Design, synthesis and biological evaluation of novel non-covalent piperidine-containing peptidyl proteasome inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.10.002

A series of novel non-covalent piperidine-containing dipeptidyl derivatives were designed, synthesized and evaluated as proteasome inhibitors. All target compounds were tested for their proteasome chymotrypsin-like inhibitory activities, and selected deri

Full text of DOI:10.1016/j.bmc.2016.10.002

Accepted Manuscript
Design, synthesis and biological evaluation of novel non-covalent piperidine-
containing peptidyl proteasome inhibitors
Jiankang Zhang, Lixin Gao, Jianjun Xi, Li Sheng, Yanmei Zhao, Lei Xu, Yidan
Shao, Shourong Liu, Rangxiao Zhuang, Yubo Zhou, Jia Li
PII:
S0968-0896(16)30896-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.002
BMC 13329
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 August 2016
1 October 2016
5 October 2016
Please cite this article as: Zhang, J., Gao, L., Xi, J., Sheng, L., Zhao, Y., Xu, L., Shao, Y., Liu, S., Zhuang, R., Zhou,
Y., Li, J., Design, synthesis and biological evaluation of novel non-covalent piperidine-containing peptidyl
proteasomeinhibitors, Bioorganic&MedicinalChemistry(2016), doi:http://dx.doi.org/10.1016/j.bmc.2016.10.002
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Design, synthesis and biological evaluation of novel non-covalent piperidine-containing peptidyl
proteasome inhibitors
Jiankang Zhanga, ┼, Lixin Gaob, ┼,Jianjun Xia, ┼, Li Shengb, Yanmei Zhaoa, Lei Xub, Yidan Shaoa,
Shourong Liua, Rangxiao Zhuanga*, Yubo Zhoub*, Jia Lib*
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
┼ These authors contributed equally to this work.
Abstract: A series of novel non-covalent piperidine-containing dipeptidyl derivatives were designed,
synthesized and evaluated as proteasome inhibitors. All target compounds were tested for their
proteasome chymotrypsin-like inhibitory activities, and selected derivatives were evaluated for the
anti-proliferation activities against two multiple myeloma (MM) cell lines RPMI 8226 and MM-1S.
Among all of these compounds, eight exhibited significant proteasome inhibitory activities with IC50
less than 20 nM, and four are more potent than the positive control Carfilzomib. Compound 28
displayed the most potent proteasome inhibitory activity (IC50: 1.4±0.1 nM) and cytotoxicities with
IC50 values at 13.9±1.8 nM and 9.5±0.5 nM against RPMI 8226 and MM-1S, respectively. Additionally,
the ex vivo blood cell proteasome inhibitory activities of compounds 24 and 27-29 demonstrated that
the enzymatic metabolism in the whole blood could be well tolerated. All these experiments confirmed
that the piperidine-containing non-covalent proteasome inhibitors are potential leads for exploring new
anti-cancer drugs.
Keywords: Proteasome inhibitors; Piperidine; Non-covalent; Anti-cancer; SARs
*Corresponding author:
Tel.: +86-21-5080-1552; Fax: +86-21-5080-0721 (Jia Li and Yubo Zhou);
Tel./Fax: +86-571-8648-1960 (Rangxiao Zhuang)
E-mail
address:
jli@simm.ac.cn
(Jia
Li);
ybzhou@simm.ac.cn
(Yubo
Zhou);
zhuangrangxiao@sina.com (Rangxiao Zhuang).
1

1. Introduction
The ubiquitin-proteasome pathway (UPP), which regulates various critical mediators of different
signaling pathways, is essential for maintaining normal cell function and cellular homeostasis.1-4 This
proteolytic pathway includes several components: ubiquitin, ubiquitin activating enzyme (E1),
ubiquitin conjugating enzyme (E2), ubiquitin protein ligase (E3), deubiquitinating enzyme (DUB) and
the dominant proteasome, most of which have been proved to be potential drug targets.5-8 The 26S
proteasome, a large protein complex with multiple proteolytic activities, is consisted of a 4 stacked
ring-formed 20S cylindrical core (α7-β7-β7-α7) and two 19S regulatory particles.9-10 Three β subunits
(β1, β2 and β5) exhibit caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (CT-L) activities,
respectively.10 Among all of these potential targets, the validated anti-cancer drug target proteasome has
played critical roles in discovery of multiple myeloma (MM) therapy drugs.11-12 To date, three
proteasome inhibitors Bortezomib, Carfilzomib and Ixazomib (Fig. 1) have been approved for the
treatment of MM.13-15 Besides, several other proteasome inhibitors are being extensively evaluated in
various clinical trials.10
Figure 1. Structures of the three approved proteasome inhibitors
Proteasome inhibitors can be classified into covalent and non-covalent types due to different structure
scaffolds and binding modes with proteasome.16 Most proteasome inhibitors approved or in clinical
trials are covalent ones. The firm covalent interactions ensure these small molecular ligands with potent
and lasting proteasome inhibitory activities, but may induce severe side effects and limit their tissue
distribution for lack of specificity and excessively reactive.17-19 Non-covalent proteasome inhibitors
may offer more therapeutic advantages due to their more widespread tissue distribution compared to
the covalent inhibitors.19-20 Although non-covalent proteasome inhibitors are less well studied, the
history of these analogues is as long as that of the covalent inhibitors. CVT-659 (Fig. 2) is the
forerunner of this kind of inhibitors with an IC50 of 140 nM.21 Additionally,
a
trimethoxy-L-phenylalanine-containing dipeptide (2, Fig. 2) was reported with potent and selective
chymotrypsin-like activity together with moderate cytotoxicity.22 Besides, Blackburn and colleagues
2

described a series of di- and tripeptides (e.g., 3 and 4, Fig. 2) with both potent constitutive proteasome
and immunoproteasome inhibitory activities.19
Figure 2. Structures of representative non-covalent proteasome inhibitors
Since most of the reported non-covalent proteasome inhibitors are short peptides, the enzymatic
stability of these compounds should be well concerned.23 Introducing a non-peptide fragment into the
peptide skeleton may solve this problem and increase the pharmacokinetic properties of the target
compounds. This is mainly owing to the specificity of proteases and peptidases against peptide bond
between peptide fragments. In this manuscript, a series of piperidine-containing non-covalent
proteasome inhibitors (Fig. 3) were synthesized and evaluated, and structure-activity relationships
(SARs) were discussed in detail.
Figure 3. Non-peptide fragment (piperidine) constructed non-covalent proteasome inhibitors
3

2. Results and discussion
2.1 Chemistry
The synthetic route for piperidine-containing fragments 9 and 10a-g are summarized in Scheme 1.
Fragment 7 can be easily obtained by condensation from pyrazine-2-carboxylic acid with
1-Boc-4-aminopiperidine. However, synthesis of the similar arylcarbamoyl piperidine derivatives 8a-g
were more difficult, in which the N-Boc-4-piperidinecarboxylic acid should first be transformed to the
acyl chloride at the presence of SOCl2, then reacted with corresponding arylamine 6a-g to afford the
products.24 Afterwards, deprotection of 7 and 8a-g with trifluoroacetic acid (TFA) resulted in piperidine
TFA salts 9 and 10a-g.
The target compounds 16-29 were synthesized following the method described in Scheme 2. Reaction
of (2-chlorophenyl)methanamine with Boc-L-hPhe furnished compound 12, which was deprotected,
treated with various Boc-protected amino acid and deprotected again to afford dipeptide TFA salts
15a-d. Subsequently, the primary amine 15a-d were first transformed to corresponding isocyanate
intermediates, which were not stable enough and were thereby reacted with piperidine fragments 9 and
10a-g to obtain target compounds 16-29.25
Scheme 1. Synthesis of piperidine-containing fragments (9 and 10a-g). Reagents and conditions: (I)
HOBt,
EDCI,
1-Boc-4-aminopiperidine,
diisopropylethylamine,
DCM,
0oC-rt;
(II)
N-Boc-4-piperidinecarboxylic acid, SOCl2, pyridine, Et3N, DMAP, DCM, rt; (III) trifluoroacetic acid,
DCM, 0oC-rt.
4

