RU2333213C2 - Beta-chain mimetics and related methods - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention describes new heterocyclic compounds of the general formula I

where A is -(CH)-, -N- or -CH₂-N-; B is -(CH₂)-, -(CH₂-CH₂)-; D is -(CH₂)- or -(C=O)-; W is -(C=O)- or is absent; X is -NH(C=O)- or is absent; Y is oxygen or sulfur; L is hydrogen, -C(O)NHR₃ or -C(=O)OR₄; R₁ is C_1-12alkyl; C_6-10aryl; C_1-6alkoxy; C_6-10aralkyl; C_6-10aralkyloxy; R₂ is saturated or non saturated C_1-10alkyl, possibly substituted; C_1-10alkenyl; C_6-10aralkyloxy; C_1-10alkanedienyl; C_6-10aryl, possibly substituted; C_6-10aralkyl, possibly substituted; benzodioxolyl; piperonyl; chromenonyl; alkyl-C(=O)-; 5-6-membered heteroaryl with one or two nitrogen atoms, or oxygen atom, or sulfur atom, and possibly substituted; R³ is a radical of the formula

or benzene; where R^a is C_1-10alkyl, possibly substituted; C_1-10alkenyl; C_3-10cycloalkyl; amino; C_6-10aralkyl; possibly substituted; R^d is hydrogen; C_6-10aralkyl, possibly substituted; R₄ is C_6-10aralkyl; on the condition that if A is -(CH)- then B is -(CH₂)-, D is -(CH₂)-, X is -NH(C=O)- and L is -C(=O)NHR₃; if A is N then B is -(CH₂-CH₂)- and X is absent; if A is -CH₂-N- then B is -(CH₂)-, D is -(CH₂)-, W is -(C=O)-, X is absent and L is hydrogen; the invention also describes a pharmaceutical composition containing the claimed compounds.

EFFECT: obtaining compound with NF-kB transcription inhibition effect, applicable in medicine.

7 cl, 3 tbl, 10 ex, 4 dwg

Description

State of the art

FIELD OF THE INVENTION

The present invention relates generally to beta chain mimetics, their chemical library, and their use.

Description of the Related Art

A randomized screening of molecules for possible activity as therapeutic agents has been carried out for many years and has led to the discovery of a number of important drugs. Although advances in molecular biology and computer chemistry have led to increased interest in what has been termed "rational drug design," such methods have not been as fast or reliable as originally intended. Therefore, in recent years, interest and a return to randomized drug screening has renewed. To this end, certain steps have been taken in new technologies based on the development of combinatorial libraries of chemical compounds and screening of such libraries in search of biologically active members.

In general, combinatorial libraries of chemical compounds are simply a collection of molecules. Such libraries are distinguished by chemical species in the library, as well as by the methods used both to form library members and to identify which members interact with biological targets of interest. Although this area is still young, the methods for obtaining and screening libraries have become completely different and improved. For example, a recent review of various combinatorial chemical libraries of compounds identified a number of such methods (Dolle, J. Com. Chem., 2 (3): 383-433, 2000), involving the use of both “labeled” and “unlabeled” library members (Janda, Proc. Natl. Acad. Sci. USA 91: 10779-10785, 1994).

Initially, combinatorial chemical libraries were mainly limited to members of peptide or nucleotide origin. To this end, the methods of Houghten et al. illustrate an example of a method called the “dual iteration” method for compiling libraries of soluble combinatorial peptides using the cleavage synthesis method (Nature (London) 354: 84-86, 1991; Biotechniques 13: 412-421, 1992; Bioorg. Med. Chem. Lett. 3: 405-412, 1993). In this way, libraries of soluble peptides containing tens of millions of members were obtained. Such libraries have been found to be effective in identifying opioid peptides such as methionine and leucine encephalin (Dooley and Houghten, Life Sci. 52, 1509-1517, 1993), and a library of N-acylated peptides was used to identify acetals that are potent antagonists opioids (Dooley et al., Proc. Natl. Acad. Sci. USA 90: 10811-10815, 1933). Most recently, a library of all D-amino acid opioid peptides was created and screened for analgesic activity against mu ("μ") opioid receptor (Dooley et al., Science, 266: 2019-2022, 1944).

Although combinatorial libraries containing members of peptide and nucleotide origin are of great value, in this area there is still a need for libraries containing members of a different origin. For example, traditional peptide libraries differ significantly only in the amino acid sequence for generating library members. Although it is generally accepted that secondary structures of peptides are important for biological activity, such peptide libraries do not impart a limited secondary structure to members of this library.

To this end, some researchers have cyclized peptides with disulfide bridges in an attempt to provide a more limited secondary structure (Timelty et al., J. Chem. Soc. 1067-68, 1994; Eichler et al., Peptide Res. 7: 300-306, 1994 ) However, such cyclized peptides are usually still quite flexible and not sufficiently bioavailable, and therefore only limited success has been achieved.

More recently, non-peptide compounds have been developed that more closely mimic the secondary structure of reverse turns found in biologically active proteins or peptides. For example, U.S. Patent No. 5,440,013 to Kahn and published PCT International Patent Application WO94 / 034494 to Rahn describes conformationally hindered non-peptide compounds that mimic the three-dimensional structure of inverted turns.

Although significant progress has been made in the synthesis and identification of conformationally hindered peptide mimetics, there remains a need for small molecules in the art that mimic the secondary structure of peptides. There is also a need in the art for libraries containing such members, as well as methods for synthesizing and screening library members against targets of interest, in particular biological targets for identifying bioactive library members. For example, US Pat. No. 5,929,237 and a partial extension of US Pat. No. 6,013,458, to Kahn, also describe conformationally hindered compounds that mimic the secondary structure of the regions of reversed turns of biologically active peptides and proteins.

The present compound also satisfies these needs and provides further related benefits by providing conformationally hindered compounds that mimic the secondary β-chain structure of biologically active peptides and proteins.

SUMMARY OF THE INVENTION

In essence, the present invention relates to conformationally hindered compounds that mimic the secondary structure of β-chain structures of biologically active peptides and proteins. The invention also describes libraries containing such compounds, as well as their synthesis and screening.

The compounds of the present invention have the following general structure (I):

where A represents - (CH) -, —N— or —CH ₂ —N—, B represents - (C = O) or - (CH ₂ ) _m -, W represents - (C = O) -, -Y (C = O) -, -NH (C = O) - or absent, X represents -NH-, -NH (C = O) - or absent, Y represents oxygen or sulfur, Z represents oxygen or hydrogen, L is hydrogen, R ₅ , —C (O) NHR ₃ or its equivalents, n is 0 or 1 and m is 1 or 2; R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are the same or different and are independently selected from hydrogen, an amino acid side chain residue or derivative thereof, a molecule residue, a linker and a solid support, and the structure of the stereoisomers of these compounds.

In one embodiment, X is absent, A is —N—, B is - (C = O) -, L is —C (O) NHR _3, and other groups have the meanings given above for structure (I), so that the compounds of the invention have the following structure (I '):

Optionally, W is absent and Z is oxygen.

In one embodiment, X is absent, A is —N—, B is - (CH ₂ ) _m -, L is —C (O) NHR ₃ - and other groups are as defined above in connection with structure (I ), so that the compounds of the invention have the following structure (I "):

Optionally, W is absent and Z is oxygen.

In one embodiment, X is —NH—, A is - (CH) -, B is - (CH ₂ ) _m -, L is —C (O) NHR _3, and other groups are as defined above. connection with the structure (I), so that the compounds of the invention have the following structure (I "'):

Optionally, when Z is oxygen, then W is absent.

In one embodiment, A is —CH ₂ —N—, B is - (CH ₂ ) _m -, L is —C (O) NHR _3, and other groups are as defined above in connection with structure (I) so that the compounds of the invention have the following structure (I ""):

Optionally, Y is oxygen and / or W is absent, and / or Z is oxygen.

The present invention also relates to libraries containing the above structures (I), (I '), (I "), (I"') and (I ""), as well as methods for synthesizing such libraries and methods for screening them for biological identification active compounds. Also described are compositions comprising a compound of this invention in combination with a pharmaceutically acceptable carrier or diluent.

These and other aspects of the present invention will be apparent from reference to the following detailed description and drawings.

A brief description of several drawings

Figures 1 and 2 show a synthetic procedure for preparing libraries of the present invention and compounds of the present invention.

Figure 3 shows a synthetic procedure for obtaining the library of the present invention and the compounds of the present invention, which are more fully described in example 9.

Figure 4 shows a synthetic procedure for obtaining the library of the present invention and the compounds of the present invention, which are more fully described in example 10.

DETAILED DESCRIPTION OF THE INVENTION

Conformationally hindered compounds that mimic the secondary structure of β-chain regions of biologically active peptides and proteins are described. Such mimetics of β-chain structures are used in a wide range of fields, including the use of diagnostic and therapeutic agents. Libraries containing mimetics of β-chain structures of the invention are also described, as well as methods for screening them to identify biologically active members.

In one aspect, the present invention relates to mimetics of β-chain structures and chemical libraries containing mimetics of β-chain structures. The β-chain structure mimetics of the present invention are useful as bioactive agents including, but not limited to, use as diagnostic, prophylactic, and / or therapeutic agents. The β-chain structure mimetic libraries of this invention are used to identify such bioactive agents. In practice of the present invention, libraries may contain tens to hundreds of thousands (or more) of individual mimetics of β-chain structures (also referred to herein as “members”).

In one aspect of the present invention, a β-chain structure mimetic is described having the following structure (I):

where A represents - (CH) -, —N— or —CH ₂ —N—, B represents - (C = O) or - (CH ₂ ) _m -, W represents - (C = O) -, -Y (C = O) -, -NH (C = O) - or absent, X represents -NH-, -NH (C = O) - or absent, Y represents oxygen or sulfur, Z represents oxygen or hydrogen (when Z is hydrogen, then C = Z is CH ₂ ), L is hydrogen, R ₅ , —C (O) NHR ₃ or its equivalents, n is 0 or 1 and m is 1 or 2; R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are the same or different and are independently selected from hydrogen, an amino acid side chain residue or derivative thereof, a molecule residue, a linker and a solid support, and stereoisomers thereof.

In one aspect of the invention, R ₁ , R ₂ , R ₃ , R ₄ and R _{5 are} independently selected from the group consisting of amino-C _2-5 alkyl, guanidino-C _2-5 alkyl, C _1-4 alkylguanidino C _2-5 -alkyl, di-C _1-4 -alkylguanidino-C _2-5 -alkyl, amidino-C _2-5 -alkyl, C _1-4 -alkylamidino-C _2-5 -alkyl, di-C _{1 -4-} alkylamidino-C _2-5 -alkyl, C _1-3 -alkoxy, phenyl, substituted phenyl (wherein the substituents are independently selected from one or more substituents from amino, amidino, guanidine, hydrazine, amidrazonyl, C _1-4 - alkylamino, C _1-4 dialkylamino, halogen, perfluoro-C _1-4 alkyl, C _1-4 alkyl, C _1-3 alkoxy, nitro, k arboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the benzyl substituents are independently selected from one or more substituents from amino, amidino, guanidino, hydrazino, amidrazonyl, C _1-4 alkylamino, C _1-4 dialkylamino, halogen, perfluoro-C _1-4 alkyl, C _1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more substituents from amino, amidino, guanidino, hydrazine amidrazone, C _1-4 alkylamino, C _1-4 dialkylamino, halogen, erftor-C _1-4 -alkyl, C _1-4 -alkyl, C _1-3 -alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bisfenilmetila substituted bisfenilmetila (where the substituents are independently selected from one or more of the substituents of amino, amidino, guanidine, hydrazine, amidrazonyl, C _1-4 alkylamino, C _1-4 dialkylamino, halogen, perfluoro-C _1-4 alkyl, C _1-4 alkyl, C _1-3 alkoxy, nitro , carboxy, cyano, sulfuryl or hydroxyl), pyridyl, substituted pyridyl (where the substituents are independently selected from one or more substituents from amino, amidino, guanidi but hydrazino, amidrazonyl, C _1-4 alkylamino, C _1-4 dialkylamino, halogen, perfluoro-C _1-4 alkyl, C _1-4 alkyl, C _1-3 alkoxy, nitro, carboxy, cyano , sulfuryl or hydroxy), pyridyl-C _1-4 -alkyl, substituted pyridyl-C _1-4 -alkyl (where the pyridine substituents are independently selected from one or more substituents from amino, amidino, guanidino, hydrazino, amidrazonyl, C _1-4 alkylamino, C _1-4 dialkylamino, halogen, perfluoro-C _1-4 alkyl, C _1-4 alkyl, C _1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidyl-C _{1 -4-} alkyl substituted with pyrimidyl-C _1-4 -alkyl (where the pyrimidine substituents are independently selected from one or more substituents from amino, amidino, guanidine, hydrazine, amidrazonyl, C _1-4 -alkylamino, C _1-4- dialkylamino, halogen, perfluoro-C _1-4 - alkyl, C _1-4 alkyl, C _1-3 alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C _1-4 alkyl, substituted triazin-2-yl-C _{1- 4-} alkyl (where the triazine substituents are independently selected from one or more substituents from amino, amidino, guanidine, hydrazine, amidrazonyl, C _1-4 alkylamino, C _1-4 dialkylamino, halogen, perfluoro -C _1-4 -alkyl, C _1-4 -alkyl, C _1-3 -alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazole-C _1-4 -alkyl substituted with imidazole-C _1-4 - alkyl (where the imidazole substituents are independently selected from one or more substituents from amino, amidino, guanidine, hydrazine, amidrazonyl, C _1-4 alkylamino, C _1-4 dialkylamino, halogen, perfluoro-C _1-4 alkyl, C _{1 -4-} alkyl, C _1-3 -alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazolinyl-C _1-4 -alkyl, N-amidinopiperazinyl-N-C _0-4 -alkyl, hydroxy-C _{2- 5-} alkyl, C _1-5 -alkylamino-C _2-5 -alkyl, C _1-5- dialkyl mino-C _2-5 alkyl; N-amidinopiperidinyl-C _1-4 alkyl; and 4-aminocyclohexyl-C _0-2 alkyl.

In one embodiment, R ₁ , R ₂ and R ₃ are the same or different and represents the remainder of the compound and R _{4 is} selected from the amino acid side chain residue or derivative thereof. In another embodiment, L is —C (═O) NHR ₃ and R ₁ , R ₂ and R ₃ are the same or different and is a residue of a compound or a residue of a side chain of an amino acid or derivative thereof and R ₄ is hydrogen.

As used herein, the term “amino acid side chain residue” is any amino acid side chain residue present in naturally occurring proteins, including (but not limited to) naturally occurring amino acid side chain residues shown in Table 1. Other side chain residues naturally occurring amino acids of this invention include (but are not limited to) side chain residues of 3,5-dibromotyrosine, 3,5-diiodotyrosine, hydroxylisine, γ-carboxyglutamate, phosphot Rosin and phosphoserine. In addition, the side chains of glycosylated amino acids, including (but not limited to) glycosylated threonine, serine and asparagine, can also be used in the practice of this invention.

In addition to the side chain residues of naturally occurring amino acids, the side chain residues of the amino acids of the present invention include various derivatives thereof. As used herein, the term “derivative” of an amino acid side chain residue includes modifications and / or variants of side chain residues of naturally occurring amino acids. For example, amino acid side chain residues of alanine, valine, leucine, isoleucine, and phenylalanine can usually be classified as lower alkyl, aryl, or aralkyl residues. Derivatives of amino acid side chain residues include others having a straight or branched chain, cyclic or non-cyclic, substituted or unsubstituted, saturated or unsaturated lower alkyl chains, aryl or arylalkyl residues.

As used herein, the terms “compound residue” and “molecule residue” are used to mean any residue, agent, compound, carrier, molecule, linker, amino acid, peptide or protein covalently attached to a mimetic of a β-chain structure. The attachment is preferably located in any of the R ₁ -, and / or R ₂ -, and / or R ₃ positions. This term also includes amino acid side chain residues and their derivatives.

As used herein, the term “lower chain alkyl residues” contains 1-12 carbon atoms, “lower aryl parts” contain 6-12 carbon atoms and “lower chain aralkyl parts” contain 7-12 carbon atoms. Thus, in one embodiment, the amino acid side chain derivative is selected from C _1-12 alkyl, C _6-12 aryl and C _7-12 arylalkyl, and in a more preferred embodiment, C _1-7 alkyl, C _6-10 aryl and C _7-11 arylalkyl.

The amino acid side chain derivatives of this invention further include substituted lower chain derivatives of alkyl residues, aryl and arylalkyl residues, wherein the substituent is selected from, but not limited to, one or more chemical residues:

-OH, -OR, -COOH, -COOR, -CONH ₂ , -NH ₂ , -NHR, -NRR, -SH, -SR, -CO ₂ R, -SO ₂ H, -SOR and halogen (including F, Cl, Br and I), where in each case, R is independently selected from straight or branched chain, cyclic or non-cyclic, substituted or unsubstituted, saturated or unsaturated lower alkyl, aryl or aralkyl residues. In one aspect, the substituent has less than 18 carbon atoms. In addition, cyclic, lower chain alkyl, aryl and arylalkyl radicals of this invention include naphthalene as well as heterocyclic compounds such as thiophene, pyrrole, furan, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine , pyrimidine, purine, quinoline, isoquinoline and carbazole. Derivatives of the amino acid side chains further include heteroalkyl derivatives of the alkyl moiety of the lower alkyl chain alkyl and aralkyl radicals, including but not limited to alkyl and aralkylphosphonates and silanes.

