BRPI0902520A2 - serine protease inhibiting pseudopeptide compounds, serine protease inhibiting compositions and pharmaceutical compositions containing such compounds - Google Patents

Abstract

Serine Protease Inhibitor Pseudopeptide Compounds, Serino Protease Inhibitor Compositions and Pharmaceutical Compositions Containing Such Compounds Abstract The present invention relates to serine protease inhibitor pseudopeptide compounds that have a rigid core derived from isomanide D-mannitol, capable of conferring rigidity to the inhibitor, and which due to its chemical nature can also act on polymerases, such as DNA or RNA polymerases, inhibiting them. These compounds that inhibit serine proteases and polymerases are the basis of compositions that inhibit serine protease, as well as pharmaceutical compositions for the development of potential antiviral drugs, against viruses of the Flaviviridae family, being mainly related to the Hepatitis C virus (HCV) and the virus of Dengue.

Description

Serino Protease Inhibitor Pseudopeptide Compounds, Serino Protease Inhibitor Compositions and Compositions

Pharmaceuticals Containing Such Compounds

Field of the Invention

The present invention relates to pseudopeptides active against flaviviridae viruses, designed as serine protease inhibitors and epotentially polymerase inhibitors characterized by having, in the central portion, a structure of the type 1,4: 3,6-dianhydromanitol. The side portions are characterized by having peptidomimetic bonds from the opening of various oxazolones. These novel compounds active against the flaviviridae family viruses are the basis for the preparation of antiviral pharmaceutical compositions capable of ceasing mainly HCV virus proliferation and dengue virus.

Background of the Invention

The family Flaviviridae comprises over 60 viruses, many of which are pathogenic to humans. These viruses include those related to Hepatitis C virus (HCV), West Nile Fever (or West Nile Fever), Yellow fever and Dengue fever. In the case of hepatitis viruses, there are at least seven types of viruses that have already been characterized: A, B, C, D, E, G and TT, which have hepatotropism in common and may lead to liver inflammation and necrosis. Hepatitis is the generic term of Greek-hepatic origin (liver) and - / f / s (inflammation), which means inflammation of the liver. Viral hepatitis, in particular, is caused by different globally distributed etiologic agents, including HCV (Craxi, A.; Laffi, G.; Zignego, AL Moi Aspects Med. 2008, 29, 85-95; Manns, MP; Foster, GR; Rockstroh, JK; Zeuzem, S.; Zoulim, F.; Houghton, M. Nat. Rev. Drug Discov.2007, 6, 991-1000).

In structural terms, HCV is an enveloped virus with an RNA genome of approximately 9.4Kb. The genome has a single open reading window encoding three structural proteins (heartwood, E1, E2) and these nonstructural proteins (p7, NS2 to NS5B). Two untranslated regions (RTUs) present at each end of the genome are important for RNA genome translation and transcription steps. A great variety in the HCV genomic sequence has been described. The different genotypes were brought together into six main groups and various subtypes. There is a different geographic distribution in relation to the different genotypes. In Brazil, the most frequent are genotypes 1, 2 and 3 with an incidence of 70; 2.5 and 28%, respectively.

The viral genome encodes a single long polyprotein of approximately 3000 amino acids. This polyprotein is subsequently processed by virus and host-encoded proteases, generating 10 mature viral proteins. The core proteins E1 and E2 are structural and are involved with viral particle assembly, while proteins NS2 through NS5B are called nonstructural and are involved with virus propagation. These non-structural proteins include the NS3 virus-specific helicase, the NS3-NS4A protease and the NS5B polymerase-RNA-dependent RNA (RdRp, or replicase).

Current therapy is based on the use of interferon alfa combined with ribavirin. IFN-alpha treatment alone has only 10-19% sustained response. Ribavirin is a synthetic guanosine analogue that directly targets RNA and DNA viruses by probable mechanism of inhibition of virus-dependent DNA polymerase. Ribavirin alone, however, has no effect on hepatitis C. Combination of interferon-alpha with Ribavirin improves sustained virological response to 38-43%, correspondingly improving histological analysis (biopsy) and possibly long-term complications. However, there are no convincing prospective studies (Heathcote, EJJ Viral Hepat. 2007, 14, Suppl 1.82-8; Koike, KJ Infect. Chemother. 2006, 12, 227-32; Toniutto, P.; Fabris, C. Pirisi, M. Expert, Opin, Pharmacother, 2006, 7, 2025-35).

Even with this therapeutic arsenal, HCV infection is responsible for over 80% of liver transplants worldwide, and in Brazil. (Consensus of the Paulista Society of Infectology (SPI) for Hepatitis C Management and Therapy, São Paulo, SP.) Several new compounds have been evaluated for the treatment of HCV infections. These compounds are in early stages of evaluation, ie in vitro or preclinical animal studies. However, most of these compounds have not yet been analyzed in screening stages involving humans (clinical studies). Overall, only 1 in 1,000 new compounds is ever evaluated in stages involving humans. Of these, only 1 in 5 is approved by the US Food and Drug Administration, FDA, US Food and Drug Administration.

Several efforts have focused on the list of treatments that have shown promise in clinical trials involving humans. Table 1 below shows some compounds with potential to inhibit HCV infection and the clinical stages that are present,

Table 1 - Compounds with potential inhibition of HCV infection and their respective clinical phases. (Soriano, V .; Madejon, A .; Vispo, E .; Labarga1 P.; Garcia-Samaniego1 J .; Martin-Carbonero, L .; Sheldon, J .; Bottecchia1 M .; Tuma1P .; Barreiro, P. Expert (Op. Emerg. Drugs 2008, 73, 1-19).

<table> table see original document page 4 </column> </row> <table> * RNA polymerase inhibitors ** protease inhibitors

The targets of anti-HCV therapy are concentrated at the different stages of viral RNA replication, and in addition to these new therapies are directed at translation (ribozymes, antisense oligos, RNAi), proteolytic processing (viral protease inhibitors) and replication steps. dogenoma RNA (viral RNA polymerase inhibitors).

Valopicitabine (Idenix Pharmaceuticals), already at an advanced stage of this clinical trial, is characterized by being a viral RNA-RNA polymerase inhibitor, and the analysis involving 79 patients showed a 90% reduction in viral load within two weeks, and most of these patients did not responded to interferon treatment. Valopicitabine will be used as a third treatment medication, along with interferon and ribavirin, to achieve a greater therapeutic response. Two other drugs, one of which should be used as a third drug in treatment are deprotease inhibitors: SCH-503034 (Schering-Plow) and VX-950 (Vertex). In a study conducted by Vertex in 34 volunteers with VX-950, showed that after two weeks of treatment the viral load decrease was 250 times greater than the pegylated Interferon combined with Ribavirin in reducing the viral load.

HCV has two potential targets for the development of new antiviral drugs. One is the (NS5) RNA-dependent RNA polymerase molecule that can replicate one RNA molecule into another. This is a RNA virus-only activity, and this activity is not found in host cells. Thus RNA-dependent RNA polymerase is a very specific target for HCV. In addition to viral replicase we also have NS3 (serine protease) which is responsible for processing viral polyprotein. Despite being an unserine protease, NS3 has a very different molecular structure than the human cell proteases, which enables the development of inhibitors specific for this protease.

The molecular weight of HCV NS3 protease monomer is 70 kDa. Its active structure is a dimer and the tertiary monomer structure has an extension at the N-terminal portion, 2 domains with 6 beta-barrels ) each, as well as an exchange domain and a alfelix. The active site of the protease is located in the N-terminal portion. Comparing the NS3 sequence of other flaviviruses we can identify three conserved residues (His-57, Asp-81, and Ser-139) that make up the centrocatalytic of this protease. The active center of the NS3 protease contains a tetrahedral oxyion stabilizing site, reaction intermediate, oxyanion / stabilization loop, and an E2 tape and preceded by an E1 tape. NS3 cleaves viral polyprotein between amino acid residues Cys / Ser (at sites 4B / 5A and 5A / 5B), Cys / Ala (no. 4A / 4B), or Thr / Ser (no. 3 / 4A).

