Terminal functionalized thiourea-containing dipeptides as multidrug-resistance reversers that target 20S proteasome and cell proliferation

Article abstract of DOI:10.1016/j.ejmech.2016.11.024

A series of inhibitors of 20S proteasome based on terminal functionalized dipeptide derivatives containing the thiourea moiety were synthesized and evaluated for inhibition of 20S proteasome and the effects of multidrug-resistance reversers. These compounds exhibited significant selectivity to the β5-subunit of the human 20S proteasome with IC₅₀values at submicromolar concentrations. A docking study of the most active compound 6i revealed key interactions between 6i and the active site of the 20S proteasome in which the thiourea moiety and a nitro group were important for improving activity. In particular, compound 6i appeared to be the most potent compound against the NCI-H460 cell line, and displayed similar efficiency in drug-sensitive versus drug-resistant cancer cell lines, at least partly, by inhibition of the activity of 20S proteasome and induce apoptosis. In addition, 6i-induced apoptosis was significantly facilitated in NCI-H460/DOX cells that had been pretreated with inhibitors of P-gp. Mechanistically, compound 6i might trigger apoptotic signalling pathway. Thus, we conclude that dipeptide derivatives containing the thiourea moiety may be the potential inhibitors of proteasome with the ability to reverse multidrug resistance.

Full text of DOI:10.1016/j.ejmech.2016.11.024

Accepted Manuscript
Terminal functionalized thiourea-containing dipeptides as multidrug-resistance
reversers that target 20S proteasome and cell proliferation
Jian-Mei Qin, Ri-Zheng Huang, Gui-Yang Yao, Zhi-Xin Liao, Ying-Ming Pan, Heng-
Shan Wang
PII:
S0223-5234(16)30962-X
10.1016/j.ejmech.2016.11.024
EJMECH 9057
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 September 2016
Revised Date: 7 November 2016
Accepted Date: 10 November 2016
Please cite this article as: J.-M. Qin, R.-Z. Huang, G.-Y. Yao, Z.-X. Liao, Y.-M. Pan, H.-S. Wang,
Terminal functionalized thiourea-containing dipeptides as multidrug-resistance reversers that target
20S proteasome and cell proliferation, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.11.024.
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Graphical abstract
Terminal functionalized thiourea-containing dipeptides as
multidrug-resistance reversers that target 20S proteasome and cell
proliferation
Jian-Mei Qin, Ri-Zheng Huang, Gui-Yang Yao, Zhi-Xin Liao, Ying-Ming Pan and Heng-Shan
Wang
Proteasome inhibition
O
H
H
H
NO2
N
N
N
N
H
O
S
CF3
6i
Induction of apoptosis
Block multidrug-resistance
Inhibit cell proliferation

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Terminal functionalized thiourea-containing dipeptides as multidrug-resistance
reversers that target 20S proteasome and cell proliferation
Jian-Mei Qina, †, Ri-Zheng Huangb, †, Gui-Yang Yaob, Zhi-Xin Liaob, Ying-Ming Pana
and Heng-Shan Wanga
a State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of
Education of China), School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin
541004, PR China
b Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University,
Nanjing 211189, China
* Corresponding author.
*Corresponding author. E-mail addresses: whengshan@163.com (H. -S. Wang),
panym2013@hotmail.com (Y.-M. Pan).
† Co-first author: These authors contributed equally to this work.

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Abstract
A series of inhibitors of 20S proteasome based on terminal functionalized dipeptide
derivatives containing the thiourea moiety were synthesized and evaluated for inhibition of 20S
proteasome and the effects of multidrug-resistance reversers. These compounds exhibited
significant selectivity to the β5-subunit of the human 20S proteasome with IC50 values at
submicromolar concentrations. A docking study of the most active compound 6i revealed key
interactions between 6i and the active site of the 20S proteasome in which the thiourea moiety and
a nitro group were important for improving activity. In particular, compound 6i appeared to be the
most potent compound against the NCI-H460 cell line, and displayed similar efficiency in
drug-sensitive versus drug-resistant cancer cell lines, at least partly, by inhibition of the activity of
20S proteasome and induce apoptosis. In addition, 6i-induced apoptosis was significantly
facilitated in NCI-H460/DOX cells that had been pretreated with inhibitors of P-gp.
Mechanistically, compound 6i might trigger apoptotic signalling pathway. Thus, we conclude that
dipeptide derivatives containing the thiourea moiety may be the potential inhibitors of proteasome
with the ability to reverse multidrug resistance.
keywords: Pseudopeptides; Thioureas; Proteasome; Anti-tumor; Multidrug resistance

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1. Introduction
Multidrug resistance (MDR), the principal mechanism by which many cancers develop
resistance to a variety of chemotherapeutic drugs, is a major impediment to effective cancer
treatment with conventional chemotherapeutic drugs and has been intensively studied for the past
three decades. The molecular mechanisms of MDR are complex and include extrusion of the drug
by cell membrane pumps, such as P-glycoprotein (P-gp) or multidrug resistance-associated protein
(MRP), modification of drug target molecules, enhanced drug detoxification, suppression of
drug-induced apoptosis, increased DNA damage repair, upregulation of lipids and other
biochemical changes [1]. As clinical drug resistance frequently hinders the treatment of neoplastic
disease, extensive research has been carried out, but to date, no useful method for reversing MDR
suitable for clinical use has been developed, thus creating the need for new classes of potent and
specific anticancer drugs.
The ubiquitin-proteasome system (UPS), responsible for regulating cellular protein homeostasis
via enzymatic cascades, plays a crucial role in mediating many normal cellular processes,
including the cell cycle, differentiation and apoptosis [2, 3]. The proteasome is a major site for
degradation of abnormal or irrelevant and regulatory proteins, such as transcription factors,
oncoproteins, cyclins and anti-apoptotic proteins, which might be responsible for various types of
drug resistance [2]. The 20S proteasome, composed of three distinct proteolytically active sites, β5,
β2 and β1, is responsible for chymotrypsin-like (ChT-L), trypsin-like (T-L), and post-glutamyl
peptide hydrolyzing (PGPH) or caspase-like proteolytic activities, respectively, and is the main
proteolytic component of this degradation system [4]. Given that the inhibition of proteasome
results in a myriad of cell responses and perturbed pathways, proteasome inhibitors provide a
solution for re-sensitizing tumor cells to chemotherapy or to delay the development of treatment
resistance [5]. Several proteasome inhibitors are in clinical trials or have been approved by the
FDA, such as bortezomib [6], carfilzomib [7] and ixazomib [8] (Fig. 1). Recent studies have
demonstrated the effectiveness of bortezomib, which is the first Food and Drug Administration
(FDA)-approved proteasome inhibitor, in the treatment of relapsed and/or refractory multiple
myeloma [9] and mantle cell lymphoma [10]. Bortezomib induces apoptosis in many types of
tumor cells through the formation of reactive oxygen species (ROS) and endoplasmic reticulum
(ER) stress [11-13]. The mitochondrial release of cytochrome c, apoptosis-inducing factor, and

