Synthesis and analgesic action of N-(substituted-ethyl)pyrrole-3,4- dicarboximides

Article abstract of DOI:10.1016/j.farmac.2004.10.002

We prepared a series of new N-[2-(4-substitutedpiperazin-1-yl)ethyl]-1-(n- butyl or phenyl)-2,5-dimethyl-3,4-pyrroledicarboximides 3 and the related products 4 and 5, nine representatives of which were evaluated as potential analgesic agents in an animal

Full text of DOI:10.1016/j.farmac.2004.10.002

IL FARMACO 60 (2005) 15–22
http://france.elsevier.com/direct/FARMAC/
Original article
Synthesis and analgesic action
of N-(substituted-ethyl)pyrrole-3,4-dicarboximides
W. Malinka a,*, M. Kaczmarz a, A. Redzicka a, B. Filipek b, J. Sapa b
a Department of Chemistry of Drugs, Wrocław Medical University, ul. Tamka 1, 50-137 Wrocław, Poland
b Laboratory of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University Medical College, ul. Medyczna 9, 30-688 Kraków, Poland
Received 2 February 2004; accepted 21 October 2004
Available online 22 December 2004
Abstract
We prepared a series of new N-[2-(4-substitutedpiperazin-1-yl)ethyl]-1-(n-butyl or phenyl)-2,5-dimethyl-3,4-pyrroledicarboximides 3 and
the related products 4 and 5, nine representatives of which were evaluated as potential analgesic agents in an animal model (mice). The new
pyrroledicarboximides were not toxic (LD50 ≥ 1466 mg/kg) and eight of them displayed analgesic activity ~1.5–5 times superior to that of
ASA in the writhing test. However, the compounds were found to be unstable in methanol solution and in dilute bases (methanol/NaOMe).
The S–A relationship is discussed.
© 2004 Elsevier SAS. All rights reserved.
Keywords: 3,4-pyrroledicarboximides; Analgesic activity
1. Introduction
In recent papers [1–4] we described the preparation and
pharmacological properties of a series of N-(4-arylpiperazin-
1-yl)alkyl]-1-substituted-2,5-dimethyl-3,4-pyrroledicar-
boximides I, shown in Fig. 1. Results of the biological evalu-
ation demonstrated that the toxicity (LD50) for most of the
Fig. 1
.
3,4-pyrroledicarboximides I tested ranged from 70 to
750 mg/kg and all compounds suppressed spontaneous and
amphetamine induced locomotor activity in mice [CNS (cen-
tral nervous system) depressive action]. Some of them (T ≠
H) additionally exhibited analgesic effect in the writhing test
after i.p. administration at a dose of up to 1/640 of LD50 and/or
weak analgesic activity at hot plate test at a dose of up to
1/80 of LD50.
The effects of the contributions of the substituent T and
the length of the central alkanyl chain (n) on toxicity, as well
as the analgesic potency in the writhing test, expressed as
ED50, for the most interesting compounds within series I are
summarized as follows:
(Ib) R = n-butyl, n = 4, T = o-OCH3—LD50 = 133 mg/kg
(ED50 = 1.06 mg/kg)
(Ic) R = Ph, n = 4, T = o-OCH3—LD50 = 383 mg/kg
(ED50 = 3.1 mg/kg).
The above data indicated that the best analgesic action was
shown by the o-methoxy derivatives, for which the effect was
observed at a 1–3 mg/kg dose, however, these compounds
were quite toxic (LD50 ~130–390 mg/kg). The identical anal-
gesic action of derivative Ia (n = 2) and its homologue Ib
with a four carbon central chain (n = 4) may suggest that Ib
in its bioactive form prefers a folded conformation rather than
an extended one.
Based on these fragmental data in this paper we undertook
further modifications of the 4-arylpiperazinyl segment of I
(Fig. 1) in order to increase analgesic properties and reduce
toxicity in selected pharmacological models. Structural modi-
(Ia) R = n-butyl, n = 2, T = o-OCH3—LD50 = 387 mg/kg
(ED50 = 1.05 mg/kg)
* Corresponding author.
E-mail address: malinka@bf.uni.wroc.pl (W. Malinka).
0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.10.002

16
W. Malinka et al. / IL FARMACO 60 (2005) 15–22
fications were achieved by varying the 4-substituent on the
piperazine moiety and the synthesis of analogues with
4-arylpiperidine or 1,2,3,4-tetrahydro-b-carboline pharma-
cophore for the structure–activity relationship (SAR) stud-
ies. In all the new compounds, we retained the two-carbon
central alkyl chain as an optimal spacer. Nine of the com-
pounds obtained were evaluated for acute toxicity and in the
writhing and hot plate analgesic tests. Additionally, the com-
pounds were evaluated for their CNS-depressive effect,
because such activity accompanied the analgesic action of
pyrroledicarboximides I tested recently.
2. Synthesis of compounds
Fig. 2
.
