Design, synthesis, and bioactivities of tasiamide B derivatives as cathepsin D inhibitors

Article abstract of DOI:10.1002/psc.3154

Cathepsin D (Cath D) is overexpressed and hypersecreted by malignant tumors and involved in the progress of tumor invasion, proliferation, metastasis, and apoptosis. Cath D has been considered as a potential target to treat cancer. Our previous studies re

Full text of DOI:10.1002/psc.3154

Received: 14 November 2018
DOI: 10.1002/psc.3154
Revised: 4 January 2019
Accepted: 15 January 2019
R E S E A R C H A R T I C L E
Design, synthesis, and bioactivities of tasiamide B derivatives
as cathepsin D inhibitors
Zhi Li1 Keting Bao2 Hao Xu2 Ping Wu2 Wei Li1 Jian Liu1 Wei Zhang2
|
|
|
|
|
|
1 School of Pharmacy, Nanjing University of
Chinese Medicine, Jiangsu Collaborative
Innovation Center of Chinese Medicinal
Resources Industrialization, Nanjing, China
Cathepsin D (Cath D) is overexpressed and hypersecreted by malignant tumors and
involved in the progress of tumor invasion, proliferation, metastasis, and apoptosis.
Cath D has been considered as a potential target to treat cancer. Our previous studies
revealed that tasiamide B derivatives TB‐9 and TB‐11 exhibited high potent inhibition
against Cath D and other aspartic proteases, but their molecular weights are still high,
and the role of each residue is unknown yet. Based on this, two series of tasiamide B
derivatives have been designed, synthesized, and evaluated for their inhibitory activ-
ity against Cath D/Cath E/BACE1. Enzymatic assays revealed that the target com-
pound 1 with lower molecule weight showed good inhibitory activity against Cath
D with IC50 of 3.29 nM and satisfactory selectivity over Cath E (72‐fold) and BACE1
(295‐fold), which could be a valuable template for the design of highly potent and
selective Cath D inhibitors.
2 School of Pharmacy, Fudan University,
Shanghai, China
Correspondence
Wei Li and Jian Liu, School of Pharmacy,
Nanjing University of Chinese Medicine, 138
Xianlin Road, Nanjing 210023, China; Jiangsu
Collaborative Innovation Center of Chinese
Medicinal Resources Industrialization, Nanjing
210023, Jiangsu, China.
Email: liwaii@126.com; liujian623@njucm.edu.cn
Wei Zhang, School of Pharmacy, Fudan
University, Shanghai 201203, China.
Email: zhangw416@fudan.edu.cn
Funding information
KEYWORDS
National Natural Science Foundation of China,
Grant/Award Number: 81573340; Postgradu-
ate Research & Practice Innovation Program
of Jiangsu Province, Grant/Award Number:
KYCX181578
Ahppa, cathepsin D, selective inhibitor, tasiamide B derivatives
|
1
INTRODUCTION
nonpeptidic Cath D inhibitors that discovered by computer‐aided drug
design or random screening have been documented previously.27 On
Cathepsin D (Cath D) is one of the aspartyl endoproteinase, involved
in different physiological processes and related with numerous patho-
logical conditions.1-6 The main physiological functions of Cath D con-
sist of metabolic degradation of intracellular proteins, activation, and
degradation of polypeptide hormones and growth factors, activation
of enzymatic precursors, processing of enzyme activators and inhibi-
tors, brain antigen processing, and regulation of programmed cell
death.7-10 However, it is overexpressed and hypersecreted by malig-
nant tumors, involved in the progress of tumor invasion, proliferation,
metastasis, and apoptosis.11-14 Cath D has been considered as a
potential target in various types of diseases15-20 and as an indepen-
dent marker of prognostic in cancer, including breast and ovarian can-
cers.12,13,19 Therefore, inhibition of Cath D has been considered as an
attractive pathway for the development of novel anticancer drugs.
Over the past 20 years, a large number of nonpeptidic and pep-
the other hand, most of the potent peptidic inhibitors contained a
statine unit (γ‐amino‐β‐hydroxy acid) or a statine‐like unit (4‐amino‐
3‐hydroxy‐5‐phenylpentanoic acid, Ahppa), which formed tetrahedron
transition‐state with two aspartic acids in the catalytic site. Pepstatin
A (Figure 1) is a typical peptidic inhibitor bearing a statine unit with
IC50s at subnanomolar level for Cath D and other aspartic prote-
ases.28-32 Tasiamide B (Figure 1), a linear peptide isolated from the
marine cyanobacteria Symploca sp.,33 was proved as a good template
for the development of aspartic proteases inhibitors.32,34 Our research
group finished the total synthesis and stereochemical reassignment of
tasiamide B and then prepared series of its derivatives.27,32,35 TB‐9
and TB‐11, two respective compounds among them, showed highly
potent inhibitory activity against Cath D. As part of the ongoing
research, herein, we would like to report the design, synthesis, and
bioactivities of two novel series of Cath D inhibitors based on TB‐9
and TB‐11 (Figure 1).
tidic Cath
D
inhibitors have been reported.21-26 Some typical
J Pep Sci. 2019;e3154.
wileyonlinelibrary.com/journal/psc
© 2019 European Peptide Society and John Wiley & Sons, Ltd.
1 of 9
https://doi.org/10.1002/psc.3154

2 of 9
LI ET AL.
