Rev. bras. farmacogn. vol.27 número6

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Chemical

constituents

from

Bauhinia

acuruana

and

their

cytotoxicity

Roberto

W.S.

Góis

a

_,

_Leôncio

_M.

_de

_Sousa

a

_,

_Horlando

_C.

_da

_Silva

a

_,

_Francisco

_E.F.

_da

_Silva

a

_,

Antonia

T.A.

Pimenta

a

_,

_Mary

_A.S.

_Lima

a

_,

_Angela

_M.C.

_Arriaga

a

_,

_Telma

_L.G.

_Lemos

a

_,

Raimundo

Braz-Filho

b,c

_,

_Gardenia

_C.G.

_Militão

d

_,

_Paulo

_B.N.

_da

_Silva

d

_,

_Francisco

_J.T.

_Gonc¸

_alves

e

_,

Gilvandete

M.P.

Santiago

f,∗

a_Departamento_de_Química_Orgânica_e_Inorgânica,_Centro_de_Ciências,_Universidade_Federal_do_Ceará,_Fortaleza,_CE,_Brazil

b_Laboratório_de_Ciências_Químicas,_Centro_de_Ciências_e_Tecnologia,_Universidade_Estadual_do_Norte_Fluminense_Darcy_Ribeiro,_Campos_dos_Goytacazes,_RJ,_Brazil c_Departamento_de_Química,_Instituto_de_Ciências_Exatas,_Universidade_Federal_Rural_do_Rio_de_Janeiro,_Seropédica,_RJ,_Brazil

d_Departamento_de_Fisiologia_e_{Farmacologia,}_Centro_de_Ciências_Biológicas,_Universidade_Federal_de_Pernambuco,_Recife,_PE,_Brazil e_Instituto_Federal_do_Mato_Grosso_do_Sul,_Campus_Nova_Andradina,_Fazenda_Santa_Bárbara,_Nova_Andradina,_MS,_Brazil

f_Departamento_de_Farmácia,_Faculdade_de_Farmácia,_Odontologia_e_Enfermagem,_Universidade_Federal_do_Ceará,_Fortaleza,_CE,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2June2017

Accepted21September2017

Availableonline31October2017

Keywords:

Fabaceae Bibenzyls

Oxepinderivatives

Flavonoids Terpenoids Cytotoxicity

a

b

s

t

r

a

c

t

Phytochemical investigation of Bauhinia acuruana Moric., Fabaceae, resulted in the isolation of sixteenconstituents,includingtwonewcompounds2′_{-hydroxy-2,3,5-trimethoxybibenzyl}₍₁_), (2R,3S)-2-(3,4′_{-dihydroxyphenyl)-5-methoxy-6-methylchroman-3,7-diol}₍₂_),_together_with_fourteen_known_ones (3–16).Thestructuresofthecompoundswereestablishedbyspectroscopicanalysisincluding HR-ESI-MS,1Dand2DNMRdata,followedbycomparisonwithpreviouslyreporteddatafromtheliterature. Compounds1,2,6,7,8and9wereevaluatedfortheircytotoxicity,whichturnedouttobemarginalina panelofsixhumancancercelllines.

Introduction

Bauhinia,Fabaceae,isalargegenuscontainingabout500species of shrubs, and small trees distributed throughout the tropical areasof Brazil,Peru,Asia,Paraguay, and Argentina (Soares and Scarminio,2008).Manyspeciesofthisgenushavebeenwidelyused infolkmedicinetotreatdiabetes,infections,painandinflammation (CechinelFilho,2009).

BauhiniaacuruanaMoric.,isashruborsubshrubthatusually growsinmountainousareasand/orwithaltitudesof600–1100m (VazandTozzi,2003).Previousstudieshaveshownthatthe essen-tialoilfromleavesof B. acuruanaand pacharin (6), compound isolatedfromtherootsofthisspecies,showedlarvicidalactivity againstAedesaegypti(Goisetal.,2011;Góisetal.,2013).Previous investigationscarriedoutwithpacharin(6)andbauhiniastatin1 (7)haveshownthatthesecompoundsexhibitedsignificantgrowth inhibitionagainstpancreasadenocarcinoma(BXPC-3),breast ade-nocarcinoma(MCF-7),CNSglioblastoma(SF268),lung largecell

∗ Correspondingauthor.

E-mail:gilvandete@pq.cnpq.br(G.M.Santiago).

(NCI-H460),andprostatecarcinoma(DU-145)humancancercell lines(Pettitetal.,2006).

InthesearchforbioactivenaturalcompoundsfromB. acuru-ana,theisolationandstructuralelucidationoftwonewcompounds (1–2), together with fourteen known compounds (3–16) are reported herein. In addition, the cytotoxicity of 2′

-hydroxy-2,3,5-trimethoxybibenzyl (1), (2R,3S)-2-(3′_,4′

-dihydroxyphenyl)-5-methoxy-6-methylchroman-3,7-diol(2),pacharin(6), bauhias-tatin1(7),fisetinidol(8),and(2R,3S)-2-(3′_,4′

-dihydroxyphenyl)-5-methoxychroman-3,7-diol (9) were assessed against colon carcinoma(HTC-116), glioblastoma(SF-295),ovarian carcinoma (OVCAR-8),breastadenocarcinoma(MCF-7),lungcarcinoma (NCI-H292) and pro-myelocytic leukemia(HL-60) humancancercell lines.