NS3 assumes a structure of two beta-stranded tape sets. The first located in the N-terminal portion contains tapes A1, B1, C1, D1, E1, and F1. The second beta barrel consists of tapes A2, B2, C2, D2, E2, and F2.

Several papers have been reported on serine protease inhibitors.

Determining how serine protease acts to enable peptide hydrolysis is an important feature in the search for effective enzyme inhibitors.

Recent studies from our group showed novel series of isomanide-derived pseudopeptide compounds (Esteia MarisFreitas Muri1, Marlito Gomes Júnior, Jerome S. Costa, Marcelo Lima Bastos, Ricardo Hernandez-Valdes, Magaly Girão Albuquerque, Ricardo B. Alencastro , OAC Antunes "Serinaprotease inhibitor compounds, process for obtaining and use for the treatment of flaviviroses" Pl0401908-3, 06/04/2004. RPI 1762 of 10/13/2004, RPI 1828 of 01/17/2006; Muri, EMF ; Gomes Jr., M .; Albuquerque, MG; Cunha, EFF; Alencastro, RB; Williamson, JS; Antunes, OAC Amino Acids 2005, 28, 413-419. Muri, EMF; Gomes Jr., M .; Costa, JS; Alencar, FL Sales Jr., A.; Bastos, ML; Hernandez-Valdes, R.; Albuquerque, MG; Cunha, EFF; Alencastro, RB; Williamson, JS; Antunes, OAC Amino Acids 2004, 27, 153- 157). In this work a molecular modeling (docking) study of an MbBBI fragment (a potent inhibitor of NS3-pro) was performed at the active site of the enzyme protease NS3, thus validating the proposed model for the study with our synthesized compounds. The structurally simplest compound of the esters series had 3 hydrogen bonds with the enzyme. A bond between the NH group of Gly133 and the oxygen atom of the bicyclic ring corresponds to the PV apposition of our compound. The other two bonds correspond to the P1 deposition, that is, the Alanine of the compound interacting with the Ser135 hydroxyl and the enzyme Asn152.

Recently, hexapeptide derivatives containing an α-keto-amide electrophilic function have been described as potent inhibitors of HCV serine protease (Arasappan, A.; Njoroge, FG; Chan, T.-Y .; Bennett, F.; Bogen, SL; Chen , K .; Gu1 H .; Hong1 L .; Jao1 E .; Liu1 Y.-T .; Lovey, RG; Parekh, T.; Pike, RE; Pinto, P.; Santhanam, B .; Venkatraman, S. ; Vaccaro, H.; Wang, H .; Yang, X .; Zhu, Z .; Mckittrick1 B.; Saksena, AK; Girijavallabhan, V.; Pichardo, J .; Butkiewicz1 .; Ingram, N.; Malcolm, B .; Prongay, A.; Yao, N.; Marten, B.; Madison, V.; Kemp, S.; Levy, O .; Lim-Wilby, M.; Tamura, S.; Ganguly, AK Bioorg. Chem. Lett. 2005, 15, 4180-4184). Still within the α-keto amide class, VX-950 and SCH503034 (Bocepravir), previously mentioned, are in clinical development. VX-950 was able to reduce the plasma viral load of patients dosed with 750mg every 8h, and in some patients the virus became undetectable after 14 days of dosing (Chen, KX; Vibulbhan, B.; Yang, W .; Cheng Liu, R. Pichardo, J. Butkiewiez, N. Njoroge, FG Bioorg.Med.Chem., 2008, doi: 10.1016 / jbmc.2007.11.002.Perni, RB; Almquist, SJ; Byrn, RA; Chandorkar, G .; Chaturvedi, PR; Courtney, LF; Decker, CJ; Dinehart, K.; Gates, CA; Harbeson, SL; Heiser, A .; Kalkeri, G .; Kolaczkowski, E .; Lin , K.; Luong, YP .; Rao, BG; Taylor1 WP; Thomson, JA; Tung, RD; Wei, Y.; Kwong, AD; Lin, C. Antimicrob. Agents Chemother. 2006, 50, 899-909) .

Summary of the Invention

The first object of the present invention relates to compound serine protease inhibitors and potentially inhibitors of RNA and DNA polymerases of formula (I) and (II): The second object of this invention relates to a serine protease inhibitor composition containing a compound serine protease inhibitor molecular formula (I):

<formula> formula see original document page 8 </formula>

and / or pharmaceutically acceptable salts thereof for the production of a medicament used with serine protease inhibitors and potentially employed as an inhibitor of DNA and RNA polymerases, as well as pharmaceutically acceptable excipients.

The third object of this invention is a pharmaceutical composition containing the compound of molecular formula (I):

<formula> formula see original document page 8 </formula>

as well as pharmaceutically acceptable salts and / or salts thereof useful in the manufacture of a medicament for the treatment of diseases caused by Flaviviridae viruses in human and non-human mammals, as well as pharmaceutically acceptable excipients.

The fourth object of this invention is a daserine protease inhibiting composition containing a compound of molecular formula (II): <formula> formula see original document page 9 </formula>

as well as derivatives and / or pharmaceutically acceptable salts thereof used for the manufacture of a medicament used as serinoprotease inhibitors and potentially inhibitors of DNA and RNA polymerases, as well as pharmaceutically acceptable excipients.

The last object of this invention is a pharmaceutical composition containing said compound of molecular formula (II),

<formula> formula see original document page 9 </formula>

as well as pharmaceutically acceptable salts and / or derivatives thereof and pharmaceutically acceptable excipients for the manufacture of a medicament used in the treatment of diseases caused by the family Flaviviridae viruses in human and non-human mammals.

Detailed Description of the Invention

The present invention relates to pseudopeptide compounds of general formula (I) and (II), designed as serine protease inhibitors and potentially polymerase inhibitors.

These pseudopeptide compounds are potentially active in flaviviridae family viruses, such compounds are active in inhibiting serine protease and potentially HCV polymerase inhibitors, yellow fever dengue virus virus, west fever virus and Nile fever virus.

The peseudopeptide compounds of general formula (I) and (II) of the present invention:

<formula> formula see original document page 10 </formula>

Where:

R1 Benzyl, carboxybenzyl, carboxy-t-butyl, hydrogen, alkyl or aryl;

R 2 phenyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl, p-trifluoromethylphenyl, p-methoxyphenyl, 2-thienyl, 3,4-methylenedioxyphenyl, 3-pyridyl, 3,4-dimethoxyphenyl, p-cyanophenyl, 2-naphthyl 4,4-methylphenyl; and,

R 3 is benzyl, carboxybenzyl, carboxy-t-butyl, hydrogen, aquyl or aryl.

Preferably,

R1 may be a phenyl or methyl;

R 2 -substituted phenyl or heterocycles; and,

R3 is benzyl (-Bn), carboxybenzyl (-Cbz) or carboxy-t-butyl (-Boc); capable of conferring stiffness to the serine protease inhibitor and which, by their nature, can also act on polymerases, such as DNA or RNA polymerases, by inhibiting them.

Table 1 below shows some of the compounds of formula (I) and (II) obtained, showing the different radicals R1 and R2 actually employed in each compound. Such compounds will be further described and exemplified later.

R2 compound

<table> table see original document page 10 </column> </row> <table> <formula> formula see original document page 11 </formula> <table> table see original document page 12 </column> </row> <table>

Said compounds of formula (I) and (II) and / or pharmaceutically salts, in addition to pharmaceutically acceptable excipients, are used in serine protease inhibiting and potentially polymerase inhibiting compositions such as DNA and RNA polymerases.

