EffEct of 3-substitutEd 1 , 4-bEnzodiazEpin-2-onEs on bradykinin-inducEd smooth musclE contraction

Biochemical properties of 3-substituted 1,4-benzodiazepine determined by the characteristics of their chemical structure. Influence of 3-substituted 1,4-benzodiazepin-2-ones on maximal normalized rate and amplitudes of isometric smooth muscle contraction in rats was investigated. Compounds MX-1775 and MX-1828 demonstrated the similar inhibition effect on bradykinin-induced contraction of smooth muscle like competitive inhibitor des-arg9-bradykinin-acetate to bradykinin B2-receptors. MX-1626 demonstrated unidirectional changes of maximal normalized rate and force of smooth muscle that proportionally depended on bradykinin concentration in the range 10-10-10-6 M. MX-1828 has statistically significant decrease of normalized rate of smooth muscle contraction for bradykinin concentrations 10-10 and 10-9 M by 20.7 and 8.6%, respectively, but for agonist concentration 10-6 M, this parameter increased by 10.7% and amplitude was reduced by 29.5%. Compounds MX-2011, MX-1785 and MX-2004 showed no natural effect on bradykinin-induced smooth muscle contraction. Compounds MX-1775, MX-1828, MX-1626 were selected for further research of their influence on kinin-kallikrein system and pain perception.

V arious peripheral mediators contribute to development and maintenance of inflammatory and neuropathic pain by few mecha nisms.Activation or excitation of nociceptive nerve endings or fibers by these substances implicates generation of action potentials which were disseminated on the central nervous system and may induce pain perception.Sensitization of nociceptors occurs in response to various stimuli of external and internal origin, including temperature, mechanical and chemical influences.Bradykinin (BK) is one of the most potent pain-producing agents formed under inflammatory conditions.Multitude of its sensiti zing and excitatory effects on peripheral nociceptors supporting its role as a prototype of peripheral pain mediators have been described [1,2,6,10].A number of bradykinin effects mediated by products of the arachidonic acidcyclooxygenase cascade tes-tify to the existence of mutual interactions between the pain mediators [12].The synthesis of bradykinin and related kallidin is carried out in two ways: intravascularly in plasma and extravascularly in tissues.Prekallikrein formed through clotting factor XII (Hagemann) by formation of kallikrein predecessors that is activated by contact with negatively charged surfaces.Plasma kallikrein acts on high molecularweight kininogen that leads to bradykinin and kallidin producing which act preferentially on B 2 bradykinin receptors.The tissue prekallikrein is transformed to kallikrein upon inflammation or tissue damage.Today two types of bradykinin and related kinin receptors B 1 and B 2 , have been described [7,9,14,16].B 2 type present in the neurons of the brain stem, basal nuclei, cortex, thalamus and hypothalamus.Immune labels of these receptors were found in the endothelium of the upper sagit-tal sinus dura matter and ependyma of the third and lateral ventricles.B 1 kinin receptors are localized in neurons of the thalamus, hypothalamus and spinal cord [13].B 1 and B 2 receptors are important mediators of cardiovascular homeostasis, inflammation and nociception.While the B 2 type is constitutively expressed in many tissues including smooth muscle of intestine and stomach [15], B 1 type synthesis is induced only in inflammatory conditions.However, B 1 is one of the central nociceptive mediators feeling that suggest permanent presence of the receptor in the brain and spinal cord [4].Bradykinin interaction with these receptors leads to activation of Gproteins and specific changes in the levels of [Ca 2+ ] cyt , involving in various systems such as phospholipase C, prostaglandins, protein kinases and phospholipase A 2 [8].Natural and artificially synthesized receptor blockers are important for investigations of action mechanisms of agonists and antagonists of the kininkallikrein system.Search for high affinity and selective non-peptide antagonists that demonstrate prolonged effect and do not decompose in a body by peptidases is very important task for pharmacology and medicine.
B2receptors activation in smooth muscle of stomach can run it contraction as described above.Therefore, the model bradykinininduced contraction of the stomach smooth muscles is simple and informative to investigate the effect of different substances on the function of kallikreinkinin system.
1,4-Benzodiazepine derivatives are synthetic inhibitors that act as highly efficient analgesics.Biochemical properties of 3-substituted 1,4-benzodiazepine are determined by features of their chemical structure.Using radicals with different chemical and physical properties to obtain derivates of basis molecules we created some compounds with affinity for specific biological targets [11].These substances can mimic the βbend that is important for their biochemical activity [5].In addition, such compounds are well sustained by patients.
Previously synthesized in A. V. Bogatsky Physi coChemical Institute of NAS of Ukraine 3-substituted 1,4-benzodiazepin-2-ones exhibiting analgesic activity at doses ranging from 0.007 to 6.6 mg/kg were studied in this paper, some representatives of these series see in Table .These substances are perspective analgesics that have sedative, spasmolytic, antiinflammatory and low toxicity properties.
The existing today compounds that act through opioid receptors cause addiction.Nonsteroidal anti inflammatory drugs have many side effects, inclu ding contribution to the development of stomach ulcers.Compounds, used in our investigation, are deprived these effects.
In view of their low toxic and high analgesic activity, the aim of the study was to investigate influen ce of 3substituted1,4dihydro benzodiazepines on bradykinininduced smooth muscle contraction, as most simple and informative model for interactions of these compounds with kallikreinkinin system.

