The study on organic nitrates, part V. New derivatives of piperazine potential NO donors

Article abstract of DOI:10.1211/0022357021778538

We have obtained a series of non-symmetrical 1,4-disubstituted derivatives of piperazine, with the structure of organic nitrates, as potential NO donors. These compounds were obtained from respective hydroxyl derivatives of piperazine in an esterification reaction by fuming nitric acid. The obtained nitrates were tested in-vitro by reaction with a sulfhydryl compound. The structure of the most active nitrate and its hydroxyl analogue was used for the calculation of geometrical optimization with the determination of 3D-QSAR by a semi-empirical method PM3 using HyperChem 4.5.

Full text of DOI:10.1211/0022357021778538

Journal of
JPP 2002, 54: 445–450
# 2002 The Authors
Received July 27, 2001
Accepted November 13, 2001
ISSN 0022-3573
The study on organic nitrates, part V. New derivatives
of piperazine potential NO donors
Lucyna Korzycka
Abstract
We have obtained a series of non-symmetrical 1,4-disubstituted derivatives of piperazine, with
the structure of organic nitrates, as potential NO donors. These compounds were obtained
from respective hydroxyl derivatives of piperazine in an esteriŽ cation reaction by fuming nitric
acid. The obtained nitrates were tested in-vitro by reaction with a sulfhydryl compound. The
structure of the most active nitrate and its hydroxyl analogue was used for the calculation of
geometrical optimization with the determination of 3D-QSAR by a semi-empirical method PM3
.
using HyperChem 4 5.
Introduction
Our earlier chemical and pharmacological studies on non-symmetrical 1,4-
disubstituted hydroxyl derivatives of piperazine showed that these compounds
aŒect vasodilatation of the circulatory system, decrease blood pressure and increase
blood ¯ ow through coronary vessels (Korzycka et al 1986). In this study a nitrate
group was introduced into the most active hydroxyl derivatives of piperazine. This
group can be decomposed in metabolic reactions, releasing nitric oxide (NO). The
formation of exogenous NO may have further positive eŒect on the circulatory
system. This assumption has been con®rmed by other authors, who demonstrated
signi®cant in¯ uence of other nitric derivatives of piperazine on the circulatory
system (Hayashi et al 1993). Endogenous NO is a multidirectional biological
transmitter, which has many roles in the body. It is thought to be the same as
EDRF, vascular spasmolytic factor (Furchgott & Zawadzki 1980; Moncada et al
1991).
The main group of drugs used in coronary heart disease and in cardiac
insu ciency are organic nitrates (e.g. glyceryl trinitrate and isosorbide-5-mono-
nitrate). The mechanism of their spasmolytic eŒect on blood vessels is based on
exogenous NO, which they provide. These compounds are NO donors (Moncada
& Higgs 1995).
Department of Pharmaceutical
Chemistry and Drug Analyses,
Medical University,
NO forms from the reaction of organic nitrates with sulfhydryl compounds. This
is a non-enzymatic trans-esteri®cation reaction, during which an unstable in-
termediate, thionitrate, is formed, which then decomposes giving NO (Feelisch &
Noack 1987; Korzycka et al 2000). Released NO is a volatile substance and is
rapidly oxygenated to nitrite and nitrate ions. The formation of these ions con®rms
the fact that organic nitrogen reacts with sulfhydrylic compounds, and thus
undergoes a reaction which determines the pharmacological activity of many
therapeutic organic nitrates.
´
´ ´
Muszynskiego 1, 90-151 odz,
Poland
Lucyna Korzycka
Correspondence: L. Korzycka,
Department of Pharmaceutical
Chemistry and Drug Analyses,
Medical University,
´
´ ´
Muszynskiego 1, 90-151 odz,
Poland.
445

