Bioactive Limonoids from the Leaves of Azaridachta indica (Neem

Feb 5, 2014 - Shradha M. Patel , Kalyan C. Nagulapalli Venkata , Piyali Bhattacharyya , Gautam Sethi , Anupam Bishayee. Seminars in Cancer Biology 201...
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Bioactive Limonoids from the Leaves of Azaridachta indica (Neem) Maria J. Gualtieri,†,‡ Nicola Malafronte,‡ Antonio Vassallo,§ Alessandra Braca,⊥ Roberta Cotugno,‡ Michele Vasaturo,‡ Nunziatina De Tommasi,*,‡ and Fabrizio Dal Piaz‡ †

Laboratorio de Medicamentos Organicos Sector Campo de Oro, Departamento de Farmacognosia y Medicamentos Organicos, Universidad de Los Andes, detras del HULA, Mérida, 5101, Venezuela ‡ Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy § Dipartimento di Scienze, Università degli Studi della Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy ⊥ Dipartimento di Farmacia, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy S Supporting Information *

ABSTRACT: Eight new limonoids (1−8) and one new phenol glycoside (9), along with six known compounds, were isolated from the leaves of Azaridachta indica. The structures of 1−9 were elucidated on the basis of spectroscopic data analysis. Compounds isolated were assayed for their cytotoxicity against different cancer cell lines. Moreover, their ability to interact with the molecular chaperone Hsp90, affecting its biological activity, was tested.

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Azadirachta indica A. Juss. (Meliaceae), commonly known as “neem”, is indigenous to India. This species is cultivated widely in many tropical areas of the world. Various parts of the neem tree have been used since ancient times for food, as medicine, and as insecticides, and many bioactive constituents including tetranortriterpenoids (limonoids) have been isolated and identified.1 Although all parts of the neem tree are recognized as conferring health benefits, neem leaves have wide-ranging medicinal utility, including immunomodulatory, anti-inflammatory, antimutagenic, anticarcinogenic,2,3 and chemopreventive activities.1 Limonoids are a class of highly oxygenated nortriterpenoids and exhibit a wide range of bioactivities such as insect antifeedant, antibacterial, antifungal, antimalarial, anti-inflammatory, and cytotoxic effects and growth regulatory properties.4,5 Recently the limonoid gedunin, isolated from A. indica, has been found to affect both the in vitro and in vivo enzymatic activity of heat shock protein 90 (Hsp90).6,7 This cytosolic chaperone is involved in the turnover, trafficking, and activity of a large number of oncoproteins.8,9 Recent preclinical and clinical studies have explored the effect of a combination of Hsp90 inhibitors and other anticancer agents in cancer therapy, and in most cases additive or synergic effects were observed.10,11 Therefore, there is a great interest in the development of new anticancer drugs targeting Hsp90. These observations prompted the present investigation of the leaves of A. indica to search for limonoids containing gedunin-like structural features in order to evaluate their anti-Hsp90 efficiency. Eight new limonoids (1−8) and one new phenol glycoside (9), as well as six known compounds, were isolated and characterized. The cytotoxicity of the isolated compounds against different cancer cell lines was assayed, and their ability to interact with the molecular chaperone Hsp90 was also investigated. © 2014 American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISCUSSION

The chloroform and methanol extracts of A. indica leaves were chromatographed by using different techniques to afford nine new (1−9) and six known compounds. Compound 1 showed a quasimolecular ion peak at m/z 507.2359 for [M + Na]+ in the HRESIMS, corresponding to the molecular formula C28H36O7, requiring 11 degrees of unsaturation. The 13C NMR spectrum (Table 1) displayed 28 signals sorted into six methyls, of which one is an acetyl group, four methylenes, nine methines, and nine quaternary carbons, indicating that 1 should have a limonoid skeleton. The 1H NMR spectrum (Table 1) showed a pair of AB doublets at δ 7.34 (H-1) and 5.86 (H-2) with a coupling constant (J = 10.0 Hz), indicating the presence of a 1-en-3-one system,12,13 which was also supported by the 13C NMR data. The 1H NMR spectrum showed also the presence of six methyl singlets, of which one is an acetyl group, in addition to two oxymethines at δ 4.32 (H-6) and 5.39 (H-7) and one trisubstituted olefinic proton at δ 5.43 (H-15). DQF-COSY, 1D-TOCSY, and HSQC experiments allowed the following connectivities: H-1−H-2 for ring A, H-5−H-7 for ring B, H-9−H-12 for ring C, and H-15− H-22 for ring D and the side chain to be established. The crosspeaks in the HMBC spectrum between H-6 and C-4, C-7, C-10; H-7 and C-5, C-9, C-30, CO (δ 172.3); and H-5 and C-4, C-7, C-10, C-28, C-29 were consistent with the presence of an oxymethine group at C-6 and an acetoxy group at C-7. The H-6 and H-7 1H NMR signals were suggestive of two α-oriented Special Issue: Special Issue in Honor of Otto Sticher Received: October 14, 2013 Published: February 5, 2014 596