FIGURE 1 The structures of pepstatin A, tasiamide B, TB‐9, and TB‐11
|
still high, and the role of each residue is unknown yet. Based on this,
the amino acid units of TB‐9 and TB‐11 were truncated at the C‐
terminus in sequence to design two series derivatives (Figure 2), in
which Val‐Me‐Gln‐Ahppa fragment was retained while other residues
were truncated one‐by‐one from the C‐terminus.
2
RESULTS AND DISCUSSION
Compounds design
|
2.1
In our previous studies,32 the lactic acid of tasiamide B was truncated,
a Cbz and Boc group were introduced to the N‐terminus yielding TB‐9
and TB‐11, respectively. The IC50s of TB‐9 were 0.0783, 0.724, and
54.2 nM against Cath D, Cath E, and BACE1. For TB‐11, the IC50s
against these three aspartic proteases were 0.126, 1.92, and
48.8 nM, respectively. Although these two compounds exhibited
highly potent inhibition against Cath D, their molecular weights are
|
2.2
Chemical synthesis
These newly designed compounds were prepared by standard
solution‐phase peptide synthesis procedures (Schemes 1 and 2).
Compounds
1
to
4
and 8a were prepared as previously
FIGURE 2 The structures of two series tasiamide B derivatives

LI ET AL.
3 of 9
SCHEME 1 Synthesis of series I compounds
SCHEME 2 Synthesis of series II compounds
described.32,35 Methyl esters 1, 2, and 3 were hydrolyzed with lith-
ium hydroxide (LiOH) to afford compounds 5, 6, and 7, respectively.
Treatment of 8a with trifluoroacetic acid (TFA) in dichloromethane
(DCM) gave compound 8. Deprotection of the Cbz group of methyl
esters 1, 2, and 3 afforded the free amine, which were then masked
with Boc group yielding compounds 9, 10, and 11, respectively.
Other target compounds 12, 13, and 14 were prepared in similar
way by hydrolyzing methyl esters 9, 10, and 11, respectively. The
structures of 5 to 14 were confirmed by nuclear magnetic resonance
(1H NMR), carbon NMR (13C NMR), and high‐resolution mass spectra
(HRMS).
|
2.3
In vitro activity assays
These two series tasiamide B derivatives (1‐14) were assessed for their
ability to inhibit the activity of Cath D, Cath E, and BACE1
(Tables 1 and 2). Enzymatic assays revealed that the inhibitory ability

4 of 9
LI ET AL.
TABLE 1 IC50s of TB‐9 analogs 1 to 8 against three aspartic proteases
In Vitro IC50, nMa
Cath Db
Compound
Structure
Cath Eb
BACE1b
TB‐9
0.0783
0.724
54.2
1
3.29
236
972
2
3
102
196
243
642
2740
5599
4
5
NAc
2105
NA
27.0
100
502
6
7
8
108
168
1330
6384
1199
more than 10 000
more than 10 000
more than 10 000
more than 10 000
aAll inhibitory values are means of at least two separate experiments.
bIC50 of positive control (pepstatin A): 0.59 nM for Cath D; 0.57 nM for Cath E; 92.4 nM for BACE1.
c“NA” means not active at 10 μM.

LI ET AL.
5 of 9
of three aspartic proteases had a sharp downtrend as the removal of
amino acid units at the C‐terminus (1‐4, 5‐8, 9‐11, 12‐14). To be
specific, the inhibition against Cath D was almost completely lost with
the lack of proline, phenylalanine, alanine, and leucine residues even
at the concentration of 10 μM (4 and 8). In series І (1‐8), the methyl
ester derivatives had slightly improved in activity and selectivity against
Cath D than corresponding carboxylic acid (1 vs 5, 2 vs 6, 3 vs 7). What
is more, the results indicated that compounds with aromatic ring at the
N‐terminus were crucial for the inhibitory potency against Cath D (1 vs
9, 2 vs 10, 3 vs 11, 5 vs 12, 6 vs 12, 7 vs 13), as same asTB‐9 vsTB‐11.
Among the tested compounds, 1, 2, 3, 5, 6, 9, and 12 showed
moderate‐to‐good inhibition against Cath D. Most notably in that
regard, we found that compound 1 is extremely potent against Cath
D with IC50 of 3.29 nM. This compound, with the lack of proline unit
compared withTB‐9, showed improved selectivity for Cath D with 72‐
fold over Cath E.
TABLE 2 IC50s of TB‐11 analogs 9 to 14 against three aspartic proteases
In Vitro IC50, nMa
Compound
Structure
Cath Db
Cath Eb
BACE1b
TB‐11
0.126
1.92
48.8
9
190
407
233
1117
10
67.3
806
11
12
more than 10 000
494
3718
131
53.4
181
13
14
903
217
91.4
more than 5000
more than 5000
NAc
aAll inhibitory values are means of at least two separate experiments.
bIC50 of positive control (pepstain A): 0.59 nM for Cath D; 0.57 nM for Cath E; 92.4 nM for BACE1.
c“NA” means not active at 10 μM.

