Structure-activity studies on the protection of Trimetazidine derivatives modified with nitroxides and their precursors from myocardial ischemia-reperfusion injury

Article abstract of DOI:10.1016/j.bmc.2006.04.040

Trimetazidine, the known anti-anginal and anti-ischemic drug, was modified by pyrroline and tetrahydropyridine nitroxides and their hydroxylamine and sterically hindered secondary amine precursors. The synthesized new compounds proved to be better superox

Full text of DOI:10.1016/j.bmc.2006.04.040

Bioorganic & Medicinal Chemistry 14 (2006) 5510–5516
Structure–activity studies on the protection of Trimetazidine
derivatives modified with nitroxides and their precursors
from myocardial ischemia–reperfusion injury
a
b
a
b
´
´
´
Tamas Kalai, Mahmood Khan, Maria Balog, Vijay Kumar Kutala,
b
a,
*
´
´
Periannan Kuppusamy and Kalman Hideg
aInstitute of Organic and Medicinal Chemistry, University of Pe´cs, H-7602 Pe´cs, PO Box 99, Hungary
bDavis Heart and Lung Research Institute, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA
Received 21 February 2006; revised 19 April 2006; accepted 24 April 2006
Available online 12 May 2006
Abstract—Trimetazidine, the known anti-anginal and anti-ischemic drug, was modified by pyrroline and tetrahydropyridine nitrox-
ides and their hydroxylamine and sterically hindered secondary amine precursors. The synthesized new compounds proved to be
better superoxide scavenger molecules compared to the parent Trimetazidine in an in vitro experiment. This reactive oxygen species
(ROS) scavenging activity was further supported by ischemia/reperfusion (I/R) studies on Langendorff-perfused rat hearts pre-
treated with Trimetazidine and with the modified Trimetazidine derivatives before ischemia. Two of the investigated compounds,
containing 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole and 4-phenyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole substituents on
the piperazine ring, provided significant protection from the cardiac dysfunction caused by I/R. The protective effect could be attrib-
uted to the combined anti-ischemic and antioxidant effects.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
it was found to be effective in the treatment of stable
angina, alone, or when combined with conventional
anti-anginal agents,8 although Trimetazidine was
reported to cause reversible parkinsonism.9
Trimetazidine (1) is widely used in heart therapy as an
anti-ischemic agent devoid of hemodynamic effects.1
This compound inhibits the 3-ketoacyl-coenzyme A thi-
olase activity and leads to a shift in energy substrate
preference: instead of fatty acid oxidation, glucose oxi-
dation is favored.2
Trimetazidine’s relatively simple structure (1-[2,3,4-tri-
methoxy benzyl] piperazine) inspired pharmaceutical
chemists to introduce substituents on the secondary
amine of the piperazine ring. The most successful deriv-
ative is probably lomerizine 1-[bis(4-fluorophenyl)meth-
yl]-4-[(2,3,4-trimethoxyphenyl)methyl] piperazine, which
was first introduced as an anti-migraine drug.10 Other
modifications include acylation of the secondary nitro-
gen of piperazine with either 2,3-dihydro-1,4-benzodiox-
in or 1,4-benzodioxin derivatives which were then used
as lipid peroxidation inhibitors.11 Trimetazidine deriva-
tives modified by a tocopherol or hydroquinone moiety
were found to be active against lipid peroxidation and
albumin oxidation.12 Flavonoid derivatives, coupled
with 2,3,4-trimethoxybenzyl piperazines, were found to
be effective modulators of multi-drug resistance.13 Two
Trimetazidine reduces intracellular acidosis and electro-
lyte abnormalities by optimizing the oxygen demand of
the mitochondria and also inhibits ATP depletion.3,4 It
was reported that Trimetazidine inhibits the formation
of reactive oxygen species (ROS) and also protects cells
from ROS-induced injuries.5 It has been shown that Tri-
metazidine protects the heart from ischemia-induced
arrhythmias, reduces infarct size, and preserves the ef-
fects of ischemic and pharmacological preconditioning.6
These anti-ischemic effects of Trimetazidine could be
useful for the treatment of ischemic heart disease7 and
experimental
Trimetazidine
derivatives
S-15176
Keywords: Nitroxides; Langendorff-perfused heart; ROS scavenging
activity; Trimetazidine.
