Influence of N,N′-Substituted Piperazines on Cytolysis

Article abstract of DOI:10.1007/s11094-016-1363-8

The hemolytic activity of N,N′-substituted piperazines and their influence on the hemolytic process initiated by radiation (red LED, 653 nm) in the presence of radachlorin (RADA-FARMA, Russia) photosensitizer and on complement-dependent hemolysis were stu

Full text of DOI:10.1007/s11094-016-1363-8

DOI 10.1007/s11094-016-1363-8
Pharmaceutical Chemistry Journal, Vol. 49, No. 11, February, 2016 (Russian Original Vol. 49, No. 11, November, 2015)
INFLUENCE OF N,N¢-SUBSTITUTED PIPERAZINES
ON CYTOLYSIS
O. S. Veselkina,1 I. L. Solovtsova,2,3 N. N. Petrishchev,2 L. V. Galebskaya,2
M. E. Borovitov,1 D. I. Nilov,1 M. A. Solov’eva,2 E. A. Vorob’ev,2 and K. S. Len’shina2
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 49, No. 11, pp. 25 – 31, November, 2015.
Original article submitted June 11, 2014.
The hemolytic activity of N,N¢-substituted piperazines and their influence on the hemolytic process initiated
by radiation (red LED, 653 nm) in the presence of radachlorin (RADA-FARMA, Russia) photosensitizer and
on complement-dependent hemolysis were studied. All compounds did not cause human RBC lysis in the
studied concentration range (0.15 – 3.0 mM) although dose-dependent inhibition of photo-induced hemolysis
that manifested as an increase of the 50% lysis time was observed. The piperazine derivatives did not substan-
tially influence RBC lysis by the activated complement. Therefore, the antihemolytic effect of the N,N¢-substi-
tuted piperazines was due to their antioxidant activity.
Keywords: N,N¢-substituted piperazines, photo-induced hemolysis, photosensitizer, antioxidants, comple-
ment system.
Piperazine derivatives have broad spectra of biological
The N,N¢-substituted piperazines were synthesized at Vertex
activity and are used successfully in various medical applica-
Co.
tions [1, 2]. The design of novel drugs based on substituted
piperazines and the comprehensive study of their properties
EXPERIMENTAL CHEMICAL PART
are of definite interest. Thus, we showed earlier [3] that
N,N¢-substituted piperazines exhibited antiaggregant, antico-
The course of reactions and purity of intermediates in
agulant, and vasodilating activity and could be used to design
each synthetic step were monitored by TLC on TLC
drugs. Thus, the cytolytic activity of drugs intended for ther-
Silicagel 60 F
(Merck) or Alugram Sil G/UV
254
254
apy of hemostatic diseases must be assessed because
cytolysis, in particular hemolysis, caused by administering
the drugs is a serious therapeutic problem [4, 5].
(Macherey-Nagel) plates with detection by UV light. PMR
and 13C NMR spectra were recorded on a DRX-500 spec-
trometer (Bruker, Germany) at operating frequency 500.13
MHz for 1H and 125.76 MHz for 13C. Residual solvent reso-
nances (CDCl , 7.28 ppm; D O, 4.80; DMSO-d , 2.50) were
Slight structural changes of physiologically active com-
pounds are known to alter considerably their biological ac-
tivity [6].
3
2
6
used as standards. Mass spectra were taken on an AmaZon
mass spectrometer (Bruker) using electrospray ionization.
The purity of the compounds was evaluated by HPLC on an
Alliance chromatograph (Waters, USA) using a Zorbax
Eclipse XDB-C18 column (3.5 mm, 3 ´ 100 mm) (Agilent
Technologies, USA). The mobile phase was a mixture
(75:25) of buffer (pH 3.0) containing sodium octanesulfonate
(0.0125 M) (Merck, #118307) and sodium dihydrogen phos-
phate (0.03 M) (Merck, #106342) and MeCN (J. Baker,
#9012) at flow rate 0.5 mL/min. The elution was isocratic
with detection at 210 nm.
The goal of the present work was to study the cytolytic
activity of novel N,N¢-substituted piperazines (I, II, III; see
Table 1) with different structures and also to assess their in-
fluence on various types of hemolysis, i.e., photo-induced
hemolysis due to oxidative stress and complement-depend-
ent hemolysis that does not involve reactive oxygen species.
1 Vertex Co., 27A 24-th Liniya, St. Petersburg, 199106 Russia.
2
Pavlov First St. Petersburg State Medical University, MH RF, 6 – 8 L’va
Tolstogo St., St. Petersburg, 197022 Russia.
3
Peter the Great St. Petersburg Polytechnic University, 5 Khlopina St., St.
Petersburg, 195251 Russia.
