Design and synthesis of tripeptidyl furylketones as selective inhibitors against the β5 subunit of human 20S proteasome

Article abstract of DOI:10.1016/j.ejmech.2020.112160

A series of tripeptidic proteasome inhibitors with furylketone as C-terminus were designed and synthesized. Biochemical evaluations against β1, β2 and β5 subunits revealed that they acted selectively on β5 subunit with IC₅₀s against chymotrypsin-like (CT-L) activity in micromolar range. LC-MS/MS analysis of the ligand-20S proteasome mixture showed that the most potent compound 11m (IC₅₀ = 0.18 μM) made no covalent modification on 20S proteasome. However, it was identified acting in a slowly reversible manner in wash-out assay and the reversibility was much lower than that of MG132, suggesting the possibility of these tripeptidic furylketones forming reversible covalent bonds with 20S proteasome. Several compounds were selected for anti-proliferative assay towards multiple cancer cell lines, and compound 11m displayed comparable potency to positive control (MG132) in all cell lines tested. Furthermore, the pharmacokinetic (PK) data in rats indicated 11m behaved similarly (C_max, 2007 μg/L; AUC_0?t, 680 μg/L·h; V_ss, 0.66 L/kg) to the clinical used agent carfilzomib. All these data suggest 11m is a good lead compound to be developed to novel anti-tumor agent.

Full text of DOI:10.1016/j.ejmech.2020.112160

Journal Pre-proof
Design and synthesis of tripeptidyl furylketones as selective inhibitors against the β5
subunit of human 20S proteasome
Qi Sun, Tongliang Zhou, Dandan Xi, Xiaona Li, Zirui Lü, Fengrong Xu, Chao Wang,
Yan Niu, Ping Xu
PII:
S0223-5234(20)30127-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112160
EJMECH 112160
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 November 2019
Revised Date: 18 February 2020
Accepted Date: 18 February 2020
Please cite this article as: Q. Sun, T. Zhou, D. Xi, X. Li, Z. Lü, F. Xu, C. Wang, Y. Niu, P. Xu, Design
and synthesis of tripeptidyl furylketones as selective inhibitors against the β5 subunit of human
20S proteasome, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112160.
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Design and synthesis of tripeptidyl furylketones
as selective inhibitors against the β5 subunit of
human 20S proteasome
ǂ,
ǂ
§
Qi Sun , Tongliang Zhou , Dandan Xi, Xiaona Li, Zirui Lü, Fengrong xu, Chao Wang, Yan
Niu* and Ping Xu*
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University
Health Science Center, Beijing 100191, China
ǂ These authors contributed equally to this work.
§ The present address can be found at the end of this paper.
*Corresponding authors: yanniu@bjmu.edu.cn (Y. Niu), pingxu@bjmu.edu.cn (P. Xu)
Abstract
A series of tripeptidic proteasome inhibitors with furylketone as C-terminus were designed
and synthesized. Biochemical evaluations against β1, β2 and β5 subunits revealed that they acted
selectively on β5 subunit with IC50s against chymotrypsin-like (CT-L) activity in micromolar
range. LC-MS/MS analysis of the ligand-20S proteasome mixture showed that the most potent
compound 11m (IC50 = 0.18 µM) made no covalent modification on 20S proteasome. However,
it was identified acting in a slowly reversible manner in wash-out assay and the reversibility was
much lower than that of MG132, suggesting the possibility of these tripeptidic furylketones
forming reversible covalent bonds with 20S proteasome. Several compounds were selected for
anti-proliferative assay towards multiple cancer cell lines, and compound 11m displayed
comparable potency to positive control (MG132) in all cell lines tested. Furthermore, the
pharmacokinetic (PK) data in rats indicated 11m behaved similarly (Cmax, 2007 µg/L; AUC0−t,
680 µg/L·h; Vss, 0.66 L/kg) to the clinical used agent carfilzomib. All these data suggest 11m is a
good lead compound to be developed to novel anti-tumor agent.
Keywords: Proteasome inhibitors, Peptidic, Furylketone, Anti-tumor
1. Introduction

In eukaryotic cells, the amount of each intracellular protein depends on the balance of its
rates of synthesis and degradation. This homeostasis is maintained by two proteolysis systems,
namely the ubiquitin-proteasome system (UPS) and the lysosomal degradation pathway [1]. The
UPS is responsible for the specific degradation of short-lived regulatory proteins and the removal
of damaged soluble proteins, and thus tightly controls a broad array of basic cellular processes
including gene transcription, signal transduction, DNA repair, cell cycle regulation, apoptosis,
and immune response [2]. The catalytic degradation of polyubiquitinated substrates in the
ubiquitin-proteasome pathway (UPP) is performed by 26S proteasome which is a barrel-shaped,
multi-catalytic protease complex consisting of a 20S core particle (CP) and two 19S regulatory
caps [3]. The 20S CP of the proteasome comprises 28 protein subunits assembled into four
heptameric rings: two outer α-rings (α1-α7) and two inner β-rings (β1-β7) [4,5]. The outer α
subunits are proteolytically inactive and serve a regulatory function, while the two inner rings
each possess three catalytic active β subunits [6-8]. Each of the active β subunits is responsible
for a single type of proteolytic activity: β1 for caspase-like (C-L), β2 for trypsin-like (T-L), and
β5 for chymotrypsin-like (CT-L) activity. Site-directed mutagenesis analysis of the yeast
proteasome has revealed that CT-L activity of proteasome had the greatest impact on proteolysis
[9], and inhibition of tumor cellular CT-L activity is a strong stimulus that induces apoptosis [10-
12]. These observations have promoted development of numerous synthetic inhibitors of the β5
subunit [13], among which bortezomib, carfilzomib and ixazomib (Fig. 1A) have been
successfully approved by the US Food and Drug Administration (FDA) in the year of 2003, 2012,
and 2015, respectively, for the treatment of multiple myeloma [14].
Recently, we have reported a series of C-terminal furylketone-based peptidic inhibitors
against β5 subunit proposing that the C-terminal furylketone could serve as a warhead that form
covalent bond with the catalytically active residue, namely Thr1, of the β5 subunit and therefore,
could produce high affinity and inhibitory potency. However, only moderate potency was
observed on the most potent compound 10b' (Fig. 1B) with IC50 of 1.64 ± 0.44 µM in enzymatic
assay [15]. Docking and dynamic simulations suggested that the distance between the C-terminal
furylketone of 10b' and the -OH of residue Thr1 was too large (4.9 Å) to form covalent bond due
to the torsional strain brought about from the P2 and P3 substitutions. To further explore our
hypothesis regarding binding mode, more active compounds are required. In the present study,
we extended substitutions on the peptidic backbone at R2 to R4 (Fig. 1C) aiming at optimized

fitting into the S2 to S4 sub-pockets of β5 subunit and therefore, enhanced potency could be
achieved as was expected for covalent inhibitors [16]. The binding mode was also discussed
based on computational simulations together with wash-out assay and LC-MS/MS analysis.
Selected compounds were evaluated for anti-proliferating effect against a panel of cancer
cell lines using MG132 (Fig. 1D) as positive control, meanwhile, their cytotoxicities were also
measured in nonmalignant cells (HEK293).
Figure 1. The structures of (A) bortezomib, carfilzomib and ixazomib cirtate; (B) Compound 10b', (C) designed
tripeptidic inhibitors, (D) MG132.
2. Results and Discussion
2.1 Chemistry.
The design of substitutions at R2 to R4 (Fig. 1C) on the tripeptidic scaffold was performed
according to the structure-activity relationship (SAR) studies on our previously described
tripeptidyl furylketones [15,17]. Overall, the best fit into the S2-S4 sub-pockets were explored.
The designed furylketone-based tripeptides (7a-7j, 11a-11o) were acquired through two
different synthetic routes. Initially, the furan-based leucine 3 was synthesized starting from
commercially available Boc-protected L-leucine according to the method described in our
previous publication (Scheme 1) [15]. The N-protected amino acids (4a-4j; 4a-4c were
commercially available, and 4d-4j were produced from peptide condensation and hydrolyzation)
were coupled with the corresponding α-amino methyl esters 5a-5c under classic peptide coupling
conditions. The obtained dipeptidic methyl esters 6a-6j were hydrolyzed to give the
corresponding acids, followed by coupling with 3 to form compounds 7a-7j (Scheme 2).

Scheme 1. The synthesis of compound 3. Reagents and conditions: a) HOBt, EDCI, DMF, NMM, rt, 12 h; b) n-
BuLi, furan, THF, 0 , 30 min, then -78 , 2.5 h; c) HCl/dioxane, 0 , 1 h.
Scheme 2. The synthesis of tripeptidic derivatives 7a-7j. Reagents and conditions: a) HOBt, EDCI, DMF, NMM, rt,
12-24 h; b) 1N LiOH, THF, rt, 2 h; c) HOBt, EDCI, DMF, NMM, rt, 12-24 h; d) 1N LiOH, THF, rt, 2 h; e) 3, HOBt,
EDCI, DMF, NMM, rt, 12-24 h.
The synthesis of 11a-11o was achieved by a convergent approach (Scheme 3) in which N-
terminal protected amino acids were coupled to furan-based C-terminal dipeptides. The synthesis
of the furylketone dipeptide started from the condensation of L-furan based leucine 3 with N-Boc
amino acids through standard amino acid coupling condition. Then, protecting group Boc was
removed under acidic condition to give dipeptide hydrochlorides 8a-8c. The N-terminal tosyl
protected amino acids (10a-10e) were obtained from free amino acids 9a-9e, among which 9a-9c
were prepared by deprotection of the N-Boc protected tert-butyl aspartate, and 9d-9e were
commercially available, followed by condensation with p-Tosyl chloride under basic condition.

