Synthesis of Novel Anti-inflammatory Psoralen Derivatives?-?Structures?with?Distinct?Anti-Inflammatory?Activities

Article abstract of DOI:10.1002/jhet.3318

As a continuum to our work with coumarins, 12 psoralens were synthesized and evaluated for their anti-inflammatory activity. Psoralens were prepared in three steps; at first, 7-hydroxycoumarins were synthesized by von Pechmann condensation and then converted to 7-(2-oxopropoxy)coumarins. In the final step, a fused furan ring was introduced in an intramolecular ring-formation reaction. Based on a SciFinder search, two out of the 12 synthesized psoralen derivatives (compounds 9 and 12) were found to be novel. The derivatives displayed anti-inflammatory activity by suppressing iNOS and IL-6 expression, but their mechanism of action seemed to be dependent on the substitution. Compound 6 with propyl side chain inhibited NF-κB mediated transcription, while compound 10 with a phenyl substituent down-regulated iNOS expression in a posttranscriptional manner. The results introduce psoralen derivatives as promising anti-inflammatory compounds with potential for treatment of conditions involving iNOS and/or IL-6-mediated adverse responses.

Full text of DOI:10.1002/jhet.3318

Month 2018
Synthesis of Novel Anti-inflammatory Psoralen Derivatives -
Structures with Distinct Anti-Inflammatory Activities
b
Juri M. Timonen,a,b,c
Katriina Vuolteenaho,
Janne Jänis,c
Tiina Leppänen,b Riina M. Nieminen,b Paula Aulaskari,c
*
*
Pirjo Vainiotalo,c and Eeva Moilanenb
aSchool of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
bThe Immunopharmacology Research Group, Faculty of Medicine and Life Sciences, University of Tampere and Tampere
University Hospital, Tampere, Finland
cDepartment of Chemistry, Faculty of Science and Forestry, University of Eastern Finland, Joensuu, Finland
*E-mail: juri.timonen@uef.fi; katriina.vuolteenaho@uta.fi
Received March 30, 2018
DOI 10.1002/jhet.3318
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
As a continuum to our work with coumarins, 12 psoralens were synthesized and evaluated for their
anti-inflammatory activity. Psoralens were prepared in three steps; at first, 7-hydroxycoumarins were
synthesized by von Pechmann condensation and then converted to 7-(2-oxopropoxy)coumarins. In the final
step, a fused furan ring was introduced in an intramolecular ring-formation reaction. Based on a SciFinder
search, two out of the 12 synthesized psoralen derivatives (compounds 9 and 12) were found to be novel.
The derivatives displayed anti-inflammatory activity by suppressing iNOS and IL-6 expression, but their
mechanism of action seemed to be dependent on the substitution. Compound 6 with propyl side chain inhibited
NF-κB mediated transcription, while compound 10 with a phenyl substituent down-regulated iNOS expression
in a posttranscriptional manner. The results introduce psoralen derivatives as promising anti-inflammatory
compounds with potential for treatment of conditions involving iNOS and/or IL-6-mediated adverse responses.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
When
screening
anti-inflammatory
candidate
compounds, inhibition of NO production is usually
considered as a positive sign of anti-inflammatory
activity. Nitric oxide (NO) is produced by the family of
NO synthases (NOSs); in inflammation, the production of
NO is enhanced due to increased expression of the
inducible isoform, iNOS. Moderation of NO production
is usually achieved either by inhibiting iNOS enzyme or
by inhibiting the expression of iNOS [10–12]. In recent
years, other mechanisms, such as preventing the
dimerization of iNOS or disrupting the stability of the
iNOS-enzyme, have also been investigated. Moreover,
NO is a radical that can be scavenged by antioxidants, for
example, coumarins [13,14].
Inflammation is the body’s defense reaction triggering a
wide range of physiological mechanisms and pathways.
However, inflammation may be detrimental if it is
inappropriately focused or regulated or if it becomes
prolonged. New drugs against inflammation are needed,
and research of natural products and their derivatives has
represented
a
growing trend in recent decades.
Coumarins are one widely studied group of natural
compounds with anti-inflammatory potential; they are a
family of secondary plant metabolites [1–6]. Recent
studies have shown that coumarins may also exert
anticancer cell activity [7] as well as displaying activity
against different infectious diseases (e.g., tuberculosis,
influenza, and HIV) [8,9].
In the present study, we synthesized 12 psoralen
derivatives and studied their effects on iNOS expression
© 2018 Wiley Periodicals, Inc.

