Design and synthesis of dye-conjugated hepsin inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.102990

Hepsin is a type II serine protease that is highly expressed in neoplastic prostate. It is an attractive biomarker for imaging metastatic prostate cancer because of its overexpression in advanced prostate cancer and the location of its active site on the

Full text of DOI:10.1016/j.bioorg.2019.102990

Accepted Manuscript
Design and synthesis of dye-conjugated hepsin inhibitors
Kyul Kim, Hongmok Kwon, Doyoung Choi, Tae Hyeong Lim, Il Minn, Sang-
Hyun Son, Youngjoo Byun
PII:
S0045-2068(19)30302-5
https://doi.org/10.1016/j.bioorg.2019.102990
102990
DOI:
Article Number:
Reference:
YBIOO 102990
Bioorganic Chemistry
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22 February 2019
16 May 2019
17 May 2019
Please cite this article as: K. Kim, H. Kwon, D. Choi, T. Hyeong Lim, I. Minn, S-H. Son, Y. Byun, Design and
synthesis of dye-conjugated hepsin inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.102990
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Design and synthesis of dye-conjugated hepsin inhibitors
Kyul Kim1,#, Hongmok Kwon1,#, Doyoung Choi1, Tae Hyeong Lim1, Il Minn2, Sang-Hyun Son1, *, and
Youngjoo Byun1, *
1College of Pharmacy, Korea University, 2511 Sejong-ro, Sejong 30019, Republic of Korea
2Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical
Institutions, Baltimore, MD 21287, United States
Corresponding Authors:
E-mail: sonsh0201@gmail.com; Fax: +82-44-860-1606; Tel: +82-44-860-1644
E-mail: yjbyun1@korea.ac.kr; Fax: +82-44-860-1606; Tel: +82-44-860-1619
# equal contribution
ABSTRACT
Hepsin is a type II serine protease that is highly expressed in neoplastic prostate. It is an
attractive biomarker for imaging metastatic prostate cancer because of its overexpression in advanced
prostate cancer and the location of its active site on the cell surface. We designed and synthesized
novel hepsin-targeted imaging probes by conjugating the hepsin-binding ligand with near-infrared
(NIR) optical dyes. The Leu-Arg dipeptides, attached to BODIPY or SulfoCy7, exhibited strong
hepsin-inhibitory activities with Ki values of 21 and 22 nM, respectively. Compound 2 showed
selective uptake and retention in hepsin-overexpressing cells. This is the first report of hepsin-targeted
optical probes with strong binding affinities and high selectivity over matriptase. Compound 2 has the
potential to be used for developing hepsin-based imaging probes and be as a prototype molecule in the
design of new hepsin inhibitors.

Key words: Hepsin; Optical dye; BODIPY; SulfoCy7
1. Introduction
Prostate cancer is the most common cancer and the second leading cause of cancer death among
men in USA and Europe [1]. Although surgery and radiation therapy improved the 5-year survival
rate of men with prostate cancer [2], it is influenced significantly by the stage of disease progression.
In particular, metastasis of locally-restricted prostate cancer into bone, lymph node, and lung
dramatically increases the mortality rate [3]. Most men are diagnosed at advanced stages of prostate
cancer, perhaps due to the lack of early and effective detection methods of metastatic prostate cancer
[4, 5]. Effective and early detection method of metastatic prostate cancer plays a crucial role in the
development of therapeutic agents for advanced prostate cancer [6, 7]. Consequently, there is an
urgent need to develop molecular imaging modalities specifically more accurate for the detection of
small lesions or metastatic sites in the early stages of prostate cancer [8].
Near-infrared (NIR) fluorescence imaging has emerged as a powerful tool in molecular imaging
and cancer biology due to high sensitivity, real- time scanning capability, high compatibility with
image-guided surgery, and cost efficiency [9]. Key to NIR fluorescence imaging is the use of NIR
fluorophores with optimal emission excitation and emission wavelength (650–900 nm), at which
biological tissues possess low absorption, minimal auto-fluorescence, and reduced scattering [10, 11].
NIR imaging has a high signal-to-noise ratio in many cases [10, 11]. Furthermore, NIR fluorescence
light is invisible to the human eye and can be applied as a sensitive and real-time imaging tool [11].
Conventional approaches that use NIR fluorophore-labeled agents for cancer imaging require them to
be chemically conjugated with cancer-specific targeting molecules such as metabolic substrates, cell-
surface peptides, growth factors, antibodies, and cancer-specific biomarkers [12-15].
Prostate-specific antigen (PSA) is the most widely used biomarker for early screening of prostate
cancer [16, 17]. However, PSA has several limitations since it is expressed by both normal prostate
cells and cancerous prostate cells [18-20]. The low specificity of PSA for prostate cancer results in
over-diagnosis of indolent cancers and unnecessary biopsies. In addition, prostate cancers with
smaller prostate glands and low circulating PSA are missed.

Hepsin is a type II transmembrane serine protease that is predominantly expressed in neoplastic
prostate compared with benign prostate [21-25]. The expression level of hepsin mRNA are
significantly elevated in > 90% of prostate cancer specimens and display > 10-fold increased levels in
metastatic prostate cancer in comparison with normal prostate or benign prostatic hyperplasia (BPH)
[23, 26-28]. Structurally, hepsin is composed of 413 amino acids and has a C-terminal protease
domain in the extracellular region [29]. Overexpression in advanced prostate cancer and the location
of its active site at the cell surface make hepsin an attractive biomarker for imaging metastatic
prostate cancer. High expression of hepsin has also been documented in several other cancers such as
ovarian, breast, and renal cancer [30-33].
In the past decade, a number of low molecular weight (M. W.) hepsin ligands have been reported
including peptide-derived analogs, benzamidines, and indolecarboxamidines [34-41]. Our group also
reported dipeptide-derived hepsin inhibitors containing a Leu-Arg amino acid sequence [35].
Although several small molecules have been identified as potential inhibitors of hepsin, only a few of
these are fluorescently labeled for selective and sensitive detection of prostate cancer [6, 42].
We, herein, report the first hepsin-targeted NIR fluorescence imaging agents based on the
structure of dipeptide-derived hepsin inhibitors. We previously published Leu-Arg hepsin ligands
with strong affinity in the sub-nanomolar Ki range [35]. For this purpose, we designed two NIR
fluorescent hepsin ligands, one labeled with borondippyrromethene (BODIPY) and the other with
SulfoCy7. The fluorophores are separated from the hepsin-binding moiety by a polyethyleneglycol
(PEG) linker, in order to decrease both steric hindrance from the bulky fluorophore and the
hydrophobicity of the conjugate compound.
2. Results and discussion
2.1. Design concept
BODIPY650/665 and SulfoCy7 were chosen because they can be used for NIR optical imaging. In
particular, BODIPY650/665 was selected as a prosthetic group for hepsin conjugation because it has

