Exploration of novel piperazine or piperidine constructed non-covalent peptidyl derivatives as proteasome inhibitors

European Journal of Medicinal Chemistry 126 (2017) 1056e1070

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Exploration of novel piperazine or piperidine constructed

non-covalent peptidyl derivatives as proteasome inhibitors

,

Rangxiao Zhuang ^{a 1}, Lixin Gao ^{b 1}, Xiaoqing Lv ^c, Jianjun Xi ^a, Li Sheng ^b, Yanmei Zhao ^a,

Ruoyu He ^a, Xiaobei Hu ^b, Yidan Shao ^a, Xuwang Pan ^a, Shourong Liu ^a, Weiwei Huang ^a,

Yubo Zhou ^b^***, Jia Li ^{b **}, Jiankang Zhang ^a

,

, *

^aDepartment of Pharmaceutical Preparation, Hangzhou Xixi Hospital, Hangzhou 310023, Zhejiang Province, China

^bNational Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai

201203, China

^cCollege of Medicine, Jiaxing University, Jiaxing 314001, Zhejiang Province, China

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of novel piperazine or piperidine-containing non-covalent peptidyl derivatives possessing a

neopentyl-asparagine residue were designed, synthesized and evaluated as proteasome inhibitors. All

target compounds were screened for their 20S proteasome chymotrypsin-like inhibitory activities, and

15 ones displayed more potent activities than carﬁlzomib with IC₅₀values lower than 10 nM. Subse-

quently, the most potent 10 analogues were tested for their cytotoxic activities against two multiple

myeloma (MM) cell lines RPMI-8226 and MM-1S. Based on these experiments, selected derivatives were

further evaluated for their ex vivo and in vivo blood cell proteasome inhibitory activities. The most po-

Received 8 November 2016

Received in revised form

15 December 2016

Accepted 16 December 2016

Available online 18 December 2016

Keywords:

tential compound 35 (proteasome inhibition IC₅₀: 1.2

0.1 nM) with potent anti-proliferation (IC₅₀:

Proteasome inhibitors

Piperazine or piperidine

Non-covalent

Peptidyl derivatives

Anti-cancer

RPMI-8226 8.4 0.8 nM; MM-1S: 6.3 0.8 nM), ex vivo and in vivo activities also had a prolonged half

life in plasma, which demonstrated that the enzymatic stabilities of this series of compounds have been

improved by constructing a six-membered ring into the peptide skeleton. All the experiments conﬁrmed

the correctness of design concept, which made this series of compounds potential leads for exploring

new anti-MM drugs.

1. Introduction

[4e8]. All of these components have been extensively studied and

the prominent proteasome has been considered and validated as

The ubiquitin-proteasome pathway (UPP) mediated protein

degradation was discovered about 40 years ago, and three scientists

Aaron Ciechanover, Avram Hershko and Irwin Rose were awarded

the Nobel Prize for Chemistry in 2004 for this important ﬁnding

[1e3]. Several components such as ubiquitin, ubiquitination related

enzymes (E1, E2 and E3), deubiquitinating enzyme (DUB) and

proteasome were included in this crucial protein degradation

pathway, which helped misfolded or damaged proteins to be

recognized, translocated, unfolded and hydrolyzed in this cascade

one of the most important drug targets [8e10]. To date, three

proteasome inhibitors have been approved by the FDA for the

treatment of multiple myeloma (MM) and mantle cell lymphoma:

peptide boronates bortezomib and ixazomib together with peptide

epoxyketone carﬁlzomib (Fig. 1A) [11e13]. Besides, several pro-

teasome inhibitors are in different stages of clinical trials, including

oprozomib (ONX-0912, Fig. 1B) and marizomib (NPI-0052, Fig. 1B)

[4]. Actually, established proteasome inhibitors are also correlated

with some other diseases including immunological [14], neurode-

generative [15], cardiovascular diseases [16], and even malaria [17].

Proteasome inhibitors can be classiﬁed into covalent and non-

covalent types according to their different binding modes with

the hydroxyl group of the N-terminal threonine residue of protea-

some [4]. All of the three approved proteasome inhibitors are co-

valent ones with a Michael acceptor as an electrophilic moiety at

the C-terminal. The ﬁrm covalent interaction with the receptor

* Corresponding author.

** Corresponding author.

*** Corresponding author.

E-mail addresses: ybzhou@simm.ac.cn (Y. Zhou), jli@simm.ac.cn (J. Li), zjk0125@

yeah.net (J. Zhang).

1

These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ejmech.2016.12.034

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

1057

Fig. 1. A: FDA approved proteasome inhibitors for cancer therapy; B: Proteasome inhibitors in clinical trials; C: Representative of non-covalent proteasome inhibitors.

leads to a prolonged biological action, but may be not beneﬁcial if

the desired proteasome function is limited and induce severe side

effects [18]. Besides, these excessively reactive inhibitors have very

short half lives, which covalently combine with the blood cell

proteasome quickly and fail to treat solid cancers [19]. In contrast,

non-covalent proteasome inhibitors are less reactive, and their

reversible interactions with proteasome ensure them with rapid

binding and dissociation kinetics [19]. Therefore, more therapeutic

advantages such as fewer side effects and broader usage for solid

cancers are probably to be achieved. Nowadays, non-covalent

proteasome inhibitors have attracted more and more attention,

and some of these compounds displayed comparable or even better

proteasome inhibitory activities [19e21]. Representative analogue

ML 16 (Fig. 1C) was developed by Blackburn and colleagues from

Millennium Pharmaceuticals, Inc., which was the most potent non-

covalent inhibitor discovered so far with IC₅₀values of 1.2 nM and

1.1 nM against constitutive proteasome and immunoproteasome,

respectively [19]. Further study of this series of compounds may

offer opportunities for discovering new anti-cancer agents.

improved enzymatic stabilities, but also good in vitro and in vivo

activities with the potential for further development.

2. Results and discussion

2.1. Chemistry

The synthetic routes for piperazine or piperidine-containing

fragments 7a-b, 8a-b, 9a-b and 10a-i are summarized in Scheme

1. An easy condensation of corresponding acid 1a-b with pro-

tected piperazine or piperidine fragment provided compounds 3a-

b, 4a-b and 5a-b. However, due to low reactive activities of various

arylamine, compounds 6a-i were synthesized from arylamine 2a-i

and N-Boc-4-piperidineformyl chloride. Afterwards, deprotection

of 3a-b, 5a-b and 6a-i with triﬂuoroacetic acid (TFA) and 4a-b with

LiOH afforded piperazine or piperidine TFA salts (7a-b, 9a-b and

10a-i) and piperidinecarboxylic acids (8a-b).

Dipeptide fragments were synthesized following the method

described in Scheme 2. Reaction of Boc-L-aspartic acid-1-benzyl

In an effort to identify novel non-covalent proteasome inhibitors

with better pharmacokinetic proﬁles, a piperidine ring was intro-

duced into the peptide skeleton in our previous research [22]. The

target compounds exhibited better enzymatic stabilities both in

plasma and blood together with good proteasome inhibitory ac-

tivities. In this manuscript, a new series of piperazine or piperidine-

containing non-covalent proteasome inhibitors with retained crit-

ical neopentylaminated aspartic acid of ML 16 were designed and

synthesized (Fig. 2), and these compounds showed not only

ester (11) with neopentylamine furnished compound 12, which

was deprotected at the presence of Pd/C (10%) to afford compound

13. 16a-f were synthesized from Boc-protected amino acid 14a-

d with condensation and deprotection, which were reacted with

fragment 13 and deprotected again to provide the dipeptide TFA

salts 18a-f. Subsequently, the target compounds 19-43 were syn-

thesized with the methods illustrated in Schemes 3 and 4. The

primary amine 18a-f were either condensed with 8a-b to afford 19-

20, or transformed to corresponding isocyanate intermediates and

Fig. 2. Design strategy of the piperazine or piperidine-containing non-covalent proteasome inhibitors.

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R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

Scheme 1. Synthesis of piperazine or piperidine-containing fragments (7a-b, 8a-b, 9a-b and 10a-i). Reagents and conditions: (I) HOBt, EDCI, N-Boc-4-aminopiperidine, diiso-

propylethylamine (DIPEA), DCM, 0^ꢀC-rt; (II) HOBt, EDCI, methyl piperidine-4-carboxylate, DIPEA, DCM, 0^ꢀC-rt; (III) HOBt, EDCI, N-Boc-piperazine, DIPEA, DCM, 0^ꢀC-rt; (IV) N-Boc-4-

piperidinecarboxylic acid, SOCl₂, pyridine, Et₃N, DMAP, DCM, rt; (V) TFA, DCM, 0^ꢀC-rt; (VI) LiOH, H₂O, acetone, rt.

reacted with 7a-b, 9a-b and 10a-i to obtain target compounds 21-

43.

potent proteasome inhibitory activities with IC₅₀values ranging

from 2.9 to 10.8 nM, they were less potent than the corresponding

2-chlorophenyl replaced analogues (IC₅₀: 29, 1.1

0.1 nM; 30,

0.6 0.1 nM). Different 6-membered rings introduced into the

2.2. Proteasome inhibitory activities

skeleton of the peptide seemed to have little impact on the inhib-

itory activity. Analogues 21 and 22 constructed form piperazine

also exhibited signiﬁcant activities with IC₅₀values of 5.5 0.2 nM

All 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.

and 3.8

0.5 nM, respectively. The linker L between the 6-

membered ring and the substituents R₁was also crucial for the

activity. Generally speaking, NHCO was more tolerable than CONH

as a linker. Most NHCO substituted compounds were more potent

than the corresponding CONH substituted analogues except two

pairs of compounds (23 versus 24; 35 versus 36). Additionally,

further study of impact of the N-terminus group (R₁) on activity

was performed. Among these compounds (30 and 36-43), most

were more potent than the positive control carﬁlzomib, and

different R₁replacements seemed to have little differences. How-

As illustrated in Table 1, almost all the target compounds

showed potent proteasome chymotrypsin-like inhibitory activities,

and even 15 compounds were more potent than the positive con-

trol carﬁlzomib with IC₅₀values lower than 10 nM. Various R₂re-

placements inﬂuenced the proteasome inhibitory activities

signiﬁcantly. Phenylethyl substituted analogues were 10e40-fold

more potent than methyl, iso-butyl and benzyl substituted com-

pounds (29 versus 23, 25 and 27; 30 versus 24, 26 and 28).

Therefore, phenylethyl was employed at R₂group for further

optimization. In order to evaluate the inﬂuences of R₃, two more

substituents were replaced. Although compounds 31-34 showed

ever,

compounds

with

4-methoxyphenyl

and

4-

triﬂuoromethylphenyl displayed weak activities with IC₅₀values

of 25.7 and 192.5 nM, respectively. Causes of this discrepancy need

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

1059

Scheme 2. Synthesis of dipeptide fragments 18a-f. Reagents and conditions: (I) HOBt, EDCI, neopentylamine hydrochloride, DIPEA, DCM, 0^ꢀC-rt; (II) Pd/C (10%), H₂, MeOH, rt; (III)

HOBt, EDCI, DIPEA, DCM, 0^ꢀC-rt; (IV) TFA, DCM, 0^ꢀC-rt.

to be further studied.

at both concentrations of 0.5

compounds (30, 35 and 39) displayed more potent activities with

inhibitory rates of more than 70% at the concentration of 2.5 M,

and compound 35 was the most potent one. After incubation in the

whole blood and various enzyme metabolisms, these compounds

still displayed potent activities, which suggested that the stabilities

of the analogues have been improved by constructing 6-membered

ring into the peptide skeleton.

mM and 2.5 mM. Besides, another 3

m

2.3. Tumor cell growth inhibitory activities

Based on the evaluation of proteasome chymotrypsin-like

inhibitory activities, 10 selected compounds (19-20, 29-30, 35-36,

39-40 and 42-43) with IC₅₀values lower than 3 nM were further

tested for their tumor cell (RPMI-8226 and MM-1S) growth

inhibitory activities in vitro by MTS assay with carﬁlzomib

employed as the positive control. According to the results sum-

marized in Table 2, all the tested compounds displayed potent

cytotoxic activities, which was consistent with the proteasome

inhibitory activities. Among them, 40 displayed the most potent

anti-proliferative activities against two MM cell lines RPMI-8226

2.5. In vivo blood cell proteasome inhibitory activities

The most potent compound 35 in the ex vivo assay was further

tested for its in vivo blood cell proteasome inhibitory activities to

verify whether this analogue has any potential for clinical appli-

cation. Carﬁlzomib was employed as the positive control. As illus-

trated in Fig. 4, compound 35 displayed the most potent

proteasome inhibitory activity with inhibitory rate of more than

50% 2 h after dosing at the dosage of 5 mg/kg. The extent of pro-

teasome inhibition induced by compound 35 was a process of ﬁrstly

enhancing and then weakening, which was different from that of

the inhibition induced by carﬁlzomib and demonstrated the non-

covalent reversible interaction between this series of compounds

and proteasome. Compared to carﬁlzomib, the in vivo activity of

compound 35 was less potent, which needs to be further optimized.

and MM-1S with IC₅₀values of 3.8

respectively.