Such pharmaceutical composition has a concentration ranging from 10μΜ to 150μΜ of said compound of molecular formula I and / or II, as well as pharmaceutically acceptable derivatives and / or salts thereof.

Preferably, the concentration of the compounds of formula (I) and (II) is from 15μΜ to 100μΜ.

The compounds of formula (I) and (II) and / or their pharmaceutically acceptable salts and pharmaceutically acceptable excipients may also be employed in pharmaceutical compositions for the preparation of medicaments for the treatment of diseases caused by flaviviridae viruses. Preferably the non-inventive falviviruses are hepatitis C viruses, dengue virus, yellow fever virus, west fever virus and Nile fever virus.

Such pharmaceutical composition has a concentration ranging from 10μΜ to 150μΜ of said compound of molecular formula I and / or II, as well as pharmaceutically acceptable derivatives and / or salts thereof.

Preferably, the concentration of the compounds of formula (I) and (II) is from 15μΜ to 100μΜ.

For purposes of this invention the excipients are all those chemical compounds which are intended to give form, volume, stability to the pharmaceutical composition. All excipients to be used herein are those of the pharmaceutical industries such as adjuvants, sweeteners, solvents, stabilizers, antioxidants, preservatives, buffers, flavorants, surfactants, humectants, lubricants, dispersants and all those known to one skilled in the art.

Such excipients may be employed in different pharmaceutical forms such as powders, tablets, capsules, nanocapsules, nanospheres, crystals, ampoules, oral solutions, transdermal patches, injectable muscle or parenteral solutions, ointments, creams, gels, suspensions, emulsions, aerosols, tinctures, elixirs, pastes, pastilles, suppositories, ointments, eggs, and all those known to a skilled artisan.

Such a pharmaceutical composition containing the compounds (I) and / or (II), as well as their pharmaceutically acceptable salts and derivatives, is employed for the manufacture of a medicament in the treatment of diseases caused by the Flaviviridae family viruses, comprising viruses of this family. hepatitis C, dengue fever, yellow fever, west fever or Nile fever, being preferably used in the treatment of diseases caused by the dengue virus and hepatitis C. In the case of hepatitis C, the following viruses A, B, C, D , E, G and TT.

The compounds of general formula (I) were produced from two mannosides as analogues to those previously published by the group, with additional analogue to pro-nucleosides, i.e. compounds capable of inhibiting polymerases. Its structural analogy with cyclic rigid dipeptides makes it an excellent chassis, rigid core, "scaffold". (Bencsik, JR; Kercher, T.; O'Sullivan, M .; Josey, JA Org. Lett. 2003, 5, 2727-2730. Dietrich, E .; Lubell1.D.D. Org. Chem. 2003, 68, 6988-6996 Furthermore, because it has a C2 axis of symmetry, it can be homologated, in two or in one position, maintaining or not the symmetry axis, via reaction with different amino acids edihydroamino acids, for example, via direct coupling catalyzed by EDC. , HOBt, and amino acids or via azalactone opening (parallel synthesis, combinatorial chemistry).

From the data obtained above, then, the desynthesis step of the predicted serine protease inhibitors was passed.

The first step in obtaining the derivatives of formula I is the conversion of isomanide (1) to the di-tosylated derivative (2) by reaction with p-toluenesulfonyl chloride in pyridine for 12h at room temperature. Treatment of di-tosylate (2) with sodium azide in [bmim] BF4 at 120 ° C for 12h provided di-azide (3) with reversal of configuration. Reduction of palladium on carbon di-azide (3) in ethanol for 12h using a pressure of 40psi resulted in diamine (4), which was the substrate for the formation of symmetrical isomanide-derived novel amides (28-45) (Scheme 1 ) (Archibald, TG; Baum, K. Synth. Comm. 1989, 19, 1493-1498. Hockett1 RC; FIetcher J., HG; Sheffield, EL; Goepp Jr., RM; Soltzberg. J. Chem. Soc. 1946, 68, 930-935 (Kuszmann, J.; Medgyes, G. Carbohyd. Res. 1980, 85, 259-269).

Scheme 1. Synthesis of intermediate compounds to obtain diamine (4).

<formula> formula see original document page 14 </formula>

Different solvents may be used in the nucleophilic substitution step, such as DMF and DMSO. However, [bmim] BF4, an ionic liquid, has many advantages over conventional organic solvents and has a better reaction yield. Ionic liquids do not have measurable vapor pressure at ambient temperature, and even at very high temperatures they only degrade above 400 ° C and can be used in a vacuum without loss of solvent. Due to these factors, besides the possibility of their reuse, ionic liquids are nowadays widely used in the concept of Green Chemistry (Andrade, CKZ; Takada, SCS; Alves, LM; Rodrigues, JP; Suarez, PAZ; Brandão, RF ; Soares, VCD Synlett 2004, 12, 2135-2138. Lancaster, NL; Salter, PA; Welton, T.; Young, GBJ Org. Chem 2002, 67, 8855-8861).

The ionic liquid, [bmim] BF4 (7) was synthesized from N-methyl imidazole (5). This reacted with 1-bromobutane forming derivative (6) which upon trochanion with NaBF4 gave rise (7). The ionic liquid was purified by column chromatography on silica gel eluting with dichloromethane, giving 83% yield as shown in scheme 2 (Lancaster, NL; Salter, PA; Welton, T.; Young, GBJ Org. Chem 2002, 67 , 8855-61).

Scheme 2. Ionic Liquid Synthesis [Bmim] + [BF4] "

<formula> formula see original document page 15 </formula>

The next step was to obtain oxazolones (or azalactones) (10-27) from commercially available glycine (8). To obtain the compound (9a), the glycine (8) was reacted with benzoyl chloride in aqueous NaOH solution. Sufficiently pure product was obtained in 95% yield. A / V-acetylglycine (9b) was obtained by the reaction of (8) with refluxing anhydridoacetic acid in aqueous medium and was obtained in 77% yield after precipitation and filtration, as observed in scheme 3 (Mesaik, AM; Rahat, S .; Khan, KM; Zia-Ullah; Choudhary, MI; Murad, S.; Ismail, Z .; Attaur-Rahman and Ahmad, A. Bioorg. Med. Chem. 2004, 12, 2049-2057).

Scheme 3. Synthesis of acylglycine derivatives (9a) and (9b) <formula> formula see original document page 16 </formula>

Derivatives (9a) and (9b) were heated with various aromatic aldehydes in the presence of acetic anhydride and sodium acetate, generating asoxazolones (or azalactones) (10-27) exclusively in Ζ, the most stable isothermodynamically stable (Scheme 4). Table 2 shows the respective yields, melting points and substituents of oxazolones (Jendralla, H.; Seuring1 B.; Herchen, J.; Kulitzscher, B.; Wunner, J.; Stiiber, W.; Koschinsky, R. Tetrahedron 1995 , 51, 12047-12068 Bautista, FM; Campeio, JM; Garcia, A.; Luna1 D.; Marinas, JM; Romero1 AAJ Chem. Soc. Perkin Trans. 2002, 2, 227-234. Meiwes, J .; Schudok Kretzschmar, G. Tetrahedron: Asymmetry, 1997, 8, 527-536. Lisichkina, LN; Ushakova, OM; Alekseeva, MO; Peregudov, AS; Belikov, VM Russ. Chem. Buli. 1999, 48.1682 -1684 Wong1 HNC; Xu, ZL; Chang, HM; Lee, CM Synthesis, 1992,793-797. Hoshina, H.; Kubo1 K.; Morita1 A.; Sakurai T. Tetrahedron, 2000, 56,2941-2951 Slater, G .; Someville, AW Tetrahedron, 1966, 22, 35-42. Paul1 S.; Nanda1 P.; Gupta1 R .; Loupy1 A. Tetrahedron Lett. 2004, 45, 425-427. Kitazawa1M .; Higuchi1 R; Takahashi MJ Phys. Chem 1995, 99, 14784-14792.

