Study of 6,7-dimethoxy-4-n-(4-cyanophenyl)aminoquinazoline as jnk inhibitor in vivo

Biological research has been conducted in vivo study of JNK activity of 6,7-dimethoxy-4-N-(4-cyanophenyl)aminoquinazolin. It has been established that this compound significantly reduces the levels of glucose and insulin in the serum, and also reduces the levels of free fatty acids and triacylglycerols in the serum and liver of rats received fructose enriched diet. Furthermore, the substance reduces the amount of p-JNK, which indicating that the inhibitory activity of JNK.

iNTRoDUCTioN. c-Jun N-terminal kinases (JNK) – group of stress activated MAPK that takes part in growth regulation, differentiation, apoptosis, inflammation and other important cellular processes [1, 2, 3]. It is known that JNK activation occurs in response to the accumulation of reactive oxygen species, in hyperglycemia etc., and involved into development of insulin resistance, metabolic syndrome, diabetes, cardiovascular diseases and other pathologies [4, 5]. Several experimental studies demonstrated positive

influence of JNK depression to type 2 diabetes mellitus, obesity, atherosclerosis [6, 7, 8]. Since these pathologies are widespread among human population, research of JNK inhibitors is the acute and topical issue.

Recently potent inhibitors of JNK containing cyano group have been found. The first of these inhibitors was developed 1,3-benzothiazol-2-yl-(2-[[2-(3-

pyridinyl)ethyl]amino}-4-pyrimidinyl)acetonitrile AS601245 (Figure. 1) [9, 10]. But lack of AS601245 is weak penetration into a cell [11, 12].

We had searched inhibitors of JNK among cyano studied against a background of incubation of cells with

derivatives of 4-aryloquinazolines that have good insight hepatotoxin acetaminophen, for the mechanism of its

into the cell and the ability to inhibit tyrosine kinase. The action is indispensable the phosphorylation and

study was conducted in vitro on hepatocytes of male rats translocation of JNK, i.e. activation of this signaling

Figure 2 - The formula of derivatives of 4-arylquinazolines: compound 6,7-dimethoxy-4-N-(4-cyanophenyl)aminoquinazolin
that showed an inhibitory effect on the activity of JNK

Conducted preliminary investigations in vitro have shown that 6,7-dimethoxy-4-N-(4-cyanophenyl)-

aminoquinazolin (IK006) produces dose-dependent decreasing of ALT activity in hepatocyte incubation medium and reduces the content of phosphorylated JNK

against a background of JNK activator acetaminophen, that reliably exhibits inhibitory effect for JNK [14].

The aim of this research was to study the potential activity of substance IK006 under experimental insulin resistance.

48

MATERIALS AND METHODS OF ANALYSIS

Experiment was carrying out on Wistar rat males. Insulin resistance was modulated by feeding animals with enriched fructose diet (60,3 % fructose, 18,3 % protein, 5,2 % fat) which is accompanied by obesity, impaired carbohydrate and lipid metabolism [15]. Experimental animals weighing 180-220 g were randomly divided into four groups: 1) intact animals received vivarium standard diet of National University of Pharmacy; 2) animals received fructose enriched diet during 6 weeks; 3) animals received fructose enriched diet during 4 weeks and 2 weeks received fructose enriched diet along with daily injections of suspension of substance to be examined IK006; 4) animals received standard diet during 4 weeks and 2 weeks received daily injections of suspension of the substance to be examined IK006. Animals were decapitated with chlorazol urethane anesthesia. Study subjects were blood serum and liver homogenate. “Ethical principles for animal experiments” (Ukraine, 2001) harmonized with “European Convention for the Protection of Backboned Animals Used for Experimental Purposes and Other Scientific ones” (Strasbourg, 1985) were committed during this experiment.

Determination of glucose, insulin, free fatty acids (FFA) and triacylglycerols (TAG) content was made with standard set manufactured by the firm «Phelisit- Diagnostic» (Ukraine) and the firm «Lachema» (Czech Republic).

Determination of total concentration of β- and pre-β- lipoproteins (apoВ-lipoproteins) and high-density lipoproteins (HDL) in blood serum was carried out by turbidity method. Determination of 2-thiobarbituric acid - reactive products (TBA-RP) content was carried out by spectrophotometric method performing reaction with thiobarbituric acid [16]. Determination of total level of JNK was performed using reagents kit (Total JNK Pan Specific DuoSet IC ELISA (R&D Systems, Inc., USA). Determination of phosphorylated JNK (р-JNK) was carrying out with reagents kit [pThr183/Tyr185] JNK1/2 EIA kit (Enzo Life Sciencies).