Scheme 2. Synthesis of piperidine-containing target compounds (16-29). Reagents and conditions: (I)
Boc-L-hPhe, HOBt, EDCI, diisopropylethylamine, DCM, 0oC-rt; (II) trifluoroacetic acid, DCM, 0oC-rt;
(III) corresponding Boc-protected amino acid, HOBt, EDCI, diisopropylethylamine, DCM, 0oC-rt; (IV)
triphosgene, diisopropylethylamine, DCM, aqueous NaHCO3, 0oC-rt.
5

2.2 Proteasome inhibitory activities
The synthesized target compounds were evaluated for their 20S proteasome chymotrypsin-like
inhibitory activities in vitro. Carfilzomib was employed as the positive control. The results are
summarized in Table 1.
As illustrated in Table 1, most target compounds showed potent proteasome inhibitory activities with
IC50 lower than 100 nM, and 4 compounds were even lower than 10 nM, which indicated that the
activities of these compounds were well maintained after introducing the piperidine ring into the
peptide skeleton. Different substituents at P2 position (R2) influenced the activity obviously. Iso-butyl
and phenylethyl substituted analogues (18, 19, 22 and 23) exhibited much more potent activities than
methyl and benzyl substituted compounds (16, 17, 20 and 21), with IC50 values of 16.6±1.7 nM,
45.7±1.1 nM, 14.7±2.4 nM and 23.7±3.5 nM, respectively. Phenylethyl showed the superiority and was
selected at the P2 position for further optimization. The linker between the piperidine ring and the
N-Cap (R1) is also essential for the inhibitory activity. Compounds with carbamoyl group (NHCO) at X
position were more potent than acylamino group (CONH) substituted compounds (16 versus 17; 18
versus 19; 20 versus 21; 22 versus 23). Additionally, the structure-activity relationship at the N-Cap (R1)
suggested that replacements at this position had little influence on their activities. Generally speaking,
replacements at R1 position displayed little impact for the proteasome inhibitory activities of this series
of compounds.
Table 1. 20S proteasome chymotrypsin-like inhibitory activities of target compounds (16-29)
O
R2
O
Cl
H
N
N
N
N
H
H
R1
O
X
Ph
16-29
Compound
R1
X
R2
IC50 (nM)a
601.4±129.0
669.9±94.9
16.6±1.7
pyrazin-2-yl
pyrazin-2-yl
pyrazin-2-yl
pyrazin-2-yl
pyrazin-2-yl
-NHCO-
-CONH-
-NHCO-
-CONH-
-NHCO-
methyl
methyl
iso-butyl
iso-butyl
benzyl
16
17
18
19
20
45.7±1.1
152.9±26.3
6

pyrazin-2-yl
pyrazin-2-yl
pyrazin-2-yl
phenyl
-CONH-
-NHCO-
-CONH-
-NHCO-
-NHCO-
-NHCO-
-NHCO-
-NHCO-
-NHCO-
-
benzyl
343.9±69.7
14.7±2.4
23.7±3.5
3.0±0.4
21
phenylethyl
phenylethyl
phenylethyl
phenylethyl
phenylethyl
phenylethyl
phenylethyl
phenylethyl
-
22
23
24
4-fluorophenyl
4-methoxyphenyl
pyridin-2-yl
pyridin-3-yl
thiazol-2-yl
-
16.9±2.0
15.7±1.3
2.4±1.0
25
26
27
1.4±0.1
28
4.3±0.9
29
Carfilzomib
8.4±0.9
a The IC50 values are an average of three independent determinations.
2.3 Tumor cell growth inhibitory activities
Based on the proteasome chymotrypsin-like inhibitory activities, selected compounds (18, 22 and
24-29) were further tested for their cytotoxic activities in vitro against two MM cell lines (RPMI 8226
and MM-1S) by MTS assay with Carfilzomib employed as the positive control. According to the
results are summarized in Table 2, 6 compounds exhibited potent anti-proliferation activities against
two MM cell lines with IC50 values in low nanomolar range, which was consistent with the proteasome
inhibitory activities. Among all of these compounds, 28 displayed the most potent cytotoxic activities
with IC50 of 13.9±1.8 nM and 9.5±0.5 nM against RPMI 8226 and MM-1S cell line, respectively.
Table 2. Cytotoxic activities of selected compounds (18, 22 and 24-29) against two MM cell lines
Compound
Cytotoxicity (IC50, nM) a
MM-1S
RPMI 8226
371.1±23.4
195.7±18.1
18.5±2.9
260.9±9.7
18
22
24
25
26
27
135.3±6.6
20.2±1.4
94.3±3.4
106.7±5.7
84.6±5.9
92.9±6.1
24.7±1.3
22.2±3.3
7

13.9±1.8
29.5±1.1
13.2±0.6
9.5±0.5
32.5±3.4
1.5±0.6
28
29
Carfilzomib
a The IC50 values are an average of three independent determinations.
2.4 Ex vivo blood cell proteasome inhibitory activities
Subsequently, four compounds (24 and 27-29) were further evaluated for the blood cell proteasome
inhibitory activities ex vivo, and Carfilzomib was used as the positive control. As illustrated in Fig. 4,
28 displayed the most potent blood cell proteasome inhibitory activities with inhibitory rates of more
than 60% at all the three concentrations (0.5 μM, 2.5μM and 12.5μM), which are comparable to that of
the Carfilzomib and is consistent with the in vitro proteasome inhibitory activity and cytotoxicities. The
impressive ex vivo activities suggested that the stability of this series of compounds were improved by
constructing a piperidine ring into the peptide skeleton, which was validated by the potent activities
after metabolism by various enzymes in the whole blood.
Figure 4. Ex vivo blood cell proteasome inhibitory activities of selected compounds (24 and 27-29)
2.5 Plasma and blood stability
The most potent compound 28 and its corresponding dipeptidyl analogue without piperidine ring
(3-pyridinyl-carbonyl-hPhe-hPhe-2-chlorobenzylamide) were further tested for the stability by
enzymatic degradation in mouse plasma and blood. As illustrated in Table 3, compound 28 showed a
8

longer half life both in plasma and blood (435.3 min and 178.7 min, respectively) compared to the
compound without a piperidine ring (285.3 min and 111.0 min, respectively), which demonstrated the
availability of piperidine ring in increasing the enzymatic stability of this series of compounds.
Table 3. Enzymatic stability of compound 28 and its corresponding dipeptidyl analogue without
piperidine ring
Half-life (T1/2, min)
Compound
plasma
435.3
blood
178.7
28
285.3
111.0
3. Conclusion
A series of novel piperidine-containing non-covalent dipeptidyl derivatives were synthesized and
evaluated for their proteasome inhibitory activities. Among these compounds, ten exhibited forceful
proteasome inhibitory activities together with anti-proliferation activities against two MM cell lines
RPMI 8226 and MM-1S, and these results are consistent with each other. In addition, compound 28
showed comparable ex vivo activity to the lead compound Carfilzomib with enhanced enzymatic
stability, which both demonstrated the validity of the design concept. Besides, the linker between
piperidine ring and the N-Cap group is also essential for the activity, which may be caused by different
hydrogen bond interaction with the proteasome. This study validated the potential of developing
non-peptide fragment constructed peptides as non-covalent proteasome inhibitors, which will be
helpful for searching new MM therapy drugs.
4. Experimental procedures
4.1 Chemistry
1H and 13C NMR spectra were recorded on Brüker 500/400MHz spectrometer (Brüker Bioscience,
Billerica, MA, USA) with CDCl3 or DMSO-d6 as solvent. Chemical shifts (δ) were reported in parts
per million (ppm) relative to internal TMS, and coupling constants (J) were reported in Hertz (Hz).
Splitting patterns were designated as singlet (s), broad singlet (brs), doublet (d), double doublet (dd),
triplet (t), quartet (q) and multiplet (m). Mass spectral data were obtained by Esquire-LC-00075
9

