Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors

The complexation of 3-alkylcarbonyloxy-7-bromo-5-(2'-chloro)phenyl-1,2-dihydro-3Н-1,4-benzodiazepine-2-ones (R=Мe (1), R=t-Bu (2)) with the central benzodiazepine receptors (CBDR) at six temperatures within the range of 0-35°С has been studied by the radioligand analysis method. It has been fou...

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Опубліковано в: :Журнал органічної та фармацевтичної хімії
Дата:2012
Автори: Smulsky, S.P., Burenkova, N.O., Andronati, S.A., Pavlovsky, V.I., Polishchuk, P.G., Andronati, K.S.
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Мова:English
Опубліковано: Інститут органічної хімії НАН України 2012
Онлайн доступ:https://nasplib.isofts.kiev.ua/handle/123456789/42067
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Цитувати:Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors / S.P. Smulsky, N.O. Burenkova, S.A. Andronati, V.I. Pavlovsky, P.G. Polishchuk, K.S. Andronati // Журнал органічної та фармацевтичної хімії. — 2012. — Т. 10, вип. 4(40). — С. 65-70. — Бібліогр.: 34 назв. — англ.

Репозитарії

Digital Library of Periodicals of National Academy of Sciences of Ukraine
id nasplib_isofts_kiev_ua-123456789-42067
record_format dspace
spelling Smulsky, S.P.
Burenkova, N.O.
Andronati, S.A.
Pavlovsky, V.I.
Polishchuk, P.G.
Andronati, K.S.
2013-03-07T18:47:58Z
2013-03-07T18:47:58Z
2012
Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors / S.P. Smulsky, N.O. Burenkova, S.A. Andronati, V.I. Pavlovsky, P.G. Polishchuk, K.S. Andronati // Журнал органічної та фармацевтичної хімії. — 2012. — Т. 10, вип. 4(40). — С. 65-70. — Бібліогр.: 34 назв. — англ.
0533-1153
https://nasplib.isofts.kiev.ua/handle/123456789/42067
547.892+ 544-971.62+ 615.31
The complexation of 3-alkylcarbonyloxy-7-bromo-5-(2'-chloro)phenyl-1,2-dihydro-3Н-1,4-benzodiazepine-2-ones (R=Мe (1), R=t-Bu (2)) with the central benzodiazepine receptors (CBDR) at six temperatures within the range of 0-35°С has been studied by the radioligand analysis method. It has been found that formation of the supramolecular complex of compound 1 with CBDR is endothermic with a rather great and unfavourable change of enthalpy (ΔН1° = +32,3 kJ/m%l), which is compensated by signifi cant change in entropy (ΔS1° = +266,7 J/(mol×К)). On the contrary, the binding of compound 2 to CBDR is exothermic (ΔН2° = -20,7 kJ/mol) and with a favourable entropy change (ΔS2° = +90,4 J/(mol×К)). The ester carbonyl groups in compounds 1 and 2 are also supposed to form different hydrogen bonds with the receptor.
Методом радіолігандного аналізу вивчено комплексоутворення 3-алкілкарбонілокси-7-бром-5-(2'-хлор)феніл-1,2-дигідро-ЗН-1,4-бенздіазепін-2-онів (R=алкіл R=Me (1), R=t-Ви (2)) з центральними бенздіазепіновими рецепторами (ЦБДР) при шести температурах у інтервалі 0-35°С. Встановлено, що утворення супрамолекулярного комплексу сполуки 1 з ЦБДР ендотермічне з доволі великою та несприятливою зміною ентальпії ΔН1°=32,3 кДж/моль), яка компенсується значною зміною ентропії (ΔS1°=266,7Дж/(моль×К)). Зв'язування сполуки 2 з ЦБДР екзотермічне (ΔН2°=-20,7 кДж/моль) зі сприятливою зміною ентропії (ΔS2°=90,4 Дж/(моль×К)). Передбачається також, що естерні карбонільні групи у сполуках 1 і 2 утворюють різні водневі зв'язки з рецептором.
