Synthesis and investigation of dihydroxychalcones as calpain and cathepsin inhibitors

Article abstract of DOI:10.1016/j.bioorg.2013.09.002

In order to identify potential calpain and cathepsin inhibitors we prepared 12 dihydroxychalcone analogues and tested their ability to inhibit μ-calpain, m-calpain, cathepsins B and L. In the calpain inhibition test, compound 10 exhibited the most active

Full text of DOI:10.1016/j.bioorg.2013.09.002

Accepted Manuscript
Synthesis and Investigation of Dihydroxychalcones as Calpain and Cathepsin
Inhibitors
Kyung Hye Baek, Radha Karki, Eung-Seok Lee, Younghwa Na, Youngjoo
Kwon
PII:
S0045-2068(13)00064-3
DOI:
http://dx.doi.org/10.1016/j.bioorg.2013.09.002
YBIOO 1679
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
9 May 2013
Please cite this article as: K.H. Baek, R. Karki, E-S. Lee, Y. Na, Y. Kwon, Synthesis and Investigation of
Dihydroxychalcones as Calpain and Cathepsin Inhibitors, Bioorganic Chemistry (2013), doi: http://dx.doi.org/
10.1016/j.bioorg.2013.09.002
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Bioorganic Chemistry
Synthesis and Investigation of Dihydroxychalcones as Calpain and
Cathepsin Inhibitors
Kyung Hye Baek1†, Radha Karki2†, Eung-Seok Lee2, Younghwa Na3*, Youngjoo Kwon1*
1College of Pharmacy, Graduate School of Pharmaceutical Sciences, Global Top5 Research
Program, Ewha Womans University, Seoul 120-750, Korea,
2College of Pharmacy, Yeungnam University, Gyeongsan, 712-749, Korea,
3College of Pharmacy, CHA University, Pocheon, 487-010, Korea
Key words: Dihydroxychalcone, Calpain inhibitor, Cathepsin B & L Inhibitor
*To whom correspondence should be addressed. Y. Na: Phone: 82-31-8017-9896. Fax: 82-31-
8017-9620. E-mail: yna7315@cha.ac.kr; Y. Kwon: Phone: 82-2-3277-4653. Fax: 82-2-3277-
3051. E-mail: ykwon@ewha.ac.kr.
† These authors equally contributed to this work.

Abstract
In order to identify potential calpain and cathepsin inhibitors we prepared 12
dihydroxychalcone analogues and tested their ability to inhibit -calpain, m-calpain,
cathepsins B and L. In the calpain inhibition test, compound 10 exhibited the most active
inhibitory activity against m-calpain with an IC50 value of 25.25 ± 0.901 M. With respect to
inhibition of cathepsins B and L, compound 13 exhibited the most potent inhibitory activity
on cathepsin L and moderate inhibitory activity on cathepsin B with IC50 values of 2.80 ±
0.100 and 11.47 ± 0.087 M, respectively. Our results suggest the possibility of developing
dual calpain and cathepsin inhibitors by properly modulating structures and/or combining the
essential aspects of the functional group effective for specific calpain and cathepsin inhibition.

1. Introduction
Cathepsins belong to a family of cysteine proteases and a class of proteolytic enzymes. A
total of 11 human cathepsins have been reported to date, several of which are highly
expressed in malignant cells [1]. Cathepsins function in a diverse set of roles after being
released into the extracellular system during tumorigenic process [2-4]. Especially, cathepsins
B and L are involved in the progression of human pancreatic cancer [5,6]. Studies performed
using the ripi-tag2 mouse model also showed that cell proliferation and tumor growth were
reduced in cathepsins B and L knockout mice, indicating that cathepsins B and L may be
potential therapeutic targets for anti-cancer drugs [5,7]. Cathepsins have also been implicated
in the pathogenesis of Alzheimer’s disease (AD) [8,9]. Specifically, neurotoxic insoluble β-
amyloids are generated through sequential cleavage of amyloid precursor protein (APP) by β-
site APP-cleaving enzyme (BACE1) and -secretase [9,10]. Cathepsins S, B and L cleave the
wild type β-site of APP faster than BACE1 [9]. In addition, cathepsin B has been identified as
a BACE1 in secretory vesicles [8]. Treatment with the cathepsin B specific inhibitor CA074
results in improved memory ability and reduction of β-amyloid formation in an AD animal
model expressing the wild type β-site of APP, which is observed in the most sporadic AD
patients [11]. The most well-studied synthetic cathepsin B inhibitors reported to date are
CA074 derivatives, namely epoxysuccinyl inhibitors, and vinyl sulfone inhibitors [12,13].
Both series of cathepsin B specific inhibitors are peptide-based inhibitors and derived based
on the tertiary structure of substrate binding pocket of cathepsin B. Cathepsin L specific
inhibitors are also developed based on its tertiary structure due to the structural similarity
between cathepsins B and L. However, the binding cleft of cathepsin L has no bulky residues
compared to that of cathepsin B [12]. Therefore, known cathepsin L inhibitors possessing
peptide-like characteristics are in general more flexible and adopt bulky side chains such as
benzyl and biphenyl groups compared to cathepsin B inhibitors [12,14].

