Optimization of piperidine constructed peptidyl derivatives as proteasome inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115867

A series of non-covalent piperidine-containing peptidyl derivatives with various substituents at side chains of different residues were designed, synthesized and evaluated as proteasome inhibitors. After proteasome inhibitory evaluations of all the synthe

Full text of DOI:10.1016/j.bmc.2020.115867

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Optimization of piperidine constructed peptidyl derivatives as proteasome in-
hibitors
Yanmei Zhao, Lei Xu, Jiankang Zhang, Mengmeng Zhang, Jingyi Lu, Ruoyu
He, Jianjun Xi, Rangxiao Zhuang, Jia Li, Yubo Zhou
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https://doi.org/10.1016/j.bmc.2020.115867
BMC 115867
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11 October 2020
1 November 2020
Please cite this article as: Y. Zhao, L. Xu, J. Zhang, M. Zhang, J. Lu, R. He, J. Xi, R. Zhuang, J. Li, Y. Zhou,
Optimization of piperidine constructed peptidyl derivatives as proteasome inhibitors, Bioorganic & Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115867
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Optimization of piperidine constructed peptidyl derivatives as proteasome inhibitors
Yanmei Zhaoa, ┼, Lei Xub,┼, Jiankang Zhanga, c, ┼ Mengmeng Zhangb, Jingyi Luc, Ruoyu Hea, Jianjun Xia,
Rangxiao Zhuanga*, Jia Lib*, Yubo Zhoub*
a
Department of pharmaceutical Preparation, Hangzhou Xixi Hospital, Hangzhou 310023, Zhejiang
Province, China
b National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
c School of Medicine, Zhejiang University City College, Hangzhou 310015, Zhejiang Province, China
┼ These authors contributed equally to this work.
Abstract: A series of non-covalent piperidine-containing peptidyl derivatives with various substituents
at side chains of different residues were designed, synthesized and evaluated as proteasome inhibitors.
After proteasome inhibitory evaluations of all the synthesized target compounds, selected ones were
tested for their anti-proliferation activities against three multiple myeloma (MM) cell lines. 8 analogues
displayed more potent activities than carfilzomib, and the most promising compound 24 showed IC50
values of 0.8±0.2 nM against 20S proteasome and 8.42±0.74 nM, 7.14±0.52 nM, 14.20±1.08 nM for
RPMI 8226, NCI-H929 and MM.1S cell lines, respectively. Additionally, mechanisms of anti-cancer
activity of representative compound 24 were further investigated. Apoptosis of RPMI-8226 cells were
achieved through accumulating polyubiquitin and inducing the cleavage of caspase and PARP. Besides,
half-life in rat plasma of compound 24 was prolonged after optimization, which would be helpful for
increasing in vivo activities of this series of derivatives. All the studies confirmed that piperidine-
containing non-covalent proteasome inhibitors can be potential leads for anti-MM drug development.
Keywords: Proteasome inhibitor, Piperidine, Peptidyl, Anti-cancer, SAR
*Corresponding authors:
Tel.: +86-571-8648-1960; Fax: +86 571 86481960 (Rangxiao Zhuang)
Tel.: +86-21-5080-1552; Fax: +86-21-5080-0721 (Jia Li and Yubo Zhou)
E-mail address: zhuangrangxiao@sina.com (Rangxiao Zhuang); jli@simm.ac.cn (Jia Li);
ybzhou@simm.ac.cn (Yubo Zhou).
1. Introduction
1

The ubiquitin-proteasome pathway (UPP) plays a critical role in recognizing and degrading abnormal
and misfolded proteins.1-3 Proteins are targeted for proteasomal degradation via covalent attachment of
a 8.5 kDa protein ubiquitin, and ubiquitination occurs with the assistance of three different enzymes.
Firstly, ubiquitin is activated by ubiquitin-activating enzyme (E1), and subsequently transferred to
ubiquitin-conjugating enzyme (E2) before it is finally coupled to the substrate protein by means of
ubiquitin-protein ligase (E3). The ubiquitinated proteins are then flattened into the 26S proteasome and
degraded in the 20s catalytic center.4 In this pathway, the 26S proteasome is the main proteolytic
component, which contains two ATP-dependent 19S regulatory particles (RPs) and one 20S core particle
(CP). The 20S proteasome is the proteolytically active key element of the UPP that directs the majority
of intracellular protein degradation in eukaryotic cells.3 It is composed of four heptameric rings stacked
in a α7-β7-β7-α7 arrangement and contains three proteolytic subunits β1, β2 and β5.5-8 All of these
components have been extensively investigated and validated as potential targets, and confirmed targeted
drugs have played important roles for the treatment of multiple myeloma and other diseases.9-10
Currently, three proteasome inhibitors Bortezomib, Carfilzomib and Ixazomib have been approved by
FDA for the treatment of multiple myeloma (Fig.1).10-12 All three are covalent peptidyl inhibitors with
an electrophilic moiety at the C-terminal end of the peptidyl backbone for covalent binding to the
catalytic terminal Thr1 residue of the proteasome β5 subunit. Besides, various short peptides and
pseudopeptides with these covalent binding groups were explored for cancer therapy.12-17 However, the
active electrophilic groups induce excessive reactivity, less specificity and instability of these inhibitors,
and the stable covalent interaction is irreversible, which is believed to be the major cause for side effects
during therapy.12, 18 Meanwhile, most of these inhibitors failed in the treatment of solid cancers, which is
mainly attributed to the quick covalent binding to the proteasome, thus limiting their widespread tissue
distribution. 12, 19-20
Figure 1. Structures of FDA approved proteasome inhibitors for multiple myeloma
In contrast, non-covalent inhibitors with reversible binding mode ensure them with rapid binding and
dissociation kinetics. These features endowed these analogues abilities for overcoming defects arising in
2

therapeutics of covalent combination drugs.21-22 Although none of the non-covalent proteasome inhibitor
has been approved, they have received more and more attention from researchers, and some of these
compounds exhibited even more potent proteasome inhibitory activities than covalent ones.12 Blackburn
and colleagues reported a series of di- and tripeptidyl non-covalent proteasome inhibitors, among which
ML 16 was the most promising one with IC50 values of 1.2 nM against constitutive proteasome and 1.1
nM against immunoproteasome (Fig.2). 19-20
Figure 2. Representative non-covalent proteasome inhibitors with potent enzymatic activities
Based on the previous studies, novel non-covalent peptidyl proteasome inhibitors containing piperidine
or piperazine were developed in our group, among which the most promising analogue (PI 35:
proteasome inhibitory activity, IC50: 1.2 ± 0.1 nM) displayed potent anti-proliferation activities (IC50:
RPMI-8226: 8.4 ± 0.8 nM; MM-1S: 6.3 ± 0.8 nM).12-13 Although the half-life of PI 35 was improved by
constructing a aliphatic heterocycle into the peptide skeleton, the blood cell proteasome inhibitory
activity was not guaranteed. In this manuscript, further optimization of PI 35 through various
substitutions replaced at side chains of each residue with retained piperidine constructed skeleton was
carried out. Promising derivatives with potent in vitro anti-cancer activities and improved stabilities were
anticipated, thus make these analogues with potential in vivo activities.
2. Synthesis
The synthetic route for piperidine-containing fragments 3a-i is summarized in Scheme 1. An easy
condensation of corresponding amine 1a-i with protected piperidine fragment provided compounds 2a-
i. Afterwards, deprotection of 2a-i with trifluoroacetic acid (TFA) afforded piperidine TFA salts (3a-i).
As shown in Scheme 1, compound 4 and 5 were treated with tert-butyl amine in the presence of EDCI
and HOBt to give the key intermediates TFA salts protected amines (6a-c), which were then deprotected
3

with TFA to afford TFA salts (7a-c). Reduction of fragments 6a-b at the presence of Pd/C (10%)
provided protected amino acid fragments 8a-b. Compounds 8a-b and 9 were then treated with 2-
chlorobenzylamine, EDCI and HOBt to afford intermediates 10a-c. Eventually, deprotection of 10a-c
with TFA afforded TFA salts 11a-c.
Scheme 1. Synthesis of piperidine-containing fragments (3a-i) and the TFA salts (7a-c and 11a-c).
Reagents and conditions: (I) HOBt, EDCI, corresponding amine, DIPEA, DCM, 0oC-rt; (II) TFA, DCM,
0oC-rt; (III) neopentylamine, HOBt, EDCI, DIPEA, DCM, 0oC-rt; (IV) tert-Butylamine, HOBt, EDCI,
4

DIPEA, DCM, 0oC-rt; (V) TFA, DCM, 0oC-rt; (VI) Pd/C (10%), H2, methanol, rt; (VII) 2-
chlorobenzylamine, HOBt, EDCI, DIPEA, DCM, 0oC-rt; (VIII) TFA, DCM, 0oC-rt.
The synthetic route for target compounds 20-23 is outlined in Scheme 2. The piperidine TFA salt 12 was
condensed with 11a to afford compound 13, which was then deprotected with TFA to afford piperidine
TFA salt 14. Subsequently, analogue 14 were transformed to corresponding isocyanate intermediates and
reacted with 3a or 3b to obtain the target compounds 20-21. As illustrated in Scheme 2, reaction of
compound 15 with 2-amino-N-(2-chlorobenzyl)-3-phenylpropanamide furnished compound 16, which
was deprotected under the condition of Pd/C (10%) to afford compound 17. Finally, compound 17 was
transformed into corresponding isocyanate intermediates and then reacted with 3a or 3b to obtain the
target compounds 22 and 23.
Scheme 2. Synthesis of piperidine-containing target compounds (20-23). Reagents and conditions: (I)
11a, HOBt, EDCI, DIPEA, DCM, 0oC-rt; (II) TFA, DCM, 0oC-rt; (III) 3a or 3b, HOBt, EDCI, DIPEA,
5

DCM, 0oC-rt; (IV) 2-amino-N-(2-chlorobenzyl)-3-phenylpropanamide, HOBt, EDCI, DIPEA, DCM,
0oC-rt; (V) Pd/C (10%), H2, methanol, rt; (VI) 3a or 3b, HOBt, EDCI, DIPEA, DCM, 0oC-rt.
The synthesis of target compounds 24-39 was summarized in Scheme 3. Compounds 7a-c were
converted into corresponding isocyanate intermediates, which were unstable and were therefore prone to
react with piperidine fragments 3a-i to obtain key intermediates 18a-m. Ultimately, the coupling of
corresponding amine with various acids 18a-m in the presence of EDCI, HOBt and DIPEA gave the
target compounds 24-39.
6

Scheme 3. Synthesis of piperidine-containing target compounds (24-39). Reagents and conditions: (I)
triphosgene, DIPEA, DCM, aqueous NaHCO3, 0oC-rt.; (II) Pd/C (10%), H2, methanol, rt; (III)
corresponding amine, HOBt, EDCI, DIPEA, DCM, 0oC-rt.
7

