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    • 专利摘要:
    Maca extract and procedure for obtaining compounds with antiviral activity from maca. The present invention relates to asymmetric cyclic compounds derived from Lepidium meyenii with antiviral activity, obtained from highly apolar organic extracts of Lepidium meyenii, commonly known as maca, by means of specific extraction methods and purifications by chromatography of columns that give rise to molecules Thiopyrazolics containing a sulfur atom and nitrogens in its nucleus (Structure A), with marked antiviral activity. The process of isolation and characterization of said compounds, as well as their use, is described. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2669921A1
    申请号:ES201601013
    申请日:2016-11-29
    公开日:2018-05-29
    发明作者:Paulina BERMEJO BENITO；María José ABAD MARTÍNEZ；Luis Miguel BEDOYA DEL OLMO；Luis Néstor APAZA TICONA；José ALCAMI PERTEJO
    申请人:Universidad Complutense de Madrid；Instituto de Salud Carlos III；
    IPC主号:
    专利说明:

    Maca extract and procedure for obtaining active compounds Antiviral from maca. 5 Technical Sector
    The present invention relates to extracts of Lepidium meyenii Walp and / or Lepidium peruvianum Chacón (commonly known as Maca) comprising asymmetric cyclic compounds with marked antiviral activity and their process of obtaining. More specifically, it refers to derivatives
    Thiadiazole imidazo obtained by extraction and subsequent isolation from maca powder, as well as its antiviral activity. State of the art
    The botanical species Lepidium meyeniiWalp and / or Lepidium peruvianum Chacón,
    15 commonly known as maca, it belongs to the Brasiccaceae (Cruciferous) family and is one of the plants that the Incas domesticated and cultivated for more than 2000 years, growing exclusively in Andean areas between 3700, 4000 and 4500 meters of altitude; It is distributed in the central Andes of Peru, Bolivia and northwestern Argentina. The underground parts (root and / or
    20 hypocotyl) of this plant have been traditionally used in these countries as food, to increase resistance to harsh working conditions and as a fertility stimulant in humans and animals. The first written testimonies of the use of maca date back to the 16th century. Cultivated maca is currently used worldwide as a supplement
    25 food and also for its traditionally described medicinal properties (Rea, J. et al. 1994, Neglected Crops: 1492 from a different perspective. Plant Production and Protection Series No.26. FAO, Rome, Italy 149-163; Hermann M. and Bernet, T. 2009. The transition of maca from neglect to market prominence: Lessons for improving use strategies and market chains of
    30 minor crops Agricultural Biodiversity and Livehoods. Discussion papers 1, Rome. Biodiversity International .; Dostert N. et al. 2013. Seven vascular plants of economic importance in Peru: Botanical sheets. Arnaldoa 20 (2): 359-432).

    Maca is described morphologically as an arrosetado bearing plant,
    5 presenting a crown of basal leaves that arise above a fleshy axis in the ground. The crop is biannual and presents a vegetative phase in which the growth and expansion of the reserve organ occurs and a reproductive phase, characterized by the production of flowers and fruits. In favorable climatic conditions (absence of frost and abundant humidity) the plants
    10 can complete their reproductive cycle in about six months (Brako L., Zarucchi JL, 1993. Catalog of the flowering plants and giymnosperms of Peru. Monogra. Syst. Bot. Missouri Bot. Gar. 45: i-xl, 1-1286 ).
    Currently, methods of cultivation and improvement of the plant species are proposed 15 Lepidium meyen ;; and its varieties, such as the one described in the patent CN 1 03650886, in order to obtain a better yield in the process.
    Likewise, methods of sequencing and molecular identification of the different ecotypes of Lepidium meyenii (CN 102952878) are known.
    20 Since the 90s of the twentieth century has increased interest in this botanical species for its energizing and aphrodisiac actions, so that there is experimental evidence of its use as a nutritional, energizing, adaptogenic, fertility-stimulating supplement (W02008012628),
    25 actions in erectile dysfunction (W02011 026500), osteoporosis, treatment of hyperlipidemia (JP2007314475), prostatic hyperplasia and dermatology (FR2885052, JP2005306768, Gonzales, GF, 2012, Ethnobiology and Ethnopharmacology of Lepidium meyen ;; from the Peruvian Highlands Evidence-Based Complementary and Alternative Medicine eCAM.
    30 2012; 2012: 193496; Gonzales, G.F. et al., 2014, Maca (Lepidium meyenii Walp.) a review of its biological properties. Rev. Peru. Med. Exp. Public Health, 31 (1): 100-110; Del Valle J. et al., 2014, Antiviral activity of maca (Lepidium
    meyenil) against human influenza virus. Asian Pacific Journal of Tropical
    Medicine 7 (1): S415-S420).
