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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Org Biomol Chem. Author manuscript; available in PMC 2010 August 25.
Published in final edited form as:
PMCID: PMC2927871
NIHMSID: NIHMS228265

Evaluation of the pharmacophoric motif of the caged Garcinia xanthones

Abstract

The combination of unique structure and potent bioactivity exhibited by several family members of the caged Garcinia xanthones, led us to evaluate their pharmacophore. We have developed a Pd(0)-catalyzed method for the reverse prenylation of catechols that, together with a Claisen/Diels–Alder reaction cascade, provides rapid and efficient access to various caged analogues. Evaluation of the growth inhibitory activity of these compounds leads to the conclusion that the intact ABC ring system containing the C-ring caged structure is essential to the bioactivity. Studies with cluvenone (7) also showed that these compounds induce apoptosis and exhibit significant cytotoxicity in multidrug-resistant leukemia cells. As such, the caged Garcinia xanthone motif represents a new and potent pharmacophore.

Introduction

The tropical trees of the genus Garcinia, found in lowland rainforests of Southeast Asia, are widely known for their use in folk medicines.1 Efforts to identify the bioactive ingredients from these plants have yielded a family of natural products structurally characterized by a xanthone backbone in which the C ring has been converted into a caged tricyclic structure. Plant-specific substitutions and oxidations of this motif produce several subfamilies, such as the morellins (1, 2),2 the gaudichaudiones (3, 4)3 and the gambogins (5, 6)4 (Fig. 1).

Fig. 1
Chemical structures of selected caged Garcinia xanthones.

The ability of several caged Garcinia xanthones to selectively inhibit tumor cell proliferation and exhibit potent cytotoxicity at low µM concentrations has been well documented.5 Moreover, gaudichaudione A (3) and gambogic acid (5) displayed strong growth inhibitory activities against both parentalmurine leukemic P388 and P388/doxorubicin-resistant cell lines, suggesting that they are not subject to the multidrug-resistance mechanisms that are typical of several relapsed cancers.6 In addition, 5 displays antitumor activity in animal models and has an appropriate therapeutic window for clinical applications as an anticancer agent.7

A recent study reported that gambogic acid binds to the Bcl-2 family of proteins resulting in apoptosis presumably by blocking the antiapoptotic activity of these proteins.8 Related mode-of-action studies have suggested that this compound binds to the transferrin receptor 1 and that this binding correlates with the induction of apoptosis.9 Structure–activity relationship studies have proposed that the caged motif plays an essential role in the cytotoxicity.10

Inspired by the therapeutic potential of the caged Garcinia xanthones, we developed a chemical strategy that allows access to these natural products and related analogues, such as cluvenone (7).11,12 This strategy relies on a biomimetic Claisen/Diels–Alder/Claisen reaction cascade that produces the caged motif from a reverse prenylated xanthone.13,14 Moreover, we have found that cluvenone (7) maintains the activity exhibited by the more structurally complex natural products of this family.12 We have also shown that this compound is equally cytotoxic to HL-60 cells and the multidrug-resistant clone, HL-60/ADR, at low µM concentrations, attesting to the pharmacologically promising caged Garcinia xanthone motif. Herein, we report our studies on the evaluation of the pharmacophoric motif of these compounds. We also present a new Pd(0)-catalyzed method for the reverse prenylation (installation of 1,1-dimethyl-2-propenyl units) of catechols, the synthetic precursors of the caged scaffolds. This reaction led to a synthesis of several caged Garcinia analogues and to an optimized synthesis of cluvenone (7).

Results and discussion

Synthesis of BC and C ring caged analogues

Our initial studies aimed to produce analogues of the caged Garcinia xanthones lacking the A ring. With this in mind, commercially available 2,3,4-trihydroxybenzoic acid (8) was treated with acetone in the presence of TFA/TFAA (Scheme 1). To minimize formation of diprotected products, the reaction was performed at 0 °C and produced dioxanone 9 in 31% yield together with starting material 8 (60% yield). Initial efforts to convert 9 to 11 were based on a previously reported two-step sequence that involves propargylation using 2-chloro-2-methyl butyne followed by Lindlar reduction of the resulting alkynes.13 However, this two-step process proved to be tedious and difficult to streamline. To overcome this problem we decided to develop an alternative method for the one-step introduction of the 1,1-dimethyl-2-propenyl unit (reverse prenyl group) to a catechol motif. Support for this reaction came from a report on the reverse prenylation of a substituted phenol using 1,1-dimethylpropenyl isobutyl carbonate (10a) under Pd(0) catalysis.15 Using 1,1-dimethylpropenyl isobutyl carbonate (10a) we obtained the desired compound 11 (62–69% yield) together with significant amounts of isobutyl addition products (5–10%). To minimize formation of the side-products we tested the allylation reaction with 1,1-dimethylpropenyl t-butyl carbonate (10b). In this case we observed the formation of 11 as the only product (94% isolated yield). Similar yields (90–92%) were obtained using the unexplored bis(1,1-dimethylpropenyl)carbonate (10c).16 We also evaluated this reaction under Rh(I) catalysis but did not observe the formation of any prenylation product.17

Scheme 1
Reagents and conditions: (a) 6.0 equiv. (CH3)2CO, 20 equiv. TFA, 10 equiv. TFAA, 19 h, 0 °C, 31% of 9, 60% RSM; (b) 10 equiv. t-butyl 2-methylbut-3-en-2-yl carbonate (10b), 10 mol% Pd(PPh3)4, THF, 20 min, 5 °C, 94%; (c) DMF, 1 h, 120 °C, ...