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126.5, δH 7.34 and 5.86 in 1) and the presence of two additional methylene groups (δC 39.5 and 33.9, δH 2.87, 2.23, 1.76, and 1.79 in 2). The relative configuration of the tetracyclic core of 2 was inferred by its ROE correlations and was the same as that obtained for compound 1. Thus, the structure of 2 was determined to be 24,25,26,27-tetranor-apotirucall-6α-hydroxy7α-acetoxy-14-en-3-one-21,24-anhydride. The molecular formula of compound 3 (C28H36O7) was established by 13C NMR and HRESIMS (m/z 507.2353 for [M + Na]+). Its NMR spectroscopic data (Table 1) suggested that the structure of 3 resembled that of 1, but differed in the nature of the side chain. The DQF-COSY, TOCSY, and HSQC spectra of compound 3 when compared to those of 1 showed the absence of the H-15−H-22 spin system in 3. In the side chain NMR spectra of 3, a double bond (δC 132.7 and 149.5; δH 7.47) and one hydroxymethyne (δC 71.8 and δH 4.91) were observed. A 22-hydroxy-20(21)-ene-21,23-γ-lactone moiety was assigned for the C-17 side chain on the basis of the HMBC cross-peaks between H-17 (δH 2.81) and C-15, C-18, C-21, C-22; H-22 and C-17, C-21; and H-21 and C-17, C-22, C-23.12,16 The relative configuration of 3 was assigned on the basis of ROESY cross-peaks and using 1H NMR coupling constant analysis. The ROESY cross-peaks of Me-19 with Me-29 and Me-30 were diagnostic of the cis relationships of these groups, and the ROE correlations H-5/H-9, H-5/Me-28, and H-9/Me-18 indicated the α-orientation of these groups. ROE observed between Me-18 and H-22 was consistent with a C-22 β-OH substituent. Thus, the structure of 3 was elucidated as 24,25,26,27-tetranor-apotirucall-6α,22-dihydroxy-7α-acetoxy1,14,20(21)-trien-3-one-21,23-olide. Compound 4 was isolated as a white, amorphous solid, with a molecular formula of C28H40O6 as determined by HRESIMS, showing a quasimolecular ion at m/z 495.2719 [M + Na]+. The negative-ion ESIMS showed a major peak at m/z 471 [M − H]− and fragments at m/z 411 [M − H − 60]− and 385 [M − H − 42 − 44]−. The NMR data (Table 1) revealed that the structure of 4 was closely related to that of 2, with the only difference being the C-17 side chain. The main difference was the absence of the carbonyl group at δC 172.5 attributed in 2 to C-21 and the presence in 4 of two additional signals for a hydroxymethylene group at δH 4.56 and 4.00 and δC 73.7 (C-21). Differences were also observed in the chemical shifts of C-17, C-20, C-21, and C-22 (Table 1). All data obtained suggested that a 21,23-lactone ring is present in 4.13,14,17 From the above evidence, 4 was assigned as 24,25,26,27-tetranorapotirucall-6α-hydroxy-7α-acetoxy-14-en-3-one-21,23-olide. Compound 5 (C28H38O7) showed a [M + Na]+ ion at m/z 509.2509. Comparison of the NMR spectroscopic data of 5 with those of 3 (Experimental Section; Tables 1 and 2) showed these to be identical in their side chains, but different in their ring portions. NMR data strongly suggested that the identity of ring A of compound 5 is the same as that described for compound 2 (Experimental Section and Table 1).2,18 Thus, the structure of 5 was elucidated as 24,25,26,27-tetranorapotirucall-6α,22-dihydroxy-7α-acetoxy-14,20(21)-dien-3-one21,23-olide. The molecular formula of compound 6 (C32H40O14) was established by 13C NMR and HRESIMS (m/z 671.2311 for [M + Na]+), indicating 13 degrees of unsaturation. The NMR data (Table 2) showed that five of the elements of unsaturation were present as double bonds: two carbon−carbon, one carboxyl group, and two ester functionalities. Therefore, the molecule was hypothesized as being octacyclic. Analysis of the NMR spectra

oxygen substituents (Table 1). A multiplet at δ 5.43 (H-15) in the 1H NMR spectrum and signals at δC 119.8 (C-15) and 160.0 (C-14) in the 13C NMR spectrum were in agreement with the presence of a Δ14,15 double bond, characteristic of tirucallane terpenoids.14,15 The molecular formula of 1 and its ESIMS fragment at m/z 339 [M − H − 44 − 100]− revealed the presence of a four-carbon side chain. These features together with the H-15−H-22 obtained spin system suggested the presence of a five-membered ring for the side chain of this compound. The relative configuration of 1 was established on the basis of the proton coupling constants and the correlation peaks in the ROESY spectrum. Thus, H-5 showed a correlation peak with Me-28 and H-9, Me-19 with Me-29 and Me-30, H-17 with H-11β, and Me-18 with H-20.2,13 The last connectivity showed that the side chain at C-17 is α-oriented. The configuration of 1 was also deduced on the basis of the high similarities of its NMR data with those of tetranor-apotirucallane derivatives. In light of these data, the structure of 1 was elucidated as 24,25,26,27-tetranor-apotirucall-6α-hydroxy-7αacetoxy-1,14-dien-3-one-21,24-anhydride. The 1H and 13C NMR spectra (Experimental Section and Table 1) of compound 2 (C28H38O7, HRESIMS at m/z 509.2511 [M + Na]+) were very similar to those of 1 except for the absence of signals due to a Δ1,2 double bond (δC 160.1 and 597

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Table 1. NMR Data of Compounds 1−5a 1 position

δH

1 2a 2b 3 4 5 6 7 8 9 10 11a 11b 12a 12b 13 14 15 16a 16b 17 18 19 20 21a 21b 22a 22b 23 28 29 30 CH3CO CH3CO

7.34 d (10.0) 5.86 d (10.0)

2.24 d (11.0) 4.32 dd (11.0, 2.0) 5.39 d (2.0) 2.27 dd (11.5, 3.0) 2.02b 1.74b 2.16b 1.74b

5.43 br m 2.57 br dd (16.0, 12.0) 2.30b 2.84 br t (7.3) 0.99 s 1.16 s 2.28b

2.00b 1.77b 1.42 1.32 1.30 2.02

s s s s

2 δC

δC

160.1 126.5

39.5 33.9

209.0 47.0 50.7 68.0 79.7 44.3 37.8 42.9 17.1

214.0 46.0 52.4 68.0 80.0 44.2 42.5 37.4 17.6

33.6

34.6

48.6 160.0 119.8 34.4

47.2 159.9 120.0 34.4

56.6 21.0 21.3 38.0 172.5

54.5 21.0 17.0 37.8 173.0

34.6

34.4

173.6 20.6 32.2 27.4 21.3 172.3

173.1 19.3 32.3 27.4 21.1 172.0

3 δH 7.38 d (9.7) 5.88 d (9.7)

