EA039991B1 - PEPTIDOMIMETIC COMPOUNDS NEUTRALIZING THE INFLUENZA VIRUS - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to the field of medicine. The present invention relates to novel peptidomimetic compounds that can bind and/or neutralize influenza viruses, in particular phylogenetic group 1 influenza A viruses, and to pharmaceutical compositions containing such compounds. The present invention also relates to the use of peptidomimetic compounds in the diagnosis, prevention and/or treatment of influenza virus infections.

State of the art

The seasonal influenza A virus is a major public health problem that kills more than 250,000 people worldwide every year and creates an economic burden for millions of people. Pandemic influenza, which occurs when a new virus emerges and infects immunocompromised or non-immune people around the world, poses an even greater threat to human health; for example, in 1918 the Spanish influenza pandemic caused an estimated 50 million deaths. A continuing concern is highly pathogenic avian influenza (HPAI), which has shown mortality rates of over 50% in infected humans. Influenza viruses of the subtype H5 as well as H7 are endemic in poultry in some parts of the world. At present, these viruses do not appear to be able to spread easily from person to person, but new data on the H5 avian influenza virus indicate that only a few amino acid changes are needed to allow the virus to spread via aerosol transmission in a model system. in vivo mammals.

Recently, antibodies have been described that have a neutralizing ability against a wide range of influenza A and/or B viruses, such as CR9114 (as disclosed in WO 2013/007770), CR6261 (disclosed in WO 2008/028946), FI6 (described in Corti et al., Science 333, 850-856 (2011)). These antibodies have been shown to interact with a range of hemagglutinin proteins and neutralize a wide range of influenza strains. Because of their efficacy and broad spectrum of action, such antibodies are now being developed for therapeutic treatment of critically ill patients and prophylactic applications in high-risk individuals. However, the relatively high cost of the products and their parenteral administration are expected to limit the use of monoclonal antibodies in a wider population.

Other currently available agents for the prevention and/or treatment of influenza virus infection also have serious limitations. Antiviral drugs such as the neuraminidase inhibitors, oseltamivir and zanamivir, and the M2 inhibitors, amantadine and rimantadine, have limited efficacy when administered late in infection, and their widespread use is likely to lead to the emergence of resistant strains of the virus. In addition, the use of oseltamivir in adults is associated with side effects such as nausea, vomiting, mental health effects, and renal events.

In addition, the efficacy of influenza vaccines has been shown to be suboptimal for patients at high risk (the elderly), and due to the constant changes in antigens in circulating influenza viruses, annual adaptation of the composition of the influenza vaccine is necessary to ensure the greatest possible match between virus strains. in the influenza vaccine and circulating strains of the influenza virus.

The search for new antiviral drugs against influenza that act on hemagglutinin (HA) as an alternative strategy for the prevention and/or treatment of influenza virus infection is also complicated by the high sequence variability of this protein. Therefore, the hemagglutinin ligands described so far are active only against a limited number of closely related strains of the influenza virus.

Given the severity of respiratory disease caused by influenza A viruses, as well as the strong economic impact of seasonal epidemics and the constant risk of pandemics, there is still a need for new effective inhibitors with a broad spectrum of activity against influenza A viruses that can be used as drugs for preventing or treating an infection caused by the influenza virus.

Brief description of the invention

The present invention provides novel peptidomimetic compounds that can specifically bind to the hemagglutinin (HA) of at least two strains of influenza A virus containing HA of different subtypes from phylogenetic group 1. In certain embodiments, the compounds can specifically bind to at least one strain an influenza virus containing HA subtype H1, such as the H1N1 strain of influenza virus, and at least one strain of influenza virus containing HA subtype H5, such as the H5N1 strain of influenza virus. At least some of these compounds can neutralize at least two strains of influenza A virus containing HA of different subtypes from phylogenetic group 1. In certain embodiments, the compounds can specifically neutralize at least one strain of influenza A virus containing HA subtype H1, such as the H1N1 strain of the influenza virus, and at least one

- 1 039991 strain of influenza virus containing HA subtype H5, such as strain H5N1 influenza virus.

In certain embodiments, the compounds have the following sequence:

where Cap1 is any amino acid sequence consisting of 0-10 residues with an N-terminal blocking group;

X1 is any L- or D-amino acid;

X2 is any L-amino acid;

X3 is an aliphatic L-amino acid with a molecular weight of less than 200 Da;

X4 is any L-amino acid;

X5 is Tyr or an L-tyrosine analogue;

X6 is Phe or an L-phenylalanine analog;

X7 is any charged or neutral hydrophilic L-amino acid;

X8 is Trp or an L-tryptophan analogue;

X9 is absent or is any hydrophilic L-amino acid;

X10 is any L-amino acid; And

Cap2 is any amino acid sequence consisting of 0-10 residues with a C-terminal blocking group.

The present invention further provides cyclized peptidomimetic compounds. Thus, in certain embodiments, the compounds have a sequence selected from

Capl-Xl-[X2-X3-X4-X5-X6-X7-X8-Leu-X9-X10]-Cap2 ,

Capl-Xl-X2-X3-[X4-X5-X6-X7-X8-Leu-X9-X10]-Cap2

Capl-Xl-[X2-X3-X4-X5-X6-X7-X8-Leu-X9-X10]-Cap2 where Cap1 is any amino acid sequence consisting of 0-10 residues with an N-terminal blocking group;

X1 is any L- or D-amino acid;

X2 is any L-amino acid;

X3 is an aliphatic L-amino acid with a molecular weight of less than 200 Da;

X4 is any L-amino acid;

X5 is Tyr or an L-tyrosine analogue;

X6 is Phe or an L-phenylalanine analog;

X7 is any charged or neutral hydrophilic L-amino acid;

X8 is Trp or an L-tryptophan analogue;

X9 is absent or is any hydrophilic L-amino acid;

X10 is any L-amino acid; And

Cap2 is any amino acid sequence consisting of 0-10 residues with a C-terminal blocking group.

In yet another aspect of the present invention, multimeric, in particular dimeric, peptidomimetic compounds are provided. Thus, in certain embodiments, the compounds have the sequence

Capl-Xl-[X2-X3-X4-X5-X6-X7-X8-Leu-X9-X10]-Cap2

I

Capl-Xl-(X2-X3-X4-X5-X6-X7-X8-Leu-X9-X10]-Cap2 I I .

where Cap1 is any amino acid sequence consisting of 0-10 residues with an N-terminal blocking group;

X1 is any L- or D-amino acid;

X2 is any L-amino acid;

X3 is an aliphatic L-amino acid with a molecular weight of less than 200 Da;

X4 is any L-amino acid;

X5 is Tyr or an L-tyrosine analogue;

X6 is Phe or an L-phenylalanine analog;

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X7 is any charged or neutral hydrophilic L-amino acid;

X8 is Trp or an L-tryptophan analogue;

X9 is absent or is any hydrophilic L-amino acid;

X10 is any L-amino acid; And

Cap2 is any amino acid sequence consisting of 0-10 residues with a C-terminal blocking group.

In addition, the present invention provides pharmaceutical compositions containing at least one compound described in this document, and a pharmaceutically acceptable carrier or diluent.

