EP1077993A1 - Generation de bibliotheques combinatoires de composes correspondant a des bibliotheques virtuelles de composes - Google Patents

Generation de bibliotheques combinatoires de composes correspondant a des bibliotheques virtuelles de composes

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Publication number
EP1077993A1
EP1077993A1 EP99922956A EP99922956A EP1077993A1 EP 1077993 A1 EP1077993 A1 EP 1077993A1 EP 99922956 A EP99922956 A EP 99922956A EP 99922956 A EP99922956 A EP 99922956A EP 1077993 A1 EP1077993 A1 EP 1077993A1
Authority
EP
European Patent Office
Prior art keywords
compounds
fragments
fragment
mixture
library
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99922956A
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German (de)
English (en)
Other versions
EP1077993A4 (fr
Inventor
Richard Griffey
Eric Swayze
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ionis Pharmaceuticals Inc
Original Assignee
Isis Pharmaceuticals Inc
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Publication date
Application filed by Isis Pharmaceuticals Inc filed Critical Isis Pharmaceuticals Inc
Publication of EP1077993A1 publication Critical patent/EP1077993A1/fr
Publication of EP1077993A4 publication Critical patent/EP1077993A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00281—Individual reactor vessels
    • B01J2219/00286—Reactor vessels with top and bottom openings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00308—Reactor vessels in a multiple arrangement interchangeably mounted in racks or blocks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00689—Automatic using computers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00695—Synthesis control routines, e.g. using computer programs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/007—Simulation or vitual synthesis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention is directed to methods for the generation of virtual combinatorial libraries of small molecules and other ligands.
  • the members or molecules of the combinatorial libraries are generated in silico, and are designed to bind to identified target molecules in silico.
  • the present invention also includes methods for docking the library members to desired target molecules whereby the library members are bound to such targets in silico.
  • the present invention also encompasses methods for interfacing the synthetic information generated in silico with instrumentation such as a parallel array synthesizer which conducts the actual synthesis of desired members of the combinatorial libraries.
  • Combinatorial chemistry is a recent addition to the toolbox of chemists and represents a field of chemistry dealing with the synthesis of a large number of chemical entities. This is generally achieved by condensing a small number of reagents together in all combinations defined by a given reaction sequence. Advances in this area of chemistry include the use of chemical software tools and advanced computer hardware which has made it possible to consider possibilities for synthesis in orders of magnitude greater than the actual synthesis of the library compounds.
  • the concept of "virtual library” is used to indicate a collection of candidate structures that would theoretically result from a combinatorial synthesis involving reactions of interest and reagents to effect those reactions. It is from this virtual library that compounds are selected to be actually synthesized.
  • Project Library (MDL Information Systems, Inc., San Leandro, CA) is said to be a desktop software system which supports combinatorial research efforts. (Practical Guide to Combinatorial Chemistry, A. W. Czarnik and S. H. DeWitt, eds., 1997, ACS, Washington, D.C.)
  • the software is said to include an information-management module for the representation and search of building blocks, individual molecules, complete combinatorial libraries, and mixtures of molecules, and other modules for computational support for tracking mixture and discrete-compound libraries.
  • Molecular Diversity Manager (Tripos, Inc., St. Louis, MO) is said to be a suite of software modules for the creation, selection, and management of compound libraries. (Practical Guide to Combinatorial Chemistry, A. W. Czarnik and S. H. DeWitt, eds., 1997, ACS, Washington, D.C.)
  • the LEGION and SELECTOR modules are said to be useful in creating libraries and characterizing molecules in terms of both 2-dimensional and 3- dimensional structural fingerprints, substituent parameters, topological indices, and physicochemical parameters.
  • Afferent Systems (San Francisco, CA) is said to offer combinatorial library software that creates virtual molecules for a database. It is said to do this by virtually reacting precursor molecules and selecting those that could be actually synthesized (Wilson, C&EN, April 27, 1998, p.32).
  • the present invention further provides methods for tracking and maintaining in databases, the fragments, reagents and unique combinations of these used for the in silico generation of the library members.
  • Methods for interfacing the information necessary for the generation of libraries in silico, as instructions designed to direct the actual synthesis of the library members on an instrument such as a parallel array synthesizer, are also provided in the present invention.
  • Figure 1 shows a compound, compound Cl, dissected into its constituent fragments
  • Figure 2 shows the various identifying characteristics of the fragments comprising compound Cl
  • Figure 3 shows the various identifying characteristics of the reagents used to introduce the corresponding fragments comprising compound Cl;
  • Figure 4 is a list of transformations that link the fragments and reagents associated with the generation of compound Cl;
  • Figure 5 is a schematic for the introduction of a common fragment using two different reagents
  • Figure 6A is a schematic for the use of a single reagent for the introduction of two different fragments into a compound
  • Figure 6B is a schematic showing the use of a common reagent for the introduction of a common fragment into the compound which can further be converted into two different fragments within the compound generated;
  • Figure 7 shows the symbolic addition of fragments yielding a symbolic compound, compound CF;
  • Figure 8 is a symbolic reagent table
  • Figure 9 is a symbolic fragment table
  • Figure 10 is a symbolic transformation table
  • Figure 11 shows the generation of individual compounds, compounds C 1 and C4, and a mixture, mixture Ml ;
  • Figure 12 shows the generation of further mixture, mixture M2
  • Figure 13 shows the generation of an additional mixture, mixture M3
  • Figures 14A and 14B show the generation of an additional mixture, mixture M4;
  • Figure 15 shows tables for tracking compound Cl by the fragments added and or transformations performed;
  • Figure 16 shows tables for tracking mixture Ml by the transformations performed
  • Figure 17 shows tables for tracking mixture M2 by the transformations performed
  • Figure 18 shows tables for tracking mixture M3 by the transformations performed
  • Figure 19 is a pictorial elevation view of an apparatus used to robotically synthesize compound
  • Figure 20 is a pictorial plan view of an apparatus used to robotically synthesize compounds
  • Figure 21 is a first synthetic reaction scheme for preparing a library of compounds
  • Figure 22 is a second synthetic reaction scheme for preparing the library of compounds of Figure 21.
  • the present invention is directed to computational methods employed for the in silico design and synthesis of combinatorial libraries of small molecules.
  • the library members are generated in silico.
  • the present invention also encompasses methods for tracking and storing the information generated during the in silico creation of library members into relational databases for later access and use of this information to synthesize chemical compounds corresponding to those generated in silico.
  • in silico refers to the creation in a computer memory, i.e., on a silicon or other like chip. Stated otherwise in silico means "virtual.”
  • each compound or library member is dissected into its component or constituent parts referred to as fragments.
  • each compound that is generated is considered to be comprised of constituent fragments such that the sum of the molecular formulas of each of the fragments when added together totals the molecular formula of the compound generated.
  • This dissection can be done in a variety of ways using chemical intuition.
  • a variety of components of fragments may be identified, each of which lend themselves to readily available reagents or reactions to generate diverse compounds.
  • each fragment is associated with at least one reagent, which represents the necessary chemical to be used to introduce that desired fragment into the compound being generated in silico.
  • Dissection of compounds is based on the ease of synthesis of the reagents, commercial availability of the reagents, or a combination of both.
  • Each of the fragments and reagents are stored in a relational database and are described in terms of identifying characteristics in the database.
  • a fragment may be available from a variety of starting materials or reaction schemes. So when a library is being generated, which entails building a database, the fragments used in building that library can be stored in the database using the corresponding set of reagents and reaction conditions. When another library is to be generated, the fragment information stored in the database is now available for use in the generation of the new library of compounds. Similarly, when a third library is being generated, an even greater quantity of fragment, reagent, and reaction information is available in the database.
  • the methods of the present invention represent a dynamic method of building a database associated with building libraries of compounds.
  • Initial library generation requires database input for fragments, reagents and transformations necessary for desired library.
  • an increasing number of fragments and reagents are available in the database, which simplifies the generation of subsequent libraries of compounds and makes for more routine combinatorial synthetic efforts which can be accomplished with increasing ease and efficacy.