743
0091-150X/15/4911-0743 © 2015 Springer Science+Business Media New York

744
O. S. Veselkina et al.
1-Carbimidamido-4-(2,3,4,5-tetramethoxybenzoyl)pi
PMR spectrum (CDCl ), d, ppm (J, Hz): 3.87 (3H,
3
perazine hemifumarate (I). 1-Carbimidamido-4-(2,3,4,5-
tetramethoxybenzoyl)piperazine acetate (IV) was prepared
as described earlier by us [3]. Compound IV (39.0 g,
CH O-), 3.95 (3H, CH O-), 4.05 (3H, CH O-), 5.5 (br.s, 1H,
3
3
3
HO-Ar), 7.17 (s, 1H, H-Ar), 10.31 (s, 1H, CO H).
2
Acid VI was 99.5% pure according to PMR spectros-
copy and TLC.
95 mmol) was dissolved in hot H O (125 mL, ~60°C) and
2
treated with a hot (~60°C) solution of fumaric acid (5.2 g,
Methyl 2-hydroxy-3,4,5-trimethoxybenzoate (VII). A
45 mmol) in H O (75 mL). The resulting precipitate was fil-
solution of VI (18.2 g, 0.080 mol) in refluxing Me CO
2
2
(150 mL) was stirred, treated in portions with anhydrous
Na CO (20.4 g, 0.192 mol) and dimethylsulfate (9.1 mL,
tered off, rinsed on the filter with H O (3 ´ 75 mL), and
2
recrystallized from hot H O to afford I as colorless crystals
2
3
2
12.1 g, 0.096 mol), refluxed with stirring for 3 h, and cooled.
The precipitate of inorganic salts was filtered off. The filtrate
was evaporated in a rotary evaporator at reduced pressure.
The obtained solid was dissolved in EtOAc (200 mL);
washed successively with saturated NaHCO solution in H O
(30.3 g, 78%). PMR spectrum (DMSO-d ), d, ppm (J, Hz):
6
3.25 (m, 2H, CH -piperazine CH ), 3.39 (m, 4H, CH -pipe-
2
2
2
razine CH and H O), 3.51 (m, 2H, CH -piperazine CH ),
2
2
2
2
3.65 (m, 5H, CH -piperazine CH and CH O-), 3.76 (m, 3H,
2
2
3
CH O-), 3.78 (m, 3H, CH O-), 3.83 (m, 3H, CH O-), 6.21 (s,
3
2
3
3
3
(2 ´ 50 mL), H O (2 ´ 50 mL), and saturated NaCl solution
1H, HO CCH=CHCO H), 6.67 (s, 1H, H-Ar), 9.43 (br.s, 4H,
2
2
2
C(NH )(N+H )). 13C NMR spectrum (DMSO-d ), d, ppm:
(50 mL); and dried over anhydrous MgSO . The desiccant
4
2
2
6
was separated by filtration. The filtrate was evaporated in a
rotary evaporator at reduced pressure. The solid was
recrystallized from heptane to afford VII as sandy-colored
40.64 (CH -piperazine CH ), 44.29 (CH -piperazine CH ),
2
2
2
2
44.82 (CH -piperazine CH ), 45.63 (CH -piperazine CH ),
2
2
2
2
56.17 (CH O-), 60.68 (CH O-), 61.06 (CH O-), 61.52
3
3
3
large crystals (11.6 g, 60%). PMR spectrum (CDCl ), d, ppm
(CH O-), 105.43 (C6, Ar), 124.54 (C1, Ar), 136.56
3
3
(J, Hz): 3.84 (s, 3H, CH O C-), 3.93 (3H, CH O-), 3.95 (3H,
(HO CCH=CHCO H), 143.04 (C2, Ar), 143.20 (C5, Ar),
3
2
3
2
2
146.48 (C4, Ar), 149.51 (C3, Ar), 157.43 (C(NH )(N+H )),
CH O-), 4.01 (3H, CH O-), 7.08 (s, 1H, H-Ar), 10.68 (s, 1H,
3
3
2
2
HO-Ar).
166.16 (CON), 172.12 (HO CCH= CHCO H). Mass spec-
2
2
trum: MH+ found, 353.20; MH+ calc., 353.18. The product
Methyl 2-(1-methylpropyl)oxy-3,4,5-trimethoxyben-
zoate (VIII). Ester VII (4.95 g, 0.20 mol), 2-bromobutane
(2.74 mL, 3.43 g, 0.025 mol), anhydrous K CO (4.15 g,
was 100% pure according to HPLC.
