Novel cathepsin K inhibitors block osteoclasts in vitro and increase spinal bone density in zebrafish

Article abstract of DOI:10.1039/c8ra10338k

Cathepsin K (Cat K) is a predominant cysteine protease and highly potent collagenase expressed in osteoclasts. Cat K inhibitors are anti-resorptive agents to treat osteoporosis. A novel scaffold of cathepsin K inhibitors, exemplified by lead compound 1x, was used as the template for designing and synthesizing a total of 61 derivatives that have not been reported before. An exploratory structure-activity relationship analysis identified the potent Cat K inhibitor A22, which displayed an IC₅₀ value of 0.44 μM against Cat K. A22 was very specific for Cat K and caused a significantly higher in vitro inhibition of the enzyme as compared to that of lead compound 1x. A surface plasmon resonance analysis confirmed in vitro binding of A22 to Cat K. Molecular docking studies indicated several favourable interaction sites for A22 within the active pocket of Cat K. Furthermore, A22 also blocked active osteoclasts in vitro and increased spinal bone density in zebrafish, in which it showed an activity that was higher than that of the marketed therapeutic bone metabolizer etidronate disodium. A22 represents a very promising lead compound for the development of novel antiresorptive agents functioning as orthosteric inhibitors of Cat K.

Full text of DOI:10.1039/c8ra10338k

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Novel cathepsin K inhibitors block osteoclasts in
vitro and increase spinal bone density in zebrafish†
Cite this: RSC Adv., 2019, 9, 8600
*
Si-tu Xue,‡ Ya-li Wang,‡ Xiao-wan Han, Hong Yi, Wei Jiang, Shu-yi Si, Hui-fang Guo
*
and Zhuo-rong Li
Cathepsin K (Cat K) is a predominant cysteine protease and highly potent collagenase expressed in
osteoclasts. Cat K inhibitors are anti-resorptive agents to treat osteoporosis. A novel scaffold of
cathepsin K inhibitors, exemplified by lead compound 1x, was used as the template for designing and
synthesizing a total of 61 derivatives that have not been reported before. An exploratory structure–
activity relationship analysis identified the potent Cat K inhibitor A22, which displayed an IC50 value of
0.44 mM against Cat K. A22 was very specific for Cat K and caused a significantly higher in vitro inhibition
of the enzyme as compared to that of lead compound 1x. A surface plasmon resonance analysis
confirmed in vitro binding of A22 to Cat K. Molecular docking studies indicated several favourable
interaction sites for A22 within the active pocket of Cat K. Furthermore, A22 also blocked active
osteoclasts in vitro and increased spinal bone density in zebrafish, in which it showed an activity that was
higher than that of the marketed therapeutic bone metabolizer etidronate disodium. A22 represents
a very promising lead compound for the development of novel antiresorptive agents functioning as
orthosteric inhibitors of Cat K.
Received 17th December 2018
Accepted 26th February 2019
DOI: 10.1039/c8ra10338k
rsc.li/rsc-advances
osteoclast (OC) cells, which is balanced by the deposition of new
bone formed by osteoblast (OB) cells. As the skeleton ages, the
1. Introduction
action of the OCs outpaces that of the OBs, causing a shi
toward excessive resorption and the development of chronic OP
associated with reduced bone mineral density (BMD) and an
increased risk of bone fractures.8,9 Cathepsin K (Cat K) is
a lysosomal cysteine protease released by OCs, which cleaves
the collagen matrix of bone tissue.10,11 Cat K knockout mice have
been shown to develop osteopetrosis with elongated bones and
vertebrae and abnormal joint morphology due to a decit in
matrix degradation,12 indicating the potential role of Cat K in
OP and other bone diseases. In recent years, Cat K inhibitors
have been intensively studied by pharmaceutical companies to
nd antiresorptive therapies for OP.13–15 The most advanced
drug candidates include odanacatib,16,17 relacatib,18,19 balica-
tib20,21 and ONO-5334 (Fig. 1).22,23 Odanacatib and balicatib are
Osteoporosis (OP) is a systemic disease associated with
a declining rigidity and mechanical stability of the bones.1
Current estimates indicate that over 200 million people world-
wide suffer from this disease, causing more than 8.9 million
fractures annually. In fact, an osteoporotic fracture is estimated
to occur every 3 s, and one out of every three women and one in
ve men over 50 years of age will have an OP-related fracture in
their lifetime.2,3 A better knowledge about the progression of
bone metabolic disorder and the development of effective
treatments will have important socio-economic and medical
consequences. The most commonly prescribed OP medications
include nitrogen-containing bisphosphonates, denosumab,
raloxifene, hormone replacement therapy, and calcitonin.4
Although these therapies improve the quality of life in patients
with OP, they have many side effects.5–7 Thus, the discovery of
novel OP medications is a priority that would provide new
options for OP patients and represent a major advancement in
the treatment of OP.