In one aspect of the invention, residues R ₁ , R _2, and R ₃ are selected from —OH, —OR, —COR, —COOR, —CONH ₂ , —CONR, —CONRR, —NH ₂ , —NHR, —NRR, —SO ₂ R and -COSR, where in each case, R has the above meanings.

In a further embodiment and in addition to an amino acid side chain residue or derivative thereof (or a compound residue in the case of R ₁ , R ₂ and R ₃ ), R ₁ , R ₂ or R ₃ may be a linker facilitating the binding of the compound to another residue or compound . For example, the compounds of this invention may be coupled to one or more known compounds, such as biotin, for use in a diagnostic or screening assay. In addition, R ₁ , R ₂ or R ₃ may be a linker that binds the compound to a solid carrier (such as the carrier used in the synthesis of the peptide in the solid phase), or, alternatively, may be the carrier itself. In this embodiment, the bond with another residue, or compound, or solid support is preferably at the R ₁ , R ₂ or R ₃ position, and more preferably at the R ₃ position.

In an embodiment, when X is absent, A is N, B is —C (═O) - and L is —C (O) NHR ₃ , the β-chain compounds of this invention have the following structure (I ′):

where R ₁ , R ₂ , R ₃ , R ₄ , W, Y, Z and n are as defined above. In a preferred embodiment, R ₂ and R ₃ are the remainder of the compound, R ₁ and R ₄ are selected from amino acid side chain residues.

In an embodiment, when X is absent, A is N, B is - (CH ₂ ) _m - and L is —C (O) NHR ₃ , mimetics of the β-chain structures of this invention include the following structure (I "):

where R ₁ , R ₂ , R ₃ , R ₄ , W, Y, Z and n are as defined above. In a preferred embodiment, R ₂ and R ₃ are the remainder of the compound, and R ₁ and R ₄ are selected from amino acid side chain residues.

In a more specific embodiment, when X is —NH—, A is - (CH) - and B is - (CH ₂ ) _m - and L is —C (O) NHR ₃ , β-chain mimetics structure have the following structure (I "'):

where R ₁ , R ₂ , R ₃ , R ₄ , W, Y, Z, m and n are as defined above.

In a more specific embodiment, when A is —CH ₂ —N—, B is - (CH ₂ ) _m - and L is —C (O) NHR ₃ , the compounds of this invention have the following structure (I "" ):

In the formula, optionally R ₁ , R ₂ , R ₃ , R ₄ , W, X, Y, Z, m, and n are as defined above, W is absent, Z is oxygen, and Y is oxygen.

Mimetics of β-chain structures of the present invention can be prepared using suitable molecules of the starting components (hereinafter referred to as “component parts”). In short, in the synthesis of mimetics of β-chain structures having structure (I '), the first and second component parts are combined to form a combined first to second intermediate compound and, if necessary, the third and / or fourth component parts are combined to form the third to fourth an intermediate (or, if it is commercially available, one third intermediate can be used), a combined first to second intermediate and a third to fourth intermediate (or t the latent intermediate) is then combined to give a first-second-third-fourth intermediate (or first-second-third intermediate), which is cyclized to form mimetics of the β-chain structures of the present invention. Alternatively, mimetics of the β-chain structures of structure (I ′) can be prepared by sequentially combining the individual component parts either stepwise in solution or by solid phase synthesis, as is commonly practiced in solid phase peptide synthesis.

In the context of the present invention, the "first component part" has the following structure 1:

where R ₂ , A and B have the meanings indicated above, and R represents a protective group suitable for use in the synthesis of peptides. Suitable R groups include alkyl groups and, in a preferred embodiment, R represents a methyl group. Such first component parts can be easily synthesized by reductive amination by combining CH (OR) ₂ - (CH ₂ ) _m -CHO with H ₂ NR ₂ or substitution in CH (OR) ₂ - (CH ₂ ) _m -Br.

The second component part according to this invention has the following structure 2:

where L and R ₄ have the meanings indicated above, P represents an amino protecting group suitable for use in peptide synthesis, and X represents a leaving group of an activated carboxylic acid group. Preferred protecting groups include tert-butyldimethylsilyl (TBDMS), BOC, FMOC, and allo (allyloxycarbonyl). When L is —C (O) NHR ₃ , —NHR ₃ may be a carboxyl protecting group. N-protected amino acids are commercially available, for example FMOC amino acids are available from various sources. The conversion of these N-protected amino acids into the second component parts of this invention can be easily achieved by activation of the carboxylic acid group of the N-protected amino acid. Suitable activated carboxylic acid groups include acid halides, where X is a halide such as chloride or bromide, acid anhydrides where X is an acyl group such as acetyl, reactive esters such as N-hydroxysuccinimide esters and pentafluorophenyl esters, and other activated intermediates, such as an active intermediate formed by coupling using carbodiimide, such as dicyclohexylcarbodiimide (DCC).

In the case of an azide derivative amino acid serving as the second component part, such compounds can be prepared from the corresponding amino acid by the reaction described by Zaloom et al. (J. Org. Chem. 46: 5173-76, 1981).

The "third component part" according to this invention has the following structure 3:

R _one -NH ₂  or R ₃ -NH ₂

where R ₁ and R ₃ have the meanings indicated above. Suitable third component parts are commercially available from various sources or can be easily obtained by standard organic synthetic methods commonly used for the synthesis of primary amines.

More specifically, mimetics of β-chain structures of the present invention having structure (I ′) are synthesized by reacting the first component part with the second component part to produce a combined first to second intermediate compound, followed by or sequentially reacting the combined first to second intermediate compound with the third component part, or with the third and fourth component parts to obtain a combined first-second-third-fourth intermediate and then cyclizing the intermediate compound to give β-mimetic chain structure.

The general synthesis of a β-chain structure mimetic having structure I ′ can be performed in the following manner. The first component part 1 is combined with the second component part 2 using a coupling reagent such as phosgene, thereby obtaining, after removal of the N-protecting group, a combined first to second intermediate compound 1-2, as shown below:

where A, B, L, R, R ₂ , R ₄ , P, X and n are as defined above. X ₂ C (= S) is an example of a coupling agent; another type of coupling agents may be used. The syntheses of representative component parts of this invention are described in the examples. Compounds-mimetics of the β-chain structure from (I ") to (I"') can be obtained by methods similar to the synthesis of the modular component described above, but with suitable modifications for the component parts.

In another aspect of the invention, libraries are described comprising mimetics of the β-chain structures of the invention. After compiling the kit, the libraries of the present invention can be screened to identify individual members with bioactivity. Such screening of libraries for bioactive members may include, for example, evaluating the binding activity of library members or evaluating the actions of library members in functional analysis. Screening is usually performed by contacting library members (or a subpopulation of library members) with a target of interest, such as, for example, an antibody, enzyme, receptor, or cell line. Library members that are capable of interacting with a target of interest are referred to herein as “bioactive library members” or “bioactive mimetics.” For example, a bioactive mimetic may be a member of a library that is capable of binding to an antibody or receptor, which is capable of inhibiting an enzyme, or which is capable of inducing or antagonizing a functional response associated, for example, with a cell line. In other words, the screening of the libraries of the present invention determines which library members are capable of interacting with one or more specific biological targets of interest. When an interaction takes place, the interaction of a bioactive mimetic (or mimetics) can be identified from among library members. Identification of one or (a limited number) of bioactive mimetic (s) from the library yields β-chain structure mimetics that are themselves biologically active and thus are useful as diagnostic, prophylactic or therapeutic agents and can additionally be used to achieve significant success in identification of major compounds in these areas.

The synthesis of peptide mimetics of the library of the present invention can be performed using known methods for the synthesis of peptides in combination with the first, second, third and, optionally, fourth component part of this invention. More specifically, any amino acid sequence may be attached to the N-terminal and / or C-terminal portion of the conformationally hindered compound. To this end, mimetics can be synthesized on a solid support (such as PAM resin) by known methods (see, for example, John M. Stewart and Janis D. Young, Solid Phase Peptide Synthesis, 1984, Pierce Chemical Comp., Rockford, III) or a silyl bound resin by addition of an alcohol (see Randolph et al., J. Am. Chem. Soc. 117: 5712-14, 1995).

In addition, a combination of synthesis methods both in solution and in the solid phase can be used to synthesize the peptide mimetics of this invention. For example, a solid support can be used to synthesize a linear peptide sequence to the point at which a conformationally hindered β chain is attached to the sequence. A suitable conformationally-hindered β-chain mimetic structure that has previously been synthesized by solution synthesis methods can then be attached as the next “amino acid” for solid-phase synthesis (ie, a conformationally-hindered β-chain mimetic that has an N-terminal portion and the C-terminal part can be used as the next amino acid to be attached to the linear peptide). By introducing the structure of the conformationally-constrained β-chain mimetic into the sequence, additional amino acids can then be attached to complete the formation of the peptide bound to the solid support. Alternatively, linear peptide sequences with a protected N-terminal and C-terminal moieties can be synthesized on a solid support, separated from the support and then coupled with mimetics of structures with a conformationally restricted β-chain in solution using known methods of coupling in solution.

In another aspect of the present invention, methods for constructing libraries are described. Traditional combinatorial chemical methods (see, for example, Gallop et al., J. Med. Chem. 37: 1233-1251, 1994) make it possible to quickly obtain a huge number of compounds by sequential addition of reagents to the main molecular framework. Combinatorial methods have been used to construct peptide libraries derived from naturally occurring amino acids. For example, by using 20 mixtures of 20 suitably protected and different amino acids and combining each with one of the 20 amino acids, a library of 400 (i.e., 20 ² ) dipeptides is created. Repeating the procedure several times results in a library of dipeptides containing approximately 20 billion (i.e. 20 ⁸ ) octapeptides.

In a further aspect, the invention provides methods for screening libraries for bioactivity and isolating bioactive library members. The libraries of the present invention can be screened for bioactivity in various ways and methods. Typically, a screening assay is carried out (1) by contacting the library with a biological target of interest, such as a receptor, and allowing binding between the library mimetics and the target and (2) detecting the binding event by a suitable analysis, such as the calorimetric analysis described by Lam et al. (Nature 354: 82-84, 1991) or Griminski et al. (Biotechnology 12: 1008-1011, 1994) (both publications are incorporated herein by reference). In a preferred embodiment, library members are in solution and the target is immobilized on a solid phase. Alternatively, the library may be immobilized on a solid phase and may be probed by contacting it with a target in solution.

The synthesis of peptide mimetics of the library of the present invention can be performed using the General scheme to obtain a library of β-chain mimetics, as shown in figure 1. The synthesis of the selected peptide targets of the bicyclic template library of the present invention was performed using the PlexChem reactor unit, which has a 96-well plate. In the above scheme, “Pol” is a 2-chlorotrityl chloride resin (Novabiochem), a detailed procedure is presented below.

Stage 1 A 2-chlorotrityl chloride resin (1 mmol / g) and a solution of Fmoc-R ₁ amino acid (1.5 equivalents) and DIEA (2 equivalents) in DCE were placed in a 96-well Robinson unit (Flexchem). The reaction mixture was shaken for 12 hours at room temperature. The resin is washed with DMF, MeOH and DCM.

Stage 2. To the resin swollen in DMF before the reaction, add 25% piperidine in DMF. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and the product mixture is washed with DMF, MeOH and then DCM. A solution of 4-R ₂ -amino-2-Fmoc-aminobutyric acid (1.5 equivalents), DIC (1.5 equivalents) and HOBT (1.5 equivalents) in NMP are added to the resin. After the reaction mixture was shaken for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

Stage 3. To the resin swollen in DMF before the reaction, add 25% piperidine in DMF. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and the product mixture is washed with DMF, MeOH and then DCM. A solution of 2- (9H-fluoren-9-ylmethoxycarbonylamino) -5,5-dimethoxypentanoic acid (1.5 eq.), DIC (1.5 eq.) And HOBT (1.5 eq.) In NMP was added to the resin. The reaction mixture was shaken for 12 hours at room temperature and then the resin was washed with DMF, MeOH and then DCM.

Stage 4 . 25% piperidine in DMF was added to the resin swollen in DMF before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and the product mixture is washed with DMF, MeOH and then DCM. A solution of commercially available R ₃ acid (1.5 eq.), DIC (1.5 eq.) And HOBT (1.5 eq.) In NMP is added to the resin. The reaction mixture was shaken for 12 hours at room temperature and then the resin was washed with DMF, MeOH and then DCM.

Stage 5 . The resin is treated with formic acid (1.2 ml per well) for 18 hours at room temperature. After that, the resin was removed by filtration, the filtrate was concentrated under reduced pressure using SpeedVac (Servant), thereby obtaining the product as an oil. These products are diluted with 50% water / acetonitrile mixture and then lyophilized after freezing.

Table 2 shows a library of mimetics of the β-chain structure that can be obtained according to the present invention, a representative preparation is given in Example 9. The compounds of table 2 illustrate one aspect of the invention, namely, compounds in which A represents - (CH) -, B is - (CH ₂ ) _m - with m = 1, W is - (C = O) -, X is —NH (C = O) -, Y is oxygen, Z is hydrogen, so C = Z is CH ₂ , L is —C (= O) NHR ₃ , n = 0, R ₄ is hydrogen, and R ₁ , R ₂ and R ₃ are the same or different and are independently selected from an amino acid side chain residue or derivative thereof, a molecule residue, a linker and a solid support, and their stereoisomers. In various embodiments of this aspect of the invention, R ₁ , R _2, and R _{3 are} independently selected from residues with a relatively low molecular weight, i.e. organic groups having molecular weights between 15 (methyl) and 1000 g / mol, and / or at least one of R ₁ , R ₂ and R ₃ is an amino acid side chain or its derivative. For example, in the compounds of Table 2, R ³ is an aspartic acid derivative. In one aspect, the compounds of the present invention have a molecular weight in the range of about 440 to 750 g / mol, the compounds of Table 2 provide numerous illustrations of such compounds.

TABLE 2

Beta Chain Mimetic Library

The synthesis of peptide mimetics in the library of the present invention can be performed using the General scheme of the library of mimetics β-chain structure, as shown in figure 2. The synthesis of selected peptide library mimetics and bicyclic templates of the present invention can be performed using the FlexChem reactor block, which has a 96-well plate. In the above scheme, “Pol” is a 2-chlorotrityl chloride resin (Novabiovhem), a detailed procedure is provided below.

Stage 1 A 2-chlorotrityl chloride resin (1 mmol / g) and a solution of Fmoc-R ₁ beta-amino acids (1.5 equivalents) and DIEA (2 equivalents) in DCE were placed in a 96-well Robinson unit (Flexchem). The reaction mixture was shaken for 12 hours at room temperature. The resin is washed with DMF, MeOH and then DCM.

Stage 2 To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and the product mixture is washed with DMF, MeOH and then DCM. A solution of 4-R ₂ -amino-2-Fmoc-aminobutyric acid (1.5 equivalents), DIC (1.5 equivalents), HOBT (1.5 equivalents) in NMP is added to the resin. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

Stage 3 . To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and the product mixture is washed with DMF, MeOH and then DCM. A solution of 2- (9H-fluoren-9-ylmethoxycarbonylamino) -5,5-dimethoxypentanoic acid (1.5 eq.), DIC (1.5 eq.) And HOBT (1.5 eq.) In NMP was added to the resin. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

Stage 4 . To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and then the product mixture is washed with DMF, MeOH and then DCM. A solution of commercially available R ₃ acid (1.5 eq.), DIC (1.5 eq.) And HOBT (1.5 eq.) In NMP is added to the resin. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

Stage 5. The resin is treated with formic acid (1.2 ml per well) for 18 hours at room temperature. After that, the resin was removed by filtration, and the filtrate was concentrated under reduced pressure using SpeedVac (Servant), thereby obtaining the product as an oil. These products are diluted with 50% water / acetonitrile mixture and then lyophilized after freezing.

The table shows a library of mimetics of the β-chain structure, which can be obtained according to the present invention and a representative receipt of which is given in example 10. The compounds of table 3 illustrate one aspect of the invention, namely, compounds in which A represents - (CH) -, B represents - (CH ₂ ) _m - with m = 1, W is absent, i.e. represents a direct bond between R ^b and N of the heterocyclic ring, X represents —NH (C = O) -, Y represents oxygen, Z represents hydrogen, so C = Z represents CH ₂ , L represents —C ( = O) NHR ₃ , n = 0, R ₄ is hydrogen, and R ₁ , R ₂ and R ₃ are the same or different and are independently selected from an amino acid side chain residue or derivative thereof, a molecule residue, a linker and a solid support, and stereoisomers. In various embodiments of this aspect of the invention, R ₁ , R ₂ and R _{3 are} independently selected from residues with a relatively low molecular weight, i.e. organic groups having molecular weights between 15 (methyl) and 1000 g / mol; and / or at least one of R ₁ , R ₂ and R ₃ is an amino acid side chain or derivative thereof. For example, in the compounds of Table 3, R ³ is glutaric acid derivatives. In one aspect, the compounds of the present invention have a molecular weight in the range of about 450-800 g / mol, the compounds of Table 3 provide numerous illustrations of such compounds.