    The process of preparing maca is important to obtain adequate biological effects. Traditionally the maca is boiled in water or extracted in alcohol before use. In experimental studies, aqueous maca extracts are only effective after boiling maca roots in water.
    Hydroalcoholic extracts have been obtained from roots of Lepidium meyenii (US6,267,995) where different components such as isothiocyanates, sterols, fatty acids and macamides (US6,428,824) are evaluated, proceeding to the structural elucidation of these last components, which present a chemical structure of N-benzyl amides, and also claiming its use in the prevention of cancer and sexual dysfunction (US RE43,005). Macamides can be isolated and studied by liquid chromatography (KR20090117284). However, these molecules have not been isolated using strongly apolar organic solvents. Another method of extraction includes the use of supercritical fluids (CN 103819570) as carbon dioxide (CN104087489), although no individual molecules are obtained, but extracts of Lepidium meyenii.
    In the state of the art there is the patent number US7,473,434 where the plant material is subjected to enzymatic digestion to obtain aqueous peptide extracts subjected to ultrafiltration and / or purification. The peptide extract comprising different amino acids is fundamentally claimed, and to which a stimulating activity of the fibroblasts is assigned, for which its use as a cosmetic is proposed. Similarly, as indicated in the CN1 patent 03892265, papain can be used as an enzymatic method obtaining extracts with vitamins, amino acids, macamides and the like. However, asymmetric heterocyclic molecules claimed in the present invention are not identified in these extracts.
    With respect to the presence of secondary metabolites of the species Lepidium meyenii Wallp, nine types of glucosinolates have been identified to date, most of them aromatic, of which the most abundant is glucotropaeolin (Dini Migliuolo, G. et al., 1994. Chemical composition of 5 Lepidium meyenii Food Chemistry 49: 347-349; Dini, l., Et al., 2002, Glucosinolates from maca (Lepidium meyenil) Biochemical Systematic and Ecology 30: 1087-1090; Li, G. , et al., 2001, Glucosinolate contents in maca (Lepidium peruvianum Chacon) seeds, sprouts, mature plants and several derived commercial products. Economic Botany 55: 255-262; Flores, HE, et 10 al., 2003, Andean root and tuber crops: Underground rainbows. Hortiscience
    38: 161-167).
    JP2008237117 A describes the uses of glucosinolates obtained from maca as additives in nutritional anti-fatigue supplements, 15 as well as their possible use in the treatment of obesity (JP2007230987).
    Maca root contains between 60 and 75% carbohydrates. From the aqueous extract of maca, polysaccharides are isolated whose structure is eluted and its antioxidant activity is demonstrated (Zha S. et al, 2014, Extraction and
    20 purification and antioxidant activity of the polisaccharides from maca (Lepidium meyeníl). Carbohydrate Polymers 111: 584-587).
    The steric fraction is important in maca roots, phytosterols have been isolated in maca, especially beta-sitosterol (Zheng, BL et al., 2000, 25 Effect of a lipidic extract of Lepidium meyenii on sexual behavior in mice and rats. Urology 55 (4): 598-602) and Dini (1994), attributing to the steric fraction benefit in the reduction of plasma cholesterol and in the prevention of menopausal problems, increasing fertility, as well as providing maca with anti-inflammatory properties and antioxidants (Lagarda, MJ et al., 2006.
    30 Analysis of phytoesterols in foods. Journal of Pharmaceutical and Biomedical Analysis 41: 1486-1496).
    Macaenos and macamides have been isolated, which are novel polyunsaturated fatty acids (Muhammad, 1. et al., 2002, Constituents of Lepidium meyenii "maca". Phytochemistry 59: 105-110; Piacente, S. et al., 2002, Investigation of the tuber constituents of maca (Lepidium meyenii Walp) Journal of Agricultural 5 and Food Chemistry 50: 5621-5625; Zhao, J. et al., 2005, New alkamides from maca (Lepidium meyenil) Journal of Agricultural and Food Chemistry 5: 690693; Chain, FE et al., 2014, Macamides from wild "Maca", Lepidium meyenii Walpers (Brassicaceae). Phytochemistry Letters 8: 145-148). In addition, Muhammad found the 1,2-dihydro-N-hydroxypyridine derivative called 10 macaridine along with the benzylated alkamides (macamides). So far, 19 macamides have been described, blaming some in particular for a neuroprotective effect by an indirect cannabimimetic mechanism (Hajdu, Z. et al., 2014, Identification of endocannabinoid system-modulating N-Alkylamines from Heliopsis helianthoides var.scabra and 15 Lepidium meyenii, Journal of Natural Products 77: 1663-1669). Eleven of these macamides have been synthesized in order to assess the in vitro enzymatic inhibition of acidic hydrolase amide (Wu, H. et al., 2013, Macamides and their synthetic analogs: Evaluation of in vitro FAAH inhibition. Bioorganic and Medicinal Chemistry 21: 5188-5197). The alcamides of the maca are the
    20 results of conservation methods performed in postharvest treatment (Esparza, E. et al., 2015, Bioactive maca (Lepidium meyenii) alkamides are a result of traditional Andean postharvest drying practices. Phytochemistry 116: 138-148).