0.5 nM and 4.3

0.7 nM,

2.4. Ex vivo blood cell proteasome inhibitory activities

Subsequently, nine compounds were selected and further

screened for the blood cell proteasome inhibitory activities ex vivo

with compound 27 employed as the negative control and carﬁl-

zomib as the positive control. As illustrated in Fig. 3 and analogues

(36, 42 and 43) showed comparable blood cell proteasome inhibi-

tory activities compared to that of the positive control carﬁlzomib

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R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

Scheme 3. Synthesis of piperazine or piperidine-containing target compounds (19-22). Reagents and conditions: (I) HOBt, EDCI, DIPEA, DCM, 0^ꢀC-rt; (II) triphosgene, DIPEA, DCM,

aqueous NaHCO₃, 0^ꢀC-rt.

Scheme 4. Synthesis of piperidine-containing target compounds (23-43). Reagents and conditions: (I) triphosgene, DIPEA, DCM, aqueous NaHCO₃, 0^oC-rt.

2.6. Plasma stability

half life of compound 35 (672.4 min) was 2-fold longer than the

compound without six-membered ring (336.4 min), which

demonstrated the enzymatic stability of this series of compounds

have been increased by constructing the six-membered ring into

the peptide skeleton.

Based on the in vitro, ex vivo and in vivo assays against protea-

some, selected compound 35 and its corresponding dipeptidyl

analogue without piperidine ring (4-ﬂuorophenyl-carbonyl- Asp

(NH-neopentyl)-hPhe-NH-2-chlorobenzyl) were tested for their

enzymatic stability in mouse plasma. As summarized in Table 3, the

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

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Table 1

20S proteasome chymotrypsin-like inhibitory activities of target compounds (19-43).

Compd.

R₁

L

X/Y

R₂

R₃

IC₅₀(nM)^a

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

pyrazin-2-yl

4-ﬂuorophenyl

pyrazin-2-yl

4-ﬂuorophenyl

pyrazin-2-yl

4-ﬂuorophenyl

4-methoxyphenyl

4-triﬂuoromethyl-

phenyl

CO

N/C

N/N

C/N

phenylethyl

methyl

iso-butyl

benzyl

2-chlorophenyl

2, 4-diﬂuorophenyl

benzo[d][1,3]dioxol-5-yl

2-chlorophenyl

2.4 0.1

2.6 0.1

5.5 0.2

3.8 0.5

13.3 3.7

19.7 3.9

21.8 0.7

11.6 0.4

41.0 6.9

18.0 1.4

1.1 0.1

0.6 0.1

10.8 0.6

6.6 1.1

10.0 0.8

2.9 0.5

1.2 0.1

1.5 0.3

25.7 1.8

192.5 18.3

CONH

NHCO

CONH

NHCO

CONH

NHCO

CONH

NHCO

CONH

NHCO

CONH

NHCO

CONH

NHCO

benzyl

phenylethyl

39

40

41

42

pyridin-3-yl

pyridin-2-yl

isoxazol-3-yl

thiazol-2-yl

thiazol-2-yl-methyl

e

NHCO

e

C/N

e

phenylethyl

e

2-chlorophenyl

e

0.7 0.1

1.0 0.1

7.0 1.3

0.9 0.2

1.0 0.1

8.4 0.9

43

Carﬁlzomib

a

The IC₅₀values are an average of three independent determinations.

Table 2

Cytotoxic activities of selected compounds against two MM cell lines.

Compound

Cytotoxicity (IC₅₀, nM)^a

RPMI 8226

MM-1S

19

20

29

30

35

36

39

40

15.9 2.4

22.7 3.1

20.3 2.7

17.1 1.5

8.4 0.8

13.9 2.4

20.8 3.8

3.8 0.5

11.8 0.7

31.8 5.4

18.8 2.6

15.8 2.9

6.3 0.8

12.5 3.7

20.7 1.4

4.3 0.7

7.2 1.1

22.5 3.6

1.5 0.6

42

43

10.2 1.6

20.7 4.1

13.2 0.6

Carﬁlzomib

a

The IC₅₀values are an average of three independent determinations.

Fig. 3. Ex vivo blood cell proteasome inhibitory activities of selected compounds.

2.7. Binding mode analysis

To explore the binding modes of target compound with the

active site of proteasome, molecular docking simulation studies

and the neopentylamino group occupied well deﬁned S1 and S3

pockets, respectively, while the homo-phenylalanine residue was

oriented toward the solvent. Besides, some critical Hydrogen bonds

were formed between the carbonyl and amino groups of the pep-

tide skeleton and Gly47, Thr21, Ala49, Ala50 and Asp114 of the

proteasome active site. These hydrogen bonds played important

roles in the inhibitory potency against proteasome. For the inactive

€

were carried out by using Glide module of Schrodinger package.

The co-crystal structure of proteasome (PDB ID code: 3MG6) was

selected as the docking template. The binding modes between

proteasome and the most potent compound 30 were shown in

Fig. 5. As depicted in Fig. 5, the C-terminal 2-chlorobenzyl group

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R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

the rational design of more potent proteasome inhibitors.

3. Conclusion

A series of novel piperazine or piperidine-containing non-co-

valent peptidyl derivatives possessing a neopentyl-asparagine

residue were synthesized and evaluated for their proteasome

inhibitory activities. Among all the synthesized compounds, 15

analogues exhibited proteasome inhibitory activities more potent

than the positive control carﬁlzomib, which also possessed forceful

anti-proliferation activities against two MM cell lines RPMI-8226

and MM-1S. In addition, 6 compounds displayed comparable or

even more potent activities against proteasome in the ex vivo blood

cell assay. Besides, the most potent compound 35 showed moder-

ate and reversible blood cell proteasome inhibitory activity in the

in vivo assay. The improved enzymatic stability of this series of

compounds could be validated by the 2-fold longer half life of

compound 35 than its corresponding dipeptidyl analogue without

piperidine ring. Compound 35 should be considered to be further

studied as a lead and deserves additional optimization.

Fig. 4. In vivo blood cell proteasome inhibitory activities of compound 35.

4. Experimental procedures

Table 3

Plasma half-lives of compound 35 and its corresponding dipeptidyl analogue

without piperazine or piperidine ring.

4.1. Chemistry

¹H and ¹³C NMR spectra were recorded on Brüker 500/400 MHz

spectrometer (Brüker Bioscience, Billerica, MA, USA) with CDCl₃or

DMSO-d₆as solvent. Chemical shifts (d) were reported in parts per

Compound

Plasma Half-life (t_1/2, min)

35

672.4

336.4

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 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

(300e400 mesh). All yields are unoptimized and generally repre-

Fig. 5. The binding mode of selected compound with chymotrypsin-like active site of 20S proteasome (PDB ID: 3MG6). A: Predicted H-bonds between compound 30 and the

protein, the H-bonds are shown as dashed magenta lines; B: Occupancy of the S3 binding pocket of the 20S proteasome by the neopentyl-asparagine residue of compound 30; C:

Overlay of the most potent compound 30 with the inactive compound 38, and the H-bonds are shown as dashed magenta lines.

compound 38, the modeling results indicated that it could bind to

proteasome with a similar binding mode of the most active com-

pound 30 (Fig. 5C). The activity difference may arise from different

properties of the two analogues. The electronegativity of N atom of

pyrazine may interact with the nearby Arg91 with positive charge.

Besides, both pyrazine and triﬂuoromethylphenyl group located in

a solvent exposed area. Pyrazine (CLogP: ꢁ0.312) may be more

favorable than the highly hydrophobic triﬂuoromethylphenyl

(CLogP: 3.025) in this area. The molecular docking study may allow

sent the result of a single experiment.

4.1.1. General procedure for the synthesis of piperidine or

piperazine-containing fragments (3a-b, 4a-b and 5a-b)

To a reaction of corresponding carboxylic acid (1a-b, 10.0 mmol)

in DCM (40.0 mL), HOBt (1.49 g, 11.0 mmol) and EDCI (2.88 g,

15.0 mmol) were added at 0 ^ꢀC. The mixture was kept at the same

temperature and was stirred for 30 min. Then corresponding

piperidine or piperazine fragment (10.0 mmol) and

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

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diisopropylethylamine (3.7 mL, 20.0 mmol) were added. After

z ¼ 373.2 [MþH]^þ.

stirring at room temperature for 3 h, the resulting mixture was

washed with aqueous NaHCO₃solution (1 ꢂ 30 mL), brine

(1 ꢂ 30 mL) and dried over Na₂SO₄. The solvent was evaporated in

vacuo and the crude product was puriﬁed by ﬂash chromatography

(ethyl acetate: petroleum ether ¼ 2:1e1:1). Compound 3a has been

reported in our previous work.

4.1.2.2. tert-Butyl 4-(isoxazol-3-ylcarbamoyl)piperidine-1-

carboxylate (6g). White solid; Yield: 84%; ¹H NMR (500 MHz,

CDCl₃):

d

¼ 9.92 (s, 1H, NH), 8.35 (d, 1H, J ¼ 2.0 Hz, isoxazole-H),

7.24 (d, 1H, J ¼ 2.0 Hz, isoxazole-H), 4.14e4.08 (m, 2H, CH₂),

2.91e2.85 (m, 2H, CH₂), 2.64e2.59 (m, 1H, CH), 1.99 (d, 2H,

J ¼ 11.0 Hz, CH₂), 1.86e1.79 (m, 2H, CH₂), 1.47 (s, 9H, CH₃); ESI-MS:

m/z ¼ 296.2 [MþH]^þ.

4.1.1.1. tert-Butyl 4-(4-ﬂuorobenzamido) piperidine-1-carboxylate

(3b). White solid; Yield: 92%; ¹H NMR (500 MHz, CDCl₃):

d

¼ 7.78 (dd, 2H, J ¼ 8.5, 5.0 Hz, Ar-H), 7.11 (t, 2H, J ¼ 8.5 Hz, Ar-H),

4.1.2.3. tert-Butyl 4-(thiazol-2-ylmethylcarbamoyl)piperidine-1-

6.08 (d, 1H, J ¼ 7.5 Hz, NH), 4.15e4.08 (m, 3H, CH þ CH₂), 2.89 (m,

2H, CH₂), 2.01 (d, 2H, J ¼ 11.0 Hz, CH₂), 1.46 (s, 9H, CH₃), 1.43e1.39

(m, 2H, CH₂); ESI-MS: m/z ¼ 323.2 [MþH]^þ.

carboxylate (6i). White form; Yield: 87%; ¹H NMR (500 MHz,

CDCl₃):

d

¼ 7.71 (d, 1H, J ¼ 3.0 Hz, thiazol-H), 7.30 (d, 1H, J ¼ 3.0 Hz,

thiazol-H), 6.58 (t, 1H, J ¼ 4.5 Hz, NH), 4.77 (d, 2H, J ¼ 5.5 Hz, CH₂),

4.23e4.04 (m, 2H, CH₂), 2.82e2.64 (m, 2H, CH₂), 2.37e2.31 (m, 1H,

CH),1.85 (d, 2H, J ¼ 11.0 Hz, CH₂), 1.72e1.64 (m, 2H, CH₂), 1.46 (s, 9H,

CH₃); ESI-MS: m/z ¼ 326.3 [MþH]^þ.

4.1.1.2. Methyl 1-(pyrazine-2-carbonyl)piperidine-4-carboxylate

(4a). White solid; Yield: 94%; ¹H NMR (500 MHz, CDCl₃):

d

¼ 8.88 (d, 1H, J ¼ 1.0 Hz, pyrazine-H), 8.61 (d, 1H, J ¼ 2.5 Hz,

pyrazine-H), 8.52 (s, 1H, pyrazine-H), 4.51 (m, 1H, CH₂), 3.91 (m, 1H,

CH₂), 3.70 (s, 3H, CH₃), 3.20 (m, 1H, CH₂), 3.09 (m, 1H, CH₂), 2.62 (m,

1H, CH), 2.04 (m, 1H, CH₂), 1.91 (m, 1H, CH₂), 1.81 (m, 2H, CH₂); ESI-

MS: m/z ¼ 250.1 [MþH]^þ.

4.1.3. General procedure for the synthesis of compounds 7a-b, 9a-b

and 10a-i

To

a suspension of protected piperidine or piperazine-

containing fragments 3a-b, 5a-b or 6a-i (5.0 mmol) in CH₂Cl₂

(20.0 mL) was added triﬂuoroacetic acid (TFA, 5.0 mL) at 0 ^ꢀC. The

reaction mixture was then warmed to room temperature and stir-

red for 1 h. The volatiles were evaporated under reduced pressure

and ether (30.0 mL) was added. White solid was precipitated, ﬁl-

trated and dried, which was put into next step without further

puriﬁcation.

4.1.1.3. Methyl 1-(4-ﬂuorobenzoyl)piperidine-4-carboxylate (4b).

White solid; Yield: 87%; ¹H NMR (500 MHz, CDCl₃):

d

¼ 7.41 (dd,

2H, J ¼ 8.5, 5.5 Hz, Ar-H), 7.10 (t, 2H, J ¼ 8.5 Hz, Ar-H), 4.48 (m, 1H,

CH₂), 3.80 (m, 1H, CH₂), 3.72 (s, 3H, CH₃), 3.06 (m, 2H, CH₂), 2.61 (m,

1H, CH), 1.96 (m, 2H, CH₂), 1.74 (m, 2H, CH₂); ESI-MS: m/z ¼ 266.1

[MþH]^þ.

