Synthetic chalcone derivatives as inhibitors of cathepsins K and B, and their cytotoxic evaluation

Article abstract of DOI:10.1002/cbdv.201200344

A series of chalcone derivatives, 1-15, were prepared by Claisen-Schmidt condensation and evaluated for their cytotoxicities on tumor cell lines and also against proteolytic enzymes such as cathepsins B and K. Of the compounds synthesized, (E)-3-(3,4-dimethoxyphenyl)-1-phenylprop-2-en-1-one (12), (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (13), (E)-3-(4-methoxyphenyl)-1- phenylprop-2-en-1-one (14), and (E)-3-(4-nitrophenyl)-1-phenylprop-2-en-1-one (15) showed significant cytotoxicities. The most effective compound was 15, which showed high cytotoxic activity with an IC₅₀ value lower than 1 μg/ml, and no selectivity on the tumor cells evaluated. Substituents at C(4) of ring B were found to be essential for cytotoxicity. In addition, it was also demonstrated that some of these chalcones are moderate inhibitors of cathepsin K and have no activity against cathepsin B. Copyright

Full text of DOI:10.1002/cbdv.201200344

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1999
Synthetic Chalcone Derivatives as Inhibitors of Cathepsins K and B, and Their
Cytotoxic Evaluation
by Suelem Demuner Ramalhoa)b), Aline Bernadesc), Giulio Demetriusa), Caridad Noda-Perez*c),
Paulo Cezar Vieirab), Caio Yu dos Santosb), James Almada da Silvab), Manoel Odorico de Moraesd),
and Kristiana Cerqueira Mousinhod)
a
´
´
) Department of Chemistry, State University of Goias, 495, Anapolis, GO, Brazil
b
˜
˜
) Department of Chemistry, Federal University of Sao Carlos, 13565-905, Sao Carlos, SP, Brazil
c
´
ˆ
) Chemical Institute, Federal University of Goias, 74001-970, Goiania, GO, Brazil (phone: þ55-
6235211094; fax: þ55-6235211167; e-mail: carynoda@yahoo.com.br)
d
´
) Department of Physiology and Pharmacology, Federal University of Ceara, 3157, Fortaleza, CE, Brazil
A series of chalcone derivatives, 1–15, were prepared by ClaisenꢀSchmidt condensation and
evaluated for their cytotoxicities on tumor cell lines and also against proteolytic enzymes such as
cathepsins B and K. Of the compounds synthesized, (E)-3-(3,4-dimethoxyphenyl)-1-phenylprop-2-en-1-
one (12), (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (13), (E)-3-(4-methoxyphenyl)-1-phenyl-
prop-2-en-1-one (14), and (E)-3-(4-nitrophenyl)-1-phenylprop-2-en-1-one (15) showed significant
cytotoxicities. The most effective compound was 15, which showed high cytotoxic activity with an IC50
value lower than 1 mg/ml, and no selectivity on the tumor cells evaluated. Substituents at C(4) of ring B
were found to be essential for cytotoxicity. In addition, it was also demonstrated that some of these
chalcones are moderate inhibitors of cathepsin K and have no activity against cathepsin B.
Introduction. – Cancer is a leading cause of death worldwide. According to the
World Health Organization (WHO), deaths from cancer are projected to continue
rising to over 11 million in 2030. Cancer arises from a change in one single cell, and this
change may be started by either external agents or inherited genetic factors [1]. Among
the methods used to control the cancer are chemotherapeutic agents, which
unfortunately possess high toxicity, multidrug resistance (MDR), and limited
effectiveness [2–4]. So, the development of new drugs plays an important role in
cancer control [5].
Most of the successful anticancer drugs acting as antimitotics originate in natural
compounds [6]. Flavonoids are an extensive and diversified group of compounds found
in edible plants with high abundance [7–9]. Among flavonoids, chalcones have been
identified as interesting compounds, which are associated with a wide range of
biological properties such as antimalarial [10], antileishmanial [11], antioxidative
activities [12], antiviral [13], analgesic [14], anti-inflammatory activities [12], and
cytotoxicity towards cancer cell lines [8][15]. They consist of open-chain flavonoids,
and one of the most widely cited mechanism is that chalcones exert their cytotoxic
activity by interfering with mitotic phase in the cell cycle [3][7][15][16]. In fact, it has
been reported that chalcones act as tubulin-polymerization inhibitors. Tubulin occurs as
a heterodimer of a- and b-subunits, and plays a vital role in various biochemical
processes of cell survival and growth [17][18].
