Synthesis and Biological Activity of New [1,3]Thiazolo[4,5-d]pyridazin-4(5H)-ones

A series of novel 2-(N-pyrrolidino, N-piperidino or N-morpholino)-7-phenyl(α-furoyl or α-thienyl)-[1,3]thiazolo[4,5-d]pyridazinones 10a–c, 14–16a,b was synthesized in 78–87% yields via the reaction of methyl 5-benzoyl(α-furoyl or α-thienyl)-2-aminosubstituted-thiazol-4-carboxylates 9a–c, 13a–e with hydrazine. These new compounds have been tested for their in vivo analgesic and anti-inflammatory activities. All compounds have been characterized by 1H-NMR, 13C-NMR spectroscopy, and liquid chromatography–mass spectrometry.

1-Pyrrolidinecarbothioamide 4a was obtained with the yield 73% through the condensation of pyrrolidine with ethyl isothiocyanatidocarbonate 2 and further hydrolysis of еster 3 with a well-known technique [3]. Key ethyl isothiocyanatidocarbonate 2 was synthesized by a modified technique [4] from ethyl chloroformate and dry potassium rhodanide in the presence of tetramethylethylenediamine (TMEDA) as a catalyst.
After the condensation of esters 9а-с and 13a-e to [1,3]thiazolo [4,5-d]pyridazinones 10a-c, 14-16a,b in the 1 H-NMR spectra, signals of the methyl ester group were not observed and appeared as broad singlet signals of the NH proton at 12.8-13.1 ppm. These signals disappeared after adding one drop of D 2 O to the sample before recording the spectra.

Pharmacology
Analgesics that are used today, whose mechanism of action is associated with the effect on COX isoenzymes, have significant side effects. Therefore, it is important to search new analgesics exceeding the existing ones in efficacy and/or safety [12]. This study was conducted to examine the possible peripheral and central action of new analgesics. The study is a fragment of the determination of the efficiency derivatives of 2-(N-pyrrolidino, N-piperidino or N-morpholino)-7-phenyl(α-furoyl or α-thienyl)- [1,3]thiazolo [4,5-d]pyridazinones in acute pain. Pain syndrome that occurs after intraperitoneal injection of acetic acid in mice is manifested through a peculiar abdominal constriction (writhing). It is associated with irritation of the peritoneum and is regarded as a model of visceral pain. In this case, intraperitoneal injection of 0.6% acetic acid solution assists in a general activation of the nociceptive system and local release of bradykinin, histamine, serotonin, prostaglandins, etc [13]. Acetic acid causes an increase in peritoneal fluid levels of prostaglandins (PGE2 and PGF2α), involving in part peritoneal receptors and inflammatory pain by inducing capillary permeability [14,15]. The above result indicates that in the model of nociceptive stimulation (writhing caused by intraperitoneal injection of acetic acid), the substance outperforms ketorolac by about 1-fold.
The hot plate model describes the supraspinal level of nociception, which may allow us to define the central component of the antinociceptive activity of the analgesic. This test characterizes the effectiveness of the compound as to its suppression of somatic superficial and acute pain. These data indicate a central component of analgesia derivatives of [1,3]thiazolo [4,5-d] pyridazinones (10b, 10c, 14b, 16a, 16b).
The substances 10a and 16a have shown an antiexudative effect (30.97% and 34.8%, respectively) in the carrageenan edema model on mice at the dose 25 mg/kg (Table 1).

Tab. 1.
Comparative evaluation of analgesic activity and antiexudative effect of derivatives [1,3] The reference medicine ketorolac induced a 41.1% edema reduction at the study dose as compared to the control group. In applying substances 10a, 16a,b in the acetic acidinduced writhing test on the mice at the dose of 25 mg/kg, a significant analgesic effect was determined 60 min after administration (Table 1).
For this purpose, the study substance and ketorolac as the reference drug were administered 60 min before injection of acetic acid and the amount of writhing in the control and treated groups was recorded. It was found that in the dose of 25 mg/kg, the compounds inhibited the amount of writhing in the range from −21.05% to −66.60%.
In the hot plate test, the five derivatives of [1,3] The basic structures with the phenyl substituent in position 7 (10b,с) provided the antinociceptive impact close to that caused by 104.9-110.6% ketorolac on the "hot plate" model which indicates the central component of the analgesic influence. Substituting the nitrogen atom for the oxygen atom in the pyridine fragment modulated the pronounced central and peripheral components of pain (10а-с).
Substituting the oxygen atom for a sulfur atom within the furane ring provided no sufficient effect.
Respective substitution of the piperidine ring for a pyrrole fragment and the furane ring for a thiophene fragment (14b) allowed registering the biggest value of the antinociceptive effect +144.2% on the "hot plate". On the contrary, substituting the thiophene fragment for the furane fragment cancelled (14а) the antinociceptive effect +49.2%.
Anti-inflammatory activity of the studied compounds was low and thus, was inferior to ketolac to which they were compared. Out of the basic structures with the phenyl substitutor in position 7 (10а-с), the 10а structure was the only one to reveal a valid antiexudative effect (−30.97%). Meanwhile, this structure was not active in the acetous "writhing" model where mediators of inflammation participate in generating nociceptive sensation. The 16а compound revealed valid antiexudative activity as well as antinociceptive (−34.8%). Therefore, we can regard it as a starting point to search further for non-steroidal anti-inflammatory drugs.