  • Fragments that are recorded in the database may be defined using identifying characteristics. Identifying characteristics defining fragments include a structural representation (as a 2-dimensional or 3-dimensional file), name, molecular weight, molecular formula, and attachment points or nodes (which denote sites of attachment or linkage of the fragment to other fragments of the compound being generated in silico).
  • 2-dimensional representations are used, which are further simplified by the use of symbolic representations without reference to any particular chemical entities.
  • the symbolic representations as used herein merely shows how fragments can be tracked to further the methods of the present invention.
  • Other identifying characteristics may also be added to the database. Any characteristic that is desired to be tracked may be included in the database, including biological data, chemical reactivity rates, or other physical or chemical properties.
  • a fragment may also be created by modifying a reagent, and such modifications can be added to the database in terms of changes made to the reagent structure. Some of the identifying characteristics associated with any fragment may be common to those of the corresponding reagent. The related fragment thus created can then be stored in the relational database.
  • Identifying characteristics defining reagents include a structural representation, name, molecular weight, molecular formula, and source, such as a commercial source or a unique compound defined by the user. In case of a commercial source for the reagent, a catalog number or a link to a web page can be provided. Some commonalities may exist between the identifying characteristics associated with a reagent and those associated with the related fragment.
  • a compound is the sum of various transformations. Transformation is the nomenclature attributed according to the present invention to a chemical synthesis. A transformation is a 1:1 link between a fragment and a reagent. Thus each transformation describes a unique conversion of a reagent into the corresponding fragment as introduced into a compound. When the compound being generated in silico is broken down into its component fragments, and the corresponding reagents have been identified, each fragment is linked to the corresponding reagent in a 1 : 1 relationship in order to describe a transformation. Thus, according to the present invention, a transformation may be viewed as the source of a fragment, thereby linking that fragment to a particular synthetic method or reaction. This description of a transformation according to the methods of the present invention also includes any auxiliary reagents or conditions used to effect the reaction denoted by the transformation, such as temperature and pressure requirements, catalysts, activators, solvents, or other additives.
  • each combination of a fragment and reagent in a 1 :1 link comprises a different transformation. Therefore, each transformation is unique.
  • the present invention allows the tracking of fragments in terms of the reaction or transformation in which those fragments are introduced into the compounds of the library.
  • the database describes not only the compounds generated in terms of their constituent fragments, but also in terms of the synthetic pathways to produce those compounds, i.e. the related transformations to generate the library compounds.
  • a user of the present invention can generate a virtual library of compounds by simply selecting the fragments desired.
  • a user can also generate the compounds by selecting the chemical pathways required for actual synthesis of the compounds. This is accomplished by selecting the appropriate transformation associated with the generation of the desired compounds.
  • Identifying characteristics defining transformations include the fragment, the reagent, and any auxiliary reagent or conditions necessary to effect the conversion of the reagent into the fragment as incorporated into the compound. For example, consider in Figure 1 the in silico generation of compound Cl according to the methods of the present invention.
  • F molecular formula of C ⁇ 2 H 18 N 2 O 5 S 1
  • F molecular formula of H 2 NO
  • F molecular formula of C 5 H 9 NO
  • F m molecular formula of C 7 H 7 O 3 S
  • F can also be a hydroxyl amine moiety linked to a solid support, i.e. P-O-NH, wherein P is a solid support.
  • P-O-NH hydroxyl amine moiety linked to a solid support
  • each of the fragments, F Cosmetic F Formula, and F ni are stored in a relational database, and are described in terms of identifying characteristics including a structural representation (which may be 2-dimensional or 3-dimensional), an identifier or name, molecular formula and attachment points or nodes which signify sites on the fragment which are linked to other fragments in compound CL Other information such as molecular weight can also be associated with the fragment in the database.
  • each of the corresponding reagents are also stored in the relational database, and described in terms of identifying characteristics. Identifying characteristics used to define the reagents include a structural representation, and identifier or name and molecular formula. As with the fragment, other associated information such as molecular weight and source (such as a commercial source verses user-supplied, amount on hand, special handling, etc.) can also be stored in database in association with the individual reagents.
  • transformation T links reagent R, with fragment F
  • Formula 4 links R substituent R, with fragment F
  • Formula 4 links R substituent R, with fragment F
  • Formula 4 links R substituent R, with fragment F
  • Formula 4 links R substituent R, with fragment F
  • Formula 4 links R substituent R, with fragment F
  • Formula 4 links R substituent R, with fragment F
  • Formula 4 links R substituent R, with fragment F
  • Formula 4 links R substituent R, with F
  • T ⁇ links R n ⁇ with F
  • F is associated with each transformation is the necessary reaction condition, so that transformation T, is associated with reaction condition alpha, T Titan with reaction condition beta, and T m with reaction condition gamma.
  • reagent R m may be a hydroxyl amine attached to a solid support so that fragment F,ute can be represented as a hydroxyl amine moiety attached to a solid support.
  • each fragment may be arrived at or generated by a unique corresponding reagent
  • the present invention also encompasses common fragments that may be generated via two or more reagents, so that two or more transformations can lead to the same fragment.
  • a common reagent may be employed to effect two or more conversions forming two or more different fragments. This then represents two or more different transformations associated with different conditions.
  • common reagent Z CH 3 -CH 2 -NH 2
  • transformation X The same common reagent Z, however, can also be employed to introduce an amide fragment into the compound by using a different set of conditions, constituting transformation Y.
  • a common reagent can introduce two or more different fragments into final compounds being generated in silico, and can be associated with two or more transformations depending upon the conditions associated with each of those transformations.
  • a fragment can be further modified and converted into yet another fragment without effecting any other chemical changes within the compound formed.
  • Common reagent Z' may be used to introduce an alkene fragment into the final compound, representing transformation X', under conditions favoring reduction and dehydration.
  • Common reagent Z' can also be used to introduce a hydroxyalkyl fragment into the final compound under conditions favoring reduction. This represents transformation Y'.
  • the present invention may be described more generally, in terms of symbolic representations. Symbolic representations are used to describe the methods of the present invention because such representations are not limited to any particular chemistry. Symbolic representations merely denote the manner of using the present invention with multiple chemical entities. Each symbol used in the representations describing the present invention may represent one compound or multiple compounds because the present invention is not limited to tracking a single compound, but may be used to track a vast variety of compounds that can be generated.
  • Figure 7 shows the symbolic addition of fragments which yields compound CL.
  • the fragments have structures F,., F Opera., and F,rise that are added sequentially to yield compound CL.
  • Structures F,., F Community>, and F . are symbolic representations of the fragments that constitute compound Cl'. These fragments can be stored in the relational database with the corresponding identifying characteristics for each of them, including the structural representation, name, molecular formula, and attachment sites or nodes.
  • a visual inspection of compounds Cl and Cl' reveals the commonality between the chemical compound Cl and the symbolic representation of a compound Cl' as well as the chemical structure of the fragments and the symbolic structure of the fragments.
  • a symbolic reagent table is shown in Figure 8.
  • Reagents Rl to RIO can be described in terms of their structure, name, molecular formula, molecular weight, and source as well as other information that might be desired to be associated with the reagents.
  • R3 and R4 are two different reagents, but may be used to introduce the same fragment into a compound. This depends upon the reaction conditions used as reagent R3 is used in a transformation associated with one set of conditions, while reagent R4 is used in another transformation associated with a different set of conditions.
  • reagent R5 is comprised of a mixture of two reagents or components. These may be (R)- and (S)-stereoisomers, D- and L-isomers, or may be two completely different reagents.
  • R5 here is represented as a mixture of only two reagents or components, it will be recognized by the art-skilled that the methods of the present invention may be practiced using a mixture of two or more reagents. Typical reagent mixtures used in constructing libraries might have four, five or more individual reagent constituting the mixture.
  • Figure 9 shows a symbolic fragment table. Fragments FI to F8 are stored in the relational database with identifying characteristics that include a structural representation, name, molecular weight, molecular formula, and attachment sites or nodes. This table depicts symbolic representations of the various fragments that are introduced into the compounds of the library by the use of reagents symbolized in Figure 8. Thus it can be seen that fragment FI can be introduced into the compound by employing reagent Rl .
