Abstract:
The present invention relates to compounds derived from biphenyl with activity as calpain inhibitors. One compound of the present invention is a 2,2′-disubstituted biphenyl, where the substituents in the 2 and 2′ positions of the biphenyl skeleton are chains containing structures related to amino acids, including fragments of aminocarbonylic compounds where at least one of the substituents in said 2- or 2′-positions is bonded to the biphenyl skeleton via a thiocarbonyl group, forming compounds with thioamide functionality. 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 disease.

Description:
FIELD OF THE ART 
     The present invention comes with the field of enzyme inhibitors with therapeutic activity, more specifically calpain inhibitors. 
     STATE OF THE ART 
     Calpains, or calcium activated neutral proteases (II) (CANP, E.C. 3.4.22.17), are a family of proteases with cysteine (“cysteine proteases”) with a very active metabolic role. Although their natural substrate has not been clearly determined, these enzymes catalyse the hydrolysis of a variety of proteins involved in the transduction of signals, in the reconstruction of the cytoskeleton, in the regulation of the cell cycle and in apoptosis ( Adv. Pharmacol.  1996, 37, 117). In mammals, the calpains family includes various tissue-specific isofoms and two ubiquitous isoenzymes: μ-calpain (or calpain I) and m-calpain (or calpain II), which require micromolar and millimolar quantities respectively of calcium (II) for their activation. Structural studies using X-ray diffraction have shown that each isoform consists of a large subunit (˜80 kDa), which presents a cysteine protease domain 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 bonding to Ca (II) (calmoduline type domain) ( FEBS Lett.  2001, 501, 111). 
     The overactivation of calpains, which can occur when the intracellular concentration of calcium (II) increases, is involved in numerous diseases, such as cerebral and cardiac ischaemias, Alzheimer, Parkinson, muscular distrophy, cataracts, demyelinating diseases (such as multiple sclerosis) and other degenerative diseases ( Pathophysiology  1999, 6, 91 ; Brain Res. Rev.  2003, 42, 169). 
     The main application of selective inhibitors of calpain is as neuroprotector agents. In the therapeutic area related to neuroprotection, a range of strategies has been used so far. Agents have been used which act on the membrane depolarisation and the entry of calcium into cells, or which prevent the production of free radicals (antioxidants), or which are antagonists of the action of neurotransmitters ( J. Clinical Neurosci.  2002, 9, 4). A great deal of attention has recently been paid to drugs capable of blocking the NMDA receptors for glutamate; nevertheless, the blocking of ionotropic receptors of excitatory amino acids cannot be an ideal method for preventing excitotoxic action since these drugs have psychotomimetic side effects ( Pharmacol. Ther.  1999, 81, 163 ; Neurobiol. Disease  2003, 12, 82). An interesting alternative for achieving neuroprotection is the blocking of “post-receptor” cell phenomena which are physiologically silenced, in other words, the search for selective blockers of catabolic cascades induced by excitotoxic agents. These potential drugs with intracellular action could, when acting on metabolic routes which are activated during neurodegeneration, foreseeably permit a more efficient and selective neuroprotector action. 
     The overactivation of calpain requires a continual increase in the intracellular concentrations of Ca (II), and this enzyme is latent in cells at rest [in other words, with “normal” Ca (II) levels]. Therefore, the inhibition of calpain is presented as a suitable treatment in neurodegenerative diseases. On the basis of its characteristics, the inhibition of calpain would foreseeably have fewer side effects in human therapeutics than the blocking of metabolic processes prior to their activation in pathological processes, as is the case with antagonism of the NMDA receptor of glutamate and aspartate, due to the fact that calpain is not activated under “normal” physiological conditions and that the action of excitatory amino acids is essential for the normal functioning of the nervous system. 
     Moreover, powerful and selective inhibitors of calpain are very useful as work tools for studying the action mechanism of this protease, along with its role in certain physiological processes. 
     Moreover, biphenyls substituted in different ways have been used as pharmacophores with a range of different biological activities ( J. Med. Chem.  2000, 43, 3443). In addition, biphenyl derivatives have been used as mesogenic fragments for the preparation of liquid crystals (EP-743293). On the other hand, amino acids and related compounds, such as aminocarbonylic compounds, possess a range of different biological properties ( J. Med. Chem.  2002, 45, 4762 ; Bioorg. Med. Chem. Lett.  2000, 10, 1497). 
     Reversible and irreversible inhibitors of calpain have been described ( Trends Mol. Medicine  2001, 7, 355; U.S. Pat. No. 5,541,290). The most frequent structural features of these inhibitors is that they are peptides or peptidomimetics with few amino acids (between 2 and 6), hydrophobic and with some electrophile functionality, among which can be mentioned α-ketophophonates, α-ketophophinates, oxides of α-ketophophines, α-ketoesters, α-ketoacids, α-ketoamides, trifluoromethylketones, aldehydes, methylsulphonium salts, epoxides, etc. These compounds apparently act on the papain type domain of the calpain, which leads to a relatively low selectivity, due to which they are frequently also inhibitors of other cysteine proteases (for example, papain) and even serine proteases. Due in part to these drawbacks, a calpain inhibitor having therapeutic utility has not yet been found. 
     There exists a need to find calpain inhibitors having therapeutic utility. One type of calpain inhibitor that has not yet been described and which has not been investigated previously consists of derivatives of 2,2′-disubstituted biphenyl, where the substituents in the 2- and 2′-positions are fragments derived from amino acids and related compounds, such as aminocarbonylic compounds where at least one of the substituents in the 2- and 2′-positions is bonded via a thiocarbonyl group, forming the thioamide functionality, are which are the object of this invention. 
     Recently, Spanish application (P-200301125) described the synthesis and biological evaluation of calpain inhibitors which are structurally derived from biphenyl with aminocarbonyl (carbamoyl) or carbonylamine substituents, indifferently, in the 2- and 2′-positions of the biphenyl system ( Bioorg. Med. Chem. Lett.  2004, 14, 2753 ; Chemistry  &amp;  Biodiversity  2004, 1, 442). 
     BRIEF DESCRIPTION OF THE INVENTION 
     The present invention relates to compounds derived from biphenyl with activity as calpain inhibitors. One compound of the present invention is a 2,2′-disubstituted biphenyl, where the substituents in the 2 and 2′ positions of the biphenyl skeleton are chains containing structures related to amino acids, including fragments of aminocarbonylic compounds where at least one of the substituents in said 2- or 2′-positions is bonded to the biphenyl skeleton via a thiocarbonyl group, forming compounds with thioamide functionality. 
     DESCRIPTION 
     The present invention relates to a compound of formula I, which has a 2,2′-disubstituted biphenyl structure, 
                                
