ES2219187B1 - CALPAINE INHIBITORS. - Google Patents

Abstract

Calpain inhibitors

The present invention relates to compounds that they have peptide-biphenyl hybrid structure and activity related compounds such as inhibitors of Calpaina A compound of the present invention is a biphenyl. 2,2'-disubstituted, being the substituents in the positions 2 and 2 'of the biphenyl skeleton chains containing structures related to amino acids, peptides and amides, which They can be the same or different. The present invention also encompasses any of the conformational isomers (atropisomers) of said compound of formula I. The compounds of formula I have application in the preventive or therapeutic treatment of a degenerative illness.

Description

Calpain inhibitors

Technical field

The present invention is framed in the field of Enzyme inhibitors with therapeutic activity, more specifically of calpain inhibitors.

Background

Calpains, or neutral calcium-activated proteases (II) (CANP, EC 3.4.22.17) are a family of cysteine proteases ("cysteine proteases") with a very active metabolic role. Although their natural substrate is not clearly determined, these enzymes catalyze the hydrolysis of a variety of proteins involved in signal transduction, cytoskeleton reconstruction, cell cycle regulation and apoptosis ( Adv. Pharmacol . 1996 , 37 , 117). In mammals, the calpain family comprises various tissue-specific isoforms and two ubiquitous isoenzymes: µ-calpain (or calpain I) and m- calpain (or calpain II), which require micromolar and millimolar amounts, respectively, of calcium (II) for activation. Structural studies by X-ray diffraction have shown that each isoform is composed of a large subunit (~ 80 kDa), which has a protease domain with cysteine of the papain type, and a small subunit (~ 30 kDa), which is common to each isoenzyme. The C-terminal ends of each subunit have domains capable of binding Ca (II) (calmodulin-like domain) ( FEBSLett . 2001 , 501 , 111).

The over-activation of calpains, which can occur by increasing the intracellular concentration of calcium (II), is implicated in numerous diseases, such as cerebral and cardiac ischemia, Alzheimer's, Parkinson's, muscular dystrophy, cataracts, demyelinating diseases (such as sclerosis multiple) and other degenerative diseases ( Pathophysiology 1999 , 6 , 91; Brain Res. Rev. 2003 , 42 , 169).

The main application of selective calpain inhibitors is as neuroprotective agents. In the therapeutic area related to neuroprotection, various strategies have been used so far. Agents that act on membrane depolarization and calcium entry into cells have been used, or that prevent the production of free radicals (antioxidants), or that are antagonistic to the action of neurotransmitters ( J. Clinical Neurosci . 2002 , 9 , 4). Recently, much attention has been given to drugs capable of blocking NMDA receptors for glutamate; however, blocking ionotropic receptors of excitatory amino acids may not be an ideal method to prevent excitotoxic action, since these drugs have psychotomimetic side effects ( Pharmacol. Ther . 1999 , 81 , 163; Neurobiol. Disease 2003 , 12 , 82 ). An interesting alternative to achieve neuroprotection is the blocking of "post-receptor" cellular phenomena that are physiologically silenced; that is, the search for selective catabolic cascade blockers induced by excitotoxic agents. It is foreseeable that these potential drugs of intracellular action, acting on metabolic pathways that are activated during neurodegeneration, could allow a more effective and selective neuroprotective action.

Calpain overactivation requires a continued increase in intracellular concentrations of Ca (II), and this enzyme is dormant in resting cells [is that is, with "normal" Ca (II) levels]. Therefore, the Calpain inhibition is presented as an appropriate treatment in neurodegenerative diseases. Based on its characteristics, it is it is expected that calpain inhibition has fewer effects side effects in human therapy that block processes metabolic prior to its activation in pathological processes, as is the case with glutamate NMDA receptor antagonism and aspartate, because calpain is not activated in conditions physiological "normal" and to which the action of amino acids excitatory is essential for the normal operation of the nervous system.

In addition, potent and selective inhibitors of calpaina are very useful as work tools to study the mechanism of action of this protease, as well as its role in certain physiological processes

On the other hand, it is well known that peptides and related compounds (peptidomimetics, peptoids, etc.) possess a wide variety of biological activities. In this specification, any of these compounds (natural or artificial peptides, peptidomimetics, peptoid, and any peptide analog) will be called peptide. There are numerous articles and patents describing biological and pharmacological activities of this type of compounds ( Peptides: Synthesis, Structures, and Applications , Academic Press, San Diego, 1995 ; Adv. Drug Res . 1997 , 29 , 1). On the other hand, differently substituted biphenyls have been used as pharmacophors in compounds with various biological activities ( J. Med. Chem . 2000 , 43 , 3443). Additionally, biphenyl derivatives have been used as mesogenic fragments for the preparation of liquid crystals (EP-743293).

Reversible and irreversible calpain inhibitors have been described ( Trends Mol. Medicine 2001 , 7 , 355; US-5541290). The most frequent structural features of these inhibitors are that they are peptides or peptidomimetics with few amino acids (between 2 and 6) hydrophobic and some electrophilic functionality, among which α-ketophosphonates, α-ketophosphinates, α-ketophosphine oxides , α-ketoesters, α-keto acids, α-ketoamides, trifluoromethyl ketones, aldehydes, methylsulfonium salts, epoxides, etc. Apparently, these compounds act on the papain-like domain of calpain, which results in a relatively low selectivity, so they are also often inhibitors of other proteases with cysteine (e.g., papain) and even proteases with serine (serine ). protect yourself ). Partly due to these inconveniences, a calpain inhibitor with therapeutic utility has not yet been found.

The compounds structurally closest to the compounds object of the present invention have been disclosed in ( Acta Chim. Hung . 1984 , 173), where a compound has been described in which a 2,2'-biphenylcarbonyl fragment is used to bind two peptide chains (with H-Pyroglu-Phe-Trp-Ser-Tyr-Lys-Leu-Arg-Pro-Gly-NH2) sequences, where the binding of the peptide chains is performed through the amino group of the chain lateral of the lysine residues); in ( Chem. Comm . 1998 , 679), where two hybrid compounds have been described between (aS) -6,6'-dimethyl-2,2'-biphenyldicarboxylic acid and L -valine and D -valine, respectively ; in ( Biorg. Med. Chem. Lett . 2002 , 12 , 57) the compound formed from 2,2'-biphenyldicarboxylic acid and L -histidine methyl ester has been described; some amides derived from 2,2'-biphenyldicarboxylic acid and aromatic amines ( J. Chem. Eng. Data 1963 , 8 , 233) and from amides derived from 2,2'-di (amino) -biphenyl and aromatic acids ( Justus Liebigs Ann. Chem . 1952 , 577 , 1; J. Org. Chem . 1975 , 22 , 873; J. Chem. Soc. C 1968 , 1017) have been described. However, none of these compounds has been linked to therapeutic activity in relation to calpain.

There is a need to find calpain inhibitors with therapeutic utility. A type of calpain inhibitors not previously described and which have not been previously investigated is constituted by peptide compounds where amino acid and / or peptide chains, or analogs, are attached to positions 2 and 2 'of the biphenyl system, resulting in what we call peptide-biphenyl hybrids and related compounds , which are the object of the present invention. A structural characteristic of the compounds object of the present invention and which have demonstrated selectivity in the inhibition of calpain is the presence of one or more additional aromatic rings in the substituents of positions 2 and / or 2 'of the biphenyl ring.

Brief Description of the Invention

The present invention relates to compounds of peptide-biphenyl hybrid structure and compounds related to activity as calpain inhibitors. A compound of the present invention is a biphenyl 2,2'-disubstituted, being the substituents in the positions 2 and 2 'of the biphenyl skeleton chains containing structures related to amino acids, peptides and amides, and They can be the same or different.

Description

The present invention relates to compounds of peptide-biphenyl hybrid structure and compounds related to activity as calpain inhibitors. In this memory any of these compounds will be called peptide natural or artificial peptides, peptidomimetics, peptoid, and any peptide analog. The present invention relates to a compound of formula I, which has biphenyl structure 2,2'-disubstituted,

one

in which the R1 and R2 may be the same or different, and may be independently selected from among groups

- NH2,

- NHR 3 in which R 3 represents a group alkyl or aryl,

- NR 4 R 5 in which R 4 and R 5 they represent two identical or different alkyl or aryl groups, or forming a cyclic system,

- CO 2 H,

- CO 2 R 6 in which R 6 is a group aryl or alkyl,

- CONH_ {2},

- CONR 7 R 8 in which R 7 and R 8 they represent two identical or different alkyl or aryl groups, or forming a cyclic system,

- a group of formula II,

2

in the that:

- Ar represents an aryl, carbocyclic or heterocyclic, other than imidazole,

- the asterisk (*) represents a stereogenic center, of configuration ( R ) or ( S ), interchangeably,

- n has a value between 0 and 6,

- X, represents a group OH, OR 9 in which R 9 represents an alkyl or aryl group, NH 2, NHR 10, wherein R 10 represents an alkyl or aryl group, NR 11 R 12 in which R 11 and R 12 represent two identical or different alkyl or aryl groups, or forming a cyclic system,

- aa is an amino acid residue, of formula

3

where the asterisk (*) indicates a stereogenic center of configuration ( R ) or ( S ), interchangeably, R 13 is selected from H, alkyl or aryl;

or of formula

4

in which the nitrogen atoms and the carbon in position α- are forming a ring between 4 and 7 links,

amino acid residues (aa) are attached to through its amino group to the chain represented by -C (O) NHCH (CH 2 Ar) CO-;

- a group of formula III,

5

in which Ar represents a group aryl;

- a group of formula IV,

6

in the that:

- Ar represents an aryl group,

- (*), and n have the meaning described previously,

- aa, has the meaning described previously, but now the amino acid residue is attached to through its carbonyl group to the chain represented by -NH C (O) CH (CH 2 Ar) NH-,

- X represents H, alkyl between 1 and 6 atoms of carbon, COR 14 where R 14 is an aryl or alkyl group, CO 2 R 15, where R 15 is an aryl or alkyl group;

- a group of formula V,

7

in which Ar represents a group aryl;

and in which at least one of the R1 groups, R2 has the structure indicated by groups II, or III, or IV, or V;

and any of the conformational isomers (atropisomers) of said compound of formula I.

Especially preferred compounds are:

- ( S, S ) -3-Phenyl-2 - {[2 '- (2-phenyl-1-methoxycarbonyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -propionate methyl (1).

- ( S, S ) -2 - {[2 '- (1-carboxy-2-phenyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -3-phenyl-propionic acid (2).

- ( S, S ) -3- (1 H -indole-3-yl) -2 - ({2 '- [2- (1 H -indole-3-yl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl- 2-carbonyl} -amino) -
methyl propionate (3).

- ( S, S, S ) -2- (2 - {[2 '- (1-Carboxy-2-phenyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -3-phenyl-propionylamino) -3- methyl methyl butyrate (4).

- ( S, S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2-phenyl-1- (2-methyl-1-methoxycarbonyl-propylcarbamoyl) -ethylcarbamoyl] -biphenyl-2 -car-
bonyl} -amino) -propionylamino] -3-methyl-butyrate-methyl (4a).

- ( S, S ) - (2-Phenyl-1- {2 '- [3-phenyl-2- (9 H -fluoren-9-ylmethoxycarbonylamino) -propionylamino] -biphenyl-2-ylcarbamoyl} -
ethyl) 9 H -fluoren-9-ylmethyl carbamate (5).

- Bis -2,2 '- [(4-nitro-benzoyl) amino] -biphenyl (6).

- 2'-Amino-2- [2- (naphthoyl) amino] biphenyl (7).

- ( S, S, S ) -2- [2 - ({2'-Amino-biphenyl-2-ylcarbamoyl) -2-phenyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -3-phenyl-propionylamino ] -3-methyl methyl butyrate (8).

- ( S, S, S, S ) -3- (1 H -Indol-3-yl) -2- {3-phenyl-2 - [(2 '- {2-phenyl-1- [2- (1 H- indol-3-yl) -1-ethoxycarbonyl-ethylcarbamoyl] -ethylcarbamoyl} -biphenyl-2-carbonyl) -amino] propionylamino} -propionate methyl (9).

- ( S ) -2 '- [2- (4-Hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carboxylic acid (10).

- ( S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -pro-
methyl pionylamino] -4-methyl-pentanoate (11).

- ( S, S, S ) -2 - ({2 '- [2-phenyl-1- (3-methyl-1-methylcarbamoyl-butylcarbamoyl) -ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -3- (4-Hydroxyphenyl) methylpropionate (12).

- ( S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -pro-
methyl pionylamino] -4-methylsulfanyl-butyrate (13).

- 2 '- {( S ) - [2-Phenyl-1- ( R ) - (2-phenyl-1-methylcarbamoyl-ethylcarbamoyl) -ethylcarbamoyl]} -biphenyl-2-carboxylic acid (14).

- ( S ) -2 - [( S ) -2 - ({2 '- ( S ) - [2-Phenyl-1- ( R ) - (2-phenyl-1-methylcarbamoyl-ethylcarbamoyl) -ethylcarbamoyl] -biphenyl Methyl -2-carbonyl} -amino) -4-methyl-pentanoylamino] -4-methylsulfanyl-butyrate (15).

- ( S ) -2 '- [2-Phenyl-1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carboxylic acid (16).

- ( R ) - (3-Phenyl-2- {[2 '- ( S ) - (2-phenyl-l-methoxycarbonyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino}) -methyl propionate (17) .

- ( S, S ) -3- (4-Hydroxy-phenyl) -2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -ami -
no) -methylpropionate (18).

- ( S, S ) - N - (1-Ferrocenylmethyl) -3-phenyl-2 - ({2 '- [2-phenyl-1- (1-ferrocenylmethylcarbamoyl)] - ethylcarbamoyl) -biphenyl-2-car-
bonil} -amino) -propionamide (19).

- N, N ' - Bis - (2-fluoro-5-nitro-phenyl) -biphenyl-2,2'-dicarboxamide (20).

- ( S, S ) -2-Amino- N - [2 '- (2-amino-3-phenyl-propionylamino) -biphenyl-2-yl] -3-phenyl-propionamide (21).