3. Pharmacology
3.1 Proteasome inhibitory activities
The obtained target compounds were screened for 20S proteasome chymotrypsin-like inhibitory
activities in vitro with Carfilzomib employed as positive control. The results are shown in Table 1. As
summarized in Table 1, the IC50 values of most target compounds were lower than 50 nM, among which
9 analogues were even lower than 10 nM, indicating that the activities of most compounds were reserved
after different substituents introduced at R1, R2 and R3 positions while retaining the dominant skeleton
containing the piperidine ring.
Different substituents at R2 position affected the activity obviously. Overall, 3-(tert-butylamino)-3-
oxopropyl, 2-(neopentylamino)-2-oxopropyl and 3-(neopentylamino)-3-oxopropyl were all tolerable.
By comparing activities of analogues 22, 23, 24, 25, 32 and 33, decreasing of activities were observed in
compounds with 4-tert-butoxybenzyl (23 and 24), and 3-(tert-butylamino)-3-oxopropyl substituted
analogues displayed the most potent enzymatic activities (IC50: 24, 0.8±0.2 nM; 25, 0.9±0.1 nM).
Compounds 20 and 21 were obtained through exchanging R2 and R3 substituents based on our previous
study, activities of which were not improved. Besides, 28 and 29 with 2-(neopentylamino)-2-oxopropyl
at R2 were also more potent than corresponding 20 and 21 with IC50 values of 5.2±0.3 and 19.9±0.3
nM, respectively, which was also an evidence for the inappropriateness of phenylethyl at R2.
In order to investigate the influences of substitution at R3, activities of analogues 26-31 were evaluated.
All of these derivatives displayed comparable proteasome inhibitory activities with IC50 values ranging
from 4.1 to 19.9 nM, which were all less potent than 24 and 25 with phenylethyl at R3. Thus, 3-(tert-
butylamino)-3-oxopropyl at R2 and phenylethyl at R3 were more preferable, which were further
employed for the screening of R1 groups.
The effects of various N-terminus groups (R1) were studied. The results indicated that different
substituted phenyl groups influenced proteasome inhibitory activities obviously. Electron-withdrawing
groups at phenyl were beneficial for the activities with IC50 values of derivatives 34-37 ranging from 9.0
to 23.7 nM, while phenyl substituted compound 39 showed moderate activity with IC50 value of 338.3
nM. Analogue 38 only displayed weak activity with electron-donating group at R1 (IC50: 4134.0±794.8
nM). Besides, R1 pyridine and pyrazine substituted derivatives (24 and 25) were much more potent than
that of R1 phenyl replaced compounds (34-39), through which more interactions might be formed with
the target.
8

Table 1. 20S proteasome chymotrypsin-like inhibitory activities of target compounds (20-39)
Proteasome inhibitory
Compound
R1
R2
R3
activities
IC50 (nM)a
20
21
22
23
24
25
26
27
28
29
pyridin-3-yl
pyrazin-2-yl
pyridin-3-yl
pyrazin-2-yl
pyridin-3-yl
pyrazin-2-yl
pyridin-3-yl
pyrazin-2-yl
pyridin-3-yl
pyrazin-2-yl
phenylethyl
2-(neopentylamino)-2-oxoethyl
2-(neopentylamino)-2-oxoethyl
phenylethyl
13.8±2.4
42.1±2.1
77.0±24.5
163.8±29.6
0.8±0.2
phenylethyl
4-tert-butoxybenzyl
4-tert-butoxybenzyl
phenylethyl
3-(tert-butylamino)-3-oxopropyl
3-(tert-butylamino)-3-oxopropyl
2-(neopentylamino)-2-oxoethyl
2-(neopentylamino)-2-oxoethyl
phenylethyl
phenylethyl
0.9±0.1
benzylthiomethyl
benzylthiomethyl
4.1±0.4
10.6±3.4
5.2±0.3
2-(neopentylamino)-2-oxoethyl 2-(neopentylamino)-2-oxoethyl
2-(neopentylamino)-2-oxoethyl 2-(neopentylamino)-2-oxoethyl
19.9±0.3
5.6±1.5
3-(tert-butylamino)-3-
2-(neopentylamino)-2-oxoethyl
oxopropyl
30
31
pyridin-3-yl
pyrazin-2-yl
3-(tert-butylamino)-3-
2-(neopentylamino)-2-oxoethyl
oxopropyl
6.2±0.6
1.4±0.1
3-(neopentylamino)-3-
phenylethyl
32
33
pyridin-3-yl
pyrazin-2-yl
2.7±0.7
oxopropyl
11.0±0.7
16.5±4.0
9.0±0.2
3-(neopentylamino)-3-
phenylethyl
oxopropyl
34
35
36
37
2-fluorophenyl
3-fluorophenyl
4-fluorophenyl
4-cyanophenyl
3-(tert-butylamino)-3-oxopropyl
3-(tert-butylamino)-3-oxopropyl
3-(tert-butylamino)-3-oxopropyl
3-(tert-butylamino)-3-oxopropyl
phenylethyl
phenylethyl
phenylethyl
phenylethyl
23.7±1.5
4134.0±794.8
338.2±75.5
9

38
39
4-methoxyphenyl 3-(tert-butylamino)-3-oxopropyl
phenylethyl
phenylethyl
-
8.4±0.9
phenyl
-
3-(tert-butylamino)-3-oxopropyl
-
Carfilzomib
a The IC50 values were an average of three independent determinations.
3.2 Tumor cell growth inhibitory activities
Based on the obtained proteasome inhibitory activities, selected compounds with IC50 values lower than
10 nM were further screened for their in vitro anti-proliferative activities against three MM cell lines
(RPMI 8226, NCI-H929 and MM.1S) by MTT-based assay with Carfilzomib used as positive control.
As summarized in Table 2, most of the tested compounds showed superior cytotoxic activities against
three MM cell lines with IC50 values lower than 20 nM, which were consistent with the proteasome
inhibitory activities. Overall, among all the tested compounds, 24 exhibited the most potent cytotoxicities
with IC50 values of 8.42±0.74, 7.14±0.52 and 14.20±1.08 nM against RPMI 8226, NCI-H929 and
MM.1S cell line, respectively. Thus, candidate 24 was then proceeded for further biological evaluation.
Table 2. Anti-proliferative activities against three different MM cell lines of selected compounds
Cytotoxicity IC50 (nM) a
Compound
RPMI8226
8.42±0.74
7.23±0.48
9.46±0.21
14.39±1.16
13.19±1.07
17.17±1.33
16.01±1.27
19.61±1.42
15.08±1.16
14.10±0.61
NCI-H929
7.14±0.52
9.01±0.67
10.06±0.14
17.45±1.23
15.72±1.17
12.20±0.94
18.49±1.38
19.57±1.37
8.92±0.56
13.90±0.52
MM.1S
24
14.20±1.08
15.88±1.12
14.90±1.04
16.33±1.19
19.36±1.52
17.12±0.86
21.82±1.75
30.39±2.17
12.50±0.88
14.35±0.73
25
26
28
30
31
32
33
36
Carfilzomib
a The IC50 values were an average of three independent determinations.
3.3 DAPI staining analysis
To further investigate the mechanism of anti-cancer activity of compound 24, cell apoptosis study by
DAPI staining in the RPMI-8226 cells was performed. RPMI-8226 cells were treated with compound 24
or Carfilzomib for 48 h. As shown in Fig. 3, the percentages of apoptotic RPMI-8226 cells were 3.10 ±
10

0.85% in control cells, 21.66 ± 5.25% with 1 nM of 24, 58.18 ± 9.79% with 5 nM of 24, 97.79 ± 3.51%
with 10 nM of 24 and 72.42 ± 4.33% with 10 nM of Carfilzomib. Much more apoptotic RPMI-8226 cells
were observed in 24 treatment group compared with Carfilzomib treatment group (10 nM of 24 vs. 10
nM of Carfilzomib, p<0.05) in dose dependent manner, indicating that analogue 24 possessed stronger
anti-apoptotic abilities than Carfilzomib in RPMI-8226 cells at the concentration of 10nM. Since 24
significantly induced apoptosis, we sought to measure the expression of apoptotic related proteins in
RPMI-8226 cells treated with 24 or Carfilzomib.
Figure 3. Compound 24 induced apoptosis in RPMI-8226 cells. RPMI-8226 cells were incubated with
24 or Carfilzomib at indicated concentrations for 48 h, and cells were collected and detected by DAPI
staining. Apoptotic nuclei were indicated by arrows, scale bar = 100 μm. 24 induced apoptosis in dose
dependent manner on RPMI-8226 cells. * p<0.05, ** p<0.01, *** p<0.001, compared with control. #
p<0.05, 10 nM of 24 vs. 10 nM of Carfilzomib.
3.4 Western blot analysis
To determine whether proteasome inhibitory activity of compound 24 in a cell-free system can be
recapitulated in vitro, we monitored the levels of polyubiquitinated proteins in treated cells by
immunoblotting, using antibody specific towards free ubiquitin and ubiquitin conjugates to form a typical
high molecular mass smear. As demonstrated in Fig. 4A and B, we observed that treatment of RPMI
8226 cells with 24 for 48 h induced a significantly greater activation of PARP and capase-3 than
Carfilzomib. To further investigate whether ubiquitin-proteasome system was involved in the anti-cancer
11

effect of 24, we observed that the efficiency of 24 on accumulating ubiquitin was stronger than
Carfilzomib, suggesting that 24 induced apoptosis via ubiquitin-proteasome pathway in RPMI-8226 cells.
However, significant increase in PARP cleavage was observed at the concentration of 1 nM and 5 nM,
while accumulation of ubiquitinated proteins was not obvious at the same concentrations, which
indicated that the mechanism of cell death might not only attributed to proteasome inhibition. Further
investigations in inducing apoptosis of this series of analogues are required.
Figure 4. Compound 24 affected apoptosis related proteins in RPMI-8226 cells. (A) RPMI-8226 cells
were incubated with 24 or Carfilzomib at indicated concentrations for 24 h, and cells were collected and
detected by western blotting. (B) Statistical analysis of the expression of cleaved-caspase-3, cleaved-
PARP and Ub. * p<0.05, ** p<0.01, *** p<0.001, compared with control. # p<0.05, 10 nM of 24 vs. 10
nM of Carfilzomib. The results are average of three independent experiments.
3.5 Plasma stability
Subsequently, 7 compounds with potent proteasome inhibitory activities and cytotoxicities were further
tested for their stabilities by measuring the half lives in rat plasma. As illustrated in Tab. 3, most of these
analogues displayed comparable stabilities with PI 35 discovered in our previous study. However, as
expected, the most potent analogue 24 was most stable after optimization, which might be beneficial for
improving in vivo anti-cancer activities of this promising analogue.
Table 3. Rat plasma stabilities of selected compounds
Compound
Plasma half-life (T1/2, min)
24
25
104.51
72.90
12

28
32
72.05
69.98
77.75
93.46
84.20
91.55
33
35
36
PI 35
3.6 Binding mode analysis
To further elucidate the binding modes of the most potent compound 24 with the active site of proteasome,
docking simulation studies were carried out. The co-crystal structure of proteasome (PDB ID code:
3MG6) was selected as the docking template. As shown in Fig. 5, the carbonyl and amino groups of the
peptide skeleton of compound 24 formed several critical hydrogen bonds with residues Thr21, Ala49,
Ala50 and Asp114 of the proteasome active site, respectively. These hydrogen bonds played important
roles in the inhibitory potency against proteasome. Additionally, the 3-(tert-butylamino)-3-oxopropyl
group occupied well defined S3 pocket, while C-terminus was located in the S1 binding pocket. The
docking result gave a rational explanation for the potency of compound 24 and it is consistent with the
previous biological results.
Figure 5. The binding mode of compound 24 with chymotrypsin-like active site of 20S proteasome (PDB
ID: 3MG6).
4. Conclusion
13

