Investigations concerning the correlation of COX-1 inhibitory and hydroxyl radical scavenging activity

Article abstract of DOI:10.1002/ardp.200700247

The aim was to study the COX-1 inhibiting efficacy in context with hydroxyl radical scavenging properties of compounds bearing a carboxylic acid and ester function, respectively. In general, the acids are more potent radical scavengers than the corresponding esters but there is no clear correlation with their COX-1 inhibiting potencies. A feasible scavenging mechanism of carboxylic acids is discussed.

Full text of DOI:10.1002/ardp.200700247

Arch. Pharm. Chem. Life Sci. 2008, 341, 281–287
M. Scholz et al.
281
Full Paper
Investigations Concerning the Correlation of COX-1 Inhibitory
and Hydroxyl Radical Scavenging Activity
Michael Scholz, Holger Ulbrich, Andreas Mattern, Jan-Peter Kramb, Werner Kiefer,
and Gerd Dannhardt
Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Johannes Gutenberg-
University, Mainz, Germany
The aim was to study the COX-1 inhibiting efficacy in context with hydroxyl radical scavenging
properties of compounds bearing a carboxylic acid and ester function, respectively. In general,
the acids are more potent radical scavengers than the corresponding esters but there is no clear
correlation with their COX-1 inhibiting potencies. A feasible scavenging mechanism of carbox-
ylic acids is discussed.
Keywords: Carboxylic acids / COX-1 / Hydroxyl radical scavenging / SAR /
Received: November 30, 2007; accepted: January 18, 2008
DOI 10.1002/ardp.200700247
The formation of hydroxyl radicals is also increased in
the progression of Parkinson's disease. High levels of
hydrogen peroxide are formed through degradation of
dopamine by monoamine oxidases in the substantia
nigra (SN). High levels of iron can be found in SN, too.
This presence of iron leads to an increased formation of
hydroxyl radicals through the Fenton reaction leading to
an increased nigral cell degeneration [3].
We describe the synthesis of pyrrole and isothiazole
derivatives with carboxylic acid and ester functions,
respectively, and their hydroxyl radical scavenging activ-
ity. To correlate 9OH radical scavenging and COX-1 inhibi-
tory potencies typical acidic COX-1 inhibitors such as
diclofenac, acetylsalicylic acid (ASA) and indomethacin
were tested, too. We also present structure-activity rela-
tionships (SAR) and a proposal for the hydroxyl radical
scavenging mechanism.
Introduction
A wide range of drugs including NSAIDs, glucocorticoids
or immunosuppressive drugs are used for therapy of
chronic inflammatory diseases like rheumatoid arthritis,
Crohn's disease and others. An additional strategy could
be the scavenging of reactive oxygen species (ROS).
In Alzheimer's disease, the accumulation of iron in the
hippocampus and cerebral cortex leads to an increased
formation of 9OH through the Fenton reaction [1] and for
this reason to an increase of oxidative damage in brain
tissue.
In rheumatoid synovitis, the cavity pressure is raised
and exceeds the capillary perfusion pressure. This causes
a collapse of blood vessels leading to a higher generation
of ROS. Super oxide anion and hydrogen peroxide do not
damage most of macromolecules but can be easily con-
verted into the highly reactive hydroxyl radical which
oxidizes IgGs, lipids and lipoproteins [2].
Chemistry
Compounds 4, 5, 7 and 8 were synthesized starting from
Correspondence: Holger Ulbrich, Institute of Pharmacy, Department of
Pharmaceutical and Medicinal Chemistry, Johannes Gutenberg-Univer-
sity, Staudingerweg 5, D-55128 Mainz, Germany.
