Structure-activity relationship studies of dipeptide-based hepsin inhibitors with Arg bioisosteres

Article abstract of DOI:10.1016/j.bioorg.2020.104521

Hepsin is a type II transmembrane serine protease (TTSP) associated with cell proliferation and overexpressed in several types of cancer including prostate cancer (PCa). Because of its significant role in cancer progression and metastasis, hepsin is an attractive protein as a potential therapeutic and diagnostic biomarker for PCa. Based on the reported Leu-Arg dipeptide-based hepsin inhibitors, we performed structural modification and determined in vitro hepsin- and matriptase-inhibitory activities. Comprehensive structure-activity relationship studies identified that the p-guanidinophenylalanine-based dipeptide analog 22a exhibited a strong hepsin-inhibitory activity (K_i = 50.5 nM) and 22-fold hepsin selectivity over matriptase. Compound 22a could be a prototype molecule for structural optimization of dipeptide-based hepsin inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.104521

Journal Pre-proofs
Structure-activity relationship studies of dipeptide-based hepsin inhibitors
with Arg bioisosteres
Hongmok Kwon, Hyunsoo Ha, Hayoung Jeon, Jaebong Jang, Sang-Hyun
Son, Kiho Lee, Song-Kyu Park, Youngjoo Byun
PII:
S0045-2068(20)31819-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.104521
YBIOO 104521
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
27 September 2020
25 November 2020
26 November 2020
Please cite this article as: H. Kwon, H. Ha, H. Jeon, J. Jang, S-H. Son, K. Lee, S-K. Park, Y. Byun, Structure-
activity relationship studies of dipeptide-based hepsin inhibitors with Arg bioisosteres, Bioorganic Chemistry
(
2020), doi: https://doi.org/10.1016/j.bioorg.2020.104521
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Structure-activity relationship studies of dipeptide-based hepsin inhibitors with
Arg bioisosteres
†
†
†
†
†
Hongmok Kwon, Hyunsoo Ha, Hayoung Jeon, Jaebong Jang, Sang-Hyun Son, Kiho
†
†
†,,*
Lee, Song-Kyu Park, Youngjoo Byun
†College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 30019,
Republic of Korea
Biomedical Research Center, Korea University Guro Hospital, 148 Gurodong-ro, Guro-gu,
Seoul 08308, Republic of Korea
*
Corresponding author
Youngjoo Byun, Ph. D.
College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 30019,
Republic of Korea
Tel.: +82-44-860-1619; Fax: +82-44-860-1607; E-mail: yjbyun1@korea.ac.kr

Abstract
Hepsin is a type II transmembrane serine protease (TTSP) associated with cell
proliferation and overexpressed in several types of cancer including prostate cancer (PCa).
Because of its significant role in cancer progression and metastasis, hepsin is an attractive
protein as a potential therapeutic and diagnostic biomarker for PCa. Based on the reported
Leu-Arg dipeptide-based hepsin inhibitors, we performed structural modification and
determined in vitro hepsin- and matriptase-inhibitory activities. Comprehensive structure-
activity relationship studies identified that the p-guanidinophenylalanine-based dipeptide
analog 22a exhibited a strong hepsin-inhibitory activity (K = 50.5 nM) and 22-fold hepsin
i
selectivity over matriptase. Compound 22a could be a prototype molecule for structural
optimization of dipeptide-based hepsin inhibitors.
Keywords: Prostate cancer, Hepsin, Dipeptide, Bioisostere, Structure-activity relationship
Abbreviations: ACN, acetonitrile; Boc, tert-butoxycarbonyl; BPH, benign prostate
hyperplasia; Cbz, benzyloxycarbonyl; DIPEA, N,N-diisopropylethylamine; DMF, N,N-
dimethylformamide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ESI,
electrospray ionization; HOBt, 1-hydroxybenzotriazole; HRMS, high resolution mass
spectrometry; NMR, nuclear magnetic resonance; PCa, prostate cancer; PMB, para-
methoxybenzyl; PTFE, polytetrafluoroethylene; RP-HPLC, reversed-phase high
performance liquid chromatography; SAR, structure-activity relationship; TFA,
trifluoroacetic
acid;
THF,
tetrahydrofuran;
TMEDA,
N,N,N’,N’,-
tetramethylethylenediamine; TTSP, type II transmembrane serine protease.

1
. Introduction
Hepatocyte Growth Factor (HGF) is a pleiotropic factor secreted by tumor-associated
fibroblasts and plays an important role in cancer metastasis [1]. It is activated through the
cleavage of inactive pro-HGF mediated by trypsin-like serine proteases such as hepsin,
hepatocyte growth factor activator (HGFA), and matriptase [2-9]. Dysregulated HGF-mediated
activity in the HGF / Met signaling pathway leads to oncogenesis, cancer cell proliferation,
invasion, and resistance to cancer therapy [10]. HGF-activating proteases, including hepsin,
matriptase, and HGFA, are upregulated in cancer cells [11-13]. HGF is inhibited by
endogenous HGF activator inhibitors (HAI-1 and HAI-2) [9, 14-16]. The poor prognosis of
patients with advanced cancer is closely associated with an increased HGF and reduced HAIs
levels [17-22]. Thus, the inhibition of the HGF activation pathway has been considered as a
potential therapeutic strategy of cancer intervention.
Hepsin, a type II transmembrane serine protease (TTSP) [23], is composed of 417
amino acids with a C-terminal serine protease domain localized on the surface [24]. Beyond
the pro-HGF activation, hepsin also contributes to the activation of matriptase, another pro-
HGF activator [25]. Furthermore, hepsin is associated with cell motility and basement
membrane components disruption, promoting cancer cell metastasis [26]. Hepsin is
predominantly overexpressed in several cancer cells including prostate, breast, ovarian cancer
[27, 28]. Especially, mRNA expression of hepsin was significantly upregulated in 90% of the
prostate cancer (PCa) specimens and the expression levels were 10-fold higher than those in
normal prostate or benign prostate hyperplasia (BPH) [12, 28-32]. The PCa-related hepsin
overexpression continues from the early to the later stages [33]. Although localized PCa can
be treated effectively with chemotherapy, surgery, and radiation, the treatment is extremely
difficult and the mortality rate increases drastically once it metastasizes to other organs
including lymph nodes or bones [34]. Therefore, hepsin is an attractive potential biomarker and

prognostic factor of PCa metastasis [31, 33] due to its structural characteristics and significant
role in metastasis [35-38].
Several low-molecular-weight (M. W.) hepsin inhibitors have been reported including
benzamidine-, indolecarboximidamide-, and peptide-derived analogs [39-47]. Moreover, some
comprehensive reviews on small molecular hepsin inhibitors were published recently [48, 49].
Previously, we reported the Leu-Arg dipeptide-based hepsin inhibitors (1–2, Fig. 1), which
exhibited strong binding affinity to hepsin with K values of nM concentration [41],
i
demonstrating that the dipeptide is the minimal structural requirement for hepsin-inhibitory
activity. Janetka and co-workers also confirmed this trend in a recent report [47]. In addition,
a NIR fluorescent dye was conjugated successfully with peptide-based hepsin inhibitors, which
were accumulated in the hepsin-overexpressing cells in vitro and in vivo [50, 51].
HN
HN
NH2
HN
HN
NH2
O
S
O
S
AcHN
N
N
AcHN
N
N
H
O
H
O
1
(Ac-LR-kbt)
2 (Ac-LR-kt)
Fig. 1. Previously reported Leu-Arg dipeptide-based hepsin inhibitors
Although peptide-based agents have many attractive aspects, they continue to have
some drawbacks as drug candidates, especially in systemic delivery [52]. Low specificity to
the target site due to the high conformational flexibility of peptide-based molecule is another
challenge to overcome [53, 54]. In addition, hydrophilic properties of peptide-based molecules
result in poor permeability through biological membranes and are responsible for rapid
clearance from the circulation by the liver and kidney [53, 54]. For these reasons, additional
structural modifications have been required for peptide-based compounds.