4.1.4. Synthesis of piperidine-4-carboxylate analogues 8a-b

To a solution of compounds 4a-b (3.0 mmol) in acetone

(10.0 mL), 6.0 mL 1 N LiOH (aq) was added dropwise. The mixture

was stirred for 1 h and acetone was evaporated in vacuo. The res-

idue was acidiﬁed to pH¼2 with 1 N HCl and extracted with ethyl

acetate (3 ꢂ 10 mL). The combined organic layers were washed

with brine (1 ꢂ 10 mL), dried over Na₂SO₄, ﬁltered and concen-

trated under reduced pressure. The crude products 8a-b were put

into next step without further puriﬁcation.

4.1.1.4. tert-Butyl 4-(pyrazine-2-carbonyl)piperazine-1-carboxylate

(5a). White solid; Yield: 76%; ¹H NMR (500 MHz, CDCl₃):

d

¼ 8.97 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.65 (d, 1H, J ¼ 2.5 Hz,

pyrazine-H), 8.54 (s, 1H, pyrazine-H), 3.79 (t, 2H, J ¼ 5.0 Hz, CH₂),

3.62 (t, 2H, J ¼ 5.0 Hz, CH₂), 3.56 (t, 2H, J ¼ 5.0 Hz, CH₂), 3.49 (t, 2H,

J ¼ 5.0 Hz, CH₂), 1.47 (s, 9H, CH₃); ESI-MS: m/z ¼ 293.2 [MþH]^þ.

4.1.1.5. tert-Butyl 4-(4-ﬂuorobenzoyl)piperazine-1-carboxylate (5b).

White solid; Yield: 97%; ¹H NMR (500 MHz, CDCl₃):

d

¼ 7.43 (dd,

2H, J ¼ 9.0, 5.5 Hz, Ar-H), 7.12 (t, 2H, J ¼ 8.5 Hz, CH₂), 3.54e3.41 (m,

4.1.5. (S)-benzyl 2-(tert-butoxycarbonylamino)-4-

(neopentylamino)-4-oxobutanoate (12)

8H, CH₂), 1.48 (s, 9H, CH₃); ESI-MS: m/z ¼ 309.2 [MþH]^þ.

To a suspension of Boc- -aspartic acid 1-benzyl ester (11, 3.23 g,

L

4.1.2. General procedure for the synthesis of piperidine-containing

fragments (6a-i)

10.0 mmol) in CH₂Cl₂(40.0 mL), HOBt (1.49 g, 11.0 mmol) and EDCI

(2.88 g, 15.0 mmol) were added at 0 ^ꢀC. The reaction mixture was

kept at 0 ^ꢀC and was stirred for 30 min. Then neopentylamine hy-

drochloride (1.24 g, 10.0 mmol) and diisopropylethylamine (3.7 mL,

20.0 mmol) were added at 0 ^ꢀC. After stirring at room temperature

for another 3 h, the resulting mixture was washed with aqueous

NaHCO₃solution (1 ꢂ 30 mL), brine (1 ꢂ 30 mL) and dried over

Na₂SO₄. The solvent was evaporated in vacuo and the crude product

was puriﬁed by ﬂash chromatography (ethyl acetate: petroleum

ether ¼ 1:3).

To a mixture of N-Boc-4-piperidinecarboxylic acid (3.65 g,

16.0 mmol), pyridine (3.4 mL, 40.0 mmol) and CH₂Cl₂(30.0 mL),

SOCl₂(1.5 mL, 19.0 mmol) was added under N₂at room tempera-

ture. The mixture was stirred for half an hour, and a solution of

corresponding amine (2a-i, 18.0 mmol), Et₃N (8.0 mL, 56.0 mmol)

and a catalytic amount of DMAP in CH₂Cl₂(30.0 mL) was added

dropwise. The suspension was stirred overnight. The organic phase

was washed with 1 N HCl (2 ꢂ 30 mL) and aqueous NaHCO₃

(2 ꢂ 30 mL), dried over Na₂SO₄(For compounds 6a, 6e and 6f, 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 ace-

tate: petroleum ether ¼ 1:2e3:1) to give compounds 6a-i. The

details of compounds 6a-c, 6e-f and 6h have been reported in our

previous work.

White solid; Yield: 99%; ¹H NMR (500 MHz, CDCl₃):

d

¼ 7.41e7.30 (m, 5H, Ar-H), 5.80 (d, 1H, J ¼ 6.5 Hz, NH), 5.66 (brs,

1H, NH), 5.17 (d, 2H, J ¼ 5.0 Hz, CH₂), 4.55e4.52 (m, 1H, CH), 3.01 (d,

2H, J ¼ 7.5 Hz, CH₂), 2.93 (dd, 1H, J ¼ 18.0, 5.0 Hz, CH₂), 2.73 (dd, 1H,

J ¼ 19.0, 5.0 Hz, CH₂), 1.42 (s, 9H, CH₃), 0.87 (s, 9H, CH₃); ESI-MS: m/

z ¼ 393.3 [MþH]^þ.

4.1.6. (S)-2-(tert-butoxycarbonylamino)-4-(neopentylamino)-4-

oxobutanoic acid (13)

Compound 12 (1.96 g, 5.0 mmol) was dissolved in methanol

(10.0 mL) in the presence of 10% palladium on carbon (10 mol%),

and the reaction mixture was stirred under an atmosphere of H₂for

2 h. The Pd/C was removed via ﬁltration through celite, and the

4.1.2.1. tert-Butyl 4-(4-(triﬂuoromethyl)phenylcarbamoyl)piperidine-

1-carboxylate (6d). Pale yellow oil; Yield: 60%; ¹H NMR (500 MHz,

CDCl₃):

d

¼ 8.09 (s, 1H, NH), 7.51e7.46 (m, 4H, Ar-H), 4.19e4.16 (m,

2H, CH₂), 2.81e2.72 (m, 2H, CH₂), 2.42e2.39 (m, 1H, CH), 1.88e1.84

(m, 2H, CH₂), 1.75e1.65 (m, 2H, CH₂), 1.48 (s, 9H, CH₃); ESI-MS: m/

1064

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

solvent was evaporated to obtain compound 13 in quantitative

yield. The crude product was used directly in the next step.

added at 0 ^ꢀC. The reaction mixture was stirred for 30 min. Then

corresponding amine hydrochloride (3.0 mmol) and diisopropyle-

thylamine (1.1 mL, 6.0 mmol) were added. After stirring at room

temperature for another 3 h, the resulting mixture was washed

with aqueous NaHCO₃solution (1 ꢂ 10 mL), brine (1 ꢂ 10 mL) and

dried over Na₂SO₄. The solvent was evaporated in vacuo and the

crude product was puriﬁed by ﬂash chromatography (ethyl acetate:

CH₂Cl₂¼ 1:5e1:1).

4.1.7. General procedure for the synthesis of C-caped amino acids

15a-f

To a suspension of corresponding N-Boc-protected amino acid

(14a-d, 5.0 mmol) in DCM (20.0 mL), HOBt (0.75 g, 5.5 mmol) and

EDCI (1.44 g, 7.5 mmol) were added. The reaction mixture was

stirred for 30 min. Then corresponding amine (5.0 mmol) and dii-

sopropylethylamine (1.9 mL, 10.0 mmol) were added and stirred for

another 3 h. The resulting mixture was washed with aqueous

NaHCO₃solution (1 ꢂ 15 mL), brine (1 ꢂ 15 mL) and dried over

Na₂SO₄. The solvent was evaporated in vacuo and the crude product

was puriﬁed by ﬂash chromatography (ethyl acetate: petroleum

ether ¼ 1:5e1:2).

4.1.9.1. Boc-Asp(NH-neopentyl)-Ala-NH-2-chlorobenzyl

White solid; Yield: 89%; ¹H NMR (500 MHz, CDCl₃):

(17a).

¼ 7.35e7.32

d

(m, 2H, Ar-H), 7.24e7.20 (m, 3H, Ar-H þ NH), 7.06 (d, 1H, J ¼ 7.5 Hz,

NH), 6.15 (d, 1H, J ¼ 6.5 Hz, NH), 6.05 (t, 1H, J ¼ 5.5 Hz, NH),

4.55e4.47 (m, 3H, CH þ CH₂), 4.41 (dd, 1H, J ¼ 11.5, 6.5 Hz, CH₂),

2.99 (dd, 1H, J ¼ 13.0, 6.5 Hz, CH₂), 2.92 (dd, 1H, J ¼ 13.5, 6.5 Hz,

CH₂), 2.79 (dd, 1H, J ¼ 14.5, 4.5 Hz, CH₂), 2.64 (dd, 1H, J ¼ 15.0,

6.5 Hz, CH₂), 1.44 (s, 9H, CH₃), 1.42 (d, 3H, J ¼ 6.0 Hz, CH₃), 0.87 (s,

9H, CH₃); ESI-MS: m/z ¼ 497.3 [MþH]^þ.

4.1.7.1. Boc-Ala-NH-2-chlorobenzyl (15a). White solid; Yield: 94%;

¹H NMR (500 MHz, CDCl₃):

d

¼ 7.38e7.36 (m, 2H, Ar-H), 7.25e7.22

(m, 2H, Ar-H), 6.75 (brs, 1H, NH), 5.05 (brs, 1H, NH), 4.54 (d, 2H,

J ¼ 5.0 Hz, CH₂), 4.25e4.18 (m, 1H, CH), 1.43 (s, 9H, CH₃), 1.38 (d, 3H,

J ¼ 7.0 Hz, CH₃); ESI-MS: m/z ¼ 313.1 [MþH]^þ.

4.1.9.2. Boc-Asp(NH-neopentyl)-Leu-NH-2-chlorobenzyl

White solid; Yield: 80%; ¹H NMR (500 MHz, CDCl₃):

(17b).

¼ 7.35e7.32

d

(m, 2H, Ar-H), 7.24e7.20 (m, 3H, Ar-H þ NH), 6.90 (d, 1H, J ¼ 8.0 Hz,

NH), 6.10 (d, 1H, J ¼ 7.0 Hz, NH), 5.93 (t, 1H, J ¼ 6.5 Hz, NH), 4.51 (dd,

2H, J ¼ 6.0, 2.0 Hz, CH₂), 4.47e4.41 (m, 2H, CH₂), 2.96e2.93 (m, 2H,

CH₂), 2.77 (dd, 1H, J ¼ 15.0, 5.0 Hz, CH₂), 2.66 (dd, 1H, J ¼ 15.0,

6.0 Hz, CH₂), 1.84e1.81 (m, 1H, CH), 1.67e1.55 (m, 2H, CH₂), 1.44 (s,

9H, CH₃), 0.92 (dd, 6H, J ¼ 22.0, 6.5 Hz, CH₂), 0.87 (s, 9H, CH₃); ESI-

MS: m/z ¼ 539.3 [MþH]^þ.

4.1.7.2. Boc-Leu-NH-2-chlorobenzyl (15b). White solid; Yield: 65%;

¹H NMR (500 MHz, CDCl₃):

d

¼ 7.39e7.36 (m, 2H, Ar-H), 7.26e7.21

(m, 2H, Ar-H), 6.70 (t,1H, J ¼ 5.0 Hz, NH), 4.93 (d,1H, J ¼ 7.5 Hz, NH),

4.55 (t, 2H, J ¼ 6.0 Hz, CH₂), 4.18e4.12 (m,1H, CH),1.76e1.66 (m, 2H,

CH₂),1.54-4-1.47 (m,1H, CH),1.46 (s, 9H, CH₃), 0.94 (t, 6H, J ¼ 7.0 Hz,

CH₃); ESI-MS: m/z ¼ 355.2 [MþH]^þ.

4.1.7.3. Boc-Phe-NH-2-chlorobenzyl (15c). White solid; Yield: 95%;

4.1.9.3. Boc-Asp(NH-neopentyl)-Phe-NH-2-chlorobenzyl

(17c).

¼ 8.59 (t,

¹H NMR (500 MHz, CDCl₃):

d

¼ 7.31 (d, 1H, J ¼ 7.5 Hz, Ar-H),

White solid; Yield: 86%; ¹H NMR (500 MHz, DMSO-d₆):

d

7.27e7.10 (m, 8H, Ar-H), 6.25 (brs, 1H, NH), 5.07 (brs, 1H, NH),

4.44 (d, 2H, J ¼ 6.0 Hz, CH₂), 4.38e4.33 (m, 1H, CH), 3.15e3.02 (m,

2H, CH₂), 1.40 (s, 9H, CH₃); ESI-MS: m/z ¼ 389.2 [MþH]^þ.

1H, J ¼ 6.0 Hz, NH), 8.16 (d, 1H, J ¼ 8.0 Hz, NH), 7.77 (t,1H, J ¼ 6.5 Hz,

NH), 7.43 (d, 1H, J ¼ 7.5 Hz, NH), 7.31e7.20 (m, 7H, Ar-H), 7.12 (d, 1H,

J ¼ 7.0 Hz, Ar-H), 6.92 (d, 1H, J ¼ 8.0 Hz, Ar-H), 4.52e4.48 (m, 1H,

CH), 4.30e4.24 (m, 3H, CH þ CH₂), 3.07 (dd, 1H, J ¼ 13.5, 5.0 Hz,

CH₂), 2.91 (dd, 1H, J ¼ 14.0, 8.0 Hz, CH₂), 2.83 (dd, 1H, J ¼ 13.0,

6.5 Hz, CH₂), 2.75 (dd, 1H, J ¼ 13.0, 6.0 Hz, CH₂), 2.49e2.39 (m, 2H,

CH₂), 1.35 (s, 9H, CH₃), 0.79 (s, 9H, CH₃); ESI-MS: m/z ¼ 573.3

[MþH]^þ.

