Bioorganic & Medicinal Chemistry Letters 24 (2014) 27732776

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

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
modication after isolation via chromatographic techniques and HPLC purication. These three acetoge-
Available online 18 April 2014
nins demonstrated an antiproliferative against human prostate cancer PC-3 cells.
2014 Published by Elsevier Ltd.
Keywords:
Annona muricata
Acetogenin
Muricin
Cytotoxicity
Human prostate cancer

Graviola (Annona muricata) is an evergreen fruit tree, indige-
nous to the warmest tropical areas in South and North America.
The bark, leaves, roots, fruit, and seeds of graviola are all regarded
as natural medicine in the indigenous tropics. The bark, leaves, and
roots have been used to manage a wide range of human diseases
including inammatory conditions, rheumatism, neuralgia, diabe-
tes, hypertension, insomnia, cystitis, parasitic infections, and
cancer. The fruit is edible and sold in local markets. The fruit pulp
is ideal for making drinks and sherbets and can be eaten out of
hand. The fruit and fruit juice are often consumed for cooling
fevers, increasing mothers milk supply after childbirth, and as an
astringent for diarrhea and dysentery. The crushed seeds are used
against internal and external parasites, head lice, and worms.1
Graviola belongs to the family Annonaceae, members of which
were found to contain a unique set of derivatives of C35 or C37
long chain fatty acids derived from the polyketide pathway2 and
characteristic of this family. These phytochemicals have been sep-
arately reported to possess signicant antitumorous properties.
They are selectively toxic to various types of cancer cells, including
multi-drug resistant cancer cell lines, at very low dosages.310
Current research on graviola is also focused on Annonaceous ace-
togenins. Specic acetogenins in graviola and/or extracts of gravi-
ola have been reported to be selectively toxic in vitro to the
following types of tumor cells: lung carcinoma cell lines, human
breast solid tumor lines, prostate adenocarcinoma, pancreatic
Corresponding author. Tel.: +1 313 577 3444; fax: +1 313 577 8616.
E-mail address: kzhou@wayne.edu (K. Zhou).
carcinoma cell lines, colon adenocarcinoma cell lines, liver cancer
cell lines, human lymphoma cell lines, and multi-drug resistant
human breast adenocarcinoma.2,7,8,1113 Studies on the pharmaco-
logical mechanisms underlying the aforementioned functions indi-
cate that Annonaceous acetogenins induce cytotoxicity by
inhibiting the mitochondrial complex I of the electron transport
chain, involved in ATP synthesis.6 Mitochondrial complex I could
be a potential target in cancer therapeutics because cancer cells
have a higher demand for ATP than normal cells. These compounds
are hence regarded as promising anti-cancer agents worthy of fur-
ther animal studies, with attempts of incorporation into new che-
motherapeutic drugs. A recent study analyzing the anti-tumor
efcacy of graviola extract revealed a direct anti-tumorigenic effect
on breast cancer cells by down-regulating the expression of the
epidermal growth factor receptor (EGFR).14 In vitro and in vivo
studies involving various prostate cancer (PC) cell lines have
reported graviola extracts as inhibitors of multiple signaling path-
ways that regulate metabolism, cell cycle, survival, and metastatic
properties of PC cells.11,15
In previous studies of graviola-derived Annonaceous acetoge-
nins, the stems, leaves, and seeds were found to contain more than
40 acetogenins,16 and more acetogenins continue to be discov-
ered.7,13 Graviola products, namely capsules and tinctures, are
becoming more widely available in the U.S. market, and now
offered under several different manufacturer labels in health food
stores. The therapeutic dosage of graviola leaf is reported to be
23 g, taken 3 or 4 times daily.11,15 We have previously reported
that the extract of graviola fruit has signicant activity against

http://dx.doi.org/10.1016/j.bmcl.2014.03.099
0960-894X/ 2014 Published by Elsevier Ltd.
2774 S. Sun et al. / Bioorg. Med. Chem. Lett. 24 (2014) 27732776

trans Comparisons of 1H and 13C nuclear magnetic resonance (NMR)

