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    • 专利摘要:
    Derivatives of 5-nitroindazole and their use as antiprotozoal agents. The present invention relates to two families of 5-nitroindazole derivatives possessing antiparasitic properties and to their use for the manufacture of a medicament, preferably for the treatment of infections caused by pathogenic protozoa of the families trypanosomatidae and trichomonadidae, such as the disease of chagas, leishmaniasis and trichomoniasis. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2614131A1
    申请号:ES201500769
    申请日:2015-10-27
    公开日:2017-05-29
    发明作者:José Antonio ESCARIO GARCIA-TREVIJANO；Alicia GÓMEZ BARRIO；Juan José NOGAL RUIZ；Alexandra IBÁÑEZ ESCRIBANO；Cristina Rosa FONSECA BERZAL；Vicente Jesús ARÁN REDÓ；Felipe REVIRIEGO PICÓN；José María CUMELLA MONTÁNCHEZ
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad Complutense de Madrid；
    IPC主号:
    专利说明:


    DERIVATIVES OF 5-NITROINDAZOL AND ITS USE AS ANTIPROTOZOAR AGENTS
    TECHNICAL FIELD The present invention relates to two families of compounds derived from 5-nitroindazole, its preparation process and its use for the manufacture of medicaments for the treatment of infections, particularly those caused by protozoa of the Trypanosomafidae and Trichomonadidae families. The invention, therefore, falls within the pharmaceutical sector.
    State of the art Protozoa are the etiologic agents of several of the main parasitic diseases. 'Many of these infections are concentrated in the poorest areas of the planet and are considered "neglected tropical diseases." The existing pharmacological treatments for many of these protozoosis are far from satisfactory and the development of a vaccine is still an unreached goal. Currently existing therapies are not adequate due essentially to several factors, such as low therapeutic rates that lead to high toxicities and unacceptable side effects, the emergence of resistant parasites, the difficulty of treatment compliance due to complex protocols, the high prices that are beyond the reach of patients in the affected countries, etc.
    Chagas disease (American trypanosomiasis), caused by the hemo-flagellated protozoan Trypanosoma cruzi (fam. Trypanosomatidae), is an anthropozoonosis transmitted primarily through the contaminated feces of hematophagous bed bugs. It is estimated that around 7 million people worldwide are infected and around 25 million more at risk of contracting the disease, which causes more than 7,000 deaths annually, mainly in 21 Latin American countries where it is endemic (WHO, Investing fo overcoming the global impact of Neglected Tropical Diseases, Third WHO report on Neglected Tropical Diseases, 2015). However, the existence of other alternative transmission routes to the vector (transfusion of contaminated blood, transplantation of infected organs or by congenital route), together with international migrations produced in recent decades, have made Chagas disease a pathology emerging in various developed countries; In Spain, it is estimated that there may be some 85,000 infected people, mainly of Bolivian origin (Schmunis, G.A. and Yadon, Z.E., Acta Trop. 2010, 115, 14-21).
    Chagas disease has an initial acute phase between 1-2 months long, usually asymptomatic or mild and with detectable parasitemia. After an undetermined phase, between 30-40% of patients evolve into a chronic phase that manifests 10-30 years after infection. This is characterized by the presence of irreversible cardiac and digestive lesions, which arise as a result of inflammatory lesions (Rassi Jr., A. et al., Lancet 2010, 375, 13881402) produced by the persistence of the parasite together with auto reactions immune system (Marin-Neto, lA. et al., Circulation 2007,115, 1109-1123).
    There is currently no vaccine or completely effective treatment for Chagas disease. The only two drugs available to date, nitroheterocycles nifurtimox and benznidazole, were developed over forty years ago (Coura, lR. Et al., Mem. Inst. Oswaldo Cruz 2009, 104, 549-554). Both are quite effective in the acute phase, but their effectiveness in the chronic phase is very limited. On the other hand, the appearance of T cruzi strains resistant to these drugs has led to variations in their efficacy linked to the geographical area. In addition, both compounds have severe side effects, but benznidazole is better tolerated by patients and is generally considered the treatment of choice for Chagas disease (Urbina, l., Acta Trop. 2010, 115, 55-68; Rassi Jr., A. et al., Lancet 2010,375, 1388-1402).
    In recent years, many compounds with antihagasic activity have been described, as well as different potential molecular targets of the parasite (Guedes, PMM et al., Expert Rev. Anti-Infect. Ther. 2011, 9, 609-620; Urbina, lA. , Acta Trop. 2010, 115, 55-68; Cerecetto, H. and González, M., Pharmaceuticals 2010, 3, 810-838; Sánchez-Sancho, F. et al., Curro Med. Chem. 2010, 17,423- 452; Soeiro, MNC and de Castro SL, Expert Opin. Ther. Targets 2009,13, 105-121).
    Of particular interest have been aroused antifungal azoles inhibiting sterile synthesis, but recent clinical studies have been very disappointing, showing that these compounds are much less effective than benznidazole, at least as a single chemotherapy (Chatelain, E., J Biomo !. Screen. 2015,20, 22-35).
    All these facts highlight the need to develop new therapeutic alternatives for the treatment of Chagas disease and, on the other hand, it has been recently pointed out that nitroheterocyclic derivatives continue to represent the only real alternative in the anti-Hagasmic struggle (Moraes, CB et al., Sci. Rep. 2014,4,4703; DOI: I0.1038 / srep04703).
    The antihagasic activity of various 5-nitroindazoles with structures and substitution patterns completely different from those possessed by the compounds included in this invention has also been described: 1-alkylindazol-3-oles (Arán, VJ et al., Bioorg. Med. Chem. 2005, 13, 3197-3207; Boiani, L. et al., Eur. J Med. Chem. 2009, 44, 1034-1040), 3-alkoxyindazoles (Montero-Torres, A. et al., Bioorg. Med. Chem. 2005, 13, 6264-6275), 3-alkoxy-l-alkylindazoles (Arán, VJ et al., Bioorg. Med. Chem. 2005,13,3197-3207; Boiani, L. et al., Eur. J Med. Chem. 2009,44,10341040; Rodríguez, 1. et al., Eur. J Med. Chem. 2009,44, 1545-1553; Rodríguez, 1. et al., Bioorg. Med. Chem. 2009, 17, 8186-8196; Vega, MC et al., Eur. J Med. Chem. 2012,58,214-227; Muro, B. et al., Eur. J Med. Chem. 2014, 74, 124-134 ), 1,2-condensed indazolin3-ones (Díaz-Urrutia, CA et al., Spectrochim. Acta A 2012, 95, 670678) and 1,1 '-hydrocarbilenbis (3-alkoxy-and 3-hydroxyindazoles) (Aguilera- Venegas, B. et al. , ¡Nt. J Electrochem. Sci. 2012, 7,5837-5863).
    The activity against Trypanosoma brucei rhodesiense, an etiological agent of African sleeping sickness, is also known for various derivatives of 5nitroindazole: 1-alkyl- and 1-arylindazol-3-oles, 2-alkylindazolin-3-ones, 3alkoxyindazoles, 3 -alkoxy-l-alkylindazoles, indazolin-3-ones 1,2-condensed, and 1,1 '
    hydrocarbylenebis (3-alkoxyindazoles) (Arán, VJ et al., Bioorg. Med. Chem. Lett. 2012, 22, 4506-4516), as well as the leishmanicidal properties of some 3-alkoxy-1alkylindozoles (Boiani, L. et al. ., Eur. J Med. Chem. 2009,44, 1034-1040; Marín, C. et al., Acta Trop. 2015, 148, 170-178) that had previously shown activity
    5 versus T cruzi.
    In the present invention, 1,2-disubstituted 5-nitroindazolin-3-ones are proposed as anthagasic agents.
    In this context, the synthesis and antihagasic properties of some related indazolinones have been described in recent years (Montero-Torres, A. et al., Bioorg. Med. Chem. 2005, 13, 6264-6275; Vega, MC et al ., Eur. J Med. Chem. 2012, 58, 214-227; Mura, F. et al., J Spectrosc. Dyn. 2013, 3, article 8; FonsecaBerzal, C. et al., Parasitol. Res. 2014 , 113, 1049-1056), as well as low activity
    15 anti-T cruzi of the isomeric 3-alkoxy-2-alkylindazoles (Vega, MC et al., Eur. J Med. Chem. 2012, 58, 214-227; Mura, F. et al., J Spectrosc. Dyn . 2013, 3, 8; Fonseca-Berzal, C. et al., Parasitol. Res. 2014, 113, 1049-1056).
    The activity against T brucei rhodesiense is also known (Arán, V.J. et al.,
    20 Bioorg. Med. Chem. Lett. 2012, 22, 4506-4516) of some substituted 1,2di 5-nitroindazolinones, as well as their anti-inflammatory properties (Marrero-Ponce, Y. et al., Eur. J Med. Chem. 2011, 46, 5736-5753).
    The previously published 1,2-disubstituted indazolin-3-ones present in position 1
    25 simple alkyl groups (methyl, propyl, isopropyl, butyl and pentyl) or benzyl. In the present invention it is shown how these groups can be substituted by complex alkyl groups, containing unsaturations or various varied functionalities (halogen, carboxyl, ester, ether, alcohol, amide, etc.) or by acyl or sulfonyl groups, being able to obtain more compounds active and less toxic than previously
    In addition to increasing the water solubility of the compounds by introducing polar groups, they can thus exhibit a more appropriate pharmacokinetic behavior.
    On the other hand, Trichomonas vaginalis (fam. Trichomonadidae) is the causative agent of trichonosis, a sexually transmitted infection (STI) responsible for more than 50% of all curable STIs in the world. According to the latest data estimated by the World Health Organization and published in 2012, more than 276 million infections occur each year (WHO, Global incidences and prevail celected of curable sexually transmitted diseases -2008, 2012).
    This protozoan is transmitted only through sexual contact. It is characterized by showing a wide range of clinical manifestations that can cause severe cases of inflammation of the genitourinary ducts accompanied by a characteristic leucorrhoea, erythema, pruritus, dysuria, infertility or the formation of small lesions called "macularisquot colpitis; in the cervix In men, this infection can lead to non-gonococcal urethritis and impaired sperm viability and mobility (Swygard, H. et al., Transmo Infect Sex 2004, 80, 91-95; Lewis, DA, Medicine (Baltimore) 2010, 38, 291-293). However, epidemiological studies reveal that at least half of women as well as 80% of infected men have no symptoms, becoming asymptomatic carriers and potential transmitters of this infection.
    Human genital uro trichomonosis has been associated with different complications such as problems during pregnancy or infants with low weight, premature births, etc. (Cotch, M.F. et al., Sex Transm. Diso 1997, 24, 353-360). Likewise, this STI also increases the risk of developing cervical neoplasia (Viikki, M. et al., Acta Onco !. 2000, 39, 71-75) and prostate (Sutc1iffe, S. et al., Cancer Epidemius !. Biomarkers Prevo 2006, 15, 939-945), as well as a greater predisposition to coinfection with other STIs of bacterial, viral, etc. (Schwebke, lR. And Burgess, D., Clin. Microbio !. Revo 2004, 17, 794-803; Cherpes, TL et al., Sex Transm. Diso 2006, 33, 747-752; Allsworth, lE et al. , Transm Sex Diso 2009, 36, 738-744; McClelland, RS et al., J Infect. Diso 2007, 195, 698-702).
    Trichomonosis is preferably treated with nitroheterocytic metronidazole, introduced in the market around 1960. Currently, metronidazole and tinidazole, both of the same family of 5-nitroimidazoles, are the only two drugs accepted by the Food and Drug Administration (FDA) to treat this STI (Crowell,
    TO THE. et al., Antimicrob. Chemother Agents 2003, 47, 1407-1409). However, there are no effective alternatives for those patients who develop side effects, who show hypersensitivity or in which their use is contraindicated. On the other hand, it is estimated that approximately 5% of cases diagnosed with trichonosis are caused by a nitroimidazole-resistant isolate (Vázquez, F. et al., Sick. Infected. Microbiol. Clin. 2001, 19, 114-124) .
    Therefore, the search for pharmacological alternatives for the treatment of this disease is necessary. In the present invention 3-alkoxy-2-alkyl-2H-indazoles are proposed.
    The tricomonicidal activity of certain 1,2-substituted indazolin-3-ones (Ibáñez Escribano, A. et al., Mem. Inst. Oswaldo Cruz 2012, 107, 637643) has been studied, as well as that of various 5-nitroindazole derivatives with structures and substitution patterns completely different from those possessed by the compounds object of this invention: l-alkylindazol-3-oles (Marrero-Ponce, Y. et al., Curro Drug Discov. Technol. 2005, 2, 245-265; Marrero-Ponce, Y. et al., Bioorg. Med Chem. 2006, 14, 6502-6524), 2-alkylindazol-3-ones, 3-alkoxyindazoles, indazol-3-ones 1,2 condensed and 1,1 '- hydrocarbylenebis (3-hydroxyindazoles) (Marrero-Ponce, Y. et al., Curro Drug Discov. Technol. 2005, 2, 245-265), and 3-alkoxy-1-alkylindazoles (Aran,
    V.J. et al., Bioorg. Med Chem. 2005, 13, 3197-3207). But the activity against T vaginalis of the 3-alkoxy-2-alkylindazoles, object of this invention, is not described in the literature.
    Detailed description of the invention
    5-nitroindazole derivatives and their use as antriprotozoal agents.
    The present invention relates to two groups of compounds derived from 5-nitroindazole, readily available, which possess biological activity against certain pathogenic protozoa.