J. M. Timonen, K. Vuolteenaho, T. Leppänen, R. M. Nieminen,
P. Aulaskari, J. Jänis, P. Vainiotalo, and E. Moilanen
Vol 000
Scheme 1. Synthesis of psoralen derivatives 1–12. Substituents (R3, R4,
R5, and R8) present in the synthetized compounds 1–12 are shown in
Table 1. Reagents and conditions: (a) (i) ethylpropiolate, 1,4-dioxane;
(ii) dry ZnCl2, reflux, 24 h; (iii) 5% HCl. (b) (i) R4COCHR3CO2Et,
H2SO4, RT, 2–12 h; (ii) H2O. (c) (i) K2CO3, (CH3)2CO; (ii)
chloroacetone, reflux, 24 h. (d) (i) 1M KOH(aq), N2(g), reflux, 16 h; (ii)
acetic acid, RT; (iii) +4°C 16 h.
used acetic acid instead of phosphoric acid. The
acidified solution was kept at +4°C overnight. After
filtration and air-drying, psoralens were purified by
recrystallization from ethanol. Their structure and purity
were confirmed with 1H NMR, 13C NMR, and
combustion analysis. According to a SciFinder search,
compounds
compounds.
9
and 12 were identified as novel
Screening for Anti-inflammatory Activity.
The anti-
inflammatory activity of the synthetized psoralens was
investigated in J774 murine macrophages, which were
exposed to bacterial lipopolysaccharide (LPS) to induce
the classically activated (M1) phenotype. First, the
toxicity of the newly synthesized compounds was
evaluated in the XTT-test. None of the psoralens exerted
cytotoxic effects when tested in similar conditions and
concentrations (10 and 30 μM) where the anti-
inflammatory activities would subsequently be examined.
The anti-inflammatory effects of the compounds were
estimated via the production of two inflammatory factors:
NO measured by the Griess method and IL-6 determined
by immunoassay. We also assessed the expression of the
inflammatory enzymes iNOS and COX-2 by QT-PCR
and/or western blotting.
As shown in Table 1, compounds 2, 5, 6, 10, and 12 were
the most active psoralen derivatives at inhibiting NO
production. These compounds inhibited NO production by
approximately 50% or more when examined at 10 μM
concentrations. Compounds 2, 4, 5, and 6 were the most
effective molecules in terms of suppressing iNOS
expression. Compounds 10 and 12 both displayed a
relatively high inhibition activity on NO production but
were only moderate inhibitors of iNOS expression.
Production of IL-6 was inhibited by over 50% by three
compounds, namely, 6, 10, and 12, where tested at 10 μM
concentrations (Table 1). In addition, the compounds were
tested for their inhibitory activity on COX-2 expression.
None of the psoralen derivatives inhibited COX-2
expression in a statistically significant manner at 10 μM
concentrations (data are presented in Table S1).
Mechanisms of anti-inflammatory activity. Altogether
six of the studied compounds (2, 4, 5, 6, 10, and 12)
inhibited at least one of the markers of anti-inflammatory
activity used in the present study (NO, iNOS, or IL-6) by
approximately 50% or more. Compound 6 was the most
versatile anti-inflammatory compound, that is, it inhibited
all three mediators by more than 50%. To study the
psoralens’ mechanisms of anti-inflammatory activity, two
of the prepared psoralens (6 and 10) with sufficiently
different structures were selected for further studies.
The mechanisms of inhibition of iNOS expression were
examined by determining the effects of compounds 6 and
10 on LPS-induced iNOS mRNA levels in macrophages
at two different time points. Compound 6 inhibited iNOS
and on the production of inflammatory mediators NO and
IL-6 in activated macrophages.
RESULTS AND DISCUSSION
Chemistry. Psoralens 1–12 were synthesized according
to Scheme 1. Substituted 7-(2-oxopropoxy)coumarins were
prepared previously from 7-hydroxycoumarins, as
described in our earlier publications [2,3]. In general,
7-hydroxycoumarins were prepared by mixing ethyl
acetoacetate and substituted resorcinol in perchloric acid
at room temperature or by refluxing resorcinol and
ethyl propiolate in 1,4-dioxane with a catalytic amount of
aluminum chloride. The prepared 7-hydroxycoumarins
were then refluxed in dry acetone with chloroacetone
and potassium carbonate to yield 7-(2-oxopropoxy)
coumarins.