160.0 126.3

2.25 d (12.3) 4.34 dd (12.3, 3.0) 5.41 br t (3.0) 2.27 dd (11.2, 3.6) 2.00b 1.77 br t (7.0) 2.01b 1.75 br t (7.0)

5.46 br t (2.2) 2.64 br dd (15.0, 11.0) 2.30b 2.81 dd (9.5, 6.5) 0.99 s 1.17 s 7.47 br s 4.91 s

1.41 1.42 1.31 2.02

4 δC

207.2 46.1 50.0 68.0 79.8 44.6 38.1 41.3 17.1 34.4 47.6 158.3 120.5 34.3 52.2 21.5 21.0 132.7 149.5 71.8 175.5 20.5 32.2 27.0 21.1 172.0

s s s s

δH 1.85 m 2.87b 2.26 m

2.24 br d (12.0) 4.19 dd (12.0, 2.9) 5.36 d (2.9) 2.19 dd (12.0, 3.5) 1.77 m 1.64 m 1.75b 1.54 m

5.38 br d (2.2) 2.18 br dd (17.0, 11.0) 2.08 dd (16.6, 8.0) 1.76b 1.11 s 0.85 s 2.79 m 4.56 br t (7.9) 4.00 br t (7.9) 2.54 dd (17.0, 8.71) 2.34b 1.33 s 1.32 s 1.19 s

5 δC

δC

39.0 33.3

39.2 33.6

222.4 48.2 51.7 67.8 79.5 44.6 41.5 38.0 16.7

212.0 47.4 52.3 68.3 79.7 41.6 42.3 37.4 17.1

34.5

34.1

47.2 160.0 120.2 34.7

47.6 159.8 120.0 34.4

59.0 19.8 16.6 38.8 73.7

52.0 21.0 17.0

34.0 180.0 19.2 31.8 25.9

133.0 149.0 71.8 176.0 19.7 32.0 26.5 21.3 172.3

a

Spectra were run in MeOH-d4 at 600 MHz (1H) and 150 MHz (13C). J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments were confirmed by COSY, 1D-TOCSY, HSQC, and HMBC experiments. bOverlapped signals.

inferred by the 1H [δH 7.39 (H-21) and 4.86 (H-22)] and 13C NMR [δC 144.3 (C-20), 148.0 (C-21), 71.1 (C-22), and 176.0 (C-23)] signals.12 The relative configuration at C-22 remained undetermined; however, the structure of 7 was assigned as 17-desfuran-17-(22-hydroxybut-20(21)-ene-21,23-γ-lactone)nimbandiol. The molecular formula of compound 8 was determined as C26H32O9 from its HRESIMS, this compound being an isomer of 7. The 1H and 13C NMR spectroscopic data (Table 2) of 8 resembled those of 7, the side chain at C-17 being the point of structural difference. The NMR spectroscopic data of 8 showed characteristics of 21-hydroxy-21,23-butenolide epimers (ratio 4:1 mixture), instead of signals for a furan ring. In this case, the presence of the major epimer showed two protons that resonated at δH 5.82 (1H, br s, H-22) and 5.38 (br s, H-21) as well as carbon signals at δC 169.0, 98.4, 120.1, and 170.0 ascribed to C-20, C-21, C-22, and C-23, respectively.24 The configuration at C-21 was not determined. Thus, compound 8 was characterized as 17-desfuran-17-(21-hydroxy-20(22)-ene21,23-γ-lactone)nimbandiol. The HRESIMS of compound 9 exhibited a sodiated molecular ion peak at m/z 495.1481 [M + Na]+. Together with the mass

allowed the assignment of most functional groups to the azadirachtin-B core.19−21 Comparison of the NMR data of 6 with those of 3-tigloylazadirachtol indicated that 6 is an azadirachtol derivative.15 In fact, the NMR data of compound 6 were similar to those of 3-tigloylazadirachtol except for few minor differences observed for resonances of the ring A protons and carbons. The presence of a tigloyl group at C-1 in 6 was supported by the chemical shifts of H-3 and H-1 (δH 4.42, and 4.93) and of C-1 and C-3 (δC 70.7 and 66.0) and HMBC correlations between H-3 and C-1, C-5 and between H-1 and C-5, C-9.22,23 Thus, compound 6 was characterized as 1-tigloylazadirachtol. Compound 7 was isolated as a white, amorphous powder. The molecular formula, C26H32O9, was deduced from its HRESIMS (m/z 511.1940 [M + Na]+). Analysis of the NMR data allowed the assignment of most functional groups to a 6-O-desacetylnimbin core.19−21 Comparison of the NMR data of 7 (Table 2) with those of 6-O-desacetylnimbin and 6-desacetylnimbandiol indicated that 7 is a nimbandiol derivative.24 In fact, the NMR data of compound 7 were found to be similar to those of 6-desacetylnimbandiol except for the absence of a furan ring at C-17. The presence of a 22-hydroxybut-20(21)-ene-21,23-γ-lactone ring at C-17 was 598

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Table 2. 1H and 13C NMR Data of Compounds 6−8a 6 δH

position 1 2a 2b 3 4 5 6 7 8 9 10 11a 11b 12 13 14 15 16a 16b 17 18 19a 19b 20 21 22 23 28a 28b 29 30 OCH3 1′ 2′ 3′ 4′ 5′

4.93 2.29 2.20 4.42

ddd (12.0, 3.8, 2.0) br dd (12.0, 3.8) br dd (12.0, 2.0) br t (3.0)

3.47 d (3.0) 4.51 dd (12.5, 2.5) 4.33 d (2.5) 3.50 d (1.3) 4.55 d (1.3)

4.84 1.75 1.26 2.28 1.85 4.38 4.02

d (8.4) ddd (12.0, 5.5, 4.0) br d (12.0) d (5.5) s d (10.0) d (10.0)

5.78 5.01 6.45 4.20 4.05

s d d d d

1.26 s 3.80 s

5.77 m 1.90 s 2.19 s

(3.0) (2.6) (8.5) (8.5)

