Synthesis and pharmacological properties of N,N-dialkyl(dialkenyl)amides of 7-methyl-3-phenyl-1-[2-hydroxy-3-(4-phenyl-1-piperazinyl)propyl]-2,4-dioxo-1, 2,3,4-tetrahydropyrido[2,3-d]pyrimidine-5-carboxylic acid

Article abstract of DOI:10.1016/S0014-827X(02)00011-3

Synthesis of N,N-dialkyl(dialkenyl)amides of 7-methyl-3-phenyl-2,4-dioxo-1, 2,3,4-tetrahydropyrido[2,3-d]pyrimidine-5-carboxylic acid (5-9) and their 1-[2-hydroxy-3-(4-phenyl-1-piperazinyl)propyl] derivatives (10-14) is described. Compounds 10-14 were tes

Full text of DOI:10.1016/S0014-827X(02)00011-3

Il Farmaco 58 (2003) 25Á32
/
www.elsevier.com/locate/farmac
Synthesis and pharmacological properties of N,N-
dialkyl(dialkenyl)amides of 7-methyl-3-phenyl-1-[2-hydroxy-3-(4-
phenyl-1-piperazinyl)propyl]-2,4-dioxo-1,2,3,4-tetrahydropyrido-
[2,3-d]pyrimidine-5-carboxylic acid
a,
Helena Sladowska *, Aleksandra Sabiniarz , Barbara Filipek , Ma lgorzata Kardasz ,
a
b
b
´
Dorota Maciag c
˛
a Department of Chemistry of Drugs, Wroc law University of Medicine, Tamka 1, Wroc law 50-137, Poland
b Laboratory of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University, Collegium Medicum, 30-688 Krako´w, Medyczna 9, Poland
c Radioligand Laboratory, Faculty of Pharmacy, Jagiellonian University, Collegium Medicum, 30-688 Krako´w, Medyczna 9, Poland
Received 9 March 2002; accepted 26 September 2002
Abstract
Synthesis of N,N-dialkyl(dialkenyl)amides of 7-methyl-3-phenyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-5-car-
boxylic acid (5Á
were tested for analgesic and sedative activities as well as for m-opioid receptors binding affinities. All the amides, being the object of
investigation, displayed an interesting analgesic action, which in case of the compounds 10Á12 and 14 was superior to that of
acetylsalicylic acid in two different tests. Furthermore all the amides (10Á14) significantly suppressed the spontaneous locomotor
/
9) and their 1-[2-hydroxy-3-(4-phenyl-1-piperazinyl)propyl] derivatives (10Á
/
14) is described. Compounds 10Á14
/
/
/
activity, prolonged barbiturate sleep in mice and showed a weak affinity to m-opioid receptors.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Pyrido[2,3-d]pyrimidines; N-2-hydroxy-3-(4-phenyl-1-piperazinyl)propyl derivatives; Amides; Synthesis; Pharmacological activity;
Affinity to m-opioid receptors
1. Introduction
Piperidinoamide 2 was the least active compound. The
results obtained indicate that the kind of amide group
influences the strength of the analgesic action. Having
regard to the above statement and continuing our
studies we synthesized then N,N-dialkyl(dialkenyl)a-
mides of 1-[2-hydroxy-3-(4-phenyl-1-piperazinyl)pro-
pyl]-7-methyl-3-phenyl-2,4-dioxo-1,2,3,4-tetrahydropyr-
It was stated previously [1] that some of 1-[2-hydroxy-
3-(4-phenyl-1-piperazinyl)propyl] derivatives of amides
of 7-methyl-3-phenyl-2,4-dioxo-1,2,3,4-tetrahydropyr-
ido[2,3-d]pyrimidine-5-carboxylic acid (1Á3) (Fig. 1)
displayed an interesting analgesic action in the ‘writhing
syndrome’ and ‘hot plate’ tests and were not toxic
/
ido[2,3-d]pyrimidine-5-carboxylic acid (10Á14) (Fig. 2)
/
(LD50ꢀ2000 mg/kg). In the case of the compounds 2
/
in order to ascertain whether the changes in the
structure of amide group would distinctly influence
their toxicity, analgesic and sedative activities. Com-
and 3 the analgesic action was associated with the weak
suppression of the spontaneous locomotor activity in
mice but only at the high doses (50Á200 mg/kg). Among
the three investigated substances, pyrrolidinylamide
derivative 1 exhibited the strongest analgesic effects.