  • X is an identifier for an attachment site. This indicates that X is the site at which FI attaches to another fragment in a compound.
  • fragment F2 may be introduced into a compound (attaching at its X site) by employing reagent R2.
  • Fragment F3 can be introduced into the compound by the use of either reagent R3 or R4. This allows for selection in the choice of the reagent used, and also allows for the consideration of the compatibility of the chemistries involved in the introduction of other fragments into the compound.
  • fragment F4 (which is a mixture of fragments) can be introduced via the use of reagent R5, which is a mixture of reagents, as shown in Figure 8.
  • Fragment F5 has two attachment sites, indicating that other fragments can attach at sites X and Y when F5 has been incorporated into a compound. The presence of two attachment sites indicates that two attachments may be undertaken to build a compound when dealing with F5.
  • F5 can be introduced into the compound using either of reagents R6 or R7, depending upon the reaction conditions used and the chemistries involved when introducing other fragments to build the compound.
  • Fragments F7 and F8 can be introduced into a compound being created in silico by employing reagents R9 and RIO, respectively. Both these fragments have three attachment sites, indicating that three attachments to other fragments can occur when using these fragments to build a compound in silico. While fragments F7 and F8 have three attachment sites, it is recognized by the art-skilled that more than three attachment sites may be present in a fragment, allowing for more attachments to the fragment upon introduction into a compound (with the use of an appropriate reagent).
  • a transformation table is created in accordance with the methods of the present invention, by linking a fragment with a reagent to form a unique transformation.
  • Figure 10 shows a symbolic transformation table where a fragment is linked to a reagent in a 1 :1 relationship.
  • the identifying characteristics describing each transformation include a 1 : 1 link (a one to one link) between a fragment and a reagent, and the reaction conditions which include, solvent, concentration, temperature and pressure requirements, or auxiliary reagents necessary to effect the introduction of the fragment into the compound by using an appropriate reagent.
  • Auxiliary reagents include catalysts, activators, acids, bases or other chemicals or additives necessary to effect the fragment introduction described. For example a base can always be added with an alkyl halide to scavenge the acid generated with use of the alkyl halide.
  • transformation TI links fragment FI with reagent Rl .
  • TI also specifies the reaction conditions ( ⁇ ) associated with this 1 : 1 link.
  • T2 links F2 with R2 under conditions ⁇ .
  • Transformations T3 and T4 are each unique transformations despite being associated with a common fragment, F3.
  • Transformation T3 links common fragment F3 with reagent R3 under conditions ⁇
  • transformation T4 links the common fragment F3 with another reagent, R4, under the different conditions, conditions ⁇ .
  • reagent R3 might be an alkyl chloride while R4 might be an alkyl iodide. While these reagents are similar (they are both alkyl halides), they might be used under different reaction conditions.
  • Transformation T5 links fragment F4 with reagent R5.
  • R5 is a mixture of reagents, such as (R)- and (S)-stereoisomers, D- and L-isomers, or two or more different reagents. As a result, use of R5 leads to the introduction of a mixture of fragments F4 into the compound.
  • R5 the multiple reagents in R5 are selected such that they are capable of being mixed together, do not react with each other, and react under similar reaction conditions.
  • R5 may be comprised of a mixture of acid halides. These do not react with each other, but do react similarly with a nucleophile under similar conditions.
  • a reagent is not limited to only one or two components or constituent reagents, but in fact may comprise of two, three, four, five or more reagents or components. When using a mixture of reagents, each of the individual component reagents may have different chemical reactivity rates.
  • Transformations T6 and T7 are similar to transformations T3 and T4 except that conditions identifying each of these transformations are different. Transformation T6 links fragment F5 with reagent R6 under conditions ⁇ , while transformation T7 links the same fragment F5 with a different reagent R7 under different conditions (condition ⁇ ). As the conditions associated with transformations T6 and T7 are different, this allows selection of compatible chemistries with other fragments during any particular synthesis being used. This is a very useful and very important consideration in actually synthesizing real libraries. When it is desired to introduce fragment F5 into the compound, the actual chemistries used to build the compound can be initially be considered in selecting transformation T6 or T7, and thus reagents R6 or R7.
  • Transformations T9 and TI 0 link fragment F7 with reagent R9 and fragment F8 with reagent RIO, respectively. Both transformations are identified to be associated with reaction conditions ⁇ . Fragments F7 and F8 have three attachment sites, but it is recognized that these fragments may have more than three attachment sites, thereby increasing the complexity of the compounds generated, and increasing the number of rounds that may be employed to attach other fragments. For the three sites illustrated, if three sets of different reagent mixtures each have five reagents in the set are used, then 125 compounds will be generated for fragment F7 and a further 125 compounds will be generated for fragment F8.
  • the methods of the present invention may be used to generate single compounds or mixtures of compounds.
  • a mixture comprises two or more compounds and may involve the use of two or more reagents (thus introduction of two or more fragments) at the outset of library generation, introduction of a mixture of reagents (thus a mixture of fragments) at a subsequent stage of library generation, or a combination of both such techniques.
  • Figures 11 and 12 illustrate this aspect of the present invention.
  • the methods of the present invention may be used to generate single compounds such as Cl and C4, or may also be used to generate a mixture of compounds, Ml, comprising compounds C2 and C3.
  • Library generation commences with selecting fragment F7 (with three attachment sites), in the first round (i.e. round n).
  • F7 is combined with fragment F2, constituting synthetic pathway P 1 a, and resulting in the formation of complex fragment CF 1.
  • F7 possesses three attachment sites (i.e. X, Y and Z).
  • round n+1 will not be complete until each of X, Y and Z have been used, if desired, to attach other fragments to.
  • Stepping around each of X, Y and Z, and attaching fragments to these sites occurs in that sequential order. Once sites X, Y and Z of the fragment selected in the first synthesis round (i.e. round n) have been exhausted, stepping around the attachment sites present in the next added fragment constitutes the next synthesis round (i.e. the third synthesis round, or round n+2).
  • the next synthesis round i.e. the third synthesis round, or round n+2
  • CF1 is next subjected to synthetic pathway Plb wherein fragment FI is introduced into CF1, thereby forming complex fragment CF2.
  • CF2 is then subjected to synthetic pathway Pic wherein fragment F5 is added to CF2, leading to the formation of complex fragment CF3.
  • fragment F5 has two attachment sites
  • CF3 has an available attachment site (i.e. site Y). Introduction of fragments to this site (Y site) constitutes synthesis round n+2 (/. e. the third round) because all the desired attachment sites on the previously added fragment have been exhausted.
  • CF3 is subjected to synthetic pathway P2 wherein fragment F4 is introduced into CF3 at attachment site Y.
  • F4 is amixture of two components, a mixture (Ml) of two compounds, C2 and C3, is generated.
  • a single compound may also be generated using the present scheme of fragment introduction.
  • compound C 1 can be generated by subj ecting CF3 to synthetic pathway Pld wherein CF3 is combined with fragment F3, which attaches to site Y in CF3.
  • the introduction of fragment F3 into CF3 constitutes the third synthesis round (i.e. round n+2), leading to the generation of Cl.
  • CF3 can be subjected to synthetic pathway P3a wherein fragment F6 is introduced into CF3 to form CF4.
  • CF4 has one more available attachment site (i. e. site Y) to which fragment F2 may be attached via synthetic pathway P3b.
  • site Y the attachment site to which fragment F2 may be attached via synthetic pathway P3b.
  • the addition of fragment F6 to CF4 constitutes the third synthesis round (i.e. round n+2).
  • Addition of fragment F2 to CF4 represents the fourth synthesis round, or round n+3, because P3b involves addition of a fragment (fragment F2) onto a site (i.e. site Y in CF4) which has been generated by adding fragment F6 to CF3, thus exhausting the available attachment sites on the previously added fragment in CF4 (i.e. fragment F5). That is, the addition of fragment F6 completed round n+2 (or the third synthesis round) because F6 attached to the last available attachment site on CF3 (i.e. site Y in CF3).