Bone health in adults depends on resorption that involves
the continuous removal of old or damaged bone tissue by
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking
Union Medical College, Beijing 100050, China. E-mail: lizhuorong@imb.pumc.edu.cn;
huifangguo@imb.pumc.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra10338k
‡ The authors contribute equally to this work.
Fig. 1 Structures of Cat K inhibitors.
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reversible covalent Cat K inhibitors carrying a cyano group that
can form a covalent bond with C25 of the active pocket in Cat K.
Odanacatib was once the most advanced drug candidate as
a Cat K inhibitor that reduced fractures in postmenopausal
women in a large multinational randomized, double-blinded
phase III clinical study. However, odanicatib was also associ-
ated with an increased risk of cerebrovascular accidents and
subsequently withdrawn from the regulatory approval process.
Thus, there is signicant value in the study of Cat K because
it is a major drug target for OP and related-bone disorders.
Our research team has been working on the development of
anti-OP drugs for many years.24,25 Our recent efforts are focused
on the discovery of Cat K inhibitors as potent antiresorptive
agents. A virtual screen of our in-house compound library
against the Cat K (PDB code: 4dmy, Fig. 2) active site identied
compound 1q (Fig. 2). 1q had a higher CDOCKER score than the
covalent Cat K inhibitor 14 which was reported by Alexander G.
Dossetter and his colleagues (Fig. 2a).26 The orientation of 1q
also showed a high tness comparable to that of 14 (Fig. 2b).
The scaffold of 1q has not been reported by others as a Cat K
inhibitor. A search of our in-house compound library identied
several structural analogues of 1q that were all tested for in vitro
Cat K inhibiting effects using an enzymatic assay. However, 1q
was not active against Cat K at 100 mM. Interestingly, we iden-
tied compound 1x (Fig. 2) with a scaffold of benzimidazole-2-
substituted pyridine propyl ketene that inhibited Cat K at
a concentration below 100 mM. Two other compounds (Fig. 3, 1o
and 1p27) with a scaffold containing benzoxazole or benzothia-
zole did not inhibit Cat K. We also found that the carbonyl
Fig. 3 Inactive compounds against Cat K.
In this study, we synthesized derivatives based on the
structure of 1x with the benzimidazole scaffold and explored
their antiresorptive potential. In total, 61 compounds contain-
ing either benzimidazole-2-substituted phenyl or pyridine
propyl ketenes were synthesized. The compounds were evalu-
ated for their potential to inhibit the kinase activity of Cat K.
Many derivatives with benzimidazole scaffold showed inhibi-
tory activity at less than 10 mM against Cat K in vitro. Then, we
tested the selectivity of 1x, A20, A22, and A32 (Table 1) against
several other cathepsins (L, S and B) and found that these
compounds were highly selective for Cat K. Modelling studies
identied several favourable interactions of A22 with the active
pocket of Cat K. Surface plasmon resonance (SPR) analysis
conrmed the high affinity of A22 to Cat K. Compounds A20,
A22 and A32 showed inhibitory activity toward OCs and also
inhibited bone resorption caused by C-telopeptide (CTX-I)
decrease observed in vitro. Moreover, A22 also increased
spinal bone density in zebrash.
2. Results and discussion
group in 1x scaffold is probably essential for the Cat K inhib- 2.1 Chemistry
iting activity because the switch to a hydroxyl group (see 1s and
The search for potential Cat K inhibitors led to the identica-
1t27 in Fig. 3) abolished the activity.