/
pounds 10Á14 were also tested for m-opioid receptors
/
binding affinities.
Newly obtained amides similarly as the compounds
1Á
group). But in the pharmacological tests they were used
in the form of racemates (9).
/
3 have an asymmetric carbon atom (bearing OH
* Corresponding author.
E-mail address: helena@bf.uni.wroc.pl (H. Sladowska).
´
/
´
0014-827X/02/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 0 0 1 1 - 3

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H. Sladowska et al. / Il Farmaco 58 (2003) 25Á32
/
The structures of all compounds synthesized were
1
confirmed by elemental and spectral analyses (IR, H
NMR).
3. Experimental
3.1. Chemistry
All the results of the C, H, N determinations (carried
out by a Carlo Erba Elemental Analyzer model NA-
1500) were within 90.4% of the theoretical values. All
/
melting points (m.p.) were uncorrected. The IR spectra,
in KBr pellets, were measured with a Zeiss Jena specord
model IR 75 and specord M 80 (Jena). 1H NMR spectra
were determined in CDCl3 on a Tesla 587 A spectro-
meter (80 MHz) using TMS as internal standard.
Fig. 1.
2. Chemistry
3.1.1. General procedure for obtaining N,N-
dialkyl(dialkenyl)amides of 7-methyl-3-phenyl-2,4-dioxo-
1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-5-carboxylic
Starting material was chloride of 7-methyl-3-phenyl-
2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-5-
carboxylic acid (4) previously synthesized [1]. It was
acid (5Á9)
/
Chloride of 7-methyl-3-phenyl-2,4-dioxo-1,2,3,4-tet-
rahydropyrido[2,3-d]pyrimidine-5-carboxylic acid (4)
(0.014 mol) was dissolved in 80 ml of anhydrous toluene.
To this solution 0.035 mol of a suitable amine (diethyl-,
diallyl-, di-n-propyl-, diisopropyl- and diisobutyla-
mines) was introduced. The mixture was then stirred
at room temperature (r.t.) for 5 h. The separated
product was collected on a filter and after drying it
transformed into suitable amides 5Á
with diethyl-, diallyl-, di-n-propyl-, diisopropyl- and
diisobutylamines in toluene solution. The amides 5Á
underwent condensation with 2-hydroxy-3-(4-phenyl-1-
piperazinyl)propyl chloride [2] in anhydrous ethanol and
in the presence of potassium ethoxide giving derivatives
/
9 in the reaction
/
9
10Á14.
/
Fig. 2.

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H. Sladowska et al. / Il Farmaco 58 (2003) 25Á
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32
27
Table 1
Properties of the investigated compounds
Comp. Formula (molecular wt) M.p. (8C) (solvent)
Yield (%) IR absorptions in KBr (cmꢁ1
CO NH or OH
)
Mono-substituted benzene
3200 700, 760
5
C
19H20N4O3 (352.38)
271Á
267Á
229Á
287Á
228Á
139Á
116Á
165Á
226Á
143Á
/
274 (ethanol)
60
58
50
49
50
55
1635, 1675, 1720 3060, 3160Á
1640, 1655, 1725 3080, 3100Á
1620, 1680, 1730 3290Á3380
1650, 1680, 1735 3080, 3140
/
6
C21H20N4O3 (376.40)
C21H24N4O3 (380.43)
/269 (ethanol)
/
3200 700, 770
700, 760
7
/231 (ethanol)
/
8
C
21H24N4O3 (380.43)
/289 (diluted ethanol)
700, 750
9
C23H28N4O3 (408.49)
C32H38N6O4 (570.67)
/230 (ethanol)
1620, 1670, 1720 3100Á
1650, 1680, 1725 3360Á
1660, 1680, 1730 3360
/
3140
700, 760
10
11
12
13
14
/141 (ethanol/ether)
/3400
700, 750Á
705, 760
700, 760
700, 750
710, 780
/760
C
C
34H38N6O4 (594.69)
34H42N6O4 (598.72)
/119 (ether/petroleum ether) 40
/
168 (ethanol)
228 (ethanol)
145 (ethanol)
45
45
44
1640, 1680, 1720 3360Á
1635, 1670, 1720 3400
1650, 1680, 1730 3390
/3400
C34H42N6O4 (598.72)
C36H46N6O4 (626.78)
/
/
was washed with 100 ml of distilled water. The obtained
substance was purified by crystallization from the
solvents given in Table 1.