E-mail: ulbrich@uni-mainz.de
3-chloro-2,3-di(4-methoxyphenyl)acrylaldehyde
1
(Scheme 1). The synthesis of 1 was described previously
[4]. Compound 1 was converted with ammonium thiocya-
nate analogous to reference [5] to 4,5-di(4-methoxy-
phenyl)-isothiazole 2. Compound 2 was N-alkylated with
ethyl 2-bromoacetate to yield 3 [6] in moderate yields
Fax: +49 6131 39-23062
Abbreviations: reactive oxygen species (ROS); 5-(diethoxyphosphoryl)-
5-methyl-1-pyrroline-N-oxide (DEPMPO); electron paramagnetic reso-
nance (EPR)
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Arch. Pharm. Chem. Life Sci. 2008, 341, 281–287
Scheme 3. A: Fenton reaction as hydroxyl radical source; B:
Spin trap reaction of DEPMPO with 9OH.
Reaction conditions: i) NH4SCN, acetone, reflux 3.5 h, NaHCO3; ii) BrCH2CO2Et,
EtOH, reflux, 6 h; iii) Et3N; iv): EtOH, KOH, 18-crown-6, 608C, 14 h.
showing additional hydroxyl radical scavenging poten-
cies.
Scheme 1. Synthesis route of compounds 1–5.
The Fenton reaction was used as hydroxyl radical
source and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-
N-oxide (DEPMPO) was used as spin trap reagent
(Scheme 3, see Experimental). To quantify the impact of
the test compounds, the spin-trap method was used [12].
Figure 1 shows the effect of thioctic acid as potent
hydroxyl radical scavenger.
Table 1 summarises the impact of the carboxylic acid
compared to the corresponding esters. Compounds 5, 8,
oxalic acid, thioctic acid and even acetic acid showed sig-
nificant 9OH scavenging efficacies in our standard elec-
tron paramagnetic resonance (EPR) assay [6, 13, 14]. In
each case, the acids are much more potent than the
esters. The highest increase in activity was found compar-
ing the ester 4 (10% inhibition at 100 lM) and the corre-
sponding acid 5 (IC50: 14 lM) as well as diethyl oxalate
(25% inhibition at 100 lM) and oxalic acid (IC50: 8.7 lM).
The esters 4, 7, diethyl oxalate and ethyl acetate pos-
sessed no or poor activity, and ethyl thioctate (IC50:
57 lM) showed a fivefold reduced potency compared to
the free acid (IC50: 12 lM). The relative high potency of
ethyl thioctate might be explained by the scavenging
potency of the 1,2-dithiolane moiety of the molecule.
Stary et al. published that a-thioctic acid can be oxidised
by singlet oxygen to its sulfoxide derivative [15] – a mech-
anism which has been also confirmed by Mattern using
the Fenton reaction [14].
Based on these results, we investigated the scavenging
mechanism of the acids. The first assumption that acids
may act as complexing agents for iron ions could be dis-
proved using instead of a FeSO4 solution a fresh prepared
FeSO4/EDTA solution in our EPR assay. EDTA forms a very
stable complex with the iron ions so the iron ions should
not be inactivated by reaction with the acids. Further-
more, the EDTA/Fe2+-complex increases the production of
9OH boosting the reduction potency of Fe2+. This leads to
intensified DEPMPOOH signals [16].
Reaction conditions: v) H2O2, glacial acid, 20 min, 808C; vi) DMF, NaH, 10 min,
BrCH2CO2Et; iv) EtOH, KOH, 18-crown-6, 608C, 14 h.
Scheme 2. Synthesis route of compounds 6–8.
(60%). Treatment of 3 with trimethylamine led to the pyr-
role derivative 4 (50%) using a method described by Rolfs
et al. and Schmidt et al. [7, 8]. This compound was previ-
ously prepared by Gupton et al. [9] using a different
method. Compound 4 was saponified using potassium
hydroxide according to ref. [10] obtaining the carboxylic
acid 5. 1H-NMR single frequency decoupling (sfd) experi-
ments and nuclear Overhauser effect (NOE) spectroscopy
studies of 5 confirmed the chemical shift of NH
(11.77 ppm) and OH (12.05 ppm) as well as both aromatic
AB systems (7.06 ppm/6.81 ppm and 6.94 ppm/6.72 ppm).