Scheme 4. Synthesis of oxazolones <formula> formula see original document page 17 </formula>

Table 2. Oxazolone substituents and melting points

<table> table see original document page 17 </column> </row> <table> <formula> formula see original document page 18 </formula>

The last step consisted of a substituted dasoxazolone (10-27) ring opening reaction with the diamine (4). The reaction was carried out at refluxing ethyl comacetate. After the time required for each reaction, it was cooled to room temperature and the precipitated product was filtered and dried (Scheme 5) (Girgis1 AS; Ellithey, M. Bioorg. Med. Chem. 2006, 14, 8527-8532. Chebanov, VA ; Desenko, SM; Kuzmenko, SA; Borovskoy, VA; Musatov, VI; Sadchikova, YV Russ. Chem. Bul .: Int. Ed., 2004, 53,2845-2849.

Synthesis of pseudopeptide derivatives <formula> formula see original document page 19 </formula>

The synthesis of the serine protease inhibitors provided for by formula II began by monotosylating isomanide (1) with p-toluenesulfonyl chloride and pyridine for 12h resulting in monotosylated compound (46). The hydroxyl group of (46) was protected with chloride of benzyl in basic medium to obtain derivative (47). A nucleophilic substitution reaction of tosyl group by azide in ionic liquid resulted in the formation of compound (48), which was reduced by catalytic hydrogenation using Pd / C in 40ps ethanol to form O-benzylated amine (49) as shown in scheme 6 ( Kumar, S.; Ramachandran, U. Tetrahedron, 2005, 6, 4141-4148. Loupy, A.; Monteux, D. Tetrahedron Lett. 1996, 37, 7023-7026. Chiappe, C.; Pieraccini, D .; Saullo Chem. 2003, 68, 6710-6715. Tamion, R. Marsais, F. Jubereau, P. Queguiner, G. Tetrahedron: Asymmetry, 1993, 4, 1879-1890).

Scheme 6. Synthesis of intermediates to obtain O-benzylated amine

<formula> formula see original document page 19 </formula>

The last step consisted of reacting O-benzylated amine (49) with asoxazolones (10-18) at reflux with ethyl acetate resulting in the formation of peptidomimetic compounds (50-58) as shown in scheme 7.

Scheme 7. Synthesis of compounds (50-58) <formula> formula see original document page 20 </formula>

In addition, diamine (4) is capable of reacting with any amino acid (D- or L-), di- or tripeptide, containing N-BOC, N-Bn or N-CBZ protecting groups, and free carboxyls, via catalyzed coupling by EDC and HOBt1 to provide the corresponding pseudopeptides.

The present invention will be described in detail through the examples given below. It should be noted that the invention is not limited to these examples and includes variations and modifications within the limits to which it applies,

Example 1: Obtaining 1.4: 3,6-Dianhydro-2,5-di-0-p-tosyl-D-mannitol.

To a solution of isomanoid (13mmol, 2g) and pyridine (54mmol, 4.43ml_) in CH 2 Cl 2 (6mL) was added TsCl (41mmol, 7.85g) at 0 ° C. The reaction mixture was stirred for 12h at room temperature. After this time, CH 2 Cl 2 (200mL) was added and then the organic phase was washed with 1N HCl solution (160mL), water (120mL) and saturated NaCl solution. After drying with Na2 SO4 and evaporating the solvent, the residue was purified by successive recrystallizations with methanol or by silica gel column chromatography using a mixture of hexane and ethyl acetate to afford the product as a white solid in 92% yield; Mp: 94 ° C, α D 20 = +96 (c, 0.1) DIVISO.

Example 2: Obtainment of 1,4: 3,6-Dianhydro-2,5-diazido-2,5-dideoxy-L-idithiol.

Di-tosylated compound (2) (7.7mmol, 3.5g) was dissolved in the [Bmim] + BF4 "(46mmol, 9.3mL) ionic liquid and then added to NaN3 (30mmol, 2g). Stirring at 120Â ° C for 12h At the end of the reaction, the flask was cooled to room temperature, ethyl ether was added and the solution was washed with water, dried with Na2SO4, evaporated solvent, the product could be purified by silica gel chromatographic column. Using a mixture of hexane and ethyl acetate afforded the product as a pale yellow oil in 78% yield.

Example 3: Obtaining 1,4: 3,6-Dianhydro-2,5-diamino-2,5-dideoxy-L-idithiol.

To a mixture of di azide (3) (1mmol, 0.25g) in ethanol (10mL) was added 10% Pd / C 10% (0.127mmol, 0.140g). It was hydrogenated at 40 psi for 12h. After this time the mixture was filtered over XAD-4 (amberliteneutral), washed with ethanol and the solvent was evaporated to give a quantitative oil product.

Example 4: Obtaining Lonic Liquid [Bmim] + BF4 "(compound 5).

To a solution of N-methyl imidazole (200mmol, 16mL) in toluene (25mL) at 0 ° C, 1-Bromobutane (230mmol, 25mL) was slowly added and comagitation. The mixture was allowed to reflux (110 ° C) for 36 h. Two phases were formed: the densest (viscous) and the lightest. They were separated. The viscous aphase was washed with ethyl ether (3 x 50mL) and ethyl acetate (3x50mL). Soon after, CH2Cfe (150mL) and NaBF4 (164mmol, 18g) were added. The mixture was stirred for 24h at room temperature. After this time, the mixture was filtered twice, the active carbon was added and left for an additional 24 hours under stirring. At the end, the mixture was filtered, evaporated and finally filtered on aluminum by washing well with CH 2 Cl 2. The product was obtained as a light yellow oil in 83% yield.

Example 5: Obtaining AZ-Benzoylglycine.

Glycine (8) (133mmol, 10g) was dissolved in a 10% NaOH (100mL) solution and then benzoyl chloride (186mmol, 21.6mL) was slowly added with vigorous stirring. Then concentrated HCl gelopic acid was added dropwise until the mixture was acidified (pH 2-3). The mixture was filtered through a Buchner funnel and the precipitate washed with ice water. The product was obtained as a white solid in 95% yield. Mp: 197 ° C.

Example 6: Obtaining AZ-Acetylglycine.

To a solution of glycine (8) (66mmol, 5g) in water (40mL), acetic anhydride was added (140mmol, 26mL). The mixture was stirred vigorously for 1h under mild reflux. At the end of this time, the reaction was cooled to room temperature and left overnight in a refrigerator. The next day, the product was filtered through a Buchner funnel and washed with ice water. The mother liquor was concentrated and filtered again yielding (9b) as a white solid in 77% yield. MP: 205 ° C.

General process for obtaining oxazolones (10-19).

A mixture of / V-benzoyl glycine (9a) (20mmol), the respective aldehyde (18mmol) and acetic anhydride (58mmol) were heated under stirring until the reagents dissolved, and soon thereafter anhydrous sodium acetate (8mmol) was added. By adding sodium acetate, a pasty mixture formed to which a further addition of acetic anhydride (58mmol) was required. The mixture remained at 60 ° C for 3h, then cooled to room temperature and water was added. The precipitate formed was filtered through a Büchner funnel and washed with plenty of ice water. Then the product was recrystallized with the appropriate solvent.

Example 7: 2-Phenyl-4- (Z) -benzylidene-4,5-dihydro-1,3-oxazol-5-one (10). MP: 169-170 ° C. Appearance: yellow solid.

Example 8: 2-Phenyl-4- (Z) -4-fluorobenzylidene-4,5-dihydro-1,3-oxazol-5-one (11) Mp: 186 ° C. Appearance: yellow solid.

Example 9: 2-Phenyl-4- (Z) -4-chlorobenzylidene-4,5-dihydro-1,3-oxazol-5-one (12) Mp: 199-200 ° C. Appearance: yellow solid.