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cleavage of caspases has been demonstrated in cells undergoing bortezomib-induced apoptosis [14,
15]. Moreover, the proteasome inhibitor, MG132, which effectively blocks the proteolytic activity
of the 26S proteasome complex, has been identified as an inhibitor of NF-κB, and suppression of
NF-κB activation causes downregulation of anti-apoptotic Bcl-2 and MDR1 (P-gp) expression
[16-18]. However, both bortezomib and the other proteasome inhibitors under clinical evaluation
are limited due to severe side effects, acquired drug resistance, peripheral neuropathy,
hematological toxicity and unsatisfactory pharmacokinetic profiles.
Due to these limitations, the development of pseudopeptides could lead to many
pharmacokinetic and pharmacodynamic advantages [19, 20]. This strategy can be realized by
introducing unnatural amino acids instead of natural residues into the peptide framework, or by
the introduction of a non-peptidic scaffold into the peptidic backbone, in order to lock a defined
conformation of the peptide [21, 22]. These novel compounds provide new perspectives in drug
design by providing an entire range of highly specific and non-toxic pharmaceuticals. In addition
to increased stability, the incorporation of a thiourea moiety into the peptide sequence also
provides access to additional binding interactions within the transition state conformation of the
enzyme/substrate complex [23, 24]. Thiourea could strongly enhance blocking since sulfur is the
weaker hydrogen bond acceptor as compared to the amide carbonyl oxygen, and simultaneously
enhance hydrogen-bonding due to the bidentate binding mode of the more acidic thiourea protons
[25, 26]. Some peptide scaffold based on thiourea or bis(thiourea) motif have been reported as
inhibitors of β-sheet aggregation and antitumor agents in recent years [24, 27]. Herein, we
designed and synthesized a series of dipeptide thiourea derivatives as multidrug-resistance
reversers which target 20S proteasome. The covalent binding mode of the designed inhibitors was
subsequently clarified by performing a docking analysis using the crystal structure of the yeast
20S proteasome. Moreover, growth inhibitory effects were evaluated against six human tumor cell
lines and doxorubicin-resistant cell lines. The molecular mechanism of apoptosis in NCI-H460
cells by the representative target compound 6i was also investigated.
2. Results and Discussion
2.1 Chemistry
The general procedures for the synthesis of functionalized dipeptide thioureas are shown in
Scheme 1. First, Boc-amide and aniline derivatives underwent acylation and deprotection, and

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each amino acid was sequentially coupled to the peptide chain from the C- to N-terminus in the
presence of HOBt, EDCI and Et3N. After obtaining the free amine dipeptide 5 by removal of the
protective group, target compounds
6
was synthesized by the condensation of
phenylisothiocyanate according to a previously published report [28] with 5 in DCM at room
temperature. Dipeptide 7 was synthesized by alkylation of 6 with methyl iodide in the presence of
K2CO3 according to a previously published study [29]. The structures of target compounds 6 and 7
were then confirmed by 1H NMR, 13C NMR and high resolution mass spectrometry (HRMS).
2.2 Biological evaluation
2.2.1 Inhibitory effect on the ChT-L, T-L and PGPH activities of human 20S proteasome
The inhibitory potency of all synthesized compounds was determined against the ChT-L
activity of the human 20S proteasome as described previously [30], and MG132 was used as a
reference compound. As shown in Table 1, most compounds showed significant inhibitory
potency against ChT-L activity. Of these compounds, 6i, 6k and 6m exhibited IC50 values at low
micromolar or submicromolar levels. The preliminary structure-activity relationships of dipeptide
thiourea derivatives for their ChT-L inhibitory activities are listed in Table 1. Analysis of the
effects of the terminal substituents at the R1 position of dipeptide thiourea derivatives showed that
a fluorine or trifluoromethyl group generally enhanced ChT-L inhibition, compared with a methyl
or methoxy group substitution. When the methoxy of compound 6a was replaced with a fluorine
group at the 3-position on the phenyl ring (compound 6d), the inhibition potency was markedly
increased, and the inhibitory activity was increased 4-fold compared with 6a. Replacing the
4-position on the phenyl ring with a fluorine or bromine group at R1 to yield compound 6g and 6f,
respectively, resulted in a marked increase in potency, thus demonstrating that the electronic
properties of the substituents may play a critical role in potent inhibitory activities against ChT-L.
However, the introduction of two methyl groups hindered the inhibitory activity. Moreover,
analogs with a methyl fragment at R1 were much less active or completely inactive. A variation at
R1 could cause a substantial difference in inhibitory potency, presumably due to different affinity
to the binding pocket. With regard to the effects of the electronic properties at R2, the ChT-L
inhibitory activities increased in the following order: bromine< trifluoromethyl < nitro. In
particular, the most potent ChT-L inhibitor, 6i, had an IC50 value against ChT-L of approximately
8.6 nM, thus illustrating that the nitro group was the optimal R2 moiety. Substitution of the

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thiourea group with methylated products markedly reduced ChT-L inhibitory activities, with the
exception of compound 7b, which may due to the positive contribution of the thiourea group to
enzyme inhibition.
Four compounds, 6i, 6k, 6m and 7b, with the most potent inhibitory activity against ChT-L
activity were further evaluated against the β1- and β2-subunits. All these compounds inhibited
PGPH activity to a lesser extent than ChT-L activity, whereas no inhibition was observed against
T-L activity. These results are particularly significant as the literature indicates that co-inhibition
of ChT-L activity together with either PGPH or T-L activity could provide optimal anticancer
agents [31], whereas inhibition of ChT-L activity alone produces only moderate effects [32], while
the cytotoxicity of inhibitors correlates with inhibition of all activities [33]. These results suggest
that this novel series of proteasome inhibitors could be selective for the β5-subunit of the 20S
proteasome, which is consistent with previous observations on linear peptide proteasome
inhibitors.
2.2.2 Molecular docking
To understand the interactions between the most active compound 6i and the target of interest
(20S proteasome), we performed molecular docking calculations on the active site of 20S
proteasome (Fig. 2) using SYBYL-X 2.0 software and the results are summarized in Table 2. The
interacting mode of compound 6i (docking score 12.63), with the best docking score and
interaction in the binding site of the 20S proteasome receptor, is described and compared with
other derivatives and is shown in Figure 2. Some key residues, such as ILE24, GLY92, GLY98
and ASP116, as well as hydrogen bonds between the selected compound and the residues are also
labeled. As shown in Figure 2a, compound 6i was stabilized predominantly by the hydrophilic or
hydrophobic group of ILE24, GLY92, GLY94 and ASP116. The nitro nitrogen and oxygen of the
compound, which are the primary important moieties, formed a hydrogen-bond interaction
network with the amino-acid residues GLY92, GLY94 and HOH of the active site. In addition, the
polar hydrogen of the thiourea moiety formed three hydrogen bonds with the backbone carbonyl
of SER115 and ASP116, which confirmed that this moiety is also crucial for binding. Furthermore,
the trifluoromethyl group in the R1 position as an acceptor established one hydrogen bond with
ILE24. As expected, the dipeptide backbone formed a hydrogen bond with the active site residue
(GLY128). In addition, the side chains of compound 6i were closely integrated with residues

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GLY47 and ASP114 of the active site. Specifically, the crucial electrostatic interactions between
compound 6i and residues ILE24, GLY92, ASP116 and GLY128 of 20S proteasome were
observed in the binding pocket. Figure 2b shows that the interacting mode of 3E47-ligand in the
binding site, which is a crucial electrostatic interaction between the ligand and residue GLY47,
was observed in the binding pocket. The binding mode of the positive control MG132 in the
ChT-L binding pocket is shown in Figure 2c. Hydrogen bonds with THR21, GLY47 and SER129
of the proteasome were observed in the docked structure.
2.2.3 Cytotoxicity Measurement
The in vitro antiproliferative activity of the dipeptide thiourea derivatives was evaluated by
MTT assay in two parental cell lines (NCI-H460 and HepG2) and the corresponding DOX
resistant cell lines (NCI-H460/DOX and HepG2/DOX), with doxorubicin (DOX) as the positive
control. The results are shown in Table 3.
As shown in Table 3, most of the test compounds exhibited high inhibitory activity against the
parental and resistant tumor cell lines, indicating that the introduction of thiourea and a nitro
moiety on the dipeptide markedly increased anti-tumor activity. Moreover, the antiproliferative
activities of the tested compounds correlated well with their ability to inhibit 20S proteasome,
with activities depending on the substituents at the R1 position, and followed the order -NO2>
-CF3 > -Cl > -Br. Most of these compounds possessed either trifluoromethyl or polymethoxy
moieties which may have had certain steric electronic properties which enhanced lipophilicity and
the ability to penetrate the cellular membrane leading to an increased antiproliferative effect. In
this regard, compound 6i with a nitro moiety at R2 and a trifluoromethyl moiety at R1 was the best
compound, depending on the cell line tested, with IC50 values of 1.70±1.16, 4.48±1.23, 6.74±1.87
and 10.18±1.53 µM against NCI-H460, HepG2, NCI-H460/DOX and HepG2/DOX cells,
respectively, and thus was more potent and effective than doxorubicin.
As the selectivity of antitumor agents for cancer cells over non-malignant cells is important to
avoid numerous severe side effects, all compounds were tested using a non-cancerous liver cell
line (HL-7702). As shown in Table 3, the cytotoxicity activities of most compounds against cancer
cells was much higher than that against HL-7702 normal cells, making them good candidates as
anticancer drugs. In summary, these modifications yielded a small increase in cytotoxicity