Compound 2 (Scheme 2) was oxidized with hydrogen
peroxide [11] to yield the isothiazole-3(2H)on-1,1-dioxide
derivative 6. In the next step, N-alkylation of 6 to 7 [6] and
subsequent saponification [10] results in formation of
the carboxylic acid 8.
Results and discussion
Compounds 4, 5, 7 and 8 have been designed in context
with diaryl isoselenazoles [4] as dual COX/LOX inhibitors
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Arch. Pharm. Chem. Life Sci. 2008, 341, 281–287
Correlation of OH Radical Scavenging and COX-1 Activity
283
Figure 1. EPR spectra: A: acetonitrile control (0% inhibition); B: thioctic acid 100 lM (100% inhibi-
tion); C: thioctic acid 10 lM (l45% inhibition), D: thioctic acid 1 lM (~0% inhibition); (for details see
Experimental).
According to these results (Table 1), no significant dif-
ference of the 9OH scavenging efficacy have been found
using the modified EPR assay indicating that 9OH scav-
enging efficacy is not due to formation of iron in com-
plexes with the acids.
Additionally, we investigated compounds 5 and 8
using a modified EPR protocol to obtain informations
about the likewise scavenging mechanism. The com-
pound concentrations were scaled up by factor 100, the
reaction was stopped after 90 s by the addition of satur-
ated sodium thiosulfate solution in order to inactivate
hydrogen peroxide. The compounds were extracted by
dichloromethane, and mass analysis was performed via
FD-MS. The FD-MS spectrum of compound 5 showed only
the molecule ion (323.7 Da). The mass analysis of 8
revealed that in addition to the molecule peak of 8
(403.5 Da, 100% relative intensity), two weak signals
(100 Da: 9.8% relative intensity, 367 Da: 6.75% relative
intensity) could be observed. This is in contrary to the
expected OH binding to these molecules. Thus, it is likely
that carboxylic acids may inactivate 9OH by an alternative
mechanism.
In order to elucidate the mechanism, we refer to a cou-
ple of publications which identified acids as 9OH-scav-
engers. This is in line for 5,6-dihydroxyindole-2-carbox-
ylic acid (DHICA) which was more powerful than 5,6-dihy-
droxyindole (DHI) on the Fenton-promoted oxidation of
deoxyribose [17].
Scheme 4. Proposed 9Oh scavenging mechanism of 5; condi-
tions: 100 lM compound 5, 200 lM DEPMPO, 25 lM H2O2 and
50 lM FeSO4 in 200 lM H2O.
Under the pH conditions used (l10.4), the formed car-
boxylate ions may react with hydroxyl radicals produc-
ing carboxylate radicals which subsequently can be
reduced by excessive Fe2+ ions (Scheme 4) which were
added in a twofold excess.
Despite the inactivation of hydroxyl radicals, there is
no correlation of the COX-1 inhibiting efficacies as sug-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 281–287
Table 1. Hydroxyl radical scavenging potencies.
Compound
9OH radical
9OH radical
COX-1 IC50
(lM)
assaya) IC50
assaya) EDTA, IC50 (lM)
(lM)
4
5
7
8
10%*
14
0%*
71%*
n.t.
11
n.t.
79%*
0.075
10.8
0.11
2.4
25%*
8.7
n.t.
7.4
n.t.
n.t.
n.t.
57
12
55
10
26% [10 lM]
0%*
n.t.
n.t.
n.t.
n.t.
54%*
83%*
n.t.
n.t.
n.t.
8.6 (89%*)
90%*
88%*
88%*
88%*
93%*
n.t.
n.t.
diclofenac
ASA
85
27
13
n.t.
n.t.
n.t.
0.001
0.43
indomethacin
0.004
a)
Values are means of three determinations.