Herein, we have performed the structure-activity relationship (SAR) studies of
compound 1 aiming at identifying a new bioisostere for Arg in the P1 position and the
optimal amino acid in the P2 position. The demonstrations in this study would support the
further development of hepsin-targeted candidate compounds for the diagnosis or treatment
of metastatic PCa.

2
2
. Results and discussion
.1. Design strategy
Fig. 2. Design strategy for the structural modification of Ac-LR-kbt (1)
The starting point of our research for structural modification was based on the
previously reported dipeptide derivative, Ac-LR-kbt (1). Based on the chemical structure of 1,
new hepsin inhibitors were designed to replace Arg with a bioisostere that enhances rotational
rigidity and increases the lipophilicity of the parent molecules. The guanidine group of
compound 1 was located at the S1 sub-pocket and formed a salt bridge with Asp189,
contributing to the potent hepsin-inhibitory activity [41]. The guanidine group of Arg interacted
with Asp189, acting as an anchor group for binding to the hepsin active site, while the other
part of the compound could restrict the catalytic triad composed of His57, Asp102, and Ser195.
Although the Arg of the dipeptide derivative at the P1 position plays a critical role for binding
to hepsin, it remains as a natural-type amino acid with high rotational flexibility at the side
chain. Therefore, we introduced a cyclic ring structure at the side chain of Arg to confer the
rotational rigidity at the P1 region (Fig. 2). As shown in Fig. 2, the cyclic ring moieties such as
aminopyrimidine, piperidine-1-carboximidamide, cyclohexylguanidine and phenylguanidine
were introduced as potential bioisosteres of the Arg in compound 1.

2
.2. Chemistry and in vitro biological evaluation
N
N(PMB)2
N
O
N(PMB)2
N
O
N(PMB)2
N
N
NH2
N
N(PMB)2
N
N
O
S
AcHN
i
ii
iii
iv
N
N
I
I
AcHN
AcHN
N
O
O
3
(50%)
4 (29%)
5 (24%)
6 (50%)
N
N(PMB)2
N
N(PMB)2
N
NH2
N
N
N
O
O
O
AcHN
OH
S
AcHN
O
S
AcHN
O
S
v, vi
N
vii
N
viii
N
H
H
H
N
N
N
7
(35%, 2 steps)
8 (52%)
9 (32%)
Scheme 1. Synthesis of aminopyrimidine-based compound 9a
a
Reagents and conditions: (i) NaH (60% in oil), 4-methoxybenzyl chloride, THF, 40 ºC, 15 h;
ii) methyl-2-acetamidoacrylate, NaHCO , Bu NCl, (o-tol) P, PdCl , DMF, 110 ºC, overnight;
(
(
3
4
3
2
iii) 10% Pd/C, H (gas), MeOH/1,4-dioxane (5/2, v/v), rt, 24 h; (iv) benzothiazole, TMEDA,
2
n-BuLi, THF, −78 ºC, 1.5 h; (v) Cp ZrHCl, THF, rt, 20 min; (vi) N-acetyl-L-leucine, HOBt,
2
EDCI∙HCl, THF, rt, 12 h; (vii) Dess-Martin periodinane, CH Cl , rt, 3 h; (viii) TFA/water
2
2
(97.5/2.5, v/v), 50 ºC, 13 h.
Compounds substituted with the Arg bioisostere were prepared as described in
Schemes 1–3. Commercial 2-amino-5-iodopyrimidine was used as a starting material for the
synthesis of compound 9 (Scheme 1). Compound 4 was prepared from the starting material by
applying para-methoxybenzyl (PMB) protection and subsequent Heck-vinylation [55]. The
double bond of 4 was reduced by catalytic hydrogenation using 10% Pd/C and H gas to afford
2
compound 5. The reaction of 5 with benzothiazolyl lithium solution afforded compound 6

yielding 50% as reported previously [41]. Deacetylation of compound 6 using Schwartz’s
reagent (Cp ZrHCl), followed by amide coupling using EDCI∙HCl and HOBt, provided
2
compound 7 in 35% yield (2 steps). Compound 8 was obtained through oxidizing the alcohol
group of 7 by a Dess-Martin periodinane treatment. The PMB groups of 8 were removed using
trifluoroacetic acid (TFA) to yield the final compound 9.
NBoc
NBoc
O
N
NBoc
N
O
S
AcHN
OH
AcHN
N
i
ii, iii
iv
v, vi
H
O
O
N
N
S
AcHN
AcHN
O
H
O
O
10 (43%)
11 (100%, 2 steps)
12 (80%)
13 (38%, 2 steps)
NH
NBoc
NH
N
NH2
O
O
O
AcHN
O
AcHN
O
S
N
H
N
AcHN
O
vii
viii
ix
H
N
H
N
S
N
N
S
14 (92%)
15 (100%)
16 (65%)
Scheme 2. Synthesis of piperidine-1-carboximidamide-based compound 16a
a
Reagents and conditions: (i) N-acetylglycine, sodium acetate, acetic anhydride, MeOH, 100
ºC, 1 min; (ii) PtO (IV), AcOH, H (gas), rt, 24 h; (iii) di-tert-butyl dicarbonate, 4-
2
2
dimethylaminopyridine, THF, rt, 2 h; (iv) benzothiazole, TMEDA, n-BuLi, THF, −78 ºC, 1.5
h; (v) Cp ZrHCl, THF, rt, 20 min; (vi) N-acetyl-L-leucine, HOBt, EDCI∙HCl, THF, rt, 12 h;
2
(
vii) Dess-Martin periodinane, CH Cl , rt, 3 h; (viii) TFA/CH Cl (1/2, v/v), rt, 1 h; (ix) 1H-
2 2 2 2
pyrazole-1-carboxamidine, DIPEA, DMF, rt, 12 h.
By applying a similar synthetic strategy to the aminopyrimidine 9 described in Scheme
, a variety of compounds with Arg bioisostere moiety at the P1 position were prepared. The
1
synthesis of piperidine-1-carboximidamide analog is described in Scheme 2. Briefly, the
dehydropyridylalanine 10 was obtained by reacting 4-pyridylaldehyde with N-acetylglycine