TABLE 3

Β-chain mimetic library

No. R ^a R ^b R ^c Molecular
weight M + H 865 Propyl Phenyl 4-methoxybenzyl 565 565 866 Propyl Phenyl 3,4-Cl ₂ -benzyl 585 585 867 Propyl Phenyl 1-Naphthyl 589 589 868 Propyl Phenyl Piperonyl 549 549 869 Propyl Phenyl 2,4,5-trimethoxyphenyl 571 571 870 Propyl Phenyl 3-hydroxybenzyl 551 551 871 Propyl Phenyl 1-Naphthylmethyl 565 565 872 Propyl Phenyl Phenethyl 578 578 873 Propyl Phenyl 3-methoxyphenyl 611 611 874 Propyl Phenyl N-benzoylaminoethyl 535 535 875 Propyl Phenyl Benzyl 551 551 876 Propyl Phenyl 4-NO ₂ -benzyl 580 580 877 Propyl Methoxy 4-methoxybenzyl 519 519 878 Propyl Methoxy 3,4-Cl ₂ -benzyl 539 539 879 Propyl Methoxy 1-Naphthyl 543 543 880 Propyl Methoxy Piperonyl 503 503 881 Propyl Methoxy 2,4,5-trimethoxyphenyl 525 525 882 Propyl Methoxy 3-hydroxybenzyl 505 505 883 Propyl Methoxy 1-naphthylmethyl 519 519 884 Propyl Methoxy Phenethyl 532 532 885 Propyl Methoxy 3-methoxyphenyl 565 565 886 Propyl Methoxy N-benzoylaminoethyl 489 489 887 Propyl Methoxy Benzyl 505 505 888 Propyl Methoxy 4-NO ₂ -benzyl 534 534 889 Isobutyl Phenyl 4-methoxybenzyl 593 593 890 Isobutyl Phenyl 3,4-Cl ₂ -benzyl 613 613 891 Isobutyl Phenyl 1-Naphthyl 618 618 892 Isobutyl Phenyl Piperonyl 577 577 893 Isobutyl Phenyl 2,4,5-trimethoxyphenyl 599 599 894 Isobutyl Phenyl 3-hydroxybenzyl 579 579 895 Isobutyl Phenyl 1-Naphthylmethyl 593 593 896 Isobutyl Phenyl Phenethyl 606 606 897 Isobutyl Phenyl 3-methoxyphenyl 639 639 898 Isobutyl Phenyl N-benzoylaminoethyl 563 563 899 Isobutyl Phenyl Benzyl 579 579 900 Isobutyl Phenyl 4-NO ₂ -benzyl 608 608 901 Isobutyl Methoxy 4-methoxybenzyl 547 547 902 Isobutyl Methoxy 3,4-Cl ₂ -benzyl 567 567 903 Isobutyl Methoxy 1-Naphthyl 571 571 904 Isobutyl Methoxy Piperonyl 531 531 905 Isobutyl Methoxy 2,4,5-trimethoxyphenyl 553 553 906 Isobutyl Methoxy 3-hydroxybenzyl 533 533 907 Isobutyl Methoxy 1-Naphthylmethyl 547 547 908 Isobutyl Methoxy Phenethyl 560 560 909 Isobutyl Methoxy 3-methoxyphenyl 593 593 910 Isobutyl Methoxy N-benzoylaminoethyl 517 517 911 Isobutyl Methoxy Benzyl 533 533 912 Isobutyl Methoxy 4-NO ₂ -benzyl 562 562 913 4-Br-benzyl Phenyl 4-methoxybenzyl 692 692 914 4-Br-benzyl Phenyl 3,4-Cl ₂ -benzyl 712 712 915 4-Br-benzyl Phenyl 1-Naphthyl 716 716 916 4-Br-benzyl Phenyl Piperonyl 676 676 917 4-Br-benzyl Phenyl 2,4,5-trimethoxyphenyl 698 698 918 4-Br-benzyl Phenyl 3-hydroxybenzyl 678 678 919 4-Br-benzyl Phenyl 1-Naphthylmethyl 692 692 920 4-Br-benzyl Phenyl Phenethyl 705 705 921 4-Br-benzyl Phenyl 3-methoxyphenyl 738 738 922 4-Br-benzyl Phenyl N-benzoylaminoethyl 662 662 923 4-Br-benzyl Phenyl Benzyl 678 678 924 4-Br-benzyl Phenyl 4-NO ₂ -benzyl 707 707 925 4-Br-benzyl Methoxy 4-methoxybenzyl 646 646 926 4-Br-benzyl Methoxy 3,4-Cl ₂ -benzyl 666 666 927 4-Br-benzyl Methoxy 1-Naphthyl 670 670 928 4-Br-benzyl Methoxy Piperonyl 630 630 929 4-Br-benzyl Methoxy 2,4,5-trimethoxyphenyl 652 652 930 4-Br-benzyl Methoxy 3-hydroxybenzyl 631 631 931 4-Br-benzyl Methoxy 1-Naphthylmethyl 645 645 932 4-Br-benzyl Methoxy Phenethyl 659 659 933 4-Br-benzyl Methoxy 3-methoxyphenyl 692 692 934 4-Br-benzyl Methoxy N-benzoylaminoethyl 615 615 935 4-Br-benzyl Methoxy Benzyl 631 631 936 4-Br-benzyl Methoxy 4-NO ₂ -benzyl 660 660 937 Benzyl Phenyl 4-methoxybenzyl 613 613 938 Benzyl Phenyl 3,4-Cl ₂ -benzyl 633 633 939 Benzyl Phenyl 1-Naphthyl 638 638 940 Benzyl Phenyl Piperonyl 597 597 941 Benzyl Phenyl 2,4,5-trimethoxyphenyl 619 619 942 Benzyl Phenyl 3-hydroxybenzyl 599 599 943 Benzyl Phenyl 1-Naphthylmethyl 613 613 944 Benzyl Phenyl Phenethyl 626 626 945 Benzyl Phenyl 3-methoxyphenyl 659 659 946 Benzyl Phenyl N-benzoylaminoethyl 583 583 947 Benzyl Phenyl Benzyl 599 599 948 Benzyl Phenyl 4-NO ₂ -benzyl 628 628 949 Benzyl Methoxy 4-methoxybenzyl 567 567 950 Benzyl Methoxy 3,4-Cl ₂ -benzyl 587 587 951 Benzyl Methoxy 1-Naphthyl 591 591 952 Benzyl Methoxy Piperonyl 551 551 953 Benzyl Methoxy 2,4,5-trimethoxyphenyl 573 573 954 Benzyl Methoxy 3-hydroxybenzyl 553 553 955 Benzyl Methoxy 1-Naphthylmethyl 567 567 956 Benzyl Methoxy Phenethyl 580 580 957 Benzyl Methoxy 3-methoxyphenyl 613 613 958 Benzyl Methoxy N-benzoylaminoethyl 537 537 959 Benzyl Methoxy Benzyl 553 553 960 Benzyl Methoxy 4-NO ₂ -benzyl 582 582 961 Propyl Benzyloxy 2,4-pentadienyl 541 541 962 Propyl Benzyloxy 3- (2,6-Cl ₂ -pyridyl) methyl 620 620 963 Propyl Benzyloxy Chromene-2-one-3-methyl 619 619 964 Propyl Benzyloxy Methoxymethyl 519 519 965 Propyl Benzyloxy Piran-2-one-4-methyl 569 569 966 Propyl Benzyloxy Ethyl 503 503 967 Propyl Benzyloxy 2-Ethyl Decanil 629 629 968 Propyl Benzyloxy Pyrazin-2-methyl 553 553 969 Propyl Benzyloxy 4-pyridylmethyl 552 552 970 Propyl Benzyloxy 4-butenyl 529 529 971 Propyl Benzyloxy 2-NO ₂ -5-Cl-phenyl 630 630 972 Propyl Benzyloxy Cyanomethyl 514 514 973 Propyl Methoxy 2,4-pentadienyl 465 465 974 Propyl Methoxy 3- (2,6-Cl ₂ -pyridyl) methyl 544 544 975 Propyl Methoxy Chromene-2-one-3-methyl 543 543 976 Propyl Methoxy Methoxymethyl 442 442 977 Propyl Methoxy Piran-2-one-4-methyl 492 492 978 Propyl Methoxy Ethyl 426 426 979 Propyl Methoxy 2-Ethyl Decanil 553 553 980 Propyl Methoxy Pyrazin-2-methyl 476 476 981 Propyl Methoxy 4-pyridylmethyl 476 476 982 Propyl Methoxy 4-butenyl 453 453 983 Propyl Methoxy 2-NO ₂ -5-Cl-phenyl 554 554 984 Propyl Methoxy Cyanomethyl 437 437 985 Isobutyl Benzyloxy 2,4-pentadienyl 569 569 986 Isobutyl Benzyloxy 3- (2,6-Cl ₂ -pyridyl) methyl 649 649 987 Isobutyl Benzyloxy Chromene-2-one-3-methyl 647 647 988 Isobutyl Benzyloxy Methoxymethyl 547 547 989 Isobutyl Benzyloxy Piran-2-one-4-methyl 597 597 990 Isobutyl Benzyloxy Ethyl 531 531 991 Isobutyl Benzyloxy 2-Ethyl Decanil 657 657 992 Isobutyl Benzyloxy Pyrazin-2-methyl 581 581 993 Isobutyl Benzyloxy 4-pyridylmethyl 580 580 994 Isobutyl Benzyloxy 4-butenyl 557 557 995 Isobutyl Benzyloxy 2-NO ₂ -5-Cl-phenyl 658 658 996 Isobutyl Benzyloxy Cyanomethyl 542 542 997 Isobutyl Methoxy 2,4-pentadienyl 493 493 998 Isobutyl Methoxy 3- (2,6-Cl ₂ -pyridyl) methyl 572 572 999 Isobutyl Methoxy Chromene-2-one-3-methyl 571 571 1000 Isobutyl Methoxy Methoxymethyl 471 471 1001 Isobutyl Methoxy Piran-2-one-4-methyl 521 521 1002 Isobutyl Methoxy Ethyl 455 455 1003 Isobutyl Methoxy 2-Ethyl Decanil 581 581 1004 Isobutyl Methoxy Pyrazin-2-methyl 505 505 1005 Isobutyl Methoxy 4-pyridylmethyl 504 504 1006 Isobutyl Methoxy 4-butenyl 481 481 1007 Isobutyl Methoxy 2-NO ₂ -5-Cl-phenyl 582 582 1008 Isobutyl Methoxy Cyanomethyl 466 466 1009 Benzyl Benzyloxy 2,4-pentadienyl 589 589 1010 Benzyl Benzyloxy 3- (2,6-Cl ₂ -pyridyl) methyl 669 669 1011 Benzyl Benzyloxy Chromene-2-one-3-methyl 667 667 1012 Benzyl Benzyloxy Methoxymethyl 567 567 1013 Benzyl Benzyloxy Piran-2-one-4-methyl 617 617 1014 Benzyl Benzyloxy Ethyl 551 551 1015 Benzyl Benzyloxy 2-Ethyl Decanil 677 677 1016 Benzyl Benzyloxy Pyrazin-2-methyl 601 601 1017 Benzyl Benzyloxy 4-pyridylmethyl 600 600 1018 Benzyl Benzyloxy 4-butenyl 577 577 1019 Benzyl Benzyloxy 2-NO ₂ -5-Cl-phenyl 678 678 1020 Benzyl Benzyloxy Cyanomethyl 562 562 1021 Benzyl Methoxy 2,4-pentadienyl 513 513 1022 Benzyl Methoxy 3- (2,6-Cl ₂ -pyridyl) methyl 592 592 1023 Benzyl Methoxy Chromene-2-one-3-methyl 591 591 1024 Benzyl Methoxy Methoxymethyl 491 491 1025 Benzyl Methoxy Piran-2-one-4-methyl 541 541 1026 Benzyl Methoxy Ethyl 475 475 1027 Benzyl Methoxy 2-Ethyl Decanil 601 601 1028 Benzyl Methoxy Pyrazin-2-methyl 525 525 1029 Benzyl Methoxy 4-pyridylmethyl 524 524 1030 Benzyl Methoxy 4-butenyl 501 501 1031 Benzyl Methoxy 2-NO ₂ -5-Cl-phenyl 602 602 1032 Benzyl Methoxy Cyanomethyl 486 486 1033 Phenylpropyl Benzyloxy 2,4-pentadienyl 617 617 1034 Phenylpropyl Benzyloxy 3- (2,6-Cl ₂ -pyridyl) methyl 697 697 1035 Phenylpropyl Benzyloxy Chromene-2-one-3-methyl 695 695 1036 Phenylpropyl Benzyloxy Methoxymethyl 595 595 1037 Phenylpropyl Benzyloxy Piran-2-one-4-methyl 645 645 1038 Phenylpropyl Benzyloxy Ethyl 579 579 1039 Phenylpropyl Benzyloxy 2-Ethyl Decanil 705 705 1040 Phenylpropyl Benzyloxy Pyrazin-2-methyl 629 629 1041 Phenylpropyl Benzyloxy 4-pyridylmethyl 628 628 1042 Phenylpropyl Benzyloxy 4-butenyl 605 605 1043 Phenylpropyl Benzyloxy 2-NO ₂ -5-Cl-phenyl 706 706 1044 Phenylpropyl Benzyloxy Cyanomethyl 590 590 1045 Phenylpropyl Methoxy 2,4-pentadienyl 541 541 1046 Phenylpropyl Methoxy 3- (2,6-Cl ₂ -pyridyl) methyl 620 620 1047 Phenylpropyl Methoxy Chromene-2-one-3-methyl 619 619 1048 Phenylpropyl Methoxy Methoxymethyl 519 519 1049 Phenylpropyl Methoxy Piran-2-one-4-methyl 569 569 1050 Phenylpropyl Methoxy Ethyl 503 503 1051 Phenylpropyl Methoxy 2-Ethyl Decanil 629 629 1052 Phenylpropyl Methoxy Pyrazin-2-methyl 553 553 1053 Phenylpropyl Methoxy 4-pyridylmethyl 552 552 1054 Phenylpropyl Methoxy 4-butenyl 529 529 1055 Phenylpropyl Methoxy 2-NO ₂ -5-Cl-phenyl 630 630 1056 Phenylpropyl Methoxy Cyanomethyl 514 514 1057 Methyl Methoxy 4-methoxybenzyl 491 491 1058 Methyl Methoxy 3,4-Cl ₂ -benzyl 515 515 1059 Methyl Methoxy 1-Naphthyl 497 497 1060 Methyl Methoxy Piperonyl 490 490 1061 Methyl Methoxy 2,4,5-trimethoxyphenyl 537 537 1062 Methyl Methoxy 3-hydroxybenzyl 476 476 1063 Methyl Methoxy 1-Naphthylmethyl 511 511 1064 Methyl Methoxy Phenethyl 475 475 1065 Methyl Methoxy 3-methoxyphenyl 476 476 1066 Methyl Methoxy N-benzoylaminoethyl 504 504 1067 Methyl Methoxy Benzyl 460 460 1068 Methyl Methoxy 4-NO ₂ -benzyl 505 505 1069 Amino Methoxy 4-methoxybenzyl 492 492 1070 Amino Methoxy 3,4-Cl ₂ -benzyl 516 516 1071 Amino Methoxy 1-Naphthyl 498 498 1072 Amino Methoxy Piperonyl 491 491 1073 Amino Methoxy 2,4,5-trimethoxyphenyl 538 538 1074 Amino Methoxy 3-hydroxybenzyl 477 477 1075 Amino Methoxy 1-Naphthylmethyl 512 512 1076 Amino Methoxy Phenethyl 476 476 1077 Amino Methoxy 3-methoxyphenyl 477 477 1078 Amino Methoxy N-benzoylaminoethyl 505 505 1079 Amino Methoxy Benzyl 461 461 1080 Amino Methoxy 4-NO ₂ -benzyl 506 506 1081 3-propenyl Methoxy 4-methoxybenzyl 517 517 1082 3-propenyl Methoxy 3,4-Cl ₂ -benzyl 541 541 1083 3-propenyl Methoxy 1-Naphthyl 523 523 1084 3-propenyl Methoxy Piperonyl 517 517 1085 3-propenyl Methoxy 2,4,5-trimethoxyphenyl 563 563 1086 3-propenyl Methoxy 3-hydroxybenzyl 503 503 1087 3-propenyl Methoxy 1-Naphthylmethyl 537 537 1088 3-propenyl Methoxy Phenethyl 501 501 1089 3-propenyl Methoxy 3-methoxyphenyl 503 503 1090 3-propenyl Methoxy N-benzoylaminoethyl 530 530 1091 3-propenyl Methoxy Benzyl 487 487 1092 3-propenyl Methoxy 4-NO ₂ -benzyl 532 532 1093 Ethanoic acid Methoxy 4-methoxybenzyl 535 535 1094 Ethanoic acid Methoxy 3,4-Cl ₂ -benzyl 559 559 1095 Ethanoic acid Methoxy 1-Naphthyl 541 541 1096 Ethanoic acid Methoxy Piperonyl 534 534 1097 Ethanoic acid Methoxy 2,4,5-trimethoxyphenyl 581 581 1098 Ethanoic acid Methoxy 3-hydroxybenzyl 521 521 1099 Ethanoic acid Methoxy 1-Naphthylmethyl 555 555 1100 Ethanoic acid Methoxy Phenethyl 519 519 1101 Ethanoic acid Methoxy 3-methoxyphenyl 521 521 1102 Ethanoic acid Methoxy N-benzoylaminoethyl 548 548 1103 Ethanoic acid Methoxy Benzyl 505 505 1104 Ethanoic acid Methoxy 4-NO ₂ -benzyl 549 549 1105 Propionic acid Methoxy 4-methoxybenzyl 549 549 1106 Propionic acid Methoxy 3,4-Cl ₂ -benzyl 573 573 1107 Propionic acid Methoxy 1-Naphthyl 555 555 1108 Propionic acid Methoxy Piperonyl 549 549 1109 Propionic acid Methoxy 2,4,5-trimethoxyphenyl 595 595 1110 Propionic acid Methoxy 3-hydroxybenzyl 535 535 1111 Propionic acid Methoxy 1-Naphthylmethyl 569 569 1112 Propionic acid Methoxy Phenethyl 533 533 1113 Propionic acid Methoxy 3-methoxyphenyl 535 535 1114 Propionic acid Methoxy N-benzoylaminoethyl 562 562 1115 Propionic acid Methoxy Benzyl 519 519 1116 Propionic acid Methoxy 4-NO ₂ -benzyl 564 564 1117 4-vinylbenzyl Methoxy 4-methoxybenzyl 593 593 1118 4-vinylbenzyl Methoxy 3,4-Cl ₂ -benzyl 617 617 1119 4-vinylbenzyl Methoxy 1-Naphthyl 599 599 1120 4-vinylbenzyl Methoxy Piperonyl 593 593 1121 4-vinylbenzyl Methoxy 2,4,5-trimethoxyphenyl 639 639 1122 4-vinylbenzyl Methoxy 3-hydroxybenzyl 579 579 1123 4-vinylbenzyl Methoxy 1-Naphthylmethyl 613 613 1124 4-vinylbenzyl Methoxy Phenethyl 577 577 1125 4-vinylbenzyl Methoxy 3-methoxyphenyl 579 579 1126 4-vinylbenzyl Methoxy N-benzoylaminoethyl 606 606 1127 4-vinylbenzyl Methoxy Benzyl 563 563 1128 4-vinylbenzyl Methoxy 4-NO ₂ -benzyl 608 608 1129 Piperonylmethyl Methoxy 4-methoxybenzyl 611 611 1130 Piperonylmethyl Methoxy 3,4-Cl ₂ -benzyl 635 635 1131 Piperonylmethyl Methoxy 1-Naphthyl 617 617 1132 Piperonylmethyl Methoxy Piperonyl 611 611 1133 Piperonylmethyl Methoxy 2,4,5-trimethoxyphenyl 657 657 1134 Piperonylmethyl Methoxy 3-hydroxybenzyl 597 597 1135 Piperonylmethyl Methoxy 1-Naphthylmethyl 631 631 1136 Piperonylmethyl Methoxy Phenethyl 595 595 1137 Piperonylmethyl Methoxy 3-methoxyphenyl 597 597 1138 Piperonylmethyl Methoxy N-benzoylaminoethyl 624 624 1139 Piperonylmethyl Methoxy Benzyl 581 581 1140 Piperonylmethyl Methoxy 4-NO ₂ -benzyl 626 626 1141 4-F-benzyl Methoxy 4-methoxybenzyl 585 585 1142 4-F-benzyl Methoxy 3,4-Cl ₂ -benzyl 609 609 1143 4-F-benzyl Methoxy 1-Naphthyl 591 591 1144 4-F-benzyl Methoxy Piperonyl 585 585 1145 4-F-benzyl Methoxy 2,4,5-trimethoxyphenyl 631 631 1146 4-F-benzyl Methoxy 3-hydroxybenzyl 571 571 1147 4-F-benzyl Methoxy 1-Naphthylmethyl 605 605 1148 4-F-benzyl Methoxy Phenethyl 569 569 1149 4-F-benzyl Methoxy 3-methoxyphenyl 571 571 1150 4-F-benzyl Methoxy N-benzoylaminoethyl 598 598 1151 4-F-benzyl Methoxy Benzyl 555 555 1152 4-F-benzyl Methoxy 4-NO ₂ -benzyl 600 600 1153 Methyl Benzyloxy 4-methoxybenzyl 567 567 1154 Methyl Benzyloxy 3,4-Cl ₂ -benzyl 591 591 1155 Methyl Benzyloxy 1-Naphthyl 573 573 1156 Methyl Benzyloxy Piperonyl 567 567 1157 Methyl Benzyloxy 2,4,5-trimethoxyphenyl 613 613 1158 Methyl Benzyloxy 3-hydroxybenzyl 553 553 1159 Methyl Benzyloxy 1-Naphthylmethyl 587 587 1160 Methyl Benzyloxy Phenethyl 551 551 1161 Methyl Benzyloxy 3-methoxyphenyl 553 553 1162 Methyl Benzyloxy N-benzoylaminoethyl 580 580 1163 Methyl Benzyloxy Benzyl 537 537 1164 Methyl Benzyloxy 4-NO ₂ -benzyl 582 582 1165 Amino Benzyloxy 4-methoxybenzyl 568 568 1166 Amino Benzyloxy 3,4-Cl ₂ -benzyl 592 592 1167 Amino Benzyloxy 1-Naphthyl 574 574 1168 Amino Benzyloxy Piperonyl 568 568 1169 Amino Benzyloxy 2,4,5-trimethoxyphenyl 614 614 1170 Amino Benzyloxy 3-hydroxybenzyl 554 554 1171 Amino Benzyloxy 1-Naphthylmethyl 588 588 1172 Amino Benzyloxy Phenethyl 552 552 1173 Amino Benzyloxy 3-methoxyphenyl 554 554 1174 Amino Benzyloxy N-benzoylaminoethyl 581 581 1175 Amino Benzyloxy Benzyl 538 538 1176 Amino Benzyloxy 4-NO ₂ -benzyl 583 583 1177 3-propenyl Benzyloxy 4-methoxybenzyl 593 593 1178 3-propenyl Benzyloxy 3,4-Cl ₂ -benzyl 617 617 1179 3-propenyl Benzyloxy 1-Naphthyl 599 599 1180 3-propenyl Benzyloxy Piperonyl 593 593 1181 3-propenyl Benzyloxy 2,4,5-trimethoxyphenyl 639 639 1182 3-propenyl Benzyloxy 3-hydroxybenzyl 579 579 1183 3-propenyl Benzyloxy 1-Naphthylmethyl 613 613 1184 3-propenyl Benzyloxy Phenethyl 577 577 1185 3-propenyl Benzyloxy 3-methoxyphenyl 579 579 1186 3-propenyl Benzyloxy N-benzoylaminoethyl 606 606 1187 3-propenyl Benzyloxy Benzyl 563 563 1188 3-propenyl Benzyloxy 4-NO ₂ -benzyl 608 608 1189 Ethanoic acid Benzyloxy 4-methoxybenzyl 611 611 1190 Ethanoic acid Benzyloxy 3,4-Cl ₂ -benzyl 635 635 1191 Ethanoic acid Benzyloxy 1-Naphthyl 617 617 1192 Ethanoic acid Benzyloxy Piperonyl 611 611 1193 Ethanoic acid Benzyloxy 2,4,5-trimethoxyphenyl 657 657 1194 Ethanoic acid Benzyloxy 3-hydroxybenzyl 597 597 1195 Ethanoic acid Benzyloxy 1-Naphthylmethyl 631 631 1196 Ethanoic acid Benzyloxy Phenethyl 595 595 1197 Ethanoic acid Benzyloxy 3-methoxyphenyl 597 597 1198 Ethanoic acid Benzyloxy N-benzoylaminoethyl 624 624 1199 Ethanoic acid Benzyloxy Benzyl 581 581 1200 Ethanoic acid Benzyloxy 4-NO ₂ -benzyl 626 626 1201 Propionic acid Benzyloxy 4-methoxybenzyl 625 625 1202 Propionic acid Benzyloxy 3,4-Cl ₂ -benzyl 649 649 1203 Propionic acid Benzyloxy 1-Naphthyl 631 631 1204 Propionic acid Benzyloxy Piperonyl 625 625 1205 Propionic acid Benzyloxy 2,4,5-trimethoxyphenyl 671 671 1206 Propionic acid Benzyloxy 3-hydroxybenzyl 611 611 1207 Propionic acid Benzyloxy 1-Naphthylmethyl 645 645 1208 Propionic acid Benzyloxy Phenethyl 609 609 1209 Propionic acid Benzyloxy 3-methoxyphenyl 611 611 1210 Propionic acid Benzyloxy N-benzoylaminoethyl 638 638 1211 Propionic acid Benzyloxy Benzyl 595 595 1212 Propionic acid Benzyloxy 4-NO ₂ -benzyl 640 640 1213 4-vinylbenzyl Benzyloxy 4-methoxybenzyl 669 669 1214 4-vinylbenzyl Benzyloxy 3,4-Cl ₂ -benzyl 694 694 1215 4-vinylbenzyl Benzyloxy 1-Naphthyl 675 675 1216 4-vinylbenzyl Benzyloxy Piperonyl 669 669 1217 4-vinylbenzyl Benzyloxy 2,4,5-trimethoxyphenyl 715 715 1218 4-vinylbenzyl Benzyloxy 3-hydroxybenzyl 655 655 1219 4-vinylbenzyl Benzyloxy 1-Naphthylmethyl 689 689 1220 4-vinylbenzyl Benzyloxy Phenethyl 653 653 1221 4-vinylbenzyl Benzyloxy 3-methoxyphenyl 655 655 1222 4-vinylbenzyl Benzyloxy N-benzoylaminoethyl 682 682 1223 4-vinylbenzyl Benzyloxy Benzyl 639 639 1224 4-vinylbenzyl Benzyloxy 4-NO ₂ -benzyl 684 684 1225 Piperonylmethyl Benzyloxy 4-methoxybenzyl 687 687 1226 Piperonylmethyl Benzyloxy 3,4-Cl ₂ -benzyl 712 712 1227 Piperonylmethyl Benzyloxy 1-Naphthyl 693 693 1228 Piperonylmethyl Benzyloxy Piperonyl 687 687 1229 Piperonylmethyl Benzyloxy 2,4,5-trimethoxyphenyl 733 733 1230 Piperonylmethyl Benzyloxy 3-hydroxybenzyl 673 673 1231 Piperonylmethyl Benzyloxy 1-Naphthylmethyl 707 707 1232 Piperonylmethyl Benzyloxy Phenethyl 671 671 1233 Piperonylmethyl Benzyloxy 3-methoxyphenyl 673 673 1234 Piperonylmethyl Benzyloxy N-benzoylaminoethyl 700 700 1235 Piperonylmethyl Benzyloxy Benzyl 657 657 1236 Piperonylmethyl Benzyloxy 4-NO ₂ -benzyl 702 702 1237 4-F-benzyl Benzyloxy 4-methoxybenzyl 661 661 1238 4-F-benzyl Benzyloxy 3,4-Cl ₂ -benzyl 686 686 1239 4-F-benzyl Benzyloxy 1-Naphthyl 667 667 1240 4-F-benzyl Benzyloxy Piperonyl 661 661 1241 4-F-benzyl Benzyloxy 2,4,5-trimethoxyphenyl 707 707