Finally, the coupling of furan-based C-terminal dipeptides 8a-8c and the N-terminal protected
10a-10i (10f-10i were commercially available) gave 11a-11o successfully.
Scheme 3. The synthesis of tripeptidic derivatives 11a-11o. Reagents and conditions: a) HOBt, EDCI, DMF, NMM,
rt, 12-24 h; b) HCl/dioxane, 0 , 1 h; c) amine, HOBt, EDCI, DMF, NMM, rt, 12-24 h; d) HCl/dioxane, 0 , 1 h; e)
p-Tosyl chloride, NaOH, dioxane; f) HOBt, EDCI, DMF, NMM, rt, 12-24 h.
2.2 Inhibitory activities against human 20S proteasome and SAR analysis

The proteasome inhibitory profiles of synthesized compounds were evaluated on the three
active subunits of human 20S proteasome using appropriate luminescent substrates as described
in our previous work [15]. None of the compounds displayed > 40% inhibition against β1 and β2
subunits, whereas almost all compounds exhibited good activities toward β5 (CT-L) during
primary screening at 50 µM (See Table S1 in the supporting information). Compounds showed
more than 50% inhibition were further evaluated for IC50s, and the results are listed in Table 1.
Table 1. Structures and the CT-L activity of proteasome inhibition potencies of target
compounds.
Compd.
R2
R3
R4
n
IC50 (µM)a
-
-
PhCH2
-
-
0.11 ± 0.01
> 50
MG132
11e
11h
7b
CH3
Cbz
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
0
0
0
0
0
0
CH3OCH2
PhCH2
Cbz
4.16 ± 0.23
>50
(4-Cl)PhCH2
CH3
PhCH2
Cbz
Boc
(4-OCH3)PhCH2
(4-OCH3)PhCH2
(4-OCH3)PhCH2
(4-OCH3)PhCH2
(4-OCH3)PhCH2
PhCH2
11.96 ± 1.88
5.86 ± 0.67
49.97 ± 1.93
1.64 ± 0.44b
8.15 ± 0.11b
45.61 ± 2.41
14.27 ± 0.33
7.26 ± 1.36
13.95 ± 0.54
2.33 ± 0.74
8.25 ± 0.52
>50
11f
11i
7a
CH3OCH2
Boc
(4-Cl)PhCH2
(4-OH)PhCH2
(4-OCH3)PhCH2
CH3
Boc
Boc
10b'
10d'
11d
11g
7c
Boc
Boc
CH3OCH2
PhCH2
Boc
(4-OH)PhCH2
(4-OH)PhCH2
(4-OH)PhCH2
(4-OH)PhCH2
(4-OCH3)PhCH2
(4-OCH3)PhCH2
(4-OCH3)PhCH2
(4-OCH3)PhCH2
(4-OCH3)PhCH2
(4-OH)PhCH2
(4-OH)PhCH2
CH3
(4-Cl)PhCH2
PhCH2
Boc
Ac
11a
11b
7f
PhCH2
p -tosyl
(4-OCH3)PhCH2
PhCH2
pyrazin-2-yl
pyrazin-2-yl
o-tolyl
7e
PhCH2
3.19 ± 0.17
>50
7g
PhCH2
2-methoxyphenyl
2-nitrophenyl
2-bromophenyl
4-(hydroxymethyl)phenyl
p -tosyl
7h
PhCH2
7.26 ± 1.94
>50
7i
PhCH2
7j
(4-OH)PhCH2
(4-OCH3)PhCH2
(CH3)3CCH2NHCOCH2
(CH3)2CHCH2NHCOCH2
(CH3)2CHCH2CH2NHCOCH2
(CH3)3CCH2NHCOCH2
(CH3)2CHCH2NHCOCH2
4.56 ± 0.39
0.54 ± 0.10
5.63 ± 0.72
3.12 ± 0.32
6.30 ± 0.35
0.18 ± 0.06
0.68 ± 0.05
7d
11c
11j
11l
11k
11m
11o
p-tosyl
CH3
p -tosyl
CH3
p -tosyl
CH3OCH2
p -tosyl
CH3OCH2
p -tosyl

CH3OCH2
(CH3)2CHCH2CH2NHCOCH2
p -tosyl
0
>50
11n
aValues represent the mean ± SD of three independent experiments, each based on three biological replicates; b See
reference 15
The substitutions with moderate size at R2 seemed more preferred when CH3OCH2- (e.g. 11h,
11i) was compared with -CH3 (e.g. 11e, 11f) or relatively bulky (4-Cl) PhCH2 (e.g. 7b, 7a).
When 4-Cl (compound 7a) replaced the electron donating 4-hydroxyl (10b') [15] or 4-methoxy
(10d') [15] groups on PhCH2, an obvious drop in potency was observed. This seemed also the
truth for R3, as can be seen from the comparison between 7c and 10b'.
The contribution of Cbz at R4 to potency was almost similar (e.g. 11e vs 11d, 11h vs 11g) to
that of Boc, which was generally consistent with our previous observation, suggesting that there
was adequate space to accommodate bulky substitutions for R4. However, the pyrazin-2-yl that
was a privilege N-terminal protecting group used in bortezomib disfavored the activities of
furylketone-based tripeptides (7f and 7e). In contrast, p-tosyl group was found an efficient N-
terminal protecting group (11b vs 11a). All together, these results revealed that their order of
activities for R4 are as follows: p-tosyl (11b) >o-tolyl (7g) >2-nitrophenyl (7i) > acetyl (11a) >
pyrazin-2-yl (7e) > 2-methoxyphenyl (7h) ≈ 2-bromophenyl (7j), and therefore p-tosyl was
selected and fixed for R4 in the following optimizations at R3. As reported by Pan et al. [18], the
S3 sub-pocket prefers to accommodate long and linear side chains. Accordingly, we replaced the
rigid PhCH2 with more flexible, as well as bulkier side chains (e.g. 11j, 11l and 11k) for R3. This
attempt was proved successful as the potencies of 11j, 11l, and 11k were comparable to 11b. The
length of the chain at R3 exhibited great impact on the potency as no inhibition was observed
with side chain longer than 7 atoms (compound 11n). The privileged order of the side chain for
R3 was as follows: 2-neopentylamino-2-oxoethyl (11m) > 2-isobutylamino-2-oxoethyl (11o) > 2-
isopentylamino-2-oxoethyl (11n). In summary, the most active compound was found in 11m
which bears the optimal isopropyl group as R1, methoxymethyl at R2, 2-neopentylamino-2-
oxoethyl for R3 and p-tosyl as the N-terminal protecting group.
The solubility of 11m was good enough in the aqueous buffers for bio-evaluations, and the
IC50 of 11m against β5 subunit was determined as 0.18 ± 0.06 µM which was comparable to that
of the positive control MG132 (IC50 = 0.11 µM). Nevertheless, its selectivity toward β5 was
much higher (showing almost no inhibitory effects on β1 or β2 subunits, Table S1 in Supporting
Information) than MG132 which also inhibits the β1 (IC50 = 12.26 µM) and β2 (IC50 = 16.30 µM)

subunits. This selectivity may enable less side effects since inhibition of all three subunits might
induce a cytotoxic effect on normal cells [19].
2.3 Binding mode analysis
To find out whether 11m interacts with 20S proteasome in covalent or noncovalent manner,
liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses was carried out to see
if covalent modified fragment(s) of 20S proteasome can be found, and to this end, the well-
known specific β5 subunit covalent inhibitor, carfilzomib [20], was used as positive control.
As shown in Figure 3, the result of vehicle 20S proteasome presented the doubly charged
unmodified TTTLAFKFR peptide (m/z 542.8114), corresponding to the N-terminal tryptic
peptide of the β5 subunit of human proteasome (Fig. 3A). The MW of the peptide fragment
covalently modified by 11m was supposed to increase by about 620 Da. However, the result (Fig.
3B) obtained from the sample treated with 11m was only observed with unmodified
TTTLAFKFR peptides (m/z 542.8116, structures shown in Fig. 3C). By contrast, a quadruply
charged peptide at m/z 451.7651 (Fig. 3D) was identified from the sample treated with
carfilzomib, corresponding to the carfilzomib-TTTLAFKFR adduct in which carfilzomib was
linked with the N-terminal Thr1 (Fig. 3E). No modification on other subunits (β1, β2) or other
site of β5 subunit of 20S proteasome was identified in the 11m treated sample according the
matching of experimental MWs to the UniProt database.

Figure 3. LC-MS/MS analysis of purified human 20S proteasome incubated with vehicle, 11m, or carfilzomib. (A)
Vehicle sample doubly charged unmodified peptide was observed at m/z 542.8114, the tandem mass spectrum of
which was matched to peptide TTTLAFKFR (the N-terminal peptide of human 20S proteasome subunit β5); (B)
11m treated sample, doubly charged unmodified peptide was observed at m/z 542.8116, the tandem mass spectrum
of which was matched to peptide TTTLAFKFR; (C) Structure of the unmodified TTTLAFKFKR tryptic peptide; (D)
Carfilzomib treated sample, quadruply charged modified peptide was observed at m/z 451.7651, the tandem mass
spectrum of which was matched to carfilzomib-modified TTTLAFKFR containing peptide; (E) Structure of the
carfilzomib-modified peptide.
The above results suggested that in contrast to carfilzomib, 11m inhibits 20S proteasome by
forming no irreversible covalent modification on it, leaving at least two possibilities regarding
binding mode. One is that 11m interacts with 20S proteasome non-covalently, and the other is
that 11m forms reversible covalent bond.
We next examined the reversibility of the binding between 11m and 20S proteasome by wash-
out assay as described by Sebti et al. [21,22]. Compound 10b', 11m, MG132, bortezomib and
carfilzomib were pre-incubated with purified human constitutive 20S proteasome and the CT-L
inhibitory activity was measured before and after wash-out. As shown in Figure 4, the catalytic
activity of β5 subunit recovered around 80% in the MG132 or bortezomib treated samples, which
was in agreement with the results reported by Tepe et al. [23] and Bolen et al. [24] that they were
reversible covalent inhibitors. Carfilzomib was irreversible with only 10% catalytic activity
recovery as reported by Bennett et al. [25].
Figure 4. CT-L activity of the human 20S proteasome treated with 10b', 11m, MG132, bortezomib or carfilzomib
before and after wash-out.
Similar to MG132 and bortizomib, the CT-L activity of the sample treated with 10b'
significantly recovered (>70%) after wash-out, which was consistent with our former observation
in dynamic simulations that 10b' formed no covalent bond due to torsion constraint [15].

However, unlike 10b', the sample treated with 11m only exhibited very low activity recovery (<
30% active) suggesting a limited reversibility regarding binding. This observation partially
supported our hypothesis that the furyl ketone warhead was electrophilic to interact with the
catalytic Thr1 of β5 subunit by forming hemiketal which endowed the inhibition with
reversibility due to the slow hydrolysis of the hemiketal intermediate (Fig. 5).
Figure 5. The hypothesized covalent interaction between furyl ketone warhead and the catalytic Thr1 of the β5
subunit of human 20S proteasome
Although the covalent modification of 11m on 20S proteasome was hard to be identified by
LC-MS/MS, the formation of hemiketal of compound 11m could be observed in high
performance liquid chromatography (HPLC) in considerable amount (~ 27%) as peaks separate
from the main product using H2O-MeOH as mobile phase. However, the small peak of methyl
hemiketal was absent when the sample was eluted with H2O-CH3CN system.
2.4 Molecular docking analysis of 11m.
In order to find out the binding mode of 11m with proteasome, we docked compound 11m
into the β5 subunit of 20S proteasome isolated from its complex with a peptidic inhibitor (PDB
code: 4NO8) using Gold 3.0.1 software. As depicted in Fig. 6A, the tripeptide backbone of 11m
generated an anti-parallel β-sheet conformation, making essential hydrogen bonds with β5 (Thr
21, Gly 47, Ala 49) and β6 (Asp 126) subunits. In this binding conformation, 11m closely
resembled the native ligand of 4NO8 (ligand 6) with its peptidomimetic chain [26], although the
side chains slightly differ in their orientations (Fig. 6B). Besides, 11m also provide the
opportunity to partially exploit the S1' specific pocket by protruding its C-terminal moiety like
ligand 6 does. The distance between the Thr1Oγ and the furylketone moiety was 2.4 Å (Fig. 6A),
which was prerequisite for hemiketal formation and in agreement with our previous SAR
discussion and LC-MS/MS results.

