Synthesis and pharmacological evaluation of N- (dimethylamino)ethyl derivatives of benzo- and pyridopyridazinones

Article abstract of DOI:10.1002/ardp.200800016

New N-(dimethylamino)ethyl derivatives of phthalazinones and pyridopyridazinones 7, 9 were synthesized and assayed as potential analgesic agents in the hot-plate, tail-flick, and writhing tests. Pharmacological assay demonstrated that eight (in ten) of th

Full text of DOI:10.1002/ardp.200800016

Arch. Pharm. Chem. Life Sci. 2009, 342, 41–47
W. Pakulska et al.
41
Full Paper
Synthesis and Pharmacological Evaluation of N-
(Dimethylamino)ethyl Derivatives of Benzo- and
Pyridopyridazinones
Wanda Pakulska1, Zbigniew Malinowski2, Aleksandra K. Szczesniak2, Elzbieta Czarnecka1, and
Jan Epsztajn2
1 Department of Pharmacodynamics, Medical University of Lodz, Lodz, Poland
2 Department of Organic Chemistry, Faculty of Chemistry, University of Lodz, Lodz, Poland
New N-(dimethylamino)ethyl derivatives of phthalazinones and pyridopyridazinones 7, 9 were
synthesized and assayed as potential analgesic agents in the hot-plate, tail-flick, and writhing
tests. Pharmacological assay demonstrated that eight (in ten) of the newly synthesized com-
pounds showed antinociceptive activity. Especially, 2-[2-(dimethylamino)ethyl]-4-phenyl-2H-
phthalazin-1-one 7a showed remarkably higher antinociceptive activity in all tests. This is con-
nected with influence on supraspinal, spinal, and peripheral structures. The decreased sensitiv-
ity to the pain stimulus in the hot-plate was higher than that of metamizole.
Keywords: Hot-plate test / Phthalazinones / Pyridopyridazinones / Tail-flick test / Writhing test /
Received: January 17, 2008; accepted: June 19, 2008
DOI 10.1002/ardp.200800016
Introduction
During the last decade, a lot of attention has been given
to the study focused on the construction of 2H-pyridazin-
3-ones type A (Fig. 1) as well as their biological activity [1].
It has been observed that the derivatives of 2H-pyrida-
zin-3-ones exhibit a number of different pharmacological
activity e.g., adenosine A1 receptor antagonist [2], a-1
adrenoreceptors antagonist [3–5], anti-aggregation activ-
ity [6], antinociceptive agents [7–9], and nonprostanoid
PGI2 agonist [10]. It has been found that some of deriv-
atives of phthalazinones exhibit potent inhibition of
phosphodiesterase (PDE4) activity [11] and agonist activ-
ity to dopamine D2 and serotonin 5-HT2 receptors [12].
The research studies of the last years also show that a few
of the analogues of phthalazinones inhibit HIV replica-
tion [13].
Figure 1. Derivatives of 2H-pyridazin-3-ones type A and of
benzo- B and pyridopyridazinones C types with a variety of sub-
stituents.
As part of the study for new analgesics and nonsteroi-
dal antiinflammatory agents (NSAIDs), we initiated a
research program based on the study related to synthesis
of new derivatives of benzo- B and pyridopyridazinones C
with variety of substituents. The well-documented anti-
nociceptive activity of the systems A which are the core
for the structures B and C (Fig. 1) dictated the selection of
these compounds, with an idea to extent the knowledge
concerned with their analgesic and NSAID pharmacolog-
ical properties. Some of them were found to have analge-
sic and nonsteroidal antiinflammatory activity, as was
shown in this preliminary communication.
Correspondence: Dr. Wanda Pakulska, Department of Pharmacody-
namics, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Po-
land.
E-mail: wpakulska@pharm.am.lodz.pl
Fax: + 48 42 677-9294
Abbreviation: percent of maximal possible effect (% MPE)
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W. Pakulska et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 41–47
Table 1. Acute toxicity of compounds 7, 8, and 9 after per-os administration.
