Phytochemistry 97 (2014) 81–87

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Phytochemistry

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Cytotoxic non-aromatic B-ring flavanones from Piper carniconnectivumC. DC.

0031-9422/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.phytochem.2013.10.012

⇑ Corresponding author.E-mail address: majokato@iq.usp.br (M.J. Kato).

Giovana C. Freitas a, João M. Batista Jr. b, Gilberto C. Franchi Jr. c, Alexandre E. Nowill c,Lydia F. Yamaguchi a, Janaina D. Vilcachagua a, Denize C. Favaro a, Maysa Furlan b,Elsie F. Guimarães d, Christopher S. Jeffrey e, Massuo J. Kato a,⇑a Research Support Center in Molecular Diversity of Natural Products, Instituto de Química, Universidade de São Paulo, CP 26077, 05599-970 São Paulo, SP, Brazilb NUBBE, Instituto de Química, Universidade Estadual Paulista, UNESP, 14800-900 Araraquara, SP, Brazilc Centro Integrado de Pesquisas Oncohematológicas na Infância, UNICAMP, CP 6141, 13083-970 Campinas, SP, Brazild Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão 2040, 22460-030 Rio de Janeiro, RJ, Brazile Department of Chemistry, University of Nevada, Reno, NV 89557, USA

a r t i c l e i n f o

Article history:Received 22 April 2013Received in revised form 26 September 2013Available online 16 November 2013

Keywords:Piper carniconnectivumPiperaceaeNon-aromatic B-ring flavanoneCytotoxic

a b s t r a c t

The EtOAc extract from the leaves of Piper carniconnectivum C. DC. was subjected to chromatographicseparation to afford two non-aromatic B-ring flavanone compounds: 5-hydroxy-2-(10-hydroxy-40-oxo-cyclohex-20-en-10-yl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (1) and 5-hydroxy-2-(10 ,20-dihydroxy-40-oxo-cyclohexyl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (2). The absoluteconfiguration of (+)-1 was unambiguously determined as 2S,10R by electronic circular dichroism (ECD)spectroscopy and comparison to simulated spectra that were calculated using time-dependent densityfunctional theory (TDDFT). This methodology allowed the assignment of the absolute configuration of(+)-2 also as 2S,10R, except for the stereogenic center at C-20 , which was assigned as R because of theevidence drawn from high resolution NMR experiments. The cytotoxic activity of both compounds and3 (hydrogenated B-ring derivative of 1) was evaluated on twelve human leukemia cell lines, and theIC50 values (<10 lM) indicated the activity of 1 against seven cell lines.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The species Piper carniconnectivum C. DC. (Piperaceae) is ende-mic to the Amazon region of Northern Brazil (Ribeiro et al.,1999). Previous phytochemical studies of its roots, resulted in theisolation of three cyclopentenediones, 2-(2H-2-chromenyliden)-4-methyl-4-cyclopenten-1,3-dione, 2-(2H-2-chromenyliden)-5-methyl-4-cyclopenten-1,3-dione, and 2-[1-hydroxyl-3-phenyl-(Z,2E)-2-propenylidene]-4-4methyl-4-cyclopentene-1,3-dione); aprenylated coumarin, xanthyletin (Facundo et al., 2004); threeC-methylated flavanones, 5-hydroxy-7-methoxy-6-methyl-2-phenyl-2,3-dihydro-4H-chromen-4-one, 5-hydroxy-7-methoxy-8-methyl-2-phenyl-2,3-dihydro-4H-chromen-4-one, and 5-hydroxy-7-methoxy-6,8-dimethyl-2-phenyl-2,3-dihydro-4H-chromen-4-one;and a C-methylated chalcone, (2E)-1-(2-hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-phenylprop-2-en-1-one (Facundo andBraz-Filho, 2004).

Flavonoids are a widespread class of secondary metabolites iso-lated from a multitude of angiosperms. Nevertheless, containing a

partial or completely hydrated non-aromatic B-ring, called protofl-avonoids, they have only been isolated from species within the Pte-ridophyte such as Thelypteridaceae (Lin et al., 2005; Wada et al.,1987) and Dryopteridaceae (Noro et al., 1969) and from one Mag-noliophyte (Ongokea gore, Olacaceae) (Jerz et al., 2005). Whileflavonoids exhibit potent antioxidant activity, their cytotoxic ef-fects are typically negligible. In contrast, protoflavonoids, such asprotoapigenone and protofarrerol, have promising cytotoxic effectsmainly against breast, ovarian, lung, liver, and prostate cancer celllines (Chang et al., 2008a,b; Wei et al., 2012).

