The synthesis and effect of fluorinated chalcone derivatives on nitric oxide production.

Article abstract of DOI:10.1016/S0960-894X(02)00317-7

Dimethoxy- and trimethoxychalcone derivatives, with various patterns of fluorination, were synthesized and evaluated for their influence on nitric oxide production. Some of them, chalcones 1, 5, 7, 10, 11 and 17, inhibited NO production with an IC(50) in the submicromolar range; 17 is especially noteworthy because of its potency (IC(50) 30nM). These effects were not the consequence of a direct inhibitory action on enzyme activity but the inhibition of enzyme expression.

Full text of DOI:10.1016/S0960-894X(02)00317-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1951–1954
The Synthesis and Effect of Fluorinated Chalcone Derivatives on
Nitric Oxide Production
Javier Rojas,a Miguel Paya,a Jose N. Dominguezb and M. Luisa Ferrandiza,*
a
´
Departamento de Farmacologıa, Universidad de Valencia, 46100 Burjasot, Valencia, Spain
b
´
Laboratorio de Sıntesis Organica, Facultad de Farmacia, Universidad Central de Venezuela, Caracas 1051, Venezuela
´
Received 9 January 2002; revised 15 April 2002; accepted 6 May 2002
Abstract—Dimethoxy- and trimethoxychalcone derivatives, with various patterns of fluorination, were synthesized and evaluated
for their influence on nitric oxide production. Some of them, chalcones 1, 5, 7, 10, 11 and 17, inhibited NO production with an IC50
in the submicromolar range; 17 is especially noteworthy because of its potency (IC50 30 nM). These effects were not the consequence
of a direct inhibitory action on enzyme activity but the inhibition of enzyme expression. # 2002 Elsevier Science Ltd. All rights
reserved.
Introduction
We have previously studied several chalcone derivatives
as potential anti-inflammatory agents9ꢀ11 and reported
that some of them are able to control NO, superoxide
and PG production in vitro as well as in vivo, having a
potential role in modulating the inflammatory process.
In the present paper, we describe the synthesis and
effects of seventeen dimethoxy- and trimethoxychalcone
derivatives, with various patterns of fluorination, on
NO production in LPS-stimulated murine RAW 264.7.
Activated macrophages play a key role in inflammatory
responses and release a variety of mediators, including
nitric oxide (NO).1 NO is a potent vasodilator that
facilitates leukocyte migration2 and formation of
edema, as well as leukocyte activity and cytokine pro-
duction.3 In addition, NO can also react with super-
oxide anion to form peroxynitrite, a potent oxidizing
molecule that contributes to tissue injury during
inflammatory responses.4 Nitric oxide is generated from
l-arginine by nitric oxide synthase (NOS).5 Neuronal
and endothelial NOS are constitutive, calcium-depen-
dent isoforms, whereas the inducible, calcium-indepen-
dent enzyme, inducible nitric oxide synthase (iNOS), is
expressed in many cell types in response to a diverse
range of inflammatory cytokines and bacterial metabo-
lites such as lipopolysaccharide (LPS).6,7 The inherent
activity exhibited by iNOS results in the production of
NO. In this regard, NO can enhance the release of
tumor necrosis factor-a (TNF-a) and interleukin-1b
(IL-1b). Thus, in addition to acting as a powerful effec-
tor molecule mediating the cytotoxic activities of mouse
macrophages, it can play a role in enhancing the
production of a variety of other inflammatory mediators,
and thus can contribute both directly and indirectly to the
immunopathology of macrophage-dependent inflamma-
tion.3,8
Chemistry
The general synthetic plan employed to prepare the
chalcone derivatives used the Claisen–Schmidt con-
densation, which has been previously reported.12 As
shown in Tables 1 and 2, a series of six dimethoxy-
chalcone13 (1–6) and eleven trimethoxychalcone14 (7–
17) derivatives were prepared by condensing aromatic
aldehydes and methyl ketones, using solid sodium
hydroxide in methanol at room temperature. In
most cases, the starting materials were commercially
available and the products were always obtained as
the trans-alkene (E-form) as determined by NMR
spectroscopy.12,15
Results and Discussion
Lipopolysaccharide stimulation of RAW 264.7 macro-
phages for 20 h induced iNOS with the consequent gen-
eration of large quantities of NO. As shown in Tables 1
*Corresponding author. Tel.: +34-9639-83284; fax: +34-9638-64943;
e-mail: luisa.ferrandiz@uv.es
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00317-7

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J. Rojas et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1951–1954
Table 1. Dimethoxychalcone derivatives
Compd
Structure
% Yield
Mp
(ꢁC)a
Nitriteb
(% inhibition)
IC50
(mM)c
1
2
3
4
5
86
73
56
86
84
93–95
82–84
77.5ꢂ3.4**
46.8ꢂ3.5**
55.7ꢂ5.1**
52.4ꢂ2.5**
99.0ꢂ0.5**
0.84 (0.46–1.81)
ND
ND
115–116
103–105
92–94
ND
0.91 (0.43–1.85)
6
64
88–90
60.1ꢂ4.0**
ND
Details of the assay procedures are provided.16
aMps are uncorrected.