The mimetics of β-chain structures of the present invention can be used as bioactive agents, such as diagnostic, prophylactic and therapeutic agents. The compounds are preferably formulated in a pharmaceutically acceptable form and then administered to a patient in need of treatment with the mimetics of the β-chain structures of the present invention.

Thus, the present invention provides a pharmaceutical composition comprising a compound with structures from (I ") to (I" '). To obtain a pharmaceutical composition containing the present compounds, a person skilled in the art can apply knowledge and methods known to the public that are known in the relevant field of technology. To obtain the composition of the present invention, commonly known varieties of carriers and other additives are used. The pharmaceutical compositions of this invention can be administered in a standard manner in a pathological condition that needs to be treated, for example, by oral, rectal or parenteral administration.

For these purposes, the compounds of the present invention can be prepared by methods known in the art, in the form of, for example, tablets, capsules, aqueous or oily solutions or suspensions, (lipid) emulsions, dispersible powders, suppositories, ointments, creams, drops and sterile injectable aqueous or oily solutions or suspensions.

A suitable pharmaceutical composition of the present invention is a composition suitable for oral administration in unit dosage form, such as, for example, a tablet or capsule, which contains from about 1 mg to about 1 g of the compound of this invention.

In another aspect, the pharmaceutical composition of the present invention is a composition suitable for intravenous, subcutaneous or intramuscular injection. The patient may receive, for example, an intravenous, subcutaneous or intramuscular dose of from about 1 μg / kg to about 1 g / kg of the compound of the present invention. An intravenous, subcutaneous and intramuscular dose may be administered by bolus injection. Alternatively, the intravenous dose may be administered by continuous infusion over a period of time.

Alternatively, the patient may receive a daily oral dose that is approximately equivalent to a daily parenteral dose, wherein the composition is administered from 1 to about 4 times per day.

The following table illustrates representative pharmaceutical dosage forms containing the compound or its pharmaceutically acceptable salt, and intended for therapeutic or prophylactic use in humans:

Tablet 1 mg / tablet Compound one hundred Lactose Ph. Eur 179 Croscarmellose sodium 12.0 Polyvinylpyrrolidone 6 Magnesium stearate 3.0

Tablet 2 mg / tablet Compound fifty Lactose Ph. Eur 229 Croscarmellose sodium 12.0 Polyvinylpyrrolidone 6 Magnesium stearate 3.0

Tablet 3 mg / tablet Compound 1,0 Lactose Ph. Eur 92 Croscarmellose sodium 4.0 Polyvinylpyrrolidone 2.0 Magnesium stearate 1,0

Capsule mg / capsule Compound 10 Lactose Ph. Eur 389 Croscarmellose sodium one hundred Magnesium stearate 1,0 Injection 1 50 (mg / ml) Compound 0.5% w / v Isotonic aqueous solution Up to 100%

A pharmaceutical composition containing a compound of general formula (I) can be used to induce a variety of biological actions, including protease inhibition in a warm-blooded animal, modulation of a cell signal transcription factor related peptide in a warm-blooded animal, and for kinase inhibition in a warm-blooded animal. These actions can be achieved by a method comprising administering to an animal in need thereof an effective amount of a compound of formula (I).

In addition, as described in detail below, mimetics of the β-chain structures of the present invention can also be effective in inhibiting MHC-I and / or MHC-II presentation of peptides to T-cell receptors in a warm-blooded animal; for inhibiting peptide binding to SH2 domains in a warm-blooded animal; to inhibit peptide binding to SH3 domains in a warm-blooded animal; for inhibiting peptide binding to PTB domains in a warm-blooded animal; for modulating the G-protein bound receptor (GPCR) and ion channel in a warm-blooded animal; and for modulating cytokines in a warm-blooded animal.

Inhibition of kinase (including inhibition of SH2 and SH3 domains)

In one aspect, the present invention provides a method for inhibiting kinase in a warm-blooded animal. The method comprises administering to an animal an amount of a compound of the present invention, wherein the amount is effective for inhibiting kinase. Kinases (also known as protein kinases) are a class of enzymes that catalyze the reaction by which a biomolecule (usually another enzyme) is phosphorylated. It is believed that up to 1000 kinases are encoded in the mammalian genome (Hunter, Cell 50: 823-829, 1987). A large number of kinases makes possible rapid amplification (amplification) of the signal and the creation of numerous regulation points.

Phosphorylation is a very common covalent modification found in signal transduction processes and causes a change in the activity of those proteins that become phosphorylated. Kinases are thus a critical component of signaling pathways. Kinases are usually organized into several modular functional regions or “domains” (Cohen, G.B., et al. Cell 80: 237-248, 1995). One domain, known as “SH3,” is a 55-70 amino acid region that binds to proline-enriched peptides, especially extended chain. Another domain known as "SH2" is a phosphotyrosine-binding region with a length of approximately 100 amino acids. It is believed that these two domains are involved in the recognition and binding of protein substrates. These, as well as other domains, including myristoylation and palmitoylation sites, are responsible for assembling polyprotein complexes that direct the catalytic domain to the correct targets (Mayer et al. Mol. Cell. Biol. 12: 609-618, 1992; and Mayer and Baltimore , Mol. Cell. Biol. 14: 2883-2894, 1994). It is known that although the SH2 and SH3 domains are present in some kinases, these domains are also present in other proteins. The compounds of the present invention can be used to inhibit SH2- or SH3-mediated binding in kinase or other proteins.

Kinases are used by the body in a very large number of different, but often interconnected, intracellular signal transduction mechanisms. For example, growth factors, transcription factors, hormones, cell cycle regulatory proteins, and many other classes of cell regulators use tyrosine kinases in their signaling pathways (see, for example, Bolen et al. FASEB J. 6: 3403-3409, 1992; and Ullrich and Schlessinger, Cell 61: 203-212, 1990). Serine / threonine kinases make up the majority of the rest of the kinase family.

