TECHNICAL FIELD

The invention relates to an agent containing carboxylic acid hydrazide-coumarin hybrid compounds with anti-inflammatory and anticonvulsant effects with various biological activity potentials.

PRIOR ART

Coumarin and hydrazides are used in biology, medicine, chemistry, paint, cosmetics, etc. It is used in various fields for different purposes (eg, antimicrobial, dyestuff, preservative). These compounds have been the focus of research in recent years due to their biological activities. Many researchers have carried out studies involving different derivatives of these compounds.

Coumarins are members of the class of heterocyclic compounds with the benzopyran-2-one structure. Due to the presence of different radical groups, they are important compounds especially for the pharmaceutical industry, as they have a wide range of biological activities. Coumarins, which are heterocyclic structures containing oxygen; It is an important group naturally found in many plant groups and produced synthetically, together with being isolated from plants for commercial use for years. It has wide usage areas such as coumarin derivatives, perfume, food, plastic, paint industry.

Hydrazides are intermediate step products mostly synthesized in organic chemistry. It is seen that the reactions occur as a result of nucleophilic attack on the carbonyl or thionyl carbon due to the fact that the three nitrogen atoms are active. When the other usage areas of hydrazides are examined, apart from being an intermediate step product, it has been seen that maleic hydrazides have been used in agriculture for many years to regulate the growth of plants.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to carboxylic acid hydrazide-coumarin hybrid compounds with anti-inflammatory and anticonvulsant effects, with various biological activity potentials. Within the scope of the invention, the anticonvulsant and anti-inflammatory effects of coumarin-hydrazide hybrid molecules, which have not been used together in the literature before, are utilized, allowing these new hybrid compounds to be used in fields such as pharmaceutical chemistry.

When the anti-inflammatory activity is evaluated in 3 different dosages of 5, 10, 25 mg I kg for 5 hours; Changes in paw edema levels and percent inhibition are observed in all groups. The most effective coumarin-hydrazide hybrid derivative was found to be N'-(2-hydroxybenzoyl)-2-oxo-2H-chromene-3-carbohydrazide. On the other hand, the pentylenetetrazol (PTZ, 80 mg/kg, i.p.) induced seizure model was used to investigate the anticonvulsant activities of six newly synthesized coumarin- hydrazide hybrid derivatives in mice. Salicylic acid hydrazide and 3-carbonyl chloride coumarin (8d) hybrid compound was determined as the most promising anticonvulsant agent among all treatment groups according to seizure onset and survival rate.

LIST OF FIGURES

Figure 1. Synthesis of Coumarin-Phenolic Acid Hydrazide Hybrid Compounds

DETAILED DESCRIPTION OF THE INVENTION

The invention is a combination of (E)-Ni-cinnamoyl-2-oxo-2H-chromene-3- carbohirazide and (E)-Ni-(3-( 4-hydroxyphenyl)acryloyl)-2-oxo-2H-chromene-3- carbohydrazide relates to N 1 -(2-hydroxybenzoyl)-2-oxo-2H-chromene-3- carbohydrazide at low doses and at all doses.

Inflammation is the body's natural defense mechanism against irritants, trauma, pathogens or microbial invasion. It occurs with symptoms such as pain, swelling, redness, heat and dysfunction in the human body (Aghasafari et al., 2019; Minhas et al., 2017; Pahwa and Jialal, 2019). Migration and activation of leukocytes causes tissue destruction and increased blood vessel permeability. Thus, inflammation may occur through activation of various enzymes (lipoxygenases and cyclooxygenases (COX), production of inflammatory mediators (leukotrienes and prostaglandins) and reactive oxy-gene species (Abdel-Lateff et al., 2020).