several advantages such as balance of hydrophilicity and hydrophobicity, sharp fluorescence peaks
with high quantum yield, tolerance to a wide range of polarity and pH, and diverse chemical reactions
for conjugating with targeting moieties or linkers [43-45]. In addition, the fluorine atom in the core
18
structure of BODIPY can be exchanged with F for PET imaging [46-49]. We previously reported
that ketobenzothiazole (kbt) or ketothiazole (kt) analogs derived from Leu-Arg dipeptide exhibited
strong hepsin inhibition [35]. In this work, we attached two NIR dyes to the dipeptide-derived hepsin
inhibitors and evaluate their biological activities in vitro (Fig 1).
<Fig. 1>
2.2. Synthesis of novel BODIPY650/665 activated ester
New BODIPY650/665 fluorophore was synthesized as described in Scheme 1 by modifying the
reported procedure [50]. Briefly, coupling of pyrrole-2-carboxaldehyde with 4-(bromomethyl)benzoic
acid methyl ester via Wittig reaction afforded the alkene 3 in 23% yield. The E-isomer was obtained
1
exclusively, which was confirmed by observing a coupling constant of 16.5 Hz in a H NMR
experiment. Compound 4 was prepared in 71% yield by reacting 3 with Vilsmeier reagent, which was
generated from phosphoryl chloride and dimethylformamide in 1,2-dichloroethane. Bipyrrole 6 was
prepared by adapting a reported procedure [51]. Condensation of 4 with 6 was achieved by treatment
with phosphoryl chloride, followed by complexation with boron trifluoride, providing BODIPY650/665
methyl ester 7 in 66% yield. Hydrolysis of compound 7 was performed by treating 4.5 N HCl solution
in THF under reflux to afford the carboxylic acid 8. Although several hydrolysis conditions including
acid catalyst (HCl, H3PO4), acid concentration (1.0 to 5.0 N), solvent (THF, CH2Cl2), and reaction
condition (rt, reflux) were explored, the best condition provided compound 8 in only 13% yield. The
carboxylic acid 8 was converted into the activated ester 9 by reacting of 8 with N-hydroxysuccinimide
(NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) in
dichloromethane. The appearance of a singlet peak at 2.93 ppm corresponding to the -CH2 group of
NHS in the 1H NMR confirmed the formation of the NHS activated ester of BODIPY650/665
.
<Scheme 1>

2.3. Synthesis of BODIPY650/665-labeled hepsin ligand (1) and SulfoCy7-labeled hepsin ligand (2)
Leu-Arg-ketothiazole (Leu-Arg-kt) hepsin ligand conjugated with PEG linker was synthesized as
shown in Scheme 2. The target compound 15 was prepared from compound 10 in 5 synthetic steps.
Initially, we attempted to conjugate 10 with Leu-Arg-kt ligand by using peptide coupling conditions
(e.g., HATU as coupling reagent, DIPEA as base, DMF as solvent, room temperature), however,
failed in obtaining the desired product. Therefore, we carried out the conjugation of Leu with PEG
linker, followed by peptide coupling between Leu and Arg-kt. Compound 12 was obtained in 71%
yield by using EDC·HCl, 1-hydroxybenzotriazole (HOBt). Hydrolysis of 12 with 25% trifluoroacetic
acid (TFA) solution in dichloromethane afforded compound 13. Coupling of 13 with H2N-Arg(Mtr)-
kt provided compound 14 in 58% yield. Global deprotection of 14 was successfully achieved in 57%
yield to afford compound 15 by a two-step procedure. The Mtr-protecting groups were removed by
the treatment of strong acid (95% TFA), followed by the deprotection of Fmoc group using 20%
piperidine in THF. Temperature was one of the key factors that contributed to the high yield of this
step. The cyclized by-product was obtained upon heating while concentrating under reduced pressure
(See supporting information).
<Scheme 2>
The BODIPY-labeled ligand 1 and the SulfoCy7-labeled ligand 2 were synthesized by
conjugating compound 15 with BODIPY650/665 NHS ester (9) or commercial SulfoCy7 NHS ester,
respectively. The coupling reaction was carried out at room temperature for 16 h in the presence of
triethylamine as a base. The final compounds were purified by reversed-phase RP-HPLC using a
linear gradient of acetonitrile and water containing 0.1% formic acid. The purities of the final
compounds were assessed using RP-HPLC. As shown in the RP-HPLC chromatograms of compounds
1-2 (see supporting information), the BODIPY-attached hepsin ligand 1 (Rt: 9.45 min) was more
hydrophobic than the corresponding SulfoCy7 ligand 2 (Rt: 3.86 min).
2.4. In vitro biological evaluation

The hepsin inhibitory activities of the conjugates 1-2 were determined by a fluorescence-based
enzymatic assay, which uses Boc-QAR-AMC as a substrate and recombinant human hepsin as an
enzyme. Ac-LR-kt and the hepsin ligand (15) without attaching an optical dye were included as
positive controls for comparison. The results of the hepsin assay are summarized in Table 1. New
hepsin-targeted optical probes showed strong hepsin-inhibitory activities (20.9 nM for 1 and 22.1 nM
for 2), which were more potent than compound 15 (42.3 nM). The other positive control Ac-LR-kt
(22.4 nM) showed similar hepsin affinity with the bulky compounds 1-2. Two diastereomers of
compound 2 were separated by HPLC and their Ki values were determined (see supporting
information). The diastereomer at 4.2 min in the HPLC chromatogram of 2 inhibited hepsin much
stronger than one at 3.86 min. Compound 18 with an iodobenzoyl moiety inhibited hepsin stronger
than compounds 1-2 with a Ki value of 6.45 nM (Fig. 2). However, both 1 and 2 showed increased
selectivity for hepsin over matriptase compared with 18 (>1000-fold for 1-2 vs. 62-fold for 18). To
our knowledge, this is the first report of hepsin-targeted imaging probes that possess Ki values in the
range of 20 nM with high selectivity for hepsin over matriptase.
< Table 1 >
< Fig. 2 >
2.5. In vitro cell uptake studies
We evaluated the hydrophilic hepsin-SulfoCy7 ligand 2 for its ability to bind to hepsin on a
surface of prostate cancer cells. Flow cytometric analyses of the ligand 2 exhibited dose-dependent
binding to hepsin-expressing PC3/ML/hepsin cells. However, it did not bind to the non-hepsin-
expressing cell PC3/ML cells, indicating that the binding of compound 2 is specific to hepsin (Fig. 2).
These data are consistent with strong hepsin-binding affinity of the ligand 2. However, the hepsin-
BODIPY ligand 1 had non-specific accumulation in both cells despite its strong binding for hepsin at
the enzyme assay. It might be due to the hydrophobic BODIPY nature of 1.
< Fig. 3 >

