Preparation and biological evaluation of soluble tetrapeptide epoxyketone proteasome inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.07.044

A series of novel tetrapeptidyl epoxyketone inhibitors of 20S proteasome was designed and synthesized. To fully understand the SAR, various groups at R¹, R², R³, R⁴ and R⁵ positions, including aromatic and aliphatic substituents were designed, synthesized and biologically assayed. Based on the enzymatic results, seven compounds were selected to evaluate their cellular activities and soluble compound 36 showed strong potency against human multiple myeloma (MM) cell lines. Microsomal stability results indicated that compound 36 was more stable in mice, rat and human microsomes than marketed carfilzomib. The in vivo activities of this compound were evaluated with the xenograft mice models of MM cell lines ARH77 and RPMI-8226 with luciferase expression and the T/C value of the two models were 49.5% and 37.6%, respectively. To evaluate the potential cardiovascular toxicity, inhibition of hERG ion channel in HEK293 cells by compound 36 and carfilzomib was carried out. The results indicated that 36 had no binding affinity for the hERG ion channel while carfilzomib could bind it with IC₅₀ of 92.1 μM.

Full text of DOI:10.1016/j.bmc.2019.07.044

Journal Pre-Proof
Preparation and Biological Evaluation of Soluble Tetrapeptide Epoxyketone
Proteasome Inhibitors
Meng Lei, Haoyang Zhang, Hang Miao, Xiao Du, Hui Zhou, Jia Wang, Xueyuan
Wang, Huayun Feng, Jingmiao Shi, Zhaogang Liu, Jian Shen, Yongqiang Zhu
PII:
S0968-0896(19)31074-0
https://doi.org/10.1016/j.bmc.2019.07.044
BMC 15028
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 June 2019
23 July 2019
26 July 2019
Please cite this article as: Lei, M., Zhang, H., Miao, H., Du, X., Zhou, H., Wang, J., Wang, X., Feng, H., Shi, J.,
Liu, Z., Shen, J., Zhu, Y., Preparation and Biological Evaluation of Soluble Tetrapeptide Epoxyketone Proteasome
Inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.044
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Preparation and Biological Evaluation of Soluble Tetrapeptide Epoxyketone
Proteasome Inhibitors
Meng Leia, *, Haoyang Zhangb, Hang Miaoa, Xiao Dub, Hui Zhoub, Jia Wangc,
Xueyuan Wangb, Huayun Fenga, Jingmiao Shic, Zhaogang Liuc, Jian Shend,
Yongqiang Zhu b, c, *
a
College of Science, Nanjing Forestry University, No. 159 Longpan Road, Nanjing
210037, PR China
b College of Life Science, Nanjing Normal University, No. 1 Wenyuan Road, Nanjing
210037, PR China
c
Jiangsu Chia Tai Fenghai Pharmaceutical Co. Ltd., No. 9 Weidi Road, Nanjing
210046, PR China
d
College of Chemistry and Materials Science, Nanjing Normal University, No. 1
Wenyuan Road, Nanjing 210046, PR China
*Corresponding authors.
Tel.:+86 25 85891591.
E-mail address: zhyqscu@hotmail.com (Y.Q. Zhu)
Tel.:+86 25 85427621.
E-mail address: hk-lm@163.com (M. Lei)

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ABSTRACT: A series of novel tetrapeptidyl epoxyketone inhibitors of 20S
proteasome was designed and synthesized. To fully understand the SAR, various
groups at R1, R2, R3, R4 and R5 positions, including aromatic, aliphatic substituents
and isomers were designed, synthesized and biologically assayed. Based on the
enzymatic results, seven compounds were selected to evaluate their cellular activities
and soluble compound 36 showed strong potency against human multiple myeloma
(MM) cell lines. Microsomal stability results indicated that compound 36 was more
stable in mice, rat and human microsomes than marketed carfilzomib. The in vivo
activities of this compound were evaluated with the xenograft mice models of MM
cell lines ARH77 and RPMI-8226 with luciferase expression and the T/C value of the
two models were 49.5% and 37.6%, respectively. To evaluate the potential
cardiovascular toxicity, inhibition of hERG ion channel in HEK293 cells by
compound 36 and carfilzomib was carried out. The results indicated that 36 had no
binding affinity for the hERG ion channel while carfilzomib could bind it with IC50 of
92.1 μM.
1. Introduction
The ubiquitin-proteasome system (UPS) plays a central role in maintaining
cellular homeostasis, such as cell-cycle regulation, activation of transcription factors,
DNA repair, obsolete proteins and apoptosis induction1. The disorder of UPS could
cause a variety of diseases including malignancies, neurodegenerative diseases and
lead to systematic autoimmunity2. As the heart of this system, 26S proteasome
functions the nonlysosomal degradation in eukaryote, which consists of one
barrel-shaped 20S proteolytic core particle capped with two 19S regulatory subunits3.
Two 19S caps are located at the two outer sides of the 20S proteolytic core and
responsible for recognizing the ubiquitinated proteins, which are degraded into
compounds consisting of small amino acids or peptides by the 20S proteasome4. The
catalytic core 20S proteasome is composed of four stacked rings covered with two
outer α-rings and two inner β-rings and shows three different types of catalytic
activities including chymotrypsin-like (CT-L, β5 subunit), trypsin-like (T-L, β2

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subunit) and caspase-like (PGPH, β1 subunit)5. And the CT-L activity is the
rate-limiting step in the degradation of intracellular proteins, including those that
determine tumor growth and survival6. The inhibitors that primarily target the CT-L
activity of the proteasome have been found to arrest cell cycle progression and to
induce apoptosis in a variety of human neoplastic cells derived from hematological
malignancies and solid tumors under experimental conditions7.
Over the past years, the academic institutes and pharmaceutical companies
around the world showed great interests in discovering and developing proteasome
inhibitors based on 20S proteasome. The first generation proteasome inhibitor
bortezomib (Velcade®, Fig. 1) was approved by the FDA in 2003 and was used for the
treatment of relapsed and refractory multiple myeloma (MM) 8, 9. The structure of this
drug was a dipeptide with the carbon terminal substituted by boronic acid as a
warhead. However, it showed serious side effects in clinical uses after several years,
especially in the peripheral nerve10, 11. In 2015, an oral dipeptidyl boronic acid
proteasome inhibitor, ixazomib was approved by the FDA. To overcome the reported
side effects of bortezomib, the second generation proteasome inhibitor carfilzomib
(Kyprolis®, Fig. 1) was developed and approved by FDA in 2012 for the treatment of
relapsed MM patients who had received at least two prior therapies including
bortezomib12, 13. The unique pharmacophore epoxyketone of carfilzomib could reduce
the serious peripheral neurotoxicity of this drug in a certain extent14, 15. However, the
solubility of this drug was too poor and additional 3000 mg of sulfobutylether
beta-cyclodextrin had to be added to help solve 60 mg of this drug in clinical usage,
which had the potential to lead to acute renal failure of patients treated with this drug
for a long time. Furthermore, during the clinical trials of carfilzomib, the most
common grade 3 or higher treatment-emergent adverse events were thrombocytopenia,
anemia, lymphoenia, neutropenia, pneumonia, fatigue and hyponatremia, especially
cardiovascular events16. The above-mentioned drawbacks limited its usage in clinic.
Thus, there is a great need to design and develop new proteasome inhibitors with
less side effects to meet the clinical need. Our group previously carried out many
studies in this area17-19. As a continuance of our work, 39 tetrapeptidyl epoxyketones

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proteasome inhibitors were synthesized and their biological activities were evaluated
in vitro and in vivo in this manuscript. The structure-activity relationships (SARs)
were discussed in detail. From the screened compounds, a structurally novel and
soluble inhibitor was selected as a clinical candidate.
Fig. 1. Chemical structures of proteasome inhibitors bortezomib and carfilzomib.
2. Results and discussion
2.1.Chemical synthesis
The synthesis of intermediate tripeptide acids 8a-8z, 8aa and 8ab was illustrated
in Scheme 1 (Detailed procedure was showed in SI file). The key epoxyketone
fragments 16a-16e were prepared in moderate to high yields according to our reported
method20 and showed in Scheme 2. With tripeptide acids 8a-8z, 8aa, 8ab and
epoxyketone fragments 16a-16e in hand, the tetrapeptide epoxyketone 17-47 were
obtained by coupling reaction in the presence of HOBt and EDCI (Scheme 3).
Scheme 1. General synthesis of tripeptide acids 8a-8z, 8aa and 8ab. a
aReagents and conditions: (i) MeOH, SOCl2, 0 ℃ to rt, 2 h, 90.5-98.9%; (ii)
BocNHCH(R3)COOH, EDCI, HOBt, DIPEA, DCM, 0 ℃ to rt, 12 h, 50.4-80.7%; (iii)
2 N TFA, DCM, 0 ℃to rt, 3 h, 85.6-93.5%; (iv) BocNHCH(R4)COOH, EDCI, HOBt,

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DIPEA, DCM, 0 ℃ to rt, 12 h, 40.4-80.9%; (v) 2 N TFA in DCM, 0 ℃ to rt, 3 h,
80.3-90.8%; (vi) R5COOH, EDCI, HOBt, DIPEA, DCM, 0 ℃ to rt, 12 h, 50.6-70.2%;
(vii) MeOH, 3N LiOH·H2O, 0 ℃to rt, 3 h, 70.9-90.1%.
Scheme 2. General synthesis of key epoxyketone fragments 16a-16e. a
R1: a=
; b =
; c =
; d =
; e =
aReagents and conditions: (i) HOBt, EDCI, N,O-dimethylhyclroxylamine
hydrochloride, DIPEA, DCM, 0 °C, 12 h, 70.4-90.8%; (ii) THF, C2H5BrMg, -20 °C, 6
h, 95.4-97.6%; (iii) piperidinium acetate, piperidine, paraformaldehyde, THF, 68 °C,
12 h, 30.1-50.6%; (iv) aluminium isopropoxide, isopropanol, toluene, 50 °C, 3 h,
60.2-80.7%; (v) tert-butylhydroperoxide solution, vanadium(III) acetylacetonate,
DCM, 0 ℃ to rt, 12 h, 40.3-50.8%; (vi) pyridine sulfur trioxide, DIPEA, dimethyl
sulfoxide, 6 h, 51.6-70.3%; (vii) 2N TFA in DCM, 0 ℃to rt, 2 h, 85.2-90.7%.
Scheme 3. General synthesis of target compounds 17-47. a
aReagents and condition: (i) HOBt, EDCI, DIPEA, DCM, 0 ℃ to rt, 12 h,
45.2-70.7%.
2.2. Biological evaluation
Inhibition of chymotrypsin-like threonine proteasome.
The target compounds were screened for their 20S proteasome
chymotrypsin-like inhibitory activities in vitro. Carfilzomib was employed as the
standard. To fully understand SAR of tetrapeptide epoxyketone proteasome inhibitors,