7 δC 70.7 33.4 66.0 54.6 35.0 75.3 74.0 47.4 44.6 52.7 80.4 178.4 70.0 69.0 78.0 26.6 48.0 19.3 71.6 83.3 108.6 109.1 147.3 74.3 175.4 21.9 55.0 170.0 158.5 116.7 27.0 20.3

δH 5.76 d (10.6) 6.57 d (10.6) 2.61 4.37 dd (11.6, 2.2) 4.02 d (2.2) 2.63 2.99 dd (16.8, 5.8) 2.32 dd (16.8, 3.5)

5.33 2.17 1.99 3.57 1.77 1.25

br t (7.0) m m br d (8.8) s s

8 δC

δH

204.3 124.4 153.0 71.6 50.3 67.7 88.9 49.4 39.6 49.1 34.5 175.4 149.6 134.7 88.5 39.9 49.0 12.4 15.8

5.81d (10.6) 6.59 d (10.6) 2.62 d (11.5) 4.38 dd (11.5, 2.0) 3.98 d (2.0) 2.65 dd (6.0, 3.5) 2.93 dd (16.8, 6.0) 2.30 dd (16.8, 3.5)

5.31 2.65 2.03 3.65 1.79 1.25

br t (7.0) m m br d (8.8) s s

δC 204.3 124.4 152.0 72.0 51.0 67.0 87.9 50.0 38.9 49.6 34.2 175.0 144.9 136.0 86.0 39.6 52.0 13.0 15.8

1.60 s

144.3 148.0 71.1 176.0 22.6

1.61 s

169.0 98.4 120.1 170.0 23.0

1.35 s 3.69 s

16.4 52.0

1.32 s 3.72 s

17.0 52.3

7.39 br s 4.86 s

5.38 br s 5.82 br s

a

Spectra were run in MeOH-d4 at 600 MHz (1H) and 150 MHz (13C). J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments were confirmed by COSY, 1D-TOCSY, HSQC, and HMBC experiments.

spectroscopic data, the 1H and 13C NMR spectra (Experimental Section) revealed a molecular formula of C21H28O12. In the 1H NMR spectrum, a singlet for two aromatic protons at δ 6.92 (2H, s), corresponding to a typical 1,3,4,5-tetrasubstituted aromatic ring, was observed. In addition, the 1H NMR spectrum also showed the signals for two trans olefinic protons at δ 7.66 (1H, d, J = 15.8 Hz) and 6.42 (1H, d, J = 15.8 Hz) and of one aromatic methoxy group at δ 3.92 (6H, s), suggesting the presence of a trans-sinapoyl moiety in compound 9. This was supported by the 13C NMR spectrum, which showed at low field signals for a sinapoyl moiety (Experimental Section). One anomeric proton was also identified in this spectrum, resonating at δ 4.62 (d, J = 7.6 Hz) and correlating with a signal at 100.2 ppm in the HSQC spectrum. Analysis of the chemical shifts, signal multiplicities, values of the coupling constants, and their magnitude in the 1H NMR spectrum, as well as the 13C NMR data, indicated the presence of a glucopyranosyl moiety with a β-configuration at the anomeric carbon. The configuration of the glucopyranosyl moiety was determined to be D by

hydrolysis of 9, trimethylsilylation, and GC analysis. In addition, from the 13C NMR spectrum, four other carbon signals were observed arising from an isobutyroyl moiety. This was substantiated in the 1H NMR spectrum, from two methyl singlets at δ 1.52 (3H, s) and 1.44 (3H, s), which correlated in the HSQC spectrum with 13C NMR signals at 24.6 and 28.3 ppm, respectively. The assignments of all protons and carbons of 9 were based on the results of the 1D-TOCSY, DQF-COSY, HSQC, and HMBC experiments. Finally, the structure of 9 was confirmed by a series of diagnostic HMBC correlations: H-3/ C-1, H-3/C-2, H-3/C-4; H-1′/C-2; H-6′/COO; H-β/C-1″, H-β/C-2″; H-2″/C-5″, H-2″/C-6″, H-2″/C-4″. Compound 9 was therefore identified as isobutyric acid 2-O-(6-O-transsinapoyl)-β-D-glucopyranoside. Six known limonoids, deacetilsalannin (10),25 azadirachtol (11),26 azadirachtolide (12),14 dehydronimonol (13),2 nimonol (14),2 and 1,3-diacetylvilasinin (15),27,28 were identified by analysis of their spectroscopic data and comparison with literature values. 599

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group at C-1 and/or C-3 appears to affect the affinity of this compound class for Hsp90. The low cytotoxicity of 10 and 15 suggests for these compounds a different mode of action from those of the classical anti-Hsp90 agents: indeed, most of described natural and synthetic nontoxic Hsp90 inhibitors interact with a protein region different from the N-terminal domain.6,31,32

The cytotoxicity of compounds 1−15 was assayed by using the HeLa (human epithelioid cervix carcinoma) and PC-3 (human prostate adenocarcinoma) cancer cell lines. In preliminary experiments, cells were exposed to 100 μM of each test compound and 15 μM phenethylisothiocyanate (PEITC) for 48 h. Under these experimental conditions, all the test compounds, except for compounds 1 and 13−15, did not display any significant cell growth inhibition activity (data not shown). Therefore, additional experiments were carried out to further characterize the cytotoxicity of these four compounds. Cells were exposed to different doses of each substance (range 20−100 μM) for 48 h. Compounds 1 and 13−15 showed weak cytotoxicity (IC50 95 ± 11, 74 ± 8, 77 ± 9, 74 ± 6 μM in HeLa and >100, 90 ± 10, 95 ± 11, 80 ± 8 μM in PC-3 cells, respectively). Finally, the interaction of compounds 1−15 with Hsp90 was investigated by a surface plasmon resonance (SPR)-based binding assay;29 the geldanamycin semisynthetic derivative 17-(allylamino)-17-demethoxygeldanamycin (17-AAG)30 was chosen as a positive control. Only two out of the 15 tested compounds interacted efficiently with the immobilized protein. As a result of fitting the relative sensorgrams to a single-site bimolecular interaction model, the thermodynamic parameters for the resulting complex formation were determined. This approach allowed the measurement of a 0.049 ± 0.019 μM KD for the Hsp90/15 complex and a 0.033 ± 0.009 μM KD for the Hsp90/10 complex. Interestingly, both compounds 10 and 15 showed an affinity toward the chaperone that was even greater than that determined for 17-AAG (KD = 0.31 ± 0.04 μM). To evaluate the effect of interacting compounds on Hsp90 biological activity, the ATPase activity of the enzyme was tested in the presence of compounds 10 and 15. Once again, 17-AAG was selected as a positive control. Compound 3 was selected as a negative control on the basis of the SPR data. Figure 1 shows