The studies carried out on P. carniconnectivum in this workresulted in the isolation and characterization of two new flavanon-es that contain an unusual non-aromatic B-ring: 5-hydroxy-2-(10-hydroxy-40-oxo-cyclohex-20-en-10-yl)-6,7-dimethoxy-2,3-dihy-dro-4H-chromen-4-one (1) and 5-hydroxy-2-(10,20-dihydroxy-40-oxo-cyclohexyl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one(2). Structural assignment of 1 and 2, required the application oflong-range coupling observed in high-field NMR (800 MHz) exper-iments to solve the relative configuration. Electronic circulardichroism (ECD) spectroscopy and time-dependent densityfunctional theory (TDDFT) calculations were used to determineabsolute configurations. The antiproliferative properties of the

2'

1

4'

2

3

5

3

3'

8

2

Fig. 1. Structures of 1, 2 and derivative 3.

82 G.C. Freitas et al. / Phytochemistry 97 (2014) 81–87

protoflavonoids 1, 2 and 3 (hydrogenated B-ring derivative of 1)were assessed against twelve human leukemia cell lines.

2. Results and discussion

A combination of vacuum liquid chromatography and columnchromatography (Sephadex LH-20) of the EtOAc extract from theleaves of P. carniconnectivum afforded two new protoflavanones:5-hydroxy-2-(1 0-hydroxy-4 0-oxo-cyclohex-2 0-en-1 0-yl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (1) and 5-hydroxy-2-(10,20-dihydroxy-40-oxo-cyclohexyl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (2). A saturated derivative 3 was obtained bycatalytic hydrogenation of 1 providing additional data regardingthe cytotoxic activity of protoflavonoids (Fig. 1).

Compound 1 was obtained as a yellow amorphous solid. Threeabsorption bands in the UV spectrum at 233, 288 and 337 nm wereevidence of a flavonoid nucleus (Harborne and Williams, 1971a,b).The molecular formula was established as C17H18O7 based onHRMS data (ESI-TOF: [M+H]+ 335.1121, calcd. 335.1131) andNMR spectroscopic data also supported the structural assignment.

Fig. 2. Optimized structures and relative energies [B3LYP/PCM(EtOH)/6-31G(d)] of the twused for ECD calculations at the B3PW91/PCM(EtOH)/TZVP level.

The 1H NMR spectrum of 1 exhibited typical resonances of anoxygenated flavanone, including a singlet of an aromatic protonfrom a pentasubstituted aromatic ring A at d 6.08, two singletsassignable to methoxy groups at d 3.83 (3H) and 3.90 (3H), and asinglet resulting from a hydrogen-bonded carbonyl group at d11.77. The location of the aryl methoxy groups were establishedvia HMBC and NOESY correlations. HMBC spectra established cor-relations from the hydrogen H-8 to the carbon at d 131.1 (C-7) anda single correlation between d 160.9 (C-6) and C-5 OH hydrogen.Further HMBC analysis demonstrated a correlation between themethoxy proton resonance (d 3.83) and carbon C-7, and the meth-oxy proton resonance (d 3.90) and C-64. NOESY correlation be-tween the methoxy group and H-8 confirmed the position of themethoxy group at C-7 (Fig. 1).

Three double of doublets resulting from an ABX spin system at d4.44 (1H, J 13.8 and 2.9 Hz, H-2), 3.09 (1H, J 17.1 and 13.8 Hz, H-3ax) and 2.67 (1H, J 17.1 and 2.9 Hz, H-3eq) led to the assignmentof the C ring of the flavanone skeleton. However, despite character-istic signals of the A- and C-rings of a flavanone, typical proton res-onances for the aromatic B-ring were absent. Instead, four aliphatichydrogen resonances at d 2.08 (ddd, J 13.7, 10.2 and 4.9 Hz, H-60ax),

o lowest-energy conformers of (2S,10R)-1 (upper frame) and (2S,10S)-1 (lower frame)

Fig. 3. Center: experimental ECD spectrum of (+)-1 in EtOH assigned as 2S,10R.Lower frame: calculated ECD spectrum [B3PW91/TZVP//B3LYP/6-31G(d) in EtOHusing PCM] of the Boltzmann average of the two lowest-energy conformers of thecorresponding (2S,10R)-1. Upper frame: calculated ECD spectrum [B3PW91/TZVP//B3LYP/6-31G(d) in EtOH using PCM] of the Boltzmann average of the two lowest-energy conformers of the corresponding (2S,10S)-1. The bars represent the rotationalstrengths for the weighted ECD spectrum.