A series of piperidine-containing non-covalent peptidyl derivatives possessing various substituents at
side chains of different residues were designed, synthesized and biologically evaluated for their
proteasome inhibitory and anti-proliferation activities. Among all the tested compounds, 8 derivatives
not only displayed proteasome inhibitory activities more excellent than the positive control Carfilzomib,
but also possessed forceful anti-proliferation activities against three MM cell lines (RPMI 8226, NCI-
H929 and MM.1S). Additionally, the most promising compound 24 showed stronger ability in inducing
apoptosis than Carfilzomib, and promoted cell apoptosis via ubiquitin-proteasome pathway in RPMI-
8226 cells. Besides, as anticipated, the most potent analogue 24 possessed the strongest stability in rat
plasma. All of these studies suggested that compound 24 could be a potentially interesting lead molecule
for further investigation.
5. Experimental procedures
5.1 Chemistry and general methods
Mass spectra (MS) were taken in the ESI mode on Esquire-LC-00075 spectrometer (Brüker Bioscience).
1H and 13C NMR spectra were obtained on Brüker 500/400MHz spectrometer (Brüker Bioscience,
Billerica, MA, USA) with TMS as an internal standard and CDCl3 or DMSO-d6 as solvent. All yields are
unoptimized and generally represent the result of a single experiment.
5.2 Synthetic procedures
5.2.1 General procedure for the synthesis of protected piperidine fragments 2a-i
N-Boc-4-piperidinecarboxylic acid (3.65g, 16.0mmol), CH2Cl2 (30.0mL) and pyridine (3.4mL,
40.0mmol) were added in the round-bottom flask in turn and then mixed and protected by N2, SOCl2
(1.5mL, 19.0mmol) was added slowly to the mixture and reacted for half an hour at room temperature.
Then the solution of catalytic amount of DMAP, amine (1a-i, 18.0mmol) and Et3N (8.0mL, 56.0mmol)
in CH2Cl2 (30.0mL) was added dropwise sequentially and stirred overnight. Upon completion of the
reaction, the organic phase was washed with 1N HCl (2×30mL) and aqueous NaHCO3 (2×30mL), dried
by Na2SO4. The solvent was evaporated under reduced pressure and the crude product was purified by
flash chromatography (ethyl acetate: petroleum ether = 1:2~3:1) to give piperidine derivatives 2a-i.
tert-Butyl 4-(Pyridin-3-ylcarbamoyl) piperidine-1-carboxylate (2a)
White solid; Yield: 63%; 1H NMR (500MHz, CDCl3): δ = 8.61 (s, 1H, pyridine-H), 8.34 (d, 1H, J = 4.0
14

Hz, pyridine-H), 8.28 (d, 1H, J = 8.5 Hz, pyridine-H), 7.81 (s, 1H, NH), 7.32 (dd, 1H, J = 8.5, 5.0 Hz,
pyridine-H), 4.19 (d, 2H, J = 13.0 Hz, CH2), 2.90-2.71 (m, 2H, CH2), 2.49-2.43 (m, 1H, CH), 1.91 (d,
2H, J = 12.0 Hz, CH2), 1.77-1.73 (m, 2H, CH2), 1.46 (s, 9H, (CH3)3); ESI-MS: m/z = 306.2 [M+H]+.
tert-Butyl 4-(Pyrazin-2-ylcarbamoyl) piperidine-1-carboxylate (2b)
White solid; Yield: 78%; 1H NMR (500MHz, CDCl3): δ = 9.55 (s, 1H, pyrazine-H), 8.35 (d, 1H, J = 2.0
Hz, pyrazine-H), 8.23 (s, 1H, pyrazine-H), 7.97 (s, 1H, NH), 4.24-4.15 (m, 2H, CH2), 2.86-2.75 (m, 2H,
CH2), 2.51-4.46 (m, 1H, CH), 1.93 (d, 2H, J = 12.5 Hz, CH2), 1.80-1.72 (m, 2H, CH2), 1.47 (s, 9H,
(CH3)3); ESI-MS: m/z = 307.2 [M+H]+.
tert-butyl 4-(2-fluorophenylcarbamoyl)piperidine-1-carboxylate (2c)
1
White solid; Yield: 88%; H NMR (500MHz, CDCl3): δ = 8.34 (s, 1H, J = 2.0 Hz, NH), 7.40 (s, 1H,
Ar-H), 7.15-7.04 (m, 3H, Ar-H), 4.22 (d, 2H, J = 12.0 Hz, CH2), 2.84-2.81 (m, 2H, CH2), 2.45-2.42 (m,
1H, CH), 1.94-1.90 (m, 2H, CH2), 1.79-1.73 (m, 2H, CH2), 1.47 (s, 9H, (CH3)3); ESI-MS: m/z = 323.2
[M+H]+.
tert-butyl 4-(3-fluorophenylcarbamoyl)piperidine-1-carboxylate (2d)
White solid; Yield: 65%; 1H NMR (500MHz, CDCl3): δ = 7.71 (m, 1H, NH), 7.52 (d, 1H, J = 13.5 Hz,
Ar-H), 7.29-7.23 (m, 1H, Ar-H), 7.14 (d, 1H, J = 10.0 Hz, Ar-H), 6.83-6.79 (m, 1H, Ar-H), 4.22-4.11
(m, 2H, CH2), 2.82-2.76 (m, 2H, CH2), 2.41-2.35 (m, 1H, CH), 1.91-1.88 (d, 2H, J = 9.5 Hz, CH2), 1.75-
1.69 (m, 2H, CH2), 1.47 (s, 9H, (CH3)3); ESI-MS: m/z = 323.2 [M+H]+.
tert-Butyl 4-(4-Fluorophenylcarbamoyl) piperidine-1-carboxylate (2e)
White solid; Yield: 75%; 1H NMR (500MHz, CDCl3): δ = 7.50-7.42 (m, 2H, Ar-H), 7.20 (s, 1H, NH),
7.01 (t, 2H, J = 8.0 Hz, Ar-H), 4.18 (d, 2H, J = 12.0 Hz, CH2), 2.80 (t, 2H, J = 12.5 Hz, CH2), 2.39-2.32
(m, 1H, CH), 1.90 (d, 2H, J = 12.5 Hz, CH2), 1.79-1.70 (m, 2H, CH2), 1.47 (s, 9H, (CH3)3); ESI-MS: m/z
= 323.3 [M+H]+.
tert-butyl 4-(4-cyanophenylcarbamoyl)piperidine-1-carboxylate (2f)
White solid; Yield: 71%; 1H NMR (500MHz, CDCl3): δ = 7.52 (d, 2H, J = 9.5 Hz, Ar-H), 7.34 (t, 2H, J
= 10.0 Hz, Ar-H), 7.13 (t, 1H, J = 9.0 Hz, NH), 4.19 (s, 2H, CH2), 2.82-2.79 (m, 2H, CH2), 2.41-2.36
(m, 1H, CH), 1.79-1.69 (m, 2H, CH2), 1.75-1.69 (m, 2H, CH2), 1.47 (s, 9H, (CH3)3); ESI-MS: m/z =
330.3 [M+H]+.
tert-butyl 4-(4-chlorophenylcarbamoyl)piperidine-1-carboxylate(2g)
White solid; Yield: 82%; 1H NMR (500MHz, CDCl3): δ = 7.49 (d, 2H, J = 9.5 Hz, Ar-H), 7.29 (s, 1H,
15

N-H), 7.27 (d, 2H, J = 9.5 Hz, Ar-H), 4.19 (s, 2H, CH2), 2.85-2.76 (m, 2H, CH2), 2.41-2.35 (m, 1H, CH),
1.91-1.88 (m, 2H, CH2), 1.79-1.68 (m, 2H, CH2), 1.47 (s, 9H, (CH3)3); ESI-MS: m/z = 339.1 [M+H]+.
tert-Butyl 4-(4-methoxyphenylcarbamoyl) piperidine-1-carboxylate (2h)
White solid; Yield: 85%; 1H NMR (500 MHz,CDCl3): δ = 7.39 (d, 2H, J = 8.5 Hz, Ar-H), 7.38 (s, 1H,
NH), 6.83 (d, 2H, J = 7.5 Hz, Ar-H), 4.16 (d, 2H, J = 13.0 Hz, CH2), 3.77 (s, 3H,CH3), 2.81-2.69 (m,
2H, CH2), 2.38-2.32 (m, 1H, CH), 1.86 (d, 2H, J = 12.0 Hz, CH2), 1.76-1.71 (m, 2H, CH2), 1.45 (s, 9H,
(CH3)3); ESI-MS: m/z = 335.2 [M+H]+.
tert-Butyl 4-(phenylcarbamoyl) piperidine-1-carboxy-late (2i)
White solid; Yield: 86%; 1H NMR (500 MHz, CDCl3): δ = 7.53 (d, 2H, J = 5.0 Hz, Ar-H), 7.39-7.30 (m,
3H, Ar-H+NH), 7.15-7.09 (m, 1H, Ar-H), 4.21 (d, 2H, J = 10.0 Hz, CH2), 2.85-2.74 (m, 2H, CH2), 2.44-
2.38 (m, 1H, CH), 1.94-1.87 (m, 2H, CH2), 1.77-1.69 (m, 2H, CH2), 1.48 (s, 9H, (CH3)3); ESI-MS: m/z
= 305.2[M+H]+.
5.2.2 General procedure for the synthesis of deprotected piperidine contained fragments 3a-i
Trifluoroacetic acid (TFA, 5.0mL) was added to the solution of protected piperidine derivatives 2a-i
(5.0mmol) in CH2Cl2 (20.0mL) at 0oC. The mixture was then stirred for 1h at room temperature. The
volatiles were evaporated under reduced pressure and white solid was precipitated immediately after the
addition of ether, filtered and dried, allowing you to move on to the next step without further purification.
5.2.3 General procedure for the synthesis of protected amino acid fragments 6a-c
Compound 4 or 5 (5.0 mmol), HOBt (0.75 g, 5.5 mmol) and EDCI (1.44 g, 7.5 mmol) were added in the
round-bottom flask in turn and then mixed in CH2Cl2 (20.0 mL) at 0 oC and stirred for 30 min under 0
oC. A solution of corresponding amine (5.0 mmol) and diisopropylethylamine (1.85mL, 10.0 mmol) were
then added sequentially at 0 oC. After stirred at room temperature for 3 hours, the solution was washed
with aqueous NaHCO3 solution (1 × 15 mL), brine (1 × 15 mL) and dried over Na2SO4. The organic
phase was then concentrated in vacuo and purified by flash chromatography (ethyl acetate: petroleum
ether = 1:3-1:2).
(S)-benzyl 2-(tert-butoxycarbonylamino)-4-(neopentylamino)-4-oxobutanoate (6a)
White solid; Yield: 99%; 1H NMR (500 MHz, CDCl3): δ = 7.41-7.30 (m, 5H, Ar-H), 5.80 (d, 1H, J = 6.5
Hz, NH), 5.66 (brs, 1H, NH), 5.17 (d, 2H, J = 5.0 Hz, CH2), 4.55-4.52 (m, 1H, CH), 3.01 (d, 2H, J = 7.5
Hz, CH2), 2.93 (dd, 1H, J = 18.0, 5.0 Hz, CH2), 1.42 (s, 9H, (CH3)3), 0.87 (s, 9H, (CH3)3); ESI-MS: m/z
= 393.3 [M+H]+.
16