Calpains are a family of intracellular Ca2+-dependent cysteine proteases that were first
discovered in the CNS [15]. Since their initial discovery, expression of calpains has been
observed in various cells and tissues [16,17]. Calpains comprises 15 isoforms that are
expressed either ubiquitously or in a tissue-specific manner, depending on the isoform [18].
Calpain 1 ( -calpain) and calpain 2 (m-calpain) require micromolar and millimolar Ca2+ ion
concentrations to reach full activation in vitro, respectively, and are the most widely studied
isoforms [19,20]. It has been known that the abnormal over-activation of calpains is related to
a number of human diseases such as brain and spinal cord injury, Alzheimer disease, cancer
and cataract formation [21,22]. The human body is consistently regulating intracellular
calcium levels, and if a cell receives a stimulus such as oxidative stress, calcium homeostasis
is perturbed and the corresponding levels of intracellular calcium are up-regulated [23]. Such
calcium influx activates calpains, which in turn cleave many cytosolic proteins including α-
spectrin, MAP-2 and tau, resulting in cytoskeletal rearrangements [17,24]. Heat shock protein
(Hsp) 70.1, which is known as a protector for lysosomal membrane integrity from oxidative
stress, is also cleaved by activated µ-calpain and carbonylated Hsp 70.1 in hippocampal
cornu Ammonis 1 (CA1) tissue induced by oxidative stress is even more susceptible to µ-
calpain-mediated cleavage leading to lysosomal rupture [25,26]. Lysosomal rupture is
accompanied by the release of cathepsins, including cathepsin B, L and D, into the cytoplasm.
These released cathepsins may in turn damage cellular constitutive proteins and cytoskeletons
[27]. Especially, the released cathepsin B causes apoptotic neuronal cell death via activation
of proapoptotic Bax and Bak and subsequent caspase activation pathway [28,29]. The
released cathepsin L induces proteolytic activation of Bid, which in turn activates caspase-9
and caspase-3 [27]. The proteolytic activation of caspase-3 can be mediated by calpains
besides cathepsin B and L and cleaved caspase-3 degrades calpastatin resulting in its
inactivation, which consequently make worse pathological over-activation of calpain and

cathepsins-mediated neuronal cell apoptosis [30,31]. Calpastatin, a well-known endogenous
calpain inhibitor, has been reported to be involved in cell survival, apoptosis, and cognitive
function in human brain by regulating calpain activation [30]. In this regard, the simultaneous
regulation of µ-calpain and cathepsin B and L may be beneficial. Synthetic calpain inhibitors
can be categorized into peptide-base inhibitors and non-peptide inhibitors [32]. Peptide-based
calpain inhibitors are developed on the basis of calpain substrate. In general, they contain L-
Val or L-Leu and are easily degraded by several proteases in cells due to their peptide-like
characteristics and presence of an aldehyde moiety [33,34]. Conversely, several non-peptide
calpain inhibitors have been reported, including chromone carboxamides, benzothiazine-1,1-
dioxide, carboxamides, and aminoquinolinones [32,35-38]. Non-peptide calpain inhibitors
may serve as better scaffolds for developing anti-neurodegenerative drugs because of their
superior pharmacokinetic profiles compared with peptide-based calpain inhibitors [32].
Chalcones obtained by synthesis and from natural resources exhibit a diverse range of
pharmacological effects, including anti-proliferative, anticancer, antioxidant, anti-
inflammatory, and antibiotic activities [39]. We previously reported that certain synthetic
chalcones act as inhibitors of -calpain and cathepsins B and L [40]. As a continuation of this
work to identify efficient calpain and cathepsins B and L inhibitors, we prepared several new
dihydroxychalcone analogues and tested their biological activity using calpains and cathepsin
B and L.
B
A
O
Chalcone
2. Results and Discussion

2.1. Synthesis
All of the dihydroxylated chalcones were synthesized by a base-catalyzed Claisen-Schmidt
condensation reaction using an appropriate aldehyde and ketone as previously described in
the literature method with modification [39]. The general synthetic procedure is shown in
scheme 1. Briefly, aldehyde and ketone reagents containing hydroxyl groups were protected
using either 2H-3,4-dihydropyran [41] or methyl chloromethyl ether (MOMCl) [42,43].
Overall the reaction proceeded smoothly with reaction yields of 64.5 ~ 80.2%; however,
compounds 6–8, 13 and 14 exhibited low reaction yields of 2.3 ~ 10.0%. We found that these
compounds are relatively hydrophilic in nature which made the usual workup process
difficult and may explain the low reaction yields of these compounds.
Activated silica-supported sodium hydrogen sulfate (NaHSO4.SiO2) [44,45] was used for the
deprotection of compound 14, since acid hydrolysis with aqueous HCl did not yield the
desired product. All of the compounds prepared showed consistent spectroscopic data
corresponding to the desired structures.

A)
O
O
HO
Ar
1) 4M-NaOH
2) 4M-HCl
+
Ar
H
CH3
O
OH
O
OH O
1 Ar = 4-pyridyl
2.Ar = 3-pyridyl
3.Ar = 2-Pyridyl
4.Ar = 2-Thiophenyl
5 Ar = 2-Furanyl
6 Ar = 4-pyridyl
7 Ar = 3-pyridyl
8 Ar = 2-Pyridyl
9. Ar = 2-Thiophenyl
10 Ar = 2-Furanyl
OH
B)
OR2
1) 4M-NaOH
H
+
OH
2) 4M-HCl
or NaHSO4SiO2
X
X
O
O
OR1
O
X = S, O
13 R = S,
14 R = O
11 R1 = H, R2 = THP
12 R1, R2 = MOM
C)
OMOM
O
OH
Ar
1) 4M-NaOH
2) 2M-HCl
+
Ar
H
CH3
MOMO
HO
O
O
15 Ar = 2-Thiophenyl
16.Ar = 2-Furanyl
17.Ar = Phenyl
18.Ar = 3-Thiophenyl
19 Ar = 3-Furanyl
20 Ar = 2-Thiophenyl
21.Ar = 2-Furanyl
22.Ar = Phenyl
23.Ar = 3-Thiophenyl
24 Ar = 3-Furanyl
Scheme 1. Synthetic methods for the prepared compounds