* Inhibition at a concentration of 100 lM.
n. t.: not tested.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 281–287
Correlation of OH Radical Scavenging and COX-1 Activity
285
started. Instrumental parameters were as follows: receiver gain
3.996105, modulation amplitude 3.0 G, time constant 0.64 ms,
conversion time 81.92 ms, attenuation 4 ms. Spectra were ana-
lysed with the WinEPR1 (Version 1.0) software. The raw data
were integrated, the background noise was subtracted and the
heights of the second peaks were analysed. Each concentration
was tested three times. Thioctic acid in test concentrations of
100, 10 and 1 lM was used as standard substance in every test
series. For an amount of 30 spectra totalling six zero values as
controls were analysed.
gested by our test results of the compounds (Table 1):
diclofenac and indomethacin behaved both as potent
COX-1 inhibitors (IC50 COX-1: 0.001 vs. 0.004 lM) but indo-
methacin inhibited the hydroxyl radicals over sixfold
more potent than diclofenac (IC50 OH radical assay: 13 lM
vs. IC50 OH radical assay: 85 lM), almost as potent as
thioctate (IC50 OH radical assay: 12 lM). Interestingly,
thioctate provided only a weak COX-1 inhibitors activity
(26% inhibition at a concentration of 10 lM). The weak
COX-1 inhibitor ASA (IC50 COX-1: 0.43 lM) showed a good
ROS inhibiting activity (IC50 OH radical assay: 27 lM).
Compound 5 (IC50 OH radical assay: 14 lM) displayed a
good ROS inhibiting activity equipotent to indomethacin
but showed a weak activity in our COX-1 assay (IC50 COX-
1: 10.8 lM). In contrast, compound 7 exhibited a potent
COX-1 inhibitory activity (IC50 COX-1: 0.1 lM) without
showing ROS scavenging activity (0% at a concentration
of 100 lM). Noticeable is the fact, that with regard to the
COX-1 inhibitory activities, the esters 4 and 7 (IC50 COX-1:
0.075 vs. IC50 COX-1: 0.11 lM) are much more potent than
the corresponding free acids 5 and 8 (IC50 COX-1: 10.8 vs.
IC50 COX-1: 2.4 lM).
Modified EPR assay
The difference of the modified EPR assay was the usage of 2 lL
fresh prepared FeSO4 (5 mM)/EDTA (5 mM) solution instead of
the FeSO4 solution.
Scaled up 9OH scavenging assay
All reagent amounts of the standard assay, except for the not
required DEPMPO solution were multiplicated by factor 100.
The following amounts were added to 19.4 ml water: 0.2 ml com-
pound solution (100 lM), 0.2 ml FeSO4 solution (50 lM) and
0.2 ml hydrogen peroxide (25 lM). The reaction was stopped
after 90 s with 2 ml saturated sodium thiosulfate solution. The
preparation was extracted with dichloromethane (3620 ml).
The organic layers were dried over Na2SO4 and concentrated
under reduced pressure. The residue was analysed by FD-MS.
In conclusion, the hydroxyl radical scavenging
potency of compounds bearing a carboxylic acid moiety
has taken into account testing potential new NSAIDs but
the impact of it to the overall inflammatory potency
depends on the complete chemical structure of the
drugs.
Chemicals, materials and spectroscopy
The EPR spectra were acquired on a Bruker EMX EPR spectrome-
ter (Bruker Bioscience, USA). Melting points were determined
with a Bꢁchi apparatus according to Dr. Tottoli and are uncor-
rected (Bꢁchi Labortechnik, Flawil, Switzerland).1H-NMR spectra
(300 MHz) were recorded on a Bruker AC 300 spectrometer
(Bruker). Column chromatography was performed with Merck
silica gel 60 (0.063–0.200 mm; Merck, Germany). The progress
of the reactions was monitored by thinlayer chromatography
(TLC) performed with Merck silica gel 60 F245 plates. All reagents
and solvents were obtained from commercial sources and used
as received. Reagents were purchased from Sigma-Aldrich
Chemie Steinheim, Germany, or Acros, Nidderau, Germany.
The authors have declared no conflict of interest.
