Polymer-assisted solution-phase (PASP) parallel synthesis of an alpha-ketothiazole library as tissue factor VIIa inhibitors

A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-L-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification protocols, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multi-step synthesis affords the desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of the tissue factor (TF) VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10e bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue factor VIIa, with some analogues demonstrating selectivity versus thrombin.     

Structure-based drug design of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex

Structure-based drug design coupled with polymer-assisted solution-phase library synthesis was utilized to develop a series of pyrazinone inhibitors of the tissue factor/Factor VIIa complex. The crystal structure of a tri-peptide ketothiazole complexed with TF/VIIa was utilized in a docking experiment that identified a benzyl-substituted pyrazinone as a P(2) surrogate for the tri-peptide. A 5-step PASP library synthesis of these aryl-substituted pyrazinones was developed. The sequence allows for attachment of a variety of P(1) and P(3) moieties, which led to synthesis pyrazinone 23. Compound 23 exhibited 16 nM IC(50) against TF/VIIa with >6250x selectivity versus Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for pre-clinical intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a primate model of thrombosis.     

Piperazinyl-glutamate-pyridines as potent orally bioavailable P2Y12 antagonists for inhibition of platelet aggregation

Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl-glutamate-pyridine as a P2Y₁₂ antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 22J with good human PRP potency, selectivity, in vivo efficacy and oral bioavailability.     

Solid-phase/solution-phase combinatorial synthesis of neuroimmunophilin ligands

A novel solid-phase/solution-phase strategy for the synthesis of neuroimmunophilin ligands based on GPI 1046 was developed. The synthesis employs a solid-phase esterification strategy followed by a solution-phase pyruvic amide formation to produce multi-milligram quantities of discrete compounds for assay. The protocol was applied to a production library of 880 discrete compounds. A highlight of the strategy is an aqueous extractive purification of the final compounds using a novel liquid/ice extraction system developed for high throughput.     

Isolation of a cDNA encoding a human serum marker for acute pancreatitis. Identification of pancreas-specific protein as pancreatic procarboxypeptidase B.

A human pancreas-specific protein (PASP), previously characterized as a serum marker for acute pancreatitis and pancreatic graft rejection, has been identified as pancreatic procarboxypeptidase B (PCPB). cDNAs encoding PASP/PCPB were isolated from a human pancreas cDNA library using a combination of nucleic acid hybridization screening and immunoscreening with antisera raised against native PASP. The deduced amino acid sequence of PASP/PCPB cDNA predicts the translation of a 416-amino acid preproenzyme with a 15-amino acid signal/leader peptide and a 95-amino acid activation peptide. The proenzyme portion of this protein has 76% identity with rat PCPB and 84% identity with bovine carboxypeptidase B. DNA and RNA blot analyses indicate that human PCPB mRNA (1,400 nucleotides) is transcribed from a single locus in the human genome in a tissue-specific fashion. N-terminal sequencing of native PASP and the specific immunoreactivity of bacterially expressed PASP/PCPB with native PASP antibodies confirm the identification of PASP as human pancreatic PCPB.     

Solid-phase synthesis of an N-(phenylalkyl)cinnamide library via Horner-Wadsworth-Emmons reaction

A readily automated solid-phase approach to the synthesis of diverse N-(phenylalkyl)cinnamides, analogues of the NR2B antagonist 2, is described. The procedure utilizes polymer supported N-(phenylalkyl)amines, (diethylphosphono)acetic acid and a wide range of commercially available hydroxybenzaldehydes. The key step, a Horner-Wadsworth-Emmons reaction is achieved under mild conditions and was found to be general for a large number of benzaldehydes. A 225-member focused library was synthesized using a Tecan Combitec synthesizer.     

A practical synthesis of the major 3-hydroxy-2-pyrrolidinone metabolite of a potent CDK2/cyclin A inhibitor

The synthesis of the major metabolite of a potent 3-aminopyrazole CDK2/cyclin A inhibitor is presented. A stereoconservative approach starting from malic acid was employed to construct the hydroxy-substituted pyrrolidinone moiety. In the key step of the synthesis the use of cyanoborohydride immobilized on Amberlyst 26 in trifluoroethanol represented a valid alternative to conventional solution-phase reducing agents.     