2.6. Molecular modeling studies
We performed molecular docking studies of compounds 1-2 with the hepsin X-ray crystal
structure (PDB code: 1O5E) by using Surflex-Dock GeomX module of Sybyl software [52] to
elucidate their binding mode at the active site of hepsin. The common dipeptide region (Leu-Arg) of
1-2 made strong hydrogen-bonding interactions with Asp189 and Gly219 (Fig. 4), which are key
amino acid residues to interact with the amidine region of 6-halo-5-amidino-indole or -benzimidazole
hepsin inhibitors [52]. The difference between 1 and 2 was the orientation of the bulky optical dye
scaffold. While the BODIPY moiety of 1 are located close to the surface of hepsin, the SulfoCy7
region of 2 was projected toward water environment. Although the orientation of the NIR dye region
was so different, hepsin-binding affinities of both compounds was almost similar, indicating that
hydrogen-bonding contacts with Asp 189 and Gly 219 is essential for binding to hepsin.
< Fig. 4 >
3. Conclusion
We have designed and synthesized new hepsin-targeted inhibitors conjugated with NIR optical
imaging dyes. The Leu-Arg-kt analogs labeled with BODIPY (1) or SulfoCy7 (2) exhibited strong in
vitro hepsin-inhibitory activities with Ki values of 20 nM range and high selectivity for hepsin over
matriptase. Cell uptake studies of 2 exhibited selective targeting and retention in hepsin-
overexpressing cells. Compound 2 has the potential to be utilized for the discovery and development
of hepsin-targeted imaging probes and provides valuable information for rational design of novel
hepsin inhibitors.
4. Experimental section
4.1. General methods
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 pre-coated silica gel plates (60 F254). Reaction progress was monitored by TLC analysis
using a UV lamp. Column chromatography was performed on silica gel (230–400 mesh, Merck,
Darmstadt, Germany). NMR spectra were recorded at room temperature on either Bruker BioSpin
Avance 300 MHz NMR or Bruker Ultrashield 600 MHz Plus spectrometer. Chemical shifts are
reported in parts per million (ppm, δ) with TMS as an internal standard. Coupling constant are given
in Hertz.13C NMR spectra were obtained by using the same NMR spectrometers and were calibrated
with CDCl3 (δ = 77.16 ppm). Splitting patterns are indicated as s, singlet; d, doublet; t, triplet; q,
1
quartet; m, multiplet; 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 a API 150EX ESI-MS spectrometer. High-performance
liquid chromatography purification were performed on Agilent 1260 Infinity (Agilent) or Shimadzu
system (system controller CMB-20A, tow pumps LC-20AR, photodiode array detector SPD-20AV).
The purity of all final compounds was measured by analytical reverse-phase high-performance liquid
chromatography(RP-HPLC) on an Agilent 1260 Infinity (Agilent) with a C18 column (Phenomenex,
150 × 4.6 mm, 3 μm, 110 Å). RP-HPLC was performed using an isocratic elution (method A) or a
linear gradient elution (method B). The isocratic elution was mobile phase consisting of
acetonitrile‒water (35:65, v/v) with 0.1% formic acid (FA). The gradient elution process included 10%
to 90% of solvent B over 20 min (A = 0.1% FA in water and B = 0.1% FA in acetonitrile). All
compounds were eluted with a flow rate of 1.0 mL/min and monitored at UV detector: 254 nm, 650
nm (for BODIPY660/665- labeled compounds), and 773 nm (for SulfoCy7- labeled compounds). Purity
of the tested compounds was > 95%.
4.2. Preparation of novel BODIPY650/665 NHS ester (9)
4.2.1. (E)-Methyl 4-(2-(1H-pyrrol-2-yl)vinyl)benzoate (3)
To a solution of methyl 4-bromomethylbenzoate (4.8 g,21.0 mmol), tributylphosphine (5.2
mL,21.0 mmol) and zinc (1.4 g, 21.0 mmol) in benzene was added pyrrole-2-carboxaldehyde (2.0 g,
21.0 mmol). The reaction mixture was stirred at 100°C for 15 h. After cooling to room temperature,

the reaction mixture was diluted with CH2Cl2 (100 mL). The organic layer was washed with water (50
mL x 3). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure.
The crude residue was purified by silica gel column chromatography (toluene–EtOAc, 50 : 1 to 40 : 1,
1
v/v) to afford compound 3 (1.1 g, 23%) as a yellow solid. Rf = 0.64 (toluene:EtOAc = 5:1, v/v). H
NMR (300 MHz, CDCl3) δ 8.41 (brs, 1H), 7.99 (d, J=8.4 Hz, 2H), 7.47 (d, J=8.4 Hz, 2H), 7.09 (d, J=
16.5 Hz, 1H), 6.86 (d, J= 0.9 Hz, 1H), 6.67 (d, J= 16.5 Hz, 1H), 6.42 (brs, 1H), 6.27 (dd, J= 2.4 and
5.9 Hz, 1H), 3.91 (s, 3H); 13C NMR (75 MHz, CDCl3) δ166.9, 142.1, 130.4, 130.1, 128.2, 125.5,
122.0, 121.3, 119.9, 110.4, 110.3, 52.0. LRMS (ESI) m/z 226.4 [M – H]–.
4.2.2. (E)-Methyl 4-(2-(5-formyl-1H-pyrrol-2-yl)vinyl)benzoate (4)
Phosphorus oxychloride (92 μL, 0.99 mmol) was added dropwise to anhydrous DMF (76 μL,
0.99 mmol) at 0°C. The solution was warmed to room temperature and stirred for 15 min. The
mixture was cooled to 0 °C and diluted with anhydrous 1,2-dichloroethane (10 mL). A solution of 3
(200mg, 0.88 mmol) in anhydrous 1,2-dichloroethane (15 mL) was added dropwise to the solution.
The reaction mixture was stirred under reflux for 1 h. The reaction mixture was cooled to room
temperature and quenched with a solution of sodium acetate trihydrate (1.3 g, 9.9 mmol) in water (5
mL). The reaction mixture was stirred under reflux for 1h. The organic layer was collected and the
aqueous layer extracted with CH2Cl2. The combined organic layer was washed with saturated aqueous
NaHCO3 solution, dried over MgSO4, and concentrated under reduced pressure. The crude residue
was purified by silica gel column chromatography (toluene–EtOAc = 40 : 1 to 5 : 1, v/v) to give
1
compound 4 (160 mg, 71%) as a yellow solid. Rf = 0.33 (toluene:EtOAc = 5:1, v/v). H NMR (300
MHz, CDCl3) δ 10.24 (brs, 1H), 9.52 (s, 1H), 8.03 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.17 (d,
J= 16.5 Hz, 1H), 7.10 (d, J= 16.5 Hz, 1H), 7.01 (dd, J= 2.4 and 3.9 Hz, 1H), 6.54 (dd, J= 2.4 and 8.0
Hz, 1H), 3.93 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 179.0, 166.9, 141.0, 138.6, 133.4, 130.3, 129.6,
129.6, 126.4, 123.1, 119.8, 111.6, 52.3.
4.2.3. 1H,1'H-2,2'-Bipyrrole (6)
To a solution of 1H-pyrrole 5 (187 μL, 2.7 mmol) in CH2Cl2 (15 mL) were added PIFA(387 mg,

