Classifications

• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
  • G16B35/10—Design of libraries
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
  • G16B15/30—Drug targeting using structural data; Docking or binding prediction
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
  • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
  • G16C20/60—In silico combinatorial chemistry
  • G16C20/62—Design of 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.
• 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.
• 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.
• 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).
• SUMMARY OF THE INVENTION there are provided methods for the generation of virtual combinatorial libraries of small molecules. These library molecules or members are generated in silico. Library members of larger molecular weight, such as those that are polymeric in nature, may also be generated using the methods of the present invention.
• 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.
• the present invention also provides methods for the in silico docking of the library members to identified target molecules. According to these methods, individual library members are allowed to bind to the desired target molecule in order to identify those library members that demonstrate high affinity binding to the targets. While there are a number of ways to identify molecular interaction sites, identify compounds likely to interact with molecular interaction sites of RNA and other biological molecules, synthesize such compounds and analyze their binding, preferred methodologies are described in U.S. Serial Numbers 09/076,405, 09/076,447, 09/076,206, 09/076,214, and 09/076,404, each of which was filed on May 12, 1998 and each assigned to the assignee of this invention. All of the foregoing applications are incorporated by reference herein in their entirety.
• 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 Cl 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;
• FIGS 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.
• 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.
• 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). For the purpose of describing this invention, 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.
• source such as a commercial source or a unique compound defined by the user.
• 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.
• 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.
• each of the fragments, F Cosmetic F, makeup and F 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.
• each of the transformations associated with the in silico generation of compound Cl are also stored in the relational database.
• transformation T links reagent R, with fragment F
• Formula 4 links R mich with F Facility
• T ⁇ n links R m with F m in a 1 : 1 relationship.
• 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 U1 with reaction condition gamma.
• reagent R n] may be a hydroxyl amine attached to a solid support so that fragment F 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 Central., and F m that are added sequentially to yield compound Cl'.
• Structures F,., F lf , and F ⁇ . are symbolic representations of the fragments that constitute compound CF. 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.
• 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.
• 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). With the fragment and reagent tables in place in the relational database, 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 Tl links fragment FI with reagent Rl.
• Tl 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.
• each of the individual component reagents may have different chemical reactivity rates. If a correction is not made for this, this could result in their products being unequally represented in the product compounds.
• This is solved by adjusting the concentration of each reagent in the reaction mixture relative to the other reagents in the mixture such that the relative rates are the same. This is effected by comparing to the reactivity of each of the reagents to a chosen standard reagent. The standardized reactivity rates can then be used to adjust the concentration of each constituent reagent in the reagent mixture to compensate for the varied reaction rates.
• a mixture of reagents with different reaction rates may be used in one reagent mixture to still generate equivalent quantities of the desired compounds in the library.
• 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 T10 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 CF1.
• 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.
• synthesis round n+1 i.e. the second round of fragment introduction, or synthesis, to build the compound.
• CF3 has an available attachment site (i.e. site Y).
• Introduction of fragments to this site (Y site) constitutes synthesis round n+2 (i.e. the third round) because all the desired attachment sites on the previously added fragment have been exhausted.
• CF3 is subj ected to synthetic pathway P2 wherein fragment F4 is introduced into CF3 at attachment site Y.
• F4 is a mixture of two components
• a mixture (Ml) of two compounds, C2 and C3 is generated.
• compound Cl can be generated by subjecting 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.
• One-pot synthesis becomes relevant, however, when the actual synthesis of libraries of compounds is to be undertaken.
• the compounds can be tracked separately for compound building in order to generate distinct chemical structures, however, they can be group together for synthesis allowing them to be made in the same "pot.”
• 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.
• fragment F5 contains two attachment sites
• site Y an attachment site 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 PI a 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 CFl , thereby forming complex fragment CF2, while Plb' employs fragment F3 which is introduced onto site Y on CFl, thereby forming complex fragment CF8.
• CF2 and CF8 employs fragment F3 which is introduced onto site Y on CFl, thereby forming complex fragment CF8.
• 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 CFl and CF5.
• 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.
• 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 Tl.
• 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 Cl 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 (i. 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 Tl, 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 Tl 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. While steps 1 and 2 involve single transformations each, 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.
• step 3 transformation Tl 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.
• DOCK allows structure-based database searches to find and identify the interactions of known molecules to a receptor of interest (Kuntz et al, Ace. Chem. Res., 1994, 27, 117; Gschwend and Kuntz, J. Compt. -Aided Mol Des., 1996, 10, 123).
• DOCK allows the screening of molecules, whose 3D structures have been generated in silico, but for which no prior knowledge of interactions with the receptor is available. DOCK, therefore, provides a tool to assist in discovering new ligands to a receptor of interest. DOCK can thus be used for docking the compounds prepared according to the methods of the present invention to desired target molecules.
• the DOCK program has been applied to protein targets and the identification of ligands that bind to them.
• the DOCK software program consists of several modules, including SPHGEN (Kuntz et al, J. Mol. Biol, 1982, 161, 269) and CHEMGRID (Meng et al, J. Comput. Chem., 1992, 13, 505).
• SPHGEN generates clusters of overlapping spheres that describe the solvent-accessible surface of the binding pocket within the target receptor. Each cluster represents a possible binding site for small molecules.
• CHEMGRID precalculates and stores in a grid file the information necessary for force field scoring of the interactions between binding molecule and target. The scoring function approximates molecular mechanics interaction energies and consists of van der Waals and electrostatic components.
• DOCK uses the selected cluster of spheres to orient ligands molecules in the targeted site on the receptor.
• Each molecule within a previously generated 3D database is tested in thousands of orientations within the site, and each orientation is evaluated by the scoring function. Only that orientation with the best score for each compound so screened is stored in the output file. Finally, all compounds of the database are ranked in order of their scores and a collection of the best candidates may then be screened experimentally.
• RNA double helices RNA plays a significant role in many diseases such as AIDS, viral and bacterial infections.
• few studies have been made on small molecules capable of specific RNA binding.
• DOCK identified several aminoglycosides as candidate ligands, characterized by shape complementarity to the RNA groove. Binding experiments then revealed that one of these aminoglycosides not only bound preferentially to RNA over B-form DNA but also that the ligand binds in the targeted RNA major groove. Recently, the application of DOCK to the problem of ligand recognition in DNA quadruplexes has also been reported (Chen et al., Proc. Natl Acad. Set, 1996, 93, 2635).
• the present invention provides a solution to this problem by allowing the building of three-dimensional models of RNA structure, the building of virtual libraries of ligands, including small molecules, polymeric compounds, oligonucleotides and other nucleic acids, screening of such virtual libraries against RNA targets in silico, scoring and identifying the best potential binders from such libraries, and finally, synthesizing such molecules in a combinatorial fashion and testing them experimentally to identify new ligands for such targets.
• the methods of the present invention aid in the drug discovery process by allowing the identification of those library members which bind with high affinity to the target molecules and, therefore, represent molecules that may be actually synthesized and developed as lead drug candidates.

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