Discovery of a benzimidazole series of ADAMTS-5 (aggrecanase-2) inhibitors by scaffold hopping

Article abstract of DOI:10.1016/j.ejmech.2013.10.075

We describe a medicinal chemistry approach to generate a series of benzimidazoles bearing thiazolidin-4-one using scaffold hopping from thiazole 1, our previously described thiazole. Our goal was to discover a potent and permeable small-molecule ADAMTS-5 inhibitor. The results suggest that small compound 22 shows promise as a potent small-molecule ADAMTS-5 inhibitor with good permeability.

Full text of DOI:10.1016/j.ejmech.2013.10.075

European Journal of Medicinal Chemistry 71 (2014) 1e9
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of a benzimidazole series of ADAMTS-5 (aggrecanase-2)
inhibitors by scaffold hopping
*
Shinya Sogame, Yoshihito Suenaga, Masakazu Atobe , Masashi Kawanishi, Eiichi Tanaka,
Shiro Miyoshi
Pharmaceutical Research Center, Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni-shi, Shizuoka 410-2321, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe a medicinal chemistry approach to generate a series of benzimidazoles bearing thiazolidin-
4-one using scaffold hopping from thiazole 1, our previously described thiazole. Our goal was to discover
a potent and permeable small-molecule ADAMTS-5 inhibitor. The results suggest that small compound
22 shows promise as a potent small-molecule ADAMTS-5 inhibitor with good permeability.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 19 May 2013
Received in revised form
29 October 2013
Accepted 30 October 2013
Available online 15 November 2013
Keywords:
ADAMTS-5
Benzimidazole
Thiazolidin-4-one
Inhibitor
Osteoarthritis
1. Introduction
Aggrecanase-1 and aggrecanase-2 are also referred to as ADAMTS-4
and ADAMTS-5, respectively [4].
Osteoarthritis (OA) is a form of arthritis caused by the break-
down and eventual loss of the cartilage in one or more joints. OA
commonly affects the hands, feet, spine, and large weight-
bearing joints such as the hips and knees. OA usually has no
known cause and is referred to as primary OA in this case. When
the cause of the OA is known, the condition is referred to as
secondary OA [1].
Cartilage is a protein substance that serves as a “cushion” be-
tween the bones of the joints. A major feature of OA is the loss of
two of the most important components of the extra cartilage ma-
trix (ECM), namely, Type II collagen and aggrecan [2]. Aggrecan
degradation is an important factor in the erosion of articular
cartilage in OA. Aggrecanase is a protease which cleaves the
aggrecan molecule at the peptide bond between Glu373 and Ala374
in the aggrecan interglobular domain [3]. Two types of aggrecanase,
aggrecanase-1 and aggrecanase-2, are found in joint tissue. These
enzymes belong to a family of enzymes called A Disintegrin
and Metalloprotease with Thrombospondin motifs (ADAMTS).
Studies on transgenic mice have confirmed the importance of
ADAMTS-5 in the development of OA [5]. As described above, there
is considerable interest in understanding the factors that lead to
increased activity of this enzyme in OA. Additionally, AGG-523, an
aggrecanases inhibitor developed by Pfizer, reduces the release of
aggrecanase-generated aggrecan fragments into joints in a rat
model of meniscal tear-induced joint instability [6]. Thus, ADAMTS-
5 inhibitors may protect cartilage from the progression of OA.
To date, several small-molecule ADAMTS-5 inhibitors have been
reported by several groups [7]. We have previously reported a se-
ries of thiazole derivatives bearing thiazolidin-4-one, described as a
non-competitive inhibitor of ADAMTS-5 [8]. 1, a representative
compound, appeared to inhibit ADAMTS-5 and to inhibit sponta-
neous aggrecan degradation in IL-1 stimulated bovine cartilage.
Unfortunately, 1 exhibits low trans-membrane permeability, so we
sought to overcome this problem. We speculated that the carbox-
ylic acid group moderates permeability [9], so this group was not a
major factor in enhancing permeability. Moreover, thiazolidin-4-
one is required for ADAMTS-5 inhibition. Our goal was to
discover a potent and permeable small-molecule ADAMTS-5 in-
hibitor. To identify new compounds with unique physicochemical
profiles, we first focused on replacing the 2-pyridyl thiazole with
other heterocyclic systems.
* Corresponding author. Tel.: þ81 558 76 8493; fax: þ81 558 76 5755.
E-mail address: atobe.mb@om.asahi-kasei.co.jp (M. Atobe).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.075

2
S. Sogame et al. / European Journal of Medicinal Chemistry 71 (2014) 1e9
2. Results and discussion
Benzimidazoles bearing the thiazolidin-4-one series were
selective against five other members of the Zn metalloproteases:
Initially, 2 was prepared as a simple molecule by removing the
ethyl carboxylic acid from 1. When tested against ADAMTS-5, 2
provided an inhibition constant similar to 1, suggesting that thia-
zolidin-4-one is important for interaction with ADAMTS-5. Struc-
tureeactivity relationship (SAR) studies were therefore initiated by
replacing 4-(2-pyridyl)-2-thiazolyl as the central ring moiety with
the thiazolidin-4-one template (Fig. 1).
Table 1 shows the results of SAR studies of the central ring
moiety. Replacement of the 4-(2-pyridyl)-2-thiazolyl ring with 4-
hydroxy-2-thiazolyl (4) resulted in moderate ADAMTS-5 inhibi-
tion activity. Substitution with one of several five-membered het-
eroaromatic rings provided thiophen-2-yl (5) and 5-imidazolyl (6),
both of which had decreased activity. A bicyclic heteroaromatic
ring, 2-benzoxazolyl (7), also showed decreased in activity,
whereas benzothiazolyl (8) showed moderate activity. In contrast,
ADAMTS-4, matrixmetaloprotease-2 (MMP-2), MMP-3, MMP-14
and tumor necrosis factor-a-converting enzyme (TACE), as
shown by representative compound 22 in Table 4. Compound
22 showed moderate selectivity towards MMP-2 and good
selectivity towards ADAMTS-4, MMP-3, MMP-14 and TACE
(Table 4).
To compare the nature of the scaffolds and their functionality,
the physicochemical properties of compounds 1 and 22 were
examined by investigating their permeability using the PAMPA
model, following a standard protocol [12]. Log D of each compound
was also measured experimentally at pH 7.4 by reverse phase-HPLC
[13]; the results are shown in Table 5. Compounds with Log D
values higher than 0 and less than 3 are categorized as belonging to
the highly permeable class [14]. Compound 1 derivatives generally
provided Log D values below 0 and thus are moderately permeable,
with the exception of compound 22, which displayed high
permeability. These data also showed a correlation between cLog P
and Log D. In the PAMPA assay, compound 22 demonstrated good
permeability. Therefore, compound 22 appears to exhibit improved
benzoimidazolyl (9) [10] showed high potent activity (0.47 mM).
