Polyisoprenylated benzophenone derivatives from the fruits of Garcinia cambogia and their absolute configuration by quantum chemical circular dichroism calculations

Tetrahedron 66 (2010) 139–145

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Polyisoprenylated benzophenone derivatives from the fruits of Garcinia cambogia and their absolute configuration by quantum chemical circular dichroism calculations Milena Masullo, Carla Bassarello, Giuseppe Bifulco *, Sonia Piacente * ` degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA), Italy Dipartimento di Scienze Farmaceutiche, Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 July 2009 Received in revised form 19 October 2009 Accepted 5 November 2009 Available online 10 November 2009

Three new tetracyclic polyisoprenylated xanthones, named oxy-guttiferones M, K2, and I, along with oxy-guttiferone K and guttiferone M, have been isolated from the fruits of Garcinia cambogia. Their structures were elucidated by MS and NMR spectroscopic experiments. The absolute configurations of oxy-guttiferone K, taken as a model of tetracyclic xanthones, and guttiferone M, as a model of polyisoprenylated benzophenones, have been determined by comparison of their experimentally measured circular dichroism (CD) curves with the TDDFT-predicted curves. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Polyisoprenylated benzophenones Oxy-guttiferones CD Quantum chemical calculations

1. Introduction Garcinol, also known as camboginol, is a polyisoprenylated benzophenone derivative occurring in Garcinia indica and Garcinia cambogia.1 This compound is reported to possess scavenging activity, playing an important role in the treatment of gastric ulcers caused by the hydroxyl radical1,2 or by a chronic infection with Helicobacter pylori.3 Garcinol shows antibiotic activity against methicillin-resistant Staphylococcus aureus comparable to that of vancomycin4 and it inhibits topoisomerases I and II at concentrations comparable to that of etoposide.5 Furthermore, garcinol has reported to possess antitumor activity against human leukemia HL60 cells, through cytochrome c release and activation of caspases6–8 and to inhibit histone acetyltransferases and p300/CPB-associated factor, both of which modulate gene expression.9 Garcinol is also able to suppress colonic aberrant crypt foci (ACF) formation in rats and to modulate tyrosine phosphorylation of FAK and subsequently to induce apoptosis in human colon cancer cells.10 Other polyisoprenylated benzophenones, named guttiferones, have shown interesting biological properties. Guttiferones A, B, E, C, and D, the latter two isolated as an inseparable mixture, were tested for anti-HIV biological activity.11 In a different study,

* Corresponding authors. Tel.: þ39 089 969741; fax: þ39 089 969602 (G.B); tel.: þ39 089 969763; fax: þ39 089 969602 (S.P.). E-mail addresses: [email protected] (G. Bifulco), [email protected] (S. Piacente). 0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2009.11.034

guttiferone F showed both cytoprotection against HIV-1 in vitro and cytotoxicity to the host cells.12 Guttiferone I is reported to inhibit the binding activity of a-liver X receptor (LXRa) but is less effective against b-receptor (LXRb).13 Our previous phytochemical study of the fruits of G. cambogia has shown the presence of garcinol and guttiferones K, I, J, M, N, along with oxy-guttiferone K.14 Continuing our investigation, here we report the isolation of further new oxy-guttiferones, namely oxy-guttiferones M, K2, and I (1–3) (see Fig. 1). Oxy-guttiferones are tetracyclic xanthones derived from the oxidation of the corresponding polyisoprenylated benzophenones. Sang et al.15 have recently reported the structure of some oxidation products of garcinol by DPPH and have proposed the mechanism for the formation of these products. The reaction of garcinol with the stable DPPH radical generates a conjugated radical whose cyclization involving the catechol ring leads to the tetracyclic xanthone derivatives. The natural occurrence of oxy-guttiferones K, K2, I, and M together with the corresponding guttiferones could be in agreement with the mechanism of cyclization proposed by Sang et al. for garcinol. Examination of the NMR spectroscopic data (1H and 13C) allows the relative stereochemistry of guttiferones to be established. The relative orientation of the C(6) substituent (axial or equatorial) can be determined on the basis of the coupling constant of H-7ax, the chemical shifts of the methyl groups at C-5, and the carbon resonance of C-7. Moreover, ROESY correlations are useful to confirm and determine the relative stereochemistry of guttiferones.3 So far,