Методом радиолигандного анализа изучено комплексообразование 3-алкилкарбонилокси-7-бром-5-(2'-хлор)фенил-1,2-дигидро-ЗН-1,4-бенздиазепин-2-онов R=Me (1), R=t-Bu (2)) с центральными бенздиазепиновыми рецепторами (ЦБДР) при шести температурах в интервале 0-35°С. Обнаружено, что образование супрамолекулярного комплекса соединения 1 с ЦБДР эндотермическое с довольно большим и неблагоприятным изменением энтальпии (ΔH1°=32,3 кДж/моль), которое компенсировано значительным изменением энтропии (ΔS1°=266,7 Дж/(моль×К)). Связывание соединения 2 с ЦБДР экзотермическое (ΔH2°=-20,7 кДж/моль) и с благоприятным изменением энтропии (ΔS2°=90,4 Дж/(моль×К)). Предполагается также, что сложноэфирные карбонильные группы в соединениях 1 и 2 образуют различные водородные связи с рецептором.
en
Інститут органічної хімії НАН України
Журнал органічної та фармацевтичної хімії
Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors
Термодинаміка комплексоутворення естерів 3-гідрокси-7-бром-5-(2'-хлор)феніл-1,2-дигідро-3H-1,4-бензодіазепін-2-онів з центральними бенздіазепіновими рецепторами
Термодинамика комплексообразования эфиров 3-гидрокси-7-бром-5-(2'-хлор) фенил-1,2-дигидро-3H-1,4-бензодиазепин-2-онов с центральными бенздиазепиновыми рецепторами
Article
published earlier
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
title Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors
spellingShingle Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors
Smulsky, S.P.
Burenkova, N.O.
Andronati, S.A.
Pavlovsky, V.I.
Polishchuk, P.G.
Andronati, K.S.
title_short Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors
title_full Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors
title_fullStr Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors
title_full_unstemmed Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors
title_sort thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3h-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors
author Smulsky, S.P.
Burenkova, N.O.
Andronati, S.A.
Pavlovsky, V.I.
Polishchuk, P.G.
Andronati, K.S.
author_facet Smulsky, S.P.
Burenkova, N.O.
Andronati, S.A.
Pavlovsky, V.I.
Polishchuk, P.G.
Andronati, K.S.
publishDate 2012
language English
container_title Журнал органічної та фармацевтичної хімії
publisher Інститут органічної хімії НАН України
format Article
title_alt Термодинаміка комплексоутворення естерів 3-гідрокси-7-бром-5-(2'-хлор)феніл-1,2-дигідро-3H-1,4-бензодіазепін-2-онів з центральними бенздіазепіновими рецепторами
Термодинамика комплексообразования эфиров 3-гидрокси-7-бром-5-(2'-хлор) фенил-1,2-дигидро-3H-1,4-бензодиазепин-2-онов с центральными бенздиазепиновыми рецепторами
description The complexation of 3-alkylcarbonyloxy-7-bromo-5-(2'-chloro)phenyl-1,2-dihydro-3Н-1,4-benzodiazepine-2-ones (R=Мe (1), R=t-Bu (2)) with the central benzodiazepine receptors (CBDR) at six temperatures within the range of 0-35°С has been studied by the radioligand analysis method. It has been found that formation of the supramolecular complex of compound 1 with CBDR is endothermic with a rather great and unfavourable change of enthalpy (ΔН1° = +32,3 kJ/m%l), which is compensated by signifi cant change in entropy (ΔS1° = +266,7 J/(mol×К)). On the contrary, the binding of compound 2 to CBDR is exothermic (ΔН2° = -20,7 kJ/mol) and with a favourable entropy change (ΔS2° = +90,4 J/(mol×К)). The ester carbonyl groups in compounds 1 and 2 are also supposed to form different hydrogen bonds with the receptor. Методом радіолігандного аналізу вивчено комплексоутворення 3-алкілкарбонілокси-7-бром-5-(2'-хлор)феніл-1,2-дигідро-ЗН-1,4-бенздіазепін-2-онів (R=алкіл R=Me (1), R=t-Ви (2)) з центральними бенздіазепіновими рецепторами (ЦБДР) при шести температурах у інтервалі 0-35°С. Встановлено, що утворення супрамолекулярного комплексу сполуки 1 з ЦБДР ендотермічне з доволі великою та несприятливою зміною ентальпії ΔН1°=32,3 кДж/моль), яка компенсується значною