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various substituents on the R1, R2, R3, R4 and R5 positions were investigated. Table 1
summarized the biological results.
Table 1. Structures of compounds 17-47 and inhibitory activities against β5 subunit.
Compd.
R5
R4
R3
R2
R1
IC50 (nM)a
17
77.1±8.1
52.2±9.1
139.6±13.9
729.0±34.2
NA
18
19
20
21
22
23
24
25
26
27
28
29
30
31
H
98.2±10.5
162.2±12.1
26.8±4.0
37.5±2.1
50.9±3.4
48.3±1.1
36.8±0.8
32.0±5.3
36.6±8.4
30.8±7.6

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NA
32
33
34
35
36
37
38
39
40
41
42
43
44
NA
44.9±6.9
34.1±3.4
36.8±3.6
20.6±4.1
60.7±8.0
25.5±3.3
56.5±5.5
30.6±7.4
41.5±3.3
43.1±9.1
38.8±8.2
H
64.8±12.9
45
52.1±8.3
NA
46
47
H
carfilzomib
23.6±8.7
a All assays were repeated three times.
b NA, no activity.
R1 modification. To investigate the influence of substituents at the R1 position
on potency, the corresponding substituents at R5, R4, R3 and R2 positions of
carfilzomib were employed in the newly designed compounds 17-20 (Table 1). The

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biological data revealed that aliphatic groups, such as ethyl (17, 77.1 nM) and butyl
(18, 52.2 nM) were more beneficial to the potency compared with aromatics groups,
such as phenyl (19, 139.6 nM) and benzyl (20, 729.0 nM). Moreover, among the
aliphatic groups, more steric substituents were more beneficial to the activity than the
less steric ones, which was proved by the comparison between isobutyl (carfilzomib,
23.6 nM) and 17, 18. These results indicated that isobutyl group played important
roles in maintaining the activities of this series of compounds. Therefore, this
substituent was employed in the design of other series of compounds.
R2 modification. Several aromatics and aliphatic groups were designed at R2
position (compounds 21-25, Table 1). The biological results showed that H atom at
this position led to the inactivity of compound 21, which suggested that substituent at
R2 position was essential to potency. Aliphatic groups at R2 position such as ethyl (22,
98.2 nM) and n-butyl (23, 162.2 nM) increased the binding affinity of molecules to β5
subunit. However, aromatic fragments such as thiazole (24, 26.8 nM), benzothiazole
(25, 37.5 nM) and phenyl (carfilzomib, 23.6 nM) were more beneficial to the activity
than aliphatic ones, which suggested that π-π interactions might occur between
inhibitors and amino acid residues of β5 subunit.
R3 modification. Modifications of the R3 position indicated that aliphatic
fragments were more beneficial to the activity than heteroaromatic ones.
Methoxymethylene (26, 50.9 nM), 2-hydroxyethyl (27, 48.3 nM), ethyl (28, 36.8 nM),
cyclopropyl (29, 32.0 nM), cyclopentylmethyl (30, 36.6 nM), cyclohexyl (31, 30.8
nM) all showed good inhibition of β5 subunit. However, substitutions with

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heteroaromatic groups (32 and 33) led to the loss of potency and the same case for H
atom substitution (47, NA vs 41, 30.6 nM). Furthermore, hydrophobic groups
(compounds 28 and 31) slightly increased the potency compared with the hydrophilic
ones (26 and 27).
R4 modification. Several aromatics and aliphatic groups were chosen for R4
modification with either morpholinylmethyl or 5-methyl-3-isoxazole as the R5
substituent and the biological results were outlined in Table 1. As for
morpholinemethyl group at R5 position, compounds with the aromatic groups at R4
position, such as thiophene (34, 44.9 nM), benzothiophene (35, 34.1 nM), thiazole (36,
36.8 nM) showed similar potency. While for the 5-methyl-3-isoxazole as the R5
substituent, aromatic 2-pyridyl (38, 60.7 nM), 2-indolyl (39, 25.5 nM) and aliphatic
methoxymethylene (41, 30.6 nM), isobutyl (42, 41.5 nM), cyclopropyl (43, 43.1 nM)
demonstrated the similar potency. Furthermore, potency of H atom at R4 position (40,
56.5 nM) also maintained, which suggested that the substituents at this position had
no crucial interactions with the residues of the active site.
R5 modification. A series of aromatic and aliphatic carboxylic acids were
attached to R5 positions and the enzymatic activities of resulting molecules were listed
in Table 1. Introduction of morpholinylmethylene (36, 36.8 nM),
5-methyl-3-isoxazole (37, 20.6 nM), pyrazinyl (44, 38.8 nM), 2,5-dichlorophenyl (45,
64.8 nM) and hydroxymethyl (46, 52.1 nM) resulted in similar potency to inhibit the
activity of β5 subunit, which suggested that this position had no obvious influence on
the potency and could be used to optimize the physicochemical properties of the

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compounds.
Cellular activities.
Based on the evaluation of proteasome chymotrypsin-like inhibitory activities,
seven compounds were selected to evaluate their cytotoxic activities against two MM
cell lines RPMI 8226 and U266B1 by CCK8 assay with carfilzomib as the positive
control. The biological results showed that all compounds showed potent cytotoxic
activities against two MM cell lines with IC50 values less than 0.1 μM (Table 2).
Among them, compound 36 exhibited potent cytotoxic activities against two MM cell
lines. So this compound was selected for further biological investigations.
Table 2. The cellular activities of compounds against MM cell lines (IC50, nM). a
Compd.
carfilzomib
24
25
28
31
34
35
36
Cell lines
76.9 86.6 47.0 14.7 34.6 53.8 27.6
±6.5 ±9.0 ±4.3 ±2.4 ±4.5 ±7.8 ±3.2
56.5 80.5 45.5 23.2 23.8 33.2 24.5
±4.5 ±8.6 ±5.7 ±3.2 ±3.5 ±5.1 ±2.4
39.1
±4.7
35.7
±4.8
RPMI8226
U226B1
aAll assays were repeated three times.
In vitro microsomal stability evaluation.
The microsomal stabilities of compound 36 was determined with various species
of liver microsomes, such as rat, mice, dog, monkey and human. And the results were
illustrated in Table 3. The marketed drug carfilzomib was selected as the standard.
The half-life (T1/2) and intrinsic clearance (CLint) data were used to evaluate the
metabolic stabilities. The results indicated that the stabilities of compound 36 and
carfilzomib were quite different in various species. Furthermore, compound 36
demonstrated better metabolic stability profiles in mice, rat and human microsomes
than carfilzomib. Especially in human microsome stability, the half-life t1/2 of
compound 36 was nearly 4 fold as that of carfilzomib, which showed compound 36
might have improved in vivo stability compared with carfilzomib.
Table 3. The microsomal stability profiles of compound 36 and carfilzomib.

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Species
Dog
Compd.
36
Parameters
Rat
1.36
1021
0.62
2231
Mice
1.10
1254
0.34
4104
Monkey
0.211
Human
1.07
T1/2 (min)
CLint (mL/min/kg)
T1/2 (min)
0.243
5712
6558
1301
0.286
4847
0.244
5673
<1.00
>1386
carfilzomib
CLint (mL/min/kg)
Due to its excellent in vitro data, compound 36 was further evaluated in vivo
antitumor efficacy against MM xenograft models.
In vivo efficacy of the luciferase expression of human MM cell RPMI8226
xenograft models.
To evaluate the inhibition of hematologic tumor, xenograft NOD/SCID mice
models inoculated with luciferase expression of RPMI8226 MM cell lines were built
and the tumor was detected by the optical in vivo imaging equipment IVIS Spectrum
(PerkinElmer life Science, Hopkinton, MA, USA). The average fluorescence intensity
and distribution were measured via Living Image 4.4 software using a region of
interest (ROI) centered of tumor tissue after mice were received D-luciferin solution
(150 mg/kg body weight) in 3 min. Once the tumor volume reached 100-150 mm3,
mice were intravenously injected with 2 mg/kg of compound 36 QD for 21 days. The
tumor was checked once a week. After 21 days, the treatment group exhibited
sustained relative tumor average radiance [p/s/cm2/sr] (Fig. 2A and 2B) and more
significant in vivo antitumor efficacy than the control group. The inhibition of tumor
growth (T/C%) for the treatment group was 49.5%.
In vivo efficacy of human MM cell ARH77 xenograft models.
To further investigate the in vivo efficacy of compound 36, human MM cell
ARH77 xenograft models were successfully developed with nude mice and the mice
were dosed with 5 mg/kg of compound 36 on day 1, 4, 8, 11, 15 and 18, and the tumor
was checked by caliper every two days. After 21 days treatment, the mice were
sacrificed with euthanasia. The results showed that compound 36 displayed
significant in vivo antitumor efficacy compared with control group and was nearly as