Figure 6. (A) Predicted binding mode of compound 11m (magenta) into the β5 (cyan)/β6 (light grey) active site of
the 20S proteasome (PDB code: 4NO8); key hydrogen bonds between the inhibitor and protein are shown as yellow
dashed lines; the distance between the carbonyl carbon atom of the furyl ketone group and the hydroxy oxygen atom
of Thr1 is marked, the value is 2.4 Å. (B) Superposition of 11m with the native ligand 6 (green) of the complex
(4NO8) within β5 (cyan)/β6 (white) active site of 20S proteasome.
2.5 Binding kinetics analysis through Surface Plasmon Resonance
We next measured the binding affinity of 11m with purified human 20S proteasome through
surface plasmon resonance (SPR) on a GE Biacore T200 apparatus. As shown in Figure 7, both
MG132 and 11m evoked specific binding and dose-dependent responses to CM5 chip [27]
linked with purified human 20S proteasome. Based on a 1:1 langmuir binding mode fit [28], the
equilibrium dissociation constants (KD) calculated from the Ka/Kd ratio for MG132 and 11m
were 17µM and 4.8 µM respectively, indicating a higher binding affinity of 11m than MG132 to
20S proteasome. Furthermore, 11m had also shown a significantly slower dissociation rate than
MG132 (Table 2) indicating a prolonged acting time of 11m, and the relatively “rapid binding
and slow dissociation” profile of 11m was supposed to be beneficial in case used in vivo due to
reduced dosing frequency.
Figure 7. SPR sensorgram of (A) MG132 and (B) 11m binding to 20S proteasome immobilized on a CM5 chip.

Table 2. Association (Ka), and dissociation (Kd) rate constants determined by SPR
Compd.
MG132
11m
Ka(1/Ms)
2716
Kd(1/s)
0.04609
0.01922
KD (µM)
17
4209
4.8
2.6 Measurement of Anti-proliferative Effects on Cancer Cell Lines
Three most potent compounds (11c, 11m and 11o) were selected for the test against a panel
of human cancer cell lines, including malignant melanoma, colorectal adenocarcinoma, and lung
carcinoma, etc. The anti-proliferation effects were measured by CellTiter-Glo® Luminescent Cell
Viability Assay (Promega Inc. Cat. # G7570) [15], and the results are summarized in Table 3. As
expected, the best potency was also achieved by 11m, which was in line with the enzymatic
assay (Table 1). Compound 11m exhibited good anti-proliferative effect which was comparable
to MG132 among all tested cancer cell lines. Interestingly, 11m was also found showing similar
single-digit micromolar IC50 as in the enzymatic assay.
Notably, the anti-proliferation effect was significant in fast proliferating cancer cells, while
the nonmalignant cell (HEK293) remained unaffected with IC50 values greater than 50 µM. This
specificity toward cancer cells promises that normal cells will not be affected when used in vivo.
Table 3. Cytotoxicity of selected compounds against a panel of solid cancer cell lines or nonmalignant cell line.
IC50
HT-29
(µ
M) a
Compd.
A375
3.87 ± 1.32
0.88 ± 0.37
2.61 ± 0.13
0.59 ± 0.06
BGC-823
8.37 ± 1.36
0.77 ± 0.28
3.96 ± 0.45
1.00 ± 0.12
Hela
6.35 ± 3.10
0.67 ± 0.08
2.47 ± 0.30
0.71 ± 0.13
A549
PC3M1E8
HCT-116
3.52 ± 1.94
0.28 ± 0.13
1.67 ± 0.44
0.40 ± 0.03
HEK293
>50
5.40 ± 3.15
0.73 ± 0.03
4.85 ± 0.81
0.78 ± 0.31
13.07 ± 3.48 4.98 ± 3.82
1.30 ± 0.17 0.57 ± 0.22
17.59 ± 6.43 3.73 ± 1.88
0.70 ± 0.02 0.96 ± 0.06
11c
11m
>50
>50
11o
>50
MG132
aValues represent the mean ± SD of three independent experiments, each based on three biological replicates.
2.7 Measurement of Effects on Cell Cycle.
To further validate the anti-proliferative effects of our furylketone derivatives, the cell cycle
arrest effects were analyzed by flow cytometry taking 11c, 11m, and 11o as representatives, in
which HCT-116 cells were first treated with either vehicle or compound before harvested and

stained with propidium iodide for analysis. As shown in Figure 8, compound 11c, 11m, and 11o
had arrested the cell cycle at G2/M phase at a concentration as low as 1 µM, just like MG132 did.
The highest percentage of cell population at G2/M phase was observed in the 11m treated group
which was in agreement with its good potency in both enzymatic and cellular assays. Therefore,
compound 11m was selected for further in vivo investigations as anti-tumor agent.
Figure 8. Human colorectal cancer cell line HCT-116 cells were treated with either vehicle, MG132, 11c, 11m, or
11o for 24 hours. The cells were stained with propidium iodide and analyzed by fluorescence activated cell sorting
to measure cellular chromosomal DNA content. Population distributions of HCT-116 cells treated with 1 µM of (A)
DMSO, (B) MG132, (C) 11c, (D) 11m, or (E) 11o.
2.8 Pharmacokinetic (PK) Evaluations of Compound 11m
The in vivo pharmacokinetic (PK) profiles of compound 11m were analyzed in rats. The
concentrations of 11m in plasma were measured after a single dose of intravenous injection (5
mg/kg). The key pharmacokinetic parameters of compound 11m are summarized in Table 4. In
rats, 11m was rapidly cleared from the plasma following i.v. administration with an average
terminal plasma half-life of ~14 min, similar to carfilzomib (~17 min) [29] that also possesses a
tripeptidic backbone.
Table 4. Pharmacokinetic parameters of 11m

AUC0-t
Cmax (µg/L)
T
(min)
Vss (L/kg)
Administrations
MRT (min)
CL (L/h/kg)
½
(µg/L·h)
11m (iv, 5 mg/kg)
2007 ± 240
680 ± 173 13.83 ± 1.80 20.20 ± 1.05
2.0 ± 0.46
0.66 ± 0.01
0.3 ± 0.1
Carfilzomib
(iv, 4 mg/kg)29
27824 ± 2304
343 ± 22
17 ± 9
11.7 ± 0.72
The area under the concentration-time curve (AUC0−t) of compound 11m was about 680
µg/L·h which was twice larger than that of carfilzomib (343 µg/L·h), indicating a broader tissue
distribution in vivo. In addition, compound 11m achieved a maximum plasma concentration
(Cmax) of 2007 µg/L within about 5 min and a volume of distribution at steady state (Vss) of 0.66
L/kg, which was much larger than blood volume (69 mL/kg in male rats) [30], suggesting
extensive tissue permeability which endow it with the possibility to be used for the treatment of
solid tumors in addition to multiple myeloma and other neoplastic hematologic disorders.
Moreover, 11m showed a lower clearance rate (CL) of 2.0 L/h/kg. These results indicated that
11m might penetrate extensively into peripheral tissues and was largely eliminated
extrahepatically [20].
2.9 In vivo anti-tumor activity of 11m
To assess the in vivo antitumor efficacy of compound 11m, HCT-116 cell xenograft nude mice
model was used. Mice bearing palpable tumor were divided into 3 groups and treated with
vehicle, carfilzomib (5 mg/kg) or 11m (10 mg/kg), respectively via intravenous injection twice
weekly for consecutive four weeks.
25
Vehicle
A
B
3000
2000
1000
0
Vehicle
11m, 10 mg/kg, i.v., D1/D4
11m, 10 mg/kg, i.v., D1/D4
Carfilzomib, 5mg/kg, i.v., D1/D4
Carfilzomib, 5mg/kg, i.v., D1/D4
20
15
10
n.s.
***
**
*
0
5
10
15
20
25
30
0
5
10
15
20
25
30
days Post-tumor Change
Days
Figure 9. 11m inhibits tumor growth in vivo. Male nude mice bearing HCT-116 tumors received 11m, carfilzomib
or vehicle alone (n= 3 mice/group). Results are expressed as mean±SEM. (A) Tumor growth curves in animals
receiving the respective treatments (n.s., non-significant; *，P ＜0.05 ；**，P ＜0.01 ；***，P ＜0.001,

significantly different from control group, two-way ANOVA followed by Bonferroni post tests); (B) Average body
weights of mice receiving the respective treatments (non-significantly different for both Carfilzomib and 11m
groups compared with control group, two-way ANOVA followed by Bonferroni post tests).
As shown in Fig. 9A, carfilzomib could inhibit tumor growth in vivo and exhibited
significant difference (P < 0.001) comparing to the vehicle treated group after 28 days’ treatment.
Although no statistically significant difference was observed during the treatment, 11m also
reduced tumor progression, albeit to a lesser extent than carfilzomib. The body weight of both
carfilzomib and 11m group did not significantly drop over the period of treatment compared with
the vehicle group (Fig. 9B), indicating 11m was well tolerated in mice without apparent systemic
toxicity. The present findings suggest that 11m demonstrates weak antitumor activity towards
solid tumor in vivo.
3.Conclusion
We designed and synthesized a series of tripeptidic furylketones as 20S proteasome
inhibitors that selectively inhibited the CT-L activity. The results of LC-MS/MS analyses and
wash-out assay indicated they inhibit the
β5 subunit in reversible manner. Docking study
suggested the distance between the C-terminal furylketone moiety and the catalytic Thr1 allowed
the formation of hemiketal which endow these furylketones “rapid loading and slow dissociation”
kinetics. Although not as potent as carfilzomib to inhibit tumor growth in tumor xenograft model
mice, the most active compound 11m exhibited greater tissue distribution and much lower
plasma clearance indicating the potential of these inhibitors to combat solid tumors when
administrated in an optimized dosing regimen.
4.Experimental Section
4.1 Chemistry
Commercially available solvents and reagents were used directly without further
purification. Reaction progress was monitored by thin-layer chromatography (TLC) performed
on silica gel GF254 purchased from Qingdao Haiyang Chemical Co. (Qingdao, China). Melting
points (mp) were obtained on an XT4A apparatus and were uncorrected.1H NMR and 13C NMR
spectra were recorded at 400/100 MHz on a Bruker Avance III 400 spectrometer with TMS as