N
S
HO
HO
N
HO
HO
N
OH
O
OH
O
OH
O
OH
OH
O
6
7
8
9
OH
OH
O
S
HO
OH
OH
HO
S
O
OH
OH
O
O
O
O
10
13
14
20
O
OH
OH
OH
S
O
HO
HO
HO
HO
O
O
O
O
21
22
23
24
Figure 1. Structure of synthesized compounds
2.2. Inhibitory activity against - and m-calpain
μ-Calpain and m-calpain inhibitory activities of compounds were determined according to
the method previously reported [37]. Briefly, fluorometric assays were performed in 96-well
plates and fluorescence intensity in each well was measured using a Microplate Fluorescence
Reader (SpectraMAX GEMINI EM, Molecular Devices); the IC50 values were obtained using
the data graphing software, TableCurve 2D (Systat software Inc.). In the assay, only
compounds 9, 10 and 13 inhibited m-calpain activity with moderate efficiency. All of the
other compounds were inactive. This result implied that 2,4-dihydroxy group on the A ring of
the chalcone structure (comparison of 9 vs 20) was effective while presence of pyridine on
the B ring in the chalcone structure hampered m-calpain inhibitory activity (comparison of 6

– 8 vs 9 and 10). Thus, it is possible that the electronic effects (thiopehene and furan vs
pyridine) of the B ring site in the chalcone structure influence m-calpain inhibitory activity,
although concrete evidence of this possibility was not obtained in the present study.
2.3. Inhibitory activity against cathepsin B and L
To determine the ability of compounds to inhibit cathepsins B and L, two substrates were
used, namely, RR-AMC (Sigma, USA) for cathepsin B and Z-FR-AMC (Sigma, USA) for
cathepsin L. Compound 13 exhibited effective inhibitory activity against both cathespin B
and L with IC50 values of 11.47 ± 0.087 and 2.80 ± 0.100 M, respectively. Compound 14 was
also a strong inhibitor of cathespin L with and an IC50 value of 2.87 ± 0.052 M. The other
compounds either mildly inhibited cathepsin function or were inactive. Together, these results
suggest that the 2,4-dihydroxy group on the B ring of the chalcone structure is important for
cathespin inhibition; however, we were unable to conclude a clear structure-activity
relationship due to the limited numbers of compounds employed in this study. Regardless of
position, the results of our study indicate that dihydroxy group in the A ring was not proper
substituent for cathepsin B and L inhibitory activity.
3. Conclusion
In the present study we designed and synthesized 12 dihydroxychalcone analogues. Among
the compounds that were prepared and tested, compound 10 exhibited the most active
inhibitory activity against m-calpain with an IC50 value of 25.25 ± 0.901 M. With respect to
inhibition of cathepsins B and L, compound 13 potently inhibited cathepsin L and moderately
inhibited cathepsin B with IC50 values of 2.80 ± 0.100 and 11.47 ± 0.087 M, respectively.
Based on our observations, the inhibitory activity of chalcone analogues on calpains and

cathepsins appeared to require different functional groups. Specifically, a 2,4-dihydroxy
group on the A ring was important for m-calpain inhibition while a 2,4-dihydroxy group on
the B ring was needed for the inhibition of cathespin B and L. Our results suggest the
possibility of developing calpain and cathepsin dual inhibitors by properly modulating
structures and/or combining the essential aspects of the functional group effective for specific
calpain and cathepsin inhibition.
4. Experimental
4.1. General Remarks
Compounds used as starting materials and reagents were obtained from Aldrich Chemical Co.,
Junsei or other chemical companies, and were utilized without further purification. HPLC
grade acetonitrile (ACN) and methanol were purchased from Burdick and Jackson, USA.
Thin-layer chromatography (TLC) and column chromatography (CC) were performed with
Kieselgel 60 F254 (Merck) and silica gel (Kieselgel 60, 230-400 mesh, Merck), respectively.
Because all of the prepared compounds contained aromatic rings, they were visualized and
detected on TLC plates with UV light (short wave, long wave or both). NMR spectra were
recorded on a Bruker AMX 250 or 300 (250 or 300 MHz, FT) for 1H NMR and 62.5 or 75.5
MHz for 13C NMR, and chemical shifts were calibrated according to TMS. Chemical shifts
(δ) were recorded in ppm and coupling constants (J) in hertz (Hz). Melting points were
determined in open capillary tubes on electrothermal 1A 9100 digital melting point apparatus
and were uncorrected.
HPLC analyses were performed using two Shimadzu LC-10AT pumps gradient-controlled
HPLC system equipped with Shimadzu system controller (SCL-10A VP) and photo diode
array detector (SPD-M10A VP) utilizing Shimadzu Class VP program. A sample volume of

10 μL was injected in a Waters X- Terra® 5 μM reverse-phase C18 column (4.6 ⅹ250 mm) with
a gradient elution of 40% to 100% of B in A for 10 min followed by 100% to 40% of B in A
for 10 min at a flow rate of 1.0 mL/min at 254 nm UV detection. The mobile phase A
consisted of double distilled water with 20 mM ammonium formate (AF) and B consisted of
90% ACN in water with 20 mM AF. The purity of each compound was described as a
percentage (%).
4.2. General synthetic method for protecting starting materials
Aldehyde or ketone containing hydroxyl groups were protected using either 2H-3,4-
dihydropyran or methyl chloromethyl ether (MOMCl).
Synthesis of 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethanone
A solution of 2',4'- dihydroxyacetophenone (7.60 g, 50.00 mmol, 1.00 equiv.), pyridinium p-
toluene sulfonate (0.50 g, 2.00 mmol, 0.04 equiv.) and 3,4-dihydro-2H-pyran (11.40 mL,
125.00 mmol, 2.50 equiv.) in methylene chloride was stirred for 4 h at room temperature.
Then the reaction mixture was then extracted with methylene chloride (100 mL) and washed
with water (30 mL x 2) and a 1 M solution of Na2CO3 (30 mL x 2). The organic layer was
separated and dried over anhydrous Na2SO4, and concentrated. When necessary, the residue
was purified by silica gel column chromatography to yield a light orange solid compound
1
(11.61 g, 98.3%). Rf = 0.38, E:H = 1:5, v/v; H NMR (250 MHz, CDCl3) δ 12.58 (s, 1H,
phenyl 2-OH), 7.65 (d, J = 8.8 Hz, 1H, phenyl H-6), 6.62 (d, J = 2.3 Hz, 1H, phenyl H-3),
6.57 (dd, J = 8.8, 2.4 Hz, 1H, phenyl H-5), 5.48 (t, J = 3.1 Hz, 1H, pyran H-2), 3.88-3.79 (m,
1H, pyran H-6), 3.64-3.59 (m, 1H, pyran H-6), 2.55 (s, 3H, CH3), 2.02-1.56 (m, 6H, pyran H-
3, H-4, H-5)