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21

20

27

19 23

22

24

18

26

17

HO

28 25

15

4

O 16

O

O

3

32

30 10

HO

O

9 34

36

O

1

2

29

12

O

O

31

38

35

33

10

1

O

37 16

1 HO

2 OH

HO

O

O

HO

O

O

HO

HO O

O

O

O

4

3

OH HO

O

O

O

OH

5 Figure 1. Compounds 1–5 isolated from G. cambogia fruits. The absolute configuration is depicted for compounds 4 and 5.

only for garcinol, isogarcinol, and guttiferone A has the absolute configuration by X-ray crystallographic analysis been determined.16,17 Guttiferone E had 1H and 13C NMR spectra virtually identical to those of garcinol. On the basis of the optical rotation [a]Dþ101, it is the opposite sign to that reported for garcinol, therefore it was considered the enantiomer of garcinol.11 Chiral molecules are of widespread interest in chemistry and biochemistry. Since the two enantiomers of a chiral compound generally possess different biological activities, it is worthwhile to establish the absolute configuration of naturally occurring molecules.18 Chiral molecules exhibit electronic optical activity, i.e., electronic circular dichroism (CD) and optical rotation (OR). Mirrorimage enantiomers of a chiral molecules exhibit mirror-image CD

and OR, which means that the CD and OR of the two enantiomers are equal in magnitude and opposite in sign.19 Recently, together with the well established NMR spectroscopic methods for the determination of the relative configuration of natural compounds,20 new efficient methods have been devised for the determination of their absolute configuration, in particular by means of calculation of circular dichroism (CD) spectra.21 CD investigations can usually only distinguish between stereoisomers with a sufficient chiroptical differentiation, whose CD curves are fully opposite and near-symmetrical. Furthermore, in the cases where more than one stereogenic element is present, the relative configuration has to be established beforehand by other methods, such as by NMR spectroscopy.22

M. Masullo et al. / Tetrahedron 66 (2010) 139–145

On this basis, we have elucidated the absolute configuration of oxy-guttiferone K, taken as a model of tetracyclic polyisoprenylated xanthones, and guttiferone M, as a model of polyisoprenylated benzophenones. 2. Results and discussion 2.1. Structural elucidation The fruits of G. cambogia were extracted with EtOH and the obtained extract was partitioned with diethyl ether–H2O (1:1). The diethyl ether extract was fractionated by semi-preparative HPLC/ DAD to yield three new compounds, oxy-guttiferones M, K2, and I (1–3) along with the known oxy-guttiferone K (4) and guttiferone M (5) (see Experimental Section).14 The 1H and 13C NMR spectra of all the compounds were recorded in CD3OD with 0.1% TFA to enhance the rate of the keto–enol interconversion of the b-hydroxy-a,b-unsaturated ketone. The 1H and 13 C NMR data in combination with the IR spectral characteristics and the known occurrence of polyisoprenylated benzophenone derivatives in the genus Garcinia suggested that these compounds were members of the guttiferone family.3 2.1.1. Oxy-guttiferone M (1). The ESI-MS spectrum of 1 showed a major ion peak at m/z 623 [MþNa]þ, suggesting the molecular