- ( S, S ) - {1- [1- (2'-Amino-biphenyl-2-ylcarbamoyl) -2-phenyl-ethylcarbamoyl] -3-methyl-butyl} -carbamate of 9 H -fluoren-9-ylmethyl (22).

- 2,2'-di- (2-naphthoyl-amino) -biphenyl (2. 3).

- 2,2'-di - [(3,5-dinitrobenzoyl) amino) -biphenyl (24).

- 2- (2-Naphthoylamino) -2 '- [(3,5-dinitrobenzoyl) amino] -biphenyl (25).

- 2- (2-Naphthoylamino) -2 '- [(4-nitrobenzoyl) amino] -biphenyl (26).

- ( S, S, S, S ) -2- (3-Phenyl-2 - {[2 '- (2-Phenyl-1- {2' - [2- (9 H -Fluoren-9-ylmethoxycarbonylamino)) - propionylamino] -biphenyl-2-ylcarbamoyl} -ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -propionylamino) -3-methyl-butyrate (27).

The synthesis of the compounds of general formula I of the present invention has been carried out by usual methods in organic synthesis, which are known to those skilled in the art ( Chemical Approaches to the Synthesis of Peptides and Proteins , CRC Press, Boca Mouse, 1997; Comprehensive Organic Synthesis , vol 6, Pergamon Press, 1991). Because the main functionality present in the R 1 and R 2 substituents of type I compounds is amide, the essential reaction in the preparation of type I compounds is an acylation reaction between a acid or acid derivative, as an electrophile, and an amine, as a nucleophile. The synthetic strategy of the compounds of formula I depends on the structure of the groups R1 and R2, as well as whether these groups are the same or different.

The amino acids, peptides, amines and derivatives thereof used in the synthesis of the compounds of formula I are commercial or are prepared by usual methods in organic synthesis. In some cases, especially with non-natural D- series amino acids, biocatalytic methods have been applied using the enzyme acylase as a biocatalyst ( Tetrahedron: Asymmetry 1998 , 9 , 1951-1965).

When peptides such as nucleophiles or electrophiles have been used in the synthesis of compounds I, their synthesis has been carried out by usual methods in amino acid and peptide chemistry. The tert -butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and fluorenylmethoxycarbonyl (Fmoc) groups have been used as protection of the amino groups; carboxyl groups have been protected as aliphatic esters (methyl, ethyl or benzyl). The coupling reaction for the synthesis of these peptides used as nucleophiles has been carried out using usual methodologies: either by activation of the carbonyl group as an acid chloride, or by formation of active esters (for example pentafluorophenyl), or through the mixed anhydride, or by " in situ " activation of the carbonyl group (by treating the acid with a combination of carbodiimide and 1-hydroxybenzotriazole or related methods) ( Chemical Approaches to the Synthesis of Peptides and Proteins , CRC Press, Boca Raton, 1997).

An essential feature of the compounds of the present invention is that they are calpain inhibitors. There are several calpain isoforms, which are very similar structurally and, as far as is known, share the same mechanism of action. The two most abundant are micro-calpain (or calpain I) and milicalpaine (or calpain-II), which differ in in vitro tests, in the concentration of calcium necessary for activation. Since the two enzyme isoforms are very similar, it has been found in many examples in the literature that calpain inhibitors are of both isoenzymes ( Adv. Synth. Catal . 2002 , 344 , 855). Thus, in the present invention when we mention calpain, we refer to the two isoforms (or isoenzymes) that are included in the definition of calpain. Therefore, another object of the present invention is the use of a compound of formula I as a calpain inhibitor. The inhibitory capacity of calpain has been quantified by the IC50 value, which is defined as the concentration of inhibitor that halves the catalytic activity of an enzyme. The lower the IC 50 value, the more potent the inhibitor. Results of inhibition of calpain I (the most relevant from a physiological point of view) of some compounds of the present invention are indicated in table 1 and in figure 1. Since calpain II, also called mili-calpain, needs more amount of calcium for activation, may not have such a relevant physiological role; because said calcium concentration would cause cell death before the milli-calpain could be activated. For this reason, inhibition assays have been performed for calpain I, but are extrapolated for calpain II.

TABLE 1 Results of compound calpain inhibition objects of this invention

Compounds IC_ {50} one 64 nM 2 70nM 3 37nM 4 10nM 5 12nM 6 187nM 7 18nM 8 87 pM (= 0.087 nM)

Studies of the structure of the peptide-biphenyl hybrids of the present invention and its biological activity, which have shown that:

1) All the compounds object of the present invention, of generic formula I, are potent inhibitors of calpain I. As representative but not limiting examples, compounds 1-8 have IC 50 values between 0.087 and 187 nM.

2) Calpain inhibition of compounds 1-4 illustrate that biological activity is virtually independent of the nature of amino acids, of the length of the peptide chain and the substitution in the ends of the peptide chains.

3) When changing the orientation of the amino acids attached to the biphenyl system, are also obtained potent calpain I inhibitors. A representative example, although not limiting is compound 5.

4) Compounds that can be considered as peptide-biphenyl hybrids with side chains Truncated are also good calpain I inhibitors. Example Illustrative of these compounds are 6 and 7.

5) The combination in a structure of the features structural compounds 4, 5 and 7 to obtain the hybrid peptide-biphenyl 8 results in an inhibitor 115 times more powerful than the previous ones, having a value of IC 50 in picomolar scale, which is the most potent inhibitor of Calpain described so far.

6) It should also be noted that these compounds have been tested as papain inhibitors (another protease with cysteine) and trypsin (a protease with serine), and it has found that these compounds are inactive against these enzymes; what is an indication of the selectivity of our compounds, a relatively difficult goal to achieve in the area of protease inhibitors.

The peptide-biphenyl hybrids shown in Figure 1 are the most potent calpain inhibitors described, and mark a path for the design of therapeutically useful compounds. Because it has been found that calpain overactivation is implicated in numerous degenerative diseases (for example, cardiac ischemia, cataracts, and a variety of neurodegenerative diseases ( Brain Res. Rev 2003 , 42 , 169)), an additional object of The present invention is the use of a compound of formula I for the treatment or prevention of degenerative diseases as well as for preparing a medicament for the preventive or therapeutic treatment of a degenerative disease, and especially when the degenerative disease is selected from cerebral ischemia, ischemia Heart disease, Alzheimer's, Parkinson's, muscular dystrophy, cataracts and demyelinating diseases, and especially if the demyelinating disease is multiple sclerosis.

Brief description of the figure

Figure 1 shows results of the study of peptide-biphenyl hybrids, hybrids amino acid-biphenyl and hybrids objects amide-biphenyl objects of the present invention and its biological activity as calpain I inhibitors.

Examples

As illustrative, but not limiting, examples Experimental procedures are indicated, data spectroscopic and analytical of some hybrids peptide-biphenyl of formula I, as well as assays of their biological activity. In addition, the examples collected below are illustrative of the variety of methods used for the synthesis of the compounds object of this invention.

Example 1 ( S, S ) -3-Phenyl-2 - {[2 '- (2-phenyl-1-methoxycarbonyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -propionate methyl (1)

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8

On a solution of diphenic acid dichloride (2.94 g, 10.53 mmol) in anhydrous CH 2 Cl 2 (100 ml), under argon, Et 3 N (4.4 'was added ml, 31.6 mmol) and L- Phe-OMe hydrochloride (5 g, 23.17 mmol) in CH2Cl2 (100 ml). The mixture was stirred at room temperature for 24 hours after which it was treated with 5% aqueous HCl solution, saturated NaHCO3 solution, washed with brine and dried over anhydrous MgSO4. Evaporation of the solvent resulted in a residue that was purified by column chromatography to give the coupling product 1 (4.8 g, 81% rto.) As a white solid.

P.f._ {1} = 67-68 ° C., P.f._ {2} = 171-172 ° C (2 phases).

[α] D = + 34 (c = 1, CHCL 3).

1 H-NMR (300 MHz, CDCl 3, 30 ° C, mixture of A + a atropisomers in a ratio of 1.2: 1) δ: 7.48 (d, 1.1 H, J = 7.5 Hz, NH, A), 7'40-728 (ni, 6H, H mom., A + a; 0'9H, NH, a), 7'24 (m, 6H, H arom., A + a) , 7'11 (m, 4H, H amm., A + a), 7.02 (m, 2H, H arom., A + a), 4.76 (m, 2H, CHα, A + a), 3.61 (s, 2.8'H, CO 2 CH 3, a), 3.54 (s, 3.2'H, CO 2 CH 3, A), 2.96 (m, 2H, C (H? H?), A), 2.77 (m, 2H, C (H?, H?), A) ppm

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 8'76 (d, 2H, J = 7.8 MHz, 2-NH), 7.34 (m, 6H, H arom.), 7'19 (m, 6H, H amm.), 7'08 (m, 4H, H arom.), 6'91 (m, 2H, H arom.), 4.39 (m, 2H, CH?, 3.48 (s, 6H, COO CH 3), 2.81 (in, 2H, C ( H ? H?) Ph), 2 '65 (m, 2H, C (H? H ?) Ph) ppm.

13 C-NMR (75 MHz, CDCl 3, 30 ° C, mixture of conformers A + a) δ: 172 '1 (s, A), 171'7 (s, a), 169'3 (s, 2C, A + a), 139'0 (s, a), 138'5 (s, A), 136'l (s, 2C, A + a), 135'7 (s, A), 135'3 (s, a), 130'1 (d, a), 129'7 (d, A), 129'3 (d), 129'2 (d), 129'0 (d), 128'5 (d), 127'8 (d), 127'5 (d), 127'2 (d), 126'9 (d), 53'6 (d, 2C, A + a), 52'2 (d, 2C, A + a), 37'9 (t, A), 37.7 (t, a) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ 171'0 (s, 2C), 168'8 (s, 2C), 138'6 (s, 2C), 136'8 (s, 2C), 1352 (s, 2C), 129'1 (d, 4C), 128'7 (d, 6C), 128'128'4C), 127'2 (d, 28C), 126'4 (d, 2C), 53'7 (d, 2C), 51'7 (c, 2C), 36'5 (t, 2C) ppm.

IR nu 3435, 3293, 1737, 1641, 1557, 1435, 1335, 1266, 1218, 757, 700 cm -1.

MS (ES +) m / e = 565 ([MH] +, 100%).

AE Calculated for C 34 H 32 N 2 O 6: C, 72'32; H, 5.71; N, 4.96. Found: C, 72'37; H, 5.12; N, 4.96.

Example 2 ( S, S ) - 2 - {[2 '- (1-Carboxy-2-phenyl-elylcarbamoyl) -biphenyl-2-carbonyl] -amino} -3-phenyl-propionic acid (2)

9

On a solution of diester 1 (4 g, 7.08 mmol) in a THF: H2O 1: 1 mixture (80 ml), was added LiOH - H 2 O (654 mg, 15.58 mmol). The mixture was stirred for 6 hours, after which THF was removed under reduced pressure. TO It was then neutralized with 5% aqueous HCl solution until pH 2-3 2-3, extracted with AcOEt (3 times), washed with brine and dried over anhydrous MgSO4 obtaining the corresponding pure diacid 2 (4.2 g, 100% yield.). Solid white.

Mp = 130-131 ° C.

[α] D = +21 (c = 1, CHCl3).

1 H-NMR (300 MHz, CDCl 3, 40 ° C, mixture of confomers, 1.8: 1, A + a) δ: 7.26 (m, 12H, H arom., 2H, 2 -NH, A + a), 7'14-7'0 (m, 6H, H arom., A + a), 4.82 (m, 1'3H, CHα, A), 4 ' 65 (m, 0.7H, CH {\ alpha}, a), 3.13 (m, 1.3H, C (H {\ alpha} {H \ beta}), a), 2.97 (m , 0.7H, C ( H ? H?), A; 1.3 H, C (H? H ?), A), 2.70 (m, 0.7H, C H? H ?), A) ppm.

1 H-NMR (300 MHz, DMSO- d 6 , 50 ° C) δ: 8'58 (d, 2H, J = 8'OHz, 2-NH), 7.71 (m, 611, H arom.), 7'19 (ni, 6H, H arom.), 7'07 (dd, 4H, J_ {ortho} = 7'7Hz, J_ {meta} = 1.8 Hz, H arom.,) , 6'92 (d, 2H, J_ ortho = 6'8Hz, H arom.), 4.35 (m, 2H, CHα), 2.84 (m, 2H, C ( H_ {? H?), 2.73 (m, 2H, C (H? H ?) ppm.

13 C-NMR (75 MHz, CDCl 3, 40 ° C, mixture of conformers, 1'8: 1, A + a) δ: 175'9 (s, A), 175'3 (s, a, 170'9 (s, 2C, A + a), 139'1 (s), 136'3 (s), 135'2 (s), 134'9 (s), 130'1 (d), 129'5 (d), 129'2 (d), 128'7 (d), 128'1 (d), 127'3 (d), 127'2 (d), 54'0 (d, A), 53'9 (d, a), 37'6  (t, A), 37'3 (t, a) ppm.

IR \ nu: 3428, 3260, 1724, 1638, 1529, 1218 cm -1.

MS (ES +) m / e = 537 ([MH] +, 100%).

AE Calculated for C 32 H 28 N 2 O 6: C, 71.63; H, 5.26; N, 5.22. Found: C, 71'32; H, 4.97; N, 4.90.

Example 3 ( S, S ) -3- (1 H -indole-3-yl) -2 - ({2 '- [2- (1 H -indole-3-yl} 1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2- carbonyl} -amino) -methylpropionate (3)

10

On a solution of diphenic acid (1 g, 4.13 mmol) in 20 ml of anhydrous DMF, L- Trp-OMe hydrochloride (2.3 g, 9.08 mmol), BOP (4.02 g) was added sequentially , 9.08 mmol) and Et 3 N (1.7 mL, 12.38 mmol). The reaction mixture was kept stirring at room temperature for 14 hours. After this time, the solvent was removed under reduced pressure, the residue was dissolved in AcOEt and washed several times with a 5% HCl solution, with saturated NaHCO3 solution and with brine. The organic phase was dried over anhydrous MgSO4 and concentrated to give a residue whose purification by column chromatography gave rise to hybrid 3 as a white solid (0.05 g, 77% rto.).

