- Email: [email protected]
Derivatives of pyrrolo[3,4-d]pyridazinone, a new class of analgesic agents
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 Wies1aw Malinka a, Aleksandra Redzicka a, *, Magdalena Jastrze˛ bska e Wie˛ sek b, Barbara Filipek b, czyk-Lipkowska e, Przemys1aw Kalicki e Ma1gorzata Dyba1a c, Zbigniew Karczmarzyk d, Zofia Urban a
Department of Chemistry of Drugs, Wrocław Medical University,1 Tamka Str., Wrocław 50-137, Poland 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 b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 December 2010 Received in revised form 2 August 2011 Accepted 2 August 2011 Available online 9 August 2011
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 exhibited affinity for the m-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. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: pyrrolo[3,4-d]pyridazinones Analgesic activity
1. Introduction Two major classes of drugs dominate in treatment of analgesia: opioids which interact with specific central receptors (m, k, d) 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 molecules 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 (4arylpiperidine, b-carboline) analogues exhibited significant analgesic action in the phenylbenzoquinone-induced ‘writhing’ test. The most active compounds were 5-times more potent than ASA used as a standard [3].
* Corresponding author. Tel.: þ48 717840400. E-mail address: [email protected] (A. Redzicka). 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.08.006
Increasing of a pyrrolidinone ring to pyridazinone in a skeleton of a lead compound is a common strategy in medicinal chemistry [4]. In this context we have synthesized series of pyrrolo[3,4-d] pyridazinodiones of general structure 4 (Scheme 1). These 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 arylpiperazinylpropyl 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). 2. Chemistry In the previous paper [11] we reported the facile conversion of the 1-phenylpyrrole-3,4-dicarboxylic acid anhydride 1 into pyrrolopyridazinone 2a by reaction with N-methylhydrazine (Scheme 1) in relatively good yield (70%). Similarly, treatment of anhydride
W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
Fig. 1.
4993
1 with b-hydroxyethylhydrazine afforded the corresponding pyrrolopyridazinone 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 hydroxyethylpyrrolopyridazine 2b was treated with thionyl chloride with formation of 2chloroethyl 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
Scheme 1.
4994
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. Alternatively compound 2b was first mesylated with formation of ester 3b. Subsequent amination of the 3b using appropriate 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 chloropropylarylpiperazines 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 corresponding 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.
2.1. Crystal structure of pyrrolopyridazinone 4c 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 energetically 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 nondegenerate Boltzmann distribution is below the threshold of the detectability 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 piperazine 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[3,4d]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
H 3C
O
N N
N
F
H 3C
O N
NH H 3C
N N
N H 3C
O
a
OH
b Fig. 3. Tautomeric forms of compounds 4c.
molecular stacks in the Z direction (Fig. 2). The p.p distances between overlapping planes are alternately 3.613(4) and 3.612(4) Å and the angle between them is 3.81.
3. Results and discussion 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 intraperitoneal 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 pyrrolopyridazines 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 antiinflammatory 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. 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). From compounds assessed in this test only pyrrolopyridazine 4f showed some affinity to m receptors comparable with that of Tramadol [15]. On the basis of these data we can suggests that 4f present its own profile of analgesic activity and the part of its 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
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]. On the basis of the above pharmacological data we can conclude that replacement of the pyrolidinone ring in analgesic pyrrole-3,4dicarboximides (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 pyrrolopyridazinone 2 diminish analgesic activity (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 represent O-substitued partial analogues of series I. To the first series was introduced heteroaryl(pyridine) analogue 4g, whereas cynnamyl 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 pyridine (4g, ED50 ¼ 6.84 mg/kg). In conclusion, we described a new and interesting series of non toxic pyrrolopyridazinones 4 with potent analgesic activity 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.
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’ test
‘hot plate’ test
Ar 4a
1.48 (0.40e5.52)
inactive
4b
3.77 (2.04e6.95)
inactive
4c
0.31 (0.13e0.71)
14.99 (10.73e20.92)
4d
8.02 (6.57e9.78)
inactive
4e
1.23 (0.27e5.65)
15.91 (9.57e26.46)
4f
0.55 (0.14e2.17)
10.31 (5.50e19.34)
4g
6.84 (3.70e12.63)
inactive
4h
0.04 (0.001e1.49)
inactive
4i
1.92 (1.04e3.54)
inactive
&O
F
H 3CO I CF3
H 3C
Cl
N
X
n
N
1
N
1
R2
2
H3C
O N
N
II
H3C
H3C III
N
X
NH
R2 N
0
4j
1.83 (0.90e3.73)
inactive
N
1
4k
1.25 (0.79e1.99)
inactive
C
1
4l
1.91 (0.68e5.39)
inactive
5a
7.9 (5.1e12.7)
inactive
5b
11.15 (8.6e14.5)
inactive
39.15 (29.10e48.40) 2.44 (1.18e5.02)
inactive at dose of 200 mg/kg 3.5 (3.0e4.1)
O
O N
N H3C
2
R3
N O
N
R3 H H3COe
N
Acetylsalicylic acid Morphine
4. Experimental 4.1. Chemistry Table 2 The IC50 and Ki values for the inhibition of the binding of [3H] dihydromorphine to m receptor. Compound
4f 4c 4e Levellorphan Tramadol
[3H] DHM IC50 [mM]
Ki [mM]
5.6 0.1 24.2 0.7 e 634.1 pM
2.6 0.1 11.1 0.2 e 2.4 1.1 [15]
4.1.1. Chemical experimental section Melting points are uncorrected. The 1H NMR spectra recorded on Bruker 300 MHz spectrometer in CDCl3 using tetramethylsilane (TMS) as internal reference (chemical shift 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 within0.4% 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
W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
reaction was monitored by TLC on silica gel plates with fluorescent indicator (Fluka) and visualized by UV light at 254 nm. 4.1.1.1. 1,2,3,4-tetrahydro-2-(2-hydroxyethyl)-5,7-dimethyl-6phenyl-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-hydroxyethylhydrazine in 40 ml of acetonitrile was refluxed for 7 h. After cooling the product separated was filtered off and purified by crystallization to give 1.94 g of 2b. 2b: Yield 65%, m.p. 262e265 C (ethanol). 1 H 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. 4.1.1.2. 1,2,3,4-tetrahydro-2-(2-chloroethyl)-5,7-dimethyl-6-phenyl6H-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 fractions containing the product of Rf ¼ 0.65 were combined and evaporated to give 0.23 g of 3a. 3a: Yield 21%, CC: ethyl acetate, Rf ¼ 0.65, m.p. 213e215 C (cyklohexane). 1 H 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-6phenyl-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) methanesulfonyl chloride in 7 ml pyridine was stirred at room 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). 1 H 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. 4.1.1.4. 1,2,3,4-tetrahydro-2-[2-(4-phenylpiperazin-1-yl)ethyl]-5,7dimethyl-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 crystallized from ethanol. 4a: from 3a and 1-phenylpiperazine 6. Yield 16%, m.p. 218e220 C (ethanol). 1 H 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. 4.1.1.5. General procedure for preparation of 1,2,3,4-tetrahydro-2-(2substituted-ethyl)-5,7-dimethyl-6-phenyl-6H-pyrrolo[3,4-d]pyridazine-1,4-diones 4ae4k. A solution of 3.77 g (10 mmol) of 1,2,3,4tetrahydro-2-[2-(mesyloxy)ethyl]-4-hydroxy-5,7-dimethyl-6-
4997
phenylpyrrolo[3,4-d]pyridazine-1,4-dione and of corresponding amine (30 mmol) in xylene was refluxed for 16 h. Than the precipitated, crude product (4ae4d, 4f, h, k,l) was filtered off and recrystallized from the appropriate solvent. In the case of compounds 4e, 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). 1 H 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, J ¼ 5.1 Hz), 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). 1 H 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. 4c: from 3b and 1-(o-fluorophenyl)piperazine. Yield 69%, m.p. 246e248 C (ethanol). 1 H 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). 1 H 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). 1 H 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), position 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. 4f: from 3b and 1-(5-chloro-2-methylphenyl)piperazine. Yield 46%, m.p. 240e241 C (ethanol). 1 H 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), position 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. 4g: from 3b and 1-(2-pyridyl)piperazine. Yield 30%, CC acetone, Rf ¼ 0.48, m.p. 239e242 C (ethanol). 1 H 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. 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
1 H NMR: 2.40 (s, 6H; 5,7eCH3), 2.63e2.82 [m, 10H; CH2N(CH2)4benzyl, 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. 238e240 C, (ethanol). 1 H 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). 1 H 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. 4k: from 3b and trans-1-cinnamylpiperazine. Yield 57%, m.p. 190e193 C (ethanol). 1 H 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). 1 H NMR: 1.49e1.55 (m, 3H; 30 50 CHax i 40 CH), 1.71 (d, 2H; 30 50 CHeq, J ¼ 11.7 Hz), 2.13 (t, 2H; 20 60 CHax, J ¼ 10.8 Hz), 2.40 (s, 6H; 5,7eCH3), 2.57 (d, 2H; 20 60 CHeq, 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-2substituted-5,7-dimethyl-6-phenyl-4-[3-(4-aryl)piperazin-1-yl)propoxy]-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 anhydrous 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. 5a: from 2a and 1-phenyl-4-(3-chloropropyl)piperazine [16]. Yield 17%, CC: ethyl acetate, Rf ¼ 0.35, m.p. 105e107 C (cyclohexane). 1 H 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. 5b: from 2a and 1-o-methoxyphenyl-4-(3-chloropropyl)piperazine [17], Yield 32%, CC: ethyl acetate, Rf ¼ 0.40, m.p. 148e150 C (cyklohexane).
1 H 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. 5c: from 2b and 1-o-methoxyphenyl-4-(3-chloropropyl)piperazine [17], Yield 15%, CC: [ethyl acetate: methanol (3:1), Rf ¼ 0.40], m.p. 124e127 C (ethanol). 1 H 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 0.06 0.03 mm, CuKa (l ¼ 1.54178 Å) radiation, 4 and u scans. The structure was solved 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 crystallization 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]. CCDC820010 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 Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: þ44(0) 1223 336 033; email: [email protected]]. 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 m3, F(000) ¼ 976, m(Cu Ka) ¼ 0.725 mm1, T ¼ 293K, 14859 measured reflections (q range 2.69e65.04 ), 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 > 2s(I). 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 crystallographic data of 4c. 4.3. Pharmacology 4.3.1. Materials 4.3.1.1. Compounds. Acetylsalicylic acid (Polopiryna, ZF Starogard ski, PL), morphine (Morphinum hydrochloricum, Polfa-Kutno, Gdan PL), phenylbenzoquinone (INC Pharmaceuticals, Inc.N.Y.).
W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
4.3.1.2. Animals. The experiments were carried out on male Albino-Swiss mice (body weight 20e24 g). The animals were housed in wire mesh cages in a room at 20 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. 4.3.1.3. Statistical analysis. The obtained results were presented as the means SEM and evaluated statistically by using Student’s ttest. Differences were considered significant when p < 0.05. 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.) 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 phenylbenzoquinone 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: % analgesic effect ¼ 100 (S of writhing incidents in experimental group/S of writhing incidents in control group) 100 The ED50 values and their confidence limits were estimated by 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. 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 injection. 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.
4999
4.3.5. Radioligand binding assay [24] 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 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 ml) consisted of 240 ml membrane suspension, 30 ml of a [3H] dihydromorphine (0.2 nM) solution and 30 ml buffer containing from seven to eight concentration (1011e104 M) of investigated compounds. For measuring unspecific binding 0.1 mM 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 scintillation cocktail. Radioactivity was measured in WALLAC 1409 DSA e liquid scintillation counter. All assays were done in dupluicates. References [1] K.W. Shyu, M.T. Lin, J. Neurol. Trans. 62 (1985) 285e293. [2] J.P. Collins, J.P. Chessel, Exp. Opin. Emerging Drugs 10 (2005) 95e1078. [3] W. Malinka, M. Kaczmarz, A. Redzicka, B. Filipek, J. Sapa, Farmaco 60 (2005) 15e22. [4] G. Patrick, Instant Notes: Medicinal Chemistry. BIOS Scientific Publishers Limited, 2001. [5] C. Biancalani, M.P. Giovannoni, S. Pieretti, N. Cesari, A. Graziano, C. Vergelli, A. Cilibrizzi, A. Di Gianuario, M. Colucci, G. Mangano, B. Garrone, L. Poenzani, V. Dal Piaz, J. Med. Chem. 52 (2009) 7397e7409. [6] N. Cesari, C. Biancalani, C. Vergelli, V. Dal Piaz, A. Graziano, P. Biagini, C. Ghelardini, N. Galeotti, M.P. Giovannoni, J. Med. Chem 49 (2006) 7826e7835. [7] M. Gokce, G. Bakir, M.F. Sahin, E. Kupeli, E. Yesilada, Arzneim.-Forsch./Drug Res. 55 (2005) 318e325. [8] M.P. Giovannoni, C. Vergelli, C. Ghelardini, N. Galeotti, A. Bartolini, J. Med. Chem. 46 (2003) 1055e1059. [9] M. Gokce, D. Dogruer, M.F. Sahin, Farmaco 56 (2001) 233e237. [10] D.S. Dogruer, M.F. Sahin, S. Unlu, S. Ito, Arch. Pharm. Pharm. Med. Chem. 333 (2000) 79e86. [11] W. Malinka, Pharmazie 56 (2001) 384e389. [12] F.H. Allen, O. Kennard, D.G. Watson, L. Brammer, A.G. Orpen, R. Taylor, J. Chem. Soc. Perkin Trans. 2 (1987) 1e19. [13] D. Cremer, J.A. Pople, J. Am. Chem. Soc. 97 (1975) 1354e1358. [14] H.G. Vogel, W.H. Vogel, Drug Discovery and Evaluation, In Pharmacological Assays. Chapter H. Analgesic, Antiinflammatory, and Antipyretic Activity. Springer, Berlin, Heidelberg, 1997, 360e420. [15] K. Minami, Y. Uezono, Y. Ueta, J. Pharmacol. Sci. 103 (2007) 253e260. [16] C. Pollard, W. Lauter, N. Nuessle, J. Org. Chem. 24 (1959) 764e767. [17] W. Malinka, M. Karczmarz, B. Filipek, J. Sapa, B. Glod, Farmaco 57 (2002) 737e746. [18] G.M. Sheldrick, Acta Cryst A64 (2008) 112e122. [19] L.J. Farrugia, J. Appl. Cryst 32 (1999) 837e838. [20] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, 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, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. AlLaham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision E.01. Gaussian, Inc., Wallingford CT, 2004. [21] N.B. Eddy, D. Leimbach, J. Phamacol Exp. Ther. 107 (1953) 385e393. [22] L.C. Hendershot, J. Forsaith, J. Pharmacol. Exp. Ther. 125 (1959) 237e240. [23] J.T. Litchfield, E. Wilcoxon, J. Pharmacol. Exp. Ther. 96 (1949) 99e113. [24] S.R. Childers, I. Creese, A.M. Snowman, S.H. Snyder, Eur. J. Pharmacol. 55 (1979) 11e18.
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 Wies1aw Malinka a, Aleksandra Redzicka a, *, Magdalena Jastrze˛ bska e Wie˛ sek b, Barbara Filipek b, czyk-Lipkowska e, Przemys1aw Kalicki e Ma1gorzata Dyba1a c, Zbigniew Karczmarzyk d, Zofia Urban a
Department of Chemistry of Drugs, Wrocław Medical University,1 Tamka Str., Wrocław 50-137, Poland 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 b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 December 2010 Received in revised form 2 August 2011 Accepted 2 August 2011 Available online 9 August 2011
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 exhibited affinity for the m-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. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: pyrrolo[3,4-d]pyridazinones Analgesic activity
1. Introduction Two major classes of drugs dominate in treatment of analgesia: opioids which interact with specific central receptors (m, k, d) 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 molecules 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 (4arylpiperidine, b-carboline) analogues exhibited significant analgesic action in the phenylbenzoquinone-induced ‘writhing’ test. The most active compounds were 5-times more potent than ASA used as a standard [3].
* Corresponding author. Tel.: þ48 717840400. E-mail address: [email protected] (A. Redzicka). 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.08.006
Increasing of a pyrrolidinone ring to pyridazinone in a skeleton of a lead compound is a common strategy in medicinal chemistry [4]. In this context we have synthesized series of pyrrolo[3,4-d] pyridazinodiones of general structure 4 (Scheme 1). These 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 arylpiperazinylpropyl 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). 2. Chemistry In the previous paper [11] we reported the facile conversion of the 1-phenylpyrrole-3,4-dicarboxylic acid anhydride 1 into pyrrolopyridazinone 2a by reaction with N-methylhydrazine (Scheme 1) in relatively good yield (70%). Similarly, treatment of anhydride
W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
Fig. 1.
4993
1 with b-hydroxyethylhydrazine afforded the corresponding pyrrolopyridazinone 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 hydroxyethylpyrrolopyridazine 2b was treated with thionyl chloride with formation of 2chloroethyl 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
Scheme 1.
4994
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. Alternatively compound 2b was first mesylated with formation of ester 3b. Subsequent amination of the 3b using appropriate 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 chloropropylarylpiperazines 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 corresponding 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.
2.1. Crystal structure of pyrrolopyridazinone 4c 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 energetically 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 nondegenerate Boltzmann distribution is below the threshold of the detectability 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 piperazine 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[3,4d]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
H 3C
O
N N
N
F
H 3C
O N
NH H 3C
N N
N H 3C
O
a
OH
b Fig. 3. Tautomeric forms of compounds 4c.
molecular stacks in the Z direction (Fig. 2). The p.p distances between overlapping planes are alternately 3.613(4) and 3.612(4) Å and the angle between them is 3.81.
3. Results and discussion 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 intraperitoneal 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 pyrrolopyridazines 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 antiinflammatory 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. 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). From compounds assessed in this test only pyrrolopyridazine 4f showed some affinity to m receptors comparable with that of Tramadol [15]. On the basis of these data we can suggests that 4f present its own profile of analgesic activity and the part of its 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
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]. On the basis of the above pharmacological data we can conclude that replacement of the pyrolidinone ring in analgesic pyrrole-3,4dicarboximides (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 pyrrolopyridazinone 2 diminish analgesic activity (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 represent O-substitued partial analogues of series I. To the first series was introduced heteroaryl(pyridine) analogue 4g, whereas cynnamyl 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 pyridine (4g, ED50 ¼ 6.84 mg/kg). In conclusion, we described a new and interesting series of non toxic pyrrolopyridazinones 4 with potent analgesic activity 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.
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’ test
‘hot plate’ test
Ar 4a
1.48 (0.40e5.52)
inactive
4b
3.77 (2.04e6.95)
inactive
4c
0.31 (0.13e0.71)
14.99 (10.73e20.92)
4d
8.02 (6.57e9.78)
inactive
4e
1.23 (0.27e5.65)
15.91 (9.57e26.46)
4f
0.55 (0.14e2.17)
10.31 (5.50e19.34)
4g
6.84 (3.70e12.63)
inactive
4h
0.04 (0.001e1.49)
inactive
4i
1.92 (1.04e3.54)
inactive
&O
F
H 3CO I CF3
H 3C
Cl
N
X
n
N
1
N
1
R2
2
H3C
O N
N
II
H3C
H3C III
N
X
NH
R2 N
0
4j
1.83 (0.90e3.73)
inactive
N
1
4k
1.25 (0.79e1.99)
inactive
C
1
4l
1.91 (0.68e5.39)
inactive
5a
7.9 (5.1e12.7)
inactive
5b
11.15 (8.6e14.5)
inactive
39.15 (29.10e48.40) 2.44 (1.18e5.02)
inactive at dose of 200 mg/kg 3.5 (3.0e4.1)
O
O N
N H3C
2
R3
N O
N
R3 H H3COe
N
Acetylsalicylic acid Morphine
4. Experimental 4.1. Chemistry Table 2 The IC50 and Ki values for the inhibition of the binding of [3H] dihydromorphine to m receptor. Compound
4f 4c 4e Levellorphan Tramadol
[3H] DHM IC50 [mM]
Ki [mM]
5.6 0.1 24.2 0.7 e 634.1 pM
2.6 0.1 11.1 0.2 e 2.4 1.1 [15]
4.1.1. Chemical experimental section Melting points are uncorrected. The 1H NMR spectra recorded on Bruker 300 MHz spectrometer in CDCl3 using tetramethylsilane (TMS) as internal reference (chemical shift 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 within0.4% 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
W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
reaction was monitored by TLC on silica gel plates with fluorescent indicator (Fluka) and visualized by UV light at 254 nm. 4.1.1.1. 1,2,3,4-tetrahydro-2-(2-hydroxyethyl)-5,7-dimethyl-6phenyl-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-hydroxyethylhydrazine in 40 ml of acetonitrile was refluxed for 7 h. After cooling the product separated was filtered off and purified by crystallization to give 1.94 g of 2b. 2b: Yield 65%, m.p. 262e265 C (ethanol). 1 H 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. 4.1.1.2. 1,2,3,4-tetrahydro-2-(2-chloroethyl)-5,7-dimethyl-6-phenyl6H-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 fractions containing the product of Rf ¼ 0.65 were combined and evaporated to give 0.23 g of 3a. 3a: Yield 21%, CC: ethyl acetate, Rf ¼ 0.65, m.p. 213e215 C (cyklohexane). 1 H 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-6phenyl-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) methanesulfonyl chloride in 7 ml pyridine was stirred at room 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). 1 H 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. 4.1.1.4. 1,2,3,4-tetrahydro-2-[2-(4-phenylpiperazin-1-yl)ethyl]-5,7dimethyl-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 crystallized from ethanol. 4a: from 3a and 1-phenylpiperazine 6. Yield 16%, m.p. 218e220 C (ethanol). 1 H 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. 4.1.1.5. General procedure for preparation of 1,2,3,4-tetrahydro-2-(2substituted-ethyl)-5,7-dimethyl-6-phenyl-6H-pyrrolo[3,4-d]pyridazine-1,4-diones 4ae4k. A solution of 3.77 g (10 mmol) of 1,2,3,4tetrahydro-2-[2-(mesyloxy)ethyl]-4-hydroxy-5,7-dimethyl-6-
4997
phenylpyrrolo[3,4-d]pyridazine-1,4-dione and of corresponding amine (30 mmol) in xylene was refluxed for 16 h. Than the precipitated, crude product (4ae4d, 4f, h, k,l) was filtered off and recrystallized from the appropriate solvent. In the case of compounds 4e, 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). 1 H 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, J ¼ 5.1 Hz), 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). 1 H 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. 4c: from 3b and 1-(o-fluorophenyl)piperazine. Yield 69%, m.p. 246e248 C (ethanol). 1 H 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). 1 H 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). 1 H 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), position 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. 4f: from 3b and 1-(5-chloro-2-methylphenyl)piperazine. Yield 46%, m.p. 240e241 C (ethanol). 1 H 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), position 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. 4g: from 3b and 1-(2-pyridyl)piperazine. Yield 30%, CC acetone, Rf ¼ 0.48, m.p. 239e242 C (ethanol). 1 H 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. 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
1 H NMR: 2.40 (s, 6H; 5,7eCH3), 2.63e2.82 [m, 10H; CH2N(CH2)4benzyl, 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. 238e240 C, (ethanol). 1 H 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). 1 H 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. 4k: from 3b and trans-1-cinnamylpiperazine. Yield 57%, m.p. 190e193 C (ethanol). 1 H 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). 1 H NMR: 1.49e1.55 (m, 3H; 30 50 CHax i 40 CH), 1.71 (d, 2H; 30 50 CHeq, J ¼ 11.7 Hz), 2.13 (t, 2H; 20 60 CHax, J ¼ 10.8 Hz), 2.40 (s, 6H; 5,7eCH3), 2.57 (d, 2H; 20 60 CHeq, 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-2substituted-5,7-dimethyl-6-phenyl-4-[3-(4-aryl)piperazin-1-yl)propoxy]-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 anhydrous 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. 5a: from 2a and 1-phenyl-4-(3-chloropropyl)piperazine [16]. Yield 17%, CC: ethyl acetate, Rf ¼ 0.35, m.p. 105e107 C (cyclohexane). 1 H 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. 5b: from 2a and 1-o-methoxyphenyl-4-(3-chloropropyl)piperazine [17], Yield 32%, CC: ethyl acetate, Rf ¼ 0.40, m.p. 148e150 C (cyklohexane).
1 H 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. 5c: from 2b and 1-o-methoxyphenyl-4-(3-chloropropyl)piperazine [17], Yield 15%, CC: [ethyl acetate: methanol (3:1), Rf ¼ 0.40], m.p. 124e127 C (ethanol). 1 H 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 0.06 0.03 mm, CuKa (l ¼ 1.54178 Å) radiation, 4 and u scans. The structure was solved 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 crystallization 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]. CCDC820010 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 Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: þ44(0) 1223 336 033; email: [email protected]]. 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 m3, F(000) ¼ 976, m(Cu Ka) ¼ 0.725 mm1, T ¼ 293K, 14859 measured reflections (q range 2.69e65.04 ), 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 > 2s(I). 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 crystallographic data of 4c. 4.3. Pharmacology 4.3.1. Materials 4.3.1.1. Compounds. Acetylsalicylic acid (Polopiryna, ZF Starogard ski, PL), morphine (Morphinum hydrochloricum, Polfa-Kutno, Gdan PL), phenylbenzoquinone (INC Pharmaceuticals, Inc.N.Y.).
W. Malinka et al. / European Journal of Medicinal Chemistry 46 (2011) 4992e4999
4.3.1.2. Animals. The experiments were carried out on male Albino-Swiss mice (body weight 20e24 g). The animals were housed in wire mesh cages in a room at 20 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. 4.3.1.3. Statistical analysis. The obtained results were presented as the means SEM and evaluated statistically by using Student’s ttest. Differences were considered significant when p < 0.05. 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.) 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 phenylbenzoquinone 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: % analgesic effect ¼ 100 (S of writhing incidents in experimental group/S of writhing incidents in control group) 100 The ED50 values and their confidence limits were estimated by 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. 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 injection. 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.
4999
4.3.5. Radioligand binding assay [24] 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 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 ml) consisted of 240 ml membrane suspension, 30 ml of a [3H] dihydromorphine (0.2 nM) solution and 30 ml buffer containing from seven to eight concentration (1011e104 M) of investigated compounds. For measuring unspecific binding 0.1 mM 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 scintillation cocktail. Radioactivity was measured in WALLAC 1409 DSA e liquid scintillation counter. All assays were done in dupluicates. References [1] K.W. Shyu, M.T. Lin, J. Neurol. Trans. 62 (1985) 285e293. [2] J.P. Collins, J.P. Chessel, Exp. Opin. Emerging Drugs 10 (2005) 95e1078. [3] W. Malinka, M. Kaczmarz, A. Redzicka, B. Filipek, J. Sapa, Farmaco 60 (2005) 15e22. [4] G. Patrick, Instant Notes: Medicinal Chemistry. BIOS Scientific Publishers Limited, 2001. [5] C. Biancalani, M.P. Giovannoni, S. Pieretti, N. Cesari, A. Graziano, C. Vergelli, A. Cilibrizzi, A. Di Gianuario, M. Colucci, G. Mangano, B. Garrone, L. Poenzani, V. Dal Piaz, J. Med. Chem. 52 (2009) 7397e7409. [6] N. Cesari, C. Biancalani, C. Vergelli, V. Dal Piaz, A. Graziano, P. Biagini, C. Ghelardini, N. Galeotti, M.P. Giovannoni, J. Med. Chem 49 (2006) 7826e7835. [7] M. Gokce, G. Bakir, M.F. Sahin, E. Kupeli, E. Yesilada, Arzneim.-Forsch./Drug Res. 55 (2005) 318e325. [8] M.P. Giovannoni, C. Vergelli, C. Ghelardini, N. Galeotti, A. Bartolini, J. Med. Chem. 46 (2003) 1055e1059. [9] M. Gokce, D. Dogruer, M.F. Sahin, Farmaco 56 (2001) 233e237. [10] D.S. Dogruer, M.F. Sahin, S. Unlu, S. Ito, Arch. Pharm. Pharm. Med. Chem. 333 (2000) 79e86. [11] W. Malinka, Pharmazie 56 (2001) 384e389. [12] F.H. Allen, O. Kennard, D.G. Watson, L. Brammer, A.G. Orpen, R. Taylor, J. Chem. Soc. Perkin Trans. 2 (1987) 1e19. [13] D. Cremer, J.A. Pople, J. Am. Chem. Soc. 97 (1975) 1354e1358. [14] H.G. Vogel, W.H. Vogel, Drug Discovery and Evaluation, In Pharmacological Assays. Chapter H. Analgesic, Antiinflammatory, and Antipyretic Activity. Springer, Berlin, Heidelberg, 1997, 360e420. [15] K. Minami, Y. Uezono, Y. Ueta, J. Pharmacol. Sci. 103 (2007) 253e260. [16] C. Pollard, W. Lauter, N. Nuessle, J. Org. Chem. 24 (1959) 764e767. [17] W. Malinka, M. Karczmarz, B. Filipek, J. Sapa, B. Glod, Farmaco 57 (2002) 737e746. [18] G.M. Sheldrick, Acta Cryst A64 (2008) 112e122. [19] L.J. Farrugia, J. Appl. Cryst 32 (1999) 837e838. [20] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, 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, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. AlLaham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision E.01. Gaussian, Inc., Wallingford CT, 2004. [21] N.B. Eddy, D. Leimbach, J. Phamacol Exp. Ther. 107 (1953) 385e393. [22] L.C. Hendershot, J. Forsaith, J. Pharmacol. Exp. Ther. 125 (1959) 237e240. [23] J.T. Litchfield, E. Wilcoxon, J. Pharmacol. Exp. Ther. 96 (1949) 99e113. [24] S.R. Childers, I. Creese, A.M. Snowman, S.H. Snyder, Eur. J. Pharmacol. 55 (1979) 11e18.