In the current study, 0.1 ml of 1 % (w/v) carrageenan, commonly used to induce a model of acute inflammation, was injected subplan-tar into the right paw of rats. Carrageenan exerts its effects on paw edema in two stages: The first stage (0-1 hour) is related to the release of serotonin, bradykinin and histamine, and the second stage (from the 2nd hour) is derived from polymorphone-clear leukocyte infiltration and high prostaglan-dins production. Free radical, tumor necrosis factor-a, reactive oxygen species, interleukin-1 b and nitric oxide amounts are also increased due to the release of polymorphonuclear leukocytes (Chauhan et al., 2018; Pasqua et al., 2019). In this study, indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), was used as the positive control group. The mechanism of action of this drug is to reduce the production of prostaglan-dins and prevent the production of prostaglandins from arachidonic acid (Lucas, 2016).

Table 1 - Synthesis of Diacyl Hydrazide-Coumarine Derivatives

In laboratory studies; To screen the anti-inflammatory profile of Diacyl Hydrazide-Coumarin derivatives (8a-f) synthesized from aryl acids, 3 different dosages (5, 10, 25 mg/kg) were tested for 5 hours. Changes in paw edema level and percent inhibition of all groups are shown in Table 2. According to the results, it was determined that the anti-inflammatory effects of all the tested compounds were dose dependent. At 1 hour, the maximum percent inhibition of carrageenan-induced paw edema volumes was demonstrated by compound 8c with the highest dose (25 mg/g dose; 57.14%), while the lowest dose (5 mg/kg) 2nd, 3rd, 4th hour, and 5 hours (65.75%; 52.38%; 36.60% and 45.29%, respectively) were found to be more effective than the control group.

Values are expressed in Mean ± SEM (n = 6 animals for each group). Two-way ANOVA was carried out followed by post hocTukey multiple comparison test. Compounds were compared to the control group and statistical significance is expressed as *p<0.05, **p<0.01 , ***p<0.001.

Table 2 - Inhibition of paw edema on paw edema volume (ml) and % carrageenan- induced inflammation model (n=6)

Indomethacin caused significant inhibition of carrageenan-induced paw edema volume at 1 , 2, 3, 4, and 5 hours (40.00%, 76.71 %, 63.80%, 58.92%, 52.99%, respectively) compared to the control group. The results show that at 1 hour, the antiinflammatory effect of 8c with a dose of 25 mg/kg (57.14%) was higher than the antiinflammatory effect of indomethacin (40.00%). However, among the compounds tested, the lowest dose 8d had the maximum mean inhibition of paw edema volume over five hours. Furthermore, the anti-inflammatory effects of 8c and 8f increased with an increase in dose administered relative to percent inhibition of paw edema volumes. Considering the structure and activity relationship, it is clear that hydrophilic substituents on the aryl subunits of diacyl-coumarin molecules significantly reduce the inhibition of carrageenan-induced paw edema volume.

Among the tests used to evaluate anticonvulsant activity, the Prothrombin Time (PTZ) test has a predictive relationship to the clinical activity spectrum of the experimental compounds. Because the Prothrombin Time (PTZ) test is assumed to identify anticonvulsant drugs effective against human generalized tonic-clonic seizures (Khoshnood-Mansoorkhani et al., 2010). Prothrombin Time (PTZ) is a non-competitive antagonist of GABAA receptors that acts through the tert-butyl-bicyclo- phosphorothionate domain of the receptor and reduces its activity.Another assumption for the Prothrombin Time (PTZ) mechanism is to alter potassium and calcium channel conductivity (Asadi-Shekaari et al., 2014). GABA is an important inhibitory neurotransmitter in the brain, and GABA is the underlying factor in epilepsy (Gowda et al., 2012). Currently used anticonvulsant drugs effectively control epileptic seizures in about 75% of patients. In addition, treatment difficulties arise from the undesirable side effects of clinically used drugs; therefore, the newer antiepileptic drugs have a greater effect than the more established anticonvulsant drugs. Therefore, newly synthesized diaryl hydrazine-coumarin derivatives are being seriously investigated for their antiepileptic potential (Alrohaimi et al., 2014; Khoshnood-Mansoorkhani et al., 2010; Zhu et al., 2014). Carbamazepine, a standard anticonvulsant drug, exerts its antiepileptic effects by increasing sodium channel inactivation by reducing the high- frequency repetitive firing of action potentials and its effect on synaptic transmission (Tolou-Ghamari et al., 2013). It is widely used as a standard drug in anticonvulsant activity studies and we used carbamazepine as a reference antiepileptic drug in our studies on the invention.