[
56]. Conversion of the pyridine into piperidine using Adam’s catalyst (PtO ) with acetic acid
2
and the subsequent tert-butoxycarbonyl (Boc) protection provided the piperidinylalanine 11 in
quantitative yield. The reaction of 11 with lithium-benzothiazole afforded compound 12 in 80%
yield. Deacetylation of 12, followed by the amide coupling, provided compound 13 in 38%
yield (2 steps). Oxidation of 13 and the subsequent Boc deprotection afforded 15 in 92% yield
(2 steps). The reaction of 15 with 1H-pyrazole-1-carboxamidine provided the final piperidine-
1
-carboximidamide compound 16 in 65% yield [57].
CbzN
X
NHCbz
NH
NO2
CbzN
X
NHCbz
NH
CbzN
X
NHCbz
NH
i, ii
iii
iv
O
S
BocHN
O
BocHN
O
O
O
BocHN
BocHN
N
OH
OH
N
1
7a: X =
(86%, 2 steps)
(64%, 2 steps)
18a: X =
18b: X =
(90%)
(85%)
19a: X =
19b: X =
(89%)
(85%)
17b: X =
CbzN
NHCbz
NH
CbzN
NHCbz
NH
HN
X
NH2
NH
X
X
O
O
v
vi
vii
O
S
AcHN
O
S
AcHN
O
S
.
TFA H N
N
N
H
2
H
N
N
N
2
0a: X =
(85%)
(63%)
21a: X =
21b: X =
(65%)
(76%)
22a: X =
(31%)
(44%)
20b: X =
22b: X =
Scheme 3. Synthesis of p-phenylguanidine-based compound 22a and cyclohexylguanidine-
based compound 22ba
a
Reagents and conditions: (i) H (gas), 10% Pd/C (for 17a) or PtO (IV) (for 17b), AcOH (for
2
2
1
7b), MeOH, rt, 3 h (for 17a) or 24 h (for 17b); (ii) N,N’-bis(carbobenzoxy)-1H-pyrazole-1-
carboxamidine, Et N, CH Cl , rt, 6 h; (iii) N,O-dimethylhydroxylamine HCl, HOBt, EDCI∙HCl,
3
2
2
DIPEA, THF, rt, 13 h; (iv) benzothiazole, TMEDA, n-BuLi, THF, −78 ºC, 1.5 h; (v) TFA,

triethylsilane, water, CH Cl , rt, 2 h; (vi) N-acetyl-L-leucine, HATU, DIPEA, rt, CH Cl , 5 h;
2
2
2
2
(vii) TFA, thioanisole, rt, 12 h.
The synthesis of phenylguanidine- and cyclohexylguanidine-based analogs were
achieved via a 7-step synthetic process (Scheme 3). The catalytic hydrogenation reaction using
Adam’s catalyst and 50% acetic acid in methanol reduced not only the nitro group but also the
benzene ring of N-(tert-butoxycarbonyl)-4-nitro-L-phenylalanine [58], while the
hydrogenation using 10% Pd/C in methanol only reduced nitro group to the amine group. The
respective reduced compounds were reacted with N,N’-bis(carbobenzoxy)-1H-pyrazole-1-
carboxamidine to give compounds 17a and 17b, respectively. During the guanidine formation,
the mono-N-Cbz-protected 1H-pyrazole-1-carboxamidine gave a detrimental effect on the
yield of the next Weinreb amide synthesis step as compared with the bis-Cbz-protected 1H-
pyrazole-1-carboxamidine. Introduction of the Weinreb amide and the subsequent addition of
lithium-benzothiazole provided compounds 19a and 19b, respectively. The Boc-protecting
groups of 19a–19b were removed with 25% TFA in CH Cl . Recrystallization of the crude
2
2
compounds 20a and 20b were successfully achieved using diethyl ether. Conjugation of 20a–
0b with N-acetyl-L-leucine was achieved using HATU as a coupling reagent. The Cbz groups
2
of 21a and 21b were removed using TFA and thioanisole to afford the final compounds 22a
and 22b, respectively [59]. The final compounds were purified by reversed-phase (RP)-HPLC
and analyzed by NMR and ESI-MS. Some compounds showed two diastereomeric peaks at
HPLC spectra due to the epimerization of the α-carbon in the P1 Arg portion during the addition
reaction with lithium–benzothiazole solution. The hepsin-inhibitory activities of the
synthesized compounds were determined by applying the reported fluorescence-based
enzymatic assay [40, 41]. To investigate the selectivity of synthesized compounds for hepsin
over matriptase, the compounds were also evaluated matriptase-inhibitory activities as

previously reported [41]. We monitored substrate proteolysis inhibition kinetically for two
hours. Hepsin and matriptase enzymatic activities of the synthesized compounds are
summarized in Table 1.
The first set of compounds (9, 16, 22a, 22b) included Leu-Arg analogs substituted with
the bioisosteres of the guanidine group (Table 1). Among them, compound 22a with the
phenylguanidine exhibited the strongest hepsin-inhibitory activities with a K value of 50.5 nM.
i
In addition, 22a showed higher hepsin selectivity over matriptase as compared to compound 1
(22-fold vs, 14-fold). However, the potencies of compounds 9 and 16 were too weak to act as
an Arg bioisostere. Compound 22b with the cyclohexylguanidine displayed moderate hepsin
inhibition (K = 505 nM).
i
Table 1. In vitro hepsin- and matriptase-inhibitory activities of the synthesized compounds
R1
O
H
O
N
R2
N
H
O
Hepsin
K (nM)
i
Matriptase Selectivity
Cmpd
9
R1
R2
cLogPa
1.06
K (nM)
i
Index
1
(Ac-LR-kbt)
11.7 ± 4.9
>10000
169 ± 38.9
>10000
14
N
NH2
S
N
1.68
N
NH
NH2
S
N
N
1
6
>10000
>10000
2.06
H
N
NH2
NH
S
N
2
2ab,c
50.5 ± 12.2
104 ± 30.4
35.2 ± 6.2
1120 ± 57.6
2190 ± 115
853 ± 98.3
>10000
22
21
2.52
2.52
2.52
2.55
0.92
0.96
H
N
NH2
NH
S
N
2
2
2a-1b,c
2a-2b,c
H
N
NH2
NH
S
N
24
H
N
NH2
NH
S
N
2
2b
6a
6b
505 ± 39.2
527 ± 92.2
2110 ± 1200
>20
>19
>4.7
H
N
NH2
NH
S
N
2
>10000
H
N
NH2
NH
S
N
2
>10000

NH
NH2
S
N
3
2
>10000
>10000
2.52
N
H
a
calculated using ChemDraw Ultra v12.0.2.1076.
HPLC retention time: 22a = 3.79 and 4.36 min; 22a-1 = 3.91 min; 22b-1 = 4.47 min.
b
c
analytical HPLC was performed with Phenomenex Gemini-NX C18 column (150×4.6 mm, 3
μm, 110 Å), isocratic elution of 35% of solvent B (A = 0.1% TFA in water and B = 0.1% TFA
in acetonitrile), flow rate of 1.0 mL/min, monitored by UV detector at 220 nm.
To clarify the effect of the absolute configuration at the P1 position on hepsin
inhibition, each epimer of the mixture compound 22a was separated and purified (See
Supplementary data). A pure epimer 22a-1 eluted at 3.91 min in the HPLC experiment and
displayed hepsin-inhibitory activity with a K value of 104 nM (Table 1). The other epimer
i
2
2a-2, which eluted at 4.47 min, exhibited a little stronger hepsin-inhibitory activity (K = 35.2
i
nM) than 22a-1. We previously reported that the -carbon epimerization of Arg at the P1
position makes a little difference in hepsin inhibition [41]. Because the difference in hepsin
inhibition and hepsin/matriptase selectivity was little between the two epimers (Table 1),
hepsin-inhibitory activity was determined using an epimeric mixture of inhibitors modified at
the P1 position.
In our previous study, Ac-LR-kt (ketothiazole) (2) was also a potent hepsin inhibitor
with possessing hepsin-inhibitory activities comparable to 1 [41]. Thus, we assumed that
dipeptide derivatives substituted C-terminus with a thiazole ring might exhibit hepsin-
inhibitory activities. Compounds 26a and 26b were synthesized to investigate the effect of the
changes from benzothiazole to thaizole on hepsin binding affinity. In addition, compound 32
with m-phenylguanidinylalanine, was synthesized to determine the effect of the orientation of
the guanidine group in 3-D space on hepsin affinity. As shown in Schemes 4 and 5, compounds
2
6a, 26b, and 32 were prepared by applying a synthetic strategy similar to 22a.