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Structural requirements for cytotoxic activity vary depending upon the mechanisms
of action. For antimitotic activity, the presence of MeO substituents is a favorable
feature, and chalcone derivatives with larger number of MeO groups have been
reported by Pati et al. [17] to be more potent than compounds with fewer similar
substituents. Conformational restraint of the chalcone template generally leads to a
decrease in cytotoxic activity [16][19].
In human diseases, cysteine proteases have been recognized as potential drug
targets due to their excessive expression in many pathological conditions [20][21]. Two
important members are the cathepsins B and K, which are also known as lysosomal
proteases [22]. In cancer, cathepsin B degrades extracellular matrix proteins (ECMs)
and thus facilitates local invasion, proliferation, and metastasis of tumor cells [23].
Cathepsin K is predominantly expressed in osteoclast and exhibits high collagenase
activity towards collagens, being strongly involved in osteoporosis progression [24]. To
the best of our knowledge, there are no reports in the literature on the inhibition of
these synthetic chalcones against cathepsin B and cathepsin K.
The aim of the current study was to evaluate the in vitro cytotoxicity of a series of
synthetic chalcone derivatives against human cancer cell lines, namely, melanoma
MDA-MB-435, colon cancer HCT-8, and central nervous system SF-295, using MTT (¼
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. In addition,
the compounds were also evaluated against cathepsins K and B, which are related with
osteoporosis and tumor processes, respectively.
Results and Discussion. – Synthesis. Chalcones were synthesized, and various
substitution patterns on the two aromatic rings were created to obtain a large number
of potential analogs [10]. Most of the chalcones, i.e., 3–15, (Table 1) were prepared by a
ClaisenꢀSchmidt condensation of suitable aldehydes with acetophenone derivatives in
the presence of methanolic KOH solution [25]. Alternatively, compounds 1 and 2,
involving hydroxybenzaldehyde were obtained under acidic conditions [26]. These
products were recrystallized from MeOH and identified by comparison of their IR, MS
1
and H-NMR data with those in the literature [26–29]. A coupling constant of ca.
16 Hz for the vinyl H-atoms in the 1H-NMR spectra confirmed the (E)-configuration.
In Table 1, structures and reaction conditions for all chalcone derivatives synthesized
are compiled.
Cytotoxicity. Fifteen synthetic chalcones were evaluated for their cytotoxic activities
in MTT assay against human tumor cells line such as MDA-MB-435, HCT-8 and SF-
295. It is already known that the cytotoxic activities of chalcones derivatives depend on
the substituent on the aromatic rings [30]. Substituents at C(4) on ring B were found to
be essential for activity. Hydroxylated chalcones were less active than the correspond-
ing alkoxylated analogs.
In general, no cytotoxicity selectivity between the different cell lines was observed
(Table 2). Four compounds, 12–15, showed cell growth inhibition higher than 85% in
at least two cell lines. Chalcones are soft electrophiles, and they would attract soft
nucleophiles like thiols. The thiol reactivity of chalcones is likely to contribute to both
cytotoxic and chemoprotective properties of these compound [16]. It has been reported
that the compound 14 already suppressed the human PBMC blastogenesis and
melanoma cell line A375 proliferation with IC50 values of 12.18 and 31.81 mm,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2001
Table 1. Structures, Reaction Conditions, and Yields of Chalcone Derivatives 1–15
Compound
X
Y
Time [h]
Temp. [8]
Yield [%]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
4-OH
4-OH
4-NO2
4-NO2
H
4-NO2
H
4-NO2
H
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
H
24
24
3
3
3
4
1
1
3
3
6
12
4
10
4
64 (reflux)
64 (reflux)
ꢀ4
45
32
87
48
54
42
75
46
75
65
68
63
42
48
78
3,4-OꢀCH2ꢀO
28 (r.t.)