Compound
7a
7b
7c
7d
7e
7f
7g
8
9a
9b
LD50 (mg/kg b.w./p.o.)
200
400
400
750
500
375
150
600
1000
300
Results and discussion
Chemistry
N-[2-(Dimethylamino)ethyl] derivatives of 2H-pyridazin-3-
one and benzo-, and pyrido- analogues (compounds 7, 8,
and 9) were synthesized as illustrated in Scheme 1. As
starting materials for the synthesis of the amines 7, 8,
and 9 the previously described 2H-phthalazin-1-ones 4
[14], pyridopyridazinones 6 [15] and commercially avail-
able 2H-pyridazin-3-one 5 were used.
Aminoalkylation of the appropriate lactams 4, 5, or 6
with the (2-chloroethyl)dimethylamine, via most com-
monly used methodology [16] afforded the final com-
pounds 7, 8, 9 in good yields. IR, NMR, HRMS, and micro-
analyses confirmed the structures of the aminoethyl
derivatives 7, 8, and 9. Melting points, yields, and spectral
data are shown in Experimental (Section 4). For pharma-
cological assays, compounds 7a–g, 8, 9a and 9b were con-
verted to their water-soluble hydrochlorides.
Pharmacology
Acute toxicity
Scheme 1. Synthesis of N-[2-(Dimethylamino)ethyl] derivatives
of 2H-pyridazin-3-one and benzo-, and pyrido- analogues (com-
pounds 7, 8 and 9).
After a single p.o. administration of the compounds at
various doses, the observations were carried out for a
period of 14 days. The LD50 values of the investigated com-
pounds 7a–g, 8, 9a, b varied and ranged from l150 to
1000 mg/kg (Table 1). The most toxic compound was 7g
(ALD50 = 150 mg/kg). The least toxic compound was 9a
(ALD50 = 1000 mg/kg). The toxicity of the remaining com-
pounds fluctuated between 200 and 750 mg/kg. For all
derivatives 1/10 LD50 was taken as the initial dose in the
further experiments. Toxic doses of the all tested com-
pounds caused sedation and depression of locomotive
activity of mice.
Compound 7a, at the dose of 20 mg/kg, significantly
prolonged the latency to pain reaction after administra-
tion. The% MPE obtained for the compound 7a was
ranged 32.45 (measured after 60 min), 35.09 (measured
after 120 min), and 39.84 (measured after 180 min). This
result was significant (p a 0.05, see Table 2) compared to
the zero measurement and comparable or higher than
metamizole. Compounds 7b and 7c, at doses 40 mg/kg,
significantly increased the time to pain reaction in ani-
mals only after 180 min (% MPE obtained for 7a = 40.86,
7c = 30.37, p a 0.05, see Table 2). Compound 7e, at the
dose of 50 mg/kg, prolonged significantly the latency of
nociceptive reaction to the hot-plate test in mice at every
measurement time. The% MPE obtained for the com-
pound was ranged 16.00 (measured after 60 min), 24.59
(measured after 120 min), and 25.99 (measured after
180 min, Table 2). Compound 7g, at the dose of 15 mg/kg,
prolonged nociceptive reaction latency after 60 and
90 min (% MPE 18.95 and 16.05, respectively). This result
was significant (p a 0.05, see Table 2) compared to the
zero measurement. Compounds 7d, 7f, 8, and 9b did not
demonstrate a significant effect in the test.
Analgesic properties
The analgesic activities of the newly synthesized com-
pounds: 7a–g, 8, 9a and 9b in this study were measured
using the hot-plate and tail-flick tests, and the acetic acid-
induced writhing assay in mice.