The present invention also relates to compounds described herein for use in the diagnosis, prevention and/or treatment of influenza.

Brief description of the figures

The drawing shows the binding curves of CP141037, CP141019 and CP141099 with HA obtained from three strains of influenza virus (A/California/07/09 (left), A/New Caledonia/20/99 (middle), A/Vietnam/1194/04 (on right)). Response units (RU) are plotted as a function of time following injection of an increasing amount of compound. The curves represent the waveform obtained from the SPR experiments, and the superimposed thin black lines represent the curves obtained by the method of best fit to the data according to the Langmuir binding model 1:1.

Detailed description of the invention

In the research that led to the present invention, new peptidomimetic compounds were developed that were guided by the structural data of HA complexes, in particular with the monoclonal antibodies CR6261, CR9114 and FI6. Peptidomimetic compounds (also referred to as peptidomimetics) are typically small protein-like molecules designed to mimic naturally occurring peptides or proteins. Peptidomimetics preferably have the ability to bind to their natural targets in the same way that natural peptides bind, and as a result, they should have the same biological effect. Additionally, it is possible to design these molecules so that they exhibit the same biological effect as the natural peptide, but with improved properties, such as higher proteolytic stability, bioavailability, selectivity and/or efficiency.

The peptidomimetic compounds of the present invention have been shown to have competitive binding activity against at least the H1 subtype HA, such as the A/California/07/2009 and A/New Caledonia/20/1999 strains of the H1N1 influenza virus, and against HA subtype H5, such as the A/Vietnam/1203/2004 strain of the H5N1 influenza virus. At least some of the compounds of the present invention have also been shown to have neutralizing activity against at least two different strains of influenza A virus, each containing HA of a distinct HA subtype from phylogenetic group 1, such as against influenza viruses containing HA subtype H1, such as strains A/California/07/2009 and A/New Caledonia/20/1999 of influenza H1N1, and for influenza viruses containing HA subtype H5, such as strain A/Vietnam/1203 /2004 H5N1 influenza virus. The compounds of the present invention offer several advantages over influenza antibodies, including small size (1.5 kDa), low chemical production cost, easy construction into multidimensional formats, and high stability with the ability to provide non-injectable routes of administration.

Thus, in a first aspect of the present invention, novel peptidomimetic compounds are provided having the following sequence:

Capl-Xl-X2-X3-X4-X5-X6-X7-X8-Leu-X9-X10-Cap2, where Cap1 is an amino acid sequence containing 0-10 residues with an N-terminal blocking group;

X1 is any L- or D-amino acid;

X2 is any L-amino acid;

X3 is an aliphatic L-amino acid with a molecular weight of less than 200 Da;

X4 is any L-amino acid;

X5 is Tyr or an L-tyrosine analogue;

X6 is Phe or an L-phenylalanine analog;

X7 is any charged or neutral hydrophilic L-amino acid;

X8 is Trp or an L-tryptophan analogue;

X9 is absent or is any hydrophilic L-amino acid;

X10 is any L-amino acid; And

Cap2 is an amino acid sequence containing 0-10 residues with a C-terminal blocking group.

As indicated above, the compounds of the present invention can specifically bind to the hemagglutinin (HA) of at least two strains of influenza A virus containing HA of different subtypes from phylogenetic group 1. In certain embodiments, the compounds can specifically bind to at least one strain of influenza virus containing HA subtype H1, such as strain H1N1 influenza virus, and at least one strain of influenza virus containing HA subtype H5, such as strain H5N1 influenza virus. In certain embodiments, the compounds can specifically bind to at least one influenza virus strain containing the H1 subtype HA, such as the H1N1 influenza virus strain, and at least one influenza virus strain containing the H5 subtype HA, such as the H5N1 influenza virus strain. .

In certain embodiments, the compounds can specifically bind to at least two, preferably at least three, more preferably at least four different influenza virus strains containing the HA subtype H1. In certain embodiments, the compounds can specifically bind to at least two, preferably at least three, more preferably at least four different influenza virus strains containing the H5 HA subtype. In certain embodiments, the compounds can neutralize at least two, preferably at least three, more preferably at least four different strains of influenza virus containing HA subtype H1.

In certain embodiments, the compounds can bind to at least one influenza virus containing another HA subtype from phylogenetic group 1, such as the H2 and/or H9 subtype.

As used herein, the term specific binding refers to compounds that bind to an epitope of a protein of interest, ie. HA, but which hardly recognize or bind other molecules in a sample containing a mixture of antigenic biological molecules. In general, the compounds of the present invention bind to group 1 influenza A virus HA with an affinity constant (Kd value) below 10 μM, preferably below 1 μM, more preferably below 0.1 μM, even more preferably below 10 nM, even more preferably below 1 nM.

As used throughout this specification, the term influenza subtype in relation to influenza A viruses refers to strains of influenza A virus that are characterized by various combinations of viral surface proteins, hemagglutinin (H) and neuraminidase (N). Influenza A virus subtypes can be named by their H number, such as influenza virus containing HA subtype H1 or H5 or influenza H1 virus, influenza H5 virus, or by a combination of H number and N number, such as influenza subtype H1N1 or H5N1. The term subtype of influenza virus includes, in particular, all individual strains of influenza virus within such subtype, which usually differ due to mutations in hemagglutinin and/or neuraminidase, and are characterized by different pathogenicity profiles, and includes natural isolates, as well as artificial mutants or reassortants, etc. .P. Such strains may also be referred to as different virus subtype isolates. Accordingly, the terms strains and isolates used herein may be used interchangeably. Influenza A virus subtypes can be further classified according to their phylogenetic group. Thus, phylogenetic analysis showed that influenza virus hemagglutinins are divided into two main groups: in particular, subtypes H1, H2, H5 and H9 belong to phylogenetic group 1 (group 1 influenza viruses), and, in particular, subtypes H3, H4, H7 and H10 belong to phylogenetic group 2 (group 2 influenza viruses).

The amino acid of the present invention may be any of the twenty naturally occurring (or standard amino acids) or variants thereof, such as, for example, D-amino acids (Denantiomers of amino acids with a chiral center) or any variants not naturally found in proteins. In table. 5 shows the abbreviations and properties of standard amino acids.

In certain embodiments of the present invention, X3 is an aliphatic L-amino acid with a molecular weight of less than 200 Da with a linear or C-γ branched side chain; X5 is Tyr or an L-tyrosine analogue having a donor and/or acceptor hydrogen bond replacing the hydroxyl group of tyrosine; X6 is Phe or an analogue of L-phenylalanine with one or more substituents selected from the group consisting of hydrogen, halogen, C ₁ -C ₄ -alkyl in the ortho and/or meta positions of the phenyl ring, halogen in the para position of the phenyl ring, L-3-naphthalene-1-yl-alanine and L-3-naphthalene-2-yl-alanine; and X8 is Tip or an L-tryptophan analogue with a halogen which is a substituent at C5, C6 and/or C7 and/or a small aliphatic substituent at C2 of the indole ring.