One important approach for determining the role and understanding of enzyme function, both in vitro and in vivo, is the use of specific enzyme inhibitors. If one or more compounds that will inhibit the enzyme can be found, the inhibitor can be used to modulate the activity of the enzyme, and the effects of such a decrease can be observed. Such approaches serve as a tool in decoding many of the pathways of intermediate metabolism and are also important in studying the kinetics of enzymes and determining catalytic mechanisms. The present invention provides such compounds.

The regulation of many immune responses is mediated through receptors that transmit signals through tyrosine kinases containing the SH2 domain. T cell activation through an antigen-specific T cell receptor (TCR) initiates a signal transduction cascade leading to lymphokine secretion and cell proliferation. One of the earliest biochemical reactions after TCR activation is an increase in tyrosine kinase activity. In particular, activation and proliferation of T cells is regulated by T cell receptor mediated p56 ^lck and p59 ^fyn tyrosine kinase activation, as well as ZAP-70 and Syk (Weiss and Litman, Cell 76: 263-274, 1994), which contain SH2 domains . Additional evidence indicates that several kinases of the src family (lck, blk, fyn) are involved in signal transduction pathways from B-cell antigen receptors and therefore can serve to integrate stimuli derived from several independent receptor structures. Thus, inhibitors that block the interaction of these kinases containing the SH2 domain with their related receptors can serve as immunosuppressive agents used in autoimmune diseases, transplant rejections or as anti-inflammatory agents, as well as anti-cancer drugs in cases of lymphocytic leukemia .

In addition, non-transmembrane PTPases containing SH2 domains are known, the nomenclature being SH-PTP1 and SH-PTP2 (Neel, Cell Biology 4: 419-432, 1993), SH-PTP1 is identical to PTP1C, HCP or SHP and SH-PTP2 is also known as PTP1D or PTP2C. SH-PTP1 is expressed at high levels in hematopoietic cells of all lines and all stages of differentiation. Since the SH-PTP1 gene has been identified as responsible for the maternal (me) phenotype of the mouse, this provides a basis for predicting the actions of inhibitors that can block its interaction with its cellular substrates. Thus, it can be expected that inhibition of SH-PTP1 function will lead to weakened T-cell responses to mitogenic stimulation, decreased NK cell function, and depletion of B-cell precursors with potential therapeutic applications, as described above.

The ability of a compound of the present invention to bind to the SH2 domain of STAT6 or to bind to the SH2 domain of protein tyrosine phosphatase SH-PTP1 can be demonstrated by the procedures described by Payne et al., P.N.A.S. USA 90: 4902-4906, 1993). A library of SH2 binding mimetics can be screened according to the procedure of Songyang et al., Cell 72: 767-778, 1993. See also the procedure of Songyang et al., Current Biology 4: 973-982, 1994) for testing the ability of the compound to act as substrate or protein kinase inhibitor.

Accordingly, in one aspect, the present invention provides a method for inhibiting phosphatase in a warm-blooded animal, the method comprising administering to the animal an amount of a compound of the present invention that is effective for inhibiting phosphatase.

In type 2 diabetes (non-insulin-dependent diabetes), tyrosine phosphatases (PTP-1b) balance the action of the kinases activated by the insulin receptor and can represent important targets for drugs. In vitro experiments show that injection of PTPase blocks insulin-stimulated phosphorylation of tyrosyl residues on endogenous proteins. Thus, the compounds of the invention can be used to modulate the effects of insulin in diabetes.

In another aspect, the present invention provides a method for inhibiting the binding of a phosphothyrosine residue in a first protein to the SH2 domain of a second protein. The method comprises contacting an amount of a compound of the present invention with a composition comprising a first and second protein. The amount is effective in loosening the binding between the first and second protein, which occurs through the SH2 domain of the second protein and the phosphotyrosine residues of the first protein.

Protease inhibition

In another aspect, the present invention provides a method for inhibiting proteases in a warm-blooded animal. The method comprises administering to the animal an amount of a compound of the present invention as described herein. The amount is effective for inhibiting protease in an animal. In various embodiments, the protease is a serine protease; the protease is a serine protease selected from thrombin, factor X, factor IX, factor VII, factor XI, urokinase, HCV protease, chymase, tryptase and kallikrein; the protease is thrombin; the protease is factor VII, and the protease is selected from aspartic acid protease, cysteine protease and metalloprotease.

Regarding protease inhibition, cathepsin B is a lysosomal cysteine protease, usually involved in enzyme processing and protein renewal. Elevated levels of activity occur in tumor metastases (Stoane, BF et al., Cancer Metastasis Rev. 9: 333-352, 1990), rheumatoid arthritis (Werb, Z. Textbook of Rheumatology, Keller, WN; Harris WD; Ruddy, S .; Siedge, CS, Eds., 1989, WB Saunder Co., Philadelphia, Pa., Pp. 300-321) and muscular dystrophy (Katunuma and Kominami, Rev. Physiol. Biochem. Pharmacol. 108: 1-20, 1987 )

Calpains are cytosolic or membrane-bound, Ca ⁺⁺ -activated proteases that are responsible for the degradation of cytoskeletal proteins in response to changes in calcium levels in the cell. They promote tissue degradation in arthritis and muscular dystrophy (see Wang and Yuen Trends Pharmacol. Sci. 15: 412-419, 1994).

An interleukin converting enzyme (ICE) breaks down pro-IL-1 beta to form IL-1 beta, a key inflammatory mediator, and therefore it can be shown that ICE inhibitors are useful in the treatment of arthritis (see, for example, Miller BE et al., J. Immunol. 154: 1331-1338, 1995). ICE or ICE-like proteases can also take part in apoptosis (programmed cell death) and, therefore, play a role in cancer, AIDS, Alzheimer's disease and other diseases in which deregulated apoptosis occurs (see Barr and Tomei, Biotechnol. 12: 487-493, 1994).

HIV protease plays a key role in the life cycle of HIV, the AIDS virus. In the final stages of viral maturation, it breaks down polyprotein precursors to functional enzymes and structural proteins of the viron nucleus. HIV protease inhibitors have quickly been identified as an excellent therapeutic tool (s) for AIDS (see Huff, JR, J. Med. Chem. 34: 2305-2314), and it has already been proven that they are useful in its treatment, which is proven recent FDA approval of ritonavir, crixivan, and sakuinavir.

Hepatitis C virus (HCV) is the leading cause of non-A and non-B hepatitis worldwide today. It is estimated that it infects up to 50 million people. Currently, there is no satisfactory treatment available to stop the development of this debilitating disease. During the life cycle of this virus, a polyprotein of approximately 3,000 amino acids is produced and it is proteolytically cleaved by the host and viral proteases to produce mature viral gene products. Serine proteinase, located in the HCV NS3 protein, cleaves at four specific sites to form non-structural proteins, which are considered essential for viral replication. Therefore, HCV protease inhibitors are attractive drug development tools and can have tremendous therapeutic benefits. (Neddermann et al., Biol. Chem. 378: 469-476, 1997).

The angiotensin converting enzyme (ACE) is part of the renin-angiotensin system, which plays a central role in the regulation of blood pressure. ACE breaks down angiotensin I into octapeptide angiltensin II, a potent, blood pressure increasing agent due to its vasoconstrictor activity. ACE inhibition has been proven to be therapeutically useful in the treatment of hypertension (Williams, G.H., N. Engl. J. Med. 319: 1517-1525, 1989).

Collagenases break down collagen, the main component of the extracellular matrix (e.g., connective tissue, skin, blood vessels). Increased collagenase activity contributes to arthritis (Krane et al., Ann. NY Acad. Sci. 580: 340-354, 1990), tumor metastasis (Flug and Korf-Maier, Acta Anat. Basel 152: 69-84, 1995) and other diseases consisting in the destruction of connective tissue.

Trypsin-like serine proteases form a large and highly selective family of enzymes involved in hemostasis / coagulation (Davie and Fujikawa, Ann. Rev. 799-829, 1975) and complement activation (Muller-Eberhard, Ann. Rev. Biochem. 44: 697- 724, 1975). Sequencing of these proteases showed the presence of a homologous trypsin-like nucleus with amino acid inserts that modify specificity and which are usually responsible for interacting with other macromolecular components (Magnusson et al., Miami Winter Symposia 11: 203-239, 1976).

Thrombin, a trypsin-like serine protease, acts to provide limited proteolysis, both in the generation of fibrin from fibrinogen and in the activation of the platelet receptor, and thus plays a critical role in thrombosis and hemostasis (Mann, KG Trends Biochem. Sci. 12: 229- 233, 1987). Thrombin exhibits marked specificity in the removal of fibrinogen peptides A and B of fibrinogen through the selective cleavage of only two Arg-Gly bonds from one hundred and eighty-one Arg- or Lys-Xaa sequences in fibrinogen (Biomback, Blood Clotting Enzymology, Seeger, WH (ed.), Academic Press New York, 1967, pp. 143-215).

Many significant pathological conditions relate to abnormal hemostasis, including acute coronary syndromes. Aspirin and heparin are widely used in the treatment of patients with acute coronary syndromes. However, these agents have several significant limitations. For example, thrombosis complicating rupture of an atherosclerotic plaque tends to be thrombin-mediated, a platelet-dependent process that is relatively resistant to inhibition by aspirin and heparin (Fuster et al., N. Engl. J. Med. 326: 242-50, 1992 )

Thrombin inhibitors prevent the formation of a thrombus at sites of vascular damage in vivo. In addition, since thrombin is also a potent growth factor that initiates proliferation of smooth muscle cells in places of mechanical damage in the coronary artery, inhibitors block this proliferative response of smooth muscle cells and reduce restenosis. Thrombin inhibitors can also reduce the inflammatory response in the vascular cell membranes (Harker et al., Am. J. Cardiol. 75: 122-16B, 1995).

In addition, at least two well-defined transcription factors, nuclear factor (NF) kB and activator protein (AP) -1, are regulated by the intracellular state (system) reduction-oxidation (redox). Regulation of gene expression by the redox system has promising therapeutic consequences. For example, the binding sites of redox-regulated transcription factors NF-kB and AP-1 are located in the promoter region of a large number of genes that are directly involved in the pathogenesis of diseases such as AIDS, cancer, atherosclerosis, and diabetic complications (Sen and Packer, FASEB Journal 10: 709-720, 1996). More specifically, the binding of transcription factors, such as NF-kB and AP-1, to consensus factors on DNA is driven by oxidative-antioxidant hemostasis, especially the thiol disulfide equilibrium.

In the case of NF-kB, a physiologically suitable thiol, which plays a critical role in regulating the function of NF-kB, restores a thioredoxin or restores a thioredoxin-like protein. Thioredoxin is an important oxidoreductase protein with antioxidant functions. Thioredoxin has been found to positively regulate the binding of DNA to activated NF-kB and thus increase gene expression (Schenk et al., Proc. Natl. Acad. Sci. USA 91: 1672-1676, 1994). Thioredoxin was involved in the restoration of activated cytosolic NF-kB (especially cys-62), which may thus contribute to its nuclear translocation and DNA binding (Hayashi et al., J. Biol. Chem. 268: 11380-11388, 1993 )

It was also found that the activity of Fos and Jun in the binding of DNA to the AP-1 complex is regulated by the redox system (Abate et al., Science 249: 1157-1162, 1990). Each protein contains one conserved cysteine (flanked by lysine and arginine) in its DNA-binding domain. It turns out that this thiol is not part of the disulfide bond and cannot exist in the form of sulfenic or sulfinic acid in oxidized form. Ref-1, a bifunctional nuclear protein that also has endonuclease activity in DNA repair, stimulates the binding of DNA to the AP-1 protein by restoring this regulatory cysteine. A Fos mutant in which critical cysteine was replaced by serine caused a three-fold increase in the activity of DNA binding to AP-1 and was no longer subject to redox control (Okuno et al., Oncogene 8: 695-701, 1993). Therefore, since all of at least four members of the fos family, 3 members of the Jun family, and at least 4 members of the ATF / CREB family of transcription factors contain this conserved cysteine, a redox regulator of transcription factors appears to be widespread .

As indicated above, the regulation of transcription factors, such as NF-kB and AP-1, has important therapeutic value. For example, AP-1 is an important mediator of tumor formation (Yoshioka et al., Proc. Natl. Acad. Sci. USA 92: 4972-4976, 1995). Thus, compounds that inhibit the transcriptional activity of AP-1 are useful in the treatment of cancer. In addition, due to its direct role in the regulation of responses to inflammatory cytokines and endotoxins, the activation of NF-kB plays an important role in the development of chronic diseases such as rheumatoid arthritis and acute pathological conditions such as septic shock. Autoimmune diseases, such as systemic lupus erythematosus (SLE) and Alzheimer's disease, are also believed to be involved in the activation of NF-kB. Similarly, NK-kB plays an important role in the activation of HIV gene expression. Other conditions that are associated with NK-kB are believed to include influenza, atherosclerosis, oncogenesis, and ataxia-telangiectasia (AT).

Oxidoreductase Inhibition

Regarding the regulation of transcription factors, the compounds of this invention regulate transcription factors whose ability to bind to DNA is regulated by the reduction of the cysteine residue by cell oxidoreductase. In one embodiment, the transcription factor is NF-kB. In this embodiment, the compounds of the present invention have activity as mediators of immune and / or inflammatory responses or serve to regulate cell growth. In another embodiment, the transcription factor is AP-1 and the cell oxidoreductase is Ref-1. In this embodiment, the compounds of the present invention have activity as anti-inflammatory and / or anti-cancer agents. In the following embodiments, the transcription factor is selected from Myb and a glucocorticoid receptor. Other transcription factors that can be regulated in the context of this invention also include: transcription factors of the NK-kB family, such as Rel-A, c-Rel, Rel-B, p50 and p52; AP-1 family transcription factors such as Fos, FosB, Fra-1, Fra-2, Jun, JunB and JunD; ATF; CREB STAT-1, -2, -3, -4, -5 and -6; NFAT-1, -2 and -4; MAF; thyroid factor; IRF Oct-1 and -2; NF-Y; Erg-1 and USF-43.

Accordingly, in one aspect, the present invention provides a method for inhibiting oxidoreductase in a warm-blooded animal, comprising administering to the animal an amount of a compound of the present invention that is effective for inhibiting oxidoreductase. Inhibition of oxidoreductase activity can be used as a means for regulating transcription.

Inhibition of CAAX

In another aspect, the present invention provides a method for inhibiting CAAX in a warm-blooded animal. The method comprises administering to the animal an amount of a compound of the present invention as described herein. The amount is effective to provide inhibition of CAAX in the animal.

Ras, the ras oncogen protein product, is a membrane-bound protein involved in signal transduction that regulates cell division and growth. Mutations in the ras gene exist among the most common genetic disorders associated with human cancers (Barbacid, M. Annu Rev Biochem 56: 779-827, 1987). These mutations lead to a growth signal that is always “on”, which leads to the formation of a cancer cell. For localization in the Ras cell membrane, prenylation of cysteine in its C-terminal CAAX sequence by farnesyl transferase (FTase) is required, where in the CAAX sequence “A” is indicated as an amino acid with a hydrophobic side chain and “X” is another amino acid. This post-translational modification is critical to its activity. Peptidyl FTase inhibitors with a CAAX sequence have been found to block or slow down the growth of tumors in cell culture and throughout the animal body (Kohl et al., Science 260: 1934-1937, 1993; Buss and Marsters, Chemistry and Biology 2: 787-791, 1995).

Methods for screening a compound for its activity of inhibiting CAAX activity are known in the art. See, for example, US Patent No. 6391574, which describes a method for identifying a compound that inhibits the proteolytic removal of the AAX tripeptide from the CAAX protein in the cell. See also US patent No. 5990277, which describes several suitable assays, and references Gibbs et al., Cell 77: 175, 1994; Gibbs, Cell 65: 1, 1991; Maltese, FASEB J. 4: 3319, 1990; Moores et al., J. Biol. Chem. 266: 14603, 1991; Goldstein et al., J. Biol. Chem . 266: 15575, 1991; European Patent 0461869 A2; Casey, J. Lipid Res. 33: 1731-1740, 1992; Cox et al., Curr. Opin . Cell Biol. 4: 1008-1016. 1992; Garcia et al., J. Biol. Chem. 268: 18415-18418, 1993; Vogt et al., J. Biol. Chem . 270: 660-664, 1995; Kohl et al., Science , 260: 1934-1937, 1993; Garcia et al., J. Biol. Chem ., 268: 18415-18418, 1993; and Vogt et al., J. Biol . Chem., 270: 660-664, 1995).

MHC molecules

In another aspect, the present invention provides a method of inhibiting the binding of an antigenic peptide to an MHC molecule of either class one or class two. The method comprises contacting a compound of the present invention with a composition comprising an antigenic peptide and an MHC molecule of either class one or class two. The compound is contacted with the antigen / molecule in an amount effective to reduce the binding affinity between the two species.