Materialsandmethods

Generalexperimentalprocedures

Meltingpoints weredeterminedona digital Mettler Toledo FP82HTapparatusandareuncorrected.Infrared(IR)spectrawere recorded ona Perkin-ElmerFT-IR 1000 spectrometerwith KBr

https://doi.org/10.1016/j.bjp.2017.09.002

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pellets. Optical rotations were obtained on a Perkin-Elmer Q-200polarimeter,at589nmand25◦_C.1_H_and13_C_NMR_(1D_and

2D)spectrawereperformedonBrukerAvanceDPXand/or DRX-500spectrometers, operatingat 300and500MHzfor 1_H _NMR, and 75 and 125MHz for 13_C _NMR, _{respectively.} _The _chemical shifts(ı)areexpressedinppm.Thehighresolutionmassspectra wererecordedonaShimadzuLCMS-IT-TOFspectrometerequipped withaZ-sprayESI(electrospray)source.Highperformanceliquid chromatography(HPLC)analysiswasperformedonaShimadzu chromatographerequippedwithaternarypump(Shimadzu LC-20AT)andUVdetector(ShimadzuSPD-M20A),usingPhenomenex RP-18 column (analytical: 250×4.6␮m, 5m; semi-preparative: 250×10mm,10␮m).HPLCgradesolventswerepurchasedfrom TediaCo.(SãoPaulo,Brazil)andHPLCgradewaterwasobtained byaMilli-Qpurificationsystem.Silicagel60(70–230mesh,Vetec, RiodeJaneiro,Brazil)andSephadexLH-20(Pharmacia)wereused for column chromatography. Thin layer chromatography (TLC) wasperformedonprecoated silicagel polyestersheets (kiesel-gel60 F254, 0.20mm, Silicycle,Quebec, Canada), and the spots werevisualizedbyUVdetectionand/orheatingaftersprayingwith vanillin/perchloricacid/EtOHsolution.Thehumantumorcelllines wereobtainedfromtheBancodeCélulasdo Rio deJaneiro(RJ, Brazil)andLaboratóriodeOncologiaExperimentaldaUniversidade FederaldoCeará(CE,Brazil).

Plantmaterial

Theleavesandstalks ofBauhiniaacuruana Moric.,Fabaceae, werecollectedinMay2008,whiletherootswerecollectedinJune 2011atTianguáCounty,StateofCeará,Brazil.Theplantmaterial wasidentifiedbyEdsonPereiraNunesfromtheHerbário Prisco Bezerra(EAC),DepartamentodeBiologia,UniversidadeFederaldo Ceará,Brazilwherevoucherspecimens(#42405and#49268)have beendeposited.

Extractionandisolation

Air-dried and finely powdered roots (1.1kg) were exhaus-tivelyextractedwithEtOH(4×8l)atroomtemperatureforthree weeks,andevaporatedunderreducedpressuretoyieldthecrude EtOH extract (50.3g), which was subjected to silica gel col-umnchromatographyelutedwithhexane,CH2Cl2,CH2Cl2/EtOAc (1:1),EtOAcand MeOHtogivehexane (0.85g),CH2Cl2 (1.59g), CH2Cl2/EtOAc (1:1)(3.89g), EtOAc(8.71g) and MeOH (21.15g) fractions.TheCH2Cl2fractionwassubmittedtosilicagelcolumn chromatography,usingahexane/CH2Cl2gradientfrom4:1to0:1, toaffordsixfractions(F1–F6).FractionF6(254.8mg;CH2Cl2)and fractionF5(136.5mg;hexane/CH2Cl2,1:4)wereindividually puri-fiedbysilicagelcolumnchromatographyelutedwithCH2Cl2 to obtaincompounds1(19.4mg)and3(22.5mg),respectively.The CH2Cl2/EtOAc(1:1)fractionwaschromatographedonasilicagel columnelutedwithagradientofhexane/EtOAc(4:1to0:1)to pro-videsevenfractions(F1–F7).SeparationoffractionF3(390.5mg; hexane/EtOAc, 4:1)by silicagel columnchromatography, using hexane/CH2Cl2 (1:1),hexane/CH2Cl2 (1:3)andCH2Cl2 aseluent, yieldedthemixtureamixtureofsitosterol(4)andstigmasterol(5) (28.8mg;hexane/CH2Cl2,1:1)andpacharin(6;26.0mg;CH2Cl2). A partof EtOAcfraction(2.4g) was subjectedto silicagel col-umnchromatography,elutedwithagradientofhexane/EtOAc(6:4 to0:1) to producesix fractions (F1-F6). Fraction F2(144.3mg; hexane/EtOAc,6:4)wasfurtherchromatographed onasilicagel column,usinghexane/EtOAc(17:3)aseluent,toafford bauhini-astatin 1 (7; 26.2mg), while fraction F6 (64.2mg, EtOAc) was submittedtosemi-preparativeRP-18HPLCanalysis,usingan iso-craticmixtureMeOH/H2O(9:11)toyieldcompounds8(29.6mg;

tR4.6min),9(4.2mg;tR5.3min)and2(21.3mg;tR7.2min).