spectrometer (Brüker Bioscience). Reagents and solvents were purchased from common commercial
suppliers and were used without further purification unless stated otherwise. Column chromatography
was performed using silica gel (300-400 mesh). All yields are unoptimized and generally represent the
result of a single experiment.
4.1.1 tert-Butyl 4-(Pyrazine-2-carboxamido) piperidine-1-carboxylate (7)
To a suspension of pyrazine-2-carboxylic acid (5, 1.24g, 10.0mmol) in DCM (40.0mL), HOBt (1.49g,
11.0mmol) and EDCI (2.88g, 15.0mmol) were added at 0oC. The reaction mixture was kept at 0oC and
was stirred for 30min. Then 1-Boc-4-aminopiperidine (2.00g, 10.0mmol) and diisopropylethylamine
(3.7mL, 20.0mmol) were added. After stirring at room temperature for 3h, the resulting mixture was
washed with aqueous NaHCO3 solution (1×30mL), brine (1×30mL) and dried over Na2SO4. The
solvent was evaporated under reduced pressure and the crude product was purified by flash
chromatography (ethyl acetate: petroleum ether = 1:1).
1
White solid; Yield: 95%; H NMR (500MHz, CDCl3): δ = 9.40 (s, 1H, pyrazine-H), 8.75 (d, 1H, J =
2.0 Hz, pyrazine-H), 8.52 (d, 1H, J = 1.5 Hz, pyrazine-H), 7.72 (d, 1H, J = 8.0 Hz, NH), 4.19-4.03 (m,
3H, CH+CH2), 2.95 (t, 2H, J = 13.5 Hz, CH2), 2.00 (d, 2H, J = 12.5 Hz, CH2), 1.54-1.43 (m, 11H,
CH2+CH3); ESI-MS: m/z = 307.2 [M+H]+.
4.1.2 General procedure for the synthesis of protected piperidine fragments (8a-g)
To a mixture of N-Boc-4-piperidinecarboxylic acid (3.65g, 16.0mmol), pyridine (3.4mL, 40.0mmol)
and CH2Cl2 (30.0mL), SOCl2 (1.5mL, 19.0mmol) was added under N2 at room temperature. The
mixture was stirred for half an hour, and a solution of corresponding amine (6a-g, 18.0mmol), Et3N
(8.0mL, 56.0mmol) and a catalytic amount of DMAP in CH2Cl2 (30.0mL) was added dropwise. The
suspension was stirred overnight. The organic phase was washed with 1N HCl (2×30mL) and aqueous
NaHCO3 (2×30mL), dried over Na2SO4 (For compounds 8e and 8f, the solvent was evaporated and
washed with aqueous NaHCO3). The solvent was evaporated under reduced pressure and the crude
product was purified by flash column chromatography (ethyl acetate: petroleum ether = 1:2~3:1) to
give compounds 8a-g.
4.1.2.1 tert-Butyl 4-(Pyrazin-2-ylcarbamoyl) piperidine-1-carboxylate (8a)
1
White solid; Yield: 78%; H 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,
10

CH3); ESI-MS: m/z = 307.2 [M+H]+.
4.1.2.2 tert-Butyl 4-(Phenylcarbamoyl) piperidine-1-carboxylate (8b)
White solid; Yield: 86%; 1H NMR (500MHz, 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); ESI-MS:
m/z = 305.2 [M+H]+.
4.1.2.3 tert-Butyl 4-(4-Fluorophenylcarbamoyl) piperidine-1-carboxylate (8c)
1
White solid; Yield: 75%; H 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);
ESI-MS: m/z = 323.3 [M+H]+.
4.1.2.4 tert-Butyl 4-(4-Methoxyphenylcarbamoyl) piperidine-1-carboxylate (8d)
White solid; Yield: 85%; 1H NMR (500MHz, 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); ESI-MS: m/z = 335.2 [M+H]+.
4.1.2.5 tert-Butyl 4-(Pyridin-2-ylcarbamoyl) piperidine-1-carboxylate (8e)
1
White solid; Yield: 65%; H NMR (500MHz, CDCl3): δ = 8.27-8.22 (m, 3H, pyridine-H+NH),
7.75-7.71 (m, 1H, pyridine-H), 7.06 (dd, 1H, J = 6.5, 5.0 Hz, pyridine-H), 4.18 (brs, 2H, CH2),
2.83-2.70 (m, 2H, CH2), 2.45-2.38 (m, 1H, CH), 1.91 (d, 2H, J = 12.5 Hz, CH2), 1.78-1.69 (m, 2H,
CH2), 1.46 (s, 9H, CH3); ESI-MS: m/z = 306.2 [M+H]+.
4.1.2.6 tert-Butyl 4-(Pyridin-3-ylcarbamoyl) piperidine-1-carboxylate (8f)
White solid; Yield: 63%; 1H NMR (500MHz, CDCl3): δ = 8.61 (s, 1H, pyridine-H), 8.34 (d, 1H, J = 4.0
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); ESI-MS: m/z = 306.2 [M+H]+.
4.1.2.7 tert-Butyl 4-(Thiazol-2-ylcarbamoyl) piperidine-1-carboxylate (8g)
White solid; Yield: 87%; 1H NMR (500MHz, CDCl3): δ = 12.10 (s, 1H, NH), 7.42 (d, 1H, J = 4.0 Hz,
thiazole-H), 7.05 (d, 1H, J = 3.5 Hz, thiazole-H), 4.29-4.11 (m, 2H, CH2), 2.93-2.80 (m, 2H, CH2),
2.67-2.62 (m, 1H, CH), 1.91 (d, 2H, J = 11.5 Hz, CH2), 1.87-1.80 (m, 2H, CH2), 1.48 (s, 9H, CH3);
11

ESI-MS: m/z = 312.1 [M+H]+.
4.1.3 General procedure for the synthesis of piperidine contained fragments (9, 10a-g)
To a solution of protected piperidine fragments 7 or 8a-g (5.0mmol) in CH2Cl2 (20.0mL) was added
trifluoroacetic acid (TFA, 5.0mL) at 0 oC. The reaction mixture was then warmed to room temperature
and stirred for 1h. The volatiles were evaporated under reduced pressure and ether (30.0mL) was added.
White solid was precipitated, filtrated and dried, which was put into next step without further
purification.
4.1.4 tert-Butyl-hPhe-2-chlorobenzylamide (12)
To a suspension of Boc-L-hPhe (1.40g, 5.0mmol) in DCM (20.0mL), HOBt (0.74g, 5.5mmol) and
EDCI (1.44g, 7.5mmol) were added at 0oC. The reaction mixture was stirred for 30min. Then
(2-chlorophenyl)methanamine (11, 0.71g, 5.0mmol) and diisopropylethylamine (1.9mL, 10.0mmol)
were added. After stirring at room temperature for 2h, the resulting mixture was washed with aqueous
NaHCO3 solution (1×30mL), brine (1×30mL) and dried over Na2SO4. The solvent was evaporated
under reduced pressure and the crude product was purified by flash chromatography (ethyl acetate:
petroleum ether = 1:4).
White solid; Yield: 89%; 1H NMR (500MHz, CDCl3): δ = 7.37-7.34 (m, 2H, Ar-H), 7.28-7.25 (m, 2H,
Ar-H), 7.24-7.17 (m, 3H, Ar-H), 7.13 (d, 2H, J = 7.0 Hz, Ar-H), 6.52 (t, 1H, J = 5.0 Hz, NH), 5.02 (d,
1H, J = 6.5 Hz, NH), 4.53 (d, 2H, J = 6.0 Hz, CH2), 4.10-4.08 (m, 1H, CH), 2.70-2.60 (m, 2H, CH2),
2.21-2.14 (m, 1H, CH2), 1.96-1.88 (m, 1H, CH2), 1.43 (s, 9H, CH3); ESI-MS: m/z = 403.2 [M+H]+.
4.1.5 hPhe-2-chlorobenzylamide TFA salt (13)
o
To a solution of compound 12 (5.0mmol) in CH2Cl2 (20.0mL) was added TFA (5.0mL) at 0 C. The
reaction mixture was then warmed to room temperature and stirred for 1h. The volatiles were
evaporated under reduced pressure and ether (30.0mL) was added. White solid was precipitated,
filtrated and dried, which was put into next step without further purification.
4.1.6 General procedure for the synthesis of protected dipeptide fragments (14a-d)
To a solution of corresponding amino-protected amino acid (5.0mmol) in DCM (20.0mL), HOBt
(0.74g, 5.5mmol) and EDCI (1.44g, 7.5mmol) were added at 0oC. The reaction mixture was stirred for
30min. Then compound 13 (1.87g, 4.5mmol) and diisopropylethylamine (1.9mL, 10.0mmol) were
added. After stirring at room temperature for 3h, the resulting mixture was washed with aqueous
NaHCO3 solution (1×30mL), brine (1×30mL) and dried over Na2SO4. The solvent was evaporated
12