The tricyclic ring compounds, (E)-2-((1H-naphtho[2,3-d]imidazol-
2-yl)methylene)thiazolidin-4-one (10) and (E)-2-((8H-indeno[1,2-
d]thiazol-2-yl)methylene)thiazolidin-4-one (11), showed moderate
activity, whereas (E)-2-((4,5-dihydronaphtho[1,2-d]thiazol-2-yl)
methylene)-thiazolidin-4-one (12) and (E)-2-((4H-thiochromeno
[4,3-d]thiazol-2-yl)methylene)-thiazolidin-4-one (13) exhibited
high activity. However, 12 and 13 have a higher molecular weight
and lower ligand efficiency (LE) [11] than 9, so benzimidazole 9 was
chosen for further optimization (Table 1).
permeability and high ADAMTS-5 inhibition compared to
(Table 5).
1
3. Chemistry
The synthesis of the benzimidazole derivatives is outlined in
Next we investigated the effect of substitution with a methyl
group; the results are shown in Table 2. Substitution at the 50 po-
sition of thiazolidin-4-one 14 (R2 ¼ Me) retained potency, while
substitution at the 30 position (15, R3 ¼ Me) led to a loss of potency.
These results suggested that the thiazolidin-4-one NH plays a key
role in the interaction with ADAMTS-5, and that the 50-methyl
group might fit into a pocket on the protein. Substitution at the 1
position of benzimidazole (16, R4 ¼ Me) provided moderate po-
tency, while substitution of the 7 position (17, R5 ¼ Me) and 6 po-
sition (18, R6 ¼ Me) reduced activity. Furthermore, introduction of a
dimethyl group (19, R6 ¼ R7 ¼ Me and 20, R4 ¼ R5 ¼ Me) led to
reduced activity. These data suggested that benzimidazole might fit
into a small pocket, so the SAR studies were narrowed to focus on
this moiety (Table 2).
Schemes 1e4. As shown in Scheme 1, reaction of (E)-2-(4-
oxothiazolidin-2-ylidene)ethanethioamide 31 and a-bromoketone
32aed in ethanol under reflux (Hanzsch reaction) resulted in the
formation of a thiazole ring to give 2 or 11e13. Reaction of (E)-2-(4-
oxothiazolidin-2-ylidene)ethanethioamide 31 and ethyl 2-
bromoacetate in dimethylformamide provided a hydroxyl thia-
zole ring to give 4 (Scheme 1).
Condensation of the heteroaromatic acetonitrile 33a, b and 2-
mercaptoacetic acid in pyridine at 100 ꢀC provided 5 and 6
(Scheme 2).
Compounds 7, 8 and 15 were prepared by condensation of ethyl
2-bromoacetate with the corresponding thioamide 34aed,
respectively (Scheme 3).
Compounds 10, 14, 16e20 and 24 (R2 ¼ H, Me and HO2CeCH2e
Since 14 (R2 ¼ Me) showed the best ADAMTS-5 activity, addi-
tional analogs were prepared (Table 3). Larger alkyl substituents on
the thiazolidin-4-one led to improved potency, with this effect
being most pronounced with compound 22, which bears an n-
propyl group. In contrast, compound 23, which bears an n-butyl
group, provided weaker activity than 22. Substitution of the polar
group, such as the carboxymethyl of 24 and the hydroxyethyl of 25,
was tolerated. Analogs of 22 were evaluated: 26 (R5 ¼ Me) and 30
(R6 ¼ F) retained activity, but 27 (R6 ¼ Me), 28 (R6 ¼ R7 ¼ Me) and
29 (R6 ¼ CF3) exhibited decreased potency (Table 3).
)
were prepared as shown in Scheme 4 using the above
route. Condensation of ethyl 2-cyanoacetate with neat commer-
cially available dianiline 35 resulted in the formation of the
benzimidazole moiety, followed by formation of the corre-
sponding 2-mercaptoacetic acid. In the case of R2 ¼ Et, n-Pr, n-Bu
and HOe(CH2)2e, thioamidation of 36 with dithiophosphoric
acid O,O-diethyl ester in H2O at 80 ꢀC provided thioamide
34, which was condensed using the corresponding a-bromoester
reagent 38 to give 21e23 and 25e30 in excellent yield
(Scheme 4).
Fig. 1. Our strategy of identifying new compounds.

S. Sogame et al. / European Journal of Medicinal Chemistry 71 (2014) 1e9
3
Table 1
Table 2
Modification of 2-pyridyl thiazole moiety.
Inhibition of ADAMTS-5 by benzimidazole derivatives.
Compound
Het
ADAMTS-5 IC50
LE
cLog P
Compound R2
R3
R4
R5
R6
R7
ADAMTS-5 %inh ADAMTS-5
(10
m
M)
IC50
9
eH eH eH eH eH eH
eMe eH eH eH eH eH
0.47
0.48
m
m
M
M
14
15
16
17
18
19
20
2
0.47
m
M
0.35
0.24
eH eMe eH eH eH eH 11%
eH eH eMe eH eH eH
eH eH eH eMe eH eH
eH eH eH eH eMe eH
eH eH eH eH eMe eMe
eH eH eH eMe eMe eH
1.7
2.7
2.5
6.0
1.5
m
M
M
M
M
M
m
m
m
m
4
5
1.4
6.0
m
M
M
0.45
0.44
ꢂ0.30
m
m
0.64
5. Experimental protocols
5.1. Synthetic chemistry
6
5.0
M
0.44
ꢂ1.18
NMR spectra were run at 300 MHz on JEOL Co, 400 MHz and
600 MHz on Varian Co. As a solvent, deuterated chloroform was
used, unless specifically described otherwise. For measurement of
chemical shift, tetramethylsilane (TMS) was employed as an in-
7
8
9
5.4
1.3
m
m
M
M
0.33
0.37
0.40
0.27
0.93
0.42
ternal standard. Chemical shifts (d) are expressed in ppm. Data for
proton spectra are reported as follows: chemical shift (multiplicity
[singlet (s), doublet (d), triplet (t), and multiplet (m)], coupling
constants [Hz], integration). Carbon spectra were recorded with
complete proton decoupling and the chemical shifts are reported in
ppm. As a mass spectrometer, Quadrupole type mass spectrometer,
UPLC/SQD system manufactured by Waters Company was used and
ionization was carried out based on an electrospray method (ESI)
for the measurement. The liquid chromatography instrument used
was Acquity Ultra Performance LC system. As a separation column,
0.47
m
M
10
11
1.7
1.7
m
M
0.29
0.34
1.59
1.48
ACQUITY UPLC BEH (C18 2.1 ꢁ 50 mm 1.7
mM, Waters Company)
was used. With respect to the Examples and the Reference
Table 3
m
m
M
M
Inhibition of ADAMTS-5 by benzimidazole derivatives.
12
0.7
0.5
0.34
0.35
1.89
1.61
Compound
R2
eMe
eEt
en-Pr
en-Bu
R5
R6
R7
ADAMTS-5 IC50
14
21
22
23
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
0.48
0.42
0.17
0.41
mM
mM
mM
mM
13
m
M
24
25
eH
eH
eH
eH
eH
eH
0.43
0.48
m
M
M
4. Conclusion
m
In conclusion, we have designed and synthesized benzimidazole
derivatives exhibiting potent ADAMTS-5 inhibition. Small com-
pound 22 holds promise as a potent small-molecule ADAMTS-5
inhibitor with good permeability. On the basis of these results,
compound 22 is currently being evaluated in a surgically induced
rat OA model involving transecting the anterior cruciate ligament.