In a further aspect of the present invention, cyclic compounds are contemplated. Cyclization reduces the flexibility of compounds and results in higher affinity and potency as well as improved properties such as higher proteolytic stability, bioavailability and/or selectivity. Thus, in certain embodiments, the compounds described herein are cyclized. In certain embodiments, the compounds are cyclized via a chemical bridge between X2 and X10 and/or between X4 and

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x10. In certain embodiments, the compounds are cyclized through the formation of an amide bond comprising residues X2 and X10. In certain embodiments, the compounds are cyclized through the formation of an amide bond, including residues X4 and X10.

Thus, in certain embodiments, the compounds have a sequence selected from the group consisting of

Capl-Xl-[X2-X3-X4-X5-X6-X7-X8-Leu-X9-X10]-Cap2,

Capl-Xl-X2-X3-[X4-X5-X6-X7-X8-Leu-X9-X10]-Cap2 I______________________________________________I and Capl-Xl-[X2-X3-X4-X5-X6-X7-X8-Leu- X9-X10]-Cap2 ,

where Cap1 is an amino acid sequence containing 0-10 residues, with an N-terminal blocking group;

X1 is any L- or D-amino acid;

X2 is any L-amino acid;

X3 is an aliphatic L-amino acid with a molecular weight of less than 200 Da;

X4 is any L-amino acid;

X5 is Tyr or an L-tyrosine analogue;

X6 is Phe or an L-phenylalanine analog;

X7 is any charged or neutral hydrophilic L-amino acid;

X8 is Trp or an L-tryptophan analogue;

X9 is absent or is any hydrophilic L-amino acid;

X10 is any L-amino acid; And

Cap2 is an amino acid sequence containing 0-10 residues with a C-terminal blocking group.

In certain embodiments, X2 and/or X4 is any L-amino acid with a functional group that can be used for cyclization, with the functional group being 2-5 atoms away from the α-C atom in the L-amino acid; and X10 is any L-amino acid with a functional group that can be used for cyclization, and the functional group is at a distance of 2-5 atoms from the α-C atom in the L-amino acid.

In certain embodiments, X2 is Lys or L-ornithine and X10 is β-alanine, 3-aminopropionic acid, 3-amino-2,2-dimethylpropionic acid, 4-aminobutanoic acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or Pro.

In certain embodiments, X2 is Asp or Glu and X10 is Lys or L-ornithine.

In certain embodiments, X4 is Lys or L-ornithine and X10 is β-alanine, 3-aminopropionic acid, 3-amino-2,2-dimethylpropionic acid, 4-aminobutanoic acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or Pro.

In certain embodiments, X4 is Asp or Glu and X10 is Lys or L-ornithine.

In certain embodiments, the compounds are cyclized via a chemical bridge between X2 and X10, and X4 and X10.

In certain embodiments, the compounds are cyclized by intramolecular crosslinking of the free amino groups introduced at residues X2 and X4 and X10 with trissuccinimidylaminotriacetate (TSAT).

In yet another aspect of the present invention, multimeric peptidomimetic compounds are provided, wherein the multimeric compounds comprise two or more of the compounds described herein. In certain embodiments, the compounds are dimeric. In certain embodiments, the implementation of the residue involved in the formation of bonds between the monomers, is in position X4. Thus, in certain embodiments, the compounds are multimerized by intermolecular crosslinking of reactive groups introduced into the X4 residue. The compounds of the present invention may be homodimers (ie containing two similar monomeric compounds) or may be heterodimers.

In certain embodiments, the compounds of the present invention have the sequence

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Capl-Xl-[X2-X3-X4-X5-X6-X7-X8-Leu-X9-X10]-Cap2

I

Capl-Xl[X2~X3~X4~X5=X6-X7"X8LeUX9-X10]-Cap2 where Cap1, X1-X10 and Cap2 are as defined above.

In some embodiments, the compounds are dimerized by intermolecular crosslinking of reactive groups introduced into the X4 residue using a linker consisting of 2-20 repeating ethylene glycol units.

As defined above, in the linear, cyclized and/or multimeric compounds of the present invention, Cap1 and/or Cap2 may be an amino acid sequence of 0-10 additional residues. Thus, according to the present invention, Cap1 and/or Cap2 may be absent, or the core sequences provided herein may have an amino acid sequence containing up to 10 additional residues (natural or non-natural amino acids) in the N-terminal region, and/or The C-terminal region may contain a sequence containing up to 10 additional residues (natural or non-natural amino acids). The C- and/or N-terminal blocking group may be any arbitrary blocking group that is known in the art. N-terminal blocking groups are, for example, acetyl or succinyl. The C-terminal blocking group is, for example, carboxamide.

In certain embodiments of the present invention, Cap1 is acetyl or succinyl.

As defined above, according to the present invention, X1 may be any L or D-amino acid. In certain embodiments, X1 is (S)-2-amino-5-phenylpentanoic acid, (2S)-3,3-dimethyl-2-amino-5-phenylpentanoic acid, or Arg.

In certain embodiments, X2 is Lys, L-ornithine, Cys, L homocysteine, or Ser.

In certain embodiments, X3 is Leu, L-norleucine, L-cyclopentylalanine, L-cyclobutylalanine, L-cyclopropylalanine, (2S,4S)-2-amino-4-methylhexanoic acid, (2S,4R)-2-amino-4- methylhexanoic acid, (2S,4S)-2-amino-4-methylheptanoic acid, (2S,4R)-2-amino-4-methylheptanoic acid, (S)-2-amino-4-ethylhexanoic acid, or an N-methylated derivative thereof.

In certain embodiments, X4 is Asp, Glu, Arg, Lys, ornithine, cysteine, homocysteine, N6-(4-carboxybutanoyl)lysine, or N5-(4-carboxybutanoyl)ornithine.

In certain embodiments, X5 is Tyr.

In certain embodiments, X6 is Phe, L-2-chlorophenylalanine, L-3-chlorophenylalanine, L-4-chlorophenylalanine, or L-3,4-dichlorophenylalanine.

As defined above, X7 may be any charged or neutral hydrophilic L-amino acid. In certain embodiments, X7 is Glu, Gln, Asp, Asn, Arg, or Lys.

In certain embodiments, X8 is Trp or L-2-methyltryptophan.

As defined above, according to the present invention, X9 may be absent or may be any hydrophilic L-amino acid. In certain embodiments, X9 is Ser or Gln.

As defined above, according to the present invention, X10 may be any La-amino acid. In certain embodiments, X10 is 4-aminobutanoic acid, β-alanine, ((2R)-3-amino-2-(3-aminopropanoylamino)-β-alanine, Lys, L-ornithine, Cys, or homocysteine.

In certain embodiments, Cap2 is absent.

In at least certain embodiments, the compounds can neutralize at least two strains of influenza A virus containing HA of different subtypes from phylogenetic group 1. In certain embodiments, the compounds can specifically neutralize at least one influenza virus strain containing HA subtype H1, such as the H1N1 strain of influenza virus; and at least one strain of influenza virus containing an HA subtype H5, such as the H5N1 strain of influenza virus.

In certain embodiments, the compounds can neutralize at least two, preferably at least three, more preferably at least four different strains of influenza virus containing HA subtype H1. In certain embodiments, the compounds can neutralize at least two, preferably at least three, more preferably at least four different strains of influenza virus containing the HA subtype H5.