Heating of 11 in DMF (120 °C, 1 h) gave rise to two caged compounds 15 and 16 in 68% and 15% yields, respectively (Scheme 1). The structure and relative stereochemistry of these compounds was unambiguously confirmed via a single-crystal X-ray analysis.18 Under these conditions we also observed the formation of phenol 14 arising from a Claisen rearrangement of 11 (10% yield). The formation of compounds 15 and 16 can be explained by considering an intramolecular Diels–Alder cycloaddition of intermediates 12 and 13 respectively, that have been formed via a Claisen rearrangement of 11. The observed site-selectivity of this reaction cascade (C5 versus C6 allylation) parallels our previous observations and favors the formation of the regular caged structure 15 versus the neo isomer 16.12,13 It is worth noting that, upon additional heating at 120 °C, phenol 14 produced caged compounds 15 and 16, supporting the reversibility of the Claisen rearrangement.19,20

Deprotection of the acetonide unit of compound 15 can open the B ring, producing C-ring caged analogues. To this end, exposure of 15 to 10% aqueous Me4NOH in MeOH provided the optimum saponification conditions and produced the desired β-hydroxy acid 17 in quantitative yield (Scheme 2). This compound was converted to amides 18–20 in good yields using standard amide coupling protocols.

Scheme 2
Reagents and conditions: (a) excess 10% NMe4OH(aq), MeOH, 24 h, 25 °C, 100%; (b) 2.0 equiv. DIPEA, 1.2 equiv. HATU, CH2Cl2, 24 h, 25 °C, 18: 54%, 19: 59%, 20: 68%.

In a similar manner, reaction of pyrrogallol (21) with acrylonitrile formed nitrile 22 (Scheme 3). Reflux of 22 in the presence of H2SO4 (50% aq.) produced dihydroxychromanone 23 (16% combined yield).21 The Pd(0)-catalyzed reverse prenylation of 23 with 10b formed compound 24 (89% yield) which upon heating gave rise to caged motif 26 in 91% yield. Single crystal X-ray analysis of 26 confirmed its chemical structure.18 In this case we did not detect the formation of the neo caged structure.

Scheme 3
Reagents and conditions: (a) 3.4 equiv. acrylonitrile, 0.3 equiv. NaOMe, 7 h, reflux, 76 °C, 34%; (b) excess 50% (w/w) H2SO4 (aq), 3 h, reflux, 100 °C, 48%; (c) 10 equiv. t-butyl 2-methylbut-3-en-2-yl carbonate (10b), 10 mol% Pd(PPh3) ...

Improved synthesis of cluvenone and synthesis of related allylic oxidation products

An improved synthesis of cluvenone (7), featuring the Pd(0)-catalyzed reverse prenylation reaction, is shown in Scheme 4. The tricyclic xanthone 31 was prepared in two steps: (a) a Friedel–Crafts acylation of pyrrogallol (29) with 2-fluorobenzoyl chloride (28) in the presence of AlCl3 and (b) a base-induced cyclization of the resulting benzophenone 30 (2 steps, 34% combined yield). Pd(0)-catalyzed reverse prenylation of 31 using 1,1-dimethylpropenyl t-butyl carbonate (10b) gave rise to compound 32 in quantitative yield. Importantly, during scale-up of this reaction to gram amounts we were able to decrease the amount of the Pd(0) to 3 mol% and the reaction time to 10 min at 25 °C. The heat-induced Claisen/Diels–Alder reaction cascade furnished cluvenone (7) in 81% yield, along with small amounts of the neo caged xanthone 33 (14% yield).

Scheme 4
Reagents and conditions: (a) 1.2 equiv. ClCOCOCl (2.0 M in DCM), DCM, DMF (cat.), 1.5 h, 0 to 25 °C, 87%; (b) 2.0 equiv. 29, 2.9 equiv. AlCl3, chloroform, DCM, 12 h, 25 °C; then 4 h, reflux, 60 °C, 45%; (c) 1.5 equiv. Na2CO3, DMF, ...

With compound 7 in hand we explored an allylic oxidation reaction (Scheme 4). Treatment of 7 with SeO2 and t-BuOOH produced aldehyde 34 as the main product, isolated in 57% yield, along with alcohol 35 (21%). The latter compound was converted to 34 via PCC oxidation in 95% yield. However, all attempts to oxidize aldehyde 34 to the corresponding acid met with failure. In all these cases the characteristic signal corresponding to the C10 proton disappeared, indicating a conjugate addition reaction of the enone bond. Under relatively mild oxidation conditions (NaClO2) we were able to isolate epoxide 36 in 70% yield, the structure of which was confirmed via a single-crystal X-ray analysis.18 This observation supports the expected reactivity of the C9–C10 enone motif as a conjugate electrophile.

Synthesis of caged Garcinia xanthone analogues modified at the C9–C10 enone bond

It has been suggested that the C9–C10 double bond of the caged Garcinia xanthones plays an essential role in the bioactivity of these molecules.10a For instance, conjugate reduction of the enone motif has yielded compounds that have reduced cytotoxicity. This has been tentatively attributed to their decreased ability to act as conjugate electrophiles. However, the C9–C10 conjugate reduction affects the chemical structure of the caged xanthone motif, which in turn could be responsible for the lack of activity. To further test this hypothesis, we sought to evaluate the bioactivity of the C10 methylated analogue 41. This compound was prepared as shown in Scheme 5. Commercially available 1,2,3-trimethoxy-5-methylbenzene (37) was demethylated with excess BBr3 to form polyphenol 38 in 59% yield.22 Friedel–Crafts acylation of 38 with 2-fluorobenzoyl chloride (28) in the presence of AlCl3 followed by Na2CO3-induced cyclization of the resulting benzophenone produced xanthone 39 in 69% combined yield. The Pd(0)-catalyzed reverse prenylation with carbonate 10b gave rise to compound 40 which, upon heating, underwent the Claisen/Diels–Alder reaction cascade to form caged xanthone 41 in 85% yield (Scheme 5).