The method used in the synthesis of the title compounds
(Scheme 1) was closely related to the general procedures that
we had already employed [1,5] to prepare pyrrolodicarbox-
imides of type I (Fig. 1). The key intermediates, imides 1,
were prepared from ethyl ␣,b-diacetylsuccinate in a five-step
synthesis according to known methods [5]. Final compounds
3a–e, 4a–d and 5a, b were synthesized from commercially
accessible 4-substituted piperazines 6a–e and piperidines
7a–d, 4-phenyl-1,2,3,6-tetrahydropyridine, or 1,2,3,4-
tetrahydro-b-carboline, prepared in accordance with a pub-
lished procedure [6], by conventional bromoalkylation of them
using 2-bromoethyl-imides 2a, b in acetonitrile in the pres-
ence of K2CO3 (35–60% yield) [1]. The 4-substitutedperazine
(piperidine) intermediates 6a–e and 7a–d used are illustrated
in Scheme 1. The structures of the new products 3a–e, 4a–d
and 5a, b were confirmed through elemental and spectral (IR,
1H NHR) analysis.
During the course of purification of the final compounds
by crystallization from methanol, we observed that the prod-
ucts slowly decompose, with cleavage of the C–N-imide bond
and formation of methyl 4-(2-substituted-ethylcarbamoyl)-
2,5-dimethylpyrrole-3-carboxylates 8a–c (Fig. 2) as a by-
product.
Scheme 1
.

W. Malinka et al. / IL FARMACO 60 (2005) 15–22
17
Table 1
any obvious behavior suggestive of neurological or auto-
nomic effects.
Acute toxicity in mice of the compounds investigated, according to Litch-
field and Wilcoxon [7]
The analgesic activities were measured using the “hot
plate” and the phenylbenzoquinone-induced “writhing” tests.
Acetylsalicylic acid (ASA) and morphine, evaluated under
identical conditions, were used as reference agents.
In the phenylbenzoquinone-induced “writhing” test
(Table 2), all the compounds tested, except for 3d, were ~1.5–
5 times more active than ASA and ~3–10 times less active
than morphine. The analgesic action of 3d (ED50 = 47.7) was
slightly weaker than that of ASA (ED50 = 39.15). The most
potent effect (ED50 = 7.6 mg/kg) was produced by com-
pound 4c, which was significantly active up to a dose of
1/320 LD50.
Compound
LD50 i.p. mg/kg
>2000
3a
3b
3c
3d
4a
4b
4c
4d
5b
1466 (771–2785)
>2000
>2000
>2000
2000
>2000
2000
>2000
The data are median lethal doses with 5% confidence limits in parentheses
(n = 6).
In spite of the limited number of compounds tested, on the
bases of the results presented in Table 2 we formulated some
SAR. From SAR examination we excluded the recently
described pyrroledicarboximides I (Fig. 1), because for these
compounds and pyrroledicarboximides 3a–e, 4a–d and 5a, b
the writhing syndrome was induced by different chemical
stimulants—0.6% acetic acid for I and 0.02% phenylbenzo-
quinone for 3–5. For SAR discussion, the compounds were
divided into two series—derivatives of piperazine (3a–e and
5b) and of piperidine (4a–d). We included compound 5b in
the piperazine group because the substructure of 1,2,3,4-
tetrahydro-b-carboline present in 5b can be considered as a
rigid analogue of the 4-arylpiperazine moiety.
The reaction of methanolysis was completed after 7 h of
heating (See Section 5.1.3.1.) and the corresponding amido-
esters 8a–c were the only isolatable products. Later exami-
nation showed that the title pyrroledicarboximides readily
undergo reaction of methanolysis at room temperature in an
alkaline medium (methanol/NaOMe), with formation of the
compounds 8a–c (See Section 5.1.3.2.), whereas they were
quite stable in an acidic environment (methanol/HCl). The
IR spectra of 8a–c showed a CO absorption for ester at 1680–
1690 cm–1, an absorption at 1620–1640 cm–1 attributed to
CO amide function, and an NH band at ~3300–3250 cm–1.
The H NMR spectra contained a methyl peak ~3.8 ppm
indicative of a methyl ester and an exchangeable signal at
~8.5 ppm for NH.
Finally, for compounds evaluated pharmacologically
(3a–d, 4a–d, 5b) the lipophilicity, expressed as logPcalc., was
determined.
1
The SAR of 3,4-pyrroledicarbiximides (Table 2) shows the
following:
Within the piperazine derivatives 3a–e, the best analgesic
effect in the writhing test had compound 3a with
o-fluorophenylpiperazine (ED50 = 9.35 mg/kg), which was
~4-times more effective than ASA (ED50 = 39.15 mg/kg).
Replacements of the o-fluorine atom with o-OCH3 (3b) reduce
the potency of analgesic activity (ED50 = 17.3 mg/kg).
Replacement of the terminal 4-arylpiperazine portion with
4-furoylpiperazine (3c) or 1,2,3,4-b-carboline (5b) leads to
compounds ~3.5 and ~2 times more effective (ED50 = 11.5 and
17.6 mg/kg, respectively) than ASA. Further modification
revealed that the 4-arylpiperazine fragment can also be
replaced by 4-diphenylmethylpiperazine, however, in this case
3. Biological results and discussion
Our recent pharmacological studies on derivatives of pyr-
roledicarboximides I (Fig. 1) demonstrated that several mem-
bers of this group (T ≠ H) had significant analgesic activity in
the writhing test [3,4]. This finding encouraged us to synthe-
size a series of related compounds 3–5 (Scheme 1) and screen
them as potential analgesic agents. For this evaluation we also
included 3b, described in our recent paper [3], due to the inter-
esting analgesic action of its n-butyl analogue Ia
(ED50 = 1.05 mg/kg). All products 3a–d, 4a–d and 5b were
first evaluated for their acute toxicity in mice (Table 1) using
standard procedure [7]. After i.p. administration, compound
(3d) the analgesic activity dropped considerably (ED50
=
47.7 mg/kg).