Psoralens were prepared by mixing substituted
7-(2-oxopropoxy)coumarin (4.5 mmol) into 45 mL of
1 M aqueous NaOH and refluxing for 16 h under a
nitrogen atmosphere. This inert gas was used to prevent
the formation of undesired angular isoforms, angelicins.
After refluxing, the reaction mixture was cooled to
room temperature and made slightly acidic to precipitate
the product. In most published methods, a small portion
(e.g., 5 mL) of orthophosphoric acid has been used for
acidifying the mixture. However, some psoralens did
not precipitate while using phosphoric acid but instead
formed an unfilterable paste. To avoid this problem, we
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Synthesis of Novel Anti-inflammatory Psoralen Derivatives
Table 1
Substituents present in the synthetized compounds 1–12 and their anti-inflammatory activities.a
Substituents
R4
Inhibition at 10 μM conc. (%)
Compound
R3
R5
R8
NO
iNOS
IL-6
1
2
3
4
5
6
7
8
—
—
—
—
—
—
CH3
—
—
—
—
—
—
33.4 1.7**
63.4 2.2**
17.2 7.4*
38.5 4.2**
53.6 3.5**
53.1 0.4**
33.4 2.4**
34.1 1.7**
22.5 2.1**
48.2 1.9**
38.8 3.5**
59.0 3.3**
41.8 5.0**
51.3 8.1**
16.6 8.7b
49.7 8.5**
53.4 7.8**
55.6 3.2**
24.3 5.4b
42.8 1.7**
38.9 4.4**
39.0 7.5**
37.4 10.1**
36.9 6.1**
41.4 2.1**
45.4 1.9**
10.2 3.1b
CH3
CH3
CH3
CH3
CH3
CH2CH3
—
—
CH3
F
22.2 2.6**
42.7 4.5**
67.5 0.9**
45.4 1.7**
44.7 0.9**
25.0 2.0**
72.1 1.9**
44.0 1.7**
59.9 2.1**
—
CH2CH2CH3
CH3
CH3
CH3
C6H5
C6H5
—
OCH3
OCH3
OCH3
—
CH3
—
9
—
—
—
—
10
11
12
—
—
—
2—FC6H5
aEffects of the 12 psoralen derivatives were investigated at 10 μM concentrations on NO, iNOS, and IL-6 production in J774 macrophages stimulated with
LPS 10 ng/mL for 24 h. Results are expressed as mean SEM, n = 4.
bNot significant.
*p < 0.05
**p < 0.01 compared with LPS-treated cells.
Figure 1. Effects of compounds 6 (A) and 10 (B) on iNOS mRNA expression in J774 macrophages stimulated with LPS 10 ng/mL for 3 or 8 h. Results
are expressed as mean + SEM, n = 4. *p < 0.05, **p < 0.01 compared with LPS-treated cells.
mRNA expression when measured at 3 h after LPS
stimulation and more clearly at 8 h, whereas compound
10 reduced iNOS mRNA levels only at 8 h time point
(Fig. 1). These results suggested that compound 10
inhibited iNOS expression at the posttranscriptional level
while compound 6 inhibited iNOS mRNA expression
also at the early transcriptional phase.
NF-κB is a major transcription factor for iNOS; therefore,
the effects of the two compounds (6 and 10) were measured
on NF-κB-mediated transcription in human HEK293 cells
that had been genetically modified to express the
luciferase gene under the control of NF-κB responsive
element. It was observed that compound 6 down-regulated
the NF-κB-mediated transcription (measured as luciferase
activity) in a statistically significant manner, whereas
psoralen 10 had no effect (Fig. 2).
Structure–activity study.
Comparison of anti-
inflammatory activity with the structures of compounds
1–12 showed that the substituent at position 4 played the
most significant role, while substitution at position 3 did
not exert any significant effect on the compound’s anti-
inflammatory potential. In
a series with aliphatic
substituents (hydrogen, methyl, and propyl) at position 4,
there was a trend that the anti-inflammatory potential
increased according to the length of alkyl chain (Fig. 3).
However, a phenyl substituent at position 4 (e.g., 10 and
12) showed generally lower activity compared with
compounds with an aliphatic substituent.