6 of 9
LI ET AL.
|
in DCM (5 mL), concentrated in vacuo again and purified by prepara-
tive TLC with DCM/MeOH (10/1) to give 8 as white solid (15.0 mg,
3
CONCLUSIONS
25
75.5%). ½αꢀD = −26.3 (c = 2.0, MeOH); 1H NMR (400 MHz, CDCl3) δ
In conclusion, two series of tasiamide B derivatives have been
designed, synthesized, and evaluated as selective Cath D inhibitors,
in which Val‐Me‐Gln‐Ahppa fragment was retained, while the amino
acid units of TB‐9 and TB‐11 were truncated at the C‐terminus in
sequence. The target compound 1 with lower molecule weight
showed highly potent inhibitory activity against Cath D with IC50 of
3.29 nM and satisfactory selectivity over Cath E (72‐fold) and BACE1
(295‐fold). These results could provide a new and good template for
the development of selective Cath D inhibitors. Further studies on
the structural optimization of tasiamide B derivatives are currently
undergoing in our laboratory and will be reported in due course.
7.35‐7.09 (m, 10H), 6.66 (s, 1H), 6.55 (s, 1H), 5.99 and 5.71 (both d,
J = 8.1 Hz, total 1H), 5.13‐4.91 (m, 3H), 4.41‐4.17 (m, 2H), 4.15‐3.99
(m, 1H), 2.97‐2.83 (m, 1H), 2.77 (d, J = 9.0 Hz, 1H), 2.70 and 2.29 (both
s, total 3H), 2.45 (s, 2H), 2.19‐2.02 (m, 3H), 1.86‐1.73 (m, 2H), 0.99‐
0.77 (m, 6H); 13C NMR (151 MHz, CDCl3) δ 176.3, 175.8, 173.5,
169.7, 156.8, 138.0, 136.2, 129.1, 128.6, 128.4, 128.3, 128.1, 128.0,
126.4, 68.7, 67.0, 56.3, 55.6, 53.7, 39.3, 37.8, 31.4, 30.6, 30.5, 23.7,
19.6, 17.5. HRESIMS calcd for C30H40N4O8 [M + Na]+ 607.2738,
found 607.2742.
|
4.2
General method for preparation of compounds
9 to 11
|
4
EXPERIMENTAL SECTION
Chemistry
(exemplified by 9)
|
4.1
|
4.2.1
Compound 9
All reactions were carried out in oven‐dried or flame‐dried glassware.
All commercial reagents were used without further purification unless
otherwise noted. Anhydrous DCM, methanol (MeOH), and tetrahydro-
furan (THF) were obtained by Solvent Purification System (PS‐MD‐5,
Innovation Technology, USA). Reactions were magnetically stirred
and monitored by thin layer chromatography (TLC) with silica gel
plates (60F‐254) and visualized by UV irradiation or by staining with
iodine absorbed on silica gel, phosphomolybdic acid/ethanol solution,
or aqueous acidic ammonium molybdate solution as appropriate. Flash
column chromatography was performed on silica gel (200‐300
meshes) with the indicated solvent system, and preparative TLC was
performed on silica gel F254 glass plates (layer thick 0.4‐0.5 mm).
Yields refer to chromatographically and spectroscopically pure com-
pounds. NMR spectra were recorded on a Bruker 400 MHz or Bruker
Avance III 600 as indicated in the data list. Chemical shifts for 1H
NMR spectra are reported in parts per million relative to the signal
residual tetramethylsilane (TMS) at 0 ppm. Chemicals shifts for carbon
13C NMR spectra are reported in parts per million relative to the cen-
ter line of the CDCl3 triplet at 77.16 ppm and the CD3OD heptet at
49.00 ppm. The abbreviations s, d, dd, t, q, br, and m stand for the res-
onance multiplicity singlet, doublet, doublet of doublets, triplet, quar-
tet, broad, and multiplet, respectively. Optical rotation was measured
on an AUTOPOL V (Na D line) by using a microcell of 1‐dm path
length. HRMS were obtained by using a quadrupole time‐of‐flight
mass spectrometer. Copies of 1H and 13C NMR spectra for com-
pounds 5‐14 may be found in the Supporting Information.
Compound 1 (93.0 mg, 0.099 mmoL) in MeOH (10 mL) was hydroge-
nated for 3 hours at room temperature in the presence of a catalytic
amount of Pd/C (10 wt%). When TLC analysis showed no starting
material remained, Pd/C was removed by filtration, and the resulting
filtrate was concentrated to give the free amine as colorless oil, which
was used without further purification.
To the cold solution (ice bath) of the amine obtained above in
DCM (5 mL) was added di‐tert‐butyl dicarbonate (Boc2O, 26 mg,
0.12 mmoL). The mixture was stirred at room temperature for 2 hours.