We recently observed that replacement of the 4-aryl-
piperazine moiety of I (Fig. 1) with similar substructures pos-
sessing one N-atom (terahydoquinoline, 4-benzylpiperidine)
leads to a loss of analgesic activity [4]. In this context, it should
be noted that the incorporation of the 4-arylpiperidnine phar-
macophore (a series 4a–d) was found to retain significant
analgesic action (ED50 ≤ 24 mg/kg), and compound 4c
(ED50 = 7.6 mg/kg) was the most potent analgesic synthe-
sized in this study.
The analgesic action of compounds 3a–e, 4a–d and 5b is
not simply related to their lipophilic properties (See Section
5). For example, the ED50 values for compounds 3c, 4a or 5b
characterized by logPcalc. ~2.8–3.0, ranged from 11.4 to
3b only displayed
a relatively low toxicity (LD50
= 1466 mg/kg). The remaining compounds were not toxic
(LD50 ≥ 2000 mg/kg), and for these compounds 200 mg/kg
(1/10 LD50) was taken to be the initial dose in the further
experiments. The above toxicological data contrast sharply
with the results described for the best analgesic agents from
series I (Ia–c), for which toxicity ranged from ~130 to
~390 mg/kg.
After intraperitoneal administration of toxic doses, the ani-
mals exhibited a decrease in locomotion, but did not have

18
W. Malinka et al. / IL FARMACO 60 (2005) 15–22
Table 2
Influence of the compounds investigated on the pain reaction in the “writhing syndrome” test in mice
Compound
Dose
mg/kg
Mean number of
writhings SEM
ED50 (mg/kg)
LD50/ED50
>213.90
Part of LD50
–
Control
3a
20.3 2.9
1.4 0.7*
4.0 2.5**
8.8 1.4***
12.2 0.8
5.2 2.2****
10.7 2.3***
13.5 0.6
–
1/40
1/80
1/160
1/320
1/40
1/80
1/160
1/320
1/40
1/80
1/160
1/320
1/40
1/80
1/160
1/320
1/40
1/80
1/160
1/320
1/40
1/80
1/160
1/320
1/40
1/80
1/160
1/320
1/40
1/80
1/160
1/320
1/40
1/80
1/160
1/320
–
50
9.35(5.84–14.9)
25
12.5
6.25
36.65
18.32
9.16
–
3b
3c
3d
4a
4b
4c
4d
5b
17.3 (12.2–24.5)
11.5 (9.3–14.3)
47.7 (38.2–59.6)
24 (20–28.8)
84.73
50
5.3 1.9****
6.3 2.2****
9.7 1.1**
12.6 3.1
10.2 4.3***
11.0 1.8***
13.8 2.4
–
>176.99
>41.92
>83.33
83.33
25
12.5
6.25
50
25
12.5
6.25
50
6.8 2.9****
10.4 1.8***
12.5 2.1
–
25
12.5
6.25
50
7.8 3.6**
10.3 3.1***
11.2 2.3***
14.5 4.7
0.8 0.4*
3.6 0.7****
7.8 1.1**
10.2 1.8
3.6 0.9*
6.7 4.0**
12.7 1.6
–
24 (18.5–31.2)
7.6 (4.2–14.3)
17.3 (15.7–19)
17.6 (11.7–26.4)
25
12.5
6.25
50
84.73
25
12.5
6.25
50
115.60
>113.63
25
12.5
6.25
50
5.5 1.9****
25
8
1.6****
12.5
6.25
100
50
11.2 1.8***
13.8 2.4
ASA
–
–
–
–
–
–
39.15 (29.1–48.4)
2.44 (1.18–5.02)
–
–
30
Morphine
–
10
–
3
–
1
Each group consisted of 6 animals. *P < 0.001; **P < 0.01; ***P < 0.02; ****P < 0.05.
24 mg/kg. A similar range of ED50 values (from 7.6 to
24 mg/kg) was observed for compounds 3a, 3b, 4b, 4c and
4d with logPcalc. ~3.8–4.5.
The above data suggest that even very profound modifica-
tions of the 4-arylpiperazinyl pharmacophore and changes in
the lipophilicity of the analgesic agents of type I (Fig. 1) may
be tolerated.
We suspected that the central analgesic effect may be espe-
cially enhanced in series 4, as the 4-arylpiperidine grouping
is a common pharmacophore in narcotic analgesics. How-
ever, the experimental data exhibited that only the piperidine
derivative 4d possesses activity (50 mg/kg; 1/40 LD50) in this
test. A similar analgesic effect was observed for morphine
when it was used at a 3 mg/kg dose.
In addition to examining pyrroledicarboximides 3a–d,
4a–d and 5b in the writhing test, all the compounds were
also evaluated in the hot plate test in mice. The writhing test
is used by many investigators to determine peripheral anal-
gesia, while the hot plate test to exhibit central analgesia [8].