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activities compaired with doxorubicin and a low, but significant improvement in selectivity of the
compounds.
2.2.4 Compound 6i induces apoptosis in NCI-H460 and NCI-H460/DOX cells
In order to confirm whether 6i-induced reduction in cell viability was responsible for the
induction of apoptosis, NCI-H460 and NCI-H460/DOX cells were co-stained with 7-AAD and
Annexin V-PE, and the number of apoptotic cells was estimated by flow cytometry (Fig. 3). Four
quadrant images were observed by flow cytometric analysis: the Q1 area represented damaged
cells which appearedduring the process of cell collection, the Q2 region showed necrotic cells and
later stage apoptotic cells; early apoptotic cells were located in the Q3 area and the Q4 area
showed normal cells. As shown in Figures 3, few (7.06%) apoptotic cells were present in the
control panel, in contrast, the percentage rose to 21.06% at the concentration of 4 µM after
treatment with 6i for 24 h against NCI-H460 cells. Compound 6i also remarkably induced
NCI-H460/DOX cells apoptosis, with 17.36% apoptotic cells as compared with control (4.27).
Interestingly, treatment of the cells with the specific P-gp inhibitor LY335979 had no significant
effects on the parental cell lines but abolished MDR in the DOX-resistent NCI-H460/DOX cell
lines, resulting in the apoptotic cells increase from 29.06 to 61.8%. This restored efficiency of this
compound in the presence of the specific P-gp inhibitor illustrating that the resistance can be
attributed to P-gp. Simultaneously, these results clearly confirmed that compared with the control,
compound 6i effectively induced apoptosis in NCI-H460 and NCI-H460/DOX cells.
3. Conclusions
A
series of dipeptide thiourea derivatives were designed and synthesized as
multidrug-resistance reversers which targeted 20S proteasome with high selectivity and an optimal
inhibition profile. These compounds exhibited significant selectivity toward the β5-subunit of the
human 20S proteasome with IC50 values at submicromolar concentrations. A docking study of the
most active compound 6i revealed key interactions between 6i and the β5-subunit of the 20S
proteasome in which the thiourea moiety and a nitro group were important for improving activity.
It is noteworthy that further antitumor activity screening revealed that some compounds exhibited
better inhibitory activity than the commercial anticancer drug doxorubicin, and selected
compounds markedly reversed multidrug-resistance in DOX-resistant cancer cells. In particular,
compound 6i (IC50 = 1.70 ± 1.16 µM) exhibited the best anticancer activity against the NCI-H460

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cell line and displayed more potent inhibitory activity than doxorubicin. Nevertheless, 6k had a
smaller resistance factor than 6i and 6m. The apoptosis-inducing activity investigated by flow
cytometry revealed that compound 6i markedly induced NCI-H460 and NCI-H460/DOX cells
apoptosis. In addition, 6i-induced apoptosis was significantly facilitated in NCI-H460/DOX cells
that had been pretreated with inhibitors of P-gp. Consequently, the rational design of dipeptide
thiourea derivatives offers significant potential for the discovery of a new class of proteasome
inhibitors with the ability to reverse multidrug resistance. The precise mechanism of this action
requires further investigation.
4. Experimental
4.1 General information
The isothiocyanate was synthesized according to the literature [23]. Compounds 5 were
synthesized according to the literature [34]. All the chemical reagents and solvents used were of
analytical grade. All amino acids were coupled as their Na-Boc-derivatives. Silica gel (300-400
mesh) used in column chromatography was provided by Tsingtao Marine Chemistry Co. Ltd.
1
Precoated silica gel plates F-254 were used for thin-layer analytical chromatography. H-NMR
and 13C-NMR spectra were recorded on a Bruker AV-400spectrometer. High-resolution mass
spectra (HRMS) were recorded on a Thermo-Scientific Exactive spectrometerusing ESI and APCI
ionization sources.
4.2 General procedure for the preparation of isothiocyanate
Toluene (20 mL) was added to the amine (10 mmol) and TEDA (30.3mmol). CS2 (30.3
mmol) were added while stirring, resulting in the precipitation of the dithiocarbamate. The
reaction mixture was stirred for 8 h at room temperature and then filtered and dried. The resulting
powder was suspended in the CH2Cl2 (20 mL), and cooled to 0°C and then triphosgene (BTC)
(3.3mmol) was added. After complete the reaction at room temperature for 1h, and keep refluxing
for 2 h. Insolubles were removed by filtration, and the solvent was purified by chromatography on
silica gel eluted with petroleum ether to offer isothiocyanate.
4.3 General procedure for the preparation of peptides
4.3.1 General procedure for compounds 2 and 4.

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To a stirred solution of Boc-L-amide (5.5 mmol) in DMF (10 mL), was added Et3N (12.5
mmol) and HOBt (7.5 mmol). The mixture was allowed to stir for 1 h and then cooled to 0 °C and
then EDCI (7.5 mmol) was added. After 1 h, a solution of aniline derivatives (5 mmol) in DMF
(10 mL) was added to the above reaction mixture. The resulting mixture was stirred for 7 h at
room temperature. After completion as monitored by TLC, the reaction was quenched with ice
water and extracted with EtOAc. The organic layer was washed with saturated brine solution,
followed by drying over anhydrous Na2SO4 and evaporating in vacuo. The crude product was
purified by column chromatography (EtOAc / petroleum ether) to give the pure intermediates 2
and 4.
4.3.2 General procedure for compounds 6.
To a round flask was added 2 or 4 and a standard 50% TFA/CH2Cl2 at 0 °C and the stirring
was continued for 3 h at room temperature. After completion as monitored by TLC, the reaction
was quenched with saturated NaHCO3 solution and extracted with CH2Cl2 (3 × 50 mL). The
organic layers were dried over anhydrous Na2SO4. After removal of the solvent, the crude residue
was purified through column chromatography on silica gel (EtOAc/petroleum ether 1: 2 as eluent)
to afford compounds 3 and 5. Compounds 6 were obtained by the condensation of isothiocyanates
with 5 in dry CH2Cl2 at room temperature for 4 h.
4.3.3 General procedure for compounds 7.
To a stirred solution of compounds 6 (1 mmol) in dry CH2Cl2 (10 mL), was added CH3I (1.5
mmol), K2CO3 (1.1mmol) and acetone (5 mL). The mixture was allowed to stir for 30 min at
0°C.Then the sample was stirred at room temperature for 24 h. After the reaction, the solvent was
evaporated under reduced pressure, and the crude product was purified by chromatography on
silicageleluted with petroleum ether/ethyl acetate (V:V=2:1) to offer compounds 7.
1
The structures were confirmed by H NMR, 13C NMR and HR-MS (see supporting
information).
4.3.3.1. N-(1-((3-methoxyphenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-
(3-(3-nitrophenyl)thioureido)pentanamide(6a)