1-Carbimidamido-4-[2-(1-methylpropyl)oxy-3,4,5-tri-
methoxybenzoyl]piperazine hemifumarate (II)
2-Bromo-3,4,5-tributoxybenzoic acid (V). A solution of
2
3
0.030 mol), and KI (0.33 g, 0.002 mol) were dissolved in
DMF (15 mL), stirred, heated to 65°C for 5 h, cooled, treated
with H O (50 mL), acidified with conc. HCl (20 mL, 24 g,
3,4,5-trimethoxybenzoic acid (106.0 g, 0.50 mol) in CHCl
2
3
~0.240 mol), and extracted with methyl-tert-butylether
(MTBE, 3 ´ 25 mL). The combined extracts were washed
successively with H O (2 ´ 30 mL), saturated NaHCO solu-
(500 mL) was treated with H O (10 mL), refluxed, stirred,
2
treated dropwise with a solution of Br (32.2 mL, 99.8 g,
2
0.626 mol) in CHCl (100 mL) over 30 min, refluxed for
3
2
3
10 h, cooled, washed with H O (3 ´ 200 mL), and dried over
tion in H O (2 ´ 30 mL), Na S O solution (10%) in H O
2
2
2
2
3
2
anhydrous MgSO . The desiccant was filtered off. The fil-
(2 ´ 20 mL), and saturated NaCl solution (30 mL) and dried
4
trate was evaporated in a rotary evaporator at reduced pres-
sure to afford the intermediate as pale-cream-colored crystals
(120.8 g, 83%) that were used in the next step without further
over anhydrous CaCl . The desiccant was separated by filtra-
2
tion. The filtrate was evaporated in a rotary evaporator at re-
duced pressure. The intermediate was obtained as a yellow
oil (5.90 g, 97%) and used in the next step without further
purification.
purification. PMR spectrum (CDCl ), d, ppm (J, Hz): 3.92
3
(3H, CH O-), 3.93 (3H, CH O-), 3.98 (3H, CH O-), 7.42 (s,
3
3
3
1H, H-Ar), 11.00 (br.s, 1H, CO H). The intermediate was
2-(1-Methylpropyl)oxy-3,4,5-trimethoxybenzoic acid
(IX). A solution of VIII (5.90 g, 0.020 mol) in aqueous
EtOH (95%, 20 mL) was treated with KOH (1.40 g,
0.025 mol) and refluxed with stirring for 1 h. The mixture
was evaporated in a rotary evaporator at reduced pressure.
2
95% pure according to PMR spectroscopy and TLC.
2-Hydroxy-3,4,5-trimethoxybenzoic acid (VI). A hot
solution of V (60.0 g, 0.206 mol) and NaOH (50.0 g,
1.25 mol) in H O (400 mL) was stirred, treated with
2
CuSO × 5H O (51.4 g, 0.206 mol, 100 mol%), refluxed for
The resulting solid was refluxed in Et O (50 mL) and cooled.
4
2
2
3.5 h [1-BuOH (10 mL) was added to the reaction mixture
2 h after the start of refluxing in order to suppress foaming],
and cooled. The resulting precipitate was filtered off, sus-
The solid was isolated by filtration, rinsed on the filter with
Et O (40 mL), and dried in air. The resulting cream-colored
2
solid was dissolved in H O (50 mL), acidified with conc.
2
pended in NaOH solution (5%) in H O (200 mL), and stirred
HCl (3 mL, 3.6 g, 0.036 mol), and extracted with Et O
2
2
for 1 h. The solid was separated by filtration (3´), suspended
(2 ´ 25 mL). The combined extracts were washed with H O
2
in H O (200 mL), and acidified with conc. HCl (50 mL,
(2 ´ 20 mL) and saturated NaCl solution (20 mL) and dried
2
60.0 g, 0.6 mol). The resulting precipitate was separated by
over anhydrous MgSO . The desiccant was separated by fil-
4
filtration, rinsed on the filter with H O (3 ´ 100 mL), and
tration. The filtrate was evaporated in a rotary evaporator at
reduced pressure to afford a yellow liquid (5.17 g, 89%) that
was used in the next step without further purification. PMR
2
dried in vacuo to afford pale-cream-colored crystals (8.46 g,
18%).

Influence of N,N¢-Substituted Piperazines on Cytolysis
745
reduced pressure. Intermediate XII (6.63 g, 97%) was used
in the next step without further purification.
spectrum (CDCl ), d, ppm (J, Hz): 1.06 (t, 3H, 6 Hz,
3
CH CH C × HCH ), 1.29 (d, 3H, 6 Hz, CH CH C × HCH ),
3
2
3
3
2
3
1-Carbimidamido-4-[2-(1-methylpropyl)oxy-3,4,5-tri
methoxybenzoyl]piperazine acetate (XIII). A solution of
XII hydrochloride (6.48 g, 17 mmol) and 1H-pyrazole-1-car-
boximidamide hydrochloride (3.66 g, 25 mmol) in DMF
(20 mL) was heated to 50°C, treated with di-isopropyl-
ethylamine (8.00 mL, 5.92 g, 46 mmol), stirred at 50°C for
10 h, and evaporated in a rotary evaporator at reduced pres-
1.8 (m, 2H, CH CH C × HCH ), 3.90 (s, 6H, 2x CH O-), 4.00
3
2
3
3
(s, 3H, CH O-), 4.73 (m, 1H, CH CH C × HCH ), 7.45 (s,
3
3
2
3
1H, H-Ar), 11.56 (br.s 1H, CO H). 13C NMR spectrum
2
(CDCl ), d, ppm: 9.92 (CH CH C × HCH ), 19.01
3
3
2
3
(CH CH C × HCH ), 29.54 (CH CH C × HCH ), 56.19
3
2
3
3
2
3
(CH O-), 61.22 (CH O-), 61.33 (CH O-), 83.51
3
3
3
(CH CH C × HCH ), 108.00 (C1, Ar), 117.20 (C6, Ar),
sure. The solid was dissolved in H O (120 mL) and extracted
3
2
3
2
144.49 (C2, Ar), 145.96 (C5, Ar), 147.92 (C3, Ar), 149.70
with MTBE (3 ´ 30 mL). The organic extracts were dis-
(C4, Ar), 165.36 (CO H). Acid IX was 99.8% pure accord-
carded. The transparent aqueous layer was made basic with
2
ing to PMR spectroscopy and TLC.