зміною ентропії (ΔS1°=266,7Дж/(моль×К)). Зв'язування сполуки 2 з ЦБДР екзотермічне (ΔН2°=-20,7 кДж/моль) зі сприятливою зміною ентропії (ΔS2°=90,4 Дж/(моль×К)). Передбачається також, що естерні карбонільні групи у сполуках 1 і 2 утворюють різні водневі зв'язки з рецептором. Методом радиолигандного анализа изучено комплексообразование 3-алкилкарбонилокси-7-бром-5-(2'-хлор)фенил-1,2-дигидро-ЗН-1,4-бенздиазепин-2-онов R=Me (1), R=t-Bu (2)) с центральными бенздиазепиновыми рецепторами (ЦБДР) при шести температурах в интервале 0-35°С. Обнаружено, что образование супрамолекулярного комплекса соединения 1 с ЦБДР эндотермическое с довольно большим и неблагоприятным изменением энтальпии (ΔH1°=32,3 кДж/моль), которое компенсировано значительным изменением энтропии (ΔS1°=266,7 Дж/(моль×К)). Связывание соединения 2 с ЦБДР экзотермическое (ΔH2°=-20,7 кДж/моль) и с благоприятным изменением энтропии (ΔS2°=90,4 Дж/(моль×К)). Предполагается также, что сложноэфирные карбонильные группы в соединениях 1 и 2 образуют различные водородные связи с рецептором.
issn 0533-1153
url https://nasplib.isofts.kiev.ua/handle/123456789/42067
citation_txt Thermodynamics of 3-hydroxy-7-bromo-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-ones esters complexation with the central benzodiazepine receptors / S.P. Smulsky, N.O. Burenkova, S.A. Andronati, V.I. Pavlovsky, P.G. Polishchuk, K.S. Andronati // Журнал органічної та фармацевтичної хімії. — 2012. — Т. 10, вип. 4(40). — С. 65-70. — Бібліогр.: 34 назв. — англ.
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fulltext Журнал органічної та фармацевтичної хімії. – 2012. – Т. 10, вип. 4 (40) 65 УДК 547.892+ 544-971.62+ 615.31 THERMODYNAMICS OF 3-HYDROXY-7-BROMO-5-(2'-CHLORO) PHENYL-1,2-DIHYDRO-3H-1,4-BENZODIAZEPINE-2-ONES ESTERS COMPLEXATION WITH THE CENTRAL BENZODIAZEPINE RECEPTORS S.P.Smulsky, N.O.Burenkova, S.A.Andronati, V.I.Pavlovsky, P.G.Polishchuk, K.S.Andronati A.V.Bogatsky Physico-Chemical Institute of the NAS of Ukraine 65080, Odessa, Lustdorfskaya doroga 86. E-mail: andronati_s@ukr.net Key words: recognition; radioligand analysis; thermodynamics of complexation; affinity; hydrogen bond; hydrophobic interactions The complexation of 3-alkylcarbonyloxy-7-bromo-5-(2'-chloro)phenyl-1,2-dihydro-3Н-1,4-ben- zodiazepine-2-ones (R=Ме (1), R=t-Bu (2)) with the central benzodiazepine receptors (CBDR) at six temperatures within the range of 0-35°С has been studied by the radioligand analysis method. It has been found that formation of the supramolecular complex of compound 1 with CBDR is endothermic with a rather great and unfavourable change of enthalpy (∆Н1° = +32,3 kJ/mоl), which is compensated by signifi cant change in entropy (∆S1° = +266,7 J/(mоl×К)). On the contrary, the binding of compound 2 to CBDR is exothermic (∆Н2° = -20,7 kJ/mоl) and with a favourable entropy change (∆S2° = +90,4 J/(mоl×К)). The ester carbonyl groups in compounds 1 and 2 are also supposed to form different hydrogen bonds with the receptor. ТЕРМОДИНАМІКА КОМПЛЕКСОУТВОРЕННЯ ЕСТЕРІВ 3-ГІДРОКСИ-7-БРОМ-5-(2'-ХЛОР)ФЕ- НІЛ-1,2-ДИГІДРО-3H-1,4-БЕНЗОДІАЗЕПІН-2-ОНІВ З ЦЕНТРАЛЬНИМИ БЕНЗДІАЗЕПІНОВИМИ РЕЦЕПТОРАМИ С.П.Смульський, Н.О.Буренкова, С.А.Андронаті, В.І.Павловський, П.Г.Поліщук, К.С.Андронаті Методом радіолігандного аналізу вивчено комплексоутворення 3-алкілкарбонілокси- 7-бром-5-(2'-хлор)феніл-1,2-дигідро-3Н-1,4-бенздіазепін-2-онів (R=алкіл R=Ме (1), R=t-Bu (2)) з центральними бенздіазепіновими рецепторами (ЦБДР) при шести температу- рах у інтервалі 0-35°С. Встановлено, що утворення супрамолекулярного комплексу сполуки 1 з ЦБДР ендотермічне з доволі великою та несприятливою зміною ентальпії (∆Н1°=32,3 кДж/моль), яка компенсується значною зміною ентропії (∆S1°=266,7 Дж/(моль×К)). Зв’язування сполуки 2 з ЦБДР екзотермічне (∆Н2°=-20,7 кДж/моль) зі сприятливою змі- ною ентропії (∆S2°=90,4 Дж/(моль×К)). Передбачається також, що естерні карбонільні групи у сполуках 1 і 2 утворюють різні водневі зв’язки з рецептором. ТЕРМОДИНАМИКА КОМПЛЕКСООБРАЗОВАНИЯ ЭФИРОВ 3-ГИДРОКСИ-7-БРОМ-5-(2'-ХЛОР) ФЕНИЛ-1,2-ДИГИДРО-3H-1,4-БЕНЗОДИАЗЕПИН-2-ОНОВ С ЦЕНТРАЛЬНЫМИ БЕНЗДИАЗЕ- ПИНОВЫМИ РЕЦЕПТОРАМИ С.