0.9 mmol) and TMSBr (233 μL, 1.8 mmol) at –78°C. The reaction mixture was stirred at the same
temperature for 1 h. After the reaction was completed, saturated aqueous NaHCO3 solution (ca. 50 mL)
was added to the reaction mixture. The reaction mixture was stirred for an additional 10 min at
ambient temperature. The organic layer was separated and the water layer was washed with CH2Cl2.
The combined organic layer was dried with MgSO4, filtered, and concentrated. The crude residue was
purified by silica gel column chromatography (toluene–EtOAc 40 : 1 to 5 : 1, v/v) to give compound 6
(116 mg, 65%) as a colorless oil. Rf = 0.32 (hexane:EtOAc = 4:1, v/v). 1H NMR (300 MHz, CDCl3) δ
8.22 (brs, 2H), 6.79‒6.74(m, 2H), 6.27‒6.19 (m, 4H). LRMS (ESI) m/z calculated for C8H8N2 [M +
H]+: 133.1, found: 133.0. 1HNMR and MS data were in complete agreement with those previously
reported [51].
4.2.4. Methyl 4-((E)-2-((Z)-2-((1-(difluoroboryl)-1H,1'H-[2,2'-bipyrrol]-5-yl)methylene)-2H-pyrrol-5-
yl)vinyl)benzoate (7)
The reaction was carried out in the dark. To a solution of 4 (260 mg, 1.02 mmol) and 6 (134
mg,1.02 mmol) in anhydrous CH2Cl2 (20 mL) was added phosphorus oxychloride (142 μL,1.53
mmol). The reaction mixture was stirred for 15 h. After the excess solvent was evaporated, the
remaining residue was dissolved in anhydrous CH2Cl2(20 mL). N,N-Diisopropylethylamine (1.7 mL,
10.2 mmol) and BF3·OEt2 (2.9 mL,10.2 mmol) were added slowly to the solution. The mixture
solution was stirred for 3 h. The reaction mixture was washed with brine, dried over MgSO4, filtered,
and concentrated under reduced pressure. The crude residue was purified by silica column
chromatography with hexane–EtOAc (2 : 1, v/v) to afford compound 7 (240 mg, 56%) as blue solid.
1
Rf = 0.31 (hexane:EtOAc = 2:1, v/v). H NMR (300 MHz, CDCl3) δ 10.50 (brs, 1H), 8.06 (d, J=8.4
Hz, 2H), 7.73 (d, J= 16.5 Hz, 1H), 7.66 (d, J=8.4 Hz, 2H), 7.31‒7.22 (m, 2H),7.07 (d, J= 4.8 Hz, 1H),
7.03 (brs, 1H), 6.98 (s, 1H), 6.95‒6.89 (m, 3H), 6.43‒6.40 (m, 1H), 3.94 (s, 3H);13C NMR (75 MHz,
CDCl3) δ166.9, 150.6, 150.3, 141.3, 138.3, 135.4, 132.9, 131.5, 130.2, 129.7, 127.1, 126.4, 126.3,
124.0, 121.70, 121.0, 118.4, 115.4, 111.9, 52.3. HRMS (ESI): calcd for C23H18BF2N3O2[M ‒ H]‒,
416.1387; found, 416.1389.

4.2.5. 4-((E)-2-((Z)-2-((1-(Difluoroboryl)-1H,1'H-[2,2'-bipyrrol]-5-yl)methylene)-2H-pyrrol-5-
yl)vinyl)benzoic acid (8)
The reaction was carried out in the dark. To a solution of 7 (400 mg, 0.96 mmol) in THF (40 mL)
was added aqueous HCl solution (4.5 M, 10 mL). The reaction mixture was stirred under reflux for 18
h. The solution was cooled to room temperature, diluted with water (ca. 50 mL), and washed with
EtOAc. The combined organic layer was dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude residue was purified by silica gel column chromatography (CH2Cl2–MeOH,30:1
to 10:1, v/v) to afford compound 8 (52 mg, 13%) as a dark blue solid. Rf = 0.33 (CH2Cl2:MeOH =
10:1, v/v). 1H NMR (300 MHz, CD3OD) δ8.05 (d, J=8.4 Hz, 2H), 7.77 (d, J= 16.5 Hz, 1H), 7.70 (d,
J=8.4 Hz, 2H), 7.41 (d, J= 16.2 Hz, 1H), 7.29 (dd, J = 1.2 and 2.4 Hz, 1H), 7.25 (dd, J = 1.5 and 3.9
Hz, 1H), 7.23 (s, 1H), 7.22 (d, J= 4.5 Hz, 1H), 7.07 (d, J= 4.5 Hz, 1H), 7.04 (d, J= 4.2 Hz, 1H), 7.00
(d, J= 4.2 Hz, 1H), 6.40 (dd, J = 2.7 and 3.9 Hz, 1H).HRMS (ESI): calcd for C22H16BF2N3O2[M ‒ H]‒,
402.1231; found, 402.1231. > 95% purity (as determined by RP-HPLC, method B, retention time =
16.84 min).
4.2.6. 2,5-Dioxopyrrolidin-1-yl 4-((E)-2-((Z)-2-((1-(difluoroboryl)-1H,1'H-[2,2'-bipyrrol]-5-
yl)methylene)-2H-pyrrol-5-yl)vinyl)benzoate (9)
To a solution of compound 8 (25 mg, 62 μmol) in dry dichloromethane (20 mL) was added N-
hydroxysuccinimide (29 mg, 0.25 mmol) and EDC·HCl (48 mmol). The reaction mixture was stirred
at room temperature for 4 h and was quenched with cooled HCl solution (1.0 M, ca. 20 mL). The
solution was with iced water and extracted with CH2Cl2(ca. 20 mL twice). The combined organic
layer was washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to
afford compound 9 (28 mg, 90%).The product was used in the next step without further purification.
1
Rf = 0.78 (hexane:EtOAc = 1:1, v/v). H NMR (300 MHz, CDCl3) δ 10.51 (brs, 1H), 8.14 (d, J=8.4
Hz, 2H), 7.78 (d, J= 16.5 Hz, 1H), 7.71 (d, J=8.4 Hz, 2H), 7.29‒7.21 (m, 2H), 7.08 (d, J= 4.8 Hz, 1H),
7.05 (brs, 1H), 6.98 (s, 1H), 6.95‒6.90 (m, 3H), 6.42 (brs, 1H), 2.93 (s, 4H). HRMS (ESI): calcd for
C26H19BF2N4O4[M ‒ H]‒, 499.1394; found, 499.1378. > 95% purity (as determined by RP-HPLC,
method B, retention time = 11.20 min).