NaOH solution (50%) in H O (35 mL) and extracted with
2
2-(1-Methylpropyl)oxy-3,4,5-trimethoxybenzoylchlor
ide (X). A solution of acid IX (5.0 g, 0.018 mol) in
CH Cl (25 mL) was treated with thionylchloride (2.50 mL,
CH Cl (3 ´ 30 mL). The combined organic extracts were
2
2
washed with H O (2 ´ 20 mL) and dried over anhydrous
2
2
2
KCO . The desiccant was separated by filtration. The filtrate
3
4.18 g, 0.035 mol) and DMF (0.01 mL), stirred at room tem-
perature for 1 h, refluxed for 2 h, and evaporated in a rotary
evaporator at reduced pressure. The intermediate was ob-
tained in quantitative yield (5.32 g, 100%) as a yellow oil
and used in the next step without further purification.
was evaporated in a rotary evaporator at reduced pressure.
The solid 1-carbimidamido-4-[2-(1-methylpropyl)oxy-
3,4,5-trimethoxybenzoyl]piperazine (3.90 g, 10 mmol) was
dissolved in CH Cl (20 mL) and treated with glacial HOAc
2
2
(0.56 mL, 0.59 g, 10 mmol). The resulting precipitate was
filtered off, rinsed on the filter successively with
CH Cl (2 ´ 5 mL) and MTBE (3 ´ 10 mL), suspended in a
1-(tert-Butoxycarbonyl)-4-[2-(1-methylpropyl)oxy-3,4, 5-
trimethoxybenzoyl]piperazine (XI). A solution of 1-(tert-
2
2
butoxycarbonyl)piperazine (3.76 g, 0.020 mol) and Et N
3
mixture of Me CO (20 mL) and H O (2 mL), stirred,
2
2
(4.20 mL, 3.02 g, 0.030 mol) in CH Cl (25 mL) was stirred,
2
2
refluxed for 1 h, and cooled. The precipitate was separated
treated dropwise with a solution of X (5.32 g, 0.018 mol) in
CH Cl (25 mL), and stirred for 3 h. The separated precipi-
by filtration, rinsed on the filter with Me CO (2 ´ 5 mL), and
2
2
2
dried in air to afford XIII as fine colorless crystals (4.00 g,
tate of Et N × HCl was filtered off. The filtrate was washed
3
53%). PMR spectrum (DMSO-d ), d, ppm (J, Hz): 0.90 (t,
6
successively with H O (2 ´ 20 mL), saturated NH Cl solu-
2
4
3H, 6 Hz, CH CH C × HCH ), 1.09 (d, 3H, 6 Hz,
3
2
3
tion (2 ´ 20 mL), H O (2 ´ 20 mL), saturated NaHCO solu-
2
3
CH CH C × HCH ), 1.64 (m, 5H, CH CH C × HCH and
3
2
3
3
2
3
tion (2 ´ 20 mL), and saturated NaCl solution (20 mL) and
CH CO -), 3.0 – 4.0 (m, 17H, 4´CH -piperazine CH and
3
2
2
2
dried over anhydrous MgSO . The desiccant was separated
4
3´CH O-), 4.12 (m, 1H, CH CH C × HCH ), 6.62 (s, 1H,
3
3
2
3
by filtration. The filtrate was evaporated in a rotary evapora-
tor at reduced pressure. The intermediate was obtained in
quantitative yield (7.95 g, 100%) as a yellowish-brown oil
that crystallized slowly upon standing into cream-colored
crystals and used in the next step without further purification.