    In addition, two imidazole alkaloids are isolated from the maca (1,3-dibenzyl-4,5-dimethyl imidazole hydrochloride and 1,3-dibenzyl-2,4,5-trimethyl imidazole hydrochloride), known as lepidin A and lepidin B (Boaling, C. et al., 2003, Imidazole alkaloids from Lepidium meyenii. Journal of Natural Products 66: 1101-1103). A new imidazole alkaloid was isolated and identified as 2
    30 benzyl-3-thioxohexahydro-1 H pyrrolo [1,2-c] imidazol-1-one (Rocabado G. et al., 2011, Isolation and identification of a new alkaloid in Lepidium meyenii Walpers. Biofarbo 19 (1): 8-14). Alkaloids derived from imidazole are known
    obtained from Lepidium meyenii, described in US 6,878,731,
    whose structure is totally symmetrical and with which studies have been carried out
    with anticancer application.
    S In the state of the art there are studies in which, synthetic compounds,
    1,3,4 thiodiazole derivatives, similar or equal to those obtained from
    Lepidium meyenii, have antimicrobial, trypanocidal activities,
    anti-inflammatory and anti-proliferative (Matysiak, J. et al, 2006, Synthesis and
    antiproliferative activity of N-substitued 2-amino-5- (2,4-dihydroxyphenyl) -1, 3,4
    10 thiadiazoles Biorganic and Medicinal Chemistry, 14 (13): 4483-4489;
    Marganakop, S.B. et al., 2015, Electronic effect of substituents present on
    carbonyl compounds: analysis of product formation in one-pot synthesis of 1, 3,
    4-thiazole ringo American Chemical Science Journal 8 (1): 1-7; De Monte, C. et
    to the. , 2015, Synthesis and pharmacological screening of a large library of 1,3,4
    1S thiadiazolines as innovative therapeutic tools for the treatment of prostate
    cancer and melanoma Eur J Med Chem. 105: 245-62) demonstrating a
    Quinesin Eg5 enzyme inhibitory activity, enzyme that plays a very role
    important in cell division (Nakai, R. et al., 2009, K858, a novel inh ibitor
    of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells.
    twenty Cancer Res. 69 (9): 3901-9; De luliis, F. et al. , 2016, The kinesin Eg5 inhibitor
    K858 induces apoptosis but also survivin-related chemoresisteance in breast
    cancer cells Investigational New Drugs 34 (4): 399-406). The 1,3,4 derivatives
    thiodiazole synthesis are postulated as anticancer molecules in the
    documents W020 15153967 and W02005061707. However, in the state of
    2S the technique has not found antiviral activity in plant extracts that
    They can contain these types of molecules.
    Regarding the imidazo tiad iazólicos derivatives studies have been carried out
    with molecules obtained by synthesis with compounds similar or equal to
    30 compound obtained from Lepidium meyenii presenting antiapoptotic activity
    and modulation of cell death by inhibition of Jun kinase activity at
    through the expression of HSP70 proteins, and antimicrobial activity (Salehi,
    A.H. et al., 2005, AEG3482 is an antiapoptotic compound that inhibits Jun kinase activity and cell death through induced expression of heat shock protein
    70. Chemistry and Biology. 13 (2): 213-23; Alagawadi, K.R. et al., 2011, Synthesis, characterization and antimicrobial activity evaluation of new 2.45 thiazolidines bearing imidazo [2, 1-b] [1, 3,4] thiadiazole moiety. Arabian Journal of Chemistry 4 (4): 465-472; Ebenezer, P.J. et al., 2010, Neuron specific toxicity of oligomeric amyloid-p: role for JUN-kinase and oxidative stress. Journal Alzheimers Disease 22 (3): 839-48; Gallo, KA, 2006, Targeting HSP90 to halt neurodegeneration. Chemistry and Biology 13 (2): 115-6). These derivatives
    Synthetic imidazo-thiodiazoles are postulated as compounds for treating neuropathic pain in patents CA 2584745 A1, WO 2007087716 and for treating neuronal alterations and proliferative diseases (WO 2004111061) And as neuroprotective compounds (WO 2003051890).