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 241 polarimeter equipped with a sodium lamp (589 nm) and a 1 dm microcell. UV spectra were recorded on a Perkin-Elmer-Lambda spectrophotometer. NMR experiments were performed on a Bruker DRX-600 spectrometer at 300 K. All the 2D-NMR spectra were acquired in methanol-d4 in the phase-sensitive mode with the transmitter set at the solvent resonance and TPPI (time proportional phase increment) used to achieve frequency discrimination in the ω1 dimension. Standard pulse sequences and phase cycling were used for DQF-COSY, TOCSY, HSQC, HMBC, and ROESY experiments. NMR data were processed on a Silicon Graphics Indigo2 workstation using XWIN-NMR software. HRESIMS were acquired in the positive-ion mode on a Q-TOF Premier spectrometer equipped with a nanospray ion source (Waters, Milford, MA, USA). Column chromatography was performed over Sephadex LH-20. HPLC separations were conducted on a Waters 590 series pumping system equipped with a Waters R 401 refractive index detector and U6K injector on a C18 μ-Bondapak column (30 cm × 7.8 mm, 10 μm, Waters; flow rate 2.0 mL/min). TLC analyses were carried out using glass-coated silica gel 60 F254 (0.20 mm thickness) plates (Merck). Fetal bovine serum (FBS) was from Gibco; Dulbecco’s modified Eagle’s medium (DMEM) and antibiotics were from Lonza BioWhittaker (NJ, USA). All other reagents were from Sigma-Aldrich. Plant Material. The leaves of A. indica were collected in April 2010 in Mérida, Venezuela, and identified by Ing. Juan Carmona. A voucher specimen (No. 01) was deposited at the Herbarium MERF of the Universidad de Los Andes, Mérida, Venezuela. Extraction and Isolation. The dried leaves of A. indica (520 g) were powdered and extracted exhaustively with n-hexane, CHCl3, CHCl3−MeOH (9:1), and MeOH using an ASE 2000 extractor to yield 4.4, 13.3, 14.3, and 29.3 g, respectively, of each residue. Part of the CHCl3 extract (7.0 g) was subjected to column chromatography using silica gel and eluting with CHCl3 followed by increasing concentrations of MeOH in CHCl3 (between 1% and 100%). Fractions of 50 mL were collected, analyzed by TLC (silica gel plates, in CHCl3 or mixtures CHCl3−MeOH 99:1, 98:2, 97:3, 9:1, 4:1; CHCl3−MeOH− H2O 40:9:1), and grouped into 13 fractions (A−O). Fraction C (610 mg) was subjected to RP-HPLC, with MeOH−H2O (7:3) as eluent, to give the pure compounds 14 (4.0 mg, tR 28 min) and 13 (5.0 mg, tR 45 min). Fraction D (82 mg) was further separated by RPHPLC with MeOH−H2O (7:3) as eluent to give compounds 1 (3.0 mg, tR 40 min) and 13 (2.6 mg, tR 45 min). Fraction F (91 mg) was chromatographed over RP-HPLC with MeOH−H2O (9:4) as eluent to yield compounds 10 (3 mg, tR 23 min) and 15 (3.5 mg, tR 53 min). Fraction G (766 mg) was subjected to RP-HPLC with MeOH−H2O (67:33) as mobile phase to yield compounds 6 (2.0 mg, tR 10 min), 3 (6.0 mg, tR 22 min), 10 (5.0 mg, tR 30 min), and 12 (4.0 mg, tR 40 min). Fraction H (229 mg) was purified by RP-HPLC with MeOH−H2O (3:2) as mobile phase to yield compounds 4 (2.0 mg, tR 35 min) and 5 (4.0 mg, tR 37 min). Fraction K (230 mg) was subjected to RP-HPLC with MeOH−H2O (55:45) as mobile phase to yield compounds 11 (1.6 mg, tR 8 min), 1 (3.5 mg, tR 28 min), and 2 (3.0 mg, tR 32 min). Fraction L (476 mg) was chromatographed over RP-HPLC with MeOH−H2O (1:1) as eluent to obtain pure compounds 8 (1.4 mg, tR 11 min) and 7 (1.1 mg, tR 27 min). The MeOH extract was partitioned between n-BuOH and H2O to give 5.7 g of a n-BuOH portion. Part of the n-BuOH-soluble fraction (2.5 g) was separated by CC using Sephadex LH-20 (5 × 100 cm), with MeOH as eluent, at a flow rate of 0.8 mL/min. Altogether, five fractions were collected (A−E). Fraction D

Figure 1. Inhibition of ATPase activity of Hsp90 α by different concentrations of compounds 10 and 15. 17-AAG and compound 3 were used as positive and negative controls, respectively. Data are the means of two independent experiments performed in triplicate.

the results obtained, indicating that both compounds 10 and 15 affect the Hsp90 ATPase activity, with their inhibitory potencies found to be similar and comparable to 17-AAG. These data allowed the effects of the different structural features of the test compounds with regard to their affinity for Hsp90 protein to be probed. In particular, the presence of a furan ring at C-17 seems crucial for the Hsp90/limonoid interactions, as demonstrated by the absence of affinity for the protein observed for compounds 7, 8, and 12, which are related structurally to the active compounds 10 and 15 but lack a C-17 furan ring. Also an ester 600