Scheme 5
Reagents and conditions: (a) excess BBr3,DCM, 3 h, 0 to 25 °C, 59%; (b) 1.5 equiv. 28, 2.0 equiv. AlCl3, chloroform, DCM, 1.5 h, 25 °C; then 6 h, 60 °C; (c) 1.5 equiv. Na2CO3, DMF, 69% (over two steps); (d) 10.0 equiv. t-butyl ...

We have also treated cluvenone (7) with piperidine and MeOH under basic conditions. These reactions led to the formation of the conjugate addition products 42 and 43 which were isolated in 86% and 41% yield, respectively (Scheme 6). The chemical structure of these compounds was determined via a single crystal X-ray analysis18 and indicated that the conjugate addition proceeded in a syn fashion across the C9–C10 enone bond.

Scheme 6
Reagents and conditions: (a) 4.0 equiv. piperidine, DCM, 6 h, 60 °C, 86%; (b) MeOH, 3 d, 65 °C, 41%.

Synthesis of amide analogues of gambogic acid

To evaluate the bioactivity of the carboxylic acid functionality of gambogic acid (5) we prepared the amide derivatives 44–46. These compounds contain affinity and fluorescent probes and can be used for studies related to receptor binding assays and subcellular localization of the caged Garcinia xanthones. The amide coupling reaction proceeded in high yields using the DIPEA and HATU protocol (Scheme 7).

Scheme 7
Reagents and conditions: (a) 2.0 equiv. DIPEA, 1.2 equiv. HATU, CH2Cl2, 24 h, 25 °C, 44: 67%, 45: 87%, 46: 77%.

Cell proliferation studies

The ability of the synthesized caged Garcinia xanthones to inhibit cancer cell growth was evaluated in a multidrug-resistant promyelocytic leukemia cell line, HL-60, using a 3H-thymidine incorporation assay. Cells were incubated with increasing concentrations of the compounds for 48 h, and then pulsed with 3H-thymidine for 6 h. Gambogic acid (5) and cluvenone (7) were the most active among all the compounds tested and exhibited IC50 values of 0.5 and 0.4 µM respectively (Table 1). Similar activity was observed for the amide analogues of gambogic acid (compounds 44, 45 and 46, entries 18–20 respectively) as well as for the oxidized analogues of cluvenone (compounds 34 and 35). Gambogin (6) also exhibited a low µM activity. These results suggest that: (a) the dihydropyran motif of 5 and 6 is not needed for the bioactivity; (b) the carboxylic acid of 5 can be functionalized without loss of function and (c) oxidation and derivatization of the prenyl group of these compounds is well tolerated during binding to the putative receptor. In contrast, the intact ABC caged ring structure is important for the bioactivity. For instance, compounds 15, 16 and 17 induce less than 10% growth inhibition at up to 10 µM concentrations. Similarly, the C-ring amide analogues 18–20 have IC50 values greater than 20 µM, while compound 26, which lacks the A ring, has an IC50 value of 10 µM. Compound 33, containing the neo caged structure, has a low micromolar activity (IC50 value of 1.3 µM) but is about 3 times less potent than cluvenone and related compounds with the regular caged motif. On the other hand, substitution of the C9–C10 enone functionality decreases substantially the bioactivity. For instance, compound 41 is almost 10 times less active than cluvenone (7), while compounds 36, 42, and 43 are about 5 times less active than 7. A similar decrease in cytotoxicity has been reported for gambogoic acid, the conjugate addition product of 5 with methanol.23 These results demonstrate the significance of the C9–C10 enone functionality for the bioactivity of these compounds. This may be due to its reactivity as a conjugate electrophile.

Table 1
Inhibition of cell proliferation by caged Garcinia xanthones and analogues in multi-drug resistant promyelocytic leukemia cells

Apoptosis studies

To determine whether the mechanism of cytotoxicity of these compounds involves the induction of apoptosis, a cell death detection ELISA which measures histone-associated DNA fragments was performed. These studies were performed with cluvenone (7) and are shown in Fig. 2. Compound 7 induced apoptosis, after 7 h of treatment of HL-60 and HL-60/ADR cells, in a dose-dependent manner with EC50 values of 0.25 and 0.32 µM respectively. These results are comparable to the apoptotic effect of gambogic acid and related caged Garcinia natural products. Specifically, the EC50 values of 5 in human breast cancer cells T47D, human colon cancer cells HCT116, and hepatocellular carcinoma cancer cells SNU398 are reported to be about 0.7 µM.10b More importantly, the similar EC50 values observed for cluvenone (7) in the HL-60 and HL-60/ADR cells parallels our previous observations12 and confirms that its cytotoxicity is not affected by the expression of P-glycoprotein which renders the HL-60/ADR cell lines multidrug-resistant.24

Fig. 2
Induction of apoptosis by cluvenone (7) in promyelocytic leukemia cells.

