WO1998008091A1

WO1998008091A1 - Normalization of combinatorial reactions

Info

Publication number: WO1998008091A1
Application number: PCT/US1997/014144
Authority: WO
Inventors: Phillip Dan Cook; Hans Gaus; Lendell L. Cummins
Original assignee: Isis Pharmaceuticals, Inc.
Priority date: 1996-08-23
Filing date: 1997-08-22
Publication date: 1998-02-26
Also published as: AU4062497A

Abstract

Improvements in the provision of combinatorial chemical libraries are provided. In accordance with preferred embodiments, sets of chemical reactants are normalized to ensure representations of all anticipated reaction products in a reaction between the set and a scaffold molecule. The processes can be practiced iteratively to yield large libraries.

Description

NORMALIZATION OF COMBINATORIAL REACTIONS

FIELD OF THE INVENTION

This invention relates to improvements in the identification of useful pharmaceutical, veterinary, agricultural, research and other chemical species through combinatorial chemical techniques. One application of the invention is the discovery of new antibiotic compounds.

BACKGROUND OF THE INVENTION From the discovery of penicillin by Fleming in the

1940' s there has been a constant search for new antibiotics, which continues to this day. Although many antibiotics have been discovered, there is an on-going need for the discovery of new antibiotic compounds because of the emergence of drug resistant strains of bacteria. Thus, research on bacterial infection is a perpetual cycle of development of new antibiotics. When penicillin was first discovered, its broad-spectrum antibiotic activity was hailed as the "magic bullet" in fighting many bacterial infections. However, over the years, many strains of bacteria have developed a resistance to penicillin and other currently available antibiotic drugs. No antibiotic drug is effective against all bacterial infections. Many antibiotic drugs available today have a narrow-spectrum of activity, that is, they are effective against only a few specific types of bacterial infections. Thus, for example, the majority of current antibiotic drugs are ineffective against both syphilis and tuberculosis. In addition, some strains of syphilis, tuberculosis and other bacteria have developed resistance to currently available antibiotic drugs, which were effective drugs in the past .

Most bacteria which are resistant to a given drug also exhibit similar resistance to chemically similar drugs. Currently, many antibiotics are based on the 3-lactam chemical core structure of penicillin . Although other chemically diverse antibiotics, such as vancomycin, are currently available, it is only a matter of time before the emergence of bacterial strains which will be resistant to all currently available antibiotic drugs. Thus, to prevent a future world-wide epidemic of drug resistant bacterial infections, there is a never ending need for the development of antibiotic drugs with novel chemical structures.

Combinatorial chemistry, known per se to the art, has emerged as a powerful method for synthesizing groups of chemical compounds that may have from a relative few to many thousands of individual compounds. These groups, denominated "libraries," can be screened for biological or other activity, for example, in antibacterial assays. Another type of biological screening assay which has recently been utilized identifies active ligands that bind molecular targets. In one recent journal article (Wrighton, N.C., et al . , Science, 1996, 273 , 458-463), combinatorial procedures have been used to synthesize libraries of peptides. From these libraries, a number of small peptides were isolated that bind to and activate the receptor for the cytokine erythropoietin. Other examples of combinatorial drug identification methodologies are described in Fodor et al . , United States patent 5,489,678; Pirrung et al . , United States patent 5,143,854; Lerner et al . , PCT patent application WO 93/20242; Lebl et al . , PCT patent application WO 94/28028; Hollis et al . , PCT patent application WO 93/22678; Brennan, United States patent 5,472,672, Nishioka, United States patent 5,449,754 and Ecker et al . , PCT patent application WO 93/04204. Each of the foregoing is incorporated herein by reference.

The application of combinatorial techniques to synthesize combinatorial libraries for the identification of antibiotics, antivirals, and other therapeutically and otherwise useful chemical compounds has created many new challenges. One such challenge is the development of analytical and preparative methods and techniques useful in preparing improved combinatorial libraries. There is also a long-felt need for methods to ensure reliable diversity in combinatorial-derived libraries.

Accordingly, it is an object of the present invention to provide improved combinatorial methods by providing normalized sets of chemical reactants for such syntheses. Sets of chemical reactants particularly useful for the synthesis of combinatorial libraries are also an object of the invention wherein the individual chemical species comprising the sets are modified in order to afford predictable behavior during combinatorialization.

Libraries formed from such sets are further objects.

These and other objects will become apparent to persons of ordinary skill in the art from a review of the present specification and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a electropherogram of a combinatorial library reaction showing differential reactivity of component reactants present as a result of a non-normalized reactant set .