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yellow solid; Yield: 78.8%; 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.90 (s, 1H), 8.85
(s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.92 (dd, J = 8.2, 1.4 Hz, 1H), 7.84 (dd,
J = 8.1, 1.1 Hz, 1H), 7.58 (t, J = 8.2 Hz, 1H), 7.31 (d, J = 7.3 Hz, 2H ), 7.24 (t, J = 7.6 Hz, 3H),
7.21-7.16 (m, 2H), 7.12 (d, J = 8.2 Hz, 1H ), 6.64 (dd, J = 8.1, 1.6 Hz, 1H), 4.89 (t, J = 6.7 Hz, 1H,
4.73 (dd, J = 13.7, 8.4 Hz, 1H), 3.72 (s, 3H), 3.09 (dd, J = 13.9, 5.1 Hz, 1H), 2.96 (dd, J = 13.8,
9.4 Hz, 1H), 1.93-1.79 (m, 1H), 1.42(ddd, J = 12.1, 7.0, 3.0 Hz, 1H), 1.13-1.03 (m, 1H), 0.83 (dd,
J = 14.8, 7.3 Hz,6H). 13C NMR (100 MHz, DMSO-d6) δ 180.5, 170.5, 169.7, 159.5, 147.5, 141.0,
139.8, 137.5, 129.7, 129.5, 129.2, 129.2, 128.1, 128.1, 127.9, 126.3, 118.0, 115.9, 111.7, 108.8,
105.3, 61.1, 54.9, 54.9, 37.5, 37.4, 24.4, 15.2, 11.4. HR-MS (m/z) (ESI): calcd for C29H34N5O5S
[M+H+]: 564.22806; found: 564.22650.
4.3.3.2. N-(1-((3,4-dimethylphenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-
(3-(3-nitrophenyl)thioureido)pentanamide(6b)
1
yellow solid; Yield: 85.70%; H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.72 (s, 1H),
8.86 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.92 (dd, J = 8.2, 1.5 Hz, 1H), 7.84
(dd, J = 8.1, 1.2 Hz, 1H), 7.58 (t, J = 8.2 Hz, 1H), 7.30 (t, J = 7.5 Hz, 4H), 7.24 (t, J = 7.4 Hz, 2H),
7.16 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 4.88 (t, J = 6.8 Hz, 1H), 4.72 (dd, J = 13.8, 8.5
Hz, 1H), 3.09 (dd, J = 13.8, 5.2 Hz, 1H), 2.95 (dd, J = 13.7, 9.3 Hz, 1H),2.16 (d, J = 5.6 Hz, 6H),
1.91-1.77 (m, 1H), 1.39 (d, J = 1.8 Hz, 1H), 1.14-1.02 (m, 1H), 0.83 (dd, J = 13.6, 6.8 Hz, 6H).
13C NMR (100 MHz, DMSO-d6) δ 180.4, 170.5, 169.3, 147.5, 141.0, 137.5, 136.4, 136.2, 131.2,
129.7, 129.5, 129.2, 129.2, 128.0, 128.0, 127.8, 126.3, 120.7, 118.0, 117.0, 115.9, 61.2, 54.8, 37.5,
37.4, 24.4, 19.6, 18.7, 15.2, 11.4. HR-MS (m/z) (ESI): calcd for C30H35N5O4S [M+Na+]:
584.23074; found: 584.22900.
4.3.3.3. N-(1-((3-chlorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-
(3-nitrophenyl)thioureido)pentanamide(6c)
1
yellow solid; Yield: 63.84%; H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H), 10.13 (s, 1H),
8.84 (s, 1H), 8.47 (d, J = 7.7 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.84 (d, J
= 8.1 Hz, 1H), 7.76 (s, 1H), 7.58 (t, J = 8.2 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.32 (dd, J = 10.4,
8.1 Hz, 3H), 7.24 (t, J = 7.4 Hz, 2H), 7.17 (t, J = 7.0 Hz, 1H), 7.11 (d, J = 7.9 Hz, 1H), 4.91 (t, J =
6.4 Hz, 1H), 4.71 (dd, J = 13.8, 7.9 Hz, 1H), 3.09(dd, J = 13.8, 5.4 Hz, 1H), 2.97 (dd, J = 13.7, 9.2
Hz, 1H), 1.86 (d, J = 6.0 Hz, 1H), 1.43(dd, J = 8.2, 4.9 Hz, 1H), 1.16-1.02 (m, 1H), 0.84 (dd, J =

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15.5, 7.3 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 180.4, 170.6, 170.1, 147.5, 141.0, 140.1,
137.3, 133.0, 130.4, 129.7, 129.1, 129.1, 128.1, 128.1, 127.9, 126.4, 123.1, 118.8, 118.0, 117.7,
115.9, 61.0, 55.0, 37.5, 37.3, 24.4, 15.2, 11.4. HR-MS (m/z) (ESI): calcd for C28H31ClN5O4S
[M+H+]: 568.17853; found: 568.17554.
4.3.3.4. N-(1-((3-fluorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-
(3-nitrophenyl)thioureido)pentanamide(6d)
yellow solid; Yield: 80%; 1H NMR (400 MHz, DMSO-d6) δ 10.17 (d, J = 3.2 Hz, 2H), 8.85 (s,
1H), 8.48 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.92 (dd, J = 8.1, 1.4 Hz, 1H), 7.83 (d, J =
8.2 Hz, 1H), 7.61-7.52 (m, 2H), 7.38-7.28 (m, 4H), 7.24 (t, J = 7.5 Hz, 2H), 7.18 (d, J = 7.2 Hz,
1H), 6.91- 6.82 (m, 1H), 4.94-4.87 (m, 1H), 4.72 (dd, J = 13.7, 8.2 Hz, 1H), 3.09(dd, J = 13.8, 5.4
Hz, 1H), 2.97 (dd, J = 13.7, 9.3 Hz, 1H), 1.90-1.83 (m, 1H), 1.49-1.40(m, 1H), 1.09 (qd, J = 14.8,
7.5 Hz, 1H), 0.89-0.78 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 180.4, 170.6, 170.1, 163.3,
160.9, 147.5, 141.0, 140.5, 140.4, 137.3, 130.4, 130.3, 129.7, 129.2, 129.2, 128.1, 128.1, 127.9,
126.4, 118.0, 115.9, 115.1, 110.0, 109.8, 106.2, 106.0, 61.0, 54.9, 37.5, 37.3, 26.3, 24.4, 15.2, 11.4.
HR-MS (m/z) (ESI): calcd for C28H30FN5O4S [M+Na+]: 574.19002; found: 574.18907.
4.3.3.5. N-(1-((4-methoxyphenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-
(3-nitrophenyl)thioureido)pentanamide(6e)
1
yellow solid; Yield: 89.1%; H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 9.00 (s, 1H), 8.92 (s,
1H), 8.46 (s, 1H), 8.36 (s, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.22 (s, 2H),
7.18-7.02 (m, 5H), 6.61 (d, J = 8.7 Hz, 2H), 5.27 (d, J = 23.3 Hz, 2H), 3.67 (s, 3H), 3.20 (s,
2H),1.96 (s, 1H), 1.62(s, 1H), 1.10 (s, 1H), 0.88 (d, J = 3.1 Hz, 3H), 0.75 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 180.7, 172.5, 171.0, 157.5, 148.0, 148.0, 140.0, 135.8, 129.9, 129.4, 129.4, 129.0,
128.8, 128.8, 123.7, 123.7, 119.1, 114.2, 114.2, 60.6, 55.4, 55.4, 47.1, 39.3, 25.8, 15.1, 15.1, 11.9,
11.9.HR-MS (m/z) (ESI): calcd for C29H32N5O5S [M-H+]: 562.21241; found: 562.21331.
4.3.3.6. N-(1-((4-bromophenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-
(3-nitrophenyl)thioureido)pentanamide(6f)
1
yellow solid; Yield: 82.3%; H NMR (500 MHz, DMSO-d6) δ 10.18 (s, 1H), 10.10 (s, 1H),
8.85 (s, 1H), 8.48 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.92 (dd, J = 8.2, 1.6 Hz, 1H), 7.83
(dd, J = 8.1, 1.4 Hz, 1H), 7.58 (t, J = 8.2 Hz, 1H), 7.55-7.51 (m, 2H), 7.50-7.46 (m, 2H), 7.30 (d, J
= 7.3 Hz, 2H), 7.24 (t, J = 7.5 Hz, 2H), 7.17 (d, J = 7.3 Hz, 1H), 4.99-4.78 (m, 1H), 4.70 (d, J =