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In certain embodiments, the compounds can neutralize at least one influenza virus containing an HA of a different subtype from phylogenetic group 1, such as subtype

H2 and/or H9. In certain embodiments of the present invention, cross-neutralizing compounds are provided.

The term neutralizing or neutralization, as used herein in relation to the compounds of the present invention, refers to the ability of a compound to inhibit influenza virus replication in vitro and/or in vivo in a subject, regardless of the mechanism by which neutralization is achieved. In certain embodiments, the compounds of the present invention neutralize the influenza virus by suppressing the fusion of the viral envelope and the cell membrane after the virus has attached to the target cell. The term cross-neutralizing or cross-neutralizing, as used herein in relation to the compounds of the present invention, refers to the ability to neutralize influenza virus strains of various subtypes of influenza A. Neutralizing activity can be measured, for example, as described herein. Alternative assays that measure neutralizing activity are described, for example, in the WHO Manual on Animal Influenza Diagnosis and Surveillance, Geneva: World Health Organization, 2005, version 2002.5. Generally, the compounds of the present invention have a neutralizing activity of 1 μM or less, preferably 100 nM or less, more preferably 10 nM or less, as determined in an in vitro virus neutralization assay (VNA), for example, as described in the examples.

In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is leucine; X4 is arginine; X5 is tyrosine; X6 is phenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is serine; X10 is β-alanine; and Cap2 is absent, where the compound is cyclized via an amide bond between the X2 residue and X10 (lactam formation from the side chain of X2 to the tail).

In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is leucine; X4 is glutamic acid; X5 is tyrosine; X6 is L-3,4-dichlorophenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is serine; X10 is β-alanine; and Cap2 is absent, where the compound is cyclized via an amide bond between X2 and X10 (lactam formation from the side chain of X2 to the tail).

In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is L-cyclopentialanine; X4 is glutamic acid; X5 is tyrosine; X6 is phenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is serine; X10 is β-alanine; and Cap2 is absent, where the compound is cyclized via an amide bond between X2 and X10 (lactam formation from the side chain of X2 to the tail).

In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is L-N-methylleucine; X4 is glutamic acid; X5 is tyrosine; X6 is phenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is serine; X10 is β-alanine; and Cap2 is absent, where the compound is cyclized via an amide bond between X2 and X10 (lactam formation from the side chain of X2 to the tail).

In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is leucine; X4 is N6-(4-carboxybutanoyl)lysine; X5 is tyrosine; X6 is phenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is serine; X10 is β-alanine; and Cap2 is absent, where the compound is cyclized via an amide bond between X2 and X10 (lactam formation from the side chain of X2 to the tail).

In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is leucine; X4 is glutamic acid; X5 is tyrosine; X6 is phenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is glutamine; X10 is β-alanine; and Cap2 is absent, where the compound is cyclized via an amide bond between X2 and X10 (lactam formation from the side chain of X2 to the tail).

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In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is leucine; X4 is N6-(4-carboxybutanoyl)lysine; X5 is tyrosine; X6 is phenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is serine; X10 is β-alanine; and Cap2 is absent, where the compound is cyclized via intramolecular bonding from the X2 side chain to the tail and dimerized via an amide bonded X4 polyethylene glycol spacer containing 13 ethylene units (PEG13).

In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is leucine; X4 is glutamic acid; X5 is tyrosine; X6 is phenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is serine; X10 is ((2R)-3-amino-2-(3aminopropanoylamino)-to-alanine; and Cap2 is absent where the compound is cyclized via an amide bond between X2 and X10 (lactam formation from the side chain of X2 to the tail).

In certain embodiments of the present invention, Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is leucine; X4 is L-ornithine; X5 is tyrosine; X6 is phenylalanine; X7 is glutamic acid; X8 is tryptophan; X9 is serine, X10 is L-ornithine, and Cap2 is a carboxamide, wherein the compound is cyclized by a TSAT (tris-succinimidyl)aminotriacetate)-mediated X2, X4 and X10 bond.

In certain embodiments, the compound has a sequence selected from the group consisting of

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The peptidomimetic compounds of the present invention may be prepared by any procedure well known in the art, in particular by conventional chemical synthesis procedures using automated solid phase peptide synthesizers followed by chromatographic purification, for example as described in the examples below.

Additionally, the present invention provides pharmaceutical compositions containing one or more of the compounds described herein, and a pharmaceutically acceptable carrier or diluent. A pharmaceutically acceptable excipient may be any inert substance which is combined with an active molecule, such as a compound of the present invention, to form a suitable composition. A pharmaceutically acceptable excipient is an excipient which, at the dosages and concentrations used, is non-toxic to patients and which is compatible with the other ingredients of the formulation. Pharmaceutically acceptable excipients are widely used and are known in the art. Pharmaceutical compositions in accordance with the present invention may further contain at least one other therapeutic, prophylactic and/or diagnostic agent. Said additional therapeutic and/or prophylactic agents, for example, may be agents which may also provide prevention and/or treatment of influenza virus infection, such as, for example, M2 inhibitors (eg amantidine, rimantadine) and/or neuraminidase inhibitors (e.g. zanamivir, oseltamivir). They can be used in combination with the compounds of the present invention. The expression in combination in this document means simultaneously in the form of separate formulations or in the form of a single combined formulation or in accordance with the scheme of sequential administration in the form of separate formulations in any order.

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In a further aspect of the present invention, the compounds described herein are provided for use in the diagnosis, prevention and/or treatment of influenza. In addition, the present invention contemplates the use of a compound described herein in the manufacture of a medicament for the diagnosis, prevention and/or treatment of influenza. As used herein, the terms influenza or influenza virus disease refer to the pathological condition resulting from the infection of a cell or subject with an influenza virus. In specific embodiments, the implementation of this term refers to a disease of the respiratory tract, the cause of which is the influenza virus. As used herein, the term "influenza virus infection" refers to the infection, multiplication, and/or presence of an influenza virus in a cell or subject. Influenza virus infections can occur in small populations, but can also spread throughout the world during seasonal epidemics or, worse, during global pandemics that put millions of people at risk. The present invention provides binding molecules that are capable of neutralizing the infection caused by strains of influenza virus that are the cause of such seasonal epidemics, as well as possible pandemics.

The present invention further provides methods for preventing and/or treating influenza in a subject, comprising administering a therapeutically effective amount of a compound described herein to a subject in need thereof. The term therapeutically effective amount refers to the amount of a compound as defined herein that is effective in preventing, alleviating and/or treating a condition resulting from infection with an influenza virus. Prevention and/or treatment may be targeted at patient groups that are susceptible to influenza infection. Such patient populations include, without limitation, for example, the elderly (eg, aged >50 years, aged >60 years, and preferably aged >65 years), young (eg, aged <5 years, aged <1 year), hospitalized patients and already infected patients who received treatment with an antiviral compound, but who had an unsatisfactory antiviral response.

The compounds of the present invention may be administered to a subject, for example, intravenously, intranasally, via oral inhalation, pulmonary route, subcutaneously, intradermally, intravitreally, orally, intramuscularly, and so on. Several factors will influence the optimal route of administration, including the physicochemical properties of the active molecules, the urgency of the clinical situation, and the relationship of plasma concentrations of the active molecules to the desired therapeutic effect.

