Design, synthesis, and structure–activity relationship study of epoxysuccinyl–peptide derivatives as cathepsin B inhibitors

Article abstract of DOI:10.1248/bpb.b17-00075

Cathepsin B is a lysosomal cysteine protease involved in many diseases. The present research demonstrates that derivatives of epoxysuccinyl–peptide are effective and selective cathepsin B inhibitors. We synthesized a series of epoxysuccinyl–peptide deriva

Full text of DOI:10.1248/bpb.b17-00075

Biological and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/bpb.b17-00075
Advance Publication
May 13, 2017
Biological and Pharmaceutical Bulletin
Regular Article
Design, synthesis, and structure-activity relationship study of epoxysuccinyl-peptide
derivatives as cathepsin B inhibitors
Xiaoye Zhanga · Xiaohong Yanga · Hongqiang Wanga · Song Lib · Kun Guoc · Dan Jiangb ·
Junhai Xiaob,* · Di Lianga,*
a School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China;
bLaboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of
c
Pharmacology and Toxicology, Beijing 100850, China; Shaanxi University of Chinese
Medicine, College of Humanities and Management, Century Avenue, The New District of
Xi’an-Xianyang, Shaanxi Province 712046, China
Corresponding author:
Di Liang, School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China;
E-Mail: zxyxiaoye@foxmail.com; liangdi123@sina.com;
Junhai Xiao, Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of
Pharmacology and Toxicology, Beijing 100850, China;
E-Mail: xiaojunhai@139.com.
Ⓒ 2017 The Pharmaceutical Society of Japan

Abstract
Cathepsin B is a lysosomal cysteine protease involved in many diseases. The present research
demonstrates that derivatives of epoxysuccinyl-peptide are effective and selective cathepsin
B inhibitors. We synthesized a series of epoxysuccinyl-peptide derivatives based on the
well-known cathepsin B inhibitor E64d. Specifically, we substituted the 2-methylpropane
group at the R1 position of E64d with a sulfane, such as ethyl(methyl) sulfane or
benzyl(methyl) sulfane. We also designed and synthesized a library of molecules with
various substituents at the R2 position of E64d to replace 2-methylbutane. By studying the
structure-activity relationships of these newly synthesized molecules as cathepsin B inhibitors,
we demonstrated that substituting ethyl(methyl) sulfane for 2-methylbutane (R2) of E64d
improves the inhibitory activity and selectivity for cathepsin B inhibition. Our new cathepsin
B inhibitors were highly effective and selective.
Keywords
Cathepsin B, lysosomal cysteine protease, structure-activity relationship, inhibitory activity,
selectivity.
Biological and Pharmaceutical Bulletin Advance Publication

Introduction
In modern society, cancer is a major threat to human health around the world. Although
most patients who are diagnosed with cancer are recommended to undergo a surgical
procedure to remove the tumors, a total resection is rarely achieved. It is necessary to follow
the surgery with extensive radio- and/or chemotherapies to control the residual cancer cells.
In addition, the total management of metastatic cancer cells is challenging; thus, a very high
1)
recurrence rate of cancers usually occurs . Cathepsin B is a member of the lysosomal
cysteine protease family encoded by the cathepsin gene in humans. It is expressed in many
organs, where it is involved in various aspects of immune responses, in the development and
2)
proliferation of various cell types, as well as in tumor invasion and metastasis . Emerging
3, 4)
evidence suggests that elevated levels of cathepsin B are present in many tumor tissues
.
Therefore, cathepsin B has been selected as a potential target for the treatment of a variety of
human cancers, including kidney cancer, lung cancer, and liver cancer 5, 6). In order to achieve
effective cancer treatment, it is of great importance to locate the active site of cathepsin B and
to develop specific inhibitors. Typically, the active site of cathepsin B is divided into multiple
subsites. Potent inhibitors of cathepsin B simultaneously bind to the S1ʹ and S2ʹ subsites 7).
In the past few years, extensive work has been conducted to develop effective cathepsin B
inhibitors for potential cancer treatment 4). For example, Katunuma has reported the synthesis
of a derivative of the epoxysuccinyl-peptide cathepsin B inhibitor by covalently linking its
ethylene oxide with the thiol group of the Cys29 residue of cathepsin B 8). In addition, Bogyo
et al. have synthesized two interesting compounds: E64d and CA-074 (1 and 2 as shown in
Figure 1) 9). Both are derivatives of the epoxysuccinyl-peptide and readily bind to the S1ʹ-S2ʹ
subsites of cathepsin B as highly selective inhibitors 10, 11). The selectivity of E64d and its
related analogs has been determined to be due to efficient hydrogen-bond interactions
Biological and Pharmaceutical Bulletin Advance Publication