Apoptosis induced in HL60/ADR cells by cluvenone (7) was also visualized by fluorescence microscopy after staining with Alexa Fluor® 488 annexin V and propidium iodide (PI) (Fig. 3). The green-fluorescent Alexa Fluor® 488 annexin V detects the externalization of phosphatidylserine, a hallmark of apoptosis (Fig. 3, middle column).25 The red-fluorescent propidium iodide stains DNA during advanced stages of apoptosis and necrosis (Fig. 3, right column). Cells in the left column of Fig. 3 have been visualized by differential interference contrast (DIC) microscopy. While untreated live cells show little or no fluorescence (Fig. 3, top row), cluvenone-treated cells undergoing early stage apoptosis show only green fluorescence after staining with both probes (Fig. 3, middle row). In the middle row is also evident the membrane blebbing, which is characteristic of apoptosis.25 Cluvenone-treated cells undergoing late stage apoptosis, at which point DNA becomes accessible to staining by PI, display both green and red fluorescence (Fig. 3, bottom row). In this row the chromatin fragmentation is also evident.

Fig. 3
Induction of apoptosis in HL-60/ADR cells by cluvenone (7) visualized by differential interference contrast microscopy (left column) and fluorescence microscopy (middle and right columns). Control untreated cells are shown in the top row. Treated cells ...

Conclusions

We present herein a study aiming to identify the pharmacophoric motif of the caged Garcinia xanthones. Our results indicate that the minimum bioactive motif of these compounds is represented by the intact ABC ring containing the C-ring caged structure. Structural changes to this motif result in substantial loss of activity. The C9–C10 enone functionality is also important to the activity, while the C5 prenyl group can be oxidized and functionalized without loss of bioactivity. In fact, this site could be used for modifications that will improve the solubility and pharmacology of these compounds. We have also developed a method for the reverse prenylation (installation of 1,1-dimethyl-2-propenyl units) of catechols. This reaction proceeds in excellent yield under Pd(0)-catalysis using 1,1-dimethylpropenyl t-butyl carbonate (10b) or bis(1,1-dimethylpropenyl)carbonate (10c) as the prenylation reagents. Application of this reaction led to an improved synthesis of lead analogue cluvenone (7). In this study, we have also demonstrated that cluvenone induces cell death via apoptosis and has similar cytotoxicity in multidrug-resistant and sensitive leukemia cells. These results support our previous findings on the pharmacological potential of the caged Garcinia xanthones, enhance our understanding of the structure–activity relationship, and pave the way for the preparation of therapeutically relevant agents.

Experimental

General notes

Gambogic acid (5), Pd(PPh3)4, and 2-fluorobenzoic acid (27) were purchased from Gaia Chemical Corporation (Gaylordsville, CT), Strem Chemicals, Inc. (Newburyport, MA), and TCI America (Portland, OR), respectively. Biotin ethylenediamine hydrobromide and BODIPY FL EDA were purchased from Invitrogen (Carlsbad, CA). The rest of the reagents were obtained (Aldrich, Acros) at highest commercial quality and used without further purification except where noted. Air- and moisture-sensitive liquids and solutions were transferred via syringe or stainless steel cannula. Organic solutions were concentrated by rotary evaporation below 45 °C at approximately 20 mmHg. All non-aqueous reactions were carried out under anhydrous conditions, i.e. using flame-dried glassware, under an argon atmosphere and in dry, freshly distilled solvents, unless otherwise noted. Dimethylformamide (DMF) and quinoline were distilled from calcium hydride under reduced pressure (20 mmHg) and stored over 4 Å molecular sieves until needed. Yields refer to chromatographically and spectroscopically (1H NMR, 13C NMR) homogeneous materials, unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-254) and visualized under UV light and/or developed by dipping in solutions of 10% ethanolic phosphomolybdic acid (PMA) or p-anisaldehyde and applying heat. E. Merck silica gel (60, particle size 0.040–0.063 mm) was used for flash chromatography. Preparative thin-layer chromatography separations were carried out on 0.25 or 0.50 mm E. Merck silica gel plates (60F-254). NMR spectra were recorded on Varian Mercury 400 and/or Unity 500 MHz instruments and calibrated using the residual undeuterated solvent as an internal reference. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. High resolution mass spectra (HRMS) were recorded on aVG7070 HS mass spectrometer under chemical ionization (CI) conditions or on a VG ZAB-ZSE mass spectrometer under fast atom bombardment (FAB) conditions. X-ray data were recorded on a Bruker SMART APEX 3 kW Sealed Tube X-ray diffraction system.

2,2-Dimethyl-7,8-bis(2-methylbut-3-en-2-yloxy)-4H-benzo[d]-[1,3]dioxin-4-one 11

To a 25 mL round-bottomed flask was added acetonide 9 (95 mg, 0.45 mmol) followed by THF (0.65 mL). The reaction vessel was degassed by argon and was placed in an ice bath. To the clear homogeneous solution was added tert-butyl 2-methylbut-3-en-2-yl carbonate 10b (0.89 mL, 4.50 mmol) via syringe, followed by Pd(PPh3)4 (52.0 mg, 45.0 µmol). The reaction vessel was stirred under argon at 5 °C for 20 min. The onset of a blue suspension indicated the formation of the desired product 11. The solvent was removed by rotary evaporation and the crude material was purified through flash column chromatography (silica, 10% EtOAc-hexane) to yield the desired product 11 (146.5 mg, 94%). 11: colorless oil; Rf = 0.61 (25% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 8.9 Hz, 1H), 6.16 (m, 2H), 5.20 (m, 3H), 5.02 (d, J = 10.9 Hz, 1H), 1.72 (s, 3H), 1.55 (s, 3H), 1.47 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 161.5, 158.6, 152.1, 143.9, 143.8, 135.6, 124.5, 114.3, 113.7, 113.0, 107.6, 106.4, 106.4, 83.3, 82.1, 27.4, 27.1, 26.1; HRMS calc. for C20H26O5 (M + H)+ 369.1672, found 369.1674.

