Convergent synthesis of combinatorial library

Details Convergent synthesis of combinatorial library Convergent synthesis of combinatorial library

1999-08-09

2003-11-25

Celsa, Bennett (Department: 1639)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C436S501000, C436S518000, C564S133000, C564S488000

Reexamination Certificate

active

06653087

ABSTRACT:

FIELD OF THE INVENTION
The invention is directed to novel C2-symmetrical and unsymmetrical chemical libraries for use in protein and receptor homodimerization and heterodimerization studies by solution phase methods. The invention further relates to novel combinatorial methods for synthesizing such libraries of compounds.
BACKGROUND
Ligand-induced receptor and protein dimerization or oligomerization has emerged as a general mechanism for signal transduction. Members of several receptor families of significance for drug discovery have been established to utilize this mode of receptor activation. These include protein tyrosine kinase receptors (homo- or heterodimerization), cytokine receptors (homo- or heterodimerization), serine/threonine kinase receptors (hetero-oligomerization) and members of the TNF-receptor family (trimerization). Within the cytokine receptor superfamily, the best studied examples are the human growth hormone (hGHr), prolactin (PRLr) and erythropoietin (EPOr) receptors, which form homodimers upon binding with their endogenous ligands. Similarly, intracellular signal transduction often proceeds by ligand-induced protein-protein homo- or heterodimerization.
The fact that the certain receptors and proteins appear to bind their ligands utilizing small clusters of residues for the majority of the binding interaction has led to the expectation that small molecules may be capable of triggering a receptor response. It has been anticipated that the generation of detailed knowledge concerning the dimerization modes and ligand binding domains of single transmembrane domain receptors will provide a basis for the design of functional agonists as well as ligand antagonists. However, the noncontiguous and multiple binding domains involved in both the protein-protein and ligand-protein interactions make it difficult to assess the dimerization mode or ligand binding domains in the absence of three-dimensional structural information. This is especially true considering the size of the typical endogenous ligands including proteins such as EPO (166 residues) which themselves contain noncontiguous binding domains which interact with both subunits of the dimerized receptor. Consequently, the search for non-protein ligands has been addressed through the use of random screening procedures.
Recently, the successful identification of cyclic polypeptides with the capacity to mimic the action of EPO was reported, together with details of the intricate receptor-ligand and receptor-receptor interactions in the bound complex (Wrighton et al. Science 1996, 273, 458; Livnah et al. Science 1996, 273, 464) Although these results represent a major achievement, the size (2 to 20 residues) and nature of ligands identified would not seem to be immediately applicable as drug candidates.
Combinatorial chemistry, introduced for polypeptide and oligonucleotide libraries, has undergone a rapid development and acceptance. It is widely recognized that this approach, when applied to generating non-peptide small molecule diversity, has provided a new paradigm for drug discovery. Perhaps as a consequence of the extension of the concept from peptide and oligonucleotide synthesis, the majority of applications have relied on solid-phase synthesis and methodological advances continue to extend common synthetic transformations to polymer-supported versions (Thompson et al. J. A. Chem. Rev. 1996, 96, 555; Früchtel et al. Angew. Chem., Int. Ed. Engl. 1996, 35, 17; Hermkens et al. Tetrahedron 1996, 52, 4527)
A less well accepted complement to adapting solution-phase chemistry to polymer-supported combinatorial synthesis is the development of protocols for solution-phase combinatorial synthesis (Han et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6419) Preceding the disclosure of our own efforts on the development of a multi-step solution-phase parallel synthesis of chemical libraries (Cheng et al. J. Am. Chem. Soc. 1996, 118, 2567; Boger et al. J. Am. Chem. Soc. 1996, 118, 2109; Cheng et al. Bioorg. Med. Chem. 1996, 4, 727), the single-step solution-phase synthesis of combinatorial libraries was detailed by at least three groups as follows. Smith and coworkers (Smith et al. Bioorg. Med. Chem. Lett. 1994, 4, 2821), prepared a library of potentially 1600 amides by reacting 40 acid chlorides with 40 nucleophiles. The library was screened as 80 sample mixtures in a matrix format, allowing immediate deconvolution.
A similar sub-library format was used by Pirrung and Chen (Pirrung et al. J. Am. Chem. Soc. 1995, 117, 1240; Pirrung et al. Chem. Biol. 1995, 2, 621) who prepared a series of carbamate mixtures which were screened for acetylcholinesterase inhibitory activity. Prior to these efforts, Rebek's group reported the single-step construction of large libraries presenting amino acid derivatives attached to rigid core templates with a reliance on amide or urea bond formation (Carell et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 2059; Carell et al. Bioorg. Med. Chem. 1996, 4, 655; Dunayevskiy et al. Anal. Chem. 1995, 67, 2906; Carell et al. Chem. Biol. 1995, 2, 171). Because of the complexity of the combinatorial libraries resulting from this approach (approaching 100,000 members), an iterative selection strategy based on structural grouping of the building blocks was devised.
In addition to recent advances in this work, substantial progress towards using solution-phase multicomponent reactions for generating combinatorial mixtures has been disclosed. For example, both Ugi and Armstrong have reported Ugi four-component condensations including the incorporation of a modifiable isocyanide in combination with resin capture strategy, to provide useful solution-phase library preparations (Ugi et al. Endeavour 1994, 18, 115; Keating et al. J. Am. Chem. Soc. 1996, 118, 2574; Armstrong et al Acc. Chem. Res. 1996, 29, 123).
Our own efforts have focused on the development of a multistep, solution-phase strategy for the preparation of chemical libraries which relies upon the simple removal of excess reactants and reagents by liquid-liquid or liquid-solid extraction procedures. The application of water-soluble coupling reagents in solution-phase peptide synthesis was introduced by Sheehan et al. J. Org. Chem. 1956, 21, 439.
The approach has been shown to dependably deliver pure, individual compounds in multi-milligram quantities, and chemical libraries of >1000 individual members have been assembled (Cheng et al. J. Am. Chem. Soc. 1996, 118, 2567; Boger et al. J. Am. Chem. Soc. 1996, 118, 2109; Cheng et al. Bioorg. Med. Chem. 1996, 4, 727; Tarby et al. In Molecular Diversity and Combinatorial Chemistry: Libraries and Drug Discovery Chaiken, I. M., Janda, K. D., Eds.; ACS: Washington, 1996; 81). Notably, it avoids the disadvantages of solid-supported synthesis including its restrictive scale, the required functionalized substrates and solid supports, compatible spacer linkers, and the requirements for othogonal attachment/detachment chemistries typically with the release of spectator functional groups. It does not require specialized protocols for monitoring the individual steps of multistep syntheses including orthogonal capping strategies for blocking unreacted substrate and does provide the purification of sequence intermediates. This latter disadvantage of solid-supported synthesis necessarily produces the released product of a multistep sequence in an impure state or requires that each reaction on each substrate proceed with an unusually high efficiency.
Ligand-induced receptor and protein dimerization or oligomerization has emerged as a general mechanism for signal transductionl and members of the important receptor superfamilies are activated by such a process. These include protein tyrosine kinase receptors (homo- or heterodimerization), class I cytokine receptors (homo- or heterodimerization), serine/threonine kinase receptors (hetero-oligomerization), and members of the TNF-receptor family (trimerization), FIG.
20
. Within the cytokine receptor superfamily, the most extensively studied examples are the human grow

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