between a free carboxylic acid on the inhibitor and two histidine residues in the occluding
loop structure found in cathepsin B 12)
.
Here, we describe the design and synthesis of highly selective and effective cathepsin B
inhibitors based on epoxysuccinyl-peptide derivatives by applying different substituents at
the R1 and R2 positions of E64d (Figure 2). When we design a new chemical structure to
replace the Leucine (Leu) of E64d, the principle is to maintain the inhibitory activity of the
new molecule to Cathepsin B while reducing its activities to other Cathepsins (such as
Cathepsin K, Cathepsin L) by different substitutions at R1 and R2 positions. Previously
Watanabe et al. reported the synthesis of a R1 derivative with oxygen atom substitutes as an
inhibitor of epoxysuccinyl-peptide cathepsin B inhibitor 13), Sadaghiani and coworkers also
synthesized a similar epoxysuccinyl-peptide cathepsin B inhibitor with a phenyl group at the
R1 position.9) Both molecules however failed to maintain the overall inhibition and selectivity
to Cathepsin B. Here we successfully synthesized a series of E64d derivatives with
Methionine (Met, contains sulfur) substitute at R1 position. As compared to Leu, Met
substitute possesses very similar characteristics in steric hindrance, size, and chain length. It
is thus hypothesized the substitution with Met will result in minimal effect on its inhibition of
Cathepsin B. As for Cysteine, it is a bulky group as compared to Leu and Met and is expected
to create a larger steric hindrance. We hope through this substitution in R1 of E64d to
understand how hindrance increase impact its overall selectivity and inhibitory activities. In
addition, we also carefully selected 5 substituents at R2 with different structure and size. We
hope through this series of comparison, we was able to understand their selectivity and
structure-activity relationships as Cathepsin B inhibitors. Interestingly, we found that the
ethyl(methyl) sulfane or benzyl(methyl) sulfane moiety at the R1 position, replacing
2-methylpropane of E64d, had improved specificity and selectivity as a cathepsin B inhibitor.
Biological and Pharmaceutical Bulletin Advance Publication

Materials and Experiments
General
Silica gel 60F254 25 aluminum sheets (20 × 20 cm) from Merck KGaA were used for TLC.
Silica gel (200–300 mesh, YanTai Huagong) was used for column chromatography. The
1H-NMR spectra (400 MHz) were recorded on a JNM-ECA-400 in DMSO and CHCl3.
Chemical shift values were reported in parts per million (δ) downfield from TMS as the
internal reference. Unless otherwise specified: br, broad; s, singlet; d, doublet; t, triplet; q,
quarter; m, multiplet; dd, double doublet. Mass spectra were obtained on an Agilent
1260-G6230A (USA).
Synthesis
Diethyl (2S,3S)- oxirane-2,3-dicarboxylate (5)
A 2-L, dry, round-bottom flask was charged with L-arginine (0.56 mol; 97.01 g) in 250
mL of warm water, to which a total of 1,078 mL of methanol solution containing 74.03 g of 3
(0.56 mol, (+/-)-trans-epoxysuccinic acid, from Alfa Aesar Chemical Co.) was then slowly
added over 20 min; the mixture was stirred at room temperature overnight. A white
precipitate was filtered and washed with 410 mL of MeOH-water (4:1) to furnish 83.50 g of
crude 4. Recrystallization of the crude material from 1,200 mL of MeOH-water (2:1) yielded
70.60 g of 4 as colorless prisms. The purified 4 (0.23 mol; 70.60 g) was then added to 67.90
g of 95% H2SO4 (0.69 mol) in 692 mL of EtOH, and the mixture was stirred for 4.5 h at
room temperature. After removing the solvent under vacuum, the mixture was poured into
400 mL of deionized water and extracted with 400 mL of AcOEt by a separatory funnel. The
combined organic layers were washed with 200 mL of NaHCO3-brine and dried over MgSO4.
The residue was purified by silica column chromatography (1:4 AcOEt:petroleum ether),
yielding 34.70 g of 5 (0.18 mol; 80%) as a colorless oil. [α]D +110.5 (c = 1.12, EtOH).
Biological and Pharmaceutical Bulletin Advance Publication

1H-NMR (CDCl3, 400 MHz) δ: 1.31 (6H, t, J= 7Hz), 3.66 (2H, s), 4.28 (4H, dq, J= 7,
2Hz). ESI-MS: m/z = 189 [M+H]+.
(2S,3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (6)
5 (0.1 mol; 18.8 g) and 85% KOH (0.1 mol; 6.72 g) were mixed in a 150-mL flask.
After adding 67 mL of EtOH at 4–6 °C, the mixture continued to stir at room temperature for
4 h. The mixture was concentrated under vacuum and then diluted with water and AcOEt.
The combined organic phase was washed with 25 mL of 6 N HCl and dried over MgSO4.
1
Distillation of AcOEt yielded 12.01 g of 6 (0.075 mol; 75%) as a colorless oil. H-NMR
(CDCl3, 400 MHz) δ: 1.17–1.27 (3H, t, J= 7Hz), 3.72–3.74 (2H, s), 4.23–4.33 (2H, dq,
J= 7, 2Hz), 10.56 (1H, s). ESI-MS: m/z = 161 [M+H]+.
Ethyl (2S,3S)-3-(((S)-1-(hexylamino)-4-(methylthio)-1-oxobutan-2-yl)carbamoyl)oxirane-2
-carboxylate (11a)
In a round-bottom flask containing 7 (Boc-L-methionine; 0.031 mol; 7.7 g), 8a
(hexylamine from Alfa Aesar; 0.031 mol; 3.13 g), and 1-hydroxybenzotriazole (0.031 mol;
4.19 g) in AcOEt, a solution of N,Nʹ-dicyclohexylcarbodiimide (0.031 mol; 6.39 g)
dissolved in AcOEt was added dropwise, and the mixture was stirred at 5 °C for 1.5 h. The
solution was then warmed to room temperature and stirred for 2.5 h. The reaction mixture
was filtered and washed with AcOEt. The organic layer was washed with 5% HCl and
saturated NaHCO3-brine solution, dried over MgSO4, and filtered. Removal of the solvent
yielded 9.99 g of 9a (0.030 mol; 96%) as a colorless solid intermediate.
Next, to a solution of 9a (0.012 mol; 3.99 g) in 15 mL of AcOEt, 10% HCl-AcOEt was
added, and the mixture was stirred for 2.5 h at room temperature. The solution was
concentrated under vacuum, and the residual volume was diluted with water and extracted
Biological and Pharmaceutical Bulletin Advance Publication