    Description of the invention The object of the invention is a maca extract comprising one or more compounds derived from 1,3,4-thiadiazole of formula A.
    (TO)
    A method for obtaining the alcoholic extract of Lepidium meyenii Walp and / or Lepidium peruvianum Chacón from maca powder is also object of the invention, as well as the separation and isolation of the different fractions by chromatographic methods. This process includes novel phases in the extraction and isolation of compounds
    30 from maca since hydroalcoholic extracts or supercritical fluids are usually used for this purpose. The extraction method used comprises:
    a) First extraction using strongly alkane type solvent
    Apolar as the first solvent. b) Concentration of the apolar extract. c) Suspension in primary alcohol not greater than 6 carbons and obtaining
    5 alcoholic extract.
    Likewise, the present invention also relates to obtaining the compounds of structure A by isolation at a later stage after obtaining the extract:
    10 d) Separation and fractionation by column chromatography.
    The compounds of structure A had not been obtained so far from plant species. These compounds contain asymmetric aromatic heterocycles that include nitrogen and sulfur molecules,
    15 whose composition and chemical structure has been determined by spectroscopic methods. Structure A is used in the present invention as a series or "core" molecule of the claimed compounds.
    Different compounds are assumed from structure A mentioned,
    Wherein R1 comprises substituents such as acetamido, sulfonamido; wherein R2 and R3 comprise substituents such as proton, methyl, ethyl, propyl, isopropyl, phenyl, oxo; and wherein R4 comprises substituents such as acetyl, 2-aminoethyl, 2-aminopropyl, 2-amino butyl, 2-aminopentanyl, 2-amino 3-methylbutyl, 2aminofenetyl, 2-aminoetenyl, 2-amino 1-propenyl, 2-amino 1 -butenyl, 2-amino
    1-Pentenyl, 2-amino 3-methyl 1-butenyl, 2-amino 2-phenyl 1-ethenyl. R2 groups. R3 and R4 have cyclisation capacity under the conditions for obtaining them.
    Asymmetric heterocyclic compounds have been isolated and characterized from Lepidium meyenii as:
    Compound 1: N- (4-acetyl-4,5-dihydro-5-methyl-5-phenyl-1, 3,4-thiadiazol-2-yl)
    acetamide
    (one)
    Compound 11: 6-phenylimidazo [2,1-b] -1, 3,4-thiadiazol-2-sulfonamide.
    (eleven)The present invention also aims at the use of the extract andisolated compounds of structure A (and / or combinations thereof and / or their salts
    15 and / or its chiral derivatives levógiro, dextrógiro and the racemic mixture) as antiviral agents.
    Finally, the invention also relates to a pharmaceutical composition comprising the extract or compounds of structure A isolated from the extract. Description of the figures
    Figure 1. Evaluation of the anti-HIV activity and cytotoxicity of compound I in the MT-2 cell line infected with the recombinant HIV virus NL4.3-REnilla.
    Figure 2. Evaluation of anti-HIV activity and cytotoxicity of compound IIin the MT-2 cell line infected with the recombinant HIV virus NL4.3-Renilla. Embodiment of the invention
    The present invention is illustrated by the following examples, which are not limiting of its scope.
    5 The roots of Lepidium meyenii Walp and / or Lepidium peruvianum Chacón are used as plant material.
    Example 1: Obtaining the extract of maca.
    From said plant material, extraction is carried out until exhaustion and
    10 the isolation of the compounds thereof. In the concrete extraction process, 10 g to 7000 g of maca powder are used, extracted by means of soxhlet, using n-solvent alkane-type solvent strongly solvent. Subsequently, the apolar extract is concentrated at controlled temperature and pressure from 20 to 50 ° C and is suspended in primary alcohol no greater than
    15 6 carbons at low temperature (between 0 and 10 ° C) for a time between 12 to 24 hours.
    Example 2: Separation of the extract obtained in different fractions
    The alcoholic extract is concentrated until the dry residue is obtained and,
    Subsequently, it is subjected to the separation of its components by column chromatography (column 1), using silica gel, as a stationary phase and as a mobile phase ethyl ether, toluene and mixtures of both in all possible proportions, acidifying to saturation. with different acids such as glacial acetic acid. The fractions obtained (1
    25 12) of column 1 are assembled according to the results of the analysis of thin layer chromatographs.