Synthesis of (2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde (11)
A solution of 2,4- dihydroxybenzaldehyde (2.76 g, 20.00 mmol, 1.00 equiv.), pyridinium p-
toluene sulfonate (0.20 g, 0.80 mmol, 0.04 equiv.) and 3,4-dihydro-2H-pyran (4.01 mL, 44.00
mmol, 2.20 equiv.) in methylene chloride (20 mL) was stirred for 4 h at room temperature.
Then the reaction mixture was extracted then with methylene chloride (70 mL) and washed
with water (25 mL x 2) and a 1 M solution of Na2CO3 (20 mL x 2). The organic layer was
separated and dried over anhydrous Na2SO4, and concentrated. When necessary, the residue
was purified by silica gel column chromatography to yield an off-white viscous compound
1
(4.01 g, 90%). Rf = 0.40, E:H = 1:5, v/v; H NMR (250 MHz, MeOD-d4) δ 9.77 (s, 1H,
CHO), 7.56 (d, J = 7.5 Hz, 1H, phenyl H-6), 6.67 (dd, J = 10.0, 2.5 Hz, 1H, phenyl H-5),
6.55 (dd, J = 2.5 Hz, 1H, phenyl H-3), 5.51 (t, J = 2.5 Hz, 1H, pyran H-2), 3.85-3.75 (m, 1H,
pyran H-6), 3.64-3.56 (m, 1H, pyran H-6), 1.90-1.55 (m, 6H, pyran H-3, H-4, H-5)
Synthesis of 2,4-bis(methoxymethoxy)benzaldehyde (12)
To a solution of 2,4- dihydroxybenzaldehyde (2.00 g, 14.48 mmol, 1.00 equiv.) in DMF (43
mL), diisopropylethylamine (10.08 mL, 57.92 mmol, 4.00 equiv.) was added followed by
dropwise addition of MOMCl (4.39 mL, 57.92 mmol, 4.00 equiv.) at room temperature. The
reaction mixture was then stirred for 5 h at room temperature. Next, the reaction mixture was
extracted with ethyl acetate (70 mL) and washed sequentially with water (15 mL x 2), a 5%
NaOH solution (10 mL x 2) and a sat. NaCl solution (10 mL x 2). The organic layer was
separated, dried over anhydrous Na2SO4, and concentrated to give a viscous compound. The
final residue was separated by silica gel column chromatography using ethyl acetate and
hexane as eluents to yield an off-white viscous compound (3.13 g, 95.7%). Rf = 0.48, E:H =
1:2, v/v; 1H NMR (250 MHz, CDCl3) δ 10.34 (s, 1H, CHO), 7.82 (d, J = 8.7 Hz, 1H, phenyl

H-6), 6.84 (d, J = 2.1 Hz, 1H, phenyl H-3), 6.77 (dd, J = 8.7, 2.1 Hz, 1H, phenyl H-5), 5.28 (s
2H, OCH2), 5.22 (s, 2H, OCH2), 3.52 (s, 3H, OCH3), 3.48 (s, 3H, OCH3)
Synthesis of 1-(3,5-bis(methoxymethoxy)phenyl)ethanone
To a solution of 3',5'- dihydroxyacetophenone (1.00 g, 6.57 mmol, 1.00 equiv.) in ACN (25
mL), diisopropylethylamine (4.57 mL, 26.28 mmol, 4.00 equiv.) was added followed by
dropwise addition of MOMCl (2.00 mL, 26.28 mmol, 4.00 equiv.) at room temperature. The
reaction mixture was then stirred for 5 h at room temperature. The reaction mixture was then
extracted with ethyl acetate (60 mL) and washed sequentially with water (10 mL x 2), a 5%
NaOH solution (10 mL x 2) and a sat. NaCl solution (10 mL). The organic layer was then
separated, dried over anhydrous Na2SO4, and concentrated to give a brown viscous
compound (1.30 g, 82.8%). Rf = 0.41, E:H = 1:2, v/v; 1H NMR (250 MHz, CDCl3) δ 7.27 (d,
J = 2.2 Hz, 2H, phenyl H-2, H-6), 6.93 (t, J = 2.2 Hz, 1H, phenyl H-4), 5.19 (s, 4H, OCH2),
3.48 (s, 6H, OCH3), 2.55 (s, 3H, CH3)
4.3. General synthetic method for hydroxylated chalcones
Method A: To an equimolar amount of the appropriate aldehyde and ketone in methanol/
methylene chloride was added 4 M NaOH (2 equiv.) and left to react for 24 hours at room
temperature. The protecting group was subsequently removed by acid hydrolysis using 4 M
HCl (3 equiv.) and reacted for 4 h at room temperautre. Water was added to the reaction
mixture (for compounds containing pyridine ring, neutralization with sat. NaHCO3 after
deprotection). The solid obtained was filtered and recrystallized in ethanol to yield desired
chalcones. If solid was not formed, the mixture was extracted with methylene chloride and
washed with water and brine. It was then dried over Na2SO4 and concentrated. The residue
was then purified by silica gel column chromatography using ethyl acetate and hexane to