Statistical processing of the received data was carried out with program STATISTICA (StatSoft Inc., USA, version 6.0). Importance of intergroup differences was estimated by Student t-test.

RESULTS AND DISCUSSIONS

After enriched fructose diet rats had two-times increase of glucose level in the blood serum (table 1). At the same time they had hyperinsulinemia (table 1) that along with in-parallel hyperglycaemia gives evidence of cells insusceptibility to insulin, development of insulin resistance. After 6 weeks of experiment increase of free fatty acids (FFA) and TAG concentration (table 1) was observed, it is the result of fat mobilization from fat tissue and intensification of liver low density lipoprotein synthesis because of depression of insulin inhibitory action to lipolysis. Accumulation of atherogenic apoВ- lipoproteins in this group of rats (table1) is compensatory reaction intended to FFA content reduction associated with increased endogenous lipoprotein synthesis and depressed utilization [17]. Otherwise there was lowering tendency towards the level of atherogenic HDL (table 1), that may be caused due to apoВ-lipoproteins remodelling disorder and katabolism increase of HDL.

Table 1 - Glucose, insulin, TAG, FFA and TBA-RP content in rats blood serum received fructose diet and/or injections of substances to be examined (M ± m, n = 6)

• - difference is truly per intact (р≤0,05),
• - difference is truly per Diets (р≤0,05), t - intact-related tendency (0,05≤р≤1)

There is accumulation of FFA and TAG in the livers of animals received high-fructose diet (table 2). FFA increase in the blood and increase of their passing to the cells of different tissues encourage further metabolic disease and enhance hyperglycemia and hyperinsulinemia. It is established that one of FFA toxicity mechanism is enhanced reactive oxygen intermediate formation and lipid peroxidation activation [17]. Our results give evidence of antioxidant-oxidant balance abnormality - accumulation of product peroxidation

(TBA-RP) was shown as in blood serum and liver of rats (table 1,2). It is known that considerable contribution of insulin resistance development makes increase of reactive oxygen species (RAS) and one of RAS mechanism of action is JNK signaling [18]. Fructose diet is not influence on the total JNK level during investigation period as shown at the table 2, however promotes its activation that show the increase of р-JNK content up to 70% in comparison with intact.

Table 2 - Triacylglycerols, FFA, TBA-RP content and levels of total and phosphorylated JNK in rats liver homogenate received fructose diet and/or injections of substances to be examined (M ± m, n = 6)

• - difference is truly per intact (р≤0,05),
• - difference is truly per Diets (р≤0,05)

IK006 injections significantly decrease glucose and insulin concentration in blood serum (table 1) and also lower the FFA and TAG level in blood serum and liver (table 1, 2). As the result of apoB-lipoproteins concentration reduced in blood serum of animals that received fructose enriched diet (table 1). It should be noted that the substance IK006 showed significant action. This compound is also exercise improved influence upon TBA-RP accumulation in both studied tissues. (table 1, 2). Fructose enriched diet related normalization tendency of р-JNK level in liver due to IK006 injections (table 2). IK006 compound has shown high potency. By the same time this compound do not show any activity for one of the researched factors. So IK006 compound didn't demonstrate directly hypoglycemic, hypolipidemic and antioxidant effects due to separate injections but it had such effects on affected by JNK activation and also reduce its activation.

We can consider a possibility of application compound as complex protection from JNK activation states.

CONCLUSIONS

Summing up what has been said the carried out researches show the followings:

1. The studied compound IK 006 for lowering the levels of glucose and insulin in blood serum related with insulin resistance
2. IK006 compound shows normalization on lipid metabolism researched factors of blood serum and liver of rats received fructose enriched diet that could lower risk of atherosclerosis due in case of high-caloric feeding.
3. IK006 compound reduces р-JNK content, lowers JNK activation induced by fructose enriched diet. IK006 showed protective effect.