H-Ar), 8.20 (br.s, 4H, C(NH )(N+H )). 13C NMR spectrum
2
2
(DMSO-d ), d, ppm: 10.31 (CH CH C × HCH ), 20.02
6
3
2
3
(CH CH C × HCH ),
25.83
(CH CO -),
30.16
3
2
3
3
2
(CH CH C × HCH ), 41.50 (CH -piperazine CH ), 45.05
3
2
3
2
2
(CH -piperazine CH ), 45.63 (CH -piperazine CH ), 46.27
2
2
2
2
PMR spectrum (CDCl , d, ppm, J, Hz) (two isomers ap-
3
(CH -piperazine CH ), 56.99 (CH O-), 61.49 (CH O-),
2
2
3
3
peared in the PMR spectrum because of hindered rotation
around the amide bond): 0.87 and 1.01 (two t, 7.4 and 7.6 Hz
respectively, 3H total, CH CH C × HCH ), 1.09 and 1.27
61.72 (CH O-), 81.38 (CH CH C × HCH ), 106.84 (C6, Ar),
3
3
2
3
126.32 (C1, Ar), 141.43 (C2, Ar), 144.31 (C5, Ar), 147.59
(C4, Ar), 150.18 (C3, Ar), 158.42 (C(NH )(N+H )), 167.44
3
2
3
2
2
(two d, 5.9 and 5.9 Hz respectively, 3H, CH CH C × HCH ),
3
2
3
(CON), 176.66 (CH CO H). The product was 99.6% pure
3
2
1.49 (s, 9H, (CH ) C-), 1.66 (m, 2H, CH CH C × HCH ),
according to HPLC with a maximum of 0.1% of an unidenti-
fied impurity and 0.3% of total unidentified impurities.
1-Carbimidamido-4-[2-(1-methylpropyl)oxy-3,4,5-tri-
methoxybenzoyl]piperazine hemifumarate (II). Acetate
3 3
3
2
3
3.0 – 4.0 (m, 17H, 4´CH -piperazine CH and 3´CH O-),
2
2
3
4.19 (m, 1H, CH CH C × HCH ), 6.60 and 6.62 (two s, 1H
3
2
3
total, H-Ar)
1-[2-(1-methylpropyl)oxy-3,4,5-trimethoxybenzoyl]pi
perazine hydrochloride (XII). Compound XI (7.95 g,
0.018 mol) was stirred with conc. HCl (50 mL, 60.0 g,
~0.6 mol) at room temperature until totally dissolved (3 h).
The mixture was evaporated in a rotary evaporator at re-
duced pressure. The solid was heated with i-PrOH (100 mL).
The insoluble impurities were removed by filtration through
Celite. The filtrate was evaporated in a rotary evaporator at
XIII (3.5 g, 7.7 mmol) was dissolved in hot H O (10 mL,
2
~60°C) and treated with a hot solution (~60°C) of fumaric
acid (0.45 g, 3.85 mmol) in H O (5 mL). The resulting pre-
2
cipitate was separated by filtration, rinsed on the filter with
H O (3 (5 mL), and recrystallized from hot H O to afford II
2
2
hemifumarate as colorless crystals (3.21 g, 92%).
PMR spectrum (DMSO-d ), d, ppm (J, Hz): 0.92 (t, 3H,
6
6 Hz,
CH CH C × HCH ),
1.11
(d,
3H,
6 Hz,
3
2
3

746
O. S. Veselkina et al.
website
http://www.organic-chemistry.org/prog/peo/
CH CH C × HCH ), 1.66 (m, 2H, CH CH C × HCH ),
3
2
3
3
2
3
(OSIRIS Property Explorer).
3.0 – 4.0 (m, 17H, 4´CH -piperazine CH and 3´CH O-),
2
2
3
4.15 (m, 1H, CH CH C × HCH ), 6.20 (s, 1H,
3
2
3
EXPERIMENTAL BIOLOGICAL PART
HO CCH=CHCO H), 6.61 (s, 1H, H-Ar), 8.30 (br.s, 4H,
2
2
C(NH )(N+H )). 13C NMR spectrum (DMSO-d ), d, ppm:
2
2
6
Blood from practically healthy people (20 – 30 years
old) and rabbits (2.5 – 3.0 kg, Rappolovo nursery) was used.
Erythrocytes were obtained from citrated blood by
centrifugation at 1,500 rpm for 10 min followed by rinsing
(3´) with normal saline. Then, the cells were stabilized for at
least 1 d at 4°C in Alsever’s solution. Erythrocytes were
rinsed three times with normal saline before use. A standard
cell suspension in Veronal—Medinal buffer (pH 7.4) was
prepared. The optical density of the standard suspension was
(0.560 ± 0.020) at 800 nm after dilution in eight times the
amount of buffer. Measurements were made on an SF 2000
spectrophotometer (LOMO) in a 5-mm cuvette. The result-
ing standard cell suspension was used in the studies.