obtain desired product.
Method B: To the equimolar amount of appropriate aldehyde and ketone in methylene
chloride was added 4 M NaOH ( 2 equiv.) dropwise and the reaction was stirred for 3-5 h at
0 °C. Water was added to the reaction mixture. The solid obtained was filtered and washed
with cold MeOH. The residue was then purified by silica gel column chromatography using
1
ethyl acetate and hexane. The compound was characterized by H NMR. The protecting
groups were subsequently removed by acid hydrolysis using activated NaHSO4.SiO2 (2.5
equiv.) and left to react for 3 h at room temperature. Then the catalyst was filtered off and
washed with methanol. The filtrate was concentrated and dried. The final residue was
separated by silica gel column chromatography to obtain the desired product.
Method C: To the equimolar amount of appropriate aldehyde and ketone in ethanol was
added 4 M NaOH (2 equiv.) and the reaction was stirred for 20-24 h at room temperature.
Then the reaction mixture was extracted with ethyl acetate and washed with water and brine.
It was then dried over Na2SO4 and concentrated. The residue was then purified by silica gel
column chromatography using ethyl acetate and hexane to obtain desired protected chalcone.
1
The compound was characterized by H NMR. The protected chalcone was dissolved in
methanol followed by the addition of 2 M HCl (3 equiv.) at 60 °C and left to react for 1 h.
The reaction mixture was submitted to the usual workup with ethyl acetate to give crude
product, which was purified by chromatography on a silica gel column to give the desired
product.
4.3.1. 1-(2,4-Dihydroxyphenyl)-3-(pyridin-4-yl)propenone (6)
Method A was employed with 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)

ethanone and 4-pyridine carboxaldehyde to obtain yellow solid compound (0.10 g, 2.4%). mp
1
238-240 °C; Rf = 0.31, E:H = 3:1, v/v; purity by HPLC: 99%; H NMR (250 MHz, DMSO-
d6) δ 12.94 (s, 1H, 1-phenyl 2-OH), 11.07 (br, 1H, 1-phenyl 4-OH), 8.95 (d, J = 7.5 Hz, 2H,
3-pyridine H-2, H-6), 8.44 (d, J = 15.0 Hz, 1H, CO-CH=CH), 8.42 (d, J = 7.5 Hz, 2H, 3-
pyridine H-3, H-5), 8.23 (d, J = 7.5 Hz, 1H, 1-phenyl H-6), 7.85 (d, J = 15.0 Hz, 1H, CO-
CH=CH), 6.51 (dd, J = 10.0, 2.5 Hz, 1H, 1-phenyl H-5), 6.38 (d, J = 2.5 Hz, 1-phenyl H-3);
13C NMR (62.5 MHz, DMSO-d6) δ 190.46, 166.37, 166.00, 149.75, 143.54, 137.78, 133.79,
131.07, 125.36, 113.40, 108.96, 102.86.
4.3.2. 1-(2,4-Dihydroxyphenyl)-3-(pyridin-3-yl)propenone (7)
Method A was employed with 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)
ethanone and 3-pyridine carboxaldehyde to obtain yellow solid compound (0.22 g, 5.3%). mp
1
247-249 °C; Rf = 0.33, E:H = 3:1, v/v; purity by HPLC: 98%; H NMR (250 MHz, DMSO-
d6) δ 13.11 (s, 1H, 1-phenyl 2-OH), 10.91 (br, 1H, 1-phenyl 4-OH), 9.32 (s, 1H, 3-pyridine
H-2), 8.88-8.81 (m, 2H, 3-pyridine H-6, H-4), 8.29 (d, J = 16.0 Hz, 1H, CO-CH=CH), 8.23
(d, J = 9.3 Hz, 1H, 1-phenyl H-6), 7.97 (dd, J = 8.0, 5.4 Hz, 1H, 3-pyridine H-5), 7.88 (d, J =
15.6 Hz, 1H, CO-CH=CH), 6.48 (dd, J = 8.8, 2.3 Hz, 1H, 1-phenyl H-5), 6.35 (d, J = 2.3 Hz,
1-phenyl H-3); 13C NMR (62.5 MHz, DMSO-d6) δ 190.85, 166.05, 144.82, 144.51, 141.76,
137.58, 133.57, 133.29, 126.41, 126.36, 113.18, 108.76, 102.84.
4.3.3. 1-(2,4-Dihydroxyphenyl)-3-(pyridin-2-yl)propenone (8)
Method A was employed with 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)
ethanone and 2-pyridine carboxaldehyde to obtain yellow solid compound (0.10 g, 4.5%). mp
207-209 °C; Rf = 0.22, E:H = 1:2, v/v; purity by HPLC: 100%; 1H NMR (250 MHz, DMSO-
d6) δ 12.99 (s, 1H, 1-phenyl 2-OH), 10.87 (br, 1H, 1-phenyl 4-OH), 8.80 (d, J = 5.0 Hz, 1H,