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column (0.32 mm × 25 m). The temperature of both the injector and detector was 200 °C. A temperature gradient system was used for the oven, starting at 100 °C for 1 min and increasing up to 180 °C at a rate of 5 °C/min. The peak of the hydrolysate was detected by comparison with retention times of authentic samples of D-glucose (Sigma Aldrich) after treatment with 1-(trimethylsilyl)imidazole in pyridine. Cell Cultures. HeLa and PC-3 cells were obtained from the American Type Culture Collection (ATTC). Cells, grown in DMEM supplemented with 10% (v/v) FBS, 100 mg/L streptomycin, and 100 IU/mL penicillin, were maintained at 37 °C in a humidified atmosphere with 5% CO2. To ensure logarithmic growth, cells were subcultured every three days. Under the given experimental conditions, control cells were able to double their number within 24 h. Cell Viability Evaluation. Stock solutions (50 mM) of purified compounds in DMSO were stored in the dark at 4 °C. Appropriate dilutions were prepared in culture medium immediately prior to use. HeLa and PC-3 cells were plated one day before the beginning of treatment at a density of 1.2 × 105/mL. After the established incubation time with different concentrations of each test compound, cell viability was determined using an MTT ([3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide]) assay.33 Mitochondria in living cells transform MTT to a formazan salt, which, upon solubilization with DMSO, can be read at 550 nm using a microplate reader (LabSystems,Vienna, VA, USA). Data were subtracted of the corresponding appropriate blank. The number of viable cells in treated samples was calculated as percentage of control samples containing an equal amount of vehicle (DMSO). To exclude any interference of test compounds with the tetrazolium salt-based assay, cell growth inhibition was randomly verified also by cytometric count (trypan blue exclusion test). PEITC was used as a positive control. Surface Plasmon Resonance Analyses. SPR experiments were performed as described elsewhere.29 Briefly, analyses were carried out using a Biacore 3000 optical biosensor equipped with research-grade CM5 sensor chips (GE Healthcare). Two separate recombinant Hsp90 surfaces, a BSA surface and an unmodified reference surface, were prepared for simultaneous analyses. Proteins (100 μg/mL in 10 mM sodium acetate, pH 5.0) were immobilized on individual sensor chip surfaces at a flow rate of 5 μL/min using standard amine-coupling protocols to obtain densities of 8−12 kRU. Compounds 1−15, as well as 17-AAG used as a positive control, were dissolved in 100% DMSO to obtain 4 mM solutions and diluted 1:200 (v/v) in PBS (10 mM NaH2PO4, 150 mM NaCl, pH 7.4) to a final DMSO concentration of 0.5%. A series of concentrations were prepared as 2-fold dilutions into running buffer: for each sample, the complete binding study was performed using a six-point concentration series, typically spanning 0.025−1 μM, and triplicate aliquots of each test compound were dispensed into single-use vials. Included in each analysis were multiple blank samples of running buffer alone. Binding experiments were performed at 25 °C, using a flow rate of 50 μL/min, with 60 s monitoring of association and 200 s monitoring of dissociation. Simple interactions were adequately fit to a single-site bimolecular interaction model (A+B = AB), yielding a single KD. Sensorgram elaborations were performed using the Biaevaluation software provided by GE Healthcare. ATP Hydrolysis Inhibition. The Discover RX ADP Hunter Plus assay kit was used, following the manufacturer’s instructions. ATPase reactions were carried out for 60 min at 40 °C in 100 mM Tris pH 7.4, 100 μM ATP, and 40 nM Hsp90 in the presence of different concentrations of compounds 3, 10, 15, and 17-AAG. ADP generation was measured using a Perkin-Elmer LS 55 fluorimeter (540 nm excitation and 620 nm emission). Fluorescence intensity values measured for Hsp90 without any test compound was assigned as 100% enzyme activity. The background reaction rate was measured in a reaction lacking enzyme or substrate and subtracted from the experimental values. Statistical Analysis. Data reported are the mean values ± SD of at least three experiments performed in duplicate. Differences between treatment groups were analyzed by Student’s t-test. Differences were considered significant when p < 0.05.

