Synthesis of new derivatives of pyrazolone and nicotinic acid and study of their effect on cytochrome P-450 activity

Full text of DOI:10.1023/A:1010355129735

Pharmaceutical Chemistry Journal
Vol. 34, No. 10, 2000
SYNTHESIS OF NEW DERIVATIVES OF PYRAZOLONE
AND NICOTINIC ACID AND STUDY OF THEIR EFFECT
ON CYTOCHROME P-450 ACTIVITY
I. V. Shemarova,1 E. B. Maizel’,1 I. V. Voznyi,1 N. P. Stepanova,1 and A. E. Khovanskikh1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 34, No. 10, pp. 17 – 18, October, 2000.
Original article submitted April 1, 1999.
At present, medicine employs preparations capable of in-
hibiting enzymes involved in the metabolism of xenobiotics;
drugs of this type include, for example, iproniazid,
imipramine, chlorcyclizine, and glutethimide [1, 2].
As is known, amidopyrine Ia (known to be a substrate for
cytochrome C-450 [4]) contains readily oxidized methyl
groups at the exocyclic atom of nitrogen. Modification of the
substrate by introducing a methine group between amino
group and pyrazolone cycle does not impart inhibiting prop-
erties to compound Ie. Compounds Ib – Id, containing
pyridylmethylene groups with variously positioned nitrogen
atoms in the pyridine cycle, exhibit a pronounced inhibiting
effect with respect to the given enzyme. This activity is prob-
ably related to a decrease in the electron density on the
pyrazolone cycle in these compounds. The maximum inhibi-
tion activity was observed for 2,3-dimethyl-1-phenyl-4-(4-
methylenepyridylamino)-3-pyrazolin-5-one (Id).
The purpose of this work was to synthesize Schiff bases
from 4-aminopyrazolones, to synthesize hydrazones from
nicotinic acid hydrazides and N-benzoyl-b-alanine, and to
study the effect of the synthesized compounds on the activity
of cytochrome P-450, which is the enzyme accelerating me-
tabolism of many antiinflammatory preparations.
RESULTS AND DISCUSSION
We have synthesized a series of 2,3-dimethyl-1-phenyl-
pyrazolin-5-one derivatives (Ib – Ie). Using the condensation
of hydrazides with aldehydes [3], we have also obtained the
hydrazones of nicotinic acid hydrazides (IIa, IIb) and N-ben-
zoyl-b-alanine (III). The yields and physicochemical charac-
teristics of the synthesized compounds are presented in Ta-
ble 1. Data on the cytochrome C-450 inhibition activity of
these compounds are summarized in Table 2.
Taking into account that a most pronounced inhibition of
the cytochrome P-450 activity is produced by the amidopy-
rine derivatives containing pyridylmethylene fragments, we
may suggest that this very fragment accounts for the en-
hanced activity. Apparently, a change in the structure renders
the base molecule less reactive as an oxidation substrate and,
as a result, the molecule interacts with the enzyme as a com-
petitive inhibitor. This is confirmed by data on the competi-
tive suppression of the process of butamine and meperidine
N-deamination by DPEA, SKF 525, and Lilly 18947 [5].
The inhibiting action with respect to monooxidases is
also typical of the derivatives of pyridinercarboxylic acid de-
rivatives [6]. In this context, it was of interest to study the in-
hibiting properties of the derivatives of nicotinic acid
NMe2
N
CHR
NMe2
N
N
N
N
O
N
O
N
Ie
O
Ib – Id
Ia
O
hydrazides and N-benzoyl-b-alanine (IIa, IIb, III) containing
–N=CH–R– groups with respect to cytochrome C-450 acting
as a terminal oxidase in reactions of the monooxygenase
type. The results of these experiments are presented in Ta-
ble 2. As is seen, the initial pyridinercarboxylic acids are not
capable of inhibiting cytochrome P-450. Introduction of the
–N=CH–R fragment into these molecules leads (as it was in
the previously studied cases) to a pronounced manifestation
of the inhibiting activity with respect to this enzyme.