wherein:
         the group X is oxygen (O) or sulphur (S), indifferently,   the groups R 1  and R 2  are the same or different and are independent selected from among the groups H, alkyl of between 1 and 10 carbon atoms, aryl, or arylalkyl, when applicable (that is, when R 1  or R 2 ≠H), the asterisk (*) represents a stereogenic center, of configuration (R) or (S), indifferently,   the groups R 3  and R 4  are the same or different and are independent selected from among the groups
           H,   alkyl of between 1 and 6 carbon atoms,   aryl,   arylalkyl,   NH 2 ,   NHR 5  in which R 5  represents an alkyl or aryl group,   NR 6 R 7  in which R 6  and R 7  represent two alkyl or aryl groups, identical or different, or forming a cyclic system,   OH,   OR 8  in which R 8  represents an alkyl or aryl group; and any of the conformational isomers (atropisomers) of said compound of formula I.   
               
     The following are preferred compounds:
     Methyl (S,S)-3-phenyl-2-{[2′-(2-phenyl-1-methoxycarbonyl-ethylthio-carbamoyl)-biphenyl-2-carbothiol]-amino}-propionate (1).   Methyl (S,S)-3-phenyl-2-{[2′-(2-phenyl-1-methoxycarbonyl-ethylthio-carbamoyl)-biphenyl-2-carbonyl]-amino}-propionate (2).   Methyl (S,S)-3-(1H-Indol-3-yl)-2-{[2′-(2-(1H-Indol-3-yl)-1-methoxycarbonyl-ethylcarbamoyl)-biphenyl-2-carbothiol]-amino}-propionate (3).   Methyl (S,S)-3-(1H-Indol-3-yl)-2-{[2′-(2-(1H-Indol-3-yl)-1-methoxycarbonyl-ethylcarbamoyl)-biphenyl-2-carbonyl]-amino}-propionate (4).   Methyl (S,S)-2-{[2′-(1-methoxycarbonyl-2-methyl-propylthiocarbamoyl)-biphenyl-2-carbothiol]-amino}-3-methyl-butyrate (5).   Methyl (S,S)-2-{[2′-(1-methoxycarbonyl-2-methyl-propylthiocarbamoyl)-biphenyl-2-carbonyl]-amino}-3-methyl-butyrate (6).   Methyl (S,S)-3-(4-hydroxy-phenyl)-2-{2′-[2-(4-hydroxy-phenyl)-1-methoxycarbonyl-ethylthiocarbamoyl]-biphenyl-2-carbothiol]-amino}-propionate (7).   Methyl (S,S)-3-(4-hydroxy-phenyl)-2-{2′-[2-(4-hydroxy-phenyl)-1-methoxycarbonyl-ethylthiocarbamoyl]-biphenyl-2-carbonyl]-amino}-propionate (8).   

     The synthesis of compounds of general formula I of the present invention has been carried out using the methods habitual in organic synthesis, which are known to experts in the art, and which involve the thionation reaction of the corresponding amides using any of the methods described in the bibliography for the synthesis of thioamide ( Topics Current Chemistry  1999, 2047, 127 ; Comprehensive Organic Synthesis . vol 6 p. 419, Pergamon Press, 1991), among which can be mentioned the use of Laweson&#39;s reagent ([2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphethane-2,4-disuphide] ( Bull. Soc. Chim. Belg.  1978, 87, 229). As substrates for the thionation reaction the corresponding amides II are used, 
     
       
                 
         
             
             
         
      
     
     which are prepared by acylation reaction between an acid or acid derivative, as electrophile, and an amine, as nucelophile, using methodologies habitual in organic synthesis, and which are known to experts in the art, ( Chemical Approaches to the Synthesis of Peptides and Proteins . CRC Press, Boca Ratón, 1997 ; Comprehensive Organic Synthesis . vol 6, Pergamon Press, 1991). 
     An essential characteristic of the compounds of the present invention is that they are calpain inhibitors. There exist various isoforms of calpain, which are structurally very similar to each other and, as far as is known, share the same action mechanism. The two most abundant are micro-calpain (or calpain I) and milli-calpain (or calpain II), which are differentiated in in vitro tests in the concentration of calcium necessary for their activation. As the two isoforms of the enzyme are very similar to each other, it has been found in many examples of the bibliography that the calpain inhibitors consist of both enzymes ( Adv. Synth. Catal.  2002, 344, 855). So, in the present invention, when we mention calpain, we are referring to the two isoforms (or isoenzymes) which are included in the definition of calpain. Therefore, another object of the present invention is the use of a compound of formula I as calpain inhibitor. 
     The calpain inhibition capacity has been quantified in terms of the value of IC 50 , which is defined as the concentration of inhibitor that reduces the catalytic activity of an enzyme by half. The lower the value of IC 50 , the more powerful the inhibitor. Inhibition results on calpain I (the most relevant from a physiological point of view) of some compounds of the present invention are shown in table 1 and in  FIG. 1 . Given that calpain II, also known as milli-calpain, needs a greater amount of calcium for activation, it might not possibly have such a relevant physiological role since such a concentration of calcium would cause cell death before the milli-calpain could become activated. For this reason, the inhibition tests have been performed for calpain I, though they can be extrapolated for calpain II. 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Inhibition results on calpain of compounds 
               