    A first aspect of the invention relates to two different types of compounds, of general formulas (1) and (11). Compounds of type (1) are 5-nitroindazolin-3-ones 1,2-disubstituted and have, in general, activity against Trypanosoma cruzi, while those of type (11) are 3-a1-coxy-2-alkyl-2H -indazoles that have activity against Trichomonas vaginalis.
    Formula (1) Formula (11)
    where R can be:
    15-an alkenyl or alkynyl group of 2-6 carbon atoms and with the multiple bond in the various possible positions; 1-allyl-2-benzyl-5-nitroindazolinone [compound type (1)] described as synthetic intermediate is specifically excluded in a patent [Qin, D. et al. (GSK), W02012162129 Al].
    20 -a polymethylene group of variable length [(CHz) n, n = 1-6] with terminal substituents of type Br, OH, COOR (R = H or 1-5 carbon alkyl), CONR1Rz (R1 and / or RZ = H or alkyl of 1-5 carbons), CN or OR (R = alkyl or acyl groups of 1-5 carbons).
    25-an alkoxycarbonyl group with the alkoxy group of 1-5 carbon atoms.
    - an aliphatic (1 to 5 carbon atoms) or aromatic (benzoyl or benzoyl or benzoyl differently substituted acyl groups in positions 2, 3 or 4 with groups such as F, CI, Br, OH, OR, NHz, NOz or CN ).
    - an aliphatic or aromatic sulfonyl group,
    and X can be any substituent of those which can usually be found in positions 2, 3 or 4 of a benzyl group, such as single or branched alkyl groups of 1-5 carbon atoms, trifluoromethyl groups (CF3), halogen atoms ( F, Cl, B), hydroxyl or alkoxy groups (OH and OR; R = alkyl), primary, secondary or tertiary amino groups (NH2, NHR, NR2; R = alkyl), nitro (N02) or cyano (CN).
    In compounds of type (1), when R = CH2COOH, derivatives with two halogen atoms are specifically excluded in the benzyl substituent that have been studied as aldose reductase inhibitors (Malamas, MS and Millen, J., J Med Chem. 1991 , 34, 1492-1503).
    In a more preferred embodiment, the present invention relates to a compound [Formula (1)] that is selected from the following list:
    2-Benzyl-5-nitro-l-propargil-l, 2-dihydro-3H-indazol-3-one (2)2-Benzyl-l- (2-bromoethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (3)2-Benzyl-l- (3-bromopropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (4)2-Benzyl-l- (methoxycarbonyl) methyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (5)2-Benzyl-l-cyanomethyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (6)2-Benzyl-l- [2- (methoxycarbonyl) ethyl] -5-nitro-l, 2-dihydro-3H-indazol-3-one (7)2-Benzyl-l- [3- (ethoxycarbonyl) propyl] -5-nitro-l, 2-dihydro-3H-indazol-3-one (8)2-Benzyl-l- (2-hydroxyethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (9)2-Benzyl-l- (3-hydroxypropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (10)2-Benzyl-l- (2-methoxyethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (11)2-Benzyl-l-ethoxycarbonyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (12)2-Benzyl-l-benzyloxycarbonyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (13)l-Acetyl-2-benzyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (14)2-Benzyl-l-benzoyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (15)
    2-Benzyl-5-nitro-l-tosyl-l, 2-dihydro-3H-indazol-3-one (16) 2-Benzyl-5-nitro-l-vinyl-l, 2-dihydro-3H-indazol- 3-one (24) 2-Benzyl-l- (2-carboxyethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (25) 2-Benzyl-l- (3-carboxypropyl) - 5-nitro-l, 2-dihydro-3H-indazol-3-one (26) 2-Benzyl-I- (3-carbamoylpropyl) -5-nitro-1, 2-dihydro-3H-indazol-3-one ( 27) 2-Benzyl-I- [3- (methylcarbamoyl) propyl] -5-nitro-1, 2-dihydro-3H-indazol-3-one (28) 2-Benzyl-l- [3- (dimethylcarbamoyl) propyl ] -5-nitro-l, 2-dihydro-3H-indazol-3-one (29) 2-Benzyl-l- (3-ethoxypropyl) -5-nitro-l, 2-dihydro-3H-indazol-3- one (30) 1- (2-Acetoxyethyl) -2-benzyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (31)
    or its solvates or prodrugs.
    In another more preferred embodiment, the present invention relates to a compound [Formula (11)] that is selected from the following list:
    2-Benzyl-3- (2-bromoethoxy) -5-nitro-2H-indazole (17)2-Benzyl-3- (3-bromopropoxy) -5-nitro-2H-indazole (18)2-Benzyl-3- (methoxycarbonyl) methoxy-5-nitro-2H-indazole (19)2-Benzyl-3- [2- (methoxycarbonyl) ethoxy] -5-nitro-2H-indazole (20)2-Benzyl-3- (2-hydroxyethoxy) -5-nitro-2H-indazole (21)2-Benzyl-3- (3-hydroxypropoxy) -5-nitro-2H-indazole (22)2-Benzyl-3- (2-methoxyethoxy) -5-nitro-2H-indazole (23)
    or its solvates or prodrugs.
    In a third aspect, the present invention relates to the process of obtaining a compound of general formula (1) or (11); As a consequence of the tautomeric nature of 5-nitroindazolin-3-one (1), some of these compounds are obtained at the same time by reacting the starting product under various conditions with appropriate alkylating, acylating or sulphonilating agents, as shown in Scheme 1. From the tautomeric form the (form "; indazolin-3-onaquot;)" the compounds of general formula (1) are obtained, while the tautomeric form lb (form "; 3
    hydroxy-2H-indazole) leads to the compounds of the general formula (11). The specific conditions for each reaction depend on the specific alkylating, acylating or sulfonylating reagents, as described in examples la-f.
    02N ~ N_8n lJ-N) 1st ~ O
    02N ~ 02N'OOR
    I --_ .. I N-Bn + :: ;; .-- N-Bn
    1 ~ ~ N '~ quot; -N' 02N ~: _8n 2-16 R
    17-23
    1 B
    5 Scheme 1. Synthetic route for the preparation of compounds 2-16 [type (1)] and 1723 [type (11)] from 2-benzyl-5-nitroindazolinone la, b.
    A fourth aspect is related to the preparation of other compounds of formula
    General (1) by chemical transformation of the chains in position 1 of the above products resulting from alkylation, using various procedures (dehydrohalogenation, hydrolysis or aminolysis of esters, etherification of alkyl halides or acylation of alcohols), as shown in Scheme 2.
    or 02N'OJ 02N ~
    I N-Bn .. I N-Bn
    ~, ~,
    N
    you
    R1 R2
    3,4,7,8,9,26 24-31
    R1 R2
    Compound Compound 3 [CH2hBr .. 24 CH = CH2 7 [CH2hCOOMe -25 [CH2hCOOH 8 [CH2hCOOEt -26 [CH2hCOOH 8 [CH2hCOOEt -27 [CH2hCONH2 8 [CH2hCOOEt -28 [CH2hCONHMe
    26 [CH2hCOOH 29 [CH2hCONMe2
    -
    ..
    4 [CH2hBr 30 [CH2hOEt 9 [CH2hOH .. 31 [CH2hOAc
    Scheme 2. Synthetic routes for the preparation of compounds 24-31 [type (11)] from compounds 3, 4, 7-9 and 26 [type (11)].
    A fifth aspect of this invention relates to the use of a compound of general formula (1) or (11) for the preparation of a medicament for the treatment of diseases caused by pathogenic protozoa of the Trypanosomatidae (Trypanosoma, Leishmania) and Trichomonadidae ( Trichomonas), preferably
    10 American trypanosomiasis (Chagas disease) and trichomonosis, caused by the T. cruzi and T. vaginalis parasites, respectively.
    A sixth aspect of the present invention relates to a pharmaceutical composition comprising a compound of general formula (1) or (11) and at least one pharmaceutically acceptable excipient (acceptable adjuvants or pharmaceutical vehicles); optionally said composition may also contain other active ingredients.
    EMBODIMENT OF THE INVENTION The present invention is further illustrated by the following examples, which are not intended to limit its scope.
    5 EXAMPLE 1. Preparation of compounds 2-16 [type (1)] and 17-23 [type (11)] from 2-benzyl-5-nitroindazolinone 1. The compounds listed in Table 1 were obtained by the alkylation, alkoxycarbonylation, acylation or sulfonylation of 2-benzyl-5-nitroindazolin-3-one 1 with the required reagents, which are mentioned in each specific case.
    10 Table 1. Compounds prepared and studied.
    02Nuj 02UOR
    I N-Bn N-Bn ~ N '~ quot; quot; N'
    I
    R 2-16,24-31 17-23
    Compound R Compound R
    2 CH2C = CH 14 Ac 3.17 [CH212Br 15 Bz
    4, 18 [CH2hBr 16 Ts S, 19 CH2COOMe 24 CH = CH2 6 CH2CN 25 [CH212COOH 7, 20 [CH212COOMe 26 [CH2hCOOH 8 [CH2hCOOEt 27 [CH2hCONH2 9, 21 [CH212OH 28 [CH2hCONHMe 10.22 [CH2hOH 29 [CH2hOH 29 [CH2hOH 29Me]
    11.23 [CH212OMe 30 [CH2hOEt 12 COOEt 31 [CH212OAc 13 COOBn
    Alkylation reactions generally lead to mixtures of the corresponding 1,2-disubstituted indazolin-3-ones [2-11; type (1)] compounds and 3-a1-coxy-2-alkyl-2H-indazoles [17-23; type compounds (11)]; The latter compounds are generally minor reaction products that in some cases were not isolated (Vega, M.C. et al., Eur. J Med. Chem. 2012,58,214-227). Many of the alkylation reactions were carried out in K2C03 / DMF alOa oC. However, the propargilation reaction leading to compound 2 gave better results using K2C03 in acetone at reflux, and alkylation of compound 1 with methyl bromoacetate or bromoacetonitrile to lead to compounds 5/19 and 6, respectively, was led to out using sodium bicarbonate in acetone at 35 oC. On the other hand, during the alkylation of compound 1 to derivatives 7/20 with methyl 3-bromopropionate in DMF alOa oC, intense dehydrogenation of the latter to methyl acrylate was observed; better results were obtained by carrying out the reaction with K2C03 in toluene / water using phase transfer catalysis; however, significant amounts of the corresponding acid 25 were also obtained, from the hydrolysis of the ester moiety of compound 7 under the basic conditions used.
    On the other hand, treatment of compound 1 with the required alkyl chloroformates or with the corresponding acyl or sulfonyl chlorides in pyridine provided l-alkoxycarbonyl (12, 13), l-benzoyl (15) or 1-tosylindazolinone (16) desired. The 1-acetyl derivative 14 was obtained by treatment of 1 with acetic anhydride in pyridine. According to some previously published results, only the corresponding 1,2disubstituted derivatives could be isolated in these processes (Baiocchi, L. et al., Synthesis 1978, 633-648).
    Example 1a. Preparation of 3- (ethoxycarbonyl) propyl (8), 2-hydroxyethyl (9/21), 3-hydroxypropyl (10/22) and 2-methoxyethyl (11/23) derivatives. A stirred mixture of the starting 2-benzylindazolinone 1 (1.00 g, 3.71 mmol), the required bromide (4.00 rmol) and K2C03 (0.55 g, 4.00 mmol) in DMF (20 mL) is heated at 100 oC until the end of the reaction [CCF; 12 h (for 11/23); 12 h followed by 1-3 additions of additional amounts of the required bromide (0.3 mmol) and, if necessary, base (0.3 mmol) every 6 h (for 8, 9/21 and 10/22) ]. The mixture was evaporated to dryness and, after the addition of water (200 mL), extracted with CHCh (3 x 50 mL). The concentrated chloroform phase was applied to a chromatography column that was eluted with chloroform / acetone mixtures (50: 1 to 25: 1) (for 8 and 11/23) or with the same mixtures (10: 1 to 5: 1 ) And then chloroform / methanol (50: 1 to 25: 1) (for 9/21 and 10/22). In all cases, the 3-alkoxy-2-benzyl-2H-indazoles (21-23) eluted first, followed by the corresponding 1-substituted 2-benzylindazolinones (8-11).
    2-Benzyl-l- [3 - (ethoxycarbonyl) propyl] -5-nitro-l, 2-dihydro-3H-indazol-3-one (8). Yield: 1.00 g (70%). Oil that solidifies over time; mp 51-53 oc. 1 H NMR [300 MHz, (CD3) 2S0]: OR 8.52 (d, J = 2.1 Hz, 1H, 4-H), 8.38 (dd, J = 9.0, 2.1 Hz , 1H, 6-H), 7.62 (d, J = 9.0 Hz, lH, 7-H), 7.25 (m, 5H, aromatic H Bn), 5.19 (s, 2H, CH2 Bn), 4.06 (t, J = 7.2 Hz, 2H, l'-H), 3.99 (e, J = 7.0 Hz, CH2 Et), 2.21 (t, J = 7.2 Hz, 2H, 3'-H), 1.53 (m, 2H, 2'-H), 1.13 (t, J = 7.0 Hz, CH3); J3C NMR [75 MHz, (CD3) 2S0]: OR 172.00 (C-4 '), 160.88 (C-3), 148.56 (C-7a), 141.32 (C-5), 136.11 (C-1 Bn), 128.70 (C-3, -5 Bn), 127.81 (C-4 Bn), 127.26 (C-2, -6 Bn), 127.19 ( C-6), 120.54 (C-4), 115.62 (C-3a), 111.88 (C-7), 59.99 (CH2 Et), 45.86 (C-l '), 44.70 (CH2 Bn), 30.11 (C-3 '), 21.40 (C-2'), 14.00 (CH3); MS (ES +): miz (%) 789 (29) ([2M + Nat), 767 (64) ([2M + Ht), 406 (37) ([M + Nat), 384 (100) ([M + Ht). Anal. tracing for C2oH21N30S (383.40): C 62.65; H 5.52; N 10.96. Found: C 62.50; H 5.72; N 11.17.