CbzN
X
NHCbz
NH
CbzN
X
NHCbz
NH
CbzN
X
NHCbz
NH
HN
X
NH2
NH
O
O
18a or 18b
i
ii
iii
iv
O
S
O
S
AcHN
O
S
AcHN
O
S
.
BocHN
TFA H2N
N
N
H
H
N
N
N
N
2
3a: X =
(from 18a, 90%)
(from 18b, 84%)
24a: X =
24b: X =
(70%)
25a: X =
25b: X =
(58%)
(80%)
26a: X =
26b: X =
(60%)
23b: X =
(92%)
(35%)
Scheme 4. Synthesis of thiazole-substituted compounds 26a and 26ba
a
Reagents and conditions: (i) Thiazole, TMEDA, n-BuLi, THF, −78 ºC, 1.5 h; (ii) TFA,
triethylsilane, water, CH Cl , rt, 2 h; (iii) N-acetyl-L-leucine, HATU, DIPEA, rt, CH Cl , 5 h;
2
2
2
2
(iv) TFA, thioanisole, rt, 12 h.
NCbz
N
NHCbz
NCbz
NHCbz
NCbz
NHCbz
H
O
S
BocHN
NO2
i, ii
N
iii
N
iv
H
H
O
O
O
O
N
BocHN
BocHN
BocHN
OH
OH
N
27 (60%, 2 steps)
28 (58%)
29 (83%)
NCbz
NHCbz
NCbz
NHCbz
NH
N
O
N
O
N
H
NH2
H
H
O
S
AcHN
O
S
AcHN
O
.
TFA H2N
N
N
v
vi
H
vii
H
N
N
N
S
30 (86%)
31 (46%)
32 (60%)
Scheme 5. Synthesis of m-phenylguanidine-based compound 32a
a
Reagents and conditions: (i) H (gas), 10% Pd/C, MeOH, rt, 3 h; (ii) N,N’-bis(carbobenzoxy)-
2
1
H-pyrazole-1-carboxamidine, Et N, CH Cl , rt, 6 h; (iii) N,O-dimethylhydroxylamine HCl,
3 2 2
HOBt, EDCI∙HCl, DIPEA, THF, rt, 13 h; (iv) benzothiazole, TMEDA, n-BuLi, THF, −78 ºC,
.5 h; (v) TFA, triethylsilane, water, CH Cl , rt, 2 h; (vi) N-acetyl-L-leucine, HATU, DIPEA,
1
2
2
rt, CH Cl , 5 h; (vii) TFA, thioanisole, rt, 12 h.
2
2
As summarized in Table 1, the thiazole-bearing compounds 26a (K = 527 nM) and
i
2
6b (K = 2110 nM) exhibited lower hepsin binding affinity than the corresponding
i

benzothiazole compounds 22a and 22b, indicating the importance for the benzothiazole moiety
at the C-terminus region. Moreover, the m-phenylguanidine 32 (K >10 μM) completely lost
i
hepsin-inhibitory activity, indicating that the positional location of the guanidine group in 3-D
space is critical for binding to hepsin.
With the p-phenylguanidinylalanine moiety fixed in the P1 position, diverse natural
and non-natural amino acids were introduced in the P2 position. As shown in Scheme 6, the
compounds 34a–34n were synthesized by applying a synthetic procedure similar to 22a. N-
acetylated non-natural amino acids were prepared as described in the Supplementary data.
During the synthetic process of compound 34j, the sulfide group of 33j acted as a soft base and
underwent a nucleophilic attack on the electron-deficient benzylic carbon of the Cbz group
[59]. According to the in vitro enzymatic studies of compounds 34a–34d with a bulky side
chain at the P2 position, 34b with a tyrosine residue displayed the most potent hepsin-inhibitory
activity (K = 129 nM, shown in Table 2), consistent with the claim that Tyr is permissive at
i
the P2 position [60]. Compounds bearing both a bulky ring moiety and hydrogen-bonding
donor capability at the P2 residue (34b, 34d) exhibited stronger hepsin-binding affinity than
3
4a and 34c (K = 149 nM for 34d vs. 357 nM for 34a; 539 nM for 34c). Next, the compounds
i
having residues with diverse lengths of carbon chain at the P2 region were synthesized.
Compounds 34e (K = 302 nM) with norleucine and 34f (K = 333 nM) with norvaline showed
i
i
weaker hepsin-inhibitory activity than 34d. In addition, hepsin inhibition decreased as the
length of linear alkyl chain residue at P2 position became shorter (K = 453 nM for 34g; 1720
i
nM for 34h; K = 2450 nM for 34i). Interestingly, hepsin selectivity over the matriptase of these
i
compounds (34h, 34i) was maintained as also reported by the Janetka and co-workers [47].
Changing the ε-carbon of 34e to the sulfur atom (34j) made little influence on hepsin-binding

affinity (K = 296 nM for 34j). As shown in Table 2, compounds bearing bifurcated carbon
i
chain residues with an additional methyl group at the β-position (34k, 34l) showed higher
hepsin-inhibitory activity (K = 162 nM for 34l; 287 nM for 34k) than those with similar lengths
i
of linear carbon chains (34f, 34g). Compound 22a, which has a carbon chain bifurcated at the
γ-position, showed higher hepsin-inhibitory activity than 34k and 34l, suggesting that the bulky
lipophilic part of the P2 residue might extend over a certain range for efficient interaction at
hepsin active site. This trend was also observed in the linear carbon length change mentioned
above. Compound 34m (K = 151 nM), with an (R)-configuration of the P2 residue of 22a,
i
displayed lower hepsin-binding affinity and selectivity over matriptase than 22a. We also
observed this trend between the phenylguanidine-conjugated compounds 34a (K = 357 nM)
i
and 34n (K = 551 nM), demonstrating that amino acids with an (S)-configuration at the P2
i
position were preferred over those with an (R)-configuration.
O
O
O
O
CbzN
NHCbz
NH
HN
NH2
NH
AcHN
AcHN
AcHN
AcHN
Y =
HN
HO
Y
Y
i
O
S
ii
O
20a
N
N
H
H
a (L-Phe)
b (L-Tyr)
c (L-Cha)
d (L-Trp)
N
N
S
O
O
O
O
O
AcHN
AcHN
AcHN
AcHN
AcHN
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3a: L-Phe (62%)
3b: L-Tyr (50%)
3c: L-Cha (30%)
3d: L-Trp (64%)
3e: L-Nle (77%)
3f: L-Nvl (72%)
3g: L-Aba (44%)
3h: L-Ala (64%)
3i: Gly (61%)
3j: L-Met (66%)
3k: L-Ile (55%)
3l: L-Val (57%)
3m: D-Leu (58%)
3n: D-Phe (70%)
34a: L-Phe (33%)
34b: L-Tyr (75%)
34c: L-Cha (55%)
34d: L-Trp (20%)
34e: L-Nle (37%)
34f: L-Nvl (42%)
34g: L-Aba (50%)
34h: L-Ala (50%)
34i: Gly (42%)
34j: L-Met (8%)
34k: L-Ile (22%)
34l: L-Val (40%)
34m: D-Leu (20%)
34n: D-Phe (24%)
e (L-Nle)
f (L-Nvl)
g (L-Aba)
h (L-Ala)
i (Gly)
O
O
O
O
O
AcHN
AcHN
AcHN
AcHN
AcHN
S
j (L-Met)
k (L-Ile)
l (L-Val)
m (D-Leu)
n (D-Phe)
Scheme 6. Synthesis of 4-phenylguanidine-based dipeptide analogsa
a
Reagents and conditions: (i) N-acetylated appropriate amino acids, HATU, DIPEA, CH Cl ,
2
2
rt, 5 h; (ii) TFA, thioanisole, rt, 12 h.