The NAD-linked aromatic alpha-hydroxy acid dehydrogenase from Trypanosoma cruzi. A new member of the cytosolic malate dehydrogenases group without malate dehydrogenase activity.

Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.     

Discovery of potent and selective phenylalanine derived CCR3 antagonists. Part 1

The discovery of a series of phenylalanine derived CCR3 antagonists is reported. Parallel, solution-phase library synthesis has been utilized to delineate the structure-activity relationship leading to the synthesis of highly potent, CCR3-selective antagonists.     

Synthesis of a 200-member library of squaric acid N-hydroxylamide amides

We report here the parallel synthesis of 200 compounds based on squaric acid template. These compounds are obtained via a one-step solution-phase procedure starting from three squaric acid N-hydroxylamide esters precursors. The set of diverse reagents qualified (amines, anilines, amino-alcohols and amino-esters) makes this strategy suitable for the search of biologically active compounds. The library was screened on the zinc metalloenzyme ADAMTS-5 and hits with IC(50) in the range of 1-50 microM were identified.     

Development of improved inhibitors of wall teichoic acid biosynthesis with potent activity against Staphylococcus aureus

A small molecule (1835F03) that inhibits Staphylococcus aureus wall teichoic acid biosynthesis, a proposed antibiotic target, has been discovered. Rapid, parallel, solution-phase synthesis was employed to generate a focused library of analogs, providing detailed information about structure–activity relationships and leading to the identification of targocil, a potent antibiotic.     

A library construction of 2,5-disubstituted pyrrole compounds by using solid/solution-phase syntheses

Using solid- and solution-phase synthesis, a library of 2,5-disubstituted pyrrole compounds was constructed. This is the first report that Stetter reaction was applied to the solid-phase synthesis for C-C bond formation. Some of 2,5-disubstituted pyrrole compounds showed the inhibitory activity of LPS-induced mouse B-lymphocyte proliferation.     

A library of novel hydroxamic acids targeting the metallo-protease family: design, parallel synthesis and screening

We report here the design and parallel synthesis of 217 compounds based on a malonic-hydroxamic acid template. These compounds are obtained via a two-step solution-phase procedure. The set of diverse building-blocks used makes this strategy suitable for the search of inhibitors of various metallo-proteases and for the investigation of the biological role of new metallo-proteases. As a proof of concept, we screened this library on Neutral Aminopeptidase (APN; EC 3.4.11.2), the prototypal enzyme of the M1 family. Several submicromolar inhibitors were identified.     

Assessment of solution-phase positional scanning libraries based on distamycin A for the discovery of new DNA binding agents

The solution-phase synthesis of two 1000-membered positional scanning libraries of distamycin A analogues is described enlisting acid/base liquid-liquid extractions for isolation and purification of all intermediates and final products. The results of their screening for functional activity (L1210 cytotoxic potency) and DNA binding affinity were compared with those derived from libraries containing the same compound members but prepared in a smaller 10-compound mixture format. The positional scanning libraries, which are substantially less demanding to prepare, allowed the accurate detection of the global observations and the clearly more potent activities, but more subtle discoveries and less distinguishable activities were not detected. This is a natural consequence of testing the larger 100-compound mixtures and the relative insensitivity of the assays to the contribution of any single, uniquely acting compound in the mixture. Thus, the disadvantages associated with the loss of some information contained within the library must be balanced against the advantages of the ease of library synthesis and judged in light of the library screening objectives.     

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.     

Synthesis of highly substituted 5-(trifluoromethyl)ketoimidazoles using a mixed-solid/solution phase motif

Using a combination of solid phase synthesis for the preparation of N-substituted-N-acylglycines 7 followed by solution-phase ring transformation of trifluoromethylacyl munchnone intermediate 8, a library of 200 trisubstituted-5-trifluoromethylketo (TFMK) imidazoles 9 was prepared. In a sublibrary, bromoacetate resin 4 was treated with 5 amines in parallel to give N-substituted glycines 5 followed by acylation with 12 acid chlorides to provide, upon cleavage from the resin, 60 individual N-substituted-N-acylglycines 7. The glycines 7 were converted to munchnones 8 by treatment with trifluoroacetic anhydride followed by reaction with benzamidine to give trisubstituted-5-TFMK-imidazoles 9. The structural content of the library was analyzed using PlateView of the LCMS results, and individual members were isolated by automated preparative LCMS.     