An important aspect of the immune system is the response of T cells. This reaction requires that T cells recognize and interact with complexes of cell surface molecules called human leukocyte antigens ("HLA") or major human histocompatibility complexes ("MHC"), and peptides (see, for example, Male et al., Advanced Immunology (JP Lipincott Company, 1987): Antigens mobilize the immune response, at least in part, by “capturing” an antigen-presenting cell (APC) that contains on its surface a class II glycoprotein encoded by a gene in the main histocompatibility complex (MHC) ). BUT the tiger is then presented to a specific helper T cell in the context of a surface-linked MHC glycoprotein and the interaction of an antigen-specific receptor for T cells with an antigen-MHC complex, the helper T cell is stimulated to mediate an antigen-specific immune response, including induction functions of cytotoxic T-cells, induction of B-cell function and secretion of a number of factors that help this reaction and support this reaction. In one aspect of the invention, the MHC molecule is HLA-A2.1, HLA-A1 or HLA-A3.1, or any other HLA allele that is present in patient melanoma.

The ability of a compound of the present invention to bind to MHC I molecules can be demonstrated essentially as described by Elliot et al., Nature 351: 402-406, 1991. Similarly, the ability of a compound of the present invention to bind to MHC II molecules can be demonstrated by the procedure Kwok et al., J. Immunol. 155: 2468-2476, 1995.

Protein with the domain 14-3-3

In another aspect, the present invention provides a method for inhibiting the binding of a first peptide to a second peptide that contains a 14-3-3 domain, where the first peptide has a binding affinity for the 14-3-3 domain of the second domain. The method comprises contacting a compound of the present invention with a composition comprising a (first) peptide that has a binding affinity for the 14-3-3 domain of the second protein.

Proteins having a 14-3-3 domain and its binding partners are described in the literature. These peptides can be used in the method of the present invention. See, for example, Dai and Murakami, J. Neurochem 2003 Jan. 84 (1): 23-34; Lim et al., J. Biol. Chem. 2002, Oct. 25, 277 (43): 40997-1008; Parvaresch et al., FEBS Let t 2002 Dec. 18, 532 (3): 357-62; Eilers et al., Mol Cell Biol 2002 Dec .; 22 (24): 8514-26; Liu et al., Cancer Res 2002 Nov. 15, 62 (22): 6475-80; Truong et al., Proteins 2002 Nov. 15, 49 (3): 321-5; Birkenfeld et al., Biochem J 2003 Jan. 1, 369 (Pt 1): 45-54; Espejo et al., Biochem J 2002 Nov. 1, 367 (Pt 3): 697-702; and Benzing et al., J Biol Chem 2002 Sep 6, 277 (36): 32954-62.

In practice, the implementation of the methods of this invention, a therapeutically effective amount of a compound of this invention is administered to a warm-blooded animal in need. For example, the compounds of this invention can be administered to a warm-blooded animal that is diagnosed or at risk of developing a condition selected from any one or more of the following diseases: Crohn’s disease, asthma, rheumatoid arthritis, ischemia, reperfusion injury, homologous disease (GVHD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, transplant rejection and T-cell leukemia in adult patients.

Tuberous Sclerosis Complex

Patients with tuberous sclerosis complex (TSC) usually develop multiple focal lesions in the brain, heart, kidneys, and other tissues (see, for example, Gomez, MR Brain Dev. 17 (suppl): 55-57, 1995). Studies on mammalian cells have found that overexpression of TSC1 (which expresses hamartin) and TSC2 (which expresses tuberin) negatively regulates cell proliferation and induces G ₁ / S delay (see, for example, Miloloza, A. et al, Hum. Mol. Genet. 9: 1721-1727, 2000). Other studies have found that hamartin and tuberin function at the level of β-catenin degradation complex and, more specifically, that these proteins negatively regulate the stability and activity of beta-catenin by participation in beta-catenin degradation complex (see, for example, Mak, B.C. ., et al. J. Biol. Chem. 278 (8): 5947-5951, 2003). Beta-catenin is a 95 kDa protein that is involved in cell adhesion by associating it with members of the cadherin family associated with membranes and in cell proliferation and differentiation as a key component of the Wnt / Wingless pathway (see, for example, Daniels, DL, et al ., Trends Biochem. Sci. 26: 672-678, 2001). It was found that rupture of this pathway is oncogenic for humans and rodents. The present invention provides compounds that modulate β-catenin activity, and especially its interaction with other proteins, and accordingly, they can be used in the treatment of TSC.

For purposes of illustration, but without limitation according to this invention, the following examples are proposed.

EXAMPLES

In preparative examples and examples, the following abbreviations are used:

BMS: Bordimethyl Sulfide

CbzOSu: Benzyloxycarbonyl-N-hydroxysuccinimide

DIC: 1,3-Diisopropylcarbodiimide

DIEA: N, N-Diisopropylethylamine

DIPEA: N, N-Diisopropylethylamine

DMAP: N, N-Dimethylaminopyridine

DMF: Dimethylformamide

DMSO: Dimethyl Sulfoxide

EA: Ethyl Acetate

EDC: 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

EDCI: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

FmocOsu: 9-Fluorenyloxycarbonyl-N-hydroxysuccinimide

HATU: [2- (1H-9-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate]

Hex .: Hexane

NOVT: N-Hydroxybenzotriazole

MS: Methylene Chloride

MeOH: Methanol

-Obn: -O-benzyl

PPTS: pyridinium p-toluenesulfonate

PyBOP: Benzotriazol-1-yloxytrispyrrolidinophosphonium hexafluorophosphate

p-TsOH: p-Toluenesulfonic acid

THF: Tetrahydrofuran

TLC: Thin layer chromatography

Preparative example 1

(1) Preparation of Naphthalene-2-Carboxylic Acid Amide

To a solution of 2-naphthoic acid (25 g, 0.145 mol) in MS (200 ml) was added oxalyl chloride (38 ml, 0.4356 mol) and a catalytic amount of DMF, and the mixture was stirred at room temperature for 2 hours. After evaporation of the solvent, the crude acyl chloride was diluted with MS (200 ml), to which ammonium hydroxide in water (160 ml) was added dropwise at the temperature of the ice bath. After stirring for 1 hour, the precipitated product was collected by suction filtration, triturated with hexane and dried, to thereby give the title compound, which was used in the next step without further purification.

(2) Obtaining naphthalene-2-ylmethylamine

To a solution of the crude amide obtained in the above step (1) in THF (100 ml), BMS (27.5 ml, 0.2904 mol) was slowly added at 0 ° C. The resulting reaction mixture was heated to 60 ° C for 3 hours, quenched with 5% HCl at 0 ° C, extracted with EA and washed with 5% HCl. The aqueous layers are combined and alkalinized 1 N. NaOH and again extracted with EA. The organic layers were combined and concentrated, to thereby give the title compound (13 g) as a white solid.

System 1 for TLC: MS / MeOH = 90:10 v / v, R _f = 0.23.

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 4.07 (s, 2H), 7.48 (m, 3H), 7.79 (m, 4H).

Preparative example 2

(1) Preparation of 1H-indole-2-carboxylic acid amide

To a solution of indole-2-carboxylic acid (1 g, 6.21 mmol) in MS (30 ml) was added oxalyl chloride (1.64 ml, 0.1862 mmol) and a catalytic amount of DMF and the mixture was stirred at room temperature for 2 hours . After evaporation of the solvent, the crude acyl chloride was diluted with MS (20 ml), to which ammonium hydroxide in water (7 ml) was added dropwise with cooling in an ice bath. After stirring for 1 hour, the precipitated product was collected by suction filtration, triturated with hexane and dried, to thereby give the title compound, which was used in the next step without further purification.

(2) Preparation of (1H-indol-2-yl) methylamine

To a solution of the crude amide obtained in the above step (1) in THF (30 ml), BMS (1.18 ml, 12.42 mol) was slowly added at 0 ° C. The resulting reaction mixture was heated to 60 ° C for 3 hours, quenched with 5% HCl at 0 ° C, extracted with EA and washed with 5% HCl. The aqueous layers are combined and alkalinized 1 N. NaOH and again extracted with EA. The organic layers were combined and concentrated, to thereby give the title compound (0.28 g) as a yellow oil.

System 1 for TLC: MS / MeOH = 90: 10 v / v, R _f = 0.15.

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 3.98 (s, 2H), 7.08 (m, 3H), 7.26 (m, 1H), 7.58 (d, 1H), 9.10 (br s, 1H).

Preparative example 3

(1) Preparation of 2-benzyloxycarbonylamino-4-oxo-butyric acid benzyl ester

To a solution of Z-Asp-OBn (10 g, 0.028 mol) in MS (200 ml) at 0 ° C. was added oxalyl chloride (2.93 ml, 0.336 mol) and a catalytic amount of DMF, and the mixture was stirred at room temperature for 2 hours. After evaporation of the solvent, the crude acyl chloride was dissolved in benzene (400 ml) and tributyltin hydride (15.1 ml, 0.056 mol) and a catalytic amount of Pd (0) were slowly added at 0 ° C and the mixture was stirred at room temperature overnight. After evaporation of the solvent, ether (100 ml) / 10% KF in water (100 ml) was added and the mixture was stirred at room temperature for 2 hours, followed by filtration, to thereby obtain a two-phase solution. The organic layer was separated and concentrated to give a crude product, which was purified by column chromatography to give the title compound, Z-Asp-OBn-aldehyde (6 g) as a pale yellow oil.

R _f : 0.29 in hexane / EA (2/1).

(2) Preparation of 2-benzyloxycarbonylamino-4,4-dimethoxybutyric acid benzyl ester

To a solution of Z-Asp-OBn-aldehyde (6 g, 17.58 mmol) obtained in the above step (1) in MeOH (100 ml), a catalytic amount of p-TsOH was added and the mixture was stirred at room temperature for 5 hours . After completion of the reaction, the solvent was evaporated to give a crude product, which was purified by column chromatography to obtain the title compound, Z-Asp-OBn-acetal (5 g) as a pale yellow oil.

R _f : 0.32 in a mixture of Hex./EA (2/1).

(3) Preparation of 2-benzyloxycarbonylamino-4,4-dimethoxybutyric acid

Z-Asp-OBn-acetal (0.5 g, 1.29 mmol) obtained in the above step (2) is dissolved in a mixture of THF (20 ml / NaOH (0.11 g, 2.1 mmol) in water (20 ml) and stirred at room temperature for 30 minutes After the complete disappearance of the starting material, the reaction mixture was concentrated by evaporation and then diluted with water / EA.The aqueous layer was separated, acidified very carefully to pH 4-5 1N HCl at 0 ° C and extracted again with EA.The organic layers are combined and concentrated to give the title compound, Z-Asp-OBn-acetal (0.27 g) as a light yellow m asla.

System 1 for TLC: hexane / EA = 20:10 v / v, R _f = 0.10.

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 2.20 (s, 2H), 3.35 (d, 6H), 4.52 (m, 2H), 5.19 (t, 2H), 5.80 (d, 1H), 7.37 (br s, 5H).

(4) Preparation of 2-amino-4,4-dimethoxybutyric acid

In a reaction vessel equipped with a hydrogen gas cylinder, add a solution of Z-Asp-OBn-acetal (2.22 g, 5.73 mmol) obtained in the above step (3) in acetic acid (20 ml) and a Pearlman catalyst and the mixture was stirred at room temperature overnight. The resulting reaction mixture was filtered, concentrated and lyophilized to give a crude product, which was used in the next step without further purification.

(5) Preparation of 2- (9H-fluoren-9-ylmethoxycarbonylamino) -4,4-dimethoxybutyric acid

To a solution of the crude Asp-OH-acetal obtained in the above step (4) in a mixture of THF (100 ml) / water (100 ml), FmocOsu (2.13 g, 6.3 mmol) / sodium bicarbonate was added (1, 93 g, 22.92 mmol) and the mixture was stirred at room temperature overnight. The resulting reaction mixture was concentrated and diluted with water / EA. The aqueous layer is separated, acidified very carefully to a pH of 4-5 1 N. HCl at 0 ° C and again extracted with EA. The organic layers were combined and concentrated to give a crude product, which was purified by column chromatography to give the title compound (1.5 g) as a foamy solid.

R _f : 0.15 in Hex./EA (2/1).

Preparative example 4

(1) Preparation of 2-benzyloxycarbonylaminopentadioic acid

To a solution of L-glutamic acid (20 g, 136 mmol) in water / THF (1/1, 400 ml) was added sodium bicarbonate (45.7 g, 544 mmol) and the mixture was cooled to 0 ° C in an ice bath. CbzOSu (37.3 g, 150 mmol) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. After completion of the reaction, the resulting reaction mixture was extracted with EA. The aqueous layer was separated, acidified to pH 2 with concentrated HCl at 0 ° C and again extracted with EA (4 times). The organic layers were concentrated to give a crude product, which was purified by column chromatography to obtain the title compound (16 g) as a colorless oil.

R _f : 0.2 in MS / MeOH (9/1).

(2) Preparation of 4- (2-carboxyethyl) -5-oxooxazolidine-3-carboxylic acid benzyl ester

The Dean-Stark apparatus was charged with N-Cbz-L-glutamic acid (4 g, 14.22 mmol) obtained in the above step (1), paraformaldehyde (5 g), a catalytic amount of pTsOH, molecular sieves (5 g) and toluene (100 ml) and the mixture was refluxed until the starting material disappeared. The resulting reaction mixture was cooled to room temperature, filtered and concentrated to give a crude product, which was purified by column chromatography, to thereby give the title compound (2 g) as a colorless oil.

R _f : 0.45 only in EA.

(3) Preparation of 5-oxo-5- (3-oxopropyl) oxazolidine-3-carboxylic acid benzyl ester

Oxalyl chloride (0.7 ml, 7.5 mmol) and a catalytic amount are added to a solution of unprotected glutamic acid (2 g, 6.82 mmol) obtained in the above step (2) in MS (200 ml) at 0 ° C. DMF and the mixture was stirred at room temperature for 2 hours. After evaporation of the solvent, the resulting crude acyl chloride was dissolved in THF (400 ml), to which tributyltin hydride (3.86 ml, 14.34 mmol) and a catalytic amount of Pd (0) were slowly added at 0 ° C, and the mixture was stirred at room temperature overnight . After evaporating the solvent, ether (100 ml) / 10% KF in water (100 ml) was added and the mixture was stirred at room temperature for 2 hours, followed by filtration to obtain a two-phase solution. The organic layer was separated and concentrated to give a crude product, which was purified by column chromatography, to thereby give the title compound (0.7 g) as a colorless oil.

R _f : 0.23 in hexane / EA (4/1)

(4) Preparation of 4- (3,3-dimethoxypropyl) -5-oxooxazolidine-3-carboxylic acid benzyl ester

To a solution of the unprotected aldehyde (0.7 g, 2.53 mmol) obtained in the above step (3) in MeOH (30 ml), a catalytic amount of pTsOH was added and the mixture was stirred at room temperature for 7 hours. After completion of the reaction, the reaction mixture was concentrated by evaporation of the solvent to obtain a crude product, which was purified by column chromatography, to thereby give the title compound (0.5 g) as a colorless oil.

R _f : 0.33 in hexane / EA (4/1).

(5) Preparation of 2-benzyloxycarbonylamino-5,5-dimethoxypentanoic acid

The deformed acetal (0.456 g, 1.411 mmol) obtained in the above step (4) is dissolved in a mixture of MeOH (20 ml) / 1 N. NaOH (10 ml) and the mixture was stirred at room temperature overnight. After complete disappearance of the starting material, the reaction mixture was concentrated by evaporation of the solvent and diluted with water / EA. The aqueous layer is separated, acidified very carefully to a pH of 4-5 1 N. HCl at 0 ° C and again extracted with EA. The organic layers were combined and concentrated, to thereby give the title compound (0.35 g) as a colorless oil.

R _f : 0.1 in a mixture of Hex./EA (1/1).

(6) Preparation of 2-amino-5,5-dimethoxypentanoic acid

A solution of Cbz-acetal (0.35 g, 1.13 mmol) obtained in the above step (5) in MeOH (10 ml) and a catalytic amount of 10% Pd / C are added to a reaction vessel equipped with a cylinder of hydrogen gas the mixture was stirred at room temperature overnight. The resulting reaction mixture was filtered and concentrated to give a crude product (0.2 g) as a colorless oil, which was used in the next step without further purification.

R _f : 0.01 in a mixture of Hex./EA (1/1).

(7) Preparation of 2- (9H-fluoren-9-ylmethoxycarbonylamino) -5,5-dimethoxypentanoic acid

To a solution of the crude Glu-OH acetal obtained in the above step (6) in a mixture of THF (10 ml) / water (10 ml), FmocOsu (0.42 g, 1.24 mmol) / sodium bicarbonate (0, 5 g, 5.9 mmol) and the mixture was stirred at room temperature overnight. After evaporation of the solvent, the resulting reaction mixture was diluted with water / EA. The aqueous layer is separated and acidified very carefully to a pH of 4-5 1 N. HCl at 0 ° C and again extracted with EA. The organic layers were combined and concentrated, to thereby give the title compound (0.19 g) as a colorless oil.

System 1 for TLC: EA only, R _f = 0.25.

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 1.75 (br.m, 4H), 3.28 (d, 6H), 3.43 (q, 1H), 4.20 ( t, 1H), 4.38 (m, 3H), 5.62 (d, 1H), 7.31 (m, 4H), 7.65 (d, 2H), 7.75 (d, 2H).