4.3. Preparation of compound 15
4.3.1. (S)-tert-Butyl 1-(9H-fluoren-9-yl)-21-isobutyl-3,19-dioxo-2,7,10,13,16-pentaoxa-4,20-
diazadocosan-22-oate (12)
To a solution of compound 10 (785 mg, 1.6mmol) and L-leucine tert-butyl ester hydrochloride
11 (291 mg, 1.3 mmol) in THF (10 mL) were added HOBt (281 mg, 2.08 mmol) and EDC·HCl (399
mg, 2.08 mmol) at 0 °C. The reaction mixture was stirred at room temperature overnight. The reaction
mixture was quenched with saturated aqueous NaHCO3 solution (ca. 10 mL) and extracted with
EtOAc (ca. 150 mL). The combined organic layer was washed with brine, dried over MgSO4, filtered,
and concentrated in vacuo. The crude residue was purified by silica gel column chromatography
(CH2Cl2:MeOH =60 : 1 to 50 : 1, v/v) to give compound 12 (604 mg, 71%) as a colorless oil. Rf =
1
0.64 (CH2Cl2:MeOH = 10:1, /v). H NMR (300 MHz, CDCl3) δ 7.76 (d, J= 7.5 Hz, 2H), 7.60 (d, J=
7.5 Hz, 2H), 7.40 (t, J= 7.2 Hz, 2H), 7.31 (t, J= 7.5 Hz, 2H), 6.59 (d, J= 7.8 Hz, 1H), 5.42 (brs, 1H),
4.54‒4.45 (m, 1H),4.40 (d, J= 6.9 Hz, 2H), 4.22 (t, J= 6.6 Hz, 1H), 3.71 (t, J= 5.7 Hz, 1H), 3.69‒3.53
(m, 15H), 3.45‒3.34 (m, 2H), 2.48 (t, J= 5.7 Hz, 2H), 1.73‒1.42 (m, 3H),1.45 (s, 9H), 0.93 (d, J= 6.3
Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 171.9, 170.9, 156.3, 143.6, 140.9, 127.3, 126.7, 124.7, 119.5,
81.0, 70.1, 70.0, 69.9, 69.8, 69.8, 66.8, 66.1, 50.9, 49.7, 46.8, 41.1, 40.5, 36.3, 27.6, 24.5, 22.5, 21.7.
HRMS (ESI): calcd for C36H52N2O9[M + Na]+, 679.3565; found, 679.3568.
4.3.2. (S)-1-(9H-Fluoren-9-yl)-21-isobutyl-3,19-dioxo-2,7,10,13,16-pentaoxa-4,20-diazadocosan-22-
oic acid (13)
To a solution of 12 (200 mg, 0.3 mmol) in CH2Cl2 (7.5 mL) was added slowly trifluoroacetic
acid (2.5 mL) at 0°C. The reaction mixture was stirred at room temperature for 5 h. The reaction
mixture was diluted with CH2Cl2 and poured into iced water. The organic layer was washed with
saturated aqueous NaHCO3 solution, followed by brine. The combined organic phase was dried over
MgSO4 and concentrated under reduced pressure to give compound 13 (192 mg, quantitative) as
1
colorless oil. Rf = 0.31 (CH2Cl2:MeOH = 10:1, v/v). H NMR (300 MHz, CDCl3) δ 9.24 (brs, 1H),
7.75 (d, J= 7.5 Hz, 2H), 7.60 (d, J= 7.5 Hz, 2H), 7.39 (t, J= 7.2 Hz, 2H), 7.33 (t, J= 7.5 Hz, 2H), 7.02