(130 mg) was purified by RP-HPLC eluting with MeOH−H2O (3.8:6.2) to give compound 9 (1.1 mg, tR 7 min). Compound 1: white, amorphous powder; [α]25 D +119.8 (c 0.1, MeOH); 1H and 13C NMR, see Table 1; ESIMS m/z 483 [M − H]−, 397 [M − H − 86]−, 339 [M − H − 44 −100]−, 485 [M + H]+, 425 [M + H − 60]+, 407 [M + H − 60 − 18]+; HRESIMS m/z 507.2359 [M + Na]+ (calcd for C28H36O7 484.2461). Compound 2: white, amorphous powder; [α]25 D +45.2 (c 0.1, MeOH); 1H NMR δ 0.87 (3H, s, Me-19), 1.00 (3H, s, Me-18), 1.28 (3H, s, Me-30), 1.30 (3H, s, Me-28), 1.31 (3H, s, Me-29), 1.64 (1H, m, H-12b), 1.66 (1H, m, H-11b), 1.76 (1H, overlapped, H-1b), 1.77 (1H, overlapped, H-11a), 1.79 (1H, m, H-1a), 1.93 (1H, m, H-12a), 2.05 (3H, s, CH3CO), 2.20 (1H, dd, J = 13.0, 3.0 Hz, H-9), 2.23 (1H, m, H-2b), 2.24 (1H, d, J = 11.0 Hz, H-5), 2.30 (1H, overlapped, H-22b), 2.30 (1H, overlapped, H-16b), 2.50 (1H, overlapped, H-16a), 2.50 (1H, overlapped, H-22a), 2.79 (1H, m, H-20), 2.85 (1H, m, H-17), 2.87 (1H, m, H-2a), 4.18 (1H, dd, J = 11.0, 3.0 Hz, H-6), 5.38 (1H, d, J = 2.2 Hz, H-7), 5.43 (1H, br d, J = 2.0 Hz, H-15); 13C NMR, see Table 1; ESIMS m/z 487 [M + H]+, 485 [M − H]−, 399 [M − H − 44 − 42]−; HRESIMS m/z 509.2511 [M + Na]+ (calcd for C28H38O7 486.2618). Compound 3: white, amorphous powder; [α]25 D +19 (c 0.1, MeOH); 1 H and 13C NMR, see Table 1; ESIMS m/z 507 [M + Na]+, 483 [M − H]−, 465 [M − H − 18]−, 441 [M − H − 42]−, 423 [M − H − 60]−; HRESIMS m/z 507.2353 [M + Na]+ (calcd for C28H36O7 484.2461). Compound 4: white, amorphous powder; [α]25 D +33.4 (c 0.1, MeOH); 1H and 13C NMR, see Table 1; ESIMS m/z 643 [M + Na]+, 471 [M − H]−, 411 [M − H − 60]−, 385 [M − H − 42 − 44]−; HRESIMS m/z 495.2719 [M + Na]+ (calcd for C28H40O6 472.2825). Compound 5: white, amorphous powder; [α]25 D +4.3 (c 0.1, MeOH); 1H NMR δ 0.87 (3H, s, Me-19), 0.93 (3H, s, Me-18), 1.25 (3H, s, Me-30), 1.31 (3H, s, Me-29), 1.33 (3H, s, Me-28), 1.68 (1H, overlapped, H-11b), 1.68 (1H, overlapped, H-12b), 1.70 (1H, overlapped, H-11a), 1.72 (1H, overlapped, H-1b), 1.74 (1H, overlapped, H-1a), 1.96 (1H, m, H-12a), 2.05 (3H, s, CH3CO), 2.17 (1H, dd, J = 11.0, 3.5 Hz, H-9), 2.23 (1H, m, H-2b), 2.24 (1H, d, J = 12.0 Hz, H-5), 2.26 (1H, overlapped, H-16b), 2.63 (1H, m, H-16a), 2.79 (1H, m, H-17), 2.87 (1H, m, H-2a), 4.18 (1H, dd, J = 12.0, 3.0 Hz, H-6), 4.92 (1H, s, H-22), 5.39 (1H, d, J = 3.0 Hz, H-7), 5.43 (1H, br m, H-15), 7.53 (1H, s, H-21); 13C NMR, see Table 2; ESIMS m/z 485 [M − H]−, 425 [M − H − 60]−; HRESIMS m/z 509.2509 [M + Na]+ (calcd for C28H38O7 486.2618). Compound 6: white, amorphous powder; [α]25 D −24.8 (c 0.1, MeOH); 1H and 13C NMR, see Table 2; HRESIMS m/z 671.2311 [M + Na]+ (calcd for C32H40O14 648.2418). Compound 7: white, amorphous powder; [α]25 D +96.9 (c 0.1, MeOH); 1H and 13C NMR, see Table 2; HRESIMS m/z 511.1940 [M + Na]+ (calcd for C26H32O9 488.2046). Compound 8: white, amorphous powder; [α]25 D +107.3 (c 0.1, MeOH); 1H and 13C NMR, see Table 2; HRESIMS m/z 511.1956 [M + Na]+ (calcd for C26H32O9 488.2046). Compound 9: yellow, amorphous powder; [α]25 D +27 (c 0.1, MeOH); 1H NMR δ 1.44 (3H, s, H-4), 1.52 (3H, s, H-3), 3.30 (1H, dd, J = 9.0, 7.6 Hz, H-2′), 3.34 (1H, t, J = 9.0 Hz, H-4′), 3.46 (1H, t, J = 9.0 Hz, H-3′), 3.56 (1H, m, H-5′), 4.34 (1H, dd, J = 12.0, 5.0 Hz, H-6′b), 3.92 (3H, s, OCH3), 4.48 (1H, dd, J = 12.0, 3.0 Hz, H-6′a), 4.62 (1H, d, J = 7.6 Hz, H-1′), 6.42 (1H, d, J = 15.8 Hz, H-α), 6.92 (2H, s, H-2″ and H-6″), 7.66 (1H, d, J = 15.8 Hz, H-β); 13C NMR δ 24.6 (C-3), 28.3 (C-4), 56.7 (OCH3), 64.9 (C-6′), 71.5 (C-4′), 75.0 (C-2′), 75.1 (C-5′), 78.3 (C-3′), 82.0 (C-2), 100.2 (C-1′), 106.0 (C-2″ and C-6″), 115.2 (C-α), 125.6 (C-1″), 139.4 (C-4″), 146.0 (C-β), 149.2 (C-3″ and C-5″), 168.0 (COO), 181.7 (COOH); HRESIMS m/z 495.1481 [M + Na]+ (calcd for C21H28O12 472.1581). Acid Hydrolysis of Compound 9. A solution of compound 9 (2.0 mg) in 1 N HCl (1 mL) was stirred at 80 °C in a stoppered reaction vial for 4 h. After cooling, the solution was evaporated under a stream of N2. The residue was dissolved in 1-(trimethylsilyl)imidazole and pyridine (0.2 mL), and the solution was stirred at 60 °C for 5 min. After drying the solution, the residue was partitioned between H2O and CHCl3. The CHCl3 layer was analyzed by GC using an