When TLC analysis showed no starting material remained, and then
the solution was concentrated in vacuo. The residue was purified by
flash column chromatography with DCM/MeOH (50:1‐20:1) to give
25
compound 9 as white solid (65.5 mg, 73.1%). ½αꢀD = −34.4 (c = 1,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 7.6 Hz, 1H), 7.50‐
7.32 (m, 1H), 7.30‐7.05 (m, 10H), 6.32 (s, 1H), 6.27 (s, 1H), 5.47‐5.31
(m, 1H), 5.25 (d, J = 8.2 Hz, 1H), 5.10‐4.94 (m, 1H), 4.86‐4.71 (m,
1H), 4.73‐4.61 (m, 1H), 4.50‐4.35 (m, 1H), 4.36‐4.24 (m, 1H), 4.07 (s,
1H), 3.74 (s, 3H), 3.45‐3.26 (m, 1H), 3.08‐2.90 (m, 2H), 2.89‐2.63 (m,
7H), 2.53‐2.40 (m, 1H), 2.38‐2.20 (m, 2H), 2.20‐2.01 (m, 2H), 2.00‐
1.77 (m, 2H), 1.74‐1.59 (m, 1H), 1.59‐1.50 (m, 2H), 1.45 (s, 9H),
1.10‐0.64 (m, 15H); 13C NMR (151 MHz, CDCl3) δ 175.1, 173.9,
173.3, 172.3, 171.8, 171.0, 170.9, 156.1, 138.4, 136.5, 129.2, 129.0,
128.8, 128.7, 128.4, 127.1, 126.4, 79.9, 69.5, 58.1, 56.3, 55.7, 54.5,
52.6, 52.1, 45.6, 41.1, 41.0, 37.1, 34.8, 32.5, 31.9, 30.8, 28.4, 24.9,
24.3, 23.2, 21.8, 19.7, 17.6, 17.5; HRESIMS calcd for C47H71N7O11
[M + Na]+ 932.5104, found 932.5108.
|
4.1.1
Compound 8
|
4.2.2
Compound 10
To the cold solution (ice bath) of compound 8a (30.0 mg, 0.034 mmoL)
in DCM (2 mL) was added TFA (2 mL) and stirred for 30 minutes. The
solution was warmed to room temperature and stirred for another
1.5 hours. When TLC analysis showed no starting material remained
the solution was concentrated in vacuo. The residue was redissolved
Compound 10 was obtained from 2 according to the similar proce-
25
dure. White solid; yield 67.0%; ½αꢀD = −67.0 (c = 0.9, CHCl3); 1H
NMR (400 MHz, CDCl3) δ 7.75 (brs, 1H), 7.50 (d, J = 6.7 Hz, 1H),
7.33 (d, J = 5.1 Hz, 1H), 7.29‐7.09 (m, 5H), 6.50 (s, 1H), 6.15 (s, 1H),

LI ET AL.
7 of 9
5.14 (d, J = 7.9 Hz, 1H), 4.98 (s, 1H), 4.54‐4.38 (m, 2H), 4.33‐4.20 (m,
1H), 4.17‐4.01 (m, 2H), 3.71 (s, 3H), 2.95 (d, J = 6.7 Hz, 2H), 2.86 (s,
3H), 2.61‐2.41 (m, 1H), 2.38‐2.19 (m, 2H), 2.08 (s, 2H), 1.94 (brs,
1H), 1.88–1.78 (m, 1H), 1.77‐1.52 (m, 3H), 1.46 (s, 9H), 1.38 (d,
J = 6.9 Hz, 3H), 0.97‐0.84 (m, 12H); 13C NMR (151 MHz, CDCl3) δ
175.2, 173.7, 173.2, 173.0, 171.9, 170.9, 156.1, 138.3, 129.1, 128.4,
126.4, 79.9, 69.9, 55.9, 55.6, 54.3, 52.4, 52.0, 48.3, 41.1, 40.8, 37.1,
30.7, 28.4, 24.8, 24.4, 23.0, 21.8, 19.6, 17.7, 17.4; HRESIMS calcd
for C37H60N6O10 [M + Na]+ 771.4263, found 771.4256.
127.4, 127.2, 79.3, 71.2, 70.0, 68.1, 67.8, 60.7, 60.3, 58.1, 57.8,
57.6, 56.9, 55.7, 53.2, 49.6, 46.8, 41.9, 41.8, 41.5, 38.4, 37.8, 35.9,
33.4, 32.7, 32.3, 31.9, 31.7, 31.5, 29.1, 26.3, 25.8, 25.3, 23.6, 21.9,
21.9, 20.1, 20.0, 19.2, 18.4, 17.4; HRESIMS calcd for C49H67N7O11
[M + H]+ 930.4971, found 930.5011.
|
4.3.2
Compound 6
Compound 6 was obtained from 2 according to the similar procedure.
25
White solid; yield 62.6%; ½αꢀD = −34.2 (c = 1.3, CHCl3); 1H NMR
(400 MHz, CD3OD, two rotamers, cat. 1:1) δ 7.45‐7.08 (m, 10H), 7.04
(d, J = 7.1 Hz, 1H), 5.23 (q, J = 12.6 Hz, 1H), 5.07 (s, 1H), 5.06‐4.98
(m, 1H), 4.48‐4.06 (m, 5H), 3.00‐2.85 (m, 1H), 2.77 and 2.17 (both s,
total 3H), 2.83‐2.66 (m, 1H), 2.51‐2.20 (m, 3H), 2.13 (s, 2H), 2.06‐1.87
(m, 2H), 1.80‐1.66 (m, 1H), 1.67‐1.52 (m, 2H), 1.38 (d, J = 7.0 Hz, 3H),
1.07‐0.90 (m, 12H); 13C NMR (151 MHz, CD3OD, two rotamers, cat.