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 10.72 (s, NH-Trp ring), 876 (d, 1H, J = 7.5, NH), 7'38-7'29 (m, 10H, H arom.), 7'05-6'91 (m, 8H, H arom.), 4.41 (m, 2H, C H α - Trp), 3.41 (s, 6H, CO 2 CH 3), 2.90 (m, 2H, C ( H ? H?) - Trp), 2 ' 75 (m, 2H, C (H? H ?) - Trp) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 171'5 (s), 169'0 (s), 139'1 (s), 136'1 (s), 135 '2 (s), 129'6 (d,), 127'3 (d), 126'9 (d), 123'5 (d), 121'0 (d), 118'4 (d), 117 '9 (d), 111'4 (d), 109'1 (d), 53.4 (d, C {\ alpha} -Trp), 51'9 (c, {2} C CO H {3 }), 26'9 (t, C H2 -Trp) ppm.

IR nu 3415, 3057, 2952.1737, 1640, 1524, 1458, 1438, 1341, 1222, 1097, 851, 744, 559 cm -1.

MS (ES +) m / e = 643 ([MH] +, 100%)

AE Calculated for C 38 H 34 N 4 O 6: C, 71.01; H, 5.33; N, 8.72. Found: C, 70'98; H, 5.31; N, 8'60.

Example 4 ( S, S, S ) -2- (2 - {[2 '- (1-Carboxy-2-phenyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -3-phenyl-propionylamino) -3-methyl -methylbutyrate (4) and ( S, S, S, S ) -2- [3-Phenyl-2 - ({2'-12-phenyl-1- (2-methyl-1-methoxycarbonyl-propylcarbamoyl)) - ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -propionylamino] -3-methyl-methyl-butyrate (4a)

On a solution of diacid 2 (1.26 g, 2.348 mmol) in anhydrous DMF (40 ml), under argon, L- ValOMe hydrochloride (358 mg, 2.13 mmol), HOBT was added sequentially (288 mg 2.13 mmol), Et3 N (0.45 ml, 3.20 mmol), EDC (409 mg, 2.13 mmol), and DMAP (26 mg, 0.213 mmol). The mixture was stirred for 19 hours after which it was treated with 5% aqueous HCl solution and extracted with AcOEt (3 times). The organic extracts were combined, washed with brine and dried over anhydrous MgSO4. By evaporation of the solvent a residue was obtained which was purified by column chromatography to give rise to the mono-coupling product 4 in the form of white solid (870 mg, 58% rto.) Together with the diaco-coupling product 4a (141 mg, 8% rto.), in the form of white solid.

( S, S, S ) -2- (2 - {[2 '- (1-Carboxy-2-phenyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -3-phenyl-propionylamino) -3-methyl -bu- methyl tylate (4)

eleven

1 H-NMR (400 MHz, DMSO- d 6 , 40 ° C) δ: 8'56 (d, 1H, J = 8'3, NH-Phe 1), 8'44 (d , 1H, J = 8'0, NH-Phe2), 8'07 (d, 1H, J = 8'3, NH-Val), 7'96 (d, 1H, J = 8'5, H arom.), 7'70 (d, 1H, J = 8'3, H arom.), 7'53 (t, 1H, J = 7'6, H atom.), 7'40 (t, 1H , J = 7'8, H atom.), 7'33-7'09 (m, 12H, H arom.), 6'88 (m, 211, H arom.), 4.59 (m, 1H, C H α -Phe 2), 4'33 (m, 1H, C H α -Phe 1), 4'12 (m, 1H, C H α -Val), 3.56 (s, 3H, CO 2 C H 3), 2.85 (m, 211, C ( H α H β) - H_ β) - Phe 1; C ( H ? H? -Phe2), 2.60 (m, 2H, C ( H ? H?) - Phe 1; C ( H ? H?) - Phe 2), 1.97 (m, 1H, C H (CH 3) 2 -Val), 0.82 (d, 3H , J = 6.5, C (CH 3) 2 Val),
0.80 (d, 311, J = 6.5, C (C H 3) 2 -Val) ppm.

13 C-NMR (75 MHz, DMSO- d 6 ) δ: 172'7 (s), 171'7 (s), 171'3 (s), 168'8 (s), 131 '7 (s), 168'6 (s), 142'9 (s), 138'5 (s), 137'7 (s), 137'6 (s), 137'4 (s), 135' 8 (s), 129'5 (d), 129'2 (d), 129'1 (d), 128'9 (d), 1282 (d), 128'0 (d), 127'8 (d ), 127'4 (d), 127'1 (d), 126'4 (d), 126'3 (d), 124'5 (d), 1192 (d), 109'6 (d), 57 '4 (d, C {? -Val), 53'7 (d, 2C, C ? -Phe1;
C α -Phe 2), 51'7 (c, CO 2 C H 3), 37'3 (t, C H 2 -Phe), 36'6 (t , C H 2 -Phe), 29'9 (d, C H (CH 3) 2 -Val), 18'9 (c, C H (QH 3) 2 -Val), 18'3 (c, CH (C C H3) 2 -Val).

IR nu 3413, 2963, 1740, 1640, 1535, 1438, 1358, 1215, 746, 700 ppm.

MS (ES +) m / e = 650 ([MH] +, 100%)

AE Calculated for C_ {38} H_ {39} N_ {O} {7}: C, 70.25; H, 6.05; N, 6'47 Found: C, 69'88; H, 6.40; N, 6'04

( S, S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2-phenyl-1- (2-methyl-1-methoxycarbonyl-propylcarbamoyl) -ethylcarbamoyl] -biphenyl-2- carbonyl} -amino) -propionylamino] -3-methyl-butyrate (4a).

12

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 8'58 (d, 2H, J = 8.5, NH-Phe), 822 (broad d, 2H, NH -Val), 7'42-7'07 (m, 16H, H arom.), 6'88 (m, 2H, H arom.), 4.59 (m, 2H, CHα -Phe) , 4.09 (m, 2H, H? -Val), 3.59 (s, 6H, CO 2 C H 3), 2.83 (m, 4H, C ( H _ {? H?) - Phe), 2.60 (m, 4H, C (H? H ?) - Phe), 1.97 (m, 2H, C H (CH 3) 2 -Val), 0.83 (d, 3H, J = 6.8, C (C H 3 ) 2 -Val), 0.80 (d, 3H, J = 6.8, C (C H 3) 2 Val) ppm.

13 C-NMR (75 MHz, DMSO- d 6 ) δ: 171'9 (s), 168'86 (s), 168'84 (s), 138'0 (s), 131 '5 (s), 129'7 (s), 129'5 (d), 129'3 (d), 128'7 (d), 128'3 (d), 127'4 (d), 127' 3 (d), 126'6 (d), 57'8 (d, Ca, Val), 54'1 (d, Cα -Phe), 52'0 (c, CO2 C H_ {3}, 377 (t, C H 2 -Phe), 30'3 (d, C H (CH 3) 2 -Val), 18'2 (c, CH (CH_ { 3) 2 -Val), 18.6 (c, CH ( C H 3) 2 -Val) ppm.

IR nu 3413, 3061, 2963.1743, 1642, 1535, 1437, 1267, 1206, 756, 700 cm -1.

MS (ES +) m / e = 763 ([MH] +, 100%).

AE Calculated for C 44 H 50 N 4 O 8: C, 69.27; H, 6.61; N, 7'34. Found: C, 68'99; H, 6.45; N, 7'12.

Example 5 ( S, S ) - (2-Phenyl-1- {2 '- [3-phenyl-2 (9 H -fluoren-9-ylmethoxycarbonylamino) -propionylamino] -biphenyl-2-ylcarbamoyl} -ethyl) - 9-carbamate H- Fluoren-9-ylmethyl (5)

13

On a 0 ° C solution of 2,2'-di (amino) -biphenyl (200 mg, 1.08 mmol) in 25 ml of anhydrous CH 2 Cl 2, Et 3 N (0 'was added 43 ml, 3.07 mmol) and L - N- Fmoc-phenylalanine chloride (685 mg, 1.68 mmol) in small portions. The reaction mixture was kept stirring at room temperature overnight. After this time, the solvent was removed under reduced pressure, and the resulting residue was purified by column chromatography to give product 5 (778 mg, 78% rto.) As a white solid.

[α] D = -35 (c = 0.5, CHCl3).

1 H-NMR (300 MHz, CDCl 3, 30 ° C, mixture of conformers A + a, 2: 1) δ: 7.77 (d, 1.33H, J = 7.88, H arom ., a), 7'74 (d, 2.66H, J = 73, H arom., A), 7.68 (s, 1.33H, NH arom., A), 7'48-7'36 (m, 9H, H arom., A + a; 0.66H, NH arom., a), 7'30-7'09 (m, 25H, H arom., A + a), 5.60 (d , 1'33H, J = 6'8, NH-Fmoc, A), 5'05 (broad d, 0.66H, NH-Fmoc, a), 4.41 (m, 1'33H, C H _ ? -Phe, A), 4.32 (m, 0.66H, C H ? -Phe, a; 0.66H, C ( H ? H?) - Fmoc, a), 4.24 (m, 1.33H, C ( H ? H?) - Fmoc, A), 04.04 (m, 2H, C H- Fmoc, A + a; 0.66H, C (H? H ?) - Fmoc, a), 3.93 (m, 1.33H, C (H? H ?) -Fmoc, A), 3.31 (m, 1.33H, C ( H ? H?) - Phe, A), 3.03 (m, 0.66H, C ( H _ {? H?) - Phe, a), 2.85 (m, 1.33H, C (H? H ?) - Phe, A; 0.33H, C (H? H ?) - Phe, a) ppm.

13 C-NMR (75 MHz, CDCl 3, mixture of 2: 1 conformers) 8: 170'1 (s, A), 169'7 (s, a), 156'3 (s, A ), 156'0 (s, a), 143'7 (s), 141'2 (s), 136'6 (s), 136'2 (s), 134'6 (s), 130'6 ( d), 130'3 (d), 129'6 (d), 129'2 (d), 129'1 (d), 128'9 (d), 128'7 (d), 127'7 (d ), 127'1 (d), 125'9 (d), 125'6 (d), 125'3 (d), 125'1 (d), 125'0 (d), 123'8 (d) , 123'3 (d), 119'9 (d), 67'3 (t, C H 2 -Fmoc, A + a), 56'8 (d, C α -Phe, a), 56'7 (d, C {\ alpha} -Phe, a), 46'9 (d, C H-Fmoc, a + a), 37'6 (t, C H {2} -Phe , a), 37'4 (t, C H2 -Phe, A) ppm.

IR nu 3410, 3063, 1696, 1585, 1516, 1450, 1246, 1049, 758, 740, 700 cm -1.

MS (ES +) m / e = 443 ([MH] +, 100%).

AE Calculated for C 60 H 50 N 4 O 6: C, 78.07; H, 5.46; N, 6.07. Found: C, 77.85; H, 5.60; N, 6.03.

Example 6 Bis 2,2 '- [(4-nitro-benzoyl) amino] -biphenyl (6)

14

On a solution at 0 ° C of 2,2'-di (amino) -biphenyl (100 mg, 0.54 mmol) in 10 ml of anhydrous CH2Cl2, was added Et 3 N (0.05 ml, 1.08 mmol) and 4- nitrobenzoyl chloride (221.5 mg, 1.19 mmol) in small portions. Reaction mixture It kept stirring at room temperature overnight. After this time the solvent was removed under pressure reduced and the residue was purified by column chromatography to give rise to the diacoplating product 6 (261 mg, 77% rto.).

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 10.04 (s, 2H, NH), 8.25 (d, 4H, J = 8'8, H- 10), 7'86 (d, 4H, J = 9'0, H-9), 7'62 (dd, 2H, J_ {1,2} = 7'8, J_ {1,3} = 0 ' 7, H-1), 7'43 (dt, 2H, J_ {2-1,3} = 7'6, J_ {2,4} = 1 /, H-2), 7'32 (dt, 2H , J_ {3- 2,4} = 7'6, J_ {3,1} = 7'6, H-3), 7'25 (dd, 2H, J_ {4,3} = 7'6, J_ {4.2} = 1.5, H-4) ppm.

13 C-NMR (50 MHz, DIVISO- d 6 ) δ: 164'9 (s), 149'9 (s), 140'5 (s), 135'9 (s), 135 '57 (s), 131'1 (d,), 129'4 (d), 129'1 (d), 127'1 (d), 127'0 (s), 124'3 (d) ppm.

MS (ES +) m / e = 505 ([MNa] +, 100%).

AE Calculated for C 26 H 18 N 4 O 6: C, 64.73; H, 3.76; N, 11.61. Found: C, 64'80; H, 3.90; N, 11.74.

Example 7 2'-Amino-2- [2- (naphthoyl) amino] biphenyl (7)

fifteen

On a solution at 0 ° C of the 2,2'-di (amino) -biphenyl (500 mg, 2.71 mmol) in 50 ml of anhydrous CH2Cl2, was added Et 3 N (0.14 ml, 1.02 mmol) and naphthoyl chloride (129.3 mg, 0.68 mmol) in small portions. The reaction mixture was maintained. stirring at room temperature for 13 hours. After this time, the solvent was removed under reduced pressure, and purified the resulting residue by column chromatography to give rise to the mono-coupling product 7 (144 mg, 63% rto.) together with the diacoplating product 23 (38 mg, 11% rto.), both in the form of white solid The analytical and spectroscopic data of Compound 7 are indicated below.