Fig. 4. Center: experimental ECD spectrum of (+)-2 in MeOH. Lower frame:calculated ECD spectrum [B3PW91/TZVP//B3LYP/6-31G(d) in MeOH using PCM] ofthe Boltzmann average of the seven lowest-energy conformers of the corresponding(2S,10R,20S)-2 (see Supplementary Data). Upper frame: calculated ECD spectrum[B3PW91/TZVP//B3LYP/6-31G(d) in MeOH using PCM] of the Boltzmann average ofthe eight lowest-energy conformers of the corresponding (2S,10R,20R)-2 (seeSupplementary Data). The bars represent the rotational strengths for the weightedECD spectrum.

G.C. Freitas et al. / Phytochemistry 97 (2014) 81–87 83

2.16 (ddd, J 13.7, 6.2 and 5.3 Hz, H-60eq), 2.50 (ddd, J 17.2, 6.2 and4.9 Hz, H-50eq) and 2.79 (ddd, J 17.2, 10.2 and 5.3 Hz, H-50ax) andtwo vinylic hydrogen resonances [d 6.11 (d, J 10.3 Hz, H-30) and7.10 (dd, J 10.3 and 1.3 Hz, H-20] provided evidence of a cyclohexe-none non-aromatic B-ring. Moreover, an additional carbonyl reso-nance at d 198.0 (C-40) and conjugated vinylic carbons at d 130.6(C-30) and 147.1 (C-20) in the 13C NMR spectrum of compound 1provided strong support of this cyclohexenone moiety. Compellingsupport for the structural assignment came from the HMBC datathat established correlations between H-20 to the carbonyl at d198.0 (C-40), 69.9 (C-10), 30.7 (C-60) and d 81.9 (C-2). HMBC corre-

lations between H-2 and the carbon at d 81.9 (C-20) and betweenH-30 and carbon resonances at d 69.9 (C-10) and 33.2 (C-50), estab-lished the connectivity between the non-aromatic B-ring and C-ring. Additional HMBC correlations combined with those in theHSQC and COSY correlations were in full accordance with thestructure depicted for the non-aromatic B-ring flavanone 1(Fig. 1). Finally, fragmentation of the non-aromatic B-ring(M-C6H8O2, m/z = 223) in the ESIMSn spectrum provided furtherevidence for the structure of flavanone 1 as 5-hydroxy-2-(10-hy-droxy-40-oxo-cyclohex-20-en-10-yl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one.

(a) (b)2'

6'

4'

3

3'

2

Fig. 5. Newman projection indicating the dihedral angle between the hydrogens atC-ring (a) and the W-coupling (b) at the B-ring of compound 2.

84 G.C. Freitas et al. / Phytochemistry 97 (2014) 81–87

Catalytic hydrogenation of 1 provided saturated product 3,which confirmed the structural assignment of 1 and also providedan additional compound for the cytotoxicity studies. The 1H and13C NMR spectroscopic data of 3 confirmed chemoselective hydro-genation of the C20–C30 alkene of compound 1 with the appearanceof new methylenic signals at d 2.80 (2H, td, J 14.0 and 6.1 Hz, H-20),1.90 (1H, td, J 14.0 and 5.0 Hz, H-30eq) and 3.02 (1H, dd, J 17.0 and14.0 Hz, H-30ax) corresponding to a fully saturated 4-hydroxycy-clohexanone. The molecular formula of 3 was confirmed asC17H20O7 based on the HRESI-MS data ([M+H]+ 337.1286, calcd.337.1287).

Compound 2, obtained as colorless needles, showed threeabsorption bands at 212, 287 and 338 nm with a shoulder at233 nm in the UV spectrum giving further confirmation of a flava-noid core. Its molecular formula was established as C17H20O8,based on the HRESI-MS data ([M + Na]+ 375.1053, calcd.375.1056), corresponding to a hydrated analog (M + 18) of 1. The1H and 13C NMR spectra for compound 2 were similar to those offlavanone 1, with a singlet at d 6.15 (H-8) corresponding to a pen-tasubstituted aromatic A-ring and two aromatic methoxy groups at3.64 (s, 3H) and 3.84 (s, 3H) at the C-6 and C-7 positions, respec-tively. The placement of the methoxy groups was confirmed byNOESY correlations between H-8 and OCH3-7.