  • a single fragment (F5) can be added to CF2 via use of either reagents R6 or R7 (as thus via the transformations associated with R6 and R7). While these additions are represented as two unique transformations for the purpose of tracking in the database on the invention, these additions in effect perform the same chemical conversion. Thus, the simultaneous tracking of compounds generated according to the methods of the invention is useful not only in working with virtual libraries of compounds, but also provide the user with a choice of synthetic pathways along which the compounds can be actually synthesized.
  • This tracking aspect of the present invention is, therefore, a novel and unique way to account for the fragments being introduced, the related transformations (or reactions) associated with the fragments, and the alternate transformations that lead to the introduction of a common fragment into the desired compounds.
  • the present invention allows not only the tracking of individual compounds that are generated by the use of multiple reagents, but also allows for the simultaneous tracking of multiple compounds that are generated via multiple transformations. While the methods described herein represent the tracking aspects of the invention in terms of symbolic representations or tables, it is recognized by the art-skilled that a variety of computer algorithmic codes and techniques may be employed for the individual or simultaneous tracking aspects described above.
  • the present invention further provides methods for the one-pot generation of mixtures of compounds by commencing the library generation using different starting fragments in a one-pot fashion.
  • One-pot generation or synthesis of compounds refers to the formation of multiple compounds in a single reaction vessel (i.e. one pot). This is possible if compatible chemistries are selected. Examples of such single vessels include but are not limited to multiple well plates, e.g. a 96-well plate, reactions flasks, e.g. a 25 mL flask, or even an industrial reactor. The reactions, or transformations, are performed in one vessel regardless of the size of the reaction vessel.
  • the concept of one-pot synthesis is irrelevant to the generation of virtual libraries of compounds as these virtual libraries are merely generated in silico.
  • FIG. 11 An example of a one-pot synthesis was shown in Figure 11 with the addition of the complex reagent R5 to form mixture Ml .
  • a further one-pot synthesis is shown in Figure 12, where a further mixture of compounds is generated.
  • Mixture M2 comprising compounds Cl and C5 can be generated by starting with fragments F7 and F8 in the first synthesis round (i.e. round n). Each of these fragments have three attachment sites onto which other fragments can be introduced.
  • subjecting the two fragments to synthetic pathway Pla wherein F7 and F8 are combined with fragment F5 at site X, results in the one-pot formation of complex fragments CF1 and CF5.
  • CF1 and CF5 are next subjected to synthetic pathway Plb wherein fragment FI is introduced into CF1 and CF5 at site Y, thereby forming complex fragments CF2 and CF6.
  • CF2 and CF6 are next subjected to synthetic pathway Pic wherein fragment F5 is introduced into these complex fragments at site Z, forming CF3 and CF7. This completes the second synthetic round (i.e. round n+1).
  • fragment F5 contains two attachment sites, after introduction into CF3 and CF7, there is still available an attachment site (i.e. site Y) for further introduction of another fragment.
  • CF3 and CF7 are converted to a mixture (M2) of compounds Cl and C5 via synthetic pathway Pld wherein CF3 and CF7 are combined with fragment F3 which attaches to the Y site on fragment F5 in CF3 and CF7.
  • the introduction of fragment F3 at site Y in CF3 and CF7 represents the third synthetic round (i.e. round n+2).
  • FIG. 13 Yet another symbolic example of the one-pot generation of mixtures of compounds, in accordance with the present invention, is shown in Figure 13.
  • silico generation of compounds commences with the selection of fragment F7, which has three sites of attachment (X, Y, and Z). This represents the first synthesis round (i.e. round n).
  • F7 is subjected to synthetic pathway Pla wherein F7 is combined with fragment F2.
  • F2 attaches to site X on fragment F7, forming complex fragment CF 1.
  • CF 1 is subj ected to two synthetic pathways, Plb and Plb'.
  • Plb employs fragment FI which is introduced onto site Y on CF1, thereby forming complex fragment CF2, while P lb' employs fragment F3 which is introduced onto site Y on CF1, thereby forming complex fragment CF8.
  • a mixture of complex fragments (CF2 and CF8) are formed.
  • Both fragments, FI and F3 can be introduced together (such as from a single reagent bottle when actual synthesis is being undertaken) for the one- pot generation of compounds if the chemistries associated with introduction of these fragments into the compounds are compatible. If not, these fragments can be introduced separately.
  • CF2 and CF8 are subjected to synthetic pathway Pic wherein both complex fragments are combined with fragment F5 which attaches to site Z on CF2 and CF8, thereby forming complex fragments CF3 and CF9.
  • the formation of CF3 and CF9 completes the second synthesis round (i.e. round n+1).
  • fragment F5 has two sites of attachment, site Y is still available for attachment to another fragment. Therefore, CF3 is subjected to synthetic pathway P3 wherein CF3 is combined with fragment F4.
  • Introduction of F4 represents the third synthesis round (i. e. round n+2).
  • F4 is a mixture of fragments (and introduced by adding a mixture of reagents), as shown in Figure 9.
  • mixture M3 is formed comprising compounds C2, C3, C7 and C8.
  • the present invention also provides methods for the generation of increasingly complex mixtures of compounds.
  • An example is shown in Figures 14a and 14b where mixture M4 is generated and comprises sixteen compounds.
  • the compounds in mixture M4 can be generated by starting with fragments F7 and F8 in the first synthesis round (i.e. round n). These fragments can then be combined with fragment F2, which is introduced at site X in each of F7 and F8, forming complex fragment CF1 and CF5. Following this, a mixture of fragments FI and F3 are introduced into CF1 and CF5 at site Y of these complex fragments, leading to the formation of four complex fragments, CF2, CF6, CF8 and CFU. These complex fragments are next combined with a mixture of fragments F5 and F6.
  • Both F5 and F6 have two attachment sites such that site X on F5 and F6 attaches to site Z on CF2, CF6, CF8 and CF11 forming a mixture of eight complex fragments, CF3, CF7, CF9, CF12, CF13, CF14, CF15 and CF16.
  • fragments F5 and F6 have two attachment sites, X and Y
  • the abovementioned eight complex fragments have one more available attachment site (i.e. site Y) onto which another fragment may be introduced. Attachment of a fragment to site Y on these eight complex fragments represents the third synthesis round (i.e. round n+2).
  • fragment F4 is introduced into CF3, CF7, CF9, CF12, CF13, CF14, CF15 and CF16.
  • fragment F4 is a mixture of two constituent fragments, sixteen compounds are generated: C2, C3, C7, C8, C9, CIO, Cl 1, C12, C13, C14, C15, C16, C17, C18, C19 and C20.
  • the present invention also provides methods for keeping track of fragment addition in the various synthesis rounds. This system of accounting is accomplished by tabulation of the synthesis rounds which are correlated with addition of fragments. While for the purposes of illustration of the invention, a tabulation method of tracking fragment addition is described herein, it will be recognized by the art-skilled that other algorithms, algorithmic codes, computer readable mediums and various software coding techniques know to those skilled in the computer arts may be used for such tracking.
  • the tables tracking fragment addition can be used to produce structural representations of compounds and create virtual libraries where actual synthesis of the compounds is not desired. Tables tracking transformations, however, can be used to synthesize compounds by selecting the appropriate transformations, and in the case of multiple transformations, selecting the preferable transformations to introduce the required fragment into the compounds being synthesized.
  • Figure 15 is descriptive of compound Cl in terms of the fragments added in each synthesis round.
  • the first synthesis round i.e. round n
  • fragments F2, FI and F5 in the second synthesis round i.e. round n+1.
  • compound Cl is generated by the addition of fragment F3 in the third synthesis round (i.e. round n+2).
  • the compounds thus generated can be stored as a 2-dimensional virtual library, or may be converted to a 3 -dimensional virtual library that can be used for in silico docking to desired target molecules.
  • Figure 15 also shows the generation of compound Cl in terms of the various transformations employed in the synthesis rounds.