(S)-benzyl 2-(tert-butoxycarbonylamino)-5-(neopentylamino)-5-oxopentanoate (6b)
White solid; Yield: 95%; 1H NMR (500 MHz, CDCl3): δ = 7.35 (s, 5H, Ar-H), 6.13 (s, 1H, NH), 5.36 (d,
1H, J = 10.0 Hz, CH), 5.22 (dd, 2H, J = 31.5, 15.0 Hz, CH2), 4.36-4.32 (m, 1H, NH), 3.06 (d, 2H, J =
7.5 Hz, CH2), 2.26-2.18 (m, 3H, CH2 + CH), 1.96-1.91 (m, 1H, CH) 1.43 (s, 9H, (CH3)3), 0.90 (s, 9H,
(CH3)3); ESI-MS: m/z = 406.2 [M+H]+.
(S)-benzyl 2-(tert-butoxycarbonylamino)-5-(tert-butylamino)-5-oxopentanoate (6c)
White solid; Yield: 98%; 1H NMR (500 MHz, CDCl3): δ = 7.36-7.33 (m, 5H, Ar-H), 5.57 (s, 1H, NH),
5.29 (d, 1H, J = 2.5 Hz, CH), 5.21 (dd, 2H, J = 33.5, 12.5 Hz, CH2), 4.31-4.28 (m, 1H, NH), 2.13-2.08
(m, 3H, CH2 + CH), 1.99-1.92 (m, 1H, CH) 1.43 (s, 9H, (CH3)3), 1.32 (s, 9H, (CH3)3); ESI-MS: m/z =
393.2 [M+H]+.
5.2.4 General procedure for the synthesis of deprotected N-caped amino acids contained fragments
7a-c
Trifluoroacetic acid (TFA, 2.5mL) was added to a solution of protected amino acid fragments 6a-c
o
(2.5mmol) in CH2Cl2 (10.0mL) at 0 C. The mixture was then stirred for 1h at room temperature. The
volatiles were evaporated under reduced pressure and white solid was precipitated immediately after the
addition of ether, filtered and dried, allowing you to move on to the next step without further purification.
5.2.5 General procedure for the synthesis of protected amino acids fragments (8a-b)
Amino acid fragments 6a-b (5.0 mmol), 10% palladium on carbon (10 mol%) and methanol (10.0 mL)
were added in the round-bottom flask in turn and then mixed together and the reaction mixture was stirred
under an atmosphere of H2 for 2 h. Filtrated to remove Pd/C and the solvent was then evaporated under
reduced pressure to get compound 8a-b. The crude product was used directly without further purification.
5.2.6 General procedure for the synthesis of protected Ortho chlorbenzamide contained fragments
10a-c
Compound 8a-b or 9 (5.0 mmol), HOBt (0.75 g, 5.5 mmol) and EDCI (1.44 g, 7.5 mmol) were added in
the round-bottom flask in turn and then mixed in CH2Cl2 (20.0 mL) at 0 oC and then stirred for 30 min.
A solution of ortho chlorbenzamide (5.0 mmol) and diisopropylethylamine (1.85mL, 10.0 mmol) were
then added sequentially at 0 oC. After stirred at room temperature for 3 hours, the solution was washed
with aqueous NaHCO3 solution (1 × 15 mL), brine (1 × 15 mL) and dried by Na2SO4. The organic phase
was then concentrated under reduced pressure and purified by flash chromatography (ethyl acetate:
petroleum ether = 1:4-1:3).
17

(S)-tert-butyl 1-(2-chlorobenzylamino)-4-(neopentylamino)-1,4-dioxobutan-2-ylcarbamate (10a)
White solid; Yield: 79%. 1H NMR (500MHz, CDCl3): δ = 7.38 (s, 1H, NH), 7.35-7.30 (m, 2H, Ar-H),
7.22-7.20 (m, 2H, Ar-H), 6.30 (d, 1H, J = 6.5Hz, NH), 6.00 (s, 1H, NH), 4.54-4.51(m, 3H, CH2 + CH),
3.05-3.01 (m, 2H, CH2), 2.97-2.91 (m, 1H, CH), 2.59-2.55 (m, 1H, CH), 1.43 (s, 9H, (CH3)3), 0.87 (s,
9H, (CH3)3); ESI-MS: m/z = 426.3 [M+H]+.
(S)-tert-butyl 5-(tert-butylamino)-1-(2-chlorobenzylamino)-1,5-dioxopentan-2-ylcarbamate(10b)
White solid; Yield: 81%. 1H NMR (500MHz, CDCl3): δ = 7.39-7.36 (m, 2H, Ar-H), 7.25-7.23 (m, 2H,
Ar-H), 7.18 (s, 1H, NH), 5.76 (s, 1H, NH), 5.64 (s, 1H, NH), 4.56 (d, 1H, J = 7.5Hz, CH2), 4.56 (d, 1H,
J = 6.5Hz, CH2), 2.33-2.18 (m, 2H, CH2), 2.11-1.94 (m, 2H, CH2), 1.43 (s, 9H, (CH3)3), 1.36 (s, 9H,
(CH3)3); ESI-MS: m/z = 426.2 [M+H]+.
(R)-tert-butyl 3-(benzylthio)-1-(2-chlorobenzylamino)-1-oxopropan-2-ylcarbamate (10c)
White solid; Yield: 83%. 1H NMR (500MHz, CDCl3): δ = 7.40-7.37 (m, 2H, Ar-H), 7.33-7.23 (m, 7H,
Ar-H), 6.73 (brs, 1H, NH), 5.30 (brs, 1H, NH), 4.60-4.52 (m, 2H, CH2), 4.28 (brs, 1H, CH), 3.72 (dd,
2H, J = 18.5, 13.5 Hz, CH2), 2.82 (ddd, 2H, J = 81.0, 14.0, 6.0 Hz, CH2), 1.50 (s, 9H, (CH3)3); ESI-MS:
m/z = 435.1 [M+H]+.
5.2.7 General procedure for the synthesis of deprotected Ortho chlorbenzamide contained
fragments 11a-c
Trifluoroacetic acid (TFA, 2.5mL) was added to a solution of protected Ortho chlorbenzamide contained
o
fragments (10a-c, 2.5mmol) in CH2Cl2 (10.0mL) at 0 C. The mixture was then stirred for 1h at room
temperature. The volatiles were evaporated under reduced pressure and white solid was precipitated
immediately after the addition of ether, filtered and dried, allowing you to move on to the next step
without further purification.
5.2.8 tert-butyl (S)-1-((S)-1-(2-chlorobenzylamino)-4-(neopentylamino)-1,4-dioxobutan-2-ylamino)-
1-oxo-4-phenylbutan-2-ylcarbamate (13)
Compound 12 (6.0 mmol), HOBt (0.82 g, 6.0 mmol) and EDCI (2.25 g, 12.0 mmol) were added in the
round-bottom flask in turn and then mixed at 0 oC in CH2Cl2 (20.0 mL) and then stirred for 30 min. A
solution of corresponding protected amino acids fragments (6.0 mmol) and diisopropylethylamine
(2.2mL, 12.0 mmol) were then added sequentially at 0 oC. After stirred for 3 hours at room temperature,
the solution was washed with aqueous NaHCO3 solution (1 × 20 mL), brine (1 × 20 mL) and dried by
Na2SO4. The organic phase was then concentrated under reduced pressure and purified by flash
18

chromatography (ethyl acetate: CH2Cl2 = 1:5-1:1).
White solid; Yield: 85%. 1H NMR (500MHz, CDCl3): δ = 8.32 (s, 1H, NH), 7.77 (s, 1H, NH), 7.32-7.29
(m, 4H, Ar-H), 7.24-7.18 (m, 5H, Ar-H), 6.24 (s, 1H, NH), 5.07 (s, 1H, NH), 4.81 (s, 1H, CH), 4.56-4.43
(m, 2H, CH2), 4.15-4.04 (m, 2H, CH2), 3.05-3.97(m, 3H, CH2 + CH), 2.75-2.72 (m, 2H, CH2), 2.55-2.52
(m, 1H, CH), 2.20-2.19 (m, 1H, CH), 1.39 (s, 9H, (CH3)3), 0.88 (s, 9H, (CH3)3); ESI-MS: m/z = 587.4
[M+H]+.
5.2.9 General procedure for the synthesis of deprotected C-caped dipeptides fragments 14
Trifluoroacetic acid (TFA, 2.5mL) was added to a solution of protected C-caped dipeptides fragments
13 (10.0 mmol) in CH2Cl2 (40.0 mL) at 0 oC. The mixture was then stirred for 1h at room temperature.
The volatiles were evaporated under reduced pressure and white solid was precipitated immediately after
the addition of ether, filtered and dried, allowing you to move on to the next step without further
purification.
5.2.10 benzyl (S)-3-(4-tert-butoxyphenyl)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-
ylamino)-1-oxopropan-2-ylcarbamate (16)
Compound 15 (6.0 mmol), HOBt (0.82 g, 6.0 mmol) and EDCI (2.25 g, 12.0 mmol) were added in the
round-bottom flask in turn and then mixed at 0 oC in CH2Cl2 (20.0 mL) and then stirred for 30 min. A
solution of chlorobenzylamine (6.0 mmol) and diisopropylethylamine (2.2mL, 12.0 mmol) were then
added sequentially at 0 oC. After stirred for 3 hours at room temperature, the solution was washed with
aqueous NaHCO3 solution (1 × 20 mL), brine (1 × 20 mL) and dried by Na2SO4. The organic phase was
then concentrated under reduced pressure and purified by flash chromatography (ethyl acetate: CH2Cl2 =
1:2-1:1).
White solid; Yield: 73%. 1H NMR (500MHz, CDCl3): δ = 8.36 (t, 1H, J = 12.0, 6.0 Hz, NH), 8.28 (d,
1H, J = 8.0 Hz, NH), 7.59 (d, 1H, J = 8.5 Hz, NH), 7.43-7.41 (m, 1H, Ar-H), 7.31-7.15 (m, 15H, Ar-H),
6.84 (d, 2H, J = 8.0 Hz, Ar-H), 4.97-4.91 (m, 2H, CH2), 4.33-4.29 (m, 4H, CH2 + CH + CH), 2.99-2.96
(m, 1H, CH), 2.75-2.71 (m, 1H, CH), 2.64-2.52 (m, 2H, CH2), 2.02-1.95 (m, 1H, CH), 1.92-1.84 (m, 1H,
CH), 1.24 (s, 9H, (CH3)3); ESI-MS: m/z = 656.3 [M+H]+.
5.2.11
(S)-2-((S)-2-amino-3-(4-tert-butoxyphenyl)propanamido)-N-(2-chlorobenzyl)-4-
phenylbutanamide (17)
Amino acid fragments 16 (5.0 mmol), 10% palladium on carbon (10 mol%) and methanol (10.0 mL)
were added in the round-bottom flask in turn and then mixed together and the reaction mixture was stirred
19