Effect of compounds on the hot-plate response
The % MPE (percent of maximal possible effect) obtained
for the all compounds are presented in Table 2. The
results of the hot-plate test after i.p. administration of
compounds 7a, 7b, 7c, 7e, and 7g are shown in Fig. 2.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 41–47
New Antinociceptive Agents
43
Figure 3. Results of the tail-flick test after i.p. administration of
compounds 7a, 7c, 7d and 9b.
Figure 2. Results of the hot-plate test after i.p. administration of
compounds 7a, 7b, 7c, 7e and 7g.
Table 3. The effects of compounds 7, 8 and 9 in the mouse tail-
flick test.
Table 2. The effects of compounds 7, 8 and 9 in the mouse hot-
plate test.
Compound Dose (mg/kg
b.w., i.p.)
Latency (% MPE) to paw licking or
jumping
Compound Dose (mg/kg Latency (% MPE) to paw licking or
b.w., i.p.)
jumping
after
after
after
60 min
120 min
180 min
after
after
after
60 min
120 min
180 min
7a
7b
7c
7d
7e
7f
7g
8
9a
9b
Met
20
40
40
75
50
37.5
15
60
19.81
4.05
7.99
13.08
9.87
3.58
0
9.22
5.69
13.69a)
12.16a)
39.61a, b)
10.45
14.84a)
23.05a)
0
5.26
0
12.36
14.22
19.02a)
22.68a)
6.28
11.72
18.26a)
27.34a)
6.45
13.26
0
7.76
7a
7b
7c
7d
7e
7f
7g
8
9a
9b
Met
20
40
40
75
50
37.5
15
60
100
30
500
32.45a, b)
0
35.09a)
10.66
10.42
1.63
39.84a, b)
40.86a, b)
30.37a)
18.85a)
25.99a)
2.88
16.00
0
3.03
12.86
3.57
16.00a)
13.97
18.95a)
0
24.59a)
12.42
16.05a)
0
100
30
500
8.94
18.32a)
14.63a)
4.98
8.87
1.10
3.29
5.27
15.58a)
30.74a)
25.54a)
Each group consisted of eight to ten animals.
a)
Significant differences versus initial value, p a 0.05.
Significant differences from the metamizole (Met)-treated
group, p a 0.05.
Each group consisted of eight to ten animals.
b)
a)
Significant differences versus initial value, p a 0.05.
Significant differences from the metamizole (Met)-treated
group, p a 0.05.
b)
23.05 (measured after 120 min) and 27.34 (measured
after 180 min, p a 0.05, Table 3). Compound 9b at the
dose of 30 mg/kg prolonged significantly the latency of
nociceptive reaction to the tail-flick test in mice et every
measurement time. The% MPE obtained for the com-
pound was ranged 13.69 (measured after 60 min), 19.02
(measured after 120 min), and 18.32 (measured after
180 min). This result was comparable to metamizole (see
Table 3). Compounds 7b, 7e, 7f, 7g, 8 and 9a did not dem-
onstrate a significant effect in the test.
Effect of compounds on the tail-flick response
The % MPE obtained for all compounds are shown in
Table 3. The results of the tail-flick test after i.p. adminis-
tration of compounds 7a, 7c, 7d and 9b are presented in
Fig. 3.
Compound 7a, at the dose of 20 mg/kg significantly
prolonged nociceptive reaction latency only 120 min
after administration (% MPE 39.61, p a 0.05, Table 3) com-
pared to the zero measurement. Compounds 7c (40 mg/
kg) and 7d (75 mg/kg) prolonged nociceptive reaction
latency after 120 and 180 min. The % MPE obtained for
the compound 7c was ranged 14.84 (measured after
120 min), 18.26 (measured after 180 min, p a 0.05, see
Table 3). The% MPE obtained for the compound 7d were
Effect of compounds of the nociceptive response to acetic
acid
Given orally at 20 mg/kg, compound 7a caused an analge-
sic effect, statistically reducing the number of abdominal
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W. Pakulska et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 41–47
structures as antinociceptive systems. The decreased sen-
sitivity to the pain stimulus in the hot-plate was higher
than to metamizole. Compound 7e demonstrated a
strong antinociceptive effect in the hot-plate test. Com-
pounds 7d and 9b showed a significant effect in the tail-
flick test. This result was comparable to metamizole.