  • Four synthesis pathways lead to the synthesis of compound Cl because of the availability of multiple transformations that can introduce the same fragment into the compound being synthesized.
  • selection of fragment F7 constitutes transformation T9 in the first synthesis round (i.e. round n).
  • fragment F2 which is achieved by employing transformation T2.
  • fragment FI is added via transformation TI.
  • Fragment F5 may be added by employing either reagent R6 via transformation T6 along synthesis paths 1 and 3, or reagent R7 via transformation T7 along synthesis paths 2 and 4.
  • the final fragment F3 can be added by using either reagent R3 via transformation T3 along synthesis paths 1 and 2, or reagent R4 via transformation T4 along synthesis paths 3 and 4.
  • Figure 15 shows that compound C 1 can be actually synthesized via one of four different synthetic schemes which can be tracked or tabulated and accounted for using the methods of the present invention.
  • Each of the four tables is completely descriptive of each of the four synthetic pathways for the preparation of Cl.
  • a user of the present invention has available all the alternate pathways of performing the same reaction (i.e. introducing the same fragment), and can select the preferable or most appropriate synthetic route to preparing the desired compounds.
  • Figure 16 shows a similar transformation tracking table for compounds C2 and C3 in mixture Ml .
  • Synthesis of compounds C2 and C3 commences with selection of fragment F7 which represents transformation T9 (step 1 in Figure 16) in the first synthesis round ( . e. round n).
  • F7 is combined with fragment F2 via transformation T2 in the second synthesis round (i.e. round n+1) (step 2).
  • fragment FI, via transformation TI, and fragment F5, via transformation T7 are added sequentially (steps 3 and 4).
  • fragment F4 is added in the third synthesis round (i. e. round n+2) .
  • Step 5 represents compounds C2 and C3.
  • Figure 17 shows a transformation tracking table for compounds C 1 and C5 in mixture M3.
  • F7 and F8 tracking begins with two parallel tables (step 1 in Figure 17).
  • F7 is selected via transformation T9
  • F8 is selected via transformation T10.
  • the second synthesis round i.e. round n+1
  • step 2 With the introduction of fragment F2 via transformation T2.
  • step 3 transformation TI introduces fragment FI into the compound.
  • step 4 transformation T7 introduces fragment F5. This completes the second synthesis round (i.e. round n+1).
  • the third synthesis round i.e.
  • transformation T4 is used to introduce fragment F3 (at step 5) producing mixture M2 comprising compounds Cl and C5.
  • the tables are duplicated early in the synthetic scheme because of the use of a mixture of fragments F7 and F8 at the outset.
  • the transformation tracking table for compounds C2, C3, C7 and C8 of mixture M3 are shown in Figure 18.
  • the synthesis of these compounds commences with the first synthesis round (i.e. round n) in which fragment F7 is selected. This represents transformation T9 (shown in step 1 in Figure 18).
  • Step 2 in Figure 18 depicts the second synthesis round (i.e. round n+1) and involves the addition of fragment F2 via transformation T2.
  • step 3 involves two different transformations because two different fragments are being introduced into the compounds through the use of two different reagents. Therefore, at step 3 the table is twice duplicated because two different reagents are being employed to introduce two different fragments via two different transformations.
  • transformation TI is used to introduce fragment FI while transformation T3 is used to introduce fragment F3.
  • the second synthesis round i.e. round n+1
  • transformation T7 which introduces fragment F5.
  • transformation T5 is used to introduce fragment F4.
  • each table at step 5 is twice duplicated for transformations T5 1 and T5 2 which represent each of the constituent fragments of F4.
  • the synthesizer described in those patents was modified to include movement in along the Y axis in addition to movement along the X axis. As so modified, a 96-well array of compounds can be synthesized by the synthesizer.
  • the synthesizer can further include temperature control and the ability to maintain an inert atmosphere during all phases of a synthesis.
  • the reagent array delivery format employs orthogonal X-axis motion of a matrix of reaction vessels and Y-axis motion of an array of reagents. Each reagent has its own dedicated plumbing system to eliminate the possibility of cross-contamination of reagents and line flushing and/or pipette washing.
  • a plate of compounds is made by permutating a set of reagents, and writing the resulting output to a text file.
  • the text file is input directly into the synthesizer and used for the synthesis of the plate of compounds.
  • the synthesizer can be interfaced with a relational database allowing data output related to the synthesized compounds to be registered in a highly efficient manner.
  • the .seq, .cmd and .tab files are built or constructed and once constructed, are stored in an appropriate database.
  • the .cmd file is a synthesis file.
  • This file can be built fresh to reflect a completely new set of machine commands reflecting a set of chemical synthesis steps (as for instance the above described transformations) or it can modify an existing file stored in a database by editing a stored file.
  • the .cmd files are built using a word processor and a command set of instructions as outlined below.
  • .tab files are built to reflect the necessary reagents used in the automatic synthesizer for the particular chemistries necessary for the library of desired compounds. Thus for each of a set of these chemistries, a .tab file is built and stored in the database.
  • an existing .tab file can be edited for use in constructing a further .tab file.
  • Both the .cmd files and the .tab files are linked together for later retrieval from the database. Linking can be as simple as using like file names to associate a .cmd file to its appropriate .tab file, e.g., syntheses.cmd is linked to syntheses.tab by use of the same preamble in their names.
  • the automated, multi-well parallel array synthesizer employs a reagent array delivery format, in which each reagent utilized has a dedicated plumbing system. As seen in Figures 19 and 20, an inert atmosphere 10 is maintained during all phases of a synthesis. Temperature is controlled via a thermal transfer plate 12, which holds an injection molded reaction block 14. The reaction plate assembly slides in the X-axis direction, while eight nozzle blocks (16, 18, 20, 22, 24, 26, 28 and 30) holding the reagent lines slide in the Y-axis direction, allowing for the extremely rapid delivery of any of 64 reagents to 96 wells.
  • a 96 well plate 44 moves in one direction along the X axis, while the series of independently controlled reagent delivery nozzles (16, 18, 20, 22, 24, 26, 28 and 30) move along the Y-axis relative to the reaction vessel 46.
  • the reaction plate 44 and reagent nozzles (16, 18, 20, 22, 24, 26, 28 and 30) can be moved independently at the same time, this arrangement facilitated the extremely rapid delivery of up to 72 reagents independently to each of the 96 reaction vessel wells.
  • the system software allows the straightforward programming of the synthesis of a large number of compounds by supplying the general synthetic procedure in the form of the command file to call upon certain reagents to be added to specific wells via lookup in the sequence file with the bottle position, flow rate, and concentration of each reagent being stored in the separate reagent table file.
  • Compounds can be synthesized on various scales ranging from small, as for example a 200 nmole scale, to larger scales, as for example a 10 ⁇ mole scale (3-5 mg).
  • the resulting crude compounds are generally >80% pure, and are utilized directly for high throughput screening assays. Alternatively, prior to use the plates can be subjected to quality control to ascertain their exact purity.
  • Use of the synthesizer results in a very efficient means for the parallel synthesis of compounds for screening.
  • the software inputs accept tab delimited text files from any text editor.
  • a typical command file, a .cmd file is shown in Example 4, Table 2.
  • a typical sequence file, a .seq files, is shown in Example 4, Table 3, and a typical reagent file, a .tab file, is shown in Example 4, Table 4.
  • Typically some of the wells of the 96 well plate may be left empty (depending on the number of compounds in the individual synthesis) or some of the well may have compounds that will serve as standards for comparison or analytical purposes.
  • the solid support for use in holding the growing compounds during synthesis is loaded into the wells of the synthesis plate 44 by pipetting the desired volume of abalanced density slurry of the support suspended in an appropriate solvent, typically an acetonitrile-methylene chloride mixture. Reactions can be run on various scales as for instance the above noted 200 nmole and 10 ⁇ mol scales.
  • Various supports can be utilized for synthesis. Particularly useful supports include medium loading polystyrene-PEG supports such as TentaGelTM or ArgoGelTM.