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formula C38H48O6Na, with 15 degrees of unsaturation. The 1H NMR spectrum of 1 showed the presence of two aromatic protons at d 6.96 and d 7.51 (singlets); two prenyl groups (two one-proton triplets at d 4.74, t, J¼6.4 Hz, and d 4.95, t, J¼6.4 Hz, and four singlet methyl groups at d 1.84, 1.66, 1.56, and 1.50), one geranyl group (two one-hydrogen triplets at d 5.02 t, J¼6.4 Hz, and d 5.10, t, J¼6.4 Hz together with three methyl groups at d 1.74, 1.63, 1.55; all singlets), and two tertiary methyl groups on a saturated carbon at d 1.24 and 0.93 (Table 1). The 1H NMR spectrum of 1 was comparable to that of the guttiferone M, except for the loss of one aromatic proton. As mentioned above, compound 1 had one more unsaturation than guttiferone M. This indicates that there is one more ring in 1 than in guttiferone M, and this was in agreement with the 13C NMR spectroscopic data. The 13C NMR spectrum showed signals for a nonconjugated ketone (d 206.5), the two quaternary carbons (d 67.1 and 66.5), an enolized 1,3-diketone (d 194.7, 119.5, and 176.0), and quaternary (d 48.7), methine (d 43.2), and methylene (d 43.5) carbons. A HSQC experiment allowed us to assign the two aromatic proton signals at d 6.96 and d 7.51, the carbon resonances at d 103.6 and at d 109.5, respectively (Table 1). HMBC correlations between the proton signals at d 6.96 and d 7.51 to the carbon resonances at d 147.0, 150.9, 154.6, together with the long-range correlations between the proton signal at d 6.96 to the carbon resonance at d 117.7, and between the proton signal at d 7.51 to the carbon resonance at d 174.1, allowed us to deduce that C-16 was oxygenated. Moreover,

Table 1 1 H and 13C NMR spectroscopic data (J in Hz) of compounds 1–3 (600 MHz, d ppm, in CD3ODþ0.1% TFA) 1

2

3

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

1 2 3 4 5 6 7

194.7 119.5 176.0 66.5 48.7 43.2 43.5

178.5 114.6 194.0 75.6 51.7 41.7 42.2

67.1 206.5 174.1 117.7 109.5 150.9 154.6 103.6 147.0 25.8

18 19 20 21 22 23 24

120.2 135.3 25.8 18.3 16.2 23.9 29.4

d d d d d 1.78, m 2.24, m 1.78, m d d d d 7.50, s d d 6.98, s d 2.74, dd (13.7, 8.8) 2.57, m 4.70, t (6.4) d 1.58, s 1.75, s 0.85, s 1.68 (2H), m 1.93 (2H), m

194.5 118.4 176.6 64.4 48.9 47.6 39.8

8 9 10 11 12 13 14 15 16 17

25 26 27 28 29

123.2 134.2 25.8 17.6 30.5

d d d d d 1.62, m 2.02, m 1.56, m d d d d 7.51, s d d 6.96, s d 3.02, dd (13.9, 9.0) 2.91, dd (13.9, 4.6) 4.74, t (6.4) d 1.50, s 1.84, s 0.93, s 1.24, s 2.12, m 1.77, m 4.95, t (6.4) d 1.66, s 1.56, s 2.57 (2H), d (6.8)

124.2 135.5 25.9 17.7 29.6

d d d d d 1.51, m 2.20, m 2.12, m d d d d 7.48, s d d 7.00, s d 2.95, dd (13.7, 8.8) 2.88, m 4.63, t (6.4) d 1.43, s 1.75, s 1.15, s 1.27, s 2.05, m 1.80, m 4.90, t (6.4) d 1.66, s 1.44, s 2.56 (2H), d (6.8)

30 31 32 33 34 35 36 37 38

120.7 138.6 40.8 27.4 124.9 131.8 25.6 17.6 16.2

5.10, t (6.4) d 1.96 (2H), m 2.04. (2H), m 5.02, t (6.4) d 1.55, s 1.63, s 1.74, s

120.5 139.0 40.9 27.7 125.1 131.9 25.8 17.6 16.2

5.27, t (6.8) d 2.02 (2H), m 2.08 (2H), m 5.10, t (6.4) d 1.59, s 1.62, s 1.72, s

58.8 207.0 174.1 118.1 109.6 151.3 154.8 103.5 146.9 26.6 121.6 134.0 25.9 18.1 15.4 36.6 24.8 125.2 132.4 25.9 17.6 29.3 123.1 134.8 25.8 17.9 31.1 119.8 135.5 25.9 18.2

5.07, t (6.4) d 1.69, s 1.62, s 2.10, m 1.88, m 4.93, t (6.4) d 1.64, s 1.58, s 2.82 (2H), d (6.8) 4.97, t (6.4) d 1.58, s 1.83, s

66.8 207.0 174.5 117.5 109.3 154.7 151.1 103.6 146.8 27.4 119.4 136.0 26.0 18.3 26.5 23.0 30.2