The compounds were also evaluated for their ability to pro-
duce a CNS-depressive effect in mice. All the derivatives
tested significantly decreased spontaneous locomotor activ-
ity during a 30 min observation period (Table 3). Compounds
3a, 4a and 4d, given at doses of 1/40–1/160, inhibited spon-

W. Malinka et al. / IL FARMACO 60 (2005) 15–22
19
taneous locomotor activity by 73–35%. The other com-
pounds significantly decreased locomotor activity by 65–37%
when administered at doses of 1/40–1/80 LD50. Moreover,
all the pyrroledicarboximides investigated, given at doses of
1/20 and 1/40 LD50, significantly prolonged barbiturate sleep
in mice by 321–119 and 160–81%, respectively (Table 4).
Analysis of the data presented in Tables 2–4 clearly reveals
that the tested compounds possess interesting analgesic prop-
erties in the “writhing syndrome” test associated with central
depressive action. However, further pharmacological studies
are needed to establish the mechanism of their analgesic action
and to optimize the structure of the basic side chain.
ally, all the new pyrroledicarboximides tested were charac-
terized by CNS-depressive action, as observed in thiopental
and locomotor activity tests. We hope that incorporation of a
polar group (i.e., OH, CO) to the central alkyl chain of the
most active analgesic agents from series I, 3 and 4 to reduce
their lipophilicity may be helpful in resolving the question of
analgesic/CNS-depressive effect selectivity and further
increase of analgesic action in pharmacological models. This
project will be the subject of continuing studies.
5. Experimental
5.1. Chemical experimental section
4. Conclusion
Melting points are uncorrected. The 1H NMR spectra were
obtained with a Tesla spectrometer 80 MHz in CDCl3; the
chemical shifts are reported in d (ppm). The IR (KBr) spectra
were recorded on Specord-75 IR spectrometer. Elemental
C,H,N analyses were run on a Carlo Erba NA-1500 analyzer,
the results were within 0.4% of the values calculated for the
corresponding formulas. Chromatographic separations were
performed on a silica gel [Kieselgel 60 (70–230 mesh),
Merck] column (CC).
In this paper we have demonstrated that incorporation of a
new 4-substitutedpiperazines, 4-arylpiperidines or 1,2,3,4-
tetrahydro-b-carboline into substructure of the side chain of
the analgesic pyrroledicarboximides of type I (Fig. 1) pro-
duced a novel series 3–5 of non-toxic (LD50 ≥ 2000 mg/kg;
for 3b = 1466 mg/kg) analgesic agents.Among the nine com-
pounds tested, eight exerted a ~1.5–5 times stronger analge-
sic effect in the writhing test after i.p. administration (ED50
= 7.6–24 mg/kg) than ASA (ED50 = 39.15 mg/kg). Addition-
Table 3
Influence of the investigated compounds on the spontaneous locomotor activity in mice
Compound
Dose
mg/kg
Number of movements
SEM during 30 min
ED50 (mg/kg)
LD50/ED50
>83.16
52.17
Part of LD50
–
Control
435 54
3a
1/40
25
116 45.4*
252 31.2**
272 41.4***
187 42.3*
262 54.8**
341 56.2
24.05 (18.5–31.2)
28.1 (23.4–33.7)
24.7 (17.6–34.6)
38.7 (25.8–58)
23.4 (18.7–29.2)
34.9 (29–41.9)
36.5 (30.4–43.8)
25 (15.4–40.5)
28 (23.3–33.6)
1/80
12.5
12.5
36.65
18.32
9.16
50
1/160
1/40
3b
3c
3d
4a
4b
4c
4d
5b
1/80
1/160
1/40
154 27.2*
197.2 42.4**
289 59.4
>80.97
>51.67
>85.47
57.3
1/80
25
1/160
1/40
12.5
50
182 32.6*
274 42.3**
372 28.4
1/80
25
1/160
1/40
12.5
50
162.6 26*
186.5 59**
282 56.4***
178.4 36.2*
266.4 56.2**
302.8 66
1/80
25
1/160
1/40
12.5
50
1/80
25
1/160
1/40
12.5
50
189.8 27.4*
241.5 46.2**
324 51.4
>54.79
80
1/80
25
1/160
1/40
12.5
50
182 22.4*
212.7 46.2**
254 56.2***
164 41.3*
221 43.4**
299 62
1/80
25
1/160
1/40
12.5
50
>71.42
1/80
25
1/160
12.5
Each group consisted of 6–8 animals. * P < 0.01; ** P < 0.02; *** P < 0.05.

20
W. Malinka et al. / IL FARMACO 60 (2005) 15–22
Table 4
5.1.1.2. N-[2-(4-o-methoxyphenylpiperazin-1-yl)ethyl]-2,5-
dimethyl-1-phenylpyrrole-3,4-dicarboximide (3b) [3]. From
N-(2-bromoethyl)-1-phenylpyrroledicarboximide 2b [2] and
1-(o-methoxyphenyl)piperazine (6b), logPcalc. = 4.11.