Discussion. In this study, we synthesized a series of 12
psoralens; of these, compounds 9 and 12 are novel
according to a SciFinder search. The anti-inflammatory
activity of the synthesized psoralens was tested, and all
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. M. Timonen, K. Vuolteenaho, T. Leppänen, R. M. Nieminen,
P. Aulaskari, J. Jänis, P. Vainiotalo, and E. Moilanen
Vol 000
Figure 2. Effects of compounds 6 (A) and 10 (B) on NF-κB-mediated transcription in HEK293 cells transfected with a luciferase reporter construct and
stimulated with TNFα 20 ng/mL for 5 h. Results are expressed as mean + SEM, n = 4. **p < 0.01 compared with TNFα-treated cells.
Figure 3. Structure–activity relationship (SAR) analysis of psoralen derivatives 1, 2, and 6 having hydrogen (H), methyl (Me), or propyl (Pr) substituents
respectively at position 4 and otherwise similar structure. Compounds were compared for their anti-inflammatory effects on NO, iNOS, and IL-6 produc-
tion in J774 macrophages stimulated with LPS 10 ng/mL for 24 h. Results are expressed as mean SEM, n = 4.
of them inhibited iNOS expression and NO production,
and all but one (compound 3) also impaired IL-6
production. Compounds 2, 5, 6, 10, and 12 were the most
potent agents in inhibiting iNOS expression as well as
the release of NO and IL-6 by approximately 50% or
more at 10 μM concentration without causing any signs
of cytotoxicity. Some of the compounds showed more
efficient inhibition of NO production than iNOS
expression (e.g., 2, 7, and 12), which suggests that
inhibition of NO is not achieved solely by inhibition of
iNOS expression. This may be due to a direct inhibitory
effect on the enzyme activity or, more likely, to the anti-
oxidative nature of psoralens: They have been shown to
scavenge NO radicals [14].
substituents (hydrogen, methyl, and propyl) of psoralens
correlated with their anti-inflammatory activity. In
addition, the substituent at position 4 seemed to be a
major factor determining which mechanism of action was
predominant: The larger compounds with a phenyl
substituent at position 4 (e.g., 10 and 12) generally
showed lower activity as compared with the compounds
with an aliphatic substituent. Our earlier studies with
7-hydroxycoumarins and 7-(2-oxoalkoxy)coumarins have
revealed a progressive increase in activity through the
same series of substituents from hydrogen to phenyl and
even to 2-fluorophenyl [2,3]. This contrast with the
structure–activity relationship results may indicate either
that psoralens act through different mechanism than
coumarins or that the active binding site of iNOS is
rather small and can bind coumarins or somewhat larger
psoralens only if they contain a smaller or more flexible
substituent than a phenyl ring at position 4.
In the structure–activity relationship analysis, the
substituent at position 4 exerted the most significant role
in determining the anti-inflammatory activity of these
psoralens. The length of the alkyl chain of aliphatic
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Synthesis of Novel Anti-inflammatory Psoralen Derivatives
We selected two active psoralens with aliphatic or phenyl
substituents at position 4 to investigate if they possessed
different mechanisms of action in their suppressive effect
on iNOS expression: psoralen 6 with a propyl substituent
and psoralen 10 with a phenyl substituent. Psoralen 6
inhibited NF-κB-mediated transcription and iNOS mRNA
at the transcriptional phase, while psoralen 10 had no
effect on NF-κB-mediated transcription and consistently
had no effect on iNOS mRNA at the transcriptional phase
(3 h) but did suppress iNOS mRNA levels at a later time
point, indicative of posttranscriptional regulation, possibly
enhanced degradation of iNOS mRNA (at 8 h). The result
suggests that these psoralens have different mechanisms
of actions depending on the substituents in their abilities
to inhibit NO production.
for 24 h under an atmosphere of nitrogen. After cooling
the reaction mixture to room temperature, acetic acid was
added to neutralize the solution. Reaction mixture was
stored at +4°C to precipitate the crude product. The
products were filtered, dried in air, and purified by
recrystallization from ethanol.
1
40-Methylpsoralen (1). Yield 72.8%; H NMR (CDCl3,
600.2 MHz): δ 2.28 (d, 3H, J = 1.3 Hz, 40-CH3), 6.38 (d,
1H, J = 9.6 Hz, 3-H), 7.43 (s, 1H, 8-H), 7.48 (d, 1H,
J = 1.3 Hz, 50-H), 7.59 (s, 1H, 5-H), 7.82 (d, 1H,
J = 9.5 Hz, 4-H); 13C NMR (CDCl3, 150.9 MHz): δ 7.8,
99.8, 114.5, 115.0, 115.5, 118.1, 126.7, 143.2, 144.2,
152.1, 156.8, 161.2. Anal. Calcd for C12H8O3: C, 72.00;
H, 4.03; found: C, 71.63; H, 4.26.