erythro threo data of compounds 13 (Fig. 1) with known acetogenins indicates
1 9 O 6 that the terminal a,b-unsaturated c-1actone unit with a 4-OH,
OH OH OH OH O typical of many Annonaceous acetogenins, exists in all three
O
compounds (see Table 1).17,18 13C NMR signals of tetrahydrofuran
trans (THF) oxymethines are usually located between d 79 and 851719,
threo threo and a count of peaks in this region revealed that all three of our
compounds had one THF ring.20,21 There are four signals from d 69
2 4 5 O 5
OH OH to 75 in each of their 13C NMR spectra, suggesting the existence of
OH OH O
O four hydroxyls in each compound.1719 High-resolution electrospray
ionization mass spectrometry (HRESIMS) produced the same quasi-
trans molecular ion with all three compounds, and the molecular compo-
threo threo
OH sitions were all C35H64O7; however, their fragmentations were
3 O different in EIMS or ESIMS spectra, resulting in diverse planar
5 8
OH OH OH O structures (Scheme 1).
O
Inference about the stereochemistry of the isolated compounds
Figure 1. Structures of Annonaceous acetogenins, muricins J (1), K (2) and L (3) could be obtained from reviewing the literature. All 4-OH acetoge-
isolated from graviola fruit. nins that have been found so far have the R conguration at C-4
and the S conguration at C-36.22 Other hydroxyls in compounds
13 were positioned by information from the 1H chemical shift
selected cancer cell lines,14 and the active fractions appear to pos- and tetra-MTPA derivatives.
sess characteristic chemical properties of Annonaceous acetoge- In the 1H and 13C NMR spectra of compound 1, the set of signals
nins. To our knowledge, most of the previously-reported natural at dH 3.83 (3H)/dC 83.2, 82.1 and 69.7, and the set of signals at dH
compounds were isolated from graviolas leaf and stem, bark, and 3.37 (2H)/dC 74.3 and 71.6, indicate that this compound is an
seeds; and there are less chemical studies on the fruit pulp. In the acetogenin with one THF ring and two anking hydroxyl groups.
current study, an extract of graviola fruit pulp underwent bioassay- Moreover, signals at dH 1.93 (1H) and 1.83 (1H)/dC 28.6 indicate
guided fractionation isolating a bioactive fraction, subfraction, and the positioning of the THF ring and the two anking hydroxyl
nally 3 pure compounds (Fig. 1) exhibiting inhibitory activities groups in a threo/trans/erythro relative conguration (Tables
against human prostate cancer PC-3 cell lines. The isolated com- 1).23,24 In addition, a signal at dH 3.56 (1H)/dC 71.5 indicates the
pounds were determined to be novel Annonaceous acetogenins. presence of another hydroxyl group in compound 1.

Table 1
NMR data ( values) of compounds 13 (400 MHz for 1H and 100 MHz for 13
C, in CDCl3)

Position Muricin J (1) Muricin K (2) Muricin L (3)

1 13 1 13 1 13
H C H C H C
1 175.1 174.7 174.3
2 131.1 131.0 131.0
3 2.38 dd (14.0, 8.0) 33.4 2.35 dd (15.6, 8.0), 2.46 dd (15.6, 2.0) 33.4 2.36 dd (14.0, 8.0), 2.47 (dd, J = 14.0, 2.0) 33.3
2.50 dd (14.0, 1.6)
4 3.83 m 69.7 3.79 m 69.6 3.79 m 69.8
5 1.231.65 m 36.937.4 1.231.60 m 36.937.4 1.221.61 m 37.3
6 1.231.65 m 25.226.0 1.231.60 m 29.329.6 1.221.61 m 29.329.7
7 1.231.65 m 29.329.6 1.231.60 m 29.329.6 1.221.61 m 29.329.7
8 1.231.65 m 29.329.6 1.231.60 m 29.329.6 1.221.61 m 29.329.7
9 1.231.65 m 29.329.6 1.231.60 m 29.329.6 1.221.61 m 29.329.7
10 1.231.65 m 29.329.6 1.231.60 m 25..425.6 1.221.61 m 29.329.7
11 1.231.65 m 26.0 1.231.60 m 31.833.4 1.221.61 m 29.329.7
12 1.231.65 m 36.937.4 3.38 m 74.074.1 1.221.61 m 29.329.7
13 3.83 m 71.571.6 3.79 m 82.682.7 1.221.61 m 25.525.7
14 1.791.99 m 31.933.4 1.611.98 m 28.8 1.221.61 m 30.433.6
15 3.83 m 82.183.2 1.611.98 m 28.8 3.40 m 74.374.7
16 3.37 m 74.3 3.79 m 82.682.7 3.79 m 79.3
17 1.231.65 m 31.933.4 3.38 m 74.074.1 1.611.99 m 26.128.4
18 1.231.65 m 31.934.4 1.231.60 m 31.933.4 1.611.99 m 26.128.4
19 3.37 m 71.571.7 1.231.60 m 25.425.7 3.79 m 81.8
20 3.83 m 82.183.2 1.231.60 m 25.425.7 3.40 m 74.474.7
21 1.231.65 m 31.933.4 1.231.60 m 29.329.7 1.301.70 m 30.433.6
22 1.231.65 m 25.226.0 1.231.60 m 29.329.7 1.301.70 m 25.526.1
23 1.231.65 m 29.329.6 1.231.60 m 29.329.7 1.301.70 m 35.4
24 1.231.65 m 29.329.6 1.231.60 m 36.937.4 3.38 m 74.474.7
25 1.231.65 m 29.329.6 3.38 m 71.6 1.221.61 m 37.2
26 1.231.65 m 29.329.6 1.231.60 m 36.937.4 1.221.61 m 25.526.1
27 1.231.65 m 29.329.6 1.231.60 m 29.329.7 1.221.61 m 29.329.7
28 1.231.65 m 29.329.6 1.231.60 m 29.329.7 1.221.61 m 29.329.7
29 1.231.65 m 29.329.6 1.231.60 m 29.329.7 1.221.61 m 29.329.7
30 1.231.65 m 31.933.4 1.231.60 m 31.933.4 1.221.61 m 30.433.6
31 1.231.65 m 22.7 1.231.60 m 22.7 1.221.61 m 22.7
32 0.85 t (6.8) 14.1 0.85 t (6.4) 14.1 0.85 t (6.4) 14.1
33 7.17 s 152.0 7.17 s 151.9 7.17 s 151.9
34 5.04 qd (6.4, 1.2) 78.0 5.03 m 78.0 5.03 qd (6.4, 1.2) 78.0
35 1.41 d (6.4) 19.1 1.40 d (7.2) 19.1 1.43d (6.8) 19.1
 S. Sun et al. / Bioorg. Med. Chem. Lett. 24 (2014) 27732776 2775