The three double of doublets from the ABX spin system ex-pected in the C-ring of a flavanone were observed at d 4.62 (1H, J13.6 and 3.0, H-2), 3.02 (1H, J 17.2 and 13.6, H-3ax) and 2.56(1H, J 17.2 and 3.0, H-3eq). While the hydrogen and carbon reso-nances from the A- and C-rings were comparable to that of 1, thoseof the double bond of the enone system were not present. Reso-nances in the 13C NMR spectrum indicated the presence of a secondnon-conjugated carbinolic center (d 69.7) with an additional meth-ylene carbon in contrast to the B-ring of 1. Further analysis of thespectroscopic data established that the double bond of the B-ringof 1 was replaced by a secondary alcohol at C-20 (d 69.7), whilemaintaining the tertiary hydroxy group at C-10 (d 72.1) and the ke-tone at C-40 (d 210.0). HMBC correlations between OH-10 and C-20,OH-20 and C-10, C-30 and C-40 were observed and these confirmedthe location of the hydroxy groups on the cyclohexanone of the Bring. With the connectivity of the C- and B-rings between hydrogen

Table 11H and 13C NMR spectroscopic data for 1 (800 and 200 MHz, CDCl3) and 2 (800 and 200 M

1

1H 13

2 4.44 dd (13.8, 2.9) 83 3.09 dd (17.1, 13.8) 3

2.67 dd (17.1, 2.9)4 – 194a – 105 – 156 – 137 – 168 6.08 s 98a – 1510 – 620 7.10 dd (10.3, 1.3) 1430 6.11 d (10.3) 13

40 – 1950 2.79 ddd (17.2, 10.2, 5.3) 3

2.50 ddd (17.2, 6.2, 4.9)60 2.08 ddd (13.7, 10.2, 4.9) 3

2.16 dddd (13.7, 6.2, 5.3, 1.3)6-OCH3 3.90 s 67-OCH3 3.83 s 55-OH 11.77 s10-OH –20-OH –

H-2 and carbons C-10, C-20 and C-60 confirmed by the HMBC-corre-lations, the structure of compound 2 was assigned as 5-hydroxy-2-(10,20-dihydroxy-40-oxo-cyclohexyl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one.

Assignment of the relative configuration of 1 and 2 by tradi-tional approaches was complicated by their conformational flexi-bility as the result of free rotation on the sigma bond connectingthe non-aromatic B-ring. The combined use of time-dependentdensity functional theory (TDDFT) calculated electronic circulardichroism (ECD) spectra with experimental ECD has been success-fully applied to determine the absolute configuration of variousnatural products (Li et al., 2010; Batista et al., 2011; Felippeet al., 2012). Monte Carlo conformational searches for the lowestenergy conformations of the two diastereoisomers of 1 (2S,10Rand 2S,10S) indicated that both compounds were predominantlyrepresented by conformations at 298 K (Fig. 2). These conformers,which differed mainly with respect to rotation of the methyl of thearomatic methoxy groups, were further optimized using B3PW91/TZVP//B3LYP/6-31G(d) level of theory and a PCM to simulate anethanolic medium and used to simulate the ECD spectra. Electronictransitions corresponding to aromatic p–p⁄ transitions (250 and280 nm, respectively) with some charge transfer (alkene to car-bonyl) contribution, as well as, ketone n-p⁄ transitions (325–350 nm) were the foci of the comparison.

The overall features of the calculated ECD spectrum for (2S,10R)-1 were consistent with the experimental ECD with positive Cotton

Hz, DMSO-d6).

2

C 1H 13C

1.9 4.62 dd (13.6, 3.0) 79.25.9 3.02 dd (17.2, 13.6) 35.5

2.56 dd (17.2, 3.0)5.8 – 197.93.0 – 102.35.1 – 153.81.1 – 129.40.9 – 160.41.5 6.15 s 91.77.6 – 158.59.9 – 72.17.1 4.42 m 69.70.6 2.88 dd (14.5, 3.3) 44.8

2.16 dt (14.5, 2.7)8.0 – 210.03.2 2.57 ddd (14.6, 13.2, 6.9) 35.7

2.04 ddd (14.6, 5.2, 2.1)0.7 1.89 td (13.2, 5.2) 27.2

1.70 ddt (13.2, 6.9, 2.1)0.9 3.64 s 60.06.3 3.84 s 56.1– 11.90 s –– 5.28 s –– 5.27 d (4.3) –

Table 2Cytotoxicity of 1, 2, and 3 against various leukemia* cancer cell lines.