Caged bicycle 15 and neo-caged bicycle 16

Alkene 11 (99 mg, 0.28 mmol) was dissolved in dry DMF (1.8 mL) and the solution was stirred under argon at 120 °C. After 1 hour, the reaction mixture was concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica, 10–17% EtOAc-hexane) to yield the caged product 15 (67 mg, 68%), neo-caged product 16 (15 mg, 15%), and compound 14 (10 mg, 10%), respectively. Caged product 15: white solid; Rf = 0.10 (25% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 6.9 Hz, 1H), 4.41 (m, 1H), 3.42 (t, J = 4.3 Hz, 1H), 2.72 (dd, J = 13.8, 10.4 Hz, 1H), 2.63 (m, 1H), 2.50 (d, J = 9.7 Hz, 1H), 2.31 (dd, J = 13.6, 4.7 Hz, 1H), 1.69 (s, 3H), 1.67 (s, 3H), 1.62 (s, 3H), 1.54 (s, 3H), 1.53 (s, 3H), 1.44 (dd, J = 13.6, 9.3 Hz, 1H), 1.23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 203.2, 159.9, 139.0, 135.6, 128.1, 118.1, 105.1, 85.0, 84.9, 82.9, 48.6, 46.8, 30.2, 29.1, 28.8, 28.5, 28.0, 26.9, 25.9, 18.4; HRMS calc. for C20H26O5 (M + Na)+ 369.1672, found 369.1675. Neo-caged product 16: white solid; Rf = 0.30 (25% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.0 Hz, 1H), 4.95 (t, J = 7.0 Hz, 1H), 3.63 (dd, J = 7.0, 4.5 Hz, 1H), 2.36–2.25 (m, 4H), 1.69 (s, 3H), 1.68 (s, 3H), 1.64–1.61 (m, 1H), 1.59 (s, 3H), 1.43 (s, 3H), 1.31 (s, 3H), 1.29 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 205.0, 159.2, 140.1, 136.7, 127.7, 117.2, 106.5, 83.6, 81.2, 80.1, 46.0, 45.5, 34.1, 30.7, 30.2, 28.6, 27.8, 27.0, 26.2, 18.2; HRMS calc. for C20H26O5 (M + Na)+ 369.1672, found 369.1686. Phenol 14: colorless oil; Rf = 0.52 (25% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.50 (s, 1H), 6.27 (s, 1H), 6.15 (dd, J = 17.5, 10.8 Hz, 1H), 5.35-5.25 (m, 2H), 5.17 (d, J = 10.9 Hz, 1H), 3.26 (d, J = 7.1 Hz, 1H), 1.74 (s, 3H), 1.72 (s, 6H), 1.69 (s, 3H) 1.48 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 161.6, 156.0, 149.1, 143.3, 133.9, 130.0, 125.6, 123.1, 121.3, 114.6, 106.4, 105.8, 83.6, 27.9, 26.9, 26.1, 18.0; HRMS calc. for C20H26O5 (M + Na)+ 369.1672, found 369.1680.

Carboxylic acid 17

To a 25 mL round-bottomed flask was added caged product 15 (41 mg, 0.12 mmol) followed by methanol (1.5 mL). The flask was placed in an ice bath and the solution was stirred at 0 °C. To the stirring solution was then added 10% NMe4OH (aq) (1.7 mL, 159 mmol) dropwise via syringe. The light yellow reaction mixture was allowed to warm to room temperature and further stirred at 25 °C for another 24 hours. Acetic acid (10 mL) was then added to neutralize the reaction mixture. The reaction mixture was partitioned between ethyl acetate (2 × 25 mL) and water (25 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated by rotary evaporation. The crude material was purified by recrystallization (DCM-hexane) to yield the acid 17 (37 mg, 100%). 17: white solid; Rf = 0.11 (67% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 7.1 Hz, 1H), 4.64 (t, J = 6.4 Hz, 1H), 3.33 (t, J = 5.3Hz, 1H), 2.69 (dd, J = 13.8, 9.8 Hz, 1H), 2.58 (dd, J = 13.8, 5.3 Hz, 1H), 2.28 (d, J = 4.5 Hz, 1H), 2.24 (d, J = 10.0 Hz, 1H), 1.60 (s, 6H), 1.57 (s, 3H), 1.40 (dd, J = 13.4, 9.6 Hz, 1H), 1.22 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 204.7, 168.4, 142.0, 135.5, 130.7, 118.8, 85.0, 84.0, 83.5, 49.5, 47.1, 30.2, 29.2, 28.7, 27.1, 26.1, 18.0; HRMS calc. for C17H22O5 (M + Na)+ 329.1359, found 329.1362.