with AcOEt. The aqueous phase was alkalified with 25% NaOH (pH > 10) and washed with
AcOEt. The combined AcOEt layers were washed with brine and dried over MgSO4.
Removal of AcOEt yielded 2.55 g of 10a (0.011 mol; 90%) as a yellow oil, which was used
directly in the next step.
Finally, in a round-bottom flask containing 6 (0.009 mol; 1.44 g), 10a (0.009 mol; 2.09
g),
and
1-hydroxybenzotriazole
dissolved
in
20
mL
of
AcOEt,
N,Nʹ-dicyclohexylcarbodiimide (0.009 mol; 1.85 g) dissolved in AcOEt was added dropwise.
After stirring at 5 °C for 3 h, the solution was warmed to room temperature and stirred for
another 15 h. The reaction mixture was then filtered and washed with AcOEt. The organic
layers were combined, washed with 100 mL of 5% HCl (v/v) and 100 mL of NaHCO3
saturated with brine, and then dried over MgSO4. After filtration, removal of the solvent
afforded the solid product 11a. Recrystallization from EtOH yielded 1.49 g of pure 11a
1
(0.0037 mol; 42%) as a white powder. [α]D +47.1 (c = 1.00, MeOH). H-NMR (DMSO,
400 MHz) δ: 0.82 (3H, t, J= 7Hz), 1.17–1.35 (11H, m), 1.79–1.88 (2H, m), 2.00 (3H, s),
2.36–2.40 (2H, m), 2.90–3.20 (2H, m), 3.61 (1H, d, J= 2Hz), 3.70 (1H, d, J= 2Hz),
4.16–4.17 (2H, m), 4.27–4.28 (1H, m), 8.00–8.02 (1H, t, J= 7Hz), 8.63 (1H, d, J= 8Hz).
13C-NMR (DMSO) δ: 170.1, 167.3, 164.8, 61.5, 53.0, 52.1, 51.2, 38.5, 31.9, 30.9, 29.5, 28.9,
25.9, 22.1, 14.6, 14.0, 13.9; ESI-MS: m/z = 375 [M+H]+ 397 [M+Na]+. HRMS calcd for
C17H30O5N2S [M+Na]+: 397.1768, Found: 397.1768.
Ethyl (2S,3S)-3-(((S)-4-(methylthio)-1-oxo-1-((3-phenylpropyl)amino)butan-2-yl)carbamoy
l)oxirane-2-carboxylate (11b)
Similarly, 11b was synthesized by starting from 8b, 3-phenyl-1-propylamine, as a white
1
powder (52%). [α]D +46.3 (c = 1.00, MeOH). H-NMR (DMSO, 400 MHz) δ: 1.17–1.27
(3H, m), 1.64–1.90 (4H, m), 2.00 (3H, s), 2.38-2.42 (2H, m), 2.53 (2H, m) 3.00–3.10
Biological and Pharmaceutical Bulletin Advance Publication