under reduced pressure and the crude product was purified by flash chromatography (ethyl acetate:
petroleum ether = 1:3~2:1).
4.1.6.1 tert-Butyl-Ala-hPhe-2-chlorobenzylamide (14a)
White solid; Yield: 92%; 1H NMR (500MHz, CDCl3): δ = 7.36-7.27 (m, 4H, Ar-H), 7.24-7.13 (m, 5H,
Ar-H), 6.75 (brs, 1H, NH), 6.54 (brs, 1H, NH), 4.75 (brs, 1H, NH), 4.53-4.45 (m, 3H, CH2+CH), 4.07
(brs, 1H, CH), 2.67 (m, 2H, CH2), 2.26-2.25 (m, 1H, CH2), 2.03 (m, 1H, CH2), 1.40 (s, 9H, CH3), 1.30
(d, 3H, J = 6.5 Hz, CH3); ESI-MS: m/z = 474.3 [M+H]+.
4.1.6.2 tert-Butyl-Leu-hPhe-2-chlorobenzylamide (14b)
White solid; Yield: 71%; 1H NMR (500MHz, CDCl3): δ = 7.35-7.27 (m, 2H, Ar-H), 7.24-7.12 (m, 7H,
Ar-H), 6.81 (brs, 1H, NH), 6.57 (brs, 1H, NH), 4.72 (brs, 1H, NH), 4.55-4.45 (m, 3H, CH2+CH), 4.03
(brs, 1H, CH), 2.65 (m, 2H, CH2), 2.24-2.22 (m, 1H, CH2), 2.02 (m, 1H, CH2), 1.60 (m, 2H, CH2),
1.43-1.41 (m, 1H, CH), 1.39 (s, 9H, CH3), 0.91 (t, 6H, J = 8.0 Hz, CH3); ESI-MS: m/z = 516.3
[M+H]+.
4.1.6.3 tert-Butyl- Phe-hPhe-2-chlorobenzylamide (14c)
White solid; Yield: 88%; 1H NMR (500MHz, CDCl3): δ = 7.35-7.33 (m, 1H, Ar-H), 7.30-7.27 (m, 3H,
Ar-H), 7.24-7.13 (m, 8H, Ar-H), 7.10-7.08 (d, 2H, J = 7.0 Hz, Ar-H), 6.61 (brs, 1H, NH), 6.36 (brs, 1H,
NH), 4.80 (brs, 1H, NH), 4.54-4.40 (m, 3H, CH+CH2), 4.30-4.27 (m, 1H, CH), 3.04 (dd, 1H, J = 13.5,
6.0 Hz, CH2), 3.02 (dd, 1H, J = 13.5, 6.0 Hz, CH2), 2.63-2.56 (m, 2H, CH2), 2.20-2.11 (m, 1H, CH2),
1.97-1.92 (m, 1H, CH2), 1.36 (s, 9H, CH3); ESI-MS: m/z = 550.3 [M+H]+.
4.1.6.4 tert-Butyl-hPhe-hPhe-2-chlorobenzylamide (14d)
White solid; Yield: 87%; 1H NMR (500MHz, CDCl3): δ = 7.33-7.26 (m, 4H, Ar-H), 7.25-7.11 (m, 10H,
Ar-H), 6.72 (brs, 1H, NH), 6.54 (brs, 1H, NH), 4.85 (brs, 1H, NH), 4.55-4.41 (m, 3H, CH+CH2), 4.01
(brs, 1H, CH), 2.67-2.61 (m, 4H, CH2), 2.24-2.18 (m, 1H, CH2), 2.11-1.96 (m, 2H, CH2), 1.87-1.79 (m,
1H, CH2), 1.41 (s, 9H, CH3); ESI-MS: m/z = 564.3 [M+H]+.
4.1.7 General procedure for the synthesis of deprotected dipeptide fragments (15a-d)
To a solution of corresponding dipeptide fragment 14a-d (3.0mmol) in CH2Cl2 (10.0mL) was added
o
TFA (3.0mL) at 0 C. The reaction mixture was then warmed to room temperature and stirred for 1h.
The volatiles were evaporated under reduced pressure and ether (20.0mL) was added. White solid was
precipitated, filtrated and dried, which was put into next step without further purification.
4.1.8 General procedure for the synthesis of target compounds 16-29
13

Corresponding dipeptide fragment 15a-d (3.0mmol) was dissolved in CH2Cl2 (5.0mL) and saturated
o
aqueous NaHCO3 (5.0mL) and was cooled to 0 C. Triphosgene (0.30g, 1.0mmol) was added in one
portion and the mixture was stirred at the same temperature for 15min. The organic layer was separated
and the aqueous layer was extracted with CH2Cl2 (2×10mL). Then the organic phase was combined,
dried over Na2SO4, evaporated in vacuo and used directly. To a solution of corresponding piperidine
contained TFA salt (9, 10a-g, 2.0mmol) was added diisopropylethylamine (0.8mL, 4.0mmol). Then the
former obtained isocyanate was dissolved in CH2Cl2 (3.0mL) and added into the reaction mixture. After
3h’s stirring, the mixture was washed with saturated aqueous NaHCO3, NH4Cl and brine. The organic
phase was dried over Na2SO4, evaporated to obtain the crude product. Purification was accomplished
by column chromatography (dichloromethane: methanol=50:1~20:1). Analysis of sample purity was
performed on an Agilent 1200 HPLC system using an Agilent ZORBAX SB-Aq, 5 μM C18 column
(250 mm × 4.6 mm). HPLC condition: linear density gradient elution with eluents MeOH/H2O =
75/25-95/5 (10 min), flow rate: 1.0 mL/min.
4.1.8.1 4-(Pyrazin-2-ylcarbamoyl) piperidine-1-carbonyl-Ala-hPhe-2-chlorobenzylamide (16)
White solid; Yield: 36%; mp: 133-135 oC; Purity: 98.7%; 1H NMR (500MHz, d6-DMSO): δ = 10.76 (s,
1H, NH), 9.34 (d, 1H, J = 1.5 Hz, pyrazine-H), 8.41-8.38 (m, 2H, NH+pyrazine-H), 8.35 (d, 1H, J =
2.5 Hz, pyrazine-H), 8.00 (d, 1H, J = 8.0 Hz, NH), 7.44-7.42 (m, 1H, Ar-H), 7.33-7.27 (m, 5H, Ar-H),
7.19-7.16 (m, 3H, Ar-H), 6.65 (d, 1H, J = 6.5 Hz, NH), 4.34 (d, 2H, J = 6.0 Hz, CH2), 4.27-4.23 (m, 1H,
CH), 4.15 (m, 1H, CH), 4.04 (d, 2H, J = 12.5 Hz, CH2), 2.75-2.53 (m, 5H, CH+CH2), 2.08-1.99 (m, 1H,
CH), 1.95-1.87 (m, 1H, CH), 1.77 (d, 2H, J = 5.0 Hz, CH2), 1.56-1.45 (m, 2H, CH2), 1.28 (d, 3H, J =
7.0 Hz, CH3); 13C NMR (100MHz, d6-DMSO): δ = 174.71, 174.27, 172.13, 157.71, 149.36, 143.03,
141.86, 140.06, 136.75, 136.66, 132.43, 129.50, 129.16, 129.03, 128.81, 128.78, 127.54, 126.28, 52.74,
50.85, 43.52, 43.48, 42.54, 33.95, 31.85, 28.44, 28.39, 18.14; ESI-MS: m/z = 606.0 [M+H]+.
4.1.8.2 4-(Pyrazine-2-carboxamido) piperidine-1-carbonyl-Ala-hPhe-2-chlorobenzylamide (17)
White solid; Yield: 29%; mp: 154-156 oC; Purity: 99.1%; 1H NMR (500MHz, d6-DMSO): δ = 9.18 (d,
1H, J = 1.5 Hz, pyrazine-H), 8.86 (d, 1H, J = 2.5 Hz, pyrazine-H), 8.75 (d, 1H, J = 8.5 Hz, NH), 8.70
(dd, 1H, J = 2.5, 1.0 Hz , pyrazine-H), 8.43 (t, 1H, J = 12.0 Hz, NH), 7.96 (d, 1H, J = 7.5 Hz, NH),
7.44-7.42 (m, 1H, Ar-H), 7.33-7.27 (m, 5H, Ar-H), 7.19-7.16 (m, 3H, Ar-H), 6.69 (d, 1H, J = 6.5 Hz,
NH), 4.36 (d, 2H, J = 6.0 Hz, CH2), 4.29-4.25 (m, 1H, CH), 4.16-4.10 (m, 1H, CH2), 4.05-3.97 (m, 3H,
J = 12.0 Hz, CH2), 2.83-2.74 (m, 2H, CH2), 2.64-2.54 (m, 2H, CH2), 2.08-2.00 (m, 1H, CH),
14