Table 2. In vitro hepsin- and matriptase-inhibitory activities of the 4-phenylguanidinylalanine-
based dipeptide analogs
H
N
NH2
NH
O
H
O
N
O
S
N
H
R1
N
Hepsin
K (nM)
i
Matriptase
K (nM)
i
Selectivity
Index
Cmpd
R1
cLogPa
1.06
2.48
1.81
3.71
2.47
2.65
2.12
1.59
1.06
0.75
1.21
2.52
1.99
2.52
2.48
1
(Ac-LR-kbt)
11.7 ± 4.9
357 ± 101
129 ± 21.1
539 ± 66.5
149 ± 9.20
302 ± 15.6
333 ± 41.7
453 ± 38.0
1720 ± 355
2450 ± 501
296 ± 26.5
287 ± 48.9
162 ± 10.0
151 ± 11.8
551 ± 37.5
169 ± 38.9
488 ± 88.3
398 ± 49.4
3020 ± 975
211 ± 19.6
396 ± 11.8
421 ± 8.0
14
1.4
3.1
5.6
1.4
1.3
1.3
0.7
0.04
0.02
1.0
8.2
2.9
18
3
4a
3
4b
HO
3
4c
4d
4e
4f
3
HN
3
3
3
3
4g
4h
312 ± 48.9
69.0 ± 1.5
46.2 ± 2.2
306 ± 15.5
2346 ± 656
465 ± 109
2780 ± 138
529 ± 92.7
3
3
4i
4j
-H
S
3
4k
4l
4m
3
3
3
4n
1.0
a
calculated using ChemDraw Ultra v12.0.2.1076

2
.3. In silico docking studies
To elucidate the binding modes of the prepared compounds, we conducted in silico virtual
docking studies with the hepsin X-ray crystal structure (PDB ID: 1O5E [61]) using the Surflex-
Dock GeomX module in SYBYL-X software (v2.1.1, Tripos Inc., NJ, USA). In the Ligand
preparation process, we fixed the configuration of P1 position of the prepared compounds to
be an (S)-configuration. The docking results revealed that compound 22a formed hydrogen
bonds with Asp189 and Gly219 as well as with Ser195, Gly193, and His57 within a range of
3
.0 Å (Fig. 3A). The benzothiazole ring of 22a made van der Waals interaction with Pro60,
Leu41, and the disulfide bridge between Cys42 and Cys58, which supported the benzothiazole
preference to the thiazole ring in dipeptide-derived hepsin inhibitors. The Leu residue of the
P2 position was projected to the aromatic amino acid residues surrounded by Trp215 and His57.
The side chain of Leu interrupted a catalytic triad which consists of Asp102, His57, and Ser195.
When the hydrophobic moieties bulkier than Leu were introduced at the side chain of the P2
residue, they made steric repulsion with Asn99 and Asp102, leading to a dramatic change of
the binding pose (See Supplementary data). The branched methyl group at the β-carbon in Val
or Ile affected the conformation of the parent molecules in the hepsin active site. However, the
non-branched linear side chain in the P2 region made a minimal effect on the surface of the
catalytic triad. In the case of 22b, the guanidine group presented strong interaction with Asp189
and Gly219 at the S1 site (Fig. 3B). However, it was located away from Ser195 out of the 3.0-Å
range. The Leu residue of P2 position of 22b was projected to Leu41 to generate lipophilic
interaction, thus the surface of catalytic triad and S2 sub-pocket were relatively more exposed
than 22a. In the case of compound 32, in which the guanidine group is substituted at meta-
position, 32 did not fully interrupt the catalytic triad and S2 sub-pocket due to the positional
change in the projection of the guanidine group (Fig. 3C). Compound 32 made several
hydrogen bonds within a 3.0-Å range; however, to a lesser extent than 22b. Furthermore, two

residues, Tyr146 and Gln192, formed strong repulsive contacts with the benzothiazole ring of
2, resulting in a decrease of hepsin-binding affinity. The orientation of the benzothiazole
3
moieties of 22b and 32 was different from that of 22a because it was located far away from the
hydrophobic residues (Leu 41 and Cys42-Cys58 disulfide bridge) due to the distortion of the
ligand dipeptide backbone.
Fig. 3. Docked poses of compounds 22a (A), 22b (B), and 32 (C) in the active site of hepsin
(PDB ID: 1O5E). S1 and S2 sub-pockets are marked in yellow and green, respectively.
In the case of molecular docking simulation with matriptase (PDB ID: 3NCL [62]),
both 22a and 1 bind to matriptase in a similar binding mode (Fig. 4). The guanidine group of
presented hydrogen bonds with the Asp202 and Gly232 of matriptase, and also made an ionic
1
interaction with Asp202 in a range of 2.00 Å. However, the hydrogen-bonding distance of 22a
was much longer than that of 1. In particular, 22a lacked hydrogen-bonding interaction with
Asp202 within the range of 2.50 Å from the matriptase active site. These results explained the
weak matriptase-inhibitory activity of 22a as compared to that of 1, indicating that the restricted
flexibility of the P1 residue enhances the selectivity of hepsin over matriptase.

Fig. 4. Docked poses of compounds 1 (A) and 22a (B) in the active site of matriptase (PDB ID:
3
NCL). S1 and S2 sub-pockets are marked in yellow and pink, respectively.
The results indicated that the flexibility of peptidomimetic compounds in the P1 region
is required for inhibitory activities of hepsin and matriptase. Although compound 22a displayed
slightly lower hepsin-inhibitory activity than 1, it possesses several merits for further structural
optimization. Compound 22a showed higher hepsin selectivity over matriptase (22-fold) than
1
(14-fold). In vitro and in silico studies indicated that flexibility in P1 residue appears to be
somewhat important for matriptase than hepsin. In addition to the reduced number of rotational
bonds, 22a was more lipophilic property than 1 (cLogP = 2.52 for 22a; 1.06 for 1). The
prolonged RP-HPLC retention time of 22a compared to 1 also supported the enhanced
lipophilicity of 22a (See Supplementary data). The parallel artificial membrane permeability
assay (PAMPA) also showed an increase in permeability of 22a compared to compound 1 (0.06
nm/s for 22a and 0.04 nm/s for 1, See Supplementary data). However, the absolute cell
permeation values of both compounds were very low due to the hydrophilic nature of 1 and
2
2a. The 4-phenylguanidine group of 22a is less basic (pKa = 10.01) than the guanidine of 1
(pKa = 11.69) [63-65], which may be favorable for membrane permeability. Therefore,

compound 22a has a potential as a prototype molecule for further structural optimization of
more potent dipeptide-based hepsin inhibitors with better lipophilicity and permeability
properties.