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active as carfilzomib (Fig. 2C), and the inhibitions of tumor growth (T/C%) for the
two compounds were 37.6% and 34.6%, respectively.
Inhibition of the human ether-a-go-go related gene (hERG) ion channel.
It was reported that carfilzomib showed cardiovascular side-effect in clinical
trial21. Generally, the human ether-a-go-go related gene (hERG) ion channel inhibition,
which may result in a concomitant risk of sudden death, must be avoided during drug
development. The inhibition of the hERG channel by compound 36 and carfilzomib
was evaluated in HEK293 cells using an in vitro Ion Works Quattro study. The results
indicated that candidate 36 had no binding with the hERG ion channel while
carfilzomib could bind the ion channel with IC50 of 92.1 μM, which suggested that
compound 36 might have less probabilities to induce QT interval prolongation than
carfilzomib for the patients.
Fig. 2. In vivo efficacy evaluation of compound 36 with human tumor cell xenograft
models: (A) Representative live animal imaging demonstrating luciferase expression
of human MM cell RPMI 8226 xenografts models in NOD/SCID mice before
initiation of treatment and at the end of experiment; (B) Effect of compound 36 on the

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luciferase expression of human MM cell RPMI-8226 xenograft models in NOD/SCID
mice after 2 mg/kg dose treatment QD; (C) Effect of compound 36 on the volume of
xenograft models in nude mice with human MM cell ARH77 after 5 mg/kg dose
treatment.
3. Conclusion
Thirty-two tetrapeptide epoxyketone proteasome inhibitors with variations of R1,
R2, R3, R4 and R5 substituents were synthesized and biologically investigated.
Comprehensive SAR analysis for such kind of compounds were performed in detail.
It showed that groups at R4 and R5 positions had no effects on the inhibition of
enzymatic activity while those at R1, R2 and R3 positions greatly affected the activity.
The cellular activity results showed that compound 36 was active against human
multiple myeloma (MM) cell lines with IC50 less than 25 nM. Microsomal stability
results indicated that compound 36 was more stable in mice, rat and human
microsomes than carfilzomib. Especially in human microsome, the half-life t1/2 of
compound 36 was nearly 4 fold as that of carfilzomib, which showed compound 36
might have improved in vivo stability compared with carfilzomib. The in vivo
xenograft mice models of human cancer cells ARH77 and luciferase expression of
RPMI-8226 showed that compound 36 effectively inhibited the tumor growth and the
T/C value of the two models were 49.5% and 37.6%, respectively. Results of
inhibiting hERG ion channel in HEK293 cells indicated that 36 did not bind the
hERG ion channel while carfilzomib could bind the ion channel with IC50 of 92.1 μM,
which suggested that compound 36 might have less probabilities to induce QT
interval prolongation than carfilzomib in clinic. Compound 36 is now being
developed as a new candidate for the treatment of MM.
4. Experimental section
4.1. General methods
Unless otherwise indicated，chemicals, solvents and reagents were purchased
from commercial suppliers and they were used without any purification. Absolutely
anhydrous solvents (CH2Cl2, THF, DMF, etc.) were purchased from Energy packaged
under nitrogen in Sure/Seal bottles. All reactions involving air or moisture-sensitive

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reagents were performed under an argon atmosphere. All reactions were detected by
thin layer chromatography on silica gel 60 plate coated with 0.25 mm layer and
spotted with UV light or iodine. All final products were purified to >95% purity. The
purity of the final products was determined by HPLC (Thermo) on an Agilent
Poroshell 120 EC-C18 column (50 mm × 4.6mm, 2.7μm) at 0.3 mL/min flow rate and
1
13
254 nm detector wavelength. H and C spectra were acquired at room temperature
on a Bruker Avance 400 spectrometer with chemical shift (δ, ppm) reported relative
to TMS as an internal standard. High-resolution mass spectra (HRMS) were recorded
on a ZAB-HS instrument using an electrospray ion source (ESI).
4.2 Chemistry
The traditional liquid peptide synthesis procedures were employed to prepare key
intermediates 8a-8z, 8aa and 8ab. A typical procedure for preparation of tripeptide
epoxyketone proteasome inhibitors of 17-47 was exemplified by the synthesis of
candidate 36.
(S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-
2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-3-(thiazo
l-4-yl)propanamido)pentanamide (36). To a cooled solution of 8q (0.7 g, 1.0 mmol)
dissolved in anhydrous CH2Cl2 (50 mL) was added HOBt (0.2 g, 1.5 mmol ). After 20
min, the temperature of the reaction system was cooled to -15 ℃and EDC·HCl (0.3 g,
1.5 mmol) was added. Finally, a solution of 16c (0.3 g, 1.0 mmol) and DIPEA (0.5 ml,
3.0 mmol) in anhydrous CH2Cl2 (5 mL) was added. The mixture was stirred at -15 ℃
for 30 min and at room temperature for 12 h, finally quenched with water. The
organic phase was washed with 10% of citric acid, 5% of NaHCO3 and saturated
brine, respectively, and dried over anhydrous Na2SO4, filtered and evaporated to
provide crude product. Column chromatography using dichloromethane/methanol
(50:1) afforded 0.3 g of pure 36. Yield 65%. 1H NMR (400 MHz, CDCl3) δ 0.88 –
0.80 (m, 6H, CH3), 0.98 – 0.90 (m, 6H, CH3), 1.25 (s, 3H, CH3), 1.33 – 1.28 (m, 2H,
CH), 1.64 – 1.50 (m, 4H, CH2), 2.87 – 2.48 (m, 4H, CH2), 3.05 – 2.97 (m, 2H, CH2),
3.10 (s, 2H, CH2), 3.26 – 3.07 (m, 2H, CH2), 3.37 – 3.27 (m, 4H, CH2), 3.72 – 3.64

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(m, 4H, CH2), 4.31 – 4.20 (m, 1H, CH), 4.74 – 4.49 (m, 3H, CH), 6.42 (d, J = 7.1 Hz,
1H, CONH), 6.57 (d, J = 8.1 Hz, 1H, CONH), 7.12 (d, J = 1.9 Hz, 1H, CONH), 7.15
(s, 1H, CONH), 7.17 (d, J = 1.4 Hz, 1H, Ph), 7.25 – 7.19 (m, 3H, Ph), 7.27 (d, J = 3.1
Hz, 1H, Ph), 8.36 (d, J = 6.4 Hz, 1H, 4-H of thiazole), 8.78 (d, J = 2.0 Hz, 1H, 2-H of
thiazole);13C NMR (100 MHz, CDCl3) δ 16.63, 21.20, 21.54, 22.89, 23.29, 24.65,
24.97, 29.61, 32.59, 36.93, 39.88, 40.32, 49.98, 52.19, 52.25, 52.75, 53.74, 53.88,
58.96, 61.70, 66.84, 116.18, 126.71, 128.39, 129.12, 136.96, 152.36, 153.42, 170.66,
170.73, 170.88, 171.78, 207.87; MS (ESI) m/z: 714.0 [M+H]+; HRMS calcd for
C36H52N6NaO7 S, [M+Na]+ 735.3550, found 735.3510.
(S)-4-methyl-N-((S)-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxobutan-2-yl)
amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenyl
butanamido)pentanamide (17)
Compound 17 was synthesized from 8a and 16a according to the general
procedure for preparing 36. Yield 73%. 1H NMR (400 MHz, DMSO-d6) δ 0.98 – 0.79
(m, 9H, CH3), 1.41 (s, 3H, CH3), 1.48 – 1.41 (m, 1H, CH), 1.70 – 1.48 (m, 4H, CH2),
1.97 – 1.74 (m, 2H, CH2), 2.50 – 2.41 (m, 4H, CH2), 2.84 – 2.71 (m, 2H, CH2), 2.97 –
2.86 (m, 2H, CH2), 3.05 – 3.01 (m, 2H, CH2), 3.22 – 3.06 (m, 2H, CH2), 3.62 – 3.45
(m, 4H, CH2), 4.32 – 4.11 (m, 2H, CH), 4.36 – 4.33 (m, 1H, CH), 4.62 – 4.50 (m, 1H,
CH), 7.32 – 7.03 (m, 10H, Ph), 7.99 – 7.91 (m, 2H, CONH), 8.11 (d, J = 7.9 Hz, 1H,
13
CONH), 8.23 (d, J = 6.9 Hz, 1H, CONH); C NMR (100 MHz, DMSO-d6) δ 10.39,
16.26, 21.58, 22.96, 23.34, 24.10, 31.40, 37.44, 40.72, 51.15, 51.41, 51.87, 52.24,
53.03, 58.88, 60.84, 65.83, 125.78, 126.14, 127.88, 128.24, 128.26, 129.08, 137.44,
141.49, 170.92, 170.96, 171.08, 171.54, 208.03; MS (ESI) m/z: 692.5 [M+H]+;
HRMS calcd for C38H53N5O7Na, [M+Na]+ 714.3837, found 714.3828.
(S)-4-methyl-N-((S)-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxohexan-2-yl)
amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenyl
butanamido)pentanamide (18)
Compound 18 was synthesized from 8a and 16b according to the general
procedure for preparing 36. Yield 87%. 1H NMR (400 MHz, DMSO-d6) δ 0.79 (d, J =
6.3 Hz, 3H, CH3), 0.85 (d, J = 6.0 Hz, 6H, CH3), 1.24 – 1.18 (m, 2H, CH2), 1.35 –