Example 10: 2-Phenyl-4- (4) -4-bromobenzylidene-4,5-dihydro-1,3-oxazol-5-one (13) Mp: 207-208 ° C. Appearance: yellow solid.

Example 11: 2-Phenyl-4- (Z) -4-trifluoromethylbenzylidene-4,5-dihydro-1,3-oxazol-5-one (14)

MP: 174-175 ° C. Appearance: yellow solid.

Example 12: 2-Phenyl-4- (Z) -4-methoxybenzylidene-4,5-dihydro-1,3-oxazol-5-one (15) Mp: 162 ° C. Appearance: yellow solid.

Example 13: 2-Phenyl-4- (Z) - (2-thiophenyl) methylidene-4,5-dihydro-1,3-oxazol-5-one (16) Mp: 176-178 ° C. Appearance: yellow solid.

Example 14: 2-Phenyl-4- (Z) -piperonylmethylidene-4,5-dihydro-1,3-oxazol-5-one (17) MP: 200 ° C. Appearance: yellow solid.

Example 15: 2-Phenyl-4- (Z) - (3-pyridyl) methylidene-4,5-dihydro-1,3-oxazol-5-one (18) MP: 149-1510 ° C. Appearance: Beige solid.Example 16: 2-Phenyl-4- (Z) -3,4-dimethoxybenzylidene-4,5-dihydro-1,3-oxazol-5-one (19)

MP: 155-156 ° C. Appearance: yellow solid.

General process for obtaining oxazolones

A mixture of / V-acetylglycine (9b) (20mmol, 2.34g), anhydrous sodium acetate (15mmol, 1.2g), acetic anhydride (64mmol, 6mL) and their aldehyde (30mmol) were slightly warmed to complete solubilization. when it was then stirred under gentle reflux for 2h. The solution was cooled to room temperature and refrigerated. After one night, the product was filtered through a Büchner funnel, washed with plenty of ice water and then recrystallized with the appropriate solvent.

Example 17: 2-Methyl-4- (Z) -benzylidene-4,5-dihydro-1,3-oxazol-5-one (20) Mp: 158 ° C. Appearance: yellow solid.

Example 18: 2-Methyl-4- (Z) -4-chlorobenzylidene-4,5-dihydro-1,3-oxazol-5-one (21) Mp: 149 ° C. Appearance: yellow solid.

Example 19: 2-Methyl-4- (Z) -4-bromobenzylidene-4,5-dihydro-1,3-oxazol-5-one (22) Mp: 135 ° C. Appearance: yellow solid.

Example 20: 2-Methyl-4- (Z) -4-trifluoromethylbenzylidene-4,5-dihydro-1,3-oxazol-5-one (23)

MP: 119 ° C. Appearance: yellow solid.

Example 21: 2-Methyl-4- (Z) -piperonylmethylidene-4,5-dihydro-1,3-oxazol-5-one (24) Mp: 191 ° C. Appearance: yellow solid.

Example 22: 2-Methyl-4- (Z) -4-cyanobenzylidene-4,5-dihydro-1,3-oxazol-5-one (25) MP: 191-192 ° C. Appearance: yellow solid.

Example 23: 2-Methyl-4- (Z) -2-naphthylmethylidene-4,5-dihydro-1,3-oxazol-5-one (26) Mp: 225 ° C. Appearance: yellow solid.

Example 24: 2-Methyl-4- (Z) -4-methylbenzylidene-4,5-dihydro-1,3-oxazol-5-one (27) Mp: 136 ° C. Appearance: solid orange.

General process of obtaining compounds

To a solution of the diamine (1mmol, 0.2g) in ethyl acetate (25mL) was added the respective oxazolone (10-27) (3mmol). In general, the mixture becomes homogeneous with heating. The reaction was kept under reflux for 24h. After being cooled to room temperature, there was a precipitated formation which was filtered and washed with ethyl acetate.

Example 25: 1.4: 3,6-Dianhydro-2,5-dip2-benzamido- (Z) -cinamamido1-2,5-dideoxy-L-idithiol (28)

MP: 169 ° C. α D 20 = + 72 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 40% .HRMS calculated for C38H34N4O6, 642.2478; found [M + 1] + 643.3465.Example 26: 1.4: 3,6-Dianhydro-2,5-dif2-benzamido- (Z) -4-fluorocinamate1-2,5-dideoxy-L-idithiol (29)

MP: 152-153 ° C. α D 20 = + 65 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 37%. HRMS calcd for C38H32F2N4O6, 678.229; found [M + 1] +679.3388.

Example 27: 1,4: 3,6-Dianhydro-2,5-dip2-benzamido- (Z) -4-chlorocinamate1-2,5-dideoxy-L-idithiol (30)

MP: 166-169 ° C. α D 20 = + 89 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 35%. HRMS calcd for C38H32Cl2N4O6, 710.1699; found [M + 1] +711.2859.

Example 28: 1.4: 3,6-Dianhydro-2,5-dip2-benzamido- (Z) -4-bromocinamate-1-2,5-dideoxy-L-idithiol (31)

MP: 172-174 ° C. α D 20 = + 80 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 40%. HRMS calcd for C38H32Br2N4O6, 798.0689; found [M + 1] + 801.1971.

Example 29: 1.4: 3,6-Dianhydro-2,5-dif2-benzamido- (Z) -4-trifluoromethylcinna-starch1-2,5-dideoxy-L-idithiol (32)

1 H NMR δ ppm (DMSO, 300 MHz): 9.99 (s, 2H); 8.54 (d, 2H. J = 6.6 Hz); 7.97 (d, 4H, J = 7.2 Hz); 7.77-7.60 (m, 8H); 7.63-7.56 (m, 2H); 7.50-7.47 (m, 4H); 7.09 (s, 2H); 4.62 (s, 2H); 4.29-4.21 (m, 2H); 4.02-3.96 (m, 2H); 3.75 (dd, 2H, J = 9.3 Hz, J = 3.0 Hz).

13 C-NMR δ ppm (DMSO, 75.4 MHz): 165.7 (-C = O); 165.0 (-C = O); 138.5 (-C); 133.3 (-C); 132.3 (-C); 131.6 (-CH); 129.6 (-CH); 128.2 (-CH); 127.7 (-CH); 125.7 (-CH); 125.1 (-CH); 112.6 (-C); 110.2 (-C); 86.4 (-CH); 71.1 (-CH 2); 56.5 (-C). IR κ cm -1 (KBr): 3274, 3065, 1646, 1580, 1519, 1477, 1324, 1281, 1168,1126, 1069, 830, 708.FP: 164-166 ° C. AD20 = + 53 (c, 0.1) DMSO Appearance: Beige solid Yield: 40% HRMS calcd for C40HaaF6N4O6, 778.2226 found [M + 1] +779.3478.

Example 30: 1,4: 3,6-Dianhydro-2,5-dip2-benzamido- (Z) -4-methoxycinnamate1-2,5-dideoxy-L-idithiol (33)

1 H NMR δ ppm (DMSO, 300 MHz): 9.80 (s, 2H); 8.27 (d, 2H, J = 7.2 Hz); 7.99 (d, 4H, J = 6.9 Hz); 7.65-7.40 (m, 10H); 7.12 (s, 2H); 6.91 (d, 4H, J = 8.7 Hz); 4.57 (s, 2H); 4.30-4.20 (m, 2H); 3.96 (dd, 2H, J = 9.3 / 5.7 Hz); 3.73 (s, 6H, -CH 3); 3.80-3.60 (m, 2H).

13 C-NMR δ ppm (DMSO, 75.4 MHz): 165.9 (-C); 165.7 (-C); 159.7 (-C); 133.8 (-C); 131.7 (-CH); 131.2 (-CH); 128.9 (-CH); 128.4 (-CH); 128.0 (-C); 127.9 (-CH); 126.7 (-C); 114.1 (-CH); 86.8 (-CH); 71.4 (-CH 2); 56.7 (-CH); 55.3 (-CH 3) .IV cm-1 (KBr): 3274, 3061, 2962, 2838, 1650, 1604, 1578, 1512, 1477, 1300,1255, 1177; 1080, 1028, 829, 707.