A residue was crystallized from the suitable solvent
(Table 1).
The properties of compounds 10Á14 are listed in
/
1
The properties of amides (5Á
/
9) are listed in Table 1
Table 1 and data concerning their H NMR spectra are
shown below:
1
but data concerning their H NMR spectra are shown
below:
1H NMR of 10: dꢀ
1.20 (2 overlapping t-6H); 2.65
(m-9H); 3.14 (m-6H,); 3.49 (q-2H,); 4.34 (m-4H); 7.15
(m-11H).
/
1H NMR of 5: dꢀ
/1.18 (2 overlapping t-6H); 2.62 (s-
3H); 3.09, 3.53 (2 q-4H); 6.85 (s-1H); 7.38 (m-5H); 11.42
(s-1H).
1H NMR of 11: dꢀ
/
2.64 (m-9H); 3.20 (m-4H); 3.70
(d-2H); 4.32 (m-6H); 5.47 (m-6H); 7.16 (m -11H).
1H NMR of 12: dꢀ
0.86 (2 overlapping t-6H); 1.66
(m-4H); 3.08 (m-17H); 4.39 (m-4H); 7.16 (m-11H).
1H NMR of 13: dꢀ
1.10 (d-6H) and 1.54 (d-6H); 2.63
(m-9H); 3.36 (m-6H); 4.39 (m-4H); 6.89 (m-11H).
1H NMR of 14: dꢀ
0.88 (m-12H); 1.98 (m-2H); 3.01
1H NMR of 6: dꢀ
2.65 (s-3H); 3.66 (d-2H); 4.13 (d-
2H) and 5.54 (m-6H); 6.85 (s-1 H); 7.35 (m-5H); 11.54
(s-1H).
/
/
1H NMR of 7: dꢀ
/0.88 (2 overlapping t-6H); 1.63 (m-
/
4H); 2.70 (s-3H); 2.98 and 3.41 (2 distorted t-4H); 6.88
(s-1H); 7.34 (m-5H); 10.77 (s-1H).
/
1H NMR of 8: dꢀ
/
1.10 (d-6H) and 1.54 (d-6H); 2.67
(m-17H); 4.44 (m-4H); 7.18 (m-11H).
(s-3H); 3.50 (m-2H); 6.82 (s-1H); 7.34 (m-5H); 10.32 (s-
1H).
3.2. Pharmacology
1H NMR of 9: dꢀ
0.88 (m-12H); 2.02 (m-2H); 3.20
(m-7H); 6.86 (s-1H); 7.34 (m-5H,); 10.81 (s-1H).
/
3.2.1. Material and methods
3.2.1.1. Substances. Acetylsalicylic acid (Polopiryna, ZF
Starogard Gdanski, PL), [3H]dihydromorphine (Amer-
sham), levallorphan (Sigma), morphine (Morphinum
hydrochloricum, Polfa-Kutno, PL), phenylbenzoqui-
none (INC Pharmaceuticals, Inc, NY), thiopental so-
3.1.2. General procedure for obtaining N,N-
˜
dialkyl(dialkenyl)amides of 7-methyl-3-phenyl-1-[2-
hydroxy-3-(4-phenyl-1-piperazinyl)propyl]-2,4-dioxo-
1,2,3,4-tetra-hydropyrido[2,3-d]pyrimidine-5-carboxylic
acid (10Á14)
/
dium (Thiopentone
Arzneimittel, Germany).
sodium,
HEFA-FRENON
Potassium (0.01 mol) was dissolved in anhydrous
ethanol (150 ml) and to this solution 0.01 mol of a
suitable amide of 7-methyl-3-phenyl-2,4-dioxo-1,2,3,4-
tetrahydropyrido[2,3-d]pyrimidine-5-carboxylic
acid
3.2.1.2. Animals. The experiments were carried out on
male Albino-Swiss mice (body weight 18Á26 g) and male
Wistar rats (body weight 120Á200 g). Animals were
housed in constant temperature facilities and exposed to
a 12 h light:12 h dark cycle and maintained on a
standard pellet diet and tap water was given ad libitum.