The IC50 of compounds 1o, 1p, 1s, and 1t against Cat K were
all above 100 mM.
tion of compound 1x with a scaffold containing benzimidazole,
a carbonyl group, and a double bond that is required for in vitro
Cat K inhibition. The design and synthesis of a series of 1x
derivatives were performed as follows. The synthesis route of
benzimidazole-2-substituted phenyl or pyridine propyl ketenes
is described in Scheme 1. In total, 61 derivatives have been
synthesized. There are different substituents on the two
aromatic rings. In this round of synthesis, we only implanted
substituents with smaller size, such as methyl, halogen or cyano
groups. The detailed structures of the compounds are reported
in Table 1.
2.2 Inhibition of Cat K in vitro and preliminary structure–
activity relationships
The inhibitory activities of our focused compound library
against Cat K are reported in Table 1 including odanacatib,
which was used as a positive control. Importantly, many deriv-
atives of scaffold A showed Cat K inhibitory effects at concen-
trations below 100 mM, which conrmed that this novel scaffold
has a high potential to serve as a template for antiresorptive
agents.
The phenyl or pyridine substitution (such as A4 and A39)
in the scaffold is favoured. The 2-substitution on the phenyl
ring seems detrimental to Cat K inhibitory activities accord-
ing to the IC50 data (>100 mM) of A11, A15, A27, and A30. The
Fig. 2 Virtual screen found Cat K inhibitor 1x. (a) The 3D model of Cat
K inhibitor 14 in docking position (PDB code 4dmy; the picture was
prepared with Pymol) in the active site of Cat K and (b) comparison of
the docking position of 1q (grey) with 14 (purple) and (c) structures,
CDOCKER scores and Cat K IC50s of 14, 1q and 1x.
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Table 1 In vitro inhibitory activities (IC50, mM) of compounds with scaffold A against Cat K
Compd.
X
R
R0
IC50
Compd.
X
R
R0
IC50
A1
A2
A3
A4
A5
A6
A7
A8
N
N
N
N
N
N
N
N
N
N
C
C
C
N
C
C
C
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
H
H
H
H
H
H
H
H
H
H
H
H
30-Cl
40-Cl
50-Cl
60-Cl
30-Me
60-Me
50-Br
60-Br
30-F
26.15
2.47
8.48
8.83
9.51
28.14
6.12
9.48
3.75
8.96
>100
7.33
9.16
25.86
>100
47.04
2.96
4.24
7.89
0.60
4.73
0.44
15.49
5.26
2.82
6.07
>100
23.42
3.06
>100
51.14
0.88
A33
A34
A35
A36
A37
A38
A39
A40
A41
A42
A43
A44
A45
A46
A47
A48
A49
A50
A51
A52
A53
A54
A55
A56
A57
A58
A59
A60
A61
1x
C
C
N
C
C
N
C
C
C
N
C
C
C
C
C
C
C
C
C
C
N
C
C
C
C
C
C
C
C
N
5-Cl
4-Cl
5,6-Dichloro
5,6-Dichloro
5,6-Dichloro
5-Br
5-Br
5-Br
5-Br
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NH2
5-NH2
5-NH2
5-NH2
5-NH2
5-NH2
5-NH2
5-NH2
5-NH2
H
40-CN
30-CN
H
2.61
6.19
6.94
7.62
5.57
7.34
6.67
2.34
0.77
30-CN
40-CN
H
30-Cl
30-CN
40-CN
H
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
60-OMe
20-CN
30-CN
40-CN
H
6.54
8.19
H
20-Cl
30-Cl
40-Cl
20-Br
30-Br
40-Br
30-Me
30-CN
11.01
10.70
26.11
11.32
9.70
15.90
25.41
3.14
22.91
4.29
8.75
9.09
4.38
6.40
18.77
4.11
4.99
9.39
10.04
0.002
H
5-Me
5-Me
5-Me
5-Me
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
20-CN
30-CN
40-CN
H
30-Cl
40-Cl
50-Cl
60-Cl
30-Me
50-Br
30-F
30-OMe
H
H
20-Cl
30-Cl
40-Cl
20-Br
30-Br
40-Br
30-Me
H
60-OMe
20-Cl
30-Cl
30-Br
30-OMe
20-CN
30-CN
Odanacatib
contribute to the activity. The molecular docking calculations of
A22 (Cat K IC50 ¼ 0.44 mM, Fig. 4) in the Cat K active pocket also
suggested that its imidazole H atom formed a hydrogen bond
with the O atom of the backbone carbonyl group at N161. This
could explain why compounds benzoxazole 1o and benzothiazole
1p did not show any Cat K inhibiting effect. They lack this key
hydrogen bond donor in their scaffold which appears to be
a necessity for this activity. The phenyl ring of benzimidazole was
coplanar with the glycine shelf (G23) and generated a hydro-
phobic interaction. The hydrophobic interaction between benz-
imidazole and C25 also enhanced the affinity of A22 and Cat K.