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 9'91 (s, 1H, NH), 8.29 (s, 1H, H-9), 7'98 (m, 3H, H-1, H-10, H-14), 7'91 (d, 1H, J = 8'0, H -15), 7'81 (dd, 1H, J_ {13.12 } = 8'5, J_ {13,11} = 1'7, H-13), 7'61 (m, 2H, H-4, H-12), 7'45 (dt, 1H, J_ {11 -10.12} = 7'1, J_ {11,13} = 2'2, H-11), 7'34 (m, 2H, H-2, H-3), 7'13 (dt, 1H , J_ {6-5.7} = 8'0, J_ {6.8} = 1'4, H-6), 7.05 (dd, 1H, J_ {8.7} = 7'6, J_ {8.6} = 1'4, H-8), 6'94 (dd, 1H, J_ {5,6} = 8'0, J_ {5,7} = 0'97, H-5), 6.72 (dt, 1H, J_ 7-6.8 = 7.3, J 7.5 = 1.2, H-7) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 165'5 (s), 145'4 (s), 136'3 (s), 134'9 (s), 134 '0 (s), 132'8 (s), 132'7 (s), 131'8 (d), 131'6 (d), 129'5 (s), 129'4 (d), 128' 9 (d), 128'6 (d), 128'4 (d), 128'3 (d), 128'1 (d), 127'6 (d), 126'2 (d), 125'4 (d), 125'1 (s), 124'4 (d), 118'5 (d), 116'6 (d) ppm.

MS (ES +) m / e = 339 ([MH] +, 100%)

AE Calculated for C 23 H 18 N 2 O: C, 81.63; H, 5.36; N, 8.28. Found: C, 81.50; H, 5.59; N, 8.41.

Example 8 ( S, S, S ) -2- [2 - ({2'-Amino-biphenyl-2-ylcarbamoyl) -2-phenyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -3-phenyl-propionyl- methyl amino] -3-methyl-butyrate (8)

16

About a dissolution of 2,2'-di (amino) -biphenyl (156 mg, 0.846 mmol) in anhydrous DMF (20 ml), under argon, acid 4 (500 mg, 0.77 mmol), HOBT was added sequentially (104 mg, 0.77 mmol), Et 3 N (0.16 ml, 1.15 mmol), EDC (147 mg, 0.07 mmol), and DMAP (9.5 mg, 0.077 mmol). The mixture was stirred for 15 hours after which it was treated with aqueous HCl solution at 5% and extracted with CH2Cl2 (3 times). Extracts organics were combined, washed with brine and dried over MgSO4 anhydrous. The solvent was evaporated to give rise to a residue that was purified by column chromatography. He coupling product 8 was obtained as a white solid (355 mg, 74% rto.) Together with 48 mg of the starting diamine.

1 H-NMR (300 MHz, DMSO- d 6 , 50 ° C) δ: 8'94 (wide s, 1H, NH-Bif.), 8'54 (wide d, 1H, M Phe_ { 1}), 8'50 (d, 1H, J = 8'8, NH-Phe2), 8'09 (d, 1H, J = 7'3, NH-Val), 7'56 (d , 1H, J = 7'8, H arom 7'40-7'84 (m, 21H, H arom.), 6'88 (m, 2H, H arum.), 6'77 (d, 1H, J = 7'8, H arom.), 6'65 (t, 1H, J 7.6, H arom ..), 4.60 (m, 1H, C H α -Phe 2) , 4.41 (m, 3H, C H α -Phe 1; NH 2 -Bif.), 4.11 (m, 1H, C H α -Val), 3.60 (s, 3H, CO 2 C H 3), 2.87 (m, 4H, C H ?, H?) - Phe 1; C H α, H β) - Phe 2), 2'63 (m, 4H, C (H α, H β) - Phe 2), 1 '98 (m, 1H, C H (CH 3) 2 -Val), 0.84 (d, 3H, J = 6.8, C (C H 3) 2 -val), 0.82 (d, 3H, J 6'8, C (C H 3} {) _ {2} -Val) ppm.

13 C-NMR (75 MHz, DMSO- d 6 , 50 ° C) δ: 171'4 (s), 168'6 (s), 168.5 (s), 168'42 (s) , 168'40 (s), 144 (s), 138'2 (s), 138'1 (s), 137'5 (s), 137'4 (s), 135'0 (s), 130 ' 7 (d), 130'5 (d), 129'3 (d), 129'2 (d), 129'0 (d), 128'8 (d), 128'4 (d), 127'9 (d), 127'8 (d), 127'3 (d), 126'9 (d), 126'8 (d), 126'1 (d), 117'0 (d), 115 (d) , 57'4 (d, C \ alpha-Val), 54'8 (d, C \ alpha-Phe_ {1}), 53'6 (d, C \ alpha-Phe_ {2}), 51.5 ( c, CO 2, C H 3), 37'2 (t, C H 2 -Phe), 36'2 (t, C H 2 -Phe), 29'8 (d, C H (CH 3) 2 -Val), 18'7 (c, CH (C C H 3) 2 -Val), 18'1 (c, CH ( C H_ { 3) 2 -Val) ppm.

IR nu: 3436, 2962, 1742, 1643, 1533, 1442, 1307, 1208, 1155, 754, 700 cm -1.

MS (ES +) m / e = 816 ([MH] +, 100%)

AE Calculated for C 50 H 49 N 5 O 6: C, 73'60; H, 6.05; N, 8'58 Found: C, 73.51; H, 6.20; N, 8'39

Example 9 ( S, S, S, S ) -3- (1 H -Indol-3-yl) -2- {3-phenyl-2 - [(2 '- {2-phenyl-1- [2- (1 H -indole-3-yl) -1-methoxycarbonyl-ethylearbamoyl] -ethylcarbamoyl} -biphenyl-2-carbonyl) -amino] propionylamino} -methyl propionate (9)

17

On a solution of diacid 2 (500 mg, 0.93 mmol) in anhydrous DMF (8 ml), under argon, L- Trp-OMe hydrochloride (523 mg, 0.05 mmol), HOBT was added sequentially (276 mg 2.05 mmol), Et 3 N (0.5 ml, 3.02 mmol), EDC (392 mg, 2.05 mmol), and DMAP (23 mg, 0.18 mmol). The mixture was stirred for 48 hours after which it was treated with 5% aqueous HCl solution and extracted with AcOEt (3 times). The organic extracts were combined, washed with brine and dried over anhydrous MgSO4. By evaporation of the solvent a residue was obtained which was purified by column chromatograse to give the product 9 as a white solid (612 mg, 70% rto.).

Mp. 1 = 115-116 ° C.

1 H-NMR (400 MHz, CDCl 3, 30 ° C, mixture of A + a conformers, 0.93: 1'07) δ: 8'90 (wide s, 0.93H, NH-Trp, a ), 8'59 (wide s, 1'07H, NH-Tip, A), 8'50 (d, 0'93H H, J = 7'8 Hz, NH-Phe, a), 7'65 (d , 1'07H, J = 7'8 Hz, NH-Phe, A), 7'48 (d, 1'07H, J = 78 Hz, H arom., A), 7'36-6'97 (m , 24H, H arom., A + a; d, 0.93H, H arom., A;), 6.96 (d, 0.93H, J = 2.2 Hz, CH-Tip, A), 6 '64 (d, 1'07H, J = 2'2 Hz, CH-Trp, a), 6'43 (d, 0'93H H, J = 7'8 Hz, NH-Trp, A), 6 ' 37 (d, 1.07H, J = 7.3 Hz, NH-Tip, a), 4.75 (m, 0.93H, CHα-Trp, a), 4.61 (m, 1 '07H, CH? -Trp, a), 4.49 (m, 2H, CH? -Phe, A + a), 3.57 (s, 3.21H, CO CH 3 , A), 3.52 (s, 2.79H, CO 2 CH 3, a), 3.30 (m, 1.07H, C ( H α -H \ \ beta Trp, A), 3.18 (m, 0.93H, C ( H ? -H?) - Trp, a), 3.11-2'91 (m, 2H, C (H {\ alpha} - H {\ beta}) - Trp, a + a; 2H, C (H {\ _ {alpha} -H \ beta}) - Phe, a + a; 0'93H, C (H? - H ?) - Phe, a), 2.65 (m, 1.07H, C (H? - H ?) - Phe, A ) ppm.

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 10.76 (broad s, 2H, NH-ring Trp), 8.53 (d, 2H, J = 8 ' 3Hz, NH-Phe), 8'29 (wide d, 2H, J = 5'8Hz, NH-Trp), 7'47 (d, 2H, J = 8'OHz, H arom.), 7'34 ( d, 2H, J = 8'OHz, H arom.), 7'19-6'97 (m, 22H, H arom.), 6'88 (m, 2H, H arom.), 4.56 (m , 2H, CH? -Phe), 4.47 (m, 2H, CH? -Trp), 3.51 (s, 6H, CO 2 CH 3), 3 ' 06 (m, 4H, CH 2 -Trp), 2.87 (m, 2H, C ( H ? -H?) - Phe), 2.61 (m, 2H, C (H {\ alpha} - H {\ beta}) - Phe) ppm.

13 C-NMR (50 MHz, CDCl 3, mixture of A + a, 3: 2) conformers: 171'7 (s, a), 171'4 (s, A), 171 ' 1 (s, A), 170'5 (s, a), 170'3 (s, a), 170'2 (s, A), 138'9 (s), 138'2 (s), 136 ' 8 (s), 136'4 (s), 136'2 (s), 136'1 (s), 135'0 (s), 130'1 (d), 129'9 (d), 129'8 (d), 129'2 (d), 128'9 (d), 28'6 (d), 128'5 (d), 128'0 (d), 127'45 (d), 127'40 ( d), 127'36 (d), 127'3 (d), 126'7 (d), 126'8 (d), 126'4 (d), 124'6 (d, A), 124'4 (d, a), 121'9 (d, a), 121'7 (d, A), 119'4 (d, a), 119'2 (d, A), 118'4 (d, A) , 118'2 (d, a), 111'4 (d, a), 111'3 (d, A), 108'7 (s, A), 108'1 (s, a), 55'6 ( C? -Phe, A), 55'1 (C? -Phe, a), 52'7 (C? -Trp, a), 52'5 (C? Trp, A), 52'3 (CO 2 CH 3, a), 52'2 (CO 2 CH 3, A), 37'5 ( CH 2 -Phe , a), 37 '1 ( CH 2 -Phe, A), 27'4 ( CH 2 -Trp, A + a) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 171'9 (s, 2C), 171'2 (s, 2C), 168'5 (CO-Biph, 2C), 137'9 (s, 2C), 138'0 (s, 2C), 137'5 (s, 2C), 136'15 (s, 2C), 136'16 (d, 2C), 136'0 (d , 2C), 129'3 (d, 2C), 129'1 (d, 2C), 128'9 (d, 2C), 127'8 (d, 2C), 127'0 (d, 2C), 126 '7 (d, 2C), 126'6 (d, 2C), 126'0 (d, 2C), 123'7 (d, 2C), 120'8 (d, 2C), 118'2 (d, 2C), 117'8 (d, 2C), 112'2 (d, 2C), 108'8 (s, 2C), 53'8 (Cα, - Phe, 2C), 53'0 ( Cα -Trp, 2C), 51.6 (CO 7 7 CH 3, 2C), 37'2 ( CH 2 -Me, 2C), 26'8 ( CH _ {2}, - Trp, 2C) ppm.

IR \ nu: 3435, 1739, 1641, 1533, 1439, 743 cm -1.

MS (ES +) m / e = 937 ([MH] +, 100%)

AE Calculated for C_ {56} H_ {52} N6 O_ {8}: C, 71.78; H, 5.59; N, 8'97. Found: C, 71.58; H, 5.70; N, 8'91.

Example 10 ( S ) -2 '- [2- (4-Hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoill-biphenyl-2-carboxylic acid (10)

18

On a solution of 2,2'-diphenic anhydride (1 g, 4.46 mmol) in 12.5 ml of anhydrous DMF was added dropwise, a solution of L- Tyr-OMe hydrochloride (1.13 g, 4.90 mmol) and 2,4,6-collidine (0.65 ml, 4.90 mmol) in 12.5 ml of DMF. The reaction mixture was kept stirring at room temperature overnight. After this time, the solvent was removed under reduced pressure, the residue was dissolved in AcOEt and washed several times with a 5% HCl solution. The organic phase was dried over anhydrous MgSO4 and concentrated to give a residue whose purification by column chromatography gave the acid 10 as a white solid (1.8 g, 99% rto.).

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 12'71 (broad s, 1H, CO 2 H), 9'15 (s, 1H, OH-Tyr ), 8'03 (d, 1H, J = 7'9, NH-Tyr), 7'79 (m, 1H, H arom.), 7'42 (m, 5H, H arom.), 7'10 (m, 1H, H arom.), 7'04 (m, 1H, H arom.), 6'86 (d, 2H, J = 8'4, H arom.-Tyr), 6'65 (d, 2H, J = 8'6, H arom.-Tyr), 4.35 (m, 1H, CHA Tyr), 3.50 (s, 3H, CO 2 CH 3), 2.70 ( m, 2H, CH2 -Tyr) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 171'5 (s), 168'8 (s), 168'2 (s), 156'0 (s), 140 '7 (s), 139'8 (s), 134'9 (s), 131'4 (s), 130'7 (d), 130'3 (d), 129'8 (d), 129' 3 (d), 127'4 (d), 127'3 (d), 127'0 (d), 126'9 (d), 115'1 (d), 54'0 (d, Ca-Tyr) , 51'8 (c, CO 2 CH 3), 35'9 (t, CH 2 -Tyr) ppm.

IR nu: 3420, 3060, 1720, 1640, 1516, 1439, 1226, 759 cm -1.

MS (ES +) m / e = 420 ([MH] +, 100%)

AE Calculated for C 24 H 21 NO 6: C, 68.73; H, 5.05; N, 3.34. Found: C, 68.44; H, 5.30; N, 3.49.

Example 11 ( S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) - methyl propionylamino] -4-methyl-pentanoate (11)

19

On a solution of the acid 10 (400 mg, 0.95 mmol) in anhydrous DMF (6 ml), under argon, the trifluoroacetic salt of L- Phe- L- Leu-OMe (503.8 mg) was added sequentially , 1.24 mmol), HOBT (167.5 mg, 1.24 mmol), Et 3 N (0.2 ml, 1.43 mmol), EDC (237.6 mg, 1.24 mmol) and DMAP (11.6 mg, 0.095 mmol). The mixture was stirred for 22 hours after which the solvent was evaporated, treated with 5% aqueous HCl solution, saturated NaHCO3 solution and extracted with ACOEt (3 times). The organic extracts were combined, washed with brine and dried over anhydrous MgSO4. The solvent was evaporated to yield a residue that was purified by column chromatography to give the coupling product 11 as a white solid (548 mg, 90% rto.) Ppm.

