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Bioorganic & Medicinal Chemistry Letters 24 (2014) 2773–2776

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Bioorganic & Medicinal Chemistry Letters
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Three new anti-proliferative Annonaceous acetogenins with
mono-tetrahydrofuran ring from graviola fruit (Annona muricata)
Shi Sun, Jingchun Liu, Hoda Kadouh, Xiuxiu Sun, Kequan Zhou ⇑
Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48201, USA

a r t i c l e i n f o a b s t r a c t

Article history: Bioassay-guided fractionation of the fruit powder of graviola (Annona muricata) yielded three novel com-
Received 14 February 2014 pounds: muricins J, K, and L. The compounds are all C35 Annonaceous acetogenins with a mono-tetrahy-
Revised 28 March 2014 drofuran ring and four hydroxyls. Their structures were elucidated by spectral methods and chemical
Accepted 31 March 2014
modification after isolation via chromatographic techniques and HPLC purification. These three acetoge-
Available online 18 April 2014
nins demonstrated an antiproliferative against human prostate cancer PC-3 cells.
Ó 2014 Published by Elsevier Ltd.
Annona muricata
Human prostate cancer

Graviola (Annona muricata) is an evergreen fruit tree, indige- carcinoma cell lines, colon adenocarcinoma cell lines, liver cancer
nous to the warmest tropical areas in South and North America. cell lines, human lymphoma cell lines, and multi-drug resistant
The bark, leaves, roots, fruit, and seeds of graviola are all regarded human breast adenocarcinoma.2,7,8,11–13 Studies on the pharmaco-
as natural medicine in the indigenous tropics. The bark, leaves, and logical mechanisms underlying the aforementioned functions indi-
roots have been used to manage a wide range of human diseases cate that Annonaceous acetogenins induce cytotoxicity by
including inflammatory conditions, rheumatism, neuralgia, diabe- inhibiting the mitochondrial complex I of the electron transport
tes, hypertension, insomnia, cystitis, parasitic infections, and chain, involved in ATP synthesis.6 Mitochondrial complex I could
cancer. The fruit is edible and sold in local markets. The fruit pulp be a potential target in cancer therapeutics because cancer cells
is ideal for making drinks and sherbets and can be eaten out of have a higher demand for ATP than normal cells. These compounds
hand. The fruit and fruit juice are often consumed for cooling are hence regarded as promising anti-cancer agents worthy of fur-
fevers, increasing mother’s milk supply after childbirth, and as an ther animal studies, with attempts of incorporation into new che-
astringent for diarrhea and dysentery. The crushed seeds are used motherapeutic drugs. A recent study analyzing the anti-tumor
against internal and external parasites, head lice, and worms.1 efficacy of graviola extract revealed a direct anti-tumorigenic effect
Graviola belongs to the family Annonaceae, members of which on breast cancer cells by down-regulating the expression of the
were found to contain a unique set of derivatives of C35 or C37 epidermal growth factor receptor (EGFR).14 In vitro and in vivo
long chain fatty acids derived from the polyketide pathway2 and studies involving various prostate cancer (PC) cell lines have
characteristic of this family. These phytochemicals have been sep- reported graviola extracts as inhibitors of multiple signaling path-
arately reported to possess significant antitumorous properties. ways that regulate metabolism, cell cycle, survival, and metastatic
They are selectively toxic to various types of cancer cells, including properties of PC cells.11,15
multi-drug resistant cancer cell lines, at very low dosages.3–10 In previous studies of graviola-derived Annonaceous acetoge-
Current research on graviola is also focused on Annonaceous ace- nins, the stems, leaves, and seeds were found to contain more than
togenins. Specific acetogenins in graviola and/or extracts of gravi- 40 acetogenins,16 and more acetogenins continue to be discov-
ola have been reported to be selectively toxic in vitro to the ered.7,13 Graviola products, namely capsules and tinctures, are
following types of tumor cells: lung carcinoma cell lines, human becoming more widely available in the U.S. market, and now
breast solid tumor lines, prostate adenocarcinoma, pancreatic offered under several different manufacturer labels in health food
stores. The therapeutic dosage of graviola leaf is reported to be
⇑ Corresponding author. Tel.: +1 313 577 3444; fax: +1 313 577 8616. 2–3 g, taken 3 or 4 times daily.11,15 We have previously reported
E-mail address: (K. Zhou). that the extract of graviola fruit has significant activity against
0960-894X/Ó 2014 Published by Elsevier Ltd.