4.1.7.4. Boc-hPhe-NH-2, 4-diﬂuorobenzyl (15e). White solid; Yield:

84%; ¹H NMR (500 MHz, CDCl₃):

d

¼ 7.34e7.29 (m, 3H, Ar-H),

7.23e7.18 (m, 1H, Ar-H), 7.16 (d, 2H, J ¼ 7.5 Hz, Ar-H), 6.87e6.80

(m, 2H, Ar-H), 6.59 (brs, 1H, NH), 5.06 (brs, 1H, NH), 4.45 (d, 2H,

J ¼ 6.0 Hz, CH₂), 4.10 (brs, 1H, CH), 2.68 (t, 2H, J ¼ 7.0 Hz, CH₂),

2.19e2.15 (m, 1H, CH₂), 1.97e1.92 (m, 1H, CH₂), 1.44 (s, 9H, CH₃);

ESI-MS: m/z ¼ 405.2 [MþH]^þ.

4.1.9.4. Boc-Asp(NH-neopentyl)-hPhe-NH-2-chlorobenzyl

(17d).

¼ 8.51 (t,

White solid; Yield: 90%; ¹H NMR (500 MHz, DMSO-d₆):

d

1H, J ¼ 6.0 Hz, NH), 8.25 (d,1H, J ¼ 8.0 Hz, NH), 7.82 (t,1H, J ¼ 6.0 Hz,

NH), 7.31e7.25 (m, 6H, Ar-H), 7.18e7.16 (m, 3H, Ar-H), 7.09 (d, 1H,

J ¼ 8.0 Hz, NH), 4.35e4.28 (m, 3H, CH₂þCH), 4.20e4.18 (m, 1H, CH),

2.82e2.71 (m, 2H, CH₂), 2.66e2.52 (m, 4H, CH₂), 2.06e1.99 (m, 1H,

CH₂), 1.90e1.88 (m, 1H, CH₂), 1.37 (s, 9H, CH₃), 0.77 (s, 9H, CH₃); ESI-

MS: m/z ¼ 587.3 [MþH]^þ.

4.1.7.5. Boc-hPhe-NH-benzo[d]

[1,3]dioxol-5-ylmethyl

(15f).

White solid; Yield: 97%; ¹H NMR (500 MHz, CDCl₃):

d

¼ 7.31e7.28

(m, 2H, Ar-H), 7.23e7.17 (m, 3H, Ar-H), 6.77e6.73 (m, 3H, Ar-H),

6.37 (brs, 1H, NH), 5.96 (s, 2H, OCH₂O), 5.04 (brs, 1H, NH), 4.36

(brs, 2H, CH₂), 4.09 (brs, 1H, CH), 2.71 (t, 2H, J ¼ 7.5 Hz, CH₂),

2.25e2.16 (m, 1H, CH₂), 2.00e1.91 (m, 1H, CH₂), 1.45 (s, 9H, CH₃);

ESI-MS: m/z ¼ 413.2 [MþH]^þ.

4.1.9.5. Boc-Asp(NH-neopentyl)-hPhe-NH-2, 4-diﬂuorobenzyl (17e).

White solid; Yield: 69%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 8.49 (t,

4.1.8. General procedure for the synthesis of deprotected C-caped

amino acids 16a-f

1H, J ¼ 5.5 Hz, NH), 8.18 (d,1H, J ¼ 8.0 Hz, NH), 7.83 (t,1H, J ¼ 6.0 Hz,

NH), 7.78 (t, 1H, J ¼ 6.5 Hz, NH), 7.37e7.15 (m, 6H, Ar-H), 7.06e7.00

(m, 2H, Ar-H), 4.37e4.23 (m, 3H, CH þ CH₂), 4.18e4.14 (m, 1H, CH),

2.89e2.78 (m, 2H, CH₂), 2.63e2.47 (m, 4H, CH₂), 2.05e2.02 (m, 1H,

CH₂), 1.89e1.82 (m, 1H, CH₂), 1.38 (s, 9H, CH₃), 0.81 (s, 9H, CH₃); ESI-

MS: m/z ¼ 589.3 [MþH]^þ.

C-caped amino acid 15a-f (5.0 mmol) dissolved in CH₂Cl₂

(20.0 mL) was added triﬂuoroacetic acid (5.0 mL) at 0 ^ꢀC. The re-

action mixture was then stirred for 1 h at room temperature. The

volatiles were evaporated under reduced pressure and ether

(30.0 mL) was added. White solid was precipitated, which was then

ﬁltrated, dried and used in the next step without further

puriﬁcation.

4.1.9.6. Boc-Asp(NH-neopentyl)-hPhe-NH-benzo[d] [1,3]dioxol-5-

ylmethyl (17f). White solid; Yield: 61%; ¹H NMR (500 MHz,

DMSO-d₆):

d

¼ 8.44 (t, 1H, J ¼ 6.0 Hz, NH), 8.13 (d, 1H, J ¼ 8.0 Hz,

4.1.9. General procedure for the synthesis of C-caped dipeptides

17a-f

To a suspension of compound 13 (0.91 g, 3.0 mmol) in CH₂Cl₂

(10.0 mL), HOBt (0.41 g, 3.0 mmol) and EDCI (1.15 g, 6.0 mmol) were

NH), 7.82 (t, 1H, J ¼ 6.0 Hz, NH), 7.26 (t, 2H, J ¼ 7.0 Hz, Ar-H),

7.19e7.14 (m, 3H, Ar-H), 7.07 (d, 1H, J ¼ 7.5 Hz, NH), 6.83e6.80

(m, 2H, Ar-H), 6.71 (d,1H, J ¼ 8.0 Hz, Ar-H), 5.97 (s, 2H, OCH₂O), 4.28

(dd, 1H, J ¼ 15.0, 7.5 Hz, CH₂), 4.23e4.11 (m, 3H, CH þ CH₂),

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

1065

2.88e2.78 (m, 2H, CH₂), 2.64e2.59 (m, 2H, CH₂), 2.58e2.48 (m, 2H,

4.1.12. General procedure for the synthesis of target compounds 21-

43

CH₂), 2.06e2.00 (m, 1H, CH₂), 1.87e1.82 (m, 1H, CH₂), 1.38 (s, 9H,

CH₃), 0.81 (s, 9H, CH₃); ESI-MS: m/z ¼ 597.3 [MþH]^þ.

Corresponding dipeptide fragment 18a-f (3.0 mmol) was dis-

solved in CH₂Cl₂(5.0 mL) and saturated aqueous NaHCO₃(5.0 mL)

and was cooled to 0 ^ꢀC. Triphosgene (0.30 g,1.0 mmol) was added in

one portion and the mixture was stirred at 0 ^ꢀC for 10 min. The

organic layer was separated and the aqueous layer was extracted

with CH₂Cl₂(2 ꢂ 10 mL). Then the organic phase was combined,

dried over Na₂SO₄, evaporated in vacuo and used directly. To a

solution of corresponding amine TFA salt (7a-b, 9a-b and 10a-i,

2.0 mmol) was added diisopropylethylamine (0.8 mL, 4.0 mmol).

Then the former obtained isocyanate was dissolved in CH₂Cl₂

(3.0 mL) and added into the reaction mixture. After 3 h'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: methanol¼50:1e10:1). Purity

test condition was the same with that of the compounds 19-20.

4.1.10. General procedure for the synthesis of deprotected dipeptide

analogues 18a-f

To a solution of compound 15a-f (5.0 mmol) in CH₂Cl₂(20.0 mL)

was added triﬂuoroacetic acid (5.0 mL) at 0 ^ꢀC. The reaction mixture

was then stirred for 1 h at room temperature. The volatiles were

evaporated under reduced pressure and ether (30.0 mL) was added.

White solid was precipitated, which was then ﬁltrated, dried and

used in the next step without further puriﬁcation.

4.1.11. General procedure for the synthesis of target compounds 19-

20

To a suspension of corresponding piperidine-4-carboxylic acid

8a-b (1.0 mmol) in CH₂Cl₂(4.0 mL), HOBt (0.15 g, 1.1 mmol) and

EDCI (0.29 g, 1.5 mmol) were added at 0 ^ꢀC. The reaction mixture

was kept at 0 ^ꢀC and was stirred for 30 min. Then compound 18d

(0.12 g, 1.0 mmol) and diisopropylethylamine (0.4 mL, 2.0 mmol)

were added at 0 ^ꢀC. After stirring at room temperature for another

3 h, the resulting mixture was washed with aqueous NaHCO₃so-

lution (1 ꢂ 10 mL), brine (1 ꢂ 5 mL) and dried over Na₂SO₄. The

solvent was evaporated in vacuo and the crude product was puri-

ﬁed by ﬂash chromatography (ethyl acetate: methanol ¼ 10:1).

Analysis of sample purity was performed on an Agilent 1200 HPLC

4.1.12.1. 4-(pyrazine-2-carbonyl)piperazine-1-carbonyl-Asp(NH-neo-

pentyl)-hPhe-NH-2-chlorobenzyl (21). White solid; Yield: 79%; mp:

205e207 ^ꢀC; Purity: 98.8%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 8.86

(d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.77 (d, 1H, J ¼ 1.5 Hz, pyrazine-H),

8.69 (dd, 1H, J ¼ 2.5, 1.5 Hz, pyrazing-H), 8.52 (t, 1H, J ¼ 6.0 Hz, NH),

8.27 (d, 1H, J ¼ 7.5 Hz, NH), 7.82 (t, 1H, J ¼ 6.0 Hz, NH), 7.43e7.41 (m,

1H, Ar-H), 7.33e7.25 (m, 5H, Ar-H), 7.20e7.15 (m, 3H, Ar-H), 6.89 (d,

1H, J ¼ 8.0 Hz, NH), 4.47 (q, 1H, J ¼ 7.5 Hz, CH), 4.32 (d, 2H,

J ¼ 6.0 Hz, CH₂), 4.20e4.16 (m, 1H, CH), 3.65e3.60 (m, 2H, CH₂),

3.47e3.32 (m, 6H, CH₂), 2.85e2.76 (m, 2H, CH₂), 2.70e2.53 (m, 4H,

CH₂), 2.10e2.02 (m, 1H, CH₂), 1.94e1.88 (m, 1H, CH₂), 0.79 (s, 9H,

system using an Agilent ZORBAX SB-Aq, 5

mM C18 column

(250 mm ꢂ 4.6 mm). HPLC condition: linear density gradient

elution with eluents MeOH/H₂O ¼ 75/25-95/5 (10 min), ﬂow rate:

1.0 mL/min.

CH₃); ¹³C NMR (100 MHz, DMSO):

d

¼ 172.73, 172.14, 170.59, 165.37,

157.46, 149.56, 146.05, 145.02, 143.68, 141.84, 136.67, 132.35, 129.44,

129.08, 128.94, 128.87, 128.74, 127.49, 126.28, 52.88, 52.44, 50.16,

46.77, 44.24, 43.67, 42.07, 37.89, 33.72, 32.28, 31.77, 27.63; ESI-MS:

m/z ¼ 705.1 [MþH]^þ.

4.1.11.1. 1-(pyrazine-2-carbonyl)piperidine-4-carbonyl-Asp(NH-neo-

pentyl)-hPhe-NH-2-chlorobenzyl (19). White solid; Yield: 45%; mp:

199e201 ^ꢀC; Purity: 98.5%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 8.81

(d, 1H, J ¼ 3.5 Hz, pyrazine-H), 8.74 (t, 1H, J ¼ 2.5 Hz, pyrazine-H),

8.65 (dt, 1H, J ¼ 12.5, 1.5 Hz, pyrazine-H), 8.54 (dd, 1H, J ¼ 8.5,

5.5 Hz, NH), 8.32 (d, 1H, J ¼ 7.5 Hz, NH), 8.23 (d, 1H, J ¼ 8.0 Hz, NH),

7.88 (q, 1H, J ¼ 6.5 Hz, NH), 7.41e7.39 (m, 1H, Ar-H), 7.31e7.23 (m,

5H, Ar-H), 7.23e7.14 (m, 3H, Ar-H), 4.61 (q, 1H, J ¼ 7.5 Hz, CH),

4.47e4.42 (m, 1H, CH₂), 4.36e4.28 (m, 2H, CH₂), 4.20e4.14 (m, 1H,

CH₂), 3.69e3.64 (m, 1H, CH), 3.11e3.07 (m, 1H, CH₂), 2.92e2.87 (m,

1H, CH₂), 2.79e2.54 (m, 7H, CH þ CH₂), 2.11e2.04 (m, 1H, CH₂),

1.93e1.76 (m, 2H, CH₂), 1.66e1.51 (m, 3H, CH₂), 0.76 (d, 9H,

4.1.12.2. 4-(4-ﬂuorobenzoyl)piperazine-1-carbonyl-Asp(NH-neo-

pentyl)-hPhe-NH-2-chlorobenzyl (22). White solid; Yield: 87%; mp:

174e176 ^ꢀC; Purity: 98.9%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 8.52

(t, 1H, J ¼ 6.0 Hz, NH), 8.27 (d, 1H, J ¼ 7.5 Hz, NH), 7.82 (t, 1H,

J ¼ 6.0 Hz, NH), 7.49e7.47 (m, 2H, Ar-H), 7.43e7.41 (m, 1H, Ar-H),

7.31e7.25 (m, 7H, Ar-H), 7.19e7.16 (m, 3H, Ar-H), 6.88 (d, 1H,

J ¼ 7.5 Hz, NH), 4.47 (q, 1H, J ¼ 7.5 Hz, CH), 4.33 (d, 2H, J ¼ 6.0 Hz,

CH₂), 4.21e4.16 (m, 1H, CH), 3.68e3.20 (m, 8H, piperazine-H),

2.85e2.75 (m, 2H, CH₂), 2.68e2.54 (m, 4H, CH₂), 2.10e2.03 (m,

1H, CH₂), 1.94e1.86 (m, 1H, CH₂), 0.79 (s, 9H, CH₃); ¹³C NMR

J ¼ 6.0 Hz, CH₃); ¹³C NMR (100 MHz, DMSO):

d

¼ 174.22, 172.08,

171.82, 170.31, 165.10, 150.12, 145.85, 144.59, 143.75, 141.71, 136.62,

132.29, 129.42, 129.02, 128.92, 128.89, 128.75, 127.48, 126.32, 52.76,

50.46, 50.08, 46.50, 41.58, 37.67, 33.61, 32.26, 31.71, 29.30, 29.23,

28.53, 28.48, 27.61; ESI-MS: m/z ¼ 704.1 [MþH]^þ.

