Solution-phase combinatorial synthesis of non-peptide bradykinin antagonists and their antagonistic activities on guinea-pig ileum

Abstract

Bradykinin은 저혈압과 염증을 유발하고, 모세혈관 투과성을 높여서 부종 및 통증을 유발하므로 bradykinin B2 antagonist는 통증 및 알러지 치료제로 개발 될 가능성이 크다. 그러나 아직 약으로 개발된 것은 없으며 최근 non-peptide 구조의 경구용 B2 antagonist에 대한 연구가 활발히 보고되고 있어 전세계적으로 개발 연구의 규모나 역사가 짧아 의약품으로 개발 될 확률이 다른 분야보다 높다. 제2세대 항히스타민제로서 최근 시판되는 cetirizine이 bradykinin B2 antagonist 효과를 갖는 것으로 보고되었으므로 이를 토대로 side chain optimization을 통하여 antibradykinin activity를 갖는 물질을 개발하고자 하였다. 다수의 다양한 화합물을 만들기 위하여 목적 화합물의 구조를 3부분으로 나누어 각각에 다른 치환체를 조합하는 solution-phase combinatorial chemistry 방법을 이용하였다. Library와 개별물질(deconvolution)은 guinea-pig ileum smooth muscle을 사용하여 functional antibradykinin activity를 확인하였다. 활성을 측정한 결과 pM6-H가 bradykinin-induced contraction을 경쟁적으로 억제하였다. 이때 IC_50 값은 1.09×10^-6M 로서 control peptide B2 antagonist인 Hoe140의 1.2×10^-8M 과 비교할 때 potency는 낮았다. 3D-구조를 통하여 pM6-H의 구조가 fold 되어 N과 N사이의 길이가 대략 6.5Å이 되는 것을 확인하였고, 이는 B2 receptor의 active binding site에 결합하기 위해 요구되는 10Å을 충족시키지 못하므로 수용체에 충분히 결합하지 못하여 potency가 낮은 것으로 사료되었다. pM4S3과 pM3S3은 bradykinin의 최고 수축력은 감소시키나 BK-induced contraction의 ED_50값은 줄어들지 않았으므로 bradykinin-induced contraction을 비경쟁적 또는 간접적으로 억제함을 확인하였다. Solution-phase combinatorial chemistry 방법을 통해서 B2 수용체에 결합하기에 요구되는 3D-구조를 디자인하여 새로운 모핵과 side chain을 도입한다면 더욱 효과가 좋은 B2 antagonist를 개발할 수 있을 것이다.;Bradykinin (BK), an endogenous linear nonapeptide hormone with the amino acid sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, is involved in a variety of physiological and pathophysiological processes. Bradykinin contributes to the inflammatory response; it produces pain, swelling, redness and heat. Therefore, BK antagonists are considered as a potential drug in allergic and inflammatory diseases. A number of peptide BK B2 antagonists including the clinically evaluated second-generation antagonists Icatibant (Hoe140) and Bradycor (CP0127) have been developed. Despite their highly potent B2 antagonistic activities, their therapeutic use is still limited because of their peptidic nature. To date, only two classes of structurally different non-peptide B2 antagonists have been identified, the phosphonium-derived WIN 64338 and the heteroaryl benzyl ether FR 173657 being the most thoroughly studied representatives. A key finding which prompted the research reported here is the synthesis of non-peptide bradykinin B2 antagonists which contain piperazine moiety, with reference to the requirement of an aromatic residue at position 8 or a D-aromatic residue in position 7 for high affinity binding and the 10Å separation of two positive charges, using the solution-phase combinatorial chemistry of iminodiacetic anhydride as template to produce large numbers of diverse compounds rapidly and efficiently. Here, we describe the pharmacological characterization of N-, N'- substituted-N"-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid triamide derivatives and N-substituted-N'-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid diamide derivatives obtained from solution-phase combinatorial chemistry for non-peptide B2 antagonists. Fifty compounds were synthesized by the 10×1×5 library (with an exception of the compound pM1S1) by the solution-phase combinatorial chemistry. Additionally 2^nd diversificated, N-tert-butyloxycarbonyl (N-Boc) deprotected compounds were synthesized. The synthesized libraries and deconvolution were tested for their functional antibradykinin activities by utilizing guinea-pig ileum smooth muscle. N-(3,4,5-trimethoxybenzyl)-N'-1-(4-chlorobenzhydryl)piperazine imi- nodiacetic acid diamide (pM6-H) antagonized BK-induced contraction with an IC_50 of 1.09×10^-6 M. The potency of pM6-H was turned out to be less than that of the potent control peptide B2 antagonist, Hoe140, of which IC_50 was observed to be 1.2×10^-8 M. pM6-H competitively antagonized the BK-induced contraction of ileal smooth muscle. When 3D-structure of the pM6-H compound was superimposed with that of FR173657, which is known as the potent non-peptide competitive B2 antagonist, the key pharmacophore of FR173657, cis-amide conformation, were nearly matched to pM6-H. Otherwise, N-phenylacetyl-N'-(4-methylbenzyl)-N"-1-(4-chlorobenzhydryl) piperazine iminodiacetic acid triamide (pM3S3) and N-phenylacetyl-N'-(4-methoxybenzyl)-N"-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid triamide (pM3S3) reduced the maximum response of BK-contractility, but showed no reduction of ED50 of BK-induced contraction of ileal smooth muscle. These compounds, therefore, appear to exhibit non-competitive or indirect antagonism. 3D-structure of FR173657 was relatively stretched conformation while those of pM6-H and pM4S3 were a U-shaped conformation. The differences of 3D-structures of pM6-H and pM4S3 from FR173657 may affect to the potency of pM6-H and pM4S3 of BK-induced contraction of ileal smooth muscle compared to the potency of FR173657. Modifying the pharmacophore to satisfy the cis-amide conformer and relatively stretched conformation to have approximately 10Å of the distance separating the positively charged by using the solution-phase combinatorial chemistry with iminodiacetic anhydride as template may be developed to the discovery of non-peptide B2 receptor antagonists.