Air-driedleaves(0.9kg) weresuccessivelyextractedatroom temperaturewithhexane(4×5l)forthreeweeks,EtOAc(4×5l) forthreeweeks,andthenwithEtOH(4×5l)forthesameperiod. Afterfiltration,thesolventswereevaporatedunderreduced pres-suretogive:hexane (21.0g), EtOAc(29.0g) andEtOH (108.0g) extracts.Apartofhexaneextract(15.7g)wasfractionedover sil-icagelbyelutionwithhexane,CH2Cl2,EtOAc,andMeOHtogive fourfractions:hexane (9.3g), CH2Cl2 (4.3g), EtOAc(0.95g) and MeOH(0.13g).Thehexanefractionwassubjectedsilicagelcolumn chromatographyelutingwiththemixtureofhexane/CH2Cl2 and CH2Cl2/EtOAcinincreasingorderofpolarity.Fractionselutedwith hexane/CH2Cl2 (1:1)werecombinedandchromatographedona silicagelcolumn,usingagradientofhexane/EtOAc(19:1to0:1)to givecompounds10(13.6mg;hexane/EtOAc,9:1),and11(7.7mg; hexane/EtOAc, 17:3). The EtOAc extract (29.0g) wassubmitted tosilicagelcolumnchromatographyelutedwithhexane/CH2Cl2, CH2Cl2,CH2Cl2/MeOH,andMeOHtoprovidetwentyfourfractions (F1-F24).FractionF20(554mg;CH2Cl2/MeOH,4:1)wassubjected torepeatedSephadexLH-20columnelutedwithCHCl3/MeOH(1:1) toobtainquercetin3-O-rhamnoside(12;7.1mg)anddaucosterol (13;9.5mg).

Air-driedandfinelypowderedstalks(1.2kg)wereexhaustively extractedwithEtOH(4×5l)atroomtemperatureforthreeweeks, andevaporatedunderreducedpressuretoyieldthecrudeEtOH extract(70g),whichwassubmittedtosilicagelcolumn chromatog-raphyelutedwithhexane,CH2Cl2,EtOAcandMeOHtogivehexane (694mg),CH2Cl2(1.17g),EtOAc(4.11g)andMeOH(36.27g) frac-tions.The hexanefractionwaschromatographed ona silicagel column elutedwitha gradient of hexane/EtOAc(9:1 to0:1)to affordlupeol(14;22.5mg),andphyscion(15;14.7mg).TheEtOAc fractionwassubjectedtosilicagelcolumnchromatographyusing aCH2Cl2/MeOHgradientfrom19:1to1:1,toobtaineleven frac-tions (F1–F11). Fraction F9(339.1mg; CH2Cl2/MeOH, 4:1) was furthersubmittedtosilicagelcolumnchromatography,elutedwith CH2Cl2/MeOH(17:3)toyieldastilbin(16;112.0mg).

Spectraldata

2′_{-Hydroxy-2,3,5-trimethoxybibenzyl}₌

2-[2-(2,3,5-trimethoxyphenyl)ethyl]phenol (1): Light brown oil; IR (KBr) √

max:3419,2960,1600,1493,1458,1202cm−1;NMRdata(CDCl3, 300and75MHz)seeTable1; HRESIMSm/z: 311.1254[M+Na]+ (calcdforC17H20NaO4+:311.1259).

(2R,3S)-2-(3′_,4′

-Dihydroxyphenyl)-5-methoxy-6-methylchroman-3,7-diol (2):Yellow solid;[␣]D20=−2.6 (c=0.1, MeOH);IR(KBr)√max:3394,1618cm−1;NMRdata(CD3OD,300 and75MHz)seeTable1;HRESIMSm/z:353.0819[M+Cl]− _calcd

forC17H18ClO6−:353.0792.

Cytotoxicityassay

The humantumor cell lines used in this work wereMCF-7 (breastadenocarcinoma),NCI-H292 (lungcarcinoma)andHL-60 (pro-myelocyticleukemia),whichwereobtainedfromtheBanco de Células do Rio de Janeiro (RJ, Brazil), and HTC-116 (colon carcinoma),SF-295(glioblastoma),OVCAR-8(ovariancarcinoma) obtained from the Laboratório de Oncologia Experimental da UniversidadeFederal doCeará(Ceará,Brazil).Cancercellswere maintainedinRPMI1640mediumorDMENsupplementedwith 10% fetal bovine serum, 2mm/l glutamine, 100U/ml penicillin, 100␮g/mlstreptomycinat37◦_C_with_5%_CO₂_._The_cytotoxic

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Table1

1_H₍₃₀₀_MHz)_and13_C_NMR₍₇₅_MHz)_data_of₁_(in_CDCl₃₎_and₂_(in_CD₃_OD),_including_data_obtained_by_HSQC_and_HMBC_{correlations.}_ı_in_ppm,_J_in_Hz.