Influence of compounds investigated on thiopental anesthesia
Compound
Dose
Duration
Prolongation
(%)
of sleeping time
(min) SEM
38 9.9
Part of LD50 Mg/kg
–
Control
5.1.1.3. N-{2-[4-(2-furoyl)piperazin-1-yl]ethyl}-1-n-butyl-
2,5-dimethylpyrrole-3,4-dicarboximide (3c). Yield 1.8 g
(40%) from N-(2-bromoethyl)-1-n-butylpyrroledicaroximide
2a [1] and 1-(2-furoyl)piperazine (6c), CC (EtOAc, Rf = 0.39),
3a
1/20
1/40
1/80
1/20
1/40
1/80
1/20
1/40
1/80
1/20
1/40
1/80
1/20
1/40
1/80
1/20
1/40
1/80
1/20
1/40
1/80
1/20
1/40
1/80
1/20
1/40
1/80
100
50
125 10.8*
97.2 16.4**
49.4 21
228
155
30
25
3b
3c
3d
4a
4b
4c
4d
5b
73.3
36.65
18.32
100
50
120 12.1*
75.8 10.4***
39.4 16.8
142 10.8*
99.8 13.1**
42.3 12.2
109.2 20.1**
119
97
o
1
mp 136–138 C (cyclohexane), logPcalc. = 2.82. H NMR:
0.98 (3H, CH3, J~5.9 Hz), 1.25–1.8 (4H,
3.68
273
160
11.3
187
t
m
NCH2CH2CH2CH3), 2.38 s (6H, 2 × CH3), 2.5–2.75 m [6H,
N(CH2)3], 3.55–3.95 m [8H, 2 × NCH2 and (CH2)2N-furoyl],
6.44–6.58 m (1H, 4′-H), 6.95 m (1H, 3′-H), 7.47 m (1H, 5′-H).
IR(KBr): 1750, 1700, 1640 (C=O).
25
100
50
88.4 22.2**** 131
25
42.4 16.8
110 5.8*
4.4
5.1.1.4. N-{2-[4-(diphenylmethyl)piperazin-1-yl)ethyl]-2,5-
dimethyl-1-phenylpyrrole-3,4-dicarboximide (3d). Yield 2.0 g
(39%) from N-(2-bromoethyl)-1-phenylpyrroledicarboximide
2b [2] and 1-(diphenylmethyl)piperazine (6d), mp 202–
100
50
189
86
71.2 13.1***
38.4 10.1
25
1.05
321
121
26.3
186
157
3.68
186
81
100
50
160 22*
o
1
84.1 14.1**
48.1 11.4
204 C (EtOH), logPcalc. = 5.83. H-NMR: 2.14 s (6H, 2 ×
CH3), 2.35–2.75 m [10H, CH2N(CH2CH2)2N], 3.72 t (2H,
NCH2, J = 7 Hz), 4.25 s (1H, CH), 7.1–7.7 m (15H, ArH).
IR(KBr): 1740,1700 (C=O).
25
100
50
103 16.2**
98 16.2***
39.4 12.1
25
100
50
109 12.2*
82.3 13.1**
40.1 13.2
5.1.1.5. N-{2-[4-(p-acetylphenyl)piperazin-1-yl]ethyl}-1-n-
butyl-2,5-dimethylpyrrole-3,4-dicarboximide (3e). Yield 1.6 g
(35%) from N-(2-bromoethyl)-1-n-butylpyrroledicarboximide
2a [1] and 1-(p-acetylphenyl)piperazine (6e), CC (EtOAc, Rf
= 0.52), mp 155–157 oC [toluene/n-heptane (1:2)]. 1H NMR:
0.9–1.15 m (3H, CH3), 1.2–1.8 m (4H, NCH2CH2CH2CH3),
2.38 s (6H, 2 × CH3), 2.51 s (3H, COCH3), 2.6–2.8 m [6H,
N(CH2)3], 3.25–3.45 m [4H, (CH2)2NAr], 3.6–3.88 m (4H, 2
× NCH2), 6.84 d (2H, ArH, J = 8.8 Hz), 7.85 d (2H, ArH,
J = 8.8 Hz). IR(KBr): 1760, 1740, 1700 (C=O).
25
5.2
100
50
101 31.2***
89.1 17****
39.4 16.2
165
134
3.68
25
Each group consisted of 6–8 animals. * P < 0.001; ** P < 0.01; *** P < 0.02;
***** P < 0.05.
5.1.1. General procedure for the preparation
of N-(2-substituted-ethyl)-1-(n-butyl or phenyl)-
2,5-dimethylpyrrole-3,4-dicarboximides 3,4 and 5
5.1.1.6. N-[2-(4-hydroxy-4-phenylpiperidin-1-yl)ethyl]-1-n-
butyl-2,5-dimethylpyrrole-3,4-dicarboximide (4a). Yield 2.5 g
(60%) from N-(2-bromoethyl)-1-n-butylylpyrroledicar-
boximide 2a [1] and 4-hydroxy-4-phenylpiperidine (7a),
CC (EtOAc, Rf = 0.21), mp 110–112 oC (cyclohexane), log-
N-(2-bromoethyl)pyrrole-3,4-dicarboximide
2
[1,2]
(0.01 mol), 1.4 g (0.01 mol) of anhydrous K2CO3 and of cor-
responding amine (001 mol) in acetonitrile were reflux with
stirring for 15 h. The hot reaction mixture was filtered, puri-
fied with charcoal and evaporated to dryness. The oily resi-
due was crystallized from an appropriate solvent to give pure
3d, 4c, 5a or 5b. Purification of 3a, 3c, 3e, 4a, 4b or 4d was
achieved by CC and further crystallization of the products
obtained from an appropriated solvent.