1:1) δ 177.7, 177.6, 176.6, 175.2, 174.6, 174.5, 173.9, 173.7, 172.0,
170.4, 159.2, 158.7, 139.9, 139.7, 138.2, 130.3, 130.3, 129.5, 129.5,
129.4, 129.1, 129.0, 128.9, 128.8, 127.4, 127.1, 71.3, 70.2, 68.1, 67.7,
60.3, 58.1, 57.8, 57.6, 56.7, 55.3, 53.3, 50.0, 42.1, 42.0, 41.8, 41.5,
38.6, 38.0, 32.7, 32.3, 31.9, 31.6, 31.4, 29.0, 26.4, 25.8, 25.3, 23.5,
22.0, 21.9, 20.1, 19.9, 19.1, 18.3, 18.0, 17.9; HRESIMS calcd for
|
4.2.3
Compound 11
Compound 11 was obtained from 3 according to the similar proce-
25
dure. White solid; yield 84.2%; ½αꢀD = −64.7 (c = 0.6, CHCl3); 1H
NMR (400 MHz, CDCl3) δ 7.43 (d, J = 6.9 Hz, 1H), 7.30‐7.10 (m,
5H), 6.20 (s, 1H), 6.11 (s, 1H), 5.16 (d, J = 7.7 Hz, 1H), 5.02 (s, 1H),
4.63‐4.50 (m, 1H), 4.38‐4.23 (m, 1H), 4.18 (d, J = 6.9 Hz, 1H), 4.07
(s, 1H), 3.71 (s, 3H), 3.01‐2.89 (m, 2H), 2.83 (s, 3H), 2.55‐2.42 (m,
1H), 2.35‐2.25 (m, 2H), 2.18‐2.06 (m, 2H), 1.96 (s, 1H), 1.90‐1.79(m,
1H), 1.77‐1.66 (m, 1H), 1.65‐1.57 (m, 2H), 1.46 (s, 9H), 1.11‐0.75 (m,
12H); 13C NMR (151 MHz, CDCl3) δ 174.9, 174.1, 173.9, 171.9,
170.7, 156.1, 138.2, 129.2, 128.4, 126.4, 80.0, 69.4, 56.0, 55.6,
54.1, 52.3, 50.8, 40.7, 37.2, 31.6, 30.6, 28.4, 24.9, 24.0, 22.9, 21.7,
19.7, 17.4; HRESIMS calcd for C34H55N5O9 [M + Na]+ 700.3892,
found 700.3896.
C
39H56N6O10 [M + H]+ 769.4131, found 769.4150.
|
4.3.3
Compound 7
Compound 7 was obtained from 3 according to the similar procedure.
|
25
White solid; yield 71.4%; ½αꢀD = −74.2 (c = 0.6, CHCl3); 1H NMR
4.3
General method for preparation of compounds
5 to 7 and 12 to 14
(400 MHz, CD3OD, two rotamers, cat. 1:1) δ 7.45‐7.09 (m, 10H),
7.05 (d, J = 7.5 Hz, 1H), 5.23 (q, J = 12.5 Hz, 1H), 5.07 (s, 1H), 5.03
(d, J = 9.0 Hz, 1H), 4.49‐4.21 (m, 3H), 4.17‐4.04 (m, 1H), 2.98‐2.88 (m,
1H), 2.81 and 2.17 (both s, total 3H), 2.77‐2.60 (m, 1H), 2.48‐2.26 (m,
3H), 2.12 (s, 2H), 2.07‐1.88 (m, 2H), 1.84‐1.70 (m, 1H), 1.68‐1.59 (m,
2H), 1.07‐0.90 (m, 12H); 13C NMR (151 MHz, CD3OD, two rotamers,
cat. 1:1) δ 177.7, 177.6, 176.6, 175.3, 174.6, 174.0, 173.7, 172.1,
170.5, 159.2, 158.7, 139.9, 139.7, 138.1, 130.3, 130.2, 129.5, 129.5,
129.4, 129.1, 129.0, 128.9, 128.8, 127.4, 127.1, 71.3, 70.1, 68.1, 67.7,
60.2, 58.0, 57.7, 57.6, 56.9, 55.7, 52.5, 49.4, 49.3, 49.1, 49.0, 48.9,
48.7, 48.6, 41.8, 41.7, 41.5, 38.4, 37.8, 32.6, 32.2, 31.9, 31.6, 31.4,
29.0, 26.3, 26.0, 25.3, 23.5, 21.9, 21.9, 20.1, 19.9, 19.2, 18.4; HRESIMS
calcd for C36H51N5O9 [M + H]+ 698.3760, found 698.3766.
(exemplified by 5)
|
4.3.1
Compound 5
Compound 1 (55.0 mg, 0.058 mmoL) in MeOH (2 mL) was treated
with a solution of LiOH monohydrate (8.0 mg, 0.19 mmoL) in water
(0.5 mL). The mixture was stirred at room temperature for 3 hours.