ACCEPTED MANUSCRIPT
5.5 Hz, 1H), 3.07(dd, J = 13.9, 5.4 Hz, 1H), 2.96 (dd, J = 13.9, 9.2 Hz, 1H), 1.97-1.72 (m, 1H),
1.57-1.28(m, 1H), 1.12-1.02m, 1H), 0.90-0.75 (m, 6H). 13C NMR (125 MHz, DMSO-d6) δ 180.4,
170.5, 169.9, 147.5, 141.0, 138.1, 137.4, 131.5, 131.5, 129.7, 129.2, 129.2, 128.1, 128.1, 127.8,
126.4, 121.3, 121.3, 118.0, 115.9, 115.0, 61.0, 54.9, 37.5, 37.3, 24.4,15.2, 11.4. HR-MS (m/z)
(ESI): calcd for C28H31BrN5O4S [M+H+]: 612.12801; found: 612.11157.
4.3.3.7. N-(1-((4-fluorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-
(3-nitrophenyl)thioureido)pentanamide(6g)
1
yellow solid; Yield: 86.56%; H NMR (500 MHz, DMSO-d6) δ 10.19 (s, 1H), 10.01 (s, 1H),
8.85 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.92 (dd, J = 8.1, 1.4 Hz, 1H), 7.83
(d, J = 7.9 Hz, 1H), 7.69-7.52 (m, 3H), 7.31 (d, J = 7.3 Hz, 2H), 7.24 (t, J = 7.4 Hz, 2H), 7.19-7.07
(m, 3H), 5.18 - 4.82 (m, 1H), 4.71 (dd, J = 14.0, 8.3 Hz, 1H), 3.08 (dd, J = 13.8, 5.4 Hz, 1H), 2.96
(dd, J = 13.7, 9.3 Hz, 1H), 1.86 (d, J = 6.2 Hz, 1H), 1.55-1.37(m, 1H), 1.11 (s, 1H), 0.91-0.74 (m,
6H). 13C NMR (125 MHz, DMSO-d6) δ 180.4, 170.5, 169.6, 157.1, 147.5, 141.0, 137.5, 135.1,
129.7, 129.2, 129.2, 128.1, 128.1, 127.9, 126.4, 121.1, 121.1, 118.0, 115.9, 115.4, 115.2, 61.0,
54.9, 37.5, 37.4, 24.4, 15.2, 11.4. HR-MS (m/z) (ESI): calcd for C28H29FN5O4S [M-H+]:
550.19243; found: 550.19342.
4.3.3.8. N-(1-((3,4-dichlorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-
(3-nitrophenyl)thioureido)pentanamide(6h)
yellow solid; Yield: 84.31%; 1H NMR (400 MHz, CD3OD) δ 8.67 (t, J = 2.1 Hz, 1H), 7.96 (ddd,
J = 8.2, 2.2, 0.9 Hz, 1H), 7.85-7.75 (m, 2H), 7.51 (t, J = 8.2 Hz, 1H), 7.36 (d, J = 1.3 Hz, 2H),
7.25 (dt, J = 14.7, 7.2 Hz, 4H), 7.16 (t, J = 7.0 Hz, 1H), 4.79 (dd, J = 8.7, 6.4 Hz, 2H), 3.24(dd, J
= 13.8, 6.4 Hz, 1H), 3.04 (dd, J = 13.8, 8.7Hz, 1H), 1.88 (dtd, J = 10.1, 6.8, 3.4 Hz, 1H), 1.44 (ddd,
J = 13.5, 7.6, 3.5 Hz, 1H), 1.16 (ddd, J = 13.6, 9.4, 7.3 Hz, 1H), 0.96-0.81 (m, 6H). 13C NMR
(100MHz, CD3OD) δ 183.4, 174.0, 171.8, 149.5, 142.1, 139.2, 138.1, 133.2, 131.4, 130.5, 130.4,
130.4, 129.7, 129.5, 129.5, 128.1, 127.9, 122.9, 120.9, 119.9, 118.5, 63.9, 56.9, 38.7, 38.5, 26.1,
16.0, 11.7. HR-MS (m/z) (ESI): calcd for C28H28Cl2N5O4S [M-H+]: 600.12391; found: 600.12485.
4.3.3.9. 3-methyl-2-(3-(3-nitrophenyl)thioureido)-N-(1-oxo-3-phenyl-1-((3-(trifluoromethyl)
phenyl)amino)propan-2-yl)pentanamide(6i)
1
yellow solid; Yield: 86.71%; H NMR (500 MHz, DMSO-d6) δ 10.31 (s, 1H), 10.19 (s, 1H),
8.85 (s, 1H), 8.53 (d, J = 7.7 Hz, 1H), 8.06-7.97 (m, 2H), 7.92 (dd, J = 8.2, 1.6 Hz, 1H), 7.84 (dd,

ACCEPTED MANUSCRIPT
J = 8.1, 1.4 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.61 - 7.52 (m, 2H), 7.40 (d, J = 7.7 Hz, 1H), 7.32
(d, J = 7.3 Hz, 2H), 7.25 (t, J = 7.5 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 4.98-4.88 (m, 1H), 4.72 (dd,
J = 14.0, 8.2 Hz, 1H), 3.11 (dd, J = 13.9, 5.5 Hz, 1H), 2.99 (dd, J = 13.9, 9.2 Hz, 1H), 1.87 (dt, J =
9.7, 8.1 Hz, 1H), 1.49-1.39(m, 1H), 1.16-0.99 (m, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (t, J = 7.4
Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 180.4, 170.7, 170.3, 147.5, 141.1, 139.5, 137.4, 130.0,
129.7, 129.2, 129.2, 128.1, 128.1, 127.9, 126.4, 125.2, 122.9, 119.8, 118.0, 115.9, 115.4, 115.4,
61.0, 55.1, 37.6, 37.3, 24.4, 15.2, 11.4. HR-MS (m/z) (ESI): calcd for C29H29F3N5O4S [M-H+]:
600.18923; found: 600.19024.
4.3.3.10. N-(1-((3-chloro-4-methylphenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-
(3-nitrophenyl)thioureido)pentanamide(6j)
1
yellow solid; Yield: 82.5%; H NMR (500 MHz, DMSO-d6) δ 10.19 (s, 1H), 10.04 (s, 1H),
8.85 (s, 1H), 8.48 (d, J = 7.5 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.84 (d, J
= 7.7 Hz, 1H), 7.75 (s, 1H), 7.58 (t, J = 8.1 Hz, 1H), 7.40-7.21 (m, 6H), 7.17 (d, J = 7.2 Hz, 1H),
5.00-4.81 (m, 1H, NCH), 4.70 (d, J = 5.4 Hz, 1H), 3.08 (dd, J = 13.7, 5.2 Hz, 1H), 2.96 (dd, J =
13.7, 9.3 Hz, 1H), 2.26 (s, 3H), 1.86 (d, J = 5.7 Hz, 1H), 1.42 (s, 1H), 1.12-1.03 (m, 1H),
0.90-0.78 (m, 6H). 13C NMR (125 MHz, DMSO-d6) δ 180.4, 170.6, 169.8, 147.5, 141.0, 137.8,
137.4, 133.0, 131.1, 130.1, 129.7, 129.2, 129.2, 128.1, 128.1, 127.8, 126.4, 119.3, 118.0, 115.9,
61.0, 54.9, 37.5, 37.4, 24.4, 18.9, 15.2, 11.4. HR-MS (m/z) (ESI): calcd for C29H31ClN5O4S
[M-H+]: 580.17853; found: 580.17948.
4.3.3.11. N-(1-((3-bromophenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-
(4(trifluoromethyl)phenyl)thioureido)pentanamide(6k)
white solid; Yield: 50.35%; 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 2H), 8.46 (d, J = 7.7
Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.93-7.81 (m, 3H), 7.65 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 7.3 Hz,
1H), 7.31 (d, J = 7.2 Hz, 2H), 7.25 (td, J = 7.8, 2.8 Hz, 4H), 7.18 (d, J = 7.2 Hz, 1H), 4.90 (t, J =
6.9 Hz, 1H), 4.71 (dd, J = 13.9, 8.2 Hz, 1H), 3.09 (dd, J = 13.8, 5.5 Hz, 1H), 2.97 (dd, J = 13.8,
9.2 Hz, 1H), 1.89-1.79 (m, 1H), 1.42(dtd, J = 12.0, 7.7, 4.1 Hz, 1H), 1.09 (dd, J = 13.7, 8.9 Hz,
1H), 0.84 (dd, J = 14.1, 7.0 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 180.8, 171.1, 170.6,
144.0, 140.8, 137.8, 131.2, 131.2, 129.6, 129.6, 128.6, 128.6, 126.9, 126.5, 126.0, 126.0, 122.2,
122.0, 121.9, 121.9, 118.6, 61.5, 55.4, 38.0, 37.8, 24.9, 15.7, 15.7, 11.9. HR-MS (m/z) (ESI):
calcd for C29H31BrF3N4O2S [M+H+]: 635.13032; found: 635.11670.