Compounds 8, 9a, and 9b exhibited an antinociceptive
effect in the writhing test. Compound 8 showed here the
strongest action.
Compound 7a showed the highest activity of all newly
synthesized compounds; this is probably due to the phe-
nyl group attached to the 4-position of the phthalazinone
ring. The observation that removal of the phenyl ring or
replacing it by a methyl group or a pyridine nucleus
caused a decrease of the antinociceptive activity suggests
this point of view.
Figure 4. Effect of compounds in the writhing test.
constrictions induced by acetic acid. The most potent
effect was produced by compounds 8 (60 mg/kg, p.o.), 9a
(100 mg/kg, p.o.), and 9b (30 mg/kg, p.o.). The strongest
action in the test was compound 8, reducing nearly two-
fold the number of abdominal constrictions in compari-
son with animals receiving acetic acid (controls), how-
ever, two times weaker in comparison with mice, which
received metamizole (Met). Compounds 7b, 7c, 7d, 7e, 7f,
and 7g did not demonstrate a significant effect in the
writhing test. The data are shown in Fig. 4.
The study was supported by the the Polish Ministry of Science
and Higher Education, Grant No. 3 T09A 015 29. The results of
this study were included in the Patent Application No. P
383550 (The Patent Office of the Republic of Poland).
The analgesic properties of the compounds obtained in
this study were assessed using three tests. In the hot-plate
test, the nociceptive reaction is coordinated predomi-
nantly by supraspinal structures, whereas the tail-flick
test allows assessing the involvement of spinal cord struc-
tures in a nociceptive reaction [17]. The writhing test
measures peripheral analgesia [18].
The efficiency of the tested compounds 7, 8, and 9
proved to be dependent upon their structures. From
amongst of the compounds, 7a appeared to be effective
in all tests and exhibited the highest antinociceptive
activity. In the hot-plate test, this compound proved to be
even more active than the known metamizole. All other
tested compounds proved to be effective in two tests or at
least in one as systematized below:
The authors have declared no conflict of interest.
Experimental
Chemistry
The melting points were determined on a Boetius hot stage appa-
ratus (VEB Analytic, Dresden, Germany) and are uncorrected. 1H-
NMR spectra were recorded at 200 MHz and 13C-NMR spectra at
50 MHz on a Varian Gemini 200 BB spectrometer (Varian Inc.,
Palo Alto, CA, USA) with TMS as an internal reference. IR spectra
were recorded on a Nexus FT-IR spectrometer (Thermo Nicolet
Co., Madison, WI, USA). Mass spectra analyses were performed
on a Mass Spectrometer MAT95 – Finnigan (Thermo Electron
Corporation, Bremen, Germany). The analytical thin layer chro-
matography tests (TLC) were carried out on Merck silica gel
plates (Kieselgel 60 F254, layer thickness 0.2 mm; Merck, Darm-
stadt, Germany) and the spots were visualized using an UV lamp.
Butyl lithium solution in hexane (Sigma-Aldrich, Poznan,
Poland) was each time titrated before use. Commercially avail-
able hydrazine monohydrate, N,N,N',N9-tetramethyl-ethylenedi-
amine, N,N-dimethylethanolamine, 2-benzoilbenzoic acid 3a, 2-
acetylbenzoic acid 3b, 2H-phthalazin-1-one 4g (all substances
Sigma-Aldrich) and 2H-pyridazin-3-one 5 (Fluka, Poznan, Poland)
were used without further purification. 3-Hydroxy-1H-isoindo-
lin-1-ones 1b–h, pyrrolopyridinones 2a–b, 2H-phthalazin-1-
ones 4a–g, pyridopyridazinones 6a–b, and (2-chloroethyl)dime-
thylammonium chloride were prepared according to an already
reported procedure [14, 15].
