Synthesis of acyl-hydrazone from usnic acid and isoniazid and its anti-Mycobacterium tuberculosis activity Síntesis de acil-hidrazona a partir del ácido úsnico e isoniazida y su actividad anti-Mycobacterium tuberculosis

Resumo ãompound usnic acid M1A6 isolated from lichen Evernia prunastri Mãajamarca0PeruA and the synthesis and characterization of its acyl0hydrazone M2A6 from the condensation reaction between usnic acid and isoniazid in an ethanol solution under reflux6 giving an overall yield of SDB6 were evaluatedμ çoth compounds were evaluated and compared with isoniazid according to its anti0 Mycobacterium tuberculosis activity based on the tetrazolium microplate assay MTQMFAμ ãompound 1 had MIã Mminimal inhibitory concentrationA value of H8μL μg/mL in each test of H72Rv Msusceptible typeA6 Tç OM S2 Mresistant wild typeA and MOR OM HLS4 Mmulti drug resistances typeA strainsμ In similar tests6 compound 2 MIã values were 3μL6 8RμL and 8RμL μg/mL respectivelyμ Se evaluó el ácido úsnico M1A6 aislado del liquen Evernia prunastri Mãajamarca0PerúA6 producto natural conocido por sus actividades biológicas6 y6 del mismo modo6 se evaluó la síntesis de su derivado acil0 hidrazona M2A6 obtenido a partir de una reacción de condensación entre el ácido úsnico y la isoniazida en solución etanólica a reflujo6 con un rendimiento global de SDBμ Fmbos compuestos fueron evaluados y comparados con la isoniazida según su actividad anti0Mycobacterium tuberculosis basada en el ensayo de susceptibilidad mediante el método TQMFμ Los resultados mostraron que el compuesto 1 presenta valores de MIã de H86L μg/mL frente a las cepas H72Rv6 Tç OM S2 y MOR OM HLS46 mientras que el compuesto 3 presenta valores de MIã de 36Lá 8R6L y 8R6L μg/mL respectivamenteμ Uoi avaliado o ácido úsnico M1A6 um produto natural conhecido pelas suas atividades biológicas6 isolado a partir do líquen Evernia prunastri Mãajamarca0PeruA6 assim mesmo foi avaliada a síntese do seu derivado6 a acil0 hidrazona M2A6 obtido a partir de uma reação de condensação com refluxo entre o ácido úsnico e a isoniazida em solução etanólica6 com um rendimento global de SDBμ F atividade anti0Mycobacterium tuberculosis de ambos compostos foi avaliada e comparada com a isoniazida mediante testes de sensibilidade obtidos pelo método TQMFμ Os resultados mostraram que o composto 1 apresenta o valor de MIã de H86L μg/mL contra variedades H72Rv6 Tç OM S2 e MOR OM HLS4á enquanto que para o composto 2 os valores de MIã são de 36Lá 8R6L e 8R6L μg/ mL6 respectivamenteμ