internal standard. Low-resolution mass spectra were obtained using a Waters ACQ-SQD LC–MS
instrument in electrospray positive and negative ionization modes. High-resolution mass
spectrometry (HRMS) data were recorded on a Bruker APEX IV FTMS system. For all final
compounds, analytical reverse phase HPLC was run using an Agilent 1260 series instrument
equipped with an Eclipse Plus C18 column (3.5 µm, 4.6×100 µm) at a flow rate of 1 mL min-1
and a gradient of solvent A (water) and solvent B (acetonitrile). HPLC results showed that the
purity of all the final products was greater than 95%.
The intermediates 3, 4a-4j and 6a-6j were synthesized according to our previous reported
methods [15]. Detailed synthesis and structural characterization data of other compounds are
indicated below.
4.1.1 tert-butyl-((S)-1-(((S)-3-(4-chlorophenyl)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-
2-yl)amino)-1-oxopropan-2-yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)carbamate
(7a): 1N LiOH was added to a solution of 6a in THF until pH 12-13, and the reaction mixture
was stirred at room temperature for 2 h, followed by acidification with 1N HCl until pH 2-3. The
aqueous phase was extracted with EtOAc (5×10 mL), and the combined organic phases were
dried with Na2SO4. The solvent was concentrated in vacuo, and the resulting residue was used in
the next step without further purification. EDC·HCl (230 mg, 1.2 mmol) and HOBt (162 mg, 1.2
mmol) were added to a solution of the crude acid obtained previously (1 mmol), and 3 (217 mg,
1 mmol) in the presence of NMM (0.34 mL, 3 mmol) in 10 mL DMF. The reaction mixture was
stirred at room temperature overnight and diluted with EtOAc and washed with 10% citric acid,
saturated aqueous NaHCO3 and brine, dried with Na2SO4, and concentrated in vacuo to give the
crude product, which was separated by silica gel chromatography to yield 7a as a white solid
1
(495 mg, 77%); mp: 176-177 ; H NMR (400 MHz, DMSO-d6):
δ = 0.89-0.92 (m, 6H, CH3),
1.28 (s, 9H, Boc), 1.49-1.55 (m, 2H, CH2), 1.64-1.67 (m, 1H, CH), 2.54-2.60 (m, 1H, CH2Ph),
2.75-2.78 (m, 2H, CH2Ph), 2.94-2.98 (m, 1H, CH2Ph), 3.70 (s, 3H, OCH3), 4.01-4.06 (m, 1H,
CH), 4.57-4.62 (m, 1H, CH), 5.00-5.06 (m, 1H, CH), 6.74 (dd, J = 1.4, 3.4 Hz, 1H, F-4), 6.78-
6.82 (m, 3H, Ph, NH), 7.08 (d, J = 8.4 Hz, 2H, Ph), 7.19-7.26 (m, 4H, Ph), 7.47 (d, J = 3.6 Hz,
1H, F-3), 7.95 (d, J = 8.6 Hz, 1H, NH), 8.05 (s, 1H, F-5), 8.50 ppm (d, J = 7.6 Hz, 1H, NH); 13C
NMR (100 MHz, DMSO-d6):
δ = 21.9, 23.5, 24.9, 28.6, 37.1, 37.6, 39.4, 39.6, 39.8, 40.0, 40.2,
40.4, 40.6, 52.9, 53.5, 55.4, 56.5, 78.6, 113.1, 113.9, 119.5, 128.3, 130.3, 130.6, 131.5, 131.6,

136, 148.7, 150.8, 155.5, 158.2, 171.1, 171.9, 187.5 ppm; HRMS (ESI) m/z [M+H]+ calcd for
C34H43ClN3O7: 640.27840, found: 640.27683; HPLC: tR= 10.29 min (96.4% purity).
4.1.2 benzyl-((S)-1-(((S)-3-(4-chlorophenyl)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (7b):
Following the same procedure as described for the synthesis of 7a except for starting from 6b,
7b was obtained in 67% yield as a white solid; mp: 184-185 ; 1H NMR (400 MHz, DMSO-d6):
δ
= 0.90-0.93 (m, 6H, CH3), 1.46-1.58 (m, 2H, CH2), 1.60-1.67 (m, 1H, CH), 2.65 (dd, J = 11.1,
13.6 Hz, 1H, CH2Ph), 2.77 (dd, J = 9.0, 22.7 Hz, 1H, CH2Ph), 2.90 (dd, J = 3.8, 14.0 Hz, 1H,
CH2Ph), 2.98 (dd, J = 4.8, 18.6 Hz, 1H, CH2Ph), 4.20-4.25 (m, 1H, CH), 4.57-4.62 (m, 1H, CH),
4.93 (s, 2H, OCH2Ph), 5.01-5.06 (m, 1H, CH), 6.73-6.74 (dd, J = 1.5, 3.4 Hz, 1H, F-4), 7.18-
7.33 (m, 14H, Ph), 7.44 (d, J = 8.8 Hz, 1H, NH), 7.47 (d, J = 3.5 Hz, 1H, F-3), 8.05 (s, 1H, F-5),
13
8.13 (d, J = 8.2 Hz, 1H,NH), 8.48 ppm (d, J = 7.6 Hz, 1H, NH); C NMR (100 MHz, DMSO-
d6): δ = 21.9, 23.5, 24.9, 37.4, 37.9, 39.4, 39.6, 39.8, 40.0, 40.2, 40.4, 40.6, 53.0, 53.7, 56.5,
65.7, 113.1, 119.5, 126.7, 127.9, 128.1, 128.4, 128.5, 128.7, 129.6, 131.5, 131.6, 136.9, 137.4,
138.5, 148.7, 150.8, 156.2, 171.1, 171.7, 187.5 ppm; HRMS (ESI) m/z [M+H]+ calcd for
C36H39ClN3O6: 644.25219, found: 644.25203; HPLC: tR = 10.45 min (96.9% purity).
4.1.3 tert-butyl-((S)-3-(4-chlorophenyl)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-
2-yl)amino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate
(7c): Following the same procedure as described for the synthesis of 7a except for starting from
6c, 7c was obtained in 76% yield as a white solid; mp: 127 ; 1H NMR (400 MHz, DMSO-d6):
δ
= 0.89-0.92 (m, 6H, CH3), 1.28 (s, 9H, Boc), 1.45-1.57 (m, 2H, CH2), 1.62-1.67 (m, 1H, CH),
2.62-2.68 (m, 2H, CH2Ph), 2.83-2.88 (m, 2H, CH2Ph), 4.02-4.11 (m, 1H, CH), 4.48-4.53 (m, 1H,
CH), 5.00-5.05 (m, 1H, CH), 6.59 (d, J = 8.3 Hz, 2H, Ph), 6.73 (dd, J = 1.5, 3.5 Hz, 1H, F-4),
6.90 (d, J = 8.9 Hz, 1H, NH), 6.97 (d, J = 8.2 Hz, 2H, Ph), 7.18 (d, J = 8.3 Hz, 2H, Ph), 7.28 (d,
J = 8.2 Hz, 2H, Ph), 7.46 (d, J = 3.6 Hz, 1H, F-3), 7.88 (d, J = 8.2 Hz, 1H, NH), 8.04 (s, 1H, F-5),
8.45 (d, J = 7.7 Hz, 1H, NH), 9.13 ppm (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6):
δ = 21.9,
23.5, 24.8, 28.5, 31.4, 37.4, 39.4, 39.6, 39.8, 40.0, 40.2, 40.4, 40.6, 52.9, 54.1, 56.1, 78.6, 113.1,
115.3, 119.5, 127.8, 128.3, 130.6, 131.3, 131.5, 137.6, 148.6, 150.9, 155.5, 156.3, 171.4, 187.5
ppm; HRMS (ESI) m/z [M+H]+ calcd for C33H41ClN3O7: 626.26275, found: 626.26338; HPLC:
tR = 9.20 min (97.8% purity).

4.1.4
N-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-
hydroxyphenyl)-1-oxopropan-2-yl)amino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)-4-
(hydroxymethyl)benzamide (7d): Following the same procedure as described for the synthesis
1
of 7a except for starting from 6d, 7d was obtained in 49% yield as a white solid; mp: 161 ; H
NMR (400 MHz, DMSO-d6):
δ = 0.90 (dd, J = 4.7, 10.0 Hz, 6H, CH3), 1.45-1.68 (m, 3H, CH2,
CH), 2.62-2.70 (m, 2H, CH2), 2.76-2.92 (m, 2H, CH2), 4.48-4.54 (m,4H, CH2, CH), 4.96-5.06
(m,1H, CH), 5.28 (s, 1H, NH), 6.55 (dd, J = 3.6, 8.1 Hz, 2H, Ph), 6.59 (dd, J = 3.7, 8.1 Hz, 2H,
Ph), 6.72 (s,1H, F-4), 6.96-7.07 (m, 4H, Ph), 7.36 (t, J = 5.6 Hz, 2H, Ph), 7.47 (t, J = 3.6 Hz, 1H,
F-3), 7.73 (d, J = 7.3 Hz, 2H, Ph), 8.03 (s, 2H, F-5, NH), 8.30-8.41 (m,2H, NH, OH), 9.10 ppm
(d, J = 4.7 Hz, 2H, OH); 13C NMR (100 MHz, DMSO-d6):
δ = 21.9, 23.5, 24.8, 36.9, 37.2, 37.3,
53.1, 54.4, 55.7, 62.9, 113.1, 115.2, 119.5, 126.3, 127.7, 128.0, 128.2, 128.8, 130.5, 130.7, 132.8 ,
146.5, 148.6, 150.8, 156.1, 156.2, 166.6, 171.5, 171.8, 187.7 ppm; HRMS (ESI) m/z [M+H]+
calcd for C36H40N3O8: 642.28099, found: 642.27997; HPLC: tR = 8.38 min (96.6% purity).
4.1.5
N-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-
methoxyphenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)pyrazine-2-
carboxamide (7e): Following the same procedure as described for the synthesis of 7a except for
1
starting from 6e, 7e was obtained in 69% yield as a white solid; mp: 165-166 ; H NMR (400
MHz, DMSO-d6): δ= 0.90 (d, J = 6.6 Hz, 3H, CH3), 0.93 (d, J = 6.5 Hz, 3H, CH3), 1.47-1.61 (m,
2H, CH2), 1.63-1.72 (m, 1H, CH), 2.67-2.72 (m, 1H, CH2Ph), 2.91-2.95 (m, 1H, CH2Ph), 3.01-
3.10 (m, 2H, CH2Ph), 3.64 (s, 3H, OCH3), 4.56-4.62 (m, 1H, CH), 4.74-4.79 (m, 1H, CH), 5.04-
5.09 (m, 1H, CH), 6.70 (d, J = 8.5 Hz, 2H, Ph), 6.74 (dd, J = 1.6, 3.5 Hz, 1H, F-4), 7.11-7.17 (m,
7H, Ph), 7.49 (d, J = 3.5 Hz, 1H, F-3), 8.05 (d, J = 0.8 Hz, 1H, F-5), 8.40 (d, J = 8.5 Hz, 1H,
NH), 8.52 (d, J = 7.7 Hz, 1H, NH), 8.63 (d, J = 8.6 Hz, 1H, NH), 8.72 (d, J = 1.5 Hz, 1H, P-5),
13
8.87 (d, J = 2.2 Hz, 1H, P-6), 9.12 ppm (d, J = 1.0 Hz, 1H, P-3); C NMR (100 MHz, DMSO-
d6): δ = 21.9, 23.5, 24.9, 37.4, 37.9, 39.4, 39.6, 39.8, 40.0, 40.2, 40.4, 40.6, 52.9, 54.1, 54.3, 55.3,
113.1, 113.8, 119.5, 126.8, 128.4, 129.7, 130.6, 137.7, 143.8, 143.9, 144.4,148.3, 148.7, 150.8,
158.2, 162.5, 170.4, 171.4, 187.5 ppm; HRMS (ESI) m/z [M+H]+ calcd for C34H38N5O6:
612.28166, found: 612.28120; HPLC: tR = 8.27 min (99.7% purity).
4.1.6
N-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-
hydroxyphenyl)-1-oxopropan-2-yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-
yl)pyrazine-2-carboxamide (7f): Following the same procedure as described for the synthesis