In the study, which is the subject of the invention, experimental studies of six newly synthesized diaryl hydrazine-coumarin derivatives were carried out with the anticonvulsant potential PTZ model at three different doses. Compounds (E) -Ni - cinamoyl-2-oxo-2H-chromene-3-carbohirazide (8b) and (E) -Ni - (3-(4- hydroxyphenyl)acryloyl)-2-oxo-2H-chromene-3 Our experimental studies have shown that -carbohydrazide (8c), at low doses and at all doses, Ni - (2-hydroxybenzoyl)-2- oxo-2H-chromene-3-carbohydrazide (8d), has significant anticonvulsant potential against PTZ-induced convulsions. The PTZ-induced seizure model in the discovery of new antiepileptic drugs has the advantages of using robust rodents as easy models to detect anticonvulsant effects regardless of their mechanism of action.

PTZ (80 mg/kg, i.p.) induced tonic-clonic seizures in all animals used. All experimental groups were compared with the model control group. Mice pretreated with diacyl hydrazide-coumarin derivatives at three different doses of 30, 100 and 300 mg/kg p.o. Convulsion onset, percentage of grades, and mortality were compared. The lowest dose (30 mg/kg) and medium dose (100 mg/kg) compounds 8b, 8c and 8d significantly delayed the onset of convulsions compared to the control group (1.07 ± 0.04).

Values are expressed in Mean ± SEM (n = 7-10 animals for each group). Two-way ANOVA was carried out followed by post hoc Tukey multiple comparison test. Compounds were compared to the control group and statistical significance is expressed as *p<0,05.

Table 3 - Effects of coumarin hydrazide hybrid derivatives on pentylenetetrazole (PTZ)-induced seizures in mice (n=7-10)

The results are given in table 3; 8b-1 (2.41 ± 0.26), 8c-1 (2.10 ± 0.54), 8d-1 (2.09 ± 1.03) and 8d-2 (2.16 ± 1.03). On the other hand, survival was significantly increased by group B (75%); 8b-1 (86%); 8c- 1 and 8c-3 (71 %); 8d-1 , 8d-2 and 8d-3 (71 %, 86%, 86%) and 8f-3 (71 %) compared to the control group (50%). Standard anti-epileptic drugs, carbamazepine (100 mg/kg) inhibited severe tonic-clonic convulsions, significantly lowering grade IV to 6.23%, though not delaying the onset of convulsions, especially compared to the control group (22.18%) and mortality in mice. Although the survival rate of carbamazepine was 75%, it was also observed that half of the animals in this group had long-term grade 5 activity for more than half of the observation period. Compound 8b donated with two hydrophobic aryl units showed a potent anticonvulsant effect at low dose but this effect was reversed at higher doses. The survival rate decreased to 29% at medium and high doses. Broadly speaking, single compound 8d has anti-convulsant potential in a dose-dependent manner. The mean site of death increased in a dose-dependent manner. Furthermore, compounds 8b and 8c may have low-dose anticonvulsant potential, while compound 8f has dose-dependent potential relative to the mean in situ death and survival rate. Seizure onset and mortality increased in these treatment groups, while several grades and mortality decreased. The anticonvulsant activity of these treatment groups may be explained by inhibited and/or attenuated PTZ-induced seizures in mice and/or neutralizing the PTZ binding site by enhancing GABA-mediated inhibition and/or reducing excitatory neurotransmission and/or blocking sodium channels.