3
. Conclusions
We designed and synthesized hepsin-targeted inhibitors derived from Leu-Arg
dipeptides and evaluated their in vitro hepsin-inhibitory activities. We introduced the Arg
bioisosteres at the P1 residue in order to restrict the rotational flexibility of the dipeptide-based
hepsin inhibitors. Comprehensive SAR studies demonstrated that the p-phenylguanidine
moiety acts as a promising Arg bioisostere at the P1 residue. For the variation of the P2 residue,
several natural and non-natural amino acids were introduced in order to investigate the effect
of the side chain and the absolute configuration of the α-carbon. The Leu with (S)-configuration
was the most potent residue for the P2 residue. Compound 22a with the p-phenylguanidine at
the P1 region and L-Leu at the P2 region exhibited the most potent hepsin binding affinity (Ki
=
50.5 nM) and a 22-fold higher hepsin selectivity compared to matriptase. In addition,
compound 22a showed a slight increase in membrane permeability compared to 1. Compound
2a can be used as a prototype molecule for structural modification of dipeptide-based hepsin
inhibitors for metastatic PCa treatment or diagnosis.
2

4
4
. Experimental section
.1. General
All the chemicals and solvents used in the reaction were purchased from Sigma-
Aldrich, TCI, or Alfa Aesar and were used without further purification. Reactions were
monitored by TLC on 0.25 mm Merck precoated silica gel plates (60 F ). Reaction progress
2
54
was monitored by TLC analysis using a UV lamp and/or KMnO staining for detection
4
purposes. Column chromatography was performed on silica gel (230−400 mesh, Merck,
1
13
Darmstadt, Germany). H and C NMR spectra were recorded at room temperature (298 K) in
CDCl (7.26 ppm/77.16 ppm), CD OD (3.31 ppm/49.00 ppm) or CD CN/D O (2.53 and 4.79
3
3
3
2
ppm/1.32 and 118.26 ppm) on either Bruker BioSpin Avance 300 MHz NMR or Bruker
Ultrashield 600 MHz Plus spectrometer and referenced to an internal solvent. NMR solvents
including CDCl , CD OD, CD CN and D O were used as received from the Eurisotop company.
3
3
3
2
Chemical shifts are reported in parts per million (ppm). Coupling constants (J) are given in
Hertz. Splitting patterns are indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
1
dd, double of doublet; dt, double of triplet; br, broad for H NMR data. High resolution mass
spectra (HRMS) were recorded on an Agilent 6530 Accurate mass Q-TOF LC/MS
spectrometer. Low resolution mass spectra (LRMS) analyses were obtained from an API
1
50EX ESI-MS spectrometer. Reversed-phase high-performance liquid chromatography (RP-
HPLC) purification using semi-preparative column (Phenomenex Gemini-NX C18, 110 Å, 150
mm × 10 mm, 5 μm) was performed on Agilent 1260 Infinity (Agilent). The purity of all final
compounds was measured by analytical RP-HPLC on an Agilent 1260 Infinity (Agilent) with
a C18 column (Phenomenex, 150 mm × 4.6 mm, 3 μm, 110 Å) using water (containing 0.1%
TFA) and acetonitrile (ACN; containing 0.1% TFA) as mobile phase. All compounds were
monitored at UV detector: 220 nm. The purities of the tested compounds were >95%.

4
.2. Synthesis
Typical procedure A for benzothiazole or thiazole addition: To benzothiazole or
thiazole (10.2 eq) and N,N,N’,N’-trimethylethylenediamine (TMEDA; 10.0 eq) in THF at –78
was added n-BuLi (1.6M in hexane, 10.0 eq) dropwise over 10 min. The lithium-
℃
benzothiazole or -thiazole solution was stirred at –78 ℃ for 35 min. The appropriate Weinreb
amide or methyl ester compound (1.0 eq) was dissolved in THF, then added via syringe to the
lithium-benzothiazole or -thiazole solution at –78 ℃ dropwise over 15 min. The reaction
mixture was stirred at –78 ℃ for 1.5 h. The reaction mixture was quenched by pouring into a
saturated aqueous ammonium chloride solution and shaking vigorously. The aqueous layer was
extracted with EtOAc and the combined organic layers were washed with brine, dried over
MgSO , filtered, and concentrated under reduced pressure. The residue was purified by flash
4
column chromatography (eluting with hexane/EtOAc) on silica gel.
Typical procedure B for Weinreb amide formation: To a solution of appropriate
carboxylic acid (1.0 eq) in dry THF (tetrahydrofuran) was added N,O-dimethylhydroxylamine
hydrochloride (2.0 eq), hydroxybenzotriazole (HOBt; 1.4 eq), and 1-(3-dimethylaminopropyl)-
3
6
-ethylcarbodiimide hydrochloride (EDCI∙HCl; 1.4 eq). N,N-diisopropylethylamine (DIPEA;
.0 eq) was added to the reaction mixture under argon atmosphere. The reaction mixture was
stirred at room temperature for 13 h. The reaction mixture was partitioned and diluted with
EtOAc and water. The organic layer was partitioned between EtOAc and brine. The combined
organic layer was dried over MgSO , filtered, and concentrated under reduced pressure. The
4
residue was purified by flash column chromatography (eluting with hexane/EtOAc) on silica
gel.
Typical procedure C for amide coupling: To a CH Cl solution of appropriate N-
2
2
acetylated amino acid (2.0 eq) and HATU (3.0 eq) was added DIPEA (4.5 eq) under argon

atmosphere. The reaction mixture was stirred at room temperature for 30 min, then was added
CH Cl suspension of Boc-deprotected Arg analog TFA salt (1.0 eq) with DIPEA (1.5 eq). The
2
2
reaction mixture was stirred at room temperature for 5 h. A saturated aqueous sodium
bicarbonate solution was added to the mixture to quench the reaction. The organic layer was
diluted by EtOAc and extracted by water and brine, then combined organic layer was dried
over MgSO , filtered, and concentrated under reduced pressure. The residue was purified by
4
flash column chromatography (eluting with hexane/EtOAc) on silica gel.
Typical procedure D for Cbz deprotection: Thioanisole (100 eq) was added to Cbz di-
protected compound (1.0 eq). TFA (560 eq) was added dropwise to reaction mixture at 0 ℃.
The reaction mixture was stirred at room temperature for 12 h then evaporated in vacuo. The
residue was added diethyl ether, then the precipitated crude product was collected by
centrifugation, followed by carefully decanting out the ether solvent. The crude product was
purified by RP-HPLC (eluting with 0.1% TFA in water/ACN).
4
.2.1. 5-Iodo-N,N-bis(4-methoxybenzyl)pyrimidin-2-amine (3)
This compound was prepared by previously reported method and the analytical data
were same as reference [55].
4
.2.2. (E)-methyl 2-acetamido-3-(2-(bis(4-methoxybenzyl)amino)pyrimidin-5-yl)acrylate (4)
This compound was prepared by previously reported method and the analytical data
were same as reference [55].
4
.2.3. Methyl 2-acetamido-3-(2-(bis(4-methoxybenzyl)amino)pyrimidin-5-yl)propanoate (5)