SUMMARY OF THE INVENTION

In accordance with the present invention, methods for preparing normalized sets of chemical reactant compounds are provided. These methods comprise selecting a set of chemical compounds, at least four in number, which are individually capable of reacting with a preselected scaffold compound. The set of reactant compounds are then simultaneously reacted with the scaffold compound to provide a mixture of reaction products. The resulting mixture of reaction products are then analyzed to determine relative reactivity of the individual members of the set of reactants. This analysis is conventionally carried out through chromatography, capillary zone electrophoresis or otherwise which conventionally quantitates the amount of individual reaction products present in the reaction mixture. The amount of reaction products and their relative abundance is related to reactivity of the reactants.

Following the determination of the relative reactivity of the individual members of the reactant set, a normalized set of chemical reactant compounds is prepared. This is done by adjusting the concentration of each of the individual members of the set of reactants generally in inverse proportion with the relative reactivity of the respective members.

A normalized set of chemical reactant compounds can then be reacted with a scaffold molecule or molecules in order to provide a library of chemical compounds which can have substantially equal representation of reaction products therein. Alternatively, it can be ensured that actual representation exists of all possible reaction products in a reaction product mixture, even though the amounts of individual reaction products are not essentially equal.

The present invention also provides methods for producing chemical libraries. These methods comprise preparing a normalized set of chemical reactant compounds by selecting a set of chemical compounds comprising at least four individual chemical species, each of which are capable of reacting with a preselected scaffold compound. The initial set of chemical reactant compounds is then simultaneously reacted with the scaffold compound to provide a mixture of reaction products. The mixture of reaction products is analyzed, such as through a preferred method of capillary zone electrophoresis, to determine relative reactivity of individual members of the set of reactant compounds. Armed with knowledge of the relative reactivity in the individual members of the set, a normalized set of chemical reactant compounds can then be prepared. This may comveniently be done by adjusting the concentration of some or all of the individual members of the set of reactants in generally inverse proportion with the relative reactivity of such members .

It is preferred that the normalized set of chemical reactants be created so that upon subsequent reaction with scaffold molecule, substantially equimolar reaction products are obtained. However, other proportions of reaction products may be satisfactory for some uses.

In accordance with other embodiments of the invention, the normalization process and subsequent reactions can be undertaken on an iterative basis. Thus, a first set of chemical reactant compounds can be normalized as described above, and reacted with a chemical scaffold molecule to yield a mixture of reaction products. This may then be followed by the normalization of a second set of reactant products which are also reactive with the scaffold molecule. Following such subsequent normalization procedure, further reaction of the scaffold molecule will yield additional reaction product mixtures with highly beneficial results being conferred in the preparation of combinatorial libraries therefrom. As will be appreciated, the normalization of the respective sets of chemical reactants may be undertaken in such a fashion that substantially equimolar amounts of all possible reaction products can be attained if desired.

The compounds which comprise the sets of chemical reactant compounds in accordance with the present invention may be any of a wide variety of species which are chemically reactive with one or more scaffold molecules as defined below. As will be appreciated by persons of ordinary skill in the art, the nature and identity of the chemical reactant compounds may be extremely broad in scope, encompassing any species which is capable of forming a covalent bond with a scaffold molecule under reasonable reaction conditions. Thus, such chemical reactants can be seen to be those which have at least one functional group which can undertake such bond formation. Nucleophilic, eletrophilic, electrochemical, photochemical, and other functional groups may be so employed. An example of one type of functional group which can be useful for this purpose is the class of alkylating agents. Compositions having a haloalkyl functionality which can be reacted with nucleophilic substituents on scaffold molecules are but one of the very many types of reactive functionalities useful in the practice of the present invention. The precise identity of the compounds forming the set of chemical reactant species is, thus, best described by what they do rather than by what they are. Such compounds are those which are capable of forming covalent bonds with one or more scaffold molecules.

The set of chemical reaction compounds preferably comprises a significant plurality of compounds, at least four, and preferably even greater numbers. It is preferred that at least six different reactive compounds be included in such sets. It is still more preferred that ten compounds comprise such sets, with greater numbers such as twenty and even more being possible for some embodiments. Any number of chemical reactant compounds may be included in chemical reactant sets in accordance with the present invention so long as they are capable of being distinguished, either directly or indirectly, in an analytical scheme to determine their respective reactivities. Scaffold molecules in accordance with the present invention may be any of a wide variety of chemical species which are capable of reacting with the chemical reactant sets and the compounds forming them. Persons of ordinary skill in the art will understand that this is an extremely broad definition. It is only necessary that such chemical species possess at least one reactive functionality which is capable of forming covalent bonds with the members of the set of chemical reactants as described above. Thus, with reference to the exemplary set of alkylating agents e.g. bromoalkyl compounds, a convenient scaffold molecule is one having at least one nucleophilic substituent, such as a primary or secondary nitrogen.