4,40-Dimethylpsoralen (2).
Yield 67.3%; 1H NMR
(CDCl3, 400.1 MHz): δ 2.30 (s, 3H, 40-CH3), 2.53 (s,
3H, 4-CH3), 6.27 (s, 1H, 3-H), 7.41 (s, 1H, 5-H), 7.47
(s, 1H, 8-H), 7.68 (s, 1H, 5-H); 13C NMR (DMSO-d6,
62.9 MHz): δ 7.5, 18.6, 99.1, 112.4, 115.5, 115.7,
116.0, 125.9, 143.8, 151.0, 153.7, 155.8, 160.0. Anal.
Calcd for C13H10O3: C, 72.89; H, 4.71; found: C, 72.61;
H, 4.84.
CONCLUSIONS
In the present study, we synthesized 12 psoralens;
according to
a SciFinder search, two compounds
(compounds 9 and 12) were novel. We also modified the
synthesis by using a weaker acid to neutralize the reaction
mixture in the last reaction step. This modification led to
improved yields of the products depending on the
derivative. The synthesized psoralen derivatives were
shown to exert anti-inflammatory properties, but their
mechanism of action seems to be dependent on the
substitution. In support of our earlier studies [2,3], psoralen
derivatives with appropriate structures are capable of
inhibiting NF-κB-mediated transcription or, as shown here
for the first time, down-regulated iNOS mRNA expression
in a posttranscriptional manner.
4,40,5-Trimethylpsoralen (3).
Yield 52.3%; 1H NMR
(CDCl3, 400.1 MHz): δ 2.41 (d, 3H, J = 1.0 Hz, 40-CH3),
2.69 (s, 3H, 4-CH3), 2.71 (d, 3H, J = 1.3 Hz, 5-CH3),
6.19 (d, 1H, J = 1.2 Hz, 3-H), 6.97 (d, 1H, J = 0.8 Hz,
8-H), 7.44 (d, 1H, J = 1.4 Hz, 50-H); 13C NMR (CDCl3,
62.9 MHz): δ 10.0, 19.1, 21.5, 104.9, 112.7, 113.0,
116.0, 123.3, 135.6, 141.3, 150.8, 151.2, 151.4, 160.6.
Anal. Calcd for C14H12O3: C, 73.67; H, 5.30; found: C,
73.52; H, 5.41.
3,4,40-Trimethylpsoralen (4).
Yield 87.3%; 1H NMR
(CDCl3, 600.2 MHz): δ 2.25 (d, 3H, J = 0.5 Hz, 3-CH3),
2.30 (d, 3H, J = 1.3 Hz, 40-CH3), 2.51 (d, 3H,
J = 0.7 Hz, 4-CH3), 7.40 (s, 1H, 8-H), 7.45 (kv, 1H,
J = 1.2 Hz, 50-H), 7.68 (s, 1H, 5-H); 13C NMR (CDCl3,
150.9 MHz): δ 7.9, 13.4, 15.6, 99.4, 114.3, 115.6, 116.8,
120.1, 126.2, 142.6, 146.4, 150.2, 155.8, 162.4. Anal.
Calcd for C14H12O3: C, 73.67; H, 5.30; found: C, 73.35;
H, 5.40.
EXPERIMENTAL
General procedures. All solvents and chemicals were
used as received unless mentioned. 7-Hydroxycoumarins
and
7-(2-oxopropoxy)coumarins
were
previously
synthesized in our laboratory as described in the literature
[2,3]. Progression of reactions was monitored using TLC.
NMR spectra were measured either with Bruker 600
3-Ethyl-4,40-dimethylpsoralen (5). Yield76.9%;1HNMR
(CDCl3, 400.1 MHz): δ 1.17 (t, 3H, J = 7.5 Hz, CH2CH3),
2.29 (s, 3H, 40-CH3), 2.50 (s, 3H, 4-CH3) 2.711 (kv, 2H,
J = 7.5 Hz, CH2CH3), 7.35 (s, 3H, 8-H), 7.44 (s, 3H, 5-H),
7.65 (s, 3H, 5’-H); 13C NMR (CDCl3, 100.6 MHz): δ 7.9,
13.2, 15.0, 21.0, 99.3, 114.4, 115.6, 116.9, 126.0, 126.2,
142.8, 145.9, 150.3, 155.8, 161.9. Anal. Calcd for
C15H14O3: C, 74.36; H, 5.82; found: C, 74.19; H, 5.91.