(2H, m), 3.61 (1H, d, J= 2Hz), 3.70 (1H, d, J= 2Hz), 4.16–4.17 (2H, m), 4.27–4.28 (1H,
m), 7.17–7.27 (5H, m), 8.13 (1H, t, J= 7Hz), 8.63 (1H, d, J= 8Hz). 13C-NMR (DMSO) δ:
170.2, 167.2, 164.8, 141.6, 128.5, 128.5, 128.2, 128.2, 125.7, 61.4, 52.9, 52.1, 51.2, 38.1,
32.3, 31.9, 30.7, 29.9, 14.6, 13.8; ESI-MS: m/z = 409[M+H]+ 431 [M+Na]+. HRMS calcd
for C20H28O5N2S [M+Na]+: 431.1610, Found: 431.1608.
Ethyl (2S,3S)-3-(((S)-1-((3-fluorophenethyl)amino)-4-(methylthio)-1-oxobutan-2-yl)carbam
oyl)oxirane-2-carboxylate (11c)
11c was synthesized by starting from 8c, 3-phenyl-1-propylamine, as a white powder
1
(44%). [α]D +44.9 (c = 1.00, MeOH). H-NMR (DMSO, 400 MHz) δ: 1.20–1.27 (3H, t,
J= 7Hz), 1.79–1.86 (2H, m), 2.00 (3H, s), 2.36-2.40 (2H, m), 2.70–2.75 (2H, m),
3.61–3.72 (4H, m), 4.16–4.17 (2H, m), 4.28–4.32 (1H, m), 7.02 (3H, m), 7.30 (1H, m),
8.17 (1H, t, J= 7Hz), 8.63 (1H, d, J= 8Hz). 13C-NMR (DMSO) δ: 170.2, 167.2, 164.8,
161.6, 142.8, 130.2, 125.2, 115.7, 113.2, 61.5, 53.2, 52.9, 52.1, 51.2, 34.7, 32.0, 29.9, 14.9,
13.9; ESI-MS: m/z = 413[M+H]+ 435 [M+Na]+. HRMS calcd for C19H25O5N2SF
[M+Na]+: 435.1326, Found: 435.1324.
Ethyl (2S,3S)-3-(((S)-4-(methylthio)-1-oxo-1-(pentan-3-ylamino)butan-2-yl)carbamoyl)oxir
ane-2-carboxylate (11d)
11d was synthesized by starting from 8d, 3-aminopentane, as a white powder (42%). [α]D
+46.9 (c = 1.00, MeOH). 1H-NMR (DMSO, 400 MHz) δ: 0.80 (6H, t, J= 7Hz), 1.21–1.45
(7H, m), 1.79–1.90 (2H, m), 2.02 (3H, s), 2.36-2.42 (2H, m), 3.49–3.52 (1H, m), 3.61
(1H, m), 3.72 (1H, d, J= 2Hz), 4.16–4.17 (2H, m), 4.27–4.28 (1H, m), 7.77 (1H, t, J=
7Hz), 8.60 (1H, d, J= 8Hz). 13C-NMR (DMSO) δ: 170.5, 167.7, 165.2, 61.5, 52.8, 52.3,
Biological and Pharmaceutical Bulletin Advance Publication

52.1, 51.2, 31.9, 29.9, 27.4, 27.2, 14.6, 13.9, 10.9, 10.8; ESI-MS: m/z = 361[M+H]+ 383
[M+Na]+. HRMS calcd for C16H28O5N2S [M+Na]+: 383.1612, Found: 383.1612.
Ethyl (2S,3S)-3-(((S)-4-(methylthio)-1-oxo-1-((4-phenylbutyl)amino)butan-2-yl)carbamoyl)
oxirane-2-carboxylate (11e)
11e was synthesized by starting from 8e, 4-phenyl-1-butylamine, as a white powder
1
(45%). [α]D +45.6 (c = 1.00, MeOH). H-NMR (DMSO, 400 MHz) δ: 1.17–1.23 (3H, m),
1.35–1.51 (4H, m), 1.74–1.88 (2H, m), 1.99 (3H, s), 2.36-2.40 (2H, m), 2.53 (2H, t, J=
7Hz), 2.96–3.09 (2H, m), 3.59 (1H, d, J= 2Hz), 3.69(1H, d, J= 2Hz), 4.12–4.17 (2H, m),
4.27–4.29 (1H, m), 7.10–7.27 (5H, m), 8.04–8.07 (1H, t, J= 7Hz), 8.63 (1H, d, J= 2Hz).
13C-NMR (DMSO) δ: 170.2, 167.2, 164.8, 141.6, 128.2, 128.2, 128.1, 128.1, 125.6, 61.5,
52.9, 52.1, 51.2, 38.2, 34.7, 31.8, 31.5, 30.7, 29.9, 14.6, 13.8; ESI-MS: m/z = 423 [M+H]+
445 [M+Na]+. HRMS calcd for C21H30O5N2S [M+Na]+: 445.1765, Found: 445.1766.
2-ethyl 3-(4-nitrophenyl) (2S,3S)- oxirane-2,3-dicarboxylate (12)
To synthesize 12, a solution of 6 (0.0625 mol; 10.00 g) and p-nitrophenol (0.0625 mol;
8.69 g) in AcOEt (40 mL) was added dropwise to N,Nʹ-dicyclohexylcarbodiimide (0.0625
mol; 12.88 g) in 60 mL of AcOEt at 4–5 °C. The reaction mixture was stirred at 4–5 °C for 3
h, then allowed to warm to room temperature, and stirred for 1 h. The reaction mixture 12.
Recrystallization from AcOEt-n-hexane yielded 11.58 g of 12 (0.0412 mol; 66%) as yellow
1
needles. [α]D +114.8 (c = 1.00, AcOEt). H-NMR (DMSO, 400 MHz) δ: 1.26 (3H, t, J=
8Hz), 4.06 (1H, d, J= 2Hz), 4.08 (1H, d, J= 2Hz), 4.24 (2H, q, J= 8Hz), 7.58 (2H, d, J=
8Hz), 8.36 (2H, d, J= 8Hz). ESI-MS: m/z = 282 [M+H]+.
Biological and Pharmaceutical Bulletin Advance Publication