    2-Benzyl-l- (2-hydroxyethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (9). Yield: 0.91 g (78%). Mp 137-139 oC (2-PrOH). 1 H NMR [300 MHz, (CD3) 2S0]: OR 8.49 (d, J = 2.1 Hz, 1H, 4-H), 8.31 (dd, J = 9.3.2.1 Hz, lH, 6-H), 7.60 (d, J = 9.3 Hz, lH, 7-H), 7.29 (m, 3H) and 7.20 (m, 2H) (aromatic H Bn) , 5.20 (s, 2H, CH2 Bn), 4.73 (t, J = 5.1 Hz, 1H, OH), 4.12 (t, J = 4.9 Hz, 2H, 1'-H ), 3.45 (m, 2H, 2'-H); 13 C NMR [75 MHz, (CD3) 2S0]: OR 160.88 (C-3), 149.23 (C-7a), 140.66 (C-5), 136.15 (Cl Bn), 128, 72 (C3, -5 Bn), 127.78 (C-4 Bn), 127.20 (C-2, -6 Bn), 126.43 (C-6), 120.29 (C-4), 114.65 (C-3a), 112.42 (C-7), 58.39 (C-2 '), 49.17 (C-1'), 44.78 (CH2 Bn); MS (lE): miz (%) 313 (100) (M +), 282 (11), 236 (6), 192 (11), 177 (9), 162 (5), 146 (8), 131 (11 ), 103 (7). Anal. tracing for Cl6HlSN304 (313.31): C 61.34; H 4.83; N 13.41. Found: C 61.57; H, 4.69; N 13.68.
    2-Benzyl-l- (3-hydroxypropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (10). Yield: 0.58 g (48%). Mp 141-143 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: J 8.52 (d, J = 2.4 Hz, lH, 4-H), 8.36 (dd, J = 9.3, 2.4 Hz, lH, 6-H), 7.62 (d, J = 9.3 Hz, lH, 7-H), 7.26 (m, 5H, aromatic H Bn), 5.18 (s, 2H, CH2 Bn), 4.58 (t, J = 4.8 Hz, 1H, OH), 4.09 (t, J = 7.0 Hz, 2H, l'-H), 3.23 (m, 2H, 3'-H), 1.45 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: J 160.88 (C-3), 148.64 (C-7a), 141.19 (C-5), 136.12 (Cl Bn), 128, 71 (C3, -5 Bn), 127.81 (C-4 Bn), 127.24 (C-2, -6 Bn), 127.05 (C-6), 120.47 (C-4), 115.46 (C-3a), 111.97 (C-7), 57.50 (C-3 '), 44.77 (CH2 Bn), 44.08 (C-1'), 29.26 ( C-2 '); MS (ES +): miz (%) 350 (40) ([M + Nat), 328 (100) ([M + Ht). Anal. cale. for C17H17N304 (327.33): C 62.38; H 5.23; N 12.84. Found: C 62.19; H 5.57; N 13.09.
    2-Benzyl-l- (2-methoxyethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (11). Yield: 0.78 g (64%). Mp 116-118 oC (2-PrOH). 1 H NMR [300 MHz, (CD3) 2S0]: J 8.50 (d, J = 2.1 Hz, 1H, 4-H), 8.32 (dd, J = 9.3, 2.1 Hz, 1H, 6-H), 7.62 (d, J = 9.3 Hz, 1H, 7-H), 7.25 (m, 5H, aromatic H Bn), 5.18 (s, 2H, CH2 Bn), 4.23 (t, J = 5.0 Hz, 2H, l'-H), 3.35 (t, J = 5.0 Hz, 2H, 2'-H), 2.98 (s , 3H, CH3); 13C NMR [75 MHz, (CD3) 2S0]: J 161.10 (C-3), 149.54 (C-7a), 141.05 (C-5), 136.13 (C-1 Bn), 128.76 (C-3, -5 Bn), 127.86 (C4 Bn), 127.27 (C-2, -6 Bn), 126.73 (C-6), 120.35 (C-4 ), 114.92 (C-3a), 112.45 (C-7), 68.92 (C-2 '), 58.15 (CH3), 47.67 (C-1'), 44.94 (CH2 Bn); MS (IE): miz (%) 327 (lOO) (M +), 282 (21), 250 (4), 236 (4), 206 (2), 177 (9), 131 (7), 103 (4 ). Anal. cale. for C17H17N304 (327.33): C 62.38; H 5.23; N 12.84. Found: C 62.57; H 5.37; N 12.57.
    2-Benzyl-3- (2-hydroxyethoxy) -5-nitro-2H-indazole (21). Yield: 0.21 g (18%). Mp 167-169 oC (2-PrOH). 1 H NMR [300 MHz, (CD3) 2S0]: J 8.90 (d, J = 2.1 Hz, 1H, 4-H), 7.91 (dd, J = 9.6, 2.1 Hz, 1H, 6-H), 7.53 (d, J = 9.6 Hz, 1H, 7-H), 7.31 (m, 5H, aromatic H Bn), 5.50 (s, 2H, CH2 Bn), 5.16 (t, J = 5.5 Hz, OH), 4.72 (t, J = 4.5 Hz, 2H, 1'-H), 3.81 (m, 2H, 2 ' -H); 13C NMR [75 MHz, (CD3) 2S0]: J 149.99 (C-3), 146.92 (C7a), 140.19 (C-5), 135.93 (C-1 Bn), 128, 61 (C-3, -5 Bn), 128.01 (C-2, -6 Bn), 127.88 (C-4 Bn), 120.86 (C-4), 119.91 (C-6 ), 118.03 (C-7), 105.33 (C-3a), 76.43 (C-1 '), 59.77 (C-2'), 51.49 (CH2 Bn); MS (lE): miz (%) 313 (100) (M +), 269 (58), 252 (14),
    222 (7), 191 (41), 164 (7), 149 (4), 103 (14). Ana!. cale. for CI6HISN304 (313.31): C
    61.34; H 4.83; N 13.41. Found: C 61.49; H 5.97; N 13.55.
    2-Benzyl-3- (3-hydroxypropoxy} -5-nitro-2H-indazole (22). Yield: 0.12 g (10%). Mp 122-124 oC (2-PrOH). 1 H NMR [300 MHz , (CD3) 2S0]: OR 8.89 (d, J = 2.1 Hz, 1H, 4-H), 7.90 (dd, J = 9.6, 2.1 Hz, lH, 6-H ), 7.53 (d, J = 9.6 Hz, 1H, 7-H), 7.32 (m, 5H, aromatic H Bn), 5.44 (s, 2H, CH2 Bn), 4, 80 (t, J = 6.1 Hz, 2H, l'-H), 4.68 (t, J = 5.2 Hz, 1H, OH), 3.60 (m, 2H, 3'-H) , 1.97 (m, 2H, 2'-H); I3C NMR [75 MHz, (CD3) 2S0]: OR 149.82 (C-3), 146.84 (C-7a), 140.14 ( C-5), 135.81 (C-1 Bn), 128.64 (C-3, -5 Bn), 127.90 (C-4 Bn), 127.82 (C-2, -6 Bn) , 121.00 (C-4), 119.90 (C-6), 118.02 (C-7), 104.94 (C-3a), 71.16 (Cl '), 56.75 (C -3 '), 51.64 (CH2 Bn), 32.36 (C-2'); MS (ES +): miz (%) 350 (27) ([M + Nat), 328 (lOO) ([M + Ht.) Anal !. for C17H17N304 (327.33): C 62.38; H 5.23; N 12.84. Found: C 62.09; H 5.52; N 13.09.
    2-Benzyl-3- (2-methoxyethoxy) -5-nitro-2H-indazole (23). Yield: 0.39 g (32%). Mp 109-111 oC (2-PrOH). IH NMR [300 MHz, (CD3) 2S0]: OR 8.88 (d, J = 2.1 Hz, lH, 4-H), 7.90 (dd, J = 9.6, 2.1 Hz, 1H, 6-H), 7.53 (d, J = 9.6 Hz, lH, 7-H), 7.30 (m, 5H, aromatic H Bn), 5.45 (s, 2H, CH2 Bn), 4.82 (m, 2H, 1'-H), 3.74 (m, 2H, 2'-H), 3.32 (s, 3H, CH3); I3C NMR [75 MHz, (CD3) 2S0]: OR 149.70 (C-3), 146.89 (C-7a), 140.28 (C5), 135.82 (C-1 Bn), 128, 62 (C-3, -5 Bn), 127.91 (C-2, -4, -6 Bn), 120.75 (C-4), 119.92 (C-6), 118.07 (C -7), 105.36 (C-3a), 73.70 (C-1 '), 70.38 (C-2'), 58.22 (CH3), 51.61 (CH2 Bn); MS (lE): miz (%) 327 (lOO) (M +), 269 (7), 252 (4), 222 (5), 191 (4), 164 (7), 149 (4), 103 (13 ). Ana!. tracing for CI7H17N304 (327.33): C 62.38; H 5.23; N 12.84. Found: C 62.50; H 4.97; N 12.63.
    Example lb. Preparation of propargyl derivative 2. A stirred mixture of 2-benzylindazolinone 1 (1.00 g, 3.71 mmol), propargyl bromide (80% by weight in toluene) (4.00 mmol) and K2C03 (0.55 g, 4.00 mmol) in acetone (50 mL) was refluxed for 12 h. The mixture was evaporated to dryness and, after the addition of water (200 mL), extracted with CHCb (3 x 50 mL). The concentrated chloroform phase was applied to a chromatography column that was eluted with chloroform / acetone mixtures (50: 1 to 30: 1) to provide compound 2.
    2-Benzyl-5-nitro-l-propargil-l, 2-dihydro-3H-indazol-3-one (2). Yield: 0.62 g (54%). Mp 163-165 oC (EtOH). lH NMR [300 MHz, (CD3) 2S0]: b 8.52 (d, J = 2.1 Hz, lH, 4-H), 8.47 (dd, J = 9.0, 2.1 Hz, lH, 6-H), 7.80 (d, J = 9.0 Hz, lH, 7-H), 7.27 (m, 5H, aromatic H Bn), 5.12 (s, 2H, CH2 Bn), 4.96 (d, J = 2.1 Hz, 2H, 1'-H), 3.16 (t, J = 2.1 Hz, lH, 3'-H); I3C NMR [75 MHz, (CD3) 2S0]: b 161.48 (C-3), 151.61 (C-7a), 143.11 (C-5), 136.09 (Cl Bn), 128, 56 (C-3, -5 Bn), 127.75 (C-4 Bn), 127.60 (C-6 and C-2, -6 Bn), 119.89 (C-4), 118.77 (C-3a), 114.02 (C-7), 76.58 (C-2 '), 75.95 (C-3'), 44.69 (CH2 Bn), 38.52 (Cl ') ; MS (lE): miz (%) 307 (100) (M +), 268 (13), 249 (5), 230 (3), 203 (4), 128 (5), 115 (4), 103 (11 ). Ana!. tracing for CI7HI3N303 (307.30): C 66.44; H 4.26; N 13.67. Found: C 66.58; H, 4.47; N 13.59.
    Example him. Preparation of the 2-bromoethyl (3117) and 3-bromopropyl (4118) derivatives.
    A stirred mixture of starting 2-benzylindazolinone 1 (2.00 g, 7.43 mmol), the required a, ro-dibromoalkane (40.00 mmol) and K2C03 (1.10 g, 8.00 mmol) in DMF (50 mL) was heated at 100 oC for 3 h. The mixture was evaporated to dryness and, after the addition of water (200 mL), extracted with CHCb (3 x 50 mL). The organic phase was dried (MgSO4), concentrated and applied to a chromatography column that was eluted with chloroform / acetone mixtures (50: 1 to 25: 1). From 1,2-dibromoethane, in this order of elution, indazole 2,3disubstituted 17, ell-vinylindazol24 [70 mg (3%)] and indazolinone 1,2-disubstituted 3 were obtained. Similarly, from 1,3-dibromopropane, the corresponding indazoI2,3-disubstituted 18 and indazolinone 1,2-disubstituted 4 were obtained.
    2-Benzyl-l- (2-bromoethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (3). Yield: 1.20 g (43%). Mp 172-174 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: b 8.52 (d, J = 2.4 Hz, lH, 4-H), 8.37 (dd, J = 9.0.2.4 Hz, lH, 6-H), 7.75 (d, J = 9.0 Hz, lH, 7-H), 7.26 (m, 5H, aromatic H Bn), 5.19 (s, 2H, CH2 Bn), 4.53 (t, J = 6.3 Hz, 2H, l'-H), 3.51 (t, J = 6.3 Hz, 2H, 2'-H); I3C NMR [75 MHz, (CD3) 2S0]: b 161.39 (C-3), 149.28 (C7a), 141.46 (C-5), 135.95 (Cl Bn), 128.75 ( C-3, -5 Bn), 127.87 (C-4 Bn), 127.26 (C2, -6 Bn), 127.02 (C-6), 120.36 (C-4), 115, 43 (C-3a), 112.59 (C-7), 47.67 (Cl '), 45.23 (CH2 Bn), 29.17 (C-2'); MS (lE): miz (%) 377 (99) ([M + 2t), 375 (100) (M +), 361 (5), 359 (5), 347 (7), 345 (7), 256 ( 9), 254 (9), 192 (8), 177 (9), 164 (9), 145 (8), 131 (17), 103 (36). Anal. tracing for Cl6Hl4BrN303 (376.20): C 51.08; H 3.75; N 11.17. Found: C 50.90; H 3.69; N 11.46.