Compound 7c in the hot-plate and tail-flick tests, 9b in
tail-flick and writhing tests, 7b and 7c only in the hot-
plate test, 7d in the tail-flick test, 8 and 9a in the writhing
test. Two of the tested compounds, 7f and 7g, did not
show any activity in the applied tests.
Conclusion
We can conclude the following: Eight (in a ten) of the
newly synthesized compounds showed antinociceptive
activity. Compound 7a (2-[2-(dimethylamino)ethyl]-4-phe-
nyl-2H-phthalazin-1-one) showed remarkably potent anti-
nociceptive activity in all (three) tests. This is connected
with influence on supraspinal, spinal, and peripheral
General procedure for preparation of N-[2-
(dimethylamino)ethyl]-derivatives 7, 8, 9
To the solution of sodium methoxide (1.975610– 2 mol) in dry
methanol (50 mL) was added 2H-phthalazin-1-one 4 or 2H-pyrida-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 41–47
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45
zin-3-one 5 or pyridopyridazinone 6 (7.897610– 3 mol) and (2-
chloroethyl)dimethylammonium chloride (1.185610– 2 mol).
The mixture was heated at reflux for 6 hours. Afterwards, the
inorganic material was collected by filtration, washed with dry
methanol and, the filtrate was evaporated to dryness. The resi-
due was subjected to column chromatography to give the pure
product.
136.8, 131.5, 130.8, 129.7, 123.4, 122.0, 120.2, 107.4, 57.3 (CH2),
55.6 (OMe), 48.7 (CH2), 45.5 (Me). MS m/z (%): 324 [M]+ (1), 254 (31),
71 (51), 58 (100); HRMS [M]+: calcd. for C18H20N4O2: 324.15862,
found: 324.15877.
2-[2-(Dimethylamino)ethyl]-6-methoxy-4-(pyridin-4-yl)-
Alternatively, in order to separate the starting lactam, the res-
idue was triturated with hydrochloric acid (c = 2 mol/L, 20 mL).
The filtrate was neutralized with aqueous solution of sodium
hydroxide and extracted with dichloromethane (3620 mL). The
combined extracts were dried over MgSO4 and concentrated to
dryness. The amines were separated by column chromatogra-
phy.
2H-phthalazin-1-one 7e
Yield 58%; m.p.: 149–1528C. Eluent: AcOEt / MeOH = 1 : 1. Rf =
0.24. FT-IR (KBr) cm– 1: 1644 (C=O). 1H-NMR (CDCl3) d: 8.81 (dd, J =
1.5, 4.4, 2H, PyH), 8.47 (d, J = 8.9, 1H, 8-ArH), 8.56 (dd, J = 1.5, 4.4,
2H, PyH), 7.36 (dd, J = 2.5, 8.9, 1H, 7-ArH), 7.02 (d, J = 2.4, 1H, 5-
ArH), 4.18 (t, J = 6.8, 2H, CH2), 3.86 (s, 3H, OMe), 2.81 (t, J = 6.8, 2H,
CH2), 2.34 (s, 6H, Me). 13C-NMR (CDCl3) d: 163.2, 158.9 (C=O),
150.3, 144.1, 143.0, 130.3, 129.7, 123.9, 123.7, 121.8, 120.3,
107.3, 56.8 (CH2), 55. 7 (OMe), 47.9 (CH2), 45.0 (Me). Anal. Calc.
(C18H20N4O2): C, 66.65; H, 6.21; N, 17.27. Found: C, 66.30; H, 6.12;
N, 17.15.