26
27
28
29
30
en-Pr
en-Pr
en-Pr
en-Pr
en-Pr
eMe
eH
eH
eH
eH
eH
eH
eH
eMe
eH
eH
0.5
1.3
1.4
4.0
m
M
M
M
M
eMe
eMe
eCF3
eF
m
m
m
0.38 mM

4
S. Sogame et al. / European Journal of Medicinal Chemistry 71 (2014) 1e9
Table 4
Activity of benzimidazole (22).
Compound ADAMTS-5 ADAMTS-4 MMP-2 MMP-3 MMP-14 TACE
1
22
0.23
0.17
m
m
M
M
23
21
m
m
M
M
>30
0.8
m
M
>30
6.4
m
M
24
4.3
m
m
M
M
>30
21
mM
M
m
M
mM
m
Scheme 2. Reagents and conditions: (a) 2-Mercaptoacetic acid, Py., 100 ꢀC (5: 96%, 6:
81%).
Table 5
Comparison of physicochemical property 1 and 22.
NMR (CDCl3, 100 MHz), d: 32.5 (CH2), 92.3 (CH2), 113.9 (CH), 120.9
TPSAb (A2) cLog Pb
a
Compound ADAMTS-5 PAMPA
inhibition
(flux, 106 cm/s)
Log D7.4
(CH),123.0 (CH),137.2 (CH),142.7 (C),149.6 (CH),151.9 (C),155.0 (C),
164.9 (C), 172.6 (C]O); RTLCMS: 1.11, Mass (ESIþ) m/z 275 [M þ H]þ;
Analytical HPLC Purity: 94%; HRMS (EI) calcd for C12H9N3OS2 (Mþ)
276.0260, found 276.0248; mp 175.5 ꢀC.
1
22
0.23
0.17
m
m
M
M
0.51
6.19
ꢂ0.21
91.12
53.49
ꢂ0.30
1.75
2.00
a
Determined by reversed phase liquid chromatography.
TPSA and clog P value was calculated with ChemBioDraw 12.0.
b
5.2.2. (E)-2-((8H-Indeno[1,2-d]thiazol-2-yl)methylene)thiazolidin-
4-one (11)
examples in which specific descriptions are given for LC condition,
the measurements were carried out by using any one of the above
described apparatuses and in accordance with the following sol-
vent condition. In addition, m/z indicates mass spectrum data
(M þ H). Flow rate: 0.6 ml/min. Solvent: Solution A ¼ water
including 0.1% (v/v) acetic acid, Solution B ¼ acetonitrile including
0.1% (v/v) acetic acid. From 0 min to 2 min: Linear gradient from
[Solution A 95% þ Solution B 5% (v/v)] to [Solution A 10% þ Solution
B 90% (v/v)]. From 2 min to 2.5 min: Linear gradient from [Solution
A 10% þ Solution B 90% (v/v)] to [Solution A 2% þ Solution B 98% (v/
v)]. From 2.5 min to 2.6 min: Linear gradient from [Solution A
2% þ Solution B 98% (v/v)] to [Solution A 95% þ Solution B 5% (v/v)].
From 2.6 min to 3.2 min: Maintain at [Solution A 95% þ Solution B
5% (v/v)]. Melting points are uncorrected.
Yield: 81%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.35 (s, 2H), 3.92 (s,
2H), 6.38 (s, 1H), 7.26 (t, 1H, J ¼ 3.0 Hz), 7.38 (t, 1H, J ¼ 3.0 Hz), 7.59
(m, 2H), 11.4 (s, 1H); RTLCMS: 1.46, Mass (ESIþ) m/z 287 [M þ H]þ;
Analytical HPLC Purity: 98%.
5.2.3. (E)-2-((4,5-Dihydronaphtho[1,2-d]thiazol-2-yl)methylene)
thiazolidin-4-one (12)
Yield: 81%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.06 (s, 4H), 3.88 (s,
2H), 6.30 (s, 1H), 7.18e7.35 (m, 3H), 7.79 (d, 1H, J ¼ 6.0 Hz), 11.37 (s,
1H); RTLCMS: 1.60, Mass (ESIþ) m/z 301 [M þ H]þ; Analytical HPLC
Purity: 92%.
5.2.4. (E)-2-((4H-Thiochromeno[4,3-d]thiazol-2-yl)methylene)
thiazolidin-4-one (13)
Yield: 81%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.60 (s, 2H), 3.88 (s,
5.2. General procedure for the Hanzsch reaction in ethanol
2H), 6.15 (s, 1H), 7.21e7.56 (m, 3H), 8.01 (d, 1H, J ¼ 9.0 Hz), 11.53 (s,
1H); RTLCMS: 1.60, Mass (ESIþ) m/z 319 [M þ H]þ; Analytical HPLC
Purity: 95%.
To a solution of (E)-2-(4-oxothiazolidin-2-ylidene)ethanethioa-
mide (31) (1.0 mmol) in ethanol (10 mL) was added a-bromoketone
(32aed) (1.0 mmol), and the mixture was stirred at reflux for 3 h.
After addition of saturated NaHCO3 (10 mL), the mixture was
extracted with EtOAc (3 ꢁ 10 mL) and the combined organic layers
were washed with brine (30 mL) and dried (MgSO4). Evaporation of
the solvent followed by chromatography (hexaneeEtOAc, 1:1) of
the residue gave thiazole (2, 11e13).
5.3. General procedure for the Hanzsch reaction in N,N-dimethyl
formamide
To a solution of thioamide (31, 34a, b, dei) (1.0 mmol) in N,N-
dimethylformamide (10 mL) was added ethyl 2-bromoacetate or 2-
bromoester (38aed) (1.0 mmol), and the mixture was stirred at
40 ꢀC for 3 h. After addition of saturated NaHCO3 (10 mL), the
mixture was extracted with EtOAc (3 ꢁ 10 mL) and the combined
organic layers were washed with brine (30 mL) and dried (MgSO4).
Evaporation of the solvent followed by chromatography (hexanee
EtOAc, 1:1) of the residue gave thiazole (4, 7, 8, 15, 21e23, 25e30).
5.2.1. (E)-2-((4-(Pyridin-2-yl)thiazol-2-yl)methylene)thiazolidin-4-
one (2)
Yield: 96%; 1H NMR (CDCl3, 400 MHz),
d: 3.86 (s, 2H), 5.77 (s,
1H), 7.25 (m, 1H), 7.81 (ddd, 1H, J ¼ 9.5, 7.6, 1.6 Hz), 7.87 (s, 1H), 8.00
(d, 1H, J ¼ 7.9 Hz), 8.61 (dd, 1H, J ¼ 4.6, 0.6 Hz), 11.5 (br s, 1H); 13C
Scheme 1. Reagents and conditions: (a) 32aed, EtOH, reflux (2: 96%, 11: 81%, 12: 81%, 13: 81%); (b) ethyl 2-bromoacetate, DMF, 40 ꢀC (59%).

S. Sogame et al. / European Journal of Medicinal Chemistry 71 (2014) 1e9
5
Scheme 3. Reagents and conditions: (a) Methylamine, EtOH, r.t. (92%); (b) ethyl 2-bromoacetate, DMF, 40 ꢀC (7: 73%, 8: 68%, 15: 80%).