of 7a except for starting from 6f, 7f was obtained in 59% yield as a white solid; mp: 115-116 ;
1H NMR (400 MHz, DMSO-d6):
= 0.83-0.95 (m, 6H, CH3), 1.49-1.59 (m, 2H, CH2), 1.64-
δ
1.73(m, 1H, CH), 2.66 (dd, J = 9.6,13.8 Hz, 1H, CH2), 2.87-3.02 (m,3H, CH2),3.67 (s, 3H, CH3),
4.54-4.61 (m,1H, CH), 4.69-4.76 (m,1H, CH), 5.05-5.11 (m,1H, CH), 6.55-6.64 (m, 2H, Ph),
6.68-6.79 (m, 3H, Ph, F-4), 7.04 (dd, J = 8.6, 16.7 Hz, 4H, Ph), 7.49 (d, J = 3.5 Hz, 1H, F-3),
8.04 (d, J = 0.8 Hz, 1H, F-5), 8.38 (d, J = 8.4 Hz, 1H, NH), 7.51 (d, J = 7.6 Hz, 1H, NH), 8.61 (d,
J = 8.5 Hz, 1H, NH), 8.72 (s, 1H, OH), 8.88 (d, J = 2.36 Hz, 1H, P-5), 9.12 ppm (d, J = 7.2 Hz,
2H, P-6, P-3); 13C NMR (100 MHz, DMSO-d6):
δ = 21.9, 23.5, 24.9, 37.1, 37.4, 53.0, 54.3, 55.3,
113.1, 113.9, 115.3, 119.5, 128.0, 129.4, 130.5, 130.8, 143.8, 143.9, 144.5, 148.3, 148.6, 148.7,
150.8, 156.2, 158.3, 162.5, 170.4, 171.6, 187.6 ppm; HRMS (ESI) m/z [M+H]+ calcd for
C34H38N5O7: 628.2785, found: 628.2781; HPLC: tR = 9.62 min (99.0% purity).
4.1.7
N-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-
methoxyphenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-methylbenzamide
(7g): Following the same procedure as described for the synthesis of 7a except for starting from
6g, 7g was obtained in 84% yield as a white solid; mp: 152-154 ; 1H NMR (400 MHz, DMSO-
d6):
δ = 0.91 (dd, J = 6.6, 10.0 Hz, 6H, CH3), 1.50-1.70 (m, 3H, CH2, CH), 2.04 (d, J = 5.0 Hz,
3H, CH3), 2.62-3.05 (m,4H, CH2), 3.66 (d, J = 8.3 Hz, 3H, OCH3), 4.62-4.71 (m, 2H, CH), 5.07
(s,1H, CH), 6.75 (s, 3H, Ph, F-4), 7.06-7.29 (m, 11H, Ph), 7.50 (s, 1H, F-4), 8.00-8.39 (m, 3H, F-
3, NH), 8.51 ppm (t, J = 7.2, 14.2 Hz, 1H, NH); 13C NMR (100 MHz, DMSO-d6):
δ = 19.5, 21.9,
23.5, 24.9, 37.4, 52.9, 53.2, 54.1, 54.8, 55.3, 113.1, 113.9, 119.6, 125.7, 126.7, 127.3, 128.5,
129.6, 129.7, 130.7, 135.8, 137.1, 138.6, 148.7, 150.9, 158.3, 169.3, 171.4, 171.6, 187.5, 187.7
ppm; HRMS (ESI) m/z [M+H]+ calcd for C37H42N3O6: 624.3074, found: 624.3079; HPLC: tR =
9.47 min (99.5% purity).
4.1.8
N-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-
methoxyphenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-
methoxybenzamide (7h): Following the same procedure as described for the synthesis of 7a
1
except for starting from 6h, 7h was obtained in 75% yield as a white solid; mp: 103-105 ; H
NMR (400 MHz, DMSO-d6):
δ = 0.86-0.95 (m, 6H, CH3), 1.49-1.69 (m, 3H, CH2, CH), 2.04 (d,
J = 5.0 Hz, 3H, CH3), 2.64-3.10(m, 4H, CH2), 3.66 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 4.51-4.62
(m, 1H, CH), 4.71-4.80 (m, 1H, CH), 5.00-5.11(m, 1H, CH), 6.71-6.82(m, 4H, Ph), 7.01-7.23 (m,
8H, Ph), 7.46-7.49(m, 2H, F-4, Ph), 7.78-7.83 (m, 1H, F-3), 7.78-7.83 (m, 1H, F-3), 8.04 (d, J =

8.4 Hz, 1H, NH), 8.22-8.29 (m, 1H, F-5), 8.45-8.49 ppm (m, 2H, NH); 13C NMR (100 MHz,
DMSO-d6): = 21.9, 23.4, 24.9, 37.1, 38.1, 53.2, 54.2, 54.5, 55.4, 56.4, 112.7, 113.1, 114.0,
119.4, 121.1, 121.9, 126.7, 128.3, 129.8, 130.1, 130.6, 131.3, 133.3, 137.4, 148.6, 150.9, 157.7,
158.2, 164.3, 170.9, 171.7, 187.7 ppm;
HRMS (ESI) m/z [M+H]+ calcd for C37H42N3O7:
640.3023, found: 640.3018; HPLC: tR = 9.46 min (99.7% purity).
δ
4.1.9
N-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-
methoxyphenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-nitrobenzamide
(7i): Following the same procedure as described for the synthesis of 7a except for starting from
1
6i, 7i was obtained in 89% yield as a white solid; mp: 193-195 ; H NMR (400 MHz, DMSO-
d6):
δ = 0.89 (d, J = 6.6 Hz, 3H, CH3), 0.92 (d, J = 6.5 Hz, 3H, CH3), 1.45-1.59 (m, 2H, CH2),
1.63-1.67 (m, 1H, CH), 2.72-2.86 (m, 2H, CH2), 2.95 (dd, J = 4.9, 14.0 Hz, 1H, CH), 3.00 (dd, J
= 4.0, 14.0 Hz, 1H, CH), 3.68 (s, 3H, CH3), 4.54-4.61 (m, 1H, CH), 4.67-4.73 (m, 1H, CH),
5.02-5.08 (m, 1H, CH), 6.73-6.76 (m, 3H, Ph, F-4), 7.12 (d, J = 8.4 Hz, 2H, Ph), 7.18-7.22 (m,
1H, Ph), 7.26-7.33 (m, 5H, Ph), 7.48(d, J = 3.4 Hz, 1H, F-3), 7.66 (t, J = 7.8 Hz, 1H, Ph), 7.74 (t,
J = 7.3, 14.7 Hz, 1H, Ph), 7.98 (d, J = 7.9, 15.3 Hz, 1H, Ph), 8.06 (s, 1H, F-5), 8.12 (d, J = 8.1
13
Hz, 1H, NH), 8.43 (d, J = 7.6 Hz, 1H, NH), 8.90ppm (d, J = 8.4 Hz, 1H, NH); C NMR (100
MHz, DMSO-d6):
δ
= 21.9, 23.5, 24.8, 37.2, 37.7, 52.9, 54.3, 54.6, 55.3, 113.1, 113.9, 119.6,
133.7, 138.2, 147.6, 148.7,
124.4, 126.7, 128.5, 129.5, 129.6, 129.7, 130.6, 131.3, 132.3,
150.8, 158.2, 165.5, 170.9, 171.3, 187.5 ppm; HRMS (ESI) m/z [M+H]+ calcd for C36H39N4O8:
655.2768, found: 655.2769; HPLC: tR = 8.92 min (98.7% purity).
4.1.10 2-bromo-N-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-
methoxyphenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide
Following the same procedure as described for the synthesis of 7a except for starting from 6j, 7j
was obtained in 84% yield as a white solid; mp: 183-185 ; 1H NMR (400 MHz, DMSO-d6):
(7j):
δ
=
0.91 (dd, J = 6.5, 11.3 Hz, 6H, CH3), 1.45-1.60 (m, 2H, CH2), 1.63-1.68 (m, 1H, CH), 2.70-2.82
(m, 2H, CH2), 2.94 (dd, J = 5.0, 13.9 Hz, 1H, CH2), 3.04(dd, J =3.9, 14.0 Hz, 1H, CH2), 3.69 (s,
3H, OCH3), 4.56-4.63 (m, 1H, CH), 4.64-4.71 (m, 1H, CH), 5.02-5.09 (m, 1H, CH), 6.75 (t, J =
1.9 Hz, 2H, Ph), 6.77 (s, 1H, F-4), 7.06 (dd, J = 1.8, 7.4 Hz, 1H, Ph), 7.12 (d, J = 8.6 Hz, 2H, Ph),
7.18-7.22 (m, 1H, Ph), 7.26-7.40 (m, 6H, Ph),7.49 (d, J = 3.5 Hz, 1H, F-3), 7.59 (d, J = 7.6 Hz,
1H, Ph), 8.01 (d, J = 8.2 Hz, 1H, NH), 8.05 (d, J = 1.04 Hz, 1H, F-5), 8.48 (d, J = 7.8 Hz, 1H,
NH), 8.57 ppm (d, J = 8.5 Hz, 1H, NH); 13C NMR (100 MHz, DMSO-d6):
δ = 21.9, 23.5, 24.8,