When TLC analysis showed no starting material remained, the mixture
was adjusted to pH = 4 with 1 M HCl and extracted with EtOAc
(20 mL × 3). The combined organic layers were washed with brine
(10 mL × 3), dried over Na2SO4, and then concentrated in vacuo. The
residue was purified by preparative TLC with DCM/MeOH (10/1) to
25
|
4.3.4
Compound 12
give 5 as white solid (47.8 mg, 88.2%). ½αꢀD = −2.8 (c = 0.1, CHCl3);
1H NMR (400 MHz, CD3OD, two rotamers, cat. 1:1) δ 7.44‐7.08 (m,
15H), 7.03 (d, J = 7.0 Hz, 1H), 5.28‐5.14 (m, 2H), 5.11‐4.99 (m, 2H),
4.65 (dd, J = 6.8, 3.0 Hz, 1H), 4.44‐4.03 (m, 4H), 3.45‐3.33 (m, 1H),
3.15‐2.98 (m, 1H), 2.91 (s, 3H), 2.84 (d, J = 3.9 Hz, 1H), 2.79 and
2.18 (both s, total 3H), 2.75‐2.58 (m, 1H), 2.52‐2.26 (m, 3H), 2.24‐
2.06 (m, 2H), 2.07‐1.85 (m, 2H), 1.74‐1.63 (m, 1H), 1.62‐1.44 (m,
2H), 1.08‐0.80 (m, 15H); 13C NMR (151 MHz, CD3OD, two rotamers,
cat. 1:1) δ 177.6, 175.2, 174.8, 174.7, 174.6, 174.2, 174.1, 173.8,
172.0, 170.4, 159.2, 158.7, 139.9, 139.7, 139., 138.2, 138.2, 130.3,
130.3, 130.1, 129.6, 129.5, 129.5, 129.1, 129.1, 128.9, 128.8, 127.6,
Compound 12 was obtained from 9 according to the similar proce-
25
dure. White solid; yield 61.5%; ½αꢀD = −2.8 (c = 1, MeOH); 1H NMR
(400 MHz, CD3OD, two rotamers, cat. 4:5) δ 7.41‐7.03 (m, 10H),
5.21 (d, J = 8.7 Hz, 1H), 5.05 (s, 1H), 4.66 (d, J = 5.8 Hz, 1H), 4.45‐
3.97 (m, 4H), 3.42‐3.34 (m, 1H), 3.15‐3.00 (m, 1H), 2.93 (s, 3H), 2.84
(s, 1H), 2.79 (s, 3H), 2.45‐2.24 (m, 3H), 2.22‐2.06 (m, 3H), 2.04‐1.84
(m, 2H), 1.79‐1.54 (m, 3H), 1.52 and 1.42 (both s, total 9), 1.08‐0.71
(m, 15H); 13C NMR (151 MHz, CD3OD, two rotamers, cat. 4:5) δ
177.8, 177.6, 175.5, 175.0, 174.8, 174.7, 174.2, 174.1, 173.8, 173.4,

8 of 9
LI ET AL.
172.0, 170.5, 158.6, 158.1, 140.0, 139.7, 139.0, 130.4, 130.2, 130.1,
129.7, 129.6, 129.5, 127.6, 127.4, 127.2, 81.0, 80.6, 71.3, 70.0,
60.6, 60.2, 57.6, 57.5, 57.2, 56.9, 55.6, 53.2, 46.7, 41.9, 41.8, 38.4,
37.8, 35.8, 33.4, 32.7, 32.3, 31.9, 31.5, 29.1, 28.7, 26.3, 25.8, 25.8,
25.4, 23.6, 21.9, 21.8, 20.1, 20.0, 19.1, 18.3, 17.4, 17.4; HRESIMS
calcd for C46H69N7O11 [M + Na]+ 918.4947, found 918.4974.
assay concentration, up to less than or equal to 2% DMSO). The
enzyme activities were detected as a time‐course measurement of
the increase in fluorescence signal (Ex 320 nM, Em 405 nM) from fluo-
rescently labeled peptide substrate for 120 minutes at room tempera-
ture. Then, we determined IC50 values with nonlinear regression in
GraphPad Prism 5.
The inhibitory activities of tasiamide B derivatives 1–14 against
these three aspartic proteases are summarized in Tables 1 and 2.
|
4.3.5
Compound 13
ACKNOWLEDGEMENTS
Compound 13 was obtained from 10 according to the similar proce-
This work was financially supported by National Natural Science
Foundation of China (81573340) and Postgraduate Research & Prac-
tice Innovation Program of Jiangsu Province (KYCX181578).
25
dure. White solid; yield 65.5%; ½αꢀD = −31.7 (c = 0.6, MeOH); 1H
NMR (400 MHz, CD3OD, two rotamers, cat. 4:5) δ 7.27‐7.00 (m,
6H), 5.03‐4.86 (m, 1H), 4.40‐3.95 (m, 5H), 2.87‐2.79 (m, 1H), 2.78‐
2.67 (m, 1H), 2.65 and 2.01 (both s, total 3H), 2.41‐2.18 (m, 3H),
2.05‐1.98 (m, 2H), 1.95‐1.76 (m, 2H), 1.69‐1.58 (m, 1H), 1.57‐1.47
(m, 2H), 1.43 and 1.33 (both s, total 9H), 1.31‐1.25 (m, 3H), 0.97‐
0.78 (m, 12H); 13C NMR (151 MHz, CD3OD, two rotamers, cat. 4:5)
δ 177.2, 176.9, 175.7, 175.5, 173.4, 173.0, 171.9, 171.6, 170.0,
168.5, 156.6, 156.0, 138.1, 137.7, 128.3, 128.2, 127.5, 127.4, 125.4,
125.1, 79.0, 78.6, 69.4, 68.0, 58.2, 55.6, 55.4, 55.1, 54.0, 51.5, 49.8,
40.0, 39.9, 36.3, 35.8, 30.8, 30.2, 29.9, 29.5, 29.4, 27.0, 26.7, 24.2,
23.9, 23.8, 23.4, 21.6, 19.9, 19.8, 18.1, 18.0, 17.5, 17.1, 16.3;
ORCID
Wei Zhang https://orcid.org/0000-0002-4684-2911
REFERENCES
1. Hasilik A, Neufeld EF. Biosynthesis of lysosomal enzymes in fibroblasts.
Synthesis as precursors of higher molecular weight. J Biol Chem.