Thus, chlorine substitution on benzimidazole of A22 is benecial
to the affinity to Cat K.
Scheme 1 Synthesis route of derivatives of 1x started from 1 or 2. (a) H2,
Pd/C, 30 ꢀC; (b) 2-hydroxypropanoic acid, 4 M HCl, H2O, 90 ꢀC;
ammonia; (c) TEMPO, NaClO, KBr, CH3CN, r.t.; (d) NaOH, CH3CH2OH, r.t.
5-halogen substitution on benzimidazole appears to be
favourable for the activity. A20, A22, A32, and A41 showed
inhibitory activities below 1 mM. Halogen and cyano group
substitution on the terminal phenyl or pyridine ring may also
2.3 Selectivity of the inhibition of cathepsins in vitro
We compared the inhibitory activities of compounds 1x, A20,
A22, and A32 against Cat K, Cat L, Cat S, and Cat B (Table 2).
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Fig. 4 Molecular docking calculations of the binding mode of A22 with Cat K (PDB code: 4dmy). (a) and (b) The calculated interactions of A22
(grey) with amino acids (green) in the active pocket of Cat K. (c) Overlay of the crystal structures of Cat L (PDB code: 6ezp), Cat S (PDB code: 4bs5)
and Cat B (PDB code: 6ay2) with Cat K. N161 (green) of Cat K located at a loop region where the conformations of the Cat L, S, B and K are
different. Compared to the orientation of Y193 (purple) of Cat S, Y67 (green) of Cat K has much less clash with pyridine ring of A22. (d) Backbone
carbonyl groups of N161 of Cat K, N286 (purple) of Cat S and D162 (yellow) of Cat L.
Table 2 Inhibitory activity against Cat K, Cat L, Cat S and Cat B in vitro
2.4 Antiresorptive effects in vitro
Type I collagen comprises about 95% of the entire collagen
content of bone.28 During skeletal renewal, type I collagen is
degraded, releasing short peptide fragments into the blood-
stream. The bone resorption process can be studied in vitro by
culturing bone cells on bone sections or dentin sheets. During
the process of bone resorption by OCs, the type I collagen C-
terminal peptide (CTX-I) is degraded into fragments and
released. In this experiment, the concentration of CTX-I
released into the cell culture medium by bone degradation
can be detected and quantied by ELISA. The in vitro anti-
resorptive effect of our compounds was tested in RANKL-
induced RAW264.7 cells. Osteoclastic features can be induced
in RAW264.7 by RANKL with high efficiency and reproduc-
ibility.29 We detected the CTX-I26,30,31 concentration released into
the culture medium from RANKL-induced RAW264.7 cells by
ELISA.
Importantly, RAW264.7 cells released much less CTX-I in the
presence of A20, A22, A32, 1ꢁ, and positive control odanacatib,
as compared to that released by untreated RAW264.7 cells. The
concentrations of all experimental compounds and positive
control drugs were 5 mM. The antiresorptive effects of all the
experimental compounds was similar to that of odanacatib,
especially the activity of A20 was much stronger than that of the
control drug. And also the efficacy of all the test compounds was
signicantly better than that of 1x on RAW264.7 cells (Fig. 5A).
IC50 (mM)
Compd.