7,8-Bis(2-methylbut-3-en-2-yloxy)chroman-4-one 24

To a 25 mL round-bottomed flask was added 7,8-dihydroxychroman-4-one 23 (50 mg, 0.28 mmol) followed by dry THF (1.5 mL). The flask was degassed by argon and was placed in an ice water bath. To the yellow homogeneous solution was added tert-butyl 2-methylbut-3-en-2-yl carbonate 10b (522 mg, 2.80 mmol), via syringe, followed by Pd(PPh3)4 (32 mg, 0.028 mmol). The reaction vessel was stirred under argon at 5 °C for 2 hours. The onset of a yellow suspension indicated the formation of the alkene 24. The solvent was removed by rotary evaporation and the crude material was purified through flash column chromatography (silica, 30–40% EtOAc-hexane) to yield 7,8-bis(2-methylbut-3-en-2-yloxy)choman-4-one 24 (79 mg, 89%). 24: yellow oil; Rf = 0.52 (30 % EtOAC-hexane); 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 9.0 Hz, 1H), 6.72 (d, J = 9.0, 1H), 6.19 (dd, J = 17.4, 10.6 Hz, 1H), 6.12 (dd, J = 17.6, 10.9 Hz, 1H), 5.13 (m, 3H), 4.98 (dd, J = 10.9, 1.1 Hz, 1H), 4.47 (t, J = 6.5 Hz, 2H), 2.71 (t, J = 6.6 Hz, 2H), 1.51 (s, 6H), 1.47 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 191.2, 157.8, 143.7, 135.8, 121.7, 116.7, 113.8, 113.6, 112.5, 82.8, 81.8, 67.1, 37.4, 27.1, 26.7; HRMS calc. for C19H24O4 (M + Na)+ 339.1567, found 339.1569.

Caged chromanone 26

A solution of compound 24 (36 mg, 0.11 mmol) in DMF (1.5 mL) was heated at 120 °C for 1.5 hours. The onset of a brown color indicated the formation of the caged xanthone 26. The reaction mixture was then cooled to 25 °C and the solvent was removed by rotary evaporation. The crude material was purified through flash column chromatography (silica, 50–55% EtOAc-hexane) to yield the caged product 26 (33 mg, 91%). 26: white solid; Rf = 0.21 (30% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.25 (d, J = 6.6 Hz, 1H), 4.41 (m, 1H), 4.17 (ddd, J = 12.1, 6.5, 1.4 Hz, 1H), 3.94 (dt, J = 12.3, 2.9 Hz, 1H), 3.34 (m, 1H), 2.63 (d, J = 8.6 Hz, 1H), 2.50 (dd, J = 12.4, 6.5 Hz, 1H), 2.42 (dd, J = 2.9, 1.4 Hz, 1H), 2.37 (m, 1H), 2.31 (dd, J = 13.6, 4.5 Hz, 1H), 1.59 (s, 3H), 1.53 (s, 3H), 1.48 (s, 3H), 1.32 (m, 1H), 1.23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 203.9, 192.0, 136.9, 135.4, 133.8, 119.2, 87.4, 84.2, 82.9, 60.0, 46.2, 44.5, 38.1, 30.1, 28.8, 27.7, 25.5, 17.9; HRMS calc. for C19H24O4 (M + Na)+ 339.1567, found 339.1571.

3,4-Dihydroxy-9H-xanthen-9-one 31

To a clean, dried 250 mL round-bottomed flask was added 2-fluorobenzoic acid 27 (5.09 g, 36.3 mmol). The flask containing 2-fluorobenzoic acid 27 and a magnetic stir bar was placed under high vacuum for about 10 min. The flask was carefully sealed and DCM (100 mL) was added by using a syringe under argon. The flask was then placed in an ice bath and the reaction mixture was stirred at 0 °C. To the stirring solution of 2-fluorobenzoic acid 27 and DCM was added a solution of oxalyl chloride (2.0 Min dichloromethane, 21.0 mL, 42.0 mmol) dropwise, via syringe, followed by a catalytic amount of DMF. The ice bath was removed and the reaction mixture was stirred at room temperature for 1.5 hours. The solution was concentrated by rotary evaporation under argon to yield a colorless oil, 2-fluorobenzoyl chloride 28 (5.01 g, 87%). To a mixture of pyrogallol 29 (6.48 g, 51.3 mmol), aluminium chloride (14.6 g, 110 mmol), chloroform (80 mL), and DCM (200 mL) in a 1 L round-bottomed flask was added a solution of 2-fluorobenzoyl chloride 28 in DCM (10 mL) dropwise via syringe. The reaction mixture was stirred at room temperature under argon for 12 hours. The reaction vessel was then equipped with a reflux condenser and stirred under argon at 80 °C for another 4 hours. The cooled, red homogeneous solution was acidified with 1 N HCl (300 mL). The reaction mixture was then partitioned between water and ethyl acetate (3 × 200 mL). The aqueous layer was back-extracted with ethyl acetate (2 × 200 mL) until the color of the aqueous layer was almost clear. The combined organic layers were dried over MgSO4, filtered, and concentrated to yield (2-fluorophenyl)(2,3,4-trihydroxy-phenyl)methanone 30 (3.54 g, 45%). To a 500 mL round-bottomed flask containing sodium carbonate (2.27 g, 21.4 mmol) and DMF(100 mL) was added (2-fluorophenyl)(2,3,4-trihydroxyphenyl)methanone 30. The reaction vessel was then equipped with a reflux condenser and stirred under argon at 90 °C for 3.5 hours. The dark reaction mixture was cooled to room temperature and acidified with 1 N HCl (300 mL). The reaction mixture was then partitioned between water and ethyl acetate (3 × 150 mL). The aqueous layer was back extracted with ethyl acetate (5 × 150 mL). The combined brown organic layers were dried over MgSO4, filtered, and concentrated by rotary evaporation. The crude material was purified through flash column chromatography (silica, 50–60% EtOAc-hexane) to yield 3,4-dihydroxy-9H-xanthen-9-one 31 (2.79 g, 86%). 31: pale yellow solid; Rf = 0.42 (90% Et2O-hexane); 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 9.43 (s, 1H), 8.15 (dd, J = 7.9, 1.7 Hz, 1H), 7.83 (ddd, J = 8.6, 7.2, 1.7 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 175.1, 155.3, 151.4, 146.2, 134.6, 132.5, 125.7, 123.8, 120.7, 117.9, 116.4, 114.6, 113.2; HRMS calc. for C13H8O4 (M + H)+ 229.0501, found 229.0509.