(5Á9) was introduced. After dissolving the solid sub-
/
/
stance, 2-hydroxy-3(4-phenyl-1-piperazinyl)propyl chlo-
ride (0.012 mol) was added. The reaction mixture was
refluxed until the alkaline reaction disappeared. After
the filtration ethanol was distilled off to about 1/4 of its
original volume and left to crystallize. The separated
/
Control and experimental groups consisted of 6Á8
/
product (12Á
/
14) was collected on a filter and purified by
animals each. The compounds investigated were admi-
nistered intraperitoneally (i.p.) as a suspension in 0.5%
methylcellulose at a constant volume of 10 ml/kg.
crystallization from the solvents given in Table 1. In case
of amides 10 and 11 ethanol was evaporated to dryness.

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H. Sladowska et al. / Il Farmaco 58 (2003) 25Á
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32
3.2.1.3. Statistical analysis. The statistical significance
was calculated using a Student’s t-test. The ED50 values
and their confidence limits were calculated according to
the method of Litchfield and Wilcoxon [3].
in the course of 10 min was counted. The analgesic effect
of individual doses was expressed in per cent:
% analgesic effectꢀ100
P
of writhing incidents in experimental group
ꢁ
ꢂ100
P
of writhing incidents in control group
3.2.1.4. Acute toxicity. Acute toxicity was assessed by
the methods of Litchfield and Wilcoxon [3] and pre-
sented as LD50 calculated from the mortality of mice
after 24 h.
The ED50 values and their confidence limits were
estimated by the method of Litchfield and Wilcoxon [3].
3.2.1.7. Spontaneous locomotor activity. Spontaneous
locomotor activity in mice was measured in circular
photoresistor actometers (32 cm in diameter). The
investigated compounds were injected i.p. at a dose-
3.2.1.5. Pain reactivity. Pain reactivity was measured in
the ‘hot plate’ test according to the method of Eddy and
Leimbach [4]. Animals were placed individually on the
metal plate heated to 56 8C. The time (s) necessary to
induce the licking reflex of the forepaws or jumping was
recorded by a stop-watch. A cut off time of 60 s was
used to present tissue damage. The experiment was
performed 30 min after administration of the investi-
gated compounds at graded doses of 25, 50, 100 and 200
mg/kg i.p.
range of 1.56Á50 mg/kg. Thirty minutes after the
/
injection of the investigated compounds mice were
placed for 30 min in the actometers. Each crossing of
the light beam was recorded automatically. The amount
of impulses was noted after 30 min.
3.2.1.8. Thiopental anesthesia. Thiopental sodium in a
narcotic dose (55 mg/kg) was injected i.p. 30 min after
administration of the tested compounds. Duration of
thiopental-induced sleep was measured from disappear-
ance to return of the righting reflex.
3.2.1.6. ‘Writhing syndrome’. ‘Writhing syndrome‘ test
was performed in mice according to Hendershot and
Forsaith [5]. Different doses of the tested compounds
ranging from 0.78 to 100 mg/kg were administered i.p.
Twenty five minutes later, 0.02% solution (ethanol:-
water, 5:95) of phenylbenzoquinone was injected i.p. in a
constant volume of 0.25 ml. Five minutes after injection
of the irritating agent, the number of ‘writhing’ episodes
3.2.1.9. Radioligand binding assay [6]. Male Wistar rats
were sacrificed by decapitation and the whole brains
were removed rapidly and placed in ice-cold TrisÁ
/
buffer. Brains were homogenized in 30 volumes of ice-
Table 2
Influence of the investigated compounds, acetylsalicylic acid and morphine on pain reactivity in the ‘hot-plate’ test in mice
Comp.
Dose (mg/kg)
Time of reaction on pain stimulus in seconds 9
/
SEM
Prolongation of the reaction time (%)
Control
10
15.59
47.39
25.99
27.09
12.79
31.69
27.59
15.39
27.89
24.09
17.39
16.09
12.39
28.89
21.89
13.89
14.59
31.39
19.69
16.29
18.49
30.69
29.69
19.49
/
1.0
200
100
50
/
10.9****
4.0***
6.5*
2.7
205.2
67.1
74.2
/
/
25
/
11
200
100
50
/
5.5***
5.2***
6.5
103.9
77.4
/
/
12
13
200
100
200
100
/
5.6***
7.1
79.3
54.8
11.6
3.2
/
/
3.8*
3.4
/
Control
/
1.4
14
200
100
50
/
6.9****
4.9**
3.0
85.8
40.6
12.2
/
/
Control
/
3.6
Acetylsalicylic acid
400
200
100
/
4.2**
4.1
116.5
35.1
11.7
/
/
4.9
Control
/
1.0
Morphine
6
3
1
/
3.9**
6.0*
2.1
66.3
60.9
5.4
/
/
Each group consisted of six to eight animals. *p B
/
0.05, **p B
/
0.02, ***p B
/
0.01, ****p B0.001.