(d, J= 7.8 Hz, 1H), 5.63 (brs, 1H), 4.68‒4.52 (m, 1H),4.39 (d, J= 6.9 Hz, 2H), 4.29‒4.13 (m,
1H)3.78‒3.51 (m, 16H), 3.47‒3.32 (m, 2H), 2.57‒2.43 (m, 2H), 1.78‒1.52 (m, 3H), 0.93 (d, J= 5.4 Hz,
6H); 13C NMR (75 MHz, CDCl3) δ174.8, 172.2, 156.8, 144.0, 141.3, 127.7, 127.1, 125.6, 125.1, 120.0,
68.0, 67.1, 66.7, 50.9, 47.2, 41.2, 40.9, 36.6, 25.6, 24.9, 23.0, 22.0. HRMS (ESI): calcd for
C32H44N2O9 [M + Na]+, 623.2939; found, 623.2940.
4.3.3. (9H-Fluoren-9-yl)methyl ((9S)-1-imino-9-isobutyl-1-(4-methoxy-2,3,6-
trimethylphenylsulfonamido)-8,11-dioxo-6-(thiazole-2-carbonyl)-14,17,20,23-tetraoxa-2,7,10-
triazapentacosan-25-yl)carbamate (14)
To a solution of 13 (384 mg, 0.64mmol) and N-(N-(4-amino-5-oxo-5-(thiazol-2-
yl)pentyl)carbamimidoyl)-4-methoxy-2,3,6-trimethylbenzenesulfonamide (233 mg, 0.51 mmol) in
THF (5 mL) were added HOBt (108 mg, 0.8 mmol) and EDC·HCl (154 mg, 0.8 mmol) at 0 °C. The
reaction mixture was allowed to slowly warm to room temperature and stirred overnight at room
temperature. The reaction mixture was quenched with saturated aqueous NaHCO3 solution (ca. 10 mL)
and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4,
and concentrated in vacuo. The crude residue was purified by silica gel column chromatography
(CH2Cl2–MeOH = 30 : 1 to 10 : 1, v/v) to give compound 14 (290 mg, 55%) as colorless oil. Rf = 0.51
1
(CH2Cl2:MeOH = 10:1, v/v). H NMR (300 MHz, CDCl3) δ 7.99 (d, J= 3.0 Hz, 1H), 7.75 (d, J= 7.5
Hz, 2H), 7.71‒7.67 (m, 1H), 7.59 (t, J= 7.2 Hz, 2H), 7.38 (t, J= 7.5 Hz, 2H), 7.30 (d, J= 7.5 Hz, 2H),
6.49 (s, 1H), 6.36 (s, 2H), 5.63‒5.51 (m, 1H), 4.54‒4.38 (m, 1H), 4.38 (d, J= 6.9 Hz, 2H), 4.20 (t, J=
6.6 Hz, 1H), 3.79 (s, 3H), 3.69‒3.53 (m, 15H), 3.52‒3.11 (m, 4H), 2.68 (s, 3H), 2.59 (s, 3H),
2.62‒2.41 (m, 1H), 2.10 (s, 3H), 2.20‒2.03 (m, 1H), 1.81‒1.42 (m, 6H), 0.88 (dd, J= 5.4and 13.1 Hz,
6H); 13C NMR (75 MHz, CDCl3) δ191.0, 172.3, 164.3, 158.2, 156.3, 156.4, 156.4, 145.3, 145.2, 144.0,
141.3, 138.5, 136.4, 134.0, 127.7, 127.1, 125.1, 124.6, 120.0, 111.6, 70.4, 70.3, 70.2, 70.1, 70.0, 67.0,
66.6, 55.4, 50.5, 47.2, 40.9, 40.4, 40.3, 25.3, 24.8, 24.7, 24.1, 23.1, 23.0, 21.8, 18.4, 12.0. HRMS
(ESI): calcd for C51H69N7O12S2[M + H]+,1036.4518; found, 1036.4519.
4.3.4. 1-Amino-N-((2S)-1-((5-guanidino-1-oxo-1-(thiazol-2-yl)pentan-2-yl)amino)-4-methyl-1-
oxopentan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide (15).

Compound 14 (104 mg, 0.1 mmol) was dissolved in TFA/thioanisole/water (95/2.5/2.5,v/v/v, 3
mL) and the solution was stirred vigorously for 3 h. The reaction mixture was quenched with iced
water and concentrated under reduced pressure at 4 °C. The crude residue was filtrated through a pad
of silica gel (CH2Cl2–MeOH, 10 : 1, v/v) and the filtrate was concentrated under reduced pressure.
To a solution of the intermediate in THF (4 mL) was added dropwise piperidine (1 mL) at 0 °C.
The reaction mixture was stirred vigorously for 10 min. The reaction mixture was quenched with iced
water and concentrated under reduced pressure at 4 °C. The crude residue was treated with cold
diethyl ether (40 mL) and the ether layer was decanted. The crude residue was dissolved with a
mixture of water and acetonitrile (1:1, 0.1% formic acid), filtered through a 0.45 μm PTFE filter, and
purified by semi-preparative RP-HPLC (using conditions: column, Phenomenex Gemini-NX C18,
110 Å, 150 mm × 10 mm, 5 μm; flow rate, 2 mL/min; mobile phase, A = 0.1% formic acid (FA) in
water and B = 0.1% FA in acetonitrile; gradient, 5% B to 90% B over 20 min; detection, 220 nm and
254 nm) to give compound 15 (retention time = 7.48 and 7.83 min) in 57% yield. 1H NMR (300 MHz,
CD3CN–D2O = 1:1, v/v) δ 8.06 (dd, J= 0.9 and 3.0 Hz, 1H), 7.99 (dd, J= 0.9 and 3.0 Hz, 1H),
5.42‒5.33 (m, 1H), 4.33‒4.27 (m, 1H), 3.73‒3.42 (m, 16H), 3.19‒2.97 (m, 4H), 2.54‒1.83 (m, 2H),
2.13‒1.89 (m, 1H), 1.87‒1.38 (m, 1H), 0.83 (dd, J= 6.3 and 13.2 Hz, 6H); 13C NMR (75 MHz,
CD3CN–D2O = 1:1, v/v) δ 192.6, 192.5, 174.9, 174.3, 174.2, 165.1, 157.6, 146.2, 129.6, 129.6, 70.5,
70.4, 67.6, 67.2, 55.8, 55.7, 53.1, 53.0, 41.4, 41.6, 40.0, 36.8, 36.7, 28.7, 28.6, 25.5, 25.5, 25.3, 25.2,
23.1, 21.9, 21.8. HRMS (ESI): calcd for C26H47N7O7S [M + H]+, 601.3258; found, 602.3344.
4.4. Preparation of BODIPY650/665-labeled hepsin ligand (1)
To a mixture of 9 (2.40 mg, 4.80 μmol) and 15 (1.72 mg, 2.86 μmol) in DMF (1 mL) was added
triethylamine (25 μL, 180 μmol). The reaction mixture was stirred at room temperature for 16 h. The
reaction mixture was quenched with a mixture of ACN and water (1:1, 0.1% formic acid, 1 mL), and
filtered through a 0.45 μm PTFE filter. The filtrate was purified by PR-HPLC as described for
1
compound 15, to afford compound 1 in 84% yield. H NMR (300 MHz, CD3CN–D2O = 1:1, v/v) δ
8.02‒8.00 (m, 1H), 7.94 (d, J= 3.0 Hz, 1H), 7.79 (d, J= 8.4 Hz, 1H), 7.65 (d, J= 8.4 Hz, 1H), 7.64 (d,