Cat K
Cat L
Cat S
Cat B
1x
A20
A22
A32
10.04
0.60
0.44
0.88
0.002
15.82
1.64
2.62
1.79
8.46
>100
>100
24.73
46.72
25.34
0.013
47.73
73.85
101.76
0.013
Odanacatib
Compounds 1x, A20, A22, and A32 showed an inhibitory effects
against the cathepsins, especially toward Cat K and Cat L. The
order of the inhibitory activities of the four test compounds
against the cathepsins were Cat K > Cat L > Cat S > Cat B. The
selectivity of A22 for Cat K over Cat S and Cat B was 106 and 168
times, respectively. According to the docking result, the
hydrogen bond between A22 and N161 of Cat K probably played
a key role of the selectivity of A22. N161 of Cat K was in a loop
region where the conformation of Cat B was highly different
from Cat L, S, and K (Fig. 4c). Different from the loops of Cat
K, L and S, it was a b-sheet of Cat B in this region. There was no
asparagine here in Cat B. The change of asparagine to histidine
and glycine distorted the binding pocket, which explained well
that A22 showed much weaker inhibitory activity against Cat B.
Similar to their counterpart N161 in Cat K, D162 of Cat L and
N286 of Cat S (Fig. 4d) also can form hydrogen bond with A22.
A22 showed high inhibiting effect against Cat L. But the higher
clash of Y193 of Cat S to A22 may alleviate the interaction
(Fig. 4c).
As compared to the inhibitory activities of 1x, the positive
control odanacatib had a higher activity against Cat K and Cat L,
but a lower activity against Cat S and Cat B. The selectivity of
odanacatib for Cat K over Cat S and Cat B was 6.5 times for both
enzymes. The order of the inhibitory activity of odanacatib
against the cathepsins was Cat K > Cat S ¼ Cat B > Cat L. These
results indicated that A22 and odanacatib differed in their
selectivity toward cathepsin family proteins and, therefore, may
differ in their activity and toxicity. Therefore, A22 has the
potential to be novel antiresorptive agents.
2.5 Inhibition of osteoclast (OC) cells
The inhibition of OCs in vitro was tested by counting the
numbers of tartrate-resistant acid phosphatase (TRAP) positive
cells. In this experiment, the RANKL-induced RAW264.7 cells
were identied by OC-specic TRAP staining specic. The
RANKL-induced RAW264.7 cells were incubated with the test
compounds added at 20 mg mLꢂ1. Aer 72 h of culture, the
TRAP staining was performed and the TRAP + cells were coun-
ted. As compared to the growth of the untreated control OCs,
the growth of the OCs incubated with the test compounds (20 mg
mLꢂ1) was reduced. The OC growth inhibition of A20, A22, and
A32 was 31.3%, 33.3%, and 42.2%, respectively (Fig. 5B).
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chromophore calcein, which binds specically to calcied skel-
etal structures, was used to indicate zebrash spine bone
density.39 Fluorescence intensities were enhanced as compared to
that of the model group, indicating that A22 treatment improved
bone density at tested concentrations. A22 also showed dose–
response relationship in OP zebrash. At the highest concen-
tration group, A22 exhibited higher activity than etidronate
disodium (ED) (Fig. 7). The OP therapeutic effect in zebrash
bone loss model showed that A22 is a promising anti-OP agent.
Fig. 5 (A) CTX-I detection of test compounds in the bone absorption
process; (B) Effects on OC differentiation by test compounds in vitro.
Three replicates were tested in each group. **Differences from the
control group with probability p < 0.05. ***Differences from the control
group with probability p < 0.001. The p value is calculated by Tukey–
Kramer multiple comparisons test. The difference is considered statis-
tically significant when probability p value is less than 0.05 and statisti-
cally highly significant when probability p value is less than 0.001.
3. Experimental section
3.1 Chemistry
3.1.1 General information. All reagents and solvents were
purchased and used without further purication unless speci-
ed. The 1H NMR and 13C NMR spectra were recorded with
a Bruker BioSpin GmbH spectrometer (400 MHz) in d6-DMSO
and the internal solvent residual peaks were used as references.