446
Lucyna Korzycka
Materials and Methods
Procedures
Melting points were measured on a Boetius apparatus
and are uncorrected. IR spectra were taken in KBr using
a Mattson In®nity MI-60 FT-IR spectrophotometer. 1H
NMR spectra were recorded on a Mercury-300 A-
300,06 MHz in d6-DMSO as solvent and tetramethyl-
silane as the internal reference. Carbon, hydrogen and
nitrogen elemental analyses were performed using a
}
Perkin Elmer 2400 series II CHNS O and agreed with
proposed structures within [GMM1]³0.3% of theor-
etical values. Computational calculations were per-
formed using the HyperChem 4.5. program.
General procedure for obtaining semi-products,
compounds 1–10
The basic substrate for synthesis of the title derivatives
was 1-(3-piperidinepropionyl) piperazine. This com-
pound was obtained by acylation of anhydrous pip-
erazine with 3-chloropropionyl chloride at pH 4. The
obtained 1-(3-chloropropionyl)piperazine, when re-
acted with anhydrous piperidine, gave the key inter-
mediate 1.
Compound 1 heated for 2 h at70°C withan equimolar
amount of 1,2-epoxypropane or 1,2-epoxy-3-chloropro-
pane in anhydrous ethanol gave derivatives 2 and 3,
respectively. Compound 3, reacted with double excess
of anhydrous piperidine at room temperature in ben-
zene, gave compound 4. Compound 3 was used in the
reaction with double excess of isopropylamine and N-
methylpiperazine. The mixture of compound 3 with
respective amine in chloroform was heated for 6 h at
60°C, giving compounds 5 and 6.
Figure 1 Schemeof synthesisof compounds2±7 and 15±20. Reagent
i, fuming HNO3 at ®15°C
Compound 1 was also reacted with an equimolar
amount of 1-chloropropanol in chloroform at 70°C.
After heating for 8 h, semi-product 7 was obtained.
For these reasons, the nitrates obtained in this study Physicochemical constants of compounds 1±7 are con-
(11±20) reacted with the hydrochloride of the ethyl ester sistent with those described in literature (Korzycka et al
of l-cysteine at 37°C in a water environment at pH 7.5. 1986).
The nitrate and nitrite ions formed in the reaction were
detected by a potentiometric method using ionoselective equimolar amount of 1,2-epoxypropane in anhydrous
nitrate and nitrite electrodes. ethanol at 65°C for 10 h to give compound 8. Its
1-(2-Hydroxyethyl)piperazine was reacted with an
The ability of the studied nitrates (11±20) to react physicochemical constants are consistent with those
with thiols is connected with their structure and physico- quoted in literature (Biel 1958).
chemical properties. For the most active nitrate and Moreover, 1-(2-hydroxyethyl)piperazine underwent
its hydroxyl analogue, QSAR parameters have been reaction with anequimolar amount of 1-chloropropanol
determined using a computer program, HyperChem in chloroform at 70°C for 8 h, and compound 9 was
4.5.
The compounds described in the title were obtained with those quoted in literature (Toth et al 1987).
in a multi-step synthesis (Figures 1 and 2). 1-(2-Hydroxyethyl)piperazine was also condensed
obtained. Its physicochemical constants are consistent

447
Nitrate derivatives of piperazine potential NO donors
Figure 2 Scheme of synthesis of compounds 8±10 and 11±14. Reagent i, fuming HNO3 at ®15°C
with an equimolar amount of 3-chloropropionyl chlor- When all the substrate was added, the reaction mixture
ide in chloroform at 0°C to give compound 10. Its was stirred for 1 h at 0°C. Then it was poured into ice-
physicochemical constants are consistent with those water, and sodium bicarbonate was added to the re-
quoted in literature (Barrett & Caldwell 1962).
action mixture until pH 9 was reached. The precipitated
product was separated by ®ltering or decantation, dried
and transformed into hydrochloride. In this way com-
pounds 11±13 and 15±20 were obtained and puri®ed by
crystallisation from 96% ethanol.
General procedure for obtaining compounds
11–20
The esteri®cation reaction of hydroxypiperazine deriva- Compound 14 was obtained by reaction of compound
tives (2±10) with fuming nitric acid was performed using 13 with anhydrous piperidine in benzene. The reagents
4 moles of nitric acid per one mole of piperazine hydroxy were used in 1:4 molar ratio (4 moles of piperidine per
derivative with one hydroxyl group.
one mole of compound 13). The reaction mixture was
Hydroxypiperazine derivatives were added in small stirred at room temperature for 16 h. Then, sedimented
portions to the fuming nitric acidat®15°C with stirring, piperidine hydrochloride was ®ltered oŒ, the solvent
so that the temperature was not higher than ®5°C. was distilled oŒ from the ®ltrate and the residue was