5.3.1. (E)-2-((4-Hydroxythiazol-2-yl)methylene)thiazolidin-4-one
(4)
5.3.5. (E)-2-((1H-Benzo[d]imidazol-2-yl)methylene)-5-ethylthiazo
lidin-4-one (21)
Yield: 59%; 1H NMR (DMSO-d6, 300 MHz),
d
: 3.86 (s, 2H), 6.00 (s,
Yield: quant; 1H NMR (DMSO-d6, 400 MHz),
d: 0.96 (t, 3H,
2H), 12.1 (br s, 2H); RTLCMS: 0.64, Mass (ESIþ) m/z 215 [M þ H]þ;
J ¼ 8.0 Hz), 1.88, (m, 2H), 4.10 (d, 1H, J ¼ 6.0, 3.0 Hz), 5.50 (s, 1H),
7.14e7.19 (m, 3H), 7.34 (br s, 1H), 9.95 (br s, 1H); RTLCMS: 0.88, Mass
(ESIþ) m/z 260 [M þ H]þ; Analytical HPLC Purity: 97%.
Analytical HPLC Purity: 91%.
5.3.2. (E)-2-(Benzo[d]oxazol-2-ylmethylene)thiazolidin-4-one (7)
5.3.6. (E)-2-((1H-Benzo[d]imidazol-2-yl)methylene)-5-propylthia
Yield: 73%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.92 (s, 2H), 6.03 (s,
zolidin-4-one (22)
1H), 7.26 (m, 2H), 7.59 (m, 2H), 11.6 (br s, 1H); RTLCMS: 1.54, Mass
Yield: quant; 1H NMR (CDCl3, 400 MHz),
d: 0.98 (t, 3H,
(ESIþ) m/z 233 [M þ H]þ; Analytical HPLC Purity: 93%.
J ¼ 7.3 Hz), 1.51 (m, 2H), 1.86 (m, 1H), 2.14 (m, 1H), 4.10 (dd, 2H,
J ¼ 9.2, 4.0 Hz), 5.46 (s, 1H), 7.20 (m, 4H), 7.46 (br s, 1H); 13C NMR
(CDCl3, 100 MHz), d: 13.6 (CH3), 20.4 (CH2), 35.5 (CH2), 48.9 (CH),
5.3.3. (E)-2-(Benzo[d]thiazol-2-ylmethylene)thiazolidin-4-one (8)
85.9 (CH), 122.6 (CH, 4 carbons),146.6 (C), 150.4 (C, 2 carbons),176.1
(C]O); RTLCMS: 1.01, Mass (ESIþ) m/z 274 [M þ H]þ; Analytical
HPLC Purity: 98%; HRMS (EI) calcd for C14H15N3OS (Mþ) 274.1009,
found 274.0999; mp 146.3 ꢀC.
Yield: 68%; 1H NMR (DMSO-d6, 400 MHz),
d: 3.94 (s, 2H), 6.46 (s,
1H), 7.38e7.43 (m, 2H), 7.92 (br s, 2H), 8.27 (br s, 1H); RTLCMS: 1.33,
Mass (ESIþ) m/z 249 [M þ H]þ; Analytical HPLC Purity: 97%.
5.3.4. (E)-2-((1H-Benzo[d]imidazol-2-yl)methylene)-3-
5.3.7. (E)-2-((1H-Benzo[d]imidazol-2-yl)methylene)-5-butylthiazo
methylthiazolidin-4-one (15)
lidin-4-one (23)
Yield: 80%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.12 (s, 3H), 3.82 (s,
Yield: quant; 1H NMR (DMSO-d6, 300 MHz),
d: 0.85 (t, 3H,
2H), 6.11 (s, 1H), 7.02e7.05 (m, 2H), 7.40 (br s, 2H); RTLCMS: 0.74,
J ¼ 3.0 Hz), 1.22e1.32 (m, 4H), 2.07 (m, 2H), 4.30 (dd, 1H, J ¼ 6.0,
Mass (ESIþ) m/z 246 [M þ H]þ; Analytical HPLC Purity: 99%.
3.0 Hz), 6.14 (s, 1H), 7.17e7.25 (m, 2H), 7.51e7.56 (m, 2H), 7.96 (s,
Scheme 4. Reagents and conditions: (a) Ethyl 2-cyanoacetate, neat, 185 ꢀC; (b) 37aec, Py, 100 ꢀC; (c) dithiophosphoric acid O,O-diethyl ester, H2O, 80 ꢀC; (d) 38aed, DMF, 40 ꢀC.
*CA ¼ commercially available.

6
S. Sogame et al. / European Journal of Medicinal Chemistry 71 (2014) 1e9
1H), 11.65 (s, 1H); RTLCMS: 1.16, Mass (ESIþ) m/z 288 [M þ H]þ;
J ¼ 3.0 Hz), 11.1 (s, 1H); RTLCMS: 1.20, Mass (ESIþ) m/z 198 [M þ H]þ;
Analytical HPLC Purity: 95%.
Analytical HPLC Purity: 97%.
5.3.8. (E)-2-((1H-Benzo[d]imidazol-2-yl)methylene)-5-(2-hydroxy
5.4.2. (E)-2-((1H-Imidazol-5-yl)methylene)thiazolidin-4-one (6)
ethyl)thiazolidin-4-one (25)
Yield: 81%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.73 (s, 2H), 5.97 (s,
Yield: quant; 1H NMR (DMSO-d6, 300 MHz),
d: 1.96 (m, 1H), 2.31
1H), 6.71 (s, 2H); RTLCMS: 0.36, Mass (ESIþ) m/z 182 [M þ H]þ;
(m, 1H), 3.65 (dd, 2H, J ¼ 9.0, 6.0 Hz), 4.54 (dd, 1H, J ¼ 9.0, 3.0 Hz),
6.23 (m,1H), 7.40 (m, 4H),12.1 (s,1H); RTLCMS: 0.67, Mass (ESIþ) m/z
276 [M þ H]þ; Analytical HPLC Purity: 98%.
Analytical HPLC Purity: 93%.
5.4.3. (E)-2-((1H-Naphtho[2,3-d]imidazol-2-yl)methylene)
thiazolidin-4-one (10)
5.3.9. (E)-2-((7-Methyl-1H-benzo[d]imidazol-2-yl)methylene)-5-
Yield: quant; 1H NMR (DMSO-d6, 300 MHz),
d: 3.94 (s, 2H), 5.15
propylthiazolidin-4-one (26)
(s, 1H), 7.31e7.34 (m, 4H), 7.91 (m, 2H), 8.00 (s, 1H); RTLCMS: 1.45,
Yield: 59%; 1H NMR (DMSO-d6, 300 MHz),
d: 0.93 (t, 3H,
Mass (ESIþ) m/z 282 [M þ H]þ; Analytical HPLC Purity: 99%.