1
Avance III (600.2 MHz for H, 150.9 MHz for 13C), with
Bruker Avance 400 FT NMR (400 MHz for 1H,
100.6 MHz for 13C), or with Bruker 250 Avance
1
(250 MHz for H, 62.9 MHz for 13C). The purity of all
tested compounds was determined by combustion
analysis using Elementar Analysesystem GmBH
varioMICROcube and was 95% or higher.
1
40-Methyl-4-propylpsoralen (6). Yield 37.1%; H NMR
(CDCl3, 600.2 MHz) δ: 1.10 (t, 3H, J = 7.6 Hz, CH2–
CH2–CH3), 1.82 (sxt. 2H, J = 7.5 Hz, CH2–CH2–CH3),
2.30 (d, 3H, J = 1.3 Hz, 40-CH3), 2.85 (td, 2H,
J1 = 0.9 Hz J2 = 8 Hz, CH2–CH2–CH3), 6.26 (s, 1H, 3-
H), 7.43 (s, 1H, 8-H), 7.47 (kv, 1H, J = 1.3 Hz, 40-H),
General
method
for
synthesis
A total of 4.5 mmol of
of
substituted
40-methylpsoralens 1–12.
substituted 7-(2-oxopropoxy)coumarin was dissolved in
45 mL of 1 M aqueous KOH. The mixture was refluxed
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. M. Timonen, K. Vuolteenaho, T. Leppänen, R. M. Nieminen,
P. Aulaskari, J. Jänis, P. Vainiotalo, and E. Moilanen
Vol 000
7.71 (s, 1H, 5-H); 13C NMR (CDCl3, 150.9 MHz): δ 7.9,
14.0, 21.3, 34.1, 100.0, 112.1, 114.4, 115.5, 115.6,
126.4, 143.1, 151.9, 156.4, 156.6, 161.5. Anal. Calcd for
C15H14O3: C, 74.36; H, 5.82; found: C, 74.02; H, 5.93.
Reagents for evaluation of anti-inflammatory activity.
The reagents were obtained as follows: 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) unless stated
otherwise.
8-Methoxy-4,40-dimethylpsoralen (7).
Yield 85.1%;
1H NMR (CDCl3, 400.1): δ 2.30 (d, 3H, J = 1.3 Hz,
40-CH3), 2.53 (d, 3H, J = 1.2 Hz, 4-CH3), 4.29 (s, 3H,
8-OCH3), 6.29 (d, 1H, J = 1.0 Hz, 3-H), 7.38 (s, 1H,
5-H), 7.49 (d, 1H, J = 1.1 Hz, 50-H); 13C NMR (CDCl3,
62.9 MHz): δ 7.8, 19.4, 61.2, 107.7, 113.2, 116.0, 117.1,
127.5, 132.5, 142.7, 142.8, 147.5, 153.1, 160.6. Anal.
Calcd for C14H12O4: C, 68.85; H, 4.95; found: C, 68.76;
H, 5.16.
Cell cultures.
J774 murine macrophages (American
Type Culture Collection, Rockville, MD, USA) were
cultured in Dulbecco’s modified Eagle’s medium with
Ultraglutamine 1. The culture medium was supplemented
with 10% heat-inactivated fetal bovine serum (Lonza
Group Ltd., Basel, Switzerland), 100 U/mL penicillin,
100 μg/mL streptomycin, and 250 ng/mL amphotericin B
(all from Invitrogen, Paisley, UK). 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 to fresh culture
medium.
8-Methoxy-3,4,40-trimethylpsoralen (8).
Yield 67.5%;
1H NMR (CDCl3, 600.2 MHz): δ 2.25 (s, 3H, 3-CH3),
2.28 (d, 3H, J = 1.0 Hz, 40-CH3), 2.49 (s, 3H, 4-CH3),
4.28 (s, 3H, 8-OCH3), 7.35 (s, 1H, 5-H), 7.45 (d, 1H,
J = 1.2 Hz, 50-H); 13C NMR (CDCl3, 150.9 MHz): δ 7.8,
13.5, 15.8, 61.2, 107.3, 115.9, 117.9, 120.3, 127.3,
132.4, 141.5, 142.6, 146.5, 146.7, 161.8. Anal. Calcd for
C15H14O4: C, 69.76; H, 5.46; found: C, 69.53%; H, 5.55%.