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HMBC correlations between the carbon resonance at d 194.7 and the proton resonances of H-7 (d 2.02 and 1.56) and H-29 (d 2.57), and between the carbon resonance at d 176.0 and the proton resonances of H-17 (d 3.02 and 2.91) allowed the assignments of the signals at d 194.7 and 176.0 to be C-1 and C-3, respectively. Consequently, it was clear that the carbonyl at C-3 was enolized and that the oxygen was attached to C-16. Thus, compound 1 represents the oxidized derivative of guttiferone M, named oxy-guttiferone M. 2.1.2. Oxy-guttiferone K2 (2). The ESI-MS spectrum of 2 showed a major ion peak at m/z 623 [MþNa]þ, again having the molecular formula C38H48O6Na, identical to 1. The 1H NMR spectrum of 2 displayed two unique aromatic proton signals at d 6.98 and d 7.50 (singlets), whose corresponding carbons were assigned by a HSQC experiment at d 103.5 and at d 109.6, respectively (Table 1). The 1H NMR spectrum of 2 revealed the presence of one tertiary methyl group (dH 0.85/dC 15.4), and four prenyl units (four one-hydrogen triplets at d 4.70, t, J¼6.4 Hz, 4.93, t, J¼6.4 Hz, 4.97, t, J¼6.4 Hz and d 5.07, t, J¼6.4 Hz, and eight methyl groups at d 1.58 (9H), 1.62, 1.64, 1.69, 1.75, and 1.83; all singlets). HMBC correlations between the proton signals at d 6.98 and d 7.50 to the carbon resonances at d 146.9, 151.3, 154.8, together with the long-range correlations between the proton signal at d 6.98 to the carbon resonance at d 118.1, and between the proton signal at d 7.50 to the carbon resonance at d 174.1, were similar to those reported for compound 1 in which the C-16 was oxygenated. The 13C NMR spectrum exhibits signals for a non-conjugated ketone (d 207.0), two quaternary carbons (d 75.6 and 58.8), an enolized 1,3-diketone (d 194.0, 114.6 and 178.5), quaternary (d 51.7), methine (d 41.7), and methylene (d 42.2) carbons. The NMR spectroscopic data of compound 2 were almost superimposable with those of oxy-guttiferone K,14 except for the chemical shift of one of the carbon atoms at d 58.8, which is absent in the spectra of both guttiferone K and oxy-guttiferone K. The cross peaks in the HMBC spectrum allowed us to establish that compound 2 is an isomer of oxy-guttiferone K. Indeed, in the HMBC spectrum of 2, the correlation between Cd194.0 and H-17 (d 2.57 and 2.74), Cd178.5 and H-7 (d 1.78 and 2.24) suggested that d 178.5 was C-1 and d 194.0 was C3, respectively. Furthermore, HMBC correlations between the

proton resonances of H-29 and the carbon resonance at d 58.8 allowed us to assign the signal at d 58.8 as C-8, thereby indicating that the carbonyl at C-1 was enolized and its oxygen attached to C16. Therefore, compound 2 represents another condensation product of guttiferone K, named oxy-guttiferone K2. As observed by Sang et al.15 concerning the antioxidant pathways/mechanisms of garcinol and its related polyisoprenylated benzophenones, the identification of oxy-guttiferone K2, together with the previously isolated oxy-guttiferone K, provides further unambiguous evidence that the oxidation reaction of these compounds involves the hydroxyl group of C-3 and C-1 of the enolized diketone and the phenolic ring part. 2.1.3. Oxy-guttiferone I (3). The ESI-MS spectrum of 3 again showed a major ion peak at m/z 623 [MþNa]þ, (molecular formula C38H48O6Na). The 1H NMR spectrum of 3 showed the presence of two aromatic protons at d 7.00 and d 7.48 (singlets); two prenyl groups (two one-proton signals at d 4.63, t, J¼6.4 Hz, and d 4.90, t, J¼6.4 Hz, and four methyl groups at d 1.75, 1.66, 1.44, and 1.43; all singlets), one geranyl group (two one-proton signals at d 5.27 t, J¼6.8 Hz, and d 5.10, t, J¼6.4 Hz, together with three methyl groups at d 1.72, 1.62, 1.59; all singlets), and two tertiary methyl groups on a saturated carbon at d 1.27 and 1.15 (Table 1). The 1H NMR spectrum of 3 was comparable to that of the guttiferone I,23 except for the loss of one aromatic proton. The 13C NMR spectrum showed signals for a non-conjugated ketone (d 207.0), two quaternary carbons (d 64.4 and 66.8), an enolized 1,3-diketone (d 194.5, 118.4 and 176.6), quaternary (d 48.9), methine (d 39.8), and methylene (d 47.6) carbons. HMBC correlations between the proton signals at d 7.00 and d 7.48 to the carbon resonances at d 146.8, 151.1, 154.7, together with cross peaks between the proton signal at d 7.00 to the carbon resonance at d 117.5, and between the proton signal at d 7.48 to the carbon resonance at d 174.5, established that the C-16 was oxygenated. The carbon resonances of C-1 and C-3 at d 194.5 and at 176.6, respectively, were determined by HMBC correlations between the carbon resonance at d 194.5 and the proton resonances of H-7 (d 2.20 and 2.12) and H-29 (d 2.56), and between the carbon resonance at d 176.6 and the proton resonances of H-17 (d 2.95 and 2.88). On this basis, the carbonyl at C-3 was enolized and its oxygen

