Derivatives of pyrrolo[3,4-d]pyridazinone, a new class of analgesic agents

Article abstract of DOI:10.1016/j.ejmech.2011.08.006

A series of N2-{2-[4-aryl(benzyl)-1-piperazinyl(piperidinyl)]ethyl} pyrrolo[3,4-d]pyridazinones 4 and related derivatives 5 were synthesized as potential analgesic agents. The structures of the new compounds were elucidated by micro, spectral and X-ray analysis. Analgesic activity of the compounds was investigated in the phenylbenzoquinone induced 'writhing' and 'hot plate' test in mice and at radioligand binding assay. At 'writhing' test all compounds, without exception, were more active than acetylsalicylic acid (ASA) with ED ₅₀ values ranging from 0.04 to 11 mg/kg (i.p.) (ED₅₀ for ASA - 39.15 mg/kg). Analgesic effect at the 'hot plate' test was observed for three compounds 4c,e,f at the dose 3-5 times higher then that of morphine (ED₅₀-3.39 mg/kg). At radioligand binding assay of 4c,e,f only compound 4f exhibited affinity for the μ-opioid receptors similar to that of Tramadol. The acute toxicity of the pyrrolopyridazinones 4, 5 were also studied and non toxic effect was observed at the 2000 mg/kg (5a 1420 mg/kg) i.p. dose level. On the basis of the available pharmacological data S-A relationship is discussed. The preferred conformational characteristic of 4, taken 4c as an example, was also described.

Full text of DOI:10.1016/j.ejmech.2011.08.006

European Journal of Medicinal Chemistry 46 (2011) 4992e4999
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Derivatives of pyrrolo[3,4-d]pyridazinone, a new class of analgesic agents
,
b
b
Wies1aw Malinka a, Aleksandra Redzicka a , Magdalena Jastrze˛bska e Wie˛sek , Barbara Filipek ,
*
c
d
e
e
ꢀ
Ma1gorzata Dyba1a , Zbigniew Karczmarzyk , Zofia Urbanczyk-Lipkowska , Przemys1aw Kalicki
a Department of Chemistry of Drugs, Wrocław Medical University,1 Tamka Str., Wrocław 50-137, Poland
b Department of Pharmacodynamics, Collegium Medicum, Jagiellonian University, 9 Medyczna Str., 30-688 Kraków, Poland
c Laboratory of Pharmacobiology, Collegium Medicum, Jagiellonian University, 9 Medyczna Str., 30-688 Kraków, Poland
d Department of Chemistry, University of Podlasie, 54 3 Maja Str., 08-110 Siedlce, Poland
e Institute of Organic Chemistry, Polish Academy of Sciences, 44/52 Kasprzaka Str., 01-224 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N2-{2-[4-aryl(benzyl)-1-piperazinyl(piperidinyl)]ethyl}pyrrolo[3,4-d]pyridazinones 4 and
related derivatives 5 were synthesized as potential analgesic agents. The structures of the new
compounds were elucidated by micro, spectral and X-ray analysis. Analgesic activity of the compounds
was investigated in the phenylbenzoquinone induced ‘writhing’ and ‘hot plate’ test in mice and at
radioligand binding assay. At ‘writhing’ test all compounds, without exception, were more active than
acetylsalicylic acid (ASA) with ED50 values ranging from 0.04 to 11 mg/kg (i.p.) (ED50 for ASA e 39.15 mg/
kg). Analgesic effect at the ‘hot plate’ test was observed for three compounds 4c,e,f at the dose 3e5 times
higher then that of morphine (ED50-3.39 mg/kg). At radioligand binding assay of 4c,e,f only compound 4f
Received 15 December 2010
Received in revised form
2 August 2011
Accepted 2 August 2011
Available online 9 August 2011
Keywords:
pyrrolo[3,4-d]pyridazinones
Analgesic activity
exhibited affinity for the m-opioid receptors similar to that of Tramadol. The acute toxicity of the pyr-
rolopyridazinones 4, 5 were also studied and non toxic effect was observed at the 2000 mg/kg (5a
1420 mg/kg) i.p. dose level. On the basis of the available pharmacological data S-A relationship is dis-
cussed. The preferred conformational characteristic of 4, taken 4c as an example, was also described.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Increasing of a pyrrolidinone ring to pyridazinone in a skeleton
of a lead compound is a common strategy in medicinal chemistry
Two major classes of drugs dominate in treatment of analgesia:
opioids which interact with specific central receptors (
[4]. In this context we have synthesized series of pyrrolo[3,4-d]
pyridazinodiones of general structure (Scheme 1). These
m,
k,
d)
4
mainly at spinal levels and cyclooxygenases inhibitors (the
enzymes that synthesize prostaglandins), which action occurs
peripherally. However the prostaglandin conception explains only
a part of the effects of these drugs [1]. As a third class of analgesic
agents may be considered antidepressants, anticonvulsants and
anesthetics used to control neuropathic pain which is very difficult
to treat [2].
For several years we are interested in synthesis of new mole-
cules as potential analgesic agents. In our previous work has been
showed that some derivatives of pyrrole-3,4-dicaboximide of
general structure shown in Fig.1 and their non-4-arylpiperazine (4-
compounds can be considered as partial analogues of our analgesic
pyrrole-3,4-dicarboximides (Fig. 1). It should be noted that
a number derivatives of pyridazinone and biheterocycles with
a pyridazinone ring bearing 4-arylpiperazinylalkyl substituents
linked to the lactam nitrogen atom of pyridiazinone exert notably
analgesic effect [5e10]. To define the importance of the substitution
of the lactam function of the pyridazinone for the analgesic activity
we synthesized also compounds 5 (Scheme 1) bearing the arylpi-
perazinylpropyl chain linked to 4eOeatom of a tautomeric form of
pyrrolopyridazinone 2. Compounds 4 and 5 were evaluated for
analgesic action in comparison to classical analgesic drugs (ASA,
Morphine).
arylpiperidine, b-carboline) analogues exhibited significant anal-
gesic action in the phenylbenzoquinone-induced ‘writhing’ test.
The most active compounds were 5-times more potent than ASA
used as a standard [3].
2. Chemistry
In the previous paper [11] we reported the facile conversion of
the 1-phenylpyrrole-3,4-dicarboxylic acid anhydride 1 into pyr-
rolopyridazinone 2a by reaction with N-methylhydrazine (Scheme
1) in relatively good yield (70%). Similarly, treatment of anhydride
* Corresponding author. Tel.: þ48 717840400.
E-mail address: redzika@wp.pl (A. Redzicka).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.006

W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
4993
1 with b-hydroxyethylhydrazine afforded the corresponding pyr-
rolopyridazinone 2b (65e70% yield). Compounds 2 were used as
key intermediates for further synthesis of the title derivatives
4 and 5.
For preparation compounds 4 first hydroxyethylpyrrolopyr-
idazine 2b was treated with thionyl chloride with formation of 2-
chloroethyl derivative 3a. The control experiments exhibited that
alkylation of N-phenylpiperazine with 3a afforded the expected
pyrrolopyridazine 4a with a very low yield (16%). The low yield
practically prevented our approach to synthesis of the planed
Fig. 1.
Scheme 1.

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W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
compounds 4 in 1e1.5 g quantity, necessary for analgesic screening
and to test toxicity in animal model.
2.1. Crystal structure of pyrrolopyridazinone 4c
Alternatively compound 2b was first mesylated with forma-
tion of ester 3b. Subsequent amination of the 3b using appro-
priate 1-substituted piperazine(piperidine) derivatives afforded
the compounds 4 with 32e69% yield and this method was used
for preparation of all the final pyrrolopyridazinones 4ael
(Scheme 1).
Our previous experiments exhibited that the substrate 2a may
react in the two tautomeric forms (Scheme 1) [11]. For preparation
of the compounds 5, possessing a 4-arylpiperazinylalkyl moiety by
4-O atom of a lactim function of the pyridazinone ring the anion of
pyrrolopyridazinone 2, obtained in reaction with sodium ethoxide
in dry ethanol, was alkylated with corresponding chloropropylar-
ylpiperazines 6 easily available (Scheme 1). Products isolated
turned out to be unplanned 4-O-alkylated derivatives 5a,b obtained
in yield of 40% or 5c obtained in 15% yield. Any trace of corre-
sponding N-isomers was detected.
The structures of the new compounds 2b, 3a,b, 4a-l and 5aec
were proved through elemental and spectral analysis (1H NMR, IR).
In the 1H NMR spectra of the most derivatives 4b,d,fel, signals for
5- and 7-methyl substituents were recorded practically as one
singlet (w2.4 ppm) integrating for six protons. The
4eOesubstitution 5 produced a downfield shift for the protons of
the 5-methyl group and the signals of the 5- and 7-methyl
substituents were separated into two singlets each for the three
protons. The 5- and 7-methyl groups of compound 5a, taken as an
example, are characterized by the following chemical shifts: 2.30 (s,
3H, 5eCH3), 2.43 (s, 3H, 7eCH3).
The different spectral behavior of the methyl groups of pyrrole
in 4 and 5 may be explain by the fact that in compounds 4, methyl
protons have a much more homogenous magnetic environment
than in their analogues 5.
Additionally the structure of 4c, taken as an example, was
unambiguously established by X-ray crystallography (Fig. 2). In
general bond lengths and angles do not show surprising features.
The compound exhibits intramolecular hydrogen bonding between
N3eH—N13 (Fig. 2) with bond length 1.98 (8) Å. This hydrogen
bound stabilize molecule 4c in terms of preferred conformation and
steric orientation of the side chain.
The structure of the molecule 4c in the crystal is shown in Fig. 2.
In the molecule investigated, the bond lengths and angles are in
normal ranges [12]. In the crystalline state, the molecule exists in
lactam tautomeric form (a, Fig. 3), as evidenced by the C4eO4,
N3eC4 and N2eN3 bond lengths of 1.249(8), 1.364(8) and
1.423(7) Å, respectively, and the position of the H atom in the
vicinity of N3 in difference electron-density map.
Theoretical calculations at the DFT/B3LYP/6-311þþG(d,p) level
show that form a of 4c (Fig. 3) obtained after energy minimization
and geometry optimization in the gaseous phase is more energet-
ically stable than form b, with a difference in the energy between
the b and a forms of 4.85 kcal/mol. Thus, the population of the
lactim tautomeric form b in vacuum estimated using a nondegen-
erate Boltzmann distribution is below the threshold of the detect-
ability of conventional analytical methods.
The 4-(o-fluorophenyl)piperazin-1-ylethyl substituent has
a gauche-gauche-gauche-trans conformation with the torsion angles
N3eN2eC1 eC12 of ꢀ54.2(8)o, N2eC11eC1 eN13 of 74.7(8)o,
C11eC12eN1 eC14 of 77.7(7)o and C11eC12eN13eC18 of
ꢀ159.8(6)o. The piperazine ring adopts a chair conformation with
puckering parameters of Q ¼ 0.587(8) Å and
q
¼ 0.0(8) [13]. The
dihedral angle between the mean planes of the phenyl and piper-
azine rings of 40.2(3)o, intermediate between co-planar and
perpendicular orientation of these rings, is forced by two opposite
effects: conjugation of the lone pair at N16 with the
p system of the
phenyl ring and the steric effect of the o-F group. In the pyrrolo[3,4-
d]pyridazine fused ring system, the six-membered ring is planar to
within 0.024(7) Å and the five-membered ring is planar to within
0.009(7) Å. These two rings are inclined at an angle of 2.8(3) . The
phenyl ring is situated nearly perpendicularly to the pyrrolo[3,4-d]
pyridazine ring with the torsion angle C5eN6eC21eC26 of
106.5(9) .
The tautomeric form and the conformation of the molecule are
stabilized by the strong intramolecular N3eH.N13 hydrogen bond
[N3eH ¼ 0.99(7), H.N13 ¼ 1.98(8), N3.N13 ¼ 2.848(8) Å and
N3eH.N13 ¼ 146(6) ].
In the crystal structure, the pyridazine rings belonging to the
molecules related by c glade planes overlap each other forming
Fig. 2. A view of the X-ray molecular structure of 4c with the atomic labeling scheme. The dashed line indicates intramolecular hydrogen bond.

W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
4995
F
F
O
N
O
N
H3C
H3C
H3C
H3C
N
N
N
N
N
NH
O
a
OH
b
Fig. 3. Tautomeric forms of compounds 4c.
molecular stacks in the Z direction (Fig. 2). The
between overlapping planes are alternately 3.613(4) and 3.612(4) Å
and the angle between them is 3.81ꢁ.
p
.
p
distances
after 1 h time of observation after i.p. injection about 45% for
compound 4f and about 20% for compounds 4c and 4e.
However to explain mechanism of action of 4c,e,f additionally
pharmacological investigations are necessary in order to determine
their possible interaction with the adrenergic or serotoninergic
system [10].
3. Results and discussion
On the basis of the above pharmacological data we can conclude
that replacement of the pyrolidinone ring in analgesic pyrrole-3,4-
dicarboximides (Fig. 1) with pyridazinone results in significant
increase of non-opioid analgesic activity observed at ‘writhing’ test.
Additionally, as exemplified at compounds 4a,d and 5a,b, remove of
the 4-arylpiperazinylalkyl moiety from lactam atom N-2 to 4-O
atom of the hydroxyl group of tautomeric form of pyrrolopyr-
idazinone 2 diminish analgesic activity (Table 1).
All pyrrolopyridazinones 4 and 5 described in this paper,
exception 5c (low yield), were first evaluated for their acute toxicity
in mice. Investigated compounds were not toxic after intraperito-
neal administration [LD50 > 2000 mg/kg (5a LD50 ¼ 1420 mg/kg)]
and all were further investigated for their analgesic action.
Because analgesic activity derivatives of pyridazinone bearing
a 4-arylpiperazinylalkyl moiety was not always related to the
mechanism of prostaglandin inhibitors [5,7,10], the analgesic effect
of our compounds was determined in two behaviorally different
tests: phenylbenzoquinone-induced ‘writhing syndrome’ test and
‘hot plate’ (thermal analgesic stimulus) test in mice. The ‘writhing’
test has been used by many investigators for measuring peripheral
analgesic activity, whereas the ‘hot plate’ test for evaluating central
analgesia [14].
In the ‘writhing syndrome’ test all investigated pyrrolopyr-
idazines 4 and 5 showed stronger statistically analgesic properties
than acetylsalicylic acid and nine of them (4a,c,e,f,hel) possessed
better activity than morphine. The most potent effect (ED50 value
weaker than 1 mg/kg i.p.) was produced by compounds 4c,f,h. The
ED50 values of the investigated compounds at ‘writhing’ test and
standards (acetylsalicylic acid, morphine) are summarized in
Table 1.
The highest analgesic effect in ‘writhing syndrome’ test was
observed for compound 4h. It showed approximately 1000-fold
less ED50 value than that of ASA (Table 1). Although additional
pharmacological investigations, especially determination of anti-
inflammatory activity, are necessary to explain mechanism of
their analgesic action observed at ‘writhing’ test.
In the ‘hot plate’ test three compounds: 4c,f and 4e produced an
analgesic effect. The most interesting compound 4f was only 3-fold
less active than morphine. The summarized data are shown in
Table 1.
3.1. SAR of pyrrolopyridazinones 4
To develop preliminary S-A relationship on the basis data
from the ‘writhing’ test compounds investigated were divided on
three series: (I) - derivatives of 4-arylpiperazine 4aef, (II) e
derivatives of 4-benzylpiperazine and its more complex
analogues 4hek, and series (III) - compounds 5a,b which repre-
sent O-substitued partial analogues of series I. To the first series
was introduced heteroaryl(pyridine) analogue 4g, whereas cyn-
namyl 4k and piperidine 4l derivatives were introduced to the
series II (Table 1).
In general, series II (derivatives of 4-benzylpiperazine), was the
most active with ED50 < 1.92 mg/kg ip. Replacement of the 4-benzyl
substituent of piperazine with the cynnamyl residue (4k)or
replacement of the 4-benzylpiperazine fragment with one-base
residue 4-benzylpiperidine (4l) maintain significant analgesic
action. The most potent compound series II (4h) was characterized
by ED50 0.04 mg/kg (ED50 for ASA e 39.15 mg/kg).
Within series I (derivatives of 4-arylpiperazine) the ED50 values
ranged from 0.31 to 8.02 mg/kg (ip.). Introduction of a substituent
to the aromatic ring of 4-phenylpiperazine moiety has got different
influence on bioactivity. For example o-F significant enhanced
analgesic effect (ED50 ¼ 0.31 mg/kg), when o-OCH3 or o-Cl
substituents markedly decreased activity (ED50 ¼ 8.02 and 3.77 mg/
kg, respectively), similarly as a replacement of the phenyl by pyri-
dine (4g, ED50 ¼ 6.84 mg/kg).
With the aim of investigation of possible opioid mechanism
action of pyrrolopyridazinones 4c,e,f which possessed strong
peripheral as well central analgesic activity radioligand binding
assay were performed (Table 2).
In conclusion, we described a new and interesting series of non
From compounds assessed in this test only pyrrolopyridazine 4f
toxic pyrrolopyridazinones 4 with potent analgesic activity
showed some affinity to
madol [15]. On the basis of these data we can suggests that 4f
present its own profile of analgesic activity and the part of its
m
receptors comparable with that of Tra-
observed in animal model (mice). In ‘writhing’ test the most active
compounds exhibited analgesic action at submiligram doses and
were 100e1000 times more active then ASA. At the ‘hot plate’ test
three of these compounds 4c,e,f were only 3e5 times less active
then morphine used as standard. The title compounds 4 are easily
synthesized and could be a promising candidates for further
chemical development. Our studies on related compounds are in
progress.
analgesic action involves a m-opioidergic mechanism.
In addition the compounds significantly active in hot plate test
4c,e,f were assessed at spontaneous locomotor activity test at doses
equaled to ED50 value obtained in ‘hot plate’ test. These compounds
decreased (diminished) spontaneous locomotor activity in mice

4996
W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
Table 1
Influence on the pain reaction in the ‘writhing syndrome’ and the ‘hot plate’ tests of the investigated compounds in mice.
Series
Structure
Substitutes
Comp.
Pain reaction ED50 (mg/kg)
‘writhing syndrome’
‘hot plate’ test
test
Ar
4a
4b
4c
4d
4e
4f
1.48 (0.40e5.52)
3.77 (2.04e6.95)
0.31 (0.13e0.71)
8.02 (6.57e9.78)
1.23 (0.27e5.65)
0.55 (0.14e2.17)
6.84 (3.70e12.63)
inactive
&O
inactive
F
14.99 (10.73e20.92)
inactive
H3CO
I
CF3
15.91 (9.57e26.46)
10.31 (5.50e19.34)
inactive
H3C
Cl
4g
N
X
n
R2
4h
4i
0.04 (0.001e1.49)
1.92 (1.04e3.54)
inactive
inactive
N
1
2
2
N
N
1
0
O
O
H3C
N
X
R2
N
II
N
NH
4j
1.83 (0.90e3.73)
inactive
H3C
N
C
1
1
4k
4l
1.25 (0.79e1.99)
1.91 (0.68e5.39)
inactive
inactive
O
H3C
N
R3
N
5a
5b
7.9 (5.1e12.7)
inactive
inactive
III
H
R3
N
H3COe
11.15 (8.6e14.5)
H3C
O
N
N
Acetylsalicylic acid
Morphine
39.15 (29.10e48.40)
2.44 (1.18e5.02)
inactive at dose of 200 mg/kg
3.5 (3.0e4.1)
4. Experimental
4.1. Chemistry
Table 2
The IC50 and Ki values for the inhibition of the binding of [3H] dihydromorphine to
receptor.
m
4.1.1. Chemical experimental section
Melting points are uncorrected. The 1H NMR spectra recorded on
Bruker 300 MHz spectrometer in CDCl3 using tetramethylsilane
Compound
[3H] DHM
IC50 M]
(TMS) as internal reference (chemical shift in
d ppm). The IR (KBr)
[
m
Ki [mM]
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 withinꢂ0.4% of the values calculated for the corresponding
formulas. Chromatographic separations were performer on a silica
gel [Kieselgel 60(70e230 mesh), Merck] kolumn (CC). Progress of the
4f
4c
4e
5.6 ꢂ 0.1
24.2 ꢂ 0.7
e
2.6 ꢂ 0.1
11.1 0.2
e
Levellorphan
Tramadol
634.1 pM
2.4 ꢂ 1.1 [15]

W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
4997
reaction was monitored by TLC on silica gel plates with fluorescent
indicator (Fluka) and visualized by UV light at 254 nm.
phenylpyrrolo[3,4-d]pyridazine-1,4-dione and of corresponding
amine (30 mmol) in xylene was refluxed for 16 h. Than the precipi-
tated, crude product (4ae4d, 4f, h, k,l) was filtered off and recrys-
tallized fromtheappropriate solvent. Inthecaseofcompounds4e, 4g
and 4j the crude product was purified by CC with appropriate eluent.
4a: from 3b and 1-phenylpiperazine. Yield 43%, m.p. 218e220 ꢁC
(ethanol).
4.1.1.1. 1,2,3,4-tetrahydro-2-(2-hydroxyethyl)-5,7-dimethyl-6-
phenyl-6H-pyrrolo [3,4-d]pyridazine-1,4-dione 2b. A mixture of
2.41 g (10 mmol) of 1-phenyl-2,5-dimethyl-3,4-pyrroledicarboxylic
acid anhydride 1 and 1.36 ml (20 mmol) of 2-hydroxyethyl-
hydrazine in 40 ml of acetonitrile was refluxed for 7 h. After cool-
ing the product separated was filtered off and purified by crystal-
lization to give 1.94 g of 2b.
1H NMR: 2.36 (s, 3H; 5eCH3), 2.43 (s, 3H; 7eCH3), 2.88e2.96 [m,
6H; CH2N(CH2)2], 3.22e3.25 [m, 4H; (CH2)2NAr], 4.14 (t, 2H; NeCH2,
J¼5.1Hz), 6.95e7.07(m, 4H;ArH),7.20e7.23(m, 2H;ArH), 6.95e7.22
(m, 3H; ArH), position of the NH proton signal was not established.
IR(KBr): 1630(CO), 3440 (NH). Anal. Calc. C26H29N5O2, (443.55): C:
70.40, H: 6.60, N: 15.78. Found: C: 70.11, H: 6.73, N: 15.39.
4b: from 3b and 1-(o-chlorophenyl)piperazine. Yield 55%, m.p.
250e252 ꢁC (toluene).
2b: Yield 65%, m.p. 262e265 ꢁC (ethanol).
1H NMR: 2.36 (s, 6H, 5,7eCH3), 3.99 (t, 2H, CH2, J ¼ 4.8 Hz), 4.16
(t, 2H, CH2 J ¼ 4.8 Hz), 7.25e7.30 (m, 2H; ArH), 7.52e7.61 (m, 3H;
ArH), position of the OH proton signal was not established. IR
(KBr):1610 (CO), 3420 (OH). Anal. Calc. for C16H17N3O3 (299.32): C:
64.20, H: 5.74, N: 14.03. Found: C: 64.19, H: 5.73, N: 14.02.
1H NMR: 2.40 (s, 6H; 5,7eCH3), 2.88e2.91 [m, 6H; CH2N(CH2)2],
3.15e3.25 [m 4H; (CH2)2NAr], 4.08 (t, 2H; NCH2, J ¼ 5.1 Hz),
7.11e7.26 (m, 6H; ArH), 7.52e7.56 (m, 3H; ArH), position of the NH
proton signal was not established. IR(KBr): 1630(CO), 3440 (NH).
Anal. Calc. C26H28ClN5O2, (477.99): C: 65.33, H: 5.91, N: 14.64.
Found: C: 65.25, H: 5.93, N: 14.25.
4.1.1.2. 1,2,3,4-tetrahydro-2-(2-chloroethyl)-5,7-dimethyl-6-phenyl-
6H-pyrrolo [3,4-d]pyridazine-1,4-dione 3a. A solution of
1 g
(3.3 mmol) of 2b and 0.4 ml of thionyl chloride in chlofororm
(15 ml) was refluxed with stirring for 2 h, then it was evaporated.
The residue was purified by CC with appropriate eluent. The frac-
tions containing the product of Rf ¼ 0.65 were combined and
evaporated to give 0.23 g of 3a.
4c: from 3b and 1-(o-fluorophenyl)piperazine. Yield 69%, m.p.
246e248 ꢁC (ethanol).
1H NMR: 2.38 (s, 3H; 5eCH3), 2.43 (s, 3H;), 2.92e2.96 [m, 6H;
CH2N(CH2)2], 3.27 [t, 4H; (CH2)2NAr, J ¼ 4.5 Hz], 4.14 (t, 2H; NeCH2,
J ¼ 5.1 Hz), 6.95e7.07 (m, 4H; ArH), 7.20e7.23 (m, 2H; ArH),
7.53e7.58 (m, 3H; ArH), position of the NH proton signal was not
established. IR(KBr): 1630 (CO), 3420 (NH). Anal. Calc. C26H28FN5O2,
(461.54): C: 67.66, H: 6.13, N: 15.17. Found: C: 68.00, H: 6.16, N: 15.07.
4d: from 3b and 1-(o-methoxyphenyl)piperazine. Yield 32%,
m.p. 244e247 ꢁC (ethanol).
3a: Yield 21%, CC: ethyl acetate, Rf ¼ 0.65, m.p. 213e215 ꢁC
(cyklohexane).
1H NMR: 2.34 (s, 6H, 5,7eCH3), 3.83 (t, 2H, CH2, J ¼ 6.6 Hz), 4.24
(t, 2H, CH2, J ¼ 6.6 Hz), 7.15e7.19 (m, 2H, ArH), 7.48e7.51 (m, 3H,
ArH). IR (KBr): 1635 (C]O). Anal. Calc. C16H16ClN3O2, (317.77): C:
60.47, H: 5.09, N: 13.22. Found: C: 60.12, H: 5.12, N: 12.82.
4.1.1.3. 1,2,3,4-tetrahydro-2-[2-(mesyloxy)ethyl]-5,7-dimethyl-6-
phenyl-6H-pyrrolo [3,4-d]pyridazine-1,4-dione 3b. A solution of 1 g
(3.3 mmol) of pyrrolopyridazinone 2b and 0.77 ml (10 mmol) meth-
anesulfonyl chloridein7 ml pyridinewasstirred atroom temperature
for 6 h. After stirring the reaction mixture was diluted with water
(20 ml). The precipitate was filtered off and crystallized to give 3b.
3b: Yield 79%, m.p. 151e153 ꢁC (ethanol).
1H NMR: 2.40 (s, 6H; 5,7eCH3), 2.41 (s, 3H; 7eCH3), 2.82e2.92
[m, 6H; CH2N(CH2)2], 3.36e3.37 [m, 4H; (CH2)2NAr], 4.10 (t, 2H;
NCH2, J ¼ 5.1 Hz), 6.95e7.07 (m, 4H; ArH), 7.21e7.25 (m, 2H; ArH),
7.53e7.59 (m, 3H; ArH), position of the NH proton signal was not
established. IR(KBr): 1615(CO), 3420 (NH). Anal. Calc. C27H31N5O3,
(473.57): C: 68.47, H: 6.61, N: 14,78. Found: C: 68.21, H: 6.63, N: 14.57.
4e: from 3b and 1-(m-trifluoromethylphenyl)piperazine. Yield
37%, CC [ethyl acetate: acetone (1:1), Rf ¼ 0.49], m.p. 220e223 ꢁC
(ethanol).
1H NMR: 2.33 (s, 3H; 5eCH3), 2.44 (s, 3H; 7eCH3), 3.51 (s, 3H;
CH3), 3.87 (t, 2H; CH2 J ¼ 6.3 Hz), 4.38 (t, 2H; CH2 J ¼ 6.3 Hz),
7.19e7.26 (m, 2H; ArH), 7.56e7.58 (m, 3H; ArH), position of the OH
proton signal was not established. IR (KBr): 1670 (C]O), 1370, 1180
(SO2), 3060 (OH). Anal. Calc. C17H19N3O5S, (377.41): C: 54.10, H:
5.08, N: 11.13. Found: C: 54.39, H: 5.12, N: 11.22.
1H NMR: 2.36 (s, 3H; 5eCH3), 2.40 (s, 3H; 7eCH3), 2.82e2.90 [m,
6H; CH2N(CH2)2], 3.36e3.37 [m, 4H; (CH2)2NAr], 4.10 (t, 2H; NCH2,
J ¼ 4.8 Hz), 7.05e7.37 (m, 6H; ArH), 7.53e7.55 (m, 3H; ArH), posi-
tion of the NH proton signal was not established. IR(KBr): 1635
(CO), 3440 (NH). Anal. Calc. C27H28F3N5O2, (511.54): C: 63.39, H:
5.53, N: 13.68. Found: C: 63.69, H: 5.68, N: 13.93.
4.1.1.4. 1,2,3,4-tetrahydro-2-[2-(4-phenylpiperazin-1-yl)ethyl]-5,7-
dimethyl-6-phenyl-6H-pyrrolo [3,4-d]pyridazine-1,4-dione 4a. A
solution of 3.18 g (10 mmol) 1,2,3,4-tetrahydro-2-(2-chloroethyl)-
5,7-dimethyl-6-phenyl-6H-pyrrolo[3,4-d]pyridazine-1,4-dione 3a
and 4.58 ml 1-phenylpiperazine in xylene was refluxed for 16 h.
Than the precipitated, crude product 4a was filtered off and crys-
tallized from ethanol.
4f: from 3b and 1-(5-chloro-2-methylphenyl)piperazine. Yield
46%, m.p. 240e241 ꢁC (ethanol).
1H NMR: 2.25 (s, 3H; AreCH3), 2.40 (s, 6H; 5,7eCH3), 2.81e2.90
[m, 6H; CH2N(CH2)2], 3.03e3.06 [m, 4H; (CH2)2NAr], 4.05e4.09 (m,
2H; NCH2), 6.95e7.22 (m, 5H; ArH), 7.53e7.56 (m, 3H; ArH), posi-
tion of the NH proton signal was not established. IR(KBr): 1635
(CO), 3450 (NH). Anal. Calc. C27H30ClN5O2, (492.02): C: 65.91, H:
6.16, N: 14.23. Found: C: 65.52, H: 6.30, N: 14.32.
4a: from 3a and 1-phenylpiperazine 6. Yield 16%, m.p.
218e220 ꢁC (ethanol).
1H NMR: 2.35 (s, 3H; 5eCH3), 2.42 (s, 3H; 7eCH3), 2.87e2.96 [m,
6H; CH2N(CH2)2], 3.22e3.26 [m, 4H; (CH2)2NAr], 4.14 (t, 2H; NeCH2,
J ¼ 5.1 Hz), 6.95e7.07 (m, 4H; ArH), 7.20e7.23 (m, 2H; ArH),
6.95e7.23 (m, 3H; ArH), position of the NH proton signal was not
established. IR(KBr): 1630(CO), 3440 (NH). Anal. Calc. C26H29N5O2,
(443.55): C:70.40, H:6.60, N: 15.78. Found: C:70.46, H:6.61, N:15.63.
4g: from 3b and 1-(2-pyridyl)piperazine. Yield 30%, CC acetone,
Rf ¼ 0.48, m.p. 239e242 ꢁC (ethanol).
1H NMR: 2.39 (s, 6H; 5,7eCH3), 2.71e2.78 [m, 4H; N(CH2)2],
2.82e2.89 (m, 2H; CH2), 3.65e3.73 [m, 4H; (CH2)2NAr], 4.05e4.11
(m, 2H; NeCH2), 6.64e6.66 (m, 2H; 2Hb), 7.19e7.21 (m, 2H; ArH),
7.49e7.55 (m, 4H; 3ArH þ Hg), 8.18e8.22 (m, 1H; Ha), position of
the NH proton signal was not established. IR(KBr): 1630 (CO), 3450
(NH). Anal. Calc. C25H28N6O2, (444.53): C: 67.54, H: 6.36, N: 18.89.
Found: C: 67.15, H: 6.21, N: 18.90.
4.1.1.5. General procedure for preparation of 1,2,3,4-tetrahydro-2-(2-
substituted-ethyl)-5,7-dimethyl-6-phenyl-6H-pyrrolo[3,4-d]pyr-
idazine-1,4-diones 4ae4k. A solution of 3.77 g (10 mmol) of 1,2,3,4-
tetrahydro-2-[2-(mesyloxy)ethyl]-4-hydroxy-5,7-dimethyl-6-
4h: from 3b and 4-benzylpiperazine. Yield 50%, m.p.
198e200 ꢁC, (ethanol).

4998
W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
1H NMR: 2.40 (s, 6H; 5,7eCH3), 2.63e2.82 [m, 10H; CH2N(CH2)4-
1H NMR: 1.37e1.50 (m, 2H; CH2), 2.29 (s, 3H; 5eCH3), 2.44 (s,
3H; 7eCH3), 2.59e2.90 [m, 6H; CH2N(CH2)2], 3.10e3.37 [m, 4H;
ArN(CH2)2], 3.59 (s, 3H; 2NCH3), 3.86 (s, 3H; ArOCH3), 4.33 (t, 2H;
4eOCH2 J ¼ 6.6 Hz), 7.30e7.58 (m, 9H; ArH). IR (KBr): 1660 (C]O).
Anal. Calc. C29H35N5O3, (501.62): C: 69.43, H: 7.05, N: 13.95. Found:
C: 69.06, H: 6.85, N: 13.60.
benzyl, 3.55 (s, 2H; CH2), 4.02 (t, 2H; NeCH2, J ¼ 4.8Hz), 6.91e7.32
(m, 7H; ArH), 7.49e7.52 (m, 3H; ArH), position of the NH proton
signal was not established. IR(KBr): 1635(CO), 3450 (NH). Anal.
Calc. C27H31N5O2, (457.57): C: 70.87, H: 6.84, N: 15.30. Found: C:
70.51, H: 6.96, N: 15.36.
4i: from 3b and 4-(piperonyl)piperazine. Yield 51%, m.p.
5c: from 2b and 1-o-methoxyphenyl-4-(3-chloropropyl)piper-
azine [17], Yield 15%, CC: [ethyl acetate: methanol (3:1), Rf ¼ 0.40],
m.p. 124e127 ꢁC (ethanol).
238e240 ꢁC, (ethanol).
1H NMR: 2.40 (s, 6H; 5,7eCH3), 2.52e2.74 [m, 6H; CH2N(CH2)2],
2.80 [m, 4H; (CH2)2NAr], 3.45 (s, 2H; CH2), 4.02 (t, 2H; NCH2,
J ¼ 4.8 Hz), 5.94 (s, 2H; OCH2O), 6.74 (s, 1H; ArH), 6.75 (s, 1H; ArH),
6.86 (s, 1H; ArH), 7.19e7.26 (m, 2H; ArH), 7.53e7.57 (m, 3H; ArH),
position of the NH proton signal was not established. IR(KBr): 1635
(CO). Anal. Calc. C28H31N5O4, (501.58): C: 67.04, H: 6.24, N: 13.96.
Found: C: 66.78, H: 6.06, N: 13.76.
4j: from 3b and 1-(diphenylmethyl)piperazine. Yield 37%, CC:
[etyl acetate: aceton (1:1) Rf ¼ 0.37], m p. 269e270 ꢁC (ethanol).
1H NMR: 2.39 (s, 6H; 5,7eCH3), 2.65e2.82 [m, 10H;
CH2N(CH2)4NAr], 4.07 (t, 2H; NCH2 J ¼ 4.8 Hz), 4.26 (s, 1H; CH),
7.17e7.21 (m, 6H; ArH), 7.33e7.37 (m, 8H; ArH), 7.42e7.44 (m, 4H;
ArH), position of the NH proton signal was not established. IR(KBr):
1630 (CO), 3420 (NH). Anal. Calc. C33H35N5O2, (533.67): C: 60.60, H:
5.05, N: 13,8. Found: C: 61.00, H: 5.12, N: 12.82.
1H NMR: 1.98e2.16 (m, 2H; CH2), 2.29 (s, 3H; 5eCH3), 2.44 (s, 3H;
7eCH3), 2.52e2.76 [m, 6H; CH2N(CH2)2], 2.96e3.17 [m, 4H;
ArN(CH2)2], 3.85 (s, 3H; ArOCH3), 3.91e4.06 (m, 2H; CH2), 4.12e4.33
(m, 4H; CH2CH2), 6.94e7.0 (m, 3H; ArH), 7.10e7.23 (m, 3H; ArH),
7.48e7.59 (m, 3H; ArH), position of the OH proton signal was not
established. IR (KBr): 1635 (CO), 3420 (OH). Anal. Calc. C30H37N5O4,
(531.65): C: 67.77, H: 7.03, N: 13.17. Found: C: 67.40, H: 6.68, N: 12.80.
4.2. Crystallography
4.2.1. X-ray structure determinations of 4c
X-ray data of 4c were collected on the Bruker SMART APEX II
CCD diffractometer; crystal sizes 0.27 ꢃ 0.06 ꢃ 0.03 mm, CuK
a
(l
¼ 1.54178 Å) radiation, and scans. The structure was solved
4
u
4k: from 3b and trans-1-cinnamylpiperazine. Yield 57%, m.p.
by direct methods using SHELXS97 [18] and refined by full-matrix
least-squares with SHELXL97 [18]. The N-bound H atom involved
in the intramolecular hydrogen bond was located by difference
Fourier synthesis and refined freely. The remaining H atoms were
positioned geometrically and treated as riding on their parent C
atoms with CeH distances of 0.93 Å (aromatic),0.97 Å (CH2) and
0.96 Å (CH3). All H atoms were refined with isotropic displacement
parameters taken as 1.5 times those of the respective parent atoms.
The relatively high values of Rint and final R parameters are caused
by the poor diffraction due to small linear sizes and unfavourable
shape of the crystals (thin colorless needles) obtained after crys-
tallization by slow evaporation of an ethanol solution. Only these
crystals were suitable for X-ray diffraction analysis. All calculations
were performed using WINGX version 1.64.05 package [19]. CCDC-
820010 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge at www.ccdc.cam.
ac.uk/conts/retrieving.html [or from the Cambridge Crystallo-
graphic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ,
UK; fax: þ44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk].
190e193 ꢁC (ethanol).
1H NMR: 2.38 (s, 6H; 5,7eCH3), 2.51e2.67 [m, 6H; CH2N(CH2)2],
2.81 [m, 4H; (CH2)2NAr], 3.18 (d, 2H; N-piperazine CH2, J ¼ 6.6 Hz),
4.03 (t, 2H; NCH2, J ¼ 4.8 Hz), 6.20e6.30 (m,1H; CH), 6.49(d,1H; CH,
J ¼ 15.6 Hz, trans), 7.18e7.38 (m, 7H; ArH), 7.47e7.56 (m, 3H; ArH),
position of the NH proton signal was not established. IR(KBr): 1630
(CO). Anal. Calc. C29H33N5O2, (483.61): C: 72.02, H: 6.89, N: 14.47.
Found: C: 71.63, H: 7.03, N: 14.60.
4l: from 3b and 4-benzylpiperidine. Yield 47%, m.p. 211e213 ꢁC
(ethanol).
1H NMR: 1.49e1.55 (m, 3H; 3050CHax i 40CH), 1.71 (d, 2H;
3050CHeq, J ¼ 11.7 Hz), 2.13 (t, 2H; 2060CHax, J ¼ 10.8 Hz), 2.40 (s, 6H;
5,7eCH3), 2.57 (d, 2H; 2060CHeq, J ¼ 6.3 Hz), 2.80 (t, 2H; CH2,
J ¼ 4.5 Hz), 3.09 (d, 2H; CH2, J ¼ 11.7 Hz) 4.03 (t, 2H; CH2, J ¼ 4.5 Hz),
7.14e7.30 (m, 6H; ArH), 7.49e7.58 (m, 4H; ArH), position of the NH
proton signal was not established. IR(KBr): 1645 (CO). Anal. Calc.,
C28H32N4O2, (456.58): C: 73.65, H: 7.08, N: 12.26. Found: C: 73.31,
H: 7.06, N: 11.96.
4.1.1.6. General procedure for preparation of 1,2-dihydro-2-
substituted-5,7-dimethyl-6-phenyl-4-[3-(4-aryl)piperazin-1-yl)pro-
poxy]-6H-pyrrolo[3,4-d]pyridazine-1-ones 5aec. To solution of
sodium ethoxide, prepared from 0.09 g of Na and 50 ml of anhy-
drous ethanol, 3.7 mmol of pyrrolopyridazinone 2a [11] or 2b and
3.7 mmol of corresponding 1-aryl-4-(3-chloropropyl)piperazine 6
were added. The solution was refluxed with stirring for 15 h, then it
was evaporated. The residue was dissolved in choroform, filtered
and the solvent was distilled off. The residue was purified by CC
with appropriate eluent.
4.2.1.1. Crystal data of 1. C26H28N5O2F, M ¼ 461.53, monoclinic,
space group P21/c, a ¼ 16.747(10), b ¼ 20.208(13), c ¼ 7.183(2) Å,
b
¼ 100.90(4)o, V ¼ 2387(2) Å3, Z ¼ 4, dcalc ¼ 1.284 Mg mꢀ3
,
F(000) ¼ 976,
m
(Cu K
a
) ¼ 0.725 mmꢀ1, T ¼ 293K, 14859 measured
reflections
(
q
range 2.69e65.04ꢁ), 3938 unique reflections
(Rint ¼ 0.154), final R ¼ 0.093, wR ¼ 0.202, S ¼ 1.013 for 1387
reflections with I > 2 (I).
s
4.2.2. Theoretical calculations
The theoretical calculations at the DFT/B3LYP level with
6e311þþG(d,p) basis set implemented in GAUSSIAN 03 [20] were
carried out to investigate the tautomeric equilibrium of 4c. The
structures of both tautomeric forms were fully optimized without
any constraint and the initial geometries were built from crystal-
lographic data of 4c.