N
R
N
H
H
N
H
N
N
N
IIà, IIb
O
O
N
III
R = 2-pyridinyl (Ib); 3-pyridinyl (Ic, IIa), 4-pyridinyl (Id); 2-pyridinyl (IIb).
1
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian
Academy of Sciences, St. Petersburg, Russia.
530
0091-150X/00/3410-0530$25.00 © 2001 Plenum Publishing Corporation

Synthesis of New Derivatives of Pyrazolone and Nicotinic Acid
531
TABLE 1. Yields and Physicochemical Characteristics of the Synthesized Compounds
Com-
pound
Empirical
formula
M.p., °C
1H NMR spectrum: d, ppm
Yield, %
Ib
C17H16N4O
C17H16N4O
C17H16N4O
C13H15N3O
C12H6N4O
C11H9N3O2
C17H16N4O2
167
2.32 (CH3); 2.95(CH3); 7.20 – 8.70 (Ph, Pyr)
2.31 (CH3); 2.93 (CH3); 7.12 – 8.14 (Ph, Pyr)
2.32 (CH3); 2.96 (CH3); 7.23 – 8.65 (Ph, Pyr)
2.30 (CH3); 3.03 (2CH3); 7.28 – 8.04 (CH, Ph)
7.13 – 9.50 (Pyr); 14.3 (NH)
74
78
84
60
80
90
71
Ic
206 – 207
241 – 242
135 – 136
219 – 220
193
Id
Ie
IIa
IIb
III
6.35 – 9.50 (Pyr, Fur); 14.25 (NH)
188
2.78 (CH2); 4.20 (CH2); 6.96 – 8.50 (Pyr, Ph); 12.61 (NH)
Thus, the results of experiments demonstrate the inhibit-
ing effect of the synthesized pyrazoline derivatives and hy-
drazones of nicotinic acid hydrazdes and N-benzoyl-b-ala-
nine with respect to cytochrome P-450. This confirms the ex-
pediency of the search for new cytochrome P-450 inhibitors
in compounds of this series.
an analogous procedure, proceeding from aminoantipyrine
and nicotinic aldehyde.
2,3-Dimethyl-1-phenyl-4-(4-pyridylmethyleneamino)-
3-pyrazolin-5-one (Id). Compound Id was synthesized simi-
larly to Ib, proceeding from aminoantipyrine and isonicotinic
aldehyde.
Pyridine-2-carbaldehyde 3-pyridinecarbohydrazone
(IIa). To a solution of 1.37 g (0.01 mole) of nicotinic acid
hydrazide in ethanol was added 1.2 ml (0.01 mole) of nico-
tinic aldehyde ethanol and the mixture was boiled for 1 h.
Upon cooling, the precipitated product was filtered and
recrystallized from ethanol.
EXPERIMENTAL CHEMICAL PART
1
The N NMR spectra of the synthesized compounds in
DMSO-d6 solutions were measured relative to TMS as the
internal standard in a Tesla BS-487c (80 MHz) spectrometer.
Purity of the target compounds was confirmed by TLC on
Silufol UV-254 plates. Compounds Ib – Ie were eluted in the
hexane–ether (3 : 1) solvent system, while compounds IIa,
IIb, and III were eluted in benzene – ethanol (8 : 1). The
spots were visualized under UV irradiation. The melting
temperatures of the synthesized compounds were determined
on a Boetius heating stage. The data of elemental analyses
agreed with the results of analytical calculations according to
the empirical formulas. The yields and some physicochemi-
cal characteristics of the synthesized compounds are presen-
ted in Table 1.
2-Furancarbaldehyde 3-pyridylcarbohydrazone (IIb).
Compound IIb was synthesized using an analogous proce-
dure, proceeding from furfural and nicotinic acid hydrazide.
Pyridine-2-carbaldehyde 2-(N-benzoylamino)ethyl-
carbohydrazone (III). Compound III was synthesized simi-
larly to IIa, proceeding from N-benzoyl-b-alanine and picoli-
nic aldehyde.