1980;255(10):4937‐4945.
2. Kornfeld S. Lysosomal enzyme targeting. Biochem Soc Trans.
1990;18(3):367‐374.
HRESIMS calcd for
757.4125.
C36H58N6O10 [M +
Na]+ 757.4107, found
3. Horikoshi T, Arany I, Rajaraman S, et al. Isoforms of cathepsin D and
human epidermal differentiation. Biochimie. 1998;80(7):605‐612.
4. Conner GE, Richo G. Isolation and characterization of a stable activa-
tion intermediate of the lysosomal aspartyl protease cathepsin.
Biochem. 1992;31(4):1142‐1147.
|
4.3.6
Compound 14
5. Erickson AH, Conner GE, Blobel G. Biosynthesis of a lysosomal
enzyme. Partial structure of two transient and functionally distinct
NH2‐terminal sequences in cathepsin D. J Biol Chem. 1981;256:11224.
Compound 14 was obtained from 11 according to the similar proce-
25
dure. White solid; yield 69.9%; ½αꢀD = −7.7 (c = 0.7, MeOH); 1H
NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.41‐7.30 (m, 1H), 7.29‐7.06
(m, 5H), 6.83 (s, 1H), 6.55 (s, 1H), 5.29 (d, J = 7.1 Hz, 1H), 4.98 (s,
1H), 4.43 (d, J = 6.2 Hz, 1H), 4.34‐3.96 (m, 3H), 3.01‐2.86 (m, 2H),
2.72 (s, 3H), 2.58‐2.42 (m, 1H), 2.44‐2.22 (m, 2H), 2.08 (s, 2H), 1.98‐
1.77 (m, 2H), 1.77‐1.53 (m, 3H), 1.45 (s, 9H), 1.02‐0.78 (m, 12H); 13C
NMR (151 MHz, CDCl3) δ 176.6, 176.1, 173.8, 172.4, 170.9, 156.2,
138.3, 129.1, 128.4, 126.4, 80.0, 69.7, 55.9, 55.7, 54.5, 51.4, 40.4,
36.9, 31.6, 30.6, 28.4, 24.9, 24.1, 23.0, 21.6, 19.6, 17.4; HRESIMS
calcd for C33H53N5O9 [M + Na]+ 686.3736, found 686.3735.
6. Lee AY, Gulnik SV, Erickson JW. Conformational switching in an
aspartic proteinase. Nat Struct Biol. 1998;5(10):866‐871.
7. Baechle D, Flad T, Cansier A, et al. Cathepsin D is present in human
eccrine sweat and involved in the postsecretory processing of the anti-
microbial peptide DCD‐1L. J Biol Chem. 2006;281(9):5406‐5415.
8. Benes P, Vetvicka V, Fusek M. Cathepsin D‐many functions of one
aspartic protease. Crit Rev Oncol Hematol. 2008;68(1):12‐28.
9. Zhao CF, Herrington DM. The function of cathepsins B, D, and X in
atherosclerosis. Am J Cardiovasc Dis. 2016;6(4):163‐170.
10. Davies DR. The structure and function of the aspartic proteinases.
Annu Rev Biophys Biophys Chern. 1990;19(1):189‐215.
11. Garcia M, Derocq D, Pujol P, Rochefort H. Overexpression of
transfected cathepsin D in transformed cells increases their malignant
phenotype and metastatic potency. Oncogene. 1990;5(12):1809‐1814.
|
4.4
Biological assays
Aspartic protease enzymatic assays to determine inhibitory activity
against Cath D, Cath E, and BACE1 were carried out as described
previously.27 Enzymes BACE1 (R&D Systems), cathepsin D (Enzo Life
Sciences), and cathepsin E (R&D systems) and corresponding sub-
strates were prepared freshly in their respective reaction buffers.
The reaction buffer for BACE1 contained 100 mM NaOAc (pH 4.0),
and the reaction buffer for cathepsins D and E contained 100 mM
NaOAc/100 mM NaCl (pH 3.5). Enzymes were added (final concentra-
tions are 30 μg/mL for BACE1, 1.0 μg/mL for cathepsin D, and
0.05 μg/mL for cathepsin E) followed by compounds dissolved in
DMSO at six different concentrations (10, 5, 1, 0.5, 0.1, 0.01 μM final
12. Ohri SS, Vashishta A, Proctor M, Fusek M, Vetvicka V. Depletion of
procathepsin D gene expression by RNA interference: a potential ther-
apeutic
target
for
breast
cancer.
Cancer
Biol
Ther.
2007;6(7):1081‐1087.
13. Ohri SS, Vashishta A, Proctor M, Fusek M, Vetvicka V. The propeptide
of cathepsin D increases proliferation, invasion and metastasis of
breast cancer cells. Int J Oncol. 2008;32:491‐498.
14. Jancekova B, Ondrouskova E, Knopfova L, Smarda J, Benes P. Enzy-
matically active cathepsin
D sensitizes breast carcinoma cells to
TRAIL. Tumour Biol. 2016;4:689‐701.