ACCEPTED MANUSCRIPT
4.3.3.12. 2-(3-(4-bromophenyl)thioureido)-N-(1-((3-chloro-4-methylphenyl)amino)-1-oxo-3-
phenylpropan-2-yl)-3-methylpentanamide(6l)
1
white solid; Yield: 55.46%; H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.84 (s, 1H), 8.40
(d, J = 7.8 Hz, 1H), 7.75 (t, J = 5.5 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.8 Hz, 2H),
7.33 (dd, J = 8.3, 1.8 Hz, 1H), 7.29 (d, J = 7.2 Hz, 2H), 7.24 (dd, J = 12.8, 5.6 Hz, 3H), 7.18 (d, J
= 7.1 Hz, 1H), 4.87 (s, 1H), 4.68 (d, J = 5.8 Hz, 1H), 3.08(dd, J = 13.8, 5.5 Hz, 1H), 2.95 (dd, J =
13.8, 9.1 Hz, 1H), 2.26 (s, 3H), 1.88-1.72 (m, 1H),1.47-1.35(m, 1H), 1.09-0.97 (m, 1H), 0.86-0.75
(m, 6H). 13C NMR (100MHz, DMSO-d6) δ 180.4, 170.7, 169.8, 139.0, 137.8, 137.4, 132.9, 131.1,
131.1, 131.1, 130.1, 129.1, 129.1, 128.1, 128.1, 126.3, 124.1, 119.3, 118.0, 115.6, 61.1, 54.9, 37.4,
37.3, 24.4, 18.9, 15.2, 15.2, 11.4. HR-MS (m/z) (ESI): calcd for C29H33BrClN4O2S [M+H+]:
615.11961; found: 615.10071.
4.3.3.13. N-(1-((3,4-dichlorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-methyl-2-(3-(3-
(trifluoromethyl)phenyl)thioureido)pentanamide(6m)
1
white solid; Yield: 70.98%; H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 10.03 (s, 1H),
8.48 (d, J = 7.6 Hz, 1H), 8.21 (s, 1H), 7.91 (dd, J = 15.0, 5.2 Hz, 2H), 7.71 (d, J = 8.2 Hz, 1H),
7.53 (t, J = 9.0 Hz, 2H), 7.46 (dd, J = 8.8, 2.2 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.30 (d, J = 7.2
Hz, 2H), 7.24 (t, J = 7.4 Hz, 2H), 7.16 (t, J = 7.1 Hz, 1H), 4.90 (s, 1H), 4.68 (dd, J = 14.0, 7.9 Hz,
1H), 3.08 (dd, J = 13.8, 5.5 Hz, 1H), 2.97 (dd, J = 13.8, 9.1 Hz, 1H), 1.94-1.76 (m, 1H),
1.49-1.37(m, 1H), 1.12 - 1.01 (m, 1H), 0.83 (dd, J = 14.5, 7.1 Hz, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 180.5, 170.7, 170.2, 140.5, 138.7, 137.2, 130.9, 130.6, 129.5, 129.1, 129.1, 128.1,
128.1, 126.4, 125.6, 124.9, 120.5, 119.3, 61.0, 59.7, 55.0, 37.5, 37.2, 24.4, 20.7, 15.2, 15.2, 14.0,
11.3. HR-MS (m/z) (ESI): calcd for C29H28F3Cl2N4O2S [M+H+]: 623.12621; found: 623.12512.
4.3.3.14. 3-methyl-N-(1-oxo-3-phenyl-1-((3-(trifluoromethyl)phenyl)amino)propan-2-yl)-2-(3-
(3-(trifluoromethyl)phenyl)thioureido)pentanamide(6n)
white solid; Yield: 38.33%; 1H NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 8.79 (s, 1H), 8.54 (s,
1H), 7.67 (s, 1H), 7.51 (d, J = 12.6 Hz, 2H), 7.42 (d, J = 8.2 Hz, 1H), 7.34 (t, J = 9.1 Hz, 1H), 7.27
(d, J = 7.7 Hz, 1H), 7.24-7.12 (m, 2H), 7.12-7.03 (m, 3H), 6.97 (d, J = 6.5 Hz, 3H), 5.06 (s, 2H),
3.22-3.08 (m, 1H), 3.09-2.97 (m, 1H), 1.81 (s, 1H), 1.43 (s, 1H), 0.99 (d, J = 6.7 Hz, 1H), 0.76 (d,
J = 6.3 Hz, 3H), 0.63 (t, J = 7.1 Hz, 3H).13C NMR (100 MHz, CDCl3) δ 180.8, 172.3, 170.8, 138.3,
137.5, 135.7, 131.6, 131.3, 129.7, 129.6, 129.3, 129.3, 128.9, 127.7, 127.4, 125.0, 123.7, 122.8,