Guiding farnesyltransferase inhibitors from an ECLiPS library to the catalytic zinc

Farnesyltransferase inhibitors identified from an ECLiPS library were optimized using solution-phase synthesis. X-ray crystallography of inhibited complexes was used to identify substructures that coordinate to the active site zinc. The X-ray structures were ultimately used to guide the design of second-generation analogs with FTase IC(50)s of less than 1.0 nM.     

Parallel synthesis of a library of bidentate protein tyrosine phosphatase inhibitors based on the alpha-ketoacid motif

Protein tyrosine phosphatases (PTPases) regulate intracellular signal transduction pathways by controlling the level of tyrosine phosphorylation in cells. These enzymes play an important role in a variety of diseases including type II diabetes and infection by the bacterium Yersinia pestis, which is the causative agent of bubonic plague. This report describes the synthesis, using parallel solution-phase methods, of a library of 104 potential inhibitors of PTPases. The library members are based on the bis(aryl alpha-ketocarboxylic acid) motif that incorporates a carboxylic acid on the central benzene linker. This carboxylic acid was coupled with a variety of different aromatic amines through an amide linkage. The aromatic component of the resulting amides is designed to make contacts with residues that surround the active site of the PTPase. The library was screened against the Yersinia PTPase and PTP1B. Based upon the screening results, four members of the library were selected for further study. These four compounds were evaluated against the Yersinia PTPase, PTP1B, TCPTP, CD45, and LAR. Compound 14 has an IC(50) value of 590nM against PTP1B and is a reversible competitive inhibitor. This affinity represents a greater than 120-fold increase in potency over compound 2, the parent structure upon which the library was based. A second inhibitor, compound 12, has an IC(50) value of 240nM against the Yersinia PTPase. In general, the selectivity of the inhibitors for PTP1B was good compared to LAR, but modest when compared to TCPTP and CD45.     

Polymer-Assisted Structural Modification on Nucleosides and Nucleotides

Nucleosides and their analogues play important roles in biological research and clinical therapeutics. Polymer-assisted structural modifications of nucleosides and nucleotides enable parallel and rapid construction of nucleoside library. For some nucleosides, higher chemical selectivity and regioselectivity can be achieved using solid-phase synthesis when compared to classic solution-phase synthesis.     

Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings

In the solid phase preparation of synthetic peptide libraries, equimolarity of the resultant peptides in the mixture simplifies the identification of active compounds. Two primary methods for the preparation of combinatorial peptide mixtures are currently used. In the first method, the starting resin is divided into equal aliquots, individual amino acids are coupled to each aliquot, and the resin is then recombined. This process is repeated for each position. However, due to the physical process, each resin bead contains only one peptide sequence. Statistically, for mixtures of longer sequences, an ever-increasing amount of resin is necessary to ensure complete representation of each peptide in the library. Thus, each peptide will be represented in the library if a sufficient number of resin beads are used. In addition, the concentration of each peptide in the library depends on both the number of mixture positions in the library and the amount of resin used. In the second method, mixtures of amino acids are coupled simultaneously at each addition step. The proportion of each amino acid in the reaction mixture is varied inversely to its reaction rate such that, ideally, an equimolar mixture of each peptide is synthesized. An advantage of this method over the previous method is that each peptide is ensured to be represented in the library, although not necessarily in equimolar amounts. It is known that not only do the coupling rates of each amino acid vary, but the coupling rates of individual amino acids also change when coupled to different amino acid resins. Consequently, in order to obtain equimolar peptide mixtures through the use of mixtures of protected amino acids, the ratio of reaction rates of one amino acid over another must be constant irrespective of the resin-bound amino acid. If this premise is true, this method of synthesis offers a significant advantage over the previous method since, theoretically, equimolar peptide libraries could be synthesized. The influence of the resin-bound amino acid on the relative reaction rates of incoming amino acids was investigated in the current study. © 1994 John Wiley & Sons, Inc.