    Example 3: Isolation of compound 1.
    A second separation of fractions 6-9 is carried out by chromatography
    30 in column (column 2), using Sephadex as a stationary phase and as a mobile phase a primary alcohol of not more than 6 carbons: dichloromethane, in a hydrophilic ratio. The fractions obtained (A-J) from column 2 are pooled according to the results of the analysis of thin layer chromatographs. A third separation of the D-J fractions is carried out by column chromatography (column 3), using silica gel as the stationary phase and as
    5 mobile phase ethyl ether, toluene and mixtures of both in all possible proportions, acidifying to saturation with different acids such as, for example, glacial acetic acid. The fractions obtained (a-h) from column 3 were pooled according to the results of the thin layer chromatography analyzes.
    10 In turn, fractions a-b are combined according to the results of the analysis of thin layer chromatographs and an extraction of their nitrogen components is carried out.
    The extracted solution is subsequently concentrated under reduced pressure. Finally, a separation of the organic extract from the nitrogen components is carried out by column chromatography (column 4), in the reverse phase, using as a mobile phase a mixture of primary alcohol of no more than 6 carbons, chloroform and ammonium hydroxide, in different gradient from
    20 polarity The fractions obtained (a-v) from column 4 are combined according to the results of the analysis of thin layer chromatographs. A white solid, re-crystallized in primary alcohol of not more than 6 carbons, which corresponds to compound I is isolated from fraction A
    Example 4: Isolation of compound 11. Fractions 2-4 are combined according to the results of the analysis of thin layer chromatographs and an extraction of their nitrogen components is carried out.
    The solution subsequently extracted is concentrated under reduced pressure. A separation of the organic extract from the nitrogen components is carried out by column chromatography (column 5), using sephadex
    as a stationary phase and as a mobile phase a primary alcohol of no more than 6 carbons: dichloromethane in hydrophilic ratio. The fractions obtained (A'-D ') are collected according to the results of the analysis of thin layer chromatographs.
    Finally, a separation of the fraction C 'is carried out by column chromatography (column 6), in reverse phase, using as a mobile phase a mixture of primary alcohol no larger than 6 carbons, chloroform and ammonium hydroxide, in different polarity gradient . The fractions obtained (a'-m ') are combined according to the results of the analysis of thin layer chromatographs. A re-crystallized white solid in primary alcohol of not more than 6 carbons, corresponding to compound 11, is isolated from fraction c '.
    Example 5: Spectroscopic methods used. Thiopyrazole alkaloids are isolated from the alcoholic extract of Lepidium meyenii Walp. and / or L. peruvianum Chacón, using the chromatographic techniques described above. These compounds are characterized by spectroscopic methods of IR, NMR-1H, NMR-13C, DEPT, HMBC, HMQC, COZY and MS.
    IR spectra are performed on a Perkin-Elmer spectrophotometer model FT -1725X. To obtain the NMR-1H and NMR-13C spectra, a 700 MHz Brucker Advance DRX-700 spectrometer is used, using no more than 6 carbons as primary alcohol solvent. For the elementary chemical analysis a CHNS combustion microanalyzer, Leca model is used
    932. Finally, for the analysis of Mass Spectrometry (EM), a Hewlett Packard model 5930 spectrometer is used.
    By these methods, asymmetric heterocyclic compounds have been isolated and characterized from Lepidium meyenii as:
    Compound 1: N- (4-acetyl-4,5-dihydro-5-methyl-5-phenyl-1, 3,4-thiadiazol-2
    il) acetamide.Compound 11: 6-Phenylimidazo [2,1-b] -1, 3,4-thiadiazol-2-sulfonamide.
    Example 6: Structural elucidation of Compound 1: N- (4-acetyl-4,5-dihydro-5-methyl-5-phenyl-1, 3,4-thiadiazol-2-yl) acetamide.
    (one)
    10 In the IR spectrum, a band at 3302.9 cm-1 is attributed to the presence of a proton associated with a secondary amide, a band at 1630.8 cm-1 due to the existence of a disubstituted amide and another band at 1407.2 cm-1 which suggests a terminal methylene of the molecule. In NMR-1H spectrum a
    15 singlet at 7.93 that integrates proton from a secondary amino group, a double at 7.44 dd J = 8.4 Hz corresponding to two pairs of aromatic protons and a triplet at 7.38 ttt J = 8.4 Hz correspond to two pairs of aromatic protons. The signal of a triplet at 7.28 J = 8.4 Hz indicates the presence of an aromatic proton. Three singles are evidenced at 2,360; 2,270 and 2,100 that
    20 indicate the presence of three methyl groups. Its 1350 DEPT spectrum shows 3 signals at 128.74; 129.53 and 125.92 corresponding to 5 aromatic tertiary carbons. The mass spectrum has a molecular ion [M +] at 278 ml with significant fragments due to the presence of two peaks at 236.08 and 229.21 which leads us to identify compound I as N- (4-acetyl- 4,5
    25 dih id ro-5-methyl-5-phenyl-1, 3,4-tiad iazol-2-yl) acetamide.