               
                 forming the object of this invention 
               
             
          
           
               
                   
                 Compounds 
                 IC 50   
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 94 
                 μM 
               
               
                   
                 2 
                 63 
                 μM 
               
             
          
           
               
                   
                 3 
                 38 pM (=0.038 nM) 
               
             
          
           
               
                   
                 4 
                 69 
                 nM 
               
               
                   
                 5 
                 71 
                 nM 
               
             
          
           
               
                   
                 6 
                 52 pM (=0.052 nM) 
               
             
          
           
               
                   
                 7 
                 796 
                 nM 
               
               
                   
                 8 
                 12 
                 μM 
               
               
                   
                   
               
             
          
         
       
     
     Some of the thioamides represented in  FIG. 1  are the most potent calpain inhibitors described, and they mark a path for the design of therapeutically useful compounds. Owing to the fact that it has been found that overactivation of calpain is involved in numerous degenerative diseases, an additional object of the present invention is the use of a compound of formula I for the treatment or prevention of degenerative diseases and for preparing a drug for the preventive or therapeutic treatment of a degenerative disease, and especially when the degenerative disease is selected from among cerebral ischaemia, cardiac ischaemia, Alzheimer, Parkinson, muscular distrophy, cataracts and demyelinating diseases, and especially if the demyelinating disease is multiple sclerosis. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURE 
         FIG. 1  shows results of the study on biphenyl derived thioamides forming the object of the present invention, and their biological activity as inhibitors of calpain I. 
     
    
    
     EXAMPLES 
     As illustrative examples, though without being limiting, the experimental procedures and spectroscopic and analytical data of some thioamides of formula I are given, along with tests on their biological activity. 
     Example 1 
     General Procedure for the Synthesis of Thioamides 
     On a solution of the corresponding bisamide derived from biphenyl (compounds of type A, 0.35 mmol, 1 equiv.) in toluene (10 ml), small portions of Lawesson&#39;s reagent were added (0.78 mml, 1.1 equiv.). The reaction mixture was stirred at reflux for 2 hours. The evaporation of the solvent gave rise to a residue which was purified by means of column chromatography in order to give the corresponding dithioamides (compounds of type B), together with monothioamides (compounds of type C), which were purified. by means of column chromatography as indicated in each case. 
     
       
                 
         
             
             
         
      
     
     Example 2 
     Methyl (S,S)-3-phenyl-2-{[2′-(2-phenyl-1-methoxycarbonyl-ethylthio-carbamoyl)-biphenyl-2-carbothiol]-amino}—propionate (1) 
     
       
                 
         
             
             
         
      
     
     Following the general procedure, it was synthesised together with compound 2, with a yield of 54% after column chromatography (hexane/AcOEt gradient: 9/1 up to hexane/AcOEt gradient: 1/1). Pale yellow solid. 
     [α] 25   D =−3 (c=0.33, MeOH). 
       1 H-NMR (300 MHz, CDCl 3 , 30° C. mixture of conformers A+a, 1.2:1) δ: 9.00 (d, 1.1H, J NH,CHα =7.0, NH, a), 9.89 (d, 1.1H, J NH,CHα =7.0, NH, A), 7.47-7.23 (m, 12H, H arom , A+a), 7.35-7.05 (m, 6H, H arom , A+a), 5.40 (m, 0.9H, C H   α -Phe, a), 5.24 (m, 1.1H, C H   α -Phe, A), 3.67 (s, 3.2H, CO 2 C H   3 , A), 3.58 (s, 2.8H, CO 2 C H   3 , a), 3.25 (A of ABX, 0.9H, J Hα,Hβ =13.9, J Hα,CHα =8.1, C( H   α H β )-Phe, a), 3.14 (B of ABX, 0.9H, J Hβ,Hα =13.9, J Hβ,CHα =5.2, C(H α   H   β )-Phe, a), 2.95 (A of ABX, 1.1H, J Hα,Hβ =13.9, J Hα,CHα =8.1, C( H   α H β )-Phe, A), 2.81 (B of ABX, 1.1H, J Hβ,Hα =5.2, J Hα,CHα =8.1, C(H α   H   β )-Phe, A), ppm 
       13 C-NMR (75 MHz, CDCl 3 , 30° C. mixture of conformers A+a) δ: 200.2 (s, CS, a), 200.0 (s, CS, A), 170.8 (s, CO, A), 170.7 (s, CO, a), 141.7 (s, C arom , a), 141.6 (s, C arom , A), 137.2 (s, C arom , A), 137.0 (s, C arom , a), 135.6 (s, 2C, C arom , A+a), 130.1 (d, C arom ,), 129,7 (d, C arom ,), 129.2 (d, C arom ,), 129.1 (d, C arom ,), 129.0 (d, C arom ,), 128.6 (d, C arom ,), 127.9 (d, C arom ,), 127.7 (d, C arom ,), 127.5 (d, C arom ,), 127.4 (d, C arom ,), 127.2 (d, C arom ,), 59.2 (d,  C H α -Phe, A), 59.0 (d,  C H α -Phe, a), 52.5 (c, CO 2   C H 3 , a), 52.3 (c, CO 2   C H 3 , A), 36.8 (t,  C H 2 -Phe, a), 36.4 (t,  C H 2 -Phe, A), ppm 
     IR v 3436, 3025, 2951, 1743, 1631, 1530, 1496, 1434, 1374, 1215, 956, 749 700 cm −1 . 
     ME (ES + ), m/e=597 ([MH] + , 100%). 
     EA calculated for C 34 H 32 N 2 O 4 S 2 : C, 68.43; H, 5.40; N, 4.69; S, 10.75. Found: C, 68.25; H, 5.19; N, 4.61; S, 10.51. 
     Example 3 
     Methyl (S,S)-3-phenyl-2-{[2′-(2-phenyl-1-methoxycarbonyl-ethylthio-carbamoyl)-biphenyl-2-carbonyl]-amino}-propionate (2) 
     