The high-resolution mass spectra (HRMS) were recorded on
a Thermo Scientic LTQ Orbitrap XL with an ESI mass selective
detector. The melting points (mp) were determined using
Mettler Toledo MP 90. Flash column chromatography using
a silica gel (200–300 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd was performed on a Combiash RF200.
3.1.2 Synthesis of A22. To a 250 mL three-necked round
bottom bottle, 4-chlorobenzene-1,2-diamine (2, 7.13 g, 0.05
mol) and 85% 2-hydroxypropanoic acid (7.95 g, 0.075 mol) were
added and mixed with 40 mL of 4 mol Lꢂ1 HCl. The mixture was
2.6 Binding affinity to Cat K tested by SPR
To determine whether the active compounds can bind to Cat K,
real-time binding analysis by SPR (Biacore 8K) was performed
using Cat K and different dilutions of A20 and A22 (Fig. 6). The
two compounds showed moderately strong affinities to Cat K.
The calculated affinities (KD) of A20 and A22 to Cat K were 53.2
ꢃ 43.7 mM and 17.2 ꢃ 5.6 mM, respectively. Moderately strong
affinities implied that the compounds may show antiresorptive
efficacy in vivo via inhibition of Cat K.
2.7 OP therapeutic activity in zebrash
ꢀ
heated to 90 C and stirred for 5 h. Aer the full conversion of
To probe into the in vivo activities, we chose A22 as a pilot
compound and evaluated its activity in zebrash32,33 because
A22 was the most potent Cat K inhibitor in vitro among our
focused library compounds and showed high selectivity and
affinity to Cat K. Etidronate disodium was used as a positive
control drug. Etidronate disodium is a bone resorption inhib-
itor and now mainly used as an antiosteoporotic medication.34
Its action mechanism involves the hydroxyapatite crystal growth
inhibition according to the in vitro chemisorption onto the
crystal surface.35 Prednisone is a glucocorticoid medication and
oen used to suppress the immune system and relieve inam-
mation. The use of prednisone can induce bone loss.36–38 In our
study, we use prednisone to set up a zebrash bone loss model.
Our dosing pilot study showed at the concentration of 1.00
mM, A22 didn't show obvious toxicity to the 30 zerash in the
group. We then tested OP therapeutic activity in zebrash of A22
at concentrations of 0.11, 0.33, and 1.00 mM. Fluorescent
the starting material (as monitored by TLC), the reaction was
cooled to 30 ꢀC. The pH of the mixture was adjusted to 8 using
ammonia water. A white precipitate formed that was collected
by ltration, washed with water, and dried under air in a culture
dish followed by a drying step performed under vacuum in an
oven. A product with the designation 1-(5-chloro-1H-benzo[d]
imidazo l-2-yl)ethan-1-ol was obtained as a white solid (3, yield
93%), MS (ESI) m/z: 197 [M + H]+
Fig. 7 Therapeutic effect of A22 in OP zebrafish. (A) Zebrafish spine
fluorescence intensity data were collected within the white rectangle
area. A22 was administered at a dose of 0.11 mM, 0.33 mM, 1.00 mM, and
etidronate disodium (ED) was administered at a dose of 600 mM; (B)
spine fluorescence intensity data were calculated based on values
obtained from 30 fish each group. (C) A22 therapeutic effect on
Fig. 6 Binding curves of A20 and A22 dilutions. The affinities (KD) zebrafish OP. ***Differences from the model group with probability p
were calibrated with the steady state affinity 1 : 1 binding model using < 0.001. The p value is calculated using Dunnett's T test and differ-
the evaluation software of Biacore 8K.
ences with probability p < 0.05 are considered statistically significant.
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1-(5-Chloro-1H-benzo[d]imidazol-2-yl)ethan-1-ol (3, 1.97 g, favourable affinity. Using the docking calculations of
0.01 mol), TEMPO (0.016 g, 0.1 mmol) and KBr (0.12 g, 1 mmol) CDOCKER, we also obtained the exact pose of the ligand in
were added to a 100 mL three-necked round bottom bottle. 4dmy.