1.95-1.87 (m, 1H, CH), 1.76-1.70 (m, 2H, CH2), 1.63-1.46 (m, 2H, CH), 1.29 (d, 3H, J = 7.0 Hz, CH3);
13C NMR (100MHz, d6-DMSO):δ = 174.23, 172.46, 172.11, 162.62, 157.64, 147.87, 145.39, 144.06,
143.68, 141.88, 136.62, 132.46, 129.52, 129.22, 129.06, 128.79, 127.56, 126.29, 52.70, 50.98, 47.12,
43.30, 43.20, 34.11, 31.84, 31.48, 18.18; ESI-MS: m/z = 606.0 [M+H]+.
4.1.8.3 4-(Pyrazin-2-ylcarbamoyl) piperidine-1-carbonyl-Leu-hPhe-2-chlorobenzylamide (18)
White solid; Yield: 26%; mp: 116-118 oC; Purity: 98.5%; 1H NMR (500MHz, d6-DMSO): δ = 10.77 (s,
1H, NH), 9.34 (d, 1H, J = 1.0 Hz, pyrazine-H), 8.40-8.35 (m, 3H, pyrazine-H+NH), 8.01 (d, 1H, J =
7.5 Hz, NH), 7.44-7.42 (m, 1H, Ar-H), 7.33-7.27 (m, 5H, Ar-H), 7.19-7.16 (m, 3H, Ar-H), 6.58 (d, 1H,
J = 7.5 Hz, NH), 4.34 (d, 2H, J = 6.0 Hz, CH2), 4.27-4.23 (m, 1H, CH), 4.18-4.13 (m, 1H, CH),
4.06-4.04 (m, 2H, CH2), 2.75-2.58 (m, 5H, CH+CH2), 2.07-1.85 (m, 2H, CH2), 1.76 (d, 2H, J = 12.0
Hz, CH2), 1.71-1.64 (m, 1H, CH), 1.64-1.44 (m, 4H, CH2), 0.89 (dd, 6H, J = 19.0, 6.5 Hz, CH3); 13C
NMR (100MHz, d6-DMSO): δ = 174.71, 174.05, 172.06, 157.87, 149.36 143.03, 141.84, 140.06,
136.76, 136.63, 132.47, 129.52, 129.22, 129.07, 128.78, 127.52, 126.29, 53.63, 52.74, 43.67, 43.61,
42.57, 33.96, 31.79, 28.44, 28.39, 24.81, 23.53, 22.14; ESI-MS: m/z = 648.1 [M+H]+.
4.1.8.4 4-(Pyrazine-2-carboxamido) piperidine-1-carbonyl-Leu-hPhe-2-chlorobenzylamide (19)
White solid; Yield: 38%; mp: 141-143 oC; Purity: 98.8%; 1H NMR (500MHz, d6-DMSO): δ = 9.18 (d,
1H, J = 1.0 Hz, pyrazine-H), 8.86 (d, 1H, J = 1.0 Hz, pyrazine-H), 8.79 (d, 1H, J = 8.0 Hz, NH), 8.69 (s,
1H, pyrazine-H), 8.43 (t, 1H, J = 5.5 Hz, NH), 8.00 (d, 1H, J = 7.5 Hz, NH), 7.43-7.37 (m, 1H, Ar-H),
7.31-7.27 (m, 5H, Ar-H), 7.19-7.16 (m, 3H, Ar-H), 6.63 (d, 1H, J = 7.0 Hz, NH), 4.35 (d, 2H, J = 5.5
Hz, CH2), 4.28-4.21 (m, 1H, CH), 4.15-4.11 (m, 1H, CH2), 4.05-3.94 (m, 3H, CH2), 2.82-2.73 (m, 2H,
CH2), 2.64-2.53 (m, 2H, CH2), 2.06-1.97 (m, 1H, CH), 1.95-1.87 (m, 1H, CH), 1.73-1.68 (m, 3H,
CH+CH2), 1.63-1.58 (m, 2H, CH2), 1.50-1.44 (m, 2H, CH2), 0.80 (dd, 6H, J = 18.5, 6.0 Hz, CH3); 13
C
NMR (100MHz, d6-DMSO): δ = 174.03, 172.05, 162.62, 157.80, 147.88, 145.41, 144.06, 143.67,
141.86, 136.59, 132.49, 129.54, 129.27, 129.10, 128.80, 128.76, 127.55, 126.31, 53.79, 52.70, 47.16,
43.43, 43.29, 34.12, 31.78, 31.50, 24.80, 23.53, 22.15; m/z = 648.1 [M+H]+.
4.1.8.5 4-(Pyrazin-2-ylcarbamoyl) piperidine-1-carbonyl-Phe-hPhe-2-chlorobenzylamide (20)
White solid; Yield: 42%; mp: 110-112 oC; Purity: 99.3%; 1H NMR (500MHz, d6-DMSO): δ = 10.74 (s,
1H, NH), 9.33 (d, 1H, J = 1.5 Hz, pyrazine-H), 8.40-8.39 (m, 1H, pyrazine-H), 8.36-8.33 (m, 2H,
NH+pyrazine-H), 8.19 (d, 1H, J = 3.0 Hz, NH), 7.45-7.43 (m, 1H, Ar-H), 7.33-7.25 (m, 9H, Ar-H),
7.20-7.17 (m, 4H, Ar-H), 6.71 (d, 1H, J = 8.0 Hz, NH), 4.39-4.34 (m, 3H, CH2+CH), 4.29-4.23 (m, 1H,
15