ãompound usnic acid M1A6 isolated from lichen Evernia prunastri Mãajamarca0PeruA and the synthesis and characterization of its acyl0hydrazone M2A6 from the condensation reaction between usnic acid and isoniazid in an ethanol solution under reflux6 giving an overall yield of SDB6 were evaluatedµ çoth compounds were evaluated and compared with isoniazid according to its anti0 Mycobacterium tuberculosis activity based on the tetrazolium microplate assay MTQMFAµ ãompound 1 had MIã Mminimal inhibitory concentrationA value of H8µL µg/mL in each test of H72Rv Msusceptible typeA6 Tç OM S2 Mresistant wild typeA and MOR OM HLS4 Mmulti drug resistances typeA strainsµ In similar tests6 compound 2 MIã values were 3µL6 8RµL and 8RµL µg/mL respectivelyµ Se evaluó el ácido úsnico M1A6 aislado del liquen Evernia prunastri Mãajamarca0PerúA6 producto natural conocido por sus actividades biológicas6 y6 del mismo modo6 se evaluó la síntesis de su derivado acil0 hidrazona M2A6 obtenido a partir de una reacción de condensación entre el ácido úsnico y la isoniazida en solución etanólica a reflujo6 con un rendimiento global de SDBµ Fmbos compuestos fueron evaluados y comparados con la isoniazida según su actividad anti0Mycobacterium tuberculosis basada en el ensayo de susceptibilidad mediante el método TQMFµ Los resultados mostraron que el compuesto 1 presenta valores de MIã de H86L µg/mL frente a las cepas H72Rv6 Tç OM S2 y MOR OM HLS46 mientras que el compuesto 3 presenta valores de MIã de 36Lá 8R6L y 8R6L µg/mL respectivamenteµ Uoi avaliado o ácido úsnico M1A6 um produto natural conhecido pelas suas atividades biológicas6 isolado a partir do líquen Evernia prunastri Mãajamarca0PeruA6 assim mesmo foi avaliada a síntese do seu derivado6 a acil0 hidrazona M2A6 obtido a partir de uma reação de condensação com refluxo entre o ácido úsnico e a isoniazida em solução etanólica6 com um rendimento global de SDBµ F atividade anti0Mycobacterium tuberculosis de ambos compostos foi avaliada e comparada com a isoniazida mediante testes de sensibilidade obtidos pelo método Despite the global availability of drugs against tuberculosis, there are still some issues to address: the long duration of the treatment, the serious side effects, and the development of multidrug-resistant strains (5,6).This reveals the urgent need to identify novel, safe, and effective candidates for the determination of an optimized and efficient drug against the M. tuberculosis.
Over the last decade, there has been extensive research on antibacterial compounds from natural products, since they are considered to be the major source of active metabolites.These may provide lead structures for the development of new drugs (7), for example, usnic acid (UA) appears to be a promising medical application.Usnic acid is a yellowish, highly functionalized dibenzofuran metabolite found in various lichen genera distributed in species of Cladonia, Usnea, Lecanora, Ramalina, Evernia and Parmotrema.It can exist as (+) and (-) enantiomers, but most of its biological activity is attributed to the (+) enantiomer (8, 9).Usnic acid displays a wide range of multiple biological effects, including: anti-bacterial, anti-parasitic, anti-viral, anti-mycotic, anti-protozoal, anti-proliferative, anti-inflammatory, anti-pyretic, and citotoxicity against human cancer cell lines (10,11).
Furthermore, earlier studies reveal that (+)-usnic acid exhibits activity against M. tuberculosis, showing a MIC value of 32 µg/mL, which was not considered potent enough (12).Considering the fact that the functional groups found within the molecule make of usnic acid a good target for structural modifications (2), a reaction between UA and INH was suggested in order to evaluate the enhancement or not of its activity against M. tuberculosis.
In this context, this research presents the synthesis and characterization of an acyl-hydrazone ( 2 obtained from a condensation reaction between usnic acid (1) and isoniazid, and the study of its anti-mycobacterial activity into M. tuberculosis strains.
Compounds 1 and 2 were analyzed and characterized in CDCl 3 and DMSO-d 6 , respectively, by 1 H-NMR 300 MHz, 1 H-NMR 600 MHz and 13 C-NMR 150 MHz with a Varian Unity Inova AS600 spectrometer.The notations used for the spectral analysis are: s (singlet), d (doublet), m (multiplet).Chemical shifts are reported in δ (ppm) using tetramethylsilane (TMS) as the internal standard.The determination of the exact molecular weights was performed by high resolution mass spectrometry (HRMS) on an Agilent Technologies Model 6210 LC-MSD-TOF (Time-of-Flight mass spectrometer) instrument.The molecular ions were protonated [M+H] + or [M-H] - for the confirmation of their empirical formula.Infrared spectra were recorded on a JASCO FT/IR-410 spectrophotometer.The optical rotation was measured using a JASCO P-1010 polarimeter.
The branched thallus of Evernia prunastri were collected at Porcon farm, located 30 km from the city of Cajamarca-Peru and identified by Dr. Luis Dávila of National University of Cajamarca, Cajamarca, Peru.
The branched dried thallus of Evernia prunastri (100 g) was crushed into a powder and extracted with an increasing polarity order of solvents using hexane (72 h (3x)), ethyl ether (72 h (x2)), chloroform (72 h (2x)), ethyl acetate (72 h (x2)), methanol (72 h (3x)), and water (72 h (3x)) at room temperature.The ethyl ether extract (4.8 g) was separated using repeatedly flash column chromatography with hexane/ethyl acetate (7:3) and hexane/toluene/ ethyl acetate (85:5:15), obtaining four fractions A, B, C and D. Fraction D was cooled at room temperature then filtered whereby a precipitate was obtained as a greenish yellow solid.This precipitate was separated out and recrystallized in ethanol 99% (v/v), from which the major compound was obtained as yellow crystals and identified as usnic acid (2.8 g), confirmed by 1 H-NMR and 13 C-NMR spectrums and by comparison with reported data (12).

Synthesis of acyl-hydrazone obtained from usnic acid and isoniazid
Usnic acid (500 mg, 1.45 mmol, 1 eq) and isoniazid (250 mg, 1.74 mmol, 1.2 eq) were added to a solution of 50 mL of absolute ethanol.The solution was stirred and heated at reflux for 12 h.The solution was cooled at room temperature and stored in a refrigerator for two days.The precipitate formed was filtered and dried in an oven at 25 °C for 24 h.The final product (acyl-hydrazone) was obtained as orange crystals (15).

Materials and methods
In general, all solvents and reagents (Acros Organics or Sigma Aldrich) were used without prior purification.The isolation and purification were monitored by thin layer chromatography (TLC) on silica gel plate (Merck 60 F 254 ), preparative chromatography (Merck 60 F 254 ), and by column chromatography using silica gel (Gel 60 silica particle size 0.063-0.200mm).