    (one)Table 1. Assignment of the 13C, 1H signals of compound I
    Atomo # 13C (ppm)1H (ppm)
    2 145.10-----
    5 79.49_._---
    one ' 144.70-----
    2 ' 129.537.44 d
    6 '129.53 7.44 d 3' 128.75 7.38 d 5 '128.75 7.38 d 4' 125.93 7, 28 t
    CO 171 .52 ----- CO 171.16 ------ CH3 49.12 2.36 s -CH3 49.00 2.27 s -CH3 48 .88 2.10 s -NH-R ------ 7.93 s
    Example 7: Structural elucidation of Compound 11: 6-Phenylimidazo [2,1
    b] -1, 3,4-thiadiazol-2-sulfonamide.
    In the IR spectrum, two bands are shown at 1375.7 cm -1 and 1291.5 cm -1 due to the presence of the sulfonamide group. In NMR-1H spectrum a singlet is observed at 8.49 which integrates for a proton of a cyclic tertiary carbon, a doublet at 7.88 dd J = 8.4 Hz corresponding to two pairs of aromatic protons, a doublet at 7.44 dd J = 8.4 Hz corresponding to two pairs of aromatic protons and additionally a triplet at 7.35 J = 8.4 Hz indicating the presence of an aromatic proton. Its 1350 DEPT spectrum shows 3 signals at 129.76; 129.14 and 126.33 corresponding to 5 aromatic tertiary carbons. The mass spectrum has a molecular ion [M +] at 281 mIz which leads us to identify compound II as 6-Phenyl imidazo [2, 1-b] -1, 3,4-thiadiazol-2-sulfonamide.
    (eleven)
    Table 2. Assignment of the 13C, 1H signals of compound 11
    Atomo # 13C (ppm) 1H (ppm) 6 165.68 ----- 8 149.04 ----- 2 147.52 -----
    5 111.55 8.49 s 1 '134.45 ----- 3' 129.83 7.44 d 5 '129.83 7.44 d 4' 129.21 7.35 t 2 '126.41 7.88 d 6' 126.41 7.88 d Example 8: Derivatives of structure A.
    5 Thus, the main structure claimed in the present invention corresponds to structure A, a substituted thiopyrazole where, in certain cases, the substituents themselves have the ability to blot. Thus, it is assumed to obtain compounds I and 11 from plant extracts, as well as other possible derivatives (table 3) that are claimed
    10 continuation. Some of these compounds have been obtained byhas indicated in the background, but never until
    (TO)
    Structure A has 4 radicals or substituents R, where R1 comprises substituents such as acetamido, sulfonamido; R2 comprises substituents such as proton, methyl, ethyl, propyl, isopropyl, phenyl, oxo; R3 comprises substituents such as proton, methyl, ethyl, propyl, isopropyl, phenyl, oxo; and R4 comprises substituents such as acetyl, 2-amino ethyl, 2-amino propyl, 2amino butyl, 2-amino pentanyl, 2-amino 3-methyl butyl, 2-amino phenethyl, 2-amino ethenyl, 2-amino 1-propenyl , 2-amino 1-butenyl, 2-amino 1-pentenyl, 2-amino 3-methyl 1-butenyl, 2-amino 2-phenyl 1-ethenyl.
    10 For compound 1, structure A has R 1 acetamido, R 2 phenyl, R 3 methyl and R 4 acetyl (compound 15 of table 3).
    Table 3Possible substituents of structure A.