under an atmosphere of H2 for 2 h. Filtrated to remove Pd/C and the solvent was then evaporated under
reduced pressure to get compound 17. The crude product was used directly without further purification.
5.2.12 General methods for the preparation of compounds 18a-m
Corresponding fragment 7a, 7b and 7c (6.0 mmol) was dissolved in saturated aqueous NaHCO3 (10.0
o
mL) and CH2Cl2 (10.0 mL) at 0 C. Triphosgene (0.60 g, 2.0mmol) was added to the mixture in one
portion and then stirred at 0 oC for 20 min. The aqueous layer was extracted with CH2Cl2 (2 × 20 mL)
and the organic phase was combined and then dried by Na2SO4, evaporated under reduced pressure and
used straightly. Diisopropylethylamine (1.6 mL, 8.0 mmol) was added to a solution of corresponding
amine TFA salt (3a-i, 4.0 mmol). The previously obtained isocyanate was dissolved in CH2Cl2 (6.0 mL)
and added to the reaction mixture. After stirring at room temperature for 3 hours, the mixture was washed
with saturated NaHCO3, NH4Cl and brine, dried by Na2SO4. The organic phase was then concentrated
under reduced pressure and purified by flash chromatography (dichloromethane: methanol=5:1-3:1).
(S)-benzyl
5-(tert-butylamino)-5-oxo-2-(4-(pyridin-3-ylcarbamoyl)piperidine-1-
carboxamido)pentanoate(18a)
1
White solid; Yield: 52%. H NMR (500MHz, CDCl3): δ = 8.97 (s, 1H, NH), 8.43 (d, 1H, J = 0.5 Hz,
pyridine-H), 8.34 (dd, 1H, J = 4.5, 1.5 Hz, pyridine-H), 8.23 (d, 1H, J = 7.5 Hz, pyridine-H), 7.41-7.39
(m, 2H, Ar-H), 7.34-7.28 (m, 4H, Ar-H + pyridine-H), 5.33 (s, 2H, CH2), 4.81(t, 1H, J = 8.5, 4.5 Hz,
CH), 4.39 (s, 1H, NH), 3.88-3.82 (m, 2H, CH + CH), 3.45-3.41 (m, 2H, CH + CH), 2.59-2.54 (m, 1H,
CH), 2.51(t, 2H, J = 11.0, 5.5 Hz, CH2), 2.38-2.65 (m, 2H, CH + CH), 2.21-2.19 (m, 1H, CH), 1.89-1.79
(m, 2H, CH + CH), 1.35 (s, 9H, (CH3)3); ESI-MS: m/z = 524.1 [M+H]+.
(S)-benzyl
5-(tert-butylamino)-5-oxo-2-(4-(pyrazin-2-ylcarbamoyl)piperidine-1-
carboxamido)pentanoate(18b)
1
White solid; Yield: 71%. H NMR (500MHz, CDCl3): δ = 9.96 (s, 1H, NH), 8.91 (s, 1H, pyridine-H),
8.39 (d, 1H, J = 7.5 Hz, pyridine-H), 8.34 (d, 1H, J = 7.5 Hz, pyridine-H), 7.42-7.39 (m, 2H, Ar-H),
7.34-7.27 (m, 3H, Ar-H), 5.42 (s, 2H, CH2), 4.84(t, 1H, J = 8.5, 4.5 Hz, CH), 3.90-3.81 (m, 2H, CH +
CH), 3.44-3.40 (m, 2H, CH + CH), 2.60-2.58 (m, 1H, CH), 2.52(t, 2H, J = 11.0, 5.5 Hz, CH2), 2.36 (dd,
1H, J = 15.0, 5.0 Hz, CH), 2.31-2.25 (m, 1H, CH), 2.22 (dd, 1H, J = 15.0, 5.0 Hz, CH), 1.88-1.83 (m,
2H, CH + CH), 1.32 (s, 9H, (CH3)3); ESI-MS: m/z = 525.3 [M+H]+.
(S)-benzyl
4-(neopentylamino)-4-oxo-2-(4-(pyridin-3-ylcarbamoyl)piperidine-1-
carboxamido)butanoate(18c)
20

1
White solid; Yield: 62%. H NMR (500MHz, CDCl3): δ = 9.41 (s, 1H, NH), 8.40 (d, 1H, J = 1.5 Hz,
pyridine-H), 8.35 (dd, 1H, J = 4.5, 1.5 Hz, pyridine-H), 8.20-8.17 (m, 1H, pyridine-H), 7.35-7.29 (m,
6H, Ar-H + pyridine-H), 6.84 (s, 1H, NH), 5.72 (s, 1H, NH), 5.54(t, 1H, J = 12.5, 6.5 Hz, CH), 5.31 (s,
2H, CH2), 3.79 (s, 1H, CH), 3.70-3.66 (m, 2H, CH + CH), 3.55-3.50 (m, 2H, CH + CH), 2.87-2.80 (m,
2H, CH + CH), 2.56-2.49 (m, 2H, CH + CH), 2.32-2.26 (m, 2H, CH + CH), 1.90-1.85 (m, 2H, CH +
CH), 0.98 (s, 9H, (CH3)3); ESI-MS: m/z = 524.2 [M+H]+.
(S)-benzyl
4-(neopentylamino)-4-oxo-2-(4-(pyrazin-2-ylcarbamoyl)piperidine-1-
carboxamido)butanoate (18d)
White solid; Yield: 59%. 1H NMR (500MHz, CDCl3): δ = 9.67 (s, 1H, NH), 9.08 (s, 1H, pyridine-H),
8.41 (d, 1H, J = 5.5 Hz, pyridine-H), 8.34 (dd, 1H, J = 4.5, pyridine-H), 7.34-7.25 (m, 5H, Ar-H), 7.17
(s, 1H, NH), 5.80 (s, 1H, NH), 5.41(t, 1H, J = 7.5, 4.0 Hz, CH), 4.05 (s, 1H, CH), 3.71-3.66 (m, 2H, CH
+ CH), 3.35-3.31 (m, 2H, CH + CH), 3.06 (s, 1H, CH), 2.92-2.89 (m, 2H, CH + CH), 2.69-2.65 (m, 1H,
CH), 2.38-2.32 (m, 2H, CH + CH), 1.89-1.83 (m, 2H, CH + CH), 1.03 (s, 9H, (CH3)3); ESI-MS: m/z =
525.4[M+H]+.
(S)-benzyl
5-(neopentylamino)-5-oxo-2-(4-(pyridin-3-ylcarbamoyl)piperidine-1-
carboxamido)pentanoate(18e)
White solid; Yield: 69%. 1H NMR (500MHz, CDCl3): δ = 8.69 (s, 1H, pyridine-H), 8.40 (d, 1H, J = 10.0
Hz, pyridine-H), 7.36-7.27 (m, 5H, Ar-H), 6.30 (t, 1H, J = 15.0, 7.5 Hz, NH), 6.25 (d, 1H, J = 10.5 Hz,
NH), 5.21-5.08 (m, 2H, CH2), 4.44-4.40 (m, 1H, CH), 4.08-4.02 (m, 1H, CH), 3.09-2.96 (m, 2H, CH2),
2.88-2.79 (m, 2H, CH2), 2.50-2.48 (m, 1H, CH), 2.34-2.14 (m, 3H, CH2 + CH), 2.05-2.00 (m, 1H, CH),
1.91-1.88 (m, 2H, CH2), 1.80-1.74(m, 2H, CH2), 0.88 (s, 9H, (CH3)3); ESI-MS: m/z = 538.4 [M+H]+.
(S)-benzyl
5-(neopentylamino)-5-oxo-2-(4-(pyrazin-2-ylcarbamoyl)piperidine-1-
carboxamido)pentanoate(18f)
1
White solid; Yield: 55%. H NMR (500MHz, CDCl3): δ = 9.55 (s, 1H, NH), 8.36 (d, 1H, J = 3.5 Hz,
pyridine-H), 8.24 (s, 1H, pyridine-H), 8.06 (s, 1H, pyridine-H), 7.36-7.30 (m, 5H, Ar-H), 6.12 (d, 1H, J
= 8.0 Hz, NH), 6.02 (t, 1H, J = 15.0, 7.5 Hz, NH), 5.24-5.12 (m, 2H, CH2), 4.48-4.44 (m, 1H, CH), 4.11-
4.06 (m, 1H, CH), 3.11-2.99 (m, 2H, CH2), 2.93-2.87 (m, 2H, CH2), 2.56-2.50 (m, 1H, CH), 2.33-2.10
(m, 4H, CH2 + CH2), 2.04-1.96 (m, 4H, CH2 + CH2), 0.89 (s, 9H, (CH3)3); ESI-MS: m/z = 539.2 [M+H]+.
(S)-benzyl
5-(tert-butylamino)-2-(4-(2-fluorophenylcarbamoyl)piperidine-1-carboxamido)-5-
oxopentanoate(18g)
21

White solid; Yield: 68%. 1H NMR (500MHz, CDCl3): δ = 8.33 (t, 1H, J = 19.0, 9.5 Hz, NH), 7.36-7.34
(m, 8H, Ar-H), 7.15-7.10 (m, 1H, Ar-H), 7.07-7.04 (m, 1H, NH), 6.22 (d, 1H, J = 8.0 Hz, NH), 5.20-
5.11 (m, 2H, CH2), 4.46-4.40 (m, 1H, CH), 4.13-4.07 (m, 2H, CH2), 2.92-2.85 (m, 2H, CH2), 2.49-2.44
(m, 1H, CH), 2.23-2.10 (m, 4H, CH2 + CH2), 1.97-1.73 (m, 4H, CH2 + CH2), 1.32 (s, 9H, (CH3)3); ESI-
MS: m/z = 541.3 [M+H]+.
(S)-benzyl
5-(tert-butylamino)-2-(4-(3-fluorophenylcarbamoyl)piperidine-1-carboxamido)-5-
oxopentanoate(18h)
White solid; Yield: 71%. 1H NMR (500MHz, CDCl3): δ = 7.60 (s, 1H, NH), 7.53 (d, 1H, J = 14.0 Hz,
Ar-H), 7.34-7.30 (m, 7H, Ar-H), 7.22-7.16 (m, 1H, Ar-H), 6.82-6.78 (m, 1H, NH), 6.29 (d, 1H, J = 7.5
Hz, NH), 5.23-5.09 (m, 2H, CH2), 4.42-4.37 (m, 1H, CH), 4.15-4.08 (m, 2H, CH2), 2.89-2.82 (m, 2H,
CH2), 2.45-2.40 (m, 1H, CH), 2.23-2.08 (m, 4H, CH2 + CH2), 1.92-1.73 (m, 4H, CH2 + CH2), 1.31 (s,
9H, (CH3)3); ESI-MS: m/z = 541.3 [M+H]+.
(S)-benzyl
5-(tert-butylamino)-2-(4-(4-fluorophenylcarbamoyl)piperidine-1-carboxamido)-5-
oxopentanoate(18i)
White solid; Yield: 57%. 1H NMR (500MHz, CDCl3): δ = 8.46 (s, 1H, NH), 7.43-7.40 (m, 2H, Ar-H),
7.33-7.27 (m, 5H, Ar-H), 6.82-6.78 (d, 1H, J = 6.5 Hz, NH), 7.16 (t, 1H, J = 7.5, 15.5 Hz, Ar-H), 5.24-
5.09 (m, 2H, CH2), 4.35-4.33 (m, 1H, CH), 4.19-4.14 (m, 2H, CH2), 2.92-2.84 (m, 2H, CH2), 2.49-2.46
(m, 1H, CH), 2.23-2.12 (m, 4H, CH2 + CH2), 1.95-1.78 (m, 4H, CH2 + CH2), 1.32 (s, 9H, (CH3)3); ESI-
MS: m/z = 541.3 [M+H]+.
(S)-benzyl
5-(tert-butylamino)-2-(4-(4-cyanophenylcarbamoyl)piperidine-1-carboxamido)-5-
oxopentanoate(18j)
White solid; Yield: 71%. 1H NMR (500MHz, CDCl3): δ = 8.16 (s, 1H, NH), 7.73 (d, 2H, J = 10.5 Hz,
Ar-H), 7.59 (d, 2H, J = 11.0 Hz, Ar-H), 7.37-7.31 (m, 5H, Ar-H), 6.82-6.78 (d, 1H, J = 6.5 Hz, NH),
5.22-5.06 (m, 2H, CH2), 4.38-4.35 (m, 1H, CH), 4.13-4.08 (m, 2H, CH2), 2.89-2.81 (m, 2H, CH2), 2.47-
2.45 (m, 1H, CH), 2.25-2.12 (m, 4H, CH2 + CH2), 1.92-1.76 (m, 4H, CH2 + CH2), 1.31 (s, 9H, (CH3)3);
ESI-MS: m/z = 548.1 [M+H]+.
(S)-benzyl 5-(tert-butylamino)-2-(4-(4-methoxyphenylcarbamoyl)piperidine-1-carboxamido)-5-
oxopentanoate(18l)
White solid; Yield: 69%. 1H NMR (500MHz, CDCl3): δ = 7.43-7.41 (m, 2H, Ar-H), 7.36-7.31 (m, 5H,
Ar-H), 6.87-6.84 (d, 2H, J = 11.0 Hz, Ar-H), 6.22 (d, 1H, J = 8.0 Hz, NH), 5.58 (s, 1H, NH), 5.23-5.10
22