It will be readily appreciated that reaction between the nitrogen functionality on such an exemplary scaffold compound with the haloalkyl group will displace the halogen and give rise to a nitrogen-carbon covalent bond. The foregoing example is only illustrative, however, and there are very many compounds which satisfy the definition of scaffold molecule.

In accordance with certain preferred embodiments, it is preferred that scaffold molecules comprise a plurality of reactive functionalities. It may be convenient in some embodiments to affix one or more protective groups to the reactive functionalities of scaffold molecules to allow sequential reaction with sets of reactive chemical compounds, with other compounds capable of reacting with the scaffold molecule, or otherwise. It is, accordingly, possible to provide iterative synthesis in accordance with the present invention. In such a case, a protective functionality is generally applied using conventional chemistry to one or more of the reactive functionalities on the scaffold molecule. At least one reactive functionality is caused to retain its ability to react with compounds of a set of reactive chemical compounds. Such set is normalized for reaction with the scaffold molecule and subsequently reacted therewith to provide an initial library. An additional reactive site on the scaffold molecule is then deprotected and caused to react with a further normalized set of reactive chemical compounds. In this context, it is understood that the normalization of the second set of reactive chemical compounds maybe either generally normalized or normalized specifically for reaction at the particular functional group on the scaffold molecule. It will be appreciated that for some embodiments of the present invention, it is preferred that the members of a chemical library prepared in accordance with this invention be present in substantially equimolar amounts. In this regard, "substantially equimolar" shall mean that such compounds are generally within the same order of magnitude of abundance within a reaction mixture as are other products from reaction of the same set of chemical reactive compounds with the scaffold molecule.

For other uses of this invention, it is not necessary that substantially equimolarity be present. Rather, it is only necessary that a sensible amount of each potential reaction product be present in a reaction mixture. In such a case, greater latitude may be employed during the normalization process such that not all members of a particular reactant set need be modified to achieve the desired goal. The normalization of chemical reactant sets is generally achieved by altering the concentration of the individual members of the set in a manner that is inversely proportional with the reactivity of such members. Such reactivity, generally reaction rate, is usually reflected by the relative abundance of a reaction product attributable to such member as a result of a reaction between an equimolar reaction set and the proposed scaffold molecule.

A number of statistical means for determining the appropriate concentration of normalized reaction sets may be employed in accordance with the present invention and any statistical paradigm which gives rise to objectives of the present invention is suitable for use herein. One method for achieving this is very straightforward. After reacting a set of chemical reactants comprising an equimolar mixture thereof with a scaffold molecule, relative abundance of the reaction products of each of the individual reactants with the scaffold is determined through chromatography or otherwise. The ratio of such abundances of individual reaction products to the average abundance of all the reaction products is determined. The set of chemical reactants is then normalized by applying an inverse ratio of the foregoing figures to determine their concentration in the normalized mixture comprising the set.

The methods of the present invention may be applied to a wide variety of chemical species and to a number of biological and other chemical reaction types. Thus, peptides, oligonucleotides, polysacchrides, polycyclic compounds, small molecules, heterocycles, and a wide variety of other material may be prepared in diverse library form using the present methodologies. It is preferred that molecules be prepared from heterocyclic compounds or cyclic compounds having a mean functionality. It has been found that libraries attained thereby have significant antibiotic effect and the same are viewed as being likely effective for antiviral, anti-inflammatory, and for many other therapeutic uses as well.

EXAMPLES

For the present examples, all investigated amines and benzyl bromides were obtained from Aldrich (Milwaukee, WI , USA). Methanol (A.C.S. grade) used for buffer preparation was purchased from Baker (Phillipsburg, NJ, USA) . Buffer salts and formic acid were obtained from Fluka (New York, NY, USA) in the highest available purity.

Capillary zone electrophoresis (CZE) separations were performed on a Beckman P/ACE capillary electrophoresis instrument (Model 5010) at 25 ^*C. An untreated fused silica capillary with 50 mm I. d. , and 360 mm o.d. was used. Detection at the cationic end of the column was performed at 214 nm. All samples were pressure injected for 3 sec (27 cm column) or 5 sec (37 cm column) . Peak areas were determined using Beckman System Gold Software on the P/ACE instrument.