JOURNAL PRE-PROOF
1.29 (m, 2H, CH2), 1.38 – 1.32 (m, 2H, CH2), 1.39 (s, 3H, CH3), 1.63 – 1.45 (m, 2H,
CH2), 1.96 – 1.71 (m, 2H, CH2), 2.46 – 2.31 (m, 4H, CH2), 2.83 – 2.70 (m, 1H, CH2),
2.95 (d, J = 4.8 Hz, 2H, CH2), 2.98 (d, J = 3.9 Hz, 1H, CH2), 3.21 – 3.06 (m, 2H,
CH2), 3.83 – 3.61 (m, 4H, CH2), 4.31 – 4.15 (m, 2H, CH), 4.40 – 4.32 (m, 1H, CH),
4.54 – 4.51 (m, 1H, CH), 7.31 – 7.03 (m, 10H, Ph), 7.94 (d, J = 7.9 Hz, 1H, CONH),
13
8.11 (d, J = 7.9 Hz, 1H, CONH), 8.23 (d, J = 6.9 Hz, 1H, CONH); C NMR (100
MHz, DMSO-d6) δ 13.68, 16.32, 21.57, 21.71, 24.09, 27.56, 29.52, 31.39, 34.32,
37.45, 40.74, 50.75, 51.13, 51.44, 51.82, 52.98, 53.13, 53.38, 58.84, 66.06, 125.76,
126.13, 127.88, 127.94, 128.24, 129.08, 137.44, 141.50, 170.84, 170.94, 171.03,
171.51, 208.03; MS (ESI) m/z: 720.6 [M+H]+，743.6 [M+Na]+; HRMS calcd for
C40H57N5O7Na, [M+Na]+ 742.4150, found 742.4159.
(S)-4-methyl-N-((S)-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-
2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenyl
butanamido)pentanamide (19)
Compound 19 was synthesized from 8a and 16d according to the general
procedure for preparing 36. Yield 80%. 1H NMR (400 MHz, DMSO-d6) δ 0.79 (d, J =
6.2 Hz, 3H, CH3), 0.84 (d, J = 6.5 Hz, 3H, CH3), 1.24 (s, 3H, CH3), 1.39 – 1.32 (m,
1H, CH), 1.56 – 1.49 (m, 2H, CH2), 1.95 – 1.74 (m, 2H, CH2), 2.46 (s, 4H, CH2), 2.56
(dd, J = 17.0, 6.8 Hz, 1H, CH2), 2.78 – 2.60 (m, 2H, CH2), 2.93 (d, J = 4.6 Hz, 2H,
CH2), 2.98 (d, J = 11.0 Hz, 1H, CH2), 3.18 – 3.01 (m, 2H, CH2), 3.61 (s, 4H, CH2),
4.28 (d, J = 4.2 Hz, 1H, CH), 4.37 (dd, J = 13.0, 7.7 Hz, 1H, CH), 4.64 – 4.55 (m, 2H,
CH), 7.32 – 6.89 (m, 15H, Ph), 7.89 (s, 1H, CONH), 7.92 (s, 1H, CONH), 8.08 (s, 1H,
CONH), 8.59 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6) δ 16.19, 21.49, 23.02,
29.02, 31.39, 35.55, 37.59, 40.59, 51.02, 51.55, 51.79, 52.11, 52.57, 53.11, 58.89,
58.94, 65.99, 125.76, 126.14, 126.56, 127.82, 127.88, 128.25, 128.99, 129.06, 129.08,
137.00, 137.34, 141.52, 170.75, 170.98, 171.05, 171.47, 207.80; MS (ESI) m/z: 754.6
[M+H]+; HRMS calcd for C43H55N5O7Na, [M+Na]+ 776.3993, found 776.4001.
(S)-4-methyl-N-((S)-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-4-phenylbutan-2
-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylb
utanamido)pentanamide (20)

JOURNAL PRE-PROOF
Compound 20 was synthesized from 8a and 16e according to the general
1
procedure for preparing 36. Yield 76%. H NMR (400 MHz, CDCl3) δ 0.85 (dd, J =
10.8, 5.7 Hz, 6H, CH3), 1.33 (s, 3H, CH3), 1.47 – 1.39 (m, 1H, CH), 1.62 – 1.49 (m,
2H, CH2), 1.71 (dd, J = 40.3, 30.2 Hz, 2H, CH2), 2.04 (dd, J = 11.7, 7.1 Hz, 4H, CH2),
2.38 – 2.18 (m, 1H, CH2), 2.74 – 2.39 (m, 6H, CH2), 2.82 (dd, J = 14.8, 4.9 Hz, 1H,
CH2), 3.10 – 3.02 (m, 2H, CH2), 3.35 – 3.11 (m, 2H, CH2), 3.73 (s, 4H, CH2), 4.67 –
4.41 (m, 3H, CH), 4.75 (d, J = 43.0 Hz, 1H, CH), 5.34 (s, 1H, CONH), 6.69 (s, 1H,
13
CONH), 7.30 – 7.06 (m, 15H, Ph), 7.35 (s, 1H, CONH), 7.54 (s, 1H, CONH); C
NMR (100 MHz, CDCl3) δ 16.69, 22.94, 24.84, 29.81, 30.32, 32.04, 32.90, 38.38,
41.67, 51.57, 52.14, 52.34, 52.56, 52.81, 53.45, 54.20, 59.12, 66.50, 126.24, 126.33,
127.07, 128.39, 128.45, 128.50, 128.56, 128.66, 129.38, 136.48, 140.73, 140.82,
171.18, 171.46, 171.71, 172.10, 207.50; MS (ESI) m/z: 768.8 [M+H]+; HRMS calcd
for C44H57N5O7Na, [M+Na]+ 790.4150, found 790.4139.
(S)-4-methyl-N-(2-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl
)amino)-2-oxoethyl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-
pentanamide (21)
Compound 21 was synthesized from 8b and 16c according to the general
procedure for preparing 36. Yield 35%. 1H NMR (400 MHz, CDCl3) δ 0.89 – 0.84 (m,
12H, CH3), 1.23 (s, 3H, CH3), 1.68 – 1.52 (m, 2H, CH), 1.86 – 1.69 (m, 2H, CH2),
2.05 – 1.93 (m, 2H, CH2), 2.08 (dd, J = 13.8, 6.7 Hz, 1H, CH2), 2.56 (dd, J = 19.0,
10.9 Hz, 1H, CH2), 2.88 (d, J = 5.0 Hz, 2H, CH2), 3.01 (d, J = 9.6 Hz, 2H, CH2), 3.31
(d, J = 5.0 Hz, 2H, CH2), 3.73 (d, J = 9.6 Hz, 2H, CH2), 4.68 – 4.45 (m, 2H, CH),
4.76 – 4.71 (m, 1H, CH), 6.62 (s, 1H, CONH), 6.99 (s, 1H, CONH), 7.24 – 7.10 (m,
13
5H, Ph), 7.63 (s, 1H, CONH), 7.72 (s, 1H, CONH); C NMR (100 MHz, CDCl3) δ
13.75, 16.74, 21.21, 22.24, 22.28, 22.77, 23.28, 24.69, 25.15, 27.33, 29.64, 31.81,
32.67, 35.07, 39.39, 42.13, 50.26, 51.54, 52.19, 52.37, 52.66, 53.56, 59.12, 66.68,
125.98, 128.04, 128.38, 140.79, 171.17, 171.75, 208.31; MS (ESI) m/z: 630.8
[M+H]+; HRMS calcd for C34H51N5O7, [M+H]+ 630.3861, found 630.3842.
(S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-

JOURNAL PRE-PROOF
2-yl)amino)-1-oxobutan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutan-
amido) pentanamide (22)
Compound 22 was synthesized from 8c and 16c according to the general
1
procedure for preparing 36. Yield 35%. H NMR (400 MHz, CDCl3) δ 0.91 (dd, J =
12.3, 6.3 Hz, 12H, CH3), 1.26 (d, J = 12.5 Hz, 3H, CH3), 1.49 – 1.35 (m, 2H, CH),
1.65 – 1.57 (m, 2H, CH2), 1.89 – 1.71 (m, 4H, CH2), 2.22 – 2.00 (m, 4H, CH2), 2.53
(d, J = 3.8 Hz, 2H, CH2), 2.71 – 2.61 (m, 2H, CH2), 3.02 (s, 2H, CH2), 3.77 – 3.68 (m,
4H, CH2), 4.06 – 3.98 (m, 2H, CH), 4.52 – 4.39 (m, 2H, CH), 6.68 (s, 1H, CONH),
13
7.23 – 7.13 (m, 5H, Ph), 7.29 (s, 1H, CONH), 7.60 (s, 1H, CONH); C NMR (100
MHz, CDCl3) δ 21.86, 22.86, 24.69, 31.85, 33.78, 40.52, 41.10, 51.65, 52.32, 52.50,
53.71, 61.67, 66.79, 126.20, 128.31, 128.52, 140.57, 170.05, 171.50, 171.94; MS (ESI)
m/z: 658.6 [M+H]+; HRMS calcd for C35H56N5O7, [M+H]+ 658.4174, found
658.4198.
(S)-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)-2-((S)-4-me
thyl-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamido)hexan-
amide (23)
Compound 23 was synthesized from 8d and 16c according to the general
procedure for preparing 36. Yield 51%. 1H NMR (400 MHz, CDCl3) δ 0.92 – 0.84 (m,
15H, CH3), 1.27 (t, J = 8.4 Hz, 3H, CH3), 1.37 – 1.30 (m, 4H, CH2), 1.53 – 1.46 (m,
2H, CH), 1.69 – 1.54 (m, 4H, CH2), 1.99 – 1.73 (m, 2H, CH2), 2.14 – 2.02 (m, 2H,
CH2), 2.54 (dd, J = 19.6, 11.4 Hz, 2H, CH2), 2.88 (d, J = 5.0 Hz, 2H, CH2), 2.99 –
2.87 (m, 4H, CH2), 3.33 (t, J = 7.3 Hz, 2H, CH2), 3.73 (t, J = 6.0 Hz, 2H, CH2), 4.56 –
4.43(m, 2H, CH), 4.68 (d, J = 7.0 Hz, 1H, CH), 4.82 (d, J = 7.1 Hz, 1H, CH), 6.72 (s,
1H, CONH), 7.03 (t, J = 10.2 Hz, 2H, Ph), 7.21 – 7.14 (m, 2H, Ph), 7.38 (s, 2H,
13
CONH), 7.73 (s, 1H, CONH); C NMR (100 MHz, CDCl3) δ 9.71, 16.73, 21.19,
22.22, 22.78, 23.29, 24.69, 25.14, 26.33, 31.82, 35.11, 39.41, 42.14, 50.29, 51.52,
52.21, 52.37, 53.63, 59.13, 66.74, 125.96, 128.04, 128.37, 140.83, 171.19, 171.61,
171.82, 208.49; MS (ESI) m/z: 686.9 [M+H]+; HRMS calcd for C38H59N5O7, [M+H]+
686.4487, found 686.4516.
(S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-