(m, 2H, CH2), 4.42-4.37 (m, 1H, CH), 4.15-4.06 (m, 2H, CH2), 2.90-2.82 (m, 2H, CH2), 2.42-2.37 (m,
1H, CH), 2.21-2.00 (m, 4H, CH2 + CH2), 1.94-1.75 (m, 4H, CH2 + CH2), 1.66 (s, 3H, CH3), 1.32 (s, 9H,
(CH3)3); ESI-MS: m/z = 553.1 [M+H]+.
(S)-benzyl
5-(tert-butylamino)-5-oxo-2-(4-(phenylcarbamoyl)piperidine-1-
carboxamido)pentanoate(18m)
White solid; Yield: 55%. 1H NMR (500MHz, CDCl3): δ = 7.53-7.51 (d, 2H, J = 9.5 Hz, Ar-H), 7.36-7.30
(m, 7H, Ar-H), 7.13 (t, 1H, J = 18.5, 9.5 Hz, Ar-H), 6.23 (d, 1H, J = 7.5 Hz, NH), 5.55 (s, 1H, NH), 5.24-
5.11 (m, 2H, CH2), 4.42-4.38 (m, 1H, CH), 4.13-4.06 (m, 2H, CH2), 2.91-2.83 (m, 2H, CH2), 2.45-2.39
(m, 1H, CH), 2.22-2.09 (m, 4H, CH2 + CH2), 1.95-1.78 (m, 4H, CH2 + CH2), 1.32 (s, 9H, (CH3)3); ESI-
MS: m/z = 523.3 [M+H]+.
5.2.13 General procedure for the synthesis of key intermediates 19a-m
The key intermediates 18a-m (5.0 mmol), 10% palladium on carbon (10 mol%) and methanol (10.0 mL)
were added in the round-bottom flask in turn and then mixed together and the reaction mixture was stirred
under an atmosphere of H2 for 2 h. Filtrated to remove Pd/C and the solvent was then evaporated under
reduced pressure to get compound 19a-m. The crude product was used straightly without further
purification.
5.2.14 General procedure for the synthesis of target compounds 20-23
Corresponding fragment 14 or 17 (6.0 mmol) was dissolved in saturated aqueous NaHCO3 (10.0 mL)and
CH2Cl2 (10.0 mL) at 0 oC. Triphosgene (0.60 g, 2.0mmol) was added to the mixture in one portion and
then stirred at 0 oC for 20 min. The aqueous layer was extracted with CH2Cl2 (2 × 20 mL) and the organic
phase was combined and then dried by Na2SO4, evaporated under reduced pressure and used straightly.
Diisopropylethylamine (1.6 mL, 8.0 mmol) was added to a solution of corresponding amine TFA salt
(3a or 3b, 4.0 mmol). The previously obtained isocyanate was dissolved in CH2Cl2 (6.0 mL) and added
to the reaction mixture. After stirring at room temperature for 3 hours, the mixture was washed with
saturated NaHCO3, NH4Cl and brine, dried by Na2SO4. The organic phase was then concentrated under
reduced pressure and purified by flash chromatography (dichloromethane: methanol=100:1-50:1).
5.2.15 General procedure for the synthesis of target compounds 24-39
Corresponding acids fragments (19a-m, 10.0 mmol), HOBt (1.48 g, 12.0 mmol) and EDCI (2.88 g, 15.0
o
mmol) were added in the round-bottom flask in turn and then mixed at 0 C in CH2Cl2 (40.0 mL) and
then stirred for 30 min. A solution of Ortho chlorbenzamide (10.0 mmol) and diisopropylethylamine
23

(3.8mL, 24.0 mmol) were then added successively at 0 oC. After stirred for 3 hours at room temperature,
the solution was washed with aqueous NaHCO3 (1 × 50 mL), brine (1 × 50 mL) and dried by Na2SO4.
The organic phase was then concentrated under reduced pressure and purified by flash chromatography
(dichloromethane: methanol=50:1-20:1).
N1-((S)-1-((S)-1-(2-chlorobenzylamino)-4-(neopentylamino)-1,4-dioxobutan-2-ylamino)-1-oxo-4-
phenylbutan-2-yl)-N4-(pyridin-3-yl)piperidine-1,4-dicarboxamide (20)
1
White solid; Yield: 37%; H NMR (500MHz, d6-DMSO): δ = 10.10 (s, 1H, NH), 8.76 (d, 1H, J = 2.5
Hz, pyrazine-H), 8.50 (d, 1H, J = 8 Hz, pyrazine-H), 8.36 (d, 1H, J = 6.0 Hz, pyrazine-H), 8.25-8.24 (m,
1H, NH), 8.07-8.04 (m, 1H, NH), 7.91 (d, 1H, J = 6.0 Hz, pyrazine-H), 7.40-7.39 (m, 1H, pyrazine-H),
7.35-7.27 (m, 4H, Ar-H), 7.22-7.19 (m, 5H, Ar-H), 6.86 (d, 1H, J = 6.0 Hz, NH), 4.53 (dd, 1H, J = 14.0,
6.0 Hz, CH), 4.39-4.34 (m, 1H, CH), 4.24-4.21 (m, 1H, CH), 4.04-3.99 (m, 2H, CH2), 3.95 (dd, 1H, J =
14.0, 7.0 Hz, CH), 2.94-2.83 (m, 2H, CH2), 2.80-2.70 (m, 4H, CH2 + CH2), 2.64-2.59 (m, 3H, CH2+ CH),
1.95-1.92 (m, 2H, CH2), 1.77-1.73 (m, 2H, CH2), 1.62-1.52 (m, 2H, CH2), 0.80 (s, 9H, (CH3) 3); 13C
NMR (100MHz, d6-DMSO): δ = 174.04, 173.44, 171.52, 170.66, 157,99, 144.47, 142.05, 141.27, 136.60,
136.42, 132.07, 129.28, 128.81, 128.72, 128.67, 127.40, 126.47, 126.25, 123.99, 55.77, 50.84, 50.10,
43.49, 43.47, 43.14, 36.64, 33.31, 32.35, 32.33, 28.47, 27.68; ESI-MS: m/z = 718.4 [M+H]+.
N1-((S)-1-((S)-1-(2-chlorobenzylamino)-4-(neopentylamino)-1,4-dioxobutan-2-ylamino)-1-oxo-4-
phenylbutan-2-yl)-N4-(pyrazin-2-yl)piperidine-1,4-dicarboxamide (21)
1
White solid; Yield: 41%; H NMR (500MHz, d6-DMSO): δ = 10.76 (s, 1H, NH), 9.36 (d, 1H, J = 1.5
Hz, NH), 8.52 (d, 1H, J = 9.0 Hz, pyrazine-H), 8.41-8.40 (m, 1H, NH), 8.38-8.35 (m, 2H, pyrazine-H+
pyrazine-H), 7.92 (t, 1H, NH), 7.40-7.38 (m, 1H, J = 6.5 Hz, NH), 7.40-7.38 (m, 1H, Ar-H), 7.31-7.28
(m, 3H, Ar-H), 7.25-7.17 (m, 5H, Ar-H), 6.86 (d, 1H, J = 5.5 Hz, Ar-H), 4.52 (dd, 1H, J = 13.5, 5.5 Hz,
CH), 4.39 (dd, 1H, J = 16.5, 6.5 Hz, CH), 4.24 (dd, 1H, J = 16.5, 6.5 Hz, CH), 4.04-3.99 (m, 2H, CH2),
3.94 (dd, 1H, J = 13.5, 6.5 Hz, CH), 2.95-2.83 (m, 2H, CH2), 2.79-2.76 (m, 1H, CH), 2.70-2.66 (m, 2H,
CH2 + CH2), 2.62-2.57 (m, 2H, CH2), 1.97-1.92 (m, 2H, CH2), 1.79-1.73 (m, 2H, CH2), 1.63-1.51 (m,
2H, CH2), 0.80 (s, 9H, (CH3) 3); 13C NMR (100MHz, d6-DMSO): δ = 174.61, 173.44, 171.52, 170.71,
157.99, 149.40, 143.02, 142.04, 140.02, 136.75, 136.60, 132.07, 129.27, 128.82, 128.72, 128.66, 127.39,
126.25, 55.80, 50.86, 50.09, 43.50, 43.45, 43.42, 42.58, 36.57, 33.27, 32.35, 28.35, 27.67; ESI-MS: m/z
= 719.4 [M+H]+.
N1-((S)-3-(4-tert-butoxyphenyl)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-
24