Mp: 167-168 ° C. α D 20 = + 86 (c, 0.1) DMSO. Appearance: Beige solid. Yield: 46%. HRMS calcd for C 40 H 38 N 4 O 8, 702.269; found [M + 1] + 703.3768.

Example 31: 1.4: 3,6-Dianhydro-2,5-dip2-benzamido- (Z) -2-thiophenylcinna-starch1-2,5-dideoxy-L-idithiol (34)

MP: 183-185 ° C. Appearance: Beige solid. Yield: 40%.

Example 32: 1.4: 3,6-Dianhydro-2,5-dif2-benzamido- (Z) -3,4-methylenedioxycinnamate-1-2,5-dideoxy-L-idithiol (35)

Mp: 246-247 ° C. α D 20 = + 77 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 70%. HRMS calcd for C 40 H 34 N 4 O 10.730.2275; found [M + 1] +731.3270.

Example 33: 1.4: 3,6-Dianhydro-2,5-dir2-benzamido- (Z) -3-pyridylcinnamalido1-2,5-dideoxy-L-idithiol (36)

MP: 167 ° C. Appearance: Beige solid. Yield: 45%.

Example 34: 1.4: 3,6-Dianhydro-2,5-dip2-benzamido- (Z) -3,4-dimethoxycinnamate1-2,5-dideoxy-L-idithiol (37)

Mp: 159-160 ° C. α D 20 = + 72 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 55%. HRMS calcd for C42H42N4O10.762.2901; found [M + 1 776.4067.Example 35: 14: 3,6-Dianhydro-2,5-dip 2-acetamido- (Z) -cinamamido1-2,5-dideoxy-L-idithiol (38)

1 H NMR δ ppm (DMSO, 300 MHz): 9.41 (s, 2H); 8.25 (d, 2H, J = 6.9 Hz); 7.53 (d, 4H, J = 7.2 Hz); 7.42-7.20 (m, 6H); 6.88 (s, 2H); 4.57 (s, 2H); 4.23-4.16 (m, 2H); 3.96 (dd, 2H, J = 9.3 Hz, J = 5.4 Hz); 3.71 (dd, 2H, J = 9.3 Hz, J = 3.6 Hz), 1.98 (s, 6H).

13 C-NMR δ ppm (DMSO, 75.4 MHz): 169.2 (-C); 165.3 (-C); 134.1 (-C); 130.1 (-C); 129.2 (-CH); 128.4 (-CH); 128.3 (-CH); 126.5 (-CH); 86.5 (-CH); 71.2 (-CH 2) 56.4 (-CH); 22.7 (-CH 3).

IR VcnT1 (KBr): 3480, 3258, 3056, 2975, 2870, 1651, 1537, 1489, 1446, 1373,1287, 1208, 1088, 1042, 932.

Mp: 191-192 ° C. α D 20 = + 71 (c, 0.1) DMSO. Appearance: Beige solid. Yield: 95%. HRMS calcd for C28H3IN4O6.519.2244; found 519.2244.

Example 36: 1.4: 3,6-Dianhydro-2,5-dir2-acetamido- (Z) -4-chlorocinamate1-2,5-dideoxy-L-idithiol (39)

MP: 168-170 ° C. α D 20 = + 85 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 96%. HRMS calcd for C28H28N4O6NaCl2, 609.1284; found 609.1282.

Example 37: 1.4: 3,6-Dianhydro-2,5-dir2-acetamido- (Z) -4-bromocina-starch1-2,5-dideoxy-L-idithiol (40)

MP: 175 ° C. α D 20 = + 81 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 58% .HRMS calculated for C28H28N4O6NaBr2, 697.0273; found 697.0306.

Example 38: 1.4: 3,6-Dianhydro-2,5-dif2-acetamido- (Z) -4-trifluoromethylcinna-starch1-2,5-dideoxy-L-idithiol (41)

MP: 209-210 ° C. α D 20 = + 51 (c, 0.1) DMSO. Appearance: Beige solid. Yield: 50%. HRMS calcd for C30H28N4O6NaF6, 677.1811; found 677.1792.

Example 39: 1.4: 3,6-Dianhydro-2,5-dip 2-acetamido- (Z) -3,4-methylenedioxycinnamamidol® .S-dideoxy-L-idithiol (42)

MP: 170-173 ° C. α D 20 = + 37 ° (c, 0.1) DMSO. Appearance: Beige solid. Yield: 70%. HRMS calcd for C 30 H 30 N 4 O 10, 606.1962; found [M + 1f607.2885.

Example 40: 1.4: 3,6-Dianhydro-2,5-di [2-acetamido- (Z) -4-cyanocyanamamido1-2,5-dideoxy-L-idithiol (43) Mp: 204-206 ° C. α D 20 = + 106 (c, 0.1) DMSO. Appearance: Beige solid. Yield: 56%. HRMS calcd for C 3 OH 2 SN 6 O 6 Na 1 591.1968; found 591.1971.

Example 41: 1 AS.e-Dianhydro-Î ”S-direacetamido-β-naphthalenacrylamidol Δ S-dideoxy-L-idithiol (44)

1 H NMR δ ppm (DMSO, 300 MHz): 9.38 (s, 2H); 8.03 (s, 2H); 7.92-7.84 (m, 8H); 7.77 (d, 2H, J = 8.1 Hz); 7.55-7.49 (m, 4H); 7.32 (s, 2H); 4.44 (s, 2H); 3.80 (dd, 2H, J = 9.0 Hz, J = 4.8 Hz); 3.60 (dd, 2H, J = 9.0 Hz, J = 4.8 Hz); 3.42 (dd, 2H, J = 4.8 Hz1 J = 2.7 Hz); 2.01 (s, 6H).

13 C-NMR δ ppm (DMSO, 75.4 MHz): 167.4 (-C); 167.2 (-C); 132.6 (-C); 132.5 (-C); 132.4 (-C); 131.2 (-C); 129.0 (-CH); 128.0 (-CH); 127.3 (-CH); 127.2 (-CH); 126.4 (-CH); 126.1 (-CH); 86.8 (-CH); 72.5 (-CH 2); 56.6 (-C); 22.8 (-CH3).

IR ν cm -1 (KBr): 3253, 3054, 2982, 2890, 1650, 2153, 1660, 1634, 1525, 1380.1322, 1275, 1149, 1050, 1015, 811, 746, 716.

Mp: 191-193 ° C. α D 20 = + 10 (c, 0.1) DMSO. Appearance: Beige solid. Yield: 86%.

Example 42: 1.4: 3,6-Dianhydro-2,5-dip2-acetamido- (Z) -4-methylcinnamate-1-2,5-dideoxy-L-idithiol (45)

MP: 174-175 ° C. α D 20 = + 83 (c, 0.1) DMSO. Appearance: Beige solid. Yield: 52%. HRMS calcd for C 30 H 34 N 4 O 6 Na, 569.2376; found 569.2361.

Example 43: Obtaining 1,4: 3,6-dianhydro-mono (4-methylbenzenesulfonate) -D-mannitol (46)

To a solution of isomanide (1g, 6mmol) and pyridine (1.1 OmL) in CH 2 Cl 2 (10mL) was added p-toluenesulfonyl chloride (1.49g, 7mmol) at 100 ° C. The reaction was stirred at room temperature for 12h and then diluted with CH 2 Cl 2 (20mL). The solution was washed with water, HClN and saturated sodium chloride solution. After evaporation of the solvent, the solid was recrystallized with a methanol / isopropanol mixture providing the product as a white solid in 40% yield. MP: 103-104 ° C.