1
Pcalc. = 3.0. H NMR: 0.85–1.1 m (3H, CH3), 1.35–2.15 m
(8H, NCH2CH2CH2CH3 and 3′,5′-CH), 2.38 s (6H, 2 × CH3),
2.5–2.95 m [6H, N(CH2)3], 3.55–3.85 m (4H, 2 × NCH2),
7.25–7.6 m, (5H, ArH), position of the OH proton signal was
not established. IR(KBr): 3500 (OH), 1750, 1695 (C=O).
5.1.1.1. N-[2-(4-o-fluorophenylpiperazin-1-yl)ethyl]-2,5-
dimethyl-1-phenylpyrrole-3,4-dicarboximide (3a). Yield 2.4 g
(55%) from N-(2-bromoethyl)-1-phenylpyrroledicarboximide
2b [2] and 1-(o-fluorophenyl)piperazine (6a), CC (EtOAc,
Rf = 0.8), mp 158–160 oC (cyclohexane), logpcalc. = 4.50. 1H
NMR: 2.17 s (6H, 2 × CH3), 2.60–2.95 m [6H, N(CH)3], 3.0–
3.25 m [4H, (CH2)2NAr], 3.75 t (2H, NCH2, J = 6.6 Hz),
6.9–7.35 m (6H, ArH), 7.45–7.7 (3H, ArH). IR(KBr):1750,
1700 (C=O).
5.1.1.7. N-{2-[4-(p-bromophenyl)-4-hydroxypiperidin-1-
yl]ethyl}-1-n-butyl-2,5-dimethylpyrrole-3,4-dicarboximide
(4b). Yield 2.4 g (48%) from N-(2-bromoethyl)-1-n-butyl-
pyrroledicarboximide 2a [1] and 4-(p-bromophenyl)-4-
hydroxypiperidine (7b), CC (EtOAc, Rf = 0.54), mp 151–
153 oC (cyclohexane), logPcalc. = 3.79. 1H-NMR: 0.87–1.1 m
(3H, CH3), 1.25–2.25 m (8H, NCH2CH2CH2CH3 and 3′,5′-
CH), 2.38 s (2 × CH3), 2.45–3.05 m [6H, N(CH2)3], 3.55–
3.85 m (4H, 2 × NCH2), 7.35–7.55 m (4H ArH), position of

W. Malinka et al. / IL FARMACO 60 (2005) 15–22
21
the OH proton signal was not established. IR(KBr): 3500
(OH), 1750, 1695 (C=O).
room temperature for 1 h. After stirring the reaction mixture
was diluted with water (2.5 ml). The precipitate formed was
filtered off and crystallized from an appropriated solvent to
give compound identical with that of 8a–c, respectively [the
5.1.1.8. N-{2-[4-(p-chlorophenyl)-4-hydroxypiperidin-1-
yl]ethyl}-2,5-dimethyl-1-phenylpyrrole-3,4-dicarboximide
1
elemental and spectral (IR, H NMR) data were identical],
(4c). Yield 2.5
g
(57%) from N-(2-bromoethyl)-1-
70–80% yield.
phenylpyrroledicarboximide 2b [2] and 4-(p-chlorophenyl)-
4-hydroxypiperidine (7c), mp 220-222 oC (EtOH), logPcalc.
= 3.99. 1H NMR: 1.6–2.25 m (10H, 2 × CH3 and 3′,5′-CH),
2.35–3.2 m [6H, N(CH2)3], 3.76 t (2H, NCH2, J~7.2 Hz) 7.1–
7.65 m (9H, ArH), position of the OH proton signal was not
established. IR(KBr): 3500 (OH), 1750, 1700 (C=O).
5.1.2.3. Methyl 4-{{N-{2-[4-(diphenylmethyl)piperazin-1-
yl)ethyl]carbamoyl}}-2,5-dimethyl-1-phenylpyrrole-3-
carboxylate (8a). Prepared from 3d, CC [acetone/methanol
(4:1), Rf = 0.58], mp 158–160 oC (cyclohexane). 1H-NMR:
2.18 s (6H, 2 × CH3), 2.35–2.7 m [10H, CH2N(CH2CH2)2N],
3.45–3.7 m (2H, NCH2), 3.79 s (3H, OCH3), 4.26 s (1H, CH),
7.15–7.6 m (15H,ArH), 8.3 br (1H, NH, D2O exchangeable).
IR(KBr): 3300 (NH), 1690 (C=O, ester), 1635 (C=O, amide).
5.1.1.9. N-[2-(4-cyano-4-phenypiperidin-1-yl)ethyl]-1-n-
butyl-2,5-dimethylpyrrole-3,4-dicarboximide (4d). Yield 2.1 g
(49%) from N-(2-bromoethyl)-1-n-butylpyrroledicarboximide
2a [1] and 4-cyano-4-phenylpiperidine (7d), CC
(EtOAc/CHCl3, Rf = 0.7), mp 134–136 oC (cyclohexane), log-
5.1.2.4. Methyl 4-{{N-{2-[4-(p-chlorophenyl)-4-hydroxy-
piperidin-1-yl]ethyl}carbamoyl}}-2,5-dimethyl-1-phenylpyr-
role-3-carboxylate (8b). Prepared from 4c, CC [acetone/
1
Pcalc. = 4.20. H NMR: 0.9–1.2 m (3H, CH3), 1.27–1.85 m
o
methanol (4:1), Rf = 0.2], m.p. 162–164 C (cyclohexane).