All 4-OH acetoge- isolated from graviola fruit.0 35 1.6–82.6 1.65 m 29. J = 14. and there are less chemical studies on the fruit pulp.3–29.61 m 30.3–29.99 m 31.38 m 71.23–1.83 m 82. 1).65 m 31.3–29. K (2) and L (3) could be obtained from reviewing the literature.6 1.3–29.22–1.5–26.83 (3H)/dC 83.6 1.83 m 69.65 m 29.61 m 29.83 (1H)/dC 28.04 qd (6.30–1.23–1.22–1.65 m 31.98 m 28. suggesting the existence of OH OH O O four hydroxyls in each compound.2–26.60 m 29. an extract of graviola fruit pulp underwent bioassay.23–1.1 33 7.3–29.9–33.60 m 29.6.30–1.50 dd (14.1 174.65 m 29.6 1.4 1.2 1.23–1. signals at dH 1.3 and 71.5 indicates the pounds were determined to be novel Annonaceous acetogenins. their fragmentations were 3 O different in EIMS or ESIMS spectra.2–26.23–1.4–33.4–25.7 13 3.7 1.1 1.85 t (6.6 1.6 1.38 m 74.4 3.3 2. / Bioorg.60 m 29.60 m 29.61 m 29.40 m 74.6 1.6 1.17.8 1.23–1.60 m 29.7.35 dd (15.23–1.7 10 1.60 m 25.6 3.61 m 30.60 m 25.4–33.3–29.0 7.22–1.70 m 25.60 m 29.4–74.65 m 29.70 m 30.37 m 71.5–71.2) 78. and tetra-MTPA derivatives.3–29.65 m 31.7 21 1.23–1.23––33.85 t (6.99 m 26.30–1.4 1. 1. a signal at dH 3.03 qd (6.79 m 81.8–33.7 1.23–1.65 m 25.61 m 29. 8. 2.2 1.0–74.0.23–1.23–1.7 1. 24 (2014) 2773–2776 trans Comparisons of 1H and 13C nuclear magnetic resonance (NMR) erythro threo data of compounds 1–3 (Fig. 1.22–1. 2. 82.3–29.60 m 36.9–37.7 16 3. muricins J (1).3–29.36 dd (14.3–29.22–1.0 5.03 m 78.61 m 37.7 3.22–1. 1) with known acetogenins indicates 1 9 O 6 that the terminal a.3–29..4 1.7 7 1.2774 S.22–1.6 1.4 1.9–37.23–1.79 m 79.23–1.6 3.9–34.65 m 29.38 m 74.6. presence of another hydroxyl group in compound 1.61 m 29. and the molecular compo- threo threo OH sitions were all C35H64O7.0 1.7 32 0.2 26 1.3–29.0).38 dd (14.65 m 36.7 1. Structures of Annonaceous acetogenins.4–25.7 28 1.4 1.6 indicate guided fractionation isolating a bioactive fraction.7 174.8 5 1. Table 1 NMR data (ä values) of compounds 1–3 (400 MHz for 1H and 100 MHz for 13 C.61 m 29.23–1.4–74.4–33.6 1.4 1.23–1.3 6 1.23–1.1 1. in CDCl3) Position Muricin J (1) Muricin K (2) Muricin L (3) 1 13 1 13 1 13 H C H C H C 1 175.0 1.6) 4 3.3–29.22–1.23–1.60 m 31.23–1.3–29.43d (6.61 m 29.40 d (7.1 .3–29.3–29.47 (dd.3–29.6 31 1. O Inference about the stereochemistry of the isolated compounds Figure 1.14 and the active fractions appear to pos.b-unsaturated c-1actone unit with a 4-OH.3–29.60 m 29.65 m 29.9 7.5–71.3 2 131.0 5.6 1.7 11 1.60 m 29.22–1.3–29.7 1.3–29.23–1.3–29.98 m 28.3–29.7 9 1. In the 1H and 13C NMR spectra of compound 1.9–33.23–1.17 s 152.23–1.6 