J ¼ 3.0 Hz), 1.31e1.55 (m, 2H), 1.78 (m, 1H), 1.99 (m, 1H), 2.50 (s, 3H),
4.02 (br s, 2H), 4.22 (br s, 1H), 5.56 (s, 1H), 6.99 (t, 1H, J ¼ 6.0 Hz),
7.08 (t, 1H, J ¼ 6.0 Hz), 7.35 (m, 1H); RTLCMS: 1.12, Mass (ESIþ) m/z
288 [M þ H]þ; Analytical HPLC Purity: 99%.
5.4.4. (E)-2-((1H-Benzo[d]imidazol-2-yl)methylene)-5-methylthia
zolidin-4-one (14)
Yield: 82%; 1H NMR (DMSO-d6, 400 MHz),
d: 1.44 (d, 3H,
J ¼ 4.9 Hz), 4.02 (br s, 1H), 5.56 (s, 1H), 7.01e7.07 (m, 4H), 7.28 (br s,
1H); RTLCMS: 1.19, Mass (ESIþ) m/z 246 [M þ H]þ; Analytical HPLC
Purity: 99%.
5.3.10. (E)-2-((6-Methyl-1H-benzo[d]imidazol-2-yl)methylene)-5-
propylthiazolidin-4-one (27)
Yield: 49%; 1H NMR (DMSO-d6, 300 MHz),
d: 0.92 (t, 3H,
J ¼ 3.0 Hz), 1.32e1.56 (m, 2H), 1.76 (m,1H), 1.98 (m, 1H), 2.45 (s, 3H),
4.02 (br s, 2H), 4.22 (br s, 1H), 5.56 (s, 1H), 7.00 (d, 1H, J ¼ 6.0 Hz),
7.32 (s, 1H), 7.42 (d, 1H, J ¼ 6.0 Hz); RTLCMS: 1.11, Mass (ESIþ) m/z
288 [M þ H]þ; Analytical HPLC Purity: 94%.
5.4.5. (E)-2-((1-Methyl-1H-benzo[d]imidazol-2-yl)methylene)
thiazolidin-4-one (16)
Yield: 88%; 1H NMR (DMSO-d6, 400 MHz),
d: 3.81 (s, 3H), 4.03 (s,
2H), 5.99 (s,1H), 7.18e7.23 (m, 2H), 7.50 (dd,1H, J ¼ 6.0, 3.0 Hz), 7.61
(dd,1H, J ¼ 6.0, 3.0 Hz), 11.5 (br s, 1H); RTLCMS: 1.23, Mass (ESIþ) m/z
246 [M þ H]þ; Analytical HPLC Purity: 96%.
5.3.11. (E)-2-((5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)methylene)-
5-propylthiazolidin-4-one (28)
Yield: 47%; 1H NMR (DMSO-d6, 400 MHz),
d: 0.93 (t, 3H,
5.4.6. (E)-2-((7-Methyl-1H-benzo[d]imidazol-2-yl)methylene)
J ¼ 3.0 Hz), 1.34e1.55 (m, 2H), 1.77 (m, 1H), 2.00 (m, 1H), 2.06 (s,
3H), 2.07 (s, 3H), 4.02 (br s, 2H), 4.22 (br s, 1H), 5.56 (s, 1H), 7.27 (br
s, 2H), 8.01 (s, 1H); RTLCMS: 1.19, Mass (ESIþ) m/z 302 [M þ H]þ;
Analytical HPLC Purity: 97%.
thiazolidin-4-one (17)
Yield: 37%; 1H NMR (DMSO-d6, 400 MHz),
d: 1.85 (s, 3H), 3.92 (s,
2H), 5.17 (s, 1H), 6.91e6.99 (m, 3H), 11.5 (br s, 1H); RTLCMS: 1.24,
Mass (ESIþ) m/z 246 [M þ H]þ; Analytical HPLC Purity: 94%.
5.3.12. (E)-5-Propyl-2-((6-(trifluoromethyl)-1H-benzo[d]imidazol-
2-yl)methylene)-thiazolidin-4-one (29)
5.4.7. (E)-2-((6-Methyl-1H-benzo[d]imidazol-2-yl)methylene)
thiazolidin-4-one (18)
Yield: 32%; 1H NMR (DMSO-d6, 400 MHz),
d
: 0.93 (t, 3H,
Yield: 45%; 1H NMR (DMSO-d6, 600 MHz),
d: 2.38 (s, 3H), 3.89 (s,
J ¼ 3.0 Hz), 1.34e1.55 (m, 2H), 1.77 (m, 1H), 2.00 (m, 1H), 4.02 (br s,
2H), 4.22 (br s, 1H), 5.56 (s, 1H), 7.52 (m, 1H), 7.70 (d, 1H, J ¼ 9.0 Hz),
7.87 (m, 1H); RTLCMS: 1.63, Mass (ESIþ) m/z 342 [M þ H]þ;
Analytical HPLC Purity: 98%.
2H), 6.92 (s, 1H), 7.31e7.46 (m, 3H), 11.9 (br s, 1H); RTLCMS: 1.18,
Mass (ESIþ) m/z 246 [M þ H]þ; Analytical HPLC Purity: 92%.
5.4.8. (E)-2-((5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)methylene)
thiazolidin-4-one (19)
5.3.13. (E)-2-((6-Fluoro-1H-benzo[d]imidazol-2-yl)methylene)-5-
Yield: 41%; 1H NMR (DMSO-d6, 600 MHz),
d: 2.18 (s, 6H), 3.88 (s,
propylthiazolidin-4-one (30)
2H), 5.34 (s,1H), 7.24 (s,1H), 7.30 (s, 1H),11.7 (br s,1H); RTLCMS: 1.27,
Yield: 59%; 1H NMR (DMSO-d6, 300 MHz),
d
: 0.93 (t, 3H,
Mass (ESIþ) m/z 260 [M þ H]þ; Analytical HPLC Purity: 91%.
J ¼ 3.0 Hz), 1.34e1.55 (m, 2H), 1.77 (m, 1H), 2.00 (m, 1H), 4.02 (br s,
2H), 4.22 (br s, 1H), 5.56 (s, 1H), 7.00 (br s, 1H), 7.34 (d, 1H,
J ¼ 6.0 Hz), 7.59 (d, 1H, J ¼ 6.0 Hz); RTLCMS: 1.25, Mass (ESIþ) m/z
292 [M þ H]þ; Analytical HPLC Purity: 93%.
5.4.9. (E)-2-((6,7-Dimethyl-1H-benzo[d]imidazol-2-yl)methylene)
thiazolidin-4-one (20)
Yield: 79%; 1H NMR (DMSO-d6, 400 MHz),
d: 2.29 (s, 3H), 2.46 (s,
3H), 3.89 (s, 2H), 5.42 (s, 1H), 6.95 (d, 1H, J ¼ 6.0 Hz), 7.27 (d, 1H,
J ¼ 6.0 Hz), 11.7 (br s, 1H); RTLCMS: 1.34, Mass (ESIþ) m/z 260
[M þ H]þ; Analytical HPLC Purity: 91%.