Preparative example 5

( 1) Preparation of 2-tert-butoxycarbonylamino-4-methoxycarbonylaminobutyric acid

To a solution of Boc-Dab-OH (3 g, 13.75 mmol) in H ₂ O (50 ml) NaOH (2.75 g, 68.75 mmol, 5 equiv.) Was slowly added to pH> 11, then methyl chloroformate was added. (2.6 g, 27.5 mmol, 2 equiv.) In toluene (50 ml). The resulting reaction mixture was stirred for 2 hours. To conduct TLC analysis, a small amount of the aqueous phase is taken and acidified with 1 N. HCl. After confirmation of the completion of the reaction by TLC, the organic phase was separated and the aqueous phase was acidified with 10% HCl and extracted with EA (5 ml × 2). The organic phases are combined, dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to give a crude product (3.277 g, 11.86 mmol, 86%) as a colorless oil.

System for TLC: EA, R _f = 0.2.

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 1.30 ~ 1.50 (br s, 9H), 2.00 ~ 2.30 (m, 2H), 3.10 ~ 3 30 (m, 2H), 3.70 (br s, 3H), 4.35 (m, 1H), 5.40 (m, 1H), 5.65 (br s, 1H).

(2) Preparation of (1-benzylcarbamoyl-3-methoxycarbonylaminopropyl) carbamic acid tert-butyl ester

To a solution of 2-tert-butoxycarbonylamino-4-methoxycarbonylaminobutyric acid (1.1 g, 3.98 mmol) obtained in the above step (1) in DMF (20 ml) at 5 ° C., EDCI (763 mg, 3 , 98 mmol, 1 equiv.), NOVT (538 mg, 3.98 mmol, 1 equiv.) And DIEA ((1.4 ml, 7.96 mmol, 2 equiv.) And the mixture was stirred for 1 hour. After confirmation of the completion of the reaction by TLC, the reaction solution was acidified with 10% HCl at 5 ° C (to pH ~ 4) and extracted with EA (20 ml). The organic phases were combined and washed with saturated NaHCO ₃ and brine, dried over anhydrous Na ₂ SO ₄ and concentrated in a vacuum, getting pr and the residue, which is solidified by the addition of EA and n-hexane and purified by column chromatography, to thereby give the title compound (620 mg, 1.7 mmol, 43%) as a white solid.

R _f = 0.7 (EA).

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 1.45 (br s, 9H), 1.75 ~ 2.10 (m, 2H), 3.05 (m, 1H), 3.45 (m, 1H), 3.65 (s, 3H), 4.25 (m, 1H), 4.45 (d, 2H, J = 5.7 Hz), 5.45 (m, 1H) ), 7.05 (m, 1H), 7.20 ~ 7.45 (m, 5H).

(3) Preparation of (3-amino-3-benzylcarbonylpropyl) carbamic acid tert-butyl ester hydrochloride

To a solution of (1-benzylcarbamoyl-3-methoxycarbonylaminopropyl) carbamic acid tert-butyl ester (1 g, 2.7 mmol) obtained in the above step (2) in 1,4-dioxane (10 ml) was added 4N. HCl in 1,4-dioxane (6.8 ml, 27 mmol) and the mixture was stirred for 2 hours. After confirming completion of the reaction, TLC the reaction solution was concentrated and dried in vacuo to give the title compound as a white solid.

Example 1

1- Benzyl-7-methyl-6-thioxohexahydropyrimido [1,6-a] pyrimidin-2-one

(A) Preparation of N-benzyl-3- [3- (2- [1,3] dioxolan-2-yl-ethyl) -3-methylthioureido] propionamide

A suspension of benzylamido-β-alanine hydrochloride (1.0 eq.) And N-methylmorpholine (2.2 eq.) In dichloromethane is treated with thiophosgene (1.2 eq.) At 0 ° C for 10 minutes. The reaction mixture was allowed to warm to room temperature and was stirred for an additional 2 hours. The clear solution was diluted with ethyl acetate and washed with 10% KHSO ₄ solution, distilled water and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue.

This product was dissolved in dichloromethane and treated with 2- (N-methyl-2-aminoethyl) -1,3-dioxolane (0.9 eq.) At 0 ° C for 10 minutes. The reaction mixture was allowed to warm to room temperature and stirred for an additional 4 hours. The reaction mixture was diluted with ethyl acetate and washed with 10% KHSO ₄ solution, distilled water, saturated NaHCO ₃ solution, distilled water and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue. The crude product is purified by column chromatography (silica gel, ethyl acetate / hexane = 5/2) to give a pure product.

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 2.02 (m, 2H), 2.60 (m, 2H), 3.18 (s, 3H), 3.82 (m, 2H), 3.88 (m, 2H), 4.03 (m, 4H), 4.44 (m, 2H), 4.91 (m, 1H), 6.84 (br s, 1H), 7.25-7.38 (m, 5H);

MS (m / z, ESI), 352 (MH ⁺ ).

(B) Preparation of 1-benzyl-7-methyl-6-thioxohexahydro-pyrimido [1,6-a] pyrimidin-2-one

The amide obtained in the above step (A) is treated with formic acid at 60 ° C. for 4 days. After evaporation of formic acid under reduced pressure, the residue was purified by preparative TLC (silica gel, ethyl acetate / methanol = 5/1) to obtain the pure title product.

¹ H-NMR (500 MHz, CDCl ₃ ) δ ppm: 2.05 (m, 1H), 2.36 (m, 1H), 2.64 (d, 1H), 2.96 (m, 1H), 3.30 (m, 3H), 3.44 (s, 3H), 4.42 (d, 1H), 4.86 (br s, 1H), 5.08 (d, 1H), 5.39 (m, 1H); 7.25-7.38 (m, 5H);

MS (m / z, ESI), 290 (MH ⁺ ), 311 (M ⁺ Na).

Example 2

1,7-Dibenzyl-6-thioxohexahydro-pyrimido [1,6-a] pyrimidin-2-one

(A) Preparation of N-benzyl-3- [3-benzyl- (3,3-diethoxypropyl) thioureido] propionamide

A suspension of benzylamido-β-alanine hydrochloride (1.0 eq.) And N-methylmorpholine (2.2 eq.) In dichloromethane is treated with thiophosgene (1.2 eq.) At 0 ° C for 10 minutes. The reaction mixture was allowed to warm to room temperature and stirred for an additional 2 hours. The clear solution was diluted with ethyl acetate and washed with 10% KHSO ₄ solution, distilled water and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue. The product is dissolved in dichloromethane and treated with 2- (N-benzyl-1-amino-3,3-diethoxypropane (0.9 equiv.) At 0 ° C for 10 minutes, the Reaction mixture was allowed to warm to room temperature and it was stirred in for an additional 6 hours, the resulting reaction mixture was diluted with ethyl acetate and washed with 10% KHSO ₄ solution, distilled water, saturated NaHCO ₃ solution, distilled water and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue. product cleanse column chromatography (silica gel, ethyl acetate / hexane = 2/1) to give the pure title product.

¹ H-NMR (500 MHz, CDCl ₃ ) δ ppm: 1.22 (t, 6H), 1.95 (m, 2H), 2.60 (m, 2H), 3.46 (m, 2H), 3.60 (br t, 2H), 3.63 (m, 2H), 3.97 (m, 2H), 4.38 (m, 2H), 4.52 (m, 1H), 5.07 (br s, 2H), 6.16 (br s, 1H), 6.98 (br s, 1H), 7.25-7.38 (m, 10H);

MS (m / z, ESI), 458 (MH ⁺ ).

(B) Preparation of 1,7-dibenzyl-6-thioxohexahydro-pyrimido [1,6-a] pyrimidin-2-one

The amide obtained in the above step (A) is treated with formic acid at 60 ° C. for 4 days. After evaporation of formic acid under reduced pressure, the residue was purified by preparative TLC (silica gel, ethyl acetate / methanol = 5/1) to obtain a pure product.

¹ H-NMR (500 MHz, CDCl ₃ ) δ ppm: 1.94 (m, 1H), 2.24 (m, 1H), 2.62 (m, 1H), 3.01 (m, 1H), 3.18 (m, 1H), 3.43 (m, 1H), 3.62 (m, 1H), 4.39 (d, 1H), 4.51 (m, 1H), 4, 91 (m, 1H), 5.02 (d, 1H), 5.26 (d, 1H), 5.53 (m, 1H), 7.25-7.40 (m, 10H);

MS (m / z, APCI), 366 (MH ⁺ ).

Example 3

1,7-Dibenzylhexahydro-pyrimido [1,6-a] pyrimidin-2,6-dione

(A) Preparation of N-benzyl-3- [3-benzyl- (3,3-diethoxypropyl) thioureido] propionamide

A suspension of benzylamido-β-alanine hydrochloride (1.0 eq.) And N-methylmorpholine (3.2 eq.) In dichloromethane was treated with triphosgene (0.7 eq.) At 0 ° C for 10 min. The reaction mixture was allowed to warm to room temperature and stirred for an additional 2 hours. The clear solution was diluted with ethyl acetate and washed with 10% KHSO ₄ solution, distilled water and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue. This product is dissolved in dichloromethane and treated with 2- (N-benzyl-1-amino-3,3-diethoxypropane (0.9 equiv.) At 0 ° C for 10 minutes, the Reaction mixture was allowed to warm to room temperature and stirred in for an additional 4 hours, The resulting reaction mixture was diluted with ethyl acetate and washed with 10% KHSO ₄ solution, distilled water, saturated NaHCO ₃ solution, distilled water and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue. This raw product chischayut column chromatography (silica gel, ethyl acetate / hexane = 2/1) to give the pure title product.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 1.23 (t, 6H), 1.87 (m, 2H), 2.55 (m, 2H), 3.24 (m, 2H), 3.49 (m, 2H), 3.59 (m, 2H), 3.65 (m, 2H), 4.45-4.58 (m, 5H), 5.62 (br s , 1H); 6.57 (br s, 1H); 7.25-7.48 (m, 10H);

MS (m / z, ESI), 442 (MH ⁺ ).

(B) Preparation of 1,7-dibenzylhexahydro-pyrimido [1,6-a] pyrimidin-2,6-dione

The amide obtained in the above step (A) is treated with formic acid at 60 ° C. for 4 days. After evaporation of formic acid under reduced pressure, the residue was purified by preparative TLC (silica gel, ethyl acetate), to thereby give the title compound.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm; 1.89 (m, 1H), 2.19 (m, 1H), 2.58 (m, 1H), 2.75 (m, 1H), 3.02 (m, 3H), 4.42 (d , j = 12.4Hz, 1H), 4.45 (d, j = 2.4Hz, 2H), 4.65 (m, 1H), 4.78 (m, 1H), 4.98 (d, j = 12.4Hz, 1H); 7.25-7.38 (m, 10H);

MS (m / z, ESI), 350 (MH ⁺ ).

Example 4

1,7-Dibenzyl-6-oxoctahydropyrimidino [1,6-a] pyrimidin-2-one

(A) Preparation of (3-bromo-1-methoxypropan-1-hydroxy) bound argogel resin

A suspension of dry argogel resin and pyridinium p-toluenesulfonate (240 mg, 0.96 mmol) in 1,2-dichloroethane (15 ml) was heated to reflux while continuously removing the solvent and traces of water. After removal of approximately 5 ml of distillate, a solution of 3-bromo-1,1-dimethoxypropane (700 mg, 3.84 mmol) in 1,2-dichloroethane (5 ml) was added and the mixture was kept at reflux for 4 hours with continuous removing EtOH / EDC, after which the resin is washed with DMF and dioxane, followed by lyophilization, to thereby obtain the desired product.

(B) Preparation of (3-benzylamino-1-methoxypropan-1-hydroxy) bound argogel resin

A solution of benzylamine (520 mg, 4.85 mmol) in DMSO (4 ml) was added to bromoacetal resin (1 g, 0.48 mmol) and the suspension was shaken at 60 ° C for 15 hours. The resulting resin was filtered, washed with DMSO, MeOH and MS and dried in vacuo overnight. The presence of a secondary amine is determined by the chloranil test.

(C) Obtaining β-alanine benzylamine urea

To a solution of β-alanine benzylamide as a salt with HCl (80 mg, 0.36 mmol) in N-methylmorpholine (120 μl) and MS (2 ml) was added triphosgene (0.72 mmol) at room temperature. After 10 minutes, the resulting isocyanate solution was added to the suspension containing the secondary amino group resin obtained in the above step (2) (100 mg, 0.048 mmol), and the suspension was kept under shaking for 3 hours at room temperature. The resin is washed with DMF, MeOH and MS and the completion of the reaction is checked by the chloranil test.

(D) Preparation of 1,7-dibenzyl-6-oxoctahydropyrimidido [1,6-a] pyrimidin-2-one

The resin of stage (C) containing the thiourea group is treated with formic acid and kept with shaking for 15 hours. The resin is filtered off and the filtrate is concentrated and purified by chromatography (silica gel) to obtain the title compound.

¹ H-NMR (500 MHz, CDCl ₃ ) δ ppm: 1.94 (m, 1H), 2.24 (m, 1H), 2.62 (m, 1H), 3.01 (m, 1H), 3.18 (m, 1H), 3.43 (m, 1H), 3.62 (m, 1H), 4.39 (d, 1H), 4.51 (m, 1H), 4, 91 (m, 1H), 5.02 (d, 1H), 5.26 (d, 1H), 5.53 (m, 1H), 7.25-7.40 (m, 10H);

MS (m / z, APCI), 366 (MH ⁺ ).

Example 5

1,7-Dibenzyl-2-oxo-6-thioxoctahydro-pyrimido [1,6-a] pyrimidine-4-carboxylic acid benzyl ester

(A) Preparation of 1-benzyl ester-4- (9H-fluoren-9-ylmethyl) ester of 2-isothiocyanatoic succinic acid

To a solution of 2-tert-butoxycarbonylamino-succinic acid 1-benzyl ester (1 g, 3.09 mmol) in MS, DIC (532 μl, 1.1 equiv.), DMAP (188 mg, 0.5 equiv.) And fluorenylmethanol ( 635 mg, 1.05 equiv.). After completion of the reaction, the resulting reaction mixture was washed with 1 N. HCl and saturated NaHCO ₃ solution and purified by column chromatography (silica gel) to obtain fluorenyl methyl ester (400 mg).

The specified ether is diluted in dioxane (10 ml) and add 4 N. a solution of HCl in dioxane and the mixture was kept under stirring for 2 hours to remove the Boc-protecting group. After completion of the reaction, the solvent was evaporated to dryness. The amine salt with HCl was diluted with MS and N-methylmorpholine and thiophosgene (1.2 eq.) Was added at approximately 0 ° C. After completion of the reaction, the mixture was washed with 10% KHSO ₄ solution, distilled water, saturated NaHCO ₃ solution, distilled water, and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue. This crude product was purified by column chromatography (silica gel, ethyl acetate / hexane = 1/1) to obtain the pure title product.

(C) Thiourea of aspartic acid benzyl fluorenyl ether

The MS solution of the isocyanate (0.5 mmol) obtained in the above step (A) with N-methylmorpholine is added to the suspension of the secondary amine-bound resin (200 mg, 0.04 mmol) obtained in step (B) of Example 4 and the mixture is kept under shaking for 3 hours at room temperature. The resulting resin is washed with DMF, MeOH and MS and the completion of the reaction is monitored by a chloranil test.

(C) Aspartic acid benzylamide thiourea

The resin obtained in the above step (C) is swelled for 30 minutes in DMF (4 ml) and a 25% piperidine solution is added to remove the fluorenylmethyl protecting group. The resulting resin is washed with DMF, MeOH and MS. The resin is dried under reduced pressure and swollen again, DIC (8 μl, 0.05 mmol), HOBt (8 mg, 0.05 mmol) and DIEA (18 μl, 0.1 mmol) are added to activate the acid. After shaking for 30 minutes, benzylamine is added and the mixture is kept shaking overnight to obtain the desired benzylamide-substituted resin.

(D) Preparation of 1,7-Dibenzyl-2-oxo-6-thioxoctahydro-pyrimido [1,6-a] pyrimidine-4-carboxylic acid benzyl ester

The resin obtained in the above step (C) is subjected to swelling in MS (4 ml), PPTS (10 mg) is added and heated for 4 hours at 60 ° C, thereby obtaining the title compound.

MS (m / z, ESI), 500 (MH ⁺ ).