CH), 4.01-3.96 (m, 2H, CH2), 3.06-3.02 (dd, 1H, J = 14.0, 4.5 Hz, CH2), 2.94-2.89 (dd, 1H, J = 13.5,
9.5 Hz, CH2), 2.70-2.51 (m, 5H, CH+CH2), 2.07-1.99 (m, 1H, CH2), 1.95-1.85 (m, 1H, CH2), 1.74-1.71
(m, 2H, CH2), 1.48-1.34 (m, 2H, CH2); 13C NMR (100MHz, d6-DMSO): δ = 174.66, 173.22, 172.03,
157.64, 149.35, 143.03, 141.84, 140.05, 139.19, 136.75, 136.63, 129.72, 129.53, 129.15, 129.06,
128.81, 128.78, 128.45, 127.55, 126.55, 126.29, 56.56, 52.91, 43.59, 43.50, 42.51, 37.25, 34.16, 31.76,
28.33; ESI-MS: m/z = 682.1 [M+H]+.
4.1.8.6 4-(Pyrazine-2-carboxamido) piperidine-1-carbonyl-Phe-hPhe-2-chlorobenzylamide (21)
White solid; Yield: 30%; mp: 105-107 oC; Purity: 97.9%; 1H NMR (500MHz, d6-DMSO): δ = 9.18 (d,
1H, J = 1.0 Hz, pyrazine-H), 8.87 (d, 1H, J = 2.5 Hz, pyrazine-H), 8.70 (m, 2H, J = 2.5 Hz,
NH+pyrazine-H), 8.36 (t, 1H, J = 6.0 Hz, NH), 8.18(d, 1H, J = 7.5 Hz, NH), 7.45-7.43 (m, 1H, Ar-H),
7.33-7.26 (m, 8H, Ar-H), 7.20-7.17 (m, 3H, Ar-H), 6.75 (d, 1H, J = 8.0 Hz, NH), 4.37-4.33 (m, 2H,
CH2), 4.30-4.26 (m, 1H, CH), 3.98-3.92 (m, 2H, CH2), 3.04 (dd, 1H, J = 14.0, 4.5 Hz, CH2), 2.92 (dd,
1H, J = 13.5, 10.0 Hz, CH2), 2.77-2.68 (m, 2H, CH2), 2.65-2.55 (m, 2H, CH2), 2.07-1.99 (m, 1H, CH),
1.96-1.89 (m, 1H, CH), 1.69 (brs, 2H, CH2), 1.56-1.48 (m, 1H, CH2), 1.41-1.24 (m, 3H, CH2); 13C
NMR (100MHz, d6-DMSO):δ= 173.20, 172.02, 162.61, 157.61, 147.89, 145.38, 144.05, 143.69,
141.85, 139.19, 136.60, 132.46, 129.72, 129.54, 129.19, 129.08, 128.80, 128.48, 127.56, 126.56,
126.30, 56.68, 52.91, 47.10, 43.28, 43.18, 37.30, 34.24, 31.76, 31.44; ESI-MS: m/z = 682.1 [M+H]+.
4.1.8.7 4-(Pyrazin-2-ylcarbamoyl) piperidine-1-carbonyl-hPhe-hPhe-2-chlorobenzylamide (22)
White solid; Yield: 43%; mp: 178-180 oC; Purity: 98.4%; 1H NMR (500MHz, d6-DMSO): δ = 10.77 (s,
1H, NH), 9.34 (s, 1H, pyrazine-H), 8.43-8.39 (m, 2H, pyrazine-H+NH), 8.35 (d, 1H, J = 2.5 Hz,
pyrazine-H), 8.07 (d, 1H, J = 7.5 Hz, NH), 7.41 (d, 1H, J = 7.0 Hz, Ar-H), 7.33-7.16 (m, 13H, Ar-H),
6.68 (d, 1H, J = 7.5 Hz, NH), 4.36-4.27 (m, 3H, CH+CH2), 4.16-4.03 (m, 3H, CH+CH2), 2.78-2.55 (m,
7H, CH+CH2), 2.05-1.94 (m, 4H, CH2), 1.80 (d, 2H, J = 11.5 Hz, CH2), 1.59-1.46 (m, 2H, CH2); 13C
NMR (100MHz, d6-DMSO): δ = 174.72, 173.55, 172.45, 172.10, 157.89, 149.37, 143.02, 142.21,
141.83, 140.06, 136.77, 136.63, 132.44, 129.49, 129.17, 129.00, 128.79, 128.74, 127.50, 126.29,
126.22, 55.03, 52.81, 43.68, 43.63, 33.98, 33.83, 32.40, 31.85, 28.45, 21.51; ESI-MS: m/z = 696.1
[M+H]+.
4.1.8.8 4-(Pyrazine-2-carboxamido) piperidine-1-carbonyl-hPhe-hPhe-2-chlorobenzylamide (23)
White solid; Yield: 39%; mp: 104-105 oC; Purity: 99.1%; 1H NMR (500MHz, d6-DMSO): δ = 9.19 (d,
1H, J = 1.5 Hz, pyrazine-H), 8.86 (d, 1H, J = 2.5 Hz, pyrazine-H), 8.77 (d, 1H, J = 8.5 Hz, NH), 8.69
16

(d, 1H, J = 2.5 Hz, pyrazine-H), 8.44 (t, 1H, J = 6.0 Hz, NH), 8.03 (d, 1H, J = 7.0 Hz, NH), 7.41-7.39
(m, 1H, Ar-H), 7.32-7.24 (m, 7H, Ar-H), 7.22-7.17 (m, 6H, Ar-H), 6.71 (d, 1H, J = 7.0 Hz, NH),
4.35-4.28 (m, 3H, CH+CH2), 4.15-4.09 (m, 1H, CH), 4.07-4.02 (m, 3H, CH2+CH), 2.86-2.54 (m, 6H,
CH2), 2.05-1.90 (m, 4H, CH2), 1.75 (d, 2H, J = 11.5 Hz, CH2), 1.63-1.51 (m, 2H, CH2); 13C NMR
(100MHz, d6-DMSO): δ = 173.53, 172.09, 162.63, 157.81, 147.87, 145.41, 144.07, 143.67, 142.21,
141.84, 136.59, 132.47, 129.51, 129.22, 129.04, 128.80, 127.52, 126.31, 126.23, 55.18, 52.78, 47.18,
43.44, 43.33, 34.14, 33.89, 32.44, 31.84, 31.53, 21.51; ESI-MS: m/z = 696.1 [M+H]+.
4.1.8.9 4-(Phenylcarbamoyl) piperidine-1-carbonyl-hPhe-hPhe-2-chlorobenzylamide (24)
White solid; Yield: 58%; mp: 154-156 oC; Purity: 98.6%; 1H NMR (500MHz, d6-DMSO): δ = 9.92 (s,
1H, NH), 8.44 (t, 1H, J = 6.0 Hz, NH), 8.10 (d, 1H, J = 7.5 Hz, NH), 7.61 (d, 2H, J = 8.0 Hz, Ar-H),
7.42-7.39 (m, 1H, Ar-H), 7.32-7.18 (m, 15H, Ar-H), 7.03 (t, 1H, J = 7.5 Hz, Ar-H), 6.70 (d, 1H, J = 7.5
Hz, NH), 4.32 (d, 2H, J = 6.0 Hz, CH2), 4.29-4.23 (m, 1H, CH), 4.16-4.04 (m, 3H, CH+CH2),
2.78-2.57 (m, 7H, CH+CH2), 2.03-1.90 (m, 4H, CH2), 1.76 (d, 2H, J = 11.0 Hz, CH2), 1.59-1.45 (m,
2H, CH2); 13C NMR (100MHz, d6-DMSO): δ = 173.59, 173.56, 172.10, 157.90, 142.22, 141.82, 139.79,
136.62, 132.44, 129.50, 129.17, 129.10, 129.01, 128.79, 128.75, 127.51, 126.30, 126.23, 123.50,
119.61, 55.03, 52.81, 43.73, 43.69, 43.27, 33.97, 33.85, 32.40, 31.85, 28.67; ESI-MS: m/z = 684.1
[M+H]+.
4.1.8.10 4-(4-Fluorophenylcarbamoyl) piperidine-1-carbonyl-hPhe-hPhe-2-chlorobenzylamide
(25)
White solid; Yield: 49%; mp: 148-150 oC; Purity: 98.6%; 1H NMR (500MHz, CDCl3): δ = 7.74 (m, 2H,
NH+NH), 7.29-7.27 (m, 2H, Ar-H), 7.25-7.13 (m, 10H, Ar-H), 7.10-7.02 (m, 6H, Ar-H), 6.28 (d, 1H, J
= 8.0 Hz, NH), 4.98 (d, 1H, J = 5.0 Hz, NH), 4.52-4.47 (m, 2H, CH2+CH), 4.42 (dd, 1H, J = 15.0, 6.0
Hz, CH2), 4.32-4.29 (m, 1H, CH), 4.12-4.06 (m, 1H, CH), 3.80 (d, 1H, J = 13.5 Hz, CH2), 3.47 (d, 1H,
J = 13.5 Hz, CH2), 2.79-2.60 (m, 6H, CH2), 2.24-2.11 (m, 2H, CH2), 2.04-1.98 (m, 2H, CH2), 1.91-1.84
(m, 2H, CH2), 1.45-1.37 (m, 2H, CH2); 13C NMR (100MHz, d6-DMSO): δ = 173.54, 172.10, 165.49,
165.01, 163.03, 157.84, 142.22, 141.83, 136.59, 132.45, 131.59, 131.56, 130.42, 130.33, 129.56,
129.51, 129.18, 129.09, 129.04, 128.78, 127.52, 126.32, 126.24, 115.63, 115.42, 55.09, 52.82, 47.35,
43.45, 43.29, 34.04, 33.95, 32.43, 31.18, 31.79; ESI-MS: m/z = 712.1 [M+H]+.
4.1.8.11 4-(4-Methoxyphenylcarbamoyl) piperidine-1-carbonyl-hPhe-hPhe-2-chlorobenzylamide
(26)
17