37.4, 37.7, 52.9, 54.2, 54.7,55.4, 113.1, 113.9, 119.3, 119.6, 126.7, 127.8, 128.5, 129.2, 129.6,
129.7, 130.7, 131.4, 133.1, 138.3, 139.0, 148.7, 150.8, 158.3, 167.3, 171.0, 171.3, 187.5 ppm;
HRMS (ESI) m/z [M+H]+ calcd for C36H39BrN3O6: 688.2022, found: 688.2027; HPLC: tR = 9.44
min (99.0% purity).
4.1.11
(S)-2-amino-N-((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)propanamide
hydrochloride (8a): EDC·HCl (230 mg, 1.2 mmol) and HOBt (162 mg, 1.2 mmol) were added
to a solution of Boc-Ala-OH (189 mg, 1 mmol), and 3 (217 mg, 1 mmol) in the presence of
NMM (0.34 mL,3 mmol) in 10 mL DMF. The reaction mixture was stirred at room temperature
overnight and diluted with EtOAc and washed with10% citric acid, saturated aqueous NaHCO3
and brine, dried with Na2SO4, and concentrated in vacuo to give the crude product, which was
separated by silica gel chromatography to yield Boc-Ala-Leu-Furan as a white solid (265 mg,
75%); mp: 118-119 ; 1H NMR (400 MHz, CDCl3):
δ = 0.91 (d, J = 6.4 Hz, 3H, CH3), 1.03 (d, J
= 6.2 Hz, 3H, CH3), 1.35 (d, J = 7.0 Hz, CH3), 1.45 (s, 9H, Boc), 1.52-1.57 (m, 1H,CH ), 1.61-
1.72 (m, 2H, CH2), 4.19 (s, 1H, CH), 5.01 (s, 1H, CH), 5.36-5.42 (m, 1H, NH), 6.57 (dd, J = 1.6,
3.5 Hz, 1H, F-4), 6.69 (d, J = 8.1 Hz, 1H, NH), 7.34 (d, J = 3.5 Hz, 1H, F-3), 7.64 ppm (d, J =
0.8 Hz, 1H, F-5); 13C NMR (100 MHz, CDCl3):
δ = 18.4, 21.7, 23.1, 24.8, 28.2, 41.5, 49.8, 52.5,
79.5, 112.5, 119.0, 147.2, 150.8, 155.3, 172.8, 187.6 ppm; HRMS (ESI) m/z [M+H]+ calcd for
C18H29N2O5: 353.20710, found: 353.20737. To a solution of Boc-Ala-Leu-Furan (265 mg, 0.75
mmol) in CH2Cl2 (0.75 mL), 4N HCl in 1, 4-dioxane (2.25 mL) was added. After 1 h, Et2O (9
mL) was added. The reaction mixture was filtered, and then the precipitate was rinsed with
enough Et2O and dried. The crude product 8a was used for the next step without further
purification.
4.1.12
(S)-2-amino-N-((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)-3-
methoxypropanamide hydrochloride (8b): Following the same procedure as described for the
synthesis of 8a except for starting from Boc-Ser(OMe)-OH, Boc-Ser(OMe)-Leu-Furan was
obtained in 75% yield as a white solid; mp: 80-82 ; 1H NMR (400MHz, DMSO-d6):
δ = 0.90 (t,
J = 6.0, 6,4 Hz, 6H, CH3), 1.38 (s, 9H,CH3), 1.48-1.59 (m, 2H, CH2), 1.66-1.69 (m, 1H, CH),
3.19 (s, 3H, OCH3), 3.39-3.46 (m, 2H, CH2), 4.16-4.21 (m, 1H, CH), 5.00-5.05 (m, 1H, CH),
6.73 (dd, J = 1.4, 3.4 Hz, 1H, F-4), 6.86 (d, J = 8.2 Hz, 1H, NH), 7.47 (d, J = 3.6 Hz, 1H, F-3),
8.03 (s, 1H, F-5), 8.28 ppm (d, J = 7.7 Hz, 1H, NH); 13C NMR (100 MHz, DMSO-d6):
δ =21.8,
23.6, 24.7, 28.6, 53.0, 54.6, 58.5, 72.3, 78.7, 113.1, 119.5, 148.6, 150.7, 155.6, 170.4, 187.6 ppm;

HRMS (ESI) m/z [M+H]+ calcd for C19H31N2O6: 383.21766, found: 383.21799. To a solution of
Boc-Ser(OMe)-Leu-Furan (287 mg, 0.75 mmol) in CH2Cl2 (0.75 mL), 4N HCl in 1,4-dioxane
(2.25 mL) was added. After1 h, Et2O (9 mL) was added. The reaction mixture was filtered, and
then the precipitate was rinsed with enough Et2O and dried. The crude product 8b was used for
the next step without further purification.
4.1.13
(S)-2-amino-N-((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)-3-(4-
hydroxyphenyl)propanamide hydrochloride (8c): Following the same procedure as described
for the synthesis of 8a except for starting from Boc-Tyr-OH, Boc-Tyr-Leu-Furan was obtained in
63% yield as a white solid; mp: 76-78 ; 1H NMR (400 MHz, DMSO-d6):
δ = 0.90-0.92 (m, 6H,
CH3), 1.31 (s, 9H, Boc), 1.48-1.61 (m, 2H, CH2), 1.69-1.71 (m, 1H, CH), 2.54-2.80 (m, 2H,
CH2Ph), 4.04-4.13 (m, 1H, CH), 5.03-5.08 (m, 1H, CH), 6.61 (d, J = 8.1 Hz, 2H, Ph), 6.74 (s, 1H,
F-4), 6.81 (d, J = 8.5 Hz, 1H, NH), 7.01 (d, J = 8.1 Hz, 2H, Ph), 7.47 (d, J = 3.3Hz, 1H, F-3),
8.04 (s, 1H, F-5), 8.26 (d, J = 7.7 Hz, 1H, NH), 9.14 ppm (s, 1H, OH); 13C NMR (100 MHz,
DMSO-d6):
δ = 21.8, 23.6, 24.7, 28.6, 36.9, 52.8, 56.3, 78.4, 113.1, 115.2, 119.5, 128.5, 130.5,
148.6, 150.8, 155.6, 156.2, 172.4, 187.8 ppm; HRMS (ESI) m/z [M+H]+ calcd for C24H33N2O6:
445.23331, found: 445.23358. To a solution of Boc-Tyr-Leu-Furan (280 mg, 0.63 mmol) in
CH2Cl2 (0.63 mL), 4N HCl in 1,4-dioxane (1.89 mL) was added. After 1 h, Et2O (8 mL) was
added. The reaction mixture was filtered, and then the precipitate was rinsed with enough Et2O
and dried. The crude product 8c was used for the next step without further purification.
4.1.14 (S)-2-amino-4-(isopentylamino)-4-oxobutanoic acid hydrochloride (9a): EDC·HCl
(230 mg, 1.2 mmol) and HOBt (162 mg, 1.2 mmol) were added to a solution of Boc-Asp-OtBu
(289 mg, 1 mmol), and isoamylamine (87 mg, 1 mmol) in the presence of NMM (0.34 mL,3
mmol) in 10 mL DMF. The reaction mixture was stirred at room temperature overnight and
diluted with EtOAc and washed with10% citric acid, saturated aqueous NaHCO3 and brine, dried
withNa2SO4, and concentrated in vacuo to give the crude product, which was separated by silica
gel chromatography to yield Boc-IsoamylAsn-OtBu as a white solid (313 mg, 87%); mp: 107-
108 ; 1H NMR (400 MHz, CDCl3):
δ = 0.90 (s, 3H, CH3), 0.91 (s, 3H, CH3), 1.38 (dd, J = 7.1,
14.8 Hz, 2H, CH2), 1.44 (s, 9H, CH3), 1.46 (s, 9H, CH3), 1.59-1.64 (m, 1H, CH), 2.65 (dd, J =
3.8, 15.0 Hz, 1H, CH2), 2.76 (dd, J = 4.9, 15.6 Hz, 1H, CH2), 3.25 (dd, J = 6.8, 13.6 Hz, 2H,
NCH2), 4.35 (t, J = 4.3, 7.2 Hz, 1H, CH), 5.66 ppm (s,2H, NH); MS (ESI) m/z(%): 359.3 (100)
[M+H]+. To a solution of Boc-IsoamylAsn-OtBu (313 mg, 0.87 mmol) in CH2Cl2 (0.87 mL), 4N

HCl in 1,4-dioxane (2.61 mL) was added. After 1 h, Et2O (10 mL) was added. The reaction
mixture was filtered, and then the precipitate was rinsed with enough Et2O and dried. The crude
product 9a was used for the next step without further purification.
4.1.15 (S)-2-amino-4-(isobutylamino)-4-oxobutanoic acid hydrochloride (9b): Following the
same procedure as described for the synthesis of 9a except for starting from isobutylamine, Boc-
1
IsobutylAsn-OtBu was obtained in 93% yield as a white solid; mp: 142 ; H NMR (400 MHz,
CDCl3): δ = 0.89 (s, 3H, CH3), 0.91 (s, 3H, CH3), 1.44 (s, 9H, CH3), 1.46 (s, 9H, CH3), 1.70-1.80
(m, 1H, CH), 2.68 (dd, J = 3.8, 15.6 Hz, 1H, CH2), 2.80 (dd, J = 4.1, 16.1 Hz, 1H, CH2), 3.06 (t,
J = 6.5, 12.8 Hz, 2H, NCH2), 4.35 (t, J = 4.3, 7.3 Hz, 1H, CH), 5.72 ppm (s, 2H, NH); MS (ESI)
m/z (%): 345.2 (100) [M+H]+. To a solution of Boc-IsobutylAsn-OtBu (1 mmol) in CH2Cl2 (1
mL), 4N HCl in 1,4-dioxane (3 mL) was added. After1 h, Et2O (12 mL) was added. The reaction
mixture was filtered, and then the precipitate was rinsed with enough Et2O and dried. The crude
product 9b was used for the next step without further purification.
4.1.16 (S)-2-amino-4-(neopentylamino)-4-oxobutanoic acid hydrochloride (9c): Following
the same procedure as described for the synthesis of 9a except for starting from neopentylamine,
Boc-NeopentylAsn-OtBu was obtained in 97% yield as a white solid; mp: 115 ; 1H NMR (400
MHz, CDCl3):
δ = 0.82 (s, 9H, CH3), 1.37 (s, 18H, CH3), 2.43-2.56 (m, 2H, CH2), 2.80 (dd, J =
5.9, 13.1 Hz, 1H, CH2), 2.92 (dd, J = 6.6, 13.1 Hz, 1H, CH2), 4.17 (dd, J = 7.7, 13.6 Hz, 1H, CH),
6.93 (d, J = 8.2 Hz, 1H, CH2), 7.73 ppm (t, J = 6.1, 12.2 Hz, 1H, CH); MS (ESI) m/z (%): 381.5
(100) [M+Na]+. To a solution of Boc-NeopentylAsn-OtBu (1 mmol) in CH2Cl2 (1 mL), 4N HCl
in 1,4-dioxane (3 mL) was added. After 1 h, Et2O (12 mL) was added. The reaction mixture was
filtered, and then the precipitate was rinsed with enough Et2O and dried. The crude product 9c
was used for the next step without further purification.
4.1.17 (S)-4-(isopentylamino)-2-(4-methylphenylsulfonamido)-4-oxobutanoic acid (10a):
Compound 9a (2.38 g, 10 mmol) was dissolved in THF (24 mL) and 1 N NaOH (12 mL), and
then cooled in ice bath. p-Toluenesulfonyl chloride (1.90 g, 10 mmol) was added portionwise to
the vigorously stirred solution. The pH of the reaction mixture was frequently adjusted to >8
with 4 N NaOH. After 2 h, the mixture was acidified with 6 M HCl and then extracted thrice
with EtOAc. The combined organic phase was washed with 1 M HCl, water, and brine, dried
with Na2SO4, and concentrated in vacuo to give 10a (3.07 g, 84%) as a white solid.