3-Fluoro-8-methoxy-4,40-dimethylpsoralen (9).
Yield
1
48.9%; H NMR (CDCl3, 600.2 MHz): δ 2.29 (d, 3H,
J = 1.3 Hz, 40-CH3), 2.470 (d, 3H, JF,H = 2.8 Hz,
4-CH3), 4.29 (s, 3H, 8-OCH3), 7.30 (s, 1H, 5-H), 7.49
(s, 1H, 50-H); 13C NMR (CDCl3, 150.9 MHz): δ 7.8, 10.7
(d), 61.3, 107.4 (d), 115.8, 116.8 (d), 128.4, 131.4 (d),
132.9, 139.6, 142.1, 143.1, 143.8, 146.8, 154.8 (d). Anal.
Calcd for C14H11O4F: C, 63.88; H, 4.59; found: C,
63.89; H, 4.42.
Human HEK293 cells (American Type Culture
Collection) were cultured at 37°C in 5% CO2 atmosphere
in Eagle’s minimal essential medium supplemented with
0.15% sodium bicarbonate, non-essential amino acids
(100 μM), sodium pyruvate (1 mM), 10% heat-
inactivated fetal bovine serum (all from Lonza), penicillin
(100 U/mL), streptomycin (100 μg/mL), and
amphotericin B (250 ng/mL).
40-Methyl-4-phenylpsoralen (10). Yield 86.3%; 1H NMR
(CDCl3, 400.1 MHz): δ 2.17 (s, 3H 40-CH3), 6.33 (s, 1H, 3-
H), 7.45 (s, 1H, 8-H), 7.49–7.56 (bs, 7H, 5-H, 50-H & 4-
C6H5); 13C NMR (CDCl3, 62.9 MHz): δ 7.3, 99.81,
113.0, 114.6, 115.4, 117.1, 125.9, 128.5, 128.9, 129.7,
135.2, 144.0, 151.7, 155.6, 155.9, 159.8. Anal. Calcd for
C18H12O3: C, 78.25; H, 4.38; found: C, 77.86; H, 4.52.
Preparation of stable HEK239-pNF-κB (luc) neo reporter
gene cell line.
HEK293 cells were stably transfected
with a luciferase reporter construct pGL4.32[luc2P/NF-κ
B-RE/Hygro] (Promega, Madison, WI, USA) using
Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s instructions. The plasmid contained five
copies of an NF-κB response element that drives
transcription of the luciferase reporter gene. Hygromycin
B (EMD Biosciences Inc., La Jolla, CA, USA) treatment
(200 μg/mL) was used for the selection of transfected
cells. After the selection, the surviving clones were
pooled to give rise to HEK293-pGL4.32[luc2P/NF-κB-
RE/Hygro] cell line. This cell line was then further
cultured in the presence of 100 μg/mL of Hygromycin
B. Cells were harvested with Trypsin–EDTA
(Invitrogen) and seeded on 24-well plates for luciferase
activity measurements.
8,40-Methyl-4-phenylpsoralen (11).
1H NMR (CDCl3, 400.1 MHz):
Yield 59.6%;
δ 2.16 (d, 3H,
J = 1.3 Hz, 40-CH3), 2.62 (s, 3H, 8-CH3), 6.31 (s, 1H,
3-H), 7.37 (s, 1H, 5-H), 7.45 (d, 1H, J = 1.4 Hz, 50-H),
7.56–7.48 (m, 5H, 4-C6H5); 13C NMR (CDCl3,
100.6 MHz): δ 7.9, 8.6, 109.9, 113.0, 114.6, 115.2,
116.0, 125.4, 128.5, 128.9, 129.5, 136.3, 142.9, 150.1,
155.9, 156.8, 161.4. Anal. Calcd for C19H14O3: C, 78.61;
H, 4.86; found: C, 78.43; H, 4.99.
40-Methyl-4-(2-fluorophenyl)psoralen (12). Yield 66.9%;
1H NMR (CDCl3, 250.1 MHz): δ 2.13 (s, 3H 40-CH3),
6.50 (s, 1H, 3-H), 7.33 (s, 1H, 8-H), 7.45 (s, 2H, 50-H &
5-H), 7.56–7.66 (m, 4H, 4-(2-FC6H4); 13C NMR (CDCl3,
62.9 MHz): δ 7.2, 99.8, 114.4, 114.7, 115.3, 115.9,
116.3, 116.7, 126.0, 125.1, 130.8, 131.9, 132.1, 144.2,
150.3, 151.3, 156.0, 159.5. Anal. Calcd for C18H11O3F:
C, 73.47; H, 3.77; found: C, 73.18; H, 3.98.