3,4-Bis(2-methylbut-3-en-2-yloxy)-9H-xanthen-9-one 32

To a 50 mL round-bottomed flask was added 3,4-dihydroxy-9Hxanthen-9-one 31 (1.0 g, 4.39 mmol) followed by dry THF(15 mL). To the yellow homogeneous solution was added tert-butyl 2-methylbut-3-en-2-yl carbonate 10b (8.18 g, 43.9 mmol), via syringe, followed by Pd(PPh3)4 (0.15 g, 0.13 mmol). The reaction vessel was stirred under argon at 25 °C for 10 minutes. The onset of a yellow suspension indicated the formation of the alkene 32. The solvent was removed by rotary evaporation and the crude material was purified through flash column chromatography (silica, 10–15% EtOAc-hexane) to yield 3,4-bis(2-methylbut-3-en-2-yloxy)-9H-xanthen-9-one 32 (1.59 g, 100%). 32: yellow solid; Rf = 0.67 (30 % EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 8.30 (dd, J = 8.0, 1.7 Hz, 1H), 7.92 (d, J = 9.1 Hz, 1H), 7.68 (ddd, J = 8.6, 7.1, 1.7 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.36 (ddd, J = 8.0, 7.2, 0.9 Hz, 1H), 7.12 (d, J = 8.9 Hz, 1H), 6.28 (dd, J = 17.5, 10.8 Hz, 1H), 6.18 (dd, J = 17.6, 10.9 Hz, 1H), 5.19 (m, 3H), 5.01 (dd, J = 10.9, 1.0 Hz, 1H), 1.58 (s, 6H), 1.56 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 176.8, 156.9, 155.9, 152.4, 143.5, 143.4, 135.7, 134.3, 126.5, 123.7, 121.5, 121.0, 117.8, 117.1, 116.8, 114.1, 113.0, 83.5, 82.1, 27.1, 26.9; HRMS calc. for C23H24O4 (M + H)+ 365.1753, found 365.1740.

Caged xanthones 7 and 33

A solution of compound 31 (350 mg, 0.96 mmol) in DMF (6 mL) was heated at 120 °C for 1.5 hours. The onset of a brown color indicated the formation of the xanthones 33 and 7. The brown reaction mixture was then cooled to room temperature and the solvent was removed by rotary evaporation. The crude material was then purified by column chromatography (silica, 20–30% Et2O-hexane) to yield a mixture of caged products 7 (285 mg, 81%) and 33 (50mg, 14%). 7: white solid; Rf = 0.28 (25% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.93 (dd, J = 8.0 Hz, 1.7 Hz, 1H), 7.51 (ddd, J = 8.9, 7.3, 1.7 Hz, 1H), 7.42 (d, J = 6.9 Hz, 1H), 7.05 (m, 2H), 4.39 (m, 1H), 3.48 (dd, J = 6.7 Hz, 4.6 Hz, 1H), 2.64 (m, 2H), 2.45 (d, J = 9.6 Hz, 1H), 2.33 (dd, J = 13.5 Hz, 4.6 Hz, 1H), 1.71 (s, 3H), 1.29 (m, 1H), 1.29 (s, 6H), 0.89 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 203.0, 176.4, 159.5, 136.1, 134.8, 134.7, 133.7, 126.8, 121.8, 118.9, 118.9, 118.0, 90.2, 84.5, 83.4, 48.7, 46.7, 30.2, 29.0, 25.2, 25.0, 16.6; HRMS calc. for C23H24O4 (M + H)+ 365.1753, found 365.1765. 33: yellow solid; Rf = 0.34 (25% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.91 (dd, J = 7.9, 1.7 Hz, 1H), 7.54 (m, 1H), 7.25 (d, J = 7.1 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H), 7.05 (ddd, J = 8.0, 7.3, 1.0 Hz, 1H), 5.02 (m, 1H), 3.76 (dd, J = 6.9, 4.6 Hz, 1H), 2.50 (m, 2H), 2.13 (m, 2H), 1.87 (dd, J = 13.2, 10.0 Hz, 1H), 1.71 (s, 3H), 1.59 (s, 3H), 1.38 (s, 3H), 1.34 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 199.7, 175.4, 160.2, 136.5, 136.1, 135.9, 134.9, 127.0, 122.0, 119.2, 118.3, 117.3, 84.1, 83.7, 78.8, 44.8, 42.1, 33.1, 30.2, 29.7, 26.8, 26.0, 18.2; HRMS calc. for C23H24O4 (M + H)+ 365.1753, found 365.1766.

1-Methyl-3,4-bis(2-methylbut-3-en-2-yloxy)-9H-xanthen-9-one 40

To a 25 mL round-bottomed flask was added methyl xanthone 39 (46 mg, 0.19 mmol) followed by dry THF (1.5 mL). The flask was degassed by argon and was placed in an ice water bath. To the yellow homogeneous solution was added tert-butyl 2-methylbut-3-en-2-yl carbonate 10b (354 mg, 1.9 mmol), via syringe, followed by Pd(PPh3)4 (22 mg, 0.019 mmol). The reaction vessel was stirred under argon at 5 °C for 2 hours. The onset of a yellow suspension indicated the formation of the desired product 40. The solvent was removed by rotary evaporation and the crude material was purified through flash column chromatography (silica, 10–15% EtOAc-hexane) to yield 1-methyl-3,4-bis(2-methylbut-3-en-2-yloxy)-9H - xanthen- 9 – one 40 (55 mg, 76%). 40: yellow oil; Rf = 0.66 (30% EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 8.24 (dd, J = 7.9, 1.5 Hz, 1H), 7.64 (ddd, J = 8.6, 7.2, 1.7 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 6.86 (s, 1H), 6.28 (dd, J = 17.5, 10.9 Hz, 1H), 6.19 (dd, J = 17.6, 10.8 Hz, 1H), 5.18 (m, 3H), 5.01 (dd, J = 10.9, 1.0 Hz, 1H), 2.80 (s, 3H), 1.56 (s, 12H); 13C NMR(100 MHz, CDCl3) δ 178.4, 155.3, 155.0, 153.5, 143.8, 143.6, 136.3, 133.9, 126.5, 123.5, 122.5, 119.1, 117.3, 115.5, 113.9, 112.8, 83.1, 82.0, 27.2, 26.9, 23.5; HRMS calc. for C24H26O4 (M + H)+ 379.1904, found 379.1911.