10.41 (CH CH C × HCH ), 20.12 (CH CH C × HCH ), 30.22
3
2
3
3
2
3
(CH CH C × HCH ), 41.51 (CH -piperazine CH ), 45.15
3
2
3
2
2
(CH -piperazine CH ), 45.66 (CH -piperazine CH ), 46.37
2
2
2
2
(CH -piperazine CH ), 57.00 (CH O-), 61.59 (CH O-),
2
2
3
3
61.75 (CH O-), 81.40 (CH CH C × HCH ), 106.88 (C6, Ar),
3
3
2
3
126.33 (C1, Ar), 136.55 (HO CCH=CHCO H), 141.45 (C2,
2
2
Ar), 144.32 (C5, Ar), 147.60 (C4, Ar), 150.21 (C3, Ar),
158.44 (C(NH )(N+H )), 167.41 (CON), 172.13 (HO CCH=
2
2
2
CHCO H). Mass spectrum: MH+ found, 395.22; MH+ calc.,
2
395.23. The product was 99.7% pure according to HPLC
with a maximum of 0.1% of an unidentified impurity and
0.2% of total unidentified impurities.
1-Carbimidamido-4-(3,4,5-trimethoxyphenylsulfonyl)-
piperazine fumarate (III). 1-Carbimidamido-4-(3,4,5-tri-
methoxyphenylsulfonyl)piperazine hydrochloride (5 g,
12.7 mmol) that was prepared as described by us earlier [3]
Cytolytic activity of the compounds was recorded at
37°C in a thermostatted spectrophotometer cuvette (5-mm
optical path length) into which a solution of the studied com-
pound (from 0.01 to 0.2 mL) that was diluted beforehand in
normal saline was placed. The volume of the mixture was ad-
justed to 0.7 mL using Veronal—Medinal buffer. Standard
erythrocyte suspension (0.1 mL) was added to the mixture
after heating for 3 min. The decrease of optical density of the
suspension was recorded at 800 nm at 5-second intervals un-
til the hemolysis was complete.
was treated with NaOH solution (50%) in H O (35 mL) and
2
extracted with CH Cl (3 ´ 30 mL). The combined organic
2
2
extracts were washed with H O (20 mL) and dried over an-
2
hydrous K CO . The desiccant was separated by filtration.
2
3
The filtrate was evaporated in a rotary evaporator. The solid
was dissolved in H O and treated with fumaric acid (1.47 g,
2
12.7 mmol). The H O was evaporated in a rotary evaporator
Antioxidant properties of the compounds were assessed
using an apparatus for studying photo-induced cytolysis by
the previously published method [7]. According to this
method, an incubation mixture containing standard erythro-
cyte suspension (0.1 mL); Veronal—Medinal buffer (pH
7.2 – 7.4); various amounts of studied compounds; and
radachlorin photosensitizer (0.35% solution for i.v. injection,
RADA-FARMA, Russia), the principal drug substance of
which was (7S,8S)-13-vinyl-5-(carboxymethyl)-7-(2-carbo-
xyethyl)-2,8,12,17-tetramethyl-18-ethyl-7H,8H-porphyrin-3-
carboxylic acid, was prepared in a shielded 5-mm cuvette.
The control contained normal saline instead of the studied
drug. The final radachlorin concentration in the sample was
6.25 mg/mL. The incubation mixture (total volume 0.8 mL)
was thermostatted in the spectrophotometer cuvette holder
for 3 min at 37°C with constant stirring and then irradiated
with monochromatic light (red LED, 653 nm, output power
12 mW, irradiation dose 1.4 J/cm2). After the irradiation was
finished, the decrease of the solution optical density at
750 nm was recorded.
2
at reduced pressure. The resulting solid was suspended in a
mixture of Me CO (40 mL) and H O (2 mL), stirred,
2
2
refluxed for 1 h, and cooled. The solid was separated by fil-
tration, rinsed on the filter with Me CO (2 ´ 10 mL), and
2
dried in vacuo to afford the product as fine colorless crystals
(3.3 g, 55%).
PMR spectrum (DMSO-d ), d, ppm (J, Hz): 3.03 (4H, m,
6
2´CH -piperazine CH ), 3.50 (4H, m, 2´CH -piperazine
2
2
2
CH ), 3.77 (s, 3H, CH O-), 3.88 (s, 6H, 2´CH O-), 6.41 (s,
2
3
3
2H, HO CCH=CHCO H), 6.89 (s, 2H, H-Ar), 8.50 (br.s, 5H,
2
2
C(NH )(N+H )). 13C NMR spectrum (DMSO-d ), d, ppm:
2
2
6
44.18 (CH -piperazine CH ), 45.22 (CH -piperazine CH ),
2
2
2
2
56.36 (2´CH O-), 104.97 (C2, Ar), 129.56 (C1, Ar), 135.34
3
(HO CCH=CHCO H), 141.36 (C4, Ar), 153.12 (C3, Ar),
2
2
156.92 (C(NH )(N+H )), 168.63 (HO CCH=CHCO H).
2
2
2
2
Mass spectrum: MH+ found, 358.15; MH+ calc., 358.13. The
product was 99.7% pure according to HPLC with a maxi-
mum of 0.1% of an unidentified impurity and 0.2% of total
unidentified impurities.