Position 1 2

ıC ıH HMBC(H→C) ıC ıH HMBC(H→C)

1 135.90 – – –

2 140.99 – CH2(7) 82.90 4.60(d,J=7.3) C(9),C(1′),CH(2′),CH(6′)

3 153.42 – 68.81 3.99(m)

4 98.73 6.42(d,J=2.9) C(2),C(3),C(5),CH(6) 28.83 2.63(dd,J=16.1,8.0) 2.91(dd,J=16.1,5.2)

CH(2),C(10)

5 156.41 – 158.80 –

6 105.29 6.32(d,J=2.9) C(2),CH(4),C(5) 111.41 –

7 32.12 2.81(s) C(1),CH(6) 156.44 –

8 32.66 2.81(s) C(1),C(1′_),_C(2′_),_CH(6′₎ _99.78 _6.15₍_s₎ _C(6),_C(7),_C(9),_C(10)

9 – – 154.33 –

10 – – 106.37 –

1′ _127.44 _– _132.35 _–

2′ _154.64 _– _115.32 _6.83₍_d_,_J₌_1.6) _CH(2),_C(4′_),_CH(6′₎

3′ _115.97 _6.90₍_d_,_J₌_6.0) _C(1′₎ _146.36 _–

4′ _128.00 _7.14₍_dt₎ _146.36 _–

5′ _120.55 _6.80₍_dt₎ _C(1′₎ _116.28 _6.76₍_d_,_J₌_8.1) _C(1′_),_C(3′₎ 6′ _130.10 _7.12₍_d_,_J₌_6.0) _CH₂_(8),_C(2′₎ _120.09 _6.70₍_dd_,_J₌_8.1,_1.6) _CH(2′_),_C(4′₎

MeO-2 61.29 3.86(s) C(2) – –

MeO-3 55.99 3.88(s) C(3) – –

MeO-5 55.79 3.78(s) C(5) 60.46 3.67(s) C(5)

Me-6 – – 8.83 2.04(s) C(5),C(6),C(7)

HSQC,HeteronuclearSingleQuantumCoherenceSpectroscopy;HMBC,HeteronuclearMultipleBondConnectivity.

adherentcellsor3×105cells/mlforleukemia).Compounds1,2,6, 7,8and9dissolvedinDMSO1%wereaddedtoeachwelland incu-batedfor72h.ControlgroupsreceivedthesameamountofDMSO. Thecompoundconcentrationsaddedtothecellsrangedfrom0.39 to25.00␮g/ml.After69hoftreatment,MTT(0.5mg/ml)wasadded, 3hlater,theMTT formazan productwasdissolvedin 100␮lof DMSO,and absorbancewasmeasuredat 570nm inplate spec-trophotometer(VarioskanFlask,ThermoScientific).Doxorubicin wasusedaspositivecontrol.IC50valuesandtheir95%confidence intervalsfortwodifferentexperimentswereobtainedbynonlinear regressionusingGraphpadPrismversion5.0forWindows (Graph-PadSoftware,SanDiego,California,USA).

Resultsanddiscussion

TheEtOHextractfromrootsofB.acuruanawassubjectedto multiplechromatographicstepstoyieldtwonewcompounds(1–2) andtheknowncompounds(3–9).Theair-driedleaveswere succes-sivelyextractedwithhexane,EtOAc,andEtOH.Chromatographic fractionationofthehexaneandEtOAcextractsofB.acuruanaleaves yieldedthecompounds(10–11)and(12–13),respectively.Three knowncompounds(14–16)wereisolatedfromtheEtOHextract fromstalksofB.acuruana.Thestructuresofknowncompounds (3–16)wereidentifiedas2′_{-hydroxy-3,5-dimethoxybibenzyl} ₍₃₎ (Takasugietal.,1987;DeSousaetal.,2016),amixtureof sitos-terol (4) and stigmasterol (5) (Da Silva et al., 2012), pacharin (6)(Pettitetal.,2006;Anjaneyuluetal.,1984),bauhiniastatin1 (7)(Pettitetal.,2006),fisetinidol(8)(Imaietal.,2008),(2R,3S

)-2-(3′_,4′_{-dihydroxyphenyl)-5-methoxychroman-3,7-diol}₍₉₎₍

Cren-Olivéetal.,2002),1␤,6␣-dihydroxy-4(14)-eudesmene(10)(Moujir etal.,2011),aromadendrane-4␤,10␣-diol(11)(Meiraetal.,2008), quercetin3-O-rhamnoside(12)(Slowingetal.,1994),daucosterol (13)(Lendletal.,2005),lupeol(14)(Imametal.,2007),physcion (15)(Danielsenetal.,1992),andastilbin(16)(Bezerraetal.,2013) bycomparisonoftheirspectroscopicdatawiththosereportedin theliterature.