JOURNAL PRE-PROOF
2-yl)amino)-1-oxo-3-(thiazol-4-yl)propan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-
phenylbutanamido)pentanamide (24)
Compound 24 was synthesized from 8e and 16c according to the general
procedure for preparing 36. Yield 55%. 1H NMR (400 MHz, CDCl3) δ 0.76 (d, J = 6.4
Hz, 3H, CH3), 0.80 (s, 3H, CH3), 0.86 (d, J = 5.9 Hz, 3H, CH3), 0.91 (d, J = 5.9 Hz,
3H, CH3), 1.24 (s, 3H, CH3), 1.42 – 1.33 (m, 2H, CH), 1.62 (dd, J = 16.8, 9.5 Hz, 2H,
CH2), 2.02 (dd, J = 12.4, 5.5 Hz, 4H, CH2), 2.71 – 2.57 (m, J = 21.4, 13.2, 6.7 Hz, 6H,
CH2), 3.13 (d, J = 13.0 Hz, 2H, CH2), 3.22 (t, J = 12.0 Hz, 2H, CH2), 3.34 (dd, J =
14.7, 5.7 Hz, 2H, CH2), 3.75 (s, 2H, CH2), 4.30 (dd, J = 10.0, 4.4 Hz, 1H, CH), 4.47
(d, J = 7.9 Hz, 1H, CH), 4.55 (s, 1H, CH), 4.75 (dd, J = 13.0, 5.5 Hz, 1H, CH), 7.06 (s,
1H, CONH), 7.13 (d, J = 7.3 Hz, 3H, Ph), 7.21 (t, J = 7.4 Hz, 2H, Ph), 7.28 (s, 1H,
CONH), 7.29 (s, 1H, CONH), 7.31 (d, J = 4.2 Hz, 1H, CONH), 7.94 (d, J = 7.2 Hz,
13
1H, 4-H of thiazole), 8.19 (s, 1H, 2-H of thiazole); C NMR (100 MHz, CDCl3) δ
16.83, 21.13, 21.54, 23.11, 23.56, 24.93, 24.99, 29.81, 31.93, 32.36, 39.54, 40.61,
50.03, 52.41, 52.99, 53.09, 53.87, 59.14, 60.53, 66.52, 66.60, 116.31, 126.60, 128.68,
128.86, 140.67, 152.91, 153.29, 170.56, 171.67, 172.17, 207.85; MS (ESI) m/z: 727.5
[M+H]+; HRMS calcd for C37H54N6O7SH, [M+H]+ 727.3847, found 727.3870.
(S)-N-((S)-3-(benzo[b]thiophen-2-yl)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-
yl)-1-oxopentan-2-yl)amino)-1-oxopropan-2-yl)-4-methyl-2-((S)-2-(2-morpholino
acetamido)-4-phenylbutanamido)pentanamide (25)
Compound 25 was synthesized from 8f and 16c according to the general
procedure for preparing 36. Yield 51%. 1H NMR (400 MHz, CDCl3) δ 0.81 – 0.77 (m,
6H, CH3), 0.93 – 0.86 (m, 6H, CH3), 1.25 (s, 3H, CH3), 1.41 – 1.36 (m, 2H, CH), 1.52
– 1.49 (m, 2H, CH2), 1.67 – 1.56 (m, 2H, CH2), 2.03 – 1.90 (m, 2H, CH2), 2.59 – 2.39
(m, 6H, CH2), 2.78 (d, J = 52.3 Hz, 2H, CH2), 2.99 (s, 2H, CH2), 3.24 (d, J = 28.1, 2H,
CH2), 3.75 (s, 2H, CH2), 4.50 (s, 1H, CH), 4.58 (d, J = 6.8 Hz, 1H, CH), 4.77 – 4.65
(m, 2H, CH), 6.01 (s, 2H, CONH), 6.93 (s, 1H, CONH), 7.04 (s, 1H, CONH), 7.18 –
7.09 (m, 5H, Ph) 7.23 (m, 1H, 3-H of benzothiophene), 7.35 – 7.29 (m, 3H, Ph), 7.83
– 7.68 (m, 2H, Ph); 13C NMR (100 MHz, CDCl3) δ 16.82, 21.45, 22.98, 23.54, 24.88,
25.17, 29.85, 37.80, 39.34, 40.11, 40.74, 52.43, 52.53, 53.59, 53.67, 54.11, 55.98,

JOURNAL PRE-PROOF
57.37, 58.51, 59.12, 66.01, 121.69, 123.22, 124.60, 124.90, 127.07, 128.64, 129.41,
130.92, 136.68, 138.67, 140.52, 170.72, 171.21, 207.96; MS (ESI) m/z: 776.4
[M+H]+; HRMS calcd for C42H57N5O7SH, [M+H]+ 776.4051, found 776.4143.
N-((S)-1-(((S)-3-methoxy-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-
1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxo-4-phenylbutan-2-yl)-5-methylisoxazole-3-carboxamide (26)
Compound 26 was synthesized from 8g and 16c according to the general
1
procedure for preparing 36. Yield 30%. H NMR (400 MHz, CDCl3) δ 0.88 (s, 3H,
CH3), 0.89 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.43 – 1.40 (m, 1H, CH), 1.50 – 1.47 (m,
2H, CH2), 2.49 (s, 3H, 5-CH3 of isoxazole), 2.87 (d, J = 4.7 Hz, 1H, CH2), 2.90 – 2.87
(m, 2H, CH2), 3.01 (d, J = 19.2 Hz, 2H, CH2), 3.21 – 3.15 (m, 2H, CH2), 3.31 (s, 3H,
OCH3), 4.27 – 4.21 (m, 1H, CH), 4.55 – 4.45 (m, 1H, CH), 4.63 – 4.58 (m, 1H, CH),
4.81 – 4.76 (m, 1H, CH), 6.38 (s, 1H, 4-H of isoxazole), 6.66 (s, 1H, CONH), 7.11 (s,
1H, CONH), 7.19 – 7.13 (m, 4H, Ph), 7.23 – 7.19 (m, 3H, Ph), 7.34 (s, 1H, Ph), 7.52
13
(d, J = 8.2 Hz, 2H, Ph), 7.74 (s, 1H, CONH), 8.52 (s, 1H, CONH); C NMR (100
MHz, CDCl3) δ 14.29, 16.87, 21.44, 21.53, 22.85, 23.13, 24.76, 36.48 , 38.56, 39.99,
40.66, 50.21, 52.47, 53.77, 54.14, 54.30, 59.22, 101.38, 126.61, 126.85, 128.60,
129.29, 129.34, 135.39, 135.50, 147.19, 151.69, 159.17, 162.48, 167.86, 171.07,
171.68, 172.26, 208.10; MS (ESI) m/z: 711.6 [M+Na]+; HRMS calcd for
C37H48N6O7Na, [M+Na]+ 711.3476 found 711.3405.
N-((S)-1-(((2S,3S)-3-hydroxy-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-
yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxobutan-2-yl)
amino)-1-oxo-4-phenylbutan-2-yl)-5-methylisoxazole-3-carboxamide (27)
Compound 27 was synthesized from 8h and 16c according to the general
procedure for preparing 36. Yield 80%. 1H NMR (400 MHz, DMSO-d6) δ 0.91 – 0.73
(m, 6H, CH3), 1.23 – 1.18 (m, 1H, CH), 1.30 (s, 3H, CH3), 1.39 (d, J = 6.9 Hz, 3H,
CH3), 1.97 (d, J = 7.2 Hz, 2H, CH2), 2.47 (s, 3H, 5-CH3 of isoxazole), 2.60 (m, 2H,
CH2), 2.90 – 2.70 (m, 2H, CH2), 3.04 – 2.90 (m, 2H, CH2), 3.23 (d, J = 5.2 Hz, 3H,
CH2), 3.45 (d, J = 5.5 Hz, 1H, CH2), 4.42 – 4.24 (m, 3H, CH), 4.54 – 4.50 (m, 2H,

JOURNAL PRE-PROOF
CH), 5.37 (s, 1H, OH), 6.57 (d, J = 6.8 Hz, 1H, 4-H of isoxazole), 7.32 – 7.02 (m,
13
10H, Ph), 8.15 (s, 1H, CONH), 8.40 (s, 2H, CONH), 8.77 (s, 1H, CONH); C NMR
(100 MHz, DMSO-d6) δ 11.83, 16.38, 21.04, 23.11, 31.61, 33.65, 37.43, 38.58, 49.17,
51.48, 52.52, 52.76, 53.25, 58.24, 58.80, 71.86, 101.40, 125.78, 126.17, 127.91,
128.29, 129.07, 129.21, 137.31, 141.37, 158.65, 162.28, 168.96, 170.76, 170.90,
171.22, 208.18; MS (ESI) m/z: 690.5 [M+H]+, 713.5 [M+Na]+; HRMS calcd for
C37H47N5O8Na, [M+Na]+ 712.3316, found 712.3320.
5-methyl-N-((S)-1-(((S)-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-
oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxobutan-2-yl)amino)-1
-oxo-4-phenylbutan-2-yl)isoxazole-3-carboxamide (28)
Compound 28 was synthesized from 8i and 16c according to the general
procedure for preparing 36. Yield 77%. 1H NMR (400 MHz, DMSO-d6) δ 0.90 – 0.71
(m, 9H, CH3), 1.31 – 1.22 (m, 1H, CH), 1.39 (d, J = 7.8 Hz, 3H, CH3), 1.70 – 1.44 (m,
2H, CH2), 2.04 – 1.92 (m, 2H, CH2), 2.47 (s, 3H, 5-CH3 of isoxazole), 2.60 – 2.56 (m,
2H, CH2), 2.92 – 2.70 (m, 2H, CH2), 2.95 – 2.84 (m, 1H, CH2), 3.16 – 3.07 (m, 1H,
CH2), 4.22 – 4.19 (m, 1H, CH), 4.42 – 4.28 (m, 1H, CH), 4.48 (m, 1H, CH), 4.64 –
4.54 (m, 1H, CH), 6.58 (s, 1H, 4-H of isoxazole), 7.32 – 7.01 (m, 10H, Ph), 8.07 –
13
7.97 (m, 2H, CONH), 8.29 (s, 2H, CONH), 8.71 (d, J = 8.0 Hz, 2H, CONH); C
NMR (100 MHz, DMSO-d6) δ 9.95, 11.82, 16.40, 23.12, 24.48, 31.64, 33.65, 37.54,
38.42, 49.26, 51.54, 52.87, 53.70, 58.80, 59.74, 101.41, 125.79, 126.17, 127.91,
128.24, 128.30, 129.05, 137.43, 141.40, 158.64, 170.29, 170.73, 170.88, 171.08,
171.21, 208.18; MS (ESI) m/z: 674.6 [M+H]+, 696.6 [M+Na]+; HRMS calcd for
C37H47N5O7Na, [M+Na]+ 696.3367, found 696.3374.
N-((S)-1-(((S)-1-cyclopropyl-2-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-
yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-
1-oxo-4-phenylbutan-2-yl)-5-methylisoxazole-3-carboxamide (29)
Compound 29 was synthesized from 8j and 16c according to the general
procedure for preparing 45. Yield 54%. 1H NMR (400 MHz, CDCl3) δ 0.69 – 0.40 (m,