  • the synthesis plate is transported back and forth in the X- direction under an array of 8 moveable banks (16, 18, 20, 22, 24, 26, 28 and 30) of 8 nozzles (64 total) in the Y-direction, and 6 banks (32, 34, 36, 38, 40 and 42) of 48 fixed nozzles, so that each well can receive the appropriate amounts of reagents and/or solvents from any reservoir (large bottle or smaller septa bottle).
  • a sliding balloon-type seal 50 surrounds this nozzle array and joins it to the reaction plate headspace 52.
  • a slow sweep of nitrogen or argon 20 at ambient pressure across the plate headspace is used to preserve an anhydrous environment.
  • the liquid contents in each well do not drip out until the headspace pressure exceeds the capillary forces on the liquid in the exit nozzle.
  • a slight positive pressure in the lower collection chamber can be added to eliminate residual slow leakage from filled wells, or to effect agitation by bubbling inert gas through the suspension.
  • the headspace gas outlet valve is closed and the internal pressure raised to about 2 psi. Normally, liquid contents are blown directly to waste 54.
  • a 96 well microtiter plate can be inserted into the lower chamber beneath the synthesis plate in order to collect the individual well eluent for spectrophotometric monitoring of reaction progress and yield.
  • the basic plumbing scheme for the machine is the gas-pressurized delivery of reagents.
  • Each reagent is delivered to the synthesis plate through a dedicated supply line, collectively identified at 56, solenoid valve collectively identified at 58 and nozzle, collectively identified at 60.
  • Reagents never cross paths until they reach the reaction well. Thus, no line needs to be washed or flushed prior to its next use and there is no possibility of cross-contamination of reagents.
  • the liquid delivery velocity is sufficiently energetic to thoroughly mix the contents within a well to form a homogeneous solution, even when employing solutions having drastically different densities. With this mixing, once reactants are in homogeneous solution, diffusion carries the individual components into and out of the solid support matrix where the desired reaction takes place.
  • Each reagent reservoir can be plumbed to either a single nozzle or any combination of up to 8 nozzles.
  • Each nozzle is also provided with a concentric nozzle washer to wash the outside of the delivery nozzles in order to eliminate problems of crystallized reactant buildup due to slow evaporation of solvent at the tips of the nozzles.
  • the nozzles and supply lines can be primed into a set of dummy wells directly to waste at any time.
  • the entire plumbing system is fabricated with Teflon tubing, and reagent reservoirs are accessed via syringe needle/septa or direct connection into the higher capacity bottles.
  • the septum vials 48 are held in removable 8-bottle racks to facilitate easy setup and cleaning.
  • the priming volume for each line is about 350 ⁇ l.
  • the minimum delivery volume is about 2 ⁇ l, and flow rate accuracy is ⁇ 5%.
  • the actual amount of material delivered depends on a timed flow of liquid.
  • the flow rate for a particular solvent will depend on its viscosity and wetting characteristics of the Teflon tubing.
  • the flow rate (typically 200-350 ⁇ l per sec) is experimentally determined, and this information is contained in the reagent table setup file.
  • Heating and cooling of the reaction block 14 is effected utilizing a recirculating heat exchanger plate 12, similar to that found in PCR thermocyclers, that nests with the polypropylene synthesis plate 44 to provide good thermal contact.
  • the liquid contents in a well can be heated or cooled at about 10°C per minute over a range of +5 to +80°C, as polypropylene begins to soften and deform at about 80°C.
  • a non-disposable synthesis plate machined from stainless steel or monel with replaceable frits might be utilized.
  • the hardware controller is designed around a set of three 1 MHZ 86332 chips.
  • This controller is used to drive the single X-axis and 8 Y-axis stepper motors as well as provide the timing functions for a total of 154 solenoid valves.
  • Each chip has 16 bidirectional timer I O and 8 interrupt channels in its timer processing unit (TPU). These are used to provide the step and direction signals, and to read 3 encoder inputs and 2 limit switches for controlling up to three motors per chip.
  • Each 86332 chip also drives a serial chain of 8 UNC5891 A darlington array chips to provide power to 64 valves with msec resolution.
  • the controller communicates with the Windows software interface program running on a PC via a 19200 Hz serial channel, and uses an elementary instruction set to communicate valve_number and time_open, and motor_number and position_data.
  • the three components of the software program that run the array synthesizer, the generalized procedure or command (.cmd) file which specifies the synthesis instructions to be performed, the sequence (.seq) file which specifies the scale of the reaction and the order in which variable groups will be added to the core synthon, and the reagent table (.tab) file which specifies the name of a chemical, its location (bottle number), flow rate, and concentration are utilized in conjunction with a basic set of command instructions.
  • the basic set of command instructions are:
  • the ADD instruction has two forms, and is intended to have the look and feel of a standard chemical equation.
  • Reagents are specified to be added by a molar amount if the number proceeds the name identifier, or by an absolute volume in micro liters if the number follows the identifier.
  • the number of reagents to be added is a parsed list, separated by the '+' sign.
  • the key word, ⁇ seq> means look in the sequence table for the identity of the reagent to be added, while the key word, ⁇ act>, means add the reagent which is associated with that particular ⁇ seq>.
  • ADD ACN 300 means: Add 300 ⁇ l of the named reagent ACN to each well of active synthesis ADD ⁇ seq> 300 means: If the sequence pointer in the .seq file is to a reagent in the list of reagents, independent of scale, add 300 ⁇ l of that particular reagent specified for that well.
  • ADD 1.1 PYR + I.0 ⁇ seq> + I.l ⁇ actl> means: If the sequence pointer in the .seq file is to a reagent in the list of acids in the Class ACIDS_1, and PYR is the name of pyridine, and ethyl chloro formate is defined in the .tab file to activate the class, ACIDS_1, then this instruction means: Add 1.1 equiv. pyridine
  • the IF command allows one to test what type of reagent is specified in the ⁇ seq> variable and process the succeeding block of commands accordingly.
  • WAIT 60 DRAIN 10 END means: Operate on those wells for which reagents contained in the Acid l class are specified, WAIT 60 sec, then operate on those wells for which reagents contained in the Acid_2 class are specified, then WAIT 60 sec longer, then DRAIN the whole plate. Note that the Acid l group has reacted for a total of 120 sec, while the Acid_2 group has reacted for only 60 sec.
  • the REPEAT command is a simple way to execute the same block of commands multiple times.
  • ADD ACN 300 DRAIN 15 END_REPEAT END means: repeats the add acetonitrile and drain sequence for each well three times.
  • the PRIME command will operate either on specific named reagents or on nozzles which will be used in the next associated ⁇ seq> operation.
  • the ⁇ l amount dispensed into a prime port is a constant that can be specified in a config.dat file.
  • the NOZZLE_WASH command for washing the outside of reaction nozzles free from residue due to evaporation of reagent solvent will operate either on specific named reagents or on nozzles which have been used in the preceding associated ⁇ seq> operation.
  • the machine is plumbed such that if any nozzle in a block has been used, all the nozzles in that block will be washed into the prime port.
  • the WAIT and DRAIN commands are by seconds, with the drain command applying a gas pressure over the top surface of the plate in order to drain the wells.
  • the LOAD and REMOVE commands are instructions for the machine to pause for operator action.
  • the NEXT SEQUENCE command increments the sequence pointer to the next group of substituents to be added in the sequence file.
  • the general form of a .seq file entry is the definition:
  • the sequence information is conveyed by a series of columns, each of which represents a variable reagent to be added at a particular position.
  • the scale ( mole) variable is included so that reactions of different scale can be run at the same time if desired.
  • the reagents are defined in a lookup table (the .tab file), which specifies the name of the reagent as referred to in the sequence and command files, its location (bottle number), flow rate, and concentration. This information is then used by the controller software and hardware to determine both the appropriate slider motion to position the plate and slider arms for delivery of a specific reagent, as well as the specific valve and time required to deliver the appropriate reagents.
  • the adept classification of reagents allows the use of conditional IF loops from within a command file to perform addition of different reagents differently during a 'single step' performed across 96 wells simultaneously.