EXAMPLE 1

N' -Benzyl-N-phenylpiperazine

To a solution of N-phenylpiperazine (0.5 mmol) and N,N-diisopropylethylamine (0.75 mmol, Hύnig' s base, 1.5 equivalents) in dry tetrahydrofuran (10 mL) was added benzyl bromide (0.5 mmol) and the reaction mixture was stirred at room temperature. At 1, 2, 3, 4, 7, 30, and 180 minutes, twenty microliter aliquots taken and quenched by diluting into methanol/water/ acetic acid (50:49.5:0.5, v/v/v) . Aliquots were analyzed by capillary zone electrophoresis using 10 mM ammonium acetate, 0.1 % formic acid in methanol as buffer. A column of 27 cm overall length, 20 cm detection length was used. Columns were conditioned by rinsing with 2 N NaOH for 15 min, water for 5 min and buffer for 5 min. Between runs, capillaries were rinsed with 0.2 N NaOH for 1 min and with buffer for 2 min. The CZE electropherogram showed complete reaction at 180 minutes.

EXAMPLE 2

N' -2-methylbenzyl-N-Phenylpiperazine The title compound was prepared as per the procedure of Example 1 using 2-methylbenzyl bromide as the alkylating agent .

EXAMPLE 3

N' -3-methylbenzyl-N-Phenylpiperazine The title compound was prepared as per the procedure of Example 1 using 3-methylbenzyl bromide as the alkylating agent .

EXAMPLE 4

N' -4-methylbenzyl-N-Phenylpiperazine The title compound was prepared as per the procedure of Example 1 using 4-methylbenzyl bromide as the alkylating agent .

EXAMPLE 5

N' -3-methoxybenzyl-N-Phenylpiperazine The title compound was prepared as per the procedure of Example 1 using 3-methoxybenzyl bromide as the alkylating agent . EXAMPLE 6

N' -3-nitrobenzyl-N-Phenylpiperazine

The title compound was prepared as per the procedure of Example 1 using 3-nitrobenzyl bromide as the alkylating agent .

EXAMPLE 7

Initial Library Synthesis; Library comprising N' -Benzyl-N- phenylpiperazine, N' -2-methylbenzyl-N-phenylpiperazine, N' - 3-methylbenzyl-N-phenylpiperazine, N' -4-methylbenzyl-N- phenylpiperazine, N' -3-methoxybenzyl-N-phenylpiperazine, N' - 3-nitrobenzyl-N-phenyl piperazine

To a solution of N-phenylpiperazine (0.5 mmol) and N,N-diisopropylethylamine (0.75 mmol, Hϋnig's base, 1.5 equivalents) in dry tetrahydrofuran (10 mL) was added benzyl bromide (0.5 mmol), 2-methylbenzyl bromide (0.5 mmol), 3- methylbenzyl bromide (0.5 mmol), 4-methylbenzyl bromide (0.5 mmol), 3-methoxybenzyl bromide (0.5 mmol), and 3-nitrobenzyl bromide (0.5 mmol). N-Phenyl-N' -acetylamidepiperazine (0.5 mmol) was added as an internal standard and the reaction mixture was stirred at room temperature. At 1, 2, 3, 4, 7, 30, and 180 minutes twenty microliter aliquots taken and quenched by diluting into methanol/water/acetic acid (50:49.5:0.5, v/v/v) . Aliquots were analyzed by capillary zone electrophoresis using 10 mM ammonium acetate, 0.1 % formic acid in methanol as buffer. A column of 27 cm overall length, 20 cm detection length was used. Columns were conditioned rinsing with 2 N NaOH for 15 min, water for 5 min and buffer for 5 min. Between runs capillaries were rinsed with 0.2 N NaOH for 1 min and with buffer for 2 min. The CZE electropherogram of Figure 1 showed complete reaction at 180 minutes. Scl is Phenyl piperazine, Scl-Fl is N' -Benzyl-N-phenylpiperazine, Scl-F2 is N' -2- Methylbenzyl-N-phenylpiperazine, Scl-F3 is N'-3- Methylbenzyl-N-phenylpiperazine, SclF4 is N' -4 -methylbenzyl- N-phenylpiperazine, Scl-F5 is N-3-methoxybenzyl-N- phenylpiperazine, and Scl-F6 is N' -3-Nitrobenzyl-N- Phenylpiperazine, and where the internal standard used was N-Phenyl-N' -acetylamidepiperazine (0.5 mmol). The final concentrations of products were: N' -benzyl-N-phenylpiperazine (0.064 mM) , N' -2 -methylbenzyl-N-phenylpiperazine (.114 mM) , N' -3 -methylbenzyl-N-phenylpiperazine (0.063 mM) , N' -4-methylbenzyl-N-phenylpiperazine (0.085 mM) , N'-3- methoxybenzyl-N-phenyl piperazine (0.070 mM) , and N'-3- nitrobenzyl-N-phenylpiperazine (.112 mM) .