5.4. General procedure for the condensation of nitrile and 2-
mercaptoacetic acid
To a solution of nitrile (33a, b, 36aef, i) (1.0 mmol) in pyridine
(2.5 mL) was added 2-mercaptoacetic acid or 37aec (1.0 mmol),
and the mixture was stirred at reflux for 8 h. After addition of H2O
(10 mL), the mixture was extracted with EtOAc (3 ꢁ 10 mL) and the
combined organic layers were washed with brine (30 mL) and dried
(MgSO4). Evaporation of the solvent followed by chromatography
(hexaneeEtOAc, 1:1) of the residue gave thiazole (5, 6, 10, 14, 16e20
and 24).
5.4.10. (E)-2-(2-((1H-benzo[d]imidazol-2-yl)methylene)-4-oxothia
zolidin-5-yl)acetic acid (24)
Yield: 60%; 1H NMR (DMSO-d6, 400 MHz),
d: 2.74 (m, 2H), 2.98
(m, 2H), 4.23 (d, 1H, J ¼ 6.0 Hz), 5.17 (s, 1H), 6.91e6.99 (m, 4H), 11.5
(br s, 1H); RTLCMS: 0.94, Mass (ESIþ) m/z 290 [M þ H]þ; Analytical
HPLC Purity: 92%.
5.5. 2-(1H-Benzo[d]imidazol-2-yl)-N-methylethanethioamide
(34d)
5.4.1. (E)-2-(Thiophen-2-ylmethylene)thiazolidin-4-one (5)
Yield: 96%; 1H NMR (DMSO-d6, 300 MHz),
1H), 6.91 (d, 1H, J ¼ 1.5 Hz), 7.00 (dd, 1H, J ¼ 3.0, 1.5 Hz), 7.38 (d, 1H,
d
: 3.95 (s, 2H), 6.30 (s,
To a solution of 2-(1H-benzo[d]imidazol-2-yl)ethanethioamide
(34c) (191.2 mg, 1.0 mmol) in ethanol (5 mL) was added 25%

S. Sogame et al. / European Journal of Medicinal Chemistry 71 (2014) 1e9
7
methylamine in H2O solution (416
m
L, 3.0 mmol), and the mixture
After the reaction was quenched with saturated aqueous NaHCO3
(10 mL), the organic phase was separated and the aqueous phase
was extracted with diethylether (3 ꢁ 30 mL). The combined organic
phases were washed with brine (30 mL), dried (MgSO4), and
concentrated in vacuo. The residue was purified by column chro-
matography (hexaneseEtOAc, 1:1) to give thioamide (34eei).
was stirred at room temperature for 24 h. After addition of H2O
(5 mL), the mixture was extracted with EtOAc (3 ꢁ 10 mL) and the
combined organic layers were washed with brine (30 mL) and dried
(MgSO4). Evaporation of the solvent followed by chromatography
(hexaneeEtOAc, 1:1) of the residue gave 34d (188 mg, 0.91 mmol).
Yield: 92%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.02 (s, 3H), 3.72 (s,
2H), 7.11e7.17 (m, 2H), 7.50 (br s, 2H); RTLCMS: 0.65, Mass (ESIþ) m/z
5.7.1. 2-(7-Methyl-1H-benzo[d]imidazol-2-yl)ethanethioamide
206 [M þ H]þ; Analytical HPLC Purity: 99%.
(34e)
Yield: quant; 1H NMR (DMSO-d6, 300 MHz),
d: 2.51 (s, 3H), 3.76
5.6. General procedure for the cyclization of benzimidazole
(s, 2H), 7.08e7.19 (m, 2H), 7.40 (br s, 1H); RTLCMS: 0.66, Mass (ESIþ)
m/z 206 [M þ H]þ; Analytical HPLC Purity: 90%.
To benzene-1,2-diamine (35aeg) (5.0 mmol) was added ethyl 2-
cyanoacetate (1.6 mL, 15.0 mmol) under neat condition, and the
mixture was stirred at 185 ꢀC for 1.5 h. After addition of diethylether
(10 mL) and saturated NaHCO3 (10 mL), the mixture was extracted
with diethylether (3 ꢁ 10 mL) and the combined organic layers
were washed with brine (30 mL) and dried (MgSO4), and concen-
trated in vacuo to give the crude benzimidazole (36cei).
5.7.2. 2-(6-Methyl-1H-benzo[d]imidazol-2-yl)ethanethioamide
(34f)
Yield: 95%; 1H NMR (DMSO-d6, 300 MHz),
d: 2.45 (s, 3H), 3.79 (s,
2H), 7.09 (d, 1H, J ¼ 6.0 Hz), 7.42 (br s, 1H), 7.54 (d, 1H, J ¼ 6.0 Hz);
RTLCMS: 0.67, Mass (ESIþ) m/z 206 [M þ H]þ; Analytical HPLC Pu-
rity: 91%.
5.6.1. 2-(7-Methyl-1H-benzo[d]imidazol-2-yl)acetonitrile (36c)
5.7.3. 2-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)ethanethioamide
Yield: 40%; 1H NMR (DMSO-d6, 300 MHz),
d: 2.50 (s, 3H), 4.37 (s,
(34g)
2H), 6.99 (t, 1H, J ¼ 6.0 Hz), 7.08 (t, 1H, J ¼ 6.0 Hz), 7.35 (m, 1H), 12.6
(br s, 1H); RTLCMS: 0.87, Mass (ESIþ) m/z 172 [M þ H]þ; Analytical
HPLC Purity: 99%.
Yield: quant; 1H NMR (DMSO-d6, 300 MHz),
d: 2.37 (s, 3H), 2.47
(s, 3H), 3.76 (s, 2H), 7.38 (br s, 2H); RTLCMS: 0.77, Mass (ESIþ) m/z
220 [M þ H]þ; Analytical HPLC Purity: 95%.
5.6.2. 2-(6-Methyl-1H-benzo[d]imidazol-2-yl)acetonitrile (36d)
5.7.4. 2-(6-(Trifluoromethyl)-1H-benzo[d]imidazol-2-yl)
ethanethioamide (34h)
Yield: quant; 1H NMR (DMSO-d6, 300 MHz),
d: 2.40 (s, 3H), 4.35
(s, 2H), 7.00 (d, 1H, J ¼ 6.0 Hz), 7.32 (s, 1H), 7.42 (d, 1H, J ¼ 6.0 Hz),
12.5 (br s, 1H); RTLCMS: 0.88, Mass (ESIþ) m/z 172 [M þ H]þ;
Analytical HPLC Purity: 99%.
Yield: 98%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.76 (s, 2H), 7.56 (br
s, 1H), 7.73 (br s, 1H), 7.96 (br s, 1H); RTLCMS: 1.06, Mass (ESIþ) m/z
261 [M þ H]þ; Analytical HPLC Purity: 91%.
5.6.3. 2-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)acetonitrile (36e)
5.7.5. 2-(6-Fluoro-1H-benzo[d]imidazol-2-yl)ethanethioamide (34i)
Yield: 58%; 1H NMR (DMSO-d6, 300 MHz),
d
: 2.29 (s, 6H), 4.49 (s,
Yield: 92%; 1H NMR (DMSO-d6, 300 MHz),
d: 3.77 (s, 2H), 7.00 (br
2H), 7.30 (s, 2H), 12.4 (br s, 1H); RTLCMS: 0.96, Mass (ESIþ) m/z 186
s, 1H), 7.34 (d, 1H, J ¼ 6.0 Hz), 7.59 (d, 1H, J ¼ 6.0 Hz); RTLCMS: 0.66,
[M þ H]þ; Analytical HPLC Purity: 99%.