Experimental
Enzyme assay
COX-1 inhibition was determined by using platelets isolated
from bovine blood, incubated with the test substance, and
stimulated by the Ca ionophor A23187. The inhibition of COX-1
was assessed by quantitative HPLC determination of the forma-
tion of 12-hydroxyheptadecatrienoic acid [18].
Chemistry
4,5-Di(4-methoxyphenyl)-isothiazole 2
Compound 2 was prepared in a manner analogous to reference
[5]. A mixture of 3-chloro-acrylaldehyde 1 (33.34 mmol) and
ammonium thiocyanate (100 mmol) in acetone (80 ml) was
heated under reflux for 3.5 h (caution HCN formation!). After
cooling to rt, the mixture was poured into a solution of satur-
ated aqueous sodium hydrogen carbonate (100 ml) and
extracted three times with ether. The combined organic layers
were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. After column chromatography (silica gel,
ethyl acetate/petroleum ether 3 : 7) 60% of the isothiazole 2 was
obtained as white solid; mp. 818C, 1H-NMR: DMSO-d6, 300 MHz, d
[ppm] = 8.61 (s, 1H, CHN), 7.245 (d, 8.69 Hz, 4H, Ar-H), 6.95 (t,
8.95 Hz, 4H, Ar-H), 3.76 (s, 3H, OCH3), 3.75 (s, 3H, OCH3); FD-MS
m/z: 297.6 [M+]. Anal. calcd. for C17H15NO2S (%): C 68.66, H 5.08, N
4.71, S 10.78. Found (%): C 69.01, H 5.00, N 4.64, S 10.62.
EPR spin trapping assay
Standard EPR assay
Assay for hydroxyl radical scavenging: DEPMPO was purchased
from Calbiochem (San Diego, CA, USA). The test solution was pre-
pared by adding 2 lL of a solution of the test substance in aceto-
nitrile (concentration 10, 1 or 0.1 mM) or acetonitrile, as zero
value, to 192 ml demineralized water in a 1.5 ml micro-centri-
fuge tube. DEPMPO solution (2 lL; 20 mM), 2 lL FeSO4 solution
(5 mM) and 2 lL hydrogen peroxide solution (2.5 mM) were put
on different points on the wall of the tube. The reaction was
started by vortexing the tube. After 10 s, a sample was gathered
with a 100 lL ringcap from Hirschmann1 Laborgerꢀte and
closed with wax. After exactly 90 s, the EPR experiment was
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Ethyl 4,5-di(4-methoxyphenyl)isothiazoliumbromid-2-
4,5-Di(4-methoxyphenyl)-isothiazole-3(2H)-on-1,1-
acetat 3
dioxide 6
Compound 3 was prepared in a manner analogous to reference
Compound 2 (20 mmol) was dissolved in glacial acetic acid and
heated to 808C. In a period of two minutes, 20 ml hydrogen per-
oxide (30% v/v) was added dropwise. The preparation was stirred
for 20 min at 808C. After this period, the solution was cooled
down. Product 6 precipitates as yellow solid. The precipitate was
filtered and washed with an acidified and saturated sodium thi-
osulfate solution (freshly produced and filtered). The product
was purified by recrystallization in diluted acetic acid. Mp.
3118C, 1H-NMR: DMSO-d6, 300 MHz, d [ppm] = 7.39 (d, 8.9 Hz, 2H,
Ar-H), 7.3 (d, 8.85 Hz, 2H, Ar-H), 7.01 (d, 8.9 Hz, 2H, Ar-H), 6.95 (d,
8.85 Hz, 2H, Ar-H), 3.77 (s, 3H, OCH3), 3.76 (s, 3H, OCH3); FD-MS
m/z: 345.4 [M+].
[6].
A mixture of 2 (10 mmol) and ethyl 2-bromoacetate
(30 mmol) in 50 ml dry ethanol was heated under reflux for 6 h.