XTT-test.
Cell viability was tested by using Cell
Proliferation Kit II, which measures the cells’ ability to
metabolize sodium 30-[1-(phenylaminocarbonyl)-3,4-
tetrazolium]-bis-(4-methoxy-6-nitro)benzene-sulphonic
acid hydrate (XTT) to formazan by mitochondrial
dehydrogenases, a reaction that only occurs in viable
cells (Boehringer Mannheim, Indianapolis, IN, USA).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Synthesis of Novel Anti-inflammatory Psoralen Derivatives
Nitrite assays. At the 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
[15] (0.1% naphthalethylenediamine 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 the
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 sodium
orthovanadate, 20 μg/mL leupeptin, 50 μg/mL aprotinin,
5 mM NaF, 2 mM sodium pyrophosphate, 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.
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 50-CCTGGTACGGGCATTGCT-30 and
50-GCTCATGCGGCCTCCTT-30 (forward and reverse
mouse iNOS primers, respectively, both 300 nM);
50-CAGCAGCGGCTCCATGACTCCC-30 (mouse iNOS
probe, 150 nM, containing 6-FAM as 50-reporter dye and
TAMRA as 30-quencher); 50-GCATGGCCTTCCGTGT
TC-30 and 50-GATGTCATCATACTTGGCAGGTTT-30
(forward and reverse mouse glyceraldehyde-3-phosphate
dehydrogenase [GAPDH] primers, respectively, both
300 nM); and 50-TCGTGGATCTGACGTGCCGCC-30
(mouse GAPDH probe, 150 nM, containing 6-FAM as
50-reporter dye and TAMRA as 30-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 calculating the
results, the iNOS mRNA levels were first normalized
against GAPDH [16].
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 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 1 h at room temperature and incubated overnight
at +4°C with 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 quantified 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 [16].
Luciferase-activity. The firefly luciferase activity was
measured using the luciferase assay reagent (Promega),
and the results were normalized to the total cellular
protein (measured by the Coomassie Blue method).
Enzyme-linked immunosorbent assay (ELISA). Culture
medium samples were kept at ꢀ20°C until assayed.
The concentration of mouse IL-6 (DuoSet ELISA,
R&D Systems Europe Ltd., Abindgon, UK) was deter-
mined by ELISA according to the manufacturer’s
instructions.
RNA extraction and quantitative reverse transcription
polymerase chain reaction (qRT-PCR).
After 3 or 8 h
incubation, cell monolayers were rapidly washed with
ice-cold phosphate-buffered saline, and cells were
homogenized using 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
Statistics.
The results are expressed as the
mean SEM. 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.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. M. Timonen, K. Vuolteenaho, T. Leppänen, R. M. Nieminen,
P. Aulaskari, J. Jänis, P. Vainiotalo, and E. Moilanen
Vol 000
[7] Mohareb, R. M.; Megally Abdo, N. Y. Chem Pharm
Bull(Tokyo) 2015, 63, 678.
[8] Keri, R. S.; Sasidhar, B. S.; Nagaraja, B. M.; Santos, M. A. Eur
J Med Chem 2015, 100, 257.
[9] Yeh, J. Y.; Coumar, M. S.; Horng, J. T.; Shiao, H. Y.; Kuo, F.
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Chen, C. J.; Shih, S. R.; Hsu, J. T.; Liao, C. C.; Chao, Y. S.; Hsieh, H.
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Acknowledgments. The University of Eastern Finland (UEF),
the Competitive Research Funding of Tampere University
Hospital, and the Paulo Foundation, Finland, are acknowledged
for the funding. Ms. Sanna Hiltunen, Mrs. Taina Nivajärvi, and
Ms. Sonja Niskanen are acknowledged for their participation in
the laboratory analyses and Mrs. Heli Määttä for her skillful
secretarial help.
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[16] Leppänen, T.; Korhonen, R.; Laavola, M.; Nieminen, R.;
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SUPPORTING INFORMATION
[5] Chen, Q.; Chen, X.; Zhu, L.; Chen, H.; Ho, H.; Yeung, W.;
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[6] Khan, S.; Shehzad, O.; Cheng, M. S.; Li, R. J.; Kim, Y. S. J
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Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet