Scientific paper
Synthesis and Evaluation of Novel Pyrrolo[2,3-^] and Thieno[2,3-^]Pyridazinones as in Vitro Antiproliferative Agents
Vittorio Dal Piaz,* Claudia Vergelli, Maria Paola Giovannoni, Claudio Biancalani, Agostino Cilibrizzi, Alessia Graziano and Nicoletta Cesari
Corresponding author: Dipartimento di Scienze Farmaceutiche, Facoltà di Farmacia, Università di Firenze, Via Ugo Schiff 6, Sesto Fiorentino 50019, Firenze, Italy.
* Corresponding author: E-mail: vittorio.dalpiaz@unifi.it; Tel.: +39-055-4573681, Fax: +39-055-4573780
Received: 25-09-2008
Dedicated to Professor Blanko Stanovnik on the occasion of his 70t' birthday
Abstract
Two different types of novel heterocyclic-fused pyridazinones like pyrrolo[2,3-d] and thieno[2,3-d] derivatives were synthesized starting from their isoxazolo[3,4-d] precursors by oxidative cleavage with CAN followed by cycloconden-sation of the five-membered system using bi-functional nucleophiles. The final compounds were preliminarily screened in vitro as antiproliferative agents under the protocols of the NCI using three human cell lines of CNS, lung and breast cancers. None of the compounds was able to reduce the growth at value < 32% which was the cut-off for a more in depth in vitro screening.
Keywords: Thieno[2,3-d]pyridazinones, pyrrolo[2,3-d]pyridazinones, synthesis, in vitro antiproliferative agents.
1. Introduction
The pyrrolopyridazinone sub-unit is well represented among heterocyclic compounds displaying antitumor
and antiviral activities.
Townsend et al.1-5 reported several series of purine nucleoside analogues containing the pyrrolo[2,3-d]pyri-dazin-7-one system as heterocyclic core. Some of these compounds, like the prototype 1 (Figure 1), showed significant antiproliferative and antiviral activities in vitro and low cytotoxicity. Very recently the same group reported a series of isosteric pyrrolotriazines active against human cytomegalovirus and herpes simplex virus.6 These compounds were synthesized as analogues of Sangivamycin 2. This natural compound, which is active against L 1210 leukemia, P 338 leukemia and Lewis lung carcinoma, has been in clinical trials against several types of human cancers.7, 8 Further examples of pyrrolopyridazinones (or fused analogues) endowed with antitumor and/or antiviral
activities (compounds 3, 4 and 5) were described by authors from different countries active both in industry9 and in academia.10-12 Our continuing interest in the chemistry and pharmacology of pyridazin-3(2^)-one derivatives and heterocyclic-fused analogues13-15 led us to undertake a research program aimed to synthesize novel examples of pyrrolo[2,3-d]pyridazin-7-ones, as well as of the corresponding thieno[2,3-d] derivatives to evaluate their in vitro antiproliferative effect. Thus in this work we report the preliminary results obtained in this area.
2. Results and Discussion
The target compounds of general structure (6) (Figure 1) were synthesized following a well established procedure described by us in some previous papers.13-15 The key intermediates are the 5-acetyl-4-nitropyridazinones 8a-h (Scheme 1) which are easily obtained from the iso-xazolopyridazinones 7a-h by oxidative cleavage of the fi-
Figure 1. Structure of compounds displaying antitumoral and antiviral activities.
Table 1. Evaluation of antiproliferative activity of compounds 10a-d, 12a-d, 13a-e and 14a,b on three different cell lines (MCF7, NCI-H460 and SF268)
Comp	R	X	Y	BREAST (MCF7)	% of growtha LUNG (NCI-H460)	CNS (SF-268)
10a	COOEt	H	H	54	89	80
10b	COOEt	H	F	93	98	121
10c	COOEt	F	H	99	97	98
10d	COOEt	H	Cl	67	90	106
12a	COOH	H	H	NTc	NTc	NTc
12b	COOH	H	F	101	98	112
12c	COOH	F	H	116	101	133
12d	COOH	H	Cl	104	99	108
13a	conh2	H	H	101	98	83
13b	conh2	H	F	105	101	96
13c	conh2	F	H	NTc	NTc	NTc
13d	conh2	H	Cl	64	100	78
13e	COZb	H	H	NTc	NTc	NTc
14a	CH2OH	H	H	103	97	124
14b	CH2OH	H	F	88	92	102
a Compounds were tested at 100 ^M; b Z = 2-methylaziridin-1-yl; c Not tested
Comp	R	Ri	X	Y	BREAST (MCF7)	% of growtha LUNG (NCI-H460)	CNS (SF-268)
11a	Et	COOEt	H	F	67	101	97
11b	Et	COOEt	F	H	75	99	75
11c	Et	COOEt	no2	H	58	93	43
11d	n-Pr	COOEt	H2	H	NTb	NTb	NTb
11e	i-Pr	COOEt	H	H	75	75	70
11f	i-Pr	COOEt	no2	H	NTb	NTb	NTb
15a	Et	COOH	H2	F	114	100	139
15b	Et	COOH	F	H	87	90	103
15c	Et	COOH	no2	H	117	101	132
15d	i-Pr	COOH	no2	H	NTb	NTb	NTb
16a	Et	conh2	H2	F	113	98	115
16b	Et	conh2	F	H	110	102	130
16c	Et	conh2	no2	H	109	92	126
16d	i-Pr	conh2	no2	H	NTb	NTb	NTb
11c 11d 11e 11f 15a 15b 15c 15d 16a 16b 16c 16d
Et
n-Pr
i-Pr
i-Pr
Et
Et
Et
i-Pr
Et
Et
Et
i-Pr
Ri
BREAST (MCF7)
% of growtha
LUNG (NCI-H460)
COOEt
COOEt
COOEt
COOEt
COOH
COOH
COOH
COOH
CONH2
CONH2
CONH2
CONH,
H
F
no2
H H
no2
H
F
no2 no2
H
F
no2 no.
F H H H H H F H H H F H H H
67 75 58 NTb 75 NTb 114 87 117 NTb 113 110 109 NTb
101
99 93
NTb 75 NTb
100 90 101 NTb
98 102 92 NTb
CNS (SF-268)
97 75 43 NTb 70 NTb 139 103 132 NTb 115 130 126 NTb
a Compounds were tested at 100 uM; b Not tested
ve-membered system with ceric ammonium nitrate (CAN). With the exception of 7h, all the precursors of type 7 were previously described by us.1415 Likewise compounds 8a-g were reported in our foregoing pa-
pers.1
^ The unknown 7h was easily prepared by alky-
lation of the 2-unsubstituted analogue14 with 2-iodopropa-ne (see experimental). In compounds 8 the nitro group is a very good leaving group and can be easily replaced under
Scheme 1. a: CAN, 65% HN03, 50% CH3COOH, 60 °C; b: glycine ethylester hydrochloride, EtOH, 45 °C; c: NaOEt/EtOH, 40 °C; d: ethyl-2-mer-captoacetate, EtOH, rt.
10-14	X	Y	Z
a	H	H	CONH2
b	H	F	CONH2
c	F	H	CONH2
d	H	C1	CONH2
e	H	H	CO—N^
Scheme 2. a: 6N NaOH, EtOH, 50 °C; b: 1) SOCl2, 60 °C, 2) NH3 or methylaziridine; c: NaBH4
Me CONH2 16a-d
Scheme 3. a: 6N NaOH, EtOH, 50 °C; b: 1) SOCl2, reflux; 2) NH3
mild conditions and in high yields by a variety of O-, N-and S-nucleophiles. In our case, treatment with glycine ethyl ester in ethanol afforded the intermediates 9a-d which, in turn, were smoothly converted into the pyrro-lo[2,3-rf]pyridazinones 10a-d by briefly heating with so-
dium ethoxide in ethanol at 40 °C. Among these compounds 9d, 10a and 10d were previously described.1417 When the precursors 8b-c and 8e-h were treated with ethyl thioglycolate in alcoholic medium the thienopyridazi-none esters 11a-f were directly isolated in good yields.
The ester group of compounds 10 was hydrolyzed using 6N NaOH in ethanol affording the corresponding car-boxylic acids 12a-d (12a was already reported19), which, in turn, were converted into the corresponding amides 13a-e (13a19) through the intermediate chlorides, by treatment with ammonia or the appropriate amine (Scheme 2). Reduction of the ester group with sodium borohydride afforded the primary alcohols 14a-b in good yields.
Several examples of amides in the thienopyridazino-ne series (compounds 16b-d) were prepared from the esters 11 (Scheme 3), using the same experimental conditions described for the conversion of the pyrrolopyridazi-nones esters 10.
Compounds 10-16 were tested in vitro as antiproli-ferative agents following the protocols optimised by the Development Therapeutic Program (DPT) of the National Cancer Institute (Bethesda, USA). On the basis on this program the novel compounds are evaluated on 60 human tumor cell lines. Since researchers from NCI found that 95% of active compounds on one of the 60 cell lines can be identified using three cell lines only, at the present the novel compounds are screened on the following cell lines: MCF7 (breast cancer), NCI-H460 (lung cancer) and SF-268 (CNS cancer).
Thus our compounds were tested at 100 micromoles concentration against the three selected cell lines and the results are depicted in Tables 1 and 2.
Unfortunately, all synthesized products showed a very low activity and they were not able to reduce the growth of anyone of the cell lines at values < 32% that is the limit given by NCI for further evaluation in the full panel of 60 cell lines.
The ester 11c was the only compound which approached the limit of 32% growth inhibition against the SF-286 cell line (CNS). Taking into account that the corresponding carboxylic acid 15c and the amide 16c were completely ineffective against all the three cell lines, it seems that lipophilicity could play a role in inducing anti-proliferative properties in the present series.
3. Conclusions
In conclusion, we synthesized a new series of pyrro-lo[2,3-d]pyridazin-7-ones and thieno[2,3-d]pyridazin-7-ones derivatives and we evaluated their in vitro antiproli-ferative effect. Unluckily, the preliminary results showed that all new products are not able to reduce the growth of the cell lines till the values given by NCI to continue evaluation. Taking into account that the compound that more closely approached the limit of 32% is the ester 11c and that its analogues bearing a polar function (the carboxylic acid 15c and the amide 16c) are inactive, we hypothize that the lipophilicity was important for activity. With this in mind the synthesis of analogues bringing the ester function and the 3-nitrophenyl fragment is in progress.
4. Experimental
All melting points were determined on a Buchi apparatus and are uncorrected. 1H-NMR spectra were recorded with Avance 400 instruments (Bruker Biospin, version 002 with SGU). Chemical shifts are reported in ppm, using the solvent as internal standard. Extracts were dried over Na2SO4 and the solvents were removed under reduced pressure. E. Merck F-254 commercial plates were used for analytical TLC to follow the course of the reaction. Silica gel 60 (Merck 70-230 mesh) was used for column chromatography.
6-Isopropyl-3-methyl-4-(3-nitrophenyl)isoxazolo[3,4-rf]pyridazin-7(6H)-one 7h. A suspension of 3-methyl-4-(3-nitrophenyl)isoxazolo[3,4-d]pyridazin-7-(6H)-one (200 mg, 0.74 mmoles), K2CO3 (250 mg, 1.8 mmoles), 2-iodopropane (305 mg, 1.8 mmoles) and anhydrous DMF (3mL) was stirred at 100 °C for 1 h. Treatment with ice-cold water (20 mL) afforded 7h as crude precipitate (87% yield). The analytical sample was obtained by crystallization from ethanol: mp 208-210 °C.
1H NMR (CDCl3): 5 8.45-8.30 (m, 2H, aromatic), 8.00 (d, J = 7.4 Hz, 1H, aromatic), 7.80-7.65 (m, 1H, aromatic), 5.40 (m, 1H, CH(CH3)2), 2.60 (s, 3H, CH3), 1.40 (d, J = 7.6 Hz, 6H, CH(CH3)2).
Anal. Calcd. for C15H14N4O4: C, 57.32; H, 4.49; N, 17.83. Found: C, 57.59; H, ^1.81; NN,17.58.
5-Acetyl-2-isopropyl-4-nitro-6-(3-nitrophenyl)pyrida-zin-3(2Ä)-one 8h. To a stirred suspension of 7h (150 mg, 0.48 mmoles) in 50% acetic acid (5 mL) and 65% HNO3 (0.45 mL), ceric ammonium nitrate (1.9 g, 3.47 mmoles) was added portionwise in 45 min, maintaining the temperature at 55-60 °C. After dilution with ice-cold H2O (50 mL) and standing for 1 h the precipitate 7h (48% yield) was collected by suction and purified by column chroma-tography (eluent: toluene/ethyl acetate 8:2); mp = 109-111 °C, crystallization solvent ethanol.
1H NMR (CDCl3): 5 8.45-8.20 (m, 2H, aromatic), 8.00 (d, J = 7.5 Hz, 1H, aromatic), 7.80-7.62 (m, 1H, aromatic), 5.58-5.30 (m, 1H, CH(CH3)2), 2.28 (s, 3H, CH3), 1.60-1.32 (m, 6H, CH(CH3)2). Anal. Calcd. for C15H14N4Og: C, 52.03; H, 4.07; N, 16.18. Found: C, 5^.93;H, 4.19; N, 16.05.
General procedure for compounds 9a-c. To a cooled solution of glycine ethylester hydrochloride (2.52 mmoles) in water (2.5 mL), 6N NaOH (0.5 mL) was added dropwi-se until pH 9.0. The solution was saturated with NH4Cl and extracted with ethyl ether (4 x 20 mL). The organic layer was dried on anhydrous sodium sulfate and evaporated in vacuo. The residual oil was dissolved in ethanol (2mL) and the appropriate 4-nitro derivative 8 (0.33 mmo-les) was added. The suspension was stirred at 45 °C for 20 min. After cooling, the crude precipitate was filtered off.
Ethyl 2-(5-acetyl-2-ethyl-3-oxo-6-phenyl-2,3-dihydrop-yridazin-4-ylamino)acetate 9a. Yield 45%, mp 116-118 °C, crystallization solvent: ethanol.
1H NMR (CDCl3): 5 7.40 (s, 5H, aromatic), 4.40 (d, J = 7.7 Hz, 2H, CH2-NH), 4.37-4.15 (m, 4H, N-CH2-CH3 and O-CH2-CH3), 1.80 (s, 3H, CH3), 1.45-1.24 (m, 6H, N-CH2-CH3 and O-CH2-CH3). Anail. Calcd. For C18 H21N3O4: C:, 62.SI6; H, 6.16; NN, 12.24. Found: C, 62.52; H, 5.97; N, 11.97.
Ethyl 2-[5-acetyl-2-ethyl-6-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazin-4-ylamino]acetate 9b. Yield 48%, mp 127-128 °C, crystallization solvent: ethanol.
1H NMR (CDCl3): 5 8.42 (exch br s, 1H, NH), 7.52-7.35 (m, 2H, arom3 atic), 7.25-7.04 (m, 2H, aromatic), 4.53-4.38 (m, 2H, CH2-NH), 4.25-4.12 (m, 4H, N-CH2-CH3 and O-CH2-CH3), 1.83 (s, 3H, CH3), 1.501.23 (m, 6H, N-CH2-CH3 and O-CH2-CH3). Anal. Calcd. For C18 H20FN3O4: C, 59.83; H, 5.58; NN, 11.63. Found: C, 60.02; H, 5.41; N, 11.87.
Ethyl 2-[5-acetyl-2-ethyl-6-(3-fluorophenyl)-3-oxo-2,3-dihydropyridazin-4-ylamino]acetate 9c. Yield 53%, mp 95-96 °C, crystallization solvent: ethanol .
1H NMR (CDCl3): 5 7.45-7.15 (m, 4H, aromatic), 4.50-4.41 (m, 2H, NH-CH2), 4.36-4.15 (m, 4H, N-CH2-CH3 and O-CH2-CH23), 1.85 (s, 3H, CH3), 1.42-1.23 (m, 6H, N-CH2-CH3 and O-CH2-CH3). Anal. Calcd. For C18H20FN3O4: C, ^9.83; H, 5.58; N, 11.63. Found: C, 60.(^5; H, 5.61; N, 11.77.
General procedure for compounds 10b,c. To a solution of the appropriate compound 9 (0.25 mmoles) in anhydrous ethanol (2 mL) a solution of sodium ethoxi-de prepared dissolving sodium (1.5 mmoles) in anhydrous ethanol (1.5 mL) was added. The mixture was stirred for 10 min at room temperature. Dilution with ice-cold water (8 mL) and acidification with 6N HCl afforded the desired 10 as a precipitate which was filtered off.
Ethyl 6-ethyl-4-(4-fluorophenyl)-3-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-rf]pyridazine-2-carboxylate
10b. Yield 83%, mp 219-220 °C, crystallization solvent: ethanol .
1H NMR (CDCl3): 5 7.56-7.43 (m, 2H, aromatic), 7.35-7.10 (m, 2H, aromatic), 4.48-4.32 (m, 4H, N-CH2-CH3 and O-CH2-CH3), 2.18 (s, 3H, CH3), 1.45-1.35 (m, 6H, N-CH2-CH3 and O-CH2-CH3). Anal. Calcd. For C18H18FN3O3: C, ^2.97; H, 5.28; N, 12.24. Found: C, 63.21; H, 5.19; N, 12.47.
Ethyl 6-ethyl-4-(3-fluorophenyl)-3-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-rf]pyridazine-2-carboxylate 10c. Yield 72%, mp 164-166 °C, crystallization solvent: ethanol.
1H NMR (CDCl3): 5 7.58-7.42 (m, 1H, aromatic), 7.35-7.15 (m, 3H, aromatic), 4.55-4.23 (m, 4H, N-CH2-CH3 and O-CH2-CH3), 2.20 (s, 3H, CH3), 1.52-1.40 (m, 6H, N-CH2-CH3 and O-CH2-CH3). Anal. Calcd. For C18H18FN3O3: C, ^2.97; H, 5.28; N, 12.24. Found: C, 63.1^1; H, 5.35l N, 12.07.
General procedure for compounds 11a-f (11c14). A solution of ethyl-2-mercaptoacetate (1.0 mmoles) in absolute ethanol (1 mL) was added to a solution of sodium etho-xide prepared dissolving sodium (1.0 mmoles) in absolute ethanol (1.5 mL). The mixture was stirred at room temperature for 20 min. After evaporation in vacuo the residue was treated with a suspension of compound 8 (0.33 mmo-les) in absolute ethanol (2 mL). After 20 min stirring the reaction mixture was diluted with ice- cold water (25 mL) and the precipitate 11 collected by suction.
Ethyl 6-ethyl-4-(4-fluorophenyl)-3-methyl-7-oxo-6,7-dihydrothieno[2,3-rf]pyridazine-2-carboxylate 11a.
Yield = 47% , mp = 185-188 °C dec., crystallization solvent: ethanol.
1H NMR (CDCl3): 5 7.50-7.38 (m, 2H, aromatic), 7.30-7.15 (m, 2H, aromatic), 4.51-4.23 (m, 4H, N-CH2CH3 and O-CH2-CH3), 2.20 (s, 3H,CH3), 1.481.32 (m, 6H, N-CH2-CH3 and O-CH2-CH3). Anal. Calcd. For C18H17FN2O3S: C, 59.99; H, 4.75; N, 7.77. Found: C, 59.73; H, 4.98; N, 8.09.
Ethyl 6-ethyl-4-(3-fluorophenyl)-3-methyl-7-oxo-6,7-dihydrothieno[2,3-rf]pyridazine-2-carboxylate 11b.
Yield = 35% , mp = 176 °C, crystallization solvent: etha-nol.
1H NMR (CDCl3): 5 7.65-7.55 (m, 1H, aromatic), 7.35-7.05 (m, 3H, aromatic), 4.39-4.18 (m, 4H, N-CH2CH3 and O-CH2-CH3), 2.50 (s, 3H, CH3), 1.56-1.25 (m, 6H, N-CH2-CH3 and O-CH2-CH3). Anal. Calcd. For C18H17FN2O3S: C, 59.99; H, 4.75; N, 7.77. Found: C, 59.85; H, 4.48; N, 7.94.
Ethyl -3-methyl-7-oxo-4-phenyl-6-propyl-6,7-dihydro-thieno[2,3-rf]pyridazine-2-carboxylate 11d. Yield = 35%, mp > 300 °C, crystallization solvent: ethanol.
1H NMR (CDCl3): 5 7.60-7.40 (H, aromatic), 4.40-4.20 (m, 4H, N-C3H2-CH2-CH3 and O-CH2-CH3), 2.18 (s, 3H, CH3), 2.00-1.80 (m, 2H, N-CH2-CH2-CH3) 1.40 (t, J = 7.6 Hz, 3H, O-CH2-CH3), 1.00 (t, J = 7.5 Hz 1H, CH2-CH2-CH3). Anal. Calcd. For C19H20N2O3S: C, 64.02; H, 5.6^; N, 7.86. Found: C, 64.24; H, 5:^5; N3 8.03.
Ethyl 6-isopropyl-3-methyl-7-oxo-4-phenyl-6,7-dihy-drothieno[2,3-rf]pyridazine-2-carboxylate 11e. Yield = 38%, mp = 153-155 °C, crystallization solvent: ethanol.
1H NMR (CDCl3): 5 7.60-7.40 (m, 5H, aromatic), 5.50-5.35 (m, 1H, CH-(CH3)2), 4.40 (q, J = 7.7 Hz, 2H, O-CH2-CH3), 2.20 (s, 3H, CH3), 1.50-1.30 (m, 9H,
(CH3)2-CH and O-CH2-CH3). Anal. Calcd. For C19H22N203S: C, 64.02; H, 5.66; N, 7.86. Found: C, 63.74; H, 5.80; N, 7.73.
Ethyl-6-isopropyl-3-methyl-4-(3-nitrophenyl)-7-oxo-6,7-dihydrothieno[2,3-d]pyridazine-2-carboxylate 11f.
Yield = 35%, mp = 164-165 °C, crystallization solvent: ethanol.
1H NMR (CDCl3): 5 8.45-8.35 (m, 2H, aromatic), 7.85-7.65 (m, 2H, aromatic), 5.54-5.46 (m, 1H, CH-(CH3)2), 4.40 (q, J = 7.6 Hz, 2H, CH2), 2.21 (s, 3H, CH3), 1.48-1.35 (m, 9H, CH2-CH3 and (CH3)2-CH). Anal. Calcd. For C19H19N3O5^: C, 56.85; H, 4.77; N, 10.47. Found: C, 56.68; H, ^4.72; N, 10.22.
General procedure for 12b-d. To a solution of the appropriate derivative 10 (0.2 mmoles) in ethanol (2 mL), 2N NaOH (4 mL) was added and the mixture was stirred at 50 °C for 5 h. After concentration in vacuo, the residue was diluted with ice-cold water. Acidification with 6N HCl afforded compound of type 12 as a precipitate which was collected by filtration.
6-Ethyl -4-(4-fluorophenyl)-3-methyl-7-oxo-6,7-dihy-dro-1H-pyrrolo[2,3-d]pyridazine-2-carboxylic acid 12b. Yield 77%, mp > 300 °C, crystallization solvent: et-hanol.
1H NMR (CDCl3): 5 7.68-7.50 (m, 2H, aromatic), 7.40-7.18 (m, 2H, aromatic), 4.20 (q, J = 7.5 Hz, 2H, CH2-CH3), 2.00 (s, 3H, CH3), 1.30 (t, J = 7.5 Hz, 3H, CH2-CH3). Anal. Calcd. For C1gH14FN3O3: C, 60.95; H, 4.4^; N, 13.33. Found: C, 61.12; H, 4.3^; NN, 13.09.
6-Ethyl -4-(3-fluorophenyl)-3-methyl-7-oxo-6,7-dihy-dro-1H-pyrrolo[2,3-d]pyridazine-2-carboxylic acid 12c.
Yield 92% , mp > 300 °C crystallization solvent: ethanol.
1H NMR (CDCl3): 5 7.64-7.45 (m, 1H, aromatic), 7.40-7.20 (m, 3H, aromatic), 4.50 (q, J = 7.5 Hz, 2H, CH2-CH3), 2.30 (s, 3H, CH3), 1.50 (t, J = 7.5 Hz, 3H, CH2-CH3). Anal. Calcd. For C1gH14FN3O3: C, 60.95; H, 4.48; N, 13.33. Found: C, 61.18 ; H, 4.60; N, 13.6.
4-Chlorophenyl-6-ethyl-3-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-d]pyridazine-2-carboxylic acid 12d.
Yield 79%, mp > 300 °C, crystallization solvent: ethanol.
1H NMR (DMSO-dg): 5 7.58 (s, 4H, aromatic), 4.20 (q, J = 7.5 Hz, 2H, CH2-CH3), 2.00 (s, 3H, CH3), 1.26 (t, J = 7.5 Hz, 3H, CH2-CH3). Anal. Calcd. For C1)H14ClN3O3: C, 57.93; H, 4.25; N, 12.67. Found: C, 58.05; 1:1, 4.30; N, 13.09.
General procedure for compounds 13b-e. The carboxy-lic acid 12 (0.16 mmoles) was suspended in SOCl2 (0.5 mmoles) and the mixture was stirred at 60 °C for 4 h. After evaporation in vacuo, the residue was cooled and treated with the opportune amine (1.5 mmoles). The obtained precipitate was washed with water and collected by filtration.
6-Ethyl-4-(4-fluorophenyl)-3-methyl-7-oxo-6,7-dihy-dro-1H-pyrrolo[2,3-d]pyridazine-2-carboxamide 13b.
Yield 68% , mp > 300 °C crystallization solvent: ethanol.
1H NMR (DMSO-d)): 5 7.70 (exch br s, 2H, NH2), 7.65-7.55 (m, 2H, aromatic), 7.45-7.25 (m, 2H, aromatic), 4.20 (q, J = 7.5 Hz, 2H, CH2-CH3), 2.00 (s, 3H, CH3), 1.30 (t, J = 7.5 Hz, 3H, CH2-CH3). Anal. Calcd. For C1)H15FN402: C, 61.14; H, 44.81; N,17.83. Found: C, 6^.38;H, 44.59; N, 17.60.
6-Ethyl-4-(3-fluorophenyl)-3-methyl-7-oxo-6,7-dihy-dro-1H-pyrrolo[2,3-d]pyridazine-2-carboxamide 13c.
Yield = 62%, mp > 300, crystallization solvent: ethanol.
1H NMR (DMSO-d.): 5 7.50-7.20 (m, 4H, aromatic), 4.20 (q, J = 7.5 Hz, 2H, CH2-CH3), 2.00 (s, 3H, CH3), 1.30 (t, J = 7.5 Hz, 3H, CH2-CH3). Anal. Calcd. For C1)H15FN402: C, 61.14; H, 4.81; N,17.83. Found: C, 6^.42; H, 44.99; N, 17.97.
4-(4-chlorophenyl)-6-ethyl-3-methyl-7-oxo-6,7-dihy-dro-1H-pyrrolo[2,3-d]pyridazine-2-carboxamide 13d.
Yield = 84%, mp > 300, crystallization solvent: ethanol.
1H NMR (DMSO-d.): 5 7.53 (s, 4H, aromatic), 4.20 (q, J = 7.5 Hz, 2H, CH2-CH3), 3.90 (exch br s, 2H, NH2), 2.05 (s, 3H, CH3), 1.20 (t, J = 7.5 Hz, 3H, CH2-CH3). Anal. Calcd. For C1)H15ClN4O2: C, 58.10; H, 4.57; N, 16.94. Found: C, 58.34; H, 4.84; N, 16.73.
6-ethyl-3-methyl-2-[(2-methylaziridin-1-yl)carbonyl]-4-phenyl-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-(6Ä)-one 13e. Yield = 53%, mp = 70-73, crystallization solvent: cyclohexane.
1H NMR (CDCl3): 5 7.50 (s, 5H, aromatic), 4.40 (q, J = 7.5 Hz, 2H, N-CH2-CH3), 2.85-2.75 (m, 1H, CHCH3), 2.55-2.44 (m, N-CH2CH), 2.22-2.18 (m, N-CH2CH), 2.18 (s, 3H, CH3), 1.40 (t, J = 7.5 Hz, 3H, N-CH2-CH3), 1.25 (d, J = 7.1 Hz, 3H, CH-CH3). Anal. Calcd. For C19H20N4O2: C, 67.84, H, 5.99; N, 16.66. Found: C, 67.79; H, 6.22; N, 16.38.
General procedure for compounds 14a,b. To a stirred and cooled solution of 11 (0.23 mmoles) in DMSO (4 mL) and H20 (0.2 mL), sodium borohydride (9.2 mmoles) was added portionwise. The mixture was stirred for additional 12 h at 110 °C. After cooling the precipitate 14 was filtered off.
6-ethyl-2-(hydroxymethyl)-3-methyl-4-phenyl-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one 14a.
Yield = 90%, mp > 300 °C, crystallization solvent: cyclohexane.
1H NMR (CDCl3): 5 7.57 (s, 5H, aromatic), 4.82 (s, 2H, CH2-0H), 4.40 (q, J = 7.5 Hz, 2H, N-CH2CH3, 1.83 (s, 3H, CH3), 1.48 (t, J = 7.5 Hz, 3H, CH2-CH3). Anal. Calcd. For C1)H17N3O2: C, 67.83; H, 6.05; NN,14.83. Found : C, 67.95; H, (5.27; N, 15.08.
6-ethyl-4-(4-fluorophenyl)-2-(hydroxymethyl)-3-methyl-1,6-dihydro-7H-pyrrolo[2,3-rf]pyridazin-7-one
14 b. Yield = 38%, mp > 300, crystallization solvent: cyclohexane.
1H NMR (CDCl3): 5 7.65-7.55 (m, 2H, aromatic), 7.38-7.24 (m, 2H, aromatic), 4.85 (s, 2H, CH2-OH), 4.80 (exch br s, 1H, OH), 4.40 (q, J = 7.5 Hz, 2H, N-CH2CH3, 1.90 (s, 3H,CH3), 1.50 (t, J = 7.5 Hz, 3H, N-CH2-CH3). Anal. Calcd. For C16H16FN302: C, 63.78; H, 5.35; 13.95. Found: C, 64.02; H, 5.16; N, 14.19.
General procedure for compounds 15a-d. To a solution of compound 10 (0.17 mmoles) in ethanol (3 mL), 6N NaOH (2 mL) was added and the mixture was stirred at 50 °C for 4 h. After concentration in vacuo the residue was diluted with ice-cold H20 (10 mL) and acidified with 6N HCl. The precipitated 15 was collected by suction.
6-ethyl-4-(4-fluorophenyl)-3-methyl-7-oxo-6,7-dihy-drothieno[2,3-rf]pyridazine-2-carboxylic acid 15a.
Yield = 58%, mp = 270 °C dec., crystallization solvent: ethanol.
1H NMR (DMSO-d6): 5 7.54-7.46 (m, 1H, aromatic), 7.38-7.25 (m, 2H, 6aromatic), 4.45-4.35 (m, 2H, N-CH2-CH3), 3.20 (exch br s, 1H, OH), 2.20 (s, 3H,CH3), 1.50 (t, J = 7.5 Hz, 3H, N-CH2-CH3). Anal. Calcd. For C16H13FN203S: C, 57.82; H, 3.94; N, 8.43. Found: C, 57.63; H, 4.19; N,8.70.
6-ethyl-4-(3-fluorophenyl)-3-methyl-7-oxo-6,7-dihy-drothieno[2.3-rf]pyridazine-2-carboxylic acid 15b.
Yield = 84%, mp = 285 °C dec., crystallization solvent: ethanol.
1H NMR (CDCl3): 5 7.60-7.40 (m, 1H, aromatic), 7.28-7.15 (m, 3H, aromatic), 4.36 (q, J = 7.6 Hz, 2H, N-CH2CH3), 2.20 (s, 3H, CH3), 1.42 (t, J = 7.6 Hz, 3H, N-CH2CH3). Anal. Calcd. For C1gH13FN2O3S: C, 57.82;
H,	3.94; N, 8.43. Found: C, 57.69; H, 4.11; N,8.47.
6-ethyl-3-methyl-7-oxo-4-(3-nitrophenyl)-6,7-dihy-drothieno[2,3-rf]pyridazine-2-carboxylic acid 15c.
Yield = 86%, mp = 255-257 °C, crystallization solvent: ethanol.
1H NMR (CDCl3): 5 8.43-8.37 (m, 2H, aromatic), 7.90-7.73 (m, 2H, aro3matic), 4.90 (exch br s, 1H, OH), 4.40 (q, J = 7.5 Hz, 2H, NCH2CH3), 2.20 (s, 3H, CH3),
I.40	(t, J = 7.5 Hz, 3H, N-CH2CH3). Anal. Calcd. for C1gH13N3O5S: C, 53.48; H, 3.65; N, 11.69. Found: C, 5^.72; H, 3.99; N,11.47.
7.84-7.62 (m, 2H, aromatic), 5.58-5.42 (m, 1H, CH(CH3)2), 3.00 (exch br s, 1H,0H ), 2.23 (s, 3H, CH3), 1.45 (d, J = 7.5 Hz, 6H, (CH3)2CH). Anal. Calcd. for C17H15N305S: C, 54.68; H, 4.05; N, 11.25. Found: 54.51; H, 4.12; N, 11.46.
General procedure for 16a-d. Compound of type 15 (0.25 mmoles) was suspended in SOCl2 (16 mmoles). The mixture was refluxed for 4 h. After cooling the excess of reagent was evaporated in vacuo and the crude residue after cooling was treated with a cold solution of 30% aqueous ammonia. The precipitate 16 was filtered off.
6-Ethyl-4-(4-fluorophenyl)-3-methyl-7-oxo-6,7-dihy-drothieno[2,3-rf]pyridazine-2-carboxamide 16a. Yield = 68%, mp = 264-265 °C, crystallization solvent: ethanol.
1H NMR (DMSO-dg): 5 8.00 (exch br s, 2H, NH), 7.60-7.56 (m, 2H, aromatic), 7.41-7.22 (m, 2H, aromatic), 4.28 (q, J = 7.5 Hz, 2H, N-CH2CH3), 1.90 (s, 3H, CH3), 1.30 (t, J = 7.5 Hz, 3H, N-CH.CH3). Anal. Calcd. for C1gH14FN3O2S: C, 57.99; H, 4.26; N, 12.68: Found: C, 58.18; H, 4.41; 12.74.
6-Ethyl-4-(3-fluorophenyl)-3-methyl-7-oxo-6,7-dihy-drothieno[2,3-rf]pyridazine-2-carboxamide 16b. Yield 75% , mp = 242-244 °C, crystallization solvent: ethanol.
1H NMR (DMSO-dg): 5 7.90 (exch br s, 2H, NH), 7.60-7.30 (m, 4H, aromatic), 4.20 (q, J = 7.5 Hz 2H, N-CH2CH3), 1.90 (s, 3H, CH3), 1.30 (t, J = 7.5 Hz, 3H, N-CH2CH3). Anal. Calcd. for C1gH14FN3O2S: C, 57.99; H, 4.2(5; N, 12.68: Found: C, 57.8:); I^, 4.^2; N, 12.86.
6-Ethyl-3-methyl-4-(3-nitrophenyl)-7-oxo-6,7-dihy-drothieno[2,3-rf]pyridazine-2-carboxamide 16c. Yield 65% , mp = 231-233, crystallization solvent ethyl ether
1H NMR (DMSO-dg): 5 8.40-7.80 (m, 4H, aromatic), 4.20 (q, J = 7.5 Hz, 2H, N-CH2CH3), 1.90 (s, 3H, CH3), 1.30 (t, J = 7.5 Hz, 3H, N-CH.CH3). Anal. Calcd. for C1gH14N4O4S: C, 53.62; H, 3.94; N, 15.63. Found: C,53.4tó; II, 44.21; N, 15.43.
6-Isopropyl-3-methyl-4-(3-nitrophenyl)-7-oxo-6,7-dihydrothieno[2,3-rf]pyridazine-2-carboxamide 16d.
Yield 65% , mp = 263-265, crystallization solvent etha-nol
1H NMR (DMSO-dg): 5 8.40-8.36 (m, 1H, aromatic), 8.05-7.50 (m, 3H, 6aromatic), 5.32-5.20 (m, 1H, (CH3)2-CH), 1.90 (s, 3H, CH3), 1.32-1.23 (m, 6H, (CH3)2-CH). Anal. Calcd. for C17H1gN4O4S: C, 54.83; H, 4.33; in, 15.04. Found: C, 55.06; H, ^4.02; N, 15.38.
6-isopropyl-3-methyl-7-oxo-4-(3-nitrophenyl)-6,7-dihydrothieno[2,3-rf]pyridazine-2-carboxylic acid 15d.
Yield = 42%, mp = 144-146 °C, crystallization solvent: ethanol.
1H NMR (CDCl3): 5 8.46-8.35 (m, 2H, aromatic),
5. Acknowledgement
The National Cancer Institute of Bethesda is gratefully acknowledged for the biological tests.
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Povzetek
Sintetizirali smo nove pirolo[2,3-d]- in tieno[2,3-d]-kondenzirane piridazinske derivate. Sinteza temelji na oksidativ-nem razcepu izoksazolo[3,4-d]piridazinskih prekurzorjev s CAN, ki mu sledi ciklokondenzacija z dinukleofili. Končni produkti so bili testirani in vitro na antiproliferacijsko aktivnost pri treh vrstah človeških celic, vendar nobena od spojin ni bistveno zmanjšala rasti.