    2-Benzyl-l- (3-bromopropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (4). Yield: 0.90 g (31%). Mp 111-113 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: b 8.52 (d, J = 2.5 Hz, lH, 4-H), 8.39 (dd, J = 9.1, 2.5 Hz, 1H, 6-H), 7.65 (d, J = 9.1 Hz, lH, 7-H), 7.28 (m, 5H, aromatic H Bn), 5.18 (s, 2H, CH2 Bn), 4.14 (t, J = 7.3 Hz, 2H, 1'-H), 3.36 (t, J = 6.6 Hz, 2H, 3'-H), 1.80 (m , 2H, 2'-H); 13 C NMR [75 MHz, (CD3) 2S0]: b 160.98 (C-3), 148.73 (C-7a), 141.52 (C-5), 136.01 (Cl Bn), 128, 72 (C-3, -5 Bn), 127.84 (C-4 Bn), 127.27 (C-6 and C-2, -6 Bn), 120.48 (C-4), 115.87 (C-3a), 112.02 (C7), 45.52 (Cl '), 44.89 (CH2 Bn), 30.81 (C-3'), 29.19 (C-2 '); MS (lE): miz (%) 391
    (98) ([M + 2t), 389 (100) ([M +]), 361 (4), 359 (4), 345 (6), 314 (6), 312 (6), 282 (7), 192 (27), 149 (23), 146 (21), 131 (25), 103 (26). Anal. tracing for C17H16BrN303 (390.23): C 52.32; H 4.13; N 10.77. Found: C 52.58; H 4.17; N 10.73.
    2-Benzyl-3- (2-bromoethoxy) -5-nitro-2H-indazole (17). Yield: 0.84 g (30%). Mp 140-142 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: b 8.88 (d, J = 2.1 Hz, lH, 4-H), 7.91 (dd, J = 9.6, 2.1 Hz, 1H, 6-H), 7.56 (d, J = 9.6 Hz, lH, 7-H), 7.32 (m, 5H, aromatic H Bn), 5.50 (s, 2H, CH2 Bn), 5.05 (m, 2H, 1'-H), 3.93 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: b 148.91 (C-3), 146.86 (C-7a), 140.46 (C-5), 135.65 (C-1 Bn), 128.63 (C-3, -5 Bn), 128.03 (C-2, -6 Bn), 127.97 (C-4 Bn), 120.50 (C-4), 119.97 (C6 ), 118.16 (C-7), 105.33 (C-3a), 73.84 (Cl '), 51.80 (CH2 Bn), 31.41 (C-2'); MS (EI): miz (%) 377 (60) ([M + 2t), 375 (61) (M +), 268 (31), 252 (58), 222 (10), 199 (65), 197 ( 67), 191 (47), 164 (14), 149 (7), 117 (51), 109 (97), 107 (10). Anal. tracing for Cl6Hl4BrN303 (376.20): C 51.08; H 3.75; N 11.17. Found: C 50.91; H 3.70; N 11.47.
    2-Benzyl-3- (3-bromopropoxy) -5-nitro-2H-indazole (18). Yield: 0.84 g (29%). Mp 124-126 oc (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: b 8.88 (d, J = 2.4 Hz, lH, 4-H), 7.90 (dd, J = 9.6, 2.4 Hz, lH, 6-H), 7.54 (d, J = 9.6 Hz, lH, 7-H), 7.31 (m, 5H, aromatic H Bn), 5.47 (s, 2H, CH2 Bn), 4.82 (t, J = 5.8 Hz, 2H, 1'-H), 3.66 (t, J = 6.6 Hz, 2H, 3'-H), 2.35 (m , 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: b 149.37 (C-3), 146.81 (C-7a), 140.28 (C-5), 135.82 (Cl Bn), 128, 63 (C-3, -5 Bn), 127.88 (C-4 Bn), 127.74 (C-2, -6 Bn), 120.76 (C-4), 119.91 (C-6 ), 118.09 (C-7), 104.92 (C-3a), 71.75 (Cl '), 51.76 (CH2 Bn), 32.02 (C-2'), 30.51 ( C-3 '); MS (lE): miz (%) 391 (99) ([M + 2t), 389 (100) (M +), 280 (7), 269 (98), 252 (43), 222 (12), 191 ( 38), 164 (14), 123 (48), 121 (50), 103 (27). Ana!. cale. for CI7HI6BrN303 (390.23): C 52.32; H 4.13; N 10.77. Found: C 52.51; H 4.36; N 10.57.
    Example Id. Preparation of (methoxycarbonyl) methyl derivatives 5119 and cyanomethyl derivative 6. A stirred mixture of 2-benzylindazolinone 1 (1.00 g, 3.71 mmol), methyl bromine acetate or bromoacetonitrile (4.5 mmol ) and NaHC03 (0.42 g, 5.00 mmol) in acetone (30 mL) was heated at 35 oC for 5 days. The mixture was evaporated to dryness and, after the addition of water (50 mL), the precipitated solid was collected by filtration. For compounds 5/19, the filtered solid was washed with acetone (2 x 5 mL) to provide pure compound 5 (1.03 g). The filtrate was concentrated to dryness, dissolved in chloroform and comatographed on a column eluted with chloroform and a mixture of chloroform / acetone (50: 1) to give, in this order of elution, compound 19 [0.04 g (3%)] And then an additional amount of compound 5 [total yield: 1.18 g (93%)]. For compound 6, the filtered solid was directly chromatographed with chloroform / acetone mixtures (50: 1 to 30: 1) to provide the pure derived l-cyanomethyl [0.84 g (73%)].
    2-Benzyl-l- (methoxycarbonyl) methyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (5). The crystals (2-PrOH) of this compound soften at 150-170 oC; then the product solidifies again showing a pfposterior at 195-197 oC. IH NMR [300 MHz, (CD3) 2S0]: () 8.54 (d, J = 2.1 Hz, 1H, 4-H), 8.40 (dd, J = 9.3, 2.1 Hz , 1H, 6-H), 7.67 (d, J = 9.3 Hz, lH, 7-H), 7.27 (m, 5H, aromatic H Bn), 5.15 (s, 2H, CH2 Bn), 5.09 (s, 2H, 1'-H), 3.41 (s, 3H, CH3); 13C NMR [75 MHz, (CD3) 2S0]: () 167.27 (C-2 '), 160.83 (C-3), 150.13 (C-7a), 141.79 (C-5) , 135.94 (C-1 Bn), 128.55 (C-3, -5 Bn), 127.72 (C-4 Bn), 127.45 (C-2, -6 Bn), 127.30 (C-6), 120.10 (C-4), 116.30 (C-3a), 112.00 (C-7), 52.00 (CH3), 47.77 (C-1 '), 44.87 (CH2 Bn); MS (ES +): miz (%) 705 (23) ([2M + Nat), 683 (25) ([2M + Ht), 364 (34) ([M + Nat), 342 (lOO) ([M + Ht). Anal. ca1c. for C17H1SN30S (341.32): C 59.82; H 4.43; N 12.31. Found: C 60.11; H 4.71; N 12.59.
    2-Benzyl-l-cyanomethyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (6). Mp 146-148 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: 8 8.57 (m, 2H, 4-, 6-H), 7.89 (d, J = 9.9 Hz, 1H, 7-H), 7 , 27 (m, 5H, aromatic H Bn), 5.43 (s, 2H, 1'-H), 5.17 (s, 2H, CH2 Bn); 13 C NMR [75 MHz, (CD3) 2S0]: 8161.86 (C-3), 151.58 (C-7a), 144.08 (C-5), 135.71 (Cl Bn), 128.66 (C-3, -5 Bn), 128.48 (C-6), 127.92 (C-4 Bn), 127.66 (C-2, -6 Bn), 120.14 (C-4), 119 , 15 (C-3a), 114.23 (C-7), 114.04 (C-2 '), 45.13 (CH2 Bn), 38.00 (C-1'); MS (lE): miz (%) 308 (87) (M +), 281 (100), 264 (3), 251 (4), 234 (4), 231 (5), 175 (14), 149 (6 ), 131 (5), 129 (5), 103 (19). Anal. tracing for C16H12N403 (308.29): C 62.33; H 3.92; N 18.17. Found: C 62.45; H 3.72; N 18.01.
    2-Benzyl-3- (methoxycarbonyl) methoxy-5-nitro-2H-indazole (19). Mp 94-96 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: 8 8.83 (d, J = 2.2 Hz, 1H, 4-H), 7.91 (dd, J = 9.6, 2.2 Hz, lH, 6-H), 7.57 (d, J = 9.6 Hz, lH, 7-H), 7.33 (m, 5H, aromat. Bn), 5.54 (s, 2H, CH2 Bn), 5.49 (s, 2H, 1'-H), 3.74 (s, 3H, CH3); 13C NMR [75 MHz, (CD3) 2S0]: b 168.38 (C-2 '), 148.71 (C-3), 146.80 (C-7a), 140.56 (C-5), 135.63 (Cl Bn), 128.58 (C-3, -5 Bn), 127.87 (C-2, -4, -6 Bn), 120.15 (C-4), 119.88 ( C-6), 118.20 (C-7), 105.15 (C-3a), 69.52 (Cl '), 52.22 (CH3), 51.84 (CH2 Bn); MS (lE): miz (%) 341
    (100) (M +), 325 (1), 282 (2), 268 (16), 252 (2), 236 (2), 222 (2), 191 (2), 164 (2), 103 (3 ). Anal. tracing for C17H1SN30S (341.32): C 59.82; H 4.43; N 12.31. Found: C 59.53; H, 4.67; N 12.57.
    Example 1e Preparation of 2- (methoxycarbonyl) ethyl derivatives 7/20 and 2-carboxy ethyl derivative 25. A stirred solution of 2-benzylindazolinone 1 (1.00 g, 3.71 mmol), K2C03 (1.38 g, 10.00 mmol) and benzyltributylammonium bromide (0.10 g) in a mixture of water (20 mL) and toluene (20 mL) was heated at 100 ° C for 30 min and then methyl 3-bromopropionate (0, 84 g, 5.03 mmol). Additional amounts of methyl 3-bromopropionate (ca. 10 x 0.25 g) and, if necessary, K2C03 to maintain the basic pH, were added for 2 days every 5-10 h. The reaction mixture was allowed to reach room temperature and then the toluene phase was separated, dried (MgSO4) and evaporated to dryness. The obtained residue was subjected to column chromatography using chloroform / acetone mixtures (50: 1 to 10: 1) to give, in this order of elution, compound 20 [0.15 g (11%)] and then the compound 7 [0.56 g (42%)].
    The remaining aqueous phase was acidified with conc. Aqueous HCl. (pH 1) And the precipitated solid was collected by filtration, dried and applied to a chromatography column that was eluted first with chloroform / methanol (50: 1 to 10: 1) and then with chloroform / methanol (10: 1 ) containing acetic acid (0.5%), to obtain, in this order of elution, the starting indazolinone 1 recovered [0.07 g (7%)] and then the acid 25 [0.33 g (26%) ].
    2-Benzyl-l- [2- (methoxycarbonyl) ethyl} -5-nitro-l, 2-dihydro-3H-indazol-3-one (7). Mp 127129 oC (2-PrOH). IH NMR [300 MHz, (CD3) 2S0]: OR 8.50 (d, J = 2.1 Hz, lH, 4-H), 8.37 (dd, J = 9.0, 2.1 Hz, lH, 6-H), 7.68 (d, J = 9.0 Hz, lH, 7-H), 7.25 (m, 5H, aromatic H Bn), 5.16 (s, 2H, CH2 Bn), 4.32 (t, J = 6.5 Hz, 2H, l'-H), 3.43 (s, 3H, CH3), 2.36 (t, J = 6.5 Hz, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: OR 170.82 (C-3 '), 161.31 (C-3), 149.04 (C-7a), 141.68 (C-5), 136.02 (Cl Bn), 128.71 (C-3, -5 Bn), 127.82 (C4 Bn), 127.28 (C-2, -6 Bn), 127.17 (C-6) , 120.29 (C-4), 116.25 (C-3a), 112.62 (C-7), 51.48 (CH3), 44.94 (CH2 Bn), 42.98 (C-l '), 30.30 (C-2'); MS (EI): miz (%) 355 (100) (M +), 282 (5), 278 (5), 177 (5), 131 (8), 103 (8). Anal. tracing for CIsHI7N30S (355.34): C 60.84; H 4.82; N 11.83. Found: C 60.77; H 4.91; N 11.57.