/

´
H. Sladowska et al. / Il Farmaco 58 (2003) 25Á
/
32
29
Table 3
Influence of the investigated compounds, acetylsalicylic acid and morphine on the pain reactivity in the ‘writhing syndrome’ test in mice
Comp.
Dose (mg/kg)
Mean number of writhings9
/
SEM
Analgesic effect (%)
ED50 (mg/kg)
Control
10
0
29.79
0.79
1.89
4.89
7.39
10.59
20.89
24.39
5.339
7.339
8.29
9.29
11.69
12.69
14.89
17.09
4.09
5.09
6.39
7.09
13.79
20.09
7.09
7.69
8.49
11.09
12.09
15.59
19.09
6.89
9.29
9.49
9.89
14.29
23.69
19.29
3.29
8.59
11.29
1.29
7.59
16.29
/
3.5
100
50
/
0.3****
1.2****
2.3****
3.8***
2.8***
2.7
97.6
93.9
83.8
75.4
64.6
30.0
18.2
82.0
75.3
72.4
69.0
60.9
57.6
50.2
42.7
86.5
83.2
78.8
76.4
53.9
32.6
76.4
74.4
71.7
63.0
59.6
47.8
36.0
77.1
69.0
68.3
67.0
52.2
20.5
5.31 (2.56Á11.0)
/
/
25
/
12
/
6.25
3.125
1.56
100
50
/
/
/2.3
11
/
2.7****
4.0***
3.0***
2.9***
3.5***
2.6**
5.2*
1.53 (0.21Á10.9)
/
/
25
/
12.5
6.25
3.125
1.56
0.78
50
/
/
/
/
/3.8
12
13
/
1.8****
1.8****
2.9****
2.3****
2.8**
3.4
2.53 (0.78Á/8.15)
25
/
12.5
6.25
3.125
1.56
50
/
/
/
/
/
3.6***
2.6***
4.0***
3.9***
4.5***
3.4*
1.93 (0.36Á/10.25)
25
/
12.5
6.25
3.125
1.56
0.78
50
/
/
/
/
/4.5
14
/
2.6***
2.7***
2.4***
2.4***
2.7**
2.3
4.8 (1.42Á16.18)
/
25
/
12.5
6.25
3.125
1.56
/
/
/
/
Control
/3.2
Acetylsalicylic acid
100
50
30
10
3
/
1.2****
1.3**
2.1
83.3
55.7
41.7
93.7
60.9
15.6
39.15 (29.1Á/48.4)
/
/
Morphine
/
0.8****
2.9**
3.51
2.44 (1.18Á/5.02)
/
1
/
Each group consisted of six to eight animals. ****p B
/
0.001, ***p B
/
0.01, **p B
/
0.02, *p B0.05.
/
cold 0.05 M TrisÁ/buffer, pH 7.7. The homogenate was
TrisÁbuffer. The radioactivity was counted in a Wallac
/
1409 DSA Instrument (Finland).
Levallorphan is used for the determination of non-
specific binding.
centrifuged at 48 000ꢂ
/
g for 15 min at 4 8C. The pellets
were suspended in the same volume of buffer and
incubated at 37 8C for 30 min, centrifuged at 4 8C for
10 min. The final pellet was resuspended in 50 volumes
of 0.05 M fresh TrisÁbuffer, to pH 7.7, and used for
/
binding studies. A 240 ml sample of the latter membrane
suspension was incubated at 25 8C for 30 min with 30 ml
of the buffer solution of the investigated compound (1
4. Results
nMÁ
100 mM) or levallorphan and 30 ml of a [3H]DHM
/
4.1. Acute toxicity
(0.5 nM). The incubation was followed by a rapid
vaccum filtration through Whatman GF/C glass filters.
The filters were washed twice with 100 ml of ice-cold
All investigated compounds were not toxic (LD50ꢀ
2000 mg/kg).
/

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H. Sladowska et al. / Il Farmaco 58 (2003) 25Á32
/
which were superior or comparable to those of mor-
phine, whereas analgetic action of amides 14 and 10 was
weaker than that of morphine. It was interesting to
observe that all compounds displayed activity superior
to that of acetylsalicylic acid (Table 3).