28 (r.t.)
28 (r.t.)
28 (r.t.)
28 (r.t.)
28 (r.t.)
28 (r.t.)
28 (r.t.)
28 (r.t.)
28 (r.t.)
64 (reflux)
28 (r.t.)
4-Cl
4-F
3,4-OꢀCH2ꢀO
2,4-(MeO)2
3,4-(MeO)2
4-F
4-MeO
3,4-(MeO)2
4-Cl
H
H
H
4-MeO
4-NO2
Table 2. Cytotoxicities of Chalcone Derivatives 1–15 against Three Tumor Cell Linesa). Cell-growth-
inhibition percentage (GI-%) at 125 mg/mlb).
Compound
MDA-MB-435
HCT-8
SF-295
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
21.5ꢁ0.6
52.6ꢁ2.3
14.4ꢁ3.3
49.7ꢁ17.6
19.9ꢁ19.1
88.6ꢁ13.4
6.7ꢁ0.8
33.3ꢁ5.3
53.9ꢁ0.9
33.3ꢁ1.3
48.9ꢁ5.7
27.3ꢁ8.6
72.5ꢁ31.8
2.6ꢁ2.7
26.2ꢁ5.3
33.8ꢁ6.3
16.3ꢁ11.1
48.4ꢁ3.5
19.8ꢁ3.1
35.6ꢁ5.9
21.6ꢁ2.2
4.5ꢁ6.9
13.9ꢁ0.9
24.1ꢁ4.9
25.2ꢁ5.8
31.5ꢁ6.7
96.6ꢁ3.1
96.3ꢁ5.4
96.7ꢁ0.9
89.2ꢁ0.1
96.6ꢁ0.1
20.3ꢁ9.4
7.5ꢁ0.1
25.1ꢁ10.4
23.3ꢁ9.3
12.7ꢁ18.4
88.1ꢁ5.8
90.3ꢁ2.8
93.1ꢁ3.0
76.6ꢁ9.9
97.4ꢁ0.1
39.8ꢁ2.9
33.0ꢁ4.5
50.5ꢁ3.4
93.3ꢁ1.
85.5ꢁ2.2
98.2ꢁ0.1
97.1ꢁ0.2
Doxorubicinc)
a) Cancer cell lines: MDA-MB-435, melanoma; HCT-8, colon cancer; SF-295, central nervous system.
b) GI-% Values shown were the average of three replicates. c) Positive control.

2002
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
respectively [31]. All four selected chalcones except 12 were already evaluated against
HSC-2 and HSC-4 human oral squamous cell carcinoma [23], kidney carcinoma cells
TK-10, human mammary adenocarcinoma cells MCF-7, and human colon adenocarci-
noma cells HT-29, and showed moderate cytotoxic potencies [3]. Nevertheless, Juvale
et al. [4] reported that chalcone 12 exhibited no inhibitory effect on MCF-7 [4].
Compounds with cytotoxic activities higher than 85% were selected for IC50
determination (Table 3). Most of the chalcones, 12–15, showed significant activities
with IC50 values in the range of 4.1–0.28 mm. Compounds with electron-withdrawing
substituents (Cl and NO2) on the ring B and no substituent on the ring A (i.e., 13 and
15) were active; however, F-containing compound 6 exhibited no activity. The
cytotoxicity progressively decreased for compounds with NO2 group in the ring A. The
presence of an electron-withdrawing group in the ring B (in particular in para-position)
enhanced the electron deficiency of the b-C-atom, and thus its reactivity to words
nucleophiles [16]. The presence of MeO group on the ring B of the chalcones 12 and 14
was found to contribute to cytotoxicity. Compounds 9 and 11, which have NO2 groups in
the ring A, were less active than those without NO2, i.e., 12 and 14.