3-pyridine H-6), 8.46 (d, J = 15.4 Hz, 1H, CO-CH=CH), 8.26-8.24 (m, 2H, 3-pyridine H-4,
H-3 ), 8.14 (d, J = 8.9 Hz, 1H, 1-phenyl H-6), 7.85 (d, J = 15.4 Hz, 1H, CO-CH=CH), 7.74
(dd, J = 8.6, 4.9 Hz, 1H, 3-pyridine H-5), 6.48 (d, J = 8.9 Hz, 1H, 1-phenyl H-5), 6.35 (d, J =
2.0 Hz, 1-phenyl H-3); 13C NMR (62.5 MHz, DMSO-d6) δ 190.61, 166.09, 165.92, 150.34,
146.69, 141.56, 137.86, 133.43, 128.20, 126.12, 125.93, 113.36, 108.95, 102.86.
4.3.4. 1-(2,4-Dihydroxyphenyl)-3-(thiophen-2-yl)propenone (9)
Method A was employed with 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)
ethanone and 2-thiophene carboxaldehyde to obtain yellow solid compound (1.20 g, 64.5%).
1
mp 140-142 °C; Rf = 0.40, E:H = 1:2, v/v; purity by HPLC: 98%; H NMR (250 MHz,
MeOD-d4) δ 8.06 (d, J = 15.1 Hz, 1H, CO-CH=CH), 7.96 (d, J = 8.9 Hz, 1H, 1-phenyl H-6),
7.66 (d, J = 5.0 Hz, 1H, 3-thiophene H-5), 7.59 (d, J = 14.9 Hz, 1H, CO-CH=CH), 7.55 (d, J
= 5.0 Hz, 1H, 3-thiophene H-3), 7.21 (dd, J = 5.0, 3.6 Hz, 1H, 3-thiophene H-4), 6.50 (dd, J =
8.9, 2.4 Hz, 1H, 1-phenyl H-5), 6.36 (d, J = 2.4 Hz, 1-phenyl H-3); 13C NMR (62.5 MHz,
MeOD-d4) δ 192.75, 167.56, 166.64, 141.61, 137.56, 133.26, 133.16, 130.50, 129.52, 120.44,
114.59, 109.31, 103.88.
4.3.5. 1-(2,4-Dihydroxyphenyl)-3-(furan-2-yl)propenone (10)
Method A was employed with 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)
ethanone and 2-furaldehyde to obtain orange solid compound (1.80 g, 78.2%). mp 123-
125 °C; Rf = 0.22, E:H = 1:3, v/v; purity by HPLC: 100%; 1H NMR (250 MHz, MeOD-d4) δ
7.95 (d, J = 8.9 Hz, 1H, 1-phenyl H-6), 7.73 (s, 1H, 3-furan H-5), 7.71 (d, J = 15.2 Hz, 1H,
CO-CH=CH), 7.62 (d, J = 15.2 Hz, 1H, CO-CH=CH), 6.93 (d, J = 3.4 Hz, 1H, 3-furan H-3),
6.65 (dd, J = 3.4, 1.8 Hz, 1H, 3-furan H-4), 6.49 (dd, J = 8.9, 2.4 Hz, 1H, 1-phenyl H-5), 6.36
(d, J = 2.4 Hz, 1-phenyl H-3); 13C NMR (62.5 MHz, MeOD-d4) δ 192.83, 167.54, 166.63,

153.18, 146.78, 133.23, 131.14, 119.13, 117.31, 114.66, 113.78, 109.30, 103.88.
4.3.6. 3-(2,4-Dihydroxyphenyl)-1-(thiophen-2-yl)propenone (13)
Method A was employed with 2-acetyl thiophene and (2-hydroxy-4-((tetrahydro-2H-pyran-2-
yl)oxy)benzaldehyde to obtain red viscous compound (0.05 g, 2.3%). Rf = 0.29, E:H = 1:1,
v/v; purity by HPLC: 96%; 1H NMR (250 MHz, MeOD-d4) δ 8.09 (d, J = 15.5 Hz, 1H, CO-
CH=CH), 7.96 (d, J = 3.7 Hz, 1H, 1-thiophene H-3), 7.80 (d, J = 4.9 Hz, 1H, 1-thiophene H-
5), 7.59 (d, J = 15.6 Hz, 1H, CO-CH=CH), 7.51 (d, J = 8.1 Hz, 1H, 3-phenyl H-6), 7.21 (t, J
= 4.4 Hz, 1H, 3-thiophene H-4), 6.36 (dd, J = 7.8, 2.0 Hz, 1H, 3-phenyl H-5), 6.32 (s, 1H, 3-
phenyl H-3); 13C NMR (62.5 MHz, MeOD-d4) δ 185.06, 163.09, 161.01, 147.30, 142.46,
134.96, 133.19, 132.32, 129.51, 118.59, 115.31, 109.14, 103.54.
4.3.7. 3-(2,4-Dihydroxyphenyl)-1-(furan-2-yl)propenone (14)
Method B was employed with 2-acetyl furan and 2,4-bis(methoxymethoxy)benzaldehyde to
obtain red viscous compound (0.02 g, 10%). Rf = 0.20, E:H = 1:1, v/v; purity by HPLC: 97%
1H NMR (250 MHz, MeOD-d4) δ 8.22 (d, J = 15.7 Hz, 1H, CO-CH=CH), 7.88 (dd, J = 1.6,
0.7 Hz, 1H, 1-furan H-5), 7.62 (d, J = 15.7 Hz, 1H, CO-CH=CH), 7.59 (d, J = 9.1 Hz, 1H, 3-
phenyl H-6), 7.49 (dd, J = 3.6, 0.7 Hz, 1H, 1-furan H-3), 6.75 (dd, J = 3.6, 1.7 Hz, 1H, 1-
furan, H-4), 6.46 (dd, J = 8.7, 2.3 Hz, 1H, 3-phenyl H-5), 6.43 (d, J = 1.1 Hz, 1H, 3-phenyl
H-3); 13C NMR (62.5 MHz, MeOD-d4) δ 180.82, 163.14, 161.03, 155.30, 148.37, 142.44,
132.24, 118.71, 118.27, 115.37, 113.55, 109.18, 103.58.
4.3.8. 1-(3,5-Dihydroxyphenyl)-3-(thiophen-2-yl)propenone (20)
Method C was employed with 1-(3,5-bis(methoxymethoxy)phenyl)ethanone and 2-thiophene
carboxaldehyde to obtain yellow solid compound (0.58 g, 78.5%). mp 177-178 °C; Rf = 0.52,