    2-Benzyl-3- [2- (methoxycarbonyljethoxy} -5-nitro-2H-indazole (20). Mp 130-132 ° C (2-PrOH). IH NMR [300 MHz, (CD3) 2S0]: OR 8, 90 (d, J = 2.1 Hz, lH, 4-H), 7.91 (dd, J = 9.6.2.1 Hz, lH, 6-H), 7.55 (d, J = 9.6 Hz, 1H, 7-H), 7.30 (m, 5H, aromatic H Bn), 5.42 (s, 2H, CH2 Bn), 4.90 (t, J = 5.7 Hz , 2H, l'-H), 3.64 (s, 3H, CH3), 2.95 (t, J = 5.7 Hz, 2'-H); 13C NMR [75 MHz, (CD3) 2S0] : OR 170.83 (C-3 '), 149.22 (C-3), 146.85 (C-7a), 140.42 (C-5), 135.69 (Cl Bn), 128.61 (C-3, -5 Bn), 127.90 (C-2, -4, -6 Bn), 120.51 (C-4), 119.95 (C-6), 118.14 (C-7) , 105.34 (C-3a), 70.18 (C-1 '), 51.68 (CH2 Bn, CH3),
    34.1 ° (C-2 '); MS (IE): miz (%) 355 (22) (M +), 324 (3), 91 (100). Anal. tracing for
    ClsH17N30S (355.34): C 60.84; H 4.82; N 11.83. Found: C 60.99; H 5.11; N 11.53.
    2-Benzyl-l- (2-carboxyethyl) -5-nitro-l, 2-dihydro-3H-indazo 1-3-one (25). Mp 224-226 oC (EtOH / H20). lH NMR [300 MHz, (CD3) 2S0]: () 12.39 (s, lH, COOH), 8.50 (d, J = 2.1 Hz, 1H, 4-H), 8.37 (dd , J = 9.3, 2.1 Hz, 1H, 6-H), 7.69 (d, J = 9.3 Hz, 1H, 7-H), 7.26 (m, 5H, aromatic H. Bn), 5.17 (s, 2H, CH2 Bn), 4.27 (t, J = 6.9 Hz, 2H, 1'-H), 2.26 (t, J = 6.9 Hz, 2H, 2 '-H); 13C NMR [75 MHz, (CD3) 2S0]: () 171.87 (C-3 '), 161.27 (C3), 149.01 (C-7a), 141.64 (C-5), 136 , 05 (Cl Bn), 128.73 (C-3, -5 Bn), 127.83 (C-4 Bn), 127.29 (C-2, -6 Bn), 127.14 (C-6 ), 120.29 (C-4), 116.22 (C-3a), 112.68 (C-7), 44.96 (CH2 Bn), 43.07 (C-1 '), 30.48 (C-2 '); MS (ES +): miz (%) 705 (51) ([2M + Nat), 683 (35) ([2M + Ht), 364 (65) ([M + Nat), 342 (100) ([M + Ht). Anal. tracing for C17HlSN30S (341.32): C 59.82; H 4.43; N 12.31. Found: C 59.60; H, 4.66; N 12.60.
    Example 1f. Alkoxycarbonylation, acylation and sulfonylation of 2-benzyl-5-nitroindazolin-3-one 1: preparation of alkoxycarboni! (12, 13), here! (14, 15) And I coughed!
    (16) derivatives. To a stirred solution of 2-benzylindazolin-3-one 1 (1.00 g; 3.71 rmol) in pyridine (15 mL), the required alkyl chloroformate (4.00 mmol) (for 12, was slowly added) 13), acetic anhydride (2.0 mL, excess) (for 14) or the corresponding acid chloride (3.90 rmol) (for 15, 16). The reaction mixture was heated at 100 oC for 3 h (for 14), or stirred at room temperature for 1 h (for 12, 13, 15 and 16), and then poured into water (100 mL). The precipitated solid was collected by filtration, washed with 2% aqueous HCl (50 mL) and with plenty of water, and air dried.
    2-Benzyl-l-ethoxycarboni! -5-nitro-l, 2-dihydro-3H-indazol-3-one (12). Yield: 1.20 g (95%). Mp 135-137 ° C (EtOH). lH NMR [300 MHz, (CD3) 2S0]: () 8.56 (d, J = 2.2 Hz, lH, 4-H), 8.52 (dd, J = 9.0, 2.2 Hz , lH, 6-H), 8.00 (d, J = 9.0 Hz, lH, 7-H), 7.26 (m, 3H) and 7.11 (m, 2H) (aromatic H Bn ), 5.33 (s, 2H, CH2 Bn), 4.39 (c, J = 7.0 Hz, 2H, CH2 Et), 1.30 (t, J = 7.0 Hz, 3H, CH3) ; 13 C NMR [75 MHz, (CD3) 2S0]: () 162.58 (C-3), 149.48 (COO), 145.57 (C-7a), 144.18 (C-5), 135, 33 (Cl Bn), 128.88 (C -6), 128.66 (C-3, -5 Bn), 127.96 (C-4 Bn), 127.41 (C-2, -6 Bn) , 119.63 (C-4), 118.19 (C-3a), 116.35 (C-7), 64.75 (CH2 Et), 49.98 (CH2 Bn), 13.83 (CH3); MS (ES +): miz (%) 705 (39) ([2M + Nat), 683 (75) ([2M + Ht), 364 (15) ([M + Nat), 342 (lOO) (M + Ht ). Anal. tracing for C17HlSN30S (341.32): C 59.82; H 4.43; N 12.31. Found: C 60.04; H 4.17; N 12.19.
    2-Benzyl-l-benzyloxycarbonyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (13).
    Yield: 1.45 g (97%). Mp 143-145 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: 8 8.56 (d, J = 2.1 Hz, lH, 4-H), 8.51 (dd, J = 8.9.2.1 Hz, lH, 6-H), 7.99 (d, J = 8.9 Hz, lH, 7-H), 7.42 (m, 6H), 7.21 (m, 2H) and 7.00 (m , 2H) (aromatic H 0- and NBn), 5.44 (s, 2H, CH2 OBn), 5.30 (s, 2H, CH2 NBn); 13C NMR [75 MHz, (CD3) 2S0]: 8 162.66 (C-3), 149.44 (COO), 145.56 (C-7a), 144.27 (C-5), 135.05 , 134.47 (Cl, and 0-y NBn), 128.95 (C-6), 128.81, 128.78, 128.64 (2 superimposed signals), 128.01, 127.49 (0- And NBn, and C-2, -3, -4, -5, -6), 119.70 (C-4), 118.29 (C-3a), 116.30 (C-7), 69.67 ( OCH2), 49.95 (NCH2); MS (ES +): miz (%) 829 (68) ([2M + Nat), 807 (36) ([2M + Ht), 426 (22) ([M + Nat), 404 (lOO) ([M + Ht). Anal. cale. for C22H17N30S (403.39): C 65.50; H 4.25; N 10.42. Found: C 65.59; H 4.06; N 10.16.
    l-Acetyl-2-benzyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (14). Yield: 1.03 g (89%). Mp 165-167 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: 8 8.56 (d, J = 2.1 Hz, lH, 4-H), 8.50 (dd, J = 9.3, 2.1 Hz, lH, 6-H), 8.30 (d, J = 9.3 Hz, lH, 7-H), 7.25 (m, 3H) and 7.11 (m, 2H) (aromatic H Bn) , 5.33 (s, 2H, CH2), 2.57 (s, 3H, CH3); 13C NMR [75 MHz, (CD3) 2S0]: 8 168.37 (CO Ac), 164.05 (C-3), 145.89 (C-7a), 144.11 (C-5), 135.12 ( Cl Bn), 128.92 (C-6), 128.67 (C-3, -5 Bn), 128.02 (C-4 Bn), 127.57 (C-2, -6 Bn), 119.69 (C-4), 118.52 (C-3a), 116.18 (C-7), 51.14 (CH2), 24.98 (CH3); MS (ES +): miz (%) 645 (44) ([2M + Nat), 623 (3) ([2M + Ht), 334 (62) ([M + Nat), 312 (100) ([M + Ht). Anal. tracing for C16H13N304 (311.29): C 61.73; H 4.21; N 13.50. Found: C 61.50; H, 4.47; N 13.59.
    2-Benzyl-l-benzoyl-5-nitro-l, 2-dihydro-3H-indazol-3-one (15). Yield: 1.36 g (98%). Mp 164-166 oC (2-PrOH). IH NMR [300 MHz, (CD3) 2S0]: 8 8.59 (d, J = 2.4 Hz, 1H, 4-H), 8.28 (dd, J = 9.3, 2.4 Hz, 1H, 6-H), 7.76 (dd, J = 7.6, 7.6 Hz, 1H, 4-H Bz), 7.57 (dd, J = 7.6, 7.6 Hz, 2H , 3-, 5-H Bz), 7.41 (d, J = 7.6 Hz, 2H, 2-, 6-H Bz), 7.27 (m, 3H) and 7.08 (m, 2H ) (H aromat. Bn), 6.48 (d, J = 9.3 Hz, 1H, 7-H), 5.27 (s, 2H, CH2); 13C NMR [75 MHz, (CD3) 2S0]: Ú 164.76 (CO Bz), 162.18 (C-3), 145.08 (C-7a), 144.03 (C-5), 134.84 ( C-1 Bn), 134.18 (C-4 Bz), 132.62 (C-1 Bz), 129.51 (C3, -5 Bz), 129.15 (C-2, -6 Bz), 128, 84 (C-3, -5 Bn), 128.53 (C-4 Bn), 128.29 (C-6), 127.72 (C-2, -6 Bn), 120.11 (C-4), 117.85 (C-3a), 114.09 (C-7), 48.84 (CH2); MS (ES +): miz (%) 769 (72) ([2M + Nat), 747 (44) ([2M + Ht), 396 (26) ([M + Nat), 374 (lOO) ([M + Ht). Anal. cale. for C21HlSN304 (373.36): C 67.56; H 4.05; N 11.25. Found: C 67.40; H 4.27; N 10.97.
    2-Benzyl-5-nitro-l-tosyl-l, 2-dihydro-3H-indazol-3-one (16). Yield: 1.54 g (98%). Mp 162-164 oC (l-PrOH). lH NMR [300 MHz, (CD3) 2S0]: Ú 8.58 (dd, J = 9.0, 2.1 Hz, 1H, 6-H), 8.40 (d, J = 2.1 Hz, 1H, 4-H), 8.14 (d, J = 9.0 Hz, 1H, 7-H), 7.44 (d, J = 8.4 Hz, 2H), 7.29 (m, 5H ) and 7.20 (m, 2H) (aromatic H, Bn and Ts), 5.34 (s, 2H, CH2), 2.28 (s, 3H, CH3); 13C NMR [75 MHz, (CD3) 2S0]: Ú 163.57 (C-3), 147.75 (C-7a), 147.23 (C-4 Ts), 146.29 (C-5), 135.11 (C-1 Bn), 130.04 (C-3, -5 Ts), 129.36 (C-6), 128.69 (C-3, -5 Bn), 128.60 (C-2 , -6 Ts), 128.20 (C-2, -6 Bn), 128.12 (C-4 Bn), 126.74 (C-1 Ts), 121.33 (C-3a), 119, 91 (C-4), 119.08 (C-7), 50.19 (CH2), 21.11 (CH3); MS (lE): miz (%) 423 (4) (M +), 268 (41), 155 (4), 91 (100). Anal. cale. for C21H17N30SS (423.44): C 59.57; H 4.05; N 9.92. Found: C 59.50; H 4.17; N 10.09
    EXAMPLE 2. Preparation of compounds 24-31 [type (1)] from compounds 3, 4, 7-9 and 26 [type (1)]. Compounds 24-31 were obtained (Scheme 2) from some of the 1,2-disubstituted indazolinones included in Scheme 1, following different procedures. Thus, the l-vinyl derivative 24 was prepared from the 2-bromoethyl derivative 3 through a dehydrohalogenation reaction. The propionic acid derivative 25 was obtained, together with ester 7, in the alkylation of indazolinone 1 with methyl 3-bromopropionate according to the procedure of Example le, by partial hydrolysis of the ester initially formed in the basic reaction medium; given its origin, compound 25 is described in Example 1. The butyric acid derivative 26 was obtained by hydrolysis (LiOH) of the corresponding ester 8. Butyramide 27 and N-methylbutyramide 28 were obtained by treatment of ethyl ester 8 with ammonia or methylamine, respectively; N, N-dimethylbutyramide 29 was prepared from acid chloride 26 (SOCh) and dimethylamine. The 3-ethoxypropyl derivative 30 was isolated from an attempt to prepare the 3-hydroxypropyl derivative 10 from bromide 4 and NaOH in ethanol / water. Finally, the 2-acetoxyethyl derivative 31 was prepared by acetylation (AC20) of the corresponding 2-hydroxyethyl derivative 9.
    Example 2a. Preparation of vinyl derivative 24. A solution of 1- (2-bromoethyl) indazolinone 3 (0.56 g, 1.49 mmol) and piperidine (0.35 g, 4.11 mmol) in ethanol (20 mL) was heated to reflux for 48 h. The solution was evaporated to dryness and, after the addition of 12% aqueous HCI (50 mL), extracted with chloroform (3 x 50 mL). The organic phase was dried (MgSO4) and evaporated to dryness to provide the desired compound; Similar results were obtained using methylamine (33% w / w in ethanolic solution). Compound 24 was also obtained as a by-product in the alkylation of 2-benzylindazolinone 1 with 1,2-dibromoethane (see above, Example 1 c).