ACCEPTED MANUSCRIPT
122.3, 121.8, 121.1, 117.4, 62.9, 56.1, 50.9, 38.5, 38.3, 25.6, 15.2, 11.5. HR-MS (m/z) (ESI):
calcd for C30H31F6N4O2S [M+H+]: 625.20719; found: 625.20496.
4.3.3.15. methyl(Z)-N'-(1-((1-((3,4-dichlorophenyl)amino)-1-oxo-3-phenylpropan-2-yl)
amino)-3-methyl-1-oxopentan-2-yl)-N-(3-(trifluoromethyl)phenyl)carbamimidothioate(7a)
white solid; Yield: 56%; 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H), 7.39 (s, 1H), 7.26 (s, 1H),
7.19 (s, 1H), 7.20 - 7.10 (m, 5H), 7.10 - 7.03 (m, 2H), 6.96 (d, J = 1.1 Hz, 2H), 6.87 (s, 1H), 6.76
(s, 1H), 4.89 (dd, J = 13.8, 6.8 Hz, 1H), 4.24 (s, 1H), 3.08 (d, J = 6.5 Hz, 2H), 2.11 (s, 3H), 1.90 (s,
1H), 1.30 (d, J = 11.1 Hz, 1H), 1.03 (d, J = 6.3 Hz, 1H), 0.83 (d, J = 6.7 Hz, 3H), 0.77 (t, J = 7.3
Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 172.4, 169.6, 169.6, 136.9, 136.1, 132.4, 131.4, 131.1,
130.2, 129.4, 129.2, 129.2, 128.9, 128.9, 127.6, 127.4, 125.5, 122.8, 121.8, 119.9, 119.4, 119.0,
55.1, 38.0, 25.0, 15.9, 15.9, 14.3, 11.5, 11.5. HR-MS (m/z) (ESI): calcd for C30H32F3Cl2N4O2S
[M+H+]: 639.15751; found: 639.15506.
4.3.3.16. methyl(Z)-N'-(3-methyl-1-oxo-1-((1-oxo-3-phenyl-1-((3-(trifluoromethyl)phenyl)
amino)propan-2-yl)amino)pentan-2-yl)-N-(3-(trifluoromethyl)phenyl)carbamimidothioate(7
b)
white solid; Yield: 53%; 1H NMR (400 MHz, CDCl3) δ 9.33 (s, 1H), 7.69 (s, 2H), 7.45 (d, J =
7.9 Hz, 1H), 7.30-7.10 (m, 10H), 7.02 (s, 1H), 6.91 (s, 1H), 5.14 (d, J = 6.6 Hz, 1H), 4.44 (s, 1H),
3.19 (d, J = 21.5 Hz, 2H), 2.18 (s, 3H), 2.01 (s, 1H), 1.45 (s, 1H), 1.15 (s, 1H), 0.95 (d, J = 6.6 Hz,
3H), 0.86 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 172.7, 170.0, 170.0, 138.1, 136.2,
129.5, 129.4, 129.3, 129.3, 128.9, 128.9, 127.4, 125.6, 125.3, 123.4, 122.9, 122.6, 121.1, 121.1,
119.7, 119.1, 117.0, 117.0, 55.3, 38.5, 37.2, 27.1, 25.1, 15.9, 14.3, 11.4. HR-MS (m/z) (ESI):
calcd for C31H33F6N4O2S [M+H+]: 639.22284; found: 639.22022.
4.3.3.17. methyl(Z)-N'-(1-((1-((3,4-dimethylphenyl)amino)-1-oxo-3-phenylpropan-2-yl)
amino)-3-methyl-1-oxopentan-2-yl)-N-(3-nitrophenyl)carbamimidothioate(7c)
1
yellow solid; Yield: 86.96%; H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.87-7.71 (m, 1H),
7.57 (s, 1H), 7.37-7.18 (m, 7H), 7.08 (d, J = 7.2 Hz, 1H), 7.03-6.95 (m, 3H), 6.91 (d, J = 8.0 Hz,
1H), 5.09 (s, 1H), 4.36 (s, 1H), 3.22 (d, J = 6.7 Hz, 1H), 3.16 (dd, J = 13.9, 6.9 Hz, 1H), 2.25 (s,
3H), 2.15 (s, 3H), 2.09 (s, 3H), 2.01 (d, J = 6.0 Hz, 1H), 1.45 (s, 1H), 1.18-1.07 (m, 1H), 0.93 (d, J
= 6.8 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ171.8, 169.0, 169.0, 148.6,
137.0, 136.5, 135.0, 133.0, 129.8, 129.4, 129.4, 128.8, 128.8, 127.2, 121.7, 118.0, 117.6, 117.2,

ACCEPTED MANUSCRIPT
61.5, 55.0, 53.6, 38.3, 37.0, 31.0, 27.0, 25.1, 19.7, 19.2, 15.8, 14.3, 11.4. HR-MS (m/z) (ESI):
calcd for C31H38N5O4S [M+H+]: 576.26445; found: 576.26318.
4. 4 Proteasome subunit inhibition assays
The 20S proteasome activity was determined using a Calbiochem proteasome assay kit
following the manufacturer's protocol. The chymotrypsin-like activity of the proteasome was
assayed by incubating the proteasome (0.5 mg/ml) with the fluorogenic substrate
Suc-LLVY-AMC (20 mM) in the presence of proteasome activators and tested compounds. The
trypsin-like and caspase-like activities of the 20S proteasome were determined using the
fluorogenic substrates Bz-VGR-AMC and (Z)-LLE-bNA, respectively. Cleavage of the substrates
by the proteasome resulted in emission of fluorescence, which was measured and expressed as the
rate of the reaction by using a Biotek fluorometer. The rate of the cleavage reaction is defined as
dF/dt, where dF/dt is the change of fluorescence intensity over time. IC50, calculated by using
CalcuSin, is the compound concentration that resulted in 50% decrease in the reaction rate.
4.5 Cytotoxicity assay
The cell lines NCI-H460, HepG2, NCI-H460/DOX, HepG2/DOX and HL-7702 were obtained
from the Shanghai Cell Bank in the Chinese Academy of Sciences. NCI-H460, HepG2,
NCI-H460/DOX, HepG2/DOX and HL-7702 cell lines were grown on 96-well microtitre plates at
a cell density of 10×105 cells/well in DMEM medium with 10% FBS. The plates were incubated
at 37 ℃ in a humidified atmosphere of 5% CO2/95% air for overnight. Therewith, the cells were
exposed to different concentrations of compounds 3, 4 and DOX, and incubated for another 48 h.
The cells were stained with 10 µl of MTT at incubator for about 4 h. The medium was thrown
away and replaced by 100 mL DMSO. The O. D. Value was read at 570/630 nmenzyme labeling
instrument.
4.6. Apoptosis analysis
NCI-H460 and NCI-H460/DOX cells were seeded at the density of 2× 106 cells/mL of the
DMEM medium with 10% FBS on 6-well platesto the final volume of 2 mL. The plates were
incubated for overnight and then treated with different consentrations compound 6i for 24 h.
Briefly, after treatment with compound 6i for 24 h, cells were collected and washed with PBS
twice, and then resuspend cells in 1×Binding Buffer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl,

ACCEPTED MANUSCRIPT
25 mM CaCl2) at a concentration of 1× 106 cells /ml. The cells were incubated with Annexin
V-PE (5 µL) (BD, Pharmingen) and 7-AAD (5 µL) in binding buffer (100 µL) using Annexin
V-PE apoptosis kit. Subsequently,the mixture was transfer to a 5 mL culture tube and incubate for
30 min at room temperature. The apoptosis ratio was quantified by system software (CellQuest;
BD Biosciences).
4.7 Molecular docking
To study the binding mode of the inhibitors in the active site of 20S proteasome protein,
molecular docking was performed using the Surflex-Dock module in SYBYL-X 2.0. The crystal
structure of the 20S proteasome complex was retrieved from the RCSB Protein Data Bank (PDB
entry code: 3E47) [35]. The ligands were docked in the corresponding protein’s binding site by an
empirical scoring function and a patented search engine in Surflex-Dock. Before the docking
process, the natural ligand was extracted; the water molecules were removed from the crystal
structure. Subsequently, the protein was prepared by using the Biopolymer module implemented
in Sybyl. The polar hydrogen atoms were added. The automated docking manner was applied in
the present work. Other parameters were established by default in the software. Surflex-Dock total
scores, which were expressed in -log10 (Kd) units to represent binding affinities, were applied to
estimate the ligand-receptor interactions of newly designed molecules.
4. 8 Statistics
The data were processed by the Student' st -test with the significance level P≤0.05 using SPSS.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (No. 81260472
21362002 and 21431001), Special Research Found for the Doctoral Program of Higher Education
(NO. 20134504110002).
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Boc
NH2
H
N
HN
H
NH2
N
1. TFA, DCM, r.t, 3h.
2. NaHCO3
Bn
Boc-L-phenylalanine, EDCI,
Bn
R1
R1
O
R1
O
HOBt, Et N, DMF, 0 , 7h.
℃
3
3
1
2
R1=F, Cl, Br, CH3, OCH3 or CF3
NH2
NCS
1. TEDA, CS2, toluene, 8h, r.t.
2. BTC, CH Cl , 0 , 30 min;
℃
2
2
R2
R2
r.t, 1h; 60 , 2h.
℃
R2=F, Cl, Br, CH3, OCH3 , NO2 or CF3
Bn
O
Bn
O
H
1. TFA, DCM, r.t, 3h.
H
N
H
N
NH2
s-Bu
N
N
N
Boc
R1
H
R1
H
2. NaHCO3
O
O
s-Bu
4
5
Bn
O
Bn
O
H
H
N
H
H
N
H
CH3I, K2CO3,
Et3N, DCM, r.t, 4h.
N
N
N
N
N
N
R1
H
R1
H
O
s-Bu S
O
s-Bu S
r.t, 24h.
R2
R2
7
6
Scheme 1.General synthetic route for compounds 6a-6n and 7a-7c.