Cell line IC50 (lM)

1 2 3 Vincristine

K562 >10 >10 >10 0.164 ± 0.066HL60 8.7 ± 1.7 >10 >10 0.0110 ± 0.0003NB4 3.4 ± 0.3 >10 >10 0.003 ± 0.001P39 >10 >10 >10 0.010 ± 0.002DAUDI >10 >10 >10 0.010 ± 0.002U937 >10 >10 >10 0.005 ± 0.002RAMOS 8.3 ± 1.0 >10 >10 0.029 ± 0.003JURKAT 5.9 ± 0.2 >10 >10 0.164 ± 0.036MOLT4 >10 >10 >10 0.009 ± 0.002B15 8.5 ± 0.2 >10 >10 0.504 ± 0.155NAMALWA 9.6 ± 1.1 >10 >10 0.077 ± 0.006NALM6 9.0 ± 0.7 >10 >10 1.707 ± 0.526

* Leukemic cell lines: K562, HL60, and NB4 human myeloid leukemia cell line; P39human leukemia myelodysplastic syndrome (MDS); NAMALWA, DAUDI, andRAMOS human Burkitt’s lymphoma; JURKAT and MOLT4 lymphoid human leuke-mia cell line T; U937 human histiocytic lymphoma; and NALM6 and B15 humanleukemia cell line lymphoid.

G.C. Freitas et al. / Phytochemistry 97 (2014) 81–87 85

effects (CE) near 325 and 250 nm, and a negative CE around280 nm (Fig. 3). The predicted transition wavelengths were notscaled, since the uniform scaling factor may be unsatisfactory forall electronic transition energies (Polavarapu et al., 2011). Compar-ison of the calculated spectra for the diastereomer (2S,10S)-1 dem-onstrated the opposite positive CE for the absorption at 250 nmwith the CE of the other two absorption bands (ca. 325 and280 nm) having similar calculated signs and intensities to that cal-culated for (2S,10R)-1. These findings, allowed the unambiguousassignment of the absolute configuration of (+)-1 as 2S,10R.

The experimental ECD spectrum of flavanone 2 was very similarto that of compound (+)-1, suggesting the absolute configuration as2S,10R. This assignment was further supported by TDDFT calcula-tions at the B3PW91/TZVP//B3LYP/6-31G(d) level in MeOH (PCM)(Fig. 4). ECD simulations of the two C-20 epimers (2S,10R,20S)- and(2S,10R,20R)-2 did not provide significant resolution to allow deter-mination of absolute configuration of C-20 (Fig. 4). This data sug-gests that the introduction of a new stereogenic center does notsignificantly perturb the chromophores of the dominant benzoyland C-40 carbonyl groups.

Further detailed analysis of long-range coupling in the high-res-olution NMR was used to assign the configuration at C-20 (Contre-ras and Peralta, 2001; Krivdin and Contreras, 2007). The magnitudeof vicinal scalar coupling constants (3JHH) between H-20 and H-30ax

(3JH20H30ax = 3.3 Hz) and H-30eq (3JH20H30ax = 2.7 Hz) of compound 2suggested an equatorial orientation of H-20 (Fig. 5), which was fur-ther confirmed by the strong NOE effect between H-20 (d 4.42) andboth H-30 diastereotopic hydrogens H-30ax (d 2.88) and H-30eq (d2.16). A long range H-C-C-C-H (W-type) coupling 4JHH of 2.1 Hz, ob-served between H-20 and H-60eq (d 1.70), provided strong evidenceof the equatorial orientation of H-20 and C-20 allowing definitiveassignment of the C-20 absolute configuration as R.

The potent cytotoxic activities (IC50 �10 lM) exhibited by theserare non-aromatic B-ring flavanones, such as protoapigenone andanalogues (Lin et al., 2007, 2005; Pouny et al., 2011), inspired theevaluation of the cytotoxic activity of compounds 1, 2 and 3 againsta panel of twelve human leukemia cell lines (Table 2). Compound 1showed inhibitory activity against human myeloid leukemia, hu-man Burkitt’s lymphoma and limphoid human leukemia cell lines,with an IC50 >10 lM and higher activity against NB4 and JURKATcell lines, with an IC50 of 3.4, 5.9 lM, respectively. Compounds 2and 3 were inactive (IC50 >100 lM), indicating that the unsatura-tion at the position C20 is essential for cytotoxic activity of 1, whichis in agreement with a previous observation (Lin et al., 2007).

