Discovery of the factor Xa inhibitor, ZK 807834 (CI-1031).

Discoveries that lead to ZK 807834 (CI-1031, 2a), a potent and selective factor Xa (fXa) inhibitor currently in clinical testing as an intravenous antithrombotic, were initiated by the identification of the potent (Z,Z)-isomer of BABCH (1c). A structure-activity relationship (SAR) was established with a series of analogues of BABCH. This SAR database, combined with computer modeling, demonstrated that binding of the second basic group in the S3/S4 pocket provided fXa potency and that a carboxylic acid group on the opposite side of the molecule resulted in selectivity versus thrombin. Simple substitution of a cyclic urea for the unsaturated ketone structure of BABCH gave disappointing results, but discovery of the bisphenoxy-pyridine analogues provided a template that could be readily optimized. The SAR established for this template is described and compared with computer modeling, REDOR NMR and X-ray crystallography studies. Inhibitor binding to fXa was increased by the introduction of a hydroxyl group on the proximal phenylamidine ring and by the introduction of fluorine atoms at C-3 and C-5 of the pyridine ring. Pharmacokinetic parameters were improved by balancing the contributions from the substituents on the distal ring and the central pyridine ring. The optimal combination was a methyl-(2H)-imidazoline group on the distal ring and a sarcosine at C-4 of the pyridine ring. The promising preclinical database for CI-1031 is described. This review relates the SAR leading to the discovery of the clinical candidate, CI-1031 directly to our best understanding of how this potent inhibitor interacts with the fXa active site.

Conformations of trypsin-bound amidine inhibitors of blood coagulant factor Xa by double REDOR NMR and MD simulations.

Double rotational-echo double resonance (double REDOR) has been used to investigate the bound conformations of (13)C,(15)N,(19)F-labeled factor Xa inhibitors to bovine trypsin. Carbon-fluorine dipolar couplings were measured by (13)C{(19)F} REDOR with natural-abundance background interferences removed by (13)C{(15)N} REDOR. The conformations of the bound inhibitors were characterized by molecular dynamics (MD) simulations of binding restrained by double REDOR-determined intramolecular C-F distances. A symmetrical bisamidine inhibitor and an asymmetrical monoamidine-monoamine inhibitor of the same general shape had distinctly different conformations in the bound state. According to the MD models, these differences arise from specific interactions of the amidine and amine groups with the active-site residues of trypsin and nearby water molecules.

Crystallographic analysis of potent and selective factor Xa inhibitors complexed to bovine trypsin.

Factor Xa is a serine protease which activates thrombin (factor IIa) and plays a key regulatory role in the blood-coagulation cascade. Factor Xa is, therefore, an important target for the design of anti-thrombotics. Both factor Xa and thrombin share sequence and structural homology with trypsin. As part of a factor Xa inhibitor-design program, a number of factor Xa inhibitors were crystallographically studied complexed to bovine trypsin. The structures of one diaryl benzimidazole, one diaryl carbazole and three diaryloxypyridines are described. All five compounds bind to trypsin in an extended conformation, with an amidinoaryl group in the S1 pocket and a second basic/hydrophobic moiety bound in the S4 pocket. These binding modes all bear a resemblance to the reported binding mode of DX-9065a in bovine trypsin and human factor Xa.

Synthesis, characterization, and structure-activity relationships of amidine-substituted (bis)benzylidene-cycloketone olefin isomers as potent and selective factor Xa inhibitors.

Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in blood coagulation linking the intrinsic and extrinsic pathways to the final common pathway of the coagulation cascade. During our initial studies, we observed facile photochemical conversion of the known FXa/tPA inhibitor, BABCH ¿(E,E)-2, 7-bis(4-amidinobenzylidene)cycloheptan-1-one, 1a, to the corresponding (Z,Z) olefin isomer, 1c (FXa K(i) = 0.66 nM), which was over 25,000 times more potent than the corresponding (E,E) isomer (1a, FXa K(i) = 17 000 nM). In order to determine the scope of this observation, we expanded on our initial investigation through the preparation of the olefin isomers in a homologous series of cycloalkanone rings, 4-substituted cyclohexanone analogues, and modified amidine derivatives. In most cases the order of potency of the olefin isomers was (Z,Z) > (E,Z) > (E,E) with the cycloheptanone analogue (1c) showing the most potent factor Xa inhibitory activity. In addition, we found that selectivity versus thrombin (FIIa) can be dramatically improved by the addition of a carboxylic acid group to the cycloalkanone ring as seen with 8c (FXa K(i) = 6.9 nM, FIIa K(i) > 50,000 nM). Compounds with one or both of the amidine groups substituted with N-alkyl substituents or replaced with amide groups led to a significant loss of activity. In this report we have demonstrated the importance of the two amidine groups, the cycloheptanone ring, and the (Z,Z) olefin configuration for maximum inhibition of FXa within the BABCH template. The results from this study provided the foundation for the discovery of potent, selective, and orally active FXa inhibitors.

High resolution solid state C NMR spectroscopy of sporopollenins from different plant taxa.

The extremely chemically resistant component of the cell wall of spores, pollens, and some microorganisms, sporopollenin, is generally accepted to be derived from carotenoids or carotenoid esters. However, we report here that (13)C NMR analyses of sporopollenin from several sources shows that this widely held view is incorrect, with one possible exception. Sporopollenin is not a unique substance but rather a series of related biopolymers derived from largely saturated precursors such as fatty acids. The biopolymers contain widely varying amounts of oxygen in the form of ether, hydroxyl, carboxylic acid, ester, and ketone groups.

Synthesis of brominated heptanones and bromoform by a bromoperoxidase of marine origin.

The presence of naturally occurring volatile halohydrocarbons in marine organisms, seawater, and the upper atmosphere has prompted a serach for their biosynthetic origin. An earlier report documented the preparation of an enzyme extract from a marine algae which catalyzed the formation of dibromomethane, tribromomethane, and 1-bromopentane from 3-oxooctanoic acid. This report did not establish a pathway nor did it examine potential intermediates involved in the synthesis of the halometabolites (Theiler, R., Cook, J., Hager, L., & Siuda, J. (1978) Science (Washington, D.C.) 202, 1094-1096). This paper shows that an extract of the green marine algae, Penicillus capitatus, which contains a potent bromoperoxidase activity, is capable of catalyzing the incorporation of bromide ion into organic combination in the presence of 3-oxooctanoic acid. By use of gas chromatography and mass spectroscopy, it has been possible to identify tribromomethane, 1-bromo-2-heptanone, 1,1-dibromo-2-heptanone, and 1,1,1-tribromo-2-heptanone as products of this reaction. The properties of the enzymatically synthesized products have been compared to authentic compounds and found to be identical. The mono- and dibromoheptanones can be utilized as precursors for the enzymatic formation of tribromoheptanone, but the final hydrolysis of the tribromoheptanone to bromoform appears to be a nonenzymatic reaction with the P. capitatus extracts.