    2-Benzyl-5-nitro-l-vinyl-l, 2-dihydro-3H-indazol-3-one (24). Yield: 0.41 g (93%). Mp 116-118 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: 8 8.53 (d, J = 3.0 Hz, 1H, 4-H), 8.40 (dd, J = 9.3, 3.0 Hz, lH, 6-H), 7.71 (d, J = 9.3 Hz, 1H, 7-H), 7.26 (m, 3H) and 7.15 (m, 2H) (aromatic H Bn) , 7.08 (dd, J = 14.7, 9.0 Hz, lH, l'-H), 5.25 (dd, J = 14.7, 1.4 Hz, lH, 2'-Htrans) , 5.20 (s, 2H, CH2 Bn), 5.10 (dd, J = 9.0, 1.4 Hz, lH, 2'-Hcis); 13C NMR [75 MHz, (CD3) 2S0]: 8 162.05 (C-3), 146.73 (C-7a), 142.10 (C-5), 135.48 (Cl Bn), 129, 93 (C-1 '), 128.66 (C-3, -5 Bn), 128.07 (C-6), 127.90 (C-4 Bn), 127.38 (C-2, -6 Bn), 120.43 (C-4), 115.46 (C-3a), 112.13 (C-7), 104.68 (C-2 '), 47.00 (CH2 Bn); MS (IE): miz (%) 295 (lOO) (M +), 280 (2), 265 (2), 218 (2), 158 (3), 145 (3), 116 (7), 104 (9) ). Anal. tracing for C16H13N303 (295.29): C 65.08; H, 4.44; N, 14.23. Found: C 65.30; H, 4.46; N 14.07.
    Example 2b Preparation of 4- (indazol-l-yl) butyric acid 26. A mixture of ethyl ester 8 (0.50 g, 1.30 mmol) and LiOH (0.16 g, 6.68 mmol) in THF / H20 ( 1: 1, 20 mL) was stirred at room temperature for 12 h. Then, THF was evaporated and the aqueous solution was acidified with conc. (pH 1). The precipitated solid was collected by filtration, washed with aqueous HCl (1%; 3 x 3 mL) and air dried.
    2-Benzyl-l- (3-carboxypropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (26). Yield: 0.42 g (91%). Mp 202-204 oC (EtOH / H20). lH NMR [300 MHz, (CD3) 2S0]: o12.20 (s, 1H, COOH), 8.52 (d, J = 2.4 Hz, lH, 4-H), 8.37 (dd, J = 9.3, 2.4 Hz, lH, 6-H), 7.62 (d, J = 9.3 Hz, 1H, 7-H), 7.27 (m, 5H, aromatic H Bn) , 5.20 (s, 2H, CH2 Bn), 4.05 (t, J = 7.5 Hz, 2H, l'-H), 2.15 (t, J = 7.2 Hz, 2H, 3'- H), 1.50 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: o173.61 (C-4 '), 160.81 (C-3), 148.53 (C7a), 141.31 (C-5), 136.13 (Cl Bn), 128.72 (C-3, -5 Bn), 127.83 (C-4 Bn), 127.27 (C2, -6 Bn), 127.17 (C-6), 120, 55 (C-4), 115.62 (C-3a), 111.89 (C-7), 45.95 (C-1 '), 44.66 (CH2 Bn), 30.15 (C-3 '), 21.40 (C-2'); MS (ES +): miz (%) 378 (20) ([M + Nat), 356 (100) ([M + Ht). Anal. tracing for ClsH17N30S (355.34): C 60.84; H 4.82; N 11.83. Found: C 60.59; H 4.97; N 11.68.
    Example 2e Preparation of amide 27 and N-methylamide 28. To obtain compound 27, a mixture of ethyl ester 8 (0.50 g, 1.30 mmol) and a saturated solution of ammonia in methanol (15 mL) was left stand at room temperature for 10 days. After removing the solvent and ammonia, in order to eliminate some by-products, the residue was chromatographed on a column that was eluted with a mixture of chloroform / methanol (25: 1) to provide the required product. To obtain compound 28, a mixture of ethyl ester 8 (0.50 g, 1.30 mmol) and 8 M methylamine in ethanol (20 mL) was allowed to stand at room temperature for 24 h. The reaction mixture was evaporated to dryness, providing the desired amide.
    2-Benzyl-l- (3-carhamoylpropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (27). Yield: 0.45 g (98%). Oil that solidifies after trituration with 2-PrOH; mp 106108 oC. lH NMR [300 MHz, (CD3) 2S0]: o8.52 (d, J = 2.1 Hz, lH, 4-H), 8.36 (dd, J = 9.0, 2.1 Hz, lH , 6-H), 7.62 (d, J = 9.0 Hz, lH, 7-H), 7.25 (m, 6H, aromatic H Bn and
    NHA), 6.79 (broad s, lH, NHs), 5.21 (s, 2H, CH2 Bn), 4.03 (t, J = 7.3 Hz, 2H, 1'-H), 1, 98 (t, J = 7.1 Hz, 2H, 3'-H), 1.51 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: () 173.16 (C-4 '), 160.73 (C-3), 148.40 (C-7a), 141.20 (C-5) , 136.16 (Cl Bn), 128.72 (C3, -5 Bn), 127.82 (C-4 Bn), 127.25 (C-2, -6 Bn), 127.09 (C-6 ), 120.54 (C-4), 115.49 (C-3a), 111.87 (C-7), 46.18 (C-l '), 44.61 (CH2 Bn), 31.04 (C-3 '), 21.77 (C-2'); MS (ES +): miz (%) 731 (15) ([2M + Nat), 709 (40) ([2M + Ht), 377 (12) ([M + Nat), 355
    (100) ([M + Ht). Anal. tracing for CI8HI8N404 (354.36): C 61.01; H 5.12; N, 15.81. Found: C 61.30; H 4.92; N 15.57.
    2-Benzyl-l- [3 - (methylcarbamoyl) propylj-5-nitro-l, 2-dihydro-3H-indazol-3-one (28).
    Yield: 0.45 g (94%). Mp 159-161 oC (2-PrOH). IH NMR [300 MHz, (CD3) 2S0]: () 8.52 (d, J = 2.1 Hz, lH, 4-H), 8.36 (dd, J = 9.3, 2.1 Hz , lH, 6-H), 7.67 (c wide, J = 4.5 Hz, lH, NH), 7.61 (d, J = 9.3 Hz, lH, 7-H), 7.25 (m, 5H, aromatic H Bn), 5.20 (s, 2H, CH2 Bn), 4.03 (t, J = 7.4 Hz, 2H, 1'-H), 2.52 (d, J = 4.5 Hz, 3H, CH3), 1.97 (t, J = 7.2 Hz, 2H, 3'-H), 1.53 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: () 171.32 (C-4 '), 160.77 (C-3), 148.45 (C-7a), 141.19 (C-5) , 136.15 (Cl Bn), 128.71 (C-3, -5 Bn), 127.81 (C-4 Bn), 127.24 (C-2, -6 Bn), 127.07 (C -6), 120.53 (C-4), 115.47 (C-3a), 111.87 (C-7), 46.16 (C-l '), 44.63 (CH2 Bn), 31.27 (C-3') , 25.40 (CH3), 22.01 (C-2 '); MS (ES +): miz (%) 759 (17) ([2M + Nat), 737 (53) ([2M + Ht), 391 (22) ([M + Na]), 369 (100) ([M + Ht). Anal. tracing for CI9H20N404 (368.39): C 61.95; H 5.47; N 15.21. Found: C 61.75; H 5.77; N 15.50.
    Example 2d. Preparation of N, N-dimethylamide 29. A suspension of acid 26 (0.53 g, 1.49 mmol) and SOCh (1.00 mL) in CHCb (30 mL) was heated at reflux for 1 h. After evaporation of the solvent and the excess of SOCh, a solution of dimethylamine hydrochloride (1.63 g, 19.99 mmol) and K2C03 (2.80 g, 20.26 mmol) in water was added to the oily residue. 20 mL) The mixture was stirred vigorously for 12 h and then the precipitated product 29 was collected by filtration, washed with water (3 x 5 mL) and air dried.
    2-Benzyl-l- [3- (dimethylcarbamoyl) propyl} -5-nitro-l, 2-dihydro-3H-indazol-3-one (29).
    Yield: 0.51 g (90%). Mp 144-146 oC (2-PrOH). IH NMR [300 MHz,
    (CD3) 2S0]: () 8.52 (d, J = 2.1 Hz, lH, 4-H), 8.37 (dd, J = 9.0, 2.1 Hz, lH, 6-H ), 7.60 (d, J = 9.0 Hz, lH, 7-H), 7.25 (m, 5H, aromatic H Bn), 5.23 (s, 2H, CH2 Bn), 4, 04 (t, J = 7.5 Hz, 2H, 1'-H), 2.80 (s, 3H, CH3 trans), 2.78 (s, 3H, CH3 cis), 2.19 (t, J = 6.6 Hz, 2H, 3'-H), 1.51 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: () 170.80 (C-4 '), 160.69 (C-3), 148.39 (C-7a), 141.19 (C-5) , 136.20 (Cl Bn), 128.71 (C-3, -5 Bn), 127.79 (C4 Bn), 127.26 (C-2, -6 Bn), 127.04 (C-6 ), 120.53 (C-4), 115.51 (C-3a), 111.79 (C-7),
    46.19 (C-l '), 44.57 (CH2 Bn), 36.39 (CH3 trans), 34.79 (CH3 cis), 28.61 (C-3'), 21.47 (C-2 '); MS (lE): miz (%) 382 (41) (M +), 337 (32), 310 (10), 291 (19), 282 (23), 268 (25), 246 (64), 232 (9 ), 218 (49), 192 (19), 172 (21), 128 (47), 114 (100), 106 (27). Ana!. cale. for C2oH22N404 (382.41): C 62.82; H 5.80; N 14.65. Found: C 62.99; H 5.96; N 14.51.
    Example 2e Preparation of 3-ethoxypropyl derivative 30. To a solution of 1- (3-bromopropyl) derivative 4 (0.59 g, 1.51 mmol) in ethanol (100 mL), NaOH (0.40 g, excess) in water was added (20 mL), and the mixture was stirred at room temperature for 3 days. The ethanol was then evaporated and the basic solution was extracted with CHCb (3 x 50 mL). The organic phase was dried (MgSO4), concentrated and column chromatographed with chloroform / acetone mixtures (50: 1 to 25: 1) to provide the ether.
    30
    2-Benzyl-l- (3-ethoxypropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one (30). Yield: 0.38 g (71%). Mp 93-95 oC (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: () 8.51 (d, J = 2.1 Hz, lH, 4-H), 8.36 (dd, J = 9.0, 2.1 Hz , lH, 6-H), 7.61 (d, J = 9.0 Hz, lH, 7-H), 7.26 (m, 5H, aromatic H Bn), 5.17 (s, 2H, CH2 Bn), 4.09 (t, J = 6.8 Hz, 2H, 1'-H), 3.17 (e, J = 7.1 Hz, CH2 Et), 3.09 (t, J = 6.0 Hz, 2H, 3'-H), 1.56 (m, 2H, 2'-H), 1.02 (t, J = 7.1 Hz, 3H, CH3); 13C NMR [75 MHz, (CD3) 2S0]: () 160.94 (C-3), 148.64 (C-7a), 141.11 (C-5), 136.11 (Cl Bn), 128 , 71 (C-3, -5 Bn), 127.80 (C-4 Bn), 127.21 (C-2, -6 Bn), 126.93 (C-6), 120.44 (C- 4), 115.34 (C-3a), 111.88 (C-7), 66.16 (C-3 '), 65.19 (CH2 Et), 44.87 (CH2 Bn), 44.09 (C-l'), 26.56 (C-2 '), 14.90 (CH3); MS (lE): miz (%) 355 (100) (M +), 282 (3), 278 (4), 268 (4), 220 (4), 192 (13), 146 (7), 131 (4 ), 106 (4), 103 (4). Anal. tracing for Cl9H21N304 (355.39): C 64.21; H 5.96; N 11.82. Found: C 63.92; H 5.77; N 11.98.
    Example 2f. Preparation of the 2-acetoxyethyl derivative 31. A suspension of 1- (2-hydroxyethyl) derivative 9 (0.47 g, 1.50 mmol) in acetic anhydride (5 mL) was heated at 100 oc for 2 h. The reaction was evaporated to dryness and the residue was triturated with 2-PrOH (2 mL); The insoluble compound was collected by filtration, washed with 2-PrOH (2 x 2 mL) and air dried.
    1- (2-Acetoxyethyl) -2-benzyl-5-nitro-I, 2-dihydro-3H-indazo1-3-one (31). Yield: 0.47 g (88%). Mp 168-170 oc (2-PrOH). lH NMR [300 MHz, (CD3) 2S0]: o8.51 (d, J = 2.4 Hz, lH, 4-H), 8.38 (dd, J = 9.0, 2.4 Hz, lH , 6-H), 7.66 (d, J = 9.0 Hz, lH, 7-H), 7.25 (m, 5H, aromatic H Bn), 5.19 (s, 2H, CH2 Bn ), 4.36 (t, J = 4.8 Hz, 2H, 1'-H), 4.07 (t, J = 4.8 Hz, 2H, 2'-H), 1.57 (s, 3H, CH3); 13C NMR [75 MHz, (CD3) 2S0]: o169.53 (CO Ac), 161.19 (C-3), 149.90 (C-7a), 141.32 (C-5), 136.04 (Cl Bn), 128.73 (C-3, 5 Bn), 127.86 (C-4 Bn), 127.32 (C-2, -6 Bn), 126.84 (C-6), 120.33 (C-4), 115.51 (C3a), 112.48 (C-7), 60.22 (C-2 '), 45.95 (C-l'), 44.81 (CH2 Bn), 20.08 (CH3); MS (EI): miz (%) 355 (10) (M +), 325 (2), 295 (5), 282 (6), 204 (6), 177 (9), 131 (10), 103 (7) ). Anal. cale. for ClsH17N305 (355.34): C 60.84; H 4.82; N 11.83. Found: C 60.60; H 5.03; N 11.57.