The present invention further provides a method for detecting influenza A virus in a sample, wherein the method comprises the steps of a) contacting said sample with a diagnostically effective amount of a compound of the present invention, and b) determining if the compound binds specifically to a molecule in the sample. The sample may be a biological sample, including, without limitation, blood, serum, tissue, or other biological material from (potentially) infected subjects. (Potentially) infected subjects may be human subjects, however, animals suspected of carrying influenza virus can also be tested for influenza virus using the compounds of the present invention.

The present invention is further illustrated by the following non-limiting examples.

Examples

Example 1 Synthesis of CP132070

The linear precursor peptide was prepared manually by solid phase peptide synthesis using Fmoc groups on a 0.5 mmol scale using β-alanine preloaded 2-chlorotrityl chloride resin (0.685 mmol/g). The functional groups of the amino acid side chains were blocked with protective groups such as N-Boc (W), O-tert-Bu (E, S, Y) and N-Pbf (R). The N-Dde group was used to orthogonally protect the amino functional group of the ornithine side chain (Boc: tert-butoxycarbonyl, tert-Bu: tert-butyl, Dde: 1-(4,4-dimethyl-2,6dioxacyclohexylidene)ethyl, Fmoc: 9-fluorenylmethoxycarbonyl , Pbf: 2,2,4,6,7pentamethyldihydrobenzofuran-5-sulfonyl). Before attaching the first amino acid, the resin was soaked in DMF (dimethylformamide) for 1 hour. A binding protocol was used using 3

- 10 039991 eq. each Fmoc-amino acid in DMF (4 ml) and an activating mixture containing HBTU ((2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, 2.85 eq.) and DIPEA (N, N-diisopropylethylamine, 6 eq.) in DMF (4 ml) The reaction mixture was shaken for 1.5-2 h. end of the peptide assembly by treating the peptide-resin complex with a mixture of acetic anhydride and 4-methylmorpholine in DMF (2:1:17, v/v/v, 20 ml) at room temperature for 1 h. Finally, the protecting group N -Dde was selectively removed by treating the peptide-resin complex with a 3% solution of hydrazine hydrate in DMF (3x10 min).

The deprotected peptide from the ornithine side chain was cleaved from the resin by agitating the peptide-resin complex for 60 minutes in a mixture of 65% (v/v) DCM (dichloromethane), 20% (v/v) hexafluoroisopropanol, 10% (v/v) trifluoroethanol and 5% (v/v) triethylsilane (10 ml/g peptide-resin complex). The resin was filtered off and washed with DCM. The peptide was precipitated from the filtrate by adding cold simple isopropyl ether and dried under vacuum. The crude peptide was used without further purification in the subsequent lactam cyclization step.

Cyclization in lactams was carried out at high dilution by dissolving the peptide with the removed protecting group from the ornithine side chain and the C-terminal carboxyl group in 150 ml of DMF, to which was added dropwise a solution of PyBOP (benzotriazol-1-yl-hydroxytripyrrolidinophosphonium hexafluorophosphate, 2 eq.) and N-methylmorpholine (6 eq.) in DMF (90 ml). The reaction mixture was stirred at room temperature until complete conversion was observed. After removal of the solvent under reduced pressure, a cyclized peptide was obtained, from which all protecting groups were removed by treatment with a mixture of 90% (vol.) TFA (trifluoroacetic acid), 5% (vol.) thioanisole, 2.5% (vol.) H ₂ O and 2.5% (v/v) EDT (ethanedithiol) at room temperature for 3 h. for prep HPLC Luna C18 (25x200 mm, 10 µm, 100 A) together with Gemini C18 prep HPLC column (30x150 mm, 50 µm, 100 A) with a mobile phase flow rate of 20 ml/min, (mobile phase A: 0, 05% TFA in water, mobile phase B: CH3CN, linear gradient applied). Lyophilization of pure fractions gave 328 mg (yield: 53%, purity: 97%) of cyclized peptidalactam as trifluoroacetate salt.

The following compounds were prepared similarly to CP132070

- 11 039991

Example 2 Synthesis of CP141032

The linear precursor peptide was prepared manually by solid phase peptide synthesis using Fmoc groups on a scale of 0.25 mmol using Sieber resin (0.69 mmol/g). The functional groups of the amino acid side chains were blocked with protective groups such as N-Boc (W), O-tert-Bu (E, S, Y) and N-Pbf (R). The N-Dde group was used for orthogonal protection of ornithine side chain amino functional groups (Boc: tert-butoxycarbonyl, tert-Bu: tert-butyl, Dde: 1-(4,4dimethyl-2,6-dioxacyclohexylidene)ethyl, Fmoc: 9-fluorenylmethoxycarbonyl , Pbf: 2,2,4,6,7pentamethyldihydrobenzofuran-5-sulfonyl). Before attaching the first amino acid, the resin was soaked in DMF for 1 hour. A binding protocol was used using 3 eq. Fmocamino acids in DMF (4 ml) and an activating mixture containing 2.85 eq. HBTU and 6 equiv. DIPEA in DMF (4 ml); while the reaction mixture was shaken for 1.5-2 h during each stage of binding. Removal of the Fmoc group was performed with a 20% solution of piperidine in DMF (2x15 min). N-terminal acetylation was performed at the end of the peptide assembly by treating the peptide-resin complex with a mixture of acetic anhydride and 4-methylmorpholine in DMF (2:1:17, v/v/v, 20 ml) at room temperature for 1 h Finally, N-Dde protecting groups were selectively removed by treating the peptide-resin complex with 3% hydrazine hydrate in DMF (3x10 min), after which the peptide-resin complex was washed with DMF and DCM.

Subsequent cleavage of the peptide from the resin was performed by agitating the peptide-resin complex for 60 minutes in a DCM solution containing 1% TFA. The resin was filtered off and washed with DCM. The filtrate was poured into cold isopropyl ether, which resulted in the precipitation of the peptide. Isolation by centrifugation, washing with cold isopropyl ether and drying under vacuum gave 348 mg (yield: 55%, purity: 80%) of the crude linear peptide, which was used without further purification in the TSAT (tris(succinimidyl)aminotriacetate) mediated cyclization step.

The side chain deprotected peptide ornithine (150 mg, 80% pure, 0.0595 mmol) was dissolved in DMF (300 mL) followed by DIPEA to pH 8-9. To this mixture was added dropwise a solution of TSAT cross-linking reagent (1.0 eq.) in DMF (50 ml), while the resulting reaction mixture was stirred at room temperature until complete conversion was observed. Removal of the solvent under reduced pressure gave a bicyclic peptide which had all protective groups removed by treatment with a mixture of 90% (v/v) TFA, 5% (v/v) thioanisole, 2.5% (v/v) H ₂ O and 2.5% (v) EDT at room temperature for 3 hours. Precipitation and washing with ice-cold tert-butyl methyl ether gave the crude cyclic peptide, which was purified by reverse phase high performance liquid chromatography (RP-HPLC) as described in Example 1. Lyophilization pure fractions gave 40 mg (yield: 34%, purity 94%) of bicyclic peptide CP141032 as trifluoroacetate salt.