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 9'14 (s, 1H, OH-Tyr), 8.66 (d, 1H, J = 7'8, NH-Tyr), 8'50 (d, 1H, J = 8'3, NH-Phe), 8'20 (d, 1H, J = 7'8, NH-Leu), 7'41-7'28 (m, 6H, H arom.), 7'25-7'18 (m, 7H, H arom.), 6'91 (m, 2H, H arom.), 6'92 (d, 2H, J = 8'0, H arom.-Tyr), 6'63 (d, 2H, J = 8'5, H arom.-Tyr), 4.54 (m, 1H, C H ? Phe), 4.27 (m, 2H, C H ? Tyr; C H ? -Leu), 3.60 (s, 3H, CO 2 CH 3), 3.42 (s, 3H, CO 2 CH 3), 2.89 (m, 1H, C ( H ? H?) - Phe), 2.66 (m, 2H, C (H? H ?) - Phe; C ( H ? H?) - Tyr), 2.47 (m, 1H, C (H?) H ?) - Tyr), 1.60 (m, 1H, C H -Leu), 1.50 (m, 2H, CH2- Leu), 0.86 (d, 3H, J = 6'1, CH 3 -Leu), 0.82 (d, 3H, J = 6'1, CH 3 -Leu) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 172'4 (s), 171'1 (s), 170'7 (s), 168'8 (s), 168 '3 (s), 155'8 (s), 138'4 (s), 137'4 (s), 135'55 (s), 135'5 (s), 129'6 (d), 129' 2 (d), 128'8 (d), 127'8 (d), 127'1 (d), 127'0 (d), 126'8 (d), 126'0 (d), 114'9 (d), 54'1 (d, C? -Tyr), 53'7 (d, C? Phe), 51'6 (c, CO 2 C H 3), ), 51'5 (c, CO 2 C H 3), 50'1 (d, C α -Leu), 40'2 (t, C H 2 -Leu), 37 '0 (t, C H 2 -Phe), 35'9 (t, C H 2 -Tyr), 24'0 (d, C H (CH 3) - Leu), 22'5 (t, C H 3 -Leu),), 22'2 (t, C H 3 -Leu) ppm.

IR nu: 3428, 2955, 1743, 1642, 1517, 1438, 1367, 1224, 757, 700, 542 cm -1.

MS (ES +) m / e = 694 ([MH] +, 100%)

AE Calculated for C 40 H 43 N 3 O 8: C, 69.25; H, 6.25; N, 6.06. Found: C, 69'32; H, 6.40; N, 6'14.

Example 12 ( S, S, S ) -2 - ({2 '- [2-phenyl-1- (3-methyl-1-methylcarbamoyl-butylcarbamoyl) -ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -3- ( 4- methyl hydroxyphenyl) -propionate (12)

twenty

On a solution of the acid 10 (160 mg, 0.38 mmol) in anhydrous DMF (3.5 ml), under an argon atmosphere, the salt was trifluoroacetic of L- Phe- L- Leu-NHMe (201 mg). , 0.49 mmol), HOBT (67 mg, 0.49 mmol), Et 3 N (0.08 ml, 0.57 mmol), EDC (95 mg, 0.49 mmol), and DMAP (4 '6 mg, 0.038 mmol). The mixture was stirred for 23 hours after which it was treated with 5% aqueous HCl solution and extracted with CH2Cl2 (3 times). The organic extracts were combined, washed with brine and dried over anhydrous MgSO4. The solvent was evaporated to give a residue that was purified by column chromatography. Coupling product 12 was obtained as a white solid (234 mg, 89% rto.).

[α] D = -31 (MeOH, c = 0.5).

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 9'15 (broad s, 1H, OH-Tyr), 8'68 (d, 1H, J = 7'7 , NH-Tyr), 8'55 (d, 1H, J = 8'0, NH-Phe), 7'85 (d, 1H, J = 8'4, NH-Leu), 7'51 (c, 1H, J = 47, NH -CH3), 7'41-7'16 (m, 11H, H arom.), 6'93 (m, 2H, H arom.), 6'85 (d, 2H, J = 8'0, H arom.-Tyr), 6'62 (d, 2H, J = 8'4, H arom.-Tyr), 4.47 (m, 1H, CHα - Phe), 4.27 (m, 1H, C H ? -Tyr), 4.21 (m, 1H, C H ? -Leu), 3.43 (s, 3H, CO_ {2} C {3} H), 2.89 (m, 1H, C (H {\ _ {alpha} -H \ beta}) - Phe), 2'65 (m, 2H, C (H { α- H ?) - Phe; C ( H ? -H?) -Tyr), 2.54 (d, 3H, J = 4.6, NH-C {3} H), 2.40 (m, 1H, C (H {\ alpha} - H {\ beta}) - Tyr), 1.50 (m, 1H, C H -Leu), 1 ' 40 (m, 2H, C H 2 -Leu), 0.85 (d, 3H, J = 6'4, C H 3 -Leu), 0.81 (d, 3H, J = 6'4, C H 3 -Leu) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 172'0 (s), 171'2 (s), 170'3 (s), 169'0 (s), 169 '0 (s), 168'6 (s), 156'0 (s), 138'6 (s), 137'6 (s), 137'5 (s), 135'5 (s), 135' 4 (s), 129'8 (d), 128'4 (d), 129'1 (d), 129'0 (d), 128'0 (d), 127'2 (d), 127'1 (d), 127'0 (d), 126'2 (d), 54.3 (d, C - Phe), 54.3 (d, C {\ alpha} Tyr), 51'7 (c, CO 2 C H 3), 51'0 (d, C -Leu), 41'1 (t, C H 2 -Leu), 37'0 (t, C H 2 -Phe ), 36'0 (t, C H 2 -Tyr), 25'6 (c, NH- C H 3), 24'1 (d, C H-Leu), 23'0 (c, C H 3 -Leu), 21.6 (c, C H 3 -Leu) ppm.

IR nu: 3411, 2955, 1743, 1644, 1516, 1438, 1229, 756 cm -1.

MS (ES +) m / e = 693 ([MH] +, 100%)

AE Calculated for C 40 H 44 N 4 O 7: C, 69'35; H, 6.40; N, 8'09. Found: C, 69'40; H, 6.50; N, 8'31.

Example 13 ( S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) - methyl propionylamino] -4-methylsulfanyl-butyrate (13)

twenty-one

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On a solution of the acid 10 (160 mg, 0.38 mmol) in anhydrous DMF (3.5 ml), under argon, the salt was trifluoroacetic L- Pe- L- Met-OMe (210 mg, 0.49 mmol), HOBT (67 mg, 0.49 mmol), Et 3 N (0.08 ml, 0.57 mmol), EDC (95 mg, 0.49 mmol) and DMAP (4.6 mg, 0.038 mmol). The mixture was stirred for 20 hours after which the solvent was evaporated, treated with 5% aqueous HCl solution and extracted with AcOEt (3 times). The organic extracts were combined, washed with brine and dried over anhydrous MgSO4. The solvent was evaporated to give a residue that was purified by column chromatography. Coupling product 13 was obtained as a white solid (255 mg, 94% rto.)

[α] D = -32 (MeOH, c = 0.47).

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 9'15 (s, 1H, OH-Tyr), 8.66 (d, 1H, J = 7.5 ', NH-Tyr), 8'53 (d, 1H, J = 8'4, NH-Phe), 7'26 (d, 1H, J = 7'7, NH-Met), 7'39-7'28 (m, 6H, H arom.),), 7'23-7'17 (m, 5H, H arom.), 6'92 (m, 2H, H arom.), 6'86 (d, 2H, J = 7'9, H arom.-Tyr), 6'63 (d, 2H, J = 8'6, H arom.-Tyr), 4.51 (m, 1H, CH {\ alpha} -Phe ), 4.35 (m, 1H, CH ? -Met), 4.27 (m, 1H, CH ? -Tyr), 3.60 (s, 3H, CO2) CH 3 -Met), 3.43 (s, 3H, CO 2 CH 3 -Tyr), 2.87 (m, 11H, C ( H ? H?? ) - Phe), 2.65 (m, 2H, C (H? H ?) - Phe; C ( H ? H?) - Tyr), 2 '50 (m, 1H, C (H? H ?) - Tyr), 2.40 (m, 2H, CH2 CH2 -Met), 2.01 (s, 3H, S CH 3), 1.92 (m, 1H, CH 2 C (H? H ?) SCH 3), 1.83 (m, 1H, CH 2 C (H? H ?) SCH 3) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 171'8 (s), 171'2 (s), 170'9 (s), 170'8 (s), 168 '8 (s), 155'9 (s), 138'5 (s), 137'5 (s), 135'5 (s), 135'49 (s), 129'7 (d), 129' 2 (d), 129'0 (d), 128'9 (d), 127'9 (d), 127'2 (d), 127'1 (d), 126'8 (d), 126'2 (d), 54'1 (d, C? -Tyr), 53'8 (d, C? -Phe), 51'8 (c, CO 2 CH 3 - Met), 51.6 (c, CO 2 CH 3 -Tyr), 50'8 (d, C α -Met), 37'1 (t, CH 2 -Phe) , 36'0 (t, CH {2} -Tyr), 30'5 (t, CH 2} {2} {CH CH, SCH {3}), 29'4 (t, CH 2} {CH {2} SCH 3, 14.5 (c, SCH 3) ppm.

IR nu: 3414, 3054, 1741, 1642, 1516, 1438, 1224, 757 cm -1.

MS (ES +) m / e = 712 ([MH] +, 100%)

AE Calculated for C 40 H 44 N 4 O 7: C, 65.81; H, 5.81; N, 5.90; S, 4.50. Found: C, 66.02; H, 6.10; N, 5.82; S, 4.61.

Example 14 2 '- {(S) - [2-Phenyl-1- ( R ) - (2-phenyl-1-methylcarbamoyl-ethylcarbamoyl) -ethylcarbamoyl]} - biphenyl-2-carboxylic acid (14)

22

On a solution of 2,2'-diphenic anhydride (200 mg, 0.89 mmol) in 4 ml of anhydrous DMF, a solution of the trifluoroacetic salt of the dipeptide L -Phe- D -Phe-NH was added dropwise -Me (431 mg, 0.98 mmol) and 2,4,6-collidine (0.13 ml, 0.98 mmol) in 4 ml of DMF. The reaction mixture was kept stirring at room temperature overnight. After this time, the solvent was removed under reduced pressure, the residue was dissolved in AcOEt and washed several times with a 5% HCl solution. The organic phase was dried over anhydrous MgSO4 and concentrated to give a residue whose purification by column chromatography resulted in acid 14 as a white solid (552 mg, 90% rto.).

[α] D = +21 (MeOH, c = 0.51).

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 8.80 (broad s, 1H, CO 2 H), 8.12 (d, 2H, J = 8 '5, NH-Phe), 7'85 (c, 1H, J = 4'1, NH- CH3 ), 7'64 (m, 1H, H arom.),), 7'63-7 '02 (m, 14H, H arom.), 6.90 (m, 3H, H arom.), 4.33 (m, 2H, CH? -Phe; CH? Phe), 2 '94 (m, 21H, C ( H ? H?) - Phe; C ( H ?, H?) - Phe), 2.66 (m, 2H , C (H? H ?) - Phe; C (H? H ?) - Phe), 2.57 (d, 311, J = 4'4, NH- CH 3) ppm.

13 C-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 170'9 (s), 168'4 (s), 140'2 (s), 137'8 (s) , 137'5 (s), 135'4 (s), 129'5 (d), 128'9 (d), 128'8 (d), 128'7 (d), 128'3 (d), 127'9 (d), 127'8 (d), 127'2 (d), 126'9 (d), 126'8 (d), 126.5 (d), 126'0 (d), 125 '9 (d), 54'5 (d, Cα -Phe), 54'0 (d, Cα -Phe), 37'4 (t, CH2 -Phe), 37.0 (t, C112-Phe), 25.6 (c, NH- CH3 ) ppm.

IR nu: 3420, 3060, 1743, 1533, 1241, 755, 700 cm -1.

MS (ES +) m / e = 550 ([MH] +, 100%)

Example 15 ( S ) -2 - [(S) -2 - ({2 '- ( S ) - [2-Phenyl-1- ( R ) - (2-phenyl-1-methylcarbamoyl-ethylcarbamoyl) -ethylcarbamoyl] -biphenyl- Methyl 2-carbonyl} - amino) -4-methyl-pentanoylamino] -4-methylsulfanyl-butyrate (15)

2. 3

On a solution of acid 14 (350 mg, 0.64 mmol) in anhydrous DMF (3.5 ml), under argon, the salt was trifluoroaceticly added L -Leu- L- Met-OMe (323mg, 0.83 mmol), HOBT (112 mg, 0.83 mmol), Et 3 N (0.13 mL, 0.95 mmol), EDC (159 mg, 0.83 mmol) and DMAP (8 mg, 0.064 mmol). The mixture was stirred for 20 hours. After this time the solvent was removed, treated with 5% aqueous HCl solution and extracted with CH2Cl2 (3 times). The organic extracts were combined, washed with brine and dried over anhydrous MgSO4. The solvent was evaporated to give a residue that was purified by column chromatography to give the coupling product 15 as a white solid (407 mg, 79% rto.).

[α] D = -11 (MeOH, c = 0'29).

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 8.68 (d, 3H, NH-Phe; NH-Leu; NH-Met), 7.67 (c, 1H, J = 4'7, NH- CH3 ), 7'38-7'13 (m, 16H, H arom.), 6'98 (m, 2H, H arom.), 4.41 (m, 2H, CH? Phe1; CH? Phe2), 4.30 (m, 1H, CH? -Met), 4.17 (m, 1H , C (H? -Leu), 3.59 (s, 3H, CO 2 CH 3), 2.99 (m, 1H, C ( H ? H_ { β) - Phe 1), 2.67 (m, 2H, C (H? H ?) - Phe 1; C ( H ? H? \ beta) - Phe2), 2.55 (d, 3H, J = 4.6, NH- CH3 ), 2.40 (m, 3H, C (H? H _ {\ beta}) - {2} Phe_; CH {CH 2} {2} CH, SCH {3}), 2.03 (s, 3H, S CH {3}), 1.86 (m, 2H, CH 2 CH 2 SCH 3) 11.24 (m, 2H, CH 2 -Leu), 0.87 (m, 1H, CH-Leu) ppm.