bResults show meanꢂSEM of percentages of inhibition at the concentration of 10 mM (n=6). *p<0.05; **p<0.01, Dunnett’s test.
cValues represent the concentration required to produce 50% inhibition of the response, along with the 95% confidence limits. ND, not
determined.
and 2, under these conditions some of the chalcones tested
inhibited the generation of this mediator at 10mM. The
IC50 values for the inhibition of nitrite accumulation of the
most active (more than 65% inhibition at 10 mM) chalcone
derivatives were determined. At the highest concentration
tested (10 mM), these compounds did not exert cytotoxic
effects (<5%) during the 20 h incubation period as
indicated by MTT reduction (data not shown).
From the structure–activity study, the following trends
can be deduced: the trimethoxychalcone derivatives
with a fluoro substituent at position C-40 (14, 17) were
found to be better inhibitors of nitrite production than
with a trifluoromethyl substituent at the same position
(13, 16). The presence of a trifluoromethyl group at C-
20, in dimethoxychalcone as well as in trimethoxy-
chalcone derivatives, is associated with a very potent
inhibition of nitrite accumulation (1, 5, 7, 11). In con-
trast, this substituent is less interesting at position C-30
(8, 12) or C-40 (2, 6, 9, 13, 16). The influence of the tri-
methoxy moiety on the activity depends strongly on the
substitution pattern of the fluorine/CF3 on the benzoyl
portion of the compound and the best two compounds
are 7 and 17.
To determine if the inhibition of nitrite production was
either due to interference with the enzyme induction by
LPS or due to direct action of this compound on NOS
activity, chalcone derivatives were incubated for 2h
with cells after the induction of the enzyme by LPS. No
significant reduction of nitrite production during these
2h was observed (data not shown). This suggests that
the inhibition of nitric oxide production by some chal-
cone derivatives in macrophages may occur at the level
of enzyme expression.
Compounds that inhibit excess production of NO by
macrophages might be of benefit for the prevention and
treatment of autoimmune diseases, septic shock and
different inflammatory pathologies.
Western blot analysis was carried out on lysates of
macrophages obtained as described. LPS induced iNOS
expression which correlated with an increase in nitrite
accumulation in the medium (Fig. 1). The addition of
selected chalcone derivatives (1, 5, 7, 10, 11, 12, 17) and
the reference compound, dexamethasone, reduced iNOS
expression as well as nitrite levels.
Figure 1. Effect of fluorinated chalcone derivatives (10 mM) on iNOS
expression in RAW 264.7 cells. B, normal cells (without LPS); C,
control (with LPS); Dex, dexamethasone.