To a solution of compound 4 (168 mg, 0.4 mmol, 1.0 eq) in 7.0 mL methanol/1,4-
dioxane (5:2, v/v) was added 10% Pd/C (4.3 mg, 0.04 mmol, 0.1 eq). The reaction mixture was
stirred at room temperature under 1 atm of hydrogen gas for 24 h. The reaction mixture was
filtered through celite then evaporated under reduced pressure. The residue was purified by
flash column chromatography (eluting with hexane/EtOAc, 2:1 to 1:2) on silica gel to afford
1
the product (40 mg, 0.1 mmol) in 24% yield. H NMR (300 MHz, CDCl ): δ ppm 8.09 (s, 2H),
3
7
1
.15 (d, J = 8.4 Hz, 4H), 6.84 (d, J = 8.4 Hz, 4H), 6.07 (d, J = 6.9 Hz, 1H), 4.84 (d, J = 6.2 Hz,
H), 4.79 (s, 4H), 3.79 (s, 9H), 3.00 (q, J = 5.1 Hz, 1H), 2.98 (q, J = 5.1 Hz, 1H), 2.05 (s, 3H).
+
+
HRMS (ESI): [M + H] calculated for C H N O , 479.2289; found, 479.2307.
2
6
31
4
5
4
.2.4.
oxopropan-2-yl)acetamide (6)
This compound was prepared in 50% yield by typical procedure A with compound 5
N-(1-(benzo[d]thiazol-2-yl)-3-(2-(bis(4-methoxybenzyl)amino)pyrimidin-5-yl)-1-
1
and benzothiazole. H NMR (300 MHz, CDCl ): δ ppm 8.22 (d, J = 7.5 Hz, 1H), 8.05 (s, 2H),
3
7
6
=
.98 (d, J = 6.9 Hz, 1H), 7.63–7.56 (m, 2H), 7.12 (d, J = 8.4 Hz, 4H), 6.82 (d, J = 8.4 Hz, 4H),
.52 (d, J = 7.5 Hz, 1H), 6.13 (q, J = 5.4 and 7.5 Hz, 1H), 4.71 (s, 4H), 3.78 (s, 6H), 3.32 (q, J
1
3
5.1Hz, 1H), 3.30 (q, J = 5.1Hz, 1H), 2.08 (s, 3H). C NMR (75 MHz, CDCl3): δ ppm 192.37,
1
1
69.80, 163.29, 161.67, 158.74, 158.43, 153.53, 137.28, 130.19, 128.91, 128.33, 127.40,
25.94, 122.47, 116.60, 113.97, 113.88, 56.41, 55.27, 48.12, 32.69, 23.29. HRMS (ESI): [M –
–
-
H] calculated for C H N O S , 580.2019; found, 580.2050.
3
2
30
5
4
4
.2.5.
(2S)-2-acetamido-N-(1-(benzo[d]thiazol-2-yl)-3-(2-(bis(4-
methoxybenzyl)amino)pyramidin-5-yl)-1-hydroxypropan-2-yl)-4-methylpentanamide (7)

To a solution of compound 6 (105 mg, 0.2 mmol, 1.0 eq) in THF (7.0 mL) was added
Schwartz’s reagent (Cp ZrHCl, 139 mg, 0.5 mmol, 3.0 eq), then the reaction mixture was
2
vigorously stirred at room temperature for 20 min. The reaction was quenched by 20 μL of
water and the mixture was filtered through short silica gel pad eluting with CH Cl /methanol
2
2
(10:1, v/v). The filterate was evaporated in vacuo, then dissolved in THF (3.0 mL). N-acetyl-
L-leucine (40 mg, 0.2 mmol, 1.3 eq) and HOBt (39 mg, 0.3 mmol, 1.6 eq) were added to the
aforementioned THF solution under argon atmosphere, then cooled to 0 ℃. To a reaction
mixture was added suspension of EDCI∙HCl (56 mg, 0.3 mmol, 1.6 eq) in THF (4.5 mL) then
stirred at room temperature for 12 h. A saturated aqueous sodium bicarbonate solution was
added to the mixture to quench the reaction. The organic layer was diluted by EtOAc and
extracted by water and brine, then combined organic layer was dried over MgSO , filtered, and
4
concentrated in vacuo. The residue was purified by flash column chromatography (eluting with
CH Cl /methanol, 100:1 to 20:1) on silica gel to afford the product (44 mg, 0.1 mmol) in overall
2
2
1
3
7
1
6
5% yield. H NMR (300 MHz, CDCl ): δ ppm 8.33–8.18 (m, 2H), 8.00–7.92 (m, 2H), 7.54–
3
.47 (m, 1H), 7.45–7.39 (m, 1H), 7.16–7.08 (m, 4H), 6.87–6.81 (m, 4H), 5.01 (d, J = 6.7 Hz,
H), 4.77–4.61 (m, 4H), 4.60–4.55 (m, 1H), 4.32 (dd, J = 5.8 and 9.8 Hz, 0.2H), 4.28 (dd, J =
.1 and 8.6 Hz, 0.8H), 3.78 (d, J = 2.8 Hz, 6H), 2.97 (dd, J = 3.8 and 14.4 Hz, 1H), 2.80–2.73
(m, 1H), 1.36–1.29 (m, 1H), 1.27–1.19 (m, 2H), 0.80 (dd, J = 4.1 and 6.5 Hz, 1H), 0.80–0.58
+
+
(
m, 6H). HRMS (ESI): [M + H] calculated for C H N O S , 697.3167; found, 697.3148.
3
8
45
6
5
4
.2.6.
(2S)-2-acetamido-N-(1-(benzo[d]thiazol-2-yl)-3-(2-(bis(4-
methoxybenzyl)amino)pyrimidin-5-yl)-1-oxopropan-2-yl)-4-methylpentanamide (8)
To a solution of compound 7 (52 mg, 0.1 mmol, 1.0 eq) in CH Cl (2.0 mL) was added
2
2
Dess-Martin periodinane (0.3M in CH Cl , 0.5 mL, 0.2 mmol, 2.0 eq), then was stirred at room
2
2