4.3. Locomotor activity
All compounds tested produced a significant decrease
in locomotor activity of mice during a 30 min observa-
tion period. Compounds 13 and 14, given at doses of
12.5, 6.25 and 3.125 mg/kg, inhibited spontaneous
Fig. 3. Comparison of analgesic effects of tested compounds to those
of acetylsalicylic acid and morphine in the ‘hot plate’ test in mice. The
columns represent prolongation of the time reaction (%) for each
tested dose of compound (mg/kg) in comparison to control.
locomotor activity in mice by 63Á
respectively. The other compounds significantly de-
creased locomotor activity in mice by 58.5Á47% when
/
67, 59 and 49Á/39%,
/
administered at doses of 50 or 25 and 12.5 mg/kg (Table
4).
4.2. Analgesic activity
Analgesic activities were evaluated in the ‘hot plate’
and in the phenylbenzoquinone-induced ‘writhing’ tests.
All compounds produced significant analgesic activity in
both tests (Tables 2 and 3).
In the ‘hot plate’ test, compound 10 exhibited a
significant effect up to the dose of 50 mg/kg, whereas
compounds 11 and 14 had analgesic action in doses up
to of 100 mg/kg. Amides 12 and 13 decreased the pain
sensitivity in mice in this test up to the dose of 200 mg/
4.4. Thiopental anesthesia
All the compounds tested significantly prolonged
barbiturate sleep in mice. The most potent effect was
produced by compounds 12 and 13 which were active up
to a dose of 12.5 mg/kg, whereas compounds 10 and 11
acted up to a dose of 25 mg/kg. Amide 14 produced a
significant effect up to a dose of 50 mg/kg (Table 5, Fig.
4).
kg. It was interesting to observe that compounds 10Á12
/
and 14 displayed activity superior to that of acetylsa-
licylic acid in the ‘hot plate’ method. The results are
summarized in Table 2 and Fig. 3.
In the phenylbenzoquinone-induced ‘writhing’ test,
compounds 11, 13 and 12 showed analgesic effects
4.5. Radioligand binding assay
Table 6 illustrates the effect of the tested compounds
on m-opioid receptors affinity, as determined by stan-
dard ligand-binding techniques. All the tested com-
Table 4
Influence of the investigated compounds on the spontaneous locomotor activity in mice
Comp.
Dose (mg/kg)
Number of movements 9
/
SEM during 30 min
% inhibition of locomotor activity
ED50 (mg/kg)
Control
10
484.09
203.89
268.09
301.89
192.49
255.09
311.89
197.29
233.89
319.89
177.29
198.89
248.29
347.09
137.59
161.59
196.29
294.49
340.29
/
58.1
50
25
/46.0***
57.8
44.6
37.6
60.2
47.3
35.6
59.3
51.7
33.9
63.4
58.9
48.7
28.3
71.6
66.6
59.5
39.2
29.7
31.4 (20.9Á47.0)
/
/78.5*
12.5
25
/80.3
11
12
13
/36.9***
14.6 (8.6Á24.8)
/
12.5
6.25
50
/53.7*
/50.5
/
38.8***
43.8**
101.4
27.5 (15.2Á
/
49.5)
5.4)
25
/
12.5
12.5
6.25
3.125
1.56
25
/
/
33.3***
48.0**
66.2*
4.5 (3.75Á
/
/
/
/27.9
14
/
10.7***
8.6***
24.1***
52.2*
6.2 (3.3Á11.8)
/
12.5
6.25
3.125
1.56
/
/
/
/75.3
Each group consisted of six to eight animals. *p B
/
0.05, **p B
/
0.01, ***p B0.001.
/

´
H. Sladowska et al. / Il Farmaco 58 (2003) 25Á
/
32
31
Table 5
Influence of the investigated compounds on the thiopental sleeping time
Comp.