J. Rojas et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1951–1954
1953
Table 2. Trimethoxychalcone derivatives
Compd
Structure
% Yield
Mp
(ꢁC)a
Nitriteb
(% inhibition)
IC50
(mM)c
7
62153–155
73.7
ꢂ3.1**
ꢂ1.5
0.28 (0.15–0.47)
8
62130–1316.7
2
ND
9
56
110–11249.3
ꢂ3.5**
ND
10
11
82108–110
81.7
ꢂ6.5**
0.64 (0.43–1.12)
1.02(0.81–1.70)
78
129–130
76.3ꢂ5.1**
12
13
14
15
72
60
83
61
123–125
75.0ꢂ2.6**
43.6ꢂ4.6*
ꢂ1.9**
1.84 (0.54–1.16)
156–158
ND
ND
ND
140–14263.9
138–140
59.0ꢂ8.7*
16
17
58
64
228–230
108–109
55.8ꢂ8.9*
ND
94.1ꢂ1.4**
0.03 (0.02–0.47)
Details of the assay procedures are provided.16
aMps are uncorrected.
bResults show meanꢂSEM of percentages of inhibition at the concentration of 10 mM (n=6). *p<0.05; **p<0.01, Dunnett’s test.
cValues represent the concentration required to produce 50% inhibition of the response, along with the 95% confidence limits. ND, not
determined.
Acknowledgements
auspices of the CYTED Program (Programa Ibero-
americano de Ciencia y Tecnologıa para el Desarrollo),
subprograma X.6 (PIBARTRI). Javier Rojas was the
recipient of a Research Fellowship from the ALFA
Program of The European Union at the University of
Valencia.
This work was supported by grants from CICYT
(SAF2001-2639), Spanish Ministerio de Ciencia y Tec-
nologıa and 06-30-4544-99 from CDCH-UCV, Vene-
zuela. The collaborative work was performed under the

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J. Rojas et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1951–1954
4H), 7.88 (d, J=8.2Hz, 1H, 6 0-H). (13): H NMR (CDCl3) d
1
References and Notes
4.53 (s, 3H, OCH3), 4.68 (s, 3H, OCH3), 4.72(s, 3H, OCH ),
3
6.38 (d, J=2.1 Hz, 1H), 6.58 (d, J=1.8 Hz, 1H), 7.22 (d,
J=16.0 Hz, 1H, a-H), 7.28–7.65 (m, 3H), 7.70 (d, J=15.6 Hz,
1H, b-H), 7.85 (d, J=8.0 Hz, 1H, 60-H). (14): 1H NMR
(CDCl3) d 3.59 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.91 (s,
3H, OCH3), 6.48 (d, J=2.1 Hz, 1H), 6.66 (d, J=2.0 Hz, 1H),
7.20 (d, J=15.8 Hz, 1H, a-H), 7.38–7.47 (m, 3H), 7.67 (d,
1. Laskin, D. L.; Pendino, K. J. Annu. Rev. Pharmacol. Tox-
icol. 1995, 35, 655.
2. Wright, C. D.; Mulsch, A.; Busse, R.; Osswald, H. Bio-
chem. Biophys. Res. Commun. 1989, 160, 813.
3. Marcinkiewicz, J.; Grabowska, A.; Chain, B. Eur. J.
Immunol. 1995, 25, 947.
J=15.9 Hz, 1H, b-H), 7.78 (d, J=8.6 Hz, 1H, 60-H). (15): H
1
4. Kaur, H.; Halliwell, B. FEBS Lett. 1994, 350, 9.
5. Knowles, R. G.; Moncada, S. Biochem. J. 1994, 298, 249.
6. MacMicking, J.; Xie, Q. W.; Nathan, C. Annu. Rev.
Immunol. 1997, 15, 323.
7. Xie, Q. W.; Nathan, C. J. Leucocyte Biol. 1994, 56, 576.
8. Alcaraz, M. J.; Guillen, I. Curr. Pharm. Des. 2002, 8, 125.
9. Herencia, F.; Ferrandiz, M. L.; Ubeda, A.; Dominguez,
J. N.; Charris, J. E.; Lobo, G. M.; Alcaraz, M. J. Bioorg. Med.
Chem. Lett. 1998, 8, 1169.
10. Herencia, F.; Ferrandiz, M. L.; Ubeda, A.; Guillen, I.;
Dominguez, J. N.; Charris, J. E.; Lobo, G. M.; Alcaraz, M. J.
FEBS Lett. 1999, 453, 129.
11. Herencia, F.; Ferrandiz, M. L.; Ubeda, A.; Guillen, I.;
Dominguez, J. N.; Charris, J. E.; Lobo, G. M.; Alcaraz, M. J.
Free Radic. Biol. Med. 2001, 30, 43.