Ethyl (2S,3S)-3-(((R)-3-(benzylthio)-1-oxo-1-(pentylamino)propan-2-yl)carbamoyl)oxirane
-2-carboxylate (17a)
To synthesize 15a, a 50-mL solution of N,Nʹ-dicyclohexylcarbodiimide (0.05 mol; 10.32
g) in AcOEt was added dropwise to a round-bottom flask containing a mixture of 13,
N-Boc-S-benzyl-L-cysteine (0.05 mol; 15.55 g), 14a n-amylamine (0.05 mol; 4.35 g), and
1-hydroxybenzotriazole (0.05 mol; 6.76 g) in 60 mL of AcOEt. After stirring at 5 °C for 1.5
h, the solution was then warmed to room temperature and stirred for an additional 2.5 h. The
reaction mixture was filtered and washed with AcOEt. The combined organic layers were
washed with 5% HCl, brine, and saturated NaHCO3 solution and brine, and dried over
MgSO4. After filtration, removal of the solvent yielded 17.10 g of 15a (0.045 mol; 90%) as a
colorless solid. It was used in the next step without further purification.
To synthesize 16a, 65 mL of 10% HCl-AcOEt (v/v) was added to 65 mL of a stirred
solution of 15a (0.045 mol; 17.10 g) in AcOEt; the mixture was stirred for 2.5 h at room
temperature. The solution was concentrated under vacuum. It was then diluted with water and
extracted with AcOEt. The aqueous phase was alkalified with 25% NaOH (pH > 10) and
washed with AcOEt. The organic layers were combined, washed with brine, and dried over
MgSO4. The AcOEt was evaporated under vacuum, yielding 10.32 g of 16a (0.037 mol;
82%) as a white oil.
To synthesize 17a, 12 (0.035 mol; 9.83 g) was dissolved in AcOEt, and an equal amount
of 16a (0.035 mol; 9.77 g) in 50 mL of AcOEt was added dropwise at room temperature
over 5 min. The mixture was then stirred at room temperature for 4 h. The reaction mixture
was filtered and washed with AcOEt. The combined organic layers were washed with 100
mL of 2% NaOH, 100 mL of brine, 100 mL of 5% HCl (v/v), and 100 mL of brine,
successively, and then dried over MgSO4. After filtration, removal of the organic solvent
under vacuum yielded a white powder. The crude 17a was finally purified by silica column
Biological and Pharmaceutical Bulletin Advance Publication

chromatography (0.020 mol; 8.84 g; 58%). 1H-NMR (DMSO, 400 MHz) δ: 0.81 (3H, t, J=
7Hz), 1.17–1.37 (9H, m), 2.51–2.68 (2H, m), 2.94–3.09 (2H, m), 3.56–3.74 (4H, m),
4.15 (2H, m), 4.49 (1H, q, J= 7Hz), 7.17–7.27 (5H, m), 8.20–8.23 (1H, t, J= 7Hz), 8.74
(1H, d, J= 8Hz). 13C-NMR (DMSO) δ: 169.2, 167.2, 164.9, 138.3, 129.0, 129.0, 128.4,
128.4, 126.9, 61.6, 52.9, 52.0, 51.3, 38.6, 34.9, 33.0, 28.6, 28.5, 21.9, 14.0, 13.9; ESI-MS:
m/z = 423 [M+H]+ 445 [M+Na]+ 867 [2M+Na]+. HRMS calcd for C21H30O5N2S [M+Na]+:
445.1767, Found: 445.1765.
Ethyl (2S,3S)-3-(((2R)-3-(benzylthio)-1-(sec-butylamino)-1-oxopropan-2-yl)carbamoyl)oxir
ane-2-carboxylate (17b)
Similarly, 17b was synthesized by starting from 14b, sec-butylamine, as a white powder
1
(65%). [α]D +15.9 (c = 1.00, MeOH). H-NMR (DMSO, 400 MHz) δ: 0.76 (3H, t, J=
7Hz), 0.99 (3H, d, J= 7Hz), 1.15–1.39 (5H, m), 2.50–2.68 (2H, m), 3.56–3.72 (4H, m),
3.96–4.16 (3H, m), 4.50 (1H, q, J= 7Hz), 7.17–7.33 (5H, m), 8.05 (1H, d, J= 7Hz), 8.70
(1H, d, J= 8Hz). 13C-NMR (DMSO) δ: 169.2, 167.2, 164.8, 138.3, 129.0, 129.0, 128.4,
128.4, 126.9, 61.6, 52.9, 52.0, 51.3, 38.6, 34.9, 33.3, 28.8, 20.3, 13.9, 10.5; ESI-MS: m/z =
409 [M+H]+ 431 [M+Na]+. HRMS calcd for C20H28O5N2S [M+Na]+: 431.1611, Found:
431.1610.
Ethyl (2S,3S)-3-(((R)-3-(benzylthio)-1-((3-fluorophenethyl)amino)-1-oxopropan-2-yl)carba
moyl)oxirane-2-carboxylate (17c).
17c was synthesized by starting from 14c, 3-fluorophenethylamine, as a white powder
1
(50%). [α]D +19.2 (c = 1.00, MeOH). H-NMR (DMSO, 400 MHz) δ: 1.15–1.21 (3H, t,
J= 7Hz), 2.60–2.72 (4H, m), 3.56–3.71 (6H, m), 3.96–4.19 (2H, m), 4.46 (1H, q, J=
7Hz), 6.98 (3H, m), 7.17–7.27 (6H, m), 8.30 (1H, t, J= 7Hz), 8.70 (1H, d, J= 8Hz).
Biological and Pharmaceutical Bulletin Advance Publication