448
Lucyna Korzycka
dissolved in chloroform. Then, ethyl ether saturated 1-(3-Piperidinepropionyl)-4-(3-
with hydrochloric acid was added drop-wise to give a nitroxypropyl)piperazine dihydrochloride (16)
pH of 6. Precipitated crystalline compound 14 was White crystals (42% ); mp 158°C; IR (KBr) m 1670 (C
1
puri®ed by crystallisation from 96% ethanol.
?O), 1590 (NO2 asym.), 1287 (NO2 sym.); H NMR
(d6-DMSO) d 1.55±2.23 (m, 8H, CH2CH2CH2, CH2),
2.42±3.45 (m, 16H, NCH2), 3.55±3.70 (t, 2H, CH2),
3.95±4.26 (t, 2H, CH2), 5.78 (bs, 2H, NH). Calculated
1-(2-Nitroxypropyl)-4-(2-nitroxyethyl)piperazine
dihydrochloride (11)
White crystals (58% ); mp 121°C; IR (KBr) m1624 (NO2 for C15H30Cl2N4O4 : C, 44.89; H, 7.53; N, 13.96. Found:
asym.), 1278 (NO2 sym.); 1H NMR (d6-DMSO) d1.18± C, 44.78; H, 7.46; N, 13.80.
1.25 (d, 3H, CH3), 2.45±3.35 (m, 12H, NCH2), 3.60±3.85
(m, 1H, CH), 4.05±4.25 (t, 2H, CH2), 5.70 (bs, 2H, NH).
Calculated for C9H20Cl2N4O6 : C, 30.78; H, 5.74; N,
15.95. Found: C, 30.62; H, 5.64; N, 15.85.
1-(3-Piperidinepropionyl)-4-(2-nitroxy-3-
chloropropyl)piperazine dihydrochloride (17)
Light yellow crystals (38% ); mp 155°C; IR (KBr) m
1665 (C?O), 1620 (NO2 asym.), 1280 (NO2 sym.); H
NMR (d6-DMSO) d 1.60±2.23 (m, 8H, CH2CH2CH2,
1
1-(3-Nitroxypropyl)-4-(2-nitroxyethyl)piperazine
dihydrochloride (12)
CH2), 2.42±3.25(m, 16H,NCH2), 3.52±3.71(t, 2H, CH2),
White crystals (69% ); mp 124°C; IR (KBr) m 1626 (NO2
3.83±3.95 (m, 1H, CH), 6.02 (bs, 2H, NH). Calculated
for C15H29Cl3N4O4 : C, 41.34; H, 6.70; N, 12.86. Found:
asym.), 1284 (NO2 sym.); 1H NMR (d6-DMSO) d 1.80±
2.15 (m, 2H, CH2CHCH2), 2.40±3.25 (m, 12H, NCH2),
C, 41.32; H, 6.72; N, 12.89.
3.54±3.73 (t, 2H, CH2), 3.80±4.05 (t, 2H, CH2), 5.62 (bs,
2H, NH). Calculated for C9H20Cl2N4O6 : C, 30.78; H,
1-(3-Piperidinepropionyl)-4-(2-nitroxy-3-
5.74; N, 15.95. Found: C, 30.72; H, 5.60; N, 15.84.
piperidinepropyl)piperazine trihydrochloride (18)
Light yellow crystals (32% ); mp 174°C; IR (KBr) m
1670 (C?O), 1642 (NO2 asym.), 1280 (NO2 sym.); H
1-(3-Chloropropionyl )-4-(2-nitroxyethyl)piperazine
1
hydrochloride (13)
NMR (d6-DMSO) d 1.42±2.45 (m, 12H, CH2CH2CH2),
White crystals (74% ); mp 136°C; IR (KBr) m 1660 (C?
2.55±3.66 (m, 22H, NCH2), 3.80±4.02 (t, 2H, CH2);
O), 1615 (NO2 asym.), 1275 (NO2 sym.); 1H NMR (d6-
4.15±4.35 (m, 1H, CH), 6.40 (bs, 3H, NH). Calculated
DMSO) d 1.75±1.95 (t, 2H, CH2), 2.24±3.05 (m, 10H,
for C20H40Cl3N5O4 : C, 46.12; H, 7.74; N, 13.44. Found:
NCH2), 3.34±3.55 (t, 2H, CH2), 3.75±3.95 (t, 2H, CH2),
C, 46.24; H, 7.62; N, 13.56.
5.45 (s, 1H, NH). Calculated for C9H17Cl2N3O4: C,
35.78; H, 5.67; N, 13.91. Found: C, 35.68; H, 5.62; N,
13.84.
1-(3-Piperidinepropionyl)-4-(2-nitroxy-3-
isopropylaminepropyl)piperazine trihydrochloride (19)
Light yellow crystals (28% ); mp 168°C; IR (KBr) m
3160 (NH), 1680 (C?O), 1645 (NO2 asym.), 1274 (NO2
sym.); 1H NMR (d6-DMSO) d 1.30±1.52 (m., 6H, CH3),
1.60±2.15 (m, 6H, CH2CH2CH2), 2.32±3.16 (m, 16H,
NCH2), 3.25±3.40 (m, 1H, CH), 3.55±3.72 (d, 2H, CH2),
3.76±3.97 (t, 2H, CH2), 4.03±4.35 (m, 1H, CH), 5.90
(bs, 1H, NH), 8.72 (s, 3H, NH). Calculated for
C18H38Cl3N5O4 : C, 43.69; H, 7.74; N, 14.15. Found: C,
43.89; H, 7.82; N, 14.38.
1-(3-Piperidinepropionyl)-4-(2-nitroxyethyl)piperazine
dihydrochloride (14)
Light yellow crystals (51% ); mp 152°C; IR (KBr) m
1
1668 (C?O), 1620 (NO2 asym.), 1210 (NO2 sym.); H
NMR (d6-DMSO) d 1.60±2.52 (m, 6H, CH2CH2CH2),
2.62±3.21 (m, 16H, NCH2), 3.48±3.55 (t, 2H, CH2),
3.68±3.97 (t, 2H, CH2), 5.72 (bs, 2H, NH). Calculated
for C14H28Cl2N4O4 : C, 43.42; H, 7.29; N, 14.47. Found:
C, 43.31; H, 7.28; N, 14.42.
1-(3-Piperidinepropionyl)-4-(2-
nitroxypropyl)piperazine dihydrochloride (15)
1-(3-Piperidinepropionyl)-4-[2-nitroxy-3-(4-
methylpiperazine)propyl]tetrahydrochloride (20)
Light yellow crystals (41% ); mp 158°C; IR (KBr) m White crystals (35% ); mp 196°C; IR (KBr) m 1675 (C?
1
1664 (C?O), 1635 (NO2 asym.), 1287 (NO2 sym.); H O), 1642 (NO2 asym.), 1276 (NO2 sym.); 1H NMR (d6-
NMR (d6-DMSO) d 1.22±1.34 (d, 3H, CH3), 1.50±2.25 DMSO) d 1.71±1.91 (m, 6H, CH2CH2CH2), 2.53±2.88
(m, 6H, CH2CH2CH2), 2.38±3.25 (m, 16H, NCH2), (m, 26 H, NCH2), 3.00 (s, 3H, CH3), 3.24±3.56 (t, 2H,
3.42±3.60 (t, 2H, CH2), 3.72±3.95 (m, 1H, CH), 5.45 (bs, CH2), 3.95±4.23 (m, 1H, CH), 4.82±5.72 (bs, 4H, NH).
2H, NH). Calculated for C15H30Cl2N4O4 : C, 44.89; H, Calculated for C20H42Cl4N6O4 : C, 41.97; H, 7.40; N
7.53; N, 13.96. Found: C, 44.82; H, 7.42; N, 13.84.
14.68. Found: C, 41.63; H, 7.28; N, 14.44.