Mass (ESIþ) m/z 210 [M þ H]þ; Analytical HPLC Purity: 91%.
5.6.4. 2-(6,7-Dimethyl-1H-benzo[d]imidazol-2-yl)acetonitrile (36f)
5.8. Biological testing
Yield: 85%; 1H NMR (DMSO-d6, 300 MHz),
d: 2.39 (s, 3H), 2.49 (s,
3H), 4.15 (s, 2H), 7.11 (d, 1H, J ¼ 6.0 Hz), 7.26 (s, 1H), 7.32 (d, 1H,
J ¼ 6.0 Hz); RTLCMS: 0.98, Mass (ESIþ) m/z 186 [M þ H]þ; Analytical
HPLC Purity: 99%.
5.8.1. Preparation of human recombinant ADAMTS-5
The enzyme protein used for measurement of ADAMTS-5 ac-
tivity was human recombinant ADAMTS-5. An insect cell line was
prepared that stably expressed human recombinant ADAMTS-5 in
which 6xHis tag was added to the C-terminal side of Asn624. The
culture supernatant of this cell line was collected, and the obtained
culture supernatant was purified using Ni-NTA agarose.
5.6.5. 2-(6-(Trifluoromethyl)-1H-benzo[d]imidazol-2-yl)acetonitrile
(36g)
Yield: 66%; 1H NMR (DMSO-d6, 300 MHz),
d: 4.48 (s, 2H), 7.52
(m, 1H), 7.70 (d, 1H, J ¼ 9.0 Hz), 7.87 (m, 1H), 7.99 (s, 1H); RTLCMS
:
1.15, Mass (ESIþ) m/z 226 [M þ H]þ; Analytical HPLC Purity: 99%.
5.8.2. Preparation of interglobular domain of human recombinant
aggrecan
5.6.6. 2-(6-Fluoro-1H-benzo[d]imidazol-2-yl)acetonitrile (36h)
A matrix peptide used for measurement of aggrecanase activity
was Interglobular domain of human recombinant aggrecan. With
reference to the method of Hughes et al. [11], the peptide of Thr331
to Gly457 of human aggrecan was contained, and the FLAG epitope
peptide was added at the N-terminal, and the C-terminal was
modified with biotin. This peptide was expressed using Escherichia
coli, and the obtained E. coli was subjected to lysis, and then the
peptide was purified with an affinity column.
Yield: 79%; 1H NMR (DMSO-d6, 300 MHz),
d: 4.39 (s, 2H), 7.06
(td, 1H, J ¼ 9.0, 3.0 Hz), 7.37 (dd, 1H, J ¼ 6.0, 3.0 Hz), 7.06 (td, 1H,
J ¼ 9.0, 6.0 Hz), 8.32 (s, 1H); RTLCMS: 0.85, Mass (ESIþ) m/z 176
[M þ H]þ; Analytical HPLC Purity: 99%.
5.6.7. 2-(1H-Naphtho[2,3-d]imidazol-2-yl)acetonitrile (36i)
Yield: 59%; 1H NMR (DMSO-d6, 300 MHz),
d: 4.51 (s, 2H), 7.38 (m,
2H), 8.00 (m, 4H); RTLCMS: 1.11, Mass (ESIþ) m/z 208 [M þ H]þ;
Analytical HPLC Purity: 99%.
5.8.3. Measurement of inhibition for ADAMTS-5
Desired concentration of the test compound was reacted in
5.7. General procedure for the conversion from nitrile to thioamide
40 mL of a mixed reaction solution containing 50 mmol/L TriseHCl
(pH 7.5), 100 mmol/L NaCl, 10 mmol/L CaCl2, 0.05% Brij35 and hu-
man recombinant ADAMTS-5 and Interglobular domain peptide of
human recombinant aggrecan at 37 ꢀC, for 60 min. To the mixed
To a stirred solution of nitrile (36cee, g and h) (4.58 mmol) in
H2O (0.92 mL) were added Dithiophosphoric acid O,O-diethyl ester
(2.6 mL, 16.5 mmol), and the mixture was stirred at 80 ꢀC for 4 h.
reaction solution was added 10 mL of 0.2 mol/L EDTA to stop the

8
S. Sogame et al. / European Journal of Medicinal Chemistry 71 (2014) 1e9
enzyme reaction. 5
m
M of this mixed reaction solution was har-
reaction. Then the fluorescence value was measured at 320 nm
excitation wavelength and 405 nm measurement wavelength with
a fluorescence plate reader.
vested and transferred to a 384 well plate. 0.2% Bovine albumin,
cryptate-labeled Streptavidin suitably diluted with PBS (ꢂ)-con-
taining 800 mmol/L potassium fluoride were added thereto so that
the amount of each of the added matters was 2.5 mM. The resultant
5.9. Permeability (PAMPA) assay
solution was incubated at room temperature. After 60 min, the
fluorescence value was measured at 340 nm excitation wavelength
and 615 nm and 665 nm measurement wavelengths with a fluo-
rescence plate reader. When the matrix peptide was not cleaved,
the fluorescence resonance energy transfer from cryptate to XL665
occurs and long life fluorescence of 665 nm wavelength is gener-
ated. Meanwhile, fluorescence resonance energy transfer dose not
occur and long life fluorescence is not generated when the matrix
peptide was cleaved. Therefore, the aggrecanase activity was
calculated using the change of the ratio of the fluorescence value at
665 nm and the fluorescence value at 615 nm.
A solution of hexadecane in hexane is prepared (5% v/v) and an
aliquot added onto the membrane of each well in the filter (donor)
plate (MultiscreenÒ filter plate for PAMPA, Millipore). The donor
plates are then allowed to dry to ensure evaporation of hexane.
Buffer (pH 7.4 or other selected pHs) containing a final DMSO
concentration of 5% is added to each well of the acceptor plates. Test
compound solutions are prepared by diluting 10 mM DMSO con-
centrates in buffer to give a final test compound concentration of
50 mM. The acceptor filter plate was carefully put on the donor plate
to form a ‘sandwich’ (consisting of the aqueous donor with test
compound on the bottom, artificial lipid membrane in the middle,
and aqueous acceptor on the top). The test compound diffused from
the donor well through the lipid membrane and into the acceptor
well. The ‘sandwich’ was left undisturbed at room temperature for
4 h while the permeation occurred. The concentration of drug in
the acceptor, the donor, and the reference wells was determined
using the LC-MS. Samples were analyzed in duplicate and the
average of the two runs was reported. Quality control standards
(Atenolol, Metoprolol, Procainamide and Propranolol) were run
with each sample set to monitor the consistency of the analysis
set [12].