REFERENCES:

1. Sehgal V. Network Motifs in JNK Signaling / V. Sehgal, P. T. Ram // Genes Cancer - 2013. - Vol. 4, № 9-10. - Р. 409-413.
2. Seki E. A liver full of JNK: signaling in regulation of cell function and disease pathogenesis, and clinical approaches / E. Seki, D. A. Brenner, M. Karin // Gastroenterology. - 2012. - Vol. 143, № 2. - P. 307-320.
3. Vlahopoulos S. JNK: a key modulator of intracellular signaling / S. Vlahopoulos, V. C. Zoumpourlis // Biochemistry Mosc. - 2004. - Vol. 69, № 8. - P. 844-854.
4. A central role for JNK in obesity and insulin resistance / J. Hirosumi, G. Tuncman, L. Chang et al. // Nature. - 2002. - Vol. 420. - P. 333.
5. Tarantino G. JNKs, insulin resistanse and inflammation: A possible link between NAFLD and coronary artery disease / G. Tarantino, A. Caputi // World J. Gastroenterol. - 2011. - Vol. 17, № 33. - P. 3785-3794.
6. Evaluation of JNK blockade as an early intervention treatment for type 1 diabetic nephropathy in hypertensive rats / A. K. Lim, F. Y. Ma, D. J. Nikolic-Paterson et al. // Am. J. Nephrol. - 2011. - Vol. 34, № 4. - P. 337-346.
7. Nakatani Y. Modulation of JNK pathway in liver affects insulin resistanse status / Y. Nakatani, H. Kaneto, D. Kawamoti // The J. Biol. Chem. - 2004. - Vol. 279, № 44. - P. 45803-45809.
8. c-Jun N-terminal kinase 2 deficiency protects against hypercholesterolemia-induced endothelial dysfunction and oxidative stress / E. Osto, C. M. Matter, A. Kouroedov et al. // Circulation. - 2008. - Vol. 118, № 20. - P. 2073-2080.
9. AS601245, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduces axon/dendrite damage and cognitive deficits after global cerebral ischaemia in gerbils / S. Carboni, U. Boschert, P. Gaillard et al. // Br .J. Pharmacol. - 2008. - Vol. 153, № 1. - P. 157-163.
10. AS601245 (1,3-benzothiazol-2-yl (2-[[2-(3-pyridinyl) ethyl] amino]-4 pyrimidinyl) acetonitrile): a c-Jun NH2-terminal protein kinase inhibitor with neuroprotective properties / S. Carboni, A. Hiver, C. Szyndralewiez et al. // J Pharmacol. Exp. Ther. - 2004. - Vol. 310, № 1. - P. 25-32.
11. Sweeney S. E., Firestein G. S. Mitogen activated protein kinase inhibitors: where are we now and where are we going? / S. E. Sweeney, G. S. Firestein // Ann. Rheum. Dis. - 2006. - Vol. 65. - P. 83-88.
12. Bubici C., Papa S. JNK signaling in cancer: in need of new, smarter therapeutic targets / C. Bubici, S. Papa // Br. J. Pharmacol. - 2014. - Vol. 171, № 1. - P. 24-37.
13. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation / [K. Du, C. D. Williams, M. R. McGill et al.] // Toxicol. Appl. Pharmacol. - 2013 - Vol. 273, № 3. - Р. 484-491.
14. Пошук інгібіторів c-jun N-кінцевих кіназ (JNK) серед 4-Ñ-(3-цɪанофенɪл)амɪно- та 4-N-(4- ціанофеніл)амінозаміщених хіназолінів / А. Л. Загайко, В. П. Филимоненко, І. Ю. Капустянський та ін. // Украïнський біофармацевтичний журнал. – 2014. – № 3 (32). – С. 55–59.
15. Endothelial dysfunction in high fructose containing diet fed rats: increased nitric oxide and decreased endothelin-1 levels in liver tissue / M. Altas, A. Var, K. Ozbilgin et al. // Dicle University Med. School. – 2010. – Vol. 37, № 3. – P. 193-198.
16. Строев Е. А. Практикум по биологической химии / Е. А. Строев, В. Г. Макарова – М.: Высшая школа, 1986. – 231 с.
17. Загайко А. Л. Метаболічний синдром: механізми розвитку та перспективи антиоксидантноï терапİï: Монографія / А. Л. Загайко, Л. М. Вороніна, К. В. Стрельченко. – Х.: Вид-во НфаУ: Золоті сторінки, 2007. – 216 с.
18. с-Jun N-terminal kinase (JNK) signaling: recent advances and challenges / M. A. Bogoyevitch, K. R. Ngoei, T. T. Zhao et al. // Biochim. Biophys. Acta. – 2010. – Vol. 1804, № 3. – P. 463-475.