449
Nitrate derivatives of piperazine potential NO donors
have a positive eŒect on the circulatory system, signi®-
cantly increasing the coronary blood ¯ ow. Compound 4
Results and Discussion
Our earlier studies revealed that the obtained series of showed the strongest eŒect. Currently, we have obtained
non-symmetrical derivatives of 1,4-disubstituted hy- nitrate analogues in this series of piperazine derivatives,
droxy derivatives of piperazine depress the CNS and which due to the presence of an ONO2 group may
provide exogenous nitric oxide and thus can be stronger
vasodilators.
The structure of compounds 11±20 was con®rmed by
spectroscopy and elementary analysis. The IR spectra
of these new compounds con®rm strong signals of
asymmetric vibrations and weaker symmetric vibrations
of an ±NO2 group. 1H NMR spectra show signals for all
protons present in the structure of these compounds.
The esteri®cation reaction of hydroxypiperazine deri-
vatives with fuming nitric acid must be carried out at
precise temperature. Too low a temperature, below
±15°C, will make the reagents solidify; too high a
"
temperature, ±5°C, will cause partial decomposition
of products. The reaction mixture must be homogenous
(vigorous stirring).
Compounds 10±20 were initially tested in-vitro. As a
model of enzymatic metabolism of therapeutic organic
nitrates we have studied the reaction of derivatives 11±20
with the ethyl ester of l-cysteine hydrochloride as the
sulfhydryl compound. The reactions were conducted in
1:3 molar ratio, using 3 moles of su¯ hydryl compound
per one mole of the studied nitrate. The reagents were
stirred in the phosphate buŒer, pH 7.5, at 37°C for 15,
30 and 45 min, respectively. The amount of nitric and
nitrate ions was measured by a potentiometric method
using nitric and nitrate ionoselective electrodes. A calo-
mel electrode was employed as the reference electrode.
Calibration curves were drawn for E (mV)¯ f (pc) for
potassium nitrite and nitrate standard solutions. On the
basis of calibration standard curves, the amount of
nitric and nitrate ions formed in the reaction of com-
pounds 11±20 with the hydrochloride of ethyl ester of l-
cysteine was calculated.
We found that all the studied nitrates, 11±20, under-
went decomposition in the reaction with the sulfhydryl
compound. Thereaction occurred during the ®rst 15 min
-
of heating and after that time the sum of released NO2
.
Figure 3 HyperChem 4 5 optimized structures of compounds 4 and
18
Table 1 Physicochemical and structure-activity relationships parameters of compounds 4 and 18
Compound
Total energy
1
Heat of formation
1
Binding energy
1
Log P
Refractivity
(L3)
Polarizability
(L3)
Hydration energy
1
-
-
-
-
(kcal mol
)
(kcal mol
)
(kcal mol
)
(kcal mol
)
4
18
®105 026.64
®124 102.13
®98.48
®9.86
®6067.28
®6158.67
0.81
®2.20
107.10
112.79
41.28
43.12
®1.46
159.18