Example 3 Synthesis of Homodimeric Peptide CP141100

- 12 039991

A linear monomeric precursor peptide was prepared by automated solid phase peptide synthesis using Fmoc groups on a 0.5 mmol scale using β-alanine preloaded 2-chlorotrityl chloride resin (0.29 mmol/g). The functional groups of the amino acid side chains were blocked with protective groups such as N-Boc (W) and O-tert-Bu (E, S, Y). The N-Mtt group was used to orthogonally protect the amino functional group of the ornithine side chain (Boc: t-butoxycarbonyl, t-Bu: t-butyl, Fmoc: 9-fluorenylmethoxycarbonyl, Mtt: methyltrityl). Before attaching the first amino acid, the resin was soaked in NMP (2x15 min). A binding protocol was used using 10 eq. Fmoc-amino acids, 9 eq. HATU (N,N,N',N'tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate, 0.45M in DMF) and 10 eq. DIPEA (2M in NMP) for each amino acid binding step (binding time: 30 min). In the case of two N-terminal amino acids, a double linking strategy was used. Removal of Fmoc was performed with 20% piperidine in NMP and monitored by UV detection. N-terminal acetylation was performed at the end of the peptide assembly by treatment with excess acetic anhydride in the presence of DIPEA (10 eq. 2M solution in NMP) for 30 min.

Cleavage of the peptide from the resin and selective removal of the Mtt protecting groups from the side chain was performed by agitating the complex of the peptide and resin for 60 min in a mixture of 65% (vol.) DCM (dichloromethane), 20% (vol.) hexafluoroisopropanol, 10% (vol.) ) trifluoroethanol and 5% (v/v) triethylsilane (10 ml/g peptide-resin complex). The resin was filtered off and washed with DCM. The peptide was precipitated from the filtrate by adding cold diethyl ether, the crude product was dried under vacuum and used without further purification in the next step.

Cyclization into lactams was performed at high dilution by dissolving the ornithine side chain deprotected peptide in 200 ml DMF followed by dropwise addition of a solution of PyBOP (2 eq.) and N-methylmorpholine (6 eq.) in DMF (60 ml). The reaction mixture was stirred at room temperature until complete conversion was observed (2 h). The solvent was removed under reduced pressure and the residue was redissolved in ethyl acetate. The organic phase was washed with aqueous 5% NaHCO ₃ and brine and concentrated under reduced pressure. All protecting groups were removed from the cyclized peptide by treatment with a mixture of 87.5% (vol.) TFA, 5% (vol.) thioanisole, 5% (vol.) H ₂ O and 2.5% (vol.) EDT at room temperature Precipitation followed by washing with ice-cold diethyl ether gave the crude peptide, which was purified by reverse phase high performance liquid chromatography (RP-HPLC) on an XBridge C18 OBD preparative HPLC column (30x250 mm, 5 µm, 140 A) with a mobile phase flow rate of 30 ml/min, (solvent A: 0.1% TFA in water+CH ₃ CN, solvent B: CH3 CN, linear gradient applied). Lyophilization of pure fractions gave 250 mg (yield 31%, purity: 83%) of fully deprotected cyclized lactam peptide as the trifluoroacetate salt.

Dimerization was carried out using a commercially available bis-PEG13-NHS ester (bis-succinimidyl-activated bis-PEG13 acid) as a coupling reagent. This was done by adding a coupling reagent (0.4 eq.) to a solution of cyclized peptidalactam (75 mg, 0.048 mmol) and Et ₃ N (5 eq.) in dry DMF, while the reaction mixture was stirred at room temperature until no further transformation was observed. The solvent was removed under reduced pressure and the resulting residue was directly purified by reverse phase high performance liquid chromatography (RP-HPLC) as described above. Lyophilization of pure fractions gave 19 mg (yield: 11%, purity 96%) of the dimeric peptide CP141100.

- 13 039991

Example 4 Synthesis of CP141066

The fully deprotected cyclized lactam peptide intermediate (50 mg, 0.032 mmol) from Example 3 was dissolved in DMF (2 mL). Et ₃ N (5 eq.) and disuccinimidylglutarate (1.0 eq.) were added and the reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the resulting residue was purified by reverse phase high performance liquid chromatography as described in Example 3. Lyophilization of pure fractions gave 28 mg (yield: 56%, 99% purity) of CP141066 peptide.

Example 5 Synthesis of Cyclized Lactam Peptide CP141099

O

A linear peptide was synthesized on a 0.3 mmol scale by automated solid phase peptide synthesis using Fmoc groups on preloaded L-Dap(Fmoc) (Dap: 2,3-diaminopropionic acid) 2-chlorotrityl chloride resin (0.15 mmol/g) . Preloaded L-Dap(Fmoc) 2-chlorotrityl chloride resin was prepared manually by treating 2-chlorotrityl chloride resin (5 g, 1.4 mmol/g, 7 mmol) with a solution of Dde-L-Dap(Fmoc)-OH (0.66 eq.) in DMF in the presence of DIPEA (2.6 eq.). The resulting reaction mixture was shaken for 90 minutes, methanol (2.5 ml) was added and shaking was continued for an additional 5 minutes. The solvent was removed by filtration and the resin was washed with DMF (5x). Finally, the NDde protecting group was removed by reaction with 2% hydrazine hydrate in DMF (5x2 min.) to give L-Dap(Fmoc) loaded 2-chlorotrityl chloride resin with a degree of substitution of 0.15 mmol/g (the degree of substitution was determined photometrically, by based on the amount of Fmoc chromophore released when the resin is treated with a solution of piperidine in DMF). Subsequent amino acid couplings and N-terminal acetylation were carried out using automated synthesis as described in example 3. It is important to note that the binding of Boc-to-alanine was performed before the removal of the Fmoc protecting groups on the preloaded L-Dap(Fmoc) 2-chlorotrityl chloride resin .

Cleavage of the peptide from the resin and selective removal of the Mtt protecting groups from the side chain was performed by agitating the complex of the peptide and resin for 60 min in a mixture of 65% (vol.) DCM, 20% (vol.) hexafluoroisopropanol, 10% (vol.) trifluoroethanol and 5% (v/v) triethylsilane (10 ml/g peptide-resin complex). The resin was filtered off and washed with DCM. The peptide was precipitated from the filtrate by adding cold diethyl ether, the crude product was dried under vacuum and used without further purification in the next step.

Cyclization into lactams was performed at high dilution by dissolving the ornithine side chain deprotected peptide in 120 ml DMF followed by dropwise addition of a solution of PyBOP (2 eq.) and N-methylmorpholine (6 eq.) in DMF (36 ml). The reaction mixture was stirred at room temperature until complete conversion (2 h). The solvent was removed under reduced pressure and the residue was redissolved in ethyl acetate. The organic phase was washed with aqueous 5% NaHCO ₃ solution and brine and concentrated under reduced pressure. Complete removal of all protecting groups was performed by treatment with a mixture of 87.5% (vol.) TFA, 5% (vol.) thioanisole, 5% (vol.) H2O and 2.5% (vol.) EDT at room temperature for 2 h. Precipitation followed by washing with ice-cold diethyl ether gave the crude peptide, which was purified by reverse phase high performance liquid chromatography on an XBridge C18 OBD preparative HPLC column (30x250 mm, 5 μm, 140 A) with a mobile phase flow rate of 20 ml/ min, (solvent A: 0.1% TFA in water+CH3CN, solvent B: MeOH, linear gradient applied). Lyophilization of pure fractions gave 36 mg (yield 7%, purity: 95%) of cyclized lactam peptide

- 14 039991

CP 141099 as trifluoroacetate salt.