Compound 1 was isolated as a brown viscous liquid and possessed a molecularformula C17H20O4 witheight degrees of unsaturationfromitshigh-resolutionelectrosprayionization spec-trum(HRESIMS)atm/z311.1254[M+Na]+_,_(calcd_311.1259)._The molecularformulawasfurthersubstantiatedbythe13_C_NMR_and distortionlessenhancementbypolarizationtransfer(DEPT)spectra of1whichshowed17carbonresonances,includingthreemethyl, twomethylene,sixmethine,andsixquaternarycarbons,including fouroxygenatedatıC140.99,153.42,154.64and156.41(Table1). TheIRspectrumshowedabsorptionbandsforhydroxylgroupat 3419cm−1_and_aromatic_ring_at₁₆₀₀_and₁₄₅₈_cm−1_._Its1_H_NMR spectrumexhibitedtwodoubletsatıH6.42(J=2.9Hz,H-4)and 6.32(J=2.9Hz,H-6)for aromaticprotons meta-positioned, indi-catingtheoccurrenceofa1,2,3,5-tetrasubstitutedbenzenering, foursignalsatıH6.90(d,J=6.0Hz,H-3′),7.14(dt,H-4′),6.80(dt, H-5′_),_and_7.12₍_d_,_J₌_6.0_Hz,_H-6′₎_which_allowed_to_postulate_the

presence ofa 1,2-disubstituted benzenering, and onesignalat

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Table2

Cytotoxicactivityofcompounds1,2,6,7,8and9.

Compound IC50(␮M)(95%confidenceintervals)a

HCT-116 SF-295 OVCAR-8 MCF-7 NCI-H292 HL-60

(coloncarcinoma) (gliobastoma) (ovariancarcinoma) (breastadenocarcinoma) (lungcarcinoma) (pro-myelocyticleukemia)

1 23.96(19.79–28.47) 37.85(27.08–52.08) 40.62(25.69–64.24) >86.80 57.63(35.42–94.44) 19.44(15.62–23.96)

2 >78.62 >78.62 >78.62 >78.62 >78.62 >78.62

6 19.26(16.30–22.96) 14.44(10.74–19.63) 23.33(18.52–30.37) 20.00(14.81–26.67) 11.11(9.63–12.59) 8.15(7.41–8.89)

7 25.70(22.53–28.87) 21.13(12.32–36.97) 22.89(18.66–27.82) 21.83(14.44–32.75) 10.91(9.51–12.68) 10.21(7.75–13.03)

8 >91.24 >91.24 >91.24 >91.24 >91.24 >91.24

9 >82.24 >82.24 >82.24 >82.24 >82.24 >82.24

Doxorubicin 0.18(0.18–0.36) 0.36(0.36–0.55) 0.55(0.36–0.55) 0.55(0.36–0.92) 0.36(0.18–0.92) 0.04(0.02–0.04)

HCT-116,coloncarcinoma;SF-295,glioblastoma;OVCAR-8,ovariancarcinoma;MCF-7,breastadenocarcinoma;NCI-H292,lungcarcinoma;HL-60,pro-myelocyticleukemia.

a_The_results_were_means_of_two_independent_experiments.

indicatedthat1wasabibenzyl(Kittakoopetal.,2000;Boonphong etal.,2007;Apisantiyakometal.,2004;Yangetal.,2014;Chen etal.,2014;Xuetal.,2014).Detailedanalysisofits1_H_and13_C NMRspectrashowedthatcompound1wassimilartocompound3

(Takasugietal.,1987;DeSousaetal.,2016),exceptforthepresence ofthesignalatıH3.86(s,3H)observedinthe1HNMRspectrum of1,whichwasassignedtoanadditionalmethoxylgroup (MeO-2).IntheHMBCspectrum,thecorrelationsfromthesignalatıH 6.42(d,J=2.9Hz,H-4)withthecarbonsignalsatıC140.99(C-2), 153.42(C-3),and156.41(C-5),andcorrelationsfromthesignalat

ıH6.32(d,J=2.9Hz,H-6)withthecarbonsignalatıC98.73(C-4) allowedtoassignthelocationofmethoxylgroupsatC-2,C-3and C-5.Similarly,thecorrelationsfromH-6′_at_ı_H_7.12₍_d_,_J₌_6.0_Hz)

withC-8 (ıC 32.66), and C-2′ (ıC 154.64), and the correlations fromH2-7atıH2.81(s)withC-1(ıC135.90),andC-6′(ıC130.10) allowedtoassignaphenolichydroxylgroupatC-2′_and_confirmed

thelocationofthe CH2CH2 unit,respectively(Table1).Onthe basisofthesespectroscopicdata,compound 1wasidentifiedas 2′_{-hydroxy-2,3,5-trimethoxybibenzyl.}

Compound 2 was obtained as yellow solid with a specific rotationof[␣]D20=−2.6.IthadthemolecularformulaC17H18O6 based on its HRESIMS (observed m/z 353.0819 [M+Cl]−_, _calcd

353.0792)and13_C_NMR_data,_indicating_nine_degrees_of unsatura-tion.ItsIRspectrumshowedabsorptionbandsforhydroxylgroup at3394cm−1_and_aromatic_ring_at₁₆₁₈_cm−1_._The1_H_and13_C_NMR

dataof2exhibitedsignalsoftwooxygenatedCHgroupsatıH/ıC 3.99(m,H-3)/68.81,and4.60(d,J=7.3Hz,H-2)/82.90,CH2groupat