(100 MHz, DMSO):

d

¼ 172.74, 172.14, 170.59, 168.82, 164.29, 161.83,

157.47, 141.84, 136.67, 132.59, 132.56, 132.35, 130.13, 130.04, 129.43,

129.06, 128.94, 128.87, 128.75, 127.49, 126.28, 115.97, 115.76, 52.89,

52.41, 50.16, 43.90, 37.89, 33.73, 32.27, 31.78, 27.63; ESI-MS: m/

z ¼ 721.1 [MþH]^þ.

4.1.11.2. 1-(4-ﬂuorobenzoyl)piperidine-4-carbonyl-Asp(NH-neo-

pentyl)-hPhe-NH-2-chlorobenzyl (20). White solid; Yield: 42%; mp:

187e189 ^ꢀC; Purity: 99.1%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 8.55

4.1.12.3. 4-(Pyrazine-2-carboxamido)piperidine-1-carbonyl-Asp(NH-

neopentyl)-Ala-NH-2-chlorobenzyl (23). White solid; Yield: 17%;

mp: 171e173 ^ꢀC; Purity: 99.3%; ¹H NMR (500 MHz, DMSO-d₆):

(t, 1H, J ¼ 6.0 Hz, NH), 8.32 (d, 1H, J ¼ 8.0 Hz, NH), 8.23 (d, 1H,

J ¼ 8.0 Hz, NH), 7.89 (t, 1H, J ¼ 6.0 Hz, NH), 7.44e7.40 (m, 3H, Ar-H),

7.32e7.24 (m, 7H, Ar-H), 7.19e7.13 (m, 3H, Ar-H), 4.61 (q, 1H,

J ¼ 8.0 Hz, CH), 4.48e4.28 (m, 3H, CH₂), 4.19e4.16 (m, 1H, CH₂),

3.64e3.56 (m, 1H, CH), 3.10e2.99 (m, 1H, CH₂), 2.85e2.55 (m, 7H,

CH þ CH₂), 2.11e2.03 (m, 1H, CH₂), 1.92e1.81 (m, 1H, CH₂),

1.79e1.43 (m, 5H, CH₂), 0.76 (s, 9H, CH₃); ¹³C NMR (100 MHz,

d

¼ 9.17 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.86 (d, 1H, J ¼ 2.5 Hz,

pyrazine-H), 8.83 (d, 1H, J ¼ 8.5 Hz, NH), 8.69 (dd, 1H, J ¼ 2.0, 1.5 Hz,

pyrazine-H), 8.53 (t, 1H, J ¼ 6.0 Hz, NH), 8.18 (d, 1H, J ¼ 7.5 Hz, NH),

7.79 (t, 1H, J ¼ 6.0 Hz, NH), 7.42e7.40 (m, 1H, Ar-H), 7.32e7.26 (m,

3H, Ar-H), 6.69 (d, 1H, J ¼ 7.5 Hz, NH), 4.39e4.25 (m, 4H, CH þ CH₂),

4.01e3.92 (m, 3H, CH₂þCH), 2.85e2.72 (m, 4H, CH₂), 2.61e2.57 (dd,

1H, J ¼ 14.5, 6.5 Hz, CH₂), 2.54e2.50 (dd, 1H, J ¼ 14.5, 8.0 Hz, CH₂),

1.73e1.71 (m, 2H, CH₂), 1.59e1.46 (m, 2H, CH₂); 1.28 (d, 3H,

J ¼ 7.0 Hz, CH₃); 0.78 (s, 9H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆):

DMSO):

d

¼ 174.27, 172.07, 171.81, 170.30, 168.56, 163.87, 161.91,

141.73, 136.61, 133.09, 132.29, 129.74, 129.67, 129.42, 129.01, 128.90,

128.74, 127.48, 126.31, 115.93, 115.76, 52.77, 50.44, 50.07, 41.92,

41.76, 37.66, 33.63, 32.26, 31.72, 30.63, 27.61; ESI-MS: m/z ¼ 720.1

[MþH]^þ.

d

¼ 172.89, 172.47, 170.57, 162.65, 157.28, 147.88, 145.42, 144.08,

1066

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

143.68, 136.64, 132.33, 129.44, 129.01, 128.45, 127.54, 52.48, 50.17,

49.02, 47.10, 43.24. 43.12, 38.17, 32.27, 31.46, 27.63, 18.38; ESI-MS:

m/z ¼ 629.1 [MþH]^þ.

CH₂), 2.52e2.44 (m, 2H, CH₂), 1.72e1.70 (m, 2H, CH₂); 1.49 (qd, 2H,

J ¼ 11.5, 3.0 Hz, CH₂), 0.79 (s, 9H, CH₃); ¹³C NMR (100 MHz, DMSO-

d₆):

d

¼ 172.58, 171.46, 170.53, 162.66, 157.38, 147.89, 145.42, 144.08,

143.68, 138.30, 136.46, 132.31, 129.64, 129.40, 129.09, 128.94,

128.59, 127.52, 126.75, 54.70, 52.47. 50.17, 47.07, 43.23, 43.09, 37.78,

37.49, 32.31, 31.41, 27.64; ESI-MS: m/z ¼ 705.1 [MþH]^þ.

4.1.12.4. 4-(Pyrazin-2-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-Ala-NH-2-chlorobenzyl (24). White solid; Yield: 43%;

mp: 186e188 ^ꢀC; Purity: 98.2%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 10.79 (s, 1H, NH), 9.33 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.51 (t, 1H,

4.1.12.8. 4-(Pyrazin-2-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-Phe-NH-2-chlorobenzyl (28). White solid; Yield: 45%;

mp: 165e167 ^ꢀC; Purity: 98.9%; ¹H NMR (500 MHz, DMSO-d₆):

J ¼ 6.0 Hz, NH), 8.40 (dd, 1H, J ¼ 2.5, 1.5 Hz, pyrazine-H), 8.35 (d, 1H,

J ¼ 2.5 Hz, pyrazine-H), 8.22 (d, 1H, J ¼ 7.5 Hz, NH), 7.78 (t, 1H,

J ¼ 6.0 Hz, NH), 7.42e7.41 (m, 1H, Ar-H), 7.33e7.25 (m, 3H, Ar-H),

6.65e6.63 (d, 1H, J ¼ 7.5 Hz, NH), 4.38 (q, 1H, J ¼ 8.0 Hz, CH), 4.31

(d, 2H, J ¼ 6.0 Hz, CH₂), 4.27 (t,1H, J ¼ 7.5 Hz, CH), 4.01e3.95 (m, 2H,

CH₂), 2.84e2.52 (m, 7H, CH₂þCH), 1.80e1.76 (m, 2H, CH₂),

1.56e1.42 (m, 2H, CH₂), 1.29 (d, 3H, J ¼ 7.0 Hz, CH₃), 0.78 (s, 9H,

d

¼ 10.80 (s, 1H, NH), 9.34 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.57 (t, 1H,

J ¼ 6.0 Hz, NH), 8.40 (dd, 1H, J ¼ 2.5, 1.5 Hz, pyrazing-H), 8.35 (d, 1H,

J ¼ 2.5 Hz, pyrazine-H), 8.16 (d, 1H, J ¼ 8.0 Hz, NH), 7.76 (t, 1H,

J ¼ 6.0 Hz, NH), 7.43e7.41 (m, 1H, Ar-H), 7.30e7.18 (m, 7H, Ar-H),

7.17e7.15 (m, 1H, Ar-H), 6.66 (d, 1H, J ¼ 7.5 Hz, NH), 4.49e4.44

(m,1H, CH), 4.38 (q,1H, J ¼ 8.0 Hz, CH₂), 4.32 (d, 2H, J ¼ 5.5 Hz, CH₂),

4.00e3.91 (m, 2H, CH þ CH₂), 3.10 (dd, 1H, J ¼ 14.0, 5.0 Hz, CH₂),

2.93 (dd, 1H, J ¼ 13.5, 8.0 Hz, CH₂), 2.85 (dd, 1H, J ¼ 13.0, 6.5 Hz,

CH₂), 2.77 (dd, 1H, J ¼ 14.0, 5.5 Hz, CH₂), 2.73e2.50 (m, 3H,

CH þ CH₂), 2.52e2.43 (m, 2H, CH₂), 1.80e1.75 (m, 2H, CH₂),

1.49e1.41 (m, 2H, CH₂), 0.80 (s, 9H, CH₃); ¹³C NMR (100 MHz,

CH₃); ¹³C NMR (100 MHz, DMSO-d₆):

d

¼ 174.67, 172.48, 170.58,

157.31, 149.35, 143.03, 140.07, 136.75, 136.68, 132.31, 129.42, 128.97,

128.91, 127.52, 52.38, 50.15, 49.06, 43.53, 43.41, 42.48, 38.22, 32.27,

28.40, 27.62, 18.52; ESI-MS: m/z ¼ 629.1 [MþH]^þ.

4.1.12.5. 4-(Pyrazine-2-carboxamido)piperidine-1-carbonyl-Asp(NH-

neopentyl)-Leu-NH-2-chlorobenzyl (25). White solid; Yield: 26%;

mp: 139e141 ^ꢀC; Purity: 98.9%; ¹H NMR (500 MHz, DMSO-d₆):

DMSO):

d

¼ 174.71, 172.62, 171.53, 170.60, 157.38, 149.30, 143.08,

140.08,138.25,136.71,136.40,132.30,129.60,129.39,129.05,128.95,

128.62, 127.52,126.79, 54.76, 52.40, 50.17, 43.45, 43.35, 42.43, 37.80,

37.38, 32.29, 28.35, 28.30, 27.60; ESI-MS: m/z ¼ 705.1 [MþH]^þ.

d

¼ 9.18 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.86 (d, 1H, J ¼ 2.5 Hz,

pyrazine-H), 8.82 (d, 1H, J ¼ 8.5 Hz, NH), 8.70 (dd, 1H, J ¼ 2.5, 1.5 Hz,

pyrazine-H), 8.52 (t, 1H, J ¼ 6.0 Hz, NH), 8.05 (d, 1H, J ¼ 8.0 Hz, NH),

7.79 (t, 1H, J ¼ 6.0 Hz, NH), 7.42e7.40 (m, 1H, Ar-H), 7.31e7.25 (m,

3H, Ar-H), 6.74 (d, 1H, J ¼ 7.5 Hz, NH), 4.39e4.25 (m, 4H, CH₂þCH),

4.02e3.91 (m, 3H, CH₂þCH), 2.86e2.71 (m, 4H, CH₂), 2.61e2.52 (m,

2H, CH₂), 1.73e1.71 (m, 2H, CH₂), 1.59e1.44 (m, 5H, CH₂þCH), 0.90

(d, 3H, J ¼ 7.0 Hz, CH₃), 0.84 (d, 3H, J ¼ 6.5 Hz, CH₃), 0.79 (s, 9H,

4.1.12.9. 4-(Pyrazine-2-carboxamido)piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-2-chlorobenzyl (29). White solid; Yield: 17%;

mp: 183e185 ^ꢀC; Purity: 97.9%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 9.17 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.86 (d, 1H, J ¼ 2.5 Hz,

pyrazine-H), 8.77 (d, 1H, J ¼ 8.5 Hz, NH), 8.70 (dd, 1H, J ¼ 2.5, 1.5 Hz,

pyrazine-H), 8.53 (t, 1H, J ¼ 6.0 Hz, NH), 8.22 (d, 1H, J ¼ 8.0 Hz, NH),

7.81 (t, 1H, J ¼ 6.0 Hz, NH), 7.42e7.40 (m, 1H, Ar-H), 7.28e7.25 (m,

5H, Ar-H), 7.18e7.17 (m, 3H, Ar-H), 6.78 (d, 1H, J ¼ 7.5 Hz, NH), 4.42

(q,1H, J ¼ 7.5 Hz, CH), 4.33 (d, 2H, J ¼ 6.0 Hz, CH₂), 4.20e4.16 (m,1H,

CH), 4.00e3.95 (m, 3H, CH₂þCH), 2.85e2.74 (m, 4H, CH₂),

2.67e2.53 (m, 4H, CH₂), 2.09e2.02 (m, 1H, CH₂), 1.93e1.86 (m, 1H,

CH₂), 1.74e1.72 (m, 2H, CH₂), 1.59e1.45 (m, 2H, CH₂), 0.78 (s, 9H,

CH₃); ¹³C NMR (100 MHz, DMSO-d₆):

d

¼ 172.78, 172.74, 170.52,

162.62, 157.39, 147.92, 145.38, 144.09, 143.70, 136.65, 132.30, 129.43,

128.95, 127.53, 52.53, 51.74, 50.13, 47.08, 43.24, 43.08, 40.75, 37.85,

32.31, 31.45, 27.62, 24.66, 23.65, 21.84; ESI-MS: m/z ¼ 671.1

[MþH]^þ.