E:H = 1:1, v/v; purity by HPLC: 96%; 1H NMR (300 MHz, MeOD-d4) δ 7.85 (d, J = 15.3 Hz,
1H, CO-CH=CH), 7.54 (d, J = 5.1 Hz, 1H, 3-thiophene H-5), 7.42 (d, J = 3.3 Hz, 3-thiophene
H-3), 7.27 (d, J = 15.6 Hz, 1H, CO-CH=CH), 7.08 (dd, J = 5.1, 3.9 Hz, 1H, 3-thiophene H-4),
6.85 (d, J = 2.1 Hz, 2H, 1-phenyl H-2, H-6), 6.45 (t, J = 2.1 Hz, 1H, 1-phenyl H-4); 13C NMR
(75.5 MHz, MeOD-d4) δ 191.77, 160.14, 141.47, 141.25, 138.48, 133.63, 130.53, 129.57,
121.74, 108.27, 107.70.
4.3.9. 1-(3,5-Dihydroxyphenyl)-3-(furan-2-yl)propenone (21)
Method C was employed with 1-(3,5-bis(methoxymethoxy)phenyl)ethanone and 2-
furaldehyde to obtain yellow solid compound (0.55 g, 80.0%). mp 189-190 °C; Rf = 0.45,
E:H = 1:1, v/v; purity by HPLC: 100%; 1H NMR (250 MHz, MeOD-d4) δ 7.65 (d, J = 1.6 Hz,
1H, 3-furan H-5), 7.54 (d, J = 15.4 Hz, 1H, CO-CH=CH), 7.36 (d, J = 15.4 Hz, 1H, CO-
CH=CH), 6.88 (d, J = 2.2 Hz, 2H, 1-phenyl H-2, H-6), 6.85 (d, J = 3.4 Hz, 3-furan H-3), 6.57
(dd, J = 3.4, 1.8 Hz, 1H, 3-furan H-4), 6.47 (t, J = 2.2 Hz, 1H, 1-phenyl H-4); 13C NMR (62.5
MHz, MeOD-d4) δ 191.86, 160.14, 152.96, 146.87, 141.32, 131.98, 120.30, 117.72, 113.85,
108.29, 107.72.
4.3.10. 1-(3,5-Dihydroxyphenyl)-3-phenylpropenone (22)
Method
C
was employed with 1-(3,5-bis(methoxymethoxy)phenyl)ethanone and
benzaldehyde obtain light yellow solid compound (0.25 g, 69.0%). mp 160-161 °C; Rf = 0.44,
E:H = 1:1, v/v; purity by HPLC: 98%; 1H NMR (250 MHz, MeOD-d4) δ 7.83 (d, J = 15.7 Hz,
1H, CO-CH=CH), 7.78-7.75 (m, 2H, 3-phenyl H-2, H-6), 7.65 (d, J = 15.7 Hz, 1H, CO-
CH=CH), 7.50-7.47 (m, 3H, 3-phenyl H-3, H-4, H-5), 7.01 (d, J = 2.1 Hz, 2H, 1-phenyl H-2,

H-6), 6.59 (t, J = 2.1 Hz, 1H, 1-phenyl H-4); 13C NMR (62.5 MHz, MeOD-d4) δ 192.59,
160.11, 146.02, 141.39, 136.28, 131.70, 130.08, 129.59, 123.44, 108.36, 107.95.
4.3.11. 1-(3,5-Dihydroxyphenyl)-3-(thiophen-3-yl)propenone (23)
Method C was employed with 1-(3,5-bis(methoxymethoxy)phenyl) ethanone and 3-thiophene
carboxaldehyde to obtain yellow solid compound (0.37 g, 77.5%). mp 127-128 °C; Rf = 0.44,
E:H = 1:1, v/v; purity by HPLC: 100%; 1H NMR (250 MHz, MeOD-d4) δ 7.88 (dd, J = 2.5,
1.2 Hz, 1H, 3-thiophene H-2),7.84 (d, J = 15.6 Hz, 1H, CO-CH=CH), 7.61 (dd, J = 5.2, 1.2
Hz, 1H, 3-thiophene H-4), 7.56 (dd, J = 5.3, 2.8 Hz, 1H, 3-thiophene H-5), 7.47 (d, J = 15.6
Hz, 1H, CO-CH=CH), 6.99 (d, J = 2.1 Hz, 2H, 1-phenyl H-2, H-6), 6.58 (t, J = 2.1 Hz, 1H,
1-phenyl H-4); 13C NMR (62.5 MHz, MeOD-d4) δ 192.96, 160.07, 141.48, 139.64, 139.60,
130.76, 128.23, 126.46, 123.03, 108.25, 107.88.
4.3.12. 1-(3,5-Dihydroxyphenyl)-3-(furan-3-yl)propenone (24)
Method C was employed with 1-(3,5-bis(methoxymethoxy)phenyl)ethanone and 3-
furaldehyde to obtain yellow solid compound (0.24 g, 80.2%). Rf = 0.38, E:H = 1:1, v/v, mp =
167-168 °C; purity by HPLC: 99%; 1H NMR (250 MHz, MeOD-d4) δ 7.97 (s, 1H, 3-furan H-
2), 7.76 (d, J = 15.5 Hz, 1H, CO-CH=CH), 7.63 (dd, J = 1.9, 1.3 Hz, 1H, 3-furan H-5), 7.36
(d, J = 15.5 Hz, 1H, CO-CH=CH), 6.97 (d, J = 2.2 Hz, 2H, 1-phenyl H-2, H-6), 6.93 (dd, J =
1.9, 0.7 Hz, 1H, 3-furan H-4), 6.56 (t, J = 2.2 Hz, 1H, 1-phenyl H-4); 13C NMR (62.5 MHz,
MeOD-d4) δ 192.63, 160.06, 147.49, 146.03, 141.39, 136.36, 124.75, 123.20, 108.49, 108.22,
107.87.
Measurement of μ- and m-calpain inhibitory activities of compounds