13 C-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 172'1 (s), 172'0 (s), 170'9 (s), 168'4 (s) , 138'3 (s), 137'8 (s), 137'7 (s), 136'2 (s), 135'6 (s), 129'5 (d), 129'2 (d), 128'0 (d), 127'1 (d), 126'2 (d), 54'4 (d, C {\ alpha} -Phe), 54'0 (d C {\ alpha} -Phe ), 51'8 (c, CO 2 C H 3), 50'9 (c, C α -Leu), 50'8 (c, C α -Met) , 43'9 (t, C H 2 -Leu), 38'3 (t, C H 2 -Phe 1), 37'8 (t, C H 2 -Phe_ {2} ), 30'4 (t, C H 2 CH 2 -Met), 29'5 (t, CH 2 C H 2 -Met), 25'6 (c, NH- C H_ {3}, 23'0 (d, C H-Leu), 21'4 (c, 2C, C H3 -Leu), 14'6 (c, C H3, Met) ppm.

IR nu: 3429, 3062, 2956.1743, 1642, 1546, 1438, 755, 700 cm -1.

MS (ES +) m / e = 808 ([MH] +, 100%)

Example 16 ( S ) -2 '- [2-Phenyl-1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carboxylic acid (16)

24

On a solution of 2,2'-diphenic anhydride (500 mg, 2,23 mmol) in 5 ml of anhydrous DMF, a solution of L- Phe-OMe hydrochloride (529 mg, 2.45 mmol) was added dropwise ) and 2,4,6-collidine (0.32 ml, 2.45 mmol) in 5 ml of DMF. The reaction mixture was kept stirring at room temperature overnight. After this time, the solvent was removed under reduced pressure, the residue was dissolved in AcOEt and washed several times with a 5% HCl solution. The organic phase was dried over anhydrous MgSO 4 and concentrated to give a residue whose purification by column chromatography gave acid 16 as a white solid (780 mg, 87% rto.).

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 8.22 (d, 1H, J = 7.8, NH-Phe), 7.78 (m, 1H, H arom.), 7'39 (m, 5H,
H arom.), 7'23 (m, 3H, H arom.), 7'10 (m, 3H, H arom.), 7'0 (m, 1H, H arom.), 4.42 (m, 1H, C H ? -Phe), 3.50 (s, 3H, CO 2 CH 3), 2.87 (m, 2H, C ( H ? H_ { β -Phe), 2.78 (m, 2H, C (H? H ? -Phe) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 171'4 (s), 168'7 (s), 168'1 (s), 140'7 (s), 139 '8 (s), 137'0 (s), 134'8 (s), 131'3 (s), 130'6 (d), 130'3 (d), 129'8 (d), 129' 24 (d), 127'19 (d), 128'9 (d), 128'2 (d), 127'3 (d), 127'2 (d), 126'9 (d), 126'5 (d), 53'6 (d, C {\ alpha} -Phe), 51'8 (c, {2} C CO H {3}), 36.5 (t, C H {2} -Phe ) ppm

IR nu: 3430, 3065, 2917, 1747, 1696, 1623, 1574, 1440, 1361, 1260, 1215, 760 cm -1.

MS (ES +) m / e = 404 ([MH] +, 100%)

AE Calculated for C 24 H 21 NO 5: C, 71.45; H, 5.25; N, 3.47. Found: C, 71'32; H, 5.40; N, 3.41.

Example 17 ( R ) - (3-Phenyl-2 - {[2 '- ( S ) - (2-phenyl-1-methoxycarbonyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino}) -methyl propionate (17)

25

On a solution of acid 16 (200 mg, 0.49 mmol) in anhydrous DMF (3 ml), under argon, D- Phe-OMe hydrochloride (139 mg, 0.64 mmol), HOBT was added sequentially (87.1 mg, 0.64 mmol), Et3 N (0.1 mL, 0.74 mmol), EDC (95 mg, 0.64 mmol) and DMAP (6 mg, 0.049 mmol) . The mixture was stirred for 20 hours. After this time the solvent was removed, treated with 5% aqueous HCl solution and saturated NaHCO 3 solution and extracted with AcOEt (3 times). The organic extracts were combined, washed with brine and dried over anhydrous MgSO4. The solvent was evaporated to give a residue that was purified by column chromatography to give the coupling product 17 as a white solid (271 mg, 97% rto.).

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 8'76 (d, 2H, J = 8'8, 2-NH), 7'37 (m, 6H, H arom.), 7'23 (m, 6H, H arom.), 7'1'0 (m, 4H, H arom.), 6'90 (m, 2H, H arom.), 4.42 ( m, 2H, CH? Phe), 3.48 (s, 6H, CO 2 CH 3), 2.84 (m, 2H, C ( H ? H \ \ beta) - Phe), 2.68 (m, 2H, C (H? H ?) - Phe) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 171'2 (s, 2C), 168'9 (s, 2C), 138'7 (s, 2C), 136 ' 9 (s, 2C), 135'2 (s, 2C), 129'3 (d), 129'2 (d), 128'9 (d), 128'2 (d), 127'3 (d) , 127'2 (d), 126'5 (d), 53'8 (d, 2C), 51'8 (c, 2C), 36'5 (t, 2C) ppm.

IR nu 3436, 3061, 1745, 1640, 1544, 1437, 1335, 1217, 757, 701 cm -1.

MS (ES +) m / e = 565 ([MH] +, 100%)

AE Calculated for C 34 H 32 N 2 O 6: C, 72'32; H, 5.71; N, 4.96. Found: C, 72'12; H, 5.60; N, 4.85.

Example 18 ( S, S ) -3- (4-Hydroxy-phenyl) -2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -methylpropionate (18)

26

On a solution of 2,2'-diphenic acid (1 g, 4.113 mmol) in anhydrous DMF (20 ml), under argon, L- Tyr-OMe hydrochloride (2.48 g, was added sequentially) 10.73mmol), HOBT (1.02 g, 9.08 mmol), Et3N (1.7 mL, 12.4 mmol), DCC (1.07 g, 9.08 mmol) and DMAP ( 101 mg, 0.041 mmol). The mixture was stirred at room temperature for 18 hours after which it was treated with 5% aqueous HCl solution, saturated NaHCO3 solution, washed with brine and dried over anhydrous MgSO4. The organic phases were combined, evaporated to give a residue that was purified by column chromatography to give the coupling product 18 (2.09 g, 85% rto.) As a white solid.

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 8.14 (s, OH), 8.70 (d, 1H, J = 7.6, NH), 7 '35 (m, 8H, H arom.), 6'84 (d, 4H, J = 8'0, H arom.-Tyr), 6'60 (d, 4H, J = 8'5, H arom. -Tyr), 4.28 (m, 2H, C H ? -Tyr), 3.44 (s, 6H, CO 2 CH 3), 2.67 (m, 2H, C ( H ? H?) - Tyr), 2.52 (m, 2H, C (H? H ?) - Tyr) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 171'3 (s), 168'8 (s), 155'9 (s), 138'7 (s), 135 '3 (s), 129'8 (d,), 129'2 (d), 127'3 (d), 127'2 (d), 126'8 (d), 54'1 (d, C _ {α), 51'7 (c, CO 2 C H 3), 35'8 (t, C H 2 -Tyr) ppm.

IR 34 3421, 3060, 2953.1738, 1639, 1516, 1440, 1362, 1226, 1174, 759 cm -1.

MS (ES +) m / e = 597 ([MH] +, 100%)

AE Calculated for C_ {34} H_ {32} N2 O_ {8}: C, 68.45; H, 5.41; N, 4.70. Found: C, 68'35; H, 5.70; N, 4.82.

Example 19 ( S, S ) - N - (1-Ferrocenylmethyl) -3-phenyl-2 - ({2 '- [2-phenyl-1- (1-ferrocenylmethylcarbamoyl)] - ethylcarbamoyl) -biphenyl-2-carbonyl} -amino) -propionamide (19)

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27

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On a solution of diacid 2 (114 mg, 0.21 mmol) and ferrocenylmethylamine (118.5 mg, 0.55 mmol) in DMF anhydrous (5 ml) and under argon atmosphere, was added sequentially HOBT (74.4 mg, 0.55 mmol), Et 3 N (0.23 ml, 1.65 mmol), EDC (105.6 mg, 0.055 mmol) and DMAP (5.2 mg, 0.042 mmol). The mixture of The reaction was stirred at room temperature for 20 hours after which solvent was removed under reduced pressure and dissolved the residue in AcOEt. Subsequently, it was treated with dissolution 5% aqueous HCl, saturated NaHCO3 solution, washed with brine and dried over anhydrous MgSO4. Evaporation of solvent resulted in a residue that was purified by column chromatography to give rise to the coupling product  19 (112 mg, 57% rto.) As an orange-yellow solid.

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 8.78 (d, 2H, J = 8.5, NH-Phe), 7.79 (broad t, 2H , NH-Fe (Cp) 2), 7'46-7'21 (m, 16H, H arom.), 6.95 (m, 2H, H arom.), 4.46 (m, 2H , C H α -Phe), 4'08 (m, 18H, H arom.-Fe (Cp) 2), 3.90 (m, 4H, C H 2 -Fe (Cp) 2), 2.84 (m, 2H, C ( H ? H?) - Phe), 2.65 (m, 2H, C (H?) H ?) - Phe) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 168'9 (s), 168'5 (s), 138'9 (s), 138'6 (s), 137 '9 (s), 129'2 (d), 129'1 (d), 129'0 (d), 128'0 (d), 127'2 (d), 127'1 (d), 126' 2 (d), 85'8 (c), 68'3 (d), 677 (d), 67'5 (d), 67'4 (d), 67'1 (d), 67'0 (d ), 54.2 (d, C {\ alpha} -Phe), 37'5 (t, C H {2} -Phe) ppm.

MS (ES +) m / e = 784 (100%), 931 ([MH] +, 48%).

Example 20 N, N ' - Bis - (2-fluoro-5-nitro-phenyl) -biphenyl-2,2'-dicarboxamide (20)

28

About an acid solution 2,2'-diphenic (100 mg, 0.41 mmol) in anhydrous DMF (3  ml), under argon, was added sequentially 2-fluoro-5-nitro-aniline  (167.5 mg, 1.07 mmol), HOBT (145 mg 1.07 mmol), Et 3 N (0.05 ml, 1.04 mmol), EDC (206 mg, 1.07 mmol) and DMAP (10 mg, 0.08 mmol). The mixture was stirred for 20 hours after which the solvent in vacuo, treated with 37% aqueous HCl solution and It was extracted with AcOEt (3 times). The organic extracts came together, they were washed with brine and dried over anhydrous MgSO4. The evaporation of the solvent resulted in a residue that was purified by column chromatography to give rise to the product of diacoplant 20 in the form of solid earth-colored (114 mg, 53% Po.).

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 7.62 (dd, 2H, J 6.4 = 2.9, J 6, F = 7 '8, H-6), 7'46-7'12 (m, 10H, NH, H arom ._ {Bif}), 7'37 (ddd, 2H, J_ {4,3} = 8'7, J_ {4, F} = 3'9, J_ {4,6} = 2'9, H-4), 7'23 (dd, 2H, J_ {3, F} = 10'7, J_ {3, 4} = 8'7, H-3) ppm.

MS (ES +) m / e = 399, 537 ([MF] +, 10%).

Example 21 ( S, S ' ) -2-Amino- N - [2' - (2-amino-3-phenyl-propionylamino) -biphenyl-2-yl] -3-phenyl-propionamide (21)

29

On a solution at 0 ° C of dicarbamate 5 (725 mg, 0.78 mmol) in 4 ml of anhydrous DMF, 1 ml was added dropwise of piperidine The reaction mixture was kept stirring at room temperature for 75 minutes. After this time, the solvent was removed under reduced pressure, and the resulting residue by column chromatography to give rise to diamine 21 (315 mg, 84% rto.) in the form of white solid.

[α] D = -68 (c = 0.5, CHCl3).

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 8.12 (m, 2H, H arom.), 7.79 (m, 2H, H arom.), 7 '27 -7'09 (m, 12H, H arom.,; 2H, NH arom.), 7'0 (m, 2H, H arom.), 3.40 (m, 2H, C H ? -Phe), 2.95 (m, 1H, C ( H ? H?) - Phe), 2.52 (m, 1H, C (H? H \ \ beta}) - Phe) ppm.

13 C-NMR (50 MHz, DMSO- d 6 , 30 ° C) δ: 172'9 (s), 172'7 (s), 138'5 (s), 1381 (s), 138 '5 (s), 138'1 (s), 135'7 (s), 135'5 (s), 130'5 (s), 130'4 (s), 129'3 (d), 129' 1 (d), 129'0 (d), 128'6 (d), 128'2 (d), 128'1 (d), 126'25 (d), 126'22 (d), 124'3 (d), 124'2 (d), 121'4 (d), 53'5 (d, C {\ alpha} -Phe), 56'2 (d, C {\ alpha} -Phe), 39'8 (t, C H 2 -Phe), 39'7 (t, C H 2 -Phe) ppm.

IR 34 3410, 3063,1696, 1585, 1516, 1450, 1246, 1049, 758, 740, 700 cm -1.

MS (ES +) m / e = 479 ([MH] +, 100%)

AE Calculated for C 30 H 30 N 4 O 2: C, 75.29; H, 6.32; N, 11'71. Found: C, 74.99; H, 6.18; N, 11'50.