(4H, NCH2CH2CH2CH3), 2.0–2.25 m (4H, 3′ and 5′-CH),
2.38 s [6H, 2 × CH3], 2.55–2.8 m (4H, 2′ and 6′-CH), 3.05–
3.35 m (2H, CH2Npiperidine), 3.6–3.95 m (4H, 2 × NCH2),
7.25–7.65 m (5H, ArH). IR(KBr): 2220 (CN), 1750, 1700
(C=O).
1H-NMR: 2.19 s (3H, CH3), 2.21 s (3H, CH3), 2.4–3.0 m
[10H, CH2N(CH2CH2)2 C(4′)], 3.5–3.75 m (2H, NCH2),
3.83 s (3H, OCH3), 7.15–7.65 m (9H, ArH), 8.65 br (1H,
NH, D2O exchangeable), position of the OH proton signal
was not established. IR(KBr): 3480 (OH), 3250 (NH), 1680
(C=O, ester), 1620 (C=O, amide).
5.1.1.10. N-[2-(4-phenyl-1,2,3,6-tetrahydropyridin-1-
yl)ethyl]-2,5-dimethyl-1-phenylpyrrole-3,4-dicarboximide
5.1.2.5. Methyl 2,5-dimethyl-1-phenyl-4-{N-[2-(4-phenyl-
1,2,3,6-tetrahydropyridin-1-yl)ethyl]carbamoyl}pyrrole-3-
carboxylate (8c). Prepared from 5a, CC (EtOAc, Rf = 0.12),
mp 97–99 oC (n-hexane). 1H NMR: 2.19 s (3H, CH3), 2.21 s
(3H, CH3), 2.55–2.9 m [6H, CH2NCH2CH2C(4′)], 3.0 d (2H,
6′-CH2, J = 1.5 Hz), 3.48–3.78 m (2H, NCH2), 3.83 s (3H,
OCH3) 6.12 t (1H, 5′-CH, J = 1.5 Hz), 7.15–7.65 m (10H,
ArH), 8.45 br (1H, NH, D2O exchangeable). IR(KBr): 3300
(NH), 1690 (C=O, ester), 1640 (C=O, amide).
(5a). Yield 2.3
g (55%) from N-(2-bromoethyl)-1-
phenylpyrroledicarboximide 2b [2] and 4-phenyl-1,2,3,6-
tetrahydropyridine, mp 115–117 oC (cyclohexane). 1H NMR:
2.16 s (6H, 2 × CH3), 2.5–2.9 m [6H, CH2NCH2CH2C(4′)],
3.0 d (2H, 6′-CH2, J = 1.5 Hz), 3.82 t (2H, NCH2, J = 6.6 Hz),
6.10 t (1H, 5′-CH, J = 1.5 Hz), 7.2–7.65 m (10H, ArH).
IR(KBr): 1745, 1700 (C=O).
5.1.1.11. N-[2-(1,2,3,4-tetrahydro-b-carbolin-2-yl)ethyl]-2,5-
dimethyl-1-phenylpyrrole-3,4-dicarboximide (5b). Yield 2.4 g
(55%) from N-(2-bromoethyl)-1-phenylpyrroledicarboximide
2b [2] and 1,2,3,4-tetrahydro-b-carboline [6], mp 276–
5.1.3. Calculation of logP
The logPcalc. values were calculated using the ChemPlus
program from Hypercube, Inc., IBM PC version, imple-
mented in HyperChem program package.
o
1
278 C (EtOH), logPcalc. = 2.85 H NMR: 2.09 s (6H, 2 ×
CH3), 2.7–3.0 m [6H, CH2NC(3′)H2C(4′)H2], 3.71 s [2H,
C(1′)H2], 3.84 t (2H, NCH2, J = 6.8 Hz), 7.0–7.65 m (9H,
ArH), 8.4 br (1H, NH, D2O exchangeable). IR(KBr): 3340
(NH), 1745, 1700 (C=O).
5.2. Pharmacological experimental section
5.2.1. Substances used
ASA (Polopiryna, ZF Starogard Gdañski, PL), morphine
(Morphinum hydrochloricum, Polfa-Kutno, PL), phenylben-
zoquinone (INC Pharmaceuticals, Inc. N.Y.) thiopental
sodium (Thiopentone sodium, HEFA-FRENON Arzneimit-
tel, Germany).
5.1.2. Methanolysis of compounds 3d, 4c and 5a
5.1.2.1. Method A. A solution of pyrroledicarboximide 3d,
4c or 5b (0.2 g) in methanol (5 ml) was refluxed for 7 h.
Methanol was then evaporated and the crude product obtained
was purified with CC and crystallized from an appropriated
solvent to give 8a-c, respectively, (50–60% yield).
5.2.2. Animals
The experiments were carried out on male Albino-Swiss
mice (body weight 18–26 g). All of the animals were housed
at constant humidity (60%) and temperature (24–25 oC) and
kept on a 12-hour light/dark cycle. Animals were fed a stan-
5.1.2.2. Method B. Pyrroledicarboximide 3d, 4c or 5a (0.2 g)
in 5 ml of methanol solution of sodium methoxide (prepared
from 2.3 ml of Na and 100 ml of methanol) was stirred at

22
W. Malinka et al. / IL FARMACO 60 (2005) 15–22
dard pellet diet with free access to tap water. All procedures
were conducted according to animal care and use committee
guidelines, and approved by the ethical committee of Jagiel-
lonian university, Kraków.