EXAMPLE 8 Normalizing Library Synthesis

The synthesis of the library of Example 7 is repeated under normalizing conditions in accordance with the present invention. The concentration of each of the reactant alkylating species comprising the set of reactants is adjusted to reflect the reactivity of that reactant as reflected in the different amounts of the different reaction products produced in Example 7.

The concentration of each reactant is adjusted such that approximately equimolar amounts of each reaction product will be produced from reaction of the set of reactants with th^'e scaffold compound. In the present case, the adjusted concentrations are:

N' -benzyl-N-phenylpiperazine 0.65 mM

N' -2 -methylbenzyl-N-phenylpiperazine 0.36 mM N' -3 -methylbenzyl-N-phenylpiperazine 0.66 mM

N' -4-methylbenzyl-N-phenylpiperazine 0.49 mM

N' -3-methoxybenzyl-N-phenylpiperazine 0.59 mM N' -3-nitrobenzyl-N-phenylpiperazine 0.37 mM

and the set of reactants having these concentrations is a normalized reactant set. This normalized set of reactants is reacted with a further quantity of N-phenylpiperazine scaffold molecule to yield the same reaction products in substantially equimolar amounts.

Claims

WHAT IS CLAIMED IS:

1. A method for producing a chemical library comprising a. reacting a mixture of at least four chemical reactant compounds with a scaffold compound to provide a mixture of reaction products; b. analyzing said mixture of reaction products to determine relative reactivity of the individual chemical reactant compounds with said scaffold; c. preparing a normalized set of said chemical reactant compounds by adjusting the concentration of each of said individual chemical reactant compounds in the set in inverse proportion to the determined relative reactivity thereof ; and d. reacting the normalized set of chemical reactant compounds with a further quantity of said scaffold molecule .

2. The method of claim 1 wherein said steps a, b, c, and d are performed at least twice .

3. The method of claim 1 wherein said scaffold molecule has a plurality of reactive sites.

4. The method of claim 1 where at least one reactive site of the scaffold molecule is protected prior to its reaction with the normalized set of chemical reactant compounds.

5. The method of claim 1 wherein the individual chemical compounds present in the library are present in substantially equimolar amounts.

6. A method for preparing a normalized set of chemical reactant compounds comprising

a. reacting a mixture of at least four chemical reactant compounds with a scaffold compound to provide a mixture of reaction products ; b. analyzing said mixture of reaction products to determine relative reactivity of the individual chemical reactant compounds with said scaffold; and c. preparing said normalized set of said chemical reactant compounds by adjusting the concentration of each of said individual chemical reactant compounds in the set in inverse proportion to the determined relative reactivity thereof.

7. The method of claim 6 wherein said set of chemical compounds has at least 6 members.

8. The method of claim 6 wherein said set of chemical compounds has at least 10 members.

9. The method of claim 6 wherein said set of chemical compounds has at least 20 members.

10. The method of claim 6 wherein said scaffold compound has a plurality of reactive sites.

11. The method of claim 10 wherein at least one of said reactive sites is a primary or secondary nitrogen.

12. The method of claim 6 wherein said analyzing step comprises capillary zone electrophoresis.

13. A method for preparing a chemical library comprising reacting the normalized set of chemical reactant compounds in accordance with claim 6 with said scaffold molecule .

14. The method of claim 13 wherein the individual chemical compounds present in the library are present in substantially equimolar amounts.

15. A method for preparing a library of chemical compounds comprising providing a scaffold molecule and reacting said scaffold molecule with a set of chemical reactant compounds to form said library; the individual members of said set of compounds being present in said set in molar concentrations inversely proportional to the reactivity of said individual members with said scaffold molecule .

16. The method of claim 15 wherein the individual chemical compounds present in the library are present in substantially equimolar amounts.

17. The method of claim 15 wherein said scaffold molecule has a plurality of reactive sites.

18. The method of claim 15 wherein at least one reactive site on said scaffold molecule is a primary or secondary nitrogen.

19. A product produced in accordance with the method of claim 1.

20. A product produced in accordance with the method of claim 6.

21. A product produced in accordance with the method of claim 13.

22. A product produced in accordance with the method of claim 15.