o
1
White solid; Yield: 45%; mp: 168-169 C; Purity: 98.2%; H NMR (500MHz, CDCl3): δ = 7.42-7.28
(m, 8H, NH+Ar-H), 7.26-7.12 (m, 10H, NH+Ar-H), 7.01-6.97 (brs, 1H, NH), 6.84 (d, 2H, J = 8.5 Hz,
Ar-H), 4.51-4.47 (m, 3H, CH+CH2), 4.25 (brs, 1H, CH), 3.78 (s, 3H, CH3), 3.65-3.58 (m, 2H, CH2),
2.75-2.64 (m, 6H, CH2), 2.37-2.34 (m, 1H, CH), 2.28-2.21 (m, 1H, CH2), 2.19-2.12 (m, 1H, CH2),
2.07-1.96 (m, 2H, CH2), 1.90-1.80 (m, 2H, CH2), 1.73-1.65 (m, 2H, CH2); 13C NMR (100MHz,
d6-DMSO): δ = 173.57, 173.07, 172.11, 157.90, 155.55, 142.22, 141.83, 136.63, 132.96, 132.45,
129.50, 129.17, 129.01, 128.79, 128.75, 127.50, 126.30, 126.23, 121.17, 114.24, 55.62, 55.04, 52.82,
43.76, 43.70, 43.16, 33.97, 33.85, 32.41, 31.85, 28.71; ESI-MS: m/z = 724.1 [M+H]+.
4.1.8.12 4-(Pyridin-2-ylcarbamoyl) piperidine-1-carbonyl-hPhe-hPhe-2-chlorobenzylamide (27)
o
1
White solid; Yield: 39%; mp: 166-167 C; Purity: 98.1%; H NMR (500MHz, CDCl3): δ = 8.73 (brs,
1H, NH), 8.26-8.23 (m, 2H, pyridine-H), 7.78 (t, 1H, J = 8.5 Hz, NH), 7.34-7.28 (m, 4H,
Ar-H+pyridine-H), 7.25-7.03 (m, 12H, Ar-H+pyridine-H), 6.76 (d, 1H, J = 8.0 Hz, NH), 4.80 (d, 1H, J
= 6.0 Hz, NH), 4.55-4.49 (m, 3H, CH+CH2), 4.23 (m, 1H, CH), 3.72 (d, 1H, J = 13.0 Hz, CH2), 3.60 (d,
1H, J = 13.0 Hz, CH2), 2.80-2.60 (m, 6H, CH2), 2.46-2.39 (m, 1H, CH), 2.33-2.22 (m, 1H, CH2),
2.17-2.12 (m, 1H, CH2), 2.01-1.95 (m, 2H, CH2), 1.87-1.78 (m, 2H, CH2), 1.72-1.60 (m, 2H, CH2); 13
C
NMR (100MHz, d6-DMSO): δ = 174.53, 173.57, 172.11, 157.89, 152.60, 148.33, 142.21, 141.82,
138.55, 136.62, 132.44, 129.49, 129.17, 129.01, 128.79, 128.75, 127.50, 126.30, 126.23, 119.73,
114.00, 55.03, 52.81, 43.73, 43.67, 42.76, 33.96, 33.83, 32.40, 31.85, 28.55, 28.52; ESI-MS: m/z =
695.3 [M+H]+.
4.1.8.13 4-(Pyridin-3-ylcarbamoyl) piperidine-1-carbonyl-hPhe-hPhe-2-chlorobenzylamide (28)
White solid; Yield: 52%; mp: 141-143 oC; Purity: 98.5%; 1H NMR (500MHz, d6-DMSO): δ = 10.17 (s,
1H, NH), 8.77 (d, 1H, J = 2.5 Hz, pyridine-H), 8.44 (t, 1H, J = 6.0 Hz, NH), 8.26 (dd, 1H, J = 4.5, 1.5
Hz, pyridine-H), 8.10-8.06 (m, 2H, NH+pyridine-H), 7.40 (dd, 1H, J = 9.5, 2.0 Hz, pyridine-H),
7.37-7.17 (m, 14H, Ar-H, pyridine-H), 6.69 (d, 1H, J = 6.5 Hz, NH), 4.33 (d, 2H, J = 6.0 Hz, CH2),
4.30-4.26 (m, 1H, CH), 4.17-4.04 (m, 3H, CH+CH2), 2.80-2.68 (m, 3H, CH+CH2), 2.66-2.53 (m, 4H,
CH2), 2.06-1.88 (m, 4H, CH2), 1.78 (d, 2H, J = 11.0 Hz, CH2), 1.57-1.49 (m, 2H, CH2); 13C NMR
(100MHz, d6-DMSO): δ = 174.17, 173.55, 172.10, 157.89, 142.21, 141.82, 141.19, 136.62, 136.40,
132.43, 129.50, 129.16, 129.11, 129.02, 128.80, 128.76, 127.51, 126.61, 126.31, 126.24, 124.08, 55.01,
52.80, 43.67, 43.62, 43.16, 33.97, 33.85, 32.40, 31.84, 28.55; ESI-MS: m/z = 695.3 [M+H]+.
4.1.8.14 4-(Thiazol-2-ylcarbamoyl) piperidine-1-carbonyl-hPhe-hPhe-2-chlorobenzylamide (29)
18