Control and experimental groups consisted of 6–8 ani-
mals each. The investigated compounds were administered
intraperitonelly as the suspension in 0.5% methylcellulose in
constant volume of 10 ml/kg.
5.2.7. Spontaneous locomotor activity
Spontaneous locomotor activity in mice was measured in
circular photoresistor actometers (32 cm in diameter). The
investigated compounds were injected intraperitoneally at
doses of 1/160–1/40 LD50. Thirty minutes after the injection
of the investigated compounds mice were placed in the acto-
meters for 30 min. Each crossing of the light beam was
recorded automatically. The amount of impulses was noted
after 30 min.
5.2.3. Statistical analysis
The statistical significance was calculated using a Stu-
dent’s t-test. The ED50 values and their confidence limits were
calculated according to the method of Litchfield and Wil-
coxon [7].
5.2.8. Thiopental anesthesia
Thiopental in a narcotic dose (55 mg/kg) was injected intra-
peritioneally 30 min after administration of the tested com-
pounds at doses of 1/80–1/20 LD50. Duration of narcotic sleep
was taken as being from disappearance to return of the right-
ing reflex.
5.2.4. Acute toxicity
Acute toxicity was assessed by the methods of Litchfield
and Wilcoxon [7] and presented as LD50 calculated from the
mortality of mice after 24 h.
References
5.2.5. Pain reactivity
[1] W. Malinka, E. Tatarczyn´ska, Synthesis and pharmacological proper-
ties of N-(4-substituted-1-piperazinylalkyl)-1-butyl-2,5-dimethyl-
pyrrole-3,4-dicarboxyimide derivatives, Farmaco 48 (1993) 933–947.
[2] W. Malinka, M. Sieklucka-Dziuba, J. Robak, Z. Kleinrok, Synthesis
and biological evaluation of derivatives of N(4-substituted-1-
piperazinylalkyl)-1-(butyl, aryl)-2,5-dimethylpyrrole-3,4-dicarboxy-
imide (Part II), Farmaco 49 (1994) 481–487.
[3] W. Malinka, M. Sieklucka-Dziuba, G. Rajtar, A. Rubaj, Z. Kleinrok,
Synthesis and pharmacological screening of some N-(4-substituted-
piperazin-1-ylalkyl)-3,4-pyrroledicarboximides, Farmaco 54 (1999)
390–401.
Pain reactivity was measured in the “hot plate” test accord-
ing to the method of Eddy and Leimbach [9]. Animals were
placed individually on the metal plate heated to 56 oC. The
time (sec) of appearance of the pain reaction (licking of the
forepaws or jumping) was recorded by a stop-watch. The
experiments were performed 30 min after administration of
the investigated compounds at graded doses of 1/40–
1/10 LD50).
[4] W. Malinka, M. Sieklucka-Dziuba, G. Rajtar, R. Rajdak, K. Rajdak,
Z. Kleinrok, Synthesis of some N-substituted 3,4-pyrroledicar-
boximides as potential CNS depressive agents, Pharmazie 55 (2000)
9–16.
[5] W. Malinka, T. Bodalski, Synthesis of some 1-substituted-2,5-
dimethylpyrrole-3,4-dicarbixyimides from ␣,b-diacetylsuccinate,
Pol. J. Chem. 68 (1994) 297–307.
5.2.6. “Writhing” syndrome in mice according to
Hendershot and Forsaith [10]
Different doses of test compounds ranging from 1/320 to
1/40 LD50. were administered intraperitonelly. Twenty five
minutes later, 0.02% phenylbenzoquinone was injected intrap-
ertioneally in a constant volume of 0.25 ml. Five minutes after
injection of the irritating agent, the number of “writhing” epi-
sodes in the course of 10 min was counted. The analgesic
effect of individual doses was expressed in percent:
[6] T. Ho Beng, K.E. Walker, 1,2,3,4-tetrahydro-b-carboline. Organic
Synthesis, vol. 51, 1971, pp. 136–138.
[7] J.T. Litchfield, F. Wilcoxon, A simplified method of evaluation dose–
effect experiments, J. Pharmacol. Exp. Ther. 96 (1949) 99–113.
[8] H.G. Vogel, W.H. Vogel, in: Drug Discovery and Evaluation. Pharma-
cological Assays, Springer, Berlin, Heidelberg, New York, 1997,
pp. 367–384.
(1)
% analgesic effect = 100−
[9] N.B. Eddy, D. Leinbach, Synthetic analgesics II. Dithienyl-butenyl
and dithienyl-butylamines, J. Pharmacol. Exp. Ther. 107 (1953) 385–
389.
[10] L.C. Hendershot, J. Forsaith, Antagonism of the frequency of
phenylquinone-induced writhing in the mouse by weak analgesics and
non-analgesics, J. Pharmacol. Exp. Ther. 125 (1959) 237–240.
of writhing incidents in experimental group
͚
× 100
of writhing incidents in control group
͚
The ED50 values and their confidence limits were esti-
mated by the method of Litchfield and Wilcoxon [7].