NMR (CDCl3) d 4.48 (s, 3H, OCH3), 4.56 (s, 3H, OCH3), 4.61
(s, 3H, OCH3), 6.85–7.90 (m, 6H), 8.02(d, J=15.8 Hz, 1H, b-
H). (16): 1H NMR (CDCl3) d 4.61 (s, 3H, OCH3), 4.86 (s, 3H,
OCH3), 4.92(s, 3H, OCH 3), 7.54–7.75 (m, 6H), 7.80 (d,
J=15.9 Hz, 1H, b-H), 7.92(d, J=8.0 Hz, 1H, 60-H). (17): H
1
NMR (CDCl3) d 3.76 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.95
(s, 3H, OCH3), 7.62(d, J=15.6 Hz, 1H, a-H), 7.64–7.75 (m,
5H), 7.78 (d, J=15.8 Hz, 1H, b-H), 7.96 (d, J=8.5 Hz, 1H, 60-
H). In addition, all compounds had IR, LSIMS and elemental
analysis in complete agreement with the assigned structures.
15. The substituted methyl ketone (1 mmol) and the aldehyde
(1 mmol) were dissolved in a minimum amount of methanol
(normally 2–4 mL) and then a single NaOH pellet was added
(about 100 mg). The reaction mixture was stirred at room
temperature until an off-white to bright yellow solid was
formed (within a few min to 24 h). The solid was collected by
filtration and washed with cold methanol. The product was
recrystallized from appropriate solvent(s) whenever necessary.
16. Cell culture: The mouse macrophage cell line RAW 264.7
was cultured in DMEM medium containing 2mM l-gluta-
mine, 100 U/mL penicillin, 100 mg/mL streptomycin and 10%
fetal bovine serum. Cells were resuspended at a concentration
of 2 ꢃ 106/mL and co-incubated with Escherichia coli LPS
(1 mg/mL) at 37 ꢁC for 20 h in the presence of test compounds
or vehicle. The nitrite concentration as a reflection of NO
release was assayed fluorometrically. The amount of nitrite
was obtained by extrapolation from a standard curve with
sodium nitrite as a standard. The mitochondrial-dependent
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) to formazan was used to assess the
possible cytotoxic effects of compounds. iNOS activity in
intact cells: RAW 264.7 macrophages stimulated for 20 h with
LPS were washed and Hank’s buffer supplemented with
l-arginine (0.5 mM) was added for 2h incubation with test
compounds to determine their effects on iNOS activity.
Supernatants were collected for the measurement of nitrite
accumulation for the last 2h fluorometrically as above. Wes-
tern blot assay: Cellular lysates from cell line RAW 264.7 (106/
well) incubated for 18 h with LPS were obtained with lysis
buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl,
and 25 mM Tris, pH 7.4). Following centrifugation (10,000ꢃ
g, 15 min), supernatant protein was determined and 25 mg
protein were loaded on 12% SDS–PAGE and transferred onto
nitrocellulose membranes for 90 min at 125 mA. Membranes
were blocked in PBS–Tween 20 containing 3% w/v unfatted
milk and incubated with anti-iNOS polyclonal antibody (1/
1000 dilution). Blots were washed and incubated with perox-
idase-conjugated goat anti-rabbit IgG (1/20,000 dilution;
Dako; Glostrup, Denmark). The immunoreactive bands were
visualized using an enhanced chemiluminescence system (ECL;
Amersham Iberica, Spain). Statistical analysis: Statistical eva-
luation included one-way analysis of variance (ANOVA) fol-
lowed by Dunnett’s test for multiple comparisons. p Values of
p<0.05 (*) or p<0.01 (**) were taken as significant. Results
are shown as meanꢂSEM for n experiments. Inhibitory con-
centration 50% (IC50) values were calculated from at least
four significant concentrations.
12. Li, R.; Kenyon, G. L.; Cohen, F. E.; Chen, X.; Gong, B.;
Dominguez, J. N.; Davidson, E.; Kurzban, G.; Miller, R. E.;
Rosenthal, P. J.; McKerrow, J. H. J. Med. Chem. 1995, 38, 5031.