White solid; Yield: 32%; mp: 144-145 oC; Purity: 98.9%; 1H NMR (500MHz, d6-DMSO): δ = 12.1 (s,
1H, NH), 8.43 (t, 1H, J = 6.0 Hz, NH), 8.09 (d, 1H, J = 8.0 Hz, NH), 7.47 (d, 1H, J = 3.5 Hz,
thiazole-H), 7.42-7.40 (m, 1H, Ar-H), 7.32-7.17 (m, 14H, thiazole-H+Ar-H), 6.69 (d, 1H, J = 7.5 Hz,
NH), 4.33 (d, 2H, J = 6.0 Hz, CH2), 4.30-4.24 (m, 1H, CH), 4.17-4.02 (m, 3H, CH+CH2), 2.79-2.54 (m,
7H, CH+CH2), 1.99-1.92 (m, 4H, CH2), 1.80 (d, 2H, J = 10.5 Hz, CH2), 1.59-1.46 (m, 2H, CH2); 13C
NMR (100MHz, d6-DMSO): δ = 173.54, 173.47, 172.10, 158.47, 157.88, 142.21, 141.82, 138.02,
136.62, 132.44, 129.49, 129.18, 129.01, 128.79, 128.75,127.50, 126.30, 126.23, 113.83, 55.03, 52.81,
43.61, 43.58, 41.84, 33.96, 33.84, 32.40, 31.84, 28.28; ESI-MS: m/z = 701.2 [M+H]+.
4.1.8.15 3-Pyridinyl-carbonyl-hPhe-hPhe-2-chlorobenzylamide
White solid; Yield: 76%; mp: 138-140 oC; Purity: 98.5%; 1H NMR (500MHz, d6-DMSO): δ = 9.08 (d,
1H, J = 2.0 Hz, pyridine-H), 8.87 (d, 1H, J = 9.0 Hz, NH), 8.74 (dd, 1H, J = 6.0, 1.5 Hz, pyridine-H),
8.40 (t, 1H, J = 7.5 Hz, NH), 8.30-8.23 (m, 2H, NH+pyridine-H), 7.55-7.52 (m, 1H, Ar-H), 7.41 (dd,
1H, J = 9.5, 2.5 Hz, pyridine-H), 7.33-7.17 (m, 13H, Ar-H), 4.58-4.52 (m, 1H, CH), 4.37-4.32 (m, 3H,
CH+CH2), 2.78-2.55 (m, 4H, CH2), 2.13-1.89 (m, 4H, CH2); 13C NMR (100MHz, d6-DMSO): δ =
172.08, 171.99, 165.88, 152.45, 149.18, 141.95, 141.78, 136.59, 135.77, 132.48, 130.16, 129.53,
129.18, 129.05, 128.79, 127.50, 126.32, 123.85, 54.07, 52.96, 40.56, 34.06, 33.82, 32.38, 31.89;
ESI-MS: m/z = 569.3 [M+H]+.
4.2 Biological evaluation
4.2.1 In vitro 20S proteasome chymotrypsin-like inhibition assay
Chymotrypsin-like enzyme activity assay was carried out in 50 μL volume and all the assay
components were diluted in Tris-HCl buffer (100 mM Tris-HCl, pH 8.0). The reaction was carried out
in black 384-well plates. 1 μL compound was added into 10 μL purified human proteasome (25 μg/mL),
a gift from Dr. Jiang-ping Wu (Notre-Dame Hospital, Montreal, Quebec, Canada), incubated for 15 min,
and then added with 39 μL synthesized substrate Suc-Leu-Leu-Val-Tyr-
Ltd., Shanghai, P.R. China) as the reference reported. And the AMC of probe was detected by
monitoring the increase of fluorescence with Envision, at 355 nm excitation and 460 nm Emission. The
IC50 data was calculated using the software GraphPad Prism, and chosen the equation “sigmoidal
dose-response (variable slope)” for curve fitting.
4.2.2 Tumor cell anti-proliferation assay
19

3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
was purchased from Promega (Madison, WI, USA). Human multiple myeloma (MM) cell lines (RPMI
8226 and MM-1S) were purchased from Invitrogen (Grand Island, NY, USA), and were grown in
RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and penicillin-Streptomycin from
Invitrogen (Grand Island, NY, USA) at 37oC in a 5% CO2 humidified atmosphere.
A 100 μL MM-1S (2×104/well) or RPMI 8226 cells (0.5×104/well) were seeded into 96-well plates.
After treated with tested compounds for 72h, cells were added with MTS at a final concentration of 0.5
mg/mL for 2 to 4 hours. Optical density was determined at 490 nm (background subtraction at 690 nm)
by SpectraMax 340 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The growth
inhibitory ratio was calculated as follows: Growth inhibitory ratio = (Acontrol–Asample)/ Acontrol. IC50
values were derived from a nonlinear regression model (curvefit) based on sigmoidal dose response
curve (variable slope) and computed using Graghpad Prism version 5.02 (Graphpad Software).
4.2.3 Ex vivo blood cell proteasome inhibition assay
Whole blood was collected from normal mice and compounds at appropriate concentrations were
added (the volume ratio of the compound to the serum was 1:50). After incubation for 40 min, the
above samples are centrifuged at 1,000 rpm for 5 min and the supernatants are removed. Blood cells
were added 100 μL EDTA (5 mM, pH=8.0) and lysed at 4ºC for 60 minutes, then centrifuged at 6,600
×g for 10 min and the supernatants were collected. Then the protein concentration of the supernatants
was measured with Bradford method and determined the activity of the proteasome. The assays were
carried out in a final volume of 50 μL containing 20 mmol/L HEPES, pH=8.0, 0.5 mmol/L EDTA, 0.05%
SDS, 50 μM substrate (Suc-Leu-Leu-Val-Tyr-AMC), 1% DMSO, and blood cell protein 100 μg, and
detected at 355 nm excitation and 460 nm Emission as the in vitro assay mentioned.
4.2.4 Enzymatic stability assay
Plasma or blood was prepared on sodium heparin. Stock solutions were prepared at 10 mM in DMSO
for the test compounds. Aliquots of the stock solutions were diluted to 0.02 mM in 0.05 M sodium
phosphate buffer containing 0.5% BSA as the dosing solution. Then 10 µL of the dosing solutions were
dosed into 90 µL of pre-warmed plasma or blood (37°C) in duplicates (n=2) in 96-well assay plates to
reach a final test concentration of 2 µM. The plates were kept in a 37°C water bath for the duration of
the experiment. At each time point, 400 µL of acetonitrile was added into corresponding wells of the
assay plates. After the final time point was quenched, the assay plates were ultrasonicated for 2 min,
20

shaken at the vibrator (IKA, MTS 2/4) for 10 min (600 rpm/min) and then centrifuged at 5594g for 15
min (Thermo Multifuge × 3R). Aliquots of the supernatant were taken, diluted 1:1 into distilled water,
and analyzed by LC-MS/MS. The peak area response ratio to internal standard (PARR) of the
compounds at different time point was compared to the PARR at time 0 to determine the percent of test
compound remaining. Half-lives (T1/2) were calculated using Excel software, fitting to a single-phase
exponential decay equation.
Conflict of interest
The authors confirm that this article content has no conflict of interest.
Acknowledgements
The authors thank the project supported by the Key Development Program of the Hangzhou Science
and Technology Committee (20152013A03), the normal project supported by the Hangzhou Science
and Technology Committee (20150733Q49) and National Natural Science Foundation of China
(81473244) for financial support.
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Figure captions:
Figure 1. Structures of the three approved proteasome inhibitors
Figure 2. Structures of representative non-covalent proteasome inhibitors
22

Figure 3. Non-peptide fragment (piperidine) constructed non-covalent proteasome inhibitors
Figure 4. Ex vivo blood cell proteasome inhibitory activities of selected compounds (24 and 27-29)
Scheme 1. Synthesis of piperidine-containing fragments (9 and 10a-g). Reagents and conditions: (I)
HOBt,
EDCI,
1-Boc-4-aminopiperidine,
diisopropylethylamine,
DCM,
0oC-rt;
(II)
N-Boc-4-piperidinecarboxylic acid, SOCl2, pyridine, Et3N, DMAP, DCM, rt; (III) trifluoroacetic acid,
DCM, 0oC-rt.
Scheme 2. Synthesis of piperidine-containing target compounds (16-29). Reagents and conditions: (I)
Boc-L-hPhe, HOBt, EDCI, diisopropylethylamine, DCM, 0oC-rt; (II) trifluoroacetic acid, DCM, 0oC-rt;
(III) corresponding Boc-protected amino acid, HOBt, EDCI, diisopropylethylamine, DCM, 0oC-rt; (IV)
triphosgene, diisopropylethylamine, DCM, aqueous NaHCO3, 0oC-rt.
23

Graphical abstract
24

Table 3. Enzymatic stability of compound 28 and its corresponding dipeptidyl analogue without
piperidine ring
Half-life (T1/2, min)
Compound
plasma
435.3
blood
178.7
28
285.3
111.0
25