13C-NMR (DMSO) δ: 169.2, 167.2, 164.8, 163.5, 142.1, 138.5, 130.9, 129.4, 129.4, 128.9,
128.9, 127.3, 125.2, 115.2, 113.7, 61.1, 53.4, 52.0, 51.5, 35.5, 35.0, 33.8, 33.2, 13.9;
ESI-MS: m/z = 475[M+H]+ 497 [M+Na]+. HRMS calcd for C24H27O5N2SF [M+Na]+:
497.1514, Found: 497.1515.
Ethyl (2S,3S)-3-(((R)-3-(benzylthio)-1-oxo-1-((4-phenylbutyl)amino)propan-2-yl)carbamoy
l)oxirane-2-carboxylate (17d).
17d was synthesized by starting from 14d, 4-phenyl-1-butylamine, as a yellow powder
(41%). 1H-NMR (DMSO, 400 MHz) δ: 1.15–1.24 (5H, m), 1.35–1.55 (4H, m), 2.54–2.69
(2H, m), 3.06–3.15 (2H, m), 3.61–3.75 (4H, m), 4.00–4.19 (2H, m), 4.53 (1H, q, J=
7Hz), 7.14–7.32 (10H, m), 8.25–8.28 (1H, t, J= 7Hz), 8.76 (1H, d, J= 8Hz). 13C-NMR
(DMSO) δ: 169.2, 167.2, 164.8, 142.1, 138.2, 128.9, 128.9, 128.8, 128.8, 128.3, 128.3, 128.2,
128.2, 126.8, 125.6, 61.5, 52.9, 52.0, 51.2, 38.3, 34.9, 32.3, 31.9, 30.7, 28.2, 13.8; ESI-MS:
m/z = 485 [M+H]+ 507 [M+Na]+. HRMS calcd for C26H32O5N2S [M+Na]+: 507.1922,
Found: 507.1921.
Pharmacology
The inhibitor potency assays were performed using inhibitor screening kits (Biovision,
USA, San Francisco, cat#: K150-100, K147-100, K161-100, and K149-100). A total of 2 µL
of 2× enzyme solution (cathepsin K, cathepsin B, cathepsin L, and cathepsin S) was added to
each well of a 384-well plate. Next, 2 µL of 10× compound (1, 2, 11a–11e, 17a–17e; 1 and 2
were purchased from Selleck Company) solution at final compound concentrations of 100,
33.3, 11.1, 3.7, 1.23, 0.41, 0.14, 0.046, 0.015, 005, and 0 µM in DMSO (two replicates for
each dose) was added. After centrifugation (1000 rpm) for 1 min and incubation for 15 min at
25 °C, 8 µL of 2.5× substrate solution (substrates were provided in the kits, and the
Biological and Pharmaceutical Bulletin Advance Publication