Figure 2. Elucidation of the absolute configuration of oxy-guttiferone K (4) by comparison of its experimentally measured CD spectrum with the TDDFT-predicted curves.

M. Masullo et al. / Tetrahedron 66 (2010) 139–145

was linked to C-16. Therefore, compound 3 is the oxidation product of guttiferone I, named oxy-guttiferone I. 2.2. Absolute configuration of oxy-guttiferone K (4) and guttiferone M (5) Following the structure determination of the above mentioned compounds, complete with the assignment of the relative stereostructures, we employed the approach proposed by Bringmann et al.21 in the determination of the absolute configuration by means of comparison between experimental and calculated circular dichroism spectra of oxy-guttiferone K and guttiferone M, taken as an example of polyisoprenylated xanthones and of polyisoprenylated benzophenones, respectively. Due to the conformational mobility of both oxy-guttiferone K and guttiferone M, a preliminary conformational analysis by means of Molecular Dynamics (MD) calculation was performed in order to locate the minimum energy conformers falling in a range of ca. 10 kcal/mol. Subsequently, the minimum energy conformers for each structure were further optimized at Density Functional Theory (DFT) level, using the mPW1PW91 functional and the 6-31G(d) basis set. From the calculated geometries, and on the basis of the Boltzmann distribution of the significant conformers, weighted averaged spectra were obtained using the Time-Dependent Density Functional Theory (TDDFT). In particular, the mPW1PW91 functional was used and the 6-31G(d,p) basis set was used for the calculationdsuch level of theory has been proven to be adequate for obtaining accurate results at an affordable computational time cost.21 In particular, the calculated Boltzmann weighted averaged CD spectrum of oxyguttiferone K, resulting from six major conformers, resulted in very good accordance with the experimental spectrum (see Fig. 2), so providing strong evidence for the absolute configuration depicted in 4. With the same confidence, it was possible to assign the absolute configuration of guttiferone M as 5, in this case considering its seven major conformers and comparing the Boltzmann weighted averaged CD spectrum with the experimental, as shown in Figure 3.