The preparation was concentrated under reduced pressure. The
remaining brown oil was dissolved in dichloromethane. In the
next step, ether was added to the solution and compound 3 crys-
tallized in green needles (yield: 60%). The crystallization process
1
has to be repeated until the product is clean; mp. 1328C, H-
NMR: DMSO-d6, 300 MHz, d [ppm] = 9.46 (s, 1H, CHN), 7.42 (d,
8.74 Hz, 2H, Ar-H), 7.29 (d, 8.71 Hz, 2H, Ar-H), 7.09 (d, 8.8 Hz, 2H,
Ar-H), 7.04 (d, 8.75 Hz, 2H, Ar-H), 5.69 (s, 2H, NCH2-COO), 4.38 (q,
7.11 Hz, 7.11 Hz, 7.14 Hz, 2H, OCH2), 3.82 (s, 3H, OCH3 ), 3.78 (s,
3H, OCH3), 1.28 (t, 7.11 Hz, 7.11 Hz, 3H, CH3).
Ethyl-2-(4,5-di(4-methoxyphenyl)-isothiazole-3(2H)-on-
1,1-dioxide-2-yl)-acetate 7
Ethyl 3,4-di(4-methoxyphenyl)-1H-pyrrol-2-carboxylate 4
Compound 4 was prepared in a manner analogous to reference
[7, 8]. Compound 3 was suspended in 5 ml trimethylamine and
heated until it dissolved. The preparation was quenched with
water. The precipitate was washed with ice cold ether and recrys-
tallized in ethanol to obtain compound 4 in yields of 50% as fine
white needles; mp. 1368C, 1H-NMR: DMSO-d6, 300 MHz, d [ppm] =
11.88 (s, 1H, NH), 7.14 (d, 3.03 Hz, 1H, CHN), 7.06 (d, 8.51 Hz, 2H,
Ar-H), 6.97 (d, 8.55 Hz, 2H, Ar-H), 6.83 (d, 8.57 Hz, 2H, Ar-H), 6.73
(d, 8.65 Hz, 2H, Ar-H), 4.06 (q, 7.05 Hz, 7.05 Hz, 7.05 Hz, 2H,
OCH2), 3.74 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 1.08 (t, 7.04 Hz,
7.04 Hz, 3H, CH3); FD-MS m/z: 351.2 [M+]. Anal. calcd. for
C21H21NO4 (%): C 71.78, H 6.02, N 3.99. Found (%): C 72.82, H 5.79,
N 3.93.
Compound 6 (5 mmol) was dissolved in 20 ml absolute DMF, and
15 mmol of sodium hydride was added slowly. The mixture
became brown and was stirred for 10 min. After this period,
7.5 mmol of ethyl 2-bromoacetate was added and the composi-
tion was stirred until the reaction was completed (TLC control).
The preparation was concentrated under reduced pressure and
purified by column chromatography (silica gel, ethyl acetate/
petroleum ether 1 : 2) to yield 45% of compound 7; m.w. 431.4;
mp. 1248C, 1H-NMR: CDCl3, 300 MHz, d [ppm] = 7.52 (d, 8.99 Hz,
2H, Ar-H), 7.38 (d, 8.95 Hz, 2H, Ar-H), 6.89 (d, 8.99 Hz, 2H, Ar-H),
6.88 (d, 8.96 Hz, 2H, Ar-H), 4.42 (s, 2H, NCH2), 4.27 (q, 7.11 Hz,
7.11 Hz, 7.09 Hz, 2H, OCH2), 3.83 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
1.31 (t, 7.13 Hz, 7.13 Hz, 3H, CH3); FD-MS m/z: 431.4 [M+]. Anal.
calcd. for C21H21NO7S (%): C 58.46, H 4.91, N 3.25, S 7,43. Found
(%): C 58.37, H 5.06, N 2.99, S 7.34.
General procedure for the synthesis of the acetic acid
derivatives 5 and 8
References
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The solution was cooled down on an ice bath and acidified with
diluted chlorine acid (2 mol/L). The preparation was extracted
with dichloromethane (3620 ml) and the combined organic
layers were concentrated under reduced pressure to obtain 5 or
8 in yields of 80%. The crystallization process has to be repeated
until the product was clean.
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