Then, 20 mL of acetrile and 20 mL of water were added. An 8%
aqueous sodium hypochlorite (13.96 g, 0.015 mol) solution was
3.3 Enzymatic activity assays in vitro
added dropwise within 20 min while keeping the temperature of
Quenched uorescent resonance energy transfer (QFRET)
technology was used to test the inhibitory activity of the
compounds against Cat K (ENZO BML-SE553) catalysing the
cleavage of the synthetic peptide Z-Phe-Arg-AMC. Compounds
ꢀ
ꢀ
the mixture between 10 C and 15 C. The mixture was stirred
for 30 min until full conversion (as monitored by TLC). The
mixture was extracted by ethyl acetate (50 mL ꢁ 3). The organic
phases were combined and washed with 60 mL of 10% sodium
thiosulfate and 60 mL of brine. The organic layer was then
separated, dried over anhydrous Na2SO4 and evaporated to
recover 1-(5-chloro-1H-benzo[d]imidazol-2-yl)ethan-1-one (4,
were tested at eight concentrations (diluted from 200 mg mLꢂ1
,
three replicates per dilution). To each test sample well on a 96-
well plate, 2 mL of test compound were mixed with 25 mL of Cat K
(0.3 ng mLꢂ1). In control wells, DMSO was added instead of the
compounds. In blank wells, DMSO was added instead of
compounds and buffer was added instead of Cat K solution. The
+
ꢀ
yield 71%). mp 156.1–157.4 C; MS (ESI) m/z: 195 [M + H] .
1-(5-Chloro-1H-benzo[d]imidazol-2-yl)ethan-1-one
(4,
607.2 mg, 3.1 mmol) and ethanol (10 mL) were added to a 25 mL
three-necked round bottom bottle. The resulting mixture was
stirred until 4 was fully dissolved. NaOH (0.12 g, 3.12 mmol) was
then added and then stirred for another 15 min. 6-Chlor-
opicolinaldehyde (0.44 g, 3.12 mmol) was added. The reaction
was stirred for 2 h until full conversion (as monitored by TLC).
The reaction was quenched with 5 mL of water while stirring. A
yellow solid precipitated from the solution. Filtration was per-
formed to collect the solid and wash it with water. The solid was
rst dried under air in a culture dish and then separated by
ash column chromatography (PE : EA ¼ 10 : 1). (E)-1-(5-chloro-
1H-benzo[d]imidazol-2-yl)-3-(6-chloropyridin-2-yl)prop-2-en-1-
one (A22, yield 54%) was obtained as yellow solid. mp 251.4–
252.7 ꢀC; 1H NMR (400 MHz, d6-DMSO) d 13.68 (s, 1H), 8.39 (d, J
¼ 15.7 Hz, 1H), 7.99–7.93 (m, 2H), 7.88 (d, J ¼ 15.7 Hz, 1H), 7.84
(d, J ¼ 7.5 Hz, 1H), 7.58 (d, J ¼ 7.6 Hz, 2H), 7.43–7.34 (m, 1H);
ꢀ
plate was incubated for 15 min at 37 C. The uorescence (F1)
was determined at a wavelength of 355 nm excitation and
460 nm emission. Then, 25 mL of 20 mM of Z-Phe-Arg-AMC was
then added and the plate was incubated for 1 h at 37 ꢀC. Then,
the uorescence (F2) was measured again at a wavelength of
355 nm excitation and 460 nm emission. The calculation was
performed as follows: inhibition (%) ¼ [control (F2–F1) ꢂ
sample (F2–F1)]/[control (F2–F1) ꢂ blank (F2–F1)] ꢁ 100%. IC50
was calculated using SPSS.
The Cat L (Sigma, C6854), Cat S (Sigma, SRP6297), and Cat B
(Sigma, C8571) inhibitory assays were carried out using
a similar protocol, except that the substrates for Cat S and Cat B
were replaced with Ac-Lys-Gln-Lys-Leu-Arg-AMC and Z-Arg-Arg-
AMC, respectively.