3. Conclusions

Two novel non-aromatic B-ring flavanones were isolated fromthe leaves of P. carniconnectivum. A comparison of experimentaland TDDFT-calculated ECD spectra combined with high-resolutionNMR experiments of these compounds established their absoluteconfiguration as (+)-(2S,10R)-1 and (+)-(2S,10R,20R)-2. Biologicalevaluation against 12 human leukemia cell lines indicated thein vitro cytotoxic activity of 1, which inhibited the growth ofimportant human leukemic cells with an IC50 <10 lM.

4. Experimental

4.1. General

NMR spectra were recorded on a Bruker Avance III 800.08 MHzspectrometer operated at 800.08 MHz for 1H and at 201.2 MHz for13C; the instrument was equipped with a TCI cryoprobe. Sampleswere prepared as solutions in CDCl3 and DMSO-d6, and TMS wasused as an internal reference. HRESI-TOFMS were measured on aBruker microTOF QII spectrometer using positive electrospray ion-ization. IR spectra were obtained for samples compacted into KBrdiscs; the spectra were recorded with a Bomen MB100 FTIR spec-trometer. UV spectra were recorded on a Shimadzu UV-1650PCspectrometer. EIMS were measured on a Shimadzu 14B/QP5050A(cell length: 1 cm). CD spectra were obtained on a Jasco J-720 cir-cular dichroism spectrophotometer (scan range: k 200–400 nm,cell path length: 0.1 cm). The [a]D values were measured at 21 �Con a Perkin Elmer model 343 polarimeter (cell path length:10 cm). Silica gel (Merck 60-230 mesh) and Sephadex LH-20(Amersham Biosciences) were used for column chromatography(CC) separations. Silica gel 60 PF254 (Merck) was used for analyticalTLC. Analytical HPLC was performed using a Shimadzu Prominencechromatograph model CMB-20A equipped with a UV–Vis detector(SPD-20A), a column oven (CTO-20A), a solvent pump (LC-20AD),and a Phenomenex C-18 column (250 � 4.6 mm; 5 lm). The sol-vents, MeCN (B) and 0.1% HCO2H (A) were used as the mobilephase in the following gradient elution: 0–5 min, 30% B; 5–30 min, 30–100% B; 30–35 min, 100% B; 35–40 min, 100–30% B;and 40–45 min, 30% B. The injection volume was 20 lL of a MeOHsolution (0.5 mg/mL), and the temperature was 40 �C.

4.2. Plant material

The specimen of P. carniconnectivum C. DC. (Piperaceae) wascollected in the Carajás National Forest, Pará State, Brazil (SISBIO15780-2) and was identified by Dr. Elsie Franklin Guimarães fromthe Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio deJaneiro, Brazil, where a voucher specimen is deposited (Kato-978).

4.3. Extraction and isolation

Dried and powdered leaves of P. carniconnnectivum (30 g) wereextracted with EtOH (3 � 700 mL, 24 h) at room temperature. Thecombined extracts were concentrated in vacuo to yield a crude ex-tract (3.5 g). The latter was subjected to silica gel CC eluted withgradient mixtures of n-hexane/EtOAc and EtOAc/MeOH, respec-tively, which afforded 43 fractions. Fractions 20-25 were submit-ted to column chromatography on Sephadex LH-20 CC to afford 1(150 mg). Fractions 31-33 yielded compound 2 (20 mg) afterrecrystallization from a solution of CH2Cl2 and MeOH.

86 G.C. Freitas et al. / Phytochemistry 97 (2014) 81–87

4.4. (2S-10R)-5-Hydroxy-2-(10-hydroxy-40-oxo-cyclohex-20-en-10-yl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (1)

Yellow amorphous solid. HPLC Rt: 9.77 min; UV (EtOH) kmax

(log e): 233 (3.39), 287 (3.38), and 338 (2.63). IR (KBr) mmax/cm�1: 3377, 3300, 2941, 2922, 2830, 1672, 1631, 1573, 1496,1448, 1298, 1207, 1118, 962, and 825. HRESI-TOFMS (positivemode) m/z: 335.1121 [M+H]+ (calcd. for C17H19O7 335,1131).[a]D

21 = + 166 (c 0.1, CHCl3). ECD (EtOH, c 5 � 10�3) kmax (De):330 (3.68), 285 (�7.20), 234 (16.74), and 220 (25.90). For 1H and13C NMR spectroscopic data: see Table 1.