J= 15.9 Hz, 1H), 7.38 (d, J= 16.5 Hz, 1H), 7.27‒7.20 (m, 4H), 7.04‒6.95 (m, 3H), 6.38 (t, J= 2.7 Hz,
1H), 5.42‒5.33 (m, 1H), 3.64‒3.46 (m, 17H), 3.09‒3.02 (m, 2H), 2.44‒2.35 (m, 2H), 1.63‒1.38 (m,
6H), 1.27‒1.81 (m, 2H), 0.83‒0.73 (m, 6H). HRMS (ESI): calcd for C48H61BF2N10O8S [M + H]+,
987.4528; found, 987.4527. > 98% purity (as determined by RP-HPLC, method A, retention time =
9.54 min and 10.66 min, respectively).
4.5. Preparation of SulfoCy7-labeled hepsin ligand (2)
To a solution of SulfoCy7 NHS ester (5.0 mg, 5.90 μmol) and 15 (4.6 mg, 7.70 μmol) in DMF (2
mL) was added triethylamine (80 μL, 570 μmol). The reaction mixture was stirred at room
temperature for 16 h. The reaction mixture was quenched with a mixture of ACN and water (1:1, 0.1%
formic acid, 1 mL) and filtered through a 0.45 μm PTFE filter. The filtrate was purified by PR-HPLC
as described for compound 15 to afford compound 2 in 50% yield. 1H NMR (300 MHz, CD3CN–D2O
= 1:1, v/v) δ 8.02 (s, 1H), 7.95 (s, 1H), 7.79‒7.70 (m, 4H), 7.64 (d, J= 15.6 Hz, 1H), 7.59 (d, J= 15.6
Hz, 1H), 7.39 (s, 1H), 7.21 (d, J= 9.0 Hz, 1H), 7.18 (d, J= 9.0 Hz, 1H), 6.08 (t, J= 14.4 Hz, 1H),
5.39‒5.31 (m, 1H), 3.64‒3.36 (m, 20H), 3.23‒3.17 (m, 2H), 3.11‒3.03 (m, 2H), 2.48‒2.35 (m, 6H),
2.12‒2.06 (m, 2H), 1.85‒1.76 (m, 2H), 1.74‒1.67 (m, 2H), 1.65‒1.57 (m, 16H), 1.57‒1.47 (m, 2H),
1.46‒1.39 (m, 2H), 1.33‒1.27 (m, 2H), 1.23‒1.17 (m, 2H), 0.79 (dd, J= 6.6 and 27.6 Hz, 6H). HRMS
(ESI): calcd for C63H88N9O14S3[M]‒, 1290.5618; found, 1290.5618. > 98% purity (as determined by
RP-HPLC, method A, retention time = 3.86 and 4.01 min).
4.6. Preparation of 4-iodobenzoic acid-labeled hepsin ligand (18)
To a solution of 4-iodobenzoic acid NHS ester (7.6 mg, 22.0μmol) and 15 (8.8 mg, 14.7 μmol) in
DMF (5 mL) was added triethylamine (25 μL, 180 μmol). The reaction mixture was stirred at room
temperature for 5 h. The reaction mixture was quenched with a mixture of ACN and water (1:1, 0.1%
formic acid, 1 mL) and filtered through a 0.45 μm PTFE filter. The filtrate was purified by PR-HPLC
1
HPLC as described for compound 15, to afford compound 18 in 63% yield. H NMR (300 MHz,
CD3CN–D2O = 1:1, v/v) δ 8.26 (dd, J= 1.5 and 3.0 Hz, 1H), 8.19 (d, J=3.0Hz, 1H), 8.02 (d, J=8.4 Hz,

1H), 8.71 (d, J=8.4 Hz, 1H), 5.62‒5.56 (m, 1H), 4.03‒3.60 (m, 17H), 3.42‒3.21 (m, 2H), 2.69‒2.52
(m, 2H), 2.01‒1.62 (m, 6H), 1.52‒1.31 (m, 1H), 1.01 (dd, J=5.4 and 13.1Hz, 6H). HRMS (ESI): calcd
for C33H50IN7O8S [M + H]+, 832.2559; found, 832.2557. > 98% purity (as determined by RP-HPLC,
method A, retention time = 6.72 and 7.53 min).
4.8. Determination of Ki values for hepsin inhibition
Hepsin inhibitors (10 nM–10 μM ) were diluted in DMSO (2% final concentration in reaction)
and mixed with activated hepsin (#4776-SE-010, R&D Systems, Minneapolis, Minnesota) to a 96-
well plate (REF 353219; BD Falcon). The final assay concentration of hepsin was 0.3 nM in TNC
buffer (25 mM Tris, 150 mM NaCl, 5 mM CaCl2, 0.01% Triton X-100, pH 8). After incubation for 30
min at room temperature, Boc-QAR-AMC substrate (#ES014, R&D Systems, Minneapolis,
Minnesota) was added to the hepsin assay solution. The final substrate concentration was 150 μM
with final reaction volume of 100 μL. Changes in fluorescence (excitation at 380 nm and emission at
460 nm) were measured at room temperature over 120 min in a Biotek Synergy 2 plate reader
(Molecular devices).
Hepsin activation: Based upon the manufacturer’s recommendations, recombinant hepsin (#4776-SE-
010, R&D Systems, Minneapolis, Minnesota) was diluted 5.5 fold in TNC buffer and incubated at
37 °C. After 24 h, the hepsin was diluted in glycerol to 50%. This stock Hepsin (1.2 μM) was stored
in a –20 °C freezer and diluted in TNC buffer for use in assays. From a plot of the mean reaction
velocity versus the inhibitor concentration, a non-linear curve fit was performed using GraphPad
Prism version 6.04 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com) to
determine the inhibitor IC50s from a plot of the mean reaction velocity versus the inhibitor
concentration. The IC50 values represent the average of three separate experimental determinations. Ki
values were calculated using the Cheng and Prusoff equation (Ki = IC50/(1 + [S]/Km).
4.9. In vitro cell uptake study
PC3/ML and PC3/ML-HPN were prepared at Johns Hopkins Medical Institutions, Baltimore,
Maryland. All the cell lines were maintained in DMEM supplemented with 10% FBS and 1x

Pen/Strep at 37 °C in a humidified chamber with 5% CO2. Cells were detached using 0.05 %
trypsin/EDTA solution and re-suspended in RPMI containing 1% FBS at 1 x 106 cells/mL cell density.
Cells were incubated with the indicated concentrations of inhibitors at 37 °C for 1 h, followed by
washing twice with RPMI containing 1% FBS. Cells were analyzed by BD™ LSRII Flow Cytometer
(BD Biosciences, San Jose, CA) and the data were analyzed using FlowJo software (FlowJo LLC,
Ashland, OR).
4.10. In silico docking studies
Ligand preparation and optimization : The 2D and 3D structure generation of all peptide ligands
were each performed by ChemBioDraw (ver. 11.0.1) and Chem3D pro (ver. 11.0.1). Boron atom of
BODIPY structure was replaced with sp3 carbon during ligand preparation process because SYBYL-X
program could not recognize boron atom for docking study. The ligands were saved as .sdf file.
‘Surflex Searching’ preparation protocol was used to ligand preparation and optimization in SYBYL-X
2.1.1 (Tripos Inc., St Louis) to clean up the structures.
Protein preparation : The PDB format protein structure was downloaded from RCSB protein data
bank (PDB ID: 1O5E). Protein structure preparation including conflicted side chains of amino acid
residues fixation was executed by SYBYL-X 2.1.1. Under the application of AMBER7 FF99 Force
Field hydrogen atoms were added and minimization process was performed by POWELL method.
Initial optimization option was set SIMPEX. Default setting was applied to other parameters.
Docking and scoring function studies : To perform the docking studies of all prepared ligands,
Surflex-Dock GeomX module was used in SYBYL-X 2.1.1. Surflex-Dock protomol as an idealized
representation of a ligand guided docking to make every potential interaction with the binding site.
For a generation of Protomol, Bloat(Å) and Threshold were set to 0.62 and 0, respectively. All other
parameters followed the default.
Conflicts of interest
The authors declare no conflict of interests.
Acknowledgments