Table 3. IC50 Values of Chalcone Derivatives 12–15 against Four Tumor Cell Linesa)
Compound
MDA-MB-435
HCT-8
SF-295
12
13
14
15
3.3 (2.6–4.1)
2.6 (2.2–3.1)
1.6 (1.1–2.2)
0.5 (0.3–0.6)
0.5 (0.4–0.7)
0.6 (0.2–1.3)
2.5 (1.4–4.4)
0.3 (0.1–0.6)
0.8 (0.6–1.2)
0.04 (0.03–0.05)
4.1 (3.3–5.1)
1.9 (1.0–3.9)
1.5 (1.3–1.8)
0.3 (0.2–0.5)
0.3 (0.2–0.4)
Doxorubicinc)
a) Cancer cell lines: MDA-MB-435, melanoma; HCT-8, colon cancer; SF-295, central nervous system.
b) IC50 (CI 95%) [mg/ml]: 50% inhibitory concentration and 95% confidence interval in mg/ml. c) Positive
control.
Compounds with IC50 values lower than 4 mg/ml were considered promising for the
search of new anticancer agents. The chalcone 15 showed strong cytotoxicity with an
IC50 value lower than 1 mg/ml and no selectivity in human tumor cells. Based on the IC50
values the promising results suggested that the structural features of compound 15
might be useful in designing new cytotoxic agents. Evaluating the selectivity, compound
12 and 14 turned out to be the most selective towards the cell line HCT-8. To investigate
a possible correlation between the cytotoxic effects exhibited by selected chalcone
derivatives, 12, 13, 14, and 15, and unspecific cell membrane damage, these compounds
were submitted to the hemolytic assay as proposed by Jimenez et al. [32]. All the
evaluated compounds showed no hemolytic activity at the concentration 50 mg/ml,
indicating that cytotoxic activity does not occur by breaking the cell membrane.
Cathepsin. By inhibition assays of cathepsin B and cathepsin K, four compounds, 4,
6, 13, and 14, among the series of chalcones were identified to have moderate potencies
as inhibitors cathepsin K. On the other hand, none of the chalcones showed significant
activity against cathepsin B (Table 4).
Cathepsin B is known to be strongly involved in the metastasis process. High levels
of this enzyme are found in different types of tumors [33–36]. Although both

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2003
Table 4. Inhibitory Activities [%] of Chalcone Derivatives against Cathepsin B and Cathepsin K at 125 mm
Compound
Cathepsin B
Cathepsin K
Compound
Cathepsin B
Cathepsin K
1
2
3
4
5
6
7
8
63.6ꢁ4.7
59.2ꢁ2.3
63.0ꢁ3.4
29.6ꢁ0.5
0.0ꢁ4.7
0.0ꢁ5.6
15.6ꢁ1.0
0.0ꢁ4.7
10.5ꢁ0.6
50.6ꢁ4.5
75.7ꢁ2.1
95.7ꢁ1.2
63.5ꢁ7.7
82.5ꢁ8.1
13.4ꢁ21.8
63.2ꢁ6.2
9
10
11
12
13
14
0.0ꢁ5.6
0.0ꢁ7.9
0.0ꢁ2.0
0.0ꢁ2.3
0.0ꢁ4.3
0.0ꢁ1.8
0.0ꢁ6.5
98.1ꢁ0.2
58.3ꢁ7.8
77.7ꢁ0.5
75.6ꢁ1.1
58.7ꢁ14.0
86.7ꢁ1.6
89.1ꢁ0.2
52.0ꢁ3.6
97.4ꢁ0.5
15
E-64c)
c) Positive control.
cytotoxicity and cathepsin B assays are closely related to the cancer process, it was
noticed that the compounds with high cytotoxic activities in tumor cells lines (Table 2)
exhibited low inhibitions or even did not inhibit the cathepsin B. These selected
chalcones, i.e., 4, 6, 13, and 14, with inhibitions higher than 80% the IC50 values were
evaluated against cathepsin K (Table 5) and showed moderate activities.
Table 5. IC50 Values of Chalcones 4, 6, 13, and 14 against Cathepsin K
Compound
IC50 Values [mm]a)
4
6
13
14
60.3ꢁ1.3
109.9ꢁ0.9
98.8ꢁ0.9
42.5ꢁ0.9
a) The values represent means of three replicatesꢁSD.