JOURNAL PRE-PROOF
4H, CH2), 1.09 – 0.92 (m, 6H, CH3), 1.25 – 1.19 (m, 1H, CH), 1.41 (d, J = 7.8 Hz, 3H,
CH3), 1.49 – 1.45 (m, 1H, CH), 1.76 – 1.61 (m, 2H, CH2), 2.25 – 2.19 (m, 2H, CH2),
2.64 (s, 3H, 5-CH3 of isoxazole), 3.00 (d, J = 4.9 Hz, 1H, CH2), 3.18 – 3.09 (m, 2H,
CH2), 3.44 (d, J = 4.9 Hz, 1H, CH2), 4.34 – 4.28 (m, 1H, CH), 4.80 – 4.72 (m, 1H,
CH), 5.18 – 5.08 (m, 2H, CH), 6.70 (s, 1H, 4-H of isoxazole), 7.39 – 7.02 (m, 10H,
Ph), 7.55 – 7.40 (s, 2H, CONH), 8.02 (s, 2H, CONH); 13C NMR (100 MHz, CDCl3) δ
3.36, 3.61, 12.60, 14.39, 14.74, 16.87, 21.79, 22.97, 23.41, 25.43, 29.64, 29.98, 32.12,
32.21, 35.05, 38.84, 40.39, 49.96, 52.53, 53.16,54.18, 56.72, 59.17, 77.00, 77.32,
77.52, 77.63, 101.89, 126.30, 127.10, 128.61, 128.65, 128.70, 129.55, 136.57, 141.08,
158.67, 159.64, 171.05, 171.09, 171.39, 171.43, 208.61; MS (ESI) m/z: 686.8
[M+H]+, 708.8 [M+Na]+; HRMS calcd for C38 H48 N5O7, [M+H]+ 686.3586, found
686.3548.
N-((S)-1-(((S)-3-cyclopentyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-y
l)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxo-4-phenylbutan-2-yl)-5-methylisoxazole-3-carboxamide (30)
Compound 30 was synthesized from 8k and 16c according to the general
procedure for preparing 36. Yield 76%. 1H NMR (400 MHz, CDCl3) δ 0.96 – 0.88 (m,
6H, CH3), 1.25 (s, 3H, CH3), 1.31 – 1.28 (m, 1H, CH), 1.32 – 1.90 (m, 10H, CH2),
2.49 (s, J = 12.2 Hz, 3H, 5-CH3 of isoxazole), 2.90 – 2.80 (m, 2H, CH2), 3.15 – 3.08
(m, 1H, CH2), 3.37 – 3.19 (m, 3H, CH2), 4.44 – 4.31 (m, 1H, CH), 4.65 – 4.52 (m, 2H,
CH), 4.80 – 4.72 (m, 1H, CH), 6.25 (s, 1H, CONH), 6.41 (s, 1H, 4-H of isoxazole),
6.56 (s, 1H, CONH), 6.70 (s, 1H, CONH), 7.02 – 6.97 (m, 2H, Ph), 7.21 – 7.11 (m,
5H, Ph), 7.37 (m, 2H, Ph), 7.52 (s, 1H, Ph), 7.71 (s, 1H, CONH), 8.46 (s, 1H, CONH);
13C NMR (100 MHz, CDCl3) δ 12.30, 14.08, 16.69, 21.30, 21.72, 22.68, 23.32, 24.51,
25.08, 29.67, 31.41, 31.90, 37.11, 39.90, 40.45, 52.33, 54.15, 59.03, 101.30, 109.89,
111.49, 118.71, 119.96, 122.55, 123.45, 123.95, 124.45, 126.84, 126.97, 128.49,
129.12, 136.31, 136.76, 158.08, 159.59,170.87, 170.96, 171.52, 171.58, 207.93; MS
(ESI) m/z: 728.0 [M+H]+, 750.1 [M+Na]+; HRMS calcd for C40H51N6O7, [M+H]+
727.3855, found 727.3813.

JOURNAL PRE-PROOF
N-((S)-1-(((S)-1-cyclohexyl-2-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl
)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-1-
oxo-4-phenylbutan-2-yl)-5-methylisoxazole-3-carboxamide (31)
Compound 31 was synthesized from 8l and 16c according to the general
procedure for preparing 36. Yield 76%. 1H NMR (400 MHz, DMSO-d6) δ 0.89 – 0.69
(m, 6H, CH3), 1.38 (d, J = 5.4 Hz, 3H, CH3), 1.44 – 1.39 (m, 1H, CH), 1.68 – 1.50 (m,
10H, CH2), 2.04 – 1.87 (m, 2H, CH2), 2.47 (s, 3H, 5-CH3 of isoxazole), 2.66 – 2.52
(m, 2H, CH2), 2.80 – 2.69 (m, 1H, CH2), 2.92 – 2.82 (m, 1H, CH2), 3.07 – 2.95 (m,
1H, CH2), 3.33 – 3.15 (m, 1H, CH2), 4.18 (t, J = 6.5 Hz, 1H, CH), 4.42 – 4.27 (m, 1H,
CH), 4.48 (d, J = 4.7 Hz, 1H, CH), 4.57 – 4.49 (m, 1H, CH), 6.57 (s, 1H, 4-H of
isoxazole), 7.30 – 6.97 (m, 10H, Ph), 7.89 (s, 1H, CONH), 8.08 (s, 1H, CONH), 8.22
(s, 1H, CONH), 8.73 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6) δ 11.84, 16.37,
20.95, 23.15, 24.42, 25.59, 27.99, 28.97, 31.69, 33.72, 37.52, 38.58, 49.10, 51.39,
52.92, 53.41, 57.08, 58.75, 101.41, 125.80, 126.11, 127.88, 128.26, 128.29, 128.94,
137.41, 141.38, 158.63, 170.28, 170.54, 170.79, 171.00, 171.24, 208.01; MS (ESI)
m/z: 728.8 [M+H]+, 750.9 [M+Na]+; HRMS calcd for C41H53N5O7Na, [M+Na]+
750.3837, found 750.3839.
5-methyl-N-((S)-1-(((S)-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-
oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-(thiazol-4-yl)
propan-2-yl)amino)-1-oxo-4-phenylbutan-2-yl)isoxazole-3-carboxamide (32)
Compound 32 was synthesized from 8m and 16c according to the general
procedure for preparing 36. Yield 39%. 1H NMR (400 MHz, CDCl3) δ 0.98 – 0.88 (m,
6H, CH3), 1.25 (s, 3H, CH3), 1.47 – 1.41 (m, 1H, CH), 1.71 – 1.69 (m, 2H, CH2), 2.12
– 2.03 (m, 2H, CH2), 2.34 – 2.23 (m, 2H, CH2), 2.47 (s, 3H, 5-CH3 of isoxazole), 2.93
(d, J = 4.9 Hz, 2H, CH2), 3.35 (d, J = 4.9 Hz, 2H, CH2), 3.49 – 3.39 (m, 2H, CH2),
4.89 – 4.66 (m, 4H, CH), 6.37 (s, 1H, 4-H of isoxazole), 7.00 (s, 1H, CONH), 7.04 (d,
J = 33.5 Hz, 10H, Ph), 7.35 (s, 2H, CONH), 7.52 (s, 1H, CONH), 8.79 (s, 1H, 4-H of
13
thiazol), 8.98 (s, 1H, 2-H of thiazol); C NMR (100 MHz, CDCl3) δ 12.31, 16.69,
22.67, 22.92, 23.31, 24.60, 24.70, 25.05, 29.09, 29.24, 29.34, 29.44, 29.58, 29.63,