temperature for 3 h. The reaction mixture was poured to silica gel directly then was purified by
flash column chromatography (eluting with hexane/EtOAc, 5:1 to 1:1). The product can be
1
afforded (27 mg, 0.04 mmol) in 52% yield. H NMR (300 MHz, CDCl ): δ ppm 8.23–8.12 (m,
3
3
6
5
H), 8.01–7.94 (m, 1H), 7.60–7.54 (m, 2H), 7.12 (d, J = 8.1 Hz, 4H), 6.82 (d, J = 8.1 Hz, 4H),
.12 (d, J = 6.1 Hz, 0.5H), 5.99 (d, J = 5.1 Hz, 1H), 5.92 (d, J = 7.7 Hz, 0.5H), 4.81–4.57 (m,
H), 3.78 (s, 6H), 3.33–3.28 (m, 1H), 3.09–3.02 (m, 1H), 1.95 (d, J = 16.8 Hz, 3H), 1.63–1.50
1
3
(
m, 3H), 0.91 (d, J = 3.9 Hz, 6H). C NMR (75 MHz, CDCl ): δ ppm 191.94, 191.81, 172.13,
3
1
1
5
71.73, 170.49, 170.40, 163.47, 161.65, 161.56, 158.70, 158.45, 153.48, 137.27, 130.34,
30.30, 128.94, 128.86, 128.25, 128.19, 127.34, 127.28, 125.85, 122.43, 116.79, 113.86, 56.47,
5.27, 51.64, 48.22, 48.08, 41.04, 40.54, 32.86, 32.58, 24.85, 24.75, 23.12, 22.97, 22.80, 22.24.
+
+
HRMS (ESI): [M + H] calculated for C H N O S , 695.3010; found, 695.2980.
3
8
43
6
5
4
.2.7.
(2S)-2-acetamido-N-(3-(2-aminopyrimidin-5-yl)-1-(benzo[d]thiazol-2-yl)-1-
oxopropan2-yl)-4-methylpentanamide (9)
To a solution of compound 8 (27 mg, 0.04 mmol) in CH Cl (2.0 mL) was added
2
2
TFA/water mixture (97.5:2.5, v/v) 2.0 mL then stirred at 50 ℃ for 18 h. The reaction mixture
was concentrated under reduced pressure and diluted with a mixture of ACN and water (1:1,
v/v). The solution was filtered through a 0.45 μm PTFE filter and the filter was washed with a
mixture of ACN and water (1:1, v/v). The filtrate was purified by RP-HPLC using 0.1% TFA
in water (A)/ACN (B) to give the product (6.0 mg, 0.01 mmol) in 32% yield as ivory power.
The mobile phase on semi-preparative RP-HPLC was consisted of 30% B at 2 mL/min flow
1
rate. R = 7.05 and 8.37 min. H NMR (600 MHz, CD OD): δ ppm 8.45 (s, 2H), 8.20 (dd, J =
t
3
8
.2 and 11.9 Hz), 8.13 (t, J = 8.3 Hz), 7.68–7.58 (m, 2H), 5.85 (q, J = 4.3 Hz, 0.5H), 5.58 (q, J
4.8 Hz, 0.5H), 4.30–4.23 (m, 1H), 3.39–3.34 (m, 1H), 3.08–3.00 (m, 1H), 1.94 (d, J = 10.6Hz,
=

1
3
3
1
1
1
2
H), 1.59–1.25 (m, 3H), 0.84–0.76 (m, 6H). C NMR (150 MHz, CD OD): δ ppm 192.42,
3
92.21, 175.13, 174.99, 173.45, 173.39, 165.34, 159.00, 158.81, 154.71, 154.62, 138.50,
38.31, 137.04, 136.87, 129.47, 129.31, 128.59, 128.49, 126.54, 126.43, 124.30, 123.83,
23.78, 123.17, 121.02, 120.76, 57.27, 56.59, 53.45, 53.31, 41.52, 41.47, 41.36, 31.61, 31.14,
+
5.73, 23.15, 23.01, 22.34, 22.30, 22.08, 21.94, 21.89. HRMS (ESI): [M + Na] calculated for
+
C H N NaO S , 477.1679; found, 477.1669.
2
2
26
6
3
4
.2.8. (E)-methyl 2-acetamido-3-(pyridin-4-yl)acrylate (10)
N-acetylglycine (1000mg, 8.5 mmol, 1.0 eq), 4-pyridinecarboxaldehyde (0.8 mL, 8.5
mmol, 1.0 eq), sodium acetate (701 mg, 8.5 mmol, 1.0 eq), and acetic anhydride (1.5 mL, 25.6
mmol, 3.0 eq) were added to dried round bottom flask. The reaction mixture was plunged
quickly at 90–100 ℃ for exactly 1 min. The mixture became deep purple color, then the
reaction mixture was cooled at room temperature. The reaction mixture was washed several
times by hexane then suspended in methanol (20 mL). The reaction mixture was heat up to 100
℃
until desolated completely. The color of mixture turned to deep orange. The solvent was
evaporated under reduced pressure and The residue was diluted by EtOAc and partitioned
between EtOAc and water. The combined organic layer was dried over MgSO , filtered, and
4
concentrated under reduced pressure. The residue was purified by flash column
chromatography (eluting with CH Cl /methanol, 80:1 to 25:1) on silica gel to afford the
2
2
1
product (816 mg, 3.7 mmol) in 43% yield. H NMR (300 MHz, CDCl ): δ ppm 8.52 (d, J = 4.2
3
Hz, 2H), 8.18 (s, 1H), 7.28 (d, J = 13.2 Hz, 2H), 7.19 (s, 1H), 3.86 (s, 3H), 2.09 (s, 3H). LRMS
+
+
(
ESI): [M + Na] calculated for C H N NaO , 243.1; found, 243.1.
11 12 2 3

4
.2.9. tert-Butyl 4-(2-acetamido-3-methoxy-3-oxopropyl)piperidine-1-carboxylate (11)
Compound 10 (240 mg, 1.1 mmol, 1.0 eq) was dissolved in acetic acid (10 mL), then
was added PtO (IV) (15 mg, 0.1 mmol, 0.06 eq). The mixture was stirred at room temperature
2
for 24 h under hydrogen gas (1 atm) atmosphere using balloon. The reaction mixture was
filtered through celite then evaporated in vacuo. The residue was washed by toluene several
times to remove acetic acid completely. The residue dissolved in THF was added DMAP (27
mg, 0.2 mmol, 0.2 eq) and di-tert-butyldicarbonate (240 mg, 1.1 mmol, 1.0 eq) then stirring at
room temperature for 2 h. The reaction mixture was diluted by EtOAc and partitioned between
EtOAc and water. The combined organic layer was dried over MgSO , filtered, and
4
concentrated under reduced pressure. The residue was purified by flash column
chromatography (eluting with CH Cl /methanol, 50:1 to 30:1) on silica gel to afford the
2
2
1
product in quantitative yield (358 mg, 1.1 mmol). H NMR (300 MHz, CDCl ): δ ppm 6.76 (d,
3
J = 8.4 Hz, 1H), 4.54–4.46 (m, 1H), 3.92 (d, J = 10.5 Hz, 2H), 3.58 (s, 3H), 2.52 (t, J = 11.4Hz,
1
3
2
H), 1.88 (s, 3H), 1.65–1.40 (m, 5H), 1.30 (s, 9H), 1.09–0.89 (m, 2H). C NMR (75 MHz,
CDCl ): δ ppm 173.23, 170.21, 154.64, 79.22, 52.41, 49.70, 38.92, 32.48, 32.14, 31.25, 28.31,
3
–
–
2
2.78. HRMS (ESI): [M – H] calculated for C H N O , 327.1920; found, 327.1928.
16 27 2 5
4
.2.10.
tert-Butyl
4-(2-acetamido-3-(benzo[d]thiazol-2-yl)-3-oxopropyl)piperidine-1-
carboxylate (12)
This compound was afforded by typical procedure A with compound 11 and
1
benzothiazole in 80% yield. H NMR (300 MHz, CDCl3): δ ppm 8.16 (dd, J = 1.4 and 7.5 Hz,
1
H), 7.95 (dd, J = 1.4 and 7.3 Hz, 1H), 7.62–7.48 (m, 2H), 6.53 (d, J = 8.6 Hz, 1H), 5.94 (dt, J
3.5 and 7.8 Hz, 1H), 4.19–3.93 (br, 2H), 2.68 (q, J = 8.5 and 11.2 Hz, 2H), 2.06 (s, 3H), 2.02–
=
1
3
1
.90 (m, 2H), 1.70–1.51 (m, 3H), 1.42 (s, 9H), 1.31–1.10 (m, 2H). C NMR (75 MHz, CDCl ):
3