concentrations used were optimized as recommended in the kit manual), a total of 250 µM/L
Ac-LR-AFC for CTSK, 250 µM/L Ac-RR-AFC for CTSB, 125 µM/L Ac-FR-AFC for CTSL,
and 250 µM/L Z-VVR-AFC for CTSS) were added and centrifuged (1000 rpm) for 1 min to
mix. Finally, the fluorescence signal (excitation: 400 nm; emission: 505 nm) was measured
by an Enspire instrument in kinetics mode at 37 °C for 120 min.
The data (duplicate measurements) were fitted to a sigmoidal dose-response curve with a
variable slope. The IC50 concentrations were calculated from the curve. The relative
inhibition was determined by the following equation: Relative inhibition % = (Slope of EC –
Slope of S)/(Slope of EC – Slope of IC) × 100, where EC indicates enzyme control, S
indicates sample, and IC indicates inhibitor control. Commercially available E64d (1) and
CA-074 (2) were assayed as controls.
Results and Discussion
Based on the previously demonstrated specificity of the general cysteine protease inhibitor
E64 9), we set out to synthesize a series of its analogs by incorporation of structural elements
at both the R1 and R2 positions in order to further investigate how these structures modulate
the specificity of the inhibitor scaffold and to understand the structure-activity relationships
of these derivatives as cathepsin B inhibitors. The Chart for synthesizing the new series of
epoxysuccinyl-peptide derivatives (11a–11e) is shown in Chart 1. First, crude 4 was obtained
by reacting 3 ((+/-)-trans-epoxysuccinic acid) with L-arginine in MeOH-water solution (4:1)
at room temperature. The product was then recrystallized from MeOH-water (2:1) solution.
Subsequently, reacting it with 95% H2SO4 in ethanol afforded the corresponding diethyl ester
(5) in 80% yield. 6 was obtained via the reaction of 5 with potassium hydroxide in ethanol
and stored under a nitrogen atmosphere at 4–6 °C.
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In parallel, an array of 10a–e was synthesized via a two-step route. It started with the
reaction between commercially available and 8a–e in the presence of
7
N,Nʹ-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, and AcOEt. By this simple
acid-amine reaction, different structures were introduced to the R2 position to form 9a–e.
Next, the butyloxycarbonyl protecting group (Boc) was removed under strongly acidic
conditions to release the unprotected amine group to react with 6 and eventually afford 11a
(42% yield). 11b–e were synthesized in a similar manner.
In addition, we evaluated the importance of the R1 diversity site by synthesizing a library
of compounds in which benzyl(methyl) sulfane substituted ethyl(methyl) sulfane at the R1
position of the above-mentioned E64 inhibitor analog. In this case, commercially available 13,
N-Boc-S-benzyl-L-cysteine, was used as a starting material to first react with n-amylamine
(14), followed with deprotection of the amino group to afford 16 (Chart 2). However, a
different route was used to synthesize the target 17. Instead of directly reacting 6 with 16 (a
similar route as shown in Chart 1), 6 was first reacted with p-nitrophenol to convert the acid
group to an active ester. The resultant 12 was then reacted with 16 to afford a group of 17
with different substitutents at R2. This intermediate active ester method successfully
increased the yield of the final products and eliminated the byproducts observed when using
the direct acid method shown in Chart 1.
In order to evaluate the inhibitory activities and to study the associated structure-activity
relationships of the newly synthesized compounds as cathepsin B inhibitors, a
pharmacological study was carried out using conventional inhibitor potency assays, and the
IC50 concentrations were calculated by the sigmoidal dose-response curve method to
determine how much of each compound is needed to effectively inhibit cathepsin B. As
shown in Table 1, ethyl(methyl) sulfane-substituted epoxysuccinyl-peptide derivatives 11a–e
appeared to be highly selective inhibitors of cathepsin B. Since cathepsins K, L, and S are
Biological and Pharmaceutical Bulletin Advance Publication

mostly related to cathepsin B in the papain family 14, 15), it1is very critical that effective
cathepsin B inhibitors have a low inhibitory activity against them. Relatively low affinities to
cathepsins K, L, and S were observed. As compared with 1 and 2 (commercially available
E64d and CA-074, respectively), 11a and 11b not only demonstrated good inhibitory
activities (low cathepsin B IC50 values) but also great selectivities, with a much larger ratio of
cathepsin B IC50 vs. cathepsin X (X: K, L, and S) IC50. Thus, these two specifically designed
compounds demonstrated the highest selectivities to cathepsin B.
The observed difference in the inhibitory performance of 11a–e was further linked to their
molecular structures at the R2 position. The hexyl substituent at R2 of 11a showed the
strongest inhibitory activity against cathepsin B (IC50 = 4.27 ± 1.51 nM) among all tested
compounds. By substituting it with the 1-ethyl propyl group, the overall inhibition and
selectivity of 11d to cathepsin B significantly decreased (cathepsin B IC50 = 74.27 ± 8.18 nM,
that is 22-fold, 129-fold, and 193-fold lower than the IC50 for cathepsins K, L, and S,
respectively), suggesting that a highly flexible linear hexyl chain is critical for cathepsin B
inhibition, compared to a branched segment even with a similar total carbon number (C6 vs.
C5). Decent potent inhibition and selectivity for cathepsin B was observed for 11b (cathepsin
B IC50 = 7.16 ± 2.26 nM, that is 116-fold, 210-fold, and 524-fold lower than the IC50 for
cathepsins K, L, and S, respectively) as well. However, the effect of the alkyl chain length to
link the benzyl group at the R2 position is complicated in this case. Simply replacing the
propyl group in 11b with a butyl group caused a significant drop in both the inhibitory
activity and selectivity of 11e. The addition of a fluorine electron affinity unit on the benzene
ring (11c) failed to show much improvement on overall inhibitory performance. Based on this
observation, it is very important to maintain a flexible alkyl chain at R2 (C6 here) to benefit
the inhibitory activity. Adding a bulky segment such as benzene at the end of the linear chain
seems to deleteriously affect its inhibitory performance.
Biological and Pharmaceutical Bulletin Advance Publication