  • Reagents can be group according to "class.” Thus all for a particular synthesis that utilizes a fragment that is based on amino acids, the class "AMLNO_ACIDS" can be created.
  • the special class ACTIVATORS defines certain reagents that always get added with a particular class of reagents (for example Betaine utilized to activate the class AMINO ACIDS).
  • the LOOP_BEGIN and LOOP_END commands define the block of commands which will continue to operate until a NEXT_SEQUENCE command points past the end of the longest list of reactants in any well.
  • a MOVE command Not included in the command set is a MOVE command.
  • the controller software and hardware determines the correct nozzle(s) and well(s) required for a particular reagent addition, then synchronizes the position of the requisite nozzle and well prior to adding the reagent.
  • a MANUAL mode is also utilized in which the synthesis plate and nozzle blocks can be 'homed' or moved to any position by the operator, the nozzles primed or washed, the various reagent bottles depressurized or washed with solvent, the chamber pressurized, etc.
  • the automatic COMMAND mode can be interrupted at any point, MANUAL commands executed, and then operation resumed at the appropriate location.
  • the sequence pointer can be incremented to restart a synthesis anywhere within a command file.
  • the compounds to be synthesized can be rearranged or grouped for optimization of synthesis. Such grouping can be effected based on any parameter that will result in optimization of synthesis.
  • One such factor considers the fragment of the compounds that are directly linked to the supporting resin. If the same fragment is to be utilized multiple times, it can be joined to the support in a batch wise manner and aliquots of this batch synthesis then loaded into the individual wells of the plate prior to start of the synthesis. Another parameter is by positioning like compounds near each other. By grouping like fragments near each other, machine movements are conserved and in doing so, overall synthesis time is shortened.
  • the position of the compounds on the synthesis plates is specified by the creation of a .seq file as described above.
  • the .seq file is associated with the respective .cmd and .tab files needed for synthesis of the particular chemistries specified for the compounds by retrieval of the .cmd and .tab files a database.
  • These files are then input into the multi well synthesizer for compound synthesis.
  • the plates can be lyophilized at this point if desired.
  • each well contains the compounds located therein as a dry compound.
  • the illustrative hydroxamic library compounds generally correspond in structure to compound Cl of Figure 1, formed from a hydroxylamine fragment, a valine fragment (the amino acid fragment) and a sulfonyl-4-methoxybenzene fragment (the sulfonyl fragment) of Figure 2. They differ from one another with respect to their amino acid fragment and their sulfonyl fragment. They have in common their hydroxyl amine fragment.
  • Compound Cl directly corresponds (they are one in the same) to compound a-x of Table 1. These compounds further corresponds to symbolic compound CF.
  • the first fragment, the hydroxyl amine fragment is the same in all members of the library. Therefore, for ease of synthesis, it is added already attached to a solid support to wells in a synthesis plate. This reduces the complexity of the synthesis by a factor of "one fragment” and in turn reduce the number of rounds by one of synthesis that must be effected on the synthesizer. In essence this eliminates the round n as described in the tables of Figures 15, 16, 17 and 18.
  • complex or single reagents can be specified in the "Reagent Name" as defined by the bottle the reagent or mixture of reagents is located. Whether it was a single reagent or a complex reagent mixture specified by a particular transformation, that information is carried over to the synthesizer instructions by the appropriate entry in the .tab file for that reagent. As for the .seq file creation, the information in the transformation tracking table can be readily converted to a .tab file. Each complex or single reagent called for in the synthesis is given a line entry in the .tab file. Additionally, the single reagent components of complex reagents may be specified in a comments section of the .tab file to facilitate preparation of complex reagents.
  • the first method of synthesis entails derivatizing commercially available ArgoGel-OHTM (which has an PEG based alcohol as the reactive functional group) with an FMOC-amino acid via a modified Mitsunobu reaction employing the sulfonamide betaine 1 as the activating species.
  • This reaction proceeded to essentially 100% completion (by FMOC) in several hours, and has the advantage over other loading procedure (symmetric anhydride/DMAP) of eliminating the potential for racemization of the amino acid. It also requires less equivalents, as one equivalent of amino acid is not wasted due to the formation of a symmetric anhydride, and the potential for FMOC loss is minimized.
  • the resin bound ester 2 was next deprotected, then sulfonylated using a sulfonyl chloride in pyridine.
  • the yield of the Mitsunobu loading step was measured by collecting the washes from the FMOC deprotection, followed by spectrophotometric determination of the amount released in a 96 well plate reader. This information was then written to a data file for import into a database, which allows a yield estimate of the synthesized compounds.
  • the procedure has the advantage that orthogonal deprotection and cleavage strategies can be employed, allowing standard peptide acid labile side chain protection (t-butyl based, trityl, PMC, etc.) to be used on the amino acid component.
  • This allows isolation of products free from side chain protection by-products in the case of commonly used trityl and sulfonyl based protection of histidine, arginine, glutamine, and asparagine.
  • the resin bound ester 4 can be treated with anhydrous TFA for 4 h on the instrument, resulting in complete side chain deprotection. If cleaned of TFA immediately after synthesis, the instrument, including lines and valves were unaffected by the extreme conditions.
  • the second method utilized the acid labile Wang based hydroxylamine support 6 ( Figure 22) to circumvent the minor problem of competitive hydrolysis, and the failure of electron deficient sulfonyl chlorides.
  • the resin was prepared in an analogous manner to the procedure described by Atheron et al., Solid Peptide Synthesis: A Practical Approach; IRL Press: Oxford, UK 1989: p 135 employing an initial Mitsunobu reaction of ArgoGel-WangTM resin with N-hydroxyphthalimide, followed by deprotection with methylhydrazine to afford 6 in quantitative yield by gel-phase 13 C ⁇ MR.
  • the hydroxylamine resin was then acylated with an FMOC-amino acid utilizing standard peptide coupling methodology to provide 7, which was deprotected then sulfonylated as before to provide resin bound hydroxamic acid 8.
  • This material was efficiently cleaved from the resin with TFA containing Et 3 SiH (5% v/v) as a scavenger to provide compounds 5.
  • ArgoGel-OHTM (360 mg, loading 0.43 mmole/g) was suspended in -16 mL solution of 3:1 CH 2 C1 2 /DMF. The suspension was distributed equally among 12 wells of a 96 well polypropylene synthesis plate (30 mg per well). The solvent was drained and the resin dried overnight in vacuo over P 2 O 5 . All solid reagents were dried in vacuo overnight over P 2 O 5 prior to use. For method 1, the Mitsunobu reagent 1 was dried, then dissolved in anhydrous CH 2 C1 2 to a concentration of 0.15M.
  • FMOC-Amino Acids (Novabiochem, Bachem CA) were dissolved to a concentration of 0.30 M in a solution of 2: 1 anhydrous CH 2 C1 2 /DMF for method 1 , and to a concentration of 0.22 M in DMF containing 0.44 M collidine for synthesis for method 2.
  • Sulfonyl chlorides were dissolved to a concentration of 0.2M in Pyridine. Pyridine proved to be an acceptable solvent for most sulfonyl chlorides, but when solubility was limited, cosolvents such as MeCN, DMSO, CH 2 C1 2 , DMF, and NMP (up to 50%) have been employed.
  • FMOC protection were removed with a solution of 10% piperidine in anhydrous DMF prepared and used the day of synthesis.
  • Low water wash solvents were employed to ensure maximum coupling efficiency of the initial amino-acid to the resin.
  • moisture sensitive reagent lines were purged with argon for 20 minutes. Reagents were dissolved to appropriate concentrations and installed on the synthesizer.
  • Large bottles (containing 8 delivery lines) were used for wash solvents and the delivery of activator.
  • Small septa bottles containing the amino acids and sulfonyl chlorides allow anhydrous preparation and efficient installation of multiple reagents by using needles to pressurize the bottle, and as a delivery path.