2-[2-(Dimethylamino)ethyl]-4-phenyl-2H-phthalazin-1-
one 7a
Yield 65%; m.p.: 99–1028C. FT-IR (KBr) cm– 1: 1653 (C=O). 1H-NMR
(CDCl3) d: 8.49–8.54 (m, 1H, 8-ArH), 7.70–7.80 (m, 3H, ArH),
7.49–7.60 (m, 5H, ArH), 4.48 (t, J = 6.8, 2H, CH2), 2.84 (t, J = 6.8,
2H, CH2), 2.35 (s, 3H, Me). 13C-NMR (CDCl3) d: 159.1 (C=O), 147.0,
135.2, 132.7, 131.3, 129.4, 129.1, 128.6, 128.2, 127.2, 126.6, 57.1
(CH2), 48.6 (CH2), 45.4 (Me). Anal. Calc. (C18H19N3O): C, 73.70; H,
6.53; N, 14.32. Found: C, 73.52; H, 6.55; N, 14.29.
2-[2-(Dimethylamino)ethyl]-4-methyl-2H-phthalazin-1-
one 7f
Yield 55%; m.p.: 30–338C. Eluent: AcOEt / MeOH = 1 : 1. Rf = 0.20.
FT-IR (KBr) cm– 1: 1652 (C=O). 1H-NMR (CDCl3) d: 8.40–8.46 (m, 1H,
8-ArH), 7.67–7.82 (m, 3H, 5,6,7-ArH), 4.31 (t, J = 7.0, 2H, CH2), 2.75
(t, J = 7.0, 2H, CH2), 2.56 (s, 3H, Me), 2.32 (s, 6H, Me). 13C-NMR
(CDCl3) d: 159.3 (C=O), 143.7, 132.7, 131.2, 129.7, 127.6, 127.0,
124.7, 57.0 (CH2), 48.2 (CH2), 45.3 (Me), 18.8 (Me). Anal. Calc.
(C13H17N3O): C, 67.51; H, 7.41; N, 18.17. Found: C, 67.49; H, 7.35;
N, 18.09.
2-[2-(Dimethylamino)ethyl]-6-methoxy-4-(2-
methoxyphenyl)-2H-phthalazin-1-one 7b
Yield 56%; m.p.: 110–1138C. Eluent: AcOEt / MeOH = 2 : 1. Rf =
0.26. FT-IR (KBr) cm– 1: 1651 (C=O). 1H-NMR (CDCl3) d: 8.40 (d, J =
8.9, 1H, 8-ArH), 7.45–7.53 (m, 1H, PhH), 7.37 (dd, J = 7.5, 1.7, 1H,
PhH), 7.28 (dd, J = 8.8, 2.5, 1H, 7-ArH), 7.03–7.15 (m, 2H, PhH),
6.66 (d, J = 2.5, 1H, 5-ArH), 4.25–4.58 (m, 2H, CH2), 3.76 (s, 3H,
OMe), 3.75 (s, 3H, OMe), 2.82 (d, J = 7.0, 2H, CH2), 2.34 (s, 6H, Me).
13C-NMR (CDCl3) d: 162.9, 159.4, 157.6, 145.0, 132.0, 131.5, 130.9,
128.9, 124.5, 121.7, 121.2, 119.9, 111.3, 108.3, 57.4, 55.51, 55.47,
48.6, 45.6. Anal. Calc. (C20H23N3O3): C, 67.97; H, 6.56; N, 11.89.
Found: C, 67.75; H, 6.68; N, 11.80.
2-[2-(Dimethylamino)ethyl]-2H-phthalazin-1-one 7g
Yield 60%; oil. Eluent: AcOEt / MeOH = 2 : 1. Rf = 0.24. FT-IR (film)
cm– 1: 1648 (C=O). 1H-NMR (CDCl3) d: 8.40–8.46 (m, 1H, 8-ArH),
8.17 (s, 1H, 4-ArH), 7.66–7.83 (m, 3H, 5,6,7-ArH), 4.38 (t, J = 6.9,
2H, CH2), 2.78 (t, J = 6.8, 2H, CH2), 2.33 (s, 6H, Me). 13C-NMR (CDCl3)
d: 159.4 (C=O), 137.8, 132.9, 131.5, 129.6, 127.8, 126.6, 125.9, 57.2
(CH2), 48.6 (CH2), 45.4 (Me). MS m/z (%): 217 [M]+ (5), 173 (14), 71
(20), 58 (100). HRMS [M]+: calcd for C12H15N3O: 217.12150, found:
217.12187.