ıH/ıC2.63(dd,J=16.1and8.0Hz,H-4a)/28.83,andıH/ıC2.91(dd,

J=16.1and5.2Hz,H-4b)/28.83,andtwosignalsofCH3groupsat

ıH/ıC2.04(s,Me-6)/8.83,and3.67(s,MeO-5)/60.46,togetherwith one1,2,3,4,5-pentasubstituted(ıH/ıC6.15(s,H-8))/99.78,andone 1,3,4-trisubstituted(ıH/ıC6.83(d,J=1.6Hz,H-2′)/115.32,6.76(d,

J=8.1Hz,H-5′_)/116.28,_and_6.70₍_dd_,_J₌_8.1_and_1.6_Hz,_H-6′_)/120.09)

benzenerings(Table1).AcomparisonoftheNMRdataof2(Table1) with those reported for (2R,3S)-2-(3′_,4′

-dihydroxyphenyl)-5-methoxychroman-3,7-diol(9)(Cren-Olivéetal.,2002)showedhigh similarity.Thedifferencebetweencompounds2and9isthe pres-enceofthesignalatıH/ıC2.04(s,3H)/8.83observedintheNMR spectraof2,whichwasassigned toamethylgroupofaromatic ring(Me-6).ThelocationofthemethylgroupatC-6was estab-lishedaccordingtoobservedcorrelationsfromthesignalatıH2.04 (s,Me-6)withthecarbonsignalsatıC111.41(C-6),156.44(C-7), and158.80(C-5),andfromthesignalatıH6.15(s,H-8)withthe car-bonsignalsatıC111.41(C-6)intheHMBCspectrum.Therelative stereochemistryof2wassubstantiatedbytheNOESYdata,which showedNOEcorrelationsbetweenthepseudoaxialhydrogenH-2 (ıH4.60(d,J=7.3Hz))andH-3(ıH3.99(m)),andbycomparisonof itsopticalrotationwiththereporteddatafor8(Imaietal.,2008) and9(Cren-Olivé etal.,2002).In addition,wereobservedNOE effectbetweenhydrogenatoms:H-2(ıH4.60(d,J=7.3Hz))and H-2′₍_ı_H_6.83₍_d_,_J₌_1.6_Hz));_H-2₍_ı_H_4.60₍_d_,_J₌_7.3_Hz))_and_H-6′₍_ı_H

6.70(dd,J=8.1and1.6Hz));andH-3(ıH3.99(m))andH-6′(ıH6.70 (dd,J=8.1and1.6Hz))(Table1).Fromtheabovedata,thestructure of 2wasdetermined tobe(2R,3S)-2-(3′_,4′

-dihydroxyphenyl)-5-methoxy-6-methylchroman-3,7-diol.

2′_{-Hydroxy-2,3,5-trimethoxybibenzyl} ₍₁_), ₍₂_R_,3_S_)-2-(3′_,4′

-dihydroxyphenyl)-5-methoxy-6-methylchroman-3,7-diol (2), pacharin (6), bauhiastatin 1 (7), fisetinidol (8), and (2R,3S )-2-(3′_,4′_{-dihydroxyphenyl)-5-methoxychroman-3,7-diol} ₍₉₎ _were

evaluated for their cytotoxic activity against colon carcinoma (HTC-116),glioblastoma(SF-295),ovariancarcinoma(OVCAR-8), breast adenocarcinoma (MCF-7), lung carcinoma (NCI-H292) andpro-myelocyticleukemia(HL-60)humancelllinesbythe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)assay(Mosmann,1983),usingdoxorubicinaspositive con-trol.Thecytotoxicactivitiesofthesecompoundsaresummarizedin Table2.Compound6,whichinpreviousstudiesshowedsignificant growth inhibition against pancreas adenocarcinoma (BXPC-3), breastadenocarcinoma(MCF-7),CNSglioblastoma(SF268),lung largecell(NCI-H460),and prostatecarcinoma (DU-145)human cancercelllines(Pettitetal.,2006),exhibitedcytotoxicityagainst pro-myelocyticleukemia(HL-60)humancelllines,withIC50value of8.15␮M,butwasinactiveagainsttheothertestedhumancell lines.Inadditioncompounds1,2,7,8and9wereinactive.These results indicated that compound 6 displays cytotoxic activity, whichareinaccordancewithotherstudiesreportingthatdifferent oxepinderivatives(Pettitetal.,2006;Boonphongetal.,2007;Li etal.,2013)canexertcytotoxicactivitiesoncancercelllines.

The phytochemical investigation of B. acuruana led to the isolationoftwonewcompounds,identifiedas2′

-hydroxy-2,3,5-trimethoxybibenzyl (1) and (2R,3S)-2-(3,4′

-dihydroxyphenyl)-5-methoxy-6-methylchroman-3,7-diol (2), along with a known bibenzyl(3), threestreoids(4,5,13), twooxepinderivatives(6,

7),fourflavonoids(8,9,12,16),twosesquitepenes(10,11),one triterpene(14)andoneanthraquinone(15).Thisisinaccordance withpreviousreportsonthechemicalconstituentsisolatedfrom theBauhiniagenus.Notably,compound9wasaflavonoidisolated forthefirsttimeasnaturalproduct,butreportedpreviouslyasa syntheticderivative(Cren-Olivéetal.,2002).