Example 44: Obtaining 1,4: 3,6-dianhydro-5-Q- (benzyl) -2- (4-methylbenzenesulfonate) -D-mannitol (47)

To a solution of (46) (1 Og1 33mmol), 50% aqueous KOH eTBAB solution (0.322g, 1mmol) in CH2 Cl2 (80mL) was added benzyl chloride (6.54g, 38mmol). After stirring for 12h, the mixture was diluted with water (10mL) and the organic phase was separated. The aqueous phase was extracted with CH 2 Cl 2 and dried with Na 2 SCV. The product was purified by chromatography on silica gel eluting with a mixture of hexane and ethyl acetate, yielding the product as a colorless oil in 75% yield.

Example 45: Obtaining 1,4: 3,6-Dianhydro-2-azido-2-deoxy-5-Q- (benzyl) -D-Glucitol (48)

A mixture of (47) (1.86g, 4mmol), [bmin] + [BF4] 'previously dried under vacuum at 90 ° C (4.8mL, 23mmol) and NaN3 (0.93g, 14mmol) was heated to 120 ° For 12h. Water was added and the aqueous phase was extracted with ethyl ether. The product was purified by flash column chromatography (hexane / ethyl acetate) to give a light yellow oil in 73% yield.

HRMS calcd for C 13 H 15 N 3 O 3 Na 1 284.1011; found 284.1025.oco 2 O = + 92 (c, 0.1) DMSO.

Example 46: Obtaining 1.4: 3,6-Dianhydro-2-amino-2-deoxy-5-0- (benzyl) -D-Glucitol (49)

To a solution of (48) (0.870g, 3mmol) in ethanol (25mL) was added 5% Pd / C (0.3mmol) and the mixture was hydrogenated under 40psi for 5h. After this time the suspension was filtered on XAD-4 and washed with ethanol. Evaporating solvent gave a colorless oil in quantitative yield. HRMS calculated for C13 H17 NO3 Na, 236.1287; found, 236.1284.

General process for obtaining compounds (50-58)

A solution of O-benzylated amine (49) (0.2g, 0.85mmol) and its oxazolone (10-18) (0.85mmol) in ethyl acetate (20mL) was refluxed for 24-48h. The reaction was monitored by c.c.f. and after its completion, it was allowed to cool to room temperature. The solid formed was filtered and washed with ethyl acetate. Some products required recrystallization with the appropriate solvent.

Example 47: 1,4: 3,6-Dianhydro-2-1,2-benzamido- (Z) -cinamamidol-2-deoxy-5-0- (benzyl) -D-Glucitol (50)

MP: 168-169 ° C. α D 20 = + 70 ° (c, 0.1) DMSO. Appearance: white solid. Yield: 50%. HRMS calcd for C 29 H 28 N 2 O 5 Na, 507.1896, found 507.1878. Example 48: 1,4: 3,6-dianhydro-2-f2-benzamido- (Z) -4-fluorocinamamido-1-2-deoxy-5-Q- (benzyl) -D-Glucitol (51)

MP: 202-203 ° C. α D 20 = + 55 ° (c, 0.1) DMSO. Appearance: white solid. Yield: 55%. HRMS calcd for C 29 H 27 FN 2 O 5 Na 1 525.1802, found 525.1784.

Example 49: 1.4: 3,6-Dianhydro-2-r 2 -benzamido- (Z) -4-chlorocinamate-1-2-deoxy-5-0- (benzyl) -D-Glucitol (52)

MP: 203-204 ° C. α D 20 = + 65 ° (c, 0.1) DMSO. Appearance: white solid. Yield: 60%. HRMS calcd for C 29 H 2 TCIN 2 O 5 Na, 541.1506, found 541.1492.

Example 50: 1,4: 3,6-Dianhydro-242-benzamido- (Z) -4-bromocina-starch1-2-deoxy-5-Q- (benzyl) -D-Glucitol (53)

Mp: 194-195 ° C. α D 20 = +62 (c, 0.1) DMSO. Appearance: white solid. Yield: 55%. HRMS calcd for C 29 H 27 BrN 2 O 5 Na, 585.10001, found 585.1000.

Example 51: 1.4: 3,6-Dianhydro-2-r 2 -benzamido- (Z) -4-trifluoromethylcinna-amido-1-2-deoxy-5-Q- (benzyl) -D-glucucol (54)

MP: 183-184 ° C. α D 20 = + 23 (c, 0.1) DMSO. Appearance: white solid. Yield: 50%. HRMS calcd for C 3 OH 27 F 3 N 2 O 5 Na, 575.1770, found 575.1771.

Example 52: 1.4: 3,6-Dianhydro-2-r2-benzamido- (Z) -4-methoxycinnamate-1-2-deoxy-5-Q- (benzyl) -D-Glucitol (55)

Mp = 167-168 ° C. α D 20 = + 75 ° (c, 0.1) DMSO. Appearance: yellow solid. Yield: 57%. HRMS calcd for C 30 H 30 N 2 O 6 Na, 537.2002, found 537.1998.

Example 53: 1.4: 3,6-Dianhydro-2-r2-benzamido- (Z) -2-thiophenylacrylamido1-2-deoxy-5-Q- (benzyl) -D-Glucitol (56)

MP: 157-158 ° C. α D 20 = + 60 ° (c, 0.1) DMSO. Appearance: white solid. Yield: 60%. HRMS calcd for C 27 H 26 N 2 O 5 SNa, 513.1460, found 513.1437.

Example 54: 1,4: 3,6-Dianhydro-2-f-2-benzamido- (Z) -3,4-methylenedioxycinnamate-1-2-deoxy-5-Q- (benzyl) -D-Glucitol (57) Mp: 140-141 ° C ° C. α D 20 = + 53 (c, 0.1) DMSO. Appearance: white solid. Yield: 60%. HRMS calcd for C 30 H 28 N 2 O 7 Na 1 551.1794, found 551.1785.

Example 55: 1,4: 3,6-Dianhydro-2-f-2-benzamido- (Z) -3-pyridylacrylamido-1-2-deoxy-5-Q- (benzyl) -D-Glucitol (58)

MP: 161-162 ° C. α D 20 = (c, 0.1) DMSO. Appearance: white solid. Yield: 62%.

In vitro activity measurement of anti-HCV drugs in HCV replicons

In the step of measuring the anti-HCV activity of the above described drugs, a subgenomic HCV RNA replicon composed of the 5 'and 3' untranslated regions of the viral genome (5'UTR and 3'UTR) and the non-structural genes NS3- NS4-NS5 preceded by a eukaryotic arytosome binding sequence (IRES) from the murine encephalomyocarditis virus (EMC-IRES). This replicon still has another cistron which is composed of the firefly yuciferase gene fused to a ubiquitilation sequence, and the antibiotic resistance gene G418. Translation of this second cistronesta under IRES control of the HCV itself located at the end of the 5'UTR region.

This replicon was named I389 / NS3-3'-LucUbiNeo ET, and has adaptive mutations in the NS5B gene that confer greater replicative capacity to these clones in established hepatocyte cell culture (Lohmann, V., Hoffmann1 S., Herian, U. (Penin, F., and Bartenschlager, R. (2003) .Cellular viral determinant of hepatitis C virus RNA replication in cell culture. J. Virol.77, 1-13). This system is absolutely safe to handle because it is not capable of generating viral particles.

The cells used in this assay are derived from a Huh7 human heptocarcinoma strain (Nakabayashi, H.; Taketa1 K.; Miyano, K.; Yamane, T.; Sato1 J. Cancer Res. 1982, 42, 3858-3863). Huh7 cells are competent to maintain replicon for more than 50 replicates in selective medium. 10% SEMEM fetal bovine serum containing 1 mg / ml G418. Introducing oreplicon into Huh7 cells requires transcription of the invitro replicon RNA. This reaction is performed using the Iinearized Plasmid I389 / NS3-3'-LucUbiNeo ET. The plasmid was Iinearized with the column epurified Spe I restriction enzyme (PCR purification Kit, Qiagen). RNAs were synthesized in a buffer containing 40 mM Tris-HCI (pH 7.9), 10 mM 12 mM NaCl1, 2 mM spermidine, 10 mM DTT, 3 mM of each nucleoside triphosphate (Invitrogen), 0.025 U pyrophosphate, 100 U RNasin1 100 U of T7RNA polymerase and 2 pg of linearized DNA for 90 minutes at 37 ° C. The final product, replicon RNA, was finally purified by extraction with Trizol LS (Invitrogen, USA) and precipitated with the addition.