Fluorescence intensity was expressed as relative fluorescence units (RFUs). RFUs were
calculated by subtracting the RFU of the no-enzyme control from all other values. To
determine percent inhibition, the percent change in RFUs between the activity of the enzyme
in the presence and absence of compounds was calculated. The value of RFU calculated in
the absence of compound represents 100% enzyme activity.
Inhibition of -calpain (Calbiochem, Germany) was assayed in the reaction buffer (50 mM
Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA and 5 mM β-mercaptoethanol, pH 7.5)
with 100 M pep1, 2.5 mM CaCl2, and 5.25 units/mL -calpain. The reaction was initiated by
sequential addition of substrate A, -calpain, each compound, and finally a CaCl2 solution.
Thereafter, the reaction mixture of 100 μL was incubated at room temperature for 30 min
with shaking. Fluorescence intensities were measured at 320 nm excitation and 420 nm
emission wavelengths. The substrate A of μ-calpain was synthesized by Peptron Corp.
(Daejeon, Korea) which is derived from the p35 cleavage site ([2-Abz]-Ser-Thr-Phe-Ala-Gln-
Pro-[3-nitrotyrosine]-NH2). Inhibition of m-calpain by the compounds was examined in the
same way as for -calpain with minor modifications [46]. m-Calpain (recombinant, High
Purity, E.Coli) was obtained from Calbiochem (Darmstadt, Germany). Substrate B, originated
from the calpain cleavage site in α-spectrin ([2-Abz]-Glu-Val-Tyr-Gly-Met-Met-[3-
nitrotyrosine]-NH2), was used as a substrate of m-calpain and was synthesized by the Peptron
Corp. (Daejeon, Korea). The assay was performed in a final volume of 100 μL. Stock
solution of substrate A, B and compounds were prepared in dimethylsulfoxide (DMSO) and
stored at -20 C until needed. m-Calpain inhibition was assayed in reaction buffer (50mM
Tris-HCl, 100mM NaCl, 5mM β-mercaptoethanol, and 1mM EDTA, pH 7.5) with 50 μM
substrate B, 5 mM CaCl2 and 2.88 U/mL m-calpain. The reaction was initiated by sequential
addition of substrate B, m-calpain, the test compound, and the CaCl2 solution. Thereafter, the

mixture was incubated with shaking at room temperature for 30min. Fluorescence intensities
were determined at 320nm for excitation and 420nm for emission wavelengths. MDL28170
(Sigma, USA) was used as a positive control for - and m-calpain inhibition.
Measurement of cathepsin B and L inhibitory activities of compounds
Cathepsin B and L inhibitory activities were evaluated in reaction buffer (50 mM NaOAc-
HCl, 2 mM dithiothreitol (DTT), 2 mM EDTA, pH 5.5 for cathepsin B and 0.1 M NaOAc-
HCl, 1 mM EDTA, 0.1% β-mercaptoethanol, pH 5.5 for cathepsin L) containing 20 μM
substrate and 1.5 nM cathepsin B or 4 nM cathepsin L. Cathepsins B and L were obtained
from Calbiochem (Germany). RR-AMC (Sigma, USA) and Z-FR-AMC (Sigma, USA) were
used as substrates for cathepsin B and L, respectively. Cathepsins were activated by
incubation in assay buffer at 37 °C for 30 min prior to initiation of the reaction by addition of
the substrate and test compound. The reaction mixtures were then incubated at room
temperature for 30 min with shaking. Fluorescence intensities were determined at 360 nm
excitation and 450 nm emission wavelengths. CA074 and Z-FF-FMK (Sigma, USA) were
used as positive controls as for inhibition of cathepsin B and L, respectively.
Acknowledgement
This work was financially supported by CHA University, Pocheon, and the Korea Basic
Science Research Program through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology (2011-0011007).

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Table 1. Inhibitory activity of compounds against - and m-calpain
Inhibitory activity as IC50 ( M)a
Compound
-Calpain
m-Calpain
MDL28170
0.1000 ± 0.0009
>100
0.1500 ± 0.0008
>100
CA074
Z-FF-FMK
0.3971±0.0004
> 100
0.418±0.0046
> 100
6
7
> 100
> 100
8
> 100
> 100
9
56.17 ± 0.048
86.74 ± 0.751
> 100
39.89 ± 1.054
25.35 ± 0.901
60.24 ± 1.949
> 100
10
13
14
20
21
22
23
24
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
97.29 ± 2.357
> 100
> 100
aEach data point represents the mean ± S.D. of three independent experiments performed in
triplicate. MDL28170 was used as a positive control for μ- and m-calpain inhibition.

Table 2. Inhibitory activity of compounds against cathepsin B and cathepsin L
Inhibitory activity as IC50 ( M)a
Compound
Cathepsin B
0.0958±0.0045
0.0044±0.00004
0.0656±0.0029
50.25 ± 0.004
> 100
Cathepsin L
0.0031±0.0003
>100
MDL28170
CA074
Z-FF-FMK
0.0057±0.00002
44.40 ± 0.216
> 100
6
7
8
> 100
> 100
9
> 100
53.01 ± 0.178
90.53 ± 0.234
2.80 ± 0.100
2.87 ± 0.052
> 100
10
13
14
20
21
22
23
24
60.63 ± 0.981
11.47 ± 0.087
49.47 ± 0.164
> 100
84.59 ± 0.375
> 100
> 100
> 100
> 100
> 100
> 100
> 100
a Each data point represents the mean ± S.D. of three independent experiments performed in
triplicate. CA074 and Z-FF-FMK were used as positive controls for inhibition of cathepsin B
and L, respectively.

Graphical abstract
Synthesis and Investigation of Dihydroxychalcones as Calpain and
Cathepsin Inhibitors
OH
O
HO
OH
S
OH
O
O
10
13
IC50 : 25.25 ± 0.901 M
m-Calpain inhibition
IC50 : 2.80 ± 0.100 M
Cathepsin L inhibition

Total 12 dihydroxychalcone analogues were synthesized and evaluated.
Compound 10 showed most active inhibitory activity against m-calpain
Compound 13 was most potent inhibitory activity on cathepsin L.