In summary, this study strongly suggests that, besides both assays being related to
the cancer disease, based on our data we could not establish a correlation between the
cytotoxic activity in tumor cell lines and the inhibition of cathepsin B. Although these
findings are preliminary, further advanced studies should be conducted to understand
this pathological process. This is the first time that the inhibition activities of these
chalcone derivatives against cathepsin B and cathepsin K had been investigated.
Conclusions. – We synthesized a number of known chalcone derivatives with
different aromatic substituents. Their structures were established by spectroscopic data
which correspond to those in the literature, and the screening for cytotoxicity revealed
that the compounds 12, 13, 14, and 15 showed significant activities against MDA-MB-
435, SF-295, and HCT-8 cell lines. Screening of chalcone derivatives against cathepsin K
disclosed compounds 4, 6, 13, and 14 to possess moderate potencies as enzyme
inhibitors. However, no compound showed high activity against cathepsin B.
The authors gratefully acknowledge CAPES, CNPq and FAPESP for financial support and
fellowship.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Experimental Part
General. All commercially available reagents were purchased from Aldrich Chemical Co. M.p.:
Micro Quꢀmica MQAPF-301 apparatus. IR Spectra: Bomem M102 spectrometer. 1H-NMR spectra:
Bruker DRX-400 (400 MHz) instrument. MS: Shimadzu GCMS-QP2000 instrument.
Chalcone Synthesis. Acidic Conditions. A soln. of the required acetophenone derivative (2.0 mmol)
and 4-hydroxybenzaldehyde (2.0 mmol) in MeOH with a cat. amount of H2SO4 was heated at reflux for
24 h, and then it was neutralized with 15% NaOH. The org. phase was extracted with AcOEt and dried
(Na2SO4) to yield compounds 1 and 2.
Chalcone Synthesis. Basic Conditions. A soln. of acetophenone derivative (2 mmol) and an
appropriate aldehyde (2 mmol) in MeOH (15 ml) with KOH 20% was stirred. The reaction time and the
temp. dependend upon the substituents in the aromatic ring. The mixture was filtered, and then the
precipitate was collected to yield compounds 3–15.
Cytotoxicity Assay. Chalcones were tested for cytotoxicity against three tumor cell lines: MDA-MB-
435 (melanoma), HCT-8 (colon cancer), and SF-295 (central nervous system). All cell lines were
obtained from National Cancer Institute (NCI) and were cultivated in RPMI 1640 medium supplemented
with 10% fetal bovine serum, 2 mm glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, at 378,
with 5% CO2. The MTTassay was used for the cytotoxicity evaluation of chalcones. The cells were placed
in 96-well plates (105 cells/well for adherent cells or 0.5ꢂ105 cells/well for those suspended in 100 ml of
medium). Doxorubicin at 100 mg/ml and DMSO at 0.005% were used as positive and negative controls,
resp. After 24 h, the chalcones were added into the wells to obtain the final concentration of 125 mg/ml.
After 72 h, plates were centrifuged (5000 rpm, 10 min), and the supernatants were removed. An aliquot
of 200 ml from the MTT soln. (0.5 mg/ml) was added into each well, and the plates were incubated for 3 h
under the same conditions described above. Then, DMSO (150 ml) was poured to dissolve the precipitate,
and the absorbance was measured at 550 nm. This experiment was run in three replicates, and all
absorbance values were converted into a cell growth inhibition percentage (GI-%) by the following
formula: GI-%¼100ꢀ[(T/C)ꢂ100%]. C is the absorbance for the negative control, and T was the
absorbance in the presence of the tested extract. Those compounds that presented more than 85% of
activity were selected to be tested at concentrations varying from 0.078 to 5 mg/ml to determine IC50
values by nonlinear regression using GraphPad Prism 4.0 software.