П.Смульский, Н.А.Буренкова, С.А.Андронати, В.И.Павловский, П.Г.Полищук, К.С.Андронати Методом радиолигандного анализа изучено комплексообразование 3-алкилкарбонил- окси-7-бром-5-(2'-хлор)фенил-1,2-дигидро-3Н-1,4-бенздиазепин-2-онов R=Ме (1), R=t-Bu (2)) с центральными бенздиазепиновыми рецепторами (ЦБДР) при шести температу- рах в интервале 0-35°С. Обнаружено, что образование супрамолекулярного комплекса соединения 1 с ЦБДР эндотермическое с довольно большим и неблагоприятным из- менением энтальпии (∆Н1°=32,3 кДж/моль), которое компенсировано значительным изменением энтропии (∆S1°=266,7 Дж/(моль×К)). Связывание соединения 2 с ЦБДР экзотермическое (∆Н2°=-20,7 кДж/моль) и с благоприятным изменением энтропии (∆S2°=90,4 Дж/(моль×К)). Предполагается также, что сложноэфирные карбонильные группы в соединениях 1 и 2 образуют различные водородные связи с рецептором. Analysis of equilibrium formation of supramolecu- lar complexes of drugs with membrane receptors within range from 0 to 35°C temperature led to certain genera- lizations. For the majority of membrane receptors, it was found ability for thermodynamic discrimination of ligands as agonists and antagonists. For 184 inde- pendent experiments and 10 receptor systems the phe- nomenon of enthalpy-entropy compensation was de- scribed [1]. The majority of membrane receptors form supramolecular complexes with drugs and endogenous Журнал органічної та фармацевтичної хімії. – 2012. – Т. 10, вип. 4 (40) 66 ligands (neurotransmitters) with the temperature in- dependent enthalpies (∆H) and entropies (∆S) [2]. Information obtained on the basis of thermody- namic analysis of ligand receptors binding is unique and is not available if equilibrium constants are mea- sured at one temperature. Cautious interpretation of thermodynamic analysis results allows conclud- ing on the mechanism of complexation and the na- ture of intermolecular interactions between a ligand and a receptor in supramolecular complexes, as well as on the causes of intrinsic activity [2, 3]. Analysis of the binding in dependence of tempe- rature allows determining free energies and, conse- quently, the equilibrium constants at different tem- peratures within a given range (typically from 0 to 35°C), including the constant at body temperature, what is the closest approximation to the conditions of pharmacological tests in experimental animals. The van’t Hoff equation is used in the thermody- namic analysis lnKА = -∆Н°/RT + ∆S°/R, where R – gas constant; T – temperature in Kelvin; KA – equilibrium association constant of ligand-re- ceptor complex; ∆Н° and ∆S° are standard enthalpy and entropy of complexation, respectively. The tem- perature of 298,15 K (25°C) and atmospheric pres- sure are the standard conditions as a rule. GABAA receptor-ionophoric complex belongs to the superfamily of ionotropic receptors and provides the Cl¯ and HCO3¯ ion transport into the cell. In ad- dition to the GABA binding sites, GABAA ionophoric complex includes binding sites of benzodiazepines, picrotoksinin, β-carbolines, barbiturates, and other li- gands [4]. 1,4-Benzodiazepine derivatives are the most common and known ligands of the central benzodi- azepine receptors and are widely used as medicine neurotropic drugs [4, 5]. Thermodynamic analysis of binding of drugs of benzodiazepine series (diazepam, clonazepam, alprazolam and others) with CBDR is described in [6-13], and results of studies [6-12], are summarized in the review [3]. The van’t Hoff plots of the ln(1/Ki) versus the (1/T)×(1000/K) for all ben- zodiazepines described in the literature are linear within the temperature range from 0 to 35°C, with the exception of certain cases, when broken lines of