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bicyclo[3.3.1]nonane ring system. Optical rotation values ([a]D) resulted to be þ20.9 for oxy-guttiferone K (4) and 29.0 for guttiferone M (5). Guttiferone A and garcinol, whose opposite absolute configurations were established by X-ray, exhibited [a]D values corresponding to þ34.0 and 125.0 , respectively, and the same absolute configuration on bicyclo[3.3.1]nonane ring system of 4 and 5, respectively. This suggests that the optical rotation value could be a useful parameter to give a preliminary indication on the absolute configuration of the guttiferones. On the basis of these considerations, since the oxidized forms of guttiferones isolated in our studies possess the same optical rotation sign of guttiferones that they are derived from, we can hypothesize for oxy-guttiferone M ([a]D 96.2) the same absolute configuration of guttiferone M ([a]D 29.0). In the same manner, for guttiferone K ([a]D 7.6) and oxy-guttiferone K2 ([a]D þ12.2) the same absolute configuration as oxy-guttiferone K ([a]D þ20.9) can be expected. 3. Experimental 3.1. General experimental procedures CD spectra were obtained using a JASCO J-810 spectrometer. Optical rotations were measured on a JASCO DIP 1000 polarimeter. IR measurements were obtained on a Bruker IFS-48 spectrometer. UV spectra were recorded on a UV-2101PC Shimadzu UV/Vis scanning spectrophotometer. NMR experiments were performed on a Bruker DRX-600 spectrometer equipped with a Bruker 5 mm TCI CryoProbe. All spectra were acquired in the phase sensitive mode and the TPPI method was used for quadrature detection in the u1 dimension. The 1H, gCOSY, ROESY, gHSQC, and gHMBC NMR experiments were run under standard conditions at 300 K dissolving each sample in 500 mL of 99.8% D CD3OD (Carlo Erba) with 0.1% TFA (1H, d¼3.34 ppm; 13C, d¼49.0 ppm). The ROESY spectra were acquired with tmix¼400 ms. ESI-MS analyses were performed using a ThermoFinnigan LCQ Deca XP Max ion trap mass spectrometer equipped with Xcalibur

Figure 3. Elucidation of the absolute configuration of guttiferone M (5) by comparison of its experimentally measured CD spectrum with the TDDFT-predicted curves.

2.3. Conclusion On the basis of the above data, oxy-guttiferone K (4) and guttiferone M (5) possess the opposite absolute configuration at the

software. Samples were dissolved in MeOH (Baker) and infused in the ESI source by using a syringe pump; the flow rate was 3 mL/min. The capillary voltage was 43 V, the spray voltage was 5 kV, and the tube lens offset was 30 V. The capillary

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temperature was 280  C. Exact masses were measured by a Voyager DE mass spectrometer (Applied Biosystems, Foster City, CA). Samples were analysed by matrix assisted laser desorption ionization (MALDI) mass spectrometry. A mixture of analyte solution and a-cyano-4-hydroxycinnamic acid (Sigma, St. Louis, MO) was applied to the metallic sample plate and dried. Mass calibration was performed with the ions from ACTH (fragment 18–39) at 2465.1989 Da and a-cyano-4-hydroxycinnamic acid at 190.0504 as internal standards. HPLC separations were carried out on a Agilent 1100 series chromatograph, equipped with a G-1312A binary pump, a G-1328B rheodyne injector, and a G-1365B photodiode array detector using a 25 cm9.4 mm i.d. Zorbax ODS semi-preparative column (Agilent Technologies, Palo Alto, CA, USA). HPLC grade acetonitrile (CH3CN), trifluoroacetic acid (TFA), MeOH, and H2O from J.T. Baker (Baker Mallinckrodt, Phillipsburg, NJ) were used for HPLC and LC–MS. TLC was performed on silica gel F254 (Merck) plates, and reagent grade chemicals (Carlo Erba) were used throughout. 3.2. Computational details In order to allow a full exploration of the conformational space of oxy-guttiferone K and guttiferone M, MM/MD calculations at different temperatures (300 K, 500 K, 700 K/10 ns) were performed using the AMBER force field (MacroModel software package).24 All the so obtained structures (in number of 100) were minimized using the Polak–Ribier Conjugate Gradient algorithm (PRCG, 1000 steps, maximum derivative less than 0.05 kcal/mol). For the quantum mechanical calculations, full geometry optimization, at the MPW1PW91/6-31g(d) level, and CD calculations, using TDDFT MPW1PW91/6-31g(d,p) level were performed using the Gaussian03 (version E.01) software package.25 CD computations were performed considering 40 lowest excited states. To simulate the CD curves, a Gaussian band-shape function was applied with the exponential half-width of 0.16 eV. The simulations and the comparisons with the experimental were performed with SpecDisc.26 3.3. Plant material G. cambogia L. fruits were collected in Ceylon in April 2006. Samples of G. cambogia were identified by Prof. Vincenzo De Feo, Dipartimento di Scienze Farmaceutiche, Universita` di Salerno. A voucher specimen (No.121) has been deposited at this Department. 3.4. Extraction and isolation G. cambogia L. dried fruits (200 g) were extracted with EtOH, (31.5 L) by maceration for 20 days at room temperature. The solvent was removed under reduced pressure to afford 88 g of crude extract. The EtOH extract (24 g) was partitioned with diethyl ether–H2O (1:1) to afford a dried diethyl ether extract (5.5 g). Part of the diethyl ether extract (120 mg) was chromatographed by semi-preparative HPLC/DAD on Zorbax ODS (injections 10 mg/ 100 mL) using H2O/0.1% TFA as eluent A and CH3CN/0.1% TFA as eluent B as mobile phase to afford compounds 1 (2.2 mg, tR¼18.4 min), 2 (2.5 mg, tR¼19.1 min), 3 (1.8 mg, tR¼19.6 min), 4 (3.8 mg, tR¼17.6 min), 5 (4.5 mg, tR¼33.5 min). The elution program started with a linear gradient of 80% of eluent B to 90% of B in 5 min and remained isocratic for 19 min at a flow rate of 2.000 mL/min, then at 24 min the flow rate was decreased to 1.500 mL/min while the elution remained isocratic for 12 min. Finally the flow rate was increased again to a flow rate of 2.000 mL/min and a linear gradient was performed to 100% B in 24 min. The detection wavelength was 360 nm.