(2,6-di-tert-butyl-4-{3-[4-(2,3,4-trimethoxy-benzyl)-
piperazin-1-yl] propylsulfanyl}phenol) and S-16950 (6-
(4-benzyl-piperazin-1-ylmethyl)-2,3-dimethoxyphenol)
*
Corresponding author. Tel.: +36 72 536220; fax: +36 72 536219;
e-mail: kalman.hideg@aok.pte.hu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.040

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T. Kalai et al. / Bioorg. Med. Chem. 14 (2006) 5510–5516
5511
were capable of inhibiting the loss of ATP synthesis
observed in isolated mitochondria exposed to cyclospor-
in A (CSA), an immunosuppressive drug.14 These two
compounds are good candidates for use in combination
with CSA because they counteract the deleterious effects
of CSA on mitochondrial membranes, without decreas-
ing the immunosuppressive effect of CSA. Considering
the advantages of the aforementioned modifications of
Trimetazidine, it was a real challenge to synthesize
new derivatives of Trimetazidine which preserved the
original activity, but exhibited better antioxidant
activity than the parent drug. The introduction of para-
magnetic five- and six-membered nitroxides (1-oxyl-
2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole or 1-oxyl-
prepared. The treatment of nitroxides 7–9A, 11A with
HCl saturated EtOH15 gave the corresponding hydrox-
ylamine/HCl salts 7B, 8B, 9B, and 11B, respectively.
The reduction of nitroxides with Fe/AcOH24 yielded
the secondary amines 7–9C and 11C, which were
also used in the biological investigations as HCl salt
(Scheme 1).
2.2. Scavenging of superoxide radical
To compare the efficiency of 1 and novel Trimetazidine
derivatives in scavenging superoxide, EPR spectroscopy
was used to study the formation of DEPMPO–OOH ad-
ducts (Fig. 1). DEPMPO will scavenge superoxide and
form DEPMPO–OOH adduct which can be measured
by EPR. The formation of superoxide radicals was gen-
erated by xanthine/xanthine oxidase. The addition of
SOD inhibited the EPR signal almost 95%, indicating
that the formed adduct was from superoxide radicals.
The adduct formed when Trimetazidine or one of its
derivatives scavenges the superoxide radicals is not
detectable by EPR. When Trimetazidine or one of its
derivatives is combined with a reaction mixture contain-
ing DEPMPO, they compete to scavenge the superoxide
radicals. When less DEPMPO–OOH is formed, the EPR
signal is inhibited. The signal was not significantly inhib-
ited by the Trimetazidine (1), however, the compounds
being investigated: 7B, 7C, 8B, 8C, 9C, 11B, and 11C
exhibited superoxide-scavenging activity because it was
shown that less DEMPO–OOH adduct was formed.
Compounds 7B, 8B, 9C, and 11C were found to have
the greatest effect, for example, both the hydroxylamines
and the sterically hindered amines were effective in scav-
enging superoxides. It was reported that sterically
hindered amines eliminate the superoxide.25,26 In the
present case, we observed that compound 1, containing
a secondary NH in the piperazine ring, does not have
significant superoxide-scavenging activity, while the ste-
rically hindered secondary amines 7C, 8C, 9C, and 11C
are better superoxide scavengers. The reaction of nitrox-
ide or hydroxylamine with superoxide is well document-
ed27,28 (Fig. 2). Nitroxides catalyze the formation of O2
and H2O2 from superoxide. All of the derivatives exhib-
ited ROS scavenging activity as shown by the reduction
in the amount of DEPMPO–OOH adduct formed.
Based on these results of the in vitro experiments, we
concluded that further ex vivo studies for both the
hydroxylamines, ‘B form’ and the sterically hindered
amines, ‘C form’ would be useful.
2,2,6,6-tetramethyl-1,2,3,6-tetrahydro-pyridine
rings)
and their reduced forms (amines or hydroxylamines)
might accomplish this. The advantage of these modifica-
tions in cardiac drugs is the protection from the cell
damage caused by ROS during ischemia.15,16 Alkylation
of the mexiletine amino group with a 2,2,5,5-teramethyl-
pyrroline-3-yl methyl group yielded a new compound
that provided more protection from ischemia–reperfu-
sion-induced contractile dysfunction in isolated hearts
compared to the parent drug, mexiletine.17 Modification
of the diethylaminoethyl side chain of amiodarone with
this same 2,2,5,5-tetramethylpyrrolin-3-ylmethyl group
resulted in a compound which has enhanced activity
and less toxicity, as its inhibitory effect on mitochondrial
permeability is similar to that of amiodarone, but with-
out biphasic characteristics.18
Our laboratory has reported very recently that the sub-
stitution of the N-phenyl group of ebselen with a,a0-tet-
ramethyl pyrroline, pyrrolidine, or tetrahydropyridine
nitroxides and their diamagnetic precursors exhibited
better glutathione peroxidase-like (GPx) activity than
the ebselen itself.19
Our current studies have been focused on alkylation or
acylation of the piperazine secondary amine group to
create a new series of paramagnetic and diamagnetic
Trimetazidine derivatives. We also investigated their
superoxide-scavenging activity and their protection of
hearts from ischemia/reperfusion-induced contractile
dysfunction.