plots for clonazepam at 21°C [8] and #lunitrazepam at 10°C were observed [9]. All 1,4 benzodiazepines described in the literature [6-13], with the exception for triazolam and dezmethylmedazepam, form exother- mic complexes with CBDR. It is known from the lit- erature that changes in the benzodiazepine chemical structure ambiguously affect the free energy (∆Н° and ∆S°) of the complexation with CBDR. For example, molecules of alprazolam and triazolam differ by a chlo- rine atom in 2 position of 5-phenyl radical, while the thermodynamic pro#iles of their interaction with CBDR are diametrically opposed. Alprazolam, containing no chlorine atom in the 5-phenyl radical, forms an exo- thermic complex with CBDR, and triazolam, which contains a chlorine atom in this position, forms an endothermic complex with the CBDR [10]. At the same time, #lunitrazenpam, diazepam and nordiaze- pam not only have close values of free binding ener- gies at 37°C (∆G°= -48±6, -45±5, -41±4 kJ/mоl), but also relatively close values of enthalpy (∆Н°=-53±3, -41±2, -44±7 kJ/mоl) and entropy (Т∆S°=-5±7, +5±5, -3±8 kJ/mоl) members of free energies [9]. Currently, there are no systematic data of studies on the relationship between the chemical structure of the 1,4-benzodiazepines, the degree of activation of GABAA receptor complex and the structures of free energies (∆Н°, ∆S°) of their complexation with CBDR. There are no works on the thermodynamic analysis of complexation of substituted in the third position 1,4-benzodiazepines with CBDR, despite the fact that this series of derivatives is very promising concern- ing the search for new neurotrophic drugs. There are well known drugs among the representatives of this series of compounds: lorazepam, oxazepam, temaze- pam, and others. For a long period of time we carry out research in molecular design, synthesis and study of relationship of structure – properties, mode of action and pharmacology of 1,4-benzodiazepine-2-one de- rivatives [14-19]. Af#inity and selectivity for central and peripheral benzodiazepine CNS receptors at the 0°С was examined for many compounds by radioli- gand analysis [17, 20-26]. However, information ob- tained at the same temperature is insuf#icient and doesn’t allow making a decision about driving forces of complexation and nature of interactions with CBDR of the investigated 1,4-benzodiazepines. Thereby (and in continuation of our ongoing research), it was in- teresting to investigate thermodynamics of comple- xation of 3-substituted 1,4-benzodiazepin-2-ones (com- Fig. 1. The van’t Hoff plots showing the affect of temperature on the association constants (1/Ki) of compounds 1, 2 in the experiment on the displacement of [3H] fl umazenil. Values 1/Ki are mean of four independent determinations, each performed in triplicate. Linear interpolation over the points connected by the continuous line (0≤t≤35 C) gives correlation coeffi cients, r, in the range of 0.98-0.99. Журнал органічної та фармацевтичної хімії. – 2012. – Т. 10, вип. 4 (40) 67 pounds 1 and 2, Fig. 1, Scheme 1) with CBDR. The compounds for which the thermodynamics of bind- ing to the GABAA receptor complex have been stud- ied in this paper have highly anxiolytic, anticonvul- sant and sedative activity and high af�inity for CBDR [24]. In this paper, compounds 1 and 2 as research objects were used. Results and Discussion The table contains inhibition constants (Ki) for the compounds 1 and 2 at six temperatures 0, 10, 20, 25, 30 and 35°C, as well as, calculated on the base of van’t Hoff plots standard enthalpies (∆H°) and entropies (∆S°) for equilibrium displacement of [3H]�lumazenil from speci�ic binding sites of CBDR. The table shows also dissociation constants for [3H] �lumazenil complex with CBDR, which were used by us for the calculation of Ki for compounds 1 and 2 by the Cheng-Prusoff equation. The van’t Hoff plots for compounds 1 and 2, in the studied