4.1.12.6. 4-(Pyrazin-2-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-Leu-NH-2-chlorobenzyl (26). White solid; Yield: 27%;

mp: 158e160 ^ꢀC; Purity: 99.5%; ¹H NMR (500 MHz, DMSO-d₆):

CH₃); ¹³C NMR (100 MHz, DMSO-d₆):

d

¼ 172.91, 172.15, 170.64,

162.65, 157.44, 147.89, 145.41, 144.06, 143.68, 141.86, 136.65, 132.36,

129.44, 129.11, 128.96, 128.88, 128.74, 127.50, 126.26, 52.85, 52.59,

50.19, 47.11, 43.26, 43.14, 37.91, 33.80, 32.27, 31.75, 31.47, 27.63; ESI-

MS: m/z ¼ 719.1 [MþH]^þ.

d

¼ 10.80 (s, 1H, NH), 9.33 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.52 (t, 1H,

J ¼ 5.5 Hz, NH), 8.40 (d, 1H, J ¼ 1.5 Hz, pyrazing-H), 8.35 (d, 1H,

J ¼ 2.5 Hz, pyrazine-H), 8.07 (d, 1H, J ¼ 8.0 Hz, NH), 7.78 (t, 1H,

J ¼ 6.0 Hz, NH), 7.43e7.40 (m, 1H, Ar-H), 7.32e7.26 (m, 3H, Ar-H),

6.71 (d, 1H, J ¼ 7.0 Hz, NH), 4.40 (q, 1H, J ¼ 8.0 Hz, CH), 4.36e4.26

(m, 3H, CH þ CH₂), 4.00e3.95 (m, 2H, CH₂), 2.86e2.75 (m, 2H, CH₂),

2.74e2.50 (m, 5H, CH þ CH₂), 1.80e1.73 (m, 2H, CH₂), 1.66e1.43 (m,

5H, CH þ CH₂), 0.90 (d, 3H, J ¼ 6.5 Hz, CH₃), 0.84 (d, 3H, J ¼ 6.5 Hz,

4.1.12.10. 4-(Pyrazin-2-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-2-chlorobenzyl (30). White solid; Yield: 15%;

mp: 191e193 ^ꢀC; Purity: 98.4%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 10.77 (s, 1H, NH), 9.33 (d, 1H, J ¼ 3.5 Hz, pyrazine-H), 8.52 (t, 1H,

CH₃), 0.80 (s, 9H, CH₃); ¹³C NMR (100 MHz, DMSO):

d

¼ 174.65,

J ¼ 6.0 Hz, NH), 8.39 (dd, 1H, J ¼ 2.5, 1.5 Hz, pyrazine-H), 8.34 (d, 1H,

J ¼ 2.5 Hz, pyrazine-H), 8.24 (d, 1H, J ¼ 7.5 Hz, NH), 7.81 (t, 1H,

J ¼ 6.0 Hz, NH), 7.42e7.40 (m, 1H, Ar-H), 7.32e7.25 (m, 5H, Ar-H),

7.18e7.15 (m, 3H, Ar-H), 6.75 (d, 1H, J ¼ 7.5 Hz, NH), 4.44 (q, 1H,

J ¼ 7.5 Hz, CH), 4.32 (d, 2H, J ¼ 5.5 Hz, CH₂), 4.19e4.15 (m, 1H, CH),

4.05e3.98 (m, 2H, CH₂), 2.85e2.54 (m, 9H, CH₂þCH), 2.10e2.03 (m,

1H, CH₂), 1.93e1.87 (m, 1H, CH₂), 1.79e1.75 (m, 2H, CH₂); 1.53e1.45

(m, 2H, CH₂), 0.78 (s, 9H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆):

172.71, 172.70, 170.60, 157.41, 149.36, 143.04, 140.06, 136.73, 136.69,

132.32, 129.41, 129.01, 128.90, 127.51, 52.40, 51.79, 50.16, 43.52,

43.43, 42.48, 37.83, 32.29, 28.40, 28.35, 27.64, 24.68, 23.61, 21.80;

ESI-MS: m/z ¼ 671.2 [MþH]^þ.

4.1.12.7. 4-(Pyrazine-2-carboxamido)piperidine-1-carbonyl-Asp(NH-

neopentyl)-Phe-NH-2-chlorobenzyl (27). White solid; Yield: 25%;

mp: 156e158 ^ꢀC; Purity: 99.2%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 174.65, 172.89, 172.15, 170.66, 157.45, 149.35, 143.03, 141.84,

d

¼ 9.18 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.86 (d, 1H, J ¼ 2.5 Hz,

140.06, 136.75, 136.67, 132.33, 129.43, 129.08, 128.93, 128.89,

128.73, 127.50, 126.25, 52.83, 52.46, 50.16, 43.55, 43.46, 42.47, 37.91,

33.71, 32.27, 31.75, 28.42, 28.37, 27.64; ESI-MS: m/z ¼ 719.1

[MþH]^þ.

pyrazine-H), 8.81 (d, 1H, J ¼ 8.5 Hz, NH), 8.70 (dd, 1H, J ¼ 2.5, 1.5 Hz,

pyrazine-H), 8.57 (t, 1H, J ¼ 6.0 Hz, NH), 8.12 (d, 1H, J ¼ 8.0 Hz, NH),

7.75 (t, 1H, J ¼ 6.0 Hz, NH), 7.42e7.40 (m, 1H, Ar-H), 7.25e7.19 (m,

7H, Ar-H), 7.16e7.14 (m, 1H, Ar-H), 6.70 (d, 1H, J ¼ 7.5 Hz, NH), 4.46

(td, 1H, J ¼ 8.5, 5.0 Hz, CH), 4.37e4.32 (m, 3H, CH₂þCH), 4.02e3.88

(m, 3H, CH₂þCH), 3.09 (dd, 1H, J ¼ 14.0, 5.0 Hz, CH₂), 2.94 (dd, 1H,

J ¼ 13.5, 9.0 Hz, CH₂), 2.86 (q, 1H, J ¼ 6.5 Hz, CH₂), 2.80e2.69 (m, 3H,

4.1.12.11. 4-(Pyrazine-2-carboxamido)piperidine-1-carbonyl-

Asp(NH-neopentyl)-hPhe-NH-2, 4-diﬂuorobenzyl (31). White solid;

Yield: 50%; mp: 153e155 ^ꢀC; Purity: 99.3%; ¹H NMR (500 MHz,

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

1067

DMSO-d₆):

d

¼ 9.18 (d, 1H, J ¼ 1.0 Hz, pyrazine-H), 8.86 (d, 1H,

120.76, 108.35, 108.27, 101.21, 52.72, 52.55, 50.20, 43.55, 43.46,

42.48, 42.35, 37.92, 33.83, 32.31, 31.75, 28.42, 27.67; ESI-MS: m/

z ¼ 729.4 [MþH]^þ.

J ¼ 2.5 Hz, pyrazine-H), 8.77 (d, 1H, J ¼ 8.0 Hz, NH), 8.71 (dd, 1H,

J ¼ 2.5, 1.5 Hz, pyrazine-H), 8.51 (t, 1H, J ¼ 5.5 Hz, NH), 8.14 (d, 1H,

J ¼ 8.0 Hz, NH), 7.82 (t, 1H, J ¼ 6.0 Hz, NH), 7.35 (q,1H, J ¼ 9.0 Hz, Ar-

H), 7.28e7.25 (m, 2H, Ar-H), 7.21e7.16 (m, 4H, Ar-H), 7.02 (dt, 1H,

J ¼ 8.5, 2.0 Hz, Ar-H), 6.76 (d, 1H, J ¼ 7.5 Hz, NH), 4.40 (q, 1H,

J ¼ 7.0 Hz, CH), 4.30e4.27 (m, 2H, CH₂), 4.18e4.13 (m, 1H, CH),

4.03e3.95 (m, 3H, CH þ CH₂), 2.87e2.78 (m, 4H, CH₂), 2.65e2.55

(m, 4H, CH₂), 2.08e2.00 (m, 1H, CH₂), 1.89e1.84 (m, 1H, CH₂),

1.76e1.73 (m, 2H, CH₂), 1.59e1.48 (m, 2H, CH₂), 0.81 (s, 9H, CH₃);

4.1.12.15. 4-(4-ﬂuorobenzamido)

piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-2-chlorobenzyl (35). White solid; Yield: 28%;

mp: 159e161 ^ꢀC; Purity: 99.3%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 8.52 (t, 1H, J ¼ 6.0 Hz, NH), 8.29 (d, 1H, J ¼ 8.0 Hz, NH), 8.23 (d,

1H, J ¼ 8.0 Hz, NH), 7.93e7.89 (m, 2H, Ar-H), 7.80 (t, 1H, J ¼ 6.0 Hz,

NH), 7.43e7.41 (m,1H, Ar-H), 7.34e7.26 (m, 7H, Ar-H), 7.20e7.16 (m,

3H, Ar-H), 6.75 (d, 1H, J ¼ 7.5 Hz, NH), 4.43 (q, 1H, J ¼ 7.5 Hz, CH),

4.33 (d, 2H, J ¼ 6.0 Hz, CH₂), 4.22e4.17 (m, 1H, CH), 4.00e3.93 (m,

3H, CH þ CH₂), 2.87e2.77 (m, 4H, CH₂), 2.68e2.55 (m, 4H, CH₂),

2.10e2.05 (m, 1H, CH₂), 1.95e1.88 (m, 1H, CH₂), 1.78e1.73 (m, 2H,

CH₂), 1.48e1.35 (m, 2H, CH₂), 0.80 (s, 9H, CH₃); ¹³C NMR (100 MHz,

¹³C NMR (100 MHz, DMSO-d₆):

d

¼ 172.83, 172.04, 170.63, 162.65,

157.44, 147.89, 145.41, 144.07, 143.68, 141.85, 131.06, 128.86, 128.73,

126.26, 122.83, 111.70, 104.22, 103.96, 103.71, 52.75, 52.60, 50.18,

47.11, 43.26, 43.14, 37.89, 36.00, 35.95, 33.80, 32.29, 31.71, 31.48,

27.64; ESI-MS: m/z ¼ 721.4 [MþH]^þ.

DMSO):

d

¼ 174.65, 172.97, 172.17, 170.62, 169.25, 165.02, 157.48,

4.1.12.12. 4-(Pyrazin-2-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-2, 4-diﬂuorobenzyl (32). White solid; Yield:

38%; mp: 168e170 ^ꢀC; Purity: 99.2%; ¹H NMR (500 MHz, DMSO-d₆):

141.85, 136.66, 132.34, 132.15, 131.57, 130.44, 129.44, 129.07, 128.90,

128.75, 127.51, 126.28, 115.64, 115.42, 53.79, 53.32, 52.86, 52.53,

50.17, 47.30, 43.29, 43.10, 37.95, 36.15, 33.74, 32.28, 31.76, 27.63; ESI-

MS: m/z ¼ 735.3 [MþH]^þ.

d

¼ 10.76 (s, 1H, NH), 9.33 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.49 (t, 1H,

J ¼ 6.0 Hz, NH), 8.40 (dd, 1H, J ¼ 2.5, 1.5 Hz, pyrazine-H), 8.35 (d, 1H,

J ¼ 2.5 Hz, pyrazine-H), 8.17 (d, 1H, J ¼ 7.5 Hz, NH), 7.81 (t, 1H,

J ¼ 6.0 Hz, NH), 7.35 (q, 1H, J ¼ 9.0 Hz, Ar-H), 7.28e7.13 (m, 6H, Ar-

H), 7.02 (dt, 1H, J ¼ 8.5, 2.0 Hz, Ar-H), 6.74 (d, 1H, J ¼ 7.5 Hz, NH),

4.42 (q, 1H, J ¼ 7.0 Hz, CH), 4.30e4.27 (m, 2H, CH₂), 4.17e4.13 (m,

1H, CH), 4.05e3.98 (m, 2H, CH₂), 2.85e2.54 (m, 9H, CH þ CH₂),

2.06e2.03 (m, 1H, CH₂), 1.89e1.77 (m, 3H, CH₂), 1.53e1.46 (m, 2H,

4.1.12.16. 4-(4-ﬂuorophenylcarbamoyl)piperidine-1-carbonyl-

Asp(NH-neopentyl)-hPhe-NH-2-chlorobenzyl (36). White solid;

Yield: 35%; mp: 161e163 ^ꢀC; Purity: 98.2%; ¹H NMR (500 MHz,

DMSO-d₆):

d

¼ 9.94 (s, 1H, NH), 8.52 (t, 1H, J ¼ 6.0 Hz, NH), 8.23 (d,

1H, J ¼ 8.0 Hz, NH), 7.80 (t, 1H, J ¼ 6.0 Hz, NH), 7.63e7.60 (m, 2H, Ar-

H), 7.43e7.41 (m, 1H, Ar-H), 7.33e7.26 (m, 5H, Ar-H), 7.19e7.11 (m,

5H, Ar-H), 6.74 (d, 1H, J ¼ 7.5 Hz, NH), 4.45 (q, 1H, J ¼ 7.5 Hz, CH),

4.33 (d, 2H, J ¼ 6.0 Hz, CH₂), 4.21e4.17 (m, 1H, CH), 4.06e3.99 (m,

2H, CH₂), 2.87e2.56 (m, 9H, CH þ CH₂), 2.08e2.06 (m, 1H, CH₂),

1.93e1.88 (m, 1H, CH₂), 1.77e1.73 (m, 2H, CH₂), 1.57e1.45 (m, 2H,

CH₂), 0.81 (s, 9H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆):

d

¼ 174.65,

172.83, 172.06, 170.65, 157.46, 149.36, 143.02, 141.84, 140.06, 136.75,

131.04, 128.87, 128.72, 126.24, 122.85, 111.69, 104.20, 103.95, 103.69,

52.74, 52.50, 50.17, 43.55, 43.46, 42.48, 37.90, 35.98, 35.95, 33.71,

32.29, 31.73, 28.42, 28.38, 27.64; ESI-MS: m/z ¼ 721.4 [MþH]^þ.