2. Results and discussion
2.1. Chemistry
2.3. Langendorff-heart experiments
The alkylation of the piperazine NH of Trimetazidine
(1) with a five-membered paramagnetic allylic bromide
2,20 a six-membered allylic bromide 3,21 and a five-mem-
bered allylic bromide with a 4-phenyl substituent 4,22 in
CHCl3 in the presence of K2CO3 gave nitroxides 7A, 8A,
and 9A, respectively. Treatment of compound 1 with an
N-methylpyrroline allylic bromide 5 in dioxane in the
presence of K2CO3 produced compound 10. The acylat-
ed derivative 11A was obtained by treatment of 1 with
paramagnetic acyl chloride23 (6) in CH2Cl2 in the pres-
ence of Et3N. To increase the water solubility of these
compounds, salts of the diamagnetic reduced forms were
Isolated rat hearts were first perfused with compounds
1, 7B, 7C, 8B, 8C, 9C, 10, 11B, or 11C (50 lM) for
1 min before ischemia and then subjected to global
ischemia followed by reperfusion. Coronary flow (CF),
left ventricular developed pressure (LVDP), heart rate
(HR), and rate pressure product (RPP, defined as a
product of heart rate and LVDP) were measured prior
to the start of global ischemia and during reperfusion.
The functional recovery of the CF, LVDP, and RPP
data obtained during reperfusion was expressed as a
percentage of pre-ischemic baseline values (Fig. 3).

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T. Kalai et al. / Bioorg. Med. Chem. 14 (2006) 5510–5516
5512
CH3O
CH3O
CH3O
N
NH
COCl
Br
N
1
R
a
Br
( )n
O
6
N
O
N
n
R
Y
Q
CH3
2-4
5
CH2
CH2
c
7A
0
0
H
H
O
b
d
7B
OH
e
CH2
CH2
H
O
7C
8A
0
1
H
H
d
CH2
CH2
CH2
CH2
CH2
8B
8C
9A
9B
9C
1
1
H
H
OH
H
e
R
0 Ph
0 Ph
0 Ph
O
d
e
n
R
OH
H
CH3O
CH3O
CH3O
N
N Y
N
Q
n( )
H
H
0
1
0
2
3
4
CH2 CH3
0
H
H
H
10
11A
11B
Ph
O
0
0
CO
CO OH
d
e
7-11
11C
0
H
CO
H
Scheme 1. Structures and synthesis of Trimetazidine and its derivatives. Reagents and conditions: (a) compounds 2–4 (1.1 equiv) K2CO3, CHCl3,
reflux, 3 h, 49–72%; (b) compound 5 (1.0 equiv) K2CO3, dioxane, and 18-crown-6 (cat.), reflux, 3 h, 39%; (c) compound 6, Et3N (1.1 equiv), CH2Cl2
0 ꢁC to room temperature, 1 h, 63%; (d) EtOH (saturated with HCl gas) reflux, 30 min then Et2O, 52–73%; (e) AcOH, Fe powder (10 equiv) 70 ꢁC,
1 h, then water, decant, K2CO3 extracted by CHCl3, 33–62%.
120
100
80
60
40
20
0
O2
+ H
(1)
+ O2
N
O
N
OH
*
*
O2
+ H
*
*
(2)
(3)
(4)
+ H2O2
*
N
N
O
**
OH
**
**
**
OOH + H
+ H2O2
N
O
N
O
Figure 1. Scavenging of superoxide by 1 and its derivatives. Superox-
ide radicals were generated by xanthine (0.5 mM) and XO (0.02 U/ml).
The addition of SOD almost completely inhibited the EPR signal,
indicating the formed adduct originated from superoxide radicals. The
reaction mixture containing DTPA (0.1 mM), DEPMPO (10 mM) in
air-saturated PBS, (pH 7.4) in the presence of 1 mM of 1 or one of its
derivatives was measured after 15 min of incubation using EPR
spectroscopy. Data represent means SD (n = 4), *p < 0.01 versus
control (CON), **p < 0.001 versus control (CON).
O2
+ O2
N
O
N
O
Figure 2. Possible reactions of a superoxide anion radical with a
nitroxide or a hydroxylamine.
the five-membered pyrroline nitroxide-containing com-
pounds 7C and 9C (data not shown). However, no sig-
nificant differences were observed in the recovery of
coronary flow when we compared the control, Trimetaz-
idine 1, and its derivatives. Trimetazidine 1 and
The pre-ischemic baseline values were as follows: CF:
17 4 ml/min; LVDP: 124 18 mmHg; and HR,
274 24 bpm. There was an insignificant drop, initially,
in the LVDP and the heart rate in hearts pretreated with

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T. Kalai et al. / Bioorg. Med. Chem. 14 (2006) 5510–5516
5513
probably due to the fact that compound 9C contains
both a lipophilic aromatic group and a hydrophilic
amino group with the ability to scavenge reactive oxy-
gen species (ROS), including superoxides, which are
formed during ischemia/reperfusion (I/R) in both the
lipid membrane and the hydrophilic cytosolic areas.