       
                 
         
             
             
         
      
     
     It was synthesised together with compound 2, following the general procedure indicated in example 1. A yield of 45% after purification in column (hexane/AcOEt gradient: 9/1 up to hexane/AcOEt gradient: 1/1). Pale yellow solid. 
     [α] D =−10 (c=0.11, MeOH). 
       1 H-NMR (400 MHz, CDCl 3 , 30° C. mixture of conformers A+a, 4:3) δ: 10.02 (d, 0.57H, J NH,CHα =7.5, NH-Phe 2 , A), 9.89 (d, 0.43H, J NH,CHα =7.5, NH-Phe 2 , a), 7.61 (dd, 0.57H, J ortho =7.7, J meta =1.5, H arom , A), 7.49 (dd, 0.43H, J ortho =7.7, J meta =1.5, H arom , a), 7.44-7.15 (m, 15H, H arom , A+a), 7.11 (dd, 0.57H, J ortho =8.1, J meta =1.5, H arom , A), 7.07 (dd, 0.43H, J ortho =7.8, J meta =1.8, H arom , a), 7.05 (dd, 0.43H, J ortho =7.8, J meta =1.9, H arom , a), 6.96 (dd, 0.57H, J ortho =8.1, J meta =1.4, H arom , A), 6.93 (d, 0.43H, J NH,CHα =7.4, NH-Phe 1 , a), 6.93 (d, 0.57H, J NH,CHα =7.4, NH-Phe 1 , A), 5.24 (X of ABX, 0.57H, C H   α -Phe 1 , A), 5.12 (X of ABX, 0.43H, C H   α -Phe 2 , a), 4.84 (X′ of A′B′X′, 1H, C H   α -Phe 1 , A+a), 3.71 (s, 1.71H, CO 2 C H   3 -Phe 2 , A), 3.66 (s, 1.29H, CO 2 C H   3 -Phe 2 , a), 3.52 (s, 1.71H, CO 2 C H   3 -Phe 1 , A), 3.40 (s, 1.29H, CO 2 C H   3 -Phe 1 , a), 3.23 (A′ of A′B′X′, 0.57H, J H, H =13.9, C( H   α H β )-Phe 1 , A), 3.04 (m, 0.57H, C(H α   H   β )-Phe 1 , A; 0.86H, C H   2 -Phe 1 , a; 0.86H, C H   2 -Phe 2 , a); 2.43 (A of ABX, 0.57H, J Hα,Hβ =13.7, J Hα,CHα =7.8, C( H   α H β )-Phe 2 , A); 2.31 (B of ABX, 0.57H, J Hβ,Hα =13.7, J Hβ,CHα =5.2, C(H α   H   β )-Phe 2 , A), ppm. 
       13 C-NMR (75 MHz, CDCl 3 , 30° C. mixture of conformers A+a, 4:3) δ: 200.4 (s, CS, a), 199.8 (s, CS, A), 171.9 (s, CO, a), 171.6 (s, CO, A), 170.7 (s, CO, A), 170.5 (s, CO, a), 169.7 (s, CO, a), 169.6 (s, CO, A), 142.3 (s, C arom , a), 142.2 (s, C arom , A), 139.7 (s, C arom , a), 139.2 (s, C arom , a), 136.2 (s, C arom , a), 136.1 (s, C arom , A), 136.0 (s, 2C, C arom , A), 135.7 (s, 2C, C arom , a), 134.9 (s, C arom , a), 134.5 (s, C arom , A), 130.4 (d, C arom ), 130.34 (d, C arom ), 130.0 (d, C arom ), 129.9 (d, C arom ), 129.4 (d, C arom ), 129.3 (d, C arom ), 129.22 (d, C arom ), 129.1 (d, C arom ), 128.9 (d, C arom ), 128.8 (d, C arom ), 128.7 (d, C arom ), 128.65 (d, C arom ), 128.62 (d, C arom ), 128.5 (d, C arom ), 128.4 (d, C arom ), 128.2 (d, C arom ), 127.9 (d, C arom ), 127.8 (d, C arom ), 127.7 (d, C arom ), 127.5 (d, C arom ), 127.3 (d, C arom ), 127.1 (d, C arom ), 126.9 (d, C arom ), 126.8 (d, C arom ), 127.6 (d, C arom ), 126.4 (d, C arom ), 59.6 (d,  C H α -Phe 2 , A), 59.5 (d,  C H α -Phe 2 , a), 53.6 (d,  C H α -Phe 1 , a), 53.4 (d,  C H α -Phe 1 , A), 52.45 (c, CO 2   C H 3 -Phe 2 , A), 52.42 c, CO 2   C H 3 -Phe 2 , a), 52.2 (c, CO 2   C H 3 -Phe 1 , a), 52.0 (c, CO 2   C H 3 -Phe 1 , A), 37.7 (t,  C H 2 -Phe 1 , a), 37.4 (t,  C H 2 -Phe 1 , A), 37.3 (t,  C H 2 -Phe 2 , a), 36.5 (t,  C H 2 -Phe 2 , A), ppm 
     IR v 3436, 3027, 2950, 1744, 1639, 1535, 1436, 1368, 1219, 754, 701 cm −1 . 
     ME (ES + ), m/e=581 ([MH] + , 100%), 603 ([MH] + , 36%), 
     EA calculated for C 34 H 32 N 2 O 5 S: C, 70.32; H, 5.55; N, 4.82; S, 5.52. Found: C, 70.25; H, 5.49; N, 4.69; S, 5.81. 
     Example 4 
     Methyl (S,S)-3-(1H-Indol-3-yl)-2-{[2′-(2-(1H-Indol-3-yl)-1-methoxycarbonyl-ethylthiocarbamoyl)-biphenyl-2-carbothioyl]-amino}-propionate (3) 
     