1-oxopropan-2-yl)-N4-(pyridin-3-yl)piperidine-1,4-dicarboxamide (22)
White solid; Yield: 45%; 1H NMR (500MHz, d6-DMSO): δ = 10.07 (s, 1H, NH), 8.72 (s, 1H, pyrazine-
H), 8.37 (t, 1H, J = 6.0 Hz, pyrazine-H), 8.22 (d, 1H, J = 4.5 Hz, pyrazine-H), 8.11 (d, 1H, J = 8.0 Hz,
pyrazine-H), 8.02 (d, 1H, J = 7.5 Hz, NH), 7.31-7.14 (m, 12H, Ar-H), 6.84 (d, 2H, J = 9.0 Hz, Ar-H +
NH), 6.68 (d, 1H, J = 9.0 Hz, NH), 4.27-4.22 (m, 4H, CH2 + CH2), 3.99-3.94 (m, 2H, CH2), 2.98-2.95
(m, 1H, CH), 2.86-2.81 (m, 1H, CH), 2.71-2.52 (m, 4H, CH2 + CH2), 1.99-1.98 (m, 1H, CH), 1.89-1.85
(m, 1H, CH), 1.70 (d, 1H, J = 11.5 Hz, CH), 1.49-1.28 (m, 4H, CH2 + CH2), 1.21 (s, 9H, (CH3) 3); 13
C
NMR (100MHz, d1-CDCl3): δ = 173.21,172.51, 171.32, 157.15, 157.11, 154.56, 144.76, 141.12, 140.83,
138.02, 135.25, 131.26, 131.22, 129.82, 129.61, 128.60, 128.56, 128.44, 127.67, 127.59, 127.38, 126.18,
124.55, 123.78, 78.72, 77.24, 56.52, 53.20, 43.43, 37.23, 33.53, 31.91, 29.71, 28.82, 28.07; ESI-MS: m/z
= 750.3 [M+H]+.
N1-((S)-3-(4-tert-butoxyphenyl)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-
1-oxopropan-2-yl)-N4-(pyrazin-2-yl)piperidine-1,4-dicarboxamide (23)
1
White solid; Yield: 37%; H NMR (500MHz, d6-DMSO): δ = 10.72 (s, 1H, NH), 9.33 (d, 1H, J = 1.5
Hz, NH), 8.30-8.38 (m, 1H, pyrazine-H), 8.37 (d, 2H, J = 3.5 Hz, pyrazine-H + pyrazine-H), 8.17 (d, 1H,
J = 9.5 Hz, NH), 7.44 (m, 1H, Ar-H), 7.30-7.17 (m, 8H, Ar-H), 6.85 (d, 2H, J = 10.5 Hz, Ar-H + NH),
6.69 (d, 1H, J = 10.0 Hz, NH), 4.35-4.25 (m, 4H, CH2 + CH2), 4.00-3.94 (m, 2H, CH2), 3.01-2.97 (m,
1H, CH), 2.89-2.83 (m, 1H, CH), 2.71-2.58 (m, 4H, CH + CH + CH2), 2.56-2.54 (m, 1H, CH), 2.07-1.86
(m, 2H, CH2), 1.72 (d, 1H, J = 13.0 Hz, CH), 1.50-1.32 (m, 3H, CH2 + CH), 1.22 (s, 9H, (CH3) 3); 13
C
NMR (100MHz, d6-DMSO): δ = 174.10, 172.73, 171.51, 157.16, 153.27, 148.87, 142.51, 141.35, 139.53,
136.23, 136.13, 133.22, 131.93, 129.86, 129.02, 128.61, 128.54, 128.30, 127.04, 125.78, 123.20, 77.48,
56.11, 52.38, 43.29, 43.04, 41.99, 36.07, 33.69, 31.26, 28.51, 27.58, 27.71; ESI-MS: m/z = 754.4 [M+H]+.
N1-((S)-5-(tert-butylamino)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-1,5-
dioxopentan-2-yl)-N4-(pyridin-3-yl)piperidine-1,4-dicarboxamide (24)
1
White solid; Yield: 35%; H NMR (500MHz, d6-DMSO): δ = 10.10 (s, 1H, NH), 8.72 (d, 1H, J = 2.5
Hz, pyrazine-H), 8.44 (m, 1H, J = 6.0 Hz, pyrazine-H), 8.36 (t, 1H, J = 4.8 Hz, pyrazine-H), 8.23-8.22
(dd, 1H, J = 4.0, 1.5 Hz, pyrazine-H), 8.04-8.01 (m, 1H, NH), 7.46 (s, 1H, NH), 7.41-7.40 (m, 1H, NH),
7.30-7.24 (m, 6H, Ar-H), 7.17-7.14 (m, 3H, Ar-H), 6.82 (d, 1H, J = 6.5 Hz, NH), 4.32 (d, 2H, J = 6.0
Hz, CH), 4.25-4.22 (m, 1H, CH), 4.01-3.98 (m, 3H, CH2 + CH), 2.77-2.64 (m, 2H, CH2), 2.62-2.51(m,
3H, CH2 + CH), 2.19-2.15 (m, 2H, CH2), 2.06-1.99 (m, 1H, CH2), 1.92-1.84 (m, 3H, CH2 + CH), 1.76-
25

1.73 (m, 2H, CH2), 1.54-1.43 (m, 2H, CH2), 1.22 (s, 9H, (CH3) 3); 13C NMR (100MHz, d6-DMSO): δ =
174.12, 173.49, 172.28, 172.13, 157.85, 144.51, 143.71, 141.84, 141.27, 140.23, 136.63, 136.37, 132.40,
129.48, 128.81, 128.77, 127.55, 126.51, 126.29, 124.02, 55.60, 52.71, 50.41, 43.54, 43.46, 43.10, 33.85,
33.40, 31.85, 28.99, 28.53, 27.64; ESI-MS: m/z = 718.4 [M+H]+.
N1-((S)-5-(tert-butylamino)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-1,5-
dioxopentan-2-yl)-N4-(pyrazin-2-yl)piperidine-1,4-dicarboxamide (25)
1
White solid; Yield: 31%; H NMR (500MHz, d6-DMSO): δ = 10.75 (s, 1H, NH), 9.31 (d, 1H, J = 1.0
Hz, NH), 8.43 (t, 1H, J = 6.0 Hz, pyrazine-H), 8.38 (t, 1H, J = 2.0 Hz, pyrazine-H), 8.33 (d, 1H, J = 2.5
Hz, pyrazine-H), 8.44 (d, 1H, J = 13.0 Hz, NH), 7.45 (s, 1H, NH), 7.41-7.39 (m, 1H, Ar-H), 7.29-7.24
(m, 5H, Ar-H), 7.17-7.14 (m, 3H, Ar-H), 6.81 (d, 1H, J = 6.5 Hz, NH), 4.32 (d, 2H, J = 5.5 Hz, CH2),
4.24-4.22 (m, 1H, CH), 4.01-3.97 (m, 3H, CH2 + CH), 2.69-2.66 (m, 3H, CH2 + CH), 2.62-2.52(m, 2H,
CH2), 2.19-2.15 (m, 2H, CH2), 2.06-1.99 (m, 1H, CH), 1.92-1.82 (m, 3H, CH2 + CH), 1.76-1.74 (m, 2H,
CH2), 1.53-1.42 (m, 2H, CH2), 1.22 (s, 9H, (CH3)3); 13C NMR (100MHz, d6-DMSO): δ = 174.68, 173.49,
172.28, 172.13, 157.86, 149.36, 143.02, 141.84,140.06, 136.75, 136.63, 132.40, 129.47, 129.12, 128.98,
128.81, 128.77, 127.54, 126.28, 55.61, 52.71, 50.41, 43.53, 43.46, 42.51, 33.84, 33.40, 31.85, 28.98,
28.40, 27.62; ESI-MS: m/z = 719.4 [M+H]+.
N1-((S)-1-((R)-3-(benzylthio)-1-(2-chlorobenzylamino)-1-oxopropan-2-ylamino)-4-
(neopentylamino)-1,4-dioxobutan-2-yl)-N4-(pyridin-3-yl)piperidine-1,4-dicarboxamide (26)
White solid; Yield: 46%; 1H NMR (500MHz, d6-DMSO): δ = 10.10 (s, 1H, NH), 8.72 (s, 1H, pyrazine-
H), 8.64 (m, 1H, NH), 8.23 (d, 1H, J = 4.0 Hz, pyrazine-H), 8.20 (d, 1H, J = 9.0 Hz, pyrazine-H), 8.03
(d, 1H, J = 11.5 Hz, NH), 7.76 (m, 1H, pyrazine-H), 7.41-7.22 (m, 10H, Ar-H + NH), 6.73 (d, 1H, J =
7.0 Hz, NH), 4.48-4.46 (m, 1H, CH), 4.44-4.40 (m, 1H, CH), 4.36 (d, 2H, J = 5.5 Hz, CH2), 3.99 (d, 2H,
J = 13.5 Hz, CH2), 3.74 (s, 2H, CH2), 2.84-2.79 (m, 2H, CH2), 2.75-2.64 (m, 4H, CH2 + CH2), 2.57-2.52
(m, 2H, CH2), 1.89 (s, 1H, CH), 1.75-1.72 (m, 2H, CH2), 1.50-1.45 (m, 2H, CH2), 0.78 (s, 9H, (CH3) 3);
13C NMR (100MHz, d6-DMSO): δ = 174.66, 172.87, 170.83, 170.46, 157.41, 149.37, 143.07, 140.09,
138.87, 136.73, 136.41, 132.35, 129.45, 129.43, 129.13, 129.01, 128.81, 127.53, 127.29, 53.16, 52.55,
50.17, 43.44, 42.47, 37.99, 35.68, 32.98, 32.34, 28.40, 28.33, 27.65; ESI-MS: m/z = 750.3 [M+H]+.
N1-((S)-1-((R)-3-(benzylthio)-1-(2-chlorobenzylamino)-1-oxopropan-2-ylamino)-4-
(neopentylamino)-1,4-dioxobutan-2-yl)-N4-(pyrazin-2-yl)piperidine-1,4-dicarboxamide (27)
1
White solid; Yield: 33%; H NMR (500MHz, d6-DMSO): δ = 10.75 (s, 1H, NH), 9.32 (d, 1H, J = 1.0
26

Hz, NH), 8.65 (t, 1H, J = 5.5 Hz, pyrazine-H), 8.38-8.37 (m, 1H, NH), 8.33 (d, 1H, J = 3.5 Hz, pyrazine-
H), 8.19 (d, 1H, J = 8.0 Hz, pyrazine-H), 8.65 (t, 1H, J = 6.0 Hz, NH), 7.42-7.20 (m, 9H, Ar-H), 6.73 (d,
1H, J = 7.5 Hz, NH), 4.50-4.40 (m, 2H, CH + CH), 4.34 (d, 2H, J = 6.0 Hz, CH2), 4.02-3.93 (m, 2H,
CH2), 3.74 (d, 2H, J = 1.5 Hz, CH2), 2.87-2.77 (m, 3H, CH2 + CH), 2.72-2.65 (m, 2H, CH2 + CH2), 2.62-
2.51 (m, 2H, CH2), 1.74-1.73 (m, 2H, CH2), 1.48-1.44 (m, 2H, CH2), 0.78 (s, 9H, (CH3) 3); 13C NMR
(100MHz, d6-DMSO): δ = 175.19, 173.47, 171.34, 171.05, 158.62, 154.23 148.11, 145.19, 141.04,
139.77, 136.65, 135.21, 131.39, 130.78, 129.69, 129.36, 129.12, 128.42, 128.05, 127.58, 55.47, 53.17,
50.86, 45.98, 42.75, 38.74, 34.22, 32.79, 31.51, 28.58, 28.13, 27.66; ESI-MS: m/z = 751.3 [M+H]+.
N1-((S)-1-((S)-1-(2-chlorobenzylamino)-4-(neopentylamino)-1,4-dioxobutan-2-ylamino)-4-
(neopentylamino)-1,4-dioxobutan-2-yl)-N4-(pyridin-3-yl)piperidine-1,4-dicarboxamide (28)
1
White solid; Yield: 45%; H NMR (500MHz, d6-DMSO): δ = 10.43 (s, 1H, NH), 8.54 (d, 1H, J = 7.5
Hz, pyrazine-H), 8.45 (m, 1H, NH), 8.30 (s, 1H, pyrazine-H), 8.10 (d, 1H, J = 10.0 Hz, pyrazine-H), 7.88
(s, 1H, NH), 7.81 (s, 1H, NH), 7.77 (t, 1H, J = 9.5 Hz, pyrazine-H), 7.41-7.22 (m, 4H, Ar-H), 7.09 (d,
1H, J = 5.5 Hz, NH), 6.81 (d, 1H, J = 6.0 Hz, NH), 4.53 (d, 1H, J = 7.5 Hz, CH), 4.38-4.23 (m, 3H, CH2
+ CH), 3.96 (d, 1H, J = 15.5 Hz, CH), 2.90-2.89 (m, 3H, CH2 + CH), 2.79-2.69 (m, 4H, CH2 + CH2),
2.66-2.58 (m, 4H, CH2 + CH2), 1.76 (t, 2H, J =15.5 Hz, CH2), 1.58-1.48 (m, 1H, CH2), 0.81 (s, 18H,
(CH3)6); 13C NMR (100MHz, d6-DMSO): δ = 173.87，172.06, 171.01,170.11, 170.02, 156.92, 152.14,
147.85, 137.98, 136.10, 131.57, 128.78, 128.26, 128.18, 126.91, 119.18, 113.43, 52.58, 50.41, 49.68,
49.60, 42.89, 42.14, 37.17, 36.23, 31.88, 31.80, 27.94, 27.90, 27.20, 27.12; ESI-MS: m/z = 741.5 [M+H]+.
N1-((S)-1-((S)-1-(2-chlorobenzylamino)-4-(neopentylamino)-1,4-dioxobutan-2-ylamino)-4-
(neopentylamino)-1,4-dioxobutan-2-yl)-N4-(pyrazin-2-yl)piperidine-1,4-dicarboxamide (29)
White solid; Yield: 38%; 1H NMR (500MHz, d6-DMSO): δ = 10.74 (s, 1H, NH), 9.35 (s, 1H, pyrazine-
H), 8.56 (d, 1H, J = 10.0 Hz, pyrazine-H), 8.44-8.39 (m, 2H, NH + NH), 8.35 (d, 1H, J = 3.0 Hz, pyrazine-
H), 7.89 (t, 1H, J = 7.5 Hz, NH), 7.83 (t, 1H, J = 7.5 Hz, NH), 7.41-7.24 (m, 4H, Ar-H),
6.83 (d, 1H, J = 8.0 Hz, NH), 4.54-4.49 (m, 1H, CH), 4.40-4.22 (m, 3H, CH2 + CH), 3.97 (d, 2H, J =16.5
Hz, CH2), 2.94-2.85 (m, 3H, CH2 + CH), 2.80-2.76 (m, 1H, CH), 2.72-2.66 (m, 4H, CH2 + CH2), 2.61-
2.55 (m, 3H, CH2 + CH), 1.79-1.73 (m, 2H, CH2), 1.61-1.48 (m, 2H, CH2), 0.81 (s, 18H, (CH3)6); 13C
NMR (100MHz, d6-DMSO): δ = 174.07, 172.06, 171.01, 171.15, 170.01, 156.90, 148.88, 142.53, 139.53,
136.21, 136.08, 131.57, 128.78, 128.26, 128.17, 126.90, 52.61, 50.41, 49.68, 49.59, 42.87, 42.81, 41.97,
37.13, 36.16, 31.87, 31.80, 27.81, 27.17, 27.11; ESI-MS: m/z = 742.7 [M+H]+.
27