2-[2-(Dimethylamino)ethyl]-6-methoxy-4-(pyridin-2-yl)-
2H-phthalazin-1-one 7c
Yield 64%; m.p.: 87–898C. Eluent: AcOEt / MeOH = 1 : 1. Rf = 0.26.
FT-IR (KBr) cm– 1: 1662 (C=O). 1H-NMR (CDCl3) d: 8.75–8.78 (m, 1H,
6-PyH), 8.43 (d, J = 8.8, 1H, 8-ArH), 7,96 (d, J = 2.6, 1H, 5-ArH),
7.88–7.92 (m, 2H, PyH), 7.38–7.45 (m, 1H, PyH), 7.33 (dd, J = 8.8,
2.4, 1H, 7-ArH), 4.47 (t, J = 6.9, 2H, CH2), 3.90 (s, 3H, OMe), 2.88 (t, J
= 6.9, 2H, CH2) 2.39 (s, 6H, Me). 13C-NMR (CDCl3) d: 162.8, 159.1,
154.9, 148.3, 143.3, 137.0, 128.8, 124.2, 123.4, 121.9, 120.1,
108.6, 57.2 (CH2), 55.4 (OMe), 48.7 (CH2), 45.4 (Me). MS m/z (%): 324
[M]+ (1), 254 (88), 58 (100). HRMS [M]+: calcd. for C18H20N4O2:
324.15862, found: 324.15897.
2-[2-(Dimethylamino)ethyl]-2H-pyridazin-3-one 8
Yield 60%; oil. FT-IR (KBr) cm– 1: 1660. 1H-NMR (CDCl3) d: 7.73 (dd,
J = 3.9, 1,7, 1H, 6Ar-H), 7.13 (dd, J = 9.4, 3.8, 1H, 5Ar-H), 6.87 (dd, J
= 9.4, 1.7, 1H, 4Ar-H), 4.25 (t, J = 6.7, 2H, CH2), 2.69 (t, J = 6.8, 2H,
CH2), 2.26 (s, 6H, Me). 13C-NMR (CDCl3) d: 160.4 (C=O), 135.9,
130.9, 129.6, 56.9, 49.3, 45.4. Anal. Calc. (C8H13N3O): C, 57.47; H,
7.84; N, 25.13. Found: C, 57.16; H, 8.04; N, 24.99.
2-[2-(Dimethylamino)ethyl]-6-methoxy-4-(pyridin-3-yl)-
7-[2-(Dimethylamino)ethyl]-7H-pyrido[2,3-d]pyridazin-8-
2H-phthalazin-1-one 7d
one 9a
Yield 60%; m.p.: 124–1278C. Eluent: AcOEt / MeOH = 1 : 1. Rf =
Yield 65%; m.p.: 77–798C. Eluent: AcOEt / MeOH = 1 : 1. FT-IR
(KBr) cm– 1: 1655. 1H-NMR (CDCl3) d: 9.11 (dd, J = 4.5, 1.5, 1H, 2Ar-
H), 8.19 (s, 1H, 5Ar-H), 8.07 (dd, J = 8.1, 1.6, 1H, 4Ar-H), 7.73 (dd, J =
8.1, 4.5, 1H, 3Ar-H), 4.63 (t, J = 6.5, 2H, CH2), 2.87 (t, J = 6.6, 2H,
CH2), 2.37 (s, 6H, Me).13C-NMR (CDCl3) d: 158.4 (C=O), 143.4, 135,9,
133.9, 127.1, 125.6, 57.0, 49.0, 45.4; Anal. Calc. (C11H14N4O): C,
60.53; H, 6.47; N, 25.67. Found: C, 60.51; H, 6.67; N, 25.42.