3.4.1. Oxy-guttiferone M (1). Yellow oil; [a]25 D 96.2 (MeOH; c 0.1); IR (KBr) nmax 3440, 2948, 2918, 2837, 1738, 1666, 1607, 1384, 1276, 1254, 1190 cm1; UV (MeOH) lmax (log 3) 230 (4.0), 285 (sh), 360 (3.6) nm; 1H NMR (CD3OD/0.1% TFA, 600 MHz) and 13C NMR (CD3OD/0.1% TFA, 150 MHz) data, see Table 1; ESI-MS m/z 623 [MþNa]þ, HR-MALDIMS m/z [MþNa]þ 623.3357 (calcd for C38H48O6Na, 623.3349). 3.4.2. Oxy-guttiferone K2 (2). Yellow oil; [a]25 D þ12.2 (MeOH; c 0.1); IR (KBr) nmax 3450, 2966, 2922, 2856, 1732, 1672, 1604, 1494, 1384, 1267, 1191 cm1; UV (MeOH) lmax (log 3) 250 (4.0), 280 (sh), 364 (3.6) nm; 1H NMR (CD3OD/0.1% TFA, 600 MHz) and 13C NMR (CD3OD/0.1% TFA, 150 MHz) data, see Table 1; ESI-MS m/z 623 [MþNa]þ, HR-MALDIMS m/z [MþNa]þ 623.3352 (calcd for C38H48O6Na, 623.3349). 3.4.3. Oxy-guttiferone I (3). Yellow oil; [a]25 D þ23.8 (MeOH; c 0.1); IR (KBr) nmax 3450, 2966, 2922, 2856, 1732, 1672, 1604, 1494, 1384, 1292, 1267, 1191 cm1; UV (MeOH) lmax (log 3) 220 (4.0), 275 (sh), 355 (3.6) nm; 1H NMR (CD3OD/0.1% TFA, 600 MHz) and 13C NMR (CD3OD/0.1% TFA, 150 MHz) data, see Table 1; ESI-MS m/z 623 [MþNa]þ, HR-MALDIMS m/z [MþNa]þ 623.3355 (calcd for C38H48O6Na, 623.3349). Acknowledgements G.B. thanks Dr. Torsten Bruhn for kindly providing SpecDisc software and suggestions on its usage. References and notes 1. Das, D.; Bandyopadhyay, D.; Bhattachariee, M.; Banerjee, R. K. Free Radical Biol. Med. 1997, 23, 8–18. 2. Das, D.; Bandyopadhyay, D.; Banerjee, R. K. Free Radical Biol. Med. 1998, 24, 460–469. 3. Ciochina, R.; Grossman, R. B. Chem. Rev. 2006, 106, 3963–3986. 4. Iinuma, M.; Tosa, H.; Tanaka, T.; Kanamaru, S.; Asai, F.; Kobayashi, Y.; Miyauchi, K.; Shimano, R. Biol. Pharm. Bull. 1996, 19, 311–314. 5. Tosa, H.; Iinuma, M.; Tanaka, T.; Nozaki, H.; Ikeda, S.; Tsutsui, K.; Yamada, M.; Fujimori, S. Chem. Pharm. Bull. 1997, 45, 418–420. 6. Pan, M. H.; Chang, W. L.; Lin-Shiau, S. Y.; Ho, C. T.; Lin, J. K. J. Agric. Food Chem. 2001, 49, 1464–1474. 7. Tanaka, T.; Kohno, H.; Shimada, A.; Koshimizu, K.; Ohigashi, H. Carcinogenesis 2000, 21, 1183–1189. 