4.5. 5-(2S-10R)-Hydroxy-2-(10,20-dihydroxy-40-oxo-cyclohexyl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (2)

Colorless needles. Mp 183–186 �C (MeOH). HPLC Rt: 5.75 min;UV (EtOH) kmax (log e): 232 (3.30), 287 (3.38), and 338 (2.63). IR(KBr) mmax/cm�1: 3380, 3309, 2969, 2911, 1717, 1644, 1632,1576, 1495, 1458, 1296, 1204, 1178, 1111, and 808. HRESI�TOFMS(positive mode) m/z: 375.1053 [M+Na]+ (calcd. for C17H20O8Na375.1056). [a]D

21 = +92 (c 0.1, MeOH), ECD (MeOH, c 5 � 10�3) kmax

(De): 333 (3.90), 287 (�10.31), and 213 (31.52). For 1H and 1H and13C NMR spectroscopic data: see Table 1.

4.6. (2S)-5-Hydroxy-2-(10-hydroxy-40-oxo-cyclohexyl)-6,7-dimethoxy-2,3-dihydro-4H-chromen-4-one (3)

A solution of 1 (20 mg) in CH2Cl2 (2 mL) was hydrogenatedusing 10% Pd/C under H2 at 5 atm pressure. After the mixturewas stirred for 4 h at room temperature (25 �C), the catalyst wasremoved by filtration using a Celite bed, and the solvent was evap-orated in vacuo. The crude product was subjected to silica gel usinggradient mixtures of hexane and EtOAc to give 3 (18 mg, 90%). Yel-low amorphous solid. HPLC Rt: 9.75 min. IR (KBr) mmax/cm�1: 3471,2931, 2855, 1712, 1642, 1455, 1299, 1111, 1018, 961, 886, and 557.HRESI�TOFMS (positive mode) m/z 337.1275 [M+H]+ (calcd. forC17H20O7H: 337.1287). 1H NMR (800 MHz, CDCl3): d 11.80 (1H, s,OH-5), 6.06 (1H, s, H-8), 4.28 (1H, dd, J = 14.0 and 2.7 Hz, H-2),3.90 (3H, s, CH3O-7), 3.83 (3H, s, CH3O-6), 3.02 (1H, dd, J = 17.0,14.0 Hz, H-3a), 2.80 (2H, td, J = 14.0, 6.0 Hz, H-20a, H-20b), 2.63(1H, dd, J = 17.0, 2.7 Hz, H-3b), 2.33�2.43 (3H, m, H-30a, H-60a, H-60b), 2.05 (1H, dquin, J = 13.7, 3.3 Hz, H-50b), 1.90 (1H, td, J = 14.0,5.0 Hz, H-30b), and 1.81 (1H, td, J = 13.7, 5.0 Hz, H-50a). 13C NMR(125 MHz, CDCl3): d 210.7 (C-40), 196.3 (C-4), 160.8 (C-7), 157.9(C-5), 154.9 (C-8a), 130.7 (C-6), 102.9 (C-4a), 91.4 (C-8), 83.0 (C-2), 70.9 (C-10), 60.9 (CH3O-6), 56.3 (CH3O-7), 36.4 (C-3), 36.2 (C-20), 36.1 (C-60), 33.7 (C-30), and 32.0 (C-50).

4.7. Computational methods

All DFT and TDDFT calculations were performed at 298 K by theGaussian 09 program package (Frisch et al., 2009). The PCM solventmodel was employed to simulate a solvent medium of eitherMeOH or EtOH. Calculations of 1 were performed for the arbitrarilychosen (2S,10R)- and (2S,10S)-1. The theoretical spectra of theirenantiomers were generated by multiplying the obtained spectraby (�1). The minimum energy configuration for each isomer wasidentified using a Monte Carlo conformational search with theMMFF force field as implemented in Spartan 08 software package.