4.1.18 (S)-4-(isobutylamino)-2-(4-methylphenylsulfonamido)-4-oxobutanoic acid (10b):
Following the same procedure as described for the synthesis of 10a except for starting from 9b,
10b was obtained in 85% yield as a white solid.
4.1.19 (S)-2-(4-methylphenylsulfonamido)-4-(neopentylamino)-4-oxobutanoic acid (10c):
Following the same procedure as described for the synthesis of 10a except for starting from 9c,
10c was obtained in 75% yield as a white solid.
4.1.20 (S)-2-(4-methylphenylsulfonamido)-3-phenylpropanoic acid (10d):
Following the same procedure as described for the synthesis of 10a except for starting from H-
Phe-OH, 10d was obtained in 90% yield as a white solid.
4.1.21 (S)-3-(4-methoxyphenyl)-2-(4-methylphenylsulfonamido)propanoic acid (10e):
Following the same procedure as described for the synthesis of 10a except for starting from H-
(4-OMe)Phe-OH, 10e was obtained in 90% yield as a white solid.
4.1.22 (S)-2-acetamido-N-((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-
(4-hydroxyphenyl)-1-oxopropan-2-yl)-3-phenylpropanamide (11a):
EDC·HCl (230 mg, 1.2 mmol) and HOBt (162 mg, 1.2 mmol) were added to a solution of Ac-
Phe-OH (207 mg, 1 mmol), and 8c (380 mg, 1 mmol) in the presence of NMM (0.34 mL, 3
mmol) in 10 mL DMF. The reaction mixture was stirred at room temperature overnight and
diluted with EtOAc and washed with10% citric acid, saturated aqueous NaHCO3 and brine, dried
with Na2SO4, and concentrated in vacuo to give the crude product, which was separated by silica
gel chromatography to yield 11a as a white solid (399 mg, 75%); mp: 197-199 ; 1H NMR (400
MHz, DMSO-d6): δ = 0.90 (d, J = 6.8 Hz, 3H, CH3), 0.92 (d, J = 6.7 Hz, 3H, CH3), 1.48-1.57 (m,
2H, CH2), 1.66-1.67 (m, 1H, CH), 1.72 (s, 3H, CH3), 2.62-2.68 (m, 2H, CH2), 2.85-2.95 (m, 2H,
CH2), 4.43-4.49 (m, 2H, CH), 5.01-5.06 (m, 1H, CH), 6.60 (d, J = 8.4 Hz, 2H, Ph), 6.73 (q, J =
1.6, 3.5 Hz, 1H, F-4), 6.98 (d, J = 8.4 Hz, 2H, Ph), 7.16-7.23 (m, 5H, Ph), 7.47 (d, J = 3.5 Hz,
1H, F-3), 8.02 (m, 3H, NH, F-5), 8.35 (d, J = 7.7 Hz, 1H, NH), 9.15 ppm (s, 1H, OH); 13C NMR
(100 MHz, DMSO-d6):
δ = 21.9, 22.9, 23.5, 24.8, 37.1, 37.8, 52.9, 54.2, 54.4, 113.1, 115.3,
119.5, 126.6, 128.0, 128.4, 129.6, 130.6, 138.5, 148.7, 150.8, 156.3, 169.5, 171.5, 171.6, 187.6
ppm; HRMS (ESI) m/z [M+H]+ calcd for C30H36N3O6: 534.25986, found: 534.25893; HPLC: tR =
8.60 min (98.8% purity).
4.1.23 (S)-N-((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)-3-(4-hydroxyphenyl)-2-((S)-2-
(4-methylphenylsulfonamido)-3-phenylpropanamido)propanamide (11b):

Following the same procedure as described for the synthesis of 11a except for starting from 10d
1
and 8c, 11b was obtained in 81% yield as a white solid; mp: 227-229 ; H NMR (400 MHz,
DMSO-d6):
δ = 0.88 (d, J = 6.8 Hz, 3H, CH3), 0.89 (d, J = 6.7 Hz, 3H, CH3), 1.43-1.53 (m, 2H,
CH2), 1.61-1.66 (m, 1H, CH), 2.29 (s, 3H, CH3), 2.54-2.58 (m, 2H, CH2), 2.74-2.82 (m, 2H,
CH2), 3.95-4.01 (m, 1H, CH), 4.23-4.29 (m, 1H, CH), 4.97-5.03 (m, 1H, CH), 6.63 (d, J = 8.4 Hz,
2H, Ph), 6.71 (dd, J = 1.6, 3.6 Hz, 1H, F-4), 6.96 (d, J = 8.4 Hz, 2H, Ph), 7.05 (d, J = 8.2 Hz, 2H,
Ph), 7.10-7.18 (m, 5H, Ph), 7.30 (d, J = 8.2 Hz, 2H, Ph), 7.45 (d, J = 3.6 Hz, 1H, F-3), 7.89 (d, J
= 9.1 Hz, 1H, NH), 8.03 (d, J = 1.1 Hz, 1H, F-5), 8.16 (d, J = 8.0 Hz, 1H, NH), 8.34 (d, J = 7.7
Hz, 1H, NH), 9.19 ppm (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6):
δ = 21.4, 21.9, 23.5, 24.8,
37.0, 38.8, 52.9, 54.4, 57.9, 113.1, 115.3, 119.5, 126.6, 126.7, 128.0, 128.4, 129.5, 129.7, 130.6,
137.7, 138.5, 142.4, 148.6, 150.8, 156.3, 170.8, 171.4, 187.5 ppm; HRMS (ESI) m/z [M+H]+
calcd for C35H40N3O7S: 646.25815, found: 646.25615; HPLC: tR = 8.51 min (98.2% purity).
4.1.24 (S)-N-((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)-3-(4-hydroxyphenyl)-2-((S)-3-
(4-methoxyphenyl)-2-(4-methylphenylsulfonamido)propanamido)propanamide (11c):
Following the same procedure as described for the synthesis of 11a except for starting from 10e
1
and 8c, 11c was obtained in 82% yield as a white solid; mp: 174-175 ; H NMR (400 MHz,
DMSO-d6):
δ = 0.89 (t, J = 6.9 Hz, 6H, CH3), 1.42-1.57 (m, 2H, CH2), 1.61-1.67 (m, 1H, CH),
2.29 (s, 3H, CH3), 2.44-2.48 (m, 1H, CH2), 2.53-2.58 (m, 1H, CH2), 2.70-2.81(m, 2H, CH2), 3.72
(s, 3H, OCH3), 3.86-3.93 (m, 1H, CH), 4.26-4.32 (m, 1H, CH), 4.97-5.04 (m, 1H, CH), 6.62 (d,J
= 8.4 Hz, 2H, Ph), 6.69 (s, 1H, F-4), 6.71-6.73 (m, 2H, Ph), 6.94-7.06 (m, 4H, Ph), 7.30 (d, J =
8.2 Hz, 2H, Ph), 7.45 (d, J = 3.5 Hz, 1H, F-3), 7.84 (d, J = 9.0 Hz, 1H, NH), 8.02 (d, J = 1.0 Hz,
1H, F-5), 8.12 (d, J = 8.0 Hz, 1H, NH), 8.33 (d, J = 7.7 Hz, 1H, NH), 9.17 ppm (s, 1H, OH); 13C
NMR (100 MHz, DMSO-d6):
δ = 21.4, 21.9, 23.5, 24.8, 37.1, 38.0, 52.9, 54.4, 55.3, 58.3, 113.1,
113.7, 115.3, 119.5, 126.7, 127.9, 129.4, 129.5, 130.6, 130.7, 138.5, 142.3, 148.6, 150.8, 156.3,
158.2, 170.9, 171.3, 187.5 ppm; HRMS (ESI) m/z [M+H]+ calcd for C36H42N3O8S: 676.2674,
found: 676.2684; HPLC: tR = 8.42 min (100% purity).
4.1.25
tert-butyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (11d):
Following the same procedure as described for the synthesis of 11a except for starting from Boc-
1
Phe-OH and 8a, 11d was obtained in 81% yield as a white solid; mp: 97-98 ; H NMR (400
MHz, DMSO-d6):
δ = 0.89 (d, J = 7.5 Hz, 3H, CH3), 0.92 (d, J = 7.2 Hz, 3H, CH3), 1.18 (d, J =

6.9 Hz, 3H, CH3), 1.29 (s, 9H, Boc), 1.44-1.57 (m, 2H, CH2), 1.66-1.69 (m, 1H, CH), 2.66-2.99
(m, 2H, CH2), 4.12-4.17 (m, 1H, CH), 4.33-4.37 (m, 1H, CH), 4.99-5.05 (m, 1H, CH), 6.74 (t, J
= 1.5, 3.5 Hz, 1H, F-4), 6.94 (d, J = 8.5 Hz, 1H, NH), 7.18-7.26 (m, 5H, Ph), 7.47 (d, J = 3.5 Hz,
1H, F-3), 7.99 (d, J = 7.3 Hz, 1H, NH), 8.04 (s, 1H, F-5), 8.35 ppm (d, J = 7.6 Hz, 1H, NH); 13C
NMR (100 MHz, DMSO-d6):
δ = 18.8, 21.8, 23.5, 24.9, 28.6, 37.7, 48.2, 53.0, 56.1, 78.5, 113.1,
119.3, 126.6, 128.4, 129.6, 138.7, 148.6, 150.8, 155.7, 171.8, 172.6, 187.8 ppm; HRMS (ESI)
m/z [M+H]+ calcd for C27H38N3O6: 500.27551, found: 500.27585; HPLC: tR = 8.52 min (99.2%
purity).
4.1.26
benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (11e):
Following the same procedure as described for the synthesis of 11a except for starting from Cbz-
Phe-OH and 8a, 11e was obtained in 81% yield as a white solid; mp: 190-191 ; 1H NMR (400
MHz, DMSO-d6):
δ = 0.90 (d, J = 7.1 Hz, 3H, CH3), 0.92 (d, J = 7.2 Hz, 3H, CH3), 1.20 (d, J =
7.0 Hz, 3H, CH3), 1.46-1.59 (m, 2H, CH2), 1.67-1.72 (m, 1H, CH), 2.70-3.03 (m, 2H, CH2),
4.24-4.30 (m, 1H, CH), 4.33-4.37 (m, 1H, CH), 4.93 (s, 2H, CH2), 5.00-5.04 (m, 1H, CH), 6.74
(dd, J = 1.6, 3.6 Hz, 1H, F-4), 7.19-7.34 (m, 10H, Ph), 7.48 (d, J = 3.2 Hz, 1H, F-3), 7.50 (s, 1H,
NH), 8.04 (d, J = 1.0 Hz, 1H, F-5), 8.17 (d, J = 7.4 Hz, 1H, NH), 8.33 ppm (d, J = 7.5 Hz, 1H,
NH); 13C NMR (100 MHz, DMSO-d6):
δ = 18.6, 21.9, 23.5, 24.9, 37.8, 48.4, 53.1, 56.5, 65.6,
113.1, 119.3, 126.7, 127.8, 128.1, 128.5, 128.7, 129.7, 137.5, 138.6, 148.6, 150.8, 156.3, 171.6,
172.6, 187.8 ppm; HRMS (ESI) m/z [M+H]+ calcd for C30H36N3O6: 534.25986, found:
534.25955; HPLC: tR = 8.70 min (99.6% purity).
4.1.27
tert-butyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxopropan-2-yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)carbamate (11f):
Following the same procedure as described for the synthesis of 11a except for starting from 4a
1
and 8a, 11f was obtained in 73% yield as a white solid; mp: 97-99 ; H NMR (400 MHz,
DMSO-d6): δ = 0.89 (d, J = 7.3 Hz, 3H, CH3), 0.92 (d, J = 7.4 Hz, 3H, CH3), 1.18 (d, J = 6.9 Hz,
3H, CH3), 1.29 (s, 9H, Boc), 1.44-1.57 (m, 2H, CH2), 1.65-1.68 (m, 1H, CH), 2.59-2.91 (m, 2H,
CH2), 3.71 (s, 3H, OCH3), 4.04-4.10 (m, 1H, CH), 4.31-4.37 (m, 1H, CH), 4.99-5.05 (m, 1H,
CH), 6.74 (dd, J = 1.5, 4.9 Hz, 1H, F-4), 6.82 (d, J = 8.3 Hz, 2H, Ph), 6.88 (d, J = 8.5 Hz, 1H,
NH), 7.17 (d, J = 8.3 Hz, 2H, Ph), 7.47 (d, J = 3.5 Hz, 1H, F-3), 7.97 (d, J = 7.4 Hz, 1H, NH),
8.04 (s, 1H, F-5), 8.34 ppm (d, J = 7.6 Hz, 1H, NH); 13C NMR (100 MHz, DMSO-d6):
δ = 18.8,