We acknowledge support from the National Research Foundation (2014R1A4A1007304 and
2017R1A2B4005036 to Y.B., and 2015R1D1A4A01016638 to S.-H.S) and from Ministry of Health
& Welfare, Republic of Korea (HI16C1827 to Y.B.).
Appendix A.
Supplementary data Supplementary data to this article can be found online at https://
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Figure Legends
Fig. 1. Design of hepsin-targeted optical imaging probes based on Ac-LR-kt, the known hepsin
inhibitor
Fig. 2. IC50 curves of compounds 1, 2, 18 and Ac-LR-kt
Fig. 3. In vitro uptake study of compound 2 in PC3/ML and PC3/ML/hepsin cell lines
Fig. 4. Docked poses of hepsin-targeted optical probes (A: compound 1, B: compound 2) at the active
site of hepsin.
Scheme 1. Synthesis of novel BODIPY650/665 activated ester
aReagents and reaction conditions: (i) PBu3, Zn, 100 ºC, 15 h, benzene, 23%; (ii) DMF, POCl3, reflux,
15 min, CH2Cl2 then NaOAc, 71%; (iii) PIFA, TMSBr, –78 ºC, 1 h, CH2Cl2, 65%; (iv) compound 4,
POCl3, rt, 5.5 h, CH2Cl2 then DIPEA, BF3·Et2O, 2 h, 65%; (v) 4.5 M HCl/THF (1/4, v/v), reflux, 18 h,
13%; (vi) EDC·HCl, N-hydroxysuccinimide, CH2Cl2, rt, 2 h.
Scheme 2. Synthesis of BODIPY650/665-labeled ligand 1 and SulfoCy7-labeled ligand 2
aReagents and reaction conditions: (i) HOBt, EDC·HCl, THF, 0 ºC to rt, 12 h, 71%; (ii) 25% TFA in
CH2Cl2, 0 ºC to rt, 5 h, quantitative; (iii) NH2Arg(Mtr)-kt, HOBt, EDC·HCl, THF, 0 ºC to rt, 12 h,
55%; (iv) TFA/thioanisole/water (95/2.5/2.5 (v/v/v)), rt, 3 h; (v) 20% piperidine in THF, 0 ºC, 10 min,
57% in two steps; (vi) compound 9 (for 1), sulfoCy7 NHS ester (for 2), or 4-iodobenzoic acid NHS
ester (for 18), Et3N, DMF, rt, 16 h (for 1 and 2) or 5 h (for 18), 84% (for 1), 50% (for 2), 63% (for 18).

Table 1. Enzyme activity of the synthesized compounds against hepsin and matriptase
Compound
hepsin Ki (nM)a
22.4 ± 0.50
20.9 ± 1.54
22.1 ± 1.37
42.3 ± 3.23
6.45 ± 0.44
matriptase Ki (nM)a
334 ± 62
hepsin selectivity
14.9
Ac-LR-kt
1
2
> 30,000
> 1,000
> 1,000
3.2
> 30,000
15
18
134 ± 34
403 ± 32
62.5
a Measurements of enzymatic activity were performed in triplicate and represent the mean ± SD at
least three experiment sets.

Scheme 1
O
O
O
H
ii
i
OMe
+
OMe
N
H
O
N
Br
OMe
O
N
H
H
H
3
4
H
N
N
7
8
9
, R1 = Me
, R1 = H
iii
iv
v
B
N
F F
vi
N
H
N
, R1 =
O
NH
H
N
O
6
5
OR1
O
Scheme 2
O
H
O
O
H
O
OH
O
N
O
N
H2N
ii
FmocHN
FmocHN
O
FmocHN
OH
i
O
4
+
4
4
O
O
O
10
11
12
13
O
H
N
NH2
HN
HN
HN
S
S
O
O
HN
O
O
S
H
H
iii
iv, v
O
N
O
N
N
N
FmocHN
N
H2N
N
H
H
4
4
O
O
O
O
14
15
I
NH2
HN
N
N
B
1
: R =
18
: R =
O
HN
F
F
NH
O
S
H
R
vi
O
N
N
N
H
N
O
H
4
O
O
-
-O3S
SO3
2
: R =
N
N
O

Figure 1
N
N
B
F
F
NH2
NH2
HN
HN
HN
HN
NH
BODIPY665/660
NIR
dye
O
N
O
S
O
S
H
H
N
O
N
-
-O3S
SO3
N
N
N
N
N
H
H
H
4
O
O
O
O
N
Ac-LR-kt
4
O
SulfoCy7
Figure 2
10000
Ac-LR-kt
cmpd 1
cmpd 2
cmpd 18
8000
6000
4000
2000
0
-10
-9
-8
-7
-6
-5
Conc.(log)

Figure 3
Figure 4

Graphical Abstract
NH2
N
F
HN
HN
N
B
NH
F
O
S
H
O
N
N
N
N
NH2
HN
HN
H
H
O
4
O
O
1
Hepsin Ki = 20.9 nM
Matriptase Ki > 20,000 nM
O
S
H
N
N
N
H
O
O
-
-O3S
SO3
NH2
HN
HN
Ac-LR-kt
Hepsin Ki = 22.4 nM
Matriptase Ki = 334 nM
N
N
O
S
H
4
N
O
N
N
N
H
H
4
O
O
O
2
Hepsin Ki = 22.1 nM
Matriptase Ki > 20,000 nM

Highlights
• Hepsin is an attractive biomarker for the diagnosis of metastatic prostate cancer.
• Novel hepsin-targeted probes labeled with optical dyes were designed and synthesized.
• The compounds labeled with BODOPY or SulfoCy7 showed strong hepsin-inhibitory activities.
• Compound 2 exhibited selective uptake and retention in hepsin-overexpressing cells.