EXPERIMENTAL BIOCHEMICAL PART
The initial cytochrome P-450 was represented by a prep-
aration of liver microsomes. The microsomes were isolated
from liver homogenates of Wistar male rats weighing
150 – 200 g by a differential centrifugal technique [7]. The
cytochrome P-450 activity was assessed by the rate of the
3,4-benz[a]pyrene hydroxylation reaction evaluated by
fluorimetric determination of the reaction product (4-hydro-
3-Methyl-1-phenyl-4-(N,N-dimethylaminomethylene)-
2-pyrazolin-5-one (Ie). A solution of 101.4 g (0.66 mole) of
POCl3 in 140 ml (1.8 mole) DMF was kept for 30 min at
12°C. To this solution was added 104.4 g (0.6 mole) of
methylphenylyrazolinone and the mixture was boiled for 3 h
on a water bath with reflux. Upon cooling to room tempera-
ture, the mixture was poured on 3 kg of ice and neutralized
with 30 % KOH. The precipitated product was filtered, dried
in air, and purified by recrystallization from ethanol.
TABLE 2. Inhibition Activity of Compounds
I – III with Respect to Cytochrome P-450 in Rat
Liver Microsomes
2,3-Dimethyl-1-phenyl-4-(2-pyridylmethyleneamino)-
3-pyrazolin-5-one (Ib). To a solution of 1.88 g (0.01 mole)
of aminoantipyrine in 30 ml of ethanol was added 1.2 ml
(0.01 mole) of picolinic aldehyde in ethanol and the mixture
was boiled for 2 h on a water bath with reflux. Upon cooling
to room temperature, the precipitated product was filtered
and recrystallized from ethanol.
Compound
MIC50, mole/liter
5.3 ´ 10 – 4
2.3 ´ 10 – 5
6.9 ´ 10 – 5
8.8 ´ 10 – 2
5.0 ´ 10 – 4
9.6 ´ 10 – 4
1.24 ´ 10 – 3
Ib
Ic
Id
Ie
IIa
IIb
III
2,3-Dimethyl-1-phenyl-4-(3-pyridylmethyleneamino)-
3-pyrazolin-5-one (Ic). Compound Ic was synthesized using

532
I. V. Shemarova et al.
2. B. Tesla and P. Jenner, Drug Metal. Reviews, 12(1), 1 – 117
(1981).
xybenz[a]pyrene) as described in [8]. The reaction was initi-
ated by adding NADP-H (Reanal) to a final concentration of
1 mM. The fluorescence measurements were performed on a
Specord MHO instrument.
3. M. M. Sprung, Chem. Rev., 26, 297 (1940).
4. B. Tesla, Biochem. Pharmacol., 37(1), 81 – 92 (1988).
5. R. E. McMahon, Pharm. Sci., 55(3), 457 – 477 (1966).
6. M. D. Mashkovskii, N. I. Andreeva, and A. I. Polezhaeva, Anti-
depressant Pharmacology [in Russian], Meditsina, Moscow
(1983).
The cytochrome P-450 inhibition activity of the synthe-
sized compounds was evaluated by the value of MIC 50, the
minimum agent concentration producing a 50% decrease in
enzyme activity. The protein content was determined by the
classical Lowry method [9].
7. A. A. Akhrem and S. A. Usanov, Dokl. Akad. Nauk SSSR, 218,
1457 – 1460 (1974).
8. K. M. Robic, Y.-N. Cha, R. E. Talleott, et al., Chem. Biol. Inter.,
12, 285 – 297 (1976).
REFERENCES
9. Q. H. Lowry, N. J. Rosenbrough, A. L. Farr, et al., J. Biol.
Chem., 193, 265 – 275 (1951).
1. P. Lesca, E. Pafidinarivo, P. Lecointe, et al., Chem. Biol. Inter-
act., 24, 189 – 198 (1979).