The T value, i.e., the time for 50% erythrocyte lysis in
50
The log P value (logarithm of the octanol—water con-
centration ratio of un-ionized compound) was used as an in-
dicator of the hydrophobicity of the synthesized compounds;
the pKa value, as an indicator of basicity (Table 1). The pKa
values were calculated using the program at the website
www.chemicalize.org (chemAxon); log P, the program at the
the incubation mixture, was determined using computer soft-
ware from the recorded smooth S-shaped hemolytic curve
[7]. The rate of hemolysis was judged from the change of
T .
50
Complement-dependent hemolysis was recorded in a
5-mm spectrophotometer cuvette thermostatted at 37°C into

Influence of N,N¢-Substituted Piperazines on Cytolysis
747
T50, s
210
190
170
150
130
110
90
which human blood serum (0.05 mL) was added. The vol-
ume was adjusted to 0.7 mL using Veronal—Medinal buffer
(5 mM, pH 7.4). The mixture was thermostatted for 3 min
and treated with standard rabbit erythrocyte suspension
(0.1 mL). The decrease of suspension optical density at
800 nm was recorded at 5-second intervals. Complement-de-
pendent hemolysis parameters, namely the induction period
duration (T-lag) in seconds and the hemolysis rate (V) ex-
pressed in millions of erythrocytes lysed per minute [8], were
determined from the hemolysis curves.
y = 4.3584x3–26.751x2+72.473x+100.67
R2 = 0.9997
y = –6.6022x2–42.721x+98.503
R2 = 0.9978
y = -5.3567x2–34.715x+99.211
R2 = 0.9989
VR-0411
VR-0511
VR-0413
0
1
2
3
Various amounts of compound in normal saline were
added to the incubation mixture during the study of the influ-
ence of the compound on complement-dependent hemolysis.
The same amount of normal saline was added as a control to
the incubation mixture.
C, mM
Fig. 1. Time for 50% erythrocyte lysis (T ) as a function of com-
50
pound concentration, % vs. control (normal saline). Control T
50
value, 87 ± 15 sec. * Differences statistically significant vs. control,
p < 0.05 (n = 5).
RESULTS AND DISCUSSION
compared with the control. This was evident in the increased
The N,N¢-substituted piperazines (Table 1) were basic
compounds, the hydrophobicity (log P) of which increased in
the order III, I, II. The compounds had practically the same
basicity (pKa).
50% erythrocyte hemolysis time (T ) (Fig. 1).
50
The results showed that the inhibitory activity of all three
compounds was polynomial and dose-dependent in nature.
The EC values for II, I, and III were 1.0, 1.6, and 2.3 mM,
All compounds in the studied concentration range
(0.15 – 3.0 mM) did not cause lysis of human erythrocytes.
It is well known [9] that erythrocyte lysis is initiated by
irradiation with UV or visible light in the presence of
photosensitizers, the most efficient of which are porphyrins
and their derivatives [10], in particular radachlorin. It was
found that generation of primarily singlet oxygen and then
other reactive oxygen species was responsible for the
photodynamic effect [11]. Binding of porphyrins to cell
membranes reduced the photolytic stability of their mem-
branes [10, 12].
50
respectively. A comparison of the physicochemical proper-
ties and activities (Table 1, Fig. 1) suggested that the inhibi-
tory activity of the compounds for photo-induced hemolysis
increased as their hydrophobicity and the number of alkoxy
substituents on the aromatic ring increased.
The influence of the N,N¢-substituted piperazines on
complement-dependent hemolysis was studied in order to
determine if their antioxidant activities or membrane-protec-
tive properties were responsible for the reduced hemolysis
(Table 2).
N,N¢-Substituted piperazines I, II, and III inhibited
radachlorin-induced hemolysis statistically significantly
Complement-dependent hemolysis does not involve re-
active oxygen species. The complement system is activated
TABLE 1. Structures and Properties of Studied N,N¢-Substituted Piperazines
Compound
Structural formula
Mol. wt.
410.15
log P
0.2
pKa
O
O
I
11.55
O
O
O
N
HO
·1/2
N
NH
OH
O
O
NH
2
II
452.50
1.42
11.53
O
O
O
O
O
N
HO
·1/2
N
NH
OH
O
O
NH
2
O
O
III
474.49
– 0.36
11.51
O
O
O
S
N
HO
·
N
NH
NH
OH
O
O
2

748
O. S. Veselkina et al.
tivity that was apparently due to protection of erythrocyte
membranes from damage by reactive oxygen species.
All studied N,N¢-substituted piperazines in the concentra-
tion range 0.15 – 3.00 mM did not exhibit hemolytic activity
for donor blood. Induced hemolysis models established that
all compounds slowed photo-induced hemolysis and did not
influence hemolysis caused by complement system activa-
tion. The activities of the compounds were not related to a di-
rect membrane-protective effect. It could be assumed based
on the photo-induced hemolysis mechanism that the studied
N,N¢-substituted piperazines protected cells from lysis as a
result of their antioxidant activities that were manifested
upon interaction with cell membranes. Compound II had the
greatest protective effect.