Example 22 ( S, S ) - {1- [1- (2'-Amino-biphenyl-2-ylcarbamoyl) -2-phenyl-ethylcarbamoyl] -3-methyl-butyl} -carbamate of 9 H -fluoren-9-ylmethyl ( 22)

30

On a solution of 2,2'-di (amino) -biphenyl (715 mg, 3.88 mmol) and Et 3 N (0.15 ml, 1.06 mmol) in CH 2 Cl 2 anhydrous (100 ml), the acid chloride, N-Fmoc- L- le- L- Phe-Cl (1.0 molar equivalent) was added little. The mixture was stirred for 19 hours after which the solvent was evaporated in vacuo. The resulting residue was purified by column chromatography to give the monocouple product 22 as a white solid (129 mg, 20% rto.).

Mp = 82-84 ° C.

[α] D = -22 (MeOH, c = 0.5).

1 H-NMR (300 MHz, DMSO- d 6 , 40 ° C) δ: 9'10 (s, 1H, NH arom.), 7.90 (d, 2H, J = 7'3, H arom.), 7'72 (d, 2H, J = 7'0, H arom.), 7'68-6'60 (m, 19H, H arom .; NH-Leu, NH-Phe), 4 '21 (m, 3H, C H ? -Phe; C H2 -Fmoc; C H -Fmoc), 2.89 (m, 1H, C ( H ? H_ { β) - Phe), 2.65 (m, 2H, C (H? H ?) - Phe), 1.49 (m, 1H, C H- Leu), 1 ' 39 (m, 2H, C H 2 -Leu), 0.77 (m, 6H, C H 3 -Leu) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 171'0 (s), 169'2 (s), 156'0 (s), 144'0 (s), 143 '7 (s), 140'7 (s), 130'7 (s), 130'6 (s), 130'5 (s), 128'5 (d), 127'6 (d), 127' 0 (d), 125'3 (d), 120'1 (d), 117'4 (d), 65'6 (t, C H {2} -Fmoc), 53'9 (d, C { \ alpha} -Phe), 46.6 (d, C {\ alpha} -Leu), 41'0 (C H {2} -Leu), 39'0 (d, C H {2} -Phe) , 24'2 (d, C H-Leu), 23'0 (c, C H3 -Leu), 21'3 (c, C H3 -Leu) ppm.

IR \ nu: 3325, 2924, 2854, 1719, 1519, 1646, 1377, 1248, 1109, 1046, 757, 740 cm -1.

MS (ES +) m / e = 520 (100%).

AE Calculated for C4 2H_ {42} N4 O4: C, 75.65; H, 6.35; N, 8.40. Found: C, 75'81; H, 6.60; N, 8.46.

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Example 23 2,2'-di- (2-naphthoyl-amino) -biphenyl (2. 3)

31

On a solution at 0 ° C of 2,2'-di (amino) -biphenyl (200 mg, 1.08 mmol) in 10 ml of anhydrous CH2Cl2, was added Et 3 N (0.6 ml, 4.32 mmol) and naphthoyl chloride (455.2 mg, 2.38 mmol) in small portions. The reaction mixture was maintained. stirring at room temperature overnight. Past at this time, the solvent was removed under reduced pressure, and purified the resulting residue by column chromatography to give rise to the diacoplating product 23 in the form of a solid white (430 mg, 81% rto.).

1 H-NMR (300 MHz, CDCl 3) δ: 8'65 (d, 2H, J = 8'3, H arom.), 8'01 (m, 4H, H arom.), 7'83 (m, 6H, H arom.), 7'64-7'49 (m, 6H, H arom.), 7'43-7'32 (m, 4H, H arom.) Ppm.

13 C-NMR (50 MHz, CDCl_3) δ: 165'9 (s), 136'3 (s), 137'1 (s), 132'7 (s), 131'7 (s), 130'7 (d,), 130'1 (d), 129'3 (d), 128'9 (d), 128'8 (s), 128'2 (d), 127'9 (d), 127'8 (d), 127'1 (d), 125'4 (d), 123'4 (d), 122.6 (d) ppm.

IR 34 3418, 3228, 3058.1735, 1643, 1516, 1496, 1432, 1306, 1237, 777 cm -1.

MS (ES +) m / e = 493 ([MH] +, 100%)

AE Calculated for C 34 H 24 N 2 O 2: C, 82'91; H, 4.91; N, 5.69. Found: C, 82.65; H, 5.10; N, 5.64.

Example 24 2,2'-di - [(3,5-dinitrobenzoyl) amino) -biphenyl (24)

32

On a solution at 0 ° C of 2,2'-di (amino) -biphenyl (30 mg, 0.16 mmol) in 1 ml of THF, Et 3 N (0.04 ml, 0.032 was added mmol) and 3,5-dinitrobenzoyl chloride (82.6 mg, 0.36 mmol) in small portions. The reaction mixture was maintained. stirring at room temperature overnight. The product 24, which precipitates as a yellow solid in the middle of reaction, filtered and dried (83 mg, 89% rto.).

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 10'13 (s, 2H, NH), 8.95 (t, 2H, J = 2'2, H- 10), 8'83 (d, 4H, J = 2'2, H-9, H-11), 7'52-7'36 (m, 8H, H-1, H-2, H-3, H-4, H-5, H-6, H-7, H-8) ppm.

13 C-NMR (75 MHz, DMSO- d 6 ) δ: 162.6 (s), 148.6 (s), 137.8 (s), 135.8 (s), 135 '5 (s), 131'8 (d,), 128'9 (d), 128'6 (d), 127'6 (d), 127'3 (s), 121'7 (d) ppm.

MS (ES +) m / e = 573 ([MH] +, 100%)

Example 25 2- (2-Naphthoylamino) -2 '- [(3,5-dinitrobenzoyl) amino] -biphenyl (25)

33

On a solution at 0 ° C of amine 7 (50 mg, 0.15 mmol) in 6 ml of anhydrous pyridine, the chloride of 2,5-dinitrobenzoyl (37.5 mg, 0.16 mmol) in little portions. The reaction mixture was kept stirring at room temperature for 14 hours. After this time, the solvent was removed under reduced pressure, and the resulting residue by column chromatography to give rise to diamide 25 (71 mg, 91% rto.) as a yellow solid.

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 10'32 (s, 1H, NH), 9'48 (s, 1H, NH), 8'93 (t , 1H, J = 1'9, H-10), 8'83 (d, 2H, J = 1'9, H-9, H-11), 8'26 (s, 1H, H-12), 7'94 (m, 3H, H-8, H-13, H-17), 7'75-7'56 (m, 5H, H-1, H-5, H-6, H-8, H -16), 7'48-7'28 (m, 6H, H-2, H-3, H-6, H-14, H-15) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 165'6 (s), 162'1 (s), 148'6 (s), 148'0 (s), 136 '9 (s), 1 135'6 (s), 135'3 (s), 135'0 (s), 134'4 (s), 134'2 (s), 131'9 (d), 131 '5 (d), 130'9 (d), 130'5 (d), 128'8 (d), 128'4 (d), 128'3 (d), 128'0 (d), 127' 8 (d), 127'7 (d), 127'6 (d), 126'9 (d), 126'8 (d), 126'7 (d), 126'1 (d), 125'8 (d), 123'9 (d), 121'1 (d) ppm.

MS (ES +) m / e = 533 ([MH] +, 100%)

AE Calculated for C 30 H 2 ON 4 O 6: C, 67.67; H, 3.79; N, 10'52. Found: C, 67.46; H, 3.60; N, 10'35.

Example 26 2- (2-Naphthoylamino) -2 '- [(4-nitrobenzoyl) amino] -biphenyl (26)

3. 4

On a solution at 0 ° C of amine 7 (50 mg, 0.15 mmol) in 6 ml of anhydrous pyridine, the chloride of 4-nitrobenzoyl (30'1 mg, 0.16 mmol) in small portions. The reaction mixture was kept stirring at temperature. atmosphere for 13 hours. After this time, the solvent under reduced pressure, and the resulting residue was purified by column chromatography to give rise to diamide 26 (71 mg, 99% rto.) in the form of a pale yellow solid.

1 H-NMR (300 MHz, DMSO- d 6 , 30 ° C) δ: 9'84 (s, 1H, NH), 9'48 (s, 1H, NH), 8'27 (d , 2H, J = 8'8, H-10, H-10 '), 8'22 (s, 1H, H-11), 7'93 (m, 3H, H-1, H-16, H- 17), 7'88 (d, 211, J = 8'8, H -9, H-9 '), 7'75-7'54 (m, 5H, H-4, 11-5, H-8 , H-12, H-15), 7'45 (m, 2H, H-13, H-14), 7'37-7'25 (m, 511, H-3, H-2, H-6 , H-7) ppm.

13 C-NMR (50 MHz, DMSO- d 6 ) δ: 165'6 (s), 164'2 (s), 149'1 (s), 139'7 (s), 135 '6 (s), 135'2 (s), 135'1 (s), 134'4 (s), 134'1 (s), 131'9 (d), 131'4 (d), 130' 3 (d), 130'1 (d), 128'7 (d), 128'5 (d), 128'3 (d), 128'0 (d), 127'7 (d), 127'5 (d), 126'8 (d), 126'5 (d), 126'2 (d), 125'8 (d), 125'6 (d), 123'6 (d), 123'5 ( d) ppm.

MS (ES +) m / e = 488 ([MH] +, 100%)

Example 27 ( S, S, S, S ) -2- (3-Phenyl-2 - {[2 '- (2-Phenyl-1- {2' - [2- (9 H- Fluoren-9-ylmethoxycarbonylamino) -propionylamino ] -biphenyl-2-ylcarbamoyl} -ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -propionylamino) -3-methyl-butyrate (27)

35

On a solution of amine 8 (100 mg, 0.12 mmol) at 0 ° C in 5 ml of anhydrous CH 2 Cl 2, Et 3 N (0.034 ml, 0.024 mmol) was added and L -Fmoc-Ala chloride, previously synthesized. The reaction mixture was kept stirring at room temperature for 22 hours. After this time, the solvent was removed under reduced pressure and the residue was purified by column chromatography to give the coupling product 27 as a white solid (126 mg, 93% rto.).

1 H-NMR (300 MHz, DMSO- d 6 , 50 ° C, mixture of conformers A + at 2: 1) δ: 8'83 (wide s, 0.33H, NH 1, a ), 8'70 (wide s, 0.66H, NH1, A), 8.51 (d, 0.66H, J = 8'4, NH-Phe, A), 8.34 (m, 0.33H, NH-Phe, a; 1H, NH-Phe, A + a), 8'09 (broad s, 1H, NH2, A + a), 8'04 (d, 0.33H, J = 8'6, NH-Val, a), 7'98 (d, 0.66H, NH-Val, A), 7'87 (d, 2H, J = 7'7, H arom.-Fmoc, A + a), 7'66 (m, 211, H arom.-Fmoc, A + a), 7'64-6'76 (m, 30H, H arom., A + a; 111, NH-Ala, A + a), 4.59 (m, 1.33H, C H ? -Phe, A), 4.38 (m, 0.66H, C H ? -Phe, a) , 4.17 (m, 411, C H α -Val, A + a; C H 2 -Fmoc, A + a; CH-Fmoc, A + a), 4'0 (m , 1H, CHα-Ala, A + a), 3.60 (s, 0.99H, CO 2 C H 3), 3.59 (s, 1.99H, CO_ 2 C 2 H), 2.84 (m, 2H, C ( H ? H?) - Phe, A + a), 2.61 (m, 2H, C ( H H ) -Phe, A + a), 1.98 (m, 1H, C H (CH 3) 2 -Val, A + a), 1.06 (m, 3H, C H _ {3} -Ala, a + a), 0.82 (m, 6H, C (C H 3} {) _ {2} Val, a + a) ppm.

13 C-NMR (75 MHz, DMSO- d 6 , 50 ° C) δ: 171'35 (s), 171'3 (s), 168.6 (s), 168.5 (s) , 168'3 (s), 168'4 (s), 138'2 (s), 138'1 (s), 137'5 (s), 137'4 (s), 135'0 (s), 130'7 (d), 130'5 (d), 129'3 (d), 129'2 (d), 129'0 (d), 128'8 (d), 128'4 (d), 127 '9 (d), 127'8 (d), 127'3 (d), 126'9 (d), 126'8 (d), 126'1 (d), 117'0 (d), 115' 7 (d), 57'4 (d, C {\ alpha} -Val), 54'8 (d, C {\ alpha} {1} -Phe_), 53'6 (d, C {\ alpha} -Phe 2), 51'5 (c, CO 2 C H 3), 37'2 (t, C H 2 -Phe), 36'2 (t, C H_ { 2 -Phe), 29'8 (d, C H (CH 3) 2 -Val), 18'7 (c, CH ( C H 3) 2 -Val), 18'1 (c, CH ( C H3) 2 -Val) ppm.

IR nu 3429, 3062, 2962, 1739, 1643, 1525, 1451, 1307, 1078, 758, 700 cm -1.

MS (ES +) m / e = 763 (100%), 1109 ([MH] +, 46%).

Example 28 Enzyme activity test: Calpain inhibition

The ability to inhibit calpain has been quantified by the value of IC_ {50}, which is defined as the inhibitor concentration that halves activity Catalytic of an enzyme. The lower the value of IC_ {50}, More potent is the inhibitor. Results of calpain inhibition I (the most relevant from a physiological point of view) of some compounds of the present invention are indicated in the table 1 and in figure 1. Since calpain II, also called milli-calpain, you need more calcium for activation, it may not have such a physiological role relevant; because said calcium concentration would cause death cell before it could activate the milli-calpain For this reason, the trials of inhibition have been performed for calpain I, but they are extrapolated for calpain II.

TABLE 1 Results of compound calpain inhibition objects of this invention

Compounds IC_ {50} one 64nM 2 70nM 3 37nM 4 10nM 5 12nM 6 187nM 7 18nM 8 87pM (= 0.087nM)

Studies of the structure of the peptide-biphenyl hybrids of the present invention and its biological activity, which have shown that:

1) All the compounds object of the present invention, of generic formula I, are potent inhibitors of calpain I. As representative but not limiting examples, compounds 1-8 have IC 50 values between 0.087 and 187 nM.

2) Calpain inhibition of compounds 1-4 illustrate that biological activity is virtually independent of the nature of amino acids, of the length of the peptide chain and the substitution in the ends of the peptide chains.