    EXAMPLE 3. In vitro study of the activity of indazole derivatives on epimastigotes of Trypanosoma cruzi. Activity tests against T cruzi are carried out in accordance with a sequential in vitro pharmacological screening protocol in which, first, simultaneous tests of activity against epimastigotes (extracellular form) and nonspecific cytotoxicity in L929 fibroblasts ( host cells of amastigotes, intracellular form of the parasite). Epimastigotes are extracellular forms present only in vector insects (triatomines), but since they are easily cultivated in vitro in the laboratory, they are used for the initial screening of antihagasic activity. Those compounds that result in epimastigote selectivity equal to or greater than benznidazole are selected for a subsequent activity test on amastigotes (Fonseca-Berzal, C. et al., Bioorg. Med Chem. Lett. 2013, 23, 4851 -4856). Amastigotes are intracellular parasites of affected mammalian cells, so the results obtained on them are much more significant from the point of view of Chagas disease in humans.
    To evaluate in vitro activity in epimastigotes, axenic cultures of T cruzi, strain CL clone B5 stably transfected with the Escherichia coli ~ galactosidase (lacZ) gene (Buckner, F. et al., Antimicrob. Chemother. 1996, 40, 2592-2597). This strain exhibits a biological behavior similar to that of the parental CL strain that makes it suitable for in vitro drug screening (Le-Senne, A. et al., Mem. Inst. Oswaldo Cruz 2002, 97, 1101-1105). Expression of the enzyme ~ -galactosidase by the parasite results in the reduction of the red chromogenic substrate of chlorophenol ~ -D-galactopyranoside (CPRG) to phenol red. Said colorimetric reaction is directly proportional to the number of live parasites, which allows estimating the percentages of activity of the compounds by photometry spectrum.
    The epimastigote cultures are maintained at 28 oC in LIT medium supplemented with 10% (v / v) with inactivated fetal bovine serum (56 oC, 30 min.), Penicillin (10 DI / mL) and streptomycin (100 µg / mL), performing weekly subcultures to keep them continuously in exponential growth phase.
    According to the protocol standardized by Vega, M. C. et al. (Parasitol. Res. 2005, 95, 296-298), subsequently modified by Fonseca-Berzal, C. et al. (Bioorg. Med Chem. Lett. 2013, 23, 4851-4856), exponentially growing cultures were distributed in 96-well plates at a rate of 250,000 epimastigotes / mL in volumes of 200 µl. Well and incubated together with the compounds to be tested for 72 h at 28 oC. All compounds were dissolved in dimethyl sulfoxide (DMSO) and added extemporaneously to the respective culture media at final concentrations of 256-0.125 µM. The final concentration of DMSO in cultures never exceeded 0.2% (v / v), with no toxic effect at this concentration for cells and parasites. In each plate each concentration was tested in triplicate and growth controls, medium and compound blanks were included, as well as an internal control with benznidazole, reference drug. After 72 hours, the test was revealed by adding 50 IlLlcillo of a 0.9% CPRG solution Triton X-lOO
    (final concentration 200 IlM, pH 7.4) and subsequent incubation of the plates for 3 h at 37 oc. Finally, the absorbance at 595 nm was determined in a plate spectrophotometer (ELx808 ELISA reader, Biotek Instruments Inc.) and the activity percentages were estimated by comparing the average value obtained in the wells treated with the average absorbance value presented by the growth control
    For each compound, the concentration necessary to inhibit 50% of the growth of epimastigotes (CIso epimastigotes) was estimated from the corresponding dose-response curve (tested concentration vs percentage of activity in epimastigotes). The activity results are expressed as the mean of the IC in epimastigotes ± standard deviation (SD), based on the values obtained in three independent experiments (n = 3). The results obtained are shown in Table 2. It can be seen that the 3-alkoxy-2-benzylindazoles [series compounds (11)] show little activity, while many of the 1,2-disubstituted indazolinones [series compounds (1)] present activities, expressed as CIso, far superior to that of benznidazole, reference drug (CIso = 22.73 IlM). Highlights include indazolinones containing 1 substituted alkyl groups such as 2-bromoethyl (3), 3-bromopropyl (4), 2-hydroxyethyl (9), 3-hydroxypropyl (10) or alkenyl groups such as vinyl (24), with values of CIso = 1,582.90 IlM. Derivatives with a carboxylic acid side chain (25, 26) as well as acylated compounds in position 1 (14, 15), on the contrary, have no activity.
    EXAMPLE 4. In vitro study of the specific cytotoxicity of indazole derivatives against murine fibroblasts L929 and determination of selectivity indices (IS). In order to rule out any toxic effects of the compounds on mammalian cells, as well as to determine their selectivity towards T cruzi, their nonspecific cytotoxicity in L929 fibroblasts was evaluated.
    Table 2. In vitro activity against extracellular (epimastigote) and intracellular (amastigote) forms of T. cruzi and nonspecific cytotoxicity in murine L929 fibroblasts, expressed as CIso and CLso, respectively.
    Compound CIso epimastigotes Ü! M)CLso L929 (tt: M2Isa eEimastigotesCIso amastigotes (tt: M2ISb amastigotes
    2 10.79 ± 4.80195.27 ± 9.7018.103.02 ± 0.5564.66
    3 2.13 ± 0.72gt; 256> 120.191.14 ± 0.14gt; 224.56
    4 2.90 ± 0.3725.19 ± 3.168.69-and
    26.17 ± 11.31 128.89 ± 10.324.92
    6 11.66 ± 2.13> 256gt; 21.953.50 ± 0.83gt; 73.14
    7 9.19 ± 1.20gt; 256gt; 27.862.93 ± 1.68gt; 87.37
    8 11.14 ± 0.5978.07 ± 8.027.01
    9 1.58 ± 0.06gt; 256gt; 162.020.22 ± 0.06gt; 1,163.64
    1.68 ± 0.36 gt; 256gt; 152.380.25 ± 0.12gt; 1,024.00
    eleven 6.56 ± 0.87gt; 256gt; 39.022.29 ± 0.79gt; 111.79
    12 49.01 ± 14.58122.03 ± 15.982.49
    13 43.59 ± 5.4186.06 ± 13.661.97
    14 > 256gt; 256NDd
    > 256 145.52 ± 6.10lt; 0.57
    16 28.76 ± 2.67> 256gt; 8.90
    17 gt; 256gt; 256ND
    18 39.04 ± 13.4259.04 ± 4.611.51
    19 144.71 ± 9.30> 256gt; 1.77
    gt; 256gt; 256 ND
    twenty-one gt; 256gt; 256ND
    22 64.91 ± 8.34> 256> 3.94
    2. 3 gt; 256gt; 256ND
    24 2.75 ± 0.09160.46 ± 14.8958.350.47 ± 0.10341.40
    gt; 256gt; 256 ND
    26 gt; 256gt; 256ND
    27 30.98 ± 3.94gt; 256gt; 8.26
    28 13.29 ± 0.92gt; 256gt; 19.261.50 ± 0.23gt; 170.67
    29 3.86 ± 0.37gt; 256gt; 66.320.54 ± 0.04gt; 474.07
    6.57 ± 0.27 170.41 ± 9.9425.943.31 ± 0.5251.48
    31 5.43 ± 1.89gt; 256> 47.140.25 ± 0.14gt; 1,024
    Benznidazo1
    22.73 ± 3.33 gt; 256 gt; 11.26 0.68 ± 0.08 gt; 376.47
    Selectivity indices for epimastigotes (IS = LC50 L929 / CI5 or epimastigotes).Selectivity indices for amastigotes (lS = LC50 L929 / CI5 or amastigotes).eNot determined.d Not evaluated in amastigotes for not reaching epimastigotes the minimum selectivityestablished by the reference drug (lScomposite <ISBenznidazoI).
    The cultures of L929 are maintained at 37 oC and 5% C02 in bottles of 75 cm2 of surface, using MEM medium without phenol red, supplemented to 10% (v / v) with inactivated fetal bovine serum (56 oC, 30 min. ), penicillin (lOO UVmL) and streptomycin (lOO¡tg / mL).
    The cytotoxicity in L929 fibroblasts was determined by fluorimetry in the presence of the resazurin substrate, a redox indicator that at the same time undergoes a color change and emits fluorescence in the presence of metabolically active cells (AlamarBlue® Assay, U.S. Patent No. 5,501,959).
    For the performance of the assay, L929 cells were detached from the culture flask by trypsinization. To do this, after removing the culture medium, a solution of EDTA-trypsin was added to the bottle and incubated for 5 min at 37 oC and 5% C02. This mixture was centrifuged 5 min at 1,500 rpm, the supernatant was removed and the cells were resuspended in MEM medium at a concentration of 15 x 04 L929 / mL, which was distributed in 96-well plates placing 100 µl. Well. After incubating the plates 3 h at 37 oC and 5% C02 to favor the adhesion of the cells, the culture medium was discarded and 200 µl of the compounds to be tested diluted in fresh MEM was added.
    The plates were incubated for 48 h at 37 oC and 5% C02. Each concentration was evaluated in triplicate and growth controls, medium and compound blanks were included, as well as an internal control with benznidazole in each plate. Finally, 20 µl of a resazurin solution in 1% PBS (2 mM, pH 7) was added and the plates were incubated for 3 h at 37 oC and 5% C02. The fluorescence intensity was determined at 535 nm (excitation) and 590 nm (emission) in a plate spectrofluorimeter (Infinite 200, Tecan). The percentage of cytotoxicity was calculated by comparing the fluorescence signal emitted by the treated cells with respect to
    to that emitted by untreated cells (Fonseca-Berzal, C. et al., Bioorg. Med. Chem. Lett. 2013,23,4851-4856).
    For each compound, the concentration necessary to inhibit 50% of cell growth (CLso L929) was estimated from the corresponding dose-response curve (concentration tested vs. percentage of cytotoxicity in fibroblasts). Activity results are expressed as the mean of the CLso ± SD, based on the values obtained in three independent experiments (n = 3). The results obtained are shown in Table 2. It can be seen that most of the active compounds against epimastigotes (3, 9, 10 and 24) have much lower toxicity than benznidazole against fibroblasts, so that their IS for epimastigotes they are far superior to those of the reference drug.
    EXAMPLE 5. In vitro study of the activity of indazole derivatives on trypanosoma cruzi amastigotes. In the same way as activity tests on epimastigotes, these tests were performed on the CL-B5 strain transfected with the Escherichia coli ~ -galactosidase (lacZ) gene and the activity of the compounds was determined by reduction of the CPRG substrate (Buckner, F. et al., Antimicrob. Agents Chemother. 1996, 40, 25922597).
    To evaluate in vitro activity in amastigotes, a clinically relevant parasitic form in Chagas disease, trypomastigotes derived from cell culture (TDC) were obtained by infecting cultures of L929 cells with epimastigotes in the stationary phase of growth. The cultures were incubated together with the infective forms for 24 h at 33 oC and 5% C02 to favor cell invasion. After this time, those parasites that had not infected the cells were removed by washing the cultures with PBS. After adding fresh MEM medium, the infected cultures were incubated under similar conditions of temperature and humidity for 7 days, at which time TDC was obtained in the supernatant.
    The tests were performed in 48-well cell culture plates (Fonseca-Berzal,
    C. et al., Parasitol. Res. 2014, 113, 1049-1056). After detaching the L929 cells from their cell culture flasks by trypsinization, they were distributed per well
    10,000 L929 in 120 IlL of MEM and the plates were incubated 2 h at 37 oC and 5% C02. Once the cells were adhered, they were infected with TDC in a 1: 6 ratio (cell: parasite) and incubated for 24 h at 33 oC and 5% C02. Then, the culture medium was removed, washed with PBS to remove the TDCs that did not penetrate the cells and dilutions of the compounds to be tested in fresh MEM were added in a final volume of 450 IlLlcillo. Plates were incubated for 7 days at 33 oC and 5% C02 and each concentration was evaluated in triplicate, including infection controls, cell growth, media and compound targets, as well as an internal control with benznidazole. At the end of the incubation time with the compounds, the test was revealed by adding 50 IlLlipocillo of a solution of CPRG in 3% Triton X-lOO (final concentration 400 IlM, pH 7.4) and subsequent incubation of the plates for 3 h at 37 oC. Finally, the absorbance at 595 nm was determined in a plate spectrofluorimeter (Infinite 200, Tecan). The activity percentages were estimated by comparing the average absorbance value obtained in the treated wells, with respect to the average absorbance of the infection controls. The absorbance corresponding to the cell growth controls (L929 cells only) was subtracted in the experimental and infection control groups.
    For each compound, the concentration necessary to inhibit 50% of the growth of amastigotes (CIso amastigotes) was estimated from the corresponding dose-response curve (concentration tested vs. percentage activity in amastigotes). The activity results are expressed as the mean of the IQ for amastigotes ± SD, based on the values found in three independent experiments (n = 3). The results obtained are shown in Table 2. It can be seen that several 1,2-disubstituted indazolinones [series compounds (1)] are also superior to benznidazole (CIso = 0.68 IlM; IS> 376.47) in Regarding activity and selectivity index against amastigotes, highlighting products 9, 10, 29 and 31 (CIso = 0.22-0.54IlM; IS> 474.07-gt; 1,163.64).
    EXAMPLE 6. In vitro study of the activity of indazole derivatives on trophozoites of Trichomonas vaginalis. The search for new tricomonicidal agents follows a sequential screening model based on several phases that, as a filter, allow only those products that show certain significant activity values to pass to the next level of study (Ibáñez-Escribano, A. et al. , J Microbio !. Methods 2014,105, 162-167).
