.61 (s, 1H, 8-
H), 6.71 (s, 1H, 6-H); 13C-NMR(DMSO-d6) δ: 20.04,
23.00,26.64, 32.61, 72.81, 99.18, 108.80,115.96, 143.18,
160.16, 171.78, 204.32; HRMS (ESI-FTICR) Calcd for
C16H19O4 (M+H+): 275.1278; found, 275.1277; Anal. Calcd for
C16H18O4: C, 70.06; H, 6.61; found: C, 70.32; H, 6.91.
7-Acetonyloxy-3-fluoro-8-methoxy-4-methylcoumarin
(17).
Pale brown powder. Yield 57%; mp 155–157°C; IR
(KBr, cmÀ1): 1723 (ν C═O, lactone), 1697 (ν C═O, R–C(O)–
R), 1606 (ν C═C, lactone), 1459 (das,s C–H, Ar–O–CH3), 1431
(das C–H, RR′C═CR″–CH3), 1383 (ds C–H, RR′C═CR″–CH3),
1364 (ds C–H, C(O)–CH3), 1282 (ν C–O, Ar–O–CH3), 1224 (ν
O–C, lactone), 1181 (ν C–F, RR′C═CR″F), 1125 (ν O–C, O–
CH2), 1060 (r C–C–C, CH2–CO–CH3), 1014 (ν O–C, Ar–O–
CH3), 813 (γ C–H, aromatic); 1H-NMR (DMSO-d6) δ: 2.19
(s, 3H, OCH2COCH3), 2.34 (d, 3H, J = 2.75 Hz 4-Me), 5.05
(s, 2H, OCH2COCH3), 7.05 (d, 1H, J = 9 Hz, 6-H), 7.42 (d, 1H,
J = 9 Hz, 5-H); 13C-NMR (DMSO-d6) δ: 11.63, 27.76, 62.40,
74.17, 112.20, 115.33, 121.65, 136.93, 141.57, 145.52, 154.44,
155.59, 156.07, 204.92; HRMS (ESI-FTICR) Calcd for
C14H14O5F (M+H+): 281.0820; found, 281.0819; Anal. Calcd
for C14H13O5F: C, 60.00; H, 4.68; found: C, 59.88; H, 4.48.
Acknowledgments. Mrs. Ritva Romppanen, Mrs. Taina Nivajärvi,
Ms. Sonja Niskanen, Mrs. Salla Hietakangas, Ms. Meiju Kukkonen,
and Ms. Petra Miikkulainen are warmly thanked for excellent
technical assistance and Mrs. Heli Määttä for skillful secretarial
help. Ms. Sanna Hiltunen is warmly thanked for personal
assistance. We are thankful for Dr. Ewen Macdonald for language
editing. The study was supported by grants from Medical
Research Fund of Tampere University Hospital, Finland, The
Academy of Finland, The National Graduate School of Organic
Chemistry and Chemical Biology, University of Joensuu, and
University of Eastern Finland.
7-Acetonyloxy-8-methoxy-4-propylcoumarin (18).
White
crystals. Yield 40%; mp 102–103°C; IR (KBr, cmÀ1): 3067 (ν
C–H, aromatic), 1730 (ν C═O, lactone), 1713 (ν C═O, R–C
(O)–R), 1447 (das C–H, R–(CH2)2–CH3), 1371 (ds C–H, R–
(CH2)2–CH3), 1348 (ds C–H, C(O)–CH3), 1281 (ν C–O, Ar–O–
CH3), 1207 (ν O–C, lactone), 1115 (ν O–C, O–CH2), 1018 (ν
C–O, Ar–O–CH3), 845 (γ C–H, RR′C═CHR″), 806 (γ C–H,
aromatic), 750 (r C–C–C, R–(CH2)2–CH3); 1H-NMR (DMSO-d6)
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