Biological activity evaluation
The activity of usnic acid] isoniazid] and their acylChydrazone against Preparation of culture inoculum °ultivation of M. tuberculosis was performed in Middlebrook NH+ broth during four weeks.The inoculation of the strains was achieved by using a sterile loop into a glass bead tube with qWW μL of mixC Tween and mixed in a vortex for L min.Three mL of qW8 [w/w4 Tween ºW was added and stirred in a vortex for LW s.The supernatant was transferred to a glass tube without beads.The turbidity was adjusted to Mc=arland Nºq with a mixCTween solution.The Mc=arland Nºq strain was combined with Middlebrook NH+ broth medium [qL mL of NH+ 2 W.j mL of the strain4 and diluted q:Lj in a =alcon tube.

Plate preparation and inoculation of drug and strain
Sterile water [LWW μL4 was added to all outer wells of sterile +FCwell plates [=alcon xWNLS /ecton Iickinson] Lincoln Park] N. J.4.Middlebrook NH+ broth [qWW μL4 was added to the wells in rows / to O in columns x to qq [labeled as commercially stamped on the plates4.Irugs solutions [usnic acid] isoniazid and their acylC hydrazone4 [qWW μL4 were added to wells in columns q and LS each two rows correspond to a different drug solution.
/y using a multichannel pipette] qWW μL of solution was transferred from column L to column x.The drug solutions were serially diluted q:L in consecutive columns through column qW] where qWW μL of excess medium was discarded.=inal drug concentration ranges were F% to W.qL μgkmL.qWW μL of the diluted q:Lj strain was added to the wells in rows / to O in columns q to qq.The wells in column qq served as inoculumConly controls.The plates were sealed with parafilm and then incubated at xN °°for five days.=ifty microliters of a q:q mixture of the tetrazoliumCTween ºW mixture was added to well /qq.The plates were reincubated at xN °°for qL h.
The following day] if well /qq turned purple] tetrazoliumCTween ºW was added to all wells and the color was recorded at L% h.If well /qq remained yellow] the plates were incubated for another L% h] after which tetrazoliumCTween ºW solution was added to well °qq before the plate was incubated for another L% h.If well °qq remained yellow] incubation was continued and tetrazoliumCTween ºW solution was added to wells Iqq] Uqq] =qq] and Oqq on days +] qq] qx] and qj] respectively.G yellow color in the well was interpreted as no growth] and a purple color was scored as growth.The MI°was defined as the lowest drug concentration] which prevented a color change from yellow to purple.

Results and discussion
Extraction, isolation and elucidation of usnic acid Usnic acid [L.º g4 was obtained as yellow crystals from dried thallus of lichen Evernia prunastri [qWW g4] using repeatedly flash column chromatography with hexanekethyl acetate [N:x4 and hexanektoluenek ethyl acetate [ºj:j:qj4.The IR spectrum showed bands at L+L+ [°HC stretching4] qFºN [elongation of the carbonyl group4] qFLº [°;°] enol ether4] qj%W] q%jj] q%Lq cm Cq [aromatic ring4.It should be mentioned that a wide band was present between xjWW and LFWW cm Cq ] due to the many hydrogen bonds present in the solid state.
The q HCNMR spectrum shows characteristic signals at δ qx.xq and δ qq.WL ppm corresponding to the hydroxyl groups °ºCOH and °qW COH respectivelyS δ j.+N ppm corresponding to the HC% from the double bondS finally δ L.FN] L.FF] L.qWS and q.Nj ppm corresponding to the methyl substituents.The hydroxyl group °xCOH appears at qº.º% ppm [=igure q4.

Biological activity against M. tuberculosis
As it is shown in Table D9 acyl5hydrazone does not ameliorate the antimycobacterial activity of usnic acid for DM /=5resistant wild type9 nor of HA=Rv5susceptible strains9 but9 contrastingly9 acyl5hydrazone showed an enhancement of its antimycobacterial activity9 as compared to usnic acid on HA=Rv5susceptibleS The comparison of D H5NMR spectrum of usnic acid and acyl5 hydrazone revealed that the chemical shift of C A 5OH is due to the presence of the imine group in the structure% the presence of this amine group also influences the electron density of the methyl group CMe5D

Conclusions
The synthesis of compound 2 Cacyl5hydrazoneO was achieved by a condensation reaction using commercial isoniazid and usnic acid9 isolated from lichen Evernia prunastri9 giving an overall yield of /[8 Cw/wOS The isolation of usnic acid was performed using standard protocols of extractions and column chromatographic purificationsS Their chemical structures were elucidated using DD NMR spectroscopy and mass spectrometry9 thus corroborating spectroscopic data reported in the literature C15518OS According to the results obtained by TEMA method, usnic acid (1) exhibited MIC value of 16.0 μg/mL for each test of H37Rv, TB DM 97, and MDR DM 1098 strains.Instead, the corresponding acylhydrazone (2) exhibited MIC values of 2.0 μg/mL against H37Rv, and 64.0 μg/mL against resistant strain and multidrug resistance from M. tuberculosis.The MIC values obtained by compound 1 provide a better prospect to be potentially active as compared to compound 2.However, both compounds exhibit less activity than isoniazid.Apparently, compound 2 does not meet initial expectations of increasing sensitivity on TB strains.However, these are preliminary in vitro results, and further in vivo studies should be carried out to confirm its antitubercular activity.