TABLE 2. Human Complement Activation Parameters in the Pres-
ence of N,N¢-Substituted Piperazines, % vs. Control (Normal Saline)
Concentration, mM
0.6 (n = 5)
T-lag
Com-
0.15 (n = 5)
1.5 (n = 5)
T-lag V
pound
T-lag
V
V
I
101 ± 10 102 ± 6
99 ± 9 107 ± 7 110 ± 10 99 ± 5
II
97 ± 11 101 ± 4 104 ± 16 101 ± 3 110 ± 14 98 ± 12
99 ± 13 106 ± 9 107 ± 12 100 ± 7 105 ± 10 103 ± 7
III
by foreign agents (rabbit erythrocytes) and attacks the mem-
brane to form complexes that are incorporated into foreign
cell membranes and cause lysis [13].
Studies of the influence of the compounds on comple-
ment-dependent hemolysis did not change T-lag or the
hemolysis rate (V).
REFERENCES
1. C. P. Meher, A. M. Rao, and M. Omar, Asian J. Pharm. Sci.
Res., 3(1), 43 – 60 (2013).
The results indicated that all studied N,N¢-substituted
piperazines possessed primary antioxidant activity that mani-
fested in the inhibition of oxidative processes at the mem-
brane level during photodynamic treatment of the erythro-
cytes.
2. A. Tomar, M. Mall, and M. Verma, Int. J. Res. Ayurveda
Pharm., 2(5), 1547 – 1548 (2011).
3. O. S. Veselkina, N. B. Viktorov, N. N. Petrishchev, and
Yu. V. Poplavskaya, RU Pat. No. 2,469,029, Jul. 10, 2010;
Byull. Izobret., No. 34 (2012).
The antioxidant activities of the N,N¢-substituted
piperazines that were studied in the present work could be
due to the three (III) or four (I and II) alkoxy groups on the
aromatic ring (Table 1). The higher antioxidant activity of II
as compared with I and III could be related to the fact that
adding a sec-butoxy substituent to the aromatic ring en-
hances the hydrophobic and membranotropic properties of
the compound and, as a result, increases the antioxidant ac-
tivity.
4. J. A. Stockman III, B. Lubin, and F. A. Oski, Pediatr. Res., 12,
927 – 931 (1978).
5. F. M. Poulet, K. Penraat, N. Collins, et al., Toxicol. Pathol., 38,
907 – 922 (2010).
6. R. B. Silverman, Organic Chemistry of Drug Design and Drug
Action, Elsevier, (2004), pp. 17 – 86.
7. http: // bankpatentov.ru / node / 204090
8. L. V. Galebskaya, E. V. Ryumina, Yu. V. Tarasova, et al., Klin.
Lab. Diagn., No. 3, 47 – 49 (2001).
9. S. Kumar, S. Devi, P. Misra, et al., Indian J. Exp. Biol., 47,
906 – 910 (2009).
Compounds containing aromatic rings with alkoxy sub-
stituents, e.g., trimethoxybenzoic acid, are known to be radi-
cal traps [14].
10. M. O. Senge and J. C. Brand, Photochem. Photobiol., 87,
1240 – 1296 (2011).
It was shown experimentally that the cytoprotective ac-
tivity of trimetazidine [1-(2,3,4-trimethoxybenzyl)piperazine-
hydrochloride] [15] was due to its ability to trap free radicals.
Antioxidant properties were also confirmed for substi-
11. S. Sandberg and I. Romslo, Clin. Chim. Acta, 109(2), 193 – 201
(1981).
12. W. An, Y. Jiao, C. Dong, et al., Dyes Pigm., 81(1), 1 – 9 (2009).
13. D. W. Michaels, A. S. Abramovitz, C. H. Hammer, et al., Proc.
Natl. Acad. Sci. USA, 73(8), 2852 – 2856 (1976).
14. L. Lagnika, F. Gbaguidi, E. Anago, et al., Int. J. Biol. Chem.
Sci., 4(3), 820 – 824 (2010).
tuted
[16].
N-(aryloxyethyl)-N¢-(2-methoxyphenyl)piperazines
The method used by us made it possible to determine the
mechanism of the antioxidant activity of the compounds
based on suppression of erythrocyte lysis as a result of oxida-
tive photo-induced degradation of membrane components in
the presence of radachlorin [17].
15. I. Maridonneau-Parini and C. Harpey, Br. J. Clin. Pharmacol.,
20, 148 – 151 (1985).
16. A. Pietrzyka, M. Stepniewski, A. Waszkielewicz, et al., Acta
Pol. Pharm. Drug Res., 63(1), 19 – 24 (2006).
17. L. V. Galebskaya, I. L. Solovtsova, E. V. Ryumina, et al.,
Uchenye Zapiski SPbGMU im. Akad. I. P. Pavlova, XVI, No. 4,
45 – 47 (2009).
The experimental results led to the conclusion that all
tested N,N¢-substituted piperazines possessed antioxidant ac-