1.0–74.9–33.23.65 m 29.1–83. and the positioning of the THF ring and the two flanking hydroxyl finally 3 pure compounds (Fig.22–1.7 3.61–1.40 m 74. however.0. exists in all three O compounds (see Table 1).6 1. Moreover.21 There are four signals from d 69 2 4 5 O 5 OH OH to 75 in each of their 13C NMR spectra.7 12 1.60 m 36.7 1.61–1.3–29.83 m 82.37 m 74. and the set of signals at dH compounds were isolated from graviola’s leaf and stem.9–37.23–1.7 3.23–1. resulting in diverse planar 5 8 OH OH OH O structures (Scheme 1).0) 33. most of the previously-reported natural at dH 3.1 1.0).1–28.22–1.1 27 1.3–29.0 1.9 34 5. 2.61–1. The isolated com.65 m 29.4 1.17–19 High-resolution electrospray ionization mass spectrometry (HRESIMS) produced the same quasi- trans molecular ion with all three compounds.4.23–1.17 s 151.38 m 74.4 1.23–1.60 m 31.4) 14.23–1.3–29. bark.6 15 3.23–1. the set of signals nins.46 dd (15.65 m 29.4 19 3.99 m 26.20.61 m 37.93 (1H) and 1.3–29.3–29.23–1.8) 19. In the acetogenin with one THF ring and two flanking hydroxyl groups.7 1.22–1.0 3 2. nins that have been found so far have the R configuration at C-4 and the S configuration at C-36.1 0.23–1.79 m 82. Sun et al.22–1.1 1.22–1.0) 33.23–1.18 13C NMR signals of tetrahydrofuran trans (THF) oxymethines are usually located between d 79 and 8517–19.4 2.65 m 26. 8.61 m 29.37 (2H)/dC 74.2) 78. OH OH OH OH O typical of many Annonaceous acetogenins.83 m 71.7 30 1.65 m 36.3–29.65 m 29. 2.4) 19.23–1.1 and 69.6 3.9–37.7 14 1.61 m 29.4 1.24 In addition. sess characteristic chemical properties of Annonaceous acetoge.22–1.61 m 25.4.60 m 29.60 m 31. indicate that this compound is an seeds.4 1.61 m 25.1–83.1–28. subfraction.4 24 1.1 131. and 3.79 m 69.7 1.60 m 36.23–1.4–25.5–25. To our knowledge.3–29. 1.65 m 29. Lett.61 m 29.60 m 25.23–1.6 1.4 18 1.41 d (6.5–26.7 8 1.4 2.3–74.65 m 25.61–1.8) 14.23–1.70 m 35.9–33. Chem.1 23 1.23–1.4) 14. 1) exhibiting inhibitory activities groups in a threo/trans/erythro relative configuration (Tables against human prostate cancer PC-3 cell lines.8 3.17 s 151.8 20 3.65 m 22.7 3.22–1.56 (1H)/dC 71.6 22 1.2) 19.6 1. 8.60 m 29.6 1.6–82.1 0.4 3.0 131.79–1.23–1.7 29 1.22–1. threo threo and a count of peaks in this region revealed that all three of our compounds had one THF ring. current study. Med.9–37.3–29.0) 33.7 1.22 Other hydroxyls in compounds 1–3 were positioned by information from the 1H chemical shift selected cancer cell lines.3 17 1.6.23–1.7 25 1.23–1.61 m 22.23–1.23–1.4 3.60 m 22.85 t (6.9–33.3 3.61 m 29.79 m 69.0.3–29.79 m 82.65 m 31.7 1.