450
Lucyna Korzycka
-
and NO3 ions did not change. The greatest amount of
nitric ions and nitrate ions was released by compound
18 and a smaller amount by compounds 20, 14, 19, 16,
15, 17, 12, 11 and 13, respectively. Compound 18
released much more nitrate and nitric ions than the
therapeutic organic nitrate isosorbide-5-mononitrate in
the same reaction. Compound 18 is a nitrate analogue of
the most pharmacologically active hydroxypiperazine
derivative, compound 4.
References
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.
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The conducted experiments show that nitrates 11±20
obtained in this study undergo the reaction that deter-
mines the pharmacological activity of therapeutic or-
ganic nitrates. Compound 18 was the most active. For
this reason, we conducted computerised comparative
analysis for compound 18 and its hydroxy analogue,
compound 4. Optimized structures of these compounds
withdescription of the chargedistributions arepresented
in Figure 3. Optimization of these structures was per-
formed by the semi-empirical method PM3, using the
programme HyperChem 4.5. We also determined and
evaluated several 3-D QSAR parameters (Table 1).
Compound 18 showed lower total energy and bond
energy than compound 4. This suggests that compound
18 is not stable and may be easily metabolised. These
parameters correlate with those obtained in the ex-
periment withthe ethyl ester of l-cysteine hydrochloride.
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18 has a nity to the water phase.
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}
295 08) 04 Nov