13. Spectral data for dimethoxychalcone derivatives: (1): 1H
NMR (CDCl3) d 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.46
(d, J=2.1 Hz, 1H, 3-H), 6.52 (dd, J=2.1 and 8.6 Hz, 1H, 5-H),
7.12(d, J=16.1 Hz, 1H, a-H), 7.32–7.51 (m, 5H), 7.70 (d,
J=16.1 Hz, 1H, b-H). (2): 1H NMR (CDCl3) d 3.82(s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 6.47 (d, J=2.1 Hz, 1H, 3-H), 6.58
(dd, J=1.9 and 8.5 Hz, 1H, 5-H), 7.14 (d, J=15.8 Hz, 1H, a-
H), 7.30–7.47 (m, 4H), 7.65 (d, J=15.9 Hz, 1H, b-H), 7.82(d,
J=8.7 Hz, 1H, 60-H). (3): 1H NMR (CDCl3) d 3.80 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 6.45 (d, J=2.0 Hz, 1H, 3-H), 6.56
(dd, J=2.0 and 8.6 Hz, 1H, 5-H), 7.10 (d, J=16.0 Hz, 1H, a-
H), 7.32–7.52 (m, 4H), 7.68 (d, J=15.9 Hz, 1H, b-H), 7.80 (d,
J=8.6 Hz, 1H, 60-H). (4): 1H NMR (CDCl3) d 3.88 (s, 3H,
OCH3), 3.90 (s, 3H, OCH3), 6.99 (d, J=8.4 Hz, 1H, 5-
1
H),7.63–8.19 (m, 5H), 8.20 (d, J=15.7 Hz, 1H, b-H). (5): H
NMR (CDCl3) d 3.78 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.91
(d, J=9.0 Hz 1H), 7.00–7.42(m, 5H), 7.76 (d, J=8.5 Hz 1H),
7.93 (d, J=15.9 Hz, 1H, b-H). (6): 1H NMR (CDCl3) d 3.86 (s,
3H, OCH3), 4.02(s, 3H, OCH ), 6.80–7.40 (m, 6H), 7.46 (d,
3
J=14.8 Hz, 1H, a-H), 7.70 (d, J=15.5 Hz, 1H, b-H), 8.05 (d,
J=8.0 Hz, 1H, 60-H). In addition, all compounds had IR,
LSIMS and elemental analysis in complete agreement with the
assigned structures.
1
14. Spectral data for trimethoxychalcone derivatives: (7): H
NMR (CDCl3) d 3.99 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 4.11
(s, 3H, OCH3), 6.68–8.02(m, 8H). ( 8): 1H NMR (CDCl3) d
4.03 (s, 3H, OCH3), 4.09 (s, 3H, OCH3), 4.14 (s, 3H, OCH3),
7.30 (d, J=14.8 Hz, 1H, a-H), 7.38–7.47 (m, 4H), 7.88 (d,
J=15.9 Hz, 1H, b-H), 7.98 (d, J=8.6 Hz, 1H, 60-H). (9): H
1
NMR (CDCl3) d 4.67 (s, 3H, OCH3), 4.78 (s, 3H, OCH3), 4.84
(s, 3H, OCH3), 6.35–7.70 (m, 5H), 7.80 (d, J=15.5 Hz, 1H, b-
0
1
H), 7.98 (d, J=8.1 Hz, 1H, 6 -H). (10): H NMR (CDCl3) d
3.92(s, 3H, OCH ), 4.15 (s, 3H, OCH3), 4.20 (s, 3H, OCH3),
3
6.89 (d, J=16.1 Hz, 1H, a-H), 7.28–7.85 (m, 6H). (11): 1H
NMR (CDCl3) d 4.65 (s, 3H, OCH3), 4.76 (s, 3H, OCH3), 4.78
(s, 3H, OCH3), 6.68–7.80 (m, 7H), 7.90 (d, J=15.5 Hz, 1H, b-
H). (12): 1H NMR (CDCl3) d 4.53 (s, 3H, OCH3), 4.62(s, 3H,
OCH3), 4.65 (s, 3H, OCH3), 6.32(d, J=2.1 Hz, 1H), 6.50 (d,
J=2.0 Hz, 1H), 7.15 (d, J=15.8 Hz, 1H, a-H), 7.25–7.50 (m,