Sincepreviousreportsdescribed thecytotoxicpropertiesfor speciesoftheBauhiniagenus,ourexpectativewasthatsomeofthe isolatedcompoundscoulddisplaythiseffect,but,unfortunately, exceptfor pacharin(6)which showedcytotoxicityagainst pro-myelocyticleukemia(HL-60)humancelllines,thecompounds1,

2,7,8and9wereinactive.

Ethicaldisclosures

(5)

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthe publicationof patientdata.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

RWSG(PhD student) did thecompound isolation procedure andcontributed incompoundidentificationbyNMRand litera-turesearch.LMS,HCS,FEFSandATAPcontributedincarryingout thelaboratorywork,andinterpretationofthespectroscopicdata. MASL,AMCA,TLGLandRBFcontributedintheinterpretationof thespectroscopic data.GCCG and PBNS contributedto biologi-calassays.FJTGcontributedtoplantcollectionandconfectionof herbarium.GMPSdesigned thestudy,supervisedthelaboratory workandwrotethemanuscript.LMScontributedtocritical read-ingofthemanuscript.Alltheauthorshavereadthefinalmanuscript andapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsaregratefultoCNPq,CAPES,FUNCAPandPRONEX forthefellowshipsandfinancialsupport.Wealsothankto CENAU-REMN (UFC)and LEMANOR for NMR and highresolution mass spectra,respectively.

References

Anjaneyulu, A.S.R., Reddy, A.V.R., Reddy, D.S.K., Ward, R.S., Adhikesavalu,D.,

Cameron,T.S.,1984.Pacharin:anewdibenzo(2,3-6,7)oxepinderivativefrom

BauhiniaracemosaLank.Tetrahedron40,4245–4252.

Apisantiyakom,S.,Kittakoop,P.,Manyum,T.,Kirtikara,K.,Bremner,J.B.,

Thebtara-nonth,Y.,2004.NovelbiologicallyactivebibenzylsfromBauhiniasaccocalyx

PIERRE.Chem.Biodivers.1,1694–1701.

Bezerra,G.P.,Góis,R.W.S.,DeBrito,T.S.,DeLima,F.J.B.,Bandeira,M.A.M.,Romero,

N.R.,Magalhães,P.J.C.,Santiago,G.M.P.,2013.Phytochemicalstudyguidedby

themyorelaxantactivityofthecrudeextract,fractionsandconstituentfrom stembarkofHymenaeacourbarilL.J.Ethnopharmacol.149,62–69.

Boonphong,S.,Puangsombat,P.,Baramee,A.,Mahidol,C.,Ruchirawat,S.,Kittakoop,

P.,2007.BioactivecompoundsfromBauhiniapurpureapossessingantimalarial,

antimycobacterial,antifungal,anti-inflammatory,andcytotoxicactivities.J.Nat. Prod.70,795–801.

CechinelFilho,V.,2009.Chemicalcompositionandbiologicalpotentialofplants

fromthegenusBauhinia.Phytother.Res.23,1347–1354.

Chen,X.-J.,Mei,W.-L.,Cai,C.-H.,Guo,Z.-K.,Song,X.-Q.,Dai,H.-F.,2014.Fournew

bibenzylderivativesfromDendrobiumsinense.Phytochem.Lett.9,107–112.

Cren-Olivé,C.,Lebrun,S.,Rolando,C.,2002.Anefficientsynthesisofthefourmono

methylatedisomersof(+)-catechinincludingthemajormetabolitesandofsome dimethylatedandtrimethylatedanaloguesthroughselectiveprotectionofthe catecholring.J.Chem.Soc.,PerkinTrans.16,821–830.

Danielsen,K.,Aksnes,D.W.,Francis,G.W.,1992.NMRstudyofsomeanthraquinones

fromRhubarb.Mag.Reson.Chem.30,359–363.

DaSilva,F.M.A.,Koolen,H.H.F.,Barison,A.,DeSouza,A.D.L.,Pinheiro,M.L.B.,2012.

SteroidsandtriterpenefromthebarkofUnonopsisguatterioidesR.E.Fr. (Anon-naceae).Int.J.Pharm.Pharm.Sci.4,522–523.

DeSousa,L.M.,DeCarvalho,J.L.,DaSilva,H.C.,Lemos,T.L.G.,Arriaga,A.M.C.,

Braz-Filho,R.,Militão,G.C.G.,Silva,T.D.S.,Ribeiro,P.R.V.,Santiago,G.M.P.,2016.New

cytotoxicbibenzylandotherconstituentsfromBauhiniaungulataL.(Fabaceae). Chem.Biodivers.13,1630–1635.