Approximately 4 pg of RNA was electroporated into 5.106 cells suspended in citomix medium (KCl 120mM, K2PO4 10mM, MgCb 5mM, HEPES25mM, CaCl2 0.15mM, EGTA 2mM, 1.9mM ATP, 4.7mM glutathione) containing 1.25% of DMSO. The cells in the medium were placed in a 0.4 cm diameter electroporation cuvette, the electrotransfer conditions were 270 ν and 960pF. After the electrical pulse, the cells were transferred to 8mL DMEM medium (10% fetal bovine serum) containing 1.25% DMSO. Cells were seeded in a 10cm diameter plate and cultured for 24 hours. After this incubation the medium was replaced with DMEM (10% fetal bovine serum), and replicon-containing cells were selected with the addition of 0.5 mg / mL G418 to be added to the culture medium. The colonies were finally visualized after 7 days in culture. The colonies were removed from the selective medium and sown in a 25cm2 culture bottle, and kept in weekly peaks in selective medium.

Antiviral assays were performed on Eagle's modified medium by Dulbecco (Gibco-Invitrogen) in the absence of G418. Cells will be seeded in 96-well plates (3,000 cells per well) and compounds tested at different concentrations (100μΜ, 20 μΜ, 4 μΜ, 1 μΜ, 0.25μΜ) will be added immediately after the cells. 4 days and after this incubation, 20µl of the Risazurin vital dye (CelITiter-Blue® Cell Viability Assay. PROMEGA, USA) was added and the same were incubated for 18 hours.The viability was measured by comparing the fluorescence units read in the wells containing drug and well control (no drug) After measuring the viability, cells were disrupted with 20 μΙ_ of an Iise buffer (25mM Tris-phosphate (pH 7.8), 2mM DTT, 2mM1,2-diaminocyclohexane- NN-N'-N'-tetraacetic acid, 10% glycerol, 1% TritonX100) and assayed for Luciferase activity by adding 100 µl of the developing solution (Luciferase Assay System, PROMEGA, USA). a luminometer (Turner-Designs - TD- EC50% of the different compounds against recombinant replicons were calculated using the Luciferase data from the control wells (100% activity). Ointerferon alfa (IFN-α) was used 300Ul / ml to 0.03 Ul / ml as a positive control in inhibition assays.

The results of the analysis of IC50% for cells and EC50% for oreplicon HCV with the different synthesized compounds are shown in Table 3.

Table 3 - Table showing ICso% and EC50% results in μΜ for oreplicon l389 / NS3-3'-LucUbiNeo ET in Huh7 cells.

<table> table see original document page 32 </column> </row> <table>

1- Concentration that inhibits 50% of huh7 cell growth in the Risazurin assay.

2- Concentration that inhibits 50% of the replicon luciferase signal from I389 / NS3-3'-LucUbiNeo ET in Huh7 cells.

3- Compound not tested.

4- Compound with low solubility in DMEM medium and cannot be tested for HCV activity.

As we can see, the compound (56) presented the most robust anti-HCV activity with EC50% in the 35 μΜ range. Compound (35) had a lower anti-HCV activity in the 95μΜ range. All drugs did not show measurable toxicity by the Risazurin technique with Huh7 cells (ICso%> 100 μΜ). The above description as well as the examples presented with by way of illustration and do not limit the invention to the form herein disclosed and exemplified. Accordingly, variations and modifications compatible with the above teachings, and the relevant technical skill or knowledge, are within the scope of the present invention. The above described and exemplified embodiments are intended to better explain the known methods for practicing the invention and to enable those skilled in the art to use the invention in such or other embodiments and various modifications necessary for the specific applications or uses of the present invention. It is intended that the present invention include all modifications and variations thereof within the scope described in the report and the appended claims.

Claims (19)

Serine protease inhibiting pseudopeptide compounds comprising general formulas (I) and (II) 1 <formula> formula see original document page 34 </formula> R1 Benzyl, carboxybenzyl, carboxy-t-butyl, hydrogen, alkyl or phenyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl, p-trifluoromethylphenyl, p-methoxyphenyl, 2-thienyl, 3,4-methylenedioxyphenyl, 3-pyridyl, 3,4-dimethoxyphenyl, p-cyanophenyl, 2 naphthyl, 4-methylphenyl; and R3 is benzyl, carboxybenzyl, carboxy-t-butyl, hydrogen, aquyl or aryl. 2. Compounds according to Claim 1, characterized in that R 1 may be phenyl or methyl, R 2 is substituted or heterocyclic phenyl; and R3 is benzyl (-Bn), carboxybenzyl (-Cbz) or carboxy-t-butyl (-Boc). Compounds according to Claim 1, characterized in that R 3 is preferably benzyl. 4. Serine protease inhibitor and potentially polymerser inhibitor composition characterized in that it consists of the compound of formula I where R1 may be a phenyl or methyl; R2 is a p-substituted phenyl or heterocycles; and R3 benzyl (-Bn), carboxybenzyl (-Cbz) or carboxy-t-butyl (-Boc) and / or pharmaceutically acceptable salts thereof in addition to pharmaceutically acceptable adjuvants. Composition according to Claim 4, characterized in that it comprises from 10μΜ to 150μΜ of a compound of formula (I). Composition according to Claim 5, characterized in that they have a preferred concentration of 10μΜ to 100 μΜ of a compound of formula (I). A pharmaceutical composition comprising the compound of formula (I) and / or its pharmaceutically acceptable salts useful in the manufacture of a medicament for the treatment of diseases caused by viruses. dafamília fíaviviridae. Composition according to Claim 7, characterized in that it comprises from 10μΜ to 150μΜ of a compound of formula (I). Composition according to Claim 8, characterized in that they have a preferential concentration of 10μΜ to 100μΜ of a compound of formula (I). Composition according to Claim 7, characterized in that it is preferably used in the preparation of a medicament for the treatment of hepatitis C virus. Composition according to Claim 7, characterized in that it is preferably used in the preparation of a medicament for the treatment of dengue. 12. Serine protease inhibitor and potentially polymerser inhibitor composition characterized in that it consists of the compound of formula (II) where R1 may be a phenyl or methyl; substituted or heterocycles; and R3 is benzyl (-Bn), carboxybenzyl (-Cbz) or carboxy-t-butyl (-Boc). and / or pharmaceutically acceptable salts thereof and pharmaceutically acceptable adjuvants. Composition according to Claim 12, characterized in that it comprises from 10μΜ to 150μΜ of a compound of formula (II). Composition according to Claim 13, characterized in that they have a preferred concentration of 10μΜ to 100μ of a compound of formula (II). 15. Pharmaceutical composition characterized in that it comprises the compound of formula (II) and / or its pharmaceutically acceptable salts, useful in the production of a medicament intended for the treatment of diseases caused by viruses. dafamilia flaviviridae. Composition according to Claim 15, characterized in that it comprises from 10μΜ to 150μΜ of a compound of formula (I). Composition according to Claim 16, characterized in that they have a preferential concentration of 10μΜ to 100μΜ of a compound of formula (II). Composition according to Claim 15, characterized in that it is preferably used in the preparation of a medicament for the treatment of hepatitis C virus. Composition according to Claim 15, characterized in that it is preferably used in the preparation of a medicament for the treatment of dengue.