Inhibition Assay of Cathepsin B and Cathepsin K. The protease inhibitor activity was carried out in
triplicate in 96-well black plate by the method described by Barret et al. [37]. Test compounds were
dissolved in DMSO, and then these inhibitors were screened against both the cathepsins B and K at initial
concentration of 125 mm. The mixture contained 192 ml of a AcONa buffer (100 mm, 5 mm EDTA, 5 mm
DTE, pH 5.5), 2 ml of 1 mm Z-Phe-Arg-MCA, 5 ml of sample, and 1 ml of cathepsin (32 nm). The enzyme
was activated during 5 min with DTT at 278, then the soln. was incubated during 5 min with the sample.
The substrate was added to start the reaction, and the fluorescence of released 7-amino-4-
methylcoumarin (AMC) was measured at lex 355 and lem 460 nm. Control assays were performed with
DMSO (negative control) and with E-64 (positive control). E-64 is an epoxide, 1-[N-[(l-3-trans-
carboxyoxirane-2-carbonyl)-l-leucyl] amino]-4-guanidinobutane, which can irreversibly inhibit a wide
range of cysteine peptidases and was first isolated and identified from Aspergillus japonicus in 1978 [38].
Those compounds that showed more than 80% of inhibition were selected for the test of IC50. The values
of IC50 were determined by rate measurements for at least seven inhibitor concentrations. Kinetics
parameters were determined from collected data employing the SigmaPlot enzyme kinetics module.
REFERENCES
[1] WHO, World Health Organization, available in: http://www.who.int/mediacentre/factsheets/fs297/
en/index.html; accessed August 6th, 2012.
[2] P. Cozzi, Farmaco 2003, 58, 213.
[3] M. Cabrera, M. Simoens, G. Falchi, M. L. Lavaggi, O. E. Piro, E. E. Castellano, A. Vidal, A.
´
´
Azqueta, A. Monge, A. L. de Cerain, G. Sagrera, G. Seoane, H. Cerecetto, M. Gonzalez, Bioorg.
Med. Chem. 2007, 15, 3356.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2005
[4] L. Juvale, V. F. S. Pape, M. Wiese, Bioorg. Med. Chem. 2012, 20, 346.
˜
[5] C. M. O. Simoes, E. P. Schenkel, G. Gosmann, J. C. P. Mello, L. A. Mentz, P. R. Petrovick,
´
ꢂFarmacognosia: da Planta ao Medicamentoꢃ, Ed. UFRGS, Florianopolis, 2007.
[6] A. L. Kiselyov, K. V. Balakin, S. E. Tkachenko, N. Savchuk, A. V. Ivachtchenko, Anti-Cancer
Agents Med. Chem. 2007, 7, 189.
˜
[7] C. Echeverria, J. F. Santibanez, O. Donoso-Tauda, C. A. Escobar, R. Ramirez-Tagle, Int. J. Mol. Sci.
2009, 10, 221.
[8] Z. Nowakowska, Eur. J. Med. Chem. 2007, 42, 125.
[9] Y. Yasuda, J. Kaletab, D. Bromme, Adv. Drug Delivery Rev. 2005, 57, 973.
[10] M. Liu, P. Wilairat, M.-L. Go, J. Med. Chem. 2001, 44, 4443.
[11] S. F. Nielsen, M. Chen, T. G. Theander, A. Kharazmi, S. B. Christensen, Bioorg. Med. Chem. Lett.
1995, 5, 449.
[12] S. Vogel, M. Barbic, G. Jꢁrgenliemk, J. Heilmann, Eur. J. Med. Chem. 2010, 45, 2206.
[13] J. C. Trivedi, J. B. Bariwal, K. D. Upadhyay, Y. T. Naliapara, S. K. Joshi, C. C. Pannecouque, E. De
Clercqd, A. K. Shah, Tetrahedron Lett. 2007, 48, 8472.
[14] G. S. B. Viana, M. A. M. Bandeira, F. J. A. Matos, Phytomedicine 2003,10, 189.
[15] R. De Vincezo, C. Ferlini, M. Distefano, C. Gaggini, A. Riva, E. Bombardelli, P. Morazzoni, P.
Valenti, F. Belluti, F. O. Ranelletti, S. Mancuso, G. Scambia, Cancer Chemother. Pharmacol. 2000,
46, 305.