-18 indicates the presence of an isolated hydroxyl group in this com-

269 327 309
253 pound.25 Examination of the EIMS fragmentation of compound 2
1 reveals peaks at m/z 257 and 339 (C-16/C-17 cleavage), and m/z
9 O 6
OH OH OH OH O 269 (C-12/C-13 cleavage), indicating that the THF ring is located
O between C-13/C-16. With ESIMS, m/z 467 (C-25/C-26 cleavage)
397 361
-18-18 (Scheme 1) suggests that the hydroxyl group is located at C-21.
467 Thus, the structure of 2 was fully determined as shown in Figure 1,
227 269 and the compound has been given the name muricin K.
2 In the 1H and 13C NMR spectra of compound 3, signals were
4 5 O 5
OH OH OH OH O observed at dH 3.743.85 (2H) and 3.56 (1H), at dC 81.8, 79.3,
O 74.5, consistent with a THF ring with one anking hydroxyl group
257 339 303
-18-18 in a threo conguration. dH 1.97 (2H) and 1.69 (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, in comparison with
OH known compounds muricatetrocin A and muricins AE.7,25,26 The
proton signal at dH 3.38 (2H) and the carbon signals at dC 74.7
5 O 8
OH OH OH O and 74.4 were suspected to represent a 1,2-diol group. By prepar-
423 O ing and examining the tetra-MTPA derivative, the conformation of
231 this vicinal diol was assigned as threo on the basis of a comparison
of its NMR chemical shifts.
Scheme 1. MS fragmentation (m/z values) of Annonaceous acetogenins, muricins J
(1), K (2) and L (3), isolated from graviola fruit.
The fragments m/z 395, 423, 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).
120 1
H NMR data of tetra-Mosher ester derivatives were assigned
100 according to regular, single-relayed and double-relayed COSY spec-
cell growth index (%)

tra. The positive value of dH(S-R) at H-13 (+0.06) suggests an S con-

80 guration at C-17. Applying the dH(S R) magnitudedistance
relationship for acetogenins, the negative value of H-20 ( 0.01)
60
indicates that it should not be dominated by the C-24-
40 MTPA and C-25-MTPA, and, thus the congurations at C24/25
were S/R.21 Thus, together with the aforementioned a,b-unsatu-
20 rated c-lactone ring, the structure (Fig. 1) of 3 was determined
and the compound was named muricin L.
0
S.C(DMSO) LLnL(1M) Compd 1 Compd 2 Compd 3 The newly isolated and elucidated muricins J, K, and L were
evaluated for antiproliferative activity against human prostate can-
Figure 2. Antiproliferative activity of graviola-derived muricins J, K, and L against cer PC-3 cells. They all showed inhibition, with the following
human prostate cancer cells (MTT assay). 60% Conuent PC3 cells were incubated potency: muricin K > muricin L > muricin J (Fig. 2), which indicates
with the compounds at 20 lg/ml or positive control (LLnL, N-acetyl-L-leucinyl-L-
that the structureactivity relationship (SAR) of acetogenins is not
norleucinal) at 1 lM for 24 h. DMSO served as solvent controls.
only affected by the number of hydroxyl groups,26 but also by the
positions of these groups.

Upon close examination of the ESIMS fragmentation of 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. Brian Shay and Dr. Lew
or C15/C16) and a peak at m/z 361 (C-20/C-21 cleavage2H2O). The
Hryhorczuk of Central Instrument Facility, Wayne State University
last hydroxyl group was present at C-13 according to ESIMS peaks
for HR-ESI-MS analysis; and Dr. James Windak and Dr. Paul Lennon
at m/z 253 (C-12/C-13 cleavage) (Scheme 1).
of University of Michigan for EI-MS analysis.
Because the amount of compound 1 that was isolated was not
sufcient to prepare a tetra-methoxy triuoromethyl phenyl acetic
Supplementary data
acid (MTPA) derivative for the determination of the absolute con-
guration, the conguration was assumed through comparison
Supplementary data associated with this article can be found,
with the known mono-THF ring of Annanoceous acetogenins bear-
in the online version, at http://dx.doi.org/10.1016/j.bmcl.
ing two anking hydroxyls. Based on the threo-trans-erythro rela-
2014.03.099.
tive relationship, the R-conguration of the anking hydroxylated
carbon is usually on the c-lactone or ketolactone side of the THF
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