Scientific paper
New Py razolo [ 1',5':1,6] py rimido [4,5-d] py ridazin-4 (3H)-ones Fluoroderivatives as Human A1 Adenosine
Receptor Ligands
Alessia Graziano,1 Maria Paola Giovannoni,1* Agostino Cilibrizzi,1 Letizia Crocetti,1 Vittorio Dal Piaz,1 Claudia Vergelli,1 Maria Letizia Trincavelli,2
Claudia Martini2 and Chiara Giacomelli2
1 Dipartimento di Scienze Farmaceutiche, via Ugo Schiff6, 50019 Sesto Fiorentino Firenze, Italy
2 Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Via Bonanno 6,
56126 Pisa, Université di Pisa
* Corresponding author: E-mail: mariapaola.giovannoni@unifi.it Tel +39-055-4573682; Fax +39-055-4573780
Received: 10-01-2012
Abstract
In this paper we report the synthesis and biological evaluation of a new series of pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones as human Aj adenosine receptor ligands. The tricyclic scaffold was modified at position 6 and 9 by introducing small alkyl chains and substituted phenyls. The most interesting compounds showed Ki for Aj in the submicromolar range (0.105-0.244 |M) and the most interesting term (compound 4c) combined an appreciable affinity for Aj (Ki = 0.132 |M) with a good selectivity toward A2A (43% inhibition at 10 |M) and A3 (46% inhibition at 10 |M).
Keywords: Adenosine receptors, A1 subtype, Ligands, Pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones.
1. Introduction
For several years, adenosine receptors have been classified in four different subtypes, Ap A2A, A2B and A3\ All these receptors have been cloned from several species and have been demonstrated to differ in their primary sequence, pharmacological effects, tissue distribution and coupling to different G proteins. A1 and A3 subtypes, other that modulate calcium levels through a Gq proteins, are coupled to Gi proteins to inhibit adenyl cyclase. On the contrary, A2A and A2B receptors are primary coupled to Gs proteins and activate adenylyl cyclase causing, in turn, an increase in intracellular cAMP production.2,3 The endogenous ligand, adenosine, interacts with all the receptor subtypes with different affinity and may elicit different effects at level of second messengers. The final effect induced by adenosine may differ in physiological and pathological conditions during which the expression levels of each subtypes are regulated and obviously these effects depend on the relative abundance of each receptor subtypes in specific tissues.
Ligand-binding properties of each adenosine receptor are primarily dictated by amino acids in the transmembrane domains of the receptors. Studies have identified certain amino acids conserved amongst adenosine receptor subtypes that are critical for ligand recognition, as well as additional residues that may differentiate between agonist and antagonist ligands and between the different receptor subtypes.4
The potential therapeutic applications of compounds able to bind these receptors have been investigated in recent years.5,6 In particular antagonists for the A1 receptor subtype may be useful for the treatment of central nervous system pathologies such as Alzheimer's and Parkinson's diseases,7 for the treatment of congestive heart failure due to their diuretic and positive inotropic effects2,8 and for the treatment of asthma since adenosine mediates broncho-constriction and inflammation in the lung.9,10
A large number of nitrogen-containing polyheterocy-cles as pyrido[2,3-d]pyrimidinediones11 (A), pyrimido[4,5-b]indole (B),12 and triazinobenzimidazolones (C)13 have been reported in literature as A1R antagonists (Figure 1).
In a recent paper14 we reported the synthesis and binding activities at human cloned A2A, A2B and A3 adenosine receptors of a new series of compounds with pyrazo-lo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones scaffold (compound D, Figure 1). Some of these compounds showed a good activity for hA1 adenosine subtypes, with values of Ki in the submicromolar range and a good selectivity versus other adenosine receptor subtypes14.
Selecting as lead the fluoroderivative 4a (Ki = 0.252 pM) from the previous series, we report here the results of further modifications on the above scaffold at the level of positions 6 and 9 by maintaining the benzyl group unchanged at 3 necessary for A1 affinity and 4-F-phenyl at position 1.
pound 5 was obtained starting from 4c by reduction of the NO2 group with SnCl2.
Scheme 2 depicts the synthesis of the final compound 10 which was obtained starting from the previously described14 pyrazolopyrimido[4,5-d]pyridazinone 8, through transformation into the corresponding 4-thione derivative 9 with Lawesson's reagent followed by alkyla-tion at position 4 with benzyl chloride in standard conditions.
2. 2. Pharmacology
All the final compounds were investigated in radioli-gand binding studies to determine their affinities for human
Figure 1: Aj receptor antagonists
2. Results and Discussion 2. 1. Chemistry
The final compounds 4-7 (4a14) were prepared following a general synthetic procedure previously described by us15 (Scheme 1).
Isoxazolo[3,4-d]-pyridazin-7(6H)-one 114 was condensed with the appropriate arylaldehydes (or N,N-di-methylformammide dimethyl acetal for compound 2g) to give the vinyl derivatives 2a-g (2a14) which treated with hydrazine hydrate furnished intermediates 3a-g (3a14) through the isoxazole opening and the following closure to pyrazole16. Finally the cyclization to pyrazo-lo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-one was carried out in different conditions depending on the substituent at position 6.
For the 6-unsubstituted 4a-f, the ring closure was performed with triethylorthoformate in anhydrous DMF in the presence of catalytic amount of concentrated sulfu-ric acid at room temperature, whereas compounds 6a-c were obtained with the opportune anhydride under reflu-xing conditions. Compound 7 was synthesized starting from 4-amino-5-pyrazolyl derivative 3a14 for treatment with levulinic acid and in the presence of 4-(dimethylami-no)pyridine and of 1-[3-(dimethylamino)propyl]-ethylcar-bodiimide hydrochloride as coupling agent. Finally, com-
Ap A2A and A3 receptors. The biological results are reported in Table 1 together with the values of affinity of our lead compound 4a14. Analysis of compounds 4b and 4d in which we modified the position 9 through introduction of a Cl or an OCH3 group in para of the phenyl ring led to products with A1 affinity and selectivity comparable to that of lead 4a (Ki = 0.244 pM and 0.233 pM for 4b and 4d respectively). Introduction of a NO2 or a NH2 group in the same position afforded compounds 4c and 5 which had higher affinity (Ki = 0.132 pM for compound 4c and Ki = 0.105 for 5), but there was loss of selectivity (Ki = 0.116 pM for A2A) for compound 5.
The replacement of the phenyl at C-9 of 4a with a 3-furyl (4e) or with a 3-pyridyl (4f) nucleus was associated with maintenance of selectivity but with of decrease in affinity for the A1 subtype (Ki = 0.886 and 0.750 pM respectively), while elimination of the phenyl group (compound 4g) resulted in a loss of potency of one order of magnitude (Ki = 2.48 pM) compared to 4a.
The introduction at position 6 of short alkyl chains (compound 6a-c) or of a functionalized alkyl chain (compound 7) led to inactive (6b,c and 7) or poorly active (6a, Ki = 0.811) compounds, suggesting that position 6 of system has to remain unsubstituted.
Finally compound 10, in which the benzyl group was shifted from N-3 of pyridazinone to the neighboring 4-position, showed less activity compared to the lead 4a,
NH,
2-4	R
a14	Ph
b	4-Cl-C6H4
c	4-N02-C6H4
d	4-OCHrC<,H4
e	3-Fury 1
f	3-Pyridyl
g	Aa
6	R,
a	/-C3H7
b	»-C3H7
c	n-C4H9
■K	XiClh). :r.|-2;Ui	il lOI'JamkiJi)	c^C,,, DMAP, DE.
Scheme 1. Synthesis of pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-one 4a-f, 5, 6a-c and 7
Regent and conditions: (a) R1CHO, CH3ONa, CH3OH reflux, 1-10 min.; for compound 2g: DMDA, 110 °C, 90 min.; (b) NH2NH2 • H2O, EtOH, r.t. -50 °C, 1-3 h; (c) CH(OC2H5)3, H2SO4 conc., DMF, r.t., 30 min.; (d) SnCl2, 37% HCl, r.t., 20 h; (e4 (RjCO)2O, H2SO4 conc., r.t., 10 min.; (f) levulinic acid, DMF,
confirming the results previously reported14 about the essential role played by carbonyl function at position 4.
3. Conclusions
In conclusion, the majority of the new tricyclic derivatives shows affinities for A1 subtypes in submicromolar concentration range (0.105-0.886) and good selectivity versus other adenosine subtypes. As regards the modifications at position 9, the most active compounds, 4c and 5, bearing a 4-NO2-Ph and a 4-NH2-Ph group respectively, are about one-fold more potent than the lead 4a, while the
worst is the 9-unsubstituted 4g. On the other hand the introduction of substituents at C-6 of the tricyclic scaffold led from low active to inactive compounds. Further studies are in progress in order to improve the potency and selectivity of these subtype ligands.
4. Experimental Section
All melting points were determined on a Buchi apparatus and are uncorrected. 1H-NMR spectra were recorded with an Avance 400 instruments (Bruker Biospin Version 002 with SGU). Chemical shifts are reported in ppm,
Regent and conditions: (a) Lawesson's reagent, toluene, 110 °C, 10 h (b) PhCH2Cl, K2CO3, anhydrous DMF, 110 °C, 1 h.
Scheme 2. Synthesis of 4-(benzylthio)pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazine 10
Table 1. Binding activity at human A1, A2a and A3 adenosine receptors
4;i-f, 5, 6a-c, 7	10
Comp.	R	R1	hA1a'b	hA2A a c	hA^
4a[14]	Ph	H	0.252±0.060	45%	6%
4b	4-Cl-Ph	H	0.244±0.024	25%	30%
4c	4-NO2-Ph	H	0.132±0.013	43%	46%
4d	4-OCH3-Ph	H	0.233±0.007	50%	48%
4e	3-Furyl	H	0.886±0.085	56%	7%
4f	3-Pyridyl	H	0.750±0.055	46%	5%
4g	H	H	2.480±0.240	30%	39%
5	4-NH2-Ph	H	0.105±0.010	0.116±0.033	39%
6a	Ph	Ï-C3H7	0.811±0.079	7%	36%
6b	Ph	n-C3H7	50%	25%	39%
6c	Ph	n-C4H9	49%	8%	37%
7	Ph	-(CH2)2COCH3	45%	9%	50%
10			1.744±0.165	20%	30%
a The binding activity is reported as Ki (|M) or percentage of inhibition at 10 |M; values are means ± DS of four separate assay, each performed in triplicate. b Displacement of [3H]DPCPX binding in CHO-Aj cells membranes. c Displacement of [3H]NECA binding in A2a CHO cells membranes. d Displacement of [125I]AB-MECA binding in A3 CHO cells membranes.
using the solvent as internal standard. Extracts were dried for analytical TLC to follow the course of reaction. Silica over Na2SO4, and the solvents were removed under redu- gel 60 (Merck 70-230 mesh) was used for column chro-ced pressure. Merck F-254 commercial plates were used matography. Microanalyses were performed with a Per-
kin-Elmer 260 elemental analyzer for C, H, and N, and the results were within ±0.4% of the theoretical values, unless otherwise stated. Reagents and starting materials were commercially available.
4. 1. Preparation of Compounds
General Procedures for 2b-f
A mixture of compound 114 (0.6 mmol), the appropriate arylaldehyde (1.5 mmol) and CH3ONa (1-2 mmol) in anhydrous methanol (1-2 mL) was refluxed under stirring for 1-5 min. After cooling, the crude solid was isolated by filtration and recrystallized by ethanol.
6-Benzyl-3-(4-chlorostyryl)-4-(4-fluorophenyl)isoxa-zolo[3,4-d]pyridazin-7(6ff)-one, 2b
Yield = 53%; mp = 217--220 °C (EtOH); 1H-NMR (CDCl3) 8 5.40 (s, 2H, CH2Ph), 6.75 (d, 1H, CH=CH), 7.30-7.40 (m, 9H, Ar), 7.55 (m, 2H, Ar), 7.65 (m, 3H: 2H, Ar; H, CH=CH); MS m/z 458 [M+]; Anal. Calcd for C26H17ClFN3O2: C, 68.20; H, 3.74; N, 9.18. Found C, 682.34; H, 3.75; N, 9.21.
6-Benzyl-4-(4-fluorophenyl)-3-(4-nitrostyryl)isoxazo-lo[3,4-d]pyridazin-7(6ff)-one, 2c
Yield = 73%; mp = 202--205 °C (EtOH); 1H-NMR (CDCl3) 8 5.40 (s, 2H, CH^h), 6.90 (d, 1H, CH=CH), 7.30-7.40 (m, 5H, Ar), 7.45-7.55 (m, 4H, Ar), 7.65 (m, 2H, Ar), 7.70 (d, 1H, CH=CH), 8.25 (d, 2H, Ar); MS m/z 469 [M+]; Anal. Calcd for C26H17FN4O4: C, 66.66; H, 3.66; N, 11.96 Found C, 66.50; H, 3.67; NT, 11.92.
6-Benzyl-4-(4-fluorophenyl)-3-(4-methoxystyryl)iso-xazolo[3,4-d]pyridazin-7(6ff)-one, 2d
Yield = 76%; mp = 228--230 °C (EtOH); 1H-NMR (CDCl3) 8 3.85 (s, 3H, OCH3), 5.40 (s,	6.65
(d, 1H, CH=CH), 6.90 (m, 2H, Ar), 7.25-7.45 (m, 9H, Ar), 7.55 (d, 1H, CH=CH), 7.60 (m, 2H, Ar); MS m/z 454 [M+]; Anal. Calcd for C^H^FN^: C, 71.51; H, 4.45; N, 9.27. Found C, 71.34; H, 4.47; N, 9.30.
6-Benzyl-4-(4-fluorophenyl)-3-[2-(furan-3-yl)vinyl]isoxazolo[3,4-d]pyridazin-7(6ff)-one, 2e
Yield = 77%; mp = 219-220 °C dec. (EtOH); 1H-NMR (CDCl3) 8 5.40 (s, 2H,CH(Ph), 6.30 (m, 1H, Ar), 6.45 (d, 1H, CH=CH), 7.25-7.40 (m2 5H, Ar), 7.45 (m, 1H, Ar), 7.50-7.60 (m, 5H: 4H, Ar; 1H CH=CH), 7.70 (s, 1H, Ar); MS m/z 414 [M+]; Anal. Calcd for C24H16FN3O3: C, 69.73; H, 3.90; N, 10.16. Found C, 69.53; H, 3.91; N, 10.20.
6-Benzyl-4-(4-fluorophenyl)-3-[2-(pyridin-3-yl)vinyl]isoxazolo[3,4-d]pyridazin-7(6ff)-one, 2f
Yield = 16%; mp = 130-131 °C dec. (EtOH); 1H-NMR (CDCl3) 8 5.40 (s, 2H, CH2Ph), 7.00 (d, 1H, CH=CH), 7.30-7.40 (m, 5H, Ar), 7.55 (m, 2H, Ar), 7.65 (m, 2H, Ar), 7.70 (d, 1H, CH=CH), 7.80 (m, 1H, Ar), 8.10
(m. 1H, Ar), 8.75 (m, 1H, Ar), 8.80 (s, 1H, Ar); MS m/z 425 [M+]; Anal. Calcd for C25H17FN4O2: C, 70.75; H, 4.04; N, 13.20. Found C, 70.95; H, 4.06; N, 13.16.
6-Benzyl-3-(2-dimethylaminovinyl)-4-(4-fluorop-henyl)isoxazolo[3,4-d]pyridazin-7(6ff)-one, 2g
A suspension of 114 (0.6 mmol) in N,N-dimethylfor-mammide dimethyl acetal (22.5 mmol) was refluxed under stirring 90 min. After cooling the precipitate was recovered by suction.
Yield = 73%; mp = 162-163 °C (EtOH); 1H-NMR (CDCl3) 8 2.90 (s, 6H, N(CH3)2), 4.75 (d, 1H, CH=CH), 5.35 (s, 2H, CH2Ph), 7.15-7.25 (m, 2H, Ar), 7.30-7.40 (m, 5H, Ar), 7.55 (m, 3H: 1H, Ar; 1H, CH=CH); MS m/z 391 [M+]; Anal. Calcd for C^H^FN^: C, 67.68; H, 4.91; N, 14.35. Found C, 67.91; H, 4.89; N, 14.32.
General Procedures for 3b-f
To a suspension of compounds 2b-g (0.45 mmol) in ethanol (3-3.5 mL), 10-15 mmol of hydrazine hydrate was added and the mixture was heated at 50-70 °C for
3-4	h. After cooling the suspension was concentrated under vacuum and the solid was recovered by suction and re-crystallizzed by ethanol.
4-Amino-2-benzyl-5-[5-(4-chlorophenyl)-2H-pyrazol-
3-yl]-6-(4-fluorophenyl)pyridazin-3(2_ff)-one,	3b
Yield = 62%; mp = 142-143 °C (EtOH); 1H-NMR (CDCl3) 8 5.45 (s, 2H, CH2Ph), 5.70 (s, 1H Ar), 6.65 (exch br s, 2H, NH2), 7.05 (m, 3H, Ar), 7.30-7.40 (m, 8H, Ar), 7.50 (m, 2H, Ar), 8.20 (exch br s, 1H, NH); MS m/z 472 [M+]; Anal. Calcd for C^H^ClFNjO: C, 66.17; H, 4.06; N, 14.84. Found C, 66.32; H, 4.07; N, 14.81.
4-Amino-2-benzyl-6-(4-fluorophenyl)-5-[5-(4-nitrop-henyl)-2H-pyrazol-3-yl]pyridazin-3(2_ff)-one, 3c
Yield = 72%; mp = 205-207 °C dec. (EtOH); 1H-NMR (CDCl3) 8 5.45 (s, 2H, CH2Ph), 6.15 (s, 1H, Ar), 6.35 (exch br s, 2H, NH2), 7.00-7.10 (m, 2H, Ar), 7.30-7.40 (m, 5H, Ar), 7.55 (d, 2H, Ar), 7.70 (d, 2H, Ar), 8.20 (exch br s, 1H, NH), 8.30 (d, 2H, Ar); MS m/z 483 [M+]; Anal. Calcd for C^H^FN^: C, 64.73; H, 3.97; N, 17.42. Found C, 64.87; H, 3.97; N, 17.47.
4-Amino-2-benzyl-6-(4-fluorophenyl)-5-[5-(4-met-hoxyphenyl)-2H-pyrazol-3-yl]pyridazin-3(2_ff)-one, 3d
Yield = 30%; mp = 168-170 °C (EtOH); 1H-NMR (CDCl3) 8 3.85 (s, 3H, CH3O), 5.40 (s, 2H,CH(Ph), 6.05 (s, 1H Ar), 6.40 (exch br s, 2H, NH2), 6.95-7.05 (m, 4H, Ar), 7.30-7.40 (m, 5H, Ar), 7.55 (m, 2H, Ar), 7.65 (m, 2H, Ar), 8.20 (exch br s, 1H, NH); MS m/z 468 [M+]; Anal. Calcd for C27H22FN5O2: C, 69.37; H, 4.74; N, 14.98. Found C, 69.56; H, 4.75; N, 14.94.
4-Amino-2-benzyl-6-(4-fluorophenyl)-5-[(5-furan-3-yl)-2H-pyrazol-3-yl]pyridazin-3(2ff)-one, 3e
Yield = 38%; mp = 132-135 °C dec. (EtOH); 1H-NMR (CDCl3) 8 5.40 (s, 2H, CH2Ph), 5.90 (s, 1H Ar), 6.30 (exch br s, 2H, NH2), 6.75 (m, 1H, Ar), 6.95-7.05 (m, 2H, Ar), 7.30-7.40 (m, 4H, Ar), 7.45 (m, 1H, Ar), 7.50 (m, 3H, Ar), 7.90 (s. 1H, Ar), 8.10 (exch br s, 1H, NH); MS m/z 428 [M+]; Anal. Calcd for C24H18FN5O2: C, 67.44; H, 4.24; N, 16.38. Found C, 67.55; H, 4.24; N, 14.30.
4-Amino-2-benzyl-6-(4-fluorophenyl)-5-[(5-pyridm-3-yl-2H-pyrazol-3-yl)]pyridazin-3(2.ff)-one, 3f
Yield = 42%; mp = 238-240 °C (EtOH); 1H-NMR (CDCl3) 8 5.40 (s, 2H, CH2Ph), 6.20 (s, 1H, Ar), 6.40 (exch br s, 2H, NH2), 7.30-7.40 (m, 6H, Ar), 7.50-7.60 (m, 3H, Ar), 7.90 (m, 1H, Ar), 8.10 (exch br s, 1H, NH), 8.40 (m, 1H, Ar), 8.60 (m. 1H, Ar), 9.75 (s, 1H, Ar); MS m/z 439 [M+]; Anal. Calcd for C25H19FN6O: C, 68.48; H, 4.37; N, 19.17. Found C, 68.34; H, 4.36; N, 19.13.
4-Amino-2-benzyl-6-(4-fluorophenyl)-5-(2H-pyrazol-
3-yl)pyridazin-3(2ff)-one, 3g
Yield = 70%; mp = 190-192 °C (EtOH); 1H-NMR (CDCl3) 8 5.40 (s, 2H, CH2Ph), 5.65 (m, 1H, Ar), 6.50 (exch br s, 2H, NH2), 7.00 (m, 2H, Ar), 7.25-7.40 (m, 5H, Ar), 7.45-7.55 (m, 3H, Ar), 8.10 (exch br s, 1H, NH); MS m/z 362 [M+]; Anal. Calcd for C20H16FN5O: C, 66.47; H, 4.46; N, 19.38. Found C, 66.62; H, 4.45; N, 19.33.
General Procedures for 4b-f
A mixture of compounds 3b-g (0.21 mmol), triethy-lorthoformate (18 mmol) and a catalytic amount of concentrated sulfuric acid in anhydrous DMF (0.5-1 mL) was stirred at room temperature for 20-30 min. The suspension was cooled and the precipitate was recovered by suction and purified by crystallization from ethanol.
3-Benzyl-9-(4-chlorophenyl)-1-(4-fluorophenyl)pyra-zolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3ff)-one, 4b
Yield = 68%; mp = 246-248 °C dec (EtOH); 1H-NMR (CDCl3) 8 5.55 (s, 2H,CH2Ph), 6.10 (s, 1H, Ar), 7.30-7.50 (m, 7H, Ar), 7.50-7.60 (m, 4H, Ar), 7.70 (m, 2H, Ar), 9.45 (s, 1H, Ar); MS m/z 482 [M+]; Anal. Calcd for C27H17ClFN5O: C, 67.29; H, 3.56; N, 14.53. Found C, 67.48; H, 3.56; N, 14.58.
3-Benzyl-1-(4-fluorophenyl)-9-(4-nitrophenyl)pyrazo-lo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3ff)-one, 4c
Yield = 69%; mp = 250-253 °C dec (EtOH); 1H-NMR (CDCl3) 8 5.50 (s, 2H,CH2Ph), 6.20 (s, 1H, Ar), 7.30-7.40 (m, 5H, Ar), 7.50-7.60 (m, 4H, Ar), 7.95 (d, 2H, Ar), 8.35 (d, 2H, Ar), 9.45 (s, 1H, Ar); MS m/z 493 [M+]; Anal. Calcd for C27H17FN6O3: C, 65.85; H, 3.48; N, 17.07. Found C, 65.66; H, 3.48; N, 17.01.
3-Benzyl-1-(4-fluorophenyl)-9-(4-methoxyphenyl) pyrazolo[1',5':1,6]pyrimido[4,5-rf]pyridazin-4(3H)-one, 4d
Yield = 96%; mp = 165-168 °C dec (THF); 1H-NMR (CDCl3) 8 3.90 (s, 3H, OCH3), 5.55 (s, 2H, CH2Ph), 6.05 (s, 1H, Ar), 6.95 (d, 2H, Ar), 7.30-7.40 (m, 5H, Ar), 7.50-7.60 (m, 4H, Ar), 7.70 (d, 2H, Ar), 9.40 (s, 1H, Ar); MS m/z 478 [M+]; Anal. Calcd for C28H20FN5O2: C, 70.43; H, 4.22; N, 14.67. Found C, 70.19; H, 4.23; N, 14.70.
3-Benzyl-1-(4-fluorophenyl)-9-(furan-3-yl)pyrazo-lo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3ff)-one, 4e
Yield = 55%; mp = 219-220 °C (EtOH); 1H-NMR (CDCl3) 8 5.55 (s, 2H, CH2Ph), 5.90 (s, 1H, Ar), 6.75 (m, 1H, Ar), 7.30-7.40 (m, 5H, Ar), 7.50-7.60 (m, 4H, Ar), 7.70 (m, 1H, Ar), 7.85 (s, 1H, Ar), 9.40 (s, 1H, Ar); MS m/z 438 [M+]; Anal. Calcd for C25H16FN5O2: C, 68.64; H, 3.69; N, 16.01. Found C, 68.81; H, 3.70; N, 16.05.
3-Benzyl-1-(4-fluorophenyl)-9-(pyridin-3-yl)pyrazo-lo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3ff)-one, 4f
Yield = 61%; mp = 200-203 °C dec. (EtOH); 1H-NMR (CDCl3) 8 5.55 (s, 2H, CH2Ph), 6.35 (s, 1H, Ar), 7.30-7.40 (m, 5H, Ar), 7.50-7.60 (m, 4H, Ar), 8.10 (m, 1H, Ar), 8.80 (m, 1H, Ar), 9.00 (m, 1H, Ar), 9.30 (s, 1H, Ar), 9.50 (s, 1H, Ar); MS m/z 449 [M+]; Anal. Calcd for C26H17FN6O: C, 69.63; H, 3.82; N, 18.74. Found C, 692.80; H, 3.81; N, 18.79.
3-Benzyl-1-(4-fluorophenyl)pyrazolo[1',5':1,6]pyrimi-do[4,5-d]pyridazin-4(3ff)-one, 4g
Yield = 81%; mp = 250-252 °C (EtOH); 1H-NMR (CDCl3) 8 5.55 (s, 2H, CH2Ph), 5.85 (m, 1H, Ar), 7.207.40 (m, 5H, Ar), 7.50-7.60 (m, 4H, Ar), 8.05 (m, 1H, Ar), 9.45 (s, 1H, Ar); MS m/z 372 [M+]; Anal. Calcd for C21H14FN5O: C, 67.92; H, 3.80; N, 18.86. Found C, 67176; H, 3.81; N, 18.89.
9-(4-Aminophenyl)-3-benzyl-1-(4-fluorophenyl)pyra-zolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3ff)-one, 5
To a solution of 4c (0.21 mmol) in 37% HCl (1 mL), a solution of SnCl2 (1.03 mmol) in 37% HCl (0.5-1 mL) was slowly added. The mixture was stirred at room temperature for 20 h. Water was then added and the mixture was neutralized with 6N NaOH. The suspension was extracted with CH2Cl2 (3 x 15 mL) and the solvent was evaporated in vacuo affording a residue oil which was purified by column chromatography using CH2Cl2/CH3OH as eluent.
Yield = 29%; mp = >300 °C (EtOH); 1H-NMR (CDCl3) 8 5.55 (s, 2H, CH2Ph), 6.10 (s, 1H, Ar), 6.65 (exch br s, 2H, NH2), 7.20 (d, 2H, Ar), 7.30-7.40 (m, 4H, Ar), 7.50-7.60 (m, 5H, Ar), 7.75 (d, 2H, Ar), 9.40 (s, 1H, Ar); MS m/z 463 [M+]; Anal. Calcd for C27H19FN6O: C, 70.12; H, 4.14; N, 18.17. Found C, 70.31; H, 4.14; N, 18.12.
General Procedures for 6a-c
A mixture of 3a14(0.14 mmol), the appropriate anhydride (4-9 mmol) and a catalytic amount of concen-
trated sulfuric acid was stirred at room temperature for 10 min. After cooling, the mixture was diluted with cold water (10 mL) and neutralized with NaHCO3. Compound 6a was filtered off and recrystallyzed from ethanol, while for compounds 6b and 6c the mixture was extracted with CH2Cl2, the solvent was evaporated under vacuum and the crude final compounds were purified by crystallization from ethanol.
3-Benzyl-1-(4-fluorophenyl)-6-isopropyl-9-phenylp-yrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3ff)-one, 6a
Yield = 89%; mp = 217-220 °C (EtOH); 1H-NMR (CDCl3) 8 1.60 (d, 6H, CH(CH3)2), 4.25 (m, 1H, CH(CH3)2), 5.50 (s, 2H, CH2Ph), 6.10 (s, 1H, Ar), 7.307.55 (m, 8H, Ar), 7.60-7.70 (m, 4H, Ar), 7.80 (m, 2H, Ar); MS m/z 490 [M+]; Anal. Calcd for C30H24FN5O: C, 73.60; H, 4.94; N, 14.31. Found C, 73.35; H, 4.93; N, 14.35.
COCH2CH2), 3.80 (t, 2H, COCH2CH2), 5.50 (s, 2H, CH2Ph), 6.10 (s, 1H, Ar), 7.30-7.40 (m, 5H, Ar), 7.407.50 (m, 3H, Ar), 7.55-7.65 (m, 4H, Ar), 7.80 (m, 2H, Ar); MS m/z 518 [M+]; Anal. Calcd for C31H24FN5O2: C, 71.94; H, 4.67; N, 13.53. Found C, 72.18; H, 4.67; N, 13.50.
1-(4-fluorophenyl)-9-phenylpyrazolo[1',5':1,6]pyrimi-do[4,5-d]pyridazin-4(3ff)-thione, 9
A mixture of 814 (0.25 mmol) and Lawesson's reagent (1.19 mmol) in toluene (4.8 mL) was heated at 110 °C for 10 h. After cooling, the precipitate was recovered by suction.
Yield = 54%; mp = 209-210 °C dec. (EtOH); 1H-NMR (CDCl3) S 6.05 (s, 1H, Ar), 7.00 (m, 3H, Ar), 7.407.80 (m, 6H, Ar), 9.80 (s, 1H, Ar), 15.00 (exch br s, 1H, NH); MS m/z 374 [M+]; Anal. Calcd for C20H12FN5S: C, 64.33; H, 3.24; N, 18.76. Found C, 64.13; H, 3.25; N, 18.79.
3-Benzyl-1-(4-fluorophenyl)-9-phenyl-6-«-propylp-yrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3ff)-one, 6b
Yield = 74%; mp = 188-191 °C (EtOH); 1H-NMR (CDCl3) 8 1.15 (t, 3H, CH3(CH2)2), 2.10 (m, 2H, CH3CH2CH2), 3.55 (t, 2H, CH3CH2CH2), 5.55 (s, 2H, CH2Ph), 6.10 (s, 1H, Ar), 7.30-7.50 (m, 8H, Ar), 7.60 (m, 4H, Ar), 7.80 (m, 2H, Ar); MS m/z 490 [M+]; Anal. Calcd for C30H24FN5O: C, 73.60; H, 4.94; N, 14.31. Found C, 73.38; H, 4.94; N, 14.28.
3-Benzyl-6-butyl-1-(4-fluorophenyl)-9-phenylpyrazo-lo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3ff)-one, 6c
Yield = 87%; mp = 190-192 °C (EtOH); 1H-NMR (CDCl3) 8 1.05 (t, 3H, CH3(CH2)3), 1.50-1.60 (m, 2H, CH3CH2(CH2)2), 2.00-2.10 (m, 2H, CH3CH2CH2CH2), 3.50-3.60 (t, 2H, CH3CH2CH2CH2), 5.55 (s, 2H,CH2Ph), 6.10 (s, 1H, Ar), 7.30-7.50 (m, 8H, Ar), 7.60-7.80 (m," 6H, Ar); MS m/z 504 [M+]; Anal. Calcd for C31H26FN5O: C, 73.94; H, 5.20; N, 13.91. Found C, 73.77; H, 55.21; N, 13.95.
3-Benzyl-1-(4-fluorophenyl)-6-(3-oxobutyl)-9-phenylpyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3tf)-one, 7
A mixture of 3a14(0.27 mmol), 0.58 mmol of levuli-nic acid, 0.41 mmol of 4-(dimethylamino)pyridine and 0.48 mmol of 1-[3-(dimethylamino)propyl]-ethylcarbo-diimide hydocloride in anhydrous CH2Cl2 (10.4 mL) and anhydrous DMF (1 mL), was refluxed for 20 h. After cooling CH2Cl2 was added (15 mL) and the organic layer was washed with 2N HCl and with 2N NaOH in turn. Evaporation in vacuum afforded the final compound 7 which was purified by column chromatography using CH2Cl2/ CH3OH 9.5:0.5 as eluent.
Yield = 15%; mp = 220-222 °C dec. (EtOH); 1H-NMR (CDCl3) 8 2.40 (s, 3H, CH3CO), 3.35 (t, 2H,
4-Benzylthio-1-(4-fluorophenyl)-9-phenylpyrazo-lo[1',5':1,6]pyrimido[4,5-rf]pyridazina, 10
A mixture of compound 9 (0.21 mmol), K2CO3 (0.66 mmol) and 0.61 mmol of benzyl chloride in anhydrous DMF (1.6 mL) was heated under stirring at 110 °C for 1 h. After cooling, cold water was added and the precipitate was isolated by filtration.
Yield = 30%; mp = 281-282 °C (Ethyl acetate); 1H-NMR (CDCl3) 8 4.70 (s, 2H, CH2S), 6.30 (s, 1H, Ar), 7.25-7.40 (m, 3H, Ar), 7.40-7.60 (m, 7H, Ar), 7.70-7.90 (m, 4H, Ar), 9.90 (s, 1H, Ar); MS m/z 464 [M+]; Anal. Calcd for C27H18FN5S: C, 69.96; H, 3.91; N, 15.11. Found C, 69.75; H,3.90; N, 15.15 .
4. 2. Adenosine Receptor Binding Assay17-19
The binding activity of each compound towards adenosine receptor subtypes was calculated by competition binding experiments. To determine the affinities of the new compounds toward human A1, A2A, and A3 ARs we evaluated the ability of different compound concentrations to displace [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, for CHO-A1), [3H]5 -N-ethylcarboxami-deadenosine ([3H]NECA, for CHO A2A), or [125I]4-ami-nobenzyl-5 -N-methylcarboxamidoadenosine ([125I]AB-MECA, for CHO-A3) binding from transfected CHO cells. Data analysis and graphic presentation were conducted using the non-linear multipurpose curve-fitting computer program Graph-Pad Prism (GraphPad, San Diego, CA). Data analysis allowed to obtain the competition curve of each compound and to calculate its affinity towards a single population of receptors expressed as Ki value. For the compounds that at 10 pM concentration showed an inhibitory effect on radioligand binding lower that 60%, the competition curve was not performed and the results were expressed as % inhibition at 10 pM.
Human A1 Adenosine Receptors. Aliquots of cell membranes (30 jg proteins) obtained from AjCHO cells were incubated at 25 °C for 180 min in 500 jL of buffer (50 mM Tris-HCl, 2 mM MgCl2, and 2 units/mL ADA, pH 7.4) containing [3H]DPCPX (3 nM) and six different concentrations of the compounds. Non-specific binding was determined in the presence of 50 jM R-PIA20 The dissociation constant (Kd) of [3H]DPCPX in A1 CHO cell membranes was 3 nM.
Human A2A Adenosine Receptors. Aliquots of cell membranes (30 jg proteins) were incubated at 25 °C for 180 min in 500 jL of buffer (50mM Tris-HCl, 2 mM Mg-Cl2, and 2 units/mL ADA, pH 7.4) in the presence of 20 n-M of [3H]NECA and six different concentrations of the synthesized compounds. Non-specific binding was determined in the presence of 100 jM R-PIA20. The dissociation constant (Kd) of [3H]NECA in A2A CHO cell membranes was 30 nM.
Human A3 Adenosine Receptors. Aliquots of cell membranes (20 jg proteins) were incubated at 25 °C for 90 min in 100 jL of buffer (50mM Tris-HCl, 10mM Mg-Cl2, 1mM EDTA, and 2 units/mL ADA, pH 7.4) in the presence of 0.14 nM [125I]AB-MECA and six different concentrations of the synthesized compounds. Non-specific binding was determined in the presence of 50 j M R-PIA20. The dissociation constant (Kd) of [125I] AB-MECA in A, CHO cell membranes was 1.4 nM.
5. References
1. B. B. Fredholm, A. P. Ijzerman, K. A. Jacobson, K. N. Klotz, J. Linden, International Union of Pharmacology. XXV. Pharmacol. Rev. 2001, 53, 527-552. 2.S. A. Poulsen, R. J. Quinn, Bioorg. Med. Chem. 1998, 6, 619-641.
3.B.	B. Fredholm, G. Arslan, L. Haldner, B. Kull, G. Schulte, W. Wasserman, Naunyn-Schmied. Arch. Pharmacol. 2000, 362, 364-374.
4.	M. E. Olah, G. L. Stiles, Pharmacol Ther. 2000, 85, 55-75
5.	S. Hess, Exp. Opin. Ther. Patents 2001, 11, 1533-1561.
6.	K. A. Jacobson, Handb. Exp. Pharmacol. 2009, 193, 1-24.
7.	J. A. Ribeiro, A. M. Sebastiao, A. De Mendica, Prog. Neuro-biol. 2003, 68, 377-392.
8.	M. M. Dohadwala, M. M. Givertz, Cardiovasc. Ther. 2008, 26, 276-286.
9.	P. Forsythe, M. Ennis, Inflamm. Res. 1999, 48, 301-307.
10.	R. A. Brown, D. Spina, C. P. Page, J. Pharmacol. 2008, 153, 446-456.
11.	J. Bulicz, D. C. G. Bertarelli, D. Baumert, F. Fulle, C. E. Muller, D. Heber, Bioorg. Med. Chem. 2006, 14, 2837-2849.
12.	S. Hess, C. E. Muller, W. Frobenius, U. Reith, K-N. Klotz, K. Eger, J. Med. Chem. 2000, 43, 4636-4646.
13.	F. Da Settimo, G. Primofiore, S. Taliani, A. M. Marini, C. La Motta, E. Novellino, G. Greco, A. La Vecchia, L. Trincavelli, C. Martini, J. Med. Chem. 2001, 44, 316-327.
14.	M. P. Giovannoni, C. Vergelli, A. Cilibrizzi, L. Crocetti, C. Biancalani, A. Graziano, V. Dal Piaz, M. I. Loza, M. I. Cada-vid, J. L. Diaz, A. Gavalda, Bioorg. Med. Chem. 2010, 18, 7890-7899.
15.	J. Feixas, M. P. Giovannoni, C. Vergelli, A. Gavalda, N. Ce-sari, A. Graziano, V. Dal Piaz, Bioorg. Med. Chem. Lett. 2005, 15, 2381-2384.
16.	V. Dal Piaz, G. Ciciani, S. Chimichi, Heterocycles 1986, 24, 3143-3148.
17.	S. Taliani, I. Pugliesi, E. Barresi, F. Simorini, S. Salerno, C. La Motta, A. M. Marini, B. Cosimelli, S. Cosconati, S. Di Maro, L. Marinelli, S. Daniele, M. L. Trincavelli, G. Greco,
E.	Novellino, C. Martini, F. Da Settimo, J. Med. Chem 2012, 55, 1490-1499.
18.	D. Poli, D. Catarzi, V. Colotta, F. Varano, G. Filacchioni, S. Daniele, M. L. Trincavelli, C. Martini, S. Paoletta, S. Moro, J. Med. Chem. 2011, 54, 2102-2113.
19.	V. Colotta, D. Catarzi, F. Varano, O. Lenzi, G. Filacchioni, C. Martini, L. Trincavelli, O. Ciampi, C. Traini, A. M. Pugliese,
F.	Pedata, E. Morizzo, S. Moro, Bioorg. Med. Chem. 2008, 16, 6086-6102.
20.	F. Da Settimo, G. Primofiore, S. Taliani, C. La Motta, E. No-vellino, G. Greco, A. Lavecchia, B. Cosimelli, M. Iadanza, K.-N. Klotz, D. Tuscano, M. L. Trincavelli, C. Martini, Drug Dev. Res. 2004, 63, 1-7.
Povzetek
Članek poroča o sintezi in biološkem ovrednotenju nove serije pirazolo[1',5':1,6]pirimido[4,5-d]piridazin-4(3H)-onov kot ligandov človeškega A1 adenozinskega receptorja. Triciklično ogrodje je bilo spremenjeno na položajih 6 in 9 z uvedbo majhnih alkilnih verig in substituiranih fenilov.
Najbolj zanimive spojine so pokazale Ki za A1 v submikromolarnem območju (0.105-0.244 |M). Najzanimivejši del (spojina 4c) je pokazal znatno afiniteto za A1 (Ki = 0.132 |M), skupaj z dobro selektivnostjo za A2A (43 % inhibicije pri 10 |M) in A3 (46 % inhibicije pri 10 |M).