Dose (mg/kg)
Duration of sleeping time9
/
SEM (min)
Prolongation (%)
Control
10
30.09
84.69
65.49
38.09
76.29
66.89
45.69
114.49
88.09
61.09
90.69
83.09
54.29
139.79
115.09
57.39
/
5.7
50
25
/
19.6**
14.9*
5.6
182.0
118.0
26.7
/
12.5
50
/
11
12
13
14
/
15.5**
15.2*
18.2
154.0
122.7
52.0
25
/
12.5
25
/
/
27.6***
20.6**
17.2
281.3
193.3
103.3
202.0
176.7
80.7
12.5
6.25
25
/
/
/
18.3***
22.8*
12.5
12.5
6.25
100
50
/
/
/
32.5***
30.1**
15.2
365.7
283.3
91.0
/
25
/
Each group consisted of six to eight animals. ****p B
/
0.001, ***p B
/
0.01, **p B
/
0.02, *p B
/
0.05.
pounds displaced [3H]dihydromorphine from binding
sites in low micromolar concentration (IC50ꢀ15.8Á46.4
showed the strongest analgesic activity. The other
compounds displayed analgesic properties at the higher
/
/
mM). This effect was considerably lower than that of
levallorphan and morphine [7], but comparable to
tramadol, which possessed a modest affinity for m-
opioid receptors (Ki values in the micromolar range [8]).
doses (100Á200 mg/kg). In all cases analgesic action was
/
associated with the suppression of the spontaneous
locomotor activity in mice. In the last test the most
active compounds appeared to be 13 and 14, i.e. amides
containing the branched alkyl groups at the amide
nitrogen atom. Moreover all compounds prolonged
the time of thiopental anesthesia. In this test the most
active amides were compounds 12 and 13 with N,N-di-
n-propyl- and diisopropyl substituents. Independently
of the differences in the structure of amide groups all
tested compounds showed a weak affinity to m-opioid
receptors.
5. Discussion
From the data presented above it follows that
submitted to investigation compounds (10Á14), inde-
/
pendently of the kind of the substituents at amide
nitrogen atom, showed activity in all tests performed
and were non-toxic. In the ‘writhing syndrome’ test
N,N-diethylamide derivative 10 proved to be the least
active compound. Replacement of ethyl groups by allyl
ones (compound 11) caused considerable increase of
analgesic action in this test. Similar effect was observed
in case of di-isopropyl- and di-n-propylamides 13 and
12. On the contrary, in the ‘hot plate’ test amide 10
Previously [1] synthesized amides 1Á3 displayed
/
analgesic properties in the ‘writhing syndrome’ test up
to the dose of 1.56 mg/kg (1), 25 mg/kg (2) and 6.25 mg/
kg (3). These data indicate that in this test, diallyl- and
diisopropylamides (11 and 13) produced the analgesic
effects in the same doses as pyrrolidinylamide 1 whereas
diethylamide 10 was comparable on that score with the
compound 3. Di-n-propyl- and diisobutylamides (12
and 14) acted weaker than compound 1 but stronger
than derivatives 2 and 3.
Table 6
Competition of tested compounds for opioid receptors labelled with
[3H]dihydromorphine
Comp.
IC509SEM (mM)
/
10
11
46.49
29.79
15.89
32.09
22.59
0.2269
/
2.5
/4.5
12
/1.5
13
/5.9
14
/3.1
Levallorphan
/0.09 nM
Fig. 4. Effect of tested compounds on the thiopental sleeping time.

´
32
H. Sladowska et al. / Il Farmaco 58 (2003) 25Á32
/
In the ‘hot plate’ test amides 1Á
analgesic activity up to the dose of 25 mg/kg (1), 100
mg/kg (2) and 50 mg/kg (3). All the newly synthesized
/
3 displayed the
plate’ test is used by many investigators for evaluation
of analgesics, acting centrally, while the ‘writhing
syndrome’ test is used for evaluation of peripheral
analgesics.
compounds (10Á14) were less active in this test than
/
amide 1. Diethylamide 10 produced the analgesic effects
up to the dose of 50 mg/kg similarly to N-methylpiper-
azinylamide 3. Amides 11 and 14 acted in this test like
piperidinoamide 2 whereas compounds 12 and 13 were
Furthermore amides 10Á14 inhibited the spontaneous
/
locomotor activity and prolonged barbiturate sleep in
mice. On the basis of the ligand binding data, we suggest
that a weak affinity to m-opioid receptors probably plays
a role in the mechanism of action of these compounds.
But the explanation of a precise mechanism of action
will demand the further pharmacological investigations.
less active than the previously synthesized substances 1Á
/
3. Amides 2 and 3 suppressed the spontaneous locomo-
tor activity in mice up to the dose of 50 mg/kg.
Compound 1 was inactive in this test. All the investi-
gated substances (10Á14) were more active in this test
/
than derivatives 2 and 3.
From the results presented it can be seen that the
described chemical changes in the structure of amide
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/
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/