[16] M. L. Go, X. Wu, X. L. Liu, Curr. Med. Chem. 2005, 12, 483.
[17] H. N. Pati, H. L. Holt Jr., R. LeBlanc, J. Dickson, M. Stewart, T. Brown, M. Lee, Med. Chem. Res.
2005, 14, 19.
[18] H. O. Saxena, U. Faridi, J. K. Kumar, S. Luqmand, M. P. Darokar, K. Shanker, C. S. Chanotiya,
M. M. Gupta, A. S. Negi, Steroids 2007, 72, 892.
[19] H. N. Pati, U. Das, H. Sakagami, M. Kawase, Q. Chu, Q Wang, J. P. Stables, J. R. Dimmock, Arch.
Pharm. 2010, 343, 535.
[20] O. Vasiljeva, B. Turk, Biochimie 2008, 90, 380.
[21] J. Reiser, B. Adair, T. Reinheckel, J. Clin. Invest. 2010, 10, 3421.
[22] G.-Z. Zeng, X.-L. Pan, N.-H. Tan, J. Xiong, Y.-M. Zhang, Eur. J. Med. Chem. 2006, 41, 1247.
[23] V. Turk, J. Kos, B. Turk, Cancer Cell 2004, 5, 409.
[24] D. Brçmme, F. Lecaille, Expert Opin. Invest. Drugs 2009, 18, 585.
[25] D. G. Powers, D. S. Casebier, D. Fokas, W. J. Ryan, J. R. Troth, D. L. Coffen, Tetrahedron 1998, 54,
4085.
[26] W. D. Seo, J. H. Kim, J. E. Kang, H. W. Ryu, M. J. Curtis-Long, H. S. Lee, M. S. Yang, K. H. Park,
Med. Chem. Lett. 2005, 15, 5514.
[27] K. N. Gusak, N. G. Kozlov, Russ. J. Gen. Chem. 2003, 73, 964.
[28] S. Iwata, T. Nishino, N. Nagata, Y. Satomi, H. Nishino, S. Shibata, Biol. Pharm. Bull. 1995, 18,
1710.
[29] M. V. B. Reddy, C.-R. Su, W.-F. Chiou, Y.-N. Liu, R. Y.-H. Chen, K. F. Bastow, K.-H. Lee, T.-S. Wu,
Bioorg. Med. Chem. 2008, 16, 7358.
[30] S. Ducki, D. Rennison, M. Woo, A. Kendall, J. F. D. Chabert, A. T. McGown, N. J. Lawrence,
Bioorg. Med. Chem. 2009, 17, 7698.
[31] K. Henmi,Y. Hiwatashi, E. Hikita, N. Toyama, T. Hirano, Biol. Pharm. Bull. 2009, 32, 1109.
[32] P. C. Jimenez, S. C. Fortier, T. M. C. Lotufo, C. Pessoa, M. E. A. Moraes, M. O. de Moraes, L. V.
Costa-Lotufo, J. Exp. Mar. Biol. Ecol. 2003, 287, 93.
[33] B. F. Sloane, J. Rozhin, K. Johnson, H. Taylor, J. D. Crissman, K. V. Honn, Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 2483.
[34] B. Turk, D. Turk, V. Turk, Biochim. Biophys. Acta – Protein Struct. Mol. Enzymol. 2000, 1477,
98.
´
[35] A. M. Eijan, E. O. Sandes, M. D. Riveros, S. Thompson, L. Pasik, H. Mallagrino, F. Celeste, A. R.
´
Casabe, Cancer 2003, 98, 262.
[36] D. Kuester, H. Lippert, A. Roessner, S. Krueger, Pathol. Res. Pract. 2008, 204, 491.

2006
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
[37] A. J. Barrett, A. A. Kembhavi, M. A. Brown, H. Kirschke, C. G. Knight, M. Tamai, K. Hanada,
Biochem. J. 1982, 201, 189.
[38] K. Hanada, M. Tamai, M. Yamagishi, S. Ohmura, J. Sawada, I. Tanaka, Agric. Biol. Chem. 1978, 42,
523.
Received October 10, 2012