Gois,R.W.S.,DeSousa,L.M.,Lemos,T.L.G.,Arriaga,A.M.C.,Andrade-Neto,M.,

Santi-ago,G.M.P.,Ferreira,Y.S.,Alves,P.B.,DeJesus,H.C.R.,2011.Chemicalcomposition

andlarvicidaleffectsofessentialoilfromBauhiniaacuruana(Moric)against Aedesaegypti.J.Essent.OilRes.23,59–62.

Góis,R.W.S.,DeSousa,L.M.,Santiago,G.M.P.,Romero,N.R.,Lemos,T.L.G.,Arriaga,

A.M.C.,Braz-Filho,R.,2013.LarvicidalactivityagainstAedesaegyptiofpacharin

fromBauhiniaacuruana.Parasitol.Res.112,2753–2757.

Imai,T.,Inoue,S.,Ohdaira,N.,Matsushita,Y.,Suzuki,R.,Sakurai,M.,DeJesus,J.M.H.,

Ozaki,S.K.,Finger,Z.,Fukushima,K.,2008.Heartwoodextractivesfromthe

Ama-zoniantreesDipteryxodorata,Hymenaeacourbaril,andAstroniumlecointeiand theirantioxidantactivities.J.WoodSci.54,470–475.

Imam,S.,Azhar,I.,Hasan,M.M.,Ali,M.S.,Ahmed,S.W.,2007.Twotriterpenes

lupanoneandlupeolisolatedandidentifiedfromTamarindusindicaLinn.Pak.J. Pharm.Sci.20,125–127.

Kittakoop,P.,Kirtikara,K.,Tanticharoen,M.,Thebtaranonth,Y.,2000.

Antimalar-ialpreracemosolsAandB,possiblebiogeneticprecursorsofracemosolfrom BauhiniamalabaricaRoxb.Phytochemistry55,349–352.

Lendl,A.,Werner,I.,Glasl,S.,Kletter,C.,Mucaji,P.,Presser,A.,Reznicek,G.,Jurenitsch,

J.,Taylor,D.W.,2005.PhenolicandterpenoidcompoundsfromChionevenosa

(SW.)Urbanvar.venosa(BoisBandé).Phytochemistry66,2381–2387.

Li,Y.-K.,Zhou,B.,Ye,Y.-Q.,Du,G.,Niu,D.-Y.,Meng,C.-Y.,Gao,X.-M.,Hu,Q.-F.,2013.

TwonewdiphenylethylenesfromArundinagraminifoliaandtheircytotoxicity. Bull.KoreanChem.Soc.34,3257–3260.

Meira,M.,David,J.M.,David,J.P.,Araújo,S.V.,Regis,T.L.,Giulietti,A.M.,DeQueiróz,

L.P.,2008. ConstituintesquímicosdeIpomoeasubincanaMeisn.

(Convolvu-laceae).Quim.Nova31,751–754.

Mosmann,T.,1983.Rapidcolorimetricassayforcellulargrowthandsurvival:

appli-cationtoproliferationandcytotoxicityassays.J.Immunol.Methods65,55–63.

Moujir,L.M.,Seca,A.M.L.,Araujo,L.,Silva,A.M.S.,Barreto,M.C.,2011.Anew

nat-uralspiroheterocycliccompoundandthecytotoxicactivityofthesecondary metabolitesfromJuniperusbrevifolialeaves.Fitoterapia82,225–229.

Pettit,G.R.,Numata,A.,Iwamoto,C.,Usami,Y.,Yamada,T.,Ohishi,H.,Cragg,G.M.,

2006.Antineoplasicagents.551.Isolationandstructuresofbauhiniastatins1–4

fromBauhiniapurpurea.J.Nat.Prod.69,323–327.

Slowing,K.,Sollhuber,M.,Carretero,E.,Villar,A.,1994.Flavonoidsglycosidesfrom

Eugeniajambos.Phytochemistry37,255–258.

Soares,P.K.,Scarminio,I.S.,2008.Multivariatechromatographicfingerprint

prepa-rationandauthenticationofplantmaterialfromthegenusBauhinia.Phytochem. Anal.19,78–85.

Takasugi,M.,Kawashima,S.,Monde,K.,Katsui,N.,Masamune,T.,Shirata,A.,1987.

AntifungalcompoundsfromDiscoreabatatas inoculatedwith Pseudomonas cichorii.Phytochemistry26,371–375.

Vaz,A.M.S.F.,Tozzi,A.M.G.A.,2003.Bauhiniaser.Cansenia(Leguminosae:

Caesalpin-ioideae)noBrasil.Rodriguésia54,55–143.

Xu,F.-Q.,Xu,F.-C.,Hou,B.,Fan,W.-W.,Zi,C.-T.,Li,Y.,Dong,F.-W.,Liu,Y.-Q.,Sheng,

J.,Zuo,Z.-L.,Hu,J.-M.,2014.Cytotoxicbibenzyldimersfromthestemsof

Den-drobiumfimbriatumHook.Bioorg.Med.Chem.Lett.24,5268–5273.

Yang,D.-S.,Wei,J.-G.,Peng,W.-B.,Wang,S.-M.,Sun,C.,Yang,Y.-P.,Liu,K.-C.,Li,

X.-L.,2014.CytotoxicprenylatedbibenzylsandflavonoidsfromMacarangakurzii.