temperature range, were strictly linear (Fig. 1). The binding of compound 1 to the CBDR of the rats cerebral cortex was accompanied by heat ab- sorption (∆Н°1 = +32,3 kJ/mоl) and relatively large increase in entropy (∆S°1 = +266,7 J/(mоl×К)). While the compound 2 complexation with CBDR was exother- mic (∆Н°2 = -20,7 kJ/mоl and ∆S°2 = +90,4 J/(mоl×К)), and was accompanied with temperature increase which was two-fold lesser. Thus, complexation of compounds 1 and 2 with CBDR are driven by differ- ent forces. Thus, the compound 1 complexation with the receptor is exclusively driven by entropy, while the compound 2 binding to the receptor is driven by both enthalpy and entropy. A well-known generalized model of the receptor complex with benzodiazepines of Huang Q. et al. [27] suggests the following features of the molecular in- teraction of 1,4-benzodiazepine-2-ones with CBDR. Carbonyl oxygen of the amide group and nitrogen atom N4 of diazepine ring form hydrogen bonds with the receptor site. Benzene ring in position 5 and the ring condensed with 1,4-diazepine cycle, as well as substituents in position 7, interact with the hydro- phobic centers of the receptor site. Taking into ac- count the given model, and keeping in mind the ac- ceptor properties of the ester carbonyl groups (the substituents in position 3 of compounds 1 and 2), one can assume the formation of different hydrogen bonds with donor regions of CBDR sites (Fig. 2), weak hy- drogen bond with the receptor for the compound 1, and stronger one in the case of compound 2. It is Table Equilibrium inhibition constants (Ki), dissociation constants (KD) at various temperatures, standard enthalpies (∆H°) and entropies (∆S°) for complexation of the compounds 1-4 and [3H]! umazenil with CBDR Compounds Ki±SEM [nM] ∆Н°±SEM (kJ/mol) ∆S°±SEM (J/(mоl×К))0°С 10°С 20°С 25°С 30°С 35°С 1 16,50 (1,2) 11,80 (1,1) 6,05 (0,4) 6,21 (0,3) 4,56 (0,2) 3,13 (0,1) +32,3 (3,0) +266,7 (10) 2 1,91 (0,04) 3,19 (0,15) 3,91 (0,1) 4,19 (0,3) 5,00 (0,2) 5,83 (0,4) -20,7 (2) +90,4 (6) 3* +35,1 +305,0 4* +23,4 +191,6 [3H]Flu-mazenil** KD±SEM [nM] 1,23 (0,05) 2,3 (0,1) 3,2 (0,1) 4,00 (0,2) 4,90 (0,2) 6,30 (0,5) * Data taken from ref. [10]; ** Data taken from ref. [33] Fig. 2. Hypothetical scheme of interactions of compounds 1 and 2 with hydrophobic sites and donor sites of hydrogen bonds (indicated by dotted lines) of CBDR site (based on Huang Q model) [30].Scheme 1 Журнал органічної та фармацевтичної хімії. – 2012. – Т. 10, вип. 4 (40) 68 possible that formation of the hydrogen bonds can occur either competing for the donor centers of the receptor with nitrogen atoms N4 or amide carbonyl groups of the 1,4-benzodiazepine ring or by the for- mation of new additional hydrogen bonds with do- nor sites of CBDR. In any case, thermodynamic pro- �iles of these compounds complexation with the re- ceptor give indirect evidence in favour of the offered assumption (Table and Fig. 3). It is generally accept- ed, that hydrogen bonds formation is accompanied by decrease of enthalpy and entropy. Therefore, in whole energetic balance (change in free energy), strong hydrogen bonds may exceed energy of non-speci�ic (hydrophobic) interactions, and that often leads to decrease of the process enthalpy and minor posi- tive changes in entropy, and in some cases, to its decrease. Weak hydrogen bonds, which are accom- panied with insigni�icant decrease of enthalpy, in many cases, aren’t able to exceed positive change of enthal- py of non-speci�ic hydrophobic interaction of ligand with its receptor. As a result, change of complexation enthalpy may turn out to be a positive one. However, it is arise of question how to explain the fact that hydrogen bond in the case of compound 1 (R = Me) is weaker then hydrogen bond of compound 2 (R = t-Bu) when they interact with their receptors. Unfortunately, the involvement of a methyl group (compound 1) and tert-butyl group (compound 