CH₂), 0.80 (s, 9H, CH₃); ¹³C NMR (100 MHz, DMSO):

d

¼ 173.40,

172.92, 172.17, 170.67, 157.47, 141.84, 136.68, 136.17, 132.34, 129.43,

129.08, 128.90, 128.73, 127.49, 126.26, 121.35, 121.28, 115.74, 115.52,

52.85, 52.48, 50.17, 43.60, 43.50, 43.11, 37.93, 33.72, 32.27, 31.77,

28.59, 27.64; ESI-MS: m/z ¼ 735.1 [MþH]^þ.

4.1.12.13. 4-(Pyrazine-2-carboxamido)piperidine-1-carbonyl-

Asp(NH-neopentyl)-hPhe-NH-benzo[d][1,3]dioxol-5-ylmethyl

White solid; Yield: 39%; mp: 174e176 ^ꢀC; Purity: 99.4%; ¹H NMR

(500 MHz, DMSO-d₆):

¼ 9.17 (d, 1H, J ¼ 1.5 Hz, pyrazine-H), 8.86

(33).

d

(d, 1H, J ¼ 2.5 Hz, pyrazine-H), 8.54 (d, 1H, J ¼ 8.5 Hz, NH), 8.69 (dd,

1H, J ¼ 2.5, 1.5 Hz, pyrazine-H), 8.44 (t, 1H, J ¼ 6.0 Hz, NH), 8.09 (d,

1H, J ¼ 8.0 Hz, NH), 7.80 (t, 1H, J ¼ 6.0 Hz, NH), 7.27e7.14 (m, 5H, Ar-

H), 6.82e6.76 (m, 3H, Ar-H), 6.70 (d, 1H, J ¼ 8.0 Hz, NH), 5.95 (s, 2H,

OCH₂O), 4.39 (q, 1H, J ¼ 7.0 Hz, CH), 4.24e4.14 (m, 3H, CH þ CH₂),

4.01e3.95 (m, 3H, CH₂þCH), 2.89e2.76 (m, 4H, CH₂), 2.65e2.52 (m,

4H, CH₂), 2.07e1.99 (m, 1H, CH₂), 1.88e1.81 (m, 1H, CH₂), 1.75e1.73

(m, 2H, CH₂), 1.59e1.48 (m, 2H, CH₂), 0.81 (s, 9H, CH₃); ¹³C NMR

4.1.12.17. 4-(4-methoxyphenylcarbamoyl)piperidine-1-carbonyl-

Asp(NH-neopentyl)-hPhe-NH-2-chlorobenzyl (37). White solid;

Yield: 16%; mp: 157e159 ^ꢀC; Purity: 98.8%; ¹H NMR (500 MHz,

DMSO-d₆):

d

¼ 9.74 (s, 1H, NH), 7.76 (t, 1H, J ¼ 6.0 Hz, NH), 7.52 (d,

2H, J ¼ 8.0 Hz, Ar-H), 7.45e7.43 (m, 1H, Ar-H), 7.35e7.16 (m, 6H, Ar-

H), 6.86 (d, 2H, J ¼ 9.0 Hz, Ar-H), 6.81 (d, 1H, J ¼ 7.5 Hz, NH),

4.46e4.36 (m, 2H, CH þ CH), 4.01e3.95 (m, 2H, CH₂), 3.72 (s, 3H,

CH₃), 3.40e3.32 (m, 2H, CH₂), 2.95 (dd, 1H, J ¼ 13.0, 6.5 Hz, CH₂),

2.82 (dd, 1H, J ¼ 13.0, 6.5 Hz, CH₂), 2.74e2.56 (m, 6H, CH₂),

2.48e2.44 (m, 1H, CH), 1.94e1.88 (m, 1H, CH₂), 1.75e1.69 (m, 2H,

CH₂), 1.52e1.46 (m, 2H, CH₂), 0.84 (s, 9H, CH₃); ¹³C NMR (100 MHz,

(100 MHz, DMSO-d₆):

d

¼ 172.74, 171.70, 170.62, 162.65, 157.43,

147.89, 147.66, 146.44, 145.41, 144.07, 143.68, 141.90, 133.77, 128.85,

128.73, 126.25, 120.78, 108.36, 108.28, 101.22, 52.72, 52.66, 50.22,

47.11, 43.28, 43.16, 42.35, 37.91, 33.94, 32.31, 31.72, 31.49, 27.66; ESI-

MS: m/z ¼ 729.3 [MþH]^þ.

DMSO):

d

¼ 175.36, 173.51, 173.01, 169.84, 157.22, 155.54, 142.38,

136.90, 132.95, 132.52, 129.56, 129.29, 129.06,128.77, 128.74,127.59,

126.17, 121.14, 114.24, 55.62, 54.80, 52.17, 51.52, 50.20, 43.61, 43.54,

43.11, 37.87, 37.27, 32.41, 31.92, 28.63, 27.66, 21.61; ESI-MS: m/

z ¼ 747.1 [MþH]^þ.

4.1.12.14. 4-(Pyrazin-2-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-

White solid; Yield: 13%; mp: 183e185 ^ꢀC; Purity: 98.9%; ¹H NMR

(500 MHz, DMSO-d₆):

¼ 10.76 (s, 1H, NH), 9.33 (d, 1H, J ¼ 1.5 Hz,

benzo[d][1,3]dioxol-5-ylmethyl

(34).

d

4.1.12.18. 4-(4-(triﬂuoromethyl)phenylcarbamoyl)piperidine-1-

pyrazine-H), 8.43 (t, 1H, J ¼ 6.0 Hz, NH), 8.40 (dd, 1H, J ¼ 2.5, 1.5 Hz,

pyrazine-H), 8.35 (d, 1H, J ¼ 2.5 Hz, pyrazine-H), 8.12 (d, 1H,

J ¼ 7.5 Hz, NH), 7.81 (t, 1H, J ¼ 6.0 Hz, NH), 7.28e7.24 (m, 2H, Ar-H),

7.18e7.15 (m, 3H, Ar-H), 6.83e6.81 (m, 2H, Ar-H), 6.75e6.71 (m, 2H,

NH þ Ar-H), 5.96 (s, 2H, OCH₂O), 4.42 (q, 1H, J ¼ 7.0 Hz, CH),

4.23e4.12 (m, 3H, CH þ CH₂), 4.05e3.99 (m, 2H, CH₂), 2.90e2.56

(m, 9H, CH þ CH₂), 2.08e2.01 (m, 1H, CH₂), 1.89e1.72 (m, 3H, CH₂),

1.52e1.43 (m, 2H, CH₂), 0.82 (s, 9H, CH₃); ¹³C NMR (100 MHz,

carbonyl-Asp(NH-neopentyl)-hPhe-NH-2-chlorobenzyl

White solid; Yield: 14%; mp: 138e140 ^ꢀC; Purity: 98.6%; ¹H NMR

(500 MHz, DMSO-d₆):

¼ 10.02 (s, 1H, NH), 8.51 (t, 1H, J ¼ 6.0 Hz,

(38).

d

NH), 8.21 (d, 1H, J ¼ 8.0 Hz, NH), 7.80 (t, 1H, J ¼ 6.0 Hz, NH), 7.56 (d,

2H, J ¼ 8.0 Hz, Ar-H), 7.43e7.41 (m, 1H, Ar-H), 7.33e7.28 (m, 6H, Ar-

H), 7.19e7.15 (m, 3H, Ar-H), 6.73 (d, 1H, J ¼ 7.5 Hz, NH), 4.44 (q, 1H,

J ¼ 7.0 Hz, CH), 4.33 (d, 2H, J ¼ 6.0 Hz, CH₂), 4.21e4.16 (m, 1H, CH),

4.06e3.99 (m, 2H, CH₂), 2.84e2.57 (m, 9H, CH þ CH₂), 2.10e2.03

(m, 1H, CH₂), 1.94e1.88 (m, 1H, CH₂), 1.76e1.73 (m, 2H, CH₂),

1.52e1.44 (m, 2H, CH₂), 0.79 (s, 9H, CH₃); ¹³C NMR (100 MHz,

DMSO-d₆):

d

¼ 174.65, 172.76, 171.71, 170.64, 157.46, 149.36, 147.67,

146.43, 143.03, 141.89, 140.06, 136.75, 133.80, 128.87, 128.71, 126.23,

1068

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

DMSO):

d

¼ 173.82, 172.90, 172.16, 170.66, 157.45, 141.84, 136.67,

2.86e2.55 (m, 9H, CH þ CH₂), 2.10e2.02 (m, 1H, CH₂), 1.95e1.86 (m,

1H, CH₂),1.81e1.73 (m, 2H, CH₂),1.56e1.44 (m, 2H, CH₂), 0.80 (s, 9H,

132.34, 130.20, 129.43, 129.08, 128.93, 128.89, 128.73, 127.49,

126.25,126.13,126.09,118.92, 52.84, 52.46, 50.17, 43.57, 43.47, 43.19,

37.92, 33.72, 32.27, 31.75, 28.53, 27.63; ESI-MS: m/z ¼ 785.1

[MþH]^þ.

CH₃); ¹³C NMR (100 MHz, DMSO):

d

¼ 173.40, 172.90, 172.16, 170.66,

158.46, 157.44, 141.84, 138.02, 136.67, 132.34, 129.43, 129.09, 128.92,

128.89, 128.73, 127.50, 126.25, 113.83, 52.85, 52.48, 50.17, 43.50,

43.41, 41.75, 37.93, 33.70, 32.27, 31.76, 28.26, 27.64; ESI-MS: m/

z ¼ 724.1 [MþH]^þ.

4.1.12.19. 4-(pyridin-3-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-2-chlorobenzyl (39). White solid; Yield: 31%;

mp: 163e165 ^ꢀC; Purity: 99.2%; ¹H NMR (500 MHz, DMSO-d₆):

4.1.12.23. 4-(thiazol-2-yl-methylcarbamoyl)piperidine-1-carbonyl-

Asp(NH-neopentyl)-hPhe-NH-2-chlorobenzyl (43). White solid;

Yield: 12%; mp: 166e168 ^ꢀC; Purity: 98.8%; ¹H NMR (500 MHz,

d

¼ 10.08 (s, 1H, NH), 8.71 (d, 1H, J ¼ 2.5 Hz, pyridine-H), 8.49 (t, 1H,

J ¼ 6.0 Hz, NH), 8.23e8.20 (m, 2H, pyridine-H þ NH), 8.02 (dt, 1H,

J ¼ 10.5, 2.0 Hz, pyridine-H), 7.78 (t, 1H, J ¼ 6.0 Hz, NH), 7.41e7.39

(m, 1H, Ar-H), 7.32e7.23 (m, 6H, Ar-H), 7.17e7.13 (m, 3H, pyridine-

H þ Ar-H), 6.72 (d, 1H, J ¼ 7.5 Hz, NH), 4.43 (q, 1H, J ¼ 6.5 Hz, CH),

4.31 (d, 2H, J ¼ 5.5 Hz, CH₂), 4.19e4.14 (m, 1H, CH), 4.04e3.97 (m,

2H, CH₂), 2.82e2.52 (m, 9H, CH þ CH₂), 2.08e2.01 (m, 1H, CH₂),

1.92e1.87 (m, 1H, CH₂), 1.77e1.73 (m, 2H, CH₂), 1.54e1.44 (m, 2H,

DMSO-d₆):

d

¼ 8.74 (t, 1H, J ¼ 6.0 Hz, NH), 8.52 (t, 1H, J ¼ 6.0 Hz,

NH), 8.24 (d, 1H, J ¼ 8.0 Hz, NH), 7.81 (t, 1H, J ¼ 6.0 Hz, NH), 7.71 (d,

1H, J ¼ 3.5 Hz, thiazol-H), 7.60 (d, 1H, J ¼ 3.0 Hz, thiazol-H),

7.43e7.41 (m, 1H, Ar-H), 7.33e7.27 (m, 5H, Ar-H), 7.26e7.17 (m,

3H, Ar-H), 6.72 (d, 1H, J ¼ 7.5 Hz, NH), 4.53 (d, 2H, J ¼ 6.0 Hz, CH₂),

4.43 (q, 1H, J ¼ 7.5 Hz, CH), 4.32 (d, 2H, J ¼ 6.0 Hz, CH₂), 4.19e4.15

(m, 1H, CH), 4.00e3.94 (m, 2H, CH₂), 2.85e2.54 (m, 8H, CH₂),

2.54e2.35 (m, 1H, CH), 2.10e2.03 (m, 1H, CH₂), 1.94e1.87 (m, 1H,

CH₂), 1.72e1.63 (m, 2H, CH₂), 1.51e1.47 (m, 2H, CH₂), 0.78 (s, 9H,

CH₂), 0.77 (s, 9H, CH₃); ¹³C NMR (100 MHz, DMSO):

d

¼ 174.09,

172.91, 172.16, 170.69, 157.47, 144.51, 141.84, 141.26, 136.67, 136.37,

132.34, 129.43, 129.08, 128.94, 128.89, 128.73,127.50, 126.51,126.26,

124.03, 52.84, 52.47, 50.17, 43.56, 43.47, 43.07, 37.93, 33.72, 32.28,

31.76, 28.54, 28.51, 27.64; ESI-MS: m/z ¼ 718.2 [MþH]^þ.