JOURNAL PRE-PROOF
29.67, 31.91, 33.88, 36.98, 39.90, 40.13, 50.11, 52.30, 52.33, 53.66, 54.14, 59.03,
101.32, 116.42, 126.73, 128.45, 129.19, 137.10, 151.92, 153.66, 158.13, 170.30,
171.02, 171.53, 171.69, 178.29, 207.84; MS (ESI) m/z: 743.3; HRMS calcd for
C39H46N6O7SNa, [M+Na]+ 766.3073, found 766.3462.
5-methyl-N-((S)-1-(((S)-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-
oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-(pyridin-2-yl)
propan-2-yl)amino)-1-oxo-4-phenylbutan-2-yl)isoxazole-3-carboxamide (33)
Compound 33 was synthesized from 8n and 16c according to the general
1
procedure for preparing 36. Yield 42%. H NMR (400 MHz, CDCl3) δ 0.95 (dd, J =
8.2, 6.5 Hz, 3H, CH3), 0.99 (dd, J = 6.3, 3.5 Hz, 3H, CH3), 1.44 – 1.41 (m, 1H, CH),
1.28 (s, 3H, CH3), 1.65 – 1.63 (m, 2H, CH2), 1.67 – 1.65 (m, 2H, CH2), 2.06 – 1.94
(m, 2H, CH2), 2.39 – 2.16 (m, 2H, CH2), 2.45 (s, 3H, 5-CH3 of isoxazole), 2.94 – 2.87
(m, 2H, CH2), 3.08 (d, J = 4.7 Hz, 1H, CH2), 3.21 (d, J = 4.7 Hz, 1H, CH2), 4.68 –
4.57 (m, 1H, CH), 4.98 – 4.86 (m, 2H, CH), 5.36 – 5.21 (m, 1H, CH), 6.20 (s, 1H,
4-H of isoxazole), 6.72 (s, 1H, CONH), 7.38 – 7.26 (m, 4H, Ph), 7.50 – 7.38 (m, 4H,
Ph), 7.62 – 7.53 (m, 4H, Ph), 7.71 (d, J = 26.2, 8.4 Hz, 1H, 4-H of pyridin), 8.04 (d, J
13
= 8.5 Hz, 2H, 6-H of pyridin), 8.07 (s, 3H, CONH); C NMR (100 MHz, CDCl3) δ
12.61, 16.52, 21.14, 21.68, 24.05, 29.46, 32.51, 35.26, 37.51, 39.12, 51.40, 54.55,
55.35, 56.79, 57.05, 57.73, 58.14, 100.35, 121.74, 122.47, 125.10, 126.07, 127.00,
127.97, 128.30, 128.67, 128.80, 129.14, 129.58, 136.10, 136.47, 142.05, 148.22,
150.08, 159.13, 160.76, 169.51, 170.09, 171.61, 171.92, 210.71; MS (ESI) m/z: 737.4
[M+H]+; HRMS calcd for C41H49N6O7, [M+H]+ 737.3657, found 737.3640.
(S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-
2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-3-(thioph
en-3-yl)propanamido)pentanamide (34)
Compound 34 was synthesized from 8o and 16c according to the general
1
procedure for preparing 36. Yield 60%. H NMR (400 MHz, CDCl3) δ 0.67 (s, 3H,
CH3), 0.77 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.86 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.43

JOURNAL PRE-PROOF
– 1.37 (m, 1H, CH), 1.51 – 1.44 (m, 2H, CH2), 2.59 – 2.46 (m, 2H, CH2), 2.88 – 2.77
(m, 4H, CH2), 3.05 – 2.93 (m, 4H, CH2), 3.15 – 3.10 (m, 2H, CH2), 3.31 – 3.20 (m,
2H, CH2), 3.75 – 3.64 (m, 4H, CH2), 4.59 – 4.35 (m, 3H, CH), 5.12 – 5.09 (s, 1H,
CH), 6.18 (s, 2H, CONH), 6.87 (s, 1H, CONH), 7.00-7.07 (m, 3H, 2, 4, 5-H of
thiophen), 7.23 – 7.14 (m, 5H, Ph), 8.41 (s, 1H, CONH); 13C NMR (100 MHz, CDCl3)
δ 16.77, 21.19, 21.38, 22.34, 22.98, 23.35, 24.81, 29.83, 31.91, 39.81, 42.04, 51.85,
52.44, 54.30, 54.51, 56.08, 57.08, 59.13, 59.28, 61.50, 66.01, 66.02, 123.17, 126.14,
126.16, 128.34, 128.55, 140.78, 140.88, 169.62, 171.28, 205.99; MS (ESI) m/z: 712.5
[M+H]+; HRMS calcd for C37H53N5O7SNa, [M+Na]+ 734.3557, found 734.3683.
(S)-2-((S)-3-(benzo[b]thiophen-2-yl)-2-(2-morpholinoacetamido)propanamido)-
4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)
amino)-1-oxo-3-phenylpropan-2-yl)pentanamide (35)
Compound 35 was synthesized from 8p and 16c according to the general
1
procedure for preparing 36. Yield 65%. H NMR (400 MHz, CDCl3) δ 0.82 (d, J =
10.9, 6H, CH3) 0.90 – 0.86 (m, 6H, CH3), 1.25 (s, 3H, CH3), 1.49 – 1.43 (m, 2H, CH),
1.86 –1.71 (m, 4H, CH2), 2.08 – 1.95 (m, 2H, CH2), 2.25 – 2.12 (m, 2H, CH2), 2.39 –
2.26 (m, 2H, CH2), 2.52 – 2.42 (m, 2H, CH2), 2.88 – 2.79 (m, 2H, CH2), 3.05 (d, J =
5.3 Hz, 2H, CH2), 3.28 – 3.17 (m, 2H, CH2), 3.48 – 3.31 (m, 2H, CH2), 4.32 – 4.25 (m,
1H, CH), 4.56 – 4.49 (m, 1H, CH), 4.84 – 4.63 (m, 2H, CH), 6.62 (d, J = 26.0 Hz, 4H,
CONH), 7.20 – 7.11 (m, 2H, Ph), 7.38 –7.22 (m, 5H, Ph), 7.59 (s, 1H, 3-H of
13
benzothiophene), 7.83 – 7.65 (m, 2H, Ph); C NMR (100 MHz, CDCl3) δ 16.82,
21.45, 22.98, 23.54, 24.88, 25.17, 29.85, 37.80, 39.34, 40.11, 40.74, 52.43, 52.53,
53.59, 53.67, 54.11, 55.98, 57.37, 58.51, 59.12, 66.01, 121.69, 123.22, 124.60, 124.90,
127.07, 128.64, 129.41, 130.92, 136.68, 138.67, 140.52, 170.72, 171.21, 207.96; MS
(ESI) m/z: 762.4 [M+H]+; HRMS calcd for C41H55N5O7SNa, [M+Na]+ 784.3714,
found 784.3858.
5-methyl-N-((S)-1-(((S)-4-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyl
oxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo
pentan-2-yl)amino)-1-oxo-3-(thiazol-4-yl)propan-2-yl)isoxazole-3-carboxamide (37)

JOURNAL PRE-PROOF
Compound 37 was synthesized from 8r and 16c according to the general
procedure for preparing 36. Yield 39%. 1H NMR (400 MHz, CDCl3) δ 0.88 – 0.98 (m,
6H, CH3), 1.25 (s, 3H, CH3), 1.47 – 1.41 (m, 2H, CH), 1.54 – 1.48 (m, 2H, CH2), 1.59
– 1.55 (m, 2H, CH2), 2.47 (s, 3H, 5-CH3 of isoxazole), 2.93 (d, J = 4.9 Hz, 2H, CH2),
3.35 (d, J = 4.9 Hz, 2H, CH2), 3.49 – 3.42 (m, 2H, CH2) , 4.89 – 4.66 (m, 4H, CH),
6.37 (s, 1H, 4-H of isoxazole), 7.00 (s, 1H, CONH), 7.04 – 7.35 (m, 5H, Ph), 7.35 (s,
3H, CONH), 7.52 (s, 1H, 4-H of thiazol), 8.79 (s, 1H, 2-H of thiazol); 13C NMR (100
MHz, CDCl3) δ 12.31, 16.69, 22.67, 22.92, 23.31, 24.60, 24.70, 25.05, 29.67, 36.98,
39.90, 40.13, 50.11, 52.30, 52.33, 53.66, 54.14, 59.03, 101.32, 116.42, 126.73, 128.45,
129.19, 137.10, 151.92, 153.66, 158.13, 170.30, 171.02, 171.53, 171.69, 178.29,
207.84; MS (ESI) m/z: 695.0; HRMS calcd for C35H46N6O7SNa, [M+Na]+ 717.3050,
found 717.3040.
5-methyl-N-((S)-1-(((S)-4-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyl
oxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-
pentan-2-yl)amino)-1-oxo-3-(pyridin-2-yl)propan-2-yl)isoxazole-3-carboxamide (38)
Compound 38 was synthesized from 8s and 16c according to the general
1
procedure for preparing 36. Yield 30%. H NMR (400 MHz, CDCl3) δ 0.73 (s, 3H，
CH3), 0.82 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.89 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.43
– 1.40 (m, 2H, CH), 1.50 – 1.47 (m, 4H, CH2), 2.49 (s, 3H, 5-CH3 of isoxazole), 2.87
(d, J = 4.7 Hz, 1H, CH2), 3.01 (d, J = 19.2 Hz, 2H, CH2), 3.21 –3.15 (m, 2H, CH2),
3.31 – 3.25 (m, 1H, CH2), 4.27 – 4.21 (m, 1H, CH), 4.55 – 4.48 (m, 1H, CH), 4.63 –
4.58 (m, 1H, CH), 4.81 – 4.76 (m, 1H, CH), 6.38 (s, 1H, 4-H of isoxazole), 6.66 (s,
1H, CONH), 7.19 – 7.11 (m, 5H, Ph), 7.23 – 7.19 (m, 3H, Ph), 7.34 (s, 1H, CONH),
7.52 (d, J = 8.2 Hz, 1H, 5-H of pyridin), 7.74 (s, 1H, CONH), 8.52 (s, 1H, CONH);
13C NMR (100 MHz, CDCl3) δ 14.29, 16.87, 21.44, 21.53, 22.85, 23.13, 24.76, 36.48 ,
38.56, 39.99, 40.66, 50.21, 52.47, 53.77, 54.14, 54.30, 59.22, 101.38, 126.61, 126.85,
128.60, 129.29, 129.34, 135.39, 135.50, 147.19, 151.69, 159.17, 162.48, 167.86,
171.07, 171.68, 172.26, 208.10; MS (ESI) m/z: 688.6 [M+H]+, 711.6 [M+Na]+;
HRMS calcd for C37H48N6O7Na, [M+Na]+ 711.3476, found 711.3605.