2) in interaction with CBDR can not be described with- in the model of Huang Q. et al. [27], because substi- tuted in the 3 position of 1,4-benzodiazepines are not taken into account at creation of this model. According to the results of our long-term research [28], pharmacological activity of 3-alkyl-substituted (alkyl = Me, Et, i-Pr and t-Bu) 1,4-benzodiazepines is inversely related to lipophilicity. Taking into account these indirect facts and thermodynamic analysis con- ducted by us on the binding of the compounds 1 and 2 with CBDR, it could be assumed that the t-Bu radical of the compound 2 does not enter into hydrophobic interactions with the receptor. The thermodynamic profiles of the interaction of compounds 1 and 2 with benzodiazepine receptor (∆Н°1 = +32,3 kJ/mоl, ∆S°1 = +266,7 J/(mоl×К), ∆Н°2 = -20,7 kJ/mоl, ∆S°2 = +90,4 J/(mоl×К)) prove this fact. It is seen in the coordinates -T∆S° versus ∆Н° (Fig. 3) that the driv- ing forces for the complexation of compound 2 with CBDR are changes in enthalpy (∆Н°) and entropy (∆S°). Compound 1, in contrast to compound 2, binds to the receptor due to the entropy change only. There are at least two examples in the literature where the driving force of benzodiazepine comple- xation with CBDR is the change in entropy. These com- pounds are triazolam (3) and dezmethylmedazepam (4), the thermodynamic analysis of the binding of which was carried out under conditions similar to conditions of our experiments (on the twice washed membranes in the presence of 0,2 M NaCl in the incubation me- dium) [10]. Thus, the compounds 1, 3 and 4 form a ligand group, which binding with CBDR differs signi- �icantly from those described in the literature and ben- zodiazepines studied by us (compound 2) (Scheme 2). Thermodynamic analysis of complexation of com- pounds 1, 3 and 4 with CBDR has demonstrated that: 1) compounds 1, 3 and 4 bind to CBDR with the heat absorption and a large favorable change in en- tropy (see Table). This means that their interaction with the receptor is mainly caused by the rearrange- ment of solvent molecules near the receptor site and near the interacting ligand molecules. It is generally accepted that such complexation thermodynamic pro- �ile mainly is a result of hydrophobic ligand-receptor interactions [29]: 2) unlike the compound 1, the compound 2 (just as benzodiazepines described in literature [6-13], with the exception of the compounds 3 and 4) forms exotermic complex with CBDR. It is known not only numerous multi-center re- lationships and interactions, but also the reorgani- zation of a solvent contribute to the free energy of ligand-protein (receptor) complexation. It is consid- ered that the only theoretical calculation is able to dif- ferentiate these contributions and to correlate them with structural fragments of molecules of ligands and receptors [30, 31].Our results on the thermodynam- ics of binding of the esters (compounds 1 and 2) to CBDR, as well as literature data, for example [10], on Fig. 3. Represent the thermodynamic data of the interaction of compound 1 (●) and 2 (▲) with CBDR in the coordinated -TDS° versus ∆Н° (were KA=104 M-1 and KA=1011 M-1 are lower and upper limits of values of association constants of the drugs, respectively, with bioreceptors, see ref. 1). Scheme 2 Журнал органічної та фармацевтичної хімії. – 2012. – Т. 10, вип. 4 (40) 69 the binding of alprazolam, triazolam and dezmethyl- medazepam to benzodiazepine receptors demonstra- te that the thermodynamics of the formation of supramo- lecular ligand-CBDR complexes can be extremely sen- sitive to changes in the ligand chemical structure. Changes of standard entropies of complexation of compounds 1, 3, 4 allow supposing that the rear- rangement of solvent molecules is the main reason for the change of the free energies of complexation of these compounds with CBDR and it demonstrates that the thermodynamics of the