3) When changing the orientation of the amino acids attached to the biphenyl system, are also obtained potent calpain I inhibitors. A representative example, although not limiting is compound 5.

4) Compounds that can be considered as peptide-biphenyl hybrids with side chains Truncated are also good calpain I inhibitors. Example Illustrative of these compounds are 6 and 7.

5) The combination in a structure of the features structural compounds 4, 5 and 7 to obtain the hybrid peptide-biphenyl 8 results in an inhibitor 115 times more powerful than the previous ones, having a value of IC 50 in picomolar scale, which is the most potent inhibitor of Calpain described so far.

To perform the inhibition test of calpaína, we use the EnzCheck® Protease Assay Kit E-338 from Molecular Probes. This kit contains casein marked with BODIPY FL®, lyophilized from phosphate buffer. Also I know used as a digestion buffer Tris-HCl at pH 7.8, containing 2 mM sodium azide.

Calpain I from swine erythrocytes or calpain Porcine kidney II used are commercial products of CALBIOCHEM®. The stock enzyme solution contains 20mM buffer imidazol-HCl, PH 6.8, 1 mM EDTA, 1 mM EGTA, 5 mM β-mercaptoethanol 30% in glycerol.

Synthetic inhibitors were dissolved in 250 µL of DMSO. The tests were performed in 96-well microplates in a final volume of 200 µL. To perform the tests, aliquots of calpain stock solution were added to 150 µL of 10 µL / µL solution in digestion buffer to obtain a final concentration of 50 ng / mL enzyme. Minimal amounts, between 5 and 20 µL of inhibitor solution in DMSO, previously diluted to achieve the desired final inhibitor concentration were added, and digestion buffer is added until a final volume of 190 µL is achieved. The assay is started by adding 10 µL of 0.05 M CaCl 2 solution. For each inhibitor blank measures were taken excluding, inhibitor, calpain and calcium solution. All measurements were performed in triplicate.

Fluorescence measurements are performed in a SPECTRAFLUOR TECAN Corp 93382 fluorimeter spectrum. Exciting 485 nm and performing the reading at 530 nm. The measurement was made during 20 cycles to complete the measurement of all wells. The agitation is of the orbital type and takes place between each cycle.

Abbreviations

The meaning of the Abbreviations used.

- AE: Elementary analysis.

- Ala: Radical corresponding to the amino acid to the girl.

- BOP: Hexafluorophosphate benzotriazol-1-yloxy-tris (dimethylamino) phosphonium.

- CANP: Calcium-activated neutral protease.

- DMAP: 4-Dimethylaminopyridine.

- DMF: N, N- Dimethylformamide.

- DMSO: Dimethylsulfoxide.

- Fmoc: 9 H- Fluoren-9-ylmethoxycarbonyl.

- EDC: 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide.

- EDTA: Ethylenediaminetetraacetic acid.

- EGTA: Ethylene bis - (oxyethylene nitrile) tetraacetic acid.

- EM: Mass spectrum.

- EN: Electro-spray .

- HOBT: 1-Hydroxybenzotriazole.

- IR: Infrared.

- Met: Radical corresponding to the amino acid methionine

- NMDA: N- methyl- D- aspartate.

- P.f .: Melting point.

- Phe: Radical corresponding to the amino acid phenylalanine

- Tris: Tris (hydroxmethyl) aminomethane.

- Trp: Radical corresponding to the amino acid tryptophan

- Tyr: Radical corresponding to the amino acid tyrosine

- Val: Radical corresponding to the amino acid valine

- 1 H-NMR: Resonance Proton nuclear magnetic.

- <13> C-NMR: Resonance Carbon-13 nuclear magnetic.

Claims (6)

1. A compound biphenyl-2,2'-disubstituted from formula I

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36

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in which the R1 and R2 may be the same or different, and may be independently selected from among groups

-

NH2,

-

NHR 3 in which R 3 represents an alkyl or aryl group,

-

NR 4 R 5 in which R 4 and R 5 represents two identical alkyl or aryl groups or different, or forming a cyclic system,

-

CO 2 H,

-

CO 2 R 6 in which R 6 is a aryl or alkyl group,

-

CONH_ {2},

-

CONR 7 R 8 in which R 7 and R 8 represents two identical alkyl or aryl groups or different, or forming a cyclic system,

-

a group of formula II,

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37

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in the that:

-

Ar represents an aryl, carbocyclic or heterocyclic group, other than  imidazole,

-

the asterisk (*) represents a stereogenic center, of configuration ( R ) or ( S ), interchangeably,

-

n It has a value between 0 and 6,

-

X, represents a group OH, OR 9 in which R 9 represents a alkyl or aryl group, NH 2, NHR 10, wherein R 10 represents an alkyl or aryl group, NR 11 R 12 in which R 11 and R 12 represent two alkyl or aryl groups, identical or different, or forming a cyclic system,

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-

aa is an amino acid residue,

of formula 38 where the asterisk (*) indicates a stereogenic center of configuration ( R ) or ( S ), interchangeably, R 13 is selected from H, alkyl or aryl; or formula 39 in which the nitrogen atoms and the carbon in position α- are forming a ring between 4 and 7 links, amino acid residues (aa) are linked through from its amino group to the chain represented by -C (O) NHCH (CH 2 Ar) CO-; - a group of formula III, 40 in which Ar represents a group aryl; - a group of formula IV, 41 in the that:

-

Ar represents an aryl group,

-

(*), Y n have the meaning described above,

-

aa It has the meaning described above, but now the residue of amino acid is attached through its carbonyl group to the chain  represented by -NHC (O) CH (CH2 Ar) NH-,

-

X represents H, alkyl between 1 and 6 carbon atoms, COR 14 where R 14 is an aryl or alkyl group, CO 2 R 15, where R 15 is an aryl or alkyl group;

-

a group of formula V,

42 in which Ar represents a group aryl; and in which at least one of the R1 groups, R2 has the structure indicated by groups II, or III, or IV, or V; and any of the conformational isomers (atropisomers) of said compound of formula I. 2. A compound biphenyl-2,2'-disubstituted from formula I 43 in which the R1 and R2 may be the same or different, and may be independently selected from among groups

-

NH2,

-

NHR 3 in which R 3 represents an alkyl or aryl group,

-

NR 4 R 5 in which R 4 and R 5 represents two identical alkyl or aryl groups or different, or forming a cyclic system,

-

CO 2 H,

-

CO 2 R 6 in which R 6 is a aryl or alkyl group,

-

CONH_ {2},

-

CONR 7 R 8 in which R 7 and R 8 represents two identical alkyl or aryl groups or different, or forming a cyclic system,

-

a group of formula II,

44

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in which:

-

Ar represents an aryl, carbocyclic or heterocyclic group, other than  imidazole,

-

the asterisk (*) represents a stereogenic center, of configuration ( R ) or ( S ), interchangeably,

-

n It has a value between 0 and 6,

-

X, represents a group OH, OR 9 in which R 9 represents a alkyl or aryl group, NH 2, NHR 10, wherein R 10 represents an alkyl or aryl group, NR 11 R 12 in which R 11 and R 12 represent two alkyl or aryl groups, identical or different, or forming a cyclic system,

-

aa is an amino acid residue of the formula

Four. Five where the asterisk (*) indicates a stereogenic center of configuration ( R ) or ( S ), interchangeably, R 13 is selected from H, alkyl or aryl; or formula 46 in which the nitrogen atoms and the carbon in position α- are forming a ring between 4 and 7 links, amino acid residues (aa) are linked through from its amino group to the chain represented by -C (O) NHCH (CH 2 Ar) CO-; - a group of formula III, 47 in which Ar represents a group aryl; - a group of formula IV, 48

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in which:

-

Ar represents an aryl group,

-

(*), Y n have the meaning described above,

-

aa It has the meaning described above, but now the residue of amino acid is attached through its carbonyl group to the chain  represented by -NHC (O) CH (CH2 Ar) NH-,

-

X represents H, alkyl between 1 and 6 carbon atoms, COR 14 where R 14 is an aryl or alkyl group, CO 2 R 15, where R 15 is an aryl or alkyl group;

-

a group of formula V,

49 in which Ar represents a group aryl; and in which at least one of the groups R 1, R 2 has the structure indicated by the groups  II, or III, or IV, or V; and any of the conformational isomers (atropisomers) of said compound of formula I; and characterized in that it is a calpain inhibitor. 3. A compound according to claim 1 characterized in that it is of formula I and selected from

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( S, S ) -3-Phenyl-2- {[2 '- (2-phenyl-1-methoxycarbonyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -methyl propionate (1),

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( S, S ) -2- [2 '- (1-carboxy-2-phenyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -3-phenyl-propionic acid (2),

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( S, S ) -3- (1 H -indole-3-yl) -2 - ({2 '- [2- (1 H -indole-3-yl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2 -carbonyl} -amino) -methylpropionate (3),

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( S, S, S ) -2- (2 - {[2 '- (1-Carboxy-2-phenyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -3-phenyl-propionylamino) -3-methyl methylbutyrate (4),

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( S, S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2-phenyl-1- (2-methyl-1-methoxycarbonyl-propylcarbamoyl) -ethylcarbamoyl] -biphenyl-2- carbonyl} -amino) -propionylamino] -3-methyl-butyrate (4a),

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( S, S ) - (2-Phenyl-1- {2 '- [3-phenyl-2- (9 H -fluoren-9-ylmethoxycarbonylamino) -propionylamino] -biphenyl-2-ylcarba-moyl} -ethyl) - 9 H -fluoren-9-ylmethyl carbamate (5),

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Bis -2,2 '- [(4-nitro-benzoyl) amino] -biphenyl (6),

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2'-Amino-2- [2- (naphthoyl) amino] biphenyl (7),

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( S, S, S ) -2- [2 - ({2'-Amino-biphenyl-2-ylcarbamoyl) -2-phenyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -3-phenyl-propionylamino] -3-methyl methyl butyrate (8),

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( S, S, S, S ) -3- (1 H -Indol-3-yl) -2- {3-phenyl-2 - [(2 '- {2-phenyl-1- [2- (1 H -indole-3-yl) -1-methoxycarbonyl-ethylcarbamo-yl] -ethylcarbamoyl} -biphenyl-2-carbonyl) -amino] propionylamino} -methyl propionate (9),

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( S ) -2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carboxylic acid (10),

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( S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) - methyl propionylamino] -4-methyl-pentanoate (11),

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( S, S, S ) -2 - ({2 '- [2-phenyl-1- (3-methyl-1-methylcarbamoyl-butylcarbamoyl) -ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -3- ( Methyl 4-hydroxyphenyl) -propionate (12),

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( S, S, S ) -2- [3-Phenyl-2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) - methyl propionylamino] -4-methylsulfanyl-butyrate (13),

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2 '- {(S) - [2-Phenyl-1- ( R ) - (2-phenyl-1-methylcarbamoyl-ethylcarbamoyl) -ethylcarbamoyl]} - biphenyl-2-carboxylic acid (14),

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( S ) -2 - [(S) -2 - ({2 '- ( S ) - [2-Phenyl-1- ( R ) - (2-phenyl-1-methylcarbamoyl-ethylcarbamoyl) -ethylcarbamoyl] -biphenyl- Methyl 2-carbonyl} -amino) -4-methyl-entanoylamino] -4-methylsulfanyl-butyrate (15),

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( S ) -2 '- [2-Phenyl-1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carboxylic acid (16),

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( R ) - (3-Phenyl-2 - {[2 '- ( S ) - (2-phenyl-1-methoxycarbonyl-ethylcarbamoyl) -biphenyl-2-carbonyl] -amino}) -methyl propionate (17),

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( S, S ) -3- (4-Hydroxy-phenyl) -2 - ({2 '- [2- (4-hydroxy-phenyl) -1-methoxycarbonyl-ethylcarbamoyl] -biphenyl-2-carbonyl} -amino) -methylpropionate (18),

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( S, S ) - N - (1-Ferrocenylmethyl) -3-phenyl-2 - ({2 '- [2-phenyl-1- (1-ferrocenylmethylcarbamoyl)] - ethylcarbamoyl) -biphenyl-2-carbonyl} -amino ) -propionamide (19),

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N, N'-Bis - (2-fluoro-5-nitro-phenyl) -biphenyl-2,2'-dicarboxamide (20),

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( S, S ) -2-Amino- N - [2 '- (2-amino-3-phenyl-propionylamino) -biphenyl-2-yl] -3-phenyl-propionamide (21),

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( S, S ) - {1- [1- (2'-Amino-biphenyl-2-ylcarbamoyl) -2-phenyl-ethylcarbamoyl] -3-methyl-butyl} -carbamate of 9 H -fluo-ren-9- ilmethyl (22),

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2,2'-di- (2-naphthoyl-amino) -biphenyl (2. 3),

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2,2'-di - [(3,5-dinitrobenzoyl) amino) -biphenyl (24),

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2- (2-Naphthoylamino) -2 '- [(3,5-dinitrobenzoyl) amino] -biphenyl (25),

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2- (2-Naphthoylamino) -2 '- [(4-nitrobenzoyl) amino] -biphenyl (26),

Y

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( S, S, S, S ) -2- (3-Phenyl-2 - {[2 '- (2-Phenyl-1- {2' - [2- (9 H- Fluoren-9-ylmethoxycarbonylamino) -propionylamino ] -biphenyl-2-ylcarbamoyl} -ethylcarbamoyl) -biphenyl-2-carbonyl] -amino} -propionylamino) -3-methyl-butyrate (27),

and because it is a calpain inhibitor. 4. Use of a calpain inhibitor compound of formula I, defined in claim 1, to prepare a medicine for the preventive or therapeutic treatment of a degenerative illness. 5. Use of a calpain inhibitor compound of formula I according to claim 4 wherein the disease degenerative is selected from cerebral ischemia, ischemia Cardiac, Alzheimer's, Parkinson's, muscular dystrophy, cataracts and demyelinating diseases. 6. Use of a calpain inhibitor compound of formula I according to claim 5 wherein the disease Degenerative demyelinating is multiple sclerosis.