For compounds (2S,10R)-1 and (2S,10S)-1, eleven and nine con-formers, respectively, with relative energies (rel E.) within10 kcal/mol of the lowest-energy conformers were selected, andfurther geometries were optimized at B3LYP/PCM/6-31G(d) levelin EtOH. Then, the two conformers of (2S,10R)-1, identified withinan energy window of 2.0 kcal/mol (corresponding to 96% of the to-tal Boltzmann distribution) and two conformers of (2S,10S)-1 (>97%

of the total Boltzmann distribution), were selected for electronicabsorption (EA) and ECD spectral calculations. The Boltzmann fac-tor for each conformer was calculated based on Gibbs free energy.Minima were confirmed via vibrational analysis at the B3LYP/PCM/6-31G(d) level in EtOH. TDDFT calculations was applied in order toobtain the excitation energy (in nm), oscillator strength (dimen-sionless), and rotatory strength R in dipole velocity (Rvel in cgsunits: 10�40 esu2 cm2) form, at the B3PW91/PCM(EtOH)/TZVP le-vel. The calculated oscillator and rotatory strengths from the first15 singlet ? singlet electronic transitions were simulated into anEA (See Supplementary Data) and ECD curve, respectively, usingGaussian band shapes and 10 nm half-width at 1/e of peak height.

Regarding compound 2, calculations were performed for thearbitrarily chosen (2S,10R,20R)-2 and (2S,10R,20S)-2. Conformationalanalyses were also carried out using the same algorithm, forcefield, and software as above described. For compounds(2S,10R,20R)-2 and (2S,10R,20S)-2, twenty eight and thirty four con-formers, respectively, with rel E. within 10 kcal/mol of the low-est-energy conformers were selected and their geometriesfurther optimized at the B3LYP/PCM/6-31G(d) level in MeOH. A to-tal of seven conformers of (2S,10R,20R)-2 (corresponding to 94% ofthe total Boltzmann distribution) and eight conformations of(2S,10R,20S)-2 (>92% of the total Boltzmann distribution), identifiedwithin an energy window of 1.5 kcal/mol, were selected for EA andECD spectral calculations at the B3PW91/PCM(MeOH)/TZVP level.The Boltzmann weighting factor for each conformer was also calcu-lated based on Gibbs free energy. Vibrational analysis at the B3LYP/PCM(MeOH)/6-31G(d) level also resulted in no imaginary frequen-cies confirming each of these conformers as true minima. TheTDDFT theoretical EA (See Supplementary Data) and ECD spectrawere simulated from the first 20 singlet ? singlet electronic tran-sitions using Gaussian band shapes and 10 nm half-width at 1/e ofpeak height. All the predicted wavelength transitions were used assuch without any scaling.

4.8. Cytotoxic activity

Cytotoxicity activities of compounds 1–3 were evaluatedagainst twelve different cell types of neoplasms. The tested celllines were K562, HL60, and NB4 of the human myeloid leukemiacell line; P39 of human leukemia myelodysplastic syndrome(MDS); NAMALWA, DAUDI, and RAMOS of the human Burkitt’slymphoma cell line; JURKAT and MOLT4 of the human T-cell lym-phoblast-like cell line; U937 of human histiocytic lymphoma; andNALM6 and B15 from human B acute lymphoblastic leukemia.Briefly, the cells were distributed in 96-well plates (100 ll cells/well with RPMI 1640 medium (Sigma R6504) supplemented with10% fetal calf serum (Gibco 16000-044), 1% penicillin (10,000 IU/mL), and streptomycin 10 mg/ml (15,070 Gibco). The establishedcells were exposed to various concentrations (100, 10, 0.1, 0.01,0.001, 0.0001, and 0.00001 lg/mL) of compounds 1–3 dissolvedin DMSO (0.1%). For titration of the cells, all steps in the assay wereautomated using an epMotion� 5070 liquid handling workstation(Eppendorf, Vaudaux, Schonenbuch, Switzerland). The plates wereincubated for 48 h at 37 �C in humidified air with 5% CO2. Cellularviability was determined by the MTT (Sigma M2128) reduction as-say using a tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-di-phenyl tetrazolium bromide). The spectrophotometric opticaldensity was measured at 570 nm (Bio-Tek Power Wave XS). TheIC50 were determined according to the concentration in whichthe activity relative to control cells was diminished 50%.

Acknowledgements

GCF and JMBJ thank FAPESP for the provision of grants (2009/51850-9) and scholarships (2008/58658-3, 2008/58717-0, and

G.C. Freitas et al. / Phytochemistry 97 (2014) 81–87 87

2011/22339-4). Research funding was provided by CNPq, FAPESP(2009/51850-9), NSF (DEB 1145609), and University of São Paulo(Research Support Center 2012.1.17646.1.8).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytochem.2013.10.012.

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