Similarly, we also evaluated the performance of compounds with benzyl(methyl) sulfane
substituted at the R1 position. From the preliminary results shown in Table 1, little cathepsin
B inhibitory activity was observed on all studied 17 (IC50 > 1000 nM). It is hypothesized that
the hydrophobic benzyl group prevented the formation of effective hydrogen-bond
interactions between the inhibitor molecule and the two histidine residues in the occluding
loop structure of cathepsin B 12)
.
Conclusions
We synthesized a library of epoxysuccinyl-peptide derivatives with various substituents at
the R1 or R2 positions of the model molecule E64d. As compared to the benyl(methyl) sulfane
at R1, compounds with an ethyl(methyl) sulfane substituent showed enhanced inhibitory
activity and selectivity against cathepsin B. In particular, when a linear hexyl linkage group
was incorporated at the R2 position, the resultant 11a exceeded the performance of the
benchmark E64d and CA-074 compounds as cathepsin B inhibitors. However, the molecules
with a benzyl(methyl) sulfane substituent at the R1 position failed to demonstrate much
cathepsin B inhibitory activity. In future work, besides continuing to study the effect of the
chain length of the linear alkyl group at the R2 position, we will also focus on studying the
size, chain length, and hydrophobicity of substituents at the R1 and R2 positions to understand
the structure-activity relationship of this model molecule as a cathepsin B inhibitor.
Biological and Pharmaceutical Bulletin Advance Publication

Conflict of Interest
The authors declare no conflict of interest
Acknowledgments
The work was supported by the National High Technology Research and Development
program of China (Grant NO. 2012AA020301) and the National Science and Technology
Major
project
(Grant
NO.
2012ZX09301003-001-002
and
Grant
NO.
2012ZX09301003-003).
Biological and Pharmaceutical Bulletin Advance Publication

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Biological and Pharmaceutical Bulletin Advance Publication

Figure legends
Figure 1. Chemical structures of the cathepsin B inhibitors E64d and CA-074.
Figure 2. Design of compound as a cathepsin B inhibitor.
Chart 1. Chart for the preparation of derivatives 11a–11e. Reagents and conditions: (a)
L-arginine, MeOH, r.t. overnight, recrystallization in MeOH-water (2:1); (b) 95% H2SO4,
EtOH, 4.5 h at r.t.; (c) 85% KOH, EtOH, 4 h at 4 °C; (d) NH2-R2 8a–e, HOBt, DCC, AcOEt,
1.5 h at 5 °C then 2.5 h at r.t.; (e) 10% HCl-AcOEt, 2.5 h at r.t.; (f) 6, DCC, HOBt, AcOEt, 3
h at 5 °C then 15 h at r.t.
Chart 2. Chart for the preparation of derivatives 17a–d. Reagents and conditions: (a)
p-nitrophenol, DCC, AcOEt, 3 h at 5 °C then 1 h at r.t.; (b) NH2-R2 14a–e, HOBt, DCC,
AcOEt, 5 °C 1.5 h then r.t., 2.5 h; (c) 10% HCl, AcOEt, r.t., 2.5 h; (d) 12, AcOEt, r.t., 4 h.
Biological and Pharmaceutical Bulletin Advance Publication

Table 1. Cathepsin B inhibition data for compounds 11a–e and 17a–d.
Compound
ID
Cathepsin B IC50
(nM)
Cathepsin B
IC50 vs.
Cathepsin K Cathepsin L Cathepsin
S
IC50 (fold)
IC50 (fold)
IC50 (fold)
1
12.03 ± 2.03
6.61 ± 0.61
4.27 ± 1.51
7.16 ± 2.26
24.09 ± 2.59
74.27 ± 8.18
23.68 ± 4.78
>1000
39
38
181
116
21
22
28
--
15
55
2
164
278
210
115
129
185
--
510
497
524
167
193
149
--
11a
11b
11c
11d
11e
17a
17b
17c
17d
>1000
--
--
--
>1000
--
--
--
>1000
--
--
--
Biological and Pharmaceutical Bulletin Advance Publication

Figure 1
Figure 2
O
H
H
H
N
N
O
H
O
O
O
1 E64d
R1
O
H
R2
H
N
N
O
H
H
O
O
R1
O
R1
S
S
S
S
O
H
O
H
H
H
N R2
H
R2
H
N
N
N
O
O
H
H
O
O
O
O
O
O
17a-17d
11a-11e
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Chart 1
COOC2H5
H
H
COOH
COOH
H
H
a
b
C2H5OOC
O
HOOC
HOOC
O
O
5
4
3
COOH
H
c
H
C2H5OOC
O
6
S
S
e
S
d
H
R2
H
N
R2
Boc
N
Boc
N
OH
H2N
N
H
O
H
O
O
10a-10e
R2
9a-9e
7
S
O
C
11a
f
H
H
R2
N
O
N
H
H
O
11b
11c
11d
11e
O
O
11a-11e
F
Biological and Pharmaceutical Bulletin Advance Publication

Chart 2
O
H
COOH
H
H
H
a
O
NO2
C2H5OOC
C2H5OOC
O
O
6
12
S
S
c
S
b
H
R2
H
N
R2
N
Boc
Boc
N
OH
H2N
N
H
O
H
O
O
16a-16d
R2
15a-15d
13
S
O
C
17a
d
H
H
R2
N
O
N
H
H
O
17b
17c
O
O
17a-17d
F
17d
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