Example 6 Peptide Analysis

UPLC (high performance liquid chromatography) and HPLC (high performance liquid chromatography) (HPLC) measurements were performed using an LC pump, diode array detector (DAD) or UV detector and column as described in the respective methods. If necessary, additional detectors were included (see table of methods below). The flow from the column was sent to a mass spectrometer (MS), which was equipped with an atmospheric pressure ionization source. It is within the skill of the art to set configurable parameters (eg, scan range, minimum measurement time) to produce ions enabling the molecular weight (MW) of a compound to be determined. Data collection was carried out using the appropriate software.

Peptides were described according to their experimental retention time (R _t ) and molecular weight (MW). All results were obtained with experimental errors that are usually associated with the method used. As used herein, MSD: Mass Selective Detector, DAD: Diode Array Detector, SQD: Single Quadrupole Detector.

Table 1

Symbol for LCMS methods (flow expressed in ml/min; column temperature (T) in °C; analysis time in minutes)

Method symbol device Column mobile phase Gradient Flow T column Analysis time A Agilent: 1200 HPLC-DAD and MSD Agilent: ZORBAX Eclipse XDB-C18 (5 MKM, 4.6*150mm) A: 0.1% TEA in H ₂ O B: 0.05% TEA in CH ₃ CN From 70% A to 40% A in 20 min. and up to 20% A in 5 min. 0.8 25 thirty IN Agilent: 1200 HPLC-DAD and MSD Agilent: ZORBAX Eclipse XDB-C18 (5 MKM, 4.6*150mm) A: 0.1% TEA in H ₂ O B: 0.05% TEA in CH ₃ CN From 90% A to 50% A in 20 min. and up to 10% A in 15 min. 0.8 25 thirty WITH Waters : Acquity® UPLC® DAD and SQD Column BEH300C4 (1.7 µm, 2.1x150 mm; Waters Acquity® A: 0.1% HCOOH + 5% CH ₃ OH in H ₂ OB: CH ₃ OH 95% A to 5% A in 14.0 min. and hold for 1 min. 0.2 15 thirty

table 2

Peptide analysis

Connection ID R _t (min.) ^# Calculated MN Purity (%) ⁵ СР141099 7.80 (C) 1556.77 95 СР141019 15.34 (A) 1539.57 100 СР141037 14.10 (A) 1484.71 99 СР141066 7.12 (C) 1583.84 99 СР132070 14.07 (A) 1497.75 97 СР141036 14.01 (A) 1496.72 98 СР141077 12.63 (C) 1511.73 99 SR141100 8.64 (C) 3594.22 96 СР141032 16.04 (V) 1633.86 94

^# Analytical methods (A, B, C) indicated in brackets are described above.

$ Peptide purity was calculated as the percentage of area of UV positive (220 nm) eluted material under the main peak of the chromatogram.

Example 7 Binding of Compounds to Influenza HA and Competition of Compounds with Other HA Binding Molecules.

Developed competitive binding studies to test compound competition

- 15 039991 associations with other well-characterized HA-binding proteins (including, for example, CR9114) with known epitopes on HA. The epitopes were either on the HA stem (proximal HA relative to the viral envelope) or, for control purposes, on the head of the HA (distal HA relative to the viral envelope). If competition was observed, both molecules were considered to bind to the same or at least overlapping epitope on the HA surface. Competition with the HA head and stem binding molecule was interpreted as non-specific binding.

For this, a competitive AlphaLISA assay (Perkin Elmer) was created, which is based on biotinylated full-length and trimeric HA proteins (Protein Sciences, 10 µl, final concentration 0.5 nM in 50 µl) bound by HA-specific binding molecules. The interaction between HA and the binding molecule was detected after 1 h incubation at room temperature (RT) using two types of beads: a streptavidin donor bead recognizing HA (10 µl 10 µg/ml) and an anti-Fc antibody bead (10 µg/ml ) that recognizes the IgG used. If, after an additional hour of incubation at RT, the excited donor bead (at 680 nm) and the acceptor bead were in close proximity, then the energy transfer (singlet oxygen production) could be measured as the luminescence signal of the acceptor bead (Envision plate reader from Perkin Elmer). The signal intensity in this homogeneous assay format is directly proportional to the binding strength (affinity/avidity) between both binding partners. A competing molecule (compound), depending on its affinity and concentration (usually tested in the range of 100 nM to 0.5 pM), could disrupt the AlphaLISA signal, resulting in a sigmoid inhibition curve that was fitted using a standard four-parameter logistic non-linear regression model in SPSS. The average calculated values of pIC ₅₀ are shown in table. 3.

Table 3

Compound competition to bind to influenza A virus HA (values are mean pIC ₅₀ , negative log half maximum inhibitory concentration, higher values indicate exponentially greater potency,

In conclusion, according to the present invention, the compounds of the present invention have been shown to have a broad spectrum of binding to influenza A group 1 viruses and specifically compete with HA stem-binding molecules, but not with control HA head-binding molecules.

Example 8 Neutralizing activity against influenza virus and cellular toxicity of the compounds.

Compounds were analyzed in a virus neutralization assay (VNA) for their ability to prevent infection of mammalian cells with influenza virus. To this end, Calu-3 cells,

- 16 039991 derived from human lung epithelium (ATCC, Cat # HTB-55) were plated in 96-well plates (4E+04 cells/well) and incubated for at least 7 days at 37°C and in an atmosphere 10% CO2 in total DMEM (containing 1x solution of non-essential amino acids MEM, 2 mM L-glutamine and 10% FBSHI, origin: Australia; Gibco). Polarized Calu-3 cells at approximately 90% confluence and tight junctions were ready for VNA. On the day of analysis, compound stock (dissolved in PBS 0.1% BSA, 0.1% Tween 5% DMSO, initial concentration 500 mM) was prepared with diluted sample plates by 2-fold dilution in incomplete DMEM (containing 2 mM L- glutamine, 1x penicillin/streptavidin). Plates with diluted samples were centrifuged (1000 g for 15 min) to remove potential aggregates. Then, 5 TCID ₅₀ /50 μl influenza virus (pre-titrated on Calu-3 cells) in incomplete DMEM was added to the diluted plate and incubated for 1 h at 37°C and 10% CO2. Media was removed from the cells and replaced with 50 μl of incomplete DMEM supplemented with 3% FBS. Then 100 μl of the virus/compound mixture was added to the cells, giving a total assay volume of 150 μl with a final concentration of 1% FBS. After incubation for 4 days at 37°C and 10% CO2, the cells were washed with PBS and fixed with 80% acetone at 200 μl/well for 15 min at room temperature (RT). The level of influenza infection was determined using ELISA against influenza nucleoproteins (NP). Primary antibody to Flu-A-NP (Abbiotec, clone 5D8) was used at 1:1000 diluted in 1% BSA in PBS and incubated for 1 hour with shaking at 300 rpm at RT. After washing the cells three times with wash buffer (PBS, 0.05% Tween), a secondary antibody (anti-mouse antibody conjugated with HRP, 1:2000) was added and incubated for 1 h with shaking at 300 rpm at RT . After washing the cells three times, POD chemiluminescent substrate was added at 50 μl/well and incubated for 2-5 min before reading the luminescence on a Synergy Neo plate reader from Biotek. The pIC ₅₀ compounds were calculated using the SPSS software. Multiple strains of influenza were tested in replicates to evaluate the neutralization spectrum (see Table 4).