15. Bornebroek M, Kumar‐Singh S. A novel drug target in Alzheimer's dis-
ease. Lancet. 2004;364(9447):1738‐1739.

LI ET AL.
9 of 9
16. Shacka JJ, Roth KA. Cathepsin D deficiency and NCL/batten disease:
28. Umezawa H, Aoyagi T, Morishima H, Matsuzaki M, Hamada M.
there's more to death than apoptosis. Autophagy. 2007;3(5):474‐476.
Pepstatin,
a new pepsin inhibitor produced by actinomycetes. J
Antibiot. 1970;23(5):259‐262.
17. Boonen M, Staudt C, Gilis F, Oorschot V, Klumperman J, Jadot M.
Cathepsin D and its newly identified transport receptor SEZ6L2 can
modulate neurite outgrowth. J Cell Sci. 2016;129(3):557‐568.
29. Baldwin ET, Bhat TN, Gulnik S, et al. Crystal structures of native and
inhibited forms of human cathepsin D: implications for lysosomal
targeting and drug design. Proc Natl Acad Sci. 1993;90(14):6796‐6800.
18. Vidoni C, Follo C, Savino M, Melome MA, Isidoro C. The role of
Cathepsin D in the pathogenesis of human neurodegenerative disor-
ders. Med Res Rev. 2016;4:845‐870.
30. Yazmin TR, Frederic B, Jonathan AE. Straightforward preparation and
assay of aspartyl protease substrates with an internal thioester linkage.
Chembiochem. 2007;8:981‐984.
19. Pranjol MD, Gutowski NJ, Hannemann M, Whatmorea JL. Cathepsin D
non‐proteolytically induces proliferation and migration in human
omental microvascular endothelial cells via activation of the ERK1/2
and PI3K/AKT pathways. Biochim Biophys Acta. 2018;1865(1):25‐33.
31. Gacko M, Minarowska A, Karwowska A, Minarowski L. Cathepsin D
inhibitors. Folia Histochem Cytobiol. 2007;45:291‐313.
32. Liu Y, Zhang W, Li L, et al. Cyanobacterial peptides as a prototype for
the design of potent β‐secretase inhibitors and the development of
selective chemical probes for other aspartic proteases. J Med Chem.
2012;55(23):10749‐10765.
20. Mehanna S, Suzuki C, Shibata M, et al. Cathepsin D in pancreatic acinar
cells is implicated in cathepsin B and L degradation, but not in autoph-
agic activity. Biochem Biophys Res Commun. 2016;469(3):405‐411.
21. Dumas J, Brittelli D, Chen J, et al. Synthesis and structure activity rela-
tionships of novel small molecule cathepsin D inhibitors. Bioorg Med
Chem Lett. 1999;9(17):2531‐2536.
33. Williams PG, Yoshida WY, Moore RE, Paul VJ. The isolation and struc-
ture elucidation of tasiamide B,
a
4‐amino‐3‐hydroxy‐5‐
phenylpentanoic acid containing peptide from the marine cyanobacte-
rium Symploca sp. J Nat Prod. 2003;66(7):1006‐1009.
22. Srivastava V, Saxena HO, Shanker K, et al. Synthesis of gallic acid
based naphthophenone fatty acid amides as cathepsin D inhibitors.
Bioorg Med Chem Lett. 2006;16(17):4603‐4608.
34. Sun T, Zhang W, Zong C, Wang P, Li Y. Total synthesis and stereo-
chemical reassignment of tasiamide B. J Pept Sci. 2010;16:364‐374.
23. Ahmed W, Khan IA, Arshad MN, Siddiqui WA, Haleem MA, Azim MK.
Identification of sulfamoylbenzamide derivatives as selective cathepsin
D inhibitors. Pak J Pharm Sci. 2013;26(4):687‐690.
35. Liu J, Chen W, Xu Y, Ren S, Zhang W, Li Y. Design, synthesis and bio-
logical evaluation of tasiamide B derivatives as BACE1 inhibitors.
Bioorg Med Chem. 2015;23(9):1963‐1974.
24. Majer P, Collins JR, Gulnik SV, Erickson JW. Structure‐based subsite
specificity mapping of human cathepsin D using statine‐based inhibi-
tors. Protein Sci. 1997;6(7):1458‐1466.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
25. Carroll CD, Johnson TO, Tao S, et al. Evaluation of a structure‐based
statine cyclic diamino amide encoded combinatorial library against
plasmepsin II and cathepsin D. Bioorg Med Chem Lett.
1998;8(22):3203‐3206.
26. Lee CE, Kick EK, Ellman JA. General solid‐phase synthesis approach to
prepare mechanism‐based aspartyl protease inhibitor libraries. Identifi-
How to cite this article: Li Z, Bao K, Xu H, et al. Design, syn-
thesis, and bioactivities of tasiamide B derivatives as cathepsin
D inhibitors. J Pep Sci. 2019;e3154. https://doi.org/10.1002/
psc.3154
cation of potent cathepsin
D
inhibitors.
J
Am Chem Soc.
1998;120(38):9735‐9747.
27. Xu H, Bao KT, Tang S, Ai J, Hu HY, Zhang W. Cyanobacterial peptides
as a prototype for the design of cathepsin D inhibitors. J Pept Sci.
2017;23(9):701‐706.