       
                 
         
             
             
         
      
     
     It was obtained, together with compound 4, following the general procedure, with a yield of 37% after column chromatography (hexane/AcOEt gradient: 9/1 up to hexane/AcOEt gradient: 3/7). Pale yellow solid. 
     m.p.=125-127° C. 
     [α] D =−31 (c=0.25, MeOH). 
       1 H-NMR (300 MHz, DMSO-d 6 ) δ: 10.82 (s, 2H, 2-NH-Indol), 10.71 (s wide, 2H, 2-NH-Trp), 7.46-7.20 (m, 10H, H arom ), 7.10-6.97 (m, 8H, H arom ), 5.07 (m, 2H, C H   α -Trp), 3.50 (s, 6H, CO 2 C H   3 ), 2.97 (m, 1H, C( H   α H β )-Trp), 2.73 (m, 1H, C(H α   H   β )-Trp), ppm. 
       13 C-NMR (75 MHz, DMSO-d 6 ) δ: 199.3 (s, 2C, CS), 170.1 (s, 2C, CO), 141.1 (s, 2C, C arom ), 136.1 (s, 6C, C arom ), 128.9 (d, 2C, C arom ), 127.6 (d, 2C, C arom ), 126.8 (d, 2C, C arom ), 123.6 (s, 4C, C arom ), 121.1 (d, 4C, C arom ), 118.5 (s, 2C, C arom ), 117.9 (d, 2C, C arom ), 111.5 (d, 2C, C arom ), 59.1 (d, 2C,  C H α -Trp), 51.0 (c, 2C, CO 2   C H 3 ), 26.5 (t, 2C,  C H 2 -Trp), ppm 
     IR v 3413, 3012, 2949, 2846, 1737, 1621, 1513, 1457, 1434, 1373, 1216, 1095, 1009, 957, 743 cm −1 . 
     ME (ES + ), m/e=675 ([MH] + , 100%), 697 ([MNa] + , 9%). 
     EA calculated for C 38 H 34 N 4 O 4 S 2 : C, 67.63; H, 5.08; N, 8.30; S, 9.50. Found: C, 67.51; H, 5.32; N, 8.04; S, 9.21. 
     Example 5 
     Methyl (S,S)-3-(1H-Indol-3-yl)-2-{[2′-(2-(1H-Indol-3-yl)-1-methoxycarbonyl-ethylthiocarbamoyl)-biphenyl-2-carbonyl]-amino}-propionate (4) 
     
       
                 
         
             
             
         
      
     
     It was obtained together with compound 3, following the general procedure, with a yield of 35% after column chromatography (hexane/AcOEt gradient: 9/1 up to hexane/AcOEt gradient: 3/7). Pale yellow solid. 
     [α] 25   D =−12 (c=0.15, MeOH). 
       1 H-NMR (300 MHz, CD 3 OD) δ: 10.80 (s, 1H, NH-Indol), 10.72 (s, 1H, NH-Indol), 10.70 (s wide, 1H, NH-Trp 2 ), 8.80 (d, 1H, J NH,CHα =6.8, NH-Trp 1 ), 7.67 (m, 2H, H arom ), 7.59-7.29 (m, 8H, H arom ), 7.28-7.03 (m, 8H, H arom ), 5.32 (m, 1H, C H   α -Trp 2 ), 4.86 (m, 1H, C H   α -Trp 1 ), 3.77 (s, 3H, CO 2 C H   3 ), 3.72 (s, 3H, CO 2 C H   3 ), 3.37 (m, 1H, C H   α H β -Trp 2 ; 1H, C H   α H β -Trp 1 ), 3.12 (m, 1H, CH α   H   β-Trp   1 ), 3.02 (m, 1H, CH α   H   β -Trp 2 ), ppm. 
       13 C-NMR (75 MHz, CD 3 OD) δ: 202.0 (s, CS), 173.6 (s, CO), 172.4 (s, CO), 172.3 (s, CO), 143.4 (s, 2C, C arom ), 138.0 (s, 4C, C arom ), 136.4 (s, C arom ), 130.8 (d, C arom ), 130.7 (d, C arom ), 130.5 (d, C arom ), 129.8 (d, C arom ), 129.0 (d, C arom ), 128.5 (d, C arom ), 128.6 (d, C arom ), 124.4 (d, C arom ), 122.4 (d, 2C, C arom ), 119.8 (d, C arom ), 119.3 (d, C arom ), 119.2 (d, C arom ), 112.4 (d, 2C, C arom ), 110.7 (s, C arom ), 110.4 (s, C arom ), 60.7 (d,  C H α -Trp 2 ), 55.2 (d,  C H α -Trp 1 ), 52.7 (c, 2C, CO 2   C H 3 ), 28.3 (t, 2C,  C H 2 -Trp 1 ), 27.9 (t,  C H 2 -Trp 2 ), ppm 
     IR v 3413, 3054, 2949, 1737, 1640, 1522, 1491, 1457, 1436, 1354, 1217, 1094,1009, 744 cm −1 . 
     ME (ES + ), m/e=659 ([MH] + , 100%), 681 ([MNa] + , 16%). 
     EA calculated for C 38 H 34 N 4 O 5 S: C, 69.28; H, 5.20; N, 8.50; S, 4.87. Found: C, 69.13; H, 5.41; N, 8.58; S, 5.01. 
     Example 6 
     Methyl (S,S)-2-{[2′-(1-methoxycarbonyl-2-methyl-propylthiocarbamoyl)-biphenyl-2-carbothioyl]-amino}-3-methyl-butyrate (5) 
     