Example 6

7-benzyl-6-thioxohexahydro-pyrimido [1,6-a] pyrimidine-1-carboxylic acid benzyl ester

(A) Preparation of {3- [3-benzyl-3- (3,3-dimethoxypropyl) thioureido] propyl} carbamic acid benzyl ester

A suspension of Cbz-diaminopropane · HCl (1.0 eq.) And N-methylmorpholine (2.2 eq.) In MS was treated with thiophosgene (1.2 eq.) At 0 ° С for 10 min. The resulting solution was allowed to warm to room temperature and stirred for an additional 2 hours. The resulting clear solution was diluted with ethyl acetate and washed with 10% KHSO ₄ , water and saturated NaCl. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue, which was dissolved in MS and treated with N-benzyl-1-amino-3,3-diethoxypropane (0.9 equiv.) At 0 ° C for 10 min and then allow it to warm to room temperature and stirred for an additional 6 hours. The resulting reaction mixture was diluted with ethyl acetate and washed with 10% KHSO ₄ solution, water, saturated NaHCO ₃ , water and saturated NaCl. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue, which was then purified by column chromatography (silica gel, ethyl acetate / hexane, 2/1) to obtain the title compound.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 1.17 (t, 6H), 1.5 (br s, 2H), 1.75 (t, 2H), 1.92 ( m, 2H), 3.20 (q, 2H), 3.45 (m, 2H), 3.60 (m, 4H), 3.75 (q, 2H), 4.51 (t, 1H), 5.06 (s, 4H); 6.75 (br s, 1H); 7.25-7.38 (m, 10H);

MS (m / z, ESI), 442 (M-OEt ⁺ ).

(B) Preparation of 7β-benzyl-6-thioxohexahydro-pyrimido [1,6-a] pyrimidine-1-carboxylic acid benzyl ester

To the amide solution obtained in the above step (A), PPTS was added to the MS and the mixture was stirred at 70 ° C. overnight. The resulting reaction mixture was concentrated under reduced pressure to give a residue, which was purified by preparative TLC (ethyl acetate only), to thereby give the title compound.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 1.89 (m, 2H), 1.95 (m, 1H), 2.63 (m, 1H), 2.80 (m, 1H), 3.10 (m, 1H), 3.45 (m, 1H), 3.89 (m, 1H), 4.01 (m, 1H), 4.39 (d, 1H), 4, 51 (m, 1H), 4.92 (m, 2H), 5.10 (m, 2H), 7.16-7.4 (m, 10H);

MS (m / z, ESI): 396 (MH ⁺ ).

Example 7

8-Acetyl-6-oxohexahydropyrazino [1,2-a] pyrimidine-1-carboxylic acid benzyl ester

(A) Obtaining [acetyl- (2,2-dimethoxyethyl) amino] acetic acid

To a solution of a benzylglycine salt with HCl (1 equiv.) In MeOH at room temperature was added dimethoxyacetaldehyde (1.05 equiv.) And then NaCNBH ₃ (1.2 equiv.) And the mixture was stirred for 5 hours. The resulting reaction mixture was concentrated under reduced pressure to obtain an oily residue, which was dissolved in MS and washed with saturated NaHCO ₃ solution, water and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue, which was dissolved in MS and treated with triethylamine (3 equiv.) And acetyl chloride (1.1 equiv.) At 0 ° C.

After completion of the reaction, the resulting reaction mixture was washed with saturated NaHCO ₃ solution, water and saturated NaCl solution. The organic layer was dried over Na ₂ SO ₄ and concentrated to give an oily residue, which was purified by column chromatography (silica gel, ethyl acetate) to give a pure product. The product is hydrogenolized using a 10% Pd / C and H ₂ -containing balloon to give the title compound, which is used in the next step without further purification.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 2.09 (s, 1H), 2.20 (s, 2H), 3.40 (d, 6H), 3.48 (d, 2H), 4.16 (s, 2H), 4.44 (m, 1H).

(B) Preparation of (3- {2- [Acetyl- (2,2-dimethoxyethyl) amino] acetylamino} propyl) carbamic acid benzyl ester

To the acid solution (1 equivalent) obtained in the above step (A), HATU (1 equivalent), DIPEA (3 equivalent) and Cbz-diaminopropane · HCl (1.0 equivalent) were added to the MS and the mixture was stirred for 3 hours at room temperature. The reaction mixture was concentrated under reduced pressure to give an oily residue, which was purified by preparative TLC, to thereby give the title compound.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 1.60 (m, 2H), 2.01 (s, 1H), 2.20 (s, 2H), 3.20 (d, 2H), 3.24 (m, 2H), 3.40 (d, 6H), 3.50 (d, 2H), 4.06 (s, 2H), 4.44 (m, 1H), 5, 08 (s, 2H), 5.18 (d, 1H), 6.91 (br.s.d, 1H), 7.16 (br.s, 5H);

MS (m / z, ESI): 396 (MH ⁺ ).

(C) Preparation of 8-Acetyl-6-oxohexahydropyrazino [1,2-a] pyrimidine-1-carboxylic acid benzyl ester

To a solution of the Cbz-protected amide precursor obtained in the above step (B) in MS, PPTS (1 eq.) Was added at room temperature and the mixture was heated to 70 ° C. for 5 hours. The resulting reaction mixture was concentrated, thus obtaining a residue, which is characterized as follows.

¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 1.90 (m, 2H), 2.10 (s, 1H), 2.30 (s, 2H), 2.61 (m, 1H), 2.82 (m, 1H), 3.15 (m, 1H) 3.50 (m, 1H), 3.9 (m, 1H), 4.0 (m, 1H), 4.2 (m, 1H), 4.3 (s, 1H), 4.47 (m, 1H), 5.08-5.18 (m, 2H), 5.28 (br s, 1H), 7, 16 (broad s, 5H);

MS (m / z, ESI): 332 (MH ⁺ ).

Example 8

7-Benzoylamino-4-benzylcarbamoyl-6-oxohexahydropyrrolo [1,2-a] pyrimidine-1-carboxylic acid methyl ester

(A) Preparation of [1- (1-benzylcarbamoyl-3-methoxycarbonylaminopropylcarbamoyl) -3,3-dimethoxypropyl] carbamic acid benzyl ester

To a solution of Cbz-protected amino acid acetal (100 mg, 1.3 equiv.) Obtained in Preparative Example 3 (3), PyBOP (1 equivalent per acid), DIPEA (6 equivalents per acid) and HOBt ( 1.3 equiv.) And the mixture is stirred for 30 minutes. An aminobenzylamide salt with HCl (71 mg, 0.27 mmol) was added to the reaction mixture, and the mixture was stirred for 7 hours. The resulting reaction mixture was washed with saturated NaHCO ₃ , water and saturated NaCl. The organic layer was dried over MgSO ₄ and concentrated to give an oily residue, which was purified by column chromatography (silica gel, ethyl acetate) to obtain the title compound (50 mg, yield: 35%).

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 2.1 (t, 2H), 3.05 (m, 1H), 3.50 (cc, 6H), 3.45 (m, 1H), 3.75 (s, 3H), 4.25 (q, 1H), 4.41 (m, 2H), 4.55 (m, 1H), 5.0 (q, 2H), 5, 3 (m, 1H); 5.95 (m, 1H); 7.2-7.4 (m, 10H).

(B) Preparation of 4-benzylcarbamoyl-7-benzyloxycarbonylamino-6-oxohexahydropyrrolo [1,2-a] pyrimidine-1-carboxylic acid methyl ester

The acetalamide cyclization product precursor (5 mg, 0.009 mmol) obtained in the above step (A) was dissolved in formic acid (1 ml) and stirred overnight. The resulting reaction mixture was concentrated to dryness and the product was used in the next step without further purification.

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 2.25 (m, 2H), 2.61 (t, 2H), 3.24 (m, 1H), 3.50 (s, 3H), 3.55 (m, 1H), 3.95 (m, 1H), 4.45 (m, 2H), 4.65 (d, 1H), 4.8 (m, 2H), 5, 3 (m, 1H), 5.7 (d, 1H), 7.15-7.4 (m, 10H), 7.85 (m, 1H).

(C) Preparation of 7-benzoylamino-4-benzylcarbamoyl-6-oxohexahydropyrrolo [1,2-a] pyrimidine-1-carboxylic acid methyl ester

At room temperature, a solution of the Cbz-bicyclic compound obtained in the above step (B) in MeOH and Pd / C (1 mg) was placed in a reaction vessel equipped with a hydrogen gas cylinder and the mixture was stirred for 2 hours. After completion of the reaction, the reaction mixture was filtered through a celite filter to remove Pd / C, and the solvent was evaporated under reduced pressure. The resulting oily residue was dissolved in MS, a solution of benzoic acid (1.1 eq.) In MS and PyBOP (1.1 eq.), HOBt (1.1 eq.) And DIPEA (3 eq.) Was added to the solution, and the mixture was stirred within 30 minutes An amine solution was added to the resulting activated acid solution, and the mixture was kept under stirring for 3 hours. The resulting reaction mixture was concentrated under reduced pressure to give an oily residue, which was purified by preparative TLC, to thereby give the title compound.

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm: 2.25 (m, 2H), 2.65 (m, 2H), 3.27 (m, 1H), 3.70 (s, 3H), 3.6 (m, 1H), 4.10 (m, 1H), 4.54 (m, 2H), 4.8 (t, 1H), 5.45 (m, 1H), 7, 15-7.42 (m, 10H), 7.9 (d, 1H), 8.31 (t, 1H).

Example 9

7-Benzoylamino-4- (1-carboxyethylcarbamoyl) -6-oxohexahydro-pyrrolo [1,2-a] pyrimidine-1-carboxylic acid methyl ester

A synthetic diagram showing the methodology of example 9 is presented in figure 3.

A 2-chlorotrityl chloride resin (200 mg, 1 mmol / g) and a solution of Fmoc-alanine (1.5 eq., Commercially available) and DIEA (2 eq.) In DCE (2 ml) were placed in a screw cap vessel. The reaction mixture was shaken at room temperature for 12 hours. The resin was collected by filtration and washed with DMF, MeOH and then DCM, thereby providing the first component portion.

To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and then the product mixture is washed with DMF, MeOH and then DCM. A solution of 2- (9H-fluoren-9-ylmethoxycarbonylamino) -4-methoxycarbonylaminobutyric acid (1.5 equivalents of the second component), DIC (1.5 equivalents), HOBT (1.5 equivalents) was added to the resin. in NMP. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and the product mixture is washed with DMF, MeOH and then DCM. A solution of 2- (9H-fluoren-9-ylmethoxycarbonylamino) -5,5-dimethoxypentanoic acid (1.5 equivalents), DIC (1.5 equivalents), HOBT (1.5 equivalents) in NMP was added to the resin. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and then the product mixture is washed with DMF, MeOH and then DCM. A solution of commercially available benzoic acid (1.5 equivalents), DIC (1.5 equivalents), HOBT (1.5 equivalents) in NMP is added to the resin. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

The resin is treated with formic acid (1.2 ml per well) for 18 hours at room temperature. After that, the resin was removed by filtration, and the filtrate was concentrated under reduced pressure, thereby obtaining the product as an oil.

¹ H-NMR (300 MHz, MeOH-d ₄ ) δ: 1.40 (d, 3H), 1.90 (m, 1H), 2.20 (m, 1H), 2.30 ~ 2.50 ( m, 2H), 3.15 (m, 2H), 3.20 (m, 1H) 3.45 (s, 3H), 3.40 ~ 3.60 (m, 1H), 4.20 ~ 4, 40 (m, 2H), 4.70 (t, 1H), 5.40 (t, 1H), 7.25 ~ 7.45 (m, 3H), 7.75 (d, 2H).

MS (m / z, ESI): 433 (MH ⁺ ), 455 (MNa ⁺ ).

Example 10

7-Benzoylamino-4- (2-carboxypropylcarbamoyl) -6-oxohexahydropyrrolo [1,2-a] pyrimidine-1-carboxylic acid methyl ester

A synthetic diagram showing the methodology of Example 10 is shown in FIG. 4.

A 2-chlorotrityl chloride resin (200 mg, 1 mmol / g) and a solution of Fmoc-beta-alanine (1.5 eq.) And DIEA (2 eq.) In DCE (2 ml) were placed in a screw cap vessel. The reaction mixture was shaken at room temperature for 12 hours. The resin was collected by filtration and washed with DMF, MeOH and then DCM, thereby providing the first component portion.

To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and the product mixture is washed with DMF, MeOH and then DCM. A solution of 2- (9H-fluoren-9-ylmethoxycarbonylamino) -4-methoxycarbonylaminobutyric acid (1.5 equivalents of the second component), DIC (1.5 equivalents) and HOBT (1.5 equivalents) was added to the resin. in NMP. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and then the product mixture is washed with DMF, MeOH and then DCM. A solution of 2- (9H-fluoren-9-ylmethoxycarbonylamino) -5,5-dimethoxypentanoic acid (1.5 equivalents), DIC (1.5 equivalents), HOBT (1.5 equivalents) in NMP was added to the resin. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

To the resin swollen in DMF, 25% piperidine in DMF was added before the reaction. After this, the reaction mixture was shaken for 30 minutes at room temperature. The deprotection step is repeated and then the product mixture is washed with DMF, MeOH and then DCM. A solution of commercially available benzoic acid (1.5 equivalents), DIC (1.5 equivalents) and HOBT (1.5 equivalents) in NMP are added to the resin. After shaking the reaction mixture for 12 hours at room temperature, the resin was washed with DMF, MeOH and then DCM.

The resin is treated with formic acid (1.2 ml per well) for 18 hours at room temperature. After that, the resin was removed by filtration, the filtrate was concentrated under reduced pressure, thereby obtaining the product as an oil.

¹ H-NMR (300 MHz, MeOH-d ₄ ) δ ppm: 1.40 (d, 3H), 1.90 (m, 1H), 2.20 (m, 1H), 2.30 ~ 2.50 (m, 2H), 3.15 (m, 2H), 3.35 (s, 3H), 3.40 ~ 3.60 (m, 3H), 4.20 ~ 4.40 (m, 2H), 4.70 (t, 1H), 5.40 (t, 1H), 7.25 ~ 7.45 (m, 3H), 7.75 (d, 2H).

MS (m / z, ESI): 447 (MH ⁺ ), 469 (MNa ⁺ ).

Various references are provided herein in which certain procedures, compounds and / or compositions are described in detail, and are incorporated by reference in their entirety.

It should be understood that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited to anything except the appended claims.

Claims (7)

1. The compound having the structure

where A represents - (CH) -, —N— or —CH ₂ —N—; B represents - (CH ₂ ) -, - (CH ₂ —CH ₂ ) -; D represents - (CH ₂ ) - or - (C = O) -; W represents - (C = O) - or is absent; X is —NH (C = O) - or absent; Y represents oxygen or sulfur; L represents hydrogen, —C (O) NHR ₃ or —C (= O) OR ₄ ; R ₁ choose from C _1-12 alkyl; C _6-10 aryl; C _1-6 alkoxy; C _6-10 aralkyl; C _6-10 aralkyloxy; R ₂ choose from saturated or unsaturated C _1-10 alkyl, which is optionally substituted with one or more substituents selected from C _1-6 alkoxy; C _1-6 alkoxycarbonyl; chromen-2-one; CN; benzyl; A 5-6 membered heteroaryl containing one or two nitrogen atoms, a sulfur or oxygen atom and which is optionally substituted with one or more substituents selected from halogen or oxo; formamidyl, which is optionally substituted with C _6-10 aryl, optionally substituted with one or more substituents selected from halogen or C _1-6 alkoxy, piperonyl, C _6-10 aralkyl, optionally substituted with one or more substituents selected from C _1-6 alkoxy , hydroxyl or nitro, C _6-10 arylcarbamoylalkyl; C _1-10 alkenyl; C _6-10 aralkyloxy; C _1-10 alkanedienyl; C _6-10 aryl, which is optionally substituted with one or more substituents selected from C _1-6 alkoxy, halogen, hydroxyl or nitro; C _6-10 aralkyl, which is optionally substituted with one or more substituents selected from C _1-6 alkoxy, halogen, nitro or hydroxyl; benzodioxolyl; piperonyl; chromenonyl; alkyl-C (= O) -; A 5-6 membered heteroaryl containing one or two nitrogen atoms, or an oxygen atom, or a sulfur atom and which is optionally substituted with one or more substituents selected from halogen or oxo; R ³ choose from

,

,

, or benzyl; where R ^a choose from C _1-10 alkyl, which is optionally substituted with one or more substituents selected from hydroxyl, C _3-10 cycloalkyl, piperonyl, amino or C _1-6 alkylthio; C _1-10 alkenyl; C _3-10 dicloalkyl; amino; C _6-10 aralkyl, which is optionally substituted with hydroxyl, halogen, nitro, C _1-6 alkyl, C _1-6 alkenyl or C _6-10 aryl; R ^d choose from hydrogen; C _6-10 aralkyl, which is optionally substituted with C _1-6 alkoxy or halogen; R ₄ represents a C _6-10 aralkyl; provided that if A is - (CH) - then B is - (CH ₂ ) -, D is - (CH ₂ ) -, X is —NH (C = O) - and L is —C (= O) NHR ₃ ; if A represents N, then B represents - (CH ₂ —CH ₂ ) - and X is absent; if A is —CH ₂ —N— then B is - (CH ₂ ) -, D is - (CH ₂ ) -, W is - (C = O) -, X is absent and L is hydrogen . 2. The compound according to claim 1, where a represents - (CH) -. 3. The compound according to claim 2, where w represents - (C = O) -. 4. The compound according to claim 2, where Y represents oxygen. 5. The compound according to claim 1, where a represents N. 6. The compound according to claim 1, A represents-CH ₂ -N-. 7. A pharmaceutical composition having an inhibitory effect on NF-kB transcription, comprising a compound according to claims 1-6 and a pharmaceutically acceptable carrier.

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