Caged xanthone 41

A solution of compound 40 (35 mg, 0.092 mmol) in DMF (1.5 mL) was heated at 120 °C under argon for 2.5 hours. The onset of a yellow color indicated the formation of the methyl caged xanthone 41. The reaction mixture was then cooled to room temperature and the solvent was removed by rotary evaporation. The crude material was purified through flash column chromatography (silica, 15–20% EtOAc-hexane) to yield the methyl caged xanthone 41 (30 mg, 85%). 41: white solid; Rf = 0.56 (30 % EtOAc-hexane); 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 7.7 Hz, 1H), 7.47 (m, 1H), 7.03 (m, 2H), 4.42 (t, J = 7.0 Hz, 1H), 3.18 (d, J = 4.4 Hz, 1H), 2.61 (m, 1H), 2.51 (s, 3H), 2.45 (d, J = 9.5 Hz, 1H), 2.28 (dd, J = 13.5, 4.7 Hz, 1H), 1.70 (s, 3H), 1.35 (s, 3H), 1.34 (m, 1H), 1.27 (s, 3H), 0.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 203.6, 179.3, 158.3, 150.7, 135.5, 134.8, 126.8, 121.7, 120.6, 118.3, 117.6, 90.5, 84.8, 83.2, 55.8, 49.2, 30.3, 29.0, 28.6, 25.6, 24.9, 19.7, 16.7; HRMS calc. for C24H26O4 (M + H)+ 379.1904, found 379.1909.

3H-Thymidine incorporation assay

Cells were plated in a 96-well plate at 10–20 × 103 cells/well in RPMI supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% penicillin/streptomycin (complete medium). The caged Garcinia xanthones were added to the cells at increasing concentrations and 0.1% DMSO was added to control cells. Cells were incubated for 48 h and then pulsed with 3H-thymidine for 6 h. Incorporation of 3H-thymidine was determined in a scintillation counter (Beckman Coulter Inc., Fullerton, CA) after cells were washed and deposited onto glass microfiber filters using a cell harvester M-24 (Brandel, Gaithersbur, MD).

Apoptosis assays

ELISA assay

The compounds were dissolved in DMSO and further diluted in complete medium to obtain final concentrations as indicated. HL-60 and HL-60/ADR cells were seeded into each well of a 96-well cell culture plate at 10,000 cells per well and incubated at 37 °C for 7 h with the indicated concentrations of each compound. Control samples were incubated in 0.1% DMSO. Each condition was in triplicate. The proapoptotic effect was detected by using the Cell Death Detection ELISAPLUS kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer instructions. This kit constitutes a photometric enzyme-immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone-associated-DNA-fragments (mono- and oligo-nucleosomes) after induced cell death. The absorption values A (A405nm – A490nm) measured give a quantitative indication of the induced amount of apoptosis.

Fluorescence microscopy of annexin V/PI stained cells

HL-60/ADR cells were plated in a 6-well plate at 1 × 106 cell/ml (4 ml) and treated with 0.5 µM 7 while control cells received 0.1% DMSO. Cells were incubated overnight and then stained with Alexa Fluor 488 annexin V and propidium iodide using the Vybrant Apoptosis Assay Kit (Molecular Probes, Eugene, OR) according to manufacturer’s recommendations. Cells were then viewed on an E800 Nikon (New York City, NY) research microscope equipped with an EXFO (Vanier, Canada) X-cite fluorescent 120 W metal halide illuminator and imaged with a DMX 1200F Nikon fluorescence sensitive digital camera.

Supplementary Material

01

Acknowledgments

Financial support from the National Institutes of Health (CA 133002) is gratefully acknowledged. We also acknowledge partial support to O.C. and W.C. from the Thailand Research Fund for a Royal Golden Jubilee Ph.D. Fellowship (Grant No. PHD/0223/2548) and Center for Petroleum, Petrochemistry and Advanced Materials. We thank the National Science Foundation for instrumentation grants CHE-9709183 and CHE-0741968. We gratefully acknowledge Dr Barbara Davids at UCSD, Department of Pathology, Division of Infectious Diseases, for performing the fluorescence microscopy. We also thank Dr A. Mrse and Dr Y. Su for NMR spectroscopic and mass spectrometric assistance respectively.

Footnotes

Electronic supplementary information (ESI) available: Experimental procedures for compounds 9–10, 18–20, 22–23, 34–36, 38–39 and 42–46. Crystallographic tables for compounds 7, 15–16, 26, 36, 42 and 43. 1H and 13C NMR spectra for compounds 7, 9, 10a, 10b, 10c, 11, 14–24, 26, 31–46. CCDC reference numbers 614936, 737621, 737622, 737623, 737624, 737625 and 737626. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/b913496d

Notes and references

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