    1 R1: -NHCOCH3 R2: -H R3: -H R4: -COCH3 2 R1: -NHCOCH3 R2: -CH3 R3: -CH3 R4: -COCH3 3 R1: -NHCOCH3 R2: -CH3 R3: -H R4: - COCH3 4 R1: -NHCOCH3 R2: -C2Hs R3: -C2Hs R4: -COCH3 5 R1: -NHCOCH3 R2: -C2Hs R3: -H R4: -COCH3 6 R1: -NHCOCH3 R2: -C3H7 R3: -C3H7 R4: -COCH3 7 R1: -NHCOCH3 R2: -C3H7 R3: -H R4: -COCH3 8 R1: -NHCOCH3 R4: -COCH3
    R2: - R3: - 9 R1: -NHCOCH3 R3: -H R4: -COCH3
    R2: - R1: -NHCOCH3 R4: -COCH3 -O-R3: -O
    R2: 11 R1: -NHCOCH3 R3: -H R4: -COCH3
    - OR
    R2: 12 R1: -NHCOCH3 R2: -C2Hs R3: -CH3 R4: -COCH3 13 R1: -NHCOCH3 R2: -C3H7 R3: -CH3 R4: -COCH3 14 R1: -NHCOCH3 R3: -CH3 R4: -COCH3
    R2: - 15 R1: -NHCOCH3 R3: -CH3 R4: -COCH3
    R2: -O
    -
    16 R1: -NHCOCH3R2: -C3H7R3: -C2HsR4: -COCH3
    17 R1: -NHCOCH3R2: - R3: -C2HsR4: -COCH3
    18 R1: -NHCOCH3-OR2: -R3: -C2HsR4: -COCH3
    19 R1: -NHCOCH3R2: - R3: -C3H7R4: -COCH3
    twenty R1: -NHCOCH3R2:-OR-R3: -C3H7R4: -COCH3
    twenty-one R1: -NHCOCH3R2: - R3:-OR-R4: -COCH3
    22 R1: -S02NH2R2Y R3: = 0) R4: H2N
    2. 3 R1: -S02NH2R2 and R3: = 0> -R4: H2 N
    24 R,: - S02NH2R2Y R3: = 0} -I R4: H2N
    25 R,: - S02NH2R2yR3: = 0~
    R4: H2N
    26 R1: -S02NH2R2Y R3: = 0> - <R4: H2N
    27 R1: -S02NH2R2 and R3: = 0~
    R4: H2N
    28 R1: -S02NH2R2 and R3: = 0~
    R4: H2N
    29 R1: -S02NH2R2Y R3: = 0~
    R4: H2N
    30 R1: -S02NH2R2 and R3: = 0-and R4: H2 N
    31 R1: -S02NH2R2Y R3: = 0~
    R4: H2N
    32 R,: - S02NH2R2Y R3: = 0~
    R4: H2N
    33 R1: -S02NH2R2Y R3: = 0~
    R4: H2N
    In the case of compound 11 substituents, the group R1 is sulfonamido R2,
    R3 and R4 are forming a cycle.
    Example 8: Pharmacological activity.
    5 The anti-HIV-1 activity and the cytotoxicity of the extracts and isolated compounds obtained are tested. The evaluation of antiviral activity is performed by infection of MT-2 cells with recombinant HIV viruses carrying renilla-Iuciferase indicator genes NL4.3-Renilla EP1752541.
    10 Table 4. Anti-HIV activity and cytotoxicity of apolar extract and isolated compounds of L. meyen ;; Concentration% IJg / ml infection% viability Apolar extract 50 ~ g / ml 36% 94%
    Excerpts
    (LMO) 54% 95%
    25 IJg / ml IC50 IJM CC50 IJM 15 Compounds Compound I 1.4> 100> 72.0 isolated
    Compound "55.7> 100> 1, 8 IS: Specificity index. Clso and CCso: 50% Inhibitory Concentration, 50% Cytotoxic Concentration
    Apolar extract inhibits viral replication in a high percentage without showing
    15 toxicity at the concentrations tested. The fractions obtained are all active. Compounds called compound I and compound 11, are capable of inhibiting viral replication with inhibitory concentrations 50 (IC50)
    1.4 ~ lM and 55.7) lM Y without showing toxicity (CC50> 100) lM).
    20 The evaluation of the anti-HIV activity and the cytotoxicity of compound I in the MT-2 cell line infected with the recombinant HIV virus NL4.3-Renilla is checked. (Figure 1). The compound called compound I has an ability to inhibit viral replication at inhibitory concentrations.
    (IC50) equal to 1, 4 ~ lM and shows no toxicity since its cytotoxic concentration
    25 50 is greater than 100 (CC50> 100 flM). Its specificity index IS is greater than
    72. (Figure 1).
    The anti-HIV activity and cytotoxicity of compound 11 in the line are evaluated
    MT-2 cell infected with the recombinant HIV virus NL4.3-Renilla. [FIG. 7].
    The compound called compound 11 has an inhibition ability
    5 of viral replication at 50 inhibitory concentrations (IC50) equal to 55.7 J. µM and shows no toxicity since its 50 cytotoxic concentration is greater than 100 (CC50> 100 J. µM). Its specificity index IS is greater than 1.8. (Figure 2).