8. Yoshida, K.; Tanaka, T.; Hirose, Y.; Yamaguchi, F.; Kohno, H.; Toida, M.; Hara, A.; Sugie, S.; Shibata, T.; Mori, H. Cancer Lett. 2005, 221, 29–39. 9. Balasubramanyam, K.; Altaf, M.; Varier, R. A.; Swaminathan, V.; Ravidran, A.; Sadhale, P. P.; Kundu, T. K. J. Biol. Chem. 2004, 279, 33716–33726. 10. Liao, C.-H.; Sang, S.; Ho, C.-T.; Lin, J.-K. J. Cell. Biochem. 2005, 96, 155–169. 11. Gustafson, K. R.; Blunt, J. W.; Munro, H. G. M.; Fuller, R. W.; McKee, C. T.; Cardellina, J. H.; McMahon, J. B.; Cragg, G. M.; Boyd, M. R. Tetrahedron 1992, 48, 10093–10102. 12. Fuller, R. W.; Blunt, J. W.; Boswell, J. L.; Cardellina, J. H.; Boyd, M. R. J. Nat. Prod. 1999, 62, 130–132. 13. Herath, K.; Jayasuriya, H.; Ondeyka, J. G.; Guan, Z. Q.; Borris, R. P.; Stijfhoorn, E.; Stevenson, D.; Wang, J. H.; Sharma, N.; MacNaul, K.; Menke, J. G.; Ali, A.; Schulman, M. J.; Singh, S. B. J. Nat. Prod. 2005, 68, 617–619. 14. Masullo, M.; Bassarello, C.; Suzuki, H.; Pizza, C.; Piacente, S. J. Agric. Food Chem. 2008, 56, 5205–5210. 15. Sang, S.; Liao, C. H.; Pan, M. H.; Rosen, R. T.; Lin-Shiau, S. Y.; Lin, J. K.; Ho, C. T. Tetrahedron 2002, 58, 10095–10102. 16. Krishnamurthy, N.; Lewis, Y. S.; Ravindranath, B. Tetrahedron Lett. 1981, 22, 793–796. 17. Martins, F. T.; Cruz, J. W., Jr.; Derogis, P. B. M. C.; dos Santos, M. H.; Veloso, M. P.; Ellena, J.; Doriguetto, A. C. J. Brazil. Chem. Soc. 2007, 18, 1515–1523. 18. Stephens, P. J.; Devlin, F. J.; Pan, J.-J. Chirality 2008, 20, 643–663. 19. Stephens, P. J.; McCann, D. M.; Butkus, E.; Stoncius, S.; Cheeseman, J. R.; Frisch, M. J. J. Org. Chem. 2004, 69, 1948–1958. 20. Bifulco, G.; Dambruoso, P.; Gomez-Paloma, L.; Riccio, R. Chem. Rev. 2007, 107, 3744–3779. 21. Bringmann, G.; Bruhn, T.; Maksimenka, K.; Hemberger, Y. Eur. J. Org. Chem. 2009, 2717–2727. 22. Bringmann, G.; Gulder, T. A. M.; Reichert, M.; Gulder, T. Chirality 2008, 20, 628–642. 23. Nilar; Nguyen, L.-H. D.; Venkatraman, G.; Sim, K.-Y.; Harrison, L. J. Phytochemistry 2005, 66, 1718–1723.

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