5a: from 2a and 1-phenyl-4-(3-chloropropyl)piperazine [16].
Yield 17%, CC: ethyl acetate, Rf
(cyclohexane).
¼
0.35, m.p. 105e107 ꢁC
1H NMR: 2.03e2.08 (m, 2H; CH2), 2.30 (s, 3H; 5eCH3), 2.46 (s,
3H; 7eCH3), 2.59e2.68 [m, 6H; N(CH2)3], 3.21e3.25 [m, 4H;
ArN(CH2)2], 3.61 (s, 3H; 2eNCH3), 4.34 (t, 2H; OCH2 J ¼ 6.3 Hz),
6.84e7.19 (m, 3H; ArH), 7.31e7.28 (m, 4H; ArH), 7.52e7.53 (m, 3H;
ArH). IR (KBr): 1660 (C]O). Anal. Calc. C28H33N5O2, (471.60): C:
71.31, H: 7.07, N: 14.84. Found: C: 70.98, H: 6.81, N: 14.50.
4.3. Pharmacology
4.3.1. Materials
5b: from 2a and 1-o-methoxyphenyl-4-(3-chloropropyl)piper-
4.3.1.1. Compounds. Acetylsalicylic acid (Polopiryna, ZF Starogard
azine [17], Yield 32%, CC: ethyl acetate, Rf ¼ 0.40, m.p. 148e150 ꢁ
C
Gdanski, PL), morphine (Morphinum hydrochloricum, Polfa-Kutno,
ꢀ
(cyklohexane).
PL), phenylbenzoquinone (INC Pharmaceuticals, Inc.N.Y.).

W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
4999
4.3.1.2. Animals. The experiments were carried out on male
4.3.5. Radioligand binding assay [24]
Albino-Swiss mice (body weight 20e24 g). The animals were
housed in wire mesh cages in a room at 20 ꢂ 2 ꢁC and exposed to
a 12 h light: 12 h dark cycle. The animals had free access to standard
pellet diet, tap water was given ad libitum. All procedures were
performed according to the Animal Care and Use Committee
Guidelines, and approved by the Ethical Committee of Jagiellonian
University, Kraków.
Control and experimental groups consisted of 6e8 animals each.
The investigated compounds were administered intraperitoneally
(i.p.) as the suspension in 0.5% methylcellulose in constant volume
of 10 ml/kg.
The experiment was carried out on the rat cerebral cortex [3H]
dihydromorphine (69 Ci/mmol) was used. Rats brains were
homogenized in 20 volumes of ice-cold 50 mM TriseHCl buffer
(pH 7.6), and centrifuged at 48,000 ꢃ g for 15 min (0e4 ꢁC). The
cell pellet was resuspended in TriseHCl buffer and centrifuged
again. This homogenate was then incubated for 30 min at 37 ꢁC to
remove endogenous opiate peptides and centrifuged again as
before. Radioligand binding assays were performed in plates
(MultiScreen/Millipore). The final incubation mixture (final
volume 300
a [3H] dihydromorphine (0.2 nM) solution and 30
taining from seven to eight concentration (10ꢀ11e10ꢀ4 M) of
investigated compounds. For measuring unspecific binding 0.1 mM
m
l) consisted of 240
m
l membrane suspension, 30
ml of
l buffer con-
m
4.3.1.3. Statistical analysis. The obtained results were presented as
the means ꢂ SEM and evaluated statistically by using Student’s t-
test. Differences were considered significant when p < 0.05.
levallorphan was applied. The incubation was terminated by rapid
filtration over glass fiber filters (Whatman GF/C) using a vacuum
manifold (Millipore). The filters were then washed 2 times with
the assay buffer and placed in scintillation vials with liquid scin-
tillation cocktail. Radioactivity was measured in WALLAC 1409
DSA e liquid scintillation counter. All assays were done in
dupluicates.
4.3.2. Pain reactivity
4.3.2.1. ‘Hot plate’ test in mice according to Eddy and Leimbach
[21]. The animals were placed individually on the metal plate
heated to 54e56 ꢁC. The animals were placed on the hot plate and
the time (s) necessary to induce the licking reflex of the forepaws or
jumping was recorded by stop-watch. The latency was recorded
before and after 30 min following i.p. administration of the tested
compound. The prolongation of the latency times comparing the
values before and after administration of the test compounds was
used for statistical comparison. A cut of time was of 45 s was used to
prevent tissue damage, as reference compounds were used
morphine (in doses 1, 3 and 6 mg/kg i.p.) and acetylsalicylic acid (in
doses 400, 200 and 100 mg/kg i.p.)
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4.3.2.2. ‘Writhing syndrome’ test in mice according to Hendershot
and Forsaith [22]. Different doses of the tested compounds ranging
from 3.125 mg/kg to 50 mg/kg were administered i.p. Thirty
minutes later, 0.02% solution (ethanol-water, 5:95 v/v) of phenyl-
benzoquinone was injected intraperitoneally in a constant volume
of 0.25 ml. Five minutes after injection of the irritating agent, the
number of writhing episodes in the course of 10 min was counted.
The analgesic effect of individual doses was expressed in per cent:
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% analgesic effect ¼ 100 ꢀ (S of writhing incidents in experimental
group/S of writhing incidents in control group) ꢃ 100
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the method of Litchfield and Wilcoxon [23].
4.3.3. Locomotor activity
The spontaneous locomotor activity of a single mouse was
measured in photoresistor actometers (circular cages, 30 cm in
diameter, provided with two photocells, and connected to the
impulse counter), in 30-min sessions.
The investigated compounds were given i.p. at a dose equal to
ED50 value fixed in ‘hot plate’ test. Thirty minutes after the injection
of compounds mice were placed separately for 30 min in the
actometers which registered the numbers of movements of the
animals.
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G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara,
K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
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4.3.4. Acute toxicity according to Litchfield and Wilcoxon [23]
The investigated compounds were injected intraperitoneally in
increasing doses up to 2000 mg/kg. Each dose was given to six
animals. The number of dead mice was assessed 24 h after injec-
tion. LD50 (lethal dose in 50% of animals) were calculated according
to the method of Litchfield and Wilcoxon [23]. The general behavior
was observed for 6 h after injection of the tested compounds.
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