5.8.4. Measurement of inhibition for ADAMTS-4
Desired concentration of the test compound was reacted in
40 mL of a mixed reaction solution containing 50 mmol/L TriseHCl
(pH 7.5), 100 mmol/L NaCl, 10 mmol/L CaCl2, 0.05% Brij35 and hu-
man recombinant ADAMTS-4 (R&D) and Interglobular domain
peptide of human recombinant aggrecan at 37 ꢀC, for 60 min. To the
mixed reaction solution was added 10 mL of 0.2 mol/L EDTA to stop
the enzyme reaction. Five 111 of this mixed reaction solution was
harvested and transferred to a 384 well plate. 0.2% Bovine albumin,
cryptate-labeled Streptavidin and XL665 labeled anti FLAGÒ (M2)
antibody suitably diluted with PBS (ꢂ)-containing 800 mmol/L
potassium fluoride were added thereto so that the amount of each
5.10. Log D determination procedure
of the added matters was 2.5 mM. The reaction solution was incu-
bated at room temperature. After 60 min, the fluorescence value
was measured at 340 nm excitation wavelength and 615 nm and
665 nm measurement wavelengths with a fluorescence plate
reader. The aggrecanase activity was calculated using the change of
the ratio of these fluorescence values.
The separations are carried out on an Inertsil, ODS-4
150 ꢁ 4.0 mm I.D., 5
mm (Shimadzu Corporation, Japan). The mo-
bile phase consists of phosphate buffered saline at pH 7.4 (mobile
phase A) and acetonitrile (mobile phase B). The HPLC analysis be-
gins with an isocratic step of 10 min at 30% B at 1.5 ml/min, fol-
lowed by a gradient from 30% to 80% B in 20 min at 1.0 ml/min. The
chromatographic system is calibrated with a set of standards with
published shake-flask Log D values. Linear regression is used to
determine the calibration line relating the retention time to Log D
for the calibration standards. This line is then used to determine the
HT HPLC Log D value (pH 7.4) from the measured retention time by
the HPLC analysis (254 nm) [13].
5.8.5. Measurement of inhibition for MMP-2
Desired concentration of the test compound was reacted in
100 mL of a mixed reaction solution containing 50 nmol/L TriseHCl
(pH 7.5), 150 mmol/L NaCl, 10 mmol/L CaCl2, 0.05% Brij35 and
activated human recombinant MMP-2 (R&D) and fluorescence
peptide matrix Mca-ProLeuGlyLeuDpaAlaArg-NH2 (R&D) at 30 ꢀC,
for 45 min. To the mixed reaction solution, 10 mL of 0.2 mol/L EDTA
was added to stop the enzyme reaction. Then the fluorescence
value was measured at 320 nm excitation wavelength and 405 nm
measurement wavelength with a fluorescence plate reader.
Acknowledgment
The authors thank Mr. Takashi Suzuki and Ms. Hiroko Arimoto
for NMR support; Mr. Keigo Yoshida, Ms. Emiko Suzuki and Ms.
Junko Hayashi for physicochemical data support. We also thank Mr.
Eisuke Toriihara for the helpful discussion during manuscript
preparation.
5.8.6. Measurement of inhibition for MMP-3
Desired concentration of the test compound was reacted in
100 mL of a mixed reaction solution containing 50 nmol/L TriseHCl
(pH7.5), 150 mmol/L NaCl, 10 mmol/L CaCl2, 0.05% Brij35 and acti-
vated human recombinant MMP-3 (R&D) and fluorescence peptide
matrix Mca-ArgProLysProValGlu-Nval-TrpArgLys(Dnp)-NH2 (R&D)
Appendix A. Supplementary data
at 30 ꢀC, for 45 min. To the mixed reaction solution,10
mL of 0.2 mol/
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.075.
L EDTA was added to stop the enzyme reaction. Then the fluores-
cence value was measured at 320 nm excitation wavelength and
405 nm measurement wavelength with a fluorescence plate reader.
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Abbreviations used
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K. Aoki, K. Yasue, H. Imai, M. Ubukata, A. Suma, M. Yokota, T. Hotta, M. Tanaka,
Y. Hase, J. Haas, A.M. Fryer, E.R. Laird, N.M. Littmann, S.W. Andrews, J.A. Josey,
T. Mimura, Y. Shinozaki, H. Yoshiuchi, T. Inaba, Discovery of (1S,2R,3R)-2,3-
dimethyl-2-phenyl-1-sulfamidocyclopropanecarboxylates: novel and highly
selective aggrecanase inhibitors, J. Med. Chem. 54 (2011) 2839;
(c) H. Deng, H. O’Keffe, C.P. Davie, K.E. Lind, R.A. Acharya, G.J. Franklin,
J. Larkin, R. Matico, M. Neeb, M.M. Thompson, T. Lohr, J.W. Gross,
P.A. Centrella, G.K. O’Donovan, M.M. Bedard (Sargent), K.V. Vloten,
S. Mataruse, S.R. Skinner, S.L. Belyanskaya, T.Y. Carpenter, T.W. Shearer,
M.A. Clark, J.W. Cuozzo, C.C. Arico-Muendel, B.A. Morgan, Discovery of highly
potent and selective small molecule ADAMTS-5 inhibitors that inhibit human
cartilage degradation via encoded library technology (ELT), J. Med. Chem. 55
(2012) 7061;
ADAMTS-5: a disintegrin and metalloproteinase with thrombospondin motifs 5
Bu: butyl
CA: commercially available
DMF: dimethylformamide
DMSO: dimethyl sulfoxide
Dnp: dinitrophenyl
ECM: extra cartilage matrix
EDTA: ethylenediamine tetraacetate
ESI: electrospray ionization
Et: ethyl
EtOAc: ethyl acetate
EtOH: ethanol
Het: heterocycle group
IL-1: interleukin-1
LE: ligand efficiency
Me: methyl
MMP-2: matrix metalloproteinase-2
MgSO4: magnesium sulfate
NaHCO3: sodium hydrogen carbonate
NMR: nuclear magnetic resonance
OA: osteoarthritis
(d) E. Nuti, S. Santamaria, F. Casalini, K. Yamamoto, L. Marinelli, V.L. Pietra,
E. Novellino, E. Orlandini, S. Nencetti, A.M. Marini, S. Salerno, S. Taliani,
F.D. Settimo, H. Nagase, A. Rossello, Arylsulfonamide inhibitors of aggreca-
nases as potential therapeutic agents for osteoarthritis: synthesis and bio-
logical evaluation, Eur. J. Med. Chem. 62 (2013) 379;
(e) M. Atobe, N. Maekawara, M. Kawanishi, H. Suzuki, E. Tanaka, S. Miyoshi,
Design, synthesis and SAR investigation of thienosultam derivatives as
ADAMTS-5 (aggrecanase-2) inhibitors, Bioorg. Med. Chem. Lett. 23 (2013)
2111;
ODS: octadecyl silyl
PAMPA: parallel artificial membrane permeability assay
PBS: phosphate buffered saline
Py: pyridine
RTLCMS: retention time
SAR: structureeactivity relationship
(f) L. Maingot, J. Elbakali, J. Dumont, D. Bosc, N. Cousaert, A. Urban, G. Deglane,
B. Villoutreix, H. Nagase, O. Sperandio, F. Leroux, B. Deprez, R. Deprez-Poulain,
Aggrecanase-2 inhibitors based on the acylthiosemicarbazide zinc-binding
group, Eur. J. Med. Chem. 69 (2013) 244.
TACE: tumor necrosis factor-
TMS: tetramethylsilane
TPSA: topological polar surface area.
a-converting enzyme