       
                 
         
             
             
         
      
     
     It was obtained together with compound 6, with a yield of 67% after column chromatography (hexane/AcOEt gradient : 9/1 up to hexane/AcOEt gradient: 4/6), using the general method described. Pale yellow solid. 
     [α] 25   D =+3 (c=0.28, MeOH). 
       1 H-NMR (300 MHz, DMSO-d 6 ) δ: 10.45 (s wide, 2H, 2-NH-Val), 7.36 (m, 6H, H arom ), 7.11 (m, 2H, H arom ), 4.72 (m, 2H, C H   α -Val), 3.60 (s, 6H, CO 2 C H   3 ), 2.03 (m, 1H, CH-Val), 0.81 (s wide, 6H, CH(C H   3 )(CH 3 )-Val), 0.72 (s wide, 6H, CH(CH 3 )(C H   3 )-Val), ppm 
       13 C-NMR (75 MHz, DMSO-d 6 ) δ: 200.3 (s, 2C, CS), 169.7 (s, 2C, CO), 141.3 (s, 2C, C arom ), 136.5 (s, 2C, C arom ), 129.3 (d, 2C, C arom ), 128.8 (d, 2C, C arom ), 128.0 (d, 2C, C arom ), 127.4 (d, 2C, C arom ), 64.2 (d, 2C,  C H α -Val), 51.7 (c, 2C, CO 2   C H 3 ), 29.8 (d, 2C, CH(CH 3 ) 2 -Val), 18.8 (c, CH( C H 3 )(CH 3 )-Val), 18.5 (c, CH(CH 3 )( C H 3 )-Val), ppm. 
     IR v 3437, 3005, 2965, 2873, 1742, 1633, 1525, 1467, 1434, 1376, 1260, 1207, 1152, 1111, 1004, 748 cm −1 . 
     ME (ES + ), m/e=501 ([MH] + , 100%), 523 ([MH] + , 35%). 
     EA calculated for C 26 H 32 N 2 O 4 S 2 : C, 62.37; H, 6.44; N, 5.60; S, 12.81. Found: C, 62.18; H, 6.61; N, 5.58; S, 12.68. 
     Example 7 
     Methyl (S,S)-2-{[2′-(1-methoxycarbonyl-2-methyl-propylthiocarbamoyl)-biphenyl-2-carbonyl]-amino}-3-methyl-butyrate (6) 
     
       
                 
         
             
             
         
      
     
     It was obtained together with compound 5, following the general procedure, with a yield of 11% after column chromatography (hexane/AcOEt gradient: 9/1 up to hexane/AcOEt gradient: 4/6). Pale yellow solid. 
       1 H-NMR (300 MHz, CD 3 OD) δ: 10.57 (s wide, 1H, NH-Val 2 ), 8.67 (d, 1H, J NH,CH =8.3, NH-Val 1 ), 7.54 (m, 1H, H arom ), 7.41 (m, 5H, H arom ), 7.27-7.09 (m, 2H, H arom ), 4.85 (m, 1H, C H   α -Val 2 ), 4.27 (m, 1H, C H   α -Val 1 ), 3.69 (s, 3H, CO 2 C H   3 ), 3.67 (s, 3H, CO 2 C H   3 ), 2.06 (m, 2H, C H -Val), 0.87 (m, 6H, CH(C H   3 ) 2 -Val), 0.75 (m, 6H, CH(C H   3 ) 2 -Val), ppm 
       13 C-NMR (75 MHz, CD 3 OD) δ: 203.0 (s, CS), 173.2 (s, CO), 172.8 (s, CO), 172.0.8 (s, CO), 143.8 (s, C arom ), 140.4 (s, C arom ), 138.7 (s, C arom ), 136.8 (s, C arom ), 131.2 (d, C arom ), 130.9 (d, 2C, C arom ), 130.0 (d, C arom ), 128.9 (d, 2C, C arom ), 128.8 (d, 2C, C arom ), 65.4 (d,  C H α -Val 2 ), 59.8 (d,  C H α -Val 1 ), 52.4 (c, 2C, CO 2   C H 3 ), 32.1 (d,  C H(CH 3 ) 2 -Val), 31.8 (d,  C H(CH 3 ) 2 -Val), 19.4 (c, CH( C H 3 )(CH 3 )-Val), 19.2 (c, CH(CH 3 )( C H 3 )-Val), 19.1 (c, CH( C H 3 )(CH 3 )-Val), 18.6 (c, CH(CH 3 )( C H 3 )-Val), ppm. 
     IR v 3430, 3021, 2963, 2923, 2873, 1742, 1642, 1532, 1468, 1435, 1376, 1315, 1262, 1207, 1155, 1100, 1020, 801, 756 cm −1 . 
     ME (ES + ) m/e=485 ([MH] + , 100%). 
     EA calculated for C 26 H 32 N 2 O 5 S: C, 64.44; H, 6.66; N, 5.78; S, 6.62. Found: C, 64.68; H, 6.76; N, 5.53; S, 6.65. 
     Example 8 
     Methyl (S,S)-3-(4-hydroxy-phenyl)-2-{2′-[2-(4-hydroxy-phenyl)-1-methoxycarbonyl-ethylthiocarbamoyl]-biphenyl-2-carbothioyl]-amino}-propionate (7) 
     