    First, indazole derivatives are evaluated against the vaginalis T isolate JH31A4, from the American Type Culture Collection (ATCC), sensitive to the reference drug metronidazole. This isolate is maintained in culture in TYM medium (tripticase, yeast extract, maltose), supplemented with L-cysteine, ascorbic acid and 10% of decomplemented fetal bovine serum. The parasite cultures are kept in an oven at 37 oC and 5% C02, with passes being made every 48 hours. In vitro screening is carried out by evaluating the growth percentage of a controlled culture after 24 hours in contact with increasing concentrations of the compound to be evaluated. To determine the inhibitory concentration 50 (CIso), a stock solution of the compounds in dimethyl sulfoxide (DMSO) is prepared, and tested in a range of six different concentrations in successive serial double dilutions, starting from a maximum concentration of 300 flM .
    First, the cultures are prepared in glass tubes with 100,000 trophozoites / mL in a final volume of 2 mL. After 5 h of incubation in an oven, 4 flL of the compounds dissolved in DMSO (final solvent volume <0.2%) are added. After 24 h of incubation at 37 oC and 5% C02, 200 flL of each culture are distributed in sterile 96-well flat bottom plates (Nunc). The culture medium is then removed after centrifuging the plates at 2,000 rpm for 5 min and the trophozoites are suspended in the same volume of sterile phosphate buffer supplemented with 0.1% glucose. Finally, the test is revealed by adding 20 flL of a 3 mM resazurine stock solution to each well. This substrate is a redox dye capable of being reduced to resorufin by viable cells. Resorufin, unlike resazurin, emits fluorescence and the percentage of cell viability can be determined by fluorometric reading. After one hour of incubation at 37 oC the fluorescence intensity is determined in an INFINITE fluorimeter (J.quot; excitation 535 nm and quot; emission 590 nm), following the protocol previously described by the research group (lbáñez Escribano, A. et al., Mem. Inst. Oswaldo Cruz 2012, 107, 637-643). All tests include a 25 IlM metronidazole control and three growth controls to which the same volume of solvent (DMSO) is added. The results are calculated from the average obtained from at least two independent experiments. All compounds are evaluated in triplicate in each test, obtaining a DS below 10%.
    Compounds that show a relevant activity against the parasitic isolate were also subjected to evaluation against the culture of IR vaginal metronidazole resistant T 78. The in vitro screening procedure is identical to that described for isolate JH31A4.
    The results obtained for the metronidazole sensitive JH31A4 T vaginalis isolate are shown in Table 3, where the concentration values that produce a 50% inhibition of parasite growth (CIso) are shown. The compounds that passed the first evaluation filter in T vaginalis, presenting values of CIso lt; 50 IlM, were 1,2-disubstituted indazolinones [type compounds (1)] 9 and 29, and 3-alkoxy-2-alkylindazoles [type compounds (11)] 18, 20, 21 and 22. Compounds should be noted of type (11) 21 and 22 [3- (2-hydroxyethoxy) and 3- (3-hydroxypropoxy) indazoles], which presented a relevant activity against the parasite with IC50 values of 9.82 and 7.25 11M, respectively . The derivative 22 showed the highest antiparasitic activity achieving a growth inhibition of more than 90% at the maximum concentration evaluated, being higher than the activity shown at the same concentration by metronidazole, reference drug. The rest of the molecules tested were not very active or did not even show any effect on the growth of the parasites, being unable to calculate their IQ values in these cases.
    Table 4 shows the results obtained against the isolate of T. vaginalis resistant to metronidazole IR 78. The value of CIso corresponding to compound 22 is very similar to that obtained with the isolate sensitive to metronidazole, which shows the lack of cross resistance between this compound and the reference drug. Thus, compound 22 can be considered a good prototype for the development of usable drugs in cases of resistance to 5-nitroimidazoles, for which there is no alternative medicine accepted by international health agencies.
    EXAMPLE 7. In vitro study of the unspecific cytotoxicity of indazole derivatives against Yero cells and determination of selectivity indices (IS).
    In this study, only those molecules that have shown significant in vitro activity against T. vaginalis (CIso <50 J.lM) are analyzed.
    This test is carried out on the Yero CCL-81 cell line (ATCC); The cells are kept in horizontal cell culture boats, in RPMI-1460 medium (SigmaAldrich) supplemented with 10% fetal bovine serum and antibiotic solution. The cells are detached using a sterile EDTA-trypsin solution. A hemocytometric count with Trypan blue is then made, adding a concentration of 50,000 cells / lOO J.lLpocillo to 96-well sterile plates. The plates are kept in an oven at 37 ° C and 5% C02 to favor the adhesion of the cells to the plastic. After incubation the compounds are added at the same concentrations that were tested against T. vaginalis. After keeping the cells in contact with the compounds for 24 h, the test is developed by adding 20 J.lL of the resazurine dye prepared at a stock concentration of 1 mM in sterile PBS solution. Fluorimetric readings are carried out after 3 hours of incubation with the redox substrate. As in the in vitro tests against T. vagina lis, the results are calculated from the average obtained after performing at least two independent experiments. Each concentration is evaluated in triplicate obtaining a DS below 10%, there is also a growth control that assumes a 0% non-specific cytotoxic activity.
    The results obtained in this study are shown in Tables 3 and 4, where the values necessary to inhibit 50% of the growth of Vero cells (CCso) are shown. Likewise, the selectivity indices (IS) that determine the selective toxicity of the molecule from the relationship between CCso and ICs have been estimated. The six compounds tested (9, 18, 20, 21, 22 and 29) showed low unspecific cytotoxic activity, with percentages of growth inhibition of Vero lt cells; 20% at 300 IlM And IS values between 6.0 and gt; 41.4. Again, it is worth highlighting the compounds of type (11) 21 and 22, which showed cytotoxicity percentages (% C) at the maximum concentration tested (300 IlM) very low (% C23 =
    10 14.7 ± 4.3; % C24 = 14.5 ± 3.2).
    Table 3. In vitro activity against T. vaginalis JH31A4 and non-specific cytotoxicity against Vero cells, expressed as CIso and CCso respectively.
    Compound CIso (¡..tM)CCso (¡..tM)IS CompoundCIso (¡..tM)CCso (¡..tM)IS
    2 260.9716151.47
    3 347.741764.75
    4 80.211818.57538.8529.0
    5 534.57twenty43.02gt; 300gt; 7.0
    6 NDtwenty-one9.82gt; 300gt; 30.5
    7 375.54227.25gt; 300gt; 41.4
    8 192.422. 3107.45
    9 17.94gt; 300gt; 16.724258.43
    10 128.4425ND
    eleven 284.6227104.13
    12 418.8628182.01
    13 588.882948.94gt; 300gt; 6.1
    14 212.9130362.18
    fifteen 197.4231265.63
    Metronidazole 1.43 gt; 600 gt; 100
    ND: Not determined by absence of antiparasitic activity
    Table 4. In vitro activity against T. vaginalis IR 78 and non-specific cytotoxicity against Vero cells, expressed as CIso and CCso, respectively.
    Compound CIso (¡..tM)CCso (¡..tM)IS
    18 39.12538.8513.8
    twenty-one 49.82gt; 300gt; 6.0
    22 9.11gt; 300gt; 32.9
    Metronidazole 5.78> 600gt; 100
    In the case of the isolate of T. vaginalis resistant to metronidazole, the values of the SI are comprised between; 6.0 Y gt; 32.9, once again presenting compound 22 as the maximum value, so it is promising as an alternative to the reference drug.
    权利要求:
    Claims (8)
    [1]
    1. Compounds derived from 5-nitroindazole of general formulas (1) and (11),
    Formula (1) Formula (11)
    where R can be:- an alkenyl or alkynyl group of 2-6 carbon atoms and with the multiple bondin any of the possible positions. 1-Allyl-2-benzyl-5-nitroindazolinone [compound type (1)] is specifically excluded.-a polymethylene group of variable length [(CH2) n, n = 1-6] with substituentsterminals of type Br, OH, COOR (R = H or alkyl of 1-5 carbon atoms),CONR1R2 (R1 and / or R2 = H or alkyl of 1-5 carbon atoms), CN, OR (R =alkyl or acyl groups of 1-5 carbon atoms).-a ethoxycarbonyl or benzyloxycarbonyl group.- an aliphatic acyl group (from 1 to 5 carbon atoms such as formyl, acetyl orpropionyl) or aromatic (benzoyl or benzoyl differently substituted in thepositions 2,3 or 4 with groups such as F, CI, Br, OH, OR, NH2, N02 or CN).- an aliphatic or aromatic sulfonyl group [methanesulfonyl or p-toluenesulfonyl(tosyl)].
    and where X can be any substituent at positions 2, 3 or 4 of a groupbenzyl, selected from a single or branched alkyl group of 1-5 atoms ofcarbon, trifluoromethyl (CF3), F, CI, Br, hydroxyl groups (OH), a1coxyl (OR; R= alkyl), amino groups (NH2, NR2), nitro (N02) or cyano (CN). In compounds oftype (1), when R = CH2COOH, derivatives with two are specifically excludedhalogen atoms in the benzyl substituent.
    or its solvates or prodrugs.
    [2]
    2. A compound of the general formula (1) according to claim 1, selected from the following list: 2-Benzyl-5-nitro-I-propargyl-1, 2-dihydro-3H-indazol-3-one 2-Benzyl- I- (2-bromoethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one 2-Benzyl-l- (3-bromopropyl) -5-nitro-l, 2-dihydro-3 H- indazol-3-one 2-Benzyl-l- (methoxycarbonyl) methyl-5-nitro-l, 2-dihydro-3H-indazol-3-one 2-Benzyl-l-cyanomethyl-5-nitro-l, 2-dihydro -3H-indazol-3-one 2-Benzyl-I- [2- (methoxycarbonyl) ethyl] -5-nitro-1, 2-dihydro-3H-indazol-3-one 2-Benzyl-I- [3- ( ethoxycarbonyl) propyl] -5-nitro-l, 2-dihydro-3H-indazol-3-one 2-Benzyl-l- (2-hydroxyethyl) -5-nitro-l, 2-dihydro-3H-indazol-3- one 2-Benzyl-l- (3-hydroxypropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one 2-Benzyl-l- (2-methoxyethyl) -5-nitro-l, 2- dihydro-3H-indazol-3-one 2-Benzyl-I-ethoxycarbonyl-5-nitro-1, 2-dihydro-3H-indazol-3-one 2-Benzyl-I-benzyloxycarbonyl-5-nitro-l, 2- dihydro-3H-indazol-3-one l-Acetyl-2-benzyl-5-nitro-I, 2-dihydro-3H-indazol-3-one 2-Benzyl-l-benzoyl-5-nitro-l, 2- dihydro-3H-in dazol-3-one 2-Benzyl-5-nitro-l-tosyl-l, 2-dihydro-3H-indazol-3-one 2-Benzyl-5-nitro-l-vinyl-l, 2-dihydro-3H- indazol-3-one 2-Benzyl-l- (2-carboxyethyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one 2-Benzyl-l- (3-carboxypropyl) -5-nitro- l, 2-dihydro-3H-indazol-3-one 2-Benzyl-l- (3-carbamoylpropyl) -5-nitro-l, 2-dihydro-3H-indazol-3-one 2-Benzyl-l- [3 - (methylcarbamoyl) propyl] -5-nitro-l, 2-dihydro-3H-indazol-3-one 2-Benzyl-l- [3- (dimethylcarbamoyl) propyl] -5-nitro-l, 2-dihydro-3H -indazol-3-one 2-Benzyl-l- (3-ethoxypropyl) -5-nitro-1, 2-dihydro-3H-indazol-3-one 1- (2-Acetoxyethyl) -2-benzyl-5-nitro -1,2-dihydro-3H-indazol-3-one
    or its solvates or prodrugs.
    [3]
    3. A compound of the general formula (11) according to claim 1, selected from the
    following list:2-Benzyl-3- (2-bromoethoxy) -5-nitro-2H-indazole2-Benzyl-3- (3-bromopropoxy) -5-nitro-2H-indazole
    2-Benzyl-3- (methoxycarbonyl) methoxy-5-nitro-2H-indazole2-Benzyl-3- [2- (methoxycarbonyl) ethoxy] -5-nitro-2H-indazole2-Benzyl-3- (2-hydroxyethoxy) -5-nitro-2H-indazole2-Benzyl-3- (3-hydroxypropoxy) -5-nitro-2H-indazole
    5 2-Benzyl-3- (2-methoxyethoxy) -5-nitro-2H-indazole
    or its solvates or prodrugs.
    [4]
    4. Procedure for the preparation of the compounds of general formula (1) and (11)
    10 by treating 5-nitroindazolinone with alkylating, alkoxycarbonylating, acylating and sulphonilating agents, as set forth in Scheme 1.
    20 Scheme 1
    [5]
    5. Process for the preparation of compounds of the general formula (1) by chemical modification of the side chain of the compounds of the general formula (1) included in claim 1, as set out in Scheme 2.
    Scheme 2
    [6]
    6. Use of the compounds of claims 1-3 for the preparation of a medicament for the treatment of diseases caused by protozoa
    pathogens of the Trypanosomatidae (Trypanosoma, Leishmania) and Trichomonadidae (Trichomonas) families.
    [7]
    7. A pharmaceutical composition that includes any of the compounds defined in claims 1-3 and at least one pharmaceutically acceptable excipient.
    [8]
    8. A pharmaceutical composition according to claim 7, which, optionally, may also contain other active ingredients.
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    公开号 | 公开日
    WO2017072374A1|2017-05-04|
    ES2614131B2|2017-10-11|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2653674B2|2017-10-20|2018-09-18|Universidad Complutense De Madrid|Amines derived from 5- Nitroindazole with antiprotozoal properties|
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