N1-((S)-1-((S)-1-(2-chlorobenzylamino)-4-(neopentylamino)-1,4-dioxobutan-2-ylamino)-4-
(neopentylamino)-1,4-dioxobutan-2-yl)-N4-(pyridin-3-yl)piperidine-1,4-dicarboxamide (30)
White solid; Yield: 39%; 1H NMR (500MHz, d6-DMSO): δ = 10.45 (s, 1H, NH), 8.50 (t, 1H, J = 7.0 Hz,
NH), 8.31 (d, 1H, J = 6.0 Hz, pyrazine-H), 8.22 (d, 1H, J = 10.0 Hz, pyrazine-H), 8.09 (d, 1H, J = 10.5
Hz, pyrazine-H), 7.83-7.74 (m, 2H, NH + pyrazine-H), 7.43-7.41 (m, 1H, NH), 7.29-7.26 (m, 4H, Ar-
H), 7.10-7.07 (m, 1H, NH), 6.89 (d, 1H, J = 8.5 Hz, NH), 4.35-4.19 (m, 3H, CH2 + CH), 4.22-4.19 (m,
1H, CH), 4.02-3.94 (m, 2H, CH2), 2.84-2.56 (m, 7H, 3CH2 + CH), 2.10-1.75 (m, 6H, 3CH2), 1.49-1.44
(m, 2H, CH2), 1.23 (s, 9H, (CH3)3), 0.79 (s, 9H, (CH3)3); 13C NMR (100MHz, d6-DMSO): δ = 173.96,
171.59, 172.37, 171.20, 170.08, 156.97, 152.10, 147.87, 138.02, 136.17, 131.80, 128.92, 128.46, 128.41,
127.01, 119.22, 113.45, 52.41, 52.24, 49.82, 49.67, 43.08, 42.96, 42.12, 37.60, 32.54, 31.77, 28.46, 28.01,
27.62, 27.13; ESI-MS: m/z = 741.5 [M+H]+.
N1-((S)-1-((S)-5-(tert-butylamino)-1-(2-chlorobenzylamino)-1,5-dioxopentan-2-ylamino)-4-
(neopentylamino)-1,4-dioxobutan-2-yl)-N4-(pyrazin-2-yl)piperidine-1,4-dicarboxamide (31)
1
White solid; Yield: 34%; H NMR (500MHz, d6-DMSO): δ = 10.77 (s, 1H, NH), 9.34 (d, 1H, J = 1.5
Hz, NH), 8.49 (t, 1H, J = 8.0 Hz, pyrazine-H), 8.40-8.39 (m, 1H, NH), 8.35 (d, 1H, J = 3.5 Hz, pyrazine-
H), 8.22 (d, 1H, J = 10.0 Hz, pyrazine-H), 7.83-7.80 (m, 1H, NH), 7.43-7.41 (m, 1H, NH), 7.33-7.26 (m,
4H, Ar-H), 6.70 (d, 1H, J = 8.5 Hz, NH), 4.39-4.28 (m, 3H, CH2 + CH), 4.23-4.18 (m, 1H, CH), 4.03-
3.95 (m, 2H, CH2), 2.86-2.55 (m, 7H, 3CH2 + CH), 2.14-1.76 (m, 6H, 3CH2), 1.53-1.45 (m, 2H, CH2),
1.23 (s, 9H, (CH3)3), 0.79 (s, 9H, (CH3)3); 13C NMR (100MHz, d6-DMSO): δ = 174.15, 172.36, 171.59,
171.22, 170.08, 156.97, 148.84, 142.54, 139.57, 136.23, 136.15, 131.80, 128.92, 128.46, 128.42, 127.01,
52.42, 52.23, 49.84, 49.67, 43.02, 42.92, 41.93, 37.59, 32.54, 31.76, 28.45, 27.90, 27.82, 27.64, 27.12;
ESI-MS: m/z = 742.7 [M+H]+.
N1-((S)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-5-(neopentylamino)-1,5-
dioxopentan-2-yl)-N4-(pyridin-3-yl)piperidine-1,4-dicarboxamide (32)
1
White solid; Yield: 29%; H NMR (500MHz, d6-DMSO): δ = 10.12 (s, 1H, NH), 8.74 (d, 1H, J = 3.0
Hz, NH), 8.47 (t, 1H, J = 7.0 Hz, pyrazine-H), 8.25 (d, 1H, J = 4.5 Hz, pyrazine-H), 8.08 (t, 1H, J = 10.0
Hz, NH), 7.80 (t, 1H, J = 7.5 Hz, pyrazine-H), 7.43-7.41 (m, 1H, Ar-H), 7.32-7.26 (m, 6H, Ar-H + NH),
7.19-7.17 (m, 3H, Ar-H), 6.82 (d, 1H, J = 8.0 Hz, NH), 4.34 (d, 2H, J = 7.5 Hz, CH2), 4.29-4.24 (m, 1H,
CH), 4.08-4.03 (m, 3H, CH2 + CH), 2.93-2.83 (m, 2H, CH2), 2.79-2.63(m, 3H, CH2 + CH), 2.61-2.56 (m,
2H, CH2), 2.28-2.26 (m, 1H, CH2), 2.03-1.87 (m, 4H, CH2 + CH2), 1.78-1.76 (m, 2H, CH2), 1.57-1.44
28

(m, 2H, CH2), 0.82 (s, 9H, (CH3)3); 13C NMR (100MHz, d6-DMSO): δ = 173.61, 172.92, 172.27, 171.63,
157.33, 144.00, 141.33, 140.76, 136.11, 135.87, 131.89, 128.98, 128.60, 128.49, 128.31, 128.27, 127.05,
125.98, 125.79, 123.52, 54.99, 52.21, 49.63, 43.05, 42.98, 42.60, 33.37, 32.20, 31.88, 31.34, 28.01, 27.31,
27.22; ESI-MS: m/z = 732.4 [M+H]+.
N1-((S)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-5-(neopentylamino)-1,5-
dioxopentan-2-yl)-N4-(pyrazin-2-yl)piperidine-1,4-dicarboxamide (33)
White solid; Yield: 41%; 1H NMR (500MHz, d6-DMSO): δ = 10.77 (s, 1H, NH), 9.33 (s, 1H, NH), 8.46
(t, 1H, J = 7.5 Hz, pyrazine-H), 8.39 (s, 1H, pyrazine-H), 8.35 (d, 1H, J = 3.0 Hz, NH), 8.07 (d, 1H, J =
10.0 Hz, NH), 7.80 (t, 1H, J = 7.5 Hz, pyrazine-H), 7.43-7.41 (m, 1H, Ar-H), 7.29-7.25 (m, 5H, Ar-H),
7.19-7.17 (m, 3H, Ar-H), 6.82 (d, 1H, J = 8.5 Hz, NH), 4.34 (d, 2H, J = 7.0 Hz, CH2), 4.27-4.24 (m, 1H,
CH), 4.08-4.03 (m, 3H, CH2 + CH), 2.91-2.83 (m, 2H, CH2), 2.77-2.64(m, 3H, CH2 + CH), 2.62-2.56 (m,
2H, CH2), 2.27-2.26 (m, 4H, CH2), 2.03-1.87 (m, 4H, CH2 + CH2), 1.78-1.76 (m, 2H, CH2), 1.56-1.46
(m, 2H, CH2), 0.82 (s, 9H, (CH3)3); 13C NMR (100MHz, d6-DMSO): δ = 174.18, 172.93, 172.27, 171.63,
157.34, 148.86, 142.53, 141.33, 139.56, 136.24, 136.12, 131.89, 128.98, 128.60, 128.49, 128.31, 128.27,
127.05, 125.78, 55.00, 52.21, 49.62, 43.04, 42.98, 42.01, 33.36, 32.20, 31.88, 31.34, 27.89, 27.35, 27.22;
ESI-MS: m/z = 733.4 [M+H]+.
N1-((S)-5-(tert-butylamino)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-1,5-
dioxopentan-2-yl)-N4-(3-fluorophenyl)piperidine-1,4-dicarboxamide (34)
White solid; Yield: 40%; 1H NMR (500MHz, d6-DMSO): δ = 10.17 (s, 1H, NH), 8.52 (t, 1H, J = 6.5 Hz,
NH), 8.12 (d, 1H, J = 10.0 Hz, NH), 7.69 (d, 1H, J = 14.5 Hz, NH), 7.53-7.48 (m, 3H, Ar-H), 7.39-7.32
(m, 5H, Ar-H), 7.26-7.23 (m, 4H, Ar-H), 6.94-6.88 (m, 2H, Ar-H + NH), 4.41-3.99 (m, 4H, CH2 + CH2),
4.33-4.29 (m, 1H, CH), 4.10-4.07 (m, 3H, CH2 + CH), 3.32-3.23 (m, 1H, CH), 2.82-2.62 (m, 6H, 3CH2),
2.27-2.23 (m, 2H, CH2), 1.82-1.79 (m, 2H, CH2), 1.59-1.45 (m, 2H, CH2), 1.30 (s, 9H, (CH3) 3); 13C
NMR (100MHz, d6-DMSO): δ = 175.66, 173.92, 172.26, 172.12, 161.38, 157.83, 142.45, 141.83, 136.95,
136.62, 132.96, 132.38, 129.54, 129.10, 128.75, 127.55, 126.29, 126.14, 115.29, 115.27, 106.46, 106.20,
55.58, 54.89, 52.69, 50.40, 43.24, 37.46, 33.84, 33.39, 31.97, 31.83, 28.98, 28.51; ESI-MS: m/z = 735.3
[M+H]+.
N1-((S)-5-(tert-butylamino)-1-((S)-1-(2-chlorobenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-1,5-
dioxopentan-2-yl)-N4-(4-fluorophenyl)piperidine-1,4-dicarboxamide (35)
1
White solid; Yield: 31%; H NMR (500MHz, d6-DMSO): δ = 7.92 (s, 1H, NH), 7.70 (d, 1H, J = 14.0
29