       
                 
         
             
             
         
      
     
     It was obtained together with compound 8, following the general procedure, with a yield of 3% after column chromatography (CH 2 Cl 2 /AcOEt gradient: 9/1 up to CH 2 Cl 2 /AcOEt gradient: 1/1). Pale yellow solid. 
     m.p.=62-65° C. [α] 25   D =−4 (c=0.15, MeOH). 
       1 H-NMR (300 MHz, CDCl 3 , 30° C., mixture of conformers A+a, 3:2) δ: 8.97 (d, 0.8H, J NH,CHα =8.0, NH, a), 7.80 (d, 1.2H, J NH,CHα =7.0, NH, A), 7.46-7.02 (m, 8H, H arom , A+a), 6.97 (d, 1.6H, J ortho =8.3, H arom -Tyr, a), 6.88 (d, 2.4H, J ortho =8.3, H arom -Tyr, A), 6.71 (d, 2.4H, J ortho =8.3, H arom -Tyr, A), 6.70 (d, 1.2H, J ortho =8.3, H arom -Tyr, A), 5.32 (m, 1.2H, C H   α -Tyr, A), 5.22 (m, 0.8H, C H   α -Tyr, a), 3.67 (s, 3.6H, CO 2 C H   3 , A), 3.56 (s, 2.4H, CO 2 C H   3 , a), 3.15 (A of ABX, 0.8H, J Hα,Hβ =14.1, J Hα,CHα =8.1, C( H   α H β )-Tyr, a), 3.14 (B of ABX, 0.8H, J Hβ,Hα =14.1, J Hβ,CHα =5.2, C(H α   H   β )-Tyr, a), 2.92 (A of ABX, 1.2H, J Hα,Hβ =14.1, J Hα,CHα =8.1, C( H   α H β )-Tyr, A), 2.74 (B of ABX, 1.2H, J Hβ,Hα =14.1, J Hβ,CHα =5.2, C(H α   H   β )-Tyr, A), ppm. 
       13 C-NMR (75 MHz, CDCl 3 , 30° C. mixture of conformers A+a, 3:2) δ: 200.2 (s, CS, a), 200.0 (s, CS, A), 170.9 (s, CO, A), 170.7 (s, CO, a), 154.7 (s, C arom , A), 153.7 (s, C arom , a), 141.6 (s, C arom , A), 141.5 (s, C arom , a), 137.2 (s, C arom , A), 137.0 (s, C arom , a), 130.4 (d, C arom , A), 130.2 (d, C arom , a), 129.9 (d, C arom , a), 129.8 (d, C arom , A), 129.2 (d, C arom , A), 129.1 (d, C arom , a), 127.8 (d, C arom , a), 127.7 (d, C arom ,), 127.7 (d, C arom , A), 127.6 (d, C arom , A), 127.5 (d, C arom , a), 115.5 (d, C arom , A), 115.4 (d, C arom , a), 59.3 (d,  C H α -Tyr, a), 59.1 (d,  C H α -Tyr, A), 52.5 (c, CO 2   C H 3 , a), 52.4 (c, CO 2   C H 3 , A), 36.0 (t,  C H 2 -Tyr, A), 35.7 (t,  C H 2 -Phe, a), ppm 
     IR v 3426, 3025, 2951, 1743, 1631, 1530, 1496, 1434, 1374, 1215, 956, 749, 700 cm −1 . 
     ME (ES + ), m/e=629 ([MH] + , 100%), 651 ([MNa] + , 15%). 
     EA calculated for C 34 H 32 N 2 O 6 S 2 : C, 64.95; H, 5.13; N, 4.46; S, 10.20. Found: C, 65.21; H, 5.28; N, 4.51; S, 10.51. 
     Example 9 
     Methyl (S,S)-3-(4-hydroxy-phenyl)-2-{2′-[2-(4-hydroxy-phenyl)-1-methoxycarbonyl-ethylthiocarbamoyl]-biphenyl-2-carbonyl]-amino}-propionate (8) 
     
       
                 
         
             
             
         
      
     