0.24. FT-IR (KBr) cm– 1: 1652 (C=O). H-NMR (CDCl3) d: 8.89–8.88
1
(m, 1H, 2-PyH), 8.76 (dd, J = 4.9, 1.7, 1H, 6-PyH), 8.46 (d, J = 8.9, 1H,
8-ArH), 7.92–7.98 (m, 1H, 4-PyH), 7.48 (ddd, J = 7.8, 4.9, 0.8, 1H, 5-
PyH), 7.35 (dd, J = 8.9, 2.5, 1H, 7-ArH), 6.99 (d, J = 2.5, 1H, 5-ArH),
2.42 (t, J = 6.9, 2H, CH2), 3.85 (s, 3H, OMe), 2.82 (t, J = 6.9, 2H, CH2),
2.34 (s, 6H, Me). 13C-NMR (CDCl3) d: 163.2, 158.9, 150.1, 143.3,
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46
W. Pakulska et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 41–47
p.o.). Acetic acid (0.1 mL/10 g b.w. of a 0.6% v/v solution) was
injected 60 min after compounds (or metamizole) into the peri-
toneal cavities of mice, which were placed in a large glass cylin-
der and the intensity of nociception was quantified by counting
the total number of writhing occurring between 0 and 30 min
after stimulus injection. Each group consisted of ten mice. The
data are presented in Fig. 3.
2-[2-(Dimethylamino)ethyl]-2H-pyrido[3,4-d]pyridazin-1-
one 9b
1
Yield 55%; oil. FT-IR (KBr) cm– 1: 1655. H-NMR (CDCl3) d: 9.15 (s,
1H, Ar-H), 8.97 (d, J = 5.3, 1H, Ar-H), 8.30 (s, 1H, Ar-H), 8.20 (d, J =
5.3, 1H, Ar-H), 4.39 (t, J = 6.6, 2H, CH2), 2.80 (t, J = 6.6, 2H, CH2),
2.34 (s, 6H, Me). Anal. Calc. (C11H14N4O): C, 60.53; H, 6.47; N,
25.67. Found: C, 60.48; H, 6.66; N, 25.31.
Statistical analysis
Pharmacology
Results are expressed as the mean l SD. The normality of distri-
bution was checked by means of Kolmogorov-Smirnov test with
Lilliefors test. The statistical evaluation was performed using
analysis of variance (ANOVA) and post hoc comparisons were
performed by means of Least Significant Differences (LSD) test. If
the data were not normally distributed, statistical evaluation
was performed by using ANOVA (Kruskall-Wallis) and Mann-
Whitney U test. Differences were considered significant when p
a 0.05.
The experiments were carried out with Balb/c male mice (18–
25 g; Animal Quarters, Medical University of Lodz, Poland). The
mice were kept in group cages under laboratory conditions at a
temperature of 20–218C, natural day / night cycle and they had
free access to commercial chow food and water. All experiments
were performed between 09.00 a.m. and 02.00 p.m. The synthe-
sized compounds 7, 8, 9 were dissolved in distilled water with
addition of one molar equivalent of hydrochloric acid and
administrated 0.1 mL/10 g, body weight (b.w.).
The analgesic effect in the hot-plate and tail-flick tests
were expressed in percent of maximal possible effect
(% MPE):
All the procedures used in these studies were approved by the
Ethics Committee of the Medical University of Lodz, Poland
(licence 115, permission L/BD/199).
ðlatency after drug administration ꢀ basic latencyÞ
100% 6
Determination of acute toxicity
ð‘‘cut off‘‘ ꢀ basic latencyÞ
The LD50 values as an indicator of acute toxicity of compounds
were determined according to the Kꢀrber method [19]. Results
are given in Table l.
Nociceptive tests
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