formation of supra- molecular ligand-CBDR complexes can be extremely sensitive to changes in the ligand chemical structure. There is no yet satisfactory explanation of the fact concerning the dominant participation of nonspeci- !ic interactions of the studied compound 1 as com- pared to compound 2, at the formation of complex with benzodiazepine receptors. These differences are dif!icult to explain without pharmacophore-recep- tor models for 3-substituted 1,4-benzodiazepines. We hope that further investigation of thermodynamics of complexation in the series of 3-substituted 1,4-ben- zodiazepines with CBDR will contribute to the clari- !ication of this interesting fact and may be used in the further development of models of the complex 1,4-benzodiazepin – CBDR. Experimental part Compounds 1-2 were synthesized using litera- ture procedures [32]. In vitro receptor binding assays. [3H]Flumaze- nil binding Adult male Wistar rats with a body weight of 180- 220 g were maintained under an arti!icial 12-h-light/dark cycle (light on 08.00 to 20.00 h). Food and water were freely available until the time of the experiment. An- imal care and handling throughout the experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The experimental protocols were ap- proved by the Animal Ethical Committee of the Uni- versity of Cagliari. Af!inity of compounds 1, 2 for CBDR of rat brain was determined by modified method and values of ІС50 were evaluated. Animals were anesthetized and decapitated, the cerebral cortex was quickly extracted and homoge- nized in 30 ml of 0,05 M ice-cold citrate buffer (pH 7,1 at 4°С) with a Dounce homogenizer. The homogen- ate was centrifuged at 20 000 g for 15 min at 4°С. The pellet was resuspended in initial volume of the same buffer and centrifuged again under the same conditions. The process of homogenization and cen- trifugation was repeated for 2 times. Supernatant was decanted, the residue was resuspended in 0,05 M of ice-cold incubation citrate buffer containing 200 mM NaCl to obtain the suspension with wet membrane con- centration of 50 mg/ml and adjusted for each tem- perature. Thermodynamic analysis of the formation of com- plexes of compounds 1, 2 with CBDR was carried out at temperatures of 0, 10, 20, 25, 30 and 35°C. Determi- nation of equilibrium binding constants (KA = 1/Ki) for the binding of compounds 1, 2 to membrane com- plex of the rats cerebral cortex was carried out in 0,5 cm3 of tris-citrate incubation buffer pH 7,1, ad- justed for each temperature. The incubation time ranged from 75 min at 0°C to 20 min at 35°C [33]. Nonspeci!ic binding (which was no more than 10%) of the radioligand [3H] !lumazenil ([3H]Ro15-1788) was determined in the presence of 1×10-6mol/dm3 cold flumazenil. To determine the semi-inhibitory concentrations (IC50) for the compounds 1, 2 eight concentrations were used for each compound, rang- ing from 0,1×10-9 to 1×10-6×mol/dm3. Inhibition con- stant Ki was calculated using the Cheng-Prusoff for- mula (Ki = IC50/(1+[L]/KD)) [34], where IC50 – con- centration of test ligand at which is observed 50% radioligand displacement from speci!ic binding sites of the receptor, [L] – total concentration of radioli- gand, KD – dissociation constant of the radioligand complex with the CBDR for each of the experimental temperatures, taken from [33]. The standard free ener- gies (∆G°) of compounds 1, 2 complexation were calcu- lated by the equation of van’t Hoff (∆G° = -RTln(1/Ki)). The standard enthalpy (∆Н°) and entropy (∆S°) of complexation were obtained by regression analysis from the slope of the van’t Hoff plots (-∆Н°/RT) and the intersection plots (-∆S°/R) with the ordinate axis, where T = 298,15 K, R = 8,314 J/(mol×K). Conclusion 1. Enthalpy and entropy of the compounds 1 and 2 complaxetion are more sensitive to changes in their chemical structure, than free energy. 2. 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