     It was obtained together with compound 7, following the general procedure, with a yield of 9% after column chromatography (CH 2 Cl 2 /AcOEt gradient : 9/1 up to CH 2 Cl 2 /AcOEt gradient: 1/1). Pale yellow solid. 
     m.p.=87-91° C. [α] 25   D =−12 (c=0.51, MeOH). 
       1 H-NMR (300 MHz, CD 3 OD) δ: 10.72 (s wide, 1H, NH-Tyr 2 ), 8.78 (d wide, 1H, NH-Tyr 1 ), 7.32 (m, 8H, H arom ), 7.02-6.76 (m, 4H, H arom -Tyr), 6.68 (d, 4H, J ortho =8.0, H arom -Tyr), 5.06 (m, 1H, C H   α -Tyr 2 ), 4.55 (m, 1H, C H   α -Tyr 1 ), 3.57 (s, 3H, CO 2 C H   3 -Tyr 2 ), 3.55 (s, 3H, CO 2 C H   3 -Tyr 1 ), 3.02-2.59 (m, 2H, C( H   α   H   β )-Tyr 2 ; 1H C( H   α H β )-Tyr 1 ), 2.37 (m, 1H, C(H α   H   β -Tyr 1 ), ppm. 
       13 C-NMR (75 MHz, CD 3 OD) δ: 202.2 (s, CS), 171.3 (s, CO), 170.2 (s, CO), 170.1 (s, CO), 155.5 (s, C arom ), 155.4 (s, C arom ), 141.7 (s, C arom ), 141.5 (s, C arom ), 138.4 (s, 3C, C arom ), 136.4 (s, C arom ), 134.4 (d, C arom ), 129.3 (d, 2C, C arom ), 129.0 (d, 2C, C arom ), 128.7 (d, C arom ), 128.0 (d, C arom ), 127.0 (d, C arom ), 126.7 (d, 2C, C arom ), 114.4 (d, 2C, C arom ), 59.7 (d,  C H α -Tyr 2 ), 53.9 (d,  C H α -Tyr 1 ), 50.8 (c, CO 2   C H 3 ), 50.7 (c, CO 2   C H 3 ), 35.6 (t,  C H 2 -Tyr 2 ), 35.1 (t,  C H 2 -Tyr 1 ), ppm 
     IR v 3427, 3014, 2949, 1735,1635, 1515, 1437, 1363, 1223, 755, 701 cm −1 . 
     ME (ES + ), m/e=613 ([MH] + , 100%), 635 ([MNa] + , 25%). 
     EA calculated for C 34 H 32 N 2 O 7 S: C, 66.65; H, 5.26; N, 4.57; S, 5.23. Found: C, 66.86; H, 5.39; N, 4.61; S, 5.51. 
     Example 10 
     Enzyme Activity Test: Inhibition of Calpain 
     The calpain inhibition capacity has been quantified in terms of the value of IC 50 , which is defined as the concentration of inhibitor that reduces the catalytic activity of an enzyme by half. The lower the value of IC 50 , the more powerful the inhibitor. Inhibition results on calpain I (the most relevant from a physiological point of view) of some compounds of the present invention are shown in table 1 and in  FIG. 1 . 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Inhibition results on calpain of compounds 
               
               
                 forming the object of this invention 
               
             
          
           
               
                   
                 Compounds 
                 IC 50   
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 94 
                 μM 
               
               
                   
                 2 
                 63 
                 μM 
               
             
          
           
               
                   
                 3 
                 38 pM (=0.038 nM) 
               
             
          
           
               
                   
                 4 
                 69 
                 nM 
               
               
                   
                 5 
                 71 
                 nM 
               
             
          
           
               
                   
                 6 
                 52 pM (=0.052 nM) 
               
             
          
           
               
                   
                 7 
                 796 
                 nM 
               
               
                   
                 8 
                 12 
                 μM 
               
               
                   
                   
               
             
          
         
       
     
     In order to carry out the inhibition test on calpains, we used the EnzCheck® Protease Assay Kit E-338 from Molecular Probes. This kit contains casein marked with BODIPY FL®, freeze-dried from phosphate buffer. Also used as a digestion buffer was Tris-HCl at pH 7.8, containing 2 mM of sodium azide. The calpain I from porcine erythrocytes or calpain II from porcine kidney that were used are commercial products from CALBIOCHEM®. The enzyme stock solution contained 20 mM of imidazol-HCl buffer, pH 6.8, 1 mM EDTA, 1 mM EGTA, 5 mM β-mercaptoethanol, 30% in glycerol. 
     The synthetic inhibitors were dissolved in 250 μL of DMSO. The tests were conducted on 96-well microplates in a final volume of 200 μL. In order to carry out the test, aliquots of stock solution of calpain were added to 150 μL of a 10 μg/μL solution in digestion buffer in order to obtain a final concentration of 50 ng/mL of enzyme. Minimum quantities were added, between 5 and 20 μL of inhibitor solution in DMSO, previously diluted in order to achieve the desired final concentration of inhibitor, and digestion buffer was added until a volume of 190 μL was achieved. The test commenced by adding 10 μL of 0.05 M CaCl 2  solution. For each inhibitor, separate measurements excluding inhibitor, calpain and calcium solution were taken as blanks. All measurements were made in triplicate. 
     The fluorescence measurements were made in a SPECTRAFLUOR TECAN Corp 93382 spectrum fluorimeter, exciting at 485 nm and taking the reading at 530 nm. The measurement was made over 20 cycles until the measurement of all wells was completed. Stirring was orbital type and took place each cycle. 
     ABBREVIATIONS 
     Stated below are the meaning of the abbreviations used:
         EA: Element analysis   CANP: Calcium activated neutral protease   DMSO: Dimethylsulphoxide   EDTA: Ethylenediaminetetraacetic acid   EGTA: Ethylene-bis-(oxyethylenenitrile)tetraacetic acid   ME: Mass spectrum   ES: Electro-spray   IR: Infrared   NMDA: N-methyl-D-aspartate   m.p.: melting point   Tris: Tris(hydroxymethyl)aminomethane     1 H-NMR: Proton nuclear magnetic resonance     13 C-NMR: Carbon-13 nuclear magnetic resonance