13C NMR (101 MHz, d6-DMSO) d 181.26, 153.43, 151.18, 141.46, 3.4 The inhibition of TRAP in RANKL-induced OC
134.07, 130.78, 126.19, 125.80, 124.38, 123.28, 121.01, 114.84,
RAW264.7 cells were seeded into a sterile 96-well plate at
+
112.88; HRMS calcd for C15H10ON3Cl2 [M + H]+ 318.01954,
a density of 1 ꢁ 102 cells per well. To the DMEM culture
found 318.01942.
medium, 10 ng mLꢂ1 RANKL was added to induce the differ-
entiation in RAW264.7 cells. Test compounds were added at 20
3.1.3 Syntheses of A1–A21 and A23–A61. The syntheses of
A1–A21 and A23–A61 used the protocol described for the
mg mLꢂ1; each compound was tested in triplicate. Wells without
synthesis of A22 and illustrated in Scheme 1. The starting
a compound were used as blanks. The cells were incubated for
72 h at 37 ꢀC in 5% CO2. Then, the culture medium was
materials differed. Specically, the syntheses of A1–A13, A14–
A17, A18–A33, A34, A35–A37, A38–A41, and A42–A61 were star-
removed. The residues in the wells were xed with xative
ted from benzene-1, 2-diamine, 4-methylbenzene-1, 2-diamine,
solution (25.5% citrate solution + 66.3% acetone + 8.2% of 37%
4-chlorobenzene-1, 2-diamine, 4-chlorobenzene-1,2-diamine, 2-
formaldehyde) for 30 s and washed with deionized water. Then
chloro-6-nitroaniline, 5-chloro-2-nitroaniline, 4-bromobenzene-
mixing solution (naphthol AS-BI phosphate solution and
1, 2-diamine and 4-nitrobenzene-1, 2-diamine, respectively. The
tartrate solution) was added and the plate was incubated at
data for the characterization of these compounds are provided
37 ꢀC in the dark. Aer a nal washing step with deionized
water, the TRAP-positive cells were counted.
in the ESI.†
3.2 Molecular docking calculations
3.5 CTX-I concentration
CDOCKER of Discovery Studio 4.5 was used to carry out the RAW264.7 cells were seeded into a sterile 12-well plates with
docking simulation of the compounds to the protein. X-ray bovine femur slices at a density of 1 ꢁ 105 cells per well. To the
crystal structure of Cat K (PDB code 4dmy) was used for the DMEM culture medium, 50 ng mLꢂ1 RANKL was added to
docking studies. The ligand was extracted from the catalytic induce the differentiation in RAW264.7 cells. Test compounds
active site of Cat K, which was dened as the docking site for were added at a concentration of 5 mM. The cells were cultured
ꢀ
binding. Both the receptor and the compounds were employed for ve days at 37 C under 5% CO2. The culture supernatants
using the CHARMm force eld before docking. A higher were collected by centrifugation for analysing the CTX-I
CDOCKER score (i.e. –DOCKER _ENERGY) indicates a more concentrations by ELISA.
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believe that A22 represents a novel lead compound for anti-
resorptive drugs that will be examined for further improve-
ments in structure-based drug design, therapeutic efficacy, and
pharmacokinetics.
3.6 Real-time binding to Cat K and analysed by SPR
SPR experiments were carried out using a Biacore 8K (GE
ꢀ
Healthcare) at 25 C. Cat K (ENZO BML-SE553) was precondi-
tioned in pH 5.0 sodium acetate and then immobilized to a CM7
chip by amine coupling. Running buffer was 50 mM NaOAc (pH
5.5), 0.01% P20, 2 mM EDTA, 100 mM DTT. When the affinities
of compounds were tested, 5% DMSO was added. Ten concen-
trations of each compound were used. A20 was diluted from 100
mM by two-fold serial dilution. A22 was diluted from 50 mM by
two-fold serial dilution. Compounds were injected to the
surface of the protein-coupled chip channels at the ow rate of
30 mL minꢂ1. The affinities (KD) were calibrated with the steady
state affinity 1 : 1 binding model with the evaluation soware of
Biacore 8K. The affinities of A20 and A22 were tested twice and
ve times, respectively.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
We gratefully acknowledge the grants from the CAMS Innova-
tion Fund for Medical Sciences (CIFMS, 2017-I2M-3-019) and
the National Natural Science Foundation of China (81473097,
81402800).
Notes and references
3.7 OP therapeutic effects in zebrash B
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water and then added to different wells to target concentration.
The zebrash larvae were incubated with the compounds for
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