CH₃); ¹³C NMR (100 MHz, DMSO):

d

¼ 174.72, 172.91, 172.16, 170.65,

170.27, 157.43, 142.66, 141.84, 136.68, 132.33, 129.43, 129.07, 128.95,

128.89, 128.74, 127.50, 126.26, 120.32, 52.82, 52.47, 50.16, 43.58,

43.45, 41.99, 37.93, 33.70, 32.27, 31.75, 31.62, 29.44, 28.54, 27.64;

ESI-MS: m/z ¼ 738.3 [MþH]^þ.

4.1.12.20. 4-(pyridin-2-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-2-chlorobenzyl (40). White solid; Yield: 26%;

mp: 140e142 ^ꢀC; Purity: 99.1%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 10.43 (s, 1H, NH), 8.51 (t, 1H, J ¼ 6.0 Hz, NH), 8.32e8.30 (m, 1H,

4.1.12.24. 4-Fluorophenyl-carbonyl-Asp(NH-neopentyl)-hPhe-NH-2-

pyridine-H), 8.22 (d, 1H, J ¼ 7.5 Hz, NH), 8.07 (d, 1H, J ¼ 8.0 Hz,

pyridine-H), 7.81e7.74 (m, 2H, NH þ pyridine-H), 7.43e7.41 (m, 1H,

Ar-H), 7.33e7.17 (m, 7H, Ar-H), 7.10e7.07 (m, 1H, pyridine-H), 6.73

(d, 1H, J ¼ 7.5 Hz, NH), 4.44 (q, 1H, J ¼ 7.0 Hz, CH), 4.32 (d, 2H,

J ¼ 6.0 Hz, CH₂), 4.21e4.16 (m, 1H, CH), 4.05e3.98 (m, 2H, CH₂),

2.87e2.55 (m, 9H, CH þ CH₂), 2.10e2.03 (m, 1H, CH₂), 1.95e1.87 (m,

1H, CH₂),1.77e1.74 (m, 2H, CH₂),1.53e1.44 (m, 2H, CH₂), 0.80 (s, 9H,

chlorobenzyl. White solid; Yield: 71%; mp: 141e143 ^ꢀC; Purity:

99.4%; ¹H NMR (500 MHz, DMSO-d₆):

d

¼ 8.77 (d, 1H, J ¼ 9.0 Hz,

NH), 8.53 (t, 1H, J ¼ 6.0 Hz, NH), 8.39 (d, 1H, J ¼ 10.0 Hz, NH),

7.99e7.90 (m, 3H, NH þ Ar-H), 7.43e7.41 (m, 1H, Ar-H), 7.35e7.21

(m, 7H, Ar-H), 7.22e7.14 (m, 3H, Ar-H), 4.82 (q, 1H, J ¼ 9.0 Hz, CH),

4.35 (d, 2H, J ¼ 7.5 Hz, CH₂), 4.27e4.21 (m, 1H, CH), 2.87e2.71 (m,

4H, CH₂), 2.64e2.53 (m, 2H, CH₂), 2.12e2.04 (m, 1H, CH₂), 1.96e1.88

(m, 1H, CH₂), 0.77 (s, 9H, CH₃); ¹³C NMR (100 MHz, DMSO):

CH₃); ¹³C NMR (100 MHz, DMSO):

d

¼ 174.45, 172.92, 172.17, 170.67,

157.45, 152.61, 148.36, 141.84, 138.52, 136.67, 132.33, 129.43, 129.08,

128.93, 128.90, 128.73, 127.50, 126.26, 119.72, 113.97, 52.84, 52.49,

52.17, 50.17, 43.61, 43.50, 42.67, 37.92, 33.70, 32.28, 31.76, 28.53,

28.48, 27.64; ESI-MS: m/z ¼ 718.1 [MþH]^þ.

d

¼ 172.07, 171.75, 170.34, 165.80, 141.77, 136.63, 132.36, 130.91,

130.88, 130.67, 130.58, 129.45, 129.09, 128.96, 128.85, 128.71, 127.51,

126.26, 115.73, 115.52, 52.97, 51.61, 50.20, 42.56, 37.62, 36.70, 34.61,

33.78, 32.26, 31.77, 27.62; ESI-MS: m/z ¼ 609.3 [MþH]^þ.

4.1.12.21. 4-(isoxazol-3-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-2-chlorobenzyl (41). White solid; Yield: 38%;

mp: 149e151 ^ꢀC; Purity: 98.9%; ¹H NMR (500 MHz, DMSO-d₆):

4.2. Biological evaluation

4.2.1. In vitro 20S proteasome chymotrypsin-like inhibition assay

d

¼ 11.00 (s, 1H, NH), 8.77 (d, 1H, J ¼ 2.0 Hz, isoxazol-H), 8.51 (t, 1H,

Chymotrypsin-like enzyme activity assay was carried out in

J ¼ 5.5 Hz, NH), 8.22 (d, 1H, J ¼ 7.5 Hz, NH), 7.79 (t, 1H, J ¼ 6.0 Hz,

NH), 7.43e7.41 (m, 1H, Ar-H), 7.34e7.25 (m, 5H, Ar-H), 7.19e7.15 (m,

3H, Ar-H), 6.92 (d, 1H, J ¼ 1.5 Hz, isoxazol-H), 6.73 (d, 1H, J ¼ 7.5 Hz,

NH), 4.44 (q, 1H, J ¼ 7.0 Hz, CH), 4.33 (d, 2H, J ¼ 6.0 Hz, CH₂),

4.21e4.16 (m, 1H, CH), 4.04e3.97 (m, 2H, CH₂), 2.84e2.55 (m, 9H,

CH þ CH₂), 2.10e2.04 (m, 1H, CH₂), 1.94e1.88 (m, 1H, CH₂),

1.77e1.73 (m, 2H, CH₂), 1.52e1.41 (m, 2H, CH₂), 0.80 (s, 9H, CH₃);

50

buffer (100 mM Tris-HCl, pH 8.0). The reaction was carried out in

black 384-well plates.1 L compound was added into 10 L puriﬁed

human proteasome (25 g/mL), a gift from Dr. Jiang-ping Wu

(Notre-Dame Hospital, Montreal, Quebec, Canada), incubated for

15 min, and then added with 39 L synthesized substrate Suc-Leu-

Leu-Val-Tyr-AMC (50 M, GL Biochem Ltd., Shanghai, P.R. China) as

mL volume and all the assay components were diluted in Tris-HCl

m

¹³C NMR (100 MHz, DMSO):

d

¼ 173.84, 172.89, 172.16, 170.66,

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 IC₅₀data was calculated using

the software GraphPad Prism, and chosen the equation “sigmoidal

dose-response (variable slope)” for curve ﬁtting.

160.43, 158.09, 157.44, 141.84, 136.67, 132.34, 129.43, 129.09, 128.94,

128.89, 128.73, 127.49, 126.26, 99.57, 52.84, 52.47, 50.17, 43.50,

43.41, 42.41, 37.92, 33.71, 32.27, 31.76, 28.30, 27.63; ESI-MS: m/

z ¼ 708.1 [MþH]^þ.

4.1.12.22. 4-(thiazol-2-ylcarbamoyl)piperidine-1-carbonyl-Asp(NH-

neopentyl)-hPhe-NH-2-chlorobenzyl (42). White solid; Yield: 39%;

mp: 157e159 ^ꢀC; Purity: 99.5%; ¹H NMR (500 MHz, DMSO-d₆):

4.2.2. Tumor cell anti-proliferation assay

3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-

(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Prom-

ega (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 37 ^ꢀC in a 5% CO₂humidiﬁed

d

¼ 12.09 (s, 1H, NH), 8.51 (t, 1H, J ¼ 6.0 Hz, NH), 8.22 (d, 1H,

J ¼ 7.5 Hz, NH), 7.80 (t, 1H, J ¼ 5.5 Hz, NH), 7.46 (d, 1H, J ¼ 2.5 Hz,

thiazol-H), 7.42 (d, 1H, J ¼ 5.0 Hz, thiazol-H), 7.31e7.15 (m, 8H, Ar-

H), 6.74 (d, 1H, J ¼ 7.5 Hz, NH), 4.44 (q, 1H, J ¼ 7.0 Hz, CH), 4.33 (d,

2H, J ¼ 5.0 Hz, CH₂), 4.19 (brs, 1H, CH), 4.04e3.98 (m, 2H, CH₂),

R. Zhuang et al. / European Journal of Medicinal Chemistry 126 (2017) 1056e1070

1069

atmosphere.

A 100

4.3. Molecular modeling

m

L MM-1S (2 ꢂ 10⁴/well) or RPMI 8226 cells (0.5 ꢂ 10⁴/

well) were seeded into 96-well plates. After treated with tested

compounds for 72 h, cells were added with MTS at a ﬁnal con-

centration of 0.5 mg/mL for 2e4 h. 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 inhibi-

tory ratio ¼ (A_controleA_sample)/A_control. IC₅₀values 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).

The molecular docking procedure was performed by using Gli-

€

deSP in Maestro (Schrodinger version 10.7) with the default option

[23,24]. The co-crystal structure of proteasome (PDB ID code:

3MG6) was selected as the docking template. For the preparation of

protein, the hydrogen atoms were added by using protein prepa-

ration Wizard module of Maestro [24], and the OPLS3 force ﬁeld

was employed. For the preparation of ligands, the 3D structures

were generated and their energy minimization was performed by

using LigPrep [25]. Conformers were generated by using ConfGen

[26]. A 30 Å docking grid was generated using centroid of ligand in

the 3MG6 crystal structure. Then the ligand was removed and

target compound was placed during the molecular docking pro-

cedure. Types of interaction of the docked proteasome with ligand

were analyzed and then the docking conformations were selected

and saved based on calculated glide docking energy score.

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 1000 rpm for 5 min and the su-

Conﬂict of interest

pernatants are removed. Blood cells were added 100

mL EDTA

(5 mM, pH¼8.0) and lysed at 4 ^ꢀC for 60 min, then centrifuged at

6600 ꢂ 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 authors conﬁrm that this article content has no conﬂict of

interest.

Acknowledgements

The assays were carried out in a ﬁnal volume of 50

20 mmol/L HEPES, pH¼8.0, 0.5 mmol/L EDTA, 0.05% SDS, 50

substrate (Suc-Leu-Leu-Val-Tyr-AMC), 1% DMSO, and blood cell

protein 100 g, and detected at 355 nm excitation and 460 nm

mL containing

mM

The authors thank the project supported by the Key Develop-

ment Program of the Hangzhou Science and Technology Committee

(20152013A03, 20142013A60), the normal project supported by the

Hangzhou Science and Technology Committee (20150733Q49) and

National Natural Science Foundation of China (81473244) for

ﬁnancial support.

m

Emission as the in vitro assay mentioned.

4.2.4. In vivo blood cell proteasome inhibition assay

6-8 weeks normal BALB/c mice were randomized into treatment

groups (3 mice per group), and administrated i.v. with vehicle,

tested compound and positive control carﬁlzomib (5 mg/kg) once.

After 1, 2, and 24 h, whole blood were collected in K2-EDTA he-

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2016.12.034.

matocrit tubes via tail vein. 50

500

L PBS, centrifuged with 3300 rpm for 10 min at 4 ^ꢀC. After

discarding the supernants, 100

mL bloods were washed once with

m

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Article Doi

Exploration of novel piperazine or piperidine constructed non-covalent peptidyl derivatives as proteasome inhibitors

DOI: 10.1016/j.ejmech.2016.12.034

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Full text of DOI:10.1016/j.ejmech.2016.12.034

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