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Tables
Table 1. Inhibition against the ChT-L, T-L and PGPH activity of the human 20S proteasome by
tested compounds
O
H
H
H
N
N
N
N
H
R1
O
S
R2
6
Compd.
R1
R2
ChT-L(µM)a
22.29
>50
T-L(µM)a
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.i.
PGPH(µM)a
n.d.
6a
6b
3-CH3O-phenyl
3, 4-CH3-phenyl
3-Cl-phenyl
3- O2N-phenyl
3- O2N-phenyl
3- O2N-phenyl
3- O2N-phenyl
3- O2N-phenyl
3- O2N-phenyl
3- O2N-phenyl
3- O2N-phenyl
3- O2N-phenyl
3- O2N-phenyl
4-CF3-phenyl
4-Br-phenyl
3-CF3-phenyl
5-CF3-phenyl
5-CF3
n.d.
6c
5.84
n.d.
6d
3-F-phenyl
3.57
n.d.
6e
4-CH3O-phenyl
4-Br-phenyl
29.03
2.71
n.d.
6f
n.d.
6g
4-F-phenyl
1.97
n.d.
6h
4, 5-Cl-phenyl
5-CF3-phenyl
1.36
n.d.
6i
0.0086
>50
34.96
n.d.
6j
4-CH3-3-Cl-phenyl
3-Br-phenyl
4-CH3-3-Cl-phenyl
3, 4-Cl-phenyl
3-CF3-phenyl
3,4-Cl
n.d.
>50
n.d.
n.i.
6k
0.022
>50
22.74
n.d.
6l
6m
6n
0.017
19.60
22.16
8.83
15.14
n.d.
n.d.
n.d.
n.i.
7a
n.d.
7b
5-CF3
5-CF3
22.63
n.d.
7c
3, 4-CH3
3- O2N
>50
n.d.
n.d.
MG-132
-
-
0.025
n.d.
a
The IC50 values are for inhibition against the ChT-L, T-L and PGPH activity of the human 20S
proteasome.
n.d. = not determined;
n.i. = no inhitition.
Table 2 Docking scores (kcal/mol) for all studied compounds
Comd.
Total
score
7.74
6.61
8.68
8.76
7.53
Crash
Polar
G_score PMF_score D-score Chem
score
Cscore
6a
6b
6c
6d
6e
-1.11
-1.57
-0.85
-2.07
-1.78
0.00
1.99
2.17
0.76
2.29
-129.58
-143.54
-139.80
-135.41
-166.21
29.58
35.50
8.33
-291.52 -17.47
-247.27 -23.68
-238.33 -21.33
-288.33 -20.11
-306.72 -31.07
2
3
2
2
4
42.51
7.31

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6f
9.02
9.24
9.43
12.63
6.11
10.23
7.00
11.51
8.59
8.12
9.50
6.97
8.73
9.22
-2.26
-0.54
-1.45
-2.97
-1.07
-1.46
-2.33
-2.50
-3.16
-1.18
-2.25
-1.74
-1.18
-2.51
1.01
3.00
0.51
0.88
1.29
1.56
0.05
0.71
0.96
0.73
0.01
0.08
0.99
2.44
-166.56
-123.18
-123.05
-157.34
-133.50
-173.49
-141.51
-155.14
-126.263
-135.19
-164.39
-177.84
184.17
20.40
-8.18
16.43
4.35
-331.18 -25.94
-207.05 -24.44
-242.50 -15.65
-331.93 -24.42
-244.21 -23.99
-309.90 -27.48
-285.44 -21.77
-327.30 -25.99
-269.87 -24.67
-102.58 -21.65
-220.20 -23.12
-190.03 -24.91
-297.49 -19.19
-304.84 -28.17
4
3
2
4
5
4
2
4
4
2
4
3
4
4
6g
6h
6i
6j
18.81
37.24
24.68
25.55
22.35
10.58
36.52
44.11
57.97
21.49
6k
6l
6m
6n
7a
7b
7c
3E47-ligand
MG-132
-169.76
Table 3. Cytotoxic effects of tested compounds on NCI-H460/DOX and HepG2/DOX Cell Lines
Compd.
IC50 (µM)a
NCI-H460 NCI-H460/DOX resistant
factor
HepG2
HepG2/DOX resistant HL-7702
factor
6c
6d
13.64±1.73
12.26±0.48
16.16±1.14
20.23±1.02
14.83±1.09
1.70±1.16
7.13±1.44
6.14±1.34
20.29±0.95
5.18±1.75
31.67±1.04
28.56±1.10
35.19±0.81
42.35±1.50
32.33±0.94
6.74±1.87
11.24±1.33
15.51±1.02
>50
2.32
2.33
2.18
2.09
2.18
3.96
1.58
2.52
-
20.72±1.32
>50
-
>50
>50
18.02±0.99 36.76±1.03
17.45±1.14 48.21±0.53
17.52±1.50 48.49±0.95
13.26±0.70 37.41±1.16
2.04
2.76
2.78
2.82
2.12
1.47
2.44
2.55
-
6f
>50
6g
>50
6h
>50
6i
4.80±1.23
10.18±1.53
>50
6k
18.62±1.32 27.43±0.79
12.12±1.12 29.61±1.68
19.31±1.54 49.18±1.13
>50
6m
7b
>50
>50
DOX
>50
-
6.91±0.96
>50
12.23±1.24

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Figures captions
Fig. 1 Chemical structures of 20S proteasome inhibitors.
Fig. 2 Binding mode of compound 6i (a), ligand (b) and MG132 (c) in the active site of 3E47,
respectively. Ligands and the important residues for binding interactions are represented by stick
and line models. The hydrogen bonds are shown as yellow dotted lines (color figure online).
Fig. 3 Apoptotic effects of compound 6i. NCI-H460 cells (A-C) and NCI-H460/DOX cells (D-F)
were treated with compound 6i or LY335979 at the indicated concentrations for 24 h. The
apoptotic effects were analyzed by flow cytometry.

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N
N
O
O
O
H
H
H
O
N
OH
N
N
N
B
N
N
N
H
H
H
O
OH
O
O
O
O
bortezomib
carfilzomib
Cl
O
H
N
O
O
N
B
O
O
H
Cl
O
OH
O
OH
ixazomib
Fig. 1 Chemical structures of 20S proteasome inhibitors.
6i (a)
3E47-ligand (b)

ACCEPTED MANUSCRIPT
MG-132 (c)
Fig. 2 Binding mode of compound 6i (a), ligand (b) and MG132 (c) in the active site of 3E47,
respectively. Ligands and the important residues for binding interactions are represented by stick
and line models. The hydrogen bonds are shown as yellow dotted lines (color figure online).
Fig. 3 Apoptotic effects of compound 6i. NCI-H460 cells (A-C) and NCI-H460/DOX cells (D-F)
were treated with compound 6i or LY335979 at the indicated concentrations for 24 h. The
apoptotic effects were analyzed by flow cytometry.