materials and methods
Tenzometric investigations were carried out using specimens ring stomach muscles of white nonbread male rats which were kept in the standard conditions in vivarium of Educational and Scientific Centre "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv.All manipu lations with animals were carried out in accordance with the "European Convention for the protection of Vertebrate Animals Used for Experimental and other Scientific purposes" and the Law of Ukraine "On protection of Animals from Cruelty".Animal weight was 240260 g.Rats were rapidly decapitated; the stomach was isolated and washed with Krebs solution.Muscle layer from antral part of the stomach was separated from the serosa and mucosa and cut into strips (size -1.52 × 10 mm).
• HighK solution where 80 mM K + was used as initial inducer of contraction for test measurements.It was prepared by replacing sodium ions equimolar number of potassium ions in the Krebs solution.
Bradykinin solutions of specified concentrations were prepared by dilution of 1 mM bradykinin solution.Action of derivatives of 3substituted 1,4benzodiazepine on smooth muscles was evaluated using experimental model of bradykinininduced contraction of smooth muscle strips.Contraction of smooth muscles caused by application of bradykinin in concentration range 10 -10 -10 -5 M. The final concentration of 3-substituted derivatives of 1,4-benzodiazepine in the incubation solution was 10 -6 M. The bradykinininduced contraction of smooth muscle strips on background applications competitive inhibi tor of bradykinin receptor desarg9 [leu8] brady kinin acetate (10 -6 М) was studied for additional evaluation of the biological effects of derivatives of 3-substituted 1,4-benzdiazepines.As derivatives of 3-substituted 1,4-benzodiazepines we used the following compounds: MX 1775, MX2011, MX2004, MX1785, MX1626, MX1828, the structural formulas of which are presented in Table .All compounds were synthesized in A. V. Bogatsky PhysicoChemical Institute, NAS of Ukraine, Odesa.

Structural formulas and molecular weights of 3-substituted 1,4-benzdiazepines and analgesic activi ty (by the method of "acetic acid writhing")
Effectiveness of 3substituted 1,4benzodiaze pines on bradykinininduced contraction we evalua ted in percent by comparison with amplitude (mN) and maximal normalized rate (Vn) of contraction upon impact of bradykinin only.
Every substance of 3substituted 1,4benzodia zepines was tested on 10 separated smooth muscle strips.
Data processing on dynamics of contraction was performed according to the method of T. Burdyha and S. Kosterin [3].Statistical analysis of experimental data was carried out using ShapiroWilk test for control of normality of distribution data.If the data did not have the normal distribution, the comparison was carried out by KruskalWallis indepen dent sample criterion.In case of normal distribution, comparison of control and experimental samples we used ANOVA (Scheffe test) (P < 0.05).

results and discussion
We analyzed the influence of 3substituted 1,4benzodiazepines on amplitude and rate of brady kinininduced smooth muscle contraction.The concentration range of bradykinin was 10 -10 -10 -6 M.
The presence of MX1775 in solution led to the statistically significant increase of Vn by 11.9% and decrease of amplitude by 13.1% (Fig. 1, A, B) for brady kinininduced contraction (10 -6 M).Maximum normalized rate of contraction decreased by 9.1% for agonist concentration 10 -7 М, but force of contraction was not changed.The reducing concentration of BK led to proportional statistically significant increase of Vn as: 10 8 M BK -13.2%, 10 -9 M BK -15.8% and 10 -10 M BK -20.7%.It demonstrates the inhibiting effect of this compound on the bradykinininduced contraction.The contraction force was increased by 9.4% for the bradykinin concentration 10 8 M.
MX2004 had no effect on Vn of contraction of smooth muscles induced by BK for all the concentration range (Fig. 1, A).However, the force of contractions for bradykinin concentrations 10 -6 and 10 -7 М was statistically significantly reduced by 8.2%, 16.6%, respectively upon influence of MX 2004 (Fig. 1, B).
We have been analyzing insufficient number derivatives 3-substituted 1,4-benzodiazepines for demonstration of biochemical effect which depends on their structure.Preliminary assumptions are follo wing: substances MX2011 and MX2004 are similar in their effect due to the presence chlorine atom.Maybe similar effects of MX1785 and MX 1828 caused by structural factors.
So, analysis data show that two 3substituted 1,4benzodiazepines MX1775 and MX1828 demon- MX1775 and MX1626 statistically significant сhanged the Vn smooth muscle contraction (Fig. 1) like as inhibitor B 2 -receptor des-arg 9 bradykinin ace tate (Fig. 2).These effects may evidence of interac tions of these compounds with bradykinin receptors or it signal transduction pathways.
MX1828 statistically significant decreased the normalized rate of smooth muscle contraction for bradykinin concentrations 10 -10 and 10 -9 M by 20.7 and 8.6%, respectively.Nevertheless, for agonist strate the similar inhibition effect on bradykinininduced contraction of smooth muscle like competitive inhibitor des-arg 9 bradykininacetate to bradykinin B 2 -receptors.These substances caused the increase of Vn of bradykinininduced contractions at 10 -6 M like as the competitive inhibitor of B 2 -receptors desarg 9 bradykininacetate.MX1626 deserves special attention, this substance demonstrates almost unidirectional changes of Vn and force smooth muscle that proportionally depend on BK concentration in range 10 -10 -10 -6 M. Compounds MX2011, MX1785 and MX2004 show no natural effect on bradykinin induced smooth muscle contraction.
So, MX1775, MX1626, MX1828 can be selected for further research of their biochemical proper ties, influence on kininbradykinin system and pain perception.