21.8, 23.5, 24.9, 28.6, 36.9, 48.2, 53.0, 55.4, 56.4, 78.5, 113.1, 113.9, 119.3, 130.5, 130.6, 148.6,
150.8, 155.7, 158.2, 171.8, 172.6, 187.8 ppm; HRMS (ESI) m/z [M+H]+ calcd for C28H40N3O7:
530.28608, found: 530.28602; HPLC: tR = 8.35 min (98.5% purity).
4.1.28
tert-butyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-
methoxy-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (11g):
Following the same procedure as described for the synthesis of 11a except for starting from Boc-
Phe-OH and 8b, 11g was obtained in 80% yield as a white solid; mp: 104-105 ; 1H NMR (400
MHz, DMSO-d6):
δ = 0.89 (d, J = 6.9 Hz, 3H, CH3), 0.92 (d, J = 7.0 Hz, 3H, CH3), 1.29 (s, 9H,
Boc), 1.45-1.58 (m, 2H, CH2), 1.64-1.69 (m, 1H, CH), 2.66-2.97 (m, 2H, CH2), 3.21 (s, 3H,
OCH3), 3.47 (d, J = 5.6 Hz, 2H, CH2), 4.17-4.22 (m, 1H, CH), 4.52-4.55 (m, 1H, CH), 5.01-5.07
(m, 1H, CH), 6.73 (dd, J = 1.7, 3.6 Hz, 1H, F-4), 6.94 (d, J = 8.5 Hz, 1H, NH), 7.18-7.27 (m, 5H,
Ph), 7.47 (d, J = 3.4 Hz, 1H, F-3), 8.03 (d, J = 1.1 Hz, 1H, F-5), 8.05 (s, 1H, NH), 8.41 ppm (d, J
= 7.8 Hz, 1H, NH); 13C NMR (100 MHz, DMSO-d6):
δ = 21.9, 23.5, 24.8, 28.6, 37.7, 52.8, 53.0,
56.1, 58.7, 72.5, 78.5, 113.1, 119.5, 126.6, 128.4, 129.7, 138.7, 148.6, 150.7, 155.7, 169.8, 172.1,
187.5 ppm; HRMS (ESI) m/z [M+H]+ calcd for C28H40N3O7: 530.28608, found: 530.28656;
HPLC: tR = 8.79 min (99.5% purity).
4.1.29
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-
methoxy-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (11h):
Following the same procedure as described for the synthesis of 11a except for starting from Cbz-
Phe-OH and 8b, 11h was obtained in 73% yield as a white solid; mp: 113 ; 1H NMR (400 MHz,
DMSO-d6):
δ = 0.89 (d, J = 7.0 Hz, 3H, CH3), 0.92 (d, J = 6.8 Hz, 3H, CH3), 1.49-1.55 (m, 2H,
CH2), 1.67-1.68 (m, 1H, CH), 2.67-3.03 (m, 2H, CH2), 3.22 (s, 3H, OCH3), 3.49 (d, J = 5.6 Hz,
2H, CH2), 4.30-4.36 (m, 1H, CH), 4.61-4.66 (m, 1H, CH), 4.93 (s, 2H, CH2), 5.01-5.07 (m, 1H,
CH), 6.73 (dd, J = 1.6, 3.5 Hz, 1H, F-4), 7.18-7.34 (m, 10H, Ph), 7.48 (d, J = 3.4 Hz, 1H, F-3),
7.51 (s, 1H, NH), 8.03 (d, J = 0.9 Hz, 1H, F-5), 8.23 (d, J = 7.8 Hz, 1H, NH), 8.38 ppm (d, J =
13
7.6 Hz, 1H, NH); C NMR (100 MHz, DMSO-d6):
δ = 21.9, 23.5, 24.8, 37.9, 53.0, 53.1, 56.4,
58.7, 65.6, 72.4, 113.1, 119.5, 126.7, 127.8, 128.1, 128.4, 128.7, 129.7, 137.5, 138.6, 148.6,
150.7, 156.3, 169.8, 172.1, 187.5 ppm; HRMS (ESI) m/z [M+H]+ calcd for C31H38N3O7:
564.27043, found: 564.26897; HPLC: tR = 8.91 min (100% purity).
4.1.30
tert-butyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-3-
methoxy-1-oxopropan-2-yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)carbamate (11i):

Following the same procedure as described for the synthesis of 11a except for starting from 4a
1
and 8b, 11i was obtained in 70% yield as a white solid; mp: 75-76 ; H NMR (400 MHz,
DMSO-d6):
δ = 0.89 (d, J = 7.1 Hz, 3H, CH3), 0.92 (d, J = 7.1 Hz, 3H, CH3), 1.30 (s, 9H, Boc),
1.46-1.58 (m, 2H, CH2), 1.64-1.67 (m, 1H, CH), 2.59-2.93 (m, 2H, CH2), 3.21 (s, 3H, OCH3),
3.46 (d, J = 5.4 Hz, 2H, CH2), 3.71 (s, 3H, OCH3), 4.10-4.15 (m, 1H, CH), 4.50-4.55 (m, 1H,
CH), 5.01-5.07 (m, 1H, CH), 6.74 (dd, J = 1.3, 3.3 Hz, 1H, F-4), 6.82 (d, J = 8.2 Hz, 2H, Ph),
6.90 (d, J = 8.6 Hz, 1H, NH), 7.18 (d, J = 8.4 Hz, 2H, Ph), 7.48 (d, J = 3.5 Hz, 1H, F-3), 8.04 (s,
1H, F-5), 8.06 (s, 1H, NH), 8.42 ppm (d, J = 7.7 Hz, 1H, NH); 13C NMR (100 MHz, DMSO-d6):
δ
= 21.9, 23.5, 24.8, 28.6, 36.9, 52.8, 53.0, 55.4, 56.4, 58.7, 72.5, 78.5, 113.1, 113.9, 119.5,
130.5, 130.7, 148.7, 150.7, 155.7, 158.2, 169.9, 172.2, 187.5 ppm; HRMS (ESI) m/z [M+H]+
calcd for C29H42N3O8: 560.29664, found: 560.29613; HPLC: tR = 7.88 min (99.0% purity).
4.1.31 (S)-N1-((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-1-oxopropan-2-
yl)-2-(4-methylphenylsulfonamido)-N4-neopentylsuccinamide (11j):
Following the same procedure as described for the synthesis of 11a except for starting from 10c
1
and 8a, 11j was obtained in 79% yield as a white solid; mp: 209-210 ; H NMR (400 MHz,
DMSO-d6):
δ = 0.79 (s, 9H, CH3), 0.88 (d, J = 3.5 Hz, 3H, CH3), 0.89 (d, J = 3.5 Hz, 3H, CH3),
1.00 (d, J = 7.1 Hz, 3H, CH3), 1.43-1.49 (m, 1H, CH), 1.59-1.68 (m, 2H, CH2), 2.23-2.55 (m, 2H,
CH2), 2.36 (s, 3H, CH3), 2.75-2.86 (m, 2H, CH2), 3.92-3.96 (m, 1H, CH), 4.04-4.09 (m, 1H, CH),
4.83-4.89 (m, 1H, CH), 6.69 (dd, J = 1.6, 3.6 Hz, 1H, F-4), 7.32 (d, J = 8.2 Hz, 2H, Ph), 7.41 (d,
J = 3.5 Hz, 1H, F-3), 7.65 (d, J = 8.2 Hz, 2H, Ph), 7.79 (t, J = 6.0, 12.0 Hz, 1H, NH), 7.97 (s, 1H,
NH), 7.99 (d, J = 1.2 Hz, 1H, F-5), 8.09 (d, J = 7.6 Hz, 1H, NH), 8.22 ppm (d, J = 7.3 Hz, 1H,
NH); 13C NMR (100 MHz, DMSO-d6):
δ = 18.0, 21.4, 21.9, 23.4, 24.8, 27.6, 32.2, 38.9, 48.3,
50.2, 53.5, 112.9, 119.2, 127.1, 129.7, 138.7, 142.9, 148.4, 150.7, 169.6, 170.0, 172.4, 187.7
ppm; HRMS (ESI) m/z [M+H]+ calcd for C29H43N4O7S: 591.28470, found: 591.28308; HPLC: tR
= 8.15 min (99.0% purity).
4.1.32 (S)-N1-((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)amino)-1-oxopropan-2-
yl)-N4-isopentyl-2-(4-methylphenylsulfonamido)succinamide (11k):
Following the same procedure as described for the synthesis of 11a except for starting from 10a
and 8a, 11k was obtained in 81% yield as a white solid; mp: 210 ; 1H NMR (400 MHz, DMSO-
d6):
δ = 0.81 (d, J = 6.5 Hz, 6H, CH3), 0.88 (d, J = 6.2 Hz, 6H, CH3), 1.01 (d, J = 7.1 Hz, 3H,
CH3), 1.19-1.25 (m, 2H, CH2), 1.41-1.54 (m, 2H, CH2), 1.61-1.73 (m, 2H, CH), 2.23-2.47 (m,