100
80
60
40
20
0
A
*
*
3. Conclusions
This work supports our previous observation that the
modification of a cardiac drug with nitroxides or
their reduced forms (hydroxylamines or sterically hin-
dered secondary amines) will be more beneficial since
these modified drugs inhibit the damages caused by
ROS during ischemia formed in statu nascendi. All
of the examined derivatives scavenged superoxide in
an in vitro assay, however the studies in the Langen-
dorff-perfused heart experiments demonstrated that
two compounds, both with a 2,2,5,5-tetramethyl-2,5-
dihydro-1H-pyrrol-3ylmethyl substituent on the
piperazine nitrogen, show promise for further
investigations.29
100
80
60
40
20
0
B
**
**
*
4. Materials and methods
Melting points were determined with a Boetius micro-
melting point apparatus and are uncorrected. Elemental
analyses (C, H, N, and S) were performed on a Fisons
EA 1110 CHNS elemental analyzer. Mass spectra were
recorded on a Thermoquest Automass Multi and VG
TRIO-2 instrument in the EI or thermospray (TSP)
100
80
60
40
20
0
C
**
1
mode. H NMR spectra were recorded with a Varian
UNITYINOVA 400 WB spectrometer. Chemical shifts
are referenced to Me4Si. Measurements were run at a
298 K probe temperature in D2O solution. EPR spectra
were taken on a Miniscope MS 200 in 10ꢀ4 M CHCl3
solution, and all monoradicals gave a triplet line
aN = 14.7–15.1 G. Flash column chromatography was
performed on a Merck Kieselgel 60 (0.040–0.063 mm).
Qualitative TLC was carried out on commercially
prepared plates (20 · 20 · 0.02 cm) coated with Merck
**
*
Kieselgel GF254
.
Compounds 1,30 2,18 3,21 4,22 5,31 and 624 were prepared
according to published procedures. All reagents and
solvents were purchased from Aldrich.
Figure 3. Effect of Trimetazidine (1) and its derivatives on the recovery
of (A) coronary flow (CF); (B) rate pressure product (RPP) and (C) left
ventricular developed pressure (LVDP). Rat hearts were pretreated with
50 lM 1 or its derivatives for 1 min before ischemia. Values obtained
from six independent experiments are expressed as means SD.
*p < 0.05 versus control (CON), **p < 0.001 versus control (CON).
4.1. General procedure for alkylation of Trimetazidine
(7A, 8A, and 9A)
A mixture of an allylic bromide 2, 3 or 4 (11.0 mmol),
K2CO3 (1.51 g, 11.0 mmol), and 1 (2.66 g, 10.0 mmol)
in CHCl3 (20 mL) was stirred and refluxed until the
starting materials were consumed (ꢁ2–3 h). After cool-
ing, the inorganic salts were filtered out, the organic
phase was washed with brine (15 mL), separated, dried
with MgSO4, filtered, evaporated, and then the residue
was purified by flash column chromatography (CHCl3/
Et2O) to give the title compounds as yellow or red oils
49–72%.
compounds 7B, 8B, 8C, 10, 11B, and 11C had influence
on the RPP and LVDP (Figs. 3b and c), but their effect
was not significant compared to control. The most
effective compound (9C) was obtained when a phenyl
substituent, that is, 4-phenyl-2,2,5,5-tetramethyl-2,5-
dihydro-1H-pyrrole, was introduced on the pyrroline
ring. This resulted in a significant enhancement of con-
tractile function as evidenced by the greatly increased
percent of recovery in the RPP and LVDP. It is

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T. Kalai et al. / Bioorg. Med. Chem. 14 (2006) 5510–5516
5514
4.1.1. 2,2,5,5-Tetramethyl-3-[4-(2,3,4-trimethoxybenzyl)-
piperazin-1-ylmethyl]-2,5-dihydro-1H-pyrrol-1-yloxyl
radical (7A, HO-2921). Yellow oil, 2.34 g, (56%). MS
(EI) m/z (%): 418 (M+, 3), 388 (10), 207 (48), 181
(100). Anal. Calcd for C23H36N3O4: C, 66.00; H, 8.67;
N, 10.04. Found: C, 65.88; H, 8.50; N, 10.01.
oil 1.36 g, (63%). MS (EI) m/z (%): 432 (M+, 1), 402 (4),
219 (14), 181 (100). Anal. Calcd for C23H34N3O5: C,
63.87; H, 7.92; N, 9.71. Found: C, 63.80; H, 7.85; N,
9.88.
4.4. General procedure for synthesis of hydroxylamines
(7B, 8B, 9B, and 11B)
4.1.2. 2,2,6,6-Tetramethyl-4-[4-(2,3,4-trimethoxybenzyl)-
piperazin-1-ylmethyl]-1,2,3,6-terahydropyridin-1-yloxyl
radical (8A, HO-2922). Red oil, 2.11 g (49%). MS (EI)
m/z (%): 432 (M+, 2), 417 (8), 221 (19), 181 (100). Anal.
Calcd for C24H38N3O4: C, 66.64; H, 8.85; N, 9.71.
Found: C, 66.59; H, 8.77; N, 9.62.
A solution of nitroxide 7A, 8A, 9A, or 11A (2.0 mmol)
was refluxed for 30 min in EtOH (15 mL) saturated with
HCl. After cooling, the solvent was evaporated off, and
then the residue was crystallized from Et2O or acetone
to give the title compounds as white crystals 52–73%.