  • Example 2 General Hydroxamic Acid Synthesis Method 1 ( Figure 21) The commercial ArgoGel-OHTM resin ( 10 ⁇ mole) was washed with CH 2 C1 2 (6x), then treated with the appropriate FMOC-amino acid (3 eq.) and 1 (3 eq.). After 30 min, the wells were drained, and the process repeated to give a total of 4 treatments (12 eq.). The resin was washed with CH 2 C1 2 (6x), DMF (4x), and the FMOC removed with 10% piperidine in DMF (4 x).
  • the resin was then washed with DMF (4x), then CH 2 C1 2 (6x), and treated with the appropriate sulfonyl chloride (4 x 6 eq. for 15 min.) in pyridine, and washed with CH 2 C1 2 (6x), DMF (6x), and CH 2 C1 2 (lOx).
  • the resin could be treated with 90:5:5 TFA H 2 O/Et 3 SiH for 4 h, then subjected to the above washing procedure to remove any side chain protection on the molecules if necessary.
  • the plates were then removed from the instrument, and individual wells treated with 4 M hydroxylamine (50% aqueous) in 1 ,4-dioxane for 24 h.
  • the filtrate was collected into a deep well 96 well plate, the samples frozen, then lyophilized to provide the desired hydroxamic acids. Addition of fresh 1 ,4-dioxane and repetition of the lyophilization process twice gave compounds free of any residual hydroxylamine (by ⁇ NMR of selected products).
  • Resin 6 was prepared from ArgoGel-Wang-OHTM resin according to published procedures and this resin (10 ⁇ mole) was washed with DMF (6x), CH 2 C1 2 (6x), then treated with the appropriate FMOC-amino acid (3 eq.) in DMF + collidine (6 eq.) and HATU (3 eq.). After 30 min, the wells were drained, and the process repeated to give a total of 4 treatments (12 eq.). The resin was washed with CH 2 C1 2 (6x), DMF (4x), and the FMOC removed with 10% piperidine in DMF (4 x).
  • the resin was washed with DMF (4x), then CH 2 C1 2 (6x), and treated with the appropriate sulfonyl chloride (4 x 6 eq. for 15 min.) in pyridine, and washed with CH 2 C1 2 (6x), DMF (8x), DMSO (8x), and CH 2 C1 2 ( 1 Ox). The plates were then removed from the instrument, and individual wells treated with 90:5:5 TFA/Et 3 SiH/H 2 O for 4 h.
  • the filtrate was collected into a deep well 96 well plate, the resin washed (3x) with TFA, and the samples concentrated in a centrifugal vacuum concentrator. Addition of fresh 1 ,4-dioxane or isopropanol and repetition of the concentration process twice, followed by drying in vacuo overnight gave the desired hydroxamic acids.
  • Example 4 Representative parallel array synthesizer input files
  • the software inputs accept tab delimited text files from any text editor.
  • Examples for the synthesis of hydroxamic acids via the procedure of Figure 21 are shown in Table 2 (.cmd file), Table 3 (.seq file), and Table 4 (.tab file). Only several wells worth of synthesis are shown for brevity. For an entire plate to be prepared, only additional sulfonyl chlorides and additional amino acids need to be added to the .tab file, and additional combinations of the two need to be added to the .seq file such that it contains 96 lines, with each line corresponding to a unique compound prepared.
  • the identity and purity of the compounds was determined by electrospray mass spectroscopy (negative mode) and thin layer chromatography on silica employing MeOH/CH 2 Cl 2 solvent mixtures (TLC).
  • TLC MeOH/CH 2 Cl 2 solvent mixtures
  • Example .cmd file (general synthesis procedure) which executes the synthesis shown in Figure 21. The cleavage from support with hydroxylamine is performed separately.
  • Pr 2 )NEt (2.61 mL, 15 mmole) and 4-napthalenesulfonyl chloride (1.36 g, 6 mmol) in CH 2 C1 2 (50 mL) was stirred at rt overnight.
  • the solution was washed with 5% NaHCO 3 , dried (Na 2 SO 4 ), concentrated, then chromatographed (CH 2 C1 2 to 1% MeOH/CH 2 Cl 2 ) and concentrated to provide 2.02 g of the sulfonamide ester.
  • This material was dissolved in 1,4- dioxane (50 mL) and 25 mL of aqueous hydroxylamine (50% w/w) was added.
  • the crude compounds were screened in a representative high throughput screening assay for antibacterial activity, and compounds 5-n-ii and 5-n-vi were found to have activities minimum inhibitory concentrations (MIC's) of 0.7-1.5 ⁇ M and 3-6 ⁇ M against E. coli, respectively. This activity was verified by manual solution synthesis of analytically pure material as described in Example 5 above, which had identical activity.
  • MIC's minimum inhibitory concentrations

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Abstract

L'invention concerne un procédé de génération de bibliothèques virtuelles de composés, et l'utilisation de ces bibliothèques pour construire des bibliothèques combinatoires. Les composés virtuels sont générés in silico. L'invention concerne également des procédés de suivi de l'adjonction de fragments, de l'utilisation de réactifs, et des transformations réalisées. L'invention concerne, en outre, des procédés permettant d'établir une interface entre les informations nécessaires à la génération de bibliothèques de composés sans instruments, ces informations conduisant la synthèse effective des composés.
EP99922956A 1998-05-12 1999-05-12 Generation de bibliotheques combinatoires de composes correspondant a des bibliotheques virtuelles de composes Withdrawn EP1077993A4 (fr)

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PCT/US1999/010383 WO1999058474A1 (fr) 1998-05-12 1999-05-12 Generation de bibliotheques combinatoires de composes correspondant a des bibliotheques virtuelles de composes

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US7295931B1 (en) 1999-02-18 2007-11-13 Cambridgesoft Corporation Deriving fixed bond information
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Publication number Priority date Publication date Assignee Title
WO1997014106A1 (fr) * 1995-10-13 1997-04-17 Terrapin Technologies, Inc. Identification d'activite chimique commune par comparaison de fragments substructuraux
WO1997027559A1 (fr) * 1996-01-26 1997-07-31 Patterson David E Procede pour creer une bibliotheque moleculaire virtuelle et procede pour y faire des recherches, en utilisant des descripteurs valides de structure moleculaire
EP0818744A2 (fr) * 1996-07-08 1998-01-14 Proteus Molecular Design Limited Procédé de sélection des compositions médicamenteuses potentielles
US5880972A (en) * 1996-02-26 1999-03-09 Pharmacopeia, Inc. Method and apparatus for generating and representing combinatorial chemistry libraries

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US5703792A (en) * 1993-05-21 1997-12-30 Arris Pharmaceutical Corporation Three dimensional measurement of molecular diversity
US5434796A (en) * 1993-06-30 1995-07-18 Daylight Chemical Information Systems, Inc. Method and apparatus for designing molecules with desired properties by evolving successive populations
US5463564A (en) * 1994-09-16 1995-10-31 3-Dimensional Pharmaceuticals, Inc. System and method of automatically generating chemical compounds with desired properties

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997014106A1 (fr) * 1995-10-13 1997-04-17 Terrapin Technologies, Inc. Identification d'activite chimique commune par comparaison de fragments substructuraux
WO1997027559A1 (fr) * 1996-01-26 1997-07-31 Patterson David E Procede pour creer une bibliotheque moleculaire virtuelle et procede pour y faire des recherches, en utilisant des descripteurs valides de structure moleculaire
US5880972A (en) * 1996-02-26 1999-03-09 Pharmacopeia, Inc. Method and apparatus for generating and representing combinatorial chemistry libraries
EP0818744A2 (fr) * 1996-07-08 1998-01-14 Proteus Molecular Design Limited Procédé de sélection des compositions médicamenteuses potentielles

Non-Patent Citations (2)

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Title
CZARNIK A W: "ENCODING METHODS FOR COMBINATORIAL CHEMISTRY" CURRENT OPINION IN CHEMICAL BIOLOGY, CURRENT BIOLOGY LTD, LONDON, GB, vol. 1, 1997, pages 60-66, XP000884345 ISSN: 1367-5931 *
See also references of WO9958474A1 *

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