Conclusion
In conclusion, we synthesized a series of new [1,3]thiazolo [4,5-d]pyridazinones. Comparative evaluations of analgesic activity and the antiexudative effect of derivatives [1,3]thiazolo [4,5-d]pyridazinones with ketorolac on the «hot plate» and «acetic acid cramps» models were studied. Compounds exceeding the analgesic activity of ketorolac were found. It was shown that the compounds of this series exhibited moderate antiinflammatory activity.

Chemistry
All solvents were purified before use. Ethyl chlorophormiate, pyrrolidine, morpholine, piperidine, acetophenone, ethyl oxalate, 2-acetylthiophene, and 2-acetylfurane were purchased from Acros Organics and used without purification. Reactions were monitored by thin-layer chromatography (TLC) using Fluka silica gel (60 F 254) plates (0.25 mm). Visualization was made with UV light. Melting points of the synthesized compounds were taken on a melting point tube. 1

Animals
Female non-linear mice (18-22 g) were used. The animals were housed in a quarantine facility for 7 days before the experiment was started. Throughout the experiment, the animals were randomised in groups of four per cage with bedding composed of wood shavings (exchanged daily). The animals had free access to a standard commercial diet and water. The animals were kept under a stable regimen of 12 h light/12 h darkness. All studies were performed under the requirements HEC of the Ministry of Health of Ukraine and the rules of the "European Convention for the Protection of Vertebrate Animals are used with Experimental and other Scientific Purposes" (Strasbourg, 1986).
The study substance was administered once orally (p.o) at the dose 25 mg/kg [16] in the form of an aqueous-ethanol emulsion using Twin-80 as an emulgator. Ketorolac was administered once orally (p.o.) in the form of an aqueous solution. The dose of 25 mg/kg was selected according to methods described in [17,18] for it will definitely be considered from the standpoint of the studied compound's therapeutic impact.

Hot Plate Test
Evaluation of analgesic activity in the experiment was carried out on thermal models of the nociceptive-stimulating "hot plate" [19]. The analgesic activity founded on the change of the latency of "paw licking" (hot plate) was evaluated. At the same time, we determined the percentage of change of the latent period of the reaction relative to the threshold of the reaction at the initial (point). The hot plate test was assessed on groups of five mice. The temperature of the metal surface was maintained at (55 ± 0.2)°C. Latency to a discomfort reaction (licking paws or jumping) was determined before and after drug administration. The cutoff time was 20 sec. The latency was recorded before and 1 hour following p.o. administration of the agents (ketorolac or study substance). The prolongation of the latency times compared with the values of the initial was used for statistical comparison.

Acetic Acid-Induced Writhing Model in Mice
The study substance was assessed by the reduction of the number of writhes induced by intraperitoneal injection of 0.6% acetic acid. The number of writhes per animal was counted for 10 min [20,21]. Inhibition of writhing was calculated by the formula below and compared with the standard drug (ketorolac): Inhibition of writhing, % = {(Wc − Wt) × 100%}/Wc where, Wc = number of writhing in the control group; Wt = number of writhing of the experimental group.

Carrageenan-Induced Inflammation in Mice
The study substance was administrated p.o. to mice 1 h before subplantar injection of 0.05 ml of 1% carrageenan [22]. The experimental group size was five animals. Three h after carrageenan injection, the mice were withdrawn from the experiment. Hind paws with swollen and non-swollen feet were amputated at the level of the hip joints. The control group was treated with a solvent. The control group size was five animals. The inhibition of edema was calculated using the formula: Inhibition of edema was calculated by the formula above and compared with the standard drug (ketorolac).