    Thus, the advantages and innovative value of this invention lie in:
    10 • The identification of new antiviral pharmacological activities with great therapeutic possibilities, far removed from traditional medicinal uses, from Lepidium meyenii, a plant cultivated and used in food by the Andean peoples.
    • The activity lies in highly apolar extracts, which had not been obtained until the moment of the present invention.
    • The isolated chemical compounds, derived from a thiopyrazole nucleus Figure 1, contain the sulfur atom inside the ring and the arrangement of the nitrogen is totally novel compared to other molecules previously isolated from L. meyenii.
    20 • The extraction method used is based on solubilization of the active ingredients in a highly apolar solvent.
    The purification of the apolar extract involves concentration, suspension and separation phases by column chromatography.
    权利要求:
    Claims (16)
    [1]
    1. Maca extract comprising at least one compound, and / or combinations of them and / or their salts and / or their chiropractic levógiro, dextrógiro and the racemic mixture, of structure A
    10 (TO)
    where R1 comprises substituents such as acetamido, sulfonamido;
    where R2 and R3 comprise substituents such as proton, methyl, ethyl,
    propyl, isopropyl, phenyl, oxo; and where R4 comprises substituents such as
    acetyl, 2-aminoethyl, 2-aminopropyl, 2-aminobutyl, 2-aminopentanyl, 2
    fifteen amino 3-methyl butyl, 2-aminofenetil, 2-aminoetenyl, 2-amino 1-propenyl, 2
    amino 1-butenyl, 2-amino 1-pentenyl, 2-amino 3-methyl-1-butenyl, 2-amino
    2-phenyl 1-ethenyl.
    [2]
    2. Maca extract according to claim 1, which comprises the compound
    twenty N- (4-acetyl-4,5-dihydro-5-methyl-5-phenyl-1, 3,4-thiadiazol-2-yl) acetamide,
    of formula 1.
    (one)
    3. Maca extract according to claim 1, comprising the compound 6phenylimidazo [2,1-b] -1, 3,4-thiadiazol-2-sulfonamide.
    (eleven)
    [4]
    4. Method of obtaining the maca extract according to claim 1, comprising: a) First extraction using strongly alkane type solvent
    Apolar as the first solvent. b) Concentration of the apolar extract. c) Suspension in primary alcohol not greater than 6 carbons and obtaining
    of the alcoholic extract.
    [5]
    5. Process for obtaining maca extract according to claim 4, wherein the first extraction with strongly apolar solvent alkane type is carried out from the plant material until depletion.
    [6]
    6. Method for obtaining maca extract, according to claim 5, wherein the plant material is formed by roots of Lepidium meyenii Walp or Lepidium peruvianum Chacón.
    [7]
    7. Procedure for obtaining maca extract according to claims 4 to 6, wherein the concentration of the apolar extract is carried out at a controlled temperature and pressure from 20 to 50 ° C.
    [8]
    8. Procedure for obtaining maca extract according to claims 4 to 7, wherein the dry residue of the first extract is suspended in alcoholic solvent keeping the suspension at a low temperature, between 0 and 10 ° C, for 12 to 24 hours.
    [9]
    9. Process for obtaining maca extract, according to claims 4 to 8, wherein the fraction solubilized in primary alcohol of not more than 6 carbons is concentrated until the dry residue is obtained.
    Method of obtaining compounds of structure A from maca which comprises obtaining the extract of maca according to claims 4 to 9 and, subsequently, separating and fractioning the extract obtained.
    [11 ]
    eleven . Procedure for obtaining compounds of structure A from
    10 according to claim 10, wherein the fractionation, separation, isolation and purification of the extract is carried out by column chromatography on silica gel and sephadex.
    [12]
    12. Use of the maca extract claimed as an antiviral agent. fifteen
    [13]
    13. Use of the claimed extract for the preparation of a medicament.
    [14]
    14. Pharmaceutical composition comprising the claimed extract.
    20 15. Use of compounds and / or combinations of them and / or their salts and / or their chiropractic levógiro, dextrógiro and racemic mixture, of structure
    (A) as antiviral agents.
    [-]
    - .. Viability
    • ... HIV> 100
    C:
    ORIVOR
    ~ 50
    Goal
    ~
    OR
    [0]
    0.01 0.1 1 10 100 1000
    IJM
    Figure 1
     Compound 11
    [-]
    - .. Viability
    • ... HIV
    c: 1 --- 1--
    ü 100
    IV
    or
    ~
    Goal 50
    ~
    ~
    or
    [0]
    0.01 0.1 1 10 100 1000
    IJM
    Figure 2
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