24 (2014) 2773–2776 2775 -18 indicates the presence of an isolated hydroxyl group in this com- 269 327 309 253 pound.. the negative value of H-20 ( 0. McLaughlin. 96.06) suggests an S con- 80 figuration at C-17. 2008. Hernandez. N-acetyl-L-leucinyl-L- that the structure–activity relationship (SAR) of acetogenins is not norleucinal) at 1 lM for 24 h. 49. 1311.. 15.4 6. / Bioorg. 71. L. P.4 were suspected to represent a 1.6 and 69. R..0 and 74.03... They all showed inhibition.. 129. H. Hernandez.8.85 (2H) and 3. James Windak and Dr. the structure of 2 was fully determined as shown in Figure 1. m/z Acknowledgments 269/(327) 309 and (397) 361 indicated that the THF ring was located C-16/C-19 based on a peak at m/z 269 (C-12/C-13 cleavage The authors would like to thank Dr. 2003. J. 60% Confluent PC3 cells were incubated potency: muricin K > muricin L > muricin J (Fig.doi. T..8. J. The Hryhorczuk of Central Instrument Facility. The positive value of dH(S-R) at H-13 (+0. DMSO served as solvent controls. in addition to signals at dH 1. two flanking hydroxyl groups in a relative threo/trans/threo config. Jones. K. D.97 (2H) and 4.. and.19. and at dH 3. 423. 1.. C. L.7 5 O 8 OH OH OH O and 74.3.. Zafra-Polo. S.26 but also by the positions of these groups. at http://dx.C(DMSO) LLnL(1µM) Compd 1 Compd 2 Compd 3 The newly isolated and elucidated muricins J. Res. signals were 4 5 O 5 OH OH OH OH O observed at dH 3.1016/j. Paul Lennon at m/z 253 (C-12/C-13 cleavage) (Scheme 1). Phytochemistry 1998. uration (Table 1). at dH 3. the R-configuration of the flanking hydroxylated carbon is usually on the c-lactone or ketolactone side of the THF References and notes ring and the S-configuration of the flanking hydroxylated carbon is on the hydrocarbon chain side. Sun et al.62 (2H)/dC 28. F. DePedro.2-diol group. 2005. 2).. 82. 147. 120 1 H NMR data of tetra-Mosher ester derivatives were assigned 100 according to regular. 2.7. Oncol. Oberlies.97 (2H) and 1.. muricins J (1). Wu. The fragments m/z 395. Chem. Austin. J. of University of Michigan for EI-MS analysis. Scheme 1. Rogers. indicating the presence of one THF ring with 5. and m/z 9 O 6 OH OH OH OH O 269 (C-12/C-13 cleavage). .. the structure of 1 was 1. which indicates with the compounds at 20 lg/ml or positive control (LLnL.26 The proton signal at dH 3. with the known mono-THF ring of Annanoceous acetogenins bear- in the online version. Pelaez.b-unsatu- 20 rated c-lactone ring.6. I. Barrachina. Royo. N. S. L. Tormo. Wayne State University last hydroxyl group was present at C-13 according to ESIMS peaks for HR-ESI-MS analysis. as shown in Figure 1.56 (1H). tive relationship. J. Kim. and L against cer PC-3 cells.40 (1H)/dC 71. in comparison with OH known compounds muricatetrocin A and muricins A–E.. Cortes. Zeng. 0 S. I. Tormo. Prod. F. Cortes..21 Thus. 227 269 and the compound has been given the name muricin K.. single-relayed and double-relayed COSY spec- cell growth index (%) tra. K (2) and L (3). T. Technical Data Report for Graviola (Annona muricata).. M. (2H)/dC 74. the conformation of 231 this vicinal diol was assigned as threo on the basis of a comparison of its NMR chemical shifts. 55. the configuration was assumed through comparison Supplementary data associated with this article can be found. S. a signal at dH 3.. TX.099. G. F. Brian Shay and Dr. By prepar- 423 O ing and examining the tetra-MTPA derivative.. Cuadrillero. C. isolated from graviola fruit.. Lew or C15/C16) and a peak at m/z 361 (C-20/C-21 cleavage–2H2O). 565. and Dr. J.74–3. Gallardo. Because the amount of compound 1 that was isolated was not sufficient to prepare a tetra-methoxy trifluoromethyl phenyl acetic Supplementary data acid (MTPA) derivative for the determination of the absolute con- figuration. 14. C. Upon close examination of the ESIMS fragmentation of 1. 2005. F. Lett. R. Based on the threo-trans-erythro rela- 2014. 2 In the 1H and 13C NMR spectra of compound 3. Applying the dH(S R) magnitude–distance relationship for acetogenins. McLaughlin.69 (2H) are the proton resonances for the two methylene groups of the mono-THF ring 3 395 (337) -18 319 and correspond to the trans conformation.7. Med. with the following human prostate cancer cells (MTT assay). thus the configurations at C24/25 were S/R. ing two flanking hydroxyls.25 Examination of the EIMS fragmentation of compound 2 1 reveals peaks at m/z 257 and 339 (C-16/C-17 cleavage). and the com. L. L.01) 60 indicates that it should not be dominated by the C-24- 40 MTPA and C-25-MTPA. Corbett. MS fragmentation (m/z values) of Annonaceous acetogenins. Res.25.38 Pelaez.22 thus. 1995. only affected by the number of hydroxyl groups. at dC 81. E. 467 Thus. O 74.bmcl. The 1H and 13C NMR spectra of compound 2 reveal two sets of 3. H. J. With ESIMS. 1) of 3 was determined and the compound was named muricin L. dH 1. consistent with a THF ring with one flanking hydroxyl group 257 339 303 -18-18 in a threo configuration. pound has been given the name muricin J. and 319 in the ESIMS spectrum and the [ion+H]+ which appeared in the positive ESIMS spectrum sug- gest that the THF ring is located between C-16/C-19 and the diol group is located at C-23/C-24 (Scheme 1).org/10. Royo. Alali. S. m/z 467 (C-25/C-26 cleavage) 397 361 -18-18 (Scheme 1) suggests that the hydroxyl group is located at C-21.. K.79 (3H)/dC 82. together with the aforementioned a. 79. indicating that the THF ring is located O between C-13/C-16. Cancer Lett.23 In addition. Sage Press: fully determined accordingly. Nat.. Antiproliferative activity of graviola-derived muricins J. I. L. Sastrodihardjo. McLaughlin.. signals. L. the structure (Fig. N. D.1. J. Taylor. Fotopoulos. P. L. and L were evaluated for antiproliferative activity against human prostate can- Figure 2. S. Oncol.38 (2H) and the carbon signals at dC 74.5. Zafra-Polo. M..

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