L-CP-Chirasil-Val

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(22) Luo, X.; Ma, Y.; Wu, S.; Wu, D. J. Nat. Prod. 1999, 62, 1022− 1024. (23) Bokel, M.; Cramer, R.; Gutzeit, H.; Reeb, S.; Kraus, W. Tetrahedron 1990, 46, 775−782. (24) Akihisa, T.; Pan, X.; Nakamura, T. K.; Takahashi, N.; Matsumoto, M.; Ogihara, E.; Fukatsu, M.; Koike, K.; Tokuda, H. Phytochemistry 2013, 89, 59−70. (25) Koul, O.; Singh, G.; Singh, R.; Singh, J.; Daniewski, W. M.; Berlozecki, S. J. Biosci. 2004, 29, 409−416. (26) Malathi, R.; Rajan, S. S.; Gopalakrishnan, G.; Suresh, G. Acta Crystallogr. C 2002, 58, o708−o710. (27) Rajab, M. S.; Bentley, M. D. J. Nat. Prod. 1988, 51, 840−844. (28) Fukuyama, Y.; Miura, I.; Ochi, M. Bull. Chem. Soc. Jpn. 1983, 56, 1139−1142. (29) Dal Piaz, F.; Vassallo, A.; Temraz, A.; Cotugno, R.; Belisario, M. A.; Bifulco, G.; Chini, M. G.; Pisano, C.; De Tommasi, N.; Braca, A. J. Med. Chem. 2013, 56, 1583−1595. (30) Clarke, P. A.; Hostein, I.; Banerji, U.; Stefano, F. D.; Maloney, A.; Walton, M.; Judson, I.; Workman, P. Oncogene 2000, 19, 4125− 4133. (31) Ansar, S.; Burlison, J. A.; Hadden, M. K.; Yu, X. M.; Desino, K. E.; Bean, J.; Neckers, L.; Audus, K. L.; Michaelis, M. L.; Blagg, B. S. Bioorg. Med. Chem. Lett. 2007, 17, 1984−1990. (32) Dal Piaz, F.; Vassallo, A.; Chini, M. G.; Cordero, F. M.; Cardona, F.; Pisano, C.; Bifulco, G.; De Tommasi, N.; Brandi, A. Plos One 2012, 7, e43316. (33) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.

ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of compounds 1−9. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39-089-969754. Fax: +39-089-969602. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry.

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REFERENCES

(1) Akihisa, T.; Noto, T.; Takahashi, A.; Fujita, Y.; Banno, N.; Tokuda, H.; Koike, K.; Suzuki, T.; Yasukawa, K.; Kimura, Y. J. Oleo Sci. 2009, 58, 581−594. (2) Manikandan, P.; Vidjaya, P.; Gopalakrishnan, M.; Nagini, S. Food Chem. Toxicol. 2008, 46, 2332−2343. (3) Kumar, H.; Mohan, C.; Rao, J.; Nagini, S. Invest. New Drugs 2009, 27, 246−252. (4) Sairam, M.; Ilavazhagan, G.; Sharma, S. K.; Dhanraj, S. A.; Suresh, B.; Parida, M. M.; Jana, A. M.; Devendra, K.; Selvamurthy, W. J. Ethnopharmacol. 2000, 71, 377−382. (5) Nanduri, S.; Thunuguntla, S. S.; Nyavanandi, V. K.; Kasu, S.; Kumar, P. M.; Ram, P. S.; Rajagopal, S.; Kumar, R. A.; Deevi, D. S.; Rajagopalan, R.; Venkateswarlu, A. Bioorg. Med. Chem. Lett. 2003, 13, 4111−4115. (6) Dal Piaz, F.; Malafronte, N.; Romano, A.; Gallotta, D.; Belisario, M. A.; Bifulco, G.; Gualtieri, M. J.; Sanogo, R.; De Tommasi, N.; Pisano, C. Phytochemistry 2012, 75, 78−89. (7) Brandt, G. E. L.; Schmidt, M. D.; Prisinzano, T. E.; Blagg, B. S. J. J. Med. Chem. 2008, 51, 6495−6502. (8) Pratt, W. B.; Toft, D. O. Exp. Biol. Med. 2003, 228, 111−133. (9) Zhang, H.; Burrows, F. J. Mol. Med. 2004, 82, 488−499. (10) Modi, S.; Stopeck, A.; Linden, H.; Solit, D.; Chandarlapaty, S.; Rosen, N.; D’Andrea, G.; Dickler, M.; Moynahan, M. E.; Sugarman, S.; Ma, W.; Patil, S.; Norton, L.; Hannah, A. L.; Hudis, C. Clin. Cancer Res. 2011, 17, 5132−5139. (11) Yao, Q.; Weigel, B.; Kersey, J. Clin. Cancer Res. 2007, 13, 1591− 1600. (12) Siddiqui, S.; Mahmood, T.; Siddiqui, B.; Faizi, S. J. Nat. Prod. 1986, 49, 1068−1073. (13) Siddiqui, B. S.; Afshan, F.; Gulzar, T.; Hanif, M. Phytochemistry 2004, 65, 2363−2367. (14) Ragasa, C. Y.; Nacpil, Z. D.; Natividad, G. M.; Tada, M.; Coll, J. C.; Rideout, J. A. Phytochemistry 1997, 46, 555−558. (15) Bohnenstengel, F. I.; Wray, V.; Witte, L.; Srivastava, R. P.; Proksch, P. Phytochemistry 1999, 50, 977−982. (16) Cortez, D. A. G.; Fernandes, J. B.; Vieria, P. C.; da Silva, M. F. G. F.; Ferreira, A. G.; Cass, Q. B.; Pirani, J. R. Phytochemistry 1998, 49, 2493−2496. (17) Rogers, L. L.; Zeng, L.; Kozlowski, J. F.; Shimada, H.; Alali, F. Q.; Johnson, H. A.; McLaughlin, J. L. J. Nat. Prod. 1998, 61, 64−70. (18) Siddiqui, B. S.; Afshan, F.; Gulzar, T.; Sultana, R.; Naqvi, S. N.; Tario, R. M. Chem. Pharm. Bull. 2003, 51, 415−417. (19) Kumar, C.; Srinivas, M.; Yakkundi, S. Phytochemistry 1996, 43, 451−455. (20) Ramji, N.; Venkatakrishnan, K.; Madyastha, K. M. Phytochemistry 1998, 49, 265−267. (21) Rossi, M.; Fernandes, M. F.; Bezerra, Q.; Fernandes, J. B.; Vieira, P. C. Anal. Methods 2010, 2, 860−869. 602

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