JOURNAL PRE-PROOF
N-((S)-3-(1H-indol-2-yl)-1-(((S)-4-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-met
hyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxope
ntan-2-yl)amino)-1-oxopropan-2-yl)-5-methylisoxazole-3-carboxamide (39)
Compound 39 was synthesized from 8t and 16c according to the general
procedure for preparing 36. Yield 44%. 1H NMR (400 MHz, CDCl3) δ 0.88 – 0.72 (m,
12H, CH3), 1.25 (s, 3H, CH3), 1.28 – 1.32 (m, 2H, CH3), 1.42 – 1.39 (m, 1H, CH),
1.51 – 1.47 (m, 1H, CH2), 1.63 – 1.55 (m, 4H, CH2), 2.49 (s, 3H, 5-CH3 of isoxazole),
2.90 – 2.80 (m, 2H, CH2), 3.08 (dd, J = 14.0, 5.9 Hz, 1H, CH2), 3.37 – 3.19 (m, 3H,
CH2), 4.31 (m, 1H, CH), 4.65 – 4.52 (m, 2H, CH), 4.80 – 4.74 (m, 1H, CH), 6.25 (s,
1H, 3-H of indol), 6.41 (s, 1H, 4-H of isoxazole), 6.56 (s, 1H, CONH), 6.70 (s, 1H,
CONH), 7.02 – 6.97 (m, 3H, Ph), 7.21 – 7.11 (m, 5H, Ph), 7.37 (d, J = 8.1 Hz, 1H,
Ph), 7.52 (s, 2H, CONH), 7.71 (d, J = 7.7 Hz, 1H, Ph), 8.46 (s, 1H, 1-H of indol); 13C
NMR (100 MHz, CDCl3) δ 12.30, 14.08, 16.69, 21.30, 21.72, 22.68, 23.32, 24.51,
25.08, 29.67, 31.41, 31.90, 37.11, 39.90, 40.45, 52.33, 54.15, 59.03, 101.30, 109.89,
111.49, 118.71, 119.96, 122.55, 123.45, 123.95, 124.45, 126.84, 126.97, 128.49,
129.12, 136.31, 136.76, 158.08, 159.59, 170.87, 170.96, 171.52, 171.58, 207.93; MS
(ESI) m/z: 728.0 [M+H]+, 750.1 [M+Na]+; HRMS calcd for C40H51N6O7, [M+H]+
727.3855, found 727.3814.
5-methyl-N-(2-(((S)-4-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-
yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopentan-2-yl)
amino)-2-oxoethyl)isoxazole-3-carboxamide (40)
Compound 40 was synthesized from 8u and 16c according to the general
procedure for preparing 36. yield 35%. 1H NMR (400 MHz, CDCl3) δ 0.87 (d, J = 4.8
Hz, 12H, CH3). 1.33 (s, 3H, CH3), 1.45 – 1.40 (m, 2H, CH), 1.58 – 1.53 (m, 4H, CH2),
2.48 (s, 3H, 5-CH3 of isoxazole), 2.86 (d, J = 3.8 Hz, 1H, CH2), 3.15 – 2.91 (m, 2H,
CH3), 3.26 (d, J = 4.2 Hz, 1H, CH2), 3.85 (s, 2H, CH2), 4.08 (s, 1H, CH), 4.61 – 4.42
(m, 2H, CH), 4.83 (d, J = 7.2 Hz, 1H, CH), 6.43 (s, 1H, 4-H of isoxazole), 7.16 – 7.01
13
(m, 5H, Ph), 7.18 (d, J = 7.0 Hz, 3H, CONH), 7.66 (s, 1H, CONH); C NMR (100
MHz, CDCl3) δ 12.30, 16.60, 22.13, 22.67, 22.74, 23.20, 24.71, 25.08, 38.13, 40.06,

JOURNAL PRE-PROOF
41.29, 43.05, 49.88, 51.92, 52.26, 54.17, 58.93, 101.36, 123.96, 126.86, 128.42,
129.28, 136.45, 158.09, 159.76, 168.13, 170.88, 171.39, 171.69, 208.15; MS (ESI)
m/z: 598.8 [M+H]+; HRMS calcd for C31H44N5O7, [M+H]+ 598.3282, found
598.3235.
N-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2
-carbonyl)-5,8,11-trioxo-2-oxa-6,9,12-triazahexadecan-4-yl)-5-methylisoxazole-3-car
boxamide (41)
Compound 41 was synthesized from 8v and 16c according to the general
1
procedure for preparing 36. Yield 82%. H NMR (400 MHz, DMSO-d6 ) δ 0.91 –
0.77 (m, 12H, CH3), 1.31 – 1.25 (m, 2H, CH), 1.40 (d, J = 3.6 Hz, 3H, CH3), 1.76
–1.58 (m, 4H, CH2), 2.46 (s, 3H, 5-CH3 of isoxazole), 2.84 – 2.71 (m, 1H, CH2), 2.98
– 2.85 (m, 1H, CH2), 3.04 – 2.99 (m, 1H, CH2), 3.17 – 3.09 (m, 1H, CH2), 3.23 (s, 3H,
CH3), 3.68 – 3.51 (m, 2H, CH2), 4.29 – 4.22 (m, 1H, CH), 4.43 – 4.30 (m, 1H, CH),
4.61 – 4.46 (m, 1H, CH), 4.73 – 4.62 (m, 1H, CH), 6.58 (d, J = 6.6 Hz, 1H, CH2, 4-H
of isoxazole), 7.25 – 7.12 (m, 5H, Ph), 8.05 (s, 1H, CONH), 8.14 (s, 1H, CONH),
13
8.34 – 8.20 (s, 1H, CONH), 8.63 (s, 1H, CONH); C NMR (100 MHz, DMSO-d6) δ
11.83, 16.40, 22.96, 23.15, 24.06, 24.43, 37.19, 38.52, 40.84, 49.23, 51.25, 51.51,
52.76, 53.37, 58.19, 58.80, 71.56, 101.36, 126.20, 127.98, 129.07, 137.64, 158.49,
168.75, 170.94, 171.05, 171.36, 171.47, 208.03, 208.12; MS (ESI) m/z: 642.2
[M+H]+, 664.2 [M+Na]+; HRMS calcd for C33H47N5O8Na, [M+Na]+ 664.3316, found
664.3315.
5-methyl-N-((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-m
ethyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo
pentan-2-yl)amino)-1-oxopentan-2-yl)isoxazole-3-carboxamide (42)
Compound 42 was synthesized from 8w and 16c according to the general
procedure for preparing 36. Yield 60%. 1H NMR (400 MHz, CDCl3) δ 0.94 – 0.81 (m,
18H, CH3),1.25 (s, 3H, CH3), 1.47 – 1.42 (m, 3H, CH), 1.68 – 1.56 (m, 6H, CH3),
2.49 (s, 3H, 5-CH3 of isoxazole), 2.86 (d, J = 4.9 Hz, 1H, CH2), 3.05 – 2.98 (m, 2H,

JOURNAL PRE-PROOF
CH2), 3.27 (d, J = 4.9 Hz, 1H, CH2), 4.39 – 4.30 (m, 1H, CH), 4.58 – 4.51 (m, 2H,
CH), 4.76 (dd, J = 14.7, 7.3 Hz, 1H, CH), 6.47 (s, 1H, 4-H of isoxazole), 6.69 (s, 1H,
CONH), 6.83 (s, 3H, CONH), 7.23 – 7.11 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3)
δ 12.32, 16.62, 21.33, 21.93, 22.77, 22.95, 23.29, 24.78, 25.00, 29.68, 40.03, 40.64,
40.91, 49.86, 52.14, 52.22, 54.03, 58.92, 101.42, 126.88, 128.43, 129.22, 136.60,
158.06, 159.65, 170.66, 171.41, 171.47, 171.60, 207.88; MS (ESI) m/z: 654.9
[M+H]+, 677.0 [M+Na]+; HRMS calcd for C35H58N6O7, [M+NH]+ 668.4052, found
668.4089.
N-((S)-1-cyclopropyl-2-(((S)-4-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyl
oxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo
pentan-2-yl)amino)-2-oxoethyl)-5-methylisoxazole-3-carboxamide (43)
Compound 43 was synthesized from 8x and 16c according to the general
procedure for preparing 36. Yield 60%. 1H NMR (400 MHz, CDCl3) δ 0.55 – 0.44 (m,
2H, CH2), 0.72 – 0.62 (m, 2H, CH2), 0.88 – 0.81 (m, 12H, CH3), 1.11 – 1.08 (m, 1H,
CH), 1.27 (s, 3H, CH3), 1.51 – 1.47 (m, 2H, CH), 1.65 – 1.63 (m, 4H, CH2), 2.50 (s,
3H, 5-CH3 of isoxazole), 2.88 (d, J = 4.9 Hz, 2H, CH2), 3.08 – 2.98 (m, 2H, CH2),
4.34 – 4.25 (m, 1H, CH), 4.52 – 4.46 (m, 1H, CH), 4.69 – 4.54 (m, 1H, CH), 5.33 –
5.27 (m, 1H, CH2), 6.43 (s, 1H, 4-H of isoxazole), 7.11 (s, 1H, CONH), 7.14 (s, 1H,
13
CONH), 7.24 – 7.18 (m, 5H, Ph), 7.46 (s, 1H, CONH), 7.53 (s, 1H, CONH); C
NMR (100 MHz, CDCl3) δ 3.36, 3.61, 12.21, 12.68, 18.45, 21.47, 21.69, 22.17, 22.88,
24.36, 24.47, 36.37, 39.31, 41.03, 50.89, 54.01, 56.42, 57.69, 58.84, 62.20, 101.03,
125.93, 126.88, 127.34, 128.03, 128.98, 137.00, 149.91, 159.81, 169.22, 170.62,
171.5, 174.38, 202.11; MS
(ESI) m/z: 638.5 [M+H]+; HRMS calcd for
C34H47N5O7Na, [M+Na]+ 660.3132, found 660.3211.
N-((S)-1-(((S)-4-methyl-1-(((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-
oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopentan-2-yl)amino)-
1-oxo-3-(thiazol-4-yl)propan-2-yl)pyrazine-2-carboxamide (44)