VNA was also performed in the absence of virus to determine the level of cellular toxicity. Cytopathic effect (CPE) was measured using the ATP Lite reagent and according to the manufacturer's protocol (Perkin Elmer) and analyzed in a similar manner (see Table 4).

Table 4

Influenza virus neutralizing compounds (values are mean pIC ₅₀ , negative logarithm of half maximum inhibitory concentration, higher values indicate exponentially greater potency, empty cells mean not tested)

Compound Toxicity VNA H1N1 A/California/ 07/09 VNA H1N1 A/New Caledonia/ 20/99 VNA H5N1 A/Vietnam/ 1194/04 СР132070 <4.00 <4.00 4.08 4.42 СР141019 <4.00 7.66 7.16 7.09 СР141036 <4.00 4.02 5.86 6, 77 СР141037 <4.00 4.13 6.29 6, 17 СР141066 <4.00 <4.00 4.38 4.32 СР141070 <4.00 4.17 4.18 <4.00 СР141077 <4.00 4.25 4.98 5.35 SR141100 <4.00 7.49 7.60 7.80 СР141032 <4.00 4.23 <4.00

In conclusion, it should be noted that the compounds of the present invention have a broad spectrum of neutralization against strains of influenza virus group 1 in the absence of detectable cellular toxicity.

- 17 039991

Table 5

Standard amino acids, abbreviations and properties

Amino acid 3 letter 1-letter Side chain polarity Side chain charge (pH 7.4) alanine Ala A non-polar Neutral arginine Arg R polar Positive asparagine As η N polar Neutral aspartic acid asp D polar Negative cysteine Cys C non-polar Neutral glutamine acid Glu E polar Negative glutamine gin Q polar Neutral glycine gly G non-polar Neutral Positive histidine His H polar (10%) ; neutral (90%) isoleucine lie I non-polar Neutral leucine Leu L non-polar Neutral lysine Lys TO polar Positive methionine Met M non-polar Neutral phenylalanine Phe F non-polar Neutral proline Pro P non-polar Neutral serine Ser S polar Neutral threonine Thr T polar Neutral tryptophan trp W non-polar Neutral tyrosine Tyr Y polar Neutral valine Vai V non-polar Neutral

Example 9 HA Direct Binding Experiments Using Surface Plasmon Resonance (SPR).

All SPR experiments were performed using a Biacore T200 instrument (GE Healthcare) operating at 25°C. HA trimeric proteins (Protein Sciences) were randomly biotinylated and covalently immobilized on streptavidin-coated carboxymethylated dextran sensor surface (SA chip, GE Healthcare). Test compounds were resuspended in running buffer (20 mM PBS, 137 mM NaCl, 0.05% P-20 surfactant, pH 7.4 (GE Healthcare) supplemented with 2% DMSO) and binding constants were obtained from a series of compound injections. in the concentration range from 0.1 nM to 10 µM at a flow rate of 30 µl/min. Single cycle kinetic data was analyzed using BIAevaluation software. Baselines were adjusted to zero for all curves and the injection start time was equalized. Reference sensograms were subtracted from the experimental sensograms to obtain curves representing specific binding followed by background subtraction (ie, double comparison with a reference). Binding kinetics were evaluated using a 1:1 binding model (Langmuir) to obtain association (ka) and dissociation (kd) rate constants. Binding affinity (KD) was estimated from the concentration dependence of observed equilibrium reactions. Experiments were performed three times to ensure reproducibility.

Binding curves in typical experiments for three compounds binding to HA from three influenza strains (A/California/07/09 (H1), A/New Caledonia/20/99 (H1) and A/Vietnam/1194/04 (H5) ) are shown in the drawing. For all three compounds, binding affinities ranging from 10 to 100 nM were found, with dissociation rates ranging from 5 s ^-1 to 20 s ^-1 depending on the HA strain, as shown in Table 1. 6.

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Table 6

pKD values with standard deviation SD pKD and corresponding dissociation rate kd [s-1 ] for CP141037, CP141019 and CP141099. Each compound was tested for binding to HA derived from three strains of influenza virus (A/California/07/09 (H1), A/New Caledonia/20/99 (H1) and ______________________A/Vietnam/1194/04 (H5)) ________________._______________.

Claims (10)

CLAIM 1. A peptidomimetic compound having the sequence Cap1-X1-X2-X3-X4-X5-X6-X7-X8-Leu-X9-X10-Cap2, where Cap1 is acetyl; X1 is (S)-2-amino-5-phenylpentanoic acid; X2 is L-ornithine; X3 is Leu, L-cyclopentylalanine, or an N-methylated derivative thereof; X4 is Glu, Arg, L-ornithine or N6-(4-carboxybutanoyl)lysine; X5 is Tyr; X6 is Phe or L-3,4-dichlorophenylalanine; X7 is Glu; X8 is Trp; X9 is Ser or Gln; X10 is β-alanine, ((2R)-3-amino-2-(3-aminopropanoylamino)-β-αlanine, or Lornithine; and Cap2 is absent, and where the compound can specifically bind to the hemagglutinin (HA) of at least two strains of influenza A virus containing HA of two different subtypes from phylogenetic group 1, and where the compound is cyclized through the formation of an amide bond comprising residues X2 and/or X4 and x10. 2. A compound according to claim 1 wherein X2 is L-ornithine and X10 is β-alanine. 3. A compound according to claim 1 wherein X4 is L-ornithine and X10 is β-alanine. 4. A compound according to claim 1, wherein the compound is cyclized by intramolecular crosslinking of the free amino groups introduced at residues X2 and X4 and X10 with tris-succinimidylaminotriacetate (TSAT). 5. Multimeric peptidomimetic compound containing at least two compounds according to any one of claims 1-4. 6. A multimeric compound according to claim 5, wherein the compound is dimeric. 7. A multimeric compound according to claim 5 or 6, wherein the compound has been dimerized by intermolecular crosslinking of reactive groups introduced into the X4 residue. 8. Pharmaceutical composition for the diagnosis, prevention and/or treatment of influenza A containing HA from phylogenetic group 1, containing a compound according to any one of paragraphs. 1-7 and a pharmaceutically acceptable carrier or diluent. 9. The use of a compound according to any one of claims 1 to 7 in the diagnosis, prevention and/or treatment of influenza caused by infection with influenza A virus containing HA from phylogenetic group 1. 10. The use of a compound according to any one of claims 1 to 7 in the preparation of a medicament for the diagnosis, prevention and/or treatment of influenza caused by infection with influenza A virus containing HA from phylogenetic group 1.