4.1.3. 4-Phenyl-2,2,5,5-tetramethyl-3-[4-(2,3,4-trimeth-
oxybenzyl)-piperazin-1-ylmethyl]-2,5-dihydro-1H-pyrrol-1-
yloxyl radical (9A, HO-3629). Yellow oil, 3.56 g (72%).
MS (EI) m/z (%): 494 (M+, 3), 464 (6), 283 (55), 181
(100). Anal. Calcd for C29H40N3O4: C, 70.42; H, 8.15;
N, 8.49. Found: C, 70.39; H, 8.02; N, 8.31.
4.4.1. 1-Hydroxy-2,2,5,5-tetramethyl-3-[4-(2,3,4-trimeth-
oxybenzyl)-piperazin-1-ylmethyl]-2,5-dihydro-1H-pyrrole
3HCl salt (7B, HO-2921/OH/3HCl). White solid,
773 mg (73%), mp 206–208 ꢁC. 1H NMR (D2O),
400 MHz: d = 7.16 (d, J = 8.8 Hz, 1H, ArH), 6.89 (d,
J = 8.8 Hz, 1H, ArH), 5.82 (s, 1H, @CH), 4.29 (s, 2H,
ArCH2N), 3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3),
3.81 (s, 3H, OCH3), 3.49 (s, 2H, CH@CH–CH2), 3.42
(br s, 4H, piperazine CH2), 3.16 (br s, 4H, piperazine
CH2), 1.50 (s, 6H, 2· CH3), 1.47 (s, 6H, 2· CH3). Anal.
Calcd for C23H40Cl3N3O4: C, 52.23; H, 7.62; N, 7.94.
Found: C, 52.11; H, 7.59; N, 7.80.
4.2. Synthesis of 1,2,2,5,5-pentamethyl-3-[4-(2,3,4-tri-
methoxybenzyl)-piperazin-1-yl methyl]-2,5-dihydro-1H-
pyrrole (10, HO-3615)
A mixture of compound 1 (1.33 g, 5.0 mmol), compound
(1.16 g, 5.0 mmol), K2CO3 (690 mg, 5.0 mmol),
5
18-crown-6 (20 mg) in dioxane (15 mL) was stirred and
refluxed for 4 h. After cooling, the inorganic salts were
filtered off, the dioxane was evaporated, the residue
was partitioned between water (10 mL) and CHCl3
(20 mL), then the organic phase was separated, dried
with MgSO4, filtered, and evaporated. The residue was
purified by column chromatography (CHCl3/Et2O) to
give the title compound as a colorless oil 811 mg, 39%.
This base was dissolved in EtOH saturated with HCl,
and after evaporation of the solvent, the residue was
crystallized from Et2O to yield a white solid, mp 173–
175 ꢁC. MS (EI) m/z (%): 431 (M+, 1), 402 (21), 181
(71), 138 (100). 1H NMR (D2O), 400 MHz: d = 7.16
(d, J = 8.8 Hz, 1H, ArH), 6.88 (d, J = 8.8 Hz, 1H,
ArH), 6.05 (s, 1H, @CH), 4.34 (s, 2H, ArCH2N), 3.88
(s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 3.72 (s, 2H, CH@CH–CH2), 3.49 (br s, 4H,
piperazine CH2), 3.32 (br s, 4H, piperazine CH2), 2.71
(s, 3H, NCH3) 1.48 (s, 3H, CH3), 1.46 (s, 3H, CH3),
1.41 (s, 3H, CH3), 1.38 (s, 3H, CH3). Anal. Calcd for
C24H42Cl3N3O3: C, 54.70; H, 8.03; N, 7.97. Found: C,
54.56; H, 7.92; N, 7.90.
4.4.2. 1-Hydroxy-2,2,6,6-tetramethyl-4-[4-(2,3,4-trimeth-
oxybenzyl)-piperazin-1-ylmethyl]-1,2,3,6-terahydropyri-
dine 3HCl salt (8B, HO-2922/OH/3HCl). White solid,
650 mg (60%), mp 192–194 ꢁC, 1H NMR (D2O),
400 MHz: d = 7.16 (d, J = 8.8 Hz, 1H, ArH), 6.88 (d,
J = 8.8 Hz, 1H, ArH), 5.97 (s, 1H, @CH), 4.34 (s, 2H,
ArCH2N), 3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3),
3.81 (s, 3H, OCH3), 3.68 (s, 2H, CH@CH–CH2N),
3.51 (br s, 4H, piperazine CH2), 3.36 (br s, 4H, pipera-
zine CH2), 2.52 (s, 2H, CH@CH–CH2), 1.46 (s, 6H, 2·
CH3), 1.41 (s, 3H, CH3), 1.37 (s, 3H, CH3). Anal. Calcd
for C24H42Cl3N3O4: C, 53.09; H, 7.80; N, 7.74. Found:
C, 53.01; H, 7.79; N, 7.59.
4.4.3.
1-Hydroxy-4-phenyl-2,2,5,5-tetramethyl-3-[4-
(2,3,4-trimethoxybenzyl)-piperazin-1-ylmethyl]-2,5-dihy-
dro-1H-pyrrole 3HCl salt (9B, HO-3629/OH/3HCl).
Hygroscopic white solid, 629 mg (52%). 1H NMR
(D2O), 400 MHz: d = 7.40 (s, 3H, ArH), 7.19 (d,
J = 7.2 Hz, 2H, ArH), 7.05 (d, J = 8.8 Hz, 1H, ArH),
6.86 (d, J = 8.8 Hz, 1H, ArH), 4.16 (s, 2H, ArCH2N),
3.83 (s, 6H, 2· OCH3), 3.80 (s, 3H, OCH3), 3.30 (s,
2H, CH@CH–CH2N), 3.10 (br s, 4H, piperazine CH2),
2.95 (br s, 4H, piperazine CH2), 1.63 (s, 6H, 2· CH3),
1.43 (s, 6H, 2· CH3). Anal. Calcd for C29H44Cl3N3O4:
C, 57.57; H, 7.33; N, 6.94. Found: C, 57.55; H, 7.27;
N, 6.89.
4.3. (1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-
3-yl)-[4-(2,3,4-trimethoxy-benzyl)-piperazin-1-yl]metha-
none radical (11A, HO-3594)
A fifteen mL solution of Et3N (555 mg, 5.5 mmol) and
acyl-chloride 6 (1.11 g, 5.5 mmol) in CH2Cl2 was added
dropwise to a stirred solution of compound 1 (1.33 g,
5.0 mmol), in CH2Cl2 at 0 ꢁC. After stirring the mixture
at room temperature for 1 h, the yellow solution was
washed with brine (10 mL), the organic phase was sepa-
rated, dried with MgSO4, filtered, and evaporated. The
residue was purified by column chromatography (hex-
ane/EtOAc) to give the title compound as a thick, yellow
4.4.4. (1-Hydroxy-2,2,5,5-tetramethyl-2,5-dihydro-1H-
pyrrol-3-yl)-[4-(2,3,4-trimethoxy-benzyl)-piperazin-1-yl]-
methanone 2HCl salt (11B, HO3594/OH/2HCl). White
solid, mp 204–206 ꢁC, 588 mg (56%). H NMR (D2O),
400 MHz: d = 7.16 (d, J = 8.8 Hz, 1H, ArH), 6.89 (d,
J = 8.8 Hz, 1H, ArH), 6.20 (s, 1H, @CH), 4.30 (s, 2H,
ArCH2N), 3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3),
3.82 (s, 3H, OCH3), 3.30 (br s, 8H, piperazine
1

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T. Kalai et al. / Bioorg. Med. Chem. 14 (2006) 5510–5516
5515
4· CH2), 1.54 (s, 6H, 2· CH3), 1.51 (s, 6H, 2· CH3).
Anal. Calcd for C23H37Cl2N3O5: C, 54.54; H, 7.36; N,
8.30; Found: C, 54.50; H, 7.22; N, 8.28.
4.5.4. (2,2,5,5-Tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)-
[4-(2,3,4- trimethoxybenzyl)-piperazin-1-yl]methanone
2HCl salt (11C, HO-3595/2HCl). White solid, 539 mg
(55%), mp 217–219 ꢁC. MS: TSP: [M + H]+: 418. 1H
NMR (D2O), 400 MHz: d = 7.16 (d, J = 8.8 Hz, 1H,
ArH), 6.89 (d, J = 8.8 Hz, 1H, ArH), 6.07 (s, 1H,
@CH), 4.29 (s, 2H, ArCH2N), 3.88 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 3.30 (br s, 8H, piperazine 4· CH2), 1.59 (s,
6H, 2· CH3), 1.55 (s, 6H, 2· CH3). Analy. Calcd for
C23H37Cl2N3O4: C, 56.32; H, 7.60; N, 8.57. Found: C,
56.28; H, 7.54; N, 8.46.
4.5. General procedure for synthesis of sterically hindered
amines (7C, 8C, 9C, 11C)
Fe powder (1.12 g, 20 mmol) was added to a solution of
nitroxide 7A, 8A, 9A, or 11A (2.0 mmol) in AcOH
(10 mL) and then the mixture was warmed to 70 ꢁC until
the reaction started. The mixture was stirred at this tem-
perature for 1 h, allowed to cool, diluted with water
(15 mL), decanted, and then the decanted aqueous solu-
tion was made alkaline by adding solid K2CO3. The
mixture was extracted with CHCl3 (3· 15 mL), dried
with MgSO4, filtered, evaporated, and after chromato-
graphic purification (CHCl3/MeOH), the title amines
7C, 8C, 9C or 11C were obtained as colorless oils 33–
62%. These bases were converted to salts by dissolving
in EtOH saturated with HCl gas and then evaporating
the solvents.
4.6. Isolated rat heart preparation
Sprague–Dawley rats (body weight 300–350 g) were first
administered 60 mg/kg sodium pentobarbital (anesthe-
sia) and 500 IU/kg heparin (anti-coagulant), intraperito-
neally. After a midline sternotomy, the hearts were
rapidly excised and then perfused retrogradely at a con-
stant perfusion pressure of 80 mmHg with a modified
Krebs solution containing NaCl (120 mM), NaHCO3
(25 mM), MgSO4 (1.2 mM), KH2PO4 (1.2 mM), CaCl2
(1.2 mM), and glucose (11 mM). The perfusate buffer
was saturated with a 95% O2 and 5% CO2 gas mixture
at 37 ꢁC. A latex balloon was inserted in the left ventri-
cle via the left atrium and inflated with 0.4 ml of distilled
water, this will produce an end diastolic pressure of 8–
12 mmHg. The contractile and hemodynamic functions
of the heart were continuously monitored with a com-
puter-based data acquisition system (PC PowerLab with
Chart 5 software, ADI Instruments, Colorado Springs,
CO). The following data were measured: coronary flow
(CF), left ventricular developed pressure (LVDP), and
heart rate (HR). The rate pressure product (RPP) was
calculated as LVDP · HR. The coronary flow rate was
measured using a flowmeter with an in-line probe (Tran-
sonic Systems Inc., Ithaca, NY).
4.5.1. 2,2,5,5-Tetramethyl-3-[4-(2,3,4-trimethoxybenzyl)-
piperazin-1-ylmethyl]-2,5-dihydro-1H-pyrrole 3HCl salt
(7C, HO-2921NH/3HCl). White solid, 600 mg (39%),
1
mp 195–197 ꢁC. MS: [M + H]+: 404. H NMR (D2O),
400 MHz: d = 7.16 (d, J = 8.8 Hz, 1H, ArH), 6.89 (d,
J = 8.8 Hz, 1H, ArH), 5.83 (s, 1H, @CH), 4.31 (s, 2H,
ArCH2N), 3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3),
3.81 (s, 3H, OCH3), 3.49 (s, 2H, CH@CH–CH2), 3.42
(br s, 4H, piperazine CH2), 3.16 (br s, 4H, piperazine
CH2), 1.51 (s, 6H, 2· CH3), 1.49 (s, 6H, 2· CH3). Anal.
Calcd for C23H40Cl3N3O3: C, 53.86; H, 7.86; N, 8.19.
Found: C, 53.83; H, 7.82; N, 8.05.
4.5.2. 2,2,6,6-Tetramethyl-4-[4-(2,3,4-trimethoxybenzyl)-
piperazin-1-ylmethyl]-1,2,3,6-tetrahydropyridine
3HCl
salt (8C, HO-2943/3HCl). White solid, 348 mg (33%),
mp 199–201 ꢁC. MS: TSP: [M+H]+: 418. 1H NMR
(D2O), 400 MHz: d = 7.16 (d, J = 8.8 Hz, 1H, ArH),
6.88 (d, J = 8.8 Hz, 1H, ArH), 5.96 (s, 1H, @CH), 4.29
(s, 2H, ArCH2N), 3.88 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 3.81 (s, 3H, OCH3), 3.68 (s, 2H, CH@CH–
CH2N), 3.50 (br s, 4H, piperazine CH2), 3.34 (br s,
4H, piperazine CH2), 2.29 (s, 2H, CH@CH–CH2), 1.44
(s, 6H, 2· CH3), 1.38 (s, 6H, 2· CH3). Analy. Calcld
for C24H42Cl3N3O3: C, 54.70; H, 8.03; N, 7.97. Found:
C, 54.66; H, 7.90; N, 7.81.
4.7. I/R experimental protocol
Isolated rat hearts were perfused for 15 min to stabilize
the functions. Then the hearts were randomly divided
into: (i) control group, received no treatment; or (ii)
pre-treated with 1, 7B, 7C, 8B, 8C, 9C, 10, 11B, or
11C. The drugs, as water soluble hydrochloride salts
(50 lM), were dissolved in Krebs buffer and then infused
through the side arm at the rate of 1 ml/min for one min
before ischemia (pH 7.4). Finally, the hearts were sub-
jected to 30-min of ischemia, followed by 45-min of
reperfusion.
4.5.3. 4-Phenyl-2,2,5,5-tetramethyl-3-[4-(2,3,4-trimeth-
oxybenzyl)-piperazin-1-ylmethyl]-2,5-dihydro-1H-pyrrole
3HCl salt (9C, HO-3630/3HCl). White solid, 730 mg
(62%), mp 178–180 ꢁC. MS (EI) m/z (%): 479 (M+, 4),
298 (41), 212 (53), 181 (100). 1H NMR (D2O),
400 MHz: d = 7.39 (s, 3H, ArH), 7.19 (d, J = 7.2 Hz,
2H, ArH), 7.07 (d, J = 8.8 Hz, 1H, ArH), 6.87 (d,
J = 8.8 Hz, 1H, ArH), 4.16 (s, 2H, ArCH2N), 3.83 (s,
6H, 2· OCH3), 3.80 (s, 3H, OCH3), 3.30 (s, 2H,
CH@CH–CH2N), 3.12 (br s, 4H, piperazine CH2),
2.96 (br s, 4H, piperazine CH2), 1.65 (s, 6H, 2· CH3),
1.45 (s, 6H, 2· CH3). Anal. Calcd for C29H44Cl3N3O3:
C, 59.13; H, 7.53; N, 7.13. Found: C, 59.08; H, 7.38;
N, 7.04.
4.8. Superoxide scavenging using in vitro study
The superoxide-scavenging properties of 1, 7B, 7C, 8B,
8C, 9C, 10, 11B, or 11C were evaluated by using EPR
spectroscopy. Xanthine (0.5 mM) and xanthine oxidase
(0.02 U/ml), in PBS, at a pH of 7.4 were used to generate
superoxide radicals. The reaction mixture contained
0.1 mM DTPA, 10 mM DEPMPO, and PBS (pH 7.4),
in presence or absence of 1 mM of either 1, 7B, 7C,
8B, 8C, 9C, 10, 11B, or 11C. In combination, these mix-
tures result in varying amounts of superoxide radicals

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T. Kalai et al. / Bioorg. Med. Chem. 14 (2006) 5510–5516
5516
´
11. Thiery, V.; Coudert, G.; Bizot-Espiard, J.-G.; Pfeiffer, B.;
which can be detected as DEPMPO–OOH adducts using
X-band EPR spectroscopy. From this, the amount (%)
of adduct in the mixture can be determined.
Renard, P.; Lindenbaum, A.; Guillaumet, G. J. Med.
Chem. 2001, 44, 3904.
12. Ancerewicz, J.; Migliavacca, E.; Carrupt, P-A.; Testa, B.;
´
Bree, F.; Zini, R.; Tillement, J.-P.; Labidalle, S.; Guyot,
4.9. Data analysis
D.; Chauvet-Monges, A-M.; Crevat, A.; Ridant, A. Free
Radical Biol. Med. 1998, 25, 113.
The statistical significance of the results of the assays
was evaluated by using ANOVA and Student’s t-test.
All of the values are expressed as means SD. A p value
<0.05 was considered significant.
´
13. Ferte, J.; Kuchnel, J-M.; Chapuis, G.; Rolland, I.; Lewin,
¨
G.; Schwaller, M. A. J. Med. Chem. 1999, 42, 478.
14. Albengres, E.; Louet, H.; d’Athis, P.; Tillement, J.-P.
Fund. Clin. Pharmacol. 2001, 15, 41.
´
15. Krishna, M. C.; Degraff, W.; Hankovszky, H. O.; Sar, P.
C.; Kalai, T.; Jeko, J.; Russo, A.; Mitchell, J. B.; Hideg, K.
´
}
J. Med. Chem. 1998, 41, 3477.
16. Halmosi, R.; Deres, P.; Berente, Z.; Kalai, T.; Sumegi, B.;
Acknowledgments
´
Hideg, K.; Toth, K. J. Cardiovasc. Pharm. 2002, 40, 854.
¨
´
17. Li, H.; Xu, K. Y.; Zhou, L.; Kalai, T.; Zweier, J. L.;
This work was supported by a grant from the Hungari-
an National Research Fund (OTKA T48334 and M
045190) and the Hungarian Ministry of Health (ETT
´
Hideg, K.; Kuppusamy, P. J. Pharmacol. Exp. Ther. 2000,
295, 563.
}
512/2003). The authors wish to thank Jozsef Jeko
(Department of Chemistry, College of Nyıregyhaza)
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18. Kalai, T.; Varbıro, G.; Bognar, Z.; Palfi, A.; Hanto, K.;
Bognar, B.; Osz, E.; Sumegi, B.; Hideg, K. Bioorg. Med.
}
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´
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Chem. 2005, 13, 2629.
´
´
for MS measurements, Zoltan Berente (Department of
Biochemistry and Medical Chemistry, University of
19. Kalai, T.; Mugesh, G.; Roy, G.; Sies, H.; Berente, Z.;
Hideg, K. Org. Biomol. Chem. 2005, 3, 3564.
20. Hankovszky, H. O.; Hideg, K.; Lex, L. Synthesis 1980,
914.
´
´
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Pecs) for NMR measurements, and Noemi Lazsanyi
for the elemental analyses. We thank Nancy Trigg for
her careful reading of the manuscript.
}
21. Cseko, J.; Hankovszky, H. O.; Hideg, K. Can. J. Chem.
1985, 63, 940.
}
´
22. Sar, C. P.; Jeko, J.; Hideg